WO2024164818A1 - 黄嘌呤类化合物及其用途 - Google Patents

黄嘌呤类化合物及其用途 Download PDF

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WO2024164818A1
WO2024164818A1 PCT/CN2024/072849 CN2024072849W WO2024164818A1 WO 2024164818 A1 WO2024164818 A1 WO 2024164818A1 CN 2024072849 W CN2024072849 W CN 2024072849W WO 2024164818 A1 WO2024164818 A1 WO 2024164818A1
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mmol
disease
int
alkyl
compound
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French (fr)
Inventor
王凯
曹征宇
沈建华
刘梦茹
宋兆祥
胡一信
许惕非
赵芳
梁化端
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China Pharmaceutical University
Shanghai Institute of Materia Medica of CAS
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China Pharmaceutical University
Shanghai Institute of Materia Medica of CAS
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Priority to JP2025546320A priority Critical patent/JP2026507486A/ja
Priority to AU2024219251A priority patent/AU2024219251A1/en
Priority to KR1020257029964A priority patent/KR20250141831A/ko
Priority to EP24752675.9A priority patent/EP4663640A1/en
Publication of WO2024164818A1 publication Critical patent/WO2024164818A1/zh
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
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    • A—HUMAN NECESSITIES
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    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
    • C07D473/06—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3
    • 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

Definitions

  • the present invention relates to the field of pharmaceutical chemistry, and in particular, to xanthine compounds, preparation methods and uses thereof.
  • the compounds are inhibitors or agonists of TRPC4 and TRPC5 ion channels, and have potential application value in preventing, delaying or treating diseases related to abnormal TRPC4/5 function or expression.
  • TRP Transient receptor potential
  • TRP ion channels are a class of six-transmembrane channel proteins widely distributed in the peripheral and central nervous systems. They are molecular receptors that sense changes in the cellular environment, transmit signals, and maintain cell homeostasis. Based on sequence similarity, mammalian TRP ion channels can be divided into six subfamilies: TRPA, TRPC, TRPM, TRPML, TRPP, and TRPV.
  • Classical transient receptor potential (TRPC) channels are non-selective cation channels that allow Ca 2+ and Na + to pass through. Mammalian TRPC consists of six members: TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7.
  • TRPC3, TRPC6, and TRPC7 are highly consistent, and the sequences of TRPC1, TRPC4, and TRPC5 are highly consistent.
  • TRPC family proteins exist in the form of homozygous or heterozygous tetramers and play the role of ion channels.
  • TRPC5 easily forms heterozygous tetramers with TRPC1 and TRPC4.
  • TRPC4/5 is expressed in multiple parts of the brain, including the hippocampus, amygdala, frontal cortex, etc., and is associated with the occurrence of neurological diseases such as anxiety, depression, epilepsy, and addiction.
  • TRPC4 or TRPC5 gene knockout, or the use of TPRC4/5 inhibitors can relieve anxiety and depression symptoms in mice.
  • a TRPC4/5 inhibitor BI-1358894 developed by Boehringer Ingelheim Pharmaceuticals is currently undergoing clinical trials for the treatment of mental illnesses such as depression, borderline personality disorder, and post-traumatic stress disorder.
  • Podocytes are special cells that form the glomerular filtration barrier. TRPC5 is also expressed in glomerular podocytes, and its dysfunction is associated with the formation of chronic kidney disease. TRPC5 is involved in the regulation of angiotensin-stimulated cell migration and actin remodeling. In a variety of animal models of kidney disease, TRPC5 gene knockout or TPRC5 inhibitors can significantly reduce proteinuria levels, protect podocytes from damage, and inhibit the progression of kidney disease. GFB-887, a TRPC4/5 inhibitor developed by Goldfinch Biotechnology, is currently undergoing clinical trials for the treatment of kidney diseases such as diabetic nephropathy and focal segmental glomerulosclerosis.
  • TRPC4/5 is expressed in skin keratinocytes and plays an important role in regulating keratinocyte differentiation. Therefore, targeted regulation of TRPC4/5 may be used to treat skin lesions with abnormal keratinocyte function as the pathological basis.
  • TRPC5 is expressed in the liver and portal vein.
  • a series of endogenous phospholipids that play an important role in the formation of liver disease, such as lysolecithin (LPC) can significantly activate TRPC5.
  • LPC lysolecithin
  • TRPC5 gene knockout can alleviate liver damage and abnormal liver lipid metabolism. It can be seen that TRPC5 is also a potential target for the treatment of liver diseases.
  • TRPC4/5 is also expressed in peripheral sensory neurons and is involved in pain perception. Compared with wild-type animals, TRPC4 -/- mice are resistant to mustard oil-induced pain and have an increased pain threshold. In various mouse models of idiopathic pain, spontaneous pain, etc., TRPC5 gene knockout or TRPC5 inhibitors can effectively alleviate mechanical allodynia, and this effect is intrinsically related to the biological function regulated by LPC.
  • TRPC4/5 is also associated with cardiovascular disease.
  • Activation of TRPC5 channels can significantly promote the proliferation and migration of vascular smooth muscle cells.
  • Inhibition of TRPC5 can significantly reduce hypoxia-ischemia-induced apoptosis and oxidative stress of endothelial cells, reduce endothelium-dependent vasoconstriction, and inhibit the formation of atherosclerotic plaques. It can also reduce the expression of myocardial hypertrophy-related genes and maintain the stability of cardiac electrical activity, thereby playing an anti-atherosclerotic, anti-myocardial hypertrophy and anti-arrhythmic role.
  • TRPC5-mediated calcium influx is a key signaling mechanism for the release of a variety of endothelial-derived contractile factors, which contributes to the development of hypertension.
  • TRPC5 promotes the proliferation of vascular smooth muscle cells and increases oxidative stress, thereby promoting the occurrence of hypertension.
  • TRPC4/5 is associated with tumor angiogenesis, tumor resistance and metastasis.
  • TRPC5 is highly expressed in colorectal cancer, promoting extracellular calcium influx to activate the Wnt5a/ ⁇ -catenin pathway, reduce tumor differentiation and increase tumor stem cells.
  • TRPC5 expression levels are negatively correlated with the prognosis of colorectal cancer patients.
  • TRPC5 can also reduce E-cadherin, increase mesenchymal marker expression, and promote tumor cell migration, invasion and proliferation.
  • TRPC5 calcium ion dysregulation caused by activation of TRPC5 can promote anaerobic inducible factor 1 (HIF-1)-mediated tumor angiogenesis, allowing cancer cells to escape from sites exposed to high doses of anticancer drugs and obtain additional nutrients to help tumors survive, thereby reducing the effect of chemotherapy.
  • HIV-1 inducible factor 1
  • inhibition of TRPC5 can reduce chemotherapy resistance.
  • activation of TRPC4/5 can also induce tumor cell death through calcium ion overload.
  • the natural product (-)-Englerin A is a potent TRPC4/5 agonist that exhibits antiproliferative effects on a variety of tumor cells, including renal cancer, lung cancer, breast cancer, and skin cancer.
  • TRPC4/5 is expressed in the smooth muscle and myenteric plexus of the small intestine, and is involved in regulating intestinal smooth muscle cell contraction, gastrointestinal motility, and enteric nerve signaling. Therefore, compounds that target the activity of TRPC4/5 channels also have potential application value in the treatment of intestinal diseases.
  • WO2014143799 discloses xanthine compounds as shown in the following general formula, wherein the 8-substituent R 2 is defined as C1-C6 alkyl, C1-C6 heteroalkyl, C1-C6 alkoxy, C3-C7 cycloalkyloxy, C6-C10 aryl, C6-C10 aryloxy, C7-C16 aryl alkoxy, heterocycloalkyl, heteroaryl, heteroaryloxy, etc.
  • Compound 260 in this application as a key research compound, has good TRPC5 inhibitory activity and also shows good efficacy in depression and anxiety mouse models.
  • compound 260 is also called HC-070, which shows in detail the activity of HC-070 in inhibiting TRPC4/5 channels and the anti-anxiety and anti-depressant effects in various animal models.
  • HC-070 lacks good selectivity for some other ion channels and can significantly inhibit hERG potassium channels and TRPC3.
  • WO2019011802 discloses compounds in which the 8-position of xanthine is substituted by "3-pyridyl” or “2-piperazinyl” as shown below. Compared with HC-070, they maintain higher TRPC5 inhibitory activity while also reducing the inhibitory effect on hERG channels.
  • the main purpose of the present invention is to provide a class of xanthine compounds used as TRPC4 and TRPC5 regulators, their preparation method and therapeutic use.
  • the present invention provides a fused pyrimidinedione compound represented by general formula (I), a stereoisomer thereof and a pharmaceutically acceptable salt thereof;
  • Ring A is a saturated or partially unsaturated C3-C10 cyclic hydrocarbon group, including monocyclic, fused, spirocyclic and bridged ring systems;
  • X is N or CH;
  • R1 is a C1-C6 alkyl group or a C1-C6 haloalkyl group;
  • n is an integer from 1 to 4, and each R2 is independently H, deuterium, a halogen atom, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, a C1-C6 haloalkoxy group, or a cyano group;
  • m is an integer from 1 to 6, and each R3 is independently selected from H, deuterium, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, a C1-C6 haloalk
  • R 4 and R 5 are each independently H, C1-C6 alkyl, or C3-C8 cycloalkyl.
  • the present invention also provides a xanthine compound represented by general formula (Ia), a stereoisomer thereof and a pharmaceutically acceptable salt thereof;
  • ring A is a saturated or partially unsaturated C3-C10 cyclic hydrocarbon group, preferably including a monocyclic, fused, spirocyclic and bridged ring systems;
  • R1 is C1-C6 alkyl or C1-C6 haloalkyl
  • n is an integer from 1 to 4, and each R 2 is independently H, deuterium, halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, or cyano;
  • each R 3 is independently selected from H, deuterium, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C8 cycloalkyl, C6-C10 aryl, 5-8 membered heteroaryl, 4-8 membered heterocyclyl, halogen, cyano, hydroxyl, -NR 4 R 5 , or R 3 and the carbon attached to the A ring together form a 4-8 membered heterocycle or C6-C10 aryl; the cycloalkyl, aryl, heteroaryl, heterocyclyl is unsubstituted or optionally substituted with 1-4 groups selected from the following group: C 1 -C 6 alkyl, C1-C 6 haloalkyl, C 1 -C 6 alkoxy, C1-C 6 haloalkoxy, halogen, cyano;
  • R 4 and R 5 are each independently H, C1-C6 alkyl, or C3-C8 cycloalkyl.
  • ring A is a saturated or partially unsaturated C3-C8 monocyclic or bridged hydrocarbon group.
  • ring A is selected from the following group:
  • ring A is cyclohexyl or cyclohexenyl (such as ).
  • the compound has a structure as shown in general formula (Ib);
  • R 1 is C1-C6 alkyl
  • n is an integer from 1 to 4, and each R 2 is independently H, CN, C1-C6 haloalkyl or a halogen atom;
  • Each R 3 is independently selected from H, deuterium, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C6-C10 aryl, C3-C8 cycloalkyl, 5-8 membered heteroaryl, 4-8 membered heterocyclic group, halogen atom, cyano, hydroxyl, -NR 4 R 5 ; or two R 3 and the carbon atoms connected to the A ring together form a 4-8 membered heterocyclic ring;
  • R 4 and R 5 are each independently H or C1-C6 alkyl.
  • R 1 is a C1-C4 alkyl group. In another preferred embodiment, R 1 is a methyl group, an ethyl group or an isopropyl group.
  • n is 1, 2 or 3, and each R 2 is independently H, deuterium, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, cyano.
  • n is 1, 2 or 3, and each R 2 is independently H, deuterium, F, Cl, Br, methyl, ethyl or isopropyl.
  • n is 1, and R 2 is F, Cl or Br, located at the para position.
  • n is 2, and each R 2 is independently F, Cl or Br, located at the para position and the meta position.
  • R 2 is -F or -Cl.
  • m is 1, 2, 3 or 4, and each R 3 is independently selected from H, deuterium, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C3-C6 cycloalkyl, phenyl, 5-6 membered heteroaryl, 5-6 membered heterocyclyl, F, Cl, Br, cyano, hydroxyl, -NR 4 R 5 , or R 3 and the carbon attached to the A ring together form a 5-6 membered heterocycle or phenyl; the cycloalkyl, phenyl, heteroaryl, heterocyclyl is unsubstituted or optionally substituted with 1-4 groups selected from the following group: C 1 -C 6 alkyl, C1-C 6 haloalkyl, C 1 -C 6 alkoxy, C1-C 6 haloalkoxy, halogen, cyano.
  • each R 3 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, trifluoromethyl, difluoromethyl, monofluoromethyl, phenyl, cyclobutyl, cyclopropyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, n-propoxy, isopropoxy, hydroxyl, F, Cl, Br, cyano, amino, methylamino, dimethylamino, oxetanyl, dioxolanyl, morpholinyl, piperazinyl, pyrrolidinyl, piperidinyl.
  • each R 3 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, phenyl, 4-8 membered heterocyclic group, halogen atom, cyano, hydroxyl, -NR 4 R 5 .
  • R 4 and R 5 are each independently H or a C1-C4 alkyl group.
  • each R 3 is independently selected from H, C1-C3 haloalkyl or halogen atom. In another preferred embodiment, R 3 is -F.
  • R3 and the carbon atoms connected to the A ring together form
  • the compound represented by general formula (I), (I-a) or (I-b) is selected from Example compounds E1 to E73-2.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one effective therapeutic dose of a compound represented by general formula (I) or (I-a) or (I-b), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • the pharmaceutical excipient is a pharmaceutically acceptable carrier, excipient, sustained-release agent, odorant, flavoring agent, etc.
  • the compound of the present invention is used as the active ingredient, and its weight accounts for 0.1 to 99.9% of the total weight of the pharmaceutical composition, and the rest are pharmaceutically acceptable excipients; the preferred ratio of the compound of the present invention to the excipients is: the compound of the present invention as the active ingredient accounts for more than 60% of the total weight, and the rest accounts for 0-40% of the total weight, and the amount of the rest is preferably 1-20%, and most preferably 1-10%.
  • the compound or pharmaceutical composition of the present invention can be prepared into various dosage forms based on the conventional processes in the field of pharmaceutical preparations, such as tablets, capsules, powders, syrups, solutions, suspensions, sprays, creams, ointments, gels, transdermal patches, etc., and can be present in suitable solid or liquid carriers or diluents.
  • the pharmaceutical composition of the present invention can also be stored in suitable sterile equipment for injection or infusion.
  • the compounds or pharmaceutical compositions of the present invention can be used in mammals, including humans and animals.
  • the administration routes include oral administration, nasal inhalation, topical administration to the skin, intravenous injection, intramuscular injection, subcutaneous injection, etc.
  • the preferred administration route of the compounds or pharmaceutical compositions of the present invention is oral administration.
  • Solid dosage forms of the compounds or pharmaceutical compositions of the present invention for oral administration include capsules, tablets, pills, powders and granules.
  • Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, etc.
  • liquid carriers include sterile water, polyethylene glycol, nonionic surfactants and edible oils (such as corn oil, peanut oil). and sesame oil), etc., as long as it is suitable for the characteristics of the active ingredient and the required specific mode of administration.
  • Adjuvants commonly used in the preparation of pharmaceutical compositions may also be advantageously included, such as flavoring agents, coloring agents, preservatives and antioxidants such as vitamin E, vitamin C, BHT and BHA.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.
  • the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices.
  • suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.
  • suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.
  • the compounds shown in the present invention are preferably in unit doses, each dose containing 0.01 mg-200 mg of the active ingredient, preferably 0.5 mg-50 mg, for single or divided use.
  • the optimal dose for an individual should be determined according to the specific treatment. Usually, it is started with a small dose and gradually increased until the most suitable dose is found.
  • the present invention provides the use of a compound represented by general formula (I) or (I-a) or (I-b), a stereoisomer thereof, a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof, wherein the compound is used as a regulator of TRPC4 and TRPC5 ion channels, and thus a drug can be prepared for preventing, delaying or treating diseases related to abnormal function or expression of TRPC4 and/or TRPC5.
  • the regulators of the TRPC4 and TRPC5 ion channels include inhibitors and agonists.
  • the regulators of TRPC4 and TRPC5 ion channels are TRPC4 inhibitors and TRPC5 inhibitors.
  • the diseases related to the abnormal function and expression of TRPC4 and/or TRPC5 include but are not limited to mental illness, neurodegenerative disease, kidney disease, pain, epilepsy, liver disease, cardiovascular disease, cancer, skin disease, intestinal disease, etc.
  • the disease related to the abnormal function and expression of TRPC4 or/and TRPC5 is a mental illness.
  • the mental illness includes diseases related to depression, such as borderline personality disorder, depression, bad mood, postpartum depression, bipolar disorder, etc.; the mental illness also includes symptoms related to anxiety and fear, such as post-traumatic stress disorder, panic disorder, agoraphobia, social phobia, generalized anxiety disorder, social anxiety disorder, separation anxiety, etc.; the mental illness also includes affective disorders and mental disorders caused by various other reasons, such as schizophrenia, mania, obsessive-compulsive disorder, apathy, neurasthenia, paranoia, etc.
  • the mental illness is borderline personality disorder, depression, anxiety, or post-traumatic stress disorder.
  • the compounds or pharmaceutical compositions of the present invention can be used alone or in combination with drugs with other mechanisms for treating mental illness.
  • the drugs with other mechanisms include but are not limited to tricyclic antidepressants (TCAs), monoamine oxidase inhibitors (MAOIs), serotonin (5-HT) reuptake inhibitors (SSRIs), serotonin and nortriptyline Adrenaline reuptake inhibitors (SNRI), norepinephrine specific 5-HT antidepressants (NaSSA), 5-HT receptor antagonists and reuptake inhibitors (SARI), etc.
  • TCAs tricyclic antidepressants
  • MAOIs monoamine oxidase inhibitors
  • SSRIs serotonin (5-HT) reuptake inhibitors
  • SNRI serotonin and nortriptyline Adrenaline reuptake inhibitors
  • NaSSA norepinephrine specific 5-HT antidepressants
  • SARI 5-HT receptor antagonists and reuptake inhibitors
  • the disease associated with the abnormal function and expression of TRPC4 and/or TRPC5 is a neurodegenerative disease.
  • the neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), memory impairment, amnesia, aphasia, chronic fatigue syndrome, Creutzfeldt-Jakob disease, dissociative amnesia, fugue amnesia, learning disabilities, sleep disorders, and other brain diseases caused by trauma or aging.
  • the disease related to the abnormal function and expression of TRPC4 or/and TRPC5 is epilepsy.
  • Epilepsy can be caused by excitotoxicity of various origins. Excessive neuronal discharges can usually drive epileptic activity. Compounds that reduce the hyperexcitability of related neuronal populations have significant potential in reducing epileptic activity. Inhibiting TRPC5-mediated calcium influx may reduce hyperexcitability and thus reduce epileptic activity.
  • the disease related to the abnormal function and expression of TRPC4 or/and TRPC5 is a kidney disease.
  • the kidney disease includes polar kidney damage and chronic kidney damage caused by various reasons, such as kidney damage caused by toxic substances, kidney damage caused by viral or bacterial infection, hypertensive nephropathy, diabetic nephropathy, glomerulonephritis, lupus nephritis, IgA nephropathy, nephrotic syndrome, membranous nephropathy, minimal change nephropathy, and also includes hereditary kidney diseases, such as polycystic kidney disease, focal segmental glomerulosclerosis, etc.
  • the disease related to the abnormal function and expression of TRPC4 and/or TRPC5 is a skin disease.
  • the skin disease is related to the abnormal function of keratinocytes, such as but not limited to psoriasis, ichthyosis, palmoplantar keratoderma, Olmsted's disease, toad skin disease or menopausal keratoderma.
  • the disease related to the abnormal function and expression of TRPC4 and/or TRPC5 is a disease related to the cardiovascular system, including but not limited to hypertension, atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, stroke, pulmonary hypertension, etc.
  • the disease related to the abnormal function and expression of TRPC4 and/or TRPC5 is liver disease, including but not limited to cholestatic liver disease, alcoholic steatohepatitis, non-alcoholic steatohepatitis, viral hepatitis, autoimmune liver disease, chemical liver injury, liver fibrosis, cirrhosis, and hepatocellular carcinoma.
  • the disease associated with the abnormal function and expression of TRPC4 and/or TRPC5 is cancer, including but not limited to renal cell carcinoma, breast cancer, colorectal cancer, etc.
  • the disease related to the abnormal function and expression of TRPC4 and/or TRPC5 is an intestinal disease, including but not limited to ulcerative colitis, short bowel syndrome, irritable bowel syndrome, intestinal cramps, diarrhea, abdominal pain, etc.
  • Lysolecithin can directly activate TRPC5, independent of other indirect activation mechanisms, such as the GPCR-PLC pathway.
  • a series of biological effects of LPC are related to its activation of TRPC5. Therefore, TRPC5 inhibition
  • the agent may be used to combat diseases associated with LPC level disorders, including but not limited to atherosclerosis, cardiovascular disease, central and peripheral nervous system diseases, osteoarthritis, hepatitis, hepatocellular carcinoma, pneumonia, obesity, gastrointestinal diseases, bacterial infections, parasitic diseases, diabetes, tumors, pain, etc.
  • the present invention provides a method for preparing the compound of the present invention, its stereoisomers, and its pharmaceutically acceptable salts, wherein the method is selected from one of the synthetic routes shown in the following Route A and Route B.
  • the compound of the present invention has a structure shown by the general formula (II-a) to (II-d), it can be prepared according to the synthetic route shown in route A; wherein halo is a halogen atom, R 3a is a C1-C6 alkyl group, THP represents a tetrahydropyran protective group, and the other substituents are defined the same as above;
  • reaction steps of route A include:
  • the intermediate INT-A1 undergoes lithium halogen exchange and nucleophilic addition reaction with a ketone raw material (commercially purchased) to generate the intermediate INT-A2; the reaction is carried out in an ultra-dry aprotic solvent; the solvent is, for example, but not limited to, dichloromethane (DCM) and tetrahydrofuran (THF), with tetrahydrofuran being the preferred solvent.
  • the reaction temperature is generally -40 to -100°C, preferably -78°C;
  • the intermediates INT-A2, INT-A3, INT-A4, and INT-A5 are deprotected from THP protecting groups to generate compounds of the general formula (II-a), (II-b), (II-c), and (II-d); the deprotection reaction of the hydroxyl group is usually carried out under the action of an acidic reagent; the acidic reagent is for example but not limited to trifluoroacetic acid, hydrogen chloride, and p-toluenesulfonic acid.
  • the compound of the present invention has a structure represented by the sub-general formula (II-e), it can be prepared according to the following route B; wherein halo is a halogen atom, THP represents a tetrahydropyran protective group, and the definitions of other substituents are the same as above;
  • reaction steps of route B include:
  • Intermediate INT-A1 undergoes Suzuki coupling reaction with boron ester raw materials (commercially purchased or synthesized according to literature methods) to generate intermediate INT-B1;
  • the Suzuki coupling reaction is usually carried out under the action of a palladium catalyst and a base;
  • the palladium catalyst is, for example, but not limited to, Pd(Ph 3 P) 2 Cl 2 , Pd(dppf)Cl 2 , Pd(Ph 3 P) 4 ;
  • the base is, for example, but not limited to, triethylamine, DIPEA, K 2 CO 3 , Cs 2 CO 3 ;
  • the reaction is carried out in a suitable organic solvent or a mixed solvent of an organic solvent and water, such as DMF or 1,4-dioxane;
  • the reaction temperature is generally room temperature to 100°C;
  • the intermediate INT-B1 undergoes a hydrogenation reduction reaction to generate the intermediate INT-B2; the reduction reaction is carried out under the catalysis of palladium/carbon; the reaction temperature is generally room temperature; the reaction solvent includes a protic solvent and aprotic solvent, such as but not limited to methanol, ethanol, ethyl acetate, THF, etc.
  • the intermediate INT-B2 is deprotected from the THP protecting group to generate a compound of the general formula (II-e).
  • the deprotection reaction of the hydroxyl group is usually carried out under the action of an acidic reagent; the acidic reagent is for example but not limited to trifluoroacetic acid, hydrogen chloride, and p-toluenesulfonic acid.
  • Figure 1 shows the effect of compound E21 on TGF- ⁇ -induced hepatic stellate cell activation ( ⁇ -SMA labeling); (A) images under a microscope; (B) statistics of the proportion of ⁇ -SMA-positive cells; **, p ⁇ 0.01; Mean ⁇ SEM.
  • the inventors of the present application have developed a xanthine compound through extensive and in-depth research, in which the 8-position is substituted by a "cycloalkyl".
  • the compound of the present invention has stronger inhibitory activity on TRPC4 and TRPC5, and weaker inhibition on hERG and TRPC3, and therefore has better selectivity.
  • the compound of the present invention also shows better metabolic stability, and therefore has better drug properties.
  • some compounds in the present invention unexpectedly show significant TRPC5 agonist activity, rather than inhibitory activity. On this basis, the present invention has been completed.
  • C1-C6 refers to having 1, 2, 3, 4, 5 or 6 carbon atoms, and so on.
  • 4-8 members refers to having 4, 5, 6, 7 or 8 ring atoms, and so on.
  • alkyl is a branched or straight-chain hydrocarbon group having a specific number of carbon atoms, and representative examples include but are not limited to methyl, ethyl, n-propyl, and isopropyl.
  • alkoxy refers to -O-alkyl.
  • C1-C6 alkoxy refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, and representative examples include but are not limited to methoxy, ethoxy, n-propoxy, isopropoxy and butoxy.
  • haloalkyl and haloalkoxy refer to groups in which the hydrogen atoms in an “alkyl” or “alkoxy” group having a specific number of carbon atoms are partially or completely replaced by "halogen atoms”.
  • the "cycloalkyl” refers to a non-aromatic cyclic aliphatic hydrocarbon group with a specific number of ring carbon atoms
  • the "C3-C10 cycloalkyl” refers to a cyclic aliphatic hydrocarbon group consisting of 3 to 10 ring carbon atoms
  • the "cycloalkyl” described in the present invention includes not only a monocyclic aliphatic hydrocarbon group, but also a cyclic, spirocyclic and bridged ring composed of multiple cyclic aliphatic hydrocarbons.
  • cycloalkyl described in the present invention includes not only aliphatic hydrocarbon groups whose carbon atoms are fully saturated (i.e., cycloalkyl groups), but also aliphatic hydrocarbon groups whose carbon atoms have unsaturated bonds (such as cycloalkenyl groups), but does not include aromatic groups that are completely composed of unsaturated carbon atoms; examples of the "cycloalkyl” described in the present invention include but are not limited to:
  • aryl is defined as a monocyclic or bicyclic ring system consisting of a specific number of carbon atoms and obeying Hückel's rule, including phenyl and naphthyl.
  • heteroaryl is defined as a monocyclic and bicyclic ring system having a specific number of ring atoms and containing 1, 2, 3 or 4 heteroatoms (selected from N, O, S) and obeying the Hückel rule; examples of “heteroaryl” include but are not limited to pyridine, pyrrole, imidazole, thiophene, benzimidazole, benzothiophene, benzofuran, etc.
  • heterocyclic group is defined as a non-aromatic monocyclic and bicyclic ring system having a specific number of ring atoms and containing 1, 2, 3 or 4 heteroatoms (selected from N, O, S), saturated or partially unsaturated; examples of “heterocyclic group” include but are not limited to oxetanyl, dioxolanyl, morpholinyl, piperazinyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, benzotetrahydrofuranyl, etc.
  • the "halogen atom” includes fluorine, chlorine, bromine and iodine.
  • substituted means replacement by one or more groups (e.g., 2, 3, 4, or 5). If it is not specified on a specific atom, it means that it can occur on any atom where the number of substituents has not yet reached saturation. When multiple substituents are selected from the same series, they can be the same or different.
  • the term "optionally" used in the present invention means that the defined group may be selected from a series of candidate groups or may not be selected.
  • “Pharmaceutically acceptable salt” of the present invention can be a salt formed by an anion and a positively charged group on the compound of formula (I), (I-a) or (I-b).
  • Suitable anions are chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronide, lactate, glutarate or maleate.
  • salts can be formed by a cation and a negatively charged group on the compound of formula (I). Suitable cations include sodium, potassium, magnesium, calcium and ammonium ions, such as tetramethylammonium ions.
  • “pharmaceutically acceptable salt” refers to a salt formed by a compound of formula (I), (Ia) or (Ib) with an acid selected from the group consisting of hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid.
  • an acid selected from the group consisting of hydrofluoric acid, hydrochloric acid, hydrobro
  • the general formula (I), (I-a) compound or (I-b) compound of the present invention or its pharmaceutically acceptable salt is distilled, crystallized or recrystallized from water or an organic solvent, and the compound may contain the solvent molecules used. In addition, different crystallization conditions may lead to different crystal forms of the compound. Therefore, the general formula (I), (I-a) compound or (I-b) compound or its pharmaceutically acceptable salt containing different chemical doses of crystallization solvents and all crystal forms are within the scope of the present invention.
  • the "effective therapeutic dose” means that the subjects receiving the treatment with the dose are cured, improved, effectively prevented, or the incidence of the disease or side effects is significantly reduced compared with the subjects who have not received the treatment with the dose; in addition, it also includes an effective dose that enhances normal physiological functions.
  • the term “modulator” includes inhibitors and agonists.
  • the term “inhibitor” can also be expressed as “antagonist” or “blocker”, which have the same meaning in the present invention and refer to compounds or mixtures that can be used to reduce or inhibit biological activity.
  • the term “agonist” can also be expressed as “activator”, which has the same meaning in the present invention and refers to drug molecules or mixtures that can be used to increase biological activity.
  • TRPC family proteins exist not only in the form of homozygous tetramers, but also often in the form of heterozygous tetramers.
  • TRPC5 it not only constitutes TRPC5:C5 homozygous channels, but also constitutes TRPC4:C5, TRPC1:C5, TRPC1:C4:C5 and other heterozygous channels. Therefore, the TRPC5 inhibitor or TRPC5 agonist of the present invention not only means to inhibit or excite TRPC5 homozygous channels, but also means to inhibit or excite TRPC5 heterozygous channels.
  • TPRC4 also has this meaning.
  • Some compounds shown in the general formula (I), (I-a), and (I-b) of the present invention have chiral centers, potential chiral centers, or unsaturated bonds, and can form various forms of stereoisomers, such as racemates, enantiomers, diastereomers, E/Z isomers, cis-trans isomers, tautomers, etc.
  • a given chemical formula or name is intended to cover all forms of stereoisomers, and mixtures consisting of individual isomers in different proportions, and pharmaceutically acceptable salts thereof.
  • a person of ordinary skill in the art can use commonly used laboratory separation methods to separate the compounds containing asymmetric centers in the present invention to obtain single isomers, but this does not destroy the novelty of the compounds of the present invention.
  • DCM dichloromethane
  • DAST diethylaminosulfur trifluoride
  • DMAP 4-dimethylaminopyridine
  • DMF N,N-dimethylformamide
  • DMSO dimethyl sulfoxide
  • TFA trifluoroacetic acid
  • THF tetrahydrofuran.
  • 6-Chlorouracil (10 g, 68.493 mmol) was dissolved in 40 mL DMSO, K 2 CO 3 (4.8 g, 34.783 mmol) and CH 3 I (12.7 mL, 205.48 mmol) were added in sequence, and stirred at room temperature for 3 h. TLC showed that the reaction was complete, 50 mL H 2 O was added, and the mixture was filtered and washed with water to obtain 7.9 g of a white solid, namely, the intermediate INT 1-1.
  • the intermediate INT 1-1 (5 g, 31.25 mmol) was dissolved in 10 mL DMF, K 2 CO 3 (8.65 g, 62.5 mmol) and 3-bromopropyl methyl ether (5.35 mL, 46.875 mmol) were added, and heated at 60°C for 3 h.
  • TLC showed that the reaction was complete, and the organic phase was separated by EA/saturated NaCl aqueous solution partitioning, and the organic phase was separated, dried over anhydrous sodium sulfate, and separated by a flash silica gel column, eluted with DCM/MeOH as the mobile phase, and the target product was collected and evaporated to dryness to obtain 4 g of a jelly, namely the intermediate INT1-2.
  • the intermediate INT 1-2 (1 g, 4.31 mmol) was dissolved in 3 mL of concentrated sulfuric acid, stirred at 0°C for 5 min, and 3 mL of concentrated nitric acid was slowly added dropwise, and stirred at 0°C for 1 h. TLC showed that the reaction was complete, and the mixture was partitioned by EA/ice water. The organic layer was washed four times with saturated brine, dried over anhydrous sodium sulfate, and separated by a flash silica gel column, eluted with DCM/MeOH as the mobile phase, and the target product was collected and evaporated to dryness to obtain 800 mg of a colloid, namely the intermediate INT 1-3.
  • the organic phase was separated, dried over anhydrous sodium sulfate, and separated by a fast silica gel column, eluted with DCM/MeOH as the mobile phase, and the target product was collected and evaporated to dryness to obtain 3.2 g of a colloid, namely the intermediate INT 1-4.
  • the intermediate INT 1-4 (3 g, 7.463 mmol) was dissolved in 15 mL of acetic acid, heated to 60 ° C, and zinc powder (7.3 g, 111.94 mmol) was added.
  • the reaction was carried out in a pressure tube at 120 ° C for 12 h. TLC showed that the reaction was complete, and the crude product was evaporated to dryness under reduced pressure. Then, it was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated. 1.8 g of a gum was obtained, namely, the intermediate INT 1-5.
  • the intermediate INT 1-5 (100 mg, 7.463 mmol) was dissolved in 3 mL of dry 1,4-dioxane, and phosphorus oxybromide (340 mg, 22.389 mmol) was added under nitrogen protection, and stirred at 100°C for 1 h. TLC showed that the reaction was complete, and the organic phase was separated by EA/saturated NaHCO 3 aqueous solution, dried over anhydrous sodium sulfate, and separated by a flash silica gel column, eluted with DCM/MeOH as the mobile phase, and the target product was collected and evaporated to dryness to obtain 90 mg of a jelly, namely the intermediate INT 1-6.
  • the intermediate INT 1-6 (90 mg, 0.286 mmol) was dissolved in 5 mL DMF, K 2 CO 3 (1.73 g, 12.5 mmol) and 4-chlorobenzyl bromide (116 mg, 0.572 mmol) were added, and heated at 60°C for 2 h. TLC showed that the reaction was complete, and the organic phase was separated by EA/saturated NaCl aqueous solution partitioning, and the organic phase was separated, dried over anhydrous sodium sulfate, and separated by a flash silica gel column, eluted with DCM/MeOH as the mobile phase, and the target product was collected and evaporated to dryness to obtain 80 mg of a white solid, namely the intermediate INT 1-7.
  • the intermediate INT 1-7 (40 mg, 0.091 mmol) was dissolved in 3 mL of dry DCM, and 182 ⁇ L of 1 M BBr 3 solution was added at 0°C, and stirred at this temperature for 1 h. TLC showed that the reaction was complete, and the crude product was evaporated under reduced pressure. Then, it was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 30 mg of a white solid, namely the intermediate INT 1-8.
  • INT 1-8 (25 mg, 0.049 mmol), 1-cyclopenteneboronic acid (13.7 mg, 0.122 mmol), Pd(dppf)Cl 2 (5.38 mg, 0.007 mmol), Cs 2 CO 3 (48 mg, 0.147 mmol), 1,4-dioxane 10 mL and H 2 O 3 mL were placed in a pressure tube, degassed with nitrogen for 3 min, the pressure tube was sealed, and heated at 100°C for 2 h. After cooling, it was diluted with ethyl acetate, the insoluble matter was filtered out, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product.
  • Example E1 The crude product was purified by a preparative liquid chromatography column, eluted with an acetonitrile/water system as the mobile phase, and the target product was collected and evaporated to dryness to obtain 15 mg of a white solid, namely Example E1.
  • Example E1 (30 mg, 0.072 mmol) was dissolved in 3 ml of methanol, and 10 mg of Pd/C was added. After hydrogen substitution, the mixture was reacted at room temperature for 2 h. After suction filtration, the filtrate was concentrated to dryness and purified by preparative liquid chromatography column with acetonitrile/water system as mobile phase. The target product was collected and evaporated to dryness to obtain 20 mg of white solid, which is Example E2.
  • INT 1-8 25 mg, 0.049 mmol
  • INT 2-2 25 mg, 0.106 mmol
  • Pd(dppf)Cl 2 5.38 mg, 0.007 mmol
  • Cs 2 CO 3 48 mg, 0.147 mmol
  • 1,4-dioxane 10 mL and H 2 O 3 mL were placed in a pressure tube, degassed with nitrogen for 3 min, the pressure tube was sealed, and heated and stirred at 100°C for 2 h. After cooling, it was diluted with ethyl acetate, the insoluble matter was filtered out, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product.
  • Example E3 The crude product was purified by a preparative liquid chromatography column, eluted with an acetonitrile/water system as the mobile phase, and the target product was collected and evaporated to dryness to obtain 15 mg of a white solid, namely Example E3.
  • Example E4 7-(4-chlorobenzyl)-8-(2-cyclopentylcyclopropyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • INT 4-2 (100 mg, 0.196 mmol), INT 2-2 (92.5 mg, 0.392 mmol), Pd(dppf)Cl 2 (14.3 mg, 0.019 mmol), Cs 2 CO 3 (191.1 mg, 0.588 mmol), 1,4-dioxane 10 mL and H 2 O 3 mL were placed in a pressure tube, degassed with nitrogen for 3 min, the pressure tube was sealed, and heated and stirred at 100°C for 2 h. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. Then, the mixture was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 80 mg of a jelly, namely the intermediate INT4-3.
  • INT 5-1 300 mg, 0.98 mmol
  • biboronic acid pinacol ester 498 mg, 1.96 mmol
  • Pd(dppf)Cl 2 71 mg, 0.097 mmol
  • KOAc 288 mg, 2.939 mmol
  • 1,4-dioxane 10 mL were placed in a pressure tube, degassed with nitrogen for 3 min, the pressure tube was sealed, and heated and stirred at 80 ° C for 2 h. After cooling, it was diluted with ethyl acetate, the insoluble matter was filtered out, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. Then, it was separated by a flash silica gel column, eluted with PE/EA as the mobile phase, and the target product was collected and evaporated to dryness to obtain 260 mg of a jelly, which is the intermediate INT 5-2.
  • INT 1-8 25 mg, 0.049 mmol
  • INT 5-2 25 mg, 0.088 mmol
  • Pd(dppf)Cl 2 5.38 mg, 0.007 mmol
  • Cs 2 CO 3 48 mg, 0.147 mmol
  • 1,4-dioxane 10 mL and H 2 O 3 mL were placed in a pressure tube, degassed with nitrogen for 3 min, the pressure tube was sealed, and heated and stirred at 100°C for 2 h. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. Then, the mixture was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 20 mg of a jelly, namely the intermediate INT 5-3.
  • Example E6 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(1,2,3,6-tetrahydro-[1,1'-biphenyl]-4-yl)-3,7-dihydro-1H-purine-2,6-dione
  • INT 4-2 (100 mg, 0.196 mmol), INT 5-2 (111.3 mg, 0.392 mmol), Pd(dppf)Cl 2 (14.3 mg, 0.019 mmol), Cs 2 CO 3 (191.1 mg, 0.588 mmol), 1,4-dioxane 10 mL and H 2 O 3 mL were placed in a pressure tube, degassed with nitrogen for 3 min, the pressure tube was sealed, and heated and stirred at 100°C for 2 h. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. Then, the mixture was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 100 mg of a jelly, namely the intermediate INT 6-1.
  • Example E7 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-8-(4-isopropoxycyclohexyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • INT 7-1 (190 mg, 0.66 mmol), bipyralidin (335 mg, 1.319 mmol), Pd(dppf)Cl 2 (50 mg, 0.068 mmol), KOAc (241 mg, 1.975 mmol), and 10 mL of 1,4-dioxane were placed in a pressure tube, degassed with nitrogen for 3 min, and the pressure tube was sealed and heated and stirred at 80°C for 2 h. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. Then, the mixture was separated by a flash silica gel column, eluted with PE/EA as the mobile phase, and the target product was collected and evaporated to dryness to obtain 150 mg of a jelly, which was the intermediate INT 7-2.
  • INT 4-2 (100 mg, 0.196 mmol), INT 7-2 (104.3 mg, 0.392 mmol), Pd(dppf)Cl 2 (14.3 mg, 0.019 mmol), Cs 2 CO 3 (191.1 mg, 0.588 mmol), 1,4-dioxane 10 mL and H 2 O 3 mL were placed in a pressure tube, degassed with nitrogen for 3 min, the pressure tube was sealed, and heated and stirred at 100°C for 2 h. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. Then, the mixture was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 120 mg of a jelly, namely the intermediate INT 7-3.
  • INT 7-3 (100 mg, 0.175 mmol) was dissolved in 5 ml of methanol, and 20 mg of Pd/C was added. After hydrogen replacement, the reaction was allowed to react at room temperature for 2 h. After filtration, the filtrate was concentrated to dryness to obtain 80 mg of a colloid, which was the intermediate INT 7-4.
  • Example E8 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-8-(4-isopropoxycyclohex-1-en-1-yl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E9 8-([1,1'-bis(cyclopropane)]-2-yl)-7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • INT 4-2 (100 mg, 0.196 mmol), INT 9-1 (81.5 mg, 0.392 mmol), Pd(dppf)Cl 2 (14.3 mg, 0.019 mmol), Cs 2 CO 3 (191.1 mg, 0.588 mmol), 1,4-dioxane 10 mL and H 2 O 3 mL were placed in a pressure tube, degassed with nitrogen for 3 min, the pressure tube was sealed, and heated and stirred at 100°C for 2 h. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. Then, the mixture was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 100 mg of a jelly, namely the intermediate INT 9-2.
  • Example E10 7-(4-chlorobenzyl)-8-(4,4-dimethylcyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. Then, the mixture was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 100 mg of a jelly, namely the intermediate INT 10-1.
  • INT 10-1 (100 mg, 0.185 mmol) was dissolved in 5 ml of methanol, and 20 mg of Pd/C was added. After hydrogen replacement, the reaction was allowed to react at room temperature for 2 h. After filtration, the filtrate was concentrated to dryness to obtain 80 mg of a colloid, which was the intermediate INT 10-2.
  • Example E11 7-(4-chlorobenzyl)-8-(4,4-dimethylcyclohex-1-en-1-yl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E12 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(4-(trifluoromethyl)cyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E12 The synthesis method of Example E12 is the same as that of Example E10, except that 4-(trifluoromethyl)-1-cyclohexene-1-boronic acid pinacol ester is used instead of 4,4-(dimethylcyclohexene-1-yl)boronic acid pinacol ester as the raw material.
  • Example E13 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(1,4-dioxacyclo[4.5]dec-8-yl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E13 The synthesis method of Example E13 is the same as that of Example E10, except that 1,4-dioxa-spiro[4,5]dec-7-ene-8-boronic acid pinacol ester is used instead of 4,4-(dimethylcyclohexen-1-yl)boronic acid pinacol ester as the starting material.
  • Example E14 8-([1,1'-bis(cyclopropane)]-2-yl)-7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-3-isopropyl-3,7-dihydro-1H-purine-2,6-dione
  • INT 14-1 (1 g, 2.825 mmol) was dissolved in 10 ml DMF, K 2 CO 3 (428 mg, 3.1 mmol) and 2-iodopropane (480 mg, 2.825 mmol) were added, and stirred at 40°C for 2 h. TLC showed that the reaction was complete, 10 ml H 2 O was added, and the mixture was filtered and dried to obtain 400 mg of a white solid, namely the intermediate INT 14-2.
  • INT 14-3 (100 mg, 0.185 mmol), INT 9-1 (81.5 mg, 0.392 mmol), Pd(dppf)Cl 2 (14.3 mg, 0.019 mmol), Cs 2 CO 3 (191.1 mg, 0.588 mmol), 1,4-dioxane 10 mL and H 2 O 3 mL were placed in a pressure tube, degassed with nitrogen for 3 min, the pressure tube was sealed, and heated and stirred at 100°C for 2 h. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. Then, the mixture was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 60 mg of a jelly, namely the intermediate INT 14-4.
  • Example E15 7-(4-chlorobenzyl)-8-(2-cyclopentylcyclopropyl)-1-(3-hydroxypropyl)-3-isopropyl-3,7-dihydro-1H-purine-2,6-dione
  • INT 14-2 (100 mg, 0.185 mmol), INT 9-1 (81.5 mg, 0.392 mmol), Pd(dppf)Cl 2 (14.3 mg, 0.019 mmol), Cs 2 CO 3 (191.1 mg, 0.588 mmol), 1,4-dioxane 10 mL and H 2 O 3 mL were placed in a pressure tube, degassed with nitrogen for 3 min, the pressure tube was sealed, and heated and stirred at 100°C for 2 h. After cooling, the mixture was diluted with ethyl acetate, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. Then, the mixture was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 80 mg of a jelly, namely the intermediate INT 15-1.
  • Example E16 7-(4-chlorobenzyl)-8-cyclopentyl-1-(3-methoxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • INT 16-1 (80 mg, 0.187 mmol) was dissolved in 5 ml methanol, 15 mg Pd/C was added, and the mixture was reacted at room temperature for 2 h after hydrogen substitution. After filtration, the filtrate was concentrated to dryness to obtain 80 mg of a colloid, which was purified by a preparative liquid chromatography column with acetonitrile/water as the mobile phase for elution. The target product was collected and evaporated to dryness to obtain 50 mg of a white solid, i.e., Example E16.
  • Example E17 7-(4-chlorobenzyl)-8-(1-fluoro-3-phenylcyclobutyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • INT 4-2 (250 mg, 0.49 mmol) was dissolved in 5 ml of ultra-dry tetrahydrofuran, and isopropylmagnesium chloride lithium chloride complex (490 ⁇ L, 0.735 mmol) was added dropwise at 0°C, stirred for 10 min, and 3-phenylcyclobutanone (130 ⁇ L, 0.98 mmol) was added, and stirred at 0°C for 2 h.
  • LC-MS showed that the reaction was complete, and the solvent was evaporated under reduced pressure. Then, it was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 100 mg of a colloid, which was the intermediate INT 17-1.
  • INT 4-2 (250 mg, 0.49 mmol) was dissolved in 5 ml of ultra-dry tetrahydrofuran, 2.5 M n-butyl lithium (235 ⁇ L, 0.588 mmol) was added dropwise at -78°C, stirred for 30 min, ultra-dry DMF (57 ⁇ L, 0.735 mmol) was added, and stirred at -78°C for 2 h.
  • LC-MS showed that the reaction was complete, and the solvent was evaporated under reduced pressure. Then, it was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 80 mg of a jelly, namely the intermediate INT 18-1.
  • NT 17-2 (40 mg, 0.075 mmol) was dissolved in 4 mL DCM, and then 1 mL TFA was added and stirred at room temperature for 1 h. TLC showed that the reaction was complete. The solvent was evaporated under reduced pressure, and the residue was purified by preparative liquid chromatography using acetonitrile/water as the mobile phase to collect the target product and evaporate to dryness to obtain 30 mg of a white solid, Example E18.
  • Example E19 The synthesis method of Example E19 is the same as that of Example E17, except that 4-isopropoxycyclohexanone is used instead of 3-phenylcyclobutanone as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • ⁇ 7.34–7.31 m, 2H
  • 7.20–7.09 m, 2H
  • 5.76 s, 2H
  • 4.21–4.13 m, 2H
  • 3.80–3.64 3.64–3.56 (m, 3H)
  • 3.55–3.37 m, 2H
  • Example E20 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-8-(1H-inden-2-yl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E20 The synthesis method of Example E20 is the same as that of Example E6, except that 1H-indene-2-boronic acid pinacol ester is used instead of 4,4,5,5-tetramethyl-2-(1,2,3,6-tetrahydro-[1,1'-biphenyl]-4-yl)-1,3,2-dioxaborane as the raw material.
  • Example 21 7-(4-chlorobenzyl)-8-(1-fluoro-4-(trifluoromethyl)cyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • INT 4-2 (250 mg, 0.49 mmol) was dissolved in 5 ml of ultra-dry tetrahydrofuran, 2.5 M n-butyl lithium (235 ⁇ L, 0.588 mmol) was added dropwise at -78°C, stirred for 30 min, 4-(trifluoromethyl) cyclohexanone (122 mg, 0.735 mmol) was added, and stirred at -78°C for 2 h.
  • LC-MS showed that the reaction was complete, and the solvent was evaporated under reduced pressure. Then, it was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 120 mg of a colloid, namely the intermediate INT 21-1.
  • Example E22 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-8-(1-methoxy-4-(trifluoromethyl)cyclohexyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • the intermediate INT 21-1 (120 mg, 0.201 mmol) was dissolved in 5 ml of ultra-dry THF, and NaH (16.08 mg, 0.402 mmol) and CH 3 I (38 ⁇ L, 0.603 mmol) were added, and the mixture was reacted at 35°C for 12 h. TLC showed that the reaction was complete, and the solvent was evaporated under reduced pressure. Then, the mixture was separated by a flash silica gel column, and eluted with MeOH/DCM as the mobile phase. The target product was collected and evaporated to dryness to obtain 80 mg of a jelly, namely the intermediate INT 22-1.
  • Example E23 7-(4-chlorobenzyl)-8-(1-hydroxy-4-(trifluoromethyl)cyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E24 7-(4-chlorobenzyl)-8-(1-fluoro-4,4-dimethylcyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E24 The synthesis method of Example E24 is the same as that of Example E21, except that 4,4-dimethylcyclohexanone is used instead of 4-(trifluoromethyl)cyclohexanone as the starting material.
  • 1 H NMR 500MHz, Chloroform-d
  • Example E25 7-(4-chlorobenzyl)-8-(1-hydroxy-4,4-dimethylcyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E26 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-8-(1-methoxy-4,4-dimethylcyclohexyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • INT 4-2 (200 mg, 0.392 mmol) was dissolved in 5 ml of 1,4-dioxane, tributyl (1-ethoxyethylene) tin (267 ⁇ L, 0.784 mmol) and Pd (PPh 3 ) 4 (45 mg, 0.039 mmol) were added under N 2 protection, and stirred at 110°C for 12 h. TLC showed that the reaction was complete, and the solvent was evaporated under reduced pressure. Then, the product was separated by flash silica gel chromatography, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 180 mg of a jelly, namely the intermediate INT27-1.
  • INT 27-1 (180 mg, 0.358 mmol) was dissolved in 3 ml of methanol, 1 ml of 1N HCl was added dropwise at 0°C, and stirred at room temperature for 12 h. TLC showed that the reaction was complete, and the solvent was evaporated under reduced pressure. Then, it was separated by a flash silica gel column, eluted with MeOH/DCM as the mobile phase, and the target product was collected and evaporated to dryness to obtain 140 mg of a colloid, which was the intermediate INT27-2.
  • INT 27-2 (140 mg, 0.295 mmol) and O-tert-butylhydroxylamine hydrochloride (74.3 mg, 0.59 mmol) were dissolved in 3 ml DCM and stirred for 12 h. TLC showed that the reaction was complete and the solvent was evaporated under reduced pressure. Then, the reaction was separated by a flash silica gel column and eluted with MeOH/DCM as the mobile phase. The target product was collected and evaporated to dryness to obtain 90 mg of a colloid, which was the intermediate INT 18-2.
  • NT 17-2 (90 mg, 0.165 mmol) was dissolved in 4 mL DCM, and then 1 mL TFA was added and stirred at room temperature for 1 h. TLC showed that the reaction was complete. The solvent was evaporated under reduced pressure, and the residue was purified by preparative liquid chromatography using acetonitrile/water as the mobile phase to collect the target product and evaporate to dryness to obtain 50 mg of a white solid, Example E27.
  • Example E28 7-(4-chlorobenzyl)-8-(2-fluoro-6,6-dimethylbicyclo[3.1.1]heptane-2-yl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E28 The synthesis method of Example E28 is the same as that of Example E21, except that (1R,5S)-6,6-dimethylbicyclo[3.1.1]heptan-2-one is used as the starting material instead of 4-(trifluoromethyl)cyclohexanone.
  • 1 H NMR 500MHz, Chloroform-d
  • 4.33–3.97 m, 2H
  • 3.55–3.49 (m, 2H) 2.64–2.
  • Example E29 7-(4-chlorobenzyl)-8-(2-fluoro-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • INT 29-1 (20 mg, 0.036 mmol) was dissolved in 4 mL DCM, and then 1 mL TFA was added and stirred at room temperature for 1 h. TLC showed that the reaction was complete. The solvent was evaporated under reduced pressure, and the residue was purified by preparative liquid chromatography with an acetonitrile/water system. The mobile phase was used for elution, and the target product was collected and evaporated to dryness to obtain 10 mg of a white solid, namely Example E29.
  • Example E30 7-(4-chlorobenzyl)-8-(4-ethyl-1-fluorocyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E30 The synthesis method of Example E30 is the same as that of Example E21, except that 4-ethylcyclohexanone is used instead of 4-(trifluoromethyl)cyclohexanone as the starting material. 1. 4 77.2[M+H] + .
  • Example E31 7-(4-chlorobenzyl)-8-(1-fluoro-4-isopropylcyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E31 The synthesis method of Example E31 is the same as that of Example E21, except that 4-isopropylcyclohexanone is used instead of 4-(trifluoromethyl)cyclohexanone as the starting material.
  • 1 H NMR 500MHz, Chloroform-d, 3:1 isomer
  • Example E32 7-(4-chlorobenzyl)-8-(1-fluoro-4-methoxycyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E32 The synthesis method of Example E32 is the same as that of Example E21, except that 4-methoxycyclohexanone is used instead of 4-(trifluoromethyl)cyclohexanone as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • 5.76 s, 2H
  • 3.61 (s, 3H) 3.56 (s, 1H), 3.54-3.49 (m, 2H), 3.42-3.39 (m, 1H), 3.38 (s, 3H), 2.46-2.29 (m, 2H), 2.08-1.90 (m, 4H), 1.88-1.78 (m, 4H).
  • Example E33 7-(4-chlorobenzyl)-8-(1-hydroxy-4-methoxycyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E34 The synthesis method of Example E34 is the same as that of Example E21, except that 4,4-dimethyl-2-cyclohexyl-1-one is used instead of 4-(trifluoromethyl)cyclohexanone as the starting material.
  • Example E35 7-(4-chlorobenzyl)-8-(1-hydroxy-4,4-dimethylcyclohex-2-en-1-yl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E35 The synthesis method of Example E35 is the same as that of Example E25, except that INT 34-1 is used as the starting material instead of INT 24-1.
  • 1 H NMR 500 MHz, Chloroform-d
  • Example E36 7-(4-chlorobenzyl)-8-(4,4-difluorocyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E36 The synthesis method of Example E36 is the same as that of Example E10, except that 2-(4,4-difluorocyclohex-1-en-1-yl)boric acid pinacol ester is used instead of 4,4-(dimethylcyclohexen-1-yl)boric acid pinacol ester as the starting material.
  • Example E37 7-(4-chlorobenzyl)-8-(4,4-difluorocyclohex-1-en-1-yl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E37 The synthesis method of Example E37 is the same as that of Example E25, except that INT 36-1 is used as the starting material instead of INT 24-1.
  • 1 H NMR 500 MHz, Chloroform-d
  • Example E38 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(1,4,4-trifluorocyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E38 The synthesis method of Example E38 is the same as that of Example E21, except that 4,4-difluorocyclohexanone is used instead of 4-(trifluoromethyl)cyclohexanone as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • Example E39 The synthesis method of Example E39 is the same as that of Example E21, except that 4-methylcyclohexanone is used instead of 4-(trifluoromethyl)cyclohexanone as the starting material.
  • 1 H NMR 500MHz, Chloroform-d, 8:1 isomer
  • Example E40 The synthesis method of Example E40 is the same as that of Example E21, except that 4-oxocyclohexanecarbonitrile is used as the starting material instead of 4-(trifluoromethyl)cyclohexanone.
  • 1 H NMR 500MHz, Chloroform-d, 7:10 isomer
  • Example E41 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(4-(trifluoromethyl)cyclohex-1-en-1-yl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E41 The synthesis method of Example E41 is the same as that of E29, except that INT 21-1 is used as the starting material instead of INT 28-1.
  • Example E42 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-3-isopropyl-8-(1,4,4-trifluorocyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E42 The synthesis method of Example E42 is the same as that of Example E38, except that INT 14-3 is used as the starting material instead of INT 4-2.
  • 1 H NMR 500 MHz, Chloroform-d
  • INT 14-3 is used as the starting material instead of INT 4-2.
  • 1 H NMR 500 MHz, Chloroform-d
  • Example E43 7-(4-chloro-3-fluorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(1,4,4-trifluorocyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E43 The synthesis method of Example E43 is the same as that of Example E38, except that INT 43-3 is used as the raw material instead of INT 4-2.
  • the synthesis method of INT 43-3 is the same as that of INT 4-2, except that 4-chloro-3-fluorobenzyl bromide is used as the raw material instead of 4-chlorobenzyl bromide.
  • Example E44 The synthesis method of Example E44 is the same as that of Example E43, except that 3,4-difluorobenzyl bromide is used instead of 4-chloro-3-fluorobenzyl bromide as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • ⁇ 7.16–7.09 m, 1H
  • 6.99–6.95 m, 1H
  • Example E45 7-(4-fluorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(1,4,4-trifluorocyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E45 The synthesis method of Example E45 is the same as that of Example E43, except that 4-fluorobenzyl bromide is used instead of 4-chloro-3-fluorobenzyl bromide as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • ⁇ 7.23–7.13 m, 2H
  • 7.04–6.97 m, 2H
  • 5.75 s, 2H
  • 4.17 (t, J 6 Hz, 2H)
  • 3.53–3.46 (m, 2H) 3.33 (s, 1H), 2.47–2.27 (m, 2H), 2.19–2.04 (m, 6H), 1.92–1.83 (m, 2H).
  • Example E46 7-(4-fluorobenzyl)-1-(3-hydroxypropyl)-3-isopropyl-8-(1,4,4-trifluorocyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E46 The synthesis method of Example E46 is the same as that of Example E45, except that 2-iodopropane is used instead of iodomethane as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • ⁇ 7.24–7.18 m, 2H
  • 7.06–6.98 m, 2H
  • 5.75 s, 2H
  • 2.46–2.27 m, 2H
  • 2.20–2.06 m, 6H
  • 1.91–1.83 m, 2H
  • Example E47 7-(3,4-difluorophenyl)-1-(3-hydroxypropyl)-3-isopropyl-8-(1,4,4-trifluorocyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E47 The synthesis method of Example E47 is the same as that of E46, except that INT 47-2 is used as the starting material instead of INT 46-2.
  • Example E48 7-(4-chlorobenzyl)-8-(4-(dimethylamino)-1-fluorocyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E48 The synthesis method of Example E48 is the same as that of Example E21, except that 4-dimethylaminocyclohexanone is used instead of 4-(trifluoromethyl)cyclohexanone as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • 3.36 (s, 1H) 2.48-2.32 (m, 2H), 2.27 (s, 6H), 2.22-2.16 (m, 1H), 1.94-1.79 (m, 8H).
  • Example E49 7-(4-chlorobenzyl)-8-(4-(dimethylamino)cyclohex-1-en-1-yl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E49 The synthesis method of Example E49 is the same as that of E29, except that INT 48-1 is used as the starting material instead of INT 28-1.
  • Example E50 7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(4-morpholinocyclohex-1-en-1-yl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E50 The synthesis method of Example E50 is the same as that of Example E6, except that 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-cyclohexene)morpholine is used instead of 4,4,5,5-tetramethyl-2-(1,2,3,6-tetrahydro-[1,1'-biphenyl]-4-yl)-1,3,2-dioxaborolan as the starting material.
  • Example E51 7-(4-chloro-3-fluorobenzyl)-3-ethyl-8-(1-fluoro-4-(trifluoromethyl)cyclohexyl)-1-(3-hydroxypropyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E51 The synthesis method of Example E51 is the same as that of Example E43, except that 2-iodopropane is used instead of iodoethane as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d, 5:11 isomer
  • ⁇ 7.41–7.34 m, 1H
  • 7.03–6.90 m, 2H
  • 5.75/5.73 2 ⁇ s, 2H
  • 4.28–4.07 m, 4H
  • 3.60–3.46 m, 2H
  • 2.49–2.39 m, 1H
  • Example E52 7-(3,4-difluorophenyl)-8-(1-fluoro-4-(trifluoromethyl)cyclohexyl)-1-(3-hydroxypropyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E52 The synthesis method of Example E52 is the same as that of E21, except that INT 44-3 is used as the starting material instead of INT 4-2.
  • 1 H NMR(500MHz,Chloroform-d,9:13 isomer) ⁇ 7.16–7.08(m,1H),7.07–6.88(m,2H),5.70/5.72(2 ⁇ s,2H),4.17/4.16(2 ⁇ t,J 7.5Hz,2H),3.57(2 ⁇ s,3H),3.54–3.47 (m,2H),3.35–3.26(m,1H),2.46–2.36(m,1H),2.32–2.19(m,1H),2.18–2.11(m,1H),2.05–1.82(m,7H),1.82–1.71(m,1H).
  • Example E53 7-(4-chlorobenzyl)-8-(1-fluoro-4-(trifluoromethyl)cyclohexyl)-1-(3-hydroxypropyl)-3-isopropyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E53 The synthesis method of Example E53 is the same as that of Example E42, except that 4-(trifluoromethyl)cyclohexanone is used instead of 4,4-difluorocyclohexanone as the raw material. material.
  • Example E54 7-(4-chlorobenzyl)-8-(1-fluoro-4-phenylcyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E54 The synthesis method of Example E54 is the same as that of Example E21, except that 4-phenylcyclohexanone is used instead of 4-(trifluoromethyl)cyclohexanone as the starting material.
  • Example E55 The synthesis method of Example E55 is the same as that of Example E21, except that 4-(1-pyrrolidinyl)cyclohexanone is used instead of 4-(trifluoromethyl)cyclohexanone as the starting material.
  • Example E56 7-(4-chloro-3-fluorobenzyl)-1-(3-hydroxypropyl)-3-isopropyl-8-(1,4,4-trifluorocyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E56 The synthesis method of Example E56 is the same as that of Example E47, except that INT 51-1 is used instead of INT 44-1 as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • INT 51-1 is used instead of INT 44-1 as the starting material.
  • Example E57 7-(4-chloro-3-fluorobenzyl)-3-ethyl-1-(3-hydroxypropyl)-8-(4-(trifluoromethyl)cyclohex-1-en-1-yl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E57 The synthesis method of Example E57 is the same as that of Example E29, except that INT 51-4 is used as the starting material instead of INT 28-1.
  • 1 H NMR 500MHz, Chloroform-d
  • INT 51-4 is used as the starting material instead of INT 28-1.
  • 1 H NMR 500MHz, Chloroform-d
  • ⁇ 7.42–7.37(m,1H),6.88(ddd,J 12.3,8.9,2.1Hz,2H)
  • Example E58 7-(3,4-difluorophenyl)-1-(3-hydroxypropyl)-3-methyl-8-(4-(trifluoromethyl)cyclohex-1-en-1-yl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E58 The synthesis method of Example E58 is the same as that of Example E29, except that INT 52-1 is used as the starting material instead of INT 28-1.
  • Example E59 8-(1-Fluoro-4-(trifluoromethyl)cyclohexyl)-7-(4-fluorobenzyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E59 The synthesis method of Example E59 is the same as that of Example E45, except that 4-(trifluoromethyl)cyclohexanone is used as the starting material instead of 4,4-difluorocyclohexanone.
  • Example E60 1-(3-Hydroxypropyl)-3-methyl-8-(1,4,4-trifluorocyclohexyl)-7-(4-(trifluoromethyl)benzyl)-3,7-dihydro-1H-purine-2,6-dione
  • the mixture was separated by a flash silica gel column using MeOH/DCM as the mobile phase for elution, and the target product was collected to obtain 300 mg of a colorless oily liquid to obtain the intermediate INT 60-2.
  • Example E60 The subsequent synthesis method of Example E60 is the same as that of Example E38, except that INT 60-2 is used instead of 4-2 as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • Example E61 7-(3,5-difluorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(1,4,4-trifluorocyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E61 The synthesis method of Example E61 is the same as that of Example E60, except that 3,5-difluorobenzyl bromide is used instead of 4-(trifluoromethyl)benzyl bromide as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • 6.70-6.64 m, 2H
  • 5.75 s, 2H
  • Example E62 The synthesis method of Example E62 is the same as that of Example E60, except that benzyl bromide is used as the starting material instead of 4-(trifluoromethyl)benzyl bromide.
  • 1 H NMR 500 MHz, Chloroform-d
  • benzyl bromide is used as the starting material instead of 4-(trifluoromethyl)benzyl bromide.
  • Example E63 The synthesis method of Example E63 is the same as that of Example E62, except that 4-(trifluoromethyl)cyclohexanone is used instead of 4,4-difluorocyclohexanone as the starting material.
  • Example E64 The synthesis method of Example E64 is the same as that of Example E60, except that 2,4-difluorobenzyl bromide is used instead of 4-(trifluoromethyl)benzyl bromide as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • ⁇ 6.89–6.84 m, 1H
  • 6.83–6.73 m, 2H
  • 5.82 s, 2H
  • Example E65 7-(2,4-difluorobenzyl)-8-(1-fluoro-4-(trifluoromethyl)cyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E65 The synthesis method of Example E65 is the same as that of Example E64, except that 4-(trifluoromethyl)cyclohexanone is used instead of 4,4-difluorocyclohexanone as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • ⁇ 6.89–6.83 m, 1H
  • 6.82–6.75 m, 1H
  • 6.72–6.64 m, 1H
  • 5.79 s, 2H
  • 3.60 s, 3H
  • 3.52–3.44 m, 2H
  • 2.45–2.33 2.31–2.21 (m, 1H), 2.02–1.82 (m, 8H.
  • Example E66 7-(2-chloro-4-fluorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(1,4,4-trifluorocyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E66 The synthesis method of Example E66 is the same as that of Example E60, except that 2-chloro-4-fluorobenzyl bromide is used instead of 4-(trifluoromethyl)benzyl bromide as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • 6.92-6.85 m, 1H
  • 6.47-6.42 m, 1H
  • Example E67 7-(2-chloro-4-fluorobenzyl)-8-(1-fluoro-4-(trifluoromethyl)cyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E67 The synthesis method of Example E67 is the same as that of Example E66, except that 4-(trifluoromethyl)cyclohexanone is used instead of 4,4-difluorocyclohexanone as the starting material.
  • Example E68 7-(3-chloro-4-fluorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(1,4,4-trifluorocyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E68 The synthesis method of Example E68 is the same as that of Example E60, except that 3-chloro-4-fluorobenzyl bromide is used instead of 4-(trifluoromethyl)benzyl bromide as the starting material.
  • Example E69 7-(3-chloro-4-fluorobenzyl)-8-(1-fluoro-4-(trifluoromethyl)cyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E69 The synthesis method of Example E69 is the same as that of Example E68, except that 4-(trifluoromethyl)cyclohexanone is used instead of 4,4-difluorocyclohexanone as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • ⁇ 7.25–7.21 m, 1H
  • 7.13–7.04 m, 2H
  • 5.69 s, 2H
  • 3.59 s, 3H
  • Example E70 7-(4-chloro-2-fluorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(1,4,4-trifluorocyclohexyl)-3,7-dihydro-1H-purine-2,6-dione
  • Example E70 The synthesis method of Example E70 is the same as that of Example E60, except that 4-chloro-2-fluorobenzyl bromide is used instead of 4-(trifluoromethyl)benzyl bromide as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • ⁇ 7.17–7.12 m, 1H
  • 7.08–7.03 m, 1H
  • 6.71–6.65 m, 1H
  • 5.82 s, 2H
  • 3.58 (s, 3H) 3.53–3.47 (m, 2H), 2.48–2.31 (m, 2H), 2.19–2.05 (m, 6H), 1.90–1.82 (m, 2H).
  • Example E71 7-(4-chloro-2-fluorobenzyl)-8-(1-fluoro-4-(trifluoromethyl)cyclohexyl)-1-(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
  • Example E71 The synthesis method of Example E71 is the same as that of Example E70, except that 4-(trifluoromethyl)cyclohexanone is used as the starting material instead of 4,4-difluorocyclohexanone.
  • Example E72 4-((1-(3-hydroxypropyl)-3-methyl-2,6-dioxo-8-(1,4,4-trifluorocyclohexyl)-1,2,3,6-tetrahydro-7H-purin-7-yl)methyl)benzonitrile
  • Example E72 The synthesis method of Example E72 is the same as that of Example E60, except that 4-cyanobenzyl bromide is used instead of 4-(trifluoromethyl)benzyl bromide as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • 5.83 s, 2H
  • Example E73 The synthesis method of Example E73 is the same as that of Example E72, except that 4-(trifluoromethyl)cyclohexanone is used instead of 4,4-difluorocyclohexanone as the starting material.
  • 1 H NMR 500 MHz, Chloroform-d
  • 4.13 (t, J 6.0 Hz, 2H)
  • 3.60 s, 3H
  • 3.52-3.47 (m, 2H) 3.19 (s, 1H), 2.47-2.36 (m, 2H), 2.33-2.21 (m, 1H), 2.04-1.80 (m, 8H).
  • the fluorescence signal of the trajectory diagram is represented by F/F 0 , where F is the fluorescence signal at different time points, and F 0 is the basic fluorescence signal, that is, the average value of the fluorescence signal at the first 10 time points.
  • HEK-293 cells expressing hTRPC5 were seeded into PDL-coated black-bottomed 96-well plates, with 2 ⁇ 10 4 cells per well. After 8 hours of culture, the original culture medium was discarded, and 60 ⁇ L of 4 ⁇ M Fluo-4/AM dye was added. The cells were incubated at 37°C for 60 minutes, and then rinsed with calcium flow detection buffer 5 times. The cell plates were placed in a 30°C preheated (Molecular Devices, Sunnyvale, CA, USA), excited at a wavelength of 488nm, and continuously recorded fluorescence signals in the range of 515-535nm at a sampling frequency of 1s.
  • the trajectory graph fluorescence signal is expressed as F/ F0 , where F is the fluorescence signal at different time points and F0 is the basic fluorescence signal, that is, the average value of the fluorescence signal at the first 10 time points.
  • CHO cells expressing hERG protein were cultured in a 175 cm2 culture flask. When the cell density grew to 60-80%, the culture medium was removed, and the cells were washed with 7 mL PBS, and then 3 mL Detachin was added for digestion. After complete digestion, 7 mL culture medium was added for neutralization, and then centrifuged, the supernatant was aspirated, and 5 mL culture medium was added for re-suspending to ensure that the cell density was 2-5 ⁇ 10 6 /mL.
  • the single-cell high-impedance sealing and whole-cell pattern formation process are all automatically completed by the Qpatch instrument. After obtaining the whole-cell recording mode, the cell is clamped at -80 mV. Before giving a 5-second +20 mV depolarizing stimulus, a 50-millisecond -50 mV pre-voltage is given, and then repolarizes to -50 mV for 5 seconds, and then returns to -80 mV. This voltage stimulus is applied every 15 seconds. After recording for 2 minutes, the extracellular solution is given for 2 minutes, and then the drug administration process begins. The compound concentration starts from the lowest test concentration, and each test concentration is given for 2 minutes. After all concentrations are given continuously, the positive control compound 10 ⁇ M Cisapride is given. At least 3 cells (n ⁇ 3) are tested for each concentration.
  • the compound stock solution was diluted with extracellular solution, 5 ⁇ L of 20mM compound stock solution was added to 2495 ⁇ L of extracellular solution, 500 times diluted to 40 ⁇ M, and then 3 times serial dilution was performed in the extracellular solution containing 0.2% DMSO to obtain the final concentration to be tested.
  • the highest test concentration of the compound was 40 ⁇ M, and the concentrations were 40, 13.33, 4.44, 1.48, 0.49 and 0.16 ⁇ M, respectively.
  • the highest test concentration of the positive compound Cisapride was 3 ⁇ M, and the concentrations were 3, 1, 0.333, 0.111, 0.037 and 0.012 ⁇ M, respectively.
  • the DMSO content in the final test concentration did not exceed 0.2%, and this concentration of DMSO had no effect on the hERG potassium channel.
  • the compound to be tested is first prepared as a stock solution with DMSO, and then diluted with water and 0.1% BSA to the working concentration when used. Buffer (final concentration 0.33 mg/mL microsomal protein), MgCl2 solution (final concentration 5 mM), test compound solution (final concentration 1 ⁇ M) and NADPH solution (final concentration 1 mM) were incubated at 37°C, and methanol was added to terminate the reaction at 0, 7, 17, 30 and 60 minutes. The residual concentration of the test compound was determined by LC/MS/MS method.
  • k e The slope of the linear regression line (absolute value of slope) in semi-logarithmic plotting. The semi-logarithm of the remaining percentage of substrate is taken and the semi-logarithmic plot is made against the reaction time.
  • the compounds of the present invention have moderate to strong inhibitory effects on TRPC5 channels, and the activity of some compounds exceeds that of the reference compound HC-070. Therefore, the compounds of the present invention have potential application value in treating diseases related to excessive activation of TRPC5 channels or overexpression of TRPC5 proteins.
  • the compounds of the present invention can be used to treat diseases related to the inhibition of TRPC5 channels and the loss of TRPC5 protein, or can activate TRPC5 channels to cause calcium ion overload and induce cell death, thereby being used to treat certain types of tumors.
  • the compounds of the present invention also have a good inhibitory effect on TRPC4, which is consistent with the biological characteristics of the high homology of TRPC4 and TRPC5. Therefore, the compounds of the present invention also have potential application value in the treatment of diseases related to excessive activation of TRPC4 channels or overexpression of TRPC4 proteins.
  • the compound of the present invention has lower inhibitory activity on TRPC3 channels, higher relative selectivity for TRPC5 channels, and lower off-target risk.
  • the compound of the present invention has lower inhibitory activity on hERG channels and higher relative selectivity on TRPC5 channels.
  • hERG channel blockade is considered an indicator of cardiac toxicity risk and should be avoided as much as possible.
  • the distinguishing structural feature of the compounds of the present invention is that the 8-position of xanthine is replaced by a "cycloalkyl" instead of an aryl, aryloxy, alkyl, alkoxy, cycloalkoxy or other groups.
  • this structural difference significantly weakens the inhibitory effect of the compound on the hERG channel, indicating that the compounds of the present invention have a lower risk of cardiac toxicity.
  • Table 3 Inhibitory activity and selectivity of compounds on TRPC3, TRPC4, and hERG channels Note: a , literature value (PLoS ONE.2018,13,e0191225.).
  • the reference compound HC-070 has moderate metabolic stability in human liver microsomes, but is rapidly metabolized in mouse liver microsomes with a short half-life. Compared with HC-070, the compound of the present invention has a longer half-life and a higher metabolic utilization rate, indicating that it is more stable in metabolic properties and has better drug-like properties.
  • MF% indicates metabolic bioavailability
  • metabolic stability criteria MF%>70%, good metabolic stability; 30% ⁇ MF% ⁇ 70%, medium metabolic stability; MF% ⁇ 30%, poor metabolic stability.
  • mice hepatic stellate cells were plated on 96-well black-bottom permeable plates for 24 hours, they were pre-incubated with 1 ⁇ M compound E21 and solvent control 0.1% DMSO for 1 hour, and then TGF- ⁇ was added to a final concentration of 10 ng/mL, and continued to be cultured in a CO 2 incubator for 24 hours.
  • the culture medium was aspirated, washed with 37°C PBS solution for 3 times, and fixed with 4% paraformaldehyde solution for 30 minutes; then 0.15% Triton X-100 solution was added for permeabilization for 10 minutes; then 5% BSA solution was added for blocking at room temperature for 1 hour; then anti- ⁇ -SMA solution (1:300) prepared with 5% BSA solution was added and incubated at 4°C overnight; the next day, Alexa-488 anti-rabbit fluorescent secondary antibody (1:1000) prepared with 5% BSA solution was added and incubated at room temperature for 1 hour in the dark.
  • the cells were washed with PBS solution for 3 times, Hoechst solution was added, and incubated at room temperature for 20 minutes in the dark. After the end, the cells were washed three times with PBS solution and fluorescence images were taken. FITC filter was used to capture green fluorescence, and DAPI filter was used to capture blue fluorescence. ImageJ software was used to count the total number of cells in each field of view, and the number of ⁇ -SMA positive cells was manually counted. GraphPad (Version 6.01) software was used for data processing and analysis.
  • mice 6-8 week old male ICR mice (Nanjing Annuokang Biotechnology Co., Ltd.) were housed in an SPF animal room with a light-to-dark time ratio of 1:1, room temperature controlled at 23 ⁇ 2°C, humidity controlled at 55%, and mice were able to eat and drink freely.
  • the test compound was prepared according to the following solvent: 2:2:96 (v/v/v) DMSO/Tween 80/0.5% (m/v) CMC-Na aqueous solution, with a drug concentration of 1 mg/mL. Then, the test compound was gavaged to mice at a dosage of 10 mL/kg, with a dose of 10 mg/kg, and 3 mice were given each compound.
  • LC-MS/MS liquid chromatography-tandem mass spectrometry
  • the compound of the present invention has higher plasma drug concentrations 1 and 4 hours after oral administration to mice, which preliminarily indicates that the compound of the present invention has better oral absorption properties and plasma exposure, and better pharmacokinetic properties.
  • IV administration the drug to be tested was dissolved in DMSO/Solutol/normal saline at a ratio of 5:5:90 (v/v/v), with a concentration of 0.2 mg/mL, and the administration volume was 5 mL/kg.
  • Oral (PO) administration the drug to be tested was dissolved in DMSO/Tween 80/0.5% CMC-Na solution at a ratio of 2:2:96 (v/v/v), with a concentration of 1 mg/mL, and the administration volume was 10 mL/kg.
  • mice Male SD rats weighing 180-280g, 3 rats in each group, fasted for 12h before the experiment, had free access to water, and ate uniformly 4h after administration.
  • the rat tail vein was administered with a dose of 1mg/kg, and blood was collected at 0.083, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 12 and 24h after administration.
  • the rat tail vein was administered with a dose of 10mg/kg, and blood was collected at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 12 and 24h after administration.
  • 0.2mL of blood was collected from the jugular vein and placed in an anticoagulant tube containing EDTA-K2. After the whole blood was collected, it was temporarily stored in an ice water bath, centrifuged at 11000rpm for 5min within 30min, the plasma was separated, and then frozen in a -70°C refrigerator for testing. Tissue distribution group, oral administration, dose of 10mg/kg, After 2 hours of drug administration, the rats were anesthetized and killed by bleeding through the abdominal aorta. The brain, heart, liver, spleen, lung, kidney, colon and muscle were immediately dissected and collected. Part of the whole blood was collected and operated in an ice bath.
  • FIG. 6 shows the distribution of compound E68 in tissues 2 hours after oral administration of 10 mg/kg to rats. It can be seen that E68 has a high concentration in each tissue, which is significantly higher than that in plasma. Therefore, the compounds of the present invention can be efficiently distributed to target tissues when used to treat mental illness, neurodegenerative diseases, liver, kidney and other diseases.
  • C max maximum plasma concentration
  • T max time to peak drug concentration
  • AUC 0- ⁇ area under the drug-time curve from zero to infinity
  • CL plasma clearance of the drug
  • V ss volume of distribution of the drug at steady state
  • t 1/2 elimination half-life
  • F oral bioavailability
  • - not calculated
  • a parameters are reported in the literature (PLoS ONE.2018,13,e0191225.).
  • the drug to be tested was dissolved in DMSO: Tween 80: distilled water at a ratio of 2:2:96 (v/v/v), and then administered to mice by gavage at a volume of 10 mL/kg.
  • the blank control group was only given 10 mL/kg of the above solvent.
  • the mouse marble burying test is a behavioral assay used for the screening of anxiolytic drugs. It exploits the natural digging behavior of mice in environments such as burrows or escape tunnels and is sensitive to small changes in behavior caused by genetic manipulation, disease, or drug treatment. The test has been shown to be effective in detecting anxiolytic effects in drug interventions and transgenic mouse models, and can accurately and sensitively measure repetitive and compulsive behaviors in rodents.
  • the mouse tail suspension test is a widely used experimental method to evaluate the effectiveness of antidepressants and other psychotropic drugs.
  • the principle of the test is to suspend a mouse by its tail and observe its behavior. The mouse will initially struggle to escape, but when it realizes that escape is impossible, it will eventually stop struggling and enter a state of immobility that indicates despair. This immobility behavior is used to measure depression-like states, and the severity of the condition is assessed by recording the duration of immobility. Antidepressants and stimulants can significantly shorten the duration of this immobility, which is why the TST is used in the initial screening of these drugs.
  • the test is sensitive to most antidepressants and the efficacy is significantly correlated with clinical efficacy, which makes it the initial testing standard for antidepressant drugs.
  • a tail suspension test box was built using a black frosted acrylic board, 55 cm high, 15 cm long, and 11.5 cm wide. The three sides and top of the box were frosted blackboards, and one side was used for video recording.
  • the mouse tail was wrapped with yellow tape 2 cm away from the tip of the tail, and the mouse was hung upside down in the middle of the top acrylic board. The tip of the mouse tail was about 3 cm away from the acrylic board, and the body could not touch the surrounding board wall.
  • the test time for each mouse lasted for 6 minutes, and the immobility time of the mouse during this time was counted.
  • the mouse was returned to the cage, and the inside of the box and the table were wiped with alcohol to remove the smell of the previous mouse to prevent affecting the next mouse experiment.
  • the criteria for immobility in the tail suspension test stop struggling, no flexion or twisting of the body, no swinging of the hind limbs, and slight swinging of the forelimbs and head were also counted as immobility time.
  • mice orally administered 0.03 mg/kg of compound E68 can significantly reduce the number of buried marbles, and the effect is more obvious when administered 0.1 mg/kg of E68, and is better than the same dose of HC-070.
  • oral administration of 0.03 mg/kg of compound E68 can also significantly reduce the immobility time of mice in the tail suspension box, and its effect is comparable to 0.1 mg/kg of HC-070. It can be seen that compared with HC-070, the compound E68 of the present invention has a more potent anti-anxiety and anti-depressant effect.

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Abstract

一种黄嘌呤类化合物及其用途，结构如式I-a所示，式中，各取代基的定义如说明书和权利要求书中所述。式I-a化合物是TRPC4和TRPC5离子通道的抑制剂或激动剂，在预防、延缓或治疗与TRPC4/5功能或表达异常相关的疾病中具有潜在的应用价值。

Description

黄嘌呤类化合物及其用途 技术领域
本发明涉及药物化学领域，具体地，本发明涉及黄嘌呤类化合物、其制备方法及其用途。所述化合物是TRPC4和TRPC5离子通道的抑制剂或激动剂，在预防、延缓或治疗与TRPC4/5功能或表达异常相关的疾病中具有潜在的应用价值。
背景技术
瞬时受体电位(TRP)离子通道是一类在外周和中枢神经系统广泛分布的六次跨膜通道蛋白，是感知细胞环境变化、传递信号、维持细胞稳态的分子感受器。根据序列相似性，哺乳动物TRP离子通道可以分为TRPA、TRPC、TRPM、TRPML、TRPP、TRPV等六个亚家族。经典瞬时受体电位(TRPC)通道是非选择性的阳离子通道，允许Ca2+、Na+透过。哺乳动物TRPC由TRPC1、TRPC3、TRPC4、TRPC5、TRPC6、TRPC7六个成员构成，其中TRPC3、TRPC6、TRPC7的氨基酸序列一致性较高，TRPC1、TRPC4、TRPC5的序列一致性较高。TRPC家族蛋白以纯合或杂合四聚体的形式存在并发挥离子通道作用，TRPC5易与TRPC1和TRPC4形成杂合四聚体。
TRPC4/5在脑内多个部位表达，包括海马体、杏仁体、额皮质等，与焦虑、抑郁、癫痫、成瘾等神经系统疾病的发生有关。TRPC4或TRPC5基因敲除、或者应用TPRC4/5抑制剂可缓解小鼠焦虑和抑郁症状。勃林格殷格翰制药公司研制的一个TRPC4/5抑制剂BI-1358894目前正在开展临床试验，用于抑郁症、边缘人格障碍、创伤后应激障碍等精神疾病的治疗。
足细胞是形成肾小球滤过屏障的特殊细胞，TRPC5也在肾小球足细胞内表达，其功能异常与慢性肾病的形成联系在一起。TRPC5参与对血管紧张素刺激的细胞迁移和肌动蛋白重塑过程的调节。在多种肾病动物模型上，TRPC5基因敲除或TPRC5抑制剂可显著降低蛋白尿水平，保护足细胞免受损伤，抑制肾病进展。Goldfinch生物技术公司研制的一个TRPC4/5抑制剂GFB-887目前正在开展临床试验，用于糖尿病肾病、局灶节段性肾小球硬化症等肾脏疾病的治疗。
TRPC4/5在皮肤角质形成细胞中表达，在调节角质细胞分化过程中起到了重要作用，因此，靶向调控TRPC4/5或许可以用于治疗以角质形成细胞功能异常为病理基础的皮肤病变。
TRPC5在肝脏以及肝门静脉血管床中表达，一系列在肝病形成过程中起到重要作用的内源性磷脂，如溶血卵磷脂(LPC)，能显著地激活TRPC5。在胆汁酸喂养诱导的胆汁淤积性肝病小鼠模型中，TRPC5基因敲除能缓解肝脏损伤及肝脂代谢异常。可见，TRPC5也是治疗肝脏疾病的潜在靶标。
TRPC4/5也在外周感觉神经元中表达，与痛觉感知有关。与野生型动物相比，TRPC4-/-小鼠对芥子油诱导的疼痛具有抵抗力，且疼痛阈值增加。在神经性疼痛、炎症 性疼痛、自发性疼痛等多种小鼠模型中，TRPC5基因敲除或者TRPC5抑制剂都能够有效缓解机械性痛觉超敏，这种作用与LPC调控的生物功能存在内在联系。
TRPC4/5也与心血管疾病有关。激活TRPC5通道可以显著促进血管平滑肌细胞的增殖和迁移。抑制TRPC5可显著减少缺氧缺血诱导的细胞凋亡与内皮细胞的氧化应激，并降低内皮依赖性血管收缩，抑制动脉粥样硬化斑块的形成。还可减少心肌肥厚相关基因的表达，维持心脏电活动的稳定性，从而起到抗动脉粥样硬化、抗心肌肥厚和抗心律失常的作用。此外，TRPC5介导的钙离子内流是多种内皮源性收缩因子释放的关键信号机制，有助于高血压的发展。TRPC5促进血管平滑肌细胞增殖，增加氧化应激，进而推动高血压的发生。
TRPC4/5与肿瘤血管形成、肿瘤耐药和转移有关。TRPC5在结直肠癌中高表达，促进细胞外钙内流从而激活Wnt5a/β-catenin通路，降低肿瘤分化层度，增加肿瘤干细胞。TRPC5表达水平与结直肠癌患者的预后呈负相关。TRPC5还可降低E-钙粘蛋白，增加间充质标志物表达，促进肿瘤细胞迁移、侵袭和增殖。此外，激活TRPC5引起的钙离子失调可促进厌氧诱导因子1(HIF-1)介导的肿瘤血管生成，使癌细胞从高剂量抗癌药物暴露的部位逃逸，并获得额外的营养以帮助肿瘤存活，从而降低化疗效果。在乳腺癌模型中，抑制TRPC5可降低化疗耐药性。但另一方面，激活TRPC4/5也可以通过钙离子过载诱导肿瘤细胞死亡。比如，天然产物(-)-Englerin A是强效的TRPC4/5激动剂，它对肾癌、肺癌、乳腺癌、皮肤癌等多种肿瘤细胞表现出抗增殖作用。
TRPC4/5在小肠平滑肌和肌间神经丛表达，参与调控肠平滑肌细胞收缩、胃肠道运动性和肠神经信号传导，因此靶向干预TRPC4/5通道活性的化合物在治疗肠道疾病中也有潜在的应用价值。
WO2014143799公开了如下通式所示的黄嘌呤类化合物，其中8-位的取代基R2定义为C1-C6烷基、C1-C6杂烷基、C1-C6烷氧基、C3-C7环烷氧基、C6-C10芳基、C6-C10芳氧基、C7-C16芳代烷氧基、杂环烷基、杂芳基、杂芳氧基等。该申请中的化合物260作为重点研究的化合物，具有较好的TRPC5抑制活性，在抑郁症和焦虑症小鼠模型上也表现出较好的药效。在另外一项对比文献中(PLoS ONE.2018,13,e0191225.)，化合物260也被称为HC-070，该文献详细展示了HC-070抑制TRPC4/5通道的活性以及在多种动物模型上的抗焦虑抗抑郁效果。但是，HC-070对其他的一些离子通道缺乏很好的选择性，它可以显著地抑制hERG钾通道和TRPC3。
WO2019011802公开了如下所示的黄嘌呤8-位被“3-吡啶基”或“2-哌嗪基”取代的化合物，与HC-070相比，它们在保持较高TRPC5抑制活性的同时也降低了对hERG通道的抑制作用。
发明内容
本发明的主要目的是提供一类用作TRPC4和TRPC5调节剂的黄嘌呤类化合物、其制备方法及治疗用途。
第一方面，本发明提供通式(I)所示的稠合嘧啶二酮类化合物、其立体异构体及其药学上可接受的盐；
其中，A环为饱和或部分不饱和的C3-C10环烃基，包括单环、并环、螺环和桥环体系在内；X为N或CH；R1为C1-C6烷基或C1-C6卤代烷基；n为1到4的整数，各R2各自独立地为H、氘、卤素原子、C1-C6烷基、C1-C6卤代烷基、C1-C6烷氧基、C1-C6卤代烷氧基、氰基；m为1到6的整数，各R3各自独立地选自H、氘、C1-C6的烷基、C1-C6卤代烷基、C1-C6的烷氧基、C1-C6卤代烷氧基、C3-C8的环烷基、C6-C10芳基、5-8元杂芳基、4-8元杂环基、卤素原子、氰基、羟基、-NR4R5，或者R3与A环上相连的碳共同形成4-8元杂环或C6-C10芳基；所述环烷基、芳基、杂芳基、杂环基未被取代或者任选地被1-4个选自下组的基团所取代：C1-C6的烷基、C1-C6的烷氧基、卤素原子、氰基；
R4、R5各自独立地为H、C1-C6的烷基、C3-C8的环烷基。
第二方面，本发明还提供通式(I-a)所示的黄嘌呤类化合物、其立体异构体及其药学上可接受的盐；
其中，A环为饱和或部分不饱和的C3-C10环烃基，较佳地包括单环、并环、螺环和桥环体系在内；
R1为C1-C6烷基或C1-C6卤代烷基；
n为1到4的整数，各R2各自独立地为H、氘、卤素、C1-C6烷基、C1-C6卤代烷基、C1-C6烷氧基、C1-C6卤代烷氧基、氰基；
m为1到6的整数，各R3各自独立地选自H、氘、C1-C6的烷基、C1-C6卤代烷基、C1-C6的烷氧基、C1-C6卤代烷氧基、C3-C8的环烷基、C6-C10芳基、5-8元杂芳基、4-8元杂环基、卤素、氰基、羟基、-NR4R5，或者R3与A环上相连的碳共同形成4-8元杂环或C6-C10芳基；所述环烷基、芳基、杂芳基、杂环基未被取代或者任选地被1-4个选自下组的基团所取代：C1-C6的烷基、C1-C6卤代烷基、C1-C6的烷氧基、C1-C6卤代烷氧基、卤素、氰基；
R4、R5各自独立地为H、C1-C6的烷基、C3-C8的环烷基。
在另一优选例中，A环为饱和或部分不饱和的C3-C8的单环或桥环烃基。
在另一优选例中，A环选自下组：
在另一优选例中，A环为环己基或环己烯基(如)。
在另一优选例中，所述的化合物具有如通式(I-b)所示的结构；
其中，R1为C1-C6烷基；
n为1到4的整数，各R2各自独立地为H、CN、C1-C6卤代烷基或卤素原子；
各R3各自独立地选自H、氘、C1-C6的烷基、C1-C6卤代烷基、C1-C6的烷氧基、C1-C6卤代烷氧基、C6-C10芳基、C3-C8的环烷基、5-8元杂芳基、4-8元杂环基、卤素原子、氰基、羟基、-NR4R5；或者两R3与A环上相连的碳共同形成4-8元杂环；
R4、R5各自独立地为各自独立地为H、C1-C6的烷基。
在另一优选例中，R1为C1-C4烷基。在另一优选例中，R1为甲基、乙基或异丙基。
在另一优选例中，n为1、2或3，各R2各自独立地为H、氘、卤素、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C1-C4卤代烷氧基、氰基。在另一优选例中，n为1、2或3，各R2各自独立地为H、氘、F、Cl、Br、甲基、乙基或异丙基。在另一优选例中，n为1，R2为F、Cl或Br，位于对位。在另一优选例中，n为2，各R2独立为F、Cl或Br，位于对位和间位
在另一优选例中，R2为-F或-Cl。
在另一优选例中，m为1、2、3或4，各R3各自独立地选自H、氘、C1-C4的烷基、 C1-C4卤代烷基、C1-C4的烷氧基、C1-C4卤代烷氧基、C3-C6的环烷基、苯基、5-6元杂芳基、5-6元杂环基、F、Cl、Br、氰基、羟基、-NR4R5，或者R3与A环上相连的碳共同形成5-6元杂环或苯基；所述环烷基、苯基、杂芳基、杂环基未被取代或者任选地被1-4个选自下组的基团所取代：C1-C6的烷基、C1-C6卤代烷基、C1-C6的烷氧基、C1-C6卤代烷氧基、卤素、氰基。
在另一优选例中，各R3各自独立地选自H、甲基、乙基、正丙基、异丙基、三氟甲基、二氟甲基、一氟甲基、苯基、环丁基、环丙基、环戊基、环己基、甲氧基、乙氧基、正丙氧基、异丙氧基、羟基、F、Cl、Br、氰基、氨基、甲氨基、二甲氨基、氧杂环丁烷基、二氧杂环戊烷基、吗啉基、哌嗪基、吡咯烷基、哌啶基。
在另一优选例中，各R3各自独立地选自H、C1-C6的烷基、C1-C6卤代烷基、C1-C6的烷氧基、C3-C8的环烷基、苯基、4-8元杂环基、卤素原子、氰基、羟基、-NR4R5。
在另一优选例中，R4、R5各自独立地为H、C1-C4的烷基。
在另一优选例中，各R3各自独立地选自H、C1-C3卤代烷基或卤素原子。在另一优选例中，R3为-F。
在另一优选例中，R3与A环上相连的碳共同形成
在另一优选例中，通式(I)、(I-a)或(I-b)所示化合物选自实施例化合物E1至E73-2。
第三方面，本发明提供一种药物组合物，其包含至少一种有效治疗剂量的通式(I)或(I-a)或(I-b)所示的化合物、其立体异构体或其药学上可接受的盐，以及药学上可接受的辅料。所述药用辅料为药学上可接受的载体、赋形剂、缓释剂、气味剂、香味剂等。
所述药物组合物中，本发明化合物作为活性组分、其重量占药物组合物总重量的0.1～99.9％，其余为药学上可接受的辅料；本发明化合物与辅料的优选比例是：本发明化合物作为活性成分占总重量60％以上，其余部分占总重量0-40％，其余部分的量优选为1-20％，最优选为1-10％。
本发明所述化合物或药物组合物可以基于药物制剂领域的惯用工艺制成多种剂型，如片剂、胶囊剂、散剂、糖浆剂、溶液剂、混悬剂、喷雾剂、乳膏、软膏、凝胶、透皮贴片等，并可以存在于适宜的固体或液体载体或稀释液中。本发明的药物组合物也可以储存在适宜的注射或滴注的消毒器具中。
本发明所述化合物或药物组合物可对哺乳动物使用，包括人和动物。给药途径包括口服、鼻腔吸入、皮肤局部给药、静脉注射、肌肉注射、皮下注射等。在另一优选例中，本发明所述化合物或药物组合物的优选给药途径为口服。
本发明所述化合物或药物组合物用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。固态载体包括：淀粉、乳糖、磷酸二钙、微晶纤维素、蔗糖和白陶土等，而液态载体包括：无菌水、聚乙二醇、非离子型表面活性剂和食用油(如玉米油、花生油 和芝麻油)等，只要适合活性成分的特性和所需的特定给药方式。在制备药物组合物中通常使用的佐剂也可有利地被包括，例如调味剂、色素、防腐剂和抗氧化剂如维生素E、维生素C、BHT和BHA。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1，3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
作为药物制剂使用时，本发明所示的化合物优选单位剂量，每剂包含有效成分0.01mg-200mg，优选0.5mg-50mg，一次或分次使用。不管用何种服用方法，个人的最佳剂量应根据具体治疗而定。通常情况下是从小剂量开始，逐渐增加剂量一直到找到最合适的剂量。
第四方面，本发明提供通式(I)或(I-a)或(I-b)所示的化合物、其立体异构体、其药学上可接受的盐或其药物组合物的用途，所述化合物用作TRPC4和TRPC5离子通道的调节剂，因此可以制备药物用于预防、延缓或治疗与TRPC4或/和TRPC5的功能、表达异常相关的疾病。
本发明中，所述TRPC4和TRPC5离子通道的调节剂包括抑制剂和激动剂。
在一优选例中，所述TRPC4和TRPC5离子通道的调节剂为TRPC4抑制剂和TRPC5抑制剂。
本发明中，所述与TRPC4或/和TRPC5的功能、表达异常相关的疾病包括但不限于精神疾病、神经退行性疾病、肾脏疾病、疼痛、癫痫、肝病、心血管疾病、癌症、皮肤疾病、肠道疾病等。
在一优选例中，所述与TRPC4或/和TRPC5的功能、表达异常相关的疾病为精神疾病。所述精神疾病包括与情绪低落相关的疾病，例如，边缘型人格障碍、抑郁症、心境恶劣、产后抑郁、双相障碍等；所述精神疾病还包括与焦虑及恐惧相关的病症，例如，创伤后应激障碍、惊恐病、广场恐怖症、社交恐惧症、广泛性焦虑症、社交焦虑症、分离焦虑等；所述精神疾病还包括其他各种原因引起的情感障碍和精神障碍，例如，精神分裂症、狂躁症、强迫症、冷漠、神经衰弱、妄想症等。
在另一优选例中，所述精神疾病为边缘型人格障碍、抑郁症、焦虑症、创伤后应激障碍。
本发明所述的化合物或其药物组合物用于治疗精神疾病时可以单独使用，也可以与其他机制的药物联用。所述其他机制的药物包括但不仅限于三环类抗抑郁药(TCA)、单胺氧化酶抑制剂(MAOI)、五羟色胺(5-HT)再摄取抑制剂(SSRI)、五羟色胺和去甲 肾上腺素再摄取抑制剂(SNRI)、去甲肾上腺素特异性5-HT抗抑郁药(NaSSA)、5-HT受体拮抗和再摄取抑制剂(SARI)等。优选SSRI和SNRI。SSRI的代表药物有氟西汀、帕罗西汀、西酞普兰、舍曲林、氟伏沙明等。SNRI的代表药物有度洛西汀、文拉法辛、米那普仑等。
在另一优选例中，所述与TRPC4或/和TRPC5的功能、表达异常相关的疾病为神经退行性疾病。所述神经退行性疾病包括阿尔茨海默病(AD)、帕金森病、亨廷顿病、肌萎缩侧索硬化(ALS)、记忆障碍、遗忘症、失语症、慢性疲劳综合征、克雅氏病、解离性遗忘症、神游遗忘症、学习障碍、睡眠障碍以及由创伤或衰老引起的其他脑部病症。
在另一优选例中，所述与TRPC4或/和TRPC5的功能、表达异常相关的疾病为疼痛症。所述疼痛症包括伤害性疼痛、机械性疼痛、炎性疼痛、癌痛及神经性疼痛(例如骨关节炎疼痛、类风湿性关节炎疼痛、疱疹后神经痛、因烧伤引起的疼痛)。所述疼痛可为慢性疼痛或急性疼痛。
在另一优选例中，所述与TRPC4或/和TRPC5的功能、表达异常相关的疾病为癫痫。癫痫可由多种起源的兴奋性中毒引起，通常过度的神经元放电可驱动癫痫活性，降低相关神经元群体的超兴奋性的化合物在降低癫痫活性中具有显著潜能。抑制TRPC5介导的钙离子内流或可以降低超兴奋性并因此降低癫痫活性。
在另一优选例中，所述与TRPC4或/和TRPC5的功能、表达异常相关的疾病为肾脏疾病。所述肾脏疾病包括各种原因引起的极性肾损伤和慢性肾损伤，如毒性物质引起的肾损伤、病毒或细菌感染引起的肾损伤、高血压肾病、糖尿病肾病、肾小球肾炎、红斑狼疮肾炎、IgA肾病、肾病综合征、膜性肾病、微小病变肾病，还包括遗传性肾病，如多囊肾、局灶节段性肾小球硬化症等。
在另一优选例中，所述与TRPC4或/和TRPC5的功能、表达异常相关的疾病为皮肤疾病。进一步，所述皮肤疾病与角质形成细胞的功能异常有关，例如但不仅限于银屑病、鱼鳞病、掌跖角化症、Olmsted症、蟾皮症或绝经期角皮症。
在另一优选例中，所述与TRPC4或/和TRPC5的功能、表达异常相关的疾病为心血管系统相关的病症。所述疾病包括但不仅限于高血压、动脉粥样硬化、冠心病、心绞痛、心肌梗塞、心率失常、中风、肺动脉高压等。
在另一优选例中，所述与TRPC4或/和TRPC5的功能、表达异常相关的疾病为肝病。所述疾病包括但不仅限于胆汁淤积性肝病、酒精性脂肪肝炎、非酒精性脂肪肝炎、病毒性肝炎、自身免疫性肝病、化学性肝损伤、肝纤维化、肝硬化、肝细胞癌。
在另一优选例中，所述与TRPC4或/和TRPC5的功能、表达异常相关的疾病为癌症。所述疾病包括但不仅限于肾细胞癌、乳腺癌、结直肠癌等。
在另一优选例中，所述与TRPC4或/和TRPC5的功能、表达异常相关的疾病为肠道疾病。所述疾病包括但不仅限于溃疡性结肠炎、短肠综合征、肠易激综合征、肠痉挛、腹泻、腹痛等。
溶血卵磷脂能(LPC)能直接激活TRPC5，不依赖于其他间接的激活机制，如GPCR-PLC通路。LPC的一系列生物效应与其对TRPC5的激活有关，因此，TRPC5抑制 剂或能用于对抗与LPC水平失调有关的疾病。所述疾病包括但不仅限于动脉粥样硬化、心血管疾病、中枢和外周神经系统疾病、骨关节炎、肝炎、肝细胞癌、肺炎、肥胖、胃肠道疾病、细菌感染、寄生虫病、糖尿病、肿瘤、疼痛等。
第五方面，本发明提供本发明化合物、其立体异构体、其药学上可接受的盐的制备方法，所述方法选自如下路线A、路线B所示合成路线中的一种。
当本发明化合物具有亚通式(II-a)至(II-d)所示的结构时，可按照路线A所示的合成路线进行制备；其中，halo为卤素原子，R3a为C1-C6烷基，THP表示四氢吡喃保护基，其他取代基定义与前相同；
路线A
路线A的反应步骤包括：
(1.1)中间INT-A1(参考WO2014143799合成)与酮原料(商业购买)发生锂卤交换与亲核加成反应，生成中间体INT-A2；反应在超干非质子性溶剂中进行；所述溶剂例如但不仅限于二氯甲烷(DCM)、四氢呋喃(THF)，优先溶剂为四氢呋喃。反应温度一般为-40至-100℃，优选温度为-78℃；
(1.2)中间INT-A2与二乙胺基三氟化硫(DAST)进行氟化反应，反应温度一般为0℃至室温，生成中间体INT-A3与中间体INT-A4；反应在非质子性溶剂如DCM中进行；反应温度一般为0℃至室温；
(1.3)中间INT-A2与R3a-halo、NaH在四氢呋喃中进行烷基化反应，反应温度一般为室温，生成中间体INT-A5；
(1.4)中间体INT-A2、INT-A3、INT-A4、INT-A5脱除THP保护基生成通式(II-a)、(II-b)、(II-c)、(II-d)所示化合物；羟基的脱保护基反应通常在酸性试剂的作用下进行；所述酸性试剂例如但不仅限于三氟乙酸、氯化氢、对甲苯磺酸。
当本发明化合物具有亚通式(II-e)所示的结构时，可按照如下路线B进行制备；其中，halo为卤素原子，THP表示四氢吡喃保护基，其他取代基定义与前述相同；
路线B
路线B的反应步骤包括：
(2.1)中间INT-A1与硼酯类原料(商业购买或根据文献方法合成)发生Suzuki偶联反应，生成中间体INT-B1；Suzuki偶联反应通常在钯催化剂和碱的作用下进行；所述钯催化剂例如但不仅限于Pd(Ph3P)2Cl2、Pd(dppf)Cl2、Pd(Ph3P)4；所述碱例如但不仅限于三乙胺、DIPEA、K2CO3、Cs2CO3；反应在适当有机溶剂或有机溶剂与水混合溶剂中进行，如DMF或1,4-dioxane；反应温度一般为室温至100℃；
(2.2)中间INT-B1进行氢化还原反应，生成中间体INT-B2；还原反应在钯/碳的催化下进行；反应温度一般为室温；反应溶剂包括质子性溶剂和非质子性溶剂，例如但不仅限于甲醇、乙醇、乙酸乙酯、THF等。
(2.3)中间体INT-B2脱除THP保护基生成通式(II-e)所示化合物，羟基的脱保护基反应通常在酸性试剂的作用下进行；所述酸性试剂例如但不仅限于三氟乙酸、氯化氢、对甲苯磺酸。
本领域一般技术人员可根据文献资料或合成过程中遇到的实际情况对反应条件进行适当调整。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。限于篇幅，在此不再一一累述。
附图说明
图1示出化合物E21对TGF-β诱导的肝星状细胞活化(α-SMA标记)的影响；(A)显微镜下图像；(B)α-SMA阳性细胞比例统计；**，p<0.01；Mean±SEM。
图2示出大鼠口服10mg/kg化合物E68后的2h分布到各个组织中的药物浓度；Mean±SD，N＝3。
图3示出在大理石埋藏试验中，小鼠口服给予Fluoxetine(10mg/kg)、HC-070(1mg/kg)和E68(0.1、0.3、1mg/kg)前后埋藏弹珠数量的差异；ns，p>0.05；*，p<0.05；**，p<0.01；Mean±SEM，N＝12-13。
图4示出在悬尾试验中，小鼠口服给予Fluoxetine(10mg/kg)、HC-070(1mg/kg)和E68(0.1、0.3、1mg/kg)前后在悬尾箱中不动时间的差异；ns，p>0.05；*，p<0.05；**，p<0.01；Mean±SEM，N＝12-13。
图5示出在大理石埋藏试验中，小鼠口服给予HC-070(0.1、0.3mg/kg)和E68(0.03、0.1mg/kg)前后埋藏弹珠数量的差异；ns，p>0.05；**，p<0.01；Mean±SEM，N＝12-13。
图6示出在悬尾试验中，小鼠口服给予HC-070(0.1、0.3mg/kg)和E68(0.03、0.1mg/kg)前后在悬尾箱中不动时间的差异；ns，p>0.05；*，p<0.05；**，p<0.01；Mean±SEM，N＝12-13。
具体实施方式
本申请的发明人经过广泛而深入地研究，研发出一种黄嘌呤类化合物，8-位被“环烷基”取代，与HC-070相比，本发明化合物对TRPC4和TRPC5的抑制活性更强，而对hERG和TRPC3的抑制更弱，因此具有更好的选择性。在肝微粒体稳定性测试中，本发明化合物也表现出更好的代谢稳定性，因此具有更好的成药性质。此外，本发明中的一些化合物出乎意料地表现出显著的TRPC5激动活性，而非抑制活性。在此基础上，完成了本发明。
术语
在本发明中，除非特别指出，所用术语具有本领域技术人员公知的一般含义。
在本发明中，术语“C1-C6”是指具有1、2、3、4、5或6个碳原子，依此类推。“4-8元”是指具有4、5、6、7或8个成环原子，依此类推。
本发明中，所述“烷基”为具有特定碳原子个数的支链和直链的烃基团，代表性的例子包括但不仅限于甲基、乙基、正丙基、异丙基。
在本发明中，所述“烷氧基”表示-O-烷基。例如术语“C1-C6烷氧基”是指具有1至6个碳原子的直链或支链烷氧基，代表性的例子包括但不仅限于甲氧基、乙氧基、正丙氧基、异丙氧基和丁氧基等。
本发明中，所述“卤代烷基”和“卤代烷氧基”是指具有特定碳原子个数的“烷基”、“烷氧基”中的氢原子被“卤素原子”部分或完全取代所形成的基团。
本发明中，所述“环烃基”代表具有特定成环碳原子个数、非芳香性的环状脂肪烃基团，“C3-C10环烃基”代表由3至10个成环碳原子构成的环状脂肪烃基团；本发明所述的“环烃基”不仅包括单环脂肪烃基，还包括由多个环状脂肪烃构成的并环、螺环和桥环体 系；本发明所述的“环烃基”不仅包括碳原子达到完全饱和的脂肪烃基(即环烷基)，还包括部分碳原子具有不饱和键的脂肪烃基(如环烯基)，但不包括完全由不饱和碳构成的芳基；本发明所述的“环烃基”的例子包括但不仅限于：
本发明中，“芳基”定义为由特定个数碳原子构成、且遵守Hückel规则的单环和双环体系，包括苯基、萘基。
本发明中，“杂芳基”定义为具有特定个数成环原子、且含有1、2、3或4个杂原子(选自N、O、S)、同时遵守Hückel规则的单环和双环体系；“杂芳基”的例子包括但不仅限于吡啶、吡咯、咪唑、噻吩、苯并咪唑、苯并噻吩、苯并呋喃等。
本发明中，“杂环基”定义为具有特定个数成环原子、且含有1、2、3或4个杂原子(选自N、O、S)、饱和或部分不饱和的非芳族单环和双环体系；“杂环基”的例子包括但不仅限于氧杂环丁烷基、二氧杂环戊烷基、吗啉基、哌嗪基、吡咯烷基、哌啶基、十氢喹啉基、苯并四氢呋喃基等。
本发明中，所述“卤素原子”包括氟、氯、溴、碘。
本发明中所述的“取代”表示被一个或多个基团(如2、3、4或5个)所替代。如没有指明发生在特定的原子上，则表示可以发生在任何取代基个数尚未达到饱和的原子上。多个取代基从同一系列中选择时，它们可以相同，也可以不同。
本发明中所述的“任选地”表示所定义基团可从一系列候选基团中进行选择，也可以不选。
本发明所述“药学上可接受的盐”可以是阴离子与式(I)、(I-a)化合物或(I-b)化合物上带正电荷的基团形成的盐。合适的阴离子为氯离子、溴离子、碘离子、硫酸根、硝酸根、磷酸根、柠檬酸根、甲基磺酸根、三氟乙酸根、乙酸根、苹果酸根、甲苯磺酸根、酒石酸根、富马酸根、谷氨酸根、葡糖醛酸根、乳酸根、戊二酸根或马来酸根。类似地，可以由阳离子与式(I)化合物上的带负电荷的基团形成盐。合适的阳离子包括钠离子、钾离子、镁离子、钙离子和铵离子，例如四甲基铵离子。
在另一优选例中，“药学上可接受的盐”是指式(I)、(I-a)化合物或(I-b)化合物同选自下组的酸形成的盐类：氢氟酸、盐酸、氢溴酸、磷酸、乙酸、草酸、硫酸、硝酸、甲磺酸、胺基磺酸、水杨酸、三氟甲磺酸、萘磺酸、马来酸、柠檬酸、醋酸、乳酸、酒石酸、琥珀酸、酢浆草酸、丙酮酸、苹果酸、谷氨酸、对甲苯磺酸、萘磺酸、乙磺酸、萘二磺酸、丙二酸、富马酸、丙酸、草酸、三氟乙酸、硬酯酸、扑酸、羟基马来酸、苯乙酸、苯甲酸、谷氨酸、抗坏血酸、对胺基苯磺酸、2-乙酰氧基苯甲酸和羟乙磺酸等；或者式(I)、(I-a)化合物或(I-b)化合物与无机碱形成的钠盐、钾盐、钙盐、铝盐或铵盐；或者通式(I)、(I-a)化合物或(I-b)化合物与有机碱形成的甲胺盐、乙胺盐或乙醇胺盐。
本发明通式(I)、(I-a)化合物或(I-b)化合物或其药学上可接受的盐是从水或有机溶剂中蒸馏析出、结晶或重结晶而来，化合物中可能包含所使用的溶剂分子。此外，不同的结晶条件可能导致化合物的晶型不同。因此，含有不同化学剂量的结晶溶剂以及所有晶型的通式(I)、(I-a)所示的化合物或(I-b)化合物或其药学上可接受的盐都在本发明的范围内。
本发明中，所述“有效治疗剂量”表示与没有接受该剂量治疗的对象相比，接受该剂量治疗的对象在病变或副作用等得到治愈、改善、有效预防或者其发生率显著降低；此外，它还包括增强正常生理功能的有效剂量。
本发明中，术语“调节剂”包含抑制剂与激动剂。术语“抑制剂”还可用“拮抗剂”或“阻断剂”来表述，在本发明中具有同等含义，是指可用于降低或抑制生物活性的化合物或混合物。术语“激动剂”还可用“激活剂”来表述，在本发明中具有同等含义，是指可用于提高生物活性的药物分子或混合物。
TRPC家族蛋白不仅以纯合四聚体的形式存在，还常以杂合四聚体的形式存在。以TRPC5为例，它不仅构成TRPC5:C5纯合通道，还构成TRPC4:C5、TRPC1:C5、TRPC1:C4:C5等杂合通道。因此，本发明所述的TRPC5抑制剂或TRPC5激动剂不仅表示抑制或激动TRPC5纯合通道，也表示抑制或激动TRPC5杂合通道。类似地，对TPRC4也具有该含义。
本发明通式(I)、(I-a)、(I-b)所示的一些化合物具有手性中心、潜手性中心或不饱和键，可形成多种形式的立体异构体，例如外消旋体、对映异构体、非对映异构体、E/Z异构体、顺反异构体、互变异构体等。除非另行说明，否则在说明书及随附权利要求书中，给定的化学式或名称意图涵盖所有形式的立体异构体、以及由不同比例的单独异构体组成的混合物、及其药学上可接受的盐。本领域的一般技术人员可采用实验室常用的分离方法对本发明中含有不对称中心的化合物进行分离以得到单一异构体，但这并不能破坏本发明化合物的新颖性。
将氢原子替换为氘原子以改变化合物的理化性质已成为本领域一般技术人员公知的结构改造方法。除非另行说明，本发明意图将通式(I)、(I-a)、(I-b)所示化合物的氘代形式也包含在发明内容中。
以下将以实施例进一步说明本发明。需要特别指出的是，这些实施例只用于举例说明本发明，而不以任何方式限制本发明。实例中的所有参数及其余说明，除另加说明外，都是以质量为依据的。柱层析分离所用填料若未说明均为硅胶。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。
缩略语：DCM：二氯甲烷；DAST：二乙胺基三氟化硫；DMAP：4-二甲氨基吡啶；DMF：N,N-二甲基甲酰胺；DMSO：二甲基亚砜；TFA：三氟乙酸；THF：四氢呋喃。
一、制备实施例
实施例E1:5-(4-氯苄基)-6-(环戊-1-烯-1-基)-3-(3-羟丙基)-1-甲基-1,5-二氢-2H-吡咯并[3,2-d]嘧啶-2,4(3H)-二酮
6-氯尿嘧啶(10g，68.493mmol)溶于40mL DMSO中，依次加入K2CO3(4.8g，34.783mmol)、CH3I(12.7mL，205.48mmol)，室温搅拌3h。TLC显示反应完毕，加入50mL H2O，抽滤，滤饼水洗后为得7.9g白色固体，即中间体INT 1-1。
中间体INT 1-1(5g，31.25mmol)溶于10mL DMF中，加入K2CO3(8.65g，62.5mmol)、3-溴丙基甲基醚(5.35mL，46.875mmol)，60℃加热3h。TLC显示反应完毕，EA/饱和NaCl水溶液分配，分出有机相，无水硫酸钠干燥后以快速硅胶色谱柱进行分离，以DCM/MeOH为流动相进行洗脱，收集目标产物部，蒸干得4g胶状物，即中间体INT1-2。
中间体INT 1-2(1g，4.31mmol)溶于3mL浓硫酸中，0℃下搅拌5min，缓慢滴加3mL浓硝酸，0℃搅拌1h。TLC显示反应完毕，EA/冰水分配，有机层用饱和食盐水洗四次，无水硫酸钠干燥后以快速硅胶色谱柱进行分离，以DCM/MeOH为流动相进行洗脱，收集目标产物部，蒸干得800mg胶状物，即中间体INT 1-3。
丙二酸二乙酯(3.24mL，21.66mmol)溶于10mL干燥的1，4-二氧六环，加入叔丁醇钾(2.16g，19.494mmol)，室温搅拌10min，再加入中间体INT 1-3(3g，10.83mmol)，室温搅拌1h。TLC显示反应完毕，2M HCl调至酸性，EA/饱和NaCl水溶液分配，分出有机相，无水硫酸钠干燥后以快速硅胶色谱柱进行分离，以DCM/MeOH为流动相进行洗脱，收集目标产物部，蒸干得3.2g胶状物，即中间体INT 1-4。
中间体INT 1-4(3g，7.463mmol)溶于15mL乙酸中，升温至60℃，加入锌粉(7.3g，111.94mmol)，120℃耐压管中反应12h。TLC显示反应完毕，减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸 干得1.8g胶状物，即中间体INT 1-5。
中间体INT 1-5(100mg，7.463mmol)溶于3mL干燥的1，4-二氧六环，氮气保护下加入三溴氧磷(340mg，22.389mmol)，100℃搅拌1h。TLC显示反应完毕，EA/饱和NaHCO3水溶液分配，分出有机相，无水硫酸钠干燥后以快速硅胶色谱柱进行分离，以DCM/MeOH为流动相进行洗脱，收集目标产物部，蒸干得90mg胶状物，即中间体INT 1-6。
中间体INT 1-6(90mg，0.286mmol)溶于5mL DMF中，加入K2CO3(1.73g，12.5mmol)、4-氯苄溴(116mg，0.572mmol)，60℃加热2h。TLC显示反应完毕，EA/饱和NaCl水溶液分配，分出有机相，无水硫酸钠干燥后以快速硅胶色谱柱进行分离，以DCM/MeOH为流动相进行洗脱，收集目标产物部，蒸干得80mg白色固体，即中间体INT 1-7。
中间体INT 1-7(40mg，0.091mmol)溶于3mL干燥DCM，0℃下加入182μL浓度为1M的BBr3溶液，该温度下搅拌1h。TLC显示反应完毕，减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得30mg白色固体，即中间体INT 1-8。
INT 1-8(25mg，0.049mmol)、1-环戊烯硼酸(13.7mg，0.122mmol)、Pd(dppf)Cl2(5.38mg，0.007mmol)、Cs2CO3(48mg，0.147mmol)、1,4-二氧六环10mL以及H2O 3mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于100℃加热2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得25mg胶状物。该粗品以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得15mg白色固体，即实施例E1。1H NMR(500MHz,Chloroform-d)δ7.31–7.28(m,2H),6.96(d,J＝8.5Hz,2H),5.98(s,1H),5.90–5.88(m,1H),5.74(s,2H),4.18(t,J＝5Hz,2H),3.52(s,3H),3.51–3.49(m,2H),2.76–2.64(m,2H),2.56–2.51(m,2H),1.99(p,J＝7.5Hz,2H),1.93–1.86(m,2H).MS(ESI):m/z 414.1[M+H]+.
实施例E2:5-(4-氯苄基)-6-环戊基-3-(3-羟丙基)-1-甲基-1,5-二氢-2H-吡咯并[3,2-d]嘧啶-2,4(3H)-二酮
E1(30mg，0.072mmol)溶于3ml甲醇中，加入10mg Pd/C，氢气置换后室温反应2h。抽滤后滤液浓缩至干，以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得20mg白色固体，即实施例E2。1H NMR(500MHz,Chloroform-d)δ7.30–7.28(m,2H),6.94(d,J＝8.5Hz,2H),5.89(s,1H),5.66–5.64(m,2H),4.19–4.16(m,2H),3.58–3.53(m,2H),3.52–3.49(m,3H),2.96(h,J＝8.1Hz,1H),2.03–1.94(m,2H),1.94–1.87(m,2H),1.85–1.77(m,2H),1.72–1.51(m,4H).MS(ESI):m/z 416.2[M+H]+.
实施例E3:5-(4-氯苄基)-6-(2-环戊基环丙基)-3-(3-羟丙基)-1-甲基-1,5-二氢-2H-吡咯并[3,2-d]嘧啶-2,4(3H)-二酮
联硼酸频那醇酯(2.026g，7.976mmol)、Cu(36mg，0.571mmol)、MeONa(62mg，1.142mmol)依次溶于EtOH，氮气保护后加入环戊基乙炔(500mg，5.319mmol)，室温搅拌12h。TLC显示反应完毕，EA/饱和NH4Cl水溶液分配，分出有机相，无水硫酸钠干燥后以快速硅胶色谱柱进行分离，以PE/EA为流动相进行洗脱，收集目标产物部，蒸干得350mg胶状物，即中间体INT 2-1。
KOH(1.5g，26.78mmol)溶于3ml H2O，加入3ml乙醚，0℃下加入甲基亚硝基脲(250mg，2.427mmol)，搅拌5min后将有机层转移至含INT 2-1(50mg，0.225mmol)、Pd(OAc)2(20mg，0.089mmol)的乙醚溶液中。0℃搅拌5min，TLC显示反应完全。过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以PE/EA为流动相进行洗脱，收集目标产物部，蒸干得30mg胶状物，即中间体INT 2-2。
INT 1-8(25mg，0.049mmol)、INT 2-2(25mg，0.106mmol)、Pd(dppf)Cl2(5.38mg，0.007mmol)、Cs2CO3(48mg，0.147mmol)、1,4-二氧六环10mL以及H2O 3mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于100℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得20mg胶状物。该粗品以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得15mg白色固体，即实施例E3。1H NMR(500MHz,Chloroform-d)δ7.31–7.29(m,2H),7.02(d,J＝8.5Hz,2H),5.83–5.72(m,1H),5.71–5.52(m,2H),4.21–4.15(m,2H),3.58–3.51(m,2H),3.49–3.43(m,3H),1.94–1.87(m,2H),1.78–1.70(m,2H),1.68–1.61(m,2H),1.59–1.51(m,2H),1.49(dt,J＝9.1,4.8Hz,1H),1.41(p,J＝8.0Hz,1H),1.34–1.19(m,2H),1.15–1.00(m,1H),0.93–0.84(m,2H).MS(ESI):m/z 456.2[M+H]+.
实施例E4：7-(4-氯苄基)-8-(2-环戊基环丙基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
8-溴-3-甲基-3,7-二氢-嘌呤-2,6-二酮(10g，40.816mmol)溶于30ml DMF中，加入KHCO3(6.18g，61.8mmol)、4-氯苄溴(8.37g，40.829mmol)，60℃搅拌2h。TLC显示反应完毕，加入100ml H2O，抽滤，滤饼烘干后得14.1g白色固体，即中间体INT 4-1。
INT 4-1(14.1g，38.3mmol)溶于30ml DMF中，加入K2CO3(10.6g，76.8mmol)、2-(3-溴丙氧基)四氢-2H-吡喃(7.8ml，45.978mmol)，60℃搅拌2h。TLC显示反应完毕，加入100ml H2O，抽滤，滤饼烘干后得20.3g白色固体，即中间体INT 4-2。
INT 4-2(100mg，0.196mmol)、INT 2-2(92.5mg，0.392mmol)、Pd(dppf)Cl2(14.3mg，0.019mmol)、Cs2CO3(191.1mg，0.588mmol)、1,4-二氧六环10mL以及H2O 3mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于100℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得80mg胶状物，即中间体INT4-3。
INT 4-3(80mg，0.148mmol)溶于2mL DCM，然后加入0.4mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得50mg白色固体，即实施例E4。1H NMR(500MHz,Chloroform-d)δ7.35(d,J＝8.5Hz,2H),7.19(d,J＝8.5Hz,2H),5.61(d,J＝4.5Hz,2H),4.19(t,J＝5Hz,2H),3.55(s,3H),3.52(t,J＝5.5Hz,2H),2.08–1.99(m,1H),1.93–1.86(m,2H),1.81–1.72(m,1H),1.71–1.60(m,4H),1.58–1.51(m,2H),1.50–1.39(m,2H),1.25–1.16(m,1H),1.03–0.98(m,1H),0.95–0.88(m,1H).MS(ESI):m/z 457.2[M+H]+.
实施例E5：5-(4-氯苄基)-3-(3-羟丙基)-1-甲基-6-(4-苯基环己基)-1,5-二氢-2H-吡咯并[3,2-d]嘧啶-2,4(3H)-二酮
4-苯基环己酮(870mg，5mmol)溶于超干DCM，加入NaCO3搅拌10min，0℃下逐滴加入Tf2O(1.55g，5.5mmol)，室温搅拌3h。TLC显示反应完全。加入5ml MeOH， 反应液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以PE/EA为流动相进行洗脱，收集目标产物部，蒸干得1g无色油状液体，即中间体INT 5-1。
INT 5-1(300mg，0.98mmol)、联硼酸频那醇酯(498mg，1.96mmol)、Pd(dppf)Cl2(71mg，0.097mmol)、KOAc(288mg，2.939mmol)、1,4-二氧六环10mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于80℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以PE/EA为流动相进行洗脱，收集目标产物部，蒸干得260mg胶状物，即中间体INT 5-2。
INT 1-8(25mg，0.049mmol)、INT 5-2(25mg，0.088mmol)、Pd(dppf)Cl2(5.38mg，0.007mmol)、Cs2CO3(48mg，0.147mmol)、1,4-二氧六环10mL以及H2O 3mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于100℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得20mg胶状物，即中间体INT 5-3。
INT 5-3(20mg，0.04mmol)溶于3ml甲醇中，加入5mg Pd/C，氢气置换后室温反应2h。抽滤后滤液浓缩至干，以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得15mg白色固体，即实施例E5。1H NMR(500MHz,Chloroform-d)δ7.38–7.34(m,1H),7.34–7.29(m,4H),7.27–7.20(m,2H),7.01–6.90(m,2H),5.93–5.89(m,1H),5.71–5.64(m,2H),4.23–4.13(m,2H),3.54–3.51(m,2H),3.49(s,3H),2.94–2.86(m,1H),2.68–2.56(m,1H),2.14–1.98(m,2H),1.96–1.81(m,4H),1.81–1.74(m,2H),1.70–1.62(m,2H).MS(ESI):m/z 506.2[M+H]+.
实施例E6：7-(4-氯苄基)-1-(3-羟丙基)-3-甲基-8-(1,2,3,6-四氢-[1,1'-联苯]-4-基)-3,7-二氢-1H-嘌呤-2,6-二酮
INT 4-2(100mg，0.196mmol)、INT 5-2(111.3mg，0.392mmol)、Pd(dppf)Cl2(14.3mg，0.019mmol)、Cs2CO3(191.1mg，0.588mmol)、1,4-二氧六环10mL以及H2O 3mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于100℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得100mg胶状物，即中间体INT 6-1。
INT 6-1(100mg，0.17mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系 为流动相进行洗脱，收集目标产物部，蒸干得80mg白色固体，即实施例E6。1H NMR(500MHz,Chloroform-d)δ7.40–7.32(m,3H),7.30–7.29(m,2H),7.27–7.25(m,2H),7.09(d,J＝8.0Hz,2H),6.24-6.20(m,1H),5.62(s,2H),4.23–4.16(m,2H),3.66–3.63(m,3H),3.56–3.52(m,2H),2.92–2.85(m,1H),2.66–2.46(m,3H),2.42–2.33(m,1H),2.19–2.08(m,1H),2.02-1.92(m,3H).MS(ESI):m/z 505.2[M+H]+.
实施例E7：7-(4-氯苄基)-1-(3-羟丙基)-8-(4-异丙氧基环己基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
4-异丙氧基环己酮(250mg，1.6mmol)溶于超干DCM，加入NaCO3搅拌10min，0℃下逐滴加入Tf2O(497mg，1.76mmol)，室温搅拌3h。TLC显示反应完全。加入5ml MeOH，反应液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以PE/EA为流动相进行洗脱，收集目标产物部，蒸干得200mg无色油状液体，即中间体INT 7-1。
INT 7-1(190mg，0.66mmol)、联硼酸频那醇酯(335mg，1.319mmol)、Pd(dppf)Cl2(50mg，0.068mmol)、KOAc(241mg，1.975mmol)、1,4-二氧六环10mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于80℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以PE/EA为流动相进行洗脱，收集目标产物部，蒸干得150mg胶状物，即中间体INT 7-2。
INT 4-2(100mg，0.196mmol)、INT 7-2(104.3mg，0.392mmol)、Pd(dppf)Cl2(14.3mg，0.019mmol)、Cs2CO3(191.1mg，0.588mmol)、1,4-二氧六环10mL以及H2O 3mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于100℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得120mg胶状物，即中间体INT 7-3。
INT 7-3(100mg，0.175mmol)溶于5ml甲醇中，加入20mg Pd/C，氢气置换后室温反应2h。抽滤后滤液浓缩至干，得80mg胶状物，即中间体INT 7-4。
INT 7-4(80mg，0.14mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得55mg白色固体，即实施例E7。1H NMR(500MHz,DMSO-d6)δ7.42(d,J＝8.5Hz,2H),7.21(d,J＝8.5Hz,2H),5.60(s,2H),4.49– 4.45(m,1H),3.95–3.85(m,2H),3.64–3.57(m,2H),3.50-3.43(m,4H),2.97–2.80(m,1H),1.87–1.73(m,4H),1.72–1.63(m,2H),1.50–1.40(m,2H),1.35–1.28(m,2H),1.08(d,J＝6.1Hz,6H).MS(ESI):m/z 489.2[M+H]+.
实施例E8：7-(4-氯苄基)-1-(3-羟丙基)-8-(4-异丙氧基环己-1-烯-1-基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
INT 7-3(40mg，0.07mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得25mg白色固体，即实施例E8。1H NMR(500MHz,Chloroform-d)δ7.32(d,J＝8.5Hz,2H),7.15(d,J＝8.5Hz,2H),6.03–5.99(m,1H),5.58(s,2H),4.20(t,J＝6.0Hz,2H),3.84–3.69(m,2H),3.62(s,3H),3.56–3.50(m,2H),3.50–3.45(m,1H),2.62–2.37(m,3H),2.34–2.16(m,1H),2.01–1.88(m,3H),1.86–1.78(m,1H),1.23–1.18(m,6H).MS(ESI):m/z 487.2[M+H]+.
实施例E9：8-([1,1'-双(环丙烷)]-2-基)-7-(4-氯苄基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
KOH(1.5g，26.78mmol)溶于3ml H2O，加入3ml乙醚，0℃下加入甲基亚硝基脲(250mg，2.427mmol)，搅拌5min后将有机层转移至(E)-2-环丙基乙烯基硼酸频那醇酯(44mg，0.225mmol)、Pd(OAc)2(20mg，0.089mmol)的乙醚溶液中。0℃搅拌5min，TLC显示反应完全。过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以PE/EA为流动相进行洗脱，收集目标产物部，蒸干得35mg胶状物，即中间体INT 9-1。
INT 4-2(100mg，0.196mmol)、INT 9-1(81.5mg，0.392mmol)、Pd(dppf)Cl2(14.3mg，0.019mmol)、Cs2CO3(191.1mg，0.588mmol)、1,4-二氧六环10mL以及H2O 3mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于100℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得100mg胶状物，即中间体INT 9-2。
INT 9-2(100mg，0.195mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得70mg白色固体，即实施例E9。1H NMR(500MHz,Chloroform-d)δ7.35(d,J＝8.5Hz,2H),7.24(d,J＝8.5Hz,2H),5.71–5.50(m,2H),4.24–4.13(m,2H),3.62–3.55(m,1H),3.54(s,3H),3.53–3.49(m,2H),1.93–1.86(m,2H),1.66(dt,J＝8.8,4.7Hz,1H),1.52-1.47(m,1H),1.37(dt,J＝9.1,4.7Hz,1H),1.02–0.88(m,1H),0.86–0.79(m,1H),0.57–0.47(m,1H),0.46–0.35(m,1H),0.22–0.06(m,2H).MS(ESI):m/z 429.2[M+H]+.
实施例E10：7-(4-氯苄基)-8-(4,4-二甲基环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
INT 4-2(100mg，0.196mmol)、4,4-(二甲基环己烯-1-基)硼酸频那醇酯(92.5mg，0.392mmol)、Pd(dppf)Cl2(14.3mg，0.019mmol)、Cs2CO3(191.1mg，0.588mmol)、1,4-二氧六环10mL以及H2O 3mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于100℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得100mg胶状物，即中间体INT 10-1。
INT 10-1(100mg，0.185mmol)溶于5ml甲醇中，加入20mg Pd/C，氢气置换后室温反应2h。抽滤后滤液浓缩至干，得80mg胶状物，即中间体INT 10-2。
INT 7-4(80mg，0.15mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得60mg白色固体，即实施例E10。1H NMR(500MHz,Chloroform-d)δ7.34(d,J＝8.5Hz,2H),7.12(d,J＝8.5Hz,2H),5.54(s,2H),4.20(t,J＝6.0Hz,2H),3.63(s,3H),3.58–3.51(m,2H),2.59(tt,J＝11.9,3.5Hz,1H),1.95–1.83(m,4H),1.51(td,J＝7.5,3.6Hz,4H),1.30–1.19(m,2H),1.02(s,3H),0.97(s,3H).MS(ESI):m/z 459.2[M+H]+.
实施例E11:7-(4-氯苄基)-8-(4,4-二甲基环己-1-烯-1-基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
INT 7-3(50mg，0.093mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得25mg白色固体，即实施例E11。1H NMR(500MHz,Chloroform-d)δ7.32(d,J＝8.5Hz,2H),7.05(d,J＝8.5Hz,2H),6.05–6.02(m,1H),5.59(s,2H),4.23–4.14(m,2H),3.64(s,3H),3.52(t,J＝5.5Hz,2H),2.42(dt,J＝6.3,3.7Hz,2H),2.02–1.99(m,2H),1.93–1.86(m,2H),1.53(t,J＝6.4Hz,2H),0.98(s,6H).MS(ESI):m/z 457.2[M+H]+.
实施例E12:7-(4-氯苄基)-1-(3-羟丙基)-3-甲基-8-(4-(三氟甲基)环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E12的合成方法同E10，除了以4-(三氟甲基)-1-环己烯-1-硼酸频哪醇酯代替4,4-(二甲基环己烯-1-基)硼酸频那醇酯为原料。1H NMR(500MHz,Chloroform-d)δ7.34(d,J＝8.5Hz,2H),7.08(d,J＝8.5Hz,2H),5.56(s,2H),4.21(t,J＝6.0Hz,2H),3.63(s,3H),3.56–3.52(m,2H),3.52–3.47(m,1H),3.07–3.02(m,1H),2.29–2.12(m,3H),1.95–1.87(m,4H),1.76–1.65(m,4H).MS(ESI):m/z 499.2[M+H]+.
实施例E13:7-(4-氯苄基)-1-(3-羟丙基)-3-甲基-8-(1,4-二氧杂环[4.5]癸-8-基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E13的合成方法同E10，除了以1,4-二氧杂-螺[4,5]癸-7-烯-8-硼酸频哪醇酯代替4,4-(二甲基环己烯-1-基)硼酸频那醇酯为原料。1H NMR(500MHz,Chloroform-d)δ7.34(d,J＝8.5Hz,2H),7.11(d,J＝8.5Hz,2H),5.56(s,2H),4.20(t,J＝6.0Hz,2H),4.00(d,J＝1.7Hz,4H),3.61(s,3H),3.53(t,J＝5.5Hz,2H),2.72(tt,J＝11.6,3.6Hz,1H),2.04(qd,J＝13.4,3.6Hz,2H),1.94–1.85(m,4H),1.73–1.66(m,2H),1.58(td,J＝13.4,4.1Hz,2H).MS(ESI):m/z 489.2[M+H]+.
实施例E14:8-([1,1'-双(环丙烷)]-2-基)-7-(4-氯苄基)-1-(3-羟丙基)-3-异丙基-3,7-二氢-1H-嘌呤-2,6-二酮
8-溴黄嘌呤(2g，8.658mmol)溶于10ml DMF中，加入KHCO3(1.298g，12.987mmol)、4-氯苄溴(1.77g，8.658mmol)，60℃搅拌2h。TLC显示反应完毕，加入10ml H2O，抽滤，滤饼烘干后得2.5g白色固体，即中间体INT 14-1。
INT 14-1(1g，2.825mmol)溶于10ml DMF中，加入K2CO3(428mg，3.1mmol)、2-碘丙烷(480mg，2.825mmol)，40℃搅拌2h。TLC显示反应完毕，加入10ml H2O，抽滤，滤饼烘干后得400mg白色固体，即中间体INT 14-2
INT 14-2(350mg，0.883mmol)溶于5ml DMF中，加入K2CO3(244mg，1.768mmol)、2-(3-溴丙氧基)四氢-2H-吡喃(236.5mg，1.06mmol)，60℃搅拌2h。TLC显示反应完毕。反应液倒入饱和NaCl水溶液中，用乙酸乙酯萃取3遍，合并有机相，再用饱和NaCl水溶液洗涤3遍。无水MgSO4干燥后用快速硅胶色谱柱分离，MeOH/DCM为流动相进行洗脱，收集目标产物部得250mg无色油状液体，得到中间体INT 14-3。
INT 14-3(100mg，0.185mmol)、INT 9-1(81.5mg，0.392mmol)、Pd(dppf)Cl2(14.3mg，0.019mmol)、Cs2CO3(191.1mg，0.588mmol)、1,4-二氧六环10mL以及H2O 3mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于100℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得60mg胶状物，即中间体INT 14-4。
INT 14-4(60mg，0.111mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得30mg白色固体，即实施例E14。1H NMR(500MHz,Chloroform-d)δ7.36(d,J＝8.5Hz,2H),7.27(d,J＝8.3Hz,2H),5.73–5.42(m,2H),5.20–5.10(m,1H),4.18(t,J＝5.9Hz,2H),3.70(t,J＝7.2Hz,1H),3.57–3.48(m,2H),1.91–1.83(m,2H),1.66(dt,J＝8.8,4.7Hz,1H),1.55(d,J＝6.9Hz,6H),1.52–1.47(m,1H),1.36(dt,J＝9.1,4.7Hz,1H),0.97–0.79(m,2H),0.57–0.38(m,2H),0.19–0.08(m,2H).MS(ESI):m/z 457.2[M+H]+.
实施例E15:7-(4-氯苄基)-8-(2-环戊基环丙基)-1-(3-羟丙基)-3-异丙基-3,7-二氢-1H-嘌呤-2,6-二酮
INT 14-2(100mg，0.185mmol)、INT 9-1(81.5mg，0.392mmol)、Pd(dppf)Cl2(14.3mg，0.019mmol)、Cs2CO3(191.1mg，0.588mmol)、1,4-二氧六环10mL以及H2O 3mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于100℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得80mg胶状物，即中间体INT 15-1。
INT 15-1(80mg，0.141mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得50mg白色固体，即实施例E15。1H NMR(500MHz,Chloroform-d)δ7.35(d,J＝8.5Hz,2H),7.23(d,J＝8.3Hz,2H),5.20–5.11(m,1H),4.17(t,J＝5.9Hz,2H),3.70(t,J＝7.2Hz,1H),3.55–3.47(m,2H),1.93–1.86(m,2H),1.81–1.74(m,1H),1.71–1.61(m,5H),1.56(d,J＝6.8,6H),1.49–1.38(m,3H),1.36–1.28(m,2H),1.26–1.17(m,1H),1.03–0.96(m,1H).MS(ESI):m/z 485.2[M+H]+.
实施例E16:7-(4-氯苄基)-8-环戊基-1-(3-甲氧基丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
INT 1-6(100mg，0.227mmol)、1-环戊烯硼酸频哪醇酯(88.1mg，0.454mmol)、Pd(dppf)Cl2(14.3mg，0.019mmol)、Cs2CO3(191.1mg，0.588mmol)、1,4-二氧六环10mL以及H2O 3mL置于耐压管中，以氮气脱气3min，将耐压管密封，并于100℃加热搅拌2h。冷却后，以乙酸乙酯稀释，过滤除去不溶物，滤液减压蒸干得粗品。然后以快 速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得80mg胶状物，即中间体INT 16-1。
INT 16-1(80mg，0.187mmol)溶于5ml甲醇中，加入15mg Pd/C，氢气置换后室温反应2h。抽滤后滤液浓缩至干，得80mg胶状物，以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得50mg白色固体，即实施例E16。1H NMR(500MHz,Chloroform-d)δ7.28(d,J＝9Hz,2H),6.95(d,J＝8.3Hz,2H),5.84(s,1H),5.68(s,2H),4.09(t,J＝7.65Hz,2H),3.49(s,3H),3.45(t,J＝6.5Hz,2H),3.32(s,3H),2.95(p,J＝8.1Hz,1H),2.00–1.90(m,,4H),1.85–1.77(m,2H),1.69–1.59(m,4H).MS(ESI):m/z 431.2[M+H]+.
实施例E17:7-(4-氯苄基)-8-(1-氟-3-苯基环丁基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
INT 4-2(250mg，0.49mmol)溶于5ml超干四氢呋喃中，0℃下逐滴加入异丙基氯化镁氯化锂络合物(490μL，0.735mmol)，搅拌10min，加入3-苯基环丁酮(130μL，0.98mmol)，0℃搅拌2h，LC-MS显示反应完毕，减压蒸除溶剂。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得100mg胶状物，即中间体INT 17-1。
中间体INT 17-1(100mg，0.173mmol)溶于3ml超干DCM，0℃下逐滴加入DAST(113μL，0.865mmol)，0℃下搅拌30min。LC-MS显示反应完毕，蒸干得80mg胶状物，即中间体INT 17-2。
INT 17-2(80mg，0.138mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得50mg白色固体，即实施例E17。1H NMR(500MHz,Chloroform-d)δ7.41–7.35(m,3H),7.34–7.30(m,4H),7.21(d,J＝8.2Hz,2H),5.68(s,2H),4.23(t,J＝6.0Hz,2H),3.68(s,3H),3.60–3.52(m,2H),3.27(p,J＝8.9Hz,1H),3.16–3.08(m,2H),2.79–2.66(m,2H),1.97–1.88(m,2H).MS(ESI):m/z 497.2[M+H]+.
实施例E18:(E)-7-(4-氯苄基)-1-(3-羟丙基)-3-甲基-2,6-二氧-2,3,6,7-四氢-1H-嘌呤-8-甲醛-O-(叔丁基)肟
INT 4-2(250mg，0.49mmol)溶于5ml超干四氢呋喃中，-78℃下逐滴加入2.5M正丁基锂(235μL，0.588mmol)，搅拌30min，加入超干DMF(57μL，0.735mmol)，-78℃搅拌2h，LC-MS显示反应完毕，减压蒸除溶剂。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得80mg胶状物，即中间体INT 18-1。
中间体INT 18-1(80mg，0.174mmol)、O-叔丁基羟胺盐酸盐(65.6mg，0.522mmol)溶于3ml DCM，搅拌12h。TLC显示反应完毕，减压蒸除溶剂。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得40mg胶状物，即中间体INT 18-2。
NT 17-2(40mg，0.075mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得30mg白色固体，即实施例E18。1H NMR(500MHz,Chloroform-d)δ8.22(s,1H),7.38–7.34(m,2H),7.22(d,J＝8.5Hz,2H),6.03(s,1H),4.28–4.20(m,2H),3.67(s,3H),3.61–3.54(m,2H),3.48–3.25(m,1H),2.07–1.83(m,2H),1.31(s,9H).MS(ESI):m/z 448.2[M+H]+.
实施例E19:7-(4-氯苄基)-8-(1-氟-4-异丙氧基环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E19的合成方法同E17，除了以4-异丙氧基环己酮代替3-苯基环丁酮为原料。1H NMR(500MHz,Chloroform-d)δ7.34–7.31(m,2H),7.20–7.09(m,2H),5.76(s,2H),4.21–4.13(m,2H),3.80–3.64(m,2H),3.64–3.56(m,3H),3.55–3.37(m,2H),2.50–2.28(m,2H),2.18–2.00(m,4H),1.97–1.79(m,4H),1.22–1.14(ms,6H).MS(ESI):m/z 507.2[M+H]+.
实施例E20:7-(4-氯苄基)-1-(3-羟丙基)-8-(1H-茚-2-基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E20的合成方法同E6，除了以1H-茚-2-硼酸频哪醇酯代替4,4,5,5-四甲基-2-(1,2,3,6-四氢-[1,1'-联苯]-4-基)-1,3,2-二氧杂硼烷为原料。1H NMR(500MHz,Chloroform-d)δ7.58–7.52(m,1H),7.47–7.42(m,1H),7.39–7.32(m,4H),7.25(s,1H),7.11(d,J＝8.5Hz,2H),5.86(s,2H),4.20(t,J＝6.0Hz,2H),4.02(s,2H),3.70(s,3H),3.54(t,J＝5.4Hz,2H),1.96–1.88(m,2H).MS(ESI):m/z 463.2[M+H]+.
实施例21:7-(4-氯苄基)-8-(1-氟-4-(三氟甲基)环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
INT 4-2(250mg，0.49mmol)溶于5ml超干四氢呋喃中，-78℃下逐滴加入2.5M正丁基锂(235μL，0.588mmol)，搅拌30min，加入4-(三氟甲基)环己酮(122mg，0.735mmol)，-78℃搅拌2h，LC-MS显示反应完毕，减压蒸除溶剂。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得120mg胶状物，即中间体INT 21-1。
中间体INT 21-1(120mg，0.201mmol)溶于3ml超干DCM，0℃下逐滴加入DAST(53μL，0.402mmol)，0℃下搅拌30min。LC-MS显示反应完毕，蒸干得80mg胶状物，即中间体INT 21-2。
INT 21-2(80mg，0.133mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得50mg白色固体，即实施例E21。1H NMR(500MHz,Chloroform-d,3:4 isomer)δ7.30/7.29(2×d,J＝7Hz,2H),7.15/7.09(2×d,J＝8.2Hz,2H),5.76/5.75(2×s,2H),4.22–4.16(m,2H),3.58/3.61(2×s,3H),3.56–3.50(m,2H),3.42–3.24(m,1H),2.46–2.36(m,1H),2.31–2.17(m,1H),2.17–2.08(m,1H),2.07–1.85(m,7H),1.82–1.71(m,1H).MS(ESI):m/z 517.2[M+H]+.
实施例E22:7-(4-氯苄基)-1-(3-羟丙基)-8-(1-甲氧基-4-(三氟甲基)环己基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
中间体INT 21-1(120mg，0.201mmol)溶于5ml超干THF中，加入NaH(16.08mg，0.402mmol)、CH3I(38μL，0.603mmol)，35℃下反应12h。TLC显示反应完毕，减压蒸除溶剂。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得80mg胶状物，即中间体INT 22-1。
INT 22-1(80mg，0.133mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得50mg白色固体，即实施例E22。1H NMR(500MHz,Chloroform-d)δ7.31(d,J＝8.5Hz,2H),6.98(d,J＝8.5Hz,2H),5.87(s,2H),4.21–4.09(m,2H),3.63(s,3H),3.54–3.49(m,2H),3.32(t,J＝7.1Hz,1H),3.00(s,3H),2.23–2.19(m,1H),1.92–1.78(m,6H),1.67(td,J＝12.4,3.6Hz,4H).MS(ESI):m/z 529.2[M+H]+.
实施例E23:7-(4-氯苄基)-8-(1-羟基-4-(三氟甲基)环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
INT 21-1(40mg，0.067mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得20mg白色固体，即实施例E23。1H NMR(500MHz,Chloroform-d)δ7.29(d,J＝8.5Hz,2H),7.01(d,J＝8.5Hz,2H),5.93(s,2H),4.18(t,J＝6.0Hz,2H),3.61(s,3H),3.51–3.46(m,2H),3.38(t,J＝7.1Hz,1H),2.17–2.04(m,3H),1.98–1.92(m,2H),1.90–1.83(m,4H),1.81–1.69(m,2H).MS(ESI):m/z 515.2[M+H]+.
实施例E24:7-(4-氯苄基)-8-(1-氟-4,4-二甲基环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E24的合成方法同E21，除了以4,4-二甲基环己酮代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d)δ7.28(d,J＝8.5Hz,2H),7.12(d,J＝8.5Hz,2H),5.76(s,2H),4.19(t,J＝6.0Hz,2H),3.61(s,3H),3.52(t,J＝5.5Hz,2H),2.26(td,J＝14.1,4.4Hz,2H),1.93–1.86(m,4H),1.64(dd,J＝13.7,4.0Hz,2H),1.38(d,J＝13.7Hz,2H),1.02(d,J＝6.0Hz,6H).MS(ESI):m/z 477.2[M+H]+.
实施例E25:7-(4-氯苄基)-8-(1-羟基-4,4-二甲基环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
INT 24-1(40mg，0.072mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得20mg白色固体，即实施例E25。1H NMR(500MHz,Chloroform-d)δ7.30(d,J＝8Hz,2H),7.04(d,J＝8.5Hz,2H),5.92(s,2H),4.16(t,J＝6.0Hz,2H),3.62(s,3H),3.54–3.47(m,2H),3.46–3.41(m,1H),2.23(td,J＝13.7,4.2Hz,2H),1.91–1.83(m,2H),1.71(d,J＝14.2Hz,2H),1.57(td,J＝13.5,3.9Hz,2H),1.41–1.29(m,2H),1.00(s,6H).MS(ESI):m/z 475.2[M+H]+.
实施例E26:7-(4-氯苄基)-1-(3-羟丙基)-8-(1-甲氧基-4,4-二甲基环己基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E26的合成方法同E22，除了以INT 24-1代替INT 21-1为原料。1H NMR(500MHz,Chloroform-d)δ7.30(d,J＝8.6Hz,2H),6.97(d,J＝9Hz,2H,2H),5.88(s,2H),4.27–4.03(m,2H),3.65(s,3H),3.56–3.48(m,2H),2.97(s,3H),2.03–1.97(m,2H),1.94–1.85(m,4H),1.50(td,J＝13.0,3.8Hz,2H),1.31–1.23(m,2H),0.97(d,J＝3.7Hz,6H).MS(ESI):m/z 489.2[M+H]+.
实施例E27:(E)-8-(1-(叔丁氧基亚胺)乙基)-7-(4-氯苄基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
INT 4-2(200mg，0.392mmol)溶于5ml 1,4-二氧六环中，N2保护下加入三丁基(1-乙氧基乙烯)锡(267μL，0.784mmol)、Pd(PPh3)4(45mg，0.039mmol)，110℃搅拌12h。TLC显示反应完毕，减压蒸除溶剂。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得180mg胶状物，即中间体INT27-1。
INT 27-1(180mg，0.358mmol)溶于3ml甲醇中，0℃下滴加1ml 1N HCl，室温搅拌12h。TLC显示反应完毕，减压蒸除溶剂。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得140mg胶状物，即中间体INT27-2。
INT 27-2(140mg，0.295mmol)、O-叔丁基羟胺盐酸盐(74.3mg，0.59mmol)溶于3ml DCM，搅拌12h。TLC显示反应完毕，减压蒸除溶剂。然后以快速硅胶色谱柱进行分离，以MeOH/DCM为流动相进行洗脱，收集目标产物部，蒸干得90mg胶状物，即中间体INT 18-2。
NT 17-2(90mg，0.165mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系为流动相进行洗脱，收集目标产物部，蒸干得50mg白色固体，即实施例E27。1H NMR(500MHz,Chloroform-d)δ7.28(d,J＝6.5Hz,2H),7.10(d,J＝8.5Hz,2H),6.00(s,2H),4.17(t,J＝6.0Hz,2H),3.64(s,3H),3.51(t,J＝5.6Hz,2H),2.38(s,3H),1.91–1.83(m,2H),1.21(s,9H).MS(ESI):m/z 462.2[M+H]+.
实施例E28:7-(4-氯苄基)-8-(2-氟-6,6-二甲基双环[3.1.1]庚烷-2-基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E28的合成方法同E21，除了以(1R,5S)-6,6-二甲基双环[3.1.1]庚烷-2-酮代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d)δ7.33(d,J＝8.5Hz,2H),7.07(d,J＝8.5Hz,2H),5.69–5.50(m,2H),4.33–3.97(m,2H),3.63(s,3H),3.55–3.49(m,2H),2.64–2.58(m,1H),2.41–2.29(m,2H),2.06–2.00(m,4H),1.92–1.84(m,3H),0.96–0.86(m,6H).MS(ESI):m/z 489.2[M+H]+.
实施例E29:7-(4-氯苄基)-8-(2-氟-6,6-二甲基双环[3.1.1]庚烷-2-基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
中间体INT 28-1(80mg，0.14mmol)溶于3ml超干DCM，0℃下逐滴加入DAST(37μL，0.28mmol)，0℃下搅拌30min。LC-MS显示反应完毕，蒸干得20mg胶状物，即中间体INT 29-1。
INT 29-1(20mg，0.036mmol)溶于4mL DCM，然后加入1mL TFA，室温搅拌1h，TLC显示反应完毕。减压蒸除溶剂，剩余物以制备液相色谱柱进行纯化，以乙腈/水体系 为流动相进行洗脱，收集目标产物部，蒸干得10mg白色固体，即实施例E29。1H NMR(500MHz,Chloroform-d)δ7.33(d,J＝8.5Hz,2H),7.11–6.92(m,2H),6.18–6.05(m,1H),5.58(s,2H),4.21–4.15(m,2H),3.63(s,3H),3.55–3.44m,2H),2.64–2.51(m,1H),2.44–2.33(m,1H),2.29–2.21(m,1H),2.07–2.22(m,1H),1.95–1.83(m,2H),1.81–1.77(m,2H),0.98–0.81(m,6H).MS(ESI):m/z 469.2[M+H]+.
实施例E30:7-(4-氯苄基)-8-(4-乙基-1-氟环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E30的合成方法同E21，除了以4-乙基环己酮代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d,3:1isomer)δ7.31(d,J＝8.5Hz,2H),7.16/7.11(2×d,J＝8.2Hz,2H),5.76/5.75(2×s,2H),4.21–4.15(m,2H),3.59/3.62(2×s,3H),3.52(t,J＝6.1Hz,2H),3.45–3.36(m,1H),2.15–1.99(m,4H),1.93–1.86(m,3H),1.78–1.72(m,2H),1.37–1.30(m,4H),0.96–0.89(m,3H).MS(ESI):m/z 477.2[M+H]+.
实施例E31:7-(4-氯苄基)-8-(1-氟-4-异丙基环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E31的合成方法同E21，除了以4-异丙基环己酮代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d,3:1 isomer)δ7.31(d,J＝8.5Hz,2H),7.16/7.10(2×d,J＝8.2Hz,2H),5.76/5.75(2×s,2H),4.21–4.13(m,2H),3.59/3.62(2×s,3H),3.55–3.46(m,2H),2.17–1.98(m,4H),1.94–1.84(m,2H),1.75–1.67(m,2H),1.55–1.48(m,1H),1.47–1.39(m,1H),1.38–1.31(m,2H),0.92(2×d,J＝7Hz,6H).MS(ESI):m/z 491.2[M+H]+.
实施例E32:7-(4-氯苄基)-8-(1-氟-4-甲氧基环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E32的合成方法同E21，除了以4-甲氧基环己酮代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d)δ7.31(d,J＝8.5Hz,2H),7.14(d,J＝8.4Hz,2H),5.76(s,2H),4.18(t,J＝6Hz,2H),3.61(s,3H),3.56(s,1H),3.54–3.49(m,2H),3.42–3.39(m,1H),3.38(s,3H),2.46–2.29(m,2H),2.08–1.90(m,4H),1.88–1.78(m,4H).MS(ESI):m/z 479.2[M+H]+.
实施例E33:7-(4-氯苄基)-8-(1-羟基-4-甲氧基环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E33的合成方法同E25，除了以INT 32-1代替INT 24-1为原料。1H NMR(500MHz,Chloroform-d)δ7.31(d,J＝8.5Hz,2H),7.05(d,J＝8.5Hz,2H),5.93(s,2H),4.16(t,J＝6.0Hz,2H),3.61(s,3H),3.53–3.47(m,2H),3.39(s,3H),3.32–3.25(m,1H),2.14(td,J＝13.5,3.9Hz,2H),2.06–2.03(m,1H),2.01–1.97(m,1H),1.97–1.90(m,2H),1.91–1.83(m,2H),1.70–1.64(m,2H).MS(ESI):m/z 477.2[M+H]+.
实施例E34:7-(4-氯苄基)-8-(1-氟-4,4-二甲基环己-2-烯-1-基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E34的合成方法同E21，除了以4,4-二甲基-2-环己基-1-酮代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d)δ7.33(d,J＝8.5Hz,2H),7.06(d,J＝8.5Hz,2H),6.00(d,J＝3.4Hz,1H),5.64(d,J＝3.2Hz2H),4.74–4.64(m,1H),4.20(t,J＝6Hz,2H),3.63(s,3H),3.57–3.51(m,2H),3.42(t,J＝7.1Hz,1H),2.54–2.33(m,2H),2.07–2.04(m,2H),1.91–1.83(m,2H),1.03(s,3H),0.94(s,3H).MS(ESI):m/z 475.2[M+H]+.
实施例E35:7-(4-氯苄基)-8-(1-羟基-4,4-二甲基环己-2-烯-1-基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E35的合成方法同E25，除了以INT 34-1代替INT 24-1为原料。1H NMR(500MHz,Chloroform-d)δ7.32(d,J＝8.5Hz,2H),7.06(d,J＝8.5Hz,2H),6.01–5.98(m,1H),5.64(d,J＝3.2Hz,2H),5.42–5.30(m,1H),4.19(t,J＝5.5Hz,2H),3.97–3.93(m,1H),3.63(s,3H),3.55–3.50(m,2H),3.42(t,J＝7Hz,1H),2.38–2.43(m,2H),2.28–2.20(m,1H),2.07–2.01(m,1H),1.91–1.83(m,2H),1.03(s,3H),0.94(s,3H).MS(ESI):m/z 473.2[M+H]+.
实施例E36:7-(4-氯苄基)-8-(4,4-二氟环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E36的合成方法同E10，除了以2-(4,4-二氟环己-1-烯-1-基)硼酸频那醇酯代替4,4-(二甲基环己烯-1-基)硼酸频那醇酯为原料。1H NMR(500MHz,Chloroform-d)δ7.35(d,J＝8.5Hz,2H),7.10(d,J＝8.5Hz,2H),5.58(s,2H),4.20(t,J＝6.0Hz,2H),3.62(s,3H),3.57–3.51(m,2H),3.49–3.45(m,1H),2.82–2.73(m,1H),2.32–2.20(m,2H),2.10–2.02(m,2H),1.93–1.86(m,2H),1.86–1.72(m,4H).MS(ESI):m/z 467.2[M+H]+.
实施例E37:7-(4-氯苄基)-8-(4,4-二氟环己-1-烯-1-基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E37的合成方法同E25，除了以INT 36-1代替INT 24-1为原料。1H NMR(500MHz,Chloroform-d)δ7.33(d,J＝8.5Hz,2H),7.09(d,J＝8.1Hz,2H),5.91(s,1H),5.59(s,2H),4.21(t,J＝6.0Hz,2H),3.62(s,3H),3.56–3.51(m,2H),3.43–3.34(m,1H),2.82–2.61(m,4H),2.27–2.15(m,2H),1.93–1.86(m,2H).MS(ESI):m/z 465.1[M+H]+.
实施例E38:7-(4-氯苄基)-1-(3-羟丙基)-3-甲基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E38的合成方法同E21，除了以4,4-二氟环已酮代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d)δ7.33(d,J＝8.5Hz,2H),7.14(d,J＝8.2Hz,2H),5.78(s,2H),4.20(t,J＝6.0Hz,2H),3.60(s,3H),3.54(t,J＝5.6Hz,2H),2.49–2.33(m,2H),2.22–2.09(m,6H),1.93–1.86(m,2H).MS(ESI):m/z 485.2[M+H]+.
实施例E39:7-(4-氯苄基)-8-(1-氟-4-甲基环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E39的合成方法同E21，除了以4-甲基环己酮代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d,8:1 isomer)δ7.31(d,J＝8.5Hz,2H),7.10/7.16(2×d,J＝8.2Hz,2H),5.75/5.76(2×s,2H),4.26–4.08(m,2H),3.62/3.60(2×s,3H),3.51(t,J＝5.5Hz,2H),3.45–3.27(m,1H),2.41–2.24(m,2H),2.12–1.97(m,1H),1.93–1.75(m,6H),1.52–1.42(m,2H),1.02(d,J＝6.7Hz,3H).MS(ESI):m/z 463.2[M+H]+.
实施例E40:4-(7-(4-氯苄基)-1-(3-羟丙基)-3-甲基-2,6-二氧-2,3,6,7-四氢-1H-嘌呤-8-基)-4-氟环己烷-1-腈
实施例E40的合成方法同E21，除了以4-氧代环己烷甲腈代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d,7:10 isomer)δ7.33/7.31(2×d,J＝8.6Hz,2H),7.14/7.12(2×d,J＝8.2Hz,2H),5.76(s,2H),4.19(t,J＝6.0Hz,2H),3.58/3.62(2×s,3H),3.53(t,J＝5.5Hz,2H),3.06(p,J＝3.9Hz,1H),2.52–2.36(m,1H),2.18–2.04(m,5H),2.03–1.99(m,2H).1.91–1.83(m,2H).MS(ESI):m/z 474.2[M+H]+.
实施例E41:7-(4-氯苄基)-1-(3-羟丙基)-3-甲基-8-(4-(三氟甲基)环己-1-烯-1-基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E41的合成方法同E29，除了以INT 21-1代替INT 28-1为原料。1H NMR(500MHz,Chloroform-d)δ7.33(d,J＝8.5Hz,2H),7.03(d,J＝8.5Hz,2H),6.08–6.04(m,1H),5.65–5.47(m,2H),4.18(t,J＝6.0Hz,2H),3.62(s,3H),3.52(t,J＝5.5Hz,2H),3.42–3.34(m,1H),2.67–2.59(m,1H),2.54–2.44(m,1H),2.44–2.24(m,3H),2.20–2.12(m,1H),1.91–1.83(m,2H),1.75–1.70(m,1H).MS(ESI):m/z 497.15[M+H]+.
实施例E42：7-(4-氯苄基)-1-(3-羟丙基)-3-异丙基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E42的合成方法同E38，除了以INT 14-3代替INT 4-2为原料。1H NMR(500MHz,Chloroform-d)δ7.33(d,J＝8.5Hz,2H),7.17(d,J＝8.2Hz,2H),5.77(s,2H),5.19(hept,J＝6.8Hz,1H),4.17(t,J＝6.0Hz,2H),3.57–3.48(m,2H),3.45–3.36(m,1H),2.54–2.24(m,2H),2.21–2.03(m,6H),1.91–1.83(m,2H)),1.59(d,J＝7.0Hz,6H).MS(ESI):m/z 513.2[M+H]+.
实施例E43：7-(4-氯-3-氟苄基)-1-(3-羟丙基)-3-甲基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E43的合成方法同E38，除了以INT 43-3代替INT 4-2为原料。其中，INT 43-3的合成方法同INT 4-2，除了以4-氯-3-氟溴苄代替4-氯苄溴为原料。1H NMR(500MHz,Chloroform-d)δ7.41–7.34(m,2H),7.01–6.90(m,2H),5.76(s,2H),4.19(t,J＝6.0Hz,2H),3.59(s,3H),3.56–3.48(m,2H),3.28(s,1H),2.55–2.32(m,2H),2.25–2.08(m,6H),1.92–1.84(m,2H).MS(ESI):m/z 503.1[M+H]+.
实施例E44：7-(3,4-二氟苯基)-1-(3-羟丙基)-3-甲基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E44的合成方法同E43，除了以3,4-二氟苄溴代替4-氯-3-氟溴苄为原料。1H NMR(500MHz,Chloroform-d)δ7.16–7.09(m,1H),7.03(ddd,J＝10.5,7.5,2.3Hz,1H),6.99–6.95(m,1H),5.72(s,2H),4.17(t,J＝6.5Hz,2H),3.57(s,3H),3.55–3.47(m,2H),3.28(s,1H),2.49–2.31(m,2H),2.23–2.05(m,6H),1.93–1.84(m,2H).MS(ESI):m/z 487.2[M+H]+.
实施例E45：7-(4-氟苄基)-1-(3-羟丙基)-3-甲基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E45的合成方法同E43，除了以4-氟溴苄代替4-氯-3-氟溴苄为原料。1H NMR(500MHz,Chloroform-d)δ7.23–7.13(m,2H),7.04–6.97(m,2H),5.75(s,2H),4.17(t,J＝6Hz,2H),3.57(s,3H),3.53–3.46(m,2H),3.33(s,1H),2.47–2.27(m,2H),2.19–2.04(m,6H),1.92–1.83(m,2H).MS(ESI):m/z 469.2[M+H]+.
实施例E46：7-(4-氟苄基)-1-(3-羟丙基)-3-异丙基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E46的合成方法同E45，除了以2-碘代丙烷代替碘甲烷为原料。1H NMR(500MHz,Chloroform-d)δ7.24–7.18(m,2H),7.06–6.98(m,2H),5.75(s,2H),5.16(h,J＝6.9Hz,1H),4.16(t,J＝6.0Hz,2H),3.54–3.47(m,2H),3.40(t,J＝7.1Hz,1H),2.46–2.27(m,2H),2.20–2.06(m,6H),1.91–1.83(m,2H),1.56(d,J＝7.0Hz,6H).MS(ESI):m/z 497.2[M+H]+.
实施例E47：7-(3,4-二氟苯基)-1-(3-羟丙基)-3-异丙基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E47的合成方法同E46，除了以INT 47-2代替INT 46-2为原料。1H NMR(500MHz,Chloroform-d)δ7.19–7.12(m,1H),7.07(ddd,J＝10.6,7.5,2.2Hz,1H),7.04–6.99(m,1H),5.74(s,2H),5.19(hept,J＝6.9Hz,1H),4.17(t,J＝6Hz,2H),3.53(t,J＝5.5Hz,2H),3.40(s,1H),2.50–2.31(m,2H),2.28–2.07(m,6H),1.94–1.84(m,2H),1.59(d,J＝6.9Hz,6H).MS(ESI):m/z 515.2[M+H]+.
实施例E48：7-(4-氯苄基)-8-(4-(二甲氨基)-1-氟环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E48的合成方法同E21，除了以4-二甲氨基环己酮代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d)δ7.28(d,J＝8.5Hz,2H),7.09(d,J＝8.4Hz,2H),5.74(s,2H),4.15(t,J＝6.0Hz,2H),3.59(s,3H),3.52–3.46(m,2H),3.36(s,1H),2.48–2.32(m,2H),2.27(s,6H),2.22–2.16(m,1H),1.94–1.79(m,8H).MS(ESI):m/z 492.2[M+H]+.
实施例E49：7-(4-氯苄基)-8-(4-(二甲氨基)环己-1-烯-1-基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E49的合成方法同E29，除了以INT 48-1代替INT 28-1为原料。1H NMR(500MHz,Chloroform-d)δ7.32(d,J＝8.5Hz,2H),6.99(d,J＝8.3Hz,2H),5.98–5.94(m,1H),5.66–5.46(m,2H),4.15(t,J＝6.1Hz,2H),3.59(s,3H),3.50(t,J＝5.5Hz,3H),3.44–3.33(m,1H),2.89–2.81(m,6H),2.73–2.63(m,1H),2.57–2.42(m,5H),2.35–2.27(m,1H),1.91–1.81(m,2H).MS(ESI):m/z 472.2[M+H]+.
实施例E50：7-(4-氯苄基)-1-(3-羟丙基)-3-甲基-8-(4-吗啉环己-1-烯-1-基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E50的合成方法同E6，除了以4-(4-(4,4,5,5-四甲基-1,3,2-二噁硼烷-2-基)-3-环己烯)吗啉代替4,4,5,5-四甲基-2-(1,2,3,6-四氢-[1,1'-联苯]-4-基)-1,3,2-二氧杂硼烷为原料。1H NMR(500MHz,Chloroform-d)δ7.30(d,J＝8.5Hz,2H),7.05(d,J＝8.3Hz,2H),6.05–6.01(m,1H),5.61–5.47(m,2H),4.20–4.12(m,2H),3.78–3.72(m,4H),3.60(s,3H),3.54–3.46(m,2H),3.41(t,J＝7.1Hz,1H),2.66–2.50(m,6H),2.45–2.30(m,2H),2.27–2.17(m,1H),2.14–2.07(m,1H),1.91–1.84(m,2H),1.57–1.53(m,1H).MS(ESI):m/z 514.2[M+H]+.
实施例E51：7-(4-氯-3-氟苄基)-3-乙基-8-(1-氟-4-(三氟甲基)环己基)-1-(3-羟丙基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E51的合成方法同E43，除了以2-碘丙烷代替碘乙烷为原料。1H NMR(500MHz,Chloroform-d,5:11 isomer)δ7.41–7.34(m,1H),7.03–6.90(m,2H),5.75/5.73(2×s,2H),4.28–4.07(m,4H),3.60–3.46(m,2H),2.49–2.39(m,1H),2.35–2.09(m,2H),2.07–1.82(m,7H),1.81–1.74(m,1H),1.37/1.36(2×t,J＝7Hz,3H).MS(ESI):m/z 549.2[M+H]+.
实施例E52：7-(3,4-二氟苯基)-8-(1-氟-4-(三氟甲基)环己基)-1-(3-羟丙基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E52的合成方法同E21，除了以INT 44-3代替INT 4-2为原料。1H NMR(500MHz,Chloroform-d,9:13 isomer)δ7.16–7.08(m,1H),7.07–6.88(m,2H),5.70/5.72(2×s,2H),4.17/4.16(2×t,J＝7.5Hz,2H),3.57(2×s,3H),3.54–3.47(m,2H),3.35–3.26(m,1H),2.46–2.36(m,1H),2.32–2.19(m,1H),2.18–2.11(m,1H),2.05–1.82(m,7H),1.82–1.71(m,1H).MS(ESI):m/z 519.2[M+H]+.
实施例E53：7-(4-氯苄基)-8-(1-氟-4-(三氟甲基)环己基)-1-(3-羟丙基)-3-异丙基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E53的合成方法同E42，除了以4-(三氟甲基)环己酮代替4,4-二氟环已酮为原 料。1H NMR(500MHz,Chloroform-d,8:1 isomer)δ7.32(d,J＝8.5Hz,2H),7.19/7.13(2×d,J＝8.2Hz,2H),5.76(s,2H),5.25–5.12(m,1H),4.17/4.16(2×t,J＝6Hz,2H),3.58–3.49(m,2H),3.48–3.39(m,1H),2.32–2.11(m,4H),2.11–2.00(m,1H),2.00–1.92(m,2H),1.91–1.85(m,2H),1.84–1.72(m,2H),1.58(d,J＝7.0Hz,6H).MS(ESI):m/z 545.2[M+H]+.
实施例E54：7-(4-氯苄基)-8-(1-氟-4-苯基环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E54的合成方法同E21，除了以4-苯基环己酮代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d,3:1 isomer)δ7.38–7.30(m,4H),7.28–7.22(m,3H),7.19/7.09(2×d,J＝8.2Hz,2H),5.79/5.77(2×s,2H),4.22–4.15(m,2H),3.61/3.65(2×s,3H),3.56–3.49(m,2H),2.83–2.64(m,1H),2.51–2.43(m,1H),2.35–2.24(m,1H),2.22–2.15(m,2H),1.99–1.86(m,6H).MS(ESI):m/z 525.2[M+H]+.
实施例E55：7-(4-氯苄基)-8-(1-氟-4-(吡咯烷-1-基)环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E55的合成方法同E21，除了以4-(1-吡咯烷基)环己酮代替4-(三氟甲基)环己酮为原料。1H NMR(500MHz,Chloroform-d)δ7.31(d,J＝8.5Hz,2H),7.08(d,J＝8.2Hz,2H),5.73(s,2H),4.17(t,J＝5.9Hz,2H),3.60(s,3H),3.51(t,J＝5.5Hz,2H),3.09–2.99(m,1H),2.87–2.75(m,2H),2.64–2.51(m,2H),2.35–2.27(m,2H),2.21–2.11(m,4H),2.09–2.01(m,4H),1.99–1.92(m,2H),1.93–1.85(m,2H).MS(ESI):m/z 518.2[M+H]+.
实施例E56：7-(4-氯-3-氟苄基)-1-(3-羟丙基)-3-异丙基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E56的合成方法同E47，除了以INT 51-1代替INT 44-1为原料。1H NMR(500MHz,Chloroform-d)δ7.39–7.35(m,1H),7.05–6.95(m,2H),5.75(s,2H),5.19(hept,J＝7.0Hz,1H),4.17(t,J＝6.0Hz,2H),3.53(t,J＝5.5Hz,2H),2.51–2.30(m,2H),2.24–2.11(m,6H),1.93–1.86(m,2H),1.59(d,J＝6.9Hz,6H).MS(ESI):m/z 531.2[M+H]+.
实施例E57：7-(4-氯-3-氟苄基)-3-乙基-1-(3-羟丙基)-8-(4-(三氟甲基)环己-1-烯-1-基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E57的合成方法同E29，除了以INT 51-4代替INT 28-1为原料。1H NMR(500MHz,Chloroform-d)δ7.42–7.37(m,1H),6.88(ddd,J＝12.3,8.9,2.1Hz,2H),6.05(s,1H),5.63–5.49(m,2H),4.28–4.09(m,4H),3.52(t,J＝5.5Hz,2H),2.72–2.63(m,1H),2.55–2.26(m,4H),2.23–2.13(m,1H),1.96–1.83(m,3H),1.39(t,J＝7.0Hz,3H).MS(ESI):m/z529.2[M+H]+.
实施例E58：7-(3,4-二氟苯基)-1-(3-羟丙基)-3-甲基-8-(4-(三氟甲基)环己-1-烯-1-基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E58的合成方法同E29，除了以INT 52-1代替INT 28-1为原料。1H NMR(500MHz,Chloroform-d)δ7.21–7.12(m,1H),6.92(ddd,J＝10.3,7.4,2.3Hz,1H),6.88–6.83(m,1H),6.10–6.04(m,1H),5.64–5.49(m,2H),4.27–4.12(m,2H),3.62(s,3H),3.57–3.47(m,2H),3.38(t,J＝7.0Hz,1H),2.69–2.61(m,1H),2.56–2.27(m,4H),2.23–2.13(m,1H),1.94–1.86(m,3H).MS(ESI):m/z 499.2[M+H]+.
实施例E59：8-(1-氟-4-(三氟甲基)环己基)-7-(4-氟苄基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E59的合成方法同E45，除了以4-(三氟甲基)环己酮代替4,4-二氟环已酮为原料。1H NMR(500MHz,Chloroform-d,3:1 isomer)δ7.23/7.16(2×m,2H),7.06–7.00(m,2H),5.77/5.75(2×s,2H),4.23–4.15(m,2H),3.58/3.61(2×s,3H),3.56–3.49(m,2H),3.39–3.30(m,1H),2.48–2.35(m,1H),2.31–2.09(m,3H),2.08–2.01(m,1H),2.00–1.85(m,5H),1.83–1.74(m,1H).MS(ESI):m/z 501.2[M+H]+.
实施例E60：1-(3-羟丙基)-3-甲基-8-(1,4,4-三氟环己基)-7-(4-(三氟甲基)苄基)-3,7-二氢-1H-嘌呤-2,6-二酮
8-溴-3-甲基-3,7-二氢-嘌呤-2,6-二酮(2g，8.13mmol)溶于10ml DMF中，加入KHCO3(1.298g，12.987mmol)、4-(三氟甲基)苄基溴(1.8g，7.531mmol)，60℃搅拌2h。TLC显示反应完毕，加入10ml H2O，抽滤，滤饼烘干后得2.5g白色固体，即中间体INT 60-1。INT 60-1(350mg，0.87mmol)溶于5ml DMF中，加入K2CO3(244mg，1.768mmol)、2-(3-溴丙氧基)四氢-2H-吡喃(236.5mg，1.06mmol)，60℃搅拌2h。TLC显示反应完毕。反应液倒入饱和NaCl水溶液中，用乙酸乙酯萃取3遍，合并有机相，再用饱和NaCl水溶液洗涤3遍。无水MgSO4干燥后用快速硅胶色谱柱分离，MeOH/DCM为流动相进行洗脱，收集目标产物部得300mg无色油状液体，得到中间体INT 60-2。
实施例E60的后续合成方法同E38，除了以INT 60-2代替4-2为原料。1H NMR(500MHz,Chloroform-d)δ7.59(d,J＝8.1Hz,2H),7.29–7.24(m,2H),5.84(s,2H),4.15(t,J＝6.0Hz,2H),3.58(s,3H),3.53–3.48(m,2H),3.26(s,1H),2.51–2.31(m,2H),2.22–2.06(m,6H),1.91–1.83(m,2H).MS(ESI):m/z 519.2[M+H]+.
实施例E61：7-(3,5-二氟苄基)-1-(3-羟丙基)-3-甲基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E61的合成方法同E60，除了以3,5-二氟溴苄代替4-(三氟甲基)苄基溴为原料。1H NMR(500MHz,Chloroform-d)δ6.74(tt,J＝2.3,8.7Hz,1H),6.70–6.64(m,2H),5.75(s,2H),4.16(t,J＝6.1Hz,2H),3.58(s,3H),3.54–3.48(m,2H),3.21(t,J＝7.0Hz,1H),2.51–2.32(m,2H),2.21–2.06(m,6H),1.93–1.83(m,2H).MS(ESI):m/z 487.2[M+H]+.
实施例E62：7-苄基-1-(3-羟丙基)-3-甲基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E62的合成方法同E60，除了以溴化苄代替4-(三氟甲基)苄基溴为原料。1H NMR(500MHz,Chloroform-d)δ7.36–7.28(m,3H),7.18–7.11(m,2H),5.80(s,2H),4.17(t,J＝6.0Hz,2H),3.57(s,3H),3.54–3.48(m,2H),2.43–2.26(m,2H),2.17–2.03(m,6H),1.91–1.84(m,2H).MS(ESI):m/z 451.2[M+H]+.
实施例E63：7-苄基-8-(1-氟-4-(三氟甲基)环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E63的合成方法同E62，除了以4-(三氟甲基)环己酮代替4,4-二氟环已酮为原料。1H NMR(500MHz,Chloroform-d)δ7.36–7.28(m,3H),7.13–7.06(m,2H),5.79(s,2H),4.15(t,J＝6.0Hz,2H),3.60(s,3H),3.52–3.45(m,2H),3.33(t,J＝7.1Hz,1H),2.45–2.34(m,2H),2.29–2.18(m,1H),1.99–1.91(m,4H),1.89–1.82(m,4H).MS(ESI):m/z 483.2[M+H]+.
实施例E64：7-(2,4-二氟苄基)-1-(3-羟丙基)-3-甲基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E64的合成方法同E60，除了以2,4-二氟溴苄代替4-(三氟甲基)苄基溴为原料。1H NMR(500MHz,Chloroform-d)δ6.89–6.84(m,1H),6.83–6.73(m,2H),5.82(s,2H),4.16(t,J＝6.0Hz,2H),3.58(s,3H),3.53–3.47(m,2H),2.47–2.30(m,2H),2.19–2.04(m,6H),1.91–1.83(m,2H).MS(ESI):m/z 487.2[M+H]+.
实施例E65：7-(2,4-二氟苄基)-8-(1-氟-4-(三氟甲基)环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E65的合成方法同E64，除了以4-(三氟甲基)环己酮代替4,4-二氟环已酮为原料。1H NMR(500MHz,Chloroform-d)δ6.89–6.83(m,1H),6.82–6.75(m,1H),6.72–6.64(m,1H),5.79(s,2H),4.14(t,J＝6.0Hz,2H),3.60(s,3H),3.52–3.44(m,2H),2.45–2.33(m,2H),2.31–2.21(m,1H),2.02–1.82(m,8H).MS(ESI):m/z 519.2[M+H]+.
实施例E66：7-(2-氯-4-氟苄基)-1-(3-羟丙基)-3-甲基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E66的合成方法同E60，除了以2-氯-4-氟溴苄代替4-(三氟甲基)苄基溴为原料。1H NMR(500MHz,Chloroform-d)δ7.19(dd,J＝2.6,8.2Hz,1H),6.92–6.85(m,1H),6.47–6.42(m,1H),5.82(s,2H),4.13(t,J＝6.0Hz,2H),3.60(s,3H),3.52–3.45(m,2H),2.50–2.31(m,2H),2.21–2.05(m,6H),1.89–1.82(m,2H).MS(ESI):m/z 503.1[M+H]+.
实施例E67：7-(2-氯-4-氟苄基)-8-(1-氟-4-(三氟甲基)环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E67的合成方法同E66，除了以4-(三氟甲基)环己酮代替4,4-二氟环已酮为原料。1H NMR(500MHz,Chloroform-d)δ7.19(dd,J＝2.6,8.2Hz,1H),6.92–6.84(m,1H),6.47–6.42(m,1H),5.80(s,2H),4.12(t,J＝6.0Hz,2H),3.62(s,3H),3.52–3.44(m,2H),3.17(s,1H),2.48–2.37(m,2H),2.30–2.19(m,1H),2.01–1.88(m,6H),1.87–1.82(m,2H).MS(ESI):m/z 535.1[M+H]+.
实施例E68：7-(3-氯-4-氟苄基)-1-(3-羟丙基)-3-甲基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E68的合成方法同E60，除了以3-氯-4-氟溴苄代替4-(三氟甲基)苄基溴为原料。1H NMR(500MHz,Chloroform-d)δ7.29–7.26(m,1H),7.10(d,J＝6.9Hz,2H),5.72(s,2H),4.17(t,J＝6.0Hz,2H),3.57(s,3H),3.54–3.47(m,2H),2.49–2.32(m,2H),2.24–2.07(m,6H),1.92–1.84(m,2H).MS(ESI):m/z 503.1[M+H]+.
实施例E69：7-(3-氯-4-氟苄基)-8-(1-氟-4-(三氟甲基)环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E69的合成方法同E68，除了以4-(三氟甲基)环己酮代替4,4-二氟环已酮为原料。1H NMR(500MHz,Chloroform-d)δ7.25–7.21(m,1H),7.13–7.04(m,2H),5.69(s,2H),4.16(t,J＝6.0Hz,2H),3.59(s,3H),3.53–3.47(m,2H),3.25(t,J＝7.0Hz,1H),2.48–2.35(m,2H),2.32–2.21(m,1H),2.11–1.96(m,4H),1.94–1.83(m,4H).MS(ESI):m/z 535.1[M+H]+.
实施例E70：7-(4-氯-2-氟苄基)-1-(3-羟丙基)-3-甲基-8-(1,4,4-三氟环己基)-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E70的合成方法同E60，除了以4-氯-2-氟苄溴代替4-(三氟甲基)苄基溴为原料。1H NMR(500MHz,Chloroform-d)δ7.17–7.12(m,1H),7.08–7.03(m,1H),6.71–6.65(m,1H),5.82(s,2H),4.14(t,J＝6.0Hz,2H),3.58(s,3H),3.53–3.47(m,2H),2.48–2.31(m,2H),2.19–2.05(m,6H),1.90–1.82(m,2H).MS(ESI):m/z 503.1[M+H]+.
实施例E71：7-(4-氯-2-氟苄基)-8-(1-氟-4-(三氟甲基)环己基)-1-(3-羟丙基)-3-甲基-3,7-二氢-1H-嘌呤-2,6-二酮
实施例E71的合成方法同E70，除了以4-(三氟甲基)环己酮代替4,4-二氟环已酮为原料。1H NMR(500MHz,Chloroform-d)δ7.13(dd,J＝2.1,9.8Hz,1H),7.08–7.03(m,1H),6.71–6.66(m,1H),5.82(s,2H),4.15(t,J＝6.0Hz,2H),3.58(s,3H),3.51–3.46(m,2H),2.26–2.10(m,4H),2.09–2.01(m,1H),1.97–1.91(m,2H),1.90–1.83(m,2H),1.79–1.67(m,2H).MS(ESI):m/z 535.1[M+H]+.
实施例E72：4-((1-(3-羟丙基)-3-甲基-2,6-二氧代-8-(1,4,4-三氟环己基)-1,2,3,6-四氢-7H-嘌呤-7-基)甲基)苄腈
实施例E72的合成方法同E60，除了以4-氰基溴化苄代替4-(三氟甲基)苄基溴为原料。1H NMR(500MHz,Chloroform-d)δ7.63(d,J＝8.2Hz,2H),7.25(d,J＝8.7Hz,2H),5.83(s,2H),4.15(t,J＝6.1Hz,2H),3.58(s,3H),3.52–3.44(m,2H),3.17(s,1H),2.50–2.32(m,2H),2.20–2.03(m,6H),1.91–1.81(m,2H).MS(ESI):m/z 476.2[M+H]+.
实施例E73：4-((8-(1-氟-4-(三氟甲基)环己基)-1-(3-羟丙基)-3-甲基-2,6-二氧代-1,2,3,6-四氢-7H-嘌呤-7-基)甲基)苄腈
实施例E73的合成方法同E72，除了以4-(三氟甲基)环己酮代替4,4-二氟环已酮为原料。1H NMR(500MHz,Chloroform-d)δ7.62(d,J＝8.0Hz,2H),7.22(d,J＝8.0Hz,2H),5.81(s,2H),4.13(t,J＝6.0Hz,2H),3.60(s,3H),3.52–3.47(m,2H),3.19(s,1H),2.47–2.36(m,2H),2.33–2.21(m,1H),2.04–1.80(m,8H).MS(ESI):m/z 508.2[M+H]+.
二、药理实施例
1.离子通道活性评价
1.1 TRPC3/4/5抑制活性测试
将表达hTRPC3、hTRPC4或hTRPC5的HEK-293细胞接种至多聚赖氨酸(PDL)包被的黑壁底透96孔板，每孔2×104个细胞，培养8小时后，弃去原培养基，随即加入60μL终浓度为4μM Fluo-4/AM染料，37℃孵育60分钟，随后用钙流检测缓冲液润洗5次，将细胞板放入30℃预热的(Molecular Devices,Sunnyvale,CA,USA)，在488nm波长下激发，在515-535nm范围内以1s的采样频率连续记录荧光信号。记录60秒后，加入溶剂对照、待测化合物和阳性抑制剂HC608(WO2014143799中化合物C31，终浓度为100nM)，继续采集信号300s，随后加入激动剂Englerin A(EA)(MedChemExpress,Shanghai,China,终浓度为0.3nM)，继续采集600秒荧光信号。轨迹图荧光信号以F/F0表示，其中F为不同时间点的荧光信号，F0为基础荧光信号，即最初10个时间点荧光信号的平均值。量效图先以F/F0＝1为基线，计算加入EA后荧光强度变化的曲线下面积，结合曲线下面积和化合物浓度的log值，采用log[Inhibitor]vs.response—Variable slope计算IC50值。
1.2 TRPC5激动活性测试
将表达hTRPC5的HEK-293细胞接种至PDL包被的黑壁底透96孔板，每孔2×104个细胞，培养8小时后，弃去原培养基，随即加入60μL终浓度为4μM Fluo-4/AM染料，37℃孵育60分钟，随后用钙流检测缓冲液润洗5次，将细胞板放入30℃预热的(Molecular Devices,Sunnyvale,CA,USA)，在488nm波长下激发，在515-535nm范围内以1s的采样频率连续记录荧光信号。记录360秒后，加入溶剂对照、待测化合物，继续采集600秒荧光信号。轨迹图荧光信号以F/F0表示，其中F为不同时间点的荧光信号，F0为基础荧光信号，即最初10个时间点荧光信号的平均值。量效图先以F/F0＝1为基线，计算加入待测化合物后荧光强度变化的曲线下面积，结合曲线下面积和化合物浓度的log值，采用log[agonist]vs.response—Variable slope计算EC50值。
1.3 hERG通道抑制作用测试
(1)细胞准备
将表达hERG蛋白的CHO细胞培养于175cm2培养瓶中，待细胞密度生长到60～80％，移走培养液，用7mL PBS洗一遍，然后加入3mL Detachin消化。待消化完全后加入7mL培养液中和，然后离心，吸走上清液，再加入5mL培养液重悬，以确保细胞密度为2～5×106/mL。
(2)溶液配制
(3)电生理记录过程
单细胞高阻抗封接和全细胞模式形成过程全部由Qpatch仪器自动完成，在获得全细胞记录模式后，细胞钳制在-80毫伏，在给予一个5秒的+20毫伏去极化刺激前，先给予一个50毫秒的-50毫伏前置电压，然后复极化到-50毫伏维持5秒，再回到-80毫伏。每15秒施加此电压刺激，记录2分钟后给予细胞外液记录2分钟，然后开始给药过程，化合物浓度从最低测试浓度开始，每个测试浓度给予2分钟，连续给完所有浓度后，给予阳性对照化合物10μM Cisapride。每个浓度至少测试3个细胞(n≥3)。
(4)化合物的准备
将化合物母液用细胞外液进行稀释，取5μL，20mM的化合物母液加入2495μL细胞外液，500倍稀释成40μM，然后在含0.2％DMSO的细胞外液中依次进行3倍连续稀释得到需要测试的最终浓度。化合物最高测试浓度为40μM，依次分别为40，13.33，4.44，1.48，0.49和0.16μM共6个浓度。阳性化合物Cisapride最高测试浓度为3μM，依次分别为3，1，0.333，0.111，0.037和0.012μM共6个浓度。最终测试浓度中的DMSO含量不超过0.2％，此浓度的DMSO对hERG钾通道没有影响。
(5)数据分析：实验数据由XLFit软件进行分析。
1.4肝微粒体稳定性测试
首先配制0.1M的pH 7.4Tris缓冲液，然后，用此缓冲液配制浓度为100mM的MgCl2溶液和浓度为10mM的辅酶因子NADPH溶液。待测化合物首先由DMSO配制成母液，随后在用时将其用水和0.1％BSA稀释至工作浓度。测定时，将肝微粒体的TRIS 缓冲液(终浓度为0.33mg/mL微粒体蛋白)、MgCl2溶液(终浓度为5mM)、待测化合物溶液(终浓度为1μM)和NADPH溶液(终浓度为1mM)在37℃下孵育，分别在0、7、17、30和60分钟加入甲醇终止反应。采用LC/MS/MS方法测定待测化合物的剩余浓度。
半衰期的计算：T1/2＝0.693/ke；
内在清除率计算：
体内清除率计算：
肝清除率计算：
代谢利用率计算：
ke：半对数作图时线性回归线的斜率(slope绝对值)，以底物的剩余百分数取半对数，对反应时间作半对数图。P：微粒体蛋白浓度(mg/mL)；Houston：Houston因子(45mg微粒体蛋白/g肝)；LW：肝重(g)；HBF：肝血流量(mL/min)；fu：未结合比例(通常fu＝1)。
1.5测试结果
(1)如表1所示，本发明化合物对TRPC5通道具有中等至较强的抑制作用，部分化合物的活性超过了参比化合物HC-070。因此，本发明化合物在治疗与TRPC5通道过度激活或TRPC5蛋白过表达有关的疾病中具有潜在的应用价值。
表1：化合物对TRPC5通道的抑制活性
(2)如表2所示，本发明部分化合物对TRPC5通道具有中等强度的激动作用。一些具有激动活性的化合物与另外一些具有抑制活性的化合物的结构十分相近，例如化合物例14与例15和例9的比较、例7和例17与例8的比较，但这种细微的结构差异意外地造成生物活性的显著变化。因此，本发明化合物可用于治疗与TRPC5通道受到抑制、TRPC5蛋白缺失有关的疾病，亦或者可以激活TRPC5通道造成钙离子过载并诱导细胞死亡，从而用于治疗某些类型的肿瘤。
表2：化合物对TRPC5通道的激动活性
(3)如表3所示，本发明化合物对TRPC4也具有较好的抑制作用，这与TRPC4与TRPC5两个蛋白同源性较高的生物特征相一致。因此，本发明化合物在治疗与TRPC4通道过度激活或TRPC4蛋白过表达有关的疾病中也具有潜在的应用价值。
另一方面，相比于HC-070，本发明化合物对TRPC3通道的抑制活性更低，对TRPC5通道的相对选择性更高，脱靶风险更低。
另一方面，相比于HC-070，本发明化合物对hERG通道的抑制活性更低，对TRPC5通道的相对选择性更高。
在药物化学领域，hERG通道阻断被认为是心脏毒性风险指标，应尽量避免。与现有公开技术相比，本发明化合物的区别结构特征在于黄嘌呤的8-位被“环烷基”取代，而非芳基、芳氧基、烷基、烷氧基、环烷氧基等基团。意料不到的是，这种结构上的差异明显地减弱了化合物对hERG通道的抑制作用，预示着本发明化合物具有更低的心脏毒性风险。
表3：化合物对TRPC3、TRPC4、hERG通道的抑制活性及选择性

注：a，文献值(PLoS ONE.2018,13,e0191225.)。
4)如表4所示，参比化合物HC-070在人肝微粒体中代谢稳定性中等，而在小鼠肝微粒体中代谢较快，半衰期较短。相比于HC-070，本发明化合物具有更长的半衰期和更高的代谢利用率，表明在代谢性质上更稳定，类药性质更好。
表4：化合物在肝微粒体中的代谢稳定性
注：MF％，表示代谢生物利用率；代谢稳定性判定标准：MF％＞70％，代谢稳定性较好；30％<MF％<70％，代谢稳定性中等；MF％<30％，代谢稳定性较差。
2.化合物抑制肝星状细胞活化作用的评价
2.1实验方法
将原代培养的小鼠肝星状细胞(HSC)铺板于96孔黑壁底透板24h后，预先孵育1μM浓度的化合物E21和溶剂对照0.1％DMSO 1小时，然后加入TGF-β，终浓度为10ng/mL，继续在CO2培养箱培养24h。然后吸弃培养基，用37℃ PBS溶液清洗3次,加入4％多聚甲醛溶液30分钟，固定；然后加入0.15％Triton X-100溶液，透化10min；然后加入5％BSA溶液，室温条件下封闭1h；再加入用5％BSA溶液配置抗α-SMA溶液(1:300),4℃孵育过夜；第二天加入用5％BSA溶液配置Alexa-488抗兔荧光二抗(1：1000)室温条件下避光孵育1h。孵育二抗结束后，用PBS溶液清洗细胞3次，加入Hoechst溶液，室温条件下避光孵育20min。结束后，用PBS溶液清洗细胞3次，进行荧光拍摄。拍摄绿色荧光使用FITC滤光片，拍摄蓝色荧光使用DAPI滤光片。使用ImageJ软件统计每个视野总细胞数，人工计数α-SMA阳性细胞个数，数据的处理和分析使用GraphPad(Version 6.01)软件。
2.2实验结果
从图1可以看出，在TGF-β刺激下、α-SMA阳性的肝星状细胞比例从0.3±0.04％增加至11.76±1.47％(P<0.01)，表明肝星状细胞活化程度增加；而TRPC4/5抑制剂E21能显著 地降低α-SMA阳性细胞比例至5.20±0.74％(P<0.01)，能够显著抑制肝星状细胞的活化，表明本发明化合物有用于治疗肝脏疾病的潜力。
3.初步药代动力学评价
3.1实验方法
6-8周龄雄性ICR小鼠(南京安诺康生物科技有限公司)饲养于SPF级动物房，光照与黑暗时间比为1:1，室温控制在23±2℃，湿度控制在55％，小鼠能够自由进食和饮水。待测化合物按照如下溶剂进行配制：2:2:96(v/v/v)的DMSO/吐温80/0.5％(m/v)CMC-Na水溶液，配药浓度为1mg/mL。然后，待测化合物以10mL/kg的给药体积对小鼠灌胃给药，剂量为10mg/kg，每个化合物给予3只小鼠。在给药后的1h和4h，从小鼠眼眶静脉丛中采血约50μL，置于含EDTA-K2抗凝剂的离心管中。血液样品经3,000rpm离心15min后取上清液，得血浆样品，然后采用液相色谱-串联质谱(LC-MS/MS)测定血浆中的药物浓度。
3.2测试结果
如表5所示，相比于HC-070，本发明化合物在小鼠口服后的1和4小时血浆药物浓度更高，初步表明本发明化合物具有更好的口服吸收性质和血浆暴露量，药代动力学性质更优。
表5：化合物对小鼠口服10mg/kg后1和4小时的血浆药物浓度(Mean±SD,N＝3)
4.大鼠药代动力学及组织分布评价
4.1药物配制
静脉注射(IV)给药：待测药物溶于5:5:90(v/v/v)的DMSO/Solutol/生理盐水，浓度为0.2mg/mL，给药体积为5mL/kg。口服(PO)给药：待测药物溶于2:2:96(v/v/v)的DMSO/吐温80/0.5％CMC-Na溶液，浓度为1mg/mL，给药体积为10mL/kg。
4.2实验方法
体重为180-280g的雄性SD大鼠，每组3只，实验前禁食12h，自由饮水，给药后4h统一进食。IV组，经大鼠尾静脉给药，剂量为1mg/kg，给药后0.083、0.25、0.5、1.0、2.0、4.0、6.0、8.0、12和24h采血。PO组，灌胃给药，剂量为10mg/kg，给药后0.25、0.5、1.0、2.0、4.0、6.0、8.0、12和24h采血。以上时间点经颈静脉取血0.2mL，置于含EDTA-K2的抗凝管中，全血采集后暂存于冰水浴中，30min内经11000rpm离心5min，分离血浆，然后于-70℃冰箱中冷冻待测。组织分布组，灌胃给药，剂量为10mg/kg，给 药2h后将大鼠麻醉，经腹主动脉放血处死，立即解剖采集脑、心、肝、脾、肺、肾、结肠、肌肉。收集部分全血，冰浴操作，全血在30min内经11000rpm离心5min，分离血浆(200μL左右)。组织和血浆收集完后于-60℃以下保存待测。每份组织准确称量后加入匀浆试剂乙腈-水(1:1,v/v)进行匀浆，组织：匀浆试剂＝1:4(g:mL)，匀浆后超声10min，离心后取上清液检测。利用LC-MS/MS法定量检测样品中的药物浓度。采用WinNonlin软件计算相关药动学参数Tmax、Cmax、AUC0-∞、t1/2等。
4.3测试结果
如表6中所示，化合物E68经大鼠口服10mg/kg后，最高血药浓度为1600ng/mL，半衰期为4.88h，生物利用率为62.2％，表现出良好的口服药代动力学性质。图2示出化合物E68经大鼠口服10mg/kg后2h在组织中的分布情况，可见E68在各组织中均有较高的浓度，明显高于血浆。因此，本发明化合物在用于治疗精神疾病、神经退行性疾病、肝脏、肾脏等的疾病时，能高效地分布到靶组织中。
表6：化合物E68对大鼠口服和静脉注射后的血浆药代动力学参数(Mean±SD,N＝3)
注释：Cmax，最大血药浓度；Tmax，药物浓度达峰时间；AUC0-∞，时间从零点至无穷时的药时曲线下面积；CL，药物的血浆清除率；Vss，药物在稳态时的分布容积；t1/2，清除半衰期；F，口服生物利用率；-，未计算；a，参数为文献报道值(PLoS ONE.2018,13,e0191225.)。
5.抗焦虑和抑郁药效实验
5.1药物配制
待测药物以2:2:96(v/v/v)的DMSO:吐温80:蒸馏水进行溶解，然后对小鼠灌胃给药，给药体积为10mL/kg。给药剂量：阳性对照Fluoxetine，10mg/kg；HC-070，0.1、0.3或1mg/kg；化合物E68，0.03、0.1、0.3或1mg/kg。空白对照组仅给予10mL/kg的上述溶剂。
5.2实验方法
(1)大理石埋藏试验
小鼠大理石埋藏试验是一种行为测定法，用于抗焦虑药物的筛选。它利用了老鼠在洞穴或逃生隧道等环境中的自然挖掘行为，对基因操纵、疾病或药物治疗引起的行为微小变化很敏感。该测试已被证明能有效检测药物干预和转基因小鼠模型中的抗焦虑作用，并能准确、灵敏地测量啮齿动物的重复和强迫行为。
在33cm×24cm×28cm的纸箱中铺5cm厚的木屑垫料，在垫料上整齐摆放4×5共20颗黑色弹珠(间隔4cm)，黑色弹珠的直径为14mm，把小鼠面对角落放进纸箱30min，30min后小心拿出小鼠，统计小鼠埋藏弹珠数量，埋藏2/3以上算整颗埋藏。首先进行空 白实验，实验开始前将小鼠拿到实验环境适应1h后开始进行实验，实验结束后将小鼠送回笼内，计小鼠埋藏弹珠数。第二天同样将小鼠拿到实验环境适应1h后开始给药，给药后再1h进行大理石埋藏实验。实验结束后将小鼠小心拿出送回笼内，防止小鼠剧烈逃窜影响实验结果，计数后将黑色弹珠取出喷酒精擦拭，垫料重新翻匀，去除上一只小鼠的气味，防止影响下一只小鼠实验。整齐摆放弹珠进行下一只小鼠的实验。
(2)悬尾试验
小鼠悬尾试验(TST)是一种广泛使用的实验方法，用于评估抗抑郁药和其他精神药物的有效性。测试的原理是将小鼠的尾巴悬吊并观察它的行为。老鼠最初会挣扎着逃跑，但当它意识到逃跑是不可能的时，最终会停止挣扎，进入一种表示绝望的静止状态。这种不动的行为被用来衡量抑郁样状态，并通过记录不动的持续时间来评估病情的严重程度。抗抑郁药和兴奋剂可以显著缩短这种不动的持续时间，这就是为什么TST被用于这些药物的初步筛选。该测试对大多数抗抑郁药敏感，且药效与临床疗效显著相关，这使其成为抗抑郁药物的初始测试标准。
同一只小鼠在大理石埋藏试验结束后立即进行悬尾试验。使用黑色磨砂亚克力板搭建悬尾实验箱，高55cm、长15cm、宽11.5cm，箱体周围三面及顶部为磨砂黑板，周围一面用于视频记录。在距小鼠尾尖2cm处用黄色胶带把小鼠尾巴缠绕起来，倒悬固定在顶部亚克力板的中间位置，小鼠尾尖距亚克力板约3cm，身体悬空不能触碰到周围的板壁。每只小鼠的测试时间持续6min，对该时间内小鼠的不动时间进行统计。实验结束后将小鼠送回笼内，对箱体内部及桌面进行酒精擦拭，去除上一只小鼠的气味，防止影响下一只小鼠实验。悬尾试验中不动的判断标准：停止挣扎，身体无屈曲或扭动，后肢无摆动，前肢头部轻微摆动也计入不动时间。
5.3实验结果
如图3和4所示，在大理石埋藏试验中，空白对照组小鼠埋藏弹珠的数量在给药前后没有显著变化；而给予Fluoxetine(10mg/kg)、HC-070(1mg/kg)和E68(0.1-1mg/kg)后，均能显著减少小鼠埋藏弹珠的个数，表明药物具有显著的抗焦虑作用。在悬尾试验中，空白对照组小鼠的不动时间在给药前后没有显著变化；而给予化合物E68、HC-070或Fluoxetine后，均能显著降低小鼠的不动时间，表明药物具有显著的抗抑郁效果。
在另外一项降低给药剂量后开展的试验中(图5和图6)，小鼠口服0.03mg/kg的化合物E68即可显著减少埋藏弹珠的数量，而在给与0.1mg/kg的E68时效果更加明显，并且优于同等剂量的HC-070。此外，口服0.03mg/kg的化合物E68也能显著降低小鼠在悬尾箱中的不动时间，其效果可比于0.1mg/kg的HC-070。可见，相比于HC-070、本发明化合物E68具有更加强效的抗焦虑、抗抑郁作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种通式(I-a)所示的化合物、其立体异构体及其药学上可接受的盐；
    其中，R1为C1-C6烷基或C1-C6卤代烷基；
    n为1到4的整数，各R2各自独立地为H、氘、卤素、C1-C6烷基、C1-C6卤代烷基、C1-C6烷氧基、C1-C6卤代烷氧基、氰基；
    A环为饱和或部分不饱和的C3-C10环烃基；
    m为1到6的整数，各R3各自独立地选自H、氘、C1-C6的烷基、C1-C6卤代烷基、C1-C6的烷氧基、C1-C6卤代烷氧基、C3-C8的环烷基、C6-C10芳基、5-8元杂芳基、4-8元杂环基、卤素、氰基、羟基、-NR4R5，或者R3与A环上相连的碳共同形成4-8元杂环或C6-C10芳基；所述环烷基、芳基、杂芳基、杂环基未被取代或者任选地被1-4个选自下组的基团所取代：C1-C6的烷基、C1-C6卤代烷基、C1-C6的烷氧基、C1-C6卤代烷氧基、卤素、氰基；
    R4、R5各自独立地为H、C1-C6的烷基、C3-C8的环烷基。
  2. 如权利要求1所述的化合物，其特征在于，A环为饱和或部分不饱和的C3-C8的单环或桥环烃基。
  3. 如权利要求1所述的化合物，其特征在于，R1为C1-C4烷基。
  4. 如权利要求1所述的化合物，其特征在于，n为1、2或3，各R2各自独立地为H、氘、卤素、C1-C4烷基、C1-C4卤代烷基、C1-C4烷氧基、C1-C4卤代烷氧基、氰基。
  5. 如权利要求1所述的化合物，其特征在于，m为1、2、3或4，各R3各自独立地选自H、氘、C1-C4的烷基、C1-C4卤代烷基、C1-C4的烷氧基、C1-C4卤代烷氧基、C3-C6的环烷基、苯基、5-6元杂芳基、5-6元杂环基、F、Cl、Br、氰基、羟基、-NR4R5，或者R3与A环上相连的碳共同形成5-6元杂环或苯基；所述环烷基、苯基、杂芳基、杂环基未被取代或者任选地被1-4个选自下组的基团所取代：C1-C6的烷基、C1-C6卤代烷基、C1-C6的烷氧基、C1-C6卤代烷氧基、卤素、氰基。
  6. 如权利要求1所述的化合物，其特征在于，具有如通式(I-b)所示的结构；
    其中，R1为C1-C6烷基；
    n为1到4的整数，各R2各自独立地为H、卤素原子、C1-C6卤代烷基或CN；
    各R3各自独立地选自H、氘、C1-C6的烷基、C1-C6卤代烷基、C1-C6的烷氧基、C1-C6卤代烷氧基、C6-C10芳基、C3-C8的环烷基、5-8元杂芳基、4-8元杂环基、卤素原子、氰基、羟基、-NR4R5；或者两R3与A环上相连的碳共同形成4-8元杂环；
    R4、R5各自独立地为各自独立地为H、C1-C6的烷基。
  7. 如权利要求1所述的化合物，其特征在于，所述化合物选自：


  8. 一种药物组合物，其特征在于，包含权利要求1至7中任一项所述的化合物、其立体异构体及其药学上可接受的盐，以及药学上可接受的载体。
  9. 如权利要求1至7所述的化合物或权利要求8所述的药物组合物的用途，其特征在于，用于制备TRPC4和TRPC5通道的抑制剂或激动剂，或用于制备预防、延缓或治疗与TRPC4或/和TRPC5的功能、表达异常相关的疾病的药物。
  10. 如权利要求9所述的用途，其特征在于，所述疾病包括精神疾病(如边缘型人格障碍、抑郁症、心境恶劣、产后抑郁、双相障碍、创伤后应激障碍、惊恐病、广场恐怖症、社交恐惧症、广泛性焦虑症、社交焦虑症、分离焦虑、精神分裂症、狂躁症、强迫症、冷漠、神经衰弱、妄想症等)、神经退行性疾病(如阿尔茨海默病、帕金森病、亨廷顿病、肌萎缩侧索硬化、记忆障碍、遗忘症、失语症、慢性疲劳综合征、克雅氏病、解离性遗忘症、神游遗忘症、学习障碍、睡眠障碍以及由创伤或衰老引起的其他脑部病症)、肾脏疾病(如由毒性物质引起的肾损伤、病毒或细菌感染引起的肾损伤、高血压肾病、糖尿病肾病、肾小球肾炎、红斑狼疮肾炎、IgA肾病、肾病综合征、膜性肾病、微小病变肾病、多囊肾、局灶节段性肾小球硬化症等)、疼痛(如伤害性疼痛、机械性疼痛、炎性疼痛、癌痛及神经性疼痛)、癫痫、肝脏疾病(如胆汁淤积性肝病、酒精性脂肪肝炎、非酒精性脂肪肝炎、病毒性肝炎、自身免疫性肝病、化学性肝损伤、肝纤维化、肝硬化、肝细胞癌)、心血管疾病(如高血压、动脉粥样硬化、冠心病、心绞痛、心肌梗塞、心率失常、中风、肺动脉高压)、癌症(如肾细胞癌、乳腺癌、结直肠癌)、皮肤疾病(如银屑病、鱼鳞病、掌跖角化症、Olmsted症、蟾皮症或绝经期角皮症等)、肠道疾病(如溃疡性结肠炎、短肠综合征、肠易激综合征、肠痉挛、腹泻、腹痛等)；在另一优选例中，所述疾病为边缘型人格障碍、抑郁症、焦虑症、创伤后应激障碍。
PCT/CN2024/072849 2023-02-08 2024-01-17 黄嘌呤类化合物及其用途 Ceased WO2024164818A1 (zh)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014143799A2 (en) 2013-03-15 2014-09-18 Hydra Biosciences, Inc. Substituted xanthines and methods of use thereof
WO2014152287A2 (en) * 2013-03-14 2014-09-25 Hydra Biosciences, Inc. Substituted xanthines and methods of use thereof
WO2019011802A1 (en) 2017-07-11 2019-01-17 Boehringer Ingelheim International Gmbh NEW SUBSTITUTED XANTHINE DERIVATIVES
WO2021067946A1 (en) * 2019-10-04 2021-04-08 Goldfinch Bio, Inc. Biomarker-based treatment of focal segmental glomerulosclerosis and diabetic kidney disease
CN113166151A (zh) * 2018-12-12 2021-07-23 勃林格殷格翰国际有限公司 经取代的黄嘌呤衍生物
CN113166150A (zh) * 2018-12-12 2021-07-23 勃林格殷格翰国际有限公司 经取代的黄嘌呤衍生物
CN115636831A (zh) * 2022-10-27 2023-01-24 中国药科大学 稠合嘧啶二酮类化合物、其用途以及药物组合物

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014152287A2 (en) * 2013-03-14 2014-09-25 Hydra Biosciences, Inc. Substituted xanthines and methods of use thereof
WO2014143799A2 (en) 2013-03-15 2014-09-18 Hydra Biosciences, Inc. Substituted xanthines and methods of use thereof
CN105143229A (zh) * 2013-03-15 2015-12-09 海德拉生物科学有限公司 取代的黄嘌呤及其使用方法
WO2019011802A1 (en) 2017-07-11 2019-01-17 Boehringer Ingelheim International Gmbh NEW SUBSTITUTED XANTHINE DERIVATIVES
CN110997675A (zh) * 2017-07-11 2020-04-10 勃林格殷格翰国际公司 新的取代的黄嘌呤衍生物
CN113166151A (zh) * 2018-12-12 2021-07-23 勃林格殷格翰国际有限公司 经取代的黄嘌呤衍生物
CN113166150A (zh) * 2018-12-12 2021-07-23 勃林格殷格翰国际有限公司 经取代的黄嘌呤衍生物
WO2021067946A1 (en) * 2019-10-04 2021-04-08 Goldfinch Bio, Inc. Biomarker-based treatment of focal segmental glomerulosclerosis and diabetic kidney disease
CN115636831A (zh) * 2022-10-27 2023-01-24 中国药科大学 稠合嘧啶二酮类化合物、其用途以及药物组合物

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PLOS ONE, vol. 13, 2018, pages 0191225
See also references of EP4663640A1

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