Inhibitors of DNA polymerase β from Schoepfia californica

Article abstract of DOI:10.1039/a807053i

Fractionation of a hexane extract prepared from the plant Schoepfia californica was carried out by bioassay guided fractionation, affording five inhibitors of DNA polymerase β; four of these were shown to be anacardic acid and structurally related derivatives, while the fifth was oleic acid.

Full text of DOI:10.1039/a807053i

Inhibitors of DNA polymerase b from Schoepfia californica
Jie Chen, Yu-Huan Zhang, Li-Kai Wang, Steven J. Sucheck, Angela M. Snow and Sidney M. Hecht*
Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901, USA.
E-mail: sidhecht@virginia.edu
Received (in Corvallis, OR, USA) 8th September 1998, Accepted 18th November 1998
OH
Fractionation of a hexane extract prepared from the plant
Schoepfia californica was carried out by bioassay guided
CO2H
fractionation, affording five inhibitors of DNA polymerase
b; four of these were shown to be anacardic acid and
structurally related derivatives, while the fifth was oleic
acid.
1
OH
In addition to their role in DNA replication, at least three
CO2H
eukaryotic DNA polymerases participate in the repair of
damaged DNA.1 Given that elements in our environment such
as sunlight and pollution provide a steady source of DNA
damage, the repair of damaged DNA must be essential to
2
maintain cell viability.2 There is, however, at least one context
in which DNA damage repair is not beneficial, namely
following the clinical administration of antitumor agents that
HO2C
function by damaging DNA. The repair of DNA damage
inflicted by these agents on cancer cells must mitigate their
therapeutic potency.
3
OH
DNA polymerase b is believed to have primary responsibility
for supporting short patch DNA base excision repair.3–5 This
CO2H
pathway has specifically been implicated in the repair of DNA
damage caused by agents such as cis-platinum, bleomycin and
neocarzinostatin.3,4,6–8 To identify non-toxic compounds that
can block the function of DNA polymerase b selectively, and
4
OH
which may thereby be able to potentiate the action of anticancer
agents such as bleomycin, we have surveyed plant extracts for
CO2H
naturally occurring inhibitors of polymerase b.9 Presently we
describe five natural inhibitory principles from Schoepfia
californica that can potentiate the action of bleomycin and cis-
platinum.
5
A hexane extract prepared from the twigs, leaves and fruit of
Schoepfia californica was partitioned between hexane and
MeOH; the hexane phase contained most of the polymerase b
inhibitory activity. Successive bioassay-guided fractionations
on two silica gel columns and then by preparative silica gel TLC
afforded an active fraction that effected 94% inhibition of
polymerase b function at 5 mg ml21 vs. 38% inhibition for the
initial extract.† Fractionation (C8 column, 80% MeOH) then
afforded pure compounds 1–5; all were obtained as colorless
needles except 3, which was a colorless oil.
The structures were determined by spectroscopic analysis.
Compound 1 was found to have a molecular ion at m/z
374.2822, establishing the molecular formula as C24H38O3. The
13C NMR spectrum of 1 displayed six resonances (d 110.31,
115.87, 122.74, 135.38, 147.76 and 163.65) assigned to an
aromatic ring and a resonance at d 175.67 assigned to a
carboxylate carbon, thus accounting for five of the six units of
unsaturation implied by the molecular formula. The presence of
three aromatic resonances in the 1H NMR spectrum at d 6.77 (d,
J 7.5), 6.87 (d, J 7.5) and 7.36 (dd, J 7.5, 7.5) indicated that the
aromatic ring was trisubstituted; the multiplicities of the proton
resonances were consistent with a 1,2,3-trisubstituted benze-
noid system.‡
163.65). The ortho relationship of this OH to the ring
carboxylate was supported by the upfield shift of the ring C
atom (d 110.31), consistent with the known (d 12.7) shielding
effect of ortho phenolic OH groups.15
These data suggested that 1 was a 6-substituted salicylic acid;
the 1H and 13C NMR spectra were consistent with an
unbranched alkyl substituent containing a single double bond.
The double bond configuration was assigned as cis based on the
13C NMR resonances of the allylic carbon atoms.16 Determina-
tion of the olefin position was made by modification of
published procedures.17
The structure assigned to 1 was confirmed by chemical
synthesis, as outlined in Scheme 1. Ethyl 2-hydroxy-6-me-
thylbenzoate 618 was protected as the corresponding MEM
ether19 and the ethyl ester was saponified, affording carboxylic
acid derivative 7 as colorless prisms (71% yield). Treatment of
7 with LDA (2 equiv., THF), followed by admixture of (9Z)-
hexadec-9-enyl bromide at 0 °C,20 afforded homologated
salicylic acid derivative 8 (68% yield). MEM deprotection19
provided 114 as colorless needles (80% yield). The synthetic and
naturally derived samples were found to be identical as judged
1
The chemical shift of the carboxylate at d 175.67 was typical
of a carboxylate attached to an aromatic ring; the downfield
shift of the aromatic H at d 7.36 (dd) suggested the para
relationship of this H to the carboxylate moiety. That the
remaining O atom was present as part of a phenolic substituent
was suggested by the downfield shift of one ring carbon (d
by their behavior on silica gel TLC, and by their identical H
and 13C NMR and mass spectra, as well as their equal abilities
to inhibit DNA polymerase b (vide infra).
Compounds 2, 4, and 5 were found to have spectroscopic
properties most similar to 1. The structures were assigned in the
same fashion as for 1, a process facilitated by previous reports
Chem. Commun., 1998, 2769–2770
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OH
OMEM
SmithKline Beecham Pharmaceuticals, for the high resolution
mass spectrometry data. We thank Dr Akio Matsukage, Aichi
Cancer Center Research Institute, Nagoya, for providing us with
a source of cloned rat DNA polymerase b. This work was
supported by NIH Research Grant CA50771 from the National
Cancer Institute.
CO2Et
CH3
CO2H
i,ii
CH3
6
7
iii,iv
OR
Notes and references
CO2H
† Inhibition of polymerase b was assayed in 62.5 m
8.6, containing 10 m MgCl2, 1 m
dithiothreitol, 50 mg ml21 of BSA, 6.25
deoxynucleotide triphosphates including [3H]thymidine triphosphate
M ammediol buffer, pH
M
M
m
M
and 12.5 mg of DNase I-treated calf thymus DNA. The reactions were
initiated with 0.2 mg of rat liver DNA polymerase b (ref. 10,11), incubated
at 37 °C for 1 h, and monitored as described (ref. 12).
8 R = MEM
1 R = H
v
Scheme 1 Reagents and conditions: i, MEMCl, Pri2NEt; ii, ButOK, 61%
over 2 steps; iii, LDA; iv, (9Z)-hexadec-9-enyl bromide, 68% over 2 steps;
v, ZnBr2, 80%.
‡ The assignments of the H3, H4 and H5 resonances in the NMR spectra of
1, 2, 4 and 5 were further supported by HMQC spectroscopy. The spectra
also proved useful for assigning proton and carbon resonances in the
aliphatic substituents of 1–5, used in combination with the full 13C NMR
assignments of a number of common fatty acids (ref. 13) and the 1H-coupled
13C NMR spectra of several compounds structurally related to 1, 2, 4 and 5
(ref. 14).
§ Also determined was the selectivity of inhibition. A number of derivatives
were found to be essentially inactive as inhibitors of calf thymus DNA
topoisomerase I, AMV reverse transcriptase, DNA polymerase I (Klenow
fragment) and restriction endonuclease HindIII at concentrations at which
those compounds strongly inhibited DNA polymerase b.
of each of these compounds.21 Verification of the structure
assignments was accomplished by total chemical synthesis of
each. The mass spectrum of 3 contained a molecular ion having
1
m/z 282. The H and 13C NMR spectra of 3 were found to be
identical with those of oleic acid.13
The activities of 1–5 as inhibitors of DNA polymerase b are
shown in Table 1. Compound 1 exhibited an IC50 of 1.4 m in
M
the absence of bovine serum albumin (BSA). BSA reduced the
IC50 to 9 m , undoubtedly reflecting the binding of 1 to this
(basic) protein. Unsaturated analogues 1 and 4 were found to
have the greatest potencies in the presence of BSA (9 and 19 m
M
1 See, e.g. T. S.-F. Wang, Annu. Rev. Biochem., 1991, 60, 513; Y.
Matsumoto and K. Kim, Science, 1995, 269, 699; A. Sancar, Annu. Rev.
Biochem., 1996, 65, 43.
2 B. N. Ames, M. K. Shigenaga and T. M. Hagen, Proc. Natl. Acad. Sci.
U.S.A., 1993, 90, 7915.
3 M. R. Miller and D. N. Chinault, J. Biol. Chem., 1982, 257, 10204.
4 J. A. DiGiuseppe and S. L. Dresler, Biochemistry, 1989, 28, 9515.
5 R. K. Singhal, R. Prasad and S. H. Wilson, J. Biol. Chem., 1995, 270,
949.
M,
respectively), and to be reproducibly more active than saturated
derivatives 2 and 5. Oleic acid, which is a simple fatty acid, was
active only at significantly higher concentration. Methylation of
the phenol or carboxylic acid moieties essentially eliminated
inhibitory activity as did conversion of the carboxylate of 5 to
the respective carboxamide, demonstrating that the anacardic
acids are specific inhibitors rather than simple denaturants.§
6 S. Seki and T. Oda, Carcinogensis, 1988, 9, 2239.
7 A. J. Fornace, Jr., B. Zmudzka, M. C. Hollander and S. H. Wilson, Mol.
Cell. Biol., 1989, 9, 851.
Table 1 Inhibition of rat liver DNA polymerase by 1–5 and structurally
related compoundsa
8 J.-S. Hoffmann, M.-J. Pillaire, G. Maga, V. Podust, U. Hübscher and G.
Villani, Proc. Natl. Acad. Sci. U.S.A., 1995, 92, 5356.
9 Polymerase b inhibitors of moderate potency have been reported,
although none has been used to potentiate the action of DNA damaging
antitumor agents. See K. Ono, H. Nakane and M. Fukushima, Eur. J.
Biochem., 1988, 172, 349; Y. Mizushina, H. Yagi, N. Tanaka, T.
Kurosawa, H. Seto, K. Katsumi, M. Onoue, H. Ishida, A. Iseki, T. Nara,
K. Morohashi, T. Horie, Y. Onomura, M. Narusawa, N. Aoyagi, K.
Takami, M. Yamaoka, Y. Inoue, A. Matsukage, S. Yoshida and K.
Sakaguchi, J. Antibiot., 1996, 49, 491; H.-D. Sun, S.-X. Qiu, L.-Z. Lin,
Z.-Y. Wang, Z.-W. Lin, T. Pengsuparp, J. M. Pezzuto, H. H. S. Fong,
G. A. Cordell and N. R. Farnsworth, J. Nat. Prod., 1996, 59, 525; H.
Ishiyama, M. Ishibashi, A. Ogawa, S. Yoshida and J. Kobayashi, J. Org.
Chem., 1997, 62, 3831; N. Tanaka, A. Kitamura, Y. Mizushina, F.
Sugawa and K. Sakaguchi, J. Nat. Prod., 1998, 61, 193.
10 T. Date, M. Yamaguchi, F. Hirose, Y. Nishimoto, K. Tanihara and A.
Matsukage, Biochemistry, 1988, 27, 2983.
11 S. G. Widen, P. Kedar and S. H. Wilson, J. Biol. Chem., 1988, 263,
16992.
12 A. M. Snow, Ph.D. Thesis, University of Virginia, 1995.
13 J. G. Batchelor, R. J. Cushley and J. H. Prestegard, J. Org. Chem., 1974,
39, 1698.
14 H. Itokawa, N. Totsuka, K. Nakahara, K. Takeya, J.-P. Lepoittevin and
Y. L. Asakawa, Chem. Pharm. Bull., 1987, 35, 3016.
15 D. F. Ewig, Org. Magn. Reson., 1979, 12, 499.
16 See, e.g. F. D. Gustone, M. R. Pollard, C. M. Scrimgeour and H. S.
Vedanayagam, Chem. Phys. Lipids, 1977, 18, 115; J. W. deHaan and
L. J. M. de Ven, Org. Magn. Reson., 1979, 5, 147; R. Rossi, A. Carpita,
M. G. Quirici and C. A. Varacini, Tetrahedron, 1982, 38, 639.
17 See e.g. J. R. Barr, R. T. Scannell and K. Yamaguchi, J. Org. Chem.,
1989, 54, 494.
18 F. M. Hauser and S. A. Pogany, Synthesis, 1980, 814.
19 E. J. Corey, J.-L. Gras and P. Ulrich, Tetrahedron Lett., 1976, 11,
809.
20 P. L. Creger, J. Am. Chem. Soc., 1970, 92, 1396. The bromide itself was
accessible from the corresponding commercially available alcohol by
treatment with Ph3PBr2 (P. E. Sonnet, Synth. Commun., 1976, 6, 21).
21 See, e.g. Y. Yamagiwa, K. Ohashi, Y. Sakamoto, S. Hirakawa, T.
Kamikawa and I. Kubo, Tetrahedron, 1987, 43, 3387; R. Zehnter and H.
Gerlach, Liebigs Ann., 1995, 2209.
Compound
IC50/mM
1
1
2
3
4
5
1.4b
9
30
72
19
25
b
a
Determined as described in footnote †. Determined in the absence of
bovine serum albumin.
Compound 1 and a few structurally related species were
evaluated in more detail as DNA polymerase b inhibitors. In
both short and long term mammalian cell culture, these
compounds potentiated the action of DNA damaging agents
such as bleomycin (Table 2) and cis-platinum, and inhibited
bleomycin-induced unscheduled DNA synthesis. They also
blocked DNA polymerase b-mediated gap filling of a DNA
duplex substrate.12 Thus specific inhibitors of DNA polymerase
b may well be able to potentiate the effects of the DNA damage
caused by clinically used antitumor agents that function at this
locus.
Table 2 Potentiation of the cytotoxicity of bleomycin (BLM) by 1a
Compounds present
—
Viable cells (% of control)
100
96
98
50 m
50 m
50 m
M
M
M
BLM
1
BLM + 50 m
M
1
68
a
P388D, cells were cultured in suspension in the presence of the test
compounds for 6 h, then assessed for viability by trypan blue exclusion
staining.
We thank Ms Shelley Starck, University of Virginia, for
biochemical evaluation of these agents and Dr Mark Hemling,
Communication 8/07053I
2770
Chem. Commun., 1998, 2769–2770