Monomeric and Dimeric Cytotoxic Guaianolide-Type

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Monomeric and Dimeric Cytotoxic Guaianolide-Type Sesquiterpenoids from the Aerial Parts of Chrysanthemum indicum Pan Luo,†,⊥ Yanfang Cheng,†,⊥ Zhiyong Yin,† Chanjuan Li,† Jun Xu,*,†,‡ and Qiong Gu*,†,‡ †

Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China ‡ Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, Guangzhou 510006, People’s Republic of China

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S Supporting Information *

ABSTRACT: Twelve new guaianolide-type sesquiterpenoids (1−12) and five known guaianolide derivatives (13−17) were isolated from an aqueous ethanol extract of the aerial parts of Chrysanthemum indicum. Their structures were determined through spectroscopic data analysis. The absolute configurations of the new compounds were assigned by X-ray crystallography and electronic circular dichroism. Compound 5 shows multiple cytotoxic activities against four human naso-pharyngeal carcinoma (NPC) cell lines (CNE1, CNE2, SUNE-1, and HONE-1) and one human intestinal epithelial cell line (HT-29) with IC50 values of 4.6, 6.0, 3.5, 4.3, and 9.6 μM, respectively. Compound 16 exhibits weak cytotoxicity against four NPC cell lines, CNE1 (IC50 = 7.3 μM), CNE2 (IC50 = 7.4 μM), HONE-1 (IC50 = 7.6 μM), and SUNE-1 (IC50 = 5.6 μM), but no cytotoxicity against HT-29 (IC50 > 10 μM). 533.2507 [M + Na]+ (calcd for C30H38O7Na, 533.2510) and C NMR data and has therefore 12 indices of hydrogen deficiency. The IR spectrum of 1 indicates the presence of hydroxy (3414 cm−1), carbonyl (1767, 1736 cm−1), and olefinic (1459 cm−1) functionalities. The 1H and 13C NMR spectroscopic data (Table 1) show 30 carbons including four methyl singlets (δH 1.26, 1.27, 1.43, and 1.84), three oxygenated methines (δC/H 68.7/4.04, 79.6/3.84, and 80.5/ 4.14), two oxygenated tertiary carbons (δC 73.4 and 75.7), an exocyclic double bond [δH 5.32, d, J = 3.2 Hz, 6.04, overlap; δC 118.8], and two carbonyl carbons (δC 171.2 and 179.5). Considering the reported structural type from C. indicum, compound 1 is most likely a guaianolide-type sesquiterpenoid dimer. Analysis of the 1D and 2D NMR spectra showed that compound 1 has the same 2D structure as deacetylhandelin, isolated from Handelia trichophylla.12 The NOESY correlations of H-7/H-5, H-5/H-1, H-6/H-8, H-8/H2-13, H3-15′/H-6′, and H-5′/H-7′ suggest that H-1, H-5, H-7, H-5′, and H-7′ could be assigned as α-oriented and H-6, H-8, H2-13, H-6′, and H3-15′ as β-oriented. Crystals of 1 were obtained in MeOH and subjected to X-ray diffraction using Cu Kα radiation (Figure 2). The absolute configuration of chrys-

Chrysanthemum indicum L. (Asteraceae), a well-known antiinflammatory and detoxifying traditional Chinese medicine, is widely spread in China and the tropics. The flowers of C. indicum L. have been used as chemotherapeutic agents against hypertension, inflammation, and cancer. A number of studies have also reported that this plant has activities against viral1−3 and bacterial infections,1 oxidation,4 osteoporosis,4 and immunomodulation.5 Previously, flavonoids4,6 and various sesquiterpenoids3,7−10 have been identified from this plant. In earlier studies, a new guaianolide-type sesquiterpenoid trimer (chrysanolide A), a dimer (chrysanolide C), and a monomer (chrysanolide B) were identified from an extract of the flowers of C. indicum.2 Recently, Kong et al. reported the isolation of a highly oxidized guaianolide-type sesquiterpenoid monomer from the aerial parts of C. indicum.11 This interesting result prompted us to investigate the chemical constituents of the aerial parts of C. indicum. Twelve new guaianolide monomers and dimers (1−12), along with five known analogues (13−17), from C. indicum were obtained. Herein, we report the isolation, structural characterization, and cytotoxicity of these isolates.

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13

RESULTS AND DISCUSSION

Compound 1, colorless crystals, has a molecular formula of C30H38O7 deduced from the sodium adduct ion at m/z © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 16, 2018

A

DOI: 10.1021/acs.jnatprod.8b00863 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

H-8/H2-13, H3-15′/H-6′, H-8′/H-6′, and H-5′/H-7′ indicate that H-1, H-5, H-7, H-5′, and H-7′ could be assigned αorientations and H-6, H-8, H2-13, H-6′, H3-15′ and H-8′ βorientations. Thus, the relative configuration of compound 3 was rel-(1R,5R,6R,7R,8S,10R,11R,1′R,4′R,5′S,6′R,7′R,8′S,10′R). Compound 4 (8-angeloyl-8′-hydroxychrysanolide D) has a molecular formula of C35H44O9 as defined by the HRESIMS ion at m/z 631.2890 [M + Na]+ (calcd for C35H44O9Na, 631.2878) and the 13C NMR data. The 1H NMR data of 4 (Table 1) are similar to those of compound 3, with the exception of an angeloyl [(2Z)-2-methylbut-2-enoyl] substituent at C-8, characterized by the key NOE cross-peaks of H-3″/H-4′′ and H-3′′/H-5′′ together with the 1H−1H COSY correlations of H-7 and H-8. In compound 4, the chemical shifts of C-4″ (δC 16.5) and C-5″ (δC 20.8) appear downfield from those of 3 (δC 14.8, 12.3), and because two methyls are cis in compound 3, that causes the γ-gauche effect. Based on the NOESY correlations of H-7/H-5, H-5/H-1, H-6/H-8, H-8/ H2-13, H3-15′/H-6′, H-8′/H-6′, and H-5′/H-7′, the relative configuration of compound 4 was elucidated as rel-(1R,5R,6R,7R,8S,10R,11R,1′R,4′R,5′S,6′R,7′R,8′S,10′R). The molecular formula of compound 5 (8,8′-ditigloylchrysanolide D) was assigned as C40H50O10 based on the HRESIMS ion at m/z 713.3300 [M + Na]+ (calcd for C40H50O10Na, 713.3296) and the 13C NMR data. Its 1D NMR data (Table 2) are similar to those of compound 3, and the additional tigloyl unit (δH 1.72, d, J = 7.2 Hz; 1.79, s, 6.85, overlap; δC 166.6, 129.0, 139.8, 14.8 and 12.2) is attached to C-8 based on the HMBC cross-peak of H-8 (δH 5.51)/C-1″ (δC 166.6). The NOESY correlations in compound 5 are identical to those of compound 3, and the relative configuration of 5 was established as rel-(1R,5R,6R,7R,8S,10R,11R,1′R,4′R,5′S,6′R,7′R,8′S,10′R). Compound 6 displays a sodium adduct ion at m/z 615.2939 [M + Na]+ (calcd for C35H44O8Na, 615.2928). Comparison of its 1D NMR data with those of 1 (Table 2) reveals the presence of a tigloyl fragment connected to C-8 in 6, and this

anolide D (1) was established as (1R,5R,6R,7R,8S,10R,11R,1′R,4′R,5′S,6′S,7′S,10′R). Based on a sodium adduct ion at m/z 633.3046 [M + Na]+ (calcd for C35H46O9Na, 633.3034) and the 13C NMR data, compound 2 has a molecular formula of C35H46O9. The NMR spectroscopic data of 2 (Table 1) are similar to those of compound 1, the exception being the absence of the exocyclic double bond and the presence of a methyl doublet and a (2E)2-methylbut-2-enoyl (tigloyl) moiety [δH 1.79, d (J = 7.2 Hz), 1.82, s, 6.85, m; δC 167.1, 128.6, 138.7, 14.8, and 12.3]. The key HMBC correlations of H-8′ (δH 5.13) with C-1″ (δC 167.1) and C-11′ (δC 42.4) indicate that the tigloyl unit in 2 is linked at C-8′. 1H−1H COSY cross-peaks of H3-13′/H-11′ and H-11′/H-7′ suggest that the methyl group in 2 is attached to C-11′, and this was confirmed by the HMBC cross-peaks of H3-13′ (δH 1.14, d, J = 7.2 Hz)/C-12′ (δC 178.8) and H3-13′ (δH 1.14)/C-7′ (δC 51.1). The relative configuration of compound 2 was defined as rel-(1R,5R,6R,7R,8S,11R,1′R,4′R,5′S,6′R,7′R,8′S,11′S) by the NOESY correlations of H-7/H-5, H-5/H-1, H-6/H-8, H-8/H2-13, H3-15′/H-6′, H-8′/H-6′, H313′/H-7′, H-11′/H-8′, and H-5′/H-7′. Biosynthetically, H-7 should have an α-orientation, similar to compound 1.2,3,11 Thus, the absolute configuration of chrysanolide E (2) was identified as (1R,5R,6R,7R,8S,10R,11R,1′R,4′R,5′S,6′R,7′R,8′S,10′R,11′S), and this was confirmed by comparison of the experimental electronic circular dichroism (ECD) spectrum of 2 in MeOH and the ECD of (10R,10′R)-2 (Figure 3). Compound 3, 8′-tigloylchrysanolide D, was determined to have a molecular formula of C35H44O9 through the MS (631.2876 [M + Na]+, calcd for C35H44O9Na, 631.2878) and 13 C NMR data. The NMR data of 3 are similar to those of 2 (Table 1), except for an exocyclic double bond (δH 5.34, d, J = 3.2 Hz; 6.06, d, J = 3.2 Hz; δC 120.7) and the absence of a methyl doublet (δH 1.14, d, J = 7.2 Hz; δC 14.8). HMBC correlations from H2-13′ (δH 5.34, 6.06) to C-7′ (δC 51.1) and C-12′ (δC 170.1) reveal the presence of the double bond at C11′. The NOESY correlations of H-7/H-5, H-5/H-1, H-6/H-8, B



Article

Table 1. 1H NMR and 13C NMR Data of Compounds 1−4 1a position

δC

1 2a 2b 3 4 5 6 7 8 9a 9b 10 11 12 13a 13b 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′a 8′b 9′a 9′b 10′ 11′ 12′ 13′a 13′b 14′ 15′ 1″ 2″ 3″ 4″ 5″

55.8 34.3 125.5 144.2 55.1 79.6 51.4 68.7 40.1 75.7 60.5 179.5 37.5 33.7 18.3 65.0 139.5 135.1 56.7 65.8 80.5 43.5 23.7 35.0 73.4 141.5 171.2 118.8 29.9 15.5

2a

δH (J in Hz) 2.45, 2.18, 2.05, 5.44,

m m m br s

2.69, 3.84, 3.17, 4.04, 2.21, 1.91,

overlap t (9.8) t (9.0) m overlap d (16.0)

2.38, 1.45, 1.26, 1.84,

d (12.0) overlap s s

6.03, overlap 5.97, d (5.2) 2.72, 4.14, 3.21, 2.19, 1.44, 1.82,

6.04, 5.32, 1.27, 1.43,

overlap t (9.8) m overlap overlap overlap

overlap d (3.2) s s

δC 56.0 34.2 125.5 144.5 55.2 79.6 52.0 68.1 40.0 72.9 60.4 179.3 37.5 33.7 18.4 64.3 140.7 134.0 56.8 65.3 76.6 51.1 74.1 44.0 75.9 42.4 178.8 14.8 30.0 15.6 167.1 128.6 138.7 14.8 12.3

3b

δH (J in Hz) 2.44, 2.19, 2.05, 5.45,

m m m br s

2.68, 3.85, 3.13, 4.11, 2.17, 2.01,

m t (10.0) t (9.0) m m m

2.44, 1.44, 1.29, 1.85,

56.1 34.2

overlap overlap s s

6.11, d (5.6) 5.91, d (5.6) 2.82, 4.14, 2.76, 5.13,

δC

d (9.6) t (10.0) m m

2.29, t (6.4) 1.88, overlap 2.26, overlap 1.14, d (7.2) 1.24, s 1.41, s

6.85, overlap 1.79, d (7.2) 1.82, s

125.5 144.6 55.3 79.6 52.2 68.0 40.0 73.1 60.0 179.1 37.2 33.7 18.5 64.5 140.6 134.0 57.1 65.6 76.2 47.9 73.1 43.1 76.0 138.8 170.1 120.7 30.0 15.6 167.1 128.6 139.0 14.8 12.3

4a

δH (J in Hz) 2.46, 2.19, 2.06, 5.46,

overlap m m br s

2.68, 3.86, 3.13, 4.14, 2.17, 2.01,

m t (10.0) t (8.8) m m m

2.46, 1.44, 1.30, 1.86,

overlap overlap s s

6.15, d (5.5) 5.90, d (5.5) 2.95, 4.17, 3.71, 5.19,

d (10.0) t (10.0) m m

2.24, dd (16.8, 5.8) 1.98, overlap

6.06, 5.34, 1.25, 1.46,

d (3.2) d (3.2) s s

6.89, m 1.80, d (7.0) 1.84, s

δC 55.0 33.7 125.9 144.6 54.7 79.0 47.4 70.4 38.5 73.7 59.8 178.3 36.8

δH (J in Hz) 2.73, 2.19, 2.05, 5.48,

m overlap m br s

2.54, 3.94, 3.63, 5.51, 2.25, 1.91,

m t (9.6) t (9.2) m overlap d (16.8)

2.22, 1.48, 1.20, 1.87,

overlap overlap s overlap

33.9 18.5 65.1 140.5 133.8 57.5 66.8 75.7 52.4 71.5

2.08, 3.99, 2.94, 3.82,

44.9

2.01, m

74.6 139.3 170.0 120.7 30.0 15.8 166.8 126.8 143.8 16.5 20.8

6.10, overlap 5.83, d (5.2)

6.10, 5.88, 1.33, 1.46,

m t (10.0) m m

overlap d (3.2) s s

6.18, m 2.00, overlap 1.86, overlap

a

Recorded in CDCl3 (1H NMR 400 MHz, 13C NMR 100 MHz). bRecorded in CDCl3 (1H NMR 500 MHz, 13C NMR 125 MHz).

Figure 1. Key 1H−1H COSY ( bold lines), HMBC (blue →), and NOESY (red dashed arrows) correlations of compound 1.

was characterized by the key HMBC cross-peaks from H-8 to C-1″ and C-11. Similar NOESY correlations suggest that compound 6 has the same relative configuration as 1. The relative configuration of 6 was defined as rel-(1R,5R,6R,7R,-

Figure 2. ORTEP drawing of compound 1.

C



Article

Figure 3. Experimental ECD spectra (190−400 nm) and TDDFT-calculated ECD spectra for compounds 2, 10, and 12.

8S,10R,11R,1′R,4′R,5′S,6′S,7′S,10′R), and the compound was named 8-tigloylchrysanolide D. Compound 7 (chrysanolide F), isolated as a colorless powder, gave a molecular formula of C15H22O4 based on an HRESIMS sodium adduct ion at m/z 289.1405 [M + Na]+ (calcd for C15H22O4Na, 289.1410) requiring five indices of hydrogen deficiency. Its 1D NMR data (Table 2) show that compound 7 is an isomer of chrysanolide B (15).2 The NOESY correlations of H3-13/H-8, H-6/H3-13, H-5/H-7, and H-5/H-1 indicate that H-1, H-5, H-7, and H-11 are α-oriented and H-6, H-8, and H3-13 (Figure 4) β-oriented. Hence, the relative configuration of 7 was concluded to be rel(1R,5R,6R,7R,8S,10R,11R) based on the same biosynthesis pathway as that of 15. Compound 8, colorless oil, has a molecular formula of C20H28O5. The 1D NMR data (Table 2) suggest that compound 8 is an isomer of 8α-angeloyloxy-10β-hydroxyslov-3-en-6,12-olide D,1 except for the angeloyl unit at C-8, which has been replaced by a tigloyl unit in compound 8 (Figures S61−S63, Supporting Information). The 8-tigloyl is supported by the key NOESY correlations of H-4′ and H-5′. Based on the NOESY cross-peaks of H3-13/H-8, H-6/H3-13, H-5/H-7, and H-5/H-1, the relative configuration of compound 8 was characterized as rel-(1R,5R,6R,7R,8S,10R,11R) and named 8-tigloylchrysanolide F. Compound 9 (chrysanolide G) possesses a molecular formula of C20H26O5 based on the HRESIMS ion at m/z 369.1656 [M + Na]+ (calcd for C20H26O5Na, 369.1672) and the 13C NMR data. The 1H and 13C NMR data (Table 3) indicate that this compound is similar to angeloylcumambrin B (16), which was identified from Chrysanthemum ornatum,13 except for the presence of the tigloyl fragment at C-8 in compound 9 instead of the angeloyl group in 16. The NOESY correlations of H-4′ and H-5′ indicated the presence of tigloyl group. Through extensive 2D NMR analysis, compound 9 was determined to be the monomer of the dimers 1−6 and 13. Based on the same biosynthesis pathway as for dimers 1−6 in combination with NOESY cross-peaks of H-5/H-7 and H-5/ H-1, the relative configuration of 9 was established as rel(1R,5R,6R,7R,8S,10R).

Compound 10 (chrysanolide H) has a molecular formula of C20H26O6. The 1H and 13C NMR data of 10 (Table 3) suggest five methyls, two oxygenated methines, an oxygenated tertiary carbon, an olefinic proton, two methylenes, three methines, and three carbonyls. These findings indicate that compound 10 is an analogue of compound 8. The ketocarbonyl group is located at C-3, and the Δ3(4) double bond has been replaced by a Δ4(5) double bond in 10 based on the key HMBC crosspeaks from H-15 to C-3 and C-5 (Figure 4). The key NOESY correlations of H3-13/H-7, H-11/H-8, H-11/H-6, H-8/H-6, and H-1/H-7 indicate that H-1 and H-7 are on the same face and are assigned α-orientations. H-6, H-8, and H-11 were assigned β-orientations. The calculated ECD spectrum of (1R,6S,7R,8S,10R,11S)-10 is consistent with the experimental data from 10 (Figure 3), and consequently its absolute configuration was defined as (1R,6S,7R,8S,10R,11S). Compound 11 has a molecular formula of C20H26O6. Its NMR data are highly similar to those of compound 10 (Table 3) and indicate that the tigloyl moiety at C-8 in 10 was substituted by an angeloyl unit in 11. Similarities of NOESY cross-peaks (Figure 4) between compounds 11 and 10 demonstrate that they have the same relative configuration. The relative configuration of compound 11 (8-angeloylchrysanolide H) was defined as rel-(1R,6S,7R,8S,10R,11S). Compound 12 (chrysanolide I) exhibits an HRESIMS sodium adduct ion at m/z 387.1790 [M + Na]+ (calcd for C20H28O6Na, 387.1778). The 1H and 13C NMR data of 12 (Table 3) display signals for five methyls, three oxygenated methines, an oxygenated tertiary carbon, an olefinic proton, two methylenes, three methines, and two carbonyls. The NMR data of compound 12 are closely similar to those of indicumolide A.8 Compound 12 differed from indicumolide A, however, in that the angeloyl unit at C-8 in indicumolide A has been replaced by a tigloyl unit in 12. This is confirmed by the key HMBC correlations shown in Figure 4. The NOESY cross-peaks of H3-13/H-7, H-11/H-8, H-11/H-6, H-6/H-8, H3-15/H-5, H-5/H-7, and H-3/H3-15 suggest that H-3, H-5, H-7, H3-13, and H3-15 should be α-oriented and H-6, H-8, and H-11 β-oriented. The absolute configuration of 12 was determined to be (3S,4R,5S,6S,7R,8S,11S) through comparD



Article


δC

1 2a 2b 3 4 5 6 7 8 9a 9b 10 11 12 13a 13b 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′a 8′b 9′a 9′b 10′ 11′ 12′ 13′a 13′b 14′ 15′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴ 3‴ 4‴ 5‴

54.5 33.7 125.8 144.7 54.8 79.1 47.6 70.9 38.4 73.7 59.9 178.4 37.1 33.9 18.5 64.6 140.5 133.8 57.3 65.9 75.6 47.3 73.0 43.1 72.6 138.9 169.5 120.6 30.0 15.6 166.6 129.0 139.8 14.8 12.2 167.0 128.7 138.8 14.8 12.4

6a

δH (J in Hz) 2.55, 2.17, 2.04, 5.48,

m overlap m br s

2.74, 3.94, 3.58, 5.51, 2.16, 1.91,

m t (10.0) t (9.2) m m m

2.35, 1.47, 1.19, 1.87,


6.10, d (5.5) 5.84, d (5.5) 2.17, 4.05, 3.47, 5.13,

overlap t (9.8) m m

2.16, overlap 1.91, overlap

6.03, 5.33, 1.22, 1.46,

d (3.4) d (3.4) s s

6.85, overlap 1.72, d (7.2) 1.79, s

δC 54.5 33.6 125.8 144.8 54.9 79.0 47.8 70.6 38.4 73.6 59.2 178.4 37.4 33.8 18.5 64.8 140.4 134.1 57.3 66.0 79.3 43.1 23.8 35.0 72.9 141.5 170.3 118.6 29.9 15.5 166.8 128.6 140.5 14.8 12.1

7a

δH (J in Hz) 2.54, 2.15, 2.02, 5.47,

m overlap m br s

2.72, 3.92, 3.51, 5.51, 2.21, 1.90,

m t (10.2) t (9.2) m dd (16.8, 4.4) overlap

2.30, 1.44, 1.17, 1.86,

55.7 34.3


6.10, d (5.6) 5.86, d (5.6) 1.91, 4.03, 2.92, 2.15, 1.38, 1.77,

overlap t (9.8) m overlap m m

5.99, 5.28, 1.25, 1.42,

d (3.4) d (3.4) s s

δC

125.5 144.3 55.7 81.8 50.6 66.9 40.7

8a

δH (J in Hz) 2.45, 2.19, 2.05, 5.44,

q (8.8) m overlap br s

2.63, 4.01, 2.99, 3.90, 2.07, 1.91,

t (8.8) t (10.4) m m overlap d (15.6)

75.5 37.6 180.3 12.4

1.22, d (7.6)

33.2 18.1

1.26, s 1.84, s

2.86, m

δC

δH (J in Hz)

53.5 33.8

2.52, q (8.3) 2.16, m

125.8 143.5 54.7 80.7 51.5 74.5 41.7

5.42, br s

73.5 42.1 178.5 15.4 31.8 17.7 167.1 128.5 138.6 14.7 12.3

2.67, 3.99, 2.80, 5.12, 2.24, 1.73,

t (9.2) t (10.3) q (10.3) m dd (16.0, 5.6) overlap

2.35, m 1.16, d (7.1) 1.14, s 1.79, s

6.82, m 1.76, d (7.0) 1.79, s

6.84, m 1.70, d (7.2) 1.75, s

6.89, overlap 1.82, d (7.2) 1.85, s

a

Recorded in CDCl3 (1H NMR 400 MHz, 13C NMR 100 MHz).

HONE-1, and HT-29 with IC50 values of 4.6, 6.0, 3.5, 4.3, and 9.6 μM, respectively. Compound 16, a sesquiterpenoid monomer, also displays weak cytotoxicity against four NPC cell lines, CNE1 (IC50 = 7.3 μM), CNE2 (IC50 = 7.4 μM), HONE-1 (IC50 = 7.6 μM), and SUNE-1 (IC50 = 5.6 μM), but no cytotoxicity in HT-29 (IC50 > 10 μM). Compound 2 selectively inhibits CNE2 (IC50 = 9.9 μM) and HONE-1 (IC50 = 9.2 μM) cell lines. Compared with 2, compound 3, with an exocyclic double bond, selectively inhibits the HONE-1 cell line with IC50 = 7.5 μM. Monomer 9 exhibits significant inhibition against CNE1 (IC50 = 7.9 μM), HONE-1 (IC50 =

ison of the experimental and calculated ECD spectral data (Figure 3). The structures of the known handelin (13),11 artanomalide C (14),15 chrysanolide B (15),2 angeloylcumambrin B (16),3,14,16 and tigloylcumambrin B (17)17 were elucidated by comparison with reported spectroscopic data. Compounds 1−17 were evaluated for their toxicity in four human NPC cell lines (CNE1, CNE2, HONE-1, and SUNE-1) and one human intestinal epithelial cell line, HT-29. Results are collated in Table 4. Compound 5, a sesquiterpenoid dimer, displays cytotoxic activity against CNE1, CNE2, SUNE-1, E



Article

Figure 4. Key 1H−1H COSY (bold lines), HMBC (blue →), and NOESY (red dashed arrows) correlations of compounds 7 and 10−12.


δC

1 2a 2b 3 4 5 6 7 8a 8b 9a 9b 10 11 12 13a 13b 14 15 1′ 2′ 3′ 4′ 5′

54.3 33.8 125.7 138.8 54.8 80.6 47.0 73.7 39.0 74.0 144.0 169.8 121.6 33.8 18.1 167.1 128.7 138.9 14.8 12.3

10a

δH (J in Hz) 2.58, 2.21, 2.08, 5.48,

m dd (15.4, 6.9) m br s

2.76, 4.00, 3.93, 5.19,

t (9.0) t (9.6) tt (9.3, 3.2) m

2.31, dd (16.7, 5.7) 1.86, overlap

6.13, 5.45, 1.21, 1.89,

d (3.1) d (3.1) s s

6.91, m 1.82, d (7.2) 1.86, s

δC 50.1 37.0 207.6 143.4 160.6 77.3 52.2 71.4 51.6 71.9 40.2 177.0 15.6 22.6 9.7 167.0 128.1 139.5 14.8 12.3

11a

δH (J in Hz)

δC

3.29, br s 2.53, m

50.1 37.0

4.98, d (10.0) 2.58, overlap 5.11, td (10.4, 3.7)

207.6 143.5 160.5 77.3 52.1 71.0

2.24, m 1.97, t (12.2)

51.6 72.0 40.1 177.0 15.4

2.59, overlap 1.29, d (6.4) 1.01, s 1.87, s

22.6 9.8 166.9 126.9 140.8 16.2 20.7

6.89, q (6.5) 1.81, overlap 1.82, s

δH (J in Hz)

12b δC

δH (J in Hz)

3.30, br s 2.54, overlap

137.3 39.2

4.98, d (10.4) 2.56, overlap 5.14, td (10.6, 3.6)

79.1 83.5 53.1 79.4 58.7 71.7

2.28, dd (12.8, 4.0) 2.01, overlap 2.62, m 1.30, d (6.8) 1.02, s 1.88, overlap

6.16, m 1.98, m 1.88, overlap

2.80, br d (16.0) 2.34, br d (16.0) 3.83, d (4.0) 2.70, 4.02, 2.26, 4.85,

42.2

d (10.1) t (10.1) q (10.6) td (10.6, 2.2)

2.46, overlap 2.17, dd (14.0, 2.5)

126.7 41.0 177.9 15.3 23.7 24.2 167.0 128.6 138.6 14.7 12.2

2.49, overlap 1.26, d (7.0) 1.74, s 1.54, s

6.87, m 1.79, m 1.82, overlap

a

Recorded in CDCl3 (1H NMR 400 MHz, 13C NMR 100 MHz). bRecorded in CDCl3 (1H NMR 500 MHz, 13C NMR 125 MHz).

7.9 μM), and SUNE-1 (IC50 = 3.0 μM). Compound 17, a monomer, selectively inhibits CNE1 (IC50 = 9.9 μM) and SUNE-1 (IC50 = 7.5 μM). In addition, compounds 2 and 16 display weak cytotoxicity against cell lines HONE-1 (IC50 = 9.2 μM; 7.6 μM) and CNE2 (IC50 = 9.9 μM; 7.4 μM). Compounds 3 and 9 show cytotoxic activity against the HONE-1 cell line with IC50 = 7.5 and 2.5 μM, respectively. Compounds 1, 4, 6, 8, and 10−15 show weak or no cytotoxicity, with IC50 > 10 μM against the five tested cell lines. As shown in Figure 5, compound 5 affects HONE-1 cell apoptosis from 1 to 20 μM. Compound 5 concentrationdependently induces G2/M cell arrest (Figure 6). Investigation

of the mechanism of action and structure−activity relation (SAR) for compound 5 is in progress in our laboratory.

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EXPERIMENTAL SECTION

General Experimental Procedures. The instruments and materials used in this study are the same as in the previous report18 and given in the Supporting Information. Plant Material. The aerial parts of C. indicum were collected from Hebe in Henan Province, People’s Republic of China, in September 2014, and their identity was authenticated by Dr. Jifeng Liu from Zhengzhou University. A voucher specimen (XG-2014003) has been deposited at the School of Pharmacy Sciences, Sun Yat-sen University. Extraction and Isolation. The air-dried aerial parts of C. indicum (6 kg) were extracted with 95% ethanol (4 × 50 L) at room temperature for 3 days to give a crude extract, which was suspended in F



Article

Table 4. Cytotoxicity of Compounds 1−17a IC50 (μM) ± SD no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 cisplatinb

CNE1 >10 >10 >10 >10 4.6 ± >10 >10 >10 7.9 ± >10 >10 >10 >10 >10 >10 7.3 ± 9.9 ± 9.0 ±

0.7

0.7

0.5 0.6 0.3

CNE2 >10 9.9 ± >10 >10 6.0 ± >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 7.4 ± >10 3.5 ±

0.2

1.3

0.4 0.8

HONE-1

SUNE-1

>10 9.2 ± 7.5 ± >10 3.5 ± >10 >10 >10 6.5 ± >10 >10 >10 >10 >10 >10 7.6 ± >10 9.0 ±

>10 >10 >10 >10 4.3 ± >10 >10 >10 3.0 ± >10 >10 >10 >10 >10 >10 5.6 ± 7.5 ± 1.7 ±

0.6 1.1 0.7

0.6

0.8 0.3

0.8

0.6

0.7 0.9 0.6

HT-29 >10 >10 >10 >10 9.6 ± 0.6 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 8.1 ± 0.5

Cytotoxicity is expressed as the mean value of three experiments ± SD. bCisplatin was used as positive control.

a

Figure 5. Effects of compound 5 on cell apoptosis in HONE-1 cells. (A) Cells were treated with compound 5 (0, 3, 5, 10, and 20 μM) for 48 h, stained with annexin-V FITC/PI, then analyzed by flow cytometry to evaluate apoptosis. (B) Histogram analysis of (A). H2O and extracted with EtOAc (3 × 1 L). The EtOAc fraction (300 g) was loaded on a silica gel (200−300 mesh) column using petroleum ether−EtOAc (10:0, 9:1, 4:1, 2:1, 0:1) as eluent to afford five fractions, A−E. Fraction D (30 g) was subjected to a C18 column (MeOH−H2O, 50% to 100%) to yield three fractions, D1−D3. Fraction D1 was chromatographed over a silica gel column eluting with petroleum ether−EtOAc (4:1 to 1:1) to give five subfractions (D1a−D1e). Compounds 8 (1 g) and 9 (600 mg) were obtained from subfration D1a by semipreparative HPLC (75% MeOH). Compound 17 (162 mg) was separated from D1c via semipreparative HPLC (70% MeOH). Fraction D2 was subjected to MPLC eluting with petroleum ether−EtOAc (5:1 to 1:1) to give four subfractions (D2a−D2d). Subfraction D2d was purified via semipreparative HPLC (75% MeOH) to yield 16 (5 mg). Fraction E (100 g) was loaded onto a C18 column (MeOH−H2O, 50% → 100%) to give fractions E1−E3. Fraction E1 was subjected to a silica gel column to give three subfractions (E1a−E1c). Subfraction E1a was subjected to a Sephadex LH-20 column with CH2Cl2−MeOH (1:1) as solvent to give E1a-1 to E1a-3. E1a-1 was subjected to semipreparative HPLC (60% MeOH) to yield 10 (29.5 mg), 11 (24.3 mg), and 12 (15 mg). Compounds 15

(34.3 mg) and 7 (15 mg) were obtained from fraction E1a-2 by semipreparative HPLC (65% MeOH). Fraction E2 was separated into five fractions (E2a−E2e) via silica gel column chromatography eluting with petroleum ether−EtOAc (4:1 to 1:2). Fraction E2b was subjected to a Sephadex LH-20 column to give subfraction E2b′, which was separated by semipreparative HPLC (80% MeOH) to afford 1 (9 mg), 2 (11 mg), 3 (10.8 mg), 4 (7 mg), 5 (24.4 mg), 6 (181 mg), 13 (10 mg), and 14 (5 mg). Chrysanolide D (1): colorless crystals; [α]20D +9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.23) nm; ECD (MeOH) λmax (log ε) 222 (−0.43) nm; IR (KBr) νmax 3414, 2963, 2927, 2858, 1767, 1736, 1459, 1378, 1261, 1088, 1025, 914, 796, 732, and 522 cm−1; 1H and 13 C NMR data, see Table 1; HRESIMS m/z 533.2507 [M + Na]+ (calcd for C30H38O7Na, 533.2510). Chrysanolide E (2): colorless oil; [α]20D +42 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.12) nm; ECD (MeOH) λmax (log ε) 214 (−0.18) nm; IR (KBr) νmax 3474, 2968, 2924, 2854, 1756, 1699, 1455, 1377, 1261, 1082, 1015, 919, 863, 799, 737, and 545 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 633.3046 [M + Na]+ (calcd for C35H46O9Na, 633.3034). G



Article

Figure 6. Compound 5 induces G2/M arrest in HONE-1 cells. (A) Cells were treated with 5 (0, 3, 5, 10, and 20 μM) for 48 h, stained with PI, then analyzed by flow cytometry to determine cell-cycle phases and distribution of cells (%). (B) Histogram analysis of (A). 8′-Tigloylchrysanolide D (3): colorless oil; [α]20D +42 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.52) nm; ECD (MeOH) λmax (log ε) 210 (−0.44), 225 (+0.13) nm; IR (KBr) νmax 3474, 2968, 2931, 2860, 1756, 1703, 1458, 1379, 1262, 1082, 1021, 913, 797, 731, and 545 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 631.2876 [M + Na]+ (calcd for C35H44O9Na, 631.2878). 8-Angeloyl-8′-hydroxychrysanolide D (4): colorless oil; [α]20D +24 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.25) nm; ECD (MeOH) λmax (log ε) 219 (−0.78) nm; IR (KBr) νmax 3482, 2968, 2927, 2854, 1748, 1705, 1457, 1375, 1354, 1261, 1157, 1091, 1022, 969, 908, 797, 732, 667, and 548 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 631.2890 [M + Na]+ (calcd for C35H44O9Na, 631.2878). 8,8′-Ditigloylchrysanolide D (5): colorless oil; [α]20D +54 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.40) nm; ECD (MeOH) λmax (log ε) 210 (−0.98), 229 (+0.25) nm; IR (KBr) νmax 3489, 2966, 2927, 2860, 1766, 1688, 1641, 1457, 1415, 1377, 1261, 1085, 1015, 866, 797, 736, and 699 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 713.3300 [M + Na]+ (calcd for C40H50O10Na, 713.3296). 8-Tigloylchrysanolide D (6): colorless oil; [α]20D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.37) nm; ECD (MeOH) λmax (log ε) 217 (−0.99) nm; IR (KBr) νmax 3499, 2963, 2933, 1753, 1699, 1648, 1454, 1378, 1345, 1260, 1146, 1086, 1030, 908, 804, 727, and 521 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 615.2939 [M + Na]+ (calcd for C35H44O8Na, 615.2928). Chrysanolide F (7): colorless solid; [α]20D +92 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 201 (3.76) nm; ECD (MeOH) λmax (log ε) 213 (−0.12) nm; IR (KBr) νmax 3258, 2966, 2921, 2854, 1772, 1684, 1454, 1384, 1261, 1217, 1146, 1033, 1000, 924, 798, 721, and 539 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 289.1405 [M + Na]+ (calcd for C15H22O4Na, 289.1410). 8-Tigloylchrysanolide F (8): colorless oil; [α]20D +86 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.07) nm; ECD (MeOH) λmax (log ε) 204 (+0.25) nm; IR (KBr) νmax 3455, 3401, 2962, 2929, 2913, 2854, 1772, 1753, 1702, 1648, 1446, 1376, 1267, 1224, 1182, 1147, 1116, 1074, 1024, 1002, 941, 806, 738, and 673 cm−1; 1H and 13 C NMR data, see Table 2; HRESIMS m/z 371.1804 [M + Na]+ (calcd for C20H28O5Na, 371.1829). Chrysanolide G (9): colorless oil; [α]20D +116 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.37) nm; ECD (MeOH) λmax (log ε) 209 (+0.42), 222 (+5.22), 264 (−0.46) nm; IR (KBr) νmax 3434, 2964, 2921, 2850, 1760, 1741, 1702, 1687, 1646, 1450, 1382, 1336, 1263, 1157, 1133, 1070, 1010, 941, 912, 815, 740, and 674 cm−1; 1H and

13

C NMR data, see Table 3; HRESIMS m/z 369.1656 [M + Na]+ (calcd. for C20H26O5Na, 369.1672). Chrysanolide H (10): colorless oil; [α]20D +144 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 201 (3.88), 230 (3.95) nm; ECD (MeOH) λmax (log ε) 236 (+0.62), 323 (−0.30) nm; IR (KBr) νmax 3493, 2969, 2936, 1787, 1693, 1643, 1459, 1373, 1253, 1192, 1095, 1060, 1009, 973, 797, 734, and 545 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 385.1613 [M + Na]+ (calcd for C20H26O6Na, 385.1622). 8-Angeloylchrysanolide H (11): colorless oil; [α]20D +114 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 201 (3.95), 231 (4.16) nm; ECD (MeOH) λmax (log ε) 208 (−0.36), 238 (+0.95), 323 (−0.06) nm; IR (KBr) νmax 3482, 2963, 2937, 2880, 1784, 1681, 1639, 1448, 1373, 1302, 1207, 1160, 1091, 1004, 949, 798, 737, and 556 cm−1; 1H and 13 C NMR data, see Table 3; HRESIMS m/z 385.1624 [M + Na]+ (calcd for C20H26O6Na, 385.1622). Chrysanolide I (12): colorless oil; [α]20D +9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.11) nm; ECD (MeOH) λmax (log ε) 206 (−0.94), 222 (+0.75) nm; IR (KBr) νmax 3570, 2968, 2942, 2856, 1756, 1700, 1505, 1446, 1383, 1259, 1171, 1083, 1048, 1014, 971, 925, 801, 733, 627, and 522 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 387.1790 [M + Na]+ (calcd for C20H28O6Na, 387.1778). X-ray Crystallographic Analysis of Compound 1. The absolute configuration of 1 was determined by single-crystal X-ray diffraction analysis. The crystal data were collected from an Xcalibur, Onyx, Nova diffractometer at 100 K. The programs XS and olex2.refine software were used to solve and refine the structure. The crystal figures was prepared using the SHELX programs with the program X-Seed as an interface. Crystal data for 1: C30H38O7 (M = 510.60); colorless plate, space group P212121 (no. 0), a = 9.74469(7) Å, b = 11.28356(7) Å, c = 11.95583(9) Å, V = 1284.314(16) Å3; Z = 2, T = 100 K, μ(Cu Kα) = 0.755 mm−1, Dcalc = 1.320 g/cm3, 24 724 reflections collected, 5045 unique (Rint = 0.0512). The final R1 was 0.0326 and wR2 was 0.0849 (all data). Flack parameter = −0.10(7). Crystallographic data for 1 (CCDC 1817347) are available from the Cambridge Crystallographic Data Centre. Bioassay. The methods for bioactivity have been detailed in the Supporting Information. H



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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00863. IR, NMR, and HRESIMS data of compounds 1−12 (PDF) X-ray crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Authors

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

Jun Xu: 0000-0002-1075-0337 Qiong Gu: 0000-0001-6011-3697 Author Contributions ⊥

P. Luo and Y.-F. Cheng contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financial supported by the Science and Technology Program of Guangzhou (201604020109) and Administration of Traditional Chinese Medicine of Guangdong Province, People’s Republic of China (20171048).

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REFERENCES

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Monomeric and Dimeric Cytotoxic Guaianolide-Type

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