Cytotoxic Tigliane Diterpenoids from Croton damayeshu | Journal of

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Cite This: J. Nat. Prod. 2019, 82, 1550−1557

Cytotoxic Tigliane Diterpenoids from Croton damayeshu Jiao-Jiao Cui,†,‡ Kai-Long Ji,† Hong-Chun Liu,† Bin Zhou,† Qun-Fang Liu,† Cheng-Hui Xu,† Jian Ding,† Jin-Xin Zhao,*,† and Jian-Min Yue*,†,‡ †

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People’s Republic of China ‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, People’s Republic of China

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S Supporting Information *

ABSTRACT: Chemical investigation of an EtOH extract of the twigs and leaves of Croton damayeshu afforded 10 new tigliane diterpenoids, crodamoids A−J (1−10), along with five known compounds. Their structures were elucidated by physical data analysis. Compounds 8, 9, and 15 displayed cytotoxic effects against two human tumor cell lines, A549 and HL-60 (IC50: 0.9−2.4 μM).

T

igliane diterpenoids, possessing a 5,7,6,3-fused tetracyclic framework, are the characteristic chemical components of the Euphorbiaceae and Thymelaeaceae plant families.1 This compound class has attracted considerable interest from the research communities of natural products, pharmacology, and synthetic chemistry due to their intriguing structures with important bioactivities, including anticancer,2,3 cyclooxygenase inhibitory,4 anti-HIV,5,6 proinflammatory,7,8 and INF γinducing activities.9 Particularly, phorbol esters of tigliane diterpenoids are well known for activating protein kinase C (PKC), which plays important physiological roles in different cellular metabolic activities and signal transduction pathways.10 Among the phorbol esters, tigilanol tiglate (Figure S0, Supporting Information) is in phase I clinical trials on patients with cutaneous or subcutaneous solid tumors,11 and phorbol 12myristate-13-acetate (Figure S0, Supporting Information), a promising agent for treating acute myeloid leukemia, is in phase II clinical trials.12 The Croton genus (Euphorbiaceae family) possesses over 1300 species that grow mostly in the tropical and subtropical zones.13 In our continuing search for biologically important tigliane diterpenoids from Croton species,2 10 new tigliane diterpenoids (1−10) and five known analogues (11− 15) were isolated for the first time from the aerial parts of Croton damayeshu, which is mainly distributed in Yunnan Province, China.14 Structural elucidation of these compounds was conducted by comprehensive analyses of the NMR, singlecrystal X-ray crystallography, and electronic circular dichroism (ECD) data. Cytotoxic tests revealed that compounds 8, 9, and 15 exhibited significant activities against lung carcinoma (A549) and human acute myeloid leukemia (HL-60) cell lines. Particularly, compound 8 displays selective activity that is comparable to adriamycin against the A549 cell line. The structure−activity relationship (SAR) for these tigliane diterpenoids is briefly discussed. © 2019 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Crodamoid A (1), [α]27D = −26 (c 0.5, MeOH), was obtained as colorless crystals. It possessed a molecular formula of C29H42O7 requiring nine indices of hydrogen deficiency (IDH) based on a sodium adduct (+)-HRESIMS ion at m/z 525.2826 (calcd 525.2828) and the 13C NMR data (Table 3). The characteristic Received: January 16, 2019 Published: May 22, 2019 1550

DOI: 10.1021/acs.jnatprod.9b00042 J. Nat. Prod. 2019, 82, 1550−1557

Journal of Natural Products

Article

Table 1. 1H NMR (400 MHz) Data of 1−5 in CDCl3 [δH, mult (J in Hz)] 1a

2a

3a

4a

5a

1 4 5β 5α 7 8 10 11 12 14 16 17 18 19 20a 20b 2′

7.06, br s 2.79, ddd (6.7, 5.1, 3.2) 2.48, dd (15.5, 5.1) 3.45, dd (15.5, 3.2) 5.12, br s 1.96, br s 3.51, m 1.69, m 5.47, d (10.4) 0.76, d (5.0) 1.17, s 1.21, s 1.08, d (6.3) 1.78, br s 4.02, dd (12.5, 5.8) 3.90, dd (12.5, 4.9) 2.43, m

7.05, br s 2.77, ddd (6.9, 5.1, 3.1) 2.48, dd (15.7, 5.1) 3.34, dd (15.7, 3.1) 5.13, br s 1.93, br s 3.49, m 1.55, dq (9.8, 6.4) 3.97, m 0.73, d (5.3) 1.18, s 1.21, s 1.27, d (6.4) 1.76, br s 4.00, br d (12.5) 3.89, br d (12.5)

7.06, br s 2.79, ddd (6.6, 5.1, 3.0) 2.48, dd (15.6, 5.1) 3.46, dd (15.6, 3.0) 5.12, br s 1.96, br s 3.51, m 1.69, m 5.48, d (10.4) 0.74, d (5.0) 1.17, s 1.21, s 1.08, d (6.4) 1.78, br s 4.02, br d (12.5) 3.90, br d (12.5) 2.43, m

7.08, br s 2.81, ddd (6.6, 5.2, 3.0) 2.50, dd (15.6, 5.2) 3.48, dd (15.6, 3.0) 5.15, br s 2.03, m 3.53, m 1.84, m 5.72, d (10.3) 0.81, d (5.0) 1.18, s 1.36, s 1.13, d (5.1) 1.80, br s 4.04, br d (12.4) 3.91, br d (12.4)

3′

1.52, m 1.70, m 0.96, t (7.4) 1.20, d (7.0)

1.52, m 1.70, m 0.96, t (7.4) 1.20, d (7.0)

8.06, m

7.04, br s 2.79, m 2.48, dd (15.6, 5.1) 3.45, dd (15.6, 3.1) 5.12, br s 1.95, br s 3.50, m 1.67, m 5.47, d (10.4) 0.73, d (5.1) 1.18, s 1.21, s 1.09, d (6.4) 1.78, br s 4.02, dd (12.5, 5.5) 3.90, dd (12.5, 4.7) 2.31, m 2.38, m 1.71, m

position

4′ 5′ 6′ 7′ 2″ 3″ 4″ 5″ 9-OH 20-OH

2.54, m 1.14, d (7.0)b,c

2.56, p (7.0) 1.15, d (7.0)b,c

1.16, d (7.0)b,c

1.16, d (7.0)b,c

5.26, s 2.37, m

2.34, m 1.70, m 1.43, m 0.91, t (7.4) 1.11, d (7.0) 5.30, s

a

7.49, dd (8.3, 7.8) 7.61, t (7.4) 7.49, dd (8.3, 7.8) 8.06, m 2.36, m 1.73, m 1.43, m 0.94, t (7.4) 1.13, d (6.8) 5.40, s

1.00, t (7.4)

2.35, m 1.70, m 1.43, m 0.92, t (7.5) 1.11, d (7.0) 5.30, s

The H atoms of the substituents at C-12 were numbered with H-n′, while those at C-13 were numbered with H-n″. assignments.

IR absorptions at 3417, 1716, and 1635 cm−1 indicated the presence of hydroxy, carbonyl, and olefinic functionalities. With the assistance of DEPT and HSQC spectra [Figure S10, Supporting Information], its NMR data (Tables 1 and 3) showed resonances for three carbonyl carbons, two trisubstituted double bonds, eight methyls, three methylenes (one oxygenated), eight methines, two oxygenated tertiary carbons, and one quaternary carbon. The carbonyls and the double bonds accounted for five out of nine IDHs, which required the presence of four rings in compound 1. Further examination of the 2D NMR spectra constructed the 2D structure of 1. First, five spin coupling units were identified via the 1H−1H COSY spectrum (Figures 1A and S11, Supporting Information). The connection of the five structural units with other functional groups was then made via the HMBC spectrum (Figures 1A and S12, Supporting Information), in which the correlations of H3-19/C-1, C-2, and C-3; H-4/C-3; H2-5/C-3, C-6, and C-7; H-7/C-9; H-10/C-9; H3-18/C-9; H14/C-9 and C-13; and H-12/C-13 permitted the construction of rings A−C and the attachment of Me-18 and Me-19. The D-ring, with two geminal methyls (Me-16/17), was fused to the C-ring according to the HMBC correlations of H3-17/C-13 and H3-16/ C-14, C-15, and C-17. The hydroxymethyl unit was located at C6 via the HMBCs of H2-5/C-20 (δC 69.5) and H2-20 (δH 3.90, 4.02)/C-6 and C-7. A hydroxy group was placed at C-9 by the HMBCs of OH-9/C-9 and C-10 and the chemical shift of C-9

b,c

Interchangeable

(δC 78.2). The 1H−1H COSY correlations of H3-5′/H-2′/H23′/H3-4′ (Figures 1A and S11, Supporting Information) confirmed the 2-methylbutanoate group (δC 176.0, 42.0, 26.9, 11.8, 17.3), and the HMBC correlation from H-12 (δH 5.47) to C-1′ (δC 176.0) indicated that this unit was located at C-12. The 13-isobutyryloxy group as identified by the key HMBC correlation of H3-4″/C-1″ was allocated by the chemical shift of C-13 (δH 65.0).15 The relative configuration of 1 was determined via the NOESY spectrum (Figures 1B and S13, Supporting Information), in which the cross-peaks of H-10/H-4, 9-OH/H-5α, 9OH/H-12, 9-OH/H-14, H-14/H3-16, H-10/H3-18, and H3-18/ H-12 suggested that all the protons were cofacial, and they were randomly assigned as α-oriented. The NOESYs of H-8/H-11, H-11/H3-17, and H-8/CH3-17 thus suggested β-orientations for H-8, H-11, and H3-17. Finally, the X-ray diffraction study of crodamoid A verified this assignment and established the absolute configuration as (4R, 8S, 9R, 10R, 11R, 12R, 13S, 14R, 2′S) (Figure 6) [Flack parameter = −0.10(9)].16 Crodamoid B (2) gave a molecular formula of C24H34O6 as deduced from the 13C NMR data (Table 3) and the sodium adduct (+)-HRESIMS ion at m/z 441.2254 (calcd 441.2253). Scrutiny of the NMR data (Tables 1 and 3) of 2 suggested the close similarity of its structure with that of 1, and the only difference was the replacement of the 2-methylbutanoate unit in 1 by a hydroxy unit at C-12 in 2 as judged by the shielded H-12 1551



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Table 2. 1H NMR (400 MHz) Data of 6−10 in CDCl3 [δH, mult (J in Hz)] 6a

7a

8a

9a

10a

7.05, br s 2.74, m 2.42, dd (16.0, 4.3) 3.34, dd (16.0, 3.5) 5.10, br s 1.90, br s 3.47, m 1.53, m 3.95, m 0.71, br d (5.3) 1.19, s 1.18, s 1.23, d (6.2) 1.73, br s a 3.96, br d (12.8) b 3.87, br d (12.8)

7.01, br s 2.84, ddd (7.7, 4.5, 4.1) 3.27, dd (16.0, 4.4) 3.08, dd (16.0, 3.2) 6.08, br s 2.25, m 3.54, m 1.63, dq (9.8, 5.5) 4.00, d (9.8) 0.82, d (5.5) 1.26, s 1.29, s 1.29, d (5.5) 1.74, br s 9.30, s

7.02, br s 2.86, ddd (7.0, 4.4, 3.0) 3.27 dd (15.8, 4.4) 3.17 dd (15.8, 3.0) 6.07, m 2.28, dd (5.3, 3.0) 3.55, m 1.78, m 5.54, d (10.3) 0.83, d (5.3) 1.24, s 1.32, s 1.09, d (6.4) 1.76, br s 9.32, s

7.01, br s 2.86, m 3.28, dd (15.9, 4.5) 3.07, dd (15.9, 3.1) 6.08, m 2.26, m 3.55, m 1.64, m 4.00, d (9.9) 0.83, d (5.5) 1.26, s 1.30, s 1.29, d (6.8) 1.75, br s 9.32, s

3′ 4′ 5′ 2″ 3″

7.06, br s 2.79, ddd (6.4, 5.1, 3.2) 2.49, dd (15.7, 5.1) 3.45, dd (15.7, 3.1) 5.13, br s 1.98, br s 3.51, m 1.75, m 5.46, d (10.3) 0.80, d (5.1) 1.18, s 1.24, s 1.09, d (6.4) 1.79,br s a 4.03, br d (13.2) b 3.91, br d (13.2) 6.93, m 1.84, br d (7.1) 1.87, m 2.48, m 4.21, m

4″ 5″ 9-OH

1.21, d (6.5) 1.13, d (7.1) 5.25, s

1.19, br d (6.5) 1.12, br d (7.1)

position 1 4 5β 5α 7 8 10 11 12 14 16 17 18 19 20

2.50, m 4.17, m

2.40, m 1.69, m 1.46, m 0.91, t (7.5) 1.14, d (7.0)

6.89, m 1.84, br d (7.3) 1.88, m 2.36, m 1.71, m 1.43, m 0.92, t (7.4) 1.11, d (7.0) 5.41, s

2.59, m 1.18, d (7.0) 1.18, d (7.0)

a

The H atoms of the substituents at C-12 were numbered with H-n′, while those at C-13 were numbered with H-n″.

highly similar to those of 4, indicating that they were structural analogues. Inspection of its NMR data showed that an nbutanoate group in 5 replaced the benzoate unit at C-12 in 4, which was supported by the HMBC (Figures S4A and S49, Supporting Information) and 1H−1H COSY correlations (Figures S4A and S48, Supporting Information), in particular the crucial HMBC correlation of H-12/C-1′ of the n-butanoate group, as well as the 1H−1H COSY correlations of H-2′/H-3′/ H-4′ within the n-butanoate group. The relative configuration of 5 was allocated as identical to that of 4 by the NOESY data (Figures S4B and S50, Supporting Information). Crodamoid F (6), colorless crystals, possessed a molecular formula of C30H42O8 as determined by the (+)-HRESIMS and the 13C NMR data (Table 3). Inspection of its 1D and 2D NMR spectra revealed that it is structurally similar to the coexisting known compound 12.17 The only difference was the presence of a 3-hydroxy-2-methylbutanoate moiety (δH 2.48, 4.21, 1.21, and 1.13; δC 177.4, 46.3, 67.5, 19.9, and 9.5) in 6 instead of the acetate group in 12. The 12-O-tigloyl group was assigned by the HMBC correlation (Figures 2A and S58, Supporting Information) from H-12 (δH 5.46) to the carbonyl carbon of the tigloyl moiety, while the attachment of the 3-hydroxy-2-methylbutanoate moiety to C-13 in 6 was established by comparison of the relevant NMR data with those of 12. The above deduction and the relative configuration of 6 were verified by a single-crystal Xray diffraction study (Figure 7) by using Ga Kα radiation [Flack parameter of −0.1(5)].16 The absolute configuration of 6 was confirmed by comparison of its ECD curves with those of 1 in the 190−400 nm range (Figure 4). Crodamoid G (7) had a molecular formula of C25H36O7 as determined by the method used above. Scrutiny of its 1D and 2D NMR data showed that it is a structurally closely related analogue of 6, and the difference was the replacement of the

(δH 3.97, m). The above assignment was supported by analysis of the HMBC spectrum (Figures S1A and S21, Supporting Information). The relative configuration of 2 was identical to 1 following analysis of the NOESY spectrum (Figures S1B and S22, Supporting Information). A molecular formula of C30H44O7 was assigned for crodamoid C (3) by the 13C NMR data (Table 3) and the sodium adduct ion at m/z 539.2972 (calcd 539.2985) in the (+)-HRESIMS. Its 1D NMR data (Tables 1 and 3) were highly similar to those of 1, and the minor differences resulted from the presence of two 2methylbutanoate groups in 3. One 2-methylbutanoate unit was attached to C-12 by the HMBC correlation from H-12 (δH 5.48) to C-1′ (δC 176.0) (Figures S2A and S30, Supporting Information), and the other one was located at C-13 (δC 65.0) by the chemical shift of C-13 and comparing the NMR data with those of 1. The relative configuration of 3 was allocated as identical to that of 1 on the basis of the NOESY spectrum (Figures S2B and S31, Supporting Information). Crodamoid D (4) displayed a molecular formula of C32H40O7 as assigned by the 13C NMR data (Table 3) and the sodium adduct ion at m/z 559.2673 (calcd 559.2672) in the (+)-HRESIMS. The 1H and 13C NMR data (Tables 1 and 3) suggested a strong resemblance of its structure to that of 3 with the differences resulting from the C-12 substituent. A benzoate unit at C-12 for 4 was identified based on the HMBC correlation (Figures S3A and S39, Supporting Information) from H-12 (δH 5.72) to C-1′ (δC 166.1) of the benzoate group. The relative configuration of 4 was assigned via the NOESY spectrum (Figures S3B and S40, Supporting Information). (+)-HRESIMS data analysis of crodamoid E (5) revealed a molecular formula of C29H42O7 based on 13C NMR data (Table 3) and the sodium adduct ion at m/z 525.2825 (calcd 525.2828). The 1D NMR data (Tables 1 and 3) of 5 were 1552



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Table 3. 13C NMR Data of 1−10 in CDCl3 position

1a,b

2 a,c

3a,c

4a,c

5a,c

6a,c

7a,c

8a,b

9a,c

10a,c

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″

156.3 143.5 213.4 49.8 25.3 137.2 126.5 40.9 78.2 47.5 43.3 75.2 65.0 37.2 25.4 24.3 16.6 12.0 10.6 69.5 176.0 42.0 26.9 11.8 17.3

156.7 143.4 213.3 49.6 25.3 136.7 126.9 40.8 78.1 47.7 45.5 75.5 67.4 36.1 26.0 24.2 16.6 12.5 10.6 69.4

156.3 143.5 213.4 49.8 25.3 137.2 126.6 40. 9 78.2 47.5 43.4 75.3 65.0 37.3 25.5 24.3 16.7 12.0 10.6 69.5 176.0 42.0 26.9 11.8 17.3

156.3 143.5 213.4 49.8 25.3 137.2 126.6 40.9 78.2 47.5 43.5 75.6 64.9 37.3 25.6 24.3 16.6 12.1 10.6 69.5 173.2 36.6 18.9 13.6

156.2 143.6 213.3 49.8 25.4 137.3 126.3 40.8 78.2 47.5 43.6 76.6 65.4 37.5 25.7 24.3 16.7 12.1 10.6 69.4 168.5 128.4 139.0 14.7 12.4

155.8 143.2 213.5 49.6 25.3 136.5 126.7 40.9 78.1 47.6 45.3 74.6 67.8 36.2 25.4 24.4 16.4 12.4 10.5 69.3

155.2 143.7 210.5 48.6 20.9 140.5 152.9 42.0 77.7 47.2 45.1 75.3 67.0 35.3 26.1 24.2 16.6 12.5 10.6 194.4

154.9 143.7 210.5 48.8 21.0 140.9 152.5 42.0 77.9 47.1 43.5 75.1 64.8 36.5 25.9 24.2 16.8 12.0 10.6 194.4 167.5 128.6 138.0 14.7 12.5

155.1 143.7 210.4 48.6 20.9 140.5 152.7 42.0 77.8 47.2 45.1 75.3 67.1 35.3 26.3 24.2 16.6 12.5 10.6 194.4

179.1 34.4 18.7d 18.6d

179.8 34.3 18.8e 19.0e

178.8 41.5 26.3 11.8 16.3

156.3 143.6 213.3 49.8 25.3 137.2 126.5 41.0 78.3 47.5 43.9 76.7 65.0 37.6 25.8 24.3 16.9 12.1 10.6 69.5 166.1 130.3 129.9 128.6 133.3 128.6 129.9 179.0 41.5 26.3 11.8 16.4

178.9 41.5 26.3 11.8 16.4

177.4 46.3 67.5 19.9 9.5

177.7 45.8 68.2 20.0 10.4

179.4 41.3 26.5 11.8 16.5

179.1 41.4 26.2 11.8 16.3

179.8 34.3 18.8f 19.0f

a

The C atoms of the substituents at C-12 were numbered with C-n′, while those at C-13 were numbered with C-n″. b,cData were measured at 150 and 125 MHz, respectively. d−fInterchangeable assignments.

suggested that it was the 12-tigloylate of 8, which was defined via the chemical shift of H-12 (δH = 5.54), the HMBC correlations within the tigloyloxy motif, and the key HMBC correlation from H-12 to the carbonyl carbon (δC 167.5) of the tigloyloxy unit (Figures S6A and S88, Supporting Information). The relative configuration of 9 was determined by analyzing the NOESY spectrum (Figures S6B and S89, Supporting Information) and comparing the NMR data with 8. Crodamoid J (10) gave a molecular formula of C24H32O6 as deduced from the 13C NMR data (Table 3) and a sodium adduct ion in the (+)-HRESIMS at m/z 439.2101 (calcd 439.2097). The 1D NMR data of 10 (Tables 2 and 3) were similar to those of 2, and the only difference was a formyl group in 10 replacing the hydroxymethyl group at C-6 in 2, based on the changes of the chemical shifts of C-20 (δC 194.4) and H-20 (δH 9.32, s). The HMBC correlations (Figures S7A and S97, Supporting Information) from H2-5 and H-7 to C-20 confirmed the above assignment. As the C-20 oxidation product of 2, the relative configuration of 10 was assigned as identical to that of 2 via the NOESY data (Figures S7B and S98, Supporting Information) and biosynthetic considerations. Both the 2-methylbutanoate substituents at C-12 in 1 and C13 in 8 possessed the (2S)-configuration. Based on the biosynthetic grounds and comparison of the relevant NMR resonances,18 all the 2-methylbutanoate groups in 3−5 were

tigloyloxy group by a hydroxy group at C-12 in 7. This conclusion was indicated by the shielded chemical shift of H-12 (ΔδH = −1.51) and analysis of the HMBC spectrum (Figures S5A and S68, Supporting Information). Crodamoid H (8) had a molecular formula of C25H34O6 as established by the 13C NMR and (+)-HRESIMS data. Its 1D NMR data (Tables 2 and 3) supported by the HSQC spectrum (Figure S76, Supporting Information) revealed that its structure showed many similarities to compound 3. A formyl group located at C-6 in 8 replaced the hydroxymethyl group in 3 based on the chemical shifts of C-20 (δC 194.4) and H-20 (δH 9.30) and the HMBC correlations from H-20 to C-6 and C-7. The H12 of 8 was strongly shielded (ΔδH = −1.48) as compared with that of 3, suggesting that an OH-12 in 8 replaced the 2methylbutanoate moiety at C-12 in 3, which was supported by the HMBC data (Figures 3A and S78, Supporting Information). The relative configuration of 8 was established by the NOESY data (Figures 3B and S79, Supporting Information). Finally, the X-ray diffraction study (Figure 8) using Ga Kα radiation of 8 determined its absolute configuration as 4R, 8S, 9R, 10R, 11R, 12R, 13S, 14R, 2″S [Flack parameter = 0.01(4)].16 Crodamoid I (9) possessed a molecular formula of C30H40O7 as determined by the 13C NMR data (Table 3) and a sodium adduct ion in the (+)-HRESIMS at m/z 535.2670 (calcd 535.2672). Analysis of its 1D NMR data (Tables 2 and 3) 1553



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Figure 3. 1H−1H COSY (A), selected HMBC (A), and NOESY (B) correlations of 8. Figure 1. 1H−1H COSY (A), selected HMBC (A), and NOESY (B) correlations of 1.

Figure 4. ECD spectra of compounds 1−7.

Figure 2. 1H−1H COSY (A), selected HMBC (A), and NOESY (B) correlations of 6.

tentatively assigned as (S)-configured. The absolute configurations of 2−5 and 7 were assigned by their similar ECD curves to those of 1 in the range of 190−400 nm (Figure 4) and biosynthetic considerations. Similarly, the absolute configurations of 9 and 10 were assigned based on their comparable ECD curves with those of 8 (Figure 5).

Figure 5. ECD spectra of compounds 8−10. 1554



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Figure 8. ORTEP drawing of compound 8.

Table 4. Cytotoxicity of Compounds 1−15 against Tumor Cell Lines (IC50 in μM)


compound

A549

HL-60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 adriamycinc

3.7 ± 0.3 IA 4.6 ± 0.8 4.1 ± 1.4 NTb IA IA 0.9 ± 0.6 1.3 ± 0.2 IA IA IA 18.3 ± 1.7 NT 1.9 ± 0.1 0.4 ± 0.1

IAa IA 18.9 ± 1.3 22.7 ± 1.7 NT IA IA IA 2.4 ± 1.0 IA IA IA IA NT 1.8 ± 0.8 0.08 ± 0.01

a

IA stands for inactive (defined as an inhibition rate < 50% at 10 μM). bNT represents compounds were not tested due to insufficient amounts. cPositive control.

methylbutanoate and an isobutyryloxy motif at C-13, respectively, showed significant activities against both the tested tumor cell lines, with IC50 values ranging from 1.3 ± 0.2 to 2.4 ± 1.0 μM. These observations suggested that the acylation patterns at C-12 and C-13 of tigliane diterpenoids will affect the cytotoxic activities, and the presence of a formyl group at C-6 seemingly favors the cytotoxic activities of this compound class.


Five known tigliane diterpenoids, 13-O-(2-methylbutyryl)-4deoxy-4α-phorbol (11),19 12-O-tigloyl-4-deoxy-4α-phorbol-13acetate (12),17 12-O-tigloyl-4-deoxy-4α-phorbol-13-2-methylbutyrate (13),20 12-O-angeloyl-4-deoxy-4α-phorbol-13-2methylbutyrate (14),21 and 4α-deoxyphorbol-12-tiglate-13isobutyrate (15),15 were also isolated and identified. All the isolates, except for compounds 5 and 14 (insufficient quantities), were tested for cytotoxic activities against the A549 and HL-60 human tumor cell lines using the CCK-8 method,22,23 and some of them showed significant activities (Table 4). Compound 8, with a formyl group at C-6 and a 2methylbutanoate unit at C-13, displayed the most potent and selective activity against the A549 cell line (IC50 = 0.9 ± 0.6 μM) that is comparable to adriamycin (IC50 = 0.4 ± 0.1 μM), while compound 11, with a hydroxymethyl group at C-6 and structurally closely related to 8, was inactive. Compounds 9 and 15, sharing a tigloyloxy unit at C-12 and possessing a 2-

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EXPERIMENTAL SECTION

General Experimental Procedures. The experiments were done by using the standard procedures with small amendments (General Experimental Procedures, Supporting Information). Plant Material. The details for the collection and the identification of the plant material of Croton damayeshu are included in the Supporting Information (Plant Material). Extraction and Isolation. The powder of C. damayeshu (6.5 kg) was extracted with 95% EtOH (3 × 20 L) at 25 °C for 2 weeks to yield a syrup (250 g), which was first suspended in water (3 L) and then extracted with EtOAc. The EtOAc extract (125 g) was chromatographed over D101-macroporous absorption resin, washing with 30%, 50%, 80%, and 95% EtOH in H2O sequentially, to give fractions 1−4. 1555



Article

3417, 2974, 2926, 2878, 1721, 1685, 1650, 1459, 1379, 1260, 1153, 1117, 1072, 1025, 736 cm−1; 1H NMR (CDCl3), see Table 2; 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 553.3 [M + Na]+, 1083.7 [2 M + Na]+; (−)-ESIMS m/z 575.8 [M + HCO2]−; (+)-HRESIMS m/z 553.2772 [M + Na]+ (calcd for C30H42O8Na, 553.2777). Crodamoid G (7): white, amorphous powder; [α]27D −37 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 232 (3.70), 212 (3.46) nm; ECD (MeOH) λ (Δε) 209 (+2.46), 235 (−3.59), 325 (+0.24); IR (KBr) νmax 3414, 2977, 2929, 2881, 1694, 1638, 1462, 1388, 1331, 1266, 1200, 1126, 1046, 995, 918, 882, 736 cm−1; 1H NMR (CDCl3), see Table 2; 13 C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 471.3 [M + Na]+, 919.5 [2 M + Na]+; (−)-ESIMS m/z 493.2 [M + HCO2]−; (+)-HRESIMS m/z 471.2357 [M + Na]+ (calcd for C25H36O7Na, 471.2359). Crodamoid H (8): colorless crystals (MeOH); mp 198−200 °C; [α]27D −1 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 224 (3.91), nm; ECD (MeOH) λ (Δε) 201 (+6.66), 228 (−13.93), 250 (+2.64), 325 (+0.82); IR (KBr) νmax 3408, 2971, 2932, 2878, 1715, 1650, 1468, 1376, 1266, 1239, 1197, 1156, 1120, 1055, 989, 736 cm−1; 1H NMR (CDCl3), see Table 2; 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/ z 431.2 [M + H]+, 883.6 [2 M + Na]+; (−)-ESIMS m/z 475.4 [M + HCO2]−; (+)-HRESIMS m/z 883.4613 [2 M + Na]+ (calcd for C50H68O12Na, 883.4608). Crodamoid I (9): white, amorphous powder; [α]27D −14 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 220 (4.14), nm; ECD (MeOH) λ (Δε) 197 (+4.98), 205 (+3.84), 228 (−15.01), 249 (+2.95), 325 (+0.78); IR (KBr) νmax 3569, 3405, 2965, 2929, 2876, 1718, 1691, 1462, 1382, 1266, 1236, 1153, 1117, 1073, 1046, 1022, 733 cm−1; 1H NMR (CDCl3), see Table 2; 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 513.4 [M + H]+, 1047.8 [2 M + Na]+; (+)-HRESIMS m/z 535.2670 [M + Na]+ (calcd for C30H40O7Na, 535.2672). Crodamoid J (10): white, amorphous powder; [α]27D −22 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 224 (3.80), nm; ECD (MeOH) λ (Δε) 202 (+4.50), 227 (−9.24), 250 (+1.70), 326 (+0.55); IR (KBr) νmax 3411, 2959, 2920, 2849, 1706, 1691, 1653, 1465, 1382, 1260, 1156, 1052, 1028, 799 cm−1; 1H NMR (CDCl3), see Table 2; 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 439.2 [M + Na]+, 855.4 [2 M + Na]+; (−)-ESIMS m/z 461.2 [M + HCO2]−; (+)-HRESIMS m/z 439.2101 [M + Na]+ (calcd for C24H32O6Na, 439.2097). X-ray Crystallographic Analysis. Compounds 1 and 8 were crystallized from MeOH at room temperature, and 6 was crystallized from EtOH at room temperature. Crystallographic data for crodamoid A (1) (Table S1, Supporting Information), crodamoid F (6) (Table S2, Supporting Information), and crodamoid H (8) (Table S3, Supporting Information) have deposition numbers CCDC 1888451 (1), CCDC 1888452 (6), and CCDC 1888453 (8) at the Cambridge Crystallographic Data Centre, respectively. The X-ray crystallography studies of these three compounds were accomplished according to a regular procedure (for details see X-ray Crystallographic Analysis, Tables S1, S2, and S3, Supporting Information). Cytotoxicity Assessment. All the isolates except for compounds 5 and 14 were assessed for the in vitro cytotoxic activities against A549 and HL-60 cell lines by using the CCK-8 method,22,23 and adriamycin was applied as the positive control (for assay details see Cytotoxicity Assessment, Supporting Information).

Fraction 3 (40 g) was separated on a column of MCI gel and eluted with gradients of MeOH/H2O (30 to 100%) to give four parts, 3A−3D. Fraction 3B (17g) was subjected to a silica gel column, eluting with petroleum ether/acetone (20:1 to 1:5, v/v), to obtain subfractions 3B1 to 3B5. Fraction 3B3 was subjected to an RP-18 silica gel column (MeOH/H2O, 40% to 100%) to give 3B3a−3B3i. After purification by semipreparative HPLC (eluted with 65%, 65%, and 70% CH3CN/H2O, respectively), fractions 3B3a, 3B3b, and 3B3d yielded compounds 10 (1 mg), 11 (10 mg), and 8 (10 mg), respectively. Fraction 3B3e was fractionated using silica gel CC with petroleum ether/CHCl3 gradient (3:1 to 1:5) to afford five parts, 3B3e1−3B3e5. Fraction 3B3e3 and 3B3e5 were separated by semipreparative HPLC (65% and 65% CH3CN/H2O) to afford compounds 12 (5 mg) and 6 (2 mg), respectively. Similarly, fraction 3B3f yielded compound 15 (5 mg) and fraction 3B3g yielded compounds 4 (3 mg) and 13 (3 mg). Fraction 3B2 was fractionated over an RP-18 silica gel column, using MeOH/ H2O (50% to 100%) as the mobile phase, to give 3B2a−3B2e. Fraction 3B2e was subjected to a silica gel column and eluted with a petroleum ether/acetone gradient (10:1 to 1:5, v/v) to give two parts, 3B2e1 and 3B2e2. Fraction 3B2e1 was further separated by semipreparative HPLC (70% CH3CN/H2O) to yield compounds 3 (15 mg) and 9 (4 mg). In similar procedures, fraction 3C (18 g) yielded compounds 5 (1 mg) and 14 (1 mg). Fraction 2 (25 g) was separated over a silica gel column using a petroleum ether/acetone gradient (20:1 to 1:5, v/v) to yield fractions 2A−2E. According to the similar procedures for fractions 3B and 3C, fractions 2B (200 mg), 2C (1 g), and 2D (2 g) yielded compounds 1 (2 mg), 2 (10 mg), and 7 (10 mg), respectively. Crodamoid A (1): colorless crystals (MeOH); mp 177−179 °C; [α]27D −26 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 204 (4.04), 232 (3.88) nm; ECD (MeOH) λ (Δε) 210 (+4.17), 242 (−4.29), 325 (+0.55); IR (KBr) νmax 3417, 2972, 2930, 2882, 1716, 1684, 1635, 1463, 1383, 1264, 1237, 1164, 1074, 739 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 525.4 [M + Na]+, 1027.7 [2 M + Na]+; (+)-HRESIMS m/z 525.2826 [M + Na]+ (calcd for C29H42O7Na, 525.2828). Crodamoid B (2): white, amorphous powder; [α]27D −36 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 232 (3.75) nm; ECD (MeOH) λ (Δε) 211 (+3.43), 249 (−2.85), 323 (+0.85); IR (KBr) νmax 3417, 2974, 2929, 2878, 1712, 1638, 1474, 1388, 1203, 1168, 1049, 995, 885, 739 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 441.3 [M + Na]+, 859.6 [2 M + Na]+; (+)-HRESIMS m/z 441.2254 [M + Na]+ (calcd for C24H34O6Na, 441.2253). Crodamoid C (3): white, amorphous powder; [α]27D −26 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 232 (3.83), 204 (3.69) nm; ECD (MeOH) λ (Δε) 210 (+4.03), 237 (−5.83), 326 (+0.26); IR (KBr) νmax 3569, 3417, 2968, 2929, 2876, 1715, 1697, 1638, 1462, 1382, 1263, 1236, 1189, 1156, 1117, 1073, 1013, 739 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 539.3 [M + Na]+, 1055.9 [2 M + Na]+; (+)-HRESIMS m/z 539.2972 [M + Na]+ (calcd for C30H44O7Na, 539.2985). Crodamoid D (4): white, amorphous powder; [α]27D −61 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 230 (4.14), 203 (4.03) nm; ECD (MeOH) λ (Δε) 210 (+2.97), 239 (−7.89), 325 (+0.26); IR (KBr) νmax 3574, 3411, 2971, 2932, 2878, 1718, 1638, 1462, 1376, 1263, 1159, 1108, 1025, 968, 733, 709 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 559.3 [M + Na]+, 1095.8 [2 M + Na]+; (−)-ESIMS m/z 581.8 [M + HCO2]−; (+)-HRESIMS m/ z 559.2673 [M + Na]+ (calcd for C32H40O7Na, 559.2672). Crodamoid E (5): white, amorphous powder; [α]27D −24 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 232 (3.72) nm; ECD (MeOH) λ (Δε) 210 (+2.43), 248 (−2.74), 325 (+0.49); IR (KBr) νmax 3414, 2974, 2932, 2878, 1739, 1715, 1635, 1462, 1385, 1248, 1194, 1093, 980, 888, 736 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 525.4 [M + Na]+, 1027.7 [2 M + Na]+; (+)-HRESIMS m/z 525.2825 [M + Na]+ (calcd for C29H42O7Na, 525.2828). Crodamoid F (6): colorless crystals (EtOH); mp 142−144 °C; [α]27D −49 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 221 (4.15), nm; ECD (MeOH) λ (Δε) 212 (+3.74), 237 (−7.56); IR (KBr) νmax 3566,

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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.jnatprod.9b00042. Selected key 2D NMR correlations of 2−5, 7, 9, and 10; X-ray crystallographic data for 1, 6, and 8; 1D and 2D NMR, IR, ESIMS, and HRESIMS spectra of 1−10 (PDF) X-ray crystallographic data of 1 (CIF) X-ray crystallographic data of 6 (CIF) X-ray crystallographic data of 8 (CIF) 1556



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Article

(22) Tominaga, H.; Ishiyama, M.; Ohseto, F.; Sasamoto, K.; Hamamoto, T.; Suzuki, K.; Watanabe, M. Anal. Commun. 1999, 36, 47−50. (23) Zhou, B.; Wu, Y.; Dalal, S.; Merino, E. F.; Liu, Q.-F.; Xu, C.-H.; Yuan, T.; Ding, J.; Kingston, D. G. I.; Cassera, M. B.; Yue, J.-M. J. Nat. Prod. 2017, 80, 96−107.

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-21-50806718. Fax: +86-21-50806718. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Bin Zhou: 0000-0001-9000-5320 Jin-Xin Zhao: 0000-0003-4754-1616 Jian-Min Yue: 0000-0002-4053-4870 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation (No. 21532007), Independent Scientific Research Projects Approved by Institute of Drug Innovation, CAS (Grant No. CASIMM0120181001), and Biological Resources Programme, CAS (Grant No. ZSTH-032).

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REFERENCES

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Cytotoxic Tigliane Diterpenoids from Croton damayeshu | Journal of

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