Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
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Triligustilides A and B: Two Pairs of Phthalide Trimers from Angelica sinensis with a Complex Polycyclic Skeleton and Their Activities Jian Zou,†,§ Guo-Dong Chen,†,§ Huan Zhao,†,‡ Ying Huang,† Xiang Luo,† Wei Xu,† Rong-Rong He,† Dan Hu,† Xin-Sheng Yao,† and Hao Gao*,† †
Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy/Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, and ‡College of Traditional Chinese Medicine, Jinan University, Guangzhou 510632, People’s Republic of China S Supporting Information *
ABSTRACT: Two pairs of enantiomeric phthalide trimers [(−)/(+) triligustilides A (1a/1b) and (−)/(+) triligustilides B (2a/2b)] with complex polycyclic skeletons simultaneously possessing bridged, fused, and spiro ring systems were isolated from Angelica sinensis, together with two pairs of new phthalide dimers. The biogenetic pathways of new phthalides were proposed, and their bioactivities were also evaluated. This is the first time optically pure polymeric phthalides have been obtained from racemates, and their absolute configurations are reported.
Angelica sinensis (Apiaceae) is a perennial herb mainly cultivated in Gansu, Yunnan, and Sichuan Provinces in China.1 Its dried root was documented in the ancient Chinese materia medica book “Shen Nong’s Herbal Classic” in the Qin Han dynasty (300 A.D.)1 and has been used as a famous traditional Chinese medicine (TCM) as Dang-Gui (Angelicae Sinensis Radix). According to classical works, it was commonly used for the treatments of gynecological disorders (e.g., menstrual disorders, amenorrhea, dysmenorrhea, anemia, premenstrual syndrome, and menopause).2 Modern pharmacology reports have demonstrated that it possesses a series of bioactivities, including antiplatelet aggregation, anti-inflammatory, anticancer, and neuroprotective effects.3 It is not only one of the most famous medicinal plants but also one of the most popular edible plants, which was widely marketed as a health food for women’s care in Asia and as a dietary supplement in Europe and America.4 A large number of phytochemical investigations have been carried out on A. sinensis, and over 150 compounds have been found from it, including phthalides, phenylpropanoids, terpenoids, alkynes, alkaloids, and other constituents.5 Among them, phthalides (such as Z-ligustilide, Z-butylidenephthalide, and nbutylphthalide) are the major and characteristic constituents in Dang-Gui.6 Modern pharmacology reports have shown that phthalides exhibit diverse bioactivities, including antiasthma, anticonvulsion, inhibition of platelet aggregation, enhancement of blood flow volume, etc.7 In particular, n-butylphthalide (NBP) has been approved as a modern drug for the treatment of ischemic stroke by the State Food and Drug Administration of China in 2005.8 Moreover, phthalides exhibit abundant structural diversity, which not only originates from the derivatization of monomeric phthalides such as hydroxylation, glycosidation, and esterification6 but also depends on the dimerization ([2 + 2] or [4 + 2] cycloaddition) with diverse linkage styles forming a number of complex polycyclic skeletons with multichiral centers.9 Phthalides have attracted much attention due to their © XXXX American Chemical Society
various bioactivities and complicated chemical structures. To date, the reported polymeric phthalides deriving from achiral monomers exist in a racemic manner. However, chiral separation of these racemic polymeric phthalides has not been carried out yet. Thus, their corresponding optically pure polymeric phthalides have not been obtained. Their absolute configurations remain unsolved. To search for interesting phthalides from nature, establish the absolute configurations of polymeric phthalides, and explore their bioactivities, a chemical investigation on Dang-Gui was carried out. The dried root of A. sinensis were extracted with EtOH−H2O (95:5 v/v). The extract was separated by continuously different column chromatography leading to the isolation of two pairs of novel enantiomeric trimeric phthalides [(−) triligustilide A (1a)/(+) triligustilide A (1b), and (−) triligustilide B (2a)/(+) triligustilide B (2b)], two pairs of new related enantiomeric dimeric phthalides [(+) tokinolide A (3a)/ (−) tokinolide A (3b), and (−) riligustilide (4a)/(+) riligustilide (4b)], and the precursor [Z-ligustilide (5)] (Figure 1). Among them, the trimeric phthalides (1a, 1b, 2a, and 2b) possess new and complex polycyclic skeleton, which was composed of three Z-ligustilide (5) units in an unique linkage style (2−4′/7−3′, 3″−8′/4′′−9′). Herein, we describe the isolation, structure elucidation, and bioassays (including anti-inflammatory, antiplatelet aggregation, cytotoxic and antimicrobial activities) of all phthalides (1−5). Moreover, the plausible biogenetic pathways of 1−4 are proposed. Triligustilide A (1) was isolated as colorless crystals. The molecular formula of 1 was established as C36H42O6 (16 degrees of unsaturation) from its HR-ESI-MS (m/z 593.2892 [M + Na]+, calcd for C36H42O6Na 593.2879). The 13C NMR spectrum Received: January 3, 2018
A
DOI: 10.1021/acs.orglett.8b00017 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
structure was established as shown in Figure 2. The geometries of the double bonds of Δ8 and Δ8″ in 1 were assigned as Z configurations by the NOESY correlations between H-9 and Ha6, between H-9″ and Ha-6′′, and between H-9′′ and Hb-6′′. The assignments of all proton and carbon resonances are provided in Table S1. In the bicyclo[4.2.0]octane system, the relative configurations of H-3″ and H-4″ should be fixed as 3″R*,4″S*. Furthermore, the NOESY correlations between H-3 and H-4′, between Ha-6 and H-3′, between Hb-6 and H-3′, between Ha-10 and Hb-11′, between Hb-10 and Hb-11′, between Hb-6′ and H9′, and between Hb-10′ and H-4″ indicated that the relative configuration of 1 was 2S*,7R*,3′R*,4′S*,8′S*,9′S*,3″R*,4″S* (Figure 3). The single-crystal X-ray diffraction experiment (Figure 4) confirmed the above deduction.
Figure 3. Key NOESY correlations of 1 and 2.
Figure 1. Chemical structures of 1−4.
showed 36 carbon signals. Combined with the DEPT-135 experiment, these carbons can be categorized as three carbonyl carbons (δC 175.7, 171.6, 167.0), 10 aromatic or olefinic carbons, three sp3 quaternary carbons [including one oxygenated (δC 92.2)], five sp3 methine carbons, 12 sp3 methylene carbons, and three methyl carbons. In the 1H NMR spectrum of 1, the characteristic protons for three methyl groups [δH 0.83 (t, J = 7.3 Hz), 0.88 (t, J = 7.2 Hz), and 0.95 (t, J = 7.4 Hz)] and four olefinic protons [δH 4.66 (dd, J = 8.4, 6.5 Hz), 5.33 (t, J = 7.9 Hz), 5.92 (br d, J = 9.7 Hz), and 6.06 (ddd, J = 9.7, 4.9, 3.6 Hz)] were observed. All of the proton resonances were assigned to relevant carbon atoms through the HSQC experiment. The 1H−1H COSY data of 1 revealed the presence of five subunits as shown in bold red lines in Figure 2. The key HMBC correlations (Figure 2) revealed the partial structure of 1. On the basis of the molecular formula information, the degree of unsaturation, and the above analyses of 1H−1H COSY and HMBC data, the planar
Figure 4. X-ray crystallographic analyses of 1 and 2.
Triligustilide B (2) was isolated as colorless crystals. The molecular formula of 2 was established as C36H42O6 (16 degrees of unsaturation) from its HR-ESI-MS (m/z 593.2875 [M + Na]+, calcd for C36H42O6Na 593.2879). The NMR data of 2 are very similar to those of 1. Further detailed NMR analyses involving 1 H−1H COSY and HMBC established the same planar structure as that of 1. The geometries of the double bonds of Δ8 and Δ8″ in 2 were assigned as Z configurations using the NOESY correlations between H-9 and Ha-6, between H-9″ and H-6′′a, and between H-9′′ and H-6′′b. The assignments of all proton and carbon resonances are provided in Table S2. In the bicyclo[4.2.0]octane system, the relative configurations of H-3″ and H-4″ should be 3″S*,4″R*. Furthermore, the NOESY correlations between H-3 and H-4′, between Ha-6 and H-3′, between Hb-6 and H-3′, between Ha-10 and H2-11′, between
Figure 2. Key 1H−1H COSY and HMBC correlations of 1. B
DOI: 10.1021/acs.orglett.8b00017 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters Hb-10 and H2-11′, between H2-6′ and H-9′, between H2-6′ and H-3″, between H-9′ and H-3′′, and between Hb-10′ and Hb-5′′ revealed the relative configuration of 2 to be 2S*,7R*,3′R*,4′S*,8′S*,9′S*,3″S*,4″R* (Figure 3). The above result was confirmed by the single-crystal X-ray diffraction experiment (Figure 4). Compound 3 was isolated as a yellow oil. The molecular formula of 3 was established as C24H28O4 (11 degrees of unsaturation) from its HR-ESI-MS (m/z 381.2060 [M + H]+, calcd for C24H29O4 381.2066). Compound 3 was identified as tokinolide A by comparison of the NMR data with literature values.10 Detailed analyses of the 1D and 2D NMR data of 3 confirmed the above deduction, and the relative configuration of 3 was first determined as 2S*,7R*,3′R*,4′S* (Table S3). Compound 4 was isolated as colorless crystals. The molecular formula of 4 was established as C24H28O4 (11 degrees of unsaturation) from its HR-ESI-MS (m/z 381.2067 [M + H]+, calcd for C24H29O4 381.2066). Compound 4 was identified as riligustilide by comparison of the NMR data with literature values.9b The single-crystal X-ray diffraction experiment confirmed the above deduction, and unambiguously determined the relative configuration of 4 as 3S*,4R*,8′S*,9′S* (Table S4). The specific optical rotations of 1−4 were close to zero, indicating that 1−4 might be mixtures of enantiomers. This deduction was confirmed by the observations of 1−4 in the chiral HPLC analyses. These four pairs of enantiomers [(−)-1a/ (+)-1b, (−)-2a/(+)-2b, (+)-3a/(−)-3b, and (−)-4a/(+)-4b] were obtained using a Phenomenex Amylose-2 column (5 μm, 250 × 4.6 mm) (Figure S1). Moreover, the quantum chemical calculations of the electronic circular dichroic (ECD) spectra were used to determine the absolute configurations of 1a and 1b. Since the flexible side chains that are far away from the chiral carbons in 1 had an insignificant effect on the ECD spectrum,11 the simplified structures of (2S,7R,3′R,4′S,8′S,9′S,3″R,4″S)-1′ and (2R,7S,3′S,4′R,8′R,9′R,3″S,4″R)-1′ (Figure S6) were used for the ECD calculations. The predicted ECD curves of (2S,7R,3′R,4′S,8′S,9′S,3″R,4″S)-1′ and (2R,7S,3′S,4′R,8′R,9′R,3″S,4″R)-1′ were almost identical to the experimental ones of 1a and 1b, respectively. Therefore, the absolute configurations of 1a and 1b were established as (2S,7R,3′R,4′S,8′S,9′S,3″R,4″S)-1a and (2R,7S,3′S,4′R,8′R,9′R,3″S,4″R)-1b, respectively (Figure 1). By the same procedure of ECD calculations as that of 1 (Figure S8), the absolute configurations of 2a and 2b were determined as (2S,7R,3′R,4′S,8′S,9′S,3″S,4″R)-2a and (2R,7S,3′S,4′R,8′R,9′R,3″R,4″S)-2b, respectively (Figure 1). The absolute configurations of 3a and 3b, 4a and 4b were established as (2S,7R,3′R,4′S)-3a and (2R,7S,3′S,4′R)-3b, (3S,4R,8′S,9′S,)-4a and (3R,4S,8′R,9′R)-4b by the same procedure as that of 1, respectively (Figures S10 and S12). According to classical effects of Dang-Gui and the established bioassay models in our group, bioassays (including antiinflammatory, antiplatelet aggregation, cytotoxic, and antimicrobial activities) of all phthalides have been carried out. In the anti-inflammatory assay in LPS-stimulated RAW264.7 macrophages, compounds 1−5 exhibited different levels of antiinflammatory effects against the production of the proinflammatory cytokines (TNF-α, IL-6). Among them, compounds 1, 4, and 5 showed inhibition in a dose-dependent manner (Figure 5). All compounds were also evaluated for their inhibition in vitro against platelet aggregation in rat plasma induced with arachidonic acid (AA). Compound 5 exhibited significant antiplatelet aggregation effects, and the results
Figure 5. Effects of compounds 1, 4, and 5 and on the inhibition of TNF-α (a) and IL-6 (b). The data represent the mean ± SD of three experiments. *p < 0.1, **p < 0.01, ***p < 0.001 compared with the LPS group; #p < 0.001 compared with the control group.
indicated that polymerization would weaken the activity of inhibition of platelet aggregation (Table 1). Table 1. Inhibition of Platelet Aggregation in Vitro Induced by AA of compounds 1−5 compd
inhibit rate (100 μM)
compd
inhibit rate (100 μM)
1 2 3
31.4 ± 5.0 27.1 ± 5.2 41.7 ± 5.9
4 5 aspirin
45.1 ± 4.6 89.9 ± 0.2 95.4 ± 0.2
Furthermore, compounds 1−5 were also evaluated for cytotoxicities against four human tumor cell line (SW480, PANC-1, MCF-7, and HepG2) and antimicrobial assays against two bacteria (Staphylococcus aureus 209P, Escherichia coli ATCC0111) and two fungi (Canidia albicans FIM709, Aspergillus niger R330). None of the compounds possessed cytotoxic and antimicrobial activities (Tables S9 and S11). As the major and characteristic constituents in Dang-Gui, phthalides not only show a variety of bioactivities but also possess abundant structural diversity.6c In particular, a large number of novel dimeric phthalides are isolated from Dang-Gui, such as E232, gelispiroloide, and sinaspirolide.9 In this present research, two pairs of novel enantiomeric trimeric phthalides 1−2 were isolated. These were composed of three Z-ligustilide (5) units in an unprecedented linkage style (2−4′/7−3′, 3″−8′/4′′−9′) forming complex polycyclic skeleton simultaneously possessing bridged-ring, fused-ring, and spiro-ring systems. In addition, two pairs of new biosynthetically related enantiomeric dimeric phthalides 3−4 were also obtained. Furthermore, bioactivities screening showed that major monomeric phthalide Z-ligustilide (5) had significant inhibition of platelet aggregation, and all compounds displayed different levels of anti-inflammatory effects, which indicatied that monomeric and polymeric phthalides are the beneficial constituents for the classical effects of Dang-Gui. The plausible biogenetic pathways of 1−4 are proposed as shown in Scheme 1. Dimeric phthalides (tokinolide A (3) and riligustilide (4)) with two different linkages (type A, 2−4′/7−3′ for 3; type B, 3−8′/4−9′ for 4) derive from two molecules of Zligustilide (5) through [2 + 2] cycloaddition,12 whereas triligustilides A (1) and B (2) with a new linkage style (type (A + B): 2−4′/7−3′, 3″−8′/4′′−9′) originate from three molecules of Z-ligustilide (5) through [2 + 2] /[2 + 2] cycloaddition. The structural shapes of polymeric phthalides are intriguingly similar to two or three flying Apsaras getting together. For the aesthetic representation of 1−2, see Figure S14. C
DOI: 10.1021/acs.orglett.8b00017 Org. Lett. XXXX, XXX, XXX−XXX
Organic Letters
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ACKNOWLEDGMENTS This work was financially supported by grants from the National Natural Science Foundation of China (81422054 and 3171101305), Chang Jiang Scholars Program (Hao Gao, 2017) from the Ministry of Education of China, the Guangdong Natural Science Funds for Distinguished Youn g Scholar (S2013050014287), Guangdong Special Support Program (2016TX03R280), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (Hao Gao, 2014), K. C. Wong Education Foundation (Hao Gao, 2016), and the high-performance computing platform of Jinan University. We are grateful to Prof. M. Banwell at the University of Australian National for the biogenetic pathway advices. Assistance with the proper usage of scientific English was provided by Dr. L. J. Sparvero at the University of Pittsburgh.
Scheme 1. Plausible Biosynthetic Pathways of Compounds 1− 4
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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.8b00017. NMR data assignments of 1−4, structural characterization of 1−4, general experimental procedures, plant materials, extraction and isolation, chiral separation of compounds 1−4, X-ray crystallographic data of 1, 2, and 4, quantum chemical ECD calculations of 1′−4′, cytotoxicity assay of 1−5, antiplatelet aggregation assay of 1−5, antiinflammatory assay of 1−5, antimicrobial assay of 1−5, the aesthetic figure of 1−2, and the 1D and 2D spectra of 1−4 (PDF) Accession Codes
CCDC 1556339−1556341 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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REFERENCES
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To date, many kinds of linkages of polymeric phthalides have been found, and all of them are considered to be related to [2 + 2] or [4 + 2] cycloaddition. All polymeric phthalides that are polymerized from achiral monomers are racemic. However, their corresponding optically pure polymeric phthalides have not been previously obtained, and their absolute configurations were unsolved. Our present investigation is the first time that the optically pure polymeric phthalides are obtained from racemates, and their absolute configurations are established.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Xin-Sheng Yao: 0000-0003-1603-4873 Hao Gao: 0000-0003-1178-0121 Author Contributions §
J.Z. and G.-D.C. contributed equally.
Notes
The authors declare no competing financial interest. D
DOI: 10.1021/acs.orglett.8b00017 Org. Lett. XXXX, XXX, XXX−XXX
