KR102265190B1 - Novel chromenone derivatives having substituted biphenyl group and a pharmaceutical composition for prevention or treatment of allergic diseases compring the same - Google Patents

Abstract

The present invention provides a novel chromenone derivative compound capable of effectively suppressing an allergic immune response by inhibiting thymic stromal lymphoprotein (TSLP)-mediated signal transduction, and fundamentally preventing various allergic diseases or using the same It relates to a therapeutic pharmaceutical composition.

Description

Novel chromenone derivatives having substituted biphenyl group and a pharmaceutical composition for prevention or treatment of allergic diseases compring the same}

The present invention relates to a novel chromenone derivative incorporating a biphenyl skeleton, and more particularly, to a chromenone derivative capable of suppressing an allergic immune response by inhibiting signal transduction mediated by thymic stromal lymphoprotein (TSLP). It relates to a menone derivative compound and a pharmaceutical composition for preventing or treating allergic diseases including the same.

The prevalence of allergic diseases such as asthma, atopic dermatitis and allergic rhinitis has steadily increased over the past few decades, and is seriously affecting the quality of life of millions of people around the world.

Allergic diseases are characterized by an abnormal inflammatory response to an allergen due to an imbalance of Th1 cell (Th1) and Th2 cell (Th2) cell responses and an overproduction of Th2 cells. Th2-mediated inflammatory responses induced by activated dendritic cells (DCs) play a key role in the pathogenesis and maintenance of allergic diseases. Upon tissue damage or exposure to various environmental allergens, epithelial cells release cytokines such as thymic stromal lymphoprotein (TSLP), IL-33, and IL-25, and these cytokines release dendritic cells (dendritic cells). , DCs), which in turn stimulates differentiation of T cells into Th2 cells and IL-4 secretion from T cells in lymphatic organs, leading to production of immunoglobulin (IgE) by B cells. In addition, these cytokines activate two groups of innate lymphoid cells (ILC2 cells) that release IL-13 for mucus secretion as well as IL-9 and IL-5 for replenishment of inflammatory response-related mast cells and eosinophils. . These cytokines promote allergic inflammation and cause allergic diseases such as asthma and atopic dermatitis.

Among these cytokines, TSLP, an IL-7-like cytokine, is recognized as a major allergenic cytokine in DCs-mediated Th2 immune responses. TSLP, which is mainly produced in epithelial cells, binds to its receptors TSLPR and IL-7Rα, resulting in STAT5 signaling, an intracellular transcription factor that leads to activation of Th2 cells and type 2 immune cells. Modulation of TSLP/TSLPR signal by immunoglobulin (TSLPR-Ig) or a soluble TSLPR fragment fused to an Fc fragment of an anti-TSLP antibody alleviated the severity of allergic disease by reducing eosinophilic airway inflammation and allergen-induced bronchoconstriction. As described above, blocking and controlling the transmission of TSLP signals is recognized as important for controlling allergic diseases, and various techniques for treating diseases by targeting TSLP have been reported in the prior art, for example, Korean Patent Laid-Open Patent Publication No. Publication No. 2008-0099330 discloses an antibody that neutralizes the activity of human TSLP and a technology for treating asthma, atopic dermatitis, allergic rhinitis, etc. using the antibody.

However, despite the urgent need for the discovery of anti-allergic drugs, there are few reports of low-molecular-weight compound inhibitors targeting TSLP signal transduction.

Accordingly, in the present invention, a chromenone derivative compound, which is a low molecular weight compound capable of preventing and treating allergic diseases by controlling intracellular signal transduction by TSLP, suppresses an allergic immune response, and including the chromenone derivative compound, which suppresses an allergic immune response and prevents allergic diseases An object of the present invention is to provide a pharmaceutical composition that can be treated and prevented.

The inventors of the present invention screen extracts of natural product libraries to develop novel low-molecular compound inhibitors that target TSLP signaling control in allergic reactions, and confirm that the characteristic active ingredient is an effective low-molecular compound that blocks the TSLP signaling pathway The present invention was completed by developing a novel TSLP inhibitor compound by conducting a structure-activity relationship (SAR) study based on the chemical structure of the active ingredient.

In order to solve the above problems, the present invention provides a chromenone derivative represented by the following [Formula 1] or [Formula 2].

[Formula 1]

[Formula 2]

In the [Formula 1] or [Formula 2],

R ^{1 To} R ^{3 Are} the same as or different from each other, and each independently represents a hydrogen or a hydroxyl group, preferably, at least two or more of ^{R 1 To} R ^{3 is a hydroxyl group.}

Also. R ⁴ may be any one selected from hydrogen, a hydroxyl group, a halogen group, and a nitro group (n is an integer of 1 to 5).

More specific structures and properties of the [Formula 1] and [Formula 2] will be described later.

The chromenone derivative represented by [Formula 1] or [Formula 2] according to the present invention inhibits the binding between thymic stromal lymphopoietin (TSLP) and TSLP receptor, and controls intracellular signal transduction by TSLP to thereby control intracellular STAT5 It is characterized in that it inhibits phosphorylation of

In addition, the present invention provides a pharmaceutical composition for preventing or treating allergic diseases, comprising the chromenone derivative represented by the above [Formula 1] or [Formula 2] as an active ingredient, and comprising a pharmaceutically acceptable carrier, , The allergic disease may be atopic dermatitis, urticaria rhinitis, allergic rhinitis, and the like.

The present invention is a novel low-molecular-weight compound drug capable of effectively suppressing an allergic immune response, which can be used to fundamentally prevent or treat various allergic diseases.

1 shows a structural modification strategy based on compound 1 for deriving novel compounds according to the present invention.
2 is a chemical formula showing Compound 2 and Compounds 3a to 3c.
3 shows the TSLP inhibitory activity results of the flavone analogs (Compounds 6a to 6d) and the flavanone analogs (Compounds 7a to 7b).
Figure 4 shows the TSLP inhibitory activity results of the flavone analogs (compounds 10a to 10i) substituted with a biphenyl moiety and the flavanone compound 11a according to an embodiment of the present invention.
Figure 5 is (a) _{HPLC chromatograms of compound 11a (R t} =16.152 min) and compound 10a (R _t =18.214 min), (b) a plot of the inhibition rate of compound 11a as determined by ELISA, (c) This is the result of Western blot analysis of pSTAT5 level. HMC-1 cells were pretreated with compound 11a (0.1, 1, 10 μM), stimulated with TSLP (20 ng/mL) for 2 hours, and the level of pSTAT5 was analyzed by Western blotting (upper panel). , and the relative density is the result of quantification by densitometry pSTAT5/STAT5×100 (lower panel).

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention confirmed the important role of human TSLP (hTSLP) in allergic reactions, and as a result of screening extracts of natural product libraries to identify novel small-molecular compound inhibitors targeting hTSLP signaling control, selective binding to hTSLP As an active ingredient that inhibits interaction with human TSLPR (hTSLPR), it was confirmed that baicalein, a major component of Scutellaria baicalensis , is an effective low-molecular compound that blocks the TSLP signaling pathway (Patent Application No. 10- 2017-0002332), to identify low-molecular compound substances that inhibit TSLP/TSLPR-mediated signaling pathways, a structure-activity relationship (SAR) study was conducted based on the chemical structure of the active ingredient to develop a novel hTSLP inhibitor compound. The present invention was completed.

Compound 1 shown in FIG. 1 is gold ( Scutellaria baicalensis ) As baicalein, a major component, it is an effective low-molecular compound that targets blocking of the TSLP signaling pathway, and it has been previously confirmed that it can be used as a promising therapeutic agent for allergic diseases.

In the SAR study, we focused on the three objectives of synthesizing novel analogs by identifying essential -OH groups based on the chemical structure of compound 1 and improving physicochemical properties.

As shown in Figure 1 below, Compound 1 has three ring regions, A, B and C regions, (i) confirmed by examining the need for three -OH groups in the A ring for TSLP inhibition, (ii) Since a hydrophobic amino acid residue was observed near the binding site of hTSLP of Compound 1 through in silico docking studies, we tried to introduce a biphenyl group instead of a phenyl group in the B ring, and (iii) the planarity of the C ring for TSLP bonding. was investigated to convert the planar flavone structure into a non-flat flavanone structure by reducing the double bond between the C-2 and C-3 carbons.

(1) In order to confirm the importance of the -OH group of the A ring for TSLP inhibition, methyl iodide and Compound 1 were reacted in acetone to convert the -OH of 3 compounds 1 into a corresponding methoxy group (Compound 2 in FIG. 2) converted. As a result of measuring hTSLP inhibitory activity with respect to compound 2 and mono-hydroxylated flavone (compounds 3a to 3c, FIG. 2 below) using ELISA, any compound inhibited > 50% at 1 mM , at least two OH groups are required to block the interaction between hTSLP and TLSPR (Table 1 below).

Compound % inhibition 0.3 mM 1 mM One 43.1 ± 1.0 57.1 ± 1.7 2 < 5 12.0 ± 2.8 3a 16.4 ± 2.7 23.3 ± 6.8 3b 22.2 ± 2.5 38.7 ± 5.3 3c 24.1 ± 5.4 16.8 ± 2.0

(2) Next, as shown in the following [Scheme 1], di-hydroxylated flavones (6a to 6d) and flavanones (7a to 7b) from 2'-hydroxy-dimethoxyacetophenone and benzaldehyde It was synthesized in 4 steps or 5 steps.

The following [Scheme 1] is a synthesis scheme of the flavone analogs (6a to 6d) having a dihydroxyl group in the A-ring and their corresponding flavanone analogs (7a to 7b).

[Scheme 1]

(a) NaOCH ₃ (1.2 eq), THF, 0 °C, rt, 8h

(b) I ₂ (1.1 eq), DMSO, 130° C., 3 h

(c) BBr ₃ (10-15 eq), CH ₂ Cl ₂ , reflux, 12h

(d) H ₂ , Pd/C, 1,4-dioxane, methanol, rt, 24h

Acetophenone and benzaldehyde (benzaldehyde for 4a and 4c and p-anisaldehyde for 4b and 4d) produced the corresponding chalcone (4a-4d) in 64-98% yield. When 4a to 4d are treated with iodine in DMSO, flavone compounds 5a to 5d are obtained in a yield of 38 to 78%. Demethylation of 5a to 5d is treated with BBr _{3 in CH 2} Cl ₂ under reflux to give flavone analogs 6a to 6d in yields of 47 to 82%. Reaction temperature and maintenance of anhydrous reaction conditions are critical for the demethylation step, and reflux conditions give fully demethylated compounds 6a-6d, whereas the reaction at room temperature yields mono-demethylated compounds as major products. The hydrogenation of compounds 6a to 6d is carried out in the presence of _{Pd/C and H 2 .} The 5,7-dihydroxylated flavones (6a-6b) are converted to the corresponding flavanones (7a-7b) in yields of 56-74%, whereas the 6,7-dihydroxylated flavones (6c-6d) ) is not the case under the same conditions. Since the intermolecular hydrogen bonding between the -OH of the A-ring and the carbonyl group of the B-ring was significantly increased in the 5,7-dihydroxylated flavones, compounds 6a to 6b were hydroxylated flavones).

The conversion from a planar flavone structure to a non-flat non-flat flavanone structure affects TSLP binding and physicochemical properties, and as shown in FIG. 3 below, flavanone analogs show weaker inhibition of TSLP activity than flavones. However, kinetic solubility in PBS is increased for flavanones compared to their corresponding flavones. Flavanone analogues 7a-7b are 111 μM and 505 μM, respectively, and 6a-6b flavones are 30 μM and 486 μM, respectively.

(3) A lipophilic ring-extended biphenyl group is introduced into the C-ring region to increase the TSLP-binding affinity. Ring-extending compounds 10a to 10i are obtained in two steps from bromoflavone 8a to 8c containing a bromo group at the 3'- or 4'-position of the B-ring as shown in [Scheme 2] below.

The following [Scheme 2] is a synthesis reaction scheme of biphenyl-introduced flavone analogs (10a to 10i) and flavonone derivatives (11a).

[Scheme 2]

(a) benzeneboronic acid, Pd(PPh ₃ ) ₄ , toluene, H ₂ O, Cs ₂ CO ₃ , 90 ℃, 5h

(b) BBr ₃ (10-15eq), CH ₂ Cl ₂ , reflux, 12h

(c) H ₂ , Pd/C, 1,4-dioxane, methanol, rt, 24h

Prepared from 2'-hydroxy-dimethoxyacetophenone and bromo benzaldehyde by applying the synthesis procedure of [Scheme 1], benzeneboronic acid (9c and 9e are benzeneboronic acid, 9d and 9f are 4-fluorobenzene boronic acid, 9b and 9h are 4-nitro benzeneboronic acid, and 9b and 9h are benzeneboronic acid) and 3'-bromoflavones (8a to 8b) or 4'-bromoflavones (for 9a, 9g and 9i) 4-methoxybenzeneboronic acid) in the presence of tetrakis(triphenylphosphine)palladium at 90° C. for 5 hours to give biphenyl compounds 9a to 9i in 23 to 78% yield. _{By treatment with BBr 3} under reflux for 12 hours, 9a to 9i were demethylated to give final compounds 10a to 10i in 13 to 46% yield, and reduction of the C-ring was performed under _{Pd/C and H 2 .} The 6,7-dihydroxylated biphenyl analogs (10e to 10i) do not occur under these conditions as identified in compounds 6c to 6d. Only compound 10a was reduced in 5,7-dihydroxylated biphenyl flavones (10a to 10d) to give compound 11a in a yield of 68%.

The high reactivity of compound 10a compared to other 5,7-dihydroxylated analogues can be explained by the stability of the carbocation resonance structure. As the -OH group of the biphenyl ring of 10a increases the electron emission effect toward the C ring, the carbocation structure of 10a is more stabilized and the single bond between C-2 and C-3 is improved over others. In addition, when acetic acid was added in the hydroreduction step to increase the reactivity of compounds 10b to 10d, the ring was broken and the desired product was not produced.

(4) Biphenyl-based flavones (10a to 10i) show increased TSLP-inhibiting activity compared to Compound 1 used as a positive control in the ELISA assay (Fig. 4 below).

In particular, the 4 compounds 10a, 10e, 10g and 10h showed >50% inhibition at 0.3 mM, and the 6,7-dihydroxy analogues (10e to 10h) were the 5,7-dihydroxy analogues (10a to 10d). ) showed stronger TSLP-inhibition. In addition, introduction of a phenyl group at the 3'-position of the B-ring (Compound 10f) favors TSLP bonding over the 4'-position of the B-ring, and the dihydroxyl groups at the 6- and 7-positions and the 3' of the B ring Combination of the -position with the phenyl ring improved the TSLP-binding affinity compared to compound 1. Compounds 10e and 10g were the most potent biphenyl analogs with _{IC 50 of 177 and 210 μM, respectively.} However, the solubility of the biphenyl compound in PBS (pH 7.4) was lower than that of compound 1.

(5) Biphenyl flavanone compounds exhibit higher kinetic solubility in PBS than the corresponding flavones as identified in compounds 7a to 7b. Hydrogenation of the C-ring under reducing conditions (Pd/C, H ₂ ) did not change the 6,7-dihydroxylated biphenyl flavones (10e to 10i) as expected. Only compound 10a was reduced to give the flavanone analogue (compound 11a) among the 5,7-dihydroxylated analogues (10a to 10d). The increased hydrophilicity of flavanone compound 11a compared to flavone compound 10a was confirmed in reverse-phase HPLC experiments (11a and 10a at 16.15 min and 18.21 min, respectively, FIG. 5a below). In addition, the kinetic solubility of flavanone compound 11a (66 μM) was equivalent to that of compound 1 (62 μM). In ELISA, the flavanone compound 11a had an IC ₅₀ value of 370 μM (Fig. 5b below), which was better than that of _{compound 1 (IC 50 =460 μM).} In addition, compound 11a strongly inhibited STAT5 phosphorylation even at 0.1 μM in a Western blot experiment (Fig. 5c below).

Therefore, the biphenyl-introduced flavanone compound 11a according to the present invention having an hTSLP-inhibiting effect and excellent water solubility can be a novel hTSLP inhibitor, and by inhibiting TSLP-mediated signal transduction, fundamental prevention and treatment of allergic diseases it becomes possible It can also serve as a prototype molecular model for further structural modifications.

In addition, the present invention provides a pharmaceutical composition for preventing and treating allergic diseases containing the chromenone derivative represented by the [Formula 1] or [Formula 2] as an active ingredient, and according to an embodiment of the present invention, the allergy The disease may be atopic dermatitis, urticarial rhinitis or allergic rhinitis.

In addition, the pharmaceutical composition according to the present invention may be administered in the form of a combination formulation together with other drugs known for the prevention or treatment of allergic diseases, or may contain other ingredients such as carriers, diluents, adjuvants and stabilizers.

In addition, the form of the composition according to the present invention may be variously selected depending on the mode to be administered, but is not limited thereto, but for example, a solid state such as a tablet, pill, powder, capsule, gel, ointment, fluid or suspension. , semi-solid or liquid dosage form, may be administered in a unit dosage form suitable for single administration of an accurate dosage, may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, It can be administered by intramuscular injection.

The composition may also contain, depending on the desired formulation, pharmaceutically acceptable carriers, diluents, adjuvants, stabilizers, defined as aqueous-based vehicles commonly used in formulating pharmaceutical compositions for human administration.

The term "carrier" refers to a substance that facilitates the addition of a compound into a cell or tissue, for example, a carbohydrate compound (eg, lactose, amylose, dextrose, water) that is commonly used in formulations. Cross, sorbitol, mannitol, starch, cellulose, etc.), gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, arabic Contains rubber, vegetable oils (e.g. corn oil, cottonseed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. However, the present invention is not limited thereto. A diluent is defined as a substance that not only stabilizes the biologically active form of the target compound, but also dissolves the compound in water. Examples of the diluent include distilled water, physiological saline, Ringer's solution, glucose solution, and It may be a Hank's solution or the like. The stabilizer may be selected from the group consisting of proteins, carbohydrates, buffers and mixtures thereof. Further, in addition to the above ingredients, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, and the like are further included, but are not limited thereto.

In addition, an effective amount of other ingredients such as carriers, diluents, adjuvants and stabilizers is an amount effective to obtain a pharmaceutically acceptable formulation in terms of solubility and biological activity of the ingredients.

As used herein, "prevention" refers to inhibiting the occurrence of a disease or disease, although it has never been diagnosed as possessing a disease or disease, and "treatment" refers to inhibiting the development of a disease or disease, the disease or disease. It is meant to include the alleviation of and the elimination of a disease or disease.

In addition, "included as an active ingredient" means that the ingredient is included in an amount necessary or sufficient to realize a desired biological effect. In actual application, the determination of the amount included as an active ingredient is an amount for treating a target disease, and may be determined in consideration of matters that do not cause other toxicity, for example, the disease or condition to be treated, the form of the composition to be administered, This may vary depending on various factors such as the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art to which this invention pertains can empirically determine the effective amount of an individual composition without undue experimentation.

In addition, "pharmaceutically acceptable" means a property that does not impair the biological activity and physical properties of a compound.

Unless otherwise defined, other terms and abbreviations used in this specification may be interpreted as commonly understood by those skilled in the art to which the present invention pertains.

Hereinafter, the present invention will be described in more detail by way of Synthesis Examples and Experimental Examples, but the following Synthesis Examples and Experimental Examples are only intended to aid understanding of the present invention, and do not limit the scope of the present invention.

Synthesis Example 1: General procedure for chalcone synthesis

To a solution of 2'-hydroxy-4',5-dimethoxyacetophenone or 2'-hydroxy-4',6'-dimethoxyacetophenone in THF at 0 °C sodium methoxide (1.1 eq) in methanol solution was added and stirred for 20 minutes. Benzaldehyde (1.2 eq) was added to the reaction mixture at the same temperature and the mixture was stirred at room temperature for 8 hours. The reaction mixture was partitioned and diluted with ethyl acetate (40 mL) and saturated ammonium chloride solution (40 mL). The organic layer was collected, dried over magnesium sulfate and concentrated under reduced pressure. The residue was recrystallized by flash column chromatography or hexane-ethyl acetate to obtain chalcone represented by compounds 4a to 4d.

(1) (E)-1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-phenylprop-2-en-1-one (4a)

Using 2'-hydroxy-4',6'-dimethoxyacetophenone and benzaldehyde as starting materials, purification by flash column chromatography (toluene-EA 50:1, v/v) gave compound 4a as a yellow solid obtained (yield 91%).

R _f = 0.33 (hexane-EA=6:1,v/v).

¹ H NMR (300 MHz, CDCl ₃ ) δ 14.18 (s, 1H), 8.85 (d, J = 1.5 Hz, 1H), 8.60 (dd, J = 1.3 and 4.7 Hz, 1H), 7.97 (d, J = 15.7 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 15.7 Hz, 1H), 7.35 (dd, J = 4.9 and 7.8 Hz, 1H), 6.12 (d, J = 2.3 Hz, 1H), 5.98 (d, J = 2.3 Hz, 1H), 3.93 (s, 3H), 3.85 (s, 3H).

HRMS (ESI) m/z calculated for C ₁₇ H ₁₆ O ₄ [M+H] ⁺ : 285.1121; found : 285.1134.

(2) (E)-1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (4b)

Using 2'-hydroxy-4',6'-dimethoxyacetophenone and benzaldehyde as starting materials, purification by flash column chromatography (toluene-EA 50:1, v/v) gave compound 4b as a yellow solid obtained (yield 78%).

R _f = 0.53 (hexane-EA=2:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ ) δ 14.45 (s, 1H), 7.81 (s, 2H), 7.57 (d, J = 7.8 Hz, 2H), 7.94 (d, J = 8.4 Hz, 1H), 6.11 (s, 1H), 5.97 (s, 1H), 3.93 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H).

HRMS (ESI) m/z calculated for C ₁₈ H ₁₈ O ₅ [M+H] ⁺ : 315.1227; found : 313.1141.

(3) (E)-1-(2-Hydroxy-4,5-dimethoxyphenyl)-3-phenylprop-2-en-1-one (4c)

Using 2'-hydroxy-4',5'-dimethoxyacetophenone and benzaldehyde as starting materials, purification by flash column chromatography (toluene-EA 30:1, v/v) gave compound 4c as a yellow solid obtained (yield 87%).

R _f = 0.20 (hexane-EA=6:1,v/v).

¹ H NMR (300 MHz, CDCl ₃ ) δ 14.18 (s, 1H), 8.85 (d, J = 1.5 Hz, 1H), 8.60 (dd, J = 1.3 and 4.7 Hz, 1H), 7.97 (d, J = 15.7 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 15.7 Hz, 1H), 7.35 (dd, J = 4.9 and 7.8 Hz, 1H), 6.12 (d, J = 2.3 Hz, 1H), 5.98 (d, J = 2.3 Hz, 1H), 3.93 (s, 3H), 3.85 (s, 3H).

HRMS (ESI) m/z calculated for C ₁₇ H ₁₆ O ₄ [M+H] ⁺ : 285.1121; found : 285.1134.

(4) (E)-1-(2-Hydroxy-4,5-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (4d)

Using 2'-hydroxy-4',5'-dimethoxyacetophenone and 4-methoxybenzaldehyde as starting materials, flash column chromatography (hexane-EA=5:1 to 3:1, v/v) to give compound 4d as a yellow solid (yield 40%).

R _f = 0.41 (hexane-EA=2:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 13.48 (s, 1H), 7.88 (d, J = 15.3 Hz, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.63 (dd, J = 4.5 and 9.6 Hz, 1H), 7.41 (d, J = 15.3 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.96 (dd, J = 4.5 and 9.6 Hz, 1H), 6.51 (s, 1H) , 3.94 (s, 3H), 3.92 (s, 3H), 3.87 (s, 3H).

HRMS (ESI) m/z calculated for C ₁₈ H ₁₈ O ₅ [M+H] ⁺ : 315.1227; found : 315.1242.

Synthesis Example 2: General procedure for flavone synthesis

To a solution of chalcone in anhydrous DMSO was added iodine powder (1.1 eq) and stirred at 130° C. for 3 h, cooled to room temperature, and then the reaction mixture was diluted with ethyl acetate. The organic layer was washed with 0.1 M aqueous sodium thiosulfate solution, and then brine was added. The organic layer was collected, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by flash column chromatography to obtain flavone compounds represented by compounds 5a to 5d.

(1) 5,7-Dimethoxy-2-phenyl-4H-chromen-4-one (5a)

The compound 4a was used as a starting material and purified by flash column chromatography (toluene-E.A=5:1 to 1:1, v/v) to give compound 5a as a white solid (yield 43%).

R _f = 0.32 (DCM-EA=4:1,v/v).

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.87 (dd, J = 1.8 and 5.4 Hz, 2H), 7.507.51 (m, 3H), 6.69 (s, 1H), 6.57 (d, J = 2.4 Hz, 1H), 6.37 (d, J = 2.1 Hz, 1H), 3.96 (s, 3H), 3.92 (s, 3H).

HRMS (ESI) m/z calculated for C ₁₇ H ₁₄ O ₄ [M+H] ⁺ : 283.0965; found : 283.0977.

(2) 5,7-Dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one (5b)

The compound 4b was used as a starting material and purified by flash column chromatography (toluene-E.A, 10:1 to 3:1, v/v) to give compound 5a as a white solid (yield 72%).

R _f = 0.55 (DCM-EA=2:1,v/v).

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.83 (d, J = 8.1 Hz, 2H), 7.01 (d, J = 8.1 Hz, 2H), 6.61 (s, 1H), 6.57 (s, 1H), 6.38 (s, 1H), 3.96 (s, 3H), 3.92 (s, 3H), 3.89 (s, 3H).

HRMS (ESI) m/z calculated for C ₁₈ H ₁₆ O ₅ [M+H] ⁺ : 313.1071; found : 313.1083.

(3) 6,7-Dimethoxy-2-phenyl-4H-chromen-4-one (5c)

The compound 4c was used as a starting material and purified by flash column chromatography (DCM-E.A, 7:1 to 3:1, v/v) to give compound 5c as a white solid (yield 38%).

R _f = 0.38 (DCM-EA=4:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.92 (s, 1H), 7.57 (s, 1H), 7.54 (s, 3H), 7.02 (s, 1H), 6.81 (s, 1H), 4.03 (s, 3H), 4.00 (s, 3H).

HRMS (ESI) m/z calculated for C ₁₇ H ₁₄ O ₄ [M+H] ⁺ : 283.0965; found : 283.0979.

(4) 6,7-Dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one (5d)

The compound 4d was used as a starting material and purified by flash column chromatography (toluene-E.A, 10:1 to 4:1, v/v) to give compound 5d as a white solid (yield 78%).

R _f = 0.33 (DCM-EA=5:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.87 (d, J = 9.0 Hz, 2H), 7.57 (d, J = 9.0 Hz, 2H), 7.00 (s, 1H), 6.72 (s, 1H), 4.02 (s, 3H), 4.00 (s, 3H), 3.90 (s, 3H).

HRMS (ESI) m/z calculated for C ₁₈ H ₁₆ O ₅ [M+H] ⁺ : 313.1071; found : 313.1086.

Synthesis Example 3: General procedure for demethylation

To a solution of the 5a-5b flavones in anhydrous dichloromethane at 0° C., 3 boron bromide per methoxy group (5 equivalents) was added under an argon atmosphere, and the reaction mixture was stirred under reflux for 12 hours. After cooling to room temperature, the reaction mixture was quenched with ice water and concentrated under reduced pressure. The residue was partitioned between ethyl acetate and water, the aqueous layer was adjusted to pH 7 and extracted with ethyl acetate. The organic layer was collected, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by flash column chromatography to obtain demethylated compounds 6a to 6d.

(1) 5,7-Dihydroxy-2-phenyl-4H-chromen-4-one (6a)

The compound 5a was used as a starting material and purified by flash column chromatography (DCM-MeOH, 30:1 to 10:1, v/v) to give compound 6a as a white solid (yield 82%).

R _f = 0.72 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, CD ₃ OD)δ 7.94 (d, J = 8.7 Hz, 2H), 7.52 (s, 3H), 6.69 (s, 1H), 6.43 (s, 1H), 6.17 (s, 1H) ).

HRMS (ESI) m/z calculated for C ₁₅ H ₁₀ O ₄ [MH] : 253.0506; found : 253.0518.

(2) 5,7-Dihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (6b)

The compound 5b was used as a starting material and purified by flash column chromatography (DCM-E.A, 10:1 to 3:1, v/v) to give compound 6b as a white solid (yield 58%).

R _f = 0.38 (DCM-EA=4:1, v/v).

¹ H NMR (300 MHz, CD ₃ OD)δ 7.86 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.7 Hz, 2H), 6.60 (s, 1H), 6.46 (s, 1H), 6.21 (s, 1H).

HRMS (ESI) m/z calculated for C ₁₅ H ₁₀ O ₅ [MH] : 269.0455; found : 269.0452.

(3) 6,7-Dihydroxy-2-phenyl-4H-chromen-4-one (6c)

The compound 5c was used as a starting material and purified by flash column chromatography (DCM-MeOH, 10:1, v/v) to give compound 6c as a white solid (yield 75%).

R _f = 0.55 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, CD ₃ OD)δ 7.85 (d, J = 4.8 Hz, 2H), 7.44 (s, 3H), 7.30 (s, 1H), 6.92 (s, 1H), 6.66 (s, 1H) ).

HRMS (ESI) m/z calculated for C ₁₅ H ₁₀ O ₄ [MH] : 253.0506; found : 253.0518.

(4) 6,7-Dihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (6d)

The compound 5d was used as a starting material and purified by flash column chromatography (DCM-MeOH, 30:1 to 10:1, v/v) to give compound 6d as a white solid (yield 48%).

R _f = 0.18 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, CD ₃ OD)δ 7.87 (d, J = 7.8 Hz, 2H), 7.39 (s, 1H), 7.02 (s, 1H), 6.94 (d, J = 7.8 Hz, 2H), 6.67 (s, 1H).

HRMS (ESI) m/z calculated for C ₁₅ H ₁₀ O ₅ [MH] : 269.0455; found : 269.0449.

Synthesis Example 4: General process for reducing flavones to flavanones

To a solution of the flavones (6a-6b) in a mixture of 1,4-dioxane and methanol (4:1) was added palladium on carbon (10%). The reaction mixture was purged with hydrogen gas and stirred at room temperature for 24 hours, the reaction mixture was filtered through a pad of celite and concentrated under reduced pressure. The residue was purified by flash column chromatography to obtain flavanone compounds 7a to 7b.

(1) 5,7-Dihydroxy-2-phenylchroman-4-one (7a)

The compound 6a was used as a starting material and purified by flash column chromatography (DCM-EA 5:1, v/v) to obtain compound 7a as a white solid (yield 76%).

R _f = 0.53 (DCM-EA=3:1, v/v).

¹ H NMR (300 MHz, (CD ₃ ) ₂ CO)δ 12.17 (s, 1H), 9.69 (bs, 1H), 7.57 (dd, J = 1.2 and 8.1 Hz, 2H), 7.37-7.49 (m, 3H) ), 5.98 (dd, J = 2.1 and 10.2 Hz, 2H), 5.58 (dd, J = 3.0 and 12.9 Hz, 1H), 3.18 (dd, J = 12.9 and 17.1 Hz, 1H), 2.81 (dd, J = 3.0 and 17.1 Hz, 1H).

HRMS (ESI) m/z calculated for C ₁₅ H ₁₂ O ₄ [MH] : 255.0663; found : 255.0672.

(2) 5,7-Dihydroxy-2-(4-hydroxyphenyl)chroman-4-one (7b)

The compound 6b was used as a starting material and purified by flash column chromatography (DCM-EA 5:1, v/v) to obtain compound 7b as a white solid (yield 56%).

R _f = 0.30 (DCM-EA=3:1, v/v).

¹ H NMR (300 MHz, (CD ₃ ) ₂ CO)δ 12.19 (s, 1H), 9.52 (bs, 1H), 8.66 (bs, 1H), 7.40 (d, J = 8.4 Hz, 2H), 6.90 ( d, J = 8.4 Hz, 2H), 5.95 (s, 2H), 5.45 (dd, J = 3.0 and 12.9 Hz, 1H), 3.19 (dd, J = 12.9 and 17.1 Hz, 1H), 2.73 (dd, J = 3.0 and 17.1 Hz, 1H).

HRMS (ESI)m/z calculated for C ₁₅ H ₁₂ O ₅ [MH] : 271.0612; found : 271.0613.

Synthesis Example 5: Brominated flavone 8a to 8c compounds were synthesized from chalcone bromide by applying the same procedure for the synthesis of compound 5a.

(1) 2-(3-Bromophenyl)-5,7-dimethoxy-4H-chromen-4-one (8a)

Using (E)-3-(3-bromophenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)pro-2-en-1-one as starting material, flash column chromatography Purification with (toluene-EA, 5:1 to 1:1, v/v) gave a white solid compound 8a (yield 94%).

R _f = 0.28 (Toluene-EA=1:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 8.04 (t, J = 2.4 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.64 (dd, J = 1.5 and 7.2 Hz, 1H), 7.38 (t, J = 1.5, 1H), 6.66 (s, 1H), 6.59 (d, J = 2.1, 1H), 6.39 (d, J = 2.1, 1H), 3.97 (s, 3H), 3.93 (s, 3H).

HRMS (ESI) m/z calculated for C ₁₇ H ₁₃ BrO ₄ [M+H] ⁺ : 361.0070; found : 361.0076.

(2) 2-(3-Bromophenyl)-6,7-dimethoxy-4H-chromen-4-one (8b)

Using (E)-3-(3-bromophenyl)-1-(2-hydroxy-4,5-dimethoxyphenyl)pro-2-en-1-one as starting material, flash column chromatography (DCM-EA, 10:1 to 4:1, v/v) to give a white solid compound 8b (yield 78%).

R _f = 0.45 (DCM-EA=4:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 8.08 (s, 1H), 7.81 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 8.7 Hz, 1H), 7.56 (s, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.03 (s, 1H), 6.78 (s, 1H), 4.04 (s, 3H), 4.00 (s, 3H).

¹³ C NMR (75 MHz, CDCl ₃ )δ 177.2, 160.7, 154.5, 152.1, 147.7, 134.1, 133.8, 130.4, 128.9, 124.5, 123.2, 117.2, 107.5, 104.1, 99.7, 56.5, 56.3.

HRMS (ESI) m/z calculated for C ₁₇ H ₁₃ BrO ₄ [M+H] ⁺ : 361.0070; found : 361.0085.

(3) 2-(4-Bromophenyl)-6,7-dimethoxy-4H-chromen-4-one (8c)

Using (E)-3-(4-bromophenyl)-1-(2-hydroxy-4,5-dimethoxyphenyl)prop-2-en-1-one as starting material, flash column chromatography (DCM) to give a white solid compound 8c (yield 52%).

R _f = 0.50 (DCM-EA=10:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 7.76 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 7.55 (s, 1H), 6.99 (s, 1H), 6.76 (s, 1H), 4.02 (s, 3H), 3.99 (s, 3H).

¹³ C NMR (75 MHz, CDCl ₃ )δ 177.4, 161.5, 154.5, 152.1, 147.7, 132.2, 130.8, 127.4, 125.9, 117.2, 107.1, 104.2, 99.7, 56.5, 56.3.

HRMS (ESI) m/z calculated for C ₁₇ H ₁₃ BrO ₄ [M+H] ⁺ : 361.0070; found : 361.0085.

Synthesis Example 6: General procedure for the synthesis of biphenyl flavones (compounds 9a to 9i)

To a toluene solution of bromoflavones (8a to 8c) under argon atmosphere were added benzenebronic acid (1.2 equivalents) and tetrakis(triphenyl phosphine)palladium (0.1 equivalents), and cesium carbonate (2M solution, 10%) was added. The reaction mixture was stirred at 90 °C for 5 hours. After cooling to room temperature, the reaction mixture was partitioned between ethyl acetate and water, and the organic layer was collected, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by flash column chromatography to obtain biphenyl flavones (compounds 9a to 9i).

(1) 5,7-Dimethoxy-2-(4'-methoxy-[1,1'-biphenyl]-3-yl)-4H-chromen-4-one (9a)

The compound 8a and 4-methoxybenzeneboronic acid were used as starting materials and purified by flash column chromatography (hexane-acetone, 5:1 to 1:1, v/v) to obtain a white solid compound 9a ( yield 50%).

R _f = 0.21 (hexane-acetone=2:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 8.02 (s, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.50-7.59 (m, 3H) , 7.02 (d, J = 8.7 Hz, 2H), 6.74 (s, 1H), 6.59 (d, J = 2.4 Hz, 1H), 6.38 (d, J = 2.4 Hz, 1H), 3.96 (s, 3H) , 3.92 (s, 3H), 3.87 (s, 3H).

¹³ C NMR (75 MHz, CDCl ₃ )δ177.6, 162.6, 159.9, 154.5, 152.3, 147.7, 143.7, 132.2, 132.0, 131.9, 130.0, 128.6, 128.4, 128.2, 127.0, 126.5, 117.4, 114.4, 106.7, 104.4, 99.8, 56.4, 56.4, 55.4.

HRMS (ESI) m/z calculated for C ₂₄ H ₂₀ O ₅ [M+H] ⁺ : 389.1384; found: 389.1419.

(2) 5,7-Dimethoxy-2-(4'-nitro-[1,1'-biphenyl]-3-yl)-4H-chromen-4-one (9b)

The compound 8a and 4-nitrobenzeneboronic acid were used as starting materials and purified by flash column chromatography (hexane-acetone, 5:1 to 1:1, v/v) to obtain a white solid compound 9b (yield) 23%).

R _f = 0.17 (hexane-acetone=2:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 8.36 (d, J = 8.7 Hz, 2H), 8.10 (s, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.75-7.82 (m, 3H) , 7.64 (t, J = 7.8 Hz, 1H), 6.76 (s, 1H), 6.61 (d, J = 2.4 Hz, 1H), 6.14 (d, J = 2.4 Hz, 1H), 3.98 (s, 3H) , 3.93 (s, 3H).

(3) 2-([1,1'-Biphenyl]-3-yl)-5,7-dimethoxy-4H-chromen-4-one (9c)

The compound 8a and phenylboronic acid were used as starting materials and purified by flash column chromatography (petroleum ether-acetone, 4:1 to 1:1, v/v) to obtain a white solid compound 9c (yield 56%). ).

R _f = 0.37 (petroleum ether-acetone = 4:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 8.05 (s, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 7.2 Hz) , 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.53-7.44 (m, 2H), 7.43-7.35 (m, 1H), 6.73 (s, 1H), 6.57 (d, J = 2.1 Hz, 1H), 6.36 (d, J = 2.1 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 3H).

¹³ C NMR (75 MHz, CDCl ₃ )δ 177.7, 164.2, 161.1, 160.7, 160.1, 142.2, 140.4, 132.2, 130.1, 129.5, 129.1, 128.0, 127.3, 124.9, 124.8, 109.4, 96.4, 93.0, 56.5, 55.9 .

HRMS (ESI)m/z calculated for C ₂₃ H ₁₈ O ₄ [M+Na] ⁺ : 381.1097; found : 381.1095.

(4) 2-(4'-Fluoro-[1,1'-biphenyl]-3-yl)-5,7-dimethoxy-4H-chromen-4-one (9d)

The compound 8a and 4-fluorobenzeneboronic acid were used as starting materials and purified by flash column chromatography (petroleum ether-acetone 4:1 to 1:1, v/v) to obtain a white solid compound 9d ( yield 91%).

R _f = 0.30 (petroleum ther-acetone = 3:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 7.99 (s, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.63-7.50 (m, 3H) , 7.17 (t, J = 8.4 Hz, 2H), 6.72 (s, 1H), 6.58 (d, J = 2.4 Hz, 1H), 6.37 (d, J = 2.4 Hz, 1H), 3.95 (s, 3H) , 3.91 (s, 3H).

¹³ C NMR (75 MHz, CDCl ₃ )δ. 177.6, 164.3, 164.2, 161.1, 161.0, 160.5, 160.0, 141.2, 136.5, 136.4,132.3, 129.8, 129.6, 129.0, 128.9, 124.9, 124.6, 116.1, 115.8, 109.4, 96.37, 93.0, 56.5, 55.9.

HRMS (ESI) m/z calculated for C ₂₃ H ₁₇ FO ₄ [M+Na] ⁺ : 399.1003; found : 399.1001.

(5) 2-([1,1'-Biphenyl]-3-yl)-6,7-dimethoxy-4H-chromen-4-one (9e)

The compound 8b and benzeneboronic acid were used as starting materials and purified by flash column chromatography (petroleum ether-acetone, 10:1 to 3:1, v/v) to obtain a white solid compound 9e (yield 63%). ).

R _f = 0.38 (petroleum ether-acetone = 4:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 8.12 (s, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.59-7.67 (m, 4H) , 7.51 (t, J = 6.9, 2H), 7.43 (d, J = 7.5 Hz, 1H), 7.03 (s, 1H), 6.87 (s, 1H), 4.04 (s, 3H), 4.01 (s, 3H) ).

¹³ C NMR (75 MHz, CDCl ₃ )δ 177.5, 162.5, 154.5, 152.2, 147.7, 142.1, 140.1, 132.4, 132.2, 132.0, 131.9, 131.9, 130.0, 129.4, 129.0, 128.6, 128.4, 127.9, 127.2, 124.8 , 124.7, 117.3, 107.2, 104.3, 99.9, 56.9, 55.9.

HRMS (ESI) m/z calculated for C ₂₃ H ₁₈ O ₄ [M+H] ⁺ : 359.1278; found:359.1294.

(6) 2-(4'-Fluoro-[1,1'-biphenyl]-3-yl)-6,7-dimethoxy-4H-chromen-4-one (9f)

The compound 8b and 4-fluorobenzeneboronic acid were used as starting materials and purified by flash column chromatography (petroleum ether-acetone, 10:1 to 3:1, v/v) to obtain a white solid compound 9f. (Yield 55%).

R _f = 0.31 (petroleum ether-Acetone = 3:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 8.06 (s, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.59-7.63 (m, 4H) , 7.19 (t, J = 8.7, 2H), 7.03 (s, 1H), 6.86 (s, 1H), 4.04 (s, 3H), 4.00 (s, 3H).

¹³ C NMR (75 MHz, CDCl ₃ )δ 177.6, 162.6, 154.6, 152.4, 147.8, 141.2, 132.6, 132.3, 132.1, 132.1, 132.0, 129.9, 129.6. 129.0, 128.9, 128.7, 128.5, 125.0, 124.7, 117.5, 116.1, 115.8, 107.4, 104.5, 99.9, 56.9, 55.9.

HRMS (ESI) m/z calculated for C ₂₃ H ₁₇ FO ₄ [M+H] ⁺ : 377.1184; found : 377.1196.

(7) 6,7-Dimethoxy-2-(4'-methoxy-[1,1'-biphenyl]-3-yl)-4H-chromen-4-one (9g)

The compound 8b and 4-methoxy benzeneboronic acid were used as starting materials and purified by flash column chromatography (petroleum ether-Acetone, 10:1 to 3:1, v/v) to obtain 9 g of a white solid compound. (yield 78%).

R _f = 0.35 (petroleumether-acetone=3:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 8.08 (s, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.54-7.61 (m, 4H) , 7.05 (s, 1H), 7.03 (s, 2H), 6.86 (s, 1H), 4.04 (s, 3H), 4.01 (s, 3H), 3.88 (s, 3H).

¹³ C NMR (75 MHz, CDCl ₃ )δ 177.7, 162.9, 159.8, 154.7, 152.5, 147.9, 141.9, 132.8, 132.6, 132.3, 132.2, 132.0, 129.8, 129.5, 128.7, 128.5, 128.4, 124.5, 124.4, 117.5 , 114.6, 56.6, 56.5, 55.5.

HRMS (ESI) m/z calculated for C ₂₄ H ₂₀ O ₅ [M+H] ⁺ : 389.1384; found : 389.1407.

(8) 6,7-Dimethoxy-2-(4'-nitro-[1,1'-biphenyl]-3-yl)-4H-chromen-4-one (9h)

The compound 8b and 4-nitrobenzeneboronic acid were used and purified by flash column chromatography (petroleumether-acetone, 5:1 to 1:1, v/v) to obtain a white solid compound 9h (yield 24%). .

R _f = 0.25 (Petroleum ether-Acetone = 3:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 8.37 (d, J = 8.4 Hz, 2H), 8.14 (s, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.77-7.83 (m, 3H) , 7.67 (t, J = 7.5 Hz, 1H), 7.59 (s, 1H), 7.04 (s, 1H), 6.88 (s, 1H), 4.04 (s, 3H), 4.01 (s, 3H).

(9) 6,7-Dimethoxy-2-(4'-methoxy-[1,1'-biphenyl]-4-yl)-4H-chromen-4-one (9i)

The compound 8c and 4-methoxy benzeneboronic acid were used as starting materials and purified by flash column chromatography (toluene-EA 10:1 to 3:1, v/v) to obtain a white solid compound 9i (yield). 63%).

R _f = 0.20 (Hexane-EA=3:1, v/v).

¹ H NMR (300 MHz, CDCl ₃ )δ 7.96 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.62 (s, 1H), 7.59 (d, J = 2.4 Hz) , 2H), 7.01-7.04 (m, 3H), 6.83 (s, 1H), 4.04 (s, 3H), 4.01 (s, 3H), 3.88 (s, 3H).

HRMS (ESI) m/z calculated for C ₂₄ H ₂₀ O ₅ [M+H] ⁺ : 389.1384; found : 389.1408.

Synthesis Example 7: General procedure for the synthesis of compounds represented by 10a to 10i

The ring-expanding flavone compound was dissolved in anhydrous dichloromethane and boron tribromide (1 M solution, 5 equivalents of methoxy functional groups) was added at 0 °C under an argon atmosphere. The reaction mixture was stirred under reflux for 12 hours, then cooled to room temperature and the reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate and water. The aqueous layer was adjusted to pH 7 and redistributed with ethyl acetate. The organic layer was collected, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by flash column chromatography to obtain final compounds (10a to 10i).

(1) 5,7-Dihydroxy-2-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4H-chromen-4-one (10a)

The compound 9a was used as a starting material and purified by flash column chromatography (DCM-MeOH, 10:1 to 3:1, v/v) to obtain a white solid compound 10a (yield 38%).

R _f = 0.40 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, tetrahydrofuran-d ₈ )δ 12.86 (brs, 1H), 8.15 (s, 1H), 7.87 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H) , 7.55 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H), 6.85 (d, J = 7.8 Hz, 3H), 6.43 (s, 1H), 6.17 (s, 1H) .

¹³ C NMR (75 MHz, tetrahydrofuran-d ₈ )δ 182.8,165.3, 164.4, 163.5, 158.8, 158.8, 142.9, 132.9, 131.7, 130.1, 129.9, 128.7, 124.7, 116.4, 106.3, 105.4, 99.6, 94.4.

HRMS (ESI) m/z calculated for C ₂₁ H ₁₄ O ₄ [MH] ^- : 345.0768; found : 345.0769.

(2) 5,7-Dihydroxy-2-(4'-nitro-[1,1'-biphenyl]-3-yl)-4H-chromen-4-one (10b)

The compound 9b was used as a starting material and purified by flash column chromatography (DCM-MeOH, 30:1 to 10:1, v/v) to obtain a white solid compound 10b (yield 13%).

R _f = 0.60 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, tetrahydrofuran-d ₈ ) δ 12.81 (s, 1H), 9.64 (brs, 1H), 8.35 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H) , 8.01 (d, J = 8.7 Hz, 2H), 7.94 (d, J = 8.4 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 6.93 (s, 1H), 6.45 (d, J = 2.1 Hz, 1H), 6.19 (d, J = 2.1 Hz, 1H).

¹³ C NMR (75 MHz, tetrahydrofuran-d ₈ ) δ 182.9, 165.6, 163.8, 163.7, 159.0, 148.7, 147.1, 140.7, 133.6, 131.3, 130.7, 129.0, 127.4, 126.1, 124.8, 106.9, 105.6, 99.9, 94.6 , 30.6.

HRMS (ESI) m/z calculated for C ₂₁ H ₁₃ NO ₆ [MH] ^- : 374.0670; found : 374.0685.

(3) 2-([1,1'-Biphenyl]-3-yl)-5,7-dihydroxy-4H-chromen-4-one (10c)

The compound 9c was used as a starting material and purified by flash column chromatography (DCM-MeOH, 50:1 to 10:1, v/v) to obtain a white solid compound 10c (yield 54%).

R _f = 0.62 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, tetrahydrofuran-d ₈ ) δ 12.85 (brs, 1H), 8.24 (s, 1H), 7.97 (d, J = 7.5 Hz, 1H), 7.81 (d, J = 7.5 Hz, 1H) , 7.61 (d, J = 6.9 Hz, 2H), 7.60 (t, J = 7.8 Hz, 1H), 7.46 (t, J = 6.9 Hz, 2H), 7.36 (t, J = 7.2 Hz, 1H), 6.87 (s, 1H), 6.45 (s, 1H), 6.19 (s, 1H).

¹³ C NMR (75 MHz, tetrahydrofuran-d ₈ ) δ 182.8, 165.4, 164.1, 163.5, 158.8, 142.9, 140.9, 133.1, 130.8, 130.1, 129.5, 128.5, 127.8, 125.8, 125.5, 106.5, 105.4, 99.7, 94.4 .

HRMS (ESI) m/z calculated for C ₂₁ H ₁₄ O ₄ [MH] ^- : 329.0819; found : 329.0819.

(4) 2-(4'-Fluoro-[1,1'-biphenyl]-3-yl)-5,7-dihydroxy-4H-chromen-4-one (10d)

The compound 9d was used as a starting material and purified by flash column chromatography (DCM-MeOH, 50:1 to 10:1, v/v) to give compound 10d as a white solid (yield 51%).

R _f = 0.59 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, tetrahydrofuran-d ₈ ) δ 12.82 (s, 1H), 9.80 (brs, 1H), 8.20 (s, 1H), 7.96 (d, J = 7.0 Hz, 1H), 7.78-7.71 ( m, 3H), 7.62-7.55 (m, 3H), 7.24-7.17 (m, 2H), 6.86 (s, 1H), 6.44 (s, 1H), 6.19 (s, 1H).

¹³ C NMR (75 MHz, tetrahydrofuran-d ₈ ) δ 183.0, 165.6, 164.2, 163.7, 159.0, 142.0, 137.3, 137.3, 133.3, 130.8, 130.4, 129.9, 129.8, 125.9, 125.5, 116.6, 116.3, 106.7, 105.6 , 99.9, 94.6.

HRMS (ESI) m/z calculated for C ₂₁ H ₁₃ FO ₄ [MH] ^- : 347.0725; found : 347.0729.

(5) 2-([1,1'-Biphenyl]-3-yl)-6,7-dihydroxy-4H-chromen-4-one (10e)

The compound 9e was used as a starting material and purified by flash column chromatography (DCM-MeOH, 30:1 to 10:1, v/v) to obtain a white solid compound 10e (yield 46%).

R _f = 0.21 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, tetrahydrofuran-d ₈ ) δ 8.26 (brs, 1H), 9.12 (brs, 1H), 8.22 (s, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 7.2 Hz, 2H), 7.57 (t, J = 7.8, 1H), 7.48-7.41 (m, 3H),7.35 (t, J = 7.2 Hz, 1H) ), 7.00 (s, 1H), 6.82 (s, 1H).

¹³ C NMR (75 MHz, tetrahydrofuran-d ₈ ) δ 176.9, 162.4, 153.1, 152.4, 145.0, 142.6, 141.0, 133.9, 130.0, 129.9, 129.4, 128.2, 127.7, 125.4, 125.1, 117.8, 108.5, 107.2, 103.5 .

HRMS (ESI) m/z calculated for C ₂₁ H ₁₄ O ₄ [MH] ^- : 329.0819; found : 329.0829.

(6) 2-(4'-Fluoro-[1,1'-biphenyl]-3-yl)-6,7-dihydroxy-4H-chromen-4-one (10f)

The compound 9f was used as a starting material and purified by flash column chromatography (DCM-MeOH, 30:1 to 10:1, v/v) to give compound 10f as a white solid (yield 33%).

R _f = 0.38 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, tetrahydrofuran-d ₈ ) δ 9.13 (brs, 1H), 8.98 (brs, 1H), 8.02 (s, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.58 (t, J = 7.8, 1H), 7.39 (s, 1H), 7.21 (t, J = 8.7, 2H), 6.97 (s, 1H), 6.80 (s, 1H).

¹³ C NMR (75 MHz, tetrahydrofuran-d ₈ ) δ 176.6, 162.2, 153.0, 152.4, 144.9, 141.6, 137.4, 134.1, 130.0, 129.9, 129.7, 129.6, 125.5, 125.1, 118.0, 116.4, 116.1, 108.6, 107.4 , 103.6, 30.5.

HRMS (ESI) m/z calculated for C ₂₁ H ₁₃ FO ₄ [MH] ^- : 347.0725; found : 347.0741.

(7) 6,7-Dihydroxy-2-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4H-chromen-4-one (10 g)

9 g of the compound was used as a starting material and purified by flash column chromatography (DCM-MeOH, 30:1 to 10:1, v/v) to obtain 10 g of a white solid compound (yield 25%).

R _f = 0.25 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, CD ₃ OD)δ 8.13 (s, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 6.6 Hz, 1H), 7.55-7.61 (m, 3H) ), 7.43 (s, 1H), 7.08 (s, 1H), 6.91 (d, J = 8.4, 2H), 6.86 (s, 1H).

HRMS (ESI) m/z calculated for C ₂₁ H ₁₄ O ₄ [MH] ^- : 345.0768; found : 345.0757.

(8) 6,7-Dihydroxy-2-(4'-nitro-[1,1'-biphenyl]-3-yl)-4H-chromen-4-one (10h)

The compound 9h was used as a starting material and purified by flash column chromatography (DCM-MeOH, 30:1 to 10:1, v/v) to give compound 10h as a white solid (yield 30%).

R _f = 0.50 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, CD ₃ OD)δ 8.46 (s, 1H), 8.40 (d, J = 8.7 Hz, 2H), 8.13-8.16 (m, 3H), 8.01 (d, J = 8.4 Hz, 1H) ), 7.77 (t, J = 7.8 Hz, 1H), 7.51 (s, 1H), 7.20 (s, 1H), 6.90 (s, 1H).

HRMS (ESI) m/z calculated for C ₂₁ H ₁₃ NO ₆ [MH] ^- : 374.0670; found : 374.0686.

(9) 6,7-Dihydroxy-2-(4'-hydroxy-[1,1'-biphenyl]-4-yl)-4H-chromen-4-one (10i)

The compound 9i was used as a starting material and purified by flash column chromatography (DCM-MeOH, 30:1 to 10:1, v/v) to give compound 10i as a white solid (yield 33%).

R _f = 0.50 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, CD ₃ OD)δ 8.09 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.7 Hz, 2H), 7.49 ( s, 1H), 7.16 (s, 1H), 6.98 (d, J = 8.7 Hz, 2H), 6.75 (s, 1H).

HRMS (ESI) m/z calculated for C ₂₁ H ₁₄ O ₄ [MH] ^- : 345.0768; found : 345.0765

Synthesis Example 8: Synthesis of 5,7-Dihydroxy-2-(4'-hydroxy-[1,1'-biphenyl]-3-yl)chroman-4-one (11a)

A mixture of compound 10a (43 mg, 0.12 mmol) and 10% Pd/C (30 mg) in 5 mL of 1,4-dioxane-EtOH (1/4, v/v) was placed in a Parr shaker apparatus at 50 psi H ₂ was stirred for 23 h to hydrogenate. After filtration, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM:MeOH, 50:1 to 10:1, v/v) to give compound 11a (68 mg, 68%) as a colorless oil. did.

R _f = 0.71 (DCM-MeOH=10:1, v/v).

¹ H NMR (300 MHz, CD ₃ OD)δ 7.47 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.44 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 7.8) Hz, 1H), 7.35 (t, J = 6.0 Hz, 1H), 6.86 (d, J = 8.7 Hz, 2H), 5.93 (dd, J = 2.2 and 14.4 Hz, 2H), 5.46 (dd, J = 3.0) and 12.3 Hz, 1H), 3.11 (dd, J = 12.8 and 17.1 Hz, 1H), 2.78 (dd, J = 3.1 and 17.1 Hz, 1H).

¹³ C NMR (75 MHz, CD ₃ OD)δ 197.3, 168.4, 165.4, 164.6, 158.4, 142.8, 140.8, 133.3, 130.1, 129.1, 128.6, 127.6, 125.4, 125.3, 116.7, 116.1, 103.4, 97.2, 96.3, 80.5, 44.2.

HRMS (ESI) m/z calculated for C ₂₁ H ₁₆ O ₅ [MH] ^- : 347.0925; found : 347.0927.

Experimental Example 1

The kinetic aqueous solubility was measured with a filter device (Whatman UniPrep Syringeless Filter Device, 1 mL, PTFE membrane, 0.45 μm pore size, Cat. No. US113UORG), high-performance liquid chromatography (Thermo UltiMate 3000 HPLC system equipped with on-line) degasser, quaternary pump, thermostatted auto-sampler, column compartment and diode array detector) and an Agilent Eclipse plus C18, 4.6 × 100 mm, 3.5 μm column.

After preparing a 10 mM solution of the test compound in DMSO, 25 µL is placed in a 1 mL uniprep vial containing 475 µL of 0.1 M potassium phosphate buffer (pH 7.4), stirred at room temperature for 90 min at 800 rpm, and then the mixture is stirred into 1 mL Mini -Filter under pressure with the top tube of a Uniprep filter (PTFE membrane, 0.45 μm). Then, 250 μL of the filtrate was transferred to an HPLC vial containing an equal volume of acetonitrile. Calibration samples for HPLC analysis were prepared using 10 mM DMSO stock solution in 50 v/v % acetonitrile in deionized water or phosphate buffer (concentrations 500, 100, 20, 5, 1 and 0 μM). The diluted filtrate and calibration samples were analyzed by HPLC, and the solubility of the test sample was calculated by multiplying the measured concentration by a dilution factor.

Experimental Example 2

The ELISA analysis was performed using a Ni-NTA HisSorb plate (Qiagen, Germany), and 100 μL of a solution containing hTSLPR with a C-terminal octa-histidine tag (TSLPR-His) was added to each well and room temperature. After incubation for 2 h in , plates were washed 3 times with 200 µL of PBS containing 0.05% Tween-20 to remove unbound TSLPR-His, and TSLP with N-terminal FLAG tag (FLAG- hTSLP) were incubated overnight (15 h) at 4 °C for every 100 μL, then the plates were washed 3 times and blocked with 100% blocking buffer (0.05% Tween 20 and 1% non-fat milk powder in PBS). The plate was washed twice to remove unbound FLAG-hTSLP, and then coated with 100 μL of monoclonal anti-FLAG antibody conjugated to HRP (Sigma-Aldrich Co., USA) at room temperature for 2 hours. After incubation, the plate was washed 5 times, and 200 μL of o-phenylene diamine dihydrochloride (Sigma-Aldrich Co., USA) solution was further treated and incubated for 30 minutes, then 1 N HCl was added to stop the reaction.

Optical density (OD) was measured at 450 nm using a microplate spectrophotometer, and the TSLP inhibitory effect was calculated using the following formula.

Inhibitory effect (%) = (1 - OD of sample / OD of control) × 100

Experimental Example 3 STAT5 analysis method

(1) cell culture

Human mast cell line-1 (HMC-1) cells were obtained from the Department of Food Technology and Inflammatory Disease Research Center (Hoseo University, Chungnam, Asan), 10% FBS and 1% Penicillin-streptomycin (PS; Gibco Industries Inc, USA) supplemented with Dulbecco's medium (IMDM, Hyclone Laboratories Inc, USA) _{, cells were cultured in 95% air humidified atmosphere and 5% CO 2} atmosphere, and recombinant hTSLP was purchased from R&D Systems (Minneapolis, USA).

(2) flow cytometry

Intracellular phospho-STAT5 (pSTAT5) staining method was performed based on a protocol obtained in the laboratory of Susan Kaech, Department of Immunobiology, Yale University School of Medicine (New Haven, Connecticut, USA), and HMC-1 cells were treated with 96-well U-bottom Plates were seeded at a density of 1×10 ⁷ cells/mL and stimulated with 100 ng/mL recombinant hTSLP alone or compound 1 for 30 minutes. After stimulation, cells were fixed with BD Cytofix/Cytoperm (BD Biosciences, USA) solution for 10 minutes. The fixed cells were then infiltrated with ice-cold methanol for 30 min, and intracellular pSTAT5 was stained with Alexa Fluor 647 mouse anti-STAT5 (pY694, BD Biosciences, USA) (1 :10 dilution in FACS buffer) for 30 min. Stained cells were further fixed with BD Cytofix/Cytoperm solution for 10 min and resuspended in FACS buffer for flow cytometry analysis. Cells were analyzed by BD LSR Fortessa Flow cytometry (BD Biosciences, USA). FACS data were analyzed using FlowJo software version 9.7 (Tree Star Inc., USA).

Experimental Example 4

Western blot analysis showed that HMC-1 cells were pretreated in the absence or presence of compounds (0.1, 1, 3, 10 μM) followed by stimulation with TSLP (20 ng/mL) for 2 h. Activated cells were lysed and separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred to nitrocellulose membranes and incubated with primary antibody (diluted 1:700 in PBS containing 0.05% Tween-20), followed by peroxidase-conjugated secondary antibody (containing 0.05% Tween-20). 1:3000 dilution in PBS) was used.

Claims (8)

A chromenone derivative represented by the following [Formula 1] or [Formula 2]:
[Formula 1]

[Formula 2]

In the [Formula 1] or [Formula 2],
R ^{1 To} R ^{3 Are} the same as or different from each other, and each independently represents a hydrogen or a hydroxyl group, and at least two or more of ^{R 1 To} R ^{3 are a hydroxyl group,}
R ⁴ is any one selected from a hydroxyl group, a halogen group and a nitro group,
The chromenone derivative inhibits the binding between thymic stromal lymphopoietin (TSLP) and the TSLP receptor,
The chromenone derivative controls intracellular signal transduction by thymic stromal lymphoprotein (TSLP) to inhibit intracellular phosphorylation of STAT5. delete delete According to claim 1,
The [Formula 1] or [Formula 2] is a chromenone derivative, characterized in that any one selected from the following formula:
[Formula 3] [Formula 4]

[Formula 5] [Formula 6]

[Formula 9] [Formula 10]

[Formula 13] [Formula 14]

[Formula 15] [Formula 16]

[Formula 17] [Formula 18]

A pharmaceutical composition for the prevention or treatment of allergic diseases, comprising the chromenone derivative according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier,
The pharmaceutical composition inhibits the binding between TSLP (thymic stromal lymphopoietin) and the TSLP receptor, and controls intracellular signal transduction by TSLP (thymic stromal lymphoprotein), characterized in that it inhibits the phosphorylation of STAT5 in the cell,
The allergic disease is a pharmaceutical composition for the prevention or treatment of allergic diseases, characterized in that atopic dermatitis, urticarial rhinitis or allergic rhinitis. delete delete delete

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