J Nat Med DOI 10.1007/s11418-014-0819-y
NOTE
Four new Amaryllidaceae alkaloids from Zephyranthes candida Nanase Shitara • Yusuke Hirasawa • Shunsuke Hasumi • Tadahiro Sasaki • Misaki Matsumoto • Chin Piow Wong Toshio Kaneda • Yoshinori Asakawa • Hiroshi Morita
•
Received: 12 December 2013 / Accepted: 17 January 2014 Ó The Japanese Society of Pharmacognosy and Springer Japan 2014
Abstract Four new Amaryllidaceae alkaloids (1–4) possessing a homolycorine-type or a crinine-type skeleton have been isolated from the aerial part of Zephyranthes candida, and their structures were elucidated on the basis of spectroscopic data. The stereochemistry was elucidated by combination of NOESY correlations and CD analyses. Keywords Zephyranthes candida Amaryllidaceae Homolycorine Crinine Zephyranthes candida (Lindl.) Herb. (Amaryllidaceae) is widely distributed in the tropical regions of the world, and is used as a medicinal plant in China [1]. The leaves and rhizomes of Z. candida show potent bitterness. Plants of the genus Zephyranthes comprise about 70 species [2], which
N. Shitara Y. Hirasawa S. Hasumi T. Sasaki M. Matsumoto C. P. Wong T. Kaneda H. Morita (&) Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41 Shinagawa-ku, Tokyo 142-8501, Japan e-mail: [email protected] Y. Asakawa Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
were reported to be rich sources of Amaryllidaceae alkaloids [3–5] such as lycorine [6], haemanthamine [7], (?)-tazettine [7], trisphaeridine [7], and galanthamine [8], among which galanthamine is used in the treatment of Alzheimer’s disease due to its selective and reversible inhibitory activity against acetylcholinesterase (AChE) [9, 10]. In our search for biogenetically interesting intermediates and new alkaloids with a novel skeleton from medicinal plants [11–19], a new homolycorine-type alkaloid, 2-hydroxyalbomaculine (1), and three new crinine-type alkaloids, 6a-hydroxyhippeastidine (2), 10-deoxy-6a-hydroxyhippeastidine (3), and 6bhydroxyhippeastidine (4), were isolated from the aerial part of Zephyranthes candida. We report here the isolation and structure elucidation of compounds 1–4, and the vasorelaxant activity against rat aortic ring of compound 1.
The molecular formula of compound 1 {½a23 D ? 107 (c 0.33, MeOH)} was determined to be C19H23NO6, as deduced by high-resolution electrospray ionization mass spectrometry (HRESIMS) [m/z 362.1606 (M ? H)?, D ?0.2 mmu], requiring 9 degrees of unsaturation. Absorption bands at 3734 and 1718 cm-1 in the IR spectrum implied the presence of OH and carbonyl functionalities, respectively.
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The 1H NMR spectrum displayed resonances for three methoxy (dH 3.84, dH 3.93, and dH 3.98), an N-methyl (dH 2.72), two olefinic protons (dH 5.85 and dH 7.12), and two oxymethines (dH 4.60 and dH 4.73). The 13C NMR spectrum showed resonances of 19 carbons attributable by the DEPT spectrum to three methoxy, an N-methyl, two methylene, six methine, a ketone, and six quaternary carbons, among which two methines (dC 79.4, dH 4.73 and dC 67.9, dH 4.60) were ascribed to those bearing an oxygen atom. The 1H–1H COSY spectrum revealed connectivity of two partial structures, a (C-1, 2, 3, 4a, and 10b) and b (C11 and C-12), as shown in Fig. 1. HMBC correlations of N-Me/C-12 (dC 56.9) and C-4a (dC 69.3), of H-3/C-4a, and of H-12 and H-10b/C-4 (dC 136.0) confirmed the presence of rings C and D. HMBC correlations between three methoxy groups (7-OMe, 8-OMe, and 9-OMe) and three aromatic carbons (dC 158.1, 144.7, and 159.7), respectively, and of H-10/C-6a (dC 111.8), C-8 (dC 144.7), and C-10b (dC 42.3), indicated that a tri-methoxyphenyl group was attached to C-10b. The molecule required an additional ring to satisfy the degree of unsaturation, suggesting that C-1 (dC 79.4) was bound to C-6a through an ester linkage. The relative configuration of 1 was deduced from a NOESY spectrum (Fig. 2). NOESY cross-peaks between H-1, H-2, and H-10b confirmed their location on the same face of the molecule. In addition, the coupling constant of H-4a/H-10b (9.6 Hz) revealed the anti-relationship between H-4a and H-10b. Thus, compound 1 was elucidated to be 2-hydroxyalbomaculine, possessing a homolycorine-type skeleton [20]. The CD spectrum of 1 in MeOH showed a similar CD curve to that of homolycorine [21]. Therefore, the absolute configurations of 1 were assigned as: 1S, 2R, 4aS, 10bS. The molecular formula, C18H25NO5, of compound 2 was established by HRESIMS [m/z 336.1824, (M ? H)?, D ?1.3 mmu]. The molecular formula accounted for 7 degrees of unsaturation. Absorption bands at 3380 and 1509 cm-1 in the IR spectrum implied the presence of OH and olefinic functionalities, respectively. 1H and 13C NMR data (Table 1) suggested the presence of three methyl, five sp3 methylene, three sp3 methine, an sp3 quaternary carbon, an sp2 methine, and five sp2 quaternary carbons. Among them, one sp3 methylene (dC 48.4, dH 3.21 and 3.81) and one sp3 methine (dC 62.6, dH 3.91) were attached to a nitrogen atom, and one sp3 methine (dC 88.7, dH 5.63) was attached to both a nitrogen atom and an oxygen atom. The 1H–1H COSY and HSQC spectra revealed connectivity of two partial structures, a (C-1–C-4, and C-4a) and b (C-11–C-12), as shown in Fig. 3. The HMBC correlations of H2-1/C-4a (dC 62.6), C-10a (dC 123.7), C-10b (dC 46.0), and C-11 (dC 31.6) indicated that C-1, C-4a,
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Fig. 1 Selected 2D NMR correlations for compound 1
Fig. 2 Selected NOESY correlations for compound 1
C-10a, and C-11 were linked with C-10b. The connectivity among C-4a, C-6, and C-12 through a nitrogen atom was deduced based on the HMBC correlations of H-6/C-4a and C-12 (dC 48.4). The existence of a tetra-substituted phenyl group at C-6 was confirmed by HMBC correlations of H-6/ C-7 (dC 104.7), and H-7/C-6a (dC 126.4), C-8 (dC 153.7), C-9 (dC 138.7), and C-10a. The position of three methoxy groups was deduced from the HMBC correlations as shown in Fig. 3. The chemical shift of C-10 (dC 149.5) suggested that this carbon was attached to a hydroxyl group. The relative configuration of 2 was deduced from a NOESY spectrum (Fig. 4). The NOESY cross-peak between H-2b and H-11a and the coupling constants of H-4b/H-4a (12.1 Hz) and H-4b/H-3 (12.1 Hz) revealed the a-orientation of H-3 and H-4a and b-orientation of C-11 and C-12, respectively. Furthermore, the relative configuration at C-6 was deduced from the NOESY correlation of H-6/H-12b. Thus, compound 2 was elucidated to be 6ahydroxyhippeastidine, possessing a crinine-type skeleton [22].
J Nat Med Table 1 1H NMR data (J, Hz) of compounds 1–4 in CD3OD at 300 K 1a
2a
3a
4.73 (1H, brs)
1.85 (1H, ddd, 14.8, 14.8, 4.4)
1.85 (1H, ddd, 13.8, 13.8, 4.6)
1.78 (1H, ddd, 14.4, 14.4, 4.4)
3.37 (1H, m)
2.63 (1H, m)
3.33 (1H, m)
4.60 (1H, brs)
2.11 (1H, brd, 13.2)
2.23 (1H, brd, 13.6)
2.07 (1H, brd, 11.6)
1.49 (1H, m)
1.59 (1H, m)
1.49 (1H, m)
5.85 (1H, brs)
3.30 (1H, m)
3.33 (1H, m)
3.25 (1H, m)
4a
2.28 (1H, m)
2.30 (1H, m)
2.36 (1H, brd, 10.8)
4b
1.43 (1H, ddd, 12.1, 12.1, 12.1)
1.48 (1H, ddd, 12.0, 12.0, 12.0)
1.42 (1H, ddd, 12.0, 12.0, 10.8)
3.91 (1H, dd, 12.1, 5.2)
3.89 (1H, m)
3.75 (1H, m)
5.63 (1H, s)
5.73 (1H, s)
6.23 (1H, s)
6.93 (1H, s)
6.59 (1H, s)
1a 1b 2a 2b 3
4a
3.93 (1H, m)
6 7
6.53 (1H, s)
4a
10 10b
7.12 (1H, s) 3.58 (1H, d, 9.6)
11a
2.75 (1H, m)
2.58 (1H, ddd, 11.8, 11.8, 7.4)
2.61 (1H, m)
2.59 (1H, ddd, 9.4, 9.4, 9.4)
11b
2.92 (1H, m)
1.89 (1H, m)
1.75 (1H, ddd, 12.6, 9.5, 3.7)
1.92 (1H, ddd, 12.2, 9.0, 2.9)
12a
3.29 (1H, m)
3.81 (1H, m)
3.81 (1H, m)
3.55 (1H, dd, 11.6, 11.6)
12b
3.88 (1H, m)
3.21 (1H, ddd, 12.8, 8.4, 8.4)
3.28 (1H, m)
3.78 (1H, m)
N-Me
2.72 (3H, s) 3.38 (3H, s)
3.41 (3H, s)
3.37 (3H, s)
3-OMe
6.89 (1H, s)
7-OMe
3.93 (3H, s)
8-OMe
3.84 (3H, s)
3.85 (3H, s)
3.84 (3H, s)
3.83 (3H, s)
9-OMe
3.98 (3H, s)
3.78 (3H, s)
3.86 (3H, s)
3.77 (3H, s)
a
Trifluoroacetate (TFA) salt
Table 2
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C NMR data of compounds 1–4 in CD3OD at 300 K 1a
Fig. 3 Selected 2D NMR correlations for compound 2
3a
4a
1
79.4
26.9
26.0
27.0
2
67.9
27.8
27.3
27.9
3
126.5
77.4
77.3
77.1
4
136.0
31.9
31.3
31.7
4a
69.3
62.6
61.5
67.8
6
163.1
88.7
88.7
87.6
6a
111.8
126.4
121.9
127.8
7 8
158.1 144.7
104.7 153.7
112.6 150.2
103.3 153.4
9
159.7
138.7
151.7
138.4
10
108.6
149.5
107.2
149.0
10a
139.0
123.7
137.3
123.7
10b
42.3
46.0
44.3
46.3
11
27.2
31.6
32.5
32.3
12
56.9
48.4
47.9
43.6
N-Me
42.3 56.2
56.1
56.2
3-OMe
Fig. 4 Selected NOESY correlations for compound 2
2a
7-OMe
62.5
8-OMe
61.5
56.4
56.3
56.3
9-OMe
57.1
61.1
56.3
61.1
a
Trifluoroacetate (TFA) salt
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Compound 3 was obtained as a colorless amorphous solid, and its molecular formula was deduced from HRESIMS [m/z 320.1870 (M ? H)?, D ?0.8 mmu) to be C18H25NO4, which implied that 3 was a deoxy form of 2. The 1H and 13C NMR (Tables 1, 2) spectra suggested that 3 had the same tetra-cyclic backbone framework as that of 2, except for the presence of an aromatic proton (dH 6.89) at C-10 (dC 107.2) instead of C-10 (dC 149.5) observed in 2. The presence of an aromatic proton at C-10 was elucidated by HMBC correlations of H-10/C-6a (dC 121.9), C-8 (dC 150.2), and C-10b (dC 44.3). Thus, compound 3 was elucidated to be 10-deoxy-6a-hydroxyhippeastidine. Compound 4 had the same molecular formula, C18H25NO5, as that of 2 by HRESIMS [m/z 336.1825 (M ? H)?, D ?1.4 mmu]. The 1H and 13C NMR data (Tables 1, 2) of 4 were very close to those of 2. Detailed analysis of 2D NMR including the 1H–1H COSY, HSQC, and HMBC spectra indicated that 4 was the C-6 epimer of 2. Furthermore, the relative configuration at C-6 was deduced from the NOESY correlation of H-4a/H-6. Since compounds 2–4 showed no significant Cotton effects, 2–4 might be racemic mixture, respectively [23]. Furthermore, compounds 2 and 4 gradually changed each other to an isomeric mixture. Compounds 1–4 were tested for cytotoxic activity against HL60 cells [24]. All of them were found to be inactive (IC50 [100 lM). Apart from cytotoxicity assessment, 1–4 were found to be inactive in inhibiting inducible nitric oxide synthase (iNOS) activity of the murine macrophage cell line, RAW246.7 cells [11]. The anti-lipid droplets accumulation activity of 1–4 on murine pre-adipocyte cell lines, MC3T3-G2/PA6 cells [25]; and antimelanin deposition activity on the murine melanoma cell line, B16-F10 cells, were also negative [26]. Compounds 1–4 were assayed for vasorelaxation effects on isolated rat aortic ring using a reported procedure [27]. Compound 1 (3 9 10-5 M) only showed relaxation responses against phenylephrine (PE, 3 9 10-7 M)-induced contraction of rat aorta strips with endothelium after achieving a maximal response (1, 57 %).
Experimental section General experimental procedures Optical rotations were measured on a JASCO DIP-1000 polarimeter. UV spectra were recorded on a Shimadzu UVmini-1240 spectrophotometer and IR spectra on a JASCO FT/IR-4100 spectrophotometer. CD spectra were recorded on a JASCO J-820 polarimeter. Mass spectra were obtained using a Waters ZQ-2000 spectrometer. 1H and 2D NMR spectra were recorded on Bruker AV 400
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spectrometers, and chemical shifts were referenced to the residual solvent peaks (dH 3.31 and dC 49.0 for methanold4). Standard pulse sequences were employed for the 2D NMR experiments. High-performance liquid chromatography (HPLC) was performed on a CAPCELL PAK C18 MG-II, 5 lm (/ 10 9 250 mm). High-resolution Electrospray ionization (ESI) Mass spectrometry (MS) was obtained on a LTQ Orbitrap XL (Thermo Scientific). Material The whole plant of Z. candida was collected from Tokushima in 2012. The botanical identification was made by Prof. Yoshinori Asakawa, Faculty of Pharmaceutical Sciences, Tokushima Bunri University. A voucher specimen (No. ZC20120113) has been deposited in the herbarium of Faculty of Pharmaceutical Sciences, Hoshi University. Extraction and isolation The aerial part of Z. candida (411 g, wet weight) was extracted with MeOH (3 9 2 L) at rt, and the extract was partitioned between EtOAc and 3 % aqueous tartaric acid. The aqueous layer was treated with saturated Na2CO3 (aq) to pH 10, and extracted with CHCl3 to give an alkaloidal fraction (310 mg). The alkaloidal fraction was subjected to an amino SiO2 column in (Hexane/EtOAc ? CHCl3/MeOH) to yield 8 fractions. The 5th fraction was applied to a SiO2 column in CHCl3/MeOH (20:1 ? 0:1). The CHCl3/MeOH (4:1) eluted fraction was separated by an ODS HPLC (23 % CH3CN aq. with 0.1 % trifluoroacetic acid (TFA), 2.0 ml/min, 256 nm) to afford 2-hydroxyalbomaculine (1, 3.4 mg, 0.0008 %, tR = 9.6 min), 6a-hydroxyhippeastidine (2, 5.0 mg, 0.001 %, tR = 19.2 min), 10-deoxy-6a-hydroxyhippeastidine (3, 1.7 mg, 0.0004 %, tR = 20.8 min), and 6b-hydroxyhippeastidine (4, 2.4 mg, 0.0006 %, tR = 23.2 min). The 6th fraction was applied to a SiO2 column in CHCl3/ MeOH (20:1 ? 0:1) to afford lycorine (100.1 mg, 0.02 %). 2-Hydroxyalbomaculine (1): colorless amorphous solid; ½a23 D ? 107 (c 0.33, MeOH); UV (MeOH) kmax (log e) 267 (3.72) and 221 (4.16); IR (Zn–Se) mmax 3734, 2938, 1718, and 1112 cm-1; CD (MeOH) kmax 299 (h -31700), 273 (-65000), 252 (?56100), and 233 (-73400); 1H and 13C NMR data (Tables 1, 2); ESIMS m/z 362 (M ? H)?; HRESIMS m/z 362.1606 (M ? H; calcd for C19H24NO6, 362.1604). 6a-hydroxyhippeastidine (2): colorless amorphous solid; UV (MeOH) kmax (log e) 370 (3.00), 340 (3.04), 228 (3.72), and 207 (4.99); IR (Zn–Se) mmax 3380, 2938, 1509, and 1251 cm-1; 1H and 13C NMR data (Tables 1, 2); ESIMS m/z 336 (M ? H)?; HRESIMS m/z 336.1824 (M ? H; calcd for C18H26NO5, 336.1811).
J Nat Med
10-deoxy-6a-hydroxyhippeastidine (3): colorless amorphous solid; UV (MeOH) kmax (log e) 370 (2.72), 341 (2.72), 285 (3.52), and 204 (4.52); IR (Zn–Se) mmax 3380, 2940, 1457, 1333, and 1129 cm-1; 1H and 13C NMR data (Tables 1, 2); ESIMS m/z 320 (M ? H)?; HRESIMS m/z 320.1870 (M ? H; calcd for C18H26NO4, 320.1862). 6b-Hydroxyhippeastidine (4): colorless amorphous solid; UV (MeOH) kmax (log e) 340 (2.66), 280 (3.39), and 207 (4.65); IR (Zn–Se) mmax 3400, 2937, 1456, 1334, and 1130 cm-1; 1H and 13C NMR data (Tables 1, 2); ESIMS m/z 336 (M ? H)?; HRESIMS m/z 336.1825 (M ? H; calcd for C18H26NO5, 336.1811). Acknowledgments This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS).
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13. Zaima K, Koga I, Iwasawa N, Hosoya T, Hirasawa Y, Kaneda T, Ismail IS, Lajis NH, Morita H (2013) Vasorelaxant activity of indole alkaloids from Tabernaemontana dichotoma. J Nat Med 67:9–16 14. Deguchi J, Motegi Y, Nakata A, Hosoya T, Morita H (2013) Cyclic diarylheptanoids as inhibitors of NO production from Acer nikoense. J Nat Med 67:234–239 15. Nugroho AE, Hirasawa Y, Piow WC, Kaneda T, Hadi AH, Shirota O, Ekasari W, Widyawaruyanti A, Morita H (2012) Antiplasmodial indole alkaloids from Leuconotis griffithii. J Nat Med 66:350–353 16. Wong CP, Shimada M, Nugroho AE, Hirasawa Y, Kaneda T, Hadi AHA, Osamu S, Morita H (2012) Ceramicines J–L, new limonoids from Chisocheton ceramicus. J Nat Med 66:566–570 17. Zaima K, Takeyama Y, Koga I, Saito A, Tamamoto H, Abd Azziz SSS, Mukhtar MR, Awang K, Hadi AHA, Morita H (2012) Vasorelaxant effect of isoquinoline derivatives from two species of Popowia perakensis and Phaeanthus crassipetalus on rat aortic artery. J Nat Med 66:421–427 18. Morita H, Mori R, Deguchi J, Oshimi S, Hirasawa Y, Ekasari W, Widyawaruyanti A, Hadi AHA (2012) Antiplasmodial decarboxyportentol acetate and 3,4-dehydrotheaspirone from Laumoniera bruceadelpha. J Nat Med 66:571–575 19. Hosoya T, Nakata A, Yamasaki F, Abas F, Shaari K, Lajis NH, Morita H (2012) Curcumin-like diarylpentanoid analogues as melanogenesis inhibitors. J Nat Med 66:166–176 20. Briggs CK, Highet PF, Highet RJ, Wildman WC (1956) Alkaloids of the Amaryllidaceae. VII. Alkaloids containing the hemiacetal or lactone group. J Am Chem Soc 78:2899–2904 21. Wagner J, Pham HL, Doepke W (1996) Alkaloids from Hippeastrum equestre herb. 5. Circular dichroism studies. Tetrahedron 52(19):6591–6600 22. Pacheco P, Silva M, Steglich W, Watson WH (1978) Alkaloids of Chilean Amaryllidaceae. I. Hippeastidine and epi-homolycorine, two novel alkaloids. Rev Latinoam Quim 9(1):28–32 23. Abou-Donia AH, Amer ME, Darwish FA, Kassem FF, Hammoda HM, Abdel-Kader MS, Zhou BN, Kingston DGI (2002) Two new alkaloids of the crinane series from Pancratium sickenbergeri. Planta Med 68:379–381 24. Janar J, Nugroho AE, Wong CP, Hirasawa Y, Kaneda T, Shirota O, Morita H (2012) Sabiperones A–F, new diterpenoids from Juniperus sabina. Chem Pharm Bull 60:154–159 25. Janar J, Fang L, Wong CP, Kaneda T, Hirasawa Y, Shahmanova BG, Abduahitovich AZ, Morita H (2012) A new galloylbergenin from Bergenia carssifolia with anti-lipid accumulation activity. Heterocycles 86:1591–1595 26. Hoshino T, Matsuda M, Yamashita Y, Takehara M, Fukuya M, Mineda K, Maji D, Ihn H, Adachi H, Sobue G, Funasaka Y, Mizushima T (2010) Suppression of melanin production by expression of HSP70. J Biol Chem 17:13254–13263 27. Morita H, Iizuka T, Choo CY, Chan KL, Takeya K, Kobayashi J (2006) Vasorelaxant activity of cyclic peptide, cyclosquamosin B, from Annona squamosa. Bioorg Med Chem Lett 16:4609–4611
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NOTE
Four new Amaryllidaceae alkaloids from Zephyranthes candida Nanase Shitara • Yusuke Hirasawa • Shunsuke Hasumi • Tadahiro Sasaki • Misaki Matsumoto • Chin Piow Wong Toshio Kaneda • Yoshinori Asakawa • Hiroshi Morita
•
Received: 12 December 2013 / Accepted: 17 January 2014 Ó The Japanese Society of Pharmacognosy and Springer Japan 2014
Abstract Four new Amaryllidaceae alkaloids (1–4) possessing a homolycorine-type or a crinine-type skeleton have been isolated from the aerial part of Zephyranthes candida, and their structures were elucidated on the basis of spectroscopic data. The stereochemistry was elucidated by combination of NOESY correlations and CD analyses. Keywords Zephyranthes candida Amaryllidaceae Homolycorine Crinine Zephyranthes candida (Lindl.) Herb. (Amaryllidaceae) is widely distributed in the tropical regions of the world, and is used as a medicinal plant in China [1]. The leaves and rhizomes of Z. candida show potent bitterness. Plants of the genus Zephyranthes comprise about 70 species [2], which
N. Shitara Y. Hirasawa S. Hasumi T. Sasaki M. Matsumoto C. P. Wong T. Kaneda H. Morita (&) Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41 Shinagawa-ku, Tokyo 142-8501, Japan e-mail: [email protected] Y. Asakawa Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
were reported to be rich sources of Amaryllidaceae alkaloids [3–5] such as lycorine [6], haemanthamine [7], (?)-tazettine [7], trisphaeridine [7], and galanthamine [8], among which galanthamine is used in the treatment of Alzheimer’s disease due to its selective and reversible inhibitory activity against acetylcholinesterase (AChE) [9, 10]. In our search for biogenetically interesting intermediates and new alkaloids with a novel skeleton from medicinal plants [11–19], a new homolycorine-type alkaloid, 2-hydroxyalbomaculine (1), and three new crinine-type alkaloids, 6a-hydroxyhippeastidine (2), 10-deoxy-6a-hydroxyhippeastidine (3), and 6bhydroxyhippeastidine (4), were isolated from the aerial part of Zephyranthes candida. We report here the isolation and structure elucidation of compounds 1–4, and the vasorelaxant activity against rat aortic ring of compound 1.
The molecular formula of compound 1 {½a23 D ? 107 (c 0.33, MeOH)} was determined to be C19H23NO6, as deduced by high-resolution electrospray ionization mass spectrometry (HRESIMS) [m/z 362.1606 (M ? H)?, D ?0.2 mmu], requiring 9 degrees of unsaturation. Absorption bands at 3734 and 1718 cm-1 in the IR spectrum implied the presence of OH and carbonyl functionalities, respectively.
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The 1H NMR spectrum displayed resonances for three methoxy (dH 3.84, dH 3.93, and dH 3.98), an N-methyl (dH 2.72), two olefinic protons (dH 5.85 and dH 7.12), and two oxymethines (dH 4.60 and dH 4.73). The 13C NMR spectrum showed resonances of 19 carbons attributable by the DEPT spectrum to three methoxy, an N-methyl, two methylene, six methine, a ketone, and six quaternary carbons, among which two methines (dC 79.4, dH 4.73 and dC 67.9, dH 4.60) were ascribed to those bearing an oxygen atom. The 1H–1H COSY spectrum revealed connectivity of two partial structures, a (C-1, 2, 3, 4a, and 10b) and b (C11 and C-12), as shown in Fig. 1. HMBC correlations of N-Me/C-12 (dC 56.9) and C-4a (dC 69.3), of H-3/C-4a, and of H-12 and H-10b/C-4 (dC 136.0) confirmed the presence of rings C and D. HMBC correlations between three methoxy groups (7-OMe, 8-OMe, and 9-OMe) and three aromatic carbons (dC 158.1, 144.7, and 159.7), respectively, and of H-10/C-6a (dC 111.8), C-8 (dC 144.7), and C-10b (dC 42.3), indicated that a tri-methoxyphenyl group was attached to C-10b. The molecule required an additional ring to satisfy the degree of unsaturation, suggesting that C-1 (dC 79.4) was bound to C-6a through an ester linkage. The relative configuration of 1 was deduced from a NOESY spectrum (Fig. 2). NOESY cross-peaks between H-1, H-2, and H-10b confirmed their location on the same face of the molecule. In addition, the coupling constant of H-4a/H-10b (9.6 Hz) revealed the anti-relationship between H-4a and H-10b. Thus, compound 1 was elucidated to be 2-hydroxyalbomaculine, possessing a homolycorine-type skeleton [20]. The CD spectrum of 1 in MeOH showed a similar CD curve to that of homolycorine [21]. Therefore, the absolute configurations of 1 were assigned as: 1S, 2R, 4aS, 10bS. The molecular formula, C18H25NO5, of compound 2 was established by HRESIMS [m/z 336.1824, (M ? H)?, D ?1.3 mmu]. The molecular formula accounted for 7 degrees of unsaturation. Absorption bands at 3380 and 1509 cm-1 in the IR spectrum implied the presence of OH and olefinic functionalities, respectively. 1H and 13C NMR data (Table 1) suggested the presence of three methyl, five sp3 methylene, three sp3 methine, an sp3 quaternary carbon, an sp2 methine, and five sp2 quaternary carbons. Among them, one sp3 methylene (dC 48.4, dH 3.21 and 3.81) and one sp3 methine (dC 62.6, dH 3.91) were attached to a nitrogen atom, and one sp3 methine (dC 88.7, dH 5.63) was attached to both a nitrogen atom and an oxygen atom. The 1H–1H COSY and HSQC spectra revealed connectivity of two partial structures, a (C-1–C-4, and C-4a) and b (C-11–C-12), as shown in Fig. 3. The HMBC correlations of H2-1/C-4a (dC 62.6), C-10a (dC 123.7), C-10b (dC 46.0), and C-11 (dC 31.6) indicated that C-1, C-4a,
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Fig. 1 Selected 2D NMR correlations for compound 1
Fig. 2 Selected NOESY correlations for compound 1
C-10a, and C-11 were linked with C-10b. The connectivity among C-4a, C-6, and C-12 through a nitrogen atom was deduced based on the HMBC correlations of H-6/C-4a and C-12 (dC 48.4). The existence of a tetra-substituted phenyl group at C-6 was confirmed by HMBC correlations of H-6/ C-7 (dC 104.7), and H-7/C-6a (dC 126.4), C-8 (dC 153.7), C-9 (dC 138.7), and C-10a. The position of three methoxy groups was deduced from the HMBC correlations as shown in Fig. 3. The chemical shift of C-10 (dC 149.5) suggested that this carbon was attached to a hydroxyl group. The relative configuration of 2 was deduced from a NOESY spectrum (Fig. 4). The NOESY cross-peak between H-2b and H-11a and the coupling constants of H-4b/H-4a (12.1 Hz) and H-4b/H-3 (12.1 Hz) revealed the a-orientation of H-3 and H-4a and b-orientation of C-11 and C-12, respectively. Furthermore, the relative configuration at C-6 was deduced from the NOESY correlation of H-6/H-12b. Thus, compound 2 was elucidated to be 6ahydroxyhippeastidine, possessing a crinine-type skeleton [22].
J Nat Med Table 1 1H NMR data (J, Hz) of compounds 1–4 in CD3OD at 300 K 1a
2a
3a
4.73 (1H, brs)
1.85 (1H, ddd, 14.8, 14.8, 4.4)
1.85 (1H, ddd, 13.8, 13.8, 4.6)
1.78 (1H, ddd, 14.4, 14.4, 4.4)
3.37 (1H, m)
2.63 (1H, m)
3.33 (1H, m)
4.60 (1H, brs)
2.11 (1H, brd, 13.2)
2.23 (1H, brd, 13.6)
2.07 (1H, brd, 11.6)
1.49 (1H, m)
1.59 (1H, m)
1.49 (1H, m)
5.85 (1H, brs)
3.30 (1H, m)
3.33 (1H, m)
3.25 (1H, m)
4a
2.28 (1H, m)
2.30 (1H, m)
2.36 (1H, brd, 10.8)
4b
1.43 (1H, ddd, 12.1, 12.1, 12.1)
1.48 (1H, ddd, 12.0, 12.0, 12.0)
1.42 (1H, ddd, 12.0, 12.0, 10.8)
3.91 (1H, dd, 12.1, 5.2)
3.89 (1H, m)
3.75 (1H, m)
5.63 (1H, s)
5.73 (1H, s)
6.23 (1H, s)
6.93 (1H, s)
6.59 (1H, s)
1a 1b 2a 2b 3
4a
3.93 (1H, m)
6 7
6.53 (1H, s)
4a
10 10b
7.12 (1H, s) 3.58 (1H, d, 9.6)
11a
2.75 (1H, m)
2.58 (1H, ddd, 11.8, 11.8, 7.4)
2.61 (1H, m)
2.59 (1H, ddd, 9.4, 9.4, 9.4)
11b
2.92 (1H, m)
1.89 (1H, m)
1.75 (1H, ddd, 12.6, 9.5, 3.7)
1.92 (1H, ddd, 12.2, 9.0, 2.9)
12a
3.29 (1H, m)
3.81 (1H, m)
3.81 (1H, m)
3.55 (1H, dd, 11.6, 11.6)
12b
3.88 (1H, m)
3.21 (1H, ddd, 12.8, 8.4, 8.4)
3.28 (1H, m)
3.78 (1H, m)
N-Me
2.72 (3H, s) 3.38 (3H, s)
3.41 (3H, s)
3.37 (3H, s)
3-OMe
6.89 (1H, s)
7-OMe
3.93 (3H, s)
8-OMe
3.84 (3H, s)
3.85 (3H, s)
3.84 (3H, s)
3.83 (3H, s)
9-OMe
3.98 (3H, s)
3.78 (3H, s)
3.86 (3H, s)
3.77 (3H, s)
a
Trifluoroacetate (TFA) salt
Table 2
13
C NMR data of compounds 1–4 in CD3OD at 300 K 1a
Fig. 3 Selected 2D NMR correlations for compound 2
3a
4a
1
79.4
26.9
26.0
27.0
2
67.9
27.8
27.3
27.9
3
126.5
77.4
77.3
77.1
4
136.0
31.9
31.3
31.7
4a
69.3
62.6
61.5
67.8
6
163.1
88.7
88.7
87.6
6a
111.8
126.4
121.9
127.8
7 8
158.1 144.7
104.7 153.7
112.6 150.2
103.3 153.4
9
159.7
138.7
151.7
138.4
10
108.6
149.5
107.2
149.0
10a
139.0
123.7
137.3
123.7
10b
42.3
46.0
44.3
46.3
11
27.2
31.6
32.5
32.3
12
56.9
48.4
47.9
43.6
N-Me
42.3 56.2
56.1
56.2
3-OMe
Fig. 4 Selected NOESY correlations for compound 2
2a
7-OMe
62.5
8-OMe
61.5
56.4
56.3
56.3
9-OMe
57.1
61.1
56.3
61.1
a
Trifluoroacetate (TFA) salt
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Compound 3 was obtained as a colorless amorphous solid, and its molecular formula was deduced from HRESIMS [m/z 320.1870 (M ? H)?, D ?0.8 mmu) to be C18H25NO4, which implied that 3 was a deoxy form of 2. The 1H and 13C NMR (Tables 1, 2) spectra suggested that 3 had the same tetra-cyclic backbone framework as that of 2, except for the presence of an aromatic proton (dH 6.89) at C-10 (dC 107.2) instead of C-10 (dC 149.5) observed in 2. The presence of an aromatic proton at C-10 was elucidated by HMBC correlations of H-10/C-6a (dC 121.9), C-8 (dC 150.2), and C-10b (dC 44.3). Thus, compound 3 was elucidated to be 10-deoxy-6a-hydroxyhippeastidine. Compound 4 had the same molecular formula, C18H25NO5, as that of 2 by HRESIMS [m/z 336.1825 (M ? H)?, D ?1.4 mmu]. The 1H and 13C NMR data (Tables 1, 2) of 4 were very close to those of 2. Detailed analysis of 2D NMR including the 1H–1H COSY, HSQC, and HMBC spectra indicated that 4 was the C-6 epimer of 2. Furthermore, the relative configuration at C-6 was deduced from the NOESY correlation of H-4a/H-6. Since compounds 2–4 showed no significant Cotton effects, 2–4 might be racemic mixture, respectively [23]. Furthermore, compounds 2 and 4 gradually changed each other to an isomeric mixture. Compounds 1–4 were tested for cytotoxic activity against HL60 cells [24]. All of them were found to be inactive (IC50 [100 lM). Apart from cytotoxicity assessment, 1–4 were found to be inactive in inhibiting inducible nitric oxide synthase (iNOS) activity of the murine macrophage cell line, RAW246.7 cells [11]. The anti-lipid droplets accumulation activity of 1–4 on murine pre-adipocyte cell lines, MC3T3-G2/PA6 cells [25]; and antimelanin deposition activity on the murine melanoma cell line, B16-F10 cells, were also negative [26]. Compounds 1–4 were assayed for vasorelaxation effects on isolated rat aortic ring using a reported procedure [27]. Compound 1 (3 9 10-5 M) only showed relaxation responses against phenylephrine (PE, 3 9 10-7 M)-induced contraction of rat aorta strips with endothelium after achieving a maximal response (1, 57 %).
Experimental section General experimental procedures Optical rotations were measured on a JASCO DIP-1000 polarimeter. UV spectra were recorded on a Shimadzu UVmini-1240 spectrophotometer and IR spectra on a JASCO FT/IR-4100 spectrophotometer. CD spectra were recorded on a JASCO J-820 polarimeter. Mass spectra were obtained using a Waters ZQ-2000 spectrometer. 1H and 2D NMR spectra were recorded on Bruker AV 400
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spectrometers, and chemical shifts were referenced to the residual solvent peaks (dH 3.31 and dC 49.0 for methanold4). Standard pulse sequences were employed for the 2D NMR experiments. High-performance liquid chromatography (HPLC) was performed on a CAPCELL PAK C18 MG-II, 5 lm (/ 10 9 250 mm). High-resolution Electrospray ionization (ESI) Mass spectrometry (MS) was obtained on a LTQ Orbitrap XL (Thermo Scientific). Material The whole plant of Z. candida was collected from Tokushima in 2012. The botanical identification was made by Prof. Yoshinori Asakawa, Faculty of Pharmaceutical Sciences, Tokushima Bunri University. A voucher specimen (No. ZC20120113) has been deposited in the herbarium of Faculty of Pharmaceutical Sciences, Hoshi University. Extraction and isolation The aerial part of Z. candida (411 g, wet weight) was extracted with MeOH (3 9 2 L) at rt, and the extract was partitioned between EtOAc and 3 % aqueous tartaric acid. The aqueous layer was treated with saturated Na2CO3 (aq) to pH 10, and extracted with CHCl3 to give an alkaloidal fraction (310 mg). The alkaloidal fraction was subjected to an amino SiO2 column in (Hexane/EtOAc ? CHCl3/MeOH) to yield 8 fractions. The 5th fraction was applied to a SiO2 column in CHCl3/MeOH (20:1 ? 0:1). The CHCl3/MeOH (4:1) eluted fraction was separated by an ODS HPLC (23 % CH3CN aq. with 0.1 % trifluoroacetic acid (TFA), 2.0 ml/min, 256 nm) to afford 2-hydroxyalbomaculine (1, 3.4 mg, 0.0008 %, tR = 9.6 min), 6a-hydroxyhippeastidine (2, 5.0 mg, 0.001 %, tR = 19.2 min), 10-deoxy-6a-hydroxyhippeastidine (3, 1.7 mg, 0.0004 %, tR = 20.8 min), and 6b-hydroxyhippeastidine (4, 2.4 mg, 0.0006 %, tR = 23.2 min). The 6th fraction was applied to a SiO2 column in CHCl3/ MeOH (20:1 ? 0:1) to afford lycorine (100.1 mg, 0.02 %). 2-Hydroxyalbomaculine (1): colorless amorphous solid; ½a23 D ? 107 (c 0.33, MeOH); UV (MeOH) kmax (log e) 267 (3.72) and 221 (4.16); IR (Zn–Se) mmax 3734, 2938, 1718, and 1112 cm-1; CD (MeOH) kmax 299 (h -31700), 273 (-65000), 252 (?56100), and 233 (-73400); 1H and 13C NMR data (Tables 1, 2); ESIMS m/z 362 (M ? H)?; HRESIMS m/z 362.1606 (M ? H; calcd for C19H24NO6, 362.1604). 6a-hydroxyhippeastidine (2): colorless amorphous solid; UV (MeOH) kmax (log e) 370 (3.00), 340 (3.04), 228 (3.72), and 207 (4.99); IR (Zn–Se) mmax 3380, 2938, 1509, and 1251 cm-1; 1H and 13C NMR data (Tables 1, 2); ESIMS m/z 336 (M ? H)?; HRESIMS m/z 336.1824 (M ? H; calcd for C18H26NO5, 336.1811).
J Nat Med
10-deoxy-6a-hydroxyhippeastidine (3): colorless amorphous solid; UV (MeOH) kmax (log e) 370 (2.72), 341 (2.72), 285 (3.52), and 204 (4.52); IR (Zn–Se) mmax 3380, 2940, 1457, 1333, and 1129 cm-1; 1H and 13C NMR data (Tables 1, 2); ESIMS m/z 320 (M ? H)?; HRESIMS m/z 320.1870 (M ? H; calcd for C18H26NO4, 320.1862). 6b-Hydroxyhippeastidine (4): colorless amorphous solid; UV (MeOH) kmax (log e) 340 (2.66), 280 (3.39), and 207 (4.65); IR (Zn–Se) mmax 3400, 2937, 1456, 1334, and 1130 cm-1; 1H and 13C NMR data (Tables 1, 2); ESIMS m/z 336 (M ? H)?; HRESIMS m/z 336.1825 (M ? H; calcd for C18H26NO5, 336.1811). Acknowledgments This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS).
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