Two new cytotoxic alkaloids from Mappianthus iodoides Hand.-Mazz.

Article abstract of DOI:10.1002/hlca.201100187

Two new alkaloids, mappine A (1) and mapposidic acid (2), together with eleven known compounds, were isolated from the stems of Mappianthus iodoides Hand.-Mazz. Their structures were elucidated by means of spectroscopic and mass-spectrometric analyses, particularly 1D- and 2D-NMR spectroscopy. The cytotoxic activities of the two new alkaloids were also evaluated. Copyright

Full text of DOI:10.1002/hlca.201100187

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Helvetica Chimica Acta – Vol. 94 (2011)
Two New Cytotoxic Alkaloids from Mappianthus iodoides Hand.-Mazz.
by Xi-Bin Xiao*a), Yue-Xing Linb), Geng-Bo Xua), Xu-Bo Gonga), Ying Gua), Jie-Feng Tonga), and
Jing Yanga)
a) Department of Hematology, Second Affiliated Hospital, Zhejiang University, No. 88 Jiefang Road,
Hangzhou 310009, P. R. China (phone: þ 86-571-87783652; e-mail: xiaoxibincsu@163.com)
b) Blood Bank, Second Affiliated Hospital, Zhejiang University, Hangzhou 310009, P. R. China
Two new alkaloids, mappine A (1) and mapposidic acid (2), together with eleven known compounds,
were isolated from the stems of Mappianthus iodoides Hand.-Mazz. Their structures were elucidated by
means of spectroscopic and mass-spectrometric analyses, particularly 1D- and 2D-NMR spectroscopy.
The cytotoxic activities of the two new alkaloids were also evaluated.
Introduction. – The genus Mappianthus (Icacinaceae family) consists of three
species which are distributed in subtropical and tropical zone. Mappianthus iodoides
Hand.-Mazz., one species occurring in China, is used by local people for the treatment
of traumatic injury, rheumatalgia, arthralgia, etc. [1].
There were no phytochemical studies on this genus except that a sesquiterpene
named (ꢀ)-cedrol had been reported from Mappianthus iodoides Hand.-Mazz. [2].
Our phytochemical investigation on the 95% EtOH extract of the title plant led to the
isolation of 13 compounds, comprising seven alkaloids, i.e., mappine A (1), mapposidic
acid (2), 1-methyl-b-carboline [3], strictosidine [4], strictosidine acid [4], 5-carboxy-
strictosidine [5], and lyalosidic acid (3) [6], three lignans, i.e., pinoresinol [7], (þ)-
(1R,2S,5R,6S)-2,6-bis(4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane [8], and pinor-
esinol dimethyl ether [9], two iridoid glycosides, i.e., dihydrocornin [10] and
secologanin [11], and one phenolic glycoside, i.e., 3,4,5-trimethoxyphenol-1-O-[b-d-
apiofuranosyl-(1 ! 6)]-b-d-glucopyranoside [12]. The above compounds were report-
ed for the first time from this genus. Here, we report on the isolation and structure
elucidation of the two new alkaloids 1 and 2, and the evaluation of their cytotoxic
activities.
Results and Discussion. – Structure Elucidation. Compound 1, isolated as an
amorphous powder, was assigned the molecular formula C18H16N2O3, on the basis of
the [M þ H]þ ion peak at m/z 309.1234 (calc. 309.1239) in HR-ESI-MS (positive-ion
1
mode). The H-NMR spectrum (DMSO) showed signals for four aromatic H-atoms
due to the A ring of the indole system at d(H) 7.70 (d, J ¼ 8.2, HꢀC(9))1), 7.15 (dd, J ¼
8.2, 8.2, HꢀC(10)), 7.33 (dd, J ¼ 8.2, 8.2, HꢀC(11)), and 7.52 (d, J ¼ 8.2, HꢀC(12)), and
for two CH2 groups at d(H) 4.87 – 4.91 (m, CH2(5)) and 3.32 – 3.36 (m, CH2(6)),
suggesting that the skeleton of 1 resembled tryptamine (Table 1). A methine C-atom
1
)
Arbitrary C-atom numbering as indicated in the formula. For systematic names, cf. the Exper. Part.
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 94 (2011)
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bearing a OH group, as indicated by the chemical shift of the corresponding H-atom at
d(H) 5.18 – 5.20 (q, J ¼ 6.5, HꢀC(18)), coupled with a Me group d(H) 1.43 (d, J ¼ 6.5,
HꢀC(19)). HMBC Correlations of HꢀC(19) with C(16) and C(18), and of HꢀC(18)
with C(15), C(16), and C(19) indicated that the 1-hydroxyethyl fragment was located
at C(16).
The signals in the 13C-NMR spectrum were attributed to one COOꢀ C-atom (d(C)
165.9 (C(20))) and five olefinic C-atoms, and the corresponding downfield signals at
d(H) 8.23 (s, HꢀC(14)) and 8.78 (s, HꢀC(17)) in the 1H-NMR spectrum of 1, together
with a strong absorption band (1606 cmꢀ1) in the IR spectrum indicated the presence of
a pyridine ring in compound 1. A characteristically highly deshielded signal at d(H)
8.78 (s, HꢀC(17)) indicated the presence of a H-atom at a C-atom adjacent to a
positively charged N-atom [13], which was verified by the HMBC experiment. HMBC
Correlations (Table 1) of HꢀC(17) with C(3), C(5), C(15), C(16), and C(18), of
HꢀC(14) with C(2), C(3) and C(20) established the structure of 1, with the chemical
shift at d(C) 165.9 indicating the C(20)OOꢀ group [13]. Accordingly, 1 was assigned an
inner salt form and named mappine.
Compound 2 was isolated as an amorphous powder, and its molecular formula was
established as C26H28N2O9, based on the HR-ESI-MS (m/z 513.1868 ([M þ H]þ; calc.
513.1873)). The 1H-NMR spectrum (CD3OD) showed signals of two H-atoms of C ring
of a b-carboline moiety at d(H) 8.11 (d, J ¼ 5.7, HꢀC(5))1) and 7.90 (d, J ¼ 5.7, HꢀC(6)),
along those of four aromatic H-atoms on the A ring (d(H) 8.12 (d, J ¼ 8.4, HꢀC(9)),
7.22 (dd, J ¼ 8.4, 8.4, HꢀC(10)), 7.52 (dd, J ¼ 8.4, 8.4, HꢀC(11)), and 7.70 (d, J ¼ 8.4,
HꢀC(12))), one acetal H-atom (d(H) 5.57 (d, J ¼ 6.9, HꢀC(21))), one anomeric H-
atom of a b-linked glucose (d(H) 4.73 (d, J ¼ 7.8, HꢀC(1’))), and one olefinic H-atom of
the acrylic part moiety (d(H) 7.30 (s, HꢀC(17))) (Table 2). The 13C-NMR spectrum
(CD3OD) showed signals for 26 C-atoms including 13 olefinic atoms, one COOH (d(C)
176.2 (C(22))), one acetal (d(C) 97.4 (C(21))), and one anomeric C-atom (d(C) 100.7
(C(1’))), and one glucose unit. The hydrolysis in acidic medium provided the free sugar,
which could be identified as d-glucose by the sign of its optical rotation and comparison

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Table 1. 1H- and 13C-NMR Data and HMBC Correlations for Compound 1. Recorded in (D6)DMSO at
400 and 100 MHz, respectively; d in ppm, J in Hz. Atom numbering as indicated in the formula.
d(H)
d(C)
HMBC (H ! C)
C(2)
C(3)
CH2(5)
CH2(6)
C(7)
138.8
141.3
55.0
4.87 – 4.91 (m)
3.32 – 3.36 (m)
C(3), C(6), C(7), C(17)
C(2), C(5), C(7), C(8)
18.9
116.0
124.7
120.3
120.4
125.6
112.6
139.1
118.7
156.5
138.8
142.8
64.6
C(8)
HꢀC(9)
HꢀC(10)
HꢀC(11)
HꢀC(12)
C(13)
7.70 (d, J ¼ 8.2)
C(7), C(11), C(13)
C(8), C(12)
C(9), C(13)
7.15 (dd, J ¼ 8.2, 8.2)
7.33 (dd, J ¼ 8.2, 8.2)
7.52 (d, J ¼ 8.2)
C(8), C(10)
HꢀC(14)
8.23 (s)
C(2), C(3), C(20)
C(15)
C(16)
HꢀC(17)
HꢀC(18)
Me(19)
C(20)
8.78 (s)
C(3), C(5), C(15), C(16), C(18)
C(15), C(16), C(19)
C(16), C(18)
5.19 (q, J ¼ 6.5)
1.43 (d, J ¼ 6.5)
24.1
165.9
of retention times (tR) of its Me3Si (TMS) ethers with the tR value of a standard sample
in gas chromatography.
The 1H- and 13C-NMR spectra of 2 were very similar to those of lyalosidic acid (3;
Table 2), except for two CH2 C-atoms in 2 instead of two olefinic C-atoms in 3. Due to
the same molecular weight of the two compounds, it may be deduced that 2 was derived
from lyalosidic acid by cyclization between C(14) and C(18), was also supported by the
HMBC correlations of HꢀC(18) with C(3) and C(20), and of HꢀC(14) with C(2),
C(16), and C(20). In the ROESY spectrum of 2, correlation peaks HꢀC(15)/HꢀC(20)
and HꢀC(14)/HꢀC(21) were observed. Thus, the iridoid part of 2 bears a loganin
skeleton, on the other hand, dihydrocornin and secologanin were also obtained in this
phytochemical study which shared the same relative configuration. Dihydrocornin [10]
and secologanin [11] were identified by comparison of their NMR data and the physical
properties with those reported in the literature. From a biogenetic view, HꢀC(15) and
HꢀC(20) of 2 should be b-oriented, while HꢀC(14) and HꢀC(21) have a-orientation.
All the above information strongly indicated that 2 was a cyclized product of lyalosidic
acid, named mapposidic acid.
The cytotoxicities of compounds 1 and 2 were evaluated. The results were listed in
Table 3. Compound 1 demonstrated inhibitory activity against K562 and Lovo cell lines
with IC50 values of less than 10 mm, and compound 2 showed cytotoxicity against the
K562, HepG2, KB, and LoVo cell lines with IC50 values of 15.3, 6.9, 2.2 and 4.2 mm,
respectively.

Helvetica Chimica Acta – Vol. 94 (2011)
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Table 2. 1H- and 13C-NMR Spectral Data and HMBC Correlations for Compounds 2 and 3. Recorded in
CD3OD at 400 and 100 MHz, respectively; d in ppm, J in Hz. Atom numbering as indicated in the
formula of 2.
2
3
d(H)
d(C) HMBC (H ! C)
d(H)
d(C)
C(2)
C(3)
HꢀC(5)
HꢀC(6)
C(7)
137.2
147.1
138.2
144.8
136.7
114.9
130.8
122.1
122.7
121.2
130.3
113.4
135.8
36.6
8.11 (d, J ¼ 5.7)
7.90 (d, J ¼ 5.7)
137.5 C(3), C(6), C(7)
114.6 C(2), C(5), C(8)
129.9
8.05 (d, J ¼ 5.6)
7.91 (d, J ¼ 5.6)
C(8)
122.8
HꢀC(9)
HꢀC(10)
HꢀC(11)
HꢀC(12)
C(13)
8.12 (d, J ¼ 8.4)
118.4 C(7), C(11), C(13)
120.7 C(8), C(12)
129.4 C(9), C(13)
113.5 C(8), C(10)
142.6
8.14 (d, J ¼ 8.4)
7.22 (dd, J ¼ 8.4, 8.4)
7.52 (dd, J ¼ 8.4, 8.4)
7.70 (d, J ¼ 8.4)
7.19 (dd, J ¼ 8.4, 8.4)
7.54 (dd, J ¼ 8.4, 8.4)
7.73 (d, J ¼ 8.4)
HꢀC(14) or 2.47 – 2.51 (m)
36.7 C(2), C(16), C(20)
2.14 – 2.17 (m),
2.35 – 2.37 (m)
2.98 – 3.01 (m)
CH2(14)
HꢀC(15)
3.66 – 3.69 (m)
36.1 C(3), C(17)
117.8
149.3 C(15), C(16), C(21) 7.71 (s)
28.7 C(3), C(20)
29.5 C(15), C(21)
36.0
114.8
151.2
118.8
C(16)
HꢀC(17)
CH2(18)
7.30 (s)
2.68 – 2.72 (m),
3.10 – 3.14 (m)
5.18 (d, J ¼ 11.0),
5.28 (d, J ¼ 17.6)
5.75 (ddd,
CH2(19) or 2.47 – 2.51 (m),
134.6
HꢀC(19)
HꢀC(20)
HꢀC(21)
C(22)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HꢀC(5’)
CH2(6’)
2.67 – 2.71 (m)
2.51 – 2.53 (m)
5.57 (d, J ¼ 6.9)
J ¼ 17.6, 11.0, 7.5)
47.1 C(14), C(16), C(21) 2.66 – 2.68 (m)
97.4 C(17), C(1’)
176.2
100.7 C(21), C(3’), C(5’)
74.9 C(3’), C(4’)
78.2 C(1’), C(5’)
71.9 C(2’), C(5’)
78.5 C(1’), C(3’), C(6’)
63.1 C(4’), C(5’)
46.4
97.6
174.7
100.4
74.6
78.4
71.7
5.78 (d, J ¼ 7.2)
4.73 (d, J ¼ 7.8)
4.72 (d, J ¼ 7.6)
3.21 (dd, J ¼ 7.8, 9.3)
3.17 (dd, J ¼ 7.6, 9.0)
3.32 (dd, J ¼ 9.0, 9.0)
3.26 – 3.29 (m)
3.17 – 3.35 (m)
3.26 (dd, J ¼ 9.3, 9.3)
3.27 – 3.35 (m)
3.27 – 3.32 (m)
78.1
63.2
3.66 (dd, J ¼ 6.6, 11.9),
3.58 (dd, J ¼ 6.9, 11.7),
3.93 (dd, J ¼ 2.1, 11.9)
3.93 (dd, J ¼ 2.1, 11.7)
Table 3. Cytotoxic Activities (IC50 [mm] ꢁ SD) of Compounds 1 and 2 against Four Tumor Cell Lines
Compound
K562
4.6 ꢁ 0.4
15.3 ꢁ 1.5
> 400b)
0.09 ꢁ 0.01
HepG2
KB
LoVo
1
2
> 100
> 100
9.8 ꢁ 0.8
6.9 ꢁ 0.6
> 400b)
0.07 ꢁ 0.0067
2.2 ꢁ 0.2
0.0011 ꢁ 0.00006
> 400b)
4.2 ꢁ 0.3
Taxoterea)
Adriamycina)
0.0021 ꢁ 0.00004
> 400b)
c) Positive control substances. b) No cytotoxic activity.
Experimental Part
General. Column chromatography (CC): silica gel (200 – 300 and 100 – 200 mesh, Qingdao Marine
Chemical, China); C18 reversed-phase (RP) silica gel (20 – 45 mm, Fuji Silica Chemical, Japan) and

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Sephadex LH-20 (Pharmacia). TLC: GF254 silica-gel plates (Yantai Marine Chemical Co., Ltd., China).
UV Spectra: Hitachi UV-3200 spectrophotometer; lmax (log e) in nm. Optical rotations: Perkin-Elmer 341
.
polarimeter. IR Spectra: Perkin-Elmer 577-IR spectrometer; in KBr; ˜n in cmꢀ1 1H- and 13C-NMR
spectra: Bruker AM-400 spectrometer; d in ppm rel. to Me4Si as internal standard, J in Hz. ESI-MS:
Finnigan LCQ-DECA instrument; HR-ESI-MS: Waters Q-TOF spectrometer; in m/z.
Plant Material. Samples of Mappianthus iodoides Hand.-Mazz. were collected in Jinxiu, Guangxi
Province, P. R. China, in July 2007. The specimen was identified by Prof. Yu Zhao, Department of
Pharmacy, Zhejiang University. A voucher specimen (No. 20070703) was deposited with the Herbarium
of the College of Agriculture and Biotechnology.
Extraction and Isolation. Powdered and air-dried stems (1.0 kg) of Mappianthus iodoides Hand.-
Mazz. were extracted exhaustively with cold 95% EtOH (3 ꢂ 4.0 l, 1.5 h each). Evaporation of EtOH
gave a residue (120 g), which was then suspended in 15% EtOH (0.8 l) and filtered. The filtrate was
concentrated (1.0 l) and partitioned sequentially with petroleum ether (3 ꢂ 1.0 l), AcOEt (3 ꢂ 1.0 l), and
BuOH (4 ꢂ 1.0 l). The AcOEt-soluble fraction (20 g) was concentrated and subjected to CC (silica gel;
CHCl3/MeOH 20 :1 – 2 :1) to yield eight fractions. Fr. 1 was purified by repeated CC (silica gel;
petroleum ether (PE)/acetone 8 :1 – 2 :1; and Sephadex LH-20; MeOH as eluent) to afford 1-methyl-b-
carboline (14 mg), pinoresinol (18 mg), (þ)-(1R,2S,5R,6S)-2,6-bis(4’-hydroxyphenyl)-3,7-dioxabicy-
clo[3.3.0]octane (14 mg), and pinoresinol dimethyl ether (9 mg). Fr. 2 was subjected to CC (silica gel;
PE/acetone 3 :1) to give dihydrocornin (18 mg). Frs. 3 – 8 were further purified similarly by CC (ODS;
MeOH/H2O with gradient increased MeOH; and Sephadex LH-20; MeOH as eluent) to give secologanin
(8 mg), strictosidine (19 mg), strictosidine acid (10 mg), 1 (10 mg), 2 (15 mg), 5-carboxystrictosidine
(19 mg), and lyalosidic acid (20 mg). The BuOH-soluble fraction (36 g) submitted to CC (first silica gel;
CHCl3/MeOH 9 :1; and then Sephadex LH-20; MeOH/H2O 1:1) to yield 3,4,5-trimethoxyphenol-1-O-
[b-d-apiofuranosyl-(1 ! 6)]-b-d-glucopyranoside (9 mg). The structures of the eleven known isolates
were identified by a combination of spectroscopic methods (MS, and 1H- and 13C-NMR) and
comparisons with data in the literature or co-TLC with authentic samples.
Mappine A (¼ 7,12-Dihydro-3-(1-hydroxyethyl)-6H-indolo[2,3-a]quinolizin-5-ium-2-carboxylate;
1). Amorphous powder. UV (DMSO): 254 (3.30), 310 (3.50). [a]2D3 ¼ þ16 (c ¼ 0.12, DMSO). IR
(KBr): 3384, 1656, 1606, 1573, 1439, 1298, 1194, 1082. 1H- and 13C-NMR: Table 1. ESI-MS: 309.3 ([M þ
H]þ). HR-ESI-MS: 309.1234 ([M þ H]þ; calc. 309.1239).
Mapposidic Acid (¼(1S,4aS,5R,7aR)-1-(b-d-Glucopyranosyloxy)-1,4a,5,6,7,7a-hexahydro-5-(9H-
pyrido[3,4-b]indol-1-yl)cyclopenta[c]pyran-4-carboxylic acid 1; 2). Amorphous powder. UV (MeOH):
231 (3.30), 298 (3.41), 311 (3.24). [a]2D3 ¼ ꢀ47 (c ¼ 0.17, MeOH). IR (KBr): 3390, 1624, 1444, 1389, 1311,
1
1066. H- and 13C-NMR: Table 2. ESI-MS: 513.2 ([M þ H]þ). HR-ESI-MS: 513.1868 ([M þ H]þ; calc.
513.1873).
Acid Hydrolysis of 2. A soln. of 2 (4 mg) in MeOH (5 ml) containing 1n HCl (2 ml) was refluxed for
4 h, concentrated under reduced pressure, diluted with H2O, and extracted with AcOEt. The aq. phase
was concentrated to obtain the sugar moiety which was identified as d-glucose by the sign of its optical
rotation ([a]2D3 ¼ þ51.8 (c ¼ 0.02, MeOH)). It was further confirmed by comparing retention times (tR) of
its Me3Si (TMS) ethers (a-anomer, 3.7 min; b-anomer, 5.1 min) with tR of a standard sample in GC.
Preparation of TMS ether and its subsequent GC was carried out according to the protocol described in
[14].
Cell Cultures. Human HepG2 (hepatocellular carcinoma), K562 (human leukemia), KB (cervix
carcinoma), and LoVo (colon adenocarcinoma) cell lines were obtained from the Shanghai Cell Bank,
Chinese Academy of Sciences. The cells were maintained in RPMI1640 medium with 10% FBS (fetal
bovine serum). In each case, 100 U/ml of penicillin and 100 U/ml of streptomycin were added.
Cytotoxicity Assay. Cells were cultured in 96-well microtiter plates for the assay. After incubation for
24 h and treatment with 10ꢀ2 to 102 mm of the test compounds for 72 h, growth inhibition of the cancer
cells was evaluated by the SRB method (adherent cells: HepG2, KB, and LoVo) or WST-1 method
(suspended cell: K562) as described in [15][16]. The activities are given as IC50 values. Results are
expressed as the mean values of triplicate data points. Adriamycin and taxotere were used as positive
controls.

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We thank the State Administration of TCM of Zhejiang, P. R. China (No. 2008YA020), for financial
support.
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Received May 3, 2011