KR102060412B1 - Synthetic method for portulacanone compounds and their derivatives and anti-inflammatory pharmaceutical compounds containing thereof - Google Patents

Abstract

We describe natural homoisoflavonoids (±) -portulacanone AC (compounds 4,8,9) and derivatives thereof isolated from Portulaca oleracea L (POL), including known portulacanone derivatives (compounds 1 and 2). (Compounds 3, 5 and 7) were synthesized. The ability to inhibit NO production in LPS-induced RAW 264.7 macrophages was assessed as an indicator of anti-inflammatory activity. All compounds tested showed no clear cytotoxicity and inhibited NO production in a concentration dependent manner in RAW 264.7 macrophages. Compound 3 (97.2% inhibition at 10 μM; IC50 = 1.26 μM) was significantly inhibited compared to Compound 1 (91.4% inhibition at 10 μM; IC50 = 1.75 μM) and Compound 7 (83.0% inhibition at 10 μM; IC50 = 2.91 μM) Indicated. The compounds of the present invention are useful for the development of NO producing targeted anti-inflammatory drugs.

Description

Synthetic method for portulacanone compounds and their derivatives and anti-inflammatory pharmaceutical compounds containing various}

The present invention relates to (±) -portulacanone AC (compounds 4,8,9) and natural homoisoflavonoid compounds isolated from Portulaca oleracea L (POL), including known portulacanone derivatives (compounds 1 and 2) and their A method of synthesizing derivatives (compounds 3, 5 and 7), and anti-inflammatory pharmaceutical compositions comprising the same.

Inflammation is a fundamental protective response of body cells / tissues to infection by pathogens, damaged cells or stimulants (1,2). Inflammation It is considered the beginning of the course of treatment and can be divided into acute or chronic inflammation. One is the host's adaptive defense mechanism against infection or injury and is self-limiting, while the other can cause various diseases including diabetes, arthritis and cancer. During the inflammatory process nitric oxide (NO), prostaglandins (PG), vascular active amines (histamines, serotonin), leukotriene (LT), cytokines (tumor necrosis factor and interleukin-1, 12) are secreted as plasma proteins or mast cells, Secreted by cells such as neutrophils, platelets and mononuclear phagocytes / macrophages. These mediators bind to specific target receptors in cells to increase vascular permeability, promote smooth muscle contraction, promote neutrophil chemotaxis, increase direct enzyme activity, induce pain and / or mediate oxidative damage ( 3).

Nitric oxide is one of the important signaling molecules synthesized from the amino acid L-arginine by nitric oxide synthase (endothelium-NOS, neuro-NOS and induced-NOS). Nitric oxide regulates a variety of physiological and pathophysiological processes and the concentration of NO plays a critical role in the development of inflammation (4). High levels of physiological NO produced by endothelial-NOS and neuronal-NOS promote cell survival and proliferation, whereas high levels of NO produced by induced-NOS stop cell cycles and promote cell death (5). The interaction of NO with active oxygen species such as superoxide and hydroxyl radicals leads to a decrease in NO concentration, impedes signaling activity, and the resulting reactive nitrogen species are oxidative and nitrifying stresses. nitrosative stress response can be increased (6). Non-steroidal anti-inflammatory drugs, steroids and antihistamines are generally preferred for the treatment of pain, inflammation and fever, but these agents have disadvantages. Therefore, pharmacological interference with the NO production cascade among various therapeutic interventions for inflammatory diseases is claimed as a promising strategy.

Subclasses of naturally occurring flavonoid compounds, especially flavonoids known as homoisoflavonoids, have shown great biological potential in recent years (7,8). Previous studies show the antioxidant, anti-inflammatory, antibacterial, antifungal, antiviral, anticytotoxic, antimutagenic and antidiabetic activity of homoisoflavonoids (8, 9). In addition, some derivatives showed enzymatic inhibition properties (10).

Tan et al. Plant formate Tula car oleic la Seah L. (Portulaca oleracea L.; POL) formate Tula canon (portulacanone) were isolated four homo iso flavonoid called AD (Fig. 1), formate Tula car oleic la Seah L. in silver It is a widely distributed medicinal plant that is used not only as an edible plant but also as a folk medicine to alleviate a wide range of diseases (11). These compounds exhibited cytotoxic activity in vitro against four human tumor cell lines including SGC-7901, NCI-H460, K-562 and SF-268 after isolation (11). To date, there are no reports on the synthesis of fortulacanone AD and the NO production of these compounds and their derivatives.

Quintans J. Immunol Cell Biol. 1994; 72: 262-264. Fullerton JN, Gilroy DW. Nat. Rev Drug Discov. 2016; 15: 551-567. Coleman JW. Clin Exp Immunol. 2002; 129: 4-10. Lyons CR. Adv Immunol. 1995; 60: 323-371. Lundberg J. et al., Nat Med. 1997; 3: 30-31. Thomas DD et al., Free Radic Biol Med. 2008; 45: 18-31. Andersen, ØM et al., Flavonoids: Chemistry, Biochemistry and Applications. Boca Raton, CRC Press; 2006. Lin L-G et al., Planta Med. 2014; 80: 1053-1066. Abegaz BM et al., Nat Prod Commun. 2007; 2: 475-498. Lin LG et al., J Med Chem. 2008; 51: 4419-4429. Jian Yan J et al., Phytochemistry 2012; 80: 37-41. Iranshahy M et al., J Ethnopharmacol. 2017; 205: 158-172. Damodar K, Kim J-K, Jun J-G. Tetrahedron Lett. 2017; 58: 50-53. Seo YH, Damodar K, Kim J-K, Jun J-G. Bioorg Med Chem Lett. 2016; 26: 1521-1524. Kontogiorgis CA, Hadjipavlou-Litina D. Med Res Rev. 2002; 22: 385-418.

It is an object of the present invention to provide a method for synthesizing portulacanone A-C and its derivatives.

It is also an object of the present invention to provide a useful medicament by studying the bioactivity of portulacanone A-C and its derivatives.

As part of a continuing study focused on the synthesis and evaluation of natural products and their analogs that are bioactive as nitric oxide inhibitors, the present invention includes (±) -portulacanone AC (compounds 4, 8 and 9). And derivatives thereof (compounds 1-3, 5 and 7) (FIG. 1).

Synthesis of portulacanone or its derivatives (Compound 1-9) began with Friedel-Crafts acylation of commercially available 1,3,5-trimethoxybenzene (Compound 10) ( 2). Compound 11 was obtained in 89% yield by treating compound 10 with acetic anhydride in the presence of boron trifluoride diethyl etherate (BF ₃ · Et ₂ O). Compound 11 using AlCl ₃ selective ortho-demethylated to give the compound 12 is (ortho -demethylation) in a yield of 87%. Another acetophenone 14 is obtained from compound 13. Acetylation and subsequent rearrangement of compound 13 yielded compound 14 in a significant yield of 93% over two steps. Compound 16 protected by ethoxymethyl (EOM) ether of salicylate (compound 15) using chloromethylethyl ether (EOM-Cl), K ₂ CO ₃ and TBAI (tetrabutylammonium iodide) was obtained in a yield of 83%. . Next, commercially available 2-hydroxy-4-methoxyacetophenone (Compound 17), Compound 12 and Compound 14 were prepared. DMF-DMA (N, N -dimethylformamide dimethyl acetal) corresponding After condensation, the resulting amino ketone to the acid treatment with 4 H-chromen-4-one (4 H -chromen-4-ones ) compound 18a-18c Were obtained in yield 88-91% respectively. Hydrogenation with a catalyst of compounds 18a-18c afforded the corresponding chroman-4-ones compounds 19a-19c.

After obtaining compounds 19a-19c and compound 16, we studied the aldol condensation reaction. At first, the reaction between compound 19b and compound 15 using p -toluenesulfonic acid ( p TsOH) and benzene was unsuccessful. Next, compound 20b was obtained in low yield (less than 20%) in the condensation reaction of compound 19b with 16 using piperidine as base in benzene solvent as well as DMF solvent at 30-100 ° C. Satisfactorily, we found that KOH in EtOH / H ₂ O (5/1) was effective for condensation of compound 19b and compound 16. Subsequently, the reaction of Compound 19a, Compound 19c, and Compound 16 yielded Compound 20a (48%) and Compound 20c (25%), respectively. Deprotection of compounds 20a-20c with 1N HCl afforded compounds 1, 3 and 7 in high yield. The hydrogenation reaction using the catalysts of compounds 1, 3 and 7 gave homoisoflavonoid compounds 2, 4 and 8 in yields 86-91%, respectively. Finally, the remaining three target compounds 5, 9 and 6 were well synthesized by the selective ortho-demethylation of compounds 4, 8 and 3 using 1.0M BCl ₃ (in CH ₂ Cl ₂ solution). The structure of all final compounds 1-9 was determined by spectral data ( ¹ H, ¹³ C NMR and MS).

The synthesized homoisoflavonoids (Compounds 1-9) were analyzed for their ability to inhibit NO production in RAW 264.7 macrophages induced by LPS. It is well known that treatment of RAW 264.7 macrophages with LPS induces NO production. ^{L-NMMA (N G -monomethyl-} L-arginine) was reported to significantly inhibit the NO production (15). In the present invention, RAW 264.7 macrophages were induced by LPS and NO production and cell viability were measured using compounds 1-9 and L-NMMA at concentrations of 0.1, 1, 10, 25, 50 and 100 μM as controls. However, at concentrations of 25, 50 and 100 μM Compounds 1-9 showed almost the same level of activity (FIG. 3). Significant changes in NO production inhibition were observed at 1-10 μM. Therefore, the present inventors analyzed the synthesized compound 1-9 only at 1 and 10 μM concentration. The concentration of NO from macrophages was determined by measuring the concentration of nitrite, a stable oxidation product of NO, in the culture supernatant using a Greases reagent.

Compound
NO Production (% inhibition) 1 μM 10 μM IC ₅₀ (μM) LPS 100.0 ± 0.8 (0.0) 100.0 ± 0.8 (0.0) - One 71.6 ± 1.4 (28.4) *** 8.6 ± 1.2 (91.4) *** 1.75 2 90.8 ± 1.7 (9.2) * 80.3 ± 6.5 (19.7) *** 14.17 3 61.5 ± 1.7 (38.5) *** 2.8 ± 0.7 (97.2) *** 1.26 4 102.8 ± 1.7 (-2.8) 79.5 ± 7.3 (20.5) *** 14.17 5 97.2 ± 1.7 (2.8) 62.0 ± 2.1 (38.0) *** 12.42 6 78.9 ± 2.0 (21.1) *** 7.5 ± 0.8 (92.5) *** 2.09 7 83.8 ± 1.9 (16.2) ** 17.0 ± 0.8 (83.0) *** 2.91 8 124.6 ± 17.6 (-24.6) 84.1 ± 2.7 (15.9) 13.43 9 111.8 ± 1.3 (-11.8) * 65.9 ± 2.9 (34.1) *** 12.16 L-NMMA 112.5 ± 4.6 (-12.5) 85.8 ± 6.4 (14.2) 16.11

All compounds 1-9 tested reduced NO production in a concentration dependent manner in RAW 264.7 macrophages (Table 1). The percentage of NO inhibition ranged from 97.2% to 15.9% at the highest (10 μM) concentration. Four of the compounds 1-9, namely Compound 3 (97.2%), Compound 6 (Fortulacanone D) (92.5%), Compound 1 (91.4%), and Compound 7 (83.0%) were most effective at 10 μM (Table 1 and Figure 3). At the lowest concentration (1 μM), compound 3 still significantly reduced NO production (38.5%) in RAW 264.7 macrophages. IC ₅₀ values of compounds 1-9 were evaluated with GraphPad Prism 4.0 software, showing 1.75, 14.17, 1.26, 14.17, 12.42, 2.09, 2.91, 13.43 and 12.16 μM, respectively (Table 1).

Compound Proliferation effect 1 μM 10 μM 50 μM 100 μM Medium (MED) 100.0 ± 2.4 100.0 ± 2.4 100.0 ± 2.4 100.0 ± 2.4 One 101.8 ± 1.5 97.7 ± 13.3 11.0 ± 0.1 *** 11.1 ± 0.2 *** 2 152.1 ± 2.4 *** 118.3 ± 1.1 49.4 ± 3.9 *** 31.5 ± 0.3 *** 3 107.6 ± 12.0 113.6 ± 5.5 13.3 ± 1.6 *** 11.7 ± 0.9 *** 4 98.6 ± 10.5 164.6 ± 8.3 ** 55.2 ± 1.0 *** 41.3 ± 1.4 *** 5 97.7 ± 0.9 81.1 ± 4.8 ** 34.2 ± 2.0 *** 12.5 ± 0.2 *** 6 94.0 ± 4.4 97.0 ± 4.6 10.7 ± 0.1 *** 11.0 ± 0.1 *** 7 94.6 ± 3.5 93.4 ± 2.8 11.8 ± 0.1 *** 10.9 ± 0.3 *** 8 89.6 ± 2.9 87.2 ± 3.2 * 58.7 ± 2.6 *** 44.0 ± 1.4 *** 9 97.6 ± 5.4 96.7 ± 2.0 41.1 ± 5.3 *** 15.5 ± 0.3 ***

^a Results are mean ± SEM for n = 3 ( ^* P <0.05, ^** P <0.01 and ^*** P <0.001).

To confirm that the NO inhibitory effect of Compound 1-9 was not due to cell death, the cytotoxicity of the compound against RAW 264.7 macrophages was tested by MTT assay. As a result, no detectable cytotoxicity was observed for all of the test compounds 1-9 at a concentration of 10 μM for 24 hours, effectively inhibiting NO production (Table 2). Although Compound 1-9 showed good inhibitory activity against NO production at 50 μM and 100 μM, this is likely due to the cytotoxic effect of Compound 1-9 on macrophages (FIG. 3 and Table 2). Cell viability at 50 μM and 100 μM was only in the range of 10.7-58.7% and 10.9-44.0%, respectively. Western blot analysis was used to assess whether this inhibitory effect was associated with the regulation of iNOS. As shown in FIG. 4, the results were consistent with those associated with NO production (Table 1 and FIG. 3), and protein expression of iNOS induced by LPS in RAW 264.7 cells was not affected by Compound 1, 3, 6 and 7 treatment. Was significantly inhibited. However, these compounds did not affect the expression of β-actin, a housekeeping gene. This indicates that reduced expression of iNOS due to this compound exposure is responsible for inhibiting NO production. As the present invention screened a limited number of compounds, ie only nine compounds, sufficient structure-activity relationship (SAR) analysis is not possible. However, the following is noteworthy: 1) Since the compounds 1, 3, 6 and 7 were most effective at inhibiting NO, the double bond between C3 and C11 appears to be crucial. 2) a compound having two methoxy groups (-OMe) (compound 3) or one methoxy group (-OMe) and one hydrate in the C3 and C11 double bonds including compounds (1, 3, 6 and 7) Compounds having a hydroxy group (-OH) (Compound 1) exhibit better NO inhibitory activity than compounds having one methoxy group (Compound 1) and compounds having three methoxy groups (Compound 7). 3) help to compound 6 (PORT NO strong inhibitory effect of Tula Canon D) is in the treatment of inflammatory diseases support the usefulness of the formate Tula car plant oleic la Seah Portulaca oleracea L. (L.), and demonstrate the traditional indication of this species Can give

In conclusion, the inventors formate Tula car oleic la Seah L. (Portulaca oleracea L.) Natural flavonoid Homo isopropyl (±) away from the-formyl Tula Canon AC (compound 4,8,9), formate Tula Canon D (Compound 6) and its derivatives (compounds 3, 5 and 7) and known derivatives (compounds 1 and 2) were synthesized from commercial compounds. These synthesized compounds were evaluated for their ability to inhibit NO production as an indicator of anti-inflammatory activity in LPS-induced RAW 264.7 macrophages. All compounds tested showed no clear cytotoxicity and inhibited NO production in a concentration dependent manner in RAW 264.7 macrophages. Compound 3 (inhibition rate 97.2% at 10 μM; IC ₅₀ = 1.26 μM), Compound 6 (portulacanone D) (inhibition rate 92.5% at 10 μM; IC ₅₀ = 2.09 μM), compound 1 (inhibition rate 91.4% at 10 μM; IC ₅₀ = 1.75 μM) and Compound 7 (inhibition rate 83.0% at 10 μM; IC ₅₀ = 2.91 μM) showed a significant inhibitory effect among the synthesized compounds. This finding is related to the inhibition of expression of iNOS induced by LPS. The information obtained in the present invention may provide a basis for considering compound 3 as a leading structure for the development of NO producing targeted anti-inflammatory drugs, and also forcing the body of Fortulaca oleracea L. as a medicinal medicinal plant to treat inflammatory diseases. Scientific evidence of the usefulness of Portulaca oleracea L.

The present invention

(A) Compound 17, Compound 12 and Compound 14 represented by the formula (12) After condensation with DMF-DMA ( N, N- dimethylformamide dimethyl acetal), each of the resulting aminoketone compounds was acid treated to give the corresponding 4H -chromen-4-one ( 4H- chromen) -4-ones) obtaining compounds 18a, 18b and 18c, respectively;

(B) the 4 H - chromen-4-one (4 H -chromen-4-ones ) compounds 18a, 18b and the corresponding chroman -4, which represented the 18c by the general formula 19 by hydrogenation using a catalyst Pd / C Obtaining -chroman-4-ones compounds 19a, 19b, 19c, respectively;

(C) Condensation reaction of the Chroman-4-ones compound 19a, 19b, 19c by adding EtOH / H ₂ O (5/1), KOH, compound 16 represented by the formula (16) Obtaining the corresponding compounds 20a, 20b, and 20c represented by 20, respectively; And

(D) deprotecting the compounds 20a, 20b, and 20c using 1N HCl to obtain the corresponding compounds 1, 3, and 7 represented by Formula 1, respectively. .

<Formula 12>

(Compound 17: R ¹ , R ² = H,

Compound 12: R ¹ = OMe, R ² = H,

Compound 14: R ¹ , R ² = OMe)

<Formula 18>

(Compound 18a: R ¹ , R ² = H,

Compound 18b: R ¹ = OMe, R ² = H,

Compound 18c: R ¹ , R ² = OMe)

<Formula 19>

(Compound 19a: R ¹ , R ² = H,

Compound 19b: R ¹ = OMe, R ² = H,

Compound 19c: R ¹ , R ² = OMe)

<Formula 16>

<Formula 20>

(Compound 20a: R ¹ , R ² = H,

Compound 20b: R ¹ = OMe, R ² = H,

Compound 20c: R ¹ , R ² = OMe)

<Formula 1>

(Compound 1: R ¹ , R ² = H,

Compound 3: R ¹ = OMe, R ² = H,

Compound 7: R ¹ , R ² = OMe)

In addition, the present invention, after the step (d) (e) hydrogenation of the compounds 1, 3 and 7 using a Pd / C catalyst to the corresponding homoisoflavonoid compounds 2, 4 and 8 represented by the formula (2), respectively It relates to a method for synthesizing a homoisoflavonoid compound, characterized in that it is further added.

<Formula 2>

(Compound 2: R ¹ , R ² = H,

Compound 4: R ¹ = OMe, R ² = H,

Compound 8: R ¹ , R ² = OMe)

In addition, the present invention is the corresponding compound 5 represented by the formula (5) by the selective ortho-demethylation reaction in CH ₂ Cl ₂ using (M) 1.0M BCl ₃ after step (e) (bar) And 9, respectively; relates to a method for synthesizing a homoisoflavonoid compound, which is further added.

<Formula 5>

(Compound 5: R ¹ = OH, R ² = H,

Compound 9: R ¹ = OH, R ² = OMe)

In addition, the present invention is a homoisoflavonoid, characterized in that the compound 16 is produced by the ethoxymethyl protecting group of the salicylic aldehyde represented by the formula (15) using chloromethylethyl ether, K ₂ CO ₃ and TBAI (tetrabutylammonium iodide) The present invention relates to a method for synthesizing a compound.

<Formula 15>

In addition, the present invention is the reaction of the compound 14 is acetylated at room temperature by adding Ac ₂ O, Et ₃ N, CH ₂ Cl ₂ to Compound 13 represented by the formula (13) and BF ₃ · Et ₂ O, AcOH It relates to a method for synthesizing a homoisoflavonoid compound, characterized in that produced by the rearrangement reaction.

<Formula 13>

In addition, the present invention is the compound 12

(A) Treating 1,3,5-trimethoxybenzene represented by the formula (10) with acetic anhydride in the presence of boron trifluoride diethyl etherate to form a Friedel-Crafts acylation (Friedel- Obtaining Compound 11 represented by Formula 11 by a crafts acylation reaction; And

(B) optionally ortho to reflux was added AlCl ₃ to the compound 11 to obtain the demethylated compound 12 with (ortho -demethylation) reaction; relates to homo-iso flavonoid compound synthesis method which comprises generated through.

<Formula 10>

<Formula 11>

In addition, the present invention is the portulacanone derivative compound 1, 3, 7 and the portulacanone derivative compound 2, the portulacanone A, the portulacanone B, and the formula represented by the formula (5). It relates to an anti-inflammatory pharmaceutical composition comprising at least one selected from canon derivative compound 5 and portulancanone C.

The present invention also relates to an anti-inflammatory pharmaceutical composition, in particular comprising at least one selected from portulacanone derivative compounds 1, 3 and 7.

In addition, the present invention relates to an anti-inflammatory pharmaceutical composition, characterized in that the IC ₅₀ value of the fortulacanone derivative compounds 1, 3 and 7 is 1 to 3 μM.

The present invention also relates to novel portulacanone derivative compounds 3, 5 and 7 synthesized for the first time in the present invention.

According to the synthesis method of the present invention can be synthesized in the formate Tula car oleic la Seah L. (Portulaca oleracea L.) derived from the formyl compound Tula Canon AC and its derivatives in high yield.

In addition, portulacanone A-C and its derivatives, especially compounds 3, 1 and 7, synthesized by the method of the present invention do not exhibit cytotoxicity and exhibit excellent anti-inflammatory activity and can be used as a therapeutic agent for inflammatory diseases.

1 is the structure of portulacanone AD (compounds 4, 8, 9 and 6) and its derivatives (compounds 1-3, 5 and 7).
Figure 2 shows the reaction scheme for the synthesis method of the portulacanone AD and its derivatives of the present invention. Reagents and conditions: (a) Ac ₂ O, BF ₃ · Et ₂ O, EtOAc, room temperature, 4 hours, 89%; (b) AlCl ₃ , CH ₂ Cl ₂ , reflux, 22 h, 87%; (c) (i) Ac ₂ O, Et ₃ N, CH ₂ Cl ₂ , room temperature, 6 hours; (ii) BF ₃ · Et ₂ O, AcOH, 70 ° C., 2 hours, 93% (2 steps total); (d) chloromethyl ethyl ether, K ₂ CO ₃ , TBAI (Tetrabutylammonium iodide), acetone, 0 ° C.-room temperature, 24 h, 83%; (e) N, N -dimethylformamide dimethyl acetal, benzene, 90 ° C., overnight reaction, 88-91%; (f) H ₂ , Pd / C (10%), MeOH, room temperature, 0.75-1 hour, 83-87%; (g) KOH, EtOH / H ₂ O (5/1), 25-30 ° C., 10-24 hours, 25-48%; (h) 1N HCl, MeOH, 55 ° C., 1 hour, 85-88%; (i) H ₂ , Pd / C (10%), MeOH, room temperature, 0.75-1 hour, 86-91%; (j) 1.0 M BCl ₃ (in CH ₂ Cl ₂ ), CH ₂ Cl ₂ , 0 ° C.-room temperature, 3 hours, 83-86%.
Figure 3 is a graph showing the inhibitory activity of homoisoflavonoids 1-9 on NO production in LPS induced RAW 264.7 macrophages.
4 shows the results of testing the effect of compound 1-9 on iNOS expression.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is obvious to those skilled in the art that the scope of the present invention is not limited only to the description of the embodiments.

Chemicals etc.

All chemicals were used without purification as purchased unless otherwise noted. All solvents used in the reaction were distilled from an appropriate dehydrating agent under nitrogen gas. All solvents used for chromatography were purchased and used directly without further purification. Thin layer chromatography (TLC) was performed using a plate coated with silica gel on a DC-Plastikfolien 60, F254 (Merck, 0.2 mm thick) plastic plate, observed using UV (254 nm) or p-anisaldehyde and / or Observed by staining with phosphomolybdic acid (PMA). Chromatographic purification was performed using Kieselgel 60 (60-120mesh, Merck). ¹ H-NMR spectra were recorded at 75 MHz for Varian Mercury-300 MHz FT-NMR and ¹³ C, chemical shifts (δ) were expressed in parts per million (ppm) for TMS, and coupling constants (J) were Hz. Quoted by. Peak cleavage patterns were abbreviated as s (singlet), d (doublet), t (triplet), dd (doublet of doublet) and m (multiplet), and CDCl ₃ / CD ₃ COCE ₃ was used as solvent and internal standard. High resolution mass spectrum electrospray ionization (HRMS-ESI) was recorded using an Agilent technologies 6220 TOF LC / MS spectrometer. Melting points were measured on a MEL-TEMP II instrument and were not corrected.

1- (2,4,6- Trimethoxyphenyl ) Ethanon  {1- (2,4,6- Trimethoxyphenyl ) ethanone } (Compound 11):

1,3,5-trimethoxybenzene (0.84 g, 5.00 mmol) and acetic anhydride (0.47 mL, 7.50 mmol) were mixed in ethyl acetate (5 mL) and BF ₃ .Et ₂ O (0.32 mL, 2.50 mmol) Was added slowly under a nitrogen atmosphere. The reaction was allowed to warm to room temperature and stirred for 4 hours. After completion of the reaction, H ₂ O (40 mL) was added and stirred for 15 minutes, then the reaction mixture was extracted with ethyl acetate (3 × 30 mL). The mixed organic solvent layer was washed with saturated NaHCO ₃ solution ( ₂ × 20 mL), H ₂ O ( ₂ × 20 mL), brine (20 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated in vacuo. The crude compound was purified by column chromatography (EtOAc / Hexane = 1 / 3-1 / 2) to give a pure white solid compound (0.94 g, 89%). Melting point 98-100 ° C. (Lit. ₁ 101-103 ° C.); ₁ H NMR (300 MHz, CDCl ₃ ) δ 6.09 (2H, s), 3.82 (3H, s), 3.79 (6H, s), 2.46 (3H, s); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 201.7, 162.3, 158.4, 113.7, 90.7, 56.0, 55.6, 32.8.

1- (2- Hydroxy -4,6- Dimethoxyphenyl ) Ethanon  {1- (2- Hydroxy -4,6-dimethoxyphenyl) ethanone} (Compound 12):

Compound 11 (0.9 g, 4.28 mmol) with anhydrous CH ₂ Cl ₂ To the stirred solution was added AlCl ₃ (0.71 g, 5.35 mmol) in a nitrogen atmosphere, and the mixture was refluxed for 22 hours. After the reaction was completed, the reaction mixture was cooled to 0 ° C., 1N HCl (6 mL) was added, and the mixture was stirred at room temperature for 15 minutes. CH ₂ Cl ₂ Extracted with (2 × 50 mL). The mixed organic solvent layer was washed with H ₂ O ( ₂ × 25 mL) and brine (20 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated in vacuo. The crude compound was purified by column chromatography (EtOAc / Hexane = 1/4) to give a pure white solid compound (0.72 g, 87%). Melting point 79-80 ° C (literally, melting point is 81-82 ° C); ₁ H NMR (300 MHz, CDCl ₃ ) δ6.03 (1H, d, J = 2.1 Hz), 5.90 (1H, d, J = 2.1 Hz), 3.84 (3H, s), 3.80 (3H, s), 2.60 (3H, s); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 203.1, 167.6, 166.1, 162.9, 106.1, 93.6, 90.8, 55.8, 33.2.

1- (6- Hydroxy -2,3,4- Trimethoxyphenyl ) Ethanon  {1- (6- Hydroxy -2,3,4-trimethoxyphenyl) ethanone} (compound 14):

3,4,5-trimethoxyphenol (Compound 13 ) (1.55 g, 8.42 mmol) was added to anhydrous CH ₂ Cl ₂ (16 mL), and Et ₃ N (2.36 mL, 16.63 mmol) was added to the stirred solution. Stir for 15 minutes. Acetic anhydride (1.2 mL, 12.62 mmol) was added and stirred at room temperature for 6 hours. After completion of reaction, CH ₂ Cl ₂ (80 mL), washed with 2N HCl (8 mL), H ₂ O ( ₂ × 20 mL), brine (20 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated in vacuo. Acetylated colorless solid compound (1.88 g, 99%) was obtained and used in the next step without further purification. Melting point 68-70 ° C .; ₁ H NMR (300 MHz, CDCl ₃ ) δ 6.32 (2H, s), 3.82 (9H, s), 2.28 (3H, s); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 169.5, 153.5, 146.8, 136.0, 99.3, 61.1, 56.4, 21.4.

The 3,4,5-trimethoxyphenyl acetate (1.88 g, 8.33 mmol) was added to AcOH (2.2 mL), and BF ₃ · Et ₂ O (3.6 mL, 29.16 mmol) was added to the stirred solution under a nitrogen atmosphere at room temperature. Dropwise. The resulting mixture was stirred at 70 ° C. for 2 hours. After the reaction was completed, the mixture was cooled to room temperature, ice water (5 mL) was added thereto, and the mixture was stirred for 15 minutes. The mixture was extracted with EtOAc (3x35 mL). The combined organic solvent layer was washed with H ₂ O (3 × 20 mL), brine ( ₂ × 20 mL), dried over anhydrous Na ₂ SO ₄ , concentrated in vacuo to give a pale yellow liquid compound 14 (1.77 g, 94%), and further purification. Used in the next step without. ₁ H NMR (300 MHz, CDCl ₃ ) δ 6.22 (1H, s), 3.98 (3H, s), 3.86 (3H, s), 3.77 (3H, s), 2.65 (3H, s); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 203.3, 161.9, 160.1, 155.3, 134.8, 108.6, 96.3, 61.2, 61.1, 56.3, 32.1.

2-( Ethoxymethoxy ) Benzaldehyde  {2-( Ethoxymethoxy ) benzaldehyde } (Compound 16):

Salicylate (0.53 mL, 5.00 mmol) was added to anhydrous acetone (12 mL) and stirred in a solution of K ₂ CO _3. (1.38 g, 20.00 mmol) was added under nitrogen atmosphere and cooled to 0 ° C. After stirring for 20 minutes, chloromethyl ethyl ether (EOM-Cl) (0.56 mL, 6.0 mmol) was added dropwise, followed by anhydrous acetone (3.0 mL) solution of TBAI (tetrabutylammonium iodide) (0.2 mmol, 0.1 equiv) Was added. The resulting mixture was allowed to warm to room temperature and stirred for 24 hours. After completion of the reaction, it was filtered through a pad of Celite®, washed with acetone (10 mL), and the filtrate was concentrated in vacuo. The crude product was dissolved in EtOAc (60 mL), washed with H ₂ O (3 × 15 mL), brine (15 mL), dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The crude compound was purified by column chromatography (EtOAc / Hexane = 1/10) to give pure EOM-protecting aldehyde Compound 16 (0.75 g, 83%) as an almost colorless oil. ₁ H NMR (300MHz, CDCl ₃ ) δ 10.47 (1H, s), 7.82 (1H, d, J = 7.5 Hz), 7.52 (1H, td, J = 8.1, 2.1 Hz), 7.23 (1H, d, J = 8.1 Hz), 7.06 (1H, td, J = 7.5, 2.1 Hz), 5.34 (2H, s), 3.77 (2H, q, J = 7.0 Hz), 1.24 (3H, t, J = 7.0 Hz); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 189.7, 159.8, 135.9, 128.3, 125.4, 121.7, 115.1, 93.4, 65.0, 15.4.

Substituted 4 H - Chromen 4-one (4 H - chromen General preparation method of -4-ones)

Substituted 2-hydroxyacetophenone (1.4 mmol) was added to anhydrous benzene (4 mL), and N , N -dimethylformamide dimethyl acetal (4.2 mmol) was added to the stirred solution under normal temperature and nitrogen atmosphere. The mixture was stirred at 90 ° C. overnight. After the reaction was completed, the reaction mixture was cooled to 0 ° C, and saturated HCl (0.85 mL) was added thereto. The resulting mixture was stirred at 55 ° C. for 1 hour. After completion of the reaction, diluted with EtOAc (40 mL), washed with H ₂ O ( ₃ × 10 mL) and brine (2 × 15 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated in vacuo. The crude compound was purified by column chromatography (EtOAc / Hexane = 1 / 1-2 / 1) to give pure substituted 4H -chromen-4-one.

7-methoxy -4 H - chromen-4-one {7- Methoxy -4 H - chromen -4 -one} ( compound 18a): Yield: 91%; Pale yellow solid; Melting point 97-99 ° C .; ₁ H NMR (300 MHz, CDCl ₃ ) δ 8.08 (1H, d, J = 8.7 Hz), 7.75 (1H, d, J = 6.0 Hz), 6.94 (1H, dd, J = 8.7, 2.4 Hz), 6.81 ( 1 H, d, J = 2.4 Hz), 6.25 (1 H, d, J = 6.0 Hz), 3.89 (3 H, s); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 177.0, 164.2, 158.4, 154.9, 127.4, 119.0, 114.7, 113.1, 100.6, 56.1.

5,7 -dimethoxy - 4H - chromen - 4-one {5,7- Dimethoxy - 4H - chromen- 4-one} (compound 18b): yield: 90%; Pale yellow solid; Melting point 131-133 ° C; ₁ H NMR (300 MHz, CDCl ₃ ) δ 7.60 (1H, d, J = 6.0 Hz), 6.41 (1H, d, J = 2.1 Hz), 6.34 (1H, d, J = 6.0 Hz), 6.17 (1H, d, J = 6.0 Hz), 3.93 (3H, s), 3.87 (3H, s); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 176.6, 163.9, 161.0, 160.1, 152.6, 114.6, 110.4, 96.3, 92.9, 56.6, 55.9.

5,6,7 -trimethoxy - 4H - chromen - 4-one {5,6,7- Trimethoxy - 4H - chromen- 4-one} (compound 18c): yield: 88%; Pale yellow solid; Melting point 113-115 ° C; ₁ H NMR (300 MHz, CDCl ₃ ) δ 7.63 (1H, d, J = 6.0 Hz), 6.65 (1H, s), 6.16 (1H, d, J = 6.0 Hz), 3.95 (3H, s), 3.93 ( 3H, s), 3.89 (3H, s); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 176.2, 157.8, 154.8, 153.0, 152.6, 140.5, 114.1, 113.9, 96.4, 62.3, 61.7, 56.4.

Substituted 4 H - Chromen General Procedure for Hydrogenation of 4-ones

Substituted 4 H -chromen-4-one (1.0 mmol) in MeOH (8 mL) was added to the stirred solution and 10% Pd / C (5% w / w relative to substituted 4 H -chromen-4-one). ) Was added under a hydrogen atmosphere at room temperature, and the mixture was stirred for 0.75-1 hour. After completion of the reaction, the mixture was filtered through a Celite® pad, washed with MeOH (10 mL), and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (EtOAc / Hexane = 1 / 3-1 / 2) to give pure substituted chromo-4-one.

7-methoxy-chroman-4-one (7- Methoxychroman -4-one) (Compound 19a): Yield: 87%; White solid; Melting point 49-50 ° C .; ₁ H NMR (300MHz, CDCl ₃ ) δ 7.80 (1H, d, J = 8.7 Hz), 6.55 (1H, dd, J = 8.7, 2.4 Hz), 6.38 (1H, d, J = 2.4 Hz), 4.50 ( 2H, t, J = 6.3 Hz), 3.82 (3H, s), 2.74 (2H, t, J = 6.3 Hz); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 190.4, 166.0, 163.8, 128.9, 115.4, 110.0, 100.9, 67.6, 55.8, 37.7.

5,7-dimethoxy-chroman-4-one (5,7- Dimethoxychroman -4-one) (Compound 19b): Yield: 83%; White solid; Melting point 92-94 ° C; ₁ H NMR (300 MHz, CDCl ₃ ) δ 6.04 (2H, s), 4.43 (2H, t, J = 6.3 Hz), 3.87 (3H, s), 3.81 (3H, s), 2.71 (2H, t, J = 6.3 Hz); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 189.1, 165.7, 165.2, 162.3, 106.4, 93.4, 93.0, 67.0, 56.3, 55.7, 39.0.

5,6,7 - Trimethoxychroman- 4-one {5,6,7- Trimethoxychroman- 4-one} (Compound 19c): yield: 84%; White solid; Melting point 58-60 ° C .; ₁ H NMR (300 MHz, CDCl ₃ ) δ 6.24 (1H, s), 4.43 (2H, t, J = 6.3 Hz), 3.91 (3H, s), 3.87 (3H, s), 3.80 (3H, s), 2.71 (2H, t, J = 6.3 Hz); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 189.1, 160.0, 159.3, 154.3, 137.4, 109.7, 96.2, 67.1, 61.7, 61.5, 56.3, 38.9.

EOM Protected Of homoisoflavonoids  General manufacturing process

Substituted Chromman-4-one (0.30 mmol) and EOM-protected salicyaldehyde compound 16 (0.07 g, 0.39 mmol) were added to EtOH / H ₂ O (1.8 mL, 5/1) and stirred in KOH. (0.05 g, 0.90 mmol) was added and stirred at 25-30 ° C. for 10-24 hours. After the reaction was completed, EtOH was removed under reduced pressure. The crude product was neutralized with 1N HCl and extracted with EtOAc (2 × 25 mL). The mixed organic solvent layer was washed with H ₂ O (3 × 10 mL) and brine (10 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated in vacuo. The crude compound was purified by column chromatography (EtOAc / Hexane = 1 / 5-1 / 3) to give pure EOM-protected homoisoflavonoid compounds.

(E) -3- (2- (ethoxy-methoxy) benzylidene) -7-methoxy chroman-4-one {(E) -3- (2- ( Ethoxymethoxy) benzylidene) -7-methoxychroman-4 -one} (Compound 20a): yield: 48%; Pale yellow liquid; ₁ H NMR (300 MHz, CDCl ₃ ) δ 7.96-7.93 (2H, m), 7.35-7.30 (1H, m), 7.20 (1H, d, J = 8.7 Hz), 7.04-6.98 (2H, m), 6.60 (1H, dd, J = 8.7, 2.4 Hz), 6.37 (1H, d, J = 2.4 Hz), 5.24 (2H, s), 5.20 (2H, d, J = 1.5 Hz), 3.81 (3H, s) , 3.71 (2H, q, J = 7.0 Hz), 1.20 (3H, t, J = 7.0 Hz); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 181.1, 165.9, 163.2, 156.2, 133.2, 130.9, 130.4, 129.7, 124.5, 121.4, 116.0, 114.9, 110.4, 100.9, 93.5, 68.5, 64.8, 55.8, 15.3.

( E ) -3- (2- ( ethoxymethoxy ) benzylidene ) -5,7 -dimethoxychroman- 4-one {( E ) -3- (2- (Ethoxymethoxy) benzylidene) -5, 7-dimethoxychroman-4-one} (Compound 20b): yield: 40%; Pale yellow liquid; ₁ H NMR (300MHz, CDCl ₃ ) δ 7.95 (1H, s), 7.37-7.31 (1H, m), 7.20 (1H, d, J = 8.1 Hz), 7.06-7.00 (2H, m), 6.12 (1H) , s), 6.07 (1H, s), 5.26 (2H, s), 5.12 (2H, s), 3.93 (3H, s), 3.84 (3H, s), 3.73 (2H, q, J = 7.0 Hz) , 1.23 (3H, t, J = 7.0 Hz); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 179.8, 165.8, 165.0, 163.0, 156.1, 132.4, 132.1, 130.7, 130.4, 125.1, 121.5, 115.0, 107.5, 93.8, 93.7, 93.6, 68.1, 64.9, 56.5, 55.9, 15.4.6

( E ) -3- (2- ( ethoxymethoxy ) benzylidene ) -5,6,7 -trimethoxychroman- 4-one {( E ) -3- (2- (Ethoxymethoxy) benzylidene)- 5,6,7-trimethoxychroman-4-one} (Compound 20c): yield: 25%; Pale yellow liquid; ₁ H NMR (300 MHz, CDCl ₃ ) δ 7.93 (1H, s), 7.30-7.26 (1H, m), 7.15 (1H, d, J = 8.1 Hz), 7.02-6.98 (1H, m), 6.22 (1H , d, J = 7.8 Hz), 6.20 (1H, s), 5.20 (2H, d, J = 1.2 Hz), 5.06 (2H, s), 3.96 (3H, s), 3.83 (3H, s), 3.77 (3H, s), 3.67 (2H, q, J = 7.0 Hz), 1.20 (3H, t, J = 7.0 Hz).

EOM - Deprotection Vaporization Homoisoflavonoids  General procedure of synthesis

EOM-protected homoisoflavonoids (0.2 mmol) were added to MeOH (5 mL), and 1N HCl (0.6 mL) was added to the stirred solution at room temperature, and the mixture was stirred at 55 ° C for 1 hour. The solvent was removed under reduced pressure. The crude product was dissolved in EtOAc (35 mL), washed with H ₂ O ( ₂ × 10 mL) and brine (10 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated in vacuo. The crude compound was purified by column chromatography (EtOAc / Hexane = 1 / 3-1 / 1) to obtain pure EOM-deprotected homoisoflavonoid compounds.

(E) -3- (2- hydroxy-benzylidene) -7-methoxy chroman-4-one {(E) -3- (2- Hydroxybenzylidene) -7-methoxychroman-4-one} ( Compound 1) : yield: 89%; Pale yellow solid; Melting point 135-137 ° C .; ₁ H NMR (300 MHz, CDCl ₃ ) δ 7.99 (1H, br s), 7.92 (1H, d, J = 8.7 Hz), 7.27 (1H, dd, J = 8.7, 2.1 Hz), 7.02-6.90 (3H, m), 6.81 (1H, s), 6.58 (1H, dd, J = 8.7, 2.4 Hz), 6.34 (1H, d, J = 2.1 Hz), 5.19 (2H, d, J = 1.5 Hz) 3.82 (3H , s); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 181.9, 166.3, 163.6, 155.4, 133.4, 131.2, 131.1, 130.3, 129.9, 122.0, 120.3, 116.7, 115.8, 110.7, 100.9, 68.5, 55.9; EI-MS m / z 282 (M &lt; + &gt;, base), 265, 253, 131; HRMS: Calcd for C ₁₇ H ₁₄ O ₄ (M &lt; + &gt;): 282.0892, found: 282.0901.

(E) -3- (2- hydroxy-benzylidene) -5,7-dimethoxy-chroman-4-one {(E) -3- (2- Hydroxybenzylidene) -5,7-dimethoxychroman-4-one } (Compound 3): yield: 86%; Pale yellow solid; ₁ H NMR (300MHz, DMSO-d6) δ 10.02 (1H, s), 7.70 (1H, s), 7.23 (1H, td, J = 8.1, 1.8 Hz), 7.03 (1H, d, J = 6.6 Hz) , 6.91 (1H, d, J = 7.8 Hz), 6.84 (1H, t, J = 7.5 Hz), 6.24 (1H, d, J = 2.1 Hz), 6.16 (1H, d, J = 2.1 Hz), 5.11 (2H, d, J = 1.5 Hz), 3.81 (3H, s), 3.80 (3H, s); ₁₃ C NMR (75 MHz, DMSO-d6) δ 177.7, 165.0, 163.8, 162.1, 156.2, 130.8, 130.7, 130.6, 130.1, 121.1, 118.7, 115.5, 106.5, 93.7, 93.4, 67.4, 55.9, 55.6; EI-MS m / z 312 (M &lt; + &gt;), 295, 180 (base), 131; HRMS: Calcd for C ₁₈ H ₁₆ O ₅ (M &lt; + &gt;): 312.0998, found: 312.0993.

(E) -3- (2- hydroxy-benzylidene) -5,6,7- trimethoxy-chroman-4-one {(E) -3- (2- Hydroxybenzylidene) -5,6,7-trimethoxychroman -4-one} (Compound 7): yield: 85%; Pale yellow solid; ₁ H NMR (300 MHz, CDCl ₃ ) δ 8.00 (1H, s), 7.81 (1H, br s), 7.21 (1H, t, J = 7.8 Hz), 7.00 (1H, d, J = 7.5 Hz), 6.96 (1H, d, J = 7.8 Hz), 6.86 (1H, t, J = 7.5 Hz), 6.19 (1H, s), 5.08 (2H, s), 3.96 (3H, s), 3.85 (3H, s ), 3.79 (3H, s); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 180.5, 160.0, 159.5, 155.6, 154.7, 137.6, 133.4, 131.3, 131.1, 130.2, 122.0, 119.9, 116.6, 110.4, 96.3, 68.0, 61.8, 61.5, 56.3; EI-MS m / z 342 (M &lt; + &gt;, base), 311, 210, 166; HRMS: Calcd for C ₁₉ H ₁₈ O ₆ (M &lt; + &gt;): 342.1103, found: 342.1110.

3- (2-hydroxy-benzyl) -7-methoxy chroman-4-one {3- (2-Hydroxybenzyl) -7 -methoxychroman-4-one} ( Compound 2): 19a-19cs the general used in the synthesis According to the procedure, compound 1 (0.04 g, 0.14 mmol) was stirred under a hydrogen (balloon) atmosphere to obtain a white solid compound 2 (0.037 g, 91%). Melting point 132-134 ° C .; ₁ H NMR (300 MHz, CDCl ₃ ) δ 8.12 (1H, br s), 7.82 (1H, d, J = 8.7 Hz), 7.10 (1H, td, J = 8.1, 1.8 Hz), 7.02 (1H, dd, J = 7.8, 1.8 Hz), 6.90 (1H, dd, J = 8.1, 1.2 Hz), 6.80 (1H, td, J = 7.5, 1.2 Hz), 6.54 (1H, dd, J = 8.7, 2.1 Hz), 6.36 (1H, d, J = 2.7 Hz), 4.51 (1H, dd, J = 11.4, 1.5 Hz), 4.21 (1H, t, J = 10.8 Hz), 3.80 (3H, s), 3.21-3.03 (2H m), 2.87-2.79 (1 H, m); 13 C NMR (75 MHz, CDCl ₃ ) δ 195.2, 166.6, 164.1, 154.8, 131.2, 129.4, 128.5, 124.8, 120.3, 117.3, 114.2, 110.4, 100.7, 70.9, 55.9, 46.6, 27.2; EI-MS m / z 284 (M &lt; + &gt;), 283 (MH), 267, 177 (base); HRMS: Calcd for C 17 H 16 O 4 (M +): 284.1049, found: 284.1055

3- (2-hydroxy-benzyl) -5,7-dimethoxy-chroman-4-one 3 - {(2-Hydroxybenzyl) -5,7-dimethoxychroman-4-one} (compound 4): 19a-19c According to the general procedure used for the synthesis, compound 3 (0.02 g, 0.06 mmol) was stirred under a hydrogen (balloon) atmosphere to obtain compound 4 (0.018 g, 87%) as a white solid. Melting point 137-139 ° C .; ₁ H NMR (300 MHz, CDCl ₃ ) δ 8.22 (1H, br s), 7.10 (1H, td, J = 7.8, 1.8 Hz), 7.01 (1H, dd, J = 7.5, 1.8 Hz), 6.90 (1H, dd, J = 8.1, 1.2 Hz), 6.79 (1H, td, J = 7.5, 1.2 Hz), 6.01 (2H, d, J = 2.1 Hz), 4.49 (1H, dd, J = 11.4, 5.4 Hz), 4.15 (1H, t, J = 11.4 Hz), 3.85 (3H, s), 3.80 (3H, s), 3.09-2.98 (2H, m), 2.80-2.72 (1H, m); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 193.8, 166.5, 165.4, 162.7, 155.0, 131.0, 128.4, 125.1, 120.2, 117.6, 105.4, 93.4, 93.1, 70.5, 56.4, 55.8, 47.8, 27.1; EI-MS m / z 314 (M &lt; + &gt;), 207, 181 (base), 132; HRMS: Calcd for C ₁₈ H ₁₈ O ₅ (M &lt; + &gt;): 314.1154, found: 314. 1150.

3- (2-hydroxybenzyl) -5,6,7- trimethoxy-chroman-4-one {3- (2-hydroxybenzyl) -5,6,7 -trimethoxychroman-4-one} ( Compound 8) : According to the general procedure used for 19a-19c synthesis, compound 7 (0.02 g, 0.06 mmol) was stirred under a hydrogen (balloon) atmosphere to obtain a white solid compound 8 (0.017 g, 86%). Melting point 102-104 ° C; ₁ H NMR (300MHz, CDCl ₃ ) δ 8.09 (1H, br s), 7.12-7.02 (2H, m), 6.91 (1H, d, J = 8.1 Hz), 6.79 (1H, t, J = 7.2 Hz) , 6.23 (1H, s), 4.43 (1H, dd, J = 11.4, 4.2 Hz), 4.19-4.12 (1H, m), 3.91 (3H, s), 3.86 (3H, s), 3.79 (3H, s ), 3.14-3.00 (2H, m), 2.78 (1H, doublet of doublets, J = 13.2, 5.7 Hz); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 193.9, 160.2, 159.9, 154.9, 154.4, 137.4, 131.1, 128.3, 124.8, 120.1, 116.9, 108.5, 96.1, 70.1, 61.8, 61.5, 56.3, 47.2, 27.4; EI-MS m / z 344 (M &lt; + &gt;, base), 327, 237, 210; HRMS: Calcd for C ₁₉ H ₂₀ O ₆ (M &lt; + &gt;): 344.1260, found: 344.1271

Of homoisoflavonoids Ortho - Demethylation  General procedure for

Homoisoflavonoids (0.12 mmol) were added to anhydrous CH ₂ Cl ₂ (4 mL), and BCl ₃ solution (1.0 M in CH ₂ Cl ₂ ; 0.36 mL) was added dropwise to the stirred solution under a nitrogen atmosphere at 0 ° C. The mixture was stirred at 0-5 ° C. for 1.5 h. After the reaction was completed, H ₂ O (5 mL) was added and stirred for 15 minutes, followed by extraction with CH ₂ Cl ₂ ( ₂ × 20 mL). The mixed organic solvent layer was washed with H ₂ O ( ₃ × 10 mL) and brine (2 × 10 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated in vacuo. The crude compound was purified by column chromatography (EtOAc / Hexane = 1 / 3-1 / 1) to obtain pure homoisoflavonoids.

5-hydroxy-3- (2-hydroxy-benzyl) -7-methoxy chroman-4-one 5-Hydroxy-3- {(2-hydroxybenzyl) -7-methoxychroman-4-one} (Compound 5) : yield: 86%; White solid; Melting point 128-130 ° C .; ₁ H NMR (300 MHz, CDCl ₃ ) δ 11.78 (1H, s), 7.12 (1H, td, J = 8.1, 1.8 Hz), 7.06-7.01 (2H, m), 6.87-6.81 (2H, m), 6.03 (1H, d, J = 2.4 Hz), 5.96 (1H, d, J = 2.4 Hz), 4.44 (1H, dd, J = 11.4, 4.5 Hz), 4.17 (1H, t, J = 11.4 Hz), 3.79 (3H, s), 3.17-3.07 (2H, m), 2.89-2.81 (1H, m); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 199.3, 168.4, 164.4, 163.2, 154.5, 131.3, 128.6, 124.3, 120.6, 116.7, 102.8, 95.2, 94.2, 70.0, 55.9, 45.6, 27.4; EI-MS m / z 300 (M &lt; + &gt;), 299 (MH), 193 (base), 165; HRMS: Calcd for C ₁₇ H ₁₆ O ₅ (M &lt; + &gt;): 300.0998, found: 300.0995

5-hydroxy-3- (2-hydroxy-benzyl) -6,7-dimethoxy-chroman-4-one {5-Hydroxy-3- (2 -hydroxybenzyl) -6,7-dimethoxychroman-4-one } (Compound 9): yield: 83%; Pale yellow solid; Melting point 135-137 ° C .; ₁ H NMR (300 MHz, CDCl ₃ ) δ 11.71 (1H, s), 7.12 (1H, t, J = 7.5 Hz), 7.06 (1H, d, J = 7.5 Hz), 6.98 (1H, br s), 6.87 -6.81 (2H, m), 6.01 (1H, s), 4.42 (1H, dd, J = 11.4, 4.2 Hz), 4.21-4.14 (1H, m), 3.86 (3H, s), 3.81 (3H, s ), 3.18-3.09 (2H, m), 2.87-2.79 (1H, m); ₁₃ C NMR (75 MHz, CDCl ₃ ) δ 199.7, 161.3, 159.2, 155.2, 154.4, 131.3, 130.6, 128.6, 124.3, 120.7, 116.8, 102.9, 91.6, 70.2, 61.1, 56.4, 45.7, 27.3; EI-MS m / z 330 (M &lt; + &gt;, base), 313, 223, 152; HRMS: Calcd for C1 ₈ H ₁₈ O ₆ (M +): 330.1103, found: 330.1095

(E) -5- hydroxy-3- (2-hydroxy-benzylidene) -7-methoxy chroman-4-one {(E) -5-Hydroxy- 3- (2-hydroxybenzylidene) -7-methoxychroman -4-one} (Compound 6): yield: 84%; Pale yellow solid; Melting point 172-174 ° C; ₁ HNMR (300 MHz, CD ₃ COCD ₃ ) δ 12.76 (1H, s), 9.09 (1H, br s), 8.02 (1H, s), 7.31 (1H, t, J = 7.8 Hz), 7.16 (1H, d , J = 7.5 Hz), 7.02 (1H, d, J = 7.8 Hz), 6.42 (1H, t, J = 7.5 Hz), 6.06 (1H, d, J = 2.1 Hz), 5.99 (1H, d, J = 2.1 Hz), 5.29 (2H, d, J = 1.8 Hz), 3.86 (3H, s); ₁₃ C NMR (75 MHz, CD ₃ COCD ₃ ) δ 186.9, 169.5, 166.6, 164.0, 157.9, 134.7, 133.0, 132.1, 130.5, 122.9, 121.1, 117.3, 104.6, 96.4, 95.1, 69.3, 57.0; EI-MS m / z 298 (M &lt; + &gt;), 281, 253, 131 (base); HRMS: Calcd for C ₁₇ H ₁₄ O ₅ (M &lt; + &gt;): 298.0841, found: 298.0847

reagent

Escherichia Coli-derived polysaccharide (LPS), DMSO (dimethylsulfoxide) and β-actin antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA). DMEM (Dulbeccos modified Eagles medium), Fetal Bovine Serum (FBS), Penicillin and Streptomycin were purchased from Hyclone (Logan, UT, USA). Cell Titer 96® Aqueous One Solution and Griess reagent system were purchased from Promega (Madison, MI, USA).

Cell Culture and Cell Viability Analysis

RAW 264.7 mouse macrophages were obtained from Korea Cell Bank (Seoul, Korea) and cultured at 37 ° C. and 5% CO ₂ in DMEM containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. The effect of various compounds on cell viability was tested by CellTiter 96® Aqueous One Solution Assay of cell proliferation (Promega, Madison, Wis., USA), which used colorimetric reactions to count viable cells. This assay was used to determine the number of viable cells remaining after the culture was completed. RAW 264.7 cells were plated in 2x10 ⁴ plates with 96-well flat bottoms. Place to cell density and each compound was added to each plate at the indicated concentration. After 24 hours of incubation, viable cell counts were counted according to the manufacturer's instructions. This assay is based on the reduction of tetrazolium compound MTS to formazan, which shows the maximum absorbance at 490 nm. Thus, the amount of product in the cell culture is represented by the optical density of formazan at 490 nm, which is directly proportional to the number of viable cells.

NO measurement

The amount of NO produced by mouse macrophages was expressed as measured in RAW 264.7 cell culture supernatants. RAW 264.7 cells were stored in ⁵ × 10 ⁴ cells in 96-well cell culture plates containing 200 μL culture medium. Cell density was added and incubated for 12 hours. The cells were put in 500ng / mL LPS and compounds 1-10 at the concentrations indicated and incubated for 18 hours. The amount of NO produced was measured according to the manufacturer's instructions using the Griess reagent system (Promega).

Immunoblotting  analysis

To determine the ability of these compounds to induce iNOS expression in RAW 264.7 cells, cells were exposed to 10 μM of each compound for 18 hours in the presence of 500 ng / mL LPS. The cells are then washed with cold PBS and 100 μl of cell lysis buffer (200 mM Tris-HCl, pH 7.5, 125 mM NaCl, 1% Triton X-100, 1 mM MgCl ₂ , 25 mM β-glycerophosphate, 50 mM NaF, 100 μM Na ₃ VO ₄ , 1 mM PMSF, 10 μg / ml leupeptin and 10 μg / ml aprotinin) were added to each sample. After the protein was denatured with sodium dodecylsulfate (SDS), the sample was transferred to nitrocellulose membrane after SDS-PAGE. Nonspecific binding was inhibited by exposing the membrane to TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% tween-20) buffer containing 5% skim milk powder at room temperature. The membrane was then incubated overnight at 4 ° C. with iNOS specific antibody (BD Biosciences) and β-actin specific antibody (Sigma). Immunoreactive bands were detected by incubating the samples with horseradish peroxidase-binding secondary antibodies and enhanced chemiluminescent agents (Amersham Biosciences, Piscataway, NJ).

4 Description

To determine the ability of these compounds to induce LPS-induced iNOS expression in mouse macrophages, RAW 264.7 cells were exposed to 500 ng / mL LPS and 10 μM of each compound or LPS alone for 18 hours. 30 μg of protein from each cell lysate was analyzed by 10% SDS-PAGE. Immunoblotting was performed as above. β-actin was used as a control. Bands were quantified using image analysis software and displayed relative intensity compared to images of carrier-treated RAW 264.7 cells.

Claims (12)

(A) Compound 17, Compound 12, or Compound 14 represented by Formula 12 DMF-DMA ( N, N- dimethylformamide dimethyl acetal) was added and reacted at 90 ° C. overnight, followed by acid treatment of the corresponding amino ketone compounds, respectively. The corresponding 4 H -chromen-4 represented by the formula (18). -one obtaining (4 H -chromen-4-ones ) compound 18a, 18b or 18c respectively;
(B) the 4 H - chromen-4-one (4 H -chromen-4-ones ) Compound 18a, 18b or 18c to a hydrogenation at normal temperatures by use of a catalyst Pd / C only the corresponding chroman of the formula 19 Obtaining -4-one (chroman-4-ones) compound 19a, 19b or 19c, respectively;
(C) 25- by adding EtOH / H ₂ O (5/1), KOH, KOH, compound 16 represented by the formula (16) to the Chroman-4-one compound 19a, 19b or 19c Condensation reaction at 30 ° C. for 10-24 hours to obtain corresponding compounds 20a, 20b, or 20c represented by Formula 20, respectively; And
(D) deprotecting the compound 20a, 20b or 20c using 1N HCl to obtain corresponding compounds 1, 3 or 7 represented by Formula 1, respectively;
Compound 12 is
(A) Treating 1,3,5-trimethoxybenzene represented by the formula (10) with acetic anhydride in the presence of boron trifluoride diethyl etherate, Friedel-Crafts acylation (Friedel- Obtaining a compound 11 represented by Chemical Formula 11 by a crafts acylation reaction; And
(B) optionally ortho to reflux was added AlCl ₃ to the compound 11 to obtain the demethylated compound 12 with (ortho -demethylation) reaction; is generated via a,
Compound 14 is produced by the reaction of acetylation at room temperature by adding Ac ₂ O, Et ₃ N, CH ₂ Cl ₂ to compound 13 represented by Formula 13 and subsequent rearrangement by adding BF ₃ · Et ₂ O, AcOH Method for synthesizing homoisoflavonoid compounds, characterized in that.
<Formula 10>

<Formula 11>

<Formula 12>

(Compound 17: R ¹ , R ² = H,
Compound 12: R ¹ = OMe, R ² = H,
Compound 14: R ¹ , R ² = OMe)
<Formula 13>

<Formula 18>

(Compound 18a: R ¹ , R ² = H,
Compound 18b: R ¹ = OMe, R ² = H,
Compound 18c: R ¹ , R ² = OMe)
<Formula 19>

(Compound 19a: R ¹ , R ² = H,
Compound 19b: R ¹ = OMe, R ² = H,
Compound 19c: R ¹ , R ² = OMe)
<Formula 16>

<Formula 20>

(Compound 20a: R ¹ , R ² = H,
Compound 20b: R ¹ = OMe, R ² = H,
Compound 20c: R ¹ , R ² = OMe)
<Formula 1>

(Compound 1: R ¹ , R ² = H,
Compound 3: R ¹ = OMe, R ² = H,
Compound 7: R ¹ , R ² = OMe)
The method according to claim 1,
After step (d)
(E) hydrogenating the compounds 1, 3 or 7 with a Pd / C catalyst to obtain the corresponding homoisoflavonoid compounds 2, 4 or 8, respectively represented by Formula 2; Method for synthesizing homoisoflavonoid compounds.
<Formula 2>

(Compound 2: R ¹ , R ² = H,
Compound 4: R ¹ = OMe, R ² = H,
Compound 8: R ¹ , R ² = OMe)
The method according to claim 2,
After step (e)
(F) obtaining the corresponding compounds 5 or 9, respectively, by the selective ortho-demethylation reaction in CH ₂ Cl ₂ using 1.0 M BCl ₃ , respectively, corresponding compounds 5 or 9; Method for synthesizing homoisoflavonoid compounds, characterized in that.
<Formula 5>

(Compound 5: R ¹ = OH, R ² = H,
Compound 9: R ¹ = OH, R ² = OMe)
The method according to claim 1,
The compound 16 is a method for synthesizing a homoisoflavonoid compound, characterized in that produced by the ethoxymethyl protecting group of the salicylic aldehyde represented by the formula (15) using chloromethyl ethyl ether, K ₂ CO ₃ and TBAI (tetrabutylammonium iodide).
<Formula 15>

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