Role of the azinomycin naphthoate and central amide in sequence-dependent DNA alkylation and cytotoxicity of epoxide-bearing substructures.

Article abstract of DOI:10.1021/ol0267275

Studies report a strong correlation between duplex DNA alkylation and in vitro cytotoxicity for a series of azinomycin partial structures 2-6 bearing the biologically relevant epoxide. Compounds lacking the naphthoate ester (e.g., 5 and 6) were poorly rea

Full text of DOI:10.1021/ol0267275

ORGANIC
LETTERS
2002
Vol. 4, No. 20
3545-3548
Role of the Azinomycin Naphthoate and
Central Amide in Sequence-Dependent
DNA Alkylation and Cytotoxicity of
Epoxide-Bearing Substructures
,†
Robert S. Coleman,* Christopher H. Burk,† Antonio Navarro,†
Robert W. Brueggemeier,‡ and Edgar S. Diaz-Cruz‡
Department of Chemistry, The Ohio State UniVersity, 100 West 18th AVenue,
Columbus, Ohio 43210, and College of Pharmacy, The Ohio State UniVersity,
496 West 12th AVenue, Columbus, Ohio 43210
coleman@chemistry.ohio-state.edu
Received August 13, 2002
ABSTRACT
Studies report a strong correlation between duplex DNA alkylation and in vitro cytotoxicity for a series of azinomycin partial structures 2−6
bearing the biologically relevant epoxide. Compounds lacking the naphthoate ester (e.g., 5 and 6) were poorly reactive toward DNA and were
biologically inactive, as were compounds bearing the naphthoate but lacking the terminal carboxamide (e.g., 2). Compounds were evaluated
for cytotoxicity against two breast cancer cell lines.
The DNA cross-linking agents azinomycins A (1a) and B
(1b) have generated considerable interest among the syn-
thetic,1 medicinal,2 and bioorganic communities.3 These
natural products possess the unusual aziridino[1,2-a]pyrro-
lidine ring system (Figure 1).4 These scarce and unstable
agents exhibit sub-micromolar in vitro cytotoxic activity and
† Department of Chemistry.
‡ College of Pharmacy.
(1) Coleman, R. S.; Li, J.; Navarro, A. Angew. Chem., Int. Ed. 2001,
40, 1736 and references therein.
Figure 1. Structure of azinomycins A and B.
(2) (a) For related structure-activity work, see: Hodgkinson, T. J.;
Kelland, L. R. Shipman, M.; Suzenet, F. Bioorg. Med. Chem. Lett. 2000,
10, 239. (b) Hartley, J. A.; Hazrati, A.; Khanim, R.; Hodgkinson, T. J.;
Shipman, M.; Suzenet, F.; Kelland, L. R. Chem. Commun. 2000, 2325. (c)
Hartley, J. A.; Hazrati, A.; Kelland, L. R.; Khanim, R.; Shipman, M.;
Suzenet, F.; Walker, L. F. Angew. Chem., Int. Ed. 2000, 39, 3467.
(3) (a) Zang, H.; Gates, K. S. Biochemistry 2000, 39, 14968. (b) Fujiwara,
T.; Saito, I.; Sugiyama, H. Tetrahedron Lett. 1999, 40, 315. (c) Armstrong,
R. W.; Salvati, M. E.; Nguyen, M. J. Am. Chem. Soc. 1992, 114, 3144.
(4) Nagaoka, K.; Matsumoto, M.; Oono, J.; Yokoi, K.; Ishizeki, S.;
Nakashima, T. J. Antibiot. 1986, 39, 1527. Yokoi, K.; Nagaoka, K.;
Nakashima, T. Chem. Pharm. Bull. 1986, 34, 4554.
promising in vivo antitumor activity5 and appear to exert
their effect by the formation of covalent interstrand cross-
links within the major groove of duplex DNA.6 We recently
reported the first total synthesis of azinomycin A,1 and we
have evaluated the sequence selectivity, noncovalent as-
(5) Ishizeki, S.; Ohtsuka, M.; Irinoda, K.; Kukita, K.; Nagaoka, K.;
Nakashima, T. J. Antibiot. 1987, 40, 60.
10.1021/ol0267275 CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/30/2002

sociation, and orientation of azinomycin B on its duplex
DNA receptor.7 We now provide further mechanistic clari-
fication on the critical role that the naphthoate and N16
carboxamide play in sequence selective DNA alkylation in
a series of partial structures, and we demonstrate a strong
correlation between the extent of covalent modification of
DNA and in vitro cytotoxicity. We find that the naphthoate
and the carboxamide increase alkylation yields and sequence
selectivity and that both are important for effective cytotoxic
activity.
Figure 3. DNA sequences.
Armstrong and co-workers reported that azinomycin B
cross-links duplex DNA in the major groove at the N7-
positions of 5′-disposed purine bases in the sequence
5′-PuNPy-3′.3c Saito and co-workers confirmed this result
and provided evidence of the orientation of the agent on the
DNA duplex.3b Modeling studies by Alcaro and Coleman8
and experimental work by Coleman and co-workers7 pro-
vided evidence that aziridine C10 alkylates the 5′ guanine
in a GGC strand in the initial monoalkylation, and cross-
linking ensues by epoxide C21 alkylation of the guanine in
the CCG strand. Computational work was consistent with
either an intercalative or non-intercalative binding mode for
the naphthoate, and experimental work by Zang and Gates
indicated that naphthoate-bearing fragments were weak
intercalating agents.3a Our studies on azinomycin B point to
a non-intercalative binding mode.7 Shipman and co-workers
provided the first evidence that the epoxide was important
for cytotoxic activity.2a We now demonstrate a strong
correlation between the alkylating ability of a series of
epoxide-bearing partial structures (Figure 2) and in vitro
cytotoxicity.
stranded and double-stranded forms of these oligodeoxy-
nucleotides.
Alkylation of GGC-strand 7 in duplex 7‚8 by azinomycin
partial structure 3 was highly effective (86%, Table 1) and
Table 1. Yield of Covalent Adduct Formationa between
Epoxyamide 3 and DNA Oligomers 7-10b
GGC (7)
CCG (8)
CGG (9)
GCC (10)
ds
ss
86%
23%
36%
17%
42%
28%
52%
12%
a Yields are the average of three measurements. Yields reported for
double-stranded oligomers represent the results obtained when that oligomer
was 32P end labeled. b Reactions were run at 8 °C for 20 h using 100 equiv
of 3 per oligodeoxynucleotides duplex.
occurred with 4:1 selectivity for the more nucleophilic 5′
guanine.9 The yield was lower with the CCG strand 8 (36%),
in accord with consideration of base nucleophilicity. (Com-
bined yields for duplex 7‚8 are greater than 100% because
of double alkylation.) With duplex 9‚10 containing an
inverted azinomycin recognition triplet where the guanine
bases are 3′-disposed, the CGG strand 9 underwent modestly
effective alkylation with 3 (42%), again with 4:1 selectivity
for the more nucleophilic 5′ guanine. The complementary
GCC strand 10 was alkylated to a similar extent by 3 (52%).
These results indicate that the partial structure 3 must interact
with duplex DNA by multiple binding modes and with
altered sequence selectivity from the natural product. In
duplexes 7‚8 and 9‚10, all guanine bases are alkylated to a
significant but differential extent by 3; azinomycin B reacts
selectively at the two distal 5′-disposed guanines in this triplet
by an apparently well-defined binding mode.7 In the duplex
7‚8, the extent of alkylation by the epoxide of 3 correlated
well with guanine nucleophilicity, but with the inverted
duplex 9‚10, the less reactive guanine in the GCC strand 10
underwent more efficient alkylation.
Figure 2. Azinomycin partial structures.
Using synthetic DNA oligomers containing the azinomycin
recognition sequence GGC‚CCG,7 and the inverted sequence
CGG‚GCC, wherein both triplets were embedded within an
unreactive A‚T tract (Figure 3), we examined the ability of
compounds 2-6 to alkylate guanine bases in both single-
Alkylation yields dropped significantly in single-stranded
oligomers, being slightly higher for 7 than 8, and similarly
for 9 compared to 10. With the GGC strand 7 and CGG
strand 9, there was no sequence selectivity for alkylation of
the two guanines (1:1) when these oligomers were single-
stranded. There was no sequence recognition of single-
stranded DNA by 3, although it is instructive to compare
the alkylation yields for single- versus double-stranded
(6) For a review of the azinomycins, see: Hodgkinson, T. J.; Shipman,
M. Tetrahedron 2001, 57, 4467. For a review on the synthesis of DNA
cross-linking agents, see: Coleman, R. S. Curr. Opin. Drug DiscoVery DeV.
2001, 4, 435. For a review on DNA cross-linking agents, see: Rajski, S.
R.; Williams, R. M. Chem. ReV. 1998, 98, 2723. For a review of agents
that covalently modify DNA, see: Gates K. S. Covalent Modification of
DNA by Natural Products. In ComprehensiVe Natural Products Chemistry;
Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.; Pergamon Press: Elsevier
Science, Oxford, U.K., 1999; p 491.
(7) Coleman, R. S.; Perez, R. J.; Burk, C. H.; Navarro, A. J. Am. Chem.
Soc. 2002, 124, in press.
(8) Alcaro, S.; Coleman, R. S. J. Med. Chem. 2000, 43, 2783. Alcaro,
S.; Ortuso, F.; Coleman, R. S. J. Med. Chem. 2002, 45, 861.
(9) Sugiyama, H.; Saito, I. J. Am. Chem. Soc. 1996, 118, 7063.
Org. Lett., Vol. 4, No. 20, 2002
3546

oligomers to establish the apparent background alkylation
and to understand the importance of noncovalent association
in driving covalent bond formation.
duplexes 7‚8 and 9‚10. Under forcing reaction conditions,
no distinct alkylated species was formed on PAGE, and the
electrophoresis profile resembled that obtained with simple
epoxides such as glycidol with less than 10% lower mobility
species formed. Piperidine-induced strand cleavage showed
that both guanines in the GGC or CGG strands (i.e., 7 or 9)
were alkylated equally. When the primary amide of 6 was
replaced with a benzyl ester as in 5, DNA alkylation was
completely abolished.
Cytotoxic activity of azinomycin partial structures was
measured against two standard breast cancer cell lines:
hormone-dependent MCF-7 cells and hormone-independent
MDA-MB-231 cells (Table 2), using the CellTiter 96
In these assays, a 100-fold excess of agents was used in
order to provide measurable alkylation yields in single-
stranded oligodeoxynucleotides. It was important to use the
same stoichiometry in all DNA systems so that a direct
comparison of yields could be made. Polyacrylamide gel
electrophoresis (PAGE) of the reaction of epoxyamide 3 (100
µΜ) and duplex 7‚8 (5 µM) in Tris buffer (pH 7.5, drug/
DNA ratio ) 20:1) showed alkylation had proceeded in
g75% yield after 20 h at 8 °C (Figure 4). With compound
3 and duplex 7‚8 using a drug/oligonucleotide ratio of 5:1,
we obtained 40-50% alkylation under the same reaction
conditions (data not shown).
Table 2. Cytotoxic Activity of Azinomycin Partial Structures
2-6 against Breast Cancer Cell Lines MCF-7 and MDA-MB
231
IC50 (MCF-7)
IC50 (MDA-MB-231)
compound
(µM)
(µM)
2
3
4
5
6
>25
2.2
4.2
naa
na
10.1
0.4
1.6
na
na
a na ) not active.
Figure 4. Alkylation of duplex 7‚8 with epoxyamide 3. Polyacryl-
amide gel electrophoresis autoradiogram. Lane 1: 5′ 32P end-labeled
DNA (strand 7). Lane 2: 5′ 32P end-labeled DNA (strand 7) +
drug (3) after incubation at 8 °C for 20 h.
aqueous nonradioactive cell proliferation assay.10 In each case
the MDA-MB-231 cells were more sensitive to the effects
of these agents. The lack of reactivity toward DNA by ester
2 relative to that of amide 3 paralleled the much weaker
cytotoxic activity exhibited by 2, and the corresponding
increase in alkylation by dimethylamide 4 correlated with
increased cytotoxicity. In both cell lines there was a marked
correlation between the extent of DNA alkylation and
cytotoxicity, with compounds 3 and 4 exhibiting the highest
levels of DNA alkylation and of cytotoxicity, indicating that
alkylation of DNA is a critical biological event in the
mechanism of action of these agents and presumably for
azinomycin B.
These results indicate the important role that the azino-
mycin naphthoate plays in increasing sequence selective
binding affinity (as evaluated by DNA alkylation yields) and
cytotoxic activity. In the absence of the naphthoate, epoxide-
bearing partial structures exhibited low reactivity toward
DNA and no sequence selectivity, with correspondingly weak
cytotoxicity. The azinomycin “left-half” partial structure 3
exhibited altered sequence selectivity compared to that of
the native agent, while retaining a substantial portion of the
cytotoxic activity: epoxide 3 alkylated both guanines in the
GGC sequence 7 to a significant extent, whereas azinomycin
B alkylates the 5′ guanine in the GGC sequence 7 with
complete selectivity.7 Even more remarkable is the ineffectual
activity of the corresponding benzyl ester 2 in DNA
Coleman and co-workers7 and Saito and co-workers3b
demonstrated that the azinomycin epoxide alkylates the
purine of the 3′-PyPyPu-5′ strand, so it appears from the
above results that azinomycin partial structure 3 binds to
duplex DNA in a mode(s) not observed with the native agent,
thus bestowing the remainder of the azinomycin molecule
with either a substantial or critical portion of the sequence
recognition ability. In duplex DNA the extent of alkylation
of guanine bases by 3 follows the order GGC . GCC >
CGG > CCG, where there is a significant difference in
alkylation yields between the GGC strand and the other three
strands.
When the native carboxamide in epoxyamide 3 was
replaced with a benzyl ester in 2, alkylation of DNA was
completely abolished. With levels of 2 as high as 104 equiv
per oligomer 7 or duplex 7‚8, there was no observable
alkylation of DNA. To determine whether this effect was
due to favorable hydrogen bond donation by the primary
amide of 3, we examined N,N-dimethylamide 4 and found
that alkylation of 7 in duplex 7‚8 was partially restored
(60%). The hydrogen bond donating C16 amide appeared
important but not critical for DNA alkylation.
With structure 6 that lacks the naphthoate completely, the
efficiency of DNA alkylation dropped dramatically, and both
the reaction temperature (37 °C) and equivalents of agent
had to be increased substantially to achieve alkylation with
(10) Barltrop, J. A.; Owen, T. C.; Cory, A. H. Bioorg. Med. Chem. Lett.
1991, 1, 611. Cory, A. H.; Owen, T. C.; Cory, J. G. Cancer Comm. 1991,
3, 207.
Org. Lett., Vol. 4, No. 20, 2002
3547

alkylation assays, where it exhibited no reactivity, and in
the cytotoxicity assays, where it was significantly less
cytotoxic than the corresponding primary carboxamide 3 or
tertiary carboxamide 4.
Acknowledgment. This work was supported by a grant
from the NIH (R01 CA65875) and by the OSU Compre-
hensive Cancer Center (P30 CA16058). A.N. was a fellow
of the Spanish Ministerio de Educacion (EX 0052672518).
E.S.D.-C. was an NIH Predoctoral Trainee (T32 GM08512).
Our results provide the first indication that covalent
modification of DNA correlates directly with cytotoxic
activity for this class of antitumor agents. These studies
provide important information about structure-activity re-
lationships2 in the left half of the azinomycins, showing that
both the naphthoate and N16 amide nitrogen are critically
important for effective interaction with duplex DNA. Un-
derstanding and utilizing such information obtained from
studies of partial structures will prove critical in designing
more effective antitumor agents based on the azinomycin
skeleton.
Supporting Information Available: Experimental pro-
cedures and spectral characterization of 2-6 and experi-
mental details for DNA alkylation and cytotoxicity assays.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL0267275
3548
Org. Lett., Vol. 4, No. 20, 2002