WO2017152725A1 - 一种视黄酸类化合物、其制备方法、中间体及应用 - Google Patents

一种视黄酸类化合物、其制备方法、中间体及应用 Download PDF

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WO2017152725A1
WO2017152725A1 PCT/CN2017/071922 CN2017071922W WO2017152725A1 WO 2017152725 A1 WO2017152725 A1 WO 2017152725A1 CN 2017071922 W CN2017071922 W CN 2017071922W WO 2017152725 A1 WO2017152725 A1 WO 2017152725A1
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wyc
mmol
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French (fr)
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曹鑫
俞飚
汪宁
陈俊威
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Shanghai Institute of Organic Chemistry of CAS
Huazhong University of Science and Technology
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Shanghai Institute of Organic Chemistry of CAS
Huazhong University of Science and Technology
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Priority claimed from CN201610310944.8A external-priority patent/CN107176945B/zh
Application filed by Shanghai Institute of Organic Chemistry of CAS, Huazhong University of Science and Technology filed Critical Shanghai Institute of Organic Chemistry of CAS
Priority to JP2018548077A priority Critical patent/JP2019509295A/ja
Priority to EP17762411.1A priority patent/EP3428155A4/en
Priority to CA3017395A priority patent/CA3017395A1/en
Priority to US16/083,879 priority patent/US10556879B2/en
Publication of WO2017152725A1 publication Critical patent/WO2017152725A1/zh
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Priority to US16/733,089 priority patent/US20200216407A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/382—Heterocyclic compounds having sulfur as a ring hetero atom having six-membered rings, e.g. thioxanthenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
    • C07C69/84—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring of monocyclic hydroxy carboxylic acids, the hydroxy groups and the carboxyl groups of which are bound to carbon atoms of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/84—Nitriles
    • C07D213/85—Nitriles in position 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D335/00—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom
    • C07D335/04—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D335/06—Benzothiopyrans; Hydrogenated benzothiopyrans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Definitions

  • the invention relates to a retinoic acid compound, a preparation method thereof, an intermediate and an application thereof.
  • Tazarotene is a retinol drug with RAR subtype selectivity (J Am Acad Dermatol. 1997, 37, S12.), mainly used for local epithelial hyperplasia of the skin (psoriasis, psoriasis, Treatment of acne, etc.).
  • Tazarotene is present as a prodrug of ethyl ester. The drug enters the body and is metabolized by enzyme to obtain the carboxyl-type active metabolite Tazarotenic acid, which can selectively interact with RAR ⁇ and RAR ⁇ receptors, and also has a certain effect on RAR ⁇ , but has a weak effect on RXR.
  • the problem to be solved by the present invention is to provide a retinoic acid compound, a preparation method thereof, an intermediate and application thereof for the purpose of overcoming defects such as poor inhibition rate of tumor cells by tazarotene, and the compound inhibits tumor cells.
  • the rate is better.
  • the present invention provides a compound, an enantiomer, a diastereomer or a pharmaceutically acceptable salt thereof, of formula I,
  • R 9a , R 9b , R 9c and R 9d are each independently hydrogen, hydroxy, nitro, cyano, halogen (for example fluorine, chlorine, bromine or iodine), C 1- C 6 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl or hexyl), halogen-substituted C 1 -C 6 Alkyl (the halogen may be fluorine, chlorine, bromine or iodine; the C 1 -C 6 alkyl group may be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl a "halo-substituted C 1 -C 6 alkyl group" such as a trifluoromethyl group, a C 1 -C
  • the R 10 , R 11 , R 12 , R 13 and R 14 are independently hydrogen or a C 1 -C 6 alkyl group (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, Isobutyl, tert-butyl, pentyl or hexyl);
  • R 2 , R 3 , R 4 and R 5 are independently hydrogen, hydroxy, halogen (for example fluorine, chlorine, bromine or iodine), C 1 -C 6 alkyl (for example methyl, ethyl, positive) Propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl or yl), C 2 -C 6 alkenyl (eg vinyl or propenyl), halogen substituted C 1 -C 6 Alkyl (the halogen may be fluorine, chlorine, bromine or iodine; the C 1 -C 6 alkyl group may be methyl, ethyl, n-propyl, isopropyl, n-butyl, iso Butyl, tert-butyl, pentyl or hexyl; said "halogen-substituted C 1 -C 6 alkyl" such as trifluoro
  • n 0, 1, 2 or 3;
  • R 1 is hydrogen, hydroxy, nitro, cyano, halogen (e.g., fluorine, chlorine, bromine or iodine), C 1 -C 6 alkyl (e.g.
  • the R 6 , R 7 and R 8 are independently hydrogen or a C 1 -C 6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl) Base, pentyl or hexyl);
  • Y is -CN, -COOR 15 or -CO 2 NHR 16 ;
  • R 15 and R 16 are independently hydrogen, C 1 -C 6 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl) Or a hexyl group, a C 2 -C 6 alkenyl group (for example, a vinyl or propenyl group) or a C 1 -C 6 acyl group (for example, a formyl group or an acetyl group);
  • said A, E, G and Z are connectable to a ring as shown below:
  • the definitions of R 2 , R 3 and R 5 are as described above.
  • At least one of the U, V, X and W (for example, 1, 2, 3 or 4) is N.
  • the compound I when two of the U, V, X and W are N, the compound I may be any of the following compounds:
  • A, E, G, Z, R 1 , m, Y, R 9a , R 9b , R 9c and R 9d are as defined above.
  • the R 15 is hydrogen or ethyl.
  • the definitions of R 2 and R 3 are as described above.
  • the Z is -S-.
  • the R 15 is a methyl group or an ethyl group.
  • R 2 and R 3 are as described above.
  • the Z is -(CR 2 R 3 )-, and R 2 and R 3 are as defined above.
  • the Z is -(CR 2 R 3 )- or -S-, and R 2 and R 3 are as defined above.
  • the definition of 3 is as described above.
  • the R 15 is hydrogen or ethyl.
  • R 2 and R 3 are as described above.
  • the Z is -S-.
  • the compound I when one of the U, V, X and W is N, the compound I may be any of the following compounds:
  • A, E, G, Z, R 1 , m, Y, R 9a , R 9b , R 9c and R 9d are as defined above.
  • the R 15 is hydrogen or ethyl.
  • the Z is -(CR 2 R 3 )-; more preferably, the A, E, G and Z are linked to a ring as shown below:
  • R 2 and R 3 are as described above.
  • the Y is CN.
  • the Z is -(CR 2 R 3 )-; more preferably, the A, E, G and Z are linked to a ring as shown below:
  • R 2 and R 3 are as described above.
  • A, E, G, Z, R 1 , m, Y, R 9a , R 9b , R 9c and R 9d are as defined above.
  • At least one of the R 9a , R 9b , R 9c and R 9d (for example, 1, 2, 3 or 4) is not hydrogen.
  • the R 15 is hydrogen, methyl or ethyl.
  • the definitions of R 2 , R 3 and R 5 are as described above.
  • the compound I is any one of the following compounds:
  • the present invention also provides a process for the preparation of a compound of formula I, which comprises the steps of: coupling a compound II and III to obtain a compound I;
  • X 1 is halogen (e.g., fluorine, chlorine, bromine or iodine).
  • the conditions of the coupling reaction may be conventional conditions of the coupling reaction in the art, and the present invention particularly preferably has the following conditions: protection of a protective gas (for example, argon).
  • a protective gas for example, argon
  • an organic solvent such as DMF
  • a catalyst such as Pd / Cu catalyst; said "Pd / Cu catalyst” such as Pd (PPh 3 ) 2 Cl 2 and CuI
  • a base such as diisopropylamine
  • the compound I can be further subjected to flexible functional group conversion and adjustment (including but not limited to chemical operations such as esterification, ester hydrolysis, reduction, acylation, oxidation, etc.) to obtain a compound I having a different functional group.
  • flexible functional group conversion and adjustment including but not limited to chemical operations such as esterification, ester hydrolysis, reduction, acylation, oxidation, etc.
  • the method for preparing the compound I may further comprise the steps of: deprotecting the compound IV to obtain the compound III;
  • the conditions of the deprotection reaction may be conventional conditions for the deprotection reaction in the art, and the present invention particularly preferably has the following conditions: in an organic solvent (for example, tetrahydrofuran), Compound IV may be subjected to a deprotection reaction in the presence of a base (for example, tetra-n-butylammonium fluoride) to obtain a compound III.
  • an organic solvent for example, tetrahydrofuran
  • Compound IV may be subjected to a deprotection reaction in the presence of a base (for example, tetra-n-butylammonium fluoride) to obtain a compound III.
  • a base for example, tetra-n-butylammonium fluoride
  • the preparation method of the compound I may further comprise the steps of: coupling the compound V and the trimethylethynyl silane to obtain the compound IV;
  • X 2 is a halogen (e.g., bromine or iodine).
  • the conditions of the coupling reaction may be conventional conditions of the coupling reaction in the art, and the present invention particularly preferably has the following conditions: protection of a protective gas (for example, argon).
  • a protective gas for example, argon
  • an organic solvent such as DMF
  • a catalyst such as Pd / Cu catalyst; said "Pd / Cu catalyst” such as Pd (PPh 3 ) 2 Cl 2 and CuI
  • a base such as diisopropylamine
  • the invention also provides a compound as shown in formula II, III, IV or V,
  • the invention also provides the use of a compound of formula I, an enantiomer, diastereomer or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a primary tumor .
  • tumors include, but are not limited to, melanoma, esophageal cancer, gastric cancer, lung cancer, liver cancer, ovarian cancer, colon cancer, renal cancer, cholangiocarcinoma, breast cancer or prostate cancer.
  • the invention also provides the use of a compound of formula I, an enantiomer, a diastereomer or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prophylaxis and/or Treatment of metastatic tumors.
  • the tumor includes, but is not limited to, melanoma, esophageal cancer, gastric cancer, lung cancer, liver cancer, ovarian cancer, colon cancer, renal cancer, cholangiocarcinoma, breast cancer, prostate cancer.
  • the invention also provides the use of a compound of formula I, an enantiomer, a diastereomer or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prophylaxis and/or Treatment, leukemia and/or lymphoma.
  • the invention also provides the use of a compound of formula I, an enantiomer, diastereomer or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in animal fetal development, One or more of internal environment stability, vision, morphogenesis, skin aging, and control of cell differentiation.
  • the invention also provides the use of a compound of formula I, an enantiomer, diastereomer or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of psoriasis .
  • the invention also provides the use of a compound, an enantiomer, a diastereomer or a pharmaceutically acceptable salt thereof, of formula I, for the manufacture of a medicament for the treatment of acne.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I, an enantiomer, a diastereomer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be administered orally, by injection, or topically.
  • the pharmaceutical composition is administered by the oral route, and the composition may be, but is not limited to, the following forms: tablets, capsules, suspensions, solutions, emulsions, microspheres or nanosphere suspensions, or controlled release. Lipid vesicles or polymer vesicles.
  • the composition may be a solution or suspension for infusion or injection.
  • the compounds of the invention are administered at a daily dose of from about 0.01 mg/kg to 100 mg/kg body weight in divided doses of 1-3.
  • the concentration of the compounds of the invention used systemically is generally from 0.01% to 10% by weight, preferably from 0.01% to 1% by weight, based on the weight of the composition.
  • the pharmaceutical composition is particularly useful for treating skin and mucosal diseases by topical administration, and may be in the form of a liquid, a paste or a solid, especially an ointment, a cream, In the form of a solution, a gel, a spray, a suspension, or a patch. It may also be a microsphere or nanosphere suspension, or a controlled release lipid vesicle or polymer vesicle or a gelled or polymeric patch.
  • the concentration of the topically applied compound is usually from 0.001% to 10% by weight, preferably from 0.01% to 1% by weight, based on the total weight of the composition.
  • compositions may also contain inert additives, or a pharmaceutically active ingredient that is positively associated with the pharmaceutical composition, or a mixture of such ingredients.
  • inert additives or a pharmaceutically active ingredient that is positively associated with the pharmaceutical composition, or a mixture of such ingredients.
  • a "C 1 -C 6 acyl” refers to an alkyl group containing 1 to 6 carbon atoms, such as an acyl group containing one carbon atom refers to HC (O) - (i.e. formyl),
  • the acyl group having 2 carbon atoms is CH 3 C(O)- (i.e., acetyl); and may also include propionyl, butyryl or valeryl.
  • alkyl refers to a saturated straight or branched monovalent hydrocarbon radical having from one to twenty carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-butyl, 2-butyl, 2-methyl-2 -propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2 -methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl , 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-g Base, 1-octyl.
  • alkenyl refers to a straight, branched or cyclic non-aromatic hydrocarbon radical containing the specified number of carbon atoms and at least one carbon to carbon double bond. Preferably there is one carbon-carbon double bond and up to four non-aromatic carbon-carbon double bonds may be present.
  • C 2 -C 12 alkenyl means an alkenyl group having 2 to 12 carbon atoms.
  • the "C 2 -C 6 alkenyl group” means an alkenyl group having 2 to 6 carbon atoms, and includes a vinyl group, a propenyl group, a butenyl group, a 2-methylbutenyl group, and a cyclohexenyl group.
  • aryl refers to any stable monocyclic or bicyclic carbon ring which may be up to 7 atoms in each ring, at least one of which is an aromatic ring.
  • aryl unit include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. It will be understood that in the case where the aryl substituent is a bicyclic substituent and one of the rings is a non-aromatic ring, the linkage is carried out through an aromatic ring.
  • arylhetero or “heteroaryl” (including when used alone and in other groups) denotes a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is an aromatic ring and Containing from 1 to 4 heteroatoms selected from O, N, and S.
  • Heteroaryl groups within the scope of this definition include, but are not limited to, acridinyl, oxazolyl, porphyrin, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furyl, thienyl , benzothienyl, benzofuranyl, quinolyl, isoquinolinyl, oxazolyl, isoxazolyl, fluorenyl, pyrazinyl, pyridazinyl, pyridyl, pyrimidinyl, Pyrrolyl, tetrahydroquinoline.
  • Heteroaryl is also understood to include any nitrogen-containing heteroaryl N-oxide derivative.
  • heteroaryl substituent is a bicyclic substituent and one ring is a non-aromatic ring or does not contain a hetero atom
  • linkage is carried out by an aromatic ring or by a hetero atom on the ring, respectively.
  • halogen includes F, Cl, Br, I.
  • pharmaceutically acceptable salt refers to a conventional acid addition or base addition salt which retains the biological effectiveness and properties of Compound I, which is formed from a suitable non-toxic organic or inorganic acid, or an organic or inorganic base.
  • acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphorous acid, sulfuric acid, phosphoric acid, and those derived from organic acids.
  • nitric acid such as formic acid, acetic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, maleic acid, lactic acid, tartaric acid, succinic acid, Fumar Acid, mandelic acid, malic acid, camphorsulfonic acid, etc.
  • base addition salts include salts derived from ammonium, potassium, sodium, calcium, and quaternary ammonium hydroxides such as tetramethylammonium hydroxide.
  • Chemical modification of a pharmaceutical compound (i.e., a drug) to a salt is a technique well known to pharmacists to achieve improved physical and chemical stability, hygroscopicity, flowability, and solubility of the compound.
  • “Pharmaceutically acceptable” in the term “pharmaceutically acceptable carrier” means that the subject to whom the particular compound is administered is pharmaceutically acceptable and substantially non-toxic.
  • the reagents and starting materials used in the present invention are commercially available.
  • the positive progress of the present invention is that the compound of the present application has a better inhibition rate against tumor cells.
  • FIG. 1 WYC-103 induces differentiation and apoptosis of melanoma B16-F1TRC cells
  • Figure 3 Inhibition of WYC-209 against lung cancer A549 tumor regenerative cells, wherein Figure 3a shows inhibition of lung cancer A549 treated with WYC-209 on day 0; Figure 3b shows treatment of lung cancer A549 with WYC-209 on day 3 Inhibition.
  • Figure 4 Inhibition of WYC-209 against breast cancer MCF-7 tumor regenerative cells, wherein Figure 4a shows inhibition of breast cancer MCF-7 treated with WYC-209 on day 0; Figure 4b shows use on day 3 WYC-209 treatment of breast cancer MCF-7 inhibition.
  • Figure 5 Inhibition of WYC-209 against melanoma MDA-MB-435S tumor regenerative cells, wherein Figure 5a shows the inhibition of treatment of melanoma MDA-MB-435S with WYC-209 on day 0; Figure 5b shows On day 3, WYC-209 was used to treat the inhibition of melanoma MDA-MB-435S.
  • Figure 6 Inhibition of WYC-209 against ovarian cancer A2780 tumor regenerative cells, wherein Figure 6a shows the inhibition of ovarian cancer A2780 treated with WYC-209 on day 0; Figure 6b shows treatment with WYC-209 on day 3 Inhibition of ovarian cancer A2780.
  • Fig. 7 Study on the inhibitory effect of WYC-209 on tumor regenerative cells of gastric cancer Hs-746T, wherein Fig. 7a shows the inhibition of treatment of gastric cancer Hs-746T with WYC-209 on day 0; Fig. 7b shows the use of WYC- on day 3 209 treatment of gastric cancer Hs-746T inhibition.
  • Figure 8 Inhibition of WYC-209 against breast cancer MDA-MB-231 tumor regenerative cells, wherein Figure 8a shows the inhibition of treatment of breast cancer MDA-MB-231 with WYC-209 on day 0; Figure 8b shows On day 3, WYC-209 was used to treat the inhibition of breast cancer MDA-MB-231.
  • Figure 9 WYC-331 against ovarian cancer A2780 and breast cancer MDA-MB-231 tumor regenerative cell inhibitory activity, wherein Figure 9a shows the inhibition of ovarian cancer A2780 treated with WYC-331 on day 3; Figure 9b shows On day 3, WYC-331 was used to treat the inhibition of breast cancer MDA-MB-231.
  • Figure 10 WYC-209 cytotoxicity study (WYC-209 inhibits B16 growth but has no significant effect on 3T3 cells); wherein, Figure 10a shows treatment of 3T3 cells with 10 ⁇ M WYC-209 for 18 hours; Figure 10b shows no WYC-209, B16 cells were treated with 10 ⁇ M WYC-209 for 48 hours.
  • Figure 11 WYC-331 cytotoxicity study; wherein, Figure 11a shows that 3T3 cells were treated with 1 ⁇ M, 10 ⁇ M WYC-331 for 24 hours; Figure 11b shows that B16 cells were treated with 1 ⁇ M, 10 ⁇ M WYC331 for 24 hours, respectively.
  • Figure 12 Inhibition of subcutaneous melanoma by WYC-103, tumor volume on day 19 of the experiment.
  • Figure 13 Study on the inhibitory effect and in vivo toxicity of WYC-103 on subcutaneous melanoma.
  • Figure 14 Inhibition of subcutaneous melanoma by WYC-103, tumor volume on day 26 of the experiment.
  • Figure 15 Inhibition of lung metastatic melanoma by WYC-103; wherein, Figure 15a shows the injection of 3000 melanoma regenerative cells, administration of WYC103, lung tissue on day 29 of the experiment; Figure 15b shows the injection of 3000 melanoma regenerative cells DMSO was administered to the lung tissue on the 29th day of the experiment; Fig. 15c shows the injection of 3000 melanoma regenerative cells, WYC103, lung tissue on the 35th day of the experiment; Fig.
  • FIG. 15d shows the injection of 3000 melanoma regenerative cells, given DMSO, the 35th day of the experiment Lung tissue;
  • Figure 15e shows lung tissue administered to WYC103 on day 37 of the experiment;
  • Figure 15f shows lung tissue on day 37 of the experiment given DMSO.
  • Figure 16 Inhibition of lung metastatic melanoma by WYC-209, wherein Figure 16a shows lung tissue administered to DMSO; Figure 16b shows lung tissue administered with 1.0 ⁇ M WYC-209; Figure 16c shows administration of 10 ⁇ M WYC-209 Lung tissue.
  • Figure 17 Single crystal structure diagram of WYC-209A.
  • Figure 18 Single crystal structure diagram of WYC-209B.
  • WYC-202 (30 mg, 0.098 mmol) was added to the flask, 2 mL of dry dichloromethane was added, and the mixture was cooled to 0 °C in an ice water bath, then mCPBA (24 mg, 0.098 mmol) was added, and the reaction was carried out for 1 hour in an ice water bath and then moved to room temperature for 2 h. , TLC tracking. After the reaction was completed, it was quenched with sodium thiosulfate solution, diluted with ethyl acetate, washed with saturated sodium hydrogen sulfate, washed with saturated sodium chloride and dried over anhydrous sodium sulfate.
  • WYC-205 (70 mg, 0.2 mmol) was added to the flask, 3 mL of dry dichloromethane was added, and the mixture was cooled to 0 °C in an ice water bath, then mCPBA (49 mg, 0.2 mmol) was added, and the reaction was carried out for 1 hour in an ice water bath and then moved to room temperature for 2 h. , TLC tracking. After the reaction was completed, it was quenched with sodium thiosulfate solution, diluted with ethyl acetate, washed with saturated sodium hydrogen sulfate, washed with saturated sodium chloride and dried over anhydrous sodium sulfate.
  • WYC-209A 2-((4,4-Dimethyl-1S-oxodithiothiopyran-6-)ethynyl)pyrimidine-5-carboxylic acid ethyl ester
  • Diethyl D-tartrate (82.7 mg, 0.4 mmol) was dissolved in anhydrous CH 2 Cl 2 (1 mL), and Ti(iso-PrO) 4 (0.2 mmol, 58 uL) was quickly added at 16 °C and stirred for 3 minutes. Water (3.6 uL, 0.2 mmol) was added dropwise and stirring was continued for 20 minutes. It was then cooled to -20 degrees, and WYC-203 (69 mg, 0.2 mmol) and cumene hydroperoxide (74 uL, 0.4 mmol) were quickly added, and the reaction was stopped after 3 hours.
  • the reaction liquid was poured into 10 mL of a mixed solution of ferrous sulfate (0.2 g), citric acid (67 mg), dioxane and diethyl ether (2:1:2), and stirred for 15 minutes.
  • WYC-209B 2-((4,4-Dimethyl-1R-oxodithiothiopyran-6-)ethynyl)pyrimidine-5-carboxylic acid ethyl ester
  • Diethyl L-tartrate (82.7 mg, 0.4 mmol) was dissolved in anhydrous CH 2 Cl 2 (1 mL), and Ti(iso-PrO) 4 (0.2 mmol, 58 uL) was quickly added at 16 °C and stirred for 3 minutes, slowly. Water (3.6 uL, 0.2 mmol) was added dropwise and stirring was continued for 20 minutes. It was then cooled to -20 degrees, and WYC-203 (69 mg, 0.2 mmol) and cumene hydroperoxide (74 uL, 0.4 mmol) were quickly added, and the reaction was stopped after 3 hours.
  • the reaction liquid was poured into 10 mL of a mixed solution of ferrous sulfate (0.2 g), citric acid (67 mg), dioxane and diethyl ether (2:1:2), and stirred for 15 minutes.
  • the aqueous layer was extracted with EtOAc.
  • ESI (+)-MS 369.4 [M+1] + .
  • the single crystal structure diagrams of WYC-209A and WYC-209B are shown in Fig. 17 and Fig. 18, respectively.
  • 6-ethynyl-4,4-dimethylbenzothiopyran (2.03 g, 10 mmol) was added to the flask, and 27 mL of dry dichloromethane was added thereto, and the mixture was cooled to 0 ° C in ice water, and then mCPBA (1.73 g, 10 mmol) was added. After reacting for 10 min in an ice water bath, the reaction was carried out to room temperature for 1 h, and then moved to room temperature for 2 h, followed by TLC.
  • WYC-216 (80 mg, 0.22 mmol) was added to the flask, 6 mg of DMAP was added, 2 mL of dry pyridine was added under argon atmosphere, cooled to 0 °C in an ice water bath, and acetyl chloride (31 ⁇ l, 0.44 mmol) was added dropwise, ice After reacting for 10 min in a water bath, it was moved to room temperature for 9 h, and was followed by TLC. After the reaction is completed, it is quenched with methanol, diluted with ethyl acetate, diluted with EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc.
  • WYC-214 (152 mg, 0.42 mmol) was added to the flask, 10 mg of DMAP was added, 5 mL of dry dichloromethane, 0.212 mL of triethylamine was added under argon atmosphere, and the mixture was cooled to 0 degree in ice water bath, and added dropwise.
  • the acid chloride (67 ⁇ l, 0.84 mmol) was reacted in an ice water bath for 10 min, then transferred to room temperature for 12 h, followed by TLC. After the reaction is completed, it is quenched with methanol, diluted with ethyl acetate, diluted with EtOAc EtOAc EtOAc EtOAc.
  • Ethyl 4-(3-bromophenyl)-2-butenoate (1.05 g, 3.92 mmol) was added to a flask, and (10% wt) dry Pd/C (220 mg, 0.194 mmol) was added and protected under argon 20 mL of ethyl acetate was added thereto, and hydrogen gas was introduced and replaced with 3 times of gas, and reacted at normal temperature and normal pressure for 24 hours, followed by TLC.
  • 2-Fluoro-4-iodobenzoic acid (1.0 g, 3.76 mmol) was added to the flask, and 10 mL of absolute ethanol was added under argon atmosphere, and the mixture was heated to 0 ° with ice water, and 0.5 mL of concentrated sulfuric acid was added dropwise. The temperature was refluxed for 4 h, and TLC was followed. After the reaction is completed, it is cooled to room temperature, and the sulfuric acid is neutralized with 1 mol/L sodium hydroxide to neutrality, washed with saturated sodium hydrogencarbonate, washed with saturated sodium chloride, and then dried over anhydrous sodium sulfate.
  • 2,3-Difluoro-4-iodobenzoic acid (0.5 g, 1.76 mmol) was added to the flask, 4 mL of absolute ethanol was added under argon, ice water was added to 0 °, and 0.3 mL of concentrated sulfuric acid was added dropwise. After the addition was completed, the temperature was refluxed for 6 h, and TLC was followed. After the reaction is completed, it is cooled to room temperature, and the sulfuric acid is neutralized with 1 mol/L sodium hydroxide to neutrality, washed with saturated sodium hydrogencarbonate, washed with saturated sodium chloride, and then dried over anhydrous sodium sulfate.
  • the compound 3-amino-4-iodobenzoic acid methyl ester (1.5 g, 5.43 mmol) was added to the flask, 20 mL of dry dichloromethane, 4.5 mL of dry triethylamine was added, and 0.77 mL of acetyl chloride was added dropwise in an ice water bath. The reaction was allowed to proceed overnight at room temperature and was followed by TLC. After the reaction was completed, it was quenched with EtOAc EtOAc (EtOAc)EtOAc.EtOAc. :1) The product was obtained as methyl 3-acetamido-4-iodobenzoate (1.34 g , 77.5%).
  • 5-bromopyridazine-2-carboxylic acid (1.0 g, 5 mmol) was added to the flask, 5 mL of absolute ethanol was added under argon atmosphere, and the mixture was cooled to 0 ° with ice water, 0.3 mL of concentrated sulfuric acid was added dropwise, and the temperature was raised after the addition was completed.
  • the reflux reaction was carried out for 6 h, followed by TLC. After the reaction is completed, it is cooled to room temperature, and the sulfuric acid is neutralized with 1 mol/L sodium hydroxide to neutrality, washed with saturated sodium hydrogencarbonate, washed with saturated sodium chloride, and then dried over anhydrous sodium sulfate.
  • WYC-209 (1.1 g, 3 mmol) was added to the flask, 20 mL of dry dichloromethane was added, and the mixture was cooled to 0 °C in an ice water bath, then mCPBA (0.663 g, 3.6 mmol) was added, and the reaction was carried out for 10 min in an ice water bath and then transferred to room temperature for 3 h. After moving to room temperature for 4 h, TLC was followed. After the reaction was completed, it was quenched with sodium thiosulfate solution, diluted with ethyl acetate, washed with saturated sodium hydrogen sulfate, washed with saturated sodium chloride and dried over anhydrous sodium sulfate.
  • WYC-316 (107 mg, 0.272 mmol) was added to the flask, and 3.5 mL of dry dichloromethane was added thereto. After cooling to 0 °C in an ice water bath, m-CPBA (67.3 mg, 0.272 mmol) was added, and the reaction was carried out for 1 hour in an ice water bath. The reaction was carried out for 2 h at room temperature and followed by TLC. After the reaction was completed, it was quenched with sodium thiosulfate solution, diluted with ethyl acetate, washed with saturated sodium hydrogen sulfate, washed with saturated sodium chloride and dried over anhydrous sodium sulfate.
  • WYC-322 (100 mg, 0.284 mmol) was added to the flask, 3.5 mL of dry dichloromethane was added, and the mixture was cooled to 0 °C in an ice water bath, then m-CPBA (49 mg, 0.284 mmol) was added, and the mixture was reacted for 1 hour in an ice water bath and then transferred to room temperature. The next reaction was 2 h, and TLC was followed. After the reaction was completed, it was quenched with sodium thiosulfate solution, diluted with ethyl acetate, washed with saturated sodium hydrogen sulfate, washed with saturated sodium chloride and dried over anhydrous sodium sulfate.
  • 6-ethynyl-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene 50 mg, 0.235 mmol
  • ethyl 2-chloropyrimidine-4-carboxylate 50.3 mg, 0.27 mmol
  • Pd(PPh 3 ) 2 Cl 2 14 mg, 0.02 mmol
  • CuI 5.6 mg, 0.03 mmol
  • DMF 0.14 mL of dry Et 3 N, heated to 70 ° C for 8 h, TLC tracking.
  • 6-ethynyl-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene 60 mg, 0.283 mmol
  • 2-chloro-5-cyanopyridine 59.5 mg, 0.325
  • Pd(PPh3)2Cl2 16.8 mg, 0.024 mmol
  • CuI 6.8 mg, 0.036 mmol
  • protecting with argon gas and replacing the gas three times to remove oxygen adding 2 mL of dry DMF with a syringe, 0.17 mL of dry Et3N was reacted at 70 ° C for 8 h, followed by TLC.
  • Example 78 Ethyl 5-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-)ethynyl)pyrimidine-2-carboxylate
  • 6-ethynyl-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene 50 mg, 0.205 mmol
  • compound c 75.3 mg, 0.271 mmol
  • Pd(PPh 3 ) 2 Cl 2 14 mg, 0.02 mmol
  • CuI 5.6 mg, 0.03 mmol
  • protected with argon and replaced with 3 times of gas to remove oxygen and 2 mL of dry DMF, 0.14 mL of dried Et. 3 N, heated to 70 ° for 8 h, TLC tracking.
  • Example 80 2-Hydroxy-4-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-)ethynyl)benzoic acid
  • Example 84 Ethyl methyl ester 5-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-)ethynyl)pyrimidine-2-carboxylate
  • the product (25 mg, 0.072 mmol) was placed in a flask, 1.5 mL of absolute ethanol was added under argon atmosphere, and the mixture was heated to 0 ° with ice water, and 5 ⁇ L of concentrated sulfuric acid was added dropwise thereto. After the completion of the dropwise addition, the mixture was heated to reflux for 4 hours, and was subjected to TLC. After the reaction is completed, it is cooled to room temperature, and the sulfuric acid is neutralized with 1 mol/L of sodium hydroxide to neutrality, washed with a saturated sodium hydrogen carbonate solution and a saturated sodium chloride solution, and the organic phase is dried over anhydrous sodium sulfate, filtered, and dried.
  • WYC-337 (25mg, 0.075mmol) was added to the flask, 1.5mL absolute ethanol was added under argon protection, ice water bath was added to 0 degree, 5 ⁇ l concentrated sulfuric acid was added dropwise, and the reaction was heated and refluxed for 4 hours after TTG tracking. TLC tracking . After the reaction is completed, it is cooled to room temperature, and the sulfuric acid is neutralized with 1 mol/L of sodium hydroxide to neutrality, washed with a saturated sodium hydrogen carbonate solution and a saturated sodium chloride solution, and the organic phase is dried over anhydrous sodium sulfate, filtered, and dried.
  • the product (25 mg, 0.075 mmol) was placed in a flask, 1.5 mL of absolute ethanol was added under argon atmosphere, and the mixture was cooled to 0 ° with ice water, and 5 ⁇ l of concentrated sulfuric acid was added dropwise. After the addition was completed, the mixture was heated to reflux for 4 hours, and was subjected to TLC. After the reaction is completed, it is cooled to room temperature, and the sulfuric acid is neutralized with 1 mol/L of sodium hydroxide to neutrality, washed with a saturated sodium hydrogen carbonate solution and a saturated sodium chloride solution, and the organic phase is dried over anhydrous sodium sulfate, filtered, and dried.
  • TRCs melanoma-derived cells
  • Melanoma B16-F1 cells were incubated with 3D fibrin glue medium (90-Pa) for 5 days, and the desired melanoma tumor regenerative cells were screened (Nat. Mater. 2012, 11, 734). Subsequently, the culture medium was separately treated with collagenase and neutral protease II to release melanoma tumor regenerative cells, and the resulting regenerated cells were transferred to a fresh preparation medium and resuspended, and maintained in a single cell state. Melanoma B16-F1 regenerative cells were inoculated in 3D fibrin glue medium (90-Pa) for 5 days, with 0.1% DMSO as a negative control group, Tazarotene and Bexarotene.
  • the drugs were added at a concentration of 10 ⁇ M, incubated for 5 days, and the tumor clone size was observed and measured, and the inhibition rate of each drug for melanoma B16-F1 tumor regenerative cells was calculated.
  • the results of the test for inhibiting cell activity of the compound comprising the compound against melanoma B16-F1 are shown in Table 2 below.
  • Table 2 Test results of melanoma B16-F1 regenerative cell inhibitory activity
  • the inhibition rate of the regenerative cells of melanoma tumor B16-F1 showed that most of the compounds showed significant tumor growth inhibitory activity at a concentration of 10 ⁇ M, such as compound WYC-103 (86.8%), WYC-207 (92.5%), WYC-209 (98.8%), WYC-209A (98.9%), WYC-209B (98.7%), WYC-212 (93.2%), WYC-217 (90.3%), WYC-218 (90.4%), WYC- 329 (94.5%), WYC-331 (96.0%), etc., the corresponding tumor clones were basically not used for growth or growth very slowly. The results were not only in sharp contrast with the negative control group, but also met or exceeded the inhibitory activity of the positive drug Tazarotene (67.6%) and Bexarotene (63.3%) on tumor clone growth.
  • a dose-effect curve study of tumor growth inhibition was then performed on compounds exhibiting significant tumor clonal growth inhibitory activity in the primary screening.
  • Tumor clones administered by equivalence gradient were fixed, DAPI nuclear staining and volume measurement, inhibition rate calculation and dose-response curve regression were performed to determine the compounds with better activity WYC-103, WYC-209, WYC-320, WYC-
  • the IC 50 of 329 and WYC-331 is shown in Table 3 below.
  • the compounds WYC-103 and WYC-209 for the inhibition and differentiation of melanoma tumor B16-F1 regenerative cells are shown in the electron micrograph (Fig. 1, Fig. 2).
  • Fig. 1 and Fig. 2 cell survival was observed by DAPI staining.
  • the above human tumor cells were separately incubated with 3D fibrin glue medium (90-Pa) for 5 days, and the desired tumor regenerative cells were selected. Subsequently, the medium was separately treated with collagenase and neutral protease II to release the tumor-regenerating cells, and then the resulting regenerated cells were transferred to a fresh preparation medium and resuspended, and maintained in a single cell state.
  • Single tumor regenerative cells were inoculated in 3D fibrin glue medium (90-Pa) for 5 days, and 0.1% DMSO was used as a negative control group (DMSO group in Fig. 3 to Fig. 8), with no drug added as a blank group ( In Fig. 3 to Fig. 8, the None group) was studied by administration from 0 days and administration from 3 days, respectively, and the tumor clone volume was calculated and measured.
  • the compound WYC-209 can effectively inhibit the proliferation of various tumor regenerative cells at 1.0 ⁇ M, which greatly blocked the volume growth of tumor clones.
  • the volume of tumor clones was only blank group. 25-30% (Fig. 3-8, Fig. 3-8 refers to drug9# for WYC-209).
  • the concentration of WYC-209 was increased to 10 ⁇ M, the proliferation of the above six tumor regenerative cells was inhibited at a higher level, and the volume of the tumor clone was even less than 10% of the volume of the blank group.
  • the use of compound WYC-209 can even reverse the growth trend of tumor clones.
  • Compound WYC-331 showed good inhibitory activity against melanoma B16-F1 tumor regenerative cell proliferation, and then we investigated the inhibitory activity of this compound against ovarian cancer A2780 and breast cancer MDA-MB-231 tumor regenerative cells.
  • the above human tumor cells were separately incubated with 3D fibrin glue medium (90-Pa) for 5 days, and the desired tumor regenerative cells were selected. Subsequently, the medium was separately treated with collagenase and neutral protease II to release the tumor-regenerating cells, and then the resulting regenerated cells were transferred to a fresh preparation medium and resuspended, and maintained in a single cell state.
  • Single tumor regenerative cells were inoculated in 3D fibrin glue medium (90-Pa) for 5 days, 0.1% DMSO as a negative control group (DMSO group in Fig. 9), and no drug addition as a blank group (Fig. 9) In the middle group, the drug was administered at a concentration of 0.1 ⁇ M, 1.0 ⁇ M, and 10 ⁇ M from the third day, and the tumor clone volume was observed and measured.
  • the compound WYC-331 could completely inhibit the proliferation of ovarian cancer A2780 tumor regenerative cells and breast cancer MDA-MB-231 tumor regenerative cells at 10 ⁇ M, which greatly blocked the tumor.
  • the volume of the clones increased, and the volume of the tumor clones was only 10-15% of the blank group.
  • the use of compound WYC-331 even reversed the growth trend of tumor clones (Fig. 9, where #31 indicates WYC-331).
  • cell staining studies found that tumor clones inhibited by WYC-331 remained viable, and only a few cells showed apoptosis.
  • mice embryonic fibroblast 3T3 cell line and melanoma cell B16-F1 were used as models to study the in vitro toxicity of compounds WYC-209 and WYC-331, respectively.
  • the effects of compounds WYC-209 and WYC-331 on the proliferation of mouse embryonic fibroblasts 3T3 and melanoma cells B16-F1 were compared at 10 ⁇ M.
  • the results showed that the compound WYC-209 did not affect the proliferation of mouse embryonic fibroblasts 3T3, nor induced apoptosis of this type of cells; at the same time, compound WYC-209 could significantly block the growth of melanoma cell clones, and at 6- 48 hours showed significant apoptosis.
  • This experiment shows that the compound WYC-209 has a certain specificity for melanoma tumor-regeneration cells, has a weak effect on ordinary cells, and has less cytotoxicity (Fig. 10).
  • the compound WYC-331 strongly inhibited the proliferation of melanoma cells B16-F1, but at the same time did not show significant apoptosis-inducing effects on mouse embryonic fibroblasts 3T3 and melanoma cells B16-F1. This shows that the compound WYC-331 also has some selectivity for melanoma-regenerating cells, and no obvious is observed. Cytotoxicity, but the mechanism of action may be different from the compound WYC-209 ( Figure 11).
  • B16-F1 cells were incubated with 3D fibrin glue medium (90-Pa) for 5 days, and the desired tumor regenerative cells were screened. Subsequently, the medium was treated with collagenase and neutral protease II to release tumor regenerative cells. The resulting regenerated cells are then pipetted into the new preparation medium and maintained in a single cell resuspended state.
  • 3D fibrin glue medium 90-Pa
  • mice Six immunocompetent mice were randomly divided into two groups: the drug-administered group and the DMSO-negative control group.
  • the melanoma model was subcutaneously implanted by subcutaneous injection of 30,000 melanoma-derived cells. Subsequently, it was administered by tail vein injection according to the body weight of the mouse and the blood volume. From day 0, the drug-administered group was treated with WYC-103 once every 2 days according to the blood concentration of 10 ⁇ M. The survival of the mice was observed by normal feeding, and the results were as follows. After 19 days of experimentation, the subcutaneous melanoma volume of the drug-administered group was significantly smaller than that of the DMSO control group. According to statistical analysis, the tumor volume of the drug-administered group was only 50% of the control group (Fig. 12).
  • the drug-administered group was injected with WYC-103 once every 2 days according to the blood concentration of 10 ⁇ M; the positive control group was administered in the same manner with BMS-453 (WYC-114) as the positive drug; 0.1% DMSO was injected once in the same manner.
  • mice in the positive drug control group died.
  • 50% of the mice in the drug-administered group and the negative control group died.
  • the tumor volume of the mice in the drug-administered group was only 50% of the negative control group, and the growth of the subcutaneous transplanted tumors of the mice in the drug-administered group was significantly inhibited.
  • the mouse body weight study showed that the body weight of the mice in the administration group was stable and balanced, and no obvious side effects were observed (Fig. 13, wherein drug3# indicates WYC-103, drug14# indicates BMS-453).
  • mice with normal immune function C57BL/6, female, 6-8 weeks were randomly selected as the matrix, and 3000 melanoma regenerative cells were injected into the mice via the tail vein to establish a lung cancer metastasis model.
  • the model mice were randomly divided into two groups: the drug-administered group and the negative control group.
  • the compounds WYC-103 (to the drug) and DMSO (negative control group) were subjected to 10 ⁇ M blood according to the tail vein injection method. The drug concentration and 0.1% of the dose were injected every two days.
  • the first control group died in the negative control group.
  • the mice in one administration group were killed at the same time.
  • the negative control group showed obvious melanoma lung metastasis tumor tissue.
  • the lung tissue of the mice in the administration group was all normal.
  • the second control group died in the negative control group, and a large number of melanoma lung metastasis tumor tissues appeared in the negative control group, while the lung tissues in the corresponding administration group were all normal.
  • 3 mice died in the negative control group, and only 1 mouse remained in the negative control group, so all the mice in both groups were sacrificed and anatomically compared.
  • 3 negative control mice showed severe melanoma lung metastasis, while the lung tissues of the mice in the administration group were all normal.
  • the experiment showed that the compound WYC-103 could effectively prevent melanoma-derived cells from metastasizing to the lungs to form secondary lung tumors at a blood concentration of 10 ⁇ M, and WYC-103 showed no obvious side effects during the whole experiment. It is hopeful that it will be further used for the prevention and treatment of human metastatic cancer.
  • B16-F1 cells were incubated with 3D fibrin glue medium (90-Pa) for 5 days, and the desired tumor regenerative cells were screened. Subsequently, the medium was treated with collagenase and neutral protease II to release tumor regenerative cells. The resulting regenerated cells were pipetted into a fresh preparation medium and maintained in a single cell resuspended state. Subsequently, 24 immunocompetent mice were randomly selected from immunocompetent mice (C57BL/6, female, 6-8 weeks), divided into low-dose group (1.0 ⁇ M) and high-dose group (10 ⁇ M).
  • DMSO negative control group Three study groups were in the DMSO negative control group, with 8 mice in each group.
  • the 30,000 melanoma regenerative cells (ten times the animal model used in the compound WYC-103) were injected into the mice via the tail vein to establish a model of melanoma B16-F1 to lung metastasis. Subsequently, it was administered by tail vein injection according to the body weight of the mouse and the blood volume. From the 5th day, the drug-administered group was intravenously injected with WYC-209 every 2 days according to the plasma concentrations of 1.0 ⁇ M and 10 ⁇ M, respectively, and the DMSO group was administered 0.1% in the same manner. DMSO was used as a negative control, and there was no significant difference in body weight between the groups. Observe the statistical results of normal feeding.
  • Figures 1 to 7 are lung tissue maps of the experiment for 30 days). In the same period, only one of the 8 mice in the 10 ⁇ M administration group developed melanoma metastasis in the lungs, and no abnormalities were found in the liver and stomach. By the 30th day, 6 mice (75%) in the DMSO group had pulmonary metastasis melanoma, 4 mice (50%) in the 1.0 ⁇ M administration group had pulmonary metastatic melanoma, and only 10 in the 10 ⁇ M administration group. Only mice (12.5%) suffered from metastatic melanoma in the lungs (see Figure 16c for details). Figures 1 to 5 show the lung tissue map for 30 days, and Figures 6-8 show experiments 29, 24, and 20 respectively. Lung tissue map). The lung tissue weight of each group also showed statistically significant differences.
  • WYC-103, WYC-209 and WYC-331 had strong inhibitory effects on melanoma-regenerating cells, and the inhibitory activity of melanin-regenerating cells reached 0.45, respectively.
  • WYC-209 has strong anti-tumor cells such as lung cancer cell A549, breast cancer cell MCF-7, melanoma cell MDA-MB-435S, ovarian cancer cell A2780, gastric cancer cell Hs-746T, breast cancer cell MDA-MB-231 Inhibition.

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Abstract

本发明公开了一种视黄酸类化合物、其制备方法、中间体及应用。本发明的视黄酸类化合物I对肿瘤细胞的抑制率较佳。

Description

一种视黄酸类化合物、其制备方法、中间体及应用
本申请要求申请日为2016年3月11日的中国专利申请CN201610141143.3和申请日为2016年5月11日的中国专利申请CN201610310944.8的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种视黄酸类化合物、其制备方法、中间体及应用。
背景技术
Figure PCTCN2017071922-appb-000001
他扎罗汀(Tazarotene)是一种具有RAR亚型选择性的视黄醇类药物(J Am Acad Dermatol.1997,37,S12.)，主要用于皮肤局部上皮增生(银屑病、牛皮癣、痤疮等)的治疗。Tazarotene是以乙酯的前药形式存在。该药物进入体内经酶代谢得到羧基型活性代谢产物Tazarotenic acid，可以高选择性地与RARβ和RARγ受体作用，对RARβ也有一定作用，但对RXR作用微弱。
发明内容
本发明所要解决的问题是为了克服他扎罗汀对肿瘤细胞抑制率较差等缺陷，而提供了一种视黄酸类化合物、其制备方法、中间体及应用，该化合物对肿瘤细胞的抑制率较佳。
本发明提供了一种如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐，
Figure PCTCN2017071922-appb-000002
其中，U为CR9a或N；V为CR9b或N；X为CR9c或N；W为CR9d或N；
所述的U、V、X和W中，R9a、R9b、R9c和R9d各自独立地为氢、羟基、硝基、氰基、卤素(例如氟、氯、溴或碘)、C1-C6的烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基)、卤素取代的C1-C6的烷基(所述的卤素可为氟、氯、溴或碘；所述的C1-C6的烷基可为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基；所述的“卤素取代的C1-C6的烷基”例如三氟甲基)、C1-C6烷氧基(例如甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、戊氧基或己氧基)、-NR10R11、
Figure PCTCN2017071922-appb-000003
或-COOR14；
所述的R10、R11、R12、R13和R14独立地为氢或C1-C6的烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基)；
AE、EG和GZ之间独立地为单键或双键；当A、E、G或Z与两个单键连接时，相应的A、E、G和Z独立地为：-(CR2R3)-、-C(＝O)-、-(NR4)-、-(N→O)-、-O-、-S-、-S(＝O)-或-SO2-；当A、E、G或Z与一个单键和一个双键连接时，相应的A、E、G和Z独立地为：-(CR5)＝或-N＝；
所述的R2、R3、R4和R5独立地为氢、羟基、卤素(例如氟、氯、溴或碘)、C1-C6的烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或基)、C2-C6的烯基(例如乙烯基或丙烯基)、卤素取代的C1-C6的烷基(所述的卤素可为氟、氯、溴或碘；所述的C1-C6的烷基可为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基；所述的“卤素取代的C1-C6的烷基”例如三氟甲基)、C1-C6的烷氧基(例如甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、戊氧基或己氧基)、C1-C6的酰基(例如乙酰基或甲酰基)、C6-C10的芳基(例如苯基)或“杂原子为氧、硫或氮原子，杂原子数为1～2个的C3-C6的杂芳基”(例如吡啶基或嘧啶基)；
m为0、1、2或3；
当有多个R1取代时，取代基相同或者不同；R1为氢、羟基、硝基、氰基、卤素(例 如氟、氯、溴或碘)、C1-C6的烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基)、卤素取代的C1-C6的烷基(所述的卤素可为氟、氯、溴或碘；所述的C1-C6的烷基可为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基；所述的“卤素取代的C1-C6的烷基”例如三氟甲基)、C1-C6烷氧基(例如甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、戊氧基或己氧基)、-NR6R7或-COOR8；
所述的R6、R7和R8独立地为氢或C1-C6的烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基)；
Y为-CN、-COOR15或-CO2NHR16；
所述的R15和R16独立地为氢、C1-C6的烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基)、C2-C6的烯基(例如乙烯基或丙烯基)或C1-C6的酰基(例如甲酰基或乙酰基)；
所述的化合物I不为
Figure PCTCN2017071922-appb-000004
较佳地，所述的A、E、G和Z可连接成如下所示的环：
Figure PCTCN2017071922-appb-000005
Figure PCTCN2017071922-appb-000006
R2、R3和R5的定义同前所述。
较佳地，所述的化合物I中，所述的U、V、X和W中至少一个(例如1个、2个、3个或4个)为N。
更佳地，所述的化合物I中，当所述的U、V、X和W中有两个为N时，所述的化合物I可为下述任一化合物：
Figure PCTCN2017071922-appb-000007
其中，A、E、G、Z、R1、m、Y、R9a、R9b、R9c和R9d的定义同前所述。
较佳地，所述的化合物A中，当Y为-COOR15时，所述的R15为氢或乙基。
较佳地，所述的化合物A中，所述的Z为-(CR2R3)-、-S-、-S(＝O)-或-SO2-；更佳地，所述的A、E、G和Z连接成如下所示的环：
Figure PCTCN2017071922-appb-000008
Figure PCTCN2017071922-appb-000009
R2和R3的定义同前所述。
更佳地，所述的化合物A中，当所述的Y为COOH时，所述的Z为-S-。
更佳地，所述的化合物A中，当所述的Y为COOEt时，所述的Z为-(CR2R3)-、-S(＝O)-或-SO2-。
较佳地，所述的化合物B中，当所述的Y为-COOR15时，所述的R15为甲基或乙基。
较佳地，所述的化合物B中，所述的Z为-(CR2R3)-、-S-或-S(＝O)-；更佳地，所述的A、E、G和Z连接成如下所示的环：
Figure PCTCN2017071922-appb-000010
R2和R3的定义同前所述。
更佳地，所述的化合物B中，当所述的Y为COOMe时，所述的Z为-(CR2R3)-，R2和R3的定义同前所述。
更佳地，所述的化合物B中，当所述的Y为COOEt时，所述的Z为-(CR2R3)-或-S-，R2和R3的定义同前所述。
更佳地，所述的化合物B中，当所述的Y为CN时，所述的Z为-(CR2R3)-、-S-或-S(＝O)-， R2和R3的定义同前所述。
较佳地，所述的化合物C中，当所述的Y为-COOR15时，所述的R15为氢或乙基。
较佳地，所述的化合物C中，所述的Z为-(CR2R3)-、-S-或-S(＝O)-；更佳地，所述的A、E、G和Z连接成如下所示的环：
Figure PCTCN2017071922-appb-000011
R2和R3的定义同前所述。
更佳地，当所述的化合物C中，当所述的Y为COOH时，所述的Z为-S-。
更佳地，当所述的化合物C中，当所述的Y为COOEt时，所述的Z为-(CR2R3)-、-S-或-S(＝O)-，R2和R3的定义同前所述。
较佳地，所述的化合物I中，当所述的U、V、X和W中有一个为N时，所述的化合物I可为下述任一化合物：
Figure PCTCN2017071922-appb-000012
其中，A、E、G、Z、R1、m、Y、R9a、R9b、R9c和R9d的定义同前所述。
较佳地，所述的化合物D中，当所述的Y为-COOR15时，所述的R15为氢或乙基。
较佳地，所述的化合物D中，所述的Z为-(CR2R3)-；更佳地，所述的A、E、G和Z连接成如下所示的环：
Figure PCTCN2017071922-appb-000013
R2和R3的定义同前所述。
较佳地，所述的化合物E中，所述的Y为CN。
较佳地，所述的化合物E中，所述的Z为-(CR2R3)-；更佳地，所述的A、E、G和Z连接成如下所示的环：
Figure PCTCN2017071922-appb-000014
R2和R3的定义同前所述。
较佳地，所述的化合物I中，当所述的U为CR9a、V为CR9b、X为CR9c、和W为 CR9d时，其结构如化合物F所示：
Figure PCTCN2017071922-appb-000015
其中，A、E、G、Z、R1、m、Y、R9a、R9b、R9c和R9d的定义同前所述。
较佳地，所述的化合物F中，所述的R9a、R9b、R9c和R9d中至少一个(例如1个、2个、3个或4个)不为氢。
较佳地，所述的化合物F中，当所述的Y为-COOR15时，所述的R15为氢、甲基或乙基。
较佳地，所述的化合物F中，所述的Z为-(CR2R3)-、-S-、-S(＝O)-或-SO2-；更佳地，所述的A、E、G和Z连接成如下所示的环：
Figure PCTCN2017071922-appb-000016
Figure PCTCN2017071922-appb-000017
R2、R3和R5的定义同前所述。
更佳地，所述的化合物I为下述任一化合物：
Figure PCTCN2017071922-appb-000018
Figure PCTCN2017071922-appb-000019
Figure PCTCN2017071922-appb-000020
本发明还提供了如式I所示的化合物的制备方法，其包括下述步骤：将化合物II和III进行偶联反应，得到化合物I即可；
Figure PCTCN2017071922-appb-000021
其中，X1为卤素(例如氟、氯、溴或碘)。
在所述的化合物I的制备方法中，所述的偶联反应的条件可为本领域该类偶联反应常规的条件，本发明特别优选下述条件：在保护气体(例如氩气)的保护下，在有机溶 剂(例如DMF)中，在催化剂(例如Pd/Cu催化剂；所述的“Pd/Cu催化剂”例如Pd(PPh3)2Cl2和CuI)和碱(例如二异丙基胺或三乙胺)存在的条件下，将化合物II和III进行偶联反应，得到化合物I即可。
所述的化合物I可进一步进行灵活的官能团转化和调整(包括但不限于酯化、酯基水解、还原、酰化、氧化等化学操作)，以得到官能团不同的化合物I。
较佳地，所述的化合物I的制备方法还可进一步包括下述步骤：将化合物IV进行脱保护反应，得到所述的化合物III即可；
Figure PCTCN2017071922-appb-000022
在所述的化合物III的制备方法中，所述的脱保护反应的条件可为本领域该类脱保护反应常规的条件，本发明特别优选下述条件：在有机溶剂(例如四氢呋喃)中，在碱(例如四正丁基氟化铵)存在的条件下，将化合物IV进行脱保护反应，得到化合物III即可。
较佳地，所述的化合物I的制备方法还可进一步包括下述步骤：将化合物V和三甲基乙炔基硅烷进行偶联反应，得到所述的化合物IV即可；
Figure PCTCN2017071922-appb-000023
其中，X2为卤素(例如溴或碘)。
在所述的化合物I的制备方法中，所述的偶联反应的条件可为本领域该类偶联反应常规的条件，本发明特别优选下述条件：在保护气体(例如氩气)的保护下，在有机溶剂(例如DMF)中，在催化剂(例如Pd/Cu催化剂；所述的“Pd/Cu催化剂”例如Pd(PPh3)2Cl2和CuI)和碱(例如二异丙基胺或三乙胺)存在的条件下，将化合物和三甲基乙炔基硅烷进行偶联反应，得到化合物IV即可。
本发明还提供了如式II、III、IV或V所示的化合物，
Figure PCTCN2017071922-appb-000024
其中，A、E、G、Z、R1、m、X1、Y、U、V、X和W的定义均如上所述。
本发明还提供了如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐在制备药物中的应用，所述的药物用于治疗原发肿瘤。所述的肿瘤包括但不限于黑色素瘤、食道癌、胃癌、肺癌、肝癌、卵巢癌、结肠癌、肾癌、胆管癌、乳腺癌或前列腺癌。
本发明还提供了如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐在制备药物中的应用，所述的药物用于预防和/或治疗转移肿瘤。所述的肿瘤包括但不限于黑色素瘤、食道癌、胃癌、肺癌、肝癌、卵巢癌、结肠癌、肾癌、胆管癌、乳腺癌、前列腺癌。
本发明还提供了如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐在制备药物中的应用，所述的药物用于预防和/或治疗、白血病和/或淋巴癌。
本发明还提供了如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐在制备药物中的应用，所述的药物用于动物胎儿发育、内环境稳定、视觉、形态发生、皮肤老化和控制细胞分化中的一种或多种。
本发明还提供了如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐在制备药物中的应用，所述的药物用于治疗银屑病。
本发明还提供了如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐在制备药物中的应用，所述的药物用于治疗痤疮。
本发明还提供了一种药物组合物，其包含如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐及可药用载体。
所述的药物组合物可以通过口服、注射、局部给药。所述的药物组合物通过口服途径给药，所述组合物可以但不限于以下形式:片剂、胶囊、混悬剂、溶液剂、乳剂、微球或纳米球混悬剂、或控释性脂囊泡或聚合物囊泡。通过注射给药，组合物可以为用于输注或注射的溶液剂或混悬剂。通常，本发明化合物每天的给药剂量为约0.01mg/kg-100mg/kg体重，分为1-3剂给予。本发明化合物系统性使用的浓度通常为相对于组合物重量的0.01％-10％(重量)、优选0.01％-1％(重量)。经由局部途径给药，所述的药用组合物特别用于治疗皮肤和粘膜疾病，并且可以为液体、糊状或固体形式，特别为软膏、乳膏、 溶液剂、凝胶、喷雾剂、混悬剂、粘贴剂形式。还可以为微球或纳米球混悬剂、或者控释性脂囊泡或聚合物囊泡或者胶凝型或聚合物型贴剂。局部应用的化合物的浓度通常是相对于组合物总重量的0.001％-10％(重量)、优选0.01％-1％(重量)。
所述的药用组合物还可以包含惰性添加剂，或者与药用组合物正相关的药效活性成分，或者这些成分的混合物。当然，本领域熟练技术人员会小心选择将加入上述组合物中的任选化合物，使得本质上属于本发明的优点不会或者基本不会受到预定的添加物的负面影响。
本申请中，所述的“C1-C6的酰基”是指含有1～6个碳原子的烷基酰基，如含1个碳原子的酰基指HC(O)-(即甲酰基)，含2个碳原子的酰基是CH3C(O)-(即乙酰基)；还可包括丙酰基、丁酰基或戊酰基。
本发明中，除非另有说明，在本发明说明书和权利要求书中出现的以下术语具有下述含义：
术语“烷基”是指具有一个到二十个碳原子的饱和的直链或支链的一价烃基。烷基的实例包括但不仅限于甲基、乙基、1-丙基、2-丙基、1-丁基、2-甲基-1-丁基、2-丁基、2-甲基-2-丙基、1-戊基、2-戊基、3-戊基、2-甲基-2-丁基、3-甲基-2-丁基、3-甲基-1-丁基、2-甲基-1-丁基、1-己基、2-己基、3-己基、2-甲基-2-戊基、3-甲基-2-戊基、4-甲基-2-戊基、3-甲基-3-戊基、2-甲基-3-戊基、2,3-二甲基-2-丁基、3,3-二甲基-2-丁基、1-庚基、1-辛基。
术语“烯基”是指含有指定数目碳原子和至少一个碳碳双键的直链、支链或者环状非芳香烃基。优选存在一个碳碳双键，并且可以存在高达四个非芳香碳碳双键。由此，“C2～C12烯基”是指具有2～12个碳原子的烯基。“C2～C6烯基”是指具有2～6个碳原子的烯基，包括乙烯基、丙烯基、丁烯基、2-甲基丁烯基和环己烯基。
术语“芳基”(包括单独使用及包含在其它基团中时)是指任何稳定的在各环中可高达7个原子的单环或者双环碳环，其中至少一个环是芳香环。上述芳基单元的实例包括苯基、萘基、四氢萘基、2,3-二氢化茚基、联苯基、菲基、蒽基或者苊基(acenaphthyl)。可以理解，在芳基取代基是二环取代基，且其中一个环是非芳香环的情况中，连接是通过芳环进行的。
术语“芳杂基”或“杂芳基”(包括单独使用及包含在其它基团中时)表示各环中可高达7个原子的稳定单环或者二环，其中至少一个环是芳香环并且含有1-4个选自O、N、和S的杂原子。在此定义范围内的杂芳基包括但不限于：吖啶基、咔唑基、噌啉基、喹喔啉基、吡唑基、吲哚基、苯并三唑基、呋喃基、噻吩基、苯并噻吩基、苯并呋喃基、喹啉基、异喹啉基、噁唑基、异噁唑基、吲哚基、吡嗪基、哒嗪基、吡啶基、嘧啶基、 吡咯基、四氢喹啉。“杂芳基”还应当理解为包括任何含氮杂芳基的N-氧化物衍生物。在其中杂芳基取代基是二环取代基并且一个环是非芳香环或者不包含杂原子的情况下，可以理解，连接分别通过芳环或者通过在环上的杂原子进行。
术语“卤素”包括F、Cl、Br、I。
术语“可药用盐”指常规的酸加成盐或碱加成盐，其保留化合物I的生物有效性和性质，其由适宜的非毒性有机或无机酸、或有机或无机碱形成。酸加成盐的例子包括衍生自无机酸和衍生自有机酸的那些盐，所述无机酸例如氢氯酸、氢溴酸、氢碘酸、硫酸、氨基磺酸、亚磷酸、硫磷酸、磷酸和硝酸，所述有机酸例如甲酸、乙酸、丙酸、对-甲苯磺酸、水杨酸、甲磺酸、草酸、琥珀酸、柠檬酸、马来酸、乳酸、酒石酸、琥珀酸、富马酸、杏仁酸、苹果酸、樟脑磺酸等。碱加成盐的例子包括衍生自铵、钾、钠、钙和季铵氢氧化物(例如氢氧化四甲铵)的盐。将药用化合物(即药物)化学改性成盐是药剂师公知的技术，用以获得改善的化合物的物理和化学稳定性、吸湿性、流动性和溶解性。
术语“可药用的载体”中的“可药用”是指对于特定化合物的给药对象是可药用的和基本上无毒的。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本申请的化合物对肿瘤细胞的抑制率较佳。
附图说明
图1：WYC-103诱导黑色素瘤B16-F1TRC细胞分化与凋亡作用研究
图2：WYC-209诱导黑色素瘤B16-F1TRC细胞分化与凋亡作用研究
图3：WYC-209针对肺癌A549肿瘤再生细胞抑制作用研究，其中，图3a表示在第0天用WYC-209处理肺癌A549的抑制作用；图3b表示在第3天用WYC-209处理肺癌A549的抑制作用。
图4：WYC-209针对乳腺癌MCF-7肿瘤再生细胞抑制作用研究，其中，图4a表示在第0天用WYC-209处理乳腺癌MCF-7的抑制作用；图4b表示在第3天用WYC-209处理乳腺癌MCF-7的抑制作用。
图5：WYC-209针对黑色素瘤MDA-MB-435S肿瘤再生细胞抑制作用研究，其中，图5a表示在第0天用WYC-209处理黑色素瘤MDA-MB-435S的抑制作用；图5b表示在第3天用WYC-209处理黑色素瘤MDA-MB-435S的抑制作用。
图6：WYC-209针对卵巢癌A2780肿瘤再生细胞抑制作用研究，其中，图6a表示在第0天用WYC-209处理卵巢癌A2780的抑制作用；图6b表示在第3天用WYC-209处理卵巢癌A2780的抑制作用。
图7：WYC-209针对胃癌Hs-746T肿瘤再生细胞抑制作用研究，其中，图7a表示在第0天用WYC-209处理胃癌Hs-746T的抑制作用；图7b表示在第3天用WYC-209处理胃癌Hs-746T的抑制作用。
图8：WYC-209针对乳腺癌MDA-MB-231肿瘤再生细胞抑制作用研究，其中，图8a表示在第0天用WYC-209处理乳腺癌MDA-MB-231的抑制作用；图8b表示在第3天用WYC-209处理乳腺癌MDA-MB-231的抑制作用。
图9：WYC-331针对卵巢癌A2780和乳腺癌MDA-MB-231肿瘤再生细胞抑制活性研究，其中，图9a表示在第3天用WYC-331处理卵巢癌A2780的抑制作用；图9b表示在第3天用WYC-331处理乳腺癌MDA-MB-231的抑制作用。
图10：WYC-209细胞毒性研究(WYC-209抑制B16生长，但对3T3细胞无明显影响)；其中，图10a表示用10μM WYC-209处理3T3细胞18小时；图10b表示不用WYC-209，或用10μM WYC-209处理B16细胞48小时。
图11：WYC-331细胞毒性研究；其中，图11a表示用1μM、10μM WYC-331处理3T3细胞24小时；图11b表示分别用1μM、10μM WYC331处理B16细胞24小时。
图12：WYC-103对皮下黑色素瘤抑制，实验第19天的肿瘤体积。
图13：WYC-103对皮下黑色素瘤抑制作用及体内毒性研究。
图14：WYC-103对皮下黑色素瘤抑制，实验第26天的肿瘤体积。
图15：WYC-103对肺部转移性黑色素瘤的抑制作用研究；其中，图15a表示注射3000黑色素瘤再生细胞，给予WYC103，实验第29天的肺组织；图15b表示注射3000黑色素瘤再生细胞，给予DMSO，实验第29天的肺组织；图15c表示注射3000黑色素瘤再生细胞，给予WYC103，实验第35天的肺组织；图15d表示注射3000黑色素瘤再生细胞，给予DMSO，实验第35天的肺组织；图15e表示给予WYC103，实验第37天的肺组织；图15f表示给予DMSO，实验第37天的肺组织。
图16：WYC-209对肺部转移性黑色素瘤的抑制作用研究，其中，图16a表示给予DMSO的肺组织；图16b表示给予1.0μM WYC-209的肺组织；图16c表示给予10μM WYC-209的肺组织。
图17：WYC-209A的单晶结构图。
图18：WYC-209B的单晶结构图。
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。
实施例1：4-((4，4-二甲基二氢苯并噻喃-6-)乙炔基)-2-羟基苯甲酸甲酯
Figure PCTCN2017071922-appb-000025
将商业化原料6-乙炔基-4,4-二甲基苯并噻喃(202.8mg,1mmol)和化合物2-羟基-4-碘代苯甲酸甲酯(292.1mg,1mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(14mg,0.02mmol)、CuI(7.6mg,0.04mmol)，用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.2mL干燥的iPr2NH，室温反应8h，TLC跟踪。反应结束后用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝100:1到20:1)得到产物WYC-101(296mg,81％)。1H NMR(400MHz,CDCl3)δ7.79(d,1H),7.53(d,1H),7.18-7.20(dd,1H),7.12(d,1H),7.06-7.07(d,1H),7.00-7.02(dd,1H),3.95(s,3H),3.03-3.06(m,2H),1.94-1.97(m,1H),1.34(s,6H)；13C NMR(101MHz,CDCl3)δ170.09,161.38,161.12,153.37,142.25,134.10,130.91,130.00,129.85,129.24,126.73,122.45,120.25,117.90,111.95,93.23,88.03,77.48,77.16,76.84,52.52,37.27,32.54,29.99,23.43；ESI(+)-MS:353.3[M+1]+。
实施例2：4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)-2-羟甲基苯甲酸甲酯
Figure PCTCN2017071922-appb-000026
将化合物6-乙炔基-4,4-二甲基苯并噻喃(202.8mg,1mmol)和化合物2-羟甲基-4-碘代苯甲酸甲酯(292mg,1mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(14mg,0.02mmol)、CuI(7.6mg,0.04mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.2mL干燥的iPr2NH，室温反应8h,TLC跟踪。反应结束后用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝100:1到50:1)得到产物WYC-102(300mg,82％)。 1H NMR(400MHz,CDCl3)δ7.78(d,1H),7.53(d,1H),7.18-7.20(dd,1H),7.11-7.13(dd,1H),7.11(d,1H),7.07(d,1H),3.98(s,3H),3.89(s,3H),3.03-3.06(m,1H),1.94-1.97(m,1H),1.34(s,6H)；13C NMR(101MHz,CDCl3)δ166.25,159.05,142.07,133.98,133.58,131.89,129.90,129.13,128.85,126.70,123.39,119.42,117.88,114.84,92.50,88.07,77.48,77.16,76.84,56.18,52.18,37.23,33.06,30.06,23.31；ESI(+)-MS:367.4[M+1]+.
实施例3：4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)-2-羟基苯甲酸乙酯
Figure PCTCN2017071922-appb-000027
将化合物6-乙炔基-4,4-二甲基苯并噻喃(202.8mg,1mmol)和化合物2-羟基-4-碘代苯甲酸乙酯(292mg,1mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(14mg,0.02mmol)、CuI(7.6mg,0.04mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.2mL干燥的iPr2NH，室温反应8h,TLC跟踪。反应结束后用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝100:1到50:1)得到产物WYC-103(300mg,82％)。1H NMR(400MHz,CDCl3)δ7.80(d,1H),7.52(s,1H),7.18-7.20(d,1H),7.12(s,1H),7.06-7.07(s,1H),7.00-7.02(s,1H),4.39-4.44(q,2H),3.04-3.06(t,2H),1.95-1.97(t,2H),1.44(t,3H),1.35(s,6H)；13C NMR(101MHz,CDCl3)δ169.97,161.45,142.27,134.07,130.80,130.02,129.91,129.26,126.74,122.37,120.25,117.94,112.22,93.19,88.02,77.48,77.36,77.16,76.84,61.71,37.30,33.12,30.11,29.85,23.37,14.34；ESI(+)-MS:367.5[M+1]+.
实施例4：4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)-2-羟甲基苯甲酸乙酯
Figure PCTCN2017071922-appb-000028
将化合物6-乙炔基-4,4-二甲基苯并噻喃(202.8mg,1mmol)和化合物2-羟甲基-4-碘代苯甲酸乙酯(306mg,1mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(14mg,0.02mmol)、CuI(7.6mg,0.04mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.2mL干燥的iPr2NH，室温反应8h,TLC跟踪。反应结束后用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝100:1到50:1)得到产物WYC-104(293mg,77％)。 1H NMR(400MHz,CDCl3)δ7.77(d,J＝7.9Hz,1H),7.53(d,J＝1.7Hz,1H),7.20(dd,J＝8.2,1.7Hz,1H),7.14–7.11(m,1H),7.11–7.05(m,2H),4.36(q,J＝7.1Hz,2H),3.93(s,3H),3.07–3.04(m,2H),1.95–1.98(m,2H),1.39(t,3H),1.35(s,6H)；13C NMR(101MHz,CDCl3)δ165.81,159.12,142.28,133.97,131.77,129.96,129.19,128.70,126.76,123.42,119.93,117.98,114.94,92.39,77.48,77.37,77.16,76.85,61.07,56.25,37.31,33.13,30.14,29.85,23.37,14.46；ESI(+)-MS:381.4[M+1]+.
实施例5：4-((4，4-二甲基二氢苯并噻喃-6-)乙炔基)-2-羟基苯甲酸
Figure PCTCN2017071922-appb-000029
将化合物WYC-103(183mg,0.5mmol)溶于5mL THF，滴入0.5M NaOH水溶液1mL，室温反应8h,TLC跟踪。反应结束后用0.5M HCl中和至pH 7，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝10:1到1:1)得到产物WYC-105(144mg,85％)。1H NMR(500MHz,cdcl3)δ10.50(s,1H),7.86-7.84(d,1H),7.53(d,1H),7.21-7.19(dd,1H),7.14(d,1H),7.08-7.06(d,1H),7.05-7.04(dd,1H),3.07-3.04(m,1H),1.98-1.95(m,1H),1.35(s,6H)；13C NMR(125MHz,CDCl3)δ171.84,161.46,142.17,131.83,130.58,129.91,129.16,126.63,120.24,117.56,110.03,89.85,37.15,32.99,29.97,29.70,23.24；ESI(-)-MS:337.1[M-1]-.
实施例6：4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)-2-羟甲基苯甲酸甲酯
Figure PCTCN2017071922-appb-000030
将化合物WYC-104(190mg,0.5mmol)溶于5mL THF，滴入0.5M NaOH水溶液1mL，室温反应8h,TLC跟踪。反应结束后用0.5M HCl中和至pH 7，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝10:1到4:1)得到产物WYC-106(151mg,86％)。1H NMR(500MHz,CDCl3)δ10.60(s,1H),8.14(d,J＝7.9Hz,1H),7.54(s,1H),7.28(m,2H),7.22(m,1H),7.08(d,J＝7.9Hz,1H),4.12(s,3H),3.06(m,2H),1.96(m,2H),1.35(s,6H)；13C NMR(101MHz,CDCl3)δ165.02,157.85,142.36,134.56,133.90,130.59,130.00,129.21,126.81,125.42, 117.51,116.99,114.42,94.06,87.56,77.48,77.36,77.16,76.84,56.98,37.22,33.12,30.10,29.83,23.37；ESI(-)-MS:351.2[M-1]-.
实施例7：4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)-3-羟基苯甲酸甲酯
Figure PCTCN2017071922-appb-000031
将化合物6-乙炔基-4,4-二甲基苯并噻喃(202.8mg,1mmol)和化合物3-羟基-4-碘代苯甲酸甲酯(278.1mg,1mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(14mg,0.02mmol)、CuI(7.6mg,0.04mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.2mL干燥的iPr2NH，室温反应8h,TLC跟踪。反应结束后用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝100:1到10:1)得到产物WYC-107(245mg,71％)。1H NMR(500MHz,cdcl3)δ7.64(d,J＝1.5Hz,1H),7.58(dd,J＝8.0,1.6Hz,1H),7.52(d,J＝1.7Hz,1H),7.46(d,J＝8.0Hz,1H),7.19(dd,J＝8.2,1.7Hz,1H),7.08(d,J＝8.1Hz,1H),6.02(s,1H),3.91(s,3H),3.06–3.04(m,2H),1.94–1.97(m,2H),1.34(s,6H)；13C NMR(101MHz,CDCl3)δ166.45,156.21,142.31,134.62,131.53,131.43,129.70,128.97,126.73,121.48,117.03,115.72,114.61,99.27,81.86,77.37,77.05,76.74,52.33,37.06,32.99,29.94,23.24；ESI(+)-MS:353.4[M+1]+。
实施例8：4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)-3-羟甲基苯甲酸甲酯
Figure PCTCN2017071922-appb-000032
将化合物6-乙炔基-4,4-二甲基苯并噻喃(202.8mg,1mmol)和化合物3-羟甲基-4-碘代苯甲酸甲酯(292.1mg,1mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(14mg,0.02mmol)、CuI(7.6mg,0.04mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.2mL干燥的iPr2NH，室温反应8h,TLC跟踪。反应结束后用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝100:1到10:1)得到产物WYC-108(275mg,75％)。1H NMR(500MHz,CDCl3)δ7.61-7.63(d,1H),7.52-7.56(m,3H),7.21-7.23(dd,1H),7.05-7.07(d,1H),3.97(s,3H),3.93(s,3H),3.03-3.06(m,1H),1.94-1.97(m,1H),1.34(s, 6H)；13C NMR(126MHz,cdcl3)δ166.59,159.56,141.99,133.50,133.19,130.64,129.80,129.14,126.49,121.75,118.27,111.25,96.82,84.38,77.26,77.01,76.76,56.07,52.29,37.24,32.97,29.98,23.22；ESI(+)-MS:367.3[M+1]+。
实施例9：4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)-3-羟甲基苯甲酸
Figure PCTCN2017071922-appb-000033
将化合物WYC-108(190mg,0.5mmol)溶于5mL THF，滴入0.5M NaOH水溶液1mL，室温反应8h,TLC跟踪。反应结束后用0.5M HCl中和至pH 7，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝10:1到2:1)得到产物WYC-110(1541mg,80％)。1H NMR(500MHz,CDCl3)δ7.69(dd,1H),7.61(s,1H),7.58–7.55(m,2H),7.23(dd,1H),7.07(d,1H),3.99(s,3H),3.06–3.04(m,2H),1.97–1.95(m,2H),1.34(s,6H)；ESI(+)-MS:339.3[M+1]+。
实施例10：2-氰基-5-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)嘧啶
Figure PCTCN2017071922-appb-000034
将商业化原料4,4-二甲基-6-炔基二氢苯并噻喃(202.8mg,1.0mmol)和2-氰基-5-氯嘧啶(93mg,0.67mmol)加入单口烧瓶中，加入Pd(PPh3)2Cl2(23mg,0.03mmol)、CuI(19mg,0.1mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF,0.25mL干燥的Et3N，加热到50度反应22h,TLC跟踪。反应结束后冷却至室温，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，分别用饱和氯化铵溶液和饱和氯化钠溶液洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝30:1)得到产物WYC-202(133mg,65％)。1H NMR(400MHz,CDCl3)δ8.89(s,2H),7.55(d,J＝1.6Hz,1H),7.21(dd,J＝8.2,1.7Hz,1H),7.11(d,J＝8.2Hz,1H),3.11–3.03(m,2H),2.01–1.93(m,2H),1.35(s,6H)；13C NMR(126MHz,CDCl3)δ159.21,142.59,141.79,136.33,130.15,129.31,126.98,122.88,116.13,115.70,101.85,81.24,37.02,33.15,30.02,23.42.ESI(+)-MS:306.3[M+1]+。
实施例11：2-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)嘧啶-5-甲酸乙酯
Figure PCTCN2017071922-appb-000035
将化合物4,4-二甲基-6-炔基二氢苯并噻喃(202.8mg,1.0mmol)和2-氯嘧啶甲酸乙酯(156mg,0.83mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(42mg,0.06mmol)、CuI(19mg,0.1mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.3mL干燥的Et3N，加热到80度反应22h,TLC跟踪。反应结束后冷却至室温，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝15:1)得到产物WYC-203(257mg,73％)。1H NMR(500MHz,CDCl3)δ9.24(s,2H),7.69(d,J＝1.7Hz,1H),7.33(dd,J＝8.2,1.8Hz,1H),7.09(d,J＝8.2Hz,1H),4.45(q,J＝7.1Hz,1H),3.11–2.99(m,2H),1.99–1.92(m,2H),1.43(t,J＝7.1Hz,3H),1.33(s,6H)；13C NMR(126MHz,CDCl3)δ163.52,158.44,155.85,142.39,136.48,131.36,130.15,126.83,121.88,115.96,92.65,87.97,62.14,37.09,33.14,30.05,23.43,14.38.ESI(+)-MS:353.2[M+1]+。
实施例12：5-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)嘧啶-2-甲酸甲酯
Figure PCTCN2017071922-appb-000036
将化合物WYC-202(50mg,0.164mmol)加入烧瓶中，加入0.5mL水和0.5mL甲醇的NaOH(20mg,0.491mmol)溶液，60度下反应5h，TLC跟踪。待原料反应完全后用1mol/L的HCl中和至中性；加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝15:1)得到产物WYC-204(39mg,74％)。1H NMR(500MHz,CDCl3)δ8.63(s,2H),7.51(d,J＝0.7Hz,1H),7.17(dd,J＝8.1,0.9Hz,1H),7.07(d,J＝8.2Hz,1H),4.04(s,3H),3.11–2.91(m,2H),2.04–1.90(m,2H),1.34(s,6H)；13C NMR(126MHz,CDCl3)δ164.02,161.24,142.32,134.10,129.72,128.97,126.77,117.65,113.33,94.71,81.81,55.37,37.28,33.12,30.11,23.36.ESI(+)-MS:339.2[M+1]+。
实施例13：5-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)嘧啶-2-甲酸乙酯
Figure PCTCN2017071922-appb-000037
将化合物WYC-202(50mg,0.164mmol)加入烧瓶中，加入乙醇钠(34mg,0.492mmol),加入5mL乙醇，45度下反应过夜，TLC跟踪。待原料反应完全后用酸性树脂中和至中性，过滤，滤液加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝8:1)得到产物WYC-205(36mg,62％)。1H NMR(500MHz,CDCl3)δ8.62(s,2H),7.51(s,1H),7.17(dd,J＝8.1,0.8Hz,1H),7.07(d,J＝8.1Hz,1H),4.45(q,J＝7.1Hz,2H),3.09–3.00(m,2H),2.00–1.92(m,2H),1.45(t,J＝7.1Hz,3H),1.34(s,6H)；13C NMR(126MHz,CDCl3)δ163.61,161.23,142.32,134.05,129.72,128.97,126.77,117.71,113.13,94.60,81.91,64.11,37.30,33.13,30.11,23.36,14.55.ESI(+)-MS:353.5[M+1]+。
实施例14：2-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)嘧啶-5-甲酸
Figure PCTCN2017071922-appb-000038
将化合物WYC-203(50mg,0.142mmol)加入烧瓶中，加入2mL 2.0mol/L的NaOH溶液中，加入2mL甲醇；45度下反应5h，TLC跟踪。待原料反应完全后用1mol/L的HCl中和至弱酸性；加入乙酸乙酯稀释、萃取，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(二氯甲烷:甲醇＝15:1)得产物WYC-206(41mg,89％)。1H NMR(400MHz,CD3OD)δ9.21(s,2H),7.71(d,J＝1.6Hz,1H),7.29(dd,J＝8.2,1.8Hz,1H),7.11(d,J＝8.2Hz,1H),3.14–3.06(m,2H),2.05–1.92(m,2H),1.36(s,6H)；13C NMR(101MHz,CD3OD)δ167.13,159.54,155.24,143.92,137.92,131.86,130.67,127.84,117.11,111.42,92.11,88.26,49.64,49.43,49.21,49.00,48.79,48.57,48.36,38.12,33.99,30.18,23.98.ESI(+)-MS:325.3[M+1]+。
实施例15：2-氰基-5-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)嘧啶
Figure PCTCN2017071922-appb-000039
将WYC-202(30mg,0.098mmol)加入烧瓶中，加入2mL干燥二氯甲烷，冰水浴下 冷却至0度后加入mCPBA(24mg,0.098mmol)，冰水浴下反应1h后移至室温下反应2h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝1:2)得产物WYC-207(27mg,86％)。1H NMR(500MHz,CDCl3)δ8.95(s,2H),7.82(d,J＝8.1Hz,1H),7.64(d,J＝1.5Hz,1H),7.55(dd,J＝8.0,1.5Hz,1H),3.30–3.08(m,2H),2.44(ddd,J＝15.0,10.1,2.0Hz,1H),1.93(ddd,J＝15.1,9.2,2.1Hz,1H),1.49(s,3H),1.36(s,3H)；13C NMR(126MHz,CDCl3)δ159.64,145.41,142.59,131.57,130.38,130.36,124.24,121.99,115.52,99.38,83.14,43.53,34.76,31.38,31.31,29.96.ESI(+)-MS:322.3[M+1]+。
实施例16：5-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)嘧啶-2-甲酸乙酯
Figure PCTCN2017071922-appb-000040
将WYC-205(70mg,0.2mmol)加入烧瓶中，加入3mL干燥二氯甲烷，冰水浴下冷却至0度后加入mCPBA(49mg,0.2mmol)，冰水浴下反应1h后移至室温下反应2h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝1:2)得产物WYC-208(68mg,93％)。1H NMR(500MHz,CDCl3)δ8.66(s,2H),7.75(d,J＝8.0Hz,1H),7.59(s,1H),7.49(d,J＝7.7Hz,1H),4.47(q,J＝7.1Hz,2H),3.16(dt,J＝20.8,11.7Hz,2H),2.46(dd,J＝14.5,10.3Hz,1H),1.90(dd,J＝14.6,8.0Hz,2H),1.48(s,3H),1.45(t,J＝7.1Hz,3H),1.34(s,3H)；13C NMR(126MHz,CDCl3)δ163.95,161.56,145.06,131.11,130.39,129.99,125.91,112.28,93.14,84.74,64.30,43.37,34.61,31.36,31.23,29.76,14.52.ESI(+)-MS:369.4[M+1]+。
实施例17：2-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)嘧啶-5-甲酸乙酯
Figure PCTCN2017071922-appb-000041
将WYC-203(48mg,0.14mmol)加入烧瓶中，加入3mL干燥二氯甲烷，冰水浴下冷却至0度后加入mCPBA(34mg,0.14mmol)，冰水浴下反应1h后移至室温下反应2h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝1:2)得 产物WYC-209(43mg,84％)。1H NMR(500MHz,CDCl3)δ9.29(s,2H),7.82–7.75(m,2H),7.66(d,J＝7.9Hz,1H),4.47(q,J＝7.1Hz,2H),3.24(t,J＝11.1Hz,1H),3.15–3.07(m,1H),2.43(dd,J＝14.0,10.4Hz,1H),1.92(dd,J＝14.9,8.8Hz,1H),1.47(s,3H),1.44(t,J＝7.2Hz,3H),1.34(s,3H)；13C NMR(126MHz,CDCl3)δ163.33,158.54,155.32,145.19,132.61,131.06,130.22,124.22,122.62,89.60,89.35,62.32,34.75,31.36,31.25,29.93,14.39.ESI(+)-MS:369.4[M+1]+；HRMS-ESI(m/z)calculated C20H21N2O3S,369.1273[M+1]+,found 369.1267.
WYC-209A:2-((4,4-二甲基-1S-氧代二氢苯并噻喃-6-)乙炔基)嘧啶-5-甲酸乙酯
Figure PCTCN2017071922-appb-000042
将D-酒石酸二乙酯(82.7mg,0.4mmol)溶于无水CH2Cl2(1mL)，16度下快速加入Ti(iso-PrO)4(0.2mmol,58uL)，搅拌3分钟，缓慢滴入水(3.6uL，0.2mmol)，继续搅拌20分钟。然后冷却至-20度，快速加入WYC-203(69mg,0.2mmol)和过氧化氢异丙苯(74uL，0.4mmol)，3小时后停止反应。将反应液倾入硫酸亚铁(0.2g)、柠檬酸(67mg)的水、二氧六环与乙醚(2：1：2)的混合溶液10mL中，搅拌15分钟。水相用乙醚萃取，合并有机层用0.5M K2CO3洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝1:2)得产物WYC-209A(43mg,84％，ee％＝90％)。再用乙醚重结晶，得WYC-209A(25mg，ee％＝99％)。1H NMR(500MHz,CDCl3)δ9.29(s,2H),7.82–7.75(m,2H),7.66(d,J＝7.9Hz,1H),4.47(q,J＝7.1Hz,2H),3.24(t,J＝11.1Hz,1H),3.15–3.07(m,1H),2.43(dd,J＝14.0,10.4Hz,1H),1.92(dd,J＝14.9,8.8Hz,1H),1.47(s,3H),1.44(t,J＝7.2Hz,3H),1.34(s,3H)；13C NMR(126MHz,CDCl3)δ163.33,158.54,155.32,145.19,132.61,131.06,130.22,124.22,122.62,89.60,89.35,62.32,34.75,31.36,31.25,29.93,14.39.ESI(+)-MS:369.4[M+1]+。ee％由手性HPLC条件测定，条件如下：Agilent 1260infinity液相色谱仪，色谱柱Lux Cellulose-1 250*4.6mm，流动相：乙腈/H2O＝80:20，20度，0.7mL/min，保留时间8.592min。
WYC-209B:2-((4,4-二甲基-1R-氧代二氢苯并噻喃-6-)乙炔基)嘧啶-5-甲酸乙酯
Figure PCTCN2017071922-appb-000043
将L-酒石酸二乙酯(82.7mg,0.4mmol)溶于无水CH2Cl2(1mL)，16度下快速加 入Ti(iso-PrO)4(0.2mmol,58uL)，搅拌3分钟，缓慢滴入水(3.6uL，0.2mmol)，继续搅拌20分钟。然后冷却至-20度，快速加入WYC-203(69mg,0.2mmol)和过氧化氢异丙苯(74uL，0.4mmol)，3小时后停止反应。将反应液倾入硫酸亚铁(0.2g)、柠檬酸(67mg)的水、二氧六环与乙醚(2：1：2)的混合溶液10mL中，搅拌15分钟。水相用乙醚萃取，合并有机层用0.5M K2CO3洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝1:2)得产物WYC-209B(43mg,84％，ee％＝90％)。再用乙醚重结晶，得WYC-209B(25mg，ee％＝99％)。1H NMR(500MHz,CDCl3)δ9.29(s,2H),7.82–7.75(m,2H),7.66(d,J＝7.9Hz,1H),4.47(q,J＝7.1Hz,2H),3.24(t,J＝11.1Hz,1H),3.15–3.07(m,1H),2.43(dd,J＝14.0,10.4Hz,1H),1.92(dd,J＝14.9,8.8Hz,1H),1.47(s,3H),1.44(t,J＝7.2Hz,3H),1.34(s,3H)；13C NMR(126MHz,CDCl3)δ163.33,158.54,155.32,145.19,132.61,131.06,130.22,124.22,122.62,89.60,89.35,62.32,34.75,31.36,31.25,29.93,14.39.ESI(+)-MS:369.4[M+1]+。其中，ee％由手性HPLC条件测定，条件如下：Agilent 1260infinity液相色谱仪，色谱柱Lux Cellulose-1 250*4.6mm，流动相：乙腈/H2O＝80:20，20度，0.7mL/min,保留时间9.147min。
WYC-209A和WYC-209B的单晶结构图分别见图17和图18，晶体属单斜晶系，空间群为P21，晶胞参数：
Figure PCTCN2017071922-appb-000044
α＝90°，β＝103.010(3)°，γ＝90°，晶胞体积
Figure PCTCN2017071922-appb-000045
晶胞内不对成单位数Z＝4，密度1.314Mg/m3。
表1 原子坐标(x104)和等价的各向同性位置参数
Figure PCTCN2017071922-appb-000046
Figure PCTCN2017071922-appb-000047
Figure PCTCN2017071922-appb-000048
Figure PCTCN2017071922-appb-000049
实施例18：2-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)嘧啶-5-甲酸
Figure PCTCN2017071922-appb-000050
将WYC-206(34mg,0.105mmol)加入烧瓶中，加入1.5mL干燥二氯甲烷，冰水浴下冷却至0℃后加入m-CPBA(26mg,0.105mmol)，冰水浴下反应1h后移至室温下反应2h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(DCM:MeOH＝12:1)得到产物WYC-210(23mg,68％)。1H NMR(500MHz,pyridine)δ9.66(s,2H),7.99–7.85(m,2H),7.65(d,J＝7.8Hz,1H),3.20(t,J＝10.7Hz,1H),3.14–3.02(m,1H),2.32(dd,J＝12.9,10.4Hz,1H),1.72(dd,J＝13.2,9.4Hz,1H),1.25(s,3H),1.18(s,3H).13C NMR(126MHz,pyridine)δ166.30,159.03,155.15,145.73,142.20,132.56,130.90,130.31,125.00,124.29,90.65,88.52,43.70,34.73,30.89,30.62,29.97.ESI(+)-MS:341.1[M+1]+。
实施例19：2-氨基-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000051
将化合物6-乙炔基-4,4-二甲基苯并噻喃(405.6mg,2mmol)和化合物2-氨基-4-碘苯 甲酸(263mg,1mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(42mg,0.06mmol)、CuI(23mg,0.12mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入10mL干燥的DMF、0.2mL干燥的Et3N，加热到75度反应12h,TLC跟踪。反应结束后冷却至室温，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝5:1)得产物WYC-212(286mg,85％)。1H NMR(500MHz,CDCl3)δ7.88(d,J＝8.3Hz,1H),7.52(d,J＝1.7Hz,1H),7.18(dd,J＝8.1,1.8Hz,1H),7.06(d,J＝8.1Hz,1H),6.84(d,J＝1.3Hz,1H),6.81(dd,J＝8.3,1.5Hz,1H),3.08–3.02(m,3H),1.99–1.94(m,2H),1.35(s,6H)；13C NMR(126MHz,CDCl3)δ165.25,165.23,163.21,161.20,142.29,134.42,133.26,133.25,131.70,131.64,129.97,129.27,126.75,125.15,125.12,117.73,117.05,116.92,116.66,116.47,98.04,98.02,81.71,61.62,37.29,33.13,30.09,23.38,14.42.ESI(+)-MS:338.3[M+1]+。
实施例20：2-乙酰氨基-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000052
将WYC-212(200mg,0.3mmol)加入烧瓶中，加入10mg的DMAP，在氩气保护下加入5mL干燥的吡啶，冰水浴下冷却至0度，滴加43μl的乙酰氯，冰水浴下反应10min后移至室温反应5h,TLC跟踪。待反应结束后用甲醇淬灭，乙酸乙酯稀释，1mol/L的盐酸洗去吡啶，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝20:1)得到产物WYC-213(195mg,87％)。1H NMR(500MHz,CDCl3)δ8.13(d,J＝8.1Hz,1H),7.64(d,J＝1.3Hz,1H),7.58(dd,J＝8.1,1.5Hz,1H),7.55(d,J＝1.7Hz,1H),7.21(dd,J＝8.1,1.8Hz,1H),7.09(d,J＝8.1Hz,1H),3.09–3.03(m,2H),2.47(s,3H),1.99–1.92(m,2H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ160.95,159.37,146.56,142.37,134.76,132.31,131.05,130.06,129.32,128.95,128.49,126.82,117.47,115.64,95.32,87.52,37.23,33.13,30.09,23.39,21.54.ESI(+)-MS:380.4[M+1]+。
实施例21：6-乙炔基-4,4-二甲基-1-氧代二氢苯并噻喃
Figure PCTCN2017071922-appb-000053
将6-乙炔基-4,4-二甲基苯并噻喃(2.03g,10mmol)加入烧瓶中，加入27mL干燥二氯甲烷，冰水浴下冷却至0度后加入mCPBA(1.73g,10mmol)，冰水浴下反应10min后 移至室温反应1h后移至室温下反应2h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝3:1)得1-氧代-6-乙炔基-4,4-二甲基苯并噻喃(1.43g,72％)。1H NMR(500MHz,CDCl3)δ7.41(d,J＝8.0Hz,1H),7.26(s,1H),7.15(d,J＝8.0Hz,1H),2.93–2.74(m,3H),2.14(ddd,J＝14.6,10.5,1.7Hz,1H),1.58(ddd,J＝15.0,8.7,1.8Hz,1H),1.14(s,3H),1.01(s,3H)；13C NMR(126MHz,CDCl3)δ144.82,138.91,131.70,130.53,130.17,125.60,82.69,79.39,43.23,34.47,31.27,31.13,29.67.ESI(+)-MS:219.3[M+1]+。
实施例22：2-氨基-4-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000054
将化合物1-氧代-6-乙炔基-4,4-二甲基苯并噻喃(263mg,1.0mmol)和化合物2-氨基-4-碘苯甲酸(262mg,1.2mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(42mg,0.06mmol)、CuI(23mg,0.0412mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入4mL干燥的DMF、0.2mL干燥的Et3N，加热到70度反应8h,TLC跟踪。反应结束后冷却至室温，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝50:1)得到产物(275mg,78％)。1H NMR(500MHz,pyridine)δ8.37(d,J＝8.1Hz,1H),7.92(d,J＝8.0Hz,1H),7.86(s,1H),7.59(s,1H),7.36(s,1H),7.03(d,J＝8.1Hz,1H),5.67(s,1H),3.20–3.14(m,1H),3.11–3.05(m,1H),2.38(ddd,J＝14.2,10.3,1.7Hz,1H),1.74–1.68(m,1H),1.26(s,3H),1.17(s,3H)；13C NMR(126MHz,pyridine)δ171.06,152.34,145.52,140.46,132.80,131.65,130.43,130.20,127.92,126.32,119.79,118.58,112.43,91.82,90.33,55.01,43.59,34.63,30.93,30.63,29.80.ESI(+)-MS:354.3[M+1]+。
实施例23：2-氨基-4-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)苯甲酸甲酯
Figure PCTCN2017071922-appb-000055
将WYC-214(150mg,0.425mmol)加入烧瓶中，加入碳酸钠(176mg,1.275mmol),在氩气保护下加入3mL干燥的DMF和碘甲烷(40μl,0.637mmol),缓慢加热到100度， 反应6h，TLC跟踪。待反应结束后冷却至室温，用乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝20:1)得产物WYC-215(143mg,92％)。1H NMR(500MHz,CDCl3)δ7.84(d,J＝8.3Hz,1H),7.73(d,J＝8.1Hz,1H),7.58(d,J＝1.5Hz,1H),7.48(dd,J＝8.0,1.6Hz,1H),6.87(d,J＝1.3Hz,1H),6.81(dd,J＝8.3,1.5Hz,1H),3.88(s,3H),3.20(ddd,J＝12.9,10.6,2.2Hz,1H),3.10(ddd,J＝13.1,8.7,2.3Hz,1H),2.46(ddd,J＝14.9,10.5,2.2Hz,1H),1.89(ddd,J＝15.1,8.7,2.2Hz,3H),1.47(s,3H),1.33(s,3H)；13C NMR(126MHz,CDCl3)δ168.19,150.04,144.92,138.59,131.48,131.30,130.34,130.18,128.13,126.44,119.72,119.64,111.06,90.93,90.26,51.84,43.28,34.57,31.36,31.21,29.73.ESI(+)-MS:368.4[M+1]+。
实施例24：2-氨基-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000056
将WYC-212(50mg,0.15mmol)加入烧瓶中，在氩气保护下加入2mL无水乙醇，冰水浴至0度，滴加0.15mL的浓硫酸，滴加完毕后升温回流反应4h，TLC跟踪。待反应完全后冷却至室温，用2mol/L的氢氧化钠中和硫酸至中性，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝10：1)得产物WYC-216(14mg,25％)。1H NMR(500MHz,CDCl3)δ7.84(d,J＝8.3Hz,1H),7.51(d,J＝1.6Hz,1H),7.17(dd,J＝8.1,1.7Hz,1H),7.06(d,J＝8.1Hz,1H),6.83(d,J＝1.3Hz,1H),6.79(dd,J＝8.3,1.4Hz,1H),4.33(q,J＝7.1Hz,2H),3.06–3.03(m,2H),1.97–1.94(m,2H),1.39(t,J＝7.1Hz,3H),1.34(s,6H).13C NMR(126MHz,CDCl3)δ167.92,150.10,142.22,133.72,131.35,129.95,129.19,129.02,126.70,119.55,119.39,118.23,110.80,91.99,88.37,60.61,37.36,33.12,30.13,23.37,14.50.ESI(+)-MS:366.4[M+1]+。
实施例25：2-乙酰氨基-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000057
将WYC-216(80mg,0.22mmol)加入烧瓶中，加入6mg的DMAP，在氩气保护下加入2mL干燥的吡啶，冰水浴下冷却至0度，滴加乙酰氯(31μl,0.44mmol)，冰水浴下反应10min后移至室温反应9h,TLC跟踪。待反应结束后用甲醇淬灭，乙酸乙酯稀释， 1mol/L的盐酸洗去吡啶，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝10:1)得到产物WYC-217(71mg,79％)。1H NMR(500MHz,CDCl3)δ11.12(s,1H),8.88(d,J＝1.4Hz,1H),7.99(d,J＝8.3Hz,1H),7.55(d,J＝1.7Hz,1H),7.20(dd,J＝3.6,1.7Hz,1H),7.18(dd,J＝3.8,1.7Hz,1H),7.06(d,J＝8.1Hz,1H),4.38(q,J＝7.1Hz,2H),3.06–3.03(m,2H),2.24(s,3H),1.97–1.94(m,2H),1.42(t,J＝7.1Hz,3H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ169.17,168.11,142.25,141.59,133.98,130.81,130.17,130.04,129.24,126.70,125.26,122.97,118.06,114.16,93.43,88.37,61.65,37.36,33.13,30.13,25.68,23.38,14.34.ESI(+)-MS:408.4[M+1]+。
实施例26：2-氨基-4-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000058
将化合物2-氨基-4-碘苯甲酸乙酯(262.0mg,1.2mmol)和化合物1-氧代-6-乙炔基-4,4-二甲基苯并噻喃(263.0mg,1mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(42mg,0.06mmol)、CuI(23mg,0.12mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入15mL干燥的DMF、0.2mL干燥的Et3N，加热到75度反应12h,TLC跟踪。反应结束后冷却至室温，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(DCM:MeOH＝40：1)得到产物WYC-218(275mg,78％)。1H NMR(500MHz,pyridine)δ8.39(d,J＝8.1Hz,1H),7.97–7.82(m,2H),7.61(s,1H),7.38(s,1H),7.05(d,J＝8.1Hz,1H),3.25–3.01(m,2H),2.40(ddd,J＝14.2,10.3,1.7Hz,1H),1.73(ddd,J＝14.8,8.8,1.5Hz,1H),1.28(s,3H),1.19(s,3H).13C NMR(126MHz,pyridine)δ171.75,153.03,150.76,150.74,150.55,150.53,150.33,150.31,146.21,141.15,133.49,132.34,131.12,130.89,128.61,127.01,120.48,119.27,113.12,92.51,91.02,44.28,35.32,31.62,31.32,30.49.ESI(+)-MS:382.4[M+1]+。
实施例27：2-甲氨基-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸甲酯
Figure PCTCN2017071922-appb-000059
将WYC-212(100mg,0.3mmol)加入烧瓶中，加入碳酸钠(125mg,0.9mmol),在氩气保护下加入3mL干燥的DMF和碘甲烷(28μl,0.445mmol),缓慢加热到100度，反应 4h，TLC跟踪。待反应结束后冷却至室温，用乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝20:1)得到产物WYC-219(43mg,39％)和3-69-2-2(45mg,43％)。1H NMR(500MHz,CDCl3)δ7.87(d,J＝8.2Hz,1H),7.54(s,1H),7.20(d,J＝8.1Hz,1H),7.06(d,J＝8.1Hz,1H),6.88(s,1H),6.78(d,J＝8.2Hz,1H),3.86(s,3H),3.08–3.02(m,2H),2.95(s,3H),1.99–1.94(m,2H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ168.77,151.15,142.24,133.75,131.64,130.02,129.68,129.24,126.71,118.37,118.24,114.31,110.05,92.11,88.89,51.79,37.38,33.14,30.15,23.39.ESI(+)-MS:366.2[M+1]+。
实施例28：2-氨基-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸甲酯
Figure PCTCN2017071922-appb-000060
将化合物WYC-212(100mg,0.3mmol)加入烧瓶中，加入碳酸钾(125mg,0.9mmol),在干燥氩气保护下加入干燥的DMF 3mL,滴加MeI(28μL，0.445mmol)100度下反应8h，待原料反应完全后，冷却至室温，加入乙酸乙酯稀释，1mol/L的HCl溶液洗涤，饱和碳酸氢钠洗涤，饱和氯化钠洗，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝20:1)得产物WYC-220(45mg,44％)。1H NMR(500MHz,cdcl3)δ7.83(d,J＝8.3Hz,1H),7.52(d,J＝1.7Hz,1H),7.18(dd,J＝8.1,1.8Hz,1H),7.07(d,J＝8.1Hz,1H),6.86(d,J＝1.4Hz,1H),6.81(dd,J＝8.3,1.5Hz,1H),3.88(s,3H),3.08–3.02(m,2H),2.00–1.93(m,2H),1.35(s,6H).13C NMR(126MHz,cdcl3)δ168.25,149.81,142.19,133.74,131.31,129.92,129.16,129.15,126.67,119.74,119.47,118.15,110.58,92.12,88.28,51.75,37.32,33.08,30.09,23.34.ESI(+)-MS:352.4[M+1]+。
实施例29：2-乙酰氨基-4-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000061
将WYC-214(152mg,0.42mmol)加入烧瓶中，加入10mg的DMAP，在氩气保护下加入5mL干燥的二氯甲烷，0.212mL的三乙胺，冰水浴下冷却至0度，滴加乙酰氯(67μl,0.84mmol)，冰水浴下反应10min后移至室温反应12h,TLC跟踪。待反应结束后用甲醇淬灭，乙酸乙酯稀释，1mol/L的盐酸洗去吡啶，饱和碳酸氢钠洗，饱和氯化钠洗涤，有 机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝1:3)得产物WYC-221(147mg,89％)。1H NMR(500MHz,CDCl3)δ8.12(d,J＝8.3Hz,1H),7.63(d,J＝1.3Hz,1H),7.57(dd,J＝8.1,1.5Hz,1H),7.54(d,J＝1.7Hz,1H),7.21(dd,J＝8.1,1.8Hz,1H),7.08(d,J＝8.1Hz,1H),3.07–3.03(m,2H),2.46(s,3H),1.99–1.94(m,2H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ160.87,159.36,146.58,142.34,134.73,132.26,131.01,130.04,129.30,128.95,128.45,126.80,117.45,115.64,95.28,87.51,37.21,33.11,30.07,23.38,21.54.ESI(+)-MS:396.2[M+1]+。
实施例30：2-(3-溴苯基)乙醛
Figure PCTCN2017071922-appb-000062
将2-(3-溴苯基)乙醇(676μl,5mmol)加入烧瓶中，加入10mL干燥的二氯甲烷，在冰水浴下DMP(2.5g,6mmol),冰水浴下反应2h后移至室温下反应0.5h，TLC跟踪。待反应结束后冷却至0度，用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝15:1)得到产物2-(3-溴苯基)乙醛(0.86g,87％)。1H NMR(300MHz,CDCl3)δ9.74(1H,t,J＝2.0),7.46–7.37(2H,m),7.30–7.14(2H,m),3.67(2H,d,J＝2.0).13C NMR(75MHz,CDCl3)δ198.9,134.5,133.0,131.0,130.9,128.7,123.3,60.8.ESI(+)-MS:199.1[M+1]+。
实施例31：4-(3-溴苯基)-2-丁烯酸乙酯
Figure PCTCN2017071922-appb-000063
将2-(3-溴苯基)乙醛(400mg,2.02mmol)和Ph3PCH＝CO2Et(703mg,2.02mmol)加入烧瓶中，在氩气保护下加入10mL干燥的甲苯，加热到90度反应8h，TLC跟踪。待反应结束后冷却至0度，乙酸乙酯稀释，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝250:1)得到产物4-(3-溴苯基)-2-丁烯酸乙酯(514mg,96％)。E-式：1H NMR(500MHz,CDCl3)δ7.36(d,J＝8.0Hz,1H),7.31(s,1H),7.17(t,J＝7.8Hz,1H),7.11–6.99(m,2H),5.80(dt,J＝15.6,1.6Hz,1H),4.17(q,J＝7.1Hz,2H),3.47(dd,J＝6.8,1.0Hz,2H),1.27(t,J＝7.1Hz,3H).13C NMR(126MHz,CDCl3)δ166.29,146.15,140.06,131.88,130.29,129.90,127.53,123.02,122.77,77.41,77.16,76.91,60.44,38.00,14.32.Z-式：1H NMR(500MHz,CDCl3)δ7.38(s,1H),7.36–7.33(m,1H),7.16(dd,J＝4.1,1.4Hz,2H),6.30(dt,J＝11.4,7.6Hz,1H),5.88(dt,J＝11.4,1.7Hz,1H),4.22(q,J＝7.1Hz,2H),4.00(dd,J＝7.6,1.4Hz,2H),1.31(t,J＝7.1Hz,3H).13C NMR(126MHz, CDCl3)δ166.33,146.76,141.84,131.76,130.25,129.60,127.41,122.74,120.73,60.23,34.73,14.39。
实施例32：4-(3-溴苯基)丁酸乙酯
Figure PCTCN2017071922-appb-000064
将4-(3-溴苯基)-2-丁烯酸乙酯(1.05g,3.92mmol)加入烧瓶中，加入(10％wt)干燥Pd/C(220mg,0.194mmol),在氩气保护下加入20mL的乙酸乙酯，通入氢气并置换3次气体，常温常压下反应24h，TLC跟踪。待反应结束后，过滤、旋干，快速柱层析(PE:EtOAc＝15:1)得到产物4-(3-溴苯基)丁酸乙酯(1.04g,98％)。1H NMR(500MHz,CDCl3)δ7.32(dd,J＝9.5,1.3Hz,2H),7.16–7.08(m,2H),4.12(q,J＝7.1Hz,2H),2.64–2.59(m,2H),2.30(t,J＝7.4Hz,2H),1.96–1.90(m,2H),1.25(t,J＝7.1Hz,3H).13C NMR(126MHz,CDCl3)δ173.27,143.87,131.59,130.01,129.18,127.22,122.51,60.40,34.82,33.57,26.36,14.33.ESI(+)-MS:271.2[M+1]+。
实施例33：5-(3-溴苯基)-2-甲基-2-戊醇
Figure PCTCN2017071922-appb-000065
将4-(3-溴苯基)丁酸乙酯(0.53g,1.96mmol)加入到烧瓶中，在氩气保护下加入15mL的四氢呋喃，冰水浴下冷却至0度，滴加甲基溴化镁(2mL,5.89mmol),在0度下反应3h，室温下反应1h,TLC跟踪。待反应结束后，冷却至0度，用冰水淬灭，乙酸乙酯稀释，加入1mol/l的盐酸中和，饱和碳酸钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝15:1)得到产物5-(3-溴苯基)-2-甲基-2-戊醇(466mg,93％)。1H NMR(400MHz,CDCl3)δ7.37–7.26(m,2H),7.22–7.10(m,2H),2.62(dt,J＝15.0,7.7Hz,2H),1.76–1.64(m,2H),1.56–1.47(m,2H),1.22(s,6H).13C NMR(126MHz,CDCl3)δ144.81,131.40,129.84,128.81,127.05,122.33,70.73,43.29,35.95,29.22,26.00.ESI(+)-MS:257.1[M+1]+。
实施例34：7-溴-4,4-二甲基-3,4二氢萘基-1-(2H)-酮
Figure PCTCN2017071922-appb-000066
将5-(3-溴苯基)-2-甲基-2-戊醇(340mg,1.33mmol)加入烧瓶中，在氩气保护下加入2mL干燥的二氯甲烷，冰水浴下冷却至0度，滴加0.2mL的浓硫酸，室温下反应4h，TLC跟踪。待反应结束后，加水稀释，用1mol/l的氢氧化钠中和硫酸，乙酸乙酯稀释，饱和 碳酸钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，得到白色固体320mg；将320mg固体加入烧瓶中，加入4.3mL的甲苯，然后将71mg的三氧化铬溶于0.648mL乙酸和0.342mL乙酸酐的混合溶液中，在冰水浴下将三氧化铬的溶液加入烧瓶中，反应2h，TLC跟踪。待反应结束后，用乙酸乙酯稀释，用水洗涤三次，饱和碳酸钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝10:1)得到产物7-溴-4,4-二甲基-3,4二氢萘基-1-(2H)-酮(268mg,80％)。1H NMR(400MHz,CDCl3)δ7.37(dd,J＝8.5,1.6Hz,1H),7.33–7.21(m,2H),2.24–2.05(m,2H),1.87–1.77(m,1H),1.58–1.49(m,1H),1.35(s,3H),1.30(s,3H).13C NMR(126MHz,CDCl3)δ197.03,151.09,136.63,132.77,130.15,128.04,120.57,36.86,35.04,33.89,29.65.ESI(+)-MS:253.1[M+1]+。
实施例35：7-溴-4,4-二甲基-1-苯基-1,2,3,4-四氢萘基-1-醇
Figure PCTCN2017071922-appb-000067
将7-溴-4,4-二甲基-3,4二氢萘基-1-(2H)-酮(340mg,1.35mmol)加入到烧瓶中，在氩气保护下加入3mL的四氢呋喃，冰水浴下冷却至0度，滴加苯基溴化镁(2.7mL,2.7mmol),在0度下反应0.5h，室温下反应18h,TLC跟踪。待反应结束后，冷却至0度，用冰水淬灭，乙酸乙酯稀释，加入1mol/l的盐酸中和，饱和碳酸钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝100:1)得到产物7-溴-4,4-二甲基-1-苯基-1,2,3,4-四氢萘基-1-醇(280mg,63％)。1H NMR(400MHz,CDCl3)δ7.39–7.08(m,8H),2.24–2.05(m,2H),1.87–1.77(m,1H),1.58–1.49(m,1H),1.35(s,3H),1.30(s,3H).13C NMR(126MHz,CDCl3)δ148.11,145.41,143.32,131.50,131.18,128.46,128.08,127.15,126.59,119.96,75.87,37.69,34.77,34.09,31.56,31.45.ESI(+)-MS:331.1[M+1]+。
实施例36：6-溴-1,1-二甲基-4-苯基-1,2-二氢萘
将7-溴-4,4-二甲基-1-苯基-1,2,3,4-四氢萘基-1-醇(260mg,0.79mmol)加入到烧瓶中，加入对甲苯磺酸(27mg,0.16mmol)，在氩气保护下加入2mL干燥甲苯，75度下反应0.5h,TLC跟踪。待反应结束后，冷却至室温，用冰水淬灭，乙酸乙酯稀释，饱和碳酸钠洗涤， 饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝250:1)得到产物6-溴-1,1-二甲基-4-苯基-1,2-二氢萘(224mg,91％)。1H NMR(500MHz,CDCl3)δ7.44–7.40(m,2H),7.39–7.33(m,4H),7.24(d,J＝8.3Hz,1H),7.17(d,J＝2.1Hz,1H),6.03(t,J＝4.7Hz,1H),2.36(d,J＝4.7Hz,2H),1.34(s,6H).13C NMR(126MHz,CDCl3)δ145.59,144.16,140.35,138.75,136.15,133.57,131.80,130.41,128.72,128.56,127.87,127.46,127.02,125.79,119.96,63.05,38.87,33.65,28.18.ESI(+)-MS:312.1[M+1]+。
实施例37：((5,5-二甲基-8-苯基-5,6-二氢萘基-2-)乙炔基)三甲基硅
Figure PCTCN2017071922-appb-000069
将化合物6-溴-1,1-二甲基-4-苯基-1,2-二氢萘(310mg,1.0mmol)和化合物三甲基乙炔基硅烷(0.28μL,2mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(21mg,0.03mmol)、CuI(11mg,0.06mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入5mL干燥的DMF、0.2mL干燥的Et3N，室温下反应过夜，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(石油醚)得到产物((5,5-二甲基-8-苯基-5,6-二氢萘基-2-)乙炔基)三甲基硅(275mg,83％)。1H NMR(500MHz,CDCl3)δ7.44–7.38(m,2H),7.36–7.32(m,4H),7.28(d,J＝7.9Hz,10H),7.11(d,J＝1.6Hz,1H),5.97(t,J＝4.7Hz,1H),2.33(d,J＝4.7Hz,2H),1.31(s,6H),0.19(s,9H).13C NMR(126MHz,CDCl3)δ146.01,140.79,139.09,133.98,131.51,129.25,128.83,128.49,127.29,127.27,123.92,120.66,105.45,93.41,38.88,33.90,28.19,0.16.ESI(+)-MS:331.2[M+1]+。
实施例38：6-乙炔基-1,1-二甲基-4-苯基-1,2-二氢萘
Figure PCTCN2017071922-appb-000070
将((5,5-二甲基-8-苯基-5,6-二氢萘基-2-)乙炔基)三甲基硅(300mg,0.91mmol)加入烧瓶中，加入TBAF(30mg,0.115mmol),干燥的四氢呋喃2mL,反应4h,TLC跟踪。反应结束后，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝1000:1)得到产物6-乙炔基-1,1-二甲基-4-苯基-1,2-二氢萘(225mg,96.6％)。1H NMR(500MHz,CDCl3)δ77.47–77.34(m,7H),77.22 (d,J＝1.6Hz,1H),76.04(t,J＝4.7Hz,1H),72.98(s,1H),72.39(d,J＝4.7Hz,2H),71.38(s,6H).13C NMR(126MHz,CDCl3)δ146.23,140.58,138.97,134.13,131.41,129.62,128.77,128.49,127.34,127.27,124.04,119.59,83.98,76.59,38.83,33.85,28.18.ESI(+)-MS:259.2[M+1]+。
实施例39：4-((5,5-二甲基-8-苯基-5,6-二氢萘基-2-)乙炔基)-2-羟基苯甲酸甲酯
Figure PCTCN2017071922-appb-000071
将化合物6-乙炔基-1,1-二甲基-4-苯基-1,2-二氢萘(116mg,0.45mmol)和2-羟基-4-碘苯甲酸甲酯(250mμl,0.9mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(9.5mg,0.0135mmol)、CuI(5.1mg,0.027mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入3mL干燥的DMF、0.2mL干燥的Et3N，75度下反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝500:1)得到产物WYC-301(155mg,79％)。1H NMR(500MHz,CDCl3)δ10.75(s,1H),7.76(d,J＝8.2Hz,1H),7.43(dd,J＝7.7,7.1Hz,7H),7.37(dd,J＝9.3,3.7Hz,4H),7.21(d,J＝1.3Hz,1H),7.09(d,J＝1.2Hz,1H),6.97(dd,J＝8.2,1.2Hz,1H),6.02(t,J＝4.6Hz,1H),3.94(s,3H),2.37(d,J＝4.7Hz,2H),1.36(s,6H).13C NMR(126MHz,CDCl3)δ170.27,161.31,146.42,140.63,138.99,134.24,131.17,130.83,129.82,129.20,128.80,128.54,127.41,127.38,124.18,122.47,120.36,120.07,111.98,92.99,87.93,52.47,38.85,33.94,28.19.ESI(+)-MS:409.3[M+1]+。
实施例40：4-((5,5-二甲基-8-苯基-5,6-二氢萘基-2-)乙炔基)-2-羟基苯甲酸乙酯
Figure PCTCN2017071922-appb-000072
将化合物WYC-301(250mg,0.613mmol)加入烧瓶中，加入乙醇钠(125mg,1.83mmol),加入5mL乙醇，室温下反应过夜，TLC跟踪。待原料反应完全后用酸性树脂中和至中性，过滤，滤液加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝100:1)得到产物WYC-302(230mg,92％)。1H NMR(500MHz,CDCl3)δ10.82(s,1H),7.77(d,J＝8.2Hz,1H),7.45–7.39(m,3H),7.38–7.34(m,4H),7.19(d,J＝1.5Hz,1H),7.07(d,J＝1.4Hz,1H),6.95(dd,J＝ 8.2,1.5Hz,1H),6.01(t,J＝4.7Hz,1H),4.40(q,J＝7.1Hz,2H),2.37(d,J＝4.7Hz,2H),1.41(t,J＝7.1Hz,3H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ169.92,161.39,146.40,140.65,139.01,134.25,131.18,130.70,129.83,129.20,128.82,128.55,127.42,127.39,124.19,122.39,120.37,120.11,112.25,92.89,87.99,61.68,38.86,33.96,28.21,14.32.ESI(+)-MS:423.4[M+1]+。
实施例41：2-氟-4-碘苯甲酸乙酯
Figure PCTCN2017071922-appb-000073
将2-氟-4-碘苯甲酸(1.0g,3.76mmol)加入烧瓶中，在氩气保护下加入10mL无水乙醇，冰水浴至0度，滴加0.5mL的浓硫酸，滴加完毕后升温回流反应4h，TLC跟踪。待反应完全后冷却至室温，用1mol/L的氢氧化钠中和硫酸至中性，饱和碳酸氢钠氯化钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝15：1)得到产物2-氟-4-碘苯甲酸乙酯(990mg,90％)。1H NMR(500MHz,CDCl3)δ7.62–7.57(m,1H),7.52(dd,J＝8.3,1.6Hz,1H),7.49(dd,J＝9.9,1.5Hz,1H),4.36(q,J＝7.1Hz,2H),1.36(t,J＝7.1Hz,3H).13C NMR(126MHz,CDCl3)δ163.87,163.84,162.18,160.06,133.48,133.45,133.05,133.04,126.54,126.34,118.76,118.68,99.59,99.52,61.59,14.29.ESI(+)-MS:294.1[M+1]+。
实施例42：2-氟-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000074
将化合物6-乙炔基-4,4-二甲基苯并噻喃(275mg,1.36mmol)和2-氟-4-碘苯甲酸乙酯(200mg,0.68mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(28.6mg,0.0408mmol)、CuI(15.5mg,0.0816mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入3mL干燥的DMF、0.3mL干燥的Et3N，80度下反应6h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝200:1)得到产物WYC-303(215mg,86％)。1H NMR(500MHz,CDCl3)δ7.90(t,J＝7.8Hz,1H),7.52(d,J＝1.7Hz,1H),7.32(dd,J＝8.1,1.4Hz,1H),7.28(d,J＝1.4Hz,1H),7.25(d,J＝1.4Hz,1H),7.19(dd,J＝8.2,1.7Hz,1H),7.07(d,J＝8.1Hz,1H),4.40(q,J＝7.1Hz,2H),3.07–3.03(m,2H),1.98–1.94(m,2H), 1.40(t,J＝7.1Hz,3H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ164.12,164.09,162.75,160.68,142.32,134.45,132.15,132.14,129.98,129.20,127.08,127.05,126.78,119.79,119.60,117.57,93.98,87.00,86.98,61.55,37.25,33.12,30.09,23.37,14.40.ESI(+)-MS:369.4[M+1]+。
实施例43：2-氟-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000075
将化合物WYC-303(15mg,0.04mmol)加入烧瓶中，加入乙醇钠(8.3mg,0.12mmol),加入1mL四氢呋喃，室温下反应过夜，TLC跟踪。待原料反应完全后用酸性树脂中和至中性，过滤，滤液加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝1:1)得到产物WYC-304(11mg,80％)。1H NMR(500MHz,CDCl3)δ7.99(t,J＝7.9Hz,1H),7.53(d,J＝1.7Hz,1H),7.35(dd,J＝8.1,1.5Hz,1H),7.30(dd,J＝11.3,1.4Hz,1H),7.19(dd,J＝8.1,1.8Hz,1H),7.08(d,J＝8.1Hz,1H),3.07–3.04(m,3H),1.98–1.95(m,2H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ167.53,163.34,161.26,142.36,134.65,132.80,131.19,131.11,130.04,129.25,127.25,127.22,126.81,119.89,119.70,117.45,116.98,94.69,86.92,37.24,33.14,30.09,23.39.ESI(+)-MS:339.3[M+1]+。
实施例44：4-((5,5-二甲基-8-苯基-5,6-二氢萘基-2-)乙炔基)-2-氟苯甲酸乙酯
Figure PCTCN2017071922-appb-000076
将化合物6-乙炔基-1,1-二甲基-4-苯基-1,2-二氢萘(180mg,0.7mmol)和2-氟-4碘-苯甲酸乙酯(410mg,1.4mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(30mg,0.042mmol)、CuI(16.0mg,0.084mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入5mL干燥的DMF、0.5mL干燥的Et3N，70度下反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝200:1)得到产物WYC-305(215mg,72％)。1H NMR(500MHz,CDCl3)δ7.87(t,J＝7.8Hz,1H),7.44–7.39(m,7H),7.37(ddd,J＝6.3,3.4,1.5Hz,4H),7.29–7.26(m,1H),7.24–7.18(m,2H),6.02(t,J＝4.7Hz,1H),4.39(q,J＝ 7.1Hz,2H),2.37(d,J＝4.7Hz,2H),1.39(t,J＝7.1Hz,3H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ164.12,164.09,162.70,160.63,146.67,140.62,138.95,134.33,132.08,132.07,131.17,129.92,129.84,129.20,128.82,128.57,127.53,127.41,127.17,127.14,124.25,119.91,119.77,119.72,118.51,118.43,93.66,86.96,86.94,61.55,38.84,33.98,28.20,14.39.ESI(+)-MS:425.5[M+1]+。
实施例45：4-((5,5-二甲基-8-苯基-5,6-二氢萘基-2-)乙炔基)-2-氟苯甲酸
Figure PCTCN2017071922-appb-000077
将化合物WYC-305(70mg,0.19mmol)加入烧瓶中，加入乙醇钠(26mg,0.38mmol),加入2mL四氢呋喃，室温下反应过夜，TLC跟踪。待原料反应完全后用酸性树脂中和至中性，过滤，滤液加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝2:1)得到产物WYC-306(53mg,75％)。1H NMR(500MHz,CDCl3)δ7.95(t,J＝7.9Hz,1H),7.44–7.39(m,7H),7.39–7.35(m,4H),7.30(dd,J＝8.2,1.4Hz,1H),7.24(d,J＝1.3Hz,1H),7.19(d,J＝1.5Hz,1H),6.02(t,J＝4.7Hz,1H),2.37(d,J＝4.7Hz,2H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ167.97,163.32,161.23,146.83,140.60,138.92,134.36,132.74,131.22,129.24,128.83,128.58,127.57,127.43,127.36,127.33,124.29,120.02,119.83,119.64,116.90,94.43,86.86,38.84,34.00,28.20.ESI(+)-MS:395.4[M+1]+。
实施例46：3-氟-4-碘苯甲酸乙酯
Figure PCTCN2017071922-appb-000078
将3-氟-4-碘苯甲酸(0.95g,3.57mmol)加入烧瓶中，在氩气保护下加入5mL无水乙醇，冰水浴至0度，滴加0.3mL的浓硫酸，滴加完毕后升温回流反应4h，TLC跟踪。待反应完全后冷却至室温，用1mol/L的氢氧化钠中和硫酸至中性，饱和碳酸氢钠氯化钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝15：1)得到产物3-氟-4-碘苯甲酸乙酯(920mg,88％)。1H NMR(500MHz,CDCl3)δ7.80(dd,J＝8.2,6.2Hz,1H),7.65(dd,J＝8.4,1.8Hz,1H),7.53(dd,J＝8.2,1.8Hz,1H),4.36(q,J＝7.1Hz,2H),1.37(t,J＝7.2Hz,3H).13C NMR(126MHz,CDCl3)δ164.99,164.97,162.64,160.67,139.61,139.59,132.94,132.89,126.51,126.48,116.46,116.26,87.88, 87.67,61.65,14.33.ESI(+)-MS:295.1[M+1]+。
实施例47：4-碘-3-硝基苯甲酸乙酯
Figure PCTCN2017071922-appb-000079
将4-碘-3-硝基苯甲酸(1.9g,6.48mmol)加入烧瓶中，在氩气保护下加入10mL无水乙醇，冰水浴至0度，滴加0.6mL的浓硫酸，滴加完毕后升温回流反应6h，TLC跟踪。待反应完全后冷却至室温，用1mol/L的氢氧化钠中和硫酸至中性，饱和碳酸氢钠氯化钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝10：1)得到产物4-碘-3-硝基苯甲酸乙酯(1.88g,90.5％)。1H NMR(500MHz,CDCl3)δ8.39(d,J＝2.0Hz,1H),8.10(d,J＝8.2Hz,1H),7.85(dd,J＝8.2,2.0Hz,1H),4.39(q,J＝7.1Hz,2H),1.39(t,J＝7.1Hz,3H).13C NMR(126MHz,CDCl3)δ164.03,153.15,142.33,133.47,131.96,125.98,92.02,62.16,14.29.ESI(+)-MS:322.1[M+1]+。
实施例48：3-氟-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000080
将化合物6-乙炔基-4,4-二甲基苯并噻喃(405.6mg,2mmol)和3-氟-4-碘苯甲酸乙酯(294mg,1.0mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(84mg,0.12mmol)、CuI(46.0mg,0.24mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入5mL干燥的DMF、0.5mL干燥的Et3N，70度下反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝500:3)得到产物WYC-307(250mg,68％)。1H NMR(500MHz,CDCl3)δ7.81(dd,J＝8.0,1.3Hz,1H),7.76(dd,J＝9.8,1.3Hz,1H),7.56(dd,J＝11.1,4.2Hz,2H),7.22(dd,J＝8.1,1.6Hz,1H),7.08(d,J＝8.1Hz,1H),4.39(q,J＝7.1Hz,2H),3.05(dd,J＝7.1,5.1Hz,2H),1.98–1.94(m,2H),1.40(t,J＝7.1Hz,3H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ165.25,165.23,163.21,161.20,142.29,134.42,133.26,133.25,131.70,131.64,129.97,129.27,126.75,125.15,125.12,117.73,117.05,116.92,116.66,116.47,98.04,98.02,81.71,61.62,37.29,33.13,30.09,23.38,14.42.ESI(+)-MS:369.3[M+1]+。
实施例49：3-硝基-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000081
将化合物6-乙炔基-4,4-二甲基苯并噻喃(405.6mg,2mmol)和3-硝基-4-碘苯甲酸乙酯(303mg,1.0mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(84mg,0.12mmol)、CuI(46.0mg,0.24mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入5mL干燥的DMF、0.5mL干燥的Et3N，70度下反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝50:1)得到产物WYC-308(384mg,97％)。1H NMR(400MHz,CDCl3)δ8.69(d,J＝1.2Hz,1H),8.20(dd,J＝8.1,1.6Hz,1H),7.74(d,J＝8.1Hz,1H),7.57(s,1H),7.23-7.25(m,1H),7.08(d,J＝8.2Hz,1H),4.42(q,J＝7.1Hz,2H),3.04(dd,J＝7.2,4.9Hz,2H),1.98–1.85(m,2H),1.41(t,J＝7.1Hz,3H),1.34(s,6H).13C NMR(126MHz,CDCl3)δ164.34,149.33,142.43,135.66,134.63,133.24,130.35,130.30,129.65,126.86,126.01,123.20,117.23,101.65,84.50,62.11,37.16,33.12,30.04,23.43,14.43.ESI(+)-MS:396.2[M+1]+。
实施例50：3-硝基-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000082
将化合物WYC-308(100mg,0.25mmol)加入烧瓶中，加入乙醇钠(35mg,0.5mmol),加入2mL乙醇，室温下反应过夜，TLC跟踪。待原料反应完全后用酸性树脂中和至中性，过滤，滤液加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(DCM:MeOH＝20:1)得到产物WYC-309(83mg,90.4％)。1H NMR(400MHz,CDCl3)δ8.78(s,1H),8.26(d,J＝8.1Hz,1H),7.78(d,J＝8.0Hz,1H),7.58(s,1H),7.24(s,1H),7.09(d,J＝8.1Hz,1H),3.10–3.04(m,2H),2.00–1.93(m,2H),1.36(s,6H).13C NMR(101MHz,CDCl3)δ168.58,149.36,142.46,135.91,134.83,133.69,130.42,129.73,126.89,126.73,124.17,117.12,102.51,84.56,37.14,33.13,30.11,30.04,23.45.ESI(+)-MS:3[M+1]+。
实施例51：3-氟-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000083
将化合物WYC-307(332mg,0.9mmol)加入烧瓶中，加入乙醇钠(83mg,1.2mmol),加入7mL乙醇，室温下反应过夜，TLC跟踪。待原料反应完全后用酸性树脂中和至中性，过滤，滤液加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝1:1)得到产物WYC-310(303mg,99％)。1H NMR(400MHz,CDCl3)δ7.85(dd,J＝8.1,1.2Hz,1H),7.80(dd,J＝9.7,1.1Hz,1H),7.61–7.56(m,1H),7.54(d,J＝1.5Hz,1H),7.21(d,J＝8.2Hz,1H),7.07(d,J＝8.2Hz,1H),3.11–2.99(m,2H),1.99–1.90(m,2H),1.34(s,6H).13C NMR(101MHz,CDCl3)δ169.23,163.46,160.95,142.34,134.64,133.45,130.03,129.32,126.79,125.79,117.62,117.27,117.04,110.16,98.73,81.63,37.28,33.15,30.10,23.40.ESI(+)-MS:341.2[M+1]+。
实施例52：3-硝基-4-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000084
将WYC-308(100mg,0.25mmol)加入烧瓶中，加入5mL干燥二氯甲烷，冰水浴下冷却至0度后加入m-CPBA(60mg,0.25mmol)，冰水浴下反应1h后移至室温下反应2h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝3:1)得到产物WYC-311(92mg,88％)。1H NMR(500MHz,CDCl3)δ8.73(d,J＝1.6Hz,1H),8.26(dd,J＝8.1,1.7Hz,1H),7.80(dd,J＝13.0,8.1Hz,2H),7.66(d,J＝1.5Hz,1H),7.57(dd,J＝8.0,1.6Hz,1H),4.44(q,J＝7.1Hz,2H),3.23(ddd,J＝12.8,10.3,2.3Hz,1H),3.12(ddd,J＝13.1,9.1,2.3Hz,1H),2.44(ddd,J＝15.1,10.3,2.3Hz,1H),1.91(ddd,J＝15.1,9.0,2.3Hz,1H),1.48(s,3H),1.43(t,J＝7.1Hz,3H),1.35(s,3H).13C NMR(126MHz,CDCl3)δ164.10,149.65,145.16,139.98,134.95,133.42,131.67,131.30,130.53,130.26,126.01,125.35,122.13,98.90,86.27,62.24,43.39,34.67,31.36,31.24,29.89,14.40.ESI(+)-MS:412.2[M+1]+。
实施例53：3-氟-4-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000085
将化合物1-氧代-6-乙炔基-4,4-二甲基苯并噻喃(223mg,1.2mmol)和3-氟-4-碘苯甲酸乙酯(200mg,0.8mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(32mg,0.0459mmol)、CuI(17.5mg,0.0918mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入3mL干燥的DMF、0.3mL干燥的Et3N，75度下反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝3:2)得到产物外WYC-312(254mg,82.5％)。1H NMR(500MHz,CDCl3)δ7.84(dd,J＝8.0,1.5Hz,1H),7.81–7.75(m,2H),7.63–7.58(m,2H),7.54(dd,J＝8.0,1.6Hz,1H),4.40(q,J＝7.1Hz,2H),3.22(ddd,J＝12.9,10.4,2.3Hz,1H),3.11(ddd,J＝13.1,8.9,2.4Hz,1H),2.46(ddd,J＝15.1,10.4,2.3Hz,1H),1.91(ddd,J＝15.1,8.9,2.3Hz,1H),1.49(s,3H),1.41(t,J＝7.1Hz,4H),1.35(s,4H).13C NMR(126MHz,CDCl33)δ165.04,163.42,161.41,145.01,139.21,133.49,132.58,132.52,132.26,132.18,131.33,130.29,130.27,128.67,128.57,125.87,125.22,125.19,116.78,116.60,116.06,115.93,96.03,96.00,84.16,61.73,43.34,34.62,31.36,31.22,29.81,14.40.ESI(+)-MS:385.2[M+1]+。
实施例54：2,3-二氟-4-碘苯甲酸乙酯
Figure PCTCN2017071922-appb-000086
将2,3-二氟-4-碘苯甲酸(0.5g,1.76mmol)加入烧瓶中，在氩气保护下加入4mL无水乙醇，冰水浴至0度，滴加0.3mL的浓硫酸，滴加完毕后升温回流反应6h，TLC跟踪。待反应完全后冷却至室温，用1mol/L的氢氧化钠中和硫酸至中性，饱和碳酸氢钠氯化钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝10：1)得到产物2,3-二氟-4-碘苯甲酸乙酯(0.48g,87％)。1H NMR(500MHz,CDCl3)δ7.55(ddd,J＝8.4,5.2,1.8Hz,1H),7.44(ddd,J＝8.4,6.3,1.8Hz,1H),4.38(q,J＝7.1Hz,2H),1.38(t,J＝7.1Hz,3H).13C NMR(126MHz,CDCl3)δ163.13,163.10,163.08,152.43,152.31,150.69,150.56,150.46,150.35,148.56,148.44,133.21,133.17,127.48,127.44,121.24,121.18,88.19,88.01,61.98,14.27.ESI(+)-MS:313.1[M+1]+。
实施例55：2,3-二氟-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000087
将化合物2,3-二氟-4-碘苯甲酸乙酯(200mg,0.64mmol)和6-乙炔基-4,4-二甲基苯并噻喃(195mg,0.96mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(20mg,0.0288mmol)、CuI(11mg,0.0576mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.2mL干燥的Et3N，75度下反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝100:1)得到产物WYC-313(215mg,82％)。1H NMR(500MHz,CDCl3)δ7.65(ddd,J＝8.3,6.5,1.8Hz,1H),7.54(d,J＝1.7Hz,1H),7.28(ddd,J＝8.0,4.9,1.9Hz,1H),7.21(dd,J＝8.2,1.8Hz,1H),7.08(d,J＝8.2Hz,1H),4.41(q,J＝7.1Hz,2H),3.07–3.03(m,2H),1.97–1.93(m,2H),1.40(t,J＝7.1Hz,3H),1.34(s,6H).13C NMR(126MHz,CDCl3)δ163.34,152.40,152.29,151.69,151.58,150.38,150.27,149.60,149.49,142.32,134.92,129.98,129.27,127.01,126.98,126.76,126.01,125.97,120.13,120.07,118.59,118.51,117.25,99.26,99.23,80.63,80.60,61.87,37.18,33.09,30.02,23.35,14.33.ESI(+)-MS:387.2[M+1]+。
实施例56：2,3-二氟-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000088
将化合物WYC-313(84mg,0.218mmol)加入烧瓶中，加入乙醇钠(44.4mg,0.653mmol),加入2mL乙醇，室温下反应过夜，TLC跟踪。待原料反应完全后用酸性树脂中和至中性，过滤，滤液加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝1:1)得到产物WYC-314(68mg,87.5％)。1H NMR(500MHz,pyridine)δ8.01(t,J＝7.4Hz,1H),7.82(s,1H),7.45(t,J＝7.2Hz,1H),7.40(d,J＝8.1Hz,1H),7.24(d,J＝8.1Hz,1H),2.95–2.89(m,2H),1.78–1.71(m,2H),1.19(s,6H).ESI(-)-MS:357.2[M-1]-。
实施例57：2,3-二氟-4-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000089
将WYC-313(90mg,0.233mmol)加入烧瓶中，加入5mL干燥二氯甲烷，冰水浴下冷却至0度后加入m-CPBA(58mg,0.233mmol)，冰水浴下反应1h后移至室温下反应2h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝3:1)得到产物WYC-315(76mg,78％)。1H NMR(500MHz,CDCl3)δ7.76(d,J＝8.0Hz,1H),7.71–7.65(m,1H),7.62(s,1H),7.53(dd,J＝8.0,0.8Hz,1H),7.32(dd,J＝10.4,3.9Hz,1H),4.41(q,J＝7.1Hz,2H),3.26–3.18(m,1H),3.15–3.07(m,1H),2.44(ddd,J＝14.8,10.3,2.0Hz,1H),1.90(ddd,J＝15.1,8.9,2.0Hz,1H),1.47(s,3H),1.40(t,J＝7.1Hz,3H),1.34(s,3H).13C NMR(126MHz,CDCl3)δ163.22,163.20,163.17,152.69,152.58,151.65,151.54,150.66,150.55,149.55,145.10,139.57,131.40,130.30,130.27,127.25,127.22,126.15,126.12,125.41,121.05,120.99,117.60,117.59,117.50,117.48,97.09,97.05,82.93,82.90,62.02,43.34,34.63,31.34,31.21,29.82,14.32.ESI(+)-MS:403.1[M+1]+。
实施例58：3-乙酰氨基-4-碘苯甲酸甲酯
Figure PCTCN2017071922-appb-000090
将化合物3-氨基-4-碘苯甲酸甲酯(1.5g,5.43mmol)加入烧瓶中，加入20mL干燥的二氯甲烷，4.5mL干燥的三乙胺，冰水浴下滴加0.77mL的乙酰氯，室温下反应过夜，TLC跟踪。待反应结束后用甲醇淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝10:1)得到产物3-乙酰氨基-4-碘苯甲酸甲酯(1.34g,77.5％)。1H NMR(500MHz,CDCl3)δ8.75(s,1H),7.86(d,J＝8.3Hz,1H),7.54–7.39(m,2H),3.90(s,3H),2.25(s,3H).13C NMR(126MHz,CDCl3)δ168.29,166.28,138.97,138.54,131.46,126.74,122.89,96.10,52.44,24.78.ESI(+)-MS:320.1[M+1]+。
实施例59：3-乙酰氨基-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸甲酯
Figure PCTCN2017071922-appb-000091
将化合物3-乙酰氨基-4-碘苯甲酸甲酯(319mg,1mmol)和6-乙炔基-4,4-二甲基苯并噻喃(304mg,1.5mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(47mg,0.0675mmol)、CuI(26mg,0.135mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入5mL干燥的DMF、0.3mL干燥的Et3N，70度下反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝10:1)得到产物WYC-316(298mg,76％)。1H NMR(500MHz,CDCl3)δ9.00(s,1H),7.97(s,1H),7.74(dd,J＝8.1,1.2Hz,1H),7.57–7.50(m,2H),7.18(dd,J＝8.1,1.7Hz,1H),7.10(d,J＝8.1Hz,1H),3.91(s,3H),3.10–3.02(m,2H),2.26(s,3H),1.98–1.93(m,2H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ168.23,166.55,142.53,138.77,135.02,131.51,130.80,129.71,128.90,126.92,124.65,120.35,117.03,110.11,99.62,83.25,52.42,37.09,33.11,30.02,25.03,23.36.ESI(+)-MS:394.2[M+1]+。
实施例60：3-乙酰氨基-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000092
将化合物WYC-316(56mg,0.142mmol)加入烧瓶中，加入乙醇钠(29mg,0.426mmol),加入2mL乙醇，室温下反应过夜，TLC跟踪。待原料反应完全后用酸性树脂中和至中性，过滤，滤液加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝20:1)得到产物WYC-317(48mg,83％)。1H NMR(500MHz,CDCl3)δ9.00(s,1H),7.97(s,1H),7.75(d,J＝8.0Hz,1H),7.53(d,J＝7.2Hz,2H),7.18(dd,J＝8.1,1.3Hz,1H),7.10(d,J＝8.1Hz,1H),4.38(q,J＝7.1Hz,2H),3.09–3.02(m,2H),2.26(s,3H),1.98–1.94(m,2H),1.39(t,J＝7.1Hz,3H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ168.22,166.08,142.53,138.76,135.00,131.47,131.21,129.72,128.91,126.93,124.64,120.38,117.07,110.12,99.54,83.31,61.39,37.11,33.12,30.04,25.03,23.36,14.44.ESI(+)-MS:408.2[M+1]+。
实施例61：3-乙酰氨基-4-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000093
将WYC-317(86mg,0.211mmol)加入烧瓶中，加入2mL干燥二氯甲烷，冰水浴下 冷却至0度后加入m-CPBA(37mg,0.211mmol)，冰水浴下反应1h后移至室温下反应2h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝1:1)得到产物WYC-318(80mg,98.6％)。1H NMR(500MHz,CDCl33)δ8.98(s,1H),7.93(s,1H),7.78(d,J＝8.0Hz,2H),7.61(d,J＝1.3Hz,1H),7.57(d,J＝8.1Hz,1H),7.50(dd,J＝8.0,1.3Hz,1H),4.38(q,J＝7.1Hz,2H),3.24(dd,J＝17.2,6.3Hz,1H),3.17–3.09(m,1H),2.45(ddd,J＝14.8,10.3,1.9Hz,1H),2.27(s,3H),1.91(ddd,J＝15.1,8.9,2.0Hz,1H),1.48(s,3H),1.39(t,J＝7.1Hz,3H),1.35(s,3H).13C NMR(126MHz,CDCl3)δ168.29,165.92,145.31,139.07,131.97,131.91,131.19,130.44,129.98,125.33,124.80,124.76,120.86,115.83,97.48,85.94,61.51,43.40,34.66,31.36,31.27,29.79,14.43.ESI(+)-MS:424.1[M+1]+。
实施例62：3-乙酰氨基-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000094
将化合物WYC-316(63mg,0.16mmol)加入烧瓶中，加入乙醇钠(14mg,0.2mmol),加入2mL四氢呋喃，0.2mL的水，室温下反应过夜，TLC跟踪。待原料反应完全后用酸性树脂中和至中性，过滤，滤液加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝1：4)得到产物WYC-319(34mg,56％)。ESI(+)-MS:380.2[M+1]+。
实施例63：5-溴哒嗪-2-甲酸乙酯
Figure PCTCN2017071922-appb-000095
将5-溴哒嗪-2-甲酸(1.0g,5mmol)加入烧瓶中，在氩气保护下加入5mL无水乙醇，冰水浴至0度，滴加0.3mL的浓硫酸，滴加完毕后升温回流反应6h，TLC跟踪。待反应完全后冷却至室温，用1mol/L的氢氧化钠中和硫酸至中性，饱和碳酸氢钠氯化钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝50:1)得到产物5-溴哒嗪-2-甲酸乙酯(0.42g,37％)。1H NMR(500MHz,CDCl3)δ9.01(d,J＝1.3Hz,1H),8.76(d,J＝1.3Hz,1H),4.47(q,J＝7.1Hz,2H),1.41(t,J＝7.1Hz,3H).13C NMR(126MHz,CDCl3)δ163.39,147.30,146.31,144.65,141.89,62.67,14.30.ESI(+)-MS:231.2[M+1]+。
实施例64：2-((4,4-二甲基-1,1-二氧代二氢苯并噻喃-6-)乙炔基)嘧啶-5-甲酸乙酯
Figure PCTCN2017071922-appb-000096
将WYC-209(1.1g,3mmol)加入烧瓶中，加入20mL干燥二氯甲烷，冰水浴下冷却至0度后加入mCPBA(0.663g,3.6mmol)，冰水浴下反应10min后移至室温反应3h后移至室温下反应4h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝1:5)得到产物WYC-320(0.99g,89％)。1H NMR(500MHz,cdcl3)δ9.29(s,2H),7.94(d,J＝8.2Hz,1H),7.76(d,J＝1.3Hz,1H),7.68(dd,J＝8.2,1.5Hz,1H),4.47(q,J＝7.1Hz,2H),3.46–3.37(m,2H),2.45–2.39(m,2H),1.44(s,6H).13C NMR(126MHz,cdcl3)δ163.22,158.51,155.10,145.17,138.59,132.27,131.31,125.41,124.24,122.74,89.85,88.71,62.32,47.06,35.49,34.47,30.71,14.35.ESI(+)-MS:385.4[M+1]+。
实施例65：3-乙酰氨基-4-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)苯甲酸甲酯
Figure PCTCN2017071922-appb-000097
将WYC-316(107mg,0.272mmol)加入烧瓶中，加入3.5mL干燥二氯甲烷，冰水浴下冷却至0度后加入m-CPBA(67.3mg,0.272mmol)，冰水浴下反应1h后移至室温下反应2h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝1:1)得到产物WYC-321(84mg,78％)。1H NMR(500MHz,CDCl3)δ8.97(s,1H),7.96(s,1H),7.80–7.72(m,2H),7.60(d,J＝1.4Hz,1H),7.56(d,J＝8.1Hz,1H),7.49(dd,J＝8.0,1.3Hz,1H),3.90(s,3H),3.14(ddt,J＝12.9,8.9,6.4Hz,2H),2.45(ddd,J＝14.8,10.3,1.7Hz,1H),2.26(s,3H),1.90(ddd,J＝15.0,8.9,1.9Hz,1H),1.47(s,3H),1.34(s,3H).13C NMR(126MHz,CDCl3)δ168.28,166.37,145.28,139.65,139.09,131.94,131.52,131.18,130.41,129.95,125.29,124.74,120.91,115.97,97.52,85.89,52.49,43.37,34.63,31.32,31.25,29.76,24.99.ESI(+)-MS:410.3[M+1]+。
实施例66：5-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)吡嗪-2-甲酸乙酯
Figure PCTCN2017071922-appb-000098
将化合物5-溴哒嗪-2-甲酸乙酯(600mg,2.61mmol)和6-乙炔基-4,4-二甲基苯并噻喃(635mg,3.13mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(66mg,0.094mmol)、CuI(36mg,0.188mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入6mL干燥的DMF、0.4mL干燥的Et3N，80度下反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝50:1)得到产物WYC-322(656mg,71.5％)。1H NMR(500MHz,CDCl3)δ9.20(d,J＝1.4Hz,1H),8.76(d,J＝1.4Hz,1H),7.58(d,J＝1.7Hz,1H),7.23(dd,J＝8.2,1.8Hz,1H),7.04(d,J＝8.2Hz,1H),4.46(q,J＝7.1Hz,2H),3.02–2.97(m,2H),1.92–1.87(m,2H),1.41(t,J＝7.1Hz,3H),1.29(s,6H).13C NMR(126MHz,CDCl3)δ163.65,146.68,145.70,143.08,142.31,140.30,136.14,130.54,129.51,126.74,116.00,97.55,85.68,62.37,36.91,32.99,29.89,23.27,14.31.ESI(+)-MS:353.5[M+1]+。
实施例67：5-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)吡嗪-2-甲酸
Figure PCTCN2017071922-appb-000099
将化合物WYC-322(50mg,0.14mmol)加入烧瓶中，加入乙醇钠(29mg,0.426mmol),加入2mL乙醇，室温下反应过夜，TLC跟踪。待原料反应完全后用酸性树脂中和至中性，过滤，滤液加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(DCM：MeOH＝15:1)得到产物WYC-323(43mg,95％)。1H NMR(500MHz,pyridine)δ9.65(s,1H),9.05(s,1H),7.84(d,J＝1.4Hz,1H),7.41(dd,J＝8.1,1.4Hz,1H),7.23(d,J＝8.1Hz,1H),2.98–2.88(m,2H),1.81–1.72(m,2H),1.19(s,6H).13C NMR(126MHz,pyridine)δ166.73,147.26,146.43,143.36,142.96,142.56,136.52,130.88,129.89,127.24,116.79,96.42,86.89,36.94,33.05,29.59,23.30.ESI(+)-MS:325.2[M+1]+。
实施例68：5-((4,4-二甲基-1-氧代二氢苯并噻喃-6-)乙炔基)吡嗪-2-甲酸乙酯
Figure PCTCN2017071922-appb-000100
将WYC-322(100mg,0.284mmol)加入烧瓶中，加入3.5mL干燥二氯甲烷，冰水浴下冷却至0度后加入m-CPBA(49mg,0.284mmol)，冰水浴下反应1h后移至室温下反应2h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝1:2)得到产物WYC-324(78mg,75％)。1H NMR(500MHz,CDCl33)δ9.29(d,J＝1.4Hz,1H),8.87(d,J＝1.4Hz,1H),7.80(d,J＝8.1Hz,1H),7.71(d,J＝1.5Hz,1H),7.61(dd,J＝8.1,1.6Hz,1H),4.52(q,J＝7.1Hz,2H),3.29–3.06(m,2H),2.43(ddd,J＝15.1,10.2,2.3Hz,1H),1.91(ddd,J＝15.1,9.2,2.3Hz,1H),1.47(dd,J＝8.8,5.5Hz,6H),1.34(s,3H).13C NMR(126MHz,CDCl3)δ163.62,147.07,145.93,145.25,142.41,141.29,140.60,132.05,130.63,130.25,124.32,94.92,87.36,62.70,43.44,34.73,31.35,31.25,29.91,14.41.ESI(+)-MS:396.2[M+1]+。
实施例69：2-氯-4-碘苯甲酸乙酯
Figure PCTCN2017071922-appb-000101
将2-氯-4-碘苯甲酸(1.0g,3.55mmol)加入烧瓶中，在氩气保护下加入7mL无水乙醇，冰水浴至0度，滴加0.4mL的浓硫酸，滴加完毕后升温回流反应5h，TLC跟踪。待反应完全后冷却至室温，用1mol/L的氢氧化钠中和硫酸至中性，饱和碳酸氢钠氯化钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝7:1)得到产物2-氯-4-碘苯甲酸乙酯(0.98g,89％)。1H NMR(500MHz,CDCl3)δ7.82(d,J＝1.6Hz,1H),7.65(dd,J＝8.2,1.6Hz,1H),7.52(d,J＝8.2Hz,1H),4.38(q,J＝7.1Hz,2H),1.39(t,J＝7.2Hz,3H).13C NMR(126MHz,CDCl3)δ165.22,139.57,135.95,134.63,132.47,129.91,98.35,61.87,14.32.ESI(+)-MS:311.3[M+1]+
实施例70：2-氯-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000102
将化合物2-氯-4-碘苯甲酸乙酯(310mg,1mmol)和6-乙炔基-4,4-二甲基苯并噻喃(244mg,1.2mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(28mg,0.04mmol)、CuI(11.4mg,0.06mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入3mL干燥的DMF、0.3mL干燥的Et3N，80度下反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝50:1)得到产物WYC-325(365mg,95％)。1H NMR(500MHz,CDCl3)δ7.81(d,J＝8.1Hz,1H),7.59(d,J＝1.5Hz,1H),7.52(d,J＝1.7Hz,1H),7.42(dd,J＝8.1,1.5Hz,1H),7.18(dd,J＝8.2,1.8Hz,1H),7.07(d,J＝8.1Hz,1H),4.40(q,J＝7.1Hz,2H),3.06–3.02(m,2H),1.97–1.93(m,2H),1.40(t,J＝7.1Hz,3H),1.34(s,6H).13C NMR(126MHz,CDCl3)δ165.27,142.26,134.37,133.89,133.65,131.41,129.92,129.38,129.30,129.14,128.27,126.72,117.56,93.73,86.76,61.71,37.21,33.06,30.04,23.32,14.33.ESI(+)-MS:385.2[M+1]+。
实施例71：2-氯-4-((4,4-二甲基二氢-1-氧代苯并噻喃-6-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000103
将WYC-325(100mg,0.26mmol)加入烧瓶中，加入3mL干燥二氯甲烷，冰水浴下冷却至0度后加入m-CPBA(60mg,0.26mmol)，冰水浴下反应1h后移至室温下反应2h，TLC跟踪。待反应结束后用硫代硫酸钠溶液淬灭，乙酸乙酯稀释，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝1:3)得到产物WYC-326(86mg,83％)。1H NMR(500MHz,CDCl3)δ7.79(d,J＝8.1Hz,1H),7.71(d,J＝8.1Hz,1H),7.59(d,J＝1.5Hz,1H),7.57(d,J＝1.5Hz,1H),7.46(dd,J＝8.0,1.5Hz,1H),7.42(dd,J＝8.1,1.6Hz,1H),4.36(q,J＝7.1Hz,2H),3.17(ddd,J＝12.7,10.5,2.1Hz,1H),3.06(ddd,J＝13.1,8.8,2.2Hz,1H),2.41(ddd,J＝14.9,10.4,2.1Hz,1H),1.85(ddd,J＝15.1,8.8,2.2Hz,1H),1.43(s,3H),1.36(t,J＝7.1Hz,3H),1.30(s,3H).13C NMR(126MHz,CDCl3)δ165.05,144.93,138.96,133.81,133.81,131.36,131.23,130.19,130.08,130.05,129.58,127.12,125.66,91.67,89.03,61.74,43.16,34.45,31.20,31.06,29.59,14.22.ESI(+)-MS:401.2[M+1]+。
实施例72：2-氯-4-((4,4-二甲基二氢苯并噻喃-6-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000104
将化合物WYC-325(100mg,0.26mmol)加入烧瓶中，加入乙醇钠(29mg,0.426mmol),加入2mL乙醇，室温下反应过夜，TLC跟踪。待原料反应完全后用酸性树脂中和至中性，过滤，滤液加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE：EA＝1:2)得到产物WYC-327(89mg,94％)。1H NMR(500MHz,CDCl3)δ7.98(d,J＝8.1Hz,1H),7.61(s,1H),7.52(d,J＝1.6Hz,1H),7.44(d,J＝7.9Hz,1H),7.18(dd,J＝8.2,1.6Hz,1H),7.07(d,J＝8.1Hz,1H),3.07–3.04(m,2H),1.98–1.94(m,2H),1.35(s,6H).13C NMR(126MHz,CDCl3)δ170.29,142.32,134.90,134.53,134.00,132.52,130.02,129.51,129.36,129.25,127.76,126.79,117.54,94.44,86.79,37.25,33.12,30.09,23.39.ESI(+)-MS:355.2[M+1]+。
实施例73：2-氰基-5-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)嘧啶
Figure PCTCN2017071922-appb-000105
将化合物6-乙炔基-1,1,4,4-四甲基-1,2,3,4-四氢萘(50mg,0.235mmol)和2-氰基-5-溴嘧啶(49.9mg,0.271mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(14mg,0.02mmol)、CuI(5.6mg,0.03mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.14mL干燥的Et3N，加热到70℃反应8h,TLC跟踪。反应结束后冷却至室温，用饱和碳酸氢铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝10:0to 10:1)得到产物WYC-329(75mg,77％)。1H NMR(400MHz,CDCl3)δ8.90(s,2H),7.52(d,J＝1.4Hz,1H),7.38–7.29(m,2H),1.70(s,4H),1.31(s,6H),1.29(s,6H).13C NMR(101MHz,CDCl3)δ159.24,147.92,145.68,141.75,130.54,129.00,127.11,122.82,117.86,115.58,101.75,80.64,77.35,77.04,76.72,34.78,34.73,34.59,34.31,31.77,31.64.ESI(+)-MS:338.4[M+1]+.ESI(+)-MS:3[M+1]+
实施例74：2-氨基-4-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000106
将6-乙炔基-1,1,4,4-四甲基-1,2,3,4-四氢萘(100mg,0.47mmol)和2-氨基-4-碘苯甲酸(141.8mg,0.54mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(28mg,0.04mmol)、CuI(11.2mg,0.059mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.28mL干燥的Et3N，加热到50度反应8h,TLC跟踪。反应结束后冷却至室温，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝50:1到10:1)得到产物WYC-330(97mg,60％)。1H NMR(400MHz,MeOD)δ7.81(s,1H),7.46(s,1H),7.33(d,J＝8.2Hz,1H),7.24(d,J＝8.2Hz,1H),6.88(s,1H),6.68(s,1H),1.72(s,4H),1.30(s,6H),1.29(s,6H).ESI(+)-MS:348.3[M+1]+.
实施例75：2-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)嘧啶-5-甲酸乙酯
Figure PCTCN2017071922-appb-000107
将6-乙炔基-1,1,4,4-四甲基-1,2,3,4-四氢萘(50mg,0.235mmol)和2-氯嘧啶-4-甲酸乙酯(50.3mg,0.27mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(14mg,0.02mmol)、CuI(5.6mg,0.03mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入1mL干燥的DMF、0.14mL干燥的Et3N，加热到70℃反应8h,TLC跟踪。反应结束后冷却至室温，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝100:1到50:1)得到产物WYC-331(67.2mg,79％)。1H NMR(500MHz,cdcl3)δ9.26(s,2H),7.67(d,J＝1.7Hz,1H),7.45(dd,J＝8.2,1.8Hz,1H),7.33(d,J＝8.2Hz,1H),4.46(q,J＝7.1Hz,2H),1.69(s,4H),1.44(t,J＝7.1Hz,3H),1.29(s,6H),1.28(s,6H).13C NMR(101MHz,CDCl3)δ163.42,158.33,155.80,148.01,145.46,131.77,129.86,126.94,121.83,117.66,92.54,87.31,62.02,34.81,34.77,34.58,34.31,31.72,31.60.ESI(+)-MS:363.3[M+1]+。
实施例76：3-羟基-4-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)苯甲酸甲酯
Figure PCTCN2017071922-appb-000108
将6-乙炔基-1,1,4,4-四甲基-1,2,3,4-四氢萘(120mg,0.566mmol)和3-羟基-4-碘苯甲酸甲酯(180.7mg,0.65mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(33.7mg,0.048mmol)、CuI(13.5mg,0.072mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入3mL干燥的DMF、0.338mL干燥的Et3N，70度下反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝50:1到10:1)得到产物WYC-332(118mg,58％)。1H NMR(400MHz,CDCl3)δ8.23(s,1H),7.95(dd,J＝8.2,1.4Hz,1H),7.85(d,J＝1.9Hz,1H),7.63(dd,J＝8.3,1.9Hz,1H),7.58(d,J＝8.1Hz,1H),7.41(d,J＝8.3Hz,1H),7.01(d,J＝0.8Hz,1H),3.96(s,3H),1.74(s,4H),1.38(s,6H),1.33(d,J＝4.9Hz,6H).13C NMR(126MHz,cdcl3)δ167.34,159.55,154.18,146.62,145.60,133.81,127.20,127.09,125.67,124.35,123.49,122.72,120.13,112.70,100.54,77.25,76.99,76.74,52.10,35.01,34.90,34.43,34.42,31.84,31.72.ESI(+)-MS:363.2[M+1]+.
实施例77：2-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)-5-氰基吡啶
Figure PCTCN2017071922-appb-000109
将6-乙炔基-1,1,4,4-四甲基-1,2,3,4-四氢萘(60mg,0.283mmol)和2-氯-5-氰基吡啶(59.5mg,0.325mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(16.8mg,0.024mmol)、CuI(6.8mg,0.036mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.17mL干燥的Et3N，70℃反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝100:1到20:1)得到产物WYC-333(59mg,67％)。1H NMR(500MHz,cdcl3)δ8.86(dd,J＝2.1,0.8Hz,1H),7.93(dd,J＝8.2,2.2Hz,1H),7.60(dd,J＝8.2,0.8Hz,1H),7.58(d,J＝1.7Hz,1H),7.36(dd,J＝8.2,1.7Hz,1H),7.32(d,J＝8.2Hz,1H),1.69(s,4H),1.29(s,6H),1.29(s,6H).13C NMR(126MHz,cdcl3)δ152.56,147.56,147.09,145.45,139.13,130.99,129.26,126.92,126.59,118.07,116.52,107.95,95.22,86.91,77.25,76.99,76.74,34.81,34.76,34.52,34.28,31.73,31.61.ESI(+)-MS:315.6[M+1]+。
实施例78：5-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)嘧啶-2-甲酸乙酯
Figure PCTCN2017071922-appb-000110
将化合物WYC-329(15mg，0.0475mmol)溶于2mL乙醇中，加入乙醇钠(9.7mg，0.143mmol)，室温搅拌过夜，反应结束后用1mol/L的HCl中和至弱酸性；加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝1000:1到20:1)得到产物WYC-334(15mg,88％)。1H NMR(400MHz,CDCl3)δ8.63(s,2H),7.47(d,J＝1.2Hz,1H),7.33–7.24(m,2H),4.45(q,J＝7.1Hz,2H),1.69(s,4H),1.45(t,J＝7.1Hz,3H),1.30(s,6H),1.28(s,6H).13C NMR(101MHz,CDCl3)δ163.53,161.19,146.22,145.28,129.93,128.61,126.82,119.29,113.07,94.57,81.42,77.35,77.04,76.72,63.94,34.91,34.85,34.41,34.25,31.77,31.68,14.42.ESI(+)-MS:362.3[M+1]+。
实施例79：2-羟基-4-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)苯甲酸甲酯
Figure PCTCN2017071922-appb-000111
将6-乙炔基-1,1,4,4-四甲基-1,2,3,4-四氢萘(50mg,0.205mmol)和化合物c(75.3mg,0.271mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(14mg,0.02mmol)、CuI(5.6mg,0.03mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.14mL干燥的Et3N，加热到70度反应8h,TLC跟踪。反应结束后冷却至室温，用饱和碳酸氢铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE)得到产物(45.2mg,61％)。1H NMR(400MHz,CDCl3)δ10.76(s,1H),7.80(d,J＝8.2Hz,1H),7.48(s,1H),7.29(d,J＝0.8Hz,2H),7.13(d,J＝1.4Hz,1H),7.03(dd,J＝8.2,1.5Hz,1H),3.96(s,3H),1.69(s,4H),1.30(s,6H),1.28(s,6H).ESI(+)-MS:363.3[M+1]+.
实施例80：2-羟基-4-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000112
将前步产物(30mg,0.082mmol)加入烧瓶中，加入2mL 2.0mol/L的NaOH溶液中， 加入2mL甲醇；55℃反应过夜。待原料反应完全后用1mol/L的HCl中和至PH为5～6；加入乙酸乙酯稀释、萃取，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝100:0to 10:1)得到产物WYC-335(26.3mg,93％)。1H NMR(400MHz,DMSO)δ7.80(d,J＝8.1Hz,1H),7.53(d,J＝1.6Hz,1H),7.39(d,J＝8.2Hz,1H),7.31(dd,J＝8.2,1.6Hz,1H),7.12–7.04(m,2H),1.65(s,4H),1.26(s,6H),1.25(s,6H).13C NMR(101MHz,DMSO)δ171.27,160.85,146.07,145.05,130.58,129.74,129.16,128.67,126.98,122.01,119.29,118.76,92.56,87.48,40.12,39.91,39.70,39.49,39.29,39.08,38.87,34.36,34.25,34.08,33.89,31.39,31.32.ESI(-)-MS:347.3[M-1]-。
实施例81：2-乙酰氨基-4-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)苯甲酸
Figure PCTCN2017071922-appb-000113
将WYC-330(52mg,0.15mmol)加入烧瓶中，加入1mg的DMAP，在氩气保护下加入3mL干燥的吡啶，冰水浴下冷却至0度，滴加7.8μl的乙酰氯，冰水浴下反应5min后移至室温反应5h,TLC跟踪。待反应结束后用甲醇淬灭，乙酸乙酯稀释，1mol/L的盐酸洗去吡啶，饱和碳酸氢钠洗，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝20:1)得到产物WYC-336(39.5mg,85％)。1H NMR(400MHz,CDCl3)δ8.13(d,J＝8.1Hz,1H),7.66(d,J＝1.2Hz,1H),7.60(dd,J＝8.1,1.5Hz,1H),7.51(s,1H),7.32(d,J＝1.0Hz,2H),2.47(s,3H),1.69(d,J＝5.7Hz,4H),1.31(s,6H),1.29(s,6H).ESI(-)-MS:388.4[M-1]-。
实施例82：5-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)吡啶-2-甲酸甲酯
Figure PCTCN2017071922-appb-000114
将6-乙炔基-1,1,4,4-四甲基-1,2,3,4-四氢萘(60mg,0.283mmol)和5-溴-吡啶-2-甲酸甲酯(70mg,0.325mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(16.8mg,0.024mmol)、CuI(6.8mg,0.036mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.17mL干燥的Et3N，70℃反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝100:1到20:1)得到产物(68.7mg,70％)。1H NMR(400 MHz,CDCl3)δ8.85(dd,J＝2.0,0.7Hz,1H),8.12(dd,J＝8.1,0.7Hz,1H),7.94(dd,J＝8.1,2.1Hz,1H),7.51(s,1H),7.32(d,J＝1.0Hz,2H),4.02(d,J＝4.5Hz,3H),1.69(d,J＝5.5Hz,4H),1.31(s,6H),1.29(s,6H).ESI(+)-MS:348.2[M+1]+。
实施例83：5-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)吡啶-2-甲酸
Figure PCTCN2017071922-appb-000115
将上步产物(50mg,0.143mmol)加入烧瓶中，加入2mL 2.0mol/L的NaOH溶液中，加入2mL甲醇；55℃下反应过夜。待原料反应完全后用1mol/L的HCl中和至PH为5～6；加入乙酸乙酯稀释、萃取，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝20:1到5:1)得到产物WYC-337(42.2mg,88％)。1H NMR(400MHz,MeOD)δ8.67(s,1H),8.00(d,J＝7.9Hz,1H),7.94(dd,J＝8.1,2.0Hz,1H),7.52(d,J＝1.6Hz,1H),7.36(d,J＝8.2Hz,1H),7.30(dd,J＝8.2,1.7Hz,1H),1.73(s,4H),1.31(s,6H),1.29(s,6H).ESI(+)-MS:334.4[M+1]+。
实施例84：5-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)嘧啶-2-甲酸乙酯甲酯
Figure PCTCN2017071922-appb-000116
将化合物WYC-329(15mg，0.0475mmol)溶于2mL甲醇中，加入甲醇钠(7.7mg，0.143mmol)，室温搅拌过夜，反应结束后用1mol/L的HCl中和至弱酸性；加入乙酸乙酯稀释，饱和碳酸氢钠洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝100:1到20:1)得到产物WYC-338(14.9mg,90％)。1H NMR(500MHz,cdcl3)δ8.64(s,2H),7.47(d,J＝1.2Hz,1H),7.34–7.27(m,2H),4.05(s,3H),1.69(s,4H),1.30(s,6H),1.28(s,6H).ESI(+)-MS:348.4[M+1]+。
实施例85：2-羟基-4-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000117
将WYC-335(15mg,0.043mmol)加入烧瓶中，在氩气保护下加入0.8mL无水乙醇，冰水浴至0℃，滴加3μl的浓硫酸，滴加完毕后升温回流反应4h，TLC跟踪。待反应完 全后冷却至室温，用1mol/L的氢氧化钠中和硫酸至中性，饱和碳酸氢钠溶液和饱和氯化钠溶液洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝100:1到10:1)得到产物WYC-339(14mg,87％)。ESI(+)-MS:377.3[M+1]+.
实施例86：3-羟基-4-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)苯甲酸乙酯
Figure PCTCN2017071922-appb-000118
将化合物WYC-332(50mg,0.143mmol)加入烧瓶中，加入2mL 2.0mol/L的NaOH溶液中，加入2mL甲醇；55度下反应过夜。待原料反应完全后用1mol/L的HCl中和至PH为5～6；加入乙酸乙酯稀释、萃取，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝20:1到5:1)得到羧酸产物(47mg,87％)。将该产物(25mg,0.072mmol)加入烧瓶中，在氩气保护下加入1.5mL无水乙醇，冰水浴至0度，滴加5μL的浓硫酸，滴加完毕后升温回流反应4h，TLC跟踪。待反应完全后冷却至室温，用1mol/L的氢氧化钠中和硫酸至中性，饱和碳酸氢钠溶液和饱和氯化钠溶液洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝50:1到10:1)得到产物WYC-340(24mg,90％)。1H NMR(400MHz,CDCl3)δ8.24(s,1H),7.95(dd,J＝8.2,1.4Hz,1H),7.84(d,J＝1.9Hz,1H),7.63(dd,J＝8.3,1.9Hz,1H),7.58(d,J＝8.2Hz,1H),7.41(d,J＝8.3Hz,1H),7.01(d,J＝0.8Hz,1H),4.42(q,J＝7.1Hz,2H),1.73(s,4H),1.43(t,J＝7.1Hz,3H),1.37(s,6H),1.32(s,6H)；13C NMR(101MHz,CDCl3)δ166.91,159.50,154.23,146.62,145.62,133.74,127.23,127.15,126.07,124.37,123.49,122.73,120.12,112.68,100.57,60.95,35.03,34.92,34.44,31.86,31.74；ESI(+)-MS:377.4[M+1]+。
实施例87：5-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)吡啶-2-甲酸乙酯
Figure PCTCN2017071922-appb-000119
将WYC-337(25mg,0.075mmol)加入烧瓶中，在氩气保护下加入1.5mL无水乙醇，冰水浴至0度，滴加5μl的浓硫酸，滴加完毕后升温回流反应4h，TLC跟踪。待反应完全后冷却至室温，用1mol/L的氢氧化钠中和硫酸至中性，饱和碳酸氢钠溶液和饱和氯化钠溶液洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝50:1到10:1)得到产物WYC-341(24mg,90％)。1H NMR(400MHz,CDCl3)δ8.85(dd,J＝2.0,0.6 Hz,1H),8.11(dd,J＝8.1,0.7Hz,1H),7.92(dd,J＝8.1,2.1Hz,1H),7.50(t,J＝1.2,1H),7.34(d,J＝2.5Hz,1H),7.26(dd,J＝8.4,2.1Hz,3H),4.49(q,J＝7.2Hz,2H),1.69(s,4H),1.45(t,J＝6.8Hz,3H),1.30(s,6H),1.28(s,6H).ESI(+)-MS:362.7[M+1]+。
实施例88：5-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)哒嗪-2-羧酸甲酯
Figure PCTCN2017071922-appb-000120
将6-乙炔基-1,1,4,4-四甲基-1,2,3,4-四氢萘(60mg,0.283mmol)和2-氯哒嗪-4-甲酸甲酯(56mg,0.325mmol)加入烧瓶中，加入Pd(PPh3)2Cl2(16.8mg,0.024mmol)、CuI(6.8mg,0.036mmol),用氩气保护并置换3次气体以排除氧气，用注射器加入2mL干燥的DMF、0.17mL干燥的Et3N，70℃反应8h，TLC跟踪。反应结束后，用饱和氯化铵溶液淬灭，加入乙酸乙酯稀释，饱和氯化铵洗涤，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝100:1到20:1)得到产物(55mg,56％)。1H NMR(400MHz,CDCl3)δ9.28(d,J＝1.4Hz,1H),8.83(d,J＝1.4Hz,1H),7.61(d,J＝1.7Hz,1H),7.40(dd,J＝8.2,1.7Hz,1H),7.34(d,J＝8.2Hz,1H),4.06(s,3H),1.70(s,4H),1.30(s,6H),1.29(s,6H).13C NMR(101MHz,CDCl3)δ164.18,147.91,146.83,145.74,145.57,143.39,140.07,131.12,129.38,127.01,117.83,97.82,85.11,77.34,77.23,77.02,76.70,53.19,34.80,34.76,34.58,34.31,31.75,31.62.ESI(+)-MS:349.2[M+1]+.
实施例89：5-((5,5,8,8-四甲基-5,6,7,8-四氢萘基-2-)乙炔基)吡嗪-2-甲酸乙酯
Figure PCTCN2017071922-appb-000121
将上步产物(35mg,0.1mmol)加入烧瓶中，加入1.5mL 2.0mol/L的NaOH溶液中，加入1.5mL甲醇；55℃下反应过夜。待原料反应完全后用1mol/L的HCl中和至PH为5～6；加入乙酸乙酯稀释、萃取，饱和氯化钠洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EA＝20:1到5:1)得到羧酸产物(28.3mg,85％)。将该产物(25mg,0.075mmol)加入烧瓶中，在氩气保护下加入1.5mL无水乙醇，冰水浴至0度，滴加5μl的浓硫酸，滴加完毕后升温回流反应4h，TLC跟踪。待反应完全后冷却至室温，用1mol/L的氢氧化钠中和硫酸至中性，饱和碳酸氢钠溶液和饱和氯化钠溶液洗涤，有机相用无水硫酸钠干燥，过滤、旋干，快速柱层析(PE:EtOAc＝50:1到10:1)得到产物WYC-342(20mg,75％)。1H NMR(400MHz,CDCl3)δ9.26(d,J＝1.4Hz,1H),8.82(d,J＝1.4Hz,1H), 7.60(d,J＝1.6Hz,1H),7.38(dd,J＝8.2,1.7Hz,1H),7.32(d,J＝8.2Hz,1H),4.51(q,J＝7.1Hz,2H),1.68(s,4H),1.45(t,J＝7.1Hz,3H),1.29(s,6H),1.27(s,6H).13C NMR(101MHz,CDCl3)δ163.72,147.84,146.83,145.72,145.53,143.21,140.41,131.09,129.37,127.00,117.87,97.61,85.15,77.38,77.07,76.75,62.42,34.81,34.76,34.56,34.30,31.75,31.62.ESI(+)-MS:363.6[M+1]+。
效果实施例1、黑色素瘤再生细胞(TRCs)体外生长抑制活性研究
采用3D纤维蛋白胶培养基(90-Pa)孵育黑色素瘤B16-F1细胞5天，筛选得到所需黑色素瘤肿瘤再生细胞(Nat.Mater.2012,11,734)。随后采用胶原酶和中性蛋白酶II对培养基分别进行处理，游离出黑色素瘤肿瘤再生细胞，将所得再生细胞移取至新制备培养基中重悬，并保持单细胞状态。将黑色素瘤B16-F1再生细胞接种于3D纤维蛋白胶培养基(90-Pa)孵育5天，以0.1％DMSO为阴性对照组，以他扎罗汀(Tazarotene)、贝沙罗汀(Bexarotene)分别为阳性对照药物，按照10μM浓度加入药物，孵育5天，观察测量计算肿瘤克隆体大小，并计算每种药物对于黑色素瘤B16-F1肿瘤再生细胞的抑制率。本发明包含化合物针对黑色素瘤B16-F1再生细胞抑制活性测试结果如下表2所示。
表2：黑色素瘤B16-F1再生细胞抑制活性测试结果
Figure PCTCN2017071922-appb-000122
Figure PCTCN2017071922-appb-000123
针对黑色素瘤肿瘤B16-F1再生细胞抑制率测试表明，在10μM浓度下，大部分化合物都表现出明显的肿瘤生长抑制活性，例如化合物WYC-103(86.8％)、WYC-207(92.5％)、WYC-209(98.8％)、WYC-209A(98.9％)、WYC-209B(98.7％)、WYC-212(93.2％)、WYC-217(90.3％)、WYC-218(90.4％)、WYC-329(94.5％)、WYC-331(96.0％)等，所对应肿瘤克隆体基本没用生长或生长非常缓慢。其结果不仅与阴性对照组形成鲜明对比，同时也都达到或超越了阳性药Tazarotene(67.6％)、Bexarotene(63.3％)对肿瘤克隆体生长的抑制活性。
随后对在初筛中表现出明显肿瘤克隆增长抑制活性的化合物进行肿瘤生长抑制的量效作用曲线研究。经对等梯度给药的肿瘤克隆体进行固定、DAPI细胞核染色与体积测量、抑制率计算和量效曲线回归，确定其中活性较好的化合物WYC-103、WYC-209、WYC-320、WYC-329、WYC-331的IC50如下表3所示。
表3：部分化合物针对黑色素瘤B16-F1再生细胞抑制IC50列表
Figure PCTCN2017071922-appb-000124
其中，化合物WYC-103与WYC-209针对黑色素瘤肿瘤B16-F1再生细胞抑制及诱导分化作用参见电镜照片(图1、图2)，图1和图2中，以DAPI染色显示细胞存活情况，以PI染色显示细胞凋亡情况，merge显示DAPI和PI染色图片叠加情况。
2、化合物WYC-209对多种肿瘤再生细胞生长抑制活性研究
以上多种化合物在对黑色素瘤B16-F1肿瘤再生细胞增殖方面表现出很好的抑制活性，其中WYC-209抑制活性尤其卓越。因此我们对该化合物针对其他6种人类肿瘤再生细胞生长抑制活性开展了进一步研究，其中包括肺癌细胞A549、乳腺癌细胞MCF-7、黑色素瘤细胞MDA-MB-435S、卵巢癌细胞A2780、胃癌细胞Hs-746T、乳腺癌细胞MDA-MB-231。
采用3D纤维蛋白胶培养基(90-Pa)分别孵育上述人肿瘤细胞5天，筛选得到所需肿瘤再生细胞。随后采用胶原酶和中性蛋白酶II对培养基分别进行处理，游离出肿瘤再生细胞，随后将所得再生细胞移取至新制备培养基中重悬，并保持单细胞状态。将单个肿瘤再生细胞接种于3D纤维蛋白胶培养基(90-Pa)孵育5天，以0.1％DMSO为阴性对照组(图3-图8中为DMSO组)，以无药物添加为空白组(图3-图8中为None组)，分别采用0天起给药和3天起给药开展研究，观察测量计算肿瘤克隆体积。
研究结果显示，与空白对照组相比化合物WYC-209在1.0μM即可有效抑制各种肿瘤再生细胞的增殖，极大阻滞了肿瘤克隆体的体积增长，肿瘤克隆体的体积仅为空白组 25-30％(图3-图8，图3-图8涉及到的drug9#表示WYC-209)。而当WYC-209浓度提高到10μM时，即可在较高水平抑制以上6种肿瘤再生细胞的增殖，肿瘤克隆体的体积甚至小于空白组体积的10％。尤其对于Hs-746T，采用化合物WYC-209甚至可逆转肿瘤克隆的生长趋势。
3、化合物WYC-331对多种肿瘤再生细胞生长抑制活性研究
化合物WYC-331在对黑色素瘤B16-F1肿瘤再生细胞增殖方面表现出良好抑制活性，接下来我们对该化合物针对卵巢癌A2780和乳腺癌MDA-MB-231肿瘤再生细胞的抑制活性进行了研究。
采用3D纤维蛋白胶培养基(90-Pa)分别孵育上述人肿瘤细胞5天，筛选得到所需肿瘤再生细胞。随后采用胶原酶和中性蛋白酶II对培养基分别进行处理，游离出肿瘤再生细胞，随后将所得再生细胞移取至新制备培养基中重悬，并保持单细胞状态。将单个肿瘤再生细胞接种于3D纤维蛋白胶培养基(90-Pa)孵育5天，以0.1％DMSO为阴性对照组(图9中为DMSO组)，，以无药物添加为空白组(图9中为None组)，第3天起分别按照0.1μM、1.0μM和10μM浓度给药，观察测量计算肿瘤克隆体积。
研究结果显示，与空白对照组相比，化合物WYC-331在10μM浓度下可以接近完全抑制卵巢癌A2780肿瘤再生细胞和乳腺癌MDA-MB-231肿瘤再生细胞的增殖，均极大阻滞了肿瘤克隆体的体积增长，肿瘤克隆体的体积仅为空白组10-15％。对于MDA-MB-231肿瘤再生细胞，采用化合物WYC-331甚至逆转了肿瘤克隆的生长趋势(图9，其中#31表示WYC-331)。同时，细胞染色研究发现，经WYC-331抑制的肿瘤克隆仍保持存活，仅极少数细胞出现细胞凋亡。
4、化合物WYC-209和WYC-331的毒性研究
我们以小鼠胚胎成纤维细胞3T3细胞系和黑色素瘤细胞B16-F1为模型，分别对化合物WYC-209和WYC-331进行了的体外毒性研究。实验选取在10μM浓度条件下，比较化合物WYC-209和WYC-331对于小鼠胚胎成纤维细胞3T3和黑色素瘤细胞B16-F1增殖的影响。研究结果表明，化合物WYC-209不影响小鼠胚胎成纤维细胞3T3的增殖，也不诱导该类细胞凋亡；同时化合物WYC-209可明显阻滞黑色素瘤细胞克隆体的生长，并在6-48小时表现出明显细胞凋亡。该实验显示化合物WYC-209对于黑色素瘤肿瘤再生细胞有一定的特异性，对普通细胞影响微弱，具有较小的细胞毒性(图10)。
在相同条件下，化合物WYC-331强烈抑制黑色素瘤细胞B16-F1的增殖，但同时对小鼠胚胎成纤维细胞3T3和黑色素瘤细胞B16-F1都没有表现出明显的诱导凋亡作用。这显示化合物WYC-331也对黑色素瘤再生细胞有一定选择性，同时也没有观察到明显的 细胞毒性，但作用机制可能与化合物WYC-209有一定差异(图11)。
5、化合物WYC-103对皮下移植黑色素瘤抑制活性的研究
我们以免疫活性小鼠(C57BL/6,雌性，6-8周)为基体，建立了黑色素瘤B16-F1的皮下植入模型，对化合物WYC-103体内原位肿瘤的抑制活性进行研究。首先采用3D纤维蛋白胶培养基(90-Pa)孵育B16-F1细胞5天，筛选得到所需肿瘤再生细胞，随后采用胶原酶和中性蛋白酶II对培养基分别进行处理，游离出肿瘤再生细胞，随后将所得再生细胞移取至新制备培养基中并保持单细胞重悬状态。
选取6只免疫活性小鼠并随机分为给药组和DMSO阴性对照组两个研究组，经皮下注射分别注入30,000黑色素瘤再生细胞建立皮下植入黑色素瘤模型。随后按照小鼠体重与血液体积，采用尾静脉注射方式给药。自第0天起，给药组按照10μM血药浓度每2天药物WYC-103一次，正常饲养观察小鼠生存情况，结果如下。经19天实验，给药组皮下黑色素瘤体积明显小于DMSO对照组，经统计分析可知给药组肿瘤体积仅为对照组50％(图12)。
为进一步证实化合物WYC-103对于黑色素瘤B16-F1的皮下植入肿瘤的抑制作用，我们重复了该实验。本次实验则选取18只免疫活性小鼠(C57BL/6,雌性,6-8周)并随机分为给药组、阳性对照组(BMS-453为阳性药)和DMSO阴性对照组三个研究组，经皮下注射分别注入30,000黑色素瘤再生细胞建立黑色素瘤皮下植入模型。随后按照小鼠体重与血液体积，采用尾静脉注射方式给药。自第0天起，给药组按照10μM血药浓度每2天注射药物WYC-103一次；阳性对照组以BMS-453(WYC-114)为阳性药以相同方式给药；阴性对照组则以相同方式注射0.1％DMSO一次。
至实验开始第20天，阳性药物对照组小鼠全部死亡。至实验开始第26天，给药组和阴性对照组各50％小鼠死亡。经统计此时给药组小鼠肿瘤体积仅为阴性对照组50％，给药组小鼠的皮下移植肿瘤生长均得到明显抑制。而对小鼠体重研究显示，给药组小鼠的体重均稳定、均衡，没有观察到明显毒副作用(图13，其中，drug3#表示WYC-103，drug14#表示BMS-453)。阳性药物对照组小鼠体重明显减轻，显示其存在较大个体毒副作用；而阴性对照组小鼠由于肿瘤体积巨大，体重上升明显(图14)。对各组小鼠进一步解剖实验显示，阳性对照组死亡小鼠腹腔内出现大量泡沫状非正常结构，可能是导致该组小鼠死亡的原因。而给药组与阴性对照组小鼠腹腔内均未发现异常。该实验显示在皮下原位黑色素瘤抑制的药效及整体毒副作用方面，WYC-103均大大优于BMS-453。
6、化合物WYC-103抑制黑色素瘤向肺部转移的活性研究
为研究化合物WYC-103是否可以抑制黑色素瘤向肺部转移从而阻止继发性肿瘤发 生，我们建立了小鼠黑色素瘤肺癌转移模型。首先，随机选取12只具有正常免疫功能的小鼠(C57BL/6,雌性,6-8周)为基体，经尾静脉注射3000黑色素瘤再生细胞进入小鼠体内建立肺癌转移模型。将模型小鼠随机分为给药组和阴性对照组两组，按照小鼠体重与血液体积，采用尾静脉注射方式，将化合物WYC-103(给药物)和DMSO(阴性对照组)按照10μM血药浓度及0.1％数量分别每两天注射一次。
至实验第29天，阴性对照组出现第1只病鼠死亡，为方便对比同时杀死1只给药组小鼠，经解剖可知，阴性对照组小鼠出现明显的黑色素瘤肺部转移肿瘤组织，而给药组小鼠肺部组织一切正常。至实验第35天，阴性对照组出现第2只病鼠死亡，阴性对照组小鼠出现大量黑色素瘤肺部转移肿瘤组织，而对应给药组小鼠肺部组织一切正常。至实验第37天，阴性对照组出现3只病鼠死亡，至此阴性对照组仅剩余1只小鼠，因此杀死两组所有小鼠，并进行解剖对比。经解剖可知，3只阴性对照组小鼠出现严重黑色素瘤肺部转移肿瘤组织，而给药组小鼠肺部组织均一切正常。
经实验统计，阴性对照组6只小鼠中5只出现了明显黑色素瘤肺部转移肿瘤组织且全部病亡，剩余1只健康存活；而给药组6只小鼠肺部组织均健康完整，且无任何黑色素瘤转移侵入迹象，以100％的有效率阻止了转移性黑色素瘤的发生。同时经统计分析，给药组小鼠肺部平均重量仅为阴性对照组1/3(图15，图15e和15f中“+”表示有转移，“-”表示没有转移)。
该实验显示，化合物WYC-103在10μM血药浓度下能够有效阻止黑色素瘤再生细胞向肺部转移形成继发性肺部肿瘤，而且在整个实验过程中WYC-103都没有表现出明显毒副作用，很有希望进一步用于人类转移性癌症的预防和治疗。
7、化合物WYC-209抑制黑色素瘤向肺部转移的活性研究
为研究化合物WYC-209是否可以抑制黑色素瘤向肺部转移从而阻止继发性肿瘤发生，我们开展了该项实验。首先采用3D纤维蛋白胶培养基(90-Pa)孵育B16-F1细胞5天，筛选得到所需肿瘤再生细胞，随后采用胶原酶和中性蛋白酶II对培养基分别进行处理，游离出肿瘤再生细胞，将所得再生细胞移取至新制备培养基中并保持单细胞重悬状态。随后以免疫活性小鼠(C57BL/6,雌性,6-8周)为基体，随机选取24只免疫活性小鼠，分为低剂量给药组(1.0μM)、高剂量给药组(10μM)和DMSO阴性对照组三个研究组，每组各8只小鼠。经尾静脉注射30000黑色素瘤再生细胞(化合物WYC-103所用动物模型的十倍)进入小鼠体内，建立黑色素瘤B16-F1向肺部转移模型。随后按照小鼠体重与血液体积，采用尾静脉注射方式给药。自第5天起，给药组分别按照1.0μM和10μM血药浓度每2天静脉注射药物WYC-209一次，DMSO组则以相同方式给予0.1％ DMSO作为阴性对照，此时各组小鼠体重无明显差异。正常饲养观察统计结果。
至实验第23天，DMSO组出现1只小鼠死亡(其肺组织图见图16a第8号图)。至第30天将剩余全部小鼠杀死，DMSO组8只小鼠中的6只小鼠肺部出现明显黑色素瘤转移组织，肝脏及胃脏未发现异常(具体见图16a第1～7号图)。同期(第29日夜)，1.0μM给药组出现1只小鼠死亡(肺组织图片见图16b第8号图)；1.0μM给药组8只小鼠中的4只小鼠肺部出现黑色素瘤转移组织，肝脏及胃脏未发现异常(图16b第1～7号图为实验30天的肺组织图)。同期，10μM给药组8只小鼠中仅1只小鼠肺部出现黑色素瘤转移组织，肝脏及胃脏未发现异常。即至第30天，DMSO组6只小鼠(75％)罹患肺部转移黑色素瘤，1.0μM给药组4只小鼠(50％)罹患肺部转移黑色素瘤，而10μM给药组仅1只小鼠(12.5％)罹患肺部转移黑色素瘤(具体见图16c，其中，第1～5号图为实验30天的肺组织图，第6～8号图分别表示实验29、24、20的肺组织图)。各组小鼠肺部组织重量同样出现明显统计学差异，DMSO组>1.0μM给药组>10μM给药组(图16中“+”表示有转移，“-”表示没有转移)。
该实验显示，化合物WYC-209在1.0μM血药浓度下即可50％有效阻止黑色素瘤再生细胞向肺部转移形成继发性肺部肿瘤，在10.0μM血药浓度下则可以87.5％的有效率阻止黑色素瘤再生细胞向肺部转移形成继发性肺部肿瘤。对照DMSO组75％的肺部转移黑色素瘤致死率，WYC-209在免疫功能完全的小鼠体内试验中表现出了强大的继发性肿瘤抑制乃至治愈活性。同时，在整个实验过程中WYC-209都没有表现出明显毒副作用，很有希望进一步用于人类多种原发或转移性癌症的预防和治疗。
总之，通过以上体外活性实验研究实施例表明，化合物WYC-103、WYC-209和WYC-331等的对黑色素瘤再生细胞都有很强的抑制作用，对黑色素再生细胞抑制活性IC50分别达到了0.45μM、0.25μM和0.017μM。WYC-209对于肺癌细胞A549、乳腺癌细胞MCF-7、黑色素瘤细胞MDA-MB-435S、卵巢癌细胞A2780、胃癌细胞Hs-746T、乳腺癌细胞MDA-MB-231等肿瘤再生细胞都有强烈抑制作用。
体内活性实验研究实施例证实，WYC-103对于小鼠皮下移植黑色素瘤具有强烈的抑制作用，与空白组对照，在10μM工作浓度条件下，瘤株体积仅为空白对照组50％。在10μM工作浓度条件下，WYC-103和WYC-209分别以100％和87％的有效率抑制了黑色素瘤再生细胞向肺部转移，为人类肿瘤的转移预防和治疗带来全新治疗方案。
体外实验实施例还证实，WYC-209和WYC-331对3T3细胞克隆体的生长没有影响，也不能诱导3T3细胞凋亡。同时，WYC-103和WYC-209的体内外活性研究实施例证实这些化合物具有很高的安全性。通过以上生物学研究实施例可知，本专利涉及化合物为 白血病、淋巴癌、原发实体瘤和转移性肿瘤的预防和治疗提供了新的可能。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (14)

  1. 一种如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐，
    Figure PCTCN2017071922-appb-100001
    其中，U为CR9a或N；V为CR9b或N；X为CR9c或N；W为CR9d或N；
    所述的U、V、X和W中，R9a、R9b、R9c和R9d独立地为氢、羟基、硝基、氰基、卤素、C1-C6的烷基、卤素取代的C1-C6的烷基、C1-C6烷氧基、-NR10R11、
    Figure PCTCN2017071922-appb-100002
    -COOR14；
    所述的R10、R11、R12、R13和R14独立地为氢或C1-C6的烷基；
    AE、EG和GZ之间独立地为单键或双键；当A、E、G或Z与两个单键连接时，相应的A、E、G和Z独立地为：-(CR2R3)-、-C(＝O)-、-(NR4)-、-(N→O)-、-O-、-S-、-S(＝O)-或-SO2-；当A、E、G或Z与一个单键和一个双键连接时，相应的A、E、G或Z独立地为：-(CR5)＝或-N＝；
    所述的R2、R3、R4和R5独立地为氢、羟基、卤素、C1-C6的烷基、C2-C6的烯基、卤素取代的C1-C6的烷基、C1-C6的烷氧基、C1-C6的酰基、C6-C10的芳基或“杂原子为氧、硫或氮原子，杂原子数为1～2个的C3-C6的杂芳基”；
    m为0、1、2或3；
    当有多个R1取代时，取代基相同或者不同；R1为氢、羟基、硝基、氰基、卤素、C1-C6的烷基、卤素取代的C1-C6的烷基、C1-C6烷氧基、-NR6R7或-COOR8；
    所述的R6、R7和R8独立地为氢或C1-C6的烷基；
    Y为-CN、-COOR15或-CO2NHR16；
    所述的R15和R16独立地为氢、C1-C6的烷基、C2-C6的烯基或C1-C6烷基酰基；
    所述的化合物I不为
    Figure PCTCN2017071922-appb-100003
  2. 如权利要求1所述的如式I所示的化合物，其特征在于，
    所述的R2、R3、R4和R5独立地为卤素时，所述的“卤素”为氟、氯、溴或碘；
    和/或，所述的R2、R3、R4和R5独立地为“C1-C6的烷基”时，所述的“C1-C6的烷基”为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基；
    和/或，所述的R2、R3、R4和R5独立地为“C2-C6的烯基”时，所述的“C2-C6的烯基”为乙烯基或丙烯基；
    和/或，所述的R2、R3、R4和R5独立地为“卤素取代的C1-C6的烷基”时，所述的“卤素取代的C1-C6的烷基”为三氟甲基；
    和/或，所述的R2、R3、R4和R5独立地为“C1-C6的烷氧基”时，所述的“C1-C6的烷氧基”为甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、戊氧基或己氧基；
    和/或，所述的R2、R3、R4和R5独立地为“C1-C6的酰基”时，所述的“C1-C6的酰基”为乙酰基或甲酰基；
    和/或，所述的R2、R3、R4和R5独立地为“C6-C10的芳基”时，所述的“C6-C10的芳基”为苯基；
    和/或，所述的R2、R3、R4和R5独立地为“杂原子为氧、硫或氮原子，杂原子数为1～2个的C3-C6的杂芳基”时，所述的“杂原子为氧、硫或氮原子，杂原子数为1～2个的C3-C6的杂芳基”为吡啶基或嘧啶基；
    和/或，所述的R1为“卤素”时，所述的“卤素”为氟、氯、溴或碘；
    和/或，所述的R1为“C1-C6的烷基”时，所述的“C1-C6的烷基”为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基；
    和/或，所述的R1为“卤素取代的C1-C6的烷基”时，所述的“卤素取代的C1-C6的烷基”为三氟甲基；
    和/或，所述的R1为“C1-C6烷氧基”时，所述的“C1-C6烷氧基”为甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、戊氧基或己氧基；
    和/或，所述的R6、R7和R8独立地为“C1-C6的烷基”时，所述的“C1-C6的烷基”为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基；
    和/或，所述的R9a、R9b、R9c和R9d独立地为“卤素”时，所述的“卤素”为氟、氯、溴或碘；
    和/或，所述的R9a、R9b、R9c和R9d独立地为“C1-C6的烷基”时，所述的“C1-C6的烷基”为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基；
    和/或，所述的R9a、R9b、R9c和R9d独立地为“卤素取代的C1-C6的烷基”时，所述的“卤素取代的C1-C6的烷基”为三氟甲基；
    和/或，所述的R9a、R9b、R9c和R9d独立地为“C1-C6烷氧基”时，所述的“C1-C6烷氧基”为甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、戊氧基或己氧基；
    和/或，所述的R10、R11、R12、R13和R14独立地为“C1-C6的烷基”时，所述的“C1-C6的烷基”为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基；
    和/或，所述的R15和R16独立地为“C1-C6的烷基”时，所述的“C1-C6的烷基”为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基；
    和/或，所述的R15和R16独立地为“C2-C6的烯基”时，所述的“C2-C6的烯基”为乙烯基或丙烯基；
    和/或，所述的R15和R16独立地为“C1-C6的酰基”时，所述的“C1-C6的酰基”为甲酰基或乙酰基。
  3. 如权利要求1或2所述的如式I所示的化合物，其特征在于，
    所述的U、V、X和W中有1个、2个、3个或4个为N，或所述的U为CR9a、V为CR9b、X为CR9c、和W为CR9d；
    和/或，所述的A、E、G和Z连接成如下所示的环：
    Figure PCTCN2017071922-appb-100004
    Figure PCTCN2017071922-appb-100005
  4. 如权利要求3所述的如式I所示的化合物，其特征在于，
    当所述的U、V、X和W中有两个为N时，所述的化合物I为下述任一化合物，
    Figure PCTCN2017071922-appb-100006
    或者，当所述的U、V、X和W中有一个为N时，所述的化合物I为下述任一化合物，
    Figure PCTCN2017071922-appb-100007
    或者，当所述的U为CR9a，V为CR9b，X为CR9c，和W为CR9d时，所述的化合物I的结构如化合物F所示，
    Figure PCTCN2017071922-appb-100008
    化合物F中，所述的R9a、R9b、R9c和R9d中有1个、2个、3个或4个不为氢。
  5. 如权利要求4所述的如式I所示的化合物，其特征在于，
    所述的化合物A中，当Y为-COOR15时，所述的R15为氢或乙基；
    和/或，所述的化合物A中，所述的Z为-(CR2R3)-、-S-、-S(＝O)-或-SO2-；较佳地，所述的A、E、G和Z连接成如下所示的环：
    Figure PCTCN2017071922-appb-100009
    Figure PCTCN2017071922-appb-100010
    和/或，所述的化合物B中，当所述的Y为-COOR15时，所述的R15为甲基或乙基；
    和/或，所述的化合物B中，所述的Z为-(CR2R3)-、-S-或-S(＝O)-；较佳地，所述的 A、E、G和Z连接成如下所示的环：
    Figure PCTCN2017071922-appb-100011
    和/或，所述的化合物C中，当所述的Y为-COOR15时，所述的R15为氢或乙基；
    和/或，所述的化合物C中，所述的Z为-(CR2R3)-、-S-或-S(＝O)-；较佳地，所述的A、E、G和Z连接成如下所示的环：
    Figure PCTCN2017071922-appb-100012
    和/或，所述的化合物D中，当所述的Y为-COOR15时，所述的R15为氢或乙基；
    和/或，所述的化合物D中，所述的Z为-(CR2R3)-；较佳地，所述的A、E、G和Z连接成如下所示的环：
    Figure PCTCN2017071922-appb-100013
    和/或，所述的化合物E中，所述的Y为CN；
    和/或，所述的化合物E中，所述的Z为-(CR2R3)-；较佳地，所述的A、E、G和Z连接成如下所示的环：
    Figure PCTCN2017071922-appb-100014
    和/或，所述的化合物F中，当所述的Y为-COOR15时，所述的R15为氢、甲基或乙基；
    和/或，所述的化合物F中，所述的Z为-(CR2R3)-、-S-、-S(＝O)-或-SO2-；较佳地，所述的A、E、G和Z连接成如下所示的环：
    Figure PCTCN2017071922-appb-100015
    Figure PCTCN2017071922-appb-100016
  6. 如权利要求5所述的如式I所示的化合物，其特征在于，
    所述的化合物A中，当所述的Y为COOH时，所述的Z为-S-；
    和/或，所述的化合物A中，当所述的Y为COOEt时，所述的Z为-(CR2R3)-、-S(＝O)-或-SO2-；
    和/或，所述的化合物B中，当所述的Y为COOMe时，所述的Z为-(CR2R3)-；
    和/或，所述的化合物B中，当所述的Y为COOEt时，所述的Z为-(CR2R3)-或-S-；
    和/或，所述的化合物B中，当所述的Y为COOEt时，所述的Z为-(CR2R3)-或-S-；
    和/或，所述的化合物B中，当所述的Y为CN时，所述的Z为-(CR2R3)-、-S-或-S(＝O)-；
    和/或，所述的化合物C中，当所述的Y为COOH时，所述的Z为-S-；
    和/或，所述的化合物C中，当所述的Y为COOEt时，所述的Z为-(CR2R3)-、-S-或-S(＝O)-。
  7. 如权利要求1所述的如式I所示的化合物，其特征在于，所述的化合物I为下述任一化合物：
    Figure PCTCN2017071922-appb-100017
    Figure PCTCN2017071922-appb-100018
    Figure PCTCN2017071922-appb-100019
  8. 如权利要求1～7任一项所述的如式I所示的化合物的制备方法，其特征在于，其包括下述步骤：将化合物II和III进行偶联反应，得到化合物I即可；
    Figure PCTCN2017071922-appb-100020
    其中，X1为卤素。
  9. 一种如式II、III、IV或V所示的化合物，
    Figure PCTCN2017071922-appb-100021
    其中，A、E、G、Z、R1、m、Y、U、V、X和W的定义均如权利要求1～7中任一项所述；X1为卤素；X2为卤素。
  10. 如权利要求1～7中任一项所述的如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐在制备药物中的应用，所述的药物用于治疗原发肿瘤。
  11. 如权利要求1～7中任一项所述的如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐在制备药物中的应用，所述的药物用于预防和/或治疗转移肿瘤。
  12. 如权利要求1～7中任一项所述的如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐在制备药物中的应用，所述的药物用于预防和/或治疗、白血病和/或淋巴癌。
  13. 如权利要求1～7中任一项所述的如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐在制备药物中的应用，所述的药物用于动物胎儿发育、内环境稳定、视觉、形态发生、皮肤老化和控制细胞分化中的一种或多种。
  14. 一种药物组合物，其包含如权利要求1～7中任一项所述的如式I所示的化合物、其对映异构体、非对映异构体或药学上可接受的盐，及可药用载体。
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WO2025172296A1 (en) * 2024-02-16 2025-08-21 University Of Durham Synthetic retinoids for use in rar activation

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