KR101997515B1 - Synthetic method for portulacanone D and anti-inflammatory pharmaceutical compounds containing thereof - Google Patents

Abstract

Inventors of the present invention synthesized natural homoisoflavonoid portulacanone D (compound 6) isolated from Portulaca oleracea L (POL). An ability to inhibit NO production in LPS-induced RAW 264.7 macrophages was assessed as an indicator of anti-inflammatory activity. The tested portulacanone D did not show clear cytotoxicity and inhibited NO production in the RAW 264.7 macrophages in a concentration dependent manner. The portulacanone D exhibits 92.5% of NO production inhibition at 10 μM and has an IC50 value of 2.09 μM. The finding has additional correlation with the suppressed expression of iNOS induced by LPS. According to the information obtained from the study of the present invention, the portulacanone D can be used in the development of anti-inflammatory drugs targeting NO production.

Description

TECHNICAL FIELD The present invention relates to a method for synthesizing a porphyran canon D and an anti-inflammatory pharmaceutical composition containing the same,

The present invention relates to a method for synthesizing a natural homoisoflavonoid compound, Portulaca cannon D (Compound 6), isolated from Portulaca oleracea L (POL) and an antiinflammatory pharmaceutical composition containing the same.

Inflammation is a basic cellular / tissue protective response to infection by pathogens, damaged cells, or stimulants (1,2). Inflammation It is thought to be the beginning of the treatment process and can be divided into acute or chronic inflammation. One is a host's adaptive defense mechanism for infection or injury and is self-limiting, while the other can cause diverse diseases including diabetes, arthritis and cancer. During the inflammation process, nitric oxide (NO), prostaglandin (PG), vasoactive amines (histamine, serotonin), leukotriene (LT), cytokines (tumor necrosis factor and interleukin- Neutrophils, platelets, and mononuclear cells / macrophages. These mediators bind to specific target receptors in cells to increase vascular permeability, promote smooth muscle contraction, promote neutrophil chemotaxis, increase direct enzymatic activity, induce pain and / or mediate oxidative damage 3).

Nitric oxide is one of the important signaling molecules synthesized from amino acid L-arginine by nitric oxide synthase (endothelial-NOS, neuron-NOS and induced-NOS). Nitric oxide controls various physiological and pathophysiological processes, and NO concentration plays a crucial role in the development of inflammation (4). High levels of physiological NO produced by endothelial-NOS and neuro-NOS stimulate cell survival and proliferation, whereas higher levels of NO induced by induction-NOS arrest cell cycle and promote apoptosis (5). The interaction of NO with reactive oxygen species, such as superoxide and hydroxyl radicals, leads to a reduction in NO concentration, inhibits signal transduction activity, and the resulting reactive nitrogen species is oxidative and nitric oxide nitrosative stress responses (6). Non-steroidal anti-inflammatory drugs, steroids and antihistamines are generally preferred for the treatment of pain, inflammation and fever, but these drugs have disadvantages. Therefore, pharmacological intervention with NO production cascade among various therapeutic interventions for inflammatory diseases has been advocated as a promising strategy.

Naturally occurring flavonoid compounds, particularly the subclasses of flavonoids known as homoisoflavonoids, have shown great biological potential in recent years (7, 8). Previous studies have shown antioxidant, anti-inflammatory, antibacterial, antifungal, antiviral, anti-cytotoxic, antimutagenic and anti-diabetic activities of homoisoflavonoids (8,9). In addition, some derivatives exhibited enzyme 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 used as a private medicine to alleviate a wide range of diseases as well as edible plants (11). These compounds exhibited in vitro cytotoxic activity on four human tumor cell lines including SGC-7901, NCI-H460, K-562 and SF-268 after isolation (11). So far, there has been no report on the synthesis of portulaca cannon AD and 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 JON 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. Kontogiorgisca, Hadjipavlou-Litina D. Med Res Rev. 2002; 22: 385-418.

It is an object of the present invention to provide a method for synthesizing Portulaca cannon D.

The present invention also aims to provide a useful drug by studying the biological activity of Portulaca cannon D.

As a continuing study focused on the synthesis and evaluation of natural products having biological activity as nitric oxide inhibitors and their analogues, the present invention synthesized naturally occurring porphylan canon D (compound 6) (Fig. 2).

The synthesis of the porutulacanone or its derivatives (compounds 1-9) was initiated with the Friedel-Crafts acylation of commercially available 1,3,5-trimethoxybenzene (compound 10) 2). Compound 10 was treated with acetic anhydride in the presence of boron trifluoride diethyl etherate (BF ₃ .Et ₂ O) to give compound 11 in 89% yield. Compound 11 using AlCl ₃ selective ortho-demethylated to give the compound 12 is (ortho -demethylation) in a yield of 87%. Another acetophenone 14 was obtained from compound 13. Compound 14 was obtained with a significant yield of 93% over two steps by acetylation of compound 13 and subsequent rearrangement. Compound 16 protected with ethoxymethyl (EOM) ether of salicylaldehyde (compound 15) was obtained in 83% yield using chloromethyl ethyl ether (EOM-Cl), K ₂ CO ₃ and TBAI (tetrabutylammonium iodide) . Next, commercially available 2-hydroxy-4-methoxyacetophenone (Compound 17), Compound 12 and Compound 14 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 Respectively, in a yield of 88-91%. Hydrogenation with the catalyst of compounds 18a-18c gave the corresponding chroman-4-ones 19a-19c.

After obtaining compounds 19a-19c and compound 16, we studied the aldol condensation reaction. Initially, the reaction between compound 19b and compound 15 was attempted using p -toluenesulfonic acid ( p TsOH) and benzene, which was unsuccessful. Next, compound 20b was obtained in a low yield (less than 20%) in the condensation reaction of compounds 19b and 16 using piperidine as base in benzene solvent as well as DMF solvent at 30-100 ° C. Satisfactory, we have found that KOH in EtOH / H ₂ O (5/1) is effective for condensation of compound 19b and compound 16. Compound 20a (48%) and compound 20c (25%) were obtained by the reaction of Compound 19a and Compound 19c with Compound 16, respectively. Compounds 1, 3 and 7 were obtained in high yield by deprotection of compounds 20a-20c using 1N HCl. Hydrogenation reaction using the catalysts of the compounds 1, 3 and 7 yielded homoisoflavonoid compounds 2, 4 and 8, respectively, in a yield of 86-91%. Finally, the three target compounds 5, 9 and 6 remaining by selective ortho-demethylation of compounds 4, 8 and 3 were synthesized well using 1.0 M 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 ability of RAW 264.7 macrophages induced by LPS to inhibit NO production on the synthesized homoiso flavonoids (Compound 1-9) was analyzed. 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 to 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 a control. However, at 25, 50 and 100 μM concentrations, compound 1-9 showed almost the same level of activity (FIG. 3). A significant change in inhibition of NO production was observed at 1 to 10 μM. Therefore, the present inventors analyzed the synthesized Compound 1-9 only at concentrations of 1 and 10 μM. 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 Griess reagent.

Compound
NO Production (% inhibition) 1 μM 10 μM IC ₅₀ ([mu] M) 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 tested compounds 1-9 decreased NO production in a concentration-dependent manner in RAW 264.7 macrophages (Table 1). The percent inhibition of NO ranged from 97.2% to 15.9% at the highest concentration (10 μM). The most effective inhibitory effect of 4 compounds of Compound 1-9, namely Compound 3 (97.2%), Compound 6 (Fortulacanone D) (92.5%), Compound 1 (91.4%) and Compound 7 (83.0% (Table 1 and Fig. 3). At the lowest concentration (1 μM), Compound 3 still significantly reduced NO production (38.5%) in RAW 264.7 macrophages. The IC ₅₀ values of the compounds 1-9 were evaluated with GraphPad Prism 4.0 software and showed 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 compounds 1-9 was not due to apoptosis, the cytotoxicity of compounds against RAW 264.7 macrophages was examined by MTT assay. As a result, no detectable cytotoxicity was observed at a concentration of 10 μM for 24 hours in all of the test compounds 1-9, and effectively suppressed NO production (Table 2). Compounds 1-9 showed excellent inhibitory activity against NO production at 50 μM and 100 μM, but this is likely due to the cytotoxic effect of compounds 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 related to the modulation of iNOS. As shown in Fig. 4, the results were in agreement with the results related to NO production (Table 1 and Fig. 3) and protein expression of LPS-induced iNOS in RAW 264.7 cells was inhibited by treatment with compounds 1, 3, 6 and 7 Lt; / RTI &gt; However, these compounds did not affect the expression of the β-actin, a housekeeping gene. This indicates that decreased expression of iNOS due to this compound exposure is responsible for inhibiting NO production. Because the present invention screened a limited number of compounds, i.e. only nine compounds, sufficient structure-activity relationship (SAR) analysis is not possible. However, the following are noteworthy: 1) Since 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 (compound 3) or one methoxy group (-OMe) having two methoxy groups (-OMe) and one hydroxymethoxy group (-OMe) in the C3 and C11 double bonds including the compounds (1,3,6 and 7) A compound having a lock time (-OH) (compound 1) exhibits an NO inhibitory activity superior to a compound having a compound having one methoxy group (compound 1) and a compound 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 .

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 commercially available compounds. The ability of these compounds to inhibit NO production as an indicator of antiinflammatory activity in RAW 264.7 macrophages induced by LPS was evaluated. All the compounds tested did not show definite cytotoxicity and inhibited NO production in a concentration dependent manner in RAW 264.7 macrophages. Compound 3 (97.2% inhibition at 10μM; IC ₅₀ = 1.26μM), compound 6 (PORT Tula Canon D) (92.5% inhibition at 10μM; IC ₅₀ = 2.09μM), Compound 1 (91.4% inhibition at 10μM; IC ₅₀ = 1.75μM) and compound 7 (83.0% inhibition at 10μM; IC ₅₀ = 2.91μM) showed a significant inhibitory effect of the synthesized compounds. This finding is related to the inhibition of LPS-induced expression of iNOS. The information obtained in the present invention can provide a basis for considering that Compound 3 can be regarded as a leading structure for the development of NO production target antiinflammatory drugs and can also be used as a medicinal plant for the treatment of inflammatory diseases such as Portulaca oleraceae L. Portulaca oleracea L. ) can provide scientific evidence for its usefulness.

The present invention relates to a method for synthesizing a homoeisoplononoid compound represented by the formula (6), PORTULA CANON D, by selective ortho-demethylation using 1.0 M BCl ₃ to the compound 3 represented by the formula (1). All the following chemical formulas refer to those shown in Fig.

&Lt; Formula 1 >

(Provided that Compound 3: R ¹ = OMe, R ² = H)

(6)

In addition, in the present invention,

(A) Compound 12 represented by the formula (12) DMF-DMA (N, N-dimethylformamide dimethyl acetal), and the resulting amino ketone was acid-treated to obtain 4H-Chromen-4-one (4H-chromen-4-ones) compound 18b;

(B) the 4 H - chromen-4-one (4 H -chromen-4-ones ) of the compound 18b chroman hydrogenated by using a catalyst represented by the formula (19) 4-one (chroman-4-ones) To obtain compound 19b;

(C) Addition of EtOH / H ₂ O (5/1), KOH and the compound 16 represented by the formula (16) to the chroman-4-ones compound 19b followed by a condensation reaction, To obtain compound 20b; And

(D) deprotecting the compound 20b with 1N HCl to obtain the compound 3 represented by the formula (1).

&Lt; Formula 12 >

(Compound 12: R ¹ = OMe, R ² = H)

&Lt; Formula 18 >

(Compound 18b: R ¹ = OMe, R ² = H)

(19)

(Provided that Compound 19b: R ¹ = OMe, R ² = H)

&Lt; Formula 16 >

(20)

(Compound 20b: R ¹ = OMe, R ² = H)

The present invention also relates to an anti-inflammatory pharmaceutical composition comprising a phytulacanone D represented by the general formula (6).

According to the synthesis method of the present invention can be synthesized in the formate Tula car oleic la years old child (Portulaca oleracea L.) L.-derived compound, formate Tula Canon D in high yield.

Furthermore, the phytulacanone D synthesized by the method of the present invention does not exhibit cytotoxicity and exhibits excellent anti-inflammatory activity and can be used as a therapeutic agent for inflammatory diseases.

Figure 1 shows the structure of Portulaca cannon AD (Compounds 4, 8, 9 and 6) and its derivatives (Compounds 1-3, 5 and 7).
Fig. 2 shows reaction schemes for the synthesis method of the present invention of portulaca cannon AD and its derivatives. Reagents and conditions: (a) Ac ₂ O, BF ₃ .Et ₂ O, EtOAc, room temperature, 4 h, 89%; (b) AlCl ₃ , CH ₂ Cl ₂ , reflux, 22h, 87%; (c) (i) Ac ₂ O, Et ₃ N, CH ₂ Cl ₂ , room temperature, 6 h; (ii) BF ₃ .Et ₂ O, AcOH, 70 ° C, 2 h, 93% (two steps overall); (d) Chloromethyl ethyl ether, K ₂ CO ₃ , TBAI (Tetrabutylammonium iodide), acetone, 0 ° C. - room temperature, 24 hours, 83%; (e) N, N -dimethylformamide dimethyl acetal, benzene, 90 [deg.] C overnight reaction, 88-91%; (f) H ₂ , Pd / C (10%), MeOH, room temperature, 0.75-1 h, 83-87%; (g) KOH, EtOH / H 2 O (5/1), 25-30 ℃, 10-24 hours, 25-48%; (h) 1N HCl, MeOH, 55 [deg.] C, 1 h, 85-88%; (i) H ₂ , Pd / C (10%), MeOH, room temperature, 0.75-1 h, 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 depicting the inhibitory activity of homoisoflavonoid 1-9 on NO production in RAW 264.7 macrophages induced by LPS.
Figure 4 shows the results of testing the effect of compounds 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 apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments.

Chemicals

All chemicals were used without purification as purchased unless otherwise noted. All solvents used in the reaction were distilled from the appropriate dehydrating agent under nitrogen gas. All solvents used in the chromatography were purchased and immediately used without further purification. Thin film chromatography (TLC) was carried out using a plate coated with silica gel on a DC-Plasticfolien 60, F254 (Merck, layer thickness 0.2 mm) plastic plate and observed with UV (254 nm) or with p-anisaldehyde and / And stained with phosphomolybdic acid (PMA). Chromatographic purification was performed using Kieselgel 60 (60-120 mesh, Merck). ^{The 1} H-NMR spectra were recorded on Varian Mercury-300 MHz FT-NMR and 75 MHz for ¹³ C, chemical shifts (δ) were expressed in parts per million (ppm) relative to TMS, coupling constants (J) Respectively. The peak splitting pattern was abbreviated as s (singlet), d (doublet), t (triplet), dd (doublet of doublet) and m (multiplet). CDCl ₃ / CD ₃ COCE ₃ was used as solvent and internal standard. High resolution mass spectroscopy electrospray ionization (HRMS-ESI) was recorded using an Agilent technologies 6220 TOF LC / MS spectrometer. Melting points were measured on the MEL-TEMP II apparatus and not calibrated.

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

1,3,5-trimethoxy benzene (0.84g, 5.00mmol) and anhydrous acetic acid (0.47mL, 7.50mmol) were mixed in ethyl acetate _{(5mL), BF 3 .Et 2} O (0.32mL, 2.50mmol) Was slowly added under a nitrogen atmosphere. The reaction was warmed to room temperature and stirred for 4 hours. After completion of the reaction, H ₂ O (40 mL) was added and after stirring for 15 minutes, the reaction mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic solvent layers were washed with saturated NaHCO ₃ solution ( ₂ x 20 mL), H ₂ O ( ₂ x 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 obtain 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 )? 6.09 (2H, s), 3.82 (3H, s), 3.79 (6H, s), 2.46 (3H, s); _{13 C NMR (75MHz, CDCl 3} ) δ201.7, 162.3, 158.4, 113.7, 90.7, 56.0, 55.6, 32.8.

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

Compound 11 (0.9 g, 4.28 mmol) was dissolved in anhydrous CH ₂ Cl ₂ (10 mL), and 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 completion of the reaction, the mixture was cooled to 0 ° C, 1N HCl (6 mL) was added, and the mixture was stirred at room temperature for 15 minutes. The mixture was extracted with CH ₂ Cl ₂ (2 x 50 mL). The combined organic solvent layers were washed with H ₂ O ( ₂ x 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 pure white solid compound (0.72 g, 87%). Melting point 79-80 占 폚 (literally melting point 81-82 占 폚); _{1 H NMR (300MHz, CDCl 3} ) δ6.03 (1H, d, J = 2.1Hz), 5.90 (1H, d, J = 2.1Hz), 3.84 (3H, s), 3.80 (3H, s), 2.60 (3H, s); _{13 C NMR (75MHz, CDCl 3} ) δ203.1, 167.6, 166.1, 162.9, 106.1, 93.6, 90.8, 55.8, 33.2.

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

3,4,5-trimethoxyphenol (compound13) (1.55 g, 8.42 mmol) was dissolved in anhydrous CH₂Cl₂(16 mL) and to the stirred solution was added Et₃N (2.36 mL, 16.63 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. Acetic anhydride (1.2 mL, 12.62 mmol) was added thereto, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction, CH₂Cl₂ (80 mL), washed with 2N HCl (8 mL), H₂O (2 x 20 mL), brine (20 mL), dried Na₂SO₄, &Lt; / RTI &gt; and concentrated in vacuo. Acetylated colorless solid compound (1.88 g, 99%) was obtained which was used in the next step without further purification. Melting point 68-70 ° C;_One&Lt; 1 &gt; H NMR (300 MHz, CDCl3₃) [delta] 6.32 (2H, s), 3.82 (9H, s), 2.28 (3H, s);₁₃C NMR (75 MHz, CDCl3₃) [delta] 169.5, 153.5, 146.8, 136.0, 99.3, 61.1, 56.4, 21.4.

BF ₃揃 Et ₂ O (3.6 mL, 29.16 mmol) was added to a solution of 3,4,5-trimethoxyphenylacetate (1.88 g, 8.33 mmol) in AcOH (2.2 mL) I went a drop. The resulting mixture was stirred at 70 &lt; 0 &gt; C for 2 hours. After completion of the reaction, the reaction 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 (3 x 35 mL). The combined organic solvent layers were washed with H ₂ O (3 x 20 mL), brine ( ₂ x 20 mL), dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to give a light yellow liquid compound 14 (1.77 g, 94% And used for the next step without. ₁ H NMR (300 MHz, CDCl ₃ )? 6.22 (1H, s), 3.98 (3H, s), 3.86 (3H, s), 3.77 (3H, s), 2.65 ₁₃ C NMR (75 MHz, CDCl ₃ )? 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):

Salicylaldehyde (0.53 mL, 5.00 mmol) was added to anhydrous acetone (12 mL) and to the stirred solution was added K ₂ CO ₃ (1.38 g, 20.00 mmol) was added under a nitrogen atmosphere and cooled to 0 &lt; 0 &gt; C. After stirring for 20 min, a solution of chloromethyl ethyl ether (EOM-Cl) (0.56 mL, 6.0 mmol) was added dropwise followed by the addition of a solution of tetrabutylammonium iodide (0.2 mmol, 0.1 eq.) In anhydrous acetone Were added. The resulting mixture was warmed to room temperature and stirred for 24 hours. After completion of the reaction, the reaction product 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 x 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-protected aldehyde compound 16 (0.75 g, 83%) as a colorless oil. _{1 H NMR (300MHz, CDCl 3} ) δ 10.47 (1H, s), 7.82 (1H, d, J = 7.5Hz), 7.52 (1H, td, J = 8.1, 2.1Hz), 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 (75MHz, 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 - Kromen (4 H - chromen -4-one)

To the stirred solution of the substituted 2-hydroxyacetophenone (1.4 mmol) in anhydrous benzene (4 mL), N , N -dimethylformamide dimethyl acetal (4.2 mmol) was added at room temperature under a nitrogen atmosphere. The mixture was stirred at 90 &lt; 0 &gt; C overnight. After completion of the reaction, the mixture was cooled to 0 ° C and saturated HCl (0.85 mL) was added. The resulting mixture was stirred at 55 &lt; 0 &gt; C for 1 hour. After completion of the reaction, it was 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 4H -chromen-4-one pure.

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

5,7-dimethoxy -4 H - chromen-4-one {5,7- Dimethoxy -4 H - chromen -4 -one} ( compound 18b): Yield: 90%; Light yellow solid; Melting point 131-133 DEG C; _{1 H NMR (300MHz, CDCl 3} ) δ 7.60 (1H, d, J = 6.0Hz), 6.41 (1H, d, J = 2.1Hz), 6.34 (1H, d, J = 6.0Hz), 6.17 (1H, d, J = 6.0 Hz), 3.93 (3H, s), 3.87 (3H, s); _{13 C NMR (75MHz, 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%; Light yellow solid; Melting point 113-115 DEG C; _{1 H NMR (300MHz, CDCl 3} ) δ 7.63 (1H, d, J = 6.0Hz), 6.65 (1H, s), 6.16 (1H, d, J = 6.0Hz), 3.95 (3H, s), 3.93 ( 3H, s), 3.89 (3H, s); ₁₃ C NMR (75 MHz, CDCl ₃ ) 隆 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 - Kromen &Lt; RTI ID = 0.0 &gt;

Substituted 4 H-chromen-4-one (1.0mmol) with 10% Pd / C (substituted 4 H placed in MeOH (8mL) was added in one-against-chromen-4-one 5% w / w ) Was added under hydrogen atmosphere at room temperature and the mixture was stirred for 0.75-1 hr. After completion of the reaction, the reaction mixture was filtered through a pad of Celite®, 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 chroman-4-one.

7-methoxy-chroman-4-one (7- Methoxychroman -4-one) (Compound 19a): Yield: 87%; White solid; Melting point 49-50 ℃; _{1 H NMR (300MHz, CDCl 3} ) δ 7.80 (1H, d, J = 8.7Hz), 6.55 (1H, dd, J = 8.7, 2.4Hz), 6.38 (1H, d, J = 2.4Hz), 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 ) 隆 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 DEG C; _{1 H NMR (300MHz, CDCl 3} ) δ 6.04 (2H, s), 4.43 (2H, t, J = 6.3Hz), 3.87 (3H, s), 3.81 (3H, s), 2.71 (2H, t, J = 6.3 Hz); ₁₃ C NMR (75 MHz, CDCl ₃ ) 隆 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 (300MHz, CDCl 3} ) δ 6.24 (1H, s), 4.43 (2H, t, J = 6.3Hz), 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 ) 隆 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 vaporized Of homoisoflavonoid  General manufacturing process

To the stirred solution of the substituted chroman-4-one (0.30 mmol) and EOM-protected vaporized salicylaldehyde compound 16 (0.07 g, 0.39 mmol) in EtOH / H ₂ O (1.8 mL, 5/1) (0.05 g, 0.90 mmol) was added thereto, followed by stirring at 25-30 ° C for 10-24 hours. After completion of the reaction, the EtOH was removed under reduced pressure. The crude product was neutralized with 1N HCl and extracted with EtOAc (2 x 25 mL). The combined organic solvent layers were 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 homoisopa flavone compound.

(E) -3- (2- (ethoxy-methoxy) benzylidene) -7-methoxy chroman-4-one {(E) -3- (2- ( Ethoxymethoxy) benzylidene) -7-methoxychroman-4 -one} (Compound 20a): Yield: 48%; Light yellow liquid; _{1 H NMR (300MHz, CDCl 3} ) δ 7.96-7.93 (2H, m), 7.35-7.30 (1H, m), 7.20 (1H, d, J = 8.7Hz), 7.04-6.98 (2H, m), 6.60 (1H, dd, J = 8.7 , 2.4Hz), 6.37 (1H, d, J = 2.4Hz), 5.24 (2H, s), 5.20 (2H, d, J = 1.5Hz), 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- (methoxy-methoxy) benzylidene in) -5,7-dimethoxy-chroman-4-one {(E) -3- (2- ( Ethoxymethoxy) benzylidene) -5, 7-dimethoxychroman-4-one} (Compound 20b): Yield: 40%; Light 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 (75MHz, 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- (ethoxy-methoxy) benzylidene) -5,6,7- trimethoxy-chroman-4-one {(E) -3- (2- ( Ethoxymethoxy) benzylidene) - 5,6,7-trimethoxychroman-4-one} (Compound 20c): Yield: 25%; Light yellow liquid; ₁ H NMR (300 MHz, CDCl ₃ )? 7.93 (1H, s), 7.30-7.26 (1H, m), 7.15 (1H, d, J = 8.1 Hz), 7.02-6.98 (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), 3.67 (2H, q, J = 7.0 Hz), 1.20 (3H, t, J = 7.0 Hz).

EOM - Deprotection vaporization Homoisoflavonoid  General procedure of synthesis

To the stirred solution of EOM-protected homoisoflavonoid (0.2 mmol) in MeOH (5 mL) was added IN HCl (0.6 mL) at room temperature and the mixture was stirred at 55 ° C for 1 h. 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 give the pure EOM-deprotected vaporized homoiso flavonoid compound.

(E) -3- (2- hydroxy-benzylidene) -7-methoxy chroman-4-one {(E) -3- (2- Hydroxybenzylidene) -7-methoxychroman-4-one} ( Compound 1) : Yield: 89%; Light yellow solid; Melting point 135-137 DEG C; _{1 H NMR (300MHz, CDCl 3} ) δ 7.99 (1H, br s), 7.92 (1H, d, J = 8.7Hz), 7.27 (1H, dd, J = 8.7, 2.1Hz), 7.02-6.90 (3H, m), 6.81 (1H, s ), 6.58 (1H, dd, J = 8.7, 2.4Hz), 6.34 (1H, d, J = 2.1Hz), 5.19 (2H, d, J = 1.5Hz) 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 +): 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%; Light yellow solid; _{1 H NMR (300MHz, DMSO-} d6) δ 10.02 (1H, s), 7.70 (1H, s), 7.23 (1H, td, J = 8.1, 1.8Hz), 7.03 (1H, d, J = 6.6Hz) , 6.91 (1H, d, J = 7.8Hz), 6.84 (1H, t, J = 7.5Hz), 6.24 (1H, d, J = 2.1Hz), 6.16 (1H, d, J = 2.1Hz), 5.11 (2H, d, J = 1.5 Hz), 3.81 (3H, s), 3.80 (3H, s); ₁₃ C NMR (75 MHz, DMSO-d 6)? 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 @ +), 295, 180 (base), 131; _{HRMS: Calcd for C 18 H 16} O 5 (M +): 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%; Light 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 (3 H, s); ₁₃ C NMR (75 MHz, CDCl ₃ ) 隆 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 +): 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 Following the procedure, Compound 1 (0.04 g, 0.14 mmol) was stirred under hydrogen (balloon) atmosphere to give a white solid compound 2 (0.037 g, 91%). Melting point 132-134 DEG C; _{1 H NMR (300MHz, CDCl 3} ) δ 8.12 (1H, br s), 7.82 (1H, d, J = 8.7Hz), 7.10 (1H, td, J = 8.1, 1.8Hz), 7.02 (1H, dd, J = 7.8, 1.8Hz), 6.90 (1H, dd, J = 8.1, 1.2Hz), 6.80 (1H, td, J = 7.5, 1.2Hz), 6.54 (1H, dd, J = 8.7, 2.1Hz), (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 , &lt; / RTI &gt; m), 2.87-2.79 (1H, 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 +), 283 (MH), 267, 177 (base); HRMS Calcd for C17H16O4 (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 Compound 3 (0.02 g, 0.06 mmol) was stirred under hydrogen (balloon) atmosphere according to the general procedure used for the synthesis to give compound 4 (0.018 g, 87%) as a white solid. Melting point 137-139 DEG C; _{1 H NMR (300MHz, CDCl 3} ) δ 8.22 (1H, br s), 7.10 (1H, td, J = 7.8, 1.8Hz), 7.01 (1H, dd, J = 7.5, 1.8Hz), 6.90 (1H, dd, J = 8.1, 1.2Hz) , 6.79 (1H, td, J = 7.5, 1.2Hz), 6.01 (2H, d, J = 2.1Hz), 4.49 (1H, dd, J = 11.4, 5.4Hz), 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 +): 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) : 19a-19c Following the general procedure used in the synthesis, Compound 7 (0.02 g, 0.06 mmol) was stirred under hydrogen (balloon) atmosphere to give a white solid compound 8 (0.017 g, 86%). Melting point 102-104 DEG C; _{1 H NMR (300MHz, CDCl 3} ) δ 8.09 (1H, br s), 7.12-7.02 (2H, m), 6.91 (1H, d, J = 8.1Hz), 6.79 (1H, t, J = 7.2Hz) , 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 ), 3.14-3.00 (2H, m), 2.78 (1H, dd, J = 13.2, 5.7 Hz); ₁₃ C NMR (75 MHz, CDCl ₃ )? 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 +): 344.1260, found: 344.1271

Of homoisoflavonoid Ortho - Demethylation  General procedure for

BCl ₃ solution (1.0 M in CH ₂ Cl ₂ ; 0.36 mL) was added dropwise to a solution of homoisoflavonoid (0.12 mmol) in anhydrous CH ₂ Cl ₂ (4 mL) at 0 ° C under nitrogen atmosphere. The mixture was stirred at &lt; RTI ID = 0.0 &gt; 0-5 C &lt; / RTI &gt; After completion of the reaction, H ₂ O (5 mL) was added and stirred for 15 minutes and then extracted with CH ₂ Cl ₂ ( ₂ × 20 mL). The combined organic solvent layers were 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 homoiso flavonoid.

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 캜; _{1 H NMR (300MHz, CDCl 3} ) δ 11.78 (1H, s), 7.12 (1H, td, J = 8.1, 1.8Hz), 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 = 2.4Hz), 4.44 (1H, dd, J = 11.4, 4.5Hz), 4.17 (1H, t, J = 11.4Hz), 3.79 (3H, s), 3.17-3.07 (2H, m), 2.89-2.81 (1H, m); _{13 C NMR (75MHz, 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 @ +), 299 (MH @ +), 193 (base), 165; _{HRMS: Calcd for C 17 H 16} O 5 (M +): 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%; Light yellow solid; Melting point 135-137 DEG C; _{1 H NMR (300MHz, CDCl 3} ) δ 11.71 (1H, s), 7.12 (1H, t, J = 7.5Hz), 7.06 (1H, d, J = 7.5Hz), 6.98 (1H, br s), 6.87 (1H, m), 6.01 (1H, s), 4.42 (1H, dd, J = 11.4, 4.2Hz), 4.21-4.14 ), 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%; Light yellow solid; Melting point 172-174 DEG C; _{1 HNMR (300MHz, CD 3 COCD} 3) δ 12.76 (1H, s), 9.09 (1H, br s), 8.02 (1H, s), 7.31 (1H, t, J = 7.8Hz), 7.16 (1H, d , J = 7.5Hz), 7.02 ( 1H, d, J = 7.8Hz), 6.42 (1H, t, J = 7.5Hz), 6.06 (1H, d, J = 2.1Hz), 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 )? 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 @ +), 281, 253, 131 (base); _{HRMS: Calcd for C 17 H 14} O 5 (M +): 298.0841, found: 298.0847

reagent

LPS, DMSO and β-actin antibodies from Escherichia coli were purchased from Sigma-Aldrich (St Louis, MO, USA). DMEM (Dulbeccos modified Eagles medium), fetal calf 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 survival analysis

RAW 264.7 mouse macrophages were cultured in DMEM containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin at 37 ° C. and 5% CO ₂ from Korean Cell Bank (Seoul, Korea). The effect of various compounds on cell viability was tested using the CellTiter 96® Aqueous One Solution Assay of Cell Proliferation (Promega, Madison, WI, USA), which uses a color reaction to count viable cells. This assay was used to measure the number of viable cells remaining after the culture process was completed. RAW 264.7 cells were 2X10 to the plate with a flat bottom of a 96-well ⁴ The cell density was set so that each compound was added to each plate at the indicated concentration. After 24 hours of culture, the number of viable cells was counted as instructed by the manufacturer. This analytical method is based on the reduction of the tetrazolium compound MTS to formazan, which exhibits maximum absorbance at 490 nm. Thus, the amount of product in the cell culture fluid 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 measured in the RAW 264.7 cell culture supernatant. RAW 264.7 cells were 5X10 on the all 96-well culture medium of the cell culture plate 200μL ⁴ Added to the cell density and incubated for 12 hours. The cells were cultured for 18 hours at a concentration of 500 ng / mL of LPS and compound 1 to 10 as indicated. The amount of NO produced was measured using the Griess reagent system (Promega) according to the manufacturer's instructions.

Immunoblotting  analysis

To examine 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. Cells were then washed with cold PBS and resuspended in 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, Na ₃ VO ₄ , 1 mM PMSF, 10 μg / ml leupeptin and 10 μg / ml aprotinin) were added to each sample. The protein was denatured with SDS (sodium dodecylsulfate), and the sample was subjected to SDS-PAGE and transferred to nitrocellulose membrane. The membrane was exposed to TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% tween-20) buffer containing 5% defatted milk powder at room temperature to inhibit nonspecific binding. The membranes were then incubated overnight at 4 ° C with iNOS-specific antibodies (BD Biosciences) and beta -actin-specific antibodies (Sigma). Immunoreactive bands were detected by incubating the samples with horseradish peroxidase-conjugated secondary antibody and enhancer chemiluminescent (Amersham Biosciences, Piscataway, NJ).

FIG.

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 each compound or LPS alone for 18 h. 30 μg of protein from each cell lysate was analyzed by 10% SDS-PAGE. Immunoblotting was performed as above. beta -actin was used as a control. The bands were quantified using image analysis software and displayed relative intensity as compared to images of carrier-treated RAW 264.7 cells.

Claims (3)

A method of synthesizing a porphyran canon D represented by the formula 6 by selective ortho-demethylation using 1.0 M BCl ₃ to the compound 3 represented by the formula (1)
&Lt; Formula 1 >

(Provided that Compound 3: R ¹ = OMe, R ² = H)
(6)

The method according to claim 1,
The compound 3
(A) Compound 12 represented by the formula (12) DMF-DMA (N, N -dimethylformamide dimethyl acetal) was condensed with, 4 H by acid treatment and the resulting amino-ketone of the formula 18-chromen-4-one (4 H -chromen-4-ones ) compound 18b;
(B) the 4 H - chromen-4-one (4 H -chromen-4-ones ) of the compound 18b chroman hydrogenated by using a catalyst represented by the formula (19) 4-one (chroman-4-ones) To obtain compound 19b;
(C) Addition of EtOH / H ₂ O (5/1), KOH and the compound 16 represented by the formula (16) to the chroman-4-ones compound 19b followed by a condensation reaction, To obtain compound 20b; And
(D) deprotecting the compound 20b with 1N HCl to obtain the compound 3 represented by the formula (1).
&Lt; Formula 12 >

(Compound 12: R ¹ = OMe, R ² = H)
&Lt; Formula 18 >

(Compound 18b: R ¹ = OMe, R ² = H)
(19)

(Provided that Compound 19b: R ¹ = OMe, R ² = H)
&Lt; Formula 16 >

(20)

(Compound 20b: R ¹ = OMe, R ² = H)
An antiinflammatory pharmaceutical composition comprising a phytulacanone D represented by the following formula (6).
(6)

Images