Design, synthesis, and evaluation of estradiol-linked genotoxicants as anti-cancer agents

Article abstract of DOI:10.1016/j.bmcl.2004.04.064

A series of bifunctional compounds was prepared consisting of 17β estradiol linked to a DNA damaging N,N-bis-(2-chloroethyl)aniline. The objective of our studies was to determine the characteristics of the linker that permitted both reaction with DNA and

Full text of DOI:10.1016/j.bmcl.2004.04.064

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3829–3833
Design, synthesis, and evaluation of estradiol-linked genotoxicants
as anti-cancer agents
U. Sharma, J. C. Marquis, A. Nicole Dinaut, S. M. Hillier, B. Fedeles, P. T. Rye,
J. M. Essigmann* and R. G. Croy*
Department of Chemistry and Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Received 27 February 2004; revised 21 April 2004; accepted 21 April 2004
Available online
Abstract—A series of bifunctional compounds was prepared consisting of 17b estradiol linked to a DNA damaging N,N-bis-(2-
chloroethyl)aniline. The objective of our studies was to determine the characteristics of the linker that permitted both reaction with
DNA and binding of the resultant covalent adducts to the estrogen receptor. Linker characteristics were pivotal determinants
underlying the ability of the compounds to kill selectively breast cancer cells that express the estrogen receptor.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
for the ER was obtained by replacing the 2PI group with
that of 17b-estradiol connected via the 7a position.6
Both the 2PI and estradiol-based compounds showed
increased toxicity toward breast cancer cells that express
the ER. Evidence was presented that the differential
toxicity of the DNA damaging agents was not due to an
anti-hormonal mechanism.
Many anti-tumor drugs produce their cytotoxic effect
through covalent damage to DNA.1;2 Repair enzymes
that remove such covalent lesions, restoring the integrity
of genetic information, limit the effectiveness of these
drugs. Inhibition of DNA repair in cancer cells is an
attractive yet not significantly explored strategy to
potentiate the therapeutic effects of alkylating chemo-
therapeutic drugs.3;4 We have previously described the
synthesis and biological activity of novel bifunctional
compounds that form covalent DNA adducts with high
affinity for the estrogen receptor (ER).5;6 The com-
pounds were designed to test the hypothesis that
adduct–ER complexes would be concealed from DNA
repair proteins and therefore be refractory to repair. The
compounds would thus exhibit greater toxicity in cancer
cells that over express the ER protein.
We investigated the molecular features of the linker that
are essential for the biological activities of the estradiol-
linked compound 1 (Fig. 1) by varying the structure of
the linker connecting the N,N-bis-(2-chloroethyl)aniline
and estradiol moiety. The roles of the amino and car-
bamate groups in the linker were of particular interest
because their substitution with different chemical groups
might lead to a less complex synthetic process and in-
crease overall yields. The modified linkers retained the
six carbon alkyl chain that appears, based on our earlier
work, to be essential for the attached ligand to fit into
The original series of compounds consisted of an N,N-
bis-chloroethylaniline connected to a 2-phenylindole
(2PI) group by alkyl-amino-carbamate linkers of vari-
ous lengths.5 Investigations with these derivatives iden-
tified several molecular characteristics of the linker that
were important for the compound to react with DNA
and bind to the ER. A compound with greater affinity
Keywords: Anti-cancer; Steroid–mustard conjugates; Breast cancer.
* Corresponding authors. Tel.: +1-617-2536227; fax: +1-617-2535445;
e-mail addresses: jessig@mit.edu; rgcroy@mit.edu
Figure 1. Structure and molecular features of lead compound.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.064

3830
U. Sharma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3829–3833
the estradiol binding site of the ER.5 Amino, amido, and
guanidino groups were incorporated into the region
connecting the hexanyl-substituted estradiol and the
aniline group. We report here the synthesis and physical
properties of these molecules and their cytotoxic effects
toward breast cancer cells.
incorporate an N,N-disubstituted guanidine moiety into
the linker. The preparation of 7 proceeded with the
initial reaction of 2 with 1,3-bis(tert-butoxycarbonyl)-2-
methyl-2-thiopseudourea under Mitsunobu conditions.7
The resulting product 6 was then allowed to react with
an excess of (N,N-bis-2-chloroethylaminophenyl)-pro-
pylamine followed by acid deprotection to furnish 7.
Compound 9 was prepared by conversion of 2 to the p-
nitrophenyl carbonate 8, which was then allowed to
react with (N,N-bis-2-chloroethylaminophenyl)-propyl-
amine.6 Removal of THP groups under acidic condi-
tions produced 9. Compound 10, the starting material
for compounds 12–15, was prepared by hydrazinolysis
of the phthalimide of 2 that was formed under Mits-
unobu conditions.8 Compound 12 containing two amide
groups was synthesized by first reacting 10 with the
NHS ester of 4(tert-butoxycarbonylamino)butyric acid.
Following removal of the THP and Boc groups the
terminal amino group was allowed to react with the
NHS ester of chlorambucil producing 12. Compound 13
in which the linker contains two amino groups was
produced by reduction of 12 with borane dimethylsul-
fide complex.9 The preparation of 14 was also accom-
plished by conversion of 2 to the phthalimide via
Mitsunobo conditions with subsequent hydrazinolysis
to obtain the amine 10. The NHS ester of chlorambucil
2. Chemistry
The synthesis of compound 1 was described in our
previous report.6 The syntheses utilized 3,17b-bis-(2-
tetrahydropyranyloxy)-7a-(6-hydroxyhexan-1-yl)-estra-
1,3,5(10) triene 2 as the starting compound (see box in
Scheme 1); its preparation has also been described.6
Construction of linkers proceeded by linear additions to
2 with final addition of the N,N-bis-(2-chloroethyl)ani-
line moiety. Compound 5 was prepared by conversion
alcohol 2 to the bromide, which was allowed to react
with a protected ethanolamine providing 3 as described
in Ref. 10. The Mitsunobo reaction7 then was used
to couple 1,3-bis-(tert-butoxycarbonyl)-2-methyl-2-thio-
pseudourea with 3. Reaction of the resulting product 4
with excess (N,N-bis-2-chloroethylaminophenyl)-prop-
ylamine followed by acid deprotection produced 5.
Procedures described by Linney et al.7 were applied to
O
m
H
H
N
O
H
H
R2
R2
(CH2)4
N
R1 (CH2)6 N (CH2)4
(CH2)4
l,n
(CH2)6
R1
R1 (CH2)6
N
(CH2)3
N
(CH2)3 R2
H
15
13
12
g,l
m
O
O
H
k
H
(CH2)6
N
(CH2)3 NHBoc
R1
R1
N
(CH2)6
(CH2)6
(CH2)3 R2
R1
NH2
14
10
11
i,j
O
(Ph)2
P
N
O
a-d
h
R1 (CH2)6 OH
R1 (H2C)6
(CH2)2 OH
NO2
O
O
R1 (CH2)6
3
e
2
8
e
Boc
(Ph)2
Boc
O
P
f,g
N
N
(CH2)6
N
O
R1
R1
N
(H2C)6
N
(CH2)2
H
S
S
R1 (CH2)6
R2
O
N (CH2)3
Boc
Boc
4
f,g
6
9
f,g
NH
H H
NH
H
H
R1 (H2C)6 N (CH2)2
N
N (CH2)3 R2
R2
(CH2)3
N
R1 (CH2)6
N
H
5
7
R1=
N
[(CH2)2Cl]2
R2=
OX
X = TPH 2,3,4,6,8
X = H 5,7,9,12,13,14,15
XO
Scheme 1. Synthesis of estradiol-linked aniline mustards. The starting compound 2 is shown in the box (center). Reagents and conditions: (a)
methanesulfonyl chloride, DIEA, THF; (b) LiBr, TEA, DMF, 60 °C; (c) Ph2P(O)NHCH2CH2OTBDMS, NaH, TBAB, Ph–H, 60 °C; (d) TBAF,
THF; (e) Boc-NHC(@N-Boc)SCH3, PPh3, DIPAD, THF; (f) 4-(N,N-bis-2-chloroethylamino-phenyl)-propylamine, THF/H2O (90:10), reflux; (g)
HCl/dioxane, CH2Cl2; (h) p-nitrophenylchloroformate, DIEA, THF; (i) phthalimide, DIPAD, PPh3, THF; (j) Hydrazine, EtOH, reflux; (k) 4-(N-
Boc)n-butyl(N-hydroxysuccinimide)ester, TEA, DMF; (l) 4-(N,N-bis-chloroethylamino-phenyl)butyl(N-hydroxysuccinimide)ester, TEA, DMF; (m)
BH3S(CH3)2, THF, HCl; (n) HCl, THF. DIEA ¼ diisopropylethyl amine; TEA ¼ triethyl amine; TBAB ¼ tetrabutylammonium bromide;
TBAF ¼ tetrabutylammonium fluoride; DIPAD ¼ diisopropylethyl azadicarboxylate; DMF ¼ dimethylformamide; THF ¼ tetrahydrofuran.

U. Sharma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3829–3833
3831
was then allowed to react with the terminal amine,
producing 14. Compound 15, containing a secondary
amino group in the linker, was prepared by reduction of
the amide in 14 using borane dimethylsulfide complex.9
The reactivity of each compound with DNA was as-
sessed by its ability to produce piperidine labile sites in
the self complementary deoxyoligonucleotide 50-
d(AATATTGGCCAATATT). The results in Table 1
(column 3) indicate the percent of the oligomer that was
cleaved by piperidine. Compound 9 in which the alkyl
linker contains a single carbamyl group produced the
lowest level of modification (i.e., 3% cleaved by piperi-
dine). Compound 14 containing an amido instead of the
carbamyl group produced approximately five times the
number of DNA adducts (14% cleaved by piperidine).
High levels of reactivity toward DNA were observed
with compounds with linkers containing secondary
amino groups. The combination of the amino and
carbamyl groups in the linker of 1 resulted in a 10-fold
increase in reactivity over 9 in which the linker contains
only the carbamyl group. The reactivity of 1 was similar
to that of 15 in which the linker contains a single sec-
ondary amine suggesting that the charged amino group
is the major determinant of reaction rate. Compound 13
in which the linker contains a diamine –NH–(CH2)4–
NH–CH2– was the most reactive (79% cleaved by
piperidine). The same is likely the case for molecules 5
and 7 in which the strongly basic guanidino groups
would be cationic under assay conditions. It is likely
that the cationic nature of these molecules gives them a
high reactivity with DNA by localizing the reactive al-
kylating group in the vicinity of nucleophilic atoms. A
similar result has been reported for a conjugate of
chlorambucil with the polyamine spermidine.12
3. Biology
In the initial characterization of the biochemical prop-
erties of new compounds 5, 7, 9, 12–15 we evaluated
their affinities for the ER. A radiometric competitive
binding assay10 with the rabbit uterine ER was used to
determine the relative binding affinity (RBA) of each
compound for the ER as compared with estradiol;
RBA ¼ 100. All of the compounds exhibited some
affinity for the ER. The data in Table 1 show that the
new compounds have RBA values for the ER ranging
from 6 to 40. Among the new compounds, 15 containing
a single amino group in the linker had an RBA of 40,
which is comparable to 1.
Although it is apparent that the original combination of
the positively charged secondary amine with the neutral
carbamyl group (compound 1) results in a bifunctional
compound with excellent affinity for the rabbit uterine
ER, compounds 7, 12, and 13 also have good affinities.
These molecules were viewed as valuable assets as we
move ahead toward probing structure–activity rela-
tionships and the biochemical mechanisms underlying
the biological activity of 1. It is likely that our 7a-linked
estradiol compounds adopt a binding mode similar to
that identified for the 7a-undecylamide estradiol analog
ICI 164,384.11 The positioning and orientation of the
estradiol moiety of ICI-164,384 within the hydrophobic
binding cavity of the ER is directed by its 7a side chain,
which protrudes out of a hydrophobic channel extend-
ing from the binding pocket. At the surface of the LBD,
a 90° flexion of the undecyl chain enables the remainder
of the linker to track closely with the surface contours of
the LBD.11 The low RBAs of compounds 5, 9, and 14
may result from surface interactions adopted by the
linkers in these molecules that do not permit optimal
alignment of the estradiol moiety within the binding
cavity.
Using an electrophoretic gel mobility shift assay,6 we
observed that covalent DNA adducts of 1, 5, 7, 13, and
15 form complexes with the portion of the ER con-
taining the ligand binding domain (ER-LBD) (Table 1,
column 4). Under conditions that allowed complex
formation, addition of the ER to the modified DNAs
resulted in the appearance of a slowly migrating band by
electrophoresis that was eliminated by addition of excess
competitor, estradiol (data not shown). The results in
Table 1 (column 4) indicate that the extent of complex
formation for 1, 7, 13, and 15 were correlated with the
RBAs of the unreacted compounds. The exception was
compound 5 in which the linker contained both amino
and guanidino groups. In this case, despite its low RBA,
virtually all of the modified oligonucleotide formed a
Table 1
Compound
RBA
Oligo modified
(%)
Oligo shifted
(%)
log P
log D
ED30 (uM)
MDA-MB231
9.6
>20
MCF-7
1
46
10
28
6
45
29
58
3
93
93
5.32
6.27
5.67
5.61
4.85
5.32
5.07
5.96
2.22
3.12
2.52
5.61
4.85
2.22
5.07
2.87
5.1
>20
>20
>20
>20
5.1
5
7
38
>20
>20
>20
9
ND
ND
38
12
13
14
15
29
29
13
40
39
79
14
44
7.9
>20
8.6
ND
67
>20
5.1
RBA ¼ relative binding affinity for the rabbit uterine ER as compared to estradiol (RBA ¼ 100); % Oligo modified ¼ percent of 16-mer cleaved by
piperidine after treatment with test compound for 24 h at 37 °C; % Oligo shifted ¼ percent of covalently modified 16-mer that formed a slowly
migrating complex with the ER-LBD under electrophoresis; ED30 ¼ concentration of compound that resulted in 30% clonal survival of cells exposed
for 2 h.

3832
U. Sharma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3829–3833
MCF-7
MDA-MB231
100
10
1
100
10
1
(5,7,9
(CMB)
(
5
,
7
,
9
12,14)
(CMB)
15
12
,14)
(
)
15)
(
)
(
13
(1)
(1)
(13)
0
5
10
15
20
0
5
10
15
20
Concentration µM
Figure 2. Survival of MCF-7 (ER+) and MDA-MB231 (ER)) breast cancer cells after 2 h exposure to estradiol-linked toxicants.
slowly migrating band. We do not know the basis for
this unexpected finding.
intended mechanisms, since the RBAs and reactivities
with DNA of 13 and 15 imply greater toxicity on ER-
positive cells. It is interesting that the results of the
electrophoretic mobility shift assay indicate that DNA
adducts of 1 have the greatest affinity for the ER-LBD;
compound 1 also shows the largest differential toxicity
between the two cell lines.
Log P and log D values can be predictive of aqueous
solubility, absorption, and permeability.13 The lipophi-
licities of 1, 5, 7, 9, 12–15 were assessed using an HPLC
method to estimate the log P of the neutral form of each
compound. Log D values at pH 7.4 were estimated using
14
ꢀ
an equation derived by Horvath et al. for basic com-
pounds. The log D values in Table 1 indicated that the
aqueous solubilities of the eight compounds span
approximately a 2500-fold range under physiological
conditions. The compounds with log D values >5
(compounds 9 and 14) had both low affinities for the ER
and low reactivity with DNA. Compounds containing
charged groups with calculated log D values <3 gener-
ally had the highest affinities for the ER along with the
greatest reactivities toward DNA. These relationships,
however, did not prove to be reliable predictors of
biological activities in cytotoxicity assays against breast
cancer cells.
4. Conclusion
We designed and synthesized a series of estradiol-aniline
mustard-linked bifunctional molecules that differ in
their relative affinities for the ER and their capacities to
covalently modify DNA. The selective cytotoxic effects
of 3 of these compounds (1, 13, 15) toward ER-positive
breast cancer cells correlated with their ability to react
with DNA and their affinity for the ER, as well as
favorable solubility. We are directing research to
determine if the interaction of the ER with DNA ad-
ducts formed by these compounds is responsible for
their selective effects.
The lethal effects of our new compounds were investi-
gated in the MCF-7 (ER+) and MDA-MB231 (ER))
breast cancer cell lines. The data shown in Figure 2 and
Table 1 indicate that most but not all of the modifica-
tions that were introduced in the linker resulted in
decreased toxicity toward both cell lines. The low
toxicity of 5 and 7, which contain guanidinium groups,
may be related to either their poor uptake by cells or
their rapid excretion once absorbed.15 Despite showing
reactivity toward DNA in vitro, neither compound
showed significant toxicity at the highest dose; that is,
20 lM. Lack of uptake may also be responsible for the
low toxicity of 9, 12, and 14, which have high log D
values that are not predictive of good absorption.13
Further work is warranted to determine if cellular
uptake is indeed limiting for these compounds.
Acknowledgements
Research was supported by NIH grant CA77743, the
Natural Sciences and Engineering Research Council of
Canada (PDF-242490-2001 Fellowship to AND), an
Award from the Susan B. Komen Foundation, and NIH
grant 1SS10RR13386-01 for support of the NMR and
the mass spectrometry facility at MIT.
References and notes
As previously reported, 1 was significantly more toxic
toward MCF-7 cells than MDA-MB231 cells.6 Com-
pounds 13 and 15 containing amino groups showed
toxicity similar to that of 1. Both of these compounds
also showed greater toxicity toward the ER-positive
MCF-7 cells than toward the ER-negative MDA-
MB231 cells. This result was consistent with our
1. Teicher, B. A. Antitumor alkylating agents. In Cancer
Principles and Practice; DeVita, V. T., Hellman, S.,
Rosenberg, S. A., Eds.; Lippincott-Raven: New York,
1997; pp 405–417.
2. Hurley, L. H. Nature Rev. Cancer 2002, 2, 188–200.
3. Barret, J. M.; Hill, B. T. Anticancer Drugs 1998, 9, 105–
123.

U. Sharma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3829–3833
3833
4. Dolan, M. E. Adv. Drug Del. Rev. 1997, 26, 105–118.
5. Rink, S. M.; Yarema, K. J.; Solomon, M. S.; Paige, L. A.;
Tadayoni-Rebek, B. M.; Essigmann, J. M.; Croy, R. G.
Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15063–15068.
6. Mitra, K.; Marquis, J. C.; Hillier, S. M.; Rye, P. T.; Zayas,
B.; Lee, A. S.; Essigmann, J. M.; Croy, R. G. J. Am.
Chem. Soc. 2002, 124, 1862–1863.
10. Korenman, S. G. Endocrinology 1970, 87, 1119–1123.
11. Pike, A. C. W.; Brzozowski, A. M.; Walton, J.; Hubbard,
ꢁ
R. E.; Thorsell, A.-G.; Li, Y.-L.; Gustafsson, J.-A.;
Carlquist, M. Structure 2001, 9, 145–153.
12. Holley, J. L.; Mather, A.; Wheelhouse, R. T.; Cullis, P.
M.; Hartley, J. A.; Bingham, J. P.; Cohen, G. M. Cancer
Res. 1992, 52, 4190–4195.
7. Linney, I. D.; Buck, I. M.; Harper, E. A.; Kalindjian, B.;
Pether, M. J.; Shankley, N. P.; Watt, G. F.; Wright, P. T.
J. Med. Chem. 2000, 43, 2362–2370.
13. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P.
J. Adv. Drug Del. Rev. 1997, 23, 3–25.
ꢀ
ꢀ ꢀ
14. Horvath, Cs.; Melander, W.; Molnar, I. Anal. Chem. 1977,
8. Hughes, D. L. Organic Reactions; John Wiley and Sons:
New York, 1992; pp 335–656.
9. Brozwn, H. C.; Heim, P. J. Org. Chem. 1973, 38, 912–916.
49, 865.
15. Zhang, L.; Brett, C. M.; Giacomini, K. M. Ann. Rev.
Pharmacol. Toxicol. 1998, 38, 431–460.