Lignan Glycosides from Urena lobata

Four new lignan glycosides; urenalignosides A–D (1–4), along with 12 known ones (5–16) were isolated from Urena lobata. Their structures were determined on the basis of extensive spectroscopic and spectrometric data (1D and 2D NMR; IR; CD; and HRESIMS). Compounds 2–4; 6; 7; 10; and 11 showed inhibition of nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophage cells with IC50 values in the range of 25.5–98.4 μM (positive control; quercetin; IC50 = 7.2 ± 0.2 μM).


Introduction
Urena lobata, belonging to the family Malvaceae, is an annually shrubby herbage widely distributed around the world, particularly in the tropical and subtropical areas of Asia, South America, and Africa [1]. This plant is also known as Caesar weed, Congo jute, and Bachita, the local name varies from region to region. In Africa, the leaves and flowers of U. lobata could be eaten as food during famine time and the bast fiber of U. lobata is used as cordage material [2]. More interestingly, U. lobata is also commonly used in folk medicines for the treatment of diabetes, abdominal colic, malaria, gonorrhea, dysentery, fever, rheumatism, and edema [3,4]. Pharmacological studies indicated that the extract of U. lobata showed significant antibacterial, antihyperglycemic, antinociceptive, antidiarrheal, anti-inflammatory, and wound healing activities [5][6][7]. In China, U. lobata is also named "Ditaohua," which is dominantly distributed in the south of China, such as Guangxi, Yunnan, and Guizhou provinces and clinically used to treat pathological leucorrhea and gonorrhea [8]. Promoted by these significant activities, great efforts have been made to clarify the bioactive constituents of U. lobata leading to the separation and elucidation of flavonoids, phenylethyl glycosides, lignans, coumarins, and triglycerides [1, [9][10][11][12][13]. In our previous report, 16 megastigmane glycosides were identified from U. lobata [14]. As an ongoing study, four new lignan glycosides, urenalignosides A-D (1-4) together with 12 known ones (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) were obtained from U. lobata (Figure 1). Herein, the isolation and structural elucidation of the new compounds, as well as their inhibitory effects on NO production on LPS-stimulated RAW264.7 macrophage cells, are described.

Results
The 95% EtOH extracts of U. lobata were suspended in H 2 O and extracted successively with petroleum ether (PE), EtOAc, and n-BuOH. The n-BuOH soluble fraction was separated by D 101 macroporous adsorption resin, silica gel, and Sephadex LH-20 column chromatography and semi-preparative HPLC to afford four new lignan glycosides (1-4) together with 12 known ones (5-16) (Figure 1).  Compound 1 was obtained as a colorless powder. Its molecular formula was assigned as C30H40O15 due to the presence of a [M − H] − ion at m/z 639.2282 (calcd for C30H39O15, 639.2294) in the HRESIMS spectrum ( Figure S1), which was also supported by the 13 C-NMR data ( Table 1). The IR spectrum of 1 showed the absorption bands contributing to hydroxy group (3385 cm −1 ), benzene ring (1615 and 1518 cm −1 ), and ester carbonyl (1735 cm −1 ) group. The NMR spectra of 1 ( Figures S2 and S3) showed the presence of two 1,3,4,5-tetrasubstituted benzene moieties [δH 6.78 (2H, s, H-2,6), 6.80 (2H, s, H-2′,6′). ]. Comparison of the above NMR data with those of icariol A2 [15], a lignan previously isolated from Epimedium sagittatum, revealing the presence of an icariol A2 moiety in 1. In addition, signals due to an acetyl group [δH 1.95 (3H, s), δC 20.7, 172.8] and a glucopyranosyl moiety were also observed in the NMR spectra of 1. The anomeric proton was presented at δH 4.36 (1H, d, J = 8.0 Hz), corresponding to the carbon at δC 104.6 assigned by HSQC experiment, and the relatively large coupling constant (J = 8.0 Hz) of the anomeric proton suggested that the glucopyranosyl moiety was in β configuration. Given that naturally occurring glucose is D-form, and limited by the small amount of 1, we tentatively determined the glucopranosyl moiet in 1 was in D-form. In the HMBC spectra of 1, the correlations between the anomeric proton δH 4.36 (1H, d, J = 8.0 Hz, H-1′′) and C-9 (δC 69.3) confirmed that the glucopyranosyl moiety was linked at C-9 ( Figure 2). The acetyl group was linked at C-9′ determined by the HMBC correlation between H-9′ and the carbonyl carbon (δC 172.8). All the protons and carbons were unambiguously assigned (Table 1)   Compound 1 was obtained as a colorless powder. Its molecular formula was assigned as C 30 Figure S1), which was also supported by the 13 C-NMR data ( Table 1). The IR spectrum of 1 showed the absorption bands contributing to hydroxy group (3385 cm −1 ), benzene ring (1615 and 1518 cm −1 ), and ester carbonyl (1735 cm −1 ) group. The NMR spectra of 1 ( Figures S2 and S3) showed the presence of two 1,3,4,5-tetrasubstituted benzene moieties [δ H 6.78 (2H, s, H-2,6), 6.80 (2H, s, H-2 ,6 ). , and four methoxyl groups [δ H 3.93 (12H, s), δ C 56.9]. Comparison of the above NMR data with those of icariol A 2 [15], a lignan previously isolated from Epimedium sagittatum, revealing the presence of an icariol A 2 moiety in 1. In addition, signals due to an acetyl group [δ H 1.95 (3H, s), δ C 20.7, 172.8] and a glucopyranosyl moiety were also observed in the NMR spectra of 1. The anomeric proton was presented at δ H 4.36 (1H, d, J = 8.0 Hz), corresponding to the carbon at δ C 104.6 assigned by HSQC experiment, and the relatively large coupling constant (J = 8.0 Hz) of the anomeric proton suggested that the glucopyranosyl moiety was in β configuration. Given that naturally occurring glucose is D-form, and limited by the small amount of 1, we tentatively determined the glucopranosyl moiet in 1 was in D-form. In the HMBC spectra of 1, the correlations between the anomeric proton δ H 4.36 (1H, d, J = 8.0 Hz, H-1 ) and C-9 (δ C 69.3) confirmed that the glucopyranosyl moiety was linked at C-9 ( Figure 2). The acetyl group was linked at C-9 determined by the HMBC correlation between H-9 and the carbonyl carbon (δ C 172.8). All the protons and carbons were unambiguously assigned (Table 1) by 1 H-1 H COSY, HSQC, and HMBC experiments ( Figures S4-S6).
The relative configuration of 1 was determined by NOESY spectrum (Figure S7), which showed the NOE correlations of H-7/H-8 and H-7 /H-8. The CD spectra ( Figure S8) of 1 showed the positive Cotton effect at 246 nm suggested that both C-7 and C-7' were in R configuration [16,17], and thus the configuration of C-8, and C-8' were assigned as 8S, 8'S. Accordingly, the structure of 1 was determined as shown in Figure 1, named as urenalignoside A.

3
The relative configuration of 1 was determined by NOESY spectrum (Figure S7), which showed the NOE correlations of H-7/H-8′ and H-7′/H-8. The CD spectra ( Figure S8) of 1 showed the positive Cotton effect at 246 nm suggested that both C-7 and C-7' were in R configuration [16,17], and thus the configuration of C-8, and C-8' were assigned as 8S, 8'S. Accordingly, the structure of 1 was determined as shown in Figure 1, named as urenalignoside A.  Figure S9), which was also supported by the 13 C-NMR -NMR data ( Table 1). The NMR spectra of 2 ( Figures S10 and S11)  In addition, signals due to a glucopyranosyl moiety were also observed in the NMR spectra of 2. The relatively large coupling constant (J = 7.5 Hz) of the anomeric proton resonated at δH 4.23 (1H, d, J = 7.5 Hz, H-1′′) suggested the glucopyranosyl moiety was in β configuration. The linkage of the glucopyranosyl moiety was determined at C-7 by the HMBC correlation between the anomeric proton and C-7 ( Figure 2). Unambiguous assignments of the protons and carbons (Table 1) were achieved by 1 H-1 H COSY, HSQC, HMBC, and NOESY experiments (Figures S12-S15).  Figure S9), which was also supported by the 13 C-NMR -NMR data ( Table 1). The NMR spectra of 2 ( Figures S10 and S11)  propane-1,3-diol, a lignan previously isolated from Bursera tonkinensis [18], suggested the occurrence of an 8-O-4 -neolignan moiety in 2. In addition, signals due to a glucopyranosyl moiety were also observed in the NMR spectra of 2. The relatively large coupling constant (J = 7.5 Hz) of the anomeric proton resonated at δ H 4.23 (1H, d, J = 7.5 Hz, H-1 ) suggested the glucopyranosyl moiety was in β configuration. The linkage of the glucopyranosyl moiety was determined at C-7 by the HMBC correlation between the anomeric proton and C-7 ( Figure 2). Unambiguous assignments of the protons and carbons (Table 1) were achieved by 1 H-1 H COSY, HSQC, HMBC, and NOESY experiments (Figures S12-S15).
It has been well reported that the relative configurations of C-7 and C-8 could be solved by the analysis of the coupling constant between H-7 and H-8. Regularly, a relatively small coupling constant (J = 3-4 Hz) between H-7 and H-8 defines the erythro configurations of C-7 and C-8, while a relatively large coupling constant (J = 6-8 Hz) give rise to the threo configurations of C-7 and C-8 [19][20][21][22][23][24]. Accordingly, the stereochemistry of C-7 and C-8 in 2 were assigned as erythro according to the small coupling constant (J = 3.0 Hz) between H-7 and H-8. The positive Cotton effect at 233 nm in the CD spectrum ( Figure S16) of 2 suggested that the configuration of C-8 was S [22,[24][25][26], and thus the configuration of C-7 was determined as R. Therefore, the structure of 2 namely urenalignoside B was elucidated as shown in Figure 1.  Figure S17). The NMR data of 3 ( Figures S18-S23) is comparable to those of 2, except the absence of one methoxy group in 3. In the HMBC spectrum of 3, the correlation between the anomeric proton [δ H 4.93 (1H, J = 7.5 Hz, H-1 )] of the glucopyranosyl moiety and the C-3' of the aglycon demonstrated that the glucopyranosyl moiety was linked at C-3' in 3 ( Figure 2). The large coupling constant (J = 8.5 Hz) between H-7 and H-8 suggested that the C-7 and C-8 were in threo orientation. The negative Cotton effect at 233 nm in the CD spectrum ( Figure S24) of 3 suggested that the configuration of C-8 was R [23,24], and thus the configuration of C-7 was 7R. Therefore, the structure of 3 namely urenalignoside C was determined as shown in Figure 1.  Figure S25). Comparison of the NMR data of 4 ( Figures S26-S31) with those of 2 revealed that these two compounds share a highly similar skeleton, except the significantly deshielded chemical shift of C-9 (δ C 69.2; ∆δ C + 6.3), suggesting that the O-glucopyranosyl moiety was linked at C-9 in 4, but not like that at C-7 in 2. The deduction was confirmed by HMBC correlation between the anomeric proton [δ H 4.32 (1H, J = 8.0 Hz, H-1 )] and C-9 ( Figure 2). The relatively large coupling constant (J = 7.0 Hz) between H-7 and H-8 suggested that the C-7 and C-8 were in threo orientation. The absolute configuration of C-8 was assigned as S based on the positive Cotton effect at 233 nm presented in the CD spectrum ( Figure S32) of 4 [22,[24][25][26], and thus the configuration of C-7 was assigned as S. Accordingly, the structure of 4 namely urenalignoside D was determined as shown in Figure 1.