Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Euphorkanlide A, a Highly Modified Ingenane Diterpenoid with a C24 Appendage from Euphorbia kansuensis Xue-Long Yan,† Jun Sang,† Shi-Xin Chen,‡ Wei Li,† Gui-Hua Tang,† Li-She Gan,‡ and Sheng Yin*,† †
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People’s Republic of China College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, People’s Republic of China
‡
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S Supporting Information *
ABSTRACT: Euphorkanlide A (1), a highly modified ingenane diterpenoid with a C24 appendage forming an additional hexahydroisobenzofuran-fused 19-membered macrocyclic bis-lactone ring system was isolated from the roots of Euphorbia kansuensis. Its structure was determined by extensive spectroscopic analysis and quantum-chemical calculations. Compound 1 showed significant cytotoxicities against a panel of cancer cell lines (IC50s < 5 μM). Mechanistic study revealed that 1 could induce the generation of ROS, leading to cell cycle arrest and cell apoptosis in drug-resistant cancer cell line HCT-15/5-FU.
The roots of E. kansuensis (3.0 kg) were extracted with 95% EtOH to give a residue, which was suspended in H2O and successively partitioned with petroleum ether, EtOAc, and nBuOH. Subsequent purification of the EtOAc fraction with various column chromatographic methods afforded 1 (3 mg).
Euphorbia (Euphorbiaceae) is one of the largest genera in flowering plants, with approximately 2160 species, distributed in both tropical and temperate mainlands.1 Plants of this genus are well-known for producing highly functionalized macrocyclic diterpenes, namely Euphorbia diterpenoids. Until now, over 700 Euphorbia diterpenoids, incorporating over 30 scaffolds, have been reported from Euphorbia plants.1 Their fascinating structures and intriguing biological activities have attracted considerable interest from both natural product chemists and pharmacologists in the past 10 years.1b For example, picato, an ingenane ester (5/7/7/3 ring system) isolated from E. peplus, is a short-term topical treatment approved by FDA in 2012 for the treatment of actinic keratosis.2 Resiniferatoxin, a daphnane orthoester (5/7/6 ring system) isolated from E. resinifera, is a potent capsaicin receptor agonist currently under phase III clinical trial for the treatment of overactive bladder and chronic pain.3 Prostratin, a phorbol ester (5/7/6/3 ring system) isolated from E. cornigera, could inhibit HIV-1 infections by down-regulating HIV-1 cellular receptors through activation of the protein kinase C (PKC) pathway.4 Euphorbia kansuensis Prokh. is a perennial herb widely distributed in northern China.5 Its roots are used in Chinese folk medicine for the treatment of pyretolysis, cholagogue, and apocenosis and as purgative.6 Previous chemical investigation of this plant led to the isolation of several lathyrane diterpennoids and ursane triterpenoids, some of which showed significant cytotoxicity.7 In our continuing efforts toward discovering structurally intriguing and biologically significant metabolites from Euphorbiaceae species,8 a highly modified ingenane diterpenoid, euphorkanlide A (1), was isolated from the roots of E. kansuensis. Compound 1 features a C24 appendage forming an additional 5/6/19 ring system. Herein, we describe the isolation, structural elucidation, plausible biosynthetic pathway, and cytotoxicity of 1. © XXXX American Chemical Society
Compound 1 was obtained as a white powder. The pseudomolecular ions in HRESIMS at m/z 749.3653 for [M + Na]+ and at m/z 761.3455 for [M + Cl]− allowed the molecular formula C44H54O9 to be assigned to 1, with 18 indices of hydrogen deficiency (IHDs). The IR spectrum exhibited absorption bands for hydroxyl (3418 cm−1) and carbonyl (1703 cm−1) functionalities. The 1H NMR data (Table 1) of 1 showed signals for two characteristic cyclopropane methines [δH 0.70 (1H, ddd, J = 9.0, 9.0, 6.4 Hz) and 0.87 (1H, dd, J = 12.0, 9.0 Hz)], five methyls [δH 1.06 (3H, s), 1.08 (3H, s), 1.07 (3H, d, J = 7.3 Hz), 0.96 (3H, t, J = 7.4 Hz), and 0.99 (3H, t, J = 7.3 Hz)], two oxygenated methylenes [δH 4.06 (1H, d, J = 13.3 Hz) and 4.12 (1H, d, J = 13.3 Hz), 4.39 (1H, d, J = 12.2 Hz) and 5.33 (1H, d, J = 12.2 Hz)], four oxymethines [δH 5.92 (1H, s), 3.89 (1H, s), 3.53 (1H, ddd, J = 10.2, 7.3, 2.5 Hz), and 4.00 (1H, m)], 14 olefinic protons (5.54−7.70 ppm), and a series of aliphatic multiplets. The 13C NMR spectrum, in combination with DEPT Received: April 15, 2019
A
DOI: 10.1021/acs.orglett.9b01315 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Spectroscopic Data of 1 in CD3OD (J in Hz, δ in ppm) no. 1 2 3 4 5 6 7 8 9 10 11 12a 12b 13 14 15 16 17 18 19a 19b 20a 20b
δH, multi (J in Hz) 6.53, s 5.92, s 3.89, s 5.99, m 4.29, dd (11.8, 2.9)
2.60, 2.37, 1.76, 0.70, 0.87,
m ddd (15.0, 9.0, 2.7) m ddd (9.0, 9.0, 6.4) dd (12.0, 9.0)
1.06, 1.08, 1.07, 4.39, 5.33, 4.06, 4.12,
s s d d d d d
(7.3) (12.2) (12.2) (13.3) (13.3)
δC
no.
140.4 135.6 82.4 87.6 75.2 143.5 125.0 44.6 208.5 72.6 40.0 32.1
1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′α 9′β 10′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″ 13″a 13″b 14″
24.3 24.5 25.4 28.9 15.8 17.5 64.1 65.6
δH, multi (J in Hz) 5.78, 7.70, 5.99, 5.63, 3.25, 1.85, 3.53, 1.76, 1.43, 0.96,
d (15.1) dd (15.1, 12.0) m dd (10.2, 10.2) ddd (10.2, 10.2, 10.2) ddd (10.2, 10.2, 10.2) ddd (10.2, 7.3, 2.5) m m t (7.4)
5.78, 6.98, 6.23, 6.69, 6.21, 5.54, 3.92, 5.58, 5.97, 2.80, 4.00, 1.45, 1.27, 0.99,
d (15.4) dd (15.4, 11.3) dd (14.5, 11.3) dd (14.5, 10.2) dd (11.8, 10.2) dd (11.8, 11.8) m ddd (10.2, 3.5, 3.5) m m m m m t (7.3)
δC 168.5 123.3 141.3 128.2 143.9 41.3 45.0 84.4 28.7 10.0 168.0 120.5 147.9 129.1 140.0 130.6 136.9 41.3 131.5 126.9 48.2 80.9 27.1 10.7
2D NMR analysis, especially 1H−1H COSY correlations (Figure 1). Two side chains, C-2′-C-10′ and C-2″-C-14″, were first established by 1H−1H COSY correlations. These two side chains were, respectively, tied to the ester carbonyls at C1′ and C-1″ by HMBC correlations (Figure 1) of H-2′/C-1′ and H-2″/C-1″. The 1H−1H COSY correlations of H-6′/H-8″ and H-7′/H-11″ fused these two side chains to form a cyclohexene ring. The HMBC correlation from an oxymethine proton at δH 4.00 (H-12″) to an oxymethine carbon at δC 84.4 (C-8′) further constructed a furan ring between the propyl tails of these two side chains to generate the hexahydroisobenzofuran core. Components A and B were finally connected by two ester bonds formed between C-19 and C-1′ and between C-3 and C-1″, as indicated by HMBC correlations of H-19/C-1′ and H-3/C-1″, generating a 19-membered macrocyclic bislactone ring. Thus, the planar structure of 1 with an unprecedented 5/6/19/5/7/7/3 ring system was constructed. The relative configuration of 1 was established by NOESY experiments (Figure 2), coupling constants analysis, as well as 1D NMR comparison. The configuration of the ingenane part (component A) was assigned to be the same as that of 2 by
experiments, resolved 44 carbon resonances attributable to one ketocarbonyl group (δC 208.5), two ester groups (δC 168.0 and 168.5), eight double bonds, five methyls, 12 sp3 methine carbons (four oxygenated), five sp3 methylenes (two oxygenated), an oxygenated sp3 tertiary carbon, and two quaternary carbons. As 11 of the 18 IHDs were accounted for by three carbonyls and eight double bonds, the remaining seven IHDs required that 1 be heptacyclic. The aforementioned data comprised the structural features of a coisolated ingenane diterpenoid, ingenol (2),9 suggesting that 1 was a highly modified ingenane-type diterpenoid with a C 24 appendage forming three additional rings. A detailed account of the structural elucidation of 1 is presented below. The ingenane diterpenoid moiety (component A, Figure 1) was readily established by comparison of its 1D NMR data
Figure 1. Key 1H−1H COSY (bold lines) and HMBC (arrows) correlations of 1.
with those of 2, as well as analysis of its 2D NMR data. In component A, the CH3-19 in 2 was oxidized, and the chemical shift of H-3 was severely downfield shifted from 4.35 to 5.92 ppm, suggesting that the C24 appendage was probably linked to C-19 and C-3 sites. The C24 appendage (component B) with a novel hexahydroisobenzofuran core was established by detailed
Figure 2. Key NOE (↔) correlations of 1. B
DOI: 10.1021/acs.orglett.9b01315 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
with a conjugated triene ester system, while the second and third Cotton effects were dominated by both part A and part B. Euphorkanlide A (1) is a highly modified ingenane diterpenoid with a C24 appendage forming an additional 5/ 6/19 ring system. Its plausible biosynthetic pathway was proposed in Scheme 1. The coisolated major component
comparison of their 1D and NOESY data. For component B, the large coupling constants of H-7′ (ddd, J = 10.2, 10.2, 10.2 Hz) on the cyclohexene ring indicated the trans-relationships of H-7′/H-6′, H-7′/H-8′, and H-7′/H-11″. Thus, H-6′, H-8′, and H-11″ were cofacial and were arbitrarily designated as βorientation. This was further supported by the NOE correlations of H-6′/H-8′ and H-11″ and H-8′/H-11″, and also revealed that H-6′, H-7′, and H-11″ were pseudoaxially oriented on the half-chair conformational cyclohexene ring. Consequently, the NOE correlations of H-7′/H-7″ and H13″b assigned H-8″and H-12″as β-oriented. The E-geometry for Δ2″, Δ4″, and Δ2′ and Z-geometry for Δ6″ and Δ4′ were determined by the coupling constants of H-2″/H-3″ (J = 15.4 Hz), H-4″/H-5″ (J = 14.5 Hz), H-2′/H-3′ (J = 15.1 Hz), H6″/H-7″ (J = 11.8 Hz), H-4′/H-5′ (J = 10.2 Hz), respectively. These assignments were also supported by the NOESY correlations of H-2″/H-4″, H-5″/H-3″, H-2′/H-4′, H-6″/H7″, and H-4′/H-5′, respectively. The absolute configuration of 1 was determined by quantum-chemical calculation methods. As 1 consists of two configurationally independent parts (A and B) connected by two ester bonds, the absolute configuration of part A was first designated as depicted based on biogenesis consideration to simplify the calculations. Therefore, two possible isomers, 1a and 1b (Figure 3), with the same absolute configuration in part
Scheme 1. Proposed Biosynthetic Pathway for 1
ingenol (2) was considered as the ingenane precursor. The fatty acids 2,4,6,8,10-tetradecapentaenoic acid (3) and 2,4,6decatrienoic acid (4), which were frequently encountered as the substitutions in ingenane diterpenoids,10 were used as the building blocks. The esterification of 3-OH in 2 by 3 generated the known compound, ingenol 2,4,6,8,10-tetradecapentaenoate (5).10b Then the oxidation of CH3-19 in 5 followed by the esterification with 4 generated the crucial intermediate i. The side chains in i underwent the oxidation and double-bond isomerizations to afford intermediate ii. The Diels−Alder reaction between Δ6′ and 8″,10″-diene in ii followed by the etherification between 8′-OH and 12″-OH finally generated 1. Compound 1 was screened for cytotoxicity (Table 2) against nine human cancer cell lines, including three drug-resistant cell Table 2. Cytotoxicity of 1 against Human Cancer Cell Lines cell lines HepG2 HepG2/DOX HCT-15 HCT-15/5-FU A549 A549/CDDP A375 RKO MDAMB-231
Figure 3. Experimental ECD spectrum of 1 and the calculated ECD spectra of 1a and 1b.
A and opposite absolute configuration in part B were proposed for theoretical ECD calculations (Supporting Information). As shown in Figure 3, in 190−400 nm region, both the experimental ECD curve of 1 and the calculated ECD curve of 1a showed the same first negative, second positive, and third negative Cotton effects around 310, 265, and 215 nm, respectively, while the calculated ECD curve of 1b showed an opposite positive first Cotton effect compared with the experimental one. Therefore, the absolute configuration of 1 was determined to be the same as 1a (3S,4S,5R,8S,10S,11R,13R,14R,6′R,7′S,8′R,8″R,11″S,12″S). In addition, the contribution of parts A and B to the experimental ECD spectrum of 1 was investigated by molecule orbital electron transition analysis (Supporting Information), which suggested that the first Cotton effect of 1 mainly contributed by part B
a
1 (IC50, μM) 12.6 11.2 5.05 4.72 12.2 11.5 2.28 3.83 5.23
± ± ± ± ± ± ± ± ±
1.52 2.68 1.31 1.70 0.48 3.78 0.42 0.73 1.95
doxorubicina (IC50, μM) 0.49 6.41 0.77 3.94 0.78 4.25 1.32 0.93 0.26
± ± ± ± ± ± ± ± ±
0.22 2.03 0.28 0.90 0.16 1.47 0.26 0.38 0.08
Positive control.
lines, HepG2/DOX, HCT-15/5-FU, and A549/CDDP. The results showed that 1 inhibited most cancer cell lines at a micromolar level, with IC50 values less than 5 μM for cell lines of HCT-15/5-FU, A375, and RKO. Interestingly, similar IC50 values for sensitive cell lines and their drug-resistant counterparts were observed, suggesting that 1 may possess a mechanism in overcoming the multidrug resistance (MDR). Thus, the mechanistic study of 1 on the 5-fluorouracil resistant cell line, HCT-15/5-FU, was further carried out. The flow cytometry analysis showed that 1 could dose-dependently induce the cell apoptosis at 5, 10, and 20 μM, and arrest the cell cycle at G2/M phase (Figure 4). As α,β-unsaturated C
DOI: 10.1021/acs.orglett.9b01315 Org. Lett. XXXX, XXX, XXX−XXX
Organic Letters
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Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Jun Sang: 0000-0003-1446-7501 Gui-Hua Tang: 0000-0002-8831-7154 Sheng Yin: 0000-0002-5678-6634 Notes
The authors declare no competing financial interest.
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Figure 4. 1 dose-dependently arrested cell cycle at G2/M phase (A) and induced cell apoptosis (B) in HCT-15/5-FU cells.
ACKNOWLEDGMENTS This work was supported by Natural Science Foundation of China (81722042 and 81573302) and Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Y093).
carbonyl moiety is generally considered as the intracellular Michael acceptor of protein thiols involving the generation of the reactive oxygen species (ROS),11 the cytotoxicity of 1 (containing two α,β-unsaturated carbonyl moieties) was probably related to the ROS pathway. To verify this, the ROS-inducible property of 1 was tested. As shown in Figure 5,
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Figure 5. (A) 1 induced intracellular ROS generation in HCT-15/5FU cells. (B) The ROS scavengers rescued the inhibition of 1 on cell viability of HCT-15/5-FU cells. ***p < 0.001, compared with 1treated group.
1 could dose-dependently induce the generation of ROS at 5, 10, and 20 μM, and the additions of ROS scavengers Nacetylcysteine (NAC) or glutathione (GSH) dramatically revived the cell viability. These results suggested that 1 could induce the generation of ROS, triggering cell apoptosis in MDR cells. Euphorkanlide A (1) represents the first example of Euphorbia diterpenoid incorporating such a large (C24) and highly modified appendage. It is possible that its unique structure renders it the ROS-induction property in cancer cell lines. Studies toward its in-depth mechanism in overcoming cancer MDR are in progress.
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REFERENCES
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b01315. Full experimental procedures, quantum chemical calculations, 1D and 2D NMR spectra, IR, and HRESIMS of 1 (PDF) D
DOI: 10.1021/acs.orglett.9b01315 Org. Lett. XXXX, XXX, XXX−XXX
