Polysubstituted Isoflavonoids from Spatholobus suberectus, Flemingia macrophylla, and Cudrania cochinchinensis

Introduction

Isoflavonoids are a large group of secondary metabolites with diverse biological activities that occur widely in plants, which can be subdivided into isoflavones, isoflavans, isoflavanones, rotenoids, pterocarpans, etc. An overwhelming number of isoflavonoids reported come from the Leguminosae family, and some non-leguminous families such as Moraceae are also relatively abundant in isoflavonoids [1, 2]. Some isoflavans and prenylated isoflavones possess potent biological activities, especially in cytotoxicity [3–6], anti-inflammatory [7], and neuroprotective activity [8]. As part of a BioBioPha (http://www.chemlib.cn) objective to assemble a large-scale natural product library valuable in the discovery of new drug leads from nature [9–12], the phytochemical investigations on Spatholobus suberectus (Leguminosae), Flemingia macrophylla (Leguminosae), and Cudrania cochinchinensis (Moraceae) led to the isolation of three new isoflavans 7,4′-dihydroxy-8,2′,3′-trimethoxyisoflavan (1), 7,2′,4′-trihydroxy-8,3′-dimethoxyisoflavan (2), and 7,2′,4′-trihydroxy-5-methoxyisoflavan (3), and one new prenylated isoflavone namely cudraisoflavone M (4), respectively (Fig. 1). Herein we report the structure elucidation of new isoflavonoid and their cytotoxicity evaluation against five human cancer cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW-480).

Fig. 1

Structures of new compounds 1–4

Results and Discussion

Compound 1 was obtained as white amorphous powder, and its molecular formula was determined to be C₁₈H₂₀O₆ from the positive HRESIMS at m/z 355.1163 [M+Na]⁺ (calcd. for C₁₈H₂₀O₆Na, 355.1158) with nine degrees of unsaturation. The ¹H NMR spectrum (Table 1) displayed five aliphatic proton signals due to two methylene groups [δ_H 2.86 (1H, ddd, J = 15.8, 5.3, 1.9 Hz) and 2.94 (1H, dd, J = 15.8, 11.3 Hz), 4.00 (1H, t, J = 10.5 Hz) and 4.37 (1H, ddd, J = 10.5, 3.7, 1.9 Hz)], and one methine [δ_H 3.53 (1H, dddd, J = 11.3, 10.5, 5.3, 3.7 Hz)], which were characteristic of an isoflavan [13]. The ¹H NMR spectrum also exhibited two pairs of ortho-coupled aromatic doublets [δ_H 6.52 (1H, d, J = 8.3 Hz), 6.72 (1H, d, J = 8.3 Hz), 6.71 (1H, d, J = 8.5 Hz), and 6.75 (1H, d, J = 8.5 Hz)], three methoxy groups [δ_H 3.87, 3.92 and 3.93 (each 3H, s)], and two phenolic hydroxy protons [δ_H 5.66 and 5.71 (each 1H, s)]. The ¹³C NMR spectrum (Table 2) showed a total of 18 carbon signals, including 12 aromatic carbons for two phenyl units (rings A and B), the ring C carbons at δ_C 31.4 (d), 31.5 (t), 70.5 (t), and three methoxy carbons at δ_C 60.6, 60.8, 61.0 (each q). The above NMR spectroscopic features were very similar to those of isoduartin (=7,2′-dihydroxy-8,3′,4′-trimethoxyisoflavan) [14], except for an obvious downfield shift for H-5′ (Δ = +0.27 ppm), which hinted a different substituted pattern in ring B. The pattern can be determined as 4′-hydroxy-2′,3′-dimethoxy by the following HMBC correlations (Fig. 2): from 4′-OH (δ_H 5.71) to C-3′ (δ_C 139.8), C-4′ (δ_C 148.5) and C-5′ (δ_C 110.5); from H-3 (δ_H 3.53) and 2′-OCH₃ (δ_H 3.87) to C-2′ (δ_C 150.8); and from 3′-OCH₃ (δ_H 3.93) to C-3′. The particular downfield shifts (Δ ≈ +5 ppm) of aromatic methoxy carbons also implied the location of these groups. According to the empirical rule, the carbon signals of the methoxy groups with substituents in both ortho positions will appear at 60–62 ppm, while those sterically non-hindered at 55–57 ppm [15]. On the basis of the above analysis, the structure of 1 was established as 7,4′-dihydroxy-8,2′,3′-trimethoxyisoflavan.

Fig. 2

Key HMBC () correlations of 1–4

Compound 2, isolated as white amorphous powder, had a molecular formula of C₁₇H₁₈O₆ by the positive HRESIMS at m/z 341.1002 [M+Na]⁺ (calcd. for C₁₇H₁₈O₆Na, 341.1001). Comparison of the ¹H and ¹³C NMR spectral data (Tables 1, 2) with those of 1 revealed that a methoxy group in the ring B was replaced by a hydroxy. From the HMBC correlations of the OH signal at δ_H 5.72 (1H, s) to C-1′ (δ_C 119.9), C-2′ (δ_C 147.1), and C-3′ (δ_C 134.5), we could conclude that the emerging hydroxy was positioned at C-2′ (Fig. 2). Hence, the structure of 2 was established as 7,2′,4′-trihydroxy-8,3′-dimethoxyisoflavan.

Compound 3 was isolated as white amorphous powder, and its molecular formula was determined to be C₁₆H₁₆O₅ according to the positive HRESIMS at m/z 289.1075 [M+H]⁺ (calcd. for C₁₆H₁₇O₅, 289.1076). Five diagnostic ring C proton signals [δ_H 2.59 (1H, dd, J = 16.3, 10.8 Hz), 2.76 (1H, ddd, J = 16.3, 5.5, 1.9 Hz), 3.34 (1H, dddd, J = 10.8, 10.2, 5.5, 3.1 Hz), 3.86 (1H, dd, J = 10.2, 10.2 Hz), and 4.16 (1H, ddd, J = 10.2, 3.1, 1.9 Hz)], an ABX-type aromatic proton system [δ_H 6.30 (1H, d, J = 2.4 Hz), 6.24 (1H, dd, J = 8.3, 2.4 Hz), and 6.85 (1H, d, J = 8.3 Hz)], two meta-coupled aromatic doublets [δ_H 5.88 (1H, d, J = 2.1 Hz) and 5.99 (1H, d, J = 2.1 Hz)], and one methoxy signal [δ_H 3.74 (3H, s)] were observed in the ¹H NMR spectrum (Table 1), which revealed that 3 was a monomethyl ether derivative of 5,7,2′,4′-tetrahydroxyisoflavan. The HMBC correlations from the methoxy and H-4 proton signals to C-5 (δ_C 160.1) confirmed the methoxy group at C-5 (Fig. 2). Therefore, the structure of 3 was established as 7,2′,4′-trihydroxy-5-methoxyisoflavan.

Absolute configurations of the isoflavans (1–3) were established by comparison of the CD curves with those reported earlier for closely related analogues. All the CD spectra showed a negative Cotton effect in the transition region (215–240 nm), similar with those of 7-O-methylisomucronulatol, (3R)-isomucronulatol, and abruquinone L [16–19], which indicated the absolute configurations at C-3 of 1–3 being R-form. And optical rotation measurements were also taken to assign the absolute configuration. We carefully examined the specific rotation values of some 2′,3′,4′-trisubstituted isoflavans (Table 3), and the results suggested that the methoxy group substituted at C-2′ had a huge impact on the specific rotation, probably on account of the spatial proximity. For 2′-methoxyisoflavans, R-/S-form result in positive/negative specific rotation values, respectively, but for 2′-hydroxyisoflavans, R-form gives negative values [14, 17, 20–25]. Our current research results were consistent with the above empirical rule.

Compound 4, yellowish amorphous powder, had a molecular formula of C₂₂H₂₂O₇ according to the positive HRESIMS at m/z 399.1440 [M+H]⁺ (calcd. for C₂₂H₂₃O₇, 399.1444). The NMR spectra (Table 4) showed an olefinic signal (δ_H 8.23, 1H, s; δ_C 154.3, d) and a chelated hydroxy group at δ_H 13.48 (1H, s), characteristic of a 5-hydroxyisoflavone skeleton [8, 13]. In addition, an ABX-type aromatic proton system [δ_H 7.15 (1H, d, J = 2.0 Hz), 6.86 (1H, d, J = 8.2 Hz), and 6.94 (1H, dd, J = 8.2, 2.0 Hz)], a prenyl group [δ_H 1.63, 1.79 (each 3H, s), 3.45 (2H, d, J = 7.1 Hz), and 5.22 (1H, br t, J = 7.1 Hz)], and a hydroxyethyl moiety [δ_H 2.97, 3.90 (each 2H, t, J = 5.2 Hz)] were also detected in the ¹H NMR spectrum. The ¹³C NMR spectrum displayed a total of 22 carbon resonances, including two methyls, three sp³ methylenes, five sp² methines, and 12 sp² quaternary carbons. These spectroscopic features were very similar to those of cudraisoflavone L, which was recently isolated from the same genus and shared the same molecular formula with 4 [26]. Their structural difference was only due to the location of the prenyl group and the hydroxyethyl moiety. The hydroxyethyl unit was located at C-6 from the HMBC correlations of H-1″ (δ_H 2.97) and 5-OH (δ_H 13.48) to C-5 (δ_C 158.5) and C-6 (δ_C 111.2), while the prenyl group at C-8 by the correlations of H-1‴ (δ_H 3.45) and H-2 (δ_H 8.23) to C-9 (δ_C 154.6). Thus the structure of 4 was established and named as cudraisoflavone M. It is worth mentioning that cudraisoflavone M (4) was also obtained in our phytochemical investigation on the plant Derris robusta (Leguminosae).

Considering the potent cytotoxic activity of some isoflavonoids, the cytotoxicity of these new isoflavonoids (1–4) was evaluated against five human cancer cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW-480) using the MTS method. DDP (cisplatin) and paclitaxel were used as positive controls. The results showed that 1 and 4 exhibited weak cytotoxic activity (Table 5), while 2 and 3 were inactive (IC₅₀ values >40 µM) for all cell lines.

Experimental Section

Plant Material and Isolation (Table 6)

The retention times (t_R) of 1–4 on an analytical HPLC Extend-C18 column (20% → 100% MeOH in H₂O over 8.0 min followed by 100% MeOH to 13.0 min, 1.0 mL/min, 25 °C) were 7.51, 6.85, 6.61 and 9.12 min, respectively.

Table 6

Plant material and isolation

7,4′-Dihydroxy-8,2′,3′-trimethoxyisoflavan (1)

White amorphous powder; UV (MeOH) λ_max: 228 (sh), 278 nm; [α]_D²⁵ +11.0 (c 0.20, MeOH); IR (KBr) ν_max: 3396, 2944, 1602, 1497, 1464, 1292, 1198, 1170, 1097, 1068, 1040, 1010, 963 cm⁻¹; ¹H NMR data: see Table 1; ¹³C NMR data: see Table 2; ESIMS (pos.): m/z 355 [M+Na]⁺; HRESIMS (pos.): m/z 355.1163 [M+Na]⁺ (calcd. for C₁₈H₂₀O₆Na, 355.1158).

7,2′,4′-Trihydroxy-8,3′-dimethoxyisoflavan (2)

White amorphous powder; UV (MeOH) λ_max: 229 (sh), 277 nm; [α]_D²⁵ −13.2 (c 0.20, MeOH); IR (KBr) ν_max: 3404, 2929, 1619, 1509, 1470, 1328, 1184, 1091, 1058, 1037, 1001, 804 cm⁻¹; ¹H NMR data: see Table 1; ¹³C NMR data: see Table 2; ESIMS (pos.): m/z 341 [M+Na]⁺; HRESIMS (pos.): m/z 341.1002 [M+Na]⁺ (calcd. for C₁₇H₁₈O₆Na, 341.1001).

7,2′,4′-Trihydroxy-5-methoxyisoflavan (3)

White amorphous powder; UV (MeOH) λ_max: 230 (sh), 279 nm; [α]_D²⁵ −24.0 (c 0.20, MeOH); IR (KBr) ν_max: 3406, 2937, 1620, 1606, 1520, 1501, 1472, 1459, 1200, 1142, 1118, 1037, 974, 819 cm⁻¹; ¹H NMR data: see Table 1; ¹³C NMR data: see Table 2; ESIMS (pos.): m/z 289 [M+H]⁺; HRESIMS (pos.): m/z 289.1075 [M+H]⁺ (calcd. for C₁₆H₁₇O₅, 289.1076).

Cudraisoflavone M (4)

Yellowish amorphous powder; UV (MeOH) λ_max: 212, 270, 346 (sh) nm; IR (KBr) ν_max: 3425, 2926, 1644, 1582, 1522, 1435, 1380, 1306, 1209, 1116, 1083, 1030 cm⁻¹; ¹H and ¹³C NMR data: see Table 4; ESIMS (pos.): m/z 399 [M+H]⁺; HRESIMS (pos.): m/z 399.1440 [M+H]⁺ (calcd. for C₂₂H₂₃O₇, 399.1444).

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