Crassins A–H, Diterpenoids from the Roots of Croton crassifolius

Article pubs.acs.org/jnp

Crassins A−H, Diterpenoids from the Roots of Croton crassifolius Qing-Qing Yuan,†,‡ Shuai Tang,† Wei-Bin Song,† Wen-Qiong Wang,† Min Huang,† and Li-Jiang Xuan*,† †

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike 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 S Supporting Information *

ABSTRACT: Phytochemical investigation of the 70% acetone extract of Croton crassifolius roots afforded eight new diterpenoids, crassins A−H (1−8), and 19 known compounds. The structures of the new compounds were determined by spectroscopic methods, and their absolute configurations were determined by electronic circular dichroism, single-crystal X-ray diffraction analysis, comparison with literature data, and biogenetic considerations. Crassins A (1) and B (2) are new ring Brearranged diterpenoids, whereas crassin C (3) is a new ring A-rearranged diterpenoid. Crassin H (8) exhibited selective cytotoxicity against A549 cells (IC50 5.2 μM) compared with HL-60 cells (IC50 11.8 μM). The known compound chettaphanin-II exhibited moderate activity against the A549 and HL-60 cell lines (IC50 8.4 and 10.5 μM, respectively).

T

he genus Croton (Euphorbiaceae) consists of approximately 1300 species that are widely distributed in tropical regions.1 Plants of this genus are known to contain diverse diterpenoids with broad biological activities, such as cytotoxic,2,3 antiviral,4 antiplasmodial,5 and anti-inflammatory activities.3,6 Croton crassifolius Geisel. is primarily distributed in southern China, Laos, Thailand, and Vietnam.7 Its roots, also known as “jiguxiang” in China, are traditionally used to treat stomachaches, rheumatism, sore throats, and cancer.8 Previous phytochemical investigations of C. crassifolius revealed that the main constituents were diterpenoids, particularly clerodane diterpenoids, with cytotoxic,9,10 antiviral,4 and antiangiogenic activities.11,12 To search for bioactive metabolites with unique structures from medicinal plants, the 70% acetone extract of C. crassifolius roots was investigated. Eight new diterpenoids (1−8) and 19 known compounds were identified. The diterpenoids were tested for their cytotoxicity against the HL60 and A549 cell lines.

Crassin A (1) possessed a molecular formula of C17H20O4 based on its 13C NMR and HRESIMS data. Typical signals of a β-substituted furan ring [δH 6.41 (1H, dd, J = 1.8, 0.9 Hz, H13), 7.44 (1H, t, J = 1.8 Hz, H-14), and 7.47 (1H, br s, H-15)], one oxygenated methine [δH 5.33 (dd, J = 9.8, 6.3 Hz, H-11)], and one methyl doublet [δH 1.07 (3H, d, J = 6.5 Hz, H3-16)] were observed in the 1H NMR data (Table 1). The 13C NMR spectra (Table 3) displayed signals corresponding to a βsubstituted furan ring (δC 108.3, 124.4, 140.0, and 144.2), one ketocarbonyl (δC 213.0), one ester carbonyl (δC 178.1), one methyl (δC 14.5), and five methylenes (δC 23.2, 25.0, 33.7, 36.2, and 39.1). The COSY cross-peaks (Figure 1) of H3-16/H-7, H7/H2-6, H2-6/H-5, H-5/H-9, H-9/H2-1, H2-1/H2-2, and H2-2/ H2-3 suggested the presence of a CH3(16)−CH(7)−CH2(6)− CH(5)−CH(9)−CH2(1)−CH2(2)−CH2(3) structural unit. The HMBC cross-peaks (Figure 1) from H2-3, H-5, and H26 to C-4 defined the location of the ketocarbonyl group as C-4. The lactone formation involving C-8, C-10, C-11, and C-17 was elaborated from the HMBC correlations of H2-10/C-8, C-9, and C-17; H-7/C-17; and H3-16/C-8. Thus, the 2D structure of 1 was assigned as shown.

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RESULTS AND DISCUSSION The 70% acetone extract of C. crassifolius roots was evaporated under reduced pressure and partitioned between CHCl3 and H2O. The CHCl3 layer was separated using repeated column chromatography to yield eight new diterpenoids, crassins A−H (1−8), and 19 known diterpenoids. © 2017 American Chemical Society and American Society of Pharmacognosy

Received: May 11, 2016 Published: February 2, 2017 254

DOI: 10.1021/acs.jnatprod.6b00425 J. Nat. Prod. 2017, 80, 254−260

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Chart 1

Table 1. 1H NMR Data (500 MHz) for Compounds 1−4 (δH in ppm, J in Hz) 1a

no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 −OMe a

2a

3a

1.32, 1.93, 2.07, 1.78, 2.36,

m m m m m

1.61, 1.89, 2.19, 1.72, 2.26, 2.40,

m m m m m m

3.28, 2.16, 1.99, 2.16,

dt (10.4,6.7) m m m

2.40, 2.00, 1.55, 1.96,

m m m m

2.77, 2.56, 2.07, 5.33,

ddd (12.3, 8.2, 5.8) dd (13.5, 6.4) dd (13.6, 9.9) dd (9.8, 6.3)

2.23, 2.81, 2.02, 5.46,

m dd (13.1, 8.3) m dd (8.2, 4.8)

6.41, 7.44, 7.47, 1.07,

dd (1.8, 0.9) t (1.8) br s d (6.5)

6.38, 7.45, 7.43, 0.97,

br s t (1.7) br s d (6.7)

4b

5.93, s

2.82, d (17.3) 3.19, d (17.3)

2.42, 2.16, 1.68, 1.47, 2.23,

4.97, t (2.7)

ddd (18.5,5.0,4.0) ddd (18.5,10.4,5.7) m m m

2.01, ddd (15.6, 3.8, 2.4) 2.11, ddd (15.7, 12.7, 3.1) 1.51, m

3.84, d (16.5) 3.16, d (16.5)

6.92, s

6.69, 7.38, 8.04, 0.94, 1.52,

6.94, 7.61, 8.61, 1.04, 1.48,

dd (2.0, 0.8) t (1.7) br s d (6.8) s

1.19, s 3.72, s

dd (1.9, 0.8) t (1.7) br s d (6.9) s

1.37, s

Recorded in CDCl3. bRecorded in pyridine-d5.

crassin A, was assigned to have the (5S,7R,8R,9S,11S) absolute configuration and, thus, the structure as shown. Crassin B (2) possessed the same molecular formula, C17H20O4, as compound 1. The 1D and 2D NMR data (Tables 1 and 3) of 2 also displayed the same arrangement as observed for 1; therefore, these compounds had the same 2D structures. However, an in-depth comparison of the 13C NMR spectra revealed some significant differences. For example, the signal of C-5 resonated at δC 55.6 for 2 but at 49.8 for 1 (ΔδC +5.8). Other differences were observed for C-8 (δC 54.8/58.2, ΔδC

The NOESY correlation (Figure 1) of H3-16/H-6α indicated that these protons were spatially close and were randomly assigned as α-oriented. Accordingly, H-5, H-7, H-9, and H-10 were β-oriented based on the NOESY cross-peaks of H-9/H6β, H-7/H-10, and H-5/H-9. The lack of an NOE cross-peak of H-11/H3-16 suggested an (11S) configuration.4,11,13 A negative Cotton effect at approximately 220 nm in the electronic circular dichroism (ECD) spectrum of 1 (Figure 2) was consistent with (8R) and (11S) configurations.4,11,14 Thus, compound 1, 255

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Table 2. 1H NMR Data (500 MHz) for Compounds 5−8 (δH in ppm, J in Hz) 5a

no. 1

6b

6.08, s

3.09, 2.84, 1.45, 1.92, 2.32,

2 3

6.07, s

4 5 6

1.59, 2.39, 1.36, 2.27, 2.02,

7 8 9 10 11

m dd (13.9, 4.6) dd (14.1, 5.1) m m

7a

td (13.4, 3.4) m m m m

5.21, t (6.6) 1.54, ddd (13.6,10.8,6.6) 2.45, dt (13.5,6.7) 2.36, m

4.88, s

3.89, d (18.8) 3.73, d (18.8)

12 13 14 15 16 17 18 19 20 −OMe a

6.44, 7.36, 7.54, 0.87, 2.17, 1.24,

d (1.3) t (1.7) br s d (7.1) s s

6.88, 7.64, 8.63, 1.12,

d (1.8) t (1.7) br s d (7.1)

1.78, 2.00, 2.16, 2.33, 6.94,

m m m m dd (5.1, 2.5)

2.50, 1.24, 1.43, 2.14, 1.61,

m m m m m

1.60, 2.31, 1.97, 2.31,

m m m m

6.27, 7.36, 7.23, 1.16,

dd (1.8, 0.9) t (1.6) br s d (6.8)

8a 1.06, m 2.06, m 1.58, m 1.98, m 1.41, m

2.06, m 1.70, m 1.43, m 1.58, m

1.62, m 2.27, m 2.00, m 5.82, s 6.00, s 0.86, d (6.2)

1.20, s

1.25, s 0.90, s 3.64, s

Recorded in CDCl3. bRecorded in pyridine-d5.

Table 3. 13C NMR Data (126 MHz) for Compounds 1−8 (δC in ppm)

a

position

1a

2a

3a

4b

5a

6b

7a

8a

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

25.0 23.2 38.1 213.0 49.8 33.7 39.1 58.2 47.0 36.2 71.7 124.4 108.3 144.2 140.0 14.5 178.1

26.2 26.8 41.2 209.9 55.6 32.4 44.0 54.8 53.0 38.6 72.2 125.7 108.2 144.3 139.2 18.00 178.8

181.9 146.5 116.3 50.8 158.5 27.3 26.0 35.0 39.5 137.1 45.7 194.2 128.9 108.8 144.0 147.4 15.9 24.4 172.6 20.7 53.3

138.0 192.9 44.1 49.7 74.0 81.5 30.2 36.3 56.3 170.8 144.6 131.7 120.7 111.5 143.3 143.7 17.9 18.0 178.8 13.3

121.4 185.6 126.1 159.8 75.6 37.6 26.6 43.3 41.2 159.0 103.9 146.6 121.4 107.4 143.3 139.3 14.1 17.6 21.7

29.2 17.4 38.7 209.1 67.7 87.9 41.0 45.4 60.6 84.8 45.2 194.4 127.3 108.9 145.7 150.3 14.9 173.8 175.3

19.5 28.0 141.1 140.4 38.4 36.6 27.2 37.2 49.9 48.7 34.0 18.1 124.6 111.0 143.1 138.7 16.7 172.8 18.2 183.0

27.7 20.1 36.4 47.9 132.7 25.2 26.9 33.8 41.1 134.9 33.0 22.8 170.8 117.0 171.9 99.3 16.2 178.5 24.5 20.8 52.1

Recorded in CDCl3. bRecorded in pyridine-d5.

−3.4), C-7 (δC 32.4/39.1, ΔδC −6.7), and C-9 (δC 53.0/47.0, ΔδC +6.0). Therefore, compounds 2 and 1 were stereoisomers differing in some stereogenic centers in close proximity to the aforementioned carbon atoms.15 The NOESY cross-peaks

(Figure 1) of H-9/H-5, H-9/H3-16, and H-7/H-10 established the relative configuration of 2. Likewise, the lack of an NOE cross-peak of H-11/H3-16 and a negative Cotton effect near 220 nm in the ECD spectrum (Figure 2) suggested (8R) and 256

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Figure 3. Key COSY and HMBC correlations of compounds 3, 5, and 6.

absolute configuration of 3 was tentatively assigned as (4S,8R,9S). Thus, compound 3, crassin C, was a ring Arearranged clerodane diterpenoid with the structure as shown. Crassin D (4) gave a molecular formula of C20H20O5 as evidenced by its HRESIMS and 13C NMR data. The NMR data of 4 (Tables 1 and 3) exhibited similarities to those of crassifoliusin A,17 except for the occurrence of an oxygenated methine carbon (δC 81.5) in 4 rather than a methoxy and a methylene group in crassifoliusin A. Therefore, the conclusion that a lactone moiety was formed to link C-6 (δC 81.5) and C19 (δC 178.8) was elaborated based on the HMBC cross-peaks of H-6/C-5, C-8, and C-10 and H3-18/C-5 and C-19. To assign the structure of 4, an X-ray diffraction experiment using graphite-monochromated Cu Kα radiation was performed and yielded an absolute structure parameter of 0.07(12), which permitted assignment of the (4S,5S,6R,8R,9R) absolute configuration for 4 (Figure 4). Therefore, the structure of compound 4, crassin D, was elucidated as shown.

Figure 1. Key COSY, HMBC, and NOESY correlations of compounds 1 and 2.

Figure 2. ECD spectra of compounds 1 and 2.

(11S) configurations of 2. The opposite sign of the Cotton effect at 270−300 nm based on the carbonyl chromophore confirmed that the C-5 configuration of compound 1 was opposite that of 2. Therefore, compound 2, crassin B, was assigned to have the (5R,7R,8R,9R,11S) absolute configuration and, thus, the structure as shown. Compounds 1 and 2 are both ring B-rearranged diterpenoids. Crassin C (3) had a molecular formula of C21H24O6 based on its HRESIMS and 13C NMR data. The 13C NMR data of 3 (Table 3) included resonances for a β-substituted furan ring, one ketocarbonyl, one hydroxycarbonyl, one methoxycarbonyl, four olefinic carbons, three methyls, and three methylene carbons. The spin system consisting of H3-17, H-8, H2-7, and H2-6 showed the connectivity of CH3(17)−CH(8)−CH2(7)− CH2(6) based on the COSY spectrum (Figure 3). The HMBC cross-peaks (Figure 3) of H-3/C-1, C-2, C-4, C-5, and C-19; H3-18/C-3, C-4, C-5, and C-19; H2-6/C-5 and C-10; OH-1/C1, C-2, C-3, and C-10; and H2-11/C-12 allowed the assignment of the methoxycarbonyl group at C-4, the hydroxycarbonyl group at C-2, and the ketocarbonyl at C-12, respectively. The ROESY cross-peaks of H3-18/H3-20 and H 3-17/H 3-20 suggested that the three methyls had a cis relationship. The co-occurrence of the rearranged diterpenoid 3 and chettaphanin-I16 in the same plant reinforced their biogenetic relationship (Scheme S1, Supporting Information). On the basis of the above relative configuration and biogenetic considerations, the

Figure 4. X-ray ORTEP drawing of compound 4. 257

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Crassin E (5) had a molecular formula of C19H20O3 based on its HRESIMS and 13C NMR data. Comparisons of the NMR data of 5 (Tables 2 and 3) with those of methyl 9-(furan-3-yl)2,7,13-trimethyl-4-oxo-10-oxatricyclo[5.3.3.0 1,6 ]trideca-5,8diene-2-carboxylate16 showed similar resonances, except for the absence of a methylene carbon (δC 45.7, C-3), a quaternary carbon (δC 51.6, C-4), and a methoxycarbonyl group (δC 52.3 and 174.0) and the occurrence of two olefinic carbons (δC 126.1 and 159.8) in 5. The presence of a Δ3,4 double bond was supported by the HMBC cross-peaks (Figure 3) of H-3/C-5 and H3-18/C-3, C-4, and C-5. The ROESY correlation between H3-19 and H3-17 indicated that the two methyl groups had a cis relationship. From a biogenetic viewpoint, compound 5 possessed the same absolute configuration as methyl 9-(furan3-yl)-2,7,13-trimethyl-4-oxo-10-oxatricyclo[5.3.3.01,6]trideca5,8-diene-2-carboxylate (Scheme S1, Supporting Information). Therefore, the absolute configuration of 5 was tentatively assigned as (5S,8R,9S). Thus, the structure of compound 5, crassin E, was assigned as shown. Crassin F (6) possessed a molecular formula of C19H18O7 based on its HRESIMS and 13C NMR data, indicating 11 indices of hydrogen deficiency. Analysis of its 13C NMR data (Table 3) showed 19 carbon signals, including a methyl group (δC 14.9), five methylenes (δC 17.4, 29.2, 38.7, 41.0, and 45.2), two methines (δC 45.4 and 87.9), two sp3 quaternary carbons (δC 60.6 and 67.7), one oxygenated tertiary carbon (δC 84.8), four olefinic carbons (δC 108.9, 127.3, 145.7, and 150.3) attributed to a β-substituted furan ring, two ester carbonyls (δC 173.8 and 175.3), and two ketocarbonyls (δC 194.4 and 209.1). The HMBC cross-peaks of H-3/C-4, H-6/C-4, and H2-11/C12 permitted the assignment of the two ketocarbonyls at C-4 and C-12, respectively (Figure 3). The cross-peaks of H-7/C-5, H-6/C-18, H-8/C-19, H2-11/C-19, and H-1/C-10 allowed the assignment of the two ester carbonyls at C-5 and C-9. Furthermore, the two ester carbonyls formed two rings with C6 and C-10, respectively, considering the indices of hydrogen deficiency. The ROESY cross-peaks of H-8/H2-11 suggested that these protons were spatially close and randomly assigned β-orientations. The J value (6.6 Hz) of H-6 suggested that H-6 was in an α-equatorial orientation. Compound 6 was assumed to have a neo-clerodane absolute configuration similar to other clerodane derivatives isolated from C. crassifolius.4,9−12 Therefore, the absolute configuration of 6 was tentatively assigned as (5R,6R,8R,9R,10R). Thus, the structure of 6, crassin F, was assigned as shown. Crassin G (7) possessed a molecular formula of C20H26O5 based on its HRESIMS and 13C NMR data. The general features of the NMR data of 7 (Tables 2 and 3) resembled those of junceic acid,18 except that an additional hydroxycarbonyl group (δC 172.8) was present in 7. The HMBC cross-peaks of H-3/C-18 and C-5, H2-2/C-18, H-8/C-20, H-9/ C-20, and H2-11/C-20 suggested that the two hydroxycarbonyl groups were attached to C-4 and C-9, respectively (Figure 5). The relative configuration of 7 was determined based on the ROESY correlations of H3-19/H-6α, H-8/H-6β, H-6β/H-10, and H-10/H2-11 (Figure 5). Both 7 and junceic acid showed negative specific rotations, indicating their (5R,8R,9R,10S) absolute configurations.18−20 Thus, the structure of 7, crassin G, was defined as shown. Crassin H (8) was obtained as colorless crystals, and its molecular formula was determined to be C21H30O5 based on its HRESIMS and 13C NMR data. The IR spectrum showed hydroxy (3442 cm−1), lactone (1742, 1168 cm−1), and ester

Figure 5. Key COSY, HMBC, and ROESY correlations of compound 7.

carbonyl (1724 cm−1) bands. The NMR data of 8 (Tables 2 and 3) resembled those of crassifolin A,4 except for the presence of an oxygenated methine carbon (δC 99.3) in 8 rather than the methylene carbon (δC 73.2) in crassifolin A. The HMBC cross-peak from H-16 (δH 6.00) to C-15 (δC 171.9) suggested that the hydroxy group was connected to C-16. The absolute configuration of 8 was defined as (4R,8R,9S,16R) on the basis of an X-ray diffraction experiment (Figure 6). Thus, the structure of 8, crassin H, was elucidated as shown.

Figure 6. X-ray ORTEP drawing of compound 8.

In addition, 19 known diterpenoids were also obtained from the extract of C. crassifolius. These diterpenoids were determined to be crassifolins A−D and F,4 crassifolin I,12 crassifolin J,11 teucvin,15 isoteucvin,15 penduliflaworosin,15 crassifoliusin A,17 6-[2-(furan-3-yl)-2-oxoethyl]-1,5,6-trimethyl-10-oxatricyclo[7.2.1.02,7]dodec-2(7)-en-11-one,16 methyl 9(furan-3-yl)-2,7,13-trimethyl-4-oxo-10-oxatricyclo[5.3.3.01,6]trideca-5,8-diene-2-carboxylate,16 chettaphanin-I,21 chettaphanin-II,21 neoclerodan-5,10-en-19,6β;20,12-diolide,22 teuscorolide,23 mollotucin D dilactone ester,24 and (12S)-15,16-epoxy6β-methoxy-19-norneoclerodane-4,13-(16),14-triene18,6α;20,12-diolide.25 All of the isolated diterpenoids were tested for their cytotoxicity against the HL-60 (human premyelocytic leuke258

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18,6α;20,12-diolide (120 mg), and chettaphanin-I (95 mg). Fr7 was separated into four subfractions (7A−7D) via silica gel (petroleum ether/EtOAc, 3:1 to 0:1). Fr7C was purified by silica gel (petroleum ether/EtOAc, 2:1 to 0:1) and Sephadex LH-20 (CHCl3/MeOH 1:1) to afford crassifolin I (12 mg) and crassifolin D (210 mg). Fr8 was separated by silica gel (petroleum CHCl3/MeOH, 20:1 to 5:1) and Sephadex LH-20 gel (CHCl3/MeOH, 1:1) to obtain isoteucvin (240 mg). Crassin A (1): amorphous powder; [α]20 D −26 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 243 (3.02) nm; ECD (MeOH) λ (Δε) 227 (−7.21), 255 (−0.23), 291 (−3.94) nm; IR (KBr) νmax 3451, 1758, 1702, 1456, 1173, 1020, 875 cm−1; 1H NMR (CDCl3, 500 MHz), see Table 1; 13C NMR (CDCl3, 126 MHz), see Table 3; HRESIMS m/z 289.1434 [M + H]+ (calcd for C17H21O4, 289.1434). Crassin B (2): amorphous powder; [α]20 D +33 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 208 (3.01) nm; ECD (MeOH) λ (Δε) 215 (−8.01), 249 (3.26), 269 (1.05), 291 (3.68) nm; IR (KBr) νmax 3449, 1759, 1712, 1450, 1176, 879 cm−1; 1H NMR (CDCl3, 500 MHz), see Table 1; 13C NMR (CDCl3, 126 MHz), see Table 3; HRESIMS m/z 289.1436 [M + H]+ (calcd for C17H21O4, 289.1434). Crassin C (3): amorphous powder; [α]20 D −54 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 196 (3.71), 260 (2.58) nm; IR (KBr) νmax 3444, 1731, 1633, 1379 cm−1; 1H NMR (CDCl3, 500 MHz), see Table 1; 13 C NMR (CDCl3, 126 MHz), see Table 3; HRESIMS m/z 395.1463 [M + Na]+ (calcd for C21H24O6 Na, 395.1465). Crassin D (4): colorless crystals (MeOH); mp 126−128 °C; [α]20 D +78 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 195 (3.72) nm; IR (KBr) νmax 3417, 1776, 1680, 1628, 1082 cm−1; 1H NMR (pyridine-d5, 500 MHz), see Table 1; 13C NMR (pyridine-d5, 126 MHz), see Table 3; HRESIMS m/z 363.1203 [M + Na]+ (calcd for C20H20O5Na, 363.1203). Crassin E (5): amorphous powder; [α]20 D −2 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 216 (3.66), 238 (3.51) nm; IR (KBr) νmax 3446, 2925, 1675, 1626 cm−1; 1H NMR (CDCl3, 500 MHz), see Table 2; 13 C NMR (CDCl3, 126 MHz), see Table 3; HRESIMS m/z 319.1307 [M + Na]+ (calcd for C19H20O3Na, 319.1305). Crassin F (6): amorphous powder; [α]20 D −81 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 240 (3.60) nm; IR (KBr) νmax 3444, 1786, 1635, 1382, 1161 cm−1; 1H NMR (pyridine-d5, 500 MHz), see Table 2; 13C NMR (pyridine-d5, 126 MHz), see Table 3; HRESIMS m/z 359.1125 [M + H]+ (calcd for C19H19O7, 359.1125). Crassin G (7): amorphous powder; [α]20 D −105 (c 0.6, CHCl3); UV (MeOH) λmax (log ε) 220 (3.49) nm; IR (KBr) νmax 3449, 2962, 1692, 1627, 1257, 869 cm−1; 1H NMR (CDCl3, 500 MHz), see Table 2; 13C NMR (CDCl3, 126 MHz), see Table 3; HRESIMS m/z 369.1676 [M + Na]+ (calcd for C20H26O5Na, 369.1672). Crassin H (8): colorless crystals (MeOH); mp 128−130 °C; [α]20 D +73 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 207 (3.52) nm; IR (KBr) νmax 3424, 2960, 1739, 1729, 1467, 1166, 961 cm−1; 1H NMR (CDCl3, 500 MHz), see Table 2; 13C NMR (CDCl3, 126 MHz), see Table 3; HRESIMS m/z 385.1992 [M + Na]+ (calcd for C21H30O5Na, 385.1985). X-ray Crystallography. The crystallographic data for compounds 4 and 8 were recorded on a Bruker APEX-II CCD diffractometer using graphite-monochromated Cu Kα radiation. Details of the X-ray diffraction data and analyses are provided in the Supporting Information. Cytotoxicity Assays. The assay was performed using the HL-60 and A549 cell lines with the MTT and SRB methods. Briefly, the cell lines were cultured in 96-well microplates for 24 h at 37 °C. The test compounds (20 μM) were added, and the microplates were incubated for 72 h. Compounds that inhibited 50% of the growth of the cancer cells were tested again at six concentrations; each concentration was evaluated in two parallel wells. Cisplatin was used as the positive control. The results were expressed as IC50 values.

mia) and A549 (human lung adenocarcinoma) cell lines using the MTT26 and SRB27 methods. As shown in Table 4, crassin Table 4. Cytotoxic Activities of the Isolated Compounds against Two Cancer Lines IC50 (μM) compound

HL-60

A549

8 chettaphanin-II cisplatin

11.8 ± 2.1 10.5 ± 0.4 3.4 ± 0.1

5.2 ± 0.4 8.4 ± 0.8 7.8 ± 0.02

H (8) exhibited selective cytotoxicity against A549 cells (IC50 5.2 μM) compared with HL-60 cells (IC50 11.8 μM). The known compound chettaphanin-II exhibited moderate activity against both cell lines (IC50 8.4 and 10.5 μM).

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EXPERIMENTAL SECTION

General Experimental Procedures. The general experiments were performed as reported previously,28 and the details are shown in the Supporting Information. Plant Material. Roots of C. crassifolius were purchased in December 2011 from Guangxi Province, China. Prof. He-Ming Yang identified the specimen, and a sample (SIMMXLJ-89) was deposited at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation. The dried roots of C. crassifolius (10 kg) were pulverized and soaked in 70% acetone (3 × 25 L, 24 h each time, rt), and the concentrated extract was partitioned between H2O and CHCl3. The CHCl3 portion (420 g) was fractionated using a silica gel column (petroleum ether/EtOAc, 25:1 to 0:1) to afford fractions 1−8. Fr3 was loaded on a silica gel column (petroleum ether/EtOAc, 50:1 to 1:1) to afford subfractions 3A−3F. Fr3B was purified by recrystallization from MeOH/CHCl3 to yield chettaphanin-II (16 mg). Fr3C was passed over a silica gel column (petroleum ether/ EtOAc, 20:1 to 5:1) to afford fractions 3C1−3C4. Fr3C1 was separated using RP C8 silica gel (MeOH/H2O, 40−100%) followed by semipreparative HPLC to obtain methyl 9-(furan-3-yl)-2,7,13trimethyl-4-oxo-10-oxatricyclo[5.3.3.01,6]trideca-5,8-diene-2-carboxylate (10 mg), penduliflaworosin (45 mg), and 5 (6 mg). Fr3C2 was purified by recrystallization from MeOH to afford crassifolin A (365 mg). Purification of Fr3D by silica gel (CHCl3/MeOH, 100:1 to 50:1) yielded 4 (25 mg). Fr4 was fractionated using a silica gel column (petroleum ether/EtOAc, 8:1 to 3:1) to yield five subfractions, 4A− 4E. Fr4C, 4D, and 4E were fractionated using a silica gel column under the same chromatographic conditions (petroleum ether/acetone, 15:1 to 3:1) to afford subfractions 4C1, 4C2, 4D1−4D5, and 4E1−4E5, respectively. Fr4C1 was purified via HPLC (75% MeOH in H2O) to yield 3 (4 mg), crassifoliusin A (20 mg), and crassifolin J (16 mg). Fr4D2 was purified by recrystallization from MeOH to afford 6-[2(furan-3-yl)-2-oxoethyl]-1,5,6-trimethyl-10-oxatricyclo[7.2.1.0 2,7]dodec-2(7)-en-11-one (245 mg). Fr4E3 was separated over a silica gel column (petroleum ether/CHCl3, 2:1 to 1:4) and further purified via semipreparative HPLC to afford neoclerodan-5,10-en-19,6β;20,12diolide (8 mg), crassifolin C (8 mg), crassifolin F (20 mg), and 8 (45 mg). Purification of Fr4E5 by Sephadex LH-20 (CHCl3/MeOH, 1:1) afforded 7 (12 mg). Fr5 was separated into five subfractions, 5A−5E, through a silica gel column (petroleum ether/EtOAc, 4:1 to 1:1). Recrystallization of Fr5A afforded crassifolin B (245 mg). Fr5D was fractionated through repeated silica gel CC to yield fractions 5D11 and 5D12. Purification of Fr5D11 via semipreparative HPLC (60% MeOH in H2O) yielded 1 (5 mg), 2 (5 mg), 6 (4 mg), and mollotucin D dilactone ester (13 mg). Fr5D12 was purified by recrystallization from MeOH to obtain teucvin (95 mg). Fr6 was separated into five subfractions (6A−6E) through a silica gel column (petroleum ether/ EtOAc, 4:1 to 1:1). Both Fr6C and Fr6D were fractionated by repeated silica gel CC to obtain teuscorolide (9 mg), (12S)-15,16epoxy-6β-methoxy-19-norneoclerodane-4,13-(16),14-triene259

DOI: 10.1021/acs.jnatprod.6b00425 J. Nat. Prod. 2017, 80, 254−260

Journal of Natural Products

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Article

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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.6b00425. IR, HRESIMS, and NMR data of compounds 1−8 (PDF) Crystallographic data of 4 (CIF) Crystallographic data of 8 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail (L.-J. Xuan): [email protected]. Phone/fax: +86 21 20231968. ORCID

Li-Jiang Xuan: 0000-0002-0593-3921 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The researchers are grateful for the financial support from the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (No. SIMM1601ZZ03).

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DOI: 10.1021/acs.jnatprod.6b00425 J. Nat. Prod. 2017, 80, 254−260