Ervatamines A–I, Anti-inflammatory Monoterpenoid Indole Alkaloids

May 29, 2015 - Ervatamine A (1) is a ring-C-contracted ibogan-type monoterpenoid indole alkaloid with an unusual 6/5/6/6/6 pentacyclic rearranged ring...
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Ervatamines A−I, Anti-inflammatory Monoterpenoid Indole Alkaloids with Diverse Skeletons from Ervatamia hainanensis Dong-Bo Zhang,† Dao-Geng Yu,‡ Meng Sun,† Xu-Xin Zhu,† Xiao-Jun Yao,† Shuang-Yan Zhou,† Jian-Jun Chen,† and Kun Gao*,† †

State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China ‡ Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Science, Danzhou 571737, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new monoterpenoid indole alkaloids, ervatamines A−I (1−9), and five known ones (10−14), were isolated from Ervatamia hainanensis. The new structures were elucidated by extensive spectroscopic analysis and comparison to known compounds. Their absolute configurations were determined by various methods including computational methods, X-ray diffraction analysis, and electronic circular dichroism spectroscopy, as well as chemical transformations. Ervatamine A (1) is a ring-C-contracted ibogan-type monoterpenoid indole alkaloid with an unusual 6/5/6/6/6 pentacyclic rearranged ring system. Ervatamines B−E (2−5) display a nitrogen-containing 9/6 ring system, which is rarely observed in nature. The epimeric ervatamines B (2) and C (3) possess a 22-nor-monoterpenoid indole alkaloid carbon skeleton, which was only found in deformylstemmadenine. Compounds 10 and 14 exhibited significant anti-inflammatory activities, with IC50 values of 25.5 and 41.5 μM, respectively, while the IC50 value of indomethacin as a positive control was found to be 42.6 μM. Additionally, compound 9 showed mild activity against 786-O and HL-60 cell lines.

T

he genus Ervatamia (Apocynaceae) consists of approximately 120 species, which are primarily distributed in the subtropical and tropical regions of Australia and Asia. Only 15 species and five varieties grow in the south of China, many of which have long been used in Traditional Chinese Medicine.1 Phytochemical studies of this genus have led to various skeletal types of monoterpenoid indole and bisindole alkaloids.2−5 Pharmacological investigations revealed promising bioactivity, including antitumor, AChE inhibitory, and MDR-reversing activities.5−8 Therefore, studies of this genus have received considerable attention.9,10 Ervatamia hainanensis Tsiang, commonly known as “HaiNan-Gou-Ya-Hua” in China, is a 1−3 m tall shrub occurring in thin forests at elevations of 100−530 m. This species is mainly distributed in the Hainan and Guangdong Provinces of China, and its root has been used in folk medicine for the treatment of snake bite, stomachache, dysentery, viral hepatitis, rheumatic arthritis, and hypertension.1 Previous studies of E. hainanensis resulted in the isolation of several bioactive monoterpenoid indole alkaloids and their dimeric forms.8,11−13 As part of our continuing search for structurally diverse and bioactive constituents,14,15 nine new indole alkaloids, ervatamines A−I (1−9), and five known ones (10−14) were isolated from E. hainanensis. Herein, the isolation, structural elucidation, and biological evaluation of these isolates are described. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 20, 2015

A

DOI: 10.1021/acs.jnatprod.5b00051 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. 1H −1H COSY (bold) and selected HMBC (arrows) correlations of 1, 2, 5, 6, 7, and 8.

Figure 2. Key NOESY correlations of 1, 3, 5, 6, 7, and 8.

■

RESULTS AND DISCUSSION The aerial parts of E. hainanensis were extracted with MeOH, and the extract was partitioned between EtOAc and 2% HCl solution. The acidic water-soluble materials, adjusted to pH 9− 10 with 10% ammonia solution, were successively extracted with CHCl3 and n-BuOH. The CHCl3 and n-BuOH extracts were fractionated by column chromatography (CC) over silica gel, Sephadex LH-20, MCI gel, ODS, and semipreparative HPLC to afford nine new monoterpenoid indole alkaloids, ervatamines A−I (1−9), which feature four types of carbon skeletons. Five known compounds (10−14) were also isolated and identified as coronaridine (10),16 heyneanine (11),17 3-(2′oxopropyl)coronaridine (12),16 3-(2′-oxopropyl)-19-epi-heyneanine (13),18 and pandine (14)19 by comparison of their physical and spectroscopic data with reported data. Ervatamine A (1) was obtained as a white, amorphous powder, [α]20D +38 (c 0.3, CHCl3), and gave a positive reaction with Dragendorff’s reagent. The molecular formula C21H24N2O4, requiring 11 indices of hydrogen deficiency, was established by HRESIMS (m/z 391.1638 [M + Na]+, calcd 391.1628) and 13C NMR data. The 1H and 13C NMR spectra of 1 revealed a typical substituted indole alkaloid moiety with signals at [δH 7.28 (1H, d, J = 7.4 Hz, H-9), 7.32 (1H, t, J = 7.4 Hz, H-10), 7.34 (1H, t, J = 7.4 Hz, H-11), and 8.14 (1H, d, J = 7.4 Hz, H-12); δC 147.7 (s, C-2), 111.5 (s, C-7), 134.2 (s, C-8), 109.2 (d, C-9), 123.4 (d, C-10), 123.5 (d, C-11), 120.1 (d, C12), and 126.7 (s, C-13)]. Additionally, the 1H NMR spectrum displayed signals for a 1-hydroxyethyl side chain [δH 4.13 (1H, q, J = 6.3 Hz, H-19) and 1.15 (3H, d, J = 6.3 Hz, H-18)], a bridgehead proton that is adjacent to a nitrogen (δH 3.36, 1H,

br s, H-21), a methoxycarbonyl group (δH 3.71, 3H, s), and one −CHO proton (δH 10.11, 1H, s). A pair of AB doublets of the isolated methylene [δH 5.21 (1H, d, J = 12.1 Hz, H-5a) and 4.98 (1H, d, J = 12.1 Hz, H-5b)] was assigned, judging from the HSQC and 1H−1H COSY spectra. In addition to the eight signals of the indole ring, the 13C NMR and DEPT spectra of 1 showed 13 remaining carbon signals, including two carbonyls (δC 172.2 and 183.4), two methyls (one oxygenated at δC 53.3), four methylenes, including two nitrogen-bearing methylenes at δC 52.3 and 65.5, respectively, four methines (one oxygenated at δC 71.3 and one nitrogen-containing at δC 58.8), and one quaternary carbon. The DEPT spectrum showed that 1 had at least 23 hydrogen atoms, whereas the molecular formula of 1 was C21H24N2O4, which implied that 1 had one exchangeable hydrogen atom that can be assigned to a hydroxy group. The IR absorption bands at 3351 cm−1 also supported this inference. All of these 1H and 13C NMR resonances account for only eight out of the 11 indices of hydrogen deficiency, indicating the structure of 1 still needed three more rings. An extended spin system a [H2-17/H-14(H2-3/N)/H2-15/H-20(H-19/H3-18)/ H-21/N] (Figure 1) was deduced from analysis of the 1H−1H COSY spectrum. The HMBC correlations from H-3 to C-5 and C-21 suggested that the CH2-3 and CH-21 of fragment a and C-5 originated from the carbons attached to N-4. The HMBC cross-peaks of H-21/C-2, C-16, C-17, C-22 and H-17/C-2, C16, C-21, C-22 implied that CH-21 and CH2-17 of fragment a and the methoxycarbonyl group were connected via C-16. These observations suggested that 1 possessed an ibogan-type monoterpenoid indole alkaloid carbon skeleton, similar to heyneanine (11), which has been previously observed from this B

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species.17 The differences between the two compounds were that a methylene resonance (δC 21.4, C-6) was absent and that the signal for C-5 (δC 65.5) in 1 was deshielded relative to 11 (δC 52.3, C-5). Moreover, the signals for H2-5 [δH 5.21 (1H, d, J = 12.1 Hz, H-5a) and 4.98 (1H, d, J = 12.1 Hz, H-5b)] were a pair of AB doublets in the 1H NMR spectrum of 1. These results indicated that the isolated methylene (C-5) may be adjacent to both a nitrogen atom (N-4) and an sp2 quaternary carbon (C-7), which was supported by the HMBC correlations from H-5 to C-8, C-2, C-3, and C-21. The linkage of C-5 to C7 confirmed that compound 1 was an unusual ring-Ccontracted ibogan-type monoterpenoid indole alkaloid. In addition, the HMBC correlations between a proton at δH 10.11 (1H, s) and C-2/C-13 revealed that the −CHO is linked to N-1. The hydroxy group was located at C-19, as indicated by HMBC correlations from H-19 to C-18, C-15, and C-21 and the chemical shift of C-19 (δC 71.3). Thus, all indices of hydrogen deficiency were satisfied, and the planar structure was deduced to be as shown in 1. The relative configuration of 1 was assigned on the basis of the NOESY analysis (Figure 2). Biogenetically H-21 of ibogantype monoterpenoid indole alkaloid derivatives isolated from Apocynaceae species is α-oriented. The NOESY correlations between H-21 and H-5α/OCH3 suggested that H-21, H-5α, and OCH3 were on the same face, whereas the correlations between H-3 and H-5β/H-15β/H-17β suggested that these protons were on the opposite side. Furthermore, the NOESY correlations of H-17α/H-20/H-15α implied that these protons possessed identical orientations. Comparison of the chemical shift values of C-15 and C-21 in 1 with those of other 19hydroxyiboga alkaloids indicated a 19S* configuration,20 which was further confirmed by the NOESY correlations of H-19/H21 and H-18/H-15. The absolute configuration of 1 was established by comparison of the optical rotations of its two enantiomers to the experimental value. The B3LYP/6-311++G(d,p) method was used to predict the optical rotation values for (14R,16S,19S,20S,21S)-1 and its enantiomer (14S,16R,19R,20R,21R)-1a.21 The calculated optical rotation for 1 was +40.37, and that of its enantiomer 1a −16.81. The former was close to the experimental value of +38 for 1, which strongly suggested that the absolute configuration of 1 was 14R,16S,19S,20S,21S. Furthermore, electronic circular dichroism (ECD) spectra calculations using the TDDFT (timedependent density functional theory) method were used to confirm this conclusion.21 The results showed that the experimental ECD spectrum of 1 was similar to the calculated spectrum of (14R,16S,19S,20S,21S)-1 rather than that of (14S,16R,19R,20R,21R)-1a (Figure 3), which was consistent with optical rotation calculations. Thus, the structure of 1 was elucidated and named ervatamine A. Ervatamine B (2) was obtained as colorless plates, [α]20D −11 (c 1.0, MeOH). The molecular formula of 2 was established as C20H24N2O2 based on the HRESIMS (m/z 325.1920 [M + H]+, calcd 325.1911) and 13C NMR data, indicating 10 indices of hydrogen deficiency. The UV absorption bands at 290, 284, and 225 nm indicated the presence of an indole chromophore.22 The IR spectrum showed absorption bands due to amino (3358 cm−1) and carboxylate (1597, 1462, 1379 cm−1) functionalities.23 The 1H NMR spectrum of 2 showed resonances for an ethylidene side chain [δH 1.89 (3H, d, J = 6.4 Hz, H-18) and 5.87 (1H, m, H19)], an ortho-disubstituted benzene ring [δH 7.43 (1H, d, J =

Figure 3. Experimental ECD spectrum of 1 and the calculated ECD spectra of (14R,16S,19S,20S,21S)-1 and (14S,16R,19R,20R,21R)-1a.

7.6 Hz, H-9), 7.05 (1H, td, J = 7.6, 1.2 Hz, H-10), 7.10 (1H, td, J = 7.6 Hz, 1.2 Hz, H-11), and 7.39 (1H, d, J = 7.6 Hz, H-12)], and an N+-methyl group [δH 2.97 (3H, s, H-23)]. The 13C NMR and DEPT spectra of 2 showed 20 carbon resonances assigned to a carbonyl, 10 olefinic carbons, two methyls, five methylenes, and two methines. In addition to the indole moiety, three structural subfragments, a [N−CH2(3)− CH2(14)−CH(15)−CH(16)], b [N−CH2(5)−CH2(6)], and c (C18−C19), as drawn with bold bonds (Figure 1), were readily established by the 1H−1H COSY spectrum. The HMBC correlations from H-23 to C-3, C-5, and C-21 implied that C-3, C-5, C-21, and CH3-23 were attached to N-4. The correlations from H-6 to C-2 and H-16 to C-7 indicated the attachment of C-6 to C-7 and C-16 to C-2. The linkage of C-19 to C-20 was confirmed by the HMBC correlations from H-21 and H-15 to C-19. With all the protons accounted for, we deduced the presence of a COO− group, which was consistent with the IR spectrum.23 The carboxylate anion was placed at C-16, which was confirmed by the HMBC correlations from H-15 and H-16 to C-17. Thus, the molecular structure of ervatamine B was deduced to be 2. The relative configuration of 2 was deduced from the NOESY experiment. The NOESY cross-peaks between H-18 and H-15/H-16 suggested that H-15 and H-16 were β-oriented (Figure 2). Correlations of H-19/H-21 and H-15/H-18 in the NOESY spectrum indicated that the configuration of the double bond between C-19 and C-20 was E. The proposed structure and relative configuration were confirmed by singlecrystal X-ray diffraction analysis using Cu Kα radiation at 173.0 K (Figure 4). Hence, the absolute configuration of 2 was defined as 4N-S,15R,16S based on a Flack absolute structure parameter of −0.02(12). Ervatamine C (3) was obtained as a colorless gum, [α]20D +80 (c 0.1, MeOH). HREIMS (m/z 325.1917 [M + H]+, calcd 325.1911) and 13C NMR data gave the molecular formula C20H24N2O2, identical to that of 2. Compound 3 showed almost the same 13C NMR spectrum as 2. Analysis of the 1D and 2D NMR data (1H−1H COSY, HSQC, and HMBC) confirmed that the planar structure of 3 was the same as that of 2. These observations suggested that compounds 2 and 3 might be a pair of epimers. The NOESY correlations of H-16 with H14b suggested that H-16 was α-oriented, in contrast to its βorientation in 2. The ECD spectrum of 3 showed a positive Cotton effect at 223 nm (Figure 5), which was similar to that of 2. On the basis of its likely biosynthesis and analysis of the ECD spectrum, the absolute configuration of 3 was assigned as 4N-S,15R,16R and named ervatamine C. C

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Figure 4. ORTEP drawings of 2 and 6.

and H-14a. Correlations of H-19/H-21b and H-15/H-18 indicated that the configuration of the C-19−C-20 double bond was E. Additionally, the ECD spectrum of 5 was in agreement with that of 2, indicating that its absolute configuration was 4N-S,15S,16R. Ervatamine F (6) was crystallized as colorless blocks from MeOH, [α]20D −117 (c 1.0, MeOH) and possessed a molecular formula of C21H26N2O4 as established by HRESIMS (m/z 371.1974 [M + H]+, calcd 371.1965) and 13C NMR data. The IR spectrum suggested the presence of amino (3349 cm−1), carbonyl (1725, 1685 cm−1), and aromatic groups (1618, 1453 cm−1). The 1H NMR spectrum of 6 showed signals for an ortho-disubstituted benzene ring [δH 7.56 (1H, d, J = 7.6 Hz, H9), 6.80 (1H, td, J = 7.6, 1.1 Hz, H-10), 7.39 (1H, td, J = 7.6, 1.1 Hz, H-11), and 6.77 (1H, d, J = 7.6 Hz, H-12)], a methoxy group (δH 3.25, 3H, s), a bridgehead proton adjacent to nitrogen (δH 4.05, 1H, d, J = 1.4 Hz, H-21), and a 1hydroxyethyl side chain [δH 4.15 (1H, qd, J = 6.4, 1.1 Hz, H19) and 1.05 (3H, d, J = 6.4 Hz, H-18)]. The 13C NMR and DEPT spectra of 6 displayed 21 resonances comprising two carbonyls, six aromatic carbons, two methyls, five methylenes, four methines, one N-containing tertiary carbon, and one quaternary carbon. The 1H and 13C NMR data of 6 closely matched those of coronaridine pseudoindoxyl,24 which was previously isolated from Ervatamia macrocarps (Apocynaceae), indicating the oxidation state of the indole ring. This suggestion was supported by the absence of carbon signals for two olefinic carbons (C-2 and C-7) of the indole ring and the presence of a carbonyl (δC 202.5) and an N-containing tertiary carbon (δC 67.0) and was further confirmed by the HMBC cross-peaks between H-6/H-9 and C-7. The most significant difference between the two compounds was the absence of a methylene (δC 28.6, C-19) and the presence of an oxygenated methine (δC 71.1), hence indicating that 6 was 19-hydroxycoronaridine pseudoindoxyl, which was confirmed by the HMBC correlations from H-19 to C-15/C-18/C-21 (Figure 1). The NOESY correlations between H-21 and H-5α/H-6α suggested that H5α, H-6α, and H-21 possessed identical orientations. The correlations between H-3 and H-5β/H-15β/H-17β and between H-6β and H-17β suggested that these protons were cofacial. Moreover, H-15α, H-17α, and H-20 were assigned on the same side on the basis of their NOESY correlations. The configuration at C-19 in 6 was determined by means of the auxiliary chiral anisotropic reagent MTPA.25 Briefly, compound

Figure 5. ECD spectra of compounds 2−5.

Ervatamine D (4) was obtained as a colorless gum, [α]20D +40 (c 0.1, MeOH). The molecular formula C21H26N2O3 was established by HREIMS (m/z 355.2028 [M + H]+, calcd 355.2016) and 13C NMR data, 30 Da higher than that of 2. The 13 C NMR and DEPT spectra showed similar patterns to those of 2, except for the absence of a methine resonance (δC 52.1, C16) and the presence of resonances for a quaternary carbon (δC 57.7) along with a hydroxymethyl group (δC 68.0, C-22), which indicated that the hydroxymethyl group might be located at C16. This inference was confirmed by the HMBC correlations from H-22 to C-16, C-15, C-2, and C-17. The NOESY correlations between H-22 and H-14a indicated the αorientation of the hydroxymethyl group. Correlations of H19/H-21b and H-15/H-18 indicated the E configuration of the double bond between C-19 and C-20. Furthermore, the ECD spectrum of 4 exhibited a Cotton effect similar to that observed for 2. Accordingly, the absolute configuration of ervatamine D was assigned as 4N-S,15S,16R. Ervatamine E (5) was obtained as a colorless gum, [α]20D +140 (c 0.1, MeOH). Its molecular formula was established as C21H26N2O4 by the HRESIMS (m/z 371.1974 [M + H]+, calcd 371.1965) and 13C NMR data. The 13C NMR and DEPT spectra showed that 5 was similar to 4, except for the absence of resonances for a methyl group (δC 14.3, C-18) and the presence of resonances for an oxymethylene (δC 58.6), indicating that there was a hydroxy group at C-18 in 5. This suggestion was supported by the molecular formula of C21H26N2O4 and HMBC cross-peaks of H-18/C-19, C-20 (Figure 1). The α-orientation of the hydroxymethyl group at C16 was deduced from the NOESY correlation between H-22 D

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and coronaridine (10)16 exhibited similar resonances, except for the absence of resonances for two methylenes (δC 32.0, C15 and 26.7, C-19) and the presence of resonances for two oxygenated methines (δC 67.1 and 70.6), indicating that 8 possessed hydroxy groups at C-15 and C-19. This was confirmed by the molecular formula of C21H26N2O4 and the HMBC correlations between H-15 and C-3/C-17/C-19/C-21 and between H-19 and C-15/C-18/C-21. The α-orientation of the 15-OH group was deduced by NOESY correlations of H-3/ H-15/H-17β (Figure 2). Like compound 1, the configuration of C-19 was assigned to be S*,20 which was confirmed by the NOESY correlations of H-19/H-21 and H-18/H-15. On the basis of the analysis of the NOESY spectrum, the relative configurations of C-14, C-16, C-20, and C-21 in 8 were the same as those in coronaridine (10).16 Finally, the absolute configuration of 8 was defined as 14R,15R,16S,20S,21S due to the agreement of the ECD spectrum of 8 with that of coronaridine (10).16 The molecular formula of ervatamine I (9) was determined to be C21H24N2O3 by the HRESIMS (m/z 353.1849 [M + H]+, calcd 353.1860) and 13C NMR data. The 13C NMR and DEPT spectra of 9 were similar to those of coronaridine (10),16 except that a methylene (26.7, C-19) in 10 was replaced with a carbonyl (δC 208.0) group. The HMBC cross-peaks between H-18/H-20/H-15/H-21 and C-19 (δC 208.0) suggested that the carbonyl group was located at C-19. The 1H NMR coupling constants and NOESY experiments confirmed that 9 and coronaridine (10) possessed the same relative configuration. Compound 9 displayed an [α]20D of −32 (c 0.6, CHCl3), while an [α]26D of −41 (c 2.0, CHCl3) was reported for coronaridine (10),16 which suggests that 9 and 10 possess the same absolute configuration. Of the new alkaloids, compound 1 is a rare ibogan-type monoterpenoid indole alkaloid in which the unusual sixmembered ring replaced the seven-membered ring of other members of this class, such as coronaridine (10)16 and heyneanine (11).17 Only flabelliformidine,26 previously isolated from Ervatamia f labelliformis Tsiang (Apocynaceae), showed a similar ring-C-contracted carbon skeleton. Ervatamines B−E (2−5) possess a nitrogen-containing 9/6 ring system that is rarely found in nature.27−30 Moreover, the C-16 epimers ervatamines B (2) and C (3) display a 22-nor-monoterpenoid indole alkaloid carbon skeleton, which was only found in deformylstemmadenine, previously isolated from Craspidospermum verticillatum (Apocynaceae).28 The anti-inflammatory activities of compounds 1−14 were assessed by a previously published protocol.31 Indomethacin served as a positive control with an IC50 value of 42.6 μM. Compounds 10 and 14 exhibited significant inhibition of lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages with IC50 values of 25.5 and 41.5 μM, respectively, while the rest of compounds showed weak or no inhibitory activities. The observed anti-inflammatory activities of compounds 7−13 indicated that the presence of substituents at C-3, C-15, and C-19 might cause a sharp decrease in the antiinflammatory activities of ibogan-type monoterpenoid indole alkaloids. This is the first report to show that ibogan-type monoterpenoid indole alkaloids, from the Ervatamia genus, exhibit significant anti-inflammatory activities. The cytotoxicities of all alkaloids against three human cancer cell lines were evaluated using the MTT method reported previously.32 However, only compound 9 showed mild

6 was treated with (R)- or (S)-MTPA-Cl, and the (S)- or (R)MTPA esters at C-19 of 6 were obtained, respectively. The S configuration at C-19 was assigned by comparison of the 1H NMR chemical shift differences between the (S)- and (R)MTPA esters of 6 (Δ values shown in Figure 6). Furthermore,

Figure 6. 1H NMR chemical shift differences Δδ (δS − δR) in ppm for the MTPA esters of 6.

X-ray diffraction analysis using Cu Kα radiation at 295.8(2) K unambiguously established the 2S,14R,16S,19S,20S,21S configuration of 6 with a Flack parameter of −0.1(3) (Figure 4). The molecular formula of ervatamine G (7) was determined to be C22H28N2O3 by HRESIMS (m/z 369.2182 [M + H]+, calcd 369.2173) and 13C NMR data, indicating 10 indices of hydrogen deficiency. The 13C NMR and DEPT spectra showed that 7 had a structure similar to that of coronaridine (10),16 except for the absence of the resonance for a methylene (δC 51.6, C-3) and the presence of resonances for a methine (δC 59.9) and a hydroxymethyl group (δC 62.4, C-24), indicating that the hydroxymethyl group might be located at C-3. This was confirmed by the HMBC correlations from H-24 to C-3/ C-14. H-3 and H-5β/H-6β/H-17β were assigned to occupy the same face of the molecule on the basis of their NOESY correlations. Analysis of the NOESY spectrum revealed that the relative configurations of C-14, C-16, C-20, and C-21 in 7 were identical to those in coronaridine (10).16 The specific rotation of 7 was [α]20D −23 (c 0.3, CHCl3), which was similar to the value of [α]26D −41 (c 2.0, CHCl3) observed for coronaridine (10).16 Additionally, the ECD spectrum of 7 showed a positive Cotton effect at 243 nm and a negative one at 280 nm (Figure 7), in agreement with those for coronaridine (10).16 Taken together, the absolute configuration of 7 was assigned as 3S,14R,16S,20S,21S. Ervatamine H (8) was obtained as a white, amorphous powder, [α]20D −60 (c 1.0, CHCl3). Its molecular formula was assigned as C21H26N2O4 by HRESIMS (m/z 371.1974 [M + H]+, calcd 371.1965) and 13C NMR data, indicating 10 indices of hydrogen deficiency. The 13C NMR and DEPT spectra of 8

Figure 7. ECD spectra of compounds 7, 8, and 10. E

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Table 1. 1H NMR Data for Compounds 1 and 6−9 (δ in ppm, J in Hz) in CDCl3 1a

position 3a 3b 5a 5b 6a 6b 9 10 11 12 14 15a 15b 17a 17b 18 19a 19b 20 21 COOCH3 CHO 24a 24b NH a

3.30 2.58 5.21 4.98

d d d d

(10.4) (10.4) (12.1) (12.1)

7.28 7.32 7.34 8.14 2.07 1.88 1.72 3.07 1.84 1.15 4.13

d (7.4) t (7.4) t (7.4) d (7.4) m ov m d (13.9) ov d (6.3) q (6.3)

1.85 ov 3.36 br s 3.71 s 10.11 s

b

6b 3.02 2.63 3.81 2.66 2.09 1.49 7.56 6.80 7.39 6.77 2.00 1.72 1.46 2.65 1.63 1.05 4.15

7a

dt (11.0, 2.4) ovc ddd (13.7, 13.5, 3.4) ov ddd (13.8, 13.4, 4.9) m d (7.6) td (7.6, 1.1) td (7.6, 1.1) d (7.6) m m m ov dt (14.1, 2.6) d (6.4) qd (6.4, 1.1)

2.94 d (5.8) 3.36 3.29 3.15 3.08 7.48 7.10 7.16 7.26 1.93 1.58 1.49 2.65 2.00 0.92 1.60 1.45 1.32 3.70 3.72

1.39 m 4.05 d (1.4) 3.25 s

8b 3.01 2.86 3.43 3.11 3.14 3.09 7.47 7.11 7.18 7.27 2.00 4.35

dt (9.4, 2.6) dd (9.4, 1.1) m ov ov ov d (7.8) t (7.8) t (7.8) d (7.8) m m

3.07 1.88 1.28 4.21

ov dd (13.9, 2.9) d (6.4) qd (6.4, 2.5)

9b 2.97 2.79 3.38 3.12 3.18 2.99 7.48 7.10 7.17 7.27 2.02 2.26 1.59 2.65 1.97 2.24

m dt (8.9, 2.2) m m m m d (7.8) t (7.8) t (7.8) d (7.8) m m m dt (13.6, 2.6) dt (13.6, 2.6) s

m m m m d (7.8) t (7.8) t (7.8) d (7.8) m m m d (14.2) dt (14.2, 2.7) t (7.3) m m m br s s

1.26 m 3.88 br s 3.76 s

2.48 m 4.27 d (1.2) 3.79 s

3.66 dd (10.8, 7.0) 3.55 dd (10.8, 2.0) 7.79 br s

7.93 br s

7.80 br s

c

Recorded at 600 MHz. Recorded at 400 MHz. ov: overlap. ammonia solution, were extracted with CHCl3 (3 × 1 L) and n-BuOH (3 × 1 L). The CHCl3 extract (6.3 g) was subjected to MCI gel column chromatography (H2O/MeOH, 1:0 to 0:1, v/v) to afford eight crude fractions (Fr. 1−8) based on TLC analysis. Fr. 6 (1.8 g) was chromatographed on silica gel CC (27 g) eluting with petroleum ether/CHCl3/EtOAc/Et2NH (4:1:1:0.006, v/v) to give 11 (9.8 mg), 14 (10.2 mg), 13 (4.2 mg), and 8 (3.8 mg). Fr. 7 (1.2 g) was separated by performing repeated Sephadex LH-20 CC eluting with CHCl3/ MeOH (1:1, v/v) to afford 10 (30.8 mg) and fraction 7A (0.4 g). Compounds 12 (3.2, mg), 9 (2.6 mg), and 6 (12.2 mg) were obtained by further separation of fraction 7A on silica gel CC (8 g) eluting with petroleum ether/acetone (4:1, v/v). Fr. 5 (0.1 g) was chromatographed on an ODS column (H2O/MeOH, 7:3 to 2:8, v/v), followed by reversed-phase preparative HPLC (MeOH/H2O, 6:4, v/v; flow rate, 2.0 mL/min), to afford 1 (1.3 mg, tR = 33.8 min) and 7 (1.1 mg, tR = 30.3 min). The n-BuOH extract (23.1 g) was subjected to MCI gel column chromatography with a H2O/MeOH (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, v/v) gradient system to give fractions M1−M6. Fr. M3 (1.2 g) was chromatographed on a reversed-phase column (30 g) using an ODS column and was eluted with 20% MeOH to afford three fractions, M3.A−M3.C. Fr. M3.B (0.12 g) was purified by reversedphase preparative HPLC (MeOH/H2O, 10:90, v/v; flow rate, 2.0 mL/ min) to yield 2 (22.1 mg, tR = 48.8 min), 3 (2.2 mg, tR = 48.6 min), 4 (0.9 mg, tR = 47.1 min), and 5 (1.8 mg, tR = 44.4 min). X-ray Diffraction Analysis of 2 and 6. All X-ray data were collected on a SuperNova, Dual, Eos diffractometer with Cu Kα radiation (λ = 1.541 84 Å). The structures were refined using fullmatrix least-squares on F2 using the ShelXL program. The crystallographic data have been deposited at the Cambridge Crystallographic Data Centre as CCDC 1040477 for 2 and CCDC 1040475 for 6. Crystal data of 2: C20H24N2O2 (fw = 324.41); orthorhombic, space group P212121; a = 6.33312(18) Å, b = 12.6666(3) Å, c = 25.6966(10) Å, α = β = γ = 90.00°; V = 2061.36(11) Å3, T = 173.0 K, Z = 4, Dc = 1.252 mg/mm3, F(000) = 840. A total of 11 382 reflections were collected in the range 3.44° ≤ θ ≤ 69.711°, of which 3517 unique reflections with I > 2σ(I) were collected for the analysis. Final R = 0.0542, Rw = 0.1452, and S = 1.096. Flack parameter = −0.02(12).

cytotoxicity against 786-O and HL-60 cell lines, with IC50 values of 27.3 and 48.5 μM, respectively.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter. Infrared spectra were recorded on a Nicolet NEXUS 670 FT-IR spectrometer. UV spectra were measured using a Shimadzu UV-260 spectrophotometer. ECD spectra were acquired with a JASCO J-720 spectropolarimeter. NMR spectra were recorded on a Varian Mercury-600BB or Bruker Avance III-400 instrument. HRESIMS data were measured on a Bruker APEXII mass spectrometer. X-ray diffraction data were collected on a SuperNova, Dual, Eos diffractometer; the structures were solved with the Superflip program using charge flipping and refined with the ShelXL program (using graphite-monochromated Cu Kα radiation). Column chromatography was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), Sephadex LH-20 (Amersham Pharmacia Biotech), MCI gel (CHP20P, 75−150 μM, Mitsubishi Chemical Industries Ltd.), and ODS (YMC, Kyoto, Japan). Semipreparative HPLC was carried out on a Waters 1525 binary pump system with a Waters 2489 detector (210 nm) using a YMC-Pack ODS-A (250 × 10 mm, 5 μm) column. Fractions were monitored by TLC, which was visualized by heating the silica gel plates after being sprayed with 5% H2SO4 in EtOH. Plant Material. The aerial parts of E. hainanensis were collected in Tunchang County, Hainan Province, People’s Republic of China, in July 2009 and identified by Dr. Qiong-Xin Zhong, Hainan Normal University, Haikou, People’s Republic of China. A voucher specimen (No. 2009021) was deposited at the Natural Product Laboratory of State Key Laboratory of Applied Organic Chemistry, Lanzhou University. Extraction and Isolation. The aerial parts of E. hainanensis (5.9 kg) were extracted with MeOH (3 × 15 L, 7 days each) at room temperature. Evaporation of the solvent gave a residue (580 g) that was partitioned between EtOAc (3 × 1 L) and 2% HCl solution (1 L). The acidic water-soluble materials, adjusted to pH 9−10 with 10% F

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Crystal data of 6: C21H26N2O4 (fw = 370.44); monoclinic, space group P1211; a = 8.82965(14) Å, b = 10.54451(18) Å, c = 9.86508(16) Å, α = γ = 90.00°, β = 90.6454(16)°; V = 918.42(3) Å3, T = 295.8(2) K, Z = 2, Dc = 1.340 mg/mm3, F(000) = 396. A total of 6779 reflections were collected in the range 6.539° ≤ θ ≤ 70.687°, of which 3364 unique reflections with I > 2σ(I) were collected for the analysis. Final R = 0.0578, Rw = 0.1514, and S = 1.039. Flack parameter = −0.1(3). Ervatamine A (1): white, amorphous powder; [α]20D +38 (c 0.3, CHCl3); IR (KBr) νmax 3351, 2925, 2854, 1734, 1654, 1597, 1453, 1247, 1209, 1040, 751 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 391.1638 [M + Na]+ (calcd for C21H24N2O4Na, 391.1628).

Ervatamine F (6): colorless blocks (MeOH); mp 189−190 °C; [α]20D −117 (c 1.0, MeOH); IR (KBr) νmax 3349, 2928, 2864, 1725, 1685, 1618, 1453, 1269, 1250, 1202, 1161, 1147, 1056, 753 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 371.1974 [M + H]+ (calcd for C21H27N2O4, 371.1965). Ervatamine G (7): white, amorphous powder; [α]20D −23 (c 0.3, CHCl3); IR (KBr) νmax 3378, 2934, 2863, 1729, 1460, 1434, 1253, 1236, 1163, 1068, 1037, 740 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 369.2182 [M + H]+ (calcd for C22H29N2O3, 369.2173). Ervatamine H (8): white, amorphous powder; [α]20D −60 (c 1.0, CHCl3); IR (KBr) νmax 3380, 2925, 2859, 1724, 1657, 1460, 1435, 1253, 1232, 1162, 1078, 1054, 741 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 371.1974 [M + H]+ (calcd for C21H27N2O4, 371.1965). Ervatamine I (9): white, amorphous powder; [α]20D −32 (c 0.6, CHCl3); IR (KBr) νmax 3376, 2930, 2862, 1712, 1460, 1435, 1257, 1235, 1175, 1063, 751 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 353.1849 [M + H]+ (calcd for C21H25N2O3, 353.1860). Preparation of (R)- and (S)-MTPA Esters of 6. (R)-MTPA chloride (10 μL) was added to a solution of 6 (4 mg) in anhydrous pyridine (100 μL). After 12 h at room temperature, the mixture was diluted with 0.5 mL of 1 M NaHCO3 and extracted with CH2Cl2. The residue of the extract was purified by reversed-phase preparative HPLC (MeOH/H2O, 1.5:0.5, v/v; flow rate, 2.0 mL/min) to yield the (S)-MTPA ester of 6 (2.1 mg, tR = 28.9 min). The (R)-MTPA ester of 6 was obtained by using the same procedure as described above. (S)-MTPA ester of 6 (6S): yellow, amorphous powder; 1H NMR (CDCl3, 400 MHz) δ 3.08 (1H, d, J = 11.0 Hz, H-3a), 2.65 (1H, m, H3b), 3.75 (1H, ddd, J = 14.3, 12.9, 3.5 Hz, H-5a), 2.68 (1H, m, H-5b), 2.07 (1H, ddd, J = 13.5, 13.2, 4.9 Hz, H-6a), 1.46 (1H, dd, J = 14.1, 2.6 Hz, H-6b), 7.54 (1H, m, H-9), 6.81 (1H, t, J = 7.8 Hz, H-10), 7.39 (1H, m, H-11), 6.77 (1H, d, J = 7.8 Hz, H-12), 1.92 (1H, m, H-14), 1.60 (1H, m, H-15a), 1.28 (1H, m, H-15b), 2.60 (1H, m, H-17a), 1.64 (1H, m, H-17b), 1.29 (1H, d, J = 6.1 Hz, H-18), 5.32 (1H, m, H-19), 1.67 (1H, m, H-20), 3.98 (1H, d, J = 2.4 Hz, H-21), 3.25 (1H, s, OMe); HRESIMS m/z 587.2358 [M + H]+ (calcd for C31H34N2F3O6, 587.2363). (R)-MTPA ester of 6 (6R): yellow, amorphous powder; 1H NMR (CDCl3, 400 MHz) δ 3.06 (1H, d, J = 11.7 Hz, H-3a), 2.63 (1H, m, H3b), 3.75 (1H, t, J = 12.7 Hz, H-5a), 2.68 (1H, m, H-5b), 2.06 (1H, ddd, J = 13.5, 13.4, 4.9 Hz, H-6a), 1.46 (1H, m, H-6b), 7.54 (1H, m, H-9), 6.81 (1H, t, J = 7.6 Hz, H-10), 7.39 (1H, m, H-11), 6.77 (1H, d, J = 7.6 Hz, H-12), 1.86 (1H, m, H-14), 1.40 (1H, m, H-15a), 1.09 (1H, d, J = 13.7 Hz, H-15b), 2.54 (1H, d, J = 13.8 Hz, H-17a), 1.62 (1H, m, H-17b), 1.42 (1H, d, J = 6.1 Hz, H-18), 5.30 (1H, m, H-19), 1.61 (1H, m, H-20), 3.99 (1H, br s, H-21), 3.26 (1H, s, OMe); HRESIMS m/z 587.2357 [M + H]+ (calcd for C31H34N2F3O6, 587.2363). NO Production Assay. Mouse monocyte-macrophage RAW264.7 cells were used in the anti-inflammatory assay. Cell culture, Griess, and MTT procedures and data analysis for the inhibition of NO production assay were the same as in the published protocol.31 Briefly, RAW 264.7 cells were cultured in RPMI 1640 medium supplemented with streptomycin (100 μg/mL), penicillin (100 U/ mL), and 10% heat-inactivated fetal bovine serum (Invitrogen, USA). The cells were harvested with trypsin-EDTA and diluted to a suspension in fresh medium. The suspended cells were seeded in 96well plates with 1 × 105 cells/well and allowed to adhere for 2 h at 37 °C in 5% CO2. The cells were treated with 1 μg/mL of LPS (Sigma, USA) for 24 h with or without various concentrations of test compounds. For the positive control group, the cells were co-incubated with indomethacin (Sigma, USA). The Griess reagent was used to determine NO production by measuring the accumulation of nitrite in the culture supernatant.33 Experiments were performed in triplicate. Cytotoxicity Assay. See ref 32.

Table 2. 1H NMR Data for Compounds 2−5 (δ in ppm, J in Hz) in Methanol-d4 2b

position 3a 3b 5a 5b 6a 6b 9 10 11 12 14a 14b 15 16 18a 18b 19 21a 21b 22a 22b 23 a

2.93 m 2.69 m 3.64 dt (12.8, 3.1) 3.51 br t (12.8) 3.73 br t (12.8) 3.25 br d (12.8) 7.43 d (7.6) 7.05 td (7.6, 1.2) 7.10 td (7.6, 1.2) 7.39 d (7.6) 2.16 m 2.09 m 3.77 m 4.14 br s 1.89 d (6.4)

3a

4a

5a

3.61 br t (11.5) 3.68 ov 3.50 m

3,67 m 3.35 m 3.73 dt (13.2, 3.0) 3.55 br t (13.2)

3.69 m 3.35 m 3.74 dt (13.0, 3.4) 3.58 br t (13.0)

3.85 ov 3.53 ov

3.88 m 3.56 ov

7.45 d (7.8) 7.01 t (7.8)

7.48 d (7.9) 7.04 t (7.9)

7.49 d (7.9) 7.05 t (7.9)

7.06 t (7.8)

7.10 t (7.9)

7.10 t (7.9)

7.31 2.49 2.29 3.98 4.05 1.78

7.35 3.17 2.44 4.24

7.36 3.18 2.44 4.12

3.86 ov 3.27 m 3.71 ov

c

d (7.8) m m m d (3.6) d (6.9)

5.87 m 4.04 ov 3.99 ov

5.45 m 3.86 ov 3.68 ov

2.97 s

3.09 s

d (7.9) m m d (12.2)

1.77 d (6.8)

5.68 4.10 3.41 3.94 3.87 3.04

q (6.8) d (14.6) m d (11.4) ov s

d (7.9) m m ov

4.23 dd (13.6, 7.8) 4.14 ov 5.74 t (6.3) 4.09 m 3.52 m 3.95 d (11.2) 3.92 ov 3.07 s

Recorded at 600 MHz. bRecorded at 400 MHz. cov: overlap.

Ervatamine B (2): colorless plates (EtOH); mp 243−244 °C; [α]20D −11 (c 1.0, MeOH); IR (KBr) νmax 3358, 2933, 2859, 1597, 1462, 1379, 1345, 1304, 1250, 1181, 750 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 325.1920 [M + H]+ (calcd for C20H25N2O2, 325.1911). Ervatamine C (3): colorless gum; [α]20D +80 (c 0.1, MeOH); IR (KBr) νmax 3378, 2926, 2855, 1594, 1463, 1380, 1344, 1301, 1260, 1186, 745 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 325.1917 [M + H]+ (calcd for C20H25N2O2, 325.1911). Ervatamine D (4): colorless gum; [α]20D +40 (c 0.1, MeOH); IR (KBr) νmax 3398, 2924, 2855, 1593, 1458, 1381, 1256, 1176, 1116, 1041, 784 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 355.2028 [M + H]+ (calcd for C21H27N2O3, 355.2016). Ervatamine E (5): colorless gum; [α]20D +140 (c 0.1, MeOH); IR (KBr) νmax 3367, 2926, 2857, 1623, 1597, 1462, 1383, 1344, 1259, 1195, 1051, 1021, 751 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 371.1974 [M + H]+ (calcd for C21H27N2O4, 371.1965). G

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Table 3. 13C NMR Data for Compounds 1−9 (δ in ppm)

a

position

1a,d

2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 COOCH3 CHO 24

147.7, C 52.3, CH2 65.5, CH2 111.5, 134.2, 109.2, 123.4, 123.5, 120.1, 126.7, 25.6, 21.9, 46.3, 36.0, 20.0, 71.3, 36.6, 58.8, 172.2,

C C CH CH CH CH C CH CH2 C CH2 CH3 CH CH CH C

2b,c 136.5, 58.2, 70.2, 23.2, 107.8, 127.6, 118.5, 120.6, 122.6, 112.6, 137.0, 23.2, 35.1, 52.1, 178.7, 14.3, 126.8, 135.8, 65.3,

C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH CH C CH3 CH C CH2

61.0, CH3

3a,c 138.1, 57.5, 70.6, 23.2, 106.9, 128.6, 118.3, 120.3, 122.4, 112.2, 136.8, 28.9, 34.5, 53.8, 178.8, 14.8, 128.5, 130.6, 66.5,

C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH CH C CH3 CH C CH2

61.9, CH3

4a,c 138.7, 59.3, 69.1, 24.2, 106.6, 129.0, 118.4, 120.5, 122.9, 112.5, 136.3, 24.5, 33.7, 57.7, 181.2, 14.0, 133.0, 129.5, 67.5, 68.0, 61.6,

5a,c

C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH C C CH3 CH C CH2 CH2 CH3

138.2, 59.3, 69.2, 24.2, 106.7, 128.9, 118.5, 120.6, 123.0, 112.5, 136.4, 24.4, 34.6, 58.9, 180.9, 58.6, 136.6, 131.1, 66.7, 67.8, 61.7,

53.3, CH3 183.4, CH

C CH2 CH2 CH2 C C CH CH CH CH C CH2 CH C C CH2 CH C CH2 CH2 CH3

6b,d 67.0, 52.5, 46.8, 25.2, 202.5, 121.1, 124.4, 119.5, 136.8, 112.3, 158.6, 26.2, 22.8, 51.6, 31.1, 19.9, 71.1, 38.2, 53.5, 174.2,

C CH2 CH2 CH2 C C CH CH CH CH C CH CH2 C CH2 CH3 CH CH CH C

52.0, CH3

7a,d 136.3, 59.9, 51.6, 21.8, 110.1, 128.6, 118.4, 119.4, 122.2, 110.4, 135.6, 30.9, 27.7, 55.0, 38.2, 11.8, 26.5, 38.4, 58.0, 175.5,

C CH CH2 CH2 C C CH CH CH CH C CH CH2 C CH2 CH3 CH2 CH CH C

52.7, CH3

8b,d 135.5, 48.8, 52.0, 21.3, 109.8, 128.4, 118.4, 119.5, 122.4, 110.5, 135.4, 35.3, 67.1, 53.8, 30.5, 20.9, 70.6, 50.8, 59.9, 174.5,

C CH2 CH2 CH2 C C CH CH CH CH C CH CH C CH2 CH3 CH CH CH C

53.0, CH3

9b,d 136.1, 50.9, 53.3, 21.7, 110.7, 128.7, 118.5, 119.5, 122.2, 110.5, 135.3, 26.8, 24.7, 54.1, 37.1, 27.7, 208.0, 50.9, 56.1, 174.9,

C CH2 CH2 CH2 C C CH CH CH CH C CH CH2 C CH2 CH3 C CH CH C

52.9, CH3

62.4, CH2

Recorded at 150 MHz. bRecorded at 100 MHz. cRecorded in methanol-d4. dRecorded in CDCl3.

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ASSOCIATED CONTENT

S Supporting Information *

Supplemetary data associated with this article (NMR, UV, IR, and HRESIMS spectra for compounds 1−9; computational details for 1). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jnatprod.5b00051.

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AUTHOR INFORMATION

Corresponding Author

*E-mail (K. Gao): [email protected]. Tel: +86-931-8912592. Fax: +86-931-8912582. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported financially by the National Basic Research Program of China (no. 2014CB138703), the NSFC (no. 31270396), and the 111 Project of China.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00051 J. Nat. Prod. XXXX, XXX, XXX−XXX