Studies on chemical structure modification and structure-activity relationship of derivatives of gambogic acid at C(39)

Article abstract of DOI:10.1002/cbdv.201100415

The natural product gambogic acid exhibits high potency in inhibiting cancer cell lines. Rational medicinal modifications on gambogic acid will improve its physicochemical properties and drug-like characters. To investigate the structure-activity relation

Full text of DOI:10.1002/cbdv.201100415

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Studies on Chemical Structure Modification and StructureꢀActivity
Relationship of Derivatives of Gambogic Acid at C(39)
by Hao-Peng Suna), Zong-Liang Liua), Xin Xuea), Yuan Gaob), Lei Zhanga), Jin-Xin Wanga),
Qing-Long Guob), and Qi-Dong You*a)
a) State Key Laboratory of Natural Medicines, Department of Medicinal Chemistry, China
Pharmaceutical University, Nanjing 210009, P. R. China
(phone/fax: þ86-25-83271351; e-mail: youqidong@gmail.com)
b) Key Laboratory of Carcinogenesis and Intervention of Jiangsu Province, China Pharmaceutical
University, Nanjing 210009, P. R. China
The natural product gambogic acid exhibits high potency in inhibiting cancer cell lines. Rational
medicinal modifications on gambogic acid will improve its physicochemical properties and drug-like
characters. To investigate the structureꢀactivity relationship of gambogic acid and also to find rational
modification position on its chemical skeleton, we designed, synthesized, and characterized 16
derivatives of gambogic acid that were modified at C(39). The structureꢀactivity relationships (SARs)
were discussed. The anti-proliferation data were accquired through MTT (¼ 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-2H-tetrazolium bromide) assays of A549, BGC823, U251, HepG2, and MDA-MB-231
cancer cell lines. Most of the synthesized compounds showed strong inhibitory effects. The SAR study
revealed that derivatives with aliphatic amino moieties at C(39) were more potent than those with other
substituents. The C(39) position can undergo different kinds of chemical modifications without leading to
loss of activity. Compounds 4 and 6 can serve as potential lead compounds for further development of
new anticancer drugs.
1. Introduction. – Gambogic acid (GA) is the major active compound of gamboge
[1][2], a dry resin exuded from various Garcinia species, which was traditionally used as
a folk remedy due to its broad spectrum of cytotoxic activity [3][4]. Although the
molecular structure of GA was established in 1966 [5], its biological mechanism has
remained ambiguous so far, and further research is needed to clarify its mode of action.
Previous studies have demonstrated that GA had a significant inhibition effect on
many kinds of cancer cell lines, including human gastric carcinoma SGC-7901 cells,
MGC-803 cells, human lung carcinoma SPC-A1 cells, and human hepatoma SMMC-
7721 cells [6–8]. GAꢀs activation on apoptotic pathways via down-regulation of
telomerase, a process which is impaired in malignant cells, has been established as the
possible mechanism for its anticancer effect [9]. GA mediates its apoptotic effect
through binding to the transferring receptor, inducing rapid apoptosis of tumor cells
[10]. It is reported that GA potentiates TNF-induced apoptosis in human leukemia
cancer cells by inhibiting the nuclear factor-kappaB (NF-kB) activity via the transferrin
receptor [11]. GA downregulates the activity of human telomerase reverse tran-
scriptase (hTERT) by reducing the level of transcription activator and subsequently
inhibiting the phosphorylation of Akt [9][12–14]. Furthermore, GA inhibits angio-
ꢁ 2012 Verlag Helvetica Chimica Acta AG, Zꢂrich

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genesis through suppression of vascular endothelial growth factor-induced tyrosine
phosphorylation of KDR/Flk-1 [9][12][15]. All studies mentioned above show that
GA is a potent anticancer agent with diverse molecular targets that act through
different mechanisms [16].
As a potential multi-target anticancer agent, the key goal now is to create more
novel derivatives of GA to study its structureꢀactivity relationships (SARs) and to
explore the molecular target. GA contains the xanthone core and a unique 4-
oxatricyclo[4.3.1.03,7]decan-2-one scaffold [4][17]. Previous reports [18–21] on the
SARs revealed that the C¼C bond of the a,b-unsaturated ketone in GA is critical for its
antitumor activity, while the HOꢀC(6), C(8)¼O, and C(30)OOH groups could tolerate
a variety of modifications. Our previous work on chemical synthesis and biological
evaluation of oxidized analogs of GA [22][23] indicated that, in preliminary SAR
studies of GA, modification of the C(32)¼C(33) or C(37)¼C(38) bonds, and of the Me
groups at C(39)/C(35) could improve the antitumor activity.
In the present study, our goal was to create further new derivatives of GA to
investigate their SARs and to further explore of its biological mechanism. This article
describes our efforts to synthesize the gambogic acid derivatives 1–16 (Fig. 1).
Antiproliferation effects of these compounds are also tested by using several cancer cell
lines.
Fig. 1. Chemical structures of gambogic acid (GA) and its derivatives 1–16
2. Results and Discussion. – 2.1. Chemistry. Gambogic acid was isolated in an overall
yield of ca. 5% from gamboge resin. It was purified by converting the crude extract
from the gamboge resin into the pyridine salt, followed by recrystallization [17]. Based
on the results reported previously by our laboratory [22][23], we planned to
regioselectively modify C(39) of GA by introducing aliphatic amino, aromatic amino,
alkoxy, and halogen moieties in this position, respectively. The synthesis of derivatives
1–16, starting from GA, are depicted in Schemes 1 and 2.

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Scheme 1
a) MeI (3.0 equiv.), K2CO3 (3.0 equiv.), acetone, 258, 6 d. b) SeO2 (0.1 equiv.), t-BuOOH (2 equiv.),
salicylic acid (0.1 equiv.), CH2Cl2, 6 h.
Scheme 2
a) N-Chlorosuccinimide (NCS; 1.3 equiv.), CCl4, reflux, 3 h. b) Different conditions.
Reaction of GAwith MeI in the presence of K2CO3 afforded the methyl ester of GA
in 95% yield in acetone at room temperature (Scheme 1). The activity of HOꢀC(6) was
very low due to the intramolecular H-bond, so the reaction required a long time. Three
compounds 18–20 could be produced from compound 17 by the oxidation with SeO2
and t-BuOOH. But, only one main product was monitored on TLC in this reaction. In

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Fig. 2. HMBC Spectrum of compound 18
the HMBC spetrum (Fig. 2), the chemical shift of the CH2 H-atoms of the CH2ꢀOH
group was d(H) 3.9, and they coupled with C(37) (d(C) 122.2). So, the structure of the
main product was confirmed as 18. The steric hindrance of C(39) was smaller than that
of C(34), so C(39) was first oxidized with SeO2 and t-BuOOH.
Compound 16 was easily obtained from 18 with NCS in CCl4 under reflux for 3 h.
Target molecules (TM) were to be prepared from 16 by introducing various
substituents. First, the reaction of 16 (R¼Cl) with K2CO3 and KI in acetone was
tried. However, the expected product was not obtained. Aromatic amines and ethers
could not be synthesized, either. For R¼aliphatic amino moiety, only a few target
compounds were obtained, and by-products, with Rf values very close to the target
compounds on the TLC, were also detected. In the 1H-NMR spetra of by-products, the
peak of HꢀC(10) disappeared, and other peaks were similar to those of products. So, we
assume that the activities of aliphatic amines were so high that the C¼C bond of the a,b-
unsaturated ketone in GA was destroyed.
Microwave-assisted reaction has been a powerful method to synthesize compounds
that cannot be obtained under common conditions. So, we attempted to construct
target compounds with microwave assistance.
When we first tried the microwave-assisted reaction, we found that compound 16
did not fully react, and the yield was only 10%, when the reaction was conducted under
microwave irradiation (100 W, 608, 5 min). So, several microwave conditions were
tested. The power of microwave irradiation was increased. Yield of compound 1 was
80% under microwave irradiation of 200 W (608, 5 min). Then, the reaction time was
prolonged. Yield of compound 1 was 70%, and a by-product was monitored on TLC

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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under microwave irradiation of 200 W (608, 10 min). Eventually, the optimal micro-
wave conditions turned out to be 200 W, 608, 5 min for aliphatic amines.
In summary, we have regioselectively synthesized 16 novel derivatives of GA
modified at C(39) (i.e., 1–16) and elucidated their structures by spectral techniques.
2.2. StructureꢀActivity Relationship. Earlier SAR studies [18–21] were mainly
focused on the C(9)¼C(10) bond of the a,b-unsaturated ketone, C(30)OOH, and
HOꢀC(6). We concentrated our synthetic efforts on modifications of the Me(39)
group, which has not been reported so far, and the structureꢀactivity relationships are
compiled in the Table. The cytotoxic activities of the derivatives 1–16 were determined
in vitro by an MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bro-
mide) assay which estimates cell viability by the remaining activity of mitochondrial
enzymes [24]. Against the A549, BGC-823, U251, HepG2, and MB-231 cell lines, the
IC50 values of the parent compound, gambogic acid, ranged between 0.2 and 2.4 mm
(Table). The novel compounds exhibited a considerable variation in their cytotoxic
activities against the same cell lines.
Table. Cytotoxicity (IC50 [mm]) Data of GA and Its Derivatives
Compound
IC50
A549
BGC823
1.94ꢁ0.05
U251
HepG2
MDA-MB-231
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
GA
1.92ꢁ0.03
2.93ꢁ0.08
1.39ꢁ0.07
3.19ꢁ0.11
1.20ꢁ0.05
2.98ꢁ0.07
7.01ꢁ0.20
81.7ꢁ4.98
1.42ꢁ0.09
3.91ꢁ0.14
2.01ꢁ0.07
1.90ꢁ0.07
3.14ꢁ0.20
3.83ꢁ0.11
4.40ꢁ0.23
2.59ꢁ0.09
2.31ꢁ0.13
1.84ꢁ0.08
3.60ꢁ0.20
1.76ꢁ0.13
0.96ꢁ0.03
1.69ꢁ0.03
1.57ꢁ0.08
2.60ꢁ0.18
4.86ꢁ0.36
2.08ꢁ0.12
4.02ꢁ0.27
1.08ꢁ0.05
1.08ꢁ0.06
2.09ꢁ0.13
3.14ꢁ0.32
3.19ꢁ0.22
1.90ꢁ0.13
0.19ꢁ0.02
0.29ꢁ0.021
0.33ꢁ0.040
0.36ꢁ0.021
0.023ꢁ0.003
0.24ꢁ0.038
0.028ꢁ0.002
0.17ꢁ0.010
0.37ꢁ0.035
0.66ꢁ0.021
0.23ꢁ0.007
0.047ꢁ0.003
0.22ꢁ0.007
0.042ꢁ0.020
0.11ꢁ0.013
0.061ꢁ0.003
0.15ꢁ0.010
0.24ꢁ0.021
4.00ꢁ0.06
4.44ꢁ0.45
1.62ꢁ0.26
0.99ꢁ0.05
1.56ꢁ0.06
0.68ꢁ0.04
1.48ꢁ0.06
5.25ꢁ0.39
4.91ꢁ0.39
3.73ꢁ0.28
4.27ꢁ0.44
0.80ꢁ0.05
2.28ꢁ0.25
1.88ꢁ0.09
5.13ꢁ0.44
1.15ꢁ0.08
0.24ꢁ0.02
2.19ꢁ0.15
0.58ꢁ0.05
2.03ꢁ0.05
1.23ꢁ0.09
1.94ꢁ0.06
5.53ꢁ0.25
122.2ꢁ8.79
1.05ꢁ0.06
4.17ꢁ0.14
1.04ꢁ0.07
1.16ꢁ0.06
2.13ꢁ0.10
3.24ꢁ0.08
5.91ꢁ0.38
1.01ꢁ0.05
1.37ꢁ0.05
As a result of cytotoxicity screening, all derivatives were identified as having a
lower effect against U251 and MB-231 than GA. Most derivatives were comparable to
GA with respect to their activities against A549 and BGC823. Of these derivatives,
compound 5 was ca. twofold more potent than GA for the inhibition of A549, and
compound 3 was two- to threefold more cytotoxic than the parent compound against
BGC823. All derivatives had an evidently higher or approximate by comparable effect
against HepG2 than GA. Against this cancer line, five compounds, 4, 6, 11, 13, and 15,
were ca. four- to tenfold more active than GA, while 4 and 6 exhibited the lowest IC50

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values of 0.023 and 0.028 mm, i.e., ca. tenfold more potent than GA. In general,
compound 8 had the lowest effect against all cancer lines.
Our newly synthesized compounds 1–16 exerted significant in vitro inhibitory
effects on growth of some cells. The selectivity of all derivatives for HepG2 was greatly
increased. Compounds 4 and 6 exhibited the lowest IC50 values, so these need further
research. In general, effect against cancer lines of derivatives with aliphatic amino
substituents was higher than that of derivatives with aromatic amino alkoxy, or halogen
moieties. Water solubility of derivatives can be the decisive factor. Aliphatic amino
groups could more efficiently enhance water solubility than aromatic amino, alkoxy, or
halogen substituents. The principle could be used to directly modify GA to search for
derivatives with ꢃdruggabilityꢀ.
3. Conclusions. – Although these experiments were preliminary, the results of the
SAR investigations suggested that promising agents with the antitumor activities may
be obtained by the conversion of Me(29) group to an aliphatic aminomethyl, aromatic
aminomethyl, alkoxymethyl, or halogenomethyl moieties. Sixteen compounds were
prepared starting from GA, and all of these derivatives had cytotoxic activities against
A549, BGC-823, U251, HepG2, and MB-231 cell lines, evaluated by an MTT assay.
Compared with GA, the selectivity of all derivatives for HepG2 was greatly increased,
and compounds 4 and 6 exhibited the lowest IC50 values, i.e., ca. tenfold more potent
than GA.
This work was supported by the Project Program of State Key Laboratory of Natural Medicines, the
National Natural Science Foundation of China (Nos. 90713038, 21072231, and 30873157), and the
National Major Science and Technology Project of China (Innovation and Development of New Drugs.
Nos. 2008ZX09401-001 and 2009ZX09501-003).
Experimental Part
General. Gambogic acid (GA) was freshly extracted from gamboges, and the contents of GA in the
extract were not less than 95% (the yield of extracts was ca. 1.5%). Column and thin-layer
chromatography (CC and TLC, resp.) were performed on silica gel (SiO2; 200–300 mesh) and SiO2
GF254, resp., supplied by Qingdao Marine Chemical Factory. IR Spectra: Nicolet Impact 410
.
spectrometer; ˜n in cmꢀ1 1H- and 13C-NMR spectra: BrukerAV-300 or AV-500 MHz instruments in
CDCl3; d in ppm rel. to Me4Si as internal standard, J in Hz. EI-MS: Shimadzu GC-MS 2050 apparatus; in
m/z (rel. %). ESI-MS: Agilent 1100 LC/MSD (70 eV) spectrometer; in m/z. HR-EI-MS: Waters Q-
Tofmicro; in m/z.
39-Hydroxy-6-methoxygambogic Acid Methyl Ester (¼ Methyl (2Z)-4-{(1R,3aS,5S,11R,14aS)-
3a,4,5,7-Tetrahydro-11-[(3E)-5-hydroxy-4-methylpent-3-en-1-yl]-8-methoxy-3,3,11-trimethyl-13-(3-
methylbut-2-en-1-yl)-7,15-dioxo-3H,11H-1,5-methanofuro[3,4-g]pyrano[3,2-b]xanthen-1-yl}-2-methyl-
but-2-enoate; 18). GA Methyl ester (17; 450 mg, 0.69 mmol) was dissolved in CH2Cl2 (2 ml) and added
dropwise to a clear soln. of SeO2 (7.6 mg, 0.068 mmol) and t-BuOOH (75%, 185 mg, 2.06 mmol) in
CH2Cl2 (10 ml) at r.t. After stirring at r.t. for 19 h, the mixture was diluted with Et2O and sequentially
with aq. 10% Na2SO3, H2O, and brine, dried, and concentrated. The resulting yellow oil was purified by
flash column chromatography (FC) on SiO2 to yield 18 (280 mg, 61%). Yellow sheet solid. IR (KBr):
3467, 3416, 2966, 2925, 1734, 1709, 1655, 1607, 1586, 1463, 1427, 1384, 1225, 1143, 1046. 1H-NMR (CDCl3,
300 MHz): 7.53 (d, J¼6.70, HꢀC(10)); 6.68 (d, J¼2.3, HꢀC(4)); 5.97–5.96 (m, HꢀC(27)); 5.53 (d, J¼6.1,
HꢀC(3)); 5.32–5.31 (m, HꢀC(37)); 5.17–5.15 (m, HꢀC(32)); 3.95 (s, CH2(39)); 3.81 (s, MeO); 3.43 (s,
COOMe); 3.42–3.40 (m, HꢀC(11)); 3.39–3.37 (m, HaꢀC(31)); 3.26–3.25 (m, HbꢀC(31)); 2.97–2.95 (m,

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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CH2(26)); 2.51 (d, J¼9.31, HꢀC(22)); 2.31–2.29 (m, HaꢀC(21)); 2.05–2.02 (m, CH2(36)); 1.75 (s,
Me(25)); 1.69 (s, Me(34)); 1.68 (s, Me(40)); 1.65 (s, Me(35)); 1.44 (s, Me(24)); 1.39–1.37 (m, HbꢀC(21));
1.28 (s, Me(19)). 13C-NMR (CDCl3, 75 MHz): 18.1 (C(34)); 20.7 (C(29)); 20.8 (C(40)); 22.1 (C(31)); 22.3
(C(36)); 25.5 (C(21)); 25.7 (C(35)); 27.9 (C(19)); 28.9 (C(24)); 29.4 (C(25)); 29.9 (C(26)); 41.7 (C(20));
46.8 (C(11)); 49.0 (C(22)); 50.9 (COOMe); 62.1 (MeO); 68.8 (C(39)); 80.5 (C(2)); 83.6 (C(13)); 83.8
(C(23)); 90.9 (C(14)); 107.4 (C(17)); 100.7 (C(7)); 112.6 (C(5)); 116.9 (C(4)); 121.9 (C(32)); 125.3
(C(37)); 126.9 (C(3)); 127.4 (C(28)); 131.9 (C(33)); 132.0 (C(38)); 134.2 (C(9)); 135.4 (C(10)); 135.6
(C(27)); 158.6 (C(16)); 159.7 (C(18)); 174.6 (C(8)); 203.9 (C(12)); 167.3 (C(30)). ESI-MS: 673.3369
([MþH]þ , C40H49Oþ9 ; calc. 673.3377).
39-Chloro-6-methoxygambogic Acid Methyl Ester (¼ Methyl (2Z)-4-{(1R,3aS,5S,11R,14aS)-11-
[(3E)-5-Chloro-4-methylpent-3-en-1-yl]-3a,4,5,7-tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methyl-
but-2-en-1-yl)-7,15-dioxo-3H,11H-1,5-methanofuro[3,4-g]pyrano[3,2-b]xanthen-1-yl}-2-methylbut-2-
enoate; 16). Compound 18 (260 mg, 0.39 mmol) was dissolved in CCl4 (5 ml), and NCS (56.7 mg,
0.42 mmol) was added. The mixture was heated to reflux for 3 h and then concentrated under reduced
pressure. The residue was purified by FC (SiO2) to yield 16 (230 mg, 86%). Yellow oil. IR (KBr): 2931,
2859, 1774, 1713, 1655, 1587, 1451, 1426, 1262, 1124, 1026, 752. 1H-NMR (CDCl3, 300 MHz): 7.48 (d, J¼
6.9, HꢀC(10)); 6.71 (d, J¼10.2, HꢀC(4)); 5.99–5.97 (m, CH2(27)); 5.55 (d, J¼10.2, HꢀC(3)); 5.49–5.48
(m, HꢀC(37)); 5.13–5.11 (m, HꢀC(32)); 3.99–3.97 (m, CH2(39)); 3.84 (s, COOMe); 3.46 (s, MeO);
3.45–3.43 (m, HꢀC(11)); 3.42–3.40 (m, HbꢀC(31)); 3.26–3.24 (m, HaꢀC(31)); 3.01–2.99 (m, CH2(26));
2.53 (d, J¼9.6, HꢀC(22)); 2.20–2.18 (m, HaꢀC(21)); 2.09–2.05 (m, CH2(36)); 1.79 (s, Me(25)); 1.75 (s,
Me(29)); 1.69 (s, Me(20)); 1.64 (s, Me(35)); 1.60 (s, Me(34)); 1.45 (s, Me(40)); 1.40–1.38 (m, HꢀC(24));
1.36–1.34 (m, HbꢀC(21)); 1.29 (s, Me(19)). ESI-MS: 691 ([MþH]þ ).
General Procedure for the Preparation of Compounds 1–7. A soln. of 16 (20 mg, 0.029 mmol) and
appropriate aliphatic amine (0.032 mmol) in acetone (3 ml) was irradiated for 5 min in a CEM single-
mode microwave cavity at 200 W and 608. The mixture was concentrated in vacuo, and the residue was
purified by FC (SiO2) to yield 1–7 (66–80%).
Methyl (2Z)-4-{(1R,3aS,5S,11R,14aS)-3a,4,5,7-Tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methyl-
but-2-en-1-yl)-11-[(3E)-4-methyl-5-(morpholin-4-yl)pent-3-en-1-yl]-7,15-dioxo-3H,11H-1,5-methano-
furo[3,4-g]pyrano[3,2-b]xanthen-1-yl}-2-methylbut-2-enoate (1). IR (KBr): 3403, 2926, 2932, 2857, 2808,
1774, 1715, 1653, 1586, 1453, 1426, 1377, 1332, 1284, 1228, 1122, 1016, 736. 1H-NMR: (CDCl3, 300 MHz):
7.43 (d, J¼6.9, HꢀC(10)); 6.67 (d, J¼10.2, HꢀC(4)); 5.97–5.95 (m, HꢀC(27)); 5.53 (d, J¼10.2,
HꢀC(3)); 5.27–5.26 (m, HꢀC(37)); 5.10–5.08 (m, HꢀC(32)); 3.87 (s, COOMe); 3.60–3.56 (m, 4 H of
morpholine); 3.43 (s, MeO); 3.38–3.36 (m, HꢀC(11)); 3.25–3.23 (m, HbꢀC(31)); 2.98–2.96 (m,
HaꢀC(31)); 2.87–2.86 (m, CH2(26)); 2.51 (d, J¼9.3, HꢀC(22)); 2.32–2.30 (m, HaꢀC(21)); 2.28 (m, 4 H
of morpholine); 2.10–2.09 (m, CH2(36)); 1.87–1.85 (m, CH2(39)); 1.79 (s, Me(25)); 1.75 (s, Me(29)); 1.69
(s, Me(20)); 1.64 (s, Me(35)); 1.60 (s, Me(34)); 1.45 (s, Me(40)); 1.40–1.38 (m, HꢀC(24)); 1.36–1.34 (m,
HbꢀC(21)); 1.29 (s, Me(19)). ESI-MS: 742 ([MþH]þ ). HR-MS: 742.3944 ([MþH]þ , C44H56NO9þ ; calc.
742.3955).
Methyl (2Z)-4-{(1R,3aS,5S,11R,14aS)-3a,4,5,7-Tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methyl-
but-2-en-1-yl)-11-[(3E)-4-methyl-5-(piperidin-1-yl)pent-3-en-1-yl]-7,15-dioxo-3H,11H-1,5-methano-
furo[3,4-g]pyrano[3,2-b]xanthen-1-yl}-2-methylbut-2-enoate (2). IR (KBr): 3403, 2931, 2932, 2859, 2386,
2276, 1775, 1715, 1652, 1587, 1451, 1426, 1376, 1328, 1262, 1227, 1124, 1026, 752. 1H-NMR (CDCl3,
300 MHz): 7.43 (d, J¼6.6, HꢀC(10)); 6.67 (d, J¼10.2, HꢀC(4)); 5.96–5.94 (m, HꢀC(27)); 5.54 (d, J¼
10.2, HꢀC(3)); 5.01–4.98 (m, HꢀC(37)); 5.10–5.08 (m, HꢀC(32)); 3.82 (s, COOMe); 3.43 (s, MeO);
3.40–3.38 (m, HꢀC(11)); 3.26–3.25 (m, HbꢀC(31)); 2.98–2.96 (m, HaꢀC(31)); 2.80–2.76 (m, CH2(26));
2.51 (d, J¼9.6, HꢀC(22)); 2.32–2.30 (m, HaꢀC(21)); 2.27 (m, 4 H of piperidine); 2.10–2.08 (m,
CH2(36)); 1.80–1.78 (m, CH2(39)); 1.75 (s, Me(25)); 1.69 (s, Me(29)); 1.68 (s, Me(20)); 1.65 (s, Me(35));
1.59 (s, Me(34)); 1.45 (s, Me(40)); 1.40–1.38 (m, HꢀC(24)); 1.36–1.34 (m, HbꢀC(21)); 1.29 (s, Me(19)).
ESI-MS: 740 ([MþH]þ ). HR-MS: 740.4168 ([MþH]þ , C45H58NOþ8 ; calc. 740.4157).
Methyl (2Z)-4-{(1R,3aS,5S,11R,14aS)-3a,4,5,7-Tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methyl-
but-2-en-1-yl)-11-[(3E)-4-methyl-5-(4-methylpiperazin-1-yl)pent-3-en-1-yl]-7,15-dioxo-3H,11H-1,5-
methanofuro[3,4-g]pyrano[3,2-b]xanthen-1-yl}-2-methylbut-2-enoate (3). IR (KBr): 3405, 2928, 2801,
2681, 2290, 2117, 1773, 1715, 1654, 1586, 1453, 1426, 1379, 1332, 1267, 1225, 1191, 1126, 1025, 735. 1H-NMR

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
(CDCl3, 300 MHz): 7.43 (d, J¼6.9, HꢀC(10)); 6.67 (d, J¼10.2, HꢀC(4)); 5.97–5.96 (m, HꢀC(27)); 5.53
(d, J¼10.2, HꢀC(3)); 5.26–5.24 (m, HꢀC(37)); 5.09–5.07 (m, HꢀC(32)); 3.81 (s, COOMe); 3.43 (s,
MeO); 3.41–3.39 (m, HꢀC(11)); 3.26–3.24 (m, HbꢀC(31)); 2.98–2.96 (m, HaꢀC(31)); 2.82–2.79 (m,
CH2(26)); 2.52–2.50 (m, HꢀC(22)); 2.46–2.44 (m, 8 H of piperazine); 2.36 (s, MeN); 2.32–2.30 (m,
HaꢀC(21)); 2.09–2.07 (m, CH2(36)); 1.81–1.79 (m, CH2(39)); 1.75 (s, Me(25)); 1.70 (s, Me(29)); 1.69 (s,
Me(20)); 1.64 (s, Me(35)); 1.57 (s, Me(34)); 1.46 (s, Me(40)); 1.41–1.39 (m, HꢀC(24)); 1.37–1.35 (m,
HbꢀC(21)), 1.34 (s, Me(19)). 13C-NMR (CDCl3, 75 MHz): 15.3 (C(40)); 20.7 (C(34)); 20.8 (C(29)); 22.0
(C(31)); 24.3 (C(36)); 25.4 (C(21)); 25.6 (C(35)); 25.7 (C(39)); 27.8 (C(19)); 28.9 (C(24)); 29.3 (C(26));
30.0 (C(25)); 42.7 (C(20)); 46.9 (C(11)); 49.0 (C(22)); 51.0 (COOMe); 62.2 (MeO); 80.2 (C(2)); 83.6
(C(13)); 83.6 (C(23)); 86.5 (C(14)); 91.1 (C(7)); 95.1 (C(5)); 107.4 (C(17)); 117.2 (C(4)); 121.7 (C(32));
122.0 (C(37)); 126.6 (C(3)); 127.4 (C(28)); 131.9 (C(33)); 132.0 (C(38)); 134.2 (C(9)); 135.4 (C(10));
135.6 (C(27)); 136.5 (C(16)); 160.0 (C(6)); 167.4 (C(18)); 173.2 (C(30)); 175.0 (C(8)); 203.9 (C(12)).
ESI-MS: 755 ([MþH]þ ). HR-MS: 755.4253 ([MþH]þ , C45H59N2Oþ8 ; calc. 755.4266).
Methyl (2Z)-4-{(1R,3aS,5S,11R,14aS)-11-[(3E)-5-(Diethylamino)-4-methylpent-3-en-1-yl]-3a,4,5,7-
tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methylbut-2-en-1-yl)-7,15-dioxo-3H,11H-1,5-methano-
furo[3,4-g]pyrano[3,2-b]xanthen-1-yl}-2-methylbut-2-enoate (4). IR (KBr): 3407, 2964, 2934, 1775, 1715,
1652, 1587, 1453, 1424, 1378, 1324, 1225, 1189, 1126, 1026, 750. 1H-NMR (CDCl3, 300 MHz): 7.43 (d, J¼
6.6, HꢀC(10)); 6.67 (d, J¼10.2, HꢀC(4)); 5.96–5.94 (m, HꢀC(27)); 5.54 (d, J¼10.2, HꢀC(3)); 5.28 (m,
HꢀC(37)); 5.10–5.08 (m, HꢀC(32)); 3.82 (s, COOMe); 3.46 (s, MeO); 3.39–3.37 (m, HꢀC(11)); 3.27–
3.25 (m, HbꢀC(31)); 2.98–2.96 (m, HaꢀC(31)); 2.91–2.89 (m, HꢀC(26)); 2.55 (d, J¼9.6, HꢀC(22));
2.54–2.52 (m, (MeCH2)2N); 2.27–2.25 (m, HaꢀC(21)); 2.10–2.08 (m, CH2(36)); 1.82–1.81 (m,
CH2(39)); 1.78 (s, Me(25)); 1.71 (s, Me(29)); 1.69 (s, Me(20)); 1.64 (s, Me(35)); 1.60 (s, Me(34)); 1.47
(s, Me(40)); 1.42–1.38 (m, HꢀC(24)); 1.36–1.34 (m, HbꢀC(21)); 1.29 (s, Me(19)); 1.05–1.01 (m,
(MeCH2)2N). ESI-MS: 728 ([MþH]þ ). HR-MS: 728.4136 ([MþH]þ , C44H58NOþ8 ; calc. 728.4157).
Methyl
(2Z)-4-{(1R,3aS,5S,11R,14aS)-11-[(3E)-5-(Dipropylamino)-4-methylpent-3-en-1-yl]-
3a,4,5,7-tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methylbut-2-en-1-yl)-7,15-dioxo-3H,11H-1,5-metha-
nofuro[3,4-g]pyrano[3,2-b]xanthen-1-yl}-2-methylbut-2-enoate (5). IR (KBr): 3408, 2961, 2928, 2384,
2242, 2096, 1777, 1717, 1652, 1586, 1456, 1428, 1380, 1266, 1225, 1188, 1125, 1028, 754. 1H-NMR (CDCl3,
300 MHz): 7.43 (d, J¼6.9, HꢀC(10)); 6.67 (d, J¼9.9, HꢀC(4)); 5.97–5.95 (m, HꢀC(27)); 5.54 (d, J¼10.2,
HꢀC(3)); 5.24–5.22 (m, HꢀC(37)); 5.11–5.09 (m, HꢀC(32)); 3.82 (s, COOMe); 3.43 (s, MeO); 3.39–
3.37 (m, HꢀC(11)); 3.28–3.26 (m, HbꢀC(31)); 2.99–2.97 (m, HꢀC(31)); 2.84–2.82 (m, CH2(26)); 2.51
(d, J¼9.0, HꢀC(22)); 2.32–2.30 (m, HaꢀC(21)); 2.31–2.29 (m, (MeCH2CH2)2N); 2.10–2.06 (m,
CH2(36)); 1.83–1.79 (m, CH2(39)); 1.75 (s, Me(25)); 1.70 (s, Me(29)); 1.68 (s, Me(20)); 1.65 (s, Me(35));
1.61 (s, Me(34)); 1.45 (s, Me(40)); 1.43–1.40 (m, (MeCH2CH2)2N); 1.40–1.38 (m, HꢀC(24)); 1.36–1.34
(m, HbꢀC(21)); 1.29 (s, Me(19)); 0.88–0.80 (m, (MeCH2CH2)2N). ESI-MS: 756 ([MþH]þ ). HR-MS:
756.4455 ([MþH]þ , C46H62NOþ8 ; calc. 756.4475).
Methyl (2Z)-4-[(1R,3aS,5S,11R,14aS)-11-{(3E)-5-[Bis(1-methylethyl)amino]-4-methylpent-3-en-1-
yl}-3a,4,5,7-tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methylbut-2-en-1-yl)-7,15-dioxo-3H,11H-1,5-
methanofuro[3,4-g]pyrano[3,2-b]xanthen-1-yl]-2-methylbut-2-enoate (6). IR (KBr): 3475, 3311, 2930,
2535, 2389, 2225, 2087, 1774, 1718, 1638, 1587, 1427, 1383, 1263, 1196, 1114, 1011, 753. 1H-NMR (CDCl3,
300 MHz): 7.43 (d, J¼6.9, HꢀC(10)); 6.67 (d, J¼10.2, HꢀC(4)); 5.97–5.95 (m, HꢀC(27)); 5.54 (d, J¼
10.2, HꢀC(3)); 5.27–5.25 (m, HꢀC(37)); 5.13–5.11 (m, HꢀC(32)); 3.82 (s, COOMe); 3.43 (s, MeO);
3.40–3.38 (m, HꢀC(11)); 3.29–3.27 (m, HbꢀC(31)); 2.98–2.96 (m, HaꢀC(31)); 2.93–2.90 (m,
(Me2CH)2N); 2.92–2.88 (m, CH2(26)); 2.52 (d, J¼9.3, HꢀC(22)); 2.32–2.30 (m, HaꢀC(21)); 2.12–
2.09 (m, CH2(36)); 1.83–1.81 (m, CH2(39)); 1.76 (s, Me(25)); 1.70 (s, Me(29)); 1.68 (s, Me(20)); 1.65 (s,
Me(35)); 1.62 (s, Me(34)); 1.45 (s, Me(40)); 1.39–1.37 (m, HꢀC(24)); 1.36–1.34 (m, HbꢀC(21)); 1.26 (s,
Me(19)); 0.99–0.91 (m, (Me2CH)2N). ESI-MS: 756 ([MþH]þ ). HR-MS: 756.4448 ([MþH]þ
,
C46H62NOþ8 ; calc. 756.4475).
Methyl (2Z)-4-{(1R,3aS,5S,11R,14aS)-11-[(3E)-5-(Dibutylamino)-4-methylpent-3-en-1-yl]-3a,4,5,7-
tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methylbut-2-en-1-yl)-7,15-dioxo-3H,11H-1,5-methano-
furo[3,4-g]pyrano[3,2-b]xanthen-1-yl}-2-methylbut-2-enoate (7). IR (KBr): 3407, 2928, 2864, 1716, 1650,
1591, 1452, 1417, 1382, 1267, 1068, 755. 1H-NMR (CDCl3, 300 MHz): 7.43 (d, J¼6.9, HꢀC(10)); 6.68 (d,
J¼10.2, HꢀC(4)); 5.97–5.95 (m, HꢀC(27)); 5.54 (d, J¼10.2, HꢀC(3)); 5.24–5.22 (m, HꢀC(37)); 5.10–

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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5.08 (m, HꢀC(32)); 3.82 (s, COOMe); 3.43 (s, MeO); 3.39–3.37 (m, HꢀC(11)); 3.28–3.26 (m,
HbꢀC(31)); 3.00–2.98 (m, HaꢀC(31)); 2.88–2.84 (m, CH2(26)); 2.52 (d, J¼9.0, HꢀC(22)); 2.34–2.32 (m,
HaꢀC(21)); 2.30–2.27 (m, (MeCH2CH2CH2)2N); 2.11–2.08 (m, CH2(36)); 1.85–1.83 (m, CH2(39)); 1.82
(s, Me(25)); 1.79 (s, Me(29)); 1.75 (s, Me(20)); 1.69 (s, Me(35)); 1.59 (s, Me(34)); 1.37–1.33 (m,
(MeCH2CH2CH2)2N); 1.45 (s, Me(40)); 1.39–1.37 (m, HꢀC(24)); 1.35–1.33 (m, HbꢀC(21)); 1.29 (s,
Me(19)); 1.26–1.23 (m, (MeCH2CH2CH2)2N); 0.92–0.87 (m, (MeCH2CH2CH2)2N). ESI-MS: 784 ([Mþ
H]þ ). HR-MS: 784.4756 ([MþH]þ , C48H66NOþ8 ; calc. 784.4783).
General Procedure for the Preparation of Compounds 8–12. A soln. of 16 (20 mg, 0.029 mmol) and
appropriate aromatic amine (0.032 mmol) in acetone (3 ml) was irradiated for 5 min in a CEM single-
mode microwave cavity at 200 W and 808. The mixture was concentrated in vacuo, and the residue was
purified by FC (SiO2) to yield 8–12 (73–79%).
Methyl (2Z)-4-[(1R,3aS,5S,11R,14aS)-3a,4,5,7-Tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methyl-
but-2-en-1-yl)-11-{(3E)-4-methyl-5-[(4-methylphenyl)amino]pent-3-en-1-yl}-7,15-dioxo-3H,11H-1,5-
methanofuro[3,4-g]pyrano[3,2-b]xanthen-1-yl]-2-methylbut-2-enoate (8). IR (KBr): 3396, 2926, 2866,
1
2741, 2389, 2229, 2103, 1890, 1771, 1719, 1654, 1587, 1519, 1451, 1380, 1266, 1121, 1031, 753. H-NMR
(CDCl3, 300 MHz): 7.43 (d, J¼6.9, HꢀC(10)); 6.96 (d, J¼8.4, 2 arom. H of ArNH); 6.66 (d, J¼10.2,
HꢀC(4)); 6.50 (d, J¼8.4, 2 arom. H of ArNH); 5.96–5.94 (m, HꢀC(27)); 5.53–5.51 (d, J¼10.2,
HꢀC(3)); 5.35–5.33 (m, HꢀC(37)); 5.09–5.07 (m, HꢀC(32)); 3.81 (s, COOMe); 3.74 (s, ArNH); 3.56 (s,
CH2(26)); 3.43 (s, MeO); 3.38–3.36 (m, HꢀC(11)); 3.27–3.25 (m, HꢀC(31)); 2.98–2.96 (m, HaꢀC(31));
2.48 (d, J¼9.3, HꢀC(22)); 2.30–2.28 (m, HaꢀC(21)); 2.22 (s, MeꢀC6H4NH); 2.12–2.08 (m, CH2(36));
1.81–1.79 (m, CH2(39)); 1.74 (s, Me(25)); 1.68 (s, Me(29)); 1.64 (s, Me(20)); 1.62 (s, Me(35)); 1.60 (s,
Me(34)); 1.44 (s, Me(40)); 1.40–1.38 (m, HꢀC(24)); 1.35–1.33 (m, HbꢀC(21)); 1.28 (s, Me(19)). ESI-MS:
762 ([MþH]þ ). HR-MS: 762.3980 ([MþH]þ , C47H56NOþ8 ; calc. 762.4006).
Methyl (2Z)-4-[(1R,3aS,5S,11R,14aS)-3a,4,5,7-Tetrahydro-8-methoxy-11-{(3E)-5-[(4-methoxyphe-
nyl)amino]-4-methylpent-3-en-1-yl}-3,3,11-trimethyl-13-(3-methylbut-2-en-1-yl)-7,15-dioxo-3H,11H-1,5-
methanofuro[3,4-g]pyrano[3,2-b]xanthen-1-yl]-2-methylbut-2-enoate (9). IR (KBr): 3342, 2929, 2947,
1
2390, 2219, 2062, 1771, 1710, 1658, 1586, 1512, 1453, 1426, 1378, 1247, 1186, 1128, 1034, 754. H-NMR
(300 MHz, CDCl3): 7.43 (d, J¼6.9, HꢀC(10)); 6.74 (d, J¼8.7, 2 arom. H of ArNH); 6.66 (d, J¼9.9,
HꢀC(4)); 6.57 (d, J¼8.7, 2 arom. H of ArNH); 5.96–5.95 (m, HꢀC(27)); 5.52 (d, J¼10.2, HꢀC(3));
5.33–5.31 (m, HꢀC(37)); 5.08–5.07 (m, HꢀC(32)); 3.81 (s, COOMe); 3.74 (s, MeOꢀC6H4NH); 3.49 (s,
HꢀC(26)); 3.43 (s, MeOꢀC(6)); 3.38–3.36 (m, HꢀC(11)); 3.25–3.24 (m, HbꢀC(31)); 2.98–2.97 (m,
HaꢀC(31)); 2.48 (d, J¼9.3, HꢀC(22)); 2.30–2.28 (m, HaꢀC(21)); 2.11–2.09 (m, CH2(36)); 1.81–1.79 (m,
CH2(39)); 1.78 (s, Me(25)); 1.74 (s, Me(29)); 1.68 (s, Me(20)); 1.63 (s, Me(35)); 1.60 (s, Me(34)); 1.44 (s,
Me(40)); 1.40–1.39 (m, HꢀC(24)); 1.36–1.35 (m, HbꢀC(21)); 1.28 (s, Me(19)). ESI-MS: 777 ([MþH]þ ).
HR-MS: 778.3909 ([MþH]þ , C47H56NO9þ ; calc. 778.3955).
Methyl (2Z)-4-[(1R,3aS,5S,11R,14aS)-11-{(3E)-5-[(4-Chlorophenyl)amino]-4-methylpent-3-en-1-
yl}-3a,4,5,7-tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methylbut-2-en-1-yl)-7,15-dioxo-3H,11H-1,5-
methanofuro[3,4-g]pyrano[3,2-b]xanthen-1-yl]-2-methylbut-2-enoate (10). IR (KBr): 3406, 2928, 2859,
2653, 2466, 2393, 2288, 2119, 1943, 1772, 1711, 1655, 1588, 1453, 1424, 1381, 1265, 1229, 1122, 1030, 754.
1H-NMR (300 MHz, CDCl3): 7.43 (d, J¼6.9, HꢀC(10)); 7.08 (d, J¼8.7, 2 arom. H of ArNH); 6.66 (d, J¼
9.9, HꢀC(4)); 6.50 (d, J¼8.7, 2 arom. H of ArNH); 5.98–5.96 (m, HꢀC(27)); 5.51 (d, J¼9.9, HꢀC(3));
5.33–5.31 (m, HꢀC(37)); 5.13–5.12 (m, HꢀC(32)); 3.81 (s, COOMe); 3.50 (s, HꢀC(26)); 3.43 (s, MeO);
3.38–3.36 (m, HꢀC(11)); 3.25–3.24 (m, HbꢀC(31)); 2.98–2.97 (m, HaꢀC(31)); 2.47 (d, J¼9.3,
HꢀC(22)); 2.30–2.28 (m, HaꢀC(21)); 2.11–2.09 (m, CH2(36)); 1.81–1.79 (m, CH2(39)); 1.73 (s, Me(25));
1.72 (s, Me(29)); 1.68 (s, Me(20)); 1.63 (s, Me(35)); 1.58 (s, Me(34)); 1.48 (s, Me(40)); 1.40–1.39 (m,
HꢀC(24)); 1.36–1.35 (m, HbꢀC(21)); 1.28 (s, Me(19)). ESI-MS: 782 ([MþH]þ ). HR-MS: 782.3434
([MþH]þ , C46H53ClNOþ8 ; calc. 782.3454).
Methyl (2Z)-4-[(1R,3aS,5S,11R,14aS)-3a,4,5,7-Tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methyl-
but-2-en-1-yl)-11-{(3E)-4-methyl-5-[(3-nitrophenyl)amino]pent-3-en-1-yl}-7,15-dioxo-3H,11H-1,5-meth-
anofuro[3,4-g]pyrano[3,2-b]xanthen-1-yl]-2-methylbut-2-enoate (11). IR (KBr): 3385, 2926, 2541, 2390,
2221, 2087, 1722, 1654, 1586, 1527, 1452, 1431, 1380, 1353, 1301, 1230, 1189, 1124, 788, 603. 1H-NMR
(300 MHz, CDCl3): 7.49–7.48 (m, 1 arom. H of ArNH); 7.43 (d, J¼6.9, HꢀC(10)); 7.36–7.34 (m, 1 arom.
H of ArNH); 7.25–7.24 (m, 1 arom. H of ArNH); 6.82–6.80 (m, 1 arom. H of ArNH); 6.66 (d, J¼10.2,

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
HꢀC(4)); 5.98–5.96 (m, HꢀC(27)); 5.52 (d, J¼10.2, HꢀC(3)); 5.38–5.36 (m, HꢀC(37)); 5.07–5.05 (m,
HꢀC(32)); 3.81 (s, COOMe); 3.65 (s, HꢀC(26)); 3.43 (s, MeO); 3.38–3.36 (m, HꢀC(11)); 3.25–3.24 (m,
HbꢀC(31)); 2.99–2.98 (m, HaꢀC(31)); 2.49 (d, J¼9.3, HꢀC(22)); 2.30–2.28 (m, HaꢀC(21)); 2.11–2.09
(m, CH2(36)); 1.81–1.79 (m, CH2(39)); 1.73 (s, Me(25)); 1.68 (s, Me(29)); 1.61 (s, Me(20)); 1.60 (s,
Me(35)); 1.58 (s, Me(34)); 1.47 (s, Me(40)); 1.42–1.39 (m, HꢀC(24)); 1.37–1.35 (m, HbꢀC(21)); 1.28 (s,
Me(19)). ESI-MS: 793 ([MþH]þ ). HR-MS: 793.3675 ([MþH]þ , C46H53N2O1þ0 ; calc. 793.3695).
Methyl (2Z)-4-{(1R,3aS,5S,11R,14aS)-3a,4,5,7-Tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methyl-
but-2-en-1-yl)-11-[(3E)-4-methyl-5-(thiazol-2-ylamino)pent-3-en-1-yl]-7,15-dioxo-3H,11H-1,5-methano-
furo[3,4-g]pyrano[3,2-b]xanthen-1-yl}-2-methylbut-2-enoate (12). IR (KBr): 3405, 3341, 3245, 2963,
2925, 2587, 2600, 2419, 2360, 2327, 2180, 1992, 1725, 1648, 1585, 1514, 1454, 1425, 1380, 1265, 1222, 1109,
1030, 754. 1H-NMR (300 MHz, CDCl3): 7.43 (d, J¼6.9, HꢀC(10)); 6.68 (d, J¼9.9, HꢀC(4)); 6.67–6.65
(m, 2 H of thiazole); 6.00–5.98 (m, HꢀC(27)); 5.52 (d, J¼10.2, HꢀC(3)); 5.08–5.06 (m, HꢀC(37));
4.80–4.78 (m, HꢀC(32)); 3.81 (s, COOMe); 3.43 (s, MeO); 3.41 (s, CH2O); 3.38–3.36 (m, HꢀC(11));
3.25–3.24 (m, HbꢀC(31)); 2.99–2.98 (m, HaꢀC(31)); 2.91 (s, CH2(26)); 2.55 (d, J¼9.3, HꢀC(22)); 2.29–
2.28 (m, HaꢀC(21)); 2.11–2.09 (m, CH2(36)); 1.83–1.82 (m, CH2(39)); 1.75 (s, Me(25)); 1.69 (s, Me(29));
1.64 (s, Me(20)); 1.63 (s, Me(35)); 1.57 (s, Me(34)); 1.46 (s, Me(40)); 1.42–1.39 (m, HꢀC(24)); 1.37–1.35
(m, HbꢀC(21)); 1.29 (s, Me(19)). ESI-MS: 755 ([MþH]þ ). HR-MS: 755.3337 ([MþH]þ , C43H51N2O8Sþ
;
calc. 755.3361).
General Procedure for the Preparation of Compounds 13–15. A soln. of 16 (20 mg, 0.029 mmol) in
appropriate alcohol (3 ml) was irradiated for 10 min in a CEM single-mode microwave cavity at 200 W
and 1008. The mixture was concentrated in vacuo, and the residue was purified by FC (SiO2) to yield 13–
15 (71–80%).
Methyl (2Z)-4-{(1R,3aS,5S,11R,14aS)-11-[(3E)-5-Ethoxy-4-methylpent-3-en-1-yl]-3a,4,5,7-tetrahy-
dro-8-methoxy-3,3,11-trimethyl-13-(3-methylbut-2-en-1-yl)-7,15-dioxo-3H,11H-1,5-methanofuro[3,4-
g]pyrano[3,2-b]xanthen-1-yl}-2-methylbut-2-enoate (13). IR (KBr): 3404, 2968, 2932, 2937, 2530, 2364,
2224, 2090, 1721, 1650, 1587, 1424, 1381, 1265, 1197, 1120, 1033, 753. 1H-NMR (300 MHz, CDCl3): 7.43 (d,
J¼6.9, HꢀC(10)); 6.68 (d, J¼10.2, HꢀC(4)); 5.97–5.94 (m, HꢀC(27)); 5.54 (d, J¼10.2, HꢀC(3)); 5.32–
5.31 (m, HꢀC(37)); 5.10–5.08 (m, HꢀC(32)); 3.82 (s, COOMe); 3.43 (s, MeO); 3.41 (s, MeCH2O); 3.40–
3.39 (m, CH2(39)); 3.36–3.35 (m, HꢀC(11)); 3.29–3.28 (m, HbꢀC(31)); 3.25–3.24 (m, HaꢀC(31)); 2.97
(s, CH2(26)); 2.51 (d, J¼9.6, HꢀC(22)); 2.30–2.28 (m, HaꢀC(21)); 2.12–2.10 (m, CH2(36)); 1.75 (s,
Me(25)); 1.69 (s, Me(29)); 1.64 (s, Me(20)); 1.63 (s, Me(35)); 1.57 (s, Me(34)); 1.46 (s, Me(40)); 1.42–1.39
(m, HꢀC(24)); 1.37–1.35 (m, HbꢀC(21)); 1.29 (s, Me(19)); 1.23–1.20 (m, MeCH2O). ESI-MS: 700 ([Mþ
H]þ ).
Methyl (2Z)-4-{(1R,3aS,5S,11R,14aS)-3a,4,5,7-Tetrahydro-8-methoxy-3,3,11-trimethyl-13-(3-methyl-
but-2-en-1-yl)-11-{(3E)-4-methyl-5-[(1-methylethyl)oxy]pent-3-en-1-yl}-7,15-dioxo-3H,11H-1,5-metha-
nofuro[3,4-g]pyrano[3,2-b]xanthen-1-yl}-2-methylbut-2-enoate (14). IR (KBr): 3381, 2974, 2944, 2391,
2289, 1722, 1629, 1592, 1436, 1381, 1315, 1267, 1204, 1108, 754. 1H-NMR (300 MHz, CDCl3): 7.46 (d, J¼
6.9, HꢀC(10)); 6.28–6.25 (m, HꢀC(4)); 6.08–6.06 (m, HꢀC(27)); 5.12–5.09 (m, HꢀC(3)); 4.90–4.88 (m,
HꢀC(37), HꢀC(32)); 3.82 (s, COOMe); 3.47–3.45 (m, CH2(39)); 3.44 (s, MeO); 3.41 (s, Me2CHO);
3.38–3.36 (m, HꢀC(11)); 3.29–3.27 (m, HbꢀC(31)); 3.25–3.23 (m, HaꢀC(31)); 2.97 (s, CH2(26)); 2.51 (d,
J¼9.6, HꢀC(22)); 2.30–2.29 (m, HaꢀC(21)); 2.12–2.09 (m, CH2(36)); 1.75 (s, Me(25)); 1.69 (s, Me(29));
1.64 (s, Me(20)); 1.63 (s, Me(35)); 1.57 (s, Me(34)); 1.46 (s, Me(40)); 1.42–1.39 (m, HꢀC(24)); 1.37–1.36
(m, HbꢀC(21)); 1.29 (s, Me(19)); 1.23–1.20 (m, Me2CHO). ESI-MS: 714 ([MþH]þ ).
Methyl (2Z)-4-{(1R,3aS,5S,11R,14aS)-11-[(3E)-5-tert-Butoxy-4-methylpent-3-en-1-yl]-3a,4,5,7-tet-
rahydro-8-methoxy-3,3,11-trimethyl-13-(3-methylbut-2-en-1-yl)-7,15-dioxo-3H,11H-1,5-methano-
furo[3,4-g]pyrano[3,2-b]xanthen-1-yl}-2-methylbut-2-enoate (15). IR (KBr): 3404, 2931, 2865, 2674,
2480, 2391, 2299, 2107, 1718, 1657, 1586, 1452, 1425, 1379, 1318, 1265, 1220, 1131, 753. 1H-NMR (300 MHz,
CDCl3): 7.47–7.43 (m, HꢀC(10)); 6.29–6.25 (m, HꢀC(4)); 6.08–6.06 (m, HꢀC(27)); 5.14–5.10 (m,
HꢀC(3)); 4.92–4.89 (m, HꢀC(32,37)); 3.82 (s, COOMe); 3.42 (s, MeO); 3.41–3.39 (m, CH2(39)); 3.38–
3.36 (m, HꢀC(11)); 3.29–3.28 (m, HbꢀC(31)); 3.25–3.24 (m, HaꢀC(31)); 2.97 (s, CH2(26)); 2.51 (d, J¼
9.6, HꢀC(22)); 2.31–2.28 (m, HaꢀC(21)); 2.13–2.11 (m, CH2(36)); 1.75 (s, Me(25)); 1.69 (s, Me(29));
1.64 (s, Me(20)); 1.63 (s, Me(35)); 1.57 (s, Me(34)); 1.46 (s, Me(40)); 1.42–1.39 (m, HꢀC(24)); 1.37–1.35
(m, HbꢀC(21)); 1.29 (s, Me(19)); 1.23–1.20 (m, Me3CO). ESI-MS: 728 ([MþH]þ ).

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1589
Biology. Compounds 1–16 and GAwere dissolved in DMSO (0.1 ml; Sigma) to a final concentration
of 10ꢀ2 m, and the soln. was stored at ꢀ208. The solns. of compounds 1–16 and GA were freshly diluted
with RPMI-1640 (GIBCO, USA) to the appropriate final concentrations at time of use.
Tumor Cells. A549, BGC-823, U251, HepG2, and MB-231 cell lines were purchased from Cell Bank
of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were grown
in RPMI-1640 medium supplemented with 10% heat-inactivated calf serum (Sijiqing, Hangzhou, P. R.
China), 100 U/ml penicillin G, and 100 U/ml streptomycin, pH 7.4, in a water-jacketed CO2 incubator
(Thermo Forma, USA) with a humidified atmosphere of 5% CO2 at 378.
Reagents. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Fluka, USA)
was dissolved in phosphate-buffered saline (PBS, 0.01m). Primary antibody of actin was obtained from
CalBiochem. Secondary antibodies were all obtained from Rockland Inc., USA. CycleTestTM Plus DNA
reagent kit was obtained from Becton-Dickinson, USA.
MTT Assay of Growth Inhibition [25]. Cells (5·103 per well in a 96-well plate) were treated with 1–
16 (0.01, 0.1, 1.0, 10, 100, and 1000 mm, resp.) and GA (0.01, 0.1, 1.0, 10, 100, and 1000 mm, resp.) for 48 h.
Then, 0.5% MTT was added into each well and incubated for 4 h at 378. The supernatant was discarded,
and 0.1 ml of DMSO was added to dissolve the precipitate. The mixture was shaken on a micro-vibrator
for 5 min, and the absorbance was measured at 570 nm with an Automated Microplate Reader ELx800
(Bio Tek). Inhibition ratio (IR%) was calculated by the following method: mean value of (A of control
groupꢀA of treated group)/A of control groupꢂ100%. Triplicate wells were analyzed at each dose. IC50
was calculated according to the Logit method.
The IC50 value was defined as the concentration that caused 50% inhibition of cell proliferation, and
was calculated by SAS statistical software.
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Received December 14, 2011