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Highly oxygenated and rearranged limonoids from the stem barks of Entandrophragma utile
Phytochemistry 172 (2020) 112282
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Highly oxygenated and rearranged limonoids from the stem barks of Entandrophragma utile
T
Ya-Lin Hu, Xiao-Meng Tian, Cheng-Cheng Wang, Quasie Olga, Dan Yan, Peng-Fei Tang, Li-Na Zhang, Jun Luo∗∗, Ling-Yi Kong∗ Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, People's Republic of China
A RT ICLE INFO
ABSTRACT
Keywords: Entandrophragma utile Meliaceae Limonoids Orthesters CD exciton chirality Multidrug resistance (MDR)
Seventeen highly oxygenated and rearranged limonoids, including nine previously undescribed phragmalin-type limonoids with 1,8,9- and 8,9,30-orthesters (entanutilins C–K, 1–9), three undescribed limonoids with rare rearranged-6/6/7/5 skeleton (entanutilins L-N, 10–12), and 5 known limonoids, were isolated from the stem barks of Entandrophragma utile from Ghana (Africa). Their structures including absolute configurations were elucidated based on comprehensive spectroscopic analyses, such as HRESIMS, 1D/2D-NMR, CD exciton chirality method, time-dependent density functional theory (TDDFT)/ECD calculations, and single-crystal X-ray diffraction. Bioactivity screenings suggested that some of these compounds effectively reversed resistance in MCF-7/DOX cells at a nontoxic concentration of 30 μM with 6- to 19-fold enhancing effects.
1. Introduction Limonoids, mainly from plants of the Meliaceae family, have attracted a broad range of interests in both agrochemistry and organic chemistry because of their enchanting structural diversity and significant biological activities (Champagne et al., 1992; Hay et al., 2007; Narender et al., 2008; Roy and Saraf, 2006; Wang et al., 2006; Yin et al., 2006; Zhang et al., 2018). Of native African meliaceous species, plants from the Entandrophragma genus include not only an economically important tropical hardwood but also a Nigerian traditional medicine used to treat gastrointestinal ulcers and sickle cell disease (SCD) (Adejumo et al., 2011; John and Onabanjo, 1990a, 1990b; 2010; John, 1994; Quasie et al., 2017; Tchouankeu et al., 1990). Previous studies on Entandrophragma utile (Dawe & Sprague) Sprague have indicated that sterols and limonoids are the main secondary metabolites (Daniewski et al., 1993, 1995; Tchouankeu et al., 1990, 1992, 1996), and our previous chemical investigations on small amounts of the bark of E. utile led to the isolation of two rare limonoids (Luo et al., 2016). The above information encouraged us to further investigate this plant as part of our research on novel and bioactive limonoids from the Meliaceae family. As a result, nine previously undescribed phragmalin-type limonoids with 1,8,9- and 8,9,30-orthesters, three undescribed limonoids with a rare rearranged-6/6/7/5 skeleton, and five known limonoids
∗
were isolated and identified from 2.0 kg of stem bark of the title plant collected in Ghana (Africa). Herein, we describe the isolation, structural elucidation including the absolute configurations, and the multidrug resistance reversal activity of these limonoids on MCF-7/DOX cells. 2. Results and discussion 2.1. Structure elucidation Entanutilin C (1) was isolated as a white amorphous powder, and its molecular formula was determined to be C41H47NO16 based on its HRESIMS ion peak at m/z 810.2972 [M + H]+ (calcd for C41H48NO16: 810.2968) in conjunction with its 13C NMR spectrum, which displayed 41 carbon resonances. Careful analyses of its NMR spectra revealed characteristic resonances of a β-furan ring (δH 6.42, 7.40 and 7.45; δC 110.0, 120.9, 141.3 and 143.2), a nicotinyl moiety (δH 7.45, 8.74, 8.79, 9.56; δC 123.2, 126.9, 138.0, 151.5, 153.2, 165.3), one methoxy signal (δH 3.72; δC 52.2), and an orthoacetate system [δH 1.78 (3H, s); δC 119.5 (C-31)]. A pair of roofed doublets at δH 1.84 (d, J = 11.0 Hz) and 1.99 (d, J = 11.0 Hz) were assigned as the H-29 protons of the characteristic phragmalin 4,29,1-ring bridge, which was confirmed by the HMBC cross-peaks (Fig. 2) of C-29 (δC 39.9)/H-3 (δH 4.96) and Me-28 (δH 0.97), H-29 (δH 1.84)/C-5 (δC 36.3). These observations showed
Corresponding author. Corresponding author. E-mail addresses: [email protected] (J. Luo), [email protected] (L.-Y. Kong).
∗∗
https://doi.org/10.1016/j.phytochem.2020.112282 Received 29 September 2019; Received in revised form 19 January 2020; Accepted 22 January 2020 0031-9422/ © 2020 Elsevier Ltd. All rights reserved.
Phytochemistry 172 (2020) 112282
Y.-L. Hu, et al.
Fig. 1. Structures of compounds 1–17.
Fig. 2. Key HMBC and ROESY correlations of 1.
2
Phytochemistry 172 (2020) 112282
Y.-L. Hu, et al.
Fig. 3. The CD spectrum of compound 1 in MeOH; the bold lines denote the electric transition dipole of the chromophores for 1.
that 1 was a phragmalin-type limonoid orthester (Lin et al., 2011). The obvious HMBC cross-peaks (Fig. 2) of H-3 (δH 4.96) and -OCOC5NH4 (δC 165.3), H-30 (δH 6.09) and -OCOCH(CH3)2 (δC 174.6), and H-12 (δH 4.53) and -OCOCH3 (δC 169.7) revealed that the nicotinyl moiety, the isobutyryl group and the acetyl group were attached to C-3, C-30 and C12, respectively. The location of the orthoacetate was determined to be 1/8/9-type based on the characteristic carbon resonances of C-1/2/8/9, although there were no direct HMBC correlations from the protons on the phragmalin skeleton to the orthoacetate carbon (C-31) (Lin et al., 2011). Thus, as shown in Fig. 1, the planar structure of 1 was determined as a phragmalin-type limonoid orthester with a rare nicotinyl moiety at C-3. The ROESY correlations (Fig. 2) of H-3/H-29 (δH 1.84), and H-29 (δH 1.99)/Me-19 and Me-32 indicated that these protons and groups were on the same side of the molecule, while the correlations (Fig. 2) of H-5/H-12, Me-28, and H-30; H-12/H-11 and H-17; and H-17/H-30 revealed that H-5, H-11, H-12, H-17, Me-28 and H-30 were on the other side. The positive chirality, resulting from the exciton coupling at 243 nm (Δε +1.610, nicotinyl moiety) and 211 nm (Δε −7.517, the furan ring) (Luo et al., 2016), can be observed in the ECD spectrum (Fig. 3), and thus the absolute configuration of 1 was elucidated as shown in Fig. 1. Finally, single crystals (Fig. 4) of 1 were obtained and subjected to X-ray diffraction using Cu Kα radiation. The high-quality crystal data [CCDD 1905734, Flack parameter of 0.06 (11)] not only confirmed the putative planar and relative structure but also the absolute configuration of 1 as 1R, 2S, 3S, 4R, 5S, 8R, 9R, 10R, 11R, 12R, 13R, 14R,17S, 30R. Entanutilin D (2) had a molecular formula of C37H46O16 as established from its HR-ESI-(+)-MS ion peak at m/z 769.2679 [M + Na]+ (calcd. 769.2678). The similarity of the NMR data (Table 1) of 2 and 1 indicated that 2 was also a pharagmalin-type limonoid with a 1/8/9orthoester. The OH-3 and OCOCH3-11 groups, the major difference between these two compounds, were revealed by the HMBC signals of δH 4.96 (H-3) to δC 14.1 (Me-28)/36.5 (C-5)/69.9 (C-30) and of δH 5.60 (H-11) to δC 169.4 (11-OCOCH3). The similar ROESY and ECD data (Fig. S85) indicated that the key asymmetric carbons of 2 and 1 had the same relative and absolute configurations. Thus, the absolute configuration of 2 was also elucidated as shown in Fig. 1. Entanutilin E (3) possessed a molecular formula of C35H42O15, based
Fig. 4. X-ray crystallographic structure of 1.
on analysis of its HRESIMS peak at m/z 725.2418 [M + Na]+. The 1H and 13C NMR data of 3 and 1 were almost the same, except for the presence of a double bond and the signals of the substituents. The HMBC correlations of δC 154.9 (C-14)/δH 5.91 (H-30) and δH 5.74 (H17) and of δH 6.81 (H-15)/δC 77.7 (C-8), δC 41.9 (C-13) and δC 164.3 (C16) suggested that the additional double bond was located at C-14/15. The HMBC correlations of δH 3.27 (OH-3)/δC 85.9 (C-3) and of δC 85.9 (C-3)/δH 1.86 (H-29) and δH 0.88 (Me-28) showed that OH-3 was free, which was another difference from 1. The relative configuration of 3 was determined to be identical to that of chubularisin B based on a detailed comparison of their ROESY data (Liu et al., 2012). The absolute configuration of compound 3 was deduced by the electronic circular dichroism (ECD) chirality method. The ECD spectrum (Fig. 5) acquired in MeOH was dominated by a negative Cotton 3
Phytochemistry 172 (2020) 112282
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Thus, the absolute configuration of 3, especially the 17-R conformation, was established as depicted in Fig. 1. Entanutilin F (4) displayed a molecular ion peak [M + Na]+ at m/z 709.2472 in its HRESIMS data, and this peak corresponded to a molecular formula of C35H42NaO14 (calcd. 709.2467). The NMR data of 4 resembled those reported for xyloccensin Q (Cui et al., 2005), with an 8/9/30-orthoacetate, except for the resonances of the acetyl and isobutyryl groups and the disappearance of the oxygenated carbon at C-6 in 4. The isobutyryl group in 4 was located at C-3 based on the HMBC correlation (Fig. 6) from H-3 (δH 3.74) and the two doublet methyl protons (δH 1.19 and 1.19) to the carbonyl signal at δC 176.6. Moreover, the methylene carbon at C-6 in 4 rather than the oxygenated carbon in xyloccensin Q was supported by the obvious correlations from H2-6 (δH 2.16, 3.11) to C-7 (δC 174.6) and C-4 (δC 44.5) in the HMBC spectrum (Cui et al., 2005). The ROESY correlations (Fig. 6) of H-29 (δH 2.70)/ Me-19 and H-3 and of Me-18/Me-19 and Me-32 showed that H-3, Me18, Me-19, H-29 and Me-32 were on the same face and they were arbitrarily assigned as α-oriented, whereas the ROESY interactions between H-5/H-12, Me-28, H-30 and H-12/H-17 suggested the β-orientations of H-5, H-12, H-17, Me-28 and H-30. Thus, the chemical structure of 4 was determined as shown. The NMR and HRMS data of 5 and 6 revealed that these two compounds were also phragmalin-type limonoids with 8/9/30-orthoacetate groups and C-14/15 double bonds as in 4. The proton resonance at δH 2.90, assignable to an –OH group, displayed HMBC correlations with C-3 (δC 86.9) and C-4 (δC 44.0), indicating the –OH group is at C-3 in 5 rather than at C-2 in 4. The above information and the characteristic 13C NMR data of C-2 (δC 84.0) indicated that the isobutyryl group was located at C-2 in 5 (Liu et al., 2012). The additional acetoxyl group in 6 was located at C-11 based on the HMBC correlations from δH 5.63 (H-11) to δC 169.9 (-OCOCH3), δC 42.2 (C-13) and δC 48.4 (C-10). The significant ROESY correlations of H-11/H-12 and H-12/H-5 suggested that 11-OCOCH3 was α-oriented; the other relative configurations of 6 and 5 were determined to be the same as that of 4. Thus, the structures of 5 and 6 were elucidated as shown. Entanutilin I (7), obtained as a white, amorphous powder, displayed a molecular formula of C35H42O15, as determined by its HRESIMS signal at m/z 725.2421 [M + Na]+ (calcd for 725.2416). Analysis of the MS and NMR data of 7 indicated that its structure was closely related to that of 12α-acetoxyswietephragmin D (Silva et al., 2008), a phragmalintype limonoid with 8,9,30-ortho-isobutyrate. OH-3 was free, as indicated by the characteristic upfield shifts of H-3 (δH 3.83) of 7 compared with H-3 (δH 4.83) of 12α-acetoxyswietephragmin D (Silva et al., 2008), respectively, and the HMBC correlations from H-3 (δH 3.83) to C-2 (δC 76.4), C-5 (δC 39.7) and C-28 (δC 14.5). An obvious HMBC correlation from H-11 (δH 5.68) to the carbon resonance at δC 170.3 (11-OCOCH3) indicated that OH-11 was acetylated as in 7. The relative configuration of 7 was assigned as the same as that of 6 based on ROESY data analysis (Silva et al., 2008). Thus, the chemical structure of 7 was determined as shown. The similarity of chromophores with △14,15α, β-unsaturated ketone and the furan ring and the ECD spectra (Fig. 5) of 3–7 indicated that the key asymmetric carbons of them had identical absolute configurations as shown in Fig. 1. Thus, the structures of 4–7 were finally elucidated. Entanutilins J (8) and K (9) were also obtained as amorphous powders, possessing molecular formulas of C41H45NO16 and C40H48O16 via analysis of their HRESIMS signals at m/z 808.2816 [M + H]+ and 807.2841 [M + Na]+, respectively. Analyses of the NMR data of 8 and 9 implied that they had the same phragmalin core as seen in 7, and the difference was the position of substituents. In compound 8, the location of the nicotinoyl moiety at C-3 was determined from the HMBC correlation between H-3 (δH 5.04) and -OCOC5NH4 (δC 165.8). For compound 9, the assignments of -OTig and –OCOCH3 at C-3 (δC 86.6) and C12 (δC 68.3), respectively, were determined based on the HMBC correlations of δH 4.82 (H-3)/δC 168.0 [3-OCOCH(CH3) = CHCH3] and δH 4.84 (H-12)/δC 170.6 (12-OCOCH3), respectively. One tertiary methyl
Table 1 1 H and 13C NMR spectroscopic data for 1–3 in CDCl3. No.
1 2 3 4 5 6
1a
2b
δH (mult; J, Hz)
4.96, s c
3.21 2.56, m 2.46, m
7 8 9 10 11 12 13 14
4.38, m 4.53, m 2.74, m 3.21c 2.70, m
15 16 17 18 19 20 21 22 23 28 29
5.44, s 1.42, s 3H 1.19, s 3H 7.45c 6.42, s 7.40, m 0.97, s 3H 1.99, d (11.0)
δC 86.0 80.4 84.6 45.3 36.3 33.7 172.4 85.4 86.2 45.6 69.7 71.9 38.6 42.5 26.8 169.9 76.7 15.9 16.7 120.9 141.3 110.0 143.2 14.1 39.9
1.84, d (11.0) 30 31 7-OCH3 R-31 OHs
6.09, s 3.72, s 3H CH3-31 1.78, s 3H
70.0 119.5 52.2 21.3
11-OAc
4.96, s 3.23, m 2.74, m 2.53, m
5.60, d (2.5) 4.60, d (2.5) 2.62, d (10.4) 3.27, m 2.78, m 5.45, s 1.34, s 3H 1.24, s 3H 7.40c 6.38, s 7.40c 0.97, s 3H 2.02, d (11.0) 1.82, d (11.0) 6.09, s 3.75, s 3H CH3-31 1.71, s 3H
2.12, s 3H
12-OAc
1.68, s, 3H
R-3
OCOC5NH4-3
R-30
δH (mult; J, Hz)
8.74, m 7.45c 8.79, m 9.56, m OCOCH(CH3)230 2.47, m 1.16, d (7.0) 0.88, d (7.0)
169.7; 20.3 165.3 126.9 138.0 123.2 153.2 151.5 174.6 34.6 19.2 17.9
1.62, s 3H
OCOCH (CH3)2-30 2.48, m 1.17, d (7.0) 0.89, d (7.0)
3a δC 85.5 80.5 84.9 45.2 36.5 33.7 172.8 86.0 85.8 45.7 69.3 70.2 38.5 43.3 26.9 169.3 76.6 15.7 16.4 120.7 141.4 109.8 143.4 14.1 40.0
69.9 119.6 52.3 21.3
169.4c; 20.9 169.4c; 20.0
174.9 34.6 19.2 17.9
δH (mult; J, Hz)
3.82, s 2.41, m 2.85, d (15.3) 2.40, m
4.30, s 4.84, s
6.81, s 5.74, s 1.75, s 3H 1.31, s 3H 7.48, s 6.59, s 7.38, s 0.88, s 3H 1.86, d (11.1) 1.75, m 5.91, s 3.73, s 3H CH3-31 1.68, s 3H OH-3: 3.27, s OH-11: 2.70, s 1.59, s 3H
OCOCH (CH3)2-30 2.60, m 1.26, d (7.0) 1.22, d (7.0)
δC 90.7 76.8 85.9 44.5 37.9 32.6 174.2 77.7 83.2 43.8 75.9 69.0 41.9 154.9 123.1 164.3 78.7 18.0 14.0 121.2 142.5 110.6 143.0 14.4 39.0
68.0 119.8 52.4 16.2
171.0; 20.0
175.3 34.2 19.8 18.8
a 1
H (600 MHz) and 13C (150 MHz) NMR data of compounds. H (500 MHz) and13C (125 MHz) NMR data of compound. Overlapped.
b 1 c
effect at 241 nm (△ε −3.690, △14,15α, β-unsaturated ketone) and a positive Cotton effect at 214 nm (△ε +4.262, the furan ring) due to the transition interaction between these two chromophores (Luo et al., 2016), which indicated an anticlockwise spatial relationship between these two chromophores. The calculated ECD spectrum by time-dependent density functional theory (TDDFT) (Bracher et al., 2004) for 3 matched fairly well with the experimental ECD spectrum (Fig. S1). 4
Phytochemistry 172 (2020) 112282
Y.-L. Hu, et al.
Fig. 5. The CD spectra of compounds 3–7 in MeOH; the bold lines denote the electric transition dipole of the chromophores for 3.
Fig. 6. Key HMBC and ROESY correlations of 4.
group (δH 1.43, 3H) in 8 was replaced by an oxygenated carbon (δC 63.6) in 9, and the other –OCOCH3 group was located at C-19, similar to velutinasin A (Zhang et al., 2014), which was supported by the HMBC cross-peaks of δH 4.50 and 4.75 (H2-19)/δC 49.2 (C-10) and δC 170.2 (19-OCOCH3) and of δC 49.2 (C-10)/δH 3.35 (H-6), δH 2.33 (H29) and δH 2.00 (H-11). The oxygenated carbon (δC 67.5) at C-11 in 8 was replaced by a methylene carbon (δC 32.2) in 9, which was confirmed by the HMBC signals from H-11 (δH 2.00; 2.21) to C-9 (δC 85.9) and C-13 (δC 42.8). The ROESY spectrum showed that the relative configurations of compounds 8 and 9 were identical to that of compound 7. Thus, the structures of 8 and 9 were elucidated. Based on the
same two chromophores, the absolute configurations of 8 and 9 were elucidated to be the same as those of 1 from their similar ECD spectra (Fig. S85). The calculated ECD data of 8 (Fig. S2) matched the experimental ECD further supporting the assignment of absolute configuration of 8 and 9 as depicted. Compounds 10–12 were isolated as white powders, and their molecular formulas were determined to be C36H44O12, C38H48O12 and C37H48O12, respectively, based on their HRESIMS data. The NMR data (Table 4) of 10–12 were similar to each other but notably different from those of 1–9, indicating that 10–12 should possess a different limonoid skeleton. Three pairs of coupled, down-shifted protons at δH 4–6, two 5
Phytochemistry 172 (2020) 112282
Y.-L. Hu, et al.
Table 2 1 H NMR Spectroscopic Data for 4–9 in CDCl3 (δ in ppm, J Values in Hz). No.
4b
3 5 6
3.74, 2.12, 3.11, 2.16, 2.31, 1.96, 4.80, 6.59, 5.89, 1.57, 1.23, 7.42, 6.54, 7.37, 0.87, 2.70, 2.32, 4.56, 1.68,
11 12 15 17 18 19 21 22 23 28 29 30 32 33 34 OHs 11-OAc 12-OAc 19-OAc 7-OCH3 R-2 or 3
5b
6b
7a
8a
s m d (16.7) m m t (13.8) dd (13.8, 4.1) s s s 3H s 3H
3.73, s 2.21, m 3.10, d (16.3) 2.30, m 2.22, m 1.94, m 4.84, dd (13.5, 4.2) 6.55c 5.92, s 1.55, s 3H 1.28, s 3H
3.73c 2.26, m 3.07, d (16.1) 2.32, m 5.63, d (3.9)
3.83, 2.29, 3.11, 2.30, 5.68,
s m d (14.9) m m
5.04, 2.85, 3.22, 2.43, 5.71,
m d (12.1) d (17.0) dd (17.0, 12.1) d (3.8)
4.96, d (3.9) 6.56c 5.93, s 1.68, s 3H 1.36, s 3H
4.89, 6.63, 5.87, 1.67, 1.35,
m s s s, 3H s, 3H
4.91, 5.89, 5.84, 1.56, 1.43,
d (3.8) s s s, 3H s, 3H
s s s s 3H d (11.9) m s s 3H
7.46, s 6.55c 7.37, s 0.87, s 3H 1.91, m 1.48, m 5.25, s 1.67, s 3H
7.48, s 6.56c 7.37, s 0.88, s 3H 1.94, d (11.4) 1.51, m 5.21, s 1.68, s 3H
OH-1: 3.25, s OH-3: 2.90, d (5.0)
OH-1: 3.21, s
7.42, s 6.55, s 7.38, m 0.84, s,3H 1.81, d (11.0) 1.64, m 4.63, s 2.18, m 1.09c 1.09c OH-1: 3.19, s
1.50, s 3H
1.50, s 3H
2.07, s 3H 1.47, s, 3H
1.47, s 3H 2.04, s 3H
3.72, s 3H OCOCH(CH3)2-3 2.54, p (6.9) 1.19c 1.19c
3.72, s 3H OCOCH(CH3)2-2 2.69, m 1.21, s 3H 1.19, s 3H
3.73, s, 3H OCOCH(CH3)2-2 2.70, p (6.9) 1.21c 1.21c
3.73, s, 3H
9b
7.49, t (1.2) 6.48, m 7.38, d (1.8) 0.93, s,3H 1.97, d (11.5) 1.86, dd (11.5, 1.5) 4.54, s 2.17, p (6.9) 1.07c 1.07c OH-1: 3.30, s OH-2: 3.65, s 2.03, s 3H 1.48, s, 3H 3.82, s, 3H OCOC5NH4-3 8.35, dd (8.0, 2.0) 7.42, dd (8.0, 5.0) 8.81, d (5.0) 9.28, d (2.0)
4.82, s 2.61, d (12.3) 3.35, d (16.5) 2.51, m 2.21, m 2.00, m 4.84, m 6.02, s 5.86, s 1.52, s, 3H 4.75, d (12.7) 4.50, m 7.41, brt (1.7) 6.54, d (1.8) 7.44, s 0.85, s,3H 2.33, d (11.2) 1.84, m 4.50, m 2.21, m 1.05c 1.05c OH-1: 3.52, s 1.52, s 3H 2.09, s 3H 3.76, s, 3H OTig-3 6.96, dd (7.0, 1.6) 1.84, s, 3H 1.73, d (7.0)
a 1
H (600 MHz) NMR data of compound. H (500 MHz) NMR data of compounds. Overlapped.
b 1 c
geminal methylenes at δH 1.5–2.5, and characteristic signals for a βsubstituted butyrolactone ring suggested that these three compounds should possess a rare, highly rearranged limonoid skeleton, similar to that of entanutilin A (13) (Luo et al., 2016). Me-19 was incorporated into the C-8/9/10/19-cyclobutyl ring, and Me-30 was inserted between C-8 and C-14 to form cycloheptyl ring C (Luo et al., 2016). Taking 10 as an example (Fig. 7), this structural assignment was confirmed by the key HMBC cross-peaks from H2-19 (δH 2.00) to C-7 (δC 72.9) and from H2-30 (δH 2.54; 2.43) to C-7 (δC 72.9), C-8 (δC 44.0), C-13 (δC 51.5), C14 (δC 175.0) and C-15 (δC 134.6). The obvious HMBC correlations between C-3 (δC 202.5) and H-1 (δH 6.08), H-5 (δH 2.43), Me-28/29 (δH 1.03; 1.07) indicated that C-3 in 10 was oxidized to the ketone. For compound 11, the only difference from 10 was that the acetoxy group at C-6 in 10 was replaced by an isobutyryl group in 11, which was confirmed by the obvious HMBC correlation from H-6 (δH 5.69) and the two doublet methyl protons (δH 1.15 and 1.17, J = 7.0 Hz) to the carbonyl signal at δC 175.8. The NMR spectra revealed that 12 was more similar to entanutilin A (13) (Luo et al., 2016) as it possessed a C–OH moiety rather than a C]O at C-3, which was confirmed by the HMBC cross-peaks between C-3 (δC 73.7) and H-1 (δH 5.24), Me-28/29 (δH 0.93; 0.80). The isovaleryl group at C-7 in 12 was assigned based on the HMBC correlations from H-7 (δH 5.49) and the methylene protons at δH 1.64 and 1.82 [-OCOCH2CH(CH3)2] to the carbonyl signal at δC 176.3 [-OCOCH2CH(CH3)2]. At this stage, the planar structures of 10–12 were determined, and they featured a highly rearranged framework with a cyclobutane and a cycloheptane rings. Their ROESY spectra indicated that the relative configurations of their basic skeletons were the same as those of entanutilin A (13) (Luo et al., 2016), and the key correlations of 10 are shown in Fig. 7. For compound 12, the ROESY correlation of H-3/Me-29 revealed the α-
orientation of the additional OH-3 moiety. The ECD spectra of 10–12 exhibited perfect Cotton effects, which can be used to determine their absolute configurations by the CD chirality method. Taking compound 10 as an example (Fig. 8), the negative chirality in the ECD spectrum at 238 nm (△ε −17.860, △14,15 α, β-unsaturated ketone) and 220 nm (△ε +15.906, △1,2 α, β-unsaturated ketone) indicated the anticlockwise spatial relationship between these two chromophores and the calculated ECD spectrum for 10 matched fairly well with the experimental ECD spectrum (Fig. S3). Thus, the absolute configuration of 10 was defined as 5R, 6R, 7S, 8S, 9R, 10S, 11S, 12R, 13S, 17S. The ECD spectra of 11 and 12 in MeOH were highly similar to 10 (Fig. 8), so the absolute configurations of 11 and 12 were assigned as shown in Fig. 1. The five reported compounds were identified as entanutilin A (13) (Luo et al., 2016), 12α-acetoxyphragmalin-3-nicotinate-30-isobutyrate (14) (Steven and David, 1988), entanutilin B (15) (Luo et al., 2016), utilin C (16) (Daniewski et al., 1994) and utilin B (17) (Daniewski et al., 1993) by comparison of their spectroscopic data with reported data. 2.2. MDR reversal activities One key obstacle in cancer treatment is the intrinsic or acquired drug resistance of certain cancers, and significant efforts have been made in clinical oncology to provide solutions to this resistance (Mellor and Callaghan, 2008). The mexicanolide-type limonoid, trichisin H from the fruits of Heynea trijuga, effectively reversed multidrug resistance in doxorubicin-resistant human breast tumour (MCF-7/DOX) cells (An et al., 2018). Therefore, all compounds (1–17) were evaluated for their MDR reversal activities on MCF-7/DOX cell line with verapamil at 10 μM as a positive control (Table 5). At 30 μM, limonoids 13, 15, 16 and 17 showed reversal fold (RF) values of 6.25, 6.52, 19.15 and 6
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Table 3 13 C NMR Spectroscopic Data for 4–9 in CDCl3 (δ in ppm).
Table 4 1 H and 13C NMR spectroscopic data for 10–12 in CDCl3.
No.
4b
5b
6b
7a
8a
9a
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 31 32 33 34 11-OAc
84.6 78.2 85.4 44.5 38.5 32.8 174.6 83.7 89.1 49.6 33.0 69.2 43.0 152.9 124.0 164.2 78.8 14.5 15.8 121.6 142.1 110.5 143.0 14.7 38.6 77.7 119.6 16.9
85.6 84.0 86.9 44.0 39.1 33.3 174.4 84.2 86.2 48.4 32.8 69.1 43.0 152.3 124.0 163.9 78.7 14.6 15.5 121.5 142.2 110.5 142.9 14.8 40.1 75.0 119.7 16.7
85.0 84.6 87.0 43.9 39.2 33.4 174.4 84.2 85.4 48.4 67.7 69.2 42.2 150.9 124.0 163.6 78.8 15.5 15.9 121.3 142.5 110.4 143.0 14.8 40.3 75.2 120.1 16.1
170.7; 20.0
170.5; 20.0
84.9 75.8 87.8 43.9 41.0 33.2 174.9 83.9 85.0 47.7 67.5 68.9 42.0 151.1 123.5 161.5 78.5 15.4 16.1 121.0 142.3 110.3 143.2 14.5 39.3 78.1 123.2 29.6 17.3 17.1 170.2; 21.0 170.3; 19.6
84.6 75.8 86.6 43.6 41.4 31.9 174.6 83.9 85.9 49.2 32.2 68.3 42.8 150.9 124.4 162.4 78.2 14.7 63.6 121.3 143.3 110.4 142.0 14.7 39.2 78.0 122.9 29.1 16.9 16.9
12-OAc
169.9; 20.9 170.2; 19.6
85.2 76.4 85.9 44.4 39.7 33.2 174.7 84.3 84.6 47.7 67.7 69.3 42.0 150.7 124.3 164.1 78.9 15.2 16.0 121.4 142.3 110.4 143.1 14.5 39.3 78.7 123.0 29.7 17.4 17.2 170.3c; 19.6 170.3c; 21.1
52.3 OCOCH (CH3)2-3 176.6 35.7 19.0 19.2
52.2 OCOCH (CH3)2-2 178.3 34.6 18.9 18.9
52.4 OCOCH (CH3)2-2 178.4 34.6 18.9c 18.9c
52.4
52.8 OCOC5NH4-3
19-OAc 7-OCH3 R-2 or 3
165.8 125.9 137.5 124.1 154.7 150.8
No.
1 2 3 4 5 6
c
δH (mult; J, Hz)
δC
δH (mult; J, Hz)
δC
6.08, d (10.3) 5.86, d (10.3)
145.6
6.08, m
145.7
5.24, d (10.0)
131.9
127.0
5.85,d (10.3)
127.0
5.80, dd (10.0, 5.9) 3.46, d (5.9)
128.1
4.83, m 4.86, m 6.08c 3.30, d (4.9) 1.14, s 3H 2.00c 2.00c 2.77c 4.43c 4.43c 2.77c 2.72, m
20 21 22 23 28 29 30 6-OAc
1.03, s 3H 1.07, s 3H 2.54, d (13.4) 2.43c 2.01, s, 3H
11-OAc
1.99, s, 3H
12-OAc
2.12, s, 3H
R-6
a 13 b
δC
2.82, dd (10.6, 4.7)
12 13 14 15 16 17 18 19
C (150 MHz) NMR data of compounds. 13 C (125 MHz) NMR data of compounds. Overlapped.
12.30, respectively, against MCF-7/DOX cells.
R-7
3. Conclusion
OCOCH (CH3)2-7 2.77c 1.30, d (7.0) 1.33, d (7.0)
In this study, twelve previously undescribed highly oxygenated and rearranged limonoids, including nine undescribed phragmalin-type limonoids with 1,8,9- and 8,9,30-orthesters (Entanutilins C–K, 1–9) and three undescribed limonoids with rare rearranged-6/6/7/5 skeleton (Entanutilins L-N, 10–12), were isolated from the stem barks of Entandrophragma utile. Moreover, bioactivity screenings suggested that some of these compounds effectively reversed resistance in MCF-7/DOX cells at a nontoxic concentration of 30 μM with 6- to 19-fold enhancing effects.
12a
δH (mult; J, Hz)
2.43 5.68, dd (9.6, 6.8) 5.54, d (6.8)
10 11
168.0 130.2 140.2 12.6 14.5
11b
c
7 8 9
170.6; 20.0 170.2; 21.4 52.5 OTig-3
10a
202.5 45.3 49.5 65.5 72.9 44.0 54.8 40.8 65.7 75.2 51.5 175.0 134.6 205.4 52.6 22.6 20.6c 34.9 72.1 32.3 176.9 21.4 21.6 33.5 169.9; 21.1 170.6; 29.6 169.4; 20.6c
176.5 34.6 19.1 19.5
2.46, m 5.69, dd (9.6, 6.8) 5.58, d (6.8) 2.83, dd (10.4, 4.8) 4.84, m 4.86, m 6.07, m 3.29, d (4.7) 1.13, s, 3H 2.01, m 1.54, m 2.74, m 4.43, m 4.42, m 2.75, m 2.71, m 1.02, s,3H 1.07, s 3H 2.50, m
202.5 41.0 49.6 65.5 72.9 44.1 54.6 45.4 65.8 75.2 51.6 175.1 134.6 205.3 52.6 22.5 29.7
2.12, s, 3H OCOCH (CH3)2-6 2.45, m 1.17, d (7.0) 1.15, d (7.0) OCOCH (CH3)2-7 2.75, m 1.35, d (7.1) 1.27, d (7.1)
2.81, dd (11.0, 5.3) 4.76, dd (11.0, 8.4) 4.86, d (8.4) 6.03, s
32.3
3.30, d (5.0) 1.11, s 3H 1.81, m 1.76, m 2.71, m 4.42c 4.42c 2.70, m
176.9 21.8 21.6 33.5
0.93, s 3H 0.80, s 3H 2.51, d (13.2)
34.9 72.1
2.44, m 1.99, s 3H
2.38, m 5.55, dd (9.9, 6.6) 5.49, d (6.6)
2.39, m 1.99c 170.5; 20.6c 169.4; 20.6c 175.8 34.2 18.8 18.6 176.3 34.5 19.6 18.9
1.99c 2.11, s 3H
OCOCH2CH (CH3)2-7 1.82, 1.64, 2.61, 1.00, 1.27,
m m q (6.9) t (7.4) d (6.9)
73.7 37.4 44.8 66.3 73.8 43.6 54.7 40.9 66.0 75.4 51.6 175.7 134.4 205.7 52.6 22.5 29.8 34.9 72.2 32.3 177.1 24.7 20.7 33.7 170.2; 20.8 170.6; 21.2 169.4; 20.7
176.3 27.0 41.8 12.1 17.1
a 1
H (600 MHz) and 13C (150 MHz) NMR data of compounds. H (500 MHz) and 13C (125 MHz) NMR data of compound. Overlapped.
b 1 c
Tensor 27 spectrometer (Bruker, Karlsruhe, Germany) and a Shimadzu UV-2450 spectrophotometer (Shimadzu, Tokyo, Japan), respectively. NMR spectra were acquired on Bruker AVIII-500 and AVIII-600 NMR instruments at 500 (1H) and 125 MHz (13C) and 600 MHz (1H) and 150 MHz (13C), respectively, using tetramethylsilane (TMS) as the internal standard. HRESI mass spectra were acquired using an Agilent 6520B UPLC-Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). ECD spectra were recorded on a JASCO J-810 spectropolarimeter (Jasco, Tokyo, Japan). RP-C18 (40–63 μm, FuJi), MPLC
4. Experimental 4.1. General experimental procedures Optical rotations were acquired on a JASCO P-1020 polarimeter (Jasco, Tokyo, Japan). IR and UV spectra were recorded on a Bruker 7
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Fig. 7. Key HMBC and ROESY correlations of 10.
Fig. 8. The CD spectra of compounds 10–12 in MeOH; the bold lines denote the electric transition dipole of the chromophores for 10.
(Beijing H&E Co., Ltd., Beijing, China), MCI gel (Mitsubishi Chemical Corp., Tokyo, Japan), Sephadex LH-20 (Pharmacia, Germany), and silica gel (100–200 mesh and 200–300 mesh, Qingdao Haiyang Chemical Co., Ltd.) were used for column chromatography. Analytical HPLC separations were carried out using an Agilent 1260 Series instrument using a DAD with a shim-pack VP-ODS column (250 × 4.6 mm, 5 μm). Semipreparative HPLC separations were conducted on a Shimadzu LC6AD instrument using an SPD-10A detector with a shim-pack RP-C18 column (20 × 200 mm, 10 μm).
Table 5 MDR reversal ability of compounds on MCF-7/DOX cells. Sample 13 15 16 17 VER
d
a
DOX+ 0 μM 12.561 12.561 12.561 12.561 12.561
± ± ± ± ±
b
3.215 3.215 3.215 3.215 3.215
c
DOX+30 μM
RF
2.010 1.926 0.656 1.021
6.25 6.52 19.15 12.30 11.60
± ± ± ±
0.309 0.112 0.231 0.278
a Serial dilutions ranging from 3.12 to 100 μM of DOX with or without sample. b Values are expressed as means ± SD. c RF = IC50 of DOX alone/IC50 of DOX with samples. d Verapamil was used as positive control at 10 μM.
4.2. Plant material The air-dried stem barks of Entandrophragma utile (Dawe & Sprague) Sprague (Meliaceae) were collected from Brong Ahafo Region of Ghana, in August 2015 and identified by Professor Mian Zhang of the Research Department of Pharmacognosy, China Pharmaceutical University. A 8
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voucher specimen (No. 2015-EU) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
4.4.3. Entanutilin E (3) white amorphous powder; [α]D25 +50.6 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 208 (4.25) nm; ECD (MeOH, Δε) 214 (+4.262), 241 (−3.690), 268 (+1.934) nm; IR (KBr) vmax 3445, 1729, 1639, 1385, 1236, 1026 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 725.2418 [M + Na]+ (calcd. for C35H42NaO15, 725.2416).
4.3. Extraction and isolation The air-dried stem bark (2.0 kg) was exhaustively extracted by refluxing with 95% ethanol three times and then cooling to room temperature. After concentration under reduced pressure, the crude extract was suspended in H2O and successively partitioned between petroleum ether and CH2Cl2. The CH2Cl2 extract (20 g) was loaded into a silica gel column and then eluted with a CH2Cl2–MeOH gradient (100:1 to 0:100, v/v), which gave five fractions (Frs. A-E) based on TLC and HPLC analysis. Fr. B (3.0 g) was separated on sequential silica gel and Sephadex LH-20 gel columns to give four subfractions (Frs. B1-4). Fr. B2 (1.1 g) was subjected to an MCI gel column (MeOH–H2O, 20:80 to 0:100, v/v) to yield three subfractions (Frs. B2a-c). Fraction B2a (60.0 mg) was separated by semipreparative HPLC with MeOH–H2O (49:51 v/v, 10 mL/min) as the mobile phase to give compound 1 (2.4 mg, tR = 10.5 min). Fr. B2c (101 mg) was separated by semipreparative HPLC using MeOH–H2O (45:55 v/v, 10 mL/min) as the mobile phase to give compound 3 (1.4 mg, tR = 12.5 min). Fr. B3 (220 mg) was also separated by semipreparative HPLC with 65% methanol in water as the eluent to give compound 8 (2.0 mg, tR = 18.2 min) and a mixture containing compounds 4 and 5. This mixture was purified using CH3CN–H2O (34:66, v/v, 10 mL/min) as the mobile phase to afford compounds 4 (4.8 mg, tR = 22.3 min) and 5 (4.2 mg, tR = 26.0 min). Fr. C (4.5 g) was separated on an ODS column using a gradient of CH3CN/H2O (10:90 to 100:0) to yield four subfractions (Frs. C1-4). Fr. C2 (2.0 g) was separated on an ODS column using a step gradient of MeOH–H2O (20:80 to 100:0, v/v), to afford four subfractions (Frs. C2a-d). Fr. C2a (860 mg) was repeatedly purified using semipreparative HPLC with a C18 column using CH3CN–H2O (38:62 v/v, 10 mL/min) as the mobile phase to afford compounds 2 (2.0 mg, tR = 9.6 min), 7 (6.0 mg, tR = 18.5 min) and 9 (1.8 mg, tR = 26.8 min). Fr. C2b (400 mg) was separated by semipreparative HPLC, with 48% methanol in water, to give compound 14 (15.0 mg, tR = 17.5 min) and a mixture containing compounds 10, 12 and 13. This mixture was purified using CH3CN–H2O (35:65, v/v, 10 mL/min) as the mobile phase to yield compounds 10 (2.1 mg, tR = 10.5 min), 12 (4.2 mg, tR = 16.9 min) and 13 (4.6 mg, tR = 21.5 min). Fr. D (3.2 g) was separated on a silica gel column eluted with a gradient of petroleum ether/acetone (20:1 to 1:2, v/v) to give four subfractions (Frs. D14). Fr. D2 (1.0 g) was subjected to an RP-C18 column using a gradient of MeOH–H2O (30:70 to 100:0, v/v), to afford three subfractions (Frs. D2a-c). Fr. D2b (85 mg) was purified by semipreparative HPLC (MeOH/ H2O 64:36, v/v) to afford compounds 6 (3.2 mg, tR = 39.8 min), and 15 (5.1 mg, tR = 45.6 min). Frs. D2c (50 mg) was separated by semipreparative HPLC with 49% methanol in water as the eluent to give compounds 11 (3.9 mg, tR = 19.8 min), 16 (16.0 mg, tR = 22.6 min) and 17 (10.0 mg, tR = 38.9 min).
4.4.4. Entanutilin F (4) white amorphous powder; [α]D25 +70.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 213 (4.02) nm; ECD (MeOH, Δε) 219 (+8.491), 261 (+2.195) nm; IR (KBr) vmax 3459, 2966, 1728, 1241, 1155, 1023 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 709.2472 [M + Na]+ (calcd. for C35H42NaO14, 709.2467). 4.4.5. Entanutilin G (5) white amorphous powder; [α]D25 +40.9 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 214 (4.10) nm; ECD (MeOH, Δε) 214 (+28.639), 261 (+7.053) nm; IR (KBr) vmax 3459, 2976, 1720, 1240, 1161, 1022 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 709.2468 [M + Na]+ (calcd. for C35H42NaO14, 709.2467). 4.4.6. Entanutilin H (6) white amorphous powder; [α]D25 +59.4 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 214 (4.10) nm; ECD (MeOH, Δε) 214 (+21.395), 261 (+5.328) nm; IR (KBr) vmax 3439, 2962, 1728, 1641, 1386, 1247, 1164, 1028 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 767.2523 [M + Na]+ (calcd. for C37H44NaO16, 767.2522). 4.4.7. Entanutilin I (7) White amorphous powder; [α]D25 +46.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 198 (2.71) nm; ECD (MeOH, Δε) 219 (+5.466), 264 (+1.763) nm; IR (KBr) vmax 3447, 1728, 1641, 1386, 1245, 1026 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 725.2421 [M + Na]+ (calcd. for C35H42NaO15, 725.2416). 4.4.8. Entanutilin J (8) white amorphous powder; [α]D25 +18.5 (c 0.26, MeOH); UV (MeOH) λmax (log ε) 214 (4.31), 263 (3.51) nm; ECD (MeOH, Δε) 205 (−17.732), 231 (+53.915) nm; IR (KBr) vmax 3461, 2980, 1740, 1636, 1478, 1384, 1278, 1025 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 808.2816 [M + H]+ (calcd. for C41H46NO16, 808.2811). 4.4.9. Entanutilin K (9) white amorphous powder; [α]D25 +50.0 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 213 (4.16) nm; ECD (MeOH, Δε) 211 (−3.086), 232 (+7.835) nm; IR (KBr) vmax 3467, 2966, 1734, 1384, 1239, 1033 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 807.2841 [M + Na]+ (calcd. for C40H48NaO16, 807.2835).
4.4. Spectroscopic data 4.4.1. Entanutilin C (1) white amorphous powder; [α]D25 -70.5 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 216 (4.05), 263 (3.41) nm; ECD (MeOH, Δε) 211 (−7.517), 243 (+1.610) nm; IR (KBr) vmax 3448, 1734, 1638, 1385, 1286, 1124, 1031 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 810.2972 [M + H]+ (calcd. for C41H48NO16, 810.2968).
4.4.10. Entanutilin L (10) white amorphous powder; [α]D25 +51.3 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 230 (4.28) nm; ECD (MeOH, Δε) 220 (+15.906), 238 (−17.860) nm; IR (KBr) vmax 3449, 1637, 1385, 1236, 1049 cm−1; 1 H and 13C NMR spectroscopic data, see Table 4; HRESIMS m/z 691.2720 [M + Na]+ (calcd. for C36H44NaO12, 691.2725).
4.42. Entanutilin D (2) white amorphous powder; [α]D25 -20.0 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 216 (3.83), 263 (3.18) nm; ECD (MeOH, Δε) 217 (−3.520), 246 (+0.758) nm nm; IR (KBr) vmax 3449, 1743, 1640, 1367, 1235, 1033 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 769.2679 [M + Na]+ (calcd. for C37H46NaO16, 769.2678).
4.4.11. Entanutilin M (11) white amorphous powder; [α]D25 +16.5 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 216 (3.93) nm; ECD (MeOH, Δε) 218 (+15.096), 9
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239 (−16.102) nm; IR (KBr) vmax 3460, 2977, 1745, 1387, 1237, 1047 cm−1; 1H and 13C NMR spectroscopic data, see Table 4; HRESIMS m/z 719.3035 [M + Na]+ (calcd. for C38H48NaO12, 719.3038).
doi.org/10.1016/j.phytochem.2020.112282.
4.4.12. Entanutilin N (12) white amorphous powder; [α]D25 -17.4 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 227 (4.03) nm; ECD (MeOH, Δε) 210 (+6.574), 230 (−16.701) nm; IR (KBr) vmax 3458, 2970, 1745, 1382, 1238, 1054 cm−1; 1H and 13C NMR spectroscopic data, see Table 4; HRESIMS m/z 707.3034 [M + Na]+ (calcd. for C37H48NaO12, 707.3038).
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Daniewski, W.M., Gumułka, M., Danikiewicz, W., Gluziński, P., Krajewski, J., Pankowska, E., Szafranski, F., 1993. Utilin B, a tetranortriterpenoid of the mexicanolide group from bark of Entandrophragma utile. Phytochemistry 33, 1534–1536. Daniewski, W.M., Gumułka, M., Danikiewicz, W., Gluziński, P., Krajewski, J., Sitkowski, J., Szafranski, F., 1994. A tetranortriterpenoid from the bark of Entandrophragma utile. Phytochemistry 36, 1001–1003. Daniewski, W.M., Gumułka, M., Danikiewicz, W., Sitkowski, J., Jacobsson, U., Norin, T., 1995. Entilin D, a heptanortriterpenoids from the bark of Entandrophragma utile. Phytochrmistry 40, 903–905. Hay, A.E., Ioset, J.R., Ahua, K.M., Diallo, D., Brun, R., Hostettmann, K., 2007. Limonoids orthoacetates and antiprotozoal compounds from the roots of Pseudocedrela kotschyi. J. Nat. Prod. 70, 9–13. John, T.A., Onabanjo, A.O., 1990a. Gastroprotective effects of an aqueous extract of Entandrophragma utile bark in experimental ethanol-induced peptic ulceration in mice and rats. J. Ethnopharmacol. 29, 87–93. John, T.A., Onabanjo, A.O., 1990b. Palazzo die Congressi. Italy, Florence. John, T.A., 1994. PhD Thesis University of Lagos, Lagos, Nigeria. John, T.A., Onabanjo, A.O., 2010. Effect of an aqueous extract of Entandrophragma utile bark on gastric acid secretion in rat and isolated ileum contractility in Guinea pig. Afr. J. Biomed. Res. 13, 197–206. Lin, B.D., Zhang, C.R., Yang, S.P., Wu, Y., Yue, J.M., 2011. Phragmalin-Type limonoid orthoesters from the twigs of Swietenia macrophylla. Chem. Pharm. Bull. 59, 458–465. Liu, H.B., Zhang, H., Li, P., Wu, Y., Gao, Z.B., Yue, J.M., 2012. Kv1.2 potassium channel inhibitors from Chukrasia tabularis. Org. Biomol. Chem. 10, 1448–1458. Luo, J., Tian, X.M., Zhang, H.J., Zhou, M.M., Li, J.H., Kong, L.Y., 2016. Two rare limonoids from the stem barks of Entandrophragma utile. Tetrahedron Lett. 57, 5334–5337. Mellor, H.R., Callaghan, R., 2008. Resistance to chemotherapy in cancer: a complex and integrated cellular response. Pharmacology 81, 275–300. Narender, T., Tanvir, K., Shweta, 2008. 13C NMR spectroscopy of D and B, D-ring secolimonoids of Meliaceae family. Nat. Prod. Res. 22, 763–800. Quasie, O., Li, H., Luo, J., Kong, L.Y., 2017. Two new phragmalin-type limonoids orthoesters from Entandrophragma candollei. Chin. J. Nat. Med. 15 0680-0683. Roy, A., Saraf, S., 2006. Limonoids: overview of significant bioactive triterpenes distributed in plants kingdom. Biol. Pharm. Bull. 29, 191–201. Steven, M.A., David, A.T., 1988. Limonoids from the seed of Entandrophragma caudatum. Phytochemistry 27, 1218–1220. Silva, M.N., Arruda, M.S.P., Castro, K.C.F., Silva, M.F.G.F., Fernandes, J.B., Vieira, P.C., 2008. Limonoids of the phragmalin type from Swietenia macrophylla and their chemotaxonomic significance. J. Nat. Prod. 71, 1983–1987. Tchouankeu, J.C., Tsamo, E., Sondengam, B.L., Connolly, J.D., Rycroft, D.S., 1990. Entilins A and B, two novel heptanortriterpenoid derivatives from Entandrophragma utile (meliaceae): structural elucidation using 2D NMR long-range δC/δH correlation experiments. Tetrahedron Lett. 31, 4505–4508. Tchouankeu, J.C., Nyasse, B., Tsamo, E., Sondengam, B.L., Morin, C., 1992. An ergostane derivative from the bark of Entandrophragma utile. Phytochemistry 31, 704–705. Tchouankeu, J.C., Nyasse, B., Tsamo, E., Connolly, J.D., 1996. 7α, 20(S)-Dihydroxy-4, 24(28)-ergostadien-3-one from Entandrophragma utile. J. Nat. Prod. 59, 958–959. Wang, X.N., Yin, S., Fan, C.Q., Wang, F.D., Lin, L.P., Ding, J., Yue, J.M., 2006. Turrapubesins A (I) and B (II), first examples of halogenated and maleimide-bearing limonoids in nature from Turraea pubescens. Org. Lett. 8, 3845–3848. Yin, S., Fan, C.Q., Wang, X.N., Lin, L.P., Ding, J., Yue, J.M., 2006. Xylogranatins A-D: novel tetranortriterpenoids with an unusual 9,10-seco scaffold from marine mangrove Xylocarpus grantum. Org. Lett. 8, 4935–4938. Zhang, F., Zhang, C.R., Tao, X., Wang, J., Chen, W.S., Yue, J.M., 2014. Phragmalin-type limonoids with NF-ҡB inhibition from Chukrasia tabularis var. velutina. Bioorg. Med. Chem. Lett 24, 3791–3796. Zhang, Q., Zheng, Q.H., Sang, Y.S., Sung, H.H.Y., Min, Z.D., 2018. New limonoids isolated from the bark of Melia toosendan. Chin. J. Nat. Med. 16 0946-0950.
References
4.5. Crystallographic data of 1 Colorless crystals of 1 were obtained from MeOH/H2O (1:1). Crystal data were obtained on a Bruker APEX-II CCD diffractometer with Cu Kα radiation (λ = 1.54178 Å) at 297 K. The structure was solved by direct methods with the ShelXT structure solution program using intrinsic phasing and refined with the ShelXL refinement package using least squares minimization. Crystal data of 1: C41H47NO16, M = 809.79, a = 10.9943 (11) Å, b = 18.8653 (19) Å, c = 18.9907 (19) Å, α = 90°, β = 90°, γ = 90°, V = 3938.9 (7) Å3, T = 297.24 K, space group P212121, Z = 4, μ(Cu Kα) = 0.889 mm−1, 19424 reflections measured, 7679 independent reflections (Rint = 0.0520). The final R1 values were 0.0514 (I > 2σ (I)). The wR (F2) values were 0.1310 (I > 2σ (I)). The final R1 values were 0.0585 (all data). The wR (F2) values were 0.1442 (all data). The goodness of fit on F2 was 1.051. Flack parameter: 0.06 (11). Crystallographic data for 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1905734). Copies of the data can be obtained free of charge via www.ccdc.cam.ac. uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, U.K. [fax (+44) 1223336-033; or e-mail: [email protected]]. 4.6. MDR reversal activity in MCF-7/DOX cells MCF-7/DOX cells (106 cells/mL) were plated in 96-well plates (5 × 103 cells per well). After incubation overnight under an atmosphere with 5% CO2 at 37 °C, the cells were treated with each test compound for 48 h and verapamil was used as the positive control. MTT reagent was added to each well and the plates were incubated for 4 h. The formed formazan crystals were dissolved by the addition of DMSO (150 μL) per well. After treatment, cell viability was evaluated, and the cytotoxicity of each isolated compound against MCF-7 cells was calculated by the MTT method. All experiments were performed in triplicate. Declaration of competing interest The authors declare no competing financial interest. Acknowledgements This research work was supported in part by the National Natural Science Foundation of China (31470416), the Outstanding Youth Fund of the Basic Research Program of Jiangsu Province (BK20160077), the 111 Project from Ministry of Education of China and the State Administration of Foreign Export Affairs of China (B18056), the “Double First-Class” University Project (CPU2018GY08), and the Drug Innovation Major Project (2018ZX09711-001-007). Appendix A. Supplementary data Supplementary data to this article can be found online at https://
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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Highly oxygenated and rearranged limonoids from the stem barks of Entandrophragma utile
T
Ya-Lin Hu, Xiao-Meng Tian, Cheng-Cheng Wang, Quasie Olga, Dan Yan, Peng-Fei Tang, Li-Na Zhang, Jun Luo∗∗, Ling-Yi Kong∗ Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, People's Republic of China
A RT ICLE INFO
ABSTRACT
Keywords: Entandrophragma utile Meliaceae Limonoids Orthesters CD exciton chirality Multidrug resistance (MDR)
Seventeen highly oxygenated and rearranged limonoids, including nine previously undescribed phragmalin-type limonoids with 1,8,9- and 8,9,30-orthesters (entanutilins C–K, 1–9), three undescribed limonoids with rare rearranged-6/6/7/5 skeleton (entanutilins L-N, 10–12), and 5 known limonoids, were isolated from the stem barks of Entandrophragma utile from Ghana (Africa). Their structures including absolute configurations were elucidated based on comprehensive spectroscopic analyses, such as HRESIMS, 1D/2D-NMR, CD exciton chirality method, time-dependent density functional theory (TDDFT)/ECD calculations, and single-crystal X-ray diffraction. Bioactivity screenings suggested that some of these compounds effectively reversed resistance in MCF-7/DOX cells at a nontoxic concentration of 30 μM with 6- to 19-fold enhancing effects.
1. Introduction Limonoids, mainly from plants of the Meliaceae family, have attracted a broad range of interests in both agrochemistry and organic chemistry because of their enchanting structural diversity and significant biological activities (Champagne et al., 1992; Hay et al., 2007; Narender et al., 2008; Roy and Saraf, 2006; Wang et al., 2006; Yin et al., 2006; Zhang et al., 2018). Of native African meliaceous species, plants from the Entandrophragma genus include not only an economically important tropical hardwood but also a Nigerian traditional medicine used to treat gastrointestinal ulcers and sickle cell disease (SCD) (Adejumo et al., 2011; John and Onabanjo, 1990a, 1990b; 2010; John, 1994; Quasie et al., 2017; Tchouankeu et al., 1990). Previous studies on Entandrophragma utile (Dawe & Sprague) Sprague have indicated that sterols and limonoids are the main secondary metabolites (Daniewski et al., 1993, 1995; Tchouankeu et al., 1990, 1992, 1996), and our previous chemical investigations on small amounts of the bark of E. utile led to the isolation of two rare limonoids (Luo et al., 2016). The above information encouraged us to further investigate this plant as part of our research on novel and bioactive limonoids from the Meliaceae family. As a result, nine previously undescribed phragmalin-type limonoids with 1,8,9- and 8,9,30-orthesters, three undescribed limonoids with a rare rearranged-6/6/7/5 skeleton, and five known limonoids
∗
were isolated and identified from 2.0 kg of stem bark of the title plant collected in Ghana (Africa). Herein, we describe the isolation, structural elucidation including the absolute configurations, and the multidrug resistance reversal activity of these limonoids on MCF-7/DOX cells. 2. Results and discussion 2.1. Structure elucidation Entanutilin C (1) was isolated as a white amorphous powder, and its molecular formula was determined to be C41H47NO16 based on its HRESIMS ion peak at m/z 810.2972 [M + H]+ (calcd for C41H48NO16: 810.2968) in conjunction with its 13C NMR spectrum, which displayed 41 carbon resonances. Careful analyses of its NMR spectra revealed characteristic resonances of a β-furan ring (δH 6.42, 7.40 and 7.45; δC 110.0, 120.9, 141.3 and 143.2), a nicotinyl moiety (δH 7.45, 8.74, 8.79, 9.56; δC 123.2, 126.9, 138.0, 151.5, 153.2, 165.3), one methoxy signal (δH 3.72; δC 52.2), and an orthoacetate system [δH 1.78 (3H, s); δC 119.5 (C-31)]. A pair of roofed doublets at δH 1.84 (d, J = 11.0 Hz) and 1.99 (d, J = 11.0 Hz) were assigned as the H-29 protons of the characteristic phragmalin 4,29,1-ring bridge, which was confirmed by the HMBC cross-peaks (Fig. 2) of C-29 (δC 39.9)/H-3 (δH 4.96) and Me-28 (δH 0.97), H-29 (δH 1.84)/C-5 (δC 36.3). These observations showed
Corresponding author. Corresponding author. E-mail addresses: [email protected] (J. Luo), [email protected] (L.-Y. Kong).
∗∗
https://doi.org/10.1016/j.phytochem.2020.112282 Received 29 September 2019; Received in revised form 19 January 2020; Accepted 22 January 2020 0031-9422/ © 2020 Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of compounds 1–17.
Fig. 2. Key HMBC and ROESY correlations of 1.
2
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Fig. 3. The CD spectrum of compound 1 in MeOH; the bold lines denote the electric transition dipole of the chromophores for 1.
that 1 was a phragmalin-type limonoid orthester (Lin et al., 2011). The obvious HMBC cross-peaks (Fig. 2) of H-3 (δH 4.96) and -OCOC5NH4 (δC 165.3), H-30 (δH 6.09) and -OCOCH(CH3)2 (δC 174.6), and H-12 (δH 4.53) and -OCOCH3 (δC 169.7) revealed that the nicotinyl moiety, the isobutyryl group and the acetyl group were attached to C-3, C-30 and C12, respectively. The location of the orthoacetate was determined to be 1/8/9-type based on the characteristic carbon resonances of C-1/2/8/9, although there were no direct HMBC correlations from the protons on the phragmalin skeleton to the orthoacetate carbon (C-31) (Lin et al., 2011). Thus, as shown in Fig. 1, the planar structure of 1 was determined as a phragmalin-type limonoid orthester with a rare nicotinyl moiety at C-3. The ROESY correlations (Fig. 2) of H-3/H-29 (δH 1.84), and H-29 (δH 1.99)/Me-19 and Me-32 indicated that these protons and groups were on the same side of the molecule, while the correlations (Fig. 2) of H-5/H-12, Me-28, and H-30; H-12/H-11 and H-17; and H-17/H-30 revealed that H-5, H-11, H-12, H-17, Me-28 and H-30 were on the other side. The positive chirality, resulting from the exciton coupling at 243 nm (Δε +1.610, nicotinyl moiety) and 211 nm (Δε −7.517, the furan ring) (Luo et al., 2016), can be observed in the ECD spectrum (Fig. 3), and thus the absolute configuration of 1 was elucidated as shown in Fig. 1. Finally, single crystals (Fig. 4) of 1 were obtained and subjected to X-ray diffraction using Cu Kα radiation. The high-quality crystal data [CCDD 1905734, Flack parameter of 0.06 (11)] not only confirmed the putative planar and relative structure but also the absolute configuration of 1 as 1R, 2S, 3S, 4R, 5S, 8R, 9R, 10R, 11R, 12R, 13R, 14R,17S, 30R. Entanutilin D (2) had a molecular formula of C37H46O16 as established from its HR-ESI-(+)-MS ion peak at m/z 769.2679 [M + Na]+ (calcd. 769.2678). The similarity of the NMR data (Table 1) of 2 and 1 indicated that 2 was also a pharagmalin-type limonoid with a 1/8/9orthoester. The OH-3 and OCOCH3-11 groups, the major difference between these two compounds, were revealed by the HMBC signals of δH 4.96 (H-3) to δC 14.1 (Me-28)/36.5 (C-5)/69.9 (C-30) and of δH 5.60 (H-11) to δC 169.4 (11-OCOCH3). The similar ROESY and ECD data (Fig. S85) indicated that the key asymmetric carbons of 2 and 1 had the same relative and absolute configurations. Thus, the absolute configuration of 2 was also elucidated as shown in Fig. 1. Entanutilin E (3) possessed a molecular formula of C35H42O15, based
Fig. 4. X-ray crystallographic structure of 1.
on analysis of its HRESIMS peak at m/z 725.2418 [M + Na]+. The 1H and 13C NMR data of 3 and 1 were almost the same, except for the presence of a double bond and the signals of the substituents. The HMBC correlations of δC 154.9 (C-14)/δH 5.91 (H-30) and δH 5.74 (H17) and of δH 6.81 (H-15)/δC 77.7 (C-8), δC 41.9 (C-13) and δC 164.3 (C16) suggested that the additional double bond was located at C-14/15. The HMBC correlations of δH 3.27 (OH-3)/δC 85.9 (C-3) and of δC 85.9 (C-3)/δH 1.86 (H-29) and δH 0.88 (Me-28) showed that OH-3 was free, which was another difference from 1. The relative configuration of 3 was determined to be identical to that of chubularisin B based on a detailed comparison of their ROESY data (Liu et al., 2012). The absolute configuration of compound 3 was deduced by the electronic circular dichroism (ECD) chirality method. The ECD spectrum (Fig. 5) acquired in MeOH was dominated by a negative Cotton 3
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Thus, the absolute configuration of 3, especially the 17-R conformation, was established as depicted in Fig. 1. Entanutilin F (4) displayed a molecular ion peak [M + Na]+ at m/z 709.2472 in its HRESIMS data, and this peak corresponded to a molecular formula of C35H42NaO14 (calcd. 709.2467). The NMR data of 4 resembled those reported for xyloccensin Q (Cui et al., 2005), with an 8/9/30-orthoacetate, except for the resonances of the acetyl and isobutyryl groups and the disappearance of the oxygenated carbon at C-6 in 4. The isobutyryl group in 4 was located at C-3 based on the HMBC correlation (Fig. 6) from H-3 (δH 3.74) and the two doublet methyl protons (δH 1.19 and 1.19) to the carbonyl signal at δC 176.6. Moreover, the methylene carbon at C-6 in 4 rather than the oxygenated carbon in xyloccensin Q was supported by the obvious correlations from H2-6 (δH 2.16, 3.11) to C-7 (δC 174.6) and C-4 (δC 44.5) in the HMBC spectrum (Cui et al., 2005). The ROESY correlations (Fig. 6) of H-29 (δH 2.70)/ Me-19 and H-3 and of Me-18/Me-19 and Me-32 showed that H-3, Me18, Me-19, H-29 and Me-32 were on the same face and they were arbitrarily assigned as α-oriented, whereas the ROESY interactions between H-5/H-12, Me-28, H-30 and H-12/H-17 suggested the β-orientations of H-5, H-12, H-17, Me-28 and H-30. Thus, the chemical structure of 4 was determined as shown. The NMR and HRMS data of 5 and 6 revealed that these two compounds were also phragmalin-type limonoids with 8/9/30-orthoacetate groups and C-14/15 double bonds as in 4. The proton resonance at δH 2.90, assignable to an –OH group, displayed HMBC correlations with C-3 (δC 86.9) and C-4 (δC 44.0), indicating the –OH group is at C-3 in 5 rather than at C-2 in 4. The above information and the characteristic 13C NMR data of C-2 (δC 84.0) indicated that the isobutyryl group was located at C-2 in 5 (Liu et al., 2012). The additional acetoxyl group in 6 was located at C-11 based on the HMBC correlations from δH 5.63 (H-11) to δC 169.9 (-OCOCH3), δC 42.2 (C-13) and δC 48.4 (C-10). The significant ROESY correlations of H-11/H-12 and H-12/H-5 suggested that 11-OCOCH3 was α-oriented; the other relative configurations of 6 and 5 were determined to be the same as that of 4. Thus, the structures of 5 and 6 were elucidated as shown. Entanutilin I (7), obtained as a white, amorphous powder, displayed a molecular formula of C35H42O15, as determined by its HRESIMS signal at m/z 725.2421 [M + Na]+ (calcd for 725.2416). Analysis of the MS and NMR data of 7 indicated that its structure was closely related to that of 12α-acetoxyswietephragmin D (Silva et al., 2008), a phragmalintype limonoid with 8,9,30-ortho-isobutyrate. OH-3 was free, as indicated by the characteristic upfield shifts of H-3 (δH 3.83) of 7 compared with H-3 (δH 4.83) of 12α-acetoxyswietephragmin D (Silva et al., 2008), respectively, and the HMBC correlations from H-3 (δH 3.83) to C-2 (δC 76.4), C-5 (δC 39.7) and C-28 (δC 14.5). An obvious HMBC correlation from H-11 (δH 5.68) to the carbon resonance at δC 170.3 (11-OCOCH3) indicated that OH-11 was acetylated as in 7. The relative configuration of 7 was assigned as the same as that of 6 based on ROESY data analysis (Silva et al., 2008). Thus, the chemical structure of 7 was determined as shown. The similarity of chromophores with △14,15α, β-unsaturated ketone and the furan ring and the ECD spectra (Fig. 5) of 3–7 indicated that the key asymmetric carbons of them had identical absolute configurations as shown in Fig. 1. Thus, the structures of 4–7 were finally elucidated. Entanutilins J (8) and K (9) were also obtained as amorphous powders, possessing molecular formulas of C41H45NO16 and C40H48O16 via analysis of their HRESIMS signals at m/z 808.2816 [M + H]+ and 807.2841 [M + Na]+, respectively. Analyses of the NMR data of 8 and 9 implied that they had the same phragmalin core as seen in 7, and the difference was the position of substituents. In compound 8, the location of the nicotinoyl moiety at C-3 was determined from the HMBC correlation between H-3 (δH 5.04) and -OCOC5NH4 (δC 165.8). For compound 9, the assignments of -OTig and –OCOCH3 at C-3 (δC 86.6) and C12 (δC 68.3), respectively, were determined based on the HMBC correlations of δH 4.82 (H-3)/δC 168.0 [3-OCOCH(CH3) = CHCH3] and δH 4.84 (H-12)/δC 170.6 (12-OCOCH3), respectively. One tertiary methyl
Table 1 1 H and 13C NMR spectroscopic data for 1–3 in CDCl3. No.
1 2 3 4 5 6
1a
2b
δH (mult; J, Hz)
4.96, s c
3.21 2.56, m 2.46, m
7 8 9 10 11 12 13 14
4.38, m 4.53, m 2.74, m 3.21c 2.70, m
15 16 17 18 19 20 21 22 23 28 29
5.44, s 1.42, s 3H 1.19, s 3H 7.45c 6.42, s 7.40, m 0.97, s 3H 1.99, d (11.0)
δC 86.0 80.4 84.6 45.3 36.3 33.7 172.4 85.4 86.2 45.6 69.7 71.9 38.6 42.5 26.8 169.9 76.7 15.9 16.7 120.9 141.3 110.0 143.2 14.1 39.9
1.84, d (11.0) 30 31 7-OCH3 R-31 OHs
6.09, s 3.72, s 3H CH3-31 1.78, s 3H
70.0 119.5 52.2 21.3
11-OAc
4.96, s 3.23, m 2.74, m 2.53, m
5.60, d (2.5) 4.60, d (2.5) 2.62, d (10.4) 3.27, m 2.78, m 5.45, s 1.34, s 3H 1.24, s 3H 7.40c 6.38, s 7.40c 0.97, s 3H 2.02, d (11.0) 1.82, d (11.0) 6.09, s 3.75, s 3H CH3-31 1.71, s 3H
2.12, s 3H
12-OAc
1.68, s, 3H
R-3
OCOC5NH4-3
R-30
δH (mult; J, Hz)
8.74, m 7.45c 8.79, m 9.56, m OCOCH(CH3)230 2.47, m 1.16, d (7.0) 0.88, d (7.0)
169.7; 20.3 165.3 126.9 138.0 123.2 153.2 151.5 174.6 34.6 19.2 17.9
1.62, s 3H
OCOCH (CH3)2-30 2.48, m 1.17, d (7.0) 0.89, d (7.0)
3a δC 85.5 80.5 84.9 45.2 36.5 33.7 172.8 86.0 85.8 45.7 69.3 70.2 38.5 43.3 26.9 169.3 76.6 15.7 16.4 120.7 141.4 109.8 143.4 14.1 40.0
69.9 119.6 52.3 21.3
169.4c; 20.9 169.4c; 20.0
174.9 34.6 19.2 17.9
δH (mult; J, Hz)
3.82, s 2.41, m 2.85, d (15.3) 2.40, m
4.30, s 4.84, s
6.81, s 5.74, s 1.75, s 3H 1.31, s 3H 7.48, s 6.59, s 7.38, s 0.88, s 3H 1.86, d (11.1) 1.75, m 5.91, s 3.73, s 3H CH3-31 1.68, s 3H OH-3: 3.27, s OH-11: 2.70, s 1.59, s 3H
OCOCH (CH3)2-30 2.60, m 1.26, d (7.0) 1.22, d (7.0)
δC 90.7 76.8 85.9 44.5 37.9 32.6 174.2 77.7 83.2 43.8 75.9 69.0 41.9 154.9 123.1 164.3 78.7 18.0 14.0 121.2 142.5 110.6 143.0 14.4 39.0
68.0 119.8 52.4 16.2
171.0; 20.0
175.3 34.2 19.8 18.8
a 1
H (600 MHz) and 13C (150 MHz) NMR data of compounds. H (500 MHz) and13C (125 MHz) NMR data of compound. Overlapped.
b 1 c
effect at 241 nm (△ε −3.690, △14,15α, β-unsaturated ketone) and a positive Cotton effect at 214 nm (△ε +4.262, the furan ring) due to the transition interaction between these two chromophores (Luo et al., 2016), which indicated an anticlockwise spatial relationship between these two chromophores. The calculated ECD spectrum by time-dependent density functional theory (TDDFT) (Bracher et al., 2004) for 3 matched fairly well with the experimental ECD spectrum (Fig. S1). 4
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Fig. 5. The CD spectra of compounds 3–7 in MeOH; the bold lines denote the electric transition dipole of the chromophores for 3.
Fig. 6. Key HMBC and ROESY correlations of 4.
group (δH 1.43, 3H) in 8 was replaced by an oxygenated carbon (δC 63.6) in 9, and the other –OCOCH3 group was located at C-19, similar to velutinasin A (Zhang et al., 2014), which was supported by the HMBC cross-peaks of δH 4.50 and 4.75 (H2-19)/δC 49.2 (C-10) and δC 170.2 (19-OCOCH3) and of δC 49.2 (C-10)/δH 3.35 (H-6), δH 2.33 (H29) and δH 2.00 (H-11). The oxygenated carbon (δC 67.5) at C-11 in 8 was replaced by a methylene carbon (δC 32.2) in 9, which was confirmed by the HMBC signals from H-11 (δH 2.00; 2.21) to C-9 (δC 85.9) and C-13 (δC 42.8). The ROESY spectrum showed that the relative configurations of compounds 8 and 9 were identical to that of compound 7. Thus, the structures of 8 and 9 were elucidated. Based on the
same two chromophores, the absolute configurations of 8 and 9 were elucidated to be the same as those of 1 from their similar ECD spectra (Fig. S85). The calculated ECD data of 8 (Fig. S2) matched the experimental ECD further supporting the assignment of absolute configuration of 8 and 9 as depicted. Compounds 10–12 were isolated as white powders, and their molecular formulas were determined to be C36H44O12, C38H48O12 and C37H48O12, respectively, based on their HRESIMS data. The NMR data (Table 4) of 10–12 were similar to each other but notably different from those of 1–9, indicating that 10–12 should possess a different limonoid skeleton. Three pairs of coupled, down-shifted protons at δH 4–6, two 5
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Table 2 1 H NMR Spectroscopic Data for 4–9 in CDCl3 (δ in ppm, J Values in Hz). No.
4b
3 5 6
3.74, 2.12, 3.11, 2.16, 2.31, 1.96, 4.80, 6.59, 5.89, 1.57, 1.23, 7.42, 6.54, 7.37, 0.87, 2.70, 2.32, 4.56, 1.68,
11 12 15 17 18 19 21 22 23 28 29 30 32 33 34 OHs 11-OAc 12-OAc 19-OAc 7-OCH3 R-2 or 3
5b
6b
7a
8a
s m d (16.7) m m t (13.8) dd (13.8, 4.1) s s s 3H s 3H
3.73, s 2.21, m 3.10, d (16.3) 2.30, m 2.22, m 1.94, m 4.84, dd (13.5, 4.2) 6.55c 5.92, s 1.55, s 3H 1.28, s 3H
3.73c 2.26, m 3.07, d (16.1) 2.32, m 5.63, d (3.9)
3.83, 2.29, 3.11, 2.30, 5.68,
s m d (14.9) m m
5.04, 2.85, 3.22, 2.43, 5.71,
m d (12.1) d (17.0) dd (17.0, 12.1) d (3.8)
4.96, d (3.9) 6.56c 5.93, s 1.68, s 3H 1.36, s 3H
4.89, 6.63, 5.87, 1.67, 1.35,
m s s s, 3H s, 3H
4.91, 5.89, 5.84, 1.56, 1.43,
d (3.8) s s s, 3H s, 3H
s s s s 3H d (11.9) m s s 3H
7.46, s 6.55c 7.37, s 0.87, s 3H 1.91, m 1.48, m 5.25, s 1.67, s 3H
7.48, s 6.56c 7.37, s 0.88, s 3H 1.94, d (11.4) 1.51, m 5.21, s 1.68, s 3H
OH-1: 3.25, s OH-3: 2.90, d (5.0)
OH-1: 3.21, s
7.42, s 6.55, s 7.38, m 0.84, s,3H 1.81, d (11.0) 1.64, m 4.63, s 2.18, m 1.09c 1.09c OH-1: 3.19, s
1.50, s 3H
1.50, s 3H
2.07, s 3H 1.47, s, 3H
1.47, s 3H 2.04, s 3H
3.72, s 3H OCOCH(CH3)2-3 2.54, p (6.9) 1.19c 1.19c
3.72, s 3H OCOCH(CH3)2-2 2.69, m 1.21, s 3H 1.19, s 3H
3.73, s, 3H OCOCH(CH3)2-2 2.70, p (6.9) 1.21c 1.21c
3.73, s, 3H
9b
7.49, t (1.2) 6.48, m 7.38, d (1.8) 0.93, s,3H 1.97, d (11.5) 1.86, dd (11.5, 1.5) 4.54, s 2.17, p (6.9) 1.07c 1.07c OH-1: 3.30, s OH-2: 3.65, s 2.03, s 3H 1.48, s, 3H 3.82, s, 3H OCOC5NH4-3 8.35, dd (8.0, 2.0) 7.42, dd (8.0, 5.0) 8.81, d (5.0) 9.28, d (2.0)
4.82, s 2.61, d (12.3) 3.35, d (16.5) 2.51, m 2.21, m 2.00, m 4.84, m 6.02, s 5.86, s 1.52, s, 3H 4.75, d (12.7) 4.50, m 7.41, brt (1.7) 6.54, d (1.8) 7.44, s 0.85, s,3H 2.33, d (11.2) 1.84, m 4.50, m 2.21, m 1.05c 1.05c OH-1: 3.52, s 1.52, s 3H 2.09, s 3H 3.76, s, 3H OTig-3 6.96, dd (7.0, 1.6) 1.84, s, 3H 1.73, d (7.0)
a 1
H (600 MHz) NMR data of compound. H (500 MHz) NMR data of compounds. Overlapped.
b 1 c
geminal methylenes at δH 1.5–2.5, and characteristic signals for a βsubstituted butyrolactone ring suggested that these three compounds should possess a rare, highly rearranged limonoid skeleton, similar to that of entanutilin A (13) (Luo et al., 2016). Me-19 was incorporated into the C-8/9/10/19-cyclobutyl ring, and Me-30 was inserted between C-8 and C-14 to form cycloheptyl ring C (Luo et al., 2016). Taking 10 as an example (Fig. 7), this structural assignment was confirmed by the key HMBC cross-peaks from H2-19 (δH 2.00) to C-7 (δC 72.9) and from H2-30 (δH 2.54; 2.43) to C-7 (δC 72.9), C-8 (δC 44.0), C-13 (δC 51.5), C14 (δC 175.0) and C-15 (δC 134.6). The obvious HMBC correlations between C-3 (δC 202.5) and H-1 (δH 6.08), H-5 (δH 2.43), Me-28/29 (δH 1.03; 1.07) indicated that C-3 in 10 was oxidized to the ketone. For compound 11, the only difference from 10 was that the acetoxy group at C-6 in 10 was replaced by an isobutyryl group in 11, which was confirmed by the obvious HMBC correlation from H-6 (δH 5.69) and the two doublet methyl protons (δH 1.15 and 1.17, J = 7.0 Hz) to the carbonyl signal at δC 175.8. The NMR spectra revealed that 12 was more similar to entanutilin A (13) (Luo et al., 2016) as it possessed a C–OH moiety rather than a C]O at C-3, which was confirmed by the HMBC cross-peaks between C-3 (δC 73.7) and H-1 (δH 5.24), Me-28/29 (δH 0.93; 0.80). The isovaleryl group at C-7 in 12 was assigned based on the HMBC correlations from H-7 (δH 5.49) and the methylene protons at δH 1.64 and 1.82 [-OCOCH2CH(CH3)2] to the carbonyl signal at δC 176.3 [-OCOCH2CH(CH3)2]. At this stage, the planar structures of 10–12 were determined, and they featured a highly rearranged framework with a cyclobutane and a cycloheptane rings. Their ROESY spectra indicated that the relative configurations of their basic skeletons were the same as those of entanutilin A (13) (Luo et al., 2016), and the key correlations of 10 are shown in Fig. 7. For compound 12, the ROESY correlation of H-3/Me-29 revealed the α-
orientation of the additional OH-3 moiety. The ECD spectra of 10–12 exhibited perfect Cotton effects, which can be used to determine their absolute configurations by the CD chirality method. Taking compound 10 as an example (Fig. 8), the negative chirality in the ECD spectrum at 238 nm (△ε −17.860, △14,15 α, β-unsaturated ketone) and 220 nm (△ε +15.906, △1,2 α, β-unsaturated ketone) indicated the anticlockwise spatial relationship between these two chromophores and the calculated ECD spectrum for 10 matched fairly well with the experimental ECD spectrum (Fig. S3). Thus, the absolute configuration of 10 was defined as 5R, 6R, 7S, 8S, 9R, 10S, 11S, 12R, 13S, 17S. The ECD spectra of 11 and 12 in MeOH were highly similar to 10 (Fig. 8), so the absolute configurations of 11 and 12 were assigned as shown in Fig. 1. The five reported compounds were identified as entanutilin A (13) (Luo et al., 2016), 12α-acetoxyphragmalin-3-nicotinate-30-isobutyrate (14) (Steven and David, 1988), entanutilin B (15) (Luo et al., 2016), utilin C (16) (Daniewski et al., 1994) and utilin B (17) (Daniewski et al., 1993) by comparison of their spectroscopic data with reported data. 2.2. MDR reversal activities One key obstacle in cancer treatment is the intrinsic or acquired drug resistance of certain cancers, and significant efforts have been made in clinical oncology to provide solutions to this resistance (Mellor and Callaghan, 2008). The mexicanolide-type limonoid, trichisin H from the fruits of Heynea trijuga, effectively reversed multidrug resistance in doxorubicin-resistant human breast tumour (MCF-7/DOX) cells (An et al., 2018). Therefore, all compounds (1–17) were evaluated for their MDR reversal activities on MCF-7/DOX cell line with verapamil at 10 μM as a positive control (Table 5). At 30 μM, limonoids 13, 15, 16 and 17 showed reversal fold (RF) values of 6.25, 6.52, 19.15 and 6
Phytochemistry 172 (2020) 112282
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Table 3 13 C NMR Spectroscopic Data for 4–9 in CDCl3 (δ in ppm).
Table 4 1 H and 13C NMR spectroscopic data for 10–12 in CDCl3.
No.
4b
5b
6b
7a
8a
9a
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 31 32 33 34 11-OAc
84.6 78.2 85.4 44.5 38.5 32.8 174.6 83.7 89.1 49.6 33.0 69.2 43.0 152.9 124.0 164.2 78.8 14.5 15.8 121.6 142.1 110.5 143.0 14.7 38.6 77.7 119.6 16.9
85.6 84.0 86.9 44.0 39.1 33.3 174.4 84.2 86.2 48.4 32.8 69.1 43.0 152.3 124.0 163.9 78.7 14.6 15.5 121.5 142.2 110.5 142.9 14.8 40.1 75.0 119.7 16.7
85.0 84.6 87.0 43.9 39.2 33.4 174.4 84.2 85.4 48.4 67.7 69.2 42.2 150.9 124.0 163.6 78.8 15.5 15.9 121.3 142.5 110.4 143.0 14.8 40.3 75.2 120.1 16.1
170.7; 20.0
170.5; 20.0
84.9 75.8 87.8 43.9 41.0 33.2 174.9 83.9 85.0 47.7 67.5 68.9 42.0 151.1 123.5 161.5 78.5 15.4 16.1 121.0 142.3 110.3 143.2 14.5 39.3 78.1 123.2 29.6 17.3 17.1 170.2; 21.0 170.3; 19.6
84.6 75.8 86.6 43.6 41.4 31.9 174.6 83.9 85.9 49.2 32.2 68.3 42.8 150.9 124.4 162.4 78.2 14.7 63.6 121.3 143.3 110.4 142.0 14.7 39.2 78.0 122.9 29.1 16.9 16.9
12-OAc
169.9; 20.9 170.2; 19.6
85.2 76.4 85.9 44.4 39.7 33.2 174.7 84.3 84.6 47.7 67.7 69.3 42.0 150.7 124.3 164.1 78.9 15.2 16.0 121.4 142.3 110.4 143.1 14.5 39.3 78.7 123.0 29.7 17.4 17.2 170.3c; 19.6 170.3c; 21.1
52.3 OCOCH (CH3)2-3 176.6 35.7 19.0 19.2
52.2 OCOCH (CH3)2-2 178.3 34.6 18.9 18.9
52.4 OCOCH (CH3)2-2 178.4 34.6 18.9c 18.9c
52.4
52.8 OCOC5NH4-3
19-OAc 7-OCH3 R-2 or 3
165.8 125.9 137.5 124.1 154.7 150.8
No.
1 2 3 4 5 6
c
δH (mult; J, Hz)
δC
δH (mult; J, Hz)
δC
6.08, d (10.3) 5.86, d (10.3)
145.6
6.08, m
145.7
5.24, d (10.0)
131.9
127.0
5.85,d (10.3)
127.0
5.80, dd (10.0, 5.9) 3.46, d (5.9)
128.1
4.83, m 4.86, m 6.08c 3.30, d (4.9) 1.14, s 3H 2.00c 2.00c 2.77c 4.43c 4.43c 2.77c 2.72, m
20 21 22 23 28 29 30 6-OAc
1.03, s 3H 1.07, s 3H 2.54, d (13.4) 2.43c 2.01, s, 3H
11-OAc
1.99, s, 3H
12-OAc
2.12, s, 3H
R-6
a 13 b
δC
2.82, dd (10.6, 4.7)
12 13 14 15 16 17 18 19
C (150 MHz) NMR data of compounds. 13 C (125 MHz) NMR data of compounds. Overlapped.
12.30, respectively, against MCF-7/DOX cells.
R-7
3. Conclusion
OCOCH (CH3)2-7 2.77c 1.30, d (7.0) 1.33, d (7.0)
In this study, twelve previously undescribed highly oxygenated and rearranged limonoids, including nine undescribed phragmalin-type limonoids with 1,8,9- and 8,9,30-orthesters (Entanutilins C–K, 1–9) and three undescribed limonoids with rare rearranged-6/6/7/5 skeleton (Entanutilins L-N, 10–12), were isolated from the stem barks of Entandrophragma utile. Moreover, bioactivity screenings suggested that some of these compounds effectively reversed resistance in MCF-7/DOX cells at a nontoxic concentration of 30 μM with 6- to 19-fold enhancing effects.
12a
δH (mult; J, Hz)
2.43 5.68, dd (9.6, 6.8) 5.54, d (6.8)
10 11
168.0 130.2 140.2 12.6 14.5
11b
c
7 8 9
170.6; 20.0 170.2; 21.4 52.5 OTig-3
10a
202.5 45.3 49.5 65.5 72.9 44.0 54.8 40.8 65.7 75.2 51.5 175.0 134.6 205.4 52.6 22.6 20.6c 34.9 72.1 32.3 176.9 21.4 21.6 33.5 169.9; 21.1 170.6; 29.6 169.4; 20.6c
176.5 34.6 19.1 19.5
2.46, m 5.69, dd (9.6, 6.8) 5.58, d (6.8) 2.83, dd (10.4, 4.8) 4.84, m 4.86, m 6.07, m 3.29, d (4.7) 1.13, s, 3H 2.01, m 1.54, m 2.74, m 4.43, m 4.42, m 2.75, m 2.71, m 1.02, s,3H 1.07, s 3H 2.50, m
202.5 41.0 49.6 65.5 72.9 44.1 54.6 45.4 65.8 75.2 51.6 175.1 134.6 205.3 52.6 22.5 29.7
2.12, s, 3H OCOCH (CH3)2-6 2.45, m 1.17, d (7.0) 1.15, d (7.0) OCOCH (CH3)2-7 2.75, m 1.35, d (7.1) 1.27, d (7.1)
2.81, dd (11.0, 5.3) 4.76, dd (11.0, 8.4) 4.86, d (8.4) 6.03, s
32.3
3.30, d (5.0) 1.11, s 3H 1.81, m 1.76, m 2.71, m 4.42c 4.42c 2.70, m
176.9 21.8 21.6 33.5
0.93, s 3H 0.80, s 3H 2.51, d (13.2)
34.9 72.1
2.44, m 1.99, s 3H
2.38, m 5.55, dd (9.9, 6.6) 5.49, d (6.6)
2.39, m 1.99c 170.5; 20.6c 169.4; 20.6c 175.8 34.2 18.8 18.6 176.3 34.5 19.6 18.9
1.99c 2.11, s 3H
OCOCH2CH (CH3)2-7 1.82, 1.64, 2.61, 1.00, 1.27,
m m q (6.9) t (7.4) d (6.9)
73.7 37.4 44.8 66.3 73.8 43.6 54.7 40.9 66.0 75.4 51.6 175.7 134.4 205.7 52.6 22.5 29.8 34.9 72.2 32.3 177.1 24.7 20.7 33.7 170.2; 20.8 170.6; 21.2 169.4; 20.7
176.3 27.0 41.8 12.1 17.1
a 1
H (600 MHz) and 13C (150 MHz) NMR data of compounds. H (500 MHz) and 13C (125 MHz) NMR data of compound. Overlapped.
b 1 c
Tensor 27 spectrometer (Bruker, Karlsruhe, Germany) and a Shimadzu UV-2450 spectrophotometer (Shimadzu, Tokyo, Japan), respectively. NMR spectra were acquired on Bruker AVIII-500 and AVIII-600 NMR instruments at 500 (1H) and 125 MHz (13C) and 600 MHz (1H) and 150 MHz (13C), respectively, using tetramethylsilane (TMS) as the internal standard. HRESI mass spectra were acquired using an Agilent 6520B UPLC-Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). ECD spectra were recorded on a JASCO J-810 spectropolarimeter (Jasco, Tokyo, Japan). RP-C18 (40–63 μm, FuJi), MPLC
4. Experimental 4.1. General experimental procedures Optical rotations were acquired on a JASCO P-1020 polarimeter (Jasco, Tokyo, Japan). IR and UV spectra were recorded on a Bruker 7
Phytochemistry 172 (2020) 112282
Y.-L. Hu, et al.
Fig. 7. Key HMBC and ROESY correlations of 10.
Fig. 8. The CD spectra of compounds 10–12 in MeOH; the bold lines denote the electric transition dipole of the chromophores for 10.
(Beijing H&E Co., Ltd., Beijing, China), MCI gel (Mitsubishi Chemical Corp., Tokyo, Japan), Sephadex LH-20 (Pharmacia, Germany), and silica gel (100–200 mesh and 200–300 mesh, Qingdao Haiyang Chemical Co., Ltd.) were used for column chromatography. Analytical HPLC separations were carried out using an Agilent 1260 Series instrument using a DAD with a shim-pack VP-ODS column (250 × 4.6 mm, 5 μm). Semipreparative HPLC separations were conducted on a Shimadzu LC6AD instrument using an SPD-10A detector with a shim-pack RP-C18 column (20 × 200 mm, 10 μm).
Table 5 MDR reversal ability of compounds on MCF-7/DOX cells. Sample 13 15 16 17 VER
d
a
DOX+ 0 μM 12.561 12.561 12.561 12.561 12.561
± ± ± ± ±
b
3.215 3.215 3.215 3.215 3.215
c
DOX+30 μM
RF
2.010 1.926 0.656 1.021
6.25 6.52 19.15 12.30 11.60
± ± ± ±
0.309 0.112 0.231 0.278
a Serial dilutions ranging from 3.12 to 100 μM of DOX with or without sample. b Values are expressed as means ± SD. c RF = IC50 of DOX alone/IC50 of DOX with samples. d Verapamil was used as positive control at 10 μM.
4.2. Plant material The air-dried stem barks of Entandrophragma utile (Dawe & Sprague) Sprague (Meliaceae) were collected from Brong Ahafo Region of Ghana, in August 2015 and identified by Professor Mian Zhang of the Research Department of Pharmacognosy, China Pharmaceutical University. A 8
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voucher specimen (No. 2015-EU) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
4.4.3. Entanutilin E (3) white amorphous powder; [α]D25 +50.6 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 208 (4.25) nm; ECD (MeOH, Δε) 214 (+4.262), 241 (−3.690), 268 (+1.934) nm; IR (KBr) vmax 3445, 1729, 1639, 1385, 1236, 1026 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 725.2418 [M + Na]+ (calcd. for C35H42NaO15, 725.2416).
4.3. Extraction and isolation The air-dried stem bark (2.0 kg) was exhaustively extracted by refluxing with 95% ethanol three times and then cooling to room temperature. After concentration under reduced pressure, the crude extract was suspended in H2O and successively partitioned between petroleum ether and CH2Cl2. The CH2Cl2 extract (20 g) was loaded into a silica gel column and then eluted with a CH2Cl2–MeOH gradient (100:1 to 0:100, v/v), which gave five fractions (Frs. A-E) based on TLC and HPLC analysis. Fr. B (3.0 g) was separated on sequential silica gel and Sephadex LH-20 gel columns to give four subfractions (Frs. B1-4). Fr. B2 (1.1 g) was subjected to an MCI gel column (MeOH–H2O, 20:80 to 0:100, v/v) to yield three subfractions (Frs. B2a-c). Fraction B2a (60.0 mg) was separated by semipreparative HPLC with MeOH–H2O (49:51 v/v, 10 mL/min) as the mobile phase to give compound 1 (2.4 mg, tR = 10.5 min). Fr. B2c (101 mg) was separated by semipreparative HPLC using MeOH–H2O (45:55 v/v, 10 mL/min) as the mobile phase to give compound 3 (1.4 mg, tR = 12.5 min). Fr. B3 (220 mg) was also separated by semipreparative HPLC with 65% methanol in water as the eluent to give compound 8 (2.0 mg, tR = 18.2 min) and a mixture containing compounds 4 and 5. This mixture was purified using CH3CN–H2O (34:66, v/v, 10 mL/min) as the mobile phase to afford compounds 4 (4.8 mg, tR = 22.3 min) and 5 (4.2 mg, tR = 26.0 min). Fr. C (4.5 g) was separated on an ODS column using a gradient of CH3CN/H2O (10:90 to 100:0) to yield four subfractions (Frs. C1-4). Fr. C2 (2.0 g) was separated on an ODS column using a step gradient of MeOH–H2O (20:80 to 100:0, v/v), to afford four subfractions (Frs. C2a-d). Fr. C2a (860 mg) was repeatedly purified using semipreparative HPLC with a C18 column using CH3CN–H2O (38:62 v/v, 10 mL/min) as the mobile phase to afford compounds 2 (2.0 mg, tR = 9.6 min), 7 (6.0 mg, tR = 18.5 min) and 9 (1.8 mg, tR = 26.8 min). Fr. C2b (400 mg) was separated by semipreparative HPLC, with 48% methanol in water, to give compound 14 (15.0 mg, tR = 17.5 min) and a mixture containing compounds 10, 12 and 13. This mixture was purified using CH3CN–H2O (35:65, v/v, 10 mL/min) as the mobile phase to yield compounds 10 (2.1 mg, tR = 10.5 min), 12 (4.2 mg, tR = 16.9 min) and 13 (4.6 mg, tR = 21.5 min). Fr. D (3.2 g) was separated on a silica gel column eluted with a gradient of petroleum ether/acetone (20:1 to 1:2, v/v) to give four subfractions (Frs. D14). Fr. D2 (1.0 g) was subjected to an RP-C18 column using a gradient of MeOH–H2O (30:70 to 100:0, v/v), to afford three subfractions (Frs. D2a-c). Fr. D2b (85 mg) was purified by semipreparative HPLC (MeOH/ H2O 64:36, v/v) to afford compounds 6 (3.2 mg, tR = 39.8 min), and 15 (5.1 mg, tR = 45.6 min). Frs. D2c (50 mg) was separated by semipreparative HPLC with 49% methanol in water as the eluent to give compounds 11 (3.9 mg, tR = 19.8 min), 16 (16.0 mg, tR = 22.6 min) and 17 (10.0 mg, tR = 38.9 min).
4.4.4. Entanutilin F (4) white amorphous powder; [α]D25 +70.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 213 (4.02) nm; ECD (MeOH, Δε) 219 (+8.491), 261 (+2.195) nm; IR (KBr) vmax 3459, 2966, 1728, 1241, 1155, 1023 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 709.2472 [M + Na]+ (calcd. for C35H42NaO14, 709.2467). 4.4.5. Entanutilin G (5) white amorphous powder; [α]D25 +40.9 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 214 (4.10) nm; ECD (MeOH, Δε) 214 (+28.639), 261 (+7.053) nm; IR (KBr) vmax 3459, 2976, 1720, 1240, 1161, 1022 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 709.2468 [M + Na]+ (calcd. for C35H42NaO14, 709.2467). 4.4.6. Entanutilin H (6) white amorphous powder; [α]D25 +59.4 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 214 (4.10) nm; ECD (MeOH, Δε) 214 (+21.395), 261 (+5.328) nm; IR (KBr) vmax 3439, 2962, 1728, 1641, 1386, 1247, 1164, 1028 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 767.2523 [M + Na]+ (calcd. for C37H44NaO16, 767.2522). 4.4.7. Entanutilin I (7) White amorphous powder; [α]D25 +46.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 198 (2.71) nm; ECD (MeOH, Δε) 219 (+5.466), 264 (+1.763) nm; IR (KBr) vmax 3447, 1728, 1641, 1386, 1245, 1026 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 725.2421 [M + Na]+ (calcd. for C35H42NaO15, 725.2416). 4.4.8. Entanutilin J (8) white amorphous powder; [α]D25 +18.5 (c 0.26, MeOH); UV (MeOH) λmax (log ε) 214 (4.31), 263 (3.51) nm; ECD (MeOH, Δε) 205 (−17.732), 231 (+53.915) nm; IR (KBr) vmax 3461, 2980, 1740, 1636, 1478, 1384, 1278, 1025 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 808.2816 [M + H]+ (calcd. for C41H46NO16, 808.2811). 4.4.9. Entanutilin K (9) white amorphous powder; [α]D25 +50.0 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 213 (4.16) nm; ECD (MeOH, Δε) 211 (−3.086), 232 (+7.835) nm; IR (KBr) vmax 3467, 2966, 1734, 1384, 1239, 1033 cm−1; 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HRESIMS m/z 807.2841 [M + Na]+ (calcd. for C40H48NaO16, 807.2835).
4.4. Spectroscopic data 4.4.1. Entanutilin C (1) white amorphous powder; [α]D25 -70.5 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 216 (4.05), 263 (3.41) nm; ECD (MeOH, Δε) 211 (−7.517), 243 (+1.610) nm; IR (KBr) vmax 3448, 1734, 1638, 1385, 1286, 1124, 1031 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 810.2972 [M + H]+ (calcd. for C41H48NO16, 810.2968).
4.4.10. Entanutilin L (10) white amorphous powder; [α]D25 +51.3 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 230 (4.28) nm; ECD (MeOH, Δε) 220 (+15.906), 238 (−17.860) nm; IR (KBr) vmax 3449, 1637, 1385, 1236, 1049 cm−1; 1 H and 13C NMR spectroscopic data, see Table 4; HRESIMS m/z 691.2720 [M + Na]+ (calcd. for C36H44NaO12, 691.2725).
4.42. Entanutilin D (2) white amorphous powder; [α]D25 -20.0 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 216 (3.83), 263 (3.18) nm; ECD (MeOH, Δε) 217 (−3.520), 246 (+0.758) nm nm; IR (KBr) vmax 3449, 1743, 1640, 1367, 1235, 1033 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 769.2679 [M + Na]+ (calcd. for C37H46NaO16, 769.2678).
4.4.11. Entanutilin M (11) white amorphous powder; [α]D25 +16.5 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 216 (3.93) nm; ECD (MeOH, Δε) 218 (+15.096), 9
Phytochemistry 172 (2020) 112282
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239 (−16.102) nm; IR (KBr) vmax 3460, 2977, 1745, 1387, 1237, 1047 cm−1; 1H and 13C NMR spectroscopic data, see Table 4; HRESIMS m/z 719.3035 [M + Na]+ (calcd. for C38H48NaO12, 719.3038).
doi.org/10.1016/j.phytochem.2020.112282.
4.4.12. Entanutilin N (12) white amorphous powder; [α]D25 -17.4 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 227 (4.03) nm; ECD (MeOH, Δε) 210 (+6.574), 230 (−16.701) nm; IR (KBr) vmax 3458, 2970, 1745, 1382, 1238, 1054 cm−1; 1H and 13C NMR spectroscopic data, see Table 4; HRESIMS m/z 707.3034 [M + Na]+ (calcd. for C37H48NaO12, 707.3038).
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References
4.5. Crystallographic data of 1 Colorless crystals of 1 were obtained from MeOH/H2O (1:1). Crystal data were obtained on a Bruker APEX-II CCD diffractometer with Cu Kα radiation (λ = 1.54178 Å) at 297 K. The structure was solved by direct methods with the ShelXT structure solution program using intrinsic phasing and refined with the ShelXL refinement package using least squares minimization. Crystal data of 1: C41H47NO16, M = 809.79, a = 10.9943 (11) Å, b = 18.8653 (19) Å, c = 18.9907 (19) Å, α = 90°, β = 90°, γ = 90°, V = 3938.9 (7) Å3, T = 297.24 K, space group P212121, Z = 4, μ(Cu Kα) = 0.889 mm−1, 19424 reflections measured, 7679 independent reflections (Rint = 0.0520). The final R1 values were 0.0514 (I > 2σ (I)). The wR (F2) values were 0.1310 (I > 2σ (I)). The final R1 values were 0.0585 (all data). The wR (F2) values were 0.1442 (all data). The goodness of fit on F2 was 1.051. Flack parameter: 0.06 (11). Crystallographic data for 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1905734). Copies of the data can be obtained free of charge via www.ccdc.cam.ac. uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, U.K. [fax (+44) 1223336-033; or e-mail: [email protected]]. 4.6. MDR reversal activity in MCF-7/DOX cells MCF-7/DOX cells (106 cells/mL) were plated in 96-well plates (5 × 103 cells per well). After incubation overnight under an atmosphere with 5% CO2 at 37 °C, the cells were treated with each test compound for 48 h and verapamil was used as the positive control. MTT reagent was added to each well and the plates were incubated for 4 h. The formed formazan crystals were dissolved by the addition of DMSO (150 μL) per well. After treatment, cell viability was evaluated, and the cytotoxicity of each isolated compound against MCF-7 cells was calculated by the MTT method. All experiments were performed in triplicate. Declaration of competing interest The authors declare no competing financial interest. Acknowledgements This research work was supported in part by the National Natural Science Foundation of China (31470416), the Outstanding Youth Fund of the Basic Research Program of Jiangsu Province (BK20160077), the 111 Project from Ministry of Education of China and the State Administration of Foreign Export Affairs of China (B18056), the “Double First-Class” University Project (CPU2018GY08), and the Drug Innovation Major Project (2018ZX09711-001-007). Appendix A. Supplementary data Supplementary data to this article can be found online at https://
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