Process for the synthesis of flavonoid dimers and oligomers and uses thereof

Abstract

A method for coupling flavonoid-containing compounds to provide flavonoid dimers, trimers, and oligomers is provided. The method includes contacting a flavonoid-containing compound with a base in the presence of air. The flavonoid dimers, trimers, and oligomers are also useful in treating diseases, such as fungal infections and diseases associated with amylolytic enzymes.

Description

Technical Field

[0001] This invention relates to a method for preparing flavonoid dimers, trimers and oligomers, and to flavonoid dimers and trimers, and their use in the treatment of diseases. Background Technology

[0002] The obvious previously published documents listed and discussed in this specification are not necessarily considered an admission that such documents are part of the prior art or common general knowledge.

[0003] Flavonoids are a variety of plant secondary metabolites with a range of biological activities. The core structure of flavonoids is characterized by a 15-carbon phenyl-chromone motif and is a preferred structure for drug discovery. Structural variations in flavonoids arise from variable substitution positions of the phenyl group, variable number and position of phenolic groups on the aromatic ring, the amount and degree of glycosylation, and the formation of flavonoid dimers and oligomers.

[0004] Although flavonoid monomers are relatively abundant in fruits and vegetables and can be extracted on an industrial scale from agri-food byproducts, flavonoid dimers and oligomers are minor components of plant biomass, and obtaining them in large quantities from natural sources is uneconomical. Synthetically, catalytic C-C bond coupling reactions, such as the Ullman reaction and Suzuki-Miyaura coupling, have been used to prepare limited quantities of biflavonoids in modest overall yields. These high-temperature reactions suffer from major drawbacks due to their use of toxic heavy metals, wasteful halogens and borate byproducts, and the need for protecting groups for phenolic functional groups. The reactions also involve multiple steps that introduce additional complexity and reduce yields. Therefore, these methods are not environmentally friendly and are unsuitable for large-scale synthesis or the synthesis of food-grade products. Furthermore, the laboratory synthesis of the three flavonoids remains relatively unexplored.

[0005] Therefore, new methods are needed to produce flavonoid dimers, trimers, and oligomers. Such methods could enable the production of entirely new compounds with useful therapeutic activities. Summary of the Invention

[0006] This invention addresses a problem in the art by providing a method for coupling flavonoid compounds using environmentally friendly reagents under advantageously mild conditions. The reaction can be carried out as a one-pot synthesis with high yield and regioselectivity under mild aqueous conditions, e.g., at room temperature using a simple base. These conditions are compatible with a variety of functional groups that can be present on flavonoids and can be used to obtain food-grade products in an environmentally friendly manner. The method of this invention can be used to produce a variety of flavonoids that were previously impossible to prepare in a laboratory setting.

[0007] The compounds prepared according to the present invention have useful activities as antifungal agents and inhibitors of starch hydrolases.

[0008] Therefore, the present invention provides the following numbered items.

[0009] 1. A method for coupling a flavonoid-containing compound, the method comprising:

[0010] (i) providing one or more flavonoid-containing compounds; and

[0011] (ii) In the presence of air, contact the one or more flavonoid-containing compounds with an alkali;

[0012] The base is selected from the group consisting of metal carbonates, metal hydroxides, and bases of the formula R4NOH, wherein each R independently represents H or C. 1-4 Alkyl groups; and

[0013] Step (ii) is carried out in a closed reaction vessel containing air and a reaction mixture containing one or more of the flavonoid-containing compounds, an alkali and water, wherein the air occupies 10 to 95% of the volume of the reaction vessel at 25°C and a pressure of 101 kPa.

[0014] 2. According to the method described in Project 1, the alkali is selected from one or more of the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and alkaline water.

[0015] Optionally, the alkali is one or more selected from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, and potassium carbonate.

[0016] 3. The method according to Project 2, wherein the base is selected from one or more of the group consisting of sodium hydroxide and potassium hydroxide.

[0017] 4. The method according to Project 3, wherein the base is potassium hydroxide.

[0018] 5. The method according to any one of the preceding items, wherein the pH of the reaction mixture in step (ii) is from about 10 to about 14, optionally, the pH is from about 11 to about 13.

[0019] 6. The method according to any one of the preceding items, wherein the temperature of the reaction mixture in step (ii) is 15 to 30°C, optionally 18 to 25°C.

[0020] 7. The method according to any one of the foregoing items, wherein step (ii) is performed for about 1 to about 20 hours, optionally for about 5 to about 15 hours, for example for about 8 to about 12 hours.

[0021] 8. The method according to any one of the foregoing items, wherein step (ii) is carried out without stirring, optionally wherein step (ii) is carried out without stirring the reaction mixture.

[0022] 9. The method according to any one of the preceding items, wherein the flavonoid-containing compound comprises a skeleton selected from the group consisting of: flavonoid skeleton, isoflavone skeleton, novel flavonoid skeleton, flavanone skeleton, flavanone skeleton, isoflavone skeleton, and isoflavone skeleton, said skeleton optionally being substituted by one or more (e.g., one to six, one to five, one to four, one to three, one or two, or one) substituents selected from the group consisting of: hydroxyl, methoxy, glycosyl, alkoxy, NO2, F, CN, SH, CF3, Cl, Br, I, =O, =CH2, C 1-18 Alkyl, C 1-18 fluoroalkyl, -OC(O)-R (where R represents C) 1-22 Alkyl), C 2-18 alkenyl, isoprene, phytyl, exocyclic C 3-6 Cycloalkyl, exocyclic C 5-6 Cycloalkenyl, phenyl, phenoxy, C 1-18 N-alkylamine group and C 1-18 N,N-dialkylaminyl

[0023]

[0024] Alternatively, the flavonoid-containing compound may comprise a dimer or trimer thereof.

[0025] 10. The method according to any one of the preceding items, wherein the flavonoid-containing compound comprises a flavonoid skeleton.

[0026] 11. The method according to any one of the preceding items, wherein the flavonoid-containing compound is a flavonoid monomer.

[0027] 12. The method according to any one of the preceding items, wherein the flavonoid-containing compound is a flavonoid dimer.

[0028] 13. The method according to any one of the preceding items, wherein the flavonoid-containing compound is a flavonoid trimer, and optionally, wherein the coupling is an intramolecular coupling.

[0029] 14. The method according to any one of the preceding items, wherein at 25°C and 101 kPa, the air occupies 30 to 90% (e.g., 40 to 80%) of the volume of the reaction vessel.

[0030] 15. The method according to any one of items 1 to 14, wherein the flavonoid is selected from the group consisting of: luteolin, apigenin, diosmetin, chrysin, wogonin, 5,6-dihydroxyflavone, genistein, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone, 3′,4′-dihydroxyflavone, dimers formed from two of the above, and trimers formed from three of the above.

[0031] 16. A compound selected from the group consisting of:

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038] And its pharmaceutically acceptable salts.

[0039] 17. Use of the compound of (a) or (b) in the preparation of a medicament for treating (i) or (ii):

[0040] (a) A dimer or trimer formed from one, two, or three flavonoids selected from the group consisting of: luteolin, apigenin, geraniol, scutellarin, 5,6-dihydroxyflavone, genistein, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone, and 3′,4′-dihydroxyflavone, or

[0041] (b) Its pharmaceutically acceptable salt.

[0042] (i) Fungal infection (e.g., Candida infection); or

[0043] (ii) Conditions or illnesses that are improved by inhibiting amylase (e.g., hyperglycemia, diabetes, and obesity).

[0044] 18. The use according to item 17, wherein the compound is the compound according to item 16 or a pharmaceutically acceptable salt.

[0045] 19. According to the use described in item 17, said compound is a compound selected from the group consisting of:

[0046]

[0047]

[0048] And its pharmaceutically acceptable salts.

[0049] 20. According to the use described in item 19, said compound is selected from digammadrugine, 3″′-deoxydammadrugine, diammonium oxyphenate, and pharmaceutically acceptable salts thereof.

[0050] Optionally, said compound is selected from digammaduramin, 3″′-deoxydammaduramin, and pharmaceutically acceptable salts thereof.

[0051] More optionally, said compound is digammadrugine or a pharmaceutically acceptable salt thereof. Attached Figure Description

[0052] Figure 1 The synthesis of naturally occurring and non-natural biflavonoids and triflavonoids is described. (A) Suzuki-Miyaura coupling reaction in the synthesis of bioflavonoids; (B) Ullmann coupling reaction in the synthesis of bioflavonoids; and (C) this work: oxygen-mediated oxidative coupling reaction in the synthesis of flavonoid oligomers; examples include 31 biflavonoids and 11 triflavonoids.

[0053] Figure 2Oxygen-mediated oxidative coupling of flavonoids is described. (A) Luteolin undergoes oxidative coupling in weakly alkaline water, with the major product being dicranolomin (2a), and minor products being philonotisflavone (2a′, structure not shown), dehydrohegoflavone B (2a″), and distichumtriluteolin (3a). Distichumtriluteolin can be obtained separately by coupling 2a with luteolin in a 42% isolated yield. (B) Intramolecular oxidative coupling of 3a produces different isomers of cyclotriluteolin (4a, 4a′, and 4a″). (C) The solid-state structure of an isomer 4a′ is determined by single-crystal X-ray diffraction.

[0054] Figure 3 The following descriptions are presented: (A) the ring rearrangement of cyclotrietrin (CTL); (B) 4a temperature-variable NMR; (C) the proposed intramolecular ring rearrangement of cyclotrietrin; and (D) the calculated Gibbs free energy of the cyclotrietrin isomers. Calculations were performed at the theoretical level of M06-2X / 6-311+G(d,p),SMD(H2O) / / M06-2X / 6-31G(d)SMD(H2O). Energy is expressed in kcal·mol⁻¹. -1 Units.

[0055] Figure 4 The characteristics of the 7,3′,4′-trihydroxyflavonoid radical anion are described. (A) Formation of the 7,3′,4′-trihydroxyflavonoid radical anion; (B) FH recorded at 295 K. -· Experimental and fitted EPR spectra; (C)F -· (D) Experimental hyperfine coupling constant; FH predicted by density functional theory (DFT, UM062 x / 6-311+G(d,p)) -· Spin density distribution in [the data].

[0056] Figure 5 Electron spin resonance (ESR) spectra of luteolin in aqueous potassium hydroxide (KOH) solutions at (A) pH 11.5 and (B) pH 12.5 are described. (i) Dissolved in H2O 16 and exposed to oxygen O2 16 (ii) Electron paramagnetic resonance (EPR) spectrum of luteolin in H2O; 16 and exposed to oxygen O2 17 EPR spectrum of luteolin in (iii) and dissolved in H2O 17 and exposed to oxygen O216 EPR spectrum of luteolin in [the sample].

[0057] Figure 6 The ESR spectrum of luteolin in KOH aqueous solution at pH 12.5 is described. (A) Dissolved in H2O 17 (A) Experimental and simulated EPR spectra of luteolin in LH2O; (B) LH2O predicted by DFT(UM062x / 6-311+G(d,p)). -. Spin density distribution in LH2O; and (C) proposed LH2O -. The formation mechanism of free radical anions.

[0058] Figure 7 The characteristics of the digamma-hydroxyflavone radical anion are described. (A) Formation of the 3′,4′-dihydroxyflavone radical anion; (B) F recorded at 295 K. -· Experimental and fitted EPR spectra; (C) The proposed digamma-hydroxyl radical anion DH8 .- The structure; and (D) the DH8 predicted by DFT(UM062x / 6-311+G(d,p)). -. Spin density distribution in [the data].

[0059] Figure 8 The yields of dimers derived from the homo cross-coupling reaction of β-catechol flavone: 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone, 7,8,3′,4′-tetrahydroxyflavone with luteolin are described.

[0060] Figure 9 The nucleophilicity of β-catechin flavonoids was described. The global nucleophilicity index (N0) was calculated using the DFT method at the theoretical level of B3LYP / 6-311++G(d,p) . Nu According to N Nu =E HOMO(Nu) -E HOMO(TCE) (LRDomingo et al., J.Org.Chem. 2008, 73, 4615-4624), with reference to the HOMO energy of tetracyanoethylene (TCE).

[0061] Figure 10 The substrate scope for oxygen-mediated cross-coupling of luteolin and flavonoids is described. (A) Luteolin as a radical precursor and acceptor; and (B) β-catechin flavonoids as radical precursors. All yields were isolated and selectivity was determined by HPLC analysis. brsm = yield based on recovery of starting material. If not specified, R = H.

[0062] Figure 11 The substrate range for oxidative coupling in the synthesis of triflavones in alkaline water is described. (A) digammaphenamine as a radical precursor and acceptor; and (B) β-catechol biflavonoids as a radical precursor. All yields are isolated yields. brsm = yield based on recovery of starting material. If not specified, R = H.

[0063] Figure 12 (A) is described for pK a (A) Sample preparation for calculating luteolin; and (B) experimental apparatus for sample preparation.

[0064] Figure 13 (A) A diagram; and (B) an image of the oxygen consumption monitoring device. Pressure changes in the reaction under stirred and unstirred conditions were recorded using a pressure gauge (NVISION, a pressure recorder with a vacuum range of 30 MPa, CRYSTAL engineering corporation).

[0065] Figure 14 The effect of pH on the yield of luteolin cross-coupling reactions with (A) luteolin; (B) apigenin (Ap); (C) dio; (D) chry; (E) baicalin (Wo); (F) 5,6-dihydroxyflavone; (G) genistein (Ge); (H) 5,3′,4′-trihydroxyflavone; (I) 6,3′,4′-trihydroxyflavone; and (J) 7,3′,4′-trihydroxyflavone was described.

[0066] Figure 15 The effect of pH on the yield of digamma-coupling reactions with (A) luteolin; (B) Ap; (C) Dio; (D) Chry; (E) Wo; (F) 5,6-dihydroxyflavone; (G) Ge; (H) 5,3′,4′-trihydroxyflavone; (I) 6,3′,4′-trihydroxyflavone; and (J) 7,3′,4′-trihydroxyflavone was described.

[0067] Figure 16 The pK of luteolin was described using nuclear magnetic resonance (NMR). a Determination of the pK of luteolin. a1 pK a2 pK a3 and PK a4 The determination is based on the (A) C4′; (B) C7; (C) C3′; and (D) C5 of luteolin. 13The dependence of C NMR chemical shift on pH. The line represents the computer fit to the Henderson-Hasselbalch equation and the pKa value; and (E) based on pKa. a The distribution curves of luteolins in aqueous solution were generated.

[0068] Figure 17 The pKa curves of luteolin and the free radicals generated are described. (A) Distribution curves of luteolins in aqueous solution. LuH4: Luteolin, LuH3 - Monovalent anion, LuH2 2- Divalent anion, LuH 3- Trivalent anions, and Lu 4- : tetravalent anion; (B)LuH ·2- Experimental and simulated EPR spectra; (C) LuH predicted by DFT(UM062X / 6-311+G(d,p)) ·2- Spin density distribution in (D)(i)LuOH ·2- (ii) in air-saturated water; 17 (iii) In H2O enriched with molecular oxygen; and in 30% 17 Experimental EPR spectra of O-enriched water; and LuOH proposed in (E) ·2- The formation mechanism of .

[0069] Figure 18 The pH-dependent ESR spectrum of luteolin (15.0 mM) in KOH aqueous solution was described. The determination was performed immediately after mixing luteolin with a specific concentration of KOH solution, and pH was determined after the ESR test.

[0070] Figure 19 The regioselectivity reaction mechanism and computational studies are described. (A) The proposed oxidative coupling reaction mechanism, with the counter cation omitted for clarity; (B) The calculated Gibbs energy difference between 2a and 2a′; and (C) The effect of the counter cation on the yield of the luteolin coupling reaction.

[0071] Figure 20 The characteristics of the 3′,4′-dihydroxyflavonoid radical anion are described. (A) Formation of the 3′,4′-dihydroxyflavonoid radical anion; (B) F recorded at 295 K. -· Experimental and fitted EPR spectra; (C)F -· The experimental hyperfine coupling constant; and (D) the F predicted by DFT(UM062x / 6-311+G(d,p)). -· Spin density distribution in [the data].

[0072] Figure 21The characteristics of the 5,3′,4′-trihydroxyflavonoid radical anion are described. (A) Formation of the 5,3′,4′-trihydroxyflavonoid radical anion; (B) FH recorded at 295 K. -· Experimental and fitted EPR spectra; (C)F -· The experimental hyperfine coupling constant; and (D) the FH predicted by DFT(UM062x / 6-311+G(d,p)). -· Spin density distribution in [the data].

[0073] Figure 22 The characteristics of the 6,3′,4′-trihydroxyflavonoid radical anion are described. (A) Formation of the 6,3′,4′-trihydroxyflavonoid radical anion; (B) FH recorded at 295 K. -· Experimental and fitted EPR spectra; (C)F -· The experimental hyperfine coupling constant; and (D) the FH predicted by DFT(UM062x / 6-311+G(d,p)). -· Spin density distribution in [the data].

[0074] Figure 23 The concentration changes of (A) luteolin (1a); (B) 2a; and (C) diammonium triamine luteolin (3a) with reaction time are described in luteolin-luteolin cross-coupling reactions carried out under stirred and unstirred conditions.

[0075] Figure 24 Oxygen consumption curves for the luteolin-luteolin cross-coupling reaction under stirred and unstirred conditions were described. Luteolin (57.2 mg, 0.1 mmol) was added to a 250 mL round flask. Then, KOH solution (50.0 mL, 0.03 M) was introduced to initiate the reaction. The reaction was carried out under real-time monitoring in an airtight system.

[0076] Figure 25The bioactivity of luteolin and flavonoid dimers and trimers is described. (A) Images of surviving *Aspergillus niger* colonies in the presence of luteolin (1a), 2a, diammonium luteolin (3a), and amphotericin B (AmB), and the degree of inhibition (%); (C) Docking of 2a in the complex with α-amylase [Protein Data Bank (PDB) 3GBN]. 2a is presented in ball-and-stick representation, and the entire amylase protein is presented in surface representation. A magnified view of the 2a binding site in the enzyme is shown. Overlap of the structure of 2a in the complex with interacting loop residues; (D) Catalytic mechanism of α-amylase. The reaction occurs with the aid of acid / base and nucleophilic interaction provided by the amino acid side chains from Asp197, Glu233, and Asp300; and (E) and (F) show polar interactions in α-amylase, and with The interaction between the FI and FII portions is measured in units of 2a [labeled in (D)].

[0077] Figure 26 The starch hydrolysis inhibitory activity of flavonoid oligomers, expressed in acarbose equivalents, is described. (A) α-amylase inhibitory activity of flavonoid oligomers; and (B) α-glucosidase inhibitory activity of flavonoid oligomers.

[0078] Figure 27a and 27b Representative kinetic and dose-response curves for α-amylase and α-glucosidase in the presence of luteolin (1a), its derivatives, biflavonoids (2a), and triflavonoids (3a) are described. (a, b, e, f, i, and j) show the starch hydrolytic activity of α-amylase with flavonoids, while (c, d, g, h, k, and i) show the starch hydrolytic activity of α-glucosidase with flavonoids.

[0079] Figure 28a and 28b Representative kinetic and dose-response curves for α-amylase and α-glucosidase in the presence of luteolin (1b), its derivatives, biflavonoids (2b), and triflavonoids (3b) are described. (a, b, e, f, i, and j) show the starch hydrolytic activity of α-amylase with flavonoids, while (c, d, g, h, k, and i) show the starch hydrolytic activity of α-glucosidase with flavonoids.

[0080] Figure 29a and 29bRepresentative kinetic and dose-response curves for α-amylase and α-glucosidase in the presence of luteolin (1c), its derivatives, biflavonoids (2c), and triflavonoids (3c) are described. (a, b, e, f, i, and j) show the starch hydrolytic activity of α-amylase with flavonoids, while (c, d, g, h, k, and i) show the starch hydrolytic activity of α-glucosidase with flavonoids.

[0081] Figure 30a and 30b Representative kinetic and dose-response curves for α-amylase and α-glucosidase in the presence of luteolin (1d), its derivatives, biflavonoids (2d), and triflavonoids (3d) are described. (a, b, e, f, i, and j) show the starch hydrolytic activity of α-amylase with flavonoids, while (c, d, g, h, k, and i) show the starch hydrolytic activity of α-glucosidase with flavonoids.

[0082] Figure 31a and 31b Representative kinetic and dose-response curves for α-amylase and α-glucosidase in the presence of luteolin (1e), its derivatives, biflavonoids (2e), and triflavonoids (3e) are described. (a, b, e, f, i, and j) show the starch hydrolytic activity of α-amylase with flavonoids, while (c, d, g, h, k, and i) show the starch hydrolytic activity of α-glucosidase with flavonoids.

[0083] Figure 32a and 32b Representative kinetic and dose-response curves for α-amylase and α-glucosidase in the presence of luteolin (1f), its derivatives, biflavonoids (2f), and triflavonoids (3f) are described. (a, b, e, f, i, and j) show the starch hydrolytic activity of α-amylase with flavonoids, while (c, d, g, h, k, and i) show the starch hydrolytic activity of α-glucosidase with flavonoids.

[0084] Figure 33a and 33b Representative kinetic and dose-response curves for α-amylase and α-glucosidase in the presence of luteolin (1 g), its derivatives, biflavonoids (2 g), and triflavonoids (3 g) are described. (a, b, e, f, i, and j) show the starch hydrolytic activity of flavonoids with α-amylase, while (c, d, g, h, k, and i) show the starch hydrolytic activity of flavonoids with α-glucosidase.

[0085] Figure 34a and 34bRepresentative kinetic and dose-response curves for α-amylase and α-glucosidase in the presence of luteolin (1h), its derivatives, biflavonoids (2h), and triflavonoids (3h) are described. (a, b, e, f, i, and j) show the starch hydrolytic activity of α-amylase with flavonoids, while (c, d, g, h, k, and i) show the starch hydrolytic activity of α-glucosidase with flavonoids.

[0086] Figure 35a and 35b Representative kinetic and dose-response curves for α-amylase and α-glucosidase in the presence of luteolin (1i), its derivatives, biflavonoids (2i), and triflavonoids (3i) are described. (a, b, e, f, i, and j) show the starch hydrolytic activity of α-amylase with flavonoids, while (c, d, g, h, k, and i) show the starch hydrolytic activity of α-glucosidase with flavonoids.

[0087] Figure 36a and 36b Representative kinetic and dose-response curves for α-amylase and α-glucosidase in the presence of luteolin (1j) and its derivatives, biflavonoids (2j), and triflavonoids (3j) are described. (a, b, e, f, i, and j) show the starch hydrolytic activity of α-amylase with flavonoids, while (c, d, g, h, k, and i) show the starch hydrolytic activity of α-glucosidase with flavonoids.

[0088] Figure 37 The inhibitory activity of the synthesized biflavonoids on amylases compared to luteolin monomers was described. AE = acarbose equivalent (based on molar concentration). In this assay, α-amylase solution (2 U / mL, 20 μL in buffer) and α-glucosidase solution (1 x 10⁻⁶ in buffer) were used. -2 The concentration of starch used in this assay was 20 mg / mL. Lu2 = bis-luteolin (2′-6 linked), Lu2' = bis-luteolin (2′-8 linked), Lu-Ap, luteolin-apigenin dimer (2′-6 linked), Lu-Dio, luteolin-geraniol dimer (2′-6 linked), Lu-Chry, luteolin-aspergillus dimer (2′-6 linked).

[0089] Figure 38 Visualizations of the docking results between amylase and ligands: acarbose, 2a, 3″′-deoxydigranoxamine (2b), 1a, and 3a are described.

[0090] Figure 39Visualizations of the docking results between amylase and ligand 2b are described. (A) Crystal structure of 2b in the complex with α-amylase [Protein Database (PDB) 1ppi]. (B) 2b presented in ball-and-stick notation; (C) Enlarged view showing the acarbose binding site in the enzyme; and (DE) Polar interactions in the 2b-1ppi complex, where... The interaction is measured in units of 2b.

[0091] Figure 40 Visualizations of the docking results between amylase and its ligand, acarbose, are described. (A) Crystal structure of 2a in the complex with α-amylase [Protein Database (PDB) 1ppi]; (B) Acarbose presented in ball-and-stick notation; (C) Enlarged view showing the acarbose binding site in the enzyme; and (DE) Polar interactions in the acarbose-1ppi complex, as shown. The interaction of acarbose was measured in units.

[0092] Figure 41 The study described (A) blood glucose concentrations in mice treated with YX2 (luteolin dimer, 2a); and (B) elevated blood glucose levels in mice treated with YX2 (luteolin dimer, 2a). Detailed Implementation

[0093] The word "comprising" in this document can be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word "comprising" can also refer to cases where only the listed components / features are intended to be presented (e.g., the word "comprising" can be replaced by the phrases "consisting of" or "substantially consisting of"). Both broader and narrower interpretations are explicitly intended to apply to all aspects and embodiments of the invention. In other words, the word "comprising" and its synonyms can be replaced by the phrases "consisting of" or "substantially consisting of" or their synonyms, and vice versa.

[0094] The phrase “consistent with…” and its pseudonym can be interpreted in this text as referring to materials that may contain small amounts of impurities. For example, the material can be more than 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0095] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a composition” includes a mixture of two or more such compositions, reference to “an oxygen support” includes a mixture of two or more such oxygen supports, reference to “the catalyst” includes a mixture of two or more such catalysts, and so on.

[0096] This invention provides a method for coupling flavonoid-containing compounds, the method comprising:

[0097] (i) providing one or more flavonoid-containing compounds; and

[0098] (ii) In the presence of air, contact the one or more flavonoid-containing compounds with an alkali;

[0099] The base is selected from the group consisting of metal hydroxides and bases of the formula R4NOH, wherein each R independently represents H or C. 1-4 Alkyl groups; and

[0100] Step (ii) is carried out in a closed reaction vessel containing air and a reaction mixture containing one or more of the flavonoid-containing compounds, the base and water, wherein the air occupies 10% to 95% of the volume of the reaction vessel at 25°C and 101 kPa.

[0101] As used herein, "flavonoid-containing compounds" are compounds comprising a flavonoid carbon skeleton, which may be substituted with additional carbon-containing functional groups. The flavonoid carbon skeleton comprises a benzene ring fused to a 6-membered heterocycle containing an oxygen atom, the heterocycle being bonded to the benzene ring via a C-C bond.

[0102]

[0103] flavonoid carbon skeleton

[0104] Carbon atoms in a six-membered heterocycle containing oxygen atoms that do not form part of a fused benzene ring can be connected by single or double bonds, as shown in the structures above (provided that any carbon atom in the heterocycle forms only part of a double bond). The oxygen atom can be bonded to a non-aromatic carbon atom via single or double bonds. When a double bond is formed, the oxygen atom will carry a positive charge, and the compound will contain a counter-charge or counter ion.

[0105] Flavonoids may contain multiple functional groups bonded to the above-described backbone, such as one or more selected from the group consisting of (e.g., 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1) substituents: hydroxyl, methoxy, glycosyl, alkoxy, NO2, F, CN, SH, CF3, Cl, Br, I, =O, =CH2, C 1-18 Alkyl, C 1-18 fluoroalkyl, C 2-18 alkenyl, -OC(O)-R (where R represents C) 1-22 Alkyl), isoprene, phytyl, exocyclic C 3-6 Cycloalkyl, exocyclic C 5-6 Cycloalkenyl, phenyl, phenoxy, C 1-18 N-alkylamine group and C 1-18 N,N-dialkylamine.

[0106] Specific examples of flavonoid skeletons that may exist in flavonoid-containing compounds include the following:

[0107]

[0108] And its dimers and trimers. As those skilled in the art will understand, herein, dimers or trimers include dimers / trimers formed of two / three identical backbones, or dimers / trimers formed of two / three different backbones.

[0109] In a specific embodiment of the present invention, the flavonoids may be selected from the group consisting of: luteolin, apigenin, geraniol, scutellarin, 5,6-dihydroxyflavone, genistein, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone, 3′,4′-dihydroxyflavone, dimers formed from two of the foregoing, and trimers formed from three of the foregoing. In other words, flavonoid-containing compounds can represent flavonoids with optional substitutions selected from the group consisting of: luteolin, apigenin, geraniol, scutellarin, 5,6-dihydroxyflavone, genistein, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone, 3′,4′-dihydroxyflavone, dimers formed from two of the foregoing, and trimers formed from three of the foregoing.

[0110] In specific embodiments of the present invention, the flavonoid-containing compound may comprise a flavonoid skeleton. In some embodiments of the present invention, the flavonoid-containing compound may be a monomer. In some embodiments of the present invention, the flavonoid-containing compound may be a dimer. In some embodiments of the present invention, the flavonoid-containing compound may be a trimer.

[0111] When the flavonoid-containing compound is a trimer, the coupling reaction of the present invention can be an intramolecular reaction to provide a cyclic trimer. Optionally (or additionally), the coupling reaction of the present invention can couple the trimer with another flavonoid-containing compound, such as a monomer, to produce a tetramer.

[0112] The method involves contacting a flavonoid-containing compound with a base selected from the group consisting of: metal carbonates, metal hydroxides, and bases of the formula R4NOH, wherein each R independently represents H or C. 1-4 Alkyl groups. Examples of bases that can be used in embodiments of the present invention include one or more of the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and alkaline water. As illustrated in the following examples, the counterionic properties of the base can affect the yield provided by the reaction. Therefore, in some embodiments, a base preferably selected from the group consisting of alkali metal hydroxides is preferred. In a further embodiment, the base may be selected from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, and cesium hydroxide. In a still further embodiment, the base may be selected from the group consisting of sodium hydroxide and potassium hydroxide. In a specific embodiment, the base may be potassium hydroxide.

[0113] The base produces an alkaline reaction mixture. The reaction mixture may have a pH of about 10 to about 14, for example, about 11 to about 13.

[0114] To avoid ambiguity, it is explicitly anticipated that the endpoints of any range herein can be combined with any endpoints of another range of the same variable. Therefore, for the pH ranges described above, the present invention explicitly considers the following pH ranges:

[0115] About 10 to about 11; about 10 to about 13; about 10 to about 14;

[0116] about 11 to about 13; about 11 to about 14; and

[0117] From approximately 13 to approximately 14.

[0118] Step (ii) of the method is carried out in a closed reaction vessel. This allows for control over the amount of oxygen exposed to the reaction mixture. If insufficient oxygen is present, the coupling reaction will not be completed, resulting in unsatisfactory yields. However, if excessive oxygen is present, over-oxidation may occur. Therefore, the present invention relates to the use of a closed reaction vessel in which 10% to 95% of the volume of the reaction vessel is occupied by air (when evaluated at 25°C and 101 kPa). In some embodiments of the invention, air may occupy 30% to 90% (e.g., 40% to 80%) of the volume of the reaction vessel at 25°C and 101 kPa. As those skilled in the art will understand, these temperature and pressure are conditions used to evaluate the volume occupied by air in the reaction vessel and do not limit the temperature or pressure at which the reaction can occur.

[0119] For similar reasons, in some embodiments, the method of the present invention can be carried out without stirring (or without agitation of the reaction mixture). Stirring the reaction mixture increases the contact between the reaction mixture and the air in the reaction vessel, thereby increasing the rate at which additional oxygen dissolves into the reaction mixture – and potentially leading to over-oxidation. The inventors have surprisingly discovered that improved yields can be obtained when the method is carried out in a sealed reaction vessel without stirring.

[0120] The reaction mixture contains water. Water can be advantageously used as a solvent because it is environmentally friendly, inexpensive, and safe. All reagents used in the method are stable in water, and food-grade products can be produced using water. However, those skilled in the art will understand that other solvents can be used in the method.

[0121] Step (ii) of the method can be performed within a certain temperature range, for example, 0°C to 50°C (provided the reaction mixture does not freeze at 0°C). In a specific embodiment of the invention, step (ii) of the method can be performed at a temperature of 15°C to 30°C, for example, 18°C ​​to 25°C. Step (ii) can also be performed for different durations, for example, about 1 to about 20 hours, about 5 to about 15 hours, for example, about 8 to about 12 hours.

[0122] This invention provides certain flavonoid compounds selected from the group consisting of:

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] And its pharmaceutically acceptable salts.

[0130] These compounds can be prepared from their constituent monomers / dimers using the methods of the present invention. In the case of cyclic trimers, they can be prepared from acyclic trimers, which themselves can be prepared from their constituent monomers / dimers using the methods of the present invention.

[0131] The dimers and trimers prepared according to the present invention can be used in medical treatment methods. Therefore, the present invention also provides:

[0132] (1) Use of the compound of (a) or (b) in the preparation of a medicament for treating (i) or (ii):

[0133] (a) A dimer or trimer formed from one, two, or three flavonoids selected from the group consisting of: luteolin, apigenin, geraniol, scutellarin, 5,6-dihydroxyflavone, genistein, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone, and 3′,4′-dihydroxyflavone, or

[0134] (b) Its pharmaceutically acceptable salt.

[0135] (i) Fungal infection (e.g., Candida infection); or

[0136] (ii) Conditions or illnesses that are improved by inhibiting amylase (e.g., hyperglycemia, diabetes, and obesity).

[0137] (2) The compound of (a) or (b) is used to treat (i) or (ii):

[0138] (a) A dimer or trimer formed from one, two, or three flavonoids selected from the group consisting of: luteolin, apigenin, geraniol, scutellarin, 5,6-dihydroxyflavone, genistein, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone, and 3′,4′-dihydroxyflavone, or

[0139] (b) Its pharmaceutically acceptable salt.

[0140] (i) Fungal infection (e.g., Candida infection); or

[0141] (ii) Conditions or illnesses that are improved by inhibiting amylase (e.g., hyperglycemia, diabetes, and obesity).

[0142] (3) A method for treating a symptom or condition selected from (i) or (ii):

[0143] (i) Fungal infection (e.g., Candida infection); or

[0144] (ii) Conditions or illnesses that are improved by inhibiting amylase (e.g., hyperglycemia, diabetes, and obesity).

[0145] The method includes administering an effective amount of compound (a) or (b) to a patient requiring such treatment:

[0146] (a) A dimer or trimer formed from one, two, or three flavonoids selected from the group consisting of: luteolin, apigenin, geraniol, scutellarin, 5,6-dihydroxyflavone, genistein, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone, and 3′,4′-dihydroxyflavone, or

[0147] (b) Its pharmaceutically acceptable salt.

[0148] In the medical uses (1), (2), and (3) described above according to the present invention, the compound may be a compound according to the present invention. For example, the compound may be a compound selected from the group consisting of:

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155] And its pharmaceutically acceptable salts.

[0156] The compound may also be a compound selected from the group consisting of:

[0157]

[0158] And its pharmaceutically acceptable salts.

[0159] In specific embodiments of the above-described medical uses (1), (2), and (3) according to the present invention, the compound may be selected from digammaduramin, 3″′-deoxydammaduramin, diammonium oxyphenate, and pharmaceutically acceptable salts thereof. For example, the compound may be selected from digammaduramin, 3″′-deoxydammaduramin, and pharmaceutically acceptable salts thereof. The compound may be digammaduramin or a pharmaceutically acceptable salt thereof.

[0160] Those skilled in the art will understand that the term "condition or ailment that is improved by inhibiting amylase" includes hyperglycemia, diabetes, and obesity.

[0161] Specific conditions or illnesses related to the aspects of the invention described above include fungal infections (e.g., Candida infections), hyperglycemia, diabetes, and obesity.

[0162] To avoid any doubt, in the context of this invention, the term "treatment" includes reference to therapeutic or palliative treatment for patients who require such treatment, as well as preventive treatment and / or diagnosis for patients susceptible to the relevant disease state.

[0163] The term "patient" includes references to mammals (e.g., humans) as patients. As used herein, the terms "subject" or "patient" are well-known in the art and are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cattle, horses, goats, sheep, pigs, camels, and most preferably humans. In some embodiments, a subject is a subject requiring treatment or a subject suffering from a disease or condition. However, in other embodiments, a subject may be a healthy subject. The term does not indicate a specific age or sex. Therefore, it is intended to cover adult and neonatal subjects, regardless of gender.

[0164] The term "effective amount" refers to the amount of a compound that imparts a therapeutic effect to the patient being treated (e.g., sufficient to treat or prevent disease). The effect can be objective (i.e. measurable by some test or marker) or subjective (i.e., the subject gives indications of the effect or feels the effect).

[0165] For the avoidance of doubt, references to flavonoid-containing compounds herein (in any aspect or embodiment of the invention) include references to such compounds themselves, to tautomers of such compounds, and to pharmaceutically acceptable salts or solvates of such compounds, or pharmaceutically functional derivatives thereof.

[0166] Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. These salts can be formed by conventional methods, such as by reacting the free acid or free base form of the compounds disclosed herein with one or more equivalents of a suitable acid or base, optionally in a solvent or in a salt-insoluble medium, followed by removal of the solvent or medium using standard techniques (e.g., in a vacuum, by freeze-drying or by filtration). The salts can also be prepared by exchanging a counter ion of the compounds disclosed herein in salt form with another counter ion, for example using a suitable ion exchange resin.

[0167] Examples of pharmaceutically acceptable salts include acid addition salts derived from inorganic and organic acids, as well as salts derived from metals such as sodium, magnesium, or preferably potassium and calcium.

[0168] Examples of acid addition salts include those formed with the following acids: acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, arylsulfonic acids (e.g., benzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, and p-toluenesulfonic acid), ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzoic acid, 4-acetaminobenzoic acid, butyric acid, (+)camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexane, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactobionic acid, gentian acid, glucoheponic acid, gluconic acid (e.g., D-gluconic acid), glucuronic acid. (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutarate, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxyethylsulfonic acid, lactic acid (e.g., (+)-L-lactic acid and (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid (e.g., (-)-L-malic acid), malonic acid, (±)-DL-mandelic acid, metaphosphoric acid, methanesulfonic acid, 1-hydroxy-2-naphthyl acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid (e.g., (+)-L-tartaric acid), thiocyanate, undecenoic acid, and valeric acid.

[0169] Specific examples of salts are those derived from: mineral acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid; organic acids, such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acid; and metals, such as sodium, magnesium, or preferably potassium and calcium.

[0170] As described above, flavonoid-containing compounds also include any solvates of the compound and its salts. Preferred solvates are those formed by incorporating a non-toxic, pharmaceutically acceptable solvent (hereinafter referred to as a solvating solvent) into the solid structure (e.g., crystal structure) of the compound of the present invention. Examples of such solvents include water, alcohols (e.g., ethanol, isopropanol, and butanol), and dimethyl sulfoxide. Solvates can be prepared by recrystallizing the compound of the present invention with a solvent or a mixture of solvents containing a solvating solvent. In any given case, whether a solvate is formed can be determined by analyzing the crystals of the compound using known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC), and X-ray crystallography.

[0171] The solvate can be stoichiometric or non-stoichiometric. Particularly preferred solvates are hydrates, including hemihydrates, monohydrates, and dihydrates.

[0172] For a more detailed discussion of solvates and the methods used to prepare and characterize them, see Bryn et al., Solid-State Chemistry of Drugs, 2nd ed., SSCI, Inc. of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0173] As defined herein, a "pharmaceutically functional derivative" of a flavonoid-containing compound includes ester derivatives and / or derivatives having or providing the same biological function and / or activity as any of the related compounds of this invention. Therefore, for the purposes of this invention, the term also includes prodrugs of the compounds of this invention. Specific examples of prodrugs of the compounds of this invention containing at least one hydroxyl group are ester derivatives, such as acetate derivatives.

[0174] However, the term "prodrug" used for the relevant compounds disclosed herein includes any compound that, after oral or parenteral administration, is metabolized in vivo to form an experimentally detectable amount of the compound within a predetermined time period (e.g., within dosing intervals of 6 to 24 hours, i.e., once to four times daily)).

[0175] Prodrugs of the compounds disclosed herein can be prepared by modifying functional groups present on the compound in such a way that the modification is cleaved in vivo when such prodrugs are administered to mammalian subjects. Modification is typically achieved by synthesizing a parent compound having prodrug substituents. Prodrugs include the compounds disclosed herein, wherein the hydroxyl, amino, mercapto, carboxyl, or carbonyl groups in the disclosed compounds are bonded to any group that can be cleaved in vivo to regenerate free hydroxyl, amino, mercapto, carboxyl, or carbonyl groups, respectively.

[0176] Examples of prodrugs include, but are not limited to, esters and carbamates with hydroxyl functional groups, esters with carboxyl functional groups, N-acyl derivatives, and N-Mannich bases. General information about prodrugs can be found, for example, in Bundegaard, H., “Design of Prodrugs” pI-92, Elsevier, New York-Oxford (1985).

[0177] For the sake of brevity, the compounds disclosed herein, as well as pharmaceutically acceptable salts, solvates, and pharmaceutically functional derivatives of these compounds, are collectively referred to below as "the compounds disclosed herein" or "the compounds of the present invention".

[0178] The compounds disclosed herein may contain exocyclic double bonds and therefore may exist as E (entgegen) and Z (zusammen) geometric isomers of each individual double bond. All such isomers and mixtures thereof are included within the scope of this invention.

[0179] The compounds disclosed herein can exist as regioisomers and can also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of this invention.

[0180] The compounds disclosed herein may contain one or more asymmetric carbon atoms and thus may exhibit optical and / or diastereomeric isomerism. Diastereomers can be separated using conventional techniques, such as chromatography or fractional crystallization. Various stereoisomers can be separated by using conventional techniques such as fractional crystallization or HPLC to separate racemic mixtures or other mixtures of the compounds. Optionally, desired optical isomers can be prepared by reacting a suitable optically active starting material under conditions that do not induce racemization or epimerization (i.e., a 'chiral pool' method), by reacting a suitable starting material with a 'chiral auxiliary' that can subsequently be removed at a suitable stage, by derivatization (i.e., resolution, including dynamic resolution) with an isochoric acid, followed by separation of the diastereomeric derivative by conventional methods such as chromatography, or by reacting with a suitable chiral reagent or chiral catalyst, all under conditions known to those skilled in the art. All stereoisomers and mixtures thereof are included within the scope of this invention.

[0181] As used herein, the term "glycosyl" refers to a group obtained by removing a hemiacetal hydroxyl group from the cyclic form of a monosaccharide, disaccharide, trisaccharide, or oligosaccharide containing six or fewer monosaccharide units. In specific cases, a glycosyl group may refer to a group obtained by removing a hemiacetal hydroxyl group from the cyclic form of a monosaccharide or disaccharide.

[0182] Unless otherwise specified, the term "alkyl" refers to an unbranched or branched, acyclic or cyclic, saturated or hydrocarbon radical, which may be substituted or unsubstituted (e.g., substituted by one or more halogen atoms). Examples of "alkyl" groups include methyl, ethyl, propyl (e.g., n-propyl or isopropyl), butyl (e.g., branched or unbranched butyl), pentyl, and methyl, for example. When the term "alkyl" is a cyclic group (possibly referring to the specified group "cycloalkyl"), it is preferably C10. 3-6 Cycloalkyl, and more preferably C10. 5-6 Cycloalkyl.

[0183] The term "fluoroalkyl" refers to an alkyl group as defined above, wherein at least one hydrogen atom is replaced by a fluorine atom. In specific instances, a fluoroalkyl group may contain 1 to 10 fluorine atoms, such as 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 fluorine atom.

[0184] Other embodiments of the invention that may be mentioned include those in which the compounds disclosed herein are isotopically labeled. However, other specific embodiments of the invention that may be mentioned include those in which the compounds disclosed herein are not isotopically labeled.

[0185] When used herein, the term "isotopically labeled" includes references to compounds of Formula I, wherein a non-natural isotope (or a non-natural distribution of the isotope) is present at one or more sites in the compound. Those skilled in the art will understand that "one or more sites in the compound" as used herein refers to one or more atoms of a compound of Formula I. Therefore, the term "isotopically labeled" includes references to compounds of Formula I that are isotopically enriched at one or more sites in the compound.

[0186] Isotopic labeling or enrichment of compounds of Formula I can be performed using any radioactive or non-radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine, and / or iodine. Specific isotopes that may be mentioned in this regard include 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 35 S, 18 F, 37 Cl、 77 Br、 82 Br and 125 I).

[0187] When a Formula I compound is labeled or enriched with a radioactive or non-radioactive isotope, the Formula I compound may be referred to as a compound that has at least one atom in the compound exhibiting an isotopic distribution in which the radioactive or non-radioactive isotope of the atom in question is present at a level at least 10% (e.g., 10% to 5000%, particularly 50% to 1000%, more particularly 100% to 500%) above the natural level of the radioactive or non-radioactive isotope.

[0188] The present invention is illustrated by the following embodiments, but these embodiments should not be construed as limiting.

[0189] Example

[0190] Material

[0191] Sodium hydroxide, potassium hydroxide (KOH), lithium hydroxide, cesium hydroxide, potato dextrose agar (PDA) powder, dimethyl sulfoxide (DMSO), and ethanol were obtained from Merck & Co., Inc. Flavonoids (FLs), including luteolin (Lu), apigenin (Ap), geraniol (Dio), chrysogenin (Chry), baicalin (Wo), genistein (Ge), 5,6-dihydroxyflavone (“56”), 5,3′,4′-trihydroxyflavone (“534”), 6,3′,4′-trihydroxyflavone (“634”), 7,3′,4′-trihydroxyflavone (“734”), and 3′,4′-dihydroxyflavone (“34”), were obtained from Indofine Chemical Co., Inc., Hillsborough, NJ, USA. Hypochlorous acid (15%) was obtained from Merck & Co., Inc. Disodium fluorescein was obtained from Aldrich (Milwaukee, WI). Flavonoids (7,8-dihydroxyflavone, baicalein, luteolin, scutellarein, fisetin, kaempferol, morin, myricetin, quercetin, 3,3′,4′-trihydroxyflavone, 3,5,7,8,3′,4′-hexahydroxyflavone, alpinetin, eriodictyol, glycyrrhizin, hesperidin, naringenin, pinocembrin, amplepsin, piperidin, catechin, epicatechin, epigallocatechin) were obtained from Nanjing Plant Origin Biological Technology Co., Ltd. 96-well polystyrene microplates and caps were purchased from VWR International Inc (Bridgeport, NJ). All aqueous solutions were prepared using 18.2 MΩ·cm ultrapure water obtained through a Millipore water purification system. Disodium tetraborate was obtained from Sigma.

[0192] Analytical techniques

[0193] Nuclear magnetic resonance

[0194] 1 H and 13C10 NMR spectra were measured using a Bruker AVANCE I 400 or 500 NMR spectrometer. Chemical shifts were reported in ppm using solvent resonance as an internal standard (DMSO-d6, δ = 2.50). The spectra are reported as follows: chemical shift (δppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, heptet = septet, m = multiplet), coupling constant (Hz), integration, and partition. Chemical shifts were reported in ppm using solvent resonance as an internal standard (DMSO, δ = 39.52).

[0195] Mass spectrometry analysis

[0196] High-resolution mass spectrometry (HRMS) was performed in ES negative mode on a Thermo Scientific LCQ Fleet ion trap mass spectrometer.

[0197] Electron paramagnetic resonance (EPR) spectroscopy

[0198] EPR spectra were recorded on an X-band EPR spectrometer (JES-TE100, JEOL, Tokyo, Japan), which was equipped with a WIN-RAD EPR data analysis system (Radical Research, Inc., Hino, Tokyo).

[0199] A 5.0 mM flavonoid solution was loaded into a capillary tube sealed with sealing putty (TERUMO CORPORATION, Tokyo, Japan). The capillary tube was then placed in an EPR tube (WILMAD QUARTZ (CFQ), 5 mm in diameter) and then placed in a TE mold cavity. EPR experiments were performed at room temperature using the following parameters: microwave frequency: 9.19 GHz, microwave power: 1 mW, central magnetic field: 328.348 mT, field scan width: ±5 mT, scan rate: 0.67 mT / min, time constant: 0.03, field modulation frequency: 500 kHz, and field modulation width: 0.025 mT. EPR data acquisition was controlled by a WIN-RAD EPR data analysis system. Spectra were simulated using JEOL IsoSimu / Fa version 2.2.0 isotropic simulation program.

[0200] fluorescence

[0201] The Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT) was used with a fluorescence filter, with an excitation wavelength of 485±20 nm and an emission wavelength of 530±25 nm. The reader was controlled by KC4 3.0 software (Revision 29). Sample dilution was performed using the Precision X automated pipetting system, managed by PrecisionPower software (Version 1.0) (Bio-Tek Instruments, Inc.).

[0202] Thin-layer chromatography (TLC) and column chromatography

[0203] Merck F254 silica gel-60 plates are used for thin-layer chromatography. Silica gel-60 (230-400 mesh) was selected as the solid phase for column chromatography.

[0204] HPLC and liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0205] The HPLC system (Waters Arc HPLC System) was equipped with a C18 column (Luna 5μm C18(2)100A, LC column 250×4.6mm). A Waters 2998 photodiode array (PDA) detector was connected to the HPLC system, with detection wavelengths of 190nm and 800nm.

[0206] The Bruker AmaZon-X was used for LC-MS and LC-MS / MS. The LC-MS system was equipped with a C18 column (Phenomenex, Luna 5u C18, 250 × 4.6 mm) guard column (4 × 3.0 mm). All mass spectra were obtained using electrospray ionization in both positive and negative ion modes. A parent ion with a width of ±2.5 Da was selected, and fragmentation was performed at a 50% setting.

[0207] Semi-preparative HPLC

[0208] The semiprep HPLC system (Waters semiprep HPLC system) is equipped with a C18 column (Phenomenex, (Luna 5μM C18(2)100A, LC column 250×10mm)) and a PDA detector.

[0209] X-ray crystallography

[0210] The narrow-frame algorithm was used, and the frames were integrated using the Bruker SAINT software package. The data was corrected for absorption effects using the SADABS (Multiple Scanning Absorption) method.

[0211] Computational research

[0212] Geometric optimization and spin density distribution

[0213] Geometric optimization and spin density distribution were calculated using Gaussian 09W software at the theoretical level of UM062X / 6-311+G(d,p) using DFT (M. Frisch et al., Gaussian 03, revision C.02; Gaussian, Inc.: Wallingford, CT, 2004). Cube files for both functions were generated using Multiwfn software (T. Lu & F. Chen, J. Comput. Chem. 2012, 33, 580-592), and isodense surface maps for non-covalent interaction studies were created using VMD visualization software (W. Humphrey, A. Dalke & K. Schulten, J. Mol. Graph. 1996, 14, 33-38). Molecular visualizations were created using CYLview (C. Legault, CYLview, 1.0b. Université de Sherbrooke 2009, 436, 437).

[0214] Gibbs free energy

[0215] The Gibbs free energy was calculated using the DFT in the Gaussian 16 program (M. Frisch et al., Gaussian 03, revision C.02; Gaussian, Inc.: Wallingford, CT, 2004.). Optimization was based on... The initial conformational search was completed using Maestro 10.6. The low-energy conformational isomer with a global minimum of 5 kcal / mol was further optimized at the M06-2X / 6-31G(d) level (Y. Zhao & D.G. Truhlar, Theor. Chem. Acc. 2008, 120, 215-241) and with a water-based SMD solvent model (AV. Marenich, C.J. Cramer & D.G. Truhlar, J. Phys. Chem. B 2009, 113, 6378-6396). Vibrational frequency analysis was performed at the same theoretical level to verify that the minimum had no imaginary frequencies, and its zero-point vibrational energy (ZPVE) and thermal correction at 298 K were evaluated. Single-point energies were calculated using a larger basis set 6-311+G(d,p) and the same solvent model.

[0216] General procedure for characterizing reaction products using HPLC and LC-MS / MS

[0217] Isocratic elution was used for the analysis of products self-coupled with luteolin. The LC-MS system was also used for the analysis of reaction products between flavonoids and HOCl. Samples (10 μL) were filtered through a 0.2 μm membrane (Merck Millipore, USA) and then injected into the HPLC system for analysis. Deionized (DI) water containing 0.1% formic acid was selected as mobile phase A, while ACN containing 0.1% formic acid was used as mobile phase B. The column was equilibrated with 71.5% mobile phase A for 10 min, followed by isocratic elution at a flow rate of 1.0 mL / min with the same percentage of mobile phase A for 0 to 35 min.

[0218] The Bruker AmaZon-X was used for characterization and fragment analysis of unknowns using LC-MS and LC-MS / MS. The LC conditions for LC-MS analysis were similar to those described above, except that the detection wavelengths were 280 and 350 nm.

[0219] General Procedure for Separating Bioflavonoids using Semi-Preparative HPLC

[0220] The reaction solution was carefully acidified to neutral pH with 1M HCl, then extracted with EA and analyzed using a semi-preparative HPLC instrument at 5 mL / min. -1Purification was performed using a flow rate of 500 μL. Compound 1a was obtained with CH3CN–water (45%:55%, 0.3% TFA v / v) at a retention time of 20.4 min. Compound 5a was separated at a retention time of 28.8 min and eluted with CH3CN–water (20%:80%, 0.3% TFA v / v). Compounds 8a and 8b were purified with CH3CN–water (45%:55%, 0.3% TFA v / v) at retention times of 36.6 min and 67.2 min, respectively. Compounds 10a, 10b, and 10c were separated with CH3CN–water (35%:65%, 0.3% TFA v / v) at retention times of 30.5 min, 42 min, and 54 min, respectively. Compounds 12a and 12b were purified from CH3CN–water (33%:67%, 0.3% TFA v / v) and obtained at retention times of 36.5 min and 53 min, respectively. The separation yields were calculated by mass using a semi-preparative HPLC with a C18 column.

[0221] Example 1. Synthesis of flavonoid cross-coupled dimers

[0222]

[0223] Luteolin (1a, 143 mg, 0.5 mmol) was added to a 50 mL centrifuge tube containing 30 mL of 0.1 M KOH aqueous solution. The resulting solution had a pH of 11.5. The tube was tightly capped to seal the reaction vessel and kept at room temperature for 10 hours without stirring. The resulting solution was then acidified with concentrated HCl (1.0 mL, 10 M) to produce a solution with a pH of 1–2. The resulting solution was then extracted with EA (3 × 50.0 mL) and the organic layers were combined. Volatiles were removed under vacuum to obtain a crude solid, which was purified by semi-preparative HPLC with an automated fractionation collection system to obtain pure 2a, 2a′, and 3a.

[0224] 6-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-2-(3,4-dihydroxyphenyl) (2a)-5,7-dihydroxy-4H-chromen-4-one (digamma-2a)

[0225] 68 mg, 48% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300 nm), t r (2a) = 19.045 min, the concentration of 2a = 1.0 mM. 1H NMR(500MHz,DMSO-d6)δ13.15(s,1H),12.79(s,1H),10.73(s,1H),10.68(s, 1H),10.13(s,1H),9.91(s,1H),9.42(s,1H),8.45(s,1H),7.48–7.40(m,2H) ,7.19(d,J=8.4Hz,1H),6.95(d,J=8.4Hz,1H),6.90(d,J=8.4Hz,1H),6.70(s ,1H),6.54(s,1H),6.09(d,J=2.1Hz,1H),6.06(s,1H),5.97(d,J=2.1Hz,1H). 13 C NMR (126MHz, DMSO) δ182.12,181.76,166.95,164.45,164.09,161.76,159.24,157.85,156.77,150.12,148.86,146.20,144.86, 124.19,121.99,120.70,120.28,119.44,116.52,114.70,113.78,108.53,106.69,103.86,103.77,103.33,99.14,93.79,93.73. C 30 H 18 O 12 The calculated HRMS (ESI-TOF) value is 569.0725, and the measured value is 569.0717.

[0226]

[0227] Crystals of compound 2a suitable for X-ray analysis were obtained by slow evaporation from MeOH. Crystals of C with dimensions approximately 0.072 mm × 0.123 mm × 0.146 mm were prepared. 30 H 18 O 12 The samples were used for X-ray crystallographic analysis. X-ray intensity data were measured. The total exposure time was 5.41 hours. Integrating the data using a triclinic crystal cell yielded a total of 26,930 reflections, with a maximum θ angle of 28.31°. (resolution), of which 7389 reflections are independent (average redundancy 3.645, integrity = 99.5%, R int =3.10%, R sig =2.95%), 5796 times (78.44%) greater than 2σ(F) 2The following final cell constants are based on a refinement of the XYZ centroids for 9938 reflections above 20σ(I), where 5.070° < 2θ < 56.58°: α=95.392(2)°, β=101.343(2)°, γ=102.776(2)°, The ratio of minimum apparent transmittance to maximum apparent transmittance is 0.920. The calculated minimum and maximum transmittance (based on crystal size) are 0.6862 and 0.7457, respectively. The Bruker SHELXTL software package was used, with space group P-1, where for the formula unit C... 30 H 18 O 12 Given Z=2, solve and refine the structure. For the observed data, F has 543 variables. 2 The final anisotropic full-matrix least-squares refinement converges to R1 = 6.80%, and for all data, wR2 = 22.47%. The goodness of fit is 0.848. The maximum peak in the final differential electron density synthesis is... The largest cavity is Where the RMS deviation is Based on the final model, the calculated density is 1.515 g / cm³. 3 And F(000), 712e - The crystallographic data has been stored at the Cambridge Crystallography Data Centre (CCDC#2044714).

[0228] Table 1. Sample and crystal data for 2a.

[0229]

[0230]

[0231] Table 2.2a. Data collection and structure refinement.

[0232]

[0233]

[0234] Table 3. Atomic coordinates and equivalent isotropic atomic displacement parameters of 2a U(eq) is defined as one-third of the trace of the orthogonalized Uij tensor.

[0235]

[0236]

[0237]

[0238] 8-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-2-(3,4-dihydroxyphenyl) 5,7-Dihydroxy-4H-chromen-4-one (Synthinol, 2a) ′ )

[0239] 11 mg, 8% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300 nm), t r (2a′) = 30.572 min, and the concentration of 2a′ = 6.5 mM. 1 H NMR(500MHz,DMSO-d6)δ13.15(s,2H),10.72(s,2H),10.15(s,4H),δ7.59(d,J=7.7Hz,2H),7.20(d,J= 8.3Hz,1H),7.01–6.89(m,3H),6.61(s,1H),6.11(d,J=2.0Hz,1H),6.05(s,1H),5.99(d,J=2.0Hz,1H). 13 C NMR (75MHz, DMSO) δ182.24,181.68,166.78,164.38,163.88,163.42,162.15,161.67,159.14,157.77,156.72,151.09,148.70,148.4 1,144.63,124.11,121.90,120.76,120.66,120.11,116.15,114.71,110.57,108.35,106.67,103.83,103.76,103.65,99.07,93.73. C 30 H 18 O 12 The calculated HRMS (ESI-TOF) value is 569.0725, and the measured value is 569.0715.

[0240] 6-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-2-(2-(2-(3,4-dihydroxyphenyl) (Hydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromene-6-yl)-3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromene- 4-Keto (Diamramite, 3a)

[0241] 90 mg, 42% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300 nm), t r (3a) = 22.742 min, concentration of 3a = 10 mM. 3a = 3a* + 3a**. Atropisomer 3a*: 1¹H NMR (500MHz, methanol-d⁴) δ 7.41 (m, 2H), 7.27 (d, J = 8.4Hz, 1H), 7.19 (d, J = 8.3Hz, 1H), 7.00 (d, J = 8.3Hz, 1H), 6.95 (d, J = 8.3Hz, 1H), 6.59 (s, 1H), 6.58 (s, 1H), 6.57 (s, 1H), 6.23 (s, 1H), 6.17 (s, 1H), 6.13 (d, 1H), 6.04 (d, 1H), 6.01 (s, 1H). Transisomer 3a**: 1 H NMR (500MHz, methanol-d4) δ7.41(m,2H),7.27(d,J=8.4Hz,1H),7.19(dd,J=8.3Hz,1H),7.00(d,J=8.3Hz,1H),6.95(d,J=8 .3Hz,1H),6.59(s,1H),6.58(s,1H),6.57(s,1H),6.19(s,1H),6.09(s,1H),6.13(d,1H),6.04(d,1H),6.01(s,1H). 13 C NMR (126MHz, DMSO) δ182.21,181.81,181.72,166.66,166.56,164.15,164.09, 162.28,161.75,159.07,157.87,157.06,156.81,150.04,148.72,146.17,144 .64,124.14,122.09,120.63,120.29,119.43,116.50,114.75,113.86,108.37 ,108.29,106.66,103.89,103.75,103.64,103.43,99.27,93.96,93.87,93.73. C 45 H 27 O 18 The calculated HRMS (ESI-TOF) value is 855.1192, and the measured value is 855.1182.

[0242] Results and Discussion

[0243] Hydroxyl-rich flavonoids (e.g., luteolin) are excellent reducing agents and are known as effective dietary antioxidants in scavenging bioassociated reactive oxygen species (N. Cotelle et al., Free Radic. Biol. Med. 1996, 20, 35-43). Furthermore, as detected by ESR spectroscopy, many weakly acidic flavonoids, including luteolin, undergo deprotonation to form phenolates under alkaline conditions. These phenolates are highly sensitive to oxidation by molecular oxygen, becoming their corresponding o-semiquinone radicals (K. Kuwabara et al., Appl. Magn. Reson. 2018, 49, 911–924; and...). RSJ Tarábek & I. Deganoca, Electrochim. Acta 2013, 110, 646–654). However, the fate of these free radicals remains unknown. We hypothesize that these electron-deficient semiquinone radicals can react with electron-rich flavonoid anions via radical-nucleophile coupling. To verify this, we performed HPLC analysis on an alkaline solution of luteolin (pH 11.5) and indeed found several products, further characterized by LC-MS as luteolin dimers and trimers. Here, we report two sp. flavonoid reactions mediated by dissolved molecular oxygen as a hydrogen atom acceptor. 2 Novel catalyst-free oxidative coupling reactions of CH bonds ( Figure 1 ).

[0244] Table 4. Aerobic oxidation of luteolin-luteolin cross-coupling a Conditional evaluation

[0245]

[0246] a Reaction conditions: Luteolin 1a (0.045 mmol) was dissolved in 3 mL of alkaline solution and incubated in a 15 mL sealed tube without stirring. b Conversion was calculated by HPLC. Isocratic elution (71.5% mobile phase A: DI water containing 0.1% formic acid and 21.5% mobile phase B: ACN containing 0.1% formic acid) was used for the analysis of luteolin-luteolin cross-coupling products. Standard curves were constructed using the separated products. c 10-gram scale. d The reaction takes place in the dark.

[0247] Following the successful self-cross-coupling of luteolin, we considered whether similar self-cross-coupling reactions could be extended to other flavonoids. Therefore, Ap, Dio, Chry, Wo, 5,6-dihydroxyflavone, and Ge were dissolved in alkaline water (pH 11.5). However, under the same conditions, the expected coupling products were not detected. Instead, only the starting materials were recovered. No radical signal was detected by EPR spectroscopy in the reaction solution, indicating that they are insensitive to oxygen. These flavonoids lack catechol groups, preventing them from forming o-semiquinone radical anions.

[0248] Example 2. Gram-scale synthesis of luteolin-coupled products

[0249] Using the conditions in Example 1, 10 g of luteolin was used in a one-pot reaction to synthesize 2a (42%, Lu-(2′-6)-Lu (this nomenclature is used to name flavonoid dimers and oligomers). For example, Lu-(2′-6)-Lu represents a luteolin (Lu) dimer linked by the C(2′) of the first luteolin to the C(6) of the second luteolin), 2a′ (Lu-(2′-8)-Lu), 2a″ (Lu-(6′-6)-Lu), and 3a (Lu-(2′-6)-Lu-(2′-6)-Lu), wherein luteolin was weighed... 10 g was dissolved in four 1-liter plastic bottles (500 mL each) of KOH solution (0.05 M, 2.0 L), and the resulting solution was neutralized with HCl (10 M) to obtain a precipitate. The mixture was extracted three times with EA (500 mL each time). The organic layers were then combined and concentrated under vacuum to obtain a crude solid product, which was dissolved in methanol and purified by semi-preparative HPLC with an automated fractionation collection system to obtain pure 2a (4.2 g, 42%), 2a′ (0.12 g, 1.2%), 2a″ (0.10 g, 1.0%), and 3a (1.0 g, 10%).

[0250] 6-(2-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-4,5-dihydroxyphenyl)-2-(3,4-dihydroxyphenyl) )-5,7-dihydroxy-4H-chromen-4-one (dehydrocyathione B, Lu-(6′-6)-Lu,2a″)

[0251]

[0252] 3 mg, 2% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300 nm), t r (2a″) = 15.922 min, the concentration of 2a″ = 3.14 mM. 1H NMR(500MHz,DMSO-d6)δ13.07(s,1H),12.72(s,1H),10.14(s,3H),9.62(s,3H),7.29(s,1H),7.12–6.97(m,2H),6.8 3(s,1H),6.76(d,J=8.4Hz,1H),6.61(s,1H),6.31(s,1H),6.08(d,J=2.1Hz,1H),5.98(s,1H),5.81(d,J=2.1Hz,1H). 13 C NMR (126MHz, DMSO) δ182.29,181.61,166.41,164.57,164.29,161.68,160.65,157.69,154.31,150.28,148.75,146.12,145.54,123. 66,123.48,123.34,121.80,120.38,119.04,116.34,116.06,114.03,107.51,106.65,103.91,103.72,102.88,99.21,98.93,93.67. C 30 H 18 O 12 The calculated HRMS (ESI-TOF) value is 569.0725, and the measured value is 569.0727.

[0253] Results and Discussion

[0254] 2a, 2a′, 2a″ and 3a were successfully synthesized in a one-pot process. Figure 2These compounds were initially isolated from mosses, among the oldest terrestrial plants, particularly *Rhizogonium distichum*, which contains all four trietramine regiomeric isomers, including *bartramiatriuteolin*, *strictatriuteolin*, and *rhizogoniatriuteolin* (H. Geiger and T. Seeger, Z. Naturforsch. 2000, 55c, 870-873). Their biosynthesis likely occurred at the neutral physiological pH of mosses, mediated by enzymes such as polyphenol oxidase. It has been proposed that high levels of flavonoids in mosses (up to 10% of their dry weight) can protect plants from biotic stresses (e.g., fungi) and abiotic stresses (temperature, water, reactive oxygen species, and ultraviolet (UV) light) (H. Wang et al., Plant Sci. 2020, 298, 110591; and M. Waterman et al., J. Nat. Prod. 2017, 80, 2224-2231). Product 3a was analyzed by HRMS, 1 H and 13 Characterization by C10 NMR spectroscopy revealed the presence of an atropisomer due to hindered rotation of the interflavonyl bond (CLCovington et al., J.Nat.Prod. 2016, 79, 2530-2537). This isomerism is common in complex natural products, including tryptorubin A (SHReisberg et al., Science 2020, 367, 458–463).

[0255] Example 3. Synthesis of Cyclotrietrin (CTL)

[0256] Triluteolin, such as 3a, has a B ring and an A ring on the terminal luteolin unit, the B ring and A ring being close to each other for intramolecular oxidative coupling. Figure 2 B).

[0257]

[0258] Except that the pH of the reaction mixture was adjusted to 12.5 with concentrated KOH before being sealed and kept at room temperature for 10 hours without stirring, cyclotrietrin (e.g., 4) was prepared from trimer (e.g., 3a) (50 mg, 0.06 mmol) in a KOH aqueous solution (10 mL, 0.1 M) in a 50 mL centrifuge tube using a similar procedure to Example 1. Figure 2 B).

[0259] (6′-6)3-Cyclotrietrin, ((6′-6)3-CTL, 4a)

[0260]

[0261] 5 mg, 10% yield, white solid. HPLC (Luna 5μm C18(2)100A, LC column 250×4.6mm, ACN / water = 71.5:28.5, flow rate 1.0mL / min, λ = 300nm), injection volume = 10μL, tr(4a) = 9.045min. 1 H NMR(500MHz,DMSO-d6)δ12.74(s,1H),12.70(s,1H),12.69(s,1H),10.56(s,1H),10.49(s,1H),9.96(s,1H),9.59(s,1H ),9.39(s,1H),8.45(s,1H),6.94(s,1H),6.89(d,J=2.4Hz,4H),6.63(s,1H),6.30–6.24(m,3H),6.06(d,J=11.1Hz,3H). 13 C NMR (126MHz, DMSO) δ182.01,181.98,181.94,168.03,167.97,167.67,163.55,16 3.46,162.95,158.62,158.49,158.46,156.90,156.67,148.23,148.19,147.99,1 45.20,145.10,145.00,126.42,126.38,125.58,123.87,120.74,120.60,119.90, 119.79,119.64,116.96,114.68,111.40,107.94,107.80,103.27,103.10,93.27. C 45 H 23 O 18 The calculated HRMS (ESI-TOF) value is 851.0890, and the measured value is 851.0901.

[0262] (2′-6)2(6′-6)-Cyclotrietrin, ((2′-6)2(6′-6)-CTL, 4b)

[0263]

[0264] 4b is obtained by crystallizing 4a. 1H NMR(500MHz,DMSO-d6)δ14.73(s,1H),13.17(s,1H),11.93(s,1H),10.69(s, 1H),10.54(s,1H),10.24(s,1H),9.98(s,1H),9.69(s,1H),9.43(s,1H),8.4 3(d,J=12.2Hz,3H),8.20(s,1H),7.24(d,J=8.5Hz,1H),7.09(s,1H),6.95–6 .84(m,4H),6.59(s,1H),6.22(s,1H),5.98(s,1H),5.72(s,1H),5.26(s,1H). 13 C NMR (126MHz, DMSO) δ195.02,183.13,182.47,169.26,168.37,165.97,161.10,160.11,159.56, 158.24,157.75,157.01,154.98,154.58,149.43,148.15,147.17,145.73,144.97,144.74,126. 59,123.16,121.80,121.47,120.83,120.08,120.02,118.56,114.75,114.63,114.05,112.68, 108.33,108.29,106.39,106.36,105.94,105.81,103.73,102.11,101.15,95.19,95.03,92.67. C 45 H 23 O 18 The calculated HRMS (ESI-TOF) value is 851.0890, and the measured value is 851.0882.

[0265] Results and Discussion

[0266] By dissolving 3a in alkaline water (pH 12.5) overnight at room temperature, the three main cyclotrietrins 4a, 4a′, and 4a″ were isolated. Figure 2B) Formed together with some luteolin monomers, and 2a was observed by HPLC in the reaction mixture. Clearly, flavonoid intermolecular isomerization occurred under these reaction conditions, and the expected (2′-6)3-triluteolin isomer was not detected. The cleavage of the flavonoid intermolecular bonds explains the formation of 1a and 2a. These cyclotriluteolins are regiomeric isomers of naturally occurring cyclobartramiatriluteolin ((2′-8)3 flavonoid intermolecular bonds) isolated from mosses (H. Geiger et al., Phytochemistry 1995, 39, 465-467).

[0267] Characterization of Example 4.4a

[0268] To confirm the structure of 4a prepared in Example 3, a single crystal was grown from its methanol solution and the molecular structure was determined.

[0269] Results and Discussion

[0270] Cyclodiamne acetoside

[0271]

[0272] Crystals of compound 4a suitable for X-ray analysis were obtained by slow evaporation from MeOH. Crystals of C4a with dimensions approximately 0.248 × 0.247 × 0.168 mm were prepared. 45 H 24 O 18 The samples were used for X-ray crystallographic analysis. X-ray intensity data were measured. Integrating the data using a triclinic unit cell yielded a total of 33,495 reflections. The final cell constants below are based on a refinement of the XYZ centroids for 4,286 reflections above 20σ(I), where 8.870° < 2θ < 133.1°: α=116.927(3)°, β=97.234(3)°, γ=100.043(3)°, The calculated minimum and maximum transmittance (based on crystal size) are 0.5865 and 0.7528, respectively. The Bruker SHELXTL software package was used, with space group P-1, where for the formula unit C... 45 H 24 O 18 Given Z=2, solve and refine the structure. For the observed data, for F... 2The final anisotropic full-matrix least-squares refinement converges to R1 = 10.5%, and for all data, wR2 = 30.28%. The goodness of fit is 1.062. The maximum peak in the final differential electron density synthesis is... The largest cavity is Based on the final model, the calculated density is 1.285 g / cm³. 3 And F(000), 1103e - The crystallographic data has been stored at the Cambridge Crystal Data Centre (CCDC#2044716).

[0273] Table 5. Sample and crystal data for 4a.

[0274]

[0275]

[0276] Data collection and structure refinement in Table S6.4a.

[0277]

[0278] Atomic coordinates and equivalent isotropic atomic displacement parameters in Table S7.4a U(eq) is defined as one-third of the trace of the orthogonalized Uij tensor.

[0279]

[0280]

[0281]

[0282]

[0283] ORTEP diagram ( Figure 2 C) shows a structure of 4a′ instead of the expected 4a. The 4a′ structure is triangular, with each corner occupied by the B ring of luteolin, and the three edges are bounded by benzopyranyl groups (length is...). Enclosed, forming an opening approximately Hydrophobic cavities. C(4)=O and C(5)-OH form intramolecular hydrogen bonds, and the benzopyran group plane is tilted at a dihedral angle of about 70° to the plane coinciding with the paper plane. In the solid state, the 4a′ molecule forms a hydrophobic channel, with the hydrophilic OH groups (C(7)OH, C(3′)-OH and C(4′)-OH) facing outwards, and the edges of C(4)=O and C(5)-OH facing inwards. Due to its unique shape and phenolic group, cyclotrietolin can complex guest molecules and metal ions. Therefore, it is an interesting building block for constructing functional covalent organic frameworks (COFs).

[0284] Isomerization of cyclotrietolin

[0285] In solution, cyclobatrimethylolamine triluterin exhibits a group of three luteolin units. 1 H and 13 C10 NMR peaks (H. Geiger et al., Phytochemistry 1995, 39, 465-467). Due to the C3 axis in 4a, its 1 1H NMR spectrum and magnetic equivalent luteolin unit C(sp) at 25℃ 2 )-H consistent ( Figure 3 A). However, due to the presence of three chiral axes and flavonoid inter-unit bonds, there exist bonds with equal strength. 1 The three rotational isomers of the H NMR signal ( Figure 3 A) Upon heating the solution to 90°C within one minute, rapid isomerization of the flavonoid inter-unit bonds occurs to produce new... 1 H signal group ( Figure 3 B). We propose that isomerization can occur via an ortho-semiquinone radical intermediate promoted under alkaline conditions or upon heating ( Figure 3 BC). We calculated the Gibbs free energy of four possible isomers of CTL and found that they have relatively similar free energies (BC). Figure 3 D), which is consistent with the experimental observation results.

[0286] Example 5. Self-cross-coupling reaction of β-catechin flavonoids

[0287] Dissolve one of 1a and 1h-k (0.045 mmol) in 3 mL of 0.1 M KOH solution in a 15 mL centrifuge tube. Adjust the pH to 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, or 13.0 with concentrated KOH, then seal the tube and leave it at room temperature overnight without stirring. Acidify the resulting solution with concentrated HCl (1.0 mL, 10 M) to obtain a solution with a pH of 1–2. Extract the resulting solution with EA (3 × 50.0 mL) and combine the organic layers. Remove volatiles under vacuum to obtain a crude solid, which is purified by semi-preparative HPLC with an automated fractionation collection system to obtain pure 2a and 2h-k.

[0288] Results and Discussion

[0289] As detected by EPR spectroscopy, treatment of trihydroxyflavones containing the B ring of catechol (including 3′,4′-dihydroxyflavones, 3′,4′,5-trihydroxyflavones, 3′,4′,6-trihydroxyflavones, and 3′,4′,7-trihydroxyflavones) in alkaline water leads to the formation of o-semiquinone radicals. Figure 4-7 However, almost no coupling reaction products were detected. Figure 8 These observations suggest that the nucleophilicity of these trihydroxyflavones is insufficient to accept the semiquinone radicals generated by their oxidation. This is consistent with calculations by DFT, which found that trihydroxyflavones have a much lower nucleophilicity than luteolin. Figure 9 Therefore, among these flavonoids, luteolin anion is unique because of its high nucleophilicity and ability to form o-semiquinone radicals, enabling it to undergo coupling reactions.

[0290] Example 6. Synthesis of β-catechol flavonoid-apigenin biflavonoid

[0291]

[0292] Except for the product extraction with 100 mL EA, 2b was prepared from 1a (0.25 mmol) and Ap (1b, 140 mg, 0.5 mmol) according to the protocol in Example 1.

[0293] 43% yield (2b), 4% yield (2b′), 10% yield (3b). Semi-preparative HPLC (DI water containing 0.1% formic acid / ACN containing 0.1% formic acid = 71.5:28.5), t r (2b) = 31.758 min, t r (2b′)=25.601min,t r (3b) = 35.711 min.

[0294] Results and Discussion

[0295] When luteolin was mixed with excess Ap (1:1.5 molar ratio), (Lu-(2′-6)-Ap, 2b), as well as trace amounts of Lu-(2′-8)-Ap 2b′, triflavonoids (Lu-(2′-6)-Lu-(2′-6)-Ap, 3b), and trace amounts of 2a were isolated in good yield (47%). The structure of 2b was confirmed by single-crystal X-ray diffraction analysis to be deoxydigammadaminozide (DGD). Figure 10A), a biflavonoid isolated from *Plagiomnium undulatum* (C. Ramendahl et al., *Phytochemistry* 1996, 41, 1621-1624). In summary, it is clear that the general rule for oxygen-mediated oxidative coupling of two flavonoids is that one flavonoid becomes a radical precursor by forming an ortho-semiquinone radical anion, while the other flavonoid is a good nucleophile under weakly basic reaction conditions. This rule is effective for the coupling of luteolin with other flavonoids, as shown in Example 7.

[0296] Table 8. Aerobic oxidation of luteolin-apigenin cross-coupling a Evaluation of conditions.

[0297]

[0298] a Reaction conditions: Luteolin 1a (0.045 mmol) and apigenin 1b (Y equivalent) were dissolved in 3 mL of KOH solution and incubated in a 15 mL sealed tube without stirring. b Conversion was calculated using HPLC. Isocratic elution (71.5% mobile phase A: DI water containing 0.1% formic acid and 21.5% mobile phase B: ACN containing 0.1% formic acid) was used for the analysis of luteolin-luteolin cross-coupling products. Standard curves were constructed using the separated products. c Separation yield. d pH 12.5 buffer (disodium tetraborate) is used as a substitute for KOH solution.

[0299] Example 7. Synthesis of luteolin-flavonoid biflavonoids

[0300]

[0301] According to the scheme in Example 1, luteolin-flavonoid biflavonoid 2b-2k was prepared from luteolin (72 mg, 0.25 mmol) and flavonoids selected from the following (0.5 mmol): Ap, Dio, Chry, Wo, 5,6-dihydroxyflavone (56), Ge, 5,3′,4′-trihydroxyflavone (534), 6,3′,4′-trihydroxyflavone (634), 7,3′,4′-trihydroxyflavone (734) and 3′,4′-dihydroxyflavone (34).

[0302] 6-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-2- (4-Hydroxyphenyl)-4H-chromen-4-one (3″′-dehydrodigamma, 2b)

[0303]

[0304] 2b was prepared from Ap. 61 mg, 44% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300 nm), t r (2b) = 30.447 min, concentration of 2b = 1.66 mM. 1 H NMR (500MHz, DMSO-d6) δ13.15(s,1H),12.80(s,1H),10.34(s,5H),7.95(d,J=8.4Hz,2H),7.17(d,J=8.4Hz, 1H),7.02–6.86(m,3H),6.77(s,1H),6.54(s,1H),6.09(d,J=2.1Hz,1H),6.03(s,1H),5.98(d,J=2.1Hz,1H). 13 C NMR (126MHz, DMSO) δ182.13,181.76,167.11,164.62,164.43,163.85,161.75,161.56,159.22,157.87,156.86,149.00,145.11,128. 92,128.92,124.21,121.72,120.61,120.47,116.43,116.43,114.52,108.77,106.60,103.85,103.52,103.26,99.13,94.09,93.81. C 30 H 17 O 11 The calculated HRMS (ESI-TOF) value is 553.0776, and the measured value is 553.0782.

[0305]

[0306] 2b crystals suitable for X-ray analysis were obtained by slow evaporation from MeOH. C crystals with dimensions of approximately 0.061 mm × 0.063 mm × 0.267 mm were used. 30 H 18 O 11 A brown, blocky sample was used for X-ray crystallography analysis. X-ray intensity data were measured. The total exposure time was 19.00 hours. Data were integrated using a triclinic crystal cell, resulting in a total of 18,025 reflections, with a maximum θ angle of 67.11°. (resolution), of which 5625 reflections are independent (average redundancy 3.204, integrity = 99.0%, R int =6.56%, R sig =6.46%), 3788 times (67.34%) greater than 2σ(F) 2The following final cell constants are based on a refinement of the XYZ centroids for 49 reflections above 20σ(I), where 8.533° < 2θ < 40.75°: α=98.306°, β=95.037°, γ=109.024°, The ratio of minimum apparent transmittance to maximum apparent transmittance is 0.883. Using the BrukerSHELXTL software package, where the unit C is... 30 H 18 O 11 Given Z=2, solve and refine the structure. For the observed data, F has 491 variables. 2 The final anisotropic full-matrix least-squares refinement converges to R1 = 4.71%, and for all data, wR2 = 12.43%. The goodness of fit is 1.029. The maximum peak in the final differential electron density synthesis is... The largest cavity is Where the RMS deviation is Based on the final model, the calculated density is 1.434 g / cm³. 3 And F(000), 720e - The crystallographic data has been stored at the Cambridge Crystallographic Data Centre (CCDC#2044715).

[0307] Table 9.2b shows the sample and crystal data.

[0308]

[0309]

[0310] Table 10.2b shows the sample and crystal data.

[0311]

[0312]

[0313] Atomic coordinates and equivalent isotropic atomic displacement parameters in Table 11.2b U(eq) is defined as one-third of the trace of the orthogonalized Uij tensor.

[0314]

[0315]

[0316]

[0317] 8-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-2- (4-Hydroxyphenyl)-4H-chromen-4-one (Lu-Ap(2′-8), 2b′)

[0318]

[0319] 2b′ was prepared from Ap. 4 mg, 3% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300 nm), t r (2b′) = 23.852 min, and the concentration of 2b′ = 8.3 mM. 1 H NMR (500MHz, DMSO-d6) δ13.01(s,1H),12.75(s,1H),10.76(s,3H),10.33(s,2H),7.53(d,J=9.1Hz,2H),7.33–7.22(d,J=8.2Hz,1H),7.03(d, J=8.2Hz,1H),6.81(d,J=9.1Hz,2H),6.76(s,1H),6.28(d,J=1.4Hz,1H),6.08(d,J=2.1Hz,1H),6.04(d,J=1.4Hz,1H),5.76(d,J=2.1Hz,1H). 13 C NMR (126MHz, DMSO) δ207.07,182.45,181.71,167.05,164.43,164.02,162.11,161.70,161.51,160.89,157.71,154.72,148.99, 144.94,128.59,124.43,121.73,121.12,119.26,116.27,115.08,106.92,104.00,103.76,103.58,102.96,99.17,98.85,93.63. C 30 H 17 O 11 The calculated HRMS (ESI-TOF) value is 553.0776, and the measured value is 553.0782.

[0320] 6-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-2- (3-Hydroxy-4-methoxyphenyl)-4H-chromen-4-one (Lu-Dio(2′-6), 2c)

[0321]

[0322] 2c was prepared from Dio. 95 mg, 65% yield, brown solid. HPLC (ACN / water = 64:36, λ = 300 nm), t r (2c) = 11.018 min, the concentration of 2c = 0.81 mM. 1H NMR (500MHz, DMSO-d6) δ13.14(s,1H),12.82(s,1H),10.52(s,4H),9.51(s,1H),7.56(dd,J=8.7,2.3Hz,1H),7.45(d,J=2.4Hz,1H),7.17(d,J=8.3Hz ,1H),7.10(d,J=8.7Hz,1H),6.92(d,J=8.3Hz,1H),6.73(s,1H),6.51(s,1 H), 6.09 (d, J = 2.1Hz, 1H), 6.04 (s, 1H), 5.99 (d, J = 2.1Hz, 1H), 3.88 (s, 3H). 13 C NMR (126MHz, DMSO) δ 182.00, 181.77, 167.22, 164.43, 163.54, 161.75, 159.21, 157.87, 156.90, 151.52, 149.11, 147.27, 145.32, 124.21, 123.59, 120.57, 119.12, 114.42, 113.35, 112.68, 109.01, 106.52, 103.88, 103.85, 103.39, 99.12, 94.19, 93.81, 56.23. For C 31 H 19 O 12 The calculated HRMS (ESI-TOF) value is 583.0882, and the measured value is 583.0898.

[0323] 6-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-2-benzene Lu-Chry(2′-6), 2d-4H-chromen-4-one

[0324]

[0325] Prepared by Chrysalis 2d. 62 mg, 46% yield, brown solid. HPLC (ACN / water = 60:40, λ = 300 nm), t r (2d) = 12.096 min, concentration at 2d = 2.40 mM. 1 H NMR(400MHz, DMSO-d6)δ13.01(s,1H),12.79(s,1H),11.14–10.09(m,4H),δ8.16–8.05(m,2H),7.68–7.53(m,3H),7.20(d, J=8.4Hz,1H),7.00(s,1H),6.95(d,J=8.4Hz,1H),6.62(s,1H),6.09(d,J=2.1Hz,1H),6.07(s,1H),5.98(d,J=2.1Hz,1H).13 C NMR (126MHz, DMSO) δ182.38,181.76,166.90,164.47,163.46,162.78,161.77,159.23,157.85,156.96,148.84,144.79,132.47,131. 20,129.63,129.63,126.91,126.91,124.19,120.75,120.13,114.79,108.75,106.74,105.68,104.13,103.86,99.16,93.97,93.80. C 30 H 17 O 10 The calculated HRMS (ESI-TOF) value is 537.0827, and the measured value is 537.0824.

[0326] 6-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-8-methyl oxy-2-phenyl-4H-chromen-4-one (Lu-Wo(2′-6), 2e)

[0327]

[0328] 2e was prepared from Wo. 121 mg, 85% yield, brown solid. HPLC (ACN / water = 60:40, λ = 300 nm), t r (2e) = 25.651 min, 2e concentration = 1.95 mM. 1 H NMR(500MHz,DMSO-d6)δ12.80(s,1H),12.73(s,1H),10.73(s,1H),10.29(s,1H),10.18(s,1H),8.53(s,1H),8.18–8.05(m,2H),7.68–7.55 (m,3H),7.22(d,J=8.4Hz,1H),7.06(s,1H),6.97(d,J=8.4Hz,1H),6.09(d,J=2.1Hz,1H),6.08(s,1H),5.91(d,J=2.1Hz,1H),3.82(s,3H). 13C NMR (126MHz, DMSO) δ 182.56, 181.77, 166.86, 164.44, 163.29, 161.76, 157.78, 155.48, 154.52, 149.05, 148.86, 144.75, 132.57, 131.28, 129.77, 127.91, 126.76, 124.07, 120.78, 119.76, 114.90, 108.89, 106.79, 105.69, 103.85, 103.82, 99.14, 93.75, 61.95. For C 31 H 19 O 11 The calculated HRMS (ESI-TOF) value is 567.0933, and the measured value is 567.0921.

[0329]

[0330] Crystals of compound 2e suitable for X-ray analysis were obtained by slow evaporation from MeOH and DMSO. Crystals of C with dimensions approximately 0.049 mm × 0.122 mm × 0.132 mm were prepared. 31 H 20 O 11 The samples were used for X-ray crystallographic analysis. X-ray intensity data were measured. The total exposure time was 17.13 hours. Integrating the data using a triclinic crystal cell yielded a total of 8055 reflections, with a maximum θ angle of 67.03°. (resolution), of which 8055 reflections are independent (average redundancy 1.000, integrity = 97.6%, R sig =10.51%), 4577 times (56.82%) greater than 2σ(F) 2 The following final cell constants are based on a refinement of the XYZ centroids for reflections above 20σ(I) at 4286, where 8.870° < 2θ < 133.1°: α=102.701(4)°, β=102.737(4)°, γ=90.020(4)°, The ratio of minimum apparent transmittance to maximum apparent transmittance is 0.637. The calculated minimum and maximum transmittance (based on crystal size) are 0.4799 and 0.7528, respectively. The Bruker SHELXTL software package was used, with space group P-1, where for the formula unit C... 31 H 20 O 11 Given Z=2, solve and refine the structure. For the observed data, F has 455 variables. 2The final anisotropic full-matrix least-squares refinement converges to R1 = 9.67%, and for all data, wR2 = 32.15%. The goodness of fit is 1.065. The maximum peak in the final differential electron density synthesis is... The largest cavity is Where the RMS deviation is Based on the final model, the calculated density is 1.475 g / cm³. 3 And F(000), 676e - The crystallographic data has been stored at the Cambridge Crystal Data Centre (CCDC#2044716).

[0331] Table 12. Sample and crystal data for 2e.

[0332]

[0333]

[0334] Table 13.2e: Data collection and structural refinement.

[0335]

[0336]

[0337] Table 14. Atomic coordinates and equivalent isotropic atomic displacement parameters of 2e U(eq) is defined as one-third of the trace of the orthogonalized Uij tensor.

[0338]

[0339]

[0340] 7-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,6-dihydroxy-2-benzene 4H-chromen-4-one (Lu-56(2′-7), 2f)

[0341]

[0342] 2f was prepared from 5,6-dihydroxyflavone. 77 mg, 57% yield, brown solid. HPLC (ACN / water = 64:36, λ = 300 nm), t r (2f) = 19.290 min, 2f concentration = 1.95 mM. 1H NMR(500MHz,DMSO-d6)δ13.25(s,1H),12.84(s,1H),11.04(s,1H),9.95(s,1H),9.58(s,1H),9.12(s,1H),7.68–7.57(m,2 H),7.52–7.44(m,1H),7.44–7.29(m,3H),7.04(s,1H),6.96–6.84(m,2H),6.71(s,1H),6.66(d,J=8.9Hz,1H),6.50(s,1H). 13 C NMR (126MHz, DMSO) δ184.38,182.45,164.32,164.10,162.30,161.40,154.81,150.11,146.87,146.39,146.10,141.03,132.5 8,131.18,129.52,126.42,126.37,121.82,118.75,116.04,113.72,111.16,110.67,104.86,104.21,103.06,102.12,99.19. C 30 H 17 O 10 The calculated HRMS (ESI-TOF) value is 537.0827, and the measured value is 537.0837.

[0343] 8-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,6-dihydroxy-2-benzene 4H-chromen-4-one (Lu-56(2′-8), 2f′)

[0344]

[0345] 2f′ was prepared from 5,6-dihydroxyflavone. 8 mg, 6% yield, brown solid. HPLC (ACN / water = 64:36, λ = 300 nm), t r (2f′) = 25.948 min, and the concentration of 2f′ = 2.50 mM. 1 H NMR (500MHz, DMSO-d6) δ13.52(s,1H),12.73(s,1H),9.51(s,4H),7.82(d,J=8.2Hz,2H),7.53(d,J=7. 2Hz,1H),7.48(q,J=8.2,7.2Hz,4H),7.25(s,1H),7.05(s,1H),6.93(s,1H),6.77(s,1H),6.73(s,1H). 13C NMR (126MHz, DMSO) δ184.37,182.28,164.42,164.23,162.54,159.59,157.07,150.30,146.54,146.27,140.78,132.59,1 31.42,129.66,126.71,126.36,121.89,119.59,116.56,113.87,111.51,111.05,106.90,104.85,103.99,103.38,93.96. C 30 H 17 O 10 The calculated HRMS (ESI-TOF) value is 537.0827, and the measured value is 537.0830.

[0346] 6-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-3- (4-Hydroxyphenyl)-4H-chromen-4-one (Lu-Ge(2′-6), 2g)

[0347]

[0348] 2g prepared from Ge. 39mg, 28% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300nm), t r (2g) = 28.867 min, the concentration of 2g = 1.13 mM. 1 H NMR(500MHz,DMSO-d6)δ13.13(s,1H),12.78(s,1H),10.73(s,3H),10.15(s,1H),9.58(s,2H),8.34(s,1H),7.42–7.34(m,2H),7.18 (d,J=8.4Hz,1H),6.94(d,J=8.4Hz,1H),6.86–6.75(m,2H),6.48(s,1H),6.10(d,J=2.1Hz,1H),6.03(s,1H),5.99(d,J=2.1Hz,1H). 13 C NMR (126MHz, DMSO) δ181.75,180.73,166.83,164.48,162.64,161.77,159.68,157.85,157.85,157.07,154.28,148.81,144.75,130. 67,130.67,124.19,122.75,121.74,120.74,120.19,115.50,115.50,114.77,108.59,106.75,104.62,103.87,99.16,93.82,93.47. C 30 H 17O 11 The calculated HRMS (ESI-TOF) value is 553.0776, and the measured value is 553.0781.

[0349] 8-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-3- (4-Hydroxyphenyl)-4H-chromen-4-one (Lu-Ge(2′-8), 2g′)

[0350]

[0351] 2 g′ was prepared from Ge. 19 mg, 14% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300 nm), t r (2g′) = 29.696 min, the concentration of 2g′ = ​​0.51 mM. 1 H NMR (500MHz, DMSO-d6) δ13.08(s,1H),12.80(s,1H),10.70(s,3H),9.59(s,2H),8.30(s,1H),7.38–7.32(m,2H),7.21(d,J=8 .4Hz,1H),6.97(d,J=8.4Hz,1H),6.84–6.77(m,2H),6.30(s,1H),6.11(d,J=2.1Hz,1H),6.08(s,1H),5.85(d,J=2.1Hz,1H). 13 C NMR (126MHz, DMSO) δ181.78,180.87,166.98,164.49,162.41,161.77,161.45,157.84,157.77,155.50,154.50,149.08,145.12,130. 57,130.57,124.42,122.46,121.66,121.10,119.26,115.51,115.51,115.00,106.92,104.78,103.83,103.48,99.18,99.12,93.74. C 30 H 17 O 11 The calculated HRMS (ESI-TOF) value is 553.0776, and the measured value is 553.0776.

[0352] 6-(2,3-dihydroxy-6-(5-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-2-(3,4-dihydroxyphenyl)- 5,7-Dihydroxy-4H-chromene-4-one (534-Lu(2′-6), 2h)

[0353]

[0354] Prepared from 5,3′,4′-trihydroxyflavone over 2 hours. 60 mg, 43% yield, brown solid. HPLC (ACN / water = 64:36, λ = 300 nm), tr (2h) = 14.195min, concentration at 2h = 4.29mM. 1 H NMR (500MHz, DMSO-d6) δ13.16(s,1H),12.64(s,1H),9.76(s,5H),7.53(t,J=8.2Hz,1H),7.48–7.40(m,2H),7.25(d,J=8. 4Hz, 1H), 6.97 (d, J = 8.4Hz, 1H), 6.91 (d, J = 8.2Hz, 1H), 6.74–6.67 (m, 2H), 6.65 (d, J = 8.4, 1H), 6.56 (s, 1H), 6.20 (s, 1H). 13 C NMR (126MHz, DMSO) δ182.95,182.16,167.91,164.19,162.43,160.24,159.28,156.83,156.36,150.19,149.14,146.22,144.86,136.1 6,124.01,121.94,121.01,120.36,119.49,116.54,114.84,113.78,111.12,110.09,108.40,107.34,107.18,103.87,103.33,93.71. C 30 H 19 O 11 The calculated HRMS (ESI-TOF) value is 553.0776, and the measured value is 553.0735.

[0355] 6-(2,3-dihydroxy-6-(6-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-5,7-dihydroxy-2-(4-hydroxy) (634-Lu(2′-6),2i)-4H-chromen-4-one

[0356]

[0357] Prepared from 6,3′,4′-trihydroxyflavonoids over 2 hours. 51 mg, 37% yield, brown solid. HPLC (ACN / water = 72.5:27.5, λ = 300 nm), t r (2i) = 9.532 min, concentration of 2i = 6.99 mM. 1H NMR(500MHz,DMSO-d6)δ13.12(s,1H),10.70(s,1H),10.06(s,1H),9.92(s,1H),9.45(s,1H),8.43(s,1H),8.14(s,1H),7 .50–7.39(m,2H),7.26–7.08(m,4H),6.96(d,J=8.4Hz,1H),6.91(d,J=8.2Hz,1H),6.69(s,1H),6.54(s,1H),6.05(s,1H). 13 C NMR (126MHz, DMSO) δ182.16,176.92,165.67,164.16,163.51,162.40,159.31,156.74,155.05,150.15,149.90,148.35,146.21,144.6 9,125.02,124.29,123.24,121.97,120.42,120.15,119.55,116.53,114.77,113.79,108.57,108.07,107.81,103.87,103.33,93.62. C 30 H 19 O 11 The calculated HRMS (ESI-TOF) value is 553.0776, and the measured value is 553.0769.

[0358] 6-(2,3-dihydroxy-6-(7-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-2-(3,4-dihydroxyphenyl)- 5,7-Dihydroxy-4H-chromene-4-one (734-Lu(2′-6), 2j)

[0359]

[0360] Prepared from 7,3′,4′-trihydroxyflavone over 2 hours. 59 mg, 43% yield, brown solid. HPLC (ACN / water = 72.5:27.5, λ = 300 nm), t r (2j) = 9.790 min, the concentration of 2j = 9.25 mM. 1H NMR(500MHz,DMSO-d6)δ13.14(s,1H),10.69(d,J=8.2Hz,2H),10.06(s,1H),9.9 4(s,1H),9.46(s,1H),8.42(s,1H),7.76(d,J=8.6Hz,1H),7.45(d,J=8.6Hz,2H) ,7.18(d,J=8.3Hz,1H),6.96(d,J=8.3Hz,1H),6.92(d,J=8.1Hz,1H),6.82(dd,J =8.1, 2.1Hz, 1H), 6.70 (s, 1H), 6.55 (s, 1H), 6.50 (d, J = 2.1Hz, 1H), 6.03 (s, 1H). 13 C NMR (126MHz, DMSO) δ207.17,182.21,176.60,165.55,164.17,162.85,162.35,159.29,157.97,156.76,150.15,148.31,146.22,144.67,1 26.83,124.93,122.02,120.47,120.11,119.49,116.56,116.22,115 .22,114.80,113.78,108.63,108.59,103.90,103.37,102.36,93.64. C 30 H 19 O 11 The calculated HRMS (ESI-TOF) value is 553.0776, and the measured value is 555.0732.

[0361] 6-(2,3-dihydroxy-6-(4-oxo-4H-chromen-2-yl)phenyl)-2-(3,4-dihydroxyphenyl)-5,7-di Hydroxy-4H-chromen-4-one (34-Lu(2′-6),34:3′,4′-dihydroxyflavone, 2k)

[0362]

[0363] 2k was prepared from 3′,4′-dihydroxyflavone. 67 mg, 50% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300 nm), t r (2k) = 18.289 min, 2k concentration = 3.25 mM. 1H NMR (400MHz, DMSO-d6) δ13.14(s,1H),9.69(s,5H),7.92(d,J=7.9Hz,1H),7.68(d,J=7.9Hz,1H),7.47–7.35(m,3H) ,7.25(dd,J=16.9,8.3Hz,2H),6.97(d,J=8.3Hz,1H),6.90(d,J=8.3Hz,1H),6.68(s,1H),6.54(s,1H),6.13(s,1H). 13 C NMR (126MHz, DMSO) δ182.14,177.05,166.20,164.14,162.64,159.31,156.78,156.17,150.17,148.62,146.22,144.82,134.46,125.6 6,125.15,124.73,123.41,121.95,120.57,120.28,119.48,118.23,116.53,114.75,113.77,108.98,108.61,103.80,103.30,93.71. C 30 H 17 O 10 The calculated HRMS (ESI-TOF) value is 537.0827, and the measured value is 537.0818.

[0364] 6-(2,3-dihydroxy-6-(5-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-5,7-dihydroxy-2-(4-hydroxy) (534-Ap(2′-6),2l)-4H-chromen-4-one

[0365]

[0366] 2L was prepared from Ap. 73 mg, 54% yield, brown solid. HPLC (ACN / water = 64:36, λ = 300 nm), t r (2l) = 21.513 min, the concentration of 2l = 15.80 mM. 1 H NMR(500MHz,DMSO-d6)δ13.19(s,1H),12.63(s,1H),10.81(s,1H),10.40(s,1H),10.26(s,1H),8.55(s,1H),7.94(d,J =8.0Hz,2H),7.49(d,J=10.1Hz,1H),7.27(d,J=8.0Hz,1H),6.98(m,3H),6.80(s,1H),6.73–6.55(m,3H),6.21(s,1H). 13C NMR (126MHz, DMSO) δ182.94,182.28,167.85,164.11,162.35,161.66,160.25,159.31,156.88,156.37,149.11,144.85,136.0 8,128.96,124.09,121.62,121.03,120.38,116.48,114.90,111.09,110.10,108.39,107.41,107.18,103.96,103.32,93.85. C 30 H 17 O 10 The calculated HRMS (ESI-TOF) value is 537.0827, and the measured value is 537.0821.

[0367] 6-(2,3-dihydroxy-6-(6-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-5,7-dihydroxy-2-(4-hydroxy) (634-Ap(2′-6), 2m)-4H-chromen-4-one

[0368]

[0369] 2m was prepared from Ap. 82 mg, 61% yield, brown solid. HPLC (ACN / water = 72.5:27.5, λ = 300 nm), t r (2m) = 14.506 min, the concentration at 2m = 13.71 mM. 1 H NMR(500MHz,DMSO-d6)δ13.14(s,1H),9.99(s,5H),7.95(d,J=8.2Hz,2H),7.25–7 .09(m,4H),6.97(dd,J=12.2,8.2Hz,3H),6.79(s,1H),6.60(s,1H),6.08(s,1H). 13 C NMR (126MHz, DMSO) δ182.29,177.02,165.76,164.10,162.51,161.66,159.34,156.84,155.09,149.96,148.42,144.75,129.0 0,125.09,124.32,123.31,121.69,120.49,120.23,119.60,116.51,114.83,108.65,108.13,107.86,103.92,103.35,93.83. C 30 H 17 O 10 The calculated HRMS (ESI-TOF) value is 537.0827, and the measured value is 537.0821.

[0370] 8-(2,3-dihydroxy-6-(6-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-5,7-dihydroxy-2-(4-hydroxy) (634-Ap(2′-8), 2m′)-4H-chromen-4-one

[0371]

[0372] 2m′ was prepared from Ap. 4 mg, 3% yield, brown solid. HPLC (ACN / water = 72.5:27.5, λ = 300 nm), t r (2m′) = 12.177 min, the concentration of 2m′ = 2.32 mM. 1 H NMR (500MHz, DMSO-d6) δ12.98(s,1H),9.94(s,5H),7.52(d,J=8.5Hz,2H),7.25(d,J=8.2Hz,1H),7.16(d,J=3.0 Hz,1H),7.10–7.01(m,2H),6.87(d,J=8.2Hz,1H),6.79(d,J=8.5Hz,2H),6.71(s,1H),6.26(s,1H),5.99(s,1H). 13 C NMR (126MHz, DMSO) δ182.38,176.80,165.90,163.89,161.52,160.85,155.03,154.73,149.67,148.73,145.16,128.61,1 25.16,124.17,123.18,121.69,120.64,119.44,119.23,116.23,114.96,108.09,107.79,103.94,103.79,102.83,99.07. C 30 H 17 O 10 The calculated HRMS (ESI-TOF) value is 537.0827, and the measured value is 537.0818.

[0373] 6-(2,3-dihydroxy-6-(7-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-5,7-dihydroxy-2-(4-hydroxy) (734-Ap(2′-6), 2n)-4H-chromen-4-one

[0374]

[0375] 2n was prepared from Ap. 65 mg, 48% yield, brown solid. HPLC (ACN / water = 72.5:27.5, λ = 300 nm), t r (2n) = 14.399 min, the concentration of 2n = 22.53 mM. 1H NMR (500MHz, DMSO-d6) δ13.13(s,1H),10.32(s,5H),7.96(d,J=8.5Hz,2H),7.75(d,J=8.5Hz,1H),7. 18(d,J=8.2Hz,1H),7.03–6.87(m,3H),6.82(d,J=8.2Hz,2H),6.58(s,1H),6.50(s,1H),6.02(s,1H). 13 C NMR (126MHz, DMSO) δ182.28,176.51,165.49,164.02,162.85,162.41,161.62,159.28,157.97,156.77,148.31,144.68,128.9 7,126.80,124.92,121.68,120.41,120.13,116.46,116.22,115.19,114.74,108.65,108.59,103.88,103.34,102.37,93.76. C 30 H 17 O 10 The calculated HRMS (ESI-TOF) value is 537.0827, and the measured value is 537.0824.

[0376] 8-(2,3-dihydroxy-6-(7-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-5,7-dihydroxy-2-(4-hydroxy) (734-Ap(2′-8), 2n′)-4H-chromen-4-one

[0377]

[0378] 2n′ was prepared from Ap. 4 mg, 3% yield, brown solid. HPLC (ACN / water = 72.5:27.5, λ = 300 nm), t r (2n′) = 12.139 min, and the concentration of 2n′ = 3.34 mM. 1 H NMR (500MHz, DMSO-d6) δ12.98(s,1H),10.42(s,5H),7.70(d,J=8.5Hz,1H),7.52(d,J=8.5Hz,2H),7.23(d,J=8.3H z, 1H), 7.02 (d, J = 8.3Hz, 1H), 6.78 (d, J = 8.5Hz, 3H), 6.72 (s, 1H), 6.26 (d, J = 8.5Hz, 1H), 6.25 (s, 1H), 5.95 (s, 1H). 13C NMR (126MHz, DMSO) δ182.38,176.36,165.70,163.88,162.78,161.50,160.81,157.78,154.71,148.63,145.08,128.59,1 26.76,125.13,121.71,120.66,119.37,116.22,116.08,115.16,114.93,108.68,103.93,103.80,102.85,102.13,99.03. C 30 H 17 O 10 The calculated HRMS (ESI-TOF) value is 537.0827, and the measured value is 537.0822.

[0379] 6-(2,3-dihydroxy-6-(4-oxo-4H-chromen-2-yl)phenyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)- 4H-chromene-4-one (34-Ap(2′-6), 2o)

[0380]

[0381] 2o was prepared from Ap. 60 mg, 46% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300 nm), t r (2o) = 31.427 min, 2o concentration = 10.06 mM. 1 H NMR (500MHz, DMSO-d6) δ13.16(s,1H),10.36(s,4H),7.93(t,J=8.6Hz,3H),7.68(t,J=8.0Hz,1H),7.42–7.32(m,1H),7.32–7 .18(m,2H),6.99(d,J=8.3Hz,1H),6.95(d,J=8.3Hz,2H),6.79(d,J=3.7Hz,1H),6.60(d,J=3.7Hz,1H),6.15(d,J=3.7Hz,1H). 13 C NMR (126MHz, DMSO) δ182.26,177.04,166.14,164.06,162.44,161.65,159.33,156.81,156.18,148.58,144.76,134.42,128.9 7,125.63,125.14,124.78,123.41,121.64,120.59,120.27,118.24,116.46,114.83,109.04,108.56,103.91,103.32,93.81. C 30 H 17The calculated HRMS (ESI-TOF) value for O9 is 521.0827, and the measured value is 521.0871.

[0382] Example 8. Reaction of luteolin with trihydroxyflavone (TFL) bearing a catechol group on ring B.

[0383] 2h-2o was prepared from one of 1a-1b (1 equivalent) and one of 1h-1k (1.5 equivalent) according to the scheme in Example 1.

[0384] 2h

[0385]

[0386] 2h was prepared from 1a and 1h. 43% yield. Semi-preparative HPLC (DI water containing 0.1% formic acid / ACN containing 0.1% formic acid = 64:36), t r (2h) = 14.273min, t r (2h′)=11.629min.

[0387] 2i

[0388]

[0389] 2i was prepared from 1a and 1i. 37% yield. Semi-preparative HPLC (DI water containing 0.1% formic acid / ACN containing 0.1% formic acid = 72.5:27.5), t r (2i) = 9.744 min.

[0390] 2j

[0391]

[0392] 2j was prepared from 1a and 1j. 43% yield. Semi-preparative HPLC (DI water containing 0.1% formic acid / ACN containing 0.1% formic acid = 72.5:27.5), t r (2j) = 9.707 min.

[0393] 2k

[0394]

[0395] 2k was prepared from 1a and 1k. 45% yield. Semi-preparative HPLC (DI water containing 0.1% formic acid / ACN containing 0.1% formic acid = 71.5:28.5), t r (2k) = 21.725min.

[0396] 2l

[0397]

[0398] 2l was prepared from 1h and 1b. 74% yield. Semi-preparative HPLC (DI water containing 0.1% formic acid / ACN containing 0.1% formic acid = 64:36), t r (2l) = 17.840 min, t r (2l′)=16.807min.

[0399] 2m

[0400]

[0401] 2m was prepared from 1i and 1b. 86% yield. Semi-preparative HPLC (DI water containing 0.1% formic acid / ACN containing 0.1% formic acid = 72.5:27.5), t r (2m) = 14.695min, t r (2m′)=12.509min.

[0402] 2n

[0403]

[0404] 2n was prepared from 1j and 1b. 86% yield. Semi-preparative HPLC (DI water containing 0.1% formic acid / ACN containing 0.1% formic acid = 72.5:27.5), t r (2n) = 13.003 min, t r (2n′)=11.015min.

[0405] 2o

[0406]

[0407] 2o was prepared from 1k and 1b. 86% yield. Semi-preparative HPLC (DI water containing 0.1% formic acid / ACN containing 0.1% formic acid = 71.5:28.5), t r (2o) = 30.602 min, t r (2o′)=28.932min.

[0408] Results and Discussion

[0409] When luteolin is mixed with TFL (1h-1j) and 3′,4′-dihydroxyflavone (1k,DFL) with catechol groups on the B ring, luteolin becomes a nucleophile, and 1h-1k is a free radical precursor, producing the corresponding biflavonoids FL-(2′-6)-Lu(2h-2k, FL = TFL and DFL). Figure 10 A). Unsurprisingly, 1h-1k coupled with other nucleophilic flavonoids such as Ap to form FL-(2′-6)-Ap(2l,2m,2n,2o) as the only product. These results expand our range of reactions for various biflavonoids containing two different monoflavonoids. Many other viable combinations of flavonoids and flavonoid glycosides exist that can satisfy this simple requirement.

[0410] Example 9. Synthesis of heterotriflavonoids

[0411]

[0412] According to the scheme in Example 1, 3b-3k was prepared from 2a (143 mg, 0.25 mmol) and flavonoids selected from the following (0.375 mmol): Ap, Dio, Chry, 5,6-dihydroxyflavone, Ge, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone and 3′,4′-dihydroxyflavone.

[0413] 2-(2-(5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromene-6-yl)-3,4-dihydroxyphenyl)- 6-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one (Lu2-Ap(2′-6), 3b)

[0414]

[0415] 3b was prepared from Ap. 109 mg, 52% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300 nm), t r (3b*) = 32.142 min, tr(3b**) = 33.539 min, 3b concentration = 0.89 mM. 3b = 3b* + 3b**. Restricted transisomer 3b*: 1¹H NMR (500MHz, DMSO-d⁶) δ 7.90 (d, J = 8.9, 2H), 7.22 (d, J = 8.4Hz, 1H), 7.15 (d, J = 8.4Hz, 1H), 6.97 (d, J = 8.5Hz, 1H), 6.94 (d, J = 8.9Hz, 2H), 6.92 (d, J = 8.5Hz, 1H), 6.69 (s, 1H), 6.57 (s, 1H), 6.21 (s, 1H), 6.09 (d, J = 7.8Hz, 1H), 6.01 (s, 1H), 5.98 (d, J = 7.8Hz, 1H), 5.97 (s, 1H). Resistive isomer 3b**: 1 H NMR (500MHz, DMSO-d6) δ7.90(d,J=8.9Hz,2H),7.22(d,J=8.4,1H),7.15(d,J=8.4Hz,1H),6.97(d,J=8.5Hz,1H),6.94(d,J=8.9Hz,2H) ,6.92(d,J=8.5Hz,1H),6.68(s,1H),6.55(s,1H),6.16(s,1H),6.09(d,J=7.8Hz,1H),5.99(s,1H),5.98(d,J=7.8Hz,1H),5.96(s,1H). 13 C NMR (126MHz, DMSO) δ182.34,181.82,181.69,166.65,166.56,164.53,164 .07,162.21,161.76,161.57,159.35,159.09,157.88,156.93,148.82,144 .74,128.98,124.18,121.77,120.63,120.15,116.42,114.78,108.41,108 .25,106.73,103.97,103.83,103.65,103.45,99.20,94.03,93.86,93.78. C 45 H 25 O 17 The calculated HRMS (ESI-TOF) value is 837.1090, and the measured value is 837.1097.

[0416]

[0417] Crystals of compound 3b suitable for X-ray analysis were obtained by slow evaporation from MeOH. C3b crystals with dimensions approximately 0.061 mm x 0.063 mm x 0.267 mm were prepared. 36 H 32 O 14A brown, blocky sample was used for X-ray crystallography analysis. X-ray intensity data were measured. The total exposure time was 19.00 hours. Data were integrated using a triclinic crystal cell, resulting in a total of 18,025 reflections, with a maximum θ angle of 67.11°. (resolution), of which 5625 reflections are independent (average redundancy 3.204, integrity = 99.0%, R int =6.56%, R sig =6.46%), 3788 times (67.34%) greater than 2σ(F) 2 The following final cell constants are based on a refinement of the XYZ centroids for 49 reflections above 20σ(I), where 8.533° < 2θ < 40.75°: α=98.306°, β=95.037°, γ=109.024°, The ratio of minimum apparent transmittance to maximum apparent transmittance is 0.883. Using the Bruker SHELXTL software package, where the unit C is... 36 H 32 O 14 Given Z=2, solve and refine the structure. For the observed data, F has 491 variables. 2 The final anisotropic full-matrix least-squares refinement converges to R1 = 4.71%, and for all data, wR2 = 12.43%. The goodness of fit is 1.029. The maximum peak in the final differential electron density synthesis is... The largest cavity is Where the RMS deviation is Based on the final model, the calculated density is 1.434 g / cm³. 3 And F(000), 720e - The crystallographic data has been stored at the Cambridge Crystallography Data Centre.

[0418] 2-(2-(5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4-oxo-4H-chromene-6-yl)-3,4-di (hydroxyphenyl)-6-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy- 4H-chromen-4-one (Lu2-Dio(2′-6),3c)

[0419]

[0420] 3c was prepared from Dio. 100 mg, 46% yield, brown solid. HPLC (ACN / water = 64:36, λ = 300 nm), t r (3c*) = 10.787 min, t r (3c**) = 10.787 min, 3c concentration = 0.50 mM. 3c = 3c* + 3c**. Restricted transisomer 3c*: 11H NMR (500 MHz, DMSO-d6) δ 13.19 (s, 1H), 13.00 (s, 1H), 12.79 (s, 1H), 10.76 (s, 4H), 10.16 (s, 2H), 8.44 (s, 2H), 7.62 (d, J = 8.6 Hz, 1H), 7.46 (m, 1H), 7.26 (d, J = 8.4, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 8.6 Hz, 1H), 6.98 (d, J = 8.5, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.78 (s, 1H), 6.60 (s, 1H), 6.28 (s, 1H), 6.12 (d, J = 8.1 Hz, 1H), 6.09 (s, 1H), 6.02 (d, J = 8.1 Hz, 1H), 5.96 (s, 1H), 3.87 (s, 3H). Atropisomer 3c**: 1 1H NMR (500 MHz, DMSO-d6) δ 13.16 (s, 1H), 12.96 (s, 1H), 12.76 (s, 1H), 10.76 (s, 4H), 10.16 (s, 2H), 8.44 (s, 2H), 7.62 (d, J = 8.6 Hz, 1H), 7.46 (m, 1H), 7.26 (d, J = 8.4, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 8.6 Hz, 1H), 6.98 (d, J = 8.5, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.78 (s, 1H), 6.56 (s, 1H), 6.12 (d, J = 8.1 Hz, 3H), 6.09 (s, 1H), 6.02 (d, J = 8.1 Hz, 1H), 5.97 (s, 1H), 5.95 (s, 1H), 3.87 (s, 3H). 13CNMR(126MHz,DMSO)δ182.33,182.26,182.18,181.86,181.75,181.69,181.63,166.68,166.63 ,166.58,166.54,166.28,164.67,164.53,164.48,164.01,163.87,163.83,162.27,162.21,16 2.07, 161.95, 161.77, 159.38, 159.32, 159.09, 157.89, 157.85, 157.80, 157.10, 156.93, 156.83, 151.61, 151.58, 151.55, 148.85, 148.79, 148.70, 147.26, 147.20, 144.73, 144.69, 144.63, 12 4.19, 124.06, 123.60, 123.57, 123.48, 120.83, 120.65, 120.19, 120.14, 120.10, 119.24, 119.20, 114.82, 114.74, 113.48, 113.42, 113.37, 112.65, 112.61, 112.55, 108.45, 108.32, 108.25, 10 8.18,106.84,106.76,106.66,104.24,104.13,104.06,104.00,103.95,103.86,103.82,103.6 8,103.62,99.36,99.31,99.20,94.40,94.35,93.90,93.85,93.80,93.47,93.29,56.22,56.17. C 46 H 27 O 18 The calculated HRMS (ESI-TOF) value is 867.1203, and the measured value is 867.1202.

[0421] 2-(2-(5,7-dihydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)-3,4-dihydroxyphenyl)-6-(6-(5, 7-Dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one (Lu2- Chry(2′-6), 3d)

[0422]

[0423] 3d was prepared by Chrysogen. 82 mg, 40% yield, brown solid. HPLC (ACN / water = 64:36, λ = 300 nm), t r (3d*) = 9.863 min, t r (3d**) = 9.863 min, 3d concentration = 2.13 mM. 3d = 3d* + 3d**. Restricted transisomer 3d*:1 ¹H NMR (500MHz, DMSO-d⁶) δ 8.07–8.00 (m, 2H), 7.65–7.53 (m, 3H), 7.23 (d, J = 8.4, Hz, 1H), 7.14 (dd, J = 8.3, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.85 (s, 1H), 6.65 (s, 1H), 6.24 (s, 1H), 6.11 (d, J = 6.1 Hz, 1H), 6.06 (s, 1H), 6.02 (d, J = 6.1 Hz, 1H), 5.96 (s, 1H). 3d** of the resisted isomer: 1 H NMR (500MHz, DMSO-d6) δ8.07–8.00(m,2H),7.65–7.53(m,3H),7.23(d,J=8.4Hz,1H),7.14(d,J=8.3Hz,1H),6.99(d,J=8.4Hz,1H),6 .93(d,J=8.3Hz,1H),6.85(s,1H),6.63(s,1H),6.16(s,1H),6.11(d,J=6.1Hz,1H),6.02(s,1H),6.01(d,J=6.1Hz,1H),5.94(s,1H). 13 C NMR (126MHz, DMSO) δ182.48,181.85,181.69,181.67,166.67,166.62,166.32,164.58,163.57,163. 52,162.20,161.75,159.32,159.08,159.04,157.89,157.08,156.98,156.93,148.79,148.70,144. 72,144.62,132.44,131.28,129.62,126.93,124.17,120.64,120.12,120.08,114.87,114.77,108. 68,108.54,108.26,108.20,106.73,105.77,104.23,103.82,103.79,103.65,99.22,93.89,93.29. C 45 H 25 O 16 The calculated HRMS (ESI-TOF) value is 821.1148, and the measured value is 821.1135.

[0424] 6-(6-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy (-4-oxo-4H-chromene-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-8-methoxy-2-phenyl-4H-chromene-4- Ketone (Lu2-Wo(2′-6), 3e)

[0425]

[0426] 3e was prepared from Wo. 115 mg, 54% yield, brown solid. HPLC (ACN / water = 64:36, λ = 300 nm), t r (3e*) = 21.393 min, t r (3e**) = 22.891 min, 3e concentration = 1.90 mM. 3e = 3e* + 3e**. Restricted transisomer 3e*: 1 H NMR(500MHz,DMSO-d6)δ12.96(s,1H),12.79(s,1H),12.75(s,1H),10.77(s,1H),10.63(s,1H ),10.31(s,1H),10.26(s,1H),8.51(s,1H),8.38(s,1H),8.09(d,J=7.7Hz,2H),7.68–7.56(m ,3H), 7.26(d,J=8.4,1H), 7.14(d,J=8.4Hz,1H), 7.04(s,1H), 6.98(d,J=8.4,1H), 6.91(d,J=8.4,1H), 6.15(s,1H), 6.12(d,J=2.1Hz,1H), 6.04(d,J=2.1Hz,1H), 6.01(s,1H), 5.95(s,1H). Resistive isomer 3e**: 1 H NMR(500MHz,DMSO-d6)δ12.96(s,1H),12.77(s,1H),12.74(s,1H),10.77(s,1H),10.57(s,1H ),10.27(s,1H),10.12(s,1H),8.51(s,1H),8.35(s,1H),8.09(d,J=7.7Hz,2H),7.68–7.56(m ,3H),7.26(d,J=8.4,1H),7.14(d,J=8.4Hz,1H),7.04(s,1H),6.98(d,J=8.4,1H),6.91(d,J= 8.4,1H),6.11(s,1H),6.08(d,J=2.1Hz,1H),5.97(s,1H),5.95(d,J=2.1Hz,1H),5.92(s,1H). 13C NMR (126MHz, DMSO) δ182.69,182.59,181.90,181.78,181.68,181.64,166.70,166.59,166.36,164.47,163.33,162.21,162 .12,161.76,159.12,159.00,157.92,157.84,157.01,156.78,155.40,154.58,149.11,148.88,148.68,144.76,144.62,13 2.52,131.41,129.75,127.99,127.87,126.78,124.30,124.15,124.01,123.93,120.74,120.58,120.14,119.80,114.91,1 14.70,108.86,108.74,108.20,106.83,106.71,105.77,103.90,103.81,103.64,99.16,93.93,93.80,93.38,93.24,61.97. C 46 H 27 O 17 The calculated HRMS (ESI-TOF) value is 851.1254, and the measured value is 851.1248.

[0427]

[0428] Crystals of compound 3e suitable for X-ray analysis were obtained by slow evaporation from MeOH and DMSO. Crystals of C with dimensions approximately 0.049 mm x 0.122 mm x 0.132 mm were prepared. 35 H 26 N2O 11 The samples were used for X-ray crystallographic analysis. X-ray intensity data were measured. The total exposure time was 17.13 hours. Integrating the data using a triclinic crystal cell yielded a total of 8055 reflections, with a maximum θ angle of 67.03°. (resolution), of which 8055 reflections are independent (average redundancy 1.000, integrity = 97.6%, R sig =10.51%), 4577 times (56.82%) greater than 2σ(F) 2 The following final cell constants are based on a refinement of the XYZ centroids for reflections above 20σ(I) at 4286, where 8.870° < 2θ < 133.1°: α=102.701(4)°, β=102.737(4)°, γ=90.020(4)°, The ratio of minimum apparent transmittance to maximum apparent transmittance is 0.637. The calculated minimum and maximum transmittance (based on crystal size) are 0.4799 and 0.7528, respectively. The Bruker SHELXTL software package was used, with space group P-1, where for the formula unit C... 35 H 26 N2O 11 Given Z=2, solve and refine the structure. For the observed data, F has 455 variables. 2 The final anisotropic full-matrix least-squares refinement converges to R1 = 9.67%, and for all data, wR2 = 32.15%. The goodness of fit is 1.065. The maximum peak in the final differential electron density synthesis is... The largest cavity is Where the RMS deviation is Based on the final model, the calculated density is 1.475 g / cm³. 3 And F(000), 676e - The crystallographic data has been stored at the Cambridge Crystallography Data Centre.

[0429] 2-(2-(5,6-dihydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)-3,4-dihydroxyphenyl)-6-(6-(5, (7-Dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one (Lu2-56) (2′-6)，3f)

[0430]

[0431] 3f was prepared from 5,6-dihydroxyflavone. 154 mg, 75% yield, brown solid. HPLC (ACN / water = 64:36, λ = 300 nm), t r (3f*)=9.731min,t r (3f**) = 9.731 min, 3f concentration = 3.50 mM. 3f = 3f* + 3f**. Restricted transisomer 3f*: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.67 (d, J = 7.1Hz, 1H), 7.59 (d, J = 7.1Hz, 1H), 7.50 (dt, J = 7.1, 3.8Hz, 1H), 7.46–7.39 (m, 2H), 7.38 (d, J = 6.9Hz, 1H), 7.33 (d, J = 7.1Hz, 1H), 7.24 (s, 1H), 7.07 (s, 1H), 6.90 (d, J = 3.8Hz, 1H), 6.70 (s, 2H), 6.60 (d, J = 6.9Hz, 1H), 6.44 (d, J = 7.1Hz, 1H), 6.37 (s, 1H), 5.89 (s, 1H). Resistive isomer 3f**: 1H NMR (400MHz, DMSO-d6) δ7.67(d,J=7.1Hz,1H),7.59(d,J=7.1Hz,1H),7.50(dt,J=7.1,3.8Hz,1H),7.46–7.39(m,2H),7.38(d,J=6.9Hz,1H),7.33(d, J=7.1Hz,1H),7.34(s,1H),6.93(s,1H),6.90(d,J=3.8Hz,1H),6.70(s,2H ), 6.60 (d, J = 6.9Hz, 1H), 6.44 (d, J = 7.1Hz, 1H), 6.43 (s, 1H), 5.81 (s, 1H). 13 C NMR (126MHz, DMSO) δ184.34,184.25,182.02,181.89,181.84,166.66,166.04,16 4.17,164.08,164.00,163.95,162.39,162.31,162.26,161.65,161.30,159.52, 158.94,156.81,156.70,155.37,155.31,150.17,150.11,148.78,148.75,146.74,146.68,146.49,146.45,146.16,144.66,144.62,141.05,140.92,132.66,132. 57,131.22,129.61,129.53,126.44,126.41,126.32,126.12,124.09,123.87,121.97,121.92,120.21,120.18,119.92,119.49,119.41,116.48,114.37,113.78, 111.13,111.08,110.67,110.56,107.74,107.47,107.00,106.81,104.88,103.9 7,103.93,103.84,103.78,103.41,103.29,102.23,102.17,98.99,93.80,93.48. C 45 H 25 O 16 The calculated HRMS (ESI-TOF) value is 821.1148, and the measured value is 821.1133.

[0432] 2-(2-(5,7-dihydroxy-3-(4-hydroxyphenyl)-4-oxo-4H-chromene-6-yl)-3,4-dihydroxyphenyl)- 6-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,3-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one (Lu2-Ge(2′-6), 3g)

[0433]

[0434] 3g prepared from Ge. 103mg, 49% yield, brown solid. HPLC (ACN / water = 64:36, λ = 300nm), t r (3g*) = 10.218 min,t r (3g**) = 11.126 min, concentration of 3g = 3.88 mM. 3g = 3g* + 3g**. Restricted transisomer 3g*: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 8.31 (s, 1H), 7.39 (d, J = 8.2Hz, 2H), 7.25 (d, J = 8.4Hz, 1H), 7.18 (d, J = 8.3Hz, 1H), 6.98 (d, J = 8.4Hz, 1H), 6.94 (d, J = 8.3Hz, 1H), 6.83 (d, J = 8.2Hz, 2H), 6.52 (s, 1H), 6.28 (s, 1H), 6.13 (d, J = 2.1Hz, 1H), 6.09 (s, 1H), 6.04 (d, J = 2.1Hz, 1H), 5.96 (s, 1H). Resistive transisomer 3g**: 1 H NMR (400MHz, DMSO-d6) δ8.34(s,1H),7.42(d,J=8.4Hz,2H),7.25(d,J=8.2Hz,1H),7.17(d,J=8.3Hz,1H),6.97(d,J=8.2Hz,1H),6.93(d ,J=8.3Hz,1H),6.83(d,J=8.2Hz,2H),6.48(s,1H),6.28(s,1H),6.11(d,J=2.1Hz,1H),6.09(s,1H),6.01(d,J=2.1Hz,1H),5.97(s,1H). 13C NMR (126MHz, DMSO) δ181.86,181.75,181.68,180.85,180.77,166.67,166.62,166.60,166.32,164.51,164.49,162.42,162.21,162.17,161.78 ,161.75,159.86,159.75,159.10,157.90,157.85,157.83,157.17,157. 09,157.06,156.91,154.27,148.82,148.77,148.71,148.68,144.70,14 4.66,144.64,144.61,130.73,130.70,130.63,124.23,124.17,124.11,122.84,122.80,121.86,121.78,120.66,120.18,120.12,115.52,115.49,114.83,108.51,108.38,108.22,106.89,106.79,106.73,104.77,104.74,103.86,103.83,103.68,99.19,93.89,93.84,93.62,93.48,93.27. C 45 H 25 O 17 The calculated HRMS (ESI-TOF) value is 837.1097, and the measured value is 837.1087.

[0435] 6-(2,3-dihydroxy-6-(5-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-2-(2-(2-(3,4-dihydroxy) (phenyl)-5,7-dihydroxy-4-oxo-4H-chromen-6-yl)-3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one (534-Lu2(2′-6), 3h)

[0436]

[0437] Prepared from 5,3′,4′-trihydroxyflavone over 3 hours. 88 mg, 42% yield, brown solid. HPLC (ACN / water = 64:36, λ = 300 nm), t r (3h*)=11.579min,t r (3h**) = 12.618 min, concentration at 3h = 3.43 mM. 3h = 3h* + 3h**. Restricted transisomer 3h*: 11H NMR (500 MHz, DMSO-d6) δ 7.46 (td, J = 8.3, 3.8 Hz, 1H), 7.44–7.37 (m, 2H), 7.22 (d, J = 8.3 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.67 (d, J = 3.8 Hz, 1H), 6.61 (s, 1H), 6.58 (s, 1H), 6.52 (d, J = 8.3 Hz, 1H), 6.24 (s, 1H), 6.15 (s, 1H), 6.08 (s, 1H). Atropisomer 3h**: 1 1H NMR (500 MHz, DMSO-d6) δ 7.46 (td, J = 8.3, 3.8 Hz, 1H), 7.44–7.37 (m, 2H), 7.22 (d, J = 8.3 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.67 (d, J = 8.1 Hz, 1H), 6.60 (s, 1H), 6.57 (s, 1H), 6.46 (d, J = 8.1 Hz, 1H), 6.19 (s, 1H), 6.15 (s, 1H), 6.02 (s, 1H). 13C NMR (126MHz, DMSO) δ182.91,182.86,182.25,182.21,181.84,181.74,167. 83,166.72,166.46,164.20,164.16,162.25,162.19,162.05,160.20,159. 27,159.05,159.00,157.07,156.91,156.85,156.77,156.24,156.19,150.13,150.08,149.05,148.81,148.73,146.18,144.72,144.68,136.03,135. 97,124.27,124.05,123.87,123.80,122.05,122.00,121.06,120.90,120.67,120.27,120.21,120.17,119.47,116.52,114.82,113.79,111.10,110. 08,110.03,108.42,108.34,108.27,108.22,107.21,107.13,107.05,106. 97,106.85,106.60,103.95,103.65,103.62,103.44,93.73,93.37,93.24. C 45 H 25 O 17 The calculated HRMS (ESI-TOF) value is 837.1097, and the measured value is 837.1091.

[0438] 6-(2,3-dihydroxy-6-(6-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-2-(2-(2-(3,4-dihydroxy) (phenyl)-5,7-dihydroxy-4-oxo-4H-chromen-6-yl)-3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one (634-Lu2(2′-6), 3i)

[0439]

[0440] 3i was prepared from 6,3′,4′-trihydroxyflavone. 85 mg, 41% yield, brown solid. HPLC (ACN / water = 72.5:27.5, λ = 300 nm), t r (3i*) = 10.218 min,t r (3i**) = 11.126 min, 3i concentration = 3.73 mM. 3i = 3i* + 3i**. Restricted transisomer 3i*: 1¹H NMR (400MHz, DMSO-d⁶) δ 7.44 (d, J = 8.5Hz, 2H), 7.24 (d, J = 8.4, 1H), 7.21 (d, J = 8.3, 1H), 7.17–7.03 (m, 3H), 6.96 (d, J = 8.4, 1H), 6.93 (d, J = 8.3, 1H), 6.90 (d, J = 8.5Hz, 1H), 6.70 (s, 1H), 6.56 (s, 1H), 6.24 (s, 1H), 6.02 (s, 1H), 5.93 (s, 1H). Resistive isomer 3i**: 1 H NMR (400MHz, DMSO-d6) δ7.44(d,J=8.5Hz,2H),7.24(d,J=8.4,11H),7.21(d,J=8.3,1H),7.17–7.03(m,3H),6.96(d,J= 8.4,1H),6.93(d,J=8.3,1H),6.90(d,J=8.5Hz,1H),6.70(s,1H),6.54(s,1H),6.09(s,1H),6.05(s,1H),5.97(s,1H). 13 C NMR (126MHz, DMSO) δ182.28,182.23,181.80,181.72,176.92,176.89,166 .52,166.31,165.61,164.24,164.19,162.33,162.17,161.97,159.36,15 9.32,159.10,159.06,157.03,156.89,156.80,155.05,150.13,150.09,1 49.87,149.83,148.83,148.77,148.30,146.19,144.71,144.68,144.58, 124.91, 124.88, 124.28, 124.23, 124.18, 124.02, 123.25, 122.09, 122.06, 120.81, 120.65, 120.41, 120.19, 120.08, 120.04, 119.50, 119.44, 116.5 2,114.81,113.87,108.50,108.39,108.28,107.97,107.92,107.82,107. 79,106.76,106.58,103.99,103.62,103.48,93.80,93.74,93.38,93.26. C 45 H 25 O 17The calculated HRMS (ESI-TOF) value is 837.1097, and the measured value is 837.1091.

[0441] 6-(2,3-dihydroxy-6-(7-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-2-(2-(2-(3,4-dihydroxy) (phenyl)-5,7-dihydroxy-4-oxo-4H-chromen-6-yl)-3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one (734-Lu2(2′-6), 3j)

[0442]

[0443] 3j was prepared from 7,3′,4′-trihydroxyflavone. 98 mg, 47% yield, brown solid. HPLC (ACN / water = 72.5:27.5, λ = 300 nm), t r (3j*)=13.331min,t r (3j**) = 13.331 min, concentration of 3j = 2.54 mM. 3j = 3j* + 3j**. Restricted transisomer 3j*: 1 H NMR(400MHz,DMSO-d6)δ7.73(td,J=8.7,6.5Hz,1H),7.49(d,J=8.1Hz,2H),7.2 4(d,J=8.4Hz,1H),7.14(d,J=8.3Hz,1H),6.97(d,J=8.4Hz,1H),6.93(d,J=8.3H z,1H),6.92(d,J=8.1Hz,1H),6.81(dd,J=8.7,6.5Hz,1H),6.71(s,1H),6.56(s, 1H), 6.52 (d, J = 6.5Hz, 1H), 6.27 (s, 1H), 6.05 (s, 1H), 5.90 (s, 1H), 3.18 (s, 1H). Transisomer 3j**: 1 H NMR(400MHz,DMSO-d6)δ7.73(td,J=8.7,6.5Hz,1H),7.49(d,J=8.1Hz,2H),7.2 4(d,J=8.4Hz,1H),7.14(d,J=8.3Hz,1H),6.97(d,J=8.4Hz,1H),6.93(d,J=8.3H z,1H),6.92(d,J=8.1Hz,1H),6.81(dd,J=8.8,6.5Hz,1H),6.70(s,1H),6.54(s, 1H), 6.52 (d, J = 6.5Hz, 1H), 6.09 (s, 1H), 5.93 (s, 1H), 5.92 (s, 1H), 3.18 (s, 1H). 13C NMR (126MHz, DMSO) δ206.96,182.26,182.19,181.80,181.71,176.37,166.50,166.20,165.23,165.15,164.20,164.16,162.87,162.28,162. 15,161.95,159.36,159.31,159.11,157.97,157.04,156.90,156.86, 156.77,150.11,150.07,148.80,148.74,148.22,148.18,146.18,144. 68,144.64,144.52,126.76,126.74,124.88,124.20,124.08,122.08, 122.04,120.27,120.21,120.06,120.02,119.47,116.53,116.18,116. 14,115.19,114.84,113.89,108.57,108.40,108.32,108.21,106.83, 106.66,103.99,103.96,103.64,103.45,102.41,93.85,93.73,93.30. C 45 H 25 O 17 The calculated HRMS (ESI-TOF) value is 837.1097, and the measured value is 837.1086.

[0444] 6-(2,3-dihydroxy-6-(7-hydroxy-4-oxo-4H-chromen-2-yl)phenyl)-2-(2-(2-(3,4-dihydroxy) (phenyl)-5,7-dihydroxy-4-oxo-4H-chromen-6-yl)-3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one (734-Lu-Ap(2′-6), 3k)

[0445]

[0446] 3k was prepared from 7,3′,4′-trihydroxyflavone. 88 mg, 43% yield, brown solid. HPLC (ACN / water = 71.5:28.5, λ = 300 nm), t r (3k) = 15.699 min, 3k concentration = 0.76 mM. 3k = 3k* + 3k**. Restricted transisomer 3k*: 1¹H NMR (500MHz, DMSO-d⁶) δ 13.20 (d, J = 13.1Hz, 1H), 12.94 (d, J = 20.1Hz, 1H), 10.37 (s, 8H), 7.94 (d, J = 8.4Hz, 2H), 7.71 (d, J = 8.4Hz, 1H), 7.22 (d, J = 8.8Hz, 1H), 7.13 (d, J = 7.9Hz, 1H), 6.93 (q, J = 10.4Hz, 3H), 6.78 (t, J = 5.9Hz, 2H), 6.53 (s, 1H), 6.44 (s, 1H), 6.22 (s, 1H), 5.98 (s, 1H), 5.86 (s, 1H). Resistive isomer 3k**: 1 H NMR (500MHz, DMSO-d6) δ13.20(d,J=13.1Hz,1H),12.94(d,J=20.1Hz,1H),10.37(s,8H),7.94(d,J=8.4Hz,2H),7.71(d,J=8.4Hz,1H),7.22(d,J=8 .8Hz,1H),7.13(d,J=7.9Hz,1H),6.93(q,J=10.4Hz,3H),6.78(t,J=5.9H z,2H),6.53(s,1H),6.44(s,1H),6.12(s,1H),5.91(s,1H),5.89(s,1H). 13 C NMR (126MHz, DMSO) δ182.23,181.77,176.41,166.57,166.38,165.29,163.97,162.50 ,161.55,159.38,159.28,159.10,157.99,156.96,148.81,148.26,144.76,144.62,1 28.96,126.68,126.67,124.76,124.20,121.80,120.55,120.24,120.12,116.41,114 .64,108.47,108.38,106.67,106.66,103.54,103.39,102.35,102.32,93.97,93.37. C 45 H 25 O 16 The calculated HRMS (ESI-TOF) value is 821.1148, and the measured value is 821.1132.

[0447] Results and Discussion

[0448] The reaction in Example 1 can be extended to synthesize novel triflavones by reacting 2a (a radical precursor) with nucleophilic flavones. These triflavones have the same type of flavonoid inter-unit bond and have the general formula Lu-(2′-6)-Lu-(2′-6)-FL( Figure 11 A). A common characteristic of these compounds is the existence of blocked transisomers due to the hindered rotation of the flavonoid intermolecular bonds, resulting in complex... 1 H and 13 C10 NMR spectra. Notably, when 2a reacts with trihydroxyflavones containing β-catechol units, 2a becomes a nucleophile and produces the product FL-(2′-6)-Lu-(2′-6)-Lu(3h, 3i, and 3j). Figure 11 A). Other biflavonoids besides 2a can also serve as coupling partners, allowing the synthesis of triflavonoids. For example, 2j has a catechol unit as a free radical precursor, which couples with nucleophilic apigenin to form FL-(2′-6)-Lu-(2′-6)-Ap,3k as the sole product. Figure 11 B). 3k is a unique triflavonoid containing three different monomeric flavonoid units. These triflavonoids all exist as a mixture of transisomers and can be separated by HPLC, but they will isomerize over time, producing complex... 1 H NMR spectrum.

[0449] Example 10. Key factors affecting reaction results

[0450] It is well known that flavonoids containing the catechol moiety are sensitive to oxidation under alkaline conditions, forming semiquinone radical intermediates (K. Kuwabara et al., Appl. Magn. Reson. 2018, 49, 911–924). However, the fate of these radicals is unclear, and they have not been used for synthetic purposes, possibly due to the formation of complex end products. Our findings are counterintuitive, thus requiring in-depth investigation of the key factors influencing the reaction results so that we can reasonably maximize the yield and selectivity for synthetic applications. These factors include the pH of the solution, counter cations, and oxygen availability.

[0451] pass 13 Determination of pK of luteolin by C NMR spectroscopy a value

[0452] Luteolin (200 mg) was added to a three-necked round flask equipped with a pH meter under argon atmosphere. Figure 12Add degassed water (20 mL) while stirring. Adjust the pH by injecting degassed KOH aqueous solution into the solution using a syringe. Transfer a small aliquot (500 μL) of the sample at the given pH to a 5 mm NMR tube (Armar Chemicals, Switzerland). Add deuterium oxide (D₂O, 5.0 μL) to the same NMR tube for deuterium locking, and then add the DMSO-d₆ solution encapsulated in the capillary ( Figure 12 The sample was placed in a tube as an external standard (for CD3SOCD3, at 2.50 ppm, HE Gottlieb, V. Kotlyar & A. Nudelman, J. Org. Chem. 1997, 62, 7512-7515). The spectra were recorded and analyzed using MestReNova (Mestrelab research, version 5.2.5-4119). (GraphPad, versions 5.0b and 5.0f) performs nonlinear curve fitting on the obtained S-shaped curve. The dissociation constant pK is calculated using the Henderson-Hasselback equation (Equations 1-4). a .

[0453]

[0454]

[0455]

[0456]

[0457] Among them, LH4 and LH3 - LH2 2- LH 3- and L 4- It is a substance containing luteolin in its fully protonated, first deprotonated, second deprotonated, third deprotonated, and 100% deprotonated forms.

[0458] The chemical shift of carbon in luteolin depends on the relative concentration of the conjugate pair. For pH values ​​at pK... a1 The solution is within the range where the substance is approximately 100% protonated (LH4), and the chemical shift is the chemical shift of the protonated substance, δ. low-1 For solutions with higher pH, the substance in the solution is the first deprotonated (LH3). - Chemical shift is the chemical shift of a deprotonated substance, δ. high-1 .

[0459]

[0460]

[0461]

[0462]

[0463] Equations 7-8 are used to determine the mole fraction of conjugated pairs based on the chemical shift of a specific carbon atom. Luteolin... 13 The assignment of C NMR is based on having a similarity to 1 The HSQC and HMBC methods were used to further confirm the 2D correlation of H NMR.

[0464] Quantitative determination of luteolin free radicals

[0465] The area under the curve (AUC) of the luteolin radical EPR spectrum. L The concentration of luteolin free radicals was linearly related to the concentration of luteolin free radicals. Then, manganese (inner sealing putty) was used as a reference and TEMPO (25.17 mM) was used as a standard to calculate the concentration of luteolin free radicals (Equation 9).

[0466]

[0467] pH-dependent EPR spectrum of luteolin free radicals

[0468] Luteolin (28.6 mg, 0.1 mmol) was dissolved in 10 mL of KOH at different concentrations, and the EPR signal of the luteolin aqueous solution was detected immediately after transfer to EPR tubes. The pH of the solution was determined after EPR measurement. Acetone and methanol were introduced to improve the solubility of the sample only in alkaline solutions with pH < 10. Statistical analysis of the numerical data was performed using Origin 8.0 software package (Origin Lab, Northampton, MA).

[0469] Time-dependent EPR spectrum of luteolin free radicals

[0470] Luteolin (28.6 mg, 0.1 mmol) was dissolved in KOH (10 mL, 0.02 M) to obtain an aqueous solution of luteolin with a pH of 11.17. The solution was evenly distributed into tubes A and B. In tube A, the solution was stirred continuously, while in tube B, it was not stirred. After being transferred to the EPR tubes, the EPR signal of the luteolin aqueous solution in both tubes was recorded immediately every 5 minutes.

[0471] Oxidative coupling reaction of luteolin

[0472] Add luteolin (214.5 mg, 0.75 mmol) to a 250 mL round flask. Introduce alkaline water (50.0 mL, 0.03 M KOH solution) into the flask to dissolve the luteolin and produce a solution with a pH of 11.78. Figure 13 As shown, the reaction was monitored in real time within a gas-tight system. Pressure changes during the reaction were recorded using a pressure gauge (NVISION, a pressure recorder with a vacuum range of 30 MPa, CRYSTAL Engineering Corporation) with and without stirring.

[0473] Results and Discussion

[0474] Optimal reaction pH

[0475] The oxidative cross-coupling of the two luteolins occurred within a narrow pH range of 9.5 to 12.5, with an optimal pH of 11.5. Figure 14 The yield decreased rapidly above pH 13.0. For cross-coupling reactions between two different flavonoids, the pH profile depended on the individual flavonoids with an optimum pH of 11.0–11.5, except for 5,6-dihydroxyflavone, which had an optimum pH of 10.0. Figure 14 (BJ). This observation suggests that the optimal pH for the reaction is determined by the different pK values ​​of the flavonoids. a The value determines this. Similar pH curves were found for the oxidative coupling reaction between 2a and flavonoids. Figure 15 Previously reported pK of luteolin. a Values ​​(G. Favaro et al., J. Fluoresc. 2007, 17, 707–714). However, the value corresponding to a specific pK is not clearly determined. a The deprotonation position of the value, and taking into account the fact that luteolin can be oxidized at alkaline pH, colorimetric pK a The measurements are affected by the oxidation products of luteolin. Therefore, we measure the luteolin content under argon atmosphere. 13 C10 NMR spectroscopy is used to determine the pK of specific phenolic protons. a Value(PKAgrawal&H.-J.Schneider,Tetrahedron Lett.1983,24,177-180)( Figure 16 ).

[0476] The first deprotonation occurs at C(7)-OH, pK a1 The value is 8.00. This value is approximately two units greater than the literature value (~6.0). RSJTarábek & I. Deganoca, Electrochim. Acta 2013, 110, 646–654). Our values ​​are consistent with observations of poor solubility of luteolin in water or weakly alkaline aqueous solutions. pK was found... a2 The pK value is 8.93 (C(4′)-OH). a3 and PK a4 They are close to each other, at 12.78 (C(3′)-OH) and 13.03 (C(5)-OH) respectively. Figure 17 A). Therefore, at the optimal reaction pH, the divalent anion of luteolin (abbreviated as LuH4, not Lu to indicate the degree of deprotonation) (LuH2) 2- ) is the main substance.

[0477] To detect the presence of luteolin radical anions, we measured the EPR spectra of air-saturated luteolin solutions at different pH values ​​and found that LuH2... 2- Oxidation occurs significantly only at pH above 9.5. Figure 18 This indicates that LuH2 2- Oxidation can only occur at pH equal to or greater than LH. 2·- PK a This occurs simultaneously, allowing electron transfer-induced deprotonation to happen concurrently:

[0478] LuH2 2- + O2 → LuH2 ·- + O2 ·- (1)

[0479] HO - + LuH2 ·- → LuH ·2- + H2O (2)

[0480] Therefore, the unobserved intermediate [LuH2] ·- ] of pK a The value determines the lower limit of the pH range for the reaction. Based on the EPR signal intensity maps for different pH values, LuH2... ·- The pKa value is estimated to be 9.65 ( Figure 19 A) This is close to the lower pH limit of the reaction. LuH detected by EPR ·2- The free radicals exhibit the same hyperfine coupling mode as reported in the literature. Figure 17 B)( RS J Tarábek & I. Deganoca, Electrochim. Acta 2013, 110, 646–654). LqCy ·2-The spin density of the figure indicates that C2′ has the highest density. Figure 17 C).

[0481] Other flavonoids with catechol B rings, including o-semiquinone radicals, were detected, and C2′ also had the highest spin density. Figure 4 and 20 -22), which is consistent with the fact that C2′ is the major site of the coupling reaction. At higher pH (>12.5), LuH ·2- The free radical signal was depleted, and a new type of free radical with a doublet splitting pattern was detected. Figure 5 To determine the nature of the new free radicals, separate studies were conducted at... 17 O-labeled oxygen and underwater EPR spectra were measured. We found 17 O2 did not change the EPR peak splitting pattern. Figure 17 D). On the other hand, 17 The EPR spectrum of luteolin (pH 12.5) in O-water (30% isotopic purity) produced a new signal due to its affinity for... 17 The hyperfine coupling of O produced a sextet, indicating that H 17 O - Add to position C2′ Figure 6 and 17 D). We believe LuH ·2- It may undergo disproportionation to yield an orthoquinone intermediate, which can react with hydroxides at high pH to produce the observed LuOH. ·2- Free radicals are harmful to coupling reactions.

[0482] Based on these observations, we propose a coupling reaction mechanism ( Figure 19 A). In alkaline water, under oxygen-limited conditions (simply without stirring), luteolin undergoes deprotonation at C(7)-OH and the catechol proton to yield LuH2. 2- LuH2 2- LuH is obtained by deprotonation involving the transfer of a single electron to coupled oxygen. ·2- The free radical anion is coupled with the divalent luteolin anion, which is the main substance under the reaction conditions. Calculations show that the C(6) of the divalent luteolin anion has a high electron density, making it a preferred reaction site, producing 2′-6-bisflavonoids as the main product. Furthermore, our calculations also show that the 2′-6 isomer diganoamine (2a) is 1.3 kcal / mol more stable than the 2′-8 isomer styraxanthin (2a′). Figure 19C). Diganamin (2a) has two catechol moieties. Notably, its reaction product with the flavonoid monomer exhibits high regiospecificity on the unreacted catechol moiety (IIB), suggesting that a semiquinone radical from IIB should be involved. Figure 19 A). EPR spectrum of the ortho-semiquinone radical of 2a ( Figure 7 The results showed a complex signal due to various free radicals, including a semiquinone formed on the IIB ring with an aH1 of 0.285 mT (Table 15). Other free radicals may be located in the IB ring with an aH1 of 0.43 mT (Table 15). The semiquinone radical at the IB ring did not participate in the coupling reaction because a linear trimer, Lu-Lu-FL, was observed and isolated, rather than a branched trimer.

[0483] Table 15. Hyperfine coupling constant (aHn), linewidth (LW), and central field (CF) of protons for flavonoid radicals. Spectra were simulated using JEOL IsoSimu / Fa version 2.2.0 isotropic simulation program.

[0484]

[0485] The effect of counter cations in coupling reactions

[0486] In order to make two divalent anions (LuH) ·2- and LuH2 2- For a reaction to occur, charge repulsion must be overcome by pairing with an ion of the countercation. Therefore, we examined the effect of different countercations on the reaction, and we found that tetramethylammonium (Me4N) reacts with the countercation. + As a Me4NOH addition, it gave the lowest yield (<20%). Lithium performed better, but not as well as cesium, sodium, and potassium (~80%). Figure 19 D). These results indicate that the counter cation not only counteracts the anionic charge but may also promote the reaction by bridging the two coupled partners to bring them closer together through weak coordination interactions. In this respect, smaller lithium ions are less effective than larger alkali metal ions. In aqueous solution, the alkali metal ion should exist as a hydrate, and the coordination bond with the phenolate of the luteolin divalent anion should be relatively weak and dynamic.

[0487] Impact of oxygen availability

[0488] Although the o-haloquinone radical of luteolin had been previously observed, the final product was found to be complex and uncharacterized. RSJ Tarábek & I. Deganoca, Electrochim. Acta 2013, 110, 646–654), possibly due to over-oxidation caused by excessive oxygen in the solution. The positive results in our example are likely due to the restriction of oxygen and the reaction proceeding without stirring, which is counterintuitive. We compared the reaction kinetics of alkaline luteolin solutions in two test tubes; one tube was vigorously magnetically stirred (resulting in an excess supply of oxygen), while the other was not stirred (oxygen availability depended on the diffusion of gaseous oxygen into the solution). After 4 hours, no coupling products were detected in the stirred tube, while in the unstirred tube, after 10 hours, both 2a and 3a showed two major products (…). Figure 23 The dissolved oxygen concentration in air-saturated water is approximately 256 μM, while the dissolved oxygen concentration in alkaline water is lower (W. Xing et al., (2014). Oxygen solubility, diffusion coefficient, and solution viscosity. In W. Xing, G. Yin & J. Zhang (eds.), Rotating Electrode Methods and Oxygen Reduction Electrocatalysts (pp. 1-31). Elsevier). At the start of the reaction, the dissolved oxygen in both tubes reacts with LH2. 2- The reaction is rapidly depleted. However, stirring replenishes dissolved oxygen, which undergoes a reaction with LH. ·2- The free radical coupling reaction leads to over-oxidation. In an unstirred tube, however, the depletion of oxygen and the slow diffusion of gaseous oxygen into the undisturbed solution prevent such a reaction, thus preventing the formation of products over time. Figure 13 and 24 Therefore, limiting oxygen utilization is a key factor in the oxygen-mediated oxidative coupling reaction of flavonoids.

[0489] Example 11. Comparison of the bioactivity of flavonoid dimers and trimers

[0490] It is believed that mosses utilize abundant biological resources in the synthesis of luteolin dimers and trimers because they need to defend against microbial stress when growing on decaying wood in damp forests (H. Geiger et al., J. Hattori Bot. Lab. 1997, 83, 273-308). To test this hypothesis, we used *Aspergillus niger* as a model fungus to measure the antifungal activity of luteolin, 2a, and 3a. Figure 25 and 26 ).

[0491] Antifungal activity study

[0492] Antifungal activity was evaluated against two fungi, *Aspergillus niger* ATCC 16888 and *Botrytis cinerea* ATCC 11542, which were isolated and identified using the ITS gene (GenBank accession number AY373852, ATaha et al., BioResources 2019, 14, 6025-6046). The bioassay was evaluated using the radial growth technique (P. Wayne, Reference method for broth dilution antifungal susceptibility testing of yeasts, approved standard. CLSI document M27-A2, 2002). Both fungal strains were aerobically cultured at 25°C on PDA medium for 14 days. Fungal spores were collected using sterile swabs and suspended in sterile water, with the concentration adjusted according to the requirements of the antifungal assay.

[0493] Assay for in vitro antifungal activity

[0494] Flavonoid oligomers were dissolved in DMSO (1.0 mg / mL) and then transferred to sterile warm PDA medium (40–45 °C) to achieve final concentrations of 1.0 mg / mL, 0.6 mg / mL, and 0.4 mg / mL. The solutions were then immediately poured into 24-well sterile plastic microtiter plates (Corning Incorporated, Costar) including flat-bottomed wells. After cooling the plates to room temperature, 10 μL of freshly prepared Aspergillus niger suspension (1.25 x 10⁻⁶ m³ / mL) was added. -6 Agar was inoculated onto each well with a concentration of 1 / mL. Drug-free agar containing 1% DMSO was used as a negative control, and amphotericin B was used as a positive control.

[0495] From 7-day-old colonies, fungi with a diameter of 9 mm were transferred to the center of treated PDA plates and controls. All plates were incubated at 26 ± 1 °C for 7 days. All test concentrations, as well as positive and negative controls, were measured in triplicate.

[0496] High-throughput assay of starch hydrolase inhibitory activity

[0497] Following the reported method, turbidity measurements were used to determine the inhibitory activity of each obtained fraction against α-amylase and α-glucosidase (Liu, Song, Wang, & Huang, 2011, Journal of Agricultural and Food Chemistry 2011, Vol. 59, No. 18, pp. 9756-9762). Corn starch (20 mg / mL) was suspended in sodium phosphate buffer (0.1 M, pH 6.9) and gelatinized at 100 °C for 2.5 min. The inhibitor solutions were diluted to appropriate concentrations in buffer. Acarbose was used as a positive control and reference standard. α-amylase solution (2 U / mL in buffer, 20 μL) or α-glucosidase solution (1 × 10⁻² U / mL in buffer) was pre-incubated with a series of inhibitor solutions (20 μL) in 96-well microplates and maintained at 37 °C for 15 min. The reaction was initiated by injecting 60 μL of gelatinized corn starch solution. Turbidity changes were recorded at 660 nm per minute for 2 hours using a Synergy HT microplate reader (Biotek Instruments Inc., Winooski, VT, USA). The percentage of inhibition was calculated using Equation 10.

[0498] Inhibition rate (%) = (AUC) 样品 -AUC 对照 ) / AUC 样品 ×100% (Formula 10)

[0499] AUC 样品 It is the area under the suppression curve, AUC. 对照 It is the area under the negative control curve. IC 50 This can be defined as the inhibitor concentration that produces 50% inhibition of enzyme activity under specific assay conditions. It is obtained by interpolating the inhibition percentage against the inhibitor concentration curve. To avoid run-to-run errors due to fluctuations in enzyme activity, the inhibitory activity of the sample is expressed using acarbose equivalents (AE) based on the following formula:

[0500] The AE of the sample = the IC50 of acarbose 50 IC of the sample 50 (Equation 11)

[0501] Molecular docking

[0502] Ligand preparation: All ligands, including acarbose, luteolin, 2a, 2b, and 3a, were selected as ligands, and their crystal structures were virtually constructed. These ligand molecules were plotted and saved as 3D conformational isomers in .cif format. The structures of these ligands were then converted to .pdb format using PyMol. Subsequently, the ligand molecules were uploaded as input files to Autodock in .pdb format, and the output was a .pdbqt file.

[0503] Protein molecule preparation: The active site 1 ppi of mammalian α-amylase was selected and obtained from RCSB as a .pdb file (M. Qian et al., Biochemistry 1994, 33, 6284-6294). The target amylase was loaded into the Autodock graphical user interface in .pdb format (GMMorris et al., J. Comput. Chem. 1998, 19, 1639-1662; and R. Huey et al., J. Comput. Chem. 2007, 28, 1145-1152). The amylase for docking was prepared by removing acarbose molecules, deleting water molecules, adding polar hydrogen atoms, and adding Coleman charges to the macromolecule. The amylase was then converted from .pdb to .pdbqt format. The grid frame was selected and adjusted to the specific size of the docking site. The output file with the grid size was saved as a .txt file.

[0504] Docking via AutoDock Vina and visualization with PyMOL: AutoDock Vina (O. Trott & A.J. Olson, J. Comput. Chem. 2010, 31, 455-461) is a virtual screening technique to predict the optimal binding conformation of a ligand to a target protein with a known structure. To perform AutoDock, prepare the ligand and amylase in .pdbqt format and set the configuration file in a .txt file. Perform docking by executing AutoDock Vina using the command prompt and analyze the docking results using PyMOL. PyMOL is a molecular visualization program widely used for the three-dimensional (3D) visualization of proteins and small molecules. The AutoDock Vina outputs a .pdbqt file and loads the amylase in .pdbqt format into the PyMOL graphical interface. There, the docked structure is visualized under the "molecular surface" and "cartoon" options. The active sites of the docking are displayed using PyMOL's "pockets" feature.

[0505] Results and Discussion

[0506] 2a and 3a can inhibit the growth of Aspergillus niger in a dose-dependent manner, IC50 50 The concentrations were 0.86 μM and 0.96 μM, respectively, compared to amphotericin B (IC50). 50 The activity was comparable to 0.50. Notably, dimer 2a showed slightly higher activity than trimer (3a). Plant flavonoids protect plants from insect ingestion by inhibiting digestive enzymes such as α-amylase and α-glucosidase. We measured the activities of selected flavonoid dimers and trimers (2a-2f, 3a-3f) in inhibiting α-amylase and α-glucosidase. Figure 26-37 Furthermore, it was found that dimers 2a and 2b exhibited comparable (2a) or even higher (2b) activity compared to acarbose, an antidiabetic drug. Molecular docking using the crystal structure of the pancreatic α-amylase-acarbose complex (M. Qian et al., Biochemistry 1994, 33, 6284-6294) revealed that the flavonoid oligomers (2a, 2b, 3a) were located at the active site, while luteolin was not, which could explain why luteolin is inactive. The active site region of α-amylase is a V-shaped depression located at the carboxyl terminus of Glu233, Asp300, and Asp197. 2a and 2b ( Figures 38-39 The molecular shape of ) is exactly V-shaped (similar to Figure 40 (Acarbose in the middle) and a B ring near the catalytically active group is well coordinated in the depression and forms hydrogen bonds with Glu233, Asp300 and Aspl97 through the catechol group.

[0507] Example 12. In vivo study

[0508] A 10% (w / v) starch solution was prepared by adding distilled water to reagent-grade corn starch and heating the solution in a boiling water bath for about 15 minutes until it was completely gelatinized.

[0509] Eight-week-old male wild-type C57BL / 6J mice were fasted overnight (with free access to water) and weighed the following day. Tail clipping was performed and blood was collected for fasting blood glucose concentration determination using a Roche glucometer.

[0510] Feeding method a, “YX2 + starch”: Luteolin dimer (2a) was administered by gavage at a dose level of 20 mg / kg body weight as a 2 mg / mL aqueous solution. After 30 minutes, a 10% (w / v) starch solution was administered by gavage at a dose level of 1 g / kg body weight.

[0511] Feeding method b, “Starch (+YX2)”: Luteolin dimer (2a) is mixed with starch at a ratio of 0.2% by dry weight and then mixed with water (1:9 by weight) to obtain a slurry labeled as starch (+YX2). Figure 41The mixture was administered to mice by gavage at a rate of 1 g / kg body weight. In contrast, luteolin (1a or YX1) was mixed with starch at 0.2% by weight. The resulting mixture was further mixed with water to obtain a 10% starch slurry labeled as starch (+YX1). Figure 41 Mice were administered the drug via gavage at a dose of 1 g / kg body weight.

[0512] Blood glucose levels were measured at 30, 60, 90, and 120 minutes to create blood glucose curves. GraphPad plotted the postprandial blood glucose response curves and calculated the area under the curve.

[0513] Results and Discussion

[0514] Blood glucose responses in mice fed different samples showed Figure 41 In this context, YX2 refers to luteolin dimer (2a), and YX1 refers to luteolin monomer (1a).

[0515] From the results ( Figure 41 A) It was clearly shown that feeding mice with starch (1 g / kg body weight, via gavage) caused blood glucose to rise to a peak after 30 minutes, and said value gradually decreased until 120 minutes.

[0516] Mice fed with luteolin dimer (YX2) at 20 mg / kg body weight alone did not exhibit a glycemic response.

[0517] If mice were fed YX2 at a dose of 20 mg / kg 30 minutes before administering starch at 1 g / kg body weight, there was no glycemic response (curve: YX2 + starch). Conversely, if mice were fed a mixture of YX2 in starch at 0.2%, the glycemic response was comparable to that of mice fed only the same dose of starch (1 g / kg body weight) (curve: starch (+YX2)).

[0518] Finally, when mice were fed a 0.2% mixture of luteolin and starch, the blood glucose response curve was almost identical to that of mice fed only the same dose of starch. This result indicates that luteolin dimer 2a needs to be administered 30 minutes before a meal in order for it to effectively inhibit the blood glucose response after starch consumption.

Claims

1. A method for coupling a flavonoid-containing compound, the method comprising: (i) Provide one or more flavonoid-containing compounds; and (ii) In the presence of air, contact the one or more flavonoid-containing compounds with an alkali; The base is selected from the group consisting of metal carbonates, metal hydroxides, and bases of the formula R4NOH, wherein each R independently represents H or C. 1-4 alkyl, Step (ii) is carried out in a closed reaction vessel containing air and a reaction mixture containing one or more of the flavonoid-containing compounds, the base, and water, wherein the air occupies 10 to 95% of the volume of the reaction vessel at 25°C and 101 kPa. The one or more flavonoid-containing compounds have a skeleton selected from the group consisting of: flavonoid skeletons, novel flavonoid skeletons, flavanone skeletons, isoflavonone skeletons, and isoflavone skeletons. Alternatively, the flavonoid-containing compound may comprise a dimer or trimer thereof.

2. The method according to claim 1, wherein the alkali is selected from one or more of the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate and alkaline water.

3. The method according to claim 2, wherein the base is selected from one or more of the group consisting of sodium hydroxide and potassium hydroxide.

4. The method according to claim 3, wherein the base is potassium hydroxide.

5. The method according to any one of the preceding claims, wherein the pH of the reaction mixture in step (ii) is 10 to 14.

6. The method according to any one of claims 1 to 4, wherein the temperature of the reaction mixture in step (ii) is 15 to 30°C.

7. The method according to any one of claims 1 to 4, wherein step (ii) 1 is performed for a period of 20 hours.

8. The method according to any one of claims 1 to 4, wherein step (ii) is performed without stirring.

9. The method according to any one of claims 1 to 4, wherein the skeleton is substituted with one or more substituents selected from the group consisting of: hydroxyl, methoxy, glycosyl, alkoxy, NO2, F, CN, SH, CF3, Cl, Br, I, =O, =CH2, C 1-18 Alkyl, C 1-18 Fluoroalkyl, -OC(O)-R, C 2-18 alkenyl, isoprene, phytyl, exocyclic C 3-6 Cycloalkyl, exocyclic C 5-6 Cycloalkenyl, phenyl, phenoxy, C 1-18 N-alkylamine group and C 1-18 N,N-dialkylamine, where R in -OC(O)-R represents C 1-22 alkyl.

10. The method according to any one of claims 1 to 4, wherein the flavonoid-containing compound comprises a flavonoid skeleton.

11. The method according to any one of claims 1 to 4, wherein the flavonoid-containing compound is a flavonoid monomer.

12. The method according to any one of claims 1 to 4, wherein the flavonoid-containing compound is a flavonoid dimer.

13. The method according to any one of claims 1 to 4, wherein the flavonoid-containing compound is a flavonoid trimer.

14. The method according to any one of claims 1 to 4, wherein at 25°C and 101 kPa, the air occupies 30 to 90% of the volume of the reaction vessel.

15. The method according to any one of claims 1 to 4, wherein the flavonoid-containing compound is selected from the group consisting of: luteolin, apigenin, geraniol, scutellarin, 5,6-dihydroxyflavone, genistein, 5,3′,4′-trihydroxyflavone, 6,3′,4′-trihydroxyflavone, 7,3′,4′-trihydroxyflavone, 3′,4′-dihydroxyflavone, dimers formed from two of the above, and trimers formed from three of the above.

16. The method according to any one of claims 1 to 4, wherein the product of the method is a compound selected from the group consisting of:

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