1. Introduction

Acorus tatarinowii Schott (Araceae), widely distributed in the northeast India and the south of China, is a famous traditional Chinese medicine (TCM), the rhizome of which is used as a remedy for various central nervous system related diseases[1]. Previous phytochemical studies indicated that the major constituents of this plant were phenylpropanoids [2, 3] and sesquiterpenoids [4, 5]. Recently, the phenylpropanoids isolated from this plant were reported to exert a broad spectrum of biological activities, such as antiepileptic and neuroprotective effects [6-8], bronchitis [8], anticardiovascular diseases [9].

In the course of our continuing efforts to discover bioactive natural products from A. tatarinowii, a series of norlignans, lignans, and neolignans, including many pairs of enantiomers, were previously reported [10, 11]. In our current study, further investigation on chemical constituents of the rhizomes of A. tatarinowii resulted in the isolation of two pairs of chlorine-containing phenylpropanoid enantiomers (1a/1b and 2a/2b). Herein, we report the extraction and isolation, planar structural elucidation, and the absolute configuration determination of the two pairs of enantiomers, as well as evaluation for their cytotoxic and antioxidant activities.

2. Results and discussion

Two pairs of chlorine-containing phenylpropanoid enantiomers (Fig. 1) were successfully isolated from the EtOAc extract of the air-dried rhizomes of A. tatarinowii through a series of chromatographic separations, including chiral HPLC.

Compound 1 (1a/1b) was isolated as colorless block crystals (MeOH), possessing the molecular formula C₁₃H₁₈ClO₅, as determined from NMR data and an HR-ESIMS [M+H]⁺ ion at m/z 313.0828 (calcd. for C₁₃H₁₉ClO₅, 313.0819) along with characteristic isotopic peaks with the ratio of 3:1 at m/z 313.0828/315.0798 suggesting the presence of a chlorine atom. Its IR spectrum indicated the presence of hydroxyl functionality (3452 cm^-1) and aromatic ring (1611 and 1510 cm^-1). The ¹³C NMR and DEPT spectra of 1 (Table 1) displayed 13 carbons, corresponding to six aromatic carbons (δ_C 119.1, 153.6, 98.9, 151.1, 144.6, and 113.8), two oxygenated methine (δ_C 78.9 and 75.7), one methylene (δ_C 46.8) and four methoxyl groups (δ_C 56.7, 56.7, 57.4 and 57.4). The ¹H NMR data (Table 1) showed signals of two aromatic protons [δ_H 6.69 (s, 1H, H-3) and 6.96 (s, 1H, H-6)] attributed to a 1, 2, 4, 5-tetrasubstituted phenyl group, two oxygenated methine protons [δ_H 4.68 (d, 1H, J = 5.1 Hz, H-1') and 3.82 (m, 1H, H-2')], one methylene group [δ_H 3.54 (dd, 1H, J = 11.2, 4.8 Hz, H-3'a) and 3.44 (dd, 1H, J = 11.2, 7.0 Hz, H-3'b)], and four methoxy protons [δ_H 3.23, 3.79, 3.82, and 3.86]. The data mentioned above suggested that the planar structure of 1 was very similar to acoraminol A [12], with the only difference that the C-2' methyl group was halogenated to be CH₂Cl. This conclusion was supported by the key HMBC correlations of H-1' to C-1, C-2, C-6, C-2' and C-3'; H-2' to C-1', C-2', and C-1; OMe-2 to C-2, OMe-4 to C-4, OMe-5 to C-5; and OMe-1' to C-1' and the ¹H-¹H COSY correlations of H-1'/H-2'/H-3'. The chemical shifts of H-3' (3.54 and 3.44) and C-3' (46.8) revealed the chlorine to be on C-3'. Thus, compound 1 was determined to be 3'-chloro-1'-methoxy-1-(2, 4, 5-trimethoxyphenyl)propan-2'-ol.

The stereochemistry configuration of 1 was determined by a single X-ray diffraction analysis coupled with the modified Mosher's method. detailed single-crystal X-ray diffraction analysis with Cu Kα irradiation revealed that compound 1 was a pair of racemic mixture with the space group P_-1 [13], which was supported by its negligible optical activity and an ECD spectrum devoid of Cotton effects. Subsequently, the chiral HPLC resolution of 1 yielded the enantiomers (-)-1a and (+)-1b with ratio of approximately 1:1 (Fig. 2a), showing specific rotations of opposite sign (1a: [α]_D²⁰ -43; 1b: [α]_D²⁰ +43) and antipodal ECD curves [1a: 234 (Δe-1.38) and 280 (Δε-0.28) nm; 1b: 232 (Δε +1.60) and 286 (Δε +0.21) nm] (Fig. 2c). In order to determine the absolute configuration of the optically pure compound 1b, a modified Mosher's experiment was performed. According to the MTPA shielding/deshielding effects[14, 15], the Δδ_H (S—R) values revealed a 2'S configuration for (+)-1b (Fig. 3), Thus, the absolute configuration of 1b was determined as 1'S, 2'R on the basis of 1', 2'-erythro configuration elucidated by single-crystal X-ray diffraction experiment (Fig. 4), and that of 1a was assigned as 1'R, 2'S.

Compound 2 (2a/2b) was obtained as colorless block crystals (MeOH). The molecular formula of 2 was established as the same as 1, based on the NMR data and HRESIMS data showing a [M + H]⁺ ion at m/z 313.0830 (calcd. for C₁₃H₁₉ClO₅, 313.0819) along with characteristic isotopic peaks with the ratio of 3:1 at m/z 313.0830/ 315.0795. A comparison of the NMR data of 2 with those of 1 (Table 1) showed that the only difference between 1 and 2 was the small change in chemical shifts from C-1' to C-3' [δ_H 4.68 (d, 1H, J = 5.1 Hz, H-1'), 3.82 (m, 1H, H-2'), 3.54 (dd, 1H, J = 11.2, 4.8 Hz, H-3'a), and 3.44 (dd, 1H, J = 11.2, 7.0 Hz, H-3'b); δC 78.9 (C-1'), 75.7 (C-2'), and 46.8 (C-3') in 1; d H 4.67 (d, 1H, J = 5.5 Hz, H-1'), 3.94 (m, 1H, H-2'), 3.65 (dd, 1H, J = 11.3, 2.6 Hz, H-3'a), and 3.54 (dd, 1H, J = 11.3, 7.8 Hz, H-3'b); d C 79.8 (C-1'), 75.3 (C-2'), and 47.5 (C-3') in 2]. These findings, combined with the 2D NMR data and the large coupling constant (J_{1', 2'} = 5.5) revealed that 2 was a stereoisomer of 1 with a threo configuration (1'R^*, 2R'^*).

Similarly, a single X-ray diffraction experiment with Mo Kα irradiation was performed to determine the absolute configuration of 2 (Fig. 4). It is interesting that the crystals of 2 had the space group C2/C [13], indicating its racemic nature, as further confirmed by [α]_D²⁰= 0. The subsequent chiral purification of 2 using a Daicel IC column in the same manner as 1, afforded the enantiomers (-)-2a and (+)-2b (Fig. 2b), displaying mirror image ECD curves [2a: 232 (Δε +0.9), 290 (Δε +0.41) nm; 1b: 232 (Δε-0.7), 282 (Δε-0.21) nm.] (Fig. 2d) and opposite specific rotations [2a:[α]_D²⁰ -34; 2b: [α]_D²⁰ +34]. Finally, the absolute configuration of 2b was determined as 1'S, 2'S on the basis of modified Mosher's method (Fig. 3) and that of 2a was assigned as 1'R, 2'R.

Compounds 1a/1b and 2a/2b were evaluated for their antioxidant activities and cytotoxic activities using the DPPH assay and MTT method, respectively. The results indicated that none of the compounds showed signally antioxidant activities at the concentration of 50.0 μg/mL, while compounds 1a and 2a exhibited weak inhibitory activities against the proliferation of the HL-60, SMMC-7721, A-549, and MCF-7 cell lines with the IC₅₀ values ranging from 23.16 μmol/L to 38.14 μmol/L (Table 2), respectively.

3. Conclusion

Compounds 1a/1b and 2a/2b, four optical isomers with two chiral centers, were obtained simultaneously from the same plant, which were rare reported in natural products. Additionally, this is the first report for natural products containing chlorine atom from the genus Acorus. Their racemic natures were discovered by X-ray diffraction and absolute configurations were determined by modified Mosher's method.

4. Experimental 4.1. General

Optical rotations were obtained on a Rudolph Research Analytical Autopol Ⅳ polarimeter equipped with a 1 dm microcell and an odium lamp (589 nm). Melting points were measured on a Beijing Tech X-5 micro-melting point apparatus. 1D and 2D NMR data were recorded on a Bruker AM-400. UV spectra were obtained on a Varian Cary 50. FT-IR spectra were determined using a Brukera Vertex 70 instrument. The ECD spectra were obtained using a JASCO J-810 spectrometer. HRESIMS were acquired in the positiveion mode on a Thermo Scientific LTQ-XL-Orbitrap mass spectrometer. The crystallographic experiments were performed on a Bruker APEX DUO diffractometer equipped with an APEX Ⅱ CCD, using Cu/Mo Kα radiation (λ = 1.54178 Å). Semi-preparative HPLC was performed on a Dionex quaternary system using an RP C₁₈ column (5 μm, 10 mm × 250 mm, SunFire^TM Prep C₁₈) and a chiralpak IC column (5 μm, 4.6 mm × 250 mm, Daicel Chiral Technologies Co., Ltd. China). MPLC was carried out with EZ Plus chromatography system (Lisure Science Co., Ltd, China). Column chromatography (CC) were performed on Sephadex LH-20 (40-70μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), silica gel (200-300 mesh; Qingdao Marine Chemical, Inc., Qingdao, China), and octadecylsilyl (ODS, 50 mm, YMC Co. Ltd., Japan). TLC (thin-layer chromatography) was carried out using glass-precoated silica gel GF254 (Merck, Germany).

4.2. Plant material

The rhizomes of A. tatarinowii were collected in Qichun County, Hubei Province, China, in September 2012, and were authenticated by Prof. J. Wang from School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology. A voucher specimen (No. 2012-0913A) has been deposited at the Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology.

4.3. Extraction and isolation

The dried rhizomes of A. tatarinowii (40.0 kg) were extracted with 95% aqueous MeOH (4 ×100 L) three times at room temperature. The solvent was concentrated under vacuum to afford the crude extract (2.6 kg), which was suspended in H₂O and successively partitioned with petroleum ether, EtOAc, and n-BuOH, respectively. The EtOAc partition was subjected to a silica gel eluting with petroleum ether-acetone (20:1-1:1, v/v), to give five fractions (Fr. 1-5).

Fr. 3 (30 g) was then chromatographed over a silica gel eluting with a gradient of (PE-EtOAc 15:1→5:1) to give four subfractions (Fr.3.1-Fr.3.4). Fr.3.2 was subjected to a Sephadex LH-20 (MeOH) column to afford three additional fractions (Fr.3.2.1 and Fr.3.2.3). Fr.3.2.2 was then chromatographed over an ODS gel column eluting with MeOH-₂O (30:50→50:30, v/v) to yield the major fraction Fr.3.2.2.1. Compounds 1 (17 mg) and 2 (7 mg) were successfully obtained from Fr.3.2.2.1 by semi-preparative HPLC eluting with 30% CH₃CN/₂O. Finally, compounds 1 was further purified by chiral HPLC using a Daicel IC column eluting with EtOH-n-hexane (20:80) to yield 1a (t_R 9.5 min) and 1b (t_R 13.2 min). Compounds 2a (t_R 12.5 min) and 2b (t_R 16.5 min) were successfully separated in the same manner.

Compound 1: Colorless block crystals (MeOH), mp 135-136 ℃; [α]_D²⁰0 (c 1.0, MeOH); UV (MeOH) λ_max (logε) 207 (4.51), 233 (4.10), and 291 (3.85) nm; IR (KBr) ν_max 3452, 2934, 2831, 1611, 1510, 1464, 1440, 1399, 1317, 1263, 1205, 1116, 1032, 856, and 762 cm^-1; ¹H NMR (methanol-d₄, 400 MHz) data, see Table 1; ¹³C NMR (methanol-d₄, 100 MHz) data, see Table 1; (+)-HR-EIMS m/z 313.0828 [M+H]⁺ (calcd. for C₁₃H₁₉ClO₅, 313.0819).

Compound 1a: White amorphous powder; [α]_D²⁰-43 (c 0.1, MeOH); ECD (MeOH) 234 (Δε -1.38) and 280 (Δε -0.28).

Compound 1b: White amorphous powder; [α]_D²⁰+43 (c 0.1, MeOH); ECD (MeOH) 232 (Δε +1.60) and 286 (Δε +0.21).

Compound 2: Colorless block crystals (MeOH), mp 138-139 ℃; [α]_D²⁰0 (c 1.0, MeOH); UV (MeOH) λ_max (loge) 208 (4.51), 232 (4.10), and 291 (3.85) nm; IR (KBr) ν_max 3443, 2932, 2830, 1610, 1509, 1463, 1398, 1273, 1205, 1108, 1032, 859, and 764 cm^-1; ¹H NMR (methanol-d₄, 400 MHz) data, see Table 1; ¹³C NMR (methanol-d₄, 100 MHz) data, see Table 1; (+)-HR-EIMS m/z 313.0830 [M+H]⁺ (calcd. for C₁₃H₁₉ClO₅, 313.0819).

Compound 2a: White amorphous powder; [α]_D²⁰-34 (c 0.1, MeOH); ECD (MeOH) 232 (Δε +0.9) and 290 (Δε +0.14).

Compound 2b: White amorphous powder; [α]_D²⁰ +34 (c 0.1, MeOH); ECD (MeOH) 232 (Δε -0.7) and 282 (Δε -0.21).

(S)-MTPA derivative of 1b: ¹H NMR (CDCl₃, 100 MHz) δ_H 7.80-7.30 (5H, overlap, aromatic protons), 6.53(s, 1H, H-2), 6.91 (s, 1H, H-5), 4.83 (d, 1H, J = 7.0 H-1'), 3.54 (dd, 1 H, J = 12.0, 7.0 Hz, H-3'a), 3.51 (dd, 1H, J = 12.0, 3.3 Hz, H-3'b), HR-ESIMS m/z 529.1205 [M +Na]⁺ (calcd. for C₂₃H₂₆ClF₃NaO₇, 529.1217).

(R)-MTPA derivative of 1b: ¹H NMR (CDCl₃, 100 MHz) δ_H 7.80-7.25 (5H, overlap, aromatic protons), 6.51 (s, 1H, H-2), 6.80 (s, 1H, H-5), 4.67 (d, 1H, J = 6.8 H-1'), 3.64 (dd, 1H, J = 12.3, 8.3 Hz, H-3'a), 3.56 (dd, 1H, J = 12.3, 3.7 Hz, H-3'b), HR-ESIMS m/z 529.1208 [M +Na]⁺ (calcd. for C₂₃H₂₆ClF₃NaO₇, 529.1217).

(S)-MTPA derivative of 2b: ¹H NMR (CDCl₃, 100 MHz) δ_H 7.80-7.25 (5H, overlap, aromatic protons), 6.37(s, 1H, H-2), 6.68 (s, 1H, H-5), 4.74 (d, 1H, J = 6.2 H-1'), 3.82 (dd, 1H, J = 12.2, 2.9 Hz, H-3'a), 3.79 (dd, 1H, J = 12.2, 6.9 Hz, H-3'b), HR-ESIMS m/z 529.1213[M +Na]⁺ (calcd. for C₂₃H₂₆ClF₃NaO₇, 529.1217).

(R)-MTPA derivative of 2b: ¹H NMR (CDCl₃, 100 MHz) δ_H 7.80-7.25 (5H, overlap, aromatic protons), 6.49 (s, 1H, H-2), 6.85 (s, 1H, H-5), 4.88 (d, 1H, J = 6.2 H-1'), 3.82 (dd, 1H, J = 12.2, 2.8 Hz, H-3'a), 3.79 (dd, 1H, J = 12.2, 7.2 Hz, H-3'b), HR-ESIMS m/z 529.1220 [M +Na]⁺ (calcd. for C₂₃H₂₆ClF₃NaO₇, 529.1217).

Crystallographic data of compound (±)-1: C₁₃H₁₉ClO₅, M = 290.73, triclinic, a = 7.7678(8) Å, b = 8.7957(10) Å, c = 11.5257 (13) Å, α = 108.221(5)°, β = 99.424(4)°, γ = 99.406(4)°, V = 718.27 (14) ų, T = 298(2) K, space group P-1, Z = 2, m(CuKα) = 2.490 mm^-1, 9866 reflections measured, 2313 independent reflections (R_int = 0.1054). The final R₁ values were 0.0477 (I > 2σ(I)). The final wR(F²) values were 0.1332 (I > 2σ(I)). The final R₁ values were 0.1128 (all data). The final wR(F²) values were 0.1457 (all data). The goodness of fit on F² was 1.052.

Crystallographic data of compound (±)-2: C₁₃H₁₉ClO₅, M = 290.73, monoclinic, a = 8.9749(18) Å, b = 14.277(3) Å, c = 22.701(5) Å, α = 90.00°, β = 93.421(3)°, γ = 90.00°, V = 2903.6 (10) ų, T = 298(2) K, space group C2/c, Z = 8, m(MoKα) = 0.276 mm^-1, 9029 reflections measured, 2271 independent reflections (Rint = 0.0345). The final R₁ values were 0.0481 (I > 2σ(I)). The final wR(F²) values were 0.1358 (I > 2σ(I)). The final R₁ values were 0.0597 (all data). The final wR(F²) values were 0.1538 (all data). The goodness of fit on F² was 1.089.

Crystallographic data for the structures of (±)-1 and (±)-2 have been deposited in the Cambridge Crystallographic Data Centre [CCDC numbers: 1028830 (1) and 1470927 (2)]. Copies of these data can be obtained free of charge from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: +44 1223 336 033; or e mail: deposit@ccdc cam ac uk

HR-ESIMS, NMR, IR and UV spectra of the compounds can be found in Supporting information.

4.4. Preparation of the R-and S-MTPA esters of 1b and 2b

Compound 1b (0.8 mg) was dissolved in 2.0 mL of anhydrous CH₂Cl₂. DMAP (dimethylaminopyridine) 30 mg, triethylamine 5 μL, and (S)-MTPA chloride (25 μL) were added in sequence. The mixture was stirred for 2.5 h at room temperature. Then, the reaction was quenched by the addition of 1 mL of aqueous CH₃OH. Finally, the reaction mixture was evaporated under reduced pressure and the residue was passed through a small silica gel column eluting with petroleum ether-acetone (100:10, v/v) to afford the (R)-MTPA ester of 1b (1.2 mg). The (S)-MTPA ester of 1b (1.1 mg) was prepared in the same manner. The MTPA esters of 2b were prepared in the same manner.

4.5. Antioxidant assay

Compounds 1a/1b and 2a/2b were evaluated for their abilities to scavenge DPPH radical using the DPPH assay [16]. Briefly, reaction mixtures included the compounds in EtOH (with 5% DMSO) (final concentrations of 3.125, 6.25, 12.5, 25, and 50 μg/mL) and DPPH (final concentrations of 100 μmol/L). After30 minutes of reaction in the dark at room temperature, the optical density (OD) was measured at 515 nm using a microplate reader. Trolox (vitamin E) was used as positive control. IC₅₀ values were calculated using Reed

4.6. Cytotoxic assay

Five human cancer cell lines (HL-60, SW-480, MCF-7, SMMC-7721, BEAS-2 B and A-549) and one noncancerous cell line (Beas-2B) were used in the antiproliferative assay employing the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] method [17, 18]. Briefly, 100 μL of adherent cells (1 ×10⁵) were dispensed into 96-well tissue culture plates and incubated in humidified atmosphere at 37 ℃ with 5% CO₂ for 24 h. Each tumor cell line was treated with compounds 1a/1b and 2a/2b at various concentrations (0.0625, 0.32, 1.6, 8, and 40 μmol/L). After incubation for 48 h, 15 μL of MTT solution (5 mg/mL) was added to each well and further incubated for 4 h. The formazan crystals produced were solubilized in DMSO. The optical density (OD) was recorded at 595 nm. The experiments were carried out in triplicate and the IC₅₀ values were yielded from the mean OD values and drug concentration curves. Both Taxol (Sigma) and DDP (cis-platin, Sigma) were used as positive controls.

Acknowledgments

The authors would like to thank the Analytical and Testing Center at Huazhong University of Science and Technology for assistance in conducting ECD and IR analyses. This work was financially supported by the Program for New Century Excellent Talents in University, State Education Ministry of China (No. NCET-2008-0224), the National Natural Science Foundation of China (Nos. 31370372, 81573316, 31570361, 31200258).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.03.024.