Accepted Manuscript A new polyoxygenated cyclohexene and a new megastigmane glycoside from Uvaria grandiflora Duc Viet Ho, Takeshi Kodama, Hien Thi Bich Le, Kiem Van Phan, Thao Thi Do, Tai Huu Bui, Anh Tuan Le, Nwet Nwet Win, Hiroshi Imagawa, Takuya Ito, Hiroyuki Morita, Hoai Thi Nguyen PII: DOI: Reference:
S0960-894X(15)00540-5 http://dx.doi.org/10.1016/j.bmcl.2015.05.066 BMCL 22756
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
9 May 2015 22 May 2015 25 May 2015
Please cite this article as: Ho, D.V., Kodama, T., Le, H.T.B., Phan, K.V., Do, T.T., Bui, T.H., Le, A.T., Win, N.N., Imagawa, H., Ito, T., Morita, H., Nguyen, H.T., A new polyoxygenated cyclohexene and a new megastigmane glycoside from Uvaria grandiflora, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/ 10.1016/j.bmcl.2015.05.066
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A new polyoxygenated cyclohexene and a new megastigmane glycoside from Uvaria grandiflora
Duc Viet Hoa, Takeshi Kodamab, Hien Thi Bich Lea, Kiem Van Phanc, Thao Thi Dod, Tai Huu Buic,e, Anh Tuan Lef, Nwet Nwet Winb, Hiroshi Imagawag, Takuya Ito b,*, Hiroyuki Moritab,*, Hoai Thi Nguyena,b,* a
Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo
Quyen, Hue City, Vietnam b
Insitute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194,
Japan c
Institute of Marine Biochemistry, VAST, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
d e f
Institute of Biotechnology, VAST, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
Quang Tri Center of Science and Technology, Mientrung Inst. for Scientific Research, 121 Ly
Thuong Kiet, Dong Ha, Quang Tri, Vietnam g
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho,
Tokushima 770-8514, Japan *To whom correspondence should be addressed: Associate Professor Takuya Ito, Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan (Tel: +8176-434-7627; Fax: +81-76-434-5059, E-mail: [email protected]), Professor Hiroyuki Morita, Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 9300194, Japan (Tel: +81-76-434-7625; Fax: +81-76-434-5059, E-mail: [email protected]), or Associate Professor Nguyen Thi Hoai, Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo Quyen, Hue City, Vietnam (Tel: +84905-974-785, E-mail: [email protected])
1
Abstract A new polyoxygenated cyclohexene, (−)-3-O-debenzoylzeylenone (1), and a new megastigmane glycoside, grandionoside A (2), were isolated from the aerial parts of Uvaria grandiflora collected in Vietnam, together with ten known compounds including polyoxygenated cyclohexenes (3−6), a triterpenoid (7), an alkaloid (8), a long chain alcohol (9), hexenyl glycopyranoside (10), and saponins (11−12). Their chemical structures were elucidated by a combination of extensive NMR spectroscopy with X-ray crystallographic analysis for 1, and chemical conversion for 2. Compound 1 exhibited significant cytotoxicity against the LU-1 and SK-Mel-2 cell lines with IC50 values of 4.68 and 3.63 µM, respectively. Remarkably, the cytotoxicity of 12 against the LU-1, KB, Hep-G2, MKN-7, and SW-480 cell lines was comparable to that of ellipticine, the positive control, with IC50 values ranging from 1.24 to 1.60 µM.
Key words: Uvaria grandiflora, polyoxygenated cyclohexene, megastigmane glycoside, cytotoxicity.
2
Uvaria grandiflora Roxb. ex Hornem (Annonaceae), also known as Unona grandiflora or Uvaria purpurea, is a long woody vine widely distributed in Southeast Asia, including Central Vietnam, India, Myanmar, China, Sri Lanka, Malaysia, and Indonesia, but is rarely cultivated.1 The plant has been used as a source of traditional medicines for cancer therapies in Vietnam. Previous phytochemical studies of this species have led to the isolation of acetogenins,2 polyoxygenated cyclohexenes,3-6 and aromatic compounds,7 with some displaying interesting anti-tumour,3, 8 anti-leishmanial,7 anti-inflammatory,8 and anti-feedant activities.9 In our previous studies focusing on the constituents of Uvaria plants and their cytotoxicity, we discovered the presence of the unique lignan glycoside, ufaside, in the aerial parts of Uvaria rufa (Dun.) Blume10 and the unique aromatic compound, coraurarin A, in the leaves of Uvaria cordata (Dun.) Wall. Ex Alston11, together with known compounds, such as an oxoaporphine alkaloid, ergosterols, and catechins. Among these constituents, oxoanolobine and ergosta-4,6,8(14),22-tetraen-3-one displayed moderate cytotoxicity against a human lung adenocarcinoma cell line (LU-1). We also observed that the methanol extract of U. grandiflora exhibits potent in vitro cytotoxicity against nine cancer cell lines (LU-1, human lung cancer cell; KB, epidermoid carcinoma; MDA-BA-231, high metastasis human breast cancer; LNCaP, human prostate cancer; Hep-G2, human hepatoma cancer; MKN-7, stomach cancer; SW-480, human colon adenocarcinoma; HL-60, human acute leukemia; SKMel-2, melanoma carcinoma). Therein, the strongest cytotoxicity was found against the MDA-BA-231, MKN-7 and KB cell lines, with IC50 values of 0.62 µM, 0.89 µM and 1.67 µM, respectively. In our continuous studies on the constituents of Uvaria plants and their cytotoxic activity, the CHCl3-, EtOAc-, and water-soluble portions were thus prepared from the active methanol extract of this plant.12 Bioassay-guided separation of the soluble portions afforded a new polyoxygenated cyclohexene, (−)-3-O-debenzoylzeylenone (1), and a new 3
megastigmane glycoside, grandionoside A (2), together with ten known compounds, pipoxide chlorohydrin (3),13 (−)-zeylenone (4),3 (−)-zeylenol (5),14 (+)-pipoxide (6),15 lupeol (7),16 velutinam (8),17 threo-octadecane-1,9,10-triol (9),18 (Z)-3-hexenyl-1-O-β-D-glucopyranoside (10),19 sakurasosaponin (11),20 and ardisiacrispin B (12)21 (Fig. 1). The spectral data of the known compounds were confirmed with the reported data. Among the isolated known compounds, 10−12 were isolated from this genus for the first time, while 9 was isolated for the first time as a natural product. Herein, the isolation and structure elucidation of the two new compounds (1 and 2), as well as the cytotoxicity of all of the isolated compounds (1−12) are described. Compound 122 was obtained as a colorless powder from the EtOAc-soluble portion.23 The ESIMS of 1 showed a quasimolecular ion peak at m/z 301 [M+Na]+. Its molecular formula was determined to be C14H14O6 by HRESIMS, in conjunction with an NMR data analysis. The IR spectrum of 1 revealed strong absorption bands corresponding to an aromatic ring (1601, 1585 cm−1), a conjugated carbonyl (1711 cm−1), an ester (1677 cm−1), and hydroxy groups (3507, 3410, 3202 cm−1). The UV spectrum supported the presence of the aromatic ring (λmax 268 nm) and an α,β-unsaturated ketone (λmax 235 nm). The 1H NMR spectrum of 1 in CDCl3 showed signals of five aromatic protons at δH 7.41-7.95 (5H), two olefinic protons [δH 6.14, dd (J = 10.5, 2.0 Hz, H-5), 6.91, dd (J = 10.5, 3.5 Hz, H-4)], oxygenated methylene protons with geminal coupling as a methine proton [δH 4.60, 4.78, both d (J = 11.5 Hz, H2-7)], and two oxygenated methine protons [δH 3.98, d (J = 5.5 Hz, H2), 4.69 (m, H-3)] (Table 1). Analyses of the
13
C NMR, DEPT, and HSQC spectra of 1
revealed 14 signals, including two carbonyl carbons (δC 195.4, 166.9), six aromatic carbons (δC 133.6, 129.9, 129.1, 128.5), two olefinic carbons (δC 148.2, 126.9), an oxygenated methylene carbon (δC 64.1), two oxygenated methine carbons (δC 74.2, 68.1), and an
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oxygenated quaternary carbon (δC 76.3) (Table 1). The set of aromatic proton signals at δH 7.41 (dd, J = 8.0, 8.0 Hz, H2-3′/5′), δH 7.56 (t, J = 8.0 Hz, H-4′), and δH 7.95 (dd, J = 8.0, 1.0 Hz, H2-2′/6′), and the HMBC correlations from H-2′/6′ to C-1′ (δC 129.1) and C-7′ (δC 166.9), suggested the presence of a benzoyl moiety in 1 (Fig. 2). However, the 1H− 1H COSY correlations of H-3 (δH 4.69, m) with H-2 (δH 3.98, d) and H-4 (δH 6.91, dd) and of H-4 with H-5 (δH 6.14, dd), and the HMBC correlations from H-2 to C-1 (δC 76.3) and C-6 (δC 195.4) and from H-5 to C-1, indicated that 1 possesses a trioxygenated cyclohexenone moiety. Further analyses of the HSQC, COSY, and HMBC spectra revealed that the remaining oxygenated methylene protons at H2-7 (δH 4.60, 4.78, both d) were connected to C-1 (δC 76.3), C-2 (δC 74.2), and C-6 (δC 195.4) on the trioxygenated cyclohexenone moiety and to C-7′ (δC 166.9) on the benzoyl moiety (Fig. 2). These results indicated that the oxygenated quaternary carbon at C-1 (δC 76.3) was directly linked to the methylene carbon at C-7, which was further connected to the benzoyl group through an oxygen atom. Furthermore, the coupling constant of 5.5 Hz between H-2 (δH 3.98, d) and H-3 (4.69, m) indicated an axial/pseudo-axial relationship between these protons as well as the 2,3-anti-dihydroxy groups. In addition, the NOESY correlation between H-2 and H2-7 and the loss of the correlation between H-2 and H3 (4.69, m) suggested the presence of 1,2-syn-dihydroxy groups (Fig. 2). Finally, the X-ray crystallographic analysis of 124 allowed us to confirm the planar and relative structure of 1 to be the same as that of the previously reported (+)-3-O-debenzoylzeylenone25 (Fig. 3). However, the optical rotation of 1 was [α]20D −13.8 (c 0.4, CHCl3), which is almost opposite to the value of [α]20D +11.7 (c 0.36, CHCl3)25 and [α]20D +12.5 (c 0.4, CHCl3)26 for (+)-3-Odebenzoylzeylenone. Compound 1 was thus verified to be the enantiomer of (+)-3-Odebenzoylzeylenone, and was identified as (−)-3-O-debenzoylzeylenone. Compound 227 was obtained as a colorless powder from the water-soluble portion.28 The ESIMS of 2 indicated a quasimolecular ion peak at m/z 411 [M+Na]+. Its molecular 5
formula was determined further to be C19H32O8, by HRESIMS and
13
C NMR data. The IR
spectrum of 2 showed strong absorption bands corresponding to conjugated carbonyl (1693 cm−1) and hydroxy (3390 cm−1) groups. The 1H NMR spectrum in CD3OD revealed the signals of two trans-olefinic protons [δH 6.07, d (J = 16.0 Hz, H-8), 6.71, dd (J = 16.0, 10.0 Hz, H-7)], an anomeric proton [δH 4.39, d (J = 8.0 Hz, H-1′)], five oxygenated methine protons [δH 4.23, ddd, (J = 3.5, 3.5, 3.0 Hz, H-3), 3.29-3.38 (overlapped, H-2′, H-3′, H-4′, H5′), and 3.25, ddd, (J = 11.0, 3.5 Hz, H-4)], oxygenated methylene protons with geminal coupling as a methine proton [δH 3.84, dd (J = 12.0, 2.5 Hz, H-6′b), 3.71, dd, (J = 12.0, 5.0 Hz, H-6′a)], two methine protons [δH 2.13, m (H-5), 1.79, m (H-6)], methylene protons with geminal coupling as a methine proton [δH 1.79, m (H-2b), 1.54, dd (J = 15.0, 3.5 Hz, H-2a)], and four methyl protons [δH 2.29, s (H3-10), 1.13, s (H3-11), 0.99, s (H3-13), and 0.83, s (H312)] (Table 1). The 13C NMR, DEPT and HSQC spectra of 2 revealed 13 signals, including a carbonyl carbon (δC 201.0), two olefinic carbons (δC 151.1 and 134.3), two oxygenated methyne carbons (δC 88.1, 70.1), two aliphatic methine carbons (δC 58.4, 32.5), a methylene carbon (δC 32.5), four methyl carbons (δC 32.2, 26.9, 24.0, and 17.1), and a quaternary carbon (δC 32.5), as well as six carbon signals of a hexose moiety (δC 105.8, 78.0, 77.7, 75.3, 71.3, 62.4) (Table 1). The 1H− 1H COSY, HSQC and HMBC spectra of the sugar moiety in 2 revealed the connectivities between the signals at H-1′, H-6′b (δH 3.84, dd), H-6′a (δH 3.71, dd), H-3′/H-4′ (δH 3.38, m), and H-2′/H-5′ (δH 3.29, m) and C-1′ (δC 105.8), C-3′ (δC 78.0), C5′ (δC 77.7), C-2′ (δC 75.3), C-4′ (δC 71.3), and C-6′ (δC 62.4), supporting the presence of a hexose moiety. The coupling constant of 8.0 Hz between H-1′ (δH 4.39, d) and H-2′ (δH 3.29, m) indicated a diaxial relationship between these two protons, as well as the β-configuration at the anomeric proton. These data suggested that the hexose moiety is a β-glucopyranose.29 Moreover, acid hydrolysis of 2, followed by its chemical conversion with L-cysteine methyl
6
ester and o-tolylisothiocyanate into a thiocarbamoyl-thiazoline derivative, clarified that the βglucopyranose moiety in 2 was the D-form.30, 31 However, the 1H− 1H COSY correlations of H-3 (δH 4.23, ddd) with H-2a (δH 1.54), H-2b (δH 1.79, m), and H-4 (δH 3.25, dd) and of H-5 (δH 2.13, m) with H-4 and H-6 (δH 1.79, m), and the HMBC correlations from H-2b to C-6 (δC 58.4), from H-3 to C-1 (δC 34.3), from H3-11 (δH 1.13, s) and H3-12 (δH 0.83, s) to C-1, C-2 (δC 45.2), and C-6, and from H3-13 (δH 0.99, s) to C-4 (δC 88.1), C-5 (δC 32.5), and C-6 indicated the presence of a 3,4-dioxygenated 1,1,5-trimethylcyclohexane moiety as a partial structure of 2 (Fig. 4). The further 1H− 1H COSY correlations of H-7 (δH 6.71, dd) with H-6 and H-8 (δH 6.07, d), and the HMBC correlations from H-7 to C-1, C-5, C-6, and C-9 (δC 201.0), from H-8 to C-6, C-9, and C10 (δC 26.9), and from H-10 (δH 2.29, s) to C-8 (δC 134.3) and C-9 confirmed that the aglycone part was 3,4-dihydroxy-7-megastigmen-9-one. The HMBC correlations from H-1′ to C-4 and from H-4 to C-1′, as well as the NOESY correlations of H-1′ and H-4, suggested that the D-β-glucopyranose moiety was linked to the hydroxyl group at C-4 in the aglycone (Fig. 4). On the basis of the 1 H NMR and NOESY experiments, the relative stereochemistry of the aglycone moiety in 2 was determined as the chair conformation (Fig. 4). The smaller coupling constants of 3.5 Hz between H-3 and H-4 confirmed the equatorial orientation at H-3. The NOESY correlations between H-4, H3-11, and H-6, as well as the coupling constants of 11.0 Hz between H-4 and H-5 (2.13, m), suggested the axial orientations of H-4, H-5, and H-6. The 1H and 13C NMR of 2 were similar to those of lasianthionoside C [(3S, 4S, 5S, 6S, 7E)-3,4-dihydroxymegastigman-7-en-9-one-4O-β-D-glucopyranoside].32 The observed significant differences were the chemical shift of C4 (2: δC 88.1; lasianthionoside C: δC 80.7) and the coupling constant values of H-4 [2: δH 3.25, dd (J = 11.0, 3.5 Hz); lasianthionoside C: δH 3.70, br s], which suggested the β configuration at C-4. Consequently, compound 2 was elucidated to be (3S, 4R, 5S, 6S, 7E)-
7
3,4-dihydroxymegastigman-7-en-9-one-4-O-β-D-glucopyranoside,
and
was
named
grandionoside A. The cytotoxicity of the isolated compounds (1−12) against the growth of nine human cancer cell lines was tested by a sulforhodamine B assay. These data revealed that 1 and 12 exhibited selective inhibitory effect against the tested human cancer cell lines with different IC50 values (Table 2). The significant cytotoxicity against the LU-1 and SK-Mel-2 cell lines (IC50 values of 4.68 and 3.63 µM, respectively), as well as the moderate cytotoxicity against the KB, Hep-G2, MKN-7, SW-480, and HL-60 cell lines (IC50 values ranging from 6.22 to 13.35 µM) was observed for 1. In addition, this compound showed very weak inhibition against the MDA-BA-231 and LNCaP cell lines (IC50 values of 64.06 and 45.43 µM, respectively). Compound 12 demonstrated potent cytotoxicity against the LU-1, KB, Hep-G2, MKN-7, and SW-480 cell lines, with IC50 values ranging from 1.24 to 1.60 µM, as well as moderate cytotoxic effects against the MDA-BA-231, LNCaP, and HL-60 cell lines with IC50 values ranging from 9.70 to 22.58 µM. Remarkably, the cytotoxicity of both compounds on the normal 3T3 cell line was significantly lower than that of the positive control, ellipticine. Based on the obtained results, the species of the genus Uvaria L. and these compounds might be selected for further studies regarding their anticancer features.
Acknowledgments This work was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (T.I. and H.M.) and for JSPS Fellows (N.T.H). Financial support for this work from the Japan Society for the Promotion of Science (ID No. P14412) is gratefully acknowledged.
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Supplementary data Supplementary data associated with this article can be found, in the online version, at ------.
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References and notes 1.
Ban, N. T. Sci-Techn. Publ. House, Hanoi, Vietnam 2000, Vol I, p 59.
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Pan, X. P.; Yu, D. Q. Chin. Chem. Lett. 1995, 6, 473.
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Liao, Y. H.; Xu, L. Z.; Yang, S. L.; Dai, J.; Zhen, Y. S.; Zhut, M.; Sun, N. J. Phytochemistry 1997, 45, 729.
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Liao, Y. H.; Zou, Z. M.; Guo, J.; Xu, L. Z.; Zhu, M.; Yang, S. L. J. Chin. Pharm. Sci. 2000, 9, 170.
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Pan, X. P.; Yu, D. Q. Phytochemistry 1995, 40, 1709.
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Pan, X. P.; Chen, R. Y.; Yu, D. Q. Phytochemistry 1998, 47, 1063.
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Ankisetty, S.; ElSohly, H. N.; Li, X. C.; Khan, S. I.; Tekwani, B. L.; Smillie, T.; Walker, L. J. Nat. Prod. 2006, 69, 692.
8.
Seangphakdee, P.; Pompimon, W.; Meepowpan, P.; Panthong, A.; Chiranthanut, N.; Banjerdpongchai, R.; Wudtiwai, B.; Nuntasaen, N.; Pitchuanchom, S. ScienceAsia 2013, 39, 610.
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Stevenson, P. C.; Veitch, N. C.; Simmonds, M. S. J. Phytochemistry 2007, 68, 1579.
10. Nguyen, T. H.; Ho, V. D.; Do, T. T.; Bui, H. T.; Phan, V. K.; Sak, K.; Raal, A. Nat. Prod. Res. 2015, 29, 247. 11. Nguyen, T. H.; Ho, V. D.; Phan, V. K. Vietnam Journal of Chemistry 2013, 51, 736. 12. Preparation of extracts: The aerial parts of U. grandiflora (6.0 kg) were extracted with MeOH (3 x 10 L) for 72 h at room temperature (25°C). After removal the solvent under reduced pressure, the MeOH extract (535 g) was then suspended in water and successively partitioned with CHCl3 and EtOAc (each, 2.5 L x 3 times) to obtain the CHCl3-soluble portion (214 g), the EtOAc-soluble portion (153 g), and the water-soluble portion (128 g).
10
13. Sumathykutty, M. A.; Rao, J. M. Phytochemistry 1991, 30, 2075. 14. Jolad, S. D.; Hoffmann, J. J.; Schram, K. H.; Cole, J. R. J. Org. Chem. 1981, 46, 4267. 15. Sigh, J.; Dhar, K. L.; Atal, C. K. Tetrahedron 1970, 26, 4403. 16. Mouffok, S.; Haba, H.; Lavaud, C.; Long, C.; Benkhaled, M. Rec. Nat. Prod. 2012, 6, 292. 17. Omar, S.; Chee, C. L.; Ahmad, F.; Ni, J. X.; Jaber, H.; Huang, J.; Nakatsu, T. Phytochemistry 1992, 31, 4395. 18. Xu, Z. B.; Qu, J. Sci. China Chem. 2011, 54, 1718. 19. Lee, I. K.; Kim, M. A.; Lee, S. Y.; Hong, J. K.; Lee, J. H.; Lee, K. R. Natural Products Sciences 2008, 14, 100. 20. Kitagawa, I.; Ikenishi, Y.; Yoshikawa, M.; Yosioka, I. Chem. Pharm. Bull. 1976, 24, 2470. 21. Jansakul, C.; Herbert, B.; Kenne, L.; Samuelsson, G. Planta Med. 1987, 405. 22. Compound 1: Colorless powder; mp 123−124 °C; [α]20 D −13.8 (c 0.4, CHCl3); IR (KBr) νmax 3507, 3410, 3202, 1711, 1677, 1601 cm−1; UV (MeOH) λmax (nm): 198, 235, 268; 1
H NMR and
13
C NMR (CDCl3): see Table 1; HRESIMS m/z 301.0679 [M+Na]+ (calcd
for C14H14O6Na, 301.0688). 23. Isolation of compound 1: The EtOAc-soluble portion (153 g) was separated by silica gel column chromatography (CHCl3–MeOH, 40:1−1:1) to obtain five fractions (B1 to B5). The fraction B5 was passed through a reversed-phase column eluted with acetone–water (1:2), to give compound 1 (39.4 mg). 24. Crystallographic data of compound 1: C14H14O6, M = 278.25, monoclinic, space group P21, a = 10.856 (2) Å, b = 5.6659 (12) Å, c = 11.104 (2) Å, β = 110.605 (2)°, V = 639.3 (2) Å3, Z = 2, Dcalcd = 1.445 g/cm3, T = 100 K, F(000) = 292, and µ (Mo Kα) = 0.114 mm−1. A total of 3953 reflections (2633 unique, Rint = 0.0189) were collected from 1.96° 11
to 29.15° in θ and index ranges of 8 ≥ h ≥ −14, 7 ≥ k ≥ −7, 14 ≥ l ≥ −11. The final stage converged to R1 = 0.0293 (wR2 = 0.0754) for 2608 observed reflections [with I > 2σ(I)] and 184 variable parameters and R1 = 0.0295 (wR2 = 0.0758) for all unique reflections, with a GoF = 1.014. Crystallographic data for the structure of 1 have been deposited with the Cambridge Crystallographic Data Centre (Deposition number CCDC 1063940). Copies of these data can be obtained, free of charge, upon application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: [email protected]). 25. Takeuchi, Y.; Wenshi, Q.; Sugiyama, T.; Oritani, T. Biosci. Biotechnol. Biochem. 2002, 66, 537. 26. Palframan, M. J.; Kohn, G. K.; Lewis, S. E. Chem. Eur. J. 2012, 18, 4766. 27. Compound 2: Colorless powder; [α]20 D −20.8 (c 0.1, MeOH); IR (KBr) ν max 3390, 1693 cm−1; 1H NMR and
13
C NMR (CD3OD): see Table 1; HRESIMS found m/z 411.1989
[M+Na]+ (calcd for C19H32O8Na, 411.1995). 28. Isolation of compound 2: The water-soluble fraction (128 g) was applied to a Diaion HP 20 column, eluted with aq. MeOH (0% to 100%) to obtain five fractions (C1 to C5). The fraction C5 (25.4 g) was chromatographed on a silica gel column elutied with CHCl3– MeOH–water (7.5:1.5:0.1) to give five sub-fractions (C5-1 to C5-5). The sub-fraction C5-3 (4.5 g) was purified by reversed-phase HPLC (MeOH–water, 1:2) to afford compound 2 (30.1 mg). 29. Roslund, M. U.; Tähtinen, P.; Niemitz, M.; Sjöholm, R. Carbohydr. Res. 2008, 343, 101. 30. Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem. Pharm. Bull. 2007, 55, 899. 31. Determination of stereochemistry of sugar moiety in compound 2: 2 (1.0 mg) was dissolved in 5% aqueous H2SO4−1,4-dioxane (1:1, v/v, 1.0 mL), and the solution was 12
heated at 90°C for 3 h and then was evaporated under reduced pressure. After extraction with EtOAc, the aqueous layer was neutralized using Amberlite IRA-400. The residue was dissolved in pyridine (0.1 mL) containing L-cysteine methyl ester hydrochloride (0.5 mg), and was heated at 60°C for 1 h. A solution of o-tolylisothiocyanate (0.5 mg) in pyridine (0.1 mL) was then added to the solution, which was incubated at 60°C for 1 h. The authentic samples were also prepared from D-glucose and L-glucose. These otolylthiocarbamoyl thiazolidine derivatives were analyzed by reversed-phase HPLC [Cosmosil 5C18-AR-II, 250×4.6 mm (Nacalai Tesque); mobile phase: MeCN−H2O in 0.1% TFA (80:20, v/v); detection: UV 250 nm; flow rate: 0.8 mL/min; column temperature: 35°C]. The peak (tR = 32.9 min) coincided with a derivative of D-glucose, as compared with the retention times of these authentic samples (D-glucose derivative, tR = 32.9 min; L-glucose derivative, tR = 29.5 min). 32. Takeda, Y.; Shimizu, H.; Masuda, T.; Hirata, E.; Shinzato, T.; Bando, M.; Otsuka, H. Phytochemistry 2004, 65, 485.
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Table 1 1
H (500 MHz) and 13C (125 MHz) NMR data for 1 and 2
Position 1 2 3 4 5 6 7 1′ 2′, 6′ 3′, 5′ 4′ 7′
1 in CDCl3 δC δH (mult., J in Hz) 76.3 74.2 3.98 (d, 5.5) 68.1 4.69 (m) 148.2 6.91 (dd, 10.5, 3.5) 126.9 6.14 (dd, 10.5, 2.0) 195.4 64.1 4.60 (d, 11.5) 4.78 (d, 11.5) 129.1 129.9 7.95 (dd, 8.0, 1.0) 128.5 7.41 (dd, 8.0, 8.0) 133.6 7.56 (t, 8.0) 166.9
Position 1 2-a 2-b 3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′-a 6′-b
2 in CD3OD δC δH (mult., J in Hz) 34.3 45.2 1.54 (dd, 15.0, 3.5) 1.79a (m) 70.1 4.23 (ddd, 3.5, 3.5, 3.0) 88.1 3.25 (dd, 11.0, 3.5) 32.5 2.13a (m) 58.4 1.79a (m) 151.1 6.71 (dd, 16.0, 10.0) 134.3 6.07 (d, 16.0) 201.0 26.9 2.29 (s) 24.0 1.13 (s) 32.2 0.83 (s) 17.1 0.99 (d, 6.5) 105.8 4.39 (d, 8.0) 75.3 3.29a (m) 78.0 3.38a (m) 71.3 3.38a (m) 77.7 3.29a (m) 62.4 3.71 (dd,12.0, 5.0) 3.84 (dd, 12.0, 2.5)
Assignments were done by HSQC, HMBC, COSY, and NOESY experiments. a overlapping signals.
14
Table 2 Cytotoxicity of 1 and 12 against human cancer cell lines Cell lines
1 4.68 13.35 64.06 45.43 13.13 8.63 11.26 6.22 3.63 25.86
LU-1 (human lung cancer) KB (epidermoid carcinoma) MDA-BA-231 (high metastasis human breast cancer) LNCaP (human prostate cancer) Hep-G2 (human hepatoma cancer) MKN-7 (stomach cancer) SW-480 (human colon adenocarcinoma) HL-60 (human acute leukemia) SK-Mel-2 (melanoma carcinoma) 3T3 (normal cell) a b
IC50 (concentration that inhibits of 50% of cell growth). Positive control.
15
IC50a (µM) 12 1.41 1.60 11.76 9.70 1.53 1.27 1.24 22.58 − 7.65
Ellipticineb 2.72 3.98 3.21 3.21 3.50 3.82 3.17 2.93 1.14 1.26
Figure legends
Figure 1. Structures of compounds 1–12 from the aerial parts of Uvaria grandiflora. Figure 2. COSY (bold lines), Key HMBC (1H→13C, arrows), and NOESY (dashed arrows) correlations for 1. Figure 3. ORTEP drawing of the X-ray crystal structure of 1. Figure 4. COSY (bold lines), Key HMBC (1H→13C, arrows), and NOESY (dashed arrows) correlations for 2.
16
17
18
19
20
21
S0960-894X(15)00540-5 http://dx.doi.org/10.1016/j.bmcl.2015.05.066 BMCL 22756
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
9 May 2015 22 May 2015 25 May 2015
Please cite this article as: Ho, D.V., Kodama, T., Le, H.T.B., Phan, K.V., Do, T.T., Bui, T.H., Le, A.T., Win, N.N., Imagawa, H., Ito, T., Morita, H., Nguyen, H.T., A new polyoxygenated cyclohexene and a new megastigmane glycoside from Uvaria grandiflora, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/ 10.1016/j.bmcl.2015.05.066
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A new polyoxygenated cyclohexene and a new megastigmane glycoside from Uvaria grandiflora
Duc Viet Hoa, Takeshi Kodamab, Hien Thi Bich Lea, Kiem Van Phanc, Thao Thi Dod, Tai Huu Buic,e, Anh Tuan Lef, Nwet Nwet Winb, Hiroshi Imagawag, Takuya Ito b,*, Hiroyuki Moritab,*, Hoai Thi Nguyena,b,* a
Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo
Quyen, Hue City, Vietnam b
Insitute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194,
Japan c
Institute of Marine Biochemistry, VAST, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
d e f
Institute of Biotechnology, VAST, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
Quang Tri Center of Science and Technology, Mientrung Inst. for Scientific Research, 121 Ly
Thuong Kiet, Dong Ha, Quang Tri, Vietnam g
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho,
Tokushima 770-8514, Japan *To whom correspondence should be addressed: Associate Professor Takuya Ito, Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan (Tel: +8176-434-7627; Fax: +81-76-434-5059, E-mail: [email protected]), Professor Hiroyuki Morita, Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 9300194, Japan (Tel: +81-76-434-7625; Fax: +81-76-434-5059, E-mail: [email protected]), or Associate Professor Nguyen Thi Hoai, Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo Quyen, Hue City, Vietnam (Tel: +84905-974-785, E-mail: [email protected])
1
Abstract A new polyoxygenated cyclohexene, (−)-3-O-debenzoylzeylenone (1), and a new megastigmane glycoside, grandionoside A (2), were isolated from the aerial parts of Uvaria grandiflora collected in Vietnam, together with ten known compounds including polyoxygenated cyclohexenes (3−6), a triterpenoid (7), an alkaloid (8), a long chain alcohol (9), hexenyl glycopyranoside (10), and saponins (11−12). Their chemical structures were elucidated by a combination of extensive NMR spectroscopy with X-ray crystallographic analysis for 1, and chemical conversion for 2. Compound 1 exhibited significant cytotoxicity against the LU-1 and SK-Mel-2 cell lines with IC50 values of 4.68 and 3.63 µM, respectively. Remarkably, the cytotoxicity of 12 against the LU-1, KB, Hep-G2, MKN-7, and SW-480 cell lines was comparable to that of ellipticine, the positive control, with IC50 values ranging from 1.24 to 1.60 µM.
Key words: Uvaria grandiflora, polyoxygenated cyclohexene, megastigmane glycoside, cytotoxicity.
2
Uvaria grandiflora Roxb. ex Hornem (Annonaceae), also known as Unona grandiflora or Uvaria purpurea, is a long woody vine widely distributed in Southeast Asia, including Central Vietnam, India, Myanmar, China, Sri Lanka, Malaysia, and Indonesia, but is rarely cultivated.1 The plant has been used as a source of traditional medicines for cancer therapies in Vietnam. Previous phytochemical studies of this species have led to the isolation of acetogenins,2 polyoxygenated cyclohexenes,3-6 and aromatic compounds,7 with some displaying interesting anti-tumour,3, 8 anti-leishmanial,7 anti-inflammatory,8 and anti-feedant activities.9 In our previous studies focusing on the constituents of Uvaria plants and their cytotoxicity, we discovered the presence of the unique lignan glycoside, ufaside, in the aerial parts of Uvaria rufa (Dun.) Blume10 and the unique aromatic compound, coraurarin A, in the leaves of Uvaria cordata (Dun.) Wall. Ex Alston11, together with known compounds, such as an oxoaporphine alkaloid, ergosterols, and catechins. Among these constituents, oxoanolobine and ergosta-4,6,8(14),22-tetraen-3-one displayed moderate cytotoxicity against a human lung adenocarcinoma cell line (LU-1). We also observed that the methanol extract of U. grandiflora exhibits potent in vitro cytotoxicity against nine cancer cell lines (LU-1, human lung cancer cell; KB, epidermoid carcinoma; MDA-BA-231, high metastasis human breast cancer; LNCaP, human prostate cancer; Hep-G2, human hepatoma cancer; MKN-7, stomach cancer; SW-480, human colon adenocarcinoma; HL-60, human acute leukemia; SKMel-2, melanoma carcinoma). Therein, the strongest cytotoxicity was found against the MDA-BA-231, MKN-7 and KB cell lines, with IC50 values of 0.62 µM, 0.89 µM and 1.67 µM, respectively. In our continuous studies on the constituents of Uvaria plants and their cytotoxic activity, the CHCl3-, EtOAc-, and water-soluble portions were thus prepared from the active methanol extract of this plant.12 Bioassay-guided separation of the soluble portions afforded a new polyoxygenated cyclohexene, (−)-3-O-debenzoylzeylenone (1), and a new 3
megastigmane glycoside, grandionoside A (2), together with ten known compounds, pipoxide chlorohydrin (3),13 (−)-zeylenone (4),3 (−)-zeylenol (5),14 (+)-pipoxide (6),15 lupeol (7),16 velutinam (8),17 threo-octadecane-1,9,10-triol (9),18 (Z)-3-hexenyl-1-O-β-D-glucopyranoside (10),19 sakurasosaponin (11),20 and ardisiacrispin B (12)21 (Fig. 1). The spectral data of the known compounds were confirmed with the reported data. Among the isolated known compounds, 10−12 were isolated from this genus for the first time, while 9 was isolated for the first time as a natural product. Herein, the isolation and structure elucidation of the two new compounds (1 and 2), as well as the cytotoxicity of all of the isolated compounds (1−12) are described. Compound 122 was obtained as a colorless powder from the EtOAc-soluble portion.23 The ESIMS of 1 showed a quasimolecular ion peak at m/z 301 [M+Na]+. Its molecular formula was determined to be C14H14O6 by HRESIMS, in conjunction with an NMR data analysis. The IR spectrum of 1 revealed strong absorption bands corresponding to an aromatic ring (1601, 1585 cm−1), a conjugated carbonyl (1711 cm−1), an ester (1677 cm−1), and hydroxy groups (3507, 3410, 3202 cm−1). The UV spectrum supported the presence of the aromatic ring (λmax 268 nm) and an α,β-unsaturated ketone (λmax 235 nm). The 1H NMR spectrum of 1 in CDCl3 showed signals of five aromatic protons at δH 7.41-7.95 (5H), two olefinic protons [δH 6.14, dd (J = 10.5, 2.0 Hz, H-5), 6.91, dd (J = 10.5, 3.5 Hz, H-4)], oxygenated methylene protons with geminal coupling as a methine proton [δH 4.60, 4.78, both d (J = 11.5 Hz, H2-7)], and two oxygenated methine protons [δH 3.98, d (J = 5.5 Hz, H2), 4.69 (m, H-3)] (Table 1). Analyses of the
13
C NMR, DEPT, and HSQC spectra of 1
revealed 14 signals, including two carbonyl carbons (δC 195.4, 166.9), six aromatic carbons (δC 133.6, 129.9, 129.1, 128.5), two olefinic carbons (δC 148.2, 126.9), an oxygenated methylene carbon (δC 64.1), two oxygenated methine carbons (δC 74.2, 68.1), and an
4
oxygenated quaternary carbon (δC 76.3) (Table 1). The set of aromatic proton signals at δH 7.41 (dd, J = 8.0, 8.0 Hz, H2-3′/5′), δH 7.56 (t, J = 8.0 Hz, H-4′), and δH 7.95 (dd, J = 8.0, 1.0 Hz, H2-2′/6′), and the HMBC correlations from H-2′/6′ to C-1′ (δC 129.1) and C-7′ (δC 166.9), suggested the presence of a benzoyl moiety in 1 (Fig. 2). However, the 1H− 1H COSY correlations of H-3 (δH 4.69, m) with H-2 (δH 3.98, d) and H-4 (δH 6.91, dd) and of H-4 with H-5 (δH 6.14, dd), and the HMBC correlations from H-2 to C-1 (δC 76.3) and C-6 (δC 195.4) and from H-5 to C-1, indicated that 1 possesses a trioxygenated cyclohexenone moiety. Further analyses of the HSQC, COSY, and HMBC spectra revealed that the remaining oxygenated methylene protons at H2-7 (δH 4.60, 4.78, both d) were connected to C-1 (δC 76.3), C-2 (δC 74.2), and C-6 (δC 195.4) on the trioxygenated cyclohexenone moiety and to C-7′ (δC 166.9) on the benzoyl moiety (Fig. 2). These results indicated that the oxygenated quaternary carbon at C-1 (δC 76.3) was directly linked to the methylene carbon at C-7, which was further connected to the benzoyl group through an oxygen atom. Furthermore, the coupling constant of 5.5 Hz between H-2 (δH 3.98, d) and H-3 (4.69, m) indicated an axial/pseudo-axial relationship between these protons as well as the 2,3-anti-dihydroxy groups. In addition, the NOESY correlation between H-2 and H2-7 and the loss of the correlation between H-2 and H3 (4.69, m) suggested the presence of 1,2-syn-dihydroxy groups (Fig. 2). Finally, the X-ray crystallographic analysis of 124 allowed us to confirm the planar and relative structure of 1 to be the same as that of the previously reported (+)-3-O-debenzoylzeylenone25 (Fig. 3). However, the optical rotation of 1 was [α]20D −13.8 (c 0.4, CHCl3), which is almost opposite to the value of [α]20D +11.7 (c 0.36, CHCl3)25 and [α]20D +12.5 (c 0.4, CHCl3)26 for (+)-3-Odebenzoylzeylenone. Compound 1 was thus verified to be the enantiomer of (+)-3-Odebenzoylzeylenone, and was identified as (−)-3-O-debenzoylzeylenone. Compound 227 was obtained as a colorless powder from the water-soluble portion.28 The ESIMS of 2 indicated a quasimolecular ion peak at m/z 411 [M+Na]+. Its molecular 5
formula was determined further to be C19H32O8, by HRESIMS and
13
C NMR data. The IR
spectrum of 2 showed strong absorption bands corresponding to conjugated carbonyl (1693 cm−1) and hydroxy (3390 cm−1) groups. The 1H NMR spectrum in CD3OD revealed the signals of two trans-olefinic protons [δH 6.07, d (J = 16.0 Hz, H-8), 6.71, dd (J = 16.0, 10.0 Hz, H-7)], an anomeric proton [δH 4.39, d (J = 8.0 Hz, H-1′)], five oxygenated methine protons [δH 4.23, ddd, (J = 3.5, 3.5, 3.0 Hz, H-3), 3.29-3.38 (overlapped, H-2′, H-3′, H-4′, H5′), and 3.25, ddd, (J = 11.0, 3.5 Hz, H-4)], oxygenated methylene protons with geminal coupling as a methine proton [δH 3.84, dd (J = 12.0, 2.5 Hz, H-6′b), 3.71, dd, (J = 12.0, 5.0 Hz, H-6′a)], two methine protons [δH 2.13, m (H-5), 1.79, m (H-6)], methylene protons with geminal coupling as a methine proton [δH 1.79, m (H-2b), 1.54, dd (J = 15.0, 3.5 Hz, H-2a)], and four methyl protons [δH 2.29, s (H3-10), 1.13, s (H3-11), 0.99, s (H3-13), and 0.83, s (H312)] (Table 1). The 13C NMR, DEPT and HSQC spectra of 2 revealed 13 signals, including a carbonyl carbon (δC 201.0), two olefinic carbons (δC 151.1 and 134.3), two oxygenated methyne carbons (δC 88.1, 70.1), two aliphatic methine carbons (δC 58.4, 32.5), a methylene carbon (δC 32.5), four methyl carbons (δC 32.2, 26.9, 24.0, and 17.1), and a quaternary carbon (δC 32.5), as well as six carbon signals of a hexose moiety (δC 105.8, 78.0, 77.7, 75.3, 71.3, 62.4) (Table 1). The 1H− 1H COSY, HSQC and HMBC spectra of the sugar moiety in 2 revealed the connectivities between the signals at H-1′, H-6′b (δH 3.84, dd), H-6′a (δH 3.71, dd), H-3′/H-4′ (δH 3.38, m), and H-2′/H-5′ (δH 3.29, m) and C-1′ (δC 105.8), C-3′ (δC 78.0), C5′ (δC 77.7), C-2′ (δC 75.3), C-4′ (δC 71.3), and C-6′ (δC 62.4), supporting the presence of a hexose moiety. The coupling constant of 8.0 Hz between H-1′ (δH 4.39, d) and H-2′ (δH 3.29, m) indicated a diaxial relationship between these two protons, as well as the β-configuration at the anomeric proton. These data suggested that the hexose moiety is a β-glucopyranose.29 Moreover, acid hydrolysis of 2, followed by its chemical conversion with L-cysteine methyl
6
ester and o-tolylisothiocyanate into a thiocarbamoyl-thiazoline derivative, clarified that the βglucopyranose moiety in 2 was the D-form.30, 31 However, the 1H− 1H COSY correlations of H-3 (δH 4.23, ddd) with H-2a (δH 1.54), H-2b (δH 1.79, m), and H-4 (δH 3.25, dd) and of H-5 (δH 2.13, m) with H-4 and H-6 (δH 1.79, m), and the HMBC correlations from H-2b to C-6 (δC 58.4), from H-3 to C-1 (δC 34.3), from H3-11 (δH 1.13, s) and H3-12 (δH 0.83, s) to C-1, C-2 (δC 45.2), and C-6, and from H3-13 (δH 0.99, s) to C-4 (δC 88.1), C-5 (δC 32.5), and C-6 indicated the presence of a 3,4-dioxygenated 1,1,5-trimethylcyclohexane moiety as a partial structure of 2 (Fig. 4). The further 1H− 1H COSY correlations of H-7 (δH 6.71, dd) with H-6 and H-8 (δH 6.07, d), and the HMBC correlations from H-7 to C-1, C-5, C-6, and C-9 (δC 201.0), from H-8 to C-6, C-9, and C10 (δC 26.9), and from H-10 (δH 2.29, s) to C-8 (δC 134.3) and C-9 confirmed that the aglycone part was 3,4-dihydroxy-7-megastigmen-9-one. The HMBC correlations from H-1′ to C-4 and from H-4 to C-1′, as well as the NOESY correlations of H-1′ and H-4, suggested that the D-β-glucopyranose moiety was linked to the hydroxyl group at C-4 in the aglycone (Fig. 4). On the basis of the 1 H NMR and NOESY experiments, the relative stereochemistry of the aglycone moiety in 2 was determined as the chair conformation (Fig. 4). The smaller coupling constants of 3.5 Hz between H-3 and H-4 confirmed the equatorial orientation at H-3. The NOESY correlations between H-4, H3-11, and H-6, as well as the coupling constants of 11.0 Hz between H-4 and H-5 (2.13, m), suggested the axial orientations of H-4, H-5, and H-6. The 1H and 13C NMR of 2 were similar to those of lasianthionoside C [(3S, 4S, 5S, 6S, 7E)-3,4-dihydroxymegastigman-7-en-9-one-4O-β-D-glucopyranoside].32 The observed significant differences were the chemical shift of C4 (2: δC 88.1; lasianthionoside C: δC 80.7) and the coupling constant values of H-4 [2: δH 3.25, dd (J = 11.0, 3.5 Hz); lasianthionoside C: δH 3.70, br s], which suggested the β configuration at C-4. Consequently, compound 2 was elucidated to be (3S, 4R, 5S, 6S, 7E)-
7
3,4-dihydroxymegastigman-7-en-9-one-4-O-β-D-glucopyranoside,
and
was
named
grandionoside A. The cytotoxicity of the isolated compounds (1−12) against the growth of nine human cancer cell lines was tested by a sulforhodamine B assay. These data revealed that 1 and 12 exhibited selective inhibitory effect against the tested human cancer cell lines with different IC50 values (Table 2). The significant cytotoxicity against the LU-1 and SK-Mel-2 cell lines (IC50 values of 4.68 and 3.63 µM, respectively), as well as the moderate cytotoxicity against the KB, Hep-G2, MKN-7, SW-480, and HL-60 cell lines (IC50 values ranging from 6.22 to 13.35 µM) was observed for 1. In addition, this compound showed very weak inhibition against the MDA-BA-231 and LNCaP cell lines (IC50 values of 64.06 and 45.43 µM, respectively). Compound 12 demonstrated potent cytotoxicity against the LU-1, KB, Hep-G2, MKN-7, and SW-480 cell lines, with IC50 values ranging from 1.24 to 1.60 µM, as well as moderate cytotoxic effects against the MDA-BA-231, LNCaP, and HL-60 cell lines with IC50 values ranging from 9.70 to 22.58 µM. Remarkably, the cytotoxicity of both compounds on the normal 3T3 cell line was significantly lower than that of the positive control, ellipticine. Based on the obtained results, the species of the genus Uvaria L. and these compounds might be selected for further studies regarding their anticancer features.
Acknowledgments This work was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (T.I. and H.M.) and for JSPS Fellows (N.T.H). Financial support for this work from the Japan Society for the Promotion of Science (ID No. P14412) is gratefully acknowledged.
8
Supplementary data Supplementary data associated with this article can be found, in the online version, at ------.
9
References and notes 1.
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Seangphakdee, P.; Pompimon, W.; Meepowpan, P.; Panthong, A.; Chiranthanut, N.; Banjerdpongchai, R.; Wudtiwai, B.; Nuntasaen, N.; Pitchuanchom, S. ScienceAsia 2013, 39, 610.
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10. Nguyen, T. H.; Ho, V. D.; Do, T. T.; Bui, H. T.; Phan, V. K.; Sak, K.; Raal, A. Nat. Prod. Res. 2015, 29, 247. 11. Nguyen, T. H.; Ho, V. D.; Phan, V. K. Vietnam Journal of Chemistry 2013, 51, 736. 12. Preparation of extracts: The aerial parts of U. grandiflora (6.0 kg) were extracted with MeOH (3 x 10 L) for 72 h at room temperature (25°C). After removal the solvent under reduced pressure, the MeOH extract (535 g) was then suspended in water and successively partitioned with CHCl3 and EtOAc (each, 2.5 L x 3 times) to obtain the CHCl3-soluble portion (214 g), the EtOAc-soluble portion (153 g), and the water-soluble portion (128 g).
10
13. Sumathykutty, M. A.; Rao, J. M. Phytochemistry 1991, 30, 2075. 14. Jolad, S. D.; Hoffmann, J. J.; Schram, K. H.; Cole, J. R. J. Org. Chem. 1981, 46, 4267. 15. Sigh, J.; Dhar, K. L.; Atal, C. K. Tetrahedron 1970, 26, 4403. 16. Mouffok, S.; Haba, H.; Lavaud, C.; Long, C.; Benkhaled, M. Rec. Nat. Prod. 2012, 6, 292. 17. Omar, S.; Chee, C. L.; Ahmad, F.; Ni, J. X.; Jaber, H.; Huang, J.; Nakatsu, T. Phytochemistry 1992, 31, 4395. 18. Xu, Z. B.; Qu, J. Sci. China Chem. 2011, 54, 1718. 19. Lee, I. K.; Kim, M. A.; Lee, S. Y.; Hong, J. K.; Lee, J. H.; Lee, K. R. Natural Products Sciences 2008, 14, 100. 20. Kitagawa, I.; Ikenishi, Y.; Yoshikawa, M.; Yosioka, I. Chem. Pharm. Bull. 1976, 24, 2470. 21. Jansakul, C.; Herbert, B.; Kenne, L.; Samuelsson, G. Planta Med. 1987, 405. 22. Compound 1: Colorless powder; mp 123−124 °C; [α]20 D −13.8 (c 0.4, CHCl3); IR (KBr) νmax 3507, 3410, 3202, 1711, 1677, 1601 cm−1; UV (MeOH) λmax (nm): 198, 235, 268; 1
H NMR and
13
C NMR (CDCl3): see Table 1; HRESIMS m/z 301.0679 [M+Na]+ (calcd
for C14H14O6Na, 301.0688). 23. Isolation of compound 1: The EtOAc-soluble portion (153 g) was separated by silica gel column chromatography (CHCl3–MeOH, 40:1−1:1) to obtain five fractions (B1 to B5). The fraction B5 was passed through a reversed-phase column eluted with acetone–water (1:2), to give compound 1 (39.4 mg). 24. Crystallographic data of compound 1: C14H14O6, M = 278.25, monoclinic, space group P21, a = 10.856 (2) Å, b = 5.6659 (12) Å, c = 11.104 (2) Å, β = 110.605 (2)°, V = 639.3 (2) Å3, Z = 2, Dcalcd = 1.445 g/cm3, T = 100 K, F(000) = 292, and µ (Mo Kα) = 0.114 mm−1. A total of 3953 reflections (2633 unique, Rint = 0.0189) were collected from 1.96° 11
to 29.15° in θ and index ranges of 8 ≥ h ≥ −14, 7 ≥ k ≥ −7, 14 ≥ l ≥ −11. The final stage converged to R1 = 0.0293 (wR2 = 0.0754) for 2608 observed reflections [with I > 2σ(I)] and 184 variable parameters and R1 = 0.0295 (wR2 = 0.0758) for all unique reflections, with a GoF = 1.014. Crystallographic data for the structure of 1 have been deposited with the Cambridge Crystallographic Data Centre (Deposition number CCDC 1063940). Copies of these data can be obtained, free of charge, upon application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: [email protected]). 25. Takeuchi, Y.; Wenshi, Q.; Sugiyama, T.; Oritani, T. Biosci. Biotechnol. Biochem. 2002, 66, 537. 26. Palframan, M. J.; Kohn, G. K.; Lewis, S. E. Chem. Eur. J. 2012, 18, 4766. 27. Compound 2: Colorless powder; [α]20 D −20.8 (c 0.1, MeOH); IR (KBr) ν max 3390, 1693 cm−1; 1H NMR and
13
C NMR (CD3OD): see Table 1; HRESIMS found m/z 411.1989
[M+Na]+ (calcd for C19H32O8Na, 411.1995). 28. Isolation of compound 2: The water-soluble fraction (128 g) was applied to a Diaion HP 20 column, eluted with aq. MeOH (0% to 100%) to obtain five fractions (C1 to C5). The fraction C5 (25.4 g) was chromatographed on a silica gel column elutied with CHCl3– MeOH–water (7.5:1.5:0.1) to give five sub-fractions (C5-1 to C5-5). The sub-fraction C5-3 (4.5 g) was purified by reversed-phase HPLC (MeOH–water, 1:2) to afford compound 2 (30.1 mg). 29. Roslund, M. U.; Tähtinen, P.; Niemitz, M.; Sjöholm, R. Carbohydr. Res. 2008, 343, 101. 30. Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem. Pharm. Bull. 2007, 55, 899. 31. Determination of stereochemistry of sugar moiety in compound 2: 2 (1.0 mg) was dissolved in 5% aqueous H2SO4−1,4-dioxane (1:1, v/v, 1.0 mL), and the solution was 12
heated at 90°C for 3 h and then was evaporated under reduced pressure. After extraction with EtOAc, the aqueous layer was neutralized using Amberlite IRA-400. The residue was dissolved in pyridine (0.1 mL) containing L-cysteine methyl ester hydrochloride (0.5 mg), and was heated at 60°C for 1 h. A solution of o-tolylisothiocyanate (0.5 mg) in pyridine (0.1 mL) was then added to the solution, which was incubated at 60°C for 1 h. The authentic samples were also prepared from D-glucose and L-glucose. These otolylthiocarbamoyl thiazolidine derivatives were analyzed by reversed-phase HPLC [Cosmosil 5C18-AR-II, 250×4.6 mm (Nacalai Tesque); mobile phase: MeCN−H2O in 0.1% TFA (80:20, v/v); detection: UV 250 nm; flow rate: 0.8 mL/min; column temperature: 35°C]. The peak (tR = 32.9 min) coincided with a derivative of D-glucose, as compared with the retention times of these authentic samples (D-glucose derivative, tR = 32.9 min; L-glucose derivative, tR = 29.5 min). 32. Takeda, Y.; Shimizu, H.; Masuda, T.; Hirata, E.; Shinzato, T.; Bando, M.; Otsuka, H. Phytochemistry 2004, 65, 485.
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Table 1 1
H (500 MHz) and 13C (125 MHz) NMR data for 1 and 2
Position 1 2 3 4 5 6 7 1′ 2′, 6′ 3′, 5′ 4′ 7′
1 in CDCl3 δC δH (mult., J in Hz) 76.3 74.2 3.98 (d, 5.5) 68.1 4.69 (m) 148.2 6.91 (dd, 10.5, 3.5) 126.9 6.14 (dd, 10.5, 2.0) 195.4 64.1 4.60 (d, 11.5) 4.78 (d, 11.5) 129.1 129.9 7.95 (dd, 8.0, 1.0) 128.5 7.41 (dd, 8.0, 8.0) 133.6 7.56 (t, 8.0) 166.9
Position 1 2-a 2-b 3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′-a 6′-b
2 in CD3OD δC δH (mult., J in Hz) 34.3 45.2 1.54 (dd, 15.0, 3.5) 1.79a (m) 70.1 4.23 (ddd, 3.5, 3.5, 3.0) 88.1 3.25 (dd, 11.0, 3.5) 32.5 2.13a (m) 58.4 1.79a (m) 151.1 6.71 (dd, 16.0, 10.0) 134.3 6.07 (d, 16.0) 201.0 26.9 2.29 (s) 24.0 1.13 (s) 32.2 0.83 (s) 17.1 0.99 (d, 6.5) 105.8 4.39 (d, 8.0) 75.3 3.29a (m) 78.0 3.38a (m) 71.3 3.38a (m) 77.7 3.29a (m) 62.4 3.71 (dd,12.0, 5.0) 3.84 (dd, 12.0, 2.5)
Assignments were done by HSQC, HMBC, COSY, and NOESY experiments. a overlapping signals.
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Table 2 Cytotoxicity of 1 and 12 against human cancer cell lines Cell lines
1 4.68 13.35 64.06 45.43 13.13 8.63 11.26 6.22 3.63 25.86
LU-1 (human lung cancer) KB (epidermoid carcinoma) MDA-BA-231 (high metastasis human breast cancer) LNCaP (human prostate cancer) Hep-G2 (human hepatoma cancer) MKN-7 (stomach cancer) SW-480 (human colon adenocarcinoma) HL-60 (human acute leukemia) SK-Mel-2 (melanoma carcinoma) 3T3 (normal cell) a b
IC50 (concentration that inhibits of 50% of cell growth). Positive control.
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IC50a (µM) 12 1.41 1.60 11.76 9.70 1.53 1.27 1.24 22.58 − 7.65
Ellipticineb 2.72 3.98 3.21 3.21 3.50 3.82 3.17 2.93 1.14 1.26
Figure legends
Figure 1. Structures of compounds 1–12 from the aerial parts of Uvaria grandiflora. Figure 2. COSY (bold lines), Key HMBC (1H→13C, arrows), and NOESY (dashed arrows) correlations for 1. Figure 3. ORTEP drawing of the X-ray crystal structure of 1. Figure 4. COSY (bold lines), Key HMBC (1H→13C, arrows), and NOESY (dashed arrows) correlations for 2.
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