Alismanin A, a Triterpenoid with a C34 Skeleton from Alisma orientale

Oct 10, 2017 - Alismanin A, a Triterpenoid with a C34 Skeleton from Alisma orientale as a Natural Agonist of Human Pregnane X Receptor. Chao Wang†âˆ...
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Letter Cite This: Org. Lett. 2017, 19, 5645-5648

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Alismanin A, a Triterpenoid with a C34 Skeleton from Alisma orientale as a Natural Agonist of Human Pregnane X Receptor Chao Wang,†,∥ Xiao-Kui Huo,†,∥ Zhi-Lin Luan,†,∥ Fei Cao,‡ Xiang-Ge Tian,§ Xin-Yu Zhao,† Cheng-Peng Sun,*,† Lei Feng,† Jing Ning,† Bao-Jing Zhang,† and Xiao-Chi Ma*,†,§ †

College of Pharmacy, College (Institute) of Integrative Medicine, and §Basic Medical College, Dalian Medical University, Dalian 116044, China ‡ Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences, Hebei University, Baoding 071002, China S Supporting Information *

ABSTRACT: Alismanin A (1), a novel aromatic triterpenoid with a C34 skeleton, was isolated from Alisma orientale together with a rearranged nor-triterpenoid (2) and a 13,17-seco triterpenoid (3). Their structures were determined by a combination of HRESIMS, 2D NMR spectra, electronic circular dichroism (ECD), theoretical calculations, and X-ray diffraction analysis. Compounds 1 and 2 displayed significant activation effects on pregnane X receptor (PXR) at 10 nM. A plausible biosynthetic pathway for 1−3 is also discussed. Alisma orientale (Sam.) Juzep. (Alismataceae), known as “Zexie” in China,1 is a traditional Chinese medicine that has been widely used to treat a variety of illnesses,2 including obesity, diuresis, diabetes, hyperlipidemia, and detumescence. Various bioactive protostane-type triterpenoids3−7 and guaiane-type and eudesmane-type sesquiterpenoids,7 such as alisols A−O, alismanols A−D, and orientalols A−C, have been isolated from the genus Alisma. Some of them displayed a wide variety of pharmacological effects, including hypolipidemic,8,9 antiproliferative,10,11 hepatoprotective,12 and immuno-enhancing activities,13,14 as well as inhibition of human carboxylesterase 23,15 and pancreatic lipase.7 In our continuing endeavor to discover structurally diverse and biologically interesting metabolites from this genus, three novel protostane-type triterpenoids, alismanins A−C (1−3), were isolated from the rhizomes of A. orientale. Their structures were established on the basis of 1D and 2D NMR, electronic circular dichroism (ECD), TD DFT calculations of ECD spectra,16,17 DFT GIAO calculations of 13CNMR chemical shift,18,19 and X-ray single crystal diffraction analysis. Alismanin A (1) possesses an unusual C34 skeleton with four six-membered and one fivemembered rings. Alismanin B (2) is reported for the first time as a rearranged nor-protostane triterpenoid, and alismanin C (3) is a novel 13,17-seco protostane (Figure 1). Compounds 1−3 were assayed for their activation effects on pregnane X receptor (PXR). Compounds 1 and 2 displayed significant agonistic effects on PXR at a concentration of 10 nM. Herein, © 2017 American Chemical Society

Figure 1. Chemical constituents from A. orientale.

the isolation, structure elucidation, bioassay, and plausible biogenetic pathways for 1−3 are described. Alismanin A (1) was obtained as a white amorphous powder. Its molecular formula was determined as C34H46O3 by the positive HRESIMS (m/z 520.3778 [M + NH4]+, calcd for C 34H 50 NO 3+ 520.3791), indicating 12 degrees of unsaturation. The 1H NMR data of 1 (Table 1) showed six methyls at δH 1.17 (3H, s), 1.08 (3H, s), 1.05 (3H, s), 1.04 (3H, s), 1.03 (3H, d, J = 7.0 Hz), and 1.02 (3H, s), an olefinic proton at δH 6.72 (1H, dd, J = 8.1, 6.9 Hz), five aromatic protons at δH 7.40 × 2 (1H, tt, J = 7.0, 1.5 Hz), 7.35 (1H, tt, J = 7.0, 1.5 Hz), and 7.13 × 2 (1H, dt, J = 7.0, 1.5 Hz), and an aldehydic proton at δH 9.53 (1H, s). The 13C NMR data of 1 (Table 1) exhibited 34 carbon resonances, including an aldehyde carbonyl carbon (δC 195.6), six aromatic carbons (δC 134.2, 130.8 × 2, 129.3 × 2, and Received: September 8, 2017 Published: October 10, 2017 5645

DOI: 10.1021/acs.orglett.7b02738 Org. Lett. 2017, 19, 5645−5648

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Organic Letters Table 1. 1H (600 MHz, MeOH-d4) and MHz, MeOH-d4) Data of 1

13

C NMR (150

Figure 3. Selected NOESY correlations of 1−3.

NMR chemical shift calculation by quantum chemistry methods represents a powerful strategy for chemical structure interpretation.16−19 To confirm the framework of 1, the gaugeindependent atomic orbital (GIAO) based 13C NMR chemical shifts calculation was performed at the B3LYP/6-311G+(2d,p) level of theory. The individual deviations, |Δδ|, between the predicted and experimental 13C chemical shifts for 1 were less than 4.9 ppm (Table S1), and the correlation coefficient (R2) was 0.9983 (Figure 4), indicating that the δC of 1 matched

129.1), four olefinic carbons (δC 157.8, 145.8, 139.3, and 136.2), and one oxygenated carbon (δC 70.6). In the HMBC spectrum of 1, the long-range correlations from Ha-2 to C-1 and C-3, Hb-2 to C-3, H-5 to C-4, C-28, and C-29, H-9 to C-8, C-10, C-18, and C-19, Ha-12 to C-9, C-11, C-13, C-14, and C-17, H-20 to C-16, C-17, and C-21, CH3-18 to C-7, C-8, and C-9, CH3-19 to C-1, C-5, C-10, CH3-29 to C-3 and C-4, and CH3-30 to C-8, C-14, and C-15 constructed rings ABCD of the triterpenoid skeleton in combination with COSY cross-peaks of Ha-1 with Ha-2 and Hb-2, H-5 with Ha-6 and Hb-6, Ha-6 with Ha-7 and Hb-7, H-9 with H-11, H-11 with Ha-12 and Hb-12, and Ha-15 with Ha-16 and Hb-16 (Figure 2), requiring that 1 should be a

Figure 4. Calculated 13C NMR chemical shifts of 1. (A) Regression analysis of experimental versus calculated 13C NMR chemical shifts of 1; linear fitting is shown as a line. (B) Comparison of calculated 13 C chemical shifts with experimental shifts.

the calculated δC very well, which confirmed the framework of 1. In addition, the ECD spectrum of 1, calculated at the B3LYP/6-311G+(2d,p) level, agreed with the experimental ECD spectrum (Figure 5A), allowing an obvious assignment

Figure 2. Selected HMBC and COSY correlations of 1−3. Figure 5. Calculated and experimental ECD spectra of 1 (A) and 3 (B) at the B3LYP/6-311G+(2d,p) level.

3

protostane. Comparison of the NMR data of 1 with those of alisol A6 suggested that their difference was the side chain at C-17. The COSY correlations between H-20 and Ha-22 with Hb-22, Ha-22 and H-23, H-26 and H-31, H-31 and H-32, H32 and H-33, and H-33 and H-34, and long-range correlations of H-23 with C-25 and C-27, H-26 with C-24 and C-34, H-31 with C-25 and C-33, and H-32 with C-26 and C-34 in the HMBC spectrum indicated the presence of an α,β-unsaturated aldehyde and a benzenic ring (ring E) located at C-24 in the side chain of C-17. The relative configuration of 1 was determined through an NOESY experiment which showed correlations from CH3-18 to H-5 and H-11, CH3-19 to CH329, CH3-30 to H-9, H-23 to H-27, and H-34 to Ha-22 and Hb-22 (Figure 3), requiring that H-9, OH-11 CH3-19, CH329, and CH3-30 were all β-oriented, H-5 and CH3-18 were both α-oriented, and the Δ23,24 double bond was E-configured. The relative configuration of C-20 was deduced to be R* on the basis of the biosynthetic background of protostanes.20

of the absolute structure to be 5R,8S,9S,10S,11S,14R,20R, which is supported by X-ray structures of the protostane skeleton 21,22 and compound 2 (Figure 6), and the biosynthetic background.20 Alismanin B (2) was assigned the molecular formula C29H48O3 according to HRESIMS (m/z 467.3492 [M + Na]+, calcd for C29H48NaO3+ 467.3501) data. The 1H NMR data of 2 (Table S2) displayed the presence of eight methyls observed at δH 1.18 (3H, s), 1.08 (3H, s), 1.06 × 2 (3H, s), 1.05 (3H, s), 1.02 (3H, d, J = 7.0 Hz), 0.87 (3H, d, J = 6.8 Hz), and 0.86 (3H, d, J = 6.8 Hz), and two oxygenated methines observed at δH 3.81 (1H, m) and 3.09 (1H, m). The 13 C NMR data of 2 (Table S2) showed 29 carbon signals, indicating that 2 was a nor-protostane.4 Compared with 1, their significant differences were the side chain at C-17. The COSY correlations of H-20 with Ha-22 and Hb-22, Ha-22 5646

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suggesting that H-23 and H-24 were β- and α-oriented, respectively, in conjunction with the coupling constant (J23,24 = 3.5 Hz) and the configuration of the biosynthetic precursor alismaketone C 23-acetate.23 The ECD spectrum of 3, calculated at the B3LYP/6-311G+(2d,p) level, fitted well with its experimental result (Figure 5B), which indicated that its absolute configuration was 5R,8S,9S,10S,14R,17S,20R,23S,24S, which is supported by the configuration of alismaketoneC 23-acetate.23 Biosynthetically, alismanins A−C could be produced from the biosynthetic precursor of triterpenoids, squalene, as shown in Scheme 1. The oxidation of alisol A, a major constituent of A. orientale, could give the 24,25,26,27-tetra-nor-protostane i similar to 24,25,26,27-tetra-nor-protostane alisolide from A. orientale.24 Condensation25 and decarboxylation of intermediate i with phenylpyruvic acid transformed from L-phenylalanine could yield compound 1. Intermediate vii was afforded by a similar biosynthetic procedure of alisol O,22 and then its decarboxylation led to the production of compound 2. Hydration and condensation of alismaketone C 23-acetate isolated from A. orientale would yield compound 3.23 Cholestatic liver disorders comprise a series of hepatobiliary diseases characterized by impaired hepatocellular secretion of bile, resulting in accumulation of cholesterol, bilirubin, and bile acids. PXR can regulate a suite of genes involved in the metabolism, transport, and elimination of their substances,25,26 such as CYP3A4; therefore, it is regarded as an important target to treat cholestatic liver disorders.27,28 Compounds 1−3 were assayed for activation effects on PXR. Compounds 1 and 2 displayed significant agonistic effects on PXR (Figure 7) at a concentration of 10 nM. The interaction mechanisms of PXR with 1 and 2 were also investigated by molecular docking, respectively. As shown in Figure S1, 1 and 2 could be well docked into the ligand binding domain of PXR, while hydroxy (OH-11) and aldehyde groups of 1 and two hydroxy (OH-11 and OH-23) groups of 2 had interactions with Met-243 and Gln-285, respectively. Their lowest binding free energies were −7.86 and −7.18 kcal/mol, respectively, indicating that 1 and 2 had high affinity for PXR. In summary, we discovered three new triterpenoids from A. orientale, including a novel protostane possessing an unusual C34 skeleton with an aromatic ring (ring E), a nor-protostane, and a 13,17-seco protostane. Compounds 1 and 2 showed

Figure 6. ORTEP drawing of compound 2.

with H-23, H-23 with H-24, and H-24 with CH3-26 and CH327 (Figure 2) in conjunction with HMBC cross-peaks of CH3-21 with C-17 and C-20, and H-20 with C-16 and C-17 established the linkage of C-20−(C-21)−C-22−C-23−C-24− (C-26)−C-27 at C-17. In the NOESY spectrum of 2, the cross-peak of H-11 with CH3-18 (Figure 3) suggested a βorientation of OH-11. The X-ray single crystal diffraction analysis of 2 (Figure 6) assigned its absolute configuration as 5R,8S,9S,10S,11S,14R,20R,23S. The molecular formula of alismanin C (3) was defined as C32H48O6 on the basis of HRESIMS (m/z 529.3525 [M + H]+, calcd for C32H49O6+ 529.3529) data. The 1H and 13C NMR data of 3 (Table S2) indicated that 3 was an unusual 13,17-seco protostane.23 The HMBC correlations of Ha-1 with C-3, Ha-2 with C-1 and C-3, H-5 with C-4, C-6, C-7, C-10, C-29, and C-30, H-9 with C-8, C-10, C-11, and C-12, H-11 with C-8 and C-13, H-12 with C-9, CH3-18 with C-7, C-8, C9, and C-14, CH3-19 with C-1, C-5, C-9, and C-10, CH3-28 and CH3-29 with C-3 and C-4, and CH3-30 with C-8, C-13, and C-14 (Figure 2) established rings A-C of 3 with an 11-en3,13-dione unit. In the HMBC spectrum, long-range correlations from Ha-15 to C-17, Hb-15 to C-14, Hb-16 to C-17, CH3-21 to C-17 and C-22, H-23 to OAc-23, C-24, and C-25, H-24 to C-17, C-22, C-26, and C-27, CH3-26 and CH327 to C-24 and C-25, and OAc-CH3 to OAc-23 (Figure 2) established the side chain at C-14. The NOESY spectrum of 3 displayed a cross-peak of H-20 with H-23 (Figure 3),

Scheme 1. Plausible Biosynthetic Pathways for Compounds 1−3

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Figure 7. Compounds 1−3 (10 nM) activate PXR to modulate PXR target gene CYP3A4 transactivation in HepG2 cells. Rifampicin (Rif, 10 μM) as a positive drug. *p < 0.05 versus control group, **p < 0.01 versus control group.

significant activation effects on PXR at a concentration of 10 nM.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b02738. Detailed experimental procedures, physical−chemical properties, 1D and 2D NMR, HRESIMS, and ECD data (except for 2) of 1−3, X-ray single-crystal diffraction data of 2, and computational data of 1 and 3 (PDF) X-ray data for 2 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Fei Cao: 0000-0002-5676-3176 Cheng-Peng Sun: 0000-0001-6801-3439 Xiao-Chi Ma: 0000-0003-4397-537X Author Contributions ∥

C.W., X.-K.H., and Z.-L.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (No. 81703679, 81622047, 81503201, and 81473334), Dalian Outstanding Youth Science and Technology Talent (2015J12JH201), Distinguished Professor of Liaoning Province, Liaoning BaiQianWan Talents Program, and the Natural Science Foundation of College (Institute) of Integrative Medicine of Dalian Medical University.

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REFERENCES

(1) Pharmacopoeia Commission of People’s Republic of China. Pharmacopoeia of the People’s Republic of China (Part 1); Chinese Medical Science and Technology Press: Beijing, 2010; pp 212. (2) Yu, Z. L.; Peng, Y. L.; Wang, C.; Cao, F.; Huo, X. K.; Tian, X. G.; Feng, L.; Ning, J.; Zhang, B. J.; Sun, C. P.; Ma, X. C. New J. Chem. 2017, DOI: 10.1039/C7NJ01806A. 5648

DOI: 10.1021/acs.orglett.7b02738 Org. Lett. 2017, 19, 5645−5648