Synthesis of a heterocyclic aza-enediyne and its DNA-cleavage properties

Article abstract of DOI:10.1016/S0960-894X(00)00516-3

The benzimidazolium salt 2, incorporating an aza-enediyne moiety, has been prepared and is shown to be a very effective DNA-cleavage agent under mild physiological conditions. Its mechanism of action is currently under investigation but may involve the generation of a diradical intermediate, DNA alkylation, or both. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00516-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2509±2512
Synthesis of a Heterocyclic Aza-Enediyne and Its DNA-Cleavage
Properties
Wendi M. David, Dalip Kumar and Sean M. Kerwin*
Division of Medicinal Chemistry, College of Pharmacy and Institute for Cellular and Molecular Biology,
University of Texas at Austin, Austin, TX 78712, USA
Received 30 June 2000; accepted 31 August 2000
AbstractÐThe benzimidazolium salt 2, incorporating an aza-enediyne moiety, has been prepared and is shown to be a very e€ective
DNA-cleavage agent under mild physiological conditions. Its mechanism of action is currently under investigation but may involve
the generation of a diradical intermediate, DNA alkylation, or both. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
electrophilic chemistry may occur via the addition of a
DNA nucleophile at the 2-alkyne position. DNA clea-
vage is expected in either case. The N-propargyl, 2-
alkynyl heterocyclic system has the potential of being
tunable; varying the heterocyclic framework and the
propargyl and alkyne substituents may modulate the
desired reactivity such that a suitable antitumor drug
candidate can be developed.
The last decade ushered in the investigation of several
potent antitumor, antibiotic natural products such as
calichemicin,1 dynemicin,2 and neocarzinostatin,3 all
of which cleave DNA upon suitable triggering of core
diradical-generating structures. These natural products
are extremely toxic and there has been much e€ort
directed towards the design of synthetic analogues capable
of cleaving DNA in a similar manner.4 There are, however,
very few examples of aza-substituted analogues. Several
groups have investigated the ability of aza-substituted
enyne allenes to undergo radical-generating cyclizations,
but none have demonstrated DNA-cleavage activity.5
Our approach utilizes mild, physiologically relevant
`triggering' mechanisms and the e€ect of heteroatom
substitution in enediyne moieties for the design of novel
DNA-cleavage warheads with the goal of increasing
antitumor selectivity without sacri®cing potency.6 Con-
tinuing our e€orts in this area, we sought to develop a
hydrolytically stable heterocyclic system incorporating
an aza-enediyne moiety that would be physiologically
viable (Fig. 1).7 The N-propargyl, 2-alkynyl heterocycle
shown may be able to undergo pH-triggered isomeriza-
tion to generate an N-allenyl, 2-alkynyl species. The
aza-enyne allene moiety may be capable of an aza-
Myers type cyclization, thereby generating a diradical
persistent enough to perform hydrogen atom abstrac-
tion under physiological conditions. Alternatively,
Figure 1. Two possible mechanisms for DNA cleavage arising from an
N-propargyl-2-alkynyl heterocyclic system: (a) isomerization, followed
by Myers cyclization to generate a diradical; (b) nucleophilic attack.
Synthesis and DNA Cleavage
We now report the synthesis and DNA-cleavage activity
of 2-phenylethynyl-1-(3-phenyl-prop-2-ynyl)-3-methyl-
1H-benzimidazolium tetra¯uoroborate or tri¯uoro
methanesulfonate salt 2. Synthesis of 2 is easily accom-
plished in two steps: condensation of phenylenediamine
with phenylpropargyl aldehyde to generate 2-phenyl-
ethynyl-1-(3-phenyl-prop-2-ynyl)-1H-benzimidazole 1,8
*Corresponding author. Tel.: +1-512-471-5074; fax: +1-512-232-
2606; e-mail: skerwin@mail.utexas.edu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00516-3

2510
W. M. David et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2509±2512
followed by alkylation with methyl tri¯ate or trimethyl-
oxonium tetra¯uoroborate9 (Scheme 1). Synthesis of the
2-phenylethynyl-1,3-dimethyl-1H-benzimidazolium tri-
¯uoromethanesulfonate salt 4 proceeds through the 2-
phenylethynyl benzimidazole 3. Palladium catalyzed
coupling of phenylacetylene to 2-bromo-1-methyl-1H-
benzimidazole,10 followed by alkylation with methyl
tri¯ate yields 411 (Scheme 1).
cleavage (data not shown). Frank single-strand DNA
cleavage is observed at concentrations as low as 1 mM,
and the amount of cleavage is unchanged after addi-
tional heating of the incubation samples. At pH 8,
100 mM concentration of 2 cleaves approximately 50%
of supercoiled DNA. In contrast, compounds 1, 4, and 6
do not show any appreciable cleavage of supercoiled
DNA, even at concentrations of 1 mM (Fig. 3).
In order to probe the ability of 1 and 2 to undergo an
aza-Myers type cyclization, thereby generating di-
radicals that might be e€ective DNA-cleavage agents,
we investigated conditions capable of promoting an N-
propargyl to N-allenyl isomerization in these benzimi-
dazoles (Scheme 2). Isomerization of the N-propargyl
substituent of 1 to an N-allene occurs only in the pre-
sence of strong base, as evidenced by the disappearance
in crude reaction mixtures of the 13C NMR alkyne
resonances at d 81.6 and 85.3 ppm and the appearance
of new resonances at d 198.3, 111.6, and 106.3 ppm.
However, the allene is too unstable to be isolated and
puri®ed; it quickly undergoes decomposition to poly-
meric products, perhaps through a process involving an
aza-Myers cyclization. Alkylation of a benzimidazole
nitrogen (as in 2) renders the 2-alkynyl substituent more
reactive to nucleophilic substitution. Treating a metha-
nolic solution of 2 with basic alumina a€ords the addi-
tion product 6 in 35% yield;12 no isomerization to an
allene is observed. In addition, interestingly, the less
nucleophilic isopropanol promotes N-demethylation of
2 under mildly basic conditions. It would appear that
the ability of these molecules to undergo either iso-
merization or electrophilic chemistry is extremely
dependent on both heterocycle substituent e€ects and
external conditions.
The DNA cleavage exhibited by 2 is remarkably sensi-
tive to temperature, pH, and the length of incubation
with supercoiled DNA (Figs. 4 and 5). At 37 ꢀC, incu-
bation for at least 8 h is required before any appreciable
cleavage is observed; nearly complete cleavage is not
observed until 24 h. There is hardly any detectable clea-
vage at 25 ꢀC, but at physiological temperature cleavage
is substantial. The pH dependence of the observed
DNA cleavage is most striking (Fig. 5). At pH 9 com-
plete cleavage is e€ected by 100 mM of 2, and complete
degradation of the supercoiled DNA is observed at
1 mM of 2. Diminished cleavage still occurs at pH 7.
This pH dependence is commensurate with a mech-
anism in which N-propargyl to N-allenyl isomerization,
followed by cyclization and diradical formation, is
operative. The ease of isomerization would increase
with increasing pH. The pH dependence is not
inconsistent with a nucleophilic mechanism whereby
Benzimidazole 2 causes extensive cleavage of super-
coiled DNA when incubated under mild conditions
(Fig. 2). Both the tetra¯uoroborate and tri¯ate salts of 2
are equipotent; the counterion has no e€ect on the
Scheme 2.
Scheme 1. Synthesis of benzimidazoles. Reagents and conditions: (a)
cat. HCO2H, toluene, 33%; (b) (Me)3OBF4 (1.3 equiv), ClCH2CH2Cl,
>99%, X=BF4 or MeOTf (1.6 equiv), ether, 86%, X=TfO ; (c)
48% HBr (1.3 equiv)/HOAc, Br2 (4 equiv), 80%; (d) (MeO)2OSO2 (1.8
equiv), 1 N NaOH (2.3 equiv), 61%; (e) Pd(OAc)2 (10 mol%), P(Ph)3
(20 mol%), CuI (15 mol%), Et3N, 60%; (f) MeOTf (1.6 equiv), ether,
69%.
Figure 2. DNA cleavage by 2. pBR322 plasmid DNA (50 mM base
pairs) was incubated with various concentrations of 2 (pH 8, 50 mM
Tris bu€er, 13% v/v DMSO, 20 h at 37 ꢀC) and analyzed by gel elec-
trophoresis (0.8% agarose, ethidium bromide stain). Lanes 1, 2, and 9,
DNA without drug; lanes 3±4, 1 mM 2; lanes 5±6 and 10±11, 100 mM
2; lane 7, 10 mM 2; lane 8, 1 mM 2; lanes 9, 10, and 11 were heated at
70 ꢀC for 90 s after incubation.

W. M. David et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2509±2512
2511
Figure 3. DNA cleavage by 1, 6, 4, and 2. ÈX174 (RF-1) phage DNA
(50 mM base pairs) was incubated with 1 mM 1, 6, 4, or 2 (pH 8,
50 mM Tris bu€er, 13% v/v DMSO, 20 h at 37 ꢀC) and analyzed by gel
electrophoresis (0.8% agarose, ethidium bromide stain).
Figure 6. E€ect of inhibitors on the DNA cleavage by 2. ÈX174 (RF-
1) phage DNA (50 mM base pairs) was incubated with 1 mM 2 and
various concentrations of inhibitors (pH 8, 50 mM Tris bu€er, 13% v/v
DMSO, 20 h at 37 ꢀC) and analyzed by gel electrophoresis (0.8%
agarose, ethidium bromide stain).
DNA alkylation is responsible for cleavage; how-
ever, the absence of any increase in the observed DNA
cleavage after heat treatment of the drug±DNA incu-
bation samples would seem to argue against DNA
alkylation.
We investigated the ability of cimetidine, glycerol,
potassium bromide, and sodium azide to inhibit clea-
vage of DNA by 2 (Fig. 6). Cimetidine and glycerol are
both radical inhibitors, while KBr and NaN3 could
function as sacri®cial nucleophiles. A comparison of the
cleavage inhibition eciencies of these four inhibitors
demonstrates that NaN3 is most e€ective, causing over
70% inhibition at equimolar concentration with 2.
Potassium bromide and cimetidine each show ꢁ10%
inhibition at 10 times the concentration of 2. Since
sodium azide is also an inhibitor of singlet oxygen we
were curious whether some oxygen-dependent species
may be involved in the DNA cleavage caused by 2.
Preliminary results, however, show no di€erence in
cleavage between anaerobic and aerobic conditions.
Figure 4. Time and temperature dependence of DNA cleavage by 2.
ÈX174 (RF-1) phage DNA (50 mM base pairs) was incubated with
1 mM 2 at various temperatures and for various time spans (pH 8,
50 mM Tris bu€er, 13% v/v DMSO) and analyzed by gel electro-
phoresis (0.8% agarose, ethidium bromide stain).
Conclusion
Clearly, based on its DNA-cleaving ability, compound 2
has the potential to function as an e€ective antitumor
agent and we are currently investigating its ecacy. The
mechanism of DNA cleavage e€ected by 2 remains to be
elucidated. If a simple electrophilic mechanism is
involved, it is curious that 2 is so much more potent
than the dimethyl benzimidazole 4. Additionally, evi-
dence of frank single-strand breakage by 2 and the steep
pH-dependence of its DNA cleavage indicate something
unique about the mechanism of action of 2 compared to
the other benzimidazoles. We are actively pursuing elu-
cidation of the mechanism of action of 2 and related
heterocycles.
Acknowledgements
Figure 5. pH dependence of DNA cleavage by 2. ÈX174 (RF-1) phage
DNA (50 mM base pairs) was incubated with various concentrations
of 2 at pH 7, 8, and 9 (50 mM Tris bu€er, 13% v/v DMSO, 20 h at
37 ꢀC) and analyzed by gel electrophoresis (0.8% agarose, ethidium
bromide stain).
We gratefully acknowledge funding from the Founda-
tion for Research and Stephanie Jones for technical
assistance.

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W. M. David et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2509±2512
7. David, W. M.; Kerwin, S. M. Abstracts of Papers, 219th
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