Novel Indane Derivatives with Antioxidant Activity from the Roots of Anisodus tanguticus

Four novel indane derivatives, anisotindans A–D (1–4), were isolated from the roots of Anisodus tanguticus. Their structures were established using comprehensive spectroscopic analyses, and their absolute configurations were determined by electronic circular dichroism (ECD) calculations and single-crystal X-ray diffraction analyses. Anisotindans C and D (3 and 4) are two unusual indenofuran analogs. ABTS•+ and DPPH•+ assays of radical scavenging activity reveal that all compounds (1–4) are active. Specifically, the ABTS•+ assay results show that anisotindan A (1) exhibits the best antioxidant activity with an IC50 value of 15.62 ± 1.85 μM (vitamin C, IC50 = 22.54 ± 5.18 μM).


Introduction
Anisodus tanguticus (Maxim.) Pascher is a folk medicine commonly used in northwest and southwest China [1,2]. The roots of A. tanguticus can relieve pain and spasms, promote blood circulation, remove blood stasis, stop bleeding, and strengthen muscles. Moreover, they are often used in the clinical treatment of pain, ulcer, colitis, gallstone, traumatic injury, catagma, hemorrhage, anesthesia, and motion sickness [3][4][5]. Due to its scarce plant resources, wild A. tanguticus was once listed as a class II protected endangered plant in the List of National Key Protected Wild Plants (the first batch). However, the implementation of long-term environmental protection strategies and the establishment of several planting bases in Ganzi and Aba (Sichuan Province) have significantly improved the plant resources of this species over the past years. Today, A. tanguticus is considered an important economic plant due to its high content of tropane-type alkaloids. Moreover, this plant is currently the natural resource of anisodamine, anisodine, hyoscine, and cuscohygrine.

Structure Elucidation
Anisotindan A (1) was obtained as colorless crystals, and based on HR-ESI-MS analysis, its molecular formula is C13H18O3, with five degrees of unsaturation (m/z 245. . The 13 C NMR and DEPT spectra exhibit carbon resonance signals that can be assigned to the protonated units listed above, as well as to five quaternary carbons (δC 154.4, 144.3, 134.7, 120.4, and 73.7). Comprehensive 2D NMR analysis reveals 1 H-1 H COSY correlations of H2-1/H-2/H2-3 and H-6/H-7, in conjunction with HMBC correlations of H2-1 with C-3, C-3a, C-7, and C-7a, and of H2-3 with C-3a and C-4 in the indane framework of 1 ( Figure 2). Based on the HMBC correlations from H2-1 and H2-3 to C-2′; from H-2 to C-1′, C-2′, and C-3′; from H2-1′ to C-2, C-2′, and C-3′; from H3-3′ to C-2, C-1′, and C-2′; from OH-2′ to C-2, C-2′, and C-3′; and from OH-1′ to C-1′ and C-2′, as well as the 1 H-1 H COSY correlation of H2-1′/OH-1′, a 1,2-dihydroxyisopropyl unit is established at C-2. The HMBC correlations of H3-4′ with C-3a, C-4, and C-5, and of OH-5 with C-4, C-5, and C-6 indicate that a methyl group and a hydroxy group are substituted at C-4 and C-5, respectively. Finally, single-crystal X-ray crystallography analysis [Flack parameter = 0.02 (13)] reveals that the absolute configuration of 1 is 2R,2′S ( Figure 3).  Anisotindan B (2) was obtained as a white powder, and its molecular formula was found to be C13H18O3, the same as 1. As shown in Table 1, the 1 H and 13 C NMR data of 2 are highly similar to those of 1, which suggests that the former might be an epimer of the latter. This is further confirmed by 2D NMR analysis (HSQC, 1 H-1 H COSY, and HMBC). Anisotindan B (2) was obtained as a white powder, and its molecular formula was found to be C 13 H 18 O 3 , the same as 1. As shown in Table 1, the 1 H and 13 C NMR data of 2 are highly similar to those of 1, which suggests that the former might be an epimer of the latter. This is further confirmed by 2D NMR analysis (HSQC, 1 H-1 H COSY, and HMBC). Thus, compound 2 may be identified as (2S,2 S)-2 or its enantiomer. A comparison of the calculated and experimental ECD data ( Figure 4) reveals that the absolute configuration of compound 2 is 2R,2 R. Table 1. 1 H and 13 C NMR data for compounds 1-4 in acetone-d 6 (δ in ppm, J in Hz). Anisotindan C (3) was obtained as colorless crystals, and it has the molecular formula C13H16O3, with six degrees of unsaturation (two fewer protons than compounds 1 and 2), as evidenced by HR-ESI-MS analysis (m/z 243.0996, calcd. for C13H16O3Na, 243.0997). The 1 H and 13 C NMR spectra of compound 3 suggest that this compound is an analog of compound 2. Indeed, comparison of the NMR data corresponding to the two compounds reveals that a methylene group in 2 is replaced by an oxymethine group [δH 5.47 (1H, d, J = 6.3 Hz), δC 88.1] in compound 3. In addition, the signal of the exchangeable hydrogen proton (OH-1′) is observed in the 1 H NMR spectrum of compound 2, but not in the spectrum of 3. This indicates that compound 3 is an ether derivative of compound 2. The 2D NMR spectra of compound 3 confirmed its planar structure, especially the HMBC correlation of H-8b to C-2 ( Figure 2). Moreover, H-3a, H-8b, and OH-3 in 3 have the same orientation, as evidenced by the enhancement of H-3a and OH-3 signals upon the irradiation of H-8b in 1D NOE spectroscopy analysis. The small coupling constant between H-3a and H-8b (J3a,8b = 6.3 Hz) also indicates that H-3a and H-8b are cis oriented [20]. Single-crystal X-ray diffraction analysis [Flack coefficient 0.08 (7)] ( Figure 3) shows that the absolute configuration of 3 is 3R,3aS,8bS.
Anisotindan D (4) is an isomer of compound 3, as indicated by HR-ESI-MS, 1 H, and 13 C NMR data. Comparison of the 1 H and 13 C NMR spectra of the two compounds reveals that the compound 4 is an isomer of compound 3. As shown in Figure 2, the 1 H-1 H COSY and HMBC correlations reveal the presence of the 3,3a,4,8b-tetrahydro-2H-indeno [1,2b]furan skeleton. In addition, the HMBC correlations of OH-3 with C-2, C-3, and C-1′; OH-7 with C-6, C-7, and C-8; and of H3-2′ with C-7, C-8, and C-8a confirm that two hydroxy groups and a methyl group are substituted at C-3, C-7, and C-8, respectively. Based on the NOE correlations of H-8b with H-3a and OH-3, as well as the small coupling constant between H-3a and H-8b (J3a,8b = 6.3 Hz), H-3a, H-8b, and OH-3 in 4 have the same orientation. As shown in Figure 4, the calculated ECD spectrum of (3S,3aR,8bR)-4 is consistent with the experimental spectrum, and thus, the absolute configuration of compound 4 is 3S,3aR,8bR.

Antioxidant Activities
As shown in Table 2, compounds 1, 2, 3, and 4 exhibit ABTS free radical scavenging activities, with IC50 values of 15.62 ± 1.85, 40.92 ± 7.02, 43.93 ± 9.35, and 32.38 ± 6.29 μM, respectively. The activity of compound 1, the most potent scavenger, is even stronger than that of vitamin C (VC, IC50 = 22.54 ± 5.18 μM). Based on the DPPH •+ assay, compound 1 also has an antioxidant effect, with an IC50 value of 68.46 ± 17.34 μM. However, the remaining compounds do not exhibit antioxidant activity, even at concentrations as high as 100 μM. Interestingly, the antioxidant activities of epimers 1 and 2 are quite different, despite the similar structures of the two compounds (differ only in the absolute configura- Anisotindan C (3) was obtained as colorless crystals, and it has the molecular formula C 13 H 16 O 3 , with six degrees of unsaturation (two fewer protons than compounds 1 and 2), as evidenced by HR-ESI-MS analysis (m/z 243.0996, calcd. for C 13 H 16 O 3 Na, 243.0997). The 1 H and 13 C NMR spectra of compound 3 suggest that this compound is an analog of compound 2. Indeed, comparison of the NMR data corresponding to the two compounds reveals that a methylene group in 2 is replaced by an oxymethine group [δ H 5.47 (1H, d, J = 6.3 Hz), δ C 88.1] in compound 3. In addition, the signal of the exchangeable hydrogen proton (OH-1 ) is observed in the 1 H NMR spectrum of compound 2, but not in the spectrum of 3. This indicates that compound 3 is an ether derivative of compound 2. The 2D NMR spectra of compound 3 confirmed its planar structure, especially the HMBC correlation of H-8b to C-2 ( Figure 2). Moreover, H-3a, H-8b, and OH-3 in 3 have the same orientation, as evidenced by the enhancement of H-3a and OH-3 signals upon the irradiation of H-8b in 1D NOE spectroscopy analysis. The small coupling constant between H-3a and H-8b (J 3a,8b = 6.3 Hz) also indicates that H-3a and H-8b are cis oriented [20]. Single-crystal X-ray diffraction analysis [Flack coefficient 0.08 (7)] (Figure 3) shows that the absolute configuration of 3 is 3R,3aS,8bS.
Anisotindan D (4) is an isomer of compound 3, as indicated by HR-ESI-MS, 1 H, and 13 C NMR data. Comparison of the 1 H and 13 C NMR spectra of the two compounds reveals that the compound 4 is an isomer of compound 3. As shown in Figure 2, the 1 H-1 H COSY and HMBC correlations reveal the presence of the 3,3a,4,8b-tetrahydro-2H-indeno [1,2-b]furan skeleton. In addition, the HMBC correlations of OH-3 with C-2, C-3, and C-1 ; OH-7 with C-6, C-7, and C-8; and of H 3 -2 with C-7, C-8, and C-8a confirm that two hydroxy groups and a methyl group are substituted at C-3, C-7, and C-8, respectively. Based on the NOE correlations of H-8b with H-3a and OH-3, as well as the small coupling constant between H-3a and H-8b (J 3a,8b = 6.3 Hz), H-3a, H-8b, and OH-3 in 4 have the same orientation. As shown in Figure 4, the calculated ECD spectrum of (3S,3aR,8bR)-4 is consistent with the experimental spectrum, and thus, the absolute configuration of compound 4 is 3S,3aR,8bR.

Antioxidant Activities
As shown in Table 2, compounds 1, 2, 3, and 4 exhibit ABTS free radical scavenging activities, with IC 50 values of 15.62 ± 1.85, 40.92 ± 7.02, 43.93 ± 9.35, and 32.38 ± 6.29 µM, respectively. The activity of compound 1, the most potent scavenger, is even stronger than that of vitamin C (VC, IC 50 = 22.54 ± 5.18 µM). Based on the DPPH •+ assay, compound 1 also has an antioxidant effect, with an IC 50 value of 68.46 ± 17.34 µM. However, the remaining compounds do not exhibit antioxidant activity, even at concentrations as high as 100 µM. Interestingly, the antioxidant activities of epimers 1 and 2 are quite different, despite the similar structures of the two compounds (differ only in the absolute configuration of C-2 ).

Discussion
Indanes are a class of small organic molecules with a benzocyclopentane skeleton that can be substituted with 4-aminobenzylidene, gallic acid, piperidine, cyclohexadienone, or nucleobase to form indane analogs with diverse structures and significant activity [21]. Many reports are available in the literature regarding the synthesis of indane analogs via Friedel-Crafts-type, Michael-type, and Heck-type cyclization reactions [21,22]. However, reports on naturally occurring indanes are scarce. Notably, A. tanguticus seems to contain several indane derivatives, including rare polyhydroxy indenofurans.
Oxidative stress, a condition induced by the excessive generation of free radicals, is considered to be an important cause of human disease and aging. Indeed, the accumulation of reactive species in cells can lead to DNA damage, as well as protein and lipid degrada-tion, which affects normal physiological functions [23]. Therefore, antioxidants play an important role in the prevention and treatment of diseases, and their development has attracted increasing attention [24][25][26]. Knowing that polyhydroxy compounds are potent antioxidants, and that they are widely present in plants [27,28], this study investigates the polyhydroxy (two or three hydroxy groups) indane derivatives in A. tanguticus. In total, four novel compounds are isolated, and their antioxidant activities are evaluated using ABTS •+ and DPPH •+ assays. The obtained results reveal that all four indane derivatives (1-4) identified herein have good free radial scavenger activities, with 1 being the most active compound. Comparison of the structures of compounds 1-4 suggests that the strong activity of compound 1 may be attributed to the OH-1 substitution and the configuration of C-2 . However, ABTS •+ and DPPH •+ assays are just simplified methods for estimating antioxidant activity, which do not reflect the actual antioxidant activity [29]. Unfortunately, further studies on the antioxidant activity of compounds 1-4 could not be carried out due to their limited sample quantities.
Indene analogs generally have significant neuroprotective effects [21]. Indeed, donepezil, a second-generation AChE inhibitor, is used to treat Alzheimer's disease due to its significant cholinesterase inhibitory activity. In light of this information, as well as the strong AChE inhibitory effect of the A. tanguticus extract [30], the AChE inhibitory activity of the compounds isolated herein is also analyzed in this study using the modified Ellman method [31] and donepezil as a positive control. However, none of the compounds show inhibitory activity against AChE, even at concentrations as high as 100 µM.

General Experimental Procedures
Optical rotation was measured using a Rudolph Autopol I automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). ECD and IR spectra were recorded on an Applied Photophysics Chirascan CD spectrometer (Applied Photophysics Ltd., Leatherhead, UK) and an Agilent Cary 600 FT-IR microscope instrument (Agilent Technologies Inc., Santa Clara, CA, USA), respectively. X-ray crystallographic analyses were performed on a Bruker D8 Quest diffractometer (Bruker Corporation, Billerica, MA, USA). Meanwhile, NMR and HR-ESI-MS spectra were acquired on a Bruker Avance NEO 600 or a Bruker Avance NEO 700 spectrometer (solvent peaks used as the references) and a Waters Synapt G2 HDMS (Waters Corporation, Milford, MA, USA) or a Bruker timsTOF MS instrument, respectively. The melting points were measured on a BÜCHI M-565 melting point apparatus (BÜCHI Labortechnik AG, Flawil, Switzerland). MPLC separations were carried out using a BÜCHI Pure C-805 instrument. HPLC separations were performed on an Agilent 1220 instrument with a Welch Ultimate XB-C 18 column (10 × 250 mm 2 , 5 µm) or a Daicel Chiralpak AD-H column (4.6 × 250 mm 2 , 5 µm). TLC was carried out using silica gel

X-ray Crystallographic Data of Compounds 1 and 3
Crystals of 1 and 3 were obtained from MeOH. Intensity data were collected on a Bruker D8 Quest diffractometer equipped with an APEX-II CCD using Cu Kα radiation. Crystallographic data for the reported structures have been deposited at the Cambridge Crystallographic Data Centre (CCDC). Copies of the data can be acquired free of charge from CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).
Crystal data for 1:

ECD Calculation
The details of ECD calculation of compounds 2 and 4 are shown in Texts S1 and S2, Figures S1 and S2, and Tables S1 and S2, Supplementary Material.

Antioxidant Activity
The ABTS and DPPH free radical scavenging assays were used to estimate the antioxidant activities of the isolated compounds.

ABTS •+ Assay
The free radical scavenging capacity of compounds 1-4 was measured using the ABTS •+ decoloration method. First, 20 of mL ABTS •+ solution (7 mM) and 20 mL of potassium persulfate solution (2.45 mM) were prepared with ultra-pure water. The prepared solutions were mixed and stored in the dark at 23 • C for 16 h to obtain ABTS •+ stock solution. Two milliliters of this solution were subsequently diluted (20 times) with 95% ethanol solution to obtain the ABTS •+ working solution with an absorbance of 0.70 ± 0.02 at 734 nm. Thereafter, compound solutions (80 µL) of varying concentrations were mixed with 400 µL of the ABTS •+ working solution and added into 96-well plates, with 150 µL in each well. After 6 min incubation in the dark at 23 • C, the absorbance (OD) of each sample was measured at 734 nm. Using vitamin C as the positive control and 95% ethanol solution as the blank control, the scavenging rate of ABTS free radicals was calculated according to the following equation: (%) = (1 − A s /A c ) × 100%, where A s and A c are the average OD values of the drug group and the blank control group, respectively. All tests were performed in triplicate.

DPPH •+ Assay
DPPH •+ solution (0.1 mM) was prepared with 95% ethanol, and 250 µL of the solution was mixed with compound solutions (250 µL) of varying concentrations. The mixtures were transferred into 96-well plates, with 150 µL in each well. After the reaction at 25 • C for 30 min, the absorbance (OD) was measured at 517 nm, and the DPPH free radical scavenging rate was calculated according to the following equation: (%) = (1 − A s /A c ) × 100%, where A s and A c are the average OD values of the drug group and the blank control group (95% ethanol solution), respectively. All tests were performed in triplicate.

Conclusions
In this study, four new indanes (1)(2)(3)(4), anisotindans A-D, were extracted from the roots of A. tanguticus. Their structures were identified by NMR and single-crystal X-ray crystallography analyses, as well as ECD calculations. Meanwhile, their antioxidant activity was estimated using ABTS and DPPH free radical scavenging assays. The obtained results show that anisotindans C and D (3 and 4) are two unusual indenofuran analogs, and that compounds 1-4 exhibit significant antioxidant capacity, especially compound 1, the ABTS radical scavenging capacity of which is greater than that of vitamin C. The preliminary structure-activity relationship analysis conducted herein suggests that the variation in antioxidant activity of indanes may be attributed to differences in OH-1 substitution and C-2 configuration.  Table S1. Energy analysis for the conformers of (2S,2 S)-2; Table S2. Energy analysis for the conformers of (3R,3aS,8bS)-4; Text S1 and S2 are the ECD calculation of compounds 2 and 4 [32][33][34][35][36].