Synthesis and biological evaluation of novel daunorubicin-estrogen conjugates

Article abstract of DOI:10.1016/S0039-128X(01)00110-6

The synthesis of two novel daunorubicin-estrogen conjugates with a steroidal and a non-steroidal ligand was undertaken in an attempt to target the cytotoxicity of anthracycline to estrogen-receptor positive cells. These conjugates (3 and 4), in contrast to their corresponding ligands, displayed weak binding affinities of 0.079 and 0.851 for the estrogen receptor. Conjugate 3 was consistently more cytotoxic than 4, which however showed some selectivity to estrogen receptor positive cell lines. Copyright

Full text of DOI:10.1016/S0039-128X(01)00110-6

Steroids 66 (2001) 785–791
Synthesis and biological evaluation of novel daunorubicin-estrogen
conjugates
Konstantinos M. Kasiotisa, Prokopios Magiatisb, Harris Pratsinisb,
Alexios-Leandros Skaltsounisb, Vasiliki Abadjic, Avgui Charalambousc,
Paraskevi Moutsatsoub, Serkos A. Haroutouniana,*
aChemistry Lab., Agric. University of Athens, Iera odos 75, Athens 11855, Greece
bDept. of Pharmacy and Medical School, University of Athens, Zografou 15771, Greece
cELPEN SA, Pharmaceutical Company, Pikermi 19009, Greece
Received 26 October 2000; received in revised form 17 January 2001; accepted 31 January 2001
Abstract
The synthesis of two novel daunorubicin-estrogen conjugates with a steroidal and a non-steroidal ligand was undertaken in an attempt
to target the cytotoxicity of anthracycline to estrogen-receptor positive cells. These conjugates (3 and 4), in contrast to their corresponding
ligands, displayed weak binding affinities of 0.079 and 0.851 for the estrogen receptor. Conjugate 3 was consistently more cytotoxic than
4, which however showed some selectivity to estrogen receptor positive cell lines. © 2001 Elsevier Science Inc. All rights reserved.
Keywords: Daunorubicin conjugates; Steroidal and non-steroidal estrogen ligand; Synthesis; Estrogen receptor binding; Cytotoxic activity
1. Introduction
Todate, several estrogen cytotoxic agents have been pre-
pared by linking steroidal or non-steroidal moieties to a
chemical function with known cytotoxic activity (nitrogen
mustards, nitrosoureas and carbamates, aziridines, vinca
alkaloids, dichloroplatinum, ellipticine etc) [8–13]. In order
for this approach to be successful, the conjugates should
exhibit antitumor activity and also possess sufficient bind-
ing affinity to the ER, which would allow their selective
accumulation in ER-rich cells [11,13]. Previous findings
based on the number of receptors per cell and the possible
drug concentration have suggested that the relative binding
affinity (RBA) value should be at least 1% comparing to
estradiol (100%) [14].
Daunorubicin 1, belongs to the anthracycline antibiotics
family and is very commonly used as a broad-spectrum
antitumor agent for the treatment of advanced cancers, ei-
ther as a single agent or in combination chemotherapy [15].
Despite its cardiotoxicity (associated with its ability to form
free radicals) [16], daunorubicin is one of the preferred
treatments for progressive metastatic breast cancers [17].
Thus, we were interested to link this cytotoxic compound to
a potent estrogen like estradiol. Such conjugate should ex-
hibit antitumor activity and can potentially bind to ER. The
initial estrogen-anthracycline (daunorubicine and doxoru-
bicine) conjugates 2, which have been reported and patented
The lack of selectivity of several antitumor agents as
well as their acute toxicity toward rapidly proliferating
tissues, constitute the major drawbacks in their use for the
treatment of human cancer. In order to circumvent this
problem, one approach is to couple these agents to carriers
which have shown selectivity towards the tumors [1,2], or
the tissues from which the tumors are derived [3–5]. This
might enable the selective delivery of a cytotoxic agent to
cells or tissues with high concentration of binding sites for
the carrier. In this context, an estrogen can be used to target
breast cancers, since it is well established that most of breast
cancers exhibit detectable levels of estrogen receptor (ER)
(estimated range ϭ 5000 to 50 000 receptor molecules/cell)
[6]. Thus, estrogens are well fitted to serve as effective
carriers of DNA-directed cytotoxic agents into the nuclei of
ER-rich cells, since it is widely accepted that the steroidal
hormones bind to a receptor dimer-DNA complex within
the nucleus, which subsequently alters the rate of transcrip-
tion by an unknown mechanism [7].
* Corresponding author. Tel: ϩ30-1-5294247; fax: ϩ30-1-5294265.
E-mail address: sehar@aua.gr (S.A. Haroutounian).
0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(01)00110-6

786
K.M. Kasiotis et al. / Steroids 66 (2001) 785–791
purchased from Aldrich (analytical reagent grades) and used
without further purification. Analytical thin-layer chroma-
tography (TLC) was conducted on Merck glass plates
coated with silica gel 60 F254 and spots were visualized with
UV light or/and an alcohol solution of anisaldehyde. Flash
column chromatography was performed using Merck silica
gel 60 (230–400 mesh ASTM). When analyses are indi-
cated by symbols of the elements, analytical results obtained
for those elements were Ϯ0.4% of their theoretical values.
2.2. Apparatus
Melting points were determined on a Bu¨chi melting point
1
apparatus and are uncorrected. H and 2D NMR spectra
were recorded at 400 MHZ on a Bruker DRX-400 spec-
trometer in the indicated solvents. The coupling constants
are recorded in Hertz (Hz) and the chemical shifts are
reported in parts per million (␦, ppm) downfield from tet-
ramethylsilane (TMS), which was used as an internal stan-
dard (by asterisk are indicated the overlapped peaks). Infra-
red spectra were obtained on a Nicolet Magna 750, series II
spectrometer.
2.3. 17␣-(Hydroxypropargyl)-3,17␤-estradiol (5)
To an ice-cold solution of estrone (0.5 g, 1.84 mmol) and
potassium ethoxide (2.25 g, 26.8 mmol) in anhydrous THF
(10 ml) was added propargyl alcohol (0.85 ml, 14.8 mmol).
The reaction was allowed to reach the room temperature and
stirred for 2 h. Then the reaction mixture was poured into
ice water, neutralized with acetic acid and extracted with
EtOAc (2 ϫ 20 ml). The combined organic layers were
washed with brine, dried over MgSO4 and concentrated
under vacuum. Flash column chromatography (EtOAc/hex-
ane 3:7) gave 0.42 g (70%) of the title compound as an off
white solid: mp 200–202°C; IR (neat) vmax 3380, 2225
cmϪ1; 1H and 13C NMR data are identical to those reported
in the literature [21]. Anal (C21H26O3) C, H.
Fig. 1. Daunorubicine 1 and daunorubicin-estrogen conjugates 2–4.
earlier by Hartman et al. [18,19], have been substituted at
carbon 17 by an imine functionality which is attached to the
anthracycline moiety. As a consequence, they show negli-
gible binding affinity for the ER and no selectivity in the
treatment of receptor positive tumors. Since it is established
by structure-activity relationship studies, that the presence
of the 17␤-OH group is essential for high binding affinity of
a steroidal estrogen to the ER [20], we have synthesized a
novel daunorubicine-estradiol conjugate 3 with a long 17␣-
linker chain, which would potentially locate the cytotoxic
molecule away from the critical for binding to ER 17␤-OH
group. Thus, we hoped that the conjugate might retain some
affinity to the ER. In addition, we have also prepared a
similar non-steroidal conjugate of daunorubicin (4). The
present report discusses the synthesis of conjugates 3 and 4,
their binding affinity to ER and their in vitro cytotoxicity
against a variety of human cell lines.
2.4. 17␣-(Bromopropargyl)-3,17␤-estradiol (6)
To an ice-cold stirred solution of 5 (0.2 g, 0.61 mmol)
and triphenylphosphine (0.32 g, 1.22 mmol) in anhydrous
THF (2 ml), carbon tetrabromide (0.31 g, 0.91 mmol) was
added in one batch. The mixture was warmed to room
temperature, stirred for 2 h and concentrated under reduced
pressure. Flash chromatographic purification (EtOAc/hex-
ane 3:7) produced 0.155 g (65%) of 6 as a white solid. Mp
68–70°C; IR (neat) vmax 3350, 2218 cmϪ1 1H NMR
;
2. Experimental
(CDCl3) ␦, 0.97 (s, 3H, CH3), 1.35–1.57 (m, 5H, H-8, H-11,
H-15), 1.74 (m, 1H, H-14), 1.77–1.84 (m, 4H, H-6, H-12),
2.01 (dt, J ϭ 4, 7 Hz, 1H, H-16␣), 2.20 (m, 1H, H-9),
2.25–2.36 (m, 3H, H-11, H-16␤), 2.78–2.81 (m, 2H, H-7),
4.02 (s, 2H, H-21), 6.55 (d, J ϭ 2.4 Hz, 1H, H-4), 6.62 (dd,
J ϭ 2.5, 8.1 Hz, 1H, H-2), 7.14 (d, J ϭ 8.1 Hz, 1H, H-1);
13C NMR (CDCl3) ␦, 12.79 (C-18), 14.82 (C-21), 22.83
2.1. General remarks
All anhydrous reactions were carried out under argon
atmosphere. Solvents were dried by distillation prior to use.
Solvent mixtures employed in chromatography were re-
ported as volume to volume ratios. Starting materials were

K.M. Kasiotis et al. / Steroids 66 (2001) 785–791
787
(C-15), 26.36 (C-11), 27.16 (C-6), 29.59 (C-7), 32.79 (C-
12), 38.70 (C-16), 39.36 (C-8), 43.48 (C-9), 47.63 (C-13),
49.54 (C-14), 80.01 (C-17), 81.15 (C-20), 90.52 (C-19),
112.68 (C-2), 115.21 (C-4), 126.53 (C-1), 132.34 (C-10),
138.18 (C-5), 153.40 (C-3). Anal (C21H25BrO2) C, H.
was stirred at room temperature for an additional 4 h. Then
the reaction was quenched with 1N HCl (10 ml) and sub-
sequently extracted with EtOAc (2ϫ 20 ml). The combined
organic layers were washed with brine, dried over MgSO4
and concentrated under reduced pressure. The resulting res-
idue was refluxed for 90 min in a mixture of ethanol (8 ml)
and 30% HCl (2 ml). The solvent was evaporated under
vacuum and the residue was partitioned in diethylether (20
ml) and water (20 ml). Workup of the reaction mixture gave
2.5. Daunorubicin-3Ј-NH-17␣-(propargyl)-3,17␤-estradiol
(3)
To a stirred solution of daunorubicin hydrochloride (0.6
mmol) and triethylamine (0.1 ml, 0.71 mmol) in anhydrous
DMF (0.5 ml) was added dropwise a solution of compound
6 (0.06 g, 0.15 mmol) in DMF (1 ml). The reaction mixture
was stirred for 6 h and the solvent was evaporated under
reduced pressure. Purification by flash chromatography
(CH2Cl2/MeOH 99.5:0.5) furnished 0.005 g (10%) of the
desired conjugate 3 as a red solid, while 71% of the unre-
1
69% of the product as a 3:1 mixture (by H NMR) of the
E/Z diastereomers. Flash chromatographic separation
(EtOAc/hexane 1:9) and recrystallization from methylene
chloride-petroleum ether resulted in the separation of the
1
pure E isomer as a pale yellow solid. Mp 163–164°C; H
NMR (CDCl3) ␦, 1.12 (t, J ϭ 7.1 Hz, 3H, H-4), 2.68 (q, J ϭ
7.2 Hz, 2H, H-3), 3.80 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),
5.17 (s, 2H, CH2-Bn), 6.68–7.62 (m, 17H, Har); 13C NMR
(CDCl3) ␦, 13.63 (C-4), 28.85 (C-3), 54.87 (OCH3), 69.72
(CH2-Bn), 112.65, 114.56 (C-3ar, C-5ar), 126.76, 131.83
(C-2ar, C-6ar), 134.51 (C-1Bn), 135.62, 137.20 (C-1ar, C-1),
140.28 (C-2), 157.10 (C-4ar). Anal. (C31H30O3) C, H.
The minor (Z) diastereomer has similar spectroscopic
data except the peaks at 3.72 (s, 3H, OCH3), 3.81 (s, 3H,
1
acted steroid was recovered. H NMR (MeOD) ␦, anthra-
cycline 2.10 (dd, J ϭ 15, 1 Hz, 1H, H-8eq), 2.31 (dd, J ϭ 15,
4 Hz, 1H, H-8ax), 2.33 (s, 3H, H-14), 2.78 (d, J ϭ 18 Hz,
1H, H-10eq), 2.96 (d, J ϭ 18 Hz, 1H, H-10ax), 3.95 (s, 3H,
OCH3), 5.03 (dd, J ϭ 4, 1 Hz, 1H, H-7), 7.45 (d, J ϭ 8 Hz,
1-H, H-3), 7.75 (t, J ϭ 8 Hz, 1H, H-2), 7.78 (d, J ϭ 8 Hz,
1H, H-1); sugar 1.27 (s, 3H, CH3), 1.80* and 1.99* (2H,
H-2Ј), 3.27 (m, 1H, H-3Ј), 3.69 (br s, 1H, H-4Ј), 4.21 (m,
1H, H-5Ј), 5.40 (br s, 1H, H-1Ј); steroid 0.69 (s, 3H, H-18Љ),
1.11* (2H, H-15Љ), 1.47* (2H, H-6Љ), 1.48* (1H, H-14Љ),
1.66* (2H, H-12Љ), 1.80* (2H, H-16Љ), 1.82* (1H, H-9Љ),
2.08* (2H, H-11Љ), 2.10* (1H, H-8Љ), 2.43* (2H, H-7Љ), 3.60
(s, 2H, H-21Љ), 6.24 (s, 1H, H-4Љ), 6.37 (d, J ϭ 8 Hz, H-2Љ),
6.80 (d, J ϭ 8 Hz, H-1Љ); 13C (MeOD) ␦, anthracycline
24.60 (C-14), 33.38 (C-10), 36.55 (C-8), 56.97 (OCH3),
71.63 (C-7), 77.66 (C-9), 112.5 (C-6a), 112.40 (C-10a),
120.15 (C-3), 120.56 (C-1), 121.70 (C-4a), 135.55 (C-12a),
135.92 (C-11a), 136.22 (C-5a), 137.13 (C-2), 156.14 (C-
11), 157.42 (C-6), 162.35 (C-4), 187.56 (C-12), 187.70
(C-5), 213.40 (C-13); sugar 17.32 (CH3), 30.61 (C-2Ј),
52.73 (C-3Ј), 68.49 (C-4Ј), 68.64 (C-5Ј), 102.16 (C-1Ј);
steroid 13.17 (C-18Љ), 23.50 (C-15Љ), 27.46 (C-11Љ), 28.33
(C-6Љ), 30.42 (C-7Љ), 33.96 (C-12Љ), 35.24 (C-21Љ), 39.73
(C-16Љ), 40.75 (C-8Љ), 44.65 (C-9Љ), 48.00 (C-13Љ), 50.54
(C-14Љ), 80.46 (C-17Љ), 82.19 (C-20Љ), 89.61 (C-19Љ),
113.61 (C-2Љ), 115.89 (C-4Љ), 127.02 (C-1Љ), 132.17 (C-
10Љ), 138.49 (C-5Љ), 155.77 (C-3Љ).
1
OCH3) and 5.03 (s, 2H, CH2-Bn) of H NMR and 54.69
(OCH3), 69.50 (CH2-Bn) of 13C NMR.
2.7. (E)-1-(p-Chloroacetylphenyl)-1,2-bis-(p-methoxyphenyl)-
1-butene (8)
Alkene 3 (0.5g, 1.13 mmol) was dissolved in ethyl ace-
tate (15 ml) and hydrogenated over 10% Pd/C (0.05 g)
under 1 atmosphere pressure for 40 min in the absence of
sunlight. The mixture was filtered over Celite, dried over
MgSO4, and evaporated furnishing (E)-1-(p-hydroxyphen-
yl)-1,2-bis-(p-methoxyphenyl)-1-butene (0.37 g, 95%) as a
pale yellowish oil. Subsequently, 0.15 g of this phenolic
intermediate (0.43 mmol) was dissolved in ice-cold anhy-
drous diethylether (1 ml) and of pyridine (7 ␮l, 0.084 mmol)
and chloroacetylchloride (44 ␮l, 0.51 mmol) were added.
The reaction was run under stirring at that temperature for 2
h, then diluted with EtOAc (5 ml). The organic layer was
separated, washed with water (10 ml) and dried over
MgSO4. Purification by flash chromatography (EtOAc/hex-
ane 1:4) afforded the desired compound as a yellowish solid
1
(0.165 g, 90%). Mp 151–152°C; H NMR (CDCl3) ␦, 0.89
2.6. (E)-1-(p-Benzyloxyphenyl)-1,2-bis-(p-methoxyphenyl)-
(t, J ϭ 7.1 Hz, 3H, H-4), 2.44 (q, J ϭ 7.2 Hz, 2H, H-3), 3.71
(s, 3H, OCH3), 3.81 (s, 3H, OCH3), 4.31 (s, 2H, CH2-Cl),
6.53–7.38 (m, 12H, Har); 13C NMR (CDCl3) ␦, 13.56 (C-4),
28.92 (C-3), 40.87 (CH2-Cl), 112.79, 113.49 (C-3ar, C-5ar),
119.81, 120.62 (C-3ar, C-5ar), 130.62 (C-2ar, C-6ar), 131.87
(C-2ar, C-6ar), 134.18 (C-1ar), 135.73 (C-1Јar), 136.68 (C-
1Љar), 141.39 (C-1), 142.16 (C-2), 148.77 (C-4ar), 158.29
(C-4Јar, C-4Љar), 165.75 (COCH2Cl). Anal. (C26H25O4Cl) C,
H.
1-butene (7)
A stirred solution of magnesium (0.1 g, 4.14 mmol) and
traces of iodine in anhydrous diethylether (40 ml) was
warmed to 40°C and p-benzyloxyphenylbromide (1.05 g,
4.14 mmol) was added dropwise. After 2 h of stirring at that
temperature, a solution of p-methoxy-2-(p-methoxyphenyl)-
butyrophenone (0.4 g, 1.38 mmol) in anhydrous diethyl-
ether (10 ml) was added via syringe. The resulting mixture

788
K.M. Kasiotis et al. / Steroids 66 (2001) 785–791
2.8. Daunorubicin-3Ј-NH-(E)-1-(p-acetylphenyl)-1,2-bis-(p-
methoxyphenyl)-1-butene (4)
1. MCF-7, derived from a mammary adenocarcinoma of
a 69-year old caucasian (ATCC: American Type Cul-
ture Collection, Rockville, USA). The cells are ex-
pressing estrogen receptors.
To an ice-cold stirred solution of daunorubicinhydro-
chloride (0.03 g, 0.06 mmol) and triethylamine (0.3 ml, 2.15
mmol) in anhydrous DMF (0.5 ml), a solution of 8 (0.08 g,
0.18 mmol) in DMF (1.5 ml) was added dropwise. The
reaction mixture was allowed to reach the room temperature
and stirred for additional 5 h. The solvent was evaporated
under reduced pressure and the remaining slurry was puri-
fied by flash chromatography (CH2Cl2/Acetone 9:1) to pro-
duce the desired conjugate 4 (0.004 g, 7%) as a red amor-
phous powder. Furthermore, 78% of the unreacted starting
material was recovered. 1H NMR (CDCl3) ␦, anthracycline
2.11 (dd, J ϭ 15, 4 Hz, 1H, H-8ax), 2.30 (dd, J ϭ 15, 1 Hz,
1H, H-8eq), 2.39 (s, 3H, H-14), 2.95 (d, J ϭ 18 Hz, 1H,
H-10ax), 3.24 (d, J ϭ 18 Hz, 1H, H-10eq), 4.07 (s, 3H,
OCH3), 5.27 (dd, J ϭ 4, 1 Hz, 1H, H-7), 7.38 (d, J ϭ 8 Hz,
1H, H-3), 7.78 (t, J ϭ 8 Hz, 1H, H-2), 8.02 (d, J ϭ 8 Hz, 1H,
H-1); sugar 1.38 (s, 3H, CH3), 1.80 (2H, H-2Ј), 3.30 (m,
1H, H-3Ј), 3.70 (br s, 1H, H-4Ј), 4.25 (m, 1H, H-5Ј), 5.56
(br s, 1H, H-1Ј); ligand 0.87 (t, J ϭ 7 Hz, 3H, H-4Љ), 2.42
(q, J ϭ 7 Hz, 2H, H-3Љ), 3.41 (d, J ϭ 15 Hz, CH2N), 3.77
(s, 3H, OCH3), 3.82 (s, 3H, OCH3), 6.51–7.22 (m, 12H,
Har); 13C (CDCl3) ␦, anthracycline 24.71 (C-14), 33.30
(C-10), 34.97 (C-8), 56.71 (OCH3), 70.46 (C-7), 77.20
(C-9), 112.89 (C-6a), 113.12 (C-10a), 118.42 (C-3), 119.80
(C-1), 122.00 (C-4a), 134.34 (C-12a), 135.08 (C-11a),
135.30 (C-5a), 135.81 (C-2), 155.98 (C-11), 156.57 (C-6),
160.09 (C-4), 187.50 (C-12 and C-5), 213.40 (C-13); sugar
16.25 (CH3), 26.79 (C-2Ј), 50.03 (C-3Ј), 65.75 (C-5Ј), 68.50
(C-4Ј), 101.00 (C-1Ј); ligand 13.60 (C-4Љ), 28.98 (C-3Љ),
49.18 (OCH2N), 112.8, 113.3, 120.8, 199.9 (Car-3,5), 130.6,
131.9 (Car-2,6), 133.9, 134.2 (Car-1), 155.8, 156.5, 157.9
(Car-4), 141.20 (C-1Љ), 142.05 (C-2Љ), 168.8 (OCOCH2).
2. MDA-MB-231, derived from a mammary adenocar-
cinoma of a 51-year old caucasian (ATCC). The cells
are estrogen-receptor negative.
3. LNCaP clone FGC, derived from a prostate carci-
noma of a 50-year old caucasian (ATCC). The cells
are expressing androgen and estrogen receptors.
4. PC-3, derived from a prostate adenocarcinoma of a
62-year old caucasian (ATCC). The cells are andro-
gen insensitive.
5. MES-SA, derived from an uterine sarcoma of a 56-
year old female (ECACC: European Collection of
Cell Cultures, Salisbury, UK).
6. MES-SA/Dx5, derived from the above cell line after
selection in the presence of doxorubicin (ECACC).
The cells are multi drug-resistant, exhibiting a 100-
fold resistance to doxorubicin.
7. BC3c, established in our laboratory [23] from a tran-
sitional cell carcinoma of the bladder of an 82-year
old caucasian female.
8. A549, derived from a lung carcinoma of a 58-year old
caucasian male (ECACC).
9. L1210, a lymphoblastic leukemia cell line derived
from an 8-month old female mouse—strain: DBA
subline 212 (ATCC).
All cell lines were routinely cultured in Dulbecco’s Minimal
Essential Medium supplemented with penicillin (100 U/ml),
streptomycin (100 ␮g/ml) and 10% Fetal Bovine Serum
(media and antibiotics from Biochrom KG, Berlin, Ger-
many) in an environment of 5% CO2, 85% humidity, 37°C.
Adherent cells were subcultured using a trypsin 0.25%-
EDTA 0.02% solution. Cytotoxicity was estimated by a
modification of the MTT assay [24]. Briefly, cells were
plated in 96-well flat-bottomed microplates at a density of
10 000 cells/well. Twenty four h after the plating, the test
compounds were added, appropriately diluted in DMSO.
After a 48 h incubation, the medium was replaced with
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] (Sigma, St. Louis, MO, USA) dissolved at a final
concentration of 1 mg/ml in serum-free, phenol-red-free
RPMI (Biochrom KG), for a further 4 h incubation. Then,
the MTT-formazan was solubilized in isopropanol and the
optical density was measured at a wavelength of 550 nm
and a reference wavelength of 690 nm. In every experiment
daunorubicin HCl was included as a positive control.
2.9. Determination of the estrogen receptor binding affinity
(RBA)
Relative binding measurements were performed as pre-
viously reported [22], using lamb uterine cytosol, diluted to
ϳ1.5 nM receptor. The protein solution was incubated with
buffer or several concentrations of unlabeled competitor
together with 10 nM [3H]estradiol at 0°C for 18–24 h. The
free ligand was removed by adsorption to dextran-coated
charcoal. Unlabeled competitors were prepared and serially
diluted in 1:1(v/v) dimethylformamide/TEA buffer (10 mM
Tris, 1.5 mM EDTA, 3 mM sodium azide, pH 7.4 at 25°C)
to ensure solubility. All data are reported relative to estra-
diol ϭ 100%.
3. Results and discussion
2.10. Antitumor activity assays
3.1. Chemistry
The following tumour cell lines were used for the assess-
ment of the cytotoxic activity of the two conjugates:
The synthesis of estradiol-linker 6 and its attachment to
daunorubicin is outlined in Fig. 2. Estrone was derivatizated

K.M. Kasiotis et al. / Steroids 66 (2001) 785–791
789
Table 1
Estrogen binding affinity of ligands and daunorubicin conjugates
Compd.
RBAa
6
3
8
4
23 Ϯ 1
0.079 Ϯ 0.014
6.0 Ϯ 0.6
0.85 Ϯ 0.04
a Relative binding affinity for the estrogen receptor determined in a
competitive radiometric assay relatively to [3H] estradiol tracer (RBA ϭ
100%), ϮSD (n ϭ 3).
Fig. 2. Synthesis of steroidal ligand 6.
at 17␣ position by reaction with propargyl alcohol in the
presence of potassium ethoxide. Then the primary hydroxy
group was selectively brominated by reaction with carbon
tetrabromide and triphenylphosphine affording the estradi-
ol-linker 6. Subsequent reaction with daunorubicin in anhy-
drous DMF resulted in its attachment to the 3Ј-N position of
daunorubicin producing the conjugate 3. The yield of this
conjugation reaction is low (10%), but during the purifica-
tion procedure almost the entire amount of the unreacted
steroid was recovered (71%).
Conjugate 4 was prepared in five steps from desoxy-
anisoine according to the synthetic sequence that is depicted
in Fig. 3. More specifically, desoxyanisoin was ethylated by
a known procedure [25] and subsequently treated with the
benzyloxyphenyl magnesium bromide to furnish the corre-
sponding Grignard product, which was dehydrated during
the workup procedure to give the desired product 7. The
yield of the reaction is satisfactory, considering the com-
peting enolization reaction of the activated, sterically hin-
dered ketone [26]. Hydrogenolysis of the benzyl group and
reaction with chloroacetylchloride produced in very good
overall yield the non-steroidal-linker 8 which was reacted
with daunorubicine furnishing the conjugate 4. In this case
the unreacted ligand was also recovered.
further adjustment to the length of the linker chain is re-
quired in order to determine its optimum length.
3.3. Antitumor activity
The dose-response curves concerning the cytotoxicity of
the conjugates 3 and 4 in comparison to that of daunorubicin
are presented in Fig. 4, where the classical estrogen positive
breast cancer cell line MCF-7 has been used as target. A
slight stimulation above control at low concentrations could
be the result of a combined hormonal and cytotoxic activity.
The IC50 values obtained from similar experiments on all
tumor-cell lines used in the present study are summarized in
Table 2. Obviously, conjugation of daunorubicin resulted in
a decrease of its cytotoxic activity, ranging from 1.4-fold to
350-fold and from 7.2-fold to 430-fold for conjugates 3 and
4, respectively. This decrease could be possibly attributed to
steric hindrance and/or to the use of the daunosamine nitro-
3.2. Estrogen receptor binding affinity (RBA)
The estrogen RBA’s of the new compounds were deter-
mined by a competitive binding assay and are shown in
Table 1. Ligands 6 and 8 displayed satisfactory binding
affinities, while their corresponding daunorubicin conju-
gates 3 and 4 exhibit low but not negligible RBA’s to the
estrogen receptor. This expected reduction of RBA is attrib-
uted to the bulk of the cytotoxic agent indicating that a
Fig. 4. Cytotoxicity of daunorubicin and of the conjugates 3 and 4. After
incubation of MCF-7 cells with the indicated concentrations of the three
substances for 48 h, cytotoxicity was determined by the MTT assay.
Details of the procedure are described in Experimental (see 2.10.). Each
point of the dose-response curves is the average of six wells. The error bars
represent standard deviations. The results shown are representative of three
experiments.
Fig. 3. Synthesis of non-steroidal ligand 8.

790
K.M. Kasiotis et al. / Steroids 66 (2001) 785–791
Table 2
Acknowledgements
In vitro cytotoxicity of daunorubicin conjugates against various cancer
cell lines
Support of this research through a grant from the General
Secretariat of Research & Technology (PAVE) and ELPEN
S.A. Pharmaceutical Company is gratefully acknowledged.
We thank Kathryn E. Carlson for the estrogen receptor
binding measurements.
Cell line
Type
IC50 (␮M)
3
4
Daunorubicin
ER ϩ : MCF-7
ER Ϫ : MDA-MB-231 Human breast
AR ϩ : LNCaP
AR Ϫ : PC-3
MES-SA
Human breast
1.2 1.6
0.4 7.0
0.22
0.22
0.02
0.40
0.07
Human prostate 7.0 7.0
Human prostate 7.0 9.0
Human uterine
sarcoma
0.1 30.0
References
MES-SA/Dx5
Human uterine
sarcoma
Human bladder
Human lung
7.0 Ͼ100.0 1.00
[1] Upesclacis J, Hinman L. Chemical modification of antibodies for
cancer chemotherapy. Ann Rep Med Chem 1988;23:151–60.
[2] Blatter WA, Lambert JM, Goldmacher VS. Realizing the full poten-
tial of immunotoxins. Cancer Cells 1989;1:50–5.
[3] Krohn K, Kuilikowski K, Leclerq G. Diethylstilbestrol-linked cyto-
toxic agents: Synthesis and binding affinities for estrogen receptors.
J Med Chem 1989;32:1532–8.
BC3c
A549
L1210
11.0 22.0
2.0 10.0
0.55
0.50
0.04
Mouse leukemia 1.5 1.2
[4] Schmidt BF, Hernandez L, Routzer C, Czerwinski G, Chmurny G,
Michejda CJ. Peptide linked 1,3-dialkyl-3-acyl-triazenes: Gastrin re-
ceptor directed antineoplastic alkylating agents. J Med Chem 1994;
37:3812–8.
[5] Varga JM, Asato N, Lande S, Lerner AB. Melanotropin-daunomycin
conjugate shows receptor-mediated cytotoxicity in cultured murine
melanoma cells. Nature 1977;267:56–8.
[6] Quivy J, Leclerq G, Deblaton M, Henrot P, Velings N, Norberg B,
Evrard G, Zeicher M. Synthesis, structure and biological properties of
Z-17␣-(2-iodovinyl)-11␤-chloromethyl estradiol-17␤ (Z-CIMIV), a
high affinity ligand for the characterization of estrogen receptor-
positive tumors. J Steroid Biochem Mol Biol 1996;59:103–17.
[7] Katzenellenbogen JA. Probes for steroid receptors-studies at the mo-
lecular, cellular, and whole organism levels. In: Proceedings of the
Robert A. Welch Foundation conference on chemical research XXXV
chemistry at the frontiers of medicine; The Robert Welch Foundation:
Houston, TX, 1991. pp 229–57.
gen for the conjugation. Apart from that, the cytotoxicity of
the two conjugates is clearly due to the daunorubicin moi-
ety, since the doxorubicin-resistant MES-SA/Dx5 cell line
exhibits a similar resistance to daunorubicin as well as to 3
and 4, in comparison to its parental (doxorubicin-sensitive)
cell line MES-SA. Furthermore, conjugate 3 was consis-
tently more potent as cytostatic agent than conjugate 4 for
most of the cell lines tested (exceptions are the murine
leukemia L1210 and the human prostate cancer LNCaP).
However, conjugate 3 showed no preferential inhibition of
the proliferation of the estrogen receptor positive MCF-7
cells in comparison to the estrogen receptor negative MDA-
MB-231. It is also noteworthy that the non-steroidal conju-
gate 4 inhibits the estrogen receptor positive MCF-7 cells
more potently than the estrogen receptor negative MDA-
MB-231 cells. These results are in agreement with the
estrogen RBA’s of the two conjugates presented above (see
3.2.).
[8] Lam HY, Goldenberg GJ, Wong CM. Interaction of estrogen-nitro-
sourea conjugates with the estrogen receptor in rat uterus. Biochem
Pharmacol 1988;37:2625–31.
[9] Ko¨hle H, Krohn K, Leclerq G. Hexestrol-linked cytotoxic agents:
Synthesis and binding affinity for estrogen receptor. J Med Chem
1989;32:1538–47.
[10] Akaza H, Imada S, Tsukamoto S, Koiso K. Inhibitory effects of
bestrabucil, a conjugate of chlorambucil and estradiol, on the produc-
tion of androgen-induced growth factors by Shiongi carcinoma 115
cells. Int J Orol 1994;1:67–73, and refs cited therein.
[11] Devraj R, Barett J, Fernandez JA, Katzenellenbogen JA, Cushman M.
Design, synthesis and biological evaluation of ellipticine-estradiol
conjugates. J Med Chem 1996;39:3367–74.
[12] Georgiadis M, Chondros KP, Haroutounian SA. Synthesis and bio-
logical studies of steroidal cis-platinum (II) complexes. Inorg Chim
Acta 1987;138:249–52.
[13] DiZio JP, Carlson KE, Bannochie CJ, Welch MJ, vonAngerer E,
Katzenellenbogen JA. Estrogen platinum-diamine complexes: Prep-
aration of a non-steroidal estrogen platinum-diamine complex labeled
with platinum-191 and study of its binding to the estrogen receptor in
vitro and its tissue distribution in vivo. J Steroid Biochem Mol Biol
1992;42:363–73.
[14] Katzenellenbogen JA, Katzenellenbogen BS. Considerations in the
design and evaluation of cytotoxic estrogens. Breast Cancer Res Treat
1982;2:347–53.
[15] Al-Omar A, Abdou S, DeRobertis L, Marsura A, Finance C. Com-
plexation study and anticellular activity enhancement by doxorubicin-
cyclodextrin complexes on a multidrug-resistant adenocarcinoma
Cell Line. Bioorg Med Chem Lett 1999;9:1115–20.
3.4. Conclusions
In summary, we have described the synthesis of two
estrogen ligands and their conjugates with cytotoxic dauno-
rubicine. Although this effort may be considered as another
unsuccessful attempt to increase the selectivity of cytotoxic
agents for cell rich in estrogen receptors, the reported results
refer to chemistry that might be useful for the preparation of
additional conjugates which may display the desired selec-
tivity. Furthermore, conjugate 4 showed some selectivity to
the estrogen receptor which was also reflected in its cyto-
toxic activity, since this compound was found to inhibit
preferentially the estrogen receptor positive cells. Thus,
further modification of linker chains by the introduction of
functional groups or by changing their length in order to
increase their binding affinities will be necessary. Addi-
tional work concerning the use of the other functional
groups of daunorubicine for the attachment to the estrogen
ligands may also be pursued.

K.M. Kasiotis et al. / Steroids 66 (2001) 785–791
791
[16] Gianni L, Corden BT, Myers CE. The biochemical basis of anthra-
cycline toxicity and antitumor activity. Rev Biochem Toxicol 1983;
5:1–82.
[22] Katzenellenbogen JA, Johnson HJ, Myers, HN. Photoaffinity labels
for the estrogen binding proteins of rat uterus. Biochemistry 1973;
13:4085–92.
[17] Epstein RJ. The clinical biology of hormone responsive breast cancer.
Cancer Treat Reports 1988;15:33–51.
[18] Hartman NG, Patterson LH, Workman P, Suarato A, Angelucci F.
Doxorubicin-3Ј-NH-oestrone-17-oxime-ethyl-carbonyl, a doxorubu-
cin-oestrone conjugate that does not redox cycle in rat liver micro-
somes. Biochem Pharmacol 1990;40:1164–7.
[23] Pratsinis H, Saetta A, Gagos S, Davaris P. Isolation and character-
ization of a novel bladder cancer cell line: inhibition by epidermal
growth factor. In Vitro Cell Dev Biol—Animal 1998;34:722–8.
[24] Denizot F, Lang R. Rapid colorimetric assay for cell growth and
survival: Modifications to the tetrazolium dye procedure giving im-
proved sensitivity and reliability. J Immunol Meth 1986;89:271–7.
[25] Haroutounian SA, Scribner AW, Katzenellenbogen JA. Derivatives
of 4-styrylpyridines: Synthesis, estrogen receptor binding affinity,
and photophysical properties. Steroids 1995;60:636–45.
[26] Meyers MJ, Carlson KE, Katzenellenbogen JA. Facile synthesis of
high affinity styrylpyridine systems as inherently fluorescent li-
gands for the estrogen receptor. Bioorg Med Chem Lett 1998;8:
3589–94.
[19] Hartman NG, Angelucci F, Suarato A. United Kingdom Patent GB
2201 419 A (application number 8704236), 1988.
[20] Zeelen FJ, Bergink EW. In: Raus J, Martens H, Leclerq G (Eds.).
Cytotoxic estrogens in hormone receptive tumors. Academic Press:
London, 1980. pp. 39–48.
[21] Dionne P, Poirier D. 13C Nuclear magnetic resonance study of 17␣-
substituted estradiols. Steroids 1995;60:830–6.