A new type of monoterpenoid indole alkaloid precursor from alstonia rostrata

Article abstract of DOI:10.1021/ol200996a

Currently, all monoterpenoid indole alkaloids (MIAs) have been derived from strictosidine, which originates from the condensation of tryptophan with secologanin in a 1:1 ratio. However, our phytochemical research on Alstonia rostrata revealed a potential new precursor for these compounds. We isolated the alstrostines A and B, and it was determined that they were derived from tryptophan and secologanin in a 1:2 ratio, which supported the presence of a new type of MIA precursor.

Full text of DOI:10.1021/ol200996a

ORGANIC
LETTERS
2011
Vol. 13, No. 14
3568–3571
A New Type of Monoterpenoid Indole
Alkaloid Precursor from Alstonia rostrata
Xiang-Hai Cai,*,† Mei-Fen Bao,† Yu Zhang,† Chun-Xia Zeng,‡ Ya-Ping Liu,† and
Xiao-Dong Luo*,†
State Key Laboratory of Phytochemistry and Plant Resources in West China and
Southwest China Germplasm Bank of Wild Species, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650204, China
xhcai@mail.kib.ac.cn; xdluo@mail.kib.ac.cn
Received April 14, 2011
ABSTRACT
Currently, all monoterpenoid indole alkaloids (MIAs) have been derived from strictosidine, which originates from the condensation of tryptophan
with secologanin in a 1:1 ratio. However, our phytochemical research on Alstonia rostrata revealed a potential new precursor for these
compounds. We isolated the alstrostines A and B, and it was determined that they were derived from tryptophan and secologanin in a 1:2 ratio,
which supported the presence of a new type of MIA precursor.
Alkaloids represent a highly diverse group of com-
pounds that are related solely by the presence of a nitrogen
atom in a heterocyclicring. Plantsare estimated toproduce
approximately 12000 different alkaloids, and they can be
organized into groups according to their carbon skeletal
structures.1 Among them, monoterpenoid indole alkaloids
(MIAs) might account for a quarter of this large group of
natural products. Both their highly complex chemical
structures and pronounced pharmacological activity have
made research on alkaloids, including elucidation of their
biosynthetic pathways, very attractive for many decades.2
MIAs have been proposed to arise from strictosidine, which
itself originates from the condensation of tryptophan with
secologanin in a 1:1 ratio.3 Currently, strictosidine is the
only key precursor recognized in the biosynthesis of
MIAs.4 However, additional ratios of tryptophan and
secologanin occur in nature (e.g., 1:2, 2:1), and this led us
to investigate whether these ratios may play a role in MIA
biosynthesis. Plants from the genera Alstonia and Melodi-
nus represent excellent systems for this work in that they
both accumulate high levels of MIAs.5 Here we describe
(4) Tietze, L. F. Angew. Chem., Int. Ed. Engl. 1983, 22, 828.
(5) (a) Cai, X. H.; Zeng, C. X.; Feng, T.; Li, Y.; Luo, X. D. Helv.
Chim. Acta 2010, 93, 2037. (b) Cai, X. H.; Shang, J. H.; Feng, T.; Luo,
X. D. Z. Naturforsch. B 2010, 65, 1164. (c) Cai, X. H.; Feng, T.; Liu,
Y. P.; Du, Z. Z.; Luo, X. D. Org. Lett. 2008, 10, 577. (d) Cai, X. H.; Du,
Z. Z.; Luo, X. D. Org. Lett. 2007, 9, 1817. (e) Feng, T.; Li, Y.; Cai, X. H.;
Luo, X. D. J. Nat. Prod. 2009, 72, 1836. (f) Feng, T.; Cai, X. H.; Zhao,
P. J.; Du, Z. Z.; Li, W. Q.; Luo, X. D. Planta Med. 2009, 75, 1537. (g)
Feng, T.; Cai, X. H.; Liu, Y. P.; Li, Y.; Wang, Y. Y.; Luo, X. D. J. Nat.
Prod. 2010, 73, 22. (h) Feng, T.; Liu, Y. P.; Cai, X. H.; Wang, Y. Y.; Luo,
X. D. Org. Lett. 2010, 12, 968. (i) Feng, T.; Li, Y.; Wang, Y. Y.; Cai,
X. H.; Liu, Y. P.; Luo, X. D. J. Nat. Prod. 2010, 73, 1075. (j) Feng, T.;
Cai, X. H.; Li, Y.; Wang, Y. Y.; Li, Y.; Liu, Y. P.; Xie, M. J.; Luo, X. D.
Org. Lett. 2009, 11, 4834.
† State Key Laboratory of Phytochemistry and Plant Resources in West
China.
‡ Southwest China Germplasm Bank of Wild Species.
(1) Ziegler, J; Facchini, P. J. Ann. Rev. Plant Biol. 2008, 59, 736.
(2) Stockigt, J.; Panjikar, S. Nat. Prod. Rep. 2007, 24, 1382.
(3) (a) Tietze, L. F.; Bachman, D. C. J.; Schul, W. Angew. Chem., Int.
Ed. Engl. 1988, 27, 971. (b) Patthy-Lukats, A.; Karolyhazy, L.; Szabo,
L. F.; Podanyi, B. J. Nat. Prod. 1997, 60, 69.
r
10.1021/ol200996a
Published on Web 06/21/2011
2011 American Chemical Society

the structural determination and proposed biosynthesis of
a new kind of MIA precursor from A. rostrata (Figure 1).
The dried and powdered leaves of A. rostrata (8.0 kg)6
were extracted with MeOH (20 L ꢀ 3) at room tempera-
ture, and the solvent was evaporated in vacuo. The residue
was dissolved in 1% HCl, and the solution was subse-
quently basified to pH 8ꢁ9, using ammonia. The basic
solution was partitioned with EtOAc, affording a two-
phase mixture including the aqueous phase and EtOAc
phase (total alkaloids). The total alkaloid fraction (78 g)
was collected and then dissolved in MeOH and was sub-
jected to column chromatography over silica gel eluting
with CHCl3ꢁMeOH [from CHCl3 to CHCl3ꢁMeOH
(1:1)] to afford six fractions (IꢁVI). Fraction IV (6.5 g)
was separated utilizing a preparative reversed-phase C18-
MPLC column with a gradient flow of 30ꢁ70% (v/v)
aqueous MeOH to yield seven subfractions IV-1ꢁ7. Sub-
fraction IV-6 (214 mg) was further separated by chromo-
tography on silica gel using CHCl3ꢁMeOH (6:1) as an
eluent to give alstrostine B (2, 11 mg). Fraction VI (5.9 g)
was purified using a preparative reversed-phase C18-MPLC
column with a gradient flow of 20ꢁ50% (v/v) aqueous
MeOH to yield six subfractions VI-1ꢁ6. Alstrostine A (1, 5
mg) was subsequently isolated from subfraction VI-6.
Compound 17 possessed a molecular formula of C44H56-
N2O19, as evidenced by a high resolution electron spray ion-
ization mass spectra (HRESIMS) at m/z917.3549 [M þ H]þ,
in combination with 1H, 13C NMR, DEPT spectra,
and appropriate for 18 degrees of unsaturation. The UV
spectrum of compound 1 demonstrated the presence of
conjugated groups by presenting maximum absorptions at
211, 231, 279, and 314 nm. Its IR spectrum indicated the
presence of hydroxyls (3430 cmꢁ1), carbonyls (1703 cmꢁ1),
existence of a five-membered pyrrolidine C ring joined to
the hydroxy-indoline framework.9 The two signals were
thus assigned to C-5 and C-6, respectively. This presump-
tion was confirmed by HMBC correlations from H-2 to
C-5/6. In addition, the remaining 34 13C NMR signals of 1
showed the presence of two terminal double bonds
[δC 120.2 (t), 135.7 (d), 119.1 (t), 137.0 (d) ppm], two R,
β-unsaturated carboxylic acid methyl esters [δC 153.0 (d),
110.6 (s), 169.6 (s), 51.9 (q), 154.0 (d), 112.5 (s), 169.0 (s),
51.9 (q) ppm], and two glucoses [δC 100.2 (d), 101.1 (d),
62.8 (t), 61.8 (t) ppm, and eight sp3 methines between δC
78.5 and 70.9 ppm]. These data support the existence of
two secologanin moieties.10,3b Both secologanin moieties
were subsitituted by condensation of their respective alde-
hyde groups, as shown by the absence of aldehyde spectral
signals. Additionally, HMBCcorrelations betweenδH 3.29
(H-3) with C-2, and between δH 6.30 (s, H-30) with C-2/C-13,
revealed that the two secologanins were jointed to N-1
and N-4, respectively. Correlations from H-30 to C-3 (δC
56.1d) and C-150 (δC 37.8, d);from H-3 toC-14(δC 33.0, t),
1
and olefin groups (1632 cmꢁ1). The H NMR spectrum
presented two doublets (δH 7.25, 6.67 ppm) and two
triplets (δH 7.13, 6.79 ppm), displaying the signals for an
unsubstituted indole alkaloid.8 In the HMBC spectrum of
1, crosspeaks between the signal (δH 7.25 ppm, H-9) with
carbon resonances [δC 149.5 (s, C-13), 130.7 (s, C-11), and
89.1 (s) ppm], allowed the assignment of the later carbon
signal to C-7. Similarly, HMBC correlations from the
signal at δC 4.90 (s) ppm to C-7/C-13 suggested the proton
signal was attributed to H-2. The presence of two methy-
lene signals (δC 50.9, 43.6 ppm) that were correlated
[δH 2.82 (1H, m)/δH 2.28 (1H, m) and 2.58 (1H, m)/δH 1.97
Figure 1. Structure of compounds 1ꢁ2.
C-140 (δC 115.2 s), and C-30; and from H-150 (δH 3.22) to
C-3 (δC 56.1, d), C-30, and C-140 in the HMBC spectrum
revealed a new six-membered ring D. The compound
numbering system corresponded in biogenetic origin to
the monoterpenoid indole alkaloids (Figure 1). Its full
assignments of 1H, 13C, spectroscopic data were determined
1
by an HMBC, HSQC 1Hꢁ H COSY spectrum (Table 1).
Compound 211 had a molecular formula of C38H46-
N2O14 according to HRESIMS (m/z 753.2886, [M ꢁ H]ꢁ),
with an absence of a glucose fraction to 1. The UV
absorption bands (211, 233, 277, and 315 nm) and IR
spectrum (3429, 1713, and 1630 cmꢁ1) of compound 2
demonstrated that it was the same type of compound as 1.
1
1
(1H, m)] in the Hꢁ H COSY spectrum and showed
HMBC correlations to C-7/C-2 (δC 81.7, s) indicated the
(6) Leaves and twigs of A. rostrata collected in Apr. 2010 in Simao of
Yunnan Province, P. R. China, and identified by Dr. Chun-Xia Zeng.
Voucher specimen (Cai100613) depositedin the State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute
of Botany, the Chinese Academy of Sciences.
1
The H and 13C NMR spectra of 2 were very similar to
(7) Alstrostine A: white powder; [R]D20 = ꢁ257° (c, 0.09, CH3OH);
UV (CH3OH) λmax 211 (ε 3310), 231(ε 3096), 279 (ε 2032), and 314 (ε
662) nm; IR (KBr) νmax 3430, 1703, and 1632 cmꢁ1; 1H and 13C NMR
data, Table 1; posiþtive ESIMS m/z [M þ H]þ 917 (100); HRESIMS m/z
917.3549 [M þ H] (calcd for C44H56N2O19, 917.3555).
those of 1, except that 2 had the signals of δC 18.1 (q), 70.0
(10) Patthy-Lukats, A.; Kocsis, A.; Szabo, L. F.; Podanyi, B. J. Nat.
Prod. 1999, 62, 1492.
(11) Alstrostine B: white powder; [R]D20 = ꢁ290° (c, 0.13, CH3OH);
UV (CH3OH) λmax 211(ε 3203), 233 (ε 3295), 277 (ε 2846), 315 (ε 708)
nm; IR (KBr) νmax 3429, 1713, and 1630 cmꢁ1; 1H and 13C NMR data,
Table 1; positive ꢁESIMS m/z [M ꢁ H]ꢁ 753 (100); HRESIMS m/z
753.2886 [M ꢁ H] (calcd for C38H45N2O14, 753.2870).
(8) Abe, F.; Chen, R. F.; Yanauchi, T.; Marubayashi, N.; Ueda, I.
Chem. Pharm. Bull. 1989, 37, 887.
(9) (a) Kawasaki, T.; Takamiya, W.; Okamota, N.; Nagaoka, M.;
Hirayama, T. Tetrahedron Lett. 2006, 47, 5379. (b) Yang, S. W.; Cordell,
G. A. J. Nat. Prod. 1997, 60, 44.
Org. Lett., Vol. 13, No. 14, 2011
3569

Table 1. 1H and 13 C NMR Assignments of Compounds 1 and 2a
b
entry
1 δC
2 δC
1 δH (J in Hz)
2 δH (J in Hz)b
2
3
5
81.7 d
56.1 d
50.9 t
81.7 d
53.1 d
49.9 t
4.90 (1H, s)
3.29 (1H, m)
2.82 (1H, m)
2.58 (1H, m)
2.28 (1H, m)
1.97 (1H, m)
5.00 (1H, s)
3.29 (1H, m)
2.90 (1H, m)
2.33 (1H, m)
2.33 (1H, m)
1.91 (1H, m)
6
43.6 t
89.1 s
43.1 t
88.6 s
7
8
9
135.7 s 136.2 s
125.0 d 124.4 d 7.25 (1H, d, 6.8)
121.4 d 120.9 d 6.79 (1H, t, 6.8)
130.7 d 129.9 d 7.13 (1H, t, 6.8
108.6 d 108.6 d 6.67 (1H, d, 6.8)
149.5 s 148.4 s
7.24 (1H, d, 6.8)
6.76 (1H, t, 6.8)
7.09 (1H, t, 6.8)
6.73 (1H, d, 6.8)
10
11
12
13
14
Figure 2. Key NOE correlations of energy-minimized confor-
mer of 1.
33.0 t
32.4 t
2.08 (1H, m)
1.97 (1H, m)
3.27 (1H, m)
1.87 (1H, m)
2.28 (1H, m)
3.33 (1H, m)
15
16
17
18
26.4 d
27.0 d
seen in 1. This suggested a rearrangement occurred from C-21
to C-19 in 2, and this was further supported by the HMBC
spectrum, with correlations of δH 4.21 (H-190) with δC 51.6
(C-200), 33.1 (C-150), 18.1(C-180), and 201.6 (C-210), and of
δH 7.69 (s, H-170) with δC 70.0 (d, C-190), 33.1 (d, C-150),
105.8 (s, C-160), and 167.3(s, C-220). Other structural parts of
2were identical to those 1, as indicated by the HMBC spectra.
H-15(150)/20(200) of secologanin units in compound 1
were at the β-position and H-21(210) at the R-position
based on the biosynthesis12 and the ROESY spectroscopic
data.10 However, ROESY correlation of H-190/H-150 in 2
placed H-190 at the β-orientation, and its rearrangement
from C-210 to C-190 resulted in inversion of configuration
H-200, R-orientation. The J values (J = 7.2, 7.8 Hz) of
the two anomeric protons of the sugar moieties revealed a
β-configuration of the glucose residues. D-Glucose was
identified in the hydrolysate by TLC comparison with
authentic samples and determination of their specific optical
rotation (þ 24°).13 Based on those data, the structures of 1
and 2 were elucidated as shown.
The relative configurations of H-2, H-3, and 7-OH of 1
and 2 were constructed from analysis of molecular models,
and the energy was minimized using density functional
theory (DFT) at the 6-31G (d,p) basis set level in Gaussian
03 overlaid with key correlations observed in the ROESY
NMR spectrum (Figure 2).14 Nuclear Overhauser Effect
(NOE) correlation of H-2/7-OH (δH 4.74/δH 5.85 of 1, and
δH 5.00/δH 5.08 of 2) in the ROESY spectrum indicated
that both protons were on the same side. In addition, the
ROESY correlations between H-3 with H-5 and their
110.6 s 111.7 s
153.0 d 152.0 d 7.44 (1H, s)
7.40 (1H, s)
120.2 t 119.7 t 5.32 (1H, d, 15.0) 5.35 (1H, d, 15.0)
5.27 (1H, d, 12.0) 5.20 (1H, d, 12.0)
19
20
21
22
135.7 d 135.8 d 5.80 (1H, m)
5.78 (1H, m)
2.80 (1H, m)
5.49 (1H, d, 3.6)
45.0 d
98.5 d
44.1 d
97.8 d
2.72 (1H, m)
5.55 (1H, d, 3.6)
169.6 s 167.9 s
22-OMe 51.9 q
7-OH
30
140
150
160
170
180
51.3 q
3.70 (3H, s)
5.85 (1H, s)c
3.65 (3H, s)
5.08 (1H, s)
6.29 (1H, s)
126.1 d 125.1 d 6.30 (1H, s)
115.2 s 115.1 s
37.8 d
112.5 s 105.8 s
154.0 d 156.8 d 7.59 (1H, s)
33.1 d
3.22 (1H, d, 3.6)
3.48 (1H, s)
7.69 (1H, s)
119.1 t 18.1 q
5.17 (1H, d, 15)
1.47 (1H, d, 6.8)
5.12 (1H, d, 12)
5.65 (1H, m)
2.54 (1H, m)
190
200
210
100
200
300
400
500
600
137.0 d 70.0 d
4.21 (1H, q, 6.8)
2.49 (1H, s)
47.1 d
98.0 d
51.6 d
201.6 d 5.48 (1H, d, 6.4)
9.67 (1H, s)
100.2 d 100.0 d 4.68 (1H, d, 7.2)
4.68 (1H, d, 7.2)
3.25 (1H, m)
3.40 (1H, m)
3.36 (1H, m)
3.40 (1H, m)
3.88 (1H, m)
3.66 (1H, m)
74.7 d
78.5 d
71.6 d
78.3 d
62.8 t
74.3 d
77.9 d
71.4 d
77.9 d
62.8 t
3.18 (1H, m)
3.34 (1H, m)
3.29 (1H, m)
3.43 (1H, m)
3.90 (1H, m)
3.68 (1H, m)
4.63 (1H, d, 7.8)
3.15 (1H, m)
3.08 (1H, m)
3.23 (1H, m)
3.34 (1H, m)
3.48 (1H, m)
3.20 (m)
1000
2000
3000
4000
5000
6000
101.1 d
74.6 d
78.1 d
70.9 d
78.0 d
61.8 t
˚
calculated interatomic distance of approximately 2.6 A in
220
169.0 s 167.3 s
51.3 q
220-OMe 51.9 q
3.66 (3H, s)
3.63 (3H, s)
the molecular model suggested the H-3 adopts another
orientation. Based on these data, there were two isomers of
1 and 2, respectively. Subsequently, for the assignment of
the configuration of 1 and 2, their optical rotation (OR)
a Data recorded in methanol-d3 (1) and acetone-d6 (2) on a Bruker
Avance 600 MHz spectrometer (1H, 13C, HSQC, HMBC, 1Hꢁ H COSY,
1
and ROESY); chemical shifts (δ) in parts per million in reference to most
downfield signal of methanol-d3 (δ 3.31 ppm) and cetone-d6 (δ 2.04 ppm)
for 1H and to center peak of downfield signal of methanol-d3 (δ 49.0 ppm)
and acetone-d6 (δ 30.0 ppm) for 13C. b Data were recorded on a Bruker
AMD-400 MHz spectrometer. c Data were recorded in DMSO.
(12) (a) Achenbach, H.; Benirschke, M. Phytochemistry 1997, 44,
1387. (b) Treimer, J. F.; Zenk, M. H. Eur. J. Biochem. 1979, 81, 225.
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M.; Mahmoud, I. I.; Long, C. J. Nat. Prod. 2005, 68, 832.
(14) Di, Y. T.; He, H. P.; Liu, H. Y.; Yi, P.; Zhang, Z.; Ren, Y. L.;
Wang, J. S.; Sun, Q. Y.; Yang, F. M.; Fang, X.; Li, S. L.; Zhu, H. J.; Hao,
X. J. J. Nat. Prod. 2007, 70, 1352.
(d), and an aldhyede at δC 201.6 (d) instead of the terminal
double bonds at C-18/C19 and signal of δC 98.0 (d, C-21)
3570
Org. Lett., Vol. 13, No. 14, 2011

which supported that H-2/7-OH were β-oriented and H-3
was R-oriented in 1 and 2.
In Figure 3, we propose a potential biosynthetic me-
chanism of compounds derived from a novel ratio of tryp-
tophan and secologanins. Like biosynthesis of strictosidine,3a
condensation of tryptophan and secologanins in a 1:2 ratio
produced compound1 witha 6/5/5/6 tetracyclic system (1).
Further degulose and rearrangement from C-21 to C-19 of
1 formed 2, which was common during biosynthesis of
MIAs, such as the ajmalicine type.17 The isolation of
alstrostines A (1) and B (2) is the first evidence to support
the presence of a new type of precursor of monoterpenoid
indole alkaloids.
Compounds 1ꢁ2 were evaluated for their cytotoxicity
against five human cancer cell lines, MCF-7 breast cancer,
SMMC-7721 hepatocellular carcinoma, HL-60 myeloid
leukemia, SW480 colon cancer, and A-549 lung cancer,
using the MTT method reported previously18 with minor
revisions.19 Unfortunately, none of them showed positive
activity (IC50 > 40 μM).
Acknowledgment. This work was supported in part by
the National Natural Science Foundation of China
(30800091), the 973 Program of Ministry of Science and
Technology of P. R. China (2009CB522300), and the
Chinese Academy of Sciences (KSCX2-EW-R-15 and
XiBuZhiGuang Project).
Figure 3. Proposed biosynthesis of 1ꢁ2.
Supporting Information Available. 1-D and 2-D NMR
data and optical rotation calculations of alstrostines
AꢁB. This material is available free of charge via the
Internet at http://pubs.acs.org.
values were calculated by using the density functional theory
(DFT) methods15 in the Gaussian 03 program package.16
The ‘self-consistent reaction field’ method (SCRF) was
employed to perform the OR calculation of the most stable
conformers of 1 and 2 with H-2 in the β-orientation in
MeOH solution at the B3LYP/6-31G (d,p) level. The calcu-
lated OR values (ꢁ296° for 1 and ꢁ270° for 2) were close to
their experimental values (ꢁ257° for 1 and ꢁ290° for 2),
(18) Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.
(19) Cytotoxicity assay. All the cells were cultured in RPMI-1640 or
DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine
serum (Hyclone, USA) in 5% CO2 at 37 °C. The cytotoxicity assay was
performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl tetrazolium bromide) method in 96-well microplates. Briefly, 100 μL
of adherent cells were seeded into each well of 96-well cell culture plates
and allowed to adhere for 12 h before drug addition, while suspended
cells were seeded just before drug addition with an initial density of 1 ꢀ
105 cells/mL. Each tumor cell line was exposed to the test compound at
concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM in triplicates for 48 h,
with cisplatin (Sigma, USA) as a positive control. After compound
treatment, cell viability was detected, and the cell growth curve was
graphed.
(15) Zhang, Y.; Di, Y. T.; Zhang, Q.; Mu, S. Z.; Tan, C. J.; Fang, X.;
He, H. P.; Li, S. L.; Hao, X. J. Org. Lett. 2009, 11, 5414.
(16) Frisch, M. J. et al. Gaussian 03, revision D.01; Gaussian, Inc.:
Wallingford, CT, 2004 (see the Supporting Information for the complete
reference).
(17) Buckingham, J. Dictionary of Natural Products on CD-ROM;
CRC Press: U.K., 2006.
Org. Lett., Vol. 13, No. 14, 2011
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