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Synthesis of 13-amino telekin derivatives and their cytotoxic activity a
a
a
a
Xiujie Wang , Xiumei Zhang , Beibei Zheng , Nan Hu , Weidong ab
Xie
& Kyungho Row
b
a
Department of Pharmacy, College of Marine Science, Shandong University, Weihai 264209, P.R. China b
Department of Chemical Engineering, Inha University, Incheon 402-751, South Korea Published online: 11 Dec 2014.
Click for updates To cite this article: Xiujie Wang, Xiumei Zhang, Beibei Zheng, Nan Hu, Weidong Xie & Kyungho Row (2015) Synthesis of 13-amino telekin derivatives and their cytotoxic activity, Natural Product Research: Formerly Natural Product Letters, 29:8, 756-763, DOI: 10.1080/14786419.2014.987143 To link to this article: http://dx.doi.org/10.1080/14786419.2014.987143
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Natural Product Research, 2015 Vol. 29, No. 8, 756–763, http://dx.doi.org/10.1080/14786419.2014.987143
Synthesis of 13-amino telekin derivatives and their cytotoxic activity Xiujie Wanga, Xiumei Zhanga, Beibei Zhenga, Nan Hua, Weidong Xiea,b* and Kyungho Rowb* a
Department of Pharmacy, College of Marine Science, Shandong University, Weihai 264209, P.R. China; Department of Chemical Engineering, Inha University, Incheon 402-751, South Korea
b
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(Received 8 September 2014; final version received 6 November 2014)
O O
O
Amine
O
Michael addition reaction OH
OH
NR1R2
Telekin Telekin is a eudesmane sesquiterpene-lactone naturally occurring in many medicinal plants with antitumour and anti-inflammatory activity. In this study, a series of 13amino derivatives of telekin have been synthesised through Michael addition reaction, and their relative configurations were exemplified by the single crystal X-ray diffraction of the dimethylamine adduct. The in vitro cytotoxicity against three tumour cell lines of these amine derivatives was evaluated. The piperidine and 4-hydroxypiperidine adducts displayed stronger cytotoxic activity than telekin. Keywords: telekin; Michael addition reaction; sesquiterpene lactone; cytotoxicity; eudesmane
1. Introduction Sesquiterpene lactones (SLs) are common secondary metabolites of plants in the Asteraceae family, some of which are often used in traditional medicine against inflammation and cancer (Mew et al. 1982; Zdero & Bohlmann 1990; Ghantous et al. 2010). In recent years, the anticancer property of various SLs has attracted a great deal of interests, and extensive research work has been carried out to characterise the anticancer activity, the structure – bioactivity relationship, the molecular mechanisms and so on (Zhang et al. 2005; Ghantous et al. 2010). Though, the poor water-solubility and extensive interactions with plasma proteins preclude most SLs further development as therapeutic agents (Ghantous et al. 2010). Up to now, only artemisinin, thapsigargin, parthenolide and some of their synthetic derivatives have been developed to be lead compounds in cancer clinical trials due to their selective targeting of specific signalling pathways of tumour and cancer stem cells (Ghantous et al. 2010). Eudesmane SLs with an a-methylene-g-lactone moiety in the structure displayed potent cytotoxicity in our researches (Cui et al. 2011; Liu et al. 2013). Telekin (1), also with a eudesmane skeleton (Scheme 1), was fist isolated from Telekia speciosa by Benesova et al. (1961). It is the primary antitumour constitution of many medicinal plants (Lee et al. 2002; Li
*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
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14 1
8
1° or 2°Amine EtOH or MeOH
O
O
O 4
11
7 OH
O Reflux OH
13
15 1
R
2a~2p
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Scheme 1. Synthesis of 13-amino telekin derivatives. Arbitrary numbering.
et al. 2011; Ma et al. 2013). We also identified telekin from Carpesium divaricatum recently (Xie et al. 2012). Subsequent investigation indicated that telekin could strongly inhibit the proliferation of several cancer cells and induce apoptosis of hepatocellular carcinoma cells associated with the mitochondria-mediated pathway (Zheng et al. 2013). It has been reported that the exo-methylene group on the lactone part is the crucial cytotoxic pharmacophore of SLs (Ghantous et al. 2010). Crooks and co-workers added the Me2N group to the exo-methylene group on lactone of parthenolide, a promising anticancer SL, aiming to overcome its poor water-solubility and low serum bioavailability limitations (Neelakantan et al. 2009; Crooks et al. 2012). The additive product dimethylamino-parthenolide (LC-1) is at present in Phase I against acute myeloid leukaemia (Ghantous et al. 2010). Inspired by the structure modification of parthenolide and other SLs (Lawrence et al. 2001; Neelakantan et al. 2009; Cantrell et al. 2010), we modified the structure of telekin by linking with amino groups at C-13 by Michael addition reaction and evaluated their cytotoxic activity in vitro.
2. Results and discussion Telekin was isolated by the procedure described in our previous work (Xie et al. 2012). From the whole plant of Carpesium divaricatum, 5.3 g pure compound was obtained. Compounds 2a– 2p were synthesised by a one-step procedure similar to that in the literature (Lawrence et al. 2001; Neelakantan et al. 2009; Cantrell et al. 2010) (Scheme 1). A variety of primary and secondary amines reacted with telekin in EtOH or MeOH to afford crude products. Then, crude products were further purified by silica gel column chromatography (CC) to yield the corresponding 13-amino telekin derivatives (Table 1). Compounds 1 and 2a– 2p were characterised by MS and 1 H NMR, and sometimes by 13C NMR. The Michael addition at the a-methylene-g-lactone of SLs with amines have been found to be highly stereo specific and yielded exclusively a single stereoisomer with the (R *)configuration at the newly formed C-12 chiral carbon (Matsuda et al. 2000; Lawrence et al. 2001; Nasim & Crooks, 2008; Neelakantan et al. 2009; Cantrell et al. 2010). This was attributed to the stereo-exclusive protonation of the enolate formed during Michael addition (Lawrence et al. 2001). Similarly in this research, only one of the possible diastereoisomers of telekin derivatives was detected in the crude reaction mixture when the reactions were observed by using thin-layer chromatography (TLC). In consideration of the absolute configuration of telekin established earlier, the X-ray crystal structure of 2g confirmed the R configuration at C-12 in 13-amino telekin derivatives (Figure 1). Using MTT assay, the in vitro cytotoxicity against human lung cancer cell line A549 and human hepatocarcinoma cell lines LX-2 and HepG-2 of telekin (1) and its 13-amino derivatives (2a– 2p) were tested. Meanwhile, adriamycin was used as a positive control.
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Table 1. In vitro cytotoxicity data of telekin (1) and 13-amino derivatives (2a– 2p). Cell lines (IC50 mmol/L) Compounds
R
A549
LX-2
HepG-2
1
–
17.6
7.9
7.1
NA
.40
NA
86.0
. 40
23.5
. 40
77.8
. 40
24.7
. 40
72.5
31.2
27.5
. 40
54.1
18.2
42.3
26.5
16.8
23.5
42.3
. 40
38.6
14.2
8.4
26.4
44.1
6.8
13.7
26.9
38.7
NA
.40
NA
17.7
NA
41.2
NA
33.4
NA
24.5
NA
29.6
15.6
12.2
. 40
38.0
22.4
28.6
22.3
20.1
28.4
9.6
2.8
22.8
2.6
12.3
3.9
71.0
26.5
19.4
24.2
37.7
1.4
0.8
0.8
2a
HN
2b
HN
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2c
HN
2d 2e
OH
HN
O
HN
2f
OH
HN
2g
N
2h
N
2i
Yield (%)
N
2j
N
2k 2l
N HO
2m
N
2n
2o
OH
N
N
N
2p Adriamycin NA, not active to tumour cells.
OH
N
O
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Figure 1. The X-ray crystal structure for compound 2g.
The products derived from primary amines are generally less active compared with the analogues obtained from secondary amines (Table 1). For example, 2b has higher IC50 values against three tumour cell lines, while the IC50 values of 2g were 14.2, 8.4 and 26.4 mmol/L against A549, LX-2 and HepG-2, respectively. This probably reflects the poorer basicity and nucleophilicity of primary amines (Lawrence et al. 2001). Hydroxy groups in the amines greatly improved the cytotoxic activity. For example, 2d exhibited stronger cytotoxicity than 2a against three tested cells. The cytotoxicity against A549 cells of 2o (IC50 2.6 mmol/L) also improved up to 11-fold compared with 2n (IC50 28.4 mmol/L). However, 2l possessing two OH groups had no obvious change in the cytotoxicity, compared with the derivative 2h, which can be explained that the OH groups hindered 2l from penetrating through the lipophilic cell membrane. The piperidine and pyrrolidine adducts of SLs always have better cytotoxic than open chain analogues (Neelakantan et al. 2009). Possessing the same number of C-atoms, the IC50 values of 2m were 22.4 mmol/L and 22.3 mmol/L against A549 and HepG-2 cells, while 2c had no effect on these two cell lines. The piperidine ring drastically enhanced the cytotoxicity of 2n against HepG-2 cells compared with the parent compound. Piperidine and its derivatives are often used as an agent in the structure modification of natural products. Srivastava et al. (2006) previously reported that the nature of substitute and its position in the piperidine ring played an important role in eliciting cytotoxicity of SLs adducts. In this research, 4-hydroxy-piperidine enhanced the cytotoxicity of 2o against A549 and HepG-2 cells significantly. Compound 2o exhibits around sevenfold and twofold stronger cytotoxicity against A549 and HepG-2 cells compared with telekin against the same cells. Though, introduction of a heteroatom, e.g. an O-atom, into the piperidine ring, usually leads to lower cytotoxicity (Srivastava et al. 2006; Neelakantan et al. 2009). For example, 2n has lower IC50 values (9.6 and 2.8 mmol/L) against LX-2 and HepG-2 cells, meanwhile the IC50 values of 2p with oxygen in the piperidine ring were 19.4 and 24.2 mmol/L, respectively.
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3. Experimental 3.1. General CC: silica gel (SiO2, 300 – 400 mesh, Qingdao Haiyang Chemical Group Co., Ltd, Qingdao, China). TLC: pre-coated SiO2 GF-254 plates (Qingdao Haiyang Chemical Group Co., Ltd, Qingdao, China). 1H and 13C NMR (DEPT) spectra: Bruker AVANCE 500 spectrometer (Bruker Corporation, Switzerland), d in ppm relative to SiMe4 as internal standard, J in Hz. EI-MS: HP5988A GC/MS instrument (Agilent Technologies Inc., Santa Clara, CA, USA). ESI-MS: MSQ Plus instrument (Thermo Fisher Scientific, Waltham, MA, USA).
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3.2. Synthesis of 13-amino telekin derivatives (2a – 2p) and isolation Synthesis of (3R,3aR,4aR,8aR,9aR)-3-((ethylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2g): Telekin (25 mg, 0.1 mmol) was dissolved in 3 mL of EtOH, with the ethylamine (0.11 mmol) added, and the mixture was stirred at 858C for 12 h. The progress of the reaction was monitored by using TLC. The solvent was evaporated under vacuum. The crude product (26.8 mg) was suspended in water and then extracted three times with 20 mL of AcOEt. The extract was dried in vacuo. Then, the residue was subjected to silica gel CC, eluted with mixtures of hexane – acetone (10:1, 5:1, 3:1, 0:1 v/v) to afford the pure compound 2g and the yield was 44.1%. The other compounds were also obtained according to the above-mentioned method. Due to the differences of nucleophilic ability of amines and stereo effects in Michael addition reactions, the yield was different clearly. Telekin (1): EI-MS m/z: 248 [M]þ. 1H NMR (500 MHz, CDCl3): d 6.15 (1H, d, J ¼ 1.1 Hz, H-13), 5.59 (1H, d, J ¼ 1.1 Hz, H-13), 4.87 (1H, brs, H-15), 4.70 (1H, brs, H-15), 4.57 (1H, ddd, J ¼ 1.5, 5.2, 5.2 Hz, H-8), 3.35 (1H, m, H-7), 0.97 (3H, s, H-14). 13C NMR (125 MHz, CDCl3): d 170.9 (s), 150.3 (s), 142.3 (s), 120.5 (t), 109.2 (t), 77.1 (d), 74.4 (s), 37.7 (d), 36.6 (s), 35.7 (t), 35.5 (t), 33.9 (t), 32.0 (t), 21.9 (q), 21.8 (t). (3R,3aR,4aR,8aR,9aR)-3-((Ethylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2a): ESI-MS m/z: 294 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.87 (1H, s, H-15), 4.67 (1H, s, H-15), 4.64 (1H, brs, H-8), 3.10 (2H, m, H-13), 2.60 (2H, m, H-10 ), 1.30 (6H, t, J ¼ 7.2 Hz, 20 -CH3), 0.94 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Propylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2b): ESI-MS m/z: 308 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.87 (1H, s, H-15), 4.69 (1H, s, H-15), 4.67 (1H, brs, H-8), 2.98 (2H, m, H-13), 2.64 (2H, m, H-10 ), 1.06 (3H, t, J ¼ 5 Hz, 30 -CH3), 0.94 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((n-Butylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2c): ESI-MS m/z: 322 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.89 (1H, s, H-15), 4.71 (1H, s, H-15), 4.61 (1H, brs, H-8), 2.90 (2H, m, H-13), 2.59 (2H, m, H-10 ), 0.96 (3H, t, J ¼ 5 Hz, 40 -CH3), 0.95 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Ethanolamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2d): ESI-MS m/z: 310 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.88 (1H, s, H-15), 4.74 (1H, s, H-15), 4.62 (1H, brs, H-8), 3.10 (2H, m, H-13), 2.38 (2H, m, H-10 ), 2.64 (2H, m, H-20 ), 0.90 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((2-(Hydroxyethoxy)ethylamino)methyl)-4a-hydroxy-8a-methyl5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2e): ESI-MS m/z: 354 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.87 (1H, s, H-15), 4.68 (1H, s, H-15), 4.61 (1H, brs, H-8), 2.92 (2H, m, H-13), 1.88 (4H, m, H-10 ,30 ), 2.64 (4H, m, H-20 ,40 ), 0.95 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Cyclohexanamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2f): ESI-MS m/z: 348 [M þ H]þ. 1H NMR
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(500 MHz, CDCl3): d 4.86 (1H, s, H-15), 4.66 (1H, s, H-15), 4.63 (1H, brs, H-8), 2.83 (2H, m, H13), 2.63 (1H, m, H-10 ), 1.85 (4H, m, H-20 ,60 ), 0.94 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Ethylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2g): EI-MS m/z: 293 [M]þ. 1H NMR (500 MHz, CDCl3): d 4.78 (1H, s, H-15), 4.62 (1H, s, H-15), 4.47 (1H, brs, H-8), 2.90 (2H, m, H-13), 2.53 (1H, m, H11), 2.25 (6H, s, 10 -CH3, 20 -CH3), 0.86 (3H, s, H-14). 13C NMR (125 MHz, CDCl3): d 176.5 (s), 150.0 (s), 107.2 (t), 77.5 (d), 73.0 (s), 53.5 (t), 44.3 (q), 43.8 (d), 35.9 (s), 34.8 (t), 34.7 (d), 34.1 (t), 30.6 (t), 25.8 (t), 20.7 (q), 20.5 (t). X-ray crystal data for 2g: See Figure 1. C17H29NO4, fw ¼ 340.16, 293 K, monoclinic, ˚ , b ¼ 6.1319 (6) A ˚ , c ¼ 12.8531 (13) A ˚ , V ¼ 856.10 (15) A ˚ 3, P212121, a ¼ 11.1066 (12) A 21 23 Z ¼ 2, m (MoKa) ¼ 0.085 mm , rcal ¼ 1.208 g cm ; crystal dimensions: 0.311 mm £ 0.157 mm £ 0.076 mm; 13101 reflections measured (umax ¼ 56.072), 3164 were unique (Rint ¼ 0.0316) and of these 1855 had I . 2s(I) for which final R1, w R2 values were 0.0598 and 0.1159, respectively, for 214 parameters. Data were collected using a Bruker Smart Apex CCD diffractometer (Bruker Analytical X-ray Instruments Inc., Madison, WI, USA) using graphitemonochromated MoKa radiation. The structure was solved by direct methods and refined by full-matrix least-squares on F2 using Bruker SHELXS-97 (Sheldrick 1997). The final R and Rw factors were 0.0789 and 0.1246, respectively. CCDC 1006336 contains the supplementary crystallographic data for dimethylamine adduct (2g). The data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. (3R,3aR,4aR,8aR,9aR)-3-((Diethylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2h): EI-MS m/z: 321 [M]þ. 1H NMR (500 MHz, CDCl3): d 4.78 (1H, s, H-15), 4.61 (1H, s, H-15), 4.49 (1H, brs, H-8), 2.90 (2H, m, H-13), 2.51 (4H, m, H10 ), 1.02 (6H, t, J ¼ 7.2 Hz, 20 -CH3, 40 -CH3), 0.84 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Diisopropylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2i): ESI-MS m/z: 350 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.90 (1H, s, H-15), 4.73 (1H, s, H-15), 4.59 (1H, brs, H-8), 3.20 (2H, m, H-13), 2.57 (2H, m, H-10 , H-40 ), 1.42 (12H, d, J ¼ 5 Hz, 20 -CH3, 30 -CH3, 50 -CH3, 60 -CH3), 0.95 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Methylpropylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2j): ESI-MS m/z: 322 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.86 (1H, s, H-15), 4.67 (1H, s, H-15), 4.66 (1H, brs, H-8), 2.97 (2H, m, H-13), 2.13 (2H, m, H-10 ), 1.88 (3H, s, H-40 ), 1.05 (3H, t, J ¼ 5 Hz, H-30 ), 0.93 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Butylmethylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2k): ESI-MS m/z: 336 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.85 (1H, s, H-15), 4.67 (1H, s, H-15), 4.65 (1H, brs, H-8), 3.01 (2H, m, H13), 2.14 (2H, m, H-10 ), 1.88 (3H, s, H-50 ), 0.99 (3H, t, J ¼ 5 Hz, H-40 ), 0.93 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Diethanolamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2l): ESI-MS m/z: 354 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.88 (1H, s, H-15), 4.70 (1H, s, H-15), 4.59 (1H, brs, H-8), 3.02 (2H, m, H-13), 1.98 (4H, m, H-10 , H-30 ), 3.67 (4H, m, H-20 ,40 ), 0.94 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Pyrroolidin-1-yl)-methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2m): ESI-MS m/z: 320 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.87 (1H, s, H-15), 4.69 (1H, s, H-15), 4.65 (1H, brs, H-8), 3.20 (2H, m, H-13), 2.10 (4H, m, H-20 ,50 ), 0.94 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Piperidin-1-yl)-methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2n): EI-MS m/z: 333 [M]þ. 1H NMR (500 MHz, CDCl3): d 4.80 (1H, s, H-15), 4.61 (1H, s, H-15), 4.47 (1H, brs, H-8), 3.07 (2H, m, H-13), 2.61 (4H, m, H-20 ,60 ), 0.86 (3H, s, H-14).
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(3R,3aR,4aR,8aR,9aR)-3-((4-Hydroxypiperidin-1-yl)-methyl)-4a-hydroxy-8a-methyl-5methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2o): EI-MS m/z: 349 [M]þ. 1H NMR (500 MHz, CDCl3): d 4.80 (1H, s, H-15), 4.63 (1H, s, H-15), 4.48 (1H, brs, H-8), 2.11 (4H, m, H20 ,60 ), 3.69 (1H, brs, H-40 ), 2.96 (2H, m, H-13), 0.86 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Morpholinomethyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2p): ESI-MS m/z: 336 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.89 (1H, s, H-15), 4.70 (1H, s, H-15), 4.67 (1H, brs, H-8), 3.01 (2H, m, H-13), 2.61 (4H, m, H-30 ,50 ), 1.98 (4H, m, H-20 ,60 ), 0.95 (3H, s, H-14).
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3.3. Cytotoxicity assays The cytotoxicity against human lung cancer cell line A549, and human hepatocarcinoma cell lines LX-2 and HepG-2 of telekin (1) and its derivatives (2a– 2p) was evaluated by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The detailed procedure has been previously described (Zheng et al. 2013). 4. Conclusions In summary, we have synthesised a series of 13-amino telekin adducts successfully. The cytotoxicity of some derivatives was improved, and they showed selectivity towards three tumour cell lines. Compounds 2o and 2n were the most potent with an IC50 value of 3.9 and 2.8 mmol/L against HepG-2 cell line, respectively. The most promising derivative is 2o, which exhibited stronger selective cytotoxicity against A549 cells than telekin. Its antitumour activity and molecular mechanisms to human lung cancer cells need to be further investigated. In addition, compound 2h is also worthy of further investigation for its lesser IC50 value of 6.8 mmol/L compared with 17.6 mmol/L of telekin against A549 cells. Funding This work was partly supported by the Basic Science Research Program of National Research Foundation from the Ministry of Education, Science and Technology of Korea [grant number 2014-002046]. Dr Xie also thanks for the support of the International Scholar Exchange Fellowship Program [grant number ISEF 2014-2015] from the Korea Foundation for Advanced Studies.
References Benesova V, Herout V, Sorm F. 1961. On terpenes. CXXIV. Structure of telekin and isotelekin, new sesquiterpenic lactones from Telekia speciosa (Schreb.) Baumg.. Collect Czech Chem Commun. 26:1350– 1357. Cantrell CL, Pridgeon JW, Fronczek FR, Becnel JJ. 2010. Structure-activity relationship studies on derivatives of eudesmanolides from Inula helenium as toxicants against Aedes aegypti larvae and adults. Chem Biodives. 7:1681–1697. Crooks PA, Jordan CT, Wei XC. 2012. Use of perthenolide derivatives as antileukemic and cytotoxic agents. USA Patent. US20120165308A1. Cui M, Zhang Y, Liu SS, Xie WD, Ji M, Lou HX, Li X. 2011. 1-Oxoeudesm-11(13)-ene-12,8a-lactone-induced apoptosis via ROS generation and mitochondria activation in MCF-7 cells. Arch Pharm Res. 34:1323–1329. Ghantous A, Gali-Muhtasib H, Vuorela H, Saliba AN, Darwiche N. 2010. What made sesquiterpene lactones reach cancer clinical trials? Drug Discov Today. 15:668–678. Lawrence NJ, McGown AT, Nduka J, Hadfield JA, Pritchard RG. 2001. Cytotoxic Michael-type amine adducts of alphamethylene lactones alantolactone and isoalantolactone. Bioorg Med Chem Lett. 11:429–431. Lee JS, Min BS, Lee SM, Min MK, Kwon BM, Lee CO, Kim YH, Bae KH. 2002. Cytotoxic sesquiterpene lactones from Carpesium abrotanoides. Plant Med. 68:745–747. Li XW, Weng L, Gao X, Zhao Y, Pang F, Liu JH, Zhang HF, Hu JF. 2011. Antiproliferative and apoptotic sesquiterpene lactones from Carpesium faberi. Bioorg Med Chem Lett. 21:366–372. Liu SS, Wang YF, Ma LS, Zheng BB, Li L, Xie WD, Li X. 2013. 1-Oxoeudesm-11(13)-eno-12,8a-lactone induces G2/M arrest and apoptosis of human glioblastoma cells in vitro. Acta Pharmacol Sin. 34:271–281.
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Ma YY, Zhao DG, Gao K. 2013. Structural investigation and biological activity of sesquiterpene lactones from the traditional Chinese herb Inula racemosa. J Nat Prod. 76:564–570. Matsuda H, Kageura T, Inoue Y, Morikawa T, Yoshikawa M. 2000. Absolute stereostructures and syntheses of saussureamines A, B, C, D and E, Amino acid-sesquiterpene conjugates with gastroprotective effect, from the roots of Saussurea lappa. Tetrahedron. 56:7763–7777. Mew D, Balza F, Towers GH, Levy JG. 1982. Anti-tumour effects of the sesquiterpene lactone parthenin. Planta Med. 45:23–27. Nasim S, Crooks PA. 2008. Antileukemic activity of aminoparthenolide analogs. Bioorg Med Chem Lett. 18:3870–3873. Neelakantan S, Nasim S, Guzman ML, Jordan CT, Crooks PA. 2009. Aminoparthenolides as novel anti-leukemic agents: discovery of the NF-kappaB inhibitor, DMAPT (LC-1). Bioorg Med Chem Lett. 19:4346 –4349. Sheldrick GM. 1997. SHELXS-97, PC version, University of Go¨ttingen, Go¨ttingen, Germany. Srivastava SK, Abraham A, Bhat B, Jaggi M, Singh AT, Sanna VK, Singh G, Agarwal SK, Mukherjee R, Burman AC. 2006. Synthesis of 13-amino costunolide derivatives as anticancer agents. Bioorg Med Chem Lett. 16:4195–4199. Xie WD, Wang XR, Ma LS, Li X, Row KH. 2012. Sesquiterpenoids from Carpesium divaricatum and their cytotoxic activity. Fitoterapia. 83:1351–1355. Zdero C, Bohlmann F. 1990. Systematics and evolution within the composita, seen with the eyes of a chemist. Plant Syst Evol. 171:1–14. Zhang SY, Won YK, Ong CN, Shen HM. 2005. Anti-cancer potential of sesquiterpene lactones: bioactivity and molecular mechanisms. Curr Med Chem Anti-Cancer Agents. 5:239–249. Zheng BB, Wu LH, Ma LS, Liu SS, Li L, Xie WD, Li X. 2013. Telekin induces apoptosis associated with the mitochondria-mediated pathway in human hepatocellular carcinoma cells. Biol Pharm Bull. 36:1118– 1125.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Synthesis of 13-amino telekin derivatives and their cytotoxic activity a
a
a
a
Xiujie Wang , Xiumei Zhang , Beibei Zheng , Nan Hu , Weidong ab
Xie
& Kyungho Row
b
a
Department of Pharmacy, College of Marine Science, Shandong University, Weihai 264209, P.R. China b
Department of Chemical Engineering, Inha University, Incheon 402-751, South Korea Published online: 11 Dec 2014.
Click for updates To cite this article: Xiujie Wang, Xiumei Zhang, Beibei Zheng, Nan Hu, Weidong Xie & Kyungho Row (2015) Synthesis of 13-amino telekin derivatives and their cytotoxic activity, Natural Product Research: Formerly Natural Product Letters, 29:8, 756-763, DOI: 10.1080/14786419.2014.987143 To link to this article: http://dx.doi.org/10.1080/14786419.2014.987143
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Natural Product Research, 2015 Vol. 29, No. 8, 756–763, http://dx.doi.org/10.1080/14786419.2014.987143
Synthesis of 13-amino telekin derivatives and their cytotoxic activity Xiujie Wanga, Xiumei Zhanga, Beibei Zhenga, Nan Hua, Weidong Xiea,b* and Kyungho Rowb* a
Department of Pharmacy, College of Marine Science, Shandong University, Weihai 264209, P.R. China; Department of Chemical Engineering, Inha University, Incheon 402-751, South Korea
b
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(Received 8 September 2014; final version received 6 November 2014)
O O
O
Amine
O
Michael addition reaction OH
OH
NR1R2
Telekin Telekin is a eudesmane sesquiterpene-lactone naturally occurring in many medicinal plants with antitumour and anti-inflammatory activity. In this study, a series of 13amino derivatives of telekin have been synthesised through Michael addition reaction, and their relative configurations were exemplified by the single crystal X-ray diffraction of the dimethylamine adduct. The in vitro cytotoxicity against three tumour cell lines of these amine derivatives was evaluated. The piperidine and 4-hydroxypiperidine adducts displayed stronger cytotoxic activity than telekin. Keywords: telekin; Michael addition reaction; sesquiterpene lactone; cytotoxicity; eudesmane
1. Introduction Sesquiterpene lactones (SLs) are common secondary metabolites of plants in the Asteraceae family, some of which are often used in traditional medicine against inflammation and cancer (Mew et al. 1982; Zdero & Bohlmann 1990; Ghantous et al. 2010). In recent years, the anticancer property of various SLs has attracted a great deal of interests, and extensive research work has been carried out to characterise the anticancer activity, the structure – bioactivity relationship, the molecular mechanisms and so on (Zhang et al. 2005; Ghantous et al. 2010). Though, the poor water-solubility and extensive interactions with plasma proteins preclude most SLs further development as therapeutic agents (Ghantous et al. 2010). Up to now, only artemisinin, thapsigargin, parthenolide and some of their synthetic derivatives have been developed to be lead compounds in cancer clinical trials due to their selective targeting of specific signalling pathways of tumour and cancer stem cells (Ghantous et al. 2010). Eudesmane SLs with an a-methylene-g-lactone moiety in the structure displayed potent cytotoxicity in our researches (Cui et al. 2011; Liu et al. 2013). Telekin (1), also with a eudesmane skeleton (Scheme 1), was fist isolated from Telekia speciosa by Benesova et al. (1961). It is the primary antitumour constitution of many medicinal plants (Lee et al. 2002; Li
*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
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14 1
8
1° or 2°Amine EtOH or MeOH
O
O
O 4
11
7 OH
O Reflux OH
13
15 1
R
2a~2p
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Scheme 1. Synthesis of 13-amino telekin derivatives. Arbitrary numbering.
et al. 2011; Ma et al. 2013). We also identified telekin from Carpesium divaricatum recently (Xie et al. 2012). Subsequent investigation indicated that telekin could strongly inhibit the proliferation of several cancer cells and induce apoptosis of hepatocellular carcinoma cells associated with the mitochondria-mediated pathway (Zheng et al. 2013). It has been reported that the exo-methylene group on the lactone part is the crucial cytotoxic pharmacophore of SLs (Ghantous et al. 2010). Crooks and co-workers added the Me2N group to the exo-methylene group on lactone of parthenolide, a promising anticancer SL, aiming to overcome its poor water-solubility and low serum bioavailability limitations (Neelakantan et al. 2009; Crooks et al. 2012). The additive product dimethylamino-parthenolide (LC-1) is at present in Phase I against acute myeloid leukaemia (Ghantous et al. 2010). Inspired by the structure modification of parthenolide and other SLs (Lawrence et al. 2001; Neelakantan et al. 2009; Cantrell et al. 2010), we modified the structure of telekin by linking with amino groups at C-13 by Michael addition reaction and evaluated their cytotoxic activity in vitro.
2. Results and discussion Telekin was isolated by the procedure described in our previous work (Xie et al. 2012). From the whole plant of Carpesium divaricatum, 5.3 g pure compound was obtained. Compounds 2a– 2p were synthesised by a one-step procedure similar to that in the literature (Lawrence et al. 2001; Neelakantan et al. 2009; Cantrell et al. 2010) (Scheme 1). A variety of primary and secondary amines reacted with telekin in EtOH or MeOH to afford crude products. Then, crude products were further purified by silica gel column chromatography (CC) to yield the corresponding 13-amino telekin derivatives (Table 1). Compounds 1 and 2a– 2p were characterised by MS and 1 H NMR, and sometimes by 13C NMR. The Michael addition at the a-methylene-g-lactone of SLs with amines have been found to be highly stereo specific and yielded exclusively a single stereoisomer with the (R *)configuration at the newly formed C-12 chiral carbon (Matsuda et al. 2000; Lawrence et al. 2001; Nasim & Crooks, 2008; Neelakantan et al. 2009; Cantrell et al. 2010). This was attributed to the stereo-exclusive protonation of the enolate formed during Michael addition (Lawrence et al. 2001). Similarly in this research, only one of the possible diastereoisomers of telekin derivatives was detected in the crude reaction mixture when the reactions were observed by using thin-layer chromatography (TLC). In consideration of the absolute configuration of telekin established earlier, the X-ray crystal structure of 2g confirmed the R configuration at C-12 in 13-amino telekin derivatives (Figure 1). Using MTT assay, the in vitro cytotoxicity against human lung cancer cell line A549 and human hepatocarcinoma cell lines LX-2 and HepG-2 of telekin (1) and its 13-amino derivatives (2a– 2p) were tested. Meanwhile, adriamycin was used as a positive control.
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Table 1. In vitro cytotoxicity data of telekin (1) and 13-amino derivatives (2a– 2p). Cell lines (IC50 mmol/L) Compounds
R
A549
LX-2
HepG-2
1
–
17.6
7.9
7.1
NA
.40
NA
86.0
. 40
23.5
. 40
77.8
. 40
24.7
. 40
72.5
31.2
27.5
. 40
54.1
18.2
42.3
26.5
16.8
23.5
42.3
. 40
38.6
14.2
8.4
26.4
44.1
6.8
13.7
26.9
38.7
NA
.40
NA
17.7
NA
41.2
NA
33.4
NA
24.5
NA
29.6
15.6
12.2
. 40
38.0
22.4
28.6
22.3
20.1
28.4
9.6
2.8
22.8
2.6
12.3
3.9
71.0
26.5
19.4
24.2
37.7
1.4
0.8
0.8
2a
HN
2b
HN
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2c
HN
2d 2e
OH
HN
O
HN
2f
OH
HN
2g
N
2h
N
2i
Yield (%)
N
2j
N
2k 2l
N HO
2m
N
2n
2o
OH
N
N
N
2p Adriamycin NA, not active to tumour cells.
OH
N
O
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Figure 1. The X-ray crystal structure for compound 2g.
The products derived from primary amines are generally less active compared with the analogues obtained from secondary amines (Table 1). For example, 2b has higher IC50 values against three tumour cell lines, while the IC50 values of 2g were 14.2, 8.4 and 26.4 mmol/L against A549, LX-2 and HepG-2, respectively. This probably reflects the poorer basicity and nucleophilicity of primary amines (Lawrence et al. 2001). Hydroxy groups in the amines greatly improved the cytotoxic activity. For example, 2d exhibited stronger cytotoxicity than 2a against three tested cells. The cytotoxicity against A549 cells of 2o (IC50 2.6 mmol/L) also improved up to 11-fold compared with 2n (IC50 28.4 mmol/L). However, 2l possessing two OH groups had no obvious change in the cytotoxicity, compared with the derivative 2h, which can be explained that the OH groups hindered 2l from penetrating through the lipophilic cell membrane. The piperidine and pyrrolidine adducts of SLs always have better cytotoxic than open chain analogues (Neelakantan et al. 2009). Possessing the same number of C-atoms, the IC50 values of 2m were 22.4 mmol/L and 22.3 mmol/L against A549 and HepG-2 cells, while 2c had no effect on these two cell lines. The piperidine ring drastically enhanced the cytotoxicity of 2n against HepG-2 cells compared with the parent compound. Piperidine and its derivatives are often used as an agent in the structure modification of natural products. Srivastava et al. (2006) previously reported that the nature of substitute and its position in the piperidine ring played an important role in eliciting cytotoxicity of SLs adducts. In this research, 4-hydroxy-piperidine enhanced the cytotoxicity of 2o against A549 and HepG-2 cells significantly. Compound 2o exhibits around sevenfold and twofold stronger cytotoxicity against A549 and HepG-2 cells compared with telekin against the same cells. Though, introduction of a heteroatom, e.g. an O-atom, into the piperidine ring, usually leads to lower cytotoxicity (Srivastava et al. 2006; Neelakantan et al. 2009). For example, 2n has lower IC50 values (9.6 and 2.8 mmol/L) against LX-2 and HepG-2 cells, meanwhile the IC50 values of 2p with oxygen in the piperidine ring were 19.4 and 24.2 mmol/L, respectively.
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3. Experimental 3.1. General CC: silica gel (SiO2, 300 – 400 mesh, Qingdao Haiyang Chemical Group Co., Ltd, Qingdao, China). TLC: pre-coated SiO2 GF-254 plates (Qingdao Haiyang Chemical Group Co., Ltd, Qingdao, China). 1H and 13C NMR (DEPT) spectra: Bruker AVANCE 500 spectrometer (Bruker Corporation, Switzerland), d in ppm relative to SiMe4 as internal standard, J in Hz. EI-MS: HP5988A GC/MS instrument (Agilent Technologies Inc., Santa Clara, CA, USA). ESI-MS: MSQ Plus instrument (Thermo Fisher Scientific, Waltham, MA, USA).
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3.2. Synthesis of 13-amino telekin derivatives (2a – 2p) and isolation Synthesis of (3R,3aR,4aR,8aR,9aR)-3-((ethylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2g): Telekin (25 mg, 0.1 mmol) was dissolved in 3 mL of EtOH, with the ethylamine (0.11 mmol) added, and the mixture was stirred at 858C for 12 h. The progress of the reaction was monitored by using TLC. The solvent was evaporated under vacuum. The crude product (26.8 mg) was suspended in water and then extracted three times with 20 mL of AcOEt. The extract was dried in vacuo. Then, the residue was subjected to silica gel CC, eluted with mixtures of hexane – acetone (10:1, 5:1, 3:1, 0:1 v/v) to afford the pure compound 2g and the yield was 44.1%. The other compounds were also obtained according to the above-mentioned method. Due to the differences of nucleophilic ability of amines and stereo effects in Michael addition reactions, the yield was different clearly. Telekin (1): EI-MS m/z: 248 [M]þ. 1H NMR (500 MHz, CDCl3): d 6.15 (1H, d, J ¼ 1.1 Hz, H-13), 5.59 (1H, d, J ¼ 1.1 Hz, H-13), 4.87 (1H, brs, H-15), 4.70 (1H, brs, H-15), 4.57 (1H, ddd, J ¼ 1.5, 5.2, 5.2 Hz, H-8), 3.35 (1H, m, H-7), 0.97 (3H, s, H-14). 13C NMR (125 MHz, CDCl3): d 170.9 (s), 150.3 (s), 142.3 (s), 120.5 (t), 109.2 (t), 77.1 (d), 74.4 (s), 37.7 (d), 36.6 (s), 35.7 (t), 35.5 (t), 33.9 (t), 32.0 (t), 21.9 (q), 21.8 (t). (3R,3aR,4aR,8aR,9aR)-3-((Ethylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2a): ESI-MS m/z: 294 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.87 (1H, s, H-15), 4.67 (1H, s, H-15), 4.64 (1H, brs, H-8), 3.10 (2H, m, H-13), 2.60 (2H, m, H-10 ), 1.30 (6H, t, J ¼ 7.2 Hz, 20 -CH3), 0.94 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Propylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2b): ESI-MS m/z: 308 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.87 (1H, s, H-15), 4.69 (1H, s, H-15), 4.67 (1H, brs, H-8), 2.98 (2H, m, H-13), 2.64 (2H, m, H-10 ), 1.06 (3H, t, J ¼ 5 Hz, 30 -CH3), 0.94 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((n-Butylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2c): ESI-MS m/z: 322 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.89 (1H, s, H-15), 4.71 (1H, s, H-15), 4.61 (1H, brs, H-8), 2.90 (2H, m, H-13), 2.59 (2H, m, H-10 ), 0.96 (3H, t, J ¼ 5 Hz, 40 -CH3), 0.95 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Ethanolamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2d): ESI-MS m/z: 310 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.88 (1H, s, H-15), 4.74 (1H, s, H-15), 4.62 (1H, brs, H-8), 3.10 (2H, m, H-13), 2.38 (2H, m, H-10 ), 2.64 (2H, m, H-20 ), 0.90 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((2-(Hydroxyethoxy)ethylamino)methyl)-4a-hydroxy-8a-methyl5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2e): ESI-MS m/z: 354 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.87 (1H, s, H-15), 4.68 (1H, s, H-15), 4.61 (1H, brs, H-8), 2.92 (2H, m, H-13), 1.88 (4H, m, H-10 ,30 ), 2.64 (4H, m, H-20 ,40 ), 0.95 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Cyclohexanamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2f): ESI-MS m/z: 348 [M þ H]þ. 1H NMR
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(500 MHz, CDCl3): d 4.86 (1H, s, H-15), 4.66 (1H, s, H-15), 4.63 (1H, brs, H-8), 2.83 (2H, m, H13), 2.63 (1H, m, H-10 ), 1.85 (4H, m, H-20 ,60 ), 0.94 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Ethylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2g): EI-MS m/z: 293 [M]þ. 1H NMR (500 MHz, CDCl3): d 4.78 (1H, s, H-15), 4.62 (1H, s, H-15), 4.47 (1H, brs, H-8), 2.90 (2H, m, H-13), 2.53 (1H, m, H11), 2.25 (6H, s, 10 -CH3, 20 -CH3), 0.86 (3H, s, H-14). 13C NMR (125 MHz, CDCl3): d 176.5 (s), 150.0 (s), 107.2 (t), 77.5 (d), 73.0 (s), 53.5 (t), 44.3 (q), 43.8 (d), 35.9 (s), 34.8 (t), 34.7 (d), 34.1 (t), 30.6 (t), 25.8 (t), 20.7 (q), 20.5 (t). X-ray crystal data for 2g: See Figure 1. C17H29NO4, fw ¼ 340.16, 293 K, monoclinic, ˚ , b ¼ 6.1319 (6) A ˚ , c ¼ 12.8531 (13) A ˚ , V ¼ 856.10 (15) A ˚ 3, P212121, a ¼ 11.1066 (12) A 21 23 Z ¼ 2, m (MoKa) ¼ 0.085 mm , rcal ¼ 1.208 g cm ; crystal dimensions: 0.311 mm £ 0.157 mm £ 0.076 mm; 13101 reflections measured (umax ¼ 56.072), 3164 were unique (Rint ¼ 0.0316) and of these 1855 had I . 2s(I) for which final R1, w R2 values were 0.0598 and 0.1159, respectively, for 214 parameters. Data were collected using a Bruker Smart Apex CCD diffractometer (Bruker Analytical X-ray Instruments Inc., Madison, WI, USA) using graphitemonochromated MoKa radiation. The structure was solved by direct methods and refined by full-matrix least-squares on F2 using Bruker SHELXS-97 (Sheldrick 1997). The final R and Rw factors were 0.0789 and 0.1246, respectively. CCDC 1006336 contains the supplementary crystallographic data for dimethylamine adduct (2g). The data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. (3R,3aR,4aR,8aR,9aR)-3-((Diethylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2h): EI-MS m/z: 321 [M]þ. 1H NMR (500 MHz, CDCl3): d 4.78 (1H, s, H-15), 4.61 (1H, s, H-15), 4.49 (1H, brs, H-8), 2.90 (2H, m, H-13), 2.51 (4H, m, H10 ), 1.02 (6H, t, J ¼ 7.2 Hz, 20 -CH3, 40 -CH3), 0.84 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Diisopropylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2i): ESI-MS m/z: 350 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.90 (1H, s, H-15), 4.73 (1H, s, H-15), 4.59 (1H, brs, H-8), 3.20 (2H, m, H-13), 2.57 (2H, m, H-10 , H-40 ), 1.42 (12H, d, J ¼ 5 Hz, 20 -CH3, 30 -CH3, 50 -CH3, 60 -CH3), 0.95 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Methylpropylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2j): ESI-MS m/z: 322 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.86 (1H, s, H-15), 4.67 (1H, s, H-15), 4.66 (1H, brs, H-8), 2.97 (2H, m, H-13), 2.13 (2H, m, H-10 ), 1.88 (3H, s, H-40 ), 1.05 (3H, t, J ¼ 5 Hz, H-30 ), 0.93 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Butylmethylamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2k): ESI-MS m/z: 336 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.85 (1H, s, H-15), 4.67 (1H, s, H-15), 4.65 (1H, brs, H-8), 3.01 (2H, m, H13), 2.14 (2H, m, H-10 ), 1.88 (3H, s, H-50 ), 0.99 (3H, t, J ¼ 5 Hz, H-40 ), 0.93 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Diethanolamino)methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2l): ESI-MS m/z: 354 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.88 (1H, s, H-15), 4.70 (1H, s, H-15), 4.59 (1H, brs, H-8), 3.02 (2H, m, H-13), 1.98 (4H, m, H-10 , H-30 ), 3.67 (4H, m, H-20 ,40 ), 0.94 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Pyrroolidin-1-yl)-methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2m): ESI-MS m/z: 320 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.87 (1H, s, H-15), 4.69 (1H, s, H-15), 4.65 (1H, brs, H-8), 3.20 (2H, m, H-13), 2.10 (4H, m, H-20 ,50 ), 0.94 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Piperidin-1-yl)-methyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2n): EI-MS m/z: 333 [M]þ. 1H NMR (500 MHz, CDCl3): d 4.80 (1H, s, H-15), 4.61 (1H, s, H-15), 4.47 (1H, brs, H-8), 3.07 (2H, m, H-13), 2.61 (4H, m, H-20 ,60 ), 0.86 (3H, s, H-14).
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(3R,3aR,4aR,8aR,9aR)-3-((4-Hydroxypiperidin-1-yl)-methyl)-4a-hydroxy-8a-methyl-5methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2o): EI-MS m/z: 349 [M]þ. 1H NMR (500 MHz, CDCl3): d 4.80 (1H, s, H-15), 4.63 (1H, s, H-15), 4.48 (1H, brs, H-8), 2.11 (4H, m, H20 ,60 ), 3.69 (1H, brs, H-40 ), 2.96 (2H, m, H-13), 0.86 (3H, s, H-14). (3R,3aR,4aR,8aR,9aR)-3-((Morpholinomethyl)-4a-hydroxy-8a-methyl-5-methylenedecahydronaphtho[2,3-b ]furan-2(3H)-one (2p): ESI-MS m/z: 336 [M þ H]þ. 1H NMR (500 MHz, CDCl3): d 4.89 (1H, s, H-15), 4.70 (1H, s, H-15), 4.67 (1H, brs, H-8), 3.01 (2H, m, H-13), 2.61 (4H, m, H-30 ,50 ), 1.98 (4H, m, H-20 ,60 ), 0.95 (3H, s, H-14).
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3.3. Cytotoxicity assays The cytotoxicity against human lung cancer cell line A549, and human hepatocarcinoma cell lines LX-2 and HepG-2 of telekin (1) and its derivatives (2a– 2p) was evaluated by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The detailed procedure has been previously described (Zheng et al. 2013). 4. Conclusions In summary, we have synthesised a series of 13-amino telekin adducts successfully. The cytotoxicity of some derivatives was improved, and they showed selectivity towards three tumour cell lines. Compounds 2o and 2n were the most potent with an IC50 value of 3.9 and 2.8 mmol/L against HepG-2 cell line, respectively. The most promising derivative is 2o, which exhibited stronger selective cytotoxicity against A549 cells than telekin. Its antitumour activity and molecular mechanisms to human lung cancer cells need to be further investigated. In addition, compound 2h is also worthy of further investigation for its lesser IC50 value of 6.8 mmol/L compared with 17.6 mmol/L of telekin against A549 cells. Funding This work was partly supported by the Basic Science Research Program of National Research Foundation from the Ministry of Education, Science and Technology of Korea [grant number 2014-002046]. Dr Xie also thanks for the support of the International Scholar Exchange Fellowship Program [grant number ISEF 2014-2015] from the Korea Foundation for Advanced Studies.
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