Synthesis and biological activities of topopyrones

Article abstract of DOI:10.1021/np200777z

Structure-activity studies were employed to investigate the stabilization of DNA-topoisomerases I and II covalent binary complexes by topopyrone analogues. The synthesis of five new topopyrone derivatives and study of their ability to stabilize DNA-topoisomerase I and DNA-topoisomerase II covalent binary complexes are described. The biochemical assays suggest that the orientation of the fused 1,4-pyrone ring and halogen substituents contribute importantly to the overall potency of the topopyrones as topoisomerase poisons.

Full text of DOI:10.1021/np200777z

Article
pubs.acs.org/jnp
Synthesis and Biological Activities of Topopyrones
Paul A. Zaleski,† Rumit Maini,†,‡ Simon J. Leiris,† Mark A. Elban,§ and Sidney M. Hecht*,†,‡
‡
†Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe,
Arizona 85287-6301, United States
§Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904-4319, United States
S
* Supporting Information
ABSTRACT: Structure−activity studies were employed to investigate
the stabilization of DNA−topoisomerases I and II covalent binary
complexes by topopyrone analogues. The synthesis of five new
topopyrone derivatives and study of their ability to stabilize DNA−
topoisomerase I and DNA−topoisomerase II covalent binary complexes
are described. The biochemical assays suggest that the orientation of the
fused 1,4-pyrone ring and halogen substituents contribute importantly to
the overall potency of the topopyrones as topoisomerase poisons.
he DNA topoisomerases are a family of nuclear enzymes
that control the topological state of DNA and are essential
activity was found to be dependent on the orientation of the
1,4-pyrone ring moiety as well as the presence and orientation
of halogen substituents.
T
to biological processes such as replication, transcription, and
chromosome segregation.1 These enzymes catalyze the
relaxation of supercoiled DNA through a breakage−rejoining
of the DNA phosphodiester bond.2,3 DNA relaxation proceeds
through the transient formation of a covalent DNA−
topoisomerase complex between the active site tyrosine of
the enzyme and the DNA phosphodiester bond of the DNA.4
DNA topoisomerases are classified by the number of
polynucleotide strand breaks generated, with type I top-
oisomerases creating single-strand DNA breaks and type II
topoisomerases creating double-strand DNA breaks.3 The
covalent binary complexes formed between DNA and
eukaryotic topoisomerases I and II are the targets of a class
of antitumor agents referred to as topoisomerase poisons.
These compounds stabilize the otherwise transient DNA−
topoisomerase covalent binary complex and are believed to
produce DNA strand breaks when advancing DNA replication
or transcription assemblies collide with the drug-stabilized
DNA−topoisomerase covalent binary complex.5 Camptothecin
(CPT) and etoposide are well-characterized topoisomerase I
and topoisomerase II poisons, respectively.6,7
Other topoisomerase I poisons that have been described
include the indenoisoquinolines,8 calothrixins,9 and topopyr-
ones.10−12 The topopyrones are natural products that were first
isolated by Kanazawa and co-workers from the culture broths of
the fungi Phoma sp. BAUA2861 and Penicillium sp.
BAUA4206.10 Structurally, the topopyrones are anthraquinone
polyphenols that contain a fused 1,4-pyrone ring in an angular
or linear orientation and that may also contain a chlorine atom,
as in topopyrones A (1) and B (2).11 In addition to being
topoisomerase I poisons, the topopyrones target and stabilize
DNA−topoisomerase II covalent binary complexes.12 Presently,
the preparation of five new topopyrone derivatives (Figure 1) is
described, as are initial assay data characterizing the compounds
as topoisomerase I and II poisons. Topoisomerase inhibitory
RESULTS AND DISCUSSION
■
The synthesis of topopyrones A, B, C, and D was reported by
Elban et al. in 2008 along with the synthesis of two novel
topopyrone derivatives (9 and 10).13 In the present study, a
similar synthetic approach was adopted to gain access to two
new topopyrone C derivatives (5 and 6), two new topopyrone
A derivatives (7 and 8), and one new topopyrone B derivative
(11). Topopyrones 9 and 10 were prepared in analogy with the
procedure reported previously.13 Highlights of the synthetic
route reported here include the Diels−Alder cycloaddition
reaction (Scheme 1), Friedel−Crafts acylation reaction at the
ortho position of a key phenolic intermediate (Scheme 1),
pyrone ring formation using a strong base (Schemes 1 and 2),
and regioselective monobromination (Scheme 2).
Diels−Alder addition of the previously reported 1,3-
dimethoxy-1-trimethylsiloxy-1,3-butadiene13,14 and commer-
cially available 2,5-dichloro-1,4-benzoquinone in the presence
of pyridine gave quinone 12 (Scheme 1). Protection of the
phenol using p-toluenesulfonyl chloride (p-TsCl) and K2CO3
in acetone at reflux afforded tosylate 13 in 90% yield. Pd/C-
catalyzed hydrogenation effected reduction of the quinone,
which was immediately methylated using dimethyl sulfate and
KOH to give 14 in 95% overall yield. Compound 15 was
obtained by removal of the tosylate group from 14 (KOH(aq)
at reflux). Although this reaction appeared to proceed cleanly,
as judged by silica gel TLC analysis, the yield of isolated
product was only moderate (56%). An acetyl group was
introduced ortho to the free hydroxyl group using TiCl4 and
acetyl chloride in 1,2-dichloroethane at reflux, affording
Received: September 24, 2011
Published: March 30, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Structures of topopyrones, etoposide, and camptothecin.
compound 16 in 96% yield.15,16 The pyrone ring was
introduced at this stage via base-catalyzed Claisen condensa-
tion; compound 16 was treated with NaH (60% dispersion in
mineral oil) in freshly distilled ethyl acetate at reflux to afford
the β-diketone. This intermediate was subsequently treated
with TFA to obtain 17 (56%, two steps). Finally, compound 17
was oxidized to quinone 18 (72% yield) using cerium(IV)
ammonium nitrate in CH3CN. A second Diels−Alder addition
reaction was used to obtain the precursors for topopyrones 5
and 6. Thus quinone 18 was treated with 1-methoxy-1-
trimethylsiloxy-3-tert-butyldimethylsiloxy-1,3-butadiene (19)13
in the presence of pyridine to facilitate the aromatization of
the intermediate Diels−Alder addition product. Compounds 20
and 21 were thereby obtained in 40% and 25% yields,
respectively. Finally, compound 20 was treated with tetra-n-
butylammonium fluoride hydrate (TBAF·H2O) to remove the
tetrabutyldimethylsilyl protecting group, followed by demethy-
lation using 1 M BBr3 in CH2Cl2. This afforded topopyrone C
derivative 5 in 23% overall yield. Similarly, topopyrone C
derivative 6 was obtained in 50% yield by the treatment of 21
with TBAF·H2O in THF.
Monobrominated intermediate 23 was obtained in 20%
overall yield by treating 2213 with N-bromosuccinimide
(Scheme 2)17,18 in the presence of (iPr)2NH at −78 °C in
freshly distilled CH2Cl2, followed by methylation of the
phenolic hydroxyl groups with dimethyl sulfate and K2CO3 in
acetone. Removal of the tosylate group, followed by O-
acetylation of the intermediate phenol afforded 24 (52%, two
steps). This was achieved using KOH in EtOH−H2O at reflux
followed by treatment with acetic anhydride in the presence of
triethylamine and 4-N,N-dimethylaminopyridine.13 Treatment
of acetylated intermediate 24 with LiH in freshly distilled THF
at reflux, followed by treatment with CF3COOH at 0 °C
(Baker−Venkataraman rearrangement)19,20 led to pyrone ring
formation, affording topopyrone 7 in 45% yield for the two
steps. Removal of the methyl groups using 1 M AlCl3 in
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Scheme 1. Synthesis of Topopyrones 5 and 6
nitrobenzene at 90 °C gave brominated topopyrone A
derivative 8 in 50% yield.13 Brominated topopyrone B
derivative 11 was obtained by rearrangement of the pyrone
ring in 8 using 1% NaOH in MeOH (50% yield).13
considerably in their efficiencies of stabilization of the DNA−
topoisomerase I covalent binary complex (Figure S2 and Table
1). Topopyrone 8 produced 25% DNA cleavage at 10 μM
concentration. In sharp contrast, topopyrone 7, a close
structural analogue of 8 containing methylated phenols, failed
to stabilize the DNA−topoisomerase I covalent binary complex
at 1 or 10 μM concentration. X-ray crystallographic analysis of
the binding of other topoisomerase I poisons to the
topoisomerase I−DNA binary complex indicates H-bonding
between the poisons and the covalent binary complex,22−24 and
it seems likely that H-bonding between one or more phenolic
OH groups in 8 contributes to its ability to stabilize the formed
ternary complex.
The amounts of the DNA−topoisomerase I covalent binary
complexes stabilized by topopyrones 3, 9, and 10 were
quantified using a nitrocellulose filter binding assay (Table
2).6 The amounts of the DNA−topoisomerase I covalent
binary complexes stabilized in the presence of CPT or the
topopyrones were measured by determination of the radio-
Biological Evaluation. The ability of novel topopyrones 5
and 6 to stabilize the DNA−topoisomerase I covalent binary
complex was investigated using a 23-base pair oligonucleotide
substrate that contained a strong topoisomerase I cleavage site
having guanosine at the +1 position and thymidine at the −1
position (Figure S1).21 Polyacrylamide gel electrophoretic
analysis indicated that topopyrone C derivatives 5 and 6 both
stabilized the DNA−topoisomerase I covalent binary complex
with similar efficiencies. Densitometric analysis was used to
determine the percentage of DNA cleavage relative to that
obtained with 1 μM CPT, which was arbitrarily assigned a value
of 100% (Table 1). At 1 and 10 μM concentrations of
topopyrone 5, 6% and 11% DNA cleavage were observed, an
amount similar to that obtained with topopyrone 6. In contrast,
brominated topopyrone A derivatives 7 and 8 varied
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Scheme 2. Synthesis of Topopyrones 7, 8, and 11
Table 1. Topoisomerase I-Mediated DNA Cleavage by CPT
and Topopyrones 5−8 Using a 3′-32P End Labeled 23-Base
Pair Oligonucleotide Substrate
Table 2. Nitrocellulose Filter Binding of CPT- or
Topopyrone-Stabilized DNA−Topoisomerase Covalent
Binary Complexes
a
a
topoisomerase I poison
% DNA cleavage
topoisomerase poison
DNA covalently bound to topoisomerase (%)
DNA alone
1
0
topoisomerase I
topoisomerase I alone
1 μM CPT
none
1.4 0.5
39 4.6
16 4.3
21 7.8
6.1 2.4
5.1 4.4
11 3.6
30 5.2
100
6
0.5 μM CPT
1 μM topopyrone 5
10 μM topopyrone 5
1 μM topopyrone 6
10 μM topopyrone 6
1 μM topopyrone 7
10 μM topopyrone 7
1 μM topopyrone 8
10 μM topopyrone 8
10 μM topopyrone C (3)
20 μM topopyrone C (3)
10 μM topopyrone 9
20 μM topopyrone 9
10 μM topopyrone 10
20 μM topopyrone 10
topoisomerase IIα
none
11
3
10
1
1
2
25
23 2.7
34 4.6
40 13
34 2.5
a
0.5 μM etoposide
1 μM topopyrone 10
5 μM topopyrone 10
Relative to CPT; determined by densitometric analysis.
activity of the 5′-32P end labeled DNA covalently bound to the
enzyme. The topopyrone-stabilized DNA−topoisomerase I
covalent binary complexes were trapped by the addition of
the protein denaturant sodium dodecyl sulfate (SDS), followed
by filtration through a nitrocellulose filter membrane that
specifically binds enzyme and not free DNA. Linearized
plasmid pBR322 DNA was used as a DNA substrate that
contains numerous topoisomerase I binding sites. Top-
a
Topoisomerase-mediated cleavage conditions described in the
experimental procedures; reaction mixtures contained 6 units of
topoisomerase I or 5 units of topoisomerase IIα. Values represent the
mean SD (n = 3).
of the DNA−topoisomerase I covalent binary complex (cf. 3 vs
9), while topopyrone 10, having the pyrone ring in a linear
orientation, restored the activity of the compound as a
topoisomerase I poison.
oisomerase I alone covalently bound 1.4
0.5% of the
linearized plasmid DNA substrate (Table 2). This increased to
39 4.6% when 0.5 μM CPT was added. Topopyrone C (3)
was found to stabilize the DNA−topoisomerase I covalent
binary complex, possibly in a dose-dependent manner (16% at
10 μM concentration vs 21% at 20 μM concentration), while
topopyrone 9 afforded only 5−6% stabilization of the DNA−
topoisomerase I covalent binary complex. In contrast, top-
opyrone 10 afforded stabilization to an extent roughly
comparable to that of topopyrone C (3) and clearly gave
dose-dependent stabilization. Thus, the chlorine substituent at
the C5 position of topopyrone 9 is detrimental to stabilization
The nitrocellulose filter binding assay was also used to
measure stabilization of the DNA−topoisomerase II covalent
binary complex by topopyrone 10 (Table 2). Human
topoisomerase II exists in two closely related isoforms,
topoisomerase IIα and topoisomerase IIβ.25 Since human
topoisomerase IIα is more closely associated with proliferating
cells,25 we used this isoform in our studies. High levels of
covalently bound DNA−topoisomerase II complexes were
observed even in the absence of any topoisomerase II poison.
In the presence of 0.5 μM etoposide, 34 4.6% of the DNA
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was covalently bound to topoisomerase IIα, an amount similar
to that achieved with 1 or 5 μM topopyrone 10.
The cleavage site specificity of the topopyrone-stabilized
DNA−topoisomerase I covalent binary complexes was analyzed
using a 3′-32P end labeled 158-base pair DNA duplex substrate
(Figure 2). The topopyrones were found to stabilize top-
I covalent binary complexes were observed at the extreme 5′-
end of the DNA duplex substrate, but could not be sequenced.
The distribution of cleavage sites was also examined for
topopyrone-stabilized DNA−topoisomerase II covalent binary
complexes using a 5′-32P end labeled 158-base pair DNA duplex
substrate (Figure 3). Topopyrone B (2) and topopyrone 11
Figure 3. Stabilization of topoisomerase II-mediated DNA cleavage
sites on a 5′-32P end labeled 158-base pair DNA duplex substrate by
etoposide and topopyrones. Lane 1, DNA alone; lane 2, DNA +
topoisomerase IIα; lane 3, 5 μM etoposide + DNA + topoisomerase
IIα; lane 4, 20 μM etoposide + DNA + topoisomerase IIα; lane 5, 5
μM topopyrone B (2) + DNA + topoisomerase IIα; lane 6, 20 μM
topopyrone B (2) + DNA + topoisomerase IIα; lane 7, 5 μM
topopyrone 11 + DNA + topoisomerase IIα; lane 8, 20 μM
topopyrone 11 + DNA + topoisomerase IIα; lane 9, G + A lane.
Figure 2. Stabilization of topoisomerase I-mediated DNA cleavage
sites on a 3′-32P end labeled 158-base pair DNA duplex substrate by
CPT and topopyrones. Upper panel: lane 1, DNA alone; lane 2, DNA
+ topoisomerase I; lane 3, 1 μM CPT + DNA + topoisomerase I; lane
4, 5 μM topopyrone (2) + DNA + topoisomerase I; lane 5, 10 μM
topopyrone B (2) + DNA + topoisomerase I; lane 6, 20 μM
topopyrone B (2) + DNA + topoisomerase I; lane 7, 5 μM
topopyrone 9 + DNA + topoisomerase I; lane 8, 10 μM topopyrone 9
+ DNA + topoisomerase I; lane 9, 20 μM topopyrone 9 + DNA +
topoisomerase I; lane 10, 5 μM topopyrone 10 + DNA +
topoisomerase I; lane 11, 10 μM topopyrone 10 + DNA +
topoisomerase I; lane 12, 20 μM topopyrone 10 + DNA +
topoisomerase I; lane 13, G + A lane. Lower panel: DNA cleavage
sites stabilized by CPT and topopyrones 2, 9, and 10.
were found to stabilize the DNA−topoisomerase II covalent
binary complex at sites similar to etoposide (5′-CA23-3′ and 5′-
CG74-3′) with varying efficiencies. While topopyrone B (2) was
able to stabilize the DNA−topoisomerase II covalent binary
complex at 5 and 20 μM concentrations, topopyrone 11 was
only active at 5 μM concentration (Figure 3, lanes 5−8),
possibly due to solubility problems.
EXPERIMENTAL SECTION
■
oisomerase I cleavage at sites similar to CPT and with varying
efficiencies. Strong stabilization of DNA−topoisomerase I
covalent binary complexes by CPT was observed at several
5′-TG-3′ and 5′-TA-3′ sites. Topopyrone B (2) displayed dose-
dependent stabilization of the DNA−topoisomerase I covalent
binary complex (Figure 2, lanes 4−6) and was more potent
than topopyrones 9 and 10. An interesting observation in this
experiment was that the three topopyrones, but not CPT,
stabilized a topoisomerase I cleavage site at 5′-TG72-3′.
Additional unique topopyrone-stabilized DNA−topoisomerase
General Experimental Procedures. Chemicals and solvents were
purchased as reagent grade from Sigma-Aldrich Chemicals and were
used without further purification. All the reactions were performed
under an argon atmosphere, unless otherwise specified. Thin-layer
chromatography (TLC) plates (precoated glass plates with silica gel 60
F254, 0.25 mm thickness) were used for analytical TLC and were
visualized by UV irradiation (254 nm). Flash chromatography was
carried out using Silicycle 200−400 mesh silica gel. H and 13C NMR
1
spectra were obtained using a Varian 400 MHz NMR spectrometer.
Chemical shifts (δ) are reported in parts per million (ppm) and are
referenced to residual CHCl3 (δ 7.26 ppm for H NMR and δ 77.16
1
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for 13C NMR) as the internal strandard. Splitting patterns are
designated as s, singlet; d, doublet; m, multiplet. High-resolution mass
spectra were obtained at the Michigan State Mass Spectrometry
Facility or the Arizona State University CLAS High Resolution Mass
Spectrometry Facility.
1-(6-Chloro-1-hydroxy-3,5,8-trimethoxynaphthalen-2-yl)-
ethan-2-one (16). To a solution containing 0.50 g (1.9 mmol) of 15
in 20 mL of 1,2-dichloroethane at −10 °C was added 2.1 mL (2.1
mmol) of TiCl4 (1.0 M in CH2Cl2). The reaction mixture was stirred
for 5 min at −10 °C, and 0.30 mL (3.8 mmol) of acetyl chloride was
added. The reaction mixture was heated at reflux for 2 h, then cooled
and quenched with 400 mL of 1:1 2 N HCl−saturated aqueous
potassium sodium tartrate, filtered through a pad of Celite, and washed
with 200 mL of ethyl acetate. The organic phase was separated, and
the aqueous phase was extracted with three 300 mL portions of ethyl
acetate. The combined organic phase was dried over anhydrous
MgSO4, filtered, and then concentrated under diminished pressure to
afford a crude residue. The residue was purified by flash
chromatography on a silica gel column (15 × 3 cm). Elution with
1:10 ethyl acetate−hexanes gave 16 as a yellow solid: yield 0.56 g
(96%); mp 128−130 °C; silica gel TLC Rf 0.61 (1:2 ethyl acetate−
hexanes); mass spectrum (ESI), m/z 311.0692 (M + H)+
(C15H16ClO5 requires m/z 311.0686).
8-Chloro-2-methyl-5,7,10-trimethoxybenzo[h]chromen-4-
one (17). To a solution containing 0.40 g (1.29 mmol) of 16 in 20 mL
of freshly distilled ethyl acetate was added 0.57 g (14.1 mmol) of NaH
(60% dispersion in mineral oil). The reaction mixture was heated at
reflux for 1 h, cooled, and quenched with 100 mL of 2 N HCl (ice
cold). The solution was extracted with three 200 mL portions of
CHCl3, dried over anhydrous MgSO4, filtered, and concentrated under
diminished pressure to afford a crude residue. The residue was
dissolved in 15 mL of TFA at 0 °C and stirred for 15 min at 0 °C and
then for 30 min at room temperature. The solvent was concentrated
under diminished pressure, and the resulting residue was co-
evaporated with two 50 mL portions of toluene. The resulting residue
was purified by flash chromatography on a silica gel column (15 × 2
cm). Step gradient elution with 1:1 ethyl acetate−hexanes → 20%
MeOH in CHCl3 gave 17 as a colorless solid: yield 0.24 g (56%); mp
182−184 °C; silica gel TLC Rf 0.43 (15% MeOH in 1:1 ethyl acetate−
hexanes); mass spectrum (ESI), m/z 335.0675 (M + H)+
(C17H16ClO5 requires m/z 335.0686).
8-Chloro-5-methoxy-2-methylbenzo[h]chromene-4,7,10-tri-
one (18). To a solution containing 0.24 g (0.71 mmol) of 17 in 50 mL
of CH3CN at 0 °C was added 0.77 g (1.4 mmol) of ceric ammonium
nitrate in 8 mL of H2O. The reaction mixture was warmed to room
temperature and stirred for 0.5 h, at which time 150 mL of H2O was
added. The reaction mixture was extracted with four 150 mL portions
of CHCl3, and the combined organic phase was dried over anhydrous
MgSO4, filtered, and concentrated under diminished pressure to afford
a crude residue. The residue was purified by flash chromatography on
a silica gel column (15 × 2 cm). Elution with CHCl3 as eluant gave 18
as a yellow solid: yield 0.16 g (72%); mp 257−259 °C; silica gel TLC
Rf 0.50 (15% MeOH in 1:1 ethyl acetate−hexanes); mass spectrum
(ESI), m/z 305.0213 (M + H)+ (C15H10O5Cl requires m/z 305.0217).
10-(tert-Butyldimethylsilyloxy)-8-hydroxy-5-methoxy-2-
methyl-1-oxabenzo[a]anthracene-4,7,12-trione (20) and 10-
(tert-Butyldimethylsilyloxy)-5,8-dimethoxy-2-methyl-1-
oxabenzo[a]anthracene-4,7,12-trione (21). To a solution con-
taining 0.11 g (0.36 mmol) of 18 in 35 mL of dry benzene was added
0.12 mL (1.5 mmol) of pyridine followed by 1.1 g (3.6 mmol) of
freshly prepared 19.13 The reaction mixture was heated at reflux for 18
h, after which the cooled reaction mixture was quenched with 350 mL
of 2 N HCl (ice cold) and extracted with three 150 mL portions of
CHCl3. The combined organic layer was dried over anhydrous
MgSO4, filtered, and concentrated under diminished pressure to afford
a crude yellow solid. The residue was purified by flash chromatography
on a silica gel column (15 × 2 cm). Elution with 2% MeOH in 1:1
ethyl acetate−hexanes gave 20 as an orange solid: yield 67 mg (40%);
mp >300 °C dec; silica gel TLC Rf 0.64 (10% MeOH in 1:1 ethyl
acetate−hexanes); mass spectrum (ESI), m/z 467.1548 (M + H)+
(C25H27O7Si requires m/z 467.1526). Further elution with 5% MeOH
in 1:1 ethyl acetate−hexanes gave 21 as a yellow solid: yield 42 mg
(25%); mp >300 °C dec; silica gel TLC Rf 0.5 (10% MeOH in 1:1
ethyl acetate−hexanes); mass spectrum (ESI), m/z 481.1698 (M +
H)+ (C26H29O7Si requires m/z 481.1683).
Biochemical Reagents. Terminal deoxynucleotidyl transferase
and Sephadex G-25 mini Quick Spin Oligo columns were purchased
from Roche Applied Science (Indianapolis, IN, USA). pBR322
plasmid DNA, Klenow fragment (3′→5′ exo−) of DNA polymerase,
T4 polynucleotide kinase, and restriction endonucleases HindIII and
EcoRV were obtained from New England Biolabs (Ipswich, MA, USA).
Thermosensitive alkaline phosphatase and the Gene-Jet PCR
purification kit were purchased from Fermentas (Glen Burnie, MD,
USA). Human topoisomerase I was purchased from Topogen, Inc.
(Access Alley, FL, USA). Human topoisomerase IIα was purchased
from USB Products (Santa Clara, CA, USA). Protran B85 nitro-
cellulose filters were obtained from Whatman (Piscataway, NJ, USA).
Liquid scintillation fluid was purchased from RPI Corp (Mount
Prospect, IL, USA). All synthetic oligonucleotides, purified by ion
exchange, were obtained from Integrated DNA Technologies
(Coralville, IA, USA). Radiolabeled nucleotides were purchased from
Perkin-Elmer Life Sciences (Waltham, MA, USA). Topopyrone and
CPT solutions were freshly prepared in 60% aqueous dimethyl
sulfoxide prior to biological evaluation.
Toluene-4-sulfonic Acid 6-Chloro-3-methoxy-5,8-dioxo-5,8-
dihydronaphthalen-1-yl Ester (13). To a solution containing 0.41
g (1.7 mmol) of 1214 in 100 mL of acetone at room temperature was
added 0.65 g (3.4 mmol) of p-toluenesulfonyl chloride (p-TsCl) and
0.47 g (3.4 mmol) of K2CO3. The reaction mixture was heated at
reflux for 6 h, and the cooled solution was filtered through a silica gel
plug. The silica gel was washed with 100 mL of ethyl acetate, and the
combined filtrate was concentrated under diminished pressure to
afford a crude residue. The residue was purified by flash
chromatography on a silica gel column (15 × 3 cm). Step gradient
elution with 1:1 CHCl3−hexanes → 3:2 CHCl3−hexanes gave 13 as a
yellow solid: yield 0.60 g (90%); mp 150−152 °C; silica gel TLC Rf
0.47 (2:1 ethyl acetate−hexanes); mass spectrum (APCI), m/z
393.0199 (M + H)+ (C18H14ClO6S requires m/z 393.0200).
Toluene-4-sulfonic Acid 6-Chloro-3,5,8-trimethoxynaphtha-
len-1-yl Ester (14). To a solution containing 1.1 g (2.9 mmol) of 13
in 50 mL of 1:1 MeOH−THF under argon was added ∼70 mg of Pd/
C. The reaction mixture was purged with H2 and stirred under H2 for
2 h at room temperature, at which time it was filtered through a pad of
silica gel and washed with 300 mL of ethyl acetate. The combined
filtrate was concentrated under diminished pressure, and the resulting
residue was dissolved in 100 mL of 1:1 THF−H2O. To this solution
was added 1.5 g (8.7 mmol) of KOH and 2.6 mL (28.6 mmol) of
(MeO)2SO2. The reaction mixture was stirred for 1 h, then quenched
with 200 mL of H2O, and extracted with three 150 mL portions of
ethyl acetate. The combined organic phase was dried over anhydrous
MgSO4, filtered, and then concentrated under diminished pressure to
afford a crude residue. The residue was purified by flash
chromatography on a silica gel column (20 × 5 cm). Elution with
1:10 ethyl acetate−hexanes gave 14 as a colorless solid: yield 1.1 g
(95%); mp 136−138 °C; silica gel TLC Rf 0.61 (2:1 ethyl acetate−
hexanes); mass spectrum (ESI), m/z 423.0676 (M + H)+
(C20H20ClO6S requires m/z 423.0669).
6-Chloro-1-hydroxy-3,5,8-trimethoxynaphthalene (15). A
solution containing 1.4 g (3.3 mmol) of 14 in 100 mL of 1:1
EtOH−H2O containing 10% (w/v) KOH was heated at reflux for 4 h.
EtOH was removed under diminished pressure, and the remaining
solution was poured into 6 N HCl (ice cold) and extracted with three
100 mL portions of ethyl acetate. The combined organic extract was
dried over anhydrous MgSO4, filtered, and concentrated under
diminished pressure to afford a crude oil. The residue was purified
by flash chromatography on a silica gel column (12 × 3 cm). Elution
with 1:7 ethyl acetate−hexanes gave 15 as a colorless solid: yield 0.50 g
(56%); mp 115−117 °C; silica gel TLC Rf 0.63 (2:1 ethyl acetate−
hexanes); mass spectrum (ESI), m/z 269.0570 (M + H)+
(C13H14ClO4 requires m/z 269.0581).
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Topopyrone 5. To a solution containing 55 mg (0.12 mmol) of 20
in 10 mL of THF at room temperature was added 34 mg (0.13 mmol)
of TBAF·H2O. The reaction mixture was stirred at room temperature
for 0.5 h, quenched with 100 mL of 2 N HCl, and extracted with three
50 mL portions of ethyl acetate and two 25 mL portions of CHCl3.
The combined organic phase was dried over anhydrous MgSO4,
filtered, and concentrated under diminished pressure to afford a crude
orange-yellow solid. The residue was dissolved in 10 mL of CH2Cl2 at
−78 °C, and 1.18 mL (1.18 mmol) of BBr3 (1.0 M in CH2Cl2) was
added. The reaction mixture was warmed to room temperature and
stirred for 18 h. The reaction was quenched by the addition of 100 mL
of 2 N HCl and extracted with three 50 mL portions of ethyl acetate.
The combined organic layer was dried over anhydrous MgSO4,
filtered, and concentrated under diminished pressure to afford a crude
orange-yellow solid. The crude product was purified by flash
chromatography on a silica gel column (25 × 2 cm). Elution with
94:5:1 CHCl3−MeOH−AcOH gave 5 as an orange solid: yield 9.0 mg
(23%); mp >150 °C dec; silica gel TLC Rf 0.51 (94:5:1 CHCl3−
MeOH−AcOH); mass spectrum (APCI), m/z 339.0505 (M + H)+
(C18H11O7 requires m/z 339.0503). All attempts at obtaining NMR
spectra of 5 met with failure in a variety of solvent systems due to
insufficient solubility. Accordingly, 5 was converted to its 3,5,7-
trimethoxy derivative to permit characterization by NMR. The
derivative was also characterized by high-resolution mass spectrometry.
To a solution containing 10.0 mg (0.03 mmol) of 5 in 5 mL of
acetone was added 0.04 mL (0.30 mmol) of (MeO)2SO2 and 42 mg
(0.30 mmol) of K2CO3. The reaction mixture was heated at reflux for
18 h, quenched with 50 mL of 1 N HCl, and extracted with two 20 mL
portions of ethyl acetate. The combined organic extract was dried over
anhydrous MgSO4, then filtered and concentrated under diminished
pressure to afford a crude residue. The crude product was purified by
flash chromatography on a silica gel column (15 × 2 cm). Elution with
2% MeOH in CHCl3 gave the 3,5,7-trimethoxy derivative of
topopyrone 5 as an orange solid: yield 5.0 mg (45%); silica gel
NMR (CDCl3) δ 19.9, 21.1, 56.8, 57.1, 104.3, 110.4, 113.1, 113.2,
114.4, 116.9, 118.3, 138.4, 139.9, 156.3, 157.7, 161.7, 164.3, 165.1,
168.3, 176.8, 179.0, and 180.2; mass spectrum (ESI), m/z 409.0931
(M + H)+ (C22H17O8 requires m/z 409.0923).
2-Acetyl-7-bromo-3,6,8-trimethoxy-1-tosyloxyanthraqui-
none (23). To a solution containing 0.05 g (0.10 mmol) of 2213 in 10
mL of CH2Cl2 at −78 °C was added 0.02 mL (0.15 mmol) of
(iPr)2NH followed by 0.02 g (0.11 mmol) of N-bromosuccinimide in
10 mL of CH2Cl2. After the additions, the reaction was quenched by
the addition of 20 mL of 1:1 H2O−CH2Cl2. The phases were
separated, and the aqueous phase was extracted with three 10 mL
portions of CH2Cl2. The combined organic extract was dried over
anhydrous MgSO4, filtered, and then concentrated under diminished
pressure to afford a crude red solid. The residue was purified by flash
chromatography on a silica gel column (15 × 2 cm). Elution with 2%
MeOH in 1:1 ethyl acetate−hexanes gave the crude monobrominated
product 23 as a yellow solid. The product obtained was dissolved in 4
mL of acetone, and 0.05 g (0.36 mmol) of K2CO3 was added, followed
by 0.03 mL (0.40 mmol) of (MeO)2SO2. The reaction mixture was
stirred at reflux for 3 h, and the cooled reaction mixture was quenched
with 50 mL of 2 N HCl and then extracted with three 50 mL portions
of ethyl acetate. The combined organic extract was dried over
anhydrous MgSO4, filtered, and then concentrated under diminished
pressure to afford a crude yellow solid. The residue was purified by
flash chromatography on a silica gel column (15 × 2 cm). Elution with
1:2 ethyl acetate−hexanes gave 23 as a yellow solid: yield 12 mg
(20%); silica gel TLC Rf 0.35 (1:1 ethyl acetate−hexanes); mass
spectrum (ESI), m/z 589.0182 (M + H)+ (C26H22BrO9S requires m/z
589.0168).
1-Acetoxy-2-acetyl-7-bromo-3,6,8-trimethoxyanthraqui-
none (24). To a solution containing 0.07 g (0.12 mmol) of 23 in 15
mL of 1:1 EtOH−H2O was added 0.06 g (1.0 mmol) of KOH. The
reaction mixture was heated at reflux for 4 h. The cooled solution was
poured into 2 N HCl and extracted with three 50 mL portions of ethyl
acetate. The combined organic extract was dried over anhydrous
MgSO4, filtered, and then concentrated under diminished pressure to
afford a crude yellow solid. The residue was purified by flash
chromatography on a silica gel column (15 × 2 cm). Elution with 1:2
ethyl acetate−hexanes afforded the product having a free hydroxy
group. This material was dissolved in 5 mL of CH2Cl2, and 0.03 mL
(0.30 mmol) of acetic anhydride was added followed by 0.04 mL (0.30
mmol) of Et3N and a catalytic amount of 4-N,N-dimethylaminopyr-
idine. The reaction mixture was stirred at room temperature for 18 h,
quenched with 50 mL of 2 N HCl, and extracted with three 50 mL
portions of ethyl acetate. The combined organic extract was dried over
anhydrous MgSO4, filtered, and then concentrated under diminished
pressure to afford a crude yellow solid. The residue was purified by
flash chromatography on a silica gel column (15 × 2 cm). Elution with
1:2 ethyl acetate−hexanes gave 24 as a yellow solid: yield 30 mg
(52%); silica gel TLC Rf 0.29 (1:1 ethyl acetate−hexanes); mass
spectrum (ESI), m/z 477.0162 (M + H)+ (C21H18BrO8 requires m/z
477.0185).
1
TLC Rf 0.48 (2% MeOH in CHCl3); H NMR (CDCl3) δ 2.48 (3H,
d, J = 0.4 Hz), 3.98 (3H, s), 4.01 (3H, s), 4.14 (3H, s), 6.20 (1H, d, J =
0.4 Hz), 6.82 (1H, d, J = 2.8 Hz), 7.35 (1H, d, J = 2.4 Hz) and 7.58
(1H, s); 13C NMR (CDCl3) δ 20.7, 56.5, 57.2, 57.5, 103.0, 103.5,
106.2, 113.5, 117.6, 118.1, 118.5, 136.3, 137.4, 158.1, 162.4, 163.3,
164.5, 165.9, 177.6, 179.6, and 183.5; mass spectrum (ESI), m/z
381.0983 (M + H)+ (C21H17O7 requires m/z 381.0974).
Topopyrone 6. To a solution containing 52 mg (0.11 mmol) of 21
in 10 mL of THF at room temperature was added 30 mg (0.11 mmol)
of TBAF·H2O. The reaction mixture was stirred for 1 h at room
temperature, quenched with 100 mL of 2 N HCl, and extracted with
three 50 mL portions of ethyl acetate and two 25 mL portions of
CHCl3. The combined organic phase was dried over anhydrous
MgSO4, filtered, and then concentrated under diminished pressure to
afford a crude orange-yellow solid. The crude product was purified by
flash chromatography on a silica gel column (20 × 2 cm). Elution with
94:5:1 CHCl3−MeOH−AcOH gave 6 as a yellow solid: yield 20 mg
(50%); mp >180 °C dec; silica gel TLC Rf 0.42 (94:5:1 CHCl3−
MeOH−AcOH); mass spectrum (ESI), m/z 367.0807 (M + H)+
(C20H15O7 requires m/z 367.0818). All attempts at obtaining NMR
spectra of 6 met with failure in a variety of solvent systems due to
insufficient solubility. Accordingly, 6 was converted to its 7-acetoxy
derivative to permit characterization by NMR. The derivative was also
characterized by high-resolution mass spectrometry.
Topopyrone 7. To a solution containing 0.04 g (0.08 mmol) of 24
in 10 mL of freshly distilled THF was added 0.02 g (2.5 mmol) of LiH.
The reaction mixture was heated at reflux for 18 h, and the cooled
reaction mixture was quenched carefully with 50 mL of 2 N HCl. The
resulting solution was extracted with three 75 mL portions of ethyl
acetate, dried over anhydrous MgSO4, filtered, and then concentrated
under diminished pressure to afford a crude residue. The residue was
dissolved in 5 mL of CF3COOH. The reaction mixture was stirred at
room temperature for 1 h and concentrated under diminished
pressure, and the residue so obtained was co-evaporated with two 5
mL portions of toluene. The resulting crude residue was purified by
flash chromatography on a silica gel column (7 × 2 cm). Gradient
elution with 1:1 ethyl acetate−hexanes → 2% MeOH in 1:1 ethyl
acetate−hexanes gave 7 as a yellow solid: yield 17 mg (45%); mp >280
A mixture of 12 mg (33 μmol) of 6, 3 mL of acetic anhydride, and
1.5 mL of pyridine was stirred at room temperature for 3 h. The
reaction mixture was quenched with 50 mL of 2 N HCl and extracted
with two 25 mL portions of ethyl acetate. The combined organic
extract was dried over anhydrous MgSO4, filtered, and concentrated
under diminished pressure to afford a crude residue. The crude
product was purified by flash chromatography on a silica gel column
(15 × 2 cm). Elution with 3% MeOH in CHCl3 gave the 7-acetoxy
derivative of topopyrone 6 as a yellow solid: yield 9.0 mg (68%); silica
1
°C dec; silica gel TLC Rf 0.21 (1:1 ethyl acetate−hexanes); H NMR
1
gel TLC Rf 0.50 (2% MeOH in CHCl3); H NMR (CDCl3) δ 2.37
(CDCl3) δ 2.48 (3H, d, J = 0.4 Hz), 4.04 (3H, s), 4.09 (3H, s), 4.16
(3H, s), 6.21 (1H, d, J = 0.8 Hz), 7.55 (1H, s) and 7.59 (1H, s); 13C
NMR (CDCl3) δ 19.9, 56.9, 57.0, 61.8, 103.1, 104.9, 112.9, 116.7,
(3H, s), 2.48 (3H, s), 4.04 (3H, s), 4.15 (3H, s), 6.22 (1H, s), 7.07
(1H, d, J = 2.0 Hz), 7.66 (1H, d, J = 2.4 Hz), and 7.69 (1H, s); 13C
583
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Journal of Natural Products
Article
117.7, 117.9, 122.3, 133.5, 136.8, 157.4, 158.9, 160.3, 163.1, 165.2,
176.8, 178.1, and 182.1; mass spectrum (ESI), m/z 459.0060 (M +
H)+ (C21H16BrO7 requires m/z 459.0079).
and either CPT or a topopyrone at the indicated concentrations were
incubated at 25 °C for 30 min. The reaction volume was 10 μL, and
the reaction was carried out in 10 mM Tris-HCl, pH 7.9, containing
0.15 M NaCl, 1 mM EDTA, 0.1% BSA, 0.1 mM spermidine, and 5%
glycerol. The reactions were stopped by the addition of SDS to a final
concentration of 0.5%. A 10 μL amount of denaturing gel loading
solution containing 80% (v/v) formamide, 2 mM EDTA, 1% (w/v)
bromophenol blue, and 1% (w/v) xylene cyanol was added to the
reaction mixture. The resulting solution was heated at 95 °C for 5 min,
followed by chilling on ice. A 5 μL portion of each sample was then
loaded onto a denaturing (7 M urea) 16% (w/v) polyacrylamide gel
and electrophoresed at 50 W for 2 h. Gels were visualized using a
phosphorimager (Molecular Dynamics) equipped with ImageQuant
version 3.2 software.
Topopyrone 8. A solution containing 14 mg (35 μmol) of 7 in 2
mL of 1.0 M AlCl3 in nitrobenzene was heated at 90 °C for 48 h. The
reaction was quenched while hot with 20 mL of 6 N HCl and stirred at
90 °C for 1 h. The reaction mixture was extracted with three 50 mL
portions of CHCl3, dried over anhydrous MgSO4, and then
concentrated under diminished pressure to afford a crude green oil.
The crude product was purified by flash chromatography on a silica gel
column (15 × 2 cm). Step gradient elution with 4:1 ethyl acetate−
hexanes → 95:4:1 CHCl3−MeOH−AcOH gave 8 as a green solid:
yield 6.5 mg (50%); mp >300 °C dec; silica gel TLC Rf 0.30 (10%
MeOH in CHCl3); mass spectrum (APCI), m/z 416.9618 (M + H)+
(C18H10O7Br requires m/z 416.9610). All attempts at obtaining NMR
spectra of 8 met with failure in a variety of solvent systems due to
insufficient solubility.
3′-32P End Labeling and Purification of a 158-Base Pair DNA
Restriction Fragment. Plasmid pBR322 DNA (25 μg) was digested
with 50 units of HindIII in 80 μL (total volume) of 10 mM Tris-HCl,
pH 7.9, containing 50 mM NaCl, 10 mM MgCl2, and 1 mM
dithiothreitol (DTT) at 37 °C for 3 h. The linearized DNA was 3′-32P
end labeled by incubation with 10 units of the Klenow fragment (3′→
5′ exo−) of DNA polymerase in 100 μL (total volume) of 10 mM Tris-
HCl, pH 7.9, containing 50 mM NaCl, 10 mM MgCl2, 1 mM DTT,
and 0.10 mCi [α-32P] dATP (3000 Ci/mmol). The reaction mixture
was incubated at 37 °C for 1 h followed by heat inactivation of the
enzyme at 75 °C for 20 min. Unincorporated [α-32P] dATP was
removed with a GeneJET PCR purification kit. The 3′-32P end labeled
DNA was digested at 37 °C for 3 h with 50 units of EcoRV in 70 μL
(total volume) of 50 mM Tris-HCl, pH 7.9, containing 0.1 M NaCl, 10
mM MgCl2 and 1 mM DTT. This digestion resulted in a 3′-32P end
labeled fragment having 158 base pairs. Purification of the restriction
fragment was performed essentially as described.26,27
Topoisomerase I-Mediated DNA Cleavage of a 158-Base
Pair DNA Duplex Substrate. A sample of 3′-32P end labeled 158-
base pair DNA (30 000 cpm), 6 units of topoisomerase I, and either
CPT or a topopyrone at the indicated concentrations were incubated
at 25 °C for 30 min in 10 μL (total volume) of 10 mM Tris-HCl, pH
7.9, containing 0.15 M NaCl, 1 mM EDTA, 0.1% BSA, 0.1 mM
spermidine, and 5% glycerol. The reactions were stopped by addition
of SDS to a final concentration of 0.5%. A 10 μL sample of denaturing
gel loading solution containing 80% (v/v) formamide, 2 mM EDTA,
1% (w/v) bromophenol blue, and 1% (w/v) xylene cyanol was added
to the reaction mixture. The resulting solution was heated at 95 °C for
5 min, followed by chilling on ice. A 5 μL amount of each sample was
then loaded onto a denaturing (7 M urea) 16% (w/v) polyacrylamide
gel and electrophoresed at 50 W for 2 h. Cleavage sites were confirmed
by comparison with the reaction products obtained by the Maxam−
Gilbert G + A sequencing protocol.28 Gels were visualized using a
phosphorimager (Molecular Dynamics) equipped with ImageQuant
version 3.2 software.
Topopyrones 9 and 10. These two compounds were synthesized
as described previously.13
Topopyrone 11. A solution containing 8.0 mg (19 μmol) of 8 in 4
mL of 1% NaOH in MeOH was heated at 60 °C for 48 h. The reaction
mixture was neutralized carefully with 1 N HCl and concentrated
under diminished pressure. The resulting residue was dissolved in 10
mL of H2O and extracted successively with three 20 mL portions of
CHCl3 and two 10 mL portions of ethyl acetate. The combined
organic phase was dried over anhydrous MgSO4, filtered, and then
concentrated under diminished pressure to afford a crude residue. The
residue was purified by flash chromatography on a silica gel column
(10 × 2 cm). Elution with 94:5:1 CHCl3−MeOH−AcOH gave 11 as a
green solid: yield 4.0 mg (50%); mp >300 °C dec; silica gel TLC Rf
0.23 (10% MeOH in CHCl3); mass spectrum (ESI), m/z 416.9613 (M
+ H)+ (C18H10BrO7 requires m/z 416.9610). All attempts at obtaining
NMR spectra of 11 met with failure in a variety of solvent systems due
to insufficient solubility. Accordingly, 11 was converted to its 1,6,8-
trimethoxy derivative to permit characterization by NMR. The
derivative was also characterized by high-resolution mass spectrometry.
To a solution containing 6.0 mg (14 μmol) of 11 in 4 mL of
acetone at room temperature was added 0.03 mL (0.23 mmol) of
(MeO)2SO2 and 32 mg (0.23 mmol) of K2CO3. The reaction mixture
was heated at reflux for 18 h, quenched with 50 mL of 1 N HCl, and
extracted with two 20 mL portions of ethyl acetate. The combined
organic extract was dried over anhydrous MgSO4, filtered, and
concentrated under diminished pressure to afford a crude residue. The
crude product was purified by flash chromatography on a silica gel
column (10 × 1 cm). Elution with 2% MeOH in CHCl3 gave the
1,6,8-trimethoxy derivative of topopyrone 11 as an orange solid: yield
1
4.0 mg (66%); silica gel TLC Rf 0.55 (2% MeOH in CHCl3); H
NMR (CDCl3) δ 2.49 (3H, s), 4.02 (3H, s), 4.10 (3H, s), 4.17 (3H, s),
6.24 (1H, s), 7.66 (1H, s), and 7.71 (1H, s); 13C NMR (CDCl3) δ
20.0, 57.1, 57.3, 61.5, 104.4, 105.9, 113.3, 114.3, 115.9, 119.7, 136.7,
139.6, 157.8, 159.3, 161.7, 164.4, 165.2, 179.19, and 179.21; mass
spectrum (ESI), m/z 459.0077 (M + H)+ (C21H16O7Br requires m/z
459.0079).
5′-32P End Labeling and Purification of a 158-Base Pair DNA
Restriction Fragment. Plasmid pBR322 DNA (25 μg) was digested
with 50 units of HindIII in 80 μL (total volume) of 10 mM Tris-HCl,
pH 7.9, containing 50 mM NaCl, 10 mM MgCl2, and 1 mM
dithiothreitol (DTT) at 37 °C for 3 h. The digest was then treated
with 25 units of thermosensitive alkaline phosphatase in 120 μL (total
volume) of 10 mM Tris-HCl, pH 8.0, containing 5 mM MgCl2, 0.1 M
KCl, 0.02% Triton X-100, 1 mM 2-mercaptoethanol, and 0.1 mg/mL
BSA. The reaction mixture was incubated at 37 °C for 30 min followed
by heat inactivation of the enzyme at 75 °C for 5 min. The reaction
mixture was then 5′-32P end labeled using 20 units of T4
polynucleotide kinase in 80 μL (total volume) of 70 mM Tris-HCl,
pH 7.6, containing 10 mM MgCl2, 5 mM DTT, and 0.10 mCi [γ-32P]
ATP (6000 Ci/mmol). The reaction mixture was incubated at 37 °C
for 1 h followed by heat inactivation of the enzyme at 65 °C for 20
min. The radiolabeled DNA restriction fragment was purified on an
8% native polyacrylamide gel, and run at 10 W for 3.5 h. The desired
DNA fragment was eluted from the gel slice in 3 M sodium acetate
overnight followed by ethanol precipitation of the radiolabeled
product.
3′-32P End Labeling and Purification of Oligonucleotides.
3′-32P end labeling was carried out by adding 10 pmol of template
DNA, 0.05 mCi [α-32P] cordycepin 5′-triphosphate (5000 Ci/mmol),
and 400 units of terminal deoxynucleotidyl transferase in 40 μL (total
volume) of 25 mM Tris-HCl, pH 6.6, containing 200 mM potassium
cacodylate, 2.5 mM CoCl2, and 0.25 mg/mL bovine serum albumin
(BSA). The reaction mixture was incubated at 37 °C for 1 h. Reactions
were terminated by the addition of SDS to a final concentration of
0.5%. The radiolabeled oligonucleotides were purified using Sephadex
G-25 Quick Spin Oligo columns.
Topoisomerase I-Mediated DNA Cleavage and Religation of
a 23-Base Pair Oligonucleotide Substrate. The 3′-32P end labeled
DNA strands were annealed with their complementary unlabeled
DNA strands in 10 mM Tris-HCl, pH 7.8, containing 100 mM NaCl
and 1 mM EDTA. The annealing process was performed by heating
the reaction mixture at 95 °C for 5 min, followed by slow cooling to 25
°C. Duplex DNA substrates (∼100 fmol), 6 units of topoisomerase I,
584
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Journal of Natural Products
Article
Topoisomerase II-Mediated DNA Cleavage of a 158-Base
Pair DNA Duplex Substrate. A sample of 5′-32P end labeled 158-
base pair DNA (30000 cpm), 10 units of DNA topoisomerase IIα, and
either etoposide or a topopyrone at the indicated concentrations were
incubated at 25 °C for 30 min. The reaction was carried out in 10 μL
(total volume) of 10 mM Tris-HCl, pH 7.9, containing 50 mM NaCl,
50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 μg/mL BSA, and 1 mM
ATP. The reactions were stopped by addition of SDS to a final
concentration of 0.5%. A 10 μL amount of denaturing gel loading
buffer containing 80% (v/v) formamide, 2 mM EDTA, 1% (w/v)
bromophenol blue, and 1% (w/v) xylene cyanol was added to the
reaction mixture. The resulting solution was heated at 95 °C for 5 min,
followed by chilling on ice. A 5 μL portion of each sample was then
loaded onto a denaturing (7 M urea) 16% (w/v) polyacrylamide gel
and electrophoresed at 50 W for 2 h. Cleavage sites were confirmed by
comparison with the reaction products obtained by the Maxam−
Gilbert G + A sequencing protocol.28 Gels were visualized using a
phosphorimager (Molecular Dynamics) equipped with ImageQuant
version 3.2 software.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Dedicated to Dr. Gordon Cragg for his pioneering work on the
development of natural product anticancer agents. The work
was supported at Arizona State University by a research grant
from the Arizona Technology and Research Initiative Fund.
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Am. Chem. Soc. 2003, 125, 8218−8227.
ASSOCIATED CONTENT
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S
* Supporting Information
1H and 13C NMR spectra values for synthetic intermediates,
figures illustrating stabilization of topoisomerase I-mediated
cleavage of a 23-base pair oligonucleotide substrate by
topopyrones, and 1H and 13C NMR spectra for new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(27) Katano, K.; An, H.; Aoyagi, Y.; Overhand, M.; Sucheck, S. J.;
Stevens, W. C. Jr.; Hess, C. D.; Zhou, X.; Hecht, S. M. J. Am. Chem.
Soc. 1998, 120, 11285−11296.
(28) Maxam, A. M.; Gilbert, W. Methods Enzymol. 1980, 65, 499−
560.
AUTHOR INFORMATION
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Corresponding Author
*Tel: (480) 965-6625. Fax: (480) 965-0038. E-mail: sid.
hecht@asu.edu.
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