Cytotoxic Tirucallane and Apotirucallane Triterpenoids from the Stems

Article pubs.acs.org/jnp

Cytotoxic Tirucallane and Apotirucallane Triterpenoids from the Stems of Picrasma quassioides Jian Xu,† Di Xiao,† Qing-Hua Lin,† Jing-Feng He,† Wen-Yuan Liu,‡ Ning Xie,§ Feng Feng,*,†,⊥ and Wei Qu*,†,⊥ †

Department of Natural Medicinal Chemistry and ‡Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, China Pharmaceutical University, Nanjing 210009, People’s Republic of China § Jiangxi Qingfeng Pharmaceutical Corporation, Ganzhou 341000, People’s Republic of China ⊥ Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing 211198, People’s Republic of China S Supporting Information *

ABSTRACT: Phytochemical investigation on the stems of Picrasma quassioides led to the isolation of a novel compound, picraquassin A (1), with an unprecedented 21,24-cycloapotirucallane skeleton, and four new apotirucallane-type triterpenoids (2−5), together with 15 new tirucallane-type triterpenoids (6−20) and 10 known tirucallane-type triterpenoids (21−30). To our knowledge, this is the first report demonstrating the presence of apotirucallane-type triterpenoids in the genus Picrasma. The structures of the new compounds were determined based on spectroscopic data interpretation. Cytotoxicities of the isolated compounds were evaluated using three human cancer cell lines, MKN-28, A-549, and MCF-7. Compound 2 exhibited the most potent activity against MKN-28 cells with an IC50 value of 2.5 μM. Flow cytometry and Western blot analysis revealed that 2 induces the apoptosis of MKN-28 cells via activating caspase-3/-9, while increasing Bax and Bad and decreasing Bcl-2 expression levels.

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compounds were further subjected to the determination of their cytotoxic activity.

ecently, several tirucallane- and apotirucallane-triterpenoids have been isolated from plants in the family Simaroubaceae.1,2 Those compounds play important roles in plant chemotaxonomy3,4 and exhibit a range of bioactivies, including cytotoxic and insecticidal effects.5−8 Picrasma quassioides (D. Don) Benn, belonging to the family Simaroubaceae, is distributed predominantly in southern mainland China, Korea, and Japan.9 The stems of this plant, designated “Ku Mu” in the Chinese Pharmacopeia, have been used traditionally for the treatment of inflammation, dysentery, microbial infection, and fever. Studies to date have revealed cytotoxic,10,11 anti-inflammatory,12,13 antihypertensive,14 and gastric mucosal protective effects15,16 of P. quassioides stems. A number of quassinoids, β-carboline alkaloids, canthin-6-one alkaloids, and triterpenoids have been isolated from this plant to date.17−19 The present study reports the isolation and characterization of 30 triterpenoid compounds from the stems of P. quassioides, including an unprecedented 21,24-cycloapotirucallane-type derivative (1), four new apotirucallane-type triterpenoids (2− 5), and 15 new tirucallane-type triterpenoids (6−20). Also isolated were the 10 known tirucallane-type triterpenoids melianodiol (21),20 (21R,23R)-epoxy-21α-ethoxy-24S,25-dihydroxytirucalla-7-en-3-one (22),21 toonaciliatin K (23),21 21methoxy-21,23-epoxytirucalla-7,24-dien-3α-ol (24),22 bourjotinolone A (25),23 sapelin B (26),24 3β,29-dihydroxytirucalla7,24-dien-21-oic acid (27),25 piscidinol A (28),26 brumollisol B (29),15 and 24S,25-dihydroxytirucall-7-en-3-one (30).27 The © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1 was isolated as a colorless oil. Its molecular formula was determined to be C30H50O5 with six degrees of unsaturation from its ammoniated molecular ion peak [M + NH4]+ at m/z 508.3999 (calcd for C30H54NO5, 508.3997) in the HRESIMS. The 1H NMR spectrum (Table 1) exhibited signals for seven tertiary methyl groups [δH 0.83, 0.88, 0.93, 1.04, 1.05, 1.26, and 1.32 (3H, s, for each)], four oxymethine protons [δH 3.39 (1H, s, H-3), 3.63 (1H, t, J ≈ 7.2 Hz, H-21), 3.89 (1H, br s, H-7), and 3.93 (1H, s, H-23)], and a typical singlet (δH 5.43, s) of an olefinic proton (H-15). All 30 carbons in the molecular structure were resolved as individual resonances (Table 2) in the 13C NMR spectrum and classified using HMQC spectroscopy as seven methyls, seven sp3 methylenes, nine sp3 methines (four oxygenated at δC 72.1, 72.6, 76.4, and 78.2), five sp3 quaternary carbons (including one oxygenated at δC 73.1), an sp2 methylene (δC 119.3), and an sp2 quaternary carbon (δC 162.7). The above data indicated that compound 1 possesses the same rings A−D as agladupol A.28 Four rings and one double bond accounted for five of the six degrees of unsaturation, with the last degree of unsaturation Received: December 24, 2015

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DOI: 10.1021/acs.jnatprod.5b01137 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

was determined as 3,7,21,23,25-pentahydroxy-21,24-cycloapotirucall-14-ene. The relative configuration of 1 was determined on the basis of the ROESY spectrum and couplings in the 1H NMR spectrum. The hydroxy groups at C-3 and C-7 were assigned as α-oriented according to the broad singlet peaks of H-3 (δH 3.39) and H-7 (δH 3.89).29 The ROESY correlations of H3-29/ H-2β (δH 1.90; δC 25.2), H-2β/H3-19, H3-19/H3-30, and H330/H-17 suggested these methyl groups are cofacial and βoriented, whereas the ROESY correlations of H3-28 (δH 0.93; δC 28.4)/H-5 and H-9/H3-18 indicated an α-orientation. Additionally, cross-peak between H3-18/H-20 indicated that H-20 is α-oriented, whereas those of H-17/H-21, H-21/H-23, and H-23/H3-26 revealed that H-21, H-23, and H-24 are β-, β-, and α-oriented, respectively. The absolute configuration of 1 was determined by the comparison of its experimental electronic circular dichroism (ECD) spectrum with the data

requiring an additional ring. A spin system, CH(17)−CH(20)− CH(21)−CH(24)−CH(23)−CH2(22)−CH(20), determined from the 1H−1H COSY spectrum, suggested associations of C-21 with C-20 and C-24, which would require an additional five-membered ring E anchored at C-17. This finding was confirmed by the HMBC correlations of H-21 (δH 3.63) with C-17, C-20, C-24, and C-25 (δC 58.6, 43.8, 66.8, and 73.1, respectively) and of H-22a (δH 1.64) and H-22b (δH 1.78) with C-17, C-20, C-24, and C-23 (δC 58.6, 43.8, 66.8, and 72.1, respectively). The HMBC correlations from the proton signals at δH 1.26 (H3-26) and 1.32 (H3-27) to the carbon signals at δC 66.8 (C-24) and 73.1 (C-25) supported the occurrence of an isopropyl alcohol group at C-24. In addition, the HMBC correlations of H3-28 (δH 1.78) with C-29, C-3, C-4, and C-5 and of H3-30 (δH 1.04) with C-7, C-8, C-9, and C-14 were consistent with the presence of OH-3, OH-7, and Δ14,15 substituents, respectively. Therefore, the planar structure of 1 B



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Table 1. 1H NMR Spectroscopic Data (δ in ppm, J in Hz) for Compounds 1−5 1a

position

2b

3 5

α 1.34 m β 1.34 m α 1.57 m β 1.90 m 3.39 br s 1.96 m

α 1.36 m β 1.36 m α 1.54 m β 1.91 m 3.39 br s 1.97 m

6

α 1.70 m

α 1.71 m

β 1.70 m

β 1.71 m

7

3.89 br s

3.89 br s

9 11

2.00 m α 1.48 m β 1.74 m α 1.83 m β 1.37 m 5.43 s

2.02 m α 1.49 m β 1.73 m α 1.82 m β 1.36 m 5.41 s

16

α 2.14 m β 2.14 m

α 2.11 m β 2.11 m

17

1.72 m

1.07 m

18 19 20 21

1.05 s 0.88 s 2.24 m 3.63 t (9.0)

22

α 1.64 m

1.07 s 0.88 s 2.37 m 4.88 d (2.0) α 1.23 m

β 1.78 m

β 2.04 m

23

3.93 s

4.68 m

24

1.85 m

26 27 28 29 30 1′

1.26 1.32 0.93 0.83 1.04

5.13 d (7.5) 1.69 s 1.72 s 0.94 s 0.84 s 1.04 s 3.43 m, 3.79 m 1.21 t (7.0)

1 2

12 15

3′ 4′ 5′ 6′ 7′

s s s s s

3c 7.18 d (10.8) 5.72 d (10.2)

2.45 dd (1.8, 13.2) α 1.82 td (2.4, 14.4) β 2.04 dt (2.4, 14.4) 4.05 t (2.4) 2.59 d (9.6) 5.54 m α 2.00 m β 2.00 m 5.54 m α 2.02 m β 2.73 ddd (0.6, 11.4, 14.4) 2.12 dd (7.2, 10.8) 1.28 s 1.28 s 4.51 s α 1.88 dd (2.4, 12.6) β 2.27 dd (9.6, 12.6) 4.54 d (8.4) 3.30 s 1.25 1.28 1.14 1.08 1.20 3.31

s s s s s s

4b

5a

α 1.54 m β 1.87 m 2.45 m

α 1.36 m β 1.86 m α 2.28 m β 2.82 m

2.10

1.71 s

1.86 m

4.43 d (2.0)

3.96 s 2.04 m α 1.57 m β 1.75 m α 2.01 m β 1.53 m 5.48 d (4.0) α 2.11 m β 2.37 m

3.79 d (2.8) 1.97 m α 1.47 m β 1.67 m α 2.03 m β 1.62 m 5.49 d (2.4) α 2.11 m β 2.39 m

1.52 m

1.54 m

1.05 s 1.01 s 1.65 m 0.98, d, 6.4 α 1.86 m

1.03 s 1.48 s 1.65 m 1.00 d (6.4) α 1.86 m

β 1.25 m

β 1.25 m

4.17 dd (5.1, 8.1) 3.96 s

4.16 dd (4.8, 8.0) 3.19, s

1.34 1.36 1.11 1.06 1.10

1.34, 1.35, 1.46, 1.20, 1.38,

s s s s s

Compound 2, obtained as a colorless oil, showed a positive molecular ion at m/z 539.3507 [M + K]+ (calcd for C32H52O4K, 539.3497) in the HRESIMS, from which a molecular formula of C32H52O4 was deduced. Its 1H and 13C NMR (Tables 1 and 2) and HSQC spectra displayed signals for two trisubstituted double bonds [δH 5.41 (1H, s) and 5.13 (1H, d, J ≈ 7.5 Hz); δC 162.4, 119.1, 124.7, and 137.0], an ethoxy group [δH 1.21 (3H, t, J ≈ 7.0 Hz), 3.43 (1H, m), and 3.79 (1H, m); δC 15.4 and 63.5], four oxygenated methines [δH 3.39 (1H, br s), 3.89 (1H, br s), 4.88 (1H, d, J ≈ 2.0 Hz), and 4.68 (1H, m); δC 76.1, 72.3, 107.7, and 73.8], and seven tertiary methyls [δH 1.07, 0.88, 1.69, 1.72, 0.94, 0.84, and 1.04; δC 19.3, 15.2, 18.3, 25.8, 22.1, 28.0, and 27.7]. The data collectively indicated that 2 is an apotirucallane-type triterpenoid with a typical Δ14,15 double bond. Comparison of the spectroscopic data of 2 and 21,23-epoxy-7α,21-dihydroxyapotirucalla-14,24dien-3-one30 revealed similarities except for the absence of the carbonyl signal at C-3 and the hydroxy group signal at C-21 in 21,23-epoxy-7α,21-dihydroxyapotirucalla-14,24-dien-3-one and the presence of an oxygenated methine and an ethoxy group in 2. These observations were supported by the HMBC data (Figure 2). The cross-peaks from the proton signals at δH 0.94 (H3-28) and 0.84 (H3-29) to the carbon signal at δC 76.1 (C-3) supported the presence of a hydroxy group at C-3, while the correlation of the oxygenated methine proton signal at δH 4.88 (H-21) with the ethoxy carbon signal at δC 63.5 (C-1′) substantiated the presence of an ethoxy group at C-21. The above data are consistent with the gross structure as depicted. The broad singlet peaks of H-3 (δH 3.39) and H-7 (δH 3.89) signified the α-orientation of OH-3 and OH-7 in compound 2.29 In addition, the ROESY correlations of H3-30/H-17, H330/H-12b (δH 1.82, m), and H-12b/H-21 confirmed the βorientation of H-17, H-12b, and H-21. The cross-peaks of H328/H-5, H-5/H-9, H-9/H3-18, H3-18/H-20, and H-20/H-23 revealed the α-orientation of H-20 and H-23. Furthermore, the ROESY correlations of H-21/H-12β and H-21/H-17 indicated the S-configuration at C-20, according to an earlier molecular model.31,32 Thus, the structure of 2 (picraquassin B) was deduced as (20S,21R,23R)-21-ethoxy-3α,7α-dihydroxy-21,23epoxyapotirucalla-14,24-diene. Compound 3 was isolated as a colorless oil. The HRESIMS displayed a protonated molecular ion at m/z 647.4160 [M + H]+ (calcd for C37H59O9, 647.4154), consistent with the molecular formula C37H58O9. The 1D-NMR data of 3 were closely related to those of brujavanone M,33 with the main differences being the absence of signals for an acetyl group at C-7 and the oxygenated methine group at C-1 in brujavanone M and the presence of a Δ1,2 double bond and an OH-20 group in 3. This conclusion was confirmed by the related HMBC correlations from the proton signal at δH 1.28 (H3-19) to the carbon signal at δC 161.8 (C-1) and from the proton signals at δH 1.88 (H-22a) and 2.27 (H-22b) to the carbon signal at δC 82.2 (C-20). ROESY experiments were conducted to determine the relative configurations at the stereocenters in 3. The ROESY correlation of H-17/H-21 indicated the β-orientation of H-21, and that between H-17 and H-22b supported the same βorientation of C-17 with H-22b in the E-ring. Since OH-20 and C-17 are on opposite faces of ring E, OH-20 was assigned as αoriented. In addition, the correlation of H-24/H-22a suggested that H-23 is β-oriented. Moreover, the hydrogen bond formation between OH-25 and the oxygen atom in the tetrahydrofuran ring conferred a limited conformational

s s s s s

2.34 dt (1.2, 7.2) 1.67 m, 2.03 m 1.35 m 1.35 m 0.94 t (7.2)

a c

Recorded in CDCl3 at 600 MHz. bRecorded in CDCl3 at 300 MHz. Recorded in MeOD at 600 MHz.

calculated using the time-dependent density functional theory (TDDFT). The experimental data were in reasonable agreement with the calculated ECD spectrum of 1a (Figure 5). Thus, 1 was structurally established as (3R,5R,7R,8R,9R,10S,13S,17S,20S,21S,23R,24S)-3,7,21,23,25-pentahydroxy-21,24-cycloapotirucall-14-ene and was conferred the trivial name picraquassin A. Compound 1 contains an unprecedented 21,24-cycloapotirucallane skeleton, which may be related biogenetically to the coisolated compounds 2−5. C



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Table 2. 13C NMR Spectroscopic Data (δ in ppm) for Compounds 1−11

a

position

1a

2b

3c

4b

5a

6d

7d

8d

9d

10b

11b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′ 7′

32.8 25.2 76.4 37.4 40.8 23.9 72.6 44.7 42.1 38.0 16.5 32.4 47.2 162.7 119.3 33.5 58.6 20.0 15.5 43.8 78.2 38.4 72.1 66.8 73.1 27.1 29.4 28.4 22.2 28.0

32.6 25.0 76.1 37.0 40.4 23.6 72.3 44.4 41.7 37.6 16.2 32.8 47.0 162.4 119.1 34.7 58.0 19.3 15.2 47.2 107.7 38.8 73.8 124.7 137.0 18.3 25.8 28.0 22.1 27.7 63.5 15.4

161.8 124.5 207.2 45.7 45.6 26.2 72.6 45.4 44.3 42.3 72.4 44.9 59.0 159.9 121.3 31.5 57.6 22.3 20.9 82.2 111.9 39.8 78.1 79.6 74.2 26.1 27.1 27.4 22.1 30.7 55.0 174.9 36.2 25.8 32.7 23.6 14.5

38.6 34.0 217.7 46.9 46.5 24.8 72.1 44.0 41.1 37.1 16.5 34.0 47.0 161.6 119.9 35.2 61.2 19.3 16.5 31.3 19.0 40.0 69.6 75.2 74.5 26.2 27.1 26. 3 21.1 27.5

41.2 34.4 216.6 48.6 51.0 72.6 76.6 43.5 42.2 37.0 16.5 34.3 47.0 162.1 120.4 35.2 61.1 19.3 16.5 31.3 19.0 40.0 69.6 75.2 74.4 27.6 26.2 24.7 24.3 26.4

37.2 27.6 79.2 39.0 50.7 24.0 118.0 145.1 48.9 35.0 17.6 31.1 43.6 50.7 34.3 27.6 45.4 23.2 13.1 45.9 104.6 35.6 73.0 126.8 134.8 18.1 25.6 27.6 14.7 27.4 54.1

37.2 27.3 79.2 38.9 50.6 23.9 118.1 145.5 48.8 35.0 17.6 31.7 43.7 50.7 33.8 27.3 50.6 22.6 13.0 49.1 108.5 39.4 73.7 124.8 136.9 18.3 25.8 27.1 14.7 27.3 55.4

31.2 25.4 76.3 37.4 44.6 23.9 118.1 145.9 48.5 34.8 17.5 31.7 43.7 51.0 33.8 27.3 50.6 22.6 12.9 49.1 108.5 39.3 73.7 124.9 136.9 18.3 25.8 27.1 21.8 27.8 55.4

37.2 27.4 79.2 39.0 50.7 24.0 118.0 145.7 48.9 35.0 17.9 31.1 43.6 50.7 34.3 27.6 45.0 23.2 13.1 47.5 104.3 36.0 75.5 128.3 134.4 17.9 25.8 27.3 14.7 27.4 54.0

37.2 27.8 79.2 38.9 50.7 23.9 118.1 145.6 49.2 35.0 17.6 31.7 43.7 50.9 33.8 27.6 50.7 22.7 13.1 49.2 107.2 39.5 73.6 124.9 136.9 18.3 25.9 27.1 14.7 27.7 63.5 15.5

37.2 27.6 79.2 38.9 50.7 24.0 118.1 145.6 48.8 34.9 17.8 32.5 43.8 51.2 33.8 27.8 48.2 22.8 13.1 48.6 106.5 36.6 75.3 128.0 134.0 17.8 25.8 27.6 14.7 27.2 62.7 15.3

Recorded in CDCl3 at 150 MHz. bRecorded in CDCl3 at 75 MHz. cRecorded in MeOD at 150 MHz. dRecorded in CDCl3 at 125 MHz.

Figure 2. Important HMBC correlations (a) and key ROESY correlations (b) for 2.

Figure 1. Important HMBC and COSY correlations (a) and key ROESY correlations (b) for 1.

mobility to the side-chain. The broad H-24 singlet in the 1H NMR spectrum indicated the gauche relationship between H-23 and H-24, 26 which was consistent with the absolute configuration at C-24 being S.5,8 Therefore, the structure of 3 (picraquassin C) was established as (20S,21R,23S,24S)-11αcaproyloxy-21-methoxy-7,24,25-trihydroxy-21,23-epoxyapotirucalla-1,14-dien-3-one. The molecular formula of compound 4, obtained as a colorless oil, was deduced as C30H50O5 from the HRESIMS at


D



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supported the assignment of OH-6 and OH-7 to β- and αorientations, respectively. Accordingly, the structure of 5 (6βhydroxypicraquassin C) was proposed as (20S,23R,24S)6β,7α,23,24,25-pentahydroxyapotirucallan-3-one. Compound 6 was obtained as a colorless oil. A protonated molecular ion peak [M + H]+ at m/z 471.3478 (calcd for C31H51O3, 471.3469) in the HRESIMS was used to assign a molecular formula of C31H50O3. Comparison of the 1D-NMR data (Tables 2 and 3) of 6 and sapinmusaponin F36 revealed 6 contains a tirucallane scaffold, with its structure consistent with the aglycone of sapinmusaponin F. This deduction was further supported by the HMBC correlations (Figure 3) from H3-28 (δH 0.98) to C-29 (δC 14.7), C-3 (δC 79.2), C-4 (δC 39.0), and C-5 (δC 50.7). In the ROESY spectrum, the correlations of H318/H-21 and H-12α/H-21 corroborated a tirucallane-type skeleton of 6,37,38 and the correlation of H-3/H3-28 signified the α-orientation of OH-3. Furthermore, similar 1D-NMR and ROESY correlations showed the relative configuration of the side-chain in 6 to be the same as in sapinmusaponin F. The absolute configuration at C-20 in tirucallane-type triterpenoids is S.26,39,40 Therefore, the structure of picraquassin E (6) was deduced as (20S,21S,23R)-21-methoxy-3β,20-dihydroxy-21,23epoxytirucalla-7,24-diene. Compounds 7−9 had the same molecular formula as 6 according to their HRESIMS data at m/z 471.3469, 471.3467, and 471.3453 [M + H]+ (calcd for C30H51O5, 471.3469), respectively. Moreover, the 1D- and 2D-NMR spectra (Tables 2 and 3) of these compounds closely resembled those of 6. A difference was the downfield-shifted Δδ 5.2 resonance of C-17 in compound 7, caused by a γ-gauche effect, which suggested the relative configuration of the side-chain is different between 7 and 6. Furthermore, the ROESY correlations of H3-18/H-20, H-24/H-20, and H-17/H-21 indicated α-, β-, and βorientations for H-20, H-21, and H-23 in 7, respectively. The substructure of 8 was similar to that of 7, with the only difference being the relative configuration of OH-3. The hydroxy group at C-3 was assigned as α-oriented according to the small coupling constants (JH‑2/H‑3 ≈ 2.7 Hz) of H-3 (δH 3.47). The structure of 9 was also closely related to that of 7, with the exception that the relative configuration of the sidechain was different. In the ROESY spectrum, the correlation networks of H3-18/H-20, H3-18/H-21, and H-20/H-23 indicated the α-orientations of H-20, H-21, and H-23. Accordingly, compounds 7 (picraquassin F), 8 (picraquassin G), and 9 (picraquassin H) were assigned as (20S,21R,23R)-21methoxy-3β,20-dihydroxy-21,23-epoxytirucalla-7,24-diene, (20S,21R,23R)-21-methoxy-3α,20-dihydroxy-21,23-epoxytirucalla-7,24-diene, and (20S,21R,23S)-21-methoxy-3β,20-dihydroxy-21,23-epoxytirucalla-7,24-diene), respectively. Compounds 10 and 11 were identified as a pair of stereoisomers with different side-chain configurations, based on their HRESIMS and 1D- and 2D-NMR spectra. The 1DNMR data (Tables 2 and 3) of 10 displayed close similarities to those of 7, except for the presence of signals for an ethoxy group [δH 3.40 (1H, m, H-1′a), 3.76 (1H, m, H-1′b), and 1.19 (3H, t, 7.2 Hz, Me-2′); δC 63.5, 15.5] at C-21 in 10 instead of the methoxy group in 7. The ROESY correlations of H3-18/H20, H-20/H-23, and H-17/H-21 suggested that H-20, H-21, and H-23 are α-, β-, and α-oriented, respectively. On the other hand, in the ROESY spectrum of 11, the correlations of H-20/ H3-18, H-21/H3-18, and H-21/H-24 were indicative of α-, α-, and β-orientations of H-20, H-21, and H-23, respectively. Therefore, 10 (picraquassin I) and 11 (picraquassin J) were


Figure 5. Measured CD spectrum (200−300 nm) of 1 and calculated ECD spectra of 1a (3R,5R,7R,8R,9R,10S,13S,17S,20S,21S,23R,24S) and the enantiomer of 1a.

m/z 491.3737 [M + H]+ (calcd for C30H51O5, 491.3731). The 1 H and 13C NMR spectra (Tables 1 and 2) along with the HSQC data supported the presence of a carbonyl group, two olefinic bonds, eight methyls, three oxygenated methines, and one oxygenated quaternary carbon, suggesting that 4 is an analogue of cumingianoside Q hydrolysate.34 The HMBC correlations from proton signals at δH 1.11 (H3-28) and 1.06 (H3-29) to the carbonyl carbon signal at δC 217.7 (C-3) revealed the hydroxy group at C-3 in the hydrolysate of cumingianoside Q is oxidized to form a ketone in 4. Thus, the planar structure of 4 was established. The broad H-7 singlet suggested its equatorial position and β-orientation. Additionally, due to the free rotation of the triol side-chain, the configurations of C-23 and C-24 were difficult to establish from the ROESY spectrum. However, a clear coupling constant, JH‑23/H‑24, of 0 Hz in the 1H NMR spectrum indicated the syngauche configuration of the side-chain in 4.26,35 Moreover, the 1 H and 13C NMR chemical shifts of 4 were consistent with those of brumollisol A7 and 24-epi-piscidinol A,26 signifying a (23R,24S)-configuration. Therefore, the overall structure of 4 (picraquassin D) was proposed as (20S,23R,24S)-7α,23,24,25tetrahydroxyapotirucallan-3-one. Compound 5 was obtained as a colorless oil with the molecular formula C30H50O6, as determined based on the positive HRESIMS data (m/z 507.3684 [M + H]+; calcd for C30H51O6, 507.3680). Examination of the 1D-NMR spectra (Tables 1 and 2) disclosed that the structure of 5 is similar to that of 4. Further analysis revealed signals for an additional hydroxy group at C-6 in 5 instead of a methylene group at the same position in 4. This difference was supported by the HMBC correlations of H-7 (δH 3.79) with δC C-5 (51.0), C-6 (72.6), and C-30 (26.4) and 1H−1H COSY correlation of H-6/ H-7. The ROESY correlations of H3-28/H-6 and H-7/H3-30 E



Article

Table 3. 1H NMR Spectroscopic Data (δ in ppm, J in Hz) for Compounds 6−11 position

6a

7a

8a

9a

10b

11a

1

α 1.15 m β 1.68 m α 1.61 m β 1.66 m 3.25 d (2.1, 6.6) 1.32 m α 1.99 m β 2.15 m 5.27 s 2.24 m α 1.52 m β 1.52 m α 1.97 m β 1.32 m α 1.50 m β 1.50 m α 1.61 m β 1.85 m 2.00 m 0.85 s 0.76 s 2.05 m 4.75 d (2.1) α 1.60 m β 1.98 m 4.80 dt (1.5, 5.1) 5.22 d (5.4) 1.71 s 1.72 s 0.98 s 0.87 s 1.00 s 3.31 s

α 1.14 m β 1.66 m α 1.27 m β 1.65 m 3.23 dd (2.1, 6.6) 1.30 m α 1.96 m β 2.13 m 5.26 s 2.24 m α 1.55 m β 1.55 m α 1.76 m β 1.56 m α 1.46 m β 1.54 m α 1.27 m β 1.86 m 1.77 m 0.86 s 0.75 s 2.20 m 4.76 d (1.8) α 1.22 m β 2.06 m 4.64 ddd (4.5, 8.0, 10.0) 5.15 d (8.0) 1.70 s 1.73 s 0.96 s 0.85 s 0.97 s 3.35 s

α 1.38 m β 1.47 m α 1.62 m β 1.94 m 3.47 t (2.7) 1.78 m α 1.96 m β 2.04 m 5.27 s 2.38 m α 1.52 m β 1.59 m α 1.77 m β 1.55 m α 1.55 m β 1.77 m α 1.28 m β 1.88 m 1.80 m 0.88 s 0.79 s 2.21 m 4.79 d (3.3) α 1.22 m β 2.08 m 4.65 ddd (4.8, 8.4, 10.8) 5.17 d (8.4) 1.72 s 1.74 s 0.94 s 0.92 s 0.99 s 3.38 s

α 1.15 m β 1.67 m α 1.59 m β 1.64 m 3.24 dd (4.0, 11.5) 1.31 m α 1.97 m β 2.14 m 5.26 d (2.5) 2.23 m α 1.53 m β 1.53 m α 1.91 m β 1.33 m α 1.51 m β 1.51 m α 1.57 m β 1.85 m 2.03 m 0.84 s 0.75 s 2.01 m 4.68 d (3.3) α 1.49 m β 1.99 m 4.73 ddd (7.0, 9.0, 15.0) 5.16 d (9.0) 1.67 s 1.72 s 0.99 s 0.86 s 0.97 s 3.30 s

α 1.14 m β 1.66 m α 1.26 m β 1.64 m 3.20 dd (3.0, 7.5) 1.29 m α 1.94 m β 2.11 m 5.22 dd (3.0, 6.6) 2.20 m α 1.51 m β 1.51 m α 1.75 m β 1.51 m α 1.44 m β 1.52 m α 1.24 m β 1.83 m 1.75 m 0.85 s 0.72 s 2.22 m 4.84 d (3.0) α 1.18 m β 2.04 m 4.63 ddd (4.8, 8.4, 10.2) 5.12 td (1.2, 8.4) 1.66 s 1.70 s 0.93 s 0.83 s 0.94 s 3.40 m, 3.76 m 1.19 t (7.2)

α 1.15 m β 1.68 m α 1.29 m β 1.65 m 3.24 d (10.0) 1.34 m α 1.97 m β 2.14 m 5.26 s 2.26 m α 1.53 m β 1.53 m α 1.81 m β 1.36 m α 1.49 m β 1.55 m α 1.34 m β 1.95 m 1.78 m 0.89 s 0.76 s 2.26 m 4.92 s α 1.78 m β 1.85 m 4.80 dd (7.0, 15.5) 5.28 d (15.5) 1.68 s 1.72 s 0.98 s 0.87 s 1.00 s 3.42 m, 3.75 m 1.21 t (7.0)

2 3 5 6 7 9 11 12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 1′ 2′ a

Recorded in CDCl3 at 500 MHz. bRecorded in CDCl3 at 300 MHz.

found to be oxidized to form a carbonyl group in 13. The HMBC correlations from the proton signal at δH 5.78 (H-7) to the carbon resonances at δC 198.4 (C-6), 170.6 (C-8), and 43.2 (C-10) supported the location of a carbonyl group at C-6 in 13. Moreover, the configuration of compound 13 was in agreement with that of 12, based on similar 1D-NMR and ROESY spectra. Consequently, compound 13 (6-oxo-21β-ethoxybourjotinolone A) was determined as (20S,21S,23S,24R)-21-ethoxy-23,25dihydroxy-21,24-epoxytirucall-7-ene-3,6-dione. Compound 14 was isolated as a colorless oil, and its molecular formula was determined as C32H50O5 on the basis of a HRESIMS peak at m/z 537.3550 [M + Na]+ (calcd for C32H50O5Na, 537.3550). Comparison of the 1D-NMR data (Tables 5 and 6) with toonaciliatin K (23)21 disclosed a close resemblance, except for the appearance of an additional Δ9(11) double bond in ring C in 14. This difference was supported by the HMBC correlations from the proton resonance at δH 1.18 (H3-19) to the carbon signal at δC 144.0 (C-9) and from the proton signal at δH 5.28 (H-11) to the carbon signals at δC 141.4 (C-8), 144.0 (C-9), 36.9 (C-12), and 44.0 (C-13). The relative and absolute configurations of 14 were the same as those of toonaciliatin K, based on the consistency of their sidechain data in the 1D-NMR and ROESY spectra. Hence, the structure of compound 14 (9,11-dehydrotoonaciliatin K) was

assigned as (20S,21R,23S)-21-ethoxy-3β,20-dihydroxy-21,23epoxytirucalla-7,24-diene and (20S,21S,23R)-21-ethoxy-3β,20dihydroxl-21,23-epoxytirucalla-7,24-diene, respectively. A molecular formula of C32H52O5 was obtained for compound 12, a colorless oil, as deduced by HRESIMS (m/z 539.2722; calcd 539.2707, [M + Na]+). Comparison of its 1DNMR (Tables 4 and 5) and HMQC data with those of bourjotinolone A (25)23 revealed that one of the protons at C21 in the latter compound is substituted with an additional ethoxy group in 12. This deduction was confirmed by the key HMBC correlations from the proton signal at δH 4.83 (H-21) to the carbon signals at δC 62.4 (C-1′), 46.6 (C-17), 35.5 (C20), 31.3 (C-22), and 72.4 (C-24). Furthermore, cross-peaks of H3-18/H-20, H-12α/H-21, H3-18/H-21, H-20/H3-26, and H326/H-23 in the ROESY spectrum were indicative of α-, α-, α-, and β-orientations, respectively. Accordingly, the structure of compound 12 (21β-ethoxybourjotinolone A) was deduced as (20S,21S,23S,24R)-21-ethoxy-23,25-dihydroxy-21,24-epoxytirucall-7-en-3-one. Compound 13 was isolated as a colorless oil and assigned a molecular formula of C32H50O6 using HRESIMS based on the peak at m/z 531.3687 [M + H]+ (calcd for C32H51O6, 531.3680). The 1D-NMR data (Tables 4 and 5) of 13 were similar to those of 12, except that the CH2-6 group in 12 was F



Article

Table 4. 1H NMR Spectroscopic Data (δ in ppm, J in Hz) for Compounds 12, 13, and 18−20 position 1 2

12a α 1.47 m β 1.96 m α 2.25 m β 2.25 m

5 6 7 9

1.71 m α 2.09 m β 2.09 m 5.30 d (2.0) 2.29 m

13b α 1.74 m β 1.98 m α 2.33 td (4.0, 14.5) β 2.75 dt (5.5, 14.5) 2.46 s

5.78 s

23

α 1.60 m β 1.68 m 4.09 br s

2.79 dd (2.5, 6.5) α 1.73 m β 1.73 m α 2.03 m β 1.63 m α 1.61 m β 1.61 m α 1.32 m β 1.94 m 1.93 m 0.89 s 1.11 s 2.15 m 4.84 d (2.0) α 1.62 m β 1.62 m 4.11 br s

24

3.34 br s

3.36 br s

26 27 28

1.30 s 1.32 s 1.11 s

1.31 s 1.33 s 1.38 s

29 30 1′

1.04 s 1.02 s 3.36 m, 3.69 m 1.20 t (7.0)

1.35 s 1.11 s 3.38 m, 3.72 m 1.22 t (7.0)

11 12 15 16 17 18 19 20 21 22

2′

α 1.56m β 1.56 m α 1.67 m β 1.44 m α 1.48 m β 1.48 m α 1.85 m β 1.85 m 1.82 m 0.86 s 1.00 s 2.10 m 4.83 br s

18c α 1.54 m β 1.99 m α 2.36 dt (0.6, 14.5) β 2.67 dt (0.6, 14.5) 1.87 m α 2.05 m β 2.15 m 5.27 s

19a

α 1.26 m β 1.26 m α 1.63 m β 1.24 m α 1.51 m β 1.51 m α 1.31 m β 1.97 m 2.03 m 0.87 s 0.97 s 2.02 m

α 1.54 m β 1.88 m 1.88 m 1.88 m 5.06 d (6.5) 1.57 s 1.68 4.06 d (11.0) 4.61 d (11.0) 1.17 s 1.00 s 3.36 s

20d

α 1.49 m β 1.49 m α 2.42 m

α 1.60 m β 2.23 m α 2.19 m

β 2.76 m

β 2.73 m

2.15 m α 2.48 m β 2.57 m

1.84 m α 2.15 m β 2.23 m 5.17 dd (2.8, 6.8) 2.38 m

2.20 m

α 2.68 m β 2.48m α 1.68 m β 2.16 m α 1.44 m β 2.13 m 1.67 m 0.91 s 1.51 1.47 m 0.95, d (5.5) α 1.26 m β 1.86 m 4.13 dd (5.7, 8.7) 3.15 br s

compound 15 (5,6,9,11-tetradehydrotoonaciliatin K) was assigned as (20S,21S,23R,24S)-21-ethoxy-24,25-dihydroxy21,23-epoxytirucalla-5,7,9(11)-trien-3-one. Compound 16 was obtained as a colorless oil, with its molecular formula assigned as C32H52O6 on the basis of the HRESIMS at m/z 555.3641 [M + Na]+ (calcd for C32H52O6Na, 555.3656). The combined analysis of the 1D-NMR (Tables 4 and 6), HMQC, and HMBC spectra of 16 revealed that it is a closely related analogue to melianodiol (21),20 except for the presence of additional OH-20 and ethoxy-21 groups in 16. The HMBC correlations from the proton resonance at δH 4.68 (H21) to the carbon signals at δC 63.9 (C-1′), 84.2 (C-20), and 39.9 (C-22) confirmed this inference. The observed ROESY correlations of H3-18/H-21, H-21/H-23, and H-24/H-22b (δH 2.17) suggested that H-21, H-23, and H-22b are α-, α-, and βoriented, respectively. The α-orientation of OH-20 was supported by the ROESY correlation of H-17/H-22b. Furthermore, the small coupling constant (JH‑23/H‑24 ≈ 2.0 Hz) indicated an S-configuration at C-24.19,21 The structure of 16 was established as (20R,21R,23R,24S)-21-ethoxy-20,24,25trihydroxy-21,23-epoxytirucall-7-en-3-one (21β-ethoxy-20α-hydroxymelianodiol). Compound 17 was isolated as a colorless oil. It gave a molecular formula of C32H54O5 from the HRESIMS at m/z 541.3860 [M + Na]+ (calcd for C32H54O5Na, 541.3863). Compound 17 showed a similar structure to meliasenin T,41 with the major difference being that OCH3-21 in meliasenin T is replaced by an ethoxy group (δH 1.20, 3.43, and 3.80; δC 15.6, 64.5) in 17. The HMBC cross-peak for H-21/C-1′ verified the presence of an ethoxy group at C-21. The ROESY correlations of H3-18/H-20, H-20/H-24, and H-23/H-21 suggested the protons of H-20, H-21, and H-23 are α-, β-, and β-oriented in 17, respectively. Moreover, the S-configuration at C-24 was confirmed on the basis of the small coupling constant (J23‑H/24‑H ≈ 2.9 Hz) in the 1H NMR spectrum. Accordingly, compound 17 (picraquassin K) was structurally determined as (20S,21R,23S,24S)-21-ethoxy-3β,24,25-trihydroxy-21,23-epoxytirucalla-7,9(11)-diene. Compound 18, a colorless oil, was assigned a molecular formula of C31H48O4, on the basis of the HRESIMS at m/z 485.3635 [M + H] + (calcd for C 31H49O4, 485.3625), representing 14 mass units more than that of xanthocerasic acid.42 On the basis of this finding, together with a detailed comparison of the NMR data of those two compounds (Tables 4 and 5), compound 18 was postulated as being a methyl ester derivative of xanthocerasic acid. This inference was confirmed by the HMBC correlations from the proton signal at δH 3.36 (H3-1′) to the carbon signals at δC 176.5 (C-21) and 47.5 (C20). Consequently, the structure of compound 18 (xanthocerasic acid methyl ester) was proposed as 29-hydroxy-3oxotirucalla-7,24-diene 21-methyl ester. Compound 19 was isolated as a colorless oil, and the molecular formula determined as C30H46O6 based on the HRESIMS at m/z 525.3173 [M + Na]+ (calcd for C30H46O6Na, 525.3187). A comparison of its 1H and 13C NMR spectra (Tables 4 and 5) with those of brumollisol A7 suggested the two compounds to be similar, except for replacement of the methylene group at C-11 in brumollisol A with a carbonyl group in 19. The HMBC correlations from the methylene proton signals at δH 2.48 (H-12a) and 2.68 (H-12b) to the conjugated carbonyl signal at δC 202.3 (C-11) verified the location of a carbonyl group at C-11. The close similarities of the 1D-NMR spectra of the side-chains in 19 and 4 indicated

α 1.50 m β 1.50 m α 1.76 m β 1.47 m α 1.46 m β 1.46 m α 1.76 m β 1.87 m 1.44 m 0.79 s 0.84 m 1.36 m 0.85 d (6.8) α 1.03 m β 1.44 m 1.92 m 2.08 m 5.27 s

1.31 s 1.33 s 1.16 s

4.12 s 1.77 s 1.28 s

1.14 s 1.11 s

1.17 s 0.95 s 4.08 q (7.2) 1.23 d (7.2)

a c

Recorded in CDCl3 at 500 MHz. bRecorded in CDCl3 at 300 MHz. Recorded in CDCl3 at 600 MHz. dRecorded in CDCl3 at 400 MHz.

assigned as (20S,21S,23R,24S)-21-ethoxy-24,25-dihydroxy21,23-epoxytirucalla-7,9(11)-dien-3-one. Compound 15, obtained as a pale yellow oil, was found to possess a molecular formula of C32H48O5 by HRESIMS at m/z 535.3398 [M + Na]+ (calcd for C32H48O5Na, 535.3394). A comparison of the 1D-NMR spectra of 15 and 14 revealed them to be structural congeners, with the only difference being the additional Δ5,6 double bond in 15. The HMBC correlations from the proton signals at δH 1.26 (H3-29) and 1.29 (H3-28) to the carbon signals at δC 214.4 (C-3), 49.2 (C-4), and 149.9 (C5) confirmed the presence of the Δ5,6 olefinic group in 15. On the basis of the supportive evidence from the ROESY spectrum, G



Article

Table 5. 13C NMR Spectroscopic Data (δ in ppm) for Compounds 12−20

a

position

12a

13b

14b

15c

16b

17d

18d

19a

20c

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′

38.5 34.9 217.0 47.9 52.3 24.4 118.0 145.7 48.4 35.0 18.2 31.9 43.4 51.2 33.9 26.8 46.6 22.8 12.8 35.5 99.2 31.3 65.9 72.4 73.7 28.0 24.9 24.5 21.6 27.5 62.4 15.2

37.6 34.1 214.8 47.1 65.3 198.4 125.0 170.6 49.5 43.2 17.6 30.9 42.9 52.4 32.9 26.3 46.2 22.8 13.9 35.3 99.0 31.2 65.6 72.5 73.8 28.1 24.8 25.2 21.7 25.0 62.4 15.2

36.9 34.9 216.5 47.8 50.1 24.2 118.6 141.4 144.0 36.2 116.1 36.9 44.0 49.1 31.4 27.2 43.4 17.7 19.9 46.5 103.5 32.0 78.6 76.7 73.0 26.3 26.4 24.5 22.2 23.2 63.8 15.2

31.1 34.4 214.4 49.2 149.9 117.2 114.9 139.2 142.6 39.2 120.4 37.8 44.5 49.0 30.9 27.1 43.3 17.9 27.7 46.4 103.5 32.0 78.6 76.6 73.0 26.3 26.4 28.6 26.8 22.8 63.8 15.2

38.4 34.9 216.7 48.5 52.4 24.4 118.4 145.2 48.1 35.2 17.8 31.8 44.8 51.3 33.8 22.9 48.2 24.5 12.5 84.2 107.9 39.9 78.3 76.9 73.0 26.3 26.3 24.5 21.5 27.7 63.9 15.2

32.7 26.6 77.1 38.5 46.0 25.2 119.7 147.3 50.0 36.0 18.8 32.7 45.0 52.2 35.5 28.5 46.8 23.7 13.7 48.0 104.9 33.9 79.6 77.9 73.8 25.3 27.6 28.7 22.5 28.0 64.5 15.6

37.4 35.6 216.4 53.2 53.5 24.7 118.2 145.8 48.0 35.0 18.0 30.2 43.1 50.9 33.7 27.1 49.8 21.9 13.5 47.5 176.5 32.4 26.1 123.6 132.1 17.6 25.7 20.6 65.8 27.4 51.1

34.6 34.3 214.3 46.9 48.0 36.2 198.8 150.2 153.5 37.9 201.8 51.3 45.2 48.0 31.8 27.5 50.0 18.2 17.3 33.7 18.8 40.3 69.5 75.2 74.3 25.4 27.5 26.3 21.2 24.0

32.7 28.9 175.3 75.6 49.8 28.5 118.0 146.4 42.2 38.3 17.3 33.7 43.6 51.4 34.4 28.3 53.0 22.2 17.8 36.2 18.5 36.7 24.8 129.4 134.1 61.9 21.5 33.7 26.2 27.5 60.5 14.5

Recorded in CDCl3 at 125 MHz. bRecorded in CDCl3 at 75 MHz. cRecorded in CDCl3 at 100 MHz. dRecorded in CDCl3 at 150 MHz.

was consistent with that of (24Z)-3α-oxa-3α-homo-27-hydroxy7,24-tirucalladien-3-one, as established based on their comparable 1D-NMR and ROESY spectra. Consequently, compound 20 (picraquassin L) was structurally determined as 4,26dihydroxy-3,4-secotirucalla-7,24-dien-3-oic acid 3-ethoxy ester. Several new compounds (2, 10−17, and 20) were found to contain an ethoxy group in their structures. To clarify the origin of these ethoxy groups, a dichloromethane extract of the stems of P. quassioides was analyzed using HPLC-QTOFMS. Protonated molecular ions of compounds 2, 10, 11, 12, 14, 15, 16, and 17 were observed in LC/HRESIMS spectra (see Supporting Information), which proved that these compounds are naturally occurring. However, no protonated molecular ion was observed in the LC/HRESIMS of either compound 13 or 20. All the isolated compounds were evaluated for cytotoxic activity against the MKN-28, A-549, and MCF-7 cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. cis-Platinum was employed as a positive control. As shown in Table 7, several compounds (2, 6, 7, 10, 13, 14, 16, 18, 25, 27, 28, and 30) exhibited inhibitory activities against all or part of the selected drug-sensitive cancer cell lines. Interestingly, these compounds exhibited more potent activities against MKN-28 cells than the other two cell lines used.

the same relative and absolute configurations. Hence, the structure of 19 (11-oxobrumollisols A) was assigned as (20S,23R,24S)-23,24,25-trihydroxytirucall-8-ene-3,7,11-trione. Compound 20, obtained as a colorless oil, was conferred a molecular formula of C32H54O4 based on the HRESIMS at m/z 525.3926 [M + Na]+ (calcd for C32H54O4Na, 525.3914). Careful analysis of the 1D-NMR data (Tables 4 and 5) in conjunction with HSQC experiments revealed that compound 20 shares the same rings B−D and side-chain unit as those of (24Z)-3α-oxa-3α-homo-27-hydroxy-7,24-tirucalladien-3-one.18 Analysis of the 1H−1H COSY and HMBC correlations was used to establish the planar structure of compound 20, as shown in Figure 4. The HMBC correlations of the proton signals at δH 1.28 (H3-28) and 1.17 (H3-29) to the carbon signals at δC 75.6 (C-4) and 49.9 (C-5) revealed the presence of an OH-4 substituent. The 1H−1H COSY correlation of H1a/H-2b and the HMBC correlations from the proton signals at δH 2.73 (H-2b) and 2.19 (H-2a) to the carbon signals at δC 175.3 (C-3), 32.7 (C-1), and 38.3 (C-10) revealed the presence of an ester carbonyl group at C-3. Moreover, the HMBC correlation from the proton signal at δH 4.08 (H-1′) to the carbon signal at δC 175.3 (C-3) supported the presence of an ethoxy group at C-3. The ROESY correlations of H3-18/H-9 and H-9/H-5 indicated the α-orientation of H-5. An additional ROESY interaction of H3-19/H3-28 suggested the α-orientation of CH3-19. The relative configuration of the side-chain in 20 H



Article

Table 6. 1H NMR Spectroscopic Data (δ in ppm, J in Hz) for Compounds 14−17 14a

position 1 2

3 5 6

α 1.82 m β 2.14 m α 2.34 m β 2.85 m

15b

16a

α 2.07 t (6.8) β 2.07 t (6.8) 2.60 m

7 9 11

5.28 d (4.5)

5.28 d (2.4, 4.4)

α 2.21 m β 2.21 m α 1.41 m β 1.70 m α 1.37 m β 1.96 m 2.17 m 0.65 s 1.18 s 1.99 m 4.85 d (3.0) α 1.96 m β 1.96 m 4.44 t (7. 5)

α 2.28 m β 1.92 m α 1.41 m β 1.73 m α 1.35 m β 1.94 m 2.17 m 0.62 s 1.05 s 1.97 m 4.83 d (4.8) α 1.94 m β 1.94 m 4.40 m

3.19 d (1.5, 8.4) 1.28 s 1.28 s 1.15 s 1.07 s 0.91 s 3.44 m, 3.78 m 1.25 t (7.0)

3.16 dd (2.0, 10.0) 1.25 s 1.25 s 1.29 s 1.26 s 0.97 s 3.42 m, 3.76 m

1.26 1.28 1.12 1.05 1.05 3.47

1.23 t (7.0)

1.24 t (7.0)

12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 1′ 2′

17c

α 1.45 m β 1.97 m α 2.25 m β 2.75 dt (5.6, 14.4)

1.67 m α 2.21 m β 2.21 m 5.42 br s

α 1.38 m β 1.54 m α 1.58 m β 1.95 m 3.39 t (2.4) 1.81 m α 1.97 m β 1.97 m 5.29 dd (3.0, 5.4) 2.42 m α 1.61 m β 1.61 m 1.94 m

1.72 t (8.8) α 2.11 m β 2.11 m 5.32 dd (3.2, 6.5) 2.29 m α 1.60 m β 1.60m α 2.02 m β 1.47 m α 1.59 m β 1.59 m α 1.84 m β 1.84 m 2.27 m 1.01 s 1.01 s

5.86 d (6.4) 5.52 d (6.4)

Activation of the mitochondrial apoptosis pathway, also known as the intrinsic apoptosis pathway, may trigger several important events in the mitochondria. The Bcl-2 protein family plays an important role in the mitochondrial pathway of apoptosis. Specifically, the activation of mitochondria and the release of intermembrane contents are under the regulatory control of a number of Bcl-2 proteins. Antiapoptotic proteins, including Bcl-2, prevent the release of cytochrome c and proapoptotic proteins, such as Bax. Cytochrome c binds to apoptotic protease activating factor 1 (Apaf-1), which recruits procaspase-9 to form an apoptosome. This complex activates caspase-9, which in turn cleaves and activates effector procaspase to yield active effector caspases, such as caspase-3.43 Data from Western blotting (Figure 7) further revealed that compound 2 induces mitochondrial apoptosis in MKN-28 cells through activating caspase-3/-9, while increasing Bax and Bad and decreasing Bcl-2 expression levels.

■

α 1.56 m β 1.56 m α 1.38 m β 1.91 m 2.07 m 0.90 s 0.81 s 2.03 m 4.85 d (3.6) α 1.80 m β 2.00 m 4.40 ddd (2.9, 6.6, 9.6) 3.16 (2.9)

4.68 s α 1.88 m β 2.17 m 4.61 ddd (3.2, 6.8, 9.2) 3.15 d (2.0) s s s s s m, 3.78 m

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter, and CD spectra obtained using an Applied Photophysics Chirascan spectrometer. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer. NMR spectra were measured on Bruker AM-300, AM-500, and AM-600 spectrometers at 25 °C. HRESIMS was performed on a Waters Micromass Q-TOF instrument. A Shimadzu LC-20 AT instrument equipped with an SPD-M20A PDA detector was used for HPLC, and a YMC-pack ODS-A column (250 × 10 mm, S-5 μm, 12 nm) applied for semipreparative HPLC separation. Silica gel (300−400 mesh, Qingdao Haiyang Chemical Co., Ltd.), C18 reversed-phase silica gel (12 nm, S-50 μm, YMC Co., Ltd.), Sephadex LH-20 gel (Amersham Biosciences), and MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.) were employed for column chromatography. All solvents used were of analytical grade (Guangzhou Chemical Reagents Co., Ltd.). Plant Material. The stems of P. quassioides were collected in August 2011 at Ganzhou, Jiangxi Province, People’s Republic of China, and authenticated by F.F. A voucher specimen (accession number Piqu-2011JX-A) has been deposited at the Department of Natural Medicinal Chemistry, China Pharmaceutical University. Extraction and Isolation. Air-dried stems (20 kg) of P. quassioides were pulverized and extracted three times with 95% aqueous EtOH (80 L) under reflux conditions. After evaporation of the combined aqueous EtOH extracts in vacuo, the resultant aqueous residue (465 g) was separated via D101 column chromatography (CC) and eluted with H2O and 30%, 50%, 70%, and 90% aqueous EtOH to generate five fractions. The 90% EtOH fraction (37 g) was subjected to passage over silica gel, by elution with petroleum ether/EtOAc (10:0−5:10, v/v), to obtain nine subfractions (A−I). Subfraction D (5 g) was passaged over silica gel CC eluted with petroleum ether/EtOAc (50:1−2:1, v/v) to yield eight fractions (D1−D8). Fraction D4 was subjected to repeated chromatography over MCI gel (MeOH/water, 7:3−0:10, v/v), silica gel (petroleum ether/EtOAc, 5:5:1, v/v), and Sephadex LH-20 (petroleum ether/CH2Cl2/MeOH, 5:5:1, v/v/v), and each of the major components was purified via semipreparative HPLC (MeOH/ water, 87:13, CH3CN/water, 91:9, v/v) to yield 4 (2.7 mg), 10 (28.2 mg), 11 (8.3 mg), 14 (11.1 mg), 15 (5.5 mg), 24 (4.5 mg), and 28

1.19 s 1.24 s 0.92 s 0.92 s 1.02 s 3.43 m, 3.80 m 1.20 t (7.0)

a

Recorded in CDCl3 at 300 MHz. bRecorded in CDCl3 at 400 MHz. c Recorded in MeOD at 600 MHz.

To elucidate the mechanism underlying cytotoxicity of compound 2 against MKN-28 cells, induction of apoptosis was investigated using the annexin V-FITC/PI staining assay and the populations of apoptotic cells quantified. Flow cytometry data (Figure 6) showed the apoptosis rate of MKN-28 including early and late apoptosis cells was increased from 6.9% to 33.4% in a dose-dependent manner after treatment with compound 2 for 48 h, clearly signifying enhanced cell death. Table 7. Cytotoxicity Data of Selected Compounds (IC50, μM)a MKN-28 A-549 MCF-7 a

2

6

7

10

13

14

16

17

18

25

27

28

30

cis-platinum

2.5 5.6 9.1

8.8 >10 >10

9.8 >10 >10

7.9 8.3 >10

8.2 >10 >10

8.3 >10 >10

9.0 >10 >10

9.1 >10 >10

8.3 >10 >10

6.7 7.0 9.9

9.1 >10 >10

6.9 8.0 8.5

9.1 >10 >10

5.2 1.6 7.8

Compounds 1, 3, 4, 5, 8, 9, 11, 12, 15, 19−24, 26, and 29 were inactive (IC50 > 10 μM) for all three cell lines. I



Article

Figure 6. Apoptosis induced by compound 2 in MKN-28 cells. (A) Flow cytometry analysis of annexin V-FITC- and PI-stained MKN-28 cells treated with compound 2 (2, 6, and 8 μM) for 48 h. (B) Quantitative analysis of the ratio of apoptotic MCF-28 cells. **p < 0.01, ***p < 0.001, oneway ANOVA, post hoc comparisons, Tukey’s test. Columns, mean; error bars, SD.

Figure 7. Western blot analysis of Bcl-2, Bax, Bad, cleaved-caspase-3, pro-caspase-3, cleaved-caspase-9, and pro-caspase-9 in MKN-28 cells treated with compound 2 (2, 6, and 8 μM) for 48 h. β-Actin served as a loading control. *p < 0.01, one-way ANOVA, post hoc comparisons, Tukey’s test. Columns, mean; error bars, SD. (11.2 mg). Fraction D5 was chromatographed repeatedly over ODS (MeOH/water, 6:4−9:1, v/v) to afford 2 (16.3 mg), 7 (15.6 mg), and 16 (5.3 mg). Fraction E was subjected to chromatography on a silica gel column (petroleum ether/EtOAc, 30:1−4:1, v/v) to yield seven major subfractions, each of which was purified using Sephadex LH-20 and semipreparative HPLC to furnish 1 (2.2 mg), 6 (3.8 mg), 19 (5.1 mg), and 20 (4.2 mg). The 70% EtOH fraction (83 g) was fractionated via a column chromatography with MCI gel, eluted with mixtures of MeOH/water (65:35−90:10, v/v), to obtain eight subfractions (a−k). Subfraction b (6.8 g) was further purified by repeated silica gel CC and elution with

petroleum ether/EtOAc (5:1−2:1, v/v), Sephadex LH-20 (petroleum ether/CH2Cl2/MeOH, 5:5:1, v/v/v), and ODS (MeOH/water, 65:35−90:10, v/v) to yield seven major subfractions, each of which was purified by semipreparative HPLC to yield 3 (2.2 mg), 8 (12.6 mg), 9 (8.7 mg), 21 (3.8 mg), 22 (1.8 mg), 23 (3.1 mg), 29 (1.8 mg), and 30 (2.7 mg). Subfraction c (4.5 g) was separated by column chromtography over ODS (MeOH/water, 55:45−85:15, v/v) and Sephadex LH-20 (petroleum ether/CH2Cl2/MeOH, 5:5:1, v/v/v), followed by semipreparative HPLC (MeOH/water, 65:35−75:25, CH3CN/water, 60:40−85:15, v/v), to yield 5 (3.3 mg), 12 (20.2 mg), J



Article

Tables 5 and 6; (+) HRESIMS m/z 541.3860 [M + Na]+ (calcd for C32H54O5Na, 541.3863). Xanthocerasic acid methyl ester (18): colorless oil; [α]25D −18.2 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 202 (4.33) nm; 1H and 13 C NMR data, see Tables 4 and 5; (+) HRESIMS m/z 485.3635 [M + H]+ (calcd for C31H49O4, 485.3625). 11-Oxobrumollisols A (19): colorless oil; [α]25D −42.2 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 219 (3.82), 273 (3.60) nm; 1H and 13C NMR data, see Tables 4 and 5; (+) HRESIMS m/z 525.3173 [M + Na]+ (calcd for C30H46O6Na, 525.3187). Picraquassin L (20): colorless oil; [α]25D −12.5 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 201 (4.22) nm; 1H and 13C NMR data, see Tables 4 and 5; (+) HRESIMS m/z 525.3926 [M + Na]+ (calcd for C32H54O4Na, 525.3914). HPLC-QTOFMS Analysis. Stems of P. quassioides powder (100 g) were passed through a 40-mesh sieve and ultrasonically extracted with 500 mL of CH2Cl2 twice (40 min each time). After evaporation of the combined aqueous CH2Cl2 extracts in vacuo, the resultant aqueous residue was subjected to chromatography over a D101 column and eluted with 50% and 90% aqueous methanol. The 90% aqueous methanol fraction was combined and evaporated to dryness, and the residue dissolved in methanol and fitered through a 0.45 μm syringe filter prior to LC-MS analysis. HPLC analysis was performed on an Agilent series system (Agilent Technolgies, Santa Clara, CA, USA) equipped with a quaternary pump, vacuum degasser, autosampler, and column heater-cooler. Samples were separated on a Hanbon Phecda RP-C18 column (Jiangsu Hanbon Science Technology, Jiangsu, People’s Republic of China) (250 × 4.6 mm, 5 μm) with a standard guard column, setting the column temperature at 35 °C. The mobile phase consisted of water (containing 2 mmol/L ammonium acetate) (A) and acetonitrile (B). The gradient elution program used was 40−100% (B) at 0−60 min. The flow rate was 1.0 mL/min, and the sample volume injected was 10 μL. The HPLC system was interfaced to an Agilent 6520 Q-TOF (Agilent Technologies) mass spectrometer equipped with an electrospray interface operating under the above conditions. The optimized MS operating conditions were as follows: positive ionization mode, scan spectra from m/z 100 to 1000, drying gas (N2) flow rate of 8.0 L/ min, drying gas temperature of 325 °C, nebulizer pressure of 40 psi, capillary voltage of 3500 V, skimmer of 65 V, and fragmentor voltage of 120 V. The sample collision energy was set at 35 V. Data were processed using MassHunter Qualitative Analysis B.04.00 (Agilent Technologies). Cytotoxicity Assays. MKN-28 human gastric carcinoma, MCF-7 human breast carcinoma, and A-549 human lung cancer cell lines were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, People’s Republic of China). Cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco Invitrogen Corp., Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou, People’s Republic of China), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO2. Cytotoxic activity was investigated using the MTT colorimetric method.44 Briefly, cells were seeded in a 96-well plate at a density of 5 × 103 cells per well in 190 μL of medium for 12 h, followed by exposure to the test compounds or positive control. After 48 h exposure, 20 μL of MTT (5 mg/mL) was added and the cells were incubated at 37 °C for an additional 4 h. Supernatant fractions were aspirated, and 150 μL of DMSO was added to each well. The absorbance was measured at 570 nm using a microplate reader (Spectramax Plus 384, Molecular Devices, Sunnyvale, CA, USA). Cytotoxic activity was expressed as IC50 values (concentration inhibiting the proliferation rate of tumor cells by 50%, compared to untreated control cells). All experiments were carried out in triplicate and repeated twice. cis-Platinum was used as the positive control. Apoptosis Assay. Cell apoptosis was analyzed using an annexin VFITC/PI staining assay kit (Biounprecedentedr Tech, Nanjing, People’s Republic of China) according to the manufacturer’s protocol. MKN-28 cells (3 × 105/well) were treated with compound 2 (2, 6,

13 (3.6 mg), 17 (1.9 mg), 18 (3.1 mg), 25 (9.1 mg), 26 (1.2 mg), and 27 (3.5 mg). Picraquassin A (1): colorless oil; [α]25D −32.7 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 201 (3.92) nm; CD (c 3.76 mM), see Figure 5; 1 H and 13C NMR data, see Tables 1 and 2; (+) HRESIMS m/z 508.3999 [M + NH4]+ (calcd for C30H54NO5, 508.3997). Picraquassin B (2): colorless oil; [α]25D −84.9 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 201 (4.16) nm; 1H and 13C NMR data, see Tables 1 and 2; (+) HRESIMS m/z 539.3507 [M + K]+ (calcd for C32H52O4K, 539.3497). Picraquassin C (3): colorless oil; [α]25D −29.6 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 202 (4.26) nm; 1H and 13C NMR data, see Tables 1 and 2; (+) HRESIMS m/z 647.4160 [M + H]+ (calcd for C37H59O9, 647.4154). Picraquassin D (4): colorless oil; [α]25D −32.9 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 201 (4.19) nm; 1H and 13C NMR data, see Tables 1 and 2; (+) HRESIMS m/z 491.3737 [M + H]+ (calcd for C30H51O5, 491.3731). 6β-Hydroxypicraquassin C (5): colorless oil; [α]25D −48.7 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 201 (4.32) nm; 1H and 13C NMR data, see Tables 1 and 2; (+) HRESIMS m/z 507.3684 [M + H]+ (calcd for C30H51O6, 507.3680). Picraquassin E (6): colorless oil; [α]25D +15.3 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 200 (4.41) nm; 1H and 13C NMR data, see Tables 2 and 3; (+) HRESIMS m/z 471.3478 [M + H]+ (calcd for C31H51O3, 471.3469). Picraquassin F (7): colorless oil; [α]25D −24.4 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 201 (4.17) nm; 1H and 13C NMR data, see Tables 2 and 3; (+) HRESIMS m/z 471.3469 [M + H]+ (calcd for C31H51O3, 471.3469). Picraquassin G (8): colorless oil; [α]25D −38.5 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 200 (4.37) nm; 1H and 13C NMR data, see Tables 2 and 3; (+) HRESIMS m/z 471.3467 [M + H]+ (calcd for C31H51O3, 471.3469). Picraquassin H (9): colorless oil; [α]25D +18.7 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 200 (4.26) nm; 1H and 13C NMR data, see Tables 2 and 3; (+) HRESIMS m/z 471.3453 [M + H]+ (calcd for C31H51O3, 471.3469). Picraquassin I (10): colorless oil; [α]25D −36.4 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 200 (4.29) nm; 1H and 13C NMR data, see Tables 2 and 3; (+) HRESIMS m/z 507.3807 [M + Na]+ (calcd for C32H52O3Na, 507.3809). Picraquassin J (11): colorless oil; [α]25D −56.4 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 200 (4.26) nm; 1H and 13C NMR data, see Tables 2 and 3; (+) HRESIMS m/z 507.3792 [M + Na]+ (calcd for C32H52O3Na, 507.3809). 21β-Ethoxybourjotinolone A (12): colorless oil; [α]25D −1.9 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 202 (4.07) nm; 1H and 13C NMR data, see Tables 4 and 5; (+) HRESIMS m/z 539.2722 [M + Na]+ (calcd for C32H52O5Na, 539.2707). 6-Oxo-21β-ethoxybourjotinolone A (13): colorless oil; [α]25D −2.4 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 200 (4.38) nm; 1H and 13 C NMR data, see Tables 4 and 5; (+) HRESIMS m/z 531.3687 [M + H]+ (calcd for C32H52O6, 531.3687). 9,11-Dehydrotoonaciliatin K (14): colorless oil; [α]25D −56.5 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 201 (4.17), 239 (4.19) nm; 1 H and 13C NMR data, see Tables 5 and 6; (+) HRESIMS m/z 537.3550 [M + Na]+ (calcd for C32H50O5Na, 537.3550). 5,6,9,11-Dehydrotoonaciliatin K (15): colorless oil; [α]25D −46.5 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 201 (4.18), 319 (3.35) nm; 1H and 13C NMR data, see Tables 5 and 6; (+) HRESIMS m/z 535.3398 [M + Na]+ (calcd for C32H48O5Na, 535.3394). 21β-Ethoxy-20α-hydroxymelianodiol (16): colorless oil; [α]25D +4.6 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 201 (4.07) nm; 1 H and 13C NMR data, see Tables 5 and 6; (+) HRESIMS m/z 555.3641 [M + Na]+ (calcd for C32H52O6Na, 555.3656). Picraquassin K (17): colorless oil; [α]25D −10.2 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 201 (4.09) nm; 1H and 13C NMR data, see K



Article

and 8 μM) for 48 h, stained with annexin V-FITC and PI solution, and examined using a FACS Calibur flow cytometer (Becton-Dickinson Co., Miami, FL, USA). Data were analyzed quantitatively with the Cell Quest program from Becton−Dickinson. Western Blotting. Bcl-2, Bax, and Bad levels and activation of caspase-3 and -9 (Abcam, Britain) were evaluated via Western blotting. MKN-28 cells (1 × 106/dish) were cultured with compound 2 (2, 6, and 8 μM) for 48 h, harvested, and lysed in RIPA buffer (0.5 M DTT, 0.1 M PMSF, 20× phosphatase inhibitor) for 30 min on ice. After centrifugation at 14000g at 4 °C for 15 min, supernatant fractions were collected as total cellular proteins and stored at −80 °C until use. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Electrophoresis and immunoblot analyses were carried out as described previously.45

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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.5b01137.

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Additional information (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel (F. Feng): +86 25 83271038. E-mail: fengsunlight@163. com. *Tel (W. Qu): +86 25 83271038. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Nos. 81373956, 81274064, 81573557, and 81502950), Fundamental Research Funds for the Central Universities (No. 2015ZD010), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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