Next Article in Journal
Exploring the Role of Phenolic Compounds in Chronic Kidney Disease: A Systematic Review
Previous Article in Journal
Metal–Flavonoid Interactions—From Simple Complexes to Advanced Systems
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 29
Issue 11
10.3390/molecules29112574
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Total Syntheses and Antibacterial Studies of Natural Isoflavones: Scandenone, Osajin, and 6,8-Diprenylgenistein

by
Hongbo Dong
1,†,
Yufei Che
1,†,
Xingtong Zhu
2,
Yi Zhong
1,
Jiafu Lin
1,
Jian Wang
1,
Weihong Du
1,* and
Tao Song
1,*
1
Anti-Infective Agent Creation Engineering Research Centre of Sichuan Province, School of Pharmacy, Chengdu University, Chengdu 610106, China
2
School of Food Science and Biological Engineering, Zhejiang Gongshang University, Hangzhou 310018, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2024, 29(11), 2574; https://doi.org/10.3390/molecules29112574
Submission received: 11 May 2024 / Revised: 28 May 2024 / Accepted: 28 May 2024 / Published: 30 May 2024
Browse Figures
Versions Notes

Abstract

:
Isoflavones are a class of natural products that exhibit a wide range of interesting biological properties, including antioxidant, hepatoprotective, antimicrobial, and anti-inflammatory activities. Scandenone (1), osajin (2), and 6,8-diprenylgenistein (3) are natural prenylated isoflavones that share the same polyphenol framework. In this research, the key intermediate 15 was used for the synthesis of the natural isoflavones 1–3, establishing a stereoselective synthetic method for both linear and angular pyran isoflavones. The antibacterial activities of 1–3 were also evaluated, and all of them displayed good antibacterial activity against Gram-positive bacteria. Among them, 2 was the most potent one against MRSA, with a MIC value of 2 μg/mL, and the SEM assay indicated that the bacterial cell membranes of both MRSA and E. faecalis could be disrupted by 2. These findings suggest that this type of isoflavone could serve as a lead for the development of novel antibacterial agents for the treatment of Gram-positive bacterial infections.

1. Introduction

Isoflavones are plant secondary metabolites characterized by a B-ring attached to the C-3 position of their C-ring (Figure 1) [1,2]. They exhibit a range of biological properties, including antioxidant [3], hepatoprotective [4], antimicrobial [5], and anti-inflammatory [6] activities. Prenylated isoflavones in particular have pronounced pharmacological effects due to their higher lipophilicity and affinity for biological cell membranes. Scandenone (1) is a natural prenylated isoflavone that has been mainly isolated from the Fabaceae and Moraceae plant families [7,8]. Till now, the biological activities of scandenone (1) have been extensively studied. It is a powerful protein kinase inhibitor and displays potent anti-inflammatory activity [9]. The antiviral [7], antibacterial [10], and insecticidal properties [11,12] of scandenone (1) have also been discussed in previously published papers. In 2006, Toker reported that scandenone (1) from the fruits of Maclura pomifera (Rafin.) Schnider (Moraceae) possessed good antibacterial activity against both Gram-positive (Staphylococcus aureus MIC = 0.5 μg/mL, Enterococcus faecalis MIC = 0.5 μg/mL) and Gram-negative (Escherichia coli, MIC = 2 μg/mL) bacterial strains [10]. In 2018, Raksat et al. found that scandenone (1) 1 from Millettia extensa displayed promising antibacterial activity against S. aureus TISTR 1466, S. epidermidis ATCC 12228, and B. subtilis TISTR 008 with the same MIC value of 2 μg/mL [13]. However, Ramasami et al. found that scandenone (1) from Erythrina addisoniae did not exhibit significant antibacterial activity against S. aureus or E. coli with MIC values > 64 μg/mL [14]. These inconsistencies may be due to the different levels of purity of 1 that were exacted from various natural sources, and they suggest that rigorous antibacterial evaluation is needed to determine its authentic activity.
Osajin (2) and 6,8-diprenylgenistein (3) (Figure 1) are natural prenylated isoflavones, that contain very close chemical structures to scandenone (1) and have been investigated over the years for their anticancer [15], anti-inflammatory [16], and antioxidant properties [17]. In particular, 6,8-diprenylgenistein (3) isolated from the roots of Glycyrrhiza uralensis has been reported to display remarkable antibacterial activity against Streptococcus mutans (MIC = 2 μg/mL) and MRSA (MIC = 8 μg/mL) [18]. We have recently become interested in exploring the pharmacological potential of flavonoids [19,20,21,22]. However, isoflavones 1–3 could only be obtained from the respective natural sources in low yields, insufficient for further detailed investigation. We therefore initiated a program to synthesize isoflavones 1–3 to investigate their antibacterial properties.

2. Results and Discussions

To our surprise, it was not until recently that the first total synthesis of Scandenone (1) and Osajin (2) was reported. As demonstrated in Scheme 1, Wang et al. [23] reported a chemoselective propargylation followed by a Claisen rearrangement to construct the crucial pyran isoflavone cores (compounds 6 and 7) of isoflavones 1 and 2. However, in our hands, the chemoselective propargylation only gave about 15% yield of pure 5, and the separation of the highly polar compounds 6 and 7 by chromatography was very difficult. To our knowledge, no synthetic approach to 6,8-diprenylgenistein (3) has been reported. The above-mentioned reasons were the impetus for our current efforts to synthesize the prenylated isoflavones 1–3 and to compare their antibacterial activities.
As shown in Scheme 2, we started our synthesis to afford natural compounds 1–3 by making a known intermediate, 3-iodochromone 12 [24]. Using previously published procedures, the selective protection of two hydroxyl groups in 8 with methoxymethyl bromide (MOMBr) and N,N-Diisopropylethylamine (DIPEA) provided MOM ether 9 (71%) [25], which was treated with dimethylacetal (10) in DMF to furnish enamino ketone 11 (75%). Enaminone 11 was subsequently subjected to intramolecular cyclization using I2 in MeOH at rt to give 3-iodochromone 12 (65%). We then utilized a palladium-catalyzed Suzuki reaction with 12 and phenylboronic acid 13 to give the desired isoflavone 14 in 76% yield [26,27]. Removal of the MOM groups of 14 provided the desired 5,7-dyhydroxyl-isoflavone derivative 15 in 98% yield [25].
With the key intermediate 15 in hand, we next investigated the di-prenylation of 15 with 1-bromo-3-methylbut-2-ene (17) or 3-methylbut-2-en-1-ol (18) to get compound 16. As shown in Table 1, we first tested previously reported conditions that used 17 as a reagent, the mixture of CH2Cl2 and H2O as a solvent, and KOH as a base (entry 1) [28]. However, when we applied these conditions to get 16, an inseparable mixture of products was obtained, and no 16 could be isolated. Replacing KOH with a weaker base such as K2CO3 and DBU [29,30], only afforded the desired product 16 in low yields (entry 2, 3). The unsuccessful attempts are likely attributable to the simultaneous occurrence of C- and O-prenylations during the reaction. To avoid the O-prenylation, we then turned to imply 3-methylbut-2-en-1-ol (18) as a reagent and appropriate organic acids as catalysts in hydrophobic solvents to obtain 16. After a few attempts, we found that the use of p-toluenesulfonic acid in CH2Cl2 could generate 16 in 14% yield (entry 4). Finally, 16 could be obtained in a satisfactory isolated yield (72%) using acidic alumina as a catalyst and 1,2-dichloroethane (DCE) as a solvent (entry 5) [31,32,33]. Compound 16 was treated with newly prepared sodium dodecane-1-thiolate in reflux DMF to generate natural product 3 in 78% yield [19].
As shown in Table 2, the synthesis of scandenone (1) and osajin (2) commenced with the stereocontrolled preparations of tetracyclic isoflavens 19 and 20, which possessed a linear or angular pyran attached to the A-ring, respectively. Our first attempt was to synthesize 19 and 20 in a one-step pyran annulation of 15. As depicted in Table 2 (entry 1 and 2), electrocyclization of 15 with α,β-unsaturated aldehyde (21) [34] or 1,1-diethoxy-3-methylbut-2-ene (22) [25] could only provide a mixture of linear and angular isomers (19 and 20). To overcome this problem, the Claisen rearrangement/cyclization reaction was explored for the stereoselective construction of the pyran rings of both 19 and 20. Treatment of 15 with 3-chloro-3-methylbut-1-yne (23) and K2CO3 in DMF in the presence of a catalytic amount of KI afforded C-7 propargyl ether 24 in a good yield (79%). As shown in Table 2, an intensive screening of reaction conditions was carried out to identify suitable reaction conditions that would lead to the regioselective Claisen rearrangement or cyclization of 24. It was observed that the solvent of the reaction significantly affected the regioselectivity. Aromatic Claisen rearrangement or cyclization in DMF afforded 20 as the main product (77%, entry 3), while in xylene, 19 was isolated as the main product in a yield of 55% (entry 4). We then found that the addition of KOH to xylene could effectively control the regioselectivity, and pure 19 was obtained in 93% yield (entry 5) [33,35], whereas the reported Claisen rearrangement/cyclization conditions of 15 only yielded a roughly 1:1 mixture of 19 and 20 under harsh conditions (entry 6) [23].
Since the NMR data of 3-(4-(benzyloxy)phenyl)-5-hydroxy-8,8-dimethyl-4H,8H-pyrano[2,3-f]chromen-4-one (19) and (4-(Benzyloxy)phenyl)-5-hydroxy-8,8-dimethyl-4H,8H-pyrano[2,3-f]chromen-4-one (20) have not been reported yet; the distinction between the two compounds was challenging and may lead to misassignment due to their structural similarity. We therefore employed 2D NMR spectroscopy to unequivocally confirm the structures of 19 and 20. The HSQC data established all 1J (1H-13C) connectivities (see the Supplementary Materials), and the key HMBC correlations are shown in Figure 2. The HMBC spectra of 19 showed a correlation of 5-OH [δH 13.18] with C-6 [δC 105.0], while there was no correlation of 5-OH with the tertiary carbon atom [C-8 (δH 6.34, δC 93.8)], suggesting that 19 is a linear isomer. The HMBC correlations between 5-OH [δH 12.95] with C-4a [δC 105.0] and tertiary carbon C-6 [δH 6.30, δC 99.3] of 20 indicated that it was an angular isomer.
Since selective debenzylation is difficult in the presence of both pyran and isopentene groups, the benzyl group in 19 was removed and replaced by the acetyl protecting group to give compound 25, which was then subjected to condensation with 18 under typical Mitsunobu conditions using triphenylphosphine (PPh3) and diethyl azodicarboxylate (DEAD) to obtain 26 (Scheme 3). Compound 26 was subjected to a europium(III)-tris(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionate) [Eu(fod)3]-catalyzed aromatic para-Claisen-Cope rearrangement conditions to afford 27 (91%) [34]. Deprotection of the -Ac group with 60% KOH in EtOH completed the synthesis of scandenone (1) (87%), the structure of which was confirmed by single-crystal X-ray analysis (see the Supplementary Materials). Our X-ray analysis data was in line with previously published results [14].
In the same manner, compound 20 was smoothly translated into Ac-protected compound 28, and compound 30 was obtained by Pd-catalyzed allylation of 28 with tert-butyl (2-methylbut-3-en-2-yl) carbonate (29), in the presence of Pd(PPh3)4 and 4Å molecular sieves in nitrogen atmosphere in 78% yield (Scheme 4) [34]. Subsequently, an [Eu(fod)3]-catalyzed aromatic ortho-Claisen rearrangement was taken to afford 31 in a yield of 84%. Finally, treatment of 31 with a KOH aqueous solution provided natural isoflavone 2 in 71% yield.
The target compounds (1–3) were evaluated for their in vitro antibacterial activities against three Gram-positive (G+) bacterial strains (S. aureus ATCC29213, MRSA ATCC33591, and Enterococcus faecalis ATCC29212,) and one Gram-negative (G-) bacterial strain (E. coli ATCC25922) using minimum inhibitory concentration (MIC) values. Ampicillin and vancomycin were used as the positive controls [33].
As shown in Table 3, the anti-bacterial activity data showed that 1–3 were active against three G+ bacteria, including a multidrug-resistant strain (MRSA ATCC33591), but not for G-strain E. coli ATCC25922, which is consistent with the results reported by Raksat et al. [13] and Nkengfack et al. [18]. Among these compounds, natural isoflavone 2 exhibited the most potent antibacterial activity against G+ bacteria, with MIC values ranging from 2 to 8 μg/mL. The MIC of natural compound 2 against S. aureus ATCC29213 was 2 μg/mL, which was 16-fold lower compared with the positive control, ampicillin. Remarkably, these natural flavones (1–3) gave low MIC values (2–4 μg/mL) against MRSA, which were much better than ampicillin. Previously published work indicated that the antibacterial activity of natural flavonoids against G+ bacteria depends on balanced lipophilicity [36]. Compared to the parent isoflavone, prenylated isoflavones 1–3 have a higher lipophilicity, which may endow them with a higher antibacterial activity against G+ bacteria. Therefore, compound 2 displayed the most potent antibacterial activity due to its well-balanced lipophilicity [37].
To further confirm the antibacterial activity of Osajin (2), we subsequently performed a scanning electron microscopy (SEM) assay [32,33], to image both MRSA ATCC33591 and E. faecalis ATCC29212 after incubation with 2 for 2 h. As shown in Figure 3, the untreated control groups (Figure 3A,C) had an intact cell structure and surface morphology, while treatment of MRSA ATCC33591 and E. faecalis ATCC29212 with 2 at a concentration of 8 × its MIC resulted in a rough and wrinkled cell membrane surface (Figure 3B,D), indicating that the bacterial cell membrane was disrupted by 2. These findings suggested that Osajin (2) could inhibit bacterial growth by disrupting the integrity of the cell membrane.

3. Materials and Methods

3.1. General Experimental Procedures

Melting points were recorded on a Büchi B-545 melting point apparatus (Sigma-Aldrich, St. Louis, MO, USA). Infrared (IR) spectra were recorded on a Thermo Scientific Nicolet iS5 FT-IR spectrometer (Waltham, MA, USA). 1H NMR, 13C NMR, HMBC, and HSQC spectra were recorded on a Bruker Avance 400 spectrometer (Billerica, MA, USA) or a JEOL Eclips-600 pectrometer (Akishima, Japan), and tetramethylsilane (TMS) was used as the internal reference. The HR-MS spectra were recorded by Thermo QExactive (Thermo Scientific) and Agilent 6545 LC/QTOF mass spectrometers (Santa Clara, CA, USA). The crystal structure was analyzed with Oxford X-calibur E four-circle X-ray diffractometry (Oxford Instruments, Oxford, UK). Column chromatography was performed on silica gel (100–200 mesh). Reagents were purchased from commercial sources and used as received, unless mentioned otherwise. The solvents were of analytical grade.

3.2. Synthesis and Characterization of the Compounds

3.2.1. 1-(2-Hydroxy-4,6-bis(methoxymethoxy)phenyl)ethan-1-one (9)

To a stirred solution of 2,4,6-trihydroxyacetophenone (5.00 g, 29.76 mmol) in CH2Cl2 (50 mL), DIPEA (10.4 mL, 59.52 mmol) was added at room temperature, and then MOMBr (5.0 mL, 59.52 mmol) was added to the reaction mixture at 0 °C. The resulting mixture was stirred at room temperature for 3 h. The reaction mixture was acidified with 10% HCl (aq.) (15 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 10 mL). The organic layer and extracts were combined, dried, and evaporated to give a red oil, which was chromatographed on silica gel (petroleum ether/EtOAc = 30/1) to give 9 (5.47 g, 21.36 mmol, 71%) as a colorless oil. 1H NMR (600 MHz, CDCl3) δ 6.26–6.18 (m, 2H, –Ph), 5.23 (s, 2H, –OCH2OCH3), 5.14 (s, 2H, –OCH2OCH3–), 3.49 (s, 3H, –OCH3), 3.44 (s, 3H, –OCH3), 2.62 (s, 3H, –COCH3); 13C NMR (150 MHz, CDCl3) δ 203.3 (C=O), 166.9, 163.5, 160.4, 106.9, 97.2, 94.5, 94.0 (2 × C), 56.7 (–OCH3), 56.5 (–OCH3), 33.0 (–CH3). The spectroscopic data corresponds to reported values [24].

3.2.2. (E)-3-(Dimethylamino)-1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)prop-2-en-1-one (11)

Compound 9 (5.47 g, 21.36 mmol) was dissolved in dry DMF (25 mL) and heated to 74 °C. After that, compound 10 (14.2 mL, 106.8 mmol) was added dropwise, and the reaction mixture was stirred for 3 h and then cooled to rt. H2O (150 mL) was added to the reaction solution with stirring, and solids precipitated out. The precipitate was filtered off to afford crude product 11. The crude material was purified by flash chromatography (PE/EtOAc = 10/1) to give 11 (4.99 g, 16.06 mmol, 75%) as a bright yellow solid: 1H NMR (400 MHz, CDCl3) δ 15.10 (s, 1H, –OH), 7.85 (d, J = 12.4 Hz, 1H, =CHN(CH3)2), 6.22 (d, J = 12.4 Hz, 1H, –CH=CHN(CH3)2), 6.19 (d, J = 2.4 Hz, 1H, –Ph), 6.10 (d, J = 2.4 Hz, 1H, –Ph), 5.14 (s, 2H, –OCH2OCH3), 5.08 (s, 2H, –OCH2OCH3), 3.45 (s, 3H, –OCH3), 3.39 (s, 3H, –OCH3), 3.09 (s, 3H, –N(CH3)2), 2.85 (s, 3H, –N(CH3)2); 13C NMR (100 MHz, CDCl3) δ 190.0 (C=O), 166.9, 161.3, 158.9, 154.5, 107.0, 97.9, 96.9, 95.2, 94.4, 94.1, 56.7 (–OCH3), 56.3 (–OCH3), 45.3 (–CH3), 37.3 (–CH3). The spectroscopic data corresponds to reported values [24].

3.2.3. 3-Iodo-5,7-bis(methoxymethoxy)-4H-chromen-4-one (12)

Iodine (6.12 g, 24.09 mmol) was added to propenone 11 (4.99 g, 16.06 mmol) in MeOH (50 mL), and the solution was stirred at room temperature for 2 h. The mixture was washed with saturated Na2S2O3 (50 mL), and the aqueous layer was extracted with EtOAc (3 × 50 mL) and dried with Na2SO4. The crude material was purified by flash chromatography (PE/EtOAc = 30/1) to give 12 (4.10 g, 10.46 mmol, 65%), light-yellowish solids: 1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H, –OCH=C–), 6.68 (d, J = 2.3 Hz, 1H, –Ph), 6.65 (d, J = 2.3 Hz, 1H, –Ph), 5.23 (s, 2H, –OCH2OCH3), 5.16 (s, 2H, –OCH2OCH3), 3.48 (s, 3H, –OCH3), 3.43 (s, 3H, –OCH3); 13C NMR (100 MHz, CDCl3) δ 171.3 (C=O), 161.6, 159.3, 158.2, 155.7, 108.8, 102.1, 96.8, 95.5, 94.4, 89.5, 56.8 (–CH3), 56.6 (–CH3). The spectroscopic data corresponds to reported values [24].

3.2.4. 3-(4-Hydroxyphenyl)-5,7-bis(methoxymethoxy)-4H-chromen-4-one (14)

To a solution of the appropriate 12 (4.10 g, 10.46 mmol) in a mixture of 1,4-dioxane (35 mL) and water (15 mL), K2CO3 (4.34 g, 31.38 mmol) and 13 (2.89 g, 20.92 mmol) were added. The mixture was purged with nitrogen for 10 min. To the mixture were then added PCy3 (235 mg, 0.84 mmol) and Pd(dba)2 (241 mg, 0.43 mmol). The mixture was warmed to 50 ℃ and then stirred at this temperature for 2 h. It was then cooled to ambient temperature. An aq. saturated solution of NH4Cl (50 mL) was added to the mixture, and the mixture was filtered. The filter cake was dissolved in ethyl acetate (EtOAC, 30 mL), poured into water (50 mL), and extracted with EtOAc. The organic phase was combined, washed with brine, and dried with anhydrous Na2SO4. The solvent was evaporated under reduced pressure, and the residue was purified by column chromatography on silica using a PE/EtOAC mixture (3:2 (v/v)) as the eluent to afford compound 14 (3.61 g, 10.08 mmol, 76%) as a colorless oil: 1H NMR (400 MHz, DMSO-d6) δ 8.22 (s, 1H, –OCH=C–), 7.50–7.29 (m, 7H, –OBn and –Ph), 7.04 (d, J = 8.8 Hz, 2H, –Ph), 6.81 (d, J = 2.2 Hz, 1H, –Ph), 6.69 (d, J = 2.2 Hz, 1H, –Ph), 5.32 (s, 2H, –OCH2OCH3), 5.26 (s, 2H, –OCH2OCH3), 5.14 (s, 2H, –OCH2Ph), 3.43 (s, 3H, –OCH3), 3.42 (s, 3H, –OCH3); 13C NMR (150 MHz, DMSO-d6) δ 174.3, 161.1, 159.2, 158.5, 158.4, 151.9, 137.6, 130.8 (2 × C), 128.9 (2 × C), 128.3, 128.1 (2 × C), 125.0, 124.9, 114.9 (2 × C), 110.9, 101.9, 97.2, 95.7, 94.6, 69.6, 56.7, 56.6; HRMS (ESI) calculated for C26H25O7+ [M + H]+ 449.1595, found 449.1598.

3.2.5. 3-(4-(Benzyloxy)phenyl)-5,7-dihydroxy-4H-chromen-4-one (15)

A solution of 14 (3.06 g, 6.84 mmol) and 3 M HCl (70 mL) in EtOH (150 mL) was stirred at 82 °C for 2 h. After the completion of the reaction, the mixture was poured into ice water and extracted with EtOAc. The organic layer was washed with a saturated NaCl solution and dried with anhydrous Na2SO4, and the solvents were removed in vacuum. The residue obtained was purified by flash column chromatography with PE/EtOAc (10:1, v/v) as the eluent to give 15 (2.43 g, 6.75 mmol, 98%) as a yellow solid: 1H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H, –OH), 11.05 (s, 1H, –OH), 8.37 (s, 1H, –OCH=C–), 7.52–7.29 (m, 7H, –OBn and –Ph), 7.07 (d, J = 8.5 Hz, 2H, –Ph), 6.43 (d, J = 1.8 Hz, 1H, –Ph), 6.26 (d, J = 1.8 Hz, 1H, –Ph), 5.15 (s, 2H, –OCH2Ph); 13C NMR (100 MHz, DMSO-d6) δ 180.5 (C=O), 164.9, 162.4, 158.6, 158.0, 154.8, 137.5, 130.6 (2 × C), 128.9 (2 × C), 128.3, 128.1 (2 × C), 123.6, 122.3, 115.1 (2 × C), 104.9, 99.5, 94.2, 69.6. HRMS (ESI) calculated for C22H17O5+ [M + H]+ 361.1071, found 361.1070.

3.2.6. 3-(4-(Benzyloxy)phenyl)-5,7-dihydroxy-6,8-bis(3-methylbut-2-en-1-yl)-4H-chromen-4-one (16)

Under a N2 atmosphere, to a suspension of compound 15 (3.20 g, 8.88 mmol), acidic Al2O3 (17.80 g), and 4 Å molecular sieves (1.00 g) in dry DCE (160 mL) was added 18 (10 eq, 9.6 mL, 88.80 mmol). The mixture was stirred at 80 ℃ for 24 h. After the reaction was completed (detected by TLC), it was cooled to ambient temperature and filtered through a Celite. The filter cake was washed with EtOAc, and the filtrate was concentrated in vacuum to obtain a residue, which was further purified by silica gel column chromatography (PE/EtOAc (25:1, v/v) to give the pure compound 16 (3.17 g, 72%) as a yellow solid: 1H NMR (400 MHz, CDCl3) δ 13.20 (s, 1H, –OH), 7.90 (s, 1H, –OCH=C–), 7.51–7.30 (m, 7H, –Ph and –OBn), 7.05 (d, J = 8.7 Hz, 2H, –Ph), 6.38 (s, 1H, –OH), 5.33–5.18 (m, 2H, –CH=C(CH3)2), 5.11 (s, 2H, –OCH2Ph), 3.48 (t, J = 6.9 Hz, 4H, –CH2–), 1.85 (d, J = 4.5 Hz, 6H, –CH3), 1.76 (d, J = 10.9 Hz, 6H, –CH3); 13C NMR (100 MHz, CDCl3) δ 180.1 (C=O), 158.5, 157.8, 156.5, 152.2, 151.4, 135.8, 134.5, 133.05, 129.1 (2 × C), 127.5 (2 × C), 126.9, 126.4 (2 × C), 122.5, 121.9, 120.5, 120.3, 113.9 (2 × C), 109.1, 104.7, 104.3, 68.99, 24.8, 24.7, 20.6 (2 × C), 16.9, 16.8. HRMS (ESI) calculated for C32H33O5+ [M + H]+ 497.2323, found 497.2322.

3.2.7. 5,7-Dihydroxy-3-(4-hydroxyphenyl)-6,8-bis(3-methylbut-2-en-1-yl)-4H-chromen-4-one (3)

Under a N2 atmosphere, a solution of 30% CH3ONa/CH3OH aqueous solution (1.8 mL, 9.68 mmol) and dodecyl mercaptan (3 eq, 2.1 mL) in DMF (35 mL) was stirred at rt for 15 min. Then 16 (1.20 g, 2.42 mmol) was added to the reaction solution with stirring and refluxing at 120 °C for 24 h.After the reaction was completed (detected by TLC), 3 M HCl was added to adjust the pH to 7, and the mixture was extracted with EtOAc. The organic layers were combined, dried over anhydrous Na2SO4, and evaporated.The residue obtained was purified over flash column chromatography with PE/EtOAc (25:1, v/v) as the eluent to give the compound 3 (767 mg, 78%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 13.11 (s, 1H, –OH), 7.89 (s, 1H, –OCH=C–), 7.33 (d, J = 8.1 Hz, 2H, –Ph), 6.82 (d, J = 8.1 Hz, 2H, –Ph), 6.40 (s, 1H, –OH), 5.32–5.18 (m, 2H, –CH=C(CH3)2), 3.47 (t, J = 6.3 Hz, 4H, –CH2–), 1.84 (d, J = 4.9 Hz, 6H, –CH3), 1.75 (d, J = 10.9 Hz, 6H, –CH3); 13C NMR (100 MHz, CDCl3) δ 180.4 (C=O), 158.6, 156.4, 155.1, 152.3, 151.7, 134.5, 133.1, 129.2 (2 × C), 122.2, 121.7, 120.4, 120.2, 114.7 (2 × C), 109.2, 104.7, 104.4, 24.8, 24.7, 20.6 (2 × C), 16.9, 16.8. HRMS (ESI) calculated for C25H27O5+ [M + H]+ 407.1853, found 407.1855.

3.2.8. 3-(4-(Benzyloxy)phenyl)-5-hydroxy-7-((2-methylbut-3-yn-2-yl)oxy)-4H-chromen-4-one (24)

Compound 15 (2.20 g, 6.11 mmol), CuI (700 mg, 1.94 mmol), K2CO3 (1.1 eq, 300 mg, 2.13 mmol), and KI (1.2 eq, 390 mg, 7.33 mmol) were suspended in DMF (20 mL). Then 23 (1.2 eq, 240 mg, 2.33 mmol) was added dropwise, and the resulting reaction mixture was stirred for 24 h at rt. The reaction mixture was quenched by the addition of 1 mol/L aqueous HCl. The organic layer was separated and then washed with a saturated aqueous NaCl solution and dried with anhydrous Na2SO4, and the solvents were removed in vacuum. The residue obtained was purified over flash column chromatography with PE/EtOAc (40:1, v/v) as the eluent to give compound 24 (550 mg, 79%): 1H NMR (400 MHz, CDCl3) δ 12.69 (s, 1H, –OH), 7.78 (s, 1H, –OCH=C–), 7.40–7.22 (m, 7H –OBn and –Ph), 6.97 (d, J = 8.8 Hz, 2H, –Ph), 6.72 (d, J = 2.2 Hz, 1H, –Ph), 6.62 (d, J = 2.2 Hz, 1H, –Ph), 5.02 (s, 2H, –OCH2Ph), 2.61 (s, 1H, –C≡CH), 1.66 (s, 6H, –CH3); 13C NMR (150 MHz, CDCl3) δ 181.0 (C=O), δ 162.1, 162.2, 159.1, 157.4, 152.9, 136.9, 130.2 (2 × C), 128.7 (2 × C), 128.1, 127.5 (2 × C), 123.7, 123.3, 115.1 (2 × C), 106.9, 102.8, 97.3, 84.8, 75.3, 72.8, 70.1, 29.6 (2 × C). HRMS (ESI) calculated for C27H23O5+ [M + H]+ 427.1540, found 427.1540.

3.2.9. 3-(4-(Benzyloxy)phenyl)-5-hydroxy-8,8-dimethyl-4H,8H-pyrano[2,3-f]chromen-4-one (19)

Under a N2 atmosphere, compound 24 (500 mg, 1.17 mmol) and KOH (391.61 mg, 6.98 mmol) were dissolved in dry p-xylene (25 mL) at rt, and the resulting mixture was stirred for 1 h at 130 oC. After the reaction was completed, the mixture was cooled, quenched with 3 mol/L HCl, and diluted with EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated in a vacuum. The residue was purified by column chromatography (silica gel, hexane/EtOAc, 80:1) to afford compound 19 (463 mg, 93%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 13.18 (s, 1H, 5-OH), 7.82 (s, 1H, H-2), 7.45 (d, J = 8.6 Hz, 4H, H-1′, H-5′ and –Bn), 7.40 (t, J = 7.3 Hz, 2H, –Bn), 7.35 (d, J = 7.1 Hz, 1H, –Bn), 7.05 (d, J = 8.7 Hz, 2H, H-2′ and H-4′), 6.73 (d, J = 10.0 Hz, 1H, H-1″), 6.34 (s, 1H, H-8), 5.63 (d, J = 10.0 Hz, 1H, H-2″), 5.11 (s, 2H, –OBn), 1.48 (s, 6H, –CH3); 13C NMR (100 MHz, CDCl3) δ 179.8 (C-4), 158.5 (C-7), 157.9 (C-3′), 156.2 (C-8a), 155.9 (C-5), 151.5 (C-2), 135.8 (–Bn), 129.1 (2 × C, C-1′ and C-5′), 127.6 (2 × C, –Bn), 127.1 (C-2″), 127.0 (–Bn), 126.4 (2 × C, –Bn), 122.4 (C-6′), 122.2 (C-3), 114.4 (C-1″), 114.0 (2 × C, C-2′ and C-4′), 105.0 (C-6), 104.5 (C-4a), 93.8 (C-8), 77.0 (C-3″), 69.0 (–OCH2Ph), 27.3 (2 × C). HRMS (ESI) calculated for C27H23O5+ [M + H]+ 427.1540, found 427.1541.

3.2.10. (4-(Benzyloxy)phenyl)-5-hydroxy-8,8-dimethyl-4H,8H-pyrano[2,3-f]chromen-4-one (20)

A solution of 24 (500 mg, 1.17 mmol) in DMF (25 mL) was heated to 130 °C for 1 h. After completion of the reaction (detected by TLC), the mixture was poured into ice-cold water and extracted with EtOAc. The organic layer was washed with a saturated aqueous NaCl solution and dried with anhydrous Na2SO4, and the solvents were removed in vacuum. The residue obtained was purified over flash column chromatography with PE as the eluent to give 20 (416 mg, 77%) and 19 (49 mg, 9%) that could also be obtained. Compound 20 was a yellow solid: 1H NMR (400 MHz, CDCl3) δ 12.95 (s, 1H, 5-OH), 7.88 (s, 1H, H-2), 7.45 (d, J = 8.7 Hz, 4H, H-1′, H-5′ and –Bn), 7.40 (t, J = 7.4 Hz, 2H, –Bn), 7.35 (dd, 1H, –Bn), 7.05 (d, J = 8.3 Hz, 2H, H-2′ and H-4′), 6.68 (d, J = 10.0 Hz, 1H, H-1″), 6.30 (s, 1H, H-6), 5.59 (d, J = 10.0 Hz, 1H, H-2″), 5.11 (s, 2H, –OCH2Bn), 1.48 (s, 6H, –CH3); 13C NMR (100 MHz, CDCl3) δ 179.9 (C-4), 161.2 (C-7), 158.5 (C-5), 157.9 (C-3′), 151.4 (C-2), 151.1 (C-8a), 135.8 (–Bn), 129.1 (2 × C, C-1′ and C-5′), 127.6 (2 × C, –Bn), 127.0 (–Bn), 126.4 (C-2″), 126.4 (2 × C, –Bn), 122.5 (C-6′), 122.09 (C-3), 113.9 (2 × C, C-2′ and C-4′), 113.5 (C-1″), 105.0 (C-4a), 100.1 (C-8a), 99.3 (C-6), 77.0 (C-3′’), 69.0 (–OCH2Bn), 27.2 (2 × C, –CH3). The spectroscopic data corresponds to reported values [35].

3.2.11. 4-Hydroxy-7-(4-hydroxyphenyl)-2,2-dimethyl-2H,6H-pyrano[3,2-g]chromen-6-one (6)

Under a N2 atmosphere, compound 19 (900 mg, 2.11 mmol) was dissolved in CH2Cl2 (30 mL), and the mixture was cooled to −78 °C. To this solution was added BCl3 (2.6 mL, 1.0 M solution in toluene, 2.53 mmol), and the reaction mixture was stirred for 0.5 h at the same temperature. The reaction mixture was quenched with a mixture of saturated aqueous NaHCO3 solution and MeOH (v/v = 1:1) and diluted with EtOAc (100 mL). The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated in a vacuum. The obtained residue was purified on a flash silica gel (PE/EtOAc, 25:1) to afford 6 (530 mg, 75%) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 13.33 (s, 1H, –OH), 9.59 (s, 1H, –OH), 8.31 (s, 1H, –OCH=C–), 7.35 (d, J = 8.6 Hz, 2H, –Ph), 6.79 (d, J = 8.6 Hz, 2H, –Ph), 6.57 (d, J = 10.1 Hz, 1H, –CH=CHC(CH3)2O–), 6.43 (s, 1H, –Ph), 5.75 (d, J = 10.1 Hz, 1H, –CH=CHC(CH3)2O–), 1.39 (s, 6H,–CH3); 13C NMR (100 MHz, DMSO-d6) δ 180.4 (C=O), 158.7, 157.4, 156.6, 155.9, 154.1, 130.1 (2 × C), 128.9, 122.3, 120.9, 115.0 (2 × C), 114.4, 105.3, 104.6, 94.5, 78.0, 27.8 (2 × C). The spectroscopic data correspond to reported values [38].

3.2.12. 4-(5-Hydroxy-2,2-dimethyl-6-oxo-2H,6H-pyrano[3,2-g]chromen-7-yl)phenyl acetate (25)

Under a N2 atmosphere, to a solution of compound 6 (800 mg, 2.38 mmol) in pyridine (10 mL), Ac2O (0.31 mL, 3.57 mmol, 1.5 eq) was added.The mixture was stirred at rt for 1 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was quenched by dilution with 50 mL of H2O and extracted with CH2Cl2. The organic layer dried over anhydrous Na2SO4, and the solvent was evaporated in vaccum. The obtained residue was purified on a flash chromatography silica gel (PE/EtOAc, 25:1) to obtain the product 25 (756 mg, 84%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 13.07 (s, 1H, –OH), 7.84 (s, 1H, –OCH=C–), 7.54 (d, J = 8.5 Hz, 2H, –Ph), 7.17 (d, J = 8.5 Hz, 2H, –Ph), 6.72 (d, J = 10.1 Hz, 1H, –CH=CHC(CH3)2O–), 6.33 (s, 1H, –Ph), 5.72 (d, J = 10.1 Hz, 1H, –CH=CHC(CH3)2O–), 2.32 (s, 3H, –CH3), 1.47 (s, 6H, –CH3); 13C NMR (150 MHz, CDCl3) δ 180.6 (C=O), 169.5, 159.7, 157.33, 156.9, 153.1, 150.8, 130.1 (2 × C), 128.5, 128., 123.1, 121.9, 115.5 (2 × C), 106.1, 105.8, 95.0, 78.2, 28.4 (2 × C), 21.2. The spectroscopic data correspond to reported values [39].

3.2.13. 4-(8,8-Dimethyl-5-((3-methylbut-2-en-1-yl)oxy)-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl acetate (26)

Under a N2 atmosphere, to a solution of 25 (720 mg, 1.92 mmol), DEAD (0.6 mL, 3.84 mmol, 2 eq) and PPh3 (1.00 g, 3.84 mmol, 2 eq) in dry THF (25 mL), was added a solution of compound 18 (332 mg, 3.84 mmol, 2 eq) in 4 mL dry THF. The resulting mixture was stirred for 4 h at rt. After the reaction was completed (TLC), the solvent was removed under reduced pressure, and the yellow viscous oil was dissolved in EtOAc and subjected to flash chromatography on silica gel (EtOAc/PE 1:50 to 1:20) to give 26 (703 mg, 82%) as a yellow solid. Compound 26 was used immediately, without further purification.

3.2.14. 4-(5-Hydroxy-2,2-dimethyl-10-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-g]chromen-7-yl)phenyl acetate (27)

A suspension of 26 (800 mg, 1.79 mmol) and Eu(fod)3 (160 mg, 0.18 mmol, 0.1 eq) in dry DCE (25 mL) was stirred at reflux for 1 h under a N2 atmosphere. The resulting orange oil was concentrated in vacuum and subjected to flash chromatography (EtOAc/PE 1:50 to 1:20) to furnish 27 (730 mg, 91%) as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ 13.02 (s, 1H, –OH), 7.93 (s, 1H, –OCH=C–), 7.56 (d, J = 8.5 Hz, 2H, –Ph), 7.18 (d, J = 8.5 Hz, 2H, –Ph), 6.74 (d, J = 10.0 Hz, 1H, –CH=CHC(CH3)2O–), 5.63 (d, J = 10.0 Hz, 1H, –CH=CHC(CH3)2O–), 5.18 (t, J = 7.1 Hz, 1H, –CH=C(CH3)2), 3.40 (d, J = 7.1 Hz, 2H, –CH2–), 2.33 (s, 3H, –Ac), 1.82 (s, 3H, –CH3), 1.69 (s, 3H, –CH3), 1.47 (s, 6H, –CH3); 13C NMR (100 MHz, CDCl3) δ 180.9 (C=O), 169.5, 157.1, 154.9, 154.6, 153.1, 150.7, 131.8, 130.1 (2 × C), 128.7, 128.1, 122.7, 121.9, 121.8 (2 × C), 115.8, 107.6, 105.9, 105.6, 77.9, 28.2 (2 × C), 25.8, 21.3, 21.2, 17.9. The spectroscopic data correspond to reported values [40].

3.2.15. 5-Hydroxy-7-(4-hydroxyphenyl)-2,2-dimethyl-10-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-g]chromen-6-one (1)

Compound 27 (400 mg, 0.89 mmol) was dissolved in 10 mL of EtOH and 0.17 mL of a 60% KOH aqueous solution (1.78 mmol). After stirring for 30 min, the mixture was acidified with 3 mol/L HCl to pH 6 and extracted with EtOAc. The organic phase was washed with brine, dried over Na2SO4, and evaporated. The residue was submitted to flash column chromatography (silica gel, EtOAc/PE 1:50 to 1:20) to afford target 1 (316 mg, 87%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 13.03 (s, 1H, –OH), 7.89 (s, 1H, –OCH=C–), 7.33 (d, J = 7.2 Hz, 2H, –Ph), 6.81 (d, J = 7.2 Hz, 2H, –Ph), 6.74 (d, J = 10.0 Hz, 1H, –CH=CHC(CH3)2O–), 5.59 (d, J = 10.0 Hz, 1H, –CH=CHC(CH3)2O–), 5.24 (t, J = 7.2 Hz, 1H, –CH=C(CH3)2), 3.35 (d, J = 7.2 Hz, 2H, –CH2–), 1.81 (s, 3H, –CH3), 1.68 (s, 3H, –CH3), 1.48 (s, 6H, –CH3); 13C NMR (100 MHz, CDCl3) δ 180.4 (C=O), 155.9, 155.0, 153.7 (2 × C), 151.7, 130.7, 129.2 (2 × C), 127.0, 122.3, 121.7, 120.8, 114.7, 114.7 (2 × C), 106.5, 104.8, 104.4, 76.8, 27.1 (2 × C), 24.7, 20.2, 16.8. HRMS (ESI) calculated for C25H25O5+ [M + H]+ 405.1697, found 405.1697.

3.2.16. 5-Hydroxy-3-((4-hydroxyphenyl)-8,8dimethyl-4H,8H-pyrano[2,3-f]chromen-4-one (7)

The method was identical to that described for the preparation of 6. Compound 7 (1.75 g, 70%) was a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 13.07 (s, 1H, –OH), 9.64 (s, 1H, –OH), 8.38 (s, 1H, –OCH=C–), 7.37 (d, J = 8.6 Hz, 2H, –Ph), 6.82 (d, J = 8.6 Hz, 2H, –Ph), 6.64 (d, J = 10.0 Hz, 1H, –CH=CHC(CH3)2O–), 6.23 (s, 1H, –Ph), 5.75 (d, J = 10.0 Hz, 1H, –CH=CHC(CH3)2O–), 1.42 (s, 6H, –CH3); 13C NMR (150 MHz, DMSO-d6) δ 181.1 (C=O), 162.0, 159.3, 158.1, 154.6, 152.1, 130.8 (2 × C), 128.7, 123.1, 121.5, 115.6 (2 × C), 114.4, 105.9, 101.2, 99.9, 78.7, 28.3 (2 × C). The spectroscopic data correspond to reported values [41].

3.2.17. 3-(4-(Benzyloxy)phenyl)-5-hydroxy-7-((2-methylbut-3-yn-2-yl)oxy)-4H-chromen-4-one (28)

The method was identical to that described for the preparation of 25. Compound 28 (744 mg, 82%) was a white solid. 1H NMR (400 MHz, CDCl3) δ 12.84 (s, 1H, –OH), 7.91 (s, 1H, –OCH=C–), 7.56–7.55 (m, 2H, –Ph), 7.17 (d, J = 8.6 Hz, 2H, –Ph), 6.68 (d, J = 10.0 Hz, 1H, –CH=CHC(CH3)2O–), 6.29 (s, 1H, –Ph), 5.59 (d, J = 10.0 Hz, 1H, –CH=CHC(CH3)2O–), 2.32 (s, 3H,–Ac), 1.47 (s, 6H, –CH3); 13C NMR (150 MHz, CDCl3) δ 180.7 (C=O), 169.6, 162.3, 159.8, 153.0, 152.2, 150.9, 130.1 (2 × C), 128.5, 127.7, 123.3, 121.9, 114.6 (2 × C), 106.1, 101.3, 100.6, 78.3, 28.3 (2 × C), 21.3. HRMS (ESI) calculated for C22H19O6+ [M + H]+ 379.1176, found 379.1176.

3.2.18. 4-(8,8-Dimethyl-5-((2-methylbut-3-en-2-yl)oxy)-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl acetate (30)

To a solution of 2-methylbut-3-en-2-ol (3.5 mL, 33.48 mmol, 1.0 eq) in THF (60 mL), n-BuLi was added in hexanes (2.5 M, 13.2 mL, 33.48 mmol, 1.0 eq) at 0 °C over the course of 10 min. The clear solution was stirred at 0 °C for 20 min and then added di-tert-butyl dicarbonate (7.32 g, 33.48 mmol, 1.0 eq). The clear solution was allowed to heat to 23 °C and further stirred for 4 h. The solvent was poured into ice-cold water, quenched with a saturated aqueous NaHCO3 solution, and extracted with EtOAc. The organic phase was separated, washed with sat. aq. NaCl (150 mL), dried over Na2SO4, and concentrated under reduced pressure to give 29 as a clear, pale, light yellow liquid (6.03 g, 96%), which was used without further purification.
To a stirred suspension of 28 (200 mg, 0.53 mmol) and pulverized 4Å molecular sieves (100 mg) in degassed THF (5 mL), 29 (500 mg, 2.65 mmol) was added at room temperature. After cooling to −20 °C, Pd(PPh3)4 (61 mg, 0.05 mmol) was added portionwise to the mixture. After stirring for 21 h, the reaction mixture was filtered through a pad of Celite and the filtrate was concentrated in a vacuum. The residue was purified by silica gel column chromatography (PE/EtOAc = 30:1) to give 30 (178 mg, 78%) as a colorless oil, which was used without further purification.

3.2.19. 4-(5-Hydroxy-8,8-dimethyl-6-(3-methylbut-2-en-1-yl)-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl acetate (31)

The method was identical to that described for the preparation of 27. Compound 31 was a yellow solid (420 mg, 75%). 1H NMR (400 MHz, CDCl3) δ 13.07 (s, 1H, –OH), 7.89 (s, 1H, –OCH=C–), 7.54 (d, J = 8.3 Hz, 2H, –Ph), 7.17 (d, J = 8.3 Hz, 2H, –Ph), 6.69 (d, J = 10.0 Hz, 1H, –CH=CHC(CH3)2O–), 5.59 (d, J = 10.0 Hz, 1H, –CH=CHC(CH3)2O–), 5.23 (t, J = 6.6 Hz, 1H, –CH=C(CH3)2), 3.35 (d, J = 6.6 Hz, 2H, –CH2–), 2.33 (s, 3H, –Ac), 1.81 (s, 3H, –CH3), 1.68 (s, 3H, –CH3), 1.48 (s, 6H, –CH3); 13C NMR (100 MHz, CDCl3) δ 180.6 (C=O), 169.5, 159.4, 157.3, 152.7, 150.7, 150.5, 131.7, 130.1 (2 × C), 128.7, 127.3, 122.9, 121.9,121.8 (2 × C), 114.9, 113.0, 105.6, 100.8, 77.9, 28.2 (2 × C), 25.8, 21.3, 21.2, 17.9. HRMS (ESI) calculated for C27H27O6+ [M + H]+ 447.1802, found 447.1803.

3.2.20. 5-Hydroxy-3-(4-hydroxyphenyl)-8,8-dimethyl-6-(3-methylbut-2-en-1-yl)-4H,8H-pyrano[2,3-f]chromen-4-one (2)

The method was identical to that described for the preparation of 1. Compound 2 was a yellow solid (320 mg, 71%). 1H NMR (400 MHz, CDCl3) δ 13.07 (s, 1H, –OH), 7.85 (s, 1H, –OCH=C–), 7.31 (d, J = 7.3 Hz, 2H, –Ph), 6.81 (d, J = 7.3 Hz, 2H, –Ph), 6.70 (d, J = 9.9 Hz, 1H, –CH=CHC(CH3)2O–), 5.59 (d, J = 9.9 Hz, 1H, –CH=CHC(CH3)2O–), 5.24 (t, J = 7.3 Hz, 1H, –CH=C(CH3)2), 3.35 (d, J = 7.3 Hz, 2H, –CH2–), 1.81 (s, 3H, –CH3), 1.68 (s, 3H, –CH3), 1.48 (s, 6H, –CH3); 13C NMR (100 MHz, CDCl3) δ 180.1 (C=O), 158.2, 156.2, 155.0, 151.3, 149.5, 130.6, 129.3 (2 × C), 126.1, 122.5, 121.8, 120.8, 114.7 (2 × C), 113.9, 111.8, 104.5, 99.7, 76.8, 27.1 (2 × C), 24.7, 20.2, 16.9. HRMS (ESI) calculated for C25H25O5+ [M + H]+ 405.1697, found 405.1696.

3.2.21. MICs Tests

S. aureus ATCC29213, E. faecalis ATCC29212, MRSA ATCC33591, and E. coli ATCC 25922 were selected to evaluate the target compounds 1–3. The bacterial strains were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and kept in our laboratory. The MICs were evaluated according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The experiment was performed as reported in the literature [32,33].

3.2.22. Scanning Electron Microscopy (SEM) Characterization

A single colony of S. aureus ATCC29213 or E. faecalis ATCC29212 was added to LB broth (1.0 mL) and incubated in a shaker (200 rpm, 37 °C) for 12 h. Then the solution was diluted to a concentration of 1 × 109 CFU/mL in PBS. After that, compound 2 (8 × MIC) was added, and the resulting mixture was incubated at 37 °C for 2 h. Subsequently, the bacterial cells were washed twice with PBS and added to 2.5% glutaraldehyde overnight at 4 °C. The bacterial cells were osmicated in 1% Osmium tetroxide for 1 h at 4 °C to further fix and stain them, then gradually eluted by an ascending graded series of EtOH, dried, and plated with gold at the critical point. Finally, the cells were visualized under SEM (Hitachi S-3400 N, Tokyo, Japan). The negative control was a bacterial suspension without compound 2 treatment [32,33].

4. Conclusions

In conclusion, the efficient syntheses of three natural isoflavones, scandenone (1), osajin (2), and 6,8-diprenylgenistein (3), were accomplished with overall yields of 10%, 6%, and 14%, respectively. Notably, natural isoflavone 3 was synthesized for the first time. The presently described synthesis of compounds 1 and 2 features a stereoselective construction of the pyran ring, and di-prenylation of key intermediate 15 is the key step to obtain isoflavone 3. The antibacterial activities of the synthetic isoflavones 1–3 against S. aureus ATCC29213, E. faecalis ATCC29212, MRSA ATCC33591, and E. coli ATCC 25922 were assessed, and it was shown that all of the natural isoflavones displayed good antibacterial activity against Gram-positive bacteria. Furthermore, the SEM assay indicated that the bacterial cell membranes of Gram-positive bacteria could be disrupted by this kind of natural isoflavone. Taken together, these results demonstrate that natural prenylated isoflavones have tremendous potential to be developed as lead compounds for further optimization to address the problem of bacterial resistance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules29112574/s1. 1H NMR and 13C NMR spectra for the synthesized compounds and HRMS spectra for new compounds. Key crystal data for compound 1, and LCMS reports for compounds 1–3 (PDF).

Author Contributions

W.D. and T.S. conceived and designed this research; H.D. synthesized compounds 1 and 2; Y.C. synthesized compound 3; Y.Z. evaluated the antibacterial activity of 1–3 and performed the scanning electron microscopy (SEM) assay; X.Z. and J.L. Writing—original draft; J.W. Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China, grant number 82003619; Sichuan Provincial Science and Technology Foundation, grant numbers 2023NSFSC0609, 2022NSFSC1252, and 2024NSFSC0258.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Al-Maharik, N. Isolation of naturally occurring novel isoflavonoids: An update. Nat. Prod. Rep. 2019, 36, 1156–1195. [Google Scholar] [CrossRef]
  2. Křížová, L.; Dadáková, K.; Kašparovská, J.; Kašparovský, T. Isoflavones. Molecules 2019, 24, 1076. [Google Scholar] [CrossRef]
  3. Qi, Y.; Xie, L.; Deng, Z.; Zhang, B.; Li, H. Stability and antioxidant activity of 10 isoflavones and anthocyanidins during in vitro digestion. Food Biosci. 2023, 56, 103189. [Google Scholar] [CrossRef]
  4. Sun, Y.; Zhang, H.; Cheng, M.; Cao, S.; Qiao, M.; Zhang, B.; Ding, L.; Qiu, F. New hepatoprotective isoflavone glucosides from Pueraria lobata (Willd.) Ohwi. Nat. Prod. Res. 2019, 33, 24. [Google Scholar] [CrossRef] [PubMed]
  5. Çiçek, S.S.; Pérez, M.G.; Wenzel-Storjohann, A.; Bezerra, R.B.; Segovia, J.F.O.; Girreser, U.; Kanzaki, I.; Tasdemir, D. Antimicrobial prenylated isoflavones from the leaves of the Amazonian medicinal plant Vatairea guianensis Aubl. J. Nat. Prod. 2022, 85, 927–935. [Google Scholar] [CrossRef] [PubMed]
  6. Seo, Y.; Jeon, J.H.; Jeong, M.; Ryu, S.M.; Jeon, W.K.; Jang, D.S.; Shim, S.H. Chemical Constituents of Apios americana tubers and their inhibitory activities on nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophages. J. Nat. Prod. 2018, 81, 1598–1603. [Google Scholar] [CrossRef] [PubMed]
  7. Ito, C.; Itoigawa, M.; Tan, H.T.; Tokuda, H.; Mou, X.Y.; Mukainaka, T.; Ishikawa, T.; Nishino, H.; Furukawa, H. Anti-tumor-promoting effects of isoflavonoids on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis. Cancer Lett. 2000, 152, 187–192. [Google Scholar] [CrossRef]
  8. Ito, C.; Murata, T.; Itoigawa, M.; Itoigawa, M.; Nakao, K.; Kumagai, M.; Kaneda, N.; Furukawa, H. Induction of apoptosis by isoflavonoids from the leaves of Millettia taiwaniana in human leukemia HL-60 Cells. Planta Med. 2006, 72, 424–429. [Google Scholar] [CrossRef]
  9. Talla, E.; Yankep, E.; Mbafor, J.T. Chemical constituents from root barks of Erythrina mildbraedii and stem barks of Erythrina addisoniae. Bull. Chem. Soc. Ethiop. 2014, 28, 155–159. [Google Scholar] [CrossRef]
  10. Ozçelik, B.; Orhan, I.; Toker, G. Antiviral and antimicrobial assessment of some selected flavonoids. Z. Naturforschung C 2006, 61, 623–638. [Google Scholar] [CrossRef]
  11. Wang, B.; Ternai, B.; Polya, G. Specific inhibition of cyclic AMP-dependent protein kinase by warangalone and robustic acid. Phytochemistry 1997, 44, 787–796. [Google Scholar] [CrossRef]
  12. Sreelatha, T.; Hymavathi, A.; Rao, V.R.S.; Devanand, P.; Rani, P.U.; Rao, J.M.; Babu, K.S. A new benzil derivative from Derris scandens: Structure-insecticidal activity study. Bioorgan. Med. Chem. 2010, 20, 549–553. [Google Scholar] [CrossRef] [PubMed]
  13. Raksat, A.; Maneerat, W.; Andersen, R.J.; Pyne, S.G.; Laphookhieo, S. Antibacterial prenylated isoflavonoids from the stems of Millettia extensa. J. Nat. Prod. 2018, 81, 1835–1840. [Google Scholar] [CrossRef] [PubMed]
  14. Peleyeju, G.B.; Emmanuel, T.; Tata, C.M.; Fotsing, M.C.D.; Niemann, N.; Rhyman, L.; Arderne, C.; Ndinteh, D.T.; Ramasami, P. Crystal structure and antibacterial activity of scandenone (warangalone) from Erythrina plants. J. Mol. Struct. 2019, 1191, 43–51. [Google Scholar] [CrossRef]
  15. Yang, R.; Hanwell, H.; Zhang, J.; Tsao, R.; Meckling, K.A. Antiproliferative activity of pomiferin in normal (MCF-10A) and transformed (MCF-7) breast epithelial cells. J. Agric. Food Chem. 2011, 59, 13328–13336. [Google Scholar] [CrossRef] [PubMed]
  16. Hošek, J.; Toniolo, A.; Neuwirth, O.; Bolego, C. Prenylated and geranylated flavonoids increase production of reactive oxygen species in mouse macrophages but inhibit the inflammatory response. J. Nat. Prod. 2013, 76, 1586–1591. [Google Scholar] [CrossRef] [PubMed]
  17. Diopan, V.; Babula, P.; Shestivska, V.; Adam, V.; Zemlicka, M.; Dvorska, M.; Hubalek, J.; Trnkova, L.; Havel, L.; Kizek, R. Electrochemical and spectrometric study of antioxidant activity of pomiferin, isopomiferin, osajin and catalposide. J. Pharmaceut. Biomed. 2008, 48, 127–133. [Google Scholar] [CrossRef] [PubMed]
  18. Nkengfack, A.E.; Kouam, J.; Vouffo, T.W.; Meyer, M.; Tempesta, M.S.; Fomum, Z.T. An isoflavanone and a coumestan from Erythrina sigmoidea. Phytochemistry 1994, 35, 521–526. [Google Scholar] [CrossRef]
  19. Dong, H.; Yu, P.; Long, B.; Peng, T.; He, Y.; Xu, B.; Liao, L.; Lu, L. Total synthesis of Kuwanons A and B and discovery of their antibacterial mechanism. J. Nat. Prod. 2023, 86, 2022–2030. [Google Scholar] [CrossRef]
  20. Dong, H.; Wu, M.; Li, Y.; Lu, L.; Qin, J.; He, Y.; Shi, Z. Total syntheses and anti-inflammatory evaluations of pongamosides A−C, natural furanoflavonoid glucosides from fruit of Pongamia pinnata (L.) Pierre. J. Nat. Prod. 2022, 85, 1118–1127. [Google Scholar] [CrossRef]
  21. Yu, P.; Long, B.; Feng, C.; Yang, T.; Jiang, X.; He, Y.; Dong, H. Total syntheses of pongaflavone and its natural analogues. J. Asian Nat. Prod. Res. 2023, 25, 1085–1096. [Google Scholar] [CrossRef]
  22. Wu, M.; Xu, L.; Li, Y.; Yu, P.; Lu, L.; Xie, S.; He, Y.; Dong, H. First total syntheses of Kanjone and its natural analogues. J. Asian Nat. Prod. Res. 2023, 25, 277–286. [Google Scholar] [CrossRef]
  23. Wang, R.; Ma, R.; Feng, K.; Lu, H.; Zhao, W.; Jin, H. Total synthesis and anti-inflammatory evaluation of osajin, scandenone and analogues. Pharmaceuticals 2024, 17, 86. [Google Scholar] [CrossRef]
  24. Wang, Y.; Yin, X.; Chen, L.; Yin, Z.; Zuo, Z. Discovery and evaluation of cytisine N-isoflavones as novel EGFR/HER2 dual inhibitors. Bioorganic Chem. 2022, 127, 105868. [Google Scholar] [CrossRef]
  25. Dong, H.; Wu, M.; Xiang, S.; Song, T.; Li, Y.; Long, B.; Feng, C.; Shi, Z. Total syntheses and antibacterial evaluations of neocyclomorusin and related flavones. J. Nat. Prod. 2022, 85, 2217–2225. [Google Scholar] [CrossRef]
  26. Luan, N.N.T.; Okada, T.; Arata, R.; Prudhvi, L.; Miyaguchi, M.; Kodama, Y.; Awale, S.; Toyooka, N. Structure-activity relationship study of 4′-O-methylgrynullarin derivatives for the development of novel anticancer agents based on anti-austerity strategy. Tetrahedron 2022, 122, 132931. [Google Scholar] [CrossRef]
  27. Kwesiga, G.; Greese, J.; Kelling, A.; Sperlich, E.; Schmidt, B. The Suzuki-Miyaura cross-coupling-Claisen rearrangement-cross-metathesis approach to prenylated isoflavones. J. Org. Chem. 2023, 88, 1649–1664. [Google Scholar] [CrossRef]
  28. Xiao, L.; Tan, W.; Li, Y. First total synthesis of (±)-Kenusanone B. Synth. Commun. 1998, 28, 2861–2869. [Google Scholar] [CrossRef]
  29. Franov, L.J.; Hart, J.D.; Pullella, G.A.; Sumby, C.J.; George, J.H. Bioinspired total synthesis of erectones A and B, and the revised structure of hyperelodione D. Angew. Chem. Int. Ed. 2022, 61, e2022004. [Google Scholar] [CrossRef]
  30. Mzozoyana, V.; Heerden, F.R. Synthesis of fluorine-containing prenylated benzophenones. Synth. Commun. 2019, 50, 2226–2235. [Google Scholar] [CrossRef]
  31. Jentsch, N.G.; Zhang, X.; Magolan, J. Efficient synthesis of cannabigerol, grifolin, and piperogalin via alumina-promoted allylation. J. Nat. Prod. 2020, 83, 2587–2591. [Google Scholar] [CrossRef]
  32. Dong, H.; Liao, L.; Yu, P.; Long, B.; Che, Y.; Lu, L.; Xu, B. Total syntheses and antibacterial evaluations of cudraflavones A-C and related flavones. Bioorganic Chem. 2023, 40, 106764. [Google Scholar] [CrossRef]
  33. Dong, H.; Liao, L.; Long, B.; Che, Y.; Peng, T.; He, Y.; Mei, L.; Xu, B. Total synthesis and antibacterial evaluation of lupinifolin and its natural analogues. J. Nat. Prod. 2024, 87, 1044–1058. [Google Scholar] [CrossRef]
  34. Zheng, S.; Li, X.; Tan, H.; Yu, C.; Zhang, J.; Shen, Z. Studies on the total synthesis of Hirtellanine A: Regioselective synthesis of benzopyran. Eur. J. Org. Chem. 2013, 2013, 1356–1366. [Google Scholar] [CrossRef]
  35. Masao, T.; Yasuhiko, K.; Hideo, T. Synthesis of 4′,5- and 3′,4′,5-oxygenated pyranoisoflavones: Alpinum isoflavone and related compounds, and revised structure of derrone. Heterocycles 1992, 34, 505–516. [Google Scholar]
  36. Song, M.; Liu, Y.; Li, T.; Liu, X.; Hao, Z.; Ding, S.; Panichayupakaranant, P.; Zhu, K.; Shen, J. Plant natural flavonoids against multidrug resistant pathogens. Adv. Sci. 2021, 8, 2100749. [Google Scholar] [CrossRef]
  37. Sychrová, A.; Škovranová, G.; Čulenová, M.; Fialová, S. Prenylated flavonoids in topical infections and wound healing. Molecules 2022, 27, 4491. [Google Scholar] [CrossRef]
  38. Lin, Y.; Kuang, Y.; Li, K.; Wang, S.; Ji, S.; Chen, K.; Song, W.; Qiao, X.; Ye, M. Nrf2 activators from Glycyrrhiza inflata and their hepatoprotective activities against CCl4-induced liver injury in mice. Bioorganic Med. Chem. 2017, 25, 5522–5530. [Google Scholar] [CrossRef]
  39. Khalid, S.A.; Waterman, P.G. Thonningine-A and thonningine-B: Two 3-phenylcoumarins from the seeds of Millettia thonningii. Phytochemistry 1983, 22, 1001–1003. [Google Scholar]
  40. Peter, A.; Stainton, P. The extractives from Derris scandens. Part II. The isolation of osajin and two new isoflavones, scandenone and scandinone. J. Chem. Soc. C 1966, 701–704. [Google Scholar] [CrossRef]
  41. Chouna, H.; Dize, D.; Kagho, D.; Bankeu, J.; Fongang, Y.; Tali, M.; Ponou, B.; Bitchagno, G.; Awantu, A.; Tapondjou, L.; et al. Constituents from ripe figs of Ficus vallis-choudae Delile (Moraceae) with antiplasmodial activity. Parasitol. Res. 2022, 121, 2121–2127. [Google Scholar] [CrossRef] [PubMed]
Molecules 29 02574 g001
Figure 1. The Structures of Isoflavone and Compounds 1–3.
Figure 1. The Structures of Isoflavone and Compounds 1–3.
Molecules 29 02574 g001
Molecules 29 02574 sch001
Scheme 1. Reported Synthetic Approaches for Isoflavones 1 and 2.
Scheme 1. Reported Synthetic Approaches for Isoflavones 1 and 2.
Molecules 29 02574 sch001
Molecules 29 02574 sch002
Scheme 2. Synthesis of the Key Intermediate 15.
Scheme 2. Synthesis of the Key Intermediate 15.
Molecules 29 02574 sch002
Molecules 29 02574 g002
Figure 2. Key HMBC correlations of 19 and 20.
Figure 2. Key HMBC correlations of 19 and 20.
Molecules 29 02574 g002
Molecules 29 02574 sch003
Scheme 3. Synthesis of Scandenone (1).
Scheme 3. Synthesis of Scandenone (1).
Molecules 29 02574 sch003
Molecules 29 02574 sch004
Scheme 4. Synthesis of Osajin (2).
Scheme 4. Synthesis of Osajin (2).
Molecules 29 02574 sch004
Molecules 29 02574 g003
Figure 3. SEM images of MRSA ATCC33591 and E. faecalis ATCC29212 bacterial cells. (A) MRSA ATCC33591 cells without treatment; scale bar: 500 nm. (B) MRSA ATCC33591 cells treated with 8 × MIC of compound 2, scale bar: 500 nm. (C) E. faecalis ATCC29212 cells without treatment; scale bar: 1 μm. (D) E. faecalis ATCC29212 cells treated with 8 × MIC of compound 2, scale bar: 1 μm.
Figure 3. SEM images of MRSA ATCC33591 and E. faecalis ATCC29212 bacterial cells. (A) MRSA ATCC33591 cells without treatment; scale bar: 500 nm. (B) MRSA ATCC33591 cells treated with 8 × MIC of compound 2, scale bar: 500 nm. (C) E. faecalis ATCC29212 cells without treatment; scale bar: 1 μm. (D) E. faecalis ATCC29212 cells treated with 8 × MIC of compound 2, scale bar: 1 μm.
Molecules 29 02574 g003
Table 1. Synthesis of Isoflavone 3.
Table 1. Synthesis of Isoflavone 3.
Molecules 29 02574 i001
EntryConditionsYields of Isolated 16 (%)
1compound 17, KOH, H2O, 0 °C to rt-
2compound 17, K2CO3, acetone, rt7
3compound 17, DBU, THF, 50 °C20
4compound 18, p-toluenesulfonic acid, CH2Cl2, rt14
5compound 18, acidic alumina, DCE, 80 °C72
Table 2. Optimization of Reaction Conditions for Compounds 19 and 20.
Table 2. Optimization of Reaction Conditions for Compounds 19 and 20.
Molecules 29 02574 i002
EntryMethodConditions19, Yield 1 (%)20, Yield 1 (%)
1Method A21, Ca(OH)2, CH3OH, 0 °C to rt2722
2Method A22, 3-picoline, toluene, 110 °C2527
3Method BDMF, 130 °C977
4Method Bxylene, 130 °C5533
5Method Bxylene, KOH, 130 °C93-
6Method Bdiethylaniline, 250 °C4140
1 Isolated yield.
Table 3. MICs of compounds 1–3.
Table 3. MICs of compounds 1–3.
CompoundMIC (μg/mL)
S. aureus
ATCC29213		E. faecalis
ATCC29212		MRSA
ATCC33591		E. coli
ATCC25922
116164>128
2282>128
38164>128
Ampicillin 321>1284
Vancomycin221128
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Dong, H.; Che, Y.; Zhu, X.; Zhong, Y.; Lin, J.; Wang, J.; Du, W.; Song, T. Total Syntheses and Antibacterial Studies of Natural Isoflavones: Scandenone, Osajin, and 6,8-Diprenylgenistein. Molecules 2024, 29, 2574. https://doi.org/10.3390/molecules29112574

AMA Style

Dong H, Che Y, Zhu X, Zhong Y, Lin J, Wang J, Du W, Song T. Total Syntheses and Antibacterial Studies of Natural Isoflavones: Scandenone, Osajin, and 6,8-Diprenylgenistein. Molecules. 2024; 29(11):2574. https://doi.org/10.3390/molecules29112574

Chicago/Turabian Style

Dong, Hongbo, Yufei Che, Xingtong Zhu, Yi Zhong, Jiafu Lin, Jian Wang, Weihong Du, and Tao Song. 2024. "Total Syntheses and Antibacterial Studies of Natural Isoflavones: Scandenone, Osajin, and 6,8-Diprenylgenistein" Molecules 29, no. 11: 2574. https://doi.org/10.3390/molecules29112574

Article Metrics

Back to TopTop