A Series of enediynes as novel inhibitors of topoisomerase I

Article abstract of DOI:10.1016/S0968-0896(01)00081-5

A series of acyclic enediynes, 2-((6-substituted)-3-hexen-1,5-diynyl)benzonitriles (8-11), display potent inhibition against topoisomerase I without the formation of active biradical intermediates and show inhibitory activity against topoisomerase I at 10

Full text of DOI:10.1016/S0968-0896(01)00081-5

Bioorganic & Medicinal Chemistry 9 (2001) 1707–1711
A Series of Enediynes as Novel Inhibitors of Topoisomerase I
Chi-Fong Lin,a Pei-Chen Hsieh,a Wen-Der Lu,a Huey-Fen Chiub and Ming-Jung Wua,*
aSchool of Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan
bGraduate Insititute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan
Received 14 December 2000; accepted 26 February 2001
Abstract—A series of acyclic enediynes, 2-((6-substituted)-3-hexen-1,5-diynyl)benzonitriles (8–11), display potent inhibition against
topoisomerase I without the formation of active biradical intermediates and show inhibitory activity against topoisomerase I at
10 mM, which is five times that of camptothecin from the results of agarose gel electrophoresis. # 2001 Elsevier Science Ltd. All
rights reserved.
Introduction
under 37 ꢀC (path b). An overview of the possible
mechanisms which anticancer drugs would proceed. The
most probable pathway of 2-(6-substituted-3(Z)-hexen-
1,5-diynyl)benzonitriles to induce the death of cancer
cells is the physiological enzyme inhibitors, especially
the topological enzymes; the topoisomerase, which was
essential for DNA replication, transcription, repair,
recombination, and chromosome segregation.4 Human
topoisomerase I (topo I) is a protein consisting of 765
amino acids, reconstituted from two fragments of the
protein involving three subdomain cores, the COOH-
terminal domains, and a positively charged surface,
complexed either covalently or noncovalently with a 22-
base pair DNA oligonucleotide. Several potent antic-
ancer therapies achieve their effects by pharmacological
inhibition of either topo I or II.5 However, cleavage of
duplex DNA by topo I is monomeric and transiently
includes nucleophilic attack by a catalytic tyrosine resi-
due on the scissile phosphodiester bond that culminates
in the formation of a covalent bond between the enzyme
and one end of the broken strand. Topo I is also the sole
target of the camptothecin family of anticancer drugs
which had two analogues of camptothecin, topotecan,
and irinotecan, has been used successfully in the treat-
ment of several human cancers.6,7 Besides camptothecin
A series of alkaloids containing enediyne cores which
were isolated from Streptomyces have manifold biologi-
cal activities1 owing to the generation of radicals. Sev-
eral biologically active synthetic enediynes are also
observed in the formation of radicals, However, besides
formation of biradical intermediates, there is little
attention to other feasible reaction modes by which
enediynes could act and their relative biological activ-
ities that enediynes could exhibit, though reports of
novel biradical reactions have begun to surface.2 There
is no investigation describing that enediynes could
deactivate topoisomerase I without the formation of
radical intermediates. During a routine screening of
cytotoxicity, it was surprising that a series of 2-(6-sub-
stituted-3(Z)-hexen-1,5-diynyl)benzonitriles
showed
potent cytotoxicity with a KB cell (see Table 1). The
high activity which 2-(6-substituted-3(Z)-hexen-1,5-diy-
nyl) benzonitriles possessed promoted our studies
toward this unexpected biological behavior.
It is considered that these molecules comprise an ene-
diyne structure but unlike the accustomed mode of ene-
diynes3 which generate biradicals and facilitate the
decease of cells (Scheme 1), there is no initiating factor
to promote the enediynes to form active biradicals via
Myers cycloaromatization (path a). Similarly, according
to Bergman’s reports, the acyclic enediynes cannot
cycloaromatize to produce biradical intermediates
Table 1. Cytotoxic of 2-(6-substituted-3(Z)-hexen-1,5-diynyl) benzo-
nitriles (IC50)
Compound/cell
KB (mM)
Hep2,2,15 (mM)
7
8
9
10
À02.1
<10
<10À2
<10À2
>10
>10
17
*Corresponding author. Tel.:+886-7-312-1101, ext 2220; fax: +886-
731-5339; e-mail: mijuwu@cc.kmu.edu.tw
>10
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00081-5

1708
C.-F. Lin et al. / Bioorg. Med. Chem. 9 (2001) 1707–1711
Scheme 1.
Results and Discussion
and its related derivatives, there are few detailed
approaches on such burgeoning topo I inhibitors,
although some natural products have also appeared to
restrain topo I.8 As a major drawback to the application
of antibiotics in clinical therapies, many mutations are
known to render topo I resistant to camptothecins, and
the continued development of topoisomerase inhibitors
is necessary.
The results of inhibition of compound (7–11, 13–14)
towards topo I were evaluated by gel electrophoresis as
shown in Figure 1. In panel (a), compounds 8–10 dis-
played inhibitory activity against topo I at 10 mM con-
centration. In panel (b), compound 11 induced a similar
amount of untwisting DNA compared with compounds
8–10 at 10 mM concn, though it showed slight activity at
1 mM, and no inhibition at 0.1 mM. Compound 13,
which has the same structure as compound 9, besides
the cyano group displayed weak inhibition of topo I at
10 mM. Moreover, there was not any supercoiled DNA
revealed at both 1 and 0.1 mM. The lack of activity of 13
related to 9 is not unexpected. It has been suggested that
there is little possibility for a hydrocarbon compound to
restrain topo I under such a low concentration without
any binding between target compound and receptor,
which is usually provided by heteroatoms. Furthermore,
compound 7 showed inactivity at 10 mM and, when
raised to 50 mM, complete fragmentation of DNA was
observed in panel (c). This might be due to the side
chain not being long enough; even if the essential length
of the side chain which restrained topo I required must
be more than one carbon, elementarily. To verify whe-
ther the enediyne core was necessary, compound 14 was
synthesized (from 10); while there was full unwinding
To explore how the requisite fundamental mainstay of
these compounds deactivates topo I, several compounds
were synthesized (11, 13–14) and were regenerated (7–
10).
Chemistry
The 2-(6-substituted-3(Z)-hexen-1,5-diynyl)benzonitriles
(7–11) were prepared by the palladium-catalyzed cou-
pling reaction of 2-ethynylbenzonitrile (1) with 1-sub-
stituted-4-chloro-3-buten-1-yne (2–6).9 Compound 13
was generated by the same coupling reaction of
phenylacetylene (12) and 1-butyl-4-chloro-3-buten-1-
yne (4). Compound 14 was obtained by the reduction
of 10 under the reaction of sodium methoxide and
Pd(dba)2 (Scheme 2).

C.-F. Lin et al. / Bioorg. Med. Chem. 9 (2001) 1707–1711
1709
Scheme 2.
DNA demonstrated, no matter the concentration of
100, 10, or 1 mM, in panel (d). This result revealed
that to exhibit restraint of topo I, the enediyne core
was imperative, even with a minor change of the
acetylenes.
or aryl group on position-6, by virtue of compounds 7,
13, and 14 displaying inactivity. As is well known for
camptothecin, deactivation of topo I to untwist super-
coiled DNA is due to a stable complex that is formed
between DNA–topo I and camptothecin. It is specu-
lated that a possible mode by which 2-(6-substituted-
3(Z)-hexen-1,5-diynyl) benzonitriles proceeds is in the
complexing of DNA–topo I by the enediyne core, while
the o-cyano group of benzene provides hydrogen bond-
ing with the COOH-terminal domain of topo I,
although more evidence is clearly required. These
investigations open the door to a novel acting mode of
enediynes that observe biological activity without the
formation of active radical intermediates. In short, this
study has provided some elementary insight into drug–
topoisomerase interactions.
Conclusion
Several preliminary structural elements of a series of 2-
(6-substituted-3(Z)-hexen-1,5-diynyl)benzonitriles are
indispensable for ceasing fragmentation of supercoiled
DNA promoted by topo I (shown in Scheme 3). Studies
on a series of analogues 7–11, 13–14 confirmed the
necessity for an enediyne core, the cyano group on the
o-substituted position of benzene and a long alkyl chain

1710
C.-F. Lin et al. / Bioorg. Med. Chem. 9 (2001) 1707–1711
Figure 1. Cleavage of supercoiled pGEM9zf(À) DNA by topoisomerase I in the presence of 2-(6-substituted-3(Z)-hexen-1,5-diynyl)benzonitriles (7--
11, 13–14). Supercoiled DNA was treated with 0.025 mg/mL topoisomerase I and compounds (7–11, 13), then analyzed on a 2% agarose gel. In panel
(a), lane 1: DNA only; lane 2: DNA+topo I; lane 3: DNA+topo I+50 mM camptothecin; lanes 4–6: DNA+topo I+10 mM compound 8, 9, 10. In
panel (b), lane 1: DNA+topo I; lane 2: DNA only; lane 3: DNA+topo I+50 mM camptothecin, lanes 4–6: DNA+topo I +compound 11; lanes 7–
9: DNA+topo I +compound 13. Each group of three lanes contained 10, 1, 0.1 mM of enediyne analogue, respectively. In panel (c), lane 1: DNA
only; lane 2: DNA+topo I; lane 3: DNA+topo I+camptothecin (50 mM); lanes 4–5: DNA+topo I+10 mM, 50 mM of compound 7. Panel (d), lane
1: DNA+topo I; lane 2: DNA only; lane 3: DNA+topo I+50 mM camptothecin; lanes 4–6: DNA+topo I+100 mM, 10 mM, 1 mM of compound 14,
respectively.
Experimental
General topoisomerase I course assay
Evalution of inhibitory concentration of camptothecin for
gel electrophoresis
All samples were kept in 20 mL total volume, which
contained Tris (pH 7.9) 50 mM HCl, 100 mM KCl,
10 mM MgCl2, 3 mg/mL BSA, 0.5 mM EDTA, 0.5 mM
dithioreitol and 0.5 mg pGEM9zf(À) DNA, 10 units of
topo I, were reacted with or without compounds 7–11,
13–14 (dissolved in DMSO, final concn of DMSO was
10% v/v), were well mixed before incubation. The tubes
were incubated in a 37 ꢀC water bath for 30 min.
Camptothecin, which showed significant topo I inhibi-
tion, was widely used as the standard for the compar-
ison of activity to the respective compounds. On behalf
of affirming the modest concn of camptothecin that
would still appear, the explicit suppression of topo I and
used in the following experimental section, various
concns were prepared, and the results were obtained by
agarose gel electrophresis. It was suggested camptothe-
cin showed less topo I inhibitory activity, when the
concn was 30 mM. On the other hand, 50 mM of camp-
tothecin seemed to be the fit observable inhibition of
topo I. Therefore, the 50 mM was used as the standard
concn of camptothecin, when the inhibitory of topo I of
2-(6-substituted-3(Z)-hexen-1,5-diynyl)benzonitriles were
proceed.
Gel electrophoresis
The reaction mixtures were electrophoresed by using
2% agarose gel in standard TBE buffer (1Â, 0.06 M
Tris, 0.06 M boric acid, 0.5 M EDTA), to which had
previously been added 2 mL of loading buffer containing
0.25% bromophenol blue, 0.25% xylene cyanol, 5%
SDS, and 0.25% sucrose. The gels were electrophoresed
at 50 volt for 1.5 h, and stained with ethidium bromide

C.-F. Lin et al. / Bioorg. Med. Chem. 9 (2001) 1707–1711
1711
Scheme 3.
94, 660. (b) Bergman, R. G. Acc. Chem. Res. 1973, 6, 25. (c)
Lockhart, T. P.; Comita, P. B.; Bergman, R. G. J. Am. Chem.
Soc. 1981, 103, 4082. (d) Myers, A. G.; Kuo, E. Y.; Finny,
N. S. J. Am. Chem. Soc. 1989, 111, 8057. (e) Myers, A. G.
Tetrahedron Lett. 1987, 28, 4493.
for 20 min. Then, it was placed on a UV box, and pho-
tographic images were made of the gels, using Polaroid
665 films.
4. (a) Champoux, J. J. In DNA Topology and its Biological
Effects: Wang, J. C., Cozzarelli, N. R., Eds.; Cold Spring
Harbor Laboratory: Cold Spring Harbor, NY, 1990; pp 217–
242. (b) Wang, J. C. Annu. Rev. Biochem. 1996, 65, 635. (c)
Champoux, J. J. Prog. Nucleic Acid Res. Mol. Biol. 1998, 60,
111.
Acknowledgements
The authors would like to thank the National Science
Council of the Republic of China for financial support.
5. Liu, L. F. Advances in Pharmacology, DNA Topoisome-
rases: Biochemistry and Molecular Biology, vol. 29A, Aca-
demic Press, San Diego, CA, 1994.
References and Notes
6. (a) Wang, H. K.; Morris-Natschke, S. L.; Lee., K. H. Med.
Res. Rev. 1997, 17, 367. (b) Hsiang, Y. H.; Hertzberg, R.;
Hecht, S.; Liu, L. F. J. Biol. Chem. 1985, 260, 14873. (c)
Bjornsti, M. A.; Benedetti, P.; Viglianti, G. A.; Wang, J. C.
Cancer Res. 1989, 49, 6318.
7. Seigo, S.; Shun-ichi, M.; Ken-ichiro, N.; Hiroshi, N.;
Tomio, F.; Teruo, Y.; Tadashi Chem. Pharmacol. Bull. 1991,
39, 3183.
1. (a) Walkers, S.; Valentine, K. G.; Kahne, D. J. Am. Chem.
Soc. 1990, 112, 6428. (b) Dark, L.; Iwasawa, N.; Danishefsky,
S.; Crother, D. M. Proc. Natl. Acad. Sci. U.S.A. 1991, 88,
7464. (c) Povirk, L. F.; Goldberg, I. H. Biochemistry. 1980, 19,
4773. (d) Kappen, L. S.; Goldberg, I. H. Nucleic Acid. Res.
1978, 5, 2959.
2. (a) Wendi, D. M.; Kerwin, S. M. J. Am. Chem. Soc. 1997,
119, 1464. (b) Tarli, A.; Wang, K. K. J. Org. Chem. 1997, 62,
8841. (c) Xu, S. L.; Moore, H. W. J. Org. Chem. 1992, 57, 326.
3. (a) Jones, R. R.; Bergman, R. G. J. Am. Chem. Soc. 1972,
8. Lackey, K.; Besterman, J. M.; Fletcher, W.; Leitner, P.;
Morton, B.; Sternbach, D. D. J. Med. Chem. 1995, 38, 906.
9. Wu, M. J.; Lin, C. F.; Chen, S. H. Organic Lett. 1999, 1, 767.