WO2021129843A1 - 三唑并三嗪衍生物在治疗疾病中的用途 - Google Patents

三唑并三嗪衍生物在治疗疾病中的用途 Download PDF

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WO2021129843A1
WO2021129843A1 PCT/CN2020/139690 CN2020139690W WO2021129843A1 WO 2021129843 A1 WO2021129843 A1 WO 2021129843A1 CN 2020139690 W CN2020139690 W CN 2020139690W WO 2021129843 A1 WO2021129843 A1 WO 2021129843A1
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compound
optionally substituted
triazine
furan
triazolo
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PCT/CN2020/139690
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English (en)
French (fr)
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孙三兴
陈正树
叶进启
赵龙
胡崇波
杨勇
管峰
潘姝花
胡宁
潘婷婷
宋国伟
侯方杰
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浙江春禾医药科技有限公司
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Priority to EP20908293.2A priority Critical patent/EP4083044A4/en
Priority to US17/789,126 priority patent/US20230048888A1/en
Priority to JP2022539212A priority patent/JP2023508182A/ja
Priority to CN202080089936.XA priority patent/CN114901665A/zh
Publication of WO2021129843A1 publication Critical patent/WO2021129843A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • 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
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152

Definitions

  • This application relates to the field of biomedicine, and specifically relates to a triazolotriazine derivative, which can treat diseases.
  • Parkinson's disease occurs as a result of chronic and progressive degeneration of neurons, the reasons for which are not yet fully understood. Its main clinical symptoms are static tremor, stiffness, bradykinesia and postural instability. Current treatment methods are mainly based on a dopamine replacement therapy that interferes with the dopaminergic and/or cholinergic signal cascade. However, these clinical symptoms often cannot be satisfactorily controlled from existing therapies. They are manifested in non-dopaminergic diseases of the brain, spinal cord and peripheral autonomic nervous system. Under existing therapies, many patients with Parkinson’s disease suffer from disabilities. . Therefore, the development and development of new therapies that can slow down, stop or reverse the progression of the disease is a top priority in Parkinson's disease research.
  • the application provides the use of a triazolotriazine derivative or a pharmaceutically acceptable solvate or salt thereof in the preparation of a medicine for the treatment of diseases, and a method for the treatment of diseases, including tumors and Parkinson's disease .
  • the compound of the present application has at least one of the following properties in the treatment of diseases: (1) good pharmacokinetic properties, higher body exposure and bioavailability; (2) improving the exercise capacity of Parkinson's subjects; And/or (4) inhibit tumor growth and reduce tumor volume.
  • the application provides the use of the compound of formula 1 in the preparation of medicines for the treatment of diseases
  • R is hydrogen or methyl;
  • Ar 1 is optionally substituted furyl, optionally substituted phenyl, or optionally substituted pyridyl; optionally optionally substituted aromatic ring is halogenated Or oxo group substitution;
  • Ar 2 is optionally substituted phenyl, optionally substituted pyridyl, or optionally substituted pyrimidinyl; optionally optionally substituted aromatic ring is halogen, Substitution by hydroxyl, cyano or methoxy;
  • X is oxygen or nitrogen;
  • Y and Z are each independently hydrogen, optionally substituted C 1-3 alkyl, optionally substituted C 1-5 cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted Substituted heterocycloalkylalkyl, optionally substituted aryl, optionally substituted C 1-3 alkylcarbonyl, optionally substituted C 1-5 cycloalkylcarbonyl, any Optionally substituted heterocycloalkylcarbonyl, optionally substituted heterocycloalkylalkylcarbonyl, optionally substituted arylcarbonyl, or optionally substituted heteroarylcarbonyl; any of The optionally substituted groups are halogen, hydroxy, methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, alkylamino, cycloalkylamino, heterocyclic, aryl, Heteroaryl, or C 1-3 alkyl polyoxyethylene substituted; alternatively, Y and Z are connected to form a hetero
  • R is hydrogen; Ar 1 is 2-furyl; Ar 2 is optionally substituted phenyl; any of the optionally substituted phenyl is substituted by halogen; X Is oxygen or nitrogen; and Y and Z are each independently hydrogen, C 2-3 alkyl optionally with substituents, heterocycloalkyl optionally with substituents, or heterocycloalkyl optionally with substituents Alkyl; any of the optionally substituted groups are substituted with methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, or C 1-2 alkyl polyoxyethylene; or Y And Z are connected to form a morpholinyl ring; or Y or Z is not present, or a pharmaceutically acceptable solvate or salt thereof.
  • R in the compound of formula 1 is hydrogen; Ar 1 is 2-furyl; Ar 2 is phenyl; X is nitrogen; and Y and Z are each independently hydrogen, optionally Substituent ethyl or optionally substituted oxetanyl; any of the optionally substituted groups is methoxy, ethoxy, trifluoromethoxy or trifluoroethoxy Group substitution; or Y and Z are connected to form a morpholinyl ring, or a pharmaceutically acceptable solvate or salt thereof.
  • the compound is selected from the group consisting of:
  • the present application provides the use of the compound of formula 2 in the preparation of drugs for the treatment of diseases
  • R is hydrogen or methyl;
  • Ar1 is optionally substituted furyl, optionally substituted phenyl, or optionally substituted pyridyl; optionally optionally substituted aromatic ring is Substituted by halogen or oxo;
  • Ar2 is optionally substituted phenyl, optionally substituted pyridinyl, or optionally substituted pyrimidinyl; optionally optionally substituted aromatic ring is halogen, Hydroxy, cyano or methoxy substitution;
  • Q is a 5-6 membered aromatic ring optionally substituted by X, an aminocarbonyl group optionally substituted by Y and Z on the nitrogen, an aminosulfonyl group optionally substituted by Y and Z on the nitrogen Group, nitro group, or cyano group;
  • X is halogen or optionally substituted C1-3 alkyl; any of the optionally substituted alkyl groups are halogen, cyano, Methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, aryl or heteroaryl substituted;
  • Y and Z are each independently hydrogen or optionally substituted C1-3 alkyl; any The optionally substituted alkyl group is substituted by halogen, hydroxy, methyl, alkylamino or cycloalkylamino; or Y and Z are connected to form an optionally substituted alkyl group having 5 to 7 ring atoms Substituted ring; any of the optionally substituted rings are substituted by hal
  • R is hydrogen;
  • Ar 1 is 2-furyl;
  • Ar 2 is phenyl or pyridyl; and
  • Q is nitro, cyano, or optionally X A substituted 5-6 membered aromatic ring;
  • X is an optionally substituted C1-3 alkyl group; any of the optionally substituted alkyl groups are halogen, cyano, methoxy, aryl or hetero Aryl substitution, or a pharmaceutically acceptable solvate or salt thereof
  • R is hydrogen; Ar1 is 2-furyl; Ar2 is phenyl; and Q is a tetrazole ring optionally substituted by X; X is optionally A substituted C1-3 alkyl group; any of the optionally substituted alkyl groups are substituted by halogen, cyano or methoxy, or a pharmaceutically acceptable solvate or salt thereof.
  • the compound is selected from the group consisting of:
  • the compound is selected from the group consisting of:
  • the pharmaceutically acceptable solvate or salt of the compound includes acetate and/or benzenesulfonate.
  • the pharmaceutically acceptable solvate or salt of the compound includes 2-(furan-2-yl)-N5-(4-(oxetan-3-ylamino)phenethyl Yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine acetate and/or N5-(4-(2- Methyl-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5 ] Triazine-5,7-diaminobenzenesulfonate.
  • the disease includes Parkinson's disease.
  • the disease includes tumors.
  • the tumor includes solid tumors and non-solid tumors.
  • the solid tumors include colon cancer and melanoma.
  • the non-solid tumor includes lymphoma, for example, B-cell lymphoma.
  • the medicament is prepared for oral administration and/or injection administration.
  • the medicament further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may include cyclodextrin.
  • the present application provides a method for treating diseases, which includes the following steps: administering a therapeutically effective dose of the compound described in the present application to a subject in need.
  • the disease includes Parkinson's disease.
  • the subject exhibits early signs of Parkinson's disease.
  • the subject has previously received or has not received drugs for the treatment of Parkinson's disease.
  • the subject receives a drug to treat Parkinson's disease before, at the same time or after administration of the compound.
  • the subject develops resistance to drugs used to treat Parkinson's disease.
  • the drug for treating Parkinson's disease includes dopamine.
  • the treatment includes reducing the rate of progression of Parkinson's disease in the subject, and/or delaying the clinical development of Parkinson's disease in the subject.
  • the rate of progression of Parkinson's disease of the subject is evaluated by UPDRS.
  • the treatment includes delaying the subject's need for symptomatic treatment against Parkinson's disease.
  • the treatment includes reducing the risk of the subject's need for symptomatic treatment against Parkinson's disease.
  • the disease includes tumors.
  • the tumor includes solid tumors and non-solid tumors.
  • the solid tumors include colon cancer and melanoma.
  • the non-solid tumor includes lymphoma, for example, B-cell lymphoma.
  • the method of administration of the compound is oral administration and/or injection administration.
  • the frequency of administration of the compound is once a day or twice a day.
  • the compound is administered at a dose of about 0.001 mg/kg to about 500 mg/kg.
  • the compound is administered at a dose of about 1 mg/kg to about 100 mg/kg.
  • the subject receives immunotherapy to treat tumors before, at the same time, or after administration of the compound.
  • the immunotherapy for treating tumors includes immune checkpoint inhibitors.
  • the immunotherapy for treating tumors includes PD-1 antibodies.
  • the present application provides a pharmaceutical combination or kit, which includes (1) the compound described in (1), and (2) a drug for treating Parkinson's disease.
  • the drug for treating Parkinson's disease includes dopamine.
  • the present application provides a pharmaceutical combination or kit, which includes (1) the compound described in, and (2) immunotherapy for the treatment of tumors.
  • the immunotherapy for treating tumors includes immune checkpoint inhibitors.
  • the immunotherapy for treating tumors includes PD-1 antibodies.
  • Figure 1 shows the XRPD results of the salt of the compound of the present application.
  • Figure 2 shows the 1 H-NMR spectrum result of the salt of the compound of the present application.
  • FIG. 3 shows the DSC and TGA results of the salt of the compound of the present application.
  • alkyl generally refers to straight-chain and branched-chain saturated aliphatic hydrocarbon groups containing a certain number of carbon atoms.
  • C4 alkyl includes n-butyl, isobutyl, sec-butyl, and tert-butyl.
  • cycloalkyl generally refers to monocyclic and bicyclic saturated aliphatic hydrocarbon groups containing a certain number of carbon atoms.
  • alkenyl generally refers to straight and branched aliphatic hydrocarbon groups containing at least one carbon-carbon double bond. For example, there is a carbon-carbon double bond.
  • aryl generally refers to monocyclic and bicyclic aromatic rings containing 5 to 14 ring atoms, and in some cases, 6 to 10 ring atoms.
  • the aryl group may be optionally substituted with one or more substituents.
  • aryl may also include monocyclic and bicyclic heteroaryl rings containing 5 to 14 ring atoms, and in certain cases, 6 to 10 ring atoms.
  • heterocycloalkyl generally refers to saturated monocyclic and bicyclic ring systems containing 3 to 14 ring atoms, for example, containing 4 to 10 ring atoms, wherein one or one of the atoms
  • the above are non-carbon elements, such as nitrogen, oxygen or sulfur. These hetero elements can exist alone or together with other hetero elements.
  • heterocycloalkyl groups include, for example, azetidinyl, hexahydroazapyridyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, tetrahydrofuranyl, thiomorpholinyl and tetrahydro Thienyl, and its N-oxide.
  • halogen is generally meant to include fluorine, chlorine, bromine and iodine.
  • trifluoromethyl generally refers to the "-CF3" group
  • hydroxyl hydroxyl or “hydroxy"
  • hydroxy generally refers to the "-OH” group.
  • anti-Parkinson's disease symptomatic treatment generally includes any therapy for the treatment of Parkinson's disease, including but not limited to bromocriptine, benztropine, levodopa, ropinirole, pramac Sodium, rotigotine, cabergoline, entacapone, tolcapone, amantadine, pergolide, apomorphine, lisuride, or selegiline.
  • the term “delayed need for symptomatic treatment of anti-Parkinson's disease” refers to the need for anti-Parkinson's disease symptomatic treatment of Parkinson's disease patients who have not received the compound described in this application in contrast to patients who have not received the compound described in this application.
  • the term "reducing the rate of progression of Parkinson's disease” generally refers to reducing the deterioration experienced by patients with Parkinson's disease. For example, this deterioration can be compared to Parkinson's disease that has not received the compound described in this application for a period of time.
  • the deterioration experienced by patients with the disease is quantified by the UPDRS score.
  • the development speed is quantified by the Total Unified Parkinson’s Disease Rating Scale (Total Unified Parkinson’s Disease Rating Scale, total UPDRS) score.
  • the increase in the total UPDRS score represents the symptom development of Parkinson's disease, and the increase in the increase of the UPDRS score over a period of time represents the progress of the symptoms of Parkinson's disease.
  • the term “functional decline” generally refers to the deterioration of the symptoms of Parkinson's disease patients over time as determined by the total UPDRS score.
  • the term "delaying the need for symptomatic treatment of anti-Parkinson's disease” generally refers to patients with Parkinson's disease who have not received the compound described in this application, and delaying Parkinson's disease patients receiving the compound against the symptomatic treatment of Parkinson's disease. The need for treatment.
  • neurological signs of Parkinson's disease generally includes one or more of the following signs:
  • the tremor is greatest when at rest, decreases when moving, and there is no tremor when sleeping;
  • the stage of Parkinson's disease in this application refers to the following five significant stages described by Hoehn and Yahr depending on the symptoms (Hoehn MM, Yahr MD, Parkinsonism: onset, progression and mortality. Neurology 1967, 17: 427 -42).
  • Stage I (Mild or early disease): Symptoms affect one side of the body.
  • Stage II Both sides of the body are affected, but the posture remains normal.
  • Stage III (moderate disease): Both sides of the body are affected, and there is a slight imbalance in standing and walking; but the patient can remain independent.
  • Stage IV (advanced disease): Both sides of the body are affected and show impaired instability during standing and walking; patients at this stage need a lot of help.
  • Stage V manifested as a serious, fully developed disease; the patient is confined to a bed or chair.
  • “early Parkinson's disease patient” refers to a patient who is at stage I or II of Parkinson's disease defined by Hoehn and Yahr, who does not require symptomatic treatment for anti-Parkinson's disease. For example, those patients do not need anti-Parkinson's symptomatic treatment for at least the next 9 months. The relevant tests can be performed to confirm those with Parkinson's disease at an early stage.
  • the term "delay the clinical development of Parkinson's disease” generally refers to the control of subjects who did not receive the compound described in this application, and the subject who received the compound has undergone the complete symptomatic effect of the compound. During the period, for example, 12 weeks after receiving the compound, a lower rate of increase of the total UPDRS score was obtained. Or control subjects who received the compound treatment with a delay, after a sufficient period of time to eliminate the changes caused by the delay in starting the compound treatment, the total UPDRS score showed a lower deterioration. Sufficient time is necessary to exceed 52 weeks (for example, at least more than 72 weeks) after initial treatment with the compound. Or control subjects who received delayed treatment with the compound, during the period after the delay initiating the symptomatic effect of the compound treatment, the total UPDRS score showed a substantially similar rate of deterioration, for example, the total UPDRS per week was within 0.15 units.
  • cancer and “tumor” are used interchangeably, and generally refer to cells that have caused malignant transformation or cellular changes that have caused abnormal or unregulated growth or hyperproliferation. Such changes or malignant transformation usually make such cells pathogenic to the host organism, and therefore are also expected to include precancerous or precancerous cells that will or may become pathogenic and require or can benefit from intervention.
  • the tumor may include solid tumors and non-solid tumors.
  • this application provides a compound represented by Formula 1, as well as its hydrates, solvates, pharmaceutically acceptable salts, prodrugs and their complexes, for use in medicines for the treatment of diseases:
  • R can be hydrogen or optionally substituted C 1-5 alkyl; any optionally substituted alkyl is halogen, cyano, hydroxy, nitro, amino, alkylamino, cycloalkane Amino, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy or trifluoroethoxy substituted;
  • Ar 1 can be a 5-6 membered aromatic ring , The 5-6 membered aromatic ring is optionally substituted by halogen, oxo, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy;
  • Ar2 can be a monocyclic or bicyclic aromatic ring , The monocyclic or bicyclic aromatic ring is optionally substituted by halogen, hydroxyl, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy;
  • X is selected from the following group: oxygen
  • Y and Z may each independently be hydrogen, optionally substituted C 1-9 alkyl, optionally substituted monocyclic or bicyclic C 1-9 cycloalkyl, optionally substituted C 1 -9 alkenyl, optionally substituted monocyclic or bicyclic C 1-9 cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl Group, optionally substituted heteroaralkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenyl, optionally substituted heterocycloalkylalkyl, optionally substituted C 1-9 alkylcarbonyl with substituents, C 1-9 cycloalkylcarbonyl with optional substituents, heterocycloalkylcarbonyl with optional substituents, heterocycloalkylalkyl with optional substituents Group carbonyl, optionally substituted arylcarbonyl, or optionally substituted heteroarylcarbonyl; the optionally substituted group may be substituted
  • R in the compound represented by Formula 1 may be hydrogen, methyl, or trifluoromethyl. In some specific cases, R in the compound represented by Formula 1 may be hydrogen or methyl. For example, R in the compound represented by Formula 1 may be hydrogen.
  • the compound represented by formula 1 Ar 1 can be substituted imidazolyl group optionally zones, triazolyl, tetrazolyl, furyl, thienyl, oxazolyl, isoxazolyl , Thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl; the aromatic ring optionally with substituents is Halogen, oxo, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy substitution.
  • the compound represented by formula 1 Ar 1 an aromatic group may be selected from Table (1).
  • Ar 1 in the compound represented by Formula 1 may be 2-furyl.
  • Ar 2 in the compound represented by Formula 1 may be an imidazolyl, triazolyl, tetrazolyl, furyl, thienyl, oxazolyl, or isoxazolyl optionally substituted.
  • Ar 2 in the compound represented by Formula 1 may be optionally substituted phenyl, pyridyl, pyridazinyl or pyrimidinyl; the optionally substituted aromatic ring may be Halogen, hydroxyl, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy substitution.
  • Ar 2 in the compound represented by Formula 1 may be phenyl or pyridyl, and the phenyl or pyridyl may be optionally substituted with halogen or hydroxy.
  • X in the compound represented by Formula 1 is oxygen or nitrogen.
  • X in the compound represented by Formula 1 is nitrogen.
  • Y and Z in the compound represented by formula 1 may each independently be hydrogen, optionally substituted C 2-5 alkyl, optionally substituted C 3-5 ring Alkyl, optionally substituted C 2-5 alkenyl, optionally substituted C 3-5 cycloalkenyl, optionally substituted aryl, optionally substituted hetero Aryl, optionally substituted aralkyl, optionally substituted heteroaralkyl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkyl Alkyl; any of the optionally substituted groups may be halogen, cyano, methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy or C 1-3 alkyl polyoxy Ethylene substitution
  • Y and Z in the compound shown in Formula 1 may each independently be hydrogen, optionally substituted C 2-3 alkyl, or optionally substituted heterocycloalkyl ; Any of the optionally substituted groups may be substituted by methoxy, ethoxy, trifluoromethoxy or trifluoroethoxy;
  • Y and Z in the compound represented by Formula 1 may each independently be hydrogen, optionally substituted ethyl, or optionally substituted oxetanyl; any The optionally substituted groups may be substituted by methoxy, ethoxy, trifluoromethoxy or trifluoroethoxy;
  • Y and Z in the compound shown in Formula 1 can be connected to form an optionally substituted ring having 4 to 8 ring atoms; any of the optionally substituted groups is
  • the substituent of can be halogen, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy or trifluoroethoxy;
  • Y and Z in the compound represented by formula 1 can be connected to form an optionally substituted heterocycloalkane having 5 to 7 ring atoms; any of the optionally substituted heterocycloalkanes The group can be substituted by halogen, oxo, methoxy, ethoxy, trifluoromethoxy or trifluoroethoxy;
  • Y and Z in the compound shown in Formula 1 may be connected to form a morpholinyl ring optionally with substituents; any of the optionally substituted groups may be substituted by halogen, methyl Oxy, ethoxy, trifluoromethoxy or trifluoroethoxy substitution.
  • the present application provides a triazolotriazine compound represented by Formula 2, as well as its hydrate, solvate, pharmaceutically acceptable salt, prodrug, and complex thereof, which are useful in medicines for the treatment of diseases use:
  • R may be hydrogen or optionally substituted C1-5 alkyl; any optionally substituted alkyl may be halogen, cyano, hydroxy, nitro, amino, alkylamino, cycloalkane Substitution of group amino, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy or trifluoroethoxy;
  • Ar 1 may be a 5-6 membered aromatic ring, and the 5-6 membered aromatic ring may be optionally substituted by halogen, oxo, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy Substitution;
  • Ar 2 can be a monocyclic or bicyclic aromatic ring, the monocyclic or bicyclic aromatic ring can be optionally halogen, hydroxyl, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy replace;
  • Q may be a monocyclic or bicyclic aromatic ring optionally substituted by X, an aminocarbonyl group optionally substituted by Y and Z on the nitrogen atom, an aminosulfonyl group optionally substituted by Y and Z on the nitrogen atom, Nitro, or cyano substitution.
  • X can be halogen, cyano, hydroxy, nitro, amino, alkylamino, methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, optionally substituted C 1-9 alkane Group, optionally substituted C 1-9 cycloalkyl, optionally substituted C 1-9 alkenyl, optionally substituted C 1-9 cycloalkenyl, optionally substituted Substituent aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted heteroaralkyl, optionally substituted heterocycloalkane Group, or optionally substituted heterocycloalkenyl; the aforementioned optionally substituted group may be halogen, cyano, hydroxy, nitro, amino, alkylamino, cycloalkylamino, aminocarbonyl, Sulfonyl, aminosulfonyl, carbonylamino, sulfonylamino, methyl, ethy
  • Y and Z may each independently be hydrogen, optionally substituted C 1-9 alkyl, optionally substituted monocyclic or bicyclic C 1-9 cycloalkyl, optionally substituted C 1-9 alkenyl group, optionally substituted monocyclic or bicyclic C 1-9 cycloalkenyl group, optionally substituted aryl group, optionally substituted heteroaryl group, any Optionally substituted aralkyl, optionally substituted heteroaralkyl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl; any of the above Optionally substituted groups can be substituted by halogen, cyano, hydroxy, nitro, amino, alkylamino, cycloalkylamino, aminocarbonyl, sulfonyl, aminosulfonyl, carbonylamino, sulfonylamino, methyl , Ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethy
  • R in the compound represented by Formula 2 may be hydrogen, methyl, or trifluoromethyl. In some specific cases, R in the compound represented by Formula 2 may be hydrogen or methyl. For example, R in the compound represented by Formula 2 is hydrogen.
  • Ar 1 in the triazolotriazine compound shown in Formula 2 may be imidazolyl, triazolyl, tetrazolyl, furyl, thienyl, oxazolyl, which may have substituents, Isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, or triazinyl; any of the substituents may be Substituted by halogen, oxo, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy.
  • Ar 1 in the compound shown in Formula 2 is selected from the aromatic groups in Table 2.
  • Ar 1 in the compound represented by Formula 2 is 2-furyl.
  • the compound of Formula 2 is Ar 2 may have a substituent group, imidazolyl optionally zones, triazolyl, tetrazolyl, furyl, thienyl, oxazolyl, isoxazolyl , Thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl, isoindolyl, indazole Group, benzimidazolyl, azaindolyl, azaindazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzothienyl, benzisoxazolyl, benzisothiazole, Benzoxazolyl, benzothiadiazole, quinolinyl, isoquinolinyl, quinoxalinyl, is
  • Ar 2 in the compound represented by formula 2 may be optionally substituted phenyl, pyridyl, pyridazinyl or pyrimidinyl; any of the optionally substituted aromatic rings may Substituted by halogen, hydroxyl, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy.
  • the compound of Formula 2 is Ar 2 may be optionally substituted with halogen or hydroxyl-substituted phenyl or pyridyl group.
  • Q in the compound represented by formula 2 may be a monocyclic or bicyclic aromatic ring optionally substituted by X;
  • X may be halogen, cyano, hydroxyl, nitro, amino, alkylamino, Methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, optionally substituted C 1-9 alkyl, optionally substituted C 1-9 cycloalkyl, any Optionally substituted C 1-9 alkenyl, optionally substituted C 1-9 cycloalkenyl, optionally substituted aryl, optionally substituted heteroaryl, any Optionally substituted aralkyl, optionally substituted heteroaralkyl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkenyl;
  • the group with optional substituents can be substituted by halogen, cyano, hydroxy, nitro, amino, alkylamino, cycloalkylamino, aminocarbonyl, sulfonyl
  • Q in the compound represented by Formula 2 may be a 5-6 membered aromatic ring optionally bearing a substituent X.
  • X can be halogen, cyano, hydroxyl, nitro, amino, alkylamino, methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, C 1-5 alkane which may have substituents Group, C 1-5 cycloalkyl which may have substituents, C 1-5 alkenyl which may have substituents, C 1-5 cycloalkenyl which may have substituents, aryl which may have substituents Groups, heteroaryl groups that may have substituents, aralkyl groups that may have substituents, heteroaralkyl groups that may have substituents, heterocycloalkyl groups that may have substituents, or substituted groups
  • the heterocycloalkenyl; the substituted groups are halogen, cyano, hydroxyl, nitro, amino, alkylamino, cycloal
  • Q in the compound represented by Formula 2 may be a tetrazole ring optionally substituted with X.
  • X may be optionally substituted C 1-3 alkyl or optionally substituted heterocycloalkyl; any of the optionally substituted base groups may be halogen, cyano, hydroxy, methyl Group, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy, trifluoroethoxy, heterocycloalkyl, aryl, or heteroaryl.
  • the compounds described in this application may exist in the form of one or more geometric isomers, enantiomers, diastereomers or tautomers.
  • the compounds described in the application may include all these forms of isomers, including racemates and other forms of mixtures.
  • the compounds described in this application may exist in the form of solvates or unsolvates.
  • solvated is used herein to describe a compound complex comprising a compound of the invention and pharmaceutically acceptable solvent molecules such as water and ethanol molecules.
  • solvent molecules such as water and ethanol molecules.
  • the compounds described in this application include all their solvated or unsolvated forms.
  • the compounds described in this application may exist in the form of pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt refers to a physiologically or toxicologically acceptable salt, and when appropriate, includes its pharmaceutically acceptable base addition salt and acid addition salt.
  • the compounds described in this application include all pharmaceutically acceptable salts thereof.
  • the pharmaceutically acceptable salt may include benzenesulfonate and/or acetate.
  • the compounds described in this application may exist in the form of pharmaceutically acceptable nanoparticles.
  • Nanoparticles containing the compound can be designed to improve the pharmacokinetics and biodistribution properties of the drug.
  • the compound can be encapsulated in liposomes, which may be able to extend the life of the drug in the process of distribution in the body. Since nanoparticles will preferentially leak out in the porous blood vessels surrounding tumor cells, nanoparticles of appropriate size can also have better safety. Doing so can also help reduce the effective dose of the drug.
  • the compounds described in this application may exist in the form of prodrugs.
  • prodrug generally refers to a compound that can be converted into the compound of the present application through a metabolic process in the body (for example, by hydrolysis, reduction, or oxidation).
  • the compounds described in this application include all forms of prodrugs.
  • the compounds described in this application also include pharmaceutically acceptable isotopic variants, that is, one or more atoms are substituted with atoms having the same atomic number but different atomic masses.
  • Atoms suitable for such isotopic replacement include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine.
  • Some isotopic variants of the compounds, such as deuterium-substituted compounds, may have better therapeutic effects in some cases because of better metabolic stability. Those skilled in the art can use conventional techniques known in the art to prepare isotopic variants of the compound.
  • the compound may include the following compounds, or pharmaceutically acceptable salts thereof:
  • the compounds described in this application can be prepared by various synthetic methods.
  • two synthetic routes for preparing the target compound are listed in synthetic route (1).
  • the first method after the intermediate (1A) is obtained by a suitable preparation method, the methylsulfonyl group on the intermediate (1A) is substituted with an alkylamino group to obtain the triazolotriazine compound (1C).
  • the second method after intermediate (1B) is obtained in an appropriate manner, the phenoxy group on intermediate (1B) is substituted with an alkylamino group to obtain the triazolotriazine compound (1C).
  • the intermediates (1A) and (1B) required in the synthetic route (1) can also be prepared by various synthetic methods.
  • a synthetic route of the intermediate compound (1A) is shown in the synthetic route (2).
  • an appropriate aryl hydrazide (2A) is reacted with S-methyl isothiourea (2B) in an aqueous sodium hydroxide solution to obtain the intermediate (2C).
  • S-methyl isothiourea (2B) in an aqueous sodium hydroxide solution to obtain the intermediate (2C).
  • the intermediate (2D) reacts with N-cyanodithioimino carbonate (2E) to obtain the sulfide intermediate (2F).
  • the intermediate (2F) is oxidized with m-chloroperoxybenzoic acid to obtain the desired intermediate sulfone (1A).
  • the target compound (1C) can be obtained by nucleophilic substitution of the methylsulfonyl group with a suitable alkylamine (J. Chem. Soc., Perkin Trans. 1 1995, 801-808; Structural Chemistry) .2013, 24, 1241–1251).
  • the intermediate (2D) in the synthetic route (2) can also be prepared by various synthetic methods.
  • synthetic route (3) shows three synthetic routes of intermediate (2D). It is worth emphasizing that among these three methods, the synthetic route starting from methyl or ethyl (3F) can generally improve efficiency and achieve higher reaction yields.
  • the intermediate (1B) in the synthetic route (1) can be prepared by the synthetic route shown in the synthetic route (4).
  • the preparation starts with cyanuric chloride (4A) and refluxes in phenol to obtain 2,4,6-triphenoxy-1,3,5-triazine (4B).
  • the next step is to react with hydrazine hydrate to obtain 2-hydrazine-4,6-diphenoxy-1,3,5-triazine (4C), which is reacted with a suitable acid chloride to obtain acylhydrazide (4D).
  • acylhydrazide (4D) undergoes cyclization reaction under dehydration conditions to obtain 2-substituted 5,7-diphenoxytriazolotriazine (4E).
  • (4E) is refluxed in methanol ammonia
  • the key intermediate (1B) can be obtained (J. Chem. Soc., Perkin Trans. 1 1995, 801-808).
  • the target compound (1C) can be prepared by reacting with (1B) with a suitable alkylamine.
  • the compounds described in this application can bind to adenosine receptors, for example, can bind to adenosine A2A receptors.
  • the compounds described in this application can bind to free adenosine receptor proteins, and can also bind to adenosine receptors on the cell surface.
  • the compounds described in the present application can selectively bind to adenosine receptors.
  • the compounds of the present application are capable of binding to adenosine A2A receptors at least 10-fold, 20-fold, 30-fold, and 40-fold to adenosine A1, A2B and A3 receptors Times, 50 times, 100 times, 200 times, 300 times, 500 times or higher.
  • the compounds of the present application is incorporated the IC 50 value is the adenosine A2A receptor binding adenosine A1, A2B and 50 at least 10-fold value IC A3 receptors, 20-fold, 30-fold , 40 times, 50 times, 100 times, 200 times, 300 times, 500 times or higher.
  • the compounds described in this application can inhibit the activity of adenosine receptors, for example, can inhibit the activity of adenosine A2A receptors.
  • the compounds described in this application can inhibit the activity of free adenosine receptor proteins and can also inhibit the activity of adenosine receptors on the cell surface.
  • the compounds described in the present application can selectively inhibit the activity of adenosine receptors.
  • the compounds of the present application can inhibit the activity of adenosine A2A receptors at least 10-fold, 20-fold, and inhibit the activity of adenosine A1, A2B, and A3 receptors. 30 times, 40 times, 50 times, 100 times, 200 times, 300 times, 500 times or higher.
  • the application of the present compounds to inhibit adenosine A2A receptor activity IC 50 values of inhibiting adenosine A1, A2B and A3 receptor activity IC 50 values at least 10-fold, 20-fold , 30 times, 40 times, 50 times, 100 times, 200 times, 300 times, 500 times or higher.
  • the compounds described in this application can be used to prepare drugs for the treatment of diseases, wherein the diseases may include Parkinson's disease.
  • the present application provides a method for treating Parkinson's disease, which may include the following steps: administering a therapeutically effective dose of the compound described in the present application to a subject in need.
  • the subject may exhibit early signs of Parkinson's disease
  • the method includes confirming that the patient exhibits early signs of Parkinson's disease, and periodically administering to the patient the determined amount of the The compound or a pharmaceutically acceptable solvate or salt thereof effectively treats patients.
  • the rate of progression of Parkinson's disease of the subject can be quantified by a Total Unified Parkinson's Disease Rating Scale (Total Unified Parkinson's Disease Rating Scale, total UPDRS) score.
  • Total Unified Parkinson's Disease Rating Scale Total Unified Parkinson's Disease Rating Scale
  • Early signs may also be olfactory disorders, depression, visual impairment and cognitive impairment, or sleep disorders.
  • a combination of different tests can be used for early diagnosis (Becker, J Neurol 249, Suppl 3, 2002 III/40; Stern, Annals of Neurol 56,2004,169).
  • the subject may exhibit functional decline in Parkinson's disease, which generally refers to the deterioration of the symptoms of Parkinson's disease patients over time as determined by the total UPDRS score.
  • the subject may exhibit fatigue of Parkinson's disease, and the functional decline generally refers to the fatigue state of a Parkinson's disease patient determined by the total UPDRS score.
  • the subject may exhibit non-motor symptoms of Parkinson's disease, and the non-motor symptoms generally refer to the non-motor symptoms of Parkinson's disease patients determined by the total UPDRS score.
  • the non-motor symptoms of Parkinson's disease can include autonomic nervous system dysfunction, neuropsychiatric disorders (including changes in mood, cognition, behavior, and thought), sensory disorders, and sleep disorders.
  • the subject has previously received or has not received treatments for Parkinson’s disease, including medications, rehabilitation (such as physical exercise, improvement of gait, slow rotation of limbs or trunk, stretching exercises, oral language Guidance, Lee Silverman voice therapy, stepping exercises, environmental modification, rhythm initiation, abdominal breathing and meditation), palliative medicine, diet therapy, repetitive transcranial magnetic stimulation, cautery, deep brain stimulation surgery, pallidectomy Surgery.
  • rehabilitation such as physical exercise, improvement of gait, slow rotation of limbs or trunk, stretching exercises, oral language Guidance, Lee Silverman voice therapy, stepping exercises, environmental modification, rhythm initiation, abdominal breathing and meditation
  • palliative medicine diet therapy
  • repetitive transcranial magnetic stimulation cautery
  • deep brain stimulation surgery pallidectomy Surgery.
  • the subject has previously received or has not received drugs for the treatment of Parkinson's disease.
  • Drugs for treating Parkinson's disease may include, but are not limited to, L-dopa, dopamine receptor agonists, monoamine oxidase inhibitors, other drugs such as amantadine and anticholinergics.
  • Drugs for the treatment of Parkinson's disease may include: L-dopa, bromocriptine, benztropine, levodopa, ropinirole, pramipexole, rotigotine, cabergoline, safinamide, Xilijilin, rasagiline, entacapone, tocapone, amantadine, pergolide, apomorphine, lisuride, or selegiline
  • the treatment may include reducing the rate of progression of Parkinson's disease in the subject, and/or delaying the clinical development of Parkinson's disease in the subject.
  • the rate of disease progression is a decrease in the average increase of the total UPDRS score.
  • the treatment may include delaying the subject's need for symptomatic treatment against Parkinson's disease. In some embodiments, the treatment may include determining that the patient is in the stage of Parkinson's disease, and periodically administering the compound or a pharmaceutically acceptable solvate or salt thereof to the patient thereby to effectively delay its resistance. The need for symptomatic treatment of Parkinson's disease.
  • the treatment may include reducing the risk of the subject's need for symptomatic treatment against Parkinson's disease.
  • the treatment includes regularly administering a certain amount of the compound described herein or a pharmaceutically acceptable solvate or salt thereof to a Parkinson's disease patient to effectively reduce the risk of anti-Parkinson's treatment needs .
  • the treatment may include reducing the functional decline of the subject.
  • the method includes determining that the patient is in the stage of Parkinson's disease, and periodically administering to the patient the determined amount of the compound or pharmaceutically acceptable solvate or salt thereof, effectively reducing its functional decline .
  • the functional decline of patients with Parkinson's disease is quantified by the Total Parkinson's Disease Unified Score Scale (Total UPDRS) score, and an increase in the total UPDRS score represents functional decline.
  • Total UPDRS Total Parkinson's Disease Unified Score Scale
  • the treatment may include reducing the fatigue of the subject.
  • the method includes determining that the patient is in the stage of Parkinson's disease, and periodically administering the compound described herein or a pharmaceutically acceptable solvate or salt thereof to the patient, thereby effectively reducing fatigue .
  • the treatment may include reducing the severity of motor symptoms in the subject.
  • the motor symptoms may mainly include tremor (such as static tremor), stiffness of the limbs, slow movement, unstable posture, hypokinesia, slow movement, abnormal posture, speech and swallowing, etc.
  • the treatment may include reducing the severity of the non-motor symptoms in the subject.
  • the method includes determining that the patient is in the stage of Parkinson's disease, and periodically administering the compound or a pharmaceutically acceptable solvate or salt thereof to the patient in such a determined amount to effectively reduce non-motor symptoms Severity.
  • the non-motor symptoms are defined by the Unified Parkinson's Disease Rating Scale (UPDRS) Fourth Edition Part One.
  • the total UPDRS (Unified Parkinson’s Disease Rating Scale) score represents the severity of Parkinson’s disease and is used to measure the change in efficacy parameters from baseline during treatment.
  • UPDRS consists of the following partial tests:
  • UPDRS consists of the following parts: Part 1: Psychological, behavioral and emotional assessment, Part 2: Self-assessment of activities of daily living (ADL), including speech, swallowing, handwriting, dressing, hygiene, falling, salivation, bed rest , Walking and Cutting Food, Part 3: Monitoring Movement Evaluation by Clinician Score, Part 4: Treatment Complications, Part 5: Hoehn and Yahr Stages of Parnson’s Disease Severity, Part 6: Schwab and England ADL Scale.
  • ADL daily living
  • the MDS-UPDRS issued by the Movement Disorder Association can also be used to score Parkinson’s disease. It contains four parts and a total of 50 subscales. They are: (1) Daily life non-exercise experience (13 items), (2) Daily life Exercise experience (13 items), (3) physical exercise examination (18 items), and (4) sports complications (6 items).
  • the compound described in this application can reduce the score of one or more UPDRS subscale items of a subject from 4 points to 3 points, 4 points to 2 points, 4 points to 1 point, and 4 points to 0 Points, 3 points are reduced to 2 points, 3 points are reduced to 1 point, 3 points are reduced to 0 points, 2 points are reduced to 1 point, 2 points are reduced to 1 point, or 1 point is reduced to 0 points.
  • the compound of the present application can extend the walking distance, walking time, upright times and upright time of a subject.
  • the walking distance of the subject using the compound of the application can be extended by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or higher, as detected in a mouse dyskinesia model induced by reserpine.
  • the walking time of the subject using the compound of the application can be prolonged by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or higher, as detected in a mouse dyskinesia model induced by reserpine.
  • the number of uprights of the subject using the compound of the application can be prolonged by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or higher, as detected in a mouse dyskinesia model induced by reserpine.
  • the upright time of the subject using the compound of the application can be prolonged by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or higher, as detected in a mouse dyskinesia model induced by reserpine.
  • the compound of the present application can reduce the subject's stick time, increase the standing time, and effectively improve the subject's stiffness symptoms.
  • a subject who uses the compound of the application can reduce at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, At least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300% or higher, as detected in the haloperidol-induced rat Parkinson’s disease model.
  • the standing time of the subject using the compound of the application can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300% or higher, such as haloperidol-induced rat Parkinson’s disease stiffness model or MPTP-induced mouse Parkinson’s model Detected.
  • the walking distance of the subject using the compound of the application can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300% or higher, as detected in the MPTP-induced mouse Parkinson's model.
  • the administered dose of the compound in the treatment of Parkinson's disease, may be about 0.001 to about 500 mg/kg, about 0.001 to about 500 mg/kg, about 0.01 to about 500 mg/kg, about 0.1 to about 500 mg/kg.
  • the administered dose of the compound may be about 1 mg/kg, about 3 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 30 mg/kg, about 50 mg/kg, or about 100 mg/kg.
  • the compound described in the present application can inhibit the drug resistance of a subject to Parkinson's drugs, for example, can inhibit the subject's resistance to levodopa (L-DOPA) or Benserazide.
  • L-DOPA levodopa
  • Benserazide levodopa
  • the compounds described in this application can also be used to prepare drugs for the treatment or prevention of host cancer and related diseases related to abnormal cell proliferation in the host.
  • the host can be any multicellular vertebrate, including humans and non-human mammals.
  • the host may be a human.
  • the diseases include tumors.
  • the tumor may include solid tumors and non-solid tumors.
  • the solid tumor may include colon cancer and melanoma.
  • the non-solid tumor may include lymphoma, for example, B-cell lymphoma.
  • the administered dose of the compound in the treatment of tumors, may be about 0.001 to about 500 mg/kg, about 0.001 to about 500 mg/kg, about 0.01 to about 500 mg/kg, about 0.1 to about 500 mg/kg, about 1 to about 500 mg/kg, about 0.001 to about 400 mg/kg, about 0.001 to about 300 mg/kg, about 0.001 to about 200 mg/kg, about 0.001 to about 100 mg/kg, about 0.001 to about 50 mg/kg, about 1 to About 200 mg/kg, about 1 to about 100 mg/kg, about 1 to about 50 mg/kg, about 1 to about 30 mg/kg, about 10 to about 30 mg/kg, about 3 to about 30 mg/kg, or about 5 to about 30 mg /kg.
  • the administered dose of the compound may be about 1 mg/kg, about 3 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 30 mg/kg, about 50 mg/kg, or about 100 mg/kg.
  • the compounds described in this application can be used to improve the anti-tumor activity of host immune cells.
  • the compound can reduce the immune incompetence of T cells or the tolerance of T cells to cancer, can make cancer cells more susceptible to immune clearance, can inhibit the proliferation of regulatory T cells, and can contribute to the formation of memory T cells.
  • the compound can not only improve the immune response of the host itself, but also improve its efficacy on various adaptive immunotherapy.
  • the drug of the present application can inhibit or delay the development or progression of the disease, can reduce the size of the tumor (or even substantially eliminate the tumor), and/or can alleviate and/or stabilize the disease state.
  • Examples of inhibiting the growth of tumors or tumor cells include: a reduction in tumor growth volume relative to the corresponding level before the intervention.
  • the present application provides a method for treating tumors, which may include the following steps: administering a therapeutically effective dose of the compound described in the present application to a subject in need.
  • the compounds described in this application can also be used in combination with other anti-tumor treatment methods (such as chemotherapy, tumor vaccines, and various immune checkpoint inhibitors) to achieve a synergistic effect.
  • anti-tumor treatment methods such as chemotherapy, tumor vaccines, and various immune checkpoint inhibitors
  • combination therapy refers to the simultaneous administration of active agents, or they can be administered sequentially in different orders.
  • the method of treating or preventing abnormal cell proliferation in the host may include the combined or alternate administration of the compound described in this application and an immune checkpoint inhibitor to the patient.
  • the immune checkpoint inhibitors here can be selected from the following group: PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, BTLA inhibitors, LAG3 inhibitors, TIM-3 inhibitors , B7-H3 inhibitor, B7-H4 inhibitor, KIR inhibitor, TIGIT inhibitor or VISTA inhibitor, etc.
  • the other anti-tumor treatment method may be the PD-1 inhibitor.
  • the method of treating or preventing abnormal cell proliferation in the host may include administering or alternating the compound described in this application and a cell vaccine to the patient in combination.
  • the cell vaccine is based on cells that match the tumor to be prevented. For example, if a host has prostate cancer, or is at risk of prostate cancer, the cell vaccine will be based on prostate cancer cells. In this case, the cells are usually irradiated with a certain amount of radioactivity or otherwise lose their replication function.
  • the cells can also be genetically modified to secrete colony stimulating factors.
  • the method of treating or preventing abnormal cell proliferation includes administering the compound and the chimeric antigen receptor CAR-T cell therapy to the patient in combination or alternately.
  • the method of treating or preventing abnormal cell proliferation may include administering the compound and one other anti-cancer drug in combination or alternately to the patient to treat abnormal cell proliferation.
  • anti-cancer drugs can be alkylating agents, anti-metabolic drugs, anthracycline derivatives, plant alkaloids, topoisomerase inhibitors, anti-tumor antibiotics, kinase inhibitors, or anti-tumor antigen monoclonal antibodies.
  • the compound of the application has good pharmacokinetic properties, for example, the compound of the application has a relatively high exposure and bioavailability. In certain embodiments, the compound of the present application has a higher oral in vivo exposure and a higher oral bioavailability.
  • the blood T 1/2 of the compound of the present application is about 0.1 h to about 10 h, for example, about 0.2 h to about 10 h, about 0.3 h to about 10 h, about 0.4 h to about 10 h, about 0.5 h to about 10 h, about 0.5h to about 9h, about 0.5h to about 8h, about 0.5h to about 7h, about 0.5h to about 6h, about 0.5h to about 5h, about 0.5h to about 3h, about 0.5h to about 2h, about 0.5 h to about 1h.
  • the compound of the present application can be formulated into an appropriate pharmaceutical composition.
  • the specific composition of the pharmaceutical composition is determined by the selected route of administration, which includes oral, parenteral, intravenous, intramuscular, nasal, oral, topical, transdermal or subcutaneous.
  • the dosage of the compound described in the application contained in the pharmaceutical composition should be sufficient to have an effective therapeutic effect, while not causing serious toxic and side effects to the host. It can be taken every day or every few days, and the medication can last for days, weeks, months, or even years.
  • a specific dose can be divided into multiple intervals for administration, such as once a day, twice a day or more, once a week, once every two weeks, once every three weeks, once a month, or once every two or more months .
  • the drug can be administered once a day or twice a day.
  • the compound can be administered at intervals before or after other anti-tumor treatment methods.
  • the interval time can be 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18. Hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months or longer.
  • the compounds described in this application can be administered by the same route of administration as other anti-tumor treatment methods or by a different route of administration.
  • the compound may be administered at intervals before or after other drugs for the treatment of Parkinson's disease.
  • the interval time can be 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 Hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months or longer.
  • the compounds described in this application can be administered by the same route of administration as other drugs for the treatment of Parkinson's disease or by a different route of administration.
  • the compound can be administered orally.
  • the ingredients of oral drugs usually include an inert diluent or an edible carrier.
  • the drug can be enclosed in gelatin capsules or compressed into tablets. Tablets, pills, capsules, lozenges and other dosage forms may contain the following ingredients: binders such as microcrystalline cellulose, tragacanth gum or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid or corn starch; lubricants such as hard Magnesium fatty acid; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate or orange flavoring agents; wetting agents or emulsifiers; preservatives; and pH buffering agents Agents, such as phosphate buffer, sodium acetate or sorbitan monolaurate.
  • a liquid carrier such as fatty oil can also be placed in it.
  • the pharmaceutical composition When the pharmaceutical composition is placed in a capsule
  • the compound of the present application can also be administered as components such as elixirs, suspensions, syrups, cakes, or chewing gum.
  • the syrup may also contain sweetening agents such as sucrose, preservatives, coloring agents and flavoring agents.
  • compositions or suspensions for oral, parenteral, intradermal, subcutaneous or topical may include sterile diluents, such as water, saline solution, Ringer's solution, fixed oil, polyethylene glycol (such as PEG400), Glycerin, propylene glycol, fatty acids such as oleic acid and its derivatives, polar solvents (such as dimethylacetamide (DMAC)) or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl p-benzene, antioxidants such as ascorbic acid or sodium sulfite , Chelating agents such as ethylene diamine tetraacetic acid; buffers such as phosphate buffer, acetic acid, citric acid or phosphate, and tonicity or osmotic pressure regulators such as sodium chloride or glucose, stabilizers such as cyclodextrin, surface active Agents such as hydroxystearate and the like.
  • the drug stock can be filled into ampoules, disposable s
  • the compound described in the present application can also be placed in a carrier capable of such a protective effect.
  • a carrier capable of such a protective effect.
  • Various other methods that can control the drug release rate can also be used, such as methods using implants and microcapsule delivery systems.
  • the compounds described in the present application can also be administered using a sprayer, a dry powder inhaler, or a metered-dose spray inhaler.
  • the drugs administered in this way can be formulated into a solution with saline and added with benzyl alcohol or other suitable preservatives, absorption enhancers, fluorocarbons and other commonly used solubilizing or dispersing agents.
  • the treatment plan and dosage to be used will depend on many factors, including the properties of the specific compound used, the patient’s age, weight, general health, gender, diet, and administration. Time, excretion rate, patient’s pathological condition, treatment goals and doctor’s judgment. If it is a combination therapy, the amount of active ingredient will also depend on which drug is used for the combination therapy.
  • the present application provides a drug combination and/or kit, which comprises (1) the compound described in the present application, and (2) immunotherapy for treating tumors.
  • the present application provides a drug combination and/or kit, which comprises (1) the compound and (2) a drug for treating Parkinson's disease.
  • MTBE methyl tert-butyl ether
  • Acetone acetone
  • MAC methyl acetate
  • IPA isopropanol
  • the solid samples were analyzed using Empyrean X-ray powder diffractometer (PANalytical) or D8 Advance X-ray powder diffractometer (Bruker).
  • the Empyrean X-ray powder diffractometer is equipped with a PIXcel 1D detector. In XRPD analysis, the 2 ⁇ scan angle is from 3 to 40°, and the scan step size is 0.013°.
  • the light tube voltage and light tube current are 45KV and 40mA, respectively.
  • the D8 Advance X-ray powder diffraction analyzer is equipped with a LynxEye detector.
  • the 2 ⁇ scan angle is from 3 to 40°, and the scan step size is 0.02°.
  • the light tube voltage and light tube current are 40KV and 40mA, respectively.
  • the instrument model of differential scanning calorimetry is DSC 250 (TA Instruments, US). After the sample is accurately weighed, it is placed in the DSC pierced sample pan, and the accurate mass of the sample is recorded. The sample was heated from 25°C to the final temperature at a temperature increase rate of 10°C/min.
  • thermogravimetric analyzer The model of the thermogravimetric analyzer is TGA 55 (TA Instruments, US). The sample is placed in a balanced open aluminum sample pan, and the mass is automatically weighed in the TGA heating furnace. The sample is heated to the final temperature at a rate of 10°C/min.
  • the instrument model used for dynamic moisture absorption and desorption analysis is Vsorp Dynamic Moisture Sorption Analyzer (ProUmid GmbH & Co. KG, Germany) or DVS Intrinsic (SMS, UK). Place the sample in the tare sample pan and automatically weigh it.
  • Vsorp Dynamic Motion Sorption Analyzer parameter settings are as follows:
  • the DVS Intrinsic instrument parameter settings are as follows:
  • the instrument model used for PLM analysis is ECLIPSE LV100POL polarizing microscope (Nikon, Japan).
  • 1 H-NMR uses Bruker AVANCE III HD 300 or 400, equipped with Sample Xpress 60 autosampler, or Bruker Advance 300, equipped with B-ACS 120 autosampler.
  • HPLC analysis method uses Agilent HPLC 1260 series instruments.
  • HPLC method for solubility and stability testing is as follows:
  • the experiment measured the solubility of free base crystal form and acetate crystal form in biological solvents (SGF (Simulated Gastric Fluid), FaSSIF (Fasted State Stimulated Intestinal Fluid) and FeSSIF (Fed State Stimulated Intestinal Fluid)) at 37°C.
  • SGF Simulated Gastric Fluid
  • FaSSIF Fested State Stimulated Intestinal Fluid
  • FeSSIF Fed State Stimulated Intestinal Fluid
  • a certain amount of acetate crystals were placed at 60°C closed and 40°C/75%RH open conditions for a maximum of 7 days. Samples were taken on days 0, 3, and 7 and diluted with diluent to ⁇ 0.3 mg/mL for HPLC purity testing. The solid sample is subjected to XRPD test to determine the crystal form.
  • the preparation reaction formula is as follows:
  • Example 3 The compound of the present application can inhibit the activity of adenosine receptor A2A
  • the inhibitory effect of the test compound on the adenosine receptor A2A was evaluated by the TR-FRET-based cAMP accumulation assay.
  • the method includes, in HEK293 cells (hADORA2A-HEK293) expressing A2AR, the production of cAMP stimulated by NECA and the inhibitory effect of A2A receptor antagonists on it. All cells were cultured in complete medium and cultured at 5% CO2 and 37°C. After digesting the cells with trypsin, the cells were collected by centrifugation at 200 g for 5 minutes. After resuspending the cells in fresh complete medium, the cell viability was counted by the trypan blue exclusion method.
  • the subsequent cAMP production measurement experiment can only be performed when the cell viability is greater than 95%.
  • HEPES 5mM
  • BSA stabilizer 0.1%)
  • Rolipram 10 ⁇ M
  • Itrafylline is a selective A2A receptor antagonist, which can improve the motor function of PD patients by changing the activity of neurons. It is clinically used to treat PD and improve the dyskinesia of PD.
  • the inhibition rate (%) was calculated according to the following formula, and the IC50 value was calculated from the concentration-inhibition (%) curve after log conversion.
  • Ratio 665/615high the high fluorescence ratio of the reference compound
  • Ratio 665/615low the low fluorescence ratio of the reference compound
  • Ratio 665/615cmpd the fluorescence ratio of the test compound
  • Example 4 The compound of the present application selectively inhibits adenosine receptors
  • the cAMP accumulation assay method detects the inhibitory activity of the compounds of this application on other adenosine receptors
  • test compound 22 The inhibitory effects of test compound 22 and Istradefylline (positive control drug) on the other three adenosine receptors A2b, A1 and A3 were evaluated by the TR-FRET-based cAMP accumulation assay.
  • the HEK293 cell line stably expressing adenosine receptor A2b was used as the research object (PerkinElmer, ES-013-C) for the experiment.
  • Cells were cultured in EMEM medium (ATCC, 30) containing 10% FBS (Hyclone, SH30406.05), 100U/mL penicillin-streptomycin mixture (Gibco, 15140-122) and 100 ⁇ g/mL G418 (Gibco, 11811031) -2003).
  • Use TrypLETM Express (Gibco, 12604-013) to digest and passage cells approximately 3 times a week, and maintain approximately 70% to 90% confluence.
  • the solution system for cell seeding was 10 ⁇ L cAMP experimental buffer.
  • the cAMP experiment buffer is prepared by adding 75 ⁇ L 1M HEPES (Gibco, 15630080), 200 ⁇ L 7.5% BSA stabilizer (PerkinElmer, TRF0263) and 7.5 ⁇ L 20mM rolipram (Sigma) in 14mL 1 ⁇ HBSS (Gibco, 14025-076) , R6520).
  • the reference agonist NECA (Sigma, E2387) and the reference inhibitor CVT-6883 (Aobious, AOB4675) were prepared at 1 mM (1000 ⁇ final concentration), and then 3-fold serial dilutions were performed in 100% DMSO (Millipore, 1029312500). The same 10 mM test compound was also diluted 3-fold in 100% DMSO. The serially diluted reference compound and test compound were added to a 384-well compound plate (Labcyte, PP-0200). Use Echo (Labcyte, 550) to transfer 10 nL of the reference agonist NECA diluted in each well to the test plate containing cells, and incubate the test plate at 37° C. for 30 minutes.
  • a CHO cell line stably expressing adenosine receptor A1 was used as the research object (PerkinElmer, ES-010-C) for the experiment.
  • Cells were cultured in F12 medium (Gibco, 11765) containing 10% FBS (Hyclone, SH30406.05), 100U/mL penicillin-streptomycin mixture (Gibco, 15140-122) and 400 ⁇ g/mL G418 (Gibco, 11811031) -062) in.
  • TrypLE TM Express (Gibco, 12604-013) was used to digest and passage cells about 3 times a week, and maintain a confluence of about 70% to 90%.
  • the cAMP experiment buffer is prepared by adding 75 ⁇ L 1M HEPES (Gibco, 15630080), 200 ⁇ L 7.5% BSA stabilizer (PerkinElmer, TRF0263) and 7.5 ⁇ L 20mM rolipram (Sigma) in 14mL 1 ⁇ HBSS (Gibco, 14025-076). , R6520).
  • the reference agonist NECA (Sigma, E2387) and the reference inhibitor DPCPX (Sigma, C101) were prepared at 1 mM (1000 ⁇ final concentration), and then 3-fold serial dilutions were performed in 100% DMSO (Millipore, 1029312500). Similarly, 10 mM test compounds were also serially diluted 3 times in 100% DMSO. 1mM forskolin (Sigma, F3917) was prepared in 100% DMSO. The reference compound, the test compound and 1 mM forskolin, which were serially diluted, were added to a 384-well compound plate (Labcyte, PP-0200).
  • Use Echo (Labcyte, 550) to transfer 10 nL of 1 mM forskolin per well and 10 nL of reference agonist NECA per well to the test plate containing cells, and incubate the test plate at 37°C for 30 minutes.
  • Use Echo to transfer 10 nL of the reference inhibitor DPCPX and the test compound in serial dilutions per well to the test plate containing the cells, and incubate the test plate at 25° C. for 20 minutes. Transfer 10 nL of 1000 ⁇ EC80 NECA per well and 10 nL of 1 mM forskolin per well to the test plate.
  • a CHO cell line stably expressing adenosine receptor A3 was used as the research object (PerkinElmer, ES-012-C) for experiments. Calculate the IC50 value of the compound.
  • Ratio High Control Reference compound high fluorescence ratio
  • Ratio Low Control Reference compound low fluorescence ratio
  • Ratio Compound Fluorescence ratio of the test compound
  • Ratio High Control Reference compound high fluorescence ratio
  • Ratio Low Control Reference compound low fluorescence ratio
  • Ratio Compound Fluorescence ratio of the test compound
  • a highly expressing adenosine receptor coupled with human Gq was used to stably transfer cell lines, and the antagonistic function of compound 22 was evaluated by testing the changes in intracellular calcium ion concentration.
  • the combination of A 2AR agonist and A 2AR can activate downstream pathways and increase the intracellular calcium ion concentration.
  • the antagonist will competitively occupy the binding site where the agonist binds to A 2AR , thereby interrupting the intracellular signal transduction and causing changes in the intracellular calcium ion concentration.
  • Antagonistic activity of ADORA 1 , ADORA 2A , ADORA 2B and ADORA 3 adenosine receptors.
  • the ADORA1, ADORA2A, ADORA2B and ADORA3 receptor stable transfection cell lines were all constructed by WuXi AppTec's Biological Department, using ThermoFisher's Lipofectamine 2000 transfection reagent to transfer the receptor plasmid and G15 plasmid into the host cell, and then pick a single clone. Construct a stable transfected cell line.
  • the specific information and training conditions are as follows:
  • Compound preparation 4 compounds to be tested were prepared into 10mM mother liquor with DMSO and stored in a refrigerator at 4°C.
  • Antagonist%Inhibition 100-(RLU-LC)/(DMSO-LC)*100
  • RLU Relative light absorption value, reading value from 1 to 90;
  • HC Mean value of fluorescence signal in DMSO group
  • LC Mean value of fluorescence signal at the highest concentration of antagonist.
  • Example 5 The compound of the application can bind to adenosine receptors
  • radioisotope ligand competitive binding technology was used to detect the binding effects of the test compound and the reference compound CGS-15943 on the adenosine receptors ADORA1, ADORA2A, ADORA2B, and ADORA3 at the biochemical level.
  • ADORA1 25mM HEPES pH 7.4, 5mM MgCl 2 , 1mM CaCl 2 , 100mM NaCl
  • ADORA2A 50mM Tris-HCl pH 7.4, 10mM MgCl 2 , 1mM EDTA
  • ADORA2B 50mM HEPES pH 7.0, 5mM MgCl 2 , 1mM EDTA
  • ADORA3 25mM HEPES pH 7.4, 10mM MgCl 2 , 1mM CaCl2, 0.5% BSA
  • ADORA1 25mM HEPES pH 7.4, 5mM MgCl 2 , 1mM CaCl 2 , 100mM NaCl.
  • ADORA2A 50mM Tris-HCl pH 7.4, 154mM NaCl
  • ADORA2B 50mM HEPES pH 6.5, 5mM MgCl 2 , 1mM EDTA, 0.2% BSA
  • ADORA3 50mM Tris-HCl pH 7.4
  • test compound preparation The test compound and reference compound are diluted with 100% DMSO.
  • the initial concentration is 10 ⁇ M, double wells, 4-fold serial dilution, 10 points, and the final working concentration is 0.038nM ⁇ 10 ⁇ M (the second and third detection of the test compound against the ADORA2A target: The initial concentration is 2 ⁇ M, double wells, 4-fold gradient dilution, 10 points, and the final working concentration is 0.0076nM ⁇ 2 ⁇ M).
  • ADORA1, ADORA2A, ADORA2B, ADORA3 reference compound the initial concentration is 1 ⁇ M, double-well, 4-fold gradient dilution, 10 points, and the final working concentration is 0.0038nM ⁇ 1 ⁇ M.
  • the ADORA receptor cell membrane is prepared with the detection buffer to the corresponding concentration.
  • Isotope preparation configure the isotope detection buffer to the corresponding concentration.
  • Target Cell membrane concentration isotope Final Isotope Concentration ADORA1 2.5 ⁇ g/hole [3H]DPCPX 1nM ADORA2A 5 ⁇ g/hole [3H]CGS-21680 6nM ADORA2B 20 ⁇ g/hole [3H]DPCPX 8nM ADORA3 2 ⁇ g/hole [3H]HEMADO 1nM
  • the high signal control wells are 0.5% DMSO, and the ADORA1, ADORA2A, ADORA2B, and ADORA3 low signal control wells are CGS-15943.
  • the 96-well plate was sealed, ADORA1, ADORA2B, and ADORA3 were incubated on a shaker (300rpm) at room temperature (22°C) for 1 hour, and ADORA2A was incubated on a shaker (300rpm) at room temperature (22°C) for 2 hours.
  • Sample_raw value the raw reading of the test compound
  • Example 6 The effect of the compound of the present application in the dyskinesia model induced by reserpine
  • Reserpine is an indole alkaloid drug. It can inhibit the reuptake of noradrenergic neuron terminals, causing slowed movement or incompetence, resulting in clinical symptoms similar to Parkinson's disease.
  • the mechanism is that reserpine can block the transport of irreversible monoamines such as DA, serotonin (5-HT) and norepinephrine (NA) in the vesicles, so that the monoamines are degraded in the cell, thereby Consumption of central and peripheral monoamines, rapid reduction of monoamine levels, resulting in dyskinesias and other Parkinson’s disease-like symptoms.
  • A2A receptor antagonist is administered to evaluate whether the test compound can improve the dyskinesia of the mice.
  • the evaluation indicators include walking distance, walking time, standing times and standing time.
  • mice Male CD1 mice weighed 29.5 ⁇ 0.1g. The total number of animals actually used is 90.
  • the animals were purchased from Beijing Weitong Lihua Experimental Technology Co., Ltd. (animal certificate number: 1100112011009984) and raised in the animal room of Shanghai Ruizhi Chemical Research Co., Ltd. (experimental animal license: SYXK (Shanghai) 2017-0018).
  • the animal room is an SPF environment.
  • the 3-channel timer, syringe, stainless steel workbench and workbench, and related record forms required for the test should be placed in the test laboratory in advance one day before the start of the test. After checking the state of the animals, no animals were found to be underweight. , Tail docking, alopecia areata, and poor physical condition. There are a total of 90 experimental animals, and a random grouping sequence is generated before the experiment. According to the random sequence, the experimental animals were randomly divided into solvent control group, model group, Istradefylline group, and 3, 10, and 30 mg/kg compound 22 acetate group on the day of the test.
  • the Laboras behavior system automatically analyzes the walking distance (m), walking time (sec), upright times and upright time (sec) of the mice for 0-30min, 30-60min, 0-60min and every 10min.
  • the results are shown in Tables 12-14.
  • the results show that oral (OP) administration of compound 22 acetate at 10 mg/kg and 30 mg/kg can significantly increase walking distance, walking time, and the number of standing uprights.
  • Tests have shown that compound 22 acetate has a therapeutic effect in a mouse dyskinesia model induced by reserpine, and the drug effect has a significant dose-effect relationship.
  • the control drug Istradefylline showed approximately the same efficacy in this trial.
  • the mouse reserpine-induced dyskinesia model test supports the application of compound 22 acetate in the treatment of Parkinson's disease.
  • test results show that the compound of the present application can significantly increase the walking distance, walking time, upright time and frequency of animals at the doses of 15 mg/kg and 30 mg/kg.
  • compound 22 acetate has a therapeutic effect in a rat dyskinesia model induced by reserpine, and there is a dose-effect relationship.
  • the control drug istradefylline showed approximately the same efficacy in this trial.
  • Rat reserpine-induced dyskinesia model test supports the application of compound 22 acetate in the treatment of Parkinson's disease.
  • Example 7 The effect of the compound of the present application on a rat model of Parkinson's disease induced by haloperidol
  • Parkinson's disease is a common degenerative disease of the nervous system.
  • the main pathological changes are the loss of substantia nigra dopaminergic neurons and the decrease of dopamine transmitter content, which leads to static tremor, muscle stiffness, and motor immobility. And postural reflex disorders and other clinical symptoms.
  • Haloperidol a dopamine D2 receptor blocker, can block the dopamine D2 receptors in the striatum, resulting in mice with dystonic posture, that is, stiffness and reduced motor symptoms.
  • This model is a recognized Parkinson’s synthesis The animal model of the levy. To evaluate the improvement effect of the test substance on the symptoms of Parkinson's disease induced by Haloperidol-induced CD-1 mice.
  • each dose of compound 22 acetate was given by gavage, the volume was 10ml/kg, and after 30min, 1mg/kg Haloperidol was injected subcutaneously, the injection volume was 10ml/kg, and the rearing and bar test were carried out 1h and 4h after the completion of the administration. The statistics were small. The number of standing times and the time the rats were in the rod were evaluated to evaluate the effect of compound 22 acetate on the improvement of Haloperidol on the symptoms of Parkinson's disease caused by CD-1 mice.
  • each dose of compound 27 was given by intragastric administration, the volume was 10ml/kg, and after 30min, 1mg/kg Haloperidol was injected subcutaneously, and the injection volume was 10ml/kg, and the detection of Rearing and Bar Test was carried out 1h and 4h after the administration, and the number of standing times of mice was counted. And during the stick time, the effect of compound 27 on the improvement of Haloperidol on the symptoms of Parkinson's disease caused by CD-1 mice was evaluated.
  • mice CD-1 mice, SPF grade, 8 weeks old/5 weeks old, male, about 40g/25g
  • laboratory animal provider Shanghai Slack Laboratory Animal Co., Ltd.
  • Reagent consumables name Item No./Batch No. supplier Methylcellulose P10995 adamas-beta Haloperidol H113852-5g/G1411016 Aladdin Sodium acetate P1246037 GENERAL-REAGENT glacial acetic acid P1480181 adamas-beta 2-hydroxypropyl- ⁇ -cyclodextrin B1922043 Aladdin Istradefylline 2PJUO-EB TCI
  • Vehicle solvent control 40% hydroxypropyl ⁇ -cyclodextrin solution
  • Vehicle solvent control acetic acid-sodium acetate (pH ⁇ 4.0) buffer containing 0.5% MC
  • MC-containing acetic acid-sodium acetate (pH ⁇ 4.0) buffer weigh out 0.0904g of sodium acetate, add 1.95mL 2M acetic acid, to a 100mL graduated cylinder, then add ultrapure water to about 80mL, pH test paper to measure the pH, about 3.5 ⁇ 4, then add ultrapure water to 100mL, then add 0.5013g MC, mix well, until MC is completely dissolved, store in a 100mL brown bottle at room temperature.
  • Compound 40 besylate solution preparation (administration volume 10ml/kg)
  • the correction factor is 1.415.
  • the working concentration at this time is 3mg/ml; the concentration of 1ml is 3mg/ml solution, add 2ml 40% HP- ⁇ -CD, the working concentration at this time is 1mg/ml; draw 0.2ml solution with a concentration of 3mg/ml, add 1.8ml 40% HP- ⁇ -CD, at this time
  • the working concentration is 0.3mg/ml; draw 0.2ml of 1mg/ml solution, add 1.8ml 40% HP- ⁇ -CD, the working concentration at this time is 0.1mg/ml.
  • the correction factor is 1.153, weigh 0.0225g compound, add 6.5ml acetic acid-sodium acetate buffer solution containing 0.5% MC, vortex at room temperature for 10min, mix well, the working concentration at this time is 3mg/ml; Draw 1ml of the solution with a concentration of 3mg/ml, add 2ml of 0.5% MC-containing acetic acid-sodium acetate buffer, the working concentration at this time is 1mg/ml; draw 0.2ml of the solution with a concentration of 3mg/ml, add 1.8ml with 0.5% %MC acetic acid-sodium acetate buffer, the working concentration at this time is 0.3mg/ml.
  • Compound 27 preparation (administration volume 10ml/kg)
  • the correction factor is 1.013, weigh 0.0182g compound, add 6ml 40% HP- ⁇ -CD, vortex at room temperature for 10min, and mix well.
  • the working concentration is 3mg/ml; the concentration of 1ml is 3mg. /ml solution, add 2ml 40% HP- ⁇ -CD, the working concentration at this time is 1mg/ml; draw 0.2ml solution with a concentration of 3mg/ml, add 1.8ml 40% HP- ⁇ -CD, at this time
  • the working concentration is 0.3mg/ml.
  • Haloperidol mice were injected subcutaneously into the abdomen, the dose was 1 mg/kg, the working solution concentration was 0.1 mg/ml, and the injection volume was 10 ml/kg.
  • mice 1h and 4h after the drug intervention, the mice were put into a white transparent plastic bucket with a diameter of 20cm and a height of 21cm, and the mice were allowed to acclimate in the bucket for 5 minutes before starting video recording. Record the spontaneous activity behavior of the mice within 10 minutes, and count the number of standing times. The more standing times, the more frequent the spontaneous exploration activities of the mice and the lower the stiffness.
  • the Bar Test is performed.
  • the mouse is placed on a long wooden stick with a diameter of 6cm and a height of 6cm.
  • the forelimbs hold the stick, and only the buttocks or hind limbs are on the ground, and the hind limbs leave the table or Stop timing when the forelimbs leave the wooden stick, record the mouse's stick time, each mouse is tested 3 times, and count the maximum stick time and the average stick time. The longer the stick time, the more severe the mouse's stiffness.
  • mice after the mice were injected with Haloperidol, they showed a state of less movement, reduced spontaneous activity, and prolonged stiffness time; the behavior of mice in the compound 40 besylate group was significantly improved, the stick time was reduced, and the number of standing times increased, showing Certain dose-effect relationship.
  • Table 19 shows that in the BarTest test, compound 22 acetate can reduce the stick time and prolong the standing time of mice, and the results show a good dose-response dependence. As the dose of compound 22 acetate increases, stiffness The time is reduced and the time on the stick is shortened. The efficacy of the 10 mg/kg group and the 30 mg/kg group is equivalent to that of the positive drug.
  • Table 20 shows that in the BarTest test, compound 27 can reduce the stick time and prolong the standing time of mice, and the results show a good dose-dependent relationship. As the compound dose increases, the rigidity time decreases, and the stick time shortens.
  • the efficacy of the 10 mg/kg group was similar to that of the positive drug, and the efficacy of the 30 mg/kg group was slightly better than that of the positive drug group.
  • test results show that in haloperidol-induced ankylosing mice, after a single oral administration of 3 test compounds, the test mice can be extremely significant, the stiffness time is reduced, and the stick time is shortened, indicating that the test compound is compounded. It can resist or prevent the symptoms of stiffness induced by haloperidol, and there is a dose-effect relationship.
  • the control drug istradefylline showed approximately the same efficacy in this trial.
  • Haloperidol-induced Parkinson's disease stiffness model test in mice supports the application of test compounds in the treatment of Parkinson's disease.
  • test animals Transport the animals needed for the day from the breeding room to the operation room in advance, and start the experiment after adapting to the environment for at least 30 minutes.
  • the test animals were divided into blank control group, model group, Istradefylline group, and 3, 10, and 30 mg/kg compound 22AC groups.
  • Istradefylline or compound 22 acetate
  • the rat’s forelimbs were gently placed vertically on a horizontal metal rod with a length of 20.5 cm, a diameter of 1.2 cm, and a height of 8.5 cm above the worktable, and then the hind limbs were gently placed On the test box, start timing.
  • the rat's forelimb moves or climbs down on the metal rod, stop the timing, and record the duration of the rat's forelimb posture on the metal rod, which is recorded as the stiffness latency. If the rat’s front paw has not been put down and the observation is terminated for 600 sec, the incubation period of the animal’s stiffness is recorded as 600 sec. When the pole climbing test was completed that day, 5 animals from each group were perfused, fixed, and whole brains were taken for preservation.
  • Stiff latency The time for the rat's two forelimbs to remain still on the metal rod. The longest latency is 600sec.
  • Rigidity rate% (Rigidity latency/600)*100%
  • the test results show that in haloperidol-induced stiffness rats, after a single oral administration of 3, 10, and 30 mg/kg of compound 22 acetate, the experimental rats can significantly reduce stiffness.
  • the incubation period shows that compound 22 acetate can resist or prevent the symptoms of rigidity induced by haloperidol, and there is a dose-effect relationship.
  • the control drug istradefylline showed approximately the same efficacy in this trial.
  • Haloperidol-induced Parkinson's disease rigidity model test supports the application of compound 22 acetate in the treatment of Parkinson's disease.
  • the intragastric administration of Vehicle, compound 40 besylate (3mg/kg, 10mg/kg, 30mg/kg), 10mg/kg of itraphylline, administration volume of 10mL/kg, start intraperitoneal injection of Haloperidol 1mg/ 30 minutes later kg, the administration volume is 5mL/kg, and the number of standing times for the Rearing test 1h after modeling. After the rearing test is completed, the rat rests for 30 minutes and then performs the BarTest test.
  • HP- ⁇ -CD 1.40% HP- ⁇ -CD: Weigh 120.2g and add about 250mL ultrapure water, stir with 900rpm magnetic force, at room temperature, completely dissolve, then add ultrapure water to 300mL, and store in a 500mL brown bottle at room temperature away from light.
  • Compound 40 benzenesulfonate prepared for immediate use, prepared with 3mg/mL, weighed 0.1777g, added 42.5mL 40% HP- ⁇ -CD, vortexed, sonicated at room temperature for 10 minutes, blown well, and then used a 20mL syringe (replaced with 2.5mL needle), blow well, mix with 1mg/mL, dilute 3 times, mix with 0.3mg/mL, dilute 10 times.
  • Istradefylline Prepare for immediate use, with 1mg/mL, weigh out 0.0312g, add 31.2mL 40% HP- ⁇ -CD, and vortex.
  • Wistar rats were given different concentrations of compound 40 besylate and itraphylline by gavage, respectively, with an administration volume of 10 mL/kg; 30 minutes later, Haloperidol was administered intraperitoneally (1 mg/kg) with an administration volume of 5 mL/kg; The number of standings in the Rearing test 1h after the administration of Haloperidol. After the rearing test is completed, the rat rests for 30 minutes and then performs the Bar Test test.
  • Bar test time To test the stiffness of the rat, use the Bar test, that is, use a wooden rod with a diameter of 8mm, which is fixed between 2 iron stands and 10cm from the table top, and then grab the rat’s tail to make the front paw Putting on a wooden stick, the hind paws or buttocks are on the ground. Generally, the rat needs to adapt several times when it is tested for the first time. To test the time for the forelimb to drop the stick or the time for the hind limb to leave the table, a rat is tested 3 times.
  • Standing times Use Rearing to detect the standing times of the rat, that is, the rat is placed in a 27*52cm cage and adapted for 5 minutes, and the spontaneous activity behavior of the rat within 10 minutes-the number of standing is recorded.
  • the rat's hind limbs are upright and the two forelimbs are raised over the shoulders and touch the ground, it is standing once, and one forelimb lift is not counted.
  • the rat will lift the forelimb to touch the cage wall. If the rat raises the forelimb over the shoulder several times without landing, it is counted only once.
  • lipid peroxidation is caused, which makes the membrane structure disorder, affects the cell function, and finally leads to the selective destruction of the substantia nigra DA neurons, leading to the massive death of the substantia nigra DA neurons.
  • Striatum tyrosine hydroxylase-positive fibers are largely lost, striatum DA and its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid levels are significantly reduced, there are also substantia nigra striatal microglia and
  • the proliferation of astrocytes and the damage of locus coeruleus, hypothalamus and other areas are basically the same as those in patients with Parkinson's disease.
  • 6-Hydroxydopamine (6-OHDA) was injected to damage the substantia nigra striatum to construct a Parkinson rat model, and then the animals were given long-term (planned 3-4 weeks) levodopa (L-DOPA)/benzyl Benserazide (BID), after seeing the animals showing resistance to levodopa, that is, when the rotation time caused by levodopa is significantly reduced (after 3-4 weeks), the animals are also given to the test Compound and levodopa/benserazide, and measure the rotation time of the animal to investigate the recovery of the test compound on the rotation symptoms of rats.
  • L-DOPA levodopa
  • BID benzyl Benserazide
  • the first stage The same batch of model rats (a total of 15) were given different doses (0, 1, 3, 10, 30 and 100 mg/kg) of compound 22 acetate for a rotation test, and counted with a rotation counter 60min later, L-DOPA(5mg/kg)/Benserazide(1.25mg/kg) was given by intraperitoneal injection, and then the rotation counter was used again to perform rotation counting for 60min. There should be at least 1 day between rotation tests for each dose. A total of 6 tests in the first stage.
  • the second stage The experimental animals in groups 2, 3, 4, 5 and 6 were intraperitoneally injected twice a day with high doses of L-DOPA (25mg/kg)/Benserazide (6.25mg/kg), the purpose is to induce emergence Resistance phenomenon, in which on the 0th day (day 0 is the day before the start of high-dose L-DOPA/Benserazide), 7, 14, 21, and 22 days, 5 rotation tests were carried out. On the day of the test, all experimental animals had only abdominal cavity L-DOPA (20mg/kg)/Benserazide (5mg/kg) given once by injection induces rotation.
  • each test group was given the vehicle, test drug or positive drug orally.
  • group 7 except for L-DOPA(25mg/kg)/Benserazide(6.25mg/kg) given by intraperitoneal injection twice a day
  • the test animals in group 8 and group 8 were also orally administered the test drug compound 22 acetate and the control drug Istradefylline twice a day, and four rotation tests were performed on days 0, 7, 14 and 21 respectively.
  • the first stage Wistar rats, 6-8 weeks, male, 178-213 grams, experimental animal provider: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., production license number: SCXK ( ⁇ ) 2016-0006, quality Certificate number: 1100112011004974;
  • the second stage Wistar rats, 6-8 weeks, male, 166-225 grams, experimental animal provider: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., production license number: SCXK ( ⁇ ) 2016-0006, quality Certificate number: 1100112011011824, 1100112011011826;
  • Parkinson rat model
  • L-DOPA injection volume each rat is injected with 4 ⁇ l of 6-OHDA solution (2.5 ⁇ g/ ⁇ l) on the left medial forebrain tract (MFB).
  • anterior fontanelle is the standard reference point, referring to the stereotactic map of rat brain, injecting the left medial forebrain tract: anterior(A), -2.5mm; lateral(L), +2.0mm; ventral( V), -8.5mm.
  • L-DOPA injection speed injection speed 1 ⁇ l/min, leave the needle in situ for 5min after injection, and then slowly withdraw the needle at a speed of 1mm/min to suture the wound.
  • the total number of rotations was recorded every 5 minutes, and the number of rotations per 5 minutes was calculated based on this, and the 5 minutes with the highest number of rotations were found out, and the definition was The number of rotations within 5min is the peak number of rotations.
  • the first 5 minutes when the number of rotations increases to 20% of the peak number of rotations is the starting point of the rotation reaction time, and the first 5 minutes when the number of rotations drops to 20% of the peak number of rotations is the end point of the rotation reaction time. The time between the start point and the end point is counted as the rotation reaction time.
  • test results are expressed as "mean ⁇ standard deviation”.
  • the data was analyzed by SPSS16.0 software package.
  • One-way analysis of variance One-way ANOVA
  • paired sample T test and independent sample T test were used for comparison.
  • P ⁇ 0.05 was statistically different.
  • the first phase of the trial aimed to evaluate the efficacy of test drug compound 22 acetate at different doses (0, 1, 3, 10, 30 and 100 mg/kg) on 6-OHDA-induced contralateral rotation in rats.
  • compound 22 acetate does not cause animal rotation to a high degree at all doses when used alone, and the efficacy of L-DOPA/Benserazide alone is not obvious at low doses, it is still effective in maintaining L-DOPA/Benserazide. In the case of the same low dose, the combination of compound 22 acetate and L-DOPA has obvious efficacy.
  • the test drug compound 22 acetate can increase the contralateral side caused by L-DOPA/Benserazide at 5 doses.
  • the number of rotations is in a dose-dependent relationship, wherein at the doses of 30mg/kg and 100mg/kg, compound 22 acetate can significantly increase the number of contralateral rotations of the animal.
  • test drug can restore or increase the rotation reaction time of the animal to varying degrees.
  • the test drug can significantly increase the rotation reaction time (compared with the 21st day) at 15 and 30 mg/k doses, and there is a dose-dependent relationship . This shows that after animals are resistant to L-DOPA/Benserazide, compound 22 acetate can effectively restore or improve the original efficacy of L-DOPA in resistant animals.
  • test results show that compound 22 acetate may also effectively prevent or slow down the resistance of 6-OHDA Parkinson's model rats to L-DOPA/Benserazide.
  • test drug compound 22 acetate had no significant effect on the body weight of the test animals.
  • the model test shows that when Parkinson's disease patients develop resistance due to long-term use of levodopa, by combining with compound 22 acetate, levodopa may restore the therapeutic efficacy of these patients with off-period Parkinson's disease. . Support the application of the claims for the treatment of Parkinson's disease.
  • MC38 mouse colon cancer cell model is a commonly used model for colon cancer drug efficacy verification. It is mostly used to study the direction of colorectal cancer occurrence and metastasis, and has become a common way for tumor drug efficacy verification. To evaluate the anti-tumor effect of the test drug in the subcutaneously transplanted female C57BL/6 mouse animal model of murine colon cancer MC38 cell line.
  • mice C57BL/6 mice, female, 7-9 weeks (the age of mice at the time of tumor cell inoculation), weighing 16.4-20.1g, purchased from Shanghai Lingchang Biotechnology Co., Ltd. (LC), production license number : SCXK (Shanghai) 2013-0018, animal certificate number: 2013001837385. Feeding environment: SPF level.
  • Anti-PD-1 antibody Lot number: 695318A1B, packaging specification: 18mg, provided by BioXcell, colorless solution, stored at 4°C.
  • Solvent preparation Weigh 16.8g of hydroxypropyl-beta-cyclodextrin (Zibo Qianhui Biological Technology Co., Ltd., batch number: 160101), add 42ml of sterilized water, and set aside after completely dissolved.
  • MC38 cells were cultured in DMEM medium containing 10% fetal bovine serum. Collect MC38 cells in the exponential growth phase and resuspend them in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.
  • mice Female mice were inoculated with 1 ⁇ 10 6 MC38 cells subcutaneously on the right side. The day of vaccination was defined as day 0. On the second day of MC38 cell inoculation (Day1), they were administered randomly according to body weight. Prepare 3 mg/mL and 10 mg/mL compound 22 solutions for use.
  • Tumor growth rate is the percentage value of tumor volume or tumor weight in the treatment group and the control group at a certain point in time. Calculated as follows:
  • T/C% T TV /C TV ⁇ 100% (T TV : the average TV of the treatment group at a specific time point; C TV : the average TV of the vehicle control group at a specific time point);
  • T/C% T TW /C TW ⁇ 100% (T TW : average tumor weight at the end of the experiment in the treatment group; C TW : average tumor weight at the end of the experiment in the vehicle control group).
  • T and C are the relative tumor volume (TV) or tumor weight (TW) at a specific time point in the treatment group and the control group, respectively).
  • test results showed that in the MC38 mouse colon cancer cell model, the test compound 22 showed good tumor suppressive activity at the dose of 30 mg/kg and 100 mg/kg on the 22nd day, and the tumor suppressor rate (TGI) was 34 respectively. % And 48%.
  • the combination of test compound 22 and Anti-PD-1 antibody also showed excellent anti-tumor activity in vivo.
  • the tumor inhibition rate (TGI) was 85%, and there was no animal death or significant change in body weight.
  • the above experiments support the application of compound 22 as a single agent or in combination with Anti-PD-1 antibody in corresponding tumor indications.
  • the B16F10 murine melanoma model is a classic melanoma drug efficacy evaluation model, which is made by inoculating the same B16F10 tumor cells into female C57BL/6 mice subcutaneously. The test substance was given to evaluate the inhibitory effect on tumor growth in the model.
  • mice Species: Rodent; Strain: C57BL/6mice; Grade: SPF; Week age: 6-8 weeks; Gender:
  • Anti PD-1 antibody provider: Kanglong Chemical (Beijing) New Drug Technology Co., Ltd.; batch: 717919M1;
  • Solvent 40% hydroxypropyl- ⁇ -cyclodextrin (SIGMA, batch: Z08D9Y76902; weigh 120g HP-beta-CD and dissolve it with distilled water, and finally use a beaker to make the volume to 300ml, and configure it to 40% hydroxypropyl- ⁇ -Cyclodextrin solution).
  • SIGMA 40% hydroxypropyl- ⁇ -cyclodextrin
  • B16F10 tumor cells were cultured in DMEM medium containing inactivated 10% fetal bovine serum, 100U/ml penicillin, 100 ⁇ g/ml streptomycin and 2mM glutamine in an incubator at 37°C and 5% CO 2 , Every 3 to 4 days when the cells are overgrown, sub-flasks for passage. Tumor cells in the logarithmic growth phase are used for tumor inoculation in vivo.
  • B16F10 tumor cells resuspended in serum-free DMEM medium were inoculated subcutaneously on the right side of experimental animals at a rate of 1 ⁇ 10 5 /100 ⁇ l.
  • 12 animals with a uniform tumor volume were selected and administered according to the experimental design.
  • the first day of tumor inoculation was D0 day
  • the first, third, and fourth groups were administered on the second day of tumor inoculation
  • the second group was administered when the tumor volume reached 75mm 3 .
  • Tumor volume Use a vernier caliper to measure the tumor volume three times a week to measure the long and short diameters of the tumor.
  • TGI Tumor growth inhibition rate
  • T/C(%) (mean value of tumor volume of the treatment group on the day-average value of tumor volume of the treatment group on the initial D0 day)/(mean value of tumor volume of the control group on the day-average value of the tumor volume of the control group on the initial D0 day) ⁇ 100.
  • the A20 mouse-derived lymphoma (B lymphocyte) allogeneic transplantation model is a classic model for evaluating the efficacy of B-lymphocyte cancer.
  • A20 tumor cells were inoculated subcutaneously into the right ribs of female BALB/c mice by the same species. to make. The test substance was given to evaluate the inhibitory effect on tumor growth in the model.
  • Solvent 40% hydroxypropyl- ⁇ -cyclodextrin (SIGMA, batch: Z08D9Y76902; weigh 120g HP-beta-CD and dissolve it with distilled water, and finally use a beaker to make the volume to 300ml, and configure it to 40% hydroxypropyl- ⁇ -Cyclodextrin solution).
  • SIGMA 40% hydroxypropyl- ⁇ -cyclodextrin
  • Tumor cells are divided into flasks for passage every 3 to 4 days after the cells are full. Tumor cells in the logarithmic growth phase are used for inoculation of tumors in vivo.
  • A20 tumor cells resuspended in serum-free RPMI 1640 culture medium were inoculated subcutaneously on the right side of experimental animals at a rate of 3 ⁇ 10 5 /100 ⁇ l, and a total of 30 animals were inoculated.
  • 24 animals with a more uniform body weight were selected according to their body weight, 12 animals in each group, and the specific dosing plan was carried out according to the experimental design.
  • Tumor volume Use a vernier caliper to measure the tumor volume three times a week to measure the long and short diameters of the tumor.
  • TGI Tumor growth inhibition rate
  • TGI,%) (1-T/C) ⁇ 100
  • TGI 100-T/C ⁇ 100
  • T/C(%) (mean value of tumor volume of the treatment group on the day-average value of tumor volume of the treatment group on the initial D0 day)/(mean value of tumor volume of the control group on the day-average value of the tumor volume of the control group on the initial D0 day) ⁇ 100.
  • the SPSS T-test test was used for statistical analysis of tumor volume between groups.
  • the test results showed that the compound 22 (50mg/kg) treatment group showed a significant anti-tumor trend, and showed significant anti-tumor effects on the 15th and 17th days after tumor inoculation (P ⁇ 0.05), and its TGI was 29.4%. , 32.6%.
  • the experimental animals in each group were in good general conditions such as activities and eating, and no obvious clinical abnormalities were found.
  • the overall body weight of the animals increased before and after treatment.
  • the above experiments support the application of compound 22 as a single agent in corresponding tumor indications.
  • Example 13 Pharmacodynamic evaluation of the compound of the application in a CT26 murine colon cancer model in an allograft model
  • the CT26 murine colon cancer allogeneic transplantation model is a classic model for evaluating the efficacy of colon cancer, which is modeled by subcutaneous inoculation of BALB/c mice with CT26. The test substance was given to evaluate the inhibitory effect on tumor growth in the model.
  • mice BALB/c mice, female, 7-8 weeks (the age of mice at the time of tumor cell inoculation), weighing 17.4-23.5g, 160 (100 plus 60 surplus mice). Purchased from Shanghai Lingchang Biological Technology Co., Ltd. (LC), production license number: SCXK (Shanghai) 2013-0018, animal certificate number: 2013001837385. Feeding environment: SPF level. ;
  • Solvent 40% hydroxypropyl- ⁇ -cyclodextrin (Zibo Qianhui Biotechnology Co., Ltd.; batch number: 160101; weigh 16.8g hydroxypropyl- ⁇ -cyclodextrin, add 42ml sterile water, and wait until it is completely dissolved use.).
  • CT26 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. CT26 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.
  • mice Female mice were inoculated subcutaneously with 5 ⁇ 10 5 CT26 cells on the right side. The day of vaccination was defined as day 0. On the second day of MC38 cell inoculation (Day1), they were administered randomly according to body weight.
  • Tumor proliferation rate is the percentage value of tumor volume or tumor weight in the treatment group and the control group at a certain point in time. Calculated as follows:
  • T/C% T TV /C TV ⁇ 100% (T TV : the average TV of the treatment group at a specific time point; C TV : the average TV of the vehicle control group at a specific time point);
  • T/C% T TW /C TW ⁇ 100% (T TW : average tumor weight at the end of the experiment in the treatment group; C TW : average tumor weight at the end of the experiment in the vehicle control group).
  • T and C are the relative tumor volume (TV) or tumor weight (TW) at a specific time point in the treatment group and the control group, respectively).
  • the test results showed that the compound 22 (50 mg/kg) treatment group showed anti-tumor effect on the 26th day, and its TGI was 19%.
  • the experimental animals in each group were in good general conditions such as activities and eating, and no obvious clinical abnormalities were found.
  • the above experiments support the application of compound 22 as a single agent in corresponding tumor indications.
  • the experimental results show that the compounds in the list have excellent pharmacokinetic performance in ICR mice.
  • Compound 27, Compound 40, Compound 22, and Compound 41 exhibited higher oral in vivo exposure and higher oral bioavailability.
  • Compound 27, Compound 22, Compound 40, and Compound 40 exhibited higher oral in vivo exposures.
  • Compound 27, Compound 22, and Compound 40 showed no significant drug accumulation under continuous oral administration at a dose of 30 mg/kg.
  • Group Test substance Dosage Blood sampling point Group 1 Compound 40 100mg/kg 0.25,0.5,1,2,4,8,24h
  • Group 2 Compound 40 besylate 100mg/kg (calculated as free alkali) 0.25,0.5,1,2,4,8,24h
  • the experimental results show that the compounds in the list have excellent pharmacokinetic performance in SD rats.
  • the T 1/2 for intravenous injection is about 0.5 to 1 h, and the T 1/2 for oral administration is about 3-5 h. , Showing higher oral body exposure and higher oral bioavailability.
  • Dosage The compounds in this group are all administered in a mixed manner, and each group of 6 compounds is a group of mixed PO administration.
  • Compound 44 and Compound 48 mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile (0.1% formic acid).
  • Electrospray ion source (Turbo spray), positive ion detection mode, select multiple reaction monitoring scan (MRM) mode for mass spectrometry analysis.
  • MRM multiple reaction monitoring scan
  • the compound was formulated according to the solvent in the test protocol.
  • Oral intragastric administration before intragastric administration, 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 8h and 24h (compound 40, compound 27, compound 40 and compound 47) after administration, or give 0.25h, 0.5h, 1h, 4h, 6h and 8h (compound 44 and compound 48) after treatment, blood was collected from the orbit in a test tube treated with sodium heparin, and the supernatant plasma was collected for LC-MS/MS analysis after centrifugation.
  • the plasma samples are processed according to the method of "Sample Processing” and then analyzed by LC-MS/MS method, accompanied by standard curve and quality control samples.
  • the time point exceeding the upper limit of quantification was diluted 10 or 100 times for analysis.
  • Table 35 and Table 36 show that the compounds in the list have excellent pharmacokinetic performance in SD rats, with a higher AUC last value, showing higher oral in vivo exposure and higher oral exposure. bioavailability.
  • the excellent rat pharmacokinetic properties of the compounds in the list can support further clinical human experiments.
  • the dosing information is as follows:
  • the dosage formulations were freshly prepared under yellow light before daily administration. Among them, the preparation of the fifth group was prepared before the first day of administration, and divided into the daily dosage, and stored at 5 ⁇ 3°C. After the preparation is taken out on the day of administration, leave it at room temperature for at least 30 minutes before administration.
  • the drug was administered according to the experimental design protocol.
  • the blood collection tube containing the anticoagulant is repeatedly inverted several times to fully mix it, and placed on wet ice before centrifugation. Within 60 minutes after blood collection, centrifuge at 2000g at 2 to 8°C for 10 minutes to separate the red blood cells to obtain a plasma sample. The plasma sample was transferred to a cryotube and stored at -75 ⁇ 15°C until analysis.
  • Verapamil was used as an internal standard compound to determine the concentration of compound 22 acetate in the plasma of SD rats.
  • the mass spectrometer was Applied Biosystems/AB Sciex Triple Quad 4500. Under the positive ion mode scanning of the electrospray ion source, the mass-to-charge ratios of the precursor and product ions of the acetate and Verapamil of compound 22 were monitored as 393.2 ⁇ 146.0 and 455.2 ⁇ 165.0, respectively.
  • the pharmacokinetic parameters of compound 22 acetate are determined by the software Phoenix TM (Version 8.1) Use non-compartmental model to calculate.
  • the linear logarithmic ladder method was used to calculate the pharmacokinetic data with a weight of 1/(Y*Y). Samples with sample concentrations below the lower limit of quantification do not participate in the calculation of pharmacokinetic parameters.
  • Use Microsoft Excel 2010 version to calculate the average value of each parameter, and the average value is determined by the pharmacokinetic parameters of each animal.
  • the average value of the peak time (T max ) is represented by the median.
  • T max The mean of T max is the median.
  • the drug exposure (AUC 0-t ) of SD rats was 42765hr*ng/mL, respectively , 111910hr*ng/mL and 241332 hr*ng/mL, the maximum blood concentration (C max ) is 18367ng/mL, 27367ng/mL and 51850ng/mL, respectively.
  • the increase ratio of AUC 0-t and C max was lower than the dose ratio.
  • the AUC 0-t values on the 1st and 7th days were 110818hr*ng/mL and 127358hr*ng/mL, respectively, on the 7th day and the 1st day
  • the exposure ratio was less than 2 times, suggesting that after continuous and repeated administration of 63 mg/kg of compound 22 acetate for 7 days, compound 22 acetate did not accumulate significantly in SD rats.
  • the concentration of compound 27, compound 22 and compound 40 in the plasma of SD rats was determined by using liquid-phase mass spectrometry technology with Verapamil as the internal standard compound.
  • the mass-to-charge ratios of the precursor and product ions of compound 27, compound 40, compound 22, and Tolbutamide were monitored by the mass spectrometer using the positive ion mode of the electrospray ion source to be 395.2 ⁇ 178.2, 404.2 ⁇ 158.9, 393.4 ⁇ 146.2 and 455.2 ⁇ 165.0, respectively.
  • the pharmacokinetic parameters of compound 27, compound 40 and compound 22 were calculated by software Phoenix TM WinNonlin version 6.1 using a non-compartmental model.
  • the pharmacokinetic data was calculated using the linear logarithmic ladder method, with a weight of 1/Y*Y. Samples with sample concentrations below the lower limit of quantification do not participate in the calculation of pharmacokinetic parameters.
  • Use Microsoft Excel 2010 version to calculate the average value of each parameter, and the average value is determined by the pharmacokinetic parameters of each animal.
  • the average value of the peak time (T max ) is represented by the median.
  • Table 38 The main PK parameters (average value) of compound 27, compound 22, and compound 40 after oral administration for 7 consecutive days
  • T max value is the median value.
  • the drug exposure AUC 0-t on the first day was 29550hr*ng/mL, 6630hr*ng/mL and 5154hr*ng/mL; the maximum blood concentration C max is 7103ng/mL, 1022ng/mL and 503ng/mL, respectively.
  • test compound has excellent pharmacokinetic parameters in SD rats, with high in vivo exposure and no significant accumulation of continuous administration.
  • the excellent rat pharmacokinetic properties of the test compound can support further clinical human experiments.
  • mice Female, 7-9 weeks (mouse age at the beginning of the experiment), weight 19.2-22.1g, 51 mice (34 mice plus 10 surplus mice). Purchased from Shanghai Lingchang Biological Technology Co., Ltd. (LC), animal certificate number: 2013001835211. Feeding environment: SPF level.
  • Test product Compound 22: Batch number: F, packaging specification: 38.0mg/vial+5045.1mg/vial, purity 98.96%, moisture 5.38%, provided by Zhejiang Chunhe Pharmaceutical Technology Co., Ltd., powder, sealed and stored at RT.
  • StudyDirector TM version number 3.1.399.19, supplier Studylog System, Inc., S. San Francisco, CA, USA was used for random grouping according to the body weight of the mice.
  • mice After a single administration, plasma was collected in each group at 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h and 24h, 3 mice at each time point, and the experiment was terminated after 24h. Ten leftover mice were used to collect blank plasma.
  • mice After a single dose of G1 and G2 mice, plasma was collected at 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h and 24h in each group to test the PK response of compound 22 in mice. The results showed that the concentration of compound 22 in the plasma of mice in the G2 group was significantly higher than the concentration of compound 22 in the plasma of the mice in the G1 group at different time points. 40% (2-hydroxypropyl)- ⁇ -cyclodextrin is a good solvent for compound 22. At the same time, under the dosage of 100mg/kg, mice have higher drug exposure and excellent pharmacokinetic performance.

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Abstract

本申请涉及一种三唑并三嗪衍生物或其药学上可接受的溶剂化物或盐在制备治疗疾病的药物中的用途，以及治疗疾病的方法。

Description

三唑并三嗪衍生物在治疗疾病中的用途 技术领域
本申请涉及生物医药领域，具体的涉及一种三唑并三嗪衍生物，其能够治疗疾病。
背景技术
帕金森氏病是由于神经元的慢性、进行性退化的结果而发生的，其原因目前还没有完全清楚。它在临床上表现出的主要症状形式有静止性震颤、僵硬、运动过缓和姿势不稳。目前的治疗方法主要基于一种多巴胺替代疗法，干扰多巴胺能和/或胆碱能的信号级联。然而，这些临床症状往往不能从现有疗法中得到令人满意的控制，体现为大脑、脊髓神经和外周植物神经系统的非多巴胺能病变，在现有疗法下，许多帕金森病患者患有残疾。因此，开发发展能减慢、制止或逆转疾病进展的新疗法是帕金森氏症研究中的当务之急。
目前，癌症仍然是主要的全球健康负担。尽管在癌症的治疗方面有所进展，但对于更有效且毒性更小的治疗仍然存在未满足的医疗需求，尤其对于患有对现有治疗具有抗性的晚期疾病或癌症的那些患者。近年来，人们已深刻认识到淋巴细胞在肿瘤免疫中的重要性。机体对肿瘤的免疫反应包括免疫监测，在识别到肿瘤相关抗原后，这种与细胞介导的免疫有关的机制可以有效地清除新生成的肿瘤细胞。虽然抗体介导的抗癌作用也通常存在，但一般都比细胞免疫介导的抗癌作用小得多。因此，亟需开发有前景的抗癌新制剂以满足医学需求。
发明内容
本申请提供了一种三唑并三嗪衍生物或其药学上可接受的溶剂化物或盐在制备治疗疾病的药物中的用途，以及治疗疾病的方法，所述疾病包括肿瘤、帕金森氏症。本申请的化合物在治疗疾病中至少具有以下一种性质：(1)良好的药代动力学性质，较高的体内暴露量和生物利用度；(2)改善帕金森受试者的运动能力；和/或(4)抑制肿瘤生长，减小肿瘤体积。
一方面，本申请提供了式1的化合物在制备治疗疾病的药物中的用途，
Figure PCTCN2020139690-appb-000001
其中：
R为氢或甲基；Ar 1为任选地带取代基的呋喃基，任选地带取代基的苯基，或任选地带取代基的吡啶基；任意的任选地带取代基的芳环被卤素或氧代基取代；Ar 2为任选地带取代基的苯基，任选地带取代基的吡啶基，或任选地带取代基的嘧啶基；任意的任选地带取代基的芳环被卤素、羟基、氰基或甲氧基取代；X为氧或氮；
且Y和Z各自独立地为氢、任选地带取代基的C 1-3烷基、任选地带取代基的C 1-5环烷基、任选地带取代基的杂环烷基、任选地带取代基的杂环烷基烷基、任选地带取代基的芳基、任选地带取代基的C 1-3烷基羰基、任选地带取代基的C 1-5环烷基羰基、任选地带取代基的杂环烷基羰基、任选地带取代基的杂环烷基烷基羰基、任选地带取代基的芳基羰基，或任选地带取代基的杂芳基羰基；任意的所述任选地带取代基的基团被卤素、羟基、甲氧基、乙氧基、三氟甲氧基、三氟乙氧基、烷基氨基、环烷基氨基、杂环基、芳基、杂芳基，或C 1-3烷基聚氧乙烯基取代；或者，Y和Z连接以形成任选地带取代基的具有5至7个环原子的杂环烷基；任意的所述任选地带取代基的环被卤素、氧代基、甲氧基、乙氧基、三氟甲氧基，或三氟乙氧基取代；或者Y或Z不存在，或其药学上可接受的溶剂化物或盐。
在某些实施方式中，的R为氢基；Ar 1为2-呋喃基；Ar 2为任选地带取代基的苯基；任意的所述任选地带取代基的苯基被卤素取代；X是氧或氮；且Y和Z各自独立地为氢、任选地带取代基的C 2-3烷基、任选地带取代基的杂环烷基，或任选地带取代基的杂环烷基烷基；任意的所述任选地取代的基团被甲氧基、乙氧基、三氟甲氧基、三氟乙氧基，或C 1-2烷基聚氧乙烯基取代；或Y和Z连接以形成吗啉基环；或Y或Z不存在，或其药学上可接受的溶剂化物或盐。
在某些实施方式中，所述式1的化合物中的R为氢；Ar 1为2-呋喃基；Ar 2为苯基；X为氮；且Y和Z各自独立地为氢、任选地带取代基的乙基或任选地带取代基的氧杂环丁烷基；任意的所述任选地带取代基的基团被甲氧基、乙氧基、三氟甲氧基或三氟乙氧基取代；或Y和Z连接以形成吗啉基环，或其药学上可接受的溶剂化物或盐。
在某些实施方式中，所述化合物选自下组：
N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)-3-吗啉丙烷酰胺；
N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)-2-吗啉丙烷酰胺；
N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)新戊酰胺；
N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)-3-羟基-2,2-二甲基丙酰胺；
N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)-2-羟基-2-甲基丙酰胺；
N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)-4-甲基四氢-2H-吡喃-4-羧酰胺；
2-(呋喃-2-基)-N5-(2-(嘧啶-4-基)乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(2-(吡啶-3-基)乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-(2-(吡咯烷-1-基)乙氧基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]-三嗪-5,7-二胺；
N5-(4-(2-(氮杂环丁烷-1-基)乙氧基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-(2-(4-甲基哌嗪-1-基)乙氧基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-(3-(4-甲基哌嗪-1-基)丙氧基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
N5-(4-(2-(3,3-二氟吡咯烷-1-基)乙氧基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
(R)-N5-(4-(2-(3-氟吡咯烷-1-基)乙氧基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
(S)-N5-(4-(2-(3-氟吡咯烷-1-基)乙氧基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-((2-吗啉代乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-((四氢-2H-吡喃-4-基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-((四氢呋喃-3-基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-(4-甲基哌嗪-1-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5，7-二胺；
N5-(4-(双(2-甲氧基乙基)氨基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-(氧杂环丁烷-3-基氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-(氧杂环丁-3-基甲基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-((1-甲氧基丙烷-2-基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-((2-甲氧基乙基)(甲基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
N5-(4-(乙基(2-甲氧基乙基)氨基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-((2-甲氧基乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-((2-(2-甲氧基乙氧基)乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-((2-(2-(2-甲氧基乙氧基)乙氧基)乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
N5-(4-((2-乙氧基乙基)氨基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-((2-(2,2,2-三氟乙氧基)乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-((3-甲氧基丙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-((4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)-苯基)氨基)乙腈；
N5-(4-(1,3-二甲氧基丙烷-2-基氨基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(3-氟苯基)-N5-(4-((2-甲氧基乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；和
2-(3-氟苯基)-N5-(4-(氧杂环丁烷-3-基氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
或其药学上可接受的溶剂化物或盐。
另一方面，本申请提供式2的化合物在制备治疗疾病的药物中的用途，
Figure PCTCN2020139690-appb-000002
其中，R为氢或甲基；Ar1为任选地带取代基的呋喃基，任选地带取代基的苯基，或任选地带取代基的吡啶基；任意的任选地带取代基的芳环被卤素或氧代基取代；Ar2为任选地带取代基的苯基，任选地带取代基的吡啶基，或任选地带取代基的嘧啶基；任意的任选地带取代基的芳环被卤素、羟基、氰基或甲氧基取代；
且Q为任选地被X取代的5-6元芳族环、任选地在氮上被Y和Z取代的氨基羰基基团、任选地在氮上被Y和Z取代的氨基磺酰基基团、硝基基团、或氰基基团；X为卤素或任选地被取代的C1-3烷基；任意的所述任选地被取代的烷基基团被卤素、氰基、甲氧基、乙氧基、三氟甲氧基、三氟乙氧基、芳基或杂芳基取代；Y和Z各自独立地为氢或任选地被取代的C1-3烷基；任意的所述任选地被取代的烷基基团被卤素、羟基、甲基、烷基氨基或环烷基氨基取代；或者Y和Z连接以形成具有5至7个环原子的任选地被取代的环；任意的所述任选地被取代的环被卤素、甲基、乙基、三氟甲基或三氟乙基取代，或其药学上可接受的溶剂化物或盐。
在某些实施方式中，所述式2的化合物中，R为氢；Ar 1为2-呋喃基；Ar2为苯基或吡啶 基；且Q为硝基、氰基，或任选地被X取代的5-6元芳族环；X为任选地被取代的C1-3烷基；任意的所述任选地被取代的烷基被卤素、氰基、甲氧基、芳基或杂芳基取代，或其药学上可接受的溶剂化物或盐
在某些实施方式中，所述式2的化合物中，R为氢；Ar1为2-呋喃基；Ar2为苯基；且Q为任选地被X取代的四唑环；X为任选地被取代的C1-3烷基；任意的所述任选地被取代的烷基中被卤素、氰基或甲氧基取代，或其药学上可接受的溶剂化物或盐。
在某些实施方式中，所述化合物选自下组：
4-(2-(7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基氨基)乙基)-N,N-二甲基苯甲酰胺；
N5-(4-(1-甲基-1H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
N5-(4-(2-甲基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
N5-(4-(2-乙基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
N5-(4-(2-异丙基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
N5-(4-(2-丙基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(5-(4-(2-(7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基氨基)乙基)-苯基)-2H-四唑-2-基)乙腈；
4-(2-(7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基氨基)乙基)-苄腈；
N5-(4-(2-(2,2,2-三氟乙基)-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
N5-(4-(2-(2-甲氧基乙基)-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-(吡啶-2-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-(吡啶-3-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-(吡啶-4-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-(嘧啶-2-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-(1-甲基-1H-1,2,4-三唑-3-基)苯乙基)-[1,2,4]三唑并[1,5-a] [1,3,5]三嗪-5,7-二胺；
N5-(4-(2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N-(4-(甲基磺酰基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-胺；
2-(3-氟苯基)-N5-(4-(2-甲基-2H-四唑-5-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；和
N5-(4-(2-甲基-2H-四唑-5-基)苯乙基)-2-苯基-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；和
2-(呋喃-2-基)-N5-(2-(6-(2-甲基-2H-四唑-5-基)吡啶-3-基)乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺，
或其药学上可接受的溶剂化物或盐。
在某些实施方式中，所述化合物选自下组：
2-(呋喃-2-基)-N5-(4-(氧杂环丁烷-3-基氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
2-(呋喃-2-基)-N5-(4-((2-甲氧基乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
N5-(4-(2-甲基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；和
N5-(4-(2-乙基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
及其药学上可接受的溶剂化物或盐。
在某些实施方式中，所述化合物的药学上可接受的溶剂化物或盐包括乙酸盐和/或苯磺酸盐。
在某些实施方式中，所述化合物的药学上可接受的溶剂化物或盐包括2-(呋喃-2-基)-N5-(4-(氧杂环丁烷-3-基氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺乙酸盐和/或N5-(4-(2-甲基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺苯磺酸盐。
在某些实施方式中，所述疾病包括帕金森氏症。
在某些实施方式中，所述疾病包括肿瘤。在某些实施方式中，所述肿瘤包括实体瘤和非实体瘤。在某些实施方式中，所述实体瘤包括结肠癌和黑色素瘤。在某些实施方式中，所述非实体瘤包括淋巴瘤，例如，B细胞淋巴瘤。
在某些实施方式中，所述药物被制备为适于口服给药和/或注射给药。
在某些实施方式中，所述药物还包含药学上可接受的载剂。例如，所述药学上可接受的载剂可包括环糊精。
另一方面，本申请提供了治疗疾病的方法，其包括以下的步骤：向有需要的受试者施用治疗有效剂量的本申请所述的化合物。
在某些实施方式中，所述疾病包括帕金森氏症。
在某些实施方式中，受试者表现出帕金森氏症的早期征兆。
在某些实施方式中，所述受试者之前接受或未接受治疗帕金森氏症的药物。
在某些实施方式中，所述受试者在施用所述化合物之前、同时或之后，接受治疗帕金森氏症的药物。
在某些实施方式中，所述受试者产生对治疗帕金森氏症的药物的耐药性。
在某些实施方式中，所述治疗帕金森氏症的药物包括多巴胺。
在某些实施方式中，所述治疗包括降低所述受试者的帕金森氏病症的发展速度，和/或延缓所述受试者的帕金森氏病症的临床发展。
在某些实施方式中，所述受试者的帕金森氏病症发展速度通过UPDRS评价。
在某些实施方式中，所述治疗包括延迟所述受试者对抗帕金森病症对症治疗的需求。
在某些实施方式中，所述治疗包括降低所述受试者对抗帕金森病症对症治疗需求的风险。
在某些实施方式中，所述疾病包括肿瘤。在某些实施方式中，所述肿瘤包括实体瘤和非实体瘤。在某些实施方式中，所述实体瘤包括结肠癌和黑色素瘤。在某些实施方式中，所述非实体瘤包括淋巴瘤，例如，B细胞淋巴瘤。
在某些实施方式中，所述化合物的给药方法为口服给药和/或注射给药。
在某些实施方式中，所述化合物的施用频率为每天一次或每天两次。
在某些实施方式中，所述化合物的给药剂量为约0.001mg/kg至约500mg/kg。
在某些实施方式中，所述化合物的给药剂量为约1mg/kg至约100mg/kg。
在某些实施方式中，所述受试者在施用所述化合物之前、同时或之后，接受治疗肿瘤的免疫疗法。
在某些实施方式中，所述治疗肿瘤的免疫疗法包括免疫检查点抑制剂。
在某些实施方式中，所述治疗肿瘤的免疫疗法包括PD-1抗体。
另一方面，本申请提供了药物组合或试剂盒，其包括(1)所述的化合物，和(2)治疗帕金森氏症的药物。
在某些实施方式中，所述治疗帕金森氏症的药物包括多巴胺。
另一方面，本申请提供了药物组合或试剂盒，其包括(1)所述的化合物，和(2)治疗肿瘤的免疫疗法。
在某些实施方式中，所述治疗肿瘤的免疫疗法包括免疫检查点抑制剂。
在某些实施方式中，所述治疗肿瘤的免疫疗法包括PD-1抗体。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请化合物的盐的XRPD结果。
图2显示的是本申请化合物的盐的 1H-NMR的谱图结果。
图3显示的是本申请化合物的盐的DSC、TGA结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“烷基”通常是指包括含有一定碳原子数的直链和支链饱和脂肪族烃基。例如，C4烷基包括正丁基、异丁基、仲丁基和叔丁基。术语“环烷基”通常是指包括含有一定碳原子数的单环和双环饱和脂肪族烃基。
在本申请中，术语“烯基”通常是指包括含有至少一个碳-碳双键的直链和支链脂肪族烃基。例如，存在一个碳-碳双键。
除非另有说明，术语“芳基”通常是指包括含有5至14个环原子的单环和双环芳环，在某些情形中，含有6至10个环原子。所述芳基可以任选地被一个或多个取代基取代。除非另有说明，术语“芳基”还可以包括含有5至14个环原子的单环和双环杂芳基环，在某西情形中，含有6至10个环原子。
在本申请中，术语“杂环烷基”通常是指包括含有3至14个环原子的饱和单环和双环系统，例如含有4至10个环原子，其中，所述原子中的一个或一个以上是非碳元素，例如氮、氧或硫，这些杂元素既可单独存在，也可与其它杂元素一起存在。杂环烷基的例子包括，例如氮杂环丁烷基、六氢氮杂吡啶基、哌嗪基、哌啶基、吡咯烷基、吗啉基、四氢呋喃基、硫代吗啉基和四氢噻吩基，以及其N-氧化物。
在本申请中，术语“卤素”通常是指包括氟、氯、溴和碘。术语“三氟甲基”通常是指“-CF3”基团，术语“羟基(“hydroxyl”或“hydroxy”)”通常是指“-OH”基团。
在本申请中，术语“抗帕金森氏症对症治疗”通常包括任何治疗帕金森氏症的疗法，包括但不限于溴隐亭、苯扎托品、左旋多巴、罗匹尼罗、普拉克索、罗替戈汀、卡麦角林、恩他卡朋、托卡朋、金刚烷胺、培高利特、阿朴吗啡、麦角乙脲或司来吉兰。术语“延迟对抗帕金森氏病对症治疗的需要”是指对照没有接受本申请所述化合物的患者，延迟接受本申请所述化合物的帕金森氏症患者对抗帕金森氏病对症治疗的需要。
在本申请中，术语“减低帕金森氏症发展速度”通常是指减低帕金森氏症患者经历的恶化，例如此恶化可通过对照在一段时间里没有接受本申请所述的化合物的帕金森氏症患者所经历的恶化，借由UPDRS评分量化。发展速度通过总帕金森氏症统一评分量表(Total  Unified Parkinson’s Disease Rating Scale，总UPDRS)评分量化。总UPDRS评分增长代表帕金森氏症的症状发展，以及在一段时间内的UPDRS评分增长的增量代表帕金森氏症症状的进展程度。
在本申请中，术语“功能衰退”通常是指在借由总UPDRS评分决定的帕金森氏症患者症状的随时间的恶化。
在本申请中，术语“延迟对抗帕金森氏病对症治疗的需要”通常是指对照没有接受本申请所述的化合物的患者，延迟接受所述化合物的帕金森氏症患者对抗帕金森氏病对症治疗的需要。
在本申请中，术语“帕金森氏症的早期征兆”通常包括以下一种或多种征兆：
a)静止时单手4-8赫兹搓丸样震颤；
b)在静止时震颤最大，在移动时减少，在睡眠时无震颤；
c)活动僵硬和缓慢(心动过缓)，活动下降(运动功能减退)，启动活动困难(失去活动能力)；
d)脸变得面具样的，口张开，眨眼减慢，这可能会与沮丧混淆；
e)体态变得弯曲；
f)启动走路困难；步态蹒跚且是小步伐，手臂弯曲至腰间以至不会随步伐摇摆；
g)有时步伐会无意的加快，患者有时会突然开始小跑以保持不跌倒(步伐慌张)；
h)由于失去姿势反射，当重心被移动了，趋于前倾(推进)或向后(逆向推进)；
i)发音变弱，特征单调，口吃，发声困难；
j)运动功能减退以及末端肌肉组织控制受损，导致写字过小症和日常活动困难增加；
k)少有的眨眼以及缺乏面部表情；
1)运动减少；
m)姿势反射障碍；和/或
n)典型的步态异常。
本申请中帕金森氏症患者所处的阶段是指由Hoehn和Yahr描述的取决于症状的以下5个显著阶段(Hoehn MM,Yahr MD,Parkinsonism:onset,progression and mortality.Neurology 1967，17:427-42)。
第I阶段：(轻度或早期的疾病)：症状影响身体的一侧。
第II阶段：身体的两侧都受影响，但体态保持正常。
第III阶段：(中度的疾病)：身体的两侧都受影响，且在站立和行走中显示轻微的不平衡；但患者能保持独立。
第IV阶段(晚期的疾病)：身体的两侧都受影响，且在站立和行走中显示受损的不稳定性；此阶段患者需要大量的帮助。
第V阶段：表现为严重的、全面发展的疾病；患者被限制在床或椅子上。
在本申请中，“帕金森氏症早期患者”是指处于由Hoehn和Yahr定义的帕金森症第I或第II阶段的患者，其不需要抗帕金森氏症对症治疗。例如，那些患者至少在随后的9个月内不 需要抗帕金森氏症对症治疗。可通过执行相关的测试来确认那些处于早期的帕金森氏症患者。
在本申请中，术语“延缓帕金森氏症的临床发展”通常是指对照没有接受本申请所述化合物的受试者，接受所述化合物的受试者在经过所述化合物完全的对症作用后的期间里，例如接受所述化合物12周后，获得更低的总UPDRS评分增长速度。或对照延迟接受所述化合物治疗的受试者，在经过一段足够的时间以消除由延迟开始所述化合物治疗引起的变化后，总UPDRS评分显示更低的恶化。足够的时间为，在初次接受所述化合物治疗后必要地超过52周(例如，至少超过72周)。或对照延迟接受所述化合物治疗的受试者，在经过延迟开始所述化合物治疗的对症作用后的期间里，总UPDRS评分显示实质相似的恶化速度，如每周总UPDRS在0.15单位以内。
在本申请中，术语“癌症”与“肿瘤”可互相交换使用，通常是指已经导致异常或无调控的生长或过度增殖的恶性转化或细胞变化的细胞。此种变化或恶性转化通常会使此类细胞对宿主有机体有致病性，因此也预期包括会或可能会变成致病性且需要或可受益于干预的前期癌变或癌前细胞。所述肿瘤可包括实体瘤和非实体瘤。
发明详述
一方面，本申请提供一种式1所示的化合物，以及其水合物、溶剂化物、药学上可接受的盐、前药和其复合物，在治疗疾病的药物中的用途：
Figure PCTCN2020139690-appb-000003
其中，R可以为氢或任选地带有取代基的C 1-5烷基；任意的任选地带取代基的烷基被卤素、氰基、羟基、硝基、氨基、烷基氨基、环烷基氨基、甲基、乙基、甲氧基、乙氧基、三氟甲基、三氟乙基、三氟甲氧基或三氟乙氧基取代；Ar 1可以为5-6元芳环，所述5-6元芳环任选地被卤素、氧代基、氰基、甲基、甲氧基、三氟甲基或三氟甲氧基取代；Ar2可以为单环或双环芳环，所述单环或双环芳环任选地被卤素、羟基、氰基、甲基、甲氧基、三氟甲基或三氟甲氧基取代；X选自下组：氧、硫、碳，和氮；
且Y和Z可以各自独立地为氢、任选地带取代基的C 1-9烷基、任选地带取代基的单环或双环C 1-9环烷基、任选地带取代基的C 1-9烯基、任选地带取代基的单环或双环C 1-9环烯基、任选地带取代基的芳基、任选地带取代基的杂芳基、任选地带取代基的芳烷基、任选地带取代基的杂芳烷基、任选地带取代基的杂环烷基、任选地带取代基的杂环烯基、任选地带取代基的杂环烷基烷基、任选地带取代基的C 1-9烷基羰基、任选地带取代基的C 1-9环烷基羰基、任选地带取代基的杂环烷基羰基、任选地带取代基的杂环烷基烷基羰基、任选地带取代基的芳基羰基、或任选地带取代基的杂芳基羰基；所述任选地带取代基的基团可以被选自下组的取代基取代：卤素、氰基、羟基、硝基、氨基、烷基氨基、环烷基氨基、杂环基、氨基羰基、磺酰基、氨基磺酰基、羰基氨基、磺酰基氨基、甲基、乙基、芳基、甲氧基、乙氧基、三氟甲 基、三氟乙基、三氟甲氧基、三氟乙氧基、聚氧乙烯、聚氧丙烯、C 1-3烷基聚氧乙烯，或C 1-3烷基聚氧丙烯；或者，Y和Z可以连接以形成任选地带取代基的具有3至10个环原子的环；任意的所述任选地带取代基的环中的取代基可以被选自下组的取代基取代：卤素、氰基、羟基、氧代基、硝基、氨基、烷基氨基、环烷基氨基、氨基羰基、磺酰基、氨基磺酰基、羰基氨基、磺酰基氨基、甲基、乙基、甲氧基、乙氧基、三氟甲基、三氟乙基、三氟甲氧基、三氟乙氧基、聚氧乙烯、聚氧丙烯、C 1-3烷基聚氧乙烯，或C 1-3烷基聚氧丙烯；或者，Y或Z在结构中可以不存在
在某些情形中，式1所示的化合物中的R可以为氢、甲基或三氟甲基。在某些具体的情形中，式1所示的化合物中的R可以为氢或甲基。例如，式1所示的化合物中的R可以为氢。
在某些情形中，式1所示的化合物中的Ar 1可以为任选地带有取代基的咪唑基、三唑基、四唑基、呋喃基、噻吩基、恶唑基、异恶唑基、噻唑基、异噻唑基、恶二唑基、噻二唑基、苯基、吡啶基、哒嗪基、嘧啶基、吡嗪基或三嗪基；所述任选地带取代基的芳香环被卤素、氧代基、氰基、甲基、甲氧基、三氟甲基或三氟甲氧基取代。
在某些情形中，式1所示的化合物中的Ar 1可以选自表(1)中的芳香基团。
表1 式1中Ar 1的结构
Figure PCTCN2020139690-appb-000004
在某些情形中，式1所示的化合物中的Ar 1可以为2-呋喃基。
在某些情形中，式1所示的化合物中的Ar 2可以为任选地带有取代基的咪唑基、三唑基、四唑基、呋喃基、噻吩基、恶唑基、异恶唑基、噻唑基、异噻唑基、恶二唑基、噻二唑基、苯基、吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、吲哚基、异吲哚基、吲唑基、苯并咪唑基、氮杂吲哚基、氮杂吲唑基、嘌呤基、苯并呋喃基、异苯并呋喃基、苯并噻吩基、苯并异恶唑基、苯并异噻唑、苯并恶唑基、苯并噻二唑、喹啉基、异喹啉基、喹喔啉基、酞嗪基、喹唑啉基、噌啉基、萘啶基、吡啶并嘧啶基、吡啶并吡嗪、或蝶啶基；所述任选地带取代基的芳香环可以被卤素、羟基、氰基、硝基、甲基、甲氧基、三氟甲基或三氟甲氧基取代。
在某些情形中，式1所示的化合物中的Ar 2可以为任选地带有取代基的苯基、吡啶基、哒嗪基或嘧啶基；所述任选地带取代基的芳香环可以被卤素、羟基、氰基、甲基、甲氧基、三氟甲基或三氟甲氧基取代。
在某些情形中，式1所示的化合物中的Ar 2可以为苯基或吡啶基，所述苯基或吡啶基可以任选地被卤素或羟基取代。
在某些情形中，式1所示的化合物中的X为氧或氮。例如，式1所示的化合物中的X为氮。
在某些情形中，式1所示的化合物中的Y和Z可以各自独立地为氢、任选地带有取代基的C 2-5烷基、任选地带有取代基的C 3-5环烷基、任选地带有取代基的C 2-5烯基、任选地带有取代基的C 3-5环烯基、任选地带有取代基的芳基、任选地带有取代基的杂芳基、任选地带有取代基的芳烷基、任选地带有取代基的杂芳烷基、任选地带有取代基的杂环烷基，或任选地带有取代基的杂环烷基烷基；任意的所述任选地带取代基的基团可以被卤素、氰基、甲氧基、乙氧基、三氟甲氧基、三氟乙氧基或C 1-3烷基聚氧乙烯取代；
在某些情形中，式1所示的化合物中的Y和Z可以各自独立地为氢、任选地带有取代基的C 2-3烷基，或任选地带有取代基的杂环烷基；任意的所述任选地带取代基的基团可以被甲氧基、乙氧基、三氟甲氧基或三氟乙氧基取代；
在某些情形中，式1所示的化合物中的Y和Z可以各自独立地为氢、任选地带有取代基的乙基，或任选地带有取代基的氧杂环丁烷基；任意的所述任选地带取代基的基团可以被甲氧基、乙氧基、三氟甲氧基或三氟乙氧基取代；
在某些情形中，式1所示的化合物中的Y和Z可以连接以形成任选地带有取代基的具有4至8个环原子的环；任意的所述任选地带取代基基团中的取代基可以为卤素、甲基、乙基、甲氧基、乙氧基、三氟甲基、三氟乙基、三氟甲氧基或三氟乙氧基；
在某些情形中，式1所示的化合物中的Y和Z可以连接以形成任选地带有取代基的具有5至7个环原子的杂环烷；任意的所述任选地带取代基的基团可以被卤素、氧代基、甲氧基、乙氧基、三氟甲氧基或三氟乙氧基取代；
在某些情形中，式1所示的化合物中的Y和Z可以连接以形成任选地带有取代基的吗啉基环；任意的所述任选地带取代基的基团可以被卤素、甲氧基、乙氧基、三氟甲氧基或三氟乙氧基取代。
另一方面，本申请提供一种式2所示的三唑并三嗪化合物，以及其水合物、溶剂化物、药学上可接受的盐、前药和其复合物，在治疗疾病的药物中的用途：
Figure PCTCN2020139690-appb-000005
其中，R可以为氢或任选地带有取代基的C1-5烷基；任意的任选地带取代基的烷基可以被卤素、氰基、羟基、硝基、氨基、烷基氨基、环烷基氨基、甲基、乙基、甲氧基、乙氧基、三氟甲基、三氟乙基、三氟甲氧基或三氟乙氧基取代；
Ar 1可以为5-6元芳环，所述5-6元芳环可以任选地被卤素、氧代基、氰基、甲基、甲氧基、三氟甲基或三氟甲氧基取代；Ar 2可以为单环或双环芳环，所述单环或双环芳环可以任选地被卤素、羟基、氰基、甲基、甲氧基、三氟甲基或三氟甲氧基取代；
Q可以为任选地被X取代的单环或双环芳环，在氮原子上任选地被Y和Z取代的氨基羰基、在氮原子上任选地被Y和Z取代的氨基磺酰基、硝基，或氰基取代。X可以为卤素、氰 基、羟基、硝基、氨基、烷基氨基、甲氧基、乙氧基、三氟甲氧基、三氟乙氧基、任选地被取代的C 1-9烷基，任选地带有取代基的C 1-9环烷基，任选地带有取代基的C 1-9烯基，任选地带有取代基的C 1-9环烯基，任选地带有取代基的芳基，任选地带有取代基的杂芳基，任选地带有取代基的芳烷基，任选地带有取代基的杂芳烷基，任选地带有取代基的杂环烷基，或任选地带有取代基的杂环烯基；前述任选地带取代基的基团可以被卤素、氰基、羟基、硝基、氨基、烷基氨基、环烷基氨基、氨基羰基、磺酰基、氨基磺酰基、羰基氨基、磺酰基氨基、甲基、乙基、甲氧基、乙氧基、三氟甲基、三氟乙基、三氟甲氧基，三氟乙氧基、杂环烷基、芳基、杂芳基、聚氧乙烯、聚氧丙烯、C 1-3烷基聚氧乙烯，或C 1-3烷基聚氧丙烯取代。Y和Z可以各自独立地为氢、任选地带有取代基的C 1-9烷基、任选地可带有取代基的单环或双环C 1-9环烷基、任选地带有取代基的C 1-9烯基、任选地带有取代基的单环或双环C 1-9环烯基、任选地带有取代基的芳基、任选地带有取代基的杂芳基、任选地带有取代基的芳烷基、任选地带有取代基的杂芳烷基、任选地带有取代基的杂环烷基，或任选地带有取代基的杂环烯基；任意所述任选地带取代基的基团可以被卤素、氰基、羟基、硝基、氨基、烷基氨基、环烷基氨基、氨基羰基、磺酰基、氨基磺酰基、羰基氨基、磺酰基氨基、甲基、乙基、甲氧基、乙氧基、三氟甲基、三氟乙基、三氟甲氧基、三氟乙氧基、聚氧乙烯、聚氧丙烯、C 1-3烷基聚氧乙烯、或C 1-3烷基聚氧丙烯取代；或Y和Z可以连接以形成任选地带有取代基的具有3至10个环原子的环；所述任选地带取代基的基团被卤素、氰基、羟基、氧代基、硝基、氨基、烷基氨基、环烷基氨基、氨基羰基、磺酰基、氨基磺酰基、羰基氨基、磺酰基氨基、甲基、乙基、甲氧基、乙氧基、三氟甲基、三氟乙基、三氟甲氧基、三氟乙氧基、聚氧乙烯、聚氧丙烯、C 1-3烷基聚氧乙烯、或C 1-3烷基聚氧丙烯取代。
在某些情形中，式2所示的化合物中的R可以为氢、甲基或三氟甲基。在某些具体的情形中，式2所示的化合物中的R可以为氢或甲基。例如，式2所示的化合物中的R为氢。
在另一些情形中，式2所示的三唑并三嗪化合物中的Ar 1可以为可带有取代基的咪唑基，三唑基，四唑基，呋喃基，噻吩基，恶唑基，异恶唑基，噻唑基，异噻唑基，恶二唑基，噻二唑基，苯基，吡啶基，哒嗪基，嘧啶基，或三嗪基；任意所述带取代基的基团可以被卤素，氧代基，氰基，甲基，甲氧基，三氟甲基或三氟甲氧基取代。
在另一些情形中，式2所示的化合物中的Ar 1选自表2中的芳香基团。例如，式2所示的化合物中的Ar 1为2-呋喃基。
表2 式2中Ar1的结构
Figure PCTCN2020139690-appb-000006
Figure PCTCN2020139690-appb-000007
在某些情形中，式2所示的化合物中的Ar 2可以为任选地带有取代基的咪唑基、三唑基、四唑基、呋喃基、噻吩基、恶唑基、异恶唑基、噻唑基、异噻唑基、恶二唑基、噻二唑基、苯基、吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、吲哚基、异吲哚基、吲唑基、苯并咪唑基、氮杂吲哚基、氮杂吲唑基、嘌呤基、苯并呋喃基、异苯并呋喃基、苯并噻吩基、苯并异恶唑基、苯并异噻唑、苯并恶唑基、苯并噻二唑、喹啉基、异喹啉基、喹喔啉基、酞嗪、喹唑啉基、噌啉基、萘啶基、吡啶并嘧啶基、吡啶并吡嗪，或蝶啶基；所述任选地带取代基的芳香环被卤素、羟基、氰基、硝基、甲基、甲氧基、三氟甲基或三氟甲氧基取代。
在某些情形中，式2所示的化合物中的Ar 2可以为任选地带有取代基的苯基、吡啶基、哒嗪基或嘧啶基；任意所述任选地带取代基的芳香环可以被卤素、羟基、氰基、甲基、甲氧基、三氟甲基或三氟甲氧基取代。
在某些情形中，式2所示的化合物中的Ar 2可以为任选地被卤素或羟基取代的苯基或吡啶基。
在某些情形中，式2所示的化合物中的Q可以为任选地被X取代的单环或双环芳环；X可以为卤素、氰基、羟基、硝基、氨基、烷基氨基、甲氧基、乙氧基、三氟甲氧基、三氟乙氧基、任选地带有取代基的C 1-9烷基、任选地带有取代基的C 1-9环烷基、任选地带有取代基的C 1-9烯基、任选地带有取代基的C 1-9环烯基、任选地带有取代基的芳基、任选地带有取代基的杂芳基、任选地带有取代基的芳烷基、任选地带有取代基的杂芳烷基、任选地带有取代基的杂环烷基、或任选地带有取代基的杂环烯基；所述任选地带取代基的基团可以被卤素、氰基、羟基、硝基、氨基、烷基氨基、环烷基氨基、氨基羰基、磺酰基、氨基磺酰基、羰基氨基、磺酰基氨基、甲基、乙基、甲氧基、乙氧基、三氟甲基、三氟乙基、三氟甲氧基、三氟乙氧基、杂环烷基、芳基、杂芳基、聚氧乙烯、聚氧丙烯、C 1-3烷基聚氧乙烯、或C 1-3烷基聚氧丙烯取代。
在某些情形中，式2所示的化合物中的Q可以为任选地带有取代基X的5-6元芳环。X可以为卤素、氰基、羟基、硝基、氨基、烷基氨基、甲氧基、乙氧基、三氟甲氧基、三氟乙氧基、可带有取代基的C 1-5烷基、可带有取代基的C 1-5环烷基、可带有取代基的C 1-5烯基、可带有取代基的C 1-5环烯基、可带有取代基的芳基、可带有取代基的杂芳基、可带有取代基的芳烷基、可带有取代基的杂芳烷基、可带有取代基的杂环烷基、或可带有取代基的杂环烯基；所述带取代基的基团被卤素、氰基、羟基、硝基、氨基、烷基氨基、环烷基氨基、氨基羰基、磺酰基、氨基磺酰基、羰基氨基、磺酰基氨基、甲基、乙基、甲氧基、乙氧基、三氟甲基、三氟乙基、三氟甲氧基、三氟乙氧基、杂环烷基、芳基、杂芳基、聚氧乙烯、聚氧丙烯、C 1-3烷基聚氧乙烯或C 1-3烷基聚氧丙烯取代。
在某些情形中，式2所示的化合物中的Q可以为任选地被X取代的四唑环。X可以为任选地带有取代基的C 1-3烷基或任选地带有取代基的杂环烷基；任意的所述任选地带取代基地基团可以被卤素、氰基、羟基、甲基、乙基、甲氧基、乙氧基、三氟甲基、三氟乙基、三氟甲 氧基、三氟乙氧基、杂环烷基、芳基、或杂芳基取代。
本申请所述化合物可以以一种或多种几何异构体、对映异构体、非对映异构体或互变异构体的形式存在。申请所述的化合物可以包括所有这些形式的异构体，包括外消旋体和其它形式的混合物。
在某些情况下，本申请所述的化合物可以以溶剂化物或非溶剂化物的形式存在。术语“溶剂化物的”在本文中用于描述包含本发明的化合物和药学上可接受的溶剂分子例如水和乙醇分子的化合物复合体。本申请中所述的化合物包括其所有的溶剂化物或非溶剂化物形式。
在某些情况下，本申请所述所示的化合物可以以药学上可接受的盐的形式存在。术语“药学上可接受的盐”是指生理上或毒理学上能够容许的盐，且在适当的时候，包括其药学上可接受的碱加成盐和酸加成盐。本申请所述的化合物包括其所有药学上可接受的盐。例如，所述药学上可接受的盐可以包括苯磺酸盐和/或乙酸盐。
在某些情况下，本申请所述的化合物可以以药学上可接受的纳米颗粒的形式存在。包含所述化合物的纳米颗粒可以被设计以改善药物的药代动力学和生物分布性能。例如，可以把所述化合物包裹在脂质体中，这可能能够延长药物在体内分布过程中的寿命。由于纳米颗粒将优先在肿瘤细胞周围的多孔状血管中泄漏出，适当尺寸的纳米颗粒还可具有更好的安全性。这么做还有助于降低药物的有效剂量。
在某些情况下，本申请中所述的化合物可以以前药的形式存在。术语“前药”通常是指能够通过体内代谢过程(例如，通过水解、还原或氧化)转化为本申请化合物的化合物。本申请所述的化合物包括其所有形式的前药。
在某些情况下，本申请所述的化合物还包括药学上可接受的同位素变体，即其中有一个或多个原子被具有相同原子序数但不同原子质量的原子取代。适合进行这种同位素置换的原子包括氢、碳、氮、氧、磷、硫、氟、碘和氯。某些所述化合物的同位素变体，例如氘取代的化合物，有可能因为具有更好的代谢稳定性而在有的情况下具有更好的治疗效果。本领域的技术人员可以用本领域已知的常规技术来制备所述化合物的同位素变体。
在本申请中，所述化合物可以包括以下化合物，或其药学上可接受的盐：
表3 本申请示例性的化合物
Figure PCTCN2020139690-appb-000008
Figure PCTCN2020139690-appb-000009
Figure PCTCN2020139690-appb-000010
Figure PCTCN2020139690-appb-000011
Figure PCTCN2020139690-appb-000012
本申请的化合物的制备
本申请所述的化合物可以通过各种合成方法来制备。作为说明性的例子，合成路线(1)中列出了两种制备目标化合物的合成路线。在第一种方法中，在通过合适的制备方法得到中间体(1A)以后，用烷基氨基取代中间体(1A)上的甲磺酰基即得到三唑并三嗪化合物(1C)。在第二种方法中，在通过适当的方式得到中间体(1B)后，用烷基氨基取代中间体(1B)上的苯氧基也可得到三唑并三嗪化合物(1C)。
Figure PCTCN2020139690-appb-000013
合成路线1
合成路线(1)中所需的中间体(1A)和(1B)也可以通过各种合成方法制备。作为说明性的例子，合成路线(2)中显示了中间体化合物(1A)的一种合成路线。在第一步，用适当的芳基酰肼(2A)与S-甲基异硫脲(2B)在氢氧化钠水溶液中反应后得到中间体(2C)。在后面的步骤中，先在水性介质中充分加热(2C)得到中间体(2D)，中间体(2D)与N-氰基二硫代亚氨基碳酸酯(2E)反应后得到硫化物中间体(2F)。之后，用间氯过氧苯甲酸氧化中间体(2F)即得到所需的中间体砜(1A)。如合成路线(1)所示，用合适的烷基胺亲核取代甲基磺酰基就可得到目标化合物(1C)(J.Chem.Soc.，Perkin Trans.1 1995，801-808；Structural Chemistry.2013，24，1241–1251)。
Figure PCTCN2020139690-appb-000014
合成路线2
合成路线(2)中的中间体(2D)也可以通过各种合成方法制备。作为说明性的例子，合成路线(3)显示了中间体(2D)的三种合成路线。值得强调的是，在这三种方法中，从甲酯或乙酯(3F)开始的合成路线通常可以提高效率，且取得更高的反应产率。
Figure PCTCN2020139690-appb-000015
合成路线3
合成路线(1)中的中间体(1B)可以用合成路线(4)所示的合成路线制备。制备首先从氰尿酰氯(4A)开始，在苯酚中回流得到2，4，6-三苯氧基-1，3，5-三嗪(4B)。下一步与水合肼反应后得到2-肼-4，6-二苯氧基-1，3，5-三嗪(4C)，其与合适的酰氯反应后得到酰基酰肼(4D)。然后，酰肼(4D)在脱水条件下进行环化反应后得到2-取代的5，7-二苯氧基三唑并三嗪(4E)，在最后一步，(4E)在甲醇氨中回流处理后就可得到关键的中间体(1B)(J.Chem.Soc.，Perkin Trans.1 1995，801-808)。如路线(1)所示，用合适的烷基胺与(1B)反应后即制成目标化合物(1C)。
Figure PCTCN2020139690-appb-000016
合成路线4
本申请所述的化合物可以结合腺苷受体，例如，可以结合腺苷A2A受体。本申请所述的化合物可以结合游离的腺苷受体蛋白质，也可以结合细胞表面的腺苷受体。本申请所述化合物可以选择性结合腺苷受体，例如，本申请化合物结合腺苷A2A受体的能力是结合腺苷A1、A2B和A3受体的至少10倍、20倍、30倍、40倍、50倍、100倍、200倍、300倍、500倍或更高。例如，使用放射性同位素配体竞争结合技术，本申请的化合物结合腺苷A2A受体的IC 50值是结合腺苷A1、A2B和A3受体的IC 50值的至少10倍、20倍、30倍、40倍、50倍、100倍、200倍、300倍、500倍或更高。
本申请所述的化合物可以抑制腺苷受体的活性，例如，可以抑制腺苷A2A受体的活性。本申请所述的化合物可以抑制游离的腺苷受体蛋白质的活性，也可以抑制细胞表面的腺苷受体的活性。本申请所述化合物可以选择性抑制腺苷受体的活性，例如，本申请化合物抑制腺苷A2A受体活性的能力是抑制腺苷A1、A2B和A3受体活性的至少10倍、20倍、30倍、40倍、50倍、100倍、200倍、300倍、500倍或更高。例如，使用cAMP累积测定法或钙流实验，本申请的化合物抑制腺苷A2A受体活性的IC 50值是抑制腺苷A1、A2B和A3受体活性的IC 50值的至少10倍、20倍、30倍、40倍、50倍、100倍、200倍、300倍、500倍或更高。
用途和方法
帕金森氏症
本申请所述的化合物可以用于制备治疗疾病的药物，其中所述疾病可包括帕金森氏症。
另一方面，本申请提供了治疗帕金森氏症的方法，其可包括以下的步骤：向有需要的受试者施用治疗有效剂量的本申请所述的化合物。
在某些实施方式中，所述受试者可以表现出帕金森氏症的早期征兆，所述方法包括确认患者表现出帕金森氏症的早期迹象，并定期给予患者由此确定量的所述化合物或其药学上可接受的溶剂化物或盐，有效地治疗患者。
早期征兆可以是脑的结构性或功能性变化，其例如在物理上是可以被检测的，例如，通过PET和SPECT研究，通过经颅声图描记(Becker,J Neurol 249,Suppl 3.2002,III/40)；Prunier C,等，Neuroimage.2003Jul；19(3):810-6)或通过检测生化标记如神经黑素(W002/31499)。
在某些实施方式中，所述受试者的帕金森氏病症发展速度可以通过总帕金森氏症统一评分量表(Total Unified Parkinson’s Disease Rating Scale，总UPDRS)评分量化。早期征兆还可能是嗅觉障碍、抑郁症、视觉障碍和认知功能损伤或睡眠障碍，可以使用不同测试的组合进行早期诊断(Becker,J Neurol 249,Suppl 3,2002III/40；Stern,Annals of Neurol 56,2004,169)。
在某些实施方式中，所述受试者可以表现出帕金森氏症的功能衰退，所述功能衰退通常是指借由总UPDRS评分决定的帕金森氏症患者症状的随时间的恶化。
在某些实施方式中，所述受试者可以表现出帕金森氏症的疲劳，所述功能衰退通常是指借由总UPDRS评分决定的帕金森氏症患者的疲劳状态。
在某些实施方式中，所述受试者可以表现出帕金森氏症的非运动症状，所述非运动症状通常是指借由总UPDRS评分决定的帕金森氏症患者的非运动症状。帕金森氏症的非运动症状可包括自主神经系统功能异常、神经精神疾患(包括情绪、认知、行为和思想改变)、感觉障碍和睡眠障碍等。
在某些实施方式中，所述受试者之前接受或未接受治疗帕金森氏症的疗法，包括药物、复健(如体能锻炼、改善步姿、缓慢的旋转四肢或躯干、伸展运动、口语引导、李·西弗曼语音治疗、踏步运动、环境改造、节律启动、腹式呼吸和冥想)、缓和医疗、饮食疗法、重复性经颅磁刺激术、烧灼术、脑深层刺激手术、苍白球切除术。
在某些实施方式中，所述受试者之前可接受或未接受治疗帕金森氏症的药物。治疗帕金森氏症的药物可包括但不限于L-多巴、多巴胺受体激动剂、单胺氧化酶抑制剂、其他药物如金刚烷胺和抗胆碱剂。治疗帕金森氏症的药物可包括：L-多巴、溴隐亭、苯扎托品、左旋多巴、罗匹尼罗、普拉克索、罗替戈汀、卡麦角林、沙芬酰胺、希利治林、雷沙吉兰、恩他卡朋、托卡朋、金刚烷胺、培高利特、阿朴吗啡、麦角乙脲或司来吉兰
在某些实施方式中，所述治疗可以包括降低所述受试者的帕金森氏病症的发展速度，和/或延缓所述受试者的帕金森氏病症的临床发展。在开始给予本申请所述的化合物或其药用可接受的溶剂化物或盐后，疾病发展速度为总UPDRS评分增长平均值降低。
在某些实施方式中，所述治疗可以包括延迟所述受试者对抗帕金森病症对症治疗的需求。在某些实施方式中，所述治疗可以包括确定患者处于帕金森氏症阶段，并定期给予患者由此确定量的所述化合物或其药学上可接受的溶剂化物或盐，有效地延迟其对抗帕金森病对症治疗的需求。
在某些实施方式中，所述治疗可以包括降低所述受试者对抗帕金森病症对症治疗需求的风险。在某些实施方式中，所述治疗包括定期给予帕金森氏症患者一定量的本申请所述化合物或其药学上可接受的溶剂化物或盐，有效地降低对抗帕金森氏症治疗需求的风险。
在某些实施方式中，所述治疗可以包括降低所述受试者的所述功能衰退。在某些实施方式中，所述方法包括确定患者处于帕金森氏症阶段，并定期给予患者由此确定量的所述化合物或其药学上可接受的溶剂化物或盐，有效地降低其功能衰退。在某些实施方式中，帕金森氏症患者的功能衰退被通过总的帕金森氏症统一评分量表(总UPDRS)评分量化，总UPDRS评分增长代表功能衰退。
在某些实施方式中，所述治疗可以包括降低所述受试者的所述疲劳。在某些实施方式中，所述方法包括确定患者处于帕金森氏症阶段，并定期给予患者由此确定量的本申请所述化合物或其药学上可接受的溶剂化物或盐，有效地减少疲劳。
在某些实施方式中，所述治疗可以包括降低所述受试者的运动症状的严重程度。所述运动症状可主要包括颤抖(如静止性颤抖)、肢体僵硬、动作迟缓、姿态不稳、运动功能减退、运动迟缓、姿态、说话与吞咽异常等。
在某些实施方式中，所述治疗可以包括降低所述受试者的所述非运动症状的严重程度。在某些实施方式中，所述方法包括确定患者处于帕金森氏症阶段，并定期给予患者由此确定量的所述化合物或其药学上可接受的溶剂化物或盐，有效地降低非运动症状的严重程度。在某些实施方式中，非运动症状是由帕金森氏症统一评分量表(UPDRS)第四版第一部分定义。
总UPDRS(帕金森氏症统一评分量表，unified Parkinson's disease rating scale)评分代表了帕金森氏病症的严重水平，被用于治疗过程中衡量疗效参数自基线的变化。UPDRS由以下部分测试组成：
UPDRS由以下部分组成：第一部分：心理，行为和情绪的评估，第二部分：对日常生活活动(ADL)的自我评估，包括言语，吞咽，手写，穿衣，卫生，摔倒，流涎，卧床，行 走和切割食物，第三部分：临床医生评分的监测运动评估，第四部分：治疗并发症，第五部分：帕恩森病严重程度的Hoehn和Yahr分期，第六部分：施瓦布和英格兰ADL量表。
运动失调协会发布的MDS-UPDRS也可用于帕金森氏病评分，其包含四部分共50项子量表内容，分别为；(1)日常生活非运动经历(13项)，(2)日常生活运动体验(13项)，(3)身体运动检查(18项)，和(4)运动并发症(6项)。每个子量表的评分为0-4，其中0＝正常，1＝轻微，2＝轻度，3＝中度和4＝严重。完成每个项目获得一个0-4的分数，其中0代表无障碍，4代表最高程度的障碍。
本申请所述的化合物可以使受试者的一个或多个UPDRS子量表项目的评分由4分降低为3分，4分降低为2分，4分降低为1分，4分降低为0分，3分降低为2分，3分降低为1分，3分降低为0分，2分降低为1分，2分降低为1分，或1分降低为0分。
本申请的化合物可以延长受试者的行走距离、行走时间、直立次数和直立时间。
例如，与未施用本申请化合物相比，使用了本申请化合物的受试者的行走距离可以延长至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％或更高，如在利血平诱导的小鼠运动障碍模型中所检测的。
例如，与未施用本申请化合物相比，使用了本申请化合物的受试者的行走时间可以延长至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％或更高，如在利血平诱导的小鼠运动障碍模型中所检测的。
例如，与未施用本申请化合物相比，使用了本申请化合物的受试者的直立次数可以延长至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％或更高，如在利血平诱导的小鼠运动障碍模型中所检测的。
例如，与未施用本申请化合物相比，使用了本申请化合物的受试者的直立时间可以延长至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％或更高，如在利血平诱导的小鼠运动障碍模型中所检测的。
本申请的化合物可以降低受试者的在棒时间、增加站立时间，有效改善受试者的僵直症状。
例如，与未施用本申请化合物相比，使用了本申请化合物的受试者的在棒时间可以减少至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％、至少200％、至少300％或更高，如氟哌啶醇诱导的大鼠帕金森病僵直模型中所检测的。
例如，与未施用本申请化合物相比，使用了本申请化合物的受试者的站立时间可以增加至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％、至少200％、至少300％或更高，如氟哌啶醇诱导的大鼠帕金森病僵直模型或MPTP诱导的小鼠帕金森模型中所检测的。
例如，与未施用本申请化合物相比，使用了本申请化合物的受试者的行走距离可以增加至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至 少90％、至少100％、至少200％、至少300％或更高，如在MPTP诱导的小鼠帕金森模型中所检测的。
在本申请中，治疗帕金森氏症中，所述化合物的施用剂量可以为约0.001至约500mg/kg、约0.001至约500mg/kg、约0.01至约500mg/kg、约0.1至约500mg/kg、约1至约500mg/kg、约0.001至约400mg/kg、约0.001至约300mg/kg、约0.001至约200mg/kg、约0.001至约100mg/kg、约0.001至约50mg/kg、约1至约200mg/kg、约1至约100mg/kg、约1至约50mg/kg、约1至约30mg/kg、约10至约30mg/kg、约3至约30mg/kg或约5至约30mg/kg。所述化合物的施用剂量可以为约1mg/kg、约3mg/kg、约5mg/kg、约7.5mg/kg、约10mg/kg、约30mg/kg、约50mg/kg或约100mg/kg。
本申请所述化合物能够抑制受试者对帕金森药物的耐药性，例如，可以抑制受试者对左旋多巴(L-DOPA)或Benserazide的耐药性。
肿瘤
本申请所述的化合物还可以用于制备治疗或预防宿主的癌症及宿主中相关的细胞异常增殖相关疾病的药物，该宿主可以是任何多细胞脊椎动物，包括人类及非人类哺乳动物。所述宿主可以是人类。其中所述疾病包括肿瘤。所述肿瘤可包括实体瘤和非实体瘤。
在本申请中，所述实体瘤可以包括结肠癌和黑色素瘤。在本申请中，所述非实体瘤可以包括淋巴癌，例如，B细胞淋巴癌。
在本申请中，治疗肿瘤中，所述化合物的施用剂量可以为约0.001至约500mg/kg、约0.001至约500mg/kg、约0.01至约500mg/kg、约0.1至约500mg/kg、约1至约500mg/kg、约0.001至约400mg/kg、约0.001至约300mg/kg、约0.001至约200mg/kg、约0.001至约100mg/kg、约0.001至约50mg/kg、约1至约200mg/kg、约1至约100mg/kg、约1至约50mg/kg、约1至约30mg/kg、约10至约30mg/kg、约3至约30mg/kg或约5至约30mg/kg。所述化合物的施用剂量可以为约1mg/kg、约3mg/kg、约5mg/kg、约7.5mg/kg、约10mg/kg、约30mg/kg、约50mg/kg或约100mg/kg。
本申请所述化合物可用于提高宿主免疫细胞的抗肿瘤活性。所述化合物能降低T细胞的免疫无能或T细胞对癌症的耐受，能使癌细胞更容易受到免疫清除，能抑制调节性T细胞的增殖，以及能够有助于记忆性T细胞的形成。所述化合物既可以提高宿主自身具有的免疫反应，也可以提高其对各种适应性免疫疗法的功效。
例如，本申请的药物可以抑制或延缓疾病的发展或进展，可以减小肿瘤大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。抑制肿瘤或肿瘤细胞生长的实例包括：相对于干预之前的相应水平，肿瘤生长体积减小。
另一方面，本申请提供了治疗肿瘤的方法，其可包括以下的步骤：向有需要的受试者施用治疗有效剂量的本申请所述的化合物。
为了使之更加有效，本申请所述化合物还可以与其它抗肿瘤治疗方法(例如化学疗法、肿瘤疫苗及各种免疫检查点抑制剂)联合使用，从而实现协同效应。术语“联合治疗”指活性试剂的同时给药，也可以按不同顺序先后给药。
在某些实施方式中，治疗或预防宿主中细胞异常增殖的方法可以包括给患者联合施用或交替施用本申请所述化合物和一种免疫检查点抑制剂。这里的免疫检查点抑制剂可以选自下组：PD-1抑制剂、PD-L1抑制剂、PD-L2抑制剂、CTLA-4抑制剂、BTLA抑制剂、LAG3抑制剂、TIM-3抑制剂、B7-H3抑制剂、B7-H4抑制剂、KIR抑制剂、TIGIT抑制剂或VISTA抑制剂等。例如，所述其它抗肿瘤治疗方法可以是所述PD-1抑制剂。
在另一个实施方式中，治疗或预防宿主中细胞异常增殖的方法可以包括给患者联合使用或交替本申请所述化合物和一种细胞疫苗。所述细胞疫苗基于与要预防的肿瘤相匹配的细胞。例如，如果一个宿主患有前列腺癌，或者有患前列腺癌的风险，所述细胞疫苗将基于前列腺癌细胞。在这种情况下，通常是给所述细胞进行一定的放射性照射，或以其它方式使其失去复制功能。也可以通过对细胞进行基因改造使其能够分泌集落刺激因子。
在另一个实施方式中，治疗或预防细胞异常增殖的方法包括给患者联合施用或交替施用所述化合物和嵌合抗原受体CAR-T细胞疗法。
在另一个实施方式中，治疗或预防细胞异常增殖的方法可以包括给患者联合施用或交替施用所述化合物和一种其它抗癌药物以治疗细胞异常增殖。这种抗癌药物可以是烷基化试剂、抗代谢药物、蒽环类衍生物、植物生物碱、拓扑异构酶抑制剂、抗肿瘤抗生素、激酶抑制剂或抗肿瘤抗原的单克隆抗体等。
本申请化合物具有良好的药代动力学性质，例如，本申请化合物具有较高的暴露量和生物利用度。在某些实施方式中，本申请化合物具有较高的口服体内暴露量和较高的口服生物利用度。例如，本申请化合物的血液T 1/2为约0.1h至约10h，例如，约0.2h至约10h，约0.3h至约10h，约0.4h至约10h，约0.5h至约10h，约0.5h至约9h，约0.5h至约8h，约0.5h至约7h，约0.5h至约6h，约0.5h至约5h，约0.5h至约3h，约0.5h至约2h，约0.5h至约1h。
药物组合物
在用药时，本申请的化合物可以配制成适当的药物组合物。药物组合物的具体组成由所选定的给药途径决定，给药途径包括口服、肠外道、静脉、肌肉内、鼻、口腔、局部、经皮或皮下。在治疗疾病(如帕金森氏症或肿瘤)中，药物组合物中所含的本申请所述化合物的剂量应足以起到有效的治疗作用，同时不会给宿主带来严重的毒副作用。每天用药或每隔几天用药都可以，用药的时间可以持续数天、数周、数月、甚至数年。某一特定剂量可分为多次间隔给药，例如每天一次、每天两次或更多、每周一次、每两周一次、每三周一次、每月一次或每两月或更多月一次。例如，所述药物可以每天一次施用或每天两次施用。
所述化合物可以在其它抗肿瘤治疗方法之前或之后间隔给药。所述间隔的时间可以为1分钟、2分钟、5分钟、10分钟、20分钟、30分钟、45分钟、1小时、2小时、3小时、4小时、5小时、6小时、12小时、18小时、1天、2天、3天、1周、2周、3周、1个月、2个月、3个月或更长。在一些实施方案中，本申请所述的化合物可以与其它抗肿瘤治疗方法以相同的给药途径给药或者以不同的给药途径给药。
所述化合物可以在其它治疗帕金森氏症的药物之前或之后间隔给药。所述间隔的时间可以为1分钟、2分钟、5分钟、10分钟、20分钟、30分钟、45分钟、1小时、2小时、3小时、4 小时、5小时、6小时、12小时、18小时、1天、2天、3天、1周、2周、3周、1个月、2个月、3个月或更长。在一些实施方案中，本申请所述的化合物可以与其它治疗帕金森氏症的药物以相同的给药途径给药或者以不同的给药途径给药。
在某些实施方式中，可以口服施用所述化合物。口服药物的成分通常包括惰性稀释剂或可食用载体。药物可封装于明胶胶囊中或压缩成片剂。药片、药丸、胶囊、锭剂以及其他剂型可含有以下成分：粘合剂如微晶纤维素、黄芪胶或明胶；辅料如淀粉或乳糖；崩解剂如海藻酸或玉米淀粉；润滑剂如硬脂酸镁；助流剂如胶质二氧化硅；甜味剂如蔗糖或糖精；调味剂如薄荷、水杨酸甲酯或橙子调味剂；润湿剂或乳化剂；防腐剂；以及pH缓冲剂等，如磷酸盐缓冲液、乙酸钠或山梨醇酐单月桂酸酯。当把药物组合物放在胶囊内时，其中还可以放入液体载体如脂肪油。此外，所述药物成分还可以包含各种其它材料，例如糖涂层、虫胶或其它的肠溶剂。
本申请化合物也可以以酏剂、悬浮液、糖浆、饼块、或口香糖等的组分施用。除了含有三唑并三嗪衍生物外，糖浆中还可以含有甜味剂如蔗糖、防腐剂、着色剂和调味剂等。用于口服、肠道外的、皮肤内的、皮下的或局部的药物溶液或悬浮液可包括无菌稀释剂，如水、盐溶液、林格溶液、固定油、聚乙二醇(如PEG400)、甘油、丙二醇、脂肪酸如油酸及其衍生物，极性溶剂(如二甲基乙酰胺(DMAC))或其它合成溶剂；抗菌剂如苄基醇或甲基对苯，抗氧化剂如抗坏血酸或亚硫酸钠，螯合剂如乙烯二胺四乙酸；缓冲剂如磷酸盐缓冲液、醋酸、柠檬酸或磷酸盐，以及张性或浸透压调节剂如氯化钠或葡萄糖，稳定剂如环糊精，表面活性剂如羟基硬脂酸酯等。药物原液可以装入安瓿，一次性的注射器，或由玻璃或塑料制成的多剂量的小瓶。
为了防止药物从体内快速清除，本申请所述化合物也可以放置在能够起到这种保护作用的载体中。也可应用其它各种能够控制药物释放速度的方法，例如使用植入物和微胶囊输送系统的方法。
本申请所述化合物也可以使用喷雾器、干粉吸入器、或定量喷雾吸入器等给药方式来实施给药。用这种方式给药的药物可以用盐水配制成溶液，并在其中加入苯甲醇或其它合适的防腐剂、吸收促进剂、碳氟化合物和其它常用的增溶或分散剂。
一般地说，对于一个患者而言，所要采用的治疗方案和给药剂量将取决于多种因素，包括所用的特定化合物的性能、患者的年龄、体重、一般健康状况、性别、饮食、给药时间、排泄率、病人的病理状况、治疗的目标和医生的判断。如果是联合治疗，活性成分的用量也会取决于使用哪种药物进行联合治疗。
另一方面，本申请提供了一种药物组合和/或试剂盒，其包含(1)本申请所述的化合物，和(2)治疗肿瘤的免疫疗法。
另一方面，本申请提供了一种药物组合和/或试剂盒，其包含(1)所述化合物，和(2)治疗帕金森氏症的药物。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的化合物、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
在本申请的实施例中，所述化合物名称对应如下表4：
表4 本申请实施例中的化合物
Figure PCTCN2020139690-appb-000017
Figure PCTCN2020139690-appb-000018
Figure PCTCN2020139690-appb-000019
Figure PCTCN2020139690-appb-000020
Figure PCTCN2020139690-appb-000021
实施例1 2-(呋喃-2-基)-N5-(4-(氧杂环丁烷-3-基氨基)苯乙基)-[1,2,4]三唑[1,5-a][1,3,5]三嗪-5,7-二胺乙酸盐(化合物22乙酸盐)的制备与分析表征
A、2-(呋喃-2-基)-N 5-(4-(氧杂环丁烷-3-基氨基)苯乙基)-[1,2,4]三唑[1,5-a][1,3,5]三嗪-5,7-二胺乙酸盐的制备如下：
Figure PCTCN2020139690-appb-000022
将500mg游离碱(A05746-093A15)分散于7.5mL乙酸乙酯中。115mg乙酸溶解于2.5mL乙酸乙酯中得到乙酸溶液。在搅拌下将乙酸溶液缓慢滴加至游离碱的混悬液中。搅拌过夜，过滤得到固体，并在40℃真空条件下干燥过夜。得到的产物进行表征测试。
按照相同步骤，使用甲基叔丁基醚(MTBE)、丙酮(Acetone)、乙酸甲酯(MAC)、异丙醇(IPA)采用相同的操作得到目标固体，进行表征测试。
B、分析方法
1)X－射线粉末衍射(XRPD)
固体样品采用Empyrean X射线粉末衍射仪(PANalytical)或D8 Advance X射线粉末衍射分析仪(Bruker)进行分析。Empyrean X射线粉末衍射仪配备了PIXcel 1D检测器。在XRPD分析中，2θ扫描角度从3到40°，扫描步长为0.013°。光管电压和光管电流分别为45KV和40mA。D8 Advance X射线粉末衍射分析仪配备了LynxEye检测器。2θ扫描角度从3到40°，扫描步长为0.02°。光管电压和光管电流分别为40KV和40mA。
2)差示扫描量热分析(DSC)
差示扫描量热分析的仪器型号为DSC 250(TA Instruments，US)。样品经精确称重后置于DSC扎孔样品盘中，并记录下样品的准确质量。样品以10℃/min的升温速率从25℃加热至最终温度。
3)热重分析(TGA)
热重分析仪的型号为TGA 55(TA Instruments，US)。将样品置于已平衡的开口铝制样品盘中，质量在TGA加热炉内自动称量。样品以10℃/min的速率加热至最终温度。
4)动态水蒸气吸附(DVS)
动态水分吸脱附分析采用的仪器型号为Vsorp动态水分吸附分析仪(ProUmid GmbH & Co.KG,Germany)或DVS Intrinsic(SMS,UK)。将样品置于已去皮的样品盘中，自动称重。
Vsorp Dynamic Moisture Sorption Analyzer仪器参数设置如下：
项目 参数
样品温度 25℃
循环时间 10min
每个循环最短时间 50min
每个循环最长时间 120min
重量限度 100％
平衡条件 0.01％/45min
循环#1 湿度0％到0％，温度40℃3小时
循环#2 温度为25℃，湿度从0％升到90％
循环#3 温度为25℃，湿度从80％回到0％
吸附 0,10,20,30,40,50,60,70,80,90
脱附 80,70,60,50,40,30,20,10,0
DVS Intrinsic仪器参数设置如下：
项目 参数
阶跃时间 60min
样品温度 25℃
循环 全循环
吸附 0,10,20,30,40,50,60,70,80,90
脱附 80,70,60,50,40,30,20,10,0
保存数据速率 5S
总流量 200sccm
总试验后流量 200sccm
5)偏光显微分析(PLM)
PLM分析采用的仪器型号为ECLIPSE LV100POL偏光显微镜(尼康，日本)。
6)核磁共振( 1H-NMR)
1H-NMR采用布鲁克AVANCE III HD 300或400，配备Sample Xpress 60自动进样器，或者布鲁克Advance 300，配备B-ACS 120自动进样器。
7)高效液相色谱法(HPLC)
HPLC分析法采用Agilent HPLC 1260系列仪器。溶解度和稳定性测试的HPLC方法如下：
Figure PCTCN2020139690-appb-000023
C、数据与结论
通过XRPD结果(图1)可知，在所有溶剂中均得到乙酸盐，具有与游离碱(free base)不同的晶体形式。在MTBE中反应得到的产物中仍含有少量的游离碱晶型。在不同溶剂中得到的乙酸盐具有相同的晶型。
从 1H-NMR的谱图(图2)，同时结合XRPD谱图(图1)可知，丙酮、乙酸乙酯、乙酸甲酯和异丙醇均能够成为乙酸盐。
DSC、TGA结果(图3)表明样品在210℃之前失重～13.4％，推测是脱去乙酸。在起始温度148.9℃处观察到一个宽的吸热峰，这是由于乙酸的脱去。而在起始温度226.0℃处的吸热峰则推测为游离碱无水晶型的溶化。
4)溶解度
实验测定了游离碱晶型和乙酸盐晶型在生物溶媒(SGF(Simulated Gastric Fluid)，FaSSIF(Fasted State Stimulated Intestinal Fluid)和FeSSIF(Fed State Stimulated Intestinal Fluid))中37℃下的溶解度。将3mg样品加入样品瓶中，加入1mL生物相关溶媒。混悬液在37℃下以500rpm的速度震荡。在0.5h和2h后分别取500μL混悬液，过滤，滤液进行HPLC测试得到溶解度。
表5 化合物22游离碱和乙酸盐形式的溶解度
Figure PCTCN2020139690-appb-000024
表5的结果显示，化合物22游离碱和乙酸盐形式的溶解度均和溶媒pH有很大关联。它们在SGF中有最大的溶解度，乙酸盐为>2.887mg/mL，游离碱0.712mg/mL。乙酸盐在三种生物相关溶媒中的溶解度均大于游离碱。
5)稳定性结果与结论
一定量乙酸盐晶体分别放置于60℃闭口和40℃/75％RH开口条件下最长7天。在0、3和7天分别取样，用稀释剂稀释至～0.3mg/mL用于HPLC纯度测试。固体样品进行XRPD测试，确定晶型。
表6 化合物22乙酸盐的XRPD结果
Figure PCTCN2020139690-appb-000025
表6的结果表明，化合物22乙酸盐在测试条件下没有发现明显的降解。乙酸盐晶型在60℃条件下，物理和化学稳定。而在40℃/75％RH条件下，部分乙酸盐晶型会转化为游离碱晶型。
实施例2 N 5-(4-(2-甲基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑[1,5-a][1,3,5]三嗪-5,7-二胺苯磺酸盐(化合物40苯磺酸盐)的制备
制备反应式如下：
Figure PCTCN2020139690-appb-000026
将苯磺酸水合物(4g,25.4mol)加入甲醇(18mL)中，常温下分批加入N 5-(4-(2-甲基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑[1,5-a][1,3,5]三嗪-5,7-二胺(2.56g,6.35mol)，固体边溶解边分批加入，加完毕后，发现有小量固体不溶解，再超声后全部溶解，搅拌5min后，有大量白色固体析出，加入50mL乙酸乙酯打浆，过滤干燥得到目标化合物为白色固体(2.0g，56％收率)。 1H NMR(500MHz,DMSO-d 6)δ9.04–8.71(m,1H),8.25(d,J＝0.7Hz,2H),8.02–7.96(m,3H),7.61(dd,J＝7.7,1.8Hz,2H),7.46(t,J＝9.7Hz.2H),7.39–7.26(m,3H),7.19(d,J＝2.8Hz,1H),6.75(s,1H),4.42(s,3H),3.65–3.53(m,2H),2.96(t,2H)。
实施例3 本申请化合物能够抑制腺苷受体A2A的活性
1.实验方法
通过基于TR-FRET的cAMP累积测定法评估了测试化合物对腺苷受体A2A的抑制作用。该方法包括，在表达有A2AR的HEK293细胞(hADORA2A-HEK293)中，NECA刺激的cAMP的产生和A2A受体拮抗剂对其的抑制作用。所有细胞均采用完全培养基，培养在5％CO2和37℃条件下进行。用胰酶消化细胞后，在200g离心5分钟收集细胞。再用新鲜完全培养基重悬细胞后采用台盼蓝排除法对细胞活力进行计数，仅当细胞活率大于95％时方可进行后续的cAMP产生测量实验。HEPES(5mM)，BSA稳定剂(0.1％)和Rolipram(10μM)的Hank's缓冲溶液稀释细胞后，将细胞加入384孔白色非透明板(细胞/孔，10μl/孔)，并在室温下与合适浓度 (11个浓度)的测试化合物孵育20分钟。然后将A2A受体激动剂NECA(最终浓度＝EC80，该数值在稍早进行的同一实验中测定)添加到样品中，并在37℃下再次孵育30分钟。最后加入Eu-cAMP示踪剂和Ulight-anti-cAMP，通过测量665nm处的TR-FRET与615nm处的荧光发射的比值来确定cAMP的产生量。选取ZM241385为参比化合物，伊曲茶碱(Istradefylline)为阳性对照化合物。伊曲茶碱是选择性的A2A受体拮抗剂，能通过改变神经元的活动而改善PD患者的运动机能，临床用于治疗PD和改善PD的运动障碍。
2.计算
根据下式计算抑制率(％)，根据从log转换后的浓度-抑制(％)曲线计算IC50值。
Figure PCTCN2020139690-appb-000027
％Inhibitor:％抑制率；
Ratio 665/615high：参照化合物高荧光比值；
Ratio 665/615low：参照化合物低荧光比值；
Ratio 665/615cmpd：测试化合物荧光比值；
利用XLfit软件拟合剂量相应曲线并确定IC 50。
3.结果与结论
表7 本申请化合物抑制腺苷受体A2A的活性
Figure PCTCN2020139690-appb-000028
从上表中的数据可以看出，列表中的本申请化合物均体现出优异的A2A受体拮抗作用，IC 50处于较低的水平，约为10nM。A2A受体抑制活性均比阳性对照化合物伊曲茶碱(Istradefylline)高。
实施例4 本申请化合物选择性抑制腺苷受体
4.1 cAMP累积测定法检测本申请化合物对其他腺苷受体的抑制活性
通过基于TR-FRET的cAMP累积测定法评估了测试化合物22和Istradefylline(阳性对照药)对其他3个腺苷受体A2b、A1和A3的抑制作用。
1.实验方法
(1)Gs偶联的腺苷受体A2b cAMP实验：
在本实验中使用稳定表达腺苷受体A2b的HEK293细胞株作为研究对象(PerkinElmer，ES-013-C)进行实验。细胞培养于含有10％FBS(Hyclone，SH30406.05)、100U/mL青霉素-链霉素混合液(Gibco，15140-122)和100μg/mL G418(Gibco，11811031)的EMEM培养基(ATCC，30-2003)中。使用TrypLETM Express(Gibco，12604-013)每周将细胞消化、传代约3次，并保持约70％至90％的汇合度。在实验当天，将细胞以500个每孔的密度接种到384孔微孔板(Corning，3570)里，细胞接种时的溶液体系为10μL cAMP实验缓冲液中。cAMP实验缓冲液的配制方法为：在14mL 1×HBSS(Gibco，14025-076)中添加75μL 1M HEPES(Gibco，15630080)、200μL 7.5％BSA稳定剂(PerkinElmer，TRF0263)和7.5μL 20mM rolipram(Sigma，R6520)。制备1mM(1000×最终浓度)的参比激动剂NECA(Sigma，E2387)和参比抑制剂CVT-6883(Aobious，AOB4675)，之后于100％DMSO(Millipore，1029312500)中进行3倍梯度稀释。同样10mM的测试化合物，也以100％DMSO中进行3倍梯度稀释。将梯度稀释的参比化合物和测试化合物加入384孔化合物板(Labcyte，PP-0200)中。使用Echo(Labcyte，550)转移10nL每孔梯度稀释的参比激动剂NECA至含有细胞的试验板中，将测试板在37℃孵育30分钟。向测试板中加入5μL每孔的Eu-cAMP示踪剂工作溶液和5μL每孔的ULight-anti-cAMP工作溶液(PerkinElmer，TRF0263)，将测试板置于25℃孵育30分钟。使用EnVision酶标仪(PerkinElmer，2009-0030)读取测试板中每孔的荧光信号值(λex＝320nm，λem＝615nm和665nm)，利用输出的参比激动剂NECA的荧光信号比值(Ratio 665/615)以及浓度计算其EC50值和EC80值。使用100％DMSO制备1000×EC80的NECA。使用Echo转移10nL每孔梯度稀释的参比抑制剂CVT-6883和测试化合物至含有细胞的测试板中。将测试板置于25℃孵育20分钟后，转移10nL每孔1000×EC80的NECA到测试板中。将测试板置于37℃孵育30分钟。向测试板中加入5μL每孔的Eu-cAMP示踪剂工作溶液和5μL每孔的ULight-anti-cAMP工作溶液，将测试板置于25℃孵育30分钟。读取测试板中每孔的荧光信号比值，利用输出的参比抑制剂和测试化合物的数据以及化合物的浓度计算其IC50值。
(2)Gi偶联的腺苷受体A1 cAMP实验：
在本实验中使用稳定表达腺苷受体A1的CHO细胞株作为研究对象(PerkinElmer，ES-010-C)进行实验。细胞培养于含有10％FBS(Hyclone，SH30406.05)、100U/mL青霉素- 链霉素混合液(Gibco，15140-122)和400μg/mL G418(Gibco，11811031)的F12培养基(Gibco，11765-062)中。使用TrypLE TM Express(Gibco，12604-013)每周将细胞消化、传代约3次，并保持约70％至90％的汇合度。在实验当天，将细胞以2000个每孔的密度接种到384孔微孔板(Corning，3570)里，细胞接种时的溶液体系为10μL cAMP实验缓冲液。cAMP实验缓冲液的配制方法为：在14mL 1×HBSS(Gibco，14025-076)中添加75μL 1M HEPES(Gibco，15630080)、200μL 7.5％BSA稳定剂(PerkinElmer，TRF0263)和7.5μL 20mM rolipram(Sigma，R6520)。制备1mM(1000×最终浓度)的参比激动剂NECA(Sigma，E2387)和参比抑制剂DPCPX(Sigma，C101)，之后于100％DMSO(Millipore，1029312500)中进行3倍梯度稀释。同样10mM的测试化合物也以100％DMSO中进行3倍梯度稀释。以100％DMSO制备1mM forskolin(Sigma，F3917)。将梯度稀释的参比化合物、测试化合物和1mM forskolin加入384孔化合物板(Labcyte，PP-0200)中。使用Echo(Labcyte，550)转移10nL每孔1mM forskolin和10nL每孔梯度稀释的参比激动剂NECA到含有细胞的测试板中，将测试板置于37℃孵育30分钟。向测试板中加入5μL每孔的Eu-cAMP示踪剂工作溶液和5μL每孔的ULight-anti-cAMP工作溶液(PerkinElmer，TRF0263)，将测试板置于25℃孵育30分钟。使用EnVision酶标仪(PerkinElmer，2009-0030)读取测试板中每孔的荧光信号值(λex＝320nm，λem＝615nm和665nm)，利用输出的参比激动剂NECA的数据以及浓度计算其EC50值和EC80值。使用100％DMSO制备1000×EC80的NECA。使用Echo转移10nL每孔梯度稀释的参比抑制剂DPCPX和测试化合物到含有细胞的测试板中，将测试板置于25℃孵育20分钟。转移10nL每孔1000×EC80的NECA和10nL每孔1mM forskolin到测试板中。将测试板置于37℃孵育30分钟。向测试板中加入5μL每孔的Eu-cAMP示踪剂工作溶液和5μL每孔的ULight-anti-cAMP工作溶液，将测试板置于25℃孵育30分钟。读取测试板中每孔的荧光信号比值，利用输出的参比抑制剂和测试化合物的数据以及化合物的浓度计算其IC50值。
(3)Gi偶联的腺苷受体A3 cAMP实验：
同样地，使用稳定表达腺苷受体A3的CHO细胞株作为研究对象(PerkinElmer，ES-012-C)进行实验。计算化合物的IC50值。
2.数据分析
1)Gs偶联的腺苷受体A2b cAMP实验：
Figure PCTCN2020139690-appb-000029
％Inhibitor:％抑制率；
Ratio High Control：参照化合物高荧光比值；
Ratio Low Control：参照化合物低荧光比值；
Ratio Compound：测试化合物荧光比值；
2)Gi偶联的腺苷受体A1和A3 cAMP实验：
Figure PCTCN2020139690-appb-000030
％Inhibitor:％抑制率；
Ratio High Control：参照化合物高荧光比值；
Ratio Low Control：参照化合物低荧光比值；
Ratio Compound：测试化合物荧光比值；
3.实验结果
表8 本申请化合物对腺苷受体的抑制活性
Figure PCTCN2020139690-appb-000031
结合实施例3中的数据，化合物22、27、40抑制腺苷A2A受体ADORA 2A活性的IC 50约为10nM，而抑制腺苷A2b、A1和A3受体活性的IC 50均大于300nM。结果表明，在本实验所用的实验条件下，化合物22、27、40和伊曲茶碱(Istradefylline)都对腺苷ADORA 2A表现出了优于其他三种腺苷受体的选择抑制活性。
4.2 钙流实验测定本申请化合物对腺苷受体的选择性抑制活性
1.试验原理：
本实验采用高表达与人Gq偶联的腺苷受体稳转细胞系，通过测试细胞内钙离子浓度的变化对化合物22的拮抗功能进行评价。A 2AR激动剂与A 2AR结合后可以激活下游通路，使细胞内钙离子浓度增加。拮抗剂会竞争性占据激动剂结合A 2AR的结合位点，从而中断胞内信号的传导，并造成细胞内钙离子浓度发生变化，通过检测细胞内钙离子信号的强弱，确定化合物对人ADORA 1、ADORA 2A、ADORA 2B和ADORA 3腺苷受体的拮抗活性。
2.细胞株信息：
ADORA1、ADORA2A、ADORA2B和ADORA3受体稳转细胞系均由药明康德生物部构建，采用ThermoFisher公司的Lipofectamine 2000转染试剂，将受体质粒和G15质粒转入宿主细胞内，然后挑单克隆，构建稳转细胞株。具体信息及培养条件如下：
靶标 参考序列 宿主细胞 培养条件
ADORA1 NM_000674 HEK293 DMEM+10％FBS；G418 300μg/mL；BS:2μg/mL
ADORA2A NM_000681 CHO F12+10％FBS；G418 300μg/mL；BS:2μg/mL
ADORA2B NM_000676.2 HEK293 DMEM+10％FBS；G418 300μg/mL；BS:2μg/mL
ADORA3 NM_000677 CHO F12+10％FBS；G418 300μg/mL；BS:2μg/mL
3.试剂和耗材：
Figure PCTCN2020139690-appb-000032
Figure PCTCN2020139690-appb-000033
4.仪器设备：
仪器 型号 厂家
FLIPR FT-0249 Molecular devices
Echo Echo555 Labcyte
细胞计数仪 AN16097 Beckman
5.试验方法：
细胞板的准备：
1)从液氮罐中取1管细胞并快速的在37℃水浴中溶解。
2)将细胞悬液加到提前加入预热的20mL培养基的离心管中。
3)在室温下1000转离心5分钟。
4)缓慢的倒掉上清，避免影响到沉降的细胞。
5)用10-30mL培养基重悬细胞，用移液器轻柔的吹打细胞。
6)用细胞计数仪计算细胞的活力及数目。
7)用培养基将细胞稀释至1.0×106cells/mL，20μL/孔种入384孔多聚赖氨酸包被细胞板，5％CO2，37℃培养箱孵育16-20小时。
Note:细胞活力达到90％以上才可用于试验。
Fluo4-Direct染料的配制：
配制250mM Probenecid溶液：按照试剂盒操作说明，在77mg probenecid中加入1mL FLIPR缓冲盐溶液，现用现配。
配制2×(8mM)Fluo-4DirectTM加样缓冲液：提前融化实验用量管数Fluo-4DirectTM，每管加入10mL FLIPR缓冲盐溶液，加入0.2mL 250mM Probenecid溶液，避光振荡涡旋>5分钟，现用现配。
化合物准备及实验操作：
1)在整个实验过程中将化合物稀释30000/900*6＝200倍
化合物的配制：将待测4个化合物用DMSO配制成10mM的母液，放于4℃冰箱保存。
2)激动剂参考化合物准备：将激动剂用Echo进行1:4梯度稀释，10个点，左右复孔，接着转移900nL至相应化合物板；然后加30μL实验用缓冲盐溶液至相应的化合物板。
3)参考和待测化合物板制备：将2mM(用DMSO将10mM的母液稀释5倍至2mM)受试化合物做1:4梯度稀释的10点剂量反应曲线。参考化合物CGS-15943用Echo做1:4梯度稀释的10点剂量反应曲线，对于ADORA1、ADORA2B和ADORA3受体CGS-15943起始浓度为200μM，对于ADORA2A受体CGS-15943起始浓度为20μM。
4)加30μL FLIPR缓冲盐溶液至相应的化合物板，备用。
5)将前一天准备好的细胞板从培养箱中取出，每孔加入20μL 2×Fluo-4DirectTM缓冲液，5％CO2，37℃培养箱孵育50分钟，室温避光放置10分钟。
6)在FLIPR仪器中放好化合物板，细胞板和枪头。
7)运行FLIPR仪器软件，按照设定程序，添加10μL实验用缓冲盐溶液到细胞板中，读取荧光信号。再添加10μL既定浓度的激动剂参考化合物到细胞板中，读取荧光信号。读数后，通过软件中“Max-Min”，“Read 91to Maximum allowed”方法导出数据，计算每个细胞系的EC80，准备6×EC80浓度的激动剂。
8)用缓冲盐溶液配制相应细胞6×EC80浓度的参考化合物激动剂，30μL/孔添加至相应的化合物板，备用。
9)运行FLIPR仪器软件，按照设定程序，添加10μL既定浓度的检测化合物及参考化合物到细胞板中，读取荧光信号。再添加10μL 6×EC80浓度的参考化合物激动剂到细胞板中，读取荧光信号。对于化合物的拮抗剂检测，通过软件中“Max-Min”，“Read 1 to 90”方法导出数据。
10)用Prism统计软件进行数据分析。
分析数据公式如下：
拮抗剂％Inhibition＝100-(RLU-LC)/(DMSO-LC)*100
RLU:相对光吸收值，1至90的读值；
HC:DMSO组荧光信号平均值；
LC:拮抗剂最高浓度点荧光信号平均值。
用GraphPad Prism 5.0中”log(inhibitor)vs response—variable slope”对数据进行拟合，得到的IC50。
5.结果与结论：
表9 本申请化合物对腺苷受体的抑制活性
Figure PCTCN2020139690-appb-000034
结果表明，化合物22和化合物40对ADORA 2A介导的信号传导的抑制能力更强，对于腺苷的其它3个受体的抑制能力较弱，即对ADORA 2A的抑制具有高选择性。
实施例5 本申请化合物能够结合腺苷受体
1.试验原理
本实验应用放射性同位素配体竞争结合技术，在生化水平上检测受试化合物和参考化合物CGS-15943对腺苷受体ADORA1、ADORA2A、ADORA2B、ADORA3的结合作用。
2.试剂/化合物和耗材
试剂耗材名称 货号 供应商
人腺苷ADORA1受体膜蛋白 ES-010-M400UA PerkinElmer
人腺苷ADORA2A受体膜蛋白 RBHA2AM400UA PerkinElmer
人腺苷ADORA2B受体膜蛋白 ES-013-M400UA PerkinElmer
人腺苷ADORA3受体膜蛋白 ES-012-M400UA PerkinElmer
[3H]DPCPX NET974001MC PerkinElmer
[3H]CGS-21680 NET1021250UC PerkinElmer
[3H]HEMADO ART1456 ARC
CGS-15943 C199-25MG Sigma
NECA E2387 Sigma
PEI P3143 Sigma
Microscint 20闪烁液 6013329 PerkinElmer
Top Seal-A封板膜 6005250 PerkinElmer
96孔锥形聚丙烯板 5042-1385 Agilent
Unifilter-96GF/C过滤板 6005174 PerkinElmer
3.仪器设备
仪器 型号 厂家
Harvester C961961 PerkinElmer
Microbeta2 2450Microplate counter PerkinElmer
4.试验方法
1)检测缓冲液配制：
ADORA1:25mM HEPES pH 7.4,5mM MgCl 2,1mM CaCl 2,100mM NaCl
ADORA2A:50mM Tris-HCl pH 7.4,10mM MgCl 2,1mM EDTA
ADORA2B:50mM HEPES pH 7.0,5mM MgCl 2,1mM EDTA
ADORA3:25mM HEPES pH 7.4,10mM MgCl 2,1mM CaCl2,0.5％BSA
2)洗液配制：
ADORA1:25mM HEPES pH 7.4,5mM MgCl 2,1mM CaCl 2,100mM NaCl.
ADORA2A:50mM Tris-HCl pH 7.4,154mM NaCl
ADORA2B:50mM HEPES pH 6.5,5mM MgCl 2,1mM EDTA,0.2％BSA
ADORA3:50mM Tris-HCl pH 7.4
3)反应体系：100μL细胞膜、100μL同位素、1μL化合物
4)化合物配制：将受试化合物及参考化合物用100％DMSO稀释。对于受试化合物：起始浓度为10μM，双副孔，4倍梯度稀释，10个点，最终工作浓度为0.038nM～10μM(受 试化合物对于ADORA2A靶点的第二次和第三次检测：起始浓度为2μM，双副孔，4倍梯度稀释，10个点，最终工作浓度为0.0076nM～2μM)。对于ADORA1、ADORA2A、ADORA2B、ADORA3参照化合物：起始浓度为1μM，双副孔，4倍梯度稀释，10个点，最终工作浓度为0.0038nM～1μM。
5)细胞膜配制：将ADORA受体细胞膜用检测缓冲液配制成相应的浓度。
6)同位素配制：将同位素用检测缓冲液配置成相应的浓度。
靶点 细胞膜浓度 同位素 同位素终浓度
ADORA1 2.5μg/孔 [3H]DPCPX 1nM
ADORA2A 5μg/孔 [3H]CGS-21680 6nM
ADORA2B 20μg/孔 [3H]DPCPX 8nM
ADORA3 2μg/孔 [3H]HEMADO 1nM
7)待测化合物和参考化合物IC50测定：
在96孔板中依次加入1μL待测样品和参考化合物。高信号对照孔为0.5％DMSO，ADORA1、ADORA2A、ADORA2B、ADORA3低信号对照孔为CGS-15943。加入100μL细胞和100μL同位素到所有实验孔中。将96孔板密封，ADORA1、ADORA2B、ADORA3室温(22℃)下于摇床上(300rpm)孵育1小时，ADORA2A室温(22℃)下于摇床上(300rpm)孵育2小时。同时，ADORA1、ADORA2A、ADORA2B、ADORA3用0.3％的PEI，室温浸泡GF/C过滤板至少30分钟，50μL/孔。孵育完成后，把孵育好的反应体系用Harvester收集到GF/C过滤板上，洗4次后在50℃烘箱干燥0.5小时。把干燥好的GF/C过滤板底部封膜，每孔加入50μL闪烁液，并用Top-seal-A膜密封。使用Microbeta读数，使用GraphPad Prism 5.0分析数据。
5.数据处理分析
％Inhibition＝100×(1-(Sample_raw value-Low control_Average)/(High control_Average-Low control_Average))
％Inhibitor:％抑制率；
Sample_raw value：测试化合物原始读数；
Low control_Average：参照化合物低水平读数；
High control_Average：参照化合物高水平读数；
用GraphPad Prism 5.0中”log(inhibitor)vs response—variable slope”对数据进行拟合，得到的IC50。
6.试验结果
表10 腺苷受体结合试验结果(IC50)
Figure PCTCN2020139690-appb-000035
表11 腺苷受体结合试验结果
Figure PCTCN2020139690-appb-000036
结果表明，化合物22和40对ADORA 2A均具有极高的结合作用，对腺苷的其他3个受体结合较弱，说明本申请化合物对ADORA 2A的结合选择性高。
实施例6 本申请化合物在小鼠利血平诱导的运动障碍模型中的作用
6.1 化合物22在小鼠利血平诱导的运动障碍模型中的作用
1.试验原理
利血平(Reserpine)是一种吲哚类生物碱药物，它可以通过抑制去甲肾上腺素能神经元末梢的再摄取，导致运动减慢或无能，从而出现了帕金森病相似的临床症状。其机制是利血平可以封闭囊泡内不可逆的单胺如DA、5-羟色胺(5-HT)和去甲肾上腺素(NA)的运输，使单胺类物质其在胞内被降解，从而消耗中枢和外周的单胺，快速降低单胺水平，导致运动障碍等帕金森病类似症状。给予A2A受体拮抗剂，评估待测化合物能否改善小鼠的运动障碍，评价指标包括行走距离、行走时间、直立次数和直立时间。
2.试验设计
第1组：溶剂对照组(Vehicle1+Vehicle2)，Vehicle1(0.1％乙酸,10mL/kg，皮下注射)+Vehicle2(含0.5％MC的乙酸-乙酸钠,10mL/kg,灌胃，行为学测试前60min给药)，n＝15；
第2组：模型组(Reserpine+Vehicle2)，Reserpine(0.6mg/kg,10mL/kg，皮下注射)+Vehicle2(含0.5％MC的乙酸-乙酸钠,10mL/kg,灌胃，行为学测试前60min给药)，n＝15；
第3组：Reserpine+Istradefylline组，Reserpine(0.6mg/kg,10mL/kg，皮下注射)+Istradefylline(10mg/kg,10mL/kg,灌胃，行为学测试前60min给药)，n＝15；
第4组：Reserpine+化合物22乙酸盐组，Reserpine(0.6mg/kg,10mL/kg,皮下注射)+化合物22(3mg/kg,10mL/kg,灌胃，行为学测试前60min给药)，n＝15；
第5组：Reserpine+化合物22乙酸盐组，Reserpine(0.6mg/kg,10mL/kg,皮下注射)+化合物22(10mg/kg,10mL/kg,灌胃，行为学测试前60min给药)，n＝15；
第6组：Reserpine+化合物22乙酸盐组，Reserpine(0.6mg/kg,10mL/kg,皮下注射)+化合物22(30mg/kg,10mL/kg,灌胃给药，行为学测试前60min给药)，n＝15；
雄性CD1种小鼠体重29.5±0.1g。实际使用动物总数90只。动物购自北京维通利华实验技术有限公司(动物合格证号：1100112011009984)，并饲养于上海睿智化学研究有限公司动物房(实验动物使用许可证：SYXK(沪)2017-0018)。动物房为SPF级环境。
3.主要仪器和试剂
名称 供应商 型号
Laboras动物自发活动平台 Metris B.V.,荷兰 NA
试剂耗材名称 货号/批号 供应商
甲基纤维素 M0430-100G/SLCB9094 SIGMA
三水乙酸钠 G70149B/P1536840 GENERAL-REAGENT
利血平 R007/UQNCN-EH TCI
Istradefylline I1100/2PJUO Tokyo Chemical Industry Co.,Ltd.
4.试验步骤
1)化合物配制
配置浓度为0.06mg/mL的利血平溶液，浓度为1mg/ml的Istradefylline溶液，浓度为3mg/ml(浓度1)、1mg/ml(浓度2)和0.3mg/ml(浓度3)的化合物22乙酸盐溶液(浓度1)。
2)试验
试验开始前一天需将试验需要的3通道计时器、注射器、不锈钢工作台和工作凳以及相关记录表格提前放置于测试实验室内备用，并检查动物状态后，未发现该批动物有体重过小、断尾、斑秃、身体状态不佳的情况。实验动物共90只，试验前产生随机分组序列。根据随机序列，在试验当天将实验动物随机分为溶剂对照组，模型组，Istradefylline组，及3、10、30mg/kg的化合物22乙酸盐组。
在试验第一天，根据预先设定的分组和时间点分别单次皮下注射0.1％乙酸或0.6mg/kg利血平，注射后将动物放回至饲养笼盒并推回原饲养间。
在试验第二天进行活动度测试，试验前需要提前校正Laboras系统，使其处于正常工作状态。然后根据预先设定的时间点(单次皮下注射0.1％乙酸或1mg/kg利血平后17hr)灌胃给予Vehicle 2(含0.5％MC的乙酸-乙酸钠(pH＝4.0)缓冲液)、Istradefylline或化合物22乙酸盐。给药后50min将小鼠放入独立的Laboras测试盒里，适应10min后启动Laboras行为学记录系统，此时动物处于末次给药后1hr，并连续记录60min。
试验结束后将实验动物放回饲养笼盒，清理Laboras测试笼盒。
试验结束后，Laboras行为系统自动分析小鼠的0-30min，30-60min，0-60min及每10min的行走距离(m)、行走时间(sec)、直立次数和直立时间(sec)。
所有计量资料均以均数±标准误(平均值±SEM)表示，采用Prism 8.0统计软件作检验分析。
5.结果与结论
表12 本申请化合物对小鼠行走距离的影响
Figure PCTCN2020139690-appb-000037
Figure PCTCN2020139690-appb-000038
表13 本申请化合物对小鼠自发行走时间的影响
Figure PCTCN2020139690-appb-000039
表14 本申请化合物对小鼠直立次数的影响
Figure PCTCN2020139690-appb-000040
结果如表12-14所示，结果显示，口服(OP)给予10mg/kg和30mg/kg的化合物22乙酸盐可以显著地增加行走距离、行走时间、直立次数。试验表明化合物22乙酸盐在利血平诱导的小鼠运动障碍模型中具有治疗作用，且药效存在明显的剂量效应关系。另外，对照药Istradefylline在该试验中显示了大致相同的药效。小鼠利血平诱导的运动障碍模型试验支持化合物22乙酸盐应用于帕金森疾病的治疗。
6.2 化合物22乙酸盐在大鼠利血平诱导的运动障碍模型中的作用
1.实验设计
第1组：溶剂对照组(Vehicle1+Vehicle2)：Vehicle1(0.1％乙酸,1mL/kg,皮下注射)+Vehicle2(含0.5％MC的乙酸-乙酸钠,10mL/kg,灌胃，行为学测试前60min给药)，n＝15；
第2组：模型组(Reserpine+Vehicle2)，Reserpine(1mg/kg,1mL/kg，皮下注射)+Vehicle2(含0.5％MC的乙酸-乙酸钠,10mL/kg,灌胃，行为学测试前60min给药)，n＝15；
第3组：Reserpine+Istradefylline组，Reserpine(1mg/kg,1mL/kg，皮下注射) +Istradefylline(15mg/kg,10mL/kg,灌胃，行为学测试前60min给药)，n＝15；
第4组：Reserpine+化合物22乙酸盐组，Reserpine(1mg/kg,1mL/kg,皮下注射)+化合物22乙酸盐(7.5mg/kg,10mL/kg,灌胃，行为学测试前60min给药)，n＝15；
第5组：Reserpine+化合物22乙酸盐组，Reserpine(1mg/kg,1mL/kg,皮下注射)+化合物22乙酸盐(15mg/kg,10mL/kg,灌胃，行为学测试前60min给药)，n＝15；
第6组：Reserpine+化合物22乙酸盐组，Reserpine(1mg/kg,1mL/kg,皮下注射)+化合物22乙酸盐(30mg/kg,10mL/kg,灌胃给药，行为学测试前60min给药)，n＝15。
2.采用与11.1相同的方法分析小鼠的0-30min，30-60min，0-60min及每10min的行走距离(m)、行走时间(sec)、直立次数和直立时间(sec)。
3.结果与结论
表15 本申请化合物对大鼠行走距离的影响
Figure PCTCN2020139690-appb-000041
表16 本申请化合物对大鼠自发行走时间的影响
Figure PCTCN2020139690-appb-000042
表17 受试化合物对大鼠直立次数的影响
Figure PCTCN2020139690-appb-000043
试验结果显示，本申请化合物在15mg/kg和30mg/kg剂量下可以显著地增加动物行走距 离、行走时间、直立时间及次数。试验表明化合物22乙酸盐在利血平诱导的大鼠运动障碍模型中具有治疗作用，且存在剂量效应关系。另外，对照药istradefylline在该试验中显示了大致相同的药效。大鼠利血平诱导的运动障碍模型试验支持化合物22乙酸盐应用于帕金森疾病的治疗。
实施例7 本申请化合物在氟哌啶醇诱导的大鼠帕金森病僵直模型上的作用
帕金森病(Parkinson’s disease，PD)是一类常见的神经系统退行性疾病，主要病理变化为黑质多巴胺能神经元丢失，多巴胺递质含量减少，导致病人出现静止性震颤、肌僵直、运动不能及姿势反射障碍等多种临床症状。多巴胺D2受体阻断剂氟哌啶醇(Haloperidol)能够阻断纹状体内的多巴胺D2受体,导致小鼠张力障碍性姿态即僵直和运动减少症状，此模型是一种公认的帕金森综合征的动物模型。评价受试物对Haloperidol诱导CD-1小鼠引起的帕金森病僵直症状的改善作用。
7.1 化合物40苯磺酸盐、化合物27和化合物22乙酸盐在氟哌啶醇诱导的大鼠帕金森病僵直模型上的作用
1.试验设计与方法
1)实验一：
57只雄性CD-1小鼠，SPF级，购入时4周龄，实验时为8周龄，体重40g左右，随机分为5组，分别为Vehicle组(n＝11)、化合物40苯磺酸盐1mg/kg组(n＝11)、化合物40苯磺酸盐3mg/kg组(n＝12)、化合物40苯磺酸盐10mg/kg组(n＝12)、化合物40苯磺酸盐30mg/kg组(n＝11)。1mg/kg Haloperidol皮下注射，注射体积10ml/kg，注射30min后灌胃各剂量化合物40，灌胃体积10ml/kg，给药完成后1h和4h分别进行Rearing和Bar Test检测，统计小鼠站立次数及在棒时间，评价化合物40对Haloperidol对CD-1小鼠引起的帕金森病僵直症状的改善作用。
2)实验二：
40只雄性CD-1小鼠，SPF级，购入时4周龄，实验时为5周龄，体重25g左右，随机分为5组，分别为Vehicle组(n＝8)、化合物22乙酸盐3mg/kg组(n＝8)、化合物22乙酸盐10mg/kg组(n＝8)、化合物22乙酸盐30mg/kg组(n＝8)和Istradefylline 10mg/kg(n＝8)。先灌胃各剂量化合物22乙酸盐，灌胃体积10ml/kg，30min后1mg/kg Haloperidol皮下注射，注射体积10ml/kg，给药完成后1h和4h分别进行Rearing和Bar Test检测，统计小鼠站立次数及在棒时间，评价化合物22乙酸盐对Haloperidol对CD-1小鼠引起的帕金森病僵直症状的改善作用。
3)实验三：
40只雄性CD-1小鼠，SPF级，购入时4周龄，实验时为5周龄，体重25-30g左右，随机分为5组，分别为Vehicle组(n＝8)、化合物27 3mg/kg组(n＝8)、化合物27 10mg/kg组(n＝8)、化合物27 30mg/kg组(n＝8)和Istradefylline 10mg/kg(n＝8)。先灌胃各剂量化合物27，灌胃体积10ml/kg，30min后1mg/kg Haloperidol皮下注射，注射体积10ml/kg，给药完成后1h和4h分别进行Rearing和Bar Test检测，统计小鼠站立次数及在棒时间，评价化合物27 对Haloperidol对CD-1小鼠引起的帕金森病僵直症状的改善作用。
2.材料、试剂/化合物信息
实验动物：CD-1小鼠，SPF级，8周龄/5周龄，雄性，40g左右/25g左右，实验动物提供商：上海斯莱克实验动物有限责任公司；生产许可证号：SCXK(沪)2017-0005；质量合格证号：20170005023704、20170005024947。
试剂耗材名称 货号/批号 供应商
甲基纤维素 P10995 adamas-beta
Haloperidol H113852-5g/G1411016 Aladdin
醋酸钠 P1246037 GENERAL-REAGENT
冰醋酸 P1480181 adamas-beta
2-羟丙基-β-环糊精 B1922043 Aladdin
Istradefylline 2PJUO-EB TCI
4.实验步骤
1)药物配制
Vehicle溶剂对照：40％羟丙基β-环糊精溶液
称取20.00g羟丙基β-环糊精，加入50ml超纯水，漩涡震荡超声20min至溶液澄清，室温保存备用。
Vehicle溶剂对照：含0.5％MC的乙酸-乙酸钠(pH≈4.0)缓冲液
配2M乙酸溶液：取11.6mL乙酸于100mL量筒，再加超纯水至100mL，即为2M乙酸，于100mL棕色瓶保存。
配含0.5％MC的乙酸-乙酸钠(pH≈4.0)缓冲液：称取乙酸钠0.0904g，加1.95mL 2M乙酸，至100mL量筒，再加超纯水至约80mL，pH试纸测pH，约3.5～4，再加超纯水至100mL，再加入0.5013g MC，混匀，至MC完全溶解，于100mL棕色瓶，室温保存。
Haloperidol配制(给药体积10ml/kg)
现配现用，称取0.0037g Haloperidol，加入7.4ml 0.9％生理盐水，漩涡振荡，加20ul 1M HCl，室温漩涡振荡超声20min，混匀，此时浓度为0.5mg/ml，稀释5倍至0.1mg/ml为工作浓度，剩余室温避光保存。
化合物40苯磺酸盐溶液配制(给药体积10ml/kg)
现用现配，校正系数为1.415，称取0.0199g化合物，加入4.7ml 40％HP-β-CD，室温涡旋超声10min，混匀，此时的工作浓度为3mg/ml；吸取1ml浓度为3mg/ml的溶液，加入2ml 40％HP-β-CD，此时的工作浓度为1mg/ml；吸取0.2ml浓度为3mg/ml的溶液，加入1.8ml 40％HP-β-CD，此时的工作浓度为0.3mg/ml；吸取0.2ml浓度为1mg/ml的溶液，加入1.8ml 40％HP-β-CD，此时的工作浓度为0.1mg/ml。
化合物22乙酸盐溶液配制(给药体积10ml/kg)
现用现配，校正系数为1.153，称取0.0225g化合物，加入6.5ml含0.5％MC的醋酸-醋酸钠缓冲液，室温涡旋超声10min，混匀，此时的工作浓度为3mg/ml；吸取1ml浓度为3mg/ml的溶液，加入2ml含0.5％MC的醋酸-醋酸钠缓冲液，此时的工作浓度为1mg/ml；吸取0.2ml 浓度为3mg/ml的溶液，加入1.8ml含0.5％MC的醋酸-醋酸钠缓冲液，此时的工作浓度为0.3mg/ml。
化合物27配制(给药体积10ml/kg)
现用现配，校正系数为1.013，称取0.0182g化合物，加入6ml 40％HP-β-CD，室温涡旋超声10min，混匀，此时的工作浓度为3mg/ml；吸取1ml浓度为3mg/ml的溶液，加入2ml 40％HP-β-CD，此时的工作浓度为1mg/ml；吸取0.2ml浓度为3mg/ml的溶液，加入1.8ml 40％HP-β-CD，此时的工作浓度为0.3mg/ml。
Istradefylline配制(给药体积10ml/kg)
现配现用，配1mg/ml，称取0.0040g，加4ml 40％HP-β-CD或者含0.5％MC的醋酸-醋酸钠缓冲液，室温涡旋超声10min，混匀。
2)Haloperidol造模
Haloperidol小鼠腹部皮下注射，剂量1mg/kg，工作液浓度为0.1mg/ml，注射体积为10ml/kg。
3)给药
按照实验设计进行。
4)Rearing检测方法
药物干预后1h及4h，将小鼠放入直径20cm，高21cm的白色透明塑料桶内，开始录像前让小鼠在桶内适应5min。记录小鼠10min内的自发活动行为，统计站立次数，站立次数越多，说明小鼠自发探索活动越频繁，僵直程度越低。
5)Bar Test检测方法
1h及4h时间点Rearing测试结束后，进行Bar Test检测，将小鼠置于直径6cm，高度为6cm的长条形木棒上，前肢握住木棒，仅臀部或者后肢着地，后肢离开台面或者前肢离开木棒时停止计时，记录小鼠在棒时间，每只小鼠测试3次，统计最大在棒时间及平均在棒时间，在棒时间越长，说明小鼠僵直情况越严重。
6)统计学分析
应用Prism GraphPad 7.0独立样本T检验对数据进行组间统计学分析，P<0.05认为有显著性差异。
5.实验结果与结论
表18 不同剂量化合物40苯磺酸盐对小鼠行为学检测结果的影响
Figure PCTCN2020139690-appb-000044
Figure PCTCN2020139690-appb-000045
Figure PCTCN2020139690-appb-000046
注：与Vehicle组相比，*P＜0.05，**P＜0.01，***P＜0.001
表18显示，小鼠注射Haloperidol之后，出现少动的状态，自发活动减少，僵直时间延长；给药化合物40苯磺酸盐组小鼠行为出现明显改善，在棒时间减少，站立次数增加，呈现一定的剂效关系。
表19 不同剂量的化合物22乙酸盐对小鼠行为学检测结果的影响
Figure PCTCN2020139690-appb-000047
Figure PCTCN2020139690-appb-000048
注：与Vehicle组相比，*P＜0.05，**P＜0.01，***P＜0.001
表19显示，在Bar Test检测中，化合物22乙酸盐能够减少在棒时间，延长小鼠站立时间，且结果呈现好的剂量效应依赖关系，随着化合物22乙酸盐剂量的升高，僵直时间减少，在棒时间缩短，10mg/kg组和30mg/kg组药效与阳性药相当。
表20 不同剂量的化合物27对小鼠行为学检测结果的影响
Figure PCTCN2020139690-appb-000049
Figure PCTCN2020139690-appb-000050
Figure PCTCN2020139690-appb-000051
注：与Vehicle组相比，*P＜0.05，**P＜0.01，***P＜0.001
表20显示，在Bar Test检测中，化合物27能够减少在棒时间，延长小鼠站立时间，且结果呈现好的剂量依赖关系，随着化合物剂量的升高，僵直时间减少，在棒时间缩短，10mg/kg组药效与阳性药相当，30mg/kg组药效稍好与阳性药组。
试验结果显示，在氟哌啶醇诱导的僵直症小鼠中，单次口服给予3个受试化合物后，试验小鼠均能极显著，僵直时间减少，在棒时间缩短，显示受试化合物化可以对抗或预防氟哌啶醇诱导的僵直症状，且存在剂量效应关系。另外，对照药istradefylline在该试验中显示了大致相同的药效。氟哌啶醇诱导的小鼠帕金森病僵直模型试验支持受试化合物应用于帕金森疾病的治疗。
7.2 化合物22乙酸盐在氟哌啶醇诱导的大鼠帕金森病僵直模型上的作用
检测以下给药方案中的化合物乙酸盐在氟哌啶醇诱导的大鼠帕金森病僵直模型上的作用。
1.实验设计
第1组：空白对照组(Vehicle1+Vehicle2)，Vehicle 1(含0.5％MC的乙酸-乙酸钠,10mL/kg,灌胃，氟哌啶醇(Haloperidol)注射前30min给药)+Vehicle 2(乙酸水溶液，5mL/kg,腹腔给药，测试前给药)，n＝10；
第2组：模型组(Vehicle1+Haloperidol)，Vehicle 1(含0.5％MC的乙酸-乙酸钠,10mL/kg,灌胃，氟哌啶醇注射前30min给药)+Haloperidol(1mg/kg,5mL/kg,腹腔给药，测试前给药)，n＝10；
第3组：Istradefylline+Haloperidol组：Istradefylline(10mg/kg,10mL/kg,灌胃,Haloperidol注射前30min给药)+Haloperidol(1mg/kg,5mL/kg,腹腔给药，测试前给药),n＝10；
第4组：化合物22乙酸盐+Haloperidol组:化合物22乙酸盐(3mg/kg,10mL/kg,灌胃,Haloperidol注射前30min给药)+Haloperidol(1mg/kg,5mL/kg,腹腔给药，测试前给药),n＝10；
第5组：化合物22乙酸盐+Haloperidol组:化合物22乙酸盐(10mg/kg,10mL/kg,灌胃,Haloperidol注射前30min给药)+Haloperidol(1mg/kg,5mL/kg,腹腔给药，测试前给药),n＝10；
第6组：化合物22乙酸盐+Haloperidol组:化合物22乙酸盐(30mg/kg,10mL/kg,灌胃, Haloperidol注射前30min给药)+Haloperidol(1mg/kg,5mL/kg,腹腔给药，测试前给药),n＝10。
2.实验方法
提前将当天所需动物从饲养间转运至操作间，并适应环境至少30min后开始试验。根据预先设定的分组，将试验动物分为空白对照组，模型组，Istradefylline组，及3、10、30mg/kg的化合物22AC组。试验动物口服给予Vehicle 1(含0.5％MC的乙酸-乙酸钠(pH＝4.0)缓冲液)、Istradefylline、或化合物22乙酸盐，然后把动物放回至原饲养笼盒。30min后，给试验动物腹腔注射Haloperidol或不含Haloperidol的Vehicle 2，并放回至原饲养笼盒。
在Haloperidol或Vehicle 2注射后60和120min时，将大鼠的前肢轻柔的垂直置于一根长20.5cm，直径1.2cm，并高于工作台8.5cm的水平金属杆上，再将后肢轻放于测试盒上，开始计时。
当大鼠的前肢在金属杆上移动或爬下时即停止计时，记录大鼠双前肢在金属杆上保持姿势的持续的时间，即记录为僵直潜伏期。如果大鼠前爪一直没有放下，600sec终止观察，则该动物的僵直潜伏期记为600sec。当天爬杆试验完成时，每组动物取5只灌流，固定并取全脑保存。
僵直潜伏期：大鼠两前肢在金属杆上保持静止不动的时间，最长的僵直潜伏期为600sec。
僵直率％＝(僵直潜伏期/600)*100％
所有计量资料均以均数±标准误(平均值±SEM)表示，采用Prism 8.0统计软件作检验分析。
3.结果与结论
表21 本申请化合物对大鼠僵直潜伏期的影响
Figure PCTCN2020139690-appb-000052
表22 本申请化合物对大鼠僵直率的影响
Figure PCTCN2020139690-appb-000053
Figure PCTCN2020139690-appb-000054
试验结果显示，在氟哌啶醇诱导的僵直症大鼠中，单次口服给予3、10和30mg/kg等三个剂量的化合物22乙酸盐后，试验大鼠均能极显著地缩短僵直潜伏期(降低僵直率)，显示化合物22乙酸盐可以对抗或预防氟哌啶醇诱导的僵直症状，且存在剂量效应关系。另外，对照药istradefylline在该试验中显示了大致相同的药效。氟哌啶醇诱导的大鼠帕金森病僵直模型试验支持化合物22乙酸盐应用于帕金森疾病的治疗。
7.3 化合物40苯磺酸盐在氟哌啶醇诱导Wistar大鼠僵直症模型中的药效学评价
1.试验设计与方法
实验共设置5组，造模组Vehicle、化合物40苯磺酸盐(3mg/kg、10mg/kg、30mg/kg)，伊曲茶碱10mg/kg，n＝8，8，9，8，8。灌胃给药Vehicle、化合物40苯磺酸盐(3mg/kg、10mg/kg、30mg/kg)，伊曲茶碱10mg/kg，给药体积为10mL/kg，30min后开始腹腔注射Haloperidol 1mg/kg，给药体积为5mL/kg，造模后1h Rearing测试站立次数，Rearing检测完成后大鼠休息30min再进行Bar Test检测。
2.实验步骤
1)药物配制
溶媒选择：40％HP-β-CD(2-羟丙基)-β-环糊精)
Vehicle溶剂对照(Ctrl)：40％HP-β-CD
1.40％HP-β-CD：称取120.2g加约250mL超纯水，900rpm磁力搅拌，室温，完全溶解，再加超纯水至300mL，室温避光保存于500mL棕色瓶。
化合物40苯磺酸盐：现配现用，配3mg/mL，称取0.1777g，加42.5mL 40％HP-β-CD，漩涡振荡，室温超声10min，吹匀，再用20mL注射器(换成2.5mL针头)，吹匀，配1mg/mL，稀释3倍，配0.3mg/mL，稀释10倍。
Istradefylline：现配现用，配1mg/mL，称取0.0312g，加31.2mL 40％HP-β-CD，漩涡振荡。
氟哌啶醇诱导：现配现用，配0.2mg/mL，称取0.0148g，加100μL乙酸，再加2mL超纯水，再加390μLl 4M NaOH，再倒至约35mL，测pH(笔式pH计)，pH＝5.86，加超纯水至74mL。
2)Bar Test
Wistar大鼠，分别灌胃给不同浓度化合物40苯磺酸盐和伊曲茶碱，给药体积10mL/kg；30min后，Haloperidol腹腔给药(1mg/kg)，给药体积为5mL/kg；Haloperidol给药后1h后Rearing测试站立次数，Rearing检测完成后大鼠休息30min再进行Bar Test检测。
Bar test时间：测试大鼠的僵直程度，用Bar test检测，即用直径8mm的木棒，固定于2个铁架台之间，离桌面高度为10cm，然后手抓大鼠尾巴，使其前爪搭在木棒上，后爪或屁股着地，一般大鼠第一次检测时，需要适应几次，测试前肢落棒时间或是后肢离开台面时间，一只大鼠测试3次。
3)Rearing
站立次数：用Rearing检测大鼠站立次数，即大鼠放入27*52cm笼子，适应5min，记录大鼠10min内的自发活动行为-站立次数。当大鼠后肢直立并将两个前肢举过肩膀并着地时，即为站立一次，一个前肢提举不计算在内。通常，大鼠会抬起前肢去触摸笼壁。如果大鼠连续几次将前肢举过肩而不落地，则只计一次。
以Bar test的时间为衡量指标，包括平均时间及最大时间。时间长说明僵直严重，时间短说明僵直轻微，药物处理后，Bar test时间越短，说明药物缓解僵直的作用越好。
Rearing统计站立次数，站立次数越多，说明大鼠自发探索活动越频繁，僵直程度越低。
4)统计分析
应用Prism GraphPad 7.0独立样本T检验对数据进行组间统计学分析，P<0.05认为有显著性差异。
5.结果与结论
表23 本申请化合物对大鼠僵直时间的影响
Figure PCTCN2020139690-appb-000055
注：与Vehicle组相比，*P＜0.05，**P＜0.01，***P＜0.001，****P＜0.0001
表23的结果显示，化合物40苯磺酸盐剂量为10mg/kg、30mg/kg治疗组、伊曲茶碱10mg/kg与Vehicle组相比，大鼠僵直平均时间和最大时间显著缩短，站立次数也明显增加，均具有显著性差异(治疗后僵直平均时间P值分别为＜0.0001，＜0.0001，＜0.0001，最大时间P值分别为＜0.0001，＜0.0001，＜0.0001，站立次数P值分别为＜0.0001，＜0.0001，＜0.0001)，而化合物40苯磺酸盐剂量为3mg/kg治疗组与Vehicle组相比，大鼠僵直平均时间和最大时间没有明显的统计学差异，而站立次数具有显著性差异(治疗后僵直平均时间，P＝0.2741；治疗后僵直最大时间，P＝0.0521；治疗后站立次数，P＝0.0351)。氟哌啶醇诱导的大鼠帕金森病僵直模型试验支持化合物40苯磺酸盐应用于帕金森疾病的治疗。
实施例8 本申请化合物在MPTP(1-甲基-4-苯基-1，2，3，6-四氢吡啶)诱导的小鼠帕金森模型中的药效学评价
1.实验原理
通过注射黑质选择性毒物MPTP，通过干预线粒体，引起脂质过氧化，使得膜结构紊 乱，影响细胞功能，最终导致选择性破坏黑质DA能神经元，导致黑质DA能神经元大量死亡，纹状体酪氨酸羟化酶阳性纤维大量丧失，纹状体DA及其代谢产物3,4-二羟基苯乙酸、高香草酸水平均明显降低，也有黑质纹状体小胶质细胞和星形细胞的增生和蓝斑、下丘脑等区的损伤，与帕金森病患者的改变基本相同。评价受试物对MPTP诱导小鼠引起的帕金森病僵直症状的改善作用。
2.实验设计与方法
组别 动物数 给药 剂量 给药方式 给药频率
第1组 6 Vehicle N/A p.o. 一次
第2组 7 化合物22 10mpk p.o. 一次
第3组 7 化合物22 30mpk p.o. 一次
3.实验步骤
选取C57BL/6雄性小鼠，8周龄，8周大的C57小鼠每天腹膜内(i.p.)注射溶于盐水中的媒介物(生理盐水)或MPTP(30mg/kg体重)，持续5天，以诱发帕金森病。
在最后一次MPTP注射后0.5小时，通过胃内给药以两种剂量(10mpk和30mpk)给药。
上次MPTP注射后1.5个小时，进行10分钟的旷场测试(总行进距离和站立次数)。
4.实验结果与结论
表24 本申请化合物对帕金森小鼠运动能力的影响
组别 运动路程(cm) 站立次数
模型组(n＝6) 139.5 0.83
化合物22(10mg/kg)(n＝6) 647.0 18.3
化合物22(30mg/kg)(n＝6) 1107.3 31.0
结果显示，在MPTP诱导的小鼠帕金森模型中，化合物22在10mg/kg和30mg/kg剂量条件下，显著减轻小鼠的帕金森运动症状，在运动路程和站立次数方面均得到明显的提升，呈现出剂量-药效关系，表现了很好的药效。MPTP诱导的小鼠帕金森模型试验支持受试化合物22应用于帕金森疾病的治疗。
实施例9 本申请化合物在6-OHDA诱导的大鼠旋转实验中的药效及对L-DOPA耐药性的克服作用评价
1.实验原理
帕金森病患者在长期使用左旋多巴以后很容易出现耐药现象(剂末现象)，即其药效持续时间往往会逐渐变短(开期缩短)，功效也会越来越弱，患者的病症也越来越不受控制，通过与受试化合物联用，能够恢复左旋多巴对已经出现剂末现象的关闭期帕金森病患者的功效。采用6-羟基多巴胺(6-OHDA)定点注射毁损黑质纹状体的方法构建帕金森大鼠模型，然后给予动物长时间(计划3-4周)的左旋多巴(L-DOPA)/苄丝肼(Benserazide)(BID)，在看到动物出现对左旋多巴的耐药现象以后，即当左旋多巴引起的旋转时间有明 显减少以后(3-4周后)，同时给予动物受试化合物和左旋多巴/苄丝肼，并测量动物的旋转时间，考察受试化合物对大鼠旋转症状的恢复情况。
2.实验设计
第一阶段：将同一批成模大鼠(共15只)分别给予不同剂量(0,1,3,10,30和100mg/kg)的化合物22乙酸盐进行旋转试验，用旋转计数仪计数60min后，腹腔注射给予L-DOPA(5mg/kg)/Benserazide(1.25mg/kg)，然后再次使用旋转计数仪进行旋转计数60min。每个剂量的旋转测试之间至少间隔1天。第一阶段总计测试6次。
第一阶段动物分组和药物处理：
Figure PCTCN2020139690-appb-000056
第二阶段：第2、3、4、5和6组的试验动物均每天腹腔注射给予两次高剂量的L-DOPA(25mg/kg)/Benserazide(6.25mg/kg)，目的是为了诱发出现耐药现象，其中在第0(第0天为开始给予高剂量L-DOPA/Benserazide的前一天)、7、14、21和22天分别进行5次旋转测试，测试当天所有试验动物均只腹腔注射给予一次的L-DOPA(20mg/kg)/Benserazide(5mg/kg)诱导旋转。在第22天，在腹腔注射给予L-DOPA(20mg/kg)/Benserazide(5mg/kg)之前60min，各试验组先分别口服给予溶媒，受试药或阳性药。为了评估化合物22乙酸盐是否能够预防动物对L-DOPA/Benserazide出现耐药现象，除了每天腹腔注射给予两次L-DOPA(25mg/kg)/Benserazide(6.25mg/kg)以外，第7组和第8组的试验动物还在每天分别口服给予两次的受试药化合物22乙酸盐和对照药Istradefylline，其中在第0、7、14和21天分别进行4次旋转测试。在测试当天，受试药和对照药均只口服给予1次；这一天所有试验动物也均只腹腔注射给予一次的L-DOPA(20mg/kg)/Benserazide(5mg/kg)诱导旋转。在第二阶段，用旋转计数仪进行旋转计数的时间均为180min。
Figure PCTCN2020139690-appb-000057
Figure PCTCN2020139690-appb-000058
3.实验试剂和动物：
第一阶段：Wistar大鼠，6-8周，雄性，178-213克，试验动物提供商：北京维通利华实验动物技术有限公司，生产许可证号：SCXK(京)2016-0006，质量合格证号：1100112011004974；
第二阶段：Wistar大鼠，6-8周，雄性，166-225克，试验动物提供商：北京维通利华实验动物技术有限公司，生产许可证号：SCXK(京)2016-0006，质量合格证号：1100112011011824、1100112011011826；
试剂耗材名称 货号/批号 供应商
甲基纤维素 079K0054V Sigma-Aldrich
冰醋酸 / 天津市光复科技发展有限公司
三水乙酸钠 WXBC8551V Sigma-Aldrich
6-羟基多巴胺 MKCH0942 Sigma-Aldrich
L-抗坏血酸 SLBS0713V Sigma-Aldrich
盐酸阿扑吗啡 / 中检所
左旋多巴 SLCB0627 Sigma-Aldrich
盐酸苄丝肼 BCBZ1809 Sigma-Aldrich
4.实验步骤与方法
帕金森大鼠造模：
L-DOPA注射量：每只大鼠左侧内侧前脑束(MFB)注射6-OHDA溶液(2.5μg/μl)4μl。
L-DOPA注射坐标：以前囟为标准参考点，参照大鼠脑立体定位图谱，注射左侧的内侧前脑束：anterior(A)，-2.5mm；lateral(L)，+2.0mm；ventral(V)，-8.5mm。
L-DOPA注射速度：注射速度1μl/min，注射后原位留针5min，再以1mm/min的速度缓慢退针，缝合伤口。
按照实验设计的方法分组和给药。
第一阶段只测试动物的旋转圈数，仅在第二阶段测试旋转反应时间。
在第二阶段，试验动物腹腔注射给予L-DOPA/Benserazide后，每5min记录一次总旋转圈数，依此计算出每5min的旋转圈数，并从中找出旋转圈数最多的5min，定义该5min内的旋转圈数为峰值旋转圈数。旋转圈数增至峰值旋转圈数20％的第一个5min为旋转反应时间的起始点，旋转圈数降至峰值旋转圈数20％的第一个5min为旋转反应时间的终止点，从起始点到终止点之间的时间算作旋转反应时间。
试验结果以“均值±标准差”表示。数据采用SPSS16.0软件包进行数据统计分析，采用单因素方差分析法(One-way ANOVA)，配对样本T检验以及独立样本T检验进行比较，P<0.05具有统计学差异。
6.结果与结论
第一阶段的结果如表25和26所示。
表25 化合物22乙酸盐单独给药后的总旋转圈数
Figure PCTCN2020139690-appb-000059
注：与溶媒组(化合物22乙酸盐,0mg/kg)相比，*P<0.05。
表26 化合物22乙酸盐与L-DOPA联用的总旋转圈数(平均值±标准差)
Figure PCTCN2020139690-appb-000060
注：化合物22乙酸盐口服1小时后腹腔注射给予L-DOPA(5mg/kg)/Benserazide(1.25mg/kg)。与溶媒组(化合物22乙酸盐，0mg/kg)相比，*P<0.05。
第一阶段试验旨在评价不同剂量(0,1,3,10,30和100mg/kg)的受试药化合物22乙酸盐对6-OHDA诱导的大鼠对侧旋转作用的药效。
结果显示，与给予溶媒(化合物22乙酸盐,0mg/kg)相比，单独给予不同剂量的受试药化合物22乙酸盐后，动物的旋转圈数在高剂量时有所增加，其中受试药在30mg/kg剂量下可显著增加对侧旋转圈数。
虽然单独用药时化合物22乙酸盐在所有剂量下引起动物旋转的程度都不高，而且L-DOPA/Benserazide在低剂量下单独起作用的药效也不明显，但在保持L-DOPA/Benserazide为同等低剂量的情况下，化合物22乙酸盐与L-DOPA联用的药效明显，受试药化合物22乙酸盐在5个剂量下均可增加L-DOPA/Benserazide所引起的对侧旋转圈数，并呈剂量依赖关系，其中在30mg/kg和100mg/kg的剂量下，化合物22乙酸盐能够显著增加动物的对侧旋转圈数。
第二阶段的结果如表27所示。
表27 各试验组动物的旋转反应时间(平均值±标准差，min)
Figure PCTCN2020139690-appb-000061
Figure PCTCN2020139690-appb-000062
结果显示，给予6-OHDA帕金森模型大鼠高剂量的L-DOPA(25mg/kg，BID)/Benserazide(6.25mg/kg,BID)后，大鼠在第7天即出现对L-DOPA的耐药性，表现为L-DOPA诱导的旋转反应时间明显缩短，并一直维持到试验结束的第22天。
在第22天，单次给予已出现耐药性的6-OHDA帕金森模型大鼠不同剂量的化合物22乙酸盐后(7.5、15和30mg/kg)，与第0天和21天相比，受试药能在不同程度上恢复或增加动 物的旋转反应时间，其中受试药在15和30mg/k剂量下能够显著增加旋转反应时间(与第21天相比)，并存在剂量依赖关系。这说明，在动物对L-DOPA/Benserazide出现耐药性以后，化合物22乙酸盐能够有效地恢复或提高L-DOPA在耐药动物中的原有药效。
试验结果也显示，化合物22乙酸盐还可能有效地预防或减缓6-OHDA帕金森模型大鼠对L-DOPA/Benserazide的耐药性。
另外，与模型对照组相比，试验期间各试验组大鼠体重无明显变化，表明受试药物化合物22乙酸盐对试验动物的体重无明显影响。
该模型试验显示，当帕金森病患者由于长期使用左旋多巴而出现耐药现象以后，通过与化合物22乙酸盐联用，左旋多巴有可能恢复对这些关闭期帕金森病患者的治疗功效。支持权利要求书中对于帕金森疾病治疗的应用。
实施例10 本申请化合物在鼠源结肠癌MC38细胞株皮下同种移植C57BL/6雌性小鼠模型中的药效学评价
1.试验原理
MC38小鼠结肠癌细胞模型为结肠癌药效验证常用模型，多用作结直肠癌发生及转移方向研究，并成为肿瘤药效验证的常用途径。评价测试药物在鼠源结肠癌MC38细胞株皮下同种移植雌性C57BL/6小鼠动物模型中的抗肿瘤作用。
2.试验设计
Figure PCTCN2020139690-appb-000063
3.试验材料和试剂
实验动物：C57BL/6小鼠，雌性，7-9周(肿瘤细胞接种时的小鼠周龄)，体重16.4-20.1g，购自上海灵畅生物科技有限公司(LC)，生产许可证号：SCXK(沪)2013-0018，动物合格证编号：2013001837385。饲养环境：SPF级。
Anti-PD-1抗体：批号：695318A1B，包装规格：18mg，由BioXcell提供，无色溶液，4℃保存。
溶媒配制：称取16.8g羟丙基-beta-环糊精(淄博千汇生物科技有限公司，批号：160101)，加入42ml灭菌水，完全溶解后待用。
4.实验方法
MC38细胞培养在含10％胎牛血清的DMEM培养液中。收集指数生长期的MC38细胞， PBS重悬至适合浓度用于小鼠皮下肿瘤接种。
雌性小鼠右侧皮下接种1×10 6MC38细胞。接种当天定义为第0天。MC38细胞接种第二天(Day1)，根据体重随机分组给药。分别配制3mg/mL和10mg/mL的化合物22溶液备用。
肿瘤体积计算公式：肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)；
肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组肿瘤体积或瘤重的百分比值。计算公式如下：
T/C％＝T TV/C TV×100％(T TV：治疗组在某一特定时间点的平均TV；C TV：溶媒对照组在某一特定时间点的平均TV)；
或T/C％＝T TW/C TW×100％(T TW：治疗组实验终结时平均瘤重；C TW：溶媒对照组实验终结时平均瘤重)。
肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(TV)或瘤重(TW))。
所有实验结果以平均瘤体积±SEM(平均标准误差)表示。不同组间的统计分析选择最佳药物治疗点(通常是在最后一次给药后)。用独立样本T检验方法比较治疗组肿瘤体积与对照组相比有无显著性差异。所有的数据均用SPSS 18.0进行分析。p<0.05为具有显著性差异。
5.实验结果与讨论
表28 本申请化合物对结肠癌小鼠模型的药效
Figure PCTCN2020139690-appb-000064
试验结果显示，在MC38小鼠结肠癌细胞模型中，测试化合物22在30mg/kg,100mg/kg的剂量条件下，第22天显示出良好的肿瘤抑制活性，肿瘤抑制率(TGI)分别为34％和48％。测试化合物22与Anti-PD-1抗体联用，同样展现出优异的抗肿瘤体内活性，肿瘤抑制率(TGI)分为85％，均未见动物死亡和体重显著变化。以上实验支持化合物22作为单药或联合Anti-PD-1抗体在相应肿瘤适应症中的应用。
实施例11 本申请化合物在B16F10鼠源黑色素瘤模型中的药效学评价
1.试验原理
B16F10鼠源黑色素瘤模型是经典的黑色素瘤药效评估模型，采用同种B16F10肿瘤细胞接种于雌性C57BL/6小鼠皮下造模而成。给与受试物，评估对模型中肿瘤生长的抑制效果。
2.实验设计
Figure PCTCN2020139690-appb-000065
3.试验材料和试剂
实验动物：种属：Rodent；品系：C57BL/6mice；等级：SPF级；周龄：6-8周；性别：
雌性；体重：18-22g；实验动物提供商：北京维通利华生物技术有限公司；生产许可证号：SCXK(京)2016-0006；质量合格证号：1100111911039475；
Anti PD-1抗体：提供单位：康龙化成(北京)新药技术股份有限公司；批次：717919M1；
溶媒：40％羟丙基-β-环糊精(SIGMA，批次：Z08D9Y76902；称取120g HP-beta-CD用蒸馏水溶解，最后用烧杯定容至300ml，配置成40％羟丙基-β-环糊精溶液)。
4.实验方法
用含有灭活的10％的胎牛血清，100U/ml的青霉素和100μg/ml的链霉素以及2mM谷氨酰胺的DMEM培养基在37℃、5％CO 2的培养箱中培养B16F10肿瘤细胞，每隔3至4天待细胞长满后分瓶传代。将处于对数生长期的肿瘤细胞用于体内肿瘤的接种。
将无血清的DMEM培养液重悬的B16F10肿瘤细胞以1×10 5/100μl接种于实验动物的右侧肋部皮下。第二组当肿瘤长至75mm 3时选出12只肿瘤体积较均一的动物按照实验设计给药。
称取31.99mg(纯度93.79％)化合物22加入6ml 40％HP-beta-CD溶液，涡旋超声混匀，配制5mg/ml浓度药物待用。
移液枪吸取7.18mg/ml Anti PD-1 0.418ml加入1.082ml PH7.0PBS溶液，轻轻倒置混匀，配制2mg/ml浓度溶液待用。
肿瘤接种的第一天为D0天，第一、三、四组从肿瘤接种的第二天开始给药，第二组当肿瘤体积达到75mm 3时开始给药。
肿瘤体积：每周使用游标卡尺对肿瘤体积进行3次的测量，测量肿瘤的长径和短径，其体积计算公式为：体积＝0.5×长径×短径 2。
肿瘤生长抑制率(TGI,％)：
肿瘤生长抑制率(TGI,％)＝(1-T/C)×100。
TGI＝100-T/C×100，
T/C(％)＝(治疗组当日肿瘤体积的均值-治疗组初始D0天肿瘤体积的均值)/(对照组当日肿瘤体积的均值-对照组初始D0天肿瘤体积的均值)×100。
应用Graphpad Prism 8.0进行生存数据分析。
5.实验结果与讨论
表29 本申请化合物对黑色素瘤小鼠模型的肿瘤抑制药效
Figure PCTCN2020139690-appb-000066
a均值±标准误； bvs.溶剂对照组；
试验结果显示，在B16F10鼠源黑色素瘤模型中，肿瘤接种后第18天的治疗结果显示，化合物22与Anti PD-1联合治疗组(平均肿瘤体积为1176mm 3)具有显著性抗肿瘤作用(TGI＝48.1％，P＝0.004，vs.溶剂对照组)；化合物22单独治疗组体现出较大的抗肿瘤趋势(平均肿瘤体积为1764mm 3，TGI＝22.1％，P＝0.173，vs.溶剂对照组)；Anti PD-1单独治疗组(平均肿瘤体积分别为2071mm 3，TGI＝8.6％，P＝0.586，vs.溶剂对照组)没有抗肿瘤作用，这与模型本身为Anti PD-1耐药模型有关。实验中均未见动物死亡和体重显著变化。以上实验支持化合物22作为单药或联合Anti-PD-1抗体在相应肿瘤适应症中的应用。
实施例12 本申请化合物在A20鼠源淋巴癌(B淋巴细胞)同种移植模型中的药效学评价
1.实验原理
A20鼠源淋巴癌(B淋巴细胞)同种移模型是经典的B淋巴细胞癌的药效评估模型，采用同种将A20肿瘤细胞接种于雌性BALB/c小鼠右侧肋部皮下造模而成。给与受试物，评估对模型中肿瘤生长的抑制效果。
2.实验设计
Figure PCTCN2020139690-appb-000067
3.试验材料和试剂
实验动物：种属：Rodent；品系：BALB/c mice；等级：SPF级；周龄：6-8周；性别：
雌性；体重：18-21g；实验动物提供商：北京维通利华生物技术有限公司；生产许可证号：SCXK(京)2016-0006；质量合格证号：1100111911036383；
溶媒：40％羟丙基-β-环糊精(SIGMA，批次：Z08D9Y76902；称取120g HP-beta-CD用蒸馏水溶解，最后用烧杯定容至300ml，配置成40％羟丙基-β-环糊精溶液)。
4.实验方法
用含有灭活的10％的胎牛血清，100U/ml的青霉素和100μg/ml的链霉素以及0.05mM2-巯基乙醇的RPMI 1640培养基在37℃、5％CO 2的培养箱中培养A20肿瘤细胞，每隔3至4天待细胞长满后分瓶传代。将处于对数生长期的肿瘤细胞用于体内肿瘤的接种。
将无血清的RPMI 1640培养液重悬的A20肿瘤细胞以3×10 5/100μl接种于实验动物的右侧肋部皮下，共接种30只动物。肿瘤细胞接种后的第一天根据体重选出24只体重较均一的动物分组给药，每组各12只，具体给药方案按照实验设计进行。
称取15.99mg(纯度93.79％)化合物22加入3ml 40％HP-beta-CD溶液，涡旋超声混匀，配制5mg/mL的化合物22溶液使用。
肿瘤体积：每周使用游标卡尺对肿瘤体积进行3次的测量，测量肿瘤的长径和短径，其体积计算公式为：体积＝0.5×长径×短径 2。
肿瘤生长抑制率(TGI,％)：
肿瘤生长抑制率(TGI,％)＝(1-T/C)×100，
TGI＝100-T/C×100；
T/C(％)＝(治疗组当日肿瘤体积的均值-治疗组初始D0天肿瘤体积的均值)/(对照组当日肿瘤体积的均值-对照组初始D0天肿瘤体积的均值)×100。
应用SPSS T-test检验对肿瘤体积进行组间统计学分析。
5.实验结果与结论
表30 本申请化合物的淋巴癌小鼠模型的肿瘤抑制药效
Figure PCTCN2020139690-appb-000068
试验结果显示，化合物22(50mg/kg)治疗组体现出明显的抗肿瘤趋势，并于肿瘤接种后第15、17天体现出显著的抗肿瘤作用(P<0.05)，其TGI分别为29.4％、32.6％。另外，给药期间，各组实验动物活动、进食等一般状态良好，并没有发现明显的临床异常，治疗前后动物整体体重均增加。以上实验支持化合物22作为单药在相应肿瘤适应症中的应用。
实施例13 本申请化合物在CT26鼠源结肠癌模型中同种移植模型中的药效学评价
1.实验原理
CT26鼠源结肠癌同种移模型是经典的结肠癌的药效评估模型，采用BALB/c小鼠皮下接种CT26造模而成。给与受试物，评估对模型中肿瘤生长的抑制效果。
2.实验设计
组别 动物数 给药组 剂量(mg/kg) 给药方 计划给药周期 实际给药周期
        式    
1 10 溶媒对照 0 p.o. QD×14or 28天 QD×25天
6 10 化合物22 100 p.o. QD×14or 28天 QD×25天
3.试验材料和试剂
实验动物：BALB/c小鼠，雌性，7-8周(肿瘤细胞接种时的小鼠周龄)，体重17.4-23.5g，160只(100只加60只富余小鼠)。购自上海灵畅生物科技有限公司(LC)，生产许可证号：SCXK(沪)2013-0018，动物合格证编号：2013001837385。饲养环境：SPF级。；
溶媒：40％羟丙基-β-环糊精(淄博千汇生物科技有限公司；批号：160101；称取16.8g羟丙基-β-环糊精，加入42ml灭菌水，完全溶解后待用。)。
4.实验方法
CT26细胞培养在含10％胎牛血清的RPMI1640培养液中。收集指数生长期的CT26细胞，PBS重悬至适合浓度用于小鼠皮下肿瘤接种。
雌性小鼠右侧皮下接种5×10 5CT26细胞。接种当天定义为第0天。MC38细胞接种第二天(Day1)，根据体重随机分组给药。
称取64.35mg化合物22(60mg free base)，加入6.0ml溶媒，涡旋超声得到6.0ml悬浊液，配制10mg/mL的化合物22溶液使用。
肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组肿瘤体积或瘤重的百分比值。计算公式如下：
T/C％＝T TV/C TV×100％(T TV：治疗组在某一特定时间点的平均TV；C TV：溶媒对照组在某一特定时间点的平均TV)；
或T/C％＝T TW/C TW×100％(T TW：治疗组实验终结时平均瘤重；C TW：溶媒对照组实验终结时平均瘤重)。
肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(TV)或瘤重(TW))。
所有的数据均用SPSS 18.0进行分析。
5.实验结果与结论
表31 本申请化合物的结肠癌小鼠模型的肿瘤抑制药效
Figure PCTCN2020139690-appb-000069
试验结果显示，化合物22(50mg/kg)治疗组在第26天体现出抗肿瘤效果，其TGI为19％。另外，给药期间，各组实验动物活动、进食等一般状态良好，并没有发现明显的临床异常。以上实验支持化合物22作为单药在相应肿瘤适应症中的应用。
实施例14 本申请化合物在ICR小鼠中的药代动力学
14.1化合物27、化合物40、化合物22和化合物41在不同给药方式中的药代动力学
1.实验信息与设计
Figure PCTCN2020139690-appb-000070
2.计算软件
采用
Figure PCTCN2020139690-appb-000071
6.4作为PK参数计算软件。
3.结果与结论
表32 本申请化合物在ICR小鼠中的药代动力学结果
Figure PCTCN2020139690-appb-000072
Figure PCTCN2020139690-appb-000073
实验结果表明，列表中的化合物在ICR小鼠中均有优异的药代动力学表现。化合物27，化合物40，化合物22，化合物41展现出较高的口服体内暴露量和较高的口服生物利用度。化合物27，化合物22，化合物40和展现出较高的口服体内暴露量，其中化合物27，化合物22和化合物40在30mg/kg的剂量下，连续口服给药均未出现明显的药物蓄积。
14.2 化合物40及其苯磺酸盐在ICR小鼠中的药代动力学
1.实验信息
Figure PCTCN2020139690-appb-000074
2.实验设计
组别 受试物 给药剂量 采血点
第1组 化合物40 100mg/kg 0.25,0.5,1,2,4,8,24h
第2组 化合物40苯磺酸盐 100mg/kg(按照游离碱计) 0.25,0.5,1,2,4,8,24h
3.实验结果与结论
表33 本申请化合物在在ICR小鼠中的药代动力学结果
Figure PCTCN2020139690-appb-000075
表33的数据显示，受试化合物在ICR小鼠体内具有优异的药代动力学参数。化合物40及其苯磺酸盐的体内暴露量均较高。
实施例15 本申请化合物在SD大鼠中的药代动力学
15.1 化合物27、化合物16、化合物14和化合物22在SD大鼠中的药代动力学
1.实验信息与设计
Figure PCTCN2020139690-appb-000076
2.计算软件
采用
Figure PCTCN2020139690-appb-000077
6.4作为PK参数计算软件。
3.结果与结论
表34 本申请化合物在SD大鼠中的药代动力学结果
Figure PCTCN2020139690-appb-000078
实验结果表明，列表中的化合物在SD大鼠中均有较为优异的药代动力学表现，静脉注射的T 1/2约在0.5至1h，口服给药的T 1/2约在3-5h，展现出较高的口服体内暴露量和较高的口服生物利用度。
15.2 受试物在SD大鼠中的药代动力学
1.试验设计
给药剂量：
Figure PCTCN2020139690-appb-000079
给药剂量：本组化合物均采用混合给药的方式进行，每6个化合物为1组混合PO给药。
Figure PCTCN2020139690-appb-000080
Figure PCTCN2020139690-appb-000081
检测模型及检测方法
Figure PCTCN2020139690-appb-000082
Figure PCTCN2020139690-appb-000083
2.试验材料与设备
1)主要试剂：
试剂名称 来源 批号
甲酸 阿拉丁 F1627037
乙腈 Merck KGaA Co. JA050830
PEG400 麦考林化学试剂有限公司 C10083707
维拉帕米 阿拉丁 K1629079
2)主要仪器
Figure PCTCN2020139690-appb-000084
3.分析方法
1)液相条件
色谱柱：ACQUITY UPLC BEH C18(2.1mm×50mm，1.7μm)
流速：化合物40，化合物27，化合物40和化合物47：0.4mL/min柱温：40℃进样量：5μL；化合物44和化合物48：0.3mL/min柱温：35℃进样量：1μL。
化合物40，化合物27，化合物40和化合物47：流动相A：水(0.1％甲酸)，流动相B：乙腈(0.075％甲酸)；
化合物44和化合物48：流动相A：水(0.1％甲酸)，流动相B：乙腈(0.1％甲酸)。
梯度洗脱，洗脱程序见下表。
Figure PCTCN2020139690-appb-000085
Figure PCTCN2020139690-appb-000086
2)质谱条件
电喷雾离子源(Turbo spray)，正离子检测模式，选择多反应监测扫描(MRM)模式进行质谱分析。质谱离子源参数及化合物检测参数见下表。
Figure PCTCN2020139690-appb-000087
4.试验方法
按照试验方案中的取血点取血。取血浆样品50μL，加入3倍体积的乙腈(含内维拉帕米定5ng/mL)，涡旋振荡30s后，15000rpm，4℃离心15min，取上清液10μL进行LC-MS/MS分析。
按照试验方案中的溶媒进行化合物配制。
SD大鼠3只/化合物，雄性，体重200-220g，给药前禁食一晚但可以饮水。
口服灌胃给药：于灌胃给药前、给药后0.083h、0.25h、0.5h、1h、2h、4h、8h和24h(化合物40，化合物27，化合物40和化合物47)，或给药后0.25h、0.5h、1h、4h、6h和8h(化合物44和化合物48)眼眶取血于肝素钠处理的试管中，离心后取上清液血浆用于LC-MS/MS分析。
将血浆样本按“样品处理”项方法处理后用LC-MS/MS法进行分析，随行标准曲线和质控样本。超过定量上限的时间点稀释10或100倍进行分析。
所有的测定数据由Analyst 1.6.3软件采集并处理，用Microsoft Excel计算和处理数据。用DAS 3.2.8软件，采用统计矩法进行药代动学参数计算。主要包括动力学参数T max、t 1/2、C max、AUC (0-t)等。
5.试验结果与结论
表35 本申请化合物在SD大鼠中的药代动力学结果
Figure PCTCN2020139690-appb-000088
表36 本申请化合物在SD大鼠中的药代动力学结果
Figure PCTCN2020139690-appb-000089
表35和表36的实验结果表明，列表中的化合物在SD大鼠中均有较为优异的药代动力学表现，AUC last数值较高，展现出较高的口服体内暴露量和较高的口服生物利用度。列表中的化合物优异的大鼠药代动力学特性可以支持进一步的临床人体实验。
15.3 化合物22乙酸盐在SD大鼠中单次和重复给药的药代动力学
1.实验设计
Figure PCTCN2020139690-appb-000090
Figure PCTCN2020139690-appb-000091
给药信息如下所示：
Figure PCTCN2020139690-appb-000092
a：溶媒为10％DMAC/15％Solutol HS 15/75％PBS(pH＝7.4)。
b：溶媒为含0.5％MC的乙酸-乙酸钠(pH＝4.0)缓冲液。
2.实验材料
动物信息：
Figure PCTCN2020139690-appb-000093
实验试剂：
名称 来源 批号
DMAC SIGMA BCBT8964
Solutol HS 15 BASF SE 69889088QO
PBS GIBCO 1948177
冰醋酸 HOheywell SZBF2670V
三水乙酸钠 SIGMA BC13W4910
甲基纤维素 SIGMA SLCB1319
去离子水 康龙化成试验室 08142019
3.实验方法
1)制剂配制
静脉注射制剂配制：精密称取7.13mg化合物22乙酸盐至容器中，加入1.5mL的DMAC，涡旋混匀使其完全溶解；加入2.25mL的Solutol HS 15并涡旋，再加入11.25mL的PBS定容至终体积(15mL)，涡旋1分钟，得到终浓度为0.4mg/mL的澄清溶液(pH＝3.96)。
口服组制剂配制(第2-5组)：第二组精密称取69.91mg化合物22乙酸盐至容器中，加入20mL含0.5％MC的乙酸-乙酸钠(pH＝4.0)缓冲液，搅拌66分钟，超声30分钟定容至终体积(28mL)，得到终浓度为2.1mg/mL的澄清溶液(PH＝3.99)。第三组精密称取209.7mg化合物22乙酸盐至容器中，加入20mL含0.5％MC的乙酸-乙酸钠(pH＝4.0)缓冲液，搅拌66分钟，超声30分钟定容至终体积(28mL)，得到终浓度为6.3mg/mL的澄清溶液(pH＝4.15)。第四组精密称取629.2mg化合物22乙酸盐至容器中，加入20mL含0.5％MC的乙酸-乙酸钠(pH＝4.0)缓冲液，搅拌66分钟，超声30分钟定容至终体积(28mL)，得到终浓度为18.9mg/mL的澄清溶液(pH＝4.35)。第五组精密称取1348.3mg化合物22乙酸盐至容器中，加入150mL含0.5％MC的乙酸-乙酸钠(pH＝4.0)缓冲液，搅拌66分钟，超声30分钟定容至终体积(180mL)，得到终浓度为6.3mg/mL的澄清溶液(pH＝4.15)。给药制剂在每天给药前于黄光灯下新鲜配制。其中，第5组的制剂在第1天给药前配制，并分装每天的给药量，在5±3℃保存。给药当天制剂取出后，给药前在室温下至少放置30分钟。
2)给药
按照实验设计方案进行给药。
3)样本处理
采血后将含抗凝剂的采血管反复颠倒数次以充分混匀，离心前放在湿冰上。采血后60分钟内，在2至8℃下2000g，离心10分钟，分离红细胞，得到血浆样品。血浆样品转移至冻存管，并保存在-75±15℃条件下直至分析。
室温下采集SD大鼠给药前和给药后24小时内的血液样本。收集到的所有样本离心前放在湿冰上并在-75±15℃条件下保存直到分析。
样品处理方法：
Figure PCTCN2020139690-appb-000094
NA：不适用
①.按照上表取所需体积转移到1.1mL塑料管中。
②.使用涡旋仪混匀样品，震荡10分钟，在4000rpm和4℃条件下离心10分钟。
③.离心后转移20μL上清到含有140μL稀释液的1.1mL塑料管中涡旋混匀，转移60μL混合样品至含有100μL稀释液的1.1mL塑料管中涡旋混匀。
④.160μL样品转移至96孔板内。
⑤.进5.0μL样品到LC-MS/MS。
4)分析方法与条件
Figure PCTCN2020139690-appb-000095
Figure PCTCN2020139690-appb-000096
质谱参数
Figure PCTCN2020139690-appb-000097
用液相质谱联用技术，以Verapamil作为内标化合物，对SD大鼠血浆中化合物22乙酸盐的浓度进行测定。质谱仪为Applied Biosystems/AB Sciex Triple Quad 4500，采用电喷雾离子源正离子模式扫描下，监测化合物22乙酸盐和Verapamil的母离子和子离子质荷比分别为393.2→146.0和455.2→165.0。
化合物22乙酸盐的药代动力学参数由软件Phoenix TM
Figure PCTCN2020139690-appb-000098
(8.1版)采用非房室模型计算。采用线性对数梯形法计算药代动力学数据，权重为1/(Y*Y)。样本浓度低于定量下限的样品不参与药代动力学参数的计算。使用Microsoft Excel 2010版计算各项参数的平均值，其平均值由每只动物的药代动力学参数确定。其中达峰时间(T max)的平均值由中位数表示。
4.实验结果与结论
表37 SD大鼠给予化合物22乙酸盐后体内的主要药代动力学参数(平均值±标准偏差)
Figure PCTCN2020139690-appb-000099
Figure PCTCN2020139690-appb-000100
NA：不适用。
a：T max均值为中位数。
*：个体数小于3不进行标准偏差计算。
结果如表36所示，静脉注射给予2.0mg/kg的化合物22乙酸盐后，化合物22乙酸盐在SD大鼠体内的药物暴露量AUC 0-t为5363hr*ng/mL，半衰期T 1/2为3.39hr，清除率(CL)为6.02mL/min/kg，远小于肝血流量(55.2mL/min/kg)，属于低清除药物。稳态表观分布容积(V ss)为0.457L/kg，接近于总体液量(0.668L/kg)，表明药物在SD大鼠体内分布较广。
SD大鼠口服给予3个剂量水平的化合物22乙酸盐(21mg/kg、63mg/kg和189mg/kg)后，SD大鼠的药物暴露量(AUC 0-t)分别是42765hr*ng/mL、111910hr*ng/mL和241332 hr*ng/mL，最大血药浓度(C max)分别为18367ng/mL、27367ng/mL和51850ng/mL。剂量由21mg/kg增加到189mg/kg时，AUC 0-t和C max的增加比例低于剂量比。
SD大鼠口服给予21、63、189mg/kg的化合物22乙酸盐后，化合物22乙酸盐的平均生物利用度分别为76.5％、65.5％和47.9％。
连续7天每天给予63mg/kg的化合物22乙酸盐后，第1天和第7天的AUC 0-t值分别为110818hr*ng/mL和127358hr*ng/mL，第7天与第1天暴露量比值小于2倍，提示在7天连续重复给予63mg/kg化合物22乙酸盐后，化合物22乙酸盐在SD大鼠体内无明显蓄积。
实验结果表明，化合物22乙酸盐在大鼠体内具有优良的药代动力学特性：低清除，分布广，体内暴露量和最大血药浓度随剂量增加而增加，具有较高的口服生物利用度，7天给药体内无明显蓄积。
15.4 化合物22化合物27和化合物40在SD大鼠连续7天口服的药代动力学
1.实验设计
12只雄性SD大鼠(给药当天约7～9周龄，283.0～298.1克)购于斯贝福(北京)生物技术有限公司，随机分为3组，每组4只，连续7天每天1次口服给药，其中第1组给予30mg/kg的化合物27，第2组给予30mg/kg的化合物22，第3组给予30mg/kg的化合物40。所有动物给药过程中自由饮水饮食。
Figure PCTCN2020139690-appb-000101
2.实验材料
1)动物信息
种属/品系/等级： Sprague-Dawley(SD)大鼠[Crl:CD(SD)](SPF/VAF)
来源： 斯贝福(北京)生物技术有限公司
试验动物生产许可证： SCXK(京)2016-0002
动物质量合格证号： 11401500037777
首次给药时年龄： 大约7～9周
首次给药时体重： 雄性283.0～298.1克
2)溶媒信息
名称 来源 批号
PEG400 Sigma BCBV8368
PBS Gibco 1951153
3)受试物信息
名称： 化合物27 化合物22 化合物40
纯度： 98.43％ 99.00％ 97.76％
失效日期： 未测 未测 未测
储存条件： 室温避光 室温避光 室温避光
性状： 白色粉末 白色粉末 白色粉末
校正因子： 1.02 1.01 1.02
校正因子＝FW/MW*1/纯度
3.实验方法
1)制剂配制步骤：
分别称取0.3366g的化合物27、0.3333g的化合物22和0.3366g的化合物40溶解于44mL的PEG400中搅拌超声溶解，加入50mL的PBS搅拌超声至完全溶解后，加PBS定容到终体积110mL，得到终浓度为3.0mg/mL的目标制剂溶液。将配制好的制剂溶液，分装为7份，每份15mL，于4℃冰箱储存，给药前将制剂放置于室温，至制剂温度恢复室温并在室温下搅拌至少10分钟，且在给药过程中持续搅拌。整个制剂配制过程在黄光下操作，配制好的制剂转移时用锡箔纸包裹避光。
2)药代动力学样品采集：
Figure PCTCN2020139690-appb-000102
3)样品分析
采用液相质谱联用技术，以Verapamil作为内标化合物，对SD大鼠血浆中化合物27，化合物22和化合物40的浓度进行测定。质谱仪采用电喷雾离子源正离子模式扫描下，监测化合物27，化合物40、化合物22和Tolbutamide的母离子和子离子质荷比分别为395.2→178.2，404.2→158.9，393.4→146.2和455.2→165.0。
化合物27，化合物40和化合物22的药代动力学参数由软件Phoenix TM WinNonlin 6.1版采用非房室模型计算。采用线性对数梯形法计算药代动力学数据，权重为1/Y*Y。样本浓度低于定量下限的样品不参与药代动力学参数的计算。使用Microsoft Excel 2010版计算各项参数的平均值，其平均值由每只动物的药代动力学参数确定。其中达峰时间(T max)的平均值由中位数表示。
4.分析方法
1)化合物27的液质联用生物分析方法
标准曲线和质控样品信息：
Figure PCTCN2020139690-appb-000103
液相条件：
Figure PCTCN2020139690-appb-000104
质谱条件：
Figure PCTCN2020139690-appb-000105
Figure PCTCN2020139690-appb-000106
2)化合物40的液质联用生物分析方法
标准曲线和质控样品信息：
Figure PCTCN2020139690-appb-000107
液相条件：
Figure PCTCN2020139690-appb-000108
Figure PCTCN2020139690-appb-000109
质谱条件
Figure PCTCN2020139690-appb-000110
3)化合物22的液质联用生物分析方法
标准曲线和质控样品信息：
Figure PCTCN2020139690-appb-000111
液相条件：
Figure PCTCN2020139690-appb-000112
Figure PCTCN2020139690-appb-000113
质谱条件：
Figure PCTCN2020139690-appb-000114
5.实验结果与结论
表38 连续7天口服化合物27，化合物22，化合物40的主要PK参数(平均值)
Figure PCTCN2020139690-appb-000115
Figure PCTCN2020139690-appb-000116
a：T max值为中位数值。
“-”：不适用。
SD雄性大鼠连续7天每天一次口服给予30mg/kg的化合物27，化合物22，化合物40后，检测其给药后第1天和第7天的血浆药物浓度。
SD雄性大鼠连续7天每天一次口服给予30mg/kg的化合物27，化合物22，化合物40后，第1天的药物暴露量AUC 0-t分别为29550hr*ng/mL，6630hr*ng/mL和5154hr*ng/mL；最大血药浓度C max分别为7103ng/mL，1022ng/mL和503ng/mL。连续给药第7天，化合物27，化合物22和化合物40在SD大鼠血浆中的药物暴露量AUC 0-t分别为22194hr*ng/mL，6953hr*ng/mL和6584hr*ng/mL；最大血药浓度C max分别为4615ng/mL，947ng/mL和581ng/mL。结果表明，连续7天每天一次口服给予30mg/kg的化合物27，化合物22，化合物40后，化合物27，化合物22，化合物40在SD大鼠体内没有明显的蓄积(表38)。
数据显示，受试化合物在SD大鼠体内具有优异的药代动力学参数，体内暴露量高，连续给药没有明显的蓄积。受试化合物优异的大鼠药代动力学特性可以支持进一步的临床人体实验。
实施例16 本申请化合物在雌性BALB/c小鼠体内的药代动力学评价
1.实验设计
组别 动物数 给药组 剂量(mg/kg) 给药方式 计划给药周期 实际给药周期
1 12 化合物22 100 p.o. QD x 1次 QD x 1次
2 12 化合物22 100 p.o. QD x 1次 QD x 1次
2.实验材料
1)试验动物
BALB/c小鼠，雌性，7-9周(实验开始时的小鼠周龄)，体重19.2-22.1g，51只(34只加10只富余小鼠)。购自上海灵畅生物科技有限公司(LC)，动物合格证编号：2013001835211。饲养环境：SPF级。
2)供试品
供试品：化合物22：批号：F，包装规格：38.0mg/vial+5045.1mg/vial，纯度98.96％，水分5.38％，由浙江春禾医药科技有限公司提供，粉末，RT密封保存。
3.实验方法
1)随机分组
在给药开始前，称量所有动物的体重，根据鼠的体重使用StudyDirector TM(版本号3.1.399.19，供应商Studylog System,Inc.,S.San Francisco,CA,USA)进行随机分组。
2)供试品和溶媒的配制
Figure PCTCN2020139690-appb-000117
4)实验观察和数据收集
本实验过程中动物实验的实验方案均通过CrownBio IACUC委员会审核并批准通过。实验过程中，动物实验操作均根据AAALAC的要求。分组后，常规监测包括了治疗对动物正常行为的影响，具体内容有实验动物的活动性，摄食和饮水情况，眼睛、被毛及其它异常情况。试验过程中观察到的临床症状均记录在原始数据中。实验中使用StudyDirector TM(版本号3.1.399.19，供应商Studylog System,Inc.)软件收集动物体重数据，原始数据由天平测量后直接导入软件，数据的任何变动都将被记录在此软件中。给药及体重称量等全部过程都在生物安全柜或超净工作台中进行。
5)实验终点
单次给药后，每组分别于0.25h,0.5h,1h,2h,4h,6h,8h和24h采集血浆，每个时间点3只小鼠，24h后终止实验。10只分剩小鼠用于采集空白血浆。
5.实验结果与讨论
表39 不同溶媒中的化合物22的药代动力学数据
Figure PCTCN2020139690-appb-000118
G1和G2小鼠在单次给药后，每组分别于0.25h,0.5h,1h,2h,4h,6h,8h和24h采集血浆检测化合物22在小鼠体内的PK反应。结果显示，不同时间点G2组小鼠血浆中化合物22浓度均明显高于G1组小鼠血浆中化合物22浓度。40％(2-羟丙基)-β-环糊精作为化合物22的溶媒效果好。同时100mg/kg的剂量条件下，小鼠体内有较高的药物暴露量，药代动力学表现优异。

Claims (43)

  1. 式1的化合物在制备治疗疾病的药物中的用途，
    Figure PCTCN2020139690-appb-100001
    其中：
    R为氢或甲基；
    Ar 1为任选地带取代基的呋喃基，任选地带取代基的苯基，或任选地带取代基的吡啶基；
    任意的任选地带取代基的芳环被卤素或氧代基取代；
    Ar 2为任选地带取代基的苯基，任选地带取代基的吡啶基，或任选地带取代基的嘧啶基；
    任意的任选地带取代基的芳环被卤素、羟基、氰基或甲氧基取代；
    X为氧或氮；且
    Y和Z各自独立地为氢、任选地带取代基的C 1-3烷基、任选地带取代基的C 1-5环烷基、任选地带取代基的杂环烷基、任选地带取代基的杂环烷基烷基、任选地带取代基的芳基、任选地带取代基的C 1-3烷基羰基、任选地带取代基的C 1-5环烷基羰基、任选地带取代基的杂环烷基羰基、任选地带取代基的杂环烷基烷基羰基、任选地带取代基的芳基羰基，或任选地带取代基的杂芳基羰基；任意的所述任选地带取代基的基团被卤素、羟基、甲氧基、乙氧基、三氟甲氧基、三氟乙氧基、烷基氨基、环烷基氨基、杂环基、芳基、杂芳基，或C 1-3烷基聚氧乙烯基取代；或者，Y和Z连接以形成任选地带取代基的具有5至7个环原子的杂环烷基；任意的所述任选地带取代基的环被卤素、氧代基、甲氧基、乙氧基、三氟甲氧基，或三氟乙氧基取代；或者Y或Z不存在，
    或其药学上可接受的溶剂化物或盐。
  2. 根据权利要求1所述的用途，其中，R为氢基；Ar 1为2-呋喃基；Ar 2为任选地带取代基的苯基；任意的所述任选地带取代基的苯基被卤素取代；X是氧或氮；且Y和Z各自独立地为氢、任选地带取代基的C 2-3烷基、任选地带取代基的杂环烷基，或任选地带取代基的杂环烷基烷基；任意的所述任选地取代的基团被甲氧基、乙氧基、三氟甲氧基、三氟乙氧基，或C 1-2烷基聚氧乙烯基取代；或Y和Z连接以形成吗啉基环；或Y或Z不存在，或其药学上可接受的溶剂化物或盐。
  3. 根据权利要求1-2中任一项所述的用途，其中R为氢；Ar 1为2-呋喃基；Ar 2为苯基；X为氮；且Y和Z各自独立地为氢、任选地带取代基的乙基或任选地带取代基的氧杂环丁烷基；任意的所述任选地带取代基的基团被甲氧基、乙氧基、三氟甲氧基或三氟乙氧基取代；或Y和Z连接以形成吗啉基环，或其药学上可接受的溶剂化物或盐。
  4. 根据权利要求1-3中任一项所述的用途，其中所述化合物选自下组：
    N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)-3-吗啉丙烷酰胺；
    N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)-2-吗啉丙烷酰胺；
    N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)新戊酰胺；
    N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)-3-羟基-2,2-二甲基丙酰胺；
    N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)-2-羟基-2-甲基丙酰胺；
    N-(4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)苯基)-4-甲基四氢-2H-吡喃-4-羧酰胺；
    2-(呋喃-2-基)-N5-(2-(嘧啶-4-基)乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(2-(吡啶-3-基)乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-(2-(吡咯烷-1-基)乙氧基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]-三嗪-5,7-二胺；
    N5-(4-(2-(氮杂环丁烷-1-基)乙氧基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-(2-(4-甲基哌嗪-1-基)乙氧基)苯乙基)-[1,2,4]三唑并[1,5-α][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-(3-(4-甲基哌嗪-1-基)丙氧基)苯乙基)-[1,2,4]三唑并[1,5-α][1,3,5]三嗪-5,7-二胺；
    N5-(4-(2-(3,3-二氟吡咯烷-1-基)乙氧基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    (R)-N5-(4-(2-(3-氟吡咯烷-1-基)乙氧基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    (S)-N5-(4-(2-(3-氟吡咯烷-1-基)乙氧基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-((2-吗啉代乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-((四氢-2H-吡喃-4-基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-((四氢呋喃-3-基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-(4-甲基哌嗪-1-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5，7-二胺；
    N5-(4-(双(2-甲氧基乙基)氨基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-(氧杂环丁烷-3-基氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-(氧杂环丁烷-3-基甲基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-((1-甲氧基丙烷-2-基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-((2-甲氧基乙基)(甲基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    N5-(4-(乙基(2-甲氧基乙基)氨基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-((2-甲氧基乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-((2-(2-甲氧基乙氧基)乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-((2-(2-(2-甲氧基乙氧基)乙氧基)乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    N5-(4-((2-乙氧基乙基)氨基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-((2-(2,2,2-三氟乙氧基)乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-((3-甲氧基丙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-((4-(2-((7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基)氨基)乙基)-苯基)氨基)乙腈；
    N5-(4-(1,3-二甲氧基丙烷-2-基氨基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(3-氟苯基)-N5-(4-((2-甲氧基乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；和
    2-(3-氟苯基)-N5-(4-(氧杂环丁烷-3-基氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    或其药学上可接受的溶剂化物或盐。
  5. 式2的化合物在制备治疗疾病的药物中的用途，
    Figure PCTCN2020139690-appb-100002
    其中，
    R为氢或甲基；
    Ar 1为任选地带取代基的呋喃基，任选地带取代基的苯基，或任选地带取代基的吡啶基；
    任意的任选地带取代基的芳环被卤素或氧代基取代；
    Ar 2为任选地带取代基的苯基，任选地带取代基的吡啶基，或任选地带取代基的嘧啶基；
    任意的任选地带取代基的芳环被卤素、羟基、氰基或甲氧基取代；且
    Q为任选地被X取代的5-6元芳族环、任选地在氮上被Y和Z取代的氨基羰基基团、任选地在氮上被Y和Z取代的氨基磺酰基基团、硝基基团、或氰基基团；X为卤素或任选地被取代的C 1-3烷基；任意的所述任选地被取代的烷基基团被卤素、氰基、甲氧基、乙氧基、三氟甲氧基、三氟乙氧基、芳基或杂芳基取代；Y和Z各自独立地为氢或任选地被取代的C 1- 3烷基；任意的所述任选地被取代的烷基基团被卤素、羟基、甲基、烷基氨基或环烷基氨基取代；或者Y和Z连接以形成具有5至7个环原子的任选地被取代的环；任意的所述任选地被取代的环被卤素、甲基、乙基、三氟甲基或三氟乙基取代，
    或其药学上可接受的溶剂化物或盐。
  6. 根据权利要求5所述的用途，其中：
    R为氢；
    Ar 1为2-呋喃基；
    Ar 2为苯基或吡啶基；且
    Q为硝基、氰基，或任选地被X取代的5-6元芳族环；X为任选地被取代的C1-3烷基；任意的所述任选地被取代的烷基被卤素、氰基、甲氧基、芳基或杂芳基取代，
    或其药学上可接受的溶剂化物或盐。
  7. 根据权利要求5-6中任一项所述的用途，其中：
    R为氢；
    Ar 1为2-呋喃基；
    Ar 2为苯基；且
    Q为任选地被X取代的四唑环；X为任选地被取代的C 1-3烷基；任意的所述任选地被取代的烷基中被卤素、氰基或甲氧基取代，
    或其药学上可接受的溶剂化物或盐。
  8. 根据权利要求5-7中任一项所述的用途，其中所述化合物选自下组：
    4-(2-(7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基氨基)乙基)-N,N-二甲基苯甲酰胺；
    N5-(4-(1-甲基-1H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    N5-(4-(2-甲基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    N5-(4-(2-乙基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    N5-(4-(2-异丙基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    N5-(4-(2-丙基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(5-(4-(2-(7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基氨基)乙基)-苯基)-2H-四唑-2-基)乙腈；
    4-(2-(7-氨基-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-基氨基)乙基)-苄腈；
    N5-(4-(2-(2,2,2-三氟乙基)-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    N5-(4-(2-(2-甲氧基乙基)-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-(吡啶-2-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-(吡啶-3-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-(吡啶-4-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-(嘧啶-2-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-(1-甲基-1H-1,2,4-三唑-3-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    N5-(4-(2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N-(4-(甲基磺酰基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5-胺；
    2-(3-氟苯基)-N5-(4-(2-甲基-2H-四唑-5-基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    N5-(4-(2-甲基-2H-四唑-5-基)苯乙基)-2-苯基-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；和
    2-(呋喃-2-基)-N5-(2-(6-(2-甲基-2H-四唑-5-基)吡啶-3-基)乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺，
    或其药学上可接受的溶剂化物或盐。
  9. 根据权利要求1-8中任一项所述的用途，其中所述化合物选自下组：
    2-(呋喃-2-基)-N5-(4-(氧杂环丁烷-3-基氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    2-(呋喃-2-基)-N5-(4-((2-甲氧基乙基)氨基)苯乙基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    N5-(4-(2-甲基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；和
    N5-(4-(2-乙基-2H-四唑-5-基)苯乙基)-2-(呋喃-2-基)-[1,2,4]三唑并[1,5-a][1,3,5]三嗪-5,7-二胺；
    及其药学上可接受的溶剂化物或盐。
  10. 根据权利要求1-9中任一项所述的用途，其中所述化合物的药学上可接受的溶剂化物或盐包括乙酸盐和/或苯磺酸盐。
  11. 根据权利要求1-10中任一项所述的用途，其中所述疾病包括帕金森氏症。
  12. 根据权利要求1-11中任一项所述的用途，其中所述疾病包括肿瘤。
  13. 根据权利要求12所述的用途，其中所述肿瘤包括实体瘤和非实体瘤。
  14. 根据权利要求13所述的用途，其中所述实体瘤包括结肠癌和黑色素瘤。
  15. 根据权利要求13所述的用途，其中所述非实体瘤包括淋巴瘤。
  16. 根据权利要求1-15中任一项所述的用途，其中所述药物被制备为适于口服给药和/或注射给药。
  17. 根据权利要求1-16中任一项所述的用途，其中所述药物还包含要学上可接受的载剂。
  18. 根据权利要求1-17中任一项所述的用途，其中所述药学上可接受的载剂包括环糊精。
  19. 治疗疾病的方法，其包括以下的步骤：向有需要的受试者施用权利要求1-18中任一项所述的化合物。
  20. 根据权利要求19所述的方法，其中所述疾病包括帕金森氏症。
  21. 根据权利要求20所述的方法，其中受试者表现出帕金森氏症的早期征兆。
  22. 根据权利要求20-21中任一项所述的方法，其中所述受试者之前接受或未接受治疗帕金森氏症的药物。
  23. 根据权利要求20-22中任一项所述的方法，其中所述受试者在施用所述化合物之前、同时或之后，接受治疗帕金森氏症的药物。
  24. 根据权利要求23所述的方法，其中所述受试者产生对治疗帕金森氏症的药物的耐药性。
  25. 根据权利要求20-24中任一项所述的方法，其中所述治疗帕金森氏症的药物包括多巴胺。
  26. 根据权利要求20-25中任一项所述的方法，其中所述治疗包括降低所述受试者的帕金森氏病症的发展速度，和/或延缓所述受试者的帕金森氏病症的临床发展。
  27. 根据权利要求20-26中任一项所述的方法，其中所述受试者的帕金森氏病症发展速度通过UPDRS评价。
  28. 根据权利要求19所述的方法，其中所述疾病包括肿瘤。
  29. 根据权利要求28所述的方法，其中所述肿瘤包括实体瘤和非实体瘤。
  30. 根据权利要求29所述的方法，其中所述实体瘤包括结肠癌和黑色素瘤。
  31. 根据权利要求30所述的方法，其中所述非实体瘤包括淋巴瘤。
  32. 根据权利要求19-31中任一项所述的方法，其包括口服给药和/或注射给药所述化合物。
  33. 根据权利要求19-32中任一项所述的方法，其中所述化合物的施用频率为每天一次或每天两次。
  34. 根据权利要求19-33中任一项所述的方法，其中所述化合物的给药剂量为约0.001mg/kg至约500mg/kg。
  35. 根据权利要求19-34中任一项所述的方法，其中所述化合物的给药剂量为约1mg/kg至约100mg/kg。
  36. 根据权利要求28-35中任一项所述的方法，其中所述受试者在施用所述化合物之前、同时或之后，接受治疗肿瘤的免疫疗法。
  37. 根据权利要求36所述的方法，其中所述治疗肿瘤的免疫疗法包括免疫检查点抑制剂。
  38. 根据权利要求37所述的方法，其中所述治疗肿瘤的免疫疗法包括PD-1抗体。
  39. 药物组合或试剂盒，其包括(1)权利要求1-10中任一项所述的化合物，和(2)治疗帕金森氏症的药物。
  40. 根据权利要求39所述的药物组合或试剂盒，其中所述治疗帕金森氏症的药物包括多巴胺。
  41. 药物组合或试剂盒，其包括(1)权利要求1-10中任一项所述的化合物，和(2)治疗肿瘤的免疫疗法。
  42. 根据权利要求41所述的药物组合或试剂盒，其中所述治疗肿瘤的免疫疗法包括免疫检查点抑制剂。
  43. 根据权利要求41所述的药物组合或试剂盒，其中所述治疗肿瘤的免疫疗法包括PD-1抗体。
PCT/CN2020/139690 2019-12-26 2020-12-25 三唑并三嗪衍生物在治疗疾病中的用途 WO2021129843A1 (zh)

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