DNA cleavage potency, cytotoxicity, and mechanism of action of a novel class of enediyne prodrugs

Article abstract of DOI:10.1021/jm015588e

We have discovered a novel class of (E)-3-acyloxy-4-(arylmethylidene)cyclodeca-1,5-diynes which exhibit promising enediyne-like DNA cleavage and cytotoxic activities. LCMS analysis of the incubation mixture (pH 8.5, 37 °C) confirmed formation of 10-member

Full text of DOI:10.1021/jm015588e

758
J . Med. Chem. 2002, 45, 758-761
Sch em e 1. Synthesis of Enediyne Derivatives 6-10a
DNA Clea va ge P oten cy, Cytotoxicity, a n d
Mech a n ism of Action of a Novel Cla ss of
En ed iyn e P r od r u gs
Wei-Min Dai,*,† Kwong Wah Lai,† Anxin Wu,†
Wataru Hamaguchi,† Mavis Yuk Ha Lee,†
Ling Zhou,‡ Atsushi Ishii,‡ and Sei-ichi Nishimoto‡
Department of Chemistry, The Hong Kong University of
Science and Technology, Clear Water Bay, Kowloon, Hong
Kong SAR, China, and Department of Energy and
Hydrocarbon Chemistry, Graduate School of Engineering,
Kyoto University, Sakyo-ku, Kyoto 606-8501, J apan
Received November 19, 2001
Abstr a ct: We have discovered a novel class of (E)-3-acyloxy-
4-(arylmethylidene)cyclodeca-1,5-diynes which exhibit promis-
ing enediyne-like DNA cleavage and cytotoxic activities. LC-
MS analysis of the incubation mixture (pH 8.5, 37 °C)
confirmed formation of 10-membered ring enediyne presum-
ably via an allylic cation and suggested that the 1,4-benzenoid
diradical might be one of the active species for DNA damage
and cytotoxicity.
a
Reagents and conditions: (a) 10 mol % Pd(PPh3)4, 20 mol %
In tr od u ction . The enediyne antitumor antibiotics
represent a family of structurally unusual natural
products that consist of a (Z)-hex-1,5-diyn-3-ene moiety
embedded in either a 9- or a 10-membered ring.1 An
apoprotein is normally associated with the labile nine-
membered ring enediyne through noncovalent interac-
tion. It prevents the enediyne moiety from decomposi-
tion and facilitates its delivery to the target DNA in the
nucleus for cleavage chemistry to occur.2 On the other
hand, the naturally occurring 10-membered ring ene-
diynes are stabilized by various “locking devices” stra-
tegically engineered in their molecular architectures.
Bioactivation is generally required for the action of these
enediyne antitumor antibiotics,1a,e,f and they are re-
ferred to as prodrugs.3
It has been well demonstrated that acyclic and cyclic
derivatives of (Z)-hex-1,5-diyn-3-ene undergo cycloaro-
matization to generate 1,4-benzenoid diradicals.1a-d,4
The latter species are capable of hydrogen atom ab-
straction from donors such as deoxyribose5 to form
benzene derivatives. The temperature for inducing
cycloaromatization depends on the enediyne structure.
As the consequence of increased strain energy in a 10-
membered ring, the parent cyclodeca-1,5-diyn-3-ene
cyclizes at 37 °C with a half-life of 18 h and causes DNA
cleavage and cell death.6 However, the relatively short
lifetime of monocyclic 10-membered ring enediynes
renders their handling, storage, and application dif-
ficult. A strategy to overcome the instability issue
targets at in situ formation of bioactive enediynes from
suitable precursors. As a matter of fact, the Maier7 and
Myers8 groups have cleverly utilized the enzymatic
redox systems to generate the 10-membered ring ene-
diynes from the cyclic 1,5-diynes. A variety of chemical
methods have been successfully developed for laboratory
CuI, Et2NH-CH3CN (1:5), 80-90 °C, 1.5 h (ref 12a); (b) 3 equiv
of CrCl2, 1 equiv of NiCl2, THF, 20 °C, 8 h (ref 12b); (c) for 5 f 6:
5 equiv of Ac2O, 10 equiv of DMAP, CH2Cl2, 20 °C, 1.5-2.5 h (6a ,
81%; 6b, 67%; 6c, 68%; 6d , 44%); for 5 f 7: 1.6 equiv of
MeOCH2CO2H, 2 equiv of DCC, 10 equiv of DMAP, CH2Cl2, 20
°C, 1 h (7a , 79%; 7b, 65%; 7c, 65%; 7d , decomposed to 9d during
chromatographic purification over silica gel, 43%); (d) for 7 f 8:
10 mol % Eu(fod)3, CHCl3, 20 °C, 24-48 h (ref 12b); (e) for 8a f
9a : 2 equiv of K2CO3, aq MeOH, 0 °C, 30 min (84%).
syntheses, but the reaction conditions are not compat-
ible with biological system.9
In a recent study, we found that the cyclic 1,5-diyne
1 cleaved DNA to an extent comparable with a structur-
ally related 10-membered ring enediyne.10 We assumed
that an allylic rearrangement occurred during incuba-
tion to convert 1, via an allylic cation 2 (R ) an-
thraquinone-2-carbonyloxy) followed by attack of H2O
at the γ position, into a cyclic enediyne. The latter
underwent the diradical-forming reaction cascade, lead-
ing to DNA damage. We now report on DNA cleavage
potency and cytotoxicity of (E)-3-acyloxy-4-(arylmeth-
ylidene)cyclodeca-1,5-diynes 6 and 7, the evidence of
allylic rearrangement through allylic cation 2 (R ) H),
and the significant substituent effects of the aryl group
(Ar) on biological activities.11
Syn th etic Ch em istr y. As shown in Scheme 1, (E)-
3-hydroxy-4-(arylmethylidene)cyclodeca-1,5-diynes 5 can
be synthesized either from the bromides 3 via an
intramolecular Sonogashira cross-coupling catalyzed by
Pd(0)-Cu(I)12a or from the aldehydes 4 via an intra-
molecular Nozaki-Hiyama-Kishi reaction using CrCl2-
NiCl2.12b Acetylation of 5 using Ac2O and DMAP in CH2-
Cl2 at 20 °C gave the acetates 6a -d in 44-81% yields.
Because the esters are quite sensitive to weak acidic
conditions, use of excess DMAP was necessary for
minimizing product decomposition. Low yield of the
p-methoxy substituted 6d is due to its ready conversion
into the allylic cation 2 (R ) H) over silica gel. We also
experienced difficulty in isolating the methoxyacetate
* To whom correspondence should be addressed. Fax: +852-2358-
1594. E-mail: chdai@ust.hk.
† Hong Kong University of Science and Technology.
‡ Kyoto University.
10.1021/jm015588e CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/17/2002

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 759
Sch em e 2. Reactions of Enediyne 9a a
a
Reagents and conditions: (a) degassed toluene:1,4-cyclohexa-
diene (5:1), 90 °C, 3 h (85%); (b) 2,2,6,6-tetramethylpiperidinooxy
[TEMPO], PhH, reflux, 6 h (42%).
F igu r e 2. Comparison of DNA cleavage potency of the alcohol
1 with the ester 7a and the enediyne 8a . ΦX174 RFI DNA
(54.3 µM/bp) was incubated with the samples at the indicated
concentrations in TEA buffer solution (pH 8.5) containing 20%
DMSO at 37 °C for 72 h and then analyzed by gel electro-
phoresis over 1% agarose gel and visualized by ethidium
bromide stain. The gel picture is shown in the bottom, and
the scanning densitometry results of the gel picture are plotted
on the top. The percentage of net DNA cleavage was calculated
by the following equation: {[(form II)s + 2 × (form III)s]/[(form
I)s + (form II)s + 2 × (form III)s] × 100} - {(form II)c/[(form
I)c + (form II)c] × 100}. The subscripts “s” and “c” refer to the
samples and controls, respectively.
F igu r e 1. Structures of the DNA-cleaving cyclic 1,5-diyne 1
and the proposed allylic cation intermediate 2.
7d which completely decomposed over silica gel during
column chromatography to give the rearranged 9d in
43% yield. The methoxyacetates 7a -c were prepared
from 5 and methoxyacetic acid using DCC-DMAP in
65-79% yields. Conversion of 7a -c into the enediynes
8a -c was carried out in the presence of 10 mol %
Eu(fod)3 in CHCl3 at 20 °C in good yields.12b,13 Alkaline
hydrolysis of 8a furnished the enediyne alcohol 9a in
84% yield.
Sch em e 3. Proposed Mechanism of Action of 6a and 7a
Cycloaromatization of 9a was carried out in a de-
gassed mixture of toluene and 1,4-cyclohexadiene (5:1)
at 90 °C for 3 h to give the product 10 in 85% yield
(Scheme 2). Kinetic studies on the cycloaromatization
of 8a were also performed at 60, 70, 80, and 90 °C,
1
respectively, in toluene-d8 and monitored by H NMR
spectroscopic analyses on a 400 MHz instrument. These
kinetic data provided an estimated half-life of ca. 62.5
h and a free energy of activation of ca. 26.0 kcal/mol for
8a at 37 °C. In contrast, heating a toluene-d8 solution
of 6a at 90 °C for 8 h did not cause any decomposition
1
as monitored by H NMR spectroscopy.
Very recently, formation of a quinone from an ene-
diyne in aqueous buffers and air was reported as a
possible alternative therapeutic mechanism.14 We syn-
thesized the quinone 11 from 9a using TEMPO in
refluxing benzene in 42% isolated yield (Scheme 2).15
Resu lts a n d Discu ssion . DNA cleavage of 8a and
related compounds was assayed using ΦX174 RFI
supercoiled DNA (form I). We found a time-dependent
DNA scission, and the potency increased with incuba-
tion time over a period of 48 h. Therefore, in the
following experiments the samples were allowed to react
with DNA for 72 h at 37 °C. The reaction mixtures were
analyzed by electrophoresis over 1% agarose gel. The
separated DNA fragments were then visualized by
ethidium bromide stain and quantified by scanning
densitometry. Figure 2 illustrates the concentration-
dependent breakage of the DNA substrate by 7a and
8a in comparison with 1. We found that the ester 7a
was ca. 100-fold much more potent than the alcohol 1.
Moreover, over the concentration range of 5-100 µM,
7a exhibited almost the same level of activity as the
enediyne 8a . These results suggested that 7a might
dissociate in the aqueous media into the allylic cation
12. The latter was trapped by H2O preferentially at the
γ position9,10 to furnish 9a (Scheme 3). According to the
known enediyne chemistry,1,4 at the incubation tem-
perature, 9a should cyclize to the diradical 13 which

760 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Letters
F igu r e 3. HPLC chromatogram of the organic extract of the
reaction mixture of 6a after incubating at 37 °C for 48 h under
N2 atmosphere in TEA buffer solution (pH 8.5) containing 10%
DMSO. The molecular structures of the marked peaks are
confirmed by LC-MS in comparison with those authentic
samples synthesized in Schemes 1 and 2. The structure 14
given in the inset is deduced from its LC-MS data. See the
Supporting Information for details of the LC-MS analysis.
F igu r e 4. DNA cleavage results of compounds 6a -d , 7a -c,
8a -c, and 9d at 20 µM, respectively. The incubation conditions
are identical to those given in Figure 2. The gel picture is
shown in the bottom, and the scanning densitometry results
of the gel picture are plotted on the top. Definition of the
percentage net DNA cleavage is the same as that given in
Figure 2.
abstracted hydrogen atoms from the sugar moiety of
DNA5 to afford the aromatic product 10.
Ta ble 1. IC50 Data (µM) against P388 Cancer Cell Linea
To confirm formation of 9a from the precursor, we
analyzed the reaction products of 6a in TEA buffer
solution (pH 8.5) containing 10% DMSO by LC-MS.
Thus, a solution of 6a was incubated in the absence of
the DNA substrate at 37 °C for 48 h under N2 atmo-
sphere, and the organic extract of the reaction mixture
was subjected to LC-MS analysis. Figure 3 shows the
HPLC chromatogram where the peaks corresponding to
5a , 6a , 9a , and 10 are identified by the retention times
and mass spectra in comparison with the authentic
samples synthesized in Schemes 1 and 2 (see the
Supporting Information for details). We found that
hydrolysis of 6a to the alcohol 5a competed against
formation of the enediyne 9a . The relationship among
6a , 5a , and 9a is proposed in Scheme 3, indicating that
both 5a and 6a can be converted into 9a through the
allylic cation 12.10 However, the route from 5a to 12
should be much slower because the acetate moiety in
6a is a better leaving group. Indeed, 6a is a much more
powerful DNA cleaver than the alcohol 5a which causes
only 22% net DNA cut at 5.0 mM. Once the enediyne
9a is formed, it enters the radical-forming reaction
cascade leading to the benzene derivative 10. The peak
intensity of 10 in Figure 3 is rather weak probably due
to the absence of an efficient hydrogen atom donor in
the buffer solution for trapping 13. We also confirmed
formation of a very polar component 14, having a
retention time of 2.0 min, by the LC-MS analysis.
Compound 14 is the trapping product of the allylic
cation 12 by tris(hydroxymethyl)aminomethane used in
the buffer. We were not able to identify the quinone 11,
possessing a retention time of 10.2 min, from the
reaction mixture of 6a . There are many unidentified
polar components having retention times less than 10
min. Mass spectra indicate that they seem not related
to the structures derived from 6a . Nevertheless, the LC-
MS data given in Figure 3 and the Supporting Informa-
tion clearly demonstrate transformation of (E)-3-acyloxy-
Ar
alcohols
acetates
esters
enediynes
Ph
5a : 25
5b: 41
5c: 3.6
5d : 5.8
6a : 48
6b: 2.4
6c: 2.8
6d : 15
7a : 23
7b: 12
7c: 28
8a : 7.7
8b: 3.8
8c: 9.5
9d : 13
1-naph
2-naph
p-MeOC6H4
a
MTT assay was used. See ref 16.
4-(arylmethylidene)-cyclodeca-1,5-diynes such as 6a into
the bioactive enediynes under the DNA assay condi-
tions.
Figure 4 shows the DNA cleavage results of 6-8 at
20 µM. In general, the phenyl derivatives 6a , 7a , and
8a are much weaker cleavers compared to the naphthyl
and p-methoxyphenyl analogues 6b-d , 7b,c, and 8b,c.
The esters 6b-d and 7b,c are 5.6-6.8-fold more potent
than 6a and 7a . Also, it was revealed that the two
naphthyl-derived enediynes 8b and 8c are 3.5- and 4.0-
fold more potent than the phenyl analogue 8a . To
examine the possibility of intercalation with DNA, we
measured the DNA binding constants K′ of 5-8 as given
in the Supporting Information. Because the K′ values
for the phenyl and naphthyl analogues are on the same
order of magnitude, a clear conclusion on the enhanced
DNA cleavage potency for 6b,c, 7b,c, and 8b,c cannot
be reached at this stage. Nevertheless, the remarkably
increased activity of the naphthyl analogues is very
promising for further development. In contrast, the
enediyne 9d gave an unusual result compared to 6d .
We suspected that 9d could dimerize via an O-methy-
lated quinone methide intermediate. This side reaction
might be suppressed when it was generated in situ from
6d . We observed such dimerization reaction of a similar
compound when the solvent of the sample solution was
evaporated to dryness.
Cytotoxicity was assayed using the P388 mouse T cell
leukemia cell line, and the results are listed in Table 1.
In general, the phenyl derivatives 5a , 6a , and 7a exhibit

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 761
reaction and trapping evidence for the 1,4-benzenediyl structure.
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less potent in vitro cytotoxicity in parallel to their low
DNA cleaving activity. The two naphthyl substituted
acetates 6b and 6c are the most promising compounds,
with IC50 values in the low micromolar range. We found
that the quinone 11 neither causes DNA cleavage at 100
µM nor exhibits cytotoxicity against the P388 cell line.
The latter result is different from the reported observa-
tion on a similar quinone compound.14
Con clu sion . We have discovered a novel series of (E)-
3-acyloxy-4-(arylmethylidene)cyclodeca-1,5-diynes 6 and
7 as the promising DNA cleaving and cytotoxic agents.
Experimental evidences have confirmed formation of 10-
membered ring enediynes from cyclic 1,5-diynes pre-
sumably through an allylic cation intermediate 2
(R ) H). It suggests that the enediyne pathway should
be one of the possible mechanisms of action for this
novel class of cyclic 1,5-diyne compounds. Manipulation
on the aryl substituent could finely tune the biological
potency although the exact factor for the enhanced DNA
damage and cytotoxicity of the naphthyl compounds is
unclear. We did not observe formation of the quinone
11 in the reaction mixture of 6a by LC-MS analysis.
Moreover, 11 failed to exhibit both DNA cleavage and
cytotoxicity. It is unlikely that our results described
above involve action of the quinone intermediates.
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Ack n ow led gm en t. Financial supports to A. Wu
through a postdoctoral fellowship from the Department
of Chemistry, HKUST, to W. Hamaguchi through an
RGC Direct Allocation Grant (DAG97/98.SC12), and to
M. Y. H. Lee through a HKUST Postdoctoral Fellowship
Matching Fund (PDF99/00) are acknowledged.
Su p p or tin g In for m a tion Ava ila ble: DNA binding con-
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LC-MS data. This information is available free of charge via
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