Article pubs.acs.org/jnp
Bioactive Limonoid Constituents of Munronia henryi Ying Yan,†,‡ Jian-Xin Zhang,† Tao Huang,† Xin-Ying Mao,† Wei Gu,† Hong-Ping He,§ Ying-Tong Di,§ Shun-Lin Li,§ Duo-Zhi Chen,§ Yu Zhang,*,§ and Xiao-Jiang Hao*,† †
The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guiyang 550002, People’s Republic of China ‡ Center for Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025, People’s Republic of China § State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, People’s Republic of China S Supporting Information *
ABSTRACT: Fourteen new limonoids, munronins A−N (1− 14), and eight known limonoids (15−22) were isolated from the whole plants of Munronia henryi. The structures of the new compounds were elucidated by 2D NMR spectroscopy and mass spectrometry, and the structure of 8 was confirmed by single-crystal X-ray diffraction analysis. Compound 1 represents the first limonoid found with a novel 7-oxabicyclo[2.2.1]heptane moiety produced by incorporating C-11 and C-14 via an oxygen atom. All compounds were evaluated for their antitobacco mosaic virus (TMV) activity and in vitro cytotoxicity against the human cancer HL-60, SMMC-7721, A-549, MCF-7, and SW-480 cell lines. Among them, compounds 2, 8, 9, 10, 11, 12, 18, and 20 showed significant anti-TMV activity, with IC50 values in the range 19.6−44.4 μg/mL. Compounds 1 and 18 exhibited cytotoxic effects for all five cancer cell lines, with IC50 values between 0.4 and 4.8 μM. substituted furan ring group (δH 7.29, 7.35, 6.37, each 1H, s; δC 139.8, 142.8, 110.7, 123.7), an exocyclic double bond (δH 5.24, 5.28, each 1H, s; δC 144.2, 109.8), three tertiary methyl groups (δH 0.42, 1.23, 1.59, each 3H, s; δC 13.9, 17.5, 19.3), and a methoxy group (δH 3.66, 3H, s; δC 52.3) in 1 indicated it to be a prieurianin-type limonoid4b with a similarity to aphanagranin D.6 The linkage of the structural fragments with quaternary carbons was achieved by analysis of the 2D NMR spectra (HSQC, 1H−1H COSY, and HMBC) (Figure 1). When compared with aphanagranin D, the main differences were the opening of the ether ring involving C-1 and C-11 and the loss of the C-15 ketone carbonyl. In the HMBC spectrum, the correlations of H-1 (δH 5.54, d, J = 6.6 Hz) to the carbonyl of the acetyl group (δC 170.3) and the ester carbonyl group C-3 (δC 173.7), as well as the correlations of the methoxy group (δH 3.66, s) to C-3, supported the locations suggested for the acetyl group (δH 2.11, s; δC 20.8, 170.3) and the ester carbonyl, respectively. The key correlations of H-11 (δH 4.99, d, J = 5.6 Hz) to C-14 (δC 101.8) and C-8 (δC 144.2) suggested an ether linkage between C-11 and C-14. Also, the correlations of H-17 (δH 3.56, t, J = 9.4 Hz) and H2-16 (δH 2.44, m; 2.75, m) to C15 (δC 68.4) indicated that the oxygenated methine group is located at C-15. Moreover, the correlations of H-12 (δH 5.29, d, J = 5.6 Hz) with the ester carbonyl C-1′ (δC 172.6) and of H-2′ (3.97, d, J = 4.8 Hz) to the carbonyl of the acetyl group (δC 172.0) supported the linkage of a 2-acetoxy-3-methylpentyryl
T
he plant genus Munronia (Meliaceae) comprises approximately 15 species that are distributed mainly in mainland China, Sri Lanka, India, Indonesia, and the Philippines,1 and they have been used for the treatment of many diseases such as tuberculosis, cough, stomachache, and sores in Chinese traditional medicine.2 Previous studies on the genus Munronia have led to the isolation of a variety of limonoids, triterpenoids, pregnane derivatives, and other types of compounds, of which some limonoids showed significant biological properties such as antifeedant, insecticidal, cytotoxic, and antiviral activities.3,4 Recently, our group has reported an array of limonoids with varied structural features and different biological activities from species in the Meliaceae family.5 In the present study, 14 new limonoids, munronins A−N (1−14), along with eight known compounds (15−22), were isolated from the whole plants of M. henryi. In this report, the isolation and structure elucidation of the new compounds, as well as the anti-tobacco mosaic virus activity and cytotoxicity evaluation against a small panel of cancer cell lines of all isolates obtained, are described.
■
RESULTS AND DISCUSSION Munronin A (1) was found to possess the molecular formula C37H50O14 on the basis of its [M + Na]+ peak at m/z 741.3100 in the HRESIMS, with 13 indices of hydrogen deficiency. The IR absorptions at 3442 and 1743 cm−1 indicated the presence of hydroxy group and ester carbonyl functionalities. All 37 carbons were well resolved in the 13C NMR and DEPT spectra (Table 1) and were classified as eight methyls, six methylenes, 12 methines, and 11 quaternary carbons. The presence of a β© XXXX American Chemical Society and American Society of Pharmacognosy
Received: December 29, 2014
A
DOI: 10.1021/np501057f J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. 1H and 13C NMR Spectroscopic Data of Compound 1 (400 and 100 MHz in CDCl3; δ in ppm, J in Hz) position
δH
δC, type
1 2α 2β 3 4 5 6a 6b 7 8 9 10 11 12 13 14 15 16α 16β 17 18 19 20 21 22 23 28 29α 29β 30a 30b OMe-3 OAc-1
5.54, d (6.6) 2.25, m 2.41, m
72.0, CH 38.5, CH2
2.89, m 2.61, m 2.78, m
3.25, s 4.99, d (5.6) 5.29, d (5.6)
4.53, 2.44, 2.75, 3.56, 0.42, 1.23,
m m m t (9.4) s s
7.29, 6.37, 7.35, 1.59, 4.13, 4.42, 5.24, 5.28, 3.66,
s s s s d (12.6) d (12.6) s s s
2.11, s OAc-2′ 2.14, s 1′ 2′ 3′ 4′ 5′a 5′b 6′
3.97, 1.65, 0.87, 1.26, 1.44, 0.84,
d (4.8) m s m m s
173.7, 87.1, 42.9, 31.6,
C C CH CH2
170.1, 144.2, 46.2, 44.0, 79.4, 79.4, 55.9, 101.8, 68.4, 37.6,
C C CH C CH CH C C CH CH2
42.9, 13.9, 17.5, 123.7, 139.8, 110.7, 142.8, 19.3, 68.1,
CH CH3 CH3 C CH CH CH CH3 CH2
(Table 3) of 2 gave 33 carbon signals including eight methyls, four methylenes, 10 methines, and 11 quaternary carbons. The NMR data of 2 were found to be similar to those of the known compound nymania 4.7 The chemical shift differences resulted from the absence of signals for a 14,15-epoxy ring and the presence of a Δ14,15 double-bond resonance (δH 5.77; δC 123.3, δC 149.2) in 2. These were confirmed by the key correlations of H-16 and H-30 to C-14 (δC 149.2) and of H-16 and H-17 to C15 (δC 123.3), respectively. Thus, the structure of compound 2 was elucidated as shown and was confirmed from the 2D NMR (HSQC, 1H−1H COSY, HMBC, and ROESY) data (Figures 1 and 2). Munronin C (3) gave the molecular formula C31H38O12 from the HREIMS, requiring 13 indices of hydrogen deficiency. The 13 C NMR data (Table 3) along with DEPT experiments showed 31 carbon signals, including seven methyls, three methylenes, 10 methines (three olefinic), and 11 quaternary carbons (two olefinic and five carbonyl) of a limonoid core that was the same as found in mulavanin A.8 The most noticeable differences were the presence of an acetyl group and the absence of a tigloyloxy unit in 3. The acetyl group was located at C-12 on the basis of the key HMBC cross-peaks of Me-18 with C-12 (δC 74.4) and H-12 (δH 5.82, d, J = 10.8) with the acetyl carbonyl (δC 170.7). The relative configurations of 3 was found to be the same as that of mulavanin A from the ROESY correlations made. Thus, compound 3 was determined as shown. Munronin D (4) was isolated as a white, amorphous solid. The molecular formula was established as C32H40O12 by HREIMS at m/z 616.2522 [M]+ (calcd 616.2520), 14 mass units greater than that of 3. Thus, the hydroxy group in 3 was found to be replaced by a methoxy group in 4 (δH 3.52, s; δC 56.9). The structure of 4 was supported by its 2D NMR (HSQC, 1H−1H COSY, HMBC, and ROESY) data (Figures S4.3−4.6, Supporting Information), especially the HMBC correlation of H-23 (δH 5.66, s) with the methoxy group (δC 56.9). Thus, 4 was deduced structurally as shown. The HREIMS and 13C NMR data of 5 (Table 3) revealed a molecular formula of C32H42O11. The 1D NMR data of the compound displayed a similar pattern to that of 4 except for the replacement of the C-21 carbonyl group with a methylene group (δC 70.9). The above observation was deduced from the key HMBC correlations of H-22 (δH 5.89, s) and H-23 (δH 5.72, m) with C-21 (δC 70.9). Accordingly, the structure of 5 was elucidated as shown, which was confirmed from its 2D NMR (HSQC, 1H−1H COSY, HMBC, and ROESY) data (Figures S5.3−5.6, Supporting Information). Munronin F (6) gave a molecular formula of C31H38O12 as determined by HREIMS at m/z 602.2352 (calcd 602.2363), identical to that of 3. The UV, IR, and NMR spectroscopic data (Tables 2 and 3) of 6 indicated that its structure is closely related to that of 3, with the only difference being in the C-17 side-chain, which also showed the presence of a mixture of two isomers. The NMR spectroscopic data supported the presence of a 21-hydroxybutenolide moiety attached to C-17, the same as that of turrapubesin E.8 The C-17 side-chain was confirmed by HMBC correlations from H-21 to C-22 (δC 145.1) and C-23 (δC 101.8) and from H-17 to C-20 (δC 136.1), C-21 (δC 169.2), and C-22 (δC 145.1) (Figures S6.3−6.6, Supporting Information). Therefore, the structure of compound 6 was determined as shown. Munronin G (7) afforded a molecular formula of C28H36O11, as deduced from its HREIMS at m/z 548.2262 (calcd
109.8, CH2 52.3, 170.3, 20.8, 172.0, 21.1, 172.6, 75.9, 38.6, 15.2, 23.6,
CH3 C CH3 C CH3 C CH CH CH3 CH2
11.1, CH3
side chain to C-12. Thus, the gross structure of 1 was constructed as depicted. The relative configuration of 1 was assigned from the ROESY spectrum. The ROESY correlations of Me-19/H-1, Me-19/H-11, H-11/H-12, and H-12/H-17 indicated these protons to be cofacial, and they were assigned arbitrarily as βoriented. In turn, H-5, H-9, Me-18, Me-28, and H-15 were assigned as α-oriented by the ROESY correlations between H5/H-9, H-5/Me-28, H-9/Me-18, and Me-18/H-15. Thus, the structure and relative configuration of compound 1 were established as shown. Munronin B (2) provided a molecular formula of C33H42O11, as determined by HRESIMS at m/z 637.2623 ([M + Na]+, calcd 637.2625). The 1H and 13C NMR data for this compound are shown in Tables 2 and 3. The 13C NMR spectroscopic data B
DOI: 10.1021/np501057f J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 1. 1H−1H COSY (bold) and key HMBC of compounds 1, 2, 8, 11, and 14.
Table 2. 1H NMR Spectroscopic Data of Compounds 2−7 in CDCl3 (δ in ppm, J in Hz) 2a
position 1 2 5 6a 6b 9 11 12 15 16α 16β 17 18 19 21a 21b 22 23 28 29 30a 30b OMe-7 OAc-11 OAc-12 OAc-1 OMe-23 a
3b
4b
5b
5.75, 3.51, 3.43, 2.17, 2.29, 3.13, 5.69, 6.06, 5.77, 1.74, 2.29, 3.07, 0.89, 1.67, 7.12,
d (13.2) m d (9.2) m m m dd (10.8, 7.2) d (10.8) s m m m s s s
6.89, 6.32, 3.34, 2.19, 2.26, 3.07, 5.60, 5.82, 3.91, 2.08, 2.30, 2.96, 0.78, 0.99,
d (13.0) d (13.0) m m m d (7.2) dd (10.8, 7.2) d (10.8) s m m m s s
6.84, 6.33, 3.33, 2.19, 2.27, 3.07, 5.58, 5.80, 3.89, 2.07, 2.30, 2.99, 0.87, 0.97,
d (13.0) d (13.0) m m m d (7.2) dd (10.8, 7.2) d (10.8) s m m m s s
6.16, 7.34, 1.36, 1.64, 5.09, 5.43, 3.73, 2.06, 1.83, 2.09,
s s s s s s s s s s
6.83, 5.60, 1.29, 1.55, 5.24, 5.37, 3.71, 2.08, 1.89,
m m s s s s s s s
6.79, 5.66, 1.28, 1.54, 5.23, 5.35, 3.71, 2.06, 1.87,
s s s s s s s s s
3.52, s
6.86, 6.24, 3.34, 2.14, 2.23, 3.06, 5.63, 5.77, 3.93, 2.12, 2.26, 3.34, 0.99, 1.04, 2.42, 3.08, 5.89, 5.72, 1.26, 1.55, 5.24, 5.36, 3.79, 2.12, 1.95,
d (12.9) d (12.9) m m m d (7.2) dd (10.8, 7.2) d (10.8) s m m m s s m m s m s s s s s s s
6a 6.88, 6.22, 3.32, 2.16, 2.24, 3.05, 5.66, 5.85, 3.92, 2.10, 2.24, 3.03, 0.94, 0.97, 5.72,
d (13.0) d (13.0) m m m d (7.2) m m s m m m s s m
7b 6.87, 6.23, 3.32, 2.18, 2.28, 3.06, 5.62, 5.84, 3.84, 2.11, 2.26, 2.78, 0.95, 0.96,
d (13.0) d (13.0) m m m d (7.2) dd (10.8, 7.2) d (10.8) s m m m s s
1.27, 1.52, 5.23, 5.34, 3.70, 2.07, 1.95,
s s s s s s s
6.81, m 1.27, 1.53, 5.26, 5.38, 3.69, 2.10, 1.95,
s s s s s s s
3.50, s
Recorded at 400 MHz. bRecorded at 600 MHz.
528.2258). Comparison of the MS and 1H and 13C NMR spectroscopic data of 7 (Tables 2 and 3) with those of 6 showed an overall similarity except for the nature of the C-17 side-chain. In the HMBC spectrum, correlations of H-16α (δH 2.11, m) and H-17 (δH 2.78, m) with a carboxylic acid carboxyl carbon resonance at δC 177.0 supported the location of this carboxylic acid carboxyl group at C-17. The structure of compound 7, possessing a rare carboxylic acid carboxyl group
instead of a more common furan or 21-hydroxybutenolide moiety at C-17, was established, therefore, as shown. The molecular formula of munronin H (8) was determined as C30H36O9 from a [M + Na]+ peak at m/z 540.2364 in the HREIMS. Further analysis of the 1H and 13C NMR spectra (Table 4) indicated 8 to be a ring A-seco limonoid with a structrural similarity to surenone.4b,9 The chemical shift differences between the compounds resulted from the absence of a hydroxy group signal and the presence of two acetyl groups C
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Table 3. 13C NMR Spectroscopic Data of Compounds 2−7 in CDCl3 (δ in ppm) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 OMe-7 OAc-11 OAc-12 OAc-1
2a 75.6, 35.0, 170.5, 84.4, 50.9, 33.1, 174.0, 136.6, 45.0, 43.3, 72.9, 72.4, 51.7, 149.2, 123.3, 37.2, 44.1, 13.1, 17.1, 122.6, 140.0, 111.1, 142.6, 29.8, 23.9, 124.0, 51.7, 170.1, 21.5, 170.8, 20.8, 171.2, 21.0,
CH CH2 C C CH CH2 C C CH C CH CH C C CH CH2 CH CH3 CH3 C CH CH CH CH3 CH3 CH2 CH3 C CH3 C CH3 C CH3
3b 148.5 (148.3), CH 122.6 (122.5), CH 167.1 (167.0), C 83.9 (83.8), C 50.2, CH 35.1, CH2 173.8 (173.7), C 136.3 (136.2), C 53.4 (53.3), CH 46.4, C 71.3 (71.2), CH 74.4, CH 46.0, C 71.1 (71.0), C 60.0 (59.7), CH 31.3, CH2 39.1 (38.5), CH 13.5, (13.4), CH3 23.0 (22.9), CH3 136.3 (136.2), C 170.1 (169.7), C 147.9 (146.8), CH 96.2 (95.8), CH 30.4, CH3 22.5, CH3 121.9, CH2 52.7, CH3 170.9 (170.8), C 20.6, CH3 170.7, C 20.8, CH3
OMe-23 a
4b 147.8, 122.5, 166.6, 83.5, 50.0, 34.8, 173.6, 136.1, 53.1, 46.1, 71.1, 74.0, 45.7, 70.9, 59.4, 31.2, 38.2, 13.3, 22.7, 136.1, 169.2, 145.1, 101.8, 30.2, 22.3, 121.6, 52.4, 170.4, 20.5, 170.4, 20.3,
5b
CH CH C C CH CH2 C C CH C CH CH C C CH CH2 CH CH3 CH3 C C CH CH CH3 CH3 CH2 CH3 C CH3 C CH3
56.9, CH3
148.2, 122.2, 166.8, 83.9, 49.9, 34.8, 173.5, 135.5, 52.8, 46.2, 71.0, 74.7, 45.9, 70.6, 59.1, 30.1, 40.3, 14.1, 22.8, 135.8, 70.9, 129.9, 107.6, 31.9, 22.2, 122.2, 52.4, 170.2, 20.9, 170.2, 20.3,
CH CH C C CH CH2 C C CH C CH CH C C CH CH2 CH CH3 CH3 C CH2 CH CH CH3 CH3 CH2 CH3 C CH3 C CH3
6a
7b
148.6 (148.3), CH 122.4 (122.2), CH 167.1 (167.0), C 84.0, C 49.9 (49.8), CH 34.8, CH2 173.5, C 135.5 (135.4), C 52.9 (52.7), CH 46.2 (46.1), C 71.1 (71.0), CH 74.5, CH 46.0 (45.8), C 70.5, C 59.5 (59.1), CH 31.5, CH2 40.2, CH 13.3 (13.0), CH3 22.7 (22.6), CH3 167.4 (167.1), C 99.5, CH 122.4 (122.2), CH 170.2 (169.8), C 30.1, CH3 22.2, CH3 122.0 (119.9), CH2 52.5, CH3 170.9 (170.8), C 20.8 (20.7), CH3 170.7, C 20.3, CH3
148.5, 122.0, 166.9, 83.7, 49.9, 34.8, 173.5, 135.7, 52.9, 46.1, 70.7, 73.3, 45.7, 70.5, 59.6, 29.7, 46.7, 12.7, 22.6, 177.0,
CH CH C C CH CH2 C C CH C CH CH C C CH CH2 CH CH3 CH3 C
30.1, 22.2, 121.8, 52.4, 170.3, 20.5, 170.0, 20.3,
CH3 CH3 CH2 CH3 C CH3 C CH3
50.8, CH3
b
Recorded at 100 MHz. Recorded at 150 MHz.
(δH 2.22, s, 1.96, s; δC 169.4, 21.0, 169.3, 21.3) and a ketone carbonyl signal (δC 206.8) in 8. The HMBC correlations from H-11 to OAc-11 (δC 169.4, 21.0) and from H-12 to OAc-12 (δC 169.3, 21.3) suggested that two acetyl groups are located at C-11 and C-12, respectively. The ketone carbonyl (δC 206.8) could be positioned at C-7 from the HMBC correlations of H-5 and H-6 with C-7. The ROESY correlations of Me-19 with Me30 and H-12 and of H-12 with H-17 indicated that those groups are cofacial, and they were assigned arbitrarily as βoriented. In turn, correlations between H-5/H-9, H-9/Me-18, Me-18/H-15, and Me-18/H-11 suggested the α-orientation of these groups. Therefore, the structure of 8 was established as shown. This was confirmed by single-crystal X-ray diffraction (Figure 3), although the presence of a disorder problem in the furan ring in 8 was evident. Moreover, its absolute configuration could not be determined by the Flack parameter [−1(3)]. The molecular formula of munronin I (9) was determined as C32H40O11 from the HREIMS. Comparison of the 1H and 13C NMR spectroscopic data (Table 4) with those of 8 indicated them to possess a similar structure except for the absence of Δ1,2 double bond and the presence of an acetyl group at C-1. This elucidation was confirmed by the HMBC correlations of Me-19 to C-1 (δC 81.3) and of H-1 (δH 5.15, s) to the acetyl
carbonyl carbon (δC 169.2). Thus, the structure of 9 was established as shown. Munronin J (10) was found to possess a molecular formula of C30H40O7, by means of HREIMS. The 1H and 13C NMR spectroscopic data of 10 (Table 4) were similar to those of carapolide I.10 The chemical shift differences resulted from the presence of an acetyl group at C-12 and the absence of an acetyl group at C-7 in 10. The observed significant downfield shifts of C-12 (δC 74.3) and upfield shifts of C-7 (δC 32.8) in the 13C NMR spectrum indicated that the acytoxy group of 10 is located at C-12 instead of C-7, as assigned by the HMBC correlations of Me-18 and H-9 to C-12 and of H-12 to the acetoxy carbonyl (δC 170.3). The observation of the 1H−1H COSY cross-peaks of H2-7/H2-6/H-5 and H-9/H2-11/H-12 verified the above proposals. Thus, the structure of 10 was established as shown, and it was assigned the same relative stereochemistry as in compounds 8 and 9. The molecular formula of munronin K (11) was deduced to be C34H46O9 from the HRESIMS. The 1H NMR spectrum (Table 5) displayed signals of a β-substituted furan ring (δH 6.46, 7.29, 7.31), a tigloyl group (δH 7.06, 1H, m; 1.87, 3H, d, J = 6.8, 1.94, 3H, s), and a methoxy group (δH 3.34, s). NMR spectroscopic data analysis of 11 (Table 5) suggested its structural similarity to the known limonoid meliatoosenin N,11 D
DOI: 10.1021/np501057f J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 2. Selected ROESY correlations of compounds 1, 2, 8, 11, and 14.
between H-1 (δH 4.82) and the carbonyl carbon (δC 166.6) of the tigloyl group. The relative configuration in 13 was ascertained by detailed 1D and 2D NMR experiments (Figures S13.1−S13.6, Supporting Information). Therefore, the structure of compound 13 (munronin M) was established as shown. Munronin N (14) gave the molecular formula C32H44O8, as deduced from the HREIMS. The NMR data (Table S1, Supporting Information) were similar to those of 6αacetoxydeoxyhavanensin.13 The only difference observed was the replacement of an acetyl group in the latter known compound by a hydroxy group in 14. In the HMBC spectrum, correlations from H-3 (δH 4.55, t, J = 3.4 Hz), Me-19 (δH 1.09, s), and H2-2 (δH 2.23, m, 1.99, m) to C-1 (δC 77.4) revealed the hydroxy group to be attached to C-1. The β-orientation of H-1 was deduced from the small coupling constant (J1,2 = 3.4 Hz) of H-1, which was confirmed by ROESY correlations of H1/Me-19. Thus, compound 14 was assigned as shown. Seven compounds, chisonimbolinin F (15),14 12-O-methylvolkensin (16),15 C-seco nimbolinin (17),16 prieurianin (18),17 mombasol (19),18 munronoid L (20),3b aphanamixoid F (21),19 and 6α-hydroxy-14,15-deoxyhavanensin triacetate (22),20 were identified by analysis of their spectroscopic data and comparison with literature values. The antiviral inhibition rates of limonoids 1−22 was tested by the half-leaf method and are listed in Table 6. The results showed that compounds 1−22 exhibited inactivation effects at 50 μg/mL against tobacco mosaic virus (TMV) replication ranging from 21.2% to 98.2%. Compounds 2, 8, 9, 10, 11, 12, 18, and 20 showed greater inhibition rates than that of the
except for a 1-tigloyl group and OH-7 group in 11 replacing a 1-acetyl and a 7-tigloyl group in the latter. The planar structure of 11 was confirmed by HMBC correlations from H-3, H-5, and Me-19 to C-1 (δC 71.3) and from H-1 to the carbonyl of the tigloyl group (δC 166.1), which was finally verified by a 2D NMR experiment. The relative configuration of 11 was consistent with that of meliatoosenin N, as inferred from a ROESY experiment. Thus, correlations between H-1/H-3 and H-1/Me-19 and Me-19/Me-30 and Me-30/H-7 indicated that both H-1 and H-7 are β-oriented. Therefore, the structure of munronin H (11) was assigned as shown. Munronin L (12) gave the same molecular formula as 11 according to the HREIMS. The 1H and 13C NMR data (Table 5) of compound 12 closely resembled those of 11, and the two compounds were found to share the same planar structure. The ROESY correlations of H-12/Me-30 and Me-30/H-15 implied that H-12 and H-15 in 12 are β-oriented. Detailed analysis of the 2D NMR (HSQC, 1H−1H COSY, HMBC, and ROESY) data (Figures S12.3−12.6, Supporting Information) confirmed that the other parts of the molecule are the same as those of 11. Therefore, compound 12 was established structurally as shown. Compound 13 gave the molecular formula C33H42O8, as deduced from the HREIMS. The 1H and 13C NMR data (Table 5) closely resembled those of 17-epi-12-dehydroxyheudebolin.12 The major differences between 13 and 17-epi-12dehydroxyheudebolin found were that the acetyl groups at C1 and C-7 in 17-epi-12-dehydroxyheudebolin should be replaced in 13 by a tigloyl moiety and a hydroxy group, respectively. Notably, a 3J HMBC correlation was observed E
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Chart 1
13 and 15−17 were inactive, suggesting that the presence of both a methoxy group of C-12 and a tigloyloxy group of C-1 might contribute to the resultant inhibitory activities. All compounds were evaluated for their in vitro growth inhibitory effects against five human cancer cell lines, namely, HL-60 (human promyelocytic leukemia cell line), SMMC-7721 (human hepatocellular carcinoma cell line), A-549 (human lung cancer cell line), MCF-7 (human breast cancer cell line), and SW480 (colorectal cancer cell line), using a previously described protocol.21 The results are summarized in Table 8. From the data obtained, compounds 1 and 18 exhibited cytotoxic activity for all five cell lines with IC50 values in the range 0.44−4.8 μM.
positive control, ningnanmycin (58.9%). The effects of compounds 1−22 at a concentration of 200 μg/mL were tested, and the results showed that all of the compounds exhibited inhibitory activities against TMV. At this same concentration, compounds 2, 8, 9, 11, 12, 16, and 18 showed the most potent antiviral activities. In addition, the protective effects of all the compounds were also evaluated at a concentration of 200 μg/mL. The results showed that compounds 2, 8, 9, 11, 12, and 16 showed protective effects. The IC50 values of the eight compounds were determined and are listed in Table 7, with ningnanmycin as the positive control. Among these, compound 8 exhibited the best activity, with an IC50 value of 19.6 μg/mL, which was 2-fold greater than that of ningnamycin (44.6 μg/mL). Preliminary structure−activity relationships of these limonoids indicated that the presence of an α,β-unsaturated lactone and an acetyl group located at C7 are important for enhancing activity. Comparison of the antiTMV activity of 8−10 and with those of 20 implied that the presence of a Δ1,2 double bond may be responsible for the higher activity of the latter compound. When considering 11− 13 and 15−17 specifically, compounds 11 and 12 showed relatively more potent inhibitory activities, whereas compounds
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 polarimeter. UV spectra were recorded on a Shimadzu UV-2401A. ECD spectra were recorded with an Applied Photophysics Chirascan spectrometer. IR spectra were determined on a Bruker Tensor-27 infrared spectrophotometer with KBr disks. 1H and 13C NMR and 2D NMR spectra were recorded on Bruker AM-400, Bruker DRX-500, and Bruker Avance III 600
F
DOI: 10.1021/np501057f J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Table 4. 1H and 13C NMR Spectroscopic Data of Compounds 8−10 in CDCl3 (δ in ppm, J in Hz) 8a position
δH
1 2 3 4 5 6α 6β 7 8 9 10 11α 11β 12 13 14 15 16α 16β 17 18 19 20 21 22 23 28 29 30 OAc-1
6.65, d (12.4) 6.05, d (12.4)
2.53, m 2.56, m 2.96, m
2.61, s 5.36, s 5.15, s
3.72, 2.07, 2.31, 2.89, 0.81, 1.57,
s m m m s s
7.12, 6.07, 7.33, 1.53, 1.44, 1.43,
s s s s s s
9a δC, type 151.5, 120.9, 166.3, 83.6, 48.3, 39.6,
CH CH C C CH CH2
206.8, 51.3, 56.4, 44.0, 75.0,
C C CH C CH
79.9, 44.7, 67.6, 56.0, 32.3,
CH C C CH CH2
41.7, 15.6, 18.6, 122.0, 140.4, 111.2, 142.7, 25.7, 31.5, 21.0,
CH CH3 CH3 C CH CH CH CH3 CH3 CH3
169.4, 21.0, 169.3, 21.3,
C CH3 C CH3
δH
δC, type
5.15, s 3.22, d (4.1)
2.59, m 2.62, m 2.81, m
2.92, s 5.28, d (5.9) 5.07, s
3.73, 2.30, 2.05, 2.89, 0.88, 1.47,
s m m m s s
7.10, 6.05, 7.31, 1.56, 1.47, 1.42,
s s s s s s
2.18, s OAc-11 2.22, s OAc-12 1.96, s a
10b
1.95, s 2.05, s
81.3, 35.1, 168.7, 84.3, 50.8, 38.6,
CH CH C C CH CH2
207.1, 51.0, 44.4, 44.1, 70.7,
C C CH C CH
74.7, 44.3, 67.9, 56.1, 32.4,
CH C C CH CH2
42.0, 14.8, 16.9, 122.1, 140.5, 111.3, 142.6, 23.2, 33.8, 21.1, 169.2, 21.3, 169.4, 20.7, 169.3, 21.0,
CH CH3 CH3 C CH CH CH CH3 CH3 CH3 C CH3 C CH3 C CH3
δH
δC, type
4.84, t (4.0) 3.15, m
70.9, 34.3, 169.8, 85.5, 51.2, 16.2,
CH CH2 C C CH CH2
32.8, 47.0, 34.9, 44.2, 26.3,
CH2 C CH C CH2
74.2, 41.9, 158.6, 119.1, 36.0,
CH C C CH CH2
2.07, s
44.1, 27.2, 15.2, 124.4, 139.6, 110.9, 142.6, 34.4, 23.6, 19.8, 170.1, 20.8,
CH CH3 CH3 C CH CH CH CH3 CH3 CH3 C CH3
2.01, s
170.3, C 21.1, CH3
2.52, 1.26, 1.45, 1.96,
m m m m
3.16, m 2.40, m 1.89, m 5.20, t (8.0)
5.37, 2.34, 2.56, 2.80, 1.21, 1.17,
dd (4.0, 2.8) m m t (8.0) s s
7.23, 6.26, 7.38, 1.40, 1.52, 0.74,
s s s s s s
Recorded at 400 and 100 MHz. bRecorded at 500 and 125 MHz. Marine Chemical Company, Qingdao, People’s Republic of China), Sephadex LH-20 (40−70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), and Lichroprep RP-C18 gel (40−63 μm, Merck, Darmstadt, Germany). TLC spots were visualized under UV light and by dipping into 5% H2SO4 in EtOH followed by heating. Plant Material. The whole plant of Munronia henryi was collected in Wenshan, Yunnan Province, People’s Republic of China, in November 2012, and was identified by Prof. Zhi-Min Fu of Guiyang College of Traditional Chinese Medicine. A voucher specimen (DHL H20121120) was deposited at the Key Laboratory of Chemistry for Natural Product of Guizhou Province and Chinese Academy of Sciences. Extraction and Isolation. The air-dried, powdered twigs of M. henryi (20.0 kg) were extracted with 95% EtOH (3 × 50 L) under reflux three times (4, 3, and 3 h, respectively). The combined EtOH extracts were concentrated under vacuum to give a crude residue (1.424 kg), which was suspended in water and then partitioned with EtOAc. The EtOAc portion (413 g) was subjected to passage over a silica gel column, eluted with a gradient of petroleum ether−acetone (from 1:0 to 2:1) and CHCl3−Me2CO (from 5:1 to 0:1), to yield nine major fractions (1−9). Fr. 4 (45 g) was then separated over an MCIgel column (MeOH−H2O from 3:7 to 10:0) to obtain five further fractions (4A−4E). Fr. 4B (11 g) was chromatographed over a C18 silica gel column, eluted with a gradient of MeOH−H2O (50:50,
Figure 3. X-ray ORTEP drawing of 8. spectrometers using TMS as an internal standard. Bruker HCT/E Squire and Waters Autospec Premier P776 mass spectrometers were used to measure ESIMS and HREIMS, respectively. X-ray data were collected using a Bruker APEX DUO instrument. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Waters XBridge C18 (4.6 × 250 mm) column. Column chromatography (CC) was performed on silica gel (90−150 μm, Qingdao G
DOI: 10.1021/np501057f J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Table 5. 1H and 13C NMR Spectroscopic Data of Compounds 11−13 in CDCl3 (δ in ppm, J in Hz) 11a δH
position
a
1 2α 2β 3 4 5 6 7 8 9 10 11 12 13 14 15 16α 16β 17 18 19 20 21 22 23 28 29 30 OMe-12
4.88, 2.13, 2.29, 4.96,
t (2.8) m m t (2.8)
OAc-3 1′ 2′ 3′ 4′ 5′
1.96, s
2.83, d (12.8) 4.03, d (2.8) 4.38, d (2.8) 2.70, d (9.2) 1.60, m 4.06, dd (10.0, 3.2)
4.47, 1.67, 1.71, 3.45, 1.73, 0.98,
d (8.0) m m m s s
7.29, 6.46, 7.31, 3.61, 1.22, 1.35, 3.34,
s s s s s s s
7.06, m 1.87, d (6.8) 1.94, s
12a δC, type 71.3, CH 27.9, CH2 71.4, 42.6, 38.6, 73.8, 73.2, 46.2, 36.9, 41.1, 32.7, 105.2, 139.3, 143.4, 81.8, 37.9,
CH C CH CH CH C CH C CH2 CH C C CH CH2
46.3, 16.1, 16.3, 128.5, 138.9, 110.5, 142.9, 77.9, 19.5, 20.8, 56.0, 170.2, 20.9, 166.1, 128.7, 138.0, 14.6, 12.1,
CH CH3 CH3 C CH CH CH CH2 CH3 CH3 CH3 C CH3 C C CH CH3 CH3
4.81, 2.10, 2.23, 4.95,
13b
δH
δC, type
m m m t (2.8)
71.6, CH 27.6, CH2
2.87, d (12.8) 4.13, d (2.8) 4.79, d (3.2) 2.75, d (8.4) 1.64, m 4.09, d (3.6)
5.15, 2.10, 2.03, 3.56, 1.86, 0.97,
t (2.8) m m d (8.8) s s
7.16, 6.08, 7.33, 3.62, 1.20, 1.20, 3.22,
s s s s s s s
1.93, s
6.99, m 1.83, d (6.8) 1.90, s
71.4, 42.6, 38.7, 73.7, 70.7, 49.7, 35.2, 40.7, 28.4, 101.2, 136.6, 142.0, 75.0, 38.2,
CH C CH CH CH C CH C CH2 CH C C CH CH2
44.7, 16.3, 16.5, 128.1, 138.8, 109.6, 143.2, 77.8, 19.3, 20.3, 54.8, 170.3, 20.8, 166.8, 128.6, 138.3, 14.6, 12.0,
CH CH3 CH3 C CH CH CH CH2 CH3 CH3 CH3 C CH3 C C CH CH3 CH3
4.82, 2.19, 2.22, 4.95,
δH
δC, type
t (2.0) m m t (2.0)
71.2, CH 27.5, CH2
2.74, d (10.0) 4.11, dd (6.0, 3.0) 4.46, d (2.4) 3.96, m 4.01, dd (8.0, 4.0) 6.06, dd (8.0, 1.6)
5.46, 1.72, 2.54, 3.50, 1.79, 0.88,
d (8.0) m m d (7.2) s s
7.26, 6.34, 7.31, 3.61, 1.21, 1.38,
s s s s s s
1.94, s
6.98, m 1.83, d (6.6) 1.91, s
71.6, 42.4, 38.8, 73.8, 71.8, 50.9, 39.4, 39.7, 97.8, 144.4, 142.7, 141.2, 86.0, 37.4,
CH C CH CH CH C CH C CH CH C C CH CH2
47.1, 15.9, 15.6, 128.1, 139.1, 110.3, 142.9, 78.0, 19.4, 20.1,
CH CH3 CH3 C CH CH CH CH2 CH3 CH3
170.3, 20.9, 166.6, 129.0, 137.3, 14.5, 12.1,
C CH3 C C CH CH3 CH3
Recorded at 400 and 100 MHz. bRecorded at 500 and 125 MHz.
60:40, and 70:30), to afford five subfractions (4B1−4B5). Fr. 4B2 (2.7 g) was purified by Sephadex LH-20 (MeOH) and then chromatographed on a silica gel column (CHCl3−Me2CO, 10:1) to obtain 1 (21 mg), 6 (5.5 mg), 7 (9.0 mg), and 21 (6.3 mg). Fr. 4C (2.5 g) was separated by Sephadex LH-20 (MeOH−CHCl3, 1:1) to obtain 2 (7.1 mg) and a major fraction (Fr. 4C1). Fr. 4C1 (500 mg) was separated by semipreparative HPLC (MeOH−H2O, 6:4) to give 3 (9.1 mg), 8 (17 mg), and 20 (5.7 mg). Fr. 4D (6 g) was chromatographed on a silica gel column, eluted with a gradient of petroleum ether−EtOAc (9:1 to 5:5), to yield 4 (25 mg), 9 (28 mg), and subfractions 4D1 (60 mg) and 4D2 (30 mg). Subfractions 4D1 and 4D2 were further purified by semipreparative HPLC (MeOH−H2O, 4:6) to afford 5 (29 mg), 10 (10 mg), and 11 (7.2 mg). Fr. 5 (30 g) was chromatographed over an MCI-gel column (MeOH−H2O from 5:5 to 10:0) to obtain four fractions (5A−5D). Fr. 5B (2.5 g) was purified using Sephadex LH-20 (MeOH) to furnish 12 (11 mg), 13 (10 mg), and the major fraction 5B1 (800 mg). The latter was separated by semipreparative HPLC using MeOH−H2O (47:53) to give 15 (10 mg), 17 (35 mg), and 19 (18 mg). Compounds 14 (13 mg) and 16 (10 mg) were isolated from Fr. 5C (800 mg) by repeated silica gel column chromatography, eluted with a gradient of CHCl3−Me2CO (from 8:1 to 5:1). Fr. 5D (4 g) was chromatographed on a C18 silica gel column,
eluted with a gradient of MeOH−H2O (60:40, 65:45, and 70:30), to afford 18 (24 mg) and 22 (10 mg). Munronin A (1): white, amorphous powder; [α]21 D +106.4 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (4.83) nm; ECD (MeOH) λmax (Δε) 204 (+91.2) nm; IR (KBr) νmax 3442, 2934, 1743, 1233, 1176, 1028 cm−1; 1H and 13C NMR data, see Table 1; positive ESIMS m/z 741 [M + Na]+; HRESIMS m/z 741.3100 [M]+ (calcd for C37H50O14Na, 741.3098). Munronin B (2): white, amorphous powder; [α]21 D +289.1 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (4.53) nm; ECD (MeOH) λmax (Δε) 228 (+82.2) nm; IR (KBr) νmax 3440, 2951, 1743, 1243, 1123, 1029 cm−1; 1H and 13C NMR data, see Tables 2 and 3; positive ESIMS m/z 637 [M + Na]+; HRESIMS m/z 637.2623 [M]+ (calcd for C33H42O11Na, 637.2625). Munronin C (3): white, amorphous powder; [α]21 D +20.0 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 206 (3.79) nm; ECD (MeOH) λmax (Δε) 216 (+2.74) nm; IR (KBr) νmax 3444, 2924, 1749, 1248, 1125, 1044 cm−1; 1H and 13C NMR data, see Tables 2 and 3; positive ESIMS m/z 625 [M + Na]+; HREIMS m/z 602.2365 [M]+ (calcd for C31H38O12, 602.2363). Munronin D (4): white, amorphous powder; [α]21 D +247.5 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 204 (4.56) nm; ECD (MeOH) λmax (Δε) 202 (+27.7), 249 (+24.0) nm; IR (KBr) νmax 3448, 2932, H
DOI: 10.1021/np501057f J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Table 8. IC50 Values (μM) of Limonoids from M. henryi for Human Tumor Cell Lines
Table 6. In Vitro and in Vivo Antiviral Activities of Limonoids 1−22 against Tobacco Mosaic Virus inhibition rate (%) compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 ningnanmycin
inactivation effectb
in vitro effecta 54.3 91.8 50.6 49.3 33.8 32.1 30.7 98.9 97.6 89.0 97.9 98.5 49.3 33.6 44.5 43.9 41.5 75.9 47.6 80.0 30.1 18.5 69.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.6 4.4 4.2 4.9 3.3 5.5 3.6 2.3 1.7 5.4 3.0 4.2 3.3 3.2 5.2 2.3 4.0 5.4 3.6 3.4 4.0 3.7 3.6
51.8 87.3 49.1 45.4 36.8 30.9 32.5 98.2 95.3 81.8 91.4 88.9 38.5 30.2 34.1 31.5 30.9 68.1 38.9 61.4 27.6 21.2 58.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.1 5.0 4.9 3.5 3.6 5.3 3.5 3.9 3.7 2.8 2.5 5.5 4.9 3.9 2.8 3.0 5.1 5.0 4.1 6.2 3.7 4.9 4.6
protection effectc 43.8 57.2 40.3 42.5 49.7 35.6 30.5 56.7 60.2 36.4 63.8 63.8 24.7 25.3 17.3 50.0 15.0 45.7 31.6 42.1 28.3 19.4 50.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.3 5.6 6.7 6.1 3.8 3.9 5.3 7.8 3.6 6.5 3.8 5.0 4.7 4.5 3.6 3.4 2.9 2.7 5.7 3.7 2.6 3.4 5.2
compounda
HL-60
SMMC-7721
A-549
MCF-7
SW480
1 18 DDPb paclitaxelb
0.44 4.8 1.7
