Extracellular Signal-Regulated Kinases (ERK ... - ACS Publications

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Extracellular Signal-Regulated Kinases (ERK) Inhibitors from Aristolochia yunnanensis Zhong-Bin Cheng,† Wei-Wei Shao,† Ye-Na Liu, Qiong Liao, Ting-Ting Lin, Xiao-Yan Shen, and Sheng Yin* School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People’s Republic of China S Supporting Information *

ABSTRACT: Six new sesquiterpenoids, aristoyunnolins A−F (1−6), an artifact of isolation [7-O-ethyl madolin W (7)], and 12 known analogues were isolated from stems of Aristolochia yunnanensis. The structures were determined by combined chemical and spectral methods, and the absolute configurations of compounds 2, 3, 5−7, 9, 14, and 17 were determined by the modified Mosher’s method and CD analysis. Compounds 1−19 were screened using a bioassay system designed to evaluate the effect on mitogen-activated protein kinases (MAPKs) signaling pathways. Among three MAPKs (ERK1/2, JNK, and p38), compounds 1, 4, 10−13, 16, 18, and 19 exhibited selective inhibition of the phosphorylation of ERK1/2. Compounds 16 and 19 were more active than the positive control PD98059, a known inhibitor of the ERK1/2 signaling pathway.

M

19). Biological tests verified that compounds 1, 4, 10−13, 16, 18, and 19 were responsible for the inhibition of the phosphorylation of ERK1/2. In particular, 16 and 19 had much stronger activity than the positive control PD98059, a known inhibitor of the ERK1/2 signaling pathway. Herein, details of the isolation, structural elucidation, and ERK1/2 phosphorylation inhibitory activity of these compounds are described.

itogen-activated protein kinase (MAPK) signaling pathways are evolutionarily conserved in multicellular organisms and play a key role in physiological events such as gene expression, mitosis, movement, metabolism, and programmed death.1−3 These pathways are a phosphorelay system composed of kinases MAPKKK, MAPKK, and MAPK. The output of these pathways is transduced via MAPK family members, which phosphorylate and regulate a wide series of substrates, including other protein kinases, phospholipases, transcription factors, and cytoskeletal proteins.1 Classic MAPKs consist of the extracellular signal-regulated kinases (ERK), the c-Jun NH2-terminal kinases (JNK), and p38.2 ERK1/2 function in the control of cell division and inhibitors of these enzymes are being explored as anticancer agents. JNKs are critical regulators of transcription, and JNK inhibitors may be effective in the control of rheumatoid arthritis. The p38 MAPKs are activated by inflammatory cytokines and environmental stresses and may contribute to diseases such as asthma and autoimmunity.3 Thus, the MAPK inhibitors are regarded as potential leads in drug development for multiform diseases. Aristolochia yunnanensis Franch. (Aristolochiaceae), endemic to Yunnan Province of China, is known as “Nan Mu Xiang” in traditional Chinese medicine for the treatment of gastrointestinal diseases, trichomoniasis, and rheumatic pain.4 Previous chemical investigation of this plant revealed four sesquiterpene lactones that showed activity in anticancer research.5 In our screening program aimed at the discovery of MAPK inhibitors from medicinal plants, a fraction of the ethanolic extract of A. yunnanensis showed significant inhibitory activity. Subsequent chemical investigation led to the isolation of seven new sesquiterpenoids (1−7) and 12 known ones (8− © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The air-dried powder of stems of A. yunnanensis was extracted with 95% EtOH at room temperature to give a crude extract, which was suspended in H2O and successively partitioned with petroleum ether, EtOAc, and n-BuOH. Various column chromatographic separations of the EtOAc extract afforded compounds 1−19. Compound 1, a white powder, had the molecular formula C15H21NO5, as determined by HREIMS and ESIMS. The IR spectrum exhibited absorption bands for OH (3309 cm−1), lactone (1753 cm−1), and nitro (1547 and 1368 cm−1) functionalities. The 1H NMR spectrum showed two methyl singlets [δH 1.06 (H3-14) and 1.80 (H3-12)], a terminal double bond [δH 4.86 (1H, br s, H-13a) and 4.87 (1H, br s, H-13b)], three protons bonded to carbons bearing heteroatoms [δH 4.21 (s, H-5), 4.61 (s, H-6), and 4.63 (dd, J = 8.0, 8.0, H-1)], and a series of aliphatic methylene multiplets. The 13C NMR spectrum, in combination with DEPT experiments, resolved 15 carbon resonances attributable to one carbonyl, one sp2 Received: December 20, 2012 Published: April 9, 2013 664

dx.doi.org/10.1021/np300887d | J. Nat. Prod. 2013, 76, 664−671

Journal of Natural Products

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unsaturation required that 1 was tricyclic. The abovementioned information was similar to that of versicolactone C (8), the first example of a hydroazulene-type sesquiterpene previously isolated from A. versicolar,6 except for the presence of a nitro group in 1. HMBC correlations from CH3-14, H2-3, and H2-9 to a severely downfield-shifted carbon (δC 95.5) revealed that the nitro group was located at C-1, as the C-1 of 8 bearing an OH resonated at δC 82.2. Detailed 2D analyses (HSQC, 1H−1H COSY, and HMBC) supported the planar structure of 1 as depicted. The relative configuration of 1 was established by analysis of the 1D NOE difference spectra. The interactions of H-5 (after irradiation) with H-1, H-6, H-7, and H-9β indicated that the skeletal rings were cis-fused, and these protons were β-oriented. The interactions of H3-14 (after irradiation) with H-2α, H-3α, H-8α, and H-9α indicated H3-14 was α-oriented (S70, Supporting Information). Compound 1, possessing a rare nitro group, was only the second example of a hydroazulene-type sesquiterpene found in nature, and it was given the trivial name aristoyunnolin A. Moreover, the initial assignments of the NMR data (at C-1, C-4, C-5, and C-6) of versicolactone C (8) were revised by detailed analyses of its 2D NMR data in this study (see Tables 1 and 2). Compound 2, a colorless oil, had the molecular formula C15H20O4, as established by HRESIMS. The 1H and 13C NMR

quaternary carbon, one sp2 methylene, two methyls, four sp3 methines (three bearing heteroatoms), four sp3 methylenes, and two sp3 quaternary carbons. As three of the six degrees of unsaturation were accounted for by a nitro group, a double bond, and a lactone, the remaining three degrees of Table 1. 1H NMR Data of Compounds 1−8a 1b

2b

4.63, dd (8.0, 8.0) 2.70, m 2.14, m 2.31, m

3.09, d (10.6)

3β 4

2.31, m

b 2.40, m

5α

4.21, s

no. 1 2α 2β 3α

2.31, m 1.87, m a 2.57, m

7.06, s

3b 5.14, dd (11.0, 5.1) 2.24, m 2.15, m 2.79, ddd (12.0, 3.5, 3.5) 2.04, m

6.20, d (9.1)

5β 6 7

4.61, s 2.35, m

8α 8β

1.55, m 1.66, dd (7.5, 3.7) 2.02, dd (15.1, 7.5) 1.76, m

9α 9β 11 12a 12b 13a

1.80, s 4.86 br s

13b

4.87 br s

14 15

1.06, s

4b

6.62, s

5b

6b

9.38, s

10.15, s

9.30, s

6.29, d (9.2)

5.93, d (10.8)

6.10, d (11.4)

4.53, dddd (9.6, 9.6, 3.8, 3.8) 2.57, m

6.20, ddd (10.8, 10.8, 4.3) 2.28, m

1.93, m

2.22, m

2.51, m

0.90, t (5.4)

8c 3.79, dd (10.3, 7.6) 1.93, m 1.67, m a 1.93, m b 2.12, m

a 2.65, d (8.8) b 2.65, d (8.8)

1.28, m

4.15, s

5.10, s 2.69, dd (12.0) 4.1) 1.73, m 1.85, m

2.36, dd (9.8, 9.1) 1.73, ddd (10.3, 9.8, 2.2) 1.86, m 0.89, m

4.85, dd (6.4, 6.4) 2.32, m 1.95, m

4.96, dd (7.3, 7.3)

4.99 br d (10.8)

2.43, d (8.4)

4.52, s 2.41, m

2.10, m 2.10, m

1.97, m 2.30, m

a 2.03, m b 1.66, m

1.58, m 1.55, m

2.41, m

2.10, m

1.97, m

2.00, m

1.99, m

a 2.31, m

1.50, m

2.18, m

2.17, m 4.70, dd (6.8, 6.8) α 2.57, m β 2.09, m 2.55, m

2.00, m 4.73, dd (7.7, 7.7)

2.16, m 4.77, dd (11.3, 3.0)

b 2.02, m 5.24 br d (10.0)

1.75, m

2.55, m 2.11, m 2.30, m

2.04, m 2.15, m 2.78, m

1.80, s 4.83, s

2.29, m

2.11, m

2.05, m

4.85, s

1.63, s 1.43, s

1.57, s 1.34, s 4-OAc:

α 2.03, m β 2.36, m α 1.72, ddd (12.8, 12.8, 3.5) β 2.78, ddd (12.8, 3.9, 3.9) 1.55, s 1.31, s 2.04, s −OCH2CH3:

1.20, s 1.44, s a 3.56, qdd (7.0, 7.0, 2.0) b 3.43, qdd (7.0, 7.0, 2.0) 1.19, t (7.0)

0.92, s

4.90, s 4.99, s 1.85, s

1.23, s 11-COOCH3:

1.41, s

9.30, s 1.25, s 3.70, s

−OCH2CH3: a

7b

Data were recorded at 400 MHz, chemical shifts are in ppm, coupling constant J is in Hz. bIn CDCl3. cIn CD3OD. 665



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Table 2. 13C NMR (100 MHz) Data (δ) of Compounds 1−8 no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

a

1a 95.5, 25.3, 23.8, 58.8, 74.1, 88.0, 50.5, 21.8, 37.2, 50.5, 145.2, 21.9, 112.7, 17.8, 177.3,

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

2a 78.3, CH 30.6, CH2 23.4, CH2 134.2, C 150.1, CH 82.4, CH 48.4, CH 25.2, CH2 66.2, CH 65.1, C 145.5, C 113.1, CH2 21.5, CH3 11.2, CH3 173.2, C −OCH3:

3a 124.9, 27.7, 23.9, 144.8, 150.4, 30.8, 36.3, 23.3, 39.2, 134.4, 27.1, 175.6, 10.0, 193.9, 17.2, 52.3,

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

4a 171.2, 135.2, 148.8, 106.3, 46.9, 128.7, 131.7, 25.2, 39.2, 135.6, 124.7, 24.1, 25.4, 18.3, 14.9,

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

5a

6a

195.9, CH 143.0, C 154.7, CH 68.3, CH 46.8, CH2 129.8, C 128.5, CH 24.8, CH2 38.4, CH2 134.0, C 125.1, CH 25.0, CH2 25.1, CH2 18.6, CH3 15.4, CH3 −OAc:

191.2, CH 141.3, C 145.0, CH 65.7, CH 45.2, CH2 128.4, C 130.7, CH 25.0, CH2 39.6, CH2 135.1, C 125.1, CH 25.8, CH2 31.7, CH2 15.9, CH3 14.9, CH3 21.2, CH3 170.1, C −OCH2CH3: −OCH2CH3:

7a 194.1, 143.0, 158.1, 25.9, 26.8, 28.9, 89.4, 29.4, 39.3, 135.8, 126.5, 27.4, 23.0, 14.3, 15.2,

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

8b 82.2, CH 29.7, CH2 22.6, CH2 58.6, C 75.4, CH 89.8, CH 51.4, CH 23.0, CH2 36.7, CH2 49.8, C 148.0, C 22.1, CH3 112. 2, CH2 15.6, CH3 182.4, C

64.6, CH2 15.7, CH3

In CDCl3. bIn CD3OD.

spectra of 2 showed signals for an α,β-unsaturated-γ-lactone [δC 173.2, 150.1, 134.2, and 82.4], an isopropenyl group [δC 145.5, 113.1, and 21.5; δH 4.90 (1H, br s), 4.99 (1H, br s), and 1.85 (3H, s)], a tertiary methyl [δH 1.23 (3H, s)], and three oxygenated carbons [δC 78.3, 66.2, and 65.1]. These data showed high similarity to those of versicolactone B (9)6 except for the presence of two additional oxygenated carbon signals [δC 65.1 (s) and 66.2 (d)] in 2 instead of signals for Δ9 in 9, indicating that 2 was a 9,10-epoxy derivative of 9. HMBC correlations from CH3-14 to these two oxygenated carbons (C9 and C-10) and from H-8 to C-10 confirmed the location of the epoxy ring. The relative configuration of 2 was determined by a NOESY experiment. NOESY correlations of H-5/H-6, H7, and H-9 and H-1/H-9 indicated that these protons were cofacial and designated as β-oriented, while the correlations of CH3-14/H-3α and H-8α assigned CH3-14 to be α-oriented (S70, Supporting Information). The chemical transformation of 9 to 2 by 3-chloroperbenzoic acid oxidation confirmed the structure of 2. As the absolute configuration at C-1 in 9 was assigned as S by the modified Mosher’s method in our current study (Figure 1), the absolute configuration of 2 was thus determined as depicted. Compound 2 was given the trivial name aristoyunnolin B. Compound 3 had a molecular formula of C16H22O3, as established by HRESIMS. The 1H and 13C NMR spectra of 3 were similar to those of (+)-isobicyclogermacrenal (12)7 except

for the absence of a tertiary methyl in 12 and the presence of a methoxycarbonyl group [δH 3.70 (3H, s); δC 175.6 and 52.3] in 3. HMBC correlations from a tertiary methyl [δH 1.41 (3H, s), CH3-13] to the carbonyl (δC 175.6, C-12) and a quaternary carbon (δC 27.1, C-11) and from an OCH3 [δH 3.70 (3H, s)] to C-12 indicated that one geminal methyl of C-11 in 12 was replaced by a methoxycarbonyl group in 3. The strong NOESY correlations of H-1/H-9, H3-15/H-2, H-14/H-5, and H-3/H-6 indicated that both Δ1,10 and Δ4 had an E configuration. The 9.8 Hz coupling constant and NOESY correlations between H6 and H-7 indicated the cis configuration of the cyclopropane moiety.8 The CH3-13 was β-oriented on the basis of NOESY correlations of CH3-13/H-5 and H-8β (S70, Supporting Information). The absolute configurations at C-6 and C-7 in 3 were assigned to be the same as those of 12 (6S and 7R), as the CD curve of 3 [λmax (Δε) 202 (+0.86), 247 (+1.72), 330 (+0.11) nm] matched well with that of 12 [λmax (Δε) 203 (+1.06), 256 (+2.88), 327 (+0.44) nm] (S69, Supporting Information). In a similar way, the absolute configuration of the known analogue madolin K (14) [λmax (Δε) 203 (+0.25), 254 (+1.99), 327 (+0.20) nm] was determined in the current study as depicted. Compound 3 was named aristoyunnolin C. Compound 4 was isolated as a white powder with the molecular formula C15H20O3, as determined by HREIMS. IR absorption bands at 3396 and 1744 cm−1 indicated the presence of OH and carbonyl groups. The 1H NMR of 4 displayed signals for two olefinic methyl singlets [δH 1.63 (s, H3-14) and 1.43 (s, H3-15)] and three olefinic protons [δH 6.62 (s, H-3), 4.85 (dd, J = 6.4, 6.4 Hz, H-7), and 4.70 (dd, J = 6.8, 6.8 Hz, H-11)]. The 15 carbon resonances were classified by DEPT experiments as two methyls, one lactone, three trisubstituted double bonds, five sp3 methylenes, and one sp3 quaternary carbon. This information was very similar to those of manshurolide (16)9 except for the absence of signals for C-4 (δH 5.10, δC 80.1) in 16 and the presence of a highly oxygenated quaternary carbon (δC 106.3, C-4) in 4. As the molecular formula of 4 showed one more O atom than that of 16, compound 4 was proposed to be a 4-hydroxylated derivative of 16. This was supported by HMBC correlations

Figure 1. ΔδH values of (S)- and (R)-MTPA esters of 5, 9, and 17, respectively. 666



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from H-5 and H-3 to C-4. Configurations of the Δ6 and Δ10 double bonds in 4 were both assigned as E by NOESY experiments and comparison of the NMR data with those of 16. As compound 4 showed no optical activity and gave a flat line in its CD spectrum, the only chiral center (C-4) was racemic. Enantiomeric separation of 4 on a chiral column failed, as the hemiketal nature of 4 resulted in a racemic equilibrium under HPLC conditions. Thus, compound 4 was 4-hydroxymanshurolide and was given the trivial name aristoyunnolin D. Compound 5 had the molecular formula C15H22O2 (by HREIMS). The UV, IR, and NMR data of 5 were similar to those of madolin L8 except for the absence of the methoxy group and the upfield-shifted carbon signal of C-4 (δC 68.3 in 5 and δC 77.0 in madolin L), implying that 5 was a 4-O-demethyl derivative of madolin L. This was supported by the molecular formula and confirmed by detailed 2D NMR analyses of 5, in which H-4 (δH 4.53) showed correlations with H-3 and H-5 in the 1H−1H COSY spectrum. The configurations of the double bonds at Δ6, Δ10, and Δ2 were assigned to be the same as those of madolin L by NOESY experiment and by comparison of their NMR data. The absolute configuration of C-4 in 5 was assigned as S by the modified Mosher’s method (Figure 1). Compound 5 was given the trivial name aristoyunnolin E. Compound 6 had the molecular formula C17H24O3. The 1H and 13C NMR spectra of 6 resembled those of madolin M,8 with the only difference being due to the presence of a C-4 acetoxyl in 6 [δH 2.04 (3H, s); δC 21.2 and 170.1] instead of a C-4 OCH3 in madolin M. The C-4 acetoxyl in 6 was confirmed by the HMBC correlation from H-4 (δH 6.20, ddd, J = 10.8, 10.8, 4.3 Hz) to the carbonyl carbon (δC 170.1). The configurations of the double bonds at Δ6, Δ10, and Δ2 were determined to be the same as those of madolin M by NOESY experiment, and comparison of the 13C NMR data with those of madolin M, especially the NOESY correlations of H-4/H-1 and H-3/H-13, assigned the Z configuration of Δ2. The absolute configuration of the only chiral center (C-4) in 6 was proposed to be R based on its positive optical rotation ([α]20D + 113), which was opposite that of 5. Compound 6 was given the trivial name aristoyunnolin F. Compound 7, obtained as a colorless oil, had the molecular formula C17H26O2, as determined by HRESIMS. The UV, IR, and NMR spectra of 7 were very similar to those of madolin W (17)10 except for the presence of an ethoxy group and the downfield-shifted C-7 carbon signal at δC 89.4 in 7 (δC 82.4 in 17), indicating that 7 was a 7-O-ethyl derivative of 17. Chemical transformation of 17 to 7 using the Williamson reaction confirmed the structure of 7. Compound 7 was considered to be an artificial product, which could not be detected in the crude extract. Moreover, the absolute configuration of madolin W (17) was first determined by the modified Mosher’s method in the current research (Figure 1). The known compounds versicolactone C (8),6 versicolactone B (9), 6 madolin U (10), 10 aristolactone (11), 8 (+)-isobicyclogermacrenal (12),7 volvalerenal D (13),11 madolin K (14),8 madolin T (15),12 manshurolide (16),9 madolin W (17),10 madolin A (18),13 and 1(10)-aristolen-2one (19)14 were identified by comparison of their NMR data with those in the literature. Compounds 1−19 were screened in a bioassay system designed to evaluate the effect on MAPK signaling pathways. To exclude cytotoxic compounds, which may also cause variations of this pathway, compounds 1−19 were first tested for cytotoxicity against HeLa cells. The data showed that

compounds 3, 6, 9, and 14 impaired the viability of HeLa cells to varying degrees at 50 μM (Figure 2). The remaining

Figure 2. Viability of HeLa cells treated with compounds 1−19. The viability of HeLa cells was inhibited by 3, 6, 9, and 14, whereas it was promoted by 4. HeLa cells were treated with 50 μM of each compound for 48 h. Cell viability was detected by a WST-8-based colorimetric assay (n = 3, *p < 0.05, **p < 0.01, ANOVA).

compounds were examined for inhibitory activity on the phosphorylation of three key MAPKs (ERK1/2, JNK, and p38 MAPK) by immunoblotting. As shown in Figure 3, cells treated with 1, 4, 10−13, 15, 16, 18, and 19 at a dose of 20 μM induced a significant decrease in phosphorylation of ERK1/2, but not of p38 and JNK, suggesting a selective inhibitory activity of these compounds on ERK1/2 activation. To evaluate the potency of the compounds, PD98059, a well-known selective inhibitor of the ERK1/2 signaling pathway, was used as the positive control for confirmation testing. Compounds 16 and 19 showed stronger activity than PD98059, while 1, 4, 10− 13, and 18 exhibited activity comparable to the positive control (Figure 4). Further investigation revealed that compounds 16 and 19 inhibited the phosphorylation of ERK1/2 in a dosedependent manner ranging from 2.5 to 40 μM, being more active than the positive control at concentrations below 10 μM (Figure 5). The MAPK signaling pathways have drawn considerable attention, especially the ERK pathway, which is becoming a promising molecular target for anticancer drug development.1−3 The present findings represent examples of natural products as selective ERK1/2 inhibitors. However, the mechanism of inhibition on the ERK1/2 phosphorylation and the potential usage of these compounds in anticancer drug development require further investigation.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer 341 polarimeter, and CD spectra were obtained on an Applied Photophysics Chirascan spectrometer. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer with KBr disks. NMR spectra were measured on a Bruker AM-400 spectrometer at 25 °C. EIMS and HREIMS (70 eV) were recorded on a Finnigan MAT 95 mass spectrometer, and ESIMS was carried out on a Finnigan LC QDECA instrument. A Shimadzu LC20 AT 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) was used for semipreparative HPLC separation. A chiral column (Phenomenex Lux, cellulose-2, 250 × 4.6 mm, 5 μm) was used for chiral analysis. 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 667



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Figure 3. Effects of nontoxic compounds on the phosphorylation of MAPKs. HeLa cells were treated with 20 μM single compounds for 3 h. Proteins (20 μg) extracted from total lysates of HeLa cells were subjected to SDS-PAGE followed by Western blot with indicated antibodies. The blots were then quantified by densitometry (n = 3, **p < 0.001, ANOVA). Compound 7 was not tested in this assay due to the limited amount and the artificial product nature.

Figure 4. Inhibitory potency of active compounds on the phosphorylation of ERK1/2. PD98059 was used as a positive control. HeLa cells were treated with 20 μM single compounds for 3 h. Proteins (20 μg) extracted from total lysates of HeLa cells were subjected to SDS-PAGE followed by Western blot with indicated antibodies. The blots were then quantified by densitometry (n = 3, *p < 0.01 vs DMSO, **p < 0.001 vs DMSO, #p < 0.01 vs PD98059, ##p < 0.001 vs PD98059, ANOVA). used for column chromatography (CC). All solvents used were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.). WST-8-based colorimetric assay used a commercial kit (Cell Counting Kit-8, CCK-8) from Beyotime Institute of Biotechnology. Antibodies against p-ERK, p-JNK, p-p38, total-ERK, and α-tublin were from Cell Signaling Technology. Anti-mouse and anti-rabbit second antibodies were from Promega, and PD98059 was from Sigma. The HeLa cell

line was obtained from the China Center for Type Culture Collection of Chinese Academy of Sciences. Plant Material. Stems of Aristolochia yunnanensis Franch. (synonym: Aristolochia grif f ithii) were collected in October 2010 from Yunnan Province, P. R. China, and were identified by Prof. YouKai Xu of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (accession number: 668



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Figure 5. Compounds 16 and 19 inhibited the phosphorylation of ERK1/2 in a dose-dependent manner. HeLa cells were treated with indicated concentrations of single compounds for 3 h. Proteins (20 μg) extracted from total lysates of HeLa cells were subjected to SDS-PAGE followed by Western blot with indicated antibodies. The blots were then quantified by densitometry (n = 3, ANOVA). PD98059 was used as the positive control. Na]+; negative ESIMS m/z 263.1 [M − H]−; HRESIMS m/z 287.1249 [M + Na]+ (calcd for C15H20O4Na, 287.1254). Aristoyunnolin C (3): white powder; [α]20D +166 (c 0.09, CHCl3); UV (MeOH) λmax (log ε) 250 (4.03) nm; CD (c 5 × 10−4 M, CH3CN), λmax (Δε) 202 (+0.86), 247 (+1.72), 330 (+0.11); IR (KBr) νmax 2927, 1717, 1677, 1626, 1213, 1137, and 1112 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 263.2 [M + H]+; HRESIMS m/z 285.1453 [M + Na]+ (calcd for C16H22O3Na, 285.1461). Aristoyunnolin D (4): white powder; [α]20D 0 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 205 (2.88) nm; IR (KBr) νmax 3396, 2917, 2851, 1744, 1435, 1130, 977 cm−1; 1H and 13C NMR data, see Tables 1 and 2; EIMS m/z 248 [M]+ (28), 230 (69), 205 (100), 175 (56); HREIMS m/z 248.1410 [M]+ (calcd for C15H20O3, 248.1407). Aristoyunnolin E (5): colorless oil; [α]20D −117 (c 0.34, CHCl3); UV (MeOH) λmax (log ε) 225 (3.95) nm; IR (KBr) νmax 3428, 2920, 2855, 1688, 1441, 1094 cm−1; 1H and 13C NMR data, see Tables 1 and 2; EIMS m/z 234 [M]+ (29), 216 (57), 191 (100), 187 (68), 173 (68); HREIMS m/z 234.1612 [M]+ (calcd for C15H22O2, 234.1614). Aristoyunnolin F (6): white powder; [α]20D +113 (c 0.04, CHCl3); UV (MeOH) λmax (log ε) 227 (3.96) nm; IR (KBr) νmax 2921, 2854, 1741, 1681, 1439, 1370, 1232, 1019, 961 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 299.2 [M + Na]+; HRESIMS m/z 299.1606 [M + Na]+ (calcd for C17H24O3Na, 299.1618). 7-O-ethylmadolin W (7): colorless oil; [α]20D −187 (c 0.07, CHCl3); UV (MeOH) λmax (log ε) 254 (3.90) nm; IR (KBr) νmax 3445, 2926, 2855, 1682, 1628, 1125 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 263.2 [M + H]+; HRESIMS m/z 285.1832 [M + Na]+ (calcd for C17H26O2Na, 285.1825). Preparation of (R)- and (S)-MTPA Esters of 5, 9, and 17. Sesquiterpenoid (5−6 mg, 0.02 mmol) was dissolved in 1 mL of pyridine and stirred at rt for 10 min. An excess of (R)- or (S)-MTPACl (6−7 mg, 0.025 mmol) was added, and the reaction was left to stir overnight at rt. The reaction mixture was evaporated in vacuo to provide the reaction crude, which was purified by C18 RP-HPLC using a gradient of MeOH/H2O (80:20 → 100:0, 20 min, 3.0 mL/min, tR of most derivatives was ca. 19 min). (S)-MTPA ester of 5 (5a): 1H NMR (CDCl3, 400 MHz) δH 9.46 (1H, s, H-1), 6.28 (1H, d, J = 10.5 Hz, H-3), 5.77 (1H, td, J = 10.5, 3.9 Hz, H-4), 5.07 (1H, t, J = 7.5 Hz, H-7), 4.82 (1H, d, J = 7.1 Hz, H-11), 2.58 (1H, m, H-5α), 2.33 (2H, m, H-12), 2.30 (1H, m, H-5β), 2.19 (2H, m, H-13), 2.13 (2H, m, H-8), 2.05 (2H, m, H-9), 1.63 (3H, s, H14), 1.37 (3H, s, H-15); 13C NMR (CDCl3, 100 MHz) δC 15.2 (CH3, C-14), 18.3 (CH3, C-15), 24.7 (CH2, C-8), 24.9 (CH2, C-13), 25.1 (CH2, C-12), 38.2 (CH2, C-9), 43.0 (CH2, C-5), 71.8 (CH, C-4), 124.9 (CH, C-11), 128.2 (C, C-6), 129.8 (CH, C-7), 133.8 (C, C-10), 146.0 (C, C-2), 147.1 (CH, C-3), 194.9 (CH, C-1); ESIMS m/z 473.1 [M + Na]+; ESIMS m/z 449.0 [M − H]+.

NMX201010) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University. Extraction and Isolation. The air-dried powder of the stems of A. yunnanensis (4.5 kg) was extracted with 95% EtOH (3 × 10 L) at room temperature (rt) to give 214 g of crude extract. The extract was suspended in H2O (1 L) and successively partitioned with petroleum ether (PE, 3 × 1 L), EtOAc (3 × 1 L), and n-BuOH (3 × 1 L). The EtOAc extract (120 g) was subjected to MCI gel CC eluted with a MeOH/H2O gradient (3:7 → 10:0) to afford five fractions (I−V). Fraction I (10.5 g) was chromatographed over C18 reversed-phase (RP-18) silica gel eluted with MeOH/H2O (5:5 → 10:0) to afford five fractions (Ia−Ie). Fraction Ie (1.6 g) was separated by silica gel CC (PE/acetone, 3:1), followed by Sephadex LH-20 CC using EtOH to give 8 (22 mg). Fraction II (12 g) was subjected to silica gel CC (PE/ EtOAc, 5:1 → 1:2) to give four fractions (IIa−IId). Fraction IIb (3.4 g) was purified on silica gel CC (PE/EtOAc, 5:1) to obtain 9 (155 mg) and 10 (12 mg). Fraction IIc (0.9 g) was applied to silica gel CC (CH2Cl2/acetone, 25:1 → 5:1) to yield 2 (46 mg). Fraction III (28 g) was subjected to silica gel CC (PE/EtOAc, 5:1 → 1:1) to give 10 fractions (IIIa−IIIj). Fraction IIIc (1.9 g) was subjected to RP-18 silica gel CC (MeOH/H2O, 6:4 → 10:0), followed by silica gel CC (PE/ acetone, 20:1 → 1:1), to afford 4 (14 mg), 5 (106 mg), and 13 (5 mg). Fraction IIIf (230 mg) was chromatographed over silica gel (CHCl3/ MeOH, 200:1) to yield 1 (13 mg). Fraction IV (35 g) was chromatographed over an MCI gel column eluted with a gradient of MeOH/H2O (v/v from 6:4 to 10:0) to give eight fractions (IVa−IVh). Fraction IVa (2.3 g) was separated by RP-18 CC using a gradient of MeOH/H2O (v/v from 6:4 to 10:0) to yield 17 (104 mg) and 14 (48 mg). Fraction IVb (8.6 g) was subjected successively to silica gel CC (PE/EtOAc, 50:1 → 5:1), RP-18 silica gel CC (MeOH/H2O, 7:3 → 10:0), and Sephadex LH-20 eluted with EtOH to yield 18 (4 mg), 11 (1.2 g), 19 (65 mg), and 16 (32 mg). Fraction IVc (2.7 g) was applied to silica gel CC (PE/EtOAc, 40:1 → 1:1) to give three fractions (IVc1−IVc3). Fraction IVc1 (840 mg) was separated by silica gel CC (PE/EtOAc, 50:1 → 30:1) to give 3 (8 mg) and 15 (46 mg). Further purification of IVc2 (1.3 g) by silica gel CC (CHCl3/PE, 3:1) afforded 7 (2 mg), 12 (150 mg), and 6 (4 mg). The purity of compounds 1−19 was estimated to be greater than 95%, as determined by 1H NMR spectra (Supporting Information). Aristoyunnolin A (1): white powder; [α]20D −50.0 (c 0.08, CHCl3); IR (KBr) νmax 3309, 2926, 2873, 1753, 1547, 1461, 1368, 1207, 1187, 958, and 899 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 296.1 [M + H]+; negative ESIMS m/z 341.2 [M + NO2]−; HREIMS m/z 295.1416 [M]+ (calcd for C15H21NO5, 295.1414). Aristoyunnolin B (2): colorless oil; [α]20D +76 (c 0.02, CHCl3); UV (MeOH) λmax (log ε) 219 (3.92) nm; IR (KBr) νmax 3474, 2963, 2872, 1749, 1446, 1076, 1033, 907, and 758 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 265.2 [M + H]+, 287.1 [M + 669



Article

(R)-MTPA ester of 5 (5b): 1H NMR (CDCl3, 400 MHz) δH 9.38 (1H, s, H-1), 6.13 (1H, d, J = 10.0 Hz, H-3), 5.76 (1H, ddd, J = 11.2, 10.0, 4.2 Hz, H-4), 5.08 (1H, t, J = 7.5 Hz, H-7), 4.81 (1H, d, J = 7.2 Hz, H-11), 2.69 (1H, m, H-5α), 2.41 (1H, m, H-5β), 2.35 (2H, m, H12), 2.20 (2H, m, H-13), 2.14 (2H, m, H-8), 2.05 (2H, m, H-9), 1.63 (3H, s, H-14), 1.37 (3H, s, H-15); 13C NMR (CDCl3, 100 MHz) δC 15.3 (CH3, C-14), 18.4 (CH3, C-15), 24.6 (CH2, C-8), 24.8 (CH2, C13), 25.1 (CH2, C-12), 38.3 (CH2, C-9), 43.1 (CH2, C-5), 72.0 (CH, C-4), 124.9 (CH, C-11), 128.2 (C, C-6), 129.8 (CH, C-7), 133.7 (C, C-10), 146.3 (C, C-2),147.0 (CH, C-3), 194.8 (CH, C-1); ESIMS m/z 449.0 [M − H]+. (S)-MTPA ester of 9 (9a): 1H NMR (CDCl3, 400 MHz) δH 6.80 (1H, t, J = 1.3 Hz, H-5), 5.50 (1H, dd, J = 11.8, 2.4 Hz, H-1), 5.10 (1H, d, J = 9.5 Hz, H-9), 5.09 (1H, d, J = 1.3 Hz, H-6), 5.01 (1H, s, H12b), 4.91 (1H, t, J = 1.4 Hz, H-12a), 2.73 (1H, m, H-8α), 2.69 (2H, m, H-3), 2.54 (1H, dd, J = 11.8, 4.4 Hz, H-7), 2.34 (1H, m, H-2α), 2.21 (1H, m, H-8β), 1.89 (3H, s, H-13), 1.62 (1H, m, H-2β), 1.58 (3H, t, J = 1.6 Hz, H-14); 13C NMR (CDCl3, 100 MHz) δC 10.8 (CH3, C-14), 21.2 (CH3, C-13), 22.5 (CH2, C-2), 23.6 (CH2, C-3), 28.0 (CH2, C-8), 45.6 (CH, C-7), 82.6 (CH, C-1), 83.4 (CH, C-6), 112.6 (CH2, C-12), 131.5 (C, C-4), 131.5 (C, C-10),134.5 (CH, C-9), 146.4 (C, C-11), 150.0 (CH, C-5), 172.8 (C, C-15); ESIMS m/z 487.1 [M + Na]+; ESIMS m/z 463.1 [M − H]+. (R)-MTPA ester of 9 (9b): 1H NMR (CDCl3, 400 MHz) δH 6.80 (1H, s, H-5), 5.49 (1H, dd, J = 11.9, 1.5 Hz, H-1), 5.09 (1H, d, J = 1.3 Hz, H-6), 5.08 (1H, d, J = 9.7 Hz, H-9), 5.01 (1H, s, H-12b), 4.91 (1H, s, H-12a), 2.71 (1H, m, H-3), 2.70 (1H, m, H-8α), 2.54 (1H, dd, J = 11.8, 4.2 Hz, H-7), 2.42 (1H, m, H-2α), 2.20 (1H, m, H-8β), 1.88 (3H, s, H-13), 1.71 (1H, m, H-2β), 1.47 (3H, s, H-14); 13C NMR (CDCl3, 100 MHz) δC 10.5 (CH3, C-14), 21.1 (CH3, C-13), 22.7 (CH2, C-2), 23.7 (CH2, C-3), 28.0 (CH2, C-8), 45.6 (CH, C-7), 82.7 (CH, C-1), 83.4 (CH, C-6), 112.5 (CH2, C-12), 131.4 (C, C-4), 131.4 (C, C-10), 134.5 (CH, C-9), 146.5 (C, C-11), 150.0 (CH, C-5), 172.8 (C, C-15); ESIMS m/z 487.1 [M + Na]+. (S)-MTPA ester of 17 (17a): 1H NMR (CDCl3, 400 MHz) δH 9.32 (1H, s, H-1), 6.05 (1H, d, J = 11.4 Hz, H-3), 5.31 (1H, dd, J = 9.2, 5.8 Hz, H-11), 4.39 (1H, d, J = 8.2 Hz, H-7), 2.82 (1H, m, H-13a), 2.24 (2H, m, H-9), 2.22 (1H, m, H-8a), 2.18 (1H, m, H-4), 2.09 (1H, m, H-13b), 2.08 (2H, m, H-12), 1.57 (1H, m, H-8b), 1.47 (3H, s, H-15), 1.33 (1H, dd, J = 8.7, 5.1 Hz, H-5α), 1.24 (3H, s, H-14), 0.85 (1H, t, J = 5.1 Hz, H-5β); 13C NMR (CDCl3, 100 MHz) δC 14.5 (CH3, C-14), 14.9 (CH3, C-15), 22.9 (CH2, C-13), 25.4 (CH2, C-5), 27.3 (CH2, C12), 27.6 (CH, C-4), 28.3 (C, C-6), 28.9 (CH2, C-8), 38.3 (CH2, C9), 86.1 (CH, C-7), 127.4 (CH, C-11), 134.5 (C, C-10), 143.4 (C, C2), 155.2 (CH, C-3), 193.6 (CH, C-1); ESIMS m/z 451 [M + H]+. (R)-MTPA ester of 17 (17b): 1H NMR (CDCl3, 400 MHz) δH 9.31 (1H, s, H-1), 6.02 (1H, d, J = 11.3 Hz, H-3), 5.32 (1H, dd, J = 9.5, 5.3 Hz, H-11), 4.39 (1H, d, J = 10.1 Hz, H-7), 2.82 (1H, m, H-13a), 2.28 (1H, m, H-8a), 2.27 (2H, m, H-9), 2.16 (1H, m, H-4), 2.09 (1H, m, H-13b), 2.07 (2H, m, H-12), 1.69 (1H, m, H-8b), 1.47 (3H, s, H-15), 1.29 (1H, dd, J = 8.7, 5.2 Hz, H-5α), 1.16 (3H, s, H-14), 0.76 (1H, t, J = 5.2 Hz, H-5β); 13C NMR (CDCl3, 100 MHz) δC 14.2 (CH3, C-14), 14.8 (CH3, C-15), 22.8 (CH2, C-13), 25.1 (CH2, C-5), 27.3 (CH, C4), 27.3 (CH2, C-12), 28.0 (C, C-6), 28.9 (CH2, C-8), 38.2 (CH2, C9), 86.1 (CH, C-7), 127.3 (CH, C-11), 134.3 (C, C-10), 143.3 (C, C2), 155.4 (CH, C-3), 193.5 (CH, C-1); ESIMS m/z 451 [M + H]+. Chemical Transformation of 9 to 2. To a stirred solution of 9 (5 mg, 0.02 mmol) in CH2Cl2 (1 mL) at 0 °C was added 3chloroperbenzoic acid (10 mg). The mixture was stirred at rt for 2 h and then evaporated. The resulting crude product was subjected to silica gel CC (CHCl3/MeOH, 40:1) to afford 2 (4 mg), which was identified by the 1H NMR and [α]20D. Chemical Transformation of 17 to 7. To a stirred solution of 17 (2.55 mg, 0.01 mmol) in DMF (1 mL) was added 2 mg of NaH. The mixture was stirred at rt for 0.5 h, and 3 μL of bromoethane (0.04 mmol) was added. The reaction was left to stir at rt for 2 h and quenched by adding 1 mL of H2O. After workup, the crude product was purified by C18 RP-HPLC using a gradient of MeOH/H2O (90:10 → 100:0, 20 min, 3.0 mL/min, tR 15 min) to afford 7 (1.5 mg), which was identified by NMR and MS data.

Cell Viability Assay. The cells were cultured in 96-well culture plates. Cell viability was analyzed with the CCK-8. A 100 μL amount of phenol red-free DMEM containing 10% WST-8 was added to each well. The optical density of each well at 450 nm was measured after 1 h of incubation at 37 °C. The effect of compounds on cell viability was calculated from the mean values of three wells. Total Protein Extraction. Cells were exposed to tested compounds at the concentration of 20 μM for 3 h. Total protein was extracted in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease and phosphatase inhibitors. The protein contents of the cell lysis were measured by the bicinchoninic acid (BCA) method. Western Blotting Assay. Proteins (20 μg) from cell lysates were denatured and loaded into 8−10% acrylamide gels and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The separated proteins were then electrotransferred to nitrocellulose membranes. Nonspecific binding sites on the membranes were blocked by 5% skimmed milk powder in TBST for 1 h at room temperature. Blocked membranes were incubated by specific primary antibodies overnight at 4 °C. After rinsing with TBST three times, secondary antibodies were chosen to bind on the membranes for 1 h at room temperature. Protein bands were detected by chemiluminescence and exposed to GE ImageQuant LAS 4000 Mini.

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ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra of 1−7, 1H and 13C NMR spectra of known compounds (8−19), and 1H NMR spectra of Mosher’s esters of 5, 9, and 17 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-20-39943090. Fax: +86-20-39943090. E-mail: [email protected]. Author Contributions †

Z.-B. Cheng and W.-W. Shao contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (No. 81102339) and Guangdong Natural Science Foundation (No. S2011040002429) for providing financial support to this work.

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REFERENCES

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