Inhibitors of DNA polymerase β: Activity and mechanism

Article abstract of DOI:10.1016/j.bmc.2008.02.071

Bioassay-guided fractionation of extracts prepared from Couepia polyandra and Edgeworthia gardneri resulted in the isolation of the DNA polymerase β (pol β) inhibitors oleanolic acid (1), edgeworin (2), betulinic acid (3), and stigmasterol (4). Study of these pol β inhibitors revealed that three of them inhibited both the lyase and polymerase activities of DNA polymerase β, while stigmasterol inhibited only the lyase activity. Further investigation indicated that the four inhibitors had substantially different effects on the DNA-pol β binary complex that is believed to be an obligatory intermediate in the lyase reaction. It was found that the inhibitors potentiated the inhibitory action of the anticancer drug bleomycin in cultured A549 cells, without any influence on the expression of pol β in the cells. The results of the unscheduled DNA synthesis assay support the thesis that the potentiation of bleomycin cytotoxicity by DNA pol β inhibitors was a result of an inhibition of DNA repair synthesis.

Full text of DOI:10.1016/j.bmc.2008.02.071

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4331–4340
Inhibitors of DNA polymerase b: Activity and mechanism
Zhijie Gao, David J. Maloney, Larisa M. Dedkova and Sidney M. Hecht*
Departments of Chemistry and Biology, University of Virginia, Charlottesville, VA 22904, USA
Received 23 January 2008; revised 20 February 2008; accepted 22 February 2008
Available online 4 March 2008
Abstract—Bioassay-guided fractionation of extracts prepared from Couepia polyandra and Edgeworthia gardneri resulted in the iso-
lation of the DNA polymerase b (pol b) inhibitors oleanolic acid (1), edgeworin (2), betulinic acid (3), and stigmasterol (4). Study of
these pol b inhibitors revealed that three of them inhibited both the lyase and polymerase activities of DNA polymerase b, while
stigmasterol inhibited only the lyase activity. Further investigation indicated that the four inhibitors had substantially different
effects on the DNA–pol b binary complex that is believed to be an obligatory intermediate in the lyase reaction. It was found that
the inhibitors potentiated the inhibitory action of the anticancer drug bleomycin in cultured A549 cells, without any influence on the
expression of pol b in the cells. The results of the unscheduled DNA synthesis assay support the thesis that the potentiation of ble-
omycin cytotoxicity by DNA pol b inhibitors was a result of an inhibition of DNA repair synthesis.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
neocarzinostatin.16 Pol b plays important roles in the
BER pathway;17 it excises the 50-terminal dRP18,19 from
a preincised apurinic or apyrimidinic (AP) site after the
modified base has been removed by DNA glycosylase
and AP endonuclease (Fig. 1). Further, polymerase b
catalyzes a template-directed nucleotidyl transfer reac-
tion to fill in the resulting gap.17,20 However, at least
one abasic lesion, a deoxyribonolactone, cannot be re-
paired efficiently, and can actually lead to crosslinking
of the repair enzyme to the DNA lesion.21,22
DNA repair in mammalian cells is a multi-pathway pro-
cess that protects the integrity of DNA in the presence
of DNA damaging agents. Many currently used antican-
cer therapeutic agents rely on the ability to create DNA
lesions, leading to cancer cell death. The ability of can-
cer cells to repair such DNA damage is a major cause of
resistance to these clinically used antitumor agents.1–3
One strategy to address this issue involves targeting pro-
teins involved in DNA repair in cancer cells, thereby
overcoming resistance to DNA damaging agents em-
ployed for anticancer therapy. Implementation of a
strategy for adjuvant chemotherapy would demand that
the agents so employed lacked significant intrinsic cyto-
toxic activity, but rather potentiated the cytotoxic action
of the DNA damaging agents.
Therefore, pol b constitutes a promising target for adju-
vant cancer therapy. It is logical to think that the effi-
cacy of clinically used DNA damaging agents could be
enhanced by blocking the repair of damaged DNA
through inhibition of this enzyme. A number of inhibi-
tors have been identified including inhibitors of both
the polymerase23–32 and lyase33–39 activities of the en-
zyme. Consistent with the thesis that the inhibition of
pol b should enhance the cytotoxicity of DNA damag-
ing agents used for cancer chemotherapy, a number of
the identified inhibitors were shown to enhance the cyto-
toxicity of the DNA damaging agent bleomycin (BLM)
Three types of excision repair have been found to target
specific DNA lesions, namely base excision repair
(BER),4 nucleotide excision repair (NER)5,6 and DNA
mismatch repair.7 BER is a major pathway employed
for the repair of damaged bases in DNA8,9 produced
by antitumor agents such as bleomycin,10,11 monofunc-
tional DNA alkylating agents,12,13 cisplatin,14,15 and
toward
cultured
mammalian
cells,24,27,30–33,35,36
although the extent of potentiation of BLM toxicity
did not seem to correlate in a simple way with the po-
tency of pol b inhibition by individual agents. However,
there are only limited data concerning the interactions
between pol b, pol b inhibitors and DNA sub-
strate,28,32,40 or regarding the effect of pol b inhibitors
on DNA synthesis or pol b expression in cultured cells.
Keywords: Polymerase inhibitor; Inhibition mechanism; Enzyme–
DNA covalent complex; Antitumor agent potentiation; Natural
products.
*
Corresponding author. Tel.: +1 434 924 3906; fax: +1 434 924
7856; e-mail: sidhecht@virginia.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.071

4332
Z. Gao et al. / Bioorg. Med. Chem. 16 (2008) 4331–4340
Figure 1. Formation of AP site and excision of the dRP group.
To facilitate an understanding of the consequences of
inhibition of the polymerase and lyase activities of pol
b, we focused on four natural products (1–4) (Fig. 2)
that were isolated recently as inhibitors of the lyase
activity of DNA polymerase b.34,36 Two of these (olean-
olic acid (1) and betulinic acid (3)), had also previously
been found to inhibit the polymerase activity of pol
b.30,31 In the present study, the pol b inhibitory activities
of these compounds were determined using an assay that
permitted each activity to be monitored under the same
reaction conditions. Also investigated under uniform
conditions was the ability of each of the four com-
pounds to potentiate the activity of bleomycin. To per-
mit an analysis of the molecular basis of the observed
effects, the expression of pol b in cultured A549 cells that
had been exposed to the pol b inhibitors was studied as
was the impact of these inhibitors on unscheduled DNA
synthesis in A549 cells. On the basis of these data, the
molecular mechanisms and the biological characteristics
of these inhibitors are discussed.
yield. Saponification gave 9, which was condensed with
2-hydroxy-4-(2-methoxyethoxymethoxy)benzaldehyde
(6) to afford MEM-protected edgeworin (10). Removal
of the protecting group afforded edgeworin (2) in 83%
yield.
2.2. Construction of 30-end labeled DNA substrate
To investigate the inhibition of pol b lyase and polymer-
ase activities, a 34-nt oligodeoxyribonucleotide having a
deoxyuridine at position 16 was employed (Fig. 3). The
DNA substrate was labeled at 30-end using
[a-32P]ddATP in the presence of terminal deoxynucleot-
idyltransferase. After purification, the substrate was ex-
posed to uracil–DNA glycosylase and AP endonuclease
to remove the uracil moiety and create an AP site. This
substrate was used to examine the pol b lyase and poly-
merase activities, as well as the formation of the DNA–
pol b binary complex.
2.3. Inhibition of pol b lyase and polymerase activities
2. Results
Previous work showed that compounds 1–4 exhibited
moderate inhibition of pol b lyase activity;34,36 two of
them (1 and 3) were also found to inhibit the polymerase
activity of the enzyme.30,31 In the earlier work, these
inhibitory activities were studied using two separate
assays carried out under quite different conditions. In
order to study the two properties under comparable
2.1. Synthesis of edgeworin (2)
Edgeworin was synthesized as outlined in Scheme 1.
Briefly 7-hydroxycoumarin (7) was treated with ethyl
bromoacetate to afford ethyl ester 8 in quantitative

Z. Gao et al. / Bioorg. Med. Chem. 16 (2008) 4331–4340
4333
O
COOH
HO
O
O
HO
O
O
2
edgeworin ( )
1
oleanolic acid ( )
COOH
HO
HO
stigmasterol (4)
3
betulinic acid ( )
Figure 2. Structures of polymerase b inhibitors 1–4.
O
O
MEMCl, (iPr)2NEt
CH2Cl2
H
H
HO
OH
MEMO
81%
OH
5
6
5% aq NaOH
EtOH, reflux
ethyl bromoacetate
EtO
HO
K2CO3, acetone
reflux
HO
O
O
O
O
O
O
O
O
O
O
9
8
7
100%
91%
O
O
O
O
bromocatechol borane
CH2Cl2
6,
Ac2O, Et3N
DMF, 110 oC
MEMO
55%
O
HO
O
O
O
83%
O
O
10
2
edgeworin ( )
Scheme 1. Synthesis of edgeworin (2).
conditions, an assay was constituted under which the
two activities could be studied in parallel (Fig. 3).40 As
shown in Figures 3C and D, oleanolic acid (1) exhibited
the strongest inhibition of both lyase and polymerase
activities of pol b under these assay conditions. Com-
pound 2 also inhibited the lyase and polymerase activi-
ties to roughly comparable extents, while compound 3
was a more potent inhibitor of the lyase activity than
the polymerase activity. Compound 4 was a weak pol
b lyase inhibitor but exhibited no detectable polymerase
inhibitory activity. The IC50 values of these compounds
for lyase and polymerase activities are summarized in
Table 1.
2.4. Effect on the interaction between DNA substrate and
pol b
The amino-terminal 8-kDa domain of DNA pol b cat-
alyzes the excision of dRP groups from 50-incised AP
sites via a b-elimination reaction following the forma-
tion of a Schiff base between an aldehyde group of
the AP site and an amino group of the enzyme

4334
Z. Gao et al. / Bioorg. Med. Chem. 16 (2008) 4331–4340
A
C
5'-CTGCAGCTGATGCGCUGTACGGATCCCCGGGTAC-32P-ddATP
3'-GACGTCGACTACGCGGCATGCCTAGGGGCCCATG
100
80
60
40
20
0
uracil DNA glycosylase
AP endonuclease
1
2
3
34mer
3’
5’
5’
3’- 32P-ddATP
19mer
15mer
OH
sugar
P
P
dRP lyase product
synthesis substrate
dRP lyase substrate
dNTPs
32P-dCTP
synthesis product
0
50
100
150
200
250
Polymerase beta inhibitor (micromole)
110
100
90
80
70
60
50
40
30
20
B
D
1
pol β
lane
-
-
1
-
+
2
+
3
+
4
+
5
+
6
+
7
1
2
3
4
dRP lyase substrate
dRP lyase product
synthesis product
0
50
100
150
200
250
polymerase beta inhibitor (micromole)
Figure 3. Inhibition of pol b lyase and polymerase activities by pol b inhibitors. The reaction conditions and analyses are described in the
Experimental. (A) Schematic representation of the substrate for the pol b lyase–polymerase assay: the 32P-labeled dRP lyase substrate, products after
AP endonuclease cleavage (19-mer having a 30-32P label and 50-sugar phosphate (AP site) + 15-mer). The 15-mer is the DNA synthesis substrate,
while the 19-mer is the lyase substrate. Also shown schematically are the products of the lyase and polymerase reactions. (B) dRP lyase and DNA
polymerase activities of pol b. Lane 1 is a control in the absence of pol b, lane 2 contained pol b without inhibitor, lanes 3–7 contained pol b with
increasing concentration of 1 (15, 31, 62, 125, and 250 lM, respectively). (C) The inhibition of pol b polymerase activity by compounds 1, 2, and 3.
(D) The inhibition of pol b dRP excision activity by compounds 1, 2, 3, and 4.
(Fig. 4).17,18,41,42 The b-elimination is relatively facile,
such that the Schiff base intermediates can be difficult
to observe and monitor. However, treatment with
NaBH4 can reduce and thereby stabilize the formed
imine, thus permitting its assay by polyacrylamide gel
electrophoresis.
Table 1. Inhibition of the catalytic activities of DNA polymerase b
Compound
Lyase inhibition
IC50 (lM)
Polymerase inhibition
IC50 (lM)
Oleanolic acid (1)
Edgeworin (2)
26.25 5.3
38.88 5.1
33.7 4.9
60.28 5.3
24.98 3.3
31.43 2.9
46.25 3.1
>250
Betulinic acid (3)
Stigmasterol (4)
To investigate the effect of inhibitors on the covalent
binding between pol b and DNA substrate, the forma-
tion of the DNA–pol b binary complex was studied
using a DNA binding mobility shift assay. Pol b was as-
sayed for its ability to bind with DNA substrates in the
presence or absence of inhibitors. A 30-32P labeled dou-
ble-stranded oligonucleotide containing a 50-incised AP
site was incubated with pol b and inhibitors. After
reducing the Schiff base intermediates by treatment with
NaBH4, the samples were analyzed by native polyacryl-
amide gel electrophoresis (Fig. 5). The result showed
that the amount of DNA–pol b binary complex was gen-
erally diminished as the concentrations of compounds
1–4 were increased (Fig. 5C). However, there were clear
differences between the four compounds. While there
Figure 4. Schiff base complex formed between incised apurinic acid
DNA lesion and lysine 72 of the N-terminal domain of polymerase b.

Z. Gao et al. / Bioorg. Med. Chem. 16 (2008) 4331–4340
4335
Compound 4 had only a slight effect on the enzyme–
DNA covalent binary complex.
oleanolic acid
A
-
-
1
-
+
2
pol
lane
+
+
4
+
5
+
6
+
7
2.5. Potentiation of the cytotoxicity of bleomycin
3
Three of the four compounds studied here were only
weakly cytotoxic to A549 cells (Table 2) while betulinic
acid (3) was moderately cytotoxic. Given that betulinic
acid has been shown to function at several critical cellu-
lar loci,43–45 the observation of greater cytotoxicity for
this compound is unsurprising. However, the modest
cytotoxicities observed for compounds 1, 2, and 4 sup-
port the thesis that inhibitors of pol b function need
not be strongly cytotoxic, and can potentially be used
for adjuvant chemotherapy. To examine the ability of
the compounds to potentiate the cytotoxicity of BLM,
each was tested at a concentration which exhibited min-
imal cytotoxic activity. A549 cells in log phase growth
were cultured with 1 mM BLM and 25 lM 1, 6 lM 2,
3 lM 3, or 6 lM 4; cell numbers were determined after
48 h. All four compounds were found to potentiate the
cytotoxic activity of blenoxane (Table 3). The extent
of potentiation of cytotoxicity ranged from a maximum
of 44% for oleanolic acid (1) to 15% for stigmasterol (4).
DNA-pol β binary complex
substrate
edgeworin
B
-
-
1
-
+
2
pol β
+
3
+
+
5
+
6
+
7
lane
4
DNA-pol β binary complex
2.6. Effect of compounds 1–4 on the expression of pol b in
A549 cells
To confirm that the potentiation of BLM cytotoxicity by
these compounds resulted from the inhibition of pol b
itself rather than an effect at the level of pol b expres-
sion, the expression of pol b was determined. Western
blot experiments performed in whole cell extracts col-
lected after 48-h incubation with 25 lM 1, 6 lM 2,
3 lM 3, and 6 lM 4, respectively, showed the same
expression of pol b in treatment groups, compared to
control. Anti-b actin antibody was used to assure the
equivalence of protein loading (Fig. 6).
substrate
C
120
1
2
3
4
110
100
90
80
70
60
50
40
30
20
10
0
2.7. Unscheduled DNA synthesis
Previous studies documented that in the presence of
2 mM nicotinamide and 10 mM hydroxyurea, optimal
Table 2. Cytotoxicity of polymerase b inhibitors toward cultured A549
cells
0
50
100
150
200
250
polymerase beta inhibitor (micromole)
Compound
Cytotoxicity IC50 (lM)
Figure 5. Inhibition of DNA–pol b binary complex by pol b inhibitors.
(A) The formation of DNA–pol b binary complex in the presence of
oleanolic acid (1). Lane 1, no pol b; lane 2, with pol b; lanes 3–7 pol b
with varying concentrations of 1 (1ane 3, 250 lM; lane 4, 200 lM; lane
5, 150 lM; lane 6, 100 lM; lane 7, 50 lM). (B) Effect of edgeworin (2)
on binary complex formation. Lane 1, no pol b; lane 2, with pol b;
lanes 3–7 pol b with varying concentration of 2 (1ane 3, 250 lM; lane
4, 200 lM; lane 5, 150 lM; lane 6, 100 lM; lane 7, 50 lM). (C)
Diminution of the concentration of the DNA–pol b binary complex by
1, 2, 3, and 4.
Oleanolic acid (1)
Edgeworin (2)
>100
80.2 2.1
6.65 2.3
98.2 2.5
Betulinic acid (3)
Stigmasterol (4)
Table 3. Potentiation of the cytotoxicity of bleomycin by polymerase b
inhibitors
Compound
Fold enhancement of growth
inhibition by 1 mM BLM
Oleanolic acid (1) (25 lM)
Edgeworin (2) (6 lM)
Betulinic acid (3) (3 lM)
Stigmasterol (4) (6 lM)
1.44 0.08
1.27 0.06
1.32 0.24
1.15 0.04
was virtually no enzyme–DNA binary complex present
at the two highest concentrations of 2 (Figs. 5B and
C), compound 1 had much less effect on the enzyme–
DNA covalent binary complex (cf Figs. 5A and B).

4336
control
Z. Gao et al. / Bioorg. Med. Chem. 16 (2008) 4331–4340
basis for the cellular effects of pol b inhibitors. More-
1
2
3
4
over, the assays employed historically for measuring
the two activities have employed rather different exper-
imental conditions,18 further complicating the analysis.
For some polymerase b inhibitors there are also ‘off-tar-
get effects’ such as DNA cleavage;54–56 while potentially
of great utility, these do not facilitate an analysis of
the cellular consequences of individual biochemical
effects.
β-actin
pol β
Figure 6. Western blot assay for quantification of pol b in A549 cells
treated with polymerase b inhibitors 1–4. The reaction conditions and
analyses are described in the Experimental section.
In the present study, we have employed four compounds
(1–4) isolated based on their lyase inhibitory activity.
Three of these compounds (1–3) also inhibited the
DNA polymerase activity of the enzyme (Fig. 3 and Ta-
ble 1). Critically, the measurement of inhibition was
made for both activities under identical experimental
conditions.
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
The use of pol b inhibitors as adjuvants for chemother-
apy with clinically used DNA damaging agents de-
mands that they be relatively non-toxic when
administered as single agents. In fact, compounds 1, 2,
and 4 were poorly cytotoxic toward cultured A549 cells
(Table 2), consistent with this requirement. Betulinic
acid (3), which functions at a number of cellular
loci,43–45 unsurprisingly exhibited somewhat greater
cytotoxicity. When these compounds were used at rela-
tively non-toxic concentrations to potentiate the cyto-
toxicity of 1 mM BLM, all of them did increase the
observed cyctotoxicity. Oleanolic acid (1), which exhib-
ited the strongest inhibition of both enzyme activities
(Table 1) and the least cytotoxicity toward A549 cells
as a single agent, afforded the best potentiation of
BLM (Table 3). Edgeworin (2) and betulinic acid (3),
which inhibited both pol b activities to comparable ex-
tents, but less potently than 1, also gave slightly less
potentiation of BLM than 1. Not surprisingly, stigmas-
terol (4), which inhibited the lyase activity of pol b
poorly, and failed to inhibit the polymerase activity
detectably, gave little potentiation of the cytotoxicity
of BLM toward A549 cells. Stigmasterol was also the
least potent of the four compounds in inhibiting
BLM-induced unscheduled DNA synthesis.
control BLM
1
-
2
-
3
-
4
-
1
+
2
+
3
+
4
+
-
+
BLM
Figure 7. Suppression of BLM-induced unscheduled DNA synthesis in
cultured A549 cells.
unscheduled DNA synthesis is measured with maximal
suppression of replicative synthesis.46,47 It has been
shown that following treatment of cells with a DNA
damaging agent, the amount of unscheduled DNA syn-
thesis represents DNA repair synthesis.48,49 This tech-
nique has been used previously to measure the increase
in cellular repair synthesis induced following BLM treat-
ment.50–53 In the present study, this technique was ap-
plied to determine the ability of DNA pol b inhibitors
to inhibit BLM-induced DNA repair synthesis. Treat-
ment with 25 lM 1 alone had little effect on unscheduled
DNA synthesis occurring in the cells. BLM, on the other
hand, induced DNA synthesis effectively following brief
treatment at 10 mM concentration. When the cells were
exposed to both 10 mM BLM and 25 lM compound 1,
there was strong inhibition of BLM-induced DNA re-
pair synthesis (Fig. 7). Compounds 2 (6 lM) and 3
(3 lM) also suppressed BLM-induced unscheduled
DNA synthesis, but a 6 lM concentration of 4 was less
effective in suppressing BLM-induced unscheduled
DNA synthesis.
The present data strongly support the thesis that inhib-
itors of polymerase b may be used to potentiate the cyto-
toxicity of DNA damaging agents in spite of their lack
of strong intrinsic cytotoxicity, and that the effect is
mediated at the level of impaired DNA repair. While
the present study does not permit a definitive analysis
of the relative effects on potentiation caused by inhibi-
tion of the polymerization and lyase activities, it may
be noted that some potentiation was obtained with stig-
masterol, a weak lyase inhibitor lacking any measurable
polymerase inhibitory activity.
3. Discussion
DNA–polymerase b is a single polypeptide chain of
39 kDa that folds into two domains, each of which con-
tains an active site. The dRP excision activity resides in
the N-terminal (8 kDa) domain, while the active site for
DNA synthesis resides within the larger (31 kDa) C-ter-
minal domain. While the lyase activity is believed to be
rate-limiting, suggesting that it may be the more logical
target for inhibition, many inhibitors of pol b can act on
both active sites,30,31,40 confounding efforts to define the
Finally, it may be noted that the compounds studied had
different effects on the stability of the DNA–pol b cova-
lent binary complex (Fig. 5), suggesting that they may
inhibit the lyase activity using somewhat different molec-
ular strategies. In this context, it may be noted that the
DNA–protein covalent binary complex formed as an
obligatory intermediate in the lyase reaction bears for-

Z. Gao et al. / Bioorg. Med. Chem. 16 (2008) 4331–4340
4337
mal analogy to the DNA–protein complex formed be-
tween topoisomerase I and its DNA substrate. This
complex is poisoned by the binding of camptothecin,
an interaction that is believed to form the basis for the
clinically useful antitumor activity of the camptothec-
ins.57 Thus, an alternative strategy for exploiting poly-
anti-b-actin antibody, hydroxyurea, nicotinamide, and
trichloroacetic acid were from Sigma Chemicals.
Ninety-six-well plates and 150 cm2 flasks were from
Fisher. Blenoxane, the clinically used mixture of ble-
omycins consisting predominantly of BLM A2 and
BLM B2, was a gift from Bristol Myers Squibb Pharma-
ceuticals. Bio-Rad protein assay dye reagent, PVDF
membrane, and Immun-Blot Assay kit were from Bio-
Rad. Whatman GF/C filters were from Fisher.
merase
b targeting for more effective antitumor
therapy might involve the identification of compounds
that bind tightly to the DNA–pol b covalent binary
complex. In fact, bioactive isomalabaricone triterpe-
noids have been found to exhibit this property, and
exhibited fairly strong cytotoxicity toward A2780 ovar-
ian cancer cells as single agents.58
Denaturing, native gels and immunoblots were quanti-
fied by use of Molecular Dynamics ImageQuant version
5.0. Scintillation counting was performed on a Beckman
LS-100C instrument using Beckman Ready Safe scintil-
lation fluid. The microplate reader was from Bio-Rad.
Distilled, deionized water from a Milli-Q system was
used for all aqueous manipulations.
4. Experimental
4.1. General methods and materials
4.2. Chemical synthesis of edgeworin (2)
Compounds 1 and 4 were purchased from Sigma Chem-
icals; compound 3 was from Aldrich. Compound 2 was
synthesized as described below (Scheme 1). Experiments
requiring anhydrous conditions were performed using
flame-dried glassware and under a nitrogen atmosphere.
Chemical reagents were purchased from Aldrich Chem-
ical Co. Anhydrous CH2Cl2 was distilled from CaH2
4.2.1. 2-Hydroxy-4-(2-methoxyethoxymethoxy)benzalde-
hyde (6). To a stirred suspension containing 3.0 g
(21.7 mmol) of 2,4-dihydroxybenzaldehyde (5) in
20 mL of dry CH2Cl2 was added 3.78 mL (2.80 g,
21.7 mmol) of N,N-diisopropylethylamine, followed by
the dropwise addition of 2.48 mL (2.71 g, 21.7 mmol)
of MEMCl over a period of 5 min. After stirring at
room temperature under nitrogen overnight, the reac-
tion mixture was partitioned in 400 mL of 1:1 methylene
chloride–water; the aqueous phase was then extracted
using three 100-mL portions of methylene chloride.
The combined organic extract was washed with
200 mL of brine solution and then dried over MgSO4,
filtered and concentrated under diminished pressure.
The residue was then purified by flash chromatography
on a silica gel column (30 · 4 cm); elution with 1:1 ethyl
acetate–hexanes gave the desired product 6 as a colorless
oil: yield 3.96 g (81%); silica gel TLC Rf 0.8 (1:1 ethyl
acetate–hexanes); 1H NMR (CDCl3) d 3.34 (s, 3H),
3.55 (m, 2H), 3.81 (m, 2H), 5.30 (s, 2H), 6.59 (s, 1H),
6.63 (dt, 1H, J = 8.7 and 1.8 Hz), 7.44 (d, 1H,
J = 8.7 Hz), 9.70 (s, 1H), and 11.34 (s, 1H); 13C NMR
(CDCl3) d 59.3, 68.5, 71.7, 93.3, 103.6, 109.2, 116.2,
135.6, 164.3, 164.5 and 194.9.
1
prior to use. H and 13C NMR spectra were recorded
on a Varian Unity 500 MHz NMR spectrometer and a
Varian Gemini 300 NMR spectrometer. All d values
are given in ppm and are relative to tetramethylsilane;
J values are recorded in Hz. High resolution mass spec-
tral data were obtained at the Michigan State University
Mass Spectrometry Facility, which is supported, in part,
by a Grant (DRR-00180) from the Biotechnology Re-
search Technology Program, National Center for Re-
search Resources, National Institutes of Health. Thin
layer chromatography was performed using Merck silica
gel F254 pre-coated plates with spots visualized using UV
light (254 nm) or by dipping the plates in a vanillin
staining reagent. Silicycle (Quebec City, Quebec) ultra
pure silica gel, mesh size 35–75 lm, was used for column
chromatography.
These compounds were initially dissolved in DMSO for
the DNA pol b lyase–polymerase assay, DNA binding
mobility shift assay, and in DMSO for the cell culture
studies with a final DMSO concentration <0.05% in
each culture. Recombinant rat liver pol b was a gift from
Dr. Xiangyang Wang and Hongge Wang, prepared as
described previously.59,60 The oligodeoxyribonucleotide
(34-mer) was purchased from Integrated DNA Technol-
ogies. [a-32P]ddATP (5000 Ci/mmol), [a-32P]dCTP
(6000 Ci/mmol) and [3H]thymidine (80 Ci/mmol) were
purchased from Amersham Pharmacia Biotech. AP
endonuclease and anti-human DNA polymerase b anti-
body were purchased from Trevigen. Uracil–DNA gly-
cosylase was from New England Biolabs; terminal
deoxynucleotidyltransferase was obtained from Invitro-
gen. The human lung carcinoma cell line A549 was ob-
tained from the American Type Culture Collection.
Kaighn’s modification of Ham’s F2 medium, Hank’s
balanced salt solution, donor horse serum, aprotinin,
phenylmethylsulfonyl fluoride (PMSF), dithiothreitol,
4.2.2. Ethyl 2-(2-oxo-2H-chromen-7-yloxy)acetate (8).
To a solution containing 5.00 g (30.9 mmol) of 7-
hydroxycoumarin (7) and 5.15 g (30.9 mmol) of ethyl
bromoacetate in 150 mL of acetone was added 4.41 g
(31.5 mmol) of anhydrous K2CO3. The reaction mixture
was heated at reflux for 4 h, after which time the cooled
reaction mixture was filtered to remove the precipitate.
The filtrate was then concentrated under diminished
pressure to afford a crude colorless solid. The crude
material was crystallized from abs EtOH to afford 8 as
colorless needles: yield 7.64 g (100%); silica gel TLC Rf
1
0.48 (1:1 ethyl acetate–hexanes); H NMR (CDCl3) d
1.15 (t, 3H, J = 6.9 Hz), 4.11 (q, 2H, J = 6.9 Hz), 4.76
(s, 2H), 6.12 (d, 1H, J = 9.6 Hz), 6.76 (d, 1H,
J = 1.8 Hz), 6.83 (dd, 1H, J = 8.7 and 2.7 Hz), 7.47 (d,
1H, J = 8.7 Hz) and 7.77 (d, 1H, J = 9.6 Hz); 13C
NMR (CDCl3) d 14.1, 61.4, 65.3, 102.2, 111.1, 113.0,
113.4, 130.0, 145.0, 154.7, 160.5, 161.0 and 167.6.

4338
Z. Gao et al. / Bioorg. Med. Chem. 16 (2008) 4331–4340
4.2.3. 2-(2-Oxo-2H-chromen-7-yloxy)acetic acid (9). To
a solution containing 7.64 g (30.8 mmol) of 8 in
150 mL of ethanol was added 22 mL of 5% aq NaOH.
The reaction mixture was stirred at reflux for 2 h and
the cooled reaction mixture was concentrated under
diminished pressure. The residue was diluted with
150 mL of H2O and acidified with 6 N HCl. Upon
becoming acidic a thick white ppt formed; this was col-
lected by filtration, dried and crystallized from abs
EtOH to afford 9 as colorless needles: yield 6.20 g
4.3. Preparation of gapped DNA substrates
The 34-mer oligodeoxyribonucleotide (9 nmol) contain-
ing a uridine at position 16 was labeled at its 30-end with
terminal deoxynucleotidyltransferase using 50 lCi of
[a-32P]ddATP (5000 Ci/mmol). The product was then
purified using a 20% denaturing polyacrylamide gel.
The band of interest was visualized by autoradiography
and excised from the gel. After recovery from the gel
matrix by the ‘crush and soak’ method, the oligodeoxy-
ribonucleotide was annealed to its complementary
strand by heating the solution at 70 ꢁC for 3 min, fol-
lowed by slow cooling to 25 ꢁC.
1
(91%); H NMR (DMSO-d6) d 4.86 (s, 2H), 6.32 (d,
1H, J = 9.6 Hz), 6.97 (s, 1H), 7.00 (s, 1H), 7.66 (d, 1H,
J = 9.0 Hz), 8.02 (d, 1H, J = 9.6 Hz) and 13.19 (br s,
1H); 13C NMR (DMSO-d6) d 65.6, 102.2, 111.3, 113.3,
113.5, 130.2, 145.0, 155.9, 161.0, 161.6 and 170.3.
An AP site was created in a reaction mixture (200 lL total
volume) that contained 40 pmol [a-32P]-labeled double-
stranded oligodeoxynucleotide having a deoxyuridine at
position 16 in 10 mM Hepes–KOH, pH 7.4, 50 mM
KCl, 5 mM MgCl2, 10 mg/mL BSA, 3 units of AP endo-
nuclease, and 2.4 U of uracil–DNA glycosylase. After
incubation at 37 ꢁC for 2 min, the [a-32P]-labeled dou-
ble-stranded oligodeoxynucleotide was ready for the
DNA pol b lyase–polymerase assay and DNA binding
mobility shift assay.
4.2.4. 7-(2-Methoxyethoxymethoxy)-3-(2-oxo-2H-chro-
men-7-yloxy)chromen-2-one (10). To a solution contain-
ing 0.480 g (2.18 mmol) of 9 were added 0.61 mL
(0.440 g, 4.36 mmol) of triethylamine and 0.668 g
(6.54 mmol) of acetic anhydride in 10 mL of dry
DMF. The reaction mixture was stirred at 110 ꢁC under
nitrogen for 18 h. The cooled reaction mixture was
poured into 150 mL of ice water, and extracted with
three 100-mL portions of ethyl acetate. The combined
organic layer was washed with two 100-mL portions
of brine and dried over MgSO4. The solvent was concen-
trated under diminished pressure. Purification by flash
chromatography on a silica gel column (28 · 3 cm), elu-
tion with 10:1 chloroform–methanol, gave the desired
product 10 as a brown solid: yield 492 mg (55%); silica
gel TLC Rf 0.51 (10:1 chloroform–methanol); 1H
NMR (DMSO-d6) d 3.28 (s, 3H), 3.56 (m, 2H), 3.80
(m, 2H), 5.43 (s, 2H), 6.43 (d, 1H, J = 9.6 Hz), 7.13
(dd, 1H, J = 8.4 and 2.1 Hz), 7.19 (d, 1H, J = 2.7 Hz),
7.21 (d, 1H, J = 2.1 Hz), 7.30 (d, 1H, J = 2.7 Hz), 7.68
(d, 1H, J = 8.7 Hz), 7.78 (d, 1H, J = 8.4 Hz), 8.00 (s,
1H) and 8.11 (d, 1H, J = 9.6 Hz); 13C NMR (DMSO-
d6) d 59.3, 68.4, 71.7, 92.6, 107.3, 109.0, 116.3, 118.8,
119.0, 119.3, 119.8, 134.9, 135.3, 136.5, 140.7, 149.6,
158.1, 159.3, 162.4, 165.1, 165.5 and 166.1; mass spec-
trum (electrospray) m/z 410.8 (M)+, theoretical m/z
410.1 (M)+.
4.4. Pol b lyase–polymerase assay
The lyase–polymerase reactions were performed as fol-
lows. The reaction mixture (10 lL total volume) con-
tained 50 mM Hepes–KOH, pH 7.4, 20 mM KCl,
2 mM DTT, 4 mM ATP, 5 mM MgCl2, 20 lM each
of dGTP, dTTP, dATP and 67 nM [a-32P]dCTP
(6000 Ci/mmol), 200 nM 30-labeled DNA, 3 nM pol b
and pol b inhibitors (15–250 lM). After incubation
at 37 ꢁC for 30 min, the reaction was terminated by
the addition of 1 lL of 0.5 M EDTA and the product
was stabilized by the addition of NaBH4 to a final
concentration of 50 mM and incubation at room tem-
perature for 10 min. After additional incubation at
75 ꢁC for 5 min the reaction products were separated
on a 20% denaturing polyacrylamide gel and visualized
by autoradiography. Lyase and polymerase products
were quantified using ImageQuant software, after
scanning the gel by the use of a Molecular Dynamics
phosphorimager.
4.2.5. 7-Hydroxy-3-(2-oxo-2H-chromen-7-yloxy)chro-
men-2-one (edgeworin) (2). To a stirred solution contain-
ing 125 mg (0.304 mmol) of 10 in 10 mL of dry CH2Cl2
was added 61 mg (0.30 mmol) of bromocatechol borane.
The reaction mixture was stirred at room temperature
under nitrogen for 3 h. The reaction mixture was then
filtered, and the precipitate was washed with three 30-
mL portions of 1 N HCl, and dried to yield 2 as a gray-
ish white solid: yield 91 mg (83%); silica gel TLC Rf 0.45
(10:1 methanol–chloroform); 1H NMR (DMSO-d6) d
6.35 (d, 1H, J = 9.0 Hz), 6.80 (s, 1H), 6.83 (d, 1H,
J = 8.0 Hz), 7.11 (dd, 1H, J = 8.5 and 1.5 Hz), 7.18 (d,
1H, J = 1.5 Hz), 7.51 (d, 1H, J = 8.5 Hz), 7.70 (d, 1H,
J = 9.5 Hz), 7.92 (s, 1H), 8.02 (d, 1H, J = 9.5 Hz) and
10.56 (br s, 1H); 13C NMR (DMSO-d6) d 107.6, 109.3,
116.3, 118.8, 119.0, 119.3, 119.8, 134.9, 135.3, 136.5,
140.7, 149.6, 158.9, 160.5, 162.4, 165.2, 165.4 and
166.1; mass spectrum (FAB), m/z 323.0557 (M+H)+
(C18H11O6 requires 323.0553).
4.5. DNA binding mobility shift assay
The affinity of pol b for a DNA substrate containing an
AP site at position 16 was studied using a gel mobility
assay18,61 in the presence and in the absence of pol b
inhibitors. Pol b (30 nM) was incubated with 200 nM
radiolabeled DNA substrate, and pol b inhibitors (30–
500 lM) in buffer (10 lL total volume) containing
10 mM Hepes–KOH, pH 7.4, 50 mM KCl, 5 mM
MgCl2, and 10 mg/mL BSA at room temperature for
5 min. The product was stabilized by the addition of
0.5 M NaBH4 to a final concentration of 50 mM. After
10 min, the samples were loaded onto a 12% non-dena-
turing polyacrylamide gel, run for 2 h at 30 W and visu-
alized by autoradiography. The 32P-labeled substrate
bound to pol b exhibited an electrophoretic mobility dis-
tinguishable from that without pol b. Bound protein was

Z. Gao et al. / Bioorg. Med. Chem. 16 (2008) 4331–4340
4339
quantified using ImageQuant software, after scanning
the gel using a phosphorimager.
4.10. Unscheduled DNA synthesis assay
The unscheduled DNA synthesis assay was based on
that described by Barra63 with some modifications.
A549 cells (ꢁ5 · 105), were placed into 25 mL flasks
and treated with 10 mM bleomycin. After incubation
at 37 ꢁC under a humidified 5% CO2 atmosphere for
30 min, the appropriate amount of dimethylsulfoxide,
10 mM hydroxyurea, 2 mM nicotinamide, and 5 lCi of
[3H]thymidine (80 Ci/mmol) was added to each flask,
and pol b inhibitors 1–4 were added to the flasks (tripli-
cates were used for each drug concentration). The sam-
ples were incubated for 3 h. Three milliliters of ice cold
0.9% saline was added to each sample to terminate the
reaction. For determination of radioactivity, the cells
were collected by centrifugation and the media were re-
moved by aspiration. Three milliliters of ice cold 5% tri-
chloroacetic acid was added and the samples were
incubated on ice for 30 min. Cell pellets were resus-
pended by brief sonication; the samples were collected
on Whatman GF/C filters and washed 5 times with ice
cold 5% trichloroacetic acid and once with ice cold
95% ethanol. Filters were dried and used for determina-
tion of radioactivity.
4.6. Cell culture conditions
Human lung carcinoma A549 cells were routinely grown
at 37 ꢁC in a 5% CO2 incubator in Kaighn’s modifica-
tion of Ham’s F12 medium with 2 mM L-glutamine,
supplemented with 1.5 g/L sodium bicarbonate and
10% fetal calf serum.
4.7. Cell growth inhibition assay
An MTT assay62 was performed to monitor cell growth
inhibition. Two hundred microliter aliquots of cell cul-
ture medium, containing approximately 1.0 · 104 A549
cells, were placed in 96-well culture plates. The following
day, the cells were exposed to the desired concentration
of bleomycin, with or without pol b inhibitors, for 1 h.
The cells were then washed with Hank’s balanced salt
solution. Fresh medium with or without pol b inhibitors
was added to the cells, which were incubated for another
48 h at 37 ꢁC under a 5% CO2 atmosphere. The culture
medium was removed and 10 lL of MTT reagent was
added to each well. Following incubation at 37 ꢁC for
4 h, 200 lL DMSO was added to each well. The con-
tents were mixed thoroughly and the OD570 value was
measured using a microplate reader. The results were ex-
pressed as ‘% control growth’ according to the formula
[(Nc ꢀ Ne)/Nc] · 100%, where Nc is the OD570 value ob-
tained in the control cultures and Ne is the OD570 value
in the treated cultures.
Acknowledgment
This work was supported by NIH Research Grant
CA50771, awarded by the National Cancer Institute.
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