Alkaloids from Alangium javanicum and Alangium grisolleoides that mediate Cu2+-dependent DNA strand scission

Article abstract of DOI:10.1021/np058013j

Crude CH₂Cl₂-MeOH extracts prepared from Alangium javanicum and A. grisolleoides were found to induce DNA strand breakage in the presence of Cu²⁺ and were subjected to bioassay-guided fractionation to permit identification of the active principle(s). Javaniside (1), a novel alkaloid possessing an unusual monoterpenoid oxindole skeleton, was identified as an active principle contributing to the DNA cleavage activity observed for the crude extract of A. javanicum. Alangiside (2), a tetrahydroisoquinoline monoterpene glucoside widely distributed in the genus Alangium, was also isolated from A. grisolleoides as a new type of Cu²⁺-dependent DNA cleavage agent. The relative configuration of the asymmetric centers in javaniside was established by analysis of ¹H-¹H coupling constants and NOESY correlations. Semisynthesis of javaniside from secologanin (3) established the absolute stereochemistry of javaniside.

Full text of DOI:10.1021/np058013j

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J. Nat. Prod. 2005, 68, 1147-1152
1147
Alkaloids from Alangium javanicum and Alangium grisolleoides that Mediate
Cu2+-Dependent DNA Strand Scission
Van Cuong Pham, Ji Ma, Shannon J. Thomas, Zhidong Xu, and Sidney M. Hecht*
Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901
Received January 24, 2005
Crude CH2Cl2-MeOH extracts prepared from Alangium javanicum and A. grisolleoides were found to
induce DNA strand breakage in the presence of Cu2+ and were subjected to bioassay-guided fractionation
to permit identification of the active principle(s). Javaniside (1), a novel alkaloid possessing an unusual
monoterpenoid oxindole skeleton, was identified as an active principle contributing to the DNA cleavage
activity observed for the crude extract of A. javanicum. Alangiside (2), a tetrahydroisoquinoline
monoterpene glucoside widely distributed in the genus Alangium, was also isolated from A. grisolleoides
as a new type of Cu2+-dependent DNA cleavage agent. The relative configuration of the asymmetric centers
in javaniside was established by analysis of 1H-1H coupling constants and NOESY correlations.
Semisynthesis of javaniside from secologanin (3) established the absolute stereochemistry of javaniside.
The recognition that DNA serves as a target for small
molecules both in the initiation of cellular disorders and
also in the therapy of certain diseases has resulted in
increased interest in the interactions of such molecules
with DNA.1-10 One such type of interaction involves DNA
strand scission, which may occur through any of several
mechanisms, including (i) oxidation of the deoxyribose
sugar ring, (ii) alkylation or oxidation of the aromatic
nucleobase, or (iii) hydrolysis of the phosphodiester back-
bone.11
Since the discovery of the bleomycins as potent DNA
strand scission agents,12,13 considerable effort has been
made to identify other naturally occurring molecules that
are capable of mediating DNA strand scission, on the
assumption that these may facilitate the identification of
species that can also serve as useful clinical antitumor
drugs. Types of natural products identified as a conse-
quence of these efforts have included members of the
flavanoid,14 aurone,14f 5-alkylresorcinol,14b,15 pterocarpan,16
biphenyl,17 stilbene,18 anthrapyrone,19 enediyne,20 macro-
ring lactam,21 lignan,14g and phenolic amide22 classes of
compounds.
successively with H2O, 1:1 MeOH-H2O, 4:1 MeOH-CH2-
Cl2, 1:1 MeOH-CH2Cl2, and 9:1 MeOH-NH4OH. The first
two fractions, from the H2O and 1:1 MeOH-H2O washes,
exhibited significant Cu2+-dependent DNA cleavage activ-
ity. These two active fractions were combined and fraction-
ated further on a C18 column using a MeOH-H2O solvent
system for elution. The 7:3 MeOH-H2O fraction possessed
the most potent activity in the DNA strand scission assay
and was subjected to further HPLC fractionation. Com-
pound 1 was finally isolated as the active principle respon-
sible for the DNA cleavage activity of the crude extract.
Compound 1 was obtained as colorless amorphous pow-
der; the positive ion CIMS displayed a quasi molecular ion
at m/z 515 [M + H]+. The HR-FAB mass spectrum of 1
exhibited a base peak at m/z 515.2015 [M + H]+, indicating
a molecular formula of C26H30N2O9. In the 1H NMR
spectrum, compound 1 displayed signals corresponding to
a 1,2-disubstituted phenyl ring at δ 7.30 (br d, J ) 7.5 Hz),
7.25 (td, J ) 7.5 and 1.2 Hz), 7.07 (td, J ) 7.5 and 1.2 Hz),
and 6.90 (br d, J ) 7.5 Hz). Also present were signals
assigned to a terminal vinyl group at δ 5.49 (dt, J ) 16.8
and 10.0 Hz), 5.19 (dd, J ) 16.8 and 1.8 Hz), and 5.16 (dd,
J ) 10.0 and 1.8 Hz), an olefinic proton at δ 7.38 (d, J )
2.4 Hz), and a hemi-acetal proton at δ 5.42 (d, J ) 1.8 Hz).
The 13C and DEPT spectra revealed the presence of two
In a preliminary report, we previously described java-
niside (1) from A. javanicum as an agent capable of Cu2+
-
dependent DNA cleavage.23 Described herein is the detailed
bioassay-guided isolation and structural elucidation of 1
from A. javanicum, as well as the identification of structur-
ally related alangiside (2) as an alkaloid from A. grisolle-
oides capable of mediating DNA cleavage. Also described
is the semisynthesis of javaniside from secologanin, thereby
establishing the absolute stereochemistry of javaniside.
Results and Discussion
During a survey of crude plant extracts for their ability
to mediate DNA cleavage in an in vitro DNA strand
scission assay, crude extracts of A. javanicum and A.
grisolleoides exhibited considerable Cu2+-dependent DNA
strand scission activity. They were thus selected for bio-
assay-guided fractionation.
A crude 1:1 CH2Cl2-MeOH extract of A. javanicum was
first applied to a polyamide column; elution was effected
* To whom correspondence should be addressed. Department of Chem-
istry, University of Virginia, Charlottesville, VA 22901. Tel: (804) 924-3906.
Fax: (804) 924-7856. E-mail: sidhecht@virginia.edu.
10.1021/np058013j CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/08/2005

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1148 Journal of Natural Products, 2005, Vol. 68, No. 8
Pham et al.
Table 1. 1H and 13C NMR Spectral Data for Javaniside (1) in
effects. The coupling constants between H-16-H-17 (J )
1.8 Hz) and H-16-H-15 (J ) 5.5 Hz) strongly suggested
the â/â/R orientation of H-15, H-16, and H-17. The â
configuration of H-3 was supported by the coupling con-
stants between H-3 and H-14 protons (J3,14R ) 11.6 Hz,
J3,14â ) 3.0 Hz). The coupling constants indicated that 1
has the relative configuration illustrated in the structural
formula above.28 This configuration was further supported
by a number of NOE signals deduced from NOESY experi-
ments.23 Further evidence for the stereochemistry of 1 came
from a comparison of its 13C NMR data with that in the
literature.25b,28a,29 On the basis of these data, the structure
of 1 was identified as that of a novel monoterpenoid
oxoindole alkaloid and was named javaniside.
Several monoterpenoid indole alkaloids have been iso-
lated from terrestrial plants.25,26,30 Javaniside (1) repre-
sents a new type of nitrogenous natural product from the
genus Alangium. Further, the occurrence of this unusual
oxindole glucoside provides some insight into the likely
biosynthesis of the monoterpenoid indole alkaloid.
The bioassay-guided fractionation of the crude extract
of A. grisolleoides was performed in a fashion similar to
that of the crude extract of A. javanicum. Following the
removal of most polyphenols on a polyamide column,
successive fractionations of the extract on C18 and C8 open
columns, and then on reversed-phase HPLC columns,
resulted in the isolation of active principle 2. Compound 2
was also obtained as a colorless, amorphous powder; its
positive ion CIMS displayed a quasi molecular ion at m/z
506 [M + H]+. 1H and 13C NMR signals revealed patterns
typical of a tetrahydroisoquinoline monoterpene glycoside.
These NMR data, compared with data from the literature,
established the structure of 2 as that of alangiside, a known
alkaloid glucoside.29,31
To confirm the structure of javaniside and to determine
its absolute configuration, a semisynthesis of javaniside
and its isomer, 7-epi-javaniside, was performed (Scheme
1). The natural compound secologanin (3), whose absolute
stereochemistry is known,32 was first acetylated in 51%
yield by treatment with Ac2O in pyridine at room temper-
ature, following the reported procedure.33 Peracylated
compound 4 was then treated at reflux in methanol with
2-oxotryptamine (5),34 the latter of which had been pre-
pared from tryptamine by treatment with dimethyl sul-
foxide and hydrochloric acid, to provide isomeric 6 (51%)
and 7 (17%) in ∼3:1 ratio by a previously reported
method.35
CD3OD (500 and 125 MHz)
b
C
δH a (J, Hz)
δC
2
180.9
65.5
45.6
3
4.09 (dd, 11.6, 3.0)
5R
5â
6R
6â
7
8
9
4.04 (ddd, 11.4, 10.5, 7.4)
3.76 (br t, 11.4, 11.2)
2.23 (dd, 13.2, 7.4)
33.4
2.40 (ddd, 13.2, 11.2, 10.5)
58.0
129.5
123.9
123.8
130.0
110.9
143.6
27.0
7.30 (br d, 7.5)
7.07 (td, 7.5, 1.2)
7.25 (td, 7.5, 1.2)
6.90 (br d, 7.5)
10
11
12
13
14R
14â
15
16
17
19
20
21
22
23
23
1′
1.27 (td, 12.2, 12.4)
1.37 (dt, 12.2, 3.8, 3.8)
2.94 (dddd, 12.4, 5.5, 3.8, 2.4)
2.56 (ddd, 9.8, 5.5, 1.8)
5.42 (d, 1.8)
28.7
44.6
97.3
148.2
108.9
165.8
133.8
120.4
7.38 (d, 2.4)
5.49 (dt, 16.8, 10.0)
5.16 (dd, 10.0, 1.8)
5.19 (dd, 16.8, 1.8)
4.64 (d, 8.0)
3.14 (dd, 9.0, 8.0)
3.36 (m)
3.26 (m)
3.28 (m)
3.63 (dd, 12.0, 5.6)
3.83 (dd, 12.0, 2.0)
99.5
74.8
77.9
71.5
78.3
62.6
2′
3′
4′
5′
6′
6′
a Recorded at 500 MHz. b Recorded at 125 MHz.
carbonyl groups (δ 180.9 and 165.8), six aromatic carbons
(δ 143.6, 130.0, 129.5, 123.9, 123.8, and 110.9), two
aliphatic terminal vinyl carbons (δ 133.8 and 120.4), and
one trisubstituted double bond (δ 148.2 and 108.9). Ad-
ditionally, signals corresponding to a glucopyranose moiety
were also identified from 13C NMR experiments (Table 1).24
1
Using H-1H DQ-COSY and TOCSY experiments, the
assignment of four separated spin-spin coupling systems
was achieved as described previously,23 including (A) H-9
(δ 7.30)-H-10 (δ 7.07)-H-11 (δ 7.25)-H-12 (δ 6.90); (B)
H-3 (δ 4.09)-H-14 (δ 1.27, 1.37)-H-15 (δ 2.94)-H-16 (δ
2.56)-H-17 (δ 5.42)-H-22 (δ 5.49)-H-23 (δ 5.16, 5.19); (C)
H-5 (δ 4.04, 4.09)-H-6 (δ 2.23, 2.40); and (D) H-1′ (δ 4.64)-
H-2′ (δ 3.14)-H-3′ (δ 3.36)-H-4′ (δ 3.26)-H-5′ (δ 3.28)-
H-6′ (δ 3.63, 3.83). The assignment of carbon signals
corresponding to each proton signal was achieved by a
HMQC experiment (Table 1). The spectral features of
coupling systems B, C and the R,â-unsaturated carboxyl
group deduced from HMBC signals indicated structural
similarity of 1 to strictosamide,25 a known monoterpenoid
indole alkaloid. However, significant differences were noted
for the indole ring carbon signals. The typical chemical
shifts of C-2 and C-7 in monoterpenoid indole alkaloids are
135 and 108 ppm, respectively, whereas the C-2 and C-7
signals of 1 were at 180.9 and 58.0 ppm, respectively, which
strongly suggested the existence of an oxoindole ring in 1
rather than the indole ring in monoterpenoid indole
The stereochemistry of isomers 6 and 7 was determined
by vicinal 1H-1H coupling constants and confirmed by
analysis of NOESY experiments.35 This analysis estab-
lished the absolute configuration as 3R,7S for 6 and 3R,7R
for 7. Compounds 6 and 7 were then treated with sodium
methoxide in methanol to provide compounds 1 (68%) and
8 (66%), respectively.36 The stereochemistry of 1 and 8 was
confirmed by analysis of vicinal 1H-1H coupling constants.
The main differences between 1 and 8 in the NOESY
experiments were the correlations of H-9 to H-3 in 1 and
of H-9 to H-5b and H-14a in 8. By comparison of NMR data
and of optical rotations, synthetic compound 1 was shown
to be identical to naturally derived javaniside. The absolute
configuration of javaniside was thus determined as 3R,7S,
15S,16R,17S.
1
alkaloids, such as strictosamide.25,26 Long range H-13C
correlations between C-2-H-3, C-2-H-5, C-2-H-6, C-7-
H-5, C-7-H-14, C-8-H-3, and C-8-H-6 suggested C-7 as
a spiro-carbon to link the oxoindole ring and the pyrrole
ring, and this linkage was further confirmed by the
comparison of the 13C NMR data with that of similar
oxoindole alkaloids.27
Plants in the genus Alangium have been recognized to
be rich in different kinds of nitrogenous secondary me-
tabolites, including ipecac alkaloids represented by emetine
and cephaeline,37 tetrahydroisoquinoline monoterpene gly-
cosides, such as alangiside,29,31 and several other types of
The stereochemistry of 1 was determined through a
careful analysis of all proton coupling constants and NOE

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Alkaloids that Mediate DNA Strand Scission
Journal of Natural Products, 2005, Vol. 68, No. 8 1149
Scheme 1. Synthesis of Javaniside from Secologanin
Table 2. Cu2+-Dependent DNA Strand Scission by Compounds
1 and 2
Table 3. Cu2+-Dependent DNA Strand Scission by Synthetic
Javaniside (1) and 7-epi-Javaniside (8)a
compound
Cu2+ (20 µM)
Form II DNA (%)
compound
Form II DNA (%)
6
none
none
-
+
-
+
+
+
+
+
-
+
+
+
+
+
2
3
3
none
javaniside (natural)
200 µM
javaniside (1, 200 µM)
javaniside (1, 200 µM)
javaniside (1, 100 µM)
javaniside (1, 50 µM)
javaniside (1, 25 µM)
javaniside (1, 10 µM)
alangiside (2, 500 µM)
alangiside (2, 500 µM)
alangiside (2, 200 µM)
alangiside (2, 100 µM)
alangiside (2, 50 µM)
alangiside (2, 10 µM)
43
28
0
58
29
20
16
10
3
42
30
25
18
14
100 µM
10 µM
javaniside (synthetic)
200 µM
35
16
0
100 µM
10 µM
7-epi-javaniside (synthetic)
200 µM
100 µM
10 µM
7
5
0
a Carried out in the presence of equimolar (epi)javaniside and
Cu2+. The data were corrected for DNA cleavage observed in the
presence of Cu2+ alone.
alkaloids.38 However, the isolation of alangiside from A.
grisolleoides has not been reported previously.
The abilities of 1 and 2 to effect DNA strand scission
were evaluated using a supercoiled, covalently closed,
circular DNA as a substrate in the absence and presence
of Cu2+. In the presence of Cu2+, dose-dependent DNA
relaxation of the supercoiled pBR322 plasmid DNA was
observed for both 1 and 2 (Table 2). Consistent with the
behavior of the crude extracts from which they were
isolated, neither 1 nor 2 exhibited DNA strand scission
activity in the presence of Fe2+, nor in the absence of added
metal ion.
In the presence of 20 µM Cu2+, compounds 1 and 2
clearly mediated DNA strand scission in a concentration-
dependent fashion. About 58% conversion of Form I to
Form II DNA was apparent when 1 was employed at 200
µM concentration, whereas 42% Form II DNA was pro-
duced when 500 µM 2 was used. However, even when the
concentrations utilized were as low as 10 µM, both 1 and
2 still possessed detectable DNA cleavage activity (Table
2). This is the first time that oxindole and tetrahydroiso-
quinoline alkaloids have been identified as agents capable
of DNA strand scission. Also compared was the extent of
DNA cleavage mediated by synthetic versus naturally
derived javaniside. As shown in Table 3, when tested in
the presence of equimolar Cu2+, the cleavage by the
synthetic and naturally derived samples was similar,
although the natural sample gave slightly more Form II
DNA. In contrast, 7-epi-javaniside effected the relaxation
of the supercoiled DNA weakly, if at all. Thus, the cleavage
of DNA by javaniside seems likely to have resulted from a
specific interaction of javaniside with the DNA substrate.
Experimental Section
General Experimental Procedures. Polyamide 6S was
purchased from Serva Electrophoresis GmbH. Silica C18 (40
µm) was obtained from J. T. Baker Chemicals. A Higgins
Kromasil 100 C18 column (250 × 10 mm, 5 µm) was used for
reversed-phase HPLC. 1H and 13C NMR spectroscopic experi-

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1150 Journal of Natural Products, 2005, Vol. 68, No. 8
Pham et al.
ments were performed on Varian Unity Inova 300 and 500
spectrometers. Low-resolution chemical ionization mass spec-
tra were recorded on a Finnigan MAT 4600 mass spectrometer.
Optical rotations were determined on a JASCO P-1020 pola-
rimeter. Ethidium bromide, bromophenol blue, and Trizma
base were purchased from Sigma Chemicals. Boric acid was
obtained from EM Sciences. (Ethylenedinitrilo)tetraacetic acid
(EDTA) disodium salt was obtained from J. T. Baker. Cupric
chloride and glycerol were from Mallinckrodt, Inc.; ultrapure
agarose was from Bethesda Research Laboratories. The pBR322
plasmid DNA was purchased from New England Biolabs.
Pierce microdialysis cassettes were used to remove EDTA from
the pBR322 plasmid DNA.
Plant Material. Leaves of A. javanicum (Wang) were
collected in Sabah, Malaysia on October 17, 1987. Branches
of A. grisolleoides (Capuron) were collected in Madagascar on
June 19, 1996. Voucher specimens of both plants (Q66O5281
and Q66V4326, respectively) are preserved in the Botany
Department of the U.S. National Arboretum Herbarium,
Washington, D.C.
Extraction and Isolation. Bioassay-Guided Fraction-
ation of a Crude Extract of A. javanicum. Dried leaves of
A. javanicum were steeped in 1:1 methylene chloride-
methanol overnight at room temperature, then drained,
washed with methanol, and freed of solvent to obtain dry crude
extract. The crude extract of A. javanicum displayed significant
Cu2+-dependent DNA cleavage activity in a plasmid relaxation
assay. In a typical experiment, the crude extract (1.5 g) was
first applied to a polyamide 6S column, which was washed
successively with H2O, 1:1 MeOH-H2O, 4:1 MeOH-CH2Cl2,
1:1 MeOH-CH2Cl2, and 9:1 MeOH-NH4OH. With the removal
of most polyphenols, the H2O and MeOH-H2O fractions (233
mg and 105 mg, respectively) induced potent DNA strand
scission in an agarose gel assay at 100 and 50 µg/mL. These
two fractions were combined and fractionated further on a C18
column using MeOH-H2O for elution. The 7:3 MeOH-H2O
fraction (30 mg) showed the greatest potency in DNA strand
scission and was applied to a C18 reversed-phase HPLC column
(250 × 10 mm, 5 µm); elution was effected with a linear
gradient of CH3CN-H2O (1:19 f 2:3) over a period of 35 min
at a flow rate of 4.0 mL/min (UV monitoring at 254 nm). One
particularly active fraction (11.3 mg) was obtained from the
reversed-phase HPLC fractionation. Purification of this active
fraction, employing the same HPLC conditions, afforded active
compound 1 (8.0 mg).
(rel int.) 516 [M + 2H]+ (23), 515 [M + H]+ (100), 353 [M + H
+ H2O - glucose]+ (21); HRFABMS m/z 515.2015 [M + H]+
(calcd for C26H31N2O9, 515.2030).
Alangiside (2): colorless amorphous powder; [R]21D -104.6°
1
(c 1.00, MeOH), lit31 [R]26 -105° (c 1.0, MeOH); H and 13C
D
NMR data were identical with the literature data;29,31 positive
CIMS m/z (rel int.) 506 [M + H]+ (100), 344 [M + H + H2O -
glucose]+ (21).
Synthesis of Javaniside and 7-epi-Javaniside from
Secologanin. O,O,O,O-Tetraacetylsecologanin (4).33
A
solution containing 215 mg (0.55 mmol) of secologanin (3) in
2 mL of dry pyridine was treated with 2 mL of Ac2O. After
stirring at 25 °C for 24 h, ice was added to the reaction
mixture. The solution was extracted with five 10-mL portions
of CH2Cl2. The combined organic extract was dried over MgSO4
and concentrated under diminished pressure. The crude
product was purified by SiO2 chromatography (7:3 hexanes-
Et2O) to provide 4 as a colorless solid: yield 157 mg (51%);
1
[R]20 -79.9° (c 1.0, CHCl3); H NMR (CDCl3) δ 1.90 (s, 3H),
D
2.00 (s, 3H), 2.02 (s, 3H), 2.09 (s, 3H), 2.38 (ddd, 1H, J ) 17.9,
7.8, 1.4 Hz), 2.80 (ddd, 1H, J ) 9.6, 5.8, 3.0 Hz), 2.91 (ddd,
1H, J ) 17.9, 5.8, 1.4 Hz), 3.29 (m, 1H), 3.67 (s, 3H), 3.73 (ddd,
1H, J ) 10.6, 4.4, 2.1 Hz), 4.14 (dd, 1H, J ) 12.3, 2.1 Hz),
4.28 (dd, 1H, J ) 12.3, 4.4 Hz), 4.88 (d, 1H, J ) 8.1 Hz), 5.01-
5.25 (m, 5H), 5.27 (d, 1H, J ) 3.0 Hz), 5.49 (m, 1H), 7.41 (d,
1H, J ) 2.1 Hz), 9.70 (dd, 1H, J ) 1.4, 1.4 Hz); mass spectrum
(EIMS), m/z 557 (M + H)+.
2-Oxo-2,3-dihydrotryptamineHydrochloride(5).34Tryptamine
(0.5 g, 3.13 mmol) was dissolved in 0.29 g (3.72 mmol) of
DMSO, and 0.36 mL (3.72 mmol) of concentrated hydrochloric
acid was added slowly. After stirring at 25 °C for 3 h, the
precipitate was collected by filtration. The solid product was
precipitated from EtOH to give 5 as a colorless solid: yield
0.43 g (65%); mp 234-235 °C; (lit34 mp 235-239 °C); 1H NMR
(DMSO-d6) δ 2.08 (dt, 2H, J ) 15.2, 6.8 Hz), 2.92 (dt, 2H, J )
12.5, 6.8 Hz), 3.60 (dd, 1H, J ) 6.8, 6.8 Hz), 6.85 (d, 1H, J )
7.7 Hz), 6.96 (t, 1H, J ) 7.7 Hz), 7.18 (t, 1H, J ) 7.7 Hz), 7.23
(d, 1H, J ) 7.7 Hz), 8.22 (br s, 3H), 10.56 (s, 1H); 13C NMR
(DMSO-d6) δ 28.7, 36.9, 43.4, 110.2, 122.2, 124.7, 128.7, 129.5,
143.3, 179.1.
O,O,O,O-Tetraacetyljavaniside (6) and O,O,O,O-Tet-
raacetyl-7-epi-javaniside (7).35 To a solution of 100 mg (0.18
mmol) of 4 in 2 mL of dry methanol were added 76 mg (0.36
mmol) of 2-oxotryptamine hydrochloride and 49 µL (0.36 mmol)
of Et3N. The mixture reaction was heated at reflux for 1 h.
The solution then was concentrated under diminished pres-
sure, and the crude product was purified by SiO2 chromatog-
raphy (7:3 CHCl3-acetone) to give compounds 6 and 7 as
colorless solids: yield 62.5 mg (51%) for 6 and 20.8 mg (17%)
for 7.
Bioassay-Guided Fractionation of a Crude Extract of
A. grisolleoides. The crude extract of A. grisolleoides was
prepared from dried branches using the same extraction
protocol for the crude extract of A. javanicum. The crude
extract of A. grisolleoides also displayed fairly strong Cu2+
-
Compound 6 (3R,7S): [R]20D -18.2° (c 1.0, CHCl3); 1H NMR
(CDCl3) δ 1.34 (m, 2H), 2.01 (s, 3H), 2.02 (s, 3H), 2.03 (s, 3H),
2.08 (s, 3H), 2.31 (m, 2H), 2.52 (ddd, 1H, J ) 10.0, 5.5, 1.8
Hz), 2.68 (m, 1H), 3.74 (ddd, 1H, J ) 9.7, 4.6, 2.2 Hz), 3.82 (br
dd, 1H, J ) 11.6, 5.8 Hz), 3.97 (dd, 1H, J ) 10.8, 3.4 Hz), 4.11
(dd, 1H, J ) 12.4, 2.2 Hz), 4.12 (m, 1H), 4.28 (dd, 1H, J )
12.4, 4.6 Hz), 4.90 (d, 1H, J ) 8.1 Hz), 4.97 (dd, 1H, J ) 9.7,
8.1 Hz), 5.05 (dd, 1H, J ) 10.0, 1.4 Hz), 5.07 (dd, 1H, J ) 9.7,
9.7 Hz), 5.13 (dd, 1H, J ) 17.3, 1.4 Hz), 5.20 (d, 1H, J ) 1.8
Hz), 5.24 (dd, 1H, J ) 9.7, 9.7 Hz), 5.44 (m, 1H), 6.90 (d, 1H,
J ) 7.3 Hz), 7.10 (t, 1H, J ) 7.3 Hz), 7.23 (d, 1H, J ) 7.3 Hz),
7.28 (t, 1H, J ) 7.3 Hz), 7.42 (d, 1H, J ) 2.4 Hz), 7.86 (s, 1H);
mass spectrum (EIMS), m/z 683 [M + H]+.
dependent DNA cleavage activity. Typically, the crude extract
(1.25 g) was first applied to a polyamide 6S column, which
was washed successively with H2O, 1:1 MeOH-H2O, 4:1
MeOH-CH2Cl2, 1:1 MeOH-CH2Cl2, and 9:1 MeOH-NH4OH.
After the removal of most polyphenols, the H2O fraction (906
mg) still induced significant in vitro DNA strand scission in
the plasmid DNA relaxation assay at 100 and 50 µg/mL. The
active fraction was fractionated further on a C18 column using
a MeOH-H2O solvent system. The 2:3 and 3:2 MeOH-H2O
fractions (19 and 10 mg, respectively) showed the most
promising result in both the fractionation experiment and the
DNA strand scission assay. Further fractionation of the
combined mixture of the two fractions on a C8 column provided
one active fraction (12 mg) that eluted from the column in the
water wash. Subsequently, the H2O fraction was applied to a
reversed-phase C18 HPLC column (250 × 10 mm, 5 µm); elution
with a linear gradient of 1:99 f 2:3 CH3CN-H2O over a period
of 35 min at 3.0 mL/min (UV monitoring at 215 nm) gave good
separation of the active principle. Purification of the active
fraction (2.5 mg) from this step, employing the same HPLC
conditions, afforded active compound 2 (2.0 mg).
Compound 7 (3R,7R): [R]20 -51.6° (c 0.6, CHCl3); 1H
D
NMR (CDCl3) δ 0.85 (ddd, 1H, J ) 12.7, 12.7, 11.4 Hz), 1.38
(ddd, 1H, J ) 12.7, 3.7, 3.7 Hz), 1.99 (s, 3H), 2.02 (s, 3H), 2.03
(s, 3H), 2.08 (s, 3H), 2.49-2.60 (m, 2H), 2.77 (m, 1H), 3.73 (ddd,
1H, J ) 10.0, 4.5, 2.2 Hz), 3.89 (m, 1H), 4.00 (ddd, 1H, J )
12.7, 12.1, 7.8 Hz), 4.10 (dd, 1H, J ) 12.1, 3.5 Hz), 4.11 (dd,
1H, J ) 12.4, 3.7 Hz), 4.12 (dd, 1H, J ) 12.5, 2.2 Hz), 4.28
(dd, 1H, J ) 12.5, 4.5 Hz), 4.89 (d, 1H, J ) 8.1 Hz), 4.99 (dd,
1H, J ) 9.5, 8.0 Hz), 5.00 (dd, 1H, J ) 9.5, 2.0 Hz), 5.06 (dd,
1H, J ) 17.1, 2.0 Hz), 5.08 (dd, 1H, J ) 9.6, 9.6 Hz), 5.21 (d,
1H, J ) 1.5 Hz), 5.26 (ddd, 1H, J ) 17.1, 9.5, 9.5 Hz), 5.29
(dd, 1H, J ) 9.6, 9.5 Hz), 6.91 (d, 1H, J ) 7.5 Hz), 6.92 (d, 1H,
Javaniside (1): colorless amorphous powder; [R]21D -50.2°
1
(c 0.2, MeOH); H NMR (CD3OD, 500 MHz), see Table 1; 13C
NMR (CD3OD, 125 MHz), see Table 1; positive ion CIMS m/z