Design and synthesis of a novel DNA-DNA interstrand adenine-guanine cross-linking agent [1]

Full text of DOI:10.1021/ja005658r

J. Am. Chem. Soc. 2001, 123, 4865-4866
4865
Design and Synthesis of a Novel DNA-DNA
Interstrand Adenine-Guanine Cross-Linking Agent
Qun Zhou,†,‡ Wenhu Duan,†,‡ Denise Simmons,§
Yuda Shayo,†,‡ Mary Ann Raymond,† Robert T. Dorr,†,‡ and
Laurence H. Hurley*,†,‡
Arizona Cancer Center, 1515 North Campbell AVenue
Tucson, Arizona 85724
College of Pharmacy, The UniVersity of Arizona
Tucson, Arizona 85721
College of Pharmacy, The UniVersity of Texas at Austin
Austin, Texas 78712
ReceiVed October 2, 2000
ReVised Manuscript ReceiVed March 29, 2001
DNA interstrand cross-linking agents that interact within the
minor groove have attracted considerable interest in the past few
years,1 and much progress has been made in the design and
synthesis of these compounds.2 These cross-linking agents belong
to two broad classes: natural products that cross-link 2-3 base
pairs apart1,3 and synthetic agents that cross-link 4-7 base pairs
apart.1,4 Both groups generally target either guanines (e.g.,
mitomycin C and DSB-120)1,3,4 or adenines (e.g., bizelesin).1,5
What is missing is the availability of minor groove DNA-DNA
interstrand cross-linking agents that alkylate guanines on one
strand and adenines on the opposite strand. This would provide
new agents capable of recognizing and binding to more extended
and mixed A‚T and G‚C sequence tracts of DNA to uniquely
define individual gene targets and hence exert biological specific-
ity. We report here the design and synthesis of the agent UTA-
6026, which contains two different alkylation moieties with the
potential to alkylate G or A.
Figure 1. Structures of (+)-CPI, DC-81, and target compound.
[1,4]benzodiazepine (P[1,4]B) family that is capable of covalently
reacting with the exocyclic 2-NH2 group of guanine7) were chosen
as the two monoalkylating subunits. By tethering them with a
suitable linker, a potential adenine-guanine interstrand cross-
linking agent could be synthesized. The linker was designed with
two objectives in mind. First, the indole was included to increase
the minor groove binding affinity without affecting the sequence
selectivity, which is primarily determined by the (+)-CPI alky-
lating subunit.8 Second, the flexible alkyl chain was chosen to
facilitate the isohelical accommodation of the P[1,4]B alkylating
subunit.2c The structure of the target compound is shown in Figure
1. To achieve the six-base-pair span, molecular modeling was
used to determine the alkyl chain length between the two
alkylating subunits. The modeling results (unpublished) show that
when n ) 3 (UTA-6026), the two units have an optimal chance
to alkylate the two desired sites, A* and G*, on opposite DNA
strands in sequence I to form the DNA-DNA interstrand cross-
link.
The strategy for the synthesis of compound UTA-6026 is shown
in Scheme 1. The main synthetic challenge is to form the active
cyclopropyl subunit in the (+)-CPI unit and the acid-sensitive
N10-C11 imine moiety in the P[1,4]B subunit in one molecule
at the same time. We used Thurston’s approach2e for synthesizing
the nucleophile-sensitive/acid-sensitive P[1,4]B analogues by
protecting the N10 with Fmoc and, after the remainder of the
molecule is assembled, removing the Fmoc group to form the
imine bond of the P[1,4]B subunit in the last step. Vanillic acid
(1), the starting material, was reacted with ethyl bromobutyrate,
followed by hydrolysis of the ester under basic conditions to give
compound 2. Nitration of 2 with nitric acid gave the nitro
compound 3. Selective esterification of 3 afforded the monoester
4, which was coupled with (2s)-(+)-pyrrolidine-2-carbaldehyde
diethyl thioacetal 12 to give 5. Reduction of 5 yielded the amino
compound 6. The amine was protected by the Fmoc group
followed by deprotection and cyclization of 7 with HgCl2/CaCO3
to give an Fmoc-protected P[1,4]B with an ether alkyl chain (8).
Hydrolysis of the methyl ester 8 under acidic conditions gave
the key intermediate acid 9.9a,b Compound 9 was coupled with
(+)-seco-CPI-indole 139c,d,e (which was converted from N-mesyl-
We chose a DNA sequence (sequence I in Figure 1) containing
an adenine and guanine six base pairs apart on opposite strands
as the potential DNA cross-linking target template. To achieve
the cross-linking, (+)-cyclopropapyrroloindole [(+)-CPI] (the
DNA-DNA alkylating moiety of (+)-CC-1065 that selectively
alkylates N3 of adenine6) and DC-81 (one member of the pyrrolo-
† Arizona Cancer Center.
‡ The University of Arizona.
§ The University of Texas at Austin.
(1) For a comprehensive review of DNA cross-linkers, see: Rajski, S. R.;
Williams, R. M. Chem. ReV. 1998, 98, 2723.
(2) (a) Mitchell, M. A.; Johnson, P. D.; Williams, M. G.; Aristoff, P. A. J.
Am. Chem. Soc. 1989, 111, 6428. (b) Mitchell, M. A.; Kelly, R. C.; Wicnienski,
N. A.; Hatzenbuhler, N. T.; Williams, M. G.; Petzold, G. L.; Slightom, J. L.;
Siemieniak, D. R. J. Am. Chem. Soc. 1991, 113, 8994. (c) Bose, D. S.;
Thompson, A. S.; Ching, J. S.; Hartley, J. A.; Berardini, M. D.; Jenkins, T.
C.; Neidle, S.; Hurley, L. H.; Thurston, D. E. J. Am. Chem. Soc. 1992, 114,
4939. (d) Thurston, D. E.; Bose, D. S.; Howard, P.; Jenkins, T. C.; Leoni, A.;
Beraldi, P. G.; Guiotto, A.; Cacciari, B.; Kelland, L. R.; Floppe, M. P.; Rault,
S. J. Med. Chem. 1999, 42, 1951. (e) Wilson, S. C.; Howard, P. W.; Forrow,
S. M.; Hartley, J. A.; Adams, L. J.; Jenkins, T. C.; Kelland, L. R.; Thurston,
D. E. J. Med. Chem. 1999, 42, 4028. (f) Mountzouris, J. A.; Wang, J. J.;
Thurston, D. E.; Hurley, L. H. J. Med. Chem. 1994, 37, 3132. (g) Gregson,
S. J.; Howard, P. W.; Jenkins, T. C.; Kelland, L. R.; Thurston, D. E. Chem.
Commun. 1999, 9, 797.
(3) (a) Norman, D.; Live, D.; Sastry, M.; Lipman, R.; Hingerty, B. E.;
Tomasz, M.; Broyde, S.; Patel, D. J. Biochemistry 1990, 29, 2861. (b) Tomasz,
M.; Palom, Y. Pharmcol. Ther. 1997, 76, 73.
(4) (a) Hurley, L. H. The Minor Groove Covalent Reactive Drugs
Anthramycin and CC-1065 and Their Interstrand Cross-linking Derivatives.
In DNA Adducts of Carcinogenic and Mutagenic Agents: Chemistry,
Identification, and Biological Significance; IARC Scientific Publications Series,
1993; pp 295-312. (b) Mountzouris, J. A.; Hurley, L. H. Small Molecule-
DNA Interactions. In Bioorganic Chemistry: Nucleic Acids; Oxford University
Press: New York, 1996; pp 288-323.
(5) (a) Ding, Z. M.; Hurley, L. H. Anti-Cancer Drug Des. 1991, 6, 427.
(b) Sun, D.; Hurley, L. H. J. Am. Chem. Soc. 1993, 115, 5925. (c) Seaman
F.; Hurley, L. H. Biochemistry 1993, 32, 12577. (d) Seaman, F. C.; Chu, J.;
Hurley, L. H. J. Am. Chem. Soc. 1996, 118, 5383. (e) Woynarowski, J. M.;
McHugh, M. M.; Gawron, L. S.; Beerman, T. A. Biochemistry 1995, 34, 13042.
(6) Hurley, L. H.; Reynolds, V. L.; Swenson, D. H.; Petzold, G. L.; Scahill,
T. A. Science 1984, 226, 843.
(7) Hurley, L. H.; Petrusek, R. L. Nature 1979, 282, 529.
(8) Hurley, L. H.; Lee, C.-S.; McGovren, J. P.; Mitchell, M.; Warpehoski,
M. A.; Kelly, R. C.; Aristoff, P. A. Biochemistry 1988, 27, 3886-3892.
10.1021/ja005658r CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/01/2001

4866 J. Am. Chem. Soc., Vol. 123, No. 20, 2001
Communications to the Editor
Scheme 1a
a (a) [i] BrCH2CH2CH2COOEt/K2CO3/DMF; [ii] NaOH/H2O/EtOH/
reflux; (b) 70% HNO3; (c) MeOH/p-TsOH/rt; (d) [i] (COCl)2/DMF/THF;
[ii] 12/Et3N/CH2Cl2; (e) SnCl2‚2H2O/MeOH/reflux; (f) Fmoc-Cl/aq Na2-
CO3/H2O/dioxane; (g) HgCl2/CaCO3/CH3CN/H2O; (h) 10% aq HCl/THF/
rt; (i) 13/EDCI/DMA; (j) Bu4N+F-/DMF/rt.
seco-CPI via six steps) by EDCI/DMA to give compound 10.
Fortuitously, removal of the Fmoc-protecting group and cycliza-
tion of the seco-CPI part were finished in one step with TBAF in
DMF to give the final target compound 11 (UTA-6026).2d The
overall yield from vanillic acid 1 to the target compound 11 was
6%.
Nondenaturating gel electrophoresis was used to explore the
DNA-DNA interstrand cross-linking ability of UTA-6026 on the
sequence shown in Figure 2C. The results in Figure 2, A and B,
show that UTA-6026 has a concentration-dependent cross-linking
ability when either strand is radiolabeled.10 To identify the sites
of alkylation on the top strand, a thermal cleavage assay was
performed.11 As expected, DNA cleavage was found at adenines
9 and 19 (Figure 2C).12 Because the P[1,4]B subunit alkylates at
N2 of guanine, no cleavage was demonstrated on the bottom
strand; however, upon substitution of the guanines purportedly
alkylated by the P[1,4]B end by inosine, which lacks the 2-amino
group, no cross-linking band equivalent to that shown in Figure
2 was found (unpublished results).
Figure 2. Concentration dependency of DNA cross-linkage by UTA-
6026 on the top (A) and bottom (B) strands. Cross-linked species ) cl;
single-stranded species ) ss. (C) Autoradiograph of products from thermal
strand breakage assay to demonstrate (+) alkylation sites at N3 of adenines
9 and 19 by UTA-6026 on the upper strand of the sequence. Lane Pu:
Maxam-Gilbert A/G sequencing reaction. Lane 1: DNA without added
drug. Lane 2: UTA-6026-reacted DNA. Arrows point to the product of
thermal cleavage, while the dashed lines indicate incomplete products
from the same reaction.11
Preliminary in vitro tests showed that UTA-6026 has remark-
ably potent cytotoxicity to several tumor cell lines (IC50 ) 0.28
nM in human non-small cell breast tumor cell line MCF-7,
(9) (a) Thurston, D. E.; Bose, D. S.; Thompson, A. S.; Howard, P. W.;
Leoni, A.; Croker, S. J.; Jenkins, T. C.; Neidle, S.; Hartley, J. A.; Hurley, L.
H. J. Org. Chem. 1996, 61, 8141. (b) Baraldi, P. G.; Balboni, G.; Cacciari,
B.; Guiotto, A.; Manfredini, S.; Romagnoli, R.; Spalluto, G.; Thurston, D.
E.; Howard P. W.; Bianchi, N.; Rutigliano, C.; Mischiati, C.; Gambari, R. J.
Med. Chem. 1999, 42, 5131. (c) Kelly, R. C.; Gebhard, I.; Wicnienski, N.;
Aristoff, P. A.; Johnson, P. D.; Martin, D. G. J. Am. Chem. Soc. 1987, 109,
6837. (d) Warpehoski, M. A.; Bradford, V. S. Tetrahedron Lett. 1986, 27,
2735. (e) Warpehoski, M. A. Tetrahedron Lett. 1986, 27, 4103.
IC50 ) 0.047 nM in colon tumor cell line SW480, and IC50
)
5.1 nM in human lung tumor cell line A549).
In summary, a unique heterobifunctional compound that forms
interstrand cross-linking between adenine and guanine six base
pairs apart was designed and synthesized. It shows mixed
sequence-specific alkylation selectivity and demonstrates potent
antitumor activity against several tumor cell lines. In vivo tests
and further cytotoxicity studies are in progress.
(10) UTA-6026 at the concentrations shown in Figure 2, A and B, was
incubated for 6 h at an ambient temperature in pH 7.2 buffer. Each reaction
was terminated with 5 µg/µL calf thymus DNA, followed by addition of 3 M
sodium acetate, pH 7.2, 5 µg/µL tRNA, and ethanol precipitation. Dried pellets
were resuspended in strand separation buffer (30% w/w DMSO in 1 mM
EDTA). Denaturation for 1 min at 70 °C was followed by immediate chilling.
Electrophoresis was performed on a 12% native polyacrylamide gel at 150 V
for 12 h at 4 °C in Tris-acetate running buffer.
Acknowledgment. This work was supported by a Grant from the
National Cancer Institute (CA49751). We thank Dr. Bob Kelly at
Pharmacia & Upjohn for providing N-mesyl-seco-CPI. We are also
grateful to Dr. Haiyong Han and Dr. Adam Siddiqui for technical
assistance and to Dr. David Bishop for proofreading, editing, and
preparing the final version of the text and figures.
(11) Reynolds, V. L.; Molineux, I. J.; Kaplan, D.; Swenson, D. H.; Hurley,
L. H. Biochemistry 1985, 24, 6228.
(12) Drug reactions were carried out using 100 µM drug with 50 ng 32P
end-labeled DNA for 6 h at ambient temperature in a final volume of 10 µL
buffer (1 mM Tris, pH 7.2, 10 mM NaCl). Reactions were stopped with 5 µg
calf thymus DNA, followed by ethanol precipitation with 3 M sodium acetate
pH 7.2 and 10 µg/µL tRNA. Ethanol precipitates were dried, resuspended in
piperidine, and boiled for 20 min to induce strand breakage. The strand
breakage products were then ethanol precipitated, resuspended in alkaline dye,
and resolved on a 20% sequencing gel.
Supporting Information Available: Experimental details and ana-
lytical data for the preparation of 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 are
provided (PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
JA005658R