Tyrosyl-DNA Phosphodiesterase i Inhibitors from the Australian Plant Macropteranthes leichhardtii

Article abstract of DOI:10.1021/acs.jnatprod.5b00211

Mass-directed isolation of the CH₂Cl₂/MeOH extract from the bark of an Australian plant, Macropteranthes leichhardtii, resulted in the purification of a new phenylpropanoid glucoside, macropteranthol (1), together with four known analogues (2-5). The structure of compound 1 was elucidated by NMR and MS data analyses and quantum chemical calculations. Compounds 3 and 5 showed inhibitory activity against tyrosyl-DNA phosphodiesterase I with IC₅₀ values of ～1.0 μM.

Full text of DOI:10.1021/acs.jnatprod.5b00211

Note
pubs.acs.org/jnp
Tyrosyl-DNA Phosphodiesterase I Inhibitors from the Australian
Plant Macropteranthes leichhardtii
†
,⊥
†
†
‡
‡
§
Li-Wen Tian, Yunjiang Feng, Trong D. Tran, Yoko Shimizu, Tom Pfeifer, Paul I. Forster,
,
†
and Ronald J. Quinn*
†Eskitis Institute for Drug Discovery, Griffith University, Brisbane, QLD 4111, Australia
‡Centre for Drug Research and Development, Vancouver, BC 2405, Canada
§Queensland Herbarium, Toowong, QLD 4066, Australia
*
S Supporting Information
ABSTRACT: Mass-directed isolation of the CH Cl /MeOH extract from the bark of an Australian plant, Macropteranthes
2
2
leichhardtii, resulted in the purification of a new phenylpropanoid glucoside, macropteranthol (1), together with four known
analogues (2−5). The structure of compound 1 was elucidated by NMR and MS data analyses and quantum chemical
calculations. Compounds 3 and 5 showed inhibitory activity against tyrosyl-DNA phosphodiesterase I with IC5 values of ∼1.0
0
μM.
yrosyl-DNA phosphodiesterase I (Tdp1), an enzyme
involved in the repair of DNA lesions, catalyzes hydrolysis
inhibitors. The library comprises over 200 000 fractions and was
constructed by fractionation of over 18 000 terrestrial and marine
biota samples. One fraction from the bark of the Australian plant
Macropteranthes leichhardtii F. Muell. ex Benth. showed activity
against Tdp1. LC-MS analysis of the active fraction showed four
molecular ions in the (+)-LRESIMS, at m/z 511, 513, 469, and
1
T
of the topoisomerase I (Top1) tyrosine residue covalently bound
2
to the 3′-phosphate moiety of DNA. Recent studies have shown
that a mutation of the human Tdp1 gene is responsible for the
3
neurological disorder spinocerebellar ataxia. Further studies
have shown that Tdp1 knockout mice are hypersensitive to
4
99, which were used to guide isolation. Ten grams of air-dried
4
−6
camptothecin (CPT), a Top1 inhibitor,
while cells over-
and ground bark was extracted by CH Cl and MeOH. Mass-
2
2
expressing Tdp1 showed resistance to CPT- and etoposide-
directed fractionation and purification of the combined CH Cl /
7
2
2
induced DNA damage. This evidence suggested that Tdp1
MeOH extracts resulted in the isolation of a new phenyl-
inhibitors could act synergistically with Top1 inhibitors in cancer
combinational therapy.
propanoid glucoside, macropteranthol (1), together with four
11
known analogues, namely, mallophenol A (2), 3,4-dimethox-
Early studies on Tdp1 led to the discovery of Tdp1 inhibitors
with millimolar activity, including aminoglycoside antibiotics and
12
yphenol-1-β-D-(6′-O-galloyl)glucopyranoside (3), 3,4,5-trime-
13
8
,9
thoxyphenol-1-β-D-(6′-O-galloyl)glucopyranoside (4), and 3-
4-hydroxy-3-methoxyphenyl)propane-1,2-diol 2-β-D-(6-O-
the ribosome inhibitors. Several new molecules including 5-
(
arylidenethioxothiazolidinones were later identified through
14
10
galloyl)glucopyranoside (5) (Figure 1). In this paper, we
high-throughput screening with improved activity. In our
continuing research on bioactive lead compounds from natural
products, a high-throughput screening assay was established and
used to screen a prefractionated natural product library for Tdp1
Received: March 8, 2015
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00211
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
report the isolation and structure elucidation of macropteranthol
(1), as well as the Tdp1 inhibitory activity of compounds 1−5.
Figure 2. gCOSY and gHMBC correlations of 1.
5
89 nm) and subsequently the Boltzmann-weighted average
based on the energy of each conformer resulted in the calculated
OR. Here two different levels, Hartree−Fock (HF) and density
functional theory (DFT), were performed to calculate the ORs.
The OR values of the (7S,8S,1′S,2′R,3′S,4′S,5′R)-stereoisomer
and its enantiomer were predicted to be +22.17 and −22.17,
respectively, at the HF level, or +14.21 and −14.21, at the DFT
level, respectively. The positive signs of the calculated ORs of the
(7S,8S,1′S,2′R,3′S,4′S,5′R)-isomer obtained via two calculation
levels were in agreement with the experimental OR value ([α]D
+2.75). Therefore, the absolute configuration of (+)-1 was
determined as (7S, 8S, 1′S, 2′R, 3′S, 4′S, 5′R).
Figure 1. Structures of compounds 1−5 isolated from M. leichhardtii.
The air-dried and ground bark of M. leichhardtii was
sequentially extracted with n-hexane, CH Cl , and MeOH. The
2
2
CH Cl and MeOH extracts were combined and chromato-
2
2
graphed using reversed-phase C -bond silica HPLC (MeOH/
1
8
H O/0.1% TFA) to give 60 fractions. Fractions 22−26 contained
2
the ions of interest [LRESIMS (+) at m/z 513, 511, 499, and 469;
LRESIMS (−) at m/z 511, 509, 497, and 467]. Further
purification of fractions 22−26 by C -bonded silica HPLC
Four known natural products, mallophenol A (2), 3,4-
dimethoxyphenol-1-β-D-(6′-O-galloyl)glucopyranoside (3),
3,4,5-trimethoxyphenol-1-β-D-(6′-O-galloyl)glucopyranoside
(4), and 3-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol 2-β-
1
8
(
MeOH/H O/0.1% TFA) afforded compounds 1−5.
2
1
Macropteranthol (1) was obtained as a pale, amorphous
D-(6-O-galloyl)glucopyranoside (5), were also isolated. Their H
and 13C NMR data were identical to reported data. Previously,
powder. HRESIMS exhibited a pseudomolecular ion peak at m/z
5
33.1266 [M + Na]+ (calcd 533.1255 for C H O Na),
mallophenol A (2) was reported only from Mallotus furetianus,
11
2
3
26 13
consistent with a molecular formula of C H O , the same as
while 3-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol 2-β-D-
(6-O-galloyl)glucopyranoside (5) was reported from the berries
of Pimentadioica, but the C-8 absolute configuration was not
2
3
26 13
that of mallophenol (2). Detailed analysis and comparison of the
1
H, gHSQC, and gCOSY NMR spectra of 1 with those of 2
14
revealed that 1 also has a galloyl group (δ 6.96; δ 109.0), a
assigned. In this study, the absolute configuration of C-8 was
assigned via acid hydrolysis and subsequent assessment of the
specific rotation of 3-(4-hydroxy-3-methoxyphenyl)propane-1,2-
H
C
1
,3,4-trisubstituted benzene moiety (δ 6.90 d, J = 1.7 Hz, 6.79
H
dd, J = 8.1, 1.7 Hz, and 6.74 d, J = 8.1 Hz), a β-glucopyranosyl
moiety with the anomeric proton at δ 4.54 (d, J = 7.7 Hz), and
diol ([α] −6.2, c 0.005, MeOH). The absolute configuration of
H
D
the tetrasubstituted 1,4-dioxane moiety (δ 4.46, 4.10; δ 78.7,
C-8 in 5 was defined as S by [α] comparison with 3-(4-hydroxy-
H
C
D
7
7.7). The obvious differences are a shielded methylene, H -6′, in
3-methoxyphenyl)propane-1,2-diol {8(S)-isomer, [α] −23, c
2
D
a glucopyranosyl moiety and a corresponding deshielded
methylene, H -9, in 1. Further gHMBC NMR data analysis
0.69, EtOH; 8(R)-isomer, [α] +18, c 0.73, EtOH} literature
D
21
values.
2
suggested that, instead of a C-6′ galloyl esterification of the
glucopyransyl moiety in 2, the galloyl group of 1 was connected
with C-9 through an ester bond by observation of the gHMBC
correlations from H-2″/H-6″ (δ 6.96) and methylene H -9 (δ
Tdp1 inhibitory activities of compounds 1−5 were evaluated
22
in vitro (Figure 3), using an assay previously described. The
assay was performed with the substrate at its K for the enzyme
m
used, and the reaction monitored using a kinetic read.
Compounds 3 and 5 were the most active, with IC50 values of
∼1.0 μM. Compound 1, its regiomer 2, and compound 4 showed
no inhibitory activity against Tdp1 at concentrations up to 40
μM. Preliminary structure−activity relationship studies sug-
gested that the relative orientations of the aromatic system on the
sugar moiety in compounds 1−5 may play a part in their
interaction with Tdp1 and, therefore, their biological activity.
Molecular modeling is currently under way to investigate the
binding of compounds 1−5 to Tdp1.
In conclusion, five compounds (1−5) were isolated from the
bark of M. leichhardtii, including the new compound 1 and
compounds 3 and 5, being active against Tdp1 (IC50 1.0 μM).
The 8(S)-absolute configuration of 5 was defined for the first
time. The current study is the first report of the chemical
constituents of a Macropteranthes species. The results provide
H
2
H
4
.05, 3.86) to the carbonyl carbon (δ 165.9). The substitution
C
position of the methoxy group and the presence of a 1,4-dioxane
ring system were similar to those of 2 and were supported by
gHMBC correlations (Figure 2). On the basis of the above
evidence, the structure of macropteranthol was established as 1.
The applications of quantum chemical calculations to the
prediction of the optical rotations (ORs) of chiral organic
molecules have greatly facilitated the reliable determination of
15,16
their absolute configurations.
The methods have been
utilized successfully to assign the absolute configurations of
1
7−20
natural products.
A conformational search for the NMR-
established relative configuration (7S,8S,1′S,2′R,3′S,4′S,5′R)-
isomer led to the identification of 14 conformers within a relative
energy window of 1 kcal/mol. Geometry optimization followed
by OR calculations at D-sodium line radiation (wavelength of
B
DOI: 10.1021/acs.jnatprod.5b00211
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Figure 3. In vitro Tdp1 inhibitory activity curves of compounds 1−5.
more information about the structure−activity relationship of
this class of compounds.
NMR spectra were recorded at 30 °C on a Varian 600 MHz Unity
INOVA spectrometer equipped with a triple-resonance cryoprobe. The
1
13
H and C NMR chemical shifts were referenced to the solvent peak for
methanol-d at δ 3.31 and δ 49.5 or DMSO-d at δ 2.50 and δ 39.5.
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured with a HORIBA SEPA-300 high-sensitive polarimeter. UV
spectra were recorded on a Jasco V650 UV/vis spectrophotometer.
4
H
C
6
H
C
■
LRESIMS data were recorded on a Waters ZQ mass spectrometer.
HRESIMS data were recorded on a Bruker Daltronics Apex III 4.7e
Fourier-transform mass spectrometer. AlltechDavisil 40−60 μmÅ C -
1
8
bonded silica was used for flash chromatography. A Waters 600 pump
equipped with a Waters 996 PDA detector and a Waters 717
autosampler was used for HPLC. A Thermo Scientific C18 Betasil 5
1
13
Table 1. H (600 MHz) and C (150 MHz) NMR Data for
Compound 1 (DMSO-d6)
μm 143 Å column (21.2 mm × 150 mm) and a Phenomenex Luna C 5
μm 143 Å column (21.2 mm × 250 mm) were used for semipreparative
HPLC separations. All solvents used for chromatography, UV, and MS
18
13C
1H (mult., J in Hz, int.)
position
HMBC correlation
4, 6, 7
were Lab-Scan HPLC grade, and the H O was Millipore Milli-Q PF
2
1
2
3
4
5
6
7
8
9
128.2
112.3
147.8
147.4
115.5
120.9
78.7
filtered. A BIOLINE orbital shaker was used for the large-scale
extraction of the sponge material.
6.90 d (1.7)
Plant Materials. Vegetative material of Macropteranthes leichhardtii
F. Muell. ex Benth. (family: Combretaceae; genus: Macropteranthes) was
collected at Palmgrove National Park (25°01 S, 149°15 E), south-central
Queensland, Australia. It was air-dried prior to freeze-drying and
extraction. A voucher sample (Forster PIF26052 (BRI AQ493829)) has
been lodged at the Queensland Herbarium, Toowong, Australia.
Extraction and Isolation. The air-dried and ground bark (10 g) was
poured into a conical flask (1 L), n-hexane (250 mL) was added, and the
flask was shaken at 200 rpm for 2 h. The n-hexane extract was filtered
under gravity, and the liquid phase discarded. CH Cl (250 mL) was
6.74 d (8.1)
1, 3
6.79 dd (1.7, 8.1)
4.46 d (9.4)
2, 4, 7
1, 2, 6, 8, 2′
7
77.7
4.10 m
63.6
4.05 dd (1.7, 12.8)
7, 7″
3
.86 dd (5.8, 12.8)
1′
2′
3′
4′
5′
6′
98.2
80.0
73.5
71.0
76.0
61.3
4.54 d (7.7)
3.03 dd (7.7, 8.9)
3.38 m
2′
1′, 3′
2
2
added to the plant material and shaken at 200 rpm for 2 h. The resulting
extract was filtered under gravity. MeOH (250 mL) was added, and the
MeOH/plant mixture was shaken for a further 2 h at 200 rpm. Following
gravity filtration the plant material was extracted with another volume of
MeOH (250 mL), while being shaken at 200 rpm for 16 h. All CH Cl /
3.28 dd (9.0, 9.0)
3.38 m
3.69 br d (12.0)
5′
2
2
3
.48 dd (5.9, 12.0)
MeOH extracts were combined and dried under reduced pressure to
yield a dark brown solid (1.35 g). The crude extract was preadsorbed to
1
2
3
4
7
3
″
119.4
109.0
146.0
139.1
165.9
55.9
C -bonded silica (1 g) and packed into a stainless steel cartridge (10 ×
″, 6″
″, 5″
″
6.96 s
3.71 s
1″, 2″, 3″, 4″, 5″, 7″
18
3
0 mm) that was subsequently attached to a C18 preparative HPLC
column. Isocratic HPLC conditions of 90% H O (0.1% TFA)/10%
2
MeOH (0.1% TFA) were initially employed for the first 10 min; then a
linear gradient to 100% MeOH (0.1% TFA) was run over 40 min,
followed by isocratic conditions of 100% MeOH (0.1% TFA) for a
″
-OCH3
3
C
DOI: 10.1021/acs.jnatprod.5b00211
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
further 10 min, all at a flow rate of 9.0 mL/min. Sixty fractions (60 × 1
min) were collected every minute from the start of the HPLC run, and
fractions 20−30 were analyzed by LC-MS. Fractions 22 (+ESIMS m/z
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00211.
5
11), 23 (+ESIMS m/z 511), 24 (+ESIMS m/z 513), 25 (+ESIMS m/z
13, 469), and 26 (+ESIMS m/z 499) showed molecular ions of
AUTHOR INFORMATION
5
■
interests. Fractions 22−26 were further purified using a C semi-
Corresponding Author
18
preparative HPLC column eluting with isocratic conditions from H O/
2
*E-mail: r.quinn@griffith.edu.au. Tel: +61-7-37356000. Fax:
MeOH (8:2) (0.1% TFA) to MeOH (0.1% TFA) within 40 min to
afford 1 (0.4 mg, 0.004% of dry wt), 2 (0.8 mg, 0.008% of dry wt), 3 (0.3
mg, 0.003% of dry wt), 4 (0.2 mg, 0.002% of dry wt), and 5 (0.3 mg,
+
61-7-37356001.
Present Address
⊥
0
.003% of dry wt).
Macropteranthol (1): pale, amorphous powder; [α] +2.75 (c 0.04,
School of Pharmaceutical Sciences, Southern Medical Uni-
D
versity, Guangzhou 510515, People’s Republic of China.
MeOH); λmax (log ε) 278 (3.76), 219 (4.12) nm; IR (KBr) ν 3242,
max
−
1
+
Notes
2
3
5
926, 2887, 1770, 1562, 1394 cm ; (+)-LRESIMS m/z 511 [M + H] ,
49 [M + H − 162] , 179 [M + H − 162 − 170] ; HRESIMS m/z
+
+
The authors declare no competing financial interest.
+
33.1266 [M + Na] (calcd for C H O Na, 533.1255).
2
3
26 13
Computational Details. A conformational search used the MMFFs
force field on Macromodel interfaced to the Maestro program.
ACKNOWLEDGMENTS
2
3
■
The authors thank H. Vu from Griffith University for acquiring
the HRESIMS data. This work was supported by the Queensland
Government Smart Futures NIRAP Program, the Australian
Research Council for NMR and MS equipment (LE0668477 and
LE0237908), and the Center for Drug Research and Develop-
ment.
Fourteen conformers having internal relative energies within 1 kcal/mol
were subjected to geometry optimization in MeOH at the HF level using
the 6-31G(d,p) basis set or at the DFT level using the B3LYP functional
and the 6-31G(d,p) basis set. Optimized conformers were subjected to
OR calculations in MeOH (SCRF) using the 6-31G(d) basis set for HF
and the B3LYP functional and the 6-31G(d) basis set for DFT in
24
Gaussian 09. Final calculated ORs were obtained as the result of the
Boltzmann-weighted average.
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ASSOCIATED CONTENT
■
(
́
*
S
Supporting Information
H, gCOSY, gHSQC, and gHMBC NMR spectra of 1,
1
1
571−1577.
computational details, and acid hydrolysis for 5 are included.
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DOI: 10.1021/acs.jnatprod.5b00211
J. Nat. Prod. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jnatprod.5b00211
J. Nat. Prod. XXXX, XXX, XXX−XXX