A rationally designed genotoxin that selectively destroys estrogen receptor-positive breast cancer cells

Article abstract of DOI:10.1021/ja017344p

We describe a novel strategy to increase the selective toxicity of genotoxic compounds. The strategy involves the synthesis of bifunctional molecules capable of forming DNA adducts that have high affinity for specific proteins in target cells. It is propo

Full text of DOI:10.1021/ja017344p

Published on Web 02/08/2002
A Rationally Designed Genotoxin that Selectively Destroys Estrogen
Receptor-Positive Breast Cancer Cells
Kaushik Mitra, John C. Marquis, Shawn M. Hillier, Peter T. Rye, Beatriz Zayas, Annie S. Lee,
John M. Essigmann,* and Robert G. Croy*
Department of Chemistry and DiVision of Bioengineering and EnVironmental Health, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139
Received October 22, 2001
Scheme 1 a
The persistence of genetic damage produced by alkylating agents1
as well as the antagonism of essential biochemical processes such
as transcription can have lethal consequences for malignant cells.2
Both mechanisms have been identified in studies to uncover the
reasons for the efficacy of cisplatin in the treatment of several can-
cers.2a,3 We describe a synthetic strategy to create bifunctional mol-
ecules that produce DNA adducts capable of binding the estrogen
receptor (ER), which is aberrantly expressed in many breast cancer
cells.4 It is speculated that DNA adducts that form complexes with
the ER will be poorly repaired in these cells because they are cam-
ouflaged from detection by DNA repair enzymes. Consequently,
the DNA lesions persist. Furthermore, the DNA adducts would be
expected to act as “molecular decoys” capable of displacing the
ER from its natural targets and antagonizing its role in malignant
growth. In healthy cells, where the abundance of the ER is minimal,
no such ER-DNA adduct complexes will be present, and the cell
should survive.5
a Conditions: (i) DHP, pyridinium p-toluenesulfonate, reflux, 92%; (ii)
n-BuLi, KOtBu, B(OMe)3, H2O2; (iii) PCC, 59%; (iv) 6-iodohexene, KOtBu,
Et3B, 47%; (v) CH3COCl/MeOH, Et3SiH, BF3.Et2O, 74%; (vi) DHP,
pyridinium p-toluenesulfonate, 77%; (vii) BH3.THF, KOH/H2O2, 66%; (viii)
methane sulfonyl chloride, LiBr, 86%; (ix) Ph2P(O)NH-CH2-CH2-OtBDMS,
NaH, catalyst tetra-n-butylammonium bromide, 73%; (x) TBAF, 73%; (xi)
p-nitrophenylchloroformate, DIEA; (xii) 4-(N,N-bis-2-chloroethylamino-
phenyl)-propylamine, DIEA; (xiii) H+, 60%.
In this report we describe the design and synthesis of compound
1, a bifunctional agent that can form covalent DNA adducts capable
of binding the ER with high affinity and specificity. We show that
1 has selective toxicity toward ER+ breast cancer cells compared
to ER- cells in vitro.
Compound 1 consists of a bis-chloroethyl aniline mustard as the
DNA alkylating unit tethered to estradiol, the natural ligand for
the ER. The site of substitution of estradiol in 1 was based on reports
that relatively large alkyl groups can be attached at the 7R position
with retention of high affinity for the ER.6 The synthetic strategy
for 1 is shown in Scheme 1. Compound 7, a key compound in the
synthesis, was prepared by a modification of a published strategy.7
Briefly, 3 was functionalized with a 6-carbon chain at the 7-position
in R-stereochemistry to provide the alkenyl steroid 4. Efficient
reduction of the 6-oxo group in 4 was achieved with Et3SiH/BF3‚
Et2O; however, this treatment also caused the loss of 3,17-tetra-
hydropyranoxy (THP) groups producing diol 5. The 3,17-OHs of
5 were reprotected with THP groups to afford 6, followed by
oxidation of the alkene at the terminus of the linker to provide
alcohol 7. Steroid alcohol 7 was converted to bromide 8, which
was subsequently allowed to react with a protected ethanolamine
to give 9. Compound 9 was desilylated with tetrabutylammonium
fluoride (TBAF) and converted to a carbonate intermediate with
p-nitrophenyl chloroformate. The carbonate was coupled to (N,N-
bis-2-chloroethylaminophenyl)propylamine that was prepared from
chlorambucil via the Curtius reaction.8 Deprotection of the product
in HCl/methanol furnished 1.
Next, the ability of 1 to modify DNA covalently was investigated.
Plasmid DNA was incubated with 100 µM [14C]-110 at 37 °C for
up to 6 h. After unbound 1 was removed by phenol-CHCl3
extraction and ethanol precipitation, the radioactivity associated with
DNA was measured. The amount of radioactivity bound to DNA
increased at a constant rate over the 6-h period indicating the
formation of covalently bound 1 (see Supporting Information). On
the basis of previous studies on DNA alkylation by nitrogen
mustards,11 it is likely that covalent adducts of 1 are formed
primarily at the N7 atom of guanines. To investigate the identity
of the covalent adducts, 1-modified DNA was treated with 0.1 N
HCl, the major product from the digested DNA was isolated by
reversed-phase HPLC and analyzed by full scan electrospray mass
spectrometry to yield a prominent molecular ion signal at M +
H+/z 813.5051. This mass is consistent with a chemical structure
in which one ethylene arm of the mustard of 1 is attached to guanine
and the other arm contains a -OH substituted for the Cl atom.12
The affinity of DNA adducts of 1 for the ER was investigated
using an Electrophoretic Mobility Shift Assay (EMSA). Substrates
for this assay were prepared by reaction of 5′[32P]-labeled self-
complementary oligonucleotide 5′-d(ATTATTGGCCAATAAT)
with 1 for 4 h at 37 °C. To assess quantitatively the level of covalent
modification under these conditions, the DNA was treated with
piperidine (1 M, 90 °C, 1 h) and the products were separated on a
denaturing 20% polyacrylamide gel (Figure 1A). This analysis
The affinity of 1 for the ER was first determined. Using a com-
petitive binding assay9 with [3H]-17â estradiol, compound 1 was
found to have a relative binding affinity (RBA) for the calf uterine
ER of 30; RBA of estradiol ) 100.
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10.1021/ja017344p CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
of selective toxicity of 1. Covalent 1-guanine adducts have been
identified in DNA isolated from treated cells (unpublished).
Therefore, 1 is stable to cell culture conditions and can form DNA
adducts in cells identical with those formed in vitro. We are
currently investigating the role of 1-DNA adducts in vivo to
determine if they are less efficiently repaired in ER+ cells and to
evaluate whether the association of the ER with DNA adducts of
1 is directly responsible for the greater sensitivity of MCF-7 cells.
Acknowledgment. We thank John Wishnok and W. Sara
Gardinar-Stillwell for helpful discussions. We are grateful to the
National Cancer Institute (1-RO1-CA77743-04) for support of this
work, to the Susan G. Komen Foundation for a fellowship to K.M.
(PDF00-000679), to the National Institutes of Health for support
of the NMR facility (1S10RR13886-01), and to the BEH Mass
Spectrometry Laboratory for use of their instruments.
Figure 1. (A) Piperidine treatment of self-complementary deoxy oligo-
nucleotide 5′-d(AATATTGGCCAATATT) treated with 1. Lane 1: untreated
oligonucleotide, Lane 2: oligonucleotide + 200 µM 1. (B) Retarded mobility
of oligonucleotide-1 in the presence of ER-LBD illustrated by EMSA, and
disappearance of the retarded band by competition with estradiol. Lane 1:
untreated oligonucleotide + ER-LBD, Lane 2: oligonucleotide modified
by 200 µM 1 + ER-LBD, Lanes 3-8: modified oligonucleotide +
ER-LBD + 10-300 nM estradiol.
Supporting Information Available: Procedures for the synthesis
of 1, hER-LBD expression and purification, RBA experiments, DNA-
damaging studies, gel shift experiments and toxicity experiments (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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Figure 2. Survival of MCF-7 (ER+) (9, 0) and MD-MB231 (ER-) (b,
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