A proposal for the mechanism-of-action of diazoparaquinone natural products

Article abstract of DOI:10.1021/ja053880w

Treatment of the representative diazoparaquinone prekinamycin dimethyl ether with Bu₃SnH/AIBN in aromatic solvents furnishes moderate-to-good yields of formal aryl adducts wherein a molecule of solvent is attached to the carbon (C(11)) previous

Full text of DOI:10.1021/ja053880w

Published on Web 10/14/2005
A Proposal for the Mechanism-of-Action of Diazoparaquinone Natural
Products
Ken S. Feldman* and Kyle J. Eastman
Department of Chemistry, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802
Received June 13, 2005; E-mail: ksf@chem.psu.edu
Scheme 1. A Proposal for the Mechanism-of-Action of the
Diazoparaquinone Antibiotics
The diazoparaquinone family of antibiotics, exemplified by the
naturally occurring species prekinamycin (1a),1 kinamycin F (2),2
and lomaiviticin A (3),3 has had a long and storied history, featuring
two major structural revisions (N-cyanocarbazole f diazofluorene
for the kinamycins,4 and diazobenzo[b]fluorene f diazobenz[a]-
fluorene for isoprekinamycin (not shown)5). Following these cor-
rections, two mechanism-of-action hypotheses have been proposed.
Jebaratnam, using diazofluorene as a model system, suggested that
oxidative activation of the diazo function might lead to DNA
damaging reactive oxygen species via a putative C(11) radical.6
Dmitrienko, employing the isoprekinamycin structure as a probe
molecule, favored an alternative mechanism whereby enhanced
diazonium character of the N2 group as a consequence of an internal
hydrogen bond network provided a site for nucleophilic attack by
DNA amino groups, possibly leading to DNA damaging C(11)
radicals as well.7 A disclosure by He et al. on the structure and
biological activity of lomaiviticin A (3) attributed its profound
cytotoxicity (IC50’s of 0.7-6.0 nM for a variety of tumor cell lines)3
to double-stranded DNA cleavage under reducing conditions.
Scheme 2. Preliminary Phenylation of Prekinamycin and
Derivatives
vene-containing intermediate 8 along with a DNA radical will be
formed. At this point in the mechanistic speculation, there are two
distinct avenues by which these two species, 8 and DNA•, might
react further to lead to the observed result with lomaiviticin A.
Generation of a DNA-based radical (C(4′), C(5′), or C(1′)) itself
may be sufficient to cause strand cleavage, as per the putative O2-
mediated mechanisms-of-action of other radical-generating DNA
damaging agents, such as bleomycin and the enediynes.10 In
addition, a rebound addition of DNA• into the enone of 8 might
also contribute to the formation of DNA lesions at low O2
concentrations.11 In addition to the potential for radical-based DNA
damage, the quinone methide moiety within 8 may interact further
with DNA to lead to strand scission products by analogy with the
documented DNA alkylating chemistry of the structurally related
hydroxymethylacylfulvenes.12 The dimeric nature of lomaiviticin
A is suggestive of double reaction to afford the observed double
strand cleavage.
A test of this hypothesis was planned using readily available
prekinamycin (1a)13 and Bu3SnH/AIBN in PhH (80 °C) as a
surrogate for the unidentified e-/H+ or H• transfer agent available
in the cellular milieu, with the anticipation that species of type 8
(or nucleophile-trapped adducts thereof) might be formed (Scheme
2).14 In fact, treatment of this substrate under standard radical-
generating conditions provided a good yield of a single characteriz-
able product, the C(11) benzene adduct 10a, following aqueous
workup and SiO2 chromatography. Presumably, loss of any attached
Neither Jebaratnam nor Dmitrienko utilized a diazoparaquinone-
containing species for their studies, and an alternative mechanism-
of-action that directly incorporates both this specific functionality
and the reductive activation observation of He can be envisioned
(Scheme 1). This proposal does not stray far from orthodox and
precedented mechanisms of known paraquinoid antitumor agents,
such as mitomycin and the anthracyclins.8 The observation that
lomaiviticin A operates through bioreductive activation is suggestive
of one-electron addition to the quinone of generic diazoparaquinone
4 to give a reactive semiquinone 5 following protonation (not
obligatory). The radical 5 so generated can be represented by the
resonance form 6. This pivotal radical (or radical anion) 5/6 can
furnish the second key intermediate of this hypothesis, a C(11)
radical 7, provided that C(11) is pyramidalized sufficiently to permit
adequate overlap between the enol (enolate) orbitals and σ*C-N for
rapid loss of N2. If this sequence occurs in proximity to DNA, then
H-atom abstraction seems plausible by analogy with the similar
reaction of a related indenyl monoradical derived from neocarzi-
nostatin and p-hydroxythiophenol,9 and the hydroxymethylacylful-
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J. AM. CHEM. SOC. 2005, 127, 15344-15345
10.1021/ja053880w CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Relative Rates of Aromatic Solvent Addition for 1c and
the Reference sp2 Radical 12
ArH
relative
ratea,b
relative
R
R1
o:m:p
rate (12)a,d
o:m:p
a
b
c
d
e
f
CH3
Cl
CN
OCH3
CH3
OCH3
CN
H
H
H
H
CH3
OCH3
CN
2.2 ( 0.1
1.5 ( 0.2
2.1 ( 0.3
3.1 ( 0.7
4.0 ( 0.9
4.2 ( 1.5
6.1 ( 0.5e
62:23:15
48:32:20
43:25:32
72:16:12
50:50:0c
31:69:0c
24:76:0c
2.1
0.9
2.0
2.2
69:23:17
49:33:19
56:18:26
63:14:23
g
a Rate relative to benzene. b Quintuple measurements with standard
deviation. c Ratio of 2:4:5 adducts. d Data from ref 15. e Extrapolated from
the results of 1:9 and 1:13 ratios of 1,3-dicyanobenzene to benzene solvent
due to solubility constraints.
Figure 1. Yield and ratio of aromatic trapping products 10c/11c upon
increasing [Bu3SnH].
tin residue and air oxidation preceded isolation of 10a. Control
experiments in which ambient light was included or omitted led to
similar yields of 10a, whereas omission of either the tin reagent,
AIBN, or heat returned starting material. Similar chemistry was
observed with the derived diacetate 1b and the dimethyl ether 1c,13
and correlations as indicated (Scheme 2) confirmed that the identical
C(11) adducts were formed in each case. Subsequent studies were
conducted with the dimethyl ether 1c as a concession to ease of
chromatographic isolation and characterization.
This latter point is significant in that it argues against the
intervention of a competing mechanism (i.e., 8 combining with the
aromatic solvent) upon formation of 11. The remainder of the
reaction mixture provided little characterizable material, but typi-
cally about 5-15% of the formal dimer of radical 7 was isolated
from many trials. The observed drop-off in overall yield of [10c +
11c] (Figure 1) only makes sense in the context of the Scheme 1
mechanism if the aryl solvent trapping rate is not too dissimilar
from the rate of sp2 radical/H-SnBu3 reaction. A rate constant for
this latter process with Ph• has been estimated to be ∼109 M-1 s-1
(80 °C),17 and given the concentration differences ([aromatic
solvents] ≈ 25-150 × [Bu3SnH]), then the rate constant for sp2
radical addition (e.g., 7) to the aromatic solvent would be on the
order of 107 M-1 s-1, a value not too far removed, given the
approximations involved, from the measured rate constant for
phenyl radical addition to chlorobenzene (∼106 M-1 s-1, 25 °C).18
Acknowledgment. Support from the General Medical Sciences
Institute of the National Institutes of Health (GM 37681) is
gratefully acknowledged.
The clean formation of the benzene adducts 10a-c under these
relative mild and neutral conditions raises the key question, what
is the reactive intermediate that precedes C(11)/PhH bond forma-
tion? Evidence that bears on this issue was gathered by examining
the relative rates of aromatic solvent incorporation (versus benzene)
for the substrate 1c in 1:1 molar mixtures of benzene with a variety
of electron-rich or electron-deficient solvents, eq 1 and Table 1.
The observed relative rates with 1c would be difficult to reconcile
with an electrophilic aromatic substitution mechanism from a
orthoquinonemethide-type electrophile as in 8, given the accelera-
tion (relative to benzene) of the electron-deficient solvents, chlo-
robenzene (entry b), cyanobenzene (entry c), and 1,3-dicyanoben-
zene (entry g). Direct addition of the putative sp2 radical of 7 to
the aromatic solvent cannot be dismissed so readily, however, in
light of the precedent provided by aromatic radical substitution data
for a related radical 1215 and also for Ph• (not shown).15 That is, a
general accelerating effect of most substituents relative to H attends
the radical aromatic substitution reactions of 12 and Ph•, as is
observed with 1c. An analysis of the o:m:p ratios provides further
evidence consistent with the radical addition mechanism. Thus,
additions that place radical density on the substituent-bearing carbon
are generally favored, whereas it is difficult to rationalize the
observed regiochemistry of addition with the electron-deficient
entries if electrophilic chemistry were operational. The reoxidation
of the presumably first-formed cyclohexadienyl radicals, even under
reducing tin hydride conditions has been described,16 although the
precise source of the oxidant in the 1c case has not been identified.
The intermediacy of a putative sp2 radical akin to 7 in the
Supporting Information Available: Experimental procedures and
characterization data for 10a-e and 11a-g. This material is available
free of charge via the Internet at http://pubs.acs.org.
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