Tetracyclic triterpenoids and terpenylated coumarins from the bark of Ailanthus altissima ( Tree of Heaven )

Article abstract of DOI:10.1016/j.phytochem.2012.10.008

Tetracyclic triterpenoids (named as altissimanins A-E, 1-5) and a terpenylated coumarin (denominated as altissimacoumarin G, 6), along with fifteen known compounds (7-21) were isolated from the bark of Ailanthus altissima. Structures of compounds 1-6 were established by spectroscopic methods and chemical transformations. Altissimanin A (1) is a tirucallane-type triterpenoid bearing an uncommon oxetane ring in the side-chain, while altissimanins D (4) and E (5) are two unprecedented dimers each consisting of one tirucallane-type and one dammarane-type triterpenoid moiety. All the isolates were evaluated for their cytotoxic effects against a small panel of human cancer cell lines.

Full text of DOI:10.1016/j.phytochem.2012.10.008

Phytochemistry 86 (2013) 159–167
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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Tetracyclic triterpenoids and terpenylated coumarins from the bark of Ailanthus
altissima (‘‘Tree of Heaven’’)
Zhi-Lai Hong a,b, Juan Xiong a, , Shi-Biao Wu b,1, Jing-Jing Zhu b, Jun-Lin Hong c, Yun Zhao b, Gang Xia b,
⇑
Jin-Feng Hu a,b,
⇑
a Department of Natural Products Chemistry, School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, PR China
b Department of Natural Products for Chemical Genetic Research, Key Laboratory of Brain Functional Genomics, Ministry of Education & Shanghai Key Laboratory of Brain
Functional Genomics, East China Normal University, No. 3663 Zhongshan Road North, Shanghai 200062, PR China
c College of Chemistry and Materials Science, Sichuan Normal University, Chengdu 610068, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2012
Received in revised form 24 September 2012
Available online 12 November 2012
Tetracyclic triterpenoids (named as altissimanins A–E, 1–5) and a terpenylated coumarin (denominated
as altissimacoumarin G, 6), along with fifteen known compounds (7–21) were isolated from the bark of
Ailanthus altissima. Structures of compounds 1–6 were established by spectroscopic methods and chem-
ical transformations. Altissimanin A (1) is a tirucallane-type triterpenoid bearing an uncommon oxetane
ring in the side-chain, while altissimanins D (4) and E (5) are two unprecedented dimers each consisting
of one tirucallane-type and one dammarane-type triterpenoid moiety. All the isolates were evaluated for
their cytotoxic effects against a small panel of human cancer cell lines.
Keywords:
Ailanthus altissima
Simaroubaceae
Triterpenoids
Altissimanins
Atissimacoumarins
Cytotoxicities
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
(21) were also encountered. We herein report the isolation and
structure elucidation of the new compounds and their
Ailanthus altissima (Miller) Swingle (family Simaroubaceae),
commonly known as the ‘Tree of Heaven’, is a deciduous tree indig-
enous to China. The bark of this tree, called ‘chu-bai-pi’ in Manda-
rin, has been used for centuries as an astringent traditional Chinese
medicine for treatment of diarrhea and bleeding (Jiangsu New
Medical College, 1986; De Feo et al., 2003). This plant has been
well-documented to be rich in quassinoids with various biological
activities (Kundu and Laskar, 2010); however, the occurrence of
tetracyclic triterpenes in ‘chu-bai-pi’ was seldom reported (Zhou
et al., 2011; Wang et al., 2010). By focusing one of our research ef-
forts on new cytotoxic tetracyclic triterpenoids from higher plants,
Melia toosendan (Wu et al., 2010) and Melia azedarach (Wu et al.,
2011) were recently studied. As a continuation of this approach,
the tetracyclic triterpenoids from ‘chu-bai-pi’ were therefore rein-
vestigated. In this study, seven tirucallane-type (1, 2, 7ꢀ11) and se-
ven dammarane-type (3, 12ꢀ17) triterpenoids together with two
triterpene dimers (4, 5) were identified. In addition, three terpeny-
lated coumarins (6, 18, 19), one quassinoid (20), and one steroid
cytotoxicities.
2. Results and discussion
2.1. Structure elucidation
Our conventional natural product research procedures (Wu et al.,
2011, 2010) applied to the methanol extract of ‘chu-bai-pi’ led to the
isolation of six (1–6) new and 15 (7–21) known compounds (Fig. 1).
Comparing their spectroscopic data and physicochemical properties
with those reported in the literature, the known compounds were
identified to be (24S)-24,25-dihydroxytirucall-7-en-3-one (7)
(Mulholland et al., 1998), niloticin (8) (Su et al., 1990), piscidinol A
(9) (McChesney et al., 1997), bourjotinolone B (10) (Wang et al.,
2008), (23E)-3b,25-dihydroxytirucalla-7,23-diene (11) (Takahashi
et al., 2007; Luo et al., 2000), isofouquierone (12) (Waterman
and Ampofo, 1985), (20S)-hydroxy-25-methoxy-dammar-23-
en- 3-one (13) (Xiong et al., 2011), 24,25-dihydroxydammar-20-
en-3-one (14) (Boar and Damps, 1977), cabralealactone (15)
(Phongmaykin et al., 2008), betulafolienediolone (16) (Asakawa
et al., 1977), (20S,24R)-12b,25-dihydroxy-20,24-epoxydammaran-
3-one (17) (Valverde et al., 1985), altissimacoumarin B (18)
(Hwang et al., 2005), altissimacoumarin D (19) (Dao et al., 2012),
⇑
Corresponding authors. Address: Department of Natural Products Chemistry,
School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203,
PR China. Tel./fax: +86 21 51980172 (J.-F. Hu).
E-mail addresses: jfhu@fudan.edu.cn, jfhu@brain.ecnu.edu.cn (J.-F. Hu).
Current address: City University of New York, Lehman College, NY 10468, USA.
1
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.10.008

160
Z.-L. Hong et al. / Phytochemistry 86 (2013) 159–167
27'
24'
OH
23'
20'
26'
R1
12
18
18
R
R2
R2
21'
H
H
O
H
H
H
11
11
19
19
5
13
19
17'
17
13
14
18
13 17
17
11'
H
16
9
16
16
14
7
9
O
19'
9
18'
13'
16'
1
1
1
9'
15
15
15
H
H
H
3
5
3 5
3
3'
1'
4'
Sh:
30
30
15'
30
H
7
O
7
R1
O
30'
H
28'
H
7'
H
H
R
29 28
29 28
29 28
29'
unit B
unit A
R1
H
R2
23
R1
R2
OH
24
R
24
OH
21
26
21
20
22
27
26
23
OSh
OSh
25
24
20
3
1
2
O
O
21
22
25
27
23
O
4a
5a
27
4
OH
20
OH
OH
OH
OH
OAc
OAc
OH
OH
H
H
12
26
OH
OH
OH
OH
5
OH
13
OCH3
OH
OH
OSh
OSh
7
8
OH
O
O
14
15
R
H
H
O
10'
6'
HO
9'
1'
O
4'
7'
OH
OH
O
3'
6
8'
5
4
4a
8a
2'
MeO
RO
5'
O
3
2
6
OH
9
O
O
OH
16
17
OH
OH
7
O
O
18
19
8
OH
OH
OMe
OH
O
10
11
OH
O
OH
OH
H
Fig. 1. Chemical structures of compounds 1–19.
shinjudilactone (20) (Kubota et al., 1996), and 7b-hydroxy-sitos-
terol (21) (Chaurasia and Wichtl, 1987), respectively.
was established by COSY correlations between OH-24 [d 1.88
(1H, br s, D2O-exchangeable)] and H-24 (d 4.35), between H-24
and H-23 (d 4.77), between H-23 and H2-22 (d 1.36/1.98), between
H2-22 and H-20 (d 1.45), and between H-20 and Me-21 (d 0.92)
(Fig. 2). The ketone at C-3, the double bond at C-7, the secondary
hydroxy group at C-24, and the ether linkage at C-23/C-25 were
all further supported by HSQC and HMBC NMR experiments.
The relative stereochemistry of the chiral centers in the C-17
side-chain of 1 was determined by a NOESY NMR experiment.
Clear NOE correlations (Fig. 2) were observed between Me-21
and H-23, between H-23 and H-24, between H-23 and Me-27,
and between H-24 and Me-27, thereby establishing that Me-21,
H-23, H-24 and Me-27 were all b-oriented. Furthermore, the NOE
between Me-18 and Me-21 was consistent with those of reported
tirucallane-type triterpenes (Liu and Abreu, 2006 Wang et al.,
2003). The absolute configuration at C-24 in 1 was established by
application of the modified Mosher’s method (Li et al., 2011; Ukiya
The molecular weight of compound 1 and its chemical formula,
C
30H48O3, was determined from HR-ESIMS, which gave an intense
quasi-molecular ion peak at m/z 479.3497 [M+Na]+ (calcd.
479.3496). The 1H NMR spectrum (Table 1) of 1 displayed signals
attributed to seven tertiary methyl groups [d 0.79 (3H, s, Me-18),
1.00 (3H, s, Me-19), 1.32 (3H, s, Me-26), 1.42 (3H, s, Me-27), 1.04
(3H, s, Me-28), 1.11 (3H, s, Me-29), 1.01 (3H, s, Me-30)], one sec-
ondary methyl group [d 0.92 (3H, d, J = 6.3 Hz, Me-21)], two oxy-
methines [d 4.35 (1H, d, J = 6.3 Hz, H-24) and 4.77 (1H, brq,
J = 6.3 Hz, H-23)], and an olefinic proton [d 5.30 (1H, brd,
J = 2.8 Hz, H-7)]. The 13C NMR spectrum of 1 exhibited thirty car-
bon resonances, which were classified by DEPT and HSQC NMR
experiments as eight sp3 methyls, eight sp3 methylenes, six sp3
methines (two oxygenated at d 73.9 and 79.5), five sp3 quaternary
(one oxygenated at d 86.1), a ketonic carbonyl (d 217.0) and two
olefinic carbons [d 117.8 (CH) and 145.9 (C)]. The above NMR spec-
troscopic data of 1 closely resembled those of niloticin (8) (Fig. 1), a
known tirucall-7-en-3-one triterpenoid derivative previously iso-
lated from the fruits of Phellodendron chinense (Su et al., 1990).
Compounds 1 and 8 were found to have the same molecular for-
mula, but the proton chemical shifts assigned to H-23 and H-24
and the carbon chemical shifts of C-22 to C-27 (Table 1) in 1 were
different from those of 8 (Su et al., 1990), implying both have a dif-
ferent internal ether linkage in the C-17 side-chain (Fig. 1). Unlike
8, which has an epoxy group at C-24/C-25, compound 1 has an
ether linkage between C-23 and C-25. This oxetane linkage in 1
et al., 2003b). Briefly, treatment of 1 with (S)- or (R)-a-methoxy-a-
trifluoromethyl-phenyl acetic acid (MTPA) in the presence of 1-
ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride
(EDCꢁHCl) and 4-dimethylaminopyridine (4-DMAP) gave rise to
its 24-O-(R)-MTPA ester (1R) and 24-O-(S)-MTPA ester (1S), respec-
tively. As shown in Fig. 3, the
d = 0.08) and Me-27 ( d = 0.01) were found to be positive,
whereas those for the H-23 ( d = ꢀ0.03) and
d = ꢀ0.02), Me-21 (
Me-18 (
Dd (dS–dR) values for Me-26
(D
D
D
D
D
d = ꢀ0.07) were negative. These data unequivocally dem-
onstrated that 1 possesses an S configuration at C-24, and the con-
figuration at C-23 was therefore accordingly deduced to be R.

Z.-L. Hong et al. / Phytochemistry 86 (2013) 159–167
161
Table 1
1H (500 MHz) and 13C (125 MHz) NMR spectroscopic data of compounds 1–3 (in CDCl3).
1
2
3
No.
1
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
1.45, m
1.99, m
2.23, m
2.74, dt (14.5, 5.5)
38.5
1.47, m
2.00, m
2.23, m
2.75, dt (14.5, 5.0)
38.5
1.50, m
1.99, m
2.44, m
2.48, m
39.9
2
34.9
34.9
34.1
3
4
5
6
7
217.0
47.9
52.3
24.3
117.8
217.0
47.8
52.3
24.3
117.8
218.1
47.4
55.4
19.6
34.5
1.71, dd (9.2, 8.3)
2.08, m
5.30, brd (2.8)
1.72, dd (9.2, 8.2)
2.09, m
5.31, brd (2.8)
1.35, m
1.55, m
1.31, m
1.58, m
8
9
10
11
145.9
48.4
35.0
18.3
145.9
48.4
35.0
18.3
40.3
50.2
36.8
22.0
2.26, m
1.55, m
2.29, m
1.58, m
1.41, m
1.30, m
1.54, m
1.31, m
1.92, m
1.73, m
12
1.63, m
1.80, m
33.6
1.66, m
1.81, m
33.6
27.5
13
14
15
43.5
51.1
34.0
43.5
51.1
34.0
42.5
50.3
31.1
1.47, m
1.49, m
1.28, m
2.00, m
1.52, m
0.79, s
1.49, m
1.09, m
1.52, m
1.56, m
1.74, m
1.75, m
1.01, s
16
28.3
1.34, m
1.98, m
1.50, m
0.81, s
1.01, s
1.40, m
0.89, d (6.4)
1.00, m
1.62, m
28.1
24.9
17
18
19
20
21
22
53.6
21.9
12.8
34.2
19.2
36.8
53.0
21.9
12.8
36.4
18.5
33.4
50.1
15.2
16.0
75.3
26.1
43.8
1.00, s
0.95, s
1.45, m
0.92, d (6.3)
1.36, m
1.98, m
4.77, q (6.3)
1.15, s
2.29, m
23
79.5
1.21, m
1.51, m
3.29, brd (9.9)
28.8
5.71, dt (15.6, 7.5)
6.20, d (15.6)
125.7
24
25
26
27
28
29
30
24-OH
4.35, d (6.3)
73.9
86.1
23.1
27.9
24.5
21.6
27.4
79.5
73.2
23.2
26.5
24.5
21.6
27.4
136.6
141.9
115.2
18.7
26.7
21.0
1.32, s
1.42, s
1.04, s
1.11, s
1.01, s
1.88, br s
1.16, s
1.22, s
1.05, s
1.12, s
1.01, s
4.91, brs
1.86, s
1.08, s
1.04, s
0.89, s
16.3
OH
O
OH
H
H
H
H
O
H
H
H
O
H
O
NOE
COSY
HMBC
H
Fig. 2. Observed key COSY, HMBC and NOE correlations for compound 1.
OR
OR
+0.04
+0.03
-0.03
OH
-0.05
-0.03
-0.07
+0.01
+0.08
O
-0.02
H
H
+0.01
H
H
H
O
O
H
2R
R = (R)-MTPA
R = (S)-MTPA
1R
1S
R = (R)-MTPA
R = (S)-MTPA
2
S
Fig. 3. Chemical shift differences (Dd) between (S)-MTPA and (R)-MTPA esters of 1 and 2.

162
Z.-L. Hong et al. / Phytochemistry 86 (2013) 159–167
Meanwhile, the S configuration at C-20 was also unambiguously
confirmed (the S configuration at C-20 was previously proposed
based upon biogenetic consideration) (McChesney et al., 1997).
Thus, the structure of 1 was elucidated to be (20S,23R,24S)-24-hy-
droxy-23,25-epoxytirucall-7-en-3-one (altissimanin A). Naturally
occurring triterpenoids with an oxetane ring in the C-17 side-chain
are quite rare. To our knowledge, only cumingianoside O, a tirucal-
lane glucoside from the leaves of Dysoxylum cumingianum, was
previously reported to possess such a four-membered ring unit
(Fujioka et al., 1997).
of 4 and 5, the mixture was treated with acetic anhydride in anhy-
drous pyridine to afford the corresponding monoacetylated deriva-
tives 4a and 5a (Fig. 1). Successful separation of 4a and 5a was then
achieved. The molecular formula of 4a was determined to be
C62H100O8 according to a pseudo-molecular ion peak at m/z
995.7316 [M+Na]+ in its HR-ESIMS, implying thirteen degrees of
unsaturation. In the highfield of the 1H NMR spectrum (Table 2)
of 4a, 13 tertiary methyls (d 0.80, 0.86, 0.92, 0.98, 0.99, 1.01, 1.05,
1.11, 1.13, 1.14, 1.16, 1.22, 1.23), a methyl doublet at d 0.95, a
vinylic methyl at d 1.74 (brs), and an acetyl methyl (d 2.07) were
presented. In addition to two characteristic signals for the acetoxy
group [d 170.4, 21.5], the 13C NMR spectrum of 4a displayed sixty
resonances, including a ketonic carbonyl (d 216.8), an ester car-
bonyl (d 173.8), and two pairs of olefinic [d 118.0 (CH), 145.7
(qC)]; [113.4 (CH2), 147.6 (qC)] carbons in the low field. Detailed
analysis of the 1D NMR data of 4a with the aid of 2D NMR tech-
niques (COSY, HSQC and HMBC) (Fig. 4) indicated that 4a was a di-
meric triterpene composed of one tirucallane-type (unit A) and one
dammarane-type (unit B) moiety. The NMR spectroscopic data of
unit B resembled those of shoreic acid, a known 20,24-epoxy-25-
hydroxy-3,4-seco-dammar-4(28)-en-3-oic acid previously reported
by one of the authors (Xiong et al., 2011). The distinct difference be-
tween unit B and shoreic acid was the chemical shift of the C-30 car-
bonyl carbon (4a: d 173.8, shoreic acid: d 179.5) (Roux et al., 1998),
indicating that the carboxylic acid group in unit B was esterified.
Meanwhile, the NMR data of unit A resembled those of com-
pound 1 (Tables 1 and 2). Careful comparison of the NMR spectro-
scopic data of unit A in 4a with those of compound 1 and another
related known tirucall-7-en-3-one analogue (piscidinol A, 9)
(McChesney et al., 1997) established that unit A was a derivative
of 9 with 23,24-dihydroxy groups both esterified, which could be
confirmed by 2D (COSY, HSQC, HMBC) NMR experiments. Correla-
tions between H-24 at d 4.88 and H-23 at d 5.42, between H-23
and H2-22 (d 1.17/1.64), between H2-22 and H-20 (d 1.42), as well
as between H-20 and Me-21 (d 0.95) were observed in the COSY
spectrum of 4a. A diagnostic 3J correlation was then found between
H-24 (d 4.88) and C-30 (d 173.8) in the HMBC spectrum of 4a (Fig. 4).
The above data unequivocally indicated that unit A was conjugated
with unit B by an ester bridge at the acyl residue as depicted in Fig. 1
and Fig. 4. Thus, the structure of 4a was established as a dimer of
23-acetyl piscidinol A (unit A) and shoreic acid (unit B), which
was also supported by ESI-MS-MS spectrum performed on the
[M+Na]+ adduct (m/z 995.7; see Supporting information). The pre-
dominant fragmentations occurred by cleavage of the acetyl group
at C-23 (m/z 935.7 [MꢀCH3COOH+Na]+) and the cleavage of ester
bond at C-24 (m/z 497.3 [Mꢀunit A+Na]+). The configuration of 4a
was determined by comparing the NMR data with those of shoreic
acid and piscidinol A (9). In fact, the configurations of these two nat-
urally occurring compounds have been previously ascertained by
single crystal X-ray diffraction analysis (Roux et al., 1998; McChes-
ney et al., 1997). For 4a, the vicinal coupling constant between H-23
and H-24 was close to zero, indicating a syn configuration between
H-23 and H-24 (Toume et al., 2011). Finally, altissimanin D (4) was
deduced to be a dimer of (23R,24S)-23,24,25-trihydroxy-tirucall-7-
en-3-one (piscidinol A) and (20S,24R)-3,4- seco-25-hydroxy-20,24-
epoxydammar-3-oic acid (shoreic acid) linked by an ester bond be-
tween C-24 and C-30 (Fig. 1).
The HR-ESIMS of compound 2 exhibited a pseudo-molecular ion
peak at m/z 481.3662 [M+Na]+ corresponding to its molecular
formula C30H50O3. The NMR spectra (Table 1) of 2 showed general
features similar to those of 1, implying that 2 was also a tirucall-7-
en-3-one derivative. Moreover, the NMR spectroscopic data of 2
were found to be almost identical with those of 24(S)-24,25-
dihydroxytirucall-7-en-3-one (7) (both 2 and 7 have the same
molecular formula), a known compound previously obtained from
the plant Owenia cepiodora (Mulholland et al., 1998). The distinct
differences between compounds 2 and 7 were the chemical shifts
of H-24 (2: d 3.29; 7: d 3.32) and C-24 (2: d 79.5, 7: d 78.6)
(Mulholland et al., 1998), suggesting that 2 might be a C-24 epimer
of 7. The absolute configuration at C-24 of 7 has been previously
ascertained to be S by using a lanthanide-shifted [e.g. adding the
Eu(dpm)3 complex] NMR procedure (Mulholland et al., 1998). An
attempt to determine the absolute configuration of the 24,25-diol
moiety in compound 2 using induced circular dichroism spectra
by Snatzke’s method (Di Bari et al., 2001) failed. Similar to 1, the
absolute configuration at C-24 in 2 was finally established to be
R by application of the modified Mosher’s method (Fig. 3). There-
fore, 2 was deduced to be (24R)-24,25-dihydroxytirucall-7-en-3-
one (altissimanin B). It is worth pointing out that the most remark-
able differences between 2 and 7 are the chemical shifts of H-24
and C-24, which are in full agreement with those of 24R and 24S
epimers of 24,25-dihydroxyhelianol (rearranged 3,4-seco-tirucal-
lane-type triterpenoids) (Ukiya et al., 2003a, 2003b).
Compound 3 was assigned a molecular formula of C30H48O2 as
determined by HR-ESIMS data. The 1H, 13C and DEPT NMR spectra
(Table 1) of 3 displayed signals for seven methyl singlets (one
vinylic methyl at dH 1.86; dc 18.7), a terminal double bond [dH
4.91 (2H, brs); dc 115.2 (CH2) and 141.9 (qC)], a disubstituted dou-
ble bond [dH 5.71 (dd, J = 15.6, 7.5 Hz) and 6.20 (brd, J = 15.6 Hz); dc
125.7 (CH) and 136.6 (CH)], and a ketonic carbonyl group [dc 218.1
(qC)]. Comparison of its NMR spectroscopic data with those of the
known dammarane-type triterpene isofouquierone (12) (Water-
man and Ampofo, 1985) suggested that both 3 and 12 had the
same dammar-3-one skeleton (Fig. 1). The obvious difference
was that the tertiary hydroxy group at C-25 in 12 was replaced
by a D25,26 double bond in the C-17 side-chain of 3. In the COSY
NMR spectrum of 3, a spin system of CH2CH@CHC(CH3)@CH2
was found between H2-22 (d 2.29) and H-23 (d 5.71), between H-
23 and H-24 (d 6.20), allylic couplings between H-24 and H-26 (d
4.91), as well as between H-26 and Me-27 (d 1.86). These data
clearly indicated that the conjugated double bonds were located
at C-23 and C-25 in 3, which was supported by the UV absorption
band at 228 nm (loge 4.05). The geometry of D
23 was assigned as E
on the basis of the large coupling constant (15.6 Hz) between H-23
and H-24. The S configuration at C-20 was determined based on the
carbon chemical shift (d 26.1) of Me-21 (the Me-21 carbon chem-
ical shift is generally around d 25.5 in 20S-isomers and about d 23.6
in 20R-isomers) (Yamashita et al., 1998). Consequently, compound
3 was elucidated to be (20S,23E)-20-hydroxydammar-23,25-dien-
3-one (altissimanin C).
Compounds 4 and 5 were obtained as a mixture in a ratio of
about 3:1 (4/5) by analysis of the 1H NMR spectrum (see Supporting
Information). Due to the difficulties encountered in the separation
Compound 5a has the same molecular formula (C62H100O8) as
4a. Comparison of the 1H and 13C NMR spectroscopic data of 5a
with those of 4a (Table 2) showed that the structures of these
two compounds were quite similar. The most obvious difference
was the proton chemical shift of the acetyl methyl group (4a: d
2.07; 5a: d 2.19), indicating that the positions of the acetyl group
and unit B (shoreic acid) were probably interchanged in these
two dimers. This was further confirmed by the HMBC NMR exam-
ination (Fig. 4). Differing from 4a, H-24 (d 4.88, brs) was found to

Z.-L. Hong et al. / Phytochemistry 86 (2013) 159–167
163
Table 2
1H (500 MHz) and 13C (125 MHz) NMR spectroscopic data of compounds 4a and 5a (in CDCl3).
No.
4a
5a
No.
4a
5a
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
1.63, m
dC
dH (J in Hz)
1.59, m
dC
1
2
1.45, m
1.98, m
2.26, m
2.74, dt (14.5, 5.3)
38.4
1.46, m
1.98, m
2.23, m
2.75, dt (14.5, 5.5)
38.5
10
20
34.7
34.5
34.9
34.9
2.23, m
2.51, m
28.6
2.10, m
2.35, m
29.1
3
4
5
6
216.8
47.8
216.8
47.9
30
40
50
60
173.8
147.6
50.8
173.6
147.5
50.7d
24.5e
1.72, m
2.09, m
52.2
1.73, m
2.08, m
52.2
2.01, m
1.39, m
1.84, m
1.28, m
1.38, m
1.97, m
1.34, m
1.80, m
1.23, m
1.44
24.3a
24.4d
24.5a
7
5.30, brs
2.25, m
117.9
5.30, brs
2.26, m
118.0
70
33.9b
33.9
8
9
10
11
12
145.7
48.4
35.0
145.7
48.4
35.0
80
90
100
110
120
39.9
41.2
39.1
22.1
27.4c
40.0
41.2
39.1
22.1
27.3
1.52, m
1.49, m
1.54, m
1.66, m
1.74, m
18.3
1.54, m
1.79, m
1.64, m
18.3
1.28, m
1.28, m
1.82, m
1.60, m
1.39, m
1.24, m
1.78, m
1.58, m
33.8b
33.8e
13
14
15
43.6
51.1
33.9b
43.6
51.2
34.0e
130
140
150
43.0
50.4
31.5
43.0
50.4
31.5
1.50, m
1.54, m
1.85, m
1.50, m
1.53, m
1.88, m
1.09, m
1.47, m
1.46, m
1.80, m
1.81, m
0.92, s
1.09, m
1.47, m
1.48, m
1.80, m
1.81, m
0.89, s
16
28.0
27.9
160
25.7
25.7
17
18
19
20
21
22
1.40, m
0.80, s
0.99, s
1.42, m
0.95, d (5.5)
1.17, m
53.6
21.9
12.8
33.1
18.3
37.9
1.43, m
0.80, s
1.00, s
1.45, m
0.95, d (5.3)
1.14, m
53.7
22.0
12.8
33.1
18.5
38.1
170
180
190
200
210
220
49.5
16.5
20.2
86.3
23.6
35.6
49.5
16.4
20.2
86.3
23.5
35.8
0.86, s
0.85, s
1.14, s
1.66, m
1.14, s
1.64, m
1.64, m
5.42, brdd (8.9, 5.0)
1.65, m
5.40, brdd (8.9, 4.8)
23
70.3
70.2
230
1.82, m
26.1
1.80, m
26.1
1.88, m
3.74, dd (7.6,7.3)
1.88, m
3.73, dd (7.6,7.4)
24
25
26
27
28
4.88, brs
76.3
72.5
27.1
26.3
24.5
4.88, brs
76.7
72.5
27.1
26.3
24.6
240
251
260
270
280
83.3
71.4
27.5c
24.3
113.4
83.3
71.4
27.5
24.3
113.5
1.16, s
1.23, s
1.05, s
1.19, s
1.23, s
1.05, s
1.22, s
1.13, s
4.70, brs
4.85, brs
1.74, brs
1.01, s
1.21, s
1.12, s
4.68, brs
4.85, brs
1.74, brs
1.00, s
29
30
OAc
1.11, s
0.98, s
2.07, s
21.6
1.11, s
0.99, s
2.19, s
21.6
27.5
20.9
170.8
290
300
23.1
15.3
23.2
15.3
27.4c
21.5
170.4
a–eAssignments might be interchangeable within the same superscript.
have a 3J HMBC correlation with the acetyl carbonyl (dc 170.8).
Thus, the acetoxy group was located at C-24, whereas unit B was
attached to C-23. Based on the above evidence, altissimanin E (5)
was assigned as a dimer with piscidinol A and shoreic acid linked
by an ester bond between C-23 and C-30 (Fig. 1).
The molecular weight of compound 6 and its chemical formula
of C21H26O7 were deduced from its positive mode HR-ESIMS, which
resulted in an [M+Na]+ ion peak at m/z 413.1561. The IR spectrum of
tertiary hydroxy group at C-30 in 6 were determined by analysis
of its 1D and 2D (COSY, HSQC, and HMBC) NMR data. The COSY
spectrum of 6 showed two proton spin systems for the C-7 side-
chain: –OCH2–CH(O)– (H2-10/H-20) and –CH2CH(O)–CH@C(Me)2
(H2-40, H-50, H-60, and allylic couplings between H-60 and Me-80/
3
Me-100) (Fig. 5). A key J correlation between H-20 (d 3.98) and C-
50 (d 74.4) was observed in the HMBC NMR experiment of 6.
The relative configurations of the chiral centers at C-20, C-30 and
C-50 in the tetrahydrofuran ring were determined by a NOESY NMR
experiment (Fig. 5). Clear NOE correlations were observed between
H2-10 (d 4.42/4.10) and Me-90 (d 1.47), between Me-90 and Hb-40 (d
1.74), between H-20 (d 3.98) and H-50 (d 4.86), between H-50 and
Ha-40 (d 2.20)/Me-100 (d 1.73), and between Ha-40 and Me-100.
These data suggested that Me-90 and Hb-40 were in the same side
of the tetrahydrofuran ring, while H-20, Ha-40, and H-50 adopted
6 showed absorption bands (mmax) at 3430 (hydroxy group), 1730
(ester carbonyl), and 1639, 1565, 1461 (aromatic ring) cmꢀ1. Its
UV spectrum exhibited absorption maxima at 225, 298 and
340 nm. Analysis of the 1H NMR spectrum established the presence
of a pair of doublets at d 6.35 and 7.61 (each 1H, d, J = 9.5 Hz)], an
aromatic proton singlet at d 6.67 (1H, s), and two methoxy signals
[d 3.88 and 4.02 (each 3H, s)] (See Experimental section). These data
indicated that 6 was an isofraxidin-type derivative related to altiss-
imacoumarin B (18), a known terpenylated coumarin previously
isolated from the bark of A. altissima by Park and coworkers (Hwang
et al., 2005). The molecular formula of 6 implied nine degrees of
unsaturation in the molecule. Apart from seven in the coumarin
framework and one olefinic bond [dH 5.19 (1H, brd, J = 8.5 Hz); dC
125.4 (CH) and 136.9 (qC)] in the C-7 oxygenated terpenyl group,
the remaining degree of unsaturation must be a ring system in
the side-chain. The tetrahydrofuran ring at C-20 and C-50 and the
the opposite orientation. Consequently, compound
6
was
characterized as 7-[(2,5)-epoxy-3-hydroxy-3,7-dimethyl-6- octen-
oxy]-6,8-dimethoxycoumarin. Compound 6 was named altissima-
coumarin G, whose trivial name was sequentially derived from
the work of two Korean groups (Hwang et al., 2005; Dao et al.,
2012). In our lab, a naturally occurring coumarin bearing a tetrahy-
drofuran ring in the monoterpenyl side-chain has been previously
obtained from the rhizomes of Notopterygium incisum (Wu et al.,
2008).

164
Z.-L. Hong et al. / Phytochemistry 86 (2013) 159–167
using the CellTiter GloTM luminescent cell viability assay (Wu
et al., 2011). Staurospirine was used as the positive control (IC50s
ranging from 0.14 to 0.58 M). In this study, some of the com-
pounds could not be tested in the cytotoxicity assay due to their
poor solubility in culture medium, and only tirucallane-type trit-
erpenoids niloticin (8) and piscidinol A (9) were found to exhibit
moderate cytotoxicities against the above BGC-823, KE-97, KB,
Huh-7 and jurkat cancer cell lines (for 8: IC50 = 51.8, 46.5, 31.0,
OH
H
l
O
H
O
O
H
H
38.1 and 26.4
25.8 M, resp.). Interestingly, these two compounds previously
showed comparable cytotoxicities against murine leukemia P388
(IC50 = 3.3 and 2.5 M, resp.) and KB (IC50 = 18.2 and 10.5 M,
lM, resp.; for 9: IC50 = 67.5, 70.0, 35.4, 40.3 and
OH
H
OAc
H
l
l
l
4a
H
resp.) cells (Itokawa et al., 1992).
O
O
3. Concluding remarks
O
H
The occurrence of tetracyclic triterpenes was seldom reported
from the genus Ailanthus. Through a comprehensive investigation,
three new tetracyclic triterpenes and two new dimeric triterpenes
were identified from the dried bark of A. altissima. Natural occur-
ring triterpenoids possessing an oxetane ring in the C-17 side-
chain (e.g. 1) are quite rare. Altissimanins D (4) and E (5) are the
first examples of triterpene dimers each composed of one tirucal-
lane-type and one dammarane-type triterpenoid moiety. Mean-
while, the known tirucallane-type triterpenoids (7, 8, 10, 11) and
the dammarane-type triterpenoids (12ꢀ15, 17) were reported
herein from the genus Ailanthus for the first time. The isolated trit-
erpenoids and terpenylated coumarins (e.g. 6, 18, and 19) could
stimulate future phytochemical/genomics studies.
OH
H
OSh
H
O
5a
Fig. 4. Key HMBC (H ? C) and NOE (H
H) correlations of 4a and 5a.
O
O
HO
O
O
O
4. Experimental
O
H
4.1. General experimental procedures
H
O
Optical rotations were determined using a Perkin-Elmer 341
polarimeter. UV absorptions were obtained on a Libra S35 PC UV-
VIS recording spectrophotometer, whereas IR spectra were mea-
sured on a Nicolet NEXUS-670 FT-IR spectrophotometer. NMR
spectra were recorded on a Bruker Avance DRX-500 spectrometer
(500 MHz). Chemical shifts are expressed in d (ppm), and are refer-
enced to the residual solvent signals. Electrospray ionization mass
spectra (ESI-MS) were measured on a Bruker Daltonics micrOTOF-
QII mass spectrometer. Semi-preparative HPLC was performed on a
Beckman system consisting of a Beckman Coulter System Gold 508
autosampler, Gold 126 gradient HPLC pumps with a Beckman Sys-
tem Gold 168 UV detector, a Sedex 80 (SEDERE, France) evapora-
tive light-scattering detector (ELSD), and a YMC-Pack ODS-A
O
O
H
O
HO
O
H
H
H
H
H
O
Fig. 5. Key COSY (—), HMBC (H ? C), and NOE (H
H) correlations of 6.
The molecular formula of compound 19 was determined to be
21H26O5 based on a pseudo-molecular ion peak at m/z 381.1675
C
[M+Na]+ in its positive mode HR-ESIMS. The 1H and 13C NMR data
of 19 resembled those of 18 (See Experimental section). Detailed
analysis of the 1D NMR data of 19 with the aid of 2D NMR tech-
niques (COSY, HSQC, HMBC and NOESY), the structure of 19 was
determined to be 7-geranyloxy-6,8-dimethoxycoumarin. Interest-
ingly, a Korean group has also reported the discovery of this com-
pound with a trivial name of altissimacoumarin D (Dao et al.,
2012). It is worth mentioning that the 1H and 13C NMR data of
the latest reported altissimacoumarin F (Dao et al., 2012) were
found to be the same as our compound 6 (altissimacoumarin G).
The assigned structure for altissimacoumarin F by Dao et al is sus-
pect, even though this structure was reported to be assured by the
Mosher’s method (Dao et al., 2012).
column (250 ꢂ 10 mm, dp 5
was performed using silica gel (200-300 mesh, Ji-Yi-Da Silysia
Chemical Ltd., Qingdao, China), MCI gel CHP20P (75-150 , Mitsu-
lM). Column chromatography (CC)
l
bishi Chemical Industries, Tokyo, Japan), and Sephadex LH-20 (GE
Healthcare Bio-Sciences AB, Uppsala, Sweden). Silica gel-precoated
plates (GF254, 0.25 mm, Kang-Bi-Nuo Silysia Chemical Ltd, Yantai,
China) were used for TLC detection. Spots were visualized using
UV light (254 and/or 365 nm) and by spraying with 5% (v/v)
H2SO4-EtOH followed by heating to 120ꢀC.
4.2. Plant material
2.2. Cytotoxicity
The dried bark of A. altissima was purchased from Shanghai Jiu-
Zhou-Tong Medicine Co. Ltd, and was originally collected in May
2009 from Nanjing, Jiangsu province of China. The plant was iden-
tified by Prof. Bao-Kang Huang (Department of Pharmacognosy,
the Second Military Medical University of China). A voucher spec-
imen (No.100114) was deposited at the Herbarium of the Shanghai
All the isolated compounds (except for 7b-hydroxy-sitosterol,
21) were evaluated for their cytotoxic effects against human cancer
cell lines of BGC-823 and KE-97 (gastric carcinoma), KB (nasophar-
ynx carcinoma), Huh-7 (liver cancer) and jurkat (T lymphoma)

Z.-L. Hong et al. / Phytochemistry 86 (2013) 159–167
165
Key Laboratory of Brain Functional Genomics, East China Normal
University.
4.3.2. Altissimanin A (1)
15
White amorphous powder; [
a
]
D
–123 (c 0.1, MeOH); IR (KBr)
m
max: 3413 (br), 2967, 2927, 1708, 1634, 1454, 1384, 1317, 1150,
1112, 1088, 1050, 949 cm-1; for 1H and 13C NMR spectroscopic
data, see Table 1; (+) ESI-MS m/z 479.3 [M+Na]+, 935.7
[2 M+Na]+; (+) HR-ESI-MS m/z 479.3497 (calcd for C30H48O3Na,
479.3496).
4.3. Extraction and isolation
The dried bark of A. altissima (10.0 kg) was extracted with
MeOH (3 ꢂ 15 L) at room temperature. The solvent was removed
under vacuum to give a crude extract (ca 980 g), which was then
subjected to silica gel CC with a petroleum ether (PE)-EtOAc gradi-
ent (15:1–1:1–0:1, v/v) and EtOAc-MeOH (15:1–1:1–0:1) to yield
twelve fractions (Fr.1–Fr.12). Fr.3 (15.5 g) was applied to a silica
gel column eluting with a CH2Cl2-EtOAc gradient (8:1–0:1) to af-
ford eight subfractions Fr.3A-Fr.3H. Fr.3B (0.8 g) was then sub-
jected to repeated silica gel CC (CH2Cl2–EtOAc, 12:1; PE-EtOAc,
8:1) to afford compound 3 (3.2 mg), which was further purified
by gel permeation chromatography (GPC) on Sephadex LH-20
(MeOH). Fr.3D (1.2 g) was applied repeatedly to a silica gel column
(PE-acetone, 10:1; PE-EtOAc, 3:1), followed by MCI gel (MeOH) to
furnish 15 (33.0 mg). Compound 19 (9.5 mg) was isolated from
Fr.3E (1.5 g) by MCI gel (MeOH) and subsequently purified by pre-
parative TLC (PE-EtOAc, 3:1). Fr.4 (55.8 g) was separated by silica
gel CC using a step gradient of CH2Cl2-EtOAc (5:1–0:1) to give six
subfractions Fr.4A–Fr.4F. Fr.4C (0.7 g) was subjected to silica gel
CC (PE-EtOAc, 3:1), and was then purified by semi-preparative re-
versed phase HPLC (CH3CN-H2O, 90:10; flow rate, 3.0 mL/min;
tR = 23.2 min) to afford 13 (4.0 mg). Compounds 12 (11.6 mg), 16
(13.5 mg) and 21 (3.5 mg) were isolated from Fr. 4D (2.8 g) through
silica gel CC with PE-EtOAc (5:1) and were further purified by
Sephadex LH-20 (MeOH). Fr.5 (32.1 g) was subjected to silica gel
CC with a CH2Cl2-EtOAc gradient (4:1–0:1) to provide Fr.5A–5D.
The purification of Fr.5B (4.2 g) yielded compounds 1 (10.1 mg),
8 (25.2 mg), 10 (17.8 mg), and 11 (12.2 mg) via silica gel CC with
PE-EtOAc (3:1), MCI-gel using a stepwise gradient elution with
MeOH-H2O (from 80% to 100% MeOH), and Sephadex LH-20 in
MeOH. Fr.6 (31.7 g) was subjected to silica gel CC (CH2Cl2-EtOAc,
2:1) to afford Fr.6A-Fr.6D. Repeated silica gel CC (CH2Cl2-acetone
10:1; PE-EtOAc, 3:1) and then Sephadex LH-20 (MeOH) of Fr.6C
(7.5 g) furnished compounds 4 and 5 (mixture, 27.3 mg), 17
(58.0 mg), and 18 (12.7 mg). Fr.6D (9.4 g) was applied to MCI-gel
using MeOH-H2O (80% MeOH) to yield Fr.6 Da- Fr.6Df. Compounds
2 (30.0 mg), 7 (13.3 mg), and 14 (10.1 mg) were isolated from
Fr.6Db (1.7 g) by silica gel CC (PE-EtOAc, 2:1), and each was further
purified by Sephadex LH-20 (MeOH). Compound 9 (7.7 mg) was
obtained by semi-preparative HPLC (MeOH-H2O, 86:14; flow rate,
3.0 mL/min; tR = 27.0 min) from Fr.6De (0.3 g). Fr.7 (6.8 g) was sub-
jected to silica gel (CH2Cl2-MeOH, 20:1) to yield Fr.7A–Fr.7G. Com-
pound 6 (10.5 mg) was purified from Fr.7D (40.5 mg) by HPLC
(MeOH-H2O, 69:31; flow rate, 3.0 mL/min; tR = 28.5 min). Fr.8
(30.9 g) was applied to a silica gel column (CH2Cl2-MeOH, 15:1)
to provide Fr.8A–Fr.8H. Crystallization of Fr.8D (1.2 g) from MeOH
yielded 20 (42.6 mg), which was further purified by Sephadex LH-
20 (CH2Cl2-MeOH, 2:1).
4.3.3. (R)-MTPA (1R) and (S)-MTPA (1S) esters of 1.
1R:
[a]
15 –71 (c 0.3, MeOH); 1H NMR data (CDCl3, 500 MHz) d 0.75
D
(3H, s, CH3-18), 0.88 (3H, d, J = 6.2 Hz, CH3-21), 0.99 (3H, s, CH3-
19), 1.00 (3H, s, CH3-30), 1.05 (3H, s, CH3-28), 1.12 (3H, s, CH3-
29), 1.22 (3H, s, CH3-26), 1.53 (3H, s, CH3-27), 3.53 (3H, s, OCH3),
4.87 (1H, q, J = 5.7 Hz H-23), 5.29 (1H, d, J = 5.7 Hz, H-24), 5.30
(1H, s, H-7), 7.41-7.42 (3H, m), 7.52-7.54 (2H, m); (+) ESI-MS m/z
15
673.3 [M+H]+, 695.2 [M+Na]+. 1S: [
a
]
D
-61 (c 0.3, MeOH); 1H
NMR data (CDCl3, 500 MHz) d 0.68 (3H, s, CH3-18), 0.85 (3H, d,
J = 6.2 Hz, CH3-21), 0.99 (3H, s, CH3-19), 1.00 (3H, s, CH3-30), 1.05
(3H, s, CH3-28), 1.12 (3H, s, CH3-29), 1.22 (3H, s, CH3-26), 1.54
(3H, s, CH3-27), 3.58 (3H, s, OCH3), 4.85 (1H, q, J = 6.3 Hz, H-23),
5.29 (1H, d, J = 6.3 Hz, H-24), 5.31 (1H, s, H-7), 7.41-7.43 (3H, m),
7.52-7.54 (2H, m); (+) ESI-MS m/z 673.3 [M+H]+.
4.3.4. Altissimanin B (2)
22
White amorphous powder; [
a
]
D
–73 (c 0.4, MeOH); IR (KBr)
m
max: 3431 (br), 2970, 2882, 1709, 1635, 1459, 1385, 1316, 1266,
1162, 1077, 1050, 967 cm-1; for 1H and 13C NMR spectroscopic
data, see Table 1; (+) ESI-MS m/z 481.4 [M+Na]+, 939.7
[2 M+Na]+; (+) HR-ESI-MS m/z 481.3662 (calcd for C30H48O2Na,
463.3552).
4.3.5. (R)-MTPA (2R) and (S)-MTPA (2S) Esters of 2.
2R:
[a]
15–61 (c 0.6, MeOH); 1H NMR data (CDCl3, 500 MHz) d 0.76
D
(3H, s, CH3-18), 0.83 (3H, d, J = 6.6 Hz, CH3-21), 0.99 (3H, s, CH3-
19), 1.00 (3H, s, CH3-30), 1.05 (3H, s, CH3-28), 1.12 (3H, s, CH3-
29), 1.17 (3H, s, CH3-26), 1.23 (3H, s, CH3-27) 3.56 (3H, s, OCH3),
4.95 (1H, d, J = 7.9 Hz H-23), 5.30 (1H, s, H-7), 7.40-7.42 (3H, m),
7.62-7.63 (2H, m); (+) ESI-MS m/z 657.3 [M+H-H2O]+, 675.3
[M+H]+, 697.3 [M+Na]+. 2S: [ 15 -53 (c 0.2, MeOH); 1H NMR data
a]
D
(CDCl3, 500 MHz) d 0.79 (3H, s, CH3-18), 0.87 (3H, d, J = 6.5 Hz, CH3-
21), 1.00 (3H, s, CH3-19), 1.00 (3H, s, CH3-30), 1.05 (3H, s, CH3-28),
1.12 (3H, s, CH3-29), 1.14 (3H, s, CH3-26), 1.18 (3H, s, CH3-27), 3.59
(3H, s, OCH3-18), 4.95 (1H, q, J = 7.7 Hz, H-23), 5.31 (1H, d,
J = 2.9 Hz, H-7), 7.37-7.41 (3H, m), 7.60-7.61 (2H, m); (+) ESI-MS
m/z 657.3 [M+H-H2O]+, 675.3 [M+H]+, 697.3 [M+Na]+.
4.3.6. Altissimanin C (3)
White amorphous powder; [
(MeOH) kmax (log ) 228 (4.05); IR (KBr) mmax: 3430 (br), 2926,
a]
15 + 22 (c 0.1, MeOH); UV
D
e
2855, 1703, 1634, 1455, 1384, 1030 cm-1; for 1H and 13C NMR
spectroscopic data, see Table 1; (+) ESI-MS m/z 463.4 [M+Na]+,
903.7 [2 M+Na]+; (+) HR-ESI-MS m/z 463.3526 (calcd for C30H48O2-
Na, 463.3547).
4.3.1. Preparation of (R)- and (S)-MTPA esters of 1 and 2 (Li et al.,
2011).
4.3.7. Altissimanins D (4) and E (5)
A solution of 1 (1.5 mg) in dried CH2Cl2 (1.5 mL) was treated
with (R)-MTPA (35 mg) [or (S)-MTPA (35 mg)] in the presence of
EDCꢁHCl (50 mg) and 4-DMAP (50 mg), and the mixture was stirred
at room temperature overnight. The residue obtained after removal
of solvent was subjected to preparative TLC (PE/EtOAc = 3:1) and
then Sephadex LH–20 in CH2Cl2/MeOH (2:1) to give pure (R)-MTPA
ester and (S)-MTPA ester of 1, respectively. Treatment of 2 with
(R)-MTPA [or (S)-MTPA] in the same manner as above gave the cor-
responding esters of 2.
Obtained as a mixture; (+) ESI-MS m/z 953.7 [M+Na]+, 1885.8
[2 M+Na]+; (+) HR-ESI-MS m/z 953.7200 (calcd for C60H98O7Na,
953.7205).
4.3.8. Acetylation of 4 and 5
The mixture (27.3 mg) of 4 and 5 were treated with dry pyridine
(2 mL) and Ac2O (2 mL) overnight at room temperature. The reac-
tion mixture was diluted with EtOAc and washed with H2O three
times. After removal of the solvent, the obtained residue was sub-

166
Z.-L. Hong et al. / Phytochemistry 86 (2013) 159–167
jected to silica gel CC (PE-EtOAc, 4:1) to afford 4a (9.7 mg) and 5a
(3.5 mg), and each was further refined by Sephadex LH–20
(MeOH).
in the DMEM medium. All media were supplemented with 10%
fetal bovine serum (FBS), 100 units/mL penicillin, and 100 units/
mL streptomycin (Invitrogen). The cells were maintained at 37 °C
in a humidified environment with 5% CO2. The cell viability was
determined by using the CellTiter GloTM luminescent cell viability
assay (Promega) (Wu et al., 2011). In order to exclude phototoxic-
ity (Colombain et al., 2001), the operation process was kept away
from bright light and the cells were incubated in a dark incubator.
The cells were also incubated in fresh cell culture medium and
washed carefully to avoid false positive results (Bruggisser et al.,
2002). Briefly, the cancer cells were seeded into 384-well plates
4.3.9. Compound 4a
22
White amorphous powder; [
a
]
D
–82 (c 0.1, MeOH); IR (KBr)
m
max: 3429 (br), 2972, 2931, 1738, 1711, 1633, 1454, 1384, 1313,
1245, 1162, 1085, 1051 cm-1; for 1H and 13C NMR spectroscopic
data, see Table 2; (+) ESI-MS m/z 995.7 [M+Na]+, 1969.7
[2 M+Na]+; (+) HR-ESI-MS m/z 995.7316 (calcd for C62H100O8Na,
995.7310).
at an initial density of 1000 cells/well in 45 lL of medium. Then
4.3.10. Compound 5a
the cells were treated with compounds at varying concentrations.
Staurosporine (Sigma-Aldrich, catalog No. S6942-200UL) was used
as a positive control. After incubation for 72 h, 10% of CellTiter
GloTM reagent was added, and luminescent signals were read on
a VeriScan reader (Thermo Fisher Scientific). The IC50 value was
calculated from the curves generated by plotting the percentage
of the viable cells versus test concentrations on a logarithmic scale
using SigmaPlot 10.0 software.
22
White amorphous powder; [
a
]
D
–76 (c 0.1, MeOH); IR (KBr)
m
max: 3431 (br), 2986, 2931, 1736, 1711, 1636, 1458, 1384, 1315,
1237, 1171, 1113, 1082, 1049 cm-1; for 1H and 13C NMR spectro-
scopic data, see Table 2; (+) ESI-MS m/z 995.7 [M+Na]+, 1969.5
[2 M+Na]+; (+) HR-EI-MS m/z 995.7255 [M+Na]+ (calcd for C62H100-
O8Na, 995.7310).
4.3.11. Altissimacoumarin G (6)
White amorphous powder, [
(MeOH) kmax (log ) 225 (3.99), 298 (3.54), 340 (3.40) nm; IR
(KBr) max: 3430 (br), 2975, 2941, 1730, 1639, 1565, 1461, 1409,
1384, 1292, 1266, 1155, 1126, 1092, 1043, 851 cm-1 1H NMR
a]
15 + 20 (c 0.10, MeOH); UV
D
Acknowledgements
e
m
The authors thank Prof. Bao-Kang Huang (Department of Phar-
macognosy, the Second Military Medical University of China) for
plant identification. This work was supported by NSFC Grants
(No. 90713040, 81273401, 81202420), MOST Grants (No.
2011ZX09307-002-01, 2013CB530700) and a STCSM Grant (No.
11DZ1921203).
;
(500 MHz, in CDCl3) d 1.47 (3H, brs, Me-90), 1.73 (3H, brs, Me-
100), 1.74 (3H, brs, Me-80), 1.74 (1H, dd, overlapped, Ha-40), 2.20
(1H, dd, J = 12.5, 7.0 Hz, Hb-40), 3.88 (3H, s, OMe-6), 3.98 (1H, dd,
J = 8.0, 4.5 Hz, H-20), 4.02 (3H, s, OMe-8), 4.10 (1H, dd, J = 10.0,
8.0 Hz, Ha-10), 4.42 (1H, dd, J = 10.0, 4.5 Hz, Hb-10), 4.86 (1H, brq,
J = 8.5 Hz, H-50), 5.19 (1H, brd, J = 8.5 Hz, H-60), 6.35 (1H, d,
J = 9.5 Hz, H-3), 6.67 (1H, s, H-5), 7.61 (1H, d, J = 9.5 Hz, H-4); 13C
NMR (125 MHz, in CDCl3) d 18.2 (C-100), 23.6 (C-90), 25.8 (C-80),
47.5 (C-40), 56.2 (OMe-6), 62.0 (OMe-8), 73.1 (C-10), 74.4 (C-50),
79.4 (C-30), 84.7 (C-20), 103.9 (C-5), 114.3 (C-4a), 115.3 (C-3),
125.4 (C-60), 136.9 (C-70), 140.7 (C-8), 143.2 (C-8a), 143.4 (C-4),
144.8 (C-7), 149.6 (C-6), 160.4 (C-2); (+) ESI-MS m/z 413.2
[M+Na]+, 803.3 [2 M+Na]+; (+) HR-ESI-MS m/z 413.1561 (calcd for
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.phytochem.
2012.10.008.
C21H26O7Na, 413.1571).
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22
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a
screening program for death-receptor expression