WO2023030043A1 - Tricyclic diterpene analogue, and synthesis and application thereof - Google Patents

Tricyclic diterpene analogue, and synthesis and application thereof Download PDF

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WO2023030043A1
WO2023030043A1 PCT/CN2022/113625 CN2022113625W WO2023030043A1 WO 2023030043 A1 WO2023030043 A1 WO 2023030043A1 CN 2022113625 W CN2022113625 W CN 2022113625W WO 2023030043 A1 WO2023030043 A1 WO 2023030043A1
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compound
tricyclic diterpene
reaction
preparation
formula
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French (fr)
Chinese (zh)
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仇文卫
宋保亮
何丽明
刘源彬
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珂阑(上海)医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/72—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms
    • C07C235/76—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
    • C07C235/78—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of an unsaturated carbon skeleton the carbon skeleton containing rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/16—Preparation of carboxylic acids or their salts, halides or anhydrides by oxidation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C53/00—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen
    • C07C53/132—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen containing rings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the technical field of medicine and chemical industry, and relates to a tricyclic diterpene analog and its synthesis and application in antitumor.
  • Hedgehog (Hh) signaling pathway plays a crucial role in embryonic development, tissue regeneration and tumorigenesis. Abnormal activation of the Hh signaling pathway can cause a series of diseases, including basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, breast cancer, lung cancer, gastric cancer, adenocarcinoma, and colon cancer. Smoothened (SMO) protein plays a key role in the Hh signaling pathway, and inhibiting the activity of SMO protein is one of the important means to inhibit the Hh signaling pathway (Nat Rev Mol Cell Biol.2013, 14, 416-29). In recent years, the research and development of drugs targeting SMO has achieved leapfrog development.
  • SMO Smoothened
  • SMO inhibitors such as Novartis’s Sonidegib (LDE225/Sonidegib, Drugs.2015,5,1559-66), Roche’s Both Moderji (GDC0449/Vismodegib, Drug Discovery.2012, 11, 437-438) and Pfizer’s Glasdegib (Clin Cancer Res. 2019, 25, 6021-6025) have been approved for marketing by the US FDA.
  • Ji and Vimodji have very significant effects in the treatment of patients with basal cell carcinoma (BCC), and Gladji can significantly prolong the life of patients in the treatment of patients with acute myeloid leukemia (AML).
  • SMO inhibitors are currently in clinical research, so SMO inhibitors have very broad application prospects in cancer treatment (Cell Chem Biol. 2017, 24, 252-280).
  • studies have shown that most of the SMO inhibitors, including the marketed Sonideji, Vemodji and Gladji, all act on the 7 transmembrane domains of SMO, which is a non-conserved region, and this region is very prone to resistance.
  • Drug mutations D473H&E518K, etc.).
  • CRD N-terminal Cysteine Rich Domain
  • the present invention provides a tricyclic diterpene analogue, a preparation method and application of the analogue.
  • the invention discloses a SMO inhibitor acting on SMO-CRD, which can not only mediate the inhibition of the Hh signaling pathway, play an anti-tumor effect, but also solve the problem of existing clinically targeted SMO drugs (Vimodeji; Sony Deji; Gladji) drug resistance problem, providing a new solution for the treatment of related cancers.
  • the present invention provides a kind of tricyclic diterpene analogue, and its general structural formula is as follows:
  • R 1 is selected from carbonyl, hydroxyl
  • R is selected from various alkane amines, heterocyclic amines, aromatic amines or hydroxyl groups;
  • n is selected from natural numbers
  • R 1 is selected from carbonyl, ⁇ -hydroxyl
  • R 2 is selected from hydroxyl –OH; alkane amines such as or heterocyclic amines such as or aromatic amines such as
  • n is selected from natural numbers 2-5.
  • a tricyclic diterpene analog provided by the present invention has a structural formula as shown in formula (2) to formula (32),
  • the present invention also provides a synthetic method for preparing tricyclic diterpene analogues, comprising the following:
  • the mother nucleus compound Q12 is obtained through reactions such as methylation, ethylene glycol protection, reduction, oxidation, Bayer-Villger oxidation, reduction, deprotection, acetylation, elimination, hydrolysis, and oxidation, and then Q12 is subjected to Amidation obtains the tricyclic diterpene analog shown in formula (2), i.e. compound 2; or, with the core compound Q12 as raw material, reduction obtains the tricyclic diterpene analog shown in formula (3), i.e. compound 3, and then the formula ( 3) Compounds are amidated to obtain tricyclic diterpene analogs shown in formula (4) to formula (25), that is, compounds 4 to 25, and the general route of the preparation method is shown in (J):
  • the method includes the following steps:
  • the compound testosterone (Testosterone) is dissolved in an organic solvent, and the methylation reagent and base used in the methylation reaction are added to carry out the methylation reaction to obtain the compound Q1.
  • the organic solvent is selected from any one or more of t-BuOH, benzene, carbon tetrachloride, tetrahydrofuran, etc.; preferably, it is t-BuOH.
  • the base is selected from any one or more of t-BuOK, KOC(Et)Me 2 and the like; preferably, it is t-BuOK.
  • the methylating agent is any one or more of CH 3 Cl, CH 3 Br, CH 3 I, etc.; preferably, it is CH 3 I.
  • step (a) the molar ratio of the compound testosterone to base is 1:(2-8); preferably, it is 1:3.
  • step (a) the molar ratio of the compound testosterone to the methylating agent is 1:(2-10); preferably, it is 1:6.
  • step (a) the reaction temperature during the addition of the methylating reagent is 0°C.
  • step (a) the temperature of the methylation reaction is 0-60°C; preferably, it is 45°C.
  • step (a) the time for the methylation reaction is 2-12 hours; preferably, 4 hours.
  • the organic solvent is selected from any one or more of tetrahydrofuran, a mixed solution of tetrahydrofuran and ethylene glycol, etc.; preferably, it is a mixed solution of tetrahydrofuran and ethylene glycol.
  • the volume ratio of tetrahydrofuran to ethylene glycol is 3:(1-3); preferably, it is 3:1.
  • step (b) the ethylene glycol is used both as a solvent and as a reactant.
  • the catalyst is selected from any one or more of p-toluenesulfonic acid, concentrated sulfuric acid, etc.; preferably, it is p-toluenesulfonic acid.
  • the water-absorbing agent is selected from any one or more of triethyl orthoformate, trimethyl orthoformate, etc.; preferably, it is triethyl orthoformate.
  • step (b) the molar ratio of the compound Q1 to the catalyst and the dehydrating agent is 1:(0.2-0.5):(5-10); preferably, it is 1:0.35:5.
  • step (b) the time for the ethylene glycol protection reaction is 2-12 hours; preferably, 3 hours.
  • step (b) the temperature of the ethylene glycol protection reaction is 25-60°C; preferably, 45°C.
  • Compound Q2 is dissolved in an organic solvent, a reducing agent is added, and a reduction reaction is carried out under pressure to obtain Compound Q3.
  • the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, dichloromethane, ethyl acetate, etc.; preferably, it is a mixed solution of tetrahydrofuran and methanol.
  • the volume ratio of the mixed solution of tetrahydrofuran and methanol is 2:(1-3); preferably, it is 2:1
  • the reducing agent is selected from one or more of palladium carbon, Raney nickel, etc.; preferably, it is palladium carbon.
  • step (c) the mass ratio of the compound Q2 to the reducing agent is 1:(0.2-1); preferably, it is 1:0.3.
  • step (c) the reaction pressure is 4MPa.
  • step (c) the temperature of the reduction reaction is 10-60°C; preferably, 25°C.
  • step (c) the reduction reaction time is 24-72 hours; preferably, 48 hours.
  • Compound Q3 is dissolved in an organic solvent, and an oxidizing agent is added to react to obtain Compound Q4.
  • the organic solvent is selected from any one or more of DMSO, toluene, tetrahydrofuran, etc.; preferably, it is a mixed solution of DMSO and tetrahydrofuran.
  • the volume ratio of the mixed solution of tetrahydrofuran and DMSO is 2:(1-3); preferably, it is 2:1.
  • the oxidizing agent is any one or more of IBX, PCC, DDQ, PDC, etc.; preferably, it is IBX.
  • step (d) the molar ratio of compound Q3 to oxidizing agent is 1:(1-3); preferably, it is 1:2.
  • step (d) the temperature of the oxidation reaction is 0-50°C; preferably, 25°C.
  • step (d) the time for the oxidation reaction is 3-8 hours; preferably, 5 hours.
  • Compound Q4 is dissolved in an organic solvent, and an oxidizing agent and a base are added to react to obtain Compound Q5.
  • the organic solvent is selected from any one or more of dichloromethane, chloroform, etc.; preferably, it is dichloromethane.
  • the oxidizing agent is any one or more of m-chloroperoxybenzoic acid, peracetic acid, potassium hydrogen persulfate, hydrogen peroxide, etc.; preferably, it is m-chloroperoxybenzoic acid.
  • the base is selected from any one or more of NaHCO 3 , Na 2 CO 3 , NaH 2 PO 4 , etc.; preferably, it is NaHCO 3 .
  • step (e) the molar ratio of the compound Q4 to the oxidizing agent is 1:(1-10); preferably, it is 1:2.5.
  • step (e) the molar ratio of the compound Q4 to the base is 1: (1-10); preferably, 1:2.7.
  • step (e) the temperature of the oxidation reaction is 10-50°C; preferably, 25°C.
  • step (e) the time for the oxidation reaction is 3-12 hours; preferably, 5 hours.
  • Compound Q5 is dissolved in an organic solvent, and a reducing agent is added to react to obtain Compound Q6.
  • the organic solvent is selected from any one or more of tetrahydrofuran, diethyl ether, methanol, ethanol, etc.; preferably, it is tetrahydrofuran.
  • the reducing agent is any one or more of LiAlH 4 , red aluminum, diisobutylaluminum hydride, etc.; preferably, it is LiAlH 4 .
  • step (f) the molar ratio of the compound Q5 to the reducing agent is 1:(2-10); preferably, it is 1:3.
  • step (f) the temperature of the reduction reaction is 0-25°C; preferably, 0°C.
  • step (f) the reduction reaction time is 0.5-2 hours; preferably, 0.5 hours.
  • the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, etc.; preferably, it is tetrahydrofuran.
  • the acid is selected from one or more of dilute sulfuric acid, dilute hydrochloric acid, hydrochloric acid gas, p-toluenesulfonic acid, etc.; preferably, it is 2mol/L dilute hydrochloric acid.
  • step (g) the volume ratio of the organic solvent to 2mol/L dilute hydrochloric acid is 5:(1-2); preferably, it is 5:1.
  • step (g) the temperature of the reaction of removing the ethylene glycol protecting group is 0-60°C; preferably, it is 25°C.
  • step (g) the reaction time for removing the ethylene glycol protecting group is 1-10 h; preferably, 5 h.
  • Compound Q7 is dissolved in an organic solvent, and an acetylating reagent is added to react to obtain Compound Q8.
  • the organic solvent is selected from one or more of pyridine, triethylamine, diethylamine, diisopropylethylamine, tetrahydrofuran, DMF, etc.; preferably, it is pyridine.
  • the acetylating agent is selected from one or more of acetic anhydride, acetyl chloride, etc.; preferably, it is acetic anhydride.
  • step (h) the molar ratio of Q7 to the acetylating agent is 1:(1-5); preferably, it is 1:1.5.
  • step (h) the temperature of the acetylation reaction is 0-25°C; preferably, 0°C.
  • step (h) the time for the acetylation reaction is 1-24 hours; preferably, 18 hours.
  • the organic solvent is selected from one or more of dichloromethane, acetonitrile, acetone, toluene, etc.; preferably, it is dichloromethane.
  • the dehydrating agent is selected from one or more of concentrated sulfuric acid, phosphoric acid, trimethylsilyl trifluoromethanesulfonate (TMSOTf) and the like; preferably, trimethylsilyl trifluoromethanesulfonic acid ester.
  • step (i) the water absorbing agent is acetic anhydride.
  • step (i) the molar ratio of Q8 to the dehydrating agent is 1:(0.1-0.4); preferably, it is 1:0.12.
  • step (i) the molar ratio of Q8 to the water-absorbing agent is 1:(1-5); preferably, it is 1:5.
  • step (i) the temperature of the elimination reaction is -10°C to 25°C; preferably, it is 0°C.
  • step (i) the time for the elimination reaction is 0.25-1 h; preferably, 0.5 h.
  • the organic solvent is selected from any one or more of methanol, tetrahydrofuran, ethanol, etc.; preferably, it is methanol.
  • the base is selected from any one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, etc.; preferably, potassium carbonate.
  • step (j) the molar ratio of the compound Q9 to the base is 1:(1-5); preferably, it is 1:4.
  • step (j) the temperature of the hydrolysis reaction is 10-70°C; preferably, 25°C.
  • step (j) the time for the hydrolysis reaction is 1-12 hours; preferably, it is 3 hours.
  • Compound Q10 is dissolved in an organic solvent, and an oxidizing agent is added to react to obtain Compound Q11.
  • the organic solvent is selected from any one or more of DMSO, toluene, tetrahydrofuran, etc.; preferably, it is a mixed solution of DMSO and tetrahydrofuran.
  • the volume ratio of the mixed solution of tetrahydrofuran and DMSO is 2:(1-3); preferably, it is 2:1.
  • the oxidizing agent is any one or more of IBX, PCC, DDQ, PDC, etc.; preferably, it is IBX.
  • step (k) the molar ratio of compound Q10 to oxidizing agent is 1:(1-2); preferably, it is 1:2.
  • step (k) the temperature of the oxidation reaction is 0-50°C; preferably, 25°C.
  • step (k) the time for the oxidation reaction is 1 to 3 hours; preferably, 1 hour.
  • Compound Q11 is dissolved in an organic solvent, and an oxidizing agent is added to react to obtain Compound Q12.
  • the organic solvent is selected from any one or more of pyridine, chloroform, dichloroethane, 1,2-dichloropropane, etc.; preferably, it is pyridine.
  • the oxidant is selected from any one or more of tetrabutylammonium permanganate, potassium permanganate, potassium dichromate, O etc .; preferably, tetrabutyl permanganese ammonium acid.
  • step (l) the molar ratio of Q11 to the oxidizing agent is 1:(1-4); preferably, it is 1:2.
  • step (l) the temperature of the oxidation reaction is 0-60°C; preferably, 0°C.
  • step (l) the time for the oxidation reaction is 1 to 2 hours; preferably, 1 hour.
  • the organic solvent is selected from any one or more of DCM, DMF, tetrahydrofuran, chloroform, etc.; preferably, it is DCM.
  • step (m) the function of the HATU and DIPEA is to promote the amidation reaction.
  • step (m) the molar ratio of the compound Q12, HATU, DIPEA and the corresponding amine is 1:(1 ⁇ 2):(1 ⁇ 2):(2 ⁇ 4); preferably, 1:1.2: 1.5:2.
  • step (m) the amidation reaction temperature is 0-40°C; preferably, 25°C.
  • step (m) the amidation reaction time is 0.5-2 hours; preferably, 1 hour.
  • Compound Q12 is dissolved in an organic solvent, and a reducing agent is added to react to obtain Compound 3.
  • the organic solvent is selected from one or more of dichloromethane, methanol, a mixed solution of dichloromethane and methanol, etc.; preferably, it is a mixed solution of methanol and dichloromethane.
  • the volume ratio of the mixed solution of methanol and dichloromethane is 1:(0.5-3); preferably 1:2.
  • the reducing agent is selected from one or more of sodium borohydride, potassium borohydride, etc.; preferably, sodium borohydride.
  • step (n) the molar ratio of the compound Q12 to the reducing agent is 1:(5-20); preferably, it is 1:10.
  • step (n) the temperature of the reduction reaction is 0-50°C; preferably, 0°C.
  • step (n) the reduction reaction time is 0.5-4 hours; preferably, 0.5 hours.
  • Described route (J) also comprises route (A), route (E), route (F), specifically as follows:
  • the present invention also proposes a preparation method of tricyclic diterpene analogues.
  • Compound Q10 is used as a starting material to obtain the compound of formula (26) through sulfonylation, substitution (cyanation), hydrolysis, reduction, and amidation reactions.
  • Show tricyclic diterpene analogue, i.e. compound 26, and described preparation method is as shown in route (K):
  • the method includes the following steps:
  • Compound Q10 is dissolved in an organic solvent, and methanesulfonyl chloride and an acid-binding agent are added to react to obtain compound Q13.
  • the organic solvent is selected from one or more of dichloromethane, tetrahydrofuran, etc.; preferably, it is dichloromethane.
  • the acid-binding agent is selected from one or more of triethylamine, tributylamine, pyridine, etc.; preferably, it is triethylamine.
  • step (a) the molar ratio of compound Q10 to methanesulfonyl chloride is 1:(1-5); preferably, it is 1:3.
  • step (a) the molar ratio of the compound Q10 to the acid-binding agent is 1:(1-5); preferably, it is 1:4.
  • step (a) the temperature of the sulfonylation reaction is -10-25°C; preferably, it is 0°C.
  • step (a) the time for the sulfonylation reaction is 1 to 4 hours; preferably, 1 hour.
  • Compound Q13 is dissolved in an organic solvent, and compound Q14 is obtained by adding a cyanation reagent and tetrabutylammonium fluoride.
  • the organic solvent is selected from one or more of DMF, tetrahydrofuran, acetonitrile, etc.; preferably, it is acetonitrile.
  • the cyanation agent is selected from one or more of sodium cyanide, trimethylsilylcyanide, etc.; preferably, it is trimethylsilylcyanide.
  • step (b) the function of tetrabutylammonium fluoride is to promote the departure of TMS group and promote the cyanation reaction.
  • step (b) the molar ratio of the compound Q13 to the cyanation reagent is 1:(1-10); preferably, it is 1:1.5.
  • step (b) the molar ratio of compound Q13 to tetrabutylammonium fluoride is 1:(1-10); preferably, it is 1:1.5.
  • step (b) the temperature of the cyanation reaction is 0-85°C; preferably, it is 82°C.
  • step (b) the time for the cyanation reaction is 0.5-4 hours; preferably, 0.5 hours.
  • Compound Q14 is dissolved in an organic solvent, and a base is added to react to obtain Compound Q15.
  • the organic solvent is selected from one or more of ethanol, ethylene glycol, methanol, etc.; preferably, it is ethylene glycol.
  • the base is selected from any one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, etc.; preferably, it is potassium hydroxide.
  • step (c) the molar ratio of the compound Q14 to the base is 1:(5-15); preferably, it is 1:10.
  • step (c) the temperature of the hydrolysis reaction is 60-170°C; preferably, it is 170°C.
  • step (c) the time for the hydrolysis reaction is 6-24 hours; preferably, it is 12 hours.
  • Described route (K) also comprises route (B), route (G), specifically as follows:
  • the present invention also proposes a preparation method of tricyclic diterpene analogues, using compound Q11 as a starting material, through Wittig reaction, reduction, hydrolysis, and amidation reactions to obtain tricyclic diterpene compounds represented by formula (27) to formula (29).
  • Diterpene analogs, i.e. compounds 27-29, the preparation method is shown in route (L):
  • the method includes the following steps:
  • Compound Q11 is dissolved in an organic solvent, and a base and Wittig reagent are added to react to obtain Compound Q17.
  • the organic solvent is selected from any one or more of tetrahydrofuran, toluene, petroleum ether, etc.; preferably, it is tetrahydrofuran.
  • the alkali is one or more of sodium methoxide, sodium ethoxide, and sodium hydride (60%); preferably, it is sodium hydride (60%).
  • the Wittig reagent is preferably triethyl phosphoroacetate.
  • step (a) the molar ratio of the compound Q11 to the base and the Wittig reagent is 1:(3-8):(3-8); preferably, it is 1:5.5:5.
  • step (a) the temperature of the Wittig reaction is -10°C to 25°C; preferably, it is 0°C.
  • step (a) the time for the Wittig reaction is 20-60 minutes; preferably, 20 minutes.
  • Compound Q17 is dissolved in an organic solvent, and a reducing agent is added to react to obtain Compound Q18.
  • the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, dichloromethane, ethyl acetate, etc.; preferably, tetrahydrofuran.
  • the reducing agent is selected from one or more of palladium carbon, Raney nickel, etc.; preferably, it is palladium carbon.
  • step (b) the mass ratio of the compound Q17 to the reducing agent is 1:(0.05-1); preferably, it is 1:0.1.
  • step (b) the reaction pressure is 1 atm.
  • step (b) the temperature of the reduction reaction is 10-60°C; preferably, 25°C.
  • step (b) the time for the reduction reaction is 0.5-10 h; preferably, 1 h.
  • the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, etc.; preferably, it is methanol.
  • the base is selected from any one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, etc.; preferably, it is sodium hydroxide.
  • step (d) the molar ratio of the compound Q19 to the base is 1:(1-10); preferably, it is 1:10.
  • step (d) the temperature of the hydrolysis reaction is 10-60°C; preferably, 25°C.
  • step (d) the time for the hydrolysis reaction is 5-12 hours; preferably, 9 hours.
  • Described route (L) also comprises route (C), route (H), specifically as follows:
  • the present invention also proposes a preparation method of tricyclic diterpene analogues, using compound Q10 as a starting material, through ethylene glycol protection, oxidation, Wittig reaction, reduction, deprotection, reduction, amidation reactions to obtain the formula (30 ) ⁇ tricyclic diterpene analogs shown in formula (32), i.e. compounds 30 ⁇ 32, the preparation method is shown in route (M):
  • the method includes the following steps:
  • step (a) the ethylene glycol is only used as a reactant.
  • the organic solvent is selected from one or more of tetrahydrofuran, toluene, etc.; preferably, it is toluene.
  • the catalyst is selected from any one or more of p-toluenesulfonic acid, concentrated sulfuric acid, etc.; preferably, p-toluenesulfonic acid.
  • the water-absorbing agent is selected from any one or more of triethyl orthoformate, trimethyl orthoformate, etc.; preferably, it is triethyl orthoformate.
  • step (a) the molar ratio of compound Q10 to ethylene glycol, catalyst, and water absorbing agent is 1: (1-10): (0.01-0.5): (1-10); preferably, 1:3 :0.05:3.
  • step (a) the temperature of the ethylene glycol protection reaction is 25-60°C; preferably, 25°C.
  • step (a) the time for the ethylene glycol protection reaction is 1 to 12 hours; preferably, 1 hour.
  • the organic solvent is selected from any one or more of tetrahydrofuran, toluene, petroleum ether, etc.; preferably, it is tetrahydrofuran.
  • step (c) the quaternary phosphorus salt is (2-carboxyethyl)triphenylphosphine bromide.
  • the base is selected from any one or more of triethylamine, sodium hydroxide, sodium hexamethyldisilazide (NaHDMS), etc., preferably, sodium hexamethyldisilazide .
  • step (c) the molar ratio of the compound Q22 to the quaternary phosphorus salt and the base is 1:(1-5):(1-5); preferably, it is 1:3.5:3.
  • step (c) the time for the Wittig reaction is 5-30 minutes; preferably, 10 minutes.
  • step (c) the temperature of the Wittig reaction is -20°C to 25°C; preferably, it is -20°C.
  • the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, etc.; preferably, it is tetrahydrofuran.
  • the acid is selected from one or more of dilute sulfuric acid, dilute hydrochloric acid, hydrochloric acid gas, p-toluenesulfonic acid, etc.; preferably, it is 2mol/L dilute hydrochloric acid.
  • step (d) the volume ratio of the organic solvent to 2mol/L dilute hydrochloric acid is 1:(1-2); preferably, it is 1:1.
  • step (e) the reaction time for removing the ethylene glycol protecting group is 1-5 hours; preferably, it is 4 hours.
  • step (e) the temperature of the reaction of removing the ethylene glycol protecting group is 0-60°C; preferably, it is 25°C.
  • the synthesis method of compound Q26 is similar to the reduction method of compound 3 in step (n) of route (J).
  • Described route (M) also comprises route (D), route (I), specifically as follows:
  • the progress of the reaction is tracked and determined by thin plate chromatography.
  • the post-processing method includes steps such as concentration, extraction, and column chromatography separation.
  • the final product is obtained by nuclear magnetic resonance spectrum and high-resolution mass spectrum verify.
  • the present invention also provides a pharmaceutical composition, which comprises the above-mentioned tricyclic diterpene analogs, and a pharmaceutically acceptable carrier.
  • the present invention also provides the application of the SMO inhibitor tricyclic diterpene analogue or the pharmaceutical composition in preparing the SMO inhibitor.
  • the tricyclic diterpene analog or pharmaceutical composition is used to inhibit the activity of wild-type SMO protein and drug-resistant SMO protein (D473H; E518K; N219D; L221R; D384N; S387N).
  • the present invention also provides the application of the tricyclic diterpene analog or the pharmaceutical composition in the preparation of antitumor drugs.
  • the tricyclic diterpene analog or pharmaceutical composition is used for inhibiting tumor growth, proliferation and metastasis, or for promoting tumor apoptosis and death.
  • Such tumors include medulloblastoma, basal cell carcinoma, and the like.
  • the tricyclic diterpene analogs involved in the present invention inhibit the Hh signaling pathway by inhibiting the activity of SMO, and also have good inhibitory ability to the growth of related medulloblastoma cells, and have good anti-tumor application prospects.
  • the advantages of the anti-tumor tricyclic diterpene analog and the preparation method thereof of the present invention include that the tricyclic diterpene lead with SMO activity has been screened in the self-owned compound library, and the structure has been modified, through amidation, A series of tricyclic diterpene analogues with novel structures were synthesized through reactions such as carbon chain extension; the compounds provided by the invention can not only inhibit the wild-type SMO protein, but also inhibit the activity of drug-resistant SMO protein, which can solve the problem of existing clinical SMO Inhibitor resistance issues.
  • Figure 1 shows the effect of Compound 8 of the present invention on cholesterol modification of drug-resistant SMO protein and the determination of IC 50 .
  • Figure a is the topological structure diagram of the transmembrane segment of SMO, the dark gray is the WT sequence, and the light gray is the mutant sequence.
  • Panel b shows the effect of the D95N mutation of SMO on the cholesterol modification of the SMO drug-resistant mutation.
  • Panel c is the effect of compound 8 on cholesterol modification of SMO drug-resistant mutations.
  • Panel d and panel e are the determination of IC 50 of vimodeji, sonideji and compound 8 on drug-resistant SMO protein.
  • Fig. 2 shows that compound 8 of the present invention binds to the cysteine-rich segment of the N-terminus of SMO protein.
  • Figure a shows that compound 8 can inhibit the binding of SMO and cholesterol in vitro.
  • Figure b shows that compound 8 can inhibit the cholesterol modification of SMO.
  • Figures c and d show that compound 8 does not affect the combination of Bodipy-Cyclopamine and SMO.
  • Figure e shows the combination of compound 8 and SMO 7TM inhibitors (vimodeji, sonideji, and Huantarget).
  • Figure f is a model diagram of compound 8 inhibiting the binding of SMO and cholesterol.
  • Figure 3 shows that Compound 8 of the present invention inhibits the growth of cerebellar neuron progenitor cells and medulloblastoma cells.
  • Figure a is the flow chart of c GNP cell growth experiment.
  • Figure b is the immunofluorescence picture of compound 8 inhibiting the growth of cerebellar neuron progenitor cells.
  • Panel c and panel d are the related quantitative analysis of panel b.
  • Panel e is the immunofluorescence picture of compound 8 inhibiting the growth of medulloblastoma cells.
  • Figures f and g are related quantitative analysis of figure e.
  • FIG. 4 shows the inhibitory effect of Compound 8 of the present invention on Hh-dependent medulloblastoma.
  • figure a is the measurement of tumor size during administration.
  • Figure b is the determination of the body weight of the mice during the administration period.
  • Panel c and panel d are the results of Gli1 mRNA and Western blot of mouse tumors extracted after administration.
  • Panel e is a picture of the mouse tumor extracted after administration.
  • Dichloromethane 500 mL was added for extraction and separation, the aqueous phase was extracted with dichloromethane (50 mL ⁇ 3), and the organic phases were combined.
  • Dissolve compound Q11 (1.12g, 0.0035mol) in pyridine (30mL), add tetrabutylammonium permanganate (2.56g, 0.0074mol) under ice bath, stir and react for 0.5h, remove the ice bath, and react After 1h, TLC detected that the reaction of the raw materials was complete, then added saturated sodium bisulfite solution until the purple color of the reaction solution faded, added 2mol/L dilute hydrochloric acid to adjust the pH to 4-5, added ethyl acetate (30mL) for extraction, separated the liquid, and the aqueous phase Wash with ethyl acetate (5 mL ⁇ 3), and combine the organic phases.
  • Dissolve compound 3 (870mg, 2.60mmol), HATU (1.29g, 3.12mmol) in dichloromethane (30mL), add DIPEA (0.7mL, 3.9mmol) and stir well, add the corresponding amine (5.2mmol), 25 Stir at °C for 1 h, TLC detects that the reaction of the raw materials is complete, and the reaction is quenched by adding hydrochloric acid.
  • the aqueous phase was extracted with DCM (20 mL ⁇ 3), and the organic phases were combined.
  • the organic phase was washed with water (5mL ⁇ 3) and saturated NaCl solution (5mL ⁇ 3), dried over anhydrous Na 2 SO 4 , concentrated under reduced pressure and subjected to silica gel column chromatography to obtain the corresponding compounds 4-25.
  • Dissolve compound Q14 (1.48g, 4.5mmol) in ethylene glycol (11mL), add 4mol/L potassium hydroxide solution (21mL), and react at 170°C for 12h. After TLC detects that the raw materials have reacted completely, wait for the reaction to cool to room temperature , add 2mol/L dilute hydrochloric acid to adjust the pH to 1-2, a white solid precipitates out, extract with ethyl acetate (40mL), separate the layers, wash the aqueous phase with ethyl acetate (10mL ⁇ 3), and combine the organic phases.
  • Dissolve compound Q20 (40mg, 0.11mmol), HATU (83.90mg, 0.22mmol) in 1mL of dichloromethane and 1mL of tetrahydrofuran, add DIPEA (0.04mL, 0.24mmol) and stir well, add the corresponding amine (0.22mmol) 25 Stir at °C for 1 h, TLC detects that the reaction of the raw materials is complete, and the reaction is quenched by adding hydrochloric acid. The aqueous phase was extracted with DCM (10 mL ⁇ 3), and the organic phases were combined.
  • Dissolve compound Q10 (500mg, 1.57mmol) in toluene (4mL), then add ethylene glycol (0.26mL, 4.7mmol), triethyl orthoformate (0.78mL, 4.7mmol), stir for 20min, then add monohydrate Toluenesulfonic acid (13.5mg, 0.078mmol) was stirred for 1h. After TLC detected that the reaction of the raw materials was complete, the reaction was quenched by adding saturated sodium bicarbonate, extracted with DCM (20mL), separated, and the aqueous phase was washed with DCM (5mL ⁇ 3).
  • Dissolve compound Q26 (40mg, 0.106mmol) in dichloromethane (1mL) and tetrahydrofuran (1mL), add HATU (48.5mg, 0.128mmol), DIPEA (0.04mL, 0.213mmol) and stir well, then add the corresponding amine (0.213mmol), stirred and reacted at 25°C for 1h, TLC detected that the reaction of raw materials was complete, and added hydrochloric acid to quench the reaction. The aqueous phase was extracted with dichloromethane (10 mL ⁇ 3), and the organic phases were combined.
  • the inhibitory activity of SMO protein in NIH3T3 cells was tested by luciferin reporter method.
  • NIH3T3 cells were inoculated into a 10 cm cell culture dish with a cell volume of 1.5 ⁇ 10 6 per plate, and cultured at 37° C. 24 hr in a 5% CO 2 incubator.
  • (2) Use liposome LTX (Invitrogen) to transfect GliSBS and pEGFP-N1 (according to 9:1, the total transfection amount is 6 ⁇ g) into NIH3T3 cells, and change back to DMEM medium (containing 10% Newborn bovine serum, double antibody); when the confluence of the cells reaches 80%, they are digested with 0.25% trypsin (containing EDTA) and blown evenly, and then inoculated into a 24-well plate at a cell volume of 1 ⁇ 10 5 per well.
  • DMEM medium containing 10% Newborn bovine serum, double antibody
  • NIH3T3 cells were inoculated into a 60mm cell culture dish with a cell volume of 1 ⁇ 10 6 per plate, and cultured in 37° C., 5% CO 2 incubator with 10% FCS medium for 24 hours;
  • ( 2) Transfect the plasmid (total transfection amount is 6 ⁇ g) into NIH3T3 cells with liposome 2000, and change back to 10% FCS medium after transfection for 6 hours;
  • (3) After continuing to cultivate for 24 hours, suck off the original Medium, washed once with PBS, replaced with cholesterol-starved medium (DMEM+P/S, 5% LPPS, 1 ⁇ M lovastatin, 10 ⁇ M mevalonic acid) and containing relevant test compounds (Vimodeji, Sonnide Ji, compound 8) was incubated overnight.
  • DMEM+P/S cholesterol-starved medium
  • LPPS 1 ⁇ M lovastatin, 10 ⁇ M mevalonic acid
  • Methods (1) Prepare 6-day-old pups (generally about 6); (2) Cut off the brain with scissors, tear off the skin on the skull with tweezers, and cut the skull from the central seam with scissors (note : do not destroy the structure of the cerebellum), take out the cerebellum, and place it in pre-cooled PBS; (3) observe with a stereoscope, carefully remove the red ribbon on the back of the cerebellum, and transfer the remaining part of the cerebellum to a new In a pre-cooled 1.5mL tube (with PBS inside); (Note: When operating a mouse, the separation of the cerebellum should be less than 6 minutes) (4) Aspirate as much supernatant (PBS) as possible with a gun , (Note: the gun first sucks the FBS back and forth twice.); (5) Add 1mL of trypsin and DNase mixture.
  • tricyclic diterpene analogs of formula (I) of the present invention can not only well inhibit the activity of wild-type SMO protein, but also inhibit the activity of Vimodeji & Sonideji & Gladji activity of drug-resistant SMO proteins.
  • These tricyclic diterpene analogs can effectively inhibit tumor cell proliferation, and have better inhibitory ability against Hedgehog-dependent medulloblastoma, and have good application prospects.
  • Table 1 The compounds prepared by the embodiments of the present invention inhibit the activity data of SMO protein
  • + means the compound has cytotoxicity
  • - means no cytotoxicity

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Abstract

Disclosed are a tricyclic diterpene analogue represented by formula (I) and a preparation method therefor. By using testosterone as a raw material, and by means of methylation, ethylene glycol protection, oxidation, Baeyer-Villiger oxidation, reduction, hydrolysis and other reactions, and passing through cyanation and a Wittig reaction to extend a carbon chain, a corresponding parent nucleus compound is obtained; and the parent core compound is subjected to amidation to obtain the tricyclic diterpene analogue. Also disclosed is an application of the tricyclic diterpene analog in inhibiting a smoothened (SMO) protein and resisting against tumors. The tricyclic diterpene analog binds to a cysteine rich domain (CRD) at an N-terminal of the SMO protein to inhibit the activity of a hedgehog (Hh) signaling pathway, which thus inhibits the growth of tumor cells and has good tumor-resisting application prospects. In addition, the present application may solve the problem of drug resistance of existing clinical SMO inhibitors, thus providing a new solution for the treatment of related diseases.

Description

一种三环二萜类似物及其合成和应用A kind of tricyclic diterpene analog and its synthesis and application 技术领域technical field
本发明属于医药化工技术领域，涉及一种三环二萜类似物及其合成和在抗肿瘤方面的应用。The invention belongs to the technical field of medicine and chemical industry, and relates to a tricyclic diterpene analog and its synthesis and application in antitumor.
背景技术Background technique
癌症治疗已成为全世界面临的重要问题。世界卫生组织国际癌症研究机构(IARC)发布的2020年全球最新癌症负担表明，中国新发癌症人数及中国癌症死亡人数均居全球第一。除了手术治疗外，药物治疗是最为重要的治疗方式。因此，研究新型抗肿瘤药物一直是医药领域的研究热点。Cancer treatment has become an important issue facing the world. According to the latest global cancer burden in 2020 released by the International Agency for Research on Cancer (IARC) of the World Health Organization, the number of new cancers and the number of cancer deaths in China rank first in the world. In addition to surgery, drug therapy is the most important treatment. Therefore, the study of new anti-tumor drugs has always been a research hotspot in the field of medicine.
Hedgehog(Hh)信号通路在胚胎发育、组织再生以及肿瘤发生中起着至关重要的作用。Hh信号通路异常激活会引起一系列的疾病，包括基底细胞癌、髓母细胞瘤、横纹肌肉瘤、乳腺癌、肺癌、胃癌、腺癌以及结肠癌等。Smoothened(SMO)蛋白在Hh信号通路上起着十分关键的作用，抑制SMO蛋白的活性是抑制Hh信号通路的重要手段之一(Nat Rev Mol Cell Biol.2013,14,416-29)。近年来，针对SMO的药物研发已经取得了跨越式的发展，三种SMO的抑制剂，如诺华公司的索尼德吉(LDE225/Sonidegib，Drugs.2015,5,1559-66)、罗氏制药的维莫德吉(GDC0449/Vismodegib，Drug Discovery.2012,11,437-438)及辉瑞制药的格拉德吉(Glasdegib，Clin Cancer Res.2019,25,6021-6025)均已被美国FDA批准上市，其中索尼德吉和维莫德吉在治疗基底细胞癌(BCC)的病人上有着非常显著的效果，格拉德吉在治疗急性髓性白血病(AML)的病人上可以明显的延长病人的寿命。针对其他癌症，目前一些SMO抑制剂也正处于临床研究中，因此SMO抑制剂在癌症治疗方面具有十分广阔的应用前景(Cell Chem Biol.2017,24,252-280)。但研究表明，大部分SMO抑制剂，包括已上市的索尼德吉、维莫德吉和格拉德吉均作用于SMO的7次跨膜结构域上，属非保守区，该区域极易产生耐药性突变(D473H&E518K等)。近期研究发现，SMO的N端Cysteine Rich Domain(CRD)不仅可以结合胆固醇(Nature.2016,535,17-522；Cell.2016,166,1176-1187)，还可以发生胆固醇修饰(Molecular Cell.2017,66,154–162)，并且该区域相对比较保守，不易产生突变。抑制SMO的CRD和胆固醇的结合可以很好的抑制Hh信号通路，进而起到抗肿瘤作用。Hedgehog (Hh) signaling pathway plays a crucial role in embryonic development, tissue regeneration and tumorigenesis. Abnormal activation of the Hh signaling pathway can cause a series of diseases, including basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, breast cancer, lung cancer, gastric cancer, adenocarcinoma, and colon cancer. Smoothened (SMO) protein plays a key role in the Hh signaling pathway, and inhibiting the activity of SMO protein is one of the important means to inhibit the Hh signaling pathway (Nat Rev Mol Cell Biol.2013, 14, 416-29). In recent years, the research and development of drugs targeting SMO has achieved leapfrog development. Three kinds of SMO inhibitors, such as Novartis’s Sonidegib (LDE225/Sonidegib, Drugs.2015,5,1559-66), Roche’s Both Moderji (GDC0449/Vismodegib, Drug Discovery.2012, 11, 437-438) and Pfizer’s Glasdegib (Clin Cancer Res. 2019, 25, 6021-6025) have been approved for marketing by the US FDA. Ji and Vimodji have very significant effects in the treatment of patients with basal cell carcinoma (BCC), and Gladji can significantly prolong the life of patients in the treatment of patients with acute myeloid leukemia (AML). For other cancers, some SMO inhibitors are currently in clinical research, so SMO inhibitors have very broad application prospects in cancer treatment (Cell Chem Biol. 2017, 24, 252-280). However, studies have shown that most of the SMO inhibitors, including the marketed Sonideji, Vemodji and Gladji, all act on the 7 transmembrane domains of SMO, which is a non-conserved region, and this region is very prone to resistance. Drug mutations (D473H&E518K, etc.). Recent studies have found that the N-terminal Cysteine Rich Domain (CRD) of SMO can not only bind cholesterol (Nature.2016,535,17-522; Cell.2016,166,1176-1187), but also undergo cholesterol modification (Molecular Cell.2017 ,66,154–162), and this region is relatively conserved and not prone to mutations. Inhibiting the combination of CRD and cholesterol of SMO can well inhibit the Hh signaling pathway, and then play an anti-tumor effect.
发明内容Contents of the invention
针对上述技术问题，本发明提供了一种三环二萜类似物，并提供了该类似物的制备方法及其应用。In view of the above technical problems, the present invention provides a tricyclic diterpene analogue, a preparation method and application of the analogue.
本发明公开了一种作用于SMO-CRD的SMO抑制剂，不仅能介导Hh信号通路的抑制， 起到抗肿瘤作用，还可以解决临床上现有靶向SMO药物(维莫德吉；索尼德吉；格拉德吉)的耐药性问题，为相关癌症的治疗提供一个新的解决方案。The invention discloses a SMO inhibitor acting on SMO-CRD, which can not only mediate the inhibition of the Hh signaling pathway, play an anti-tumor effect, but also solve the problem of existing clinically targeted SMO drugs (Vimodeji; Sony Deji; Gladji) drug resistance problem, providing a new solution for the treatment of related cancers.
本发明提供了一种三环二萜类似物，其结构通式如下：The present invention provides a kind of tricyclic diterpene analogue, and its general structural formula is as follows:
Figure PCTCN2022113625-appb-000001
Figure PCTCN2022113625-appb-000001
其中，in,
R 1选自羰基、羟基； R 1 is selected from carbonyl, hydroxyl;
R 2选自各种烷烃胺、杂环胺、芳香胺或羟基； R is selected from various alkane amines, heterocyclic amines, aromatic amines or hydroxyl groups;
n选自自然数；n is selected from natural numbers;
优选地，Preferably,
R 1选自羰基、β-羟基； R 1 is selected from carbonyl, β-hydroxyl;
R 2选自羟基–OH；烷烃胺如
Figure PCTCN2022113625-appb-000002
Figure PCTCN2022113625-appb-000003
Figure PCTCN2022113625-appb-000004
或者杂环胺如
Figure PCTCN2022113625-appb-000005
或者芳香胺如
Figure PCTCN2022113625-appb-000006
Figure PCTCN2022113625-appb-000007
R 2 is selected from hydroxyl –OH; alkane amines such as
Figure PCTCN2022113625-appb-000002
Figure PCTCN2022113625-appb-000003
Figure PCTCN2022113625-appb-000004
or heterocyclic amines such as
Figure PCTCN2022113625-appb-000005
or aromatic amines such as
Figure PCTCN2022113625-appb-000006
Figure PCTCN2022113625-appb-000007
n选自自然数2～5。n is selected from natural numbers 2-5.
本发明提供的一种三环二萜类似物，其结构式如式(2)～式(32)所示，A tricyclic diterpene analog provided by the present invention has a structural formula as shown in formula (2) to formula (32),
Figure PCTCN2022113625-appb-000008
Figure PCTCN2022113625-appb-000008
本发明还提供了一种制备三环二萜类似物的合成方法，包括以下：The present invention also provides a synthetic method for preparing tricyclic diterpene analogues, comprising the following:
(一)：式(2)～式(25)所示三环二萜类似物的制备：(1): Preparation of tricyclic diterpene analogs shown in formula (2) to formula (25):
以睾酮为起始原料，经甲基化、乙二醇保护、还原、氧化、Bayer-Villger氧化、还原、脱保护、乙酰化、消除、水解、氧化等反应得到母核化合物Q12，然后Q12经酰胺化得到式(2)所示三环二萜类似物即化合物2；或，以母核化合物Q12为原料，还原得到式(3)所 示三环二萜类似物即化合物3，然后式(3)化合物经酰胺化得到式(4)～式(25)所示三环二萜类似物，即化合物4～25，所述制备方法的总路线如(J)所示：Using testosterone as the starting material, the mother nucleus compound Q12 is obtained through reactions such as methylation, ethylene glycol protection, reduction, oxidation, Bayer-Villger oxidation, reduction, deprotection, acetylation, elimination, hydrolysis, and oxidation, and then Q12 is subjected to Amidation obtains the tricyclic diterpene analog shown in formula (2), i.e. compound 2; or, with the core compound Q12 as raw material, reduction obtains the tricyclic diterpene analog shown in formula (3), i.e. compound 3, and then the formula ( 3) Compounds are amidated to obtain tricyclic diterpene analogs shown in formula (4) to formula (25), that is, compounds 4 to 25, and the general route of the preparation method is shown in (J):
Figure PCTCN2022113625-appb-000009
Figure PCTCN2022113625-appb-000009
具体地，所述方法包括以下步骤：Specifically, the method includes the following steps:
a：4,4-二甲基化反应a: 4,4-dimethylation reaction
将化合物睾酮(Testosterone)溶于有机溶剂中，加入甲基化反应所用的甲基化试剂和碱，进行甲基化反应，得到化合物Q1。The compound testosterone (Testosterone) is dissolved in an organic solvent, and the methylation reagent and base used in the methylation reaction are added to carry out the methylation reaction to obtain the compound Q1.
步骤(a)中，所述有机溶剂选自t-BuOH、苯、四氯化碳、四氢呋喃等中的任意一种或多种；优选地，为t-BuOH。In step (a), the organic solvent is selected from any one or more of t-BuOH, benzene, carbon tetrachloride, tetrahydrofuran, etc.; preferably, it is t-BuOH.
步骤(a)中，所述碱选自t-BuOK、KOC(Et)Me 2等中的任意一种或多种；优选地，为t-BuOK。 In step (a), the base is selected from any one or more of t-BuOK, KOC(Et)Me 2 and the like; preferably, it is t-BuOK.
步骤(a)中，所述甲基化试剂为CH 3Cl、CH 3Br、CH 3I等中的任意一种或多种；优选地，为CH 3I。 In step (a), the methylating agent is any one or more of CH 3 Cl, CH 3 Br, CH 3 I, etc.; preferably, it is CH 3 I.
步骤(a)中，所述化合物睾酮与碱的摩尔比为1：(2～8)；优选地，为1：3。In step (a), the molar ratio of the compound testosterone to base is 1:(2-8); preferably, it is 1:3.
步骤(a)中，所述化合物睾酮与甲基化试剂的摩尔比为1：(2～10)；优选地，为1：6。In step (a), the molar ratio of the compound testosterone to the methylating agent is 1:(2-10); preferably, it is 1:6.
步骤(a)中，所述加入甲基化试剂期间反应的温度为0℃。In step (a), the reaction temperature during the addition of the methylating reagent is 0°C.
步骤(a)中，所述甲基化反应的温度为0～60℃；优选地，为45℃。In step (a), the temperature of the methylation reaction is 0-60°C; preferably, it is 45°C.
步骤(a)中，所述甲基化反应的时间为2～12h；优选地，为4h。In step (a), the time for the methylation reaction is 2-12 hours; preferably, 4 hours.
b：乙二醇保护反应b: Ethylene glycol protection reaction
将化合物Q1溶于有机溶剂，加入催化剂、吸水剂，反应得到化合物Q2。Dissolving compound Q1 in an organic solvent, adding a catalyst and a water-absorbing agent, and reacting to obtain compound Q2.
步骤(b)中，所述有机溶剂选自四氢呋喃、四氢呋喃和乙二醇的混合溶液等中的任意一种或多种；优选地，为四氢呋喃和乙二醇的混合溶液。In step (b), the organic solvent is selected from any one or more of tetrahydrofuran, a mixed solution of tetrahydrofuran and ethylene glycol, etc.; preferably, it is a mixed solution of tetrahydrofuran and ethylene glycol.
所述四氢呋喃与乙二醇的体积比为3：(1～3)；优选地，为3：1。The volume ratio of tetrahydrofuran to ethylene glycol is 3:(1-3); preferably, it is 3:1.
步骤(b)中，所述乙二醇既做为溶剂也作为反应物。In step (b), the ethylene glycol is used both as a solvent and as a reactant.
步骤(b)中，所述催化剂选自对甲苯磺酸、浓硫酸等中的任意一种或多种；优选地，为对甲苯磺酸。In step (b), the catalyst is selected from any one or more of p-toluenesulfonic acid, concentrated sulfuric acid, etc.; preferably, it is p-toluenesulfonic acid.
步骤(b)中，所述吸水剂选自原甲酸三乙酯、原甲酸三甲酯等中的任意一种或多种；优选地，为原甲酸三乙酯。In step (b), the water-absorbing agent is selected from any one or more of triethyl orthoformate, trimethyl orthoformate, etc.; preferably, it is triethyl orthoformate.
步骤(b)中，所述化合物Q1与催化剂、脱水剂的摩尔比为1：(0.2～0.5)：(5～10)；优选地，为1：0.35：5。In step (b), the molar ratio of the compound Q1 to the catalyst and the dehydrating agent is 1:(0.2-0.5):(5-10); preferably, it is 1:0.35:5.
步骤(b)中，所述乙二醇保护反应的时间为2～12h；优选地，为3h。In step (b), the time for the ethylene glycol protection reaction is 2-12 hours; preferably, 3 hours.
步骤(b)中，所述乙二醇保护反应的温度为25～60℃；优选地，为45℃。In step (b), the temperature of the ethylene glycol protection reaction is 25-60°C; preferably, 45°C.
c：还原反应c: reduction reaction
将化合物Q2溶于有机溶剂中，加入还原试剂，加压下进行还原反应得到化合物Q3。Compound Q2 is dissolved in an organic solvent, a reducing agent is added, and a reduction reaction is carried out under pressure to obtain Compound Q3.
步骤(c)中，所述有机溶剂选自甲醇、乙醇、四氢呋喃、二氯甲烷、乙酸乙酯等中的一种或多种；优选地，为四氢呋喃和甲醇的混合溶液。In step (c), the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, dichloromethane, ethyl acetate, etc.; preferably, it is a mixed solution of tetrahydrofuran and methanol.
所述四氢呋喃和甲醇混合溶液的体积比为2：(1～3)；优选地，为2：1The volume ratio of the mixed solution of tetrahydrofuran and methanol is 2:(1-3); preferably, it is 2:1
步骤(c)中，所述还原试剂选自钯碳、雷尼镍等中的一种或多种；优选地，为钯碳。In step (c), the reducing agent is selected from one or more of palladium carbon, Raney nickel, etc.; preferably, it is palladium carbon.
步骤(c)中，所述化合物Q2与还原试剂的质量比为1：(0.2～1)；优选地，为1：0.3。In step (c), the mass ratio of the compound Q2 to the reducing agent is 1:(0.2-1); preferably, it is 1:0.3.
步骤(c)中，所述反应压力为4MPa。In step (c), the reaction pressure is 4MPa.
步骤(c)中，所述还原反应的温度为10～60℃；优选地，为25℃。In step (c), the temperature of the reduction reaction is 10-60°C; preferably, 25°C.
步骤(c)中，所述还原反应的时间为24～72h；优选地，为48h。In step (c), the reduction reaction time is 24-72 hours; preferably, 48 hours.
d：氧化反应d: oxidation reaction
将化合物Q3溶于有机溶剂，加入氧化剂，反应得到化合物Q4。Compound Q3 is dissolved in an organic solvent, and an oxidizing agent is added to react to obtain Compound Q4.
步骤(d)中，所述有机溶剂选自DMSO、甲苯、四氢呋喃等中的任意一种或多种；优选 地，为DMSO和四氢呋喃的混合溶液。In step (d), the organic solvent is selected from any one or more of DMSO, toluene, tetrahydrofuran, etc.; preferably, it is a mixed solution of DMSO and tetrahydrofuran.
所述四氢呋喃和DMSO混合溶液的体积比为2：(1～3)；优选地，为2：1。The volume ratio of the mixed solution of tetrahydrofuran and DMSO is 2:(1-3); preferably, it is 2:1.
步骤(d)中，所述氧化剂为IBX、PCC、DDQ、PDC等中的任意一种或多种；优选地，为IBX。In step (d), the oxidizing agent is any one or more of IBX, PCC, DDQ, PDC, etc.; preferably, it is IBX.
步骤(d)中，化合物Q3与氧化剂的摩尔比为1：(1～3)；优选地，为1：2。In step (d), the molar ratio of compound Q3 to oxidizing agent is 1:(1-3); preferably, it is 1:2.
步骤(d)中，所述氧化反应的温度为0～50℃；优选地，为25℃。In step (d), the temperature of the oxidation reaction is 0-50°C; preferably, 25°C.
步骤(d)中，所述氧化反应的时间为3～8h；优选地，为5h。In step (d), the time for the oxidation reaction is 3-8 hours; preferably, 5 hours.
e：Bayer-Villiger氧化反应e: Bayer-Villiger oxidation reaction
将化合物Q4溶于有机溶剂，加入氧化剂、碱，反应得到化合物Q5。Compound Q4 is dissolved in an organic solvent, and an oxidizing agent and a base are added to react to obtain Compound Q5.
步骤(e)中，所述有机溶剂选自二氯甲烷、三氯甲烷等中的任意一种或多种；优选地，为二氯甲烷。In step (e), the organic solvent is selected from any one or more of dichloromethane, chloroform, etc.; preferably, it is dichloromethane.
步骤(e)中，所述氧化剂为间氯过氧苯甲酸、过氧乙酸、过硫酸氢钾、双氧水等中的任意一种或多种；优选地，为间氯过氧苯甲酸。In step (e), the oxidizing agent is any one or more of m-chloroperoxybenzoic acid, peracetic acid, potassium hydrogen persulfate, hydrogen peroxide, etc.; preferably, it is m-chloroperoxybenzoic acid.
步骤(e)中，所述碱选自NaHCO 3、Na 2CO 3、NaH 2PO 4等中任意一种或多种；优选地，为NaHCO 3。 In step (e), the base is selected from any one or more of NaHCO 3 , Na 2 CO 3 , NaH 2 PO 4 , etc.; preferably, it is NaHCO 3 .
步骤(e)中，所述化合物Q4与氧化剂的摩尔比为1：(1～10)；优选地，为1：2.5。In step (e), the molar ratio of the compound Q4 to the oxidizing agent is 1:(1-10); preferably, it is 1:2.5.
步骤(e)中，所述化合物Q4与碱的摩尔比为1：(1～10)；优选地，为1：2.7。In step (e), the molar ratio of the compound Q4 to the base is 1: (1-10); preferably, 1:2.7.
步骤(e)中，所述氧化反应的温度为10～50℃；优选地，为25℃。In step (e), the temperature of the oxidation reaction is 10-50°C; preferably, 25°C.
步骤(e)中，所述氧化反应的时间为3～12h；优选地，为5h。In step (e), the time for the oxidation reaction is 3-12 hours; preferably, 5 hours.
f：还原反应f: reduction reaction
将化合物Q5溶于有机溶剂，加入还原剂，反应得到化合物Q6。Compound Q5 is dissolved in an organic solvent, and a reducing agent is added to react to obtain Compound Q6.
步骤(f)中，所述有机溶剂选自四氢呋喃、乙醚、甲醇、乙醇等中的任意一种或多种；优选地，为四氢呋喃。In step (f), the organic solvent is selected from any one or more of tetrahydrofuran, diethyl ether, methanol, ethanol, etc.; preferably, it is tetrahydrofuran.
步骤(f)中，所述还原剂为LiAlH 4、红铝、二异丁基氢化铝等中的任意一种或多种；优选地，为LiAlH 4。 In step (f), the reducing agent is any one or more of LiAlH 4 , red aluminum, diisobutylaluminum hydride, etc.; preferably, it is LiAlH 4 .
步骤(f)中，所述化合物Q5与还原剂的摩尔比为1：(2～10)；优选地，为1：3。In step (f), the molar ratio of the compound Q5 to the reducing agent is 1:(2-10); preferably, it is 1:3.
步骤(f)中，所述还原反应的温度为0～25℃；优选地，为0℃。In step (f), the temperature of the reduction reaction is 0-25°C; preferably, 0°C.
步骤(f)中，所述还原反应的时间为0.5～2h；优选地，为0.5h。In step (f), the reduction reaction time is 0.5-2 hours; preferably, 0.5 hours.
g：脱乙二醇保护基g: removal of ethylene glycol protecting group
将化合物Q6溶于有机溶剂，加入酸，反应得到化合物Q7。Dissolve compound Q6 in an organic solvent, add acid, and react to obtain compound Q7.
步骤(g)中，所述有机溶剂选自甲醇、乙醇、四氢呋喃等中的一种或多种；优选地，为 四氢呋喃。In step (g), the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, etc.; preferably, it is tetrahydrofuran.
步骤(g)中，所述酸选自稀硫酸、稀盐酸、盐酸气、对甲苯磺酸等中的一种或多种；优选地，为2mol/L稀盐酸。In step (g), the acid is selected from one or more of dilute sulfuric acid, dilute hydrochloric acid, hydrochloric acid gas, p-toluenesulfonic acid, etc.; preferably, it is 2mol/L dilute hydrochloric acid.
步骤(g)中，所述有机溶剂与2mol/L稀盐酸的体积比为5：(1～2)；优选地，为5：1。In step (g), the volume ratio of the organic solvent to 2mol/L dilute hydrochloric acid is 5:(1-2); preferably, it is 5:1.
步骤(g)中，所述脱乙二醇保护基反应的温度为0～60℃；优选地，为25℃。In step (g), the temperature of the reaction of removing the ethylene glycol protecting group is 0-60°C; preferably, it is 25°C.
步骤(g)中，所述脱乙二醇保护基反应的时间为1～10h；优选地，为5h。In step (g), the reaction time for removing the ethylene glycol protecting group is 1-10 h; preferably, 5 h.
h：乙酰化反应h: acetylation reaction
将化合物Q7溶于有机溶剂，加入乙酰化试剂，反应得到化合物Q8。Compound Q7 is dissolved in an organic solvent, and an acetylating reagent is added to react to obtain Compound Q8.
步骤(h)中，所述有机溶剂选自吡啶、三乙胺、二乙胺、二异丙基乙基胺、四氢呋喃、DMF等中的一种或多种；优选地，为吡啶。In step (h), the organic solvent is selected from one or more of pyridine, triethylamine, diethylamine, diisopropylethylamine, tetrahydrofuran, DMF, etc.; preferably, it is pyridine.
步骤(h)中，所述乙酰化试剂选自乙酸酐、乙酰氯等中的一种或多种；优选地，为乙酸酐。In step (h), the acetylating agent is selected from one or more of acetic anhydride, acetyl chloride, etc.; preferably, it is acetic anhydride.
步骤(h)中，所述Q7和乙酰化试剂的摩尔比为1：(1～5)；优选地，为1：1.5。In step (h), the molar ratio of Q7 to the acetylating agent is 1:(1-5); preferably, it is 1:1.5.
步骤(h)中，所述乙酰化反应的温度为0～25℃；优选地，为0℃。In step (h), the temperature of the acetylation reaction is 0-25°C; preferably, 0°C.
步骤(h)中，所述乙酰化反应的时间为1～24h；优选地，为18h。In step (h), the time for the acetylation reaction is 1-24 hours; preferably, 18 hours.
i：消除反应i: Elimination reaction
将化合物Q8溶于有机溶剂，加入脱水剂、吸水剂，反应得到化合物Q9。Dissolving compound Q8 in an organic solvent, adding a dehydrating agent and a water-absorbing agent, and reacting to obtain compound Q9.
步骤(i)中，所述有机溶剂选自二氯甲烷、乙腈、丙酮、甲苯等中的一种或多种；优选地，为二氯甲烷。In step (i), the organic solvent is selected from one or more of dichloromethane, acetonitrile, acetone, toluene, etc.; preferably, it is dichloromethane.
步骤(i)中，所述脱水剂选自浓硫酸、磷酸、三甲硅基三氟甲磺酸酯(TMSOTf)等中的一种或多种；优选地，为三甲硅基三氟甲磺酸酯。In step (i), the dehydrating agent is selected from one or more of concentrated sulfuric acid, phosphoric acid, trimethylsilyl trifluoromethanesulfonate (TMSOTf) and the like; preferably, trimethylsilyl trifluoromethanesulfonic acid ester.
步骤(i)中，所述吸水剂为乙酸酐。In step (i), the water absorbing agent is acetic anhydride.
步骤(i)中，所述Q8和脱水剂的摩尔比为1：(0.1～0.4)；优选地，为1：0.12。In step (i), the molar ratio of Q8 to the dehydrating agent is 1:(0.1-0.4); preferably, it is 1:0.12.
步骤(i)中，所述Q8和吸水剂的摩尔比为1：(1～5)；优选地，为1：5。In step (i), the molar ratio of Q8 to the water-absorbing agent is 1:(1-5); preferably, it is 1:5.
步骤(i)中，所述消除反应的温度为-10℃～25℃；优选地，为0℃。In step (i), the temperature of the elimination reaction is -10°C to 25°C; preferably, it is 0°C.
步骤(i)中，所述消除反应的时间为0.25～1h；优选地，为0.5h。In step (i), the time for the elimination reaction is 0.25-1 h; preferably, 0.5 h.
j：水解反应j: hydrolysis reaction
将化合物Q9溶于有机溶剂，加入碱，反应得到化合物Q10。Compound Q9 is dissolved in an organic solvent, and a base is added to react to obtain Compound Q10.
步骤(j)中，所述有机溶剂选自甲醇、四氢呋喃、乙醇等中的任意一种或多种；优选地，为甲醇。In step (j), the organic solvent is selected from any one or more of methanol, tetrahydrofuran, ethanol, etc.; preferably, it is methanol.
步骤(j)中，所述碱选自氢氧化锂、氢氧化钠、氢氧化钾、碳酸钾等中的任意一种或多 种；优选地，为碳酸钾。In step (j), the base is selected from any one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, etc.; preferably, potassium carbonate.
步骤(j)中，所述化合物Q9与碱的摩尔比为1：(1～5)；优选地，为1：4。In step (j), the molar ratio of the compound Q9 to the base is 1:(1-5); preferably, it is 1:4.
步骤(j)中，所述水解反应的温度为10～70℃；优选地，为25℃。In step (j), the temperature of the hydrolysis reaction is 10-70°C; preferably, 25°C.
步骤(j)中，所述水解反应的时间为1～12h；优选地，为3h。In step (j), the time for the hydrolysis reaction is 1-12 hours; preferably, it is 3 hours.
k：氧化反应k: oxidation reaction
将化合物Q10溶于有机溶剂，加入氧化剂，反应得到化合物Q11。Compound Q10 is dissolved in an organic solvent, and an oxidizing agent is added to react to obtain Compound Q11.
步骤(k)中，所述有机溶剂选自DMSO、甲苯、四氢呋喃等中的任意一种或多种；优选地，为DMSO和四氢呋喃的混合溶液。In step (k), the organic solvent is selected from any one or more of DMSO, toluene, tetrahydrofuran, etc.; preferably, it is a mixed solution of DMSO and tetrahydrofuran.
所述四氢呋喃和DMSO混合溶液的体积比为2：(1～3)；优选地，为2：1。The volume ratio of the mixed solution of tetrahydrofuran and DMSO is 2:(1-3); preferably, it is 2:1.
步骤(k)中，所述氧化剂为IBX、PCC、DDQ、PDC等中的任意一种或多种；优选地，为IBX。In step (k), the oxidizing agent is any one or more of IBX, PCC, DDQ, PDC, etc.; preferably, it is IBX.
步骤(k)中，化合物Q10与氧化剂的摩尔比为1：(1～2)；优选地，为1：2。In step (k), the molar ratio of compound Q10 to oxidizing agent is 1:(1-2); preferably, it is 1:2.
步骤(k)中，所述氧化反应的温度为0～50℃；优选地，为25℃。In step (k), the temperature of the oxidation reaction is 0-50°C; preferably, 25°C.
步骤(k)中，所述氧化反应的时间为1～3h；优选地，为1h。In step (k), the time for the oxidation reaction is 1 to 3 hours; preferably, 1 hour.
l：氧化反应l: Oxidation reaction
将化合物Q11溶于有机溶剂，加入氧化剂，反应得到化合物Q12。Compound Q11 is dissolved in an organic solvent, and an oxidizing agent is added to react to obtain Compound Q12.
步骤(l)中，所述有机溶剂选自吡啶、三氯甲烷、二氯乙烷、1，2-二氯丙烷等中的任意一种或多种；优选地，为吡啶。In step (1), the organic solvent is selected from any one or more of pyridine, chloroform, dichloroethane, 1,2-dichloropropane, etc.; preferably, it is pyridine.
步骤(l)中，所述氧化剂选自四丁基高锰酸铵、高锰酸钾、重铬酸钾、O 3等中的任意一种或多种；优选地，为四丁基高锰酸铵。 In step (1), the oxidant is selected from any one or more of tetrabutylammonium permanganate, potassium permanganate, potassium dichromate, O etc .; preferably, tetrabutyl permanganese ammonium acid.
步骤(l)中，所述Q11与氧化剂的摩尔比为1：(1～4)；优选地，为1：2。In step (l), the molar ratio of Q11 to the oxidizing agent is 1:(1-4); preferably, it is 1:2.
步骤(l)中，所述氧化反应的温度为0～60℃；优选地，为0℃。In step (l), the temperature of the oxidation reaction is 0-60°C; preferably, 0°C.
步骤(l)中，所述氧化反应的时间为1～2h；优选地，为1h。In step (l), the time for the oxidation reaction is 1 to 2 hours; preferably, 1 hour.
m：酰胺化反应m: amidation reaction
将化合物Q12溶于有机溶剂中，加入HATU、DIPEA及相应的胺，得到化合物2。Compound Q12 is dissolved in an organic solvent, and HATU, DIPEA and the corresponding amine are added to obtain compound 2.
步骤(m)中，所述有机溶剂选自DCM、DMF、四氢呋喃、氯仿等中的任意一种或多种；优选地，为DCM。In step (m), the organic solvent is selected from any one or more of DCM, DMF, tetrahydrofuran, chloroform, etc.; preferably, it is DCM.
步骤(m)中，所述HATU、DIPEA的作用为促进酰胺化反应。In step (m), the function of the HATU and DIPEA is to promote the amidation reaction.
步骤(m)中，所述化合物Q12、HATU、DIPEA及相应的胺的摩尔比为1：(1～2)：(1～2)：(2～4)；优选地，为1：1.2：1.5：2。In step (m), the molar ratio of the compound Q12, HATU, DIPEA and the corresponding amine is 1:(1~2):(1~2):(2~4); preferably, 1:1.2: 1.5:2.
步骤(m)中，所述酰胺化反应的温度为0～40℃；优选地，为25℃。In step (m), the amidation reaction temperature is 0-40°C; preferably, 25°C.
步骤(m)中，所述酰胺化反应的时间为0.5～2h；优选地，为1h。In step (m), the amidation reaction time is 0.5-2 hours; preferably, 1 hour.
n：还原反应n: reduction reaction
化合物Q12溶于有机溶剂，加入还原剂，反应得到化合物3。Compound Q12 is dissolved in an organic solvent, and a reducing agent is added to react to obtain Compound 3.
步骤(n)中，所述有机溶剂选自二氯甲烷、甲醇、二氯甲烷和甲醇的混合溶液等中的一种或多种；优选地，为甲醇和二氯甲烷的混合溶液。In step (n), the organic solvent is selected from one or more of dichloromethane, methanol, a mixed solution of dichloromethane and methanol, etc.; preferably, it is a mixed solution of methanol and dichloromethane.
所述甲醇和二氯甲烷的混合溶液的体积比为1：(0.5～3)；优选地；为1：2。The volume ratio of the mixed solution of methanol and dichloromethane is 1:(0.5-3); preferably 1:2.
步骤(n)中，所述还原剂选自硼氢化钠、硼氢化钾等中的一种或多种；优选地，为硼氢化钠。In step (n), the reducing agent is selected from one or more of sodium borohydride, potassium borohydride, etc.; preferably, sodium borohydride.
步骤(n)中，所述化合物Q12与还原剂的摩尔比为1：(5～20)；优选地，为1：10。In step (n), the molar ratio of the compound Q12 to the reducing agent is 1:(5-20); preferably, it is 1:10.
步骤(n)中，所述还原反应的温度为0～50℃；优选地，为0℃。In step (n), the temperature of the reduction reaction is 0-50°C; preferably, 0°C.
步骤(n)中，所述还原反应的时间为0.5～4h；优选地，为0.5h。In step (n), the reduction reaction time is 0.5-4 hours; preferably, 0.5 hours.
o:酰胺化反应o: amidation reaction
化合物4～25的合成类似于路线(J)步骤(m)中化合物2的酰胺化方法。The synthesis of compounds 4-25 is similar to the amidation method of compound 2 in step (m) of route (J).
所述路线(J)还包含路线(A)、路线(E)、路线(F)，具体如下：Described route (J) also comprises route (A), route (E), route (F), specifically as follows:
Figure PCTCN2022113625-appb-000010
Figure PCTCN2022113625-appb-000010
Figure PCTCN2022113625-appb-000011
Figure PCTCN2022113625-appb-000011
(二)：式(26)所示三环二萜类似物的制备(2): Preparation of tricyclic diterpene analogs shown in formula (26)
本发明还提出了一种三环二萜类似物的制备方法，以化合物Q10为起始原料，经过磺酰化、取代(氰基化)、水解、还原、酰胺化反应得到式(26)所示三环二萜类似物，即化合物26，所述制备方法如路线(K)所示：The present invention also proposes a preparation method of tricyclic diterpene analogues. Compound Q10 is used as a starting material to obtain the compound of formula (26) through sulfonylation, substitution (cyanation), hydrolysis, reduction, and amidation reactions. Show tricyclic diterpene analogue, i.e. compound 26, and described preparation method is as shown in route (K):
Figure PCTCN2022113625-appb-000012
Figure PCTCN2022113625-appb-000012
具体地，所述方法包括以下步骤：Specifically, the method includes the following steps:
a：磺酰化反应a: Sulfonylation reaction
化合物Q10溶于有机溶剂，加入甲磺酰氯、缚酸剂，反应得到化合物Q13。Compound Q10 is dissolved in an organic solvent, and methanesulfonyl chloride and an acid-binding agent are added to react to obtain compound Q13.
步骤(a)中，所述有机溶剂选自二氯甲烷、四氢呋喃等中的一种或多种；优选地，为二氯甲烷。In step (a), the organic solvent is selected from one or more of dichloromethane, tetrahydrofuran, etc.; preferably, it is dichloromethane.
步骤(a)中，所述缚酸剂选自三乙胺、三丁胺、吡啶等中的一种或多种；优选地，为三乙胺。In step (a), the acid-binding agent is selected from one or more of triethylamine, tributylamine, pyridine, etc.; preferably, it is triethylamine.
步骤(a)中，所述化合物Q10与甲磺酰氯的摩尔比为1：(1～5)；优选地，为1：3。In step (a), the molar ratio of compound Q10 to methanesulfonyl chloride is 1:(1-5); preferably, it is 1:3.
步骤(a)中，所述化合物Q10与缚酸剂的摩尔比为1：(1～5)；优选地，为1：4。In step (a), the molar ratio of the compound Q10 to the acid-binding agent is 1:(1-5); preferably, it is 1:4.
步骤(a)中，所述磺酰化反应的温度为-10～25℃；优选地，为0℃。In step (a), the temperature of the sulfonylation reaction is -10-25°C; preferably, it is 0°C.
步骤(a)中，所述磺酰化反应的时间为1～4h；优选地，为1h。In step (a), the time for the sulfonylation reaction is 1 to 4 hours; preferably, 1 hour.
b：取代(氰基化)反应b: Substitution (cyanation) reaction
化合物Q13溶于有机溶剂，加入氰基化试剂、四丁基氟化铵，反应得到化合物Q14。Compound Q13 is dissolved in an organic solvent, and compound Q14 is obtained by adding a cyanation reagent and tetrabutylammonium fluoride.
步骤(b)中，所述有机溶剂选自DMF、四氢呋喃、乙腈等中的一种或多种；优选地，为乙腈。In step (b), the organic solvent is selected from one or more of DMF, tetrahydrofuran, acetonitrile, etc.; preferably, it is acetonitrile.
步骤(b)中，所述氰基化试剂选自氰化钠、三甲基硅氰等中的一种或多种；优选地，为三甲基硅氰。In step (b), the cyanation agent is selected from one or more of sodium cyanide, trimethylsilylcyanide, etc.; preferably, it is trimethylsilylcyanide.
步骤(b)中，四丁基氟化铵的作用为促进TMS基团离去，促进氰基化反应。In step (b), the function of tetrabutylammonium fluoride is to promote the departure of TMS group and promote the cyanation reaction.
步骤(b)中，所述化合物Q13与氰基化试剂的摩尔比为1：(1～10)；优选地，为1：1.5。In step (b), the molar ratio of the compound Q13 to the cyanation reagent is 1:(1-10); preferably, it is 1:1.5.
步骤(b)中，所述化合物Q13与四丁基氟化铵的摩尔比为1：(1～10)；优选地，为1：1.5。In step (b), the molar ratio of compound Q13 to tetrabutylammonium fluoride is 1:(1-10); preferably, it is 1:1.5.
步骤(b)中，所述氰基化反应的温度为0～85℃；优选地，为82℃。In step (b), the temperature of the cyanation reaction is 0-85°C; preferably, it is 82°C.
步骤(b)中，所述氰基化反应的时间为0.5～4h；优选地，为0.5h。In step (b), the time for the cyanation reaction is 0.5-4 hours; preferably, 0.5 hours.
c：水解反应c: hydrolysis reaction
化合物Q14溶于有机溶剂，加入碱，反应得到化合物Q15。Compound Q14 is dissolved in an organic solvent, and a base is added to react to obtain Compound Q15.
步骤(c)中，所述有机溶剂选自乙醇、乙二醇、甲醇等中的一种或多种；优选地，为乙二醇。In step (c), the organic solvent is selected from one or more of ethanol, ethylene glycol, methanol, etc.; preferably, it is ethylene glycol.
步骤(c)中，所述碱选自氢氧化锂、氢氧化钠、氢氧化钾、碳酸钾等中的任意一种或多种；优选地，为氢氧化钾。In step (c), the base is selected from any one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, etc.; preferably, it is potassium hydroxide.
步骤(c)中，所述化合物Q14与碱的摩尔比为1：(5～15)；优选地，为1：10。In step (c), the molar ratio of the compound Q14 to the base is 1:(5-15); preferably, it is 1:10.
步骤(c)中，所述水解反应的温度为60～170℃；优选地，为170℃。In step (c), the temperature of the hydrolysis reaction is 60-170°C; preferably, it is 170°C.
步骤(c)中，所述水解反应的时间为6～24h；优选地，为12h。In step (c), the time for the hydrolysis reaction is 6-24 hours; preferably, it is 12 hours.
d：还原反应d: reduction reaction
化合物Q16的合成类似于路线(J)步骤(n)中化合物3的还原反应方法。The synthesis of compound Q16 is similar to the reduction reaction method of compound 3 in step (n) of route (J).
e：酰胺化反应e: amidation reaction
化合物26的合成类似于路线(J)步骤(m)中化合物2的酰胺化方法。The synthesis of compound 26 is similar to the amidation method of compound 2 in step (m) of route (J).
所述路线(K)还包含路线(B)、路线(G)，具体如下：Described route (K) also comprises route (B), route (G), specifically as follows:
Figure PCTCN2022113625-appb-000013
Figure PCTCN2022113625-appb-000013
(三)：式(27)～式(29)所示三环二萜类似物的制备(3): Preparation of tricyclic diterpene analogs shown in formula (27) to formula (29)
本发明还提出了一种三环二萜类似物的制备方法，以化合物Q11为起始原料，经过Wittig反应、还原、水解、酰胺化反应得到式(27)～式(29)所示三环二萜类似物，即化合物27～29，所述制备方法如路线(L)所示：The present invention also proposes a preparation method of tricyclic diterpene analogues, using compound Q11 as a starting material, through Wittig reaction, reduction, hydrolysis, and amidation reactions to obtain tricyclic diterpene compounds represented by formula (27) to formula (29). Diterpene analogs, i.e. compounds 27-29, the preparation method is shown in route (L):
Figure PCTCN2022113625-appb-000014
Figure PCTCN2022113625-appb-000014
具体地，所述方法包括以下步骤：Specifically, the method includes the following steps:
a：Wittig反应a: Wittig reaction
化合物Q11溶于有机溶剂，加入碱、Wittig试剂，反应得到化合物Q17。Compound Q11 is dissolved in an organic solvent, and a base and Wittig reagent are added to react to obtain Compound Q17.
步骤(a)中，所述有机溶剂选自四氢呋喃、甲苯、石油醚等中的任意一种或多种；优选地，为四氢呋喃。In step (a), the organic solvent is selected from any one or more of tetrahydrofuran, toluene, petroleum ether, etc.; preferably, it is tetrahydrofuran.
步骤(a)中，所述碱为甲醇钠、乙醇钠、氢化钠(60％)中的一种或多种；优选地，为 氢化钠(60％)。In step (a), the alkali is one or more of sodium methoxide, sodium ethoxide, and sodium hydride (60%); preferably, it is sodium hydride (60%).
步骤(a)中，所述Wittig试剂优选为磷酰基乙酸三乙酯。In step (a), the Wittig reagent is preferably triethyl phosphoroacetate.
步骤(a)中，所述化合物Q11与碱、Wittig试剂的摩尔比为1：(3～8)：(3～8)；优选地，为1：5.5：5。In step (a), the molar ratio of the compound Q11 to the base and the Wittig reagent is 1:(3-8):(3-8); preferably, it is 1:5.5:5.
步骤(a)中，所述Wittig反应的温度为-10℃～25℃；优选地，为0℃。In step (a), the temperature of the Wittig reaction is -10°C to 25°C; preferably, it is 0°C.
步骤(a)中，所述Wittig反应的时间为20～60min；优选地，为20min。In step (a), the time for the Wittig reaction is 20-60 minutes; preferably, 20 minutes.
b：还原反应b: reduction reaction
将化合物Q17溶于有机溶剂中，加入还原剂，反应得到化合物Q18。Compound Q17 is dissolved in an organic solvent, and a reducing agent is added to react to obtain Compound Q18.
步骤(b)中，所述有机溶剂选自甲醇、乙醇、四氢呋喃、二氯甲烷、乙酸乙酯等中的一种或多种；优选地，为四氢呋喃。In step (b), the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, dichloromethane, ethyl acetate, etc.; preferably, tetrahydrofuran.
步骤(b)中，所述还原剂选自钯碳、雷尼镍等中的一种或多种；优选地，为钯碳。In step (b), the reducing agent is selected from one or more of palladium carbon, Raney nickel, etc.; preferably, it is palladium carbon.
步骤(b)中，所述化合物Q17与还原剂的质量比为1：(0.05～1)；优选地，为1：0.1。In step (b), the mass ratio of the compound Q17 to the reducing agent is 1:(0.05-1); preferably, it is 1:0.1.
步骤(b)中，所述反应压力为1atm。In step (b), the reaction pressure is 1 atm.
步骤(b)中，所述还原反应的温度为10～60℃；优选地，为25℃。In step (b), the temperature of the reduction reaction is 10-60°C; preferably, 25°C.
步骤(b)中，所述还原反应的时间为0.5～10h；优选地，为1h。In step (b), the time for the reduction reaction is 0.5-10 h; preferably, 1 h.
c：还原反应c: reduction reaction
化合物Q19的合成类似于路线(J)步骤(n)中化合物3的还原反应方法。The synthesis of compound Q19 is similar to the reduction reaction method of compound 3 in step (n) of route (J).
d：水解反应d: Hydrolysis reaction
将化合物Q19溶于有机溶剂中，加入碱，反应得到化合物Q20。Compound Q19 is dissolved in an organic solvent, and a base is added to react to obtain compound Q20.
步骤(d)中，所述有机溶剂选自甲醇、乙醇、四氢呋喃等中的一种或多种；优选地，为甲醇。In step (d), the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, etc.; preferably, it is methanol.
步骤(d)中，所述碱选自氢氧化锂、氢氧化钠、氢氧化钾、碳酸钾等中的任意一种或多种；优选地，为氢氧化钠。In step (d), the base is selected from any one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, etc.; preferably, it is sodium hydroxide.
步骤(d)中，所述化合物Q19与碱的摩尔比为1：(1～10)；优选地，为1：10。In step (d), the molar ratio of the compound Q19 to the base is 1:(1-10); preferably, it is 1:10.
步骤(d)中，所述水解反应的温度为10～60℃；优选地，为25℃。In step (d), the temperature of the hydrolysis reaction is 10-60°C; preferably, 25°C.
步骤(d)中，所述水解反应的时间为5～12h；优选地，为9h。In step (d), the time for the hydrolysis reaction is 5-12 hours; preferably, 9 hours.
e：酰胺化反应e: amidation reaction
化合物27～29的合成类似于路线(J)步骤(m)中化合物2的酰胺化反应方法。The synthesis of compounds 27-29 is similar to the amidation reaction method of compound 2 in step (m) of route (J).
所述路线(L)还包含路线(C)、路线(H)，具体如下：Described route (L) also comprises route (C), route (H), specifically as follows:
Figure PCTCN2022113625-appb-000015
Figure PCTCN2022113625-appb-000015
(四)：式(30)～式(32)所示三环二萜类似物的制备(4): Preparation of tricyclic diterpene analogs shown in formula (30) to formula (32)
本发明还提出了一种三环二萜类似物的制备方法，以化合物Q10为起始原料，经过乙二醇保护、氧化、Wittig反应、还原、脱保护、还原、酰胺化反应得到式(30)～式(32)所示三环二萜类似物，即化合物30～32，所述制备方法如路线(M)所示：The present invention also proposes a preparation method of tricyclic diterpene analogues, using compound Q10 as a starting material, through ethylene glycol protection, oxidation, Wittig reaction, reduction, deprotection, reduction, amidation reactions to obtain the formula (30 )～tricyclic diterpene analogs shown in formula (32), i.e. compounds 30～32, the preparation method is shown in route (M):
Figure PCTCN2022113625-appb-000016
Figure PCTCN2022113625-appb-000016
具体地，所述方法包括以下步骤：Specifically, the method includes the following steps:
a：乙二醇保护反应a: Ethylene glycol protection reaction
将化合物Q10溶于有机溶剂，加入催化剂、吸水剂、乙二醇，反应得到化合物Q21。Dissolving compound Q10 in an organic solvent, adding catalyst, water absorbing agent, and ethylene glycol, and reacting to obtain compound Q21.
步骤(a)中，所述乙二醇只作为反应物。In step (a), the ethylene glycol is only used as a reactant.
步骤(a)中，所述有机溶剂选自四氢呋喃、甲苯等中的一种或多种；优选地，为甲苯。In step (a), the organic solvent is selected from one or more of tetrahydrofuran, toluene, etc.; preferably, it is toluene.
步骤(a)中，所述催化剂选自为对甲苯磺酸、浓硫酸等中的任意一种或多种；优选地，为对甲苯磺酸。In step (a), the catalyst is selected from any one or more of p-toluenesulfonic acid, concentrated sulfuric acid, etc.; preferably, p-toluenesulfonic acid.
步骤(a)中，所述吸水剂选自原甲酸三乙酯、原甲酸三甲酯等中的任意一种或多种；优选地，为原甲酸三乙酯。In step (a), the water-absorbing agent is selected from any one or more of triethyl orthoformate, trimethyl orthoformate, etc.; preferably, it is triethyl orthoformate.
步骤(a)中，所述化合物Q10与乙二醇、催化剂、吸水剂的摩尔比为1：(1～10)：(0.01～0.5)：(1～10)；优选地，为1：3：0.05：3。In step (a), the molar ratio of compound Q10 to ethylene glycol, catalyst, and water absorbing agent is 1: (1-10): (0.01-0.5): (1-10); preferably, 1:3 :0.05:3.
步骤(a)中，所述乙二醇保护反应的温度为25～60℃；优选地，为25℃。In step (a), the temperature of the ethylene glycol protection reaction is 25-60°C; preferably, 25°C.
步骤(a)中，所述乙二醇保护反应的时间为1～12h；优选地，为1h。In step (a), the time for the ethylene glycol protection reaction is 1 to 12 hours; preferably, 1 hour.
b：氧化反应b: oxidation reaction
化合物Q22的合成类似于路线(J)步骤(k)中Q11的氧化反应方法。The synthesis of compound Q22 is similar to the oxidation reaction method of Q11 in step (k) of route (J).
c：Wittig反应c: Wittig reaction
将季磷盐、碱溶于有机溶剂中，向其中滴加Q22的溶液，经Wittig反应得到化合物Q23。Dissolve quaternary phosphorus salt and alkali in an organic solvent, add the solution of Q22 dropwise thereto, and obtain compound Q23 through Wittig reaction.
步骤(c)中，所述有机溶剂选自四氢呋喃、甲苯、石油醚等中的任意一种或多种；优选地，为四氢呋喃。In step (c), the organic solvent is selected from any one or more of tetrahydrofuran, toluene, petroleum ether, etc.; preferably, it is tetrahydrofuran.
步骤(c)中，所述季磷盐为(2-羧乙基)三苯基溴化膦。In step (c), the quaternary phosphorus salt is (2-carboxyethyl)triphenylphosphine bromide.
步骤(c)中，所述碱选自三乙胺、氢氧化钠、六甲基二硅胺钠(NaHDMS)等中的任意一种或多种，优选地，为六甲基二硅胺钠。In step (c), the base is selected from any one or more of triethylamine, sodium hydroxide, sodium hexamethyldisilazide (NaHDMS), etc., preferably, sodium hexamethyldisilazide .
步骤(c)中，所述化合物Q22与季磷盐、碱的摩尔比为1：(1～5)：(1～5)；优选地，为1：3.5：3。In step (c), the molar ratio of the compound Q22 to the quaternary phosphorus salt and the base is 1:(1-5):(1-5); preferably, it is 1:3.5:3.
步骤(c)中，所述Wittig反应的时间为5～30min；优选地，为10min。In step (c), the time for the Wittig reaction is 5-30 minutes; preferably, 10 minutes.
步骤(c)中，所述Wittig反应的温度为-20℃～25℃；优选地，为-20℃。In step (c), the temperature of the Wittig reaction is -20°C to 25°C; preferably, it is -20°C.
d：还原反应d: reduction reaction
化合物Q24的合成类似于路线(L)步骤(b)中Q18的合成方法。The synthesis of compound Q24 is similar to the synthesis method of Q18 in step (b) of route (L).
e：脱乙二醇保护基e: removal of ethylene glycol protecting group
将化合物Q24溶于有机溶剂中，加入酸，反应得到化合物Q25。Dissolve compound Q24 in an organic solvent, add acid, and react to obtain compound Q25.
步骤(e)中，所述有机溶剂选自甲醇、乙醇、四氢呋喃等中的一种或多种；优选地，为 四氢呋喃。In step (e), the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, etc.; preferably, it is tetrahydrofuran.
步骤(e)中，所述酸选自稀硫酸、稀盐酸、盐酸气、对甲苯磺酸等中的一种或多种；优选地，为2mol/L稀盐酸。In step (e), the acid is selected from one or more of dilute sulfuric acid, dilute hydrochloric acid, hydrochloric acid gas, p-toluenesulfonic acid, etc.; preferably, it is 2mol/L dilute hydrochloric acid.
步骤(d)中，所述有机溶剂与2mol/L稀盐酸的体积比为1：(1～2)；优选地，为1：1。In step (d), the volume ratio of the organic solvent to 2mol/L dilute hydrochloric acid is 1:(1-2); preferably, it is 1:1.
步骤(e)中，所述脱乙二醇保护基反应的时间为1～5h；优选地，为4h。In step (e), the reaction time for removing the ethylene glycol protecting group is 1-5 hours; preferably, it is 4 hours.
步骤(e)中，所述脱乙二醇保护基反应的温度为0～60℃；优选地，为25℃。In step (e), the temperature of the reaction of removing the ethylene glycol protecting group is 0-60°C; preferably, it is 25°C.
f：还原反应f: reduction reaction
化合物Q26的合成方法类似于路线(J)步骤(n)中化合物3的还原方法。The synthesis method of compound Q26 is similar to the reduction method of compound 3 in step (n) of route (J).
g：酰胺化反应g: amidation reaction
化合物30～32的合成类似于路线(J)步骤(m)中化合物2的合成方法。The synthesis of compounds 30-32 is similar to the synthesis method of compound 2 in step (m) of route (J).
所述路线(M)还包含路线(D)、路线(I)，具体如下：Described route (M) also comprises route (D), route (I), specifically as follows:
Figure PCTCN2022113625-appb-000017
Figure PCTCN2022113625-appb-000017
本发明制备方法中，上述反应通过薄板层析法跟踪测定反应的进度，反应完毕后采用的后处理方法包括浓缩、萃取、柱层析分离等步骤，最终产物以核磁共振谱及高分辨质谱来验证。In the preparation method of the present invention, the progress of the reaction is tracked and determined by thin plate chromatography. After the reaction is completed, the post-processing method includes steps such as concentration, extraction, and column chromatography separation. The final product is obtained by nuclear magnetic resonance spectrum and high-resolution mass spectrum verify.
本发明还提供了一种药物组合物，其包含如上所述的三环二萜类似物，及药学上可接受 的载体。The present invention also provides a pharmaceutical composition, which comprises the above-mentioned tricyclic diterpene analogs, and a pharmaceutically acceptable carrier.
本发明还提供了所述SMO抑制剂三环二萜类似物或药物组合物在制备SMO抑制剂中的应用。The present invention also provides the application of the SMO inhibitor tricyclic diterpene analogue or the pharmaceutical composition in preparing the SMO inhibitor.
所述三环二萜类似物或药物组合物用于抑制野生型SMO蛋白和耐药性SMO蛋白(D473H；E518K；N219D；L221R；D384N；S387N)的活性。The tricyclic diterpene analog or pharmaceutical composition is used to inhibit the activity of wild-type SMO protein and drug-resistant SMO protein (D473H; E518K; N219D; L221R; D384N; S387N).
本发明还提供了所述三环二萜类似物或药物组合物在制备抗肿瘤药物中的应用。The present invention also provides the application of the tricyclic diterpene analog or the pharmaceutical composition in the preparation of antitumor drugs.
所述三环二萜类似物或药物组合物用于抑制肿瘤的生长、增殖、转移，或用于促进肿瘤的凋亡和灭亡。The tricyclic diterpene analog or pharmaceutical composition is used for inhibiting tumor growth, proliferation and metastasis, or for promoting tumor apoptosis and death.
所述肿瘤包括髓母细胞瘤、基底细胞癌等。Such tumors include medulloblastoma, basal cell carcinoma, and the like.
本发明涉及的三环二萜类似物通过抑制SMO活性从而抑制Hh信号通路，对相关髓母细胞瘤细胞的生长也有较好抑制能力，具有良好的抗肿瘤应用前景。The tricyclic diterpene analogs involved in the present invention inhibit the Hh signaling pathway by inhibiting the activity of SMO, and also have good inhibitory ability to the growth of related medulloblastoma cells, and have good anti-tumor application prospects.
本发明涉及的抗肿瘤三环二萜类似物及其制备方法的优点包括，在自有化合物库中筛选得到了具有SMO活性的三环二萜先导物，并进行了结构修饰，通过酰胺化、延长碳链等反应合成了一系列结构新颖的三环二萜类似物；本发明提供的化合物不仅可以抑制野生型SMO蛋白，而且可以抑制耐药性SMO蛋白的活性，可解决现有临床上SMO抑制剂的耐药性问题。The advantages of the anti-tumor tricyclic diterpene analog and the preparation method thereof of the present invention include that the tricyclic diterpene lead with SMO activity has been screened in the self-owned compound library, and the structure has been modified, through amidation, A series of tricyclic diterpene analogues with novel structures were synthesized through reactions such as carbon chain extension; the compounds provided by the invention can not only inhibit the wild-type SMO protein, but also inhibit the activity of drug-resistant SMO protein, which can solve the problem of existing clinical SMO Inhibitor resistance issues.
附图说明Description of drawings
图1为本发明化合物8对耐药性SMO蛋白的胆固醇修饰的影响及IC 50的测定。其中，图a为SMO跨膜区段的拓朴结构图，深灰色为WT序列，浅灰色为突变序列。图b为SMO的D95N突变对SMO抗药突变的胆固醇修饰的影响。图c为化合物8对SMO抗药突变的胆固醇修饰的影响。图d和图e为维莫德吉，索尼德吉以及化合物8对耐药性SMO蛋白的IC 50的测定。 Figure 1 shows the effect of Compound 8 of the present invention on cholesterol modification of drug-resistant SMO protein and the determination of IC 50 . Among them, Figure a is the topological structure diagram of the transmembrane segment of SMO, the dark gray is the WT sequence, and the light gray is the mutant sequence. Panel b shows the effect of the D95N mutation of SMO on the cholesterol modification of the SMO drug-resistant mutation. Panel c is the effect of compound 8 on cholesterol modification of SMO drug-resistant mutations. Panel d and panel e are the determination of IC 50 of vimodeji, sonideji and compound 8 on drug-resistant SMO protein.
图2为本发明化合物8结合SMO蛋白N端的半胱氨酸富集区段。其中，图a为化合物8可以体外抑制SMO和胆固醇的结合。图b为化合物8可以抑制SMO的胆固醇修饰。图c和图d为化合物8不影响Bodipy-Cyclopamine和SMO的结合。图e为化合物8和SMO 7TM抑制剂(维莫德吉，索尼德吉，环靶眀)的联合用药。图f为化合物8抑制SMO和胆固醇结合的模型图。Fig. 2 shows that compound 8 of the present invention binds to the cysteine-rich segment of the N-terminus of SMO protein. Among them, Figure a shows that compound 8 can inhibit the binding of SMO and cholesterol in vitro. Figure b shows that compound 8 can inhibit the cholesterol modification of SMO. Figures c and d show that compound 8 does not affect the combination of Bodipy-Cyclopamine and SMO. Figure e shows the combination of compound 8 and SMO 7TM inhibitors (vimodeji, sonideji, and Huantarget). Figure f is a model diagram of compound 8 inhibiting the binding of SMO and cholesterol.
图3为本发明化合物8抑制小脑神经元祖细胞和髓母细胞瘤细胞的生长。其中，图a为c GNP细胞生长实验的流程图。图b为化合物8抑制小脑神经元祖细胞生长的免疫荧光图片。图c和图d为图b的相关定量分析。图e为化合物8抑制髓母细胞瘤细胞生长的免疫荧光图片。图f和图g为图e的相关定量分析。Figure 3 shows that Compound 8 of the present invention inhibits the growth of cerebellar neuron progenitor cells and medulloblastoma cells. Among them, Figure a is the flow chart of c GNP cell growth experiment. Figure b is the immunofluorescence picture of compound 8 inhibiting the growth of cerebellar neuron progenitor cells. Panel c and panel d are the related quantitative analysis of panel b. Panel e is the immunofluorescence picture of compound 8 inhibiting the growth of medulloblastoma cells. Figures f and g are related quantitative analysis of figure e.
图4为本发明化合物8对Hh依赖型的髓母细胞瘤的抑制效果。其中，图a为给药期间 肿瘤大小的测定。图b为给药期间小鼠体重的测定。图c和图d为给药后提取的小鼠肿瘤的Gli1 mRNA和蛋白质印迹的结果。图e为给药后提取的小鼠肿瘤的图片。Fig. 4 shows the inhibitory effect of Compound 8 of the present invention on Hh-dependent medulloblastoma. Wherein, figure a is the measurement of tumor size during administration. Figure b is the determination of the body weight of the mice during the administration period. Panel c and panel d are the results of Gli1 mRNA and Western blot of mouse tumors extracted after administration. Panel e is a picture of the mouse tumor extracted after administration.
具体实施方式Detailed ways
结合以下具体实施例和附图，对本发明作进一步的详细说明。实施本发明的过程、条件、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。The present invention will be further described in detail in conjunction with the following specific embodiments and accompanying drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the content specifically mentioned below, are common knowledge and common knowledge in this field, and the present invention has no special limitation content.
下述实施例中化合物结构由核磁共振仪测定；试剂主要由上海国药化学试剂公司提供；产品纯化主要通过柱色谱，硅胶(200-300目)由青岛海洋化工厂生产。The structures of the compounds in the following examples were determined by nuclear magnetic resonance; reagents were mainly provided by Shanghai Sinopharm Chemical Reagent Company; product purification was mainly through column chromatography, and silica gel (200-300 mesh) was produced by Qingdao Ocean Chemical Factory.
实施例1化合物Q1～Q5的制备The preparation of embodiment 1 compound Q1～Q5
将化合物睾酮(50g，0.173mol)溶于t-BuOH(500mL)，冰浴下，分批加入t-BuOK(58.36g，0.52mol)，缓慢滴加CH 3I(64mL，1.04mol)，移去冰浴，加热至45℃搅拌反应4h，TLC检测原料反应完全后，加入1L水搅拌，过滤，滤饼用饱和亚硫酸钠溶液洗涤(50mL)，干燥，得到化合物Q1，未经纯化直接投下一步。 The compound testosterone (50 g, 0.173 mol) was dissolved in t-BuOH (500 mL), under ice cooling, t-BuOK (58.36 g, 0.52 mol) was added in batches, CH 3 I (64 mL, 1.04 mol) was slowly added dropwise, and the Remove from the ice bath, heat to 45°C and stir to react for 4 hours. After the reaction of the raw materials was detected by TLC, 1L of water was added and stirred, filtered, and the filter cake was washed with saturated sodium sulfite solution (50mL) and dried to obtain compound Q1, which was directly used in the next step without purification.
将化合物Q1溶于四氢呋喃(250mL)和乙二醇(100mL)中，加入原甲酸三乙酯(117g，0.790mol)，45℃下，搅拌45min后，加入p-TsOH·H 2O(10.5g，0.055mol)，搅拌反应3h，TLC检测原料反应完全后，加入饱和碳酸氢钠调节pH至弱碱性。加入二氯甲烷(500mL)萃取分液，水相用二氯甲烷(50mL×3)萃取，合并有机相。有机相分别用水(100mL×3)、饱和氯化钠溶液(100mL×3)洗涤，无水硫酸钠干燥，减压浓缩，打浆纯化(Et 2O：EA＝15mL：15mL)，得化合物Q2(白色固体，30.5g，两步产率48.8％)。 Dissolve compound Q1 in tetrahydrofuran (250mL) and ethylene glycol (100mL), add triethyl orthoformate (117g, 0.790mol), stir at 45°C for 45min, add p-TsOH·H 2 O (10.5g , 0.055mol), stirred for 3 hours, and after the reaction of the raw materials was detected by TLC, saturated sodium bicarbonate was added to adjust the pH to weakly alkaline. Dichloromethane (500 mL) was added for extraction and separation, the aqueous phase was extracted with dichloromethane (50 mL×3), and the organic phases were combined. The organic phase was washed with water (100mL×3) and saturated sodium chloride solution (100mL×3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by beating (Et 2 O:EA=15mL:15mL) to obtain compound Q2 ( White solid, 30.5 g, 48.8% yield over two steps).
将化合物Q2(5g，13.88mmol)置于高压釜中，四氢呋喃(75mL)和甲醇(25mL)搅拌溶解，加入10％钯碳(1.5g)，密封高压釜，N 2置换3次，充氢气至4MPa下搅拌反应48h，TLC检测原料反应完全后，抽滤，滤饼用DCM(20mL×3)洗涤，减压浓缩得到化合物Q3，未经纯化，直接投下一步。 Compound Q2 (5g, 13.88mmol) was placed in an autoclave, THF (75mL) and methanol (25mL) were stirred and dissolved, 10% palladium on carbon (1.5g) was added, the autoclave was sealed, N2 was replaced 3 times, and hydrogen was charged to Stirring and reacting at 4MPa for 48 hours, TLC detected that the reaction of the raw materials was complete, suction filtration, the filter cake was washed with DCM (20mL×3), and concentrated under reduced pressure to obtain compound Q3, which was directly used in the next step without purification.
将化合物Q3(4.95g，0.0154mol)溶于四氢呋喃(70mL)和DMSO(35mL)中，加入IBX(8.65g，0.0309mmol)，25℃搅拌反应5h，TLC检测原料反应完全后，加水，抽滤，滤液中加入DCM(100mL)萃取分液，水相用二氯甲烷(20mL×3)萃取，合并有机相。有机相分别用氢氧化钠溶液(10mL×3)、水(10mL×3)、饱和氯化钠溶液(10mL×3)洗涤，无水硫酸钠干燥，减压浓缩得到化合物Q4，直接投下一步。Dissolve compound Q3 (4.95g, 0.0154mol) in tetrahydrofuran (70mL) and DMSO (35mL), add IBX (8.65g, 0.0309mmol), stir and react at 25°C for 5h, TLC detects that the raw materials have reacted completely, add water, and filter with suction , DCM (100 mL) was added to the filtrate for extraction and separation, the aqueous phase was extracted with dichloromethane (20 mL×3), and the organic phases were combined. The organic phase was washed with sodium hydroxide solution (10mL×3), water (10mL×3), and saturated sodium chloride solution (10mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound Q4, which was directly injected into the next step.
将化合物Q4(5.68g，0.0158mol)溶于二氯甲烷(50mL)中，加入m-CPBA(5.46g，0.0317mol)，NaHCO 3(2.9g，0.0348mol)，25℃搅拌反应5h，TLC检测原料反应完全后，加入饱和亚硫酸氢钠溶液淬灭反应至淀粉碘化钾试纸不变色，加入DCM(100mL)萃取分 液，水相用二氯甲烷(20mL×3)萃取，合并有机相。有机相分别用碳酸氢钠饱和溶液(20mL×3)、水(10mL×3)、饱和氯化钠溶液(10mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝6：1)得到化合物Q5(白色固体，4.1g，三步产率78.8％)。 1H NMR(400MHz,Chloroform-d)δ4.01–3.87(m,4H),2.71–2.50(m,2H),2.03–1.87(m,3H),1.83–1.69(m,2H),1.63–1.58(m,2H),1.54–1.40(m,3H),1.37–1.31(m,3H),1.28(s,4H),1.20–1.11(m,2H),0.94(s,4H),0.92(s,1H),0.85(d,J＝13.9Hz,6H). 13C NMR(100MHz,CDCl 3)δ171.66,113.00,83.37,64.92,64.81,54.87,52.26,46.37,42.02,39.15,37.48,36.21,35.57,31.38,28.67,26.92,22.93,21.44,21.17,20.10,20.07,19.80,14.10. Compound Q4 (5.68g, 0.0158mol) was dissolved in dichloromethane (50mL), m-CPBA (5.46g, 0.0317mol), NaHCO 3 (2.9g, 0.0348mol) were added, stirred at 25°C for 5h, detected by TLC After the reaction of raw materials is complete, add saturated sodium bisulfite solution to quench the reaction until starch potassium iodide test paper does not change color, add DCM (100 mL) for extraction and separation, extract the aqueous phase with dichloromethane (20 mL×3), and combine the organic phases. The organic phase was washed with saturated sodium bicarbonate solution (20mL×3), water (10mL×3), saturated sodium chloride solution (10mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure for silica gel column chromatography (PE: EA=6:1) to obtain compound Q5 (white solid, 4.1 g, three-step yield 78.8%). 1 H NMR (400MHz, Chloroform-d) δ4.01–3.87(m,4H),2.71–2.50(m,2H),2.03–1.87(m,3H),1.83–1.69(m,2H),1.63– 1.58(m,2H),1.54–1.40(m,3H),1.37–1.31(m,3H),1.28(s,4H),1.20–1.11(m,2H),0.94(s,4H),0.92( s,1H),0.85(d,J=13.9Hz,6H). 13 C NMR(100MHz,CDCl 3 )δ171.66,113.00,83.37,64.92,64.81,54.87,52.26,46.37,42.02,39.15,37.48,36.21, 35.57, 31.38, 28.67, 26.92, 22.93, 21.44, 21.17, 20.10, 20.07, 19.80, 14.10.
实施例2化合物Q6～Q7的制备The preparation of embodiment 2 compound Q6～Q7
将化合物Q5(8.55g，22.70mmol)溶于四氢呋喃(80mL)中，于0℃下，缓慢加入LiAlH4(2.58g，67.98mmol)，0℃下搅拌反应0.5h，TLC检测原料反应完全后，分别加入，2.6mL水、5.2mL 15％氢氧化钠溶液，7.8mL水淬灭反应。搅拌0.5h后，过滤，滤液减压浓缩得化合物Q6，未经纯化直接投下一步。Compound Q5 (8.55g, 22.70mmol) was dissolved in tetrahydrofuran (80mL), at 0°C, LiAlH4 (2.58g, 67.98mmol) was slowly added, stirred and reacted at 0°C for 0.5h, after TLC detected that the raw materials were completely reacted, respectively Add, 2.6mL water, 5.2mL 15% sodium hydroxide solution, 7.8mL water to quench the reaction. After stirring for 0.5h, it was filtered, and the filtrate was concentrated under reduced pressure to obtain compound Q6, which was directly used in the next step without purification.
将上述化合物Q6溶于四氢呋喃(80mL)中，加入2mol/L稀盐酸(16mL)，25℃搅拌反应5h，TLC检测反应完全，加入乙酸乙酯(200mL)萃取，分液，水相用乙酸乙酯(20mL×3)洗涤，合并有机相。有机相分别用水(20mL×3)、饱和氯化钠溶液(20mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝3：1)得到化合物Q7(白色固体，5.91g，81％)。 1H NMR(400MHz,Chloroform-d)δ3.66(d,J＝20.9Hz,2H),2.63–2.46(m,3H),2.37–2.27(m,1H),2.06(d,J＝13.0Hz,1H),1.99–1.90(m,1H),1.73(d,J＝10.8Hz,3H),1.61(t,J＝12.3Hz,2H),1.46–1.38(m,3H),1.32(d,J＝12.4Hz,1H),1.25–1.13(m,3H),1.09(s,3H),1.04(d,J＝9.1Hz,6H),0.97(s,3H),0.94–0.77(m,2H). Dissolve the above compound Q6 in tetrahydrofuran (80mL), add 2mol/L dilute hydrochloric acid (16mL), stir and react at 25°C for 5h, TLC detects that the reaction is complete, add ethyl acetate (200mL) for extraction, separate the layers, and wash the aqueous phase with ethyl acetate The ester (20 mL×3) was washed, and the organic phases were combined. The organic phase was washed with water (20mL×3) and saturated sodium chloride solution (20mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure by silica gel column chromatography (PE:EA=3:1) to obtain compound Q7 (white solid , 5.91g, 81%). 1 H NMR (400MHz, Chloroform-d) δ3.66 (d, J = 20.9Hz, 2H), 2.63–2.46 (m, 3H), 2.37–2.27 (m, 1H), 2.06 (d, J = 13.0Hz ,1H),1.99–1.90(m,1H),1.73(d,J=10.8Hz,3H),1.61(t,J=12.3Hz,2H),1.46–1.38(m,3H),1.32(d, J＝12.4Hz, 1H), 1.25–1.13(m, 3H), 1.09(s, 3H), 1.04(d, J＝9.1Hz, 6H), 0.97(s, 3H), 0.94–0.77(m, 2H ).
实施例3化合物Q8的制备The preparation of embodiment 3 compound Q8
将化合物Q7(2.1g，6.24mmol)溶于吡啶(20mL)中，冰浴下，缓慢滴加乙酸酐(0.956g，9.36mmol)，搅拌反应18h，TLC检测原料反应完全后，加入2mol/L稀盐酸调节pH为4～5，加入乙酸乙酯(50mL)萃取，分液，水相用乙酸乙酯(10mL×3)洗涤，合并有机相。有机相分别用水(10mL×3)、饱和氯化钠溶液(10mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝6：1)得到化合物Q8(白色固体，1.99g，产率84％)。 1H NMR(400MHz,Chloroform-d)δ4.04(s,2H),2.68–2.53(m,1H),2.41–2.27(m,1H),2.04(s,4H),1.99–1.91(m,1H),1.76(d,J＝12.3Hz,2H),1.63(d,J＝8.8Hz,5H),1.45–1.30(m,4H),1.25–1.12(m,3H),1.07(d,J＝6.7Hz,6H),1.03(s,3H),0.98(s,3H),0.94–0.90(m,1H),0.81(t,J＝11.7Hz,1H). 13C NMR(100MHz,CDCl 3)δ217.16,171.26,73.24,64.82,54.93,54.80,53.51,47.60,42.42,40.07,37.97,36.53,34.58,32.64,30.08,25.84,24.60,22.71,22.48,21.79,21.14,21.06,13.93. Compound Q7 (2.1g, 6.24mmol) was dissolved in pyridine (20mL), under ice-cooling, acetic anhydride (0.956g, 9.36mmol) was slowly added dropwise, and stirred for 18h. After the reaction of the raw materials was detected by TLC, 2mol/L Adjust the pH to 4-5 with dilute hydrochloric acid, add ethyl acetate (50 mL) for extraction, separate the layers, wash the aqueous phase with ethyl acetate (10 mL×3), and combine the organic phases. The organic phase was washed with water (10mL×3) and saturated sodium chloride solution (10mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure by silica gel column chromatography (PE:EA=6:1) to obtain compound Q8 (white solid , 1.99 g, yield 84%). 1 H NMR (400MHz, Chloroform-d) δ4.04(s,2H),2.68–2.53(m,1H),2.41–2.27(m,1H),2.04(s,4H),1.99–1.91(m, 1H), 1.76(d, J＝12.3Hz, 2H), 1.63(d, J＝8.8Hz, 5H), 1.45–1.30(m, 4H), 1.25–1.12(m, 3H), 1.07(d,J ＝6.7Hz, 6H), 1.03(s, 3H), 0.98(s, 3H), 0.94–0.90(m, 1H), 0.81(t, J＝11.7Hz, 1H). 13 C NMR (100MHz, CDCl 3 )δ 217.16, 171.26, 73.24, 64.82, 54.93, 54.80, 53.51, 47.60, 42.42, 40.07, 37.97, 36.53, 34.58, 32.64, 30.08, 25.84, 24.60, 22.71, 22.48, 21.3949, 21
实施例4化合物Q9的制备The preparation of embodiment 4 compound Q9
将化合物Q8(316.9mg，0.837mmol)溶于二氯甲烷(12mL)中，25℃下，加入乙酸酐(0.37mL，4.18mmol)，将0.02mL TMSOTf溶于二氯甲烷(2mL)中，取1mL滴入体系中，25℃下搅拌反应0.5h。TLC检测原料反应完全后，加入饱和碳酸氢钠淬灭反应，DCM(20mL)萃取，分液，水相用DCM(5mL×3)洗涤，合并有机相，有机相分别用水(5mL×3)、饱和氯化钠(5mL×3)洗涤，无水硫酸钠干燥。减压浓缩硅胶柱层析(PE：EA＝80：1)得到化合物Q9(无色油状物，148mg，产率43.9％)。 1H NMR(600MHz,Chloroform-d)δ5.21(dd,J＝6.8,2.0Hz,1H),4.10–3.93(m,2H),2.20–2.11(m,5H),2.09(t,J＝7.2Hz,1H),2.05(d,J＝4.0Hz,3H),2.04–1.97(m,1H),1.97–1.88(m,2H),1.85(dd,J＝16.8,2.0Hz,1H),1.79–1.67(m,2H),1.67–1.61(m,2H),1.59(d,J＝13.3Hz,3H),1.47–1.39(m,1H),1.37–1.31(m,1H),1.26(d,J＝18.2Hz,1H),1.20–1.11(m,1H),1.01(d,J＝4.8Hz,3H),0.96–0.89(m,7H),0.88–0.80(m,1H). Compound Q8 (316.9mg, 0.837mmol) was dissolved in dichloromethane (12mL), at 25°C, acetic anhydride (0.37mL, 4.18mmol) was added, 0.02mL TMSOTf was dissolved in dichloromethane (2mL), and 1mL was dropped into the system, and stirred at 25°C for 0.5h. After TLC detects that the raw materials have reacted completely, add saturated sodium bicarbonate to quench the reaction, extract with DCM (20mL), separate the liquids, wash the aqueous phase with DCM (5mL×3), combine the organic phases, and use the organic phases with water (5mL×3), respectively Washed with saturated sodium chloride (5 mL×3), dried over anhydrous sodium sulfate. Silica gel column chromatography (PE:EA=80:1) was concentrated under reduced pressure to obtain compound Q9 (colorless oil, 148 mg, yield 43.9%). 1 H NMR (600MHz, Chloroform-d) δ5.21 (dd, J = 6.8, 2.0 Hz, 1H), 4.10–3.93 (m, 2H), 2.20–2.11 (m, 5H), 2.09 (t, J = 7.2Hz,1H),2.05(d,J=4.0Hz,3H),2.04–1.97(m,1H),1.97–1.88(m,2H),1.85(dd,J=16.8,2.0Hz,1H), 1.79–1.67(m,2H),1.67–1.61(m,2H),1.59(d,J＝13.3Hz,3H),1.47–1.39(m,1H),1.37–1.31(m,1H),1.26( d, J＝18.2Hz, 1H), 1.20–1.11(m, 1H), 1.01(d, J＝4.8Hz, 3H), 0.96–0.89(m, 7H), 0.88–0.80(m, 1H).
实施例5化合物Q10的制备The preparation of embodiment 5 compound Q10
将化合物Q9(2.11g，0.0052mol)溶于甲醇(20mL)中，加入碳酸钾(2.90g，0.021mol)，25℃搅拌反应3h，TLC检测原料反应完全后，加2mol/L稀盐酸淬灭反应，乙酸乙酯(50mL)萃取，分液，水相用乙酸乙酯(10mL×3)洗涤三次，合并有机相。有机相分别用水(10mL×3)、饱和氯化钠溶液(10mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝7：1)得到化合物Q10(无色油状物，1.44g，产率86.7％)。 1HNMR(500MHz,Chloroform-d)δ3.63(d,J＝4.5Hz,2H),2.66–2.56(m,1H),2.41–2.34(m,1H),2.21–2.14(m,2H),2.12–1.97(m,4H),1.96–1.88(m,1H),1.62(s,6H),1.57–1.51(m,1H),1.49–1.40(m,2H),1.37(dd,J＝12.4,2.8Hz,1H),1.17–1.09(m,1H),1.05(d,J＝13.4Hz,6H),0.96(s,5H). 13C NMR(125MHz,CDCl 3)δ217.80,132.43,127.62,63.23,54.78,53.38,47.68,38.19,37.77,36.50,34.65,33.58,31.79,31.61,26.07,25.44,23.27,22.35,21.57,19.64,13.99. Compound Q9 (2.11g, 0.0052mol) was dissolved in methanol (20mL), potassium carbonate (2.90g, 0.021mol) was added, and the reaction was stirred at 25°C for 3h. After the reaction of the raw materials was detected by TLC, 2mol/L dilute hydrochloric acid was added to quench Reaction, extraction with ethyl acetate (50 mL), liquid separation, the aqueous phase was washed three times with ethyl acetate (10 mL×3), and the organic phases were combined. The organic phase was washed with water (10mL×3) and saturated sodium chloride solution (10mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure by silica gel column chromatography (PE:EA=7:1) to obtain compound Q10 (colorless Oil, 1.44 g, 86.7% yield). 1 H NMR (500MHz, Chloroform-d) δ3.63 (d, J = 4.5Hz, 2H), 2.66–2.56 (m, 1H), 2.41–2.34 (m, 1H), 2.21–2.14 (m, 2H), 2.12–1.97(m,4H),1.96–1.88(m,1H),1.62(s,6H),1.57–1.51(m,1H),1.49–1.40(m,2H),1.37(dd,J=12.4 ,2.8Hz,1H),1.17–1.09(m,1H),1.05(d,J＝13.4Hz,6H),0.96(s,5H). 13 C NMR(125MHz,CDCl 3 )δ217.80,132.43,127.62, 63.23,54.78,53.38,47.68,38.19,37.77,36.50,34.65,33.58,31.79,31.61,26.07,25.44,23.27,22.35,21.57,19.64,13.99.
实施例6化合物Q11～Q12的制备The preparation of embodiment 6 compound Q11～Q12
将化合物Q10(1.40g，4.4mmol)溶于四氢呋喃(30mL)和DMSO(15mL)中，加入IBX(2.46g，8.8mmol)，25℃下搅拌反应1h，TLC检测原料反应完全后，加水，抽滤。滤液用乙酸乙酯(30mL)萃取，分液，水相用乙酸乙酯(5mL×3)洗涤，合并有机相。有机相分别用饱和碳酸氢钠溶液(5mL×3)、水(5mL×3)、饱和氯化钠溶液(5mL×3)洗涤，无水硫酸钠干燥，减压浓缩得到化合物Q11，未经纯化直接投下一步。Compound Q10 (1.40g, 4.4mmol) was dissolved in tetrahydrofuran (30mL) and DMSO (15mL), and IBX (2.46g, 8.8mmol) was added, and the reaction was stirred at 25°C for 1h. filter. The filtrate was extracted with ethyl acetate (30 mL), separated, the aqueous phase was washed with ethyl acetate (5 mL×3), and the organic phases were combined. The organic phase was washed with saturated sodium bicarbonate solution (5mL×3), water (5mL×3), saturated sodium chloride solution (5mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound Q11 without purification Go straight to the next step.
将化合物Q11(1.12g，0.0035mol)溶于吡啶(30mL)中，冰浴下，加入四丁基高锰酸铵(2.56g，0.0074mol)，搅拌反应0.5h后，移去冰浴，反应1h，TLC检测原料反应完 全后，加入饱和亚硫酸氢钠溶液至反应液紫红色褪去，加入2mol/L稀盐酸调节pH至4～5，加入酸乙酯(30mL)萃取，分液，水相用乙酸乙酯(5mL×3)洗涤，合并有机相。有机相分别用水(5mL×3)、饱和氯化钠溶液(5mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝6：1)得到化合物Q12(白色固体，657mg，产率56.6％)。 1H NMR(500MHz,Chloroform-d)δ2.65–2.57(m,1H),2.49–2.28(m,5H),2.20–2.13(m,1H),2.07–1.97(m,3H),1.95–1.87(m,1H),1.71–1.64(m,2H),1.63(s,3H),1.51–1.40(m,2H),1.37(dd,J＝12.5,2.8Hz,1H),1.25(s,1H),1.18–1.11(m,1H),1.06(s,3H),1.04(s,3H),0.97(s,3H),0.95–0.92(m,1H). 13C NMR(125MHz,)δ202.79,169.18,126.91,125.38,60.42,59.17,54.25,45.95,45.55,44.46,42.85,41.88,41.71,40.30,35.37,34.03,32.88,31.99,31.44,29.68,24.81. Dissolve compound Q11 (1.12g, 0.0035mol) in pyridine (30mL), add tetrabutylammonium permanganate (2.56g, 0.0074mol) under ice bath, stir and react for 0.5h, remove the ice bath, and react After 1h, TLC detected that the reaction of the raw materials was complete, then added saturated sodium bisulfite solution until the purple color of the reaction solution faded, added 2mol/L dilute hydrochloric acid to adjust the pH to 4-5, added ethyl acetate (30mL) for extraction, separated the liquid, and the aqueous phase Wash with ethyl acetate (5 mL×3), and combine the organic phases. The organic phase was washed with water (5mL×3) and saturated sodium chloride solution (5mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure by silica gel column chromatography (PE:EA=6:1) to obtain compound Q12 (white solid , 657 mg, yield 56.6%). 1 H NMR (500MHz, Chloroform-d) δ2.65–2.57(m,1H),2.49–2.28(m,5H),2.20–2.13(m,1H),2.07–1.97(m,3H),1.95– 1.87(m,1H),1.71–1.64(m,2H),1.63(s,3H),1.51–1.40(m,2H),1.37(dd,J＝12.5,2.8Hz,1H),1.25(s, 1H),1.18–1.11(m,1H),1.06(s,3H),1.04(s,3H),0.97(s,3H),0.95–0.92(m,1H). 13 C NMR(125MHz,)δ202 .79, 169.18, 126.91, 125.38, 60.42, 59.17, 54.25, 45.95, 45.55, 44.46, 42.85, 41.88, 41.71, 40.30, 35.37, 34.03, 32.88, 31.99, 31.44, 29.61.8, 24.8
实施例7化合物2的制备The preparation of embodiment 7 compound 2
将化合物Q12(50mg，0.15mmol)、HATU(68.7mg，0.18mmol)溶于二氯甲烷(2mL)中，加入DIPEA(0.04mL，0.225mmol)搅拌均匀，加入甲胺盐酸盐(20.3mg，0.3mmol)，25℃搅拌1h，TLC检测原料反应完全，加入盐酸淬灭反应。用DCM(5mL×3)萃取水相，合并有机相。有机相分别用水(5mL×3)、饱和NaCl溶液(5mL×3)洗涤，无水Na 2SO 4干燥，经减压浓缩后硅胶柱层析(PE：EA＝6：1)得到化合物2(白色固体，36.4mg，产率70.2％)。 Dissolve compound Q12 (50mg, 0.15mmol), HATU (68.7mg, 0.18mmol) in dichloromethane (2mL), add DIPEA (0.04mL, 0.225mmol) and stir well, add methylamine hydrochloride (20.3mg, 0.3 mmol), stirred at 25°C for 1 h, TLC detected that the reaction of the raw materials was complete, and the reaction was quenched by adding hydrochloric acid. The aqueous phase was extracted with DCM (5 mL×3), and the organic phases were combined. The organic phase was washed with water (5mL×3) and saturated NaCl solution (5mL×3), dried over anhydrous Na 2 SO 4 , concentrated under reduced pressure and subjected to silica gel column chromatography (PE:EA=6:1) to obtain compound 2 ( White solid, 36.4 mg, 70.2% yield).
实施例8化合物3的制备The preparation of embodiment 8 compound 3
将化合物Q12(0.9g，0.0026mol)溶于二氯甲烷(15mL)和甲醇(7mL)中，冰浴下，加入硼氢化钠(0.98g，0.026mol)，搅拌反应0.5h，TLC检测原料反应完全后，加入2mol/L稀盐酸调节pH至3～4，乙酸乙酯(20mL)萃取，分液，水相用乙酸乙酯(5mL×3)洗涤，合并有机相。有机相分别用水(5mL×3)、饱和氯化钠溶液(5mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝6：1)得到化合物3(白色固体，870mg，产率96.1％)。 1H NMR(500MHz,Chloroform-d)δ3.25(dd,J＝11.6,4.6Hz,1H),2.49–2.29(m,4H),2.20–2.12(m,1H),2.03–1.87(m,3H),1.81–1.76(m,1H),1.73–1.67(m,2H),1.67–1.62(m,4H),1.42–1.34(m,1H),1.27(d,J＝2.6Hz,1H),1.14–1.03(m,2H),0.98(s,3H),0.94–0.89(m,1H),0.89–0.86(m,1H),0.85(dd,J＝6.0,2.2Hz,1H),0.83(s,3H),0.81(s,3H). 13C NMR(125MHz,CDCl 3)δ179.29,131.21,128.74,79.17,54.28,53.90,38.80,37.93,36.92,36.49,33.48,33.27,32.02,28.04,27.54,24.43,21.89,21.72,19.45,15.39,14.26. Compound Q12 (0.9g, 0.0026mol) was dissolved in dichloromethane (15mL) and methanol (7mL), under ice cooling, sodium borohydride (0.98g, 0.026mol) was added, and the reaction was stirred for 0.5h, and the reaction of raw materials was detected by TLC After completion, add 2 mol/L dilute hydrochloric acid to adjust the pH to 3-4, extract with ethyl acetate (20 mL), separate the layers, wash the aqueous phase with ethyl acetate (5 mL×3), and combine the organic phases. The organic phase was washed with water (5mL×3) and saturated sodium chloride solution (5mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure by silica gel column chromatography (PE:EA=6:1) to obtain compound 3 (white solid , 870 mg, yield 96.1%). 1 H NMR (500MHz, Chloroform-d) δ3.25 (dd, J=11.6, 4.6Hz, 1H), 2.49–2.29(m, 4H), 2.20–2.12(m, 1H), 2.03–1.87(m, 3H),1.81–1.76(m,1H),1.73–1.67(m,2H),1.67–1.62(m,4H),1.42–1.34(m,1H),1.27(d,J＝2.6Hz,1H) ,1.14–1.03(m,2H),0.98(s,3H),0.94–0.89(m,1H),0.89–0.86(m,1H),0.85(dd,J＝6.0,2.2Hz,1H),0.83 (s,3H),0.81(s,3H). 13 C NMR(125MHz,CDCl 3 )δ179.29,131.21,128.74,79.17,54.28,53.90,38.80,37.93,36.92,36.49,33.48,33.27,32.02,28.04, 27.54, 24.43, 21.89, 21.72, 19.45, 15.39, 14.26.
实施例9化合物4～25的制备The preparation of embodiment 9 compound 4～25
将化合物3(870mg，2.60mmol)、HATU(1.29g，3.12mmol)溶于二氯甲烷(30mL)中，加入DIPEA(0.7mL，3.9mmol)搅拌均匀，加入相应的胺(5.2mmol)，25℃搅拌1h， TLC检测原料反应完全，加入盐酸淬灭反应。用DCM(20mL×3)萃取水相，合并有机相。有机相分别用水(5mL×3)、饱和NaCl溶液(5mL×3)洗涤，无水Na 2SO 4干燥，经减压浓缩后硅胶柱层析，得到相应的化合物4～25。 Dissolve compound 3 (870mg, 2.60mmol), HATU (1.29g, 3.12mmol) in dichloromethane (30mL), add DIPEA (0.7mL, 3.9mmol) and stir well, add the corresponding amine (5.2mmol), 25 Stir at ℃ for 1 h, TLC detects that the reaction of the raw materials is complete, and the reaction is quenched by adding hydrochloric acid. The aqueous phase was extracted with DCM (20 mL×3), and the organic phases were combined. The organic phase was washed with water (5mL×3) and saturated NaCl solution (5mL×3), dried over anhydrous Na 2 SO 4 , concentrated under reduced pressure and subjected to silica gel column chromatography to obtain the corresponding compounds 4-25.
化合物4，白色固体，产率40％。 1HNMR(600MHz,Chloroform-d)δ5.52(s,1H),3.21(dd,J＝11.7,4.5Hz,1H),2.79(d,J＝4.8Hz,3H),2.43–2.30(m,2H),2.26–2.20(m,1H),2.17–2.06(m,2H),1.97–1.92(m,1H),1.89(dd,J＝17.3,5.2Hz,1H),1.77–1.73(m,1H),1.71–1.63(m,3H),1.61(d,J＝2.6Hz,3H),1.59–1.56(m,1H),1.37–1.31(m,1H),1.24(s,1H),1.08–0.99(m,2H),0.95(s,3H),0.90–0.86(m,1H),0.84–0.82(m,1H),0.81(d,J＝3.2Hz,1H),0.80(s,3H),0.78(s,3H). 13C NMR(150MHz,CDCl3)δ173.78,131.97,128.56,79.17,54.41,54.06,38.94,38.15,37.06,36.62,35.71,33.63,32.24,28.17,27.73,26.45,25.35,22.02,21.91,19.63,15.50,14.40. Compound 4, white solid, yield 40%. 1 HNMR (600MHz, Chloroform-d) δ5.52(s, 1H), 3.21(dd, J＝11.7, 4.5Hz, 1H), 2.79(d, J＝4.8Hz, 3H), 2.43–2.30(m, 2H),2.26–2.20(m,1H),2.17–2.06(m,2H),1.97–1.92(m,1H),1.89(dd,J＝17.3,5.2Hz,1H),1.77–1.73(m, 1H),1.71–1.63(m,3H),1.61(d,J＝2.6Hz,3H),1.59–1.56(m,1H),1.37–1.31(m,1H),1.24(s,1H),1.08 –0.99(m,2H),0.95(s,3H),0.90–0.86(m,1H),0.84–0.82(m,1H),0.81(d,J＝3.2Hz,1H),0.80(s,3H) ),0.78(s,3H) .13 C NMR(150MHz,CDCl3)δ173.78,131.97,128.56,79.17,54.41,54.06,38.94,38.15,37.06,36.62,35.71,33.63,32.24,25.17,27.453,25. ,22.02,21.91,19.63,15.50,14.40.
化合物5，白色固体，产率58.5％。 1HNMR(600MHz,Chloroform-d)δ5.46(s,1H),3.34–3.24(m,2H),3.21(dd,J＝11.7,4.5Hz,1H),2.46–2.39(m,1H),2.36–2.29(m,1H),2.26–2.18(m,1H),2.18–2.06(m,2H),2.03–1.89(m,3H),1.80–1.73(m,2H),1.72–1.64(m,3H),1.62(s,3H),1.38–1.32(m,1H),1.25(d,J＝2.9Hz,1H),1.12(t,J＝7.3Hz,3H),1.08–1.00(m,2H),0.96(s,3H),0.91–0.86(m,1H),0.84(dd,J＝3.5,1.9Hz,1H),0.80(s,3H),0.78(s,3H). 13C NMR(150MHz,CDCl3)δ172.97,132.01,128.62,79.18,54.42,54.07,38.95,38.04,37.06,36.63,35.70,34.46,33.67,32.24,28.17,27.74,25.26,22.03,21.92,19.69,15.50,15.03,14.41. Compound 5, white solid, yield 58.5%. 1 HNMR (600MHz, Chloroform-d) δ5.46(s,1H),3.34–3.24(m,2H),3.21(dd,J＝11.7,4.5Hz,1H),2.46–2.39(m,1H), 2.36–2.29(m,1H),2.26–2.18(m,1H),2.18–2.06(m,2H),2.03–1.89(m,3H),1.80–1.73(m,2H),1.72–1.64(m ,3H),1.62(s,3H),1.38–1.32(m,1H),1.25(d,J＝2.9Hz,1H),1.12(t,J＝7.3Hz,3H),1.08–1.00(m, 2H), 0.96(s, 3H), 0.91–0.86(m, 1H), 0.84(dd, J=3.5, 1.9Hz, 1H), 0.80(s, 3H), 0.78(s, 3H). 13 C NMR (150MHz,CDCl3)δ172.97,132.01,128.62,79.18,54.42,54.07,38.95,38.04,37.06,36.63,35.70,34.46,33.67,32.24,28.17,27.74,25.26,22.03,21.92,19.69,15.50,15.03,14.41 .
化合物6，白色固体，产率65.9％。 1H NMR(500MHz,DMSO-d 6)δ7.75(t,J＝5.7Hz,1H),4.33(d,J＝5.1Hz,1H),3.04–2.97(m,2H),2.97–2.86(m,1H),2.32–2.23(m,1H),2.16–2.09(m,2H),2.09–2.04(m,1H),2.00–1.89(m,3H),1.86–1.79(m,1H),1.66(t,J＝3.6Hz,1H),1.65–1.57(m,3H),1.55(s,3H),1.53–1.45(m,2H),1.42–1.33(m,3H),1.02–0.91(m,3H),0.87(s,3H),0.82(t,J＝7.4Hz,3H),0.78(s,1H),0.76(s,3H),0.68(s,3H). 13C NMR(125MHz,DMSO)δ171.70,132.29,126.67,76.92,53.92,53.52,40.21,38.47,37.43,36.58,34.74,33.08,31.70,28.19,27.44,25.12,22.45,21.59,21.40,19.30,15.82,14.10,11.43. Compound 6, white solid, yield 65.9%. 1 H NMR (500MHz, DMSO-d 6 ) δ7.75(t, J=5.7Hz, 1H), 4.33(d, J=5.1Hz, 1H), 3.04–2.97(m, 2H), 2.97–2.86( m,1H),2.32–2.23(m,1H),2.16–2.09(m,2H),2.09–2.04(m,1H),2.00–1.89(m,3H),1.86–1.79(m,1H), 1.66(t,J＝3.6Hz,1H),1.65–1.57(m,3H),1.55(s,3H),1.53–1.45(m,2H),1.42–1.33(m,3H),1.02–0.91( m,3H),0.87(s,3H),0.82(t,J＝7.4Hz,3H),0.78(s,1H),0.76(s,3H),0.68(s,3H). 13 C NMR (125MHz ,DMSO) δ171.70,132.29,126.67,76.92,53.92,53.52,40.21,38.47,37.43,36.58,34.74,33.08,31.70,28.19,27.44,25.12,22.45,21.59,21.40,148.3,148.3
化合物7，白色固体，产率60.6％。 1H NMR(600MHz,DMSO-d 6)δ7.74(t,J＝5.9Hz,1H),4.32(d,J＝5.1Hz,1H),3.05–2.98(m,1H),2.93–2.87(m,1H),2.83–2.76(m,1H),2.35–2.28(m,1H),2.19–2.08(m,3H),2.05–1.99(m,1H),1.98–1.89(m,2H),1.86–1.79(m,1H),1.69–1.59(m,5H),1.56(s,3H),1.53–1.47(m,2H),1.38–1.30(m,1H),1.03–0.93(m,3H),0.88(s,3H),0.82(d,J＝6.7Hz,6H),0.79–0.76(m,4H),0.69(s,3H). 13C NMR(125MHz,DMSO)δ171.79,132.28,126.65,76.93,53.92,53.51,45.95,38.48,37.31,36.58,36.10,34.67,33.11,31.70, 28.20,28.13,27.45,25.12,21.59,21.38,20.12,19.34,15.83,14.11. Compound 7, white solid, yield 60.6%. 1 H NMR (600MHz, DMSO-d 6 ) δ7.74(t, J=5.9Hz, 1H), 4.32(d, J=5.1Hz, 1H), 3.05–2.98(m, 1H), 2.93–2.87( m,1H),2.83–2.76(m,1H),2.35–2.28(m,1H),2.19–2.08(m,3H),2.05–1.99(m,1H),1.98–1.89(m,2H), 1.86–1.79(m,1H),1.69–1.59(m,5H),1.56(s,3H),1.53–1.47(m,2H),1.38–1.30(m,1H),1.03–0.93(m,3H ),0.88(s,3H),0.82(d,J＝6.7Hz,6H),0.79–0.76(m,4H),0.69(s,3H). 13 C NMR(125MHz,DMSO)δ171.79,132.28,126.65 .
化合物8，白色固体，产率50.6％。 1H NMR(400MHz,DMSO-d 6)δ7.72(s,1H),4.33(d,J＝4.7Hz,1H),2.99(s,3H),2.34–2.22(m,1H),2.19–2.02(m,3H),2.00–1.88(m,3H),1.87–1.79(m,1H),1.62(q,J＝9.7,6.2Hz,3H),1.55(s,3H),1.49(q,J＝6.5,5.5Hz,2H),1.33(q,J＝7.0,6.4Hz,3H),1.25(q,J＝7.3,6.8Hz,2H),1.02–0.91(m,2H),0.90–0.81(m,6H),0.76(s,6H),0.68(s,3H). 13C NMR(100MHz,CDCl 3)δ173.04,131.98,128.56,79.10,54.37,54.01,39.33,38.91,37.96,37.02,36.58,35.65,33.65,32.19,31.84,28.15,27.69,25.23,21.99,21.86,20.18,19.67,15.49,14.37,13.88. Compound 8, white solid, yield 50.6%. 1 H NMR (400MHz,DMSO-d 6 )δ7.72(s,1H),4.33(d,J＝4.7Hz,1H),2.99(s,3H),2.34–2.22(m,1H),2.19– 2.02(m,3H),2.00–1.88(m,3H),1.87–1.79(m,1H),1.62(q,J＝9.7,6.2Hz,3H),1.55(s,3H),1.49(q, J=6.5,5.5Hz,2H), 1.33(q,J=7.0,6.4Hz,3H),1.25(q,J=7.3,6.8Hz,2H),1.02–0.91(m,2H),0.90–0.81 (m,6H),0.76(s,6H),0.68(s,3H). 13 C NMR(100MHz,CDCl 3 )δ173.04,131.98,128.56,79.10,54.37,54.01,39.33,38.91,37.96,37.02,36.58 ,35.65,33.65,32.19,31.84,28.15,27.69,25.23,21.99,21.86,20.18,19.67,15.49,14.37,13.88.
化合物9，白色固体，产率68.3％。 1H NMR(600MHz,DMSO-d 6)δ7.71(t,J＝5.7Hz,1H),3.20(s,1H),3.06–2.92(m,3H),2.32–2.25(m,1H),2.18–2.10(m,2H),2.10–2.04(m,1H),2.00–1.89(m,3H),1.86–1.80(m,1H),1.67–1.58(m,3H),1.55(s,3H),1.50(s,2H),1.39–1.33(m,3H),1.30–1.19(m,4H),1.02–0.92(m,2H),0.88–0.84(m,6H),0.82–0.72(m,6H),0.69(s,3H). 13C NMR(150MHz,DMSO)δ171.64,132.29,126.64,76.91,53.91,53.51,38.46,38.36,37.39,36.58,36.08,34.72,33.09,31.69,28.89,28.62,28.17,27.43,25.05,21.87,21.58,21.39,19.29,15.80,14.09,13.90. Compound 9, white solid, yield 68.3%. 1 H NMR (600MHz,DMSO-d 6 )δ7.71(t,J=5.7Hz,1H),3.20(s,1H),3.06–2.92(m,3H),2.32–2.25(m,1H), 2.18–2.10(m,2H),2.10–2.04(m,1H),2.00–1.89(m,3H),1.86–1.80(m,1H),1.67–1.58(m,3H),1.55(s,3H ),1.50(s,2H),1.39–1.33(m,3H),1.30–1.19(m,4H),1.02–0.92(m,2H),0.88–0.84(m,6H),0.82–0.72(m ,6H),0.69(s,3H). 13 C NMR (150MHz,DMSO)δ171.64,132.29,126.64,76.91,53.91,53.51,38.46,38.36,37.39,36.58,36.08,34.72,33.09,31.69,28.629,2 ,28.17,27.43,25.05,21.87,21.58,21.39,19.29,15.80,14.09,13.90.
化合物10，白色固体，产率96.1％。 1H NMR(600MHz,DMSO-d 6)δ7.71(t,J＝5.6Hz,1H),4.31(dd,J＝5.2,1.8Hz,1H),3.08–2.92(m,3H),2.69(d,J＝1.8Hz,1H),2.32–2.23(m,1H),2.18–2.10(m,2H),2.10–2.04(m,1H),2.00–1.89(m,3H),1.86–1.79(m,1H),1.68–1.59(m,3H),1.55(s,3H),1.50(t,J＝5.7Hz,2H),1.37–1.33(m,2H),1.29–1.21(m,6H),1.02–0.90(m,3H),0.91–0.85(m,5H),0.84(d,J＝1.9Hz,1H),0.80–0.74(m,5H),0.68(s,3H). 13C NMR(150MHz,DMSO)δ171.62,132.31,126.63,76.90,53.90,53.51,38.45,38.38,37.39,36.57,36.07,34.72,33.07,31.68,31.02,29.15,28.17,27.43,26.07,25.05,22.04,21.58,21.38,19.28,15.79,14.09,13.90. Compound 10, white solid, yield 96.1%. 1 H NMR (600MHz, DMSO-d 6 ) δ7.71(t, J=5.6Hz, 1H), 4.31(dd, J=5.2, 1.8Hz, 1H), 3.08–2.92(m, 3H), 2.69( d,J＝1.8Hz,1H),2.32–2.23(m,1H),2.18–2.10(m,2H),2.10–2.04(m,1H),2.00–1.89(m,3H),1.86–1.79( m,1H),1.68–1.59(m,3H),1.55(s,3H),1.50(t,J＝5.7Hz,2H),1.37–1.33(m,2H),1.29–1.21(m,6H) 13 C NMR (150MHz,DMSO)δ171.62,132.31,126.63,76.90,53.90,53.51,38.45,38.38,37.39,36.57,36.07,34.72,33.07,31.68,31.02,29.15,28.17,27.43,26.07,25.05,22.04,21.58,21.38 ,19.28,15.79,14.09,13.90.
化合物11，无色油状物，产率75.9％。 1H NMR(600MHz,DMSO-d 6)δ7.70(t,J＝5.7Hz,1H),4.30(s,1H),3.07–2.91(m,3H),2.33–2.25(m,1H),2.18–2.10(m,2H),2.10–2.03(m,1H),2.00–1.89(m,3H),1.83(dd,J＝16.4,5.0Hz,1H),1.67–1.58(m,4H),1.55(s,3H),1.50–1.48(m,1H),1.38–1.33(m,3H),1.28–1.19(m,10H),0.98–0.93(m,2H),0.87(d,J＝5.4Hz,3H),0.85(d,J＝7.1Hz,2H),0.79–0.77(m,1H),0.76(s,4H),0.69(s,3H). 13C NMR(150MHz,DMSO)δ171.63,132.30,126.63,76.90,53.90,53.51,38.46,38.37,37.39,36.57,36.08,34.71,33.08,31.69,31.24,29.19,28.44,28.17,27.43,26.36,25.04,22.06,21.58,21.39,19.29,15.80,14.10,13.94. Compound 11, colorless oil, yield 75.9%. 1 H NMR (600MHz,DMSO-d 6 )δ7.70(t,J=5.7Hz,1H),4.30(s,1H),3.07–2.91(m,3H),2.33–2.25(m,1H), 2.18–2.10(m,2H),2.10–2.03(m,1H),2.00–1.89(m,3H),1.83(dd,J＝16.4,5.0Hz,1H),1.67–1.58(m,4H), 1.55(s,3H),1.50–1.48(m,1H),1.38–1.33(m,3H),1.28–1.19(m,10H),0.98–0.93(m,2H),0.87(d,J＝5.4 Hz,3H),0.85(d,J=7.1Hz,2H),0.79–0.77(m,1H),0.76(s,4H),0.69(s,3H). 13 C NMR(150MHz,DMSO)δ171. 63,132.30,126.63,76.90,53.90,53.51,38.46,38.37,37.39,36.57,36.08,34.71,33.08,31.69,31.24,29.19,28.44,28.17,27.43,26.36,25.04,22.06,21.58,21.39,19.29,15.80, 14.10, 13.94.
化合物12，无色油状物，产率30％。 1H NMR(500MHz,DMSO-d 6)δ7.71(t,J＝5.6Hz,1H),4.31(d,J＝5.1Hz,1H),3.09–2.89(m,3H),2.36–2.22(m,1H),2.19–2.03(m,3H),2.01–1.88(m,3H),1.87–1.78(m,1H),1.68–1.57(m,3H),1.55(s,3H),1.52–1.45(m,2H),1.39–1.32(m,3H),1.27–1.20(m,11H),1.00–0.92(m,2H),0.87(d,J＝3.9Hz,3H),0.85(d,J＝7.1Hz,2H),0.81–0.74(m,6H),0.68(s,3H). 13C NMR(125MHz,DMSO)δ171.63,132.29,126.62,76.91,53.91,53.51,38.45,38.38,37.39,36.58,36.07,34.72,33.09,31.69,31.26,29.19,28.77,28.67,28.16,27.43,26.41,25.04,22.11,21.59,21.40,19.28,15.78,14.09,13.93. Compound 12, colorless oil, yield 30%. 1 H NMR (500MHz, DMSO-d 6 ) δ7.71(t, J=5.6Hz, 1H), 4.31(d, J=5.1Hz, 1H), 3.09-2.89(m, 3H), 2.36-2.22( m,1H),2.19–2.03(m,3H),2.01–1.88(m,3H),1.87–1.78(m,1H),1.68–1.57(m,3H),1.55(s,3H),1.52– 1.45(m,2H),1.39–1.32(m,3H),1.27–1.20(m,11H),1.00–0.92(m,2H),0.87(d,J＝3.9Hz,3H),0.85(d, J＝7.1Hz, 2H), 0.81–0.74(m, 6H), 0.68(s, 3H). 13 C NMR (125MHz, DMSO) δ171.63, 132.29, 126.62, 76.91, 53.91, 53.51, 38.45, 38.38, 37.39, 36.58,36.07,34.72,33.09,31.69,31.26,29.19,28.77,28.67,28.16,27.43,26.41,25.04,22.11,21.59,21.40,19.28,15.78,14.09,13.93.
化合物13，无色油状物，产率55％。 1H NMR(600MHz,Chloroform-d)δ5.45(d,J＝6.7Hz,1H),3.29–3.12(m,3H),2.47–2.39(m,1H),2.37–2.29(m,1H),2.27–2.20(m,1H),2.18–2.08(m,2H),2.02–1.87(m,3H),1.79–1.73(m,1H),1.69–1.65(m,3H),1.62(s,3H),1.59(d,J＝3.6Hz,1H),1.47(t,J＝7.1Hz,3H),1.40–1.32(m,2H),1.30–1.24(m,11H),1.08–1.02(m,2H),0.96(s,3H),0.89–0.87(m,2H),0.87–0.85(m,1H),0.85–0.83(m,1H),0.83–0.81(m,1H),0.81(s,3H),0.78(s,3H). 13C NMR(125MHz,CDCl 3)δ173.02,132.02,128.59,79.14,54.41,54.06,39.67,38.94,38.00,37.05,36.61,35.69,33.68,32.23,31.97,29.79,29.62,29.44,29.36,28.16,27.73,27.06,25.26,22.77,22.02,21.91,19.69,15.49,14.40,14.22. Compound 13, colorless oil, yield 55%. 1 H NMR (600MHz, Chloroform-d) δ5.45 (d, J=6.7Hz, 1H), 3.29–3.12 (m, 3H), 2.47–2.39 (m, 1H), 2.37–2.29 (m, 1H) ,2.27–2.20(m,1H),2.18–2.08(m,2H),2.02–1.87(m,3H),1.79–1.73(m,1H),1.69–1.65(m,3H),1.62(s, 3H), 1.59(d, J＝3.6Hz, 1H), 1.47(t, J＝7.1Hz, 3H), 1.40–1.32(m, 2H), 1.30–1.24(m, 11H), 1.08–1.02(m ,2H),0.96(s,3H),0.89–0.87(m,2H),0.87–0.85(m,1H),0.85–0.83(m,1H),0.83–0.81(m,1H),0.81(s ,3H),0.78(s,3H). 13 C NMR(125MHz,CDCl 3 )δ173.02,132.02,128.59,79.14,54.41,54.06,39.67,38.94,38.00,37.05,36.61,35.69,33.68,32.273,31.9 29.79, 29.62, 29.44, 29.36, 28.16, 27.73, 27.06, 25.26, 22.77, 22.02, 21.91, 19.69, 15.49, 14.40, 14.22.
化合物14，无色油状物，产率54.8％。 1H NMR(500MHz,Chloroform-d)δ5.47(d,J＝5.8Hz,1H),3.21(q,J＝6.2Hz,3H),2.48–2.29(m,2H),2.28–2.06(m,3H),2.05–1.92(m,2H),1.93–1.85(m,1H),1.78–1.72(m,2H),1.69–1.65(m,2H),1.62(s,3H),1.50–1.43(m,2H),1.42–1.33(m,2H),1.33–1.26(m,7H),1.25(s,8H),1.03(t,J＝8.8Hz,2H),0.96(s,3H),0.87(t,J＝6.8Hz,4H),0.84(s,1H),0.80(s,3H),0.78(s,3H). 13C NMR(125MHz,CDCl 3)δ173.02,132.01,128.53,79.09,54.39,54.03,39.65,38.91,37.98,37.04,36.59,35.66,33.66,32.20,31.98,29.76,29.64,29.42,29.40,28.15,27.70,27.05,25.24,22.77,22.00,21.88,19.66,15.48,14.37,14.21. Compound 14, a colorless oil, yield 54.8%. 1 H NMR (500MHz, Chloroform-d) δ5.47(d, J=5.8Hz, 1H), 3.21(q, J=6.2Hz, 3H), 2.48–2.29(m, 2H), 2.28–2.06(m ,3H),2.05–1.92(m,2H),1.93–1.85(m,1H),1.78–1.72(m,2H),1.69–1.65(m,2H),1.62(s,3H),1.50–1.43 (m,2H),1.42–1.33(m,2H),1.33–1.26(m,7H),1.25(s,8H),1.03(t,J＝8.8Hz,2H),0.96(s,3H), 0.87(t,J=6.8Hz,4H),0.84(s,1H),0.80(s,3H),0.78(s,3H). 13 C NMR(125MHz,CDCl 3 )δ173.02,132.01,128.53,79.09, 54.39,54.03,395,38.91,37.98,37.04,36.66,33.66,32.20,398,29.64,29.40,27.7.7.05, 22.77,77,8.8.8.8.8.8. 14.21.
化合物15，无色油状物，产率43％。 1HNMR(500MHz,Chloroform-d)δ5.49(t,J＝5.7Hz,1H),3.29–3.12(m,3H),2.47–2.37(m,1H),2.37–2.27(m,1H),2.27–2.18(m,1H),2.17–2.08(m,2H),2.03–1.87(m,3H),1.83–1.70(m,3H),1.69–1.64(m,3H),1.61(s,3H),1.45(q,J＝7.0Hz,2H),1.36(dd,J＝13.0,3.7Hz,1H),1.27(d,J＝5.3Hz,5H),1.24(s,13H),1.07–1.01(m,2H),0.96(s,3H),0.87(t,J＝6.9Hz,4H),0.84–0.82(m,1H),0.80(s,3H),0.78(s,3H). 13C NMR(125MHz,CDCl 3)δ173.01,132.02,128.57,79.13,54.41,54.05,39.66,38.93,37.99,37.05,36.61,35.68,33.68,32.22,32.02,29.78,29.76,29.74,29.70,29.66,29.46,29.44,28.16,27.72,27.06,25.26,22.79,22.02,21.90,19.68,15.49,14.39,14.23. Compound 15, a colorless oil, yield 43%. 1 HNMR (500MHz, Chloroform-d) δ5.49(t, J=5.7Hz, 1H), 3.29–3.12(m, 3H), 2.47–2.37(m, 1H), 2.37–2.27(m, 1H), 2.27–2.18(m,1H),2.17–2.08(m,2H),2.03–1.87(m,3H),1.83–1.70(m,3H),1.69–1.64(m,3H),1.61(s,3H ),1.45(q,J=7.0Hz,2H),1.36(dd,J=13.0,3.7Hz,1H),1.27(d,J=5.3Hz,5H),1.24(s,13H),1.07–1.01 (m,2H),0.96(s,3H),0.87(t,J＝6.9Hz,4H),0.84–0.82(m,1H),0.80 ( s,3H),0.78(s,3H). NMR(125MHz,CDCl 3 )δ173.01,132.02,128.57,79.13,54.41,54.05,39.66,38.93,37.99,37.05,36.61,35.68,33.68,32.22,32.02,29.78,29.76,29.74,29.70,29.66,29.46,29.44 ,28.16,27.72,27.06,25.26,22.79,22.02,21.90,19.68,15.49,14.39,14.23.
化合物16，白色固体，产率97％。 1H NMR(600MHz,DMSO-d 6)δ4.31(d,J＝5.2Hz,1H),3.45–3.40(m,1H),3.40–3.34(m,3H),3.02–2.97(m,1H),2.33–2.21(m,2H),2.21–2.14(m,2H),2.10–2.05(m,1H),1.94(q,J＝7.5,4.0Hz,2H),1.88–1.81(m,1H),1.65–1.59(m,2H),1.59–1.53(m,5H),1.52–1.44(m,4H),1.42–1.37(m,2H),1.35–1.28(m,1H),1.28–1.21(m,2H),1.02–0.93(m,2H),0.87(s,3H),0.81(dd,J＝12.1,4.3Hz,1H),0.78–0.75(m,4H),0.68(s,3H). 13C NMR(150MHz,DMSO)δ170.06,132.41,126.77,76.91,53.89,53.48,46.01,41.85,38.46,37.58,36.58,36.07,33.02,31.72,31.58,28.19,27.43,26.18,25.29,24.70,24.08,21.57,21.37,19.27,15.80,14.10. Compound 16, white solid, yield 97%. 1 H NMR (600MHz,DMSO-d 6 )δ4.31(d,J=5.2Hz,1H),3.45–3.40(m,1H),3.40–3.34(m,3H),3.02–2.97(m,1H ),2.33–2.21(m,2H),2.21–2.14(m,2H),2.10–2.05(m,1H),1.94(q,J＝7.5,4.0Hz,2H),1.88–1.81(m,1H ),1.65–1.59(m,2H),1.59–1.53(m,5H),1.52–1.44(m,4H),1.42–1.37(m,2H),1.35–1.28(m,1H),1.28–1.21 (m,2H),1.02–0.93(m,2H),0.87(s,3H),0.81(dd,J＝12.1,4.3Hz,1H),0.78–0.75(m,4H),0.68(s,3H ). 13 C NMR (150MHz, DMSO) δ170.06, 132.41, 126.77, 76.91, 53.89, 53.48, 46.01, 41.85, 38.46, 37.58, 36.58, 36.07, 33.02, 31.72, 31.58, 28.19, 27.48, 24.46 24.08, 21.57, 21.37, 19.27, 15.80, 14.10.
化合物17，白色固体，产率99％。 1H NMR(600MHz,DMSO-d 6)δ4.30(s,1H),3.56–3.51(m,4H),3.42(d,J＝11.1Hz,4H),3.00(dd,J＝9.5,6.5Hz,1H),2.69(s,1H),2.34–2.28(m,1H),2.28–2.23(m,1H),2.22–2.16(m,2H),2.07(dd,J＝12.6,3.5Hz,1H),1.98–1.90(m,2H),1.85(dd,J＝16.1,5.1Hz,1H),1.64–1.59(m,2H),1.56(s,3H),1.52–1.46(m,2H),1.40–1.31(m,1H),1.28–1.17(m,1H),1.03–0.91(m,2H),0.87(s,3H),0.81(dd,J＝12.6,4.0Hz,1H),0.77(s,4H),0.68(s,3H). 13C NMR(150MHz,DMSO)δ170.70,132.30,126.83,76.91,66.16,66.09,53.89,53.47,45.43,41.41,38.46,37.55,36.57,36.07,33.02,31.70,31.26,28.19,27.43,24.40,21.56,21.38,19.28,15.80,14.10. Compound 17, white solid, yield 99%. 1 H NMR (600MHz, DMSO-d 6 )δ4.30(s,1H),3.56–3.51(m,4H),3.42(d,J=11.1Hz,4H),3.00(dd,J=9.5,6.5 Hz,1H),2.69(s,1H),2.34–2.28(m,1H),2.28–2.23(m,1H),2.22–2.16(m,2H),2.07(dd,J＝12.6,3.5Hz, 1H),1.98–1.90(m,2H),1.85(dd,J＝16.1,5.1Hz,1H),1.64–1.59(m,2H),1.56(s,3H),1.52–1.46(m,2H) ,1.40–1.31(m,1H),1.28–1.17(m,1H),1.03–0.91(m,2H),0.87(s,3H),0.81(dd,J＝12.6,4.0Hz,1H),0.77 (s,4H),0.68(s,3H). 13 C NMR(150MHz,DMSO)δ170.70,132.30,126.83,76.91,66.16,66.09,53.89,53.47,45.43,41.41,38.46,37.55,36.57,36.07,33.02 ,31.70,31.26,28.19,27.43,24.40,21.56,21.38,19.28,15.80,14.10.
化合物18，白色固体，产率99％。 1H NMR(600MHz,DMSO-d 6)δ4.44(d,J＝13.0Hz,1H),4.32(d,J＝5.1Hz,1H),3.89(d,J＝13.5Hz,1H),3.46–3.23(m,2H),3.05–2.91(m,2H),2.59(s,3H),2.49–2.44(m,1H),2.31(dd,J＝14.1,8.0Hz,1H),2.28–2.21(m,1H),2.21–2.14(m,2H),2.08(d,J＝12.5Hz,1H),1.99–1.91(m,2H),1.88–1.70(m,3H),1.67–1.45(m,12H),1.41(s,2H),1.34(dd,J＝12.9,3.4Hz,1H),1.27(d,J＝40.2Hz,2H),1.04–0.92(m,2H),0.88(s,3H),0.86–0.81(m,1H),0.80–0.74(m,5H),0.69(s,3H). 13C NMR(150MHz,DMSO)δ170.13,132.38,132.33,126.82,76.91,61.88,53.89,53.49,53.46,49.44,48.58,44.33,40.37,40.06,38.46,37.66,37.52,36.58,36.07,33.03,31.72,31.53,31.45,28.19,27.43,24.62,21.58,21.37,19.28,15.80,14.10. Compound 18, white solid, yield 99%. 1 H NMR (600MHz, DMSO-d 6 )δ4.44(d, J=13.0Hz, 1H), 4.32(d, J=5.1Hz, 1H), 3.89(d, J=13.5Hz, 1H), 3.46 –3.23(m,2H),3.05–2.91(m,2H),2.59(s,3H),2.49–2.44(m,1H),2.31(dd,J＝14.1,8.0Hz,1H),2.28–2.21 (m,1H),2.21–2.14(m,2H),2.08(d,J＝12.5Hz,1H),1.99–1.91(m,2H),1.88–1.70(m,3H),1.67–1.45(m ,12H),1.41(s,2H),1.34(dd,J＝12.9,3.4Hz,1H),1.27(d,J＝40.2Hz,2H),1.04–0.92(m,2H),0.88(s, 3H),0.86–0.81(m,1H),0.80–0.74(m,5H),0.69(s,3H). 13 C NMR(150MHz,DMSO)δ170.13,132.38,132.33,126.82,76.91,61.88,53.89, 53.49, 53.46, 49.44, 48.58, 44.33, 40.37, 40.06, 38.46, 37.66, 37.52, 36.58, 36.07, 33.03, 31.72, 31.53, 31.45, 28.19, 27.43, 24.62, 21.58, 241.2808, 24.62, 21.58, 241.2808,
化合物19，淡黄色固体，产率63.5％。 1H NMR(600MHz,Chloroform-d)δ7.48(d,J＝8.1Hz,2H),7.31(t,J＝7.8Hz,3H),7.09(t,J＝7.4Hz,1H),3.22(dd,J＝11.7,4.4Hz,1H),2.59–2.50(m,1H),2.48–2.39(m,2H),2.37–2.28(m,1H),2.18(dd,J＝12.9,3.5Hz,1H),2.02(d,J＝13.3Hz,2H),1.94(dd,J＝17.0,4.7Hz,1H),1.80–1.73(m,1H),1.71–1.69(m,1H),1.69–1.67(m,1H),1.66(s,3H),1.62(q,J＝4.0Hz,1H),1.40–1.34(m,1H),1.25(s,1H),1.11–1.01(m,2H),0.97(s,3H),0.95–0.91(m,1H),0.87–0.83(m,2H),0.80(s,3H),0.79(s,3H). 13C NMR (150MHz,CDCl3)δ171.38,138.15,131.85,129.33,129.15,124.27,119.77,79.19,54.41,54.07,38.96,38.01,37.06,36.64,36.54,33.75,32.28,28.18,27.74,25.04,22.03,21.95,19.77,15.50,14.41. Compound 19, light yellow solid, yield 63.5%. 1 H NMR (600MHz, Chloroform-d) δ7.48(d, J=8.1Hz, 2H), 7.31(t, J=7.8Hz, 3H), 7.09(t, J=7.4Hz, 1H), 3.22( dd,J＝11.7,4.4Hz,1H),2.59–2.50(m,1H),2.48–2.39(m,2H),2.37–2.28(m,1H),2.18(dd,J＝12.9,3.5Hz, 1H), 2.02(d, J＝13.3Hz, 2H), 1.94(dd, J＝17.0, 4.7Hz, 1H), 1.80–1.73(m, 1H), 1.71–1.69(m, 1H), 1.69–1.67 (m,1H),1.66(s,3H),1.62(q,J＝4.0Hz,1H),1.40–1.34(m,1H),1.25(s,1H),1.11–1.01(m,2H), 0.97(s,3H),0.95–0.91(m,1H),0.87–0.83(m,2H),0.80(s,3H),0.79(s,3H). 13 C NMR (150MHz,CDCl3)δ171.38,138.15 ,131.85,129.33,129.15,124.27,119.77,79.19,54.41,54.07,38.96,38.01,37.06,36.64,36.54,33.75,32.28,28.18,27.74,25.04,21.03,29.450,1
化合物20，白色固体，产率54％。 1H NMR(600MHz,DMSO-d 6)δ9.69(s,1H),7.53–7.42(m,2H),6.95–6.77(m,2H),4.32(d,J＝5.1Hz,1H),3.70(s,3H),3.06–2.96(m,1H),2.42–2.34(m,1H),2.33–2.22(m,2H),2.22–2.16(m,2H),1.96(q,J＝15.2,13.5Hz,2H),1.87–1.80(m,1H),1.67–1.55(m,6H),1.52–1.43(m,2H),1.39–1.28(m,1H),1.03–0.92(m,2H),0.88(s,3H),0.86–0.82(m,1H),0.80–0.76(m,2H),0.75(s,3H),0.69(s,3H). 13C NMR(150MHz,DMSO)δ170.44,154.96,132.54,132.14,126.96,120.57,120.46,113.73,113.71,76.92,55.11,53.92,53.53,38.47,37.52,36.57,36.08,35.61,33.08,31.73,28.17,27.44,24.91,21.60,21.41,19.33,15.80,14.08. Compound 20, white solid, yield 54%. 1 H NMR (600MHz,DMSO-d 6 )δ9.69(s,1H),7.53–7.42(m,2H),6.95–6.77(m,2H),4.32(d,J＝5.1Hz,1H), 3.70(s,3H),3.06–2.96(m,1H),2.42–2.34(m,1H),2.33–2.22(m,2H),2.22–2.16(m,2H),1.96(q,J=15.2 ,13.5Hz,2H),1.87–1.80(m,1H),1.67–1.55(m,6H),1.52–1.43(m,2H),1.39–1.28(m,1H),1.03–0.92(m,2H ),0.88(s,3H),0.86–0.82(m,1H),0.80–0.76(m,2H),0.75(s,3H),0.69(s,3H). 13 C NMR(150MHz,DMSO)δ170 .44,154.96,132.54,132.14,126.96,120.57,120.46,113.73,113.71,76.92,55.11,53.92,53.53,38.47,37.52,36.57,36.08,35.61,33.08,31.73,28.17,27.44,24.91,21.60,21.41,19.33 ,15.80,14.08.
化合物21，白色固体，产率80.3％。 1HNMR(600MHz,DMSO-d 6)δ10.08(s,1H),7.81(d,J＝2.2Hz,1H),7.43(dd,J＝8.2,1.9Hz,1H),7.30(t,J＝8.1Hz,1H),7.06(dd,J＝7.9,2.1Hz,1H),4.30(s,1H),3.00(dd,J＝9.7,6.2Hz,1H),2.42–2.32(m,2H),2.29–2.20(m,2H),2.16(dd,J＝12.6,3.6Hz,1H),1.99–1.90(m,2H),1.83(dd,J＝16.8,5.0Hz,1H),1.66–1.57(m,6H),1.53–1.44(m,2H),1.36–1.28(m,1H),0.99–0.91(m,2H),0.87(s,3H),0.84–0.81(m,1H),0.80–0.72(m,5H),0.68(s,3H). 13C NMR(150MHz,DMSO)δ171.43,140.78,132.99,131.97,130.31,127.17,122.60,118.50,117.35,76.92,53.91,53.52,38.46,37.51,36.56,36.07,35.78,33.08,31.72,28.17,27.44,24.67,21.59,21.39,19.33,15.79,14.05. Compound 21, white solid, yield 80.3%. 1 HNMR (600MHz, DMSO-d 6 ) δ10.08(s, 1H), 7.81(d, J=2.2Hz, 1H), 7.43(dd, J=8.2, 1.9Hz, 1H), 7.30(t, J =8.1Hz,1H),7.06(dd,J=7.9,2.1Hz,1H),4.30(s,1H),3.00(dd,J=9.7,6.2Hz,1H),2.42–2.32(m,2H) ,2.29–2.20(m,2H),2.16(dd,J＝12.6,3.6Hz,1H),1.99–1.90(m,2H),1.83(dd,J＝16.8,5.0Hz,1H),1.66–1.57 (m,6H),1.53–1.44(m,2H),1.36–1.28(m,1H),0.99–0.91(m,2H),0.87(s,3H),0.84–0.81(m,1H),0.80 –0.72(m,5H),0.68(s,3H). 13 C NMR(150MHz,DMSO)δ171.43,140.78,132.99,131.97,130.31,127.17,122.60,118.50,117.35,76.92,53.91,53.52,378.516, ,36.56,36.07,35.78,33.08,31.72,28.17,27.44,24.67,21.59,21.39,19.33,15.79,14.05.
化合物22，白色固体，产率58.3％。 1HNMR(600MHz,DMSO-d 6)δ10.02(s,1H),7.95(t,J＝2.0Hz,1H),7.48–7.45(m,1H),7.27–7.18(m,2H),4.31(d,J＝5.1Hz,1H),3.04–2.96(m,1H),2.44–2.31(m,2H),2.30–2.20(m,2H),2.19–2.13(m,1H),1.99–1.90(m,2H),1.87–1.80(m,1H),1.66–1.57(m,6H),1.52–1.46(m,2H),1.37–1.28(m,1H),1.00–0.93(m,2H),0.88(s,3H),0.83(dd,J＝13.2,3.9Hz,1H),0.79–0.75(m,2H),0.74(s,3H),0.68(s,3H). 13C NMR(150MHz,DMSO)δ171.39,140.89,131.96,130.64,127.18,125.52,121.49,121.36,117.73,76.91,53.90,53.51,38.46,37.49,36.56,36.07,35.79,33.07,31.72,28.17,27.43,24.66,21.59,21.39,19.32,15.79,14.05. Compound 22, white solid, yield 58.3%. 1 HNMR (600MHz, DMSO-d 6 )δ10.02(s,1H),7.95(t,J=2.0Hz,1H),7.48–7.45(m,1H),7.27–7.18(m,2H),4.31 (d,J=5.1Hz,1H),3.04–2.96(m,1H),2.44–2.31(m,2H),2.30–2.20(m,2H),2.19–2.13(m,1H),1.99–1.90 (m,2H),1.87–1.80(m,1H),1.66–1.57(m,6H),1.52–1.46(m,2H),1.37–1.28(m,1H),1.00–0.93(m,2H) ,0.88(s,3H),0.83(dd,J＝13.2,3.9Hz,1H),0.79–0.75(m,2H),0.74(s,3H),0.68(s,3H). 13 C NMR (150MHz ,DMSO)δ171.39,140.89,131.96,130.64,127.18,125.52,121.49,121.36,117.73,76.91,53.90,53.51,38.46,37.49,36.56,36.07,35.79,33.07,31.72,28.17,27.43,24.66,21.59,21.39 ,19.32,15.79,14.05.
化合物23，白色固体，产率89.7％。 1H NMR(600MHz,DMSO-d 6)δ9.75(s,1H),7.43(s,1H),7.38(d,J＝8.2Hz,1H),7.17(t,J＝7.8Hz,1H),6.86(d,J＝7.6Hz,1H),4.32(s,1H),3.04–2.97(m,1H),2.55(q,J＝7.6Hz,2H),2.42–2.29(m,2H),2.29–2.15(m,3H),1.96(q,J＝15.2,11.9Hz,2H),1.84(dd,J＝17.1,4.9Hz,1H),1.67–1.62(m,2H),1.59(s,3H),1.53–1.46(m,2H), 1.38–1.29(m,1H),1.24(d,J＝14.3Hz,1H),1.15(t,J＝7.6Hz,3H),1.01–0.93(m,2H),0.88(s,3H),0.84(dd,J＝13.0,3.2Hz,1H),0.76(d,J＝17.6Hz,5H),0.69(s,3H). 13C NMR(150MHz,DMSO)δ170.94,144.11,139.33,132.15,128.51,127.02,122.46,118.44,116.53,76.92,53.91,53.54,39.52,37.54,36.57,36.08,35.78,33.07,31.73,28.27,28.18,27.43,24.84,21.60,21.39,19.32,15.80,15.52,14.07. Compound 23, white solid, yield 89.7%. 1 H NMR (600MHz,DMSO-d 6 )δ9.75(s,1H),7.43(s,1H),7.38(d,J=8.2Hz,1H),7.17(t,J=7.8Hz,1H) ,6.86(d,J=7.6Hz,1H),4.32(s,1H),3.04–2.97(m,1H),2.55(q,J=7.6Hz,2H),2.42–2.29(m,2H), 2.29–2.15(m,3H),1.96(q,J=15.2,11.9Hz,2H),1.84(dd,J=17.1,4.9Hz,1H),1.67–1.62(m,2H),1.59(s, 3H),1.53–1.46(m,2H), 1.38–1.29(m,1H),1.24(d,J＝14.3Hz,1H),1.15(t,J＝7.6Hz,3H),1.01–0.93(m , 2H), 0.88(s, 3H), 0.84(dd, J＝13.0, 3.2Hz, 1H), 0.76(d, J＝17.6Hz, 5H), 0.69(s, 3H). 13 C NMR (150MHz, DMSO)δ170.94,144.11,139.33,132.15,128.51,127.02,122.46,118.44,116.53,76.92,53.91,53.54,39.52,37.54,36.57,36.08,35.78,33.07,31.73,28.27,28.18,27.43,24.84,21.60, 21.39, 19.32, 15.80, 15.52, 14.07.
化合物24，白色固体，产率73.7％。 1H NMR(500MHz,DMSO-d 6)δ8.22(t,J＝6.0Hz,1H),7.14(d,J＝8.2Hz,2H),6.86(d,J＝8.1Hz,2H),4.33(d,J＝5.2Hz,1H),4.25–4.07(m,2H),3.72(s,3H),3.03–2.93(m,1H),2.37–2.27(m,1H),2.21–2.09(m,3H),2.11–2.00(m,2H),1.94(t,J＝12.5Hz,2H),1.86–1.79(m,1H),1.61(d,J＝14.6Hz,2H),1.55(s,3H),1.50(d,J＝9.0Hz,2H),1.35–1.28(m,1H),1.24(d,J＝11.3Hz,1H),0.96(dd,J＝12.6,5.1Hz,2H),0.87(s,3H),0.80–0.72(m,5H),0.68(s,3H). 13C NMR(125MHz,DMSO)δ171.76,158.15,132.19,131.65,128.51,126.81,113.63,76.93,55.06,53.92,53.51,41.44,39.52,37.38,36.58,36.10,34.63,33.09,31.70,28.21,27.45,25.11,21.59,21.37,19.33,15.84,14.11. Compound 24, white solid, yield 73.7%. 1 H NMR (500MHz, DMSO-d 6 ) δ8.22(t, J=6.0Hz, 1H), 7.14(d, J=8.2Hz, 2H), 6.86(d, J=8.1Hz, 2H), 4.33 (d,J=5.2Hz,1H),4.25–4.07(m,2H),3.72(s,3H),3.03–2.93(m,1H),2.37–2.27(m,1H),2.21–2.09(m ,3H),2.11–2.00(m,2H),1.94(t,J＝12.5Hz,2H),1.86–1.79(m,1H),1.61(d,J＝14.6Hz,2H),1.55(s, 3H), 1.50(d, J=9.0Hz, 2H), 1.35–1.28(m, 1H), 1.24(d, J=11.3Hz, 1H), 0.96(dd, J=12.6, 5.1Hz, 2H), 0.87(s,3H),0.80–0.72(m,5H),0.68(s,3H) .13C NMR(125MHz,DMSO)δ171.76,158.15,132.19,131.65,128.51,126.81,113.63,76.93,55.06,53.92 ,53.51,41.44,39.52,37.38,36.58,36.10,34.63,33.09,31.70,28.21,27.45,25.11,21.59,21.37,19.33,15.84,14.11.
化合物25，淡黄色固体，产率66.6％。 1HNMR(600MHz,DMSO-d 6)δ10.97(s,1H),9.72(s,1H),7.95(s,1H),7.40(d,J＝8.4Hz,1H),7.23(t,J＝2.7Hz,1H),7.01(dd,J＝8.4,1.8Hz,1H),6.33(t,J＝2.4Hz,1H),4.32(d,J＝5.1Hz,1H),3.04–2.97(m,1H),2.46–2.39(m,1H),2.39–2.33(m,1H),2.30(d,J＝12.2Hz,1H),2.26(t,J＝4.2Hz,1H),2.25–2.20(m,1H),2.04–1.92(m,2H),1.84(dd,J＝16.8,4.8Hz,1H),1.67–1.59(m,6H),1.54–1.47(m,2H),1.41–1.32(m,1H),1.01–0.92(m,2H),0.89(s,3H),0.84(dd,J＝12.5,3.8Hz,1H),0.81–0.75(m,5H),0.69(s,3H). 13C NMR(150MHz,DMSO)δ170.51,135.90,133.52,132.22,126.93,124.71,123.69,119.64,112.29,102.11,100.86,76.95,53.94,53.56,38.48,37.51,36.59,36.10,35.78,33.11,31.76,28.20,27.45,24.98,21.63,21.44,19.38,15.82,14.12. Compound 25, light yellow solid, yield 66.6%. 1 HNMR (600MHz, DMSO-d 6 )δ10.97(s,1H),9.72(s,1H),7.95(s,1H),7.40(d,J＝8.4Hz,1H),7.23(t,J ＝2.7Hz, 1H), 7.01(dd, J＝8.4, 1.8Hz, 1H), 6.33(t, J＝2.4Hz, 1H), 4.32(d, J＝5.1Hz, 1H), 3.04–2.97(m ,1H),2.46–2.39(m,1H),2.39–2.33(m,1H),2.30(d,J＝12.2Hz,1H),2.26(t,J＝4.2Hz,1H),2.25–2.20( m,1H),2.04–1.92(m,2H),1.84(dd,J＝16.8,4.8Hz,1H),1.67–1.59(m,6H),1.54–1.47(m,2H),1.41–1.32( m,1H),1.01–0.92(m,2H),0.89(s,3H),0.84(dd,J＝12.5,3.8Hz,1H),0.81–0.75(m,5H),0.69(s,3H) . 13 C NMR(150MHz,DMSO)δ170.51,135.90,133.52,132.22,126.93,124.71,123.69,119.64,112.29,102.11,100.86,76.95,53.94,53.56,38.48,37.51,36.59,36.10,35.78,33.11,31.76 ,28.20,27.45,24.98,21.63,21.44,19.38,15.82,14.12.
实施例10化合物Q13～Q14的制备The preparation of embodiment 10 compound Q13～Q14
将化合物Q10(2.46g，7.72mmol)溶于二氯甲烷(75mL)中，加入三乙胺(4.3mL，30.89mmol)。冰浴下，加入甲磺酰氯(1.8mL，23.17mmol)，搅拌反应1h，TLC检测原料反应完全后，加水淬灭，二氯甲烷(50mL)萃取，分液，水相用乙酸乙酯(10mL×3)洗涤三次，合并有机相。有机相分别用饱和氯化钠溶液(10mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(DCM：CH 3OH＝100：1)得到化合物Q13(无色油状物，2.94g，产率96％)。 Compound Q10 (2.46 g, 7.72 mmol) was dissolved in dichloromethane (75 mL), and triethylamine (4.3 mL, 30.89 mmol) was added. Under ice bath, add methanesulfonyl chloride (1.8mL, 23.17mmol), stir the reaction for 1h, after TLC detects that the reaction of raw materials is complete, add water to quench, extract with dichloromethane (50mL), separate layers, and use ethyl acetate (10mL) for the aqueous phase ×3) Wash three times, and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (10mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Silica gel column chromatography (DCM:CH 3 OH=100:1) gave compound Q13 (colorless oil, 2.94 g, yield 96%).
将化合物Q13(808mg,2.04mmol)溶于乙腈(20mL)，加入TMSCN(0.4mL，3.06mmol)，TBAF·3H 2O(965.5mg，3.06mmol)，82℃下搅拌反应30min，TLC检测原料反应完全后，减压浓缩硅胶柱层析(PE：EA＝10：1)得到化合物Q14(无色油状物，389mg，产率58.3％)。 1HNMR(600MHz,Chloroform-d)δ2.66–2.56(m,1H),2.41–2.35(m,1H),2.33–2.27(m,2H),2.25–2.18(m,2H),2.15–2.10(m,1H),2.04–1.98(m,2H),1.97–1.94(m,1H),1.78–1.72(m,1H),1.71–1.65(m,2H),1.64–1.61(m,3H),1.50–1.46(m,1H),1.45–1.42(m,1H),1.41(s,1H),1.36(dd,J＝12.5,2.8Hz,1H),1.13(dd,J＝12.3,5.3Hz,1H),1.06(s,3H),1.03(s,3H),0.97(s,3H),0.96–0.94(m,1H),0.94–0.92(m,1H),0.89–0.81(m,1H). Dissolve compound Q13 (808mg, 2.04mmol) in acetonitrile (20mL), add TMSCN (0.4mL, 3.06mmol), TBAF·3H 2 O (965.5mg, 3.06mmol), stir and react at 82°C for 30min, TLC detects the reaction of raw materials After completion, silica gel column chromatography (PE:EA=10:1) was concentrated under reduced pressure to obtain compound Q14 (colorless oil, 389 mg, yield 58.3%). 1 HNMR (600MHz, Chloroform-d) δ2.66–2.56(m,1H),2.41–2.35(m,1H),2.33–2.27(m,2H),2.25–2.18(m,2H),2.15–2.10 (m,1H),2.04–1.98(m,2H),1.97–1.94(m,1H),1.78–1.72(m,1H),1.71–1.65(m,2H),1.64–1.61(m,3H) ,1.50–1.46(m,1H),1.45–1.42(m,1H),1.41(s,1H),1.36(dd,J＝12.5,2.8Hz,1H),1.13(dd,J＝12.3,5.3Hz ,1H),1.06(s,3H),1.03(s,3H),0.97(s,3H),0.96–0.94(m,1H),0.94–0.92(m,1H),0.89–0.81(m,1H ).
实施例11化合物Q15的制备The preparation of embodiment 11 compound Q15
将化合物Q14(1.48g，4.5mmol)溶于乙二醇(11mL)，加入4mol/L氢氧化钾溶液(21mL)，170℃下，反应12h，TLC检测原料反应完全后，待反应冷却至室温，加2mol/L稀盐酸调节pH至1～2，析出白色固体，乙酸乙酯(40mL)萃取，分液，水相用乙酸乙酯(10mL×3)洗涤，合并有机相。有机相分别用饱和氯化钠溶液(10mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝6：1)得到化合物Q15(无色油状物，736mg，产率49％)。Dissolve compound Q14 (1.48g, 4.5mmol) in ethylene glycol (11mL), add 4mol/L potassium hydroxide solution (21mL), and react at 170°C for 12h. After TLC detects that the raw materials have reacted completely, wait for the reaction to cool to room temperature , add 2mol/L dilute hydrochloric acid to adjust the pH to 1-2, a white solid precipitates out, extract with ethyl acetate (40mL), separate the layers, wash the aqueous phase with ethyl acetate (10mL×3), and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (10mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure by silica gel column chromatography (PE:EA=6:1) to obtain compound Q15 (colorless oil, 736mg, yield rate 49%).
实施例12化合物Q16的制备The preparation of embodiment 12 compound Q16
将化合物Q15(595mg，1.72mmol)溶于二氯甲烷(10mL)和甲醇(5mL)，冰浴下，加入硼氢化钠(649.6mg，17.2mmol)，搅拌反应0.5h，TLC检测原料反应完全后，加入2mol/L稀盐酸调pH至4～5，乙酸乙酯(30mL)萃取，分液，水相用乙酸乙酯(10mL×3)洗涤，合并有机相。有机相用饱和氯化钠溶液(10mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝6：1)得到化合物Q16(无色油状物，287mg，产率48％)。Compound Q15 (595mg, 1.72mmol) was dissolved in dichloromethane (10mL) and methanol (5mL), and sodium borohydride (649.6mg, 17.2mmol) was added under ice-cooling, and the reaction was stirred for 0.5h. TLC detected that the raw materials were completely reacted , add 2 mol/L dilute hydrochloric acid to adjust the pH to 4-5, extract with ethyl acetate (30 mL), separate the layers, wash the aqueous phase with ethyl acetate (10 mL×3), and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (10mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Silica gel column chromatography (PE:EA=6:1) gave compound Q16 (colorless oil, 287mg, yield 48%).
实施例13化合物26的制备The preparation of embodiment 13 compound 26
将化合物Q16(46mg，0.132mmol)、HATU(60.3mg，0.159mmol)溶于四氢呋喃(1mL)和二氯甲烷中(1mL)，加入DIPEA(0.04mL，0.26mmol)搅拌均匀，加入正己胺(0.03mL，0.264mmol)，25℃搅拌反应1h，TLC检测原料反应完全后，加入2mol/L稀盐酸淬灭反应，DCM(15mL)萃取，分液，水相用DCM(5mL×3)洗涤，合并有机相。有机相用饱和氯化钠(5mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝6：1)得到化合物26(白色固体，36mg，产率63.3％)。 1H NMR(500MHz,DMSO-d 6)δ4.31(d,J＝5.2Hz,1H),3.05–2.95(m,3H),2.11(dd,J＝12.6,3.5Hz,1H),2.04–1.79(m,7H),1.68–1.58(m,3H),1.54(d,J＝1.7Hz,4H),1.52–1.46(m,2H),1.36(p,J＝7.3Hz,3H),1.29(d,J＝6.0Hz,1H),1.28–1.20(m,7H),1.04–0.92(m,2H),0.89–0.83(m,6H),0.82–0.73(m,6H),0.68(s,3H). 13C NMR(125MHz,CDCl3)δ173.07,132.67,127.60,79.17,54.41,54.06,39.66,38.92,37.97,37.07,36.82,36.61,33.65,32.20,31.61,29.78,28.72,28.16,27.74,26.72,24.73,22.66,22.03,21.96,19.70,15.49,14.39,14.13. Compound Q16 (46mg, 0.132mmol), HATU (60.3mg, 0.159mmol) were dissolved in tetrahydrofuran (1mL) and dichloromethane (1mL), added DIPEA (0.04mL, 0.26mmol) and stirred well, added n-hexylamine (0.03 mL, 0.264mmol), stirred and reacted at 25°C for 1h, after the reaction of the raw materials was detected by TLC, 2mol/L dilute hydrochloric acid was added to quench the reaction, extracted with DCM (15mL), separated, the aqueous phase was washed with DCM (5mL×3), and combined The organic phase. The organic phase was washed with saturated sodium chloride (5mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Silica gel column chromatography (PE:EA=6:1) gave compound 26 (white solid, 36mg, yield 63.3%) . 1 H NMR (500MHz, DMSO-d 6 ) δ4.31 (d, J=5.2Hz, 1H), 3.05–2.95 (m, 3H), 2.11 (dd, J=12.6, 3.5Hz, 1H), 2.04– 1.79(m,7H),1.68–1.58(m,3H),1.54(d,J=1.7Hz,4H),1.52–1.46(m,2H),1.36(p,J=7.3Hz,3H),1.29 (d,J＝6.0Hz,1H),1.28–1.20(m,7H),1.04–0.92(m,2H),0.89–0.83(m,6H),0.82–0.73(m,6H),0.68(s ,3H) .13C NMR(125MHz,CDCl3)δ173.07,132.67,127.60,79.17,54.41,54.06,39.66,38.92,37.97,37.07,36.82,36.61,33.65,32.20,31.61,29.78,28.72 26.72, 24.73, 22.66, 22.03, 21.96, 19.70, 15.49, 14.39, 14.13.
实施例14化合物Q17～Q18的制备The preparation of embodiment 14 compound Q17～Q18
将NaH(246.9mg，10.29mmol)加入无水四氢呋喃(20mL)中，氮气保护，冰浴下加入膦酰基乙酸三乙酯(2.04mL，10.29mmol)，再加入5mL的化合物Q11(592mg，1.87mmol)的四氢呋喃溶液，冰浴下搅拌反应20min，TLC检测原料反应完全后，加入5mL饱和氯化铵溶液淬灭反应，乙酸乙酯(30mL)萃取，分液，水相用乙酸乙酯(10mL×3)洗涤三次，合并有机相。有机相用饱和氯化钠溶液(10mL×3)洗涤，无水硫酸钠干燥，减压浓缩得化合物Q17(无色油状物，582mg)，直接进行下一步。Add NaH (246.9mg, 10.29mmol) into anhydrous tetrahydrofuran (20mL), under nitrogen protection, add triethyl phosphonoacetate (2.04mL, 10.29mmol) under ice-cooling, then add 5mL of compound Q11 (592mg, 1.87mmol ) in tetrahydrofuran solution, stirred and reacted under ice bath for 20min, after TLC detected that the raw materials were completely reacted, 5mL saturated ammonium chloride solution was added to quench the reaction, extracted with ethyl acetate (30mL), separated, and the aqueous phase was purified with ethyl acetate (10mL× 3) Wash three times, and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (10 mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound Q17 (colorless oil, 582 mg), which was directly carried out to the next step.
将化合物Q17(582mg，1.61mmol)溶于四氢呋喃(12mL)中，加入58.2mg 10％Pd/C，氮气置换3次，氢气压力为1atm，25℃下搅拌反应1h，TLC检测原料反应完全后，抽滤，滤液减压浓缩硅胶柱层析(PE：EA＝10：1)得到化合物Q18(无色油状物，434mg，产率74％)。 1H NMR(600MHz,Chloroform-d)δ4.09(q,J＝7.2Hz,2H),2.62–2.51(m,1H),2.38–2.31(m,1H),2.26(q,J＝6.9,6.3Hz,2H),2.13–2.07(m,1H),2.04–1.92(m,4H),1.91–1.84(m,1H),1.66–1.49(m,7H),1.45–1.35(m,3H),1.34(dd,J＝5.8,2.5Hz,1H),1.28–1.17(m,5H),1.14–1.07(m,1H),1.02(d,J＝5.2Hz,3H),1.00(s,3H),0.97(d,J＝2.1Hz,1H),0.93(s,3H),0.91–0.88(m,1H). Compound Q17 (582mg, 1.61mmol) was dissolved in tetrahydrofuran (12mL), 58.2mg of 10% Pd/C was added, nitrogen was replaced 3 times, the hydrogen pressure was 1atm, and the reaction was stirred at 25°C for 1h. After the reaction of the raw materials was detected by TLC, Suction filtration, the filtrate was concentrated under reduced pressure and subjected to silica gel column chromatography (PE:EA=10:1) to obtain compound Q18 (colorless oil, 434 mg, yield 74%). 1 H NMR (600MHz, Chloroform-d) δ4.09 (q, J = 7.2Hz, 2H), 2.62–2.51 (m, 1H), 2.38–2.31 (m, 1H), 2.26 (q, J = 6.9, 6.3Hz, 2H), 2.13–2.07(m, 1H), 2.04–1.92(m, 4H), 1.91–1.84(m, 1H), 1.66–1.49(m, 7H), 1.45–1.35(m, 3H) ,1.34(dd,J=5.8,2.5Hz,1H),1.28–1.17(m,5H),1.14–1.07(m,1H),1.02(d,J=5.2Hz,3H),1.00(s,3H) ),0.97(d,J＝2.1Hz,1H),0.93(s,3H),0.91–0.88(m,1H).
实施例15化合物Q19的制备The preparation of embodiment 15 compound Q19
将化合物Q18(434mg，1.117mmol)溶于二氯甲烷(4mL)和甲醇(2mL)中，冰浴下加入硼氢化钠(422.5mg，11.17mmol)，搅拌反应0.5h，TLC检测原料反应完全后，加入2mol/L稀盐酸淬灭反应，DCM(20mL)萃取，分液，水相用DCM(5mL×3)洗涤，合并有机相，有机相分别用饱和碳酸氢钠(5mL×3)、水(5mL×3)、饱和氯化钠(5mL×3)洗涤，无水硫酸钠干燥。减压浓缩硅胶柱层析(PE：EA＝10：1)得到化合物Q19(无色油状物，346mg，产率79.3％)。 1H NMR(600MHz,Chloroform-d)δ4.10(q,J＝7.2,3.1Hz,2H),3.19(dd,J＝11.6,4.5Hz,1H),2.27(q,J＝7.8Hz,2H),2.12–2.05(m,1H),2.04–1.82(m,4H),1.76–1.71(m,1H),1.70–1.55(m,9H),1.42–1.29(m,2H),1.28–1.20(m,4H),1.20–1.09(m,1H),1.08–0.97(m,2H),0.94(s,3H),0.85(dd,J＝12.7,4.1Hz,1H),0.83–0.80(m,1H),0.80(s,1H),0.78(s,3H),0.76(s,3H). Compound Q18 (434mg, 1.117mmol) was dissolved in dichloromethane (4mL) and methanol (2mL), and sodium borohydride (422.5mg, 11.17mmol) was added under ice-cooling, and the reaction was stirred for 0.5h. TLC detected that the raw materials were completely reacted , add 2mol/L dilute hydrochloric acid to quench the reaction, extract with DCM (20mL), separate the layers, wash the aqueous phase with DCM (5mL×3), combine the organic phase, and wash the organic phase with saturated sodium bicarbonate (5mL×3), water (5mL×3), washed with saturated sodium chloride (5mL×3), and dried over anhydrous sodium sulfate. Silica gel column chromatography (PE:EA=10:1) was concentrated under reduced pressure to obtain compound Q19 (colorless oil, 346 mg, yield 79.3%). 1 H NMR (600MHz, Chloroform-d) δ 4.10 (q, J = 7.2, 3.1 Hz, 2H), 3.19 (dd, J = 11.6, 4.5 Hz, 1H), 2.27 (q, J = 7.8 Hz, 2H ),2.12–2.05(m,1H),2.04–1.82(m,4H),1.76–1.71(m,1H),1.70–1.55(m,9H),1.42–1.29(m,2H),1.28–1.20 (m,4H),1.20–1.09(m,1H),1.08–0.97(m,2H),0.94(s,3H),0.85(dd,J＝12.7,4.1Hz,1H),0.83–0.80(m ,1H),0.80(s,1H),0.78(s,3H),0.76(s,3H).
实施例16化合物Q20的制备The preparation of embodiment 16 compound Q20
将化合物Q19(274mg，0.7mmol)溶于甲醇(3mL)中，加入氢氧化钠(28mg，7.01mmol)，25℃搅拌反应9h，TLC检测原料反应完全后，加入2mol/L稀盐酸调节pH至4～5，乙酸乙酯(30mL)萃取，分液，水相用乙酸乙酯(10mL×3)洗涤三次，合并有机相。有机相用饱和氯化钠溶液(10mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝4：1)得到化合物Q20(白色固体，160mg，产率63％)。Compound Q19 (274mg, 0.7mmol) was dissolved in methanol (3mL), sodium hydroxide (28mg, 7.01mmol) was added, and the reaction was stirred at 25°C for 9h. After the reaction of the raw materials was detected by TLC, 2mol/L dilute hydrochloric acid was added to adjust the pH to 4-5, extract with ethyl acetate (30 mL), separate the layers, wash the aqueous phase with ethyl acetate (10 mL×3) three times, and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (10mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Silica gel column chromatography (PE:EA=4:1) gave compound Q20 (white solid, 160mg, yield 63% ).
实施例17化合物27～29的制备The preparation of embodiment 17 compound 27～29
将化合物Q20(40mg，0.11mmol)、HATU(83.90mg，0.22mmol)溶于1mL二氯甲烷和1mL四氢呋喃中，加入DIPEA(0.04mL，0.24mmol)搅拌均匀，加入相应的胺(0.22mmol)25℃搅拌1h，TLC检测原料反应完全，加入盐酸淬灭反应。用DCM(10mL×3)萃取水相，合并有机相。有机相分别用水(10mL)、饱和NaCl溶液(10mL)洗涤，无水硫酸钠干燥，经减压浓缩后硅胶柱层析(PE：EA＝6：1)，得到相应的化合物27～29。Dissolve compound Q20 (40mg, 0.11mmol), HATU (83.90mg, 0.22mmol) in 1mL of dichloromethane and 1mL of tetrahydrofuran, add DIPEA (0.04mL, 0.24mmol) and stir well, add the corresponding amine (0.22mmol) 25 Stir at ℃ for 1 h, TLC detects that the reaction of the raw materials is complete, and the reaction is quenched by adding hydrochloric acid. The aqueous phase was extracted with DCM (10 mL×3), and the organic phases were combined. The organic phase was washed with water (10 mL) and saturated NaCl solution (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then chromatographed on a silica gel column (PE:EA=6:1) to obtain the corresponding compounds 27-29.
化合物27，白色固体，产率86.9％。 1H NMR(500MHz,DMSO-d 6)δ7.70(t,J＝5.6Hz,1H),4.31(d,J＝5.1Hz,1H),3.08–2.95(m,3H),2.11–2.01(m,3H),2.00–1.75(m,5H),1.69–1.58(m,3H),1.54(d,J＝1.7Hz,3H),1.52–1.42(m,4H),1.38–1.33(m,2H),1.32(t,J＝3.9Hz,1H),1.30–1.23(m,3H),1.20–1.10(m,1H),1.03–0.91(m,2H),0.89–0.83(m,6H),0.81–0.72(m,6H),0.68(s,3H). 13C NMR(125MHz,DMSO)δ171.81,132.96,125.85,76.90,53.91,53.52,38.44,38.02,37.66,36.58,36.03,35.32,33.02,31.79,31.35,28.56,28.14,27.76,27.42,25.61,21.64,21.46,19.57,19.30,15.76,14.06,13.67. Compound 27, white solid, yield 86.9%. 1 H NMR (500MHz, DMSO-d 6 ) δ7.70(t, J=5.6Hz, 1H), 4.31(d, J=5.1Hz, 1H), 3.08–2.95(m, 3H), 2.11–2.01( m,3H),2.00–1.75(m,5H),1.69–1.58(m,3H),1.54(d,J＝1.7Hz,3H),1.52–1.42(m,4H),1.38–1.33(m, 2H), 1.32(t, J＝3.9Hz, 1H), 1.30–1.23(m, 3H), 1.20–1.10(m, 1H), 1.03–0.91(m, 2H), 0.89–0.83(m, 6H) ,0.81–0.72(m,6H),0.68(s,3H). 13 C NMR(125MHz,DMSO)δ171.81,132.96,125.85,76.90,53.91,53.52,38.44,38.02,37.66,36.58,36.03,35.32,33.02 ,31.79,31.35,28.56,28.14,27.76,27.42,25.61,21.64,21.46,19.57,19.30,15.76,14.06,13.67.
化合物28，白色固体，产率99.7％。 1H NMR(500MHz,DMSO-d 6)δ7.71(t,J＝5.6Hz,1H),4.31(d,J＝5.1Hz,1H),3.04–2.95(m,3H),2.11–1.83(m,7H),1.63(dd,J＝18.1,13.4Hz,3H),1.57–1.52(m,3H),1.53–1.39(m,5H),1.36(t,J＝7.0Hz,2H),1.30–1.21(m,8H),1.15(d,J＝7.3Hz,1H),1.01–0.92(m,2H),0.90–0.83(m,6H),0.81–0.72(m,6H),0.68(s,3H). 13C NMR(150MHz,DMSO)δ171.80,132.97,125.88,76.90,53.90,53.52,38.45,38.34,37.65,36.58,36.04,35.30,33.01,31.80,31.04,29.19,28.56,28.16,27.70,27.42,26.10,25.60,22.08,21.65,21.45,19.32,15.79,14.07,13.92. Compound 28, white solid, yield 99.7%. 1 H NMR (500MHz, DMSO-d 6 ) δ7.71(t, J=5.6Hz, 1H), 4.31(d, J=5.1Hz, 1H), 3.04–2.95(m,3H), 2.11–1.83( m,7H), 1.63(dd,J=18.1,13.4Hz,3H),1.57–1.52(m,3H),1.53–1.39(m,5H),1.36(t,J=7.0Hz,2H),1.30 –1.21(m,8H),1.15(d,J＝7.3Hz,1H),1.01–0.92(m,2H),0.90–0.83(m,6H),0.81–0.72(m,6H),0.68(s ,3H) .13C NMR(150MHz,DMSO)δ171.80,132.97,125.88,76.90,53.90,53.52,38.45,38.34,37.65,36.58,36.04,35.30,33.01,31.80,31.04,29.19,28.16,28.56, 27.42, 26.10, 25.60, 22.08, 21.65, 21.45, 19.32, 15.79, 14.07, 13.92.
化合物29，无色油状物，产率67％。 1H NMR(500MHz,Chloroform-d)δ5.43(q,J＝6.9,5.8Hz,1H),3.28–3.15(m,3H),2.16(t,J＝7.6Hz,2H),2.12–2.07(m,1H),2.05–1.84(m,4H),1.78–1.72(m,1H),1.73–1.55(m,10H),1.51–1.45(m,2H),1.42–1.35(m,2H),1.31–1.23(m,11H),1.10–0.99(m,2H),0.97(d,J＝8.2Hz,3H),0.87(t,J＝6.8Hz,4H),0.83(d,J＝2.5Hz,1H),0.81(d,J＝5.8Hz,4H),0.78(s,3H). Compound 29, colorless oil, yield 67%. 1 H NMR (500MHz, Chloroform-d) δ5.43 (q, J=6.9, 5.8Hz, 1H), 3.28–3.15 (m, 3H), 2.16 (t, J=7.6Hz, 2H), 2.12–2.07 (m,1H),2.05–1.84(m,4H),1.78–1.72(m,1H),1.73–1.55(m,10H),1.51–1.45(m,2H),1.42–1.35(m,2H) ,1.31–1.23(m,11H),1.10–0.99(m,2H),0.97(d,J=8.2Hz,3H),0.87(t,J=6.8Hz,4H),0.83(d,J=2.5 Hz,1H),0.81(d,J=5.8Hz,4H),0.78(s,3H).
实施例18化合物Q21的制备The preparation of embodiment 18 compound Q21
将化合物Q10(500mg，1.57mmol)溶于甲苯(4mL)，再加入乙二醇(0.26mL，4.7mmol)，原甲酸三乙酯(0.78mL，4.7mmol)，搅拌20min后，加入一水合对甲苯磺酸(13.5mg，0.078mmol)，搅拌1h，TLC检测原料反应完全后，加入饱和碳酸氢钠淬灭反应，DCM(20mL)萃取，分液，水相用DCM(5mL×3)洗涤，合并有机相，有机相分别用水(5mL×3)、饱和氯化钠溶液(5mL×3)洗涤，无水硫酸钠干燥。减压浓缩硅胶柱层析(PE：EA＝10：1)得到 化合物Q21(无色油状物，381mg，产率67％)。Dissolve compound Q10 (500mg, 1.57mmol) in toluene (4mL), then add ethylene glycol (0.26mL, 4.7mmol), triethyl orthoformate (0.78mL, 4.7mmol), stir for 20min, then add monohydrate Toluenesulfonic acid (13.5mg, 0.078mmol) was stirred for 1h. After TLC detected that the reaction of the raw materials was complete, the reaction was quenched by adding saturated sodium bicarbonate, extracted with DCM (20mL), separated, and the aqueous phase was washed with DCM (5mL×3). The organic phases were combined, washed with water (5 mL×3) and saturated sodium chloride solution (5 mL×3), and dried over anhydrous sodium sulfate. Silica gel column chromatography (PE:EA=10:1) was concentrated under reduced pressure to obtain compound Q21 (colorless oil, 381 mg, yield 67%).
实施例19化合物Q22～Q23的制备The preparation of embodiment 19 compound Q22～Q23
将化合物Q21(381mg，1.05mmol)溶于DMSO(2mL)和四氢呋喃(2mL)，加入IBX(588.9mg，2.10mmol)，25℃搅拌1h，TLC检测原料反应完全后，加水，抽滤。滤液用乙酸乙酯(20mL)萃取，分液，水相用乙酸乙酯(5mL×3)洗涤，合并有机相。有机相分别用饱和碳酸氢钠溶液(5mL×3)、水(5mL×3)、饱和氯化钠溶液(5mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝20：1)得到化合物Q22(无色油状物，300mg，产率79.2％)。Compound Q21 (381mg, 1.05mmol) was dissolved in DMSO (2mL) and tetrahydrofuran (2mL), added IBX (588.9mg, 2.10mmol), stirred at 25°C for 1h, after the reaction of the raw materials was detected by TLC, water was added, and suction filtered. The filtrate was extracted with ethyl acetate (20 mL), separated, the aqueous phase was washed with ethyl acetate (5 mL×3), and the organic phases were combined. The organic phase was washed with saturated sodium bicarbonate solution (5mL×3), water (5mL×3), saturated sodium chloride solution (5mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure for silica gel column chromatography (PE: EA=20:1) Compound Q22 (colorless oil, 300 mg, yield 79.2%) was obtained.
将(2-羧乙基)三苯基溴化膦(927.9mg，2.234mmol)加入四氢呋喃(10mL)中形成悬浊液，氮气保护，-20℃下，加入NaHDMS(0.96mL，1.975mmol)，搅拌至橙黄色，加入Q22(230mg，0.638mmol)的10mL四氢呋喃的溶液，溶液黄色褪去，TLC检测原料反应完全后，加入饱和氯化铵溶液淬灭反应，乙酸乙酯(30mL)萃取，分液，水相用乙酸乙酯(10mL×3)洗涤三次，合并有机相。有机相用饱和氯化钠溶液(10mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝6：1)得到化合物Q23(无色油状物，255mg，产率96％)。 1H NMR(600MHz,Chloroform-d)δ5.64–5.55(m,1H),5.55–5.43(m,1H),4.03–3.80(m,4H),3.20–3.02(m,2H),2.13–2.07(m,3H),2.07–2.00(m,1H),1.99–1.92(m,2H),1.91–1.79(m,2H),1.68–1.62(m,2H),1.60–1.55(m,3H),1.55–1.49(m,1H),1.39–1.35(m,1H),1.34–1.29(m,3H),1.28(s,1H),1.27–1.24(m,1H),1.10–1.00(m,1H),0.96–0.91(m,4H),0.87–0.79(m,6H). Add (2-carboxyethyl)triphenylphosphine bromide (927.9mg, 2.234mmol) into tetrahydrofuran (10mL) to form a suspension, under nitrogen protection, add NaHDMS (0.96mL, 1.975mmol) at -20°C, Stir until orange-yellow, add a solution of Q22 (230 mg, 0.638 mmol) in 10 mL of tetrahydrofuran, the yellow color of the solution fades, and after TLC detects that the raw materials have reacted completely, add saturated ammonium chloride solution to quench the reaction, extract with ethyl acetate (30 mL), and separate , the aqueous phase was washed three times with ethyl acetate (10 mL×3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Silica gel column chromatography (PE:EA=6:1) gave compound Q23 (colorless oil, 255mg, yield 96%). 1 H NMR (600MHz, Chloroform-d) δ5.64–5.55(m,1H),5.55–5.43(m,1H),4.03–3.80(m,4H),3.20–3.02(m,2H),2.13– 2.07(m,3H),2.07–2.00(m,1H),1.99–1.92(m,2H),1.91–1.79(m,2H),1.68–1.62(m,2H),1.60–1.55(m,3H ),1.55–1.49(m,1H),1.39–1.35(m,1H),1.34–1.29(m,3H),1.28(s,1H),1.27–1.24(m,1H),1.10–1.00(m ,1H),0.96–0.91(m,4H),0.87–0.79(m,6H).
实施例20化合物Q24～Q25的制备The preparation of embodiment 20 compound Q24～Q25
将化合物Q23(295mg，0.708mmol)溶于四氢呋喃(6mL)中，加入29.5mg 10％Pd/C，氢气压力为1atm，25℃下搅拌反应1h，TLC检测原料反应完全后，过滤，滤液减压浓缩得化合物Q24，未经纯化直接进行下一步。Compound Q23 (295 mg, 0.708 mmol) was dissolved in tetrahydrofuran (6 mL), 29.5 mg of 10% Pd/C was added, the hydrogen pressure was 1 atm, and the reaction was stirred at 25 ° C for 1 h. After the reaction of the raw materials was detected by TLC, it was filtered and the filtrate was decompressed. Concentration gave compound Q24, which was directly carried to the next step without purification.
将化合物Q24溶于四氢呋喃(4mL)中，加入2mol/L稀盐酸(4mL)，25℃搅拌反应4h，TLC检测原料反应完全后，乙酸乙酯(30mL)萃取，分液，水相用乙酸乙酯(10mL×3)洗涤三次，合并有机相。有机相分别用饱和碳酸氢钠(10mL×3)、水(10mL×3)、饱和氯化钠溶液(10mL×3)洗涤，无水硫酸钠干燥，减压浓缩硅胶柱层析(PE：EA＝6：1)得到化合物Q25(无色油状物，197.5mg，两步产率74％)。 1HNMR(500MHz,Chloroform-d)δ2.68–2.56(m,1H),2.39–2.32(m,2H),2.17–2.11(m,1H),2.08–1.95(m,4H),1.91(dd,J＝16.4,5.3Hz,1H),1.74–1.61(m,4H),1.59(d,J＝1.6Hz,3H),1.52–1.40(m,3H),1.39–1.33(m,2H),1.32(s,1H),1.29–1.24(m,2H),1.13(dd,J＝12.1,5.3Hz,1H),1.06(d,J＝3.5Hz,3H),1.04(s,3H),1.00(s,1H),0.97(s,3H),0.95–0.90(m,2H). Dissolve compound Q24 in tetrahydrofuran (4mL), add 2mol/L dilute hydrochloric acid (4mL), stir and react at 25°C for 4h, after TLC detects that the raw materials have reacted completely, extract with ethyl acetate (30mL), separate the layers, and use ethyl acetate for the aqueous phase The ester (10 mL×3) was washed three times, and the organic phases were combined. The organic phase was washed with saturated sodium bicarbonate (10mL×3), water (10mL×3), saturated sodium chloride solution (10mL×3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure by silica gel column chromatography (PE: EA =6:1) Compound Q25 (colorless oil, 197.5 mg, yield 74% in two steps) was obtained. 1 HNMR (500MHz, Chloroform-d) δ2.68–2.56(m,1H),2.39–2.32(m,2H),2.17–2.11(m,1H),2.08–1.95(m,4H),1.91(dd ,J=16.4,5.3Hz,1H),1.74–1.61(m,4H),1.59(d,J=1.6Hz,3H),1.52–1.40(m,3H),1.39–1.33(m,2H), 1.32(s,1H),1.29–1.24(m,2H),1.13(dd,J=12.1,5.3Hz,1H),1.06(d,J=3.5Hz,3H),1.04(s,3H),1.00 (s,1H),0.97(s,3H),0.95–0.90(m,2H).
实施例21化合物Q26的制备Preparation of Example 21 Compound Q26
将化合物Q25(183mg，0.489mmol)溶于二氯甲烷(6mL)和甲醇(3mL)，冰浴下，加入硼氢化钠(185mg，4.89mmol)，搅拌反应5min，TLC检测原料反应完全后，加水淬灭反应，2mol/L稀盐酸调节pH至4～5，二氯甲烷(15mL)萃取，分液，水相用二氯甲烷(5mL×3)洗涤，合并有机相，有机相分别用水(5mL×3)、饱和氯化钠(5mL×3)洗涤，无水硫酸钠干燥。减压浓缩硅胶柱层析(PE：EA＝3：1)得到化合物Q26(白色固体，110mg，产率59.8％)。 1H NMR(600MHz,Chloroform-d)δ3.23(dd,J＝11.7,4.5Hz,1H),2.34(t,2H),2.14–2.08(m,1H),2.03–1.84(m,4H),1.79–1.74(m,1H),1.69–1.60(m,5H),1.58(d,J＝1.9Hz,3H),1.39–1.36(m,1H),1.35(d,J＝4.0Hz,1H),1.33(s,2H),1.28(s,2H),1.25(s,1H),1.08–1.00(m,2H),0.97(d,J＝9.0Hz,3H),0.90–0.85(m,1H),0.84(d,J＝2.4Hz,1H),0.82(d,J＝2.5Hz,1H),0.81(s,3H),0.79(s,3H). Compound Q25 (183mg, 0.489mmol) was dissolved in dichloromethane (6mL) and methanol (3mL), under ice bath, sodium borohydride (185mg, 4.89mmol) was added, and the reaction was stirred for 5min. After the reaction of the raw materials was detected by TLC, water was added Quench the reaction, adjust the pH to 4-5 with 2mol/L dilute hydrochloric acid, extract with dichloromethane (15mL), separate the layers, wash the aqueous phase with dichloromethane (5mL×3), combine the organic phases, and separate the organic phases with water (5mL ×3), washed with saturated sodium chloride (5mL×3), and dried over anhydrous sodium sulfate. Silica gel column chromatography (PE:EA=3:1) was concentrated under reduced pressure to obtain compound Q26 (white solid, 110 mg, yield 59.8%). 1 H NMR (600MHz, Chloroform-d) δ3.23 (dd, J = 11.7, 4.5Hz, 1H), 2.34 (t, 2H), 2.14–2.08 (m, 1H), 2.03–1.84 (m, 4H) ,1.79–1.74(m,1H),1.69–1.60(m,5H),1.58(d,J＝1.9Hz,3H),1.39–1.36(m,1H),1.35(d,J＝4.0Hz,1H ),1.33(s,2H),1.28(s,2H),1.25(s,1H),1.08–1.00(m,2H),0.97(d,J＝9.0Hz,3H),0.90–0.85(m, 1H),0.84(d,J=2.4Hz,1H),0.82(d,J=2.5Hz,1H),0.81(s,3H),0.79(s,3H).
实施例22化合物30～32的制备The preparation of embodiment 22 compound 30～32
将化合物Q26(40mg，0.106mmol)溶于二氯甲烷(1mL)和四氢呋喃(1mL)中，加入HATU(48.5mg，0.128mmol)，DIPEA(0.04mL，0.213mmol)搅拌均匀，再加入相应的胺(0.213mmol)，25℃搅拌反应1h，TLC检测原料反应完全，加入盐酸淬灭反应。用二氯甲烷(10mL×3)萃取水相，合并有机相。有机相分别用水(10mL)、饱和NaCl溶液(10mL)洗涤，无水硫酸钠干燥，经减压浓缩后硅胶柱层析，得到相应的化合物30～32。Dissolve compound Q26 (40mg, 0.106mmol) in dichloromethane (1mL) and tetrahydrofuran (1mL), add HATU (48.5mg, 0.128mmol), DIPEA (0.04mL, 0.213mmol) and stir well, then add the corresponding amine (0.213mmol), stirred and reacted at 25°C for 1h, TLC detected that the reaction of raw materials was complete, and added hydrochloric acid to quench the reaction. The aqueous phase was extracted with dichloromethane (10 mL×3), and the organic phases were combined. The organic phase was washed with water (10 mL) and saturated NaCl solution (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure and then chromatographed on a silica gel column to obtain the corresponding compounds 30-32.
化合物30，白色固体，产率87.1％。 1H NMR(600MHz,DMSO-d 6)δ7.70(t,J＝5.6Hz,1H),4.31(d,J＝5.1Hz,1H),3.08–2.91(m,3H),2.10–2.05(m,1H),2.02(t,J＝7.3Hz,2H),1.99–1.89(m,3H),1.84(dd,J＝17.1,4.9Hz,1H),1.68–1.58(m,3H),1.54(s,3H),1.51–1.42(m,4H),1.38–1.32(m,3H),1.30(dd,J＝9.2,3.7Hz,1H),1.28–1.22(m,3H),1.22–1.13(m,3H),1.02–0.89(m,3H),0.91–0.83(m,6H),0.79–0.73(m,5H),0.69(s,3H). 13C NMR(150MHz,DMSO)δ171.86,133.05,125.83,76.93,53.92,53.54,38.47,38.02,37.66,36.60,36.06,35.41,33.04,31.83,31.31,28.89,28.68,28.18,27.94,27.44,25.27,21.64,21.49,19.56,19.33,15.81,14.09,13.68. Compound 30, white solid, yield 87.1%. 1 H NMR (600MHz, DMSO-d 6 ) δ7.70(t, J=5.6Hz, 1H), 4.31(d, J=5.1Hz, 1H), 3.08-2.91(m, 3H), 2.10-2.05( m,1H),2.02(t,J=7.3Hz,2H),1.99–1.89(m,3H),1.84(dd,J=17.1,4.9Hz,1H),1.68–1.58(m,3H),1.54 (s,3H),1.51–1.42(m,4H),1.38–1.32(m,3H),1.30(dd,J＝9.2,3.7Hz,1H),1.28–1.22(m,3H),1.22–1.13 (m,3H),1.02–0.89(m,3H),0.91–0.83(m,6H),0.79–0.73(m,5H),0.69(s,3H). 13 C NMR(150MHz,DMSO)δ171. 86,133.05,125.83,76.93,53.92,53.54,38.47,38.02,37.66,36.60,36.06,35.41,33.04,31.83,31.31,28.89,28.68,28.18,27.94,27.44,25.27,21.64,21.49,19.56,19.33,15.81, 14.09, 13.68.
化合物31，无色油状物，产率50.5％。 1HNMR(600MHz,DMSO-d 6)δ7.70(t,J＝5.6Hz,1H),4.31(s,1H),3.00(q,J＝6.7Hz,3H),2.08(dd,J＝12.7,3.8Hz,1H),2.02(t,J＝7.2Hz,2H),1.98–1.88(m,3H),1.84(dd,J＝17.1,4.9Hz,1H),1.68–1.57(m,3H),1.54(s,3H),1.52–1.42(m,4H),1.39–1.32(m,3H),1.31–1.29(m,1H),1.28–1.21(m,8H),1.19–1.13(m,2H),1.03–0.90(m,2H),0.86(s,6H),0.81(d,J＝4.1Hz,1H),0.80–0.73(m,5H),0.69(s,3H). 13C NMR(150MHz,DMSO)δ171.81,133.04,125.81,76.90,53.91,53.52,38.45,38.31,37.65,36.58,36.05,35.42,33.02,31.82,31.01,29.14,28.87,28.68,28.16,27.94,27.43,26.08,25.26,22.08,21.63, 21.47,19.31,15.79,14.07,13.91. Compound 31, colorless oil, yield 50.5%. 1 HNMR (600MHz, DMSO-d 6 )δ7.70(t, J=5.6Hz, 1H), 4.31(s, 1H), 3.00(q, J=6.7Hz, 3H), 2.08(dd, J=12.7 ,3.8Hz,1H),2.02(t,J=7.2Hz,2H),1.98–1.88(m,3H),1.84(dd,J=17.1,4.9Hz,1H),1.68–1.57(m,3H) ,1.54(s,3H),1.52–1.42(m,4H),1.39–1.32(m,3H),1.31–1.29(m,1H),1.28–1.21(m,8H),1.19–1.13(m, 13 C NMR (150MHz,DMSO)δ171.81,133.04,125.81,76.90,53.91,53.52,38.45,38.31,37.65,36.58,36.05,35.42,33.02,31.82,31.01,29.14,28.87,28.68,28.16,27.94,27.43,26.08,25.26 ,22.08,21.63, 21.47,19.31,15.79,14.07,13.91.
化合物32，无色油状物，产率：52.9％。 1H NMR(600MHz,Chloroform-d)δ5.44(d,J＝20.9Hz,1H),3.32–3.15(m,3H),2.15(t,J＝7.5Hz,2H),2.13–2.08(m,1H),2.03–1.84(m,4H),1.79–1.73(m,1H),1.70–1.60(m,7H),1.60–1.56(m,3H),1.48(t,J＝7.1Hz,2H),1.39–1.35(m,1H),1.33(s,2H),1.31–1.24(m,13H),1.08–1.01(m,2H),0.97(s,3H),0.90–0.85(m,4H),0.84(d,J＝2.5Hz,1H),0.82–0.77(m,6H). Compound 32, colorless oil, yield: 52.9%. 1 H NMR (600MHz, Chloroform-d) δ5.44(d, J=20.9Hz, 1H), 3.32–3.15(m, 3H), 2.15(t, J=7.5Hz, 2H), 2.13–2.08(m ,1H),2.03–1.84(m,4H),1.79–1.73(m,1H),1.70–1.60(m,7H),1.60–1.56(m,3H),1.48(t,J＝7.1Hz,2H ),1.39–1.35(m,1H),1.33(s,2H),1.31–1.24(m,13H),1.08–1.01(m,2H),0.97(s,3H),0.90–0.85(m,4H ),0.84(d,J＝2.5Hz,1H),0.82–0.77(m,6H).
由于化合物2-32的基础母核结构类似，在图1-4中，本发明以化合物8的应用效果为例可以体现本发明中化合物的效果。Since the basic core structures of compounds 2-32 are similar, in Figures 1-4, the present invention takes the application effect of compound 8 as an example to reflect the effects of the compounds in the present invention.
实施例23 SMO抑制剂抑制SMO蛋白活性测试Example 23 SMO inhibitor inhibits SMO protein activity test
采用荧光素报告法对NIH3T3细胞进行SMO蛋白抑制活性测试。The inhibitory activity of SMO protein in NIH3T3 cells was tested by luciferin reporter method.
1.实验方法：(1)将NIH3T3细胞以每盘1.5×10 6的细胞量接种到10cm的细胞培养皿中，用DMEM培养基(含有10％新生牛血清，双抗)培养于37℃、5％CO 2培养箱中24小时。(2)用脂质体LTX(英杰)将GliSBS和pEGFP-N1(按9：1，总转染量为6μg)转染进NIH3T3细胞中，转染6小时换回DMEM培养基(含有10％新生牛血清，双抗)；待细胞汇合度达到80％时，用0.25％胰酶(含有EDTA)消化吹匀后以每孔1×10 5的细胞量接种到24孔板中。(3)继续培养24小时后，吸掉原有的培养基，用PBS缓冲液(137mM NaCl，2.7mM KCl，10mM Na 2HPO 4，2mM KH 2PO 4)洗一遍，换上含有不同浓度的本发明实施例制备的化合物(2-32)的饥饿培养基(含有本发明制备的Shh-N(人的音猬因子的cDNA 1-198)培养上清液)。在Shh-N的刺激下，可以最大程度上激活Hh信号通路。(4)培养24小时后，吸掉培养基，用PBS缓冲液洗一遍，每孔加入100μL配置好的裂解缓冲液(普洛麦格E166A)，放置于-80℃冰箱中冻结5min(或者过夜)，再室温解冻后，放置于涡旋仪上，以800rpm振荡混匀30min，达到充分裂解细胞的目的。(5)裂解好的细胞用萤火虫萤光素酶试剂盒(普洛麦格E1501)测定自发光的数值，用Cytation3多功能酶标仪测定EGFP的数值(激发光为485，接收光为528)，以自发光的读值和EGFP的读值的比值，即单位荧光值作为各测试化合物对Hh信号通路的抑制能力。IC 50值见表1，表1为本发明所述化合物2-32在NIH3T3细胞上抑制Hh信号通路活性数据(IC 50)。实验重复三次，IC 50取其平均值。各测试化合物对Hh信号通路抑制IC 50值见表1。 1. Experimental method: (1) NIH3T3 cells were inoculated into a 10 cm cell culture dish with a cell volume of 1.5×10 6 per plate, and cultured at 37° C. 24 hr in a 5% CO 2 incubator. (2) Use liposome LTX (Invitrogen) to transfect GliSBS and pEGFP-N1 (according to 9:1, the total transfection amount is 6μg) into NIH3T3 cells, and change back to DMEM medium (containing 10% Newborn bovine serum, double antibody); when the confluence of the cells reaches 80%, they are digested with 0.25% trypsin (containing EDTA) and blown evenly, and then inoculated into a 24-well plate at a cell volume of 1×10 5 per well. (3) After continuing to cultivate for 24 hours, suck off the original medium, wash it once with PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na 2 HPO 4 , 2mM KH 2 PO 4 ), and replace it with The starvation medium of the compound (2-32) prepared in the example of the present invention (containing the culture supernatant of Shh-N (cDNA 1-198 of human sonic hedgehog) prepared in the present invention). Under the stimulation of Shh-N, the Hh signaling pathway can be activated to the greatest extent. (4) After culturing for 24 hours, suck off the medium, wash it once with PBS buffer, add 100 μL of prepared lysis buffer (Promega E166A) to each well, and place it in a -80°C refrigerator for 5 minutes (or overnight). ), and then thawed at room temperature, placed on a vortex instrument, oscillating and mixing at 800rpm for 30min, to achieve the purpose of fully lysing the cells. (5) Use the firefly luciferase kit (Promega E1501) to measure the value of autoluminescence of the lysed cells, and measure the value of EGFP with Cytation3 multifunctional microplate reader (the excitation light is 485, and the received light is 528) , the ratio of the read value of autoluminescence to the read value of EGFP, that is, the unit fluorescence value, was used as the inhibitory ability of each test compound to the Hh signaling pathway. The IC 50 values are shown in Table 1, and Table 1 shows the activity data (IC 50 ) of compound 2-32 of the present invention inhibiting the Hh signaling pathway on NIH3T3 cells. The experiment was repeated three times, and the average value of IC 50 was taken. See Table 1 for the IC 50 values of each test compound on Hh signaling pathway inhibition.
实验结果表明：本发明制备的一系列三环二萜类似物(2-32)对SMO蛋白均有较好的抑制效果，如表1所示，化合物14抑制效果最佳。The experimental results show that: a series of tricyclic diterpene analogs (2-32) prepared by the present invention have good inhibitory effect on SMO protein. As shown in Table 1, compound 14 has the best inhibitory effect.
实施例24 SMO胆固醇修饰实验Example 24 SMO cholesterol modification experiment
实验方法：(1)将NIH3T3细胞以每盘1×10 6的细胞量接种到60mm的细胞培养皿中， 用10％FCS培养基培养于37℃、5％CO 2培养箱中24小时；(2)用脂质体2000将质粒(总转染量为6μg)转染进NIH3T3细胞中，转染6小时换回10％FCS培养基；(3)继续培养24小时后，吸去原有的培养基，用PBS洗一次，换上胆固醇饥饿培养基(DMEM+P/S，5％LPPS，1μM洛伐他汀，10μM甲羟戊酸)并含有相关测试的化合物(维莫德吉，索尼德吉，化合物8)培养过夜。(4)吸去原有培养基，换上含有化合物(维莫德吉，索尼德吉，化合物8)的胆固醇饥饿培养基继续处理8小时；(5)刮下细胞，4℃，1000g，离心5min；(6)弃去上清，加入800μl预冷的PBS，并转移至1.5mL ep管中，1000rpm，4℃，离心5min；(7)弃去上清，用200μl RIPA裂解液(50mM Tris-HCl，pH＝8.0，150mM NaCl，0.1％SDS，1.5％NP-40，2mM MgCl 2，0.5％脱氧胆酸钠，5μg/mL抑肽素，10μg/mL亮肽素，5μM MG132，1mM苯甲基磺酰氟，50mM二硫苏糖醇)重悬细胞，用7号针头来回吸吹10次，132000rpm，4℃，离心10min；(8)取一定量的上清到新的EP管中，按顺序逐步加入biotin(终浓度100μM)、CuSO 4(终浓度1mM)及维生素C钠盐(终浓度2.5mM)，加完之后来回混匀一下后，再加入TBTA(终浓度1mM)，全部加完后再来回混匀一下。27℃，水平震荡仪上震荡反应1.5hrs；(9)反应完后的样品，13200rpm，4℃离心10min，取一定量上清到10k的透析管中，4℃透析两小时或过夜，透析两次；(10)透析完后的样品，取一定量上清到含有20μL树脂包被的中性亲和素磁珠(beads使用前用RIPA裂解缓冲液洗一次)，IP 6小时或者过夜；(11)IP完后的样品用RIPA裂解缓冲液洗3次，后用2×蛋白上样缓冲液，95℃，离心10min后，取上清，并加入等体积的跨膜蛋白溶解缓冲液，-20℃保存。实验结果如图1所示。 Experimental method: (1) NIH3T3 cells were inoculated into a 60mm cell culture dish with a cell volume of 1×10 6 per plate, and cultured in 37° C., 5% CO 2 incubator with 10% FCS medium for 24 hours; ( 2) Transfect the plasmid (total transfection amount is 6 μg) into NIH3T3 cells with liposome 2000, and change back to 10% FCS medium after transfection for 6 hours; (3) After continuing to cultivate for 24 hours, suck off the original Medium, washed once with PBS, replaced with cholesterol-starved medium (DMEM+P/S, 5% LPPS, 1 μM lovastatin, 10 μM mevalonic acid) and containing relevant test compounds (Vimodeji, Sonnide Ji, compound 8) was incubated overnight. (4) Suck off the original medium and replace it with a cholesterol-starved medium containing compounds (Vimodeji, Sonideji, Compound 8) and continue to treat for 8 hours; (5) Scrape off the cells, centrifuge at 4°C, 1000g 5min; (6) Discard the supernatant, add 800μl pre-cooled PBS, and transfer to a 1.5mL ep tube, 1000rpm, 4°C, centrifuge for 5min; (7) Discard the supernatant, use 200μl RIPA lysate (50mM Tris -HCl, pH=8.0, 150 mM NaCl, 0.1% SDS, 1.5% NP-40, 2 mM MgCl 2 , 0.5% sodium deoxycholate, 5 μg/mL peptistatin, 10 μg/mL leupeptin, 5 μM MG132, 1 mM benzene Methylsulfonyl fluoride, 50mM dithiothreitol) to resuspend the cells, blow back and forth 10 times with a No. 7 needle, centrifuge at 132000rpm, 4°C for 10min; (8) Take a certain amount of supernatant into a new EP tube , gradually add biotin (final concentration 100μM), CuSO 4 (final concentration 1mM) and vitamin C sodium salt (final concentration 2.5mM) in order, after adding, mix back and forth, then add TBTA (final concentration 1mM), all Mix back and forth after adding. 27°C, shaking reaction on a horizontal shaker for 1.5hrs; (9) After the reaction, the sample was centrifuged at 13200rpm, 4°C for 10min, and a certain amount of supernatant was taken into a 10k dialysis tube, and dialyzed at 4°C for two hours or overnight, and dialyzed for two hours. (10) After the dialyzed sample, take a certain amount of supernatant to contain 20 μL resin-coated neutravidin magnetic beads (beads are washed once with RIPA lysis buffer before use), IP for 6 hours or overnight; ( 11) After IP, the sample was washed 3 times with RIPA lysis buffer, and then washed with 2× protein loading buffer, 95°C, centrifuged for 10 minutes, and the supernatant was taken, and an equal volume of transmembrane protein lysis buffer was added, - Store at 20°C. The experimental results are shown in Figure 1.
2.实验结果表明：本发明实施例制备的三环二萜类似物(8)对SMO的胆固醇修饰有很好的抑制效果，且具有浓度梯度效应。2. The experimental results show that the tricyclic diterpene analog (8) prepared in the embodiment of the present invention has a good inhibitory effect on the cholesterol modification of SMO, and has a concentration gradient effect.
实施例25 MTT细胞毒性测试Example 25 MTT Cytotoxicity Test
1.实验方法：(1)将293T细胞以每孔5×10 3的细胞量接种到96孔板中，用10％FBS培养基培养于37℃、5％CO 2培养箱中24小时。(2)吸掉原有的培养基，用PBS缓冲液洗一遍，换上含有不同浓度的本发明实施例制备的化合物(2-32)的DMEM培养基(含有Shh-N培养上清液，双抗)处理24小时。(3)每孔加入20μL MTT溶液(5mg/mL，即0.5％MTT)，继续培养4h。(4)终止培养，小心吸去孔内培养液。(5)每孔加入150μL二甲基亚砜(DMSO)，置摇床上低速振荡10min，使结晶物充分溶解。在酶联免疫检测仪OD490nm处测量各孔的吸光值。实验结果如表1(细胞毒性)所示。 1. Experimental method: (1) 293T cells were inoculated into a 96-well plate at a cell volume of 5×10 3 per well, and cultured in 10% FBS medium in a 37°C, 5% CO 2 incubator for 24 hours. (2) Suck off the original medium, wash it once with PBS buffer, and replace it with DMEM medium (containing Shh-N culture supernatant, double antibody) treatment for 24 hours. (3) 20 μL of MTT solution (5 mg/mL, ie 0.5% MTT) was added to each well, and culture was continued for 4 h. (4) Terminate the culture, and carefully suck off the culture medium in the well. (5) Add 150 μL of dimethyl sulfoxide (DMSO) to each well, shake on a shaker at low speed for 10 min, and fully dissolve the crystals. The absorbance of each well was measured at OD490nm in an enzyme-linked immunosorbent assay instrument. The experimental results are shown in Table 1 (cytotoxicity).
2.实验结果表明：本发明实施例制备的一系列三环二萜类似物(2-32)大多数都不具有细胞毒性。2. The experimental results show that most of the series of tricyclic diterpene analogs (2-32) prepared in the examples of the present invention have no cytotoxicity.
实施例26氟硼荧-环靶眀细胞结合实验Example 26 Fluoroborofluorescein-ring target cell binding experiment
1.实验方法：(1)将293T细胞以每孔6×10 6的细胞量接种到12孔板(内含有细胞爬片)中，用10％FBS培养基培养于37℃、5％CO 2培养箱中24小时；用LPEI将质粒(Hus-SMO-C-mCherry，总转染量为1μg)转染进293T细胞中，转染6小时后换回10％FBS培养基；(2)待细胞汇合度达到80％左右时，吸掉原有的培养基，用PBS缓冲液洗一遍，换上含有不同浓度本发明实施例制备的化合物8和对照化合物(环靶眀)的DMEM培养基(含有10％FBS，100unit/mL双抗，10μM氟硼荧-环靶眀)处理1小时。实验结果如图2所示。 1. Experimental method: (1) 293T cells were inoculated into 12-well plates (containing cell slides) at a cell volume of 6×10 6 per well, and cultured with 10% FBS medium at 37°C and 5% CO 2 24 hours in the incubator; transfect the plasmid (Hus-SMO-C-mCherry, the total transfection amount is 1 μg) into 293T cells with LPEI, and change back to 10% FBS medium after 6 hours of transfection; (2) When the confluence of the cells reached about 80%, the original culture medium was sucked off, washed once with PBS buffer, and replaced with DMEM culture medium ( Containing 10% FBS, 100unit/mL double antibody, 10μM fluborfluorescein-cyclotarget) for 1 hour. The experimental results are shown in Figure 2.
2.实验结果表明：本发明三环二萜类似物(8)不会竞争抑制氟硼荧-环靶眀和SMO的结合，说明本发明实施例制备的三环二萜类似物与传统的SMO抑制剂的结合位点不一样。2. The experimental results show that: the tricyclic diterpene analog (8) of the present invention will not compete to inhibit the combination of fluborfluorin-cyclophanate and SMO, illustrating that the tricyclic diterpene analog prepared by the embodiments of the present invention is compatible with traditional SMO Inhibitors have different binding sites.
实施例27小鼠小脑的神经元祖细胞(cGNP)分离实验和髓母细胞瘤小鼠移植实验Example 27 Neuron Progenitor Cell (cGNP) Isolation Experiment of Mouse Cerebellum and Medulloblastoma Mouse Transplantation Experiment
1.小鼠小脑的神经元祖细胞(cGNP)分离实验1. Mouse cerebellar neuron progenitor cells (cGNP) isolation experiment
方法：(1)准备刚出生6天的幼鼠(一般6只左右)；(2)用剪刀减掉脑子，并用镊子撕开头骨上的外皮，用剪刀从中心缝的位置剪开头盖骨(注意：不要破坏了小脑的结构)，取出小脑，并置于预冷的PBS里；(3)用体视镜观察，小心的取掉小脑背面的红丝带，并将剩余部分的小脑转移至一个新的预冷1.5mL的管里(里面装有PBS)；(注意：每操作一只小鼠时，小脑的分离应小于6分钟)(4)用枪吸走尽可能多的上清(PBS)，(注意：枪先来回吸取FBS两次。)；(5)加入1mL胰酶和DNA酶的混合液。37℃孵育5-10min；(6)用枪吸走尽可能多的上清(注意：枪先来回吸取FBS两次。)；(7)加入1mL DNA酶溶液，用枪反复吸吹10次即可。(注意不要有气泡)；(8)使悬浮液静置1min，将上清转移至新的1.5mL EP管中(不要吸到底部沉淀部分)；(9)800g，4℃，5min。速度调至最低；(10)去掉上清，加入50μL DNA酶，2mL神经细胞培养基(0.1mL 500 X双抗，0.5mL 100 X谷氨酰胺，0.5mL浓度为45％的D+-葡萄糖，0.5mL of 100 X SPITE，0.5mL of 100 X油酸白蛋白/亚油酸，1mL 50X B27，50μL 1000X N-乙酰半胱氨酸，用神经基础培养基补平至50mL)重悬细胞；(11)用尼龙网过滤细胞；(12)在多聚右旋赖氨酸(Millipore)包被的10cm培养皿里预培养20min，5％CO 2，37℃培养箱。轻拍平皿。并吸取上清至新的培养皿里；(13)5％CO 2，37℃，预培养20min；(14)将上清收集后，200g，5min，离心。用神经细胞培养基重悬细胞；(15)铺板。实验结果如图3所示。 Methods: (1) Prepare 6-day-old pups (generally about 6); (2) Cut off the brain with scissors, tear off the skin on the skull with tweezers, and cut the skull from the central seam with scissors (note : do not destroy the structure of the cerebellum), take out the cerebellum, and place it in pre-cooled PBS; (3) observe with a stereoscope, carefully remove the red ribbon on the back of the cerebellum, and transfer the remaining part of the cerebellum to a new In a pre-cooled 1.5mL tube (with PBS inside); (Note: When operating a mouse, the separation of the cerebellum should be less than 6 minutes) (4) Aspirate as much supernatant (PBS) as possible with a gun , (Note: the gun first sucks the FBS back and forth twice.); (5) Add 1mL of trypsin and DNase mixture. Incubate at 37°C for 5-10 minutes; (6) suck as much supernatant as possible with a gun (note: the gun first sucks the FBS back and forth twice.); (7) add 1mL DNase solution, and use the gun to repeatedly suck and blow 10 times Can. (Be careful not to have air bubbles); (8) Allow the suspension to stand for 1 min, and transfer the supernatant to a new 1.5 mL EP tube (do not suck the sediment at the bottom); (9) 800g, 4°C, 5 min. Adjust the speed to the lowest; (10) remove the supernatant, add 50μL DNase, 2mL nerve cell culture medium (0.1mL 500X double antibody, 0.5mL 100X glutamine, 0.5mL 45% D+-glucose, 0.5 mL of 100 X SPITE, 0.5 mL of 100 X oleic acid albumin/linoleic acid, 1 mL of 50X B27, 50 μL of 1000X N-acetylcysteine, fill up to 50 mL with neural basal medium) to resuspend cells; (11 ) Filter the cells with a nylon mesh; (12) Pre-cultivate the cells in a 10 cm culture dish coated with poly-D-lysine (Millipore) for 20 min, 5% CO 2 , in a 37° C. incubator. Tap the plate lightly. And pipette the supernatant into a new petri dish; (13) 5% CO 2 , 37°C, pre-culture for 20 min; (14) collect the supernatant, centrifuge at 200 g for 5 min. Resuspend cells with neuron culture medium; (15) plate. The experimental results are shown in Figure 3.
2.髓母细胞瘤小鼠移植实验2. Medulloblastoma mouse transplantation experiment
实验方法：(1)冻存的肿瘤块或长有肿瘤的小鼠致死后，用工具剪碎并切细。(在预冷的PBS中)；(2)迅速将组织转移至50％细胞消化液中，进一步剪碎，切细3min；(3)将剪碎后的样品(一般至于皿中)，放置培养箱中，4min；(4)用1ml的移液枪来回吸吹4min；(5)将消化后的组织悬液转移到15mL的离心管中，加入3倍体积的PBS，1000g/5min； (6)去上清，用预冷的PBS重悬沉淀，计数；(7)调整细胞浓度到1×10 5K/mL。(计数可以稀的记，然后往高浓度浓缩)；(8)加入等体积的生长因子人工基质(BD)，混匀并置于冰上；(9)以每只小鼠0.2mL/只的量，打在小鼠的右前肢下的肢夹窝处。(打之前用酒精先擦拭一下)；(10)每隔两天观察一下小鼠的成瘤情况。实验结果如图4所示。 Experimental methods: (1) After the frozen tumor mass or mice with tumors were killed, they were shredded and finely chopped with tools. (in pre-cooled PBS); (2) quickly transfer the tissue to 50% cell digestion solution, further chop it up, and mince it for 3 minutes; (3) place the chopped sample (usually in a dish) for culture (4) Use a 1ml pipette gun to suck back and forth for 4min; (5) Transfer the digested tissue suspension to a 15mL centrifuge tube, add 3 times the volume of PBS, 1000g/5min; (6 ) remove the supernatant, resuspend the pellet with pre-cooled PBS, and count; (7) adjust the cell concentration to 1×10 5 K/mL. (The counts can be recorded dilutely, and then concentrated to a high concentration); (8) Add an equal volume of growth factor artificial matrix (BD), mix well and place on ice; (9) Add 0.2mL/mouse To measure the amount, hit the right forelimb of the mouse at the limb clamp socket. (wipe it with alcohol before beating); (10) Observe the tumor formation of mice every two days. The experimental results are shown in Figure 4.
3.细胞生长实验3. Cell Growth Experiment
实验方法：(1)将分离的小鼠小脑的神经元祖细胞或者分离培养的髓母细胞瘤细胞以每盘1×10 6的细胞量接种到12孔板(内含有细胞爬片)中，用神经细胞培养液培养于37℃、5％CO 2培养箱中24小时；(2)吸去原有的培养基，换上含有不同浓度本发明制备的化合物(8)的神经细胞培养基(加上本发明制备的Shh-N培养上清液)继续培养24小时。(3)吸去原有的培养基，换上含有不同浓度本发明实施例制备的化合物(8)和BrdU(10μM，Thermo)的神经细胞培养基(加上本发明制备的Shh-N培养上清液)继续培养12小时。(4)弃去细胞培养液，加入200μL/每孔4％PFA/PBS，室温固定15分钟。(5)弃去固定液，200μL每孔3％BSA/PBS洗细胞2遍，每遍5分钟。(6)弃去清洗液，加入200μL每孔0.2％Triton X-100/PBS透化，室温孵化20分钟。(7)弃去透化液，500μL每孔3％BSA/PBS洗细胞2遍，每遍5分钟。(8)EdU的点击化学反应。1×Click-iT缓冲液215μL，CuSO 410μL，Alexa Fluor叠氮化物0.6μL，添加剂25μL，总体积/孔250μL。弃去清洗液，加入250μL每孔1×Click-iT反应液，轻轻晃匀，室温避光孵育30分钟。(9)弃去反应液，500μL每孔3％BSA/PBS洗细胞1遍，每遍5分钟。(10)去离子水润洗一次。(11)封片。 Experimental method: (1) Inoculate isolated mouse cerebellar neuron progenitor cells or isolated and cultured medulloblastoma cells in a 12-well plate (containing cell slides) at a cell volume of 1×10 6 per plate, and use Nerve cell culture solution was cultivated at 37° C., 5% CO in the incubator for 24 hours; (2) absorb the original culture medium and replace it with the nerve cell culture medium containing the compound (8) prepared by the present invention in different concentrations (adding Shh-N culture supernatant prepared by the present invention) was continued to culture for 24 hours. (3) Aspirate the original culture medium, and replace it with a nerve cell culture medium containing different concentrations of compound (8) and BrdU (10 μM, Thermo) prepared in the embodiments of the present invention (plus Shh-N cultured in the present invention) supernatant) for a further 12 hours. (4) Discard the cell culture medium, add 200 μL/well of 4% PFA/PBS, and fix at room temperature for 15 minutes. (5) The fixative was discarded, and the cells were washed twice with 200 μL of 3% BSA/PBS per well for 5 minutes each time. (6) Discard the washing solution, add 200 μL per well of 0.2% Triton X-100/PBS to permeabilize, and incubate at room temperature for 20 minutes. (7) The permeabilization solution was discarded, and the cells were washed twice with 500 μL per well of 3% BSA/PBS for 5 minutes each time. (8) Click chemical reaction of EdU. 1× Click-iT buffer 215 μL, CuSO 4 10 μL, Alexa Fluor azide 0.6 μL, additive 25 μL, total volume/well 250 μL. Discard the washing solution, add 250 μL of 1×Click-iT reaction solution per well, shake gently, and incubate at room temperature for 30 minutes in the dark. (9) The reaction solution was discarded, and the cells were washed once with 500 μL per well of 3% BSA/PBS for 5 minutes each time. (10) Rinse once with deionized water. (11) Seal the film.
实验表明，本发明式(I)三环二萜类似物(以本发明制备的8号化合物为例)不仅能很好的抑制野生型SMO蛋白活性，也可以抑制维莫德吉&索尼德吉&格拉德吉药物抵抗型SMO蛋白的活性。这些三环二萜类似物(以本发明制备的8号化合物为例)能有效地抑制肿瘤细胞增殖，且对于Hedgehog依赖型的髓母细胞瘤具有较好抑制能力，具有良好的应用前景。Experiments have shown that the tricyclic diterpene analogs of formula (I) of the present invention (taking compound No. 8 prepared by the present invention as an example) can not only well inhibit the activity of wild-type SMO protein, but also inhibit the activity of Vimodeji & Sonideji & Gladji activity of drug-resistant SMO proteins. These tricyclic diterpene analogs (taking compound No. 8 prepared by the present invention as an example) can effectively inhibit tumor cell proliferation, and have better inhibitory ability against Hedgehog-dependent medulloblastoma, and have good application prospects.
表1本发明实施例制备的化合物抑制SMO蛋白的活性数据Table 1 The compounds prepared by the embodiments of the present invention inhibit the activity data of SMO protein
Figure PCTCN2022113625-appb-000018
Figure PCTCN2022113625-appb-000018
Figure PCTCN2022113625-appb-000019
Figure PCTCN2022113625-appb-000019
Figure PCTCN2022113625-appb-000020
Figure PCTCN2022113625-appb-000020
Figure PCTCN2022113625-appb-000021
Figure PCTCN2022113625-appb-000021
Figure PCTCN2022113625-appb-000022
Figure PCTCN2022113625-appb-000022
a：化合物有细胞毒性时，无IC 50，以—表示 a: When the compound is cytotoxic, there is no IC 50 , represented by -
b：+表示化合物有细胞毒性，—为无细胞毒性b: + means the compound has cytotoxicity, - means no cytotoxicity
本发明的保护内容不局限于以上实施例。在不背离本发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the concept of the present invention, changes and advantages conceivable by those skilled in the art are all included in the present invention, and the appended claims are the protection scope.

Claims (15)

  1. 一种三环二萜类似物，其特征在于，其结构如式(Ⅰ)所示：A tricyclic diterpene analog, characterized in that its structure is as shown in formula (I):
    Figure PCTCN2022113625-appb-100001
    Figure PCTCN2022113625-appb-100001
    其中，in,
    R 1选自羰基、羟基； R 1 is selected from carbonyl, hydroxyl;
    R 2选自烷烃胺、杂环胺、芳香胺或羟基； R is selected from alkane amines, heterocyclic amines, aromatic amines or hydroxyl groups;
    n选自自然数。n is selected from natural numbers.
  2. 如权利要求1所述的三环二萜类似物，其特征在于，R 1选自羰基、β-羟基； The tricyclic diterpene analog as claimed in claim 1, wherein R is selected from carbonyl, β-hydroxyl;
    R 2选自羟基–OH、烷烃胺、杂环胺、芳香胺；其中，所述烷烃胺选自
    Figure PCTCN2022113625-appb-100002
    Figure PCTCN2022113625-appb-100003
    Figure PCTCN2022113625-appb-100004
    所述杂环胺选自
    Figure PCTCN2022113625-appb-100005
    Figure PCTCN2022113625-appb-100006
    所述芳香胺选自
    Figure PCTCN2022113625-appb-100007
    Figure PCTCN2022113625-appb-100008
     R2 is selected from hydroxyl-OH, alkane amines, heterocyclic amines, aromatic amines; wherein, the alkane amines are selected from
    Figure PCTCN2022113625-appb-100002
    Figure PCTCN2022113625-appb-100003
    Figure PCTCN2022113625-appb-100004
    The heterocyclic amine is selected from
    Figure PCTCN2022113625-appb-100005
    Figure PCTCN2022113625-appb-100006
    The aromatic amine is selected from
    Figure PCTCN2022113625-appb-100007
    Figure PCTCN2022113625-appb-100008
    n选自自然数2～5。n is selected from natural numbers 2-5.
  3. 如权利要求1所述的三环二萜类似物，其特征在于，所述三环二萜类似物选自如式(2)～式(32)所示的三环二萜类似物：The tricyclic diterpene analogue according to claim 1, wherein the tricyclic diterpene analogue is selected from the tricyclic diterpene analogues shown in formula (2) to formula (32):
    Figure PCTCN2022113625-appb-100009
    Figure PCTCN2022113625-appb-100009
  4. 一种三环二萜类似物的制备方法，其特征在于，以睾酮为原料，通过甲基化、乙二醇保护、还原、氧化、Bayer-Villger氧化、还原、脱保护、乙酰化、消除、水解、氧化得到Q12，然后所述Q12经还原反应得到式(3)所示的母核化合物；或，以所述Q10为原料，经磺酰化、取代、水解、还原反应得到Q16所示的母核化合物；或，以所述Q11为原料，经Wittig、还原、水解反应得到Q20所示的母核化合物；或，以所述Q10为原料，经乙二醇保护、氧化、 Wittig、还原、脱保护、还原反应得到Q26所示的母核化合物，所述制备方法的反应路线如下：A preparation method of a tricyclic diterpene analog, characterized in that, using testosterone as a raw material, through methylation, ethylene glycol protection, reduction, oxidation, Bayer-Villger oxidation, reduction, deprotection, acetylation, elimination, Hydrolyze, oxidize to obtain Q12, then said Q12 obtains the parent nucleus compound shown in formula (3) through reduction reaction; Or, using the Q11 as a raw material, the parent nucleus compound shown in Q20 is obtained through Wittig, reduction, and hydrolysis reactions; or, using the Q10 as a raw material, through ethylene glycol protection, oxidation, Wittig, reduction, Deprotection and reduction reactions obtain the parent nucleus compound shown in Q26, and the reaction scheme of the preparation method is as follows:
    Figure PCTCN2022113625-appb-100010
    Figure PCTCN2022113625-appb-100010
    路线(A)；route(A);
    Figure PCTCN2022113625-appb-100011
    Figure PCTCN2022113625-appb-100011
    路线(B)；route(B);
    Figure PCTCN2022113625-appb-100012
    Figure PCTCN2022113625-appb-100012
    路线(C)；Route (C);
    Figure PCTCN2022113625-appb-100013
    Figure PCTCN2022113625-appb-100013
    路线(D)。route (D).
  5. 一种三环二萜类似物的制备方法，其特征在于，以如权利要求4所述的方法中的所述母核化合物Q12所示的三环二萜类似物为原料，在HATU、DIPEA催化下，通过酰胺化反应得到式(2)所示的三环二萜类似物，所述制备方法的反应路线如下：A preparation method of tricyclic diterpene analogs, characterized in that, using the tricyclic diterpene analogs shown in the parent nucleus compound Q12 in the method as claimed in claim 4 as raw material, catalyzed by HATU and DIPEA Next, the tricyclic diterpene analog shown in formula (2) is obtained by amidation reaction, and the reaction scheme of the preparation method is as follows:
    Figure PCTCN2022113625-appb-100014
    Figure PCTCN2022113625-appb-100014
    路线(E)。Route (E).
  6. 一种三环二萜类似物的制备方法，其特征在于，以如权利要求4所述的方法中的所述式(3)所示三环二萜类似物为原料，在HATU、DIPEA催化下，通过酰胺化反应分别得到式(4)～式(25)所示的三环二萜类似物，所述制备方法的反应路线如下：A preparation method of tricyclic diterpene analogs, characterized in that, using the tricyclic diterpene analogs shown in the formula (3) in the method as claimed in claim 4 as raw material, under the catalysis of HATU and DIPEA , respectively obtain the tricyclic diterpene analog shown in formula (4)～formula (25) by amidation reaction, the reaction route of described preparation method is as follows:
    Figure PCTCN2022113625-appb-100015
    Figure PCTCN2022113625-appb-100015
    路线(F)。Route (F).
  7. 一种三环二萜类似物的制备方法，其特征在于，以如权利要求4所述的方法中的所述母核化合物Q16所示的三环二萜类似物为原料，在HATU、DIPEA催化下，通过酰胺化反应得到 式(26)所示的三环二萜类似物，所述制备方法的反应路线如下：A preparation method of tricyclic diterpene analogs, characterized in that, using the tricyclic diterpene analogs shown in the parent nucleus compound Q16 in the method as claimed in claim 4 as raw material, catalyzed by HATU and DIPEA Next, the tricyclic diterpene analog shown in formula (26) is obtained by amidation reaction, and the reaction scheme of the preparation method is as follows:
    Figure PCTCN2022113625-appb-100016
    Figure PCTCN2022113625-appb-100016
    路线(G)。Route (G).
  8. 一种三环二萜类似物的制备方法，其特征在于，以如权利要求4所述的方法中的所述母核化合物Q20所示的三环二萜类似物为原料，在HATU、DIPEA催化下，通过酰胺化反应分别得到如式(27)～式(29)所示的三环二萜类似物，所述制备方法的反应路线如下：A preparation method of tricyclic diterpene analogs, characterized in that, using the tricyclic diterpene analogs shown in the parent nucleus compound Q20 in the method as claimed in claim 4 as raw material, catalyzed by HATU and DIPEA Next, the tricyclic diterpene analogs shown in formula (27) to formula (29) are respectively obtained by amidation reaction, and the reaction scheme of the preparation method is as follows:
    Figure PCTCN2022113625-appb-100017
    Figure PCTCN2022113625-appb-100017
    路线(H)。Route (H).
  9. 一种三环二萜类似物的制备方法，其特征在于，以如权利要求4所述的方法中的所述母核化合物Q26所示的三环二萜类似物为原料，在HATU、DIPEA催化下，通过酰胺化反应分别得到式(30)～式(32)所示的三环二萜类似物，所述制备方法的反应路线如下：A preparation method for tricyclic diterpene analogs, characterized in that, using the tricyclic diterpene analogs shown in the parent nucleus compound Q26 in the method as claimed in claim 4 as raw material, catalyzed by HATU and DIPEA Next, tricyclic diterpene analogs shown in formula (30) to formula (32) are respectively obtained by amidation reaction, and the reaction scheme of the preparation method is as follows:
    Figure PCTCN2022113625-appb-100018
    Figure PCTCN2022113625-appb-100018
    路线(I)。Route (I).
  10. 一种药物组合物，其特征在于，其包含如权利要求1-3之任一项所述的三环二萜类似物，及药学上可接受的载体。A pharmaceutical composition, characterized in that it comprises the tricyclic diterpene analog according to any one of claims 1-3, and a pharmaceutically acceptable carrier.
  11. 如权利要求1-3之任一项所述的三环二萜类似物或如权利要求10所述的药物组合物在制备SMO抑制剂中的应用。Use of the tricyclic diterpene analog as claimed in any one of claims 1-3 or the pharmaceutical composition as claimed in claim 10 in the preparation of SMO inhibitors.
  12. 如权利要求11所述的应用，其特征在于，所述三环二萜类似物或药物组合物用于抑制野生型SMO蛋白和耐药性SMO蛋白的活性，抑制Hedgehog信号通路活性。The application according to claim 11, wherein the tricyclic diterpene analog or pharmaceutical composition is used to inhibit the activity of wild-type SMO protein and drug-resistant SMO protein, and inhibit the activity of Hedgehog signaling pathway.
  13. 如权利要求1-3任一项所述的三环二萜类似物或如权利要求10所述的药物组合物在制备抗肿瘤药物中的应用。Use of the tricyclic diterpene analog as claimed in any one of claims 1-3 or the pharmaceutical composition as claimed in claim 10 in the preparation of antitumor drugs.
  14. 如权利要求13所述的应用，其特征在于，所述三环二萜类似物或药物组合物用于抑制肿瘤的生长、增殖、转移，或用于促进肿瘤的凋亡和灭亡。The use according to claim 13, characterized in that the tricyclic diterpene analogue or the pharmaceutical composition is used for inhibiting tumor growth, proliferation and metastasis, or for promoting tumor apoptosis and death.
  15. 如权利要求13所述的应用，其特征在于，所述肿瘤包括髓母细胞瘤、基底细胞癌、白血病、其它Hedgehog通路依赖型肿瘤。The use according to claim 13, characterized in that the tumor comprises medulloblastoma, basal cell carcinoma, leukemia, and other Hedgehog pathway-dependent tumors.
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