Indole alkaloid glycosides with a 1'-(phenyl)ethyl unit from Isatis indigotica leaves.

Seven indole alkaloid glycosides containing a 1'-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl unit (1-7) were isolated from an aqueous extract of Isatis indigotica leaves (da qing ye). Their structures were determined by spectroscopic data analysis combined with enzymatic hydrolysis as well as comparison of their experimental CD (circular dichroism) and calculated ECD (electrostatic circular dichroism) spectra. Based on analysis of [ α ] D 20 and/or Cotton effect (CE) data of 1-7, two simple roles to assign location and/or configuration of β-glycopyranosyloxy and 1'-(phenyl)ethyl units in the indole alkaloid glycosides are proposed. Stereoselectivity in plausible biosynthetic pathways of 1-7 is discussed. Compounds 3 and 4 and their mixture in a 3:2 ratio showed activity against KCNQ2 in CHO cells. The mixture of 5 and 6 (3:2) exhibited antiviral activity against influenza virus H1N1 PR8 with IC50 64.7 μmol/L (ribavirin, IC50 54.3 μmol/L), however, the individual 5 or 6 was inactive. Preliminary structure-activity relationships were observed.

Introduction

Isatis indigotica Fort. (Cruciferae) is a widely cultivated medicinal plant. The dried leaves and roots of this plant, named “da qing ye” and “ban lan gen” in Chinese, respectively, are used in traditional Chinese medicines for the treatment of influenza and other infections. Diverse bioactive constituents have been reported from ethanol or methanol extracts of the I. indigotica leaves and roots2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. However, few works were done on the chemical constituents of decoctions of the two herbal medicines7, 13, 14, 16, 18, 24 though the decoctions are practically applied in traditional Chinese medicine. Therefore, the aqueous extracts of “ban lan gen” and “da qing ye” were successively investigated as part of our program to systematically assess the chemical diversity and pharmacological activity of traditional Chinese medicines focusing on the minor components26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39. From the “ban lan gen” extract, 57 new alkaloids including 22 indole and bisindole alkaloid glycosides were isolated, and some of them showed antiviral activity40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55. Previously we reported two pairs of unusual scalemic enantiomers isatidifoliumindolinones A−D from the “da qing ye” extract, this paper deals with isolation, structural elucidation, biosynthetic postulation, and biological activity of seven unusual indole alkaloid glycosides (1–7), including three pairs of epimers named isatidifoliumosides/epiisatidifoliumosides A (1/2), B (3/4), and C (5/6), respectively, and an isomer named isatidifoliumoside D (7) (Fig. 1). The new isolates contain a 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl moiety at different positions (N-1, C-2, or C-3) of the indole nucleus, biogenetically associating to isatidifoliumindolinones A–D. Analysis of specific rotations [α]D and Cotton effects (CEs) of 1–7 resulted in preliminary roles for assignment of location and configuration of the β-glycopyranosyloxy and 1′-(phenyl)ethyl units in this group of indole alkaloids. Especially, 5 and 6 are the first indole alkaloid β-d-allopyranosides from nature, while the mixture of 5 and 6 (3:2) showed antiviral activity against influenza virus H1N1 PR8, which represents the first bioactive indole glycosides supporting clinic application of the herbal medicine.

Figure 1

The structures of compounds 1–7.

Results and discussion

Compound 1 was isolated as a white amorphous powder with –23.7 (c 0.1, MeOH). The IR spectrum of 1 exhibited absorption bands assignable to hydroxy (3303 cm−1) and aromatic ring (1613, 1519, and 1460 cm−1) functional groups. Its molecular formula of C24H29NO9 was determined by HR-ESI-MS combined with the NMR spectroscopic data (Table 1, Table 2). The NMR spectrum of 1 in CD3OD showed resonances attributable to two ortho-disubstituted benzene rings at δH 7.69 (brd, J = 7.8 Hz, H-4), 6.97 (dt, J = 7.8, 1.2 Hz, H-5), 7.06 (ddd, J = 8.4, 7.8, 1.2 Hz, H-6), and 7.25 (brd, J = 8.4 Hz, H-7); a 1′,1′-disubstituted ethyl unit at δH 5.60 (q, J = 7.2 Hz, H-1′) and 1.84 (d, J = 7.2 Hz, H3-2′); a 4″-hydroxy-3″,5″-dimethoxyphenyl at δH 3.73 (s, OCH3-3″,5″) and 6.46 (s, H-2″/6″); and a trisubstituted double bond at δH 7.31 (s, H-2). It also displayed diagnostic resonances for a β-glucopyranosyl moiety (Table 1). The 13C NMR and DEPT spectra showed carbon resonances corresponding to the above units (Table 2). The presence of the β-glucopyranosyl unit was confirmed by enzymatic hydrolysis of 1 with snailase. The sugar isolated from the hydrolysate exhibited retention factor (Rf) on TLC, specific rotation { +40.2 (c 0.08, H2O)}, and 1H NMR spectroscopic data identical to those of an authentic glucopyranose (see Experimental Section and Supporting Information Figs. S120 and S124).

Table 1

1H NMR spectroscopic data for compounds 1–7 in CD3ODa.

No.	1	2	3	4	5	6	7
2	7.31 s	7.31 s					6.99 s
4	7.69 brd (7.8)	7.69 brd (7.8)	7.70 brd (7.8)	7.74 brd (7.8)	7.72 brd (7.8)	7.76 brd (7.8)
5	6.97 dt (7.8, 1.2)	6.97 dt (7.8, 1.2)	6.94 dt (7.8, 1.2)	6.94 dt (7.8, 1.2)	6.94 dt (7.8, 1.2)	6.95 dt (7.8, 1.2)	6.62 brd (7.2)
6	7.06 ddd (8.4, 7.8, 1.2)	7.07 ddd (8.4, 7.8, 1.2)	7.00 dt (7.8, 1.2)	7.00 ddd (8.4, 7.8, 1.2)	7.00 dt (7.8, 1.2)	7.00 dt (7.8, 1.2)	6.94 t (7.2)
7	7.25 brd (8.4)	7.25 brd (8.4)	7.22 brd (7.8)	7.21 brd (8.4)	7.21 d (7.8)	7.21 brd (7.8)	6.97 dd (7.2, 1.2)
1′	5.60 q (7.2)	5.61 q (7.2)	4.74 q (7.2)	4.73 q (7.2)	4.74 q (7.2)	4.74 q (7.8)	4.87 q (7.2)
2′	1.84 d (7.2)	1.84 d (7.2)	1.68 d (7.2)	1.65 d (7.2)	1.67 d (7.2)	1.66 d (7.8)	1.59 d (7.2)
2″	6.46 s	6.45s	6.71 s	6.64 s	6.68 s	6.64 s	6.56 s
6″	6.46 s	6.45 s	6.71 s	6.64 s	6.68 s	6.64 s	6.56 s
1‴	4.72 d (7.2)	4.71 d (7.8)	4.62 d (8.4)	4.66 d (7.8)	5.05 d (7.8)	5.04 d (8.4)	5.06 d (7.8)
2‴	3.50 dd (9.0, 7.2)	3.50 dd (7.8, 9.0)	3.53 dd (9.0, 8.4)	3.50 dd (9.0, 7.8)	3.65 dd (7.8,3.0)	3.63 dd (8.4,3.0)	3.58 dd (9.0, 7.8)
3‴	3.44 t (9.0)	3.43 t (9.0)	3.40 t (9.0)	3.41 t (9.0)	4.17 t (3.0)	4.15 t (3.0)	3.46 t (9.0)
4‴	3.39 t (9.0)	3.38 t (9.0)	3.47 t (9.0)	3.46 t (9.0)	3.67 dd (9.6,3.0)	3.66 dd (9.6,3.0)	3.28 t (9.0)
5‴	3.36 m	3.35 m	3.14 m	3.16 m	3.62 m	3.61 m	3.41 m
6‴a	3.89 dd (12.0, 2.4)	3.89 dd (12.0, 2.4)	3.82 dd (11.4, 1.8)	3.66 m	3.83 dd (11.4, 2.4)	3.67 m	3.83 dd (12.0, 2.4)
6‴b	3.70 dd (12.0, 5.4)	3.70 dd (12.0, 5.4)	3.73 dd (11.4, 4.8)	3.66 m	3.74 dd (11.4, 4.8)	3.67 m	3.60 dd (12.0, 6.0)
3″,5″-OCH3	3.73 s	3.73 s	3.80 s	3.79 s	3.80 s	3.79 s	3.75 s

Data (δH) were measured for 1–7 at 600 MHz. Coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H–1H COSY, HSQC, and HMBC experiments.

Table 2

13C NMR spectroscopic data for compounds 1–7 in CD3ODa.

No.	1	2	3	4	5	6	7
2	113.1	112.9	133.7	132.9	133.6	132.8	121.6
3	138.7	138.8	133.0	133.8	133.1	133.9	122.2
3a	122.3	122.2	122.8	122.7	122.7	122.8	118.6
4	119.0	119.0	118.6	118.7	118.7	118.8	153.5
5	119.8	119.8	119.7	119.7	119.6	119.6	103.4
6	122.9	122.9	121.9	122.0	121.9	121.9	123.0
7	111.1	111.0	111.9	111.9	111.9	111.9	106.7
7a	135.1	135.1	134.7	134.6	134.7	134.6	140.2
1′	56.0	55.9	35.6	35.7	35.5	35.6	38.2
2′	22.0	22.0	20.7	21.0	20.6	20.9	24.2
1″	135.4	135.4	137.4	137.2	137.2	137.2	141.5
2″	104.5	104.5	105.9	106.1	105.9	106.1	106.0
3″	149.3	149.3	149.1	149.1	149.1	149.1	148.8
4″	135.8	135.8	134.8	134.9	134.8	134.9	134.0
5″	149.3	149.3	149.1	149.1	149.1	149.1	148.8
6″	104.5	104.5	105.9	106.1	105.9	106.1	106.0
1‴	105.9	105.9	107.4	107.7	105.0	105.3	101.6
2‴	75.1	75.1	75.4	75.4	72.6	72.6	75.2
3‴	78.1	78.1	78.2	78.2	73.2	73.4	78.7
4‴	71.6	71.7	71.5	71.4	68.8	68.7	71.6
5‴	78.2	78.3	77.9	77.9	75.4	75.4	78.1
6‴	62.7	62.7	62.6	62.5	63.1	62.9	62.7
3″,5″-OCH3	56.8	56.7	56.8	56.8	56.8	56.8	56.8

Data (δC) were measured in for 1–7 at 150 MHz. The assignments were based on DEPT, 1H–1H COSY, HSQC, and HMBC experiments.

As compared with those of the previously reported compounds from I. indigotica40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, the above spectroscopic data suggested that 1 was an unusual indole alkaloid β-d-glucoside containing the 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl moiety. The structure was further elucidated by 2D NMR data analysis. In the 1H–1H COSY spectrum of 1, homonuclear vicinal coupling cross-peaks of H-4/H-5/H-6/H-7 (Fig. 2), combined with the chemical shifts and coupling constants of these protons, confirmed the presence of the ortho-disubstituted benzene ring. In the HMBC spectrum, two and three-bond correlations from H-7 to C-3a and C-5; from H-4 to C-7a and C-6; from H-5 to C-7 and C-3a; from H-6 to C-4 and C-7a; and from H-2 to C-3, C-7a, and C-3a; along with their chemical shifts, proved that there was a 3-substituted indole moiety in 1. The HMBC correlations from H-1‴ to C-3, combined with the 1H–1H COSY correlations of H-1‴/H-2‴/H-3‴/H-4‴/H-5‴/H2-6‴ (Fig. 2) as well as chemical shifts of these proton and carbon resonances, located a β-d-glucopyranosyloxy unit at the C-3 of the indole moiety. In addition, the HMBC correlations from H-1′ to C-2, C-2′, C-2″/6″, and C-7a; from H3-2′ to C-1′ and C-1″; along with the 1H–1H COSY cross-peaks between H-1′ and H3-2′ and their chemical shifts, located the 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl moiety at the N atom of the indole moiety. Accordingly, the planar structure of 1 was elucidated as shown. The CD (circular dichroism) spectrum of 1 displayed two positive Cotton effects at 206 and 292 nm and a negative Cotton effect at 234 nm, arising from overlapped transitions of the indole and benzene chromophores in the molecule. The 1′S configuration of 1 was assigned by calculations of ECD (electronic circular dichroism) spectra using the time-dependent density functional theory (TDDFT) method. The calculated ECD spectrum of 1 was in well agreement with the experimental CD spectrum (Supporting Information Fig. S3). Therefore, the structure of compound 1 was determined and named isatidifoliumoside A.

Figure 2

Main 1H–1H COSY (thick lines) and three-bond HMBC (arrows, from 1H to 13C) correlations of compounds 1–7.

Compound 2, white amorphous powder { –50.5 (c 0.1, MeOH)}, was separated by chiral HPLC from a mixture of 1 and 2 with peak integrations in around 1:1 ratio (Supporting Information Fig. S26). Comparison of the NMR spectroscopic data of 2 and 1 (Table 1, Table 2) demonstrated that the C-2 resonance in 2 was shielded by ΔδC −0.2 ppm, while the other proton and carbon resonances were shifted by ΔδH≤±0.01 and ΔδC≤±0.1 ppm, respectively. This suggested that 2 was the 1′-epimer of 1, which was confirmed by HR-ESI-MS and 2D NMR data analysis (Fig. 2) as well as by the specific rotation and CEs (Fig. 3) of 2. Especially, the experimental CD and calculated ECD spectra of 2 were consistent with each other (Fig. S3). Thus, the structure of compound 2 was determined and named epiisatidifoliumoside A.

Figure 3

The overlaid experimental CD spectra of 1−7.

Compound 3, a white amorphous powder with +4.7 (c 0.1, MeOH), is another isomer of 1 as indicated by (+)-HR-ESI-MS, NMR, and IR spectroscopic data. Comparison of the NMR data of 3 with those of 1 (Table 1, Table 2) indicated that the two compounds mainly differed in substitution of the methine unit (C-2) in 1 by a quaternary carbon (δC 133.0) in 3. In addition, as compared with those of 1, the H-1′ and C-1′ resonances in 3 were shielded by ΔδH −0.86 and ΔδC −20.4 ppm. This suggested that the 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl moiety at the N atom in 1 was migrated to C-2 in 3. The suggestion was verified by 2D NMR data analysis (Fig. 2). Especially the HMBC correlation from H-1‴ to C-3, from H-1′ to C-2, C-1″ and C-2″/6″, and from H-2′ to C-2 and C-1″ proved the locations of the β-glucopyranosyloxy and 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl moieties at C-3 and C-2 in 3, respectively. Using the aforementioned protocol, the d-configuration of β-glucopyranosyl in 3 was verified. Comparison of the experimental CD and calculated ECD spectra supported the 1′R configuration of 3 (Supporting Information Fig. S6). Therefore, the structure of compound 3 was determined and named isatidifoliumoside B.

Compound 4, a white amorphous powder with –22.4 (c 0.1, MeOH), showed similar spectroscopic data as those of 3. Comparison of the NMR spectroscopic data of the two compounds (Table 1, Table 2) demonstrated that H-2″/6″, H-6‴a and H-6‴b, and C-2 in 4 were shielded by ΔδH −0.07, −0.16 and −0.07, and ΔδC −0.8 ppm, respectively, whereas C-3 was deshielded by ΔδC +0.8 ppm. This, together with nearly opposite CEs of the two compounds (Fig. 3), suggested that 4 was 1′-epimer of 3, which was further confirmed by 2D NMR data analysis (Fig. 2) as well as by comparison of the experimental CD and calculated ECD spectra of 4 (Fig. S6). Thus, the structure of compound 4 was determined and named epiisatidifoliumoside B.

Compound 5, a white amorphous powder with +8.6 (c 0.1, MeOH), is the isomer of 3 and 4 having a different sugar unit, as indicated by spectroscopic data and confirmed by 2D NMR data analysis. Especially, in the HMBC spectrum, correlations from H-1‴ to C-3 and from H-2′ to C-2 (Fig. 2) confirmed that the sugar and 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl units located at C-3 and C-2 of the indole nucleus in 5, respectively. In the 1H NMR spectrum of 5, the coupling constants (J1‴,2‴ = 7.8 Hz, J2‴,3‴ = J3‴,4‴ = 3.0 Hz, and J4‴,5‴ = 9.6 Hz) indicated that the sugar unit was β-allopyranosyl only differing from β-glycopyranosyl in the 3‴-configuration. This deduction was proved by enzymatic hydrolysis of 5 with snailase. The sugar isolated from the hydrolysate of 5 exhibited retention factor (Rf) on TLC, specific rotation { +12.0 (c 0.07, H2O)} (see Experimental Section), and 1H NMR spectroscopic data completely identical to those of an authentic d-allopyranose (Supporting Information Figs. S122 and S125). Similarity of the CD curves between 5 and 3 (Fig. 3) demonstrated that the two compounds possessed the same 1′R configuration, which was supported by comparison of the experimental CD and the calculated ECD spectra of 5 (Supporting Information Fig. S9). Therefore, the structure of compound 5 was determined and named isatidifoliumoside C.

Compound 6 was obtained as a white amorphous powder with −35.6 (c 0.1, MeOH). Comparison of the NMR spectroscopic data of 6 and 5 (Table 1, Table 2) demonstrated that the H-6‴a and H-6‴b, and C-2 resonances in 6 were shielded by ΔδH −0.16, −0.07 and ΔδC −0.8 ppm, respectively, whereas the C-3 resonance was deshielded by ΔδC +0.8 ppm. The differences suggested that 6 is the 1′-epimer of 5, which was supported by the reverse CEs in the CD spectrum of 6 as compared with that of 5 (Fig. 3). The elucidation was verified by 2D NMR data analysis (Fig. 2) as well as by consistence of the experimental CD and calculated ECD spectra of 6 (Fig. S9). Thus, the structure of compound 6 was determined and named epiisatidifoliumoside C.

Compound 7 was obtained as a white amorphous powder with –67.5 (c 0.1, MeOH). The spectroscopic data demonstrated that 7 was one more isomer of 3 and 4 having the same β-d-glucopyranosyl unit. The presence of β-d-glucopyranosyl in 7 was verified by enzymatic hydrolysis (see Experimental Section). As compared, the NMR spectroscopic data (Table 1, Table 2) indicated replacement of the 2,3-disubstited indole nucleus in 3 and 4 by a 3,4-disubstited indole in 7. This was confirmed by 2D NMR spectroscopic data analysis of 7 (Fig. 2). In particular, the HMBC spectrum of 7 showed the correlations from H-1′ to C-1″, C-2, C-3, C-3a, and C-2″/6″; from H-1‴ to C-4; and from H3-2′ to C-1″ and C-3. These correlations revealed that the β-d-glucopyranosyloxy and 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl units substituted at the C-4 and C-3 of the indole nucleus in 7, respectively. The 1′S configuration of 7 was assigned by consistence of the experimental CD and calculated ECD spectra (Supporting Information Fig. S12). Therefore, the structure of 7 was determined and named isatidifoliumoside D.

Adducting and deducting analysis of the specific rotation data of the three pairs of epimers showed that the glycosidic indole moieties contributed the values of −37.1, −8.9, and −13.4 to 1/2, 3/4, and 5/6, respectively, while the (1′S)-/(1′R)-1′- and (1′R)-/(1′S)-1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl unit contributed +/−13.4 to 1/2 and +/−13.5 and +/−22.0 to 3/4 and 5/6. These data revealed that the contributions of the 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl unit to the specific rotation data depended upon the C-1′ configuration as well as the location of the unit at the glycosidic indole nucleus. At the N-1 atom, the (1′S)-1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl had the positive contribution (1), and at C-2 (4 and 6) the negative. In contrast, (1′R)-1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl at N-1 (2) had the negative contribution, and at C-2 (3 and 5) the positive. As compared with those of 1, 2, 4, and 6, the specific rotation value of 7 suggested that the (1′S)-1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl unit at C-3 had the negative contribution though the 1′R epimer of 7 was not obtained in this study. In addition, comparison of the CD spectra of 1–6 showed that signs of the CEs were dominated by the C-1′ configuration and location of the 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl unit (Fig. 3). The CD spectra of 1–6 display three CEs around 210, 236, and 288 nm, arising from the overlapped 1Ba/1La, 1Bb/1Lb, and 1Lb/transitions of the substituted indole/benzene chromophores, respectively. The (1′S)-1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl unit at the N atom (1) dominated the positive 1Ba/1La (210 nm) and 1Lb/(293 nm) and negative 1Bb/1Lb (236 nm) CEs, whereas at C-2 (4 and 6) it gave the corresponding reverse CEs. Meanwhile, the (1′R)-1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl unit at the N atom (2) dominated the negative 1Ba/1La and 1Lb/and positive 1Bb/1Lb CEs, and at C-2 (3 and 5) the positive 1Ba/1La and 1Lb/and negative1Bb/1Lb CEs. Adduction of the CD data between the epimeric pairs indicated that the glycosidic indole moieties in 1–6 had the relatively weak contributions to the 1Ba/1La (positive) and 1Lb (negative) CEs. This was supported by high consistence of the experimental CD and calculated ECD spectra of 1–6 (Figs. S3, S6, and S9) as well as by the calculated ECD spectra of aglycones 1a–6a (Supporting Information Figs. S14, S16, and S17) and the proposed biogenetic precursors 8 and 9 (Supporting Information Figs. S23−25). The CD spectrum of 7 exhibited the positive 1Ba/1La (200 nm) and 1Lb/(274 nm) and negative 1Bb/1Lb (225 nm) CEs. As compared with those of 3 and 4, wavelengths of the CE maximums of 7 were blue-shifted by Δλmax 10 nm (see Fig. 3), which would be due to the location change of the substituents at the indole ring. Further comparison of the experimental CD and calculated ECD spectra supported that the 1′S configuration dominated the CEs of 7. This was also supported by the calculated ECD spectra of the aglycone 7a and the proposed biogenetic precursor 10 (Supporting Information Figs. S19 and S25).

Based on the above analysis, two preliminary rules to assign location and configuration of the β-d-glucopyranosyloxy, β-d-allopyranosyloxy, and 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl in these indole glycosides are disclosed: (a) β-d-glucopyranosyloxy at C-3 or C-4 as well as β-d-allopyranosyloxy at C-3 of the indole nucleus have the negative contributions to both the specific rotation (>8.0) and 1Bb CE (230 ± 7 nm); and (b) the (1′S)-1′-(phenyl)ethyl units at the N atom or C-3 and the (1′R)-1′-(phenyl)ethyl units at C-2 positively contributes to the specific rotation and 1Lb/CE (284 ± 10 nm) but negatively to the 1Bb/1Lb (230 ± 7 nm) CE, whereas the (1′R)-1′-(phenyl)ethyl units at the N atom or C-3 and the (1′S)-1′-(phenyl)ethyl units at C-2 negatively contributes to the specific rotation and 1Lb/CE (284 ± 10 nm) but positively to the 1Bb/1Lb (230 ± 7 nm) CE. The roles simply using the specific rotation and/or CD data may be validated to determination of the location and configuration of other β-glycopyranosyloxy and 1′-(phenyl)ethyl units in the related indole glycosides.

Compounds 1–7, together with isatidifoliumindolinones A–D from the same extract, represent the first examples of natural products with the novel carbon skeleton derived from coupling between the indole or indolin-3-one and phenylethyl units, especially 1–7 are the first glycosidic forms. Based on the molecular architecture, the biosynthetic precursors of 1–7 are traced to 1H-indol-3-ol glycosides (8–10) and sinapic acid (11), among them 8 was reported from the roots of the plant and 11 from this study. A plausible biosynthetic pathway for 1–7 is postulated in Scheme 1. Decarboxylation of 11 gives an intermediate 2,6-dimethoxy-4-vinylphenol (12), which undergoes nucleophilic addition with 8, 9, or 10 to generate 1–7. Interestingly, in this plant the epimers occur in the relative ratios of 1:1 for 1/2 and 3:2 for 3/4 and 5/6, as indicated by chiral HPLC separation (Supporting Information Figs. S26–28). This demonstrates that the nucleophilic addition of 12 with 8 to produce 1/2 is non-stereoselective, while the addition of 12 with 8 or 9 to give 3/4 or 5/6 is partially stereoselective. However, the addition of 12 with 10 to yield 7 is fully stereoselective. These facts suggest that location of β-d-glucopyranosyloxy/β-d-allopyranosyloxy and nucleophilic center on the indole nucleus play important roles in stereoselective biosynthesis of 1–7. In addition, 3 and/or 4 is the potential precursors of isatidifoliumindolinones A–D that may be produced by hydrolysis of 3 and/or 4, followed by successive or simultaneous oxidation and methylation/methoxylation (not shown in Scheme 1).

Scheme 1

Proposed biosynthetic pathways of 1–7.

In the preliminary in vitro assays, compounds 3 and 4 as well as their mixture in 1:1 showed inhibitory activity against the K+ channel KCNQ2 in CHO cells with IC50 19.15, 11.57, and 13.27 μmol/L, respectively (the positive control ML252, IC50 0.27 ± 0.2 μmol/L), indicating that 4 with the 1′S configuration is relatively more active than 3 with the 1′R configuration. However, in this assay 1, 2, and 5–7 were inactive at a concentration of 10−4 mol/L. In addition, the mixture of 5 and 6 in the 3:2 ratio exhibited antiviral activity against influenza virus H1N1 PR8 with IC50 64.7 μmol/L (the positive control ribavirin, IC50 54.3 μmol/L), while the individual epimers 5 and 6 were inactive (10−4 mol/L). This suggested that the epimers had a synergistic effect against influenza virus H1N1 PR8. The results provide preliminary clues for activity–structure relationships of these indole glycosides. For KCNQ2 inhibition, β-d-glucopyranosyloxy at C-3 is required while the 1′S configuration may increase activity, and for anti-influenza virus H1N1 PR8, β-d-allopyranosyloxy at C-3 and a synergistic effect of the 1′S and 1′R configurations play roles.

Conclusions

Seven indole alkaloid glycosides containing the 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl unit (1–7) were isolated from the I. indigotica leaves (da qing ye) decoction. The aglycones, together with that of the stereoisomer isatidifoliumindolinones A−D from the same decoction, represent a novel group of the indole alkaloid natural products. Based on structure-specific rotation/Cotton effect analysis of 1–7, two preliminary roles are proposed for assignment of the location and configuration of the β-glycopyranosyloxy and 1′-(phenyl)ethyl units in the indole alkaloid glycosides. Compounds 5 and 6 are the first indole alkaloid β-d-allopyranosides. Especially the two allopyranoside epimers showed the synergistic effect against influenza virus H1N1 PR8, representing the novel antiviral constituents from the decoction of I. indigotica leaves9, 23, 59 that support clinical application of the herbal medicine and the theory of multiple active components of traditional Chinese medicine.

Experimental

General experimental procedures

See Supporting Information.

Plant material

See Ref..

Extraction and isolation

For preliminary extraction and isolation, see Ref. 56. Subfraction B2-4-1-21 (1.5 g) was chromatographed over silica gel, eluting with CHCl2/MeOH (15:1), to give B2-4-1-21-1−B2-4-1-21-10, of which B2-4-1-21-8 (60 mg) was further separated by RP-HPLC (25% acetonitrile, 3.0 mL/min) to afford a mixture (20 mg, tR = 35.1 min). The mixture was separated by chiral HPLC with a Chiralpak AD-H column (250 mm × 10 mm) using a mobile phase of EtOH/n-hexane (27:73, flow rate 1.0 mL/min) to yield 5 (5.0 mg, tR = 34.1 min) and 6 (3.5 mg, tR = 37.8 min). Subfraction B2-4-2-9 (500 mg) was further fractionated by CC over HW-40C (MeOH) to yield B2-4-2-9-1−B2-4-2-9-4, of which subfraction B2-4-2-9-2 (16 mg) was separated by RP-HPLC (40% MeOH, 3.0 mL/min) afforded 7 (4.0 mg, tR = 25.0 min). Fraction B2-4-2 (20 g) was fractionated by CC over RP (C18) silica gel (460 mm × 36 mm, 150 g) eluting with a gradient of increasing MeOH concentration (0%–100%) in H2O to yield B2-4-2-1−B2-4-2-28. Purification of B2-4-2-25 (30 mg) by RP-HPLC (8% acetonitrile, 3.0 mL/min) afforded a mixture (12 mg, tR = 22.2 min), which was further separated by chiral HPLC with a Chiralpak AD-H column (250 mm × 10 mm, EtOH/n-hexane 3:7, flow rate 1.5 mL/min) to obtain 1 (4.0 mg, tR = 22.3 min) and 2 (3.5 mg, tR = 37.2 min). Separation of B2-4-2-19 (300 mg) by RP-HPLC (30% MeOH, 3.0 mL/min) afforded the mixture (10 mg, tR = 77.0 min), which were isolated by chiral HPLC with a Chiralpak AD-H column (250 mm × 10 mm, EtOH/n-hexane 1:3, flow rate 2.5 mL/min) to obtain 3 (4.5 mg, tR = 30.6 min) and 4 (3.0 mg, tR = 35.7 min).

Isatidifoliumoside A (1)

White amorphous powder; –23.7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (4.59), 228 (4.40), 281 (3.64) nm; CD (MeOH) λmax (Δε) 206 (+39.5), 234 (−28.1), 292 (+3.53) nm; IR νmax 3403, 2935, 1613, 1554, 1519, 1460, 1427, 1361, 1331, 1225, 1157, 1115, 911, 836, 800, 743, 670, 615 cm−1; see 1H NMR (CD3OD, 600 MHz) data in Table 1; See 13C NMR (CD3OD, 150 MHz) data in Table 2; (+)-ESI-MS m/z 476 [M+H]+, 498 [M+Na]+; (−)-ESI-MS m/z 474 [M–H]−; (+)-HR-ESI-MS m/z 498.1746 [M+Na]+ (Calcd. for C24H29NO9Na, 498.1735).

Episatidifoliumoside A (2)

White amorphous powder; –50.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (4.74), 228 (4.54), 281 (3.84) nm; CD (MeOH) λmax (Δε) 210 (−26.9), 236 (+22.1), 293 (−3.73) nm; IR νmax 3402, 2934, 1613, 1554, 1519, 1460, 1427, 1361, 1331, 1226, 1157, 1116, 911, 836, 800, 743, 670, 614 cm−1; see 1H NMR (CD3OD, 600 MHz) data in Table 1; see 13C NMR (CD3OD, 150 MHz) data in Table 2; (+)-ESI-MS m/z 498 [M+Na]+; (−)-ESI-MS m/z 474 [M–H]−; (+)-HR-ESI-MS m/z 498.1739 [M+Na]+ (Calcd. for C24H29NO9Na, 498.1735).

Isatidifoliumoside B (3)

White amorphous powder; +4.7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.67), 227 (4.40), 283 (3.85) nm; CD λmax (Δε) 210 (+13.9), 235 (−29.5), 284 (+5.78) nm; IR νmax 3359, 2971, 2932, 1701, 1617, 1518, 1459, 1427, 1326, 1220, 1157, 1115, 1072, 941, 901, 838, 797, 750, 588 cm−1; see 1H NMR (CD3OD, 600 MHz) data in Table 1; see 13C NMR (CD3OD, 150 MHz) data in Table 2; (+)-ESI-MS m/z 498 [M+Na]+; (−)-ESI-MS m/z 474 [M–H]−; (+)-HR-ESI-MS m/z 476.1922 [M+H]+ (Calcd. for C24H30NO9, 476.1915).

Episatidifoliumoside B (4)

White amorphous powder; –22.4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.51), 226 (4.38), 281 (3.79) nm; CD λmax (Δε) 211 (−10.8), 236 (+22.3), 284 (−4.11) nm; IR νmax 3352, 2972, 2931, 1702, 1616, 1518, 1459, 1426, 1326, 1220, 1116, 1075, 1047, 941, 880, 837, 798, 749, 636 cm−1; see 1H NMR (CD3OD, 600 MHz) data in Table 1; see 13C NMR (CD3OD, 150 MHz) data in Table 2; (+)-ESI-MS m/z 498 [M+Na]+; (−)-ESI-MS m/z 474 [M–H]−; (+)-HR-ESI-MS m/z 476.1925 [M+H]+ (Calcd. for C24H30NO9, 476.1915).

Isatidifoliumoside C (5)

White amorphous powder; +8.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.57), 226 (4.55), 282 (4.01) nm; CD λmax (Δε) 210 (+20.7), 236 (−24.8), 285 (+5.24) nm; IR νmax 3373, 2932, 1616, 1518, 1459, 1426, 1326, 1218, 1115, 1041, 941, 910, 837, 797, 735, 633 cm−1; see 1H NMR (CD3OD, 600 MHz) data in Table 1; see 13C NMR (CD3OD, 150 MHz) data in Table 2; (+)-ESI-MS m/z 498 [M+Na]+; (+)-HR-ESI-MS m/z 476.1929 [M+H]+ (Calcd. for C24H30NO9, 476.1915).

Episatidifoliumoside C (6)

White amorphous powder; –35.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.62), 227 (4.37), 282 (3.77) nm; CD λmax (Δε) 210 (−13.7), 236 (+18.4), 287 (−3.37) nm; IR νmax 3375, 2927, 1966, 1616, 1518, 1459, 1426, 1325, 1218, 1115, 1039, 941, 910, 837, 797, 736 cm−1; see 1H NMR (CD3OD, 600 MHz) data in Table 1; see 13C NMR (CD3OD, 150 MHz) data in Table 2; (+)-ESI-MS m/z 498 [M+Na]+; (−)-ESI-MS m/z 474 [M–H]−; (+)-HR-ESI-MS m/z 476.1928 [M+H]+ (Calcd. for C24H30NO9, 476.1915).

Isatidifoliumoside D (7)

White amorphous powder; –67.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.68), 222 (4.62), 271 (3.93) nm; CD λmax (Δε) 225 (−28.1), 274 (+5.33) nm; IR νmax 3370, 2968, 2934, 1677, 1617, 1514, 1460, 1426, 1356, 1325, 1229, 1079, 913, 838, 800, 743, 655 cm−1; see 1H NMR (CD3OD, 600 MHz) data in Table 1; see 13C NMR (CD3OD, 150 MHz) data in Table 2; (+)-ESI-MS m/z 498 [M+Na]+; (−)-ESI-MS m/z 474 [M–H]−; (+)-HR-ESI-MS m/z 498.1726 [M+Na]+ (Calcd. for C24H29NO9Na 498.1735).

Enzymatic hydrolysis of 1, 3, 5, and 7

Compounds 1, 3, 5, and 7 (2.0–3.0 mg) were separately hydrolyzed in H2O (5 mL) with snailase (5.0 mg, CODE S0100, Beijing Biodee Biotech Co., Ltd., Beijing, China) at 37 °C for 24 h. The hydrolysate was evaporated under reduced pressure, then chromatographed over silica gel eluting with CH3CN/H2O (8:1), to yield sugar. The sugar (0.8–1.2 mg) from the hydrolysates of 1, 3, and 7 gave retention factor (Rf ∼0.41) on TLC (CH2Cl2/MeOH/H2O, 7:3.5:1), +36.5–+40.2 (c 0.06–0.08, H2O), and 1H NMR (D2O, 600 MHz) data, consistent with those of the authentic d-glucose (Figs. S120, S121, S123, and S124). The sugar (0.7 mg) from the hydrolysate of 5 gave retention factor (Rf ∼0.47) on TLC (CH2Cl2/MeOH/H2O, 7:3.5:1), +12.0 (c 0.07, H2O), and 1H NMR (D2O, 600 MHz) data, consistent with those of the authentic d-allose (Figs. S122 and S125).

ECD calculations of 1–10 and aglycones 1a−7a

For details, see Supporting Information. Briefly, conformational analysis was conducted by Monte Carlo searching with the MMFF94 molecular mechanics force field using the Molecular Operating Environment (MOE) software for 1–6 and Gaussian 16 program package for 7, 1a−7a, and 8–10. The lowest-energy conformers having relative energies within 3 kcal/mol were optimized with the Gaussian 09 or Gaussian 16 program. Subsequently, the conformers were re-optimized using DFT at the B3LYP/6-31 + G (d, p) level for 1–7 and at APFD/6-31 + G (d, p) for 8–10 and aglycones 1a−7a, with the solvent effects considered using the dielectric constant of MeOH (ε = 32.6) via conductor-like polarizable continuum model (CPCM). The energies, oscillator strengths, and rotational strengths of the excitations were calculated using the TDDFT methodology at the B3LYP/6–311++G (2d, 2p) level in vacuum for 1–7 and at APFD/6-311 + G (2d, p) for 8–10 and aglycones 1a−7a. The re-optimized conformers showed relative Gibbs free energies (ΔG) under 3 kcal/mol were used for ECD spectra simulation. The ECD spectra were simulated by the Gaussian function (σ = 0.28 eV). To obtain the final spectra, the simulated spectra of the lowest energy conformers were averaged on the basis of the Boltzmann distribution theory and their relative Gibbs free energy (ΔG).