WO2020001166A1 - 糖苷类化合物、其制备方法、组合物、应用及中间体 - Google Patents

糖苷类化合物、其制备方法、组合物、应用及中间体 Download PDF

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WO2020001166A1
WO2020001166A1 PCT/CN2019/085823 CN2019085823W WO2020001166A1 WO 2020001166 A1 WO2020001166 A1 WO 2020001166A1 CN 2019085823 W CN2019085823 W CN 2019085823W WO 2020001166 A1 WO2020001166 A1 WO 2020001166A1
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substituted
unsubstituted
group
alkoxy group
independently
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PCT/CN2019/085823
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English (en)
French (fr)
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林国强
张建革
田平
冯陈国
詹常森
周俊杰
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上海和黄药业有限公司
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Priority to EP19825863.4A priority Critical patent/EP3819303A4/en
Priority to JP2020566939A priority patent/JP7248707B2/ja
Priority to US17/054,124 priority patent/US11325937B2/en
Publication of WO2020001166A1 publication Critical patent/WO2020001166A1/zh
Priority to JP2023042119A priority patent/JP7442712B2/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/23—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
    • C07C69/12—Acetic acid esters
    • C07C69/16—Acetic acid esters of dihydroxylic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/08—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to sulfur, selenium or tellurium
    • C07H5/10—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to sulfur, selenium or tellurium to sulfur

Definitions

  • the invention relates to a glycoside compound, a preparation method, a composition, an application and an intermediate thereof.
  • Angiogenesis has become a potential biological target for drug intervention in the treatment of cancer, vascular disease and chronic inflammation.
  • Drug development such as anti-angiogenesis inhibitors designed to reduce tumor angiogenesis, has become a successful therapy for many advanced and aggressive cancers.
  • the discovery of promoting angiogenesis has provided a new direction for the effective treatment of ischemic vascular diseases, and it has also become a research hotspot in the medical field.
  • cardio-cerebral vascular diseases such as angina pectoris, myocardial infarction, cerebral infarction, stroke
  • lower limb ischemia such as diabetic lower limb vascular disease, thrombo-occlusive vasculitis
  • Cerebrovascular disease refers to a group of diseases in which cerebral arteries or cervical arteries that dominate the brain cause lesions in the intracranial blood circulation and damage to brain tissue. Clinically, the main manifestations are sudden fainting, unconsciousness, or distorted mouth and eyes, unfavorable speech, and hemiplegia.
  • Ischemic cerebrovascular disease mainly refers to cerebral thrombosis, cerebral embolism, multiple cerebral infarction, etc .; its disease is characterized by sudden onset, rapid progress, critical condition, and because it is more common in the elderly, it is easy to be complicated by multiple organ damage. The prognosis is poor and the mortality rate is high. Ischemic cerebrovascular disease is called cerebral infarction due to ischemic necrosis of the brain tissue in the corresponding area due to acute interruption of blood flow to the cerebral arteries.
  • Cerebral stroke is also known as "stroke” and cerebral vascular accident (CVA). It is one of the most important fatal diseases in the world. It is caused by the sudden rupture of blood vessels in the brain or the inability of blood to flow into the brain due to blocked blood vessels. A group of diseases that cause brain tissue damage, including ischemic and hemorrhagic stroke. The incidence of ischemic stroke is higher than that of hemorrhagic stroke. Ischemic stroke is caused by a variety of causes of regional blood supply regional brain tissue regional blood supply disorders, leading to necrosis of ischemic hypoxic lesions in the brain tissue, resulting in clinical response. The loss of neurological function seriously affects the quality of life of patients.
  • the research focus on pathological intervention of ischemic injury is mainly to improve cerebral blood circulation and neuroprotection.
  • the measures to improve cerebral blood circulation are mainly antithrombotic therapy.
  • Antithrombotic drugs are divided into thrombolytic drugs, antiplatelet aggregation drugs and anticoagulants.
  • Neuroprotective drugs currently include calcium antagonists, glutamate antagonists, glutamate release inhibitors, GABA receptor agonists, free radical scavengers, and cell membrane stabilizers.
  • Angiogenesis can promote neuron survival after stroke, improve neurological deficits and quality of life after stroke, but the influencing factors and regulation mechanism of angiogenesis after stroke are complicated. Recent studies have found that PAR1 is involved in microangiogenesis and nerve repair after stroke. Angiogenesis refers to the formation of new capillaries through budding and / or non-budding based on the original blood vessels.
  • the main processes of angiogenesis include: increased vascular permeability; production of proteolytic enzymes, degradation of the extracellular matrix, and promotion of endothelial cell proliferation; endothelial cells are separated from the basement membrane, migrate to the space around the blood vessel, and adhere, proliferate, and rebuild, It composes a three-dimensional lumen; differentiates into new capillaries; interstitial cells enter the blood vessel wall under the induction of intermediary molecules, so that the blood vessels are stable and mature. Under normal physiological conditions, blood vessels in the body remain highly stable once they are formed, and are regulated by many key molecules that have positive or negative regulatory effects (that is, angiogenic and angiogenic inhibitors).
  • angiogenesis The initiation of angiogenesis is only turned on briefly with the appearance of the stimulus signal, and then closed, maintaining the dynamic balance of angiogenesis and decline.
  • Factors affecting microangiogenesis after stroke include: local blood supply and oxygen supply; changes in thrombin and its concentration; levels of pro-angiogenic factors such as hypoxia-inducible factor 1 ⁇ (HIF-1 ⁇ ), vascular endothelial growth factor (VEGF), matrix Metalloproteinases (MMPs), Angiopoietin 1 (Ang-1), Angiopoietin 2 (Ang-2), etc.
  • HIF-1 ⁇ hypoxia-inducible factor 1 ⁇
  • VEGF vascular endothelial growth factor
  • MMPs matrix Metalloproteinases
  • Angiopoietin 1 Ang-1
  • Angiopoietin 2 Ang-2
  • PAR1 usually interacts with pro-angiogenic factors and plays a role in promoting angiogenesis.
  • VEGF is currently recognized as a
  • VEGF vascular endothelial cell proliferation and migration, increases vascular permeability, and enhances the expression of factors that degrade extracellular matrix.
  • pathological processes such as inflammation, tumor, wound healing, ischemia, hypoxia, etc.
  • Increased expression of VEGF in neurons and glial cells surrounding stroke lesions in patients with stroke By specifically binding to endothelial cell surface receptors, it promotes vascular endothelial cell proliferation and migration, increases vascular permeability, and enhances the expression of factors that degrade extracellular matrix. To promote microangiogenesis.
  • the technical problem to be solved by the present invention is to fill the gaps in the market for drugs with clear therapeutic angiogenesis and difficulties in drug preparation in the prior art, so as to provide a glycoside compound, its preparation method, composition, and application And intermediates.
  • the glycoside compound provided by the present invention has a simple preparation method, can significantly increase the expression of VEGF-A mRNA, and can be used for preparing angiogenesis-promoting.
  • ischemic cardio-cerebral-vascular disease especially cerebral infarction Stroke
  • myocardial infarction myocardial infarction
  • lower limb ischemia Research on drugs for circulatory disorders provides a reliable guarantee.
  • the present invention solves the above technical problems through the following technical solutions.
  • the present invention provides a glycoside compound represented by the following formula III, its tautomer, optical isomer, solvate, polymorph, pharmaceutically acceptable salt, ester, pharmaceutically acceptable Prodrugs or derivatives:
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from hydrogen, hydroxyl, mercapto, substituted or unsubstituted C 1 to C 20 alkoxy, nitro or halogen;
  • any two adjacent carbon atoms of R 1 , R 2 , R 3 , R 4, and R 5 form a 5- to 7-membered heterocyclic ring, and the hetero atom of the heterocyclic ring is O or S;
  • the number of heteroatoms is one or more; when the number of heteroatoms is multiple, the heteroatoms are the same or different;
  • the substituent in the substituted or unsubstituted C 1 -C 20 alkoxy group is selected from the group consisting of a C 3 -C 20 cycloalkyl group, a C 1 -C 20 alkenyl group, and a substituted or unsubstituted C 6 -C 20 aryl group. , Halogen, substituted or unsubstituted C 2 -C 20 heteroaryl, C 3 -C 6 cycloalkoxy, C 1 -C 20 alkoxy, or C 1 -C 20 alkyl;
  • the substituted or unsubstituted C 6 -C 20 aryl group and the substituted or unsubstituted C 2 -C 20 heteroaryl group are each independently a halogen or halogen-substituted C 1 -C 20 alkyl group;
  • X is CH 2 , NR 7 , O or S;
  • Y is CH 2 , NR 8 , O or S;
  • Z is O or S
  • R 7 and R 8 are each independently hydrogen, aryl-substituted C 1 -C 6 alkoxycarbonyl, or C 1 -C 6 alkoxycarbonyl;
  • Each R 6 is independently hydrogen or glycosyl
  • n 2, 3 or 4;
  • R 1 , R 4 and R 5 are H, R 2 and R 3 are not OCH 3 and OH at the same time;
  • R 1 , R 2 and R 5 are H, R 3 and R 4 are not OH and OCH 3 at the same time;
  • R 2 , R 4 and R 5 are H, R 1 and R 3 are not OH at the same time;
  • R 1, R 2 and R 4 are H, R 3 and R 5 are not simultaneously OH.
  • the present invention provides a glycoside compound represented by the following formula I, which is a tautomer, optical isomer, solvate, polymorph, pharmaceutically acceptable salt, ester, pharmaceutically acceptable Prodrugs or derivatives:
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from hydrogen, hydroxyl, thiol, substituted or unsubstituted C 1 to C 20 alkoxy (eg, substituted or unsubstituted C 1 to C 10 Alkoxy, such as substituted or unsubstituted C 1 -C 6 alkoxy,), nitro or halogen;
  • any two adjacent phases of R 1 , R 2 , R 3 , R 4 and R 5 form a 5-7 membered heterocyclic ring with the carbon atom connected to its benzene ring (for example, R 2 and R 3 form 5-7 Heterocycle), the hetero atom of the heterocycle is O or S (for example, O); the number of the heteroatoms is one or more (for example, two); when the number of heteroatoms is more, the hetero The atoms are the same or different;
  • the substituent in the substituted or unsubstituted C 1 -C 20 alkoxy group is selected from C 3 -C 20 cycloalkyl (for example, C 3 -C 10 cycloalkyl, and for example, C 3 -C 6 cycloalkyl Base), C 2 -C 20 alkenyl (e.g. C 2 -C 6 alkenyl, e.g.
  • C 2 -C 4 alkenyl ), Substituted or unsubstituted C 6 -C 20 aryl (for example, substituted or unsubstituted C 6 -C 10 aryl, for example, substituted or unsubstituted aryl or naphthyl), halogen (for example, fluorine, chlorine, Bromine or iodine), substituted or unsubstituted C 2 -C 20 heteroaryl.
  • the hetero atom in the substituted or unsubstituted C 2 -C 20 heteroaryl may be selected from one or more of N, S and O. (For example, the number of heteroatoms is one; for example, the heteroatom is N; for example, the substituted or unsubstituted C 2 -C 20 heteroaryl group may be an arylhetero group having only one ring).
  • substituted or unsubstituted C 2 -C 10 heteroaryl for example, substituted or unsubstituted C 2 -C 6 heteroaryl
  • C 3 -C 6 cycloalkoxy for example
  • C 1 to C 20 alkoxy such as C 1 to C 10 alkoxy, such as C 1 to C 6 alkoxy, and then methoxy, ethoxy, or propoxy
  • C 1 ⁇ C 20 alkyl group e.g., C 1 ⁇ C 10 alkyl group; for example, C 1 ⁇ C 6 alkyl group; further example C 1 ⁇ C 3 alkyl group
  • C 1 ⁇ C 10 alkyl group for example, C 1 ⁇ C 6 alkyl group; further example C 1 ⁇ C 3 alkyl group
  • the substituted or unsubstituted C 1 -C 20 aryl group and the substituents in the substituted or unsubstituted C 2 -C 20 heteroaryl group are each independently selected from halogen (for example, fluorine, chlorine, bromine or iodine) or Halo (such as fluorine, chlorine, bromine or iodine) substituted C 1 to C 20 alkyl (the number of substituents in the halogen substituted C 1 to C 20 alkyl may be one or more, when it is When there are multiple substituents, each substituent may be the same or different; for example, halogen-substituted C 1 to C 10 alkyl; for example, halogen-substituted C 1 to C 6 alkyl; for example, halogen-substituted C 1 to C 3 alkyl ; For example -CF 3 , -CHF 2 or -CH 2 F);
  • X is CH 2 , NR 7 , O or S;
  • Y is CH 2 , NR 8 , O or S;
  • Z is O or S
  • R 7 and R 8 are each independently hydrogen, aryl-substituted C 1 -C 6 alkoxycarbonyl (for example, benzyloxycarbonyl) or C 1 -C 6 alkoxycarbonyl (for example, tert-butoxycarbonyl);
  • R 6 is hydrogen or
  • n 2, 3 or 4.
  • R 1 , R 4 and R 5 are H, R 2 and R 3 are not OCH 3 and OH at the same time;
  • R 1 , R 2 and R 5 are H, R 3 and R 4 are not OH and OCH 3 at the same time.
  • the substituted or unsubstituted C 2 -C 20 heteroaryl group may be substituted or unsubstituted pyrrole, substituted or unsubstituted furan, substituted or unsubstituted thiophene, substituted or unsubstituted pyrazole, Substituted or unsubstituted imidazole, substituted or unsubstituted oxazole, substituted or unsubstituted thiazole, substituted or unsubstituted isoxazole, substituted or unsubstituted pyridine, substituted or unsubstituted pyridazine, substituted or unsubstituted Pyrimidine, or substituted or unsubstituted pyrazine, such as substituted or substituted pyridyl.
  • the unsubstituted C 1 -C 20 alkoxy group may be
  • the C 3 -C 20 cycloalkyl group may be a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, or a cyclohexyl group.
  • the substituted C 6 -C 20 aryl group may be
  • the substituted C 1 -C 20 alkoxy group may be any suitable substituted C 1 -C 20 alkoxy group.
  • the unsubstituted C 2 -C 20 heteroaryl group may be
  • the 5-7 membered heterocyclic ring may be
  • R 1 may be hydrogen
  • the R 2 may be hydrogen, a substituted or unsubstituted C 1 to C 20 alkoxy group, or the R 2 and R 3 may form
  • the definitions of the substituted or unsubstituted C 1 to C 20 alkoxy groups and the substituents thereof are as described above.
  • the R 3 may be hydrogen, a hydroxyl group, a mercapto group, a substituted or unsubstituted C 1 to C 20 alkoxy group, a hydroxyl group, a nitro group, or a halogen; the substituted or unsubstituted C 1 to C 20 alkyl group
  • the definition of the oxy group and its substituents, and the halogen is as described above.
  • the R 4 may be hydrogen, or a substituted or unsubstituted C 1 to C 20 alkoxy group; the definition of the substituted or unsubstituted C 1 to C 20 alkoxy group and the substituents thereof is as above. As described.
  • R 5 may be hydrogen
  • each group is as follows (the definition of the unmentioned groups is as described above): the X is CH 2 , and the Y is CH 2 , NR 8 , S, or O; The R 8 is hydrogen, aryl-substituted alkoxycarbonyl or alkoxycarbonyl.
  • each group is as follows (the definitions of the unmentioned groups are as described above): the X is NR 7 , the Y is CH 2 , and R 7 is hydrogen, Aryl substituted alkoxycarbonyl or alkoxycarbonyl.
  • each group is as follows (the definition of the unmentioned groups is as described above): the X is O or S, and the Y is CH 2 .
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from hydrogen, hydroxyl, mercapto, substituted or unsubstituted C 1 to C 20 alkoxy, nitro or halogen
  • the number of the substituted or unsubstituted C 1 to C 20 alkoxy groups may be one or more (for example, one, two, or three; for example, one).
  • said Can be any of the following structures:
  • n 2.
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from hydrogen, substituted or unsubstituted C 1 to C 20 alkoxy, nitro or halogen.
  • R 6 is hydrogen
  • optical isomers preferably, among the compounds represented by the formula I
  • the optical isomers can be:
  • the substituent of the compound described by Formula I may be the following (the definition of the substituent not mentioned is as described above):
  • n 2, 3 or 4;
  • X is CH 2 , NR 7 , O or S.
  • the substituent of the compound described by Formula I may be the following (the definition of the substituent not mentioned is as described above):
  • n 2, 3 or 4;
  • Y is CH 2 , NR 8 , O, or S.
  • the substituent of the compound described by Formula I may be the following (the definition of the substituent not mentioned is as described above):
  • R 1 is hydrogen
  • the R 2 is hydrogen, a substituted or unsubstituted C 1 to C 20 alkoxy group, or the R 2 and R 3 are formed.
  • the definitions of the substituted or unsubstituted C 1 to C 20 alkoxy groups and their substituents are as described above;
  • the R 3 is hydrogen, hydroxyl, mercapto, substituted or unsubstituted C 1 to C 20 alkoxy, hydroxyl, nitro or halogen; the substituted or unsubstituted C 1 to C 20 alkoxy and substituted And the definition of the halogen is as described above;
  • the R 4 is hydrogen, or a substituted or unsubstituted C 1 to C 20 alkoxy group; the definition of the substituted or unsubstituted C 1 to C 20 alkoxy group and the substituent thereof is as described above;
  • the R 5 is hydrogen.
  • the substituent of the compound described by Formula I may be the following (the definition of the substituent not mentioned is as described above):
  • n 2;
  • the Y is CH 2 ;
  • the invention also provides compounds of any of the following structures:
  • these compounds are:
  • the invention also provides a method for preparing a glycoside compound, which is characterized by comprising the steps of: performing a reduction reaction of a compound represented by Formula V in a solvent to obtain a compound represented by Formula VI;
  • R 9 ′ , R 10 ′ and R 11 ′ are each independently hydrogen or Ac, and at least one of them is Ac;
  • R 6 ′ is hydrogen, Ac or R 12 ' , R 13' , R 14 ' , and R 15' are each independently hydrogen, Bz or Ac; n, X, Y, Z, R 1 , R 2 , R 3 , R 4 , R 5 and R
  • n X, Y, Z, R 1 , R 2 , R 3 , R 4 , R 5 and R
  • the definition of 6 is as described in any of the above; when R 6 ′ is hydrogen or Ac, R 6 is hydrogen; when R 6 ′ is When R 6 is
  • the present invention also provides a method for preparing a compound represented by Formula I, which includes the following steps: a compound represented by Formula II is subjected to a reduction reaction in a solvent to obtain a compound represented by Formula I;
  • R 9 ′ , R 10 ′ and R 11 ′ are each independently hydrogen or Ac, and at least one of them is Ac;
  • R 6 ′ is hydrogen, Ac or R 12 ' , R 13' , R 14 ' , and R 15' are each independently hydrogen, Bz or Ac;
  • n X, Y, Z, R 1 , R 2 , R 3 , R 4 , R 5 and R
  • 6 is as described above; when R 6 ′ is hydrogen or Ac, R 6 is hydrogen; when R 6 ′ is When R 6 is
  • the present invention also provides the glycoside compounds shown above, or the compounds shown below, which are tautomers, optical isomers, solvates, polymorphs, pharmaceutically acceptable salts, esters, pharmaceutically The use of an acceptable prodrug or derivative in the preparation of pro-angiogenic drugs;
  • the present invention also provides a glycoside compound represented by Formula I, compound TG-052, compound TG-053, compound TG-054 or TG-057, its tautomers, optical isomers, solvates, polymorphs
  • a glycoside compound represented by Formula I compound TG-052, compound TG-053, compound TG-054 or TG-057, its tautomers, optical isomers, solvates, polymorphs
  • steroids pharmaceutically acceptable salts, esters, pharmaceutically acceptable prodrugs or derivatives in the preparation of pro-angiogenic drugs
  • the present invention also provides a glycoside compound shown above, a compound represented by the following formula, its tautomer, optical isomer, solvate, polymorph, pharmaceutically acceptable salt, ester, pharmaceutically acceptable Acceptable prodrugs or derivatives in the preparation of ischemic cardio-cerebral vascular diseases, especially cerebral infarction (stroke), myocardial infarction, lower extremity ischemic Application of medicines for circulatory disorders;
  • ischemic cardio-cerebral vascular diseases especially cerebral infarction (stroke), myocardial infarction, lower extremity ischemic Application of medicines for circulatory disorders;
  • the present invention also provides a glycoside compound represented by formula I, compound TG-052, compound TG-053 or compound TG-054, its tautomer, optical isomer, solvate, polymorph, pharmacy
  • ischemic cardio-cerebral vascular diseases especially cerebral infarction (stroke), myocardial infarction, lower limb ischemic Application of medicines for circulatory disorders;
  • the ischemic cardio-cerebral vascular disease involves all symptoms and / or pathological changes in the early to late stages of ischemia in the heart tissue and brain tissue caused by vascular disease caused by vascular wall lesions, changes in blood components and / or hemodynamic changes , Such as cerebral infarction (stroke) or myocardial infarction.
  • stroke cerebral infarction
  • myocardial infarction myocardial infarction
  • the present invention also provides an intermediate compound of the glycoside compound shown above:
  • the present invention also provides an intermediate compound of a glycoside compound represented by Formula I:
  • the present invention further provides the aforementioned glycoside compound, its tautomer, optical isomer, solvate, polymorph, pharmaceutically acceptable salt, ester, pharmaceutically acceptable prodrug or derivative, Its application in the prevention and / or treatment of angiogenic drugs, preferably in ischemic vascular disease, and more preferably in ischemic cardio-cerebral vascular disease, especially cerebral infarction (stroke), myocardial infarction, Used in lower limb ischemic diseases.
  • angiogenic drugs preferably in ischemic vascular disease, and more preferably in ischemic cardio-cerebral vascular disease, especially cerebral infarction (stroke), myocardial infarction, Used in lower limb ischemic diseases.
  • This aspect can be expressed as the aforementioned glycoside compounds, their tautomers, optical isomers, solvates, polymorphs, pharmaceutically acceptable salts, esters, pharmaceutically acceptable prodrugs or derivatives
  • ischemic cardiovascular and cerebrovascular diseases especially cerebral infarction (stroke), myocardial infarction, and lower limb ischemic diseases.
  • a method for promoting angiogenesis in a patient in need preferably a method for treating and / or preventing a mammal (including a human) including or susceptible to ischemic vascular disease, further referring to suffering from Ischemic cardio-cerebral vascular disease, especially cerebral infarction (stroke), myocardial infarction, ischemic lower extremity Method for mammals (including humans) of circulatory disorders, said method comprising administering to said patient a therapeutically effective amount of said glycoside compound of the present invention, its tautomers, optical isomers, solvates, polymorphs Type, pharmaceutically acceptable salt, ester, pharmaceutically acceptable prodrug or derivative, together with a pharmaceutically acceptable excipient or carrier.
  • a mammal including a human
  • ischemic vascular disease especially cerebral infarction (stroke), myocardial infarction, ischemic lower extremity Method for mammals (including humans) of circulatory disorders
  • said method comprising administering to said patient a therapeutically effective amount of said glycoside compound of the
  • glycoside compound of the present invention is used for tautomers, optical isomers, solvates, polymorphs, pharmaceutically acceptable salts, esters, pharmaceutically acceptable prodrugs or derivatives. Use for preventing and / or treating ischemic cerebrovascular disease.
  • ischemic cerebrovascular diseases include: stroke, atherosclerotic cerebral thrombosis, cardiogenic cerebral thrombosis, acute ischemic cerebrovascular syndrome, small vessel disease (also known as lacunar stroke) , Multiple cerebral infarction, large-scale cerebral infarction, watershed cerebral infarction, hemorrhagic cerebral infarction, asymptomatic cerebral infarction, cerebral venous and venous sinus thrombosis, abnormal skull base vascular network disease, ischemic brain caused by other reasons Stroke.
  • the ischemic cerebrovascular disease is caused by atherosclerotic thrombosis, which is the formation of thrombus on the basis of atherosclerosis and various vascular diseases such as arteritis. Lead to luminal occlusion, causing clinical syndromes such as cerebral ischemia, hypoxia, necrosis, and softening.
  • the ischemic cerebrovascular disease is formed by cardiogenic cerebral embolism
  • cardiogenic cerebral embolism refers to the cardiogenic embolus that migrates distally into the cerebral arterial system through the blood flow path Cerebral infarction caused by vascular occlusion.
  • the ischemic cerebrovascular disease is formed by acute ischemic cerebrovascular syndrome, which includes three diseases: transient ischemic attack, reversible deficiency Bloody neurological deficits and complete recovery stroke.
  • cerebrovascular disease refers to stroke, which is a group of diseases that cause brain tissue damage due to sudden rupture of blood vessels in the brain or the inability of blood to flow into the brain due to vascular occlusion, including ischemic and Hemorrhagic stroke.
  • a therapeutically effective amount of the above-mentioned glycoside compound of the present invention is formulated according to the usual route of administration and according to methods known in the art in conventional pharmaceutical compositions (the pharmaceutical composition comprising an effective amount of the active ingredient and a suitable pharmaceutically acceptable carrier) and dosage form for administration to a patient in need of such treatment. .
  • a “therapeutically effective amount” refers to an amount of the active ingredient which, when administered, is sufficient to prevent the development of one or more symptoms of the disease in question, or to some extent alleviate the one or more symptoms.
  • the specific dose of the compound administered according to the invention will be determined by the circumstances surrounding the case, including the compound administered, the route of administration, the specific condition being treated, and similar considerations.
  • a therapeutically effective amount of a compound "Means an amount of a compound that is sufficient to prevent or to some extent alleviate one or more ischemic vascular diseases including ischemic cardio-cerebral vascular disease and especially cerebral infarction (stroke) , Myocardial infarction, ischemic diseases of the lower limbs, etc.
  • ischemic cerebrovascular diseases include stroke, atherosclerotic thrombosis, cardiogenic cerebral thrombosis, acute ischemic cerebrovascular syndrome, and small vessel disease ( (Also known as lacunar stroke), multiple cerebral infarction, large-scale cerebral infarction, watershed cerebral infarction, hemorrhagic cerebral infarction, asymptomatic cerebral infarction, cerebral vein and venous sinus thrombosis Formation, abnormal vascular network disease of the skull base, ischemic stroke caused by other reasons.
  • small vessel disease (Also known as lacunar stroke)
  • multiple cerebral infarction large-scale cerebral infarction
  • watershed cerebral infarction watershed cerebral infarction
  • hemorrhagic cerebral infarction hemorrhagic cerebral infarction
  • asymptomatic cerebral infarction cerebral vein and venous sinus thrombosis Formation
  • abnormal vascular network disease of the skull base ischemic stroke caused by other reasons.
  • the single dose and daily dose are different. Therefore, an accurate single dose will be determined according to standard medical principles under the guidance of a physician.
  • An effective daily dose for use of a glycoside compound of the present invention in the treatment of ischemic vascular disease is 0.01 mg to about 1000 mg, about 1 mg to about 500 mg, or about 10 mg to about 200 mg and optionally about 0.01 mg to about 2000 mg,
  • glycoside compound of the present invention may be alone Used or in combination or in combination therapy with other therapeutic agents.
  • the above glycoside compound, its tautomer, optical isomer, solvate, polymorph, pharmaceutically acceptable salt, ester, pharmaceutically acceptable precursor is for use in the prevention and / or treatment of ischemic vascular disease, wherein the prevention or treatment includes administration as the sole active ingredient.
  • a prodrug or derivative is for use in the prevention and / or treatment of ischemic vascular disease, wherein the prevention or treatment includes administration in combination therapy with a therapeutic agent selected from the group consisting of: another Glycoside compounds of the present invention, their tautomers, optical isomers, solvates, polymorphs, pharmaceutically acceptable salts, esters, pharmaceutically acceptable prodrugs or derivatives, and antihypertensive drugs , Lipid lowering drugs, thrombolytic drugs, antiplatelet aggregation drugs, anticoagulants, neuroprotective drugs, calcium antagonists, glutamate antagonists, glutamate release inhibitors, GABA receptor agonists, free Base scavenger, cell membrane stabilizer.
  • the additional therapeutic agent is selected from the group consisting of another glycoside compound of the invention, its tautomer, optical isomer, solvate, polymorph, Pharmaceutically acceptable salts, esters, pharmaceutically acceptable prodrugs or derivatives, reteplase, rantiporase, monteplase, tempeh fibrinolytic enzyme, new earthworm fibrinolytic enzyme, nattokinase , Snake venom plasminogen activator, aspirin, ticlopidine, clopidogrel, prasugrel, ticagrelor, cangrelor, sagrelate hydrochloride, vorapaxar, atopaxar, heparin, low molecular weight heparin, warfarin , Rivaroxaban, bifarudin, dabigatran etexilate, edaravone, statins.
  • another glycoside compound of the invention its tautomer, optical isomer, solvate, polymorph
  • glycoside compounds of the present invention includes the glycoside compounds of the present invention, tautomers, optical isomers, solvates, polymorphs, pharmaceutically acceptable salts, esters, pharmaceutically acceptable
  • the combination of the present invention of the received prodrug or derivative with another therapeutic agent is effective not only when these active ingredients are used in a single composition, but also in two different compositions (simultaneously, sequentially, or after a period of time) Separately applied) Also effective when used.
  • glycoside compounds of the present invention can be prescribed for use with other active ingredients in combination therapies to prevent and / or treat ischemic vascular disease.
  • the combination therapy comprises the simultaneous, sequential or separate administration of a glycoside compound of the invention, its tautomer, optical isomer, solvate, polymorph, pharmacy Pharmaceutically acceptable salts, esters, pharmaceutically acceptable prodrugs or derivatives, and additional therapeutic agents.
  • the combination therapy comprises administering to a subject a glycoside compound of the invention, a tautomer, an optical isomer, a solvate, a polymorph, a pharmaceutically acceptable salt thereof, in a single composition.
  • Esters, pharmaceutically acceptable prodrugs or derivatives, and additional therapeutic agents are examples of the combination therapy.
  • the glycoside compound of the present invention in one embodiment of the present invention, its tautomer, optical isomer, solvate, polymorph, pharmaceutically acceptable salt, ester, pharmaceutically acceptable Prodrugs or derivatives can be conveniently administered to a patient. Therefore, the compound for use in the present invention may be an effective amount of a glycoside compound of the present invention in combination with a pharmaceutically acceptable excipient or carrier, its tautomer, optical isomer, solvate, polymorph The form of a pharmaceutical composition of a type, a pharmaceutically acceptable salt, an ester, a pharmaceutically acceptable prodrug or derivative.
  • This aspect can also be expressed as comprising an effective amount of a glycoside compound of the present invention in combination with a pharmaceutically acceptable excipient or carrier, a tautomer, an optical isomer, a solvate, a polymorph, a pharmaceutical Compositions of acceptable salts, esters, pharmaceutically acceptable prodrugs or derivatives are used in the prevention and / or treatment of: cerebral infarction, stroke, atherosclerotic thrombosis, cardiogenic cerebral thrombosis , Acute ischemic cerebrovascular syndrome, small vessel disease (also known as lacunar stroke), multiple cerebral infarction, large-scale cerebral infarction, watershed cerebral infarction, hemorrhagic cerebral infarction, asymptomatic cerebral infarction, brain Venous and sinus thrombosis, abnormal vascular network disease of the skull base, ischemic stroke due to other reasons.
  • the compounds used may be administered orally, injection, subcutaneously, respiratory, transdermal, parenteral, rectal, topical, intravenous, intramuscular, or by other Way to give.
  • the pharmaceutical composition can be formulated into any medicinal form, such as: tablets, granules, injections, gels, pills, capsules, suppositories, implants, nano-formulations, powder injections.
  • Some dosage forms such as tablets and capsules may be subdivided into suitable dosage unit dosage forms containing appropriate amounts of the active ingredients such as effective amounts to achieve the desired purpose.
  • the glycoside compounds of the present invention for use in the present invention are an injection preparation to be administered to a patient to be treated.
  • An injection suitable for the present invention refers to a sterilized or non-sterilized drug that is made of a drug and a suitable solvent or dispersion medium for injection into a human body.
  • Bacterial solutions, emulsions or suspensions, and powdered sterile preparations which are formulated into solutions or suspensions before use.
  • the injection includes an injection solution (wherein a large volume injection solution for intravenous drip infusion is also called an intravenous infusion), a sterile powder for injection, and a concentrated solution for injection.
  • Carriers include excipients and diluents and must be of sufficiently high purity and very low toxicity to make them suitable for administration to a patient to be treated.
  • the carrier may be inert or it may itself have pharmaceutical benefits.
  • the type of carrier includes, but is not limited to, diluents such as fillers and bulking agents, adhesives, lubricants, anti-caking agents, disintegrants, sweeteners, buffers, preservatives, solubilizers, isotonic agents, suspensions And dispersants, wetting or emulsifying agents, flavoring and fragrance agents, thickeners and vehicles.
  • diluents such as fillers and bulking agents, adhesives, lubricants, anti-caking agents, disintegrants, sweeteners, buffers, preservatives, solubilizers, isotonic agents, suspensions And dispersants, wetting or emulsifying agents, flavoring and fragrance agents, thickeners and vehicles.
  • Some carriers can be listed in more than one category, for example, vegetable oils can be used as lubricants in some formulations and as diluents in other formulations.
  • Exemplary pharmaceutical carriers include sugar, starch, cellulose, malt, gelatin, talc,
  • the positive progress effect of the present invention is that the glycoside compounds provided by the present invention have a simple preparation method, can significantly increase the expression of VEGF-A mRNA, and can be used to prepare proangiogenesis.
  • VEGF-A mRNA can significantly increase the expression of VEGF-A mRNA
  • proangiogenesis In order to seek cerebral infarction and heart with proangiogenic activity, Infarction, lower limb ischemia, etc. Research on drugs for circulatory disorders provides a reliable guarantee.
  • the compound or salt of the present application may be the sole active agent administered or may be administered in conjunction with other active agents.
  • a “stereoisomer,” “optical isomer,” or “optical isomer” is a compound that has the same chemical composition but a different arrangement of atoms or groups in space. It includes “diastereomers” and “enantiomers”
  • Diastereomers are stereoisomers that have two or more centers of chirality and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral characteristics, and reactivity. In the presence of a resolving agent or chromatography, a mixture of diastereomers can be separated using high resolution analytical steps such as electrophoresis, crystallization using a chiral HPLC column.
  • Enantiomers refer to two stereoisomers of a compound without overlapping mirror images of each other.
  • a 50:50 mixture of enantiomers is called a racemic mixture or a racemate, which can occur in a chemical reaction or process without stereoselectivity or stereospecificity.
  • Alkyl includes both branched and straight-chain saturated aliphatic hydrocarbon groups and has a specified number of carbon atoms, typically 1 to about 12 carbon atoms.
  • C1-C6 alkyl as used herein, means an alkyl group having from 1 to about 6 carbon atoms.
  • the (phenyl) C0-C4 alkyl group is taken as an example.
  • the specified group, in this case, the phenyl group is through a single covalent bond (C0 ) Is directly bonded or connected through an alkyl chain having a specified number of carbon atoms (in this case, 1 to about 4 carbon atoms).
  • alkyl examples include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, tert-butyl, n-pentyl, and sec-pentyl.
  • alkenyl refers to straight and branched hydrocarbon chains including one or more unsaturated carbon-carbon bonds, which can occur at any stable point along the chain.
  • the alkenyl groups described herein typically have 2 to about 12 carbon atoms.
  • Preferred alkenyl groups are lower alkenyl groups, those alkenyl groups having 2 to about 8 carbon atoms, such as: C 2 -C 8 , C 2 -C 6 , and C 2 -C 4 alkenyl.
  • alkenyl include vinyl, propenyl, and butenyl.
  • Alkoxy refers to an alkyl group, as defined above, having a specified number of carbon atoms connected through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, 3-hexyloxy, and 3-methylpentyloxy.
  • heterocycle means a 5- to 8-membered saturated ring, a partially unsaturated ring, or an aromatic ring containing 1 to about 4 heteroatoms selected from N, O, and S and the remaining ring atoms are carbon, or Is a 7 to 11 membered saturated ring, a partially unsaturated ring, or an aromatic heterocyclic ring system and a 10 to 15-membered tricyclic ring system, the system containing at least 1 heteroatom selected from a polycyclic ring system of N, O, and S And each ring in the polycyclic ring system contains up to about 4 heteroatoms independently selected from N, O, and S.
  • a heterocycle may be attached to a group that it substitutes at any heteroatom and carbon atom and results in a stable structure.
  • the heterocycles described herein may be substituted on a carbon or nitrogen atom, so long as the resulting compound is stable.
  • the nitrogen atom in the heterocyclic ring may optionally be quaternized.
  • the total number of heteroatoms in the heterocyclyl group is preferably not more than 4 and the total number of S and O atoms in the heterocyclyl group is preferably not more than 2, and more preferably not more than 1.
  • heterocyclic groups include: pyridyl, indolyl, pyrimidinyl, pyridizinyl, pyrazinyl, imidazolyl, oxazolyl, furyl, phenylthio, thiazolyl, triazolyl, tetra Oxazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, benz [b] phenylthio (benz [b] thiophenyl), isoquinolinyl, quinazolinyl, quinoxalinyl, Thienyl, isoindolyl, dihydroisoindolyl, 5,6,7,8-tetrahydroisoquinoline, pyridyl, pyrimidinyl, furyl, thienyl, pyrrolyl, pyrazolyl, pyrrolidine , Morpholinyl, piperazinyl, piperidin
  • Aryl or “heteroaryl” means a stable 5- or 6-membered monocyclic ring containing 1 to 4 or preferably 1 to 3 heteroatoms selected from N, O and S and the remaining ring atoms are carbon Or polycyclic.
  • the total number of S and O atoms in the heteroaryl group exceeds 1, these heteroatoms are not adjacent to each other.
  • the total number of S and O atoms in the heteroaryl group is preferably not more than two. It is particularly preferred that the total number of S and O atoms in the heteroaryl group is not more than one.
  • the nitrogen atom in the heterocyclic ring may optionally be quaternized. When specified, these heteroaryl groups may also be substituted with carbon or non-carbon atoms or groups.
  • substitutions may include condensation with a 5 to 7-membered saturated cyclic group optionally containing 1 or 2 heteroatoms independently selected from N, O, and S, thereby forming, for example, [1,3] dioxin Zolo [4,5-c] pyridyl.
  • heteroaryl groups include, but are not limited to: pyridyl, indolyl, pyrimidinyl, pyridazinyl, pyrazinyl, imidazolyl, oxazolyl, furyl, phenylthio, thiazolyl, triazolyl, tetrazolyl Oxazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, benzo [b] phenylthio, isoquinolinyl, quinazolinyl, quinoxalinyl, thienyl, isoindolyl , And 5,6,7,8-tetrahydroisoquinoline.
  • “Pharmaceutically acceptable salt” or “salt of a compound” is a derivative of the disclosed compound, wherein the parent compound is modified by preparing a non-toxic acid or a base addition salt thereof, and also refers to these compounds and these salts Of medicinal solvates, including hydrates.
  • Examples of pharmaceutically acceptable salts include, but are not limited to: inorganic or organic acid addition salts of basic residues such as amines; base or organic addition salts of acidic residues such as carboxylic acids; and the like, and include one or more A combination of the above salts.
  • Pharmaceutically acceptable salts include non-toxic salts and quaternary ammonium salts such as the parent compound formed from non-toxic inorganic or organic acids.
  • non-toxic acid salts include those derived from inorganic acids, such as: hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc .; other acceptable inorganic salts include metal salts such as sodium, potassium, Cesium salts and the like; alkaline earth metal salts such as calcium salts, magnesium salts, etc., and combinations including one or more of the above salts.
  • Organic salts of compounds include compounds such as acetic acid, trifluoroacetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, acetic acid, maleic acid, and hydroxymaleic acid.
  • glycosyl includes monosaccharides, disaccharides, oligosaccharides, polysaccharides, etc., where the monosaccharides include, but are not limited to, erythrosyl, threosyl, arabinyl, ribosyl, xylose, Lexosyl, glucosyl, mannosyl, fructosyl, galactosyl and the like.
  • Disaccharides include, but are not limited to, sucrose, maltosyl, cellobiosyl, isomaltosyl, gentiobiosyl, trehalosyl, and lactosyl.
  • Prodrug refers to a compound that is metabolized in the host, such as hydrolysis or oxidation, to a compound of the invention.
  • Typical examples of prodrugs include compounds having biologically labile protective groups on the functional group of the active compound. Prodrugs can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrated, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated to produce Compounds of active compounds.
  • Derivative refers to a more complex product derived from the replacement of a hydrogen atom or group of atoms by another atom or group of atoms.
  • Fig. 1 is a schematic diagram of compounds promoting the expression of VEGF-A mRNA in an animal model of sponge implantation.
  • Figure 2 shows the infarct rate (%) of MCAO acute cerebral infarction model with different drugs.
  • Figure 3 is the weight change rate (%) of MCAO acute cerebral infarction model of different drugs.
  • Figure 4 is a schematic diagram of the Longa neural scoring criteria. From left to right: 1 point, which means that the contralateral front claw cannot be fully extended; 2 points, which means turning to the opposite side; 3 points, which means dumping to the opposite side; 4 points, which means that it cannot be released by itself, meaning is lost.
  • FIG. 5 is a schematic diagram of Longa nerve score of MCAO acute cerebral infarction model of different drugs.
  • Figure 6 shows the measurement of brain water content and blood-brain barrier integrity of MCAO acute cerebral infarction models of different drugs.
  • Figure 7 shows the rate of change in body weight of the drug on a 2VO chronic cerebral ischemia rat model.
  • Figure 8 shows the results of a Morris water maze localization navigation experiment on a 2VO chronic cerebral ischemia rat model.
  • FIG. 9 is a Morris water maze space exploration experiment result of the drug on a 2VO chronic cerebral ischemia rat model.
  • FIG. 10 shows the results of HE staining and Nissl staining of brain sections after chronic cerebral ischemia.
  • FIG. 11 is a TTC staining result of the inhibitory effect of VEGF protein on MCAO acute cerebral infarction model for 3 days on cerebral infarction.
  • Figure 12 shows the infarction rate of VEGF protein in MCAO acute cerebral infarction model.
  • Figure 13 shows the infarct inhibition rate of VEGF protein in MCAO acute cerebral infarction model.
  • FIG. 14 shows the effect of samples of each group on HUVEC-12 cell proliferation in Effect Example 4.
  • FIG. 15 shows the effect of each group of samples on HUVEC-12 cell migration in Effect Example 4.
  • FIG. 16 is a result of a scratch test of each group of samples in Effect Example 4.
  • FIG. 17 is an inverted microscope view of the effect of each group of samples on tubule formation of HUVEC-12 cells in Effect Example 4.
  • FIG. 18 shows the effect of samples of each group on tubule formation in HUVEC-12 cells in Example 4.
  • Figure 19 shows the effect of salidroside (TG-056) alone at different doses on the expression of VEGF-A mRNA in sponge implanted animal models.
  • Figure 20 shows the infarct rate and Longa nerve score of TG-028 and TG-057 on MCAO acute cerebral infarction model.
  • TG-002, TG-003, TG-004, TG-005, TG-006, TG-007, TG-008, TG-057 were synthesized using a similar synthetic route to compound TG-001, and their structures are as follows.
  • the compounds TG-010, TG-011, and TG-012 were synthesized using a similar synthetic route to compound TG-009.
  • the structures and identification data of compounds TG-009, TG-010, TG-011 and TG-012 are shown below:
  • Route C compounds TG-013, TG-014, TG-015, TG-016, TG-017, TG-018, TG-019, TG-020, TG-021, TG-022, TG-024, TG- Synthesis of 025, TG-026, TG-027, TG-049, TG-050, TG-051 and TG-023
  • Fragment 24a Compounds 28 (2.48 g, 0.02 mol) and 29 (3.336 g, 0.024 mol) were added to DMF (30 mL), K 2 CO 3 (13.8 g, 0.1 mol) was added at room temperature, and the mixture was stirred at room temperature for 48 hours. Water was added for extraction, and the extracts were combined and washed with water, and dried over anhydrous sodium sulfate. The sand core funnel was filtered and concentrated under reduced pressure. Recrystallization from a mixed solution of ethyl acetate and petroleum ether gave 24a: 3.1 g of 85% yield.
  • Fragment 24b 31 (3.72 g, 60 mmol) was added to THF, and NaH (0.624 g, 26 mmol) was added in portions. 30 (3.132 g, 20 mmol) of Bu 4 NI (738 mg, 2 mmol) were sequentially added, heated to 70 ° C., and stirred for 5 h. The reaction solution was lowered to room temperature, washed once with saturated ammonium chloride, and concentrated under reduced pressure. Purified by silica gel column chromatography to obtain 24b: 2.9 g, 80% yield.
  • Fragment 24c 33 (6.1 g, 0.1 mol) was added to MeOH, and 32 (13.6 g, 0.1 mol) was heated to 70 ° C at room temperature and stirred overnight. Cool to room temperature, add NaBH 4 (3.8 g, 0.1 mol) in portions, then heat to 70 ° C. and stir for 2 h. Cool to room temperature and extract with ethyl acetate. The combined extracts were washed with water and dried over anhydrous sodium sulfate. Filtered on a sand core funnel, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 34 (13.4 g, 88% yield).
  • Fragment 24d 34 (3.6 g, 23.8 mmol) was added to dichloromethane, and CbzCl (6.63 g, 0.39 mmol) and Et 3 N (4.5 g, 0.45 mmol) were sequentially added dropwise on an ice bath and stirred for 5 h. The reaction solution was washed once with water, saturated NaHCO 3 solution, and saturated brine in this order. It was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 24d (6g, 63.5% yield).
  • Fragment 24e 35 (6.15 g, 50 mmol), 29 (13.9 g, 100 mol), and K 2 CO 3 (13.8 g, 100 mmol) were sequentially added to n-butanol (40 mL), iodine (50 mg) was added, and the reaction was refluxed for 30 h . It was concentrated under reduced pressure and purified by silica gel column chromatography to obtain 36 8.5 g of the product, 93% yield. 36 (3.3 g, 18.2 mmol) was added to dichloromethane, K 2 CO 3 (4.9 g, 36 mmol) was added, and Cbz-Cl (4 g, 23.7 mmol) was added dropwise in an ice bath and stirred at room temperature overnight. It was washed once with water and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 24e (3 g, 72% yield).
  • TG-046 identification data is as follows:
  • the preparation method of TG-047 refers to the preparation method of TG-048, and its structure is as follows:
  • the specific steps of activity screening are: placing 1 to 10 ⁇ 10 6 logarithmic growth phase macrophages (RAW264.7) in a cell culture dish, and placing the test compound (the above compounds TG-001, TG- 002, TG-003, TG-004, TG-005, TG-006, TG-007, TG-008, TG-009, TG-010, TG-011, TG-012, TG-013, TG-014, TG-015, TG-016, TG-017, TG-018, TG-019, TG-020, TG-021, TG-022, TG-023, TG-024, TG-025, TG-026, TG- 027, TG-028, TG-029, TG-030, TG-031, TG-032, TG-033, TG-034, TG-035, TG-036, TG-037, TG-045, TG-039, TG-040TG-041,
  • RNA reverse transcription reaction system RNA reverse transcription reaction system
  • RT EasyTM I Forward first-strand cDNA synthesis
  • Table 3 RNA reverse transcription reaction system
  • the obtained cDNA was prepared as a fluorescent quantitative PCR reaction solution according to Table 3.
  • Set up the real-time PCR reaction conditions according to Table 4 and use the reverse transcription reaction cDNA as a template to perform the real-time PCR reaction (Real time PCR EasyTM-SYBR Green real-time PCR kit, Chengdu Fuji Biotechnology Co., Ltd.).
  • the Ct value of each sample was calculated using the blank control group as the control and ⁇ -Actin as the internal reference to calculate the relative expression of the target gene 2- ⁇ Ct .
  • TG-052, TG-053, and TG-054 can be synthesized by referring to the method described in Chemical & Pharmaceutical Bulletin, 2010, 58, 1627-1629.
  • Compound TG-056 was purchased from (Shanghai Yuanye Biotechnology Co., Ltd.), and its structure is:
  • the expression of VEGF-A mRNA in macrophages without drugs is set to 1, which is used clinically in the field of cardiovascular and cerebrovascular diseases.
  • Salvia polyphenolate promoted the maximum expression level of VEGF-A mRNA in macrophages 6 (see Table 5 for the results), and the compound with the maximum expression level greater than 4 was the target compound.
  • test substance when the maximum expression level of VEGF-A mRNA in the macrophages promoted by the test substance is more than 4 times higher than the expression level of VEGF-A mRNA in macrophages without the test substance added, the test substance
  • the test substance is the preliminary target pro-angiogenic active substance.
  • Table 6 The results show that most of the analytes provided by the present invention have a good effect of promoting VEGF-A mRNA expression at a certain dose.
  • the target pro-angiogenic compounds TG-052, TG-053, TG-054, TG-028, and TG-055 obtained in Example 1 which exhibited good cell level were used in the "sponge” of inflammatory angiogenesis.
  • Implanted animal model to further screen for active molecules that promote VEGF-A mRNA expression. Relevant research shows that in the "sponge implant animal model", about 75% of the cells that can be recruited to the sponge in the initial stage of inflammation are macrophages, and this model can well reflect the VEGF recruited to the macrophages around the inflammation. -A mRNA expression.
  • the above-mentioned compound obtained by screening in effect example 1 was weighed in two weeks and then mixed with a gel matrix agent and a solvent to prepare a compound sustained-release gel (the preparation method and model establishment of the sustained-release gel and For details of the detection method, refer to the patent application CN201610168493.9).
  • the specific steps are: Weigh a certain amount of the compound obtained in Example 1 into a 1.5mL EP tube, and then weigh a certain amount of L-polylactic acid with a weight-average molecular weight of less than 10,000.
  • PLLA PLLA mixed with it (the mass ratio of the obtained compound to PLLA is between 0.5: 1 to 2: 1), and finally add a certain amount of DMSO (less than 100 microliters), and vortex to seal with a sealing film.
  • Sustained-release gel of the compound was obtained at 200W power and continuous ultrasonication for 5 hours at 25 ° C, and stored at 4 ° C until use.
  • the prepared sustained-release gel can release the drug completely within 2 weeks.
  • the Ct value of each sample was calculated using the blank control group as the control and ⁇ -Actin as the internal reference to calculate the relative expression of the target gene 2- ⁇ Ct . To observe the expression level of VEGF-A mRNA in the cells extracted from the sponge under the action time of 2 weeks.
  • TG-055 (19.48mg / kg / d) is less than TG-052 (41.37mg / kg / d), and its activity is better than TG-052;
  • the expression levels of TG-053 and TG-054VEGF-A mRNA were slightly lower than those of Danshen polyphenate (60mg / kg / d);
  • TG-028 was at the doses of 11.74mg / kg / d and 23.48mg / kg / d
  • the expression of VEGF-A mRNA was more than 6 times that of the blank control without drug addition, and the activity was significantly higher than the activity of salvia dofenate (60mg / kg / d).
  • the activity result can be expressed as: TG-028> TG-055> TG-052> danshendofenate> TG-053> TG-054.
  • the results show that most of the compounds screened by the in vitro cell experiment in Example 1 have better activity in the in vivo animal screening model than the commercially available drug salvia polyphenolate or equivalent.
  • VEGF-A mRNA As shown in FIG. 19, the expression of VEGF-A mRNA was very low in sponge implanted animal models using salidroside (TG-056) alone at different doses.
  • salidroside and tyrosol LC the main component in salidroside injection
  • salidroside 20.62mg /(kg.d)+tyrosol 3.08mg / (kg.d) can promote the expression of VEGF-A mRNA, which is not as effective as salvia polyphenolate. Therefore, we will not consider the salidroside in subsequent experiments. effect.
  • the rat cerebral infarction model was used for further verification.
  • the methods described in Longa, EZ, Longa, EZ, Weinstein, PR, Carlson, S. & Commin, R. Remsible, ceramic, arterial, and occlusion without craniectomy in rats [J] .Stroke, 1989.1: 84–91 Arterial occlusion (MCAO) acute cerebral infarction model, and according to C. dela Torre, T. Fortin, GAS Park, KS Butler, P. Kozlowski, BAPappas, H. de Socarraz, JKSaunders and MTRichard.
  • TG-028 and TG-055 which show high activity in Example 2, were selected for activity verification.
  • the control drug used was edaravone (purchased from Zhejiang Shengtong Biotechnology Co., Ltd. Company), clopidogrel hydrogen sulfate (purchased from Wuhan Yuancheng Gongchuang Technology Co., Ltd.) and salvia polyphenolate (purchased from Henan Provincial People's Hospital), of which clopidogrel hydrogen sulfate is a platelet aggregation inhibitor.
  • Daravone is a brain protectant (free radical scavenger).
  • the rat MCAO model was made by using the suture method.
  • the rats used in this effect example were SD rats, male, 280-320 g, purchased from Shanghai Slark Experimental Animal Co., Ltd. Five SD (Sprague Dawley) rats in each group were administered by intraperitoneal injection, 2 hours before surgery, and every 24 hours after modeling. The specific doses are shown in Table 7.
  • infarct rate (right brain slice Non-infarcted area-left infarcted non-infarcted area) / total area of brain slice * 100%, according to ttests test, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 compared with model group (MCAO) The results are shown in Figure 2.
  • the experimental rats were scored for neurological function according to the Longa neural score standard.
  • the schematic diagram of the score standard is shown in FIG. 4. Among them, 0 points indicate normal and no nerve damage; 1 point indicates that the contralateral forelimb cannot be fully extended; 2 points indicate a circle to the opposite side; 3 points indicate a dump to the opposite side; 4 points indicate that the spontaneous release cannot be performed, meaning is lost.
  • the experimental rats were scored for nerve function, and Longa nerve scores of different doses of different drugs were obtained.
  • the results are shown in FIG. 5.
  • the results of the Longa neural scoring standard show that TG-028 and TG-055 have neuroprotective effects after trauma to the brain of rats as compared to edaravone, clopidogrel hydrogen sulfate, and salvia polyphenolate.
  • TG-057 On the basis of the above experiments, the activity of TG-057 was also tested. As shown in Figure 20, at the same dosage (5mg / kg) as TG-028, TG-057 also inhibited the brain of rats. Infarct effect, Longa nerve score results show that TG-057 has protective effects on nerves after brain injury in rats, but its effect is not as strong as TG-028.
  • mice were tested for the optimal dose of each drug: TG-028 (5mg / Kg), salvia polyphenolate (20mg / Kg), clopidogrel hydrogen sulfate (40mg / Kg) Kg), edaravone (40 mg / Kg).
  • FIG. 6A shows (Sham is a sham operation group) that TG-028 reduces cerebral edema after MCAO acute brain injury in rats is better than edaravone, clopidogrel bisulfate, and salvia polyphenolate.
  • Figure 6B shows that TG-028 protects the blood-brain barrier better than edaravone and salvia polyphenolate after MCAO acute brain injury in rats.
  • TG-028 5mg / kg was administered daily by intraperitoneal injection for a total of 3 weeks, 5 in each group.
  • edaravone 40 mg / kg
  • the weight change of rats in 3 weeks is shown in Fig. 7 (Sham is a sham operation group).
  • the TG-028 and edaravone groups were compared with the sham operation group. All had weight loss, but after 1 week, there was no difference in body weight between the TG-028 group and the sham operation group, indicating that TG-028 has a protective effect on body weight, while the edaravone group compared with the sham operation group.
  • the weight gain is not obvious, and the protective effect on body weight is low.
  • the brain sections after 3 weeks of chronic cerebral ischemia were subjected to HE staining (hematoxylin-eosin staining) and Nissl staining (Nissl staining).
  • the results are shown in Fig. 10 (Sham is a sham operation group).
  • the results showed that compared with the 2VO + NaCl group, the neurons in the hippocampal CA3 region of the 2VO + TG-028 group were neatly arranged, the particle size was uniform and clear, and the cell morphology of the 2VO + edarav group was also improved. It is shown that both TG-028 and edaravone can protect and repair damaged neurons and promote the improvement of neural function.
  • TG-028 has a large and safe treatment window. Sex is good.
  • TG-028 acts on macrophages, it can promote endothelial cell proliferation, migration, and tubule formation, thereby affecting the formation of new blood vessels.
  • TG-028 can promote the proliferation, migration, and tubule formation of endothelial cells by acting on macrophages, thereby affecting the formation of new blood vessels.
  • the experiment was divided into seven groups.
  • the first group was pure DMEM culture medium (Gibcol, Shanghai) plus 10% FBS (SERANA, Germany) and directly acted on HUVEC-12 endothelial cells (purchased from Shanghai Rongchuang Biological Co., Ltd.).
  • the macrophages in the group were cultured with DMEM medium and 10% FBS for 24 hours, and the supernatant was applied to endothelial cells (s-control).
  • the third group was TG-028, which directly affected endothelial cells cultured with DMEM and 10% FBS.
  • the fourth group was TG-028 acting on macrophages (DMEM culture medium plus 10% FBS) and co-cultivating the supernatant after 24 hours acting on endothelial cells (s-TG-028, 10 ⁇ M);
  • the fifth group is Cpd-18 (danshen polyphenolate) directly acting on DMEM culture medium plus 10% FBS cultured endothelial cells (cpd-18, 10 ⁇ M);
  • the sixth group is Cpd-18 (danshen polyphenol) (10 ⁇ M) on macrophages (DMEM culture medium plus 10% FBS) after 24 hours of culture, the supernatant acts on endothelial cells (s-cpd18);
  • the seventh group is VEGF protein directly acting on DMEM culture medium plus 10 % FBS cultured endothelial cells (Recombinant Human VEGF 165, 10 ng / mL).
  • HUVEC-12 cells in logarithmic growth phase were taken for cell counting, the cell density was adjusted to 2.5 ⁇ 10 4 cells / mL, and 100 ⁇ L per well was seeded on a 96-well culture plate. Put it in the incubator overnight and completely adhere to the wall. According to the above different groups, HUVEC-12 endothelial cells were processed, and the 96-well plates were placed in a 37 ° C, 5% CO2 incubator for 24 hours. Remove the 96-well plate and add 5 mg / mL MTT, 20 ⁇ L / well, and continue to culture for 4 h. After 4 hours, the supernatant was discarded and DMSO was added.
  • HUVEC-12 cells Take human umbilical vein endothelial cells HUVEC-12 cells in logarithmic growth phase for cell counting, adjust the cell density to 5 ⁇ 10 5 cells / mL, and inoculate 1 mL per well to a 6-well plate. Put it into an incubator for routine culture to form a 90% confluent monolayer cell. Take out the cells covered with a six-well plate, and use a 200L pipette tip to be perpendicular to the cell surface and draw from the end of the well to the other end as much as possible. Scratches. At this moment, a chevron-shaped scratch on the cell surface can be clearly observed on the surface of the culture dish.
  • the TG-028 group ie, the third group
  • the VEGF group ie, the seventh group
  • s-TG-028 the fourth group
  • s-cpd18 the sixth group
  • the VEGF group Compared with the effect of the supernatant of macrophages cultured for 24 hours on the endothelial cell (s-control) group (ie, the second group), the VEGF group, s-TG-028 and s-cpd18 all had better migration effects on endothelial cells.
  • the s-control group it was shown that the compounds TG-028 and salvia polyphenolate can promote the migration of endothelial cells by acting on macrophages.
  • the 96-well plate and the sterile yellow pipette tip were refrigerated at -20 ° C overnight. Put the divided Matrigel Matrigel in the refrigerator at 4 ° C overnight before thawing. After Matrigel Matrigel freeze-thaw the next day, centrifuge for several minutes. Put the pre-chilled Matrigel Matrigel on a clean bench in a 96-well plate on ice, add 60 ⁇ L per well, and place it in the incubator for 1 h.
  • Matrigel Matrigel After Matrigel Matrigel has solidified, take HUVEC-12 cells in logarithmic growth phase, adjust the cell density, mix with each group of culture solution at a density of 5 ⁇ 10 4 cells / well and inoculate them into 96-well plates, place at 37 ° C, 5 After 3 hours in the% CO 2 incubator, remove the cell culture plate, and start to continuously observe and take pictures under an inverted microscope (magnification 50 ⁇ , German Leica DMi1 inverted microscope). Randomly select 5 fields of vision for each hole to observe the formation of cell connections. The tube-like structure was analyzed with ImageJ image analysis software, and the number of master junction quantitative index was used to evaluate the tube-forming ability. The results are shown in Figs. 17 and 18.
  • the compound TG-028 selected by the activity screening method of the present invention can promote the proliferation, migration, and tubule formation of endothelial cells by acting on macrophages, thereby affecting the formation of new blood vessels.
  • the in vitro macrophage experiments of the present invention have screened out preliminary target pro-angiogenic active substances from a large number of test compounds, which have been shown in subsequent semi-in vivo animal screening models.
  • a good angiogenic active substance was produced, and the activity of compound TG-028 was even better than that of salvia polyphenolate. That is, the present invention can screen the target pro-angiogenic active substance from the molecular level, and further subsequent in vivo animal model experiments have verified the accuracy and stability of the activity screening method of the present invention.
  • the compound TG-028 can promote the proliferation, migration, and tubule formation of endothelial cells mainly by acting on macrophages, thereby affecting the formation of new blood vessels, which in turn contradicts the selection of macrophages in vitro.
  • the compound screened out according to the activity screening method of the present invention is the expected compound, and can achieve the promotion of angiogenesis and thereby inhibit cerebral infarction, neuroprotective function, reduce cerebral edema, protect the integrity of the blood-brain barrier after injury, and affect body weight after brain injury.

Abstract

公开了一种如式（III）所示的糖苷类化合物、其制备方法、组合物、应用及中间体。所提供的糖苷类化合物其制备方法简单，且能显著提高VEGF-A mRNA的表达，可有效促进血管生成，为寻求具有促血管生成活性治疗脑梗死、心梗、下肢缺血性等微循环障碍疾病药物的研究提供了可靠的保障。

Description

糖苷类化合物、其制备方法、组合物、应用及中间体 技术领域
本发明涉及糖苷类化合物、其制备方法、组合物、应用及中间体。
背景技术
近年来针对血管生成的药物研究备受人们关注，血管生成已成为在癌症、血管疾病和慢性炎症的治疗中药物干预的潜在的生物靶点。如旨在减少肿瘤血管生成的抗血管生成抑制剂的药物研发，已成为许多晚期和侵袭性癌症的成功疗法。而促血管新生的发现已为有效治疗缺血性血管疾病提供了新的方向，目前也已成为医学领域的研究热点。通过促进血管生成作用，可望改善微循环障碍所致的心脑血管疾病(如心绞痛、心肌梗死、脑梗死、中风)及下肢缺血(如糖尿病下肢血管病变、血栓闭塞性脉管炎)等危害人类健康的重大疾病。
其中脑血管疾病是指脑部动脉或支配脑的颈部动脉发生病变，从而引起颅内血液循环障碍，脑组织受损的一组疾病。临床上常以猝然昏倒、不省人事、或伴有口眼歪斜、言语不利和偏瘫为主要表现。缺血性脑血管病主要是指脑血栓形成、脑栓塞、多发性脑梗死等；其疾病特点为发病突然，进展迅速，病情危重，又因多发于老年人，易合并多发脏器功能损伤，预后不佳，死亡率较高。缺血性脑血管病由于脑动脉的血流急性中断致相应区域的脑组织缺血坏死，故称脑梗死。
脑卒中(cerebral stroke)又称“中风”、脑血管意外(cerebralvascular accident,CVA)，是世界上最重要的致死性疾病之一，是由于脑部血管突然破裂或因血管阻塞导致血液不能流入大脑而引起脑组织损伤的一组疾病，包括缺血性和出血性卒中。缺血性卒中的发病率高于出血性卒中，缺血性脑卒中系由各种原因所致的局部脑组织区域血液供应障碍，导致脑组织缺血缺氧性病变坏死，进而产生临床上对应的神经功能缺失表现，严重影响着患者的生存质量。
对于缺血损伤病理干预的研究热点主要是改善脑血循环和神经保护。其中，改善脑血循环的措施目前主要是抗血栓治疗。抗血栓药分为溶栓药、抗血小板聚集药和抗凝血药。神经保护药物目前主要有钙离子拮抗剂、谷氨酸拮抗剂、谷氨酸释放抑制剂、GABA受体激动剂、自由基清除剂、细胞膜稳定剂等。
近年促血管新生的发现为有效治疗缺血性血管疾病提供了新的方向，目前也已成为医学领域的研究热点。血管新生能促进脑卒中后神经元存活，改善患者神经功能缺损及卒中后生存质量，但脑卒中后血管新生的影响因素及调控机制复杂。近年研究发现，PA R1参与卒中后微血管新生、神经修复等过程。血管新生是指在原有血管的基础上，通过芽生和(或)非芽生方式形成新的毛细血管。血管新生的主要过程包括:血管通透性增加；产生蛋白水解酶，降解细胞外基质，促进内皮细胞增殖；内皮细胞从基底膜上分离，迁移到血管周围间隙，通过黏附—增殖—重构，组成三维管腔；分化为新的毛细血管；间质细胞在中介分子诱导下进入血管壁，使血管稳定成熟。在正常生理状态下，机体内的血管一旦生成就保持高度的稳定性，并且受许多具有正向或负向调节作用的关键分子(即促血管生长因子和抑血管生长因子)调控。血管新生的启动仅随刺激信号的出现而短暂开启，随即关闭，维持血管新生与减退的动态平衡。脑卒中后影响微血管新生的因素包括:局部供血供氧情况；凝血酶及其浓度变化；促血管生成因子水平，如缺氧诱导因子1α(HIF-1α)、血管内皮生长因子(VEGF)、基质金属蛋白酶(MMPs)、促血管生成素1(Ang-1)、促血管生成素2(Ang-2)等。PAR1通常与促血管生成因子相互作用，发挥促进血管新生的作用。VEGF是目前公认的对血管新生起关键作用的因子，正常情况下VEGF仅少量表达，以维持生理状态下的血管密度和通透性。而一些病理过程如炎症、肿瘤、创伤愈合、缺血、缺氧等可促进VEGF表达。脑卒中患者卒中病灶周围神经元、胶质细胞中VEGF表达增加，通过与内皮细胞表面受体特异性结合，促进血管内皮细胞增殖和迁徙、增加血管通透性、增强降解细胞外基质的因子表达，从而促进微血管新生。
但是具有明确的治疗性血管生成作用的药物目前仍处于从实验室向临床过渡的阶段。例如美国Cardium Therapeutics公司的产品Generx是血管生成FGF4基因疗法，用于治疗心肌微血管功能不全，目前处于III期临床研究阶段。
因此，如何寻求一种工艺简单稳定且有效的具有促血管生成活性的药物是本领域技术人员的研发热点与难点。
发明内容
本发明所要解决的技术问题是为了填补现有技术中具有明确治疗性血管生成作用的药物的市场空白及药物制备困难等问题，从而提供了一种糖苷类化合物、其制备方法、组合物、应用及中间体。本发明所提供的糖苷类化合物制备方法简单，能显著提高VEGF-A mRNA的表达，可用于制备促血管生成，为寻求具有促血管生成活性的治疗缺血性心脑血管疾病尤其是脑梗死(脑卒中)、心梗，下肢缺血性
Figure PCTCN2019085823-appb-000001
循环障碍疾病的药物的研究提供了可靠的保障。
本发明是通过以下技术方案解决上述技术问题的。
本发明提供了一种如下式III所示的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物：
Figure PCTCN2019085823-appb-000002
R 1、R 2、R 3、R 4和R 5各自独立地选自氢、羟基、巯基、取代或未取代的C 1～C 20烷氧基、硝基或卤素；
或者R 1、R 2、R 3、R 4和R 5中任意相邻两个与其苯环上相连的碳原子形成5-7元杂环，所述杂环的杂原子为O或S；所述杂原子的数目为1个或多个；当杂原子数目多个时，所述杂原子相同或不同；
所述取代或未取代的C 1～C 20烷氧基中的取代基选自C 3-C 20环烷基、C 1-C 20烯烃基、取代或未取代的C 6-C 20芳基、卤素、取代或未取代的C 2-C 20杂芳基、C 3-C 6的环烷氧基、C 1～C 20的烷氧基或C 1～C 20烷基；
所述取代或未取代的C 6-C 20的芳基和取代或未取代的C 2-C 20杂芳基中的取代基各自独立地为卤素或卤素取代的C 1～C 20烷基；
X为CH 2、NR 7、O或S；
Y为CH 2、NR 8、O或S；
Z为O或S；
R 7和R 8各自独立地为氢、芳基取代的C 1-C 6烷氧羰基，或C 1-C 6烷氧羰基；
每个R 6各自独立地为氢或糖基；
n为2，3或4；
优选地：
当X为CH 2，Y为CH 2，n＝2，3或4时，R 1-R 5不同时是H；
当n＝2，R 1、R 4和R 5是H时，R 2和R 3不同时是OCH 3和OH；
当n＝2，R 1、R 2和R 5是H时，R 3和R 4不同时是OH和OCH 3；
当n＝2，R 2、R 4和R 5是H时，R 1和R 3不同时是OH；
当n＝2，R 1、R 2和R 4是H时，R 3和R 5不同时是OH。
本发明提供了一种如下式I所示的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物：
Figure PCTCN2019085823-appb-000003
R 1、R 2、R 3、R 4和R 5各自独立地选自氢、羟基、巯基、取代或未取代的C 1～C 20烷氧基(例如取代或未取代的C 1～C 10烷氧基，例如取代或未取代的C 1～C 6烷氧基，)、硝基或卤素；
或者R 1、R 2、R 3、R 4和R 5中任意相邻两个相与其苯环上相连的碳原子形成5-7元杂环(例如所述R 2和R 3形成5-7元杂环)，所述杂环的杂原子为O或S(例如O)；所述杂原子的数目为1个或多个(例如两个)；当杂原子数目多个时，所述杂原子相同或不同；
所述取代或未取代的C 1～C 20烷氧基中的取代基选自C 3-C 20环烷基(例如C 3-C 10环烷基，再例如为C 3-C 6环烷基)、C 2-C 20烯烃基(例如C 2-C 6烯烃基，例如C 2-C 4烯烃基，再例如
Figure PCTCN2019085823-appb-000004
)、取代或未取代的C 6-C 20芳基(例如取代或未取代的C 6-C 10芳基，再例如取代或未取代的芳基或萘基)、卤素(例如氟、氯、溴或碘)、取代或未取代的C 2-C 20杂芳基所述取代或未取代的C 2-C 20杂芳基中的杂原子可选自N、S和O中的一个或多个，(例如杂原子数目为1个；再例如所述杂原子为N；例如，所述取代或未取代的C 2-C 20杂芳基中可为仅有一个环的芳杂基)。
例如取代或未取代的C 2-C 10杂芳基，再例如取代或未取代的C 2-C 6杂芳基)、C 3-C 6的环烷氧基(例如
Figure PCTCN2019085823-appb-000005
))、C 1～C 20的烷氧基(例如C 1～C 10烷氧基，例如C 1～C 6烷氧基，再例如甲氧基、乙氧基或丙氧基)或C 1～C 20烷基(例如C 1～C 10烷基；例如C 1～C 6 烷基；再例如C 1～C 3烷基)；
所述取代或未取代的C 1-C 20的芳基和取代或未取代的C 2-C 20杂芳基中的取代基各自独立地选自卤素(例如氟、氯、溴或碘)或卤素(例如氟、氯、溴或碘)取代的C 1～C 20烷基(所述卤素取代的C 1～C 20烷基中的取代基的个数可为1个或多个，当为多个取代基时，各取代基可相同或不同；例如为卤素取代的C 1～C 10烷基；例如卤素取代的C 1～C 6烷基；例如卤素取代的C 1～C 3烷基；再例如-CF 3、-CHF 2或-CH 2F)；
X为CH 2、NR 7、O或S；
Y为CH 2、NR 8、O或S；
Z为O或S；
R 7和R 8各自独立地为氢、芳基取代的C 1-C 6烷氧羰基(例如苄氧羰基)或C 1-C 6烷氧羰基(例如叔丁氧羰基)；
R 6为氢或
Figure PCTCN2019085823-appb-000006
n为2，3或4。
优选地：
当X为CH 2，Y为CH 2，n＝2，3或4时，R 1-R 5不同时是H；
当n＝2，R 1、R 4和R 5是H时，R 2和R 3不同时是OCH 3和OH；
当n＝2，R 1、R 2和R 5是H时，R 3和R 4不同时是OH和OCH 3。
本发明中，所述取代或未取代的C 2-C 20杂芳基可为取代或未取代的吡咯，取代或未取代的呋喃，取代或未取代的噻吩，取代或未取代的吡唑，取代或未取代的咪唑，取代或未取代的噁唑，取代或未取代的噻唑，取代或未取代的异噁唑，取代或未取代的吡啶，取代或未取代的哒嗪，取代或未取代的嘧啶，或取代或未取代的吡嗪，例如取代或取代的吡啶基。
本发明中，所述未取代的C 1～C 20烷氧基可为
Figure PCTCN2019085823-appb-000007
Figure PCTCN2019085823-appb-000008
本发明中，所述C 3-C 20环烷基可为环丙基、环丁基、环戊基或环己基。
本发明中，所述取代的C 6-C 20的芳基可为
Figure PCTCN2019085823-appb-000009
本发明中，所述的取代的C 1～C 20烷氧基可为
Figure PCTCN2019085823-appb-000010
Figure PCTCN2019085823-appb-000011
本发明中，所述未取代的C 2-C 20杂芳基可为
Figure PCTCN2019085823-appb-000012
本发明中，所述5-7元杂环可为
Figure PCTCN2019085823-appb-000013
本发明中，所述R 1可为氢。
本发明中，所述R 2可为氢，取代或未取代的C 1～C 20烷氧基，或所述R 2和R 3形成
Figure PCTCN2019085823-appb-000014
所述取代或未取代的C 1～C 20烷氧基及其取代基的定义如前所述。
本发明中，所述R 3可为氢、羟基、巯基、取代或未取代的C 1～C 20烷氧基、羟基、硝基或卤素；所述取代或未取代的C 1～C 20烷氧基及其取代基、和所述卤素的定义如前所述。
本发明中，所述R 4可为氢，或取代或未取代的C 1～C 20烷氧基；所述取代或未取代的C 1～C 20烷氧基及其取代基的定义如前所述。
本发明中，所述R 5可为氢。
本发明一实施例方式中，各基团定义如下所述(未提及的基团定义如前所述)：所述X为CH 2，所述Y为CH 2、NR 8、S或O；所述R 8为氢、芳基取代的烷氧羰基或烷氧羰基。
本发明一实施例方式中，各基团定义如下所述(未提及的基团定义如前所述)：所述X为NR 7，所述Y为CH 2；所述R 7为氢、芳基取代的烷氧羰基或烷氧羰基。
本发明一实施例方式中，各基团定义如下所述(未提及的基团定义如前所述)：所述X为O或S，所述Y为CH 2。
本发明中，当所述R 1、R 2、R 3、R 4和R 5各自独立地选自氢、羟基、巯基、取代或未取代的C 1～C 20烷氧基、硝基或卤素时，所述取代或未取代的C 1～C 20烷氧基的个数可为一个或多个(例如1个，2个，或3个；再例如1个)。
本发明中，所述
Figure PCTCN2019085823-appb-000015
可为以下任一结构：
Figure PCTCN2019085823-appb-000016
Figure PCTCN2019085823-appb-000017
Figure PCTCN2019085823-appb-000018
本发明中，较佳地，所述n＝2。
本发明中，较佳地，所述R 1、R 2、R 3、R 4和R 5各自独立地选自氢、取代或未取代的C 1～C 20烷氧基、硝基或卤素。
本发明中，较佳地，所述R 6为氢。
本发明中，较佳地，所述式I所示的化合物中的
Figure PCTCN2019085823-appb-000019
的光学异构体可为：
Figure PCTCN2019085823-appb-000020
更佳地为
Figure PCTCN2019085823-appb-000021
在本发明某一实施方式中，所述式I所述的化合物的取代基可为以下情况(未提及的取代基的定义如前所述)：
所述n为2，3或4；
所述X为CH 2、NR 7、O或S。
在本发明某一实施方式中，所述式I所述的化合物的取代基可为以下情况(未提及的取代基的定义如前所述)：
所述n为2，3或4；
Y为CH 2、NR 8、O或S。
在本发明某一实施方式中，所述式I所述的化合物的取代基可为以下情况(未提及的取代基的定义如前所述)：
所述R 1为氢；
所述R 2为氢，取代或未取代的C 1～C 20烷氧基，或所述R 2和R 3形成
Figure PCTCN2019085823-appb-000022
所述取代或未取代的C 1～C 20烷氧基及其取代基的定义如前所述；
所述R 3为氢、羟基、巯基、取代或未取代的C 1～C 20烷氧基、羟基、硝基或卤素；所述取代或未取代的C 1～C 20烷氧基及其取代基、和所述卤素的定义如前所述；
所述R 4为氢，或取代或未取代的C 1～C 20烷氧基；所述取代或未取代的C 1～C 20烷氧基及其取代基的定义如前所述；
所述R 5为氢。
在本发明某一实施方式中，所述式I所述的化合物的取代基可为以下情况(未提及的取代基的定义如前所述)：
所述n为2；
所述X为CH 2时，所述Y为CH 2、NR 8、S或O；
所述所述X为NR 7时，所述Y为CH 2；
所述X为O或S时，所述Y为CH 2。
本发明还提供了以下任一结构的化合物：
Figure PCTCN2019085823-appb-000023
Figure PCTCN2019085823-appb-000024
优选地，这些化合物是：
Figure PCTCN2019085823-appb-000025
Figure PCTCN2019085823-appb-000026
Figure PCTCN2019085823-appb-000027
Figure PCTCN2019085823-appb-000028
本发明还提供了糖苷类化合物的制备方法，其特征在于，包含以下步骤：溶剂中，将式V所示化合物进行还原反应，得到式VI所示化合物；
Figure PCTCN2019085823-appb-000029
R 9’，R 10’和R 11’各自独立地为氢或Ac，且其中至少一个为Ac；R 6’为氢、Ac或者
Figure PCTCN2019085823-appb-000030
R 12’,R 13’,R 14’,和R 15’各自独立地为氢、Bz或Ac；n,X，Y，Z，R 1，R 2，R 3，R 4，R 5和R 6的定义如上任一项中所述；当R 6’为氢或Ac时，R 6为氢；当R 6’为
Figure PCTCN2019085823-appb-000031
时，R 6为
Figure PCTCN2019085823-appb-000032
本发明还提供了式I所示化合物的制备方法，其包含以下步骤：溶剂中，将式II所示化合物进行还原反应，得到式I所示化合物，即可；
Figure PCTCN2019085823-appb-000033
R 9’，R 10’和R 11’各自独立地为氢或Ac，且其中至少一个为Ac；R 6’为氢、Ac或者
Figure PCTCN2019085823-appb-000034
R 12’,R 13’,R 14’,和R 15’各自独立地为氢、Bz或Ac；n,X，Y，Z，R 1，R 2，R 3，R 4，R 5和R 6的定义如前所述；当R 6’为氢或Ac时,R 6为氢；当R 6’为
Figure PCTCN2019085823-appb-000035
时，R 6为
Figure PCTCN2019085823-appb-000036
本发明还提供了上述所示的糖苷类化合物，或如下所示化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，在制备促血管生成药物中的应用；
Figure PCTCN2019085823-appb-000037
本发明还提供了式I所示的糖苷类化合物，化合物TG-052、化合物TG-053、化合物TG-054或TG-057，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物在制备促血管生成药物中的应用；
Figure PCTCN2019085823-appb-000038
本发明还提供了上述所示的糖苷类化合物，下式所示化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，在制备治疗缺血性心脑血管疾病尤其是脑梗死(脑卒中)、心梗，下肢缺血性
Figure PCTCN2019085823-appb-000039
循环障碍疾病的药物中的应用；
Figure PCTCN2019085823-appb-000040
本发明还提供了式I所示的糖苷类化合物，化合物TG-052、化合物TG-053或化合物TG-054，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物在制备治疗缺血性心脑血管疾病尤其是脑梗死(脑卒中)、心梗，下肢缺血性
Figure PCTCN2019085823-appb-000041
循环障碍疾病的药物中的应用；
Figure PCTCN2019085823-appb-000042
所述缺血性心脑血管疾病涉及血管壁病变、血液成分改变和/或血流动力学变化导致的血管病变引发的心脏组织、脑部组织缺血的初期至晚期所有症状和/或病理变化，例如脑梗死(脑卒中)或心梗。
本发明还提供了上述所示糖苷类化合物的中间体化合物：
Figure PCTCN2019085823-appb-000043
Figure PCTCN2019085823-appb-000044
本发明还提供了式I所示糖苷类化合物的中间体化合物：
Figure PCTCN2019085823-appb-000045
Figure PCTCN2019085823-appb-000046
Figure PCTCN2019085823-appb-000047
Figure PCTCN2019085823-appb-000048
本发明进一步提供上述糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多 晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，其用于在预防和/或治疗促血管生成的药物中的应用，优选在缺血性血管病中使用，进一步优选在缺血性心脑血管疾病尤其是脑梗死(脑卒中)、心梗，下肢缺血性疾病中使用。这一方面可以表达为上述糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物用于制备药物的用途，所述药物通过促血管生成用于预防和/或治疗哺乳动物(包括人)的缺血性血管疾病。进一步优选在缺血性心脑血管疾病尤其是脑梗死(脑卒中)、心梗，下肢缺血性疾病中使用。另一方面还可以表达为促进有需要的患者血管生成的方法，优选为治疗和/或预防包括患有或易于患有缺血性血管疾病的哺乳动物(包括人)的方法，进一步是指患有缺血性心脑血管病尤其是脑梗死(脑卒中)、心梗，下肢缺血性
Figure PCTCN2019085823-appb-000049
循环障碍疾病的哺乳动物(包括人)的方法，所述方法包括向所述患者施用治疗有效量的本发明上述糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，连同药用赋形剂或载体。
进一步本发明的上述糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物用于预防和/或治疗缺血性脑血管疾病的用途。缺血性脑血管疾病的实例包括：脑卒中、动脉粥样硬化性脑血栓形成、心源性脑血栓、急性缺血性脑血管综合征、小血管病变(也称为腔隙性脑卒中)、多发性脑梗死、大面积脑梗死、分水岭脑梗死、出血性脑梗死、无症状的脑梗死、脑静脉及静脉窦血栓形成、颅底异常血管网病、其他原因所致的缺血性脑卒中。
在一个优选的实施方案中，缺血性脑血管疾病是由动脉粥样硬化性血栓形成，动脉粥样硬化性血栓形成即在动脉粥样硬化及各类动脉炎等血管病变的基础上形成血栓导致管腔闭塞，引起脑组织缺血缺氧、坏死、软化等临床综合征。
在另一个优选的实施方案中，缺血性脑血管疾病是由心源性脑栓塞形成，心源性脑栓塞是指心源性栓子循血流径路向远端迁移进入脑动脉系统后导致血管闭塞引起的脑梗死。
在另一个优选的实施方案中，缺血性脑血管疾病是由急性缺血性脑血管综合征形成，急性缺血性脑血管综合征包括3种疾病：短暂性脑缺血发作、可逆性缺血性神经功能缺失和完全恢复性脑卒中。
在另一个优选的实施方案中，脑血管疾病是指脑卒中，脑卒中是由于脑部血管突然破裂或因血管阻塞导致血液不能流入大脑而引起脑组织损伤的一组疾病，包括缺血性和出血性卒中。
优选地将治疗有效量的本发明上述糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物根据通常给药途径并且根据本领域已知方法以常规药物组合物(该药物组合物包含有效量的活性成分和合适的药用载体)和剂型配制而施用至需要这样治疗的患者。
治疗有效量”是指这样的活性成分的量，当施用时，其足以防止所针对的疾病的一种或多种症状的发展，或在某种程度上缓解所述一种或多种症状。根据本发明施用的化合物的具体剂量将由围绕该病例的具体情况确定，所述情况包括所施用的化合物、给药途径、治疗的具体病况以及类似的考虑因素。特别地，“治疗有效量的化合物”是指足以防止或在某种程度上缓解一种或多种缺血性血管疾病的化合物的量，所述缺血性血管疾病包括缺血性心脑血管病尤其是脑梗死(脑卒中)、心梗，下肢缺血性疾病等，进一步，缺血性脑血管疾病包括脑卒中、动脉粥样硬化性血栓形成、心源性脑血栓、急性缺血性脑血管综合征、小血管病变(也称为腔隙性脑卒中)、多发性脑梗死、大面积脑梗死、分水岭脑梗死、出血性脑梗死、无症状的脑梗死、脑静脉及静脉窦血栓形成、颅底异常血管网病、其他原因所致的缺血性脑卒中。
进一步根据待治疗的缺血性血管疾病的类型和严重性以及具体患者对药物治疗的反应，单个剂量以及日剂量不同。因此，将根据在医生的指导下的标准医学原理来确定准确的单个剂量。
用于在治疗缺血性血管疾病中使用本发明糖苷类化合物的有效日剂量为0.01mg至约1000mg、约1mg至约500mg、或约10mg至约200mg以及可选的约0.01mg至约2000mg、 约10mg至约1000mg、约100mg至约800mg、或约200mg至约600mg的其他活性剂的口服剂型。
本发明的上述糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物可以单独使用或组合使用或与其他治疗剂的联合疗法使用。
在本发明的一个实施方案中，上述糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物用于在预防和/或治疗缺血性血管疾病中使用，其中所述预防或治疗包括施用作为唯一活性成分使用。
在本发明的另一个实施方案中，上述糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物用于在预防和/或治疗缺血性血管疾病中使用，其中所述预防或治疗包括以与选自由以下各项组成的组中的治疗剂的联合疗法施用：另一种本发明糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物、降血压药、降低血脂的药物、溶解血栓的药物、抗血小板聚集的药物、抗凝药物、神经保护药物、钙离子拮抗剂、谷氨酸拮抗剂、谷氨酸释放抑制剂、GABA受体激动剂、自由基清除剂、细胞膜稳定剂。在一个优选的实施方案中，另外的治疗剂选自由以下各项组成的组：另一种本发明糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物、瑞替普酶、兰替普酶、孟替普酶、豆豉纤溶酶、新型蚯蚓纤溶酶、纳豆激酶、蛇毒纤溶酶原激活剂、阿司匹林、噻氯匹定、氯吡格雷、普拉格雷、替卡格雷、坎格雷洛、盐酸沙格雷酯、vorapaxar、atopaxar、肝素、低分子肝素、华法林、利伐沙班、比法卢定、达比加群酯、依达拉奉、他汀类药物。
如对于本领域技术人员将明显的是，包含本发明糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或 衍生物与另外的治疗剂的本发明的组合不仅在这些活性成分以单一组合物使用时是有效的，而且在以两个不同组合物(同时、依次或在一段时间之后分开地施用)使用时也是有效的。此外，本领域技术人员将理解，本发明糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物、可以开处方为与联合疗法中的其他活性成分一起使用，以预防和/或治疗缺血性血管疾病。
在一个特别的实施方案中，联合疗法包括向受试者同时、依次或分开地施用本发明的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物和另外的治疗剂。备选地，联合疗法包括向受试者施用在单一组合物中的本发明糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物和另外的治疗剂。
在本发明的一个实施方案中，本发明的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物可以方便地施用至患者。因此，用于本发明的用途的化合物可以为包含与药用赋形剂或载体组合的有效量的本发明的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物的药物组合物的形式。这一方面也可以表达为，包含与药用赋形剂或载体组合的有效量的本发明的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物的组合物用于在预防和/或治疗：脑梗死、脑卒中、动脉粥样硬化性血栓形成、心源性脑血栓、急性缺血性脑血管综合征、小血管病变(也称为腔隙性脑卒中)、多发性脑梗死、大面积脑梗死、分水岭脑梗死、出血性脑梗死、无症状的脑梗死、脑静脉及静脉窦血栓形成、颅底异常血管网病、其他原因所致的缺血性脑卒中。
在本发明的一个实施方案中，使用的化合物可以以包含常规药用载体的剂量单位制剂通过口腔、注射、皮下、呼吸道、透皮、非肠道、直肠、局部外用、静脉、肌肉或通过其它方式来给予。可以将药物组合物配制成任何药用形式，如：片剂、颗粒剂、注射 剂、凝胶剂、丸剂、胶囊剂、栓剂、植入剂、纳米制剂、粉针剂。诸如片剂和胶囊剂的一些剂型可以再分成包含诸如达到期望目的的有效量的适当量活性组分的适当剂量单位剂型。
在另一个实施方案中，用于本发明的用途的本发明的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物为待施用至要治疗的患者的注射制剂，适用于本发明的注射剂是指药物与适宜的溶剂或分散介质制成供注入人体内的灭菌或无菌溶液、乳状液或悬浮液，以及供使用前配制成溶液或悬浮液的粉末无菌制剂。所述注射剂包括注射液(其中供静脉滴注用的大体积注射液也称静脉输液)、注射用无菌粉末与注射用浓溶液。
载体包括赋形剂和稀释剂，并且必须具有足够高的纯度和十分低的毒性以使它们适于被给予待治疗的患者。载体可以是惰性的或其可以本身具有药用益处。
载体的种类包括但不限于：稀释剂如填料和疏松剂、粘合剂、润滑剂、抗结块剂、崩解剂、增甜剂、缓冲剂、防腐剂、增溶剂、等张剂、悬浮剂和分散剂、润湿剂或乳化剂、调味剂和芳香剂、增稠剂和媒介物。一些载体可以列在多于一种的类别中，如：植物油可以在一些制剂中用作滑润剂并在其他制剂中用作稀释剂。示例性药用载体包括糖、淀粉、纤维素、麦芽、明胶、滑石和植物油。可选的活性剂可以包括在药物组合物中，其基本上不影响本发明的化合物的活性。
本发明的积极进步效果在于：本发明所提供的糖苷类化合物制备方法简单，能显著提高VEGF-A mRNA的表达，可用于制备促血管生成，为寻求具有促血管生成活性治疗的脑梗死、心梗、下肢缺血性等
Figure PCTCN2019085823-appb-000050
循环障碍疾病的药物的研究提供了可靠的保障。
本申请的化合物或盐可以是被给予的唯一活性剂或可以连同其他活性剂被给予。
术语约定：
“立体异构体”、“光学异构体”或“旋光异构体”是具有相同化学组成但原子或基团在空间中的排布不同的化合物。其包括“非对映异构体”和“对映异构体”
“非对映异构体”是具有两个或更多手性中心并且其分子不是彼此的镜像的立体异构体。非对映异构体具有不同物理性能，例如：熔点、沸点、谱特性和反应活性。在拆分剂或色谱存在的情况下，使用诸如手性HPLC柱，可以在诸如电泳、结晶的高分辨分析步骤下分离非对映异构体的混合物。
“对映异构体”指代彼此无重叠镜像的一种化合物的两个立体异构体。对映异构体的50:50的混合称为外消旋混合物或外消旋体，其在化学反应或处理过程中可以出现在已经没有立体选择性或立体定向性的情况下。
“烷基”包括支链和直链饱和脂肪族烃基两者，并具有指定数量的碳原子数量，一般1至约12个碳原子。如在本文中使用的术语C1-C6烷基表示具有1至约6个碳原子的烷基。当本文中结合另一基团使用C0-Cn烷基时，以(苯基)C0-C4烷基为例，指定的基团，在这种情况下，苯基是通过单个共价键(C0)直接键合或通过具有指定的碳原子数(在这种情况下，1至约4个碳原子)的烷基链连接。烷基的实例包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、3-甲基丁基、叔丁基、正戊基、和仲戊基。
“烯基”或“烯烃基”指包括一个或多个不饱和的碳-碳键的直链和支链烃链，碳-碳键可以出现在沿着链的任一稳定点。本文中所述的烯基通常具有2至约12个碳原子。优选烯基是低级烯基，那些烯基具有2至约8个碳原子，如：C 2-C 8、C 2-C 6、和C 2-C 4烯基。烯基的实例包括乙烯基、丙烯基、和丁烯基。
“烷氧基”是指具有通过氧桥连接的指定数量的碳原子的如上所定义的烷基。烷氧基的实例包括但不限于甲氧基、乙氧基、3-己氧基、和3-甲基戊氧基。
术语“杂环”表示5-至8-元饱和环、部分不饱和环、或包含选自N、O和S的1至约4个杂原子且剩余的环原子是碳的芳族环，或是7至11元饱和环、部分不饱和环、或芳族杂环系统和10至15-元三环系统，该系统包含选自N、O和S的多环系统中的至少1个杂原子并且在多环系统中的各环中包含独立地选自N、O和S的至多约4个杂原子。除非另外指明，否则杂环可以连接至它在任何杂原子和碳原子处取代并且产生稳定结构的基团。当指明时，本文中所述的杂环可以在碳或氮原子上被取代，只要得到的化合物是稳定的。可以可选地季铵化杂环中的氮原子。优选杂环基中杂原子的总数不大于4而且优选杂环基中S和O原子的总数不大于2，更优选不大于1。杂环基的实例包括：吡 啶基、吲哚基、嘧啶基、哒嗪基(pyridizinyl)、吡嗪基、咪唑基、噁唑基、呋喃基、苯硫基、噻唑基、三唑基、四唑基、异噁唑基、喹啉基、吡咯基、吡唑基、苯并[b]苯硫基(benz[b]thiophenyl)、异喹啉基、喹唑啉基、喹喔啉基、噻吩基、异吲哚基、二氢异吲哚基、5,6,7,8-四氢异喹啉、吡啶基、嘧啶基、呋喃基、噻吩基、吡咯基、吡唑基、吡咯烷基、吗啉基、哌嗪基、哌啶基、和吡咯烷基。
“芳基”或“杂芳基”表示包含选自N、O和S的1至4个、或优选1至3个杂原子并且剩余环原子为碳的稳定的5-或6-元单环或多环。当杂芳基中S和O原子的总数超过1时，这些杂原子不彼此邻近。优选杂芳基中S和O原子的总数不大于2。尤其优选杂芳基中S和O原子的总数不大于1。可以可选地季铵化杂环中的氮原子。当指明时，这些杂芳基还可以用碳或非碳原子或基团取代。这种取代可以包括与可选地包含独立地选自N、O和S的1或2个杂原子的5至7-元饱和的环基的稠合，从而形成例如[1,3]二噁唑并[4,5-c]吡啶基。杂芳基的实例包括但不限于：吡啶基、吲哚基、嘧啶基、哒嗪基、吡嗪基、咪唑基、噁唑基、呋喃基、苯硫基、噻唑基、三唑基、四唑基、异噁唑基、喹啉基、吡咯基、吡唑基、苯并[b]苯硫基、异喹啉基、喹唑啉基、喹喔啉基、噻吩基、异吲哚基、和5,6,7,8-四氢异喹啉。
“药学上可接受的盐”或“化合物的盐”是所公开的化合物的衍生物，其中，母体化合物通过制备无毒的酸或其碱加成盐改性，并且还指这些化合物和这些盐的药用溶剂化物，包括水合物。药用盐的实例包括但不限于：碱性残基如胺类的无机或有机酸加成盐；酸性残基如羧酸的碱或有机加成盐；等等，以及包括一种或多种上述盐的组合。药用盐包括诸如从无毒无机或有机酸形成的母体化合物的无毒盐和季铵盐。例如，无毒酸性盐包括衍生自无机酸的那些，例如：盐酸、氢溴酸、硫酸、氨基磺酸、磷酸、硝酸等；其他可接受的无机盐包括金属盐如：钠盐、钾盐、铯盐等；碱土金属盐如：钙盐、镁盐等，以及包括一种或多种上述盐的组合。
化合物的有机盐包括由诸如乙酸、三氟乙酸、丙酸、丁二酸、乙醇酸、硬脂酸、乳酸、苹果酸、酒石酸、柠檬酸、抗坏血酸、扑酸、马来酸、羟基马来酸、苯乙酸、谷氨酸、苯甲酸、水杨酸、甲磺酸、乙磺酸、苯磺酸、对氨基苯磺酸、2-乙酸基苯酸、富马酸、对甲苯磺酸、甲磺酸、乙烷二磺酸、草酸、羟乙磺酸、HOOC-(CH2)n-COOH(其中n为0至4)等的有机酸制备的盐；有机胺盐，如：三乙胺盐、吡啶盐、甲基吡啶盐、乙醇胺盐、三乙醇胺盐、二环己基胺盐、N,N'-二苄基乙二胺盐等；和氨基酸盐，如：精氨酸盐、天冬氨酸盐、谷氨酸盐等，以及包括一种或多种上述盐的组合。
“糖基”包括单糖基、双糖基、寡糖基、多糖基等，其中单糖基包括但不限于赤藓糖基、苏力糖基、阿拉伯糖基、核糖基、木糖基、来苏糖基、葡萄糖基、甘露糖基、果糖基、半乳糖基等。双糖基包括但不限于蔗糖基、麦芽糖基、纤维二糖基、异麦芽糖基、龙胆二糖基、海藻糖基和乳糖基等。
“前药”是指在宿主中代谢，如水解或氧化成本发明化合物的化合物，前药典型的例子包括在活性化合物的官能团上具有生物不稳定保护基团的化合物。前药包括可被氧化、还原、胺化、脱胺化、羟基化、脱羟基化、水解、脱水、烷基化、脱烷基化、酰基化、脱酰基、磷酸化、脱磷酸化以产生活性化合物的化合物。
“衍生物”是指化合物的氢原子或原子团被其他原子或原子团取代而衍生的较复杂的产物。
附图说明
图1为海绵植入动物模型上化合物促进VEGF-A mRNA表达作用的示意图。
图2为不同药物在MCAO急性脑梗模型上的梗死率(％)。
图3为不同药物在MCAO急性脑梗模型上的体重变化率(％)。
图4为Longa神经评分标准的示意图。从左向右依次为：1分，表示不能完全伸展对侧前爪；2分，表示向对侧转圈；3分，表示向对侧倾倒；4分，表示不能自发行走，意思丧失。
图5为不同药物在MCAO急性脑梗模型上的Longa神经评分的示意图。
图6为不同药物在MCAO急性脑梗模型上的脑含水量及血脑屏障完整性的测定。
图7为药物在2VO慢性脑缺血大鼠模型上的体重变化率。
图8为药物在2VO慢性脑缺血大鼠模型上的Morris水迷宫定位航行实验结果。
图9为药物在2VO慢性脑缺血大鼠模型上的Morris水迷宫空间探索实验结果。
图10为慢性脑缺血后的脑切片的HE染色及Nissl染色结果。
图11为VEGF蛋白在MCAO急性脑梗模型上作用3天时对于脑梗死的抑制作用的TTC染色结果。
图12为VEGF蛋白在MCAO急性脑梗模型上的梗死率。
图13为VEGF蛋白在MCAO急性脑梗模型上的梗死抑制率。
图14为效果实施例4中各组样品对HUVEC-12细胞增殖的影响。
图15为效果实施例4中各组样品对HUVEC-12细胞迁移的影响。
图16为效果实施例4中各组样品的划痕实验结果。
图17为效果实施例4中各组样品对HUVEC-12细胞小管生成的影响的倒置显微镜 图。
图18为效果实施例4中各组样品对HUVEC-12细胞小管生成的影响。
图19为单独使用不同剂量的红景天苷(TG-056)在海绵植入动物模型上对VEGF-A mRNA表达量的影响。
图20为TG-028和TG-057在MCAO急性脑梗模型上的梗死率和Longa神经评分。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
路线A：化合物TG-001,TG-002,TG-003,TG-004,TG-005,TG-006,TG-007,TG-008，TG-057的合成
Figure PCTCN2019085823-appb-000051
路线a实验操作：
将化合物4a(1eq)、Pd(PPh 3) 2Cl 2(2mol％)、CuI(2mol％)加入到反应瓶中，在N 2保护下，向体系中依次加入THF(0.2M)、化合物5(1.2eq)、Et 3N(9.6eq)，然后在室温下反应20h。用硅藻土(上海大合化学品有限公司分析纯AR)过滤后直接旋干过柱(石油醚/乙酸乙酯＝2：1)，得化合物6a(yield 89％)。将化合物6a(1eq)、Pd/C(10％)加入MeOH(0.1M)中，在60atm的H 2条件下，室温反应20h。硅藻土过滤后，减压浓缩，硅胶柱层析提纯，得化合物7a(yield 94％)。
向化合物8a(1eq)中滴加33％的HBr/AcOH(4eq)溶液，在室温条件下反应4h。直接旋干，用Et 2O和正己烷重结晶，得化合物9a(76g,yield 98％)。
将化合物7a(1eq)、9a(1.2eq)加入到反应瓶中，在N 2保护下，向体系中加入二氯甲烷(0.3M)，室温条件下反应30min。再向体系中加入Ag 2CO 3(1.2eq)，室温条件下反应20h，过滤后直接旋干过柱(洗脱剂：石油醚/乙酸乙酯＝4：1)，得化合物10a(yield40％)。将化合物10a(1eq)加入到反应瓶中，分别加入MeOH(4M)、H 2O(4M)、Et 3N(2eq)，室温条件下反应20h，减压浓缩，硅胶柱过柱提纯(洗脱剂：二氯甲烷:甲醇＝9:1)，得化合物TG-001(yield 70％)。
化合物TG-002,TG-003,TG-004,TG-005,TG-006,TG-007,TG-008,TG-057的合成使用与化合物TG-001类似的合成路线，其结构如下。
Figure PCTCN2019085823-appb-000052
化合物TG-001，TG-002,TG-003,TG-004,TG-005,TG-006,TG-007,TG-008，TG-057的结构鉴定数据如下：
TG-001的鉴定数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.17–7.06(m,2H),6.88(d,J＝8.0Hz,1H),6.82(td,J＝7.4,0.9Hz,1H),4.23(d,J＝7.8Hz,1H),3.91(m,1H),3.85(dd,J＝11.9,2.0Hz,1H),3.80(s,3H),3.66(dd,J＝11.9,5.3Hz,1H),3.55(m,1H),3.37–3.21(m,3H),3.19–3.12(m,1H),2.61(t,J＝7.0Hz,2H),1.64(m,4H).
LRMS(ESI):[M+Na] +365.1；HRMS(ESI):[M+H] +计算值C 17H 27O 7 +343.1751，实测值为343.1748。
TG-002的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)6.35(d,J＝2.2Hz,2H),6.28(t,J＝2.2Hz,1H),4.24(d,J＝7.8Hz,1H),3.96–3.91(m,1H),3.85(dd,J＝11.8,1.7Hz,1H),3.74(s,6H),3.66(dd,J＝11.9,5.2Hz,1H),3.58-3.53(m,1H),3.38–3.21(m,3H),3.19–3.11(m,1H),2.57(t,J＝7.3Hz,2H),1.75–1.58(m,4H).
LRMS(ESI):[M+Na] +395.1；HRMS(ESI):[M+NH 4] +计算值为C 18H 32O 8N +390.2122，实测值为390.2122。
TG-003的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)6.84(d,J＝8.2Hz,1H),6.79(d,J＝1.9Hz,1H),6.72(dd,J＝8.1,1.9Hz,1H),4.24(d,J＝7.8Hz,1H),3.98-3.88(m,1H),3.86(dd,J＝11.9,1.9Hz,1H),3.81(s,3H),3.79(s,1H),3.66(dd,J＝11.9,5.3Hz,1H),3.61–3.51(m,1H),3.38–3.21(m,3H),3.20–3.12(m,1H),2.58(t,J＝7.2Hz,2H),1.75–1.56(m,4H).
LRMS(ESI):[M+Na] +395.1；HRMS(ESI):[M+NH 4] +计算值为C 18H 32O 8N +390.2122，实测值为390.2118。
TG-005的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.07(d,J＝8.8Hz,2H),6.80(d,J＝8.8Hz,2H),4.23(d,J＝7.8Hz,1H),3.93–3.82(m,2H),3.75(s,3H),3.66(dd,J＝11.9,5.3Hz,1H),3.52(dt,J＝9.5,6.7Hz,1H),3.38–3.22(m,3H),3.20–3.12(m,1H),2.54(t,J＝7.6Hz,2H),1.68-1.61(m,4H),1.47–1.31(m,2H).
LRMS(ESI):[M+Na] +375.1；HRMS(ESI):[M+H] +计算值为C 18H 29O 7 +357.1908，实测值为357.1906。
TG-006的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.06(d,J＝8.6Hz,2H),6.84–6.80(d,J＝8.6Hz,2H),4.23(d,J＝7.8Hz,1H),3.93–3.82(m,2H),3.75(s,3H),3.66(dd,J＝11.9,5.2Hz,1H),3.52(dt,J＝9.5,6.7Hz,1H),3.38–3.21(m,3H),3.20–3.11(m,1H),2.53(t,J＝7.6Hz,2H),1.68–1.50(m,4H),1.47–1.25(m,4H).
LRMS(ESI):[M+Na] +393.1；HRMS(ESI):[M+NH 4] +计算值C 19H 34O 7N +388.2330,实测值为388.2327。
终产物TG-004的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.15(t,J＝7.8Hz,1H),6.79–6.67(m,3H),4.24(d,J＝7.8Hz,1H),3.92(dt,J＝9.4,6.4Hz,1H),3.85(dd,J＝11.9,1.8Hz,1H),3.76(s,3H),3.66(dd,J＝11.9,5.2Hz,1H),3.60–3.51(m,1H),3.39–3.21(m,3H),3.19–3.12(m,1H),2.60(t,J＝7.3Hz,2H),1.78–1.56(m,4H).
LRMS(ESI):[M+Na] +365.1；HRMS(ESI):[M+NH 4] +计算值C 17H 30O 7N +360.2017,实测值为为360.2016。
TG-007的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)6.49(s,2H),4.24(d,J＝7.8Hz,1H),3.97–3.83(m,2H),3.81(s,6H),3.72(s,3H),3.69–3.52(m,2H),3.37–3.21(m,3H),3.20–3.13(m,1H),2.59(t,J＝7.3Hz,2H),1.79–1.55(m,4H).
LRMS(ESI):[M+Na] +425.1；HRMS(ESI):[M+NH 4] +计算值C 19H 34O 9N +420.2228，实测值为为420.2226。
TG-008的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)6.71-6.67(m,2H),6.65-6.61(m,1H),5.87(s,2H),4.23(d,J＝7.8Hz,1H),3.95-3.88(m,1H),3.85(m,1H),3.66(dd,J＝11.8,5.2Hz,1H),3.60–3.48(m,1H),3.38–3.20(m,3H),3.19–3.12(m,1H),2.55(t,J＝7.1Hz,2H),1.73–1.53(m,4H).
LRMS(ESI):[M+Na] +379.0；HRMS(ESI):[M+Na] +计算值C 17H 24O 8Na +379.1374,实测值为379.1362。
TG-057的数据：
1H NMR(400MHz,CDCl 3)δ(ppm)6.83(d,1H,J＝7.6Hz),6.70–6.64(m,2H),6.26(br,1H),4.83(br,8H),4.00(s,1H),3.88(s,3H),3.74(t,2H,J＝6.4Hz),3.55(s,2H),2.58(t,2H,J＝7.2Hz),1.69–1.62(m,4H).
LRMS(ESI):[M+Na] +381.2。
该实验过程中的各反应中间体的结构如下：
Figure PCTCN2019085823-appb-000053
上述中间体10a-10h的鉴定数据为：
10a:
1H NMR(400MHz,CDCl 3)δ6.32(d,J＝2.2Hz,2H),6.29(t,J＝2.2Hz,1H),5.20(t,J＝9.5Hz,1H),5.08(t,J＝9.7Hz,1H),4.98(dd,J＝9.6,8.0Hz,1H),4.48(d,J＝8.0Hz,1H),4.26(dd,J＝12.3,4.7Hz,1H),4.13(dd,J＝12.3,2.3Hz,1H),3.89(d,J＝9.5Hz,1H),3.78(s,6H),3.68(dd,J＝9.9,2.2Hz,1H),3.49(d,J＝9.4Hz,1H),2.55(t,J＝6.6Hz,2H),2.08(s,3H),2.05–1.97(m,9H),1.63(dd,J＝11.5,4.3Hz,4H).
LRMS(ESI):[M+Na] +533.5。
10b：
1H NMR(400MHz,CDCl 3)δ6.34–6.28(m,3H),5.20(t,J＝9.5Hz,1H),5.08(t,J＝9.7Hz,1H),4.98(dd,J＝9.6,8.0Hz,1H),4.48(d,J＝8.0Hz,1H),4.26(dd,J＝12.3,4.7Hz,1H),4.13(dd,J＝12.3,2.3Hz,1H),3.89(d,J＝9.5Hz,1H),3.78(s,6H),3.68(dd,J＝9.9,2.2Hz,1H),3.49(d,J＝9.4Hz,1H),2.55(t,J＝6.6Hz,2H),2.10–2.07(s,3H),2.03(s,3H),2.00(m,6H),1.63(dd,J＝11.5,4.3Hz,4H).
LRMS(ESI):[M+Na] +563.5。
10c：
1H NMR(300MHz,CDCl 3)δ6.82–6.59(m,3H),5.27–4.83(m,3H),4.46(d,J＝7.9Hz,1H),4.32–3.99(m,2H),3.83(m,7H),3.74–3.35(m,2H),2.53(m,2H),1.99(m,12H),1.59(m,4H).
LRMS(ESI):[M+Na] +563.5。
10d：
1H NMR(300MHz,CDCl 3)δ7.19(t,J＝8.1Hz,1H),6.81–6.65(m,3H),5.28–4.88(m,3H),4.48(d,J＝8.0Hz,1H),4.32–4.06(m,2H),3.88(m,1H),3.80(s,3H),3.68(d,J＝8.2Hz,1H),3.51(m,1H),2.59(m,2H),2.03(m,12H),1.64(m,4H).
LRMS(ESI):[M+Na] +533.5。
10e：
1H NMR(300MHz,CDCl 3)δ7.08(d,J＝6.9Hz,2H),6.82(d,J＝6.8Hz,2H),5.69(d,J＝4.2Hz,1H),5.19(m,1H),4.91(d,J＝9.3Hz,1H),4.30(m,1H),4.20(m,2H),3.96(m,1H),3.79(s,3H),3.46(t,J＝5.7Hz,2H),2.55(t,J＝7.0Hz,2H),2.09(m,9H),1.71(s,3H),1.66–1.48(m,4H),1.36(d,J＝6.7Hz,2H).
LRMS(ESI):[M+Na] +547.5。
10f：
1H NMR(400MHz,CDCl 3)δ7.08(d,J＝8.6Hz,2H),6.83(t,J＝5.7Hz,2H),5.20(t,J＝9.5Hz,1H),5.08(t,J＝9.7Hz,1H),4.98(dd,J＝9.6,8.0Hz,1H),4.48(d,J＝8.0Hz,1H),4.26(dd,J＝12.3,4.7Hz,1H),4.12(m,1H),3.86(dt,J＝9.6,6.3Hz,1H),3.79(s,3H),3.68(ddd,J＝9.9,4.6,2.4Hz,1H),3.46(dt,J＝9.5,6.8Hz,1H),2.58–2.49(m,2H),2.08(s,3H),2.02(m,9H),1.62–1.49(m,4H),1.36–1.28(m,4H).
LRMS(ESI):[M+Na] +561.5。
10g:
LRMS(ESI):[M+Na] +593.5。
10h：
LRMS(ESI):[M+Na] +547.5。
路线B：化合物TG-009,TG-010,TG-011,TG-012的合成
Figure PCTCN2019085823-appb-000054
路线B实验操作：
将化合物12i(1eq)溶于THF(0.2M)中，0℃下向体系中缓慢加入LiAlH 4(2eq)，室温条件下反应2～20h。0℃下依次向体系中缓慢加入H 2O(1倍THF溶剂体积)、15％NaOH溶液(1倍THF溶剂体积)、H 2O(3倍THF溶剂体积)。以乙酸乙酯萃取，萃取液合并后，饱和食盐水洗涤，无水硫酸钠干燥。减压蒸除溶剂后，硅胶柱过柱提纯(洗脱剂：石油醚/乙酸乙酯＝4:1)，得化合物7i(yield 98％)。
将化合物7i(1eq)、9a(1.2eq)加入到反应瓶中，在N 2保护下，向体系中加入二氯甲烷(0.3M)，室温条件下反应30min。再向体系中加入Ag 2CO 3(1.2eq)，室温条件下反应20h。硅藻土过滤后，减压浓缩，硅胶柱层析提纯，得化合物10i(yield 26％)。将化合物10i(1eq)加入到反应瓶中，分别加入MeOH(4M)、H 2O(4M)、Et 3N(2eq)，室温条件下反应20h，硅胶柱过柱提纯(洗脱剂：二氯甲烷:甲醇＝9:1)，得化合物TG-009(yield 75％)。
化合物TG-010,TG-011,TG-012的合成使用与化合物TG-009类似的合成路线。化合物TG-009、TG-010,TG-011和TG-012的结构及鉴定数据如下所示：
Figure PCTCN2019085823-appb-000055
TG-009的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)4.24(d,J＝7.8Hz,1H),3.95–3.81(m,2H),3.66(dd,J＝11.9,5.2Hz,1H),3.53(dt,J＝9.5,6.7Hz,1H),3.38–3.21(m,3H),3.20–3.11(m,1H),1.81–1.51(m,7H),1.46–1.08(m,8H),0.95–0.80(m,2H).
LRMS(ESI):[M+Na] +341.1；HRMS(ESI):[M+Na] +计算值C 16H 30O 6Na +341.1946,实测值为341.1934。
TG-012的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.38(d,J＝8.3Hz,2H),7.12(d,J＝8.3Hz,2H),4.24(d,J＝7.8Hz,1H),3.92(dt,J＝9.5,6.4Hz,1H),3.85(dd,J＝11.9,1.6Hz,1H),3.66(dd,J＝11.8,5.2Hz,1H),3.56(dt,J＝9.6,6.3Hz,1H),3.31(m,3H),3.19–3.11(m,1H),2.61(t,J＝7.4Hz,2H),1.77–1.55(m,4H).
LRMS(ESI):[M+Na] +414.9；HRMS(ESI):[M+Na] +计算值C 16H 23O 6BrNa +413.0570,实测值为413.0568。
TG-011的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)8.14(d,J＝8.7Hz,2H),7.45(d,J＝8.7Hz,2H),4.25(d,J＝7.8Hz,1H),4.00–3.82(m,2H),3.70–3.50(m,2H),3.37–3.22(m,3H),3.21–3.12(m,1H),2.78(t,J＝7.6Hz,2H),1.85–1.57(m,4H).
LRMS(ESI):[M+Na] +380.0；HRMS(ESI):[M+Na] +计算值C 16H 23O 8NNa +380.1327,实测值为380.1314。
TG-010的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.18(dd,J＝8.5,5.6Hz,2H),7.00–6.91(m,2H),4.24(d,J＝7.8Hz,1H),3.92(dt,J＝9.6,6.3Hz,1H),3.87-3.82(m,1H),3.66(dd,J＝11.8, 5.2Hz,1H),3.56(dt,J＝9.5,6.3Hz,1H),3.37–3.22(m,3H),3.20–3.11(m,1H),2.62(t,J＝7.3Hz,2H),1.78–1.54(m,4H).
LRMS(ESI):[M+COOH] -375.0；HRMS(ESI):[M+Cl] -计算值C 16H 23O 6FCl -365.1173,实测值为365.1173。
该实验过程中的各反应中间体的结构如下：
Figure PCTCN2019085823-appb-000056
上述中间体10i-10l的鉴定数据为：
10i：
LRMS(ESI):[M+Na] +509.5。
10j：
LRMS(ESI):[M+Na] +521.5。
10k：
LRMS(ESI):[M+Na] +548.5。
10l：
LRMS(ESI):[M+Na] +582.4。
路线C:化合物TG-013,TG-014，TG-015,TG-016,TG-017,TG-018,TG-019,TG-020,TG-021,TG-022,TG-024,TG-025,TG-026，TG-027，TG-049、TG-050、TG-051和TG-023的合成
Figure PCTCN2019085823-appb-000057
路线C实验操作：
将化合物13(1eq)、Et 3N(1.5eq)加入二氯甲烷(0.5M)中，在0℃的条件下滴加Ac 2O(1.2eq)，0℃至室温条件下反应20h后，分别用饱和NH 4Cl、NaCl溶液洗涤，无水Na 2SO4干燥后减压浓缩，得化合物14(16g,yield 92％)。
将化合物14(1eq)加入二氯甲烷(0.5M)中，在-78℃的条件下缓慢滴加1M的 BBr 3/二氯甲烷(2.5eq)，0℃下反应5h，用饱和NaHCO 3溶液淬灭，再用二氯甲烷萃取，无水Na 2SO4干燥后减压浓缩，硅胶柱过柱提纯(洗脱剂：石油醚/乙酸乙酯＝2:1)，得化合物15(8.7g,yield 78％)。
将化合物15(1eq)加入MeOH(0.5M)中，向体系中加入1M的NaOH溶液(3eq)，室温条件下反应2h，2N HCl溶液调节PH至3，再用乙酸乙酯萃取，无水Na 2SO4干燥后减压浓缩，硅胶柱过柱提纯(洗脱剂：石油醚/乙酸乙酯＝1:1)，得化合物16(6g,yield85％)。
将化合物16(1eq)、9a(1.2eq)加入到反应瓶中，在N 2保护下，向体系中加入二氯甲烷(0.3M)，室温条件下反应30min。再向体系中加入Ag 2CO 3(2.2eq)，室温条件下反应20h。硅藻土过滤后，减压浓缩，硅胶柱过柱提纯(洗脱剂：石油醚/乙酸乙酯＝1:2)，得化合物17(7.6g,yield 32％)。
将化合物17(1eq)、PPh 3(1.5eq)加入到反应瓶中，室温下向体系中缓慢滴加DIAD(1.5eq)，继续反应30min后向体系中滴加R 3’OH(1.2eq)，65℃条件下反应2～10h。减压浓缩，硅胶柱过柱提纯，得化合物18a～j(yield 55～90％)。
将化合物18a或17(1eq)加入到反应瓶中，分别加入MeOH(4M)、H 2O(4M)、Et 3N(2eq)，室温条件下反应20h。减压浓缩，硅胶柱过柱提纯(洗脱剂：二氯甲烷:甲醇＝9:1)，分别得化合物TG-013(yield 59％)和TG-023(yield 13％)。
化合物TG-014,TG-015,TG-016,TG-017,TG-018,TG-019,TG-020,TG-021,TG-022,TG-024,TG-025,TG-026,TG-027、TG-049、TG-050、TG-051和TG-023的合成使用与化合物TG-013类似的合成路线。
化合物TG-013，TG-014,TG-015,TG-016,TG-017,TG-018,TG-019,TG-020,TG-021,TG-022,TG-024,TG-025,TG-026,TG-027、TG-049、TG-050、TG-051和TG-023结构及鉴定数据如下所示：
Figure PCTCN2019085823-appb-000058
TG-013的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.34(m,5H),7.09(d,J＝8.3Hz,2H),6.88(d,J＝8.4Hz,2H),5.03(s,2H),4.23(d,J＝7.8Hz,1H),4.01–3.82(m,2H),3.66(dd,J＝11.8,5.0Hz,1H),3.59–3.47(m,1H),3.39–3.22(m,3H),3.16(t,J＝8.3Hz,1H),2.56(t,J＝6.9Hz,2H),1.65(d,J＝4.1Hz,4H).
LRMS(ESI):[M+Na] +441.1；HRMS(ESI):[M+Na] +计算值C 23H 30O 7Na +441.1884,实测值为441.1880。
TG-015的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.08(d,J＝8.6Hz,2H),6.82(d,J＝8.6Hz,2H),6.12–5.96(m,1H),5.37(dd,J＝17.3,1.6Hz,1H),5.22(dd,J＝10.6,1.4Hz,1H),4.55– 4.44(m,2H),4.23(d,J＝7.8Hz,1H),3.97–3.80(m,2H),3.66(dd,J＝11.9,5.2Hz,1H),3.60–3.50(m,1H),3.38–3.21(m,3H),3.20–3.12(m,1H),2.56(t,J＝7.1Hz,2H),1.73–1.55(m,4H).
LRMS(ESI):[M+Na] +391.0；HRMS(ESI):[M+Na] +计算值C 19H 28O 7Na +391.1727,实测值为391.1726。
TG-016的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.06(d,J＝8.5Hz,2H),6.78(d,J＝8.6Hz,2H),4.23(d,J＝7.8Hz,1H),3.95–3.82(m,2H),3.72(d,J＝6.4Hz,2H),3.66(dd,J＝11.9,5.2Hz,1H),3.59–3.50(m,1H),3.37–3.20(m,3H),3.19–3.12(m,1H),2.56(t,J＝7.1Hz,2H),1.86(d,J＝13.1Hz,2H),1.81–1.57(m,8H),1.38–1.16(m,3H),1.09(m,2H).
LRMS(ESI):[M+Na] +447.2；HRMS(ESI):[M+Na] +计算值C 23H 36O 7Na +447.2353,实测值为447.2352。
TG-017的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)8.65(d,J＝1.2Hz,1H),8.52(dd,J＝4.9,1.2Hz,1H),7.96(d,J＝7.9Hz,1H),7.48(dd,J＝7.8,5.0Hz,1H),7.14(d,J＝8.5Hz,2H),6.94(d,J＝8.6Hz,2H),5.14(s,2H),4.27(d,J＝7.8Hz,1H),3.99–3.85(m,2H),3.69(dd,J＝11.9,5.2Hz,1H),3.58(dt,J＝9.4,6.2Hz,1H),3.43–3.24(m,3H),3.23–3.16(m,1H),2.60(t,J＝7.1Hz,2H),1.76–1.60(m,4H).
LRMS(ESI):[M+Na] +442.1；HRMS(ESI):[M+Na] +计算值C 22H 29O 7NNa +442.1836,实测值为442.1834。
TG-018的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.10(d,J＝8.5Hz,2H),6.82(d,J＝8.6Hz,2H),4.27(d,J＝7.8Hz,1H),3.99–3.86(m,2H),3.83(d,J＝6.9Hz,2H),3.69(dd,J＝11.8,5.2Hz,1H),3.62–3.54(m,1H),3.41–3.24(m,3H),3.21-3.17(m,1H),2.59(t,J＝7.0Hz,2H),2.40-2.30(m,1H),1.93-1.78(m,2H),1.76–1.57(m,8H),1.47–1.35(m,2H).
LRMS(ESI):[M+Na] +433.2；HRMS(ESI):[M+Na] +计算值C 22H 34O 7Na +433.2197，实测值为433.2192。
TG-019的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)8.66–8.44(m,2H),7.54(d,J＝5.9Hz,2H),7.15(d,J＝8.6Hz,2H),6.93(d,J＝8.6Hz,2H),5.18(s,2H),4.26(d,J＝7.8Hz,1H),3.95(dt,J＝9.5,6.3Hz,1H),3.89(dd,J＝11.9,1.9Hz,1H),3.69(dd,J＝11.8,5.2Hz,1H),3.62–3.54 (m,1H),3.41–3.24(m,3H),3.23–3.16(m,1H),2.61(t,J＝7.1Hz,2H),1.77–1.58(m,4H).
LRMS(ESI):[M+H] +420.2；HRMS(ESI):[M+H] +计算值C 22H 30O 7N +420.2017，实测值为420.2014。
TG-020的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.11(d,J＝8.5Hz,2H),6.83(d,J＝8.5Hz,2H),4.27(d,J＝7.8Hz,1H),4.05–3.86(m,4H),3.82(d,J＝6.3Hz,2H),3.69(dd,J＝11.8,5.2Hz,1H),3.58(dt,J＝9.5,6.2Hz,1H),3.49(td,J＝12.0,1.7Hz,2H),3.40–3.24(m,3H),3.19(t,J＝8.4Hz,1H),2.60(t,J＝7.1Hz,2H),2.14–1.98(m,1H),1.85–1.60(m,6H),1.47(qd,J＝12.3,4.5Hz,2H).
LRMS(ESI):[M+Na] +449.2；HRMS(ESI):[M+H] +计算值C 22H 35O 8 +427.2326,实测值为427.2323。
TG-021的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.10(d,J＝8.5Hz,2H),6.83(d,J＝8.5Hz,2H),4.27(d,J＝7.8Hz,1H),3.99–3.85(m,4H),3.69(dd,J＝11.8,5.2Hz,1H),3.58(dt,J＝9.4,6.2Hz,1H),3.41–3.24(m,3H),3.19(t,J＝8.4Hz,1H),2.85–2.71(m,1H),2.59(t,J＝7.1Hz,2H),2.24–2.08(m,2H),2.08–1.85(m,4H),1.70(m,4H).
LRMS(ESI):[M+Na] +419.2。
TG-014的数据：
1H NMR(400MHz,CD 3OD)δ(ppm)7.07(d,J＝8.5Hz,2H),6.79(d,J＝8.6Hz,2H),4.23(d,J＝7.8Hz,1H),3.96–3.80(m,4H),3.66(dd,J＝11.9,5.2Hz,1H),3.59–3.49(m,1H),3.38–3.21(m,3H),3.20–3.11(m,1H),2.56(t,J＝7.1Hz,2H),1.75(m,2H),1.70–1.57(m,4H),1.02(t,J＝7.4Hz,3H).
LRMS(ESI):[M+Na] +393.1；HRMS(ESI):[M+Na] +计算值C 19H 30O 7Na +393.1884,实测值为393.1880。
TG-022的数据：
1H NMR(400MHz,CD 3OD)δ8.53(d,J＝4.4Hz,1H),7.86(td,J＝7.8,1.6Hz,1H),7.59(d,J＝7.9Hz,1H),7.36(dd,J＝7.0,5.4Hz,1H),7.10(d,J＝8.6Hz,2H),6.90(d,J＝8.6Hz,2H),5.13(s,2H),4.23(d,J＝7.8Hz,1H),3.95–3.88(m,1H),3.85(dd,J＝11.9,1.7Hz,1H),3.66(dd,J＝11.9,5.2Hz,1H),3.59–3.51(m,1H),3.35-3.12(m,4H),2.57(t,J＝7.1Hz,2H),1.81–1.52(m,4H)。
LRMS(ESI):[M+Na] +442.0；HRMS(ESI):[M+Na] +计算值C 22H 29O 7NNa +442.1836, 实测值为442.1834。
TG-024的数据：
1H NMR(400MHz,CD 3OD)δ7.10(d,J＝8.5Hz,2H),6.82(d,J＝8.6Hz,2H),4.27(d,J＝7.8Hz,1H),4.02(q,J＝7.0Hz,2H),3.98–3.92(m,1H),3.89(dd,J＝11.8,1.6Hz,1H),3.69(dd,J＝11.8,5.2Hz,1H),3.58(dt,J＝9.5,6.2Hz,1H),3.41–3.29(m,3H),3.23–3.13(m,1H),2.59(t,J＝7.1Hz,2H),1.78–1.56(m,4H),1.39(t,J＝7.0Hz,3H).
LRMS(ESI):[M+Na] +379.1。
TG-025的数据：
1H NMR(400MHz,CD 3OD)δ7.13(d,J＝8.5Hz,2H),6.88(d,J＝8.6Hz,2H),4.97–4.83(m,2H),4.62(t,J＝6.0Hz,2H),4.27(d,J＝7.8Hz,1H),4.19(d,J＝6.4Hz,2H),3.95(dt,J＝6.3,3.3Hz,1H),3.89(dd,J＝11.8,1.7Hz,1H),3.69(dd,J＝11.8,5.2Hz,1H),3.63–3.54(m,1H),3.52–3.41(m,1H),3.41–3.24(m,3H),3.23–3.15(m,1H),2.61(t,J＝7.1Hz,2H),1.81–1.57(m,4H).
LRMS(ESI):[M+Na] +421.1。
TG-026的数据：
1H NMR(400MHz,CD 3OD)δ7.68(dd,J＝18.9,8.3Hz,4H),7.14(d,J＝8.6Hz,2H),6.93(d,J＝8.6Hz,2H),5.17(s,2H),4.27(d,J＝7.8Hz,1H),3.95(dt,J＝6.3,3.3Hz,1H),3.89(dd,J＝11.8,1.7Hz,1H),3.69(dd,J＝11.8,5.2Hz,1H),3.62–3.54(m,1H),3.42–3.24(m,3H),3.23–3.15(m,1H),2.61(t,J＝7.1Hz,2H),1.83–1.52(m,4H).
LRMS(ESI):[M+Na] +509.2。
TG-027的数据：
H NMR(400MHz,CD 3OD)δ7.40(dt,J＝7.9,5.9Hz,1H),7.26(d,J＝7.7Hz,1H),7.20(d,J＝9.9Hz,1H),7.13(d,J＝8.6Hz,2H),7.05(td,J＝8.5,2.3Hz,1H),6.91(d,J＝8.6Hz,2H),5.09(s,2H),4.27(d,J＝7.8Hz,1H),3.95(dt,J＝9.4,6.3Hz,1H),3.89(dd,J＝11.9,1.8Hz,1H),3.69(dd,J＝11.9,5.2Hz,1H),3.62–3.54(m,1H),3.41–3.24(m,3H),3.23–3.15(m,1H),2.60(t,J＝7.1Hz,2H),1.90–1.55(m,4H).
LRMS(ESI):[M+Na] +459.2。
TG-049的鉴定数据
1H NMR(400MHz,CD 3OD)δ7.74(s,1H),7.69(d,J＝7.4Hz,1H),7.62-7.53(m,2H),7.11(d,J＝8.6Hz,2H),6.91(d,J＝8.6Hz,2H),5.13(s,2H),4.23(d,J＝7.8Hz,1H),3.92(dt,J＝9.4,6.3Hz,1H),3.85(dd,J＝11.9,1.7Hz,1H),3.66(dd,J＝11.8,5.2Hz,1H),3.55 (dt,J＝9.6,6.2Hz,1H),3.37-3.32(m,1H),3.26(t,J＝5.9Hz,2H),3.20–3.11(m,1H),2.57(t,J＝7.1Hz,2H),1.80-1.49(m,4H).
LRMS(ESI):[M+Na] +509.20
TG-050的鉴定数据
1H NMR(400MHz,CD 3OD)δ7.53(td,J＝7.5,1.3Hz,1H),7.42-7.34(m,1H),7.21(td,J＝7.5,1.0Hz,1H),7.18-7.10(m,1H),7.14(d,J＝8.6Hz,2H),6.92(d,J＝8.6Hz,2H),5.12(s,2H),4.27(d,J＝7.8Hz,1H),3.95(dt,J＝9.4,6.3Hz,1H),3.89(dd,J＝11.9,1.8Hz,1H),3.69(dd,J＝11.9,5.2Hz,1H),3.58(dt,J＝9.5,6.3Hz,1H),3.39-3.25(m,3H),3.24-3.16(m,1H),2.61(t,J＝7.1Hz,2H),1.78-1.59(m,4H).
LRMS(ESI):[M+Na] +459.20
TG-051的数据
1H NMR(400MHz,CD 3OD)δ7.47(dd,J＝8.5,5.5Hz,2H),7.13(d,J＝8.6Hz,4H),7.14-7.09(m,1H),6.91(d,J＝8.6Hz,2H),5.04(s,2H),4.27(d,J＝7.8Hz,1H),3.95(dt,J＝9.3,6.2Hz,1H),3.89(dd,J＝11.8,1.7Hz,1H),3.69(dd,J＝11.8,5.2Hz,1H),3.58(dt,J＝9.4,6.2Hz,1H),3.-3.24(m,3H),3.23-3.14(m,1H),2.60(t,J＝7.1Hz,2H),1.91-1.56(m,4H).
LRMS(ESI):[M+Na] +459.2
TG-023的数据：
1H NMR(400MHz,CD 3OD)δ6.99(d,J＝8.5Hz,2H),6.70–6.63(m,2H),4.23(d,J＝7.8Hz,1H),3.96–3.82(m,2H),3.66(dd,J＝11.9,5.2Hz,1H),3.54(dt,J＝9.5,6.1Hz,1H),3.38–3.21(m,3H),3.16(dd,1H),2.53(t,J＝7.0Hz,2H),1.70–1.56(m,4H).
LRMS(ESI):[M+Na] +351.1；HRMS(ESI):[M+NH 4] +计算值C 16H 28O 7N +346.1860,实测值为346.1857。
该实验过程中的各反应中间体的结构如下：
Figure PCTCN2019085823-appb-000059
上述中间体18a-18q的鉴定数据为：
18a：
LRMS(ESI):[M+Na] +609.6。
18b：
LRMS(ESI):[M+Na] +561.5。
18c：
LRMS(ESI):[M+Na] +559.5。
18d：
LRMS(ESI):[M+Na] +615.6。
18e：
LRMS(ESI):[M+Na] +610.6。
18f：
LRMS(ESI):[M+Na] +601.6。
18g：
LRMS(ESI):[M+Na] +610.6。
18h：
LRMS(ESI):[M+Na] +617.6。
18i：
LRMS(ESI):[M+Na] +587.6。
18j：
LRMS(ESI):[M+Na] +610.2。
18k：
LRMS(ESI):[M+Na] +547.2。
18l：
LRMS(ESI):[M+Na] +589.2。
18m：
LRMS(ESI):[M+Na] +677.2。
18n：
LRMS(ESI):[M+Na] +627.2。
18o：
LRMS(ESI):[M+Na] +677.23。
18p：
LRMS(ESI):[M+Na] +654.23。
18q：
LRMS(ESI):[M+Na] +654.23。
路线D：终产物TG-028,TG-029,TG-030的合成
Figure PCTCN2019085823-appb-000060
路线D实验操作：
将20a(3g，16.7mmol)加入40ml吡啶中，滴加Ac 2O，反应3天。减压浓缩，硅胶柱层析提纯(洗脱剂：酸乙酯：石油醚＝1：4)得产品8a，产率95％。向化合物8a中滴加33％的HBr/AcOH(4eq)溶液，在室温条件下反应4h。减压浓缩，粗产物以Et 2O和正己烷重结晶，得化合物9a。将9a(1eq)加入三颈瓶，加入4A分子筛和无水二氯甲烷/Et 2O混合溶液。混合液搅拌均匀后，加入原料13(0.95eq)。继续搅拌30min后加入Ag 2CO 3(1.2eq)，反应过夜。硅藻土过滤，减压浓缩，硅胶柱层析提纯得产品22a(19％yield)。
将化合物22a(1eq)加入到反应瓶中，分别加入MeOH(4M)、H 2O(4M)、Et 3N(2eq)，室温条件下反应20h。硅藻土过滤后，减压浓缩，硅胶柱层析提纯，得化合物TG-028(64％yield)。
化合物TG-029和TG-030的合成使用与化合物TG-028类似的合成路线。
化合物TG-028、TG-029和TG-030的结构及鉴定数据如下所示：
Figure PCTCN2019085823-appb-000061
TG-028的数据：
H NMR(400MHz,DMSO)δ7.10(d,J＝8.5Hz,2H),6.83(d,J＝8.5Hz,2H),5.10–4.78(m,3H),4.45(t,J＝5.9Hz,1H),4.09(d,J＝7.8Hz,1H),3.77(m,J＝12.5,6.4Hz,1H),3.71(s,3H),3.65(dd,J＝10.8,6.0Hz,1H),3.49–3.37(m,2H),3.17–2.98(m,3H),2.92(td,J＝8.3,5.1Hz,1H),2.55-2.5(m,2H)1.69–1.41(m,4H).
LRMS(ESI):[M+Na] +365.0；HRMS(ESI):[M+Na] +计算值C 17H 26O 7Na +365.1571,实测值为365.1569。
TG-029的数据
1H NMR(400MHz,CD 3OD)δ7.08(d,J＝8.5Hz,2H),6.80(d,J＝8.6Hz,2H),4.23(d,J＝7.8Hz,1H),3.91(m,1H),3.85(dd,J＝11.9,1.7Hz,1H),3.76(s,3H),3.66(dd,J＝11.8,5.2Hz,1H),3.55(dt,J＝9.5,6.1Hz,1H),3.37–3.1(m,4H),2.56(t,J＝7.1Hz,2H),1.78–1.53(m,4H).
LRMS(ESI):[M+Na] +365.0；HRMS(ESI):[M+Na] +计算值C 17H 26O 7Na +365.1571,实测值为365.1570。
TG-030的数据
1H NMR(400MHz,CD 3OD)δ7.12(d,J＝8.3Hz,2H),6.83(d,J＝8.4Hz,2H),4.22(d,J＝7.3Hz,1H),3.95(dd,J＝10.9,4.9Hz,1H),3.85(d,J＝2.4Hz,1H),3.83–3.71(m,5H),3.64–3.44(m,4H),2.59(t,J＝6.9Hz,2H),1.68(d,J＝3.7Hz,4H).
LRMS(ESI):[M+Na] +365.1；HRMS(ESI):[M+NH 4] +计算值C 17H 30O 7N +360.2017,实测值为360.2015。
该实验过程中的各反应中间体的结构如下：
Figure PCTCN2019085823-appb-000062
上述中间体22a-22c的鉴定数据为：
22a：
1H NMR(400MHz,CDCl 3)δ7.07(d,J＝8.6Hz,2H),6.82(t,J＝5.7Hz,2H),5.20(t,J＝9.5Hz,1H),5.08(t,J＝9.7Hz,1H),4.98(t,J＝9.6,8.0Hz,1H),4.48(d,J＝8.0Hz,1H),4.26(dd,J＝12.3,4.7Hz,1H),4.16–4.07(m,1H),3.89(dd,J＝5.8,3.7Hz,1H),3.78(s,3H),3.68(m,J＝9.9,4.6,2.4Hz,1H),3.49(dt,J＝9.4,6.1Hz,1H),2.55(t,J＝6.6Hz,2H),2.08(s,3H),2.05–1.96(m,9H),1.68–1.52(m,4H).
LRMS(ESI):[M+Na] +533.2。
22b：
LRMS(ESI):[M+Na] +533.2。
22c：
LRMS(ESI):[M+Na] +533.2。
路线E：化合物TG-031TG-032TG-033TG-034TG-035TG-036TG-037的合成
Figure PCTCN2019085823-appb-000063
其中片段24a～e的合成如下所示：
Figure PCTCN2019085823-appb-000064
路线E实验操作：
片段24a：将化合物28(2.48g,0.02mol)和29(3.336g，0.024mol)分别加入DMF(30mL)中，室温加K 2CO 3(13.8g,0.1mol)，室温搅拌48小时。加水用乙酸乙酯萃取，萃取液合并水洗，以无水硫酸钠干燥。砂芯漏斗过滤，减压浓缩。以乙酸乙酯和石油醚混合液重结晶得24a：3.1g 85％yield。
片段24b：将31(3.72g，60mmol)加入THF中，分批加入NaH(0.624g，26mmol)依次加入30(3.132g，20mmol)Bu 4NI(738mg，2mmol)加热到70℃，搅拌5h。反应液降低到室温，用饱和氯化铵洗涤一次，减压浓缩。硅胶柱层析提纯，得到24b：2.9g，80％yield。
片段24c：将33(6.1g，0.1mol)加入MeOH中，室温下32(13.6g，0.1mol)加热到70℃，搅拌过夜。冷却到室温，分批加入NaBH 4(3.8g,0.1mol)，然后加热到70℃，搅拌2h。冷却到室温，采用乙酸乙酯萃取。萃取液合并后用水洗，无水硫酸钠干燥。砂芯漏斗过滤，减压浓缩，硅胶柱层析提纯得34(13.4g，88％yield)。
将34(3.6g，23.8mmol)加入二氯甲烷中，冰浴滴加Boc 2O(6.23g，28.6mmol)。滴加完毕后，室温搅拌反应过夜，减压浓缩，硅胶柱层析提纯得24c(4.9g，73％yield)。
片段24d：将34(3.6g，23.8mmol)加入二氯甲烷中，冰浴依次滴加CbzCl(6.63g，0.39mmol)和Et 3N(4.5g，0.45mmol)搅拌5h。反应液依次以水、NaHCO 3饱和液、饱和食盐水洗涤一次。无水硫酸钠干燥，减压浓缩，硅胶柱层析提纯得24d(6g，63.5％yield)。
片段24e：将35(6.15g，50mmol)，29(13.9g,100mol),和K 2CO 3(13.8g，100mmol)依次加入正丁醇(40mL)中，加碘(50mg),回流反应30h。减压浓缩，硅胶柱层析提纯得产品36 8.5g，93％yield。将36(3.3g，18.2mmol)加入二氯甲烷,加K 2CO 3(4.9g，36mmol)，冰浴滴加Cbz-Cl(4g，23.7mmol)室温搅拌过夜。依次以水、饱和食盐水洗涤一次，无水硫酸钠干燥，减压浓缩，硅胶柱层析提纯得24e(3g，72％yield)。
将9a(1eq)和24a～e(0.95mmol)，4A分子筛依次加入反应瓶。加干燥的二氯甲烷(0.25mol/L),搅拌30min。氩气氛保护下加Ag 2CO 3(1.2eq)，室温搅拌过夜，硅藻土过滤。滤液减压浓缩，以硅胶柱层析提纯得产品25a～e。将25a～e(1eq)各自加入单颈瓶，向各单颈瓶中均加入MeOH(6eq)，H 2O(6eq)，Et 3N(3eq)，搅拌过夜，减压浓缩，硅胶柱层析提纯(洗脱剂：甲醇/二氯甲烷＝1:15)得产品TG-031(产率24％),TG-032(产率26％),TG-033(产率24％),TG-034(产率20％),TG-035(产率12％)。
将TG-033(1g，2.25mmol)和2,6-Lutidine(481mg，4.5mmol)加入二氯甲烷,冰浴下滴加TBDMSOTf(893mg，3,38mmol)，0℃搅拌2小时，TLC显示反应完全。用0.5M的HCl将PH调到5，旋干，过柱(甲醇/二氯甲烷＝1:20)，得TG-036 400mg，产率：46.8％。
将TG-035(700mg，1.46mmol)，加入MeOH(30ml)加Pd/C，用氢气抽气换气，40个大气压常温搅拌过夜。硅藻土过滤，减压浓缩，硅胶柱层析提纯(洗脱剂：甲醇/二氯甲烷＝1:12)得产品TG-037(320mg，63％yield)。
化合物TG-031，TG-032，TG-033，TG-034，TG-035，TG-036和TG-037的结构及鉴定数据如下所示：
Figure PCTCN2019085823-appb-000065
TG-031的数据：
1H NMR(400MHz,DMSO)δ6.89–6.81(m,1H),4.90(s,1H),4.46(s,1H),4.46(s,1H),4.13(d,J＝7.8Hz,1H),3.99(t,J＝6.4Hz,1H),3.90(dt,J＝9.9,6.3Hz,1H),3.66–3.55(m,1H),3.43(dd,J＝11.8,5.5Hz,1H),3.08(dq,J＝23.5,8.9Hz,1H),2.94(t,J＝8.3Hz,1H),1.94(q,J＝6.6Hz,1H).
LRMS(ESI):[M+Na] +367.1；HRMS(ESI):[M+NH 4] +计算值C 16H 28O 8N +362.1809,实测值为362.1807。
TG-032的数据：
1H NMR(300MHz,CD 3OD)δ7.25(d,J＝8.3Hz,2H),6.86(d,J＝8.4Hz,2H),4.46(s,2H),4.25(d,J＝7.7Hz,1H),4.06–3.91(m,1H),3.87–3.54(m,7H),3.36–3.10(m,4H).
LRMS(ESI):[M+Na] +367.1；HRMS(ESI):[M+NH 4] +计算值C 16H 28O 8N +362.1809,实测值为362.1806。
TG-033的数据：
1H NMR(400MHz,DMSO)δ7.18(d,J＝8.4Hz,2H),6.90(d,J＝8.1Hz,2H),5.02(bs,2H),4.59–4.28(m,3H),4.11(t,1H),3.73(s,4H),3.66(d,J＝11.6Hz,1H),3.61–3.50(m,1H),3.44(dd,J＝11.6,4.7Hz,1H),3.39–3.00(m,6H),2.94(t,J＝8.3Hz,1H),1.41(d,J＝14.2Hz,9H)。
TG-034的数据：
1H NMR(300MHz,DMSO)δ7.35(d,J＝13.0Hz,5H),7.17(d,J＝15.0Hz,2H),6.88(t,2H),5.09(dd,J＝14.9Hz,3H),4.95(dd,J＝14.2Hz,2H),4.54–4.41(m,3H),4.17–4.04(m,1H),3.87–3.51(m,5H),3.50–3.20(m,4H),3.18–2.87(m,5H).
LRMS(ESI):[M+Na] +500.2；HRMS(ESI):[M+H] +计算值C 24H 32O 9N +478.2072,实测值为478.2065。
TG-035的数据：
1H NMR(400MHz,CD 3OD)δ7.55–7.02(m,7H),6.92(d,J＝8.9Hz,2H),5.08(s,2H),4.17(d,J＝7.7Hz,1H),3.98–3.87(m,1H),3.86–3.70(m,6H),3.64(dd,J＝11.9,5.3Hz,1H),3.60–3.50(m,1H),3.36–3.19(m,3H),3.14(t,J＝8.4Hz,1H),1.93–1.74(m,2H).
LRMS(ESI):[M+Na] +500.0；HRMS(ESI):[M+Na] +计算值C 24H 31O 9NNa +500.1891，实测值为500.1889。
TG-036的数据：
1H NMR(400MHz,CD 3OD)δ7.43(d,J＝8.7Hz,2H),7.00(d,J＝8.7Hz,2H),4.35(d,J＝7.8Hz,1H),4.20(s,2H),4.09(s,1H),3.90(m,J＝14.0,4.4Hz,2H),3.82(s,3H),3.65(dd,J＝11.7,6.1Hz,1H),3.40–3.32(m,2H),3.24(dt,J＝11.5,9.1Hz,4H).
LRMS(ESI):[M+H] +344.1；HRMS(ESI):[M+H] +计算值C 16H 26O 7N +344.1704,实测值为344.1701。
TG-037的数据：
1H NMR(400MHz,CD 3OD)δ6.78–6.72(m,2H),6.72–6.65(m,2H),4.28(d,J＝7.8Hz,1H),4.07–3.96(m,1H),3.87(dd,J＝11.8,1.4Hz,1H),3.70(s,3H),3.69–3.63(m,2H),3.34(t,J＝4.5Hz,1H),3.29–3.25(m,2H),3.23–3.14(m,3H),1.89(t,J＝6.4Hz,2H).
LRMS(ESI):[M+Na] +366.1
该实验过程中的各反应中间体的结构如下：
Figure PCTCN2019085823-appb-000066
上述中间体25a-25e的鉴定数据为:
25a：
LRMS(ESI):[M+Na] +535.1。
25b：
LRMS(ESI):[M+Na] +535.1。
25c：
LRMS(ESI):[M+Na] +634.2。
25d：
LRMS(ESI):[M+Na] +668.2。
25e：
LRMS(ESI):[M+Na] +668.2。
路线F：化合物TG-038，TG-039，TG-040，TG-041，TG-042，TG-043和TG-044的合成
Figure PCTCN2019085823-appb-000067
路线F的实验操作：
将15(1eq)和PPh 3(1.3eq)加入反应瓶，加干燥的THF(0.5mol/L)，加37a～f(1eq)，0℃下滴加DIAD(1.1eq)。升至60℃搅拌，TLC监测反应结束。减压浓缩，硅胶柱层析提纯39a～f。
将37g(1eq)加入THF(0.2mol/L)，冰浴下滴加PPh 3(2eq)，咪唑(2eq)，I 2(2.5eq)，升至室温搅拌过夜。硅藻土过滤，减压浓缩，硅胶柱层析提纯(洗脱剂：乙酸乙酯：石油醚＝1：9)，得产品38g(5.7g，93％yield)。将38g(1.2eq)，15(1eq)和K 2CO 3(2eq)加入DMF,加热到110℃，搅拌10h。硅藻土过滤，滤液用乙酸乙酯萃取。萃取液合并后水洗，无水硫酸钠干燥。减压浓缩得粗品39g 3.8g。
将39a～g(1eq)加入MeOH(0.5mol/L),室温滴加1M的NaOH水溶液(2.5eq)，搅拌4小时。以2M的HCl中和反应体系，然后乙酸乙酯萃取，萃取液以无水硫酸钠干燥。减压浓缩，硅胶柱层析提纯(洗脱液：乙酸乙酯：石油醚＝1:5)得产品40a～g。
将8a(120g，0.3mol)500ml的DMF中，室温中加(NH 4) 2CO 3(60g，0.6mol)，将 反应液加热到45℃，搅拌5h。硅藻土过滤，滤液加水，以乙酸乙酯萃取。萃取液合并后以依次以水、饱和食盐水洗涤，无水硫酸钠干燥。减压浓缩得粗产品41(95g)，直接用于下一步反应。将41(95g，0.273mol)和K 2CO 3(49g,0.355mol)加入二氯甲烷(400mL)，冰浴下滴加CCl 3CN(157g，1.1mol)升至室温搅拌过夜。硅藻土过滤，减压浓缩，硅胶柱层析提纯得42(95g，71％yield)。
将40a～g(1.1eq)和42(1eq)，4A 分子筛加入反应瓶，加干燥的二氯甲烷(0.2mol/L)，将反应冷却至-20℃滴加TMSOTf(0.5eq)。TLC检测反应结束，加Et 3N(1.5eq)淬灭反应。减压浓缩，硅胶柱层析提纯得(洗脱剂：乙酸乙酯：石油醚＝1:5)得产品43a～g。
将43a/43b/43e(1eq)分别加入单颈瓶，加入MeOH(6eq)，H 2O(6eq)，Et 3N(3eq)，搅拌过夜。减压浓缩，硅胶柱层析提纯得(洗脱剂：甲醇/二氯甲烷＝1:15)得产品TG-038(产率63％)/TG-039(产率60％)/TG-042(产率36％)。
将43c/43d/43f/43g(1eq)分别加入MeOH(0.5mol/L)，冰浴滴加0.2M的MeONa(2.5eq)升至室温搅拌1h。TLC检测反应结束，减压浓缩，硅胶柱层析提纯得TG-040(产率15％)/TG-041(产率64％)/TG-043(产率55％)/TG-044(产率16％)。
化合物TG-038，TG-039，TG-040，TG-041，TG-042，TG-043和TG-044的结构及鉴定数据如下所示：
Figure PCTCN2019085823-appb-000068
TG-038的数据：
1H NMR(400MHz,CD 3OD)δ7.07(d,J＝8.6Hz,2H),6.89–6.70(m,2H),4.52(dt,J＝12.1,6.1Hz,1H),4.23(d,J＝7.8Hz,1H),3.95–3.88(m,1H),3.85(dd,J＝11.9,1.9Hz,1H),3.66(dd,J＝11.8,5.3Hz,1H),3.62–3.51(m,1H),3.36–3.33(m,1H),3.28–3.20(m,2H),3.17(dd,J＝15.1,7.2Hz,1H),2.56(t,J＝7.1Hz,2H),1.75–1.54(m,4H),1.27(d,J＝6.0Hz,6H).
LRMS(ESI):[M+Na] +393.0；HRMS(ESI):[M+Na] +计算值C 19H 30O 7Na +393.1884,实测值为393.1880。
TG-039的数据：
1H NMR(400MHz,CD 3OD)δ7.07(d,J＝8.6Hz,2H),6.79(d,J＝8.6Hz,2H),4.23(d,J＝7.8Hz,1H),3.91(m,1H),3.88–3.82(m,1H),3.77(d,J＝6.8Hz,2H),3.67(dd,J＝12.0,5.3Hz,1H),3.55(m,1H),3.33-3.17(m,4H),2.56(t,J＝7.1Hz,2H),1.76–1.54(m,4H),1.27–1.16(m,1H),0.59(dd,J＝8.1,1.4Hz,2H),0.38–0.24(m,2H).
LRMS(ESI):[M+Na] +405.0；HRMS(ESI):[M+Na] +计算值C 20H 30O 7Na +405.1884，实测值为405.1883。
TG-040的数据：
1H NMR(400MHz,CD 3OD)δ7.07(d,J＝8.5Hz,2H),6.79(d,J＝8.6Hz,2H),4.23(d,J＝7.8Hz,1H),3.99(t,J＝7.1Hz,2H),3.91(dt,J＝9.3,6.3Hz,1H),3.85(dd,J＝11.9,1.9Hz,1H),3.67(dd,J＝11.9,5.3Hz,1H),3.57-3.52(m,1H),3.26-3.11(m,4H),2.56(t,J＝7.1Hz,2H),1.75–1.53(m,6H),0.99(s,9H).
LRMS(ESI):[M+Na] +435.1；HRMS(ESI):[M+Na] +计算值C 22H 36O 7Na +435.2353，实测值为435.2352。
TG-041的数据：
1H NMR(400MHz,CD 3OD)δ7.10(d,J＝8.5Hz,2H),6.82(d,J＝8.5Hz,2H),4.27(d,J＝7.8Hz,1H),4.01–3.82(m,2H),3.71(d,J＝5.2Hz,1H),3.60(m,3H),3.42–3.24(m,4H),3.20(t,J＝8.4Hz,1H),2.59(t,J＝7.0Hz,2H),1.78–1.53(m,4H),1.06(s,9H).
LRMS(ESI):[M+Na] +421.1；HRMS(ESI):[M+Na] +计算值C 21H 34O 7Na +421.2197，实测值为421.2194。
TG-042的数据：
Figure PCTCN2019085823-appb-000069
1H NMR(400MHz,CD 3OD)δ6.85(m,2H),6.58(d,J＝8.9Hz,1H),4.23(d,J＝7.8Hz,1H),3.95–3.88(m,1H),3.85(dd,J＝11.9,1.8Hz,1H),3.66(dd,J＝11.9,5.2Hz,1H),3.58–3.51(m,1H),3.36-3.26(m,2H),3.25-3.13(m,2H),2.74(t,J＝6.8Hz,2H),2.51(t,J＝6.9Hz,2H),1.77(t,J＝6.8Hz,2H),1.71–1.56(m,4H),1.28(s,6H)。
LRMS(ESI):[M+Na] +419.1；HRMS(ESI):[M+Na] +计算值C 21H 32O 7Na +419.2040，实测值为419.2041。
TG-043的数据：
1H NMR(400MHz,CD 3OD)δ7.07(d,J＝8.6Hz,2H),6.79(d,J＝8.6Hz,2H),4.23(d,J＝7.8Hz,1H),3.92(dd,J＝7.9,5.1Hz,2H),3.85(dd,J＝11.9,1.9Hz,1H),3.66(dd,J＝11.9,5.2Hz,1H),3.55(m,1H),3.34–3.21(m,4H),2.56(t,J＝7.1Hz,2H),1.70(m,6H),1.45(m,2H),1.41–1.20(m,24H),0.99(s,1H),0.89(t,J＝6.8Hz,3H).
LRMS(ESI):[M+Na]575.3；HRMS(ESI):[M+Na] +计算值C 32H 56O 7Na +,575.3918，实测值为575.3917。
TG-044的数据：
1H NMR(400MHz,CD 3OD)δ7.08(d,J＝8.5Hz,2H),6.83(d,J＝8.6Hz,2H),4.23(d,J＝7.8Hz,1H),4.15–4.02(m,2H),3.91(m,1H),3.85(dd,J＝11.8,1.7Hz,1H),3.83–3.78(m,2H),3.71–3.47(m,14H),3.36-3.17(m,7H),2.56(t,J＝7.0Hz,2H),1.66(m,4H).
LRMS(ESI):[M+Na] +541.2；HRMS(ESI):[M+Na] +计算值C 25H 42O 11Na 541.2619,found 541.2618。
该实验过程中的各反应中间体的结构如下：
Figure PCTCN2019085823-appb-000070
上述中间体39a-39f的鉴定数据为:
39a：
LRMS(ESI):[M+Na] +273.1。
39b：
LRMS(ESI):[M+Na] +285.1。
39c：
LRMS(ESI):[M+Na] +315.2。
39d：
LRMS(ESI):[M+Na] +301.1。
39e：
LRMS(ESI):[M+Na] +299.1。
39f：
LRMS(ESI):[M+Na] +455.6。
39g：
LRMS(ESI):[M+Na] +421.2。
Figure PCTCN2019085823-appb-000071
40b：
LRMS(ESI):[M+Na] +243.1。
40c：
LRMS(ESI):[M+Na] +273.1。
40d：
LRMS(ESI):[M+Na] +259.1。
40e：
LRMS(ESI):[M+Na] +257.1。
40f：
LRMS(ESI):[M+Na] +413.3。
40g：
LRMS(ESI):[M+Na] +329.2。
Figure PCTCN2019085823-appb-000072
43a：
LRMS(ESI):[M+Na] +561.2。
43b：
LRMS(ESI):[M+Na] +573.2。
43c：
LRMS(ESI):[M+Na] +603.2。
43d：
LRMS(ESI):[M+Na] +589.2。
43e：
LRMS(ESI):[M+Na] +587.2。
43f：
LRMS(ESI):[M+Na] +743.4。
43g：
LRMS(ESI):[M+Na] +709.3。
TG-045的合成
Figure PCTCN2019085823-appb-000073
实验操作：
将原料45和Py加入体系中，搅拌溶解，体系降至0℃滴加BzCl，滴加完毕自然升至室温搅拌12h，减压蒸出溶剂。加大量H 2O,二氯甲烷萃取3次，合并有机相。有机相依次用NaHCO 3x 2、饱和NaCl x 1洗，Na 2SO 4干燥。减压蒸除溶剂，硅胶柱柱层析提纯(洗脱剂：石油醚/乙酸乙酯＝2：1)得无色液体化合物46，产率100％。
将46和NH 2NH 2.AcOH加入体系中，DMF搅拌溶解，60℃搅拌7h。加大量H 2O,二氯甲烷萃取3次，合并有机相。有机相依次用NaHCO 3x 2、饱和NaCl x 1洗，Na 2SO 4干燥。减压蒸除溶剂，硅胶柱柱层析提纯(洗脱剂：石油醚/乙酸乙酯＝1：1)得无色液体化合物47，产率78％。
将47和二氯甲烷加入体系中，0℃搅拌依次滴加DBU、CCl 3CN，滴加完毕自然升至室温搅拌3h。减压蒸除溶剂，硅胶柱柱层析提纯(洗脱剂：石油醚/乙酸乙酯＝2：1)得白色固体化合物48，产率72％。
将化合物48、醇13、二氯甲烷和4A分子筛加入体系中，搅拌溶解，0℃滴加TMSOTf，滴加完毕自然升至室温搅拌12h。抽滤，减压蒸除溶剂，硅胶柱柱层析提纯(洗脱剂：石油醚/乙酸乙酯＝5：1)得无色液体化合物49,产率85％。
将49、MeOH加入体系中，搅拌溶解，0℃加入NaOMe，加完毕自然升至室温搅拌 12h。减压蒸除溶剂，硅胶柱柱层析提纯(洗脱剂：二氯甲烷/MeOH＝5：1)得白色固体终产物TG-045，产率75％。
Figure PCTCN2019085823-appb-000074
TG-045的鉴定数据：
1H NMR(400MHz,CD 3OD)δ7.25-7.01(m,2H),6.95-6.75(m,2H),4.41-4.26(m,2H),3.90-3.38(m,16H),3.31-3.22(m,1H),2.60-2.36(m,2H),1.65-1.42(m,4H).
LRMS(ESI):[M+Na] +527.2。
该实验过程中的各反应中间体的结构如下：
Figure PCTCN2019085823-appb-000075
46的鉴定数据：
LRMS(ESI):[M+Na] +1197.3。
Figure PCTCN2019085823-appb-000076
47的鉴定数据：
LRMS(ESI):[M+Na] +1093.3。
Figure PCTCN2019085823-appb-000077
48的鉴定数据：
LRMS(ESI):[M+Na] +1236.2。
Figure PCTCN2019085823-appb-000078
49的鉴定数据：
LRMS(ESI):[M+Na] +1255.4。
TG-046的合成
Figure PCTCN2019085823-appb-000079
将化合物50(9g,50mmol)和13(27g，150mmol)，TsOH.H 2O(1g,5.26mmol)加入反应瓶，加热到80℃，搅拌18小时。冷却至室温，加H 2O(50mL)，搅拌1小时，加乙酸乙酯(100mL)萃取三次。萃取液合并，无水硫酸钠干燥。减压蒸除溶剂，硅胶柱柱层析提纯得到粗产品3.1g，结晶得纯品TG-046(500mg)。
TG-046鉴定数据如下所示：
1H NMR(400MHz,CD 3OD)δ7.12(d,J＝8.3Hz,2H),6.84(d,J＝8.3Hz,2H),4.79(d,J＝3.5Hz,1H),3.88-3.73(m,2H),3.78(s,3H),3.73-3.64(m,2H),3.62-3.54(m,1H), 3.53-3.45(m,1H),3.41(dd,J＝9.7,3.7Hz,1H),3.29(m,1H),2.63-2.59(m,2H),1.75-1.63(m,4H).
LRMS(ESI):[M+Na] +365.1
TG-047和TG-048的合成
Figure PCTCN2019085823-appb-000080
将化合物9a(16.4g，40mmol)，KSAc(13.28g，80mmol)，依次加入丙酮(150mL)中，室温搅拌4小时，TLC监测基本反应完全。将反应液倒入150mL水中，乙酸乙酯萃取，萃取液合并，无水硫酸钠干燥。减压蒸除溶剂，硅胶柱柱层析提纯(洗脱剂：乙酸乙酯:石油醚＝1:6)得化合物51 12.7g。
将化合物51(12.7g，31.2mmol)、52(6.26g，40.7mmol)和NaHCO 3(0.262g，3.12mmol)加入DMF(100mL)中室温搅拌1小时。加水150mL，用乙酸乙酯萃取。合并有机相，氯化钠水溶液洗，无水硫酸钠干燥。减压蒸除溶剂，硅胶柱柱层析提纯(洗脱剂：乙酸乙酯/石油醚＝1:4)得化合物53 10g。
将化合物13(10mmol)、PPh 3(4.3g，13mmol)加入加入25mL二氯甲烷中。室温下分批加入CBr 4(4.3g，13mmol)，室温搅拌1小时。减压蒸除溶剂，硅胶柱柱层析提纯得化合物54(2g)
将化合物53(1.2g，3.34mmol)54(3.674mmol)和Et 3N(334mg，3.34mmol)依次加入乙腈(20mL)中，室温搅拌1小时，加水20mL，用乙酸乙酯萃取，饱和NaCl水溶液洗，无水硫酸钠干燥。减压蒸除溶剂，硅胶柱柱层析提纯得化合物55(1.1g)
Figure PCTCN2019085823-appb-000081
LRMS(ESI):[M+Na] +549.2
将化合物55(1.1g)加入MeOH(5mL)中，加H 2O(5mL)，Et 3N(2.5mL)，室温搅拌过夜，TLC显示反应完全。减压蒸除溶剂，硅胶柱柱层析提纯得TG-048(500mg)。
Figure PCTCN2019085823-appb-000082
TG-048鉴定数据
1H NMR(400MHz,CD 3OD)δ7.12(d,J＝8.6Hz,2H),6.85(d,J＝5.7Hz,2H),4.35(d,J＝9.7Hz,1H),3.86(dd,J＝12.0,2.0Hz,1H),3.78(s,3H),3.67(dd,J＝11.8,5.7Hz,1H),3.43-3.16(m,4H),2.76(m,2H),2.60(t,J＝7.2Hz,2H),1.88-1.54(m,4H).
LRMS(ESI):[M+Na] +381.1
TG-047的制备方法参考TG-048的制备方法，其结构如下所示：
Figure PCTCN2019085823-appb-000083
TG-047鉴定数据
1H NMR(400MHz,CD3OD)δ7.18(d,J＝8.6Hz,2H),6.85(d,J＝8.6Hz,2H),4.38(d,J＝9.5Hz,1H),3.90(dd,J＝12.0,1.8Hz,1H),3.78(s,3H),3.69(dd,J＝12.0,5.4Hz,1H),3.36-3.26(m,3H),3.22(t,J＝8.7Hz,1H),3.07-2.86(m,4H).
LRMS(ESI):[M+COOH]-375.0。
效果实施例
效果实施例1、体外巨噬细胞实验测试化合物的促VEGF-A mRNA表达活性
1.1、活性筛选的具体步骤为：分别将1～10×10 6对数生长期的巨噬细胞(RAW264.7) 置于细胞培养皿中，将待测化合物(上述化合物TG-001,TG-002,TG-003,TG-004,TG-005,TG-006,TG-007,TG-008，TG-009,TG-010,TG-011,TG-012，TG-013,TG-014,TG-015,TG-016,TG-017,TG-018,TG-019,TG-020,TG-021,TG-022,TG-023，TG-024，TG-025，TG-026，TG-027，TG-028,TG-029,TG-030，TG-031，TG-032，TG-033，TG-034，TG-035，TG-036，TG-037，TG-045，TG-039，TG-040TG-041，TG-042，TG-043，TG-044，TG-045、TG-052、TG-053、TG-054、TG-047、TG-048、TG-046、TG-049、TG-050、TG-051、TG-055和TG-056)分别稀释成不同的浓度，分别加入到上述的含有巨噬细胞的培养皿中，在37℃、5％CO 2细胞培养箱中于完全培养液(RPMI 1640培养基：胎牛血清：三抗(即青霉素-链霉素-庆大霉素，北京雷根生物技术有限公司)体积比＝89:10:1)中培养3h，Trizol(康为世纪生物科技有限公司)裂解细胞提取RNA，使用超微量核酸蛋白测定仪(Nanodrop2000c)测定RNA浓度。根据RNA浓度计算所需RNA体积V，根据表1进行RNA逆转录反应体系配制，然后根据表2所述条件进行RNA逆转录(cDNA合成)(RT EasyTM I(For first-strand cDNA synthesis)反转录试剂盒，成都福际生物技术有限公司)反应。所得cDNA按照表3配制荧光定量PCR反应溶液。按照表4设置荧光定量PCR反应条件，以逆转录反应的cDNA为模板进行荧光定量PCR反应(Real time PCR EasyTM-SYBR Green荧光定量试剂盒，成都福际生物技术有限公司)。每个样品的Ct值以空白对照组为对照，以β-Actin为内参，计算目标基因的相对表达量2 －ΔΔCt。
化合物TG-052、TG-053和TG-054可参考Chemical&Pharmaceutical Bulletin,2010，58,1627-1629记载的方法进行合成得到。
Figure PCTCN2019085823-appb-000084
化合物TG-056购于(上海源叶生物科技有限公司)，其结构为：
Figure PCTCN2019085823-appb-000085
表1 RT反应体系配制
Figure PCTCN2019085823-appb-000086
表2 cDNA合成反应条件
Figure PCTCN2019085823-appb-000087
表3 QPCR反应溶液
Figure PCTCN2019085823-appb-000088
表4 QPCR反应条件
Figure PCTCN2019085823-appb-000089
根据药物在一定浓度下能够促进VEGF-A mRNA表达的最大量来判断其活性，规定未加药物时巨噬细胞VEGF-A mRNA表达量为1，以临床上用于心脑血管疾病领域的一款中药注射液丹参多酚酸盐促进巨噬细胞VEGF-A mRNA最大表达量6(结果见表5)为判断标准，最大表达量大于4的化合物为目标化合物。即，当所述待测物质促进巨噬细胞中VEGF-A mRNA的最大表达量比未加所述待测物质时巨噬细胞中VEGF-A mRNA的表达量提高4倍以上时，所述待测物质即为初步的目标促血管生成活性物质。结果如表6所示，结果显示本发明提供的待测物大部分在一定剂量下具有良好的促VEGF-A mRNA表达的作用。
表5 不同浓度的丹参多酚酸盐作用于巨噬细胞时VEGF-A mRNA的表达量
Figure PCTCN2019085823-appb-000090
表6 不同浓度的化合物作用于巨噬细胞时VEGF-A mRNA的表达量
Figure PCTCN2019085823-appb-000091
Figure PCTCN2019085823-appb-000092
“-”代表未检测。
效果实施例2 利用“海绵(sponge)植入动物模型”筛选促进VEGF-A mRNA表达的活性分子
选用效果实施例1中所获得的在细胞水平表现良好的目标促血管生成化合物TG-052、TG-053、TG-054、TG-028、TG-055，在炎症性血管生成的“海绵(sponge)植入动物模型”上进一步筛选促进VEGF-A mRNA表达的活性分子。相关研究表明，在“海绵植入动物模型”中，炎症初期能募集到海绵中的细胞约有75％以上为巨噬细胞，该模型可以很好地反映募集到炎症周围的巨噬细胞中VEGF-A mRNA的表达量。
通过Zhang J,Modi Y,Yarovinsky T,et al.Macrophageβ2integrin-mediated,HuR-dependent stabilization of angiogenic factor-encoding mRNAs in inflammatory angiogenesis.[J].American Journal of Pathology,2012.180(4):1751-1760中所述方法建立小鼠海绵植入动物模型，本发明所用小鼠为C57/BL6小鼠，雄性，周龄为6-8周，体重为20-25克，购自上海斯莱克实验动物有限公司，生产许可证号SCXK(沪)2013-0016。将效果实施例1中筛选得到的上述化合物按两周用量进行称量，然后与凝胶基质剂及溶剂混合制得化合物缓释凝胶剂(所述缓释凝胶的制备方法及模型建立和检测方法详见专利申请CN201610168493.9)，具体步骤为：称取一定量的效果实施例1所得化合物置于1.5mL EP管中，再称取一定量的重均分子量小于1万的左旋聚乳酸(PLLA)与之混合(所得化合物与PLLA的质量比值在0.5:1到2:1之间)，最后加入一定量的DMSO(小于100微升)，涡旋混匀后用封口膜封口，在200W功率，25℃条件下连续超声5h后得到该化合物的缓释凝胶剂，4℃保存备用。
所制得的缓释凝胶剂可以在2周内将药物释放完全。将该缓释凝胶剂与海绵同时植入到小鼠的背部皮下，2周后将海绵取出，除去多余组织和皮毛后，转移到10mL含胶原酶的DMEM培养液的细胞培养皿中(胶原酶：DMEM＝10mg/10mL)，将海绵切碎，并置于细胞培养箱中孵育1h。然后将上述含海绵碎屑的培养液过滤，得到的滤液离心，弃去上清液，往沉淀中加入500μL Trizol试剂，吹散混合均匀后将液体转移到1.5mL EP管中，-80℃保存。按照常规的方法提取RNA，使用超微量核酸蛋白测定仪(Nanodrop2000c)测定RNA浓度。根据RNA浓度计算所需RNA体积V，根据表1进行RNA逆转录反应体系配制，然后根据表2所述条件进行RNA逆转录(cDNA合成)反应。所得cDNA按照表3配制荧光定量qPCR反应溶液。按照表4设置荧光定量qPCR反应条件，以逆转录反应的cDNA为模板进行荧光定量PCR反应。每个样品的Ct值以空白对照组为对照，以β-Actin为内参，计算目标基因的相对表达量2 －ΔΔCt。观察在2周作用时间下，由海绵所提取的细胞中的VEGF-A mRNA的表达水平。
在“海绵植入动物模型”上化合物分别选择了高、中、低三个不同的给药剂量，60mg/kg/d组的丹参多芬酸盐(购于河南省人民医院)为阳性对照(实验表明该剂量为丹参多芬酸盐在此模型上能够促进VEGF-A mRNA表达的最佳剂量，如图1A所示)，未加 药的空白对照缓释凝胶为空白对照，图1为通过RT-qPCR实验所得的结果。
与未加药的空白对照相比，结果发现TG-052(41.37mg/kg/d)、TG-055(19.48mg/kg/d)VEGF-A mRNA表达量增加了约3倍，与丹参多芬酸盐(60mg/kg/d)的活性相当，TG-055(19.48mg/kg/d)的剂量要小于TG-052(41.37mg/kg/d)，其活性要优于TG-052；TG-053与TG-054VEGF-A mRNA表达量与丹参多芬酸盐(60mg/kg/d)相比，活性稍微降低；TG-028在11.74mg/kg/d及23.48mg/kg/d剂量下，促进VEGF-A mRNA表达量为未加药的空白对照的6倍以上，活性明显高于丹参多芬酸盐(60mg/kg/d)的活性。因此，活性结果可以表述为：TG-028＞TG-055＞TG-052＞丹参多芬酸盐＞TG-053＞TG-054。结果表明，效果实施例1中利用体外细胞实验筛选出的化合物在体内动物筛选模型上大部分活性优于商购的药物丹参多酚酸盐或与其相当。
如图19所示，单独使用不同剂量的红景天苷(TG-056)在海绵植入动物模型上对VEGF-A mRNA表达量非常低。同时，在海绵植入动物模型上我们考察红景天苷和酪醇LC(红景天苷注射液里面的主要成分)一起作用对VEGF-A mRNA表达的影响，发现在红景天苷20.62mg/(kg.d)+酪醇3.08mg/(kg.d)可以促进VEGF-A mRNA 的表达，作用不如丹参多酚酸盐，因此，在后续实验中我们就不再考察红景天苷的作用。
效果实施例3 筛选出的活性分子的抑制脑梗作用和对脑损伤神经保护功能的验证
在炎症性血管生成动物模型上获得能够促进VEGF-A mRNA表达的活性分子后，运用大鼠脑梗模型进行进一步验证。根据Longa,E.Z.,Longa,E.Z.,Weinstein,P.R.,Carlson,S.&Commin,R.Reversible middle cerebral artery occlusion without craniectomy in rats.[J].Stroke,1989.1:84–91所述方法建立大鼠大脑中动脉阻塞(MCAO)急性脑梗模型，和根据C.dela Torre,T.Fortin,G.A.S.Park,K.S.Butler,P.Kozlowski,B.A.Pappas,H.de Socarraz,J.K.Saunders and M.T.Richard.[J].Brain Research,1992.582:186-195所述方法建立大鼠双侧颈总动脉永久结扎(2VO)慢性脑缺血大鼠模型，对效果实施例2中筛选出的活性分子进行抑制脑梗作用和对脑损伤神经保护功能的验证。
在大鼠MCAO急性脑梗模型上，选择效果实施例2中显示活性较高的TG-028及TG-055进行活性验证，所用对照药为：依达拉奉(购于浙江盛通生物科技有限公司)、硫酸氢氯吡格雷(购于武汉远成共创科技有限公司)及丹参多酚酸盐(购于河南省人民医院)，其中硫酸氢氯吡格雷是一种血小板聚集抑制剂，依达拉奉是一种脑保护剂(自由基清除剂)。运用线栓法制作大鼠MCAO模型，本效果实施例所用大鼠为SD大鼠，雄性，280-320克，购自于上海斯莱克实验动物有限公司。每组5只SD(Sprague Dawley)大鼠，腹腔注射给药，术前2h给药，造模后每隔24h给药，具体给药剂量如表7所示。
表7 所用药品的给药剂量
Figure PCTCN2019085823-appb-000093
三天后取脑，对大鼠大脑进行TTC染色(2，3，5—氯化三苯基四氮唑染色，用于检测细胞活力)后拍照，计算梗死率(％)＝(右侧脑切片非梗死面积-左侧脑切片非梗死面积)/脑切片总面积*100％，根据t tests检验，*P<0.05,**P<0.01,***P<0.001与模型组(MCAO)比较，结果如图2所示。计算不同药物剂量的梗死抑制率(％)＝(模型组梗死率-给药组梗死率)/模型组梗死率*100％，根据不同药物剂量的梗死抑制率作图，计算获得药物的ED50，结果如表8所示。
表8 不同药物在MCAO急性脑梗模型上的梗死抑制率(％)的ED50值
Figure PCTCN2019085823-appb-000094
由表8结果可知，化合物TG-028在大鼠大脑中动脉阻塞(MCAO)急性脑梗模型上抑制大鼠大脑梗死的作用最强，要优于目前市场上常用的药物。
同时，记录大鼠的体重，计算体重变化率(％)＝(最后体重-初始体重)/最后体重*100％，得到不同药物不同给药量的体重变化率，如图3所示。由大鼠在大脑损伤三天后的体重变化率可以看到，TG-028在大鼠大脑损伤后对于大鼠体重的保护作用明显，要优于TG-055、依达拉奉、硫酸氢氯吡格雷、丹参多酚酸盐。
在大鼠MCAO急性脑梗模型三天时，按照Longa神经评分标准对实验大鼠进行神经功能评分，评分标准的示意图如图4所示。其中0分表示正常，无神经损伤；1分表示不能完全伸展对侧前爪；2分表示向对侧转圈；3分表示向对侧倾倒；4分表示不能自发行走，意思丧失。
按照Longa神经评分标准对实验大鼠进行神经功能评分，获得不同药物不同剂量的Longa神经评分，结果如图5所示。Longa神经评分标准结果显示：TG-028、TG-055在 大鼠大脑损伤后对于神经的保护作用与依达拉奉、硫酸氢氯吡格雷、丹参多酚酸盐相当。
从上述大鼠MCAO急性脑梗模型上药物的梗死抑制率(％)，体重的变化率及Longa神经评分的结果发现TG-028要明显优于TG-055，这与前面的在sponge植入动物模型上的实验结果相一致，说明本发明筛选模型的可靠性。
在上述实验的基础上，同时也测试了TG-057的活性，如图20所示，在与TG-028相同给药剂量(5mg/kg)的情况下，TG-057也具有抑制大鼠大脑梗死的作用，Longa神经评分结果显示TG-057在大鼠大脑损伤后对于神经具有保护作用，但是其作用都不如TG-028强。
大鼠MCAO急性脑梗造模3天后，取出完整脑组织，去除嗅球及额极前部4mm的脑组织，取之后相邻2mm层厚的脑组织，进行脑含水量测定，结果如图6A所示。脑组织含水量的计算公式为：(WW-DW)/WW×100％，其中WW为湿重(Wet weight)，DW为干重(Dry weight)。大鼠MCAO急性脑梗造模3天后，脑组织用伊文思蓝(Evans Blue，EB，一种常用的偶氮染料制剂)评价血脑屏障(BBB)通透性，对其进行血脑屏障完整性的评估，结果如图6B所示。这两个实验均用之前测定的每个药物中最佳剂量的老鼠来进行检测：TG-028(5mg/Kg)，丹参多酚酸盐(20mg/Kg)，硫酸氢氯吡格雷(40mg/Kg)，依达拉奉(40mg/Kg)。
图6A显示(Sham为假手术组)，大鼠MCAO急性脑损伤后TG-028对于脑水肿的减轻作用要优于依达拉奉、硫酸氢氯吡格雷、丹参多酚酸盐。图6B显示，大鼠MCAO急性脑损伤后TG-028对于血脑屏障的保护作用要优于依达拉奉、丹参多酚酸盐。
在本发明后期，建立了双侧颈总动脉永久结扎(2VO)慢性脑缺血大鼠模型，通过腹腔注射，每天给药TG-028(5mg/kg)，共给药3周，每组5只SD大鼠，观察TG-028及对照药依达拉奉(40mg/kg)在慢性脑缺血大鼠模型上的作用。
在3周内大鼠的体重变化如图7(Sham为假手术组)所示，大鼠脑缺血慢性损伤3天时，给药组TG-028及依达拉奉组与假手术组相比均有体重减轻，但是在1周以后，给药组TG-028与假手术组相比体重变化没有差别，说明TG-028对体重有保护作用，而依达拉奉组与假手术组相比体重增加不明显，对体重的保护作用低。
在2VO慢性脑缺血大鼠模型建立3周后，对实验大鼠进行Morris水迷宫实验来判断药物对于动物学习记忆的影响。首先需要对实验大鼠进行5天周围定位和记忆的训练，训练至第五天后，进行定位航行实验，依次记录各组大鼠从不同入水点面向池壁放入水中的找到平台时间，即逃避潜伏期。第六天撤去平台，进行空间探索实验，从目标象限的对侧象限入水记录120s内，大鼠在目标象限的游泳时间，目标象限的停留时间，目标象限的路程，经过平台的次数等。水迷宫定位航行实验结果如图8(Sham为假手术组) 所示。大鼠在2VO慢性脑缺血给药3周之后进行Morris水迷宫定位航行实验的结果显示，大鼠逃避潜伏期所用时间，与2VO+NaCl组比较，2VO+TG-028组与2VO+依达拉奉组的逃避潜伏期时间与总路程在训练后实验第三天开始出现差异性，到第五天时差异性最大。TG-028组与依达拉奉组相比作用相当，说明大鼠的记忆能力得到了改善，认知能力得到提升。
双侧颈总动脉永久结扎三周后，进行5天定位训练，训练结束后进行Morris水迷宫空间探索实验，结果如图9(Sham为假手术组)所示。与2VO+NaCl组比较，2VO+TG-028组经过平台次数显著性增加，而2VO+依达拉奉组的并没有显著性差异，说明在给药之后，2VO+TG-028组对空间的探索能力增强，认知能力得到改善。Data were Mean±SEM(n＝7)。**P﹤0.01，*P﹤0.05，ns：No Significant Different。
3周慢性脑缺血后的脑切片进行HE染色(苏木精-伊红染色)及Nissl染色(尼氏染色)结果如图10(Sham为假手术组)所示。结果显示与2VO+NaCl组比较，2VO+TG-028组的大鼠海马CA3区神经元细胞排列整齐密集，颗粒大小均匀且清晰，同时2VO+依达拉奉组细胞形态也得到改善。说明了TG-028和依达拉奉均具有保护与修复损伤的神经元促进神经功能的改善。
除此之外，还进行了TG-028的急性毒性实验，测得其LD50为616.59mg/kg，治疗指数LD50/ED50＝616.59/2.61＝236，该数值说明TG-028的治疗窗口大，安全性好。
最后，我们还直接将Recombinant Human VEGF165蛋白(PEPROTECH，美国)作用于大鼠大脑中动脉阻塞(MCAO)模型上，并观察3天后其对于脑梗死的抑制作用。大鼠MCAO模型的制备方法和相关的实验所用动物和实验步骤均与前述相同。具体给药剂量和TTC染色结果如图11中所示。相关梗死率和梗死抑制率的结果如图12和13所示。结果显示，VEGF具有抑制大鼠大脑梗死的作用，直接证明了VEGF是脑缺血后促血管生成的重要因子。
效果实施例4 TG-028作用于巨噬细胞后可以促进内皮细胞的增殖、迁移和小管形成，从而影响新生血管的形成
此外，进一步验证了：在体外细胞实验上，发现TG-028通过作用于巨噬细胞，可以促进内皮细胞的增殖、迁移、小管形成，从而影响新生血管的形成。
该实验分为七组，第一组为单纯DMEM培养液(Gibcol，上海)加10％FBS(SERANA，德国)直接作用于HUVEC-12内皮细胞(购于上海荣创生物，control)；第二组巨噬细胞用DMEM培养液加10％FBS培养24小时后的上清作用于内皮细胞(s-control)；第三组为TG-028直接作用于DMEM培养液加10％FBS培养的内皮细胞组(TG-028，10μΜ)； 第四组为TG-028作用于巨噬细胞(DMEM培养液加10％FBS)并共培养24小时后的上清作用于内皮细胞(s-TG-028，10μM)；第五组为Cpd-18(丹参多酚酸盐)直接作用于DMEM培养液加10％FBS培养的内皮细胞(cpd-18，10μM)；第六组为Cpd-18(丹参多酚酸盐，10μM)作用于巨噬细胞(DMEM培养液加10％FBS)培养24小时后的上清作用于内皮细胞(s-cpd18)；第七组为VEGF蛋白直接作用于DMEM培养液加10％FBS培养的内皮细胞(Recombinant Human VEGF165，10ng/mL)。
(1)TG-028作用巨噬细胞24h后的上清对内皮细胞增殖的影响
取对数生长期的人脐静脉内皮细胞HUVEC-12细胞进行细胞计数，调整细胞密度为2.5×10 4个/mL，以每孔100μL接种于96孔培养板。放入培养箱内过夜培养后完全贴壁。根据以上不同分组，处理HUVEC-12内皮细胞，将96孔板置于37℃，5％CO2培养箱中培养24小时。取出96孔板加入5mg/mL MTT，20μL/孔，继续培养4h。4h后弃上清加DMSO，150μL/孔，摇床上摇10min，使紫色结晶物充分溶解，DMSO溶后10min置酶联免疫检测仪上测定波长570nm下的OD值。(酶标仪，Thermo Fisher公司)。根据所测得的吸光度OD值，以单纯DMEM培养液直接作用于HUVEC-12内皮细胞(control)为空白对照(即第一组)，计算各组细胞增加的百分比，并进行对比研究。结果发现：除了Cpd-18(丹参多酚酸盐，10μM)直接作用于内皮细胞(即第五组)对内皮细胞的增殖没有显著性的差异以外，其他各组均显著性地促进了内皮细胞的增殖，而且TG-028作用于巨噬细胞培养24小时后的上清作用于内皮细胞(s-TG-028，10μM)组(即第四组)与Cpd-18(丹参多酚酸盐，10μM)作用于巨噬细胞培养24小时后的上清作用于内皮细胞组(即第六组)，这两组均比VEGF直接作用于内皮细胞组(即第七组)促进内皮细胞的增殖作用强，具有显著性差异(##P<0.01)，而巨噬细胞培养24小时后的上清作用于内皮细胞(s-control)组(即第二组)与VEGF直接作用于内皮细胞组(即第七组)相比对内皮细胞的增殖没有显著性差异(ns)，由此说明，TG-028与Cpd-18(丹参多酚酸盐)可以通过作用于巨噬细胞从而促进内皮细胞的增殖，并且不同于VEGF直接对内皮细胞的增殖作用。如图14所示。
(2)TG-028作用巨噬细胞24h后的上清对内皮细胞迁移的影响
取对数生长期的人脐静脉内皮细胞HUVEC-12细胞进行细胞计数，调整细胞密度为5×10 5个/mL，以每孔1mL接种至6孔板。放入培养箱内常规培养形成90％融合的单层细胞，取出铺满六孔板的细胞，用200L枪头垂直于细胞表面由孔一端划向另一端，尽可能垂直于板底的横线划痕，此刻可以清楚地在培养皿表面观察到细胞表面呈井字形划痕。
吸去旧的培养基，用PBS洗掉划下的细胞。同样根据上面的七个分组，处理上述培 养好的HUVEC-12细胞，每组两个副孔，放入37℃，5％CO 2饱和温度培养箱培养。按0h、24h取样，拍照，观察不同处理组的划痕宽度。根据细胞划痕法检测内皮细胞的迁移情况并计算相对迁移率。相对迁移率＝(0h划痕宽度-24h划痕宽度)/0h划痕宽度。划痕实验结果如图15和16所示。
在24小时内与单纯DMEM培养液直接作用于HUVEC-12内皮细胞(control)组(即第一组)相比，TG-028组(即第三组)、VEGF组(即第七组)、s-TG-028(即第四组)及s-cpd18(即第六组)对于内皮细胞的迁移具有显著性的差异(*P<0.05，***P<0.001)，s-TG-028及s-cpd18对内皮细胞的迁移作用与VEGF相当。与巨噬细胞培养24小时后的上清作用于内皮细胞(s-control)组(即第二组)相比，VEGF组、s-TG-028及s-cpd18对内皮细胞的迁移作用均优于s-control组，说明化合物TG-028及丹参多酚酸盐可以通过作用于巨噬细胞从而促进内皮细胞的迁移。
(3)TG-028作用巨噬细胞24h后的上清对内皮细胞小管形成的影响
将96孔板和无菌黄枪头放入-20℃冰箱冷藏过夜。将分装好的Matrigel基质胶于实验前放入4℃冰箱融化过夜，第二天待Matrigel基质胶冻融后，离心数分钟。把预冷的Matrigel基质胶在超净台中在冰上加入96孔板，每孔加60μL，然后放到培养箱中放置1h。待Matrigel基质胶凝固后，取对数生长期的HUVEC-12细胞，调整细胞密度，以5×10 4个/孔密度与各组培养液混匀接种到96孔板，置于37℃，5％CO 2培养箱中3h之后，取出细胞培养板，开始连续在倒置显微镜下观察并拍照(放大倍数50×，德国Leica DMi1倒置显微镜)，每孔随机选取5个视野拍照观察细胞间连接形成的管样结构并采用ImageJ图像分析软件分析管样结构，用number of master junction量化指标评价成管能力。结果如图17和图18所示。
在成管实验中，与单纯DMEM培养液直接作用于HUVEC-12内皮细胞(control)组相比，TG-028组、VEGF组、s-TG-028及s-cpd18对于内皮细胞的成管具有显著性的差异(*P<0.05,***P<0.001)，s-TG-028及s-cpd18对内皮细胞的成管作用与VEGF相当。单独巨噬细胞培养24小时后的上清作用于内皮细胞(s-control)组对于内皮细胞的成管没有作用，但是加入药物TG-028及丹参多酚酸盐的s-TG-028及s-cpd18组却大大提高了内皮细胞的成管活性，说明化合物TG-028及丹参多酚酸盐可以通过巨噬细胞活化促进内皮细胞的成管作用。
因此，通过本发明的活性筛选方法筛选出的化合物TG-028，其通过作用于巨噬细胞可以促进内皮细胞的增殖、迁移、小管形成，从而影响新生血管的形成。
以上各效果实施例的结果可以看出，本发明的体外巨噬细胞实验从大批量待测化合物中筛选出了初步的目标促血管生成活性物质，其在后续的半体内动物筛选模型上都表 现出很好的促血管生成活性物质，其中化合物TG-028的活性甚至优于丹参多酚酸盐。即，本发明从分子水平就能筛选出目标促血管生成活性物质，并且进一步后续的体内动物模型实验都验证了本发明活性筛选方法的准确性和稳定性。此外，还验证了化合物TG-028主要是通过作用于巨噬细胞后，可以促进内皮细胞的增殖、迁移、小管形成，从而影响新生血管的形成，从而从机理上又反证了选用体外巨噬细胞、海绵植入动物模型检测VEGF-A mRNA作为促血管生成活性物质的筛选模型的可靠性。按照本发明的活性筛选方法筛选出来的化合物是预期化合物，并能达到促进血管新生进而抑制脑梗死、对神经保护功能、减轻脑水肿、保护损伤后血脑屏障的完整性、脑损伤后对体重的保护作用以及改善大鼠的记忆功能、保护与修复损伤的神经元促进神经功能的改善的效果。

Claims (25)

  1. 一种如式III所示的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物：
    Figure PCTCN2019085823-appb-100001
    R 1、R 2、R 3、R 4和R 5各自独立地选自氢、羟基、巯基、取代或未取代的C 1～C 20烷氧基、硝基或卤素；
    或者R 1、R 2、R 3、R 4和R 5中任意相邻两个与其苯环上相连的碳原子形成5-7元杂环，所述杂环的杂原子为O或S；所述杂原子的数目为1个或多个；当杂原子数目多个时，所述杂原子相同或不同；
    所述取代或未取代的C 1～C 20烷氧基中的取代基选自C 3-C 20环烷基、C 1-C 20烯烃基、取代或未取代的C 6-C 20芳基、卤素、取代或未取代的C 2-C 20杂芳基、C 3-C 6的环烷氧基、C 1～C 20的烷氧基或C 1～C 20烷基；
    所述取代或未取代的C 6-C 20的芳基和取代或未取代的C 2-C 20杂芳基中的取代基各自独立地为卤素或卤素取代的C 1～C 20烷基；
    X为CH 2、NR 7、O或S；
    Y为CH 2、NR 8、O或S；
    Z为O或S；
    R 7和R 8各自独立地为氢、芳基取代的C 1-C 6烷氧羰基，或C 1-C 6烷氧羰基；
    每个R 6各自独立地为氢或糖基；
    n为2，3或4；
    其条件是：
    当X为CH 2，Y为CH 2，n＝2，3或4时，R 1-R 5不同时是H；
    当n＝2，R 1、R 4和R 5是H时，R 2和R 3不同时是OCH 3和OH；
    当n＝2，R 1、R 2和R 5是H时，R 3和R 4不同时是OH和OCH 3；
    当n＝2，R 2、R 4和R 5是H时，R 1和R 3不同时是OH；
    当n＝2，R 1、R 2和R 4是H时，R 3和R 5不同时是OH。
  2. 一种如式I所示的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物：
    Figure PCTCN2019085823-appb-100002
    R 1、R 2、R 3、R 4和R 5各自独立地选自氢、羟基、巯基、取代或未取代的C 1～C 20烷氧基、硝基或卤素；
    或者R 1、R 2、R 3、R 4和R 5中任意相邻两个与其苯环上相连的碳原子形成5-7元杂环，所述杂环的杂原子为O或S；所述杂原子的数目为1个或多个；当杂原子数目多个时，所述杂原子相同或不同；
    所述取代或未取代的C 1～C 20烷氧基中的取代基选自C 3-C 20环烷基、C 1-C 20烯烃基、取代或未取代的C 6-C 20芳基、卤素、取代或未取代的C 2-C 20杂芳基、C 3-C 6的环烷氧基、C 1～C 20的烷氧基或C 1～C 20烷基；
    所述取代或未取代的C 6-C 20的芳基和取代或未取代的C 2-C 20杂芳基中的取代基各自独立地为卤素或卤素取代的C 1～C 20烷基；
    X为CH 2、NR 7、O或S；
    Y为CH 2、NR 8、O或S；
    Z为O或S；
    R 7和R 8各自独立地为氢、芳基取代的C 1-C 6烷氧羰基，或C 1-C 6烷氧羰基；
    R 6为氢或
    Figure PCTCN2019085823-appb-100003
    n为2，3或4；
    其条件是：
    当X为CH 2，Y为CH 2，n＝2，3或4时，R 1-R 5不同时是H；
    当n＝2，R 1、R 4和R 5是H时，R 2和R 3不同时是OCH 3和OH；
    当n＝2，R 1、R 2和R 5是H时，R 3和R 4不同时是OH和OCH 3。
  3. 如权利要求1或2所述的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，其特征在于，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基时，所述取代或未取代的C 1～C 20烷氧基为取代或未取代的C 1～C 10烷氧基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为卤素时，所述卤素为氟、氯、溴 或碘；
    和/或，当所述R 1、R 2、R 3、R 4和R 5中任意相邻两个与其苯环上相连的碳原子形成5-7元杂环时，所述5-7元杂环的杂原子为O；
    和/或，当所述R 1、R 2、R 3、R 4和R 5中任意相邻两个与其苯环上相连的碳原子形成5-7元杂环时，所述5-7元杂环的杂原子个数为2个；
    和/或，R 1、R 2、R 3、R 4和R 5中任意相邻两个与其相连的苯环上相连的碳原子形成5-7元杂环时，所述R 2和R 3形成5-7元杂环；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 3-C 20环烷基时，所述C 3-C 20环烷基为C 3-C 10环烷基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 1-C 20烯烃基时，所述C 1-C 20烯烃基为C 1-C 6烯烃基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 6-C 20芳基时，所述取代或未取代的C 6-C 20芳基为取代或未取代的C 6-C 10芳基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为卤素时，所述卤素选自氟、氯、溴和碘中的一种或多种；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 2-C 20杂芳基时，所述取代或未取代的C 2-C 20杂芳基为取代或未取代的C 2-C 10杂芳基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 1～C 20的烷氧基时，所述C 1～C 20的烷氧基为C 1～C 10的烷氧基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 1～C 20烷基时，所述C 1～C 20烷基为C 1～C 10烷基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 6-C 20芳基，所述取代或未取代的C 6-C 20芳基的取代基为卤素或卤素取代的C 1～C 20烷基时，所述卤素为氟、氯、溴或碘；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代C 2-C 20杂芳基，所述取代或未取代的C 2-C 20杂芳基的取代基为卤素或卤素取代的C 1～C 20烷基时，所述卤素为氟、氯、溴或碘；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 6-C 20芳基，所述取代或未取代的C 6-C 20芳基的取代基为卤素取代的C 1～C 20烷基时，所述卤素取代的C 1～C 20烷基为卤素取代的C 1～C 10烷基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 2-C 20杂芳基，所述取代或未取代的C 2-C 20杂芳基的取代基为卤素取代的C 1～C 20烷基时，所述卤素取代的C 1～C 20烷基为卤素取代的C 1～C 10烷基；
    和/或，所述R 7和R 8各自独立地为芳基取代的C 1-C 6烷氧羰基时，所述芳基取代的C 1-C 6烷氧羰基为苄氧羰基；
    和/或，所述R 7和R 8各自独立地为C 1-C 6烷氧羰基时，所述C 1-C 6烷氧羰基为叔丁氧羰基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地选自氢、羟基、巯基、取代或未取代的C 1～C 20烷氧基、硝基或卤素时，所述取代或未取代的C 1～C 20烷氧基的个数为1个；
    和/或，所述式I所示的化合物中的
    Figure PCTCN2019085823-appb-100004
    的光学异构体为：
    Figure PCTCN2019085823-appb-100005
    和/或，所述R 1、R 2、R 3、R 4和R 5各自独立地为氢、取代或未取代的C 1～C 20烷氧基、硝基或卤素。
  4. 如权利要求1或2所述的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，其特征在于，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基时，所述取代或未取代的C 1～C 20烷氧基为取代或未取代的C 1～C 6烷氧基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为卤素时，所述卤素为氟、氯或溴；
    和/或，当所述R 1、R 2、R 3、R 4和R 5中任意相邻两个与其苯环上相连的碳原子形成 5-7元杂环时，所述5-7元杂环为
    Figure PCTCN2019085823-appb-100006
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 3-C 20环烷基时，所述C 3-C 20环烷基为C 3-C 6环烷基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 1-C 20烯烃基时，所述C 1-C 20烯烃基为C 1-C 4烯烃基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 6-C 20芳基时，所述取代或未取代的C 6-C 20芳基为取代或未取代的芳基或萘基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为卤素时，所述卤素选自氟、氯或溴中的一种或多种；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 2-C 20杂芳基时，所述取代或未取代的C 2-C 20杂芳基为取代或未取代的C 2-C 6杂芳基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 1～C 20的烷氧基时，所述C 1～C 20的烷氧基为C 1～C 6的烷氧基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 1～C 20烷基时，所述C 1～C 20烷基为C 1～C 6烷基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 6-C 20芳基，所述取代或未取代的C 6-C 20芳基的取代基为卤素或卤素取代的C 1～C 20烷基时，所述卤素为氟、氯或溴；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代C 2-C 20杂芳基，所述取代或未取代的C 2-C 20杂芳基的取代基为卤素或卤素取代的C 1～C 20烷基时，所述卤素为氟、氯、或溴；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基， 所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 6-C 20芳基，所述取代或未取代的C 6-C 20芳基的取代基为卤素取代的C 1～C 20烷基时，所述卤素取代的C 1～C 20烷基为卤素取代的C 1～C 6烷基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 2-C 20杂芳基，所述取代或未取代的C 2-C 20杂芳基的取代基为卤素取代的C 1～C 20烷基时，所述卤素取代的C 1～C 20烷基为卤素取代的C 1～C 6烷基。
  5. 如权利要求1或2所述的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，其特征在于，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基时，所述取代或未取代的C 1～C 20烷氧基为
    Figure PCTCN2019085823-appb-100007
    Figure PCTCN2019085823-appb-100008
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 3-C 20环烷基时，所述C 3-C 20环烷基为环丙基、环丁基、环戊基或环己基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 2-C 20烯烃基时，所述C 2-C 20烯烃基为
    Figure PCTCN2019085823-appb-100009
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 6-C 20芳基时，所述取代 或未取代的C 6-C 20芳基为
    Figure PCTCN2019085823-appb-100010
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为取代或未取代的C 2-C 20杂芳基时，所述取代或未取代的C 2-C 20杂芳基为取代或未取代的吡咯，取代或未取代的呋喃，取代或未取代的噻吩，取代或未取代的吡唑，取代或未取代的咪唑，取代或未取代的噁唑，取代或未取代的噻唑，取代或未取代的异噁唑，取代或未取代的吡啶，取代或未取代的哒嗪，取代或未取代的嘧啶或取代或未取代的吡嗪；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 1～C 20的烷氧基时，所述C 1～C 20的烷氧基为甲氧基、乙氧基或丙氧基；
    和/或，当所述R 1、R 2、R 3、R 4和R 5各自独立地为取代或未取代的C 1～C 20烷氧基，所述取代或未取代的C 1～C 20烷氧基的取代基为C 1～C 20烷基时，所述C 1～C 20烷基为C 1～C 3烷基；
    和/或，所述
    Figure PCTCN2019085823-appb-100011
    为以下任一结构：
    Figure PCTCN2019085823-appb-100012
    Figure PCTCN2019085823-appb-100013
    Figure PCTCN2019085823-appb-100014
  6. 如权利要求1或2所述的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，其特征在于，所述R 1为氢；
    和/或，所述R 2为氢，取代或未取代的C 1～C 20烷氧基，或所述R 2和R 3形成
    Figure PCTCN2019085823-appb-100015
    和/或，所述R 3为氢、羟基、巯基、取代或未取代的C 1～C 20烷氧基、羟基、硝基或卤素；
    和/或，所述R 4为氢，或取代或未取代的C 1～C 20烷氧基；
    和/或，所述R 5为氢；
    和/或，所述X为CH 2，所述Y为CH 2、NR 8、S或O；所述R 8为氢、芳基取代的烷氧羰基或烷氧羰基；
    和/或，所述X为NR 7，所述Y为CH 2；所述R 7为氢、芳基取代的烷氧羰基或烷氧羰基；
    和/或，所述X为O或S，所述Y为CH 2；
    和/或，所述R 6为氢；
    和/或，所述式I所示的化合物中
    Figure PCTCN2019085823-appb-100016
    的光学异构体为：
    Figure PCTCN2019085823-appb-100017
  7. 一种糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，所述糖苷类化合物选自如下任一结构的化合物：
    Figure PCTCN2019085823-appb-100018
    Figure PCTCN2019085823-appb-100019
    Figure PCTCN2019085823-appb-100020
  8. 一种糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，所述糖苷类化合物选自如下任一结构的化合物：
    Figure PCTCN2019085823-appb-100021
    Figure PCTCN2019085823-appb-100022
  9. 如权利要求1-8任一项所述的糖苷类化合物的制备方法，其特征在于，包含以下步骤：溶剂中，将式V所示化合物进行还原反应，得到式VI所示化合物；
    Figure PCTCN2019085823-appb-100023
    R 9’，R 10’和R 11’各自独立地为氢或Ac，且其中至少一个为Ac；R 6’为氢、Ac或者
    Figure PCTCN2019085823-appb-100024
    R 12’,R 13’,R 14’,和R 15’各自独立地为氢、Bz或Ac；n,X，Y，Z，R 1，R 2，R 3，R 4，R 5和R 6的定义如权利要求1-5中任一项中所述；当R 6’为氢或Ac时，R 6为氢；当R 6’为
    Figure PCTCN2019085823-appb-100025
    时，R 6为
    Figure PCTCN2019085823-appb-100026
  10. 如权利要求1-8任一项所述的糖苷类化合物的制备方法，其特征在于，包含以下步骤：溶剂中，将式II所示化合物进行还原反应，得到式I所示化合物，即可；
    Figure PCTCN2019085823-appb-100027
    R 9’，R 10’和R 11’各自独立地为氢或Ac，且其中至少一个为Ac；R 6’为氢、Ac或者
    Figure PCTCN2019085823-appb-100028
    R 12’,R 13’,R 14’,和R 15’各自独立地为氢、Bz或Ac；n,X，Y，Z，R 1，R 2，R 3，R 4，R 5和R 6的定义如权利要求1-5中任一项中所述；当R 6’为氢或Ac时，R 6为氢；当R 6’为
    Figure PCTCN2019085823-appb-100029
    时，R 6为
    Figure PCTCN2019085823-appb-100030
  11. 如权利要求1-8任一项中所述的糖苷类化合物，或如下所示化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、药学上可接受的前体药物或衍生物，在制备促血管生成药物中的应用；
    Figure PCTCN2019085823-appb-100031
    Figure PCTCN2019085823-appb-100032
  12. 如权利要求1-8任一项中所述的糖苷类化合物，下式所示化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，在制备促血管生成药物中的应用；
    Figure PCTCN2019085823-appb-100033
  13. 如权利要求1-8任一项所述的糖苷类化合物，下式所示化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，在制备治疗缺血性心脑血管疾病或下肢缺血性等
    Figure PCTCN2019085823-appb-100034
    循环障碍疾病的药物中的应用；
    Figure PCTCN2019085823-appb-100035
  14. 如权利要求1-8任一项中所述的式I所示的糖苷类化合物，下式所示化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，在制备治疗缺血性心脑血管疾病或下肢缺血性等
    Figure PCTCN2019085823-appb-100036
    循环障碍疾病的药物中的应用；
    Figure PCTCN2019085823-appb-100037
    Figure PCTCN2019085823-appb-100038
  15. 如权利要求11-14任一项所述的应用，其中缺血性脑血管疾病是指脑卒中、动脉粥样硬化性血栓形成、心源性脑血栓、急性缺血性脑血管综合征、小血管病变(也称为腔隙性脑卒中)、多发性脑梗死、大面积脑梗死、分水岭脑梗死、出血性脑梗死、无症状的脑梗死、脑静脉及静脉窦血栓形成、颅底异常血管网病、其他原因所致的缺血性脑卒中使用。
  16. 如权利要求11-15任一项所述的应用，其中所述预防或治疗包括施用作为唯一活性成分的所述糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物。
  17. 如权利要求11-15任一项所述的应用，其中所述预防或治疗包括在与选自由以下各项组成的组中的另外的治疗剂的联合疗法中施用：另一种本发明所述糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物、降血压药、降低血脂的药物、溶解血栓的药物、抗血小板聚集的药物、抗凝药物、神经保护药物、钙离子拮抗剂、谷氨酸拮抗剂、谷氨酸释放抑制剂、GABA受体激动剂、自由基清除剂、细胞膜稳定剂。
  18. 如权利要求17所述的应用，其中所述另外的治疗剂选自由以下各项组成的组：瑞替普酶、兰替普酶、孟替普酶、豆豉纤溶酶、新型蚯蚓纤溶酶、纳豆激酶、蛇毒纤溶酶原激活剂、阿司匹林、噻氯匹定、氯吡格雷、普拉格雷、替卡格雷、坎格雷洛、盐酸沙格雷酯、vorapaxar、atopaxar、肝素、低分子肝素、华法林、利伐沙班、比法卢定、达比加群酯、依达拉奉、他汀类药物。
  19. 如权利要求16或17所述的应用，其中所述联合疗法包括向受试者同时、依次或分开地施用所述糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物和所述另外的治疗剂。
  20. 如权利要求11-19任一项所述的应用，其中所述药物组合物是片剂、颗粒剂、注 射剂、凝胶剂、丸剂、胶囊剂、栓剂、植入剂、纳米制剂。
  21. 如下式所示的中间体化合物：
    Figure PCTCN2019085823-appb-100039
    Figure PCTCN2019085823-appb-100040
  22. 如下式所示的中间体化合物：
    Figure PCTCN2019085823-appb-100041
    Figure PCTCN2019085823-appb-100042
    Figure PCTCN2019085823-appb-100043
    Figure PCTCN2019085823-appb-100044
  23. 一种药物组合物，包括权利要求1-8任一项所述的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物 或衍生物，任选地包含药学上可接受的载体和/或其他活性剂。
  24. 一种促进血管生成的方法，其特征在于给与患者权利要求1-8任一项所述的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，或者给与患者权利要求23的药物组合物。
  25. 一种治疗心脑血管疾病或下肢缺血性
    Figure PCTCN2019085823-appb-100045
    循环障碍疾病的方法，其特征在于给与患者权利要求1-8任一项所述的糖苷类化合物，其互变异构体、光学异构体、溶剂化物、多晶型物、药学上可接受的盐、酯、药学上可接受的前体药物或衍生物，或者给与患者权利要求23的药物组合物。
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