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

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.


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.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.

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.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.

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 I 2 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].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-dyhydroxylisoflavone 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 Oprenylations during the reaction.To avoid the O-prenylation, we then turned to imply 3methylbut-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].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 CH 2 Cl 2 and H 2 O 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 K 2 CO 3 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 Cand 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 CH 2 Cl 2 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-3methylbut-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].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 K 2 CO 3 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].
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 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 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.
terial 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 wellbalanced 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-(4-Hydroxyphenyl)-5,7-bis(methoxymethoxy)-4H-chromen-4-one (14)
To a solution of the appropriate 12 (4.10g, 10.46 mmol) in a mixture of 1,4-dioxane (35 mL) and water (15 mL), K 2 CO 3 (4.34g, 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 PCy 3 (235 mg, 0.84 mmol) and Pd(dba) 2 (241 mg, 0.43 mmol).The mixture was warmed to 50 °C and then stirred at this temperature for 2 h.It was then cooled to ambient temperature.An aq.saturated solution of NH 4 Cl (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 Na 2 SO 4 .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

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].

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 × 10 9 CFU/mL in PBS.After that, compound 2 (8 × MIC)

Table 2 .
Optimization of Reaction Conditions for Compounds 19 and 20.

Table 2 .
Optimization of Reaction Conditions for Compounds 19 and 20.