Two new alkaloids from Crinum asiaticum var. sinicum

Article abstract of DOI:10.1248/cpb.59.1545

Two new alkaloids (1, 2) were isolated from the whole plants of Crinum asiaticum var. sinicum together with seven known alkaloids. The structures of the new alkaloids were elucidated by spectroscopic analyses and chemical conversions from known alkaloids. New alkaloid 1 was isolated for the first time as a natural product, although it has been prepared as a synthetic product.

Full text of DOI:10.1248/cpb.59.1545

December 2011
Note
Chem. Pharm. Bull. 59(12) 1545—1548 (2011)
1545
Two New Alkaloids from Crinum asiaticum var. sinicum
Noriyuki KOGURE, Noriko KATSUTA, Mariko KITAJIMA, and Hiromitsu TAKAYAMA*
Graduate School of Pharmaceutical Sciences, Chiba University; 1–8–1 Inohana, Chuo-ku, Chiba 260–8675, Japan.
Received June 3, 2011; accepted September 1, 2011; published online September 7, 2011
Two new alkaloids (1, 2) were isolated from the whole plants of Crinum asiaticum var. sinicum together with
seven known alkaloids. The structures of the new alkaloids were elucidated by spectroscopic analyses and chemi-
cal conversions from known alkaloids. New alkaloid 1 was isolated for the first time as a natural product,
although it has been prepared as a synthetic product.
Key words Amaryllidaceae; alkaloid; isopowellamine; crinamine
Amaryllidaceae alkaloids having structural diversity as H2-6, H2-11, H2-12), including one benzylic methylene [d
well as significant pharmacological activities, such as acetyl- 4.24 (1H, d, Jꢁ17.6 Hz, H-6b), d 3.70 (1H, d, Jꢁ17.6 Hz, H-
choline esterase (AChE) inhibitory activity, have been exten- 6a)], were found.
sively studied for many years.1—3) Quite recently, galanth-
From the above analyses, 1 has a crinine-type skeleton de-
amine (3), one of the principal Amaryllidaceae alkaloids, spite having o-oriented aromatic protons like powellamine,14)
was approved as a therapeutic agent for Alzheimer’s disease. an alkaloid isolated from Crinum powellii var. harlemense
A comprehensive study on the chemical constituents of (Fig. 2). To elucidate the relative stereochemistry, nuclear
Crinum asiaticum var. sinicum has been executed by utilizing Overhauser effect (NOE) experiment was executed. NOE
HPLC-diode array detector (DAD)-solid-phase extraction correlations between H-4a/H-11 and H-12 suggested that H-
(SPE)-NMR4) and 21 alkaloids have been characterized. As 4a and the bridge (C10b-C11-C12-N) were syn oriented (Fig.
part of our continuing chemical studies on the Amaryl- 3). This unique stereochemistry is characteristic of the
lidaceae alkaloids,5,6) we were able to isolate new alkaloids 1 isopowellamine skeleton.
and 2 from the whole plant of C. asiaticum var. sinicum to-
To confirm the structure, chemical conversion from gal-
gether with seven known alkaloids, crinamine (4),7) norgal- anthamine (3) was conducted15) (Chart 1). First, the allylic al-
anthamine,8) hamayne,9) 3-O-acetylhamayne,10) lycorine,11) cohol of 3 was oxidized with MnO2 to give the natural alka-
hippadine,12) and platorinine.13) In this paper, we describe loid, narwedine (5),16) in 86% yield. Then, the N-Me group
the structure elucidation of these new alkaloids.
was removed from 5 via N-oxide derivative 6 to give nornar-
The molecular formula of new alkaloid 1, seco- wedine (7) in 29% yield (2 steps), together with byproduct 8
isopowellaminone, was established as C16H17NO3 on the (23%). Finally, 7 was converted into 1 via the retro-Michael–
basis of high resolution-electron ionization (HR-EI)-MS data Michael-addition cascade by refluxing in aqueous EtOH in
[m/z 271.1211 (Mꢀ)]. The 1H-NMR spectrum showed o-cou- the presence of CaCl2.17)
pled aromatic proton signals [d 6.72 and d 6.53 (each d,
The NMR data of semi-synthetic 1 was identical with
Jꢁ8.2 Hz, H-7, H-8)], and one methoxy proton signal [d those of natural 1 and therefore, the structure was estab-
3.86 (3H, s)]. The presence of an a,b-unsaturated ketone lished. However, semi-synthetic 1 was obtained as a racemic
residue was suggested by the characteristic signals at dH 7.93 compound, formed probably via achiral intermediate 9.
(1H, d, Jꢁ9.8 Hz, H-1), dH 5.98 (1H, d, Jꢁ9.8 Hz, H-2), dC Therefore, the absolute configuration of natural product
198.9 (C-3), dC 154.8 (C-1), and dC 128.2 (C-2). Further- ([a]D ꢀ11.1°) was not elucidated.
more, signals assignable to four methylene protons (H2-4,
seco-Isopowellaminone (1) was biogenetically considered
to be generated via the pathway shown in Chart 2. The o, p-
phenol coupling from norbelladine (10), a common interme-
diate of Amaryllidaceae alkaloids, generates intermediate 9.
Michael addition from nitrogen atom gives 1 and that from
oxygen atom gives galanthamine-type alkaloids. This is the
first example of isolation of an alkaloid having the
isopowellaminone skeleton from nature, although it has been
prepared as a synthetic product.17)
The HR-FAB-MS spectrum of new alkaloid 2 gave a pro-
Fig. 1. Structures of New Alkaloids 1 and 2
tonated molecular ion peak at m/z 318.1353 ([MꢀH]ꢀ),
Fig. 2. Structures of Crinine Type of Amaryllidaceae Alkaloids
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: htakayam@p.chiba-u.ac.jp

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Vol. 59, No. 12
which corresponded to the molecular formula C17H20NO5 signals [d 5.98 (1H, d, Jꢁ1.5 Hz, –OCH2O–), d 5.97 (1H, d,
(m/z 318.1341). UV absorption bands at 291.5, 240.0, and Jꢁ1.5 Hz, –OCH2O–)], benzylic methylene proton signals [d
210.0 nm are similar to those of crinine-type alkaloids.
4.78 (1H, d, Jꢁ15.9 Hz, H-6b), d 4.65 (1H, d, Jꢁ15.9 Hz, H-
1
The H-NMR spectrum of 2 showed two singlet aromatic 6a)], and one methoxy proton signal [d 3.45 (3H, s)]. Fur-
proton signals [d 6.81 and d 6.56 (each s, H-10, H-7)], two thermore, 2 has two oxymethine proton signals [d 4.21 (1H,
cis-olefinic proton signals [d 6.28 (1H, d, Jꢁ10.1 Hz, H-2), d m, H-11), d 4.02 (1H, m, H-3)].
1
6.11 (1H, dd, Jꢁ10.2, 1.7 Hz, H-1)], methylenedioxy proton
The H- and 13C-NMR data of 2 were similar to those of
crinamine (4)6) except for the chemical shifts of proton and
carbon signals ascribable to C4a, C6, and C12 positions. The
above data indicated that 2 must be an N-oxide derivative of
crinamine (4).
To confirm the structure, the oxidation of crinamine (4)
was executed (Chart 3). 4 was treated with 1.1 eq of meta-
chloroperbenzoic acid (MCPBA) to give desired crinamine
N-oxide (2). All the spectroscopic data, including the CD
spectrum, of synthetic 2 were identical with those of natural
2 and therefore, the structure, including the absolute configu-
ration, was established.
Fig. 3. NOE Experiment of seco-Isopowellaminone (1)
Chart 1. Chemical Conversion from Galanthamine (3) to seco-Isopowellaminone (1)
Chart 2. Plausible Biosynthetic Pathway of seco-Isopowellaminone (1)

December 2011
1547
Chart 3. N-Oxidation of Crinamine (4)
Experimental
1), 121.5 (C-6a), 106.2 (C-7), 103.5 (C-10), 101.6 (–OCH2O–), 76.7 (C-12),
76.3 (C-4a), 75.7 (C-11), 75.4 (C-6), 75.2 (C-3), 56.2 (OMe), 52.3 (C-10b),
25.1 (C-4). UV (MeOH) lmax nm (log e): 291.5 (3.53), 240.0 (3.41), 210.0
General Procedure 1H- and 13C-NMR spectra: JEOL JNM A-500 at
500 MHz (1H-NMR) and at 125 MHz (13C-NMR), using tetramethylsilane
(TMS) as an internal reference. UV: JASCO V-560. EI-MS and HR-EI-MS: (4.16). CD (cꢁ0.24 mmol/l, MeOH, 24 °C) De (nm): 0 (313), ꢀ1.48 (289),
JEOL GC-mate. FAB-MS: JEOL JMS-AX500. HR-FAB-MS: JEOL JMS- 0 (264), ꢃ1.17 (243), 0 (239), ꢀ29.3 (208). [a]D24 ꢀ72.7° (cꢁ0.10, CHCl3).
HX110. Optical rotation: JASCO P-1020. CD: JASCO J-720WI. TLC: Pre-
coated silica gel 60 F254 plates (Merck, 0.25 mm thick). Column chromatog-
Oxidation of Galanthamine (3) MnO2 (93.3 mg, 1.07 mmol) was
added to a solution of galanthamine (3, 15.2 mg, 0.053 mmol) in CH2Cl2
raphy: Silica gel 60 (Merck, 70—230 mesh), Al2O3 (Merck, Aluminium (2.0 ml) under cooling with ice and the mixture was stirred vigorously for
oxide 90 standardized). Flash column chromatography: Silica gel 60N
(Kanto Chemical, 40—50 mm). Amino silica gel column chromatography:
NH-DM1020 (Fuji Silysia Chemical, Japan).
Plant Material Crinum asiaticum var. sinicum was harvested from the
medicinal plant garden of Chiba University, Japan. A voucher specimen was
deposited at the Faculty of Pharmaceutical Sciences, Chiba University,
Japan.
Extraction and Isolation The whole plant of Crinum asiaticum var.
sinicum (7.4 kg, wet weight) was chopped with a blender and extracted with
MeOH (total 37 l, two times at room temperature and two times under re-
flux). The extract was evaporated to give the crude MeOH extract (525.15 g,
wet weight). The MeOH extract was dissolved in H2O and extracted with n-
hexane (750 ml, 500 mlꢂ2) to give the n-hexane extract (13.62 g). The aque-
14.5 h at r.t. The reaction mixture was passed through Celite and the filtrate
was evaporated. The crude product was purified by SiO2 flash chromatogra-
phy (5% MeOH/CHCl3) to give narwedine (5, 13.0 mg, 86%).
Demethylation of Narwedine (5) A solution of MCPBA (10.9 mg,
0.048 mmol) in CH2Cl2 (1.0 ml) was added dropwise to a solution of narwe-
dine (5, 12.5 mg, 0.044 mmol) in CH2Cl2 (0.5 ml) at r.t. and the mixture was
stirred for 1.5 h under Ar. The reaction mixture was passed through Al2O3
short column chromatography (5% MeOH/CHCl3) to give crude narwedine
N-oxide (6) mixture (16.3 mg), which was subjected to the next reaction
without purification.
6: FAB-MS (NBA) m/z: 302 (MꢀHꢀ).
To the above crude
6 (13.2 mg) in MeOH (0.5 ml) was added
FeSO4·7H2O (24.4 mg, 0.088 mmol) under cooling with ice and the mixture
ous layer was successively extracted with AcOEt (1.0 lꢂ4), 5% MeOH/ was stirred for 3 h at 10 °C and another 2 h at r.t.
CHCl3 (1.2 lꢂ4), and n-BuOH (1.0 lꢂ4) to give AcOEt extract (4.03 g),
5% MeOH/CHCl3 extract (1.40 g), and n-BuOH extract (36.58 g), respec-
tively.
The 5% MeOH/CHCl3 extract (1.40 g) was separated by SiO2 flash col-
umn chromatography with CHCl3/MeOH gradient to give eight fractions: fr.
A CHCl3 (12.6 mg); fr. B 0—2% MeOH/CHCl3 (437.0 mg); fr. C 2—5%
MeOH/CHCl3 (50.7 mg); fr. D 5—7% MeOH/CHCl3 (297.9 mg); fr. E 10—
15% MeOH/CHCl3 (308.2 mg); fr. F 15—20% MeOH/CHCl3 (74.9 mg); fr.
Ethylenediamine (3.0 ml), 1 M aq. NaOH (88 ml), and CHCl3 were added to
the mixture and the whole was stirred for 1 h. The reaction mixture was
passed through Celite and the filtrate was extracted with CHCl3, washed
with brine, filtered, and evaporated. The crude product was purified by SiO2
flash chromatography (10% MeOH/CHCl3) to give nornarwedine (7, 3.4 mg,
29%, 2 steps) and byproduct 8 (3.0 mg, 23%).
Byproduct 8 EI-MS m/z (%): 301 (M+, 100). 1H-NMR (500 MHz, CDCl3)
d: 9.97 (1H, s, CHO), 7.49 (1H, d, Jꢁ8.4 Hz, H-7), 6.97 (1H, d, Jꢁ8.4 Hz,
G 30% MeOH/CHCl3 (27.1 mg); and fr. H 30% MeOH/CHCl3 and MeOH H-8), 4.77 (1H, dd, Jꢁ2.9, 2.9 Hz, H-4a), 3.98 (3H, s, OMe), 3.91 (1H, m,
(116.2 mg).
H-1), 3.19 (1H, dd, Jꢁ8.2, 8.2 Hz), 3.12 (1H, dd, Jꢁ8.5, 3.3 Hz), 3.00 (1H,
dd, Jꢁ18.1, 8.8 Hz), 2.91 (1H, dd, Jꢁ18.4, 2.7 Hz), 2.78—2.71 (1H, m),
Fr. D was purified successively by SiO2 flash column chromatography
(MeOH/CHCl3 gradient and MeOH/AcOEt gradient) to afford seco- 2.48 (1H, dd, Jꢁ17.0, 3.5 Hz), 2.25 (3H, s, NMe), 2.24 (1H, dd, Jꢁ17.0,
isopowellaminone (1, 0.3 mg). Fr. H was purified successively by SiO2 flash
column chromatography (MeOH/CHCl3 gradient) and amino silica gel open
column chromatography (10% MeOH/CHCl3) to give crinamine N-oxide (2,
1.9 mg).
2.4 Hz), 1.77 (1H, m, H-12).
Chemical Conversion of 7 into 1 via retro-Michael–Michael-Addition
Cascade CaCl2 (2.5 mg, 0.023 mmol) was added to a solution of nornar-
wedine (7, 3.4 mg, 0.013 mmol) in 70% EtOH (0.5 ml) and the mixture was
seco-Isopowellaminone (1) EI-MS m/z (%): 271 (Mꢀ, 100), 221 (24), heated at 80 °C for 4 h. The reaction mixture was extracted with CHCl3,
187 (45). HR-EI-MS m/z: 271.1211 (Mꢀ, Calcd for C16H17NO3: 271.1208). washed with brine, filtered, and evaporated. The crude product was purified
1H-NMR (500 MHz, CDCl3) d: 7.93 (1H, d, Jꢁ9.8 Hz, H-1), 6.72 (1H, d,
Jꢁ8.2 Hz, H-8), 6.53 (1H, d, Jꢁ8.2 Hz, H-7), 5.98 (1H, d, Jꢁ9.8 Hz, H-2),
by SiO2 column chromatography (25% MeOH/CHCl3), amino silica gel
open column chromatography (CHCl3), and SiO2 column chromatography
1
4.24 (1H, d, Jꢁ17.6 Hz, H-6b), 3.86 (3H, s, OMe), 3.70 (1H, d, Jꢁ17.6 Hz, (10% MeOH/AcOEt) to give seco-isopowellaminone (1, 0.8 mg, 22%). H-
H-6a), 3.44 (1H, ddd, Jꢁ13.0, 10.2, 3.1 Hz, H-12exo), 3.17 (1H, dd, NMR and UV spectra were identical with those of the natural product.
Jꢁ14.4, 4.1 Hz, H-4a), 2.92 (1H, ddd, Jꢁ15.2, 8.4, 6.8 Hz, H-12endo), 2.85
(1H, dd, Jꢁ16.7, 3.8 Hz, H-4a), 2.53 (1H, dd, Jꢁ16.4, 14.2 Hz, H-4b), 2.35
N-Oxidation of Crinamine (3) MCPBA (4.1 mg, 0.018 mmol) was
added to a solution of crinamine (3, 4.9 mg, 0.016 mmol) in CH2Cl2 (1.0 ml)
(1H, ddd, Jꢁ11.7, 8.4, 4.3 Hz, H-11endo), 1.99 (1H, ddd, Jꢁ10.8, 10.8, and the reaction mixture was stirred for 1 h at 0 °C under Ar. The mixture
6.8 Hz, H-11exo). 13C-NMR (125 MHz, CDCl3) d: 198.9 (C-3), 154.8 (C-1),
145.6 (C-9), 142.8 (C-10), 128.2 (C-2), 126.0 (C-10a), 125.1 (C-6a), 116.9
(C-7), 109.4 (C-8), 65.2 (C-4a), 56.2 (OMe), 54.7 (C-6), 53.5 (C-12), 43.6
(C-10b), 40.3 (C-11), 38.8, (C-4). UV (MeOH) lmax nm (log e): 280.0
(3.25), 228.5 (sh, 3.85), 205.0 (4.26). [a]D19 ꢀ11.1° (cꢁ0.027, CHCl3).
Crinamine N-Oxide (2) FAB-MS (NBA) m/z: 318 (MꢀHꢀ), HR-FAB-
was purified by Al2O3 column chromatography (10% MeOH/CHCl3) to give
desired crinamine N-oxide (2, 6.4 mg, quant.). All of the spectroscopic data
(1H-NMR, 13C-NMR, FAB-MS, UV, [a]D, and CD) were identical with those
of the natural product.
Acknowledgement This work was supported by a Grant-in-Aid for Sci-
MS (NBA/PEG) m/z: 318.1363 (MꢀHꢀ, Calcd for C17H20NO5: 318.1341). entific Research from Japan Society for the Promotion of Science.
1H-NMR (500 MHz, CDCl3) d: 6.81 (1H, s, H-10), 6.56 (1H, s, H-7), 6.28
(1H, d, Jꢁ10.1 Hz, H-2), 6.11 (1H, dd, Jꢁ10.2, 1.7 Hz, H-1), 5.98 (1H, d,
Jꢁ1.5 Hz, –OCH2O–), 5.97 (1H, d, Jꢁ1.5 Hz, –OCH2O–), 4.78 (1H, d,
Jꢁ15.9 Hz, H-6b), 4.65 (1H, d, Jꢁ15.9 Hz, H-6a), 4.21 (1H, m, H-11), 4.02
(1H, m, H-3), 3.98—3.95 (2H, overlapped, H-11, 12), 3.58 (1H, dd, Jꢁ14.7,
3.7 Hz, H-4a), 3.45 (3H, s, OMe), 3.08 (1H, ddd, Jꢁ10.9, 5.2, 5.2 Hz, H-
4b), 2.31 (1H, ddd, Jꢁ12.5, 11.5, 11.5 Hz, H-4a). 13C-NMR (125 MHz,
CDCl3) d: 148.1 (C-9), 147.5 (C-8), 136.8 (C-2), 131.9 (C-10a), 121.6 (C-
References
1) Unver N., Phytochem. Rev., 6, 125—135 (2007), and references cited
therein.
2) Jin Z., Nat. Prod. Rep., 24, 886—905 (2007), and references cited
therein.
3) Hoshino O., “The Alkaloids,” Vol. 51, ed. by Cordell G. A., Academic
Press, San Diego, 1998, pp. 323—424, and references cited therein.

1548
4) Chen C.-K., Lin F.-H., Tseng L.-H., Jiang C.-L., Lee S.-S., J. Nat.
Vol. 59, No. 12
10) Viladomat F., Bastida J., Codina C., Cambell W.-E., Mathee S., Phyto-
chemistry, 35, 809—812 (1994).
Prod., 74, 411—419 (2011).
5) Toriizuka Y., Kinoshita E., Kogure N., Kitajima M., Ishiyama A.,
Otoguro K., Yamada H., Omura S., Takayama H., Bioorg. Med. Chem.,
16, 10182—10189 (2008).
6) Kitajima M., Kinoshita E., Kogure N., Takayama H., Heterocycles, 77,
1389—1396 (2009).
11) Kobayashi S., Tokumoto T., Kihara M., Imakura Y., Shingu T., Taira
Z., Chem. Pharm. Bull., 32, 3015—3022 (1984).
12) Youssef D.-T.-A., Frahm A.-W., Planta Med., 64, 669—670 (1998).
13) Ghosal S., Lao P.-H., Jaiswal D.-K., Kumar Y., Frahm A.-W., Phyto-
chemistry, 20, 2003—2007 (1981).
7) Mason L.-H., Puschett E.-R., Wildman W.-C., J. Am. Chem. Soc., 77,
1253—1256 (1955).
8) Zhang F.-M., Tu Y.-Q., Liu J.-D., Fan X.-H., Shi L., Hu X.-D., Wang
S.-H., Zang Y.-Q., Tetrahedron, 62, 9446—9455 (2006).
9) Li H.-Y., Ma G.-E., Xu Y., Hong S.-H., Planta Med., 53, 259—261
(1987).
14) Boit H.-G., Dopke W., Naturwissenschaften, 46, 475 (1959).
15) Treu M., Welzig S., Jordis U., Heterocycles, 55, 2397—2404 (2001).
16) Kuenburg B., Czollner L., Frohlich J., Jordis U., Org. Process Res.
Dev., 3, 425—431 (1999).
17) Hirnschall M., Mereirer K., Froehlich J., Jordis U., J. Heterocycl.
Chem., 39, 1265—1270 (2002).