Toosendanin relatives, trypanocidal principles from Meliae Cortex

Article abstract of DOI:10.1007/s11418-020-01422-9

Africa Trypanosomiasis remains a serious health problem, but the approved drugs for this disease are so few that novel trypanocidal compounds are demanded. In search for trypanocidal principles from medicinal plants, we found MeOH extracts of Meliae Cortex with potent activity through the screening from about 300 kinds of methanolic extract. By bioassay-guided fractionation from this extract through the liquid–liquid partition and subsequent chromatographic technique using silica gel and ODS, finally we disclosed toosendanin (1) and its relatives as active principles. These active congeners showed not only potent trypanocidal activity but also little cytotoxicity to display the excellent selective index. Taking the isolated amount as well as trypanocidal activity into consideration, 1 was disclosed to be the responsible active principle in Meliae Cortex. Additionally, the derivatives of 1 were chemically prepared from 1 and bioactivity of them were also evaluated. Through the comparison with their trypanocidal activity among the isolated relatives and the synthesized derivatives of 1, the epoxide moiety was revealed to be essential for their potent trypanocidal activity. Furthermore, 3-O-acetyl group and 7-hydroxyl group were presumed to be important functional groups and introduction of methylpropionyl group into hemiacetal hydroxy moiety was clarified to enhance their typanocidal activity.

Full text of DOI:10.1007/s11418-020-01422-9

Journal of Natural Medicines
https://doi.org/10.1007/s11418-020-01422-9
ORIGINAL PAPER
Toosendanin relatives, trypanocidal principles from Meliae Cortex
Michel N. Mifundu1,2 · Nobutoshi Murakami · Tomikazu Kawano · Satoru Tamura
1
3
1,3
Received: 3 April 2020 / Accepted: 7 June 2020
©
The Japanese Society of Pharmacognosy 2020
Abstract
Africa Trypanosomiasis remains a serious health problem, but the approved drugs for this disease are so few that novel tryp-
anocidal compounds are demanded. In search for trypanocidal principles from medicinal plants, we found MeOH extracts of
Meliae Cortex with potent activity through the screening from about 300 kinds of methanolic extract. By bioassay-guided
fractionation from this extract through the liquid–liquid partition and subsequent chromatographic technique using silica gel
and ODS, ꢀnally we disclosed toosendanin (1) and its relatives as active principles. These active congeners showed not only
potent trypanocidal activity but also little cytotoxicity to display the excellent selective index. Taking the isolated amount
as well as trypanocidal activity into consideration, 1 was disclosed to be the responsible active principle in Meliae Cortex.
Additionally, the derivatives of 1 were chemically prepared from 1 and bioactivity of them were also evaluated. Through the
comparison with their trypanocidal activity among the isolated relatives and the synthesized derivatives of 1, the epoxide
moiety was revealed to be essential for their potent trypanocidal activity. Furthermore, 3-O-acetyl group and 7-hydroxyl
group were presumed to be important functional groups and introduction of methylpropionyl group into hemiacetal hydroxy
moiety was clariꢀed to enhance their typanocidal activity.
Keywords Toosendanin · Trypanocidal activity · Meliae Cortex · SAR analysis
Introduction
century, thus anyone cannot deny next one. WHO estimated
that sleeping sickness threatens the population in 36 coun-
tries in sub-Saharan Africa and some 65 million people lived
in infectious-risk areas. The people in the areas with limited
access to appropriate health service are especially aꢂected.
In the last 10 years, over 70% of reported cases occurred in
D. R. Congo [2]. The clinical treatment for trypanosoma
disease had been formerly limited to the only four existing
drugs (suramin, pentamidine, melarsoprol, and eꢁornithine)
with the severe drawbacks due to their high toxicity, emerg-
ing susceptibility to parasite resistance, and poor drug-like
properties [3]. Recently, nifurtimox and fexinidazole were
recognized as new tripanocidal agents and their eꢂective-
ness on putting down T. brucei were veriꢀed [4, 5]. How-
ever, nifurtimox is diꢃcult to be applied to the patients
with severe renal or hepatic dysfunction [6] and much more
clinical data are required for fexinidazole [7]. All these prob-
lems enhanced the serious demand to develop new drugs
against this disease. In this circumstance, we were engaged
in search for trypanocidal principles from medicinal plants.
Previously, we already disclosed the trypanocidal eꢂects of
Central African medicinal plant, Cola acuminata and its
active principles proanthocyanidins [8]. Further exploration
Human Africa Trypanosomiasis, also known as sleeping
sickness, has been resurgent in Sub-Saharan Africa since the
late 1900s [1]. The African trypanosomiasis is pathogenic to
humans and domestic animals by infection of Trypanosoma
brucei, a parasite transmitted by tsetse ꢁies (Glossina spp.).
The number of reported new infections was declined year by
year, but that in 2018 was still estimated to be about 1000
cases by World Health Organization (WHO). Furthermore,
there have been several epidemics in Africa over the last
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11418-020-01422-9) contains
supplementary material, which is available to authorized users.
*
Satoru Tamura
satamura@iwate-med.ac.jp
1
2
3
Graduate School of Pharmaceutical Sciences, Osaka
University, 1-6 Yamada-oka Suita, Osaka 565-0871, Japan
Department of Chemistry, Faculty of Sciences, University
of Kinshasa, Kinshasa, Democratic Republic of the Congo
School of Pharmacy, Iwate Medical University, 1-1-1
Idai-dori Yahaba-cho Siwa-gun, Iwate 019-3694, Japan
Vol.:(0
1
123456789)
3

Journal of Natural Medicines
for trypanocidal compounds from the other medicinal plants
allowed us to ꢀnd promising active constituents, toosendain
and its relatives, from Meliae Cortex, called as “Kurem-
pi” in Japan. Here, we deal with not only the identiꢀcation
of trypanocidal toosendain (1) along with its relatives but
also the structure–activity relationship analysis among the
isolated relatives and the derivatives chemically converted
from 1.
(0.4 mg), A-4-1 (2.2 mg), A-4-2 (3.2 mg), A-5-1 (23 mg),
and A-6-1 (3.1 mg) as the active compounds, respectively.
All trypanocidal principles except for A-3-1 showed more
than 80% inhibition at a concentration of 30 ng/mL whereas
A-3-1 displayed a little weaker potency than them (Fig. S1).
The most abundant two fractions, A-2-1 and -2, showed
1
13
the superimposable H and C NMR spectra indicative of
a mixture of two compounds. Although the two fractions
were given from completely distinguished single peaks by
the HPLC separation, both these puriꢀed fractions showed,
respectively, two peaks at the same retention time corre-
sponding to the two fractions by HPLC analysis. On the
basis of these ꢀndings, they were intensively suggested to
be a mixture of tautomers or two interchangeable or con-
formational isomers; therefore, the structure elucidation
of A-2-1 and -2 was performed as a mixture. In addition
to the IR spectrum and the molecular formula C H O
Result and discussion
As a result of screening of about 300 kinds of extracts of
medicinal plants by use of the trypanocidal assay, the MeOH
extract of Meliae Cortex was found as the promising candi-
date, which exhibited potent trypanocidal activity with little
cytotoxicity. Thus, we started bioassay-guided separation to
search for the trypanocidal principles from this extract. To
remove too high polar constituents, ꢀrst, the MeOH extract
3
0
38 11
+
determined by HR-FAB-MS ([M+H] m/z 575.2488, calcd
575.2494), detailed analysis of NMR spectra (1D and 2D)
and the comparison of spectroscopic data with reported ones
[9, 10] revealed A-2-1 and -2 to be toosendanin (1), a hemi-
acetal triterpenoid derivative (Fig. 1, Table S1). Toosenda-
(
60 g) was subjected to DIAION HP-20, one of the synthetic
adsorbents, column chromatography to furnish MeOH elu-
tion (40 g) after washing with H O. The succeeding frac-
2
tionation of the MeOH elution (4.2 g) through SiO with
nin (1) showed potent trypanocidal activity (IC : 4.0 nM)
2
50
CHCl -MeOH-H O (10:3:1, lower layer) and MeOH as
with excellent selectivity against cytotoxicity for HeLa cells
(IC : 0.79 μM). Toosendanin (1) is one of the representative
3
2
elution furnished seven fractions. The most potent fraction
among them, Fr. A, was subjected to ODS column chro-
matography gave the active fractions A-2 (110 mg), A-3
50
natural compounds in Melia azedarach, an original plant
of Meliae Cortex, which was reported to show the various
bioactivity; anti-tumor [11, 12], inducing apoptosis [13–20],
insecticidal [21, 22], anti-botulismic [23–28], inhibition for
epithelial-mesenchymal transition [29, 30], inhibition for
(
130 mg), A-4 (35 mg), A-5 (240 mg), and A-6 (280 mg).
Finally, ODS HPLC puriꢀcation was applied to each active
fraction to yield A-2-1 (30.1 mg), A-2-2 (30.7 mg), A-3-1
Fig. 1 Isolated toosendanin relatives from Meliae Cortex
1
3

Journal of Natural Medicines
adipogenesis [31], anti-inꢁammatory [32], anti-infection of
inꢁuenza A virus [33], modulation for drug resistance of
tumor cells [34] and so on. However, trypanocidal activity
of 1 has never reported so far; thus, this is the ꢀrst study.
Next, the other trypanocidal principles were also exam-
Table 1 Trypanocidal activity and cytotoxicity of toosendanin rela-
tives
Compound Trypanocidal activity Cytotoxicity Selective index
(
IC /nM)
(IC /μM)
5
0
50
1
13
1
2
3
4
5
6
4.0
0.79
20
0.22
0.72
0.45
0.82
198
67
220
157
62
ined for their structure. The H- and C-NMR of fraction
A-3-1 resembled those of 1 except for the hemiacetal moiety
and the epoxy portion. The HR-FAB MS analysis for A-3-1
clariꢀed its molecular formula as C H O , which sug-
300
1.0
4.6
7.3
3
1
40 11
gested the methylation of 1. Both a keto-carbonyl function
instead of the epoxy moiety of 1 and a methoxyl group at
C-29 were disclosed by intensive analysis for 2D-NMR, and
10.3
80
ꢀ
nally the chemical structure was determined as 2, depicted
in Fig. 1 [35]. However, this compound 2 was presumed to
constituents, 3 showed most potent activity, but 1 was
suggested to be a responsible principle for the activity of
Meliae Cortex taking into account the yield.
be an artifact derived from 1 by MeOH treatment in SiO
2
column chromatography since 1 was easily converted to 2
by MeOH in acidic condition. With regard to fraction A-4-1
For the further investigation about the relationship
between structure and activity, we synthesized some ana-
logs from 1 and examined their trypanocidal activity. At
ꢀrst, a hydrolysis of 1 by aqueous NaOH in 1,4-dioxane
removed two acetyl groups, giving bisdeaceteyltoosenda-
nin (7). Inversely, acetylation of 1 was performed by acetic
anhydride in pyridine at 0 ℃ to furnish (29S)-29-O-acetyl-
toosendanin (8) and (29S)-7,29-O-diacetyltoosendanin (9).
Next, lactone derivative (10) was prepared by selective
oxidation of hemiacetal hydroxy group in 1 by treatment
of Dess–Martin periodinane. Finally, expoxide moiety in
1 was readily converted into keto-carbonyl partial struc-
ture through 1,2-hydride rearrangement by 80% aqueous
acetic acid at 50 ℃ to aꢂord 15-keto-derivative (11) in
99% yield (Scheme 1). The synthesized derivatives of 1
were evaluated for their trypanocidal activity and cyto-
toxicity (Table 2). Unfortunately, all of them except for 8
display more than 100-fold weaker activity as compared
with 1. Considering the result of the structure–activity
relationship using the chemically prepared derivatives as
well as the isolated analogs of 1, epoxide sub-structure
was revealed to be essential for potent activity after all. In
addition, 3-O-acetyl group, 7-hydroxyl group, and hemi-
acetal portion were disclosed to be important for tryp-
anocidal activity of 1. On the other hand, introduction of
acyl group to hydroxy moiety of hemiacetal portion was
found to give the diꢂerent eꢂect on their potency, increase/
decrease, depended on the acyl size.
1
13
and -2, the H- and C-NMR were very similar to that of 1
except for down ꢀeld shift of H- and C-29 signals and the
existence of acyl moieties. The comparison of spectroscopic
data with reported ones and HR-FAB MS revealed A-4-1
and -2 to be 12-O-acetylazedarachin B (3) and 12-O-acet-
ylazedarachin A (4), respectively [10, 36]. Both of frac-
tions A-5-1 and A-6-1 seemed to be congeners of 4 bearing
an acetoxyl group at C-2 by analysis for their NMR data.
By detailed analysis for their spectra, an additional acetyl
moiety was detected at 1-hydroxyl group in case of A-5-1
whereas A-6-1 was disclosed to lack an acetoxyl group at
C-12. Eventually, by comparison with reported spectral data,
[
36, 37] they were identiꢀed to be 1,12-O-diacetyltrichilin
B (5) and trichilin D (6), respectively. (Fig. 1) Among these
active principles, 4, 5, and 6 possessed 2-methylbutyrate
moiety, but the absolute configurations of chiral center
in 2-methylbutyrate were not determined in any previous
reports. Thus, these compounds (4~6) were hydrolyzed by
aq. NaOH and resulting 2-methylbutyric acid was subjected
to HPLC analysis by chiral column. In comparison with
authentic sample, it was revealed that all of them (4 ~ 6)
entirely possess (S)-2-methylbutyrate moieties.
The isolated toosendanin congeners (1–6) were evalu-
ated for their trypanocidal activity and cytotoxicity. As
shown in Table 1, although all relatives showed good
selectivity, 2 lacking the epoxide moiety exhibited sig-
niꢀcantly weaker activity than the others. Thus, epoxy
group was disclosed to be crucial for potent trypanocidal
activity. In comparison of the activity among 1, 3, and 4,
inducing methylpropionyl group into the hemiacetal moi-
ety was disclosed to enhance the trypanocial activity but
In conclusion, during the exploration for trypanocidal
principles from Meliae Cortex, we disclosed toosendanin
(1) and its relatives as promising principles which exhib-
ited the potent trypanocidal activity with weak cytotoxicity.
Moreover, by structure–activity relationship analysis among
the ꢀve isolated congeners along with ꢀve analogs derived
from 1, we clariꢀed the necessary sub-structure and the
some other important functional groups for its trypanocidal
potency.
2
-methylbutyryl group wasn’t. Thus, appropriate size acyl
group on the hydroxy moiety at C29 might be suitable for
trypanocidal activity. Judging from the outcome of 5 and
6
, the acetoxy group at C2 was presumed not to increase
the potency, at least (Table 1). Meanwhile, among these
1
3

Journal of Natural Medicines
Scheme 1 Preparation for toosendanin derivatives. Reagents and conditions: a aq. NaOH, 1,4-dioxane, 40 °C, 33%; b Ac O, pyridine, 0 °C, 38%
2
for 8, 25% for 9; c Dess-Martin periodinane, CH Cl , 46%; d 80% aq. AcOH, 50 ℃, 99%
2
2
1
13
Table 2 Trypanocidal activity and cytotoxicity of toosendanin deriva-
for H- and C-NMR. The observed chemical shift val-
ues (δ) were given in ppm and coupling constant (J) was
represented in Hz. Splitting patterns were designated as
s (singlet), d (doublet), t (triplet), q (quartet), m (multi-
plet) and br (broadened). IR spectra were recorded on a
JASCO FT/IR-5300 infrared spectrometer. HR FAB-MS
data were acquired on a JEOL JMS SX-102 mass spec-
trometer using m-nitrobenzyl alcohol as a matrix. For
column chromatography, silica gel (BW-200, Fuji Silysia
Chemical), ODS (Cosmosil 75C OPN, Nacalai tesque)
tives
Compound Trypanocidal activity Cytotoxicity Selective index
(
IC /μM)
(IC /μM)
5
0
50
1
7
8
9
1
1
0.004
14
0.014
1.4
1.6
0.72
0.79
>100
0.21
3.4
>100
17
198
>7.1
15
2.4
>63
24
0
1
1
8
were used. The TLC analyses were performed over normal
phase pre-coated plates (Kiesel gel 60F , MERCK) and
2
54
reversed phase high-performance thin layer chromatogra-
phy (HP-TLC) plates (RP-18 WF
, MERCK). Spots on
254S
Materials and methods
TLC were detected under UV light (254 nm) and stained
with p-anisaldehyde/H SO or phosphomolybdic acid fol-
General procedure
2
4
lowed by heating.
1
13
H- and C-NMR spectra were recorded on a JEOL JNM
1
13
Trypanocidal assay
LA-500 ( H: 500 MHz, C: 125 MHz) and/or a Varian
1
inova600 ( H: 600 MHz) spectrometer. Tetramethylsilane
Trypanosoma brucei brucei of blood stream form was axeni-
(
δ 0.00 ppm, δ 0.0 ppm) was used as an internal standard
H C
cally cultivated in Iscove’s modiꢀed Dulbecco’s medium
1
3

Journal of Natural Medicines
(
Gibco, Thermo Fisher Scientiꢀc K.K.) supplemented with
in MeOH for 6 h (×4 times). Removal of MeOH from the
whole of gathered maceration and extractions gave MeOH
extract (60 g). First, the MeOH extract (60 g) was eluted
over synthetic adsorbents DIAION HP-20 resin (Mitsubishi
Chemical Corporation, 1 kg) column using 100% MeOH
l-glutamine (4 mM), HEPES (25 mM), 15% heat-inactivated
fetal bovine serum, bathocuproine disulfonic acid diso-
dium salt (0.05 mM), l-cysteine (1.5 mM), hypoxanthine
(
1.0 mM), thymidine (0.16 mM), 2-mercaptoethanol, and
sodium pyruvate (1.0 mM). Trypanosomes at late exponen-
after washed by H O to aꢂord the MeOH elute (40 g). This
2
4
tial growth level were harvested and re-supended as 1×10
MeOH elute (40 g) was fractionated by silica gel column
cells/mL by appropriate dilution in culture medium. Aliquots
of 90 μL of T. b. brucei suspension were transferred to well
of 96-well microculture plate. Then, 10 μL of test sample
solution in 10% DMSO at the corresponding concentration
was added to each well (ꢀnal concentration of DMSO was
chromatography (SiO : 1 kg) using CHCl -MeOH-H O
2
3
2
(10: 3: 1, lower layer) and MeOH as elution to aꢂord ꢀve
fractions: A (12.3 g), B (0.7 g), C (0.7 g), D (2.3 g), and E
(20 g). Subsequently, ODS column chromatography (ODS:
50 g) was applied to Fr. A (1.3 g), then we obtained seven
fractions; Fr. A-1 (220 mg) and Fr. A-2 (110 mg) as 20%
1
%). After incubation under 5% CO atmosphere at 37 ℃ for
2
4
8 h, the viability of parasites was determined by counting
aqueous CH CN elution (300 mL), Fr. A-3 (130 mg) and
3
survival number on hemocytometer under microscope. Dur-
ing isolation scheme, the trypanocidal activity of each frac-
tion was evaluated and translated into mathematic symbols
Fr. A-4 (35 mg) as 40% aqueous CH CN elution (150 mL
3
each), Fr. A-5 (240 mg) and Fr. A-6 (280 mg) as 60% aque-
ous CH CN elution (150 mL each), and Fr. A-7 (300 mg) as
3
[
(−):<80%, (±):>80%, and (+): 100% growth inhibition
80% aqueous (300 mL) and 100% CH CN elution (300 mL).
3
level, respectively]. The commercial drug, pentamidine, was
used as positive control, which showed normally (+) at 5 μg/
mL and (±) at 1~0.3 μg/mL inhibition.
Because Fr. A-2~A-6 showed potent trypanocidal activity,
each fraction was subjected to ꢀnal ODS HPLC puriꢀcation.
Fraction A-2 (110 mg) was subjected to HPLC [column:
Cosmosil 5C AR-II 20 mm id×250 mm (Nacalai tesque),
1
8
Cytotoxicity assay
mobile phase: 40% CH CN, ꢁow rate: 9.9 mL/min, detec-
3
tion: UV (λ = 210 nm)] leading to isolation of Fr. A-2–1
(30.1 mg, Rt: 20.6 min) and A-2–2 (30.7 mg, Rt: 25.2 min).
Both of them were clariꢀed to be toosendanin (1) through
the analyses of physical and spectroscopic data [9, 36].
Standardized MTT assay procedure was applied. HeLa cell,
a human cervix epithelial carcinoma cell-line, was seeded in
a 96-well microculture plate as 100 μL of suspension in the
5
culture medium at 1×10 cells/mL density. Then, 100 μL of
Toosendanin (1): a colorless amorphous powder. [α] +3.2°
D
−1
1
test sample solution at the corresponding concentration in
(c 0.38, CHCl ). IR (KBr)ν: 3503, 1740, 1722 cm . H- and
3
1
3
2
% DMSO was added into respective wells. After incubation
C-NMR data were shown in Table S1. HR-FAB-MS m/z
under 5% CO atmosphere 37 ℃ for 72 h, 25 μL of MTT rea-
[M+H]: 575.2488 (Calcd for C H O : 575.2494).
2
30 39 11
gent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] was added to each well. Further 3 h incubation
at 37 ℃ allowed the accumulation of MTT-formazan, then
supernatants were removed, followed by addition of 200 μL
of DMSO. After shaking for 5 min to dissolve the formazan
crystals, each well of absorbance (ABS) at 540 nm was
measured. The cytotoxicity was calculated by the formula:
Fraction A-3 (130 mg) was subjected to HPLC [col-
umn: Cosmosil 5C AR-II 20 mm id × 250 mm, mobile
1
8
phase: 40% CH CN, ꢁow rate: 9.9 mL/min, detection: UV
3
(λ=210 nm)] leading to isolation of Fr. A-3–1 (0.4 mg, Rt:
29.6 min), which was clariꢀed to be deepoxy-29-O-methyl-
15-oxotoosendanin (2) through the analyses of physical and
spectroscopic data [35].
[1-(ABS of sample treated/ABS of control)]×100 (%). Each
Fraction A-4 (35 mg) was subjected to HPLC [col-
umn: Cosmosil 5C AR-II 10 mm id × 250 mm, mobile
condition was performed in triplicate. The commercial drug,
mitomycin C, was used as positive control, which showed
normally 66.6± 5.0% (average± SD) inhibition at 1 μg/mL
concentration and 31.7 ± 5.0% (average ± SD) at 0.05 μg/
mL.
1
8
phase: 45% CH CN, ꢁow rate: 4.0 mL/min, detection: UV
3
(λ=210 nm)] leading to isolation of Fr. A-4–1 (2.2 mg, Rt:
25.4 min) and A-4–2 (3.2 mg, Rt: 27.3 min), which were
clariꢀed to be 12-O-acetylazedarachin B (3) and 12-O-acet-
ylazedarachin A (4) through the analyses of physical and
spectroscopic data, respectively [10, 36].
Isolation of trypanocidal compounds from Meliae
Cortex
Fraction A-5 (240 mg) was subjected to HPLC [col-
umn: Cosmosil 5C AR-II 20 mm id × 250 mm, mobile
1
8
Meliae Cortex (Kurem-pi) was purchased from Maechu
Co, which was harvested in Guangxi on 2005 winter as lot
phase: 55% CH CN, ꢁow rate: 9.9 mL/min, detection: UV
3
(λ=210 nm)] leading to isolation of Fr. A-5–1 (23.0 mg, Rt:
16.1 min), which was clariꢀed to be 1,12-diacetyltrichilin B
(5) through the analyses of physical and spectroscopic data
[36, 37].
#
400-2533. The dried and ground Meliae Cortex (1 kg) was
macerated in MeOH during 24 h and ꢀltered through a ꢀlter
paper. The resultant residue was then extracted under reꢁux
1
3

Journal of Natural Medicines
Fraction A-6 (280 mg) was subjected to HPLC [col-
6.51 (1H, s), 4.80 (0.5H, s), 4.79 (0.5H, s), 4.49 (1H, s),
4.23 (0.5H, d, J = 12.8 Hz), 4.18 (0.5H, br), 4.12 (0.5H,
d, J=12.2 Hz), 4.01 (0.5H, d, J=12.2 Hz), 3.90 (0.5H, d,
J=12.8 Hz), 3.83 (0.5H, br), 3.72 (1H, s), 3.54 (2H, m) 3.53
(1H, m), 2.84 (1H, dd, J=6.1, 12.2 Hz), 2.74 (1H, m), 2.64
(1H, m), 2.19 (1H m), 1.89 (1H, m), 1.77 (2H, m), 1.60 (1H,
m), 1.07 (3H, s), 1.01 (3H, s), 0.85 (3H, s). HR-FAB MS
umn: Cosmosil 5C AR-II 20 mm id × 250 mm, mobile
1
8
phase: 55% CH CN, ꢁow rate: 9.9 mL/min, detection: UV
3
(
λ=210 nm)] leading to isolation of Fr. A-6–1 (3.1 mg, Rt:
2
0.2 min), which was clariꢀed to be trichilin D (6) through
the analyses of physical and spectroscopic data [36].
+
Determination of absolute conꢀguration
of 2‑methylbutyryl esters
m/z [M+Li] : 495.2202 (Calcd for C H O Li: 495.2206).
2
6
32
9
Preparation for (29S)‑29‑O‑acetyltoosendanin (8)
and (29S)‑7,29‑O‑diacetyltoosendanin (9)
To a solution of 4 (1.5 mg) in MeOH (45 μL) was added
1
0% aqueous NaOH (5 μL). After stirring for 30 min at
room temperature, the reaction was quenched by addition
of a drop of concetrated HCl and the whole was mixed
well. Next, Na SO (100 mg) and CH Cl (200 μL) were
To a solution of toosendanin (1, 5.0 mg, 8.7 μmol) in pyri-
dine (0.5 mL) was added Ac O (0.4 mL) at 0 ℃. After stir-
2
2
4
2
2
ring for 4 h at 0 ℃, the reaction was quenched by addition of
added and the mixture was subjected to vortex mixer for
ice-cold H O. Then, the mixture was extracted with CH Cl
2
2
2
1
5 s. After centrifuge (6,200 rpm) for 15 s, the superna-
(
× 3) and the combined organic layers were washed with
tant was transferred to a new tube. The resulted residue was
5
% HCl, saturated aqueous NaHCO , and brine, dried over
3
extracted again with CH Cl (200 μL). The supernatant
2
2
MgSO , ꢀltered and concentrated under reduced pressure.
4
and the extract were gathered, then CH Cl was removed
2
2
The resultant residue was puriꢀed by silica gel column chro-
under Ar stream. The obtained residue (0.15 mg) contain-
ing 2-methylbutyric acid was dissolved with n-hexane, then
the solution was analyzed by HPLC [column: CHIRALCEL
OJ-H 4.6 mm id × 250 mm (Daicel Corporation), mobile
phase: n-hexane:2-propanol=99: 1, ꢁow rate: 1.0 mL/min,
detection: UV (λ=210 nm)]. In the same manner, 5 (1.5 mg)
and 6 (1.5 mg) also gave the ꢀnal residue (0.13 mg and
matography (CHCl : MeOH: H O=30: 3: 1<lower layer>)
3
2
and subsequently HPLC [column: Cosmosil 5C AR-II
1
8
1
0 mm id×250 mm, mobile phase: 40% aqueous CH CN,
3
ꢁ
8
ow rate: 3.5 mL/min, detection: UV (λ=210 nm)] to give
(2.0 mg, 38%) and 9 (1.4 mg, 25%). The chemical struc-
tures of (29S)-29-O-acetyltoosendanin (8) and (29S)-7,29-O-
diacetyltoosendanin (9) were determined by the comparison
of spectroscopic data with reported ones [9, 38].
0
.15 mg), respectively. By the comparison of retention time
with those of authentic (S)- and (R)-2-methylbutyric acid,
absolute conꢀguration of 2-methylbutyric ester portion in 4,
5
, and 6 were all clariꢀed to be S.
Preparation for lactone derivative (10)
Preparation for bisdeacetyltoosendanin (7)
To a solution of toosendanin (1, 6.0 mg, 10 μmol) in CH Cl
2
2
(
1.3 mL) was added Dess–Martin periodinane (45 mg,
To a solution of toosendanin (1, 6.0 mg, 10 μmol) in
0.11 mmol) at room temperature. After stirring for 42 h at
room temperature, the reaction was quenched by addition of
saturated aqueous Na S O and saturated aqueous NaHCO .
1
,4-dioxane (0.8 mL) was added 1.0 M aqueous NaOH
(
0.6 mL) at room temperature. After stirring for 30 min at
2
2
3
3
4
0 °C, 1,4-dioxane was removed under the reduced pressure.
Then, the mixture was extracted with EtOAc (×3) and the
Thereafter, H O (0.5 mL) and cation exchange resin Dowex
combined organic layers were dried over MgSO , ꢀltered
2
4
HCR-W2-H (FUJIFILM Wako Pure Chemical Corporation)
were added to the residue and all are stirred until pH 2. Then,
the mixture was extracted with CH Cl (×3) and the com-
and concentrated under reduced pressure. The resultant resi-
due was puriꢀed by HPLC [column: Cosmosil 5C AR-II
1
8
10 mm id×250 mm, mobile phase: 50% aqueous CH CN,
2
2
3
bined organic layers were washed with 5% HCl, saturated
flow rate: 3.5 mL/min, detection: UV (λ = 210 nm)] to
give 10 (2.8 mg, 46%). Lactone derivative (10): a color-
less amorphous powder. [α] + 15.2° (c 0.11, CHCl ). IR
aqueous NaHCO , and brine, dried over MgSO , ꢀltered and
3
4
concentrated under reduced pressure. The resultant residue
D
3
−
1 1
was puriꢀed by silica gel column chromatography (CHCl :
(KBr) ν: 3488, 1742, 1720, 1711 cm . H-NMR (CDCl ,
3
3
MeOH: H O = 50: 3: 1 < lower layer >) and subsequently
500 MHz)δ: 7.36 (1H, s), 7.16 (1H, s), 6.20 (1H, s), 5.10
(2H, m), 4.96 (1H, d, J=12.2 Hz), 4.68 (1H, s), 4.49 (1H,
d, J = 12.2 Hz), 4.14 (1H, m), 3.75 (1H, s), 3.66 (1H, m),
2.99 (1H, m), 2.90 (1H, m), 2.85 (1H, m), 2.28 (1H, m),
1.86 (1H, m), 1.69 (1H, m), 1.31 (3H, s), 1.19 (3H, s), 1.18
2
HPLC [column: Cosmosil 5C AR-II 10 mm id×250 mm,
1
8
mobile phase: 40% aqueous CH CN, ꢁow rate: 3.5 mL/min,
3
detection: UV (λ=210 nm)] to give 7 (3.2 mg, 33%). Bis-
deacetyltoosendanin (7): a pale yellow amorphous powder.
−
1
+
[
α] -37.2° (c 0.10, CHCl ). IR (KBr) ν: 3504, 1720 cm .
(3H, s). HR-FAB MS m/z [M + H] : 573.2333 (Calcd for
D
3
1
H-NMR (CDCl , 500 MHz) δ: 7.26 (1H, s), 7.24 (1H, s),
C H O : 573.2336).
3
30 37 11
1
3

Journal of Natural Medicines
1
1
0. Zhou J-B, Minami Y, Yagi F, Tadera K, Nakatani M (1997)
Preparation for 15‑keto derivative (11)
Antifeeding Limonoids from Melia toosendan. Heterocylces
4
5:1781–1786. https://doi.org/10.3987/COM-97-7831
A solution of toosendanin (1, 9.8 mg, 17 μmol) in 80%
aqueous AcOH (0.8 mL) was stirred for 3 h at 50 ℃. After
cooling down, the reaction was quenched by neutraliza-
1. Zhang T, Li J, Yin F, Lin B, Wang Z, Xu J, Wang H, Zuo D,
Wang G, Hua Y, Cai Z (2017) Toosendanin demonstrates prom-
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tion using saturated aqueous NaHCO . Then, the mixture
3
1
1
1
was extracted with CH Cl (×3) and the combined organic
2
2
layers were dried over MgSO , ꢀltered and concentrated
4
1
6
under reduced pressure. The resultant residue was puri-
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growth of neuronal processes and apoptosis in PC12 cells.
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fied by HPLC [column: Cosmosil 5C AR-II 10 mm
1
8
id × 250 mm, mobile phase: 40% aqueous CH CN, ꢁow
3
rate: 4.0 mL/min, detection: UV (λ = 210 nm)] to give 11
4. Zhang B, Wang ZF, Tang MZ, Shi YL (2005) Growth inhibition
and apoptosis-induced eꢂect on human cancer cells of toosenda-
nin, a triterpenoid derivative from Chinese traditional medicine.
Invest New Drugs 23:547–553. https://doi.org/10.1007/s1063
(
9.7 mg, 99%) as tautomeric mixture. The chemical struc-
tures of 15-keto derivative (11) was determined by the
comparison of spectroscopic data with reported ones [10].
7
-005-0909-5
Acknowledgements This work was supported by Research funds from
15. He Y, Wang J, Liu X, Zhang L, Yi G, Li C, He X, Wang P, Jiang
H (2010) Toosendanin inhibits hepatocellular carcinoma cells
by inducing mitochondria-dependent apoptosis. Planta Med
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Conflict of interest The authors declare no conꢁict of interest.
:
//doi.org/10.1016/j.tiv.2012.09.013
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7. Wang G, Feng CC, Chu SJ, Zhang R, Lu YM, Zhu JS, Zhang
J (2015) Toosendanin inhibits growth and induces apoptosis
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