Total synthesis and DNA-cleaving properties of thiarubrine C

Full text of DOI:10.1021/jo981849p

8644
J . Org. Chem. 1998, 63, 8644-8645
Sch em e 1
Sch em e 2
Tota l Syn th esis a n d DNA-Clea vin g P r op er ties
of Th ia r u br in e C
Yamin Wang and Masato Koreeda*,†
Department of Chemistry, The University of Michigan,
Ann Arbor, Michigan 48109-1055
Tonika Chatterji and Kent S. Gates*,‡
Departments of Chemistry and Biochemistry, University of
MissourisColumbia, Columbia, Missouri 65211
Received September 11, 1998
The thiarubrines are structurally intriguing antibiotics
that have been isolated from a variety of plant sources and
have been found to possess interesting antibacterial, anti-
fungal, anticancer, and antiviral properties.1,2 The mode of
action of 1,2-dithiins such as the thiarubrines has been
considered;1,3,4 however, the cellular target(s) involved in the
antibiotic action of these compounds remains uncertain.
It has been reported that thiarubrine C5 causes DNA
strand scission,6 although to our knowledge relevant experi-
mental conditions and data supporting this statement have
not been published. It was further suggested that this
process might involve an enediyne-like7 cyclization of the
diene-yne side-chain of thiarubrine C that generates, with
or without loss of a hydrogen radical, DNA-cleaving radical-
(s) (see Scheme 1).6 Intrigued by the unusual, seemingly
high-energy process proposed to explain DNA damage by
thiarubrine C, we examined DNA cleavage by this class of
natural products. We report here on the first total synthesis
and DNA-cleaving properties of thiarubrine C.
The synthesis of thiarubrine C (1) was achieved following
the strategy employed for the synthesis of thiarubrine A.8
Thus, the element of dissymmetry was introduced by
monoalkylation of a symmetrical intermediate such as 2 via
its dianion. The precursor to this dianion, tetrabromide 3,
was obtained in three steps in 75% overall yield from 2,4-
hexadiyne-1,6-diol (Scheme 2).8 Treatment of the dianion 4,
generated in situ from 3 with 4.0 equiv of n-BuLi, with 1.1
equiv of MeI, provided monomethyl derivative 5 in 40% yield
from 3, together with dimethylated and nonmethylated,
protonated products in 18 and 15% yield, respectively. This
apparent lack of control for the selective monomethylation
of the dilithio acetylide generated from tetrabromide 3 must
be the reflection of ineffective orbital overlap between the
two carbanionic orbitals with the rest of the diyne-diene
π-system. However, in view of its directness and overall
yield, this approach was judged preferable over an alternate
selective sequence which would have added a few extra
steps. The sequential Pd(0)-mediated alkenylation of 5
furnished the (Z)-diene-yne 7 in 59% overall yield. Depro-
tection to regenerate the two thiol groups proved quite
problematic. Treatment of 7 with excess TBAF resulted in
only the mono-deprotection, and the subsequent I2-catalyzed
oxidation provided the two dimeric disulfides. This problem
of sluggish reactivity toward fluoride ion-mediated depro-
tection was circumvented by activation through acylation
of the sulfur atoms. Thus, treatment of 7 with TBAF and
trifluoroacetic anhydride followed by oxidation of the result-
ing bis-thioenolate with iodine provided thiarubrine C (1)
in 24-34% overall yield from 7. Apparently, the bis-SCOCF3
intermediate was readily hydrolyzable to the bis-thioenolate
during the aqueous sodium bicarbonate treatment prior to
the oxidation.
† Masato Koreeda: phone (734) 764-7371; fax (734) 764-7371; e-mail:
koreeda@umich.edu.
‡ Kent S. Gates: phone (573) 882-6763; fax (573) 882-2754; e-mail:
GatesK@missouri.edu.
(1) Bierer, D. E.; Dener, J . M.; Dubenko, L. G.; Gerber, R. E.; Litvak, J .;
Peterli, S.; Peterli-Roth, P.; Truong, T. V.; Mao, G.; Bauer, B. E. J . Med.
Chem. 1995, 38, 2628-2648.
(2) Towers, G. H. N.; Page, J . E.; Hudson, J . B. Curr. Org. Chem. 1997,
1, 395-414.
(3) Guillet, G.; Philogene, J . R.; O’Meara, J .; Durst, T.; Arnason, J . T.
Phytochemistry 1997, 46, 495-498.
The DNA-cleaving properties of thiarubrine C thus ob-
tained were examined using a plasmid-based assay in which
strand scission causes the conversion of supercoiled DNA
(form I) to the open circular form (form II). It was found that
thiarubrine C (concentrations up to 1 mM) does not ef-
ficiently cause DNA strand scission in our assays, thus
suggesting that the previously proposed cyclization mech-
anism6 is not significant under these conditions (Figure 1,
(4) Constabel, C. P.; Towers, G. H. N. Planta Med. 1989, 55, 35-37.
(5) (a) Norton, R. A.; Finlayson, A. J .; Towers, G. H. N. Phytochemistry
1985, 24, 356-357. (b) Constabel, C. P.; Balza, F.; Towers, G. H. N. Ibid.
1988, 27, 3533-3535.
(6) Freeman, F.; Aregullin, M.; Rodriguez, E. Rev. Heteroatom Chem.
1993, B, 1-19.
(7) Nicolaou, K. C.; Smith, A. L.; Yue, E. W. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 5881-5888.
(8) Koreeda, M.; Yang, W. J . Am. Chem. Soc. 1994, 116, 10793-10794.
10.1021/jo981849p CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/10/1998

Communications
J . Org. Chem., Vol. 63, No. 24, 1998 8645
involving reduction of molecular oxygen to superoxide radi-
cal, followed by spontaneous disproportionation of superox-
ide to hydrogen peroxide which ultimately undergoes a trace
metal-catalyzed Fenton reaction to yield DNA-cleaving
species such as hydroxyl radical.13-15
The nature of the products formed in the reaction of
thiarubrine C with thiols remains under investigation;
however, one can imagine that the chemistry underlying
thiol-dependent formation of oxygen radicals by thiarubrine
C is conceptually similar to the thiol-driven production of
DNA-cleaving oxygen radicals reported previously for the
epidithiapiperazine-2,5-dione-containing antibiotics gliotoxin
and sporidesmin.16,17 In the case of the epidithiapiperazine-
2,5-diones, reaction of thiols with the disulfide linkage18 of
the antibiotic is thought to yield a thiol species that is able
to efficiently react with adventitious trace metals and
molecular oxygen to produce reactive oxygen species.19-21
The notion that the 1,2-dithiin moiety of thiarubrine C may
be the key site of thiol attack is supported by the observation
that the simple 1,2-dithiin analogue 1022 is comparable to
the natural product in its ability to serve as a thiol-
dependent DNA-cleaving agent. It is noteworthy that the
alicyclic six-membered disulfide trans-1,2-dithiane-4,5-diol
(11, oxidized DTT) is a relatively poor DNA-cleaving agent
under the conditions employed here.
In conclusion, our work provides evidence that the thiaru-
brines can produce DNA-cleaving reactive oxygen species
under physiologically relevant conditions. This is the first
report of thiol-dependent DNA cleavage by these antibiotics.
In general, the production of reactive oxygen species has
important biological consequences;13 however, it must be
noted that the efficiency of DNA cleavage by thiarubrine C
in our assays is not comparable to some other agents known
to derive activity through DNA damage,23 and our results
should not necessarily be taken as evidence that DNA
represents a biologically relevant target for these antibiotics.
Finally, our findings do not support the previous postulation6
that the diene-yne substituent found in the thiarubrines can
efficiently cleave DNA involving a cyclization mechanism.
F igu r e 1. Thiol-dependent DNA Cleavage by various concentra-
tions of thiarubrine C (1). Supercoiled pBR322 DNA (38 µM bp)
was incubated for 14 h at 37 °C with various concentrations of
thiarubrine C and 20 equiv of 2-mercaptoethanol in sodium
phosphate buffer (50 mM, pH 7) containing 10% acetonitrile (by
volume). Agarose gel electrophoresis was performed as described
in the Supporting Information. The reactions were prepared under
red (darkroom) light and were incubated in the dark. The number
in parentheses following the description of each lane below
indicates the S-value (mean number of strand breaks per plasmid
molecule) for each lane and was calculated using the equation S
) -ln fI, where fI is the fraction of plasmid in a given lane that is
present as form I. Values reported here represent the average of
multiple experiments, and the standard error in these measure-
ments is less than 2%. Lane 1, DNA alone (0.2); lane 2, 100 µM
thiarubrine C (0.2); lane 3, 2 mM 2-mercaptoethanol (0.2); lane 4,
1 µM thiarubrine C + thiol (0.2); lane 5, 5 µM thiarubrine C +
thiol (0.3); lane 6, 10 µM thiarubrine C + thiol (0.3); lane 7, 25
µM thiarubrine C + thiol (0.4); lane 8, 50 µM thiarubrine C +
thiol (0.6); lane 9, 100 µM thiarubrine C + thiol (0.8).
lane 2).9 The notion that the diene-yne substituent of
thiarubrine C is not capable of causing significant DNA
damage was further corroborated by our observation that
other non-1,2-dithiin derivatives with the same diene-yne
side chain, 810 and 911 (100 µM concentrations), do not induce
significant DNA cleavage in our assays.
Ack n ow led gm en t. We thank the National Institutes
of Health (GM51565, K.S.G.) for partial support of this
work.
Interestingly, it was found that, in the presence of
biologically relevant12 concentrations of thiol, thiarubrine C
does cause DNA strand scission (Figure 1, lanes 4-9). Some
insight regarding the chemical mechanism of thiol-depend-
ent DNA cleavage by thiarubrine C was provided by
performing the reaction in the presence of various additives.
The hydrogen peroxide-destroying enzyme catalase, the
chelator of adventitious trace metals desferal, and the known
radical scavengers ethanol and mannitol all significantly
inhibit thiol-mediated DNA cleavage by thiarubrine C. The
enzyme superoxide dismutase, which catalyzes the dispro-
portionation of superoxide into hydrogen peroxide and
molecular oxygen, has little effect on the cleavage reaction.
When considered together, our experiments suggest that
DNA cleavage in this system results from a pathway
Su p p or tin g In for m a tion Ava ila ble: Experimental details for
the synthesis of thiarubrine C (1), from the known tetrabromide
3, and yne-diene analogues 8 and 9; characterization data for
compounds 5, 7, 1, 8, and 9; experimental procedures for DNA
cleavage by thiarubrine C and 10 (19 pages).
J O981849P
(13) Halliwell, B.; Gutteridge, J . M. C. Methods Enzymol. 1990, 186,
1-85.
(14) Breen, A. P.; Murphy, J . A. Free Radical Biol. Med. 1995, 18, 1033-
1077.
(15) It has previously been reported (ref 6) that thiarubrines can produce
superoxide; however, to the best of our knowledge experimental conditions
and data supporting this statement have not been published.
(16) Munday, R. J . Appl. Toxicol. 1987, 7, 17-22.
(17) Eichner, R. D.; Waring, P.; Geue, A. M.; Braithwaite, A. W.;
Mullbacher, A. J . Biol. Chem. 1988, 263, 3772-3777.
(18) For a consideration of factors that control the reaction of thiols with
disulfides, see: Burns, J . A.; Whitesides, G. M. J . Am. Chem. Soc. 1990,
112, 6296-6303.
(19) Misra, H. P. J . Biol. Chem. 1974, 249, 2151-2155.
(20) The observed metal dependence in our reactions (indicated by
inhibition of DNA cleavage by the metal chelator desferal) may reflect the
requirement for transition metal ions such as copper or iron in an initial
thiol oxidiation reaction as well as in the subsequent Fenton reaction. See
ref 13.
(21) For DNA damage by thiols, see: (a) Rosenkrantz, H. S.; Rosenkrantz,
S. Arch. Biochem. Biophys. 1971, 146, 483-487. Bode, V. C. J . Mol. Biol.
1967, 26, 125-129. (b) Spear, N.; Aust, S. D. J . Biochem. Mol. Toxicol. 1998,
12, 125-132.
(9) All of our assays were prepared under red light (darkroom) and were
incubated in the dark, as it is known that the thiarubrines are photoreac-
tive: Block, E.; Page, J .; Toscano, J . P.; Wang, C.-X.; Zhang, X.; DeOrazio,
R.; Guo, C.; Sheridan, R. S.; Towers, C. H. N. J . Am. Chem. Soc. 1996, 118,
4719-4720. See also ref 1.
(10) Prepared from phenylacetylene in two steps [1. cis-1,2-dichloroethene
(1.25 equiv), Pd(PPh3)4 (5 mol %), CuI (10 mol %), n-BuNH2 (2.5 equiv)/
benzene, rt, 1.5 h; 2. H2CdCHMgBr (2.0 equiv)/THF, Pd(PPh3)4 (5 mol %)/
benzene, rt, 12 h] in 85% overall yield.
(11) Prepared from 2-iodothiophene in four steps [1. (trimethylsilyl)-
acetylene (1.2 equiv), Pd(PPh3)2Cl2 (4 mol %), CuI (8 mol %), n-BuNH2 (4.0
equiv)/benzene, rt, 12 h; 2. KOH (1.0 equiv)/H2O/MeOH, rt, 2 h; 3. cis-1,2-
dichloroethene (2.0 equiv), Pd(PPh3)4 (5 mol %), CuI (10 mol %), n-BuNH2
(2.0 equiv)/benzene, rt, 1.5 h; 4. H2CdCHMgBr (2.0 equiv)/THF, Pd(PPh3)4
(5 mol %)/benzene, rt, 12 h] in 35% overall yield.
(22) (a) Koreeda, M.; Yang, W. Synlett 1994, 201-203. (b) Koreeda, M.;
Wang, Y. J . Org. Chem. 1997, 62, 446-447.
(23) For example, see: Epstein, J . L.; Zhang, X.; Doss, G. A.; Liesch, J .
M.; Krishnan, B.; Stubbe, J .; Kozarich, J . W. J . Am. Chem. Soc. 1997, 119,
6731-6738.
(12) Meister, A.; Anderson, M. E. Annu. Rev. Biochem. 1983, 52, 711-
760.