Letter pubs.acs.org/OrgLett
Artemisians A−D, Diseco-guaianolide Involved Heterodimeric [4 + 2] Adducts from Artemisia argyi Gui-Min Xue,† Chao Han,† Chen Chen,† Ling-Nan Li,† Xiao-Bing Wang,† Ming-Hua Yang,† Yu-Cheng Gu,‡ Jian-Guang Luo,*,† and Ling-Yi Kong*,† †
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China ‡ Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom S Supporting Information *
ABSTRACT: Artemisians A−D (1−4), the first examples of [4 + 2] Diels−Alder type adducts presumably biosynthesized from a rare 1, 10-4, 5-diseco-guaianolide and a guaianolide diene, along with their possible precursor 5, were isolated from the traditional Chinese medicine Artemisia argyi. The structures of 1−4 were elucidated by extensive spectroscopic analyses and calculated electronic circular dichroism. Compound 2, with an IC50 value of 3.21 μM, exhibited significant antiproliferative activity via apoptosis induction and G2/M arrest in MDA-MB-468 cells.
I
mg, 0.004‰ yield; 2, 14.0 mg, 0.007‰ yield; 3, 5.2 mg, 0.0026‰ yield; 4, 7.6 mg, 0.004‰ yield; 5, 10.0 mg, 0.005‰ yield) were isolated from the leaves of A. argyi (2.0 kg) with the help of LC-MS [(Supporting Information, (SI)]. These four compounds are not common α-methylene (Δ11−13) involved DA-type adducts and represent unprecedented structures with a 1, 10-4, 5-disecoguaianolide unit. Herein, the isolation, structural elucidation, plausible biosynthetic pathway, and antiproliferative activity of compounds 1−4 are described. Artemisian A (1), a colorless gum, afforded the molecular formula C30H36O8 by (+)-HRESIMS ion at m/z 542.2741 ([M +NH4]+, calcd, 542.2748), implying 13 indices of hydrogen deficiency. The IR spectrum of 1 showed characteristic absorption bands at 1764 and 1705 cm−1 corresponding to C O stretching of ester and ketone moieties, respectively, and a band at 1642 cm−1 attributed to CC. The 1H NMR spectrum of 1 exhibited signals of four methyls (1.47, s; 1.73, s; 2.20, s; 2.32, s), two pairs of terminal double bonds [(5.46, d, J = 3.1 Hz; 6.18, d, J = 3.5 Hz); (5.67, d, J = 1.8 Hz; 6.34, d, J = 2.2 Hz)], and two oxymethine protons (4.65, dt, J = 6.5, 4.0 Hz; 4.69, d, J = 10.0 Hz) (Table S1). The 13C NMR data, interpreted with the help of an HSQC experiment, revealed 30 carbon resonances including 4 methyls, 8 methylenes, 7 methines, and 11 quaternary carbons. Among these resonances, three ketone carbonyls (δC 206.9, 207.5, 210.1), two ester carboxyls (δC169.6, 169.7), and six olefinic carbons were clearly observed. These described NMR characteristic signals and MS data suggested that 1 should be a
n recent years, dimeric sesquiterpenoids (DSs) have attracted considerable attention owing to their unique structures and widely ranging bioactivities, such as cytotoxic, antiplasmodial, and anti-inflammatory activities.1−3 As is well-known, Diels−Alder (DA), hetero-DA, and radical reactions, among others, provide prevalent ways of biosynthesizing DSs by connecting two monomeric units.4 Investigations show that in the Asteraceae family no less than 150 DSs have been identified and a large amount of them were considered to be produced via regular DA cycloaddition reaction through linkage of one monomer with a cyclopentadiene and another in the presence of an electrondeficient carbon−carbon double bond to generate a cyclohexene.5 Guaianolide sesquiterpenoids, usually with a 5/7/5 tricyclic system, occur as possible precursors, which contain both cyclopentadiene and α-methylene-γ-lactone (αMγL) functionalities in forms ready for a DA cycloaddition.6 Artemisia argyi, a famous traditional Chinese medicine (Aiye in Chinese), is commonly used for treatment of inflammation, diarrhea, eczema, tuberculosis, and menstruation-related symptoms.7 Phytochemical investigations have revealed that A. argyi contains an abundance of electron-deficient Δ11,13-didehydro guaianolide sesquiterpenoids,8 suggesting their possibility of polymerizing into more complex structures and prompting us to research deeper into the diverse biological components. Generally, guaianolide dimers are frequently isolated from Artemisia genus, including A. argy,9 but seco-guaianolide involved DA dimers have seldom been identified. In our continuing efforts to search for novel bioactive polymeric terpenes,10 two pairs of hetero head-tohead/tail sesquiterpenoid dimers, artemisians A−D (1−4; 1, 8.0 © 2017 American Chemical Society
Received: August 28, 2017 Published: September 28, 2017 5410
DOI: 10.1021/acs.orglett.7b02681 Org. Lett. 2017, 19, 5410−5413
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Organic Letters sesquiterpene dimer (Figure 1). The characteristic α-methylene protons in the 1H NMR spectrum and upfield ester carboxyl
guaianolide. Furthermore, the combined spin-coupling systems of H-3′−H-2′−H-2′−H-2−H-3 in the 1H−1H COSY spectrum with HMBC correlations of H-2′/C-1, C-2, and C-3, of H-2/C-1, C-1′, and C-2′, of H-3/C-4, C-4′, and C-5′, and of H-15′/C-3 and C-5′ demonstrated that unit I was fused with unit II through C-2− C-2′ and C-3−C-4′ to form a new five-membered ring D (Figure 2). Thus, the planar structure of 1 is confirmed as in Figure 1. The relative configuration of 1 was determined by interpretation of the observed ROESY correlations, as well as coupling constants. The ROESY cross-peak of H-7′/H-14′ suggested they were situated at the α-side of the ring B′. H-6′ was assigned to be β-configured owing to the large coupling constant between H-6′ and H-7′ (J6′, 7′ = 10.0 Hz).11 The five-membered ring D fused with ring A′ to form a rigid bicyclo[2.2.1]heptene core by sharing a CH2 bridge between C-2′ and C-4′. The successive ROESY correlations of H-14′/H-2′, 14′/H-3′, H-3′/ H-15′, and H-3′/H-3 revealed H-3, H-2′, H-3′, and H-15′ to be on the cofacial position of the cyclohexene (C-1′−C-2′−C-2−C3−C-4′−C-5′), while the obvious ROESY correlations of H-6′/ H-9′b, H-9′b/H-2 indicated the opposite side of H-2. Besides, the ROESY correlations of H-5/H-7, H-6/H-8 were indicative of the trans-relationship of H-6 and H-7, which was further defined by the coupling constant J6, 7 = 4.0 Hz.11b Thus, the structure of 1 should be deduced as shown in Figure 1. Artemisian B (2) possessed the same molecular formula as 1 according to their consistent molecular ion peaks in HRESIMS. Detailed analysis of the 1D and 2D NMR data (SI, Figure S7) of 2 indicated that its structure was also established to consist of I and II units, but in a different linking mode. The HMBC correlations from H-2′ to C-3, C-4, and C-10′; H-3 to C-1′ and C-4 indicated the linkage between C-3 and C-2′, while the cross-peaks of H-2/ C-4′, C-5′, C-1, and C-5; H-15′/C-2 suggested the connectivity between C-2 and C-4′ (Figure 3). This was further consolidated
Figure 1. Structures of compounds 1−4.
resonances occurred at δC 169.6, 169.7 suggesting the presence of two αMγL functionalities.11 Starting from a typical proton resonance at δH 4.69 (d, J = 10.0 Hz, H-6′), the HMBC corrections of H-6′/C-1′, C-8′, C-11′, and C-12′; H-7′/C-5′, C-9′, C-11′, and C-12′; H-13′/C-7′ and C-12′; H-14′/C-1′ and C-9′; and H-15′/C-3′ and C-5′, coupled with the successive 1H−1H COSY correlations of H-6′−H-7′−H-8′−H9′ suggested unit II was a 6,7-trans-fused guaianolide with an αMγL moiety and double olefinic carbons between C-1′ and C-5′ (Figure 1).11a The remaining signals for unit I were speculated on the basis of the HMBC and 1H−1H COSY correlations (Figure 2). The HMBC correlations from H-6 and H-7 to C-11 and C-12
Figure 3. Selected NMR correlations of 2.
by the ROESY correlations of H-2′ with H-14′ and H-15 and of H15′ with H-2 and H-5 (Figure 3). Subsequently, the ROESY correlations of H7′/H-14′, H-14′/H-2′, H-14′/H-3′, H-3′/H-2, H-5/H-7, H-6/H-8, H-6′/H-9′b, and H-9′b/H-2, together with the large coupling constant between H-6′ and H-7′, suggested the stereochemistry of 2 was deduced as shown in Figure 3. The HRESIMS of artemisians C (3) and D (4) displayed the same molecular ion peaks as 1 and 2. Comprehensive analysis of the 1D and 2D NMR spectra gave rise to the presence of a disecoguaianolide for unit I and a complete guaianolide scaffold similar to unit II in 3 and 4. Notably, the downfield methyl chemical shift at δH 2.03 (H-15′) and simultaneous HMBC correlations of H15′ with olefinic carbons C-4′ (δC 137.8) and C-5′ (δC 138.0) suggested the translocation of a double bond from Δ1′‑5′ in II to Δ4′‑5′ as shown in unit III (Figure 1). The following HMBC correlations from H-2 to C-5, C-5′, and C-10′, H-3 to C-3′ and C4′, and H-3′ to C-4 (Figure 4) confirmed the linkage of unit I and III for 3 via C−C single bonds of C-1′−C-2 and C-3′−C-3,
Figure 2. Key NMR correlations for 1.
and from H-13 to C-7 and C-12 indicated the presence of an additional αMγL moiety in unit I. The 1H−1H COSY correlations of H-7−H-8−H-9 and HMBC correlations of H-8/C-6, C-10 and C-11, H-9/C-7, and H-14/C-10 (Figure 2) suggested that a 2butanone moiety (SI, Figure S1] was located at C-7 of ring C. The other side chain, according to the 1H−1H COSY correlations of H-5−H-6−H-7, H-2−H-3, and HMBC correlations of H-2, H-3, and H-5 with C-1 and H-2, H-3, and H-15 with C-4 (δC 210.1) (Figure 2), thus was deduced to be a hexane-2, 5-dione moiety (SI, Figure S1). The aforementioned NMR data allowed unit I in 1 to be a rare C-1/C-10 and C-4/C-5 simultaneously cracked 5411
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Organic Letters Scheme 1. Plausible Biosynthetic Pathway of 1−4
Figure 4. Key HMBC and ROESY correlations of 3 and 4.
respectively. Besides, the results were further supported by ROESY cross-peaks of H-2/H-14′, H-15/H-3′ for 3 and ROESY correlations of H-3/H-14′, H-5/H-2, and H-5/H-3′ for 4 (Figure 4). In the ROESY spectrum of 3, the consecutive correlations of H-7′/H-9′a, H-9′a/H-2′, H-2′/H-2, H-2′/H-3′, H-2/H-14′, and H-3/15′, together with trans-orientation of H-6′ and H-7′ (J6′, 7′ = 10.0 Hz), demonstrated the α-side of H-7′ and H-14′ in ring B′, βdirection of H-3, and α-orientation of H-2 and the CH2 bridge in cyclohexene (C-1′−C-2−C-3−C-3′−C-4′−C-5′). The trans configuration of H-6/H7 was deduced by the same ROESY correlations as 1. The stereochemistry of 4 was finally defined by comparison with 3 and comprehensive analysis of ROESY correlations data. Thus, the structures for 3 and 4 are defined as shown in Figure 1. Finally, it is concluded that artemisians A−D (1−4) are two pairs of sesquiterpenoid dimers linked in head-to-head (1 and 3) and head-to-tail (2 and 4) formation by units I/II and I/III, respectively. The flexible structure in unit I of 1−4 resulted in failing to receive a single crystal for X-ray analysis. Hitherto, biosynthetic logic-based elucidation for absolute configuration determinations is a very beneficial approach in natural products.12 As shown in Scheme 1, co-occurrence of 1−4, along with the arglabin (5), of which the 7S stereostructure was previously identified by an X-ray crystal isolated from A. glabella,13 inspired us to propose a biosynthetic pathway for 1−4. Initially, 5 would undergo hydrolysis and elimination reactions to generate the intermediate IV (Δ1(2), 3(4)), whose analogues had been isolated from Asteraceae plants.14 Then, IV (Δ1(2), 3(4)) would undergo a sigmatropic rearrangement to afford dienes II (Δ1(2), 4(5)) and III (Δ1(5), 3(4)).15 Besides, the [4 + 2] DA reaction of IV with singlet oxygen produced a endoperoxide,16 and further ring cleavage of the endoperoxide between C-1 and C-10 gave possible biogenetic precursor 6, which was previously isolated from A. gilvescens.11c The cycloreversion reaction of 6 between C-4 and C-5 yielded the 1, 10-4, 5-diseco-guaianolide for unit I with a 2-ene-1, 4-dione chain. Intermediate I would undergo a DA reaction with unit II or III by head-to-head and head-to-tail approaches to provide four cycloadditive products 1−4. Summarized from the biosynthetic pathway, the products of units I, II, and III with the 6/7 transfusion of the αMγL are retained with a 7S configuration, which is in agreement with other same typical guaianolides.11 Therefore, to a large extent, the absolute configurations of 1−4 are speculated as shown in Figure 1. In order to consolidate their stereochemistry, the absolute configurations of 1−4 were evaluated by the ECD quantum chemical calculation using the TD-SCF
method using the B3LYP/6-311+G(d, 2p) level with the Gaussian 09 program package.17 As shown in Figure 5, the
Figure 5. Experimental ECD spectra for 1−4 in MeOH along with their possible absolute configurations of calculated ECD spectra based on internal energies with free energy corrections after optimization at the B3LYP/6-311+G (d, 2p) level with the polarizable continuum model in MeOH.
similarities between the experimental and the calculated ECD spectra of 1−4 indicated the 2R, 3S, 6R, 7S, 2′S, 4′R, 6′S, 7′S, 10′S configurations for 1; 2S, 3R, 6R, 7S, 2′S, 4′R, 6′S, 7′S, 10′S configurations for 2; 2R, 3S, 6R, 7S, 1′R, 3′R, 6′S, 7′S, 10′S configurations for 3; and 2S, 3R, 6R, 7S, 1′R, 3′R, 6′S, 7′S, 10′S configurations for 4. These results are also in agreement with the 5412
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Organic Letters ORCID
above-mentioned biosynthetic prediction. Therefore, the structures of 1−4 are assigned as shown in Figure 1. Compounds 1−4 were primarily evaluated for cytotoxic effects toward the human breast (MDA-MB-468, MDA-MB-231, MCF7) and colon (HCT-116) cancer cell lines by the MTT assay with oxaliplatin as the positive control. All compounds exhibited antiproliferative activity in those cell lines (Table 1). Note-
Ling-Yi Kong: 0000-0001-9712-2618 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research work was financially supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Syngenta Ph.D. Fellowship awarded to G.M.X.
Table 1. Cytotoxicity of Compounds 1−4 and 7 in Four Cancer Cell Lines IC50 (μM) compd
MDA-MB-468
MDA-MB-231
MCF-7
HCT-116
1 2 3 4 7 oxaa
3.56 ± 0.62 3.21 ± 0.48 7.66 ± 1.22 5.67 ± 0.58 >50 8.91 ± 1.12
10.51 ± 1.51 9.72 ± 0.52 17.52 ± 1.62 14.76 ± 1.23 >50 11.20 ± 0.88
16.51 ± 1.12 13.26 ± 0.90 17.81 ± 1.76 10.22 ± 0.78 >50 12.3 ± 1.51
16.21 ± 0.62 17.05 ± 0.48 15.19 ± 1.22 24.55 ± 0.58 >50 11.45 ± 1.11
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oxa (oxaliplatin): positive control. Each value represents the mean ± SD from three independent experiments. a
worthily, compounds 1−4 revealed good activity against the MDA-MB-468 cell line with IC50 values of 3.21−7.66 μM, with 2 displaying the best activity. Additionally, compound 2 was catalyzed by 10% palladium/carbon to yield 11, 13-11′, 13′dimethyls artemisian B (7) (Scheme S1). Compound 7 showed no cytotoxicity (IC50 > 50 μM, Table 1), suggesting that the αMγL functionality, usually as a Michael acceptor, plays an important role in the activities of electrophilic natural products.18 The antiproliferative effect of 2 was further demonstrated using the Annexin V-FITC/PI staining assay. As shown in Figure S4 (SI), 2 dose-dependently increased the apoptotic rate in MDA-MB-468 cells and showed better activity than the positive control (oxaliplatin) at a concentration of 5 μM. Subsequently, cell cycle analysis showed 2 significantly increased the number of G2/ M phase cells in a dose-dependent manner (SI, Figure S5), thus demonstrating that the inhibition of the cell cycle is an important way for 2 to suppress MDA-MB-468 cell proliferation. In summary, artemisians A−D, the first four unprecedented carbon frameworks consisting of a rare 1, 10-4, 5-disecoguaianolide and a Δ1(2), 4(5) or a Δ1(5), 3(4) guaianolide diene, were isolated from the famous traditional Chinese medicine, A. argyi. In consideration of the complexities and the potent cytotoxicities of 1−4, we believe that they will be of great interest to chemists and pharmacologists for further research on their synthesis, modification, and pharmacological mechanisms.
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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.7b02681. Details of isolation, synthesis and biological experimental procedures, HRESIMS, UV, IR, and NMR data for new compounds (PDF)
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Corresponding Authors
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[email protected] (L.-Y.K.). *E-mail:
[email protected] (J.-G.L.). 5413
DOI: 10.1021/acs.orglett.7b02681 Org. Lett. 2017, 19, 5410−5413
