Synthesis, in vitro progesterone receptors affinity of gadolinium containing mifepristone conjugates and estimation of binding sites in human breast cancer cells

Article abstract of DOI:10.1016/j.bmc.2010.01.048

Novel gadolinium-based mifepristone conjugates were synthesised using various synthetic routes. Moderate antiprogestagenic activity of the new conjugates was observed in human breast cancer cells (T47-D cells) using AP (alkaline phosphatase) assay. The amount of incorporated Gd determined by inductively coupled plasma mass spectroscopy (ICPMS) indicates the number of binding sites per cell. These conjugates might be important compounds to develop receptor-targeted MRI contrast agents as well as other anti-breast cancer therapeutics.

Full text of DOI:10.1016/j.bmc.2010.01.048

Bioorganic & Medicinal Chemistry 18 (2010) 1891–1898
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, in vitro progesterone receptors affinity of gadolinium containing
mifepristone conjugates and estimation of binding sites in human breast
cancer cells
b
c
c
d
b
Pijus Saha a, , Claudia Hödl , Wolfgang S. L. Strauss , Rudolf Steiner , Walter Goessler , Olaf Kunert ,
*
Alexander Leitner e, Ernst Haslinger b, H. Wolfgang Schramm b
a Department of Chemistry-Biology, University of Quebec at Trois-Rivieres, 3351-boul. Des Forges, Trois-Rivieres, Quebec, Canada QC G8Y 4L9
b Institute of Pharmaceutical Sciences, Pharmaceutical Chemistry, Karl-Franzens- University Graz, Universitätsplatz 1, A-8010 Graz, Austria
c Institute for Lasertechnology in Medicine and Metrology at the University of Ulm, Helmholtzstraße 12, D-89081 Ulm, Germany
d Institute of Chemistry, Karl-Franzens-Universität Graz, Universitätsplatz 1, A-8010 Graz, Austria
e Institute for Analytical Chemistry and Food Chemistry, University of Vienna, Währinger Straße 38, 1090 Wien, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel gadolinium-based mifepristone conjugates were synthesised using various synthetic routes. Mod-
erate antiprogestagenic activity of the new conjugates was observed in human breast cancer cells (T47-D
cells) using AP (alkaline phosphatase) assay. The amount of incorporated Gd determined by inductively
coupled plasma mass spectroscopy (ICPMS) indicates the number of binding sites per cell. These conju-
gates might be important compounds to develop receptor-targeted MRI contrast agents as well as other
anti-breast cancer therapeutics.
Received 19 November 2009
Revised 14 January 2010
Accepted 16 January 2010
Available online 25 January 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Gadolinium-based mifepristone conjugates
Antiprogestagenic activity
Proton relaxation
Progesterone binding sites
T47-D human breast cancer cells
1. Introduction
paramagnetic label and several reports have been published
describing such agents that contain a significant number of gadolin-
In continuation of our studies of novel, high affinity PR (Proges-
terone Receptor) antagonists, which are selectively enriched in hu-
man breast cancer cells, we synthesised mifepristone conjugates
containing Gd.1 Such conjugates could be used as contrast agents
for magnetic resonance imaging (MRI) or can be model compounds
for radioligand containing conjugates.2,3 Approximately more than
50% of all MRI contrast agents used for scanning are based on Gd
complexes.4 Over the last two decades in vivo application of para-
magnetic gadolinium complexes as MRI contrast agents has be-
come an important tool in modern medical diagnostics.5,6
Complexes of the highly paramagnetic Gd(III) ion with polyamino
carboxylate ligands are the most widely utilised MRI contrast
agents and complexes with DOTA [1,4,7,10-tetraaza-1,4,7,10-tetra-
kis(carboxymethyl)cyclododecane] and DTPA (diethylene triamine
pentaacetic acid) are currently used in clinical practice.5
ium atoms.7–9 Gd(III) accelerates proton relaxation in the surround-
ing water through dipolar interactions between the unpaired
electron spin of the metal ion and the proton nuclei of the water mol-
ecules resulting MRI contrast enhancement.10 Also numerous conju-
gates based on radiometals for diagnostic and therapeutic purposes
have been described.11–14 Present development of drugs selectively
interacting with the PR in human tumour cells include: (i) selective
PR antagonists15,16 expected to be useful for the treatment of chronic
conditions, for example, endometriosis or leiomyomas and (ii) radio
ligands17–19 to enable determination of PR levels in vivo for assess-
ing the hormone responsiveness of breast cancer without tissue
biopsy. PR status of breast cancer was found to be an independent
predictive factor for benefit from adjuvant endocrine therapy. Pa-
tients with steroid hormone receptor-positive tumours, expressing
ER (Estrogen Receptor) as well as PR, have the most favourable prog-
nosis.20 Therefore for reliable determination of PR status as well as
getting clear picture of the breast tumour needs a high selective
and harmless MRI contrast agent.
Paramagnetic metals have also been attached to antibodies or
other tissue-specific molecules to impart disease-specific MRI
agents.6 It is however important to deliver sufficient quantity of
Nitrobenzyl-DOTA has been used as a precursor for the synthe-
sis of a new chelate conjugate for pre-targeted diagnosis and
therapy for its superior stability with radiometals as well as lan-
* Corresponding author. Tel.: +1 819 376 5011x3398; fax: +1 819 376 5084.
E-mail address: pijus.saha@uqtr.ca (P. Saha).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.048

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P. Saha et al. / Bioorg. Med. Chem. 18 (2010) 1891–1898
thanides.12,21,22 Lee et al. published the synthesis of a conjugate
MRI contrast agent with Gd-containing residue attached to the 3-
keto position of mifepristone.23 They however determined
in vitro a very small influence on proton relaxation at extremely
high concentrations, which are far beyond the limit of therapeutic
or diagnostic useful concentrations. This might be due to the fact
that attachment of the Gd-cage at the 3-keto-position reduces con-
siderably the PR affinity. We have reported a novel high affinity
fluorescent PR antagonist that is accumulated in the nucleus of tu-
mour cells.1 This conjugate successfully crossed cell membranes
and modulated gene transcription. It has been used for the fluores-
cence microscopic investigation of progesterone receptors in T47-
D tumour cells. Aim of this work was to retain this selectivity
and synthesise MRI agents, which will analogously selectively
accumulate in PR positive breast cancer as well as other PR positive
tumours such as uterine fibroid tumours and their metastatic
tumours.24
Scheme 3 shows the synthesis of conjugates 16 and 17 from the
coupling reaction of 9 with 12 and 13 respectively. Mifepristone
derivatives 12 or 13 were activated with isobutyl chloroformate
and triethylamine in THF. Coupling the gadolinium complex 9
afforded the desired conjugates 16 and 17.
Structures of all compounds were established by NMR except
for conjugate 16 and 17. Due to paramagnetic nature of Gd(III), it
was not possible to characterise the conjugates by NMR spectros-
copy and the structure of 16 and 17 was confirmed by high resolu-
tion mass spectroscopy. In addition, the structure of compounds 8
and 9 was also established by high resolution mass spectros-
copy.
The mass spectrum of compound 8 in positive mode showed
signals corresponding to [M+H]+ and [M+Na]+ ions as expected
from the proposed structure. Further signals can be attributed to
sodium chloride adducts as [M+NaCl+H]+ and [M+NaCl+Na]+, prob-
ably due to presence of a small amount of NaCl in the sample. At m/
z 452.0 a signal corresponding to the trialkylated derivative (pro-
posed as a by-product) was observed but seems to be only of minor
abundance. In negative mode, all major signals can be assigned to
the main compound: [MꢀH]ꢀ, [M+Naꢀ2H]ꢀ, [M+NaClꢀH]ꢀ and
[M+NaCl+Naꢀ2H]ꢀ.
2. Results and discussion
2.1. Chemistry
For the compound 9, the unique isotope distribution of gado-
linium facilitates the identification of Gd-containing species in
the sample. In positive mode, the most abundant signal corre-
sponds to the [M+2Na]+ ion at m/z 708.9 (most abundant isotope).
The signal at m/z 766.9 corresponds to [M+NaCl+2Na]+, while the
trialkyl impurity only appears at very low abundance at m/z
628.9.
For conjugate 16, signals were observed in negative mode only.
The signal corresponding to [M]ꢀ was the most abundant one. It
was accompanied by a peak at 14 units lower; this could result
from partial demethylation of the N-methyl group during ionisa-
tion step.
Scheme 1 shows the synthesis of the Gd chelating part, (p-NH2-
Bn-DOTA) via seven steps starting from the nitration of S-phenyl-
alanine (1) and concluded with hydrogenation of tetrabenzyl ester
7 by 5%Pd/C/H2 gas as described in the literature.21,25–30
Scheme 2 illustrates the synthetic pathway1,31,32 of attaching
linkers to the antiprogestin mifepristone (10, RU486). Mifepristone
was converted to desmethylmifepristone (11) by demethylation
with CaO and I2 in MeOH/THF mixture. Alkylation of the methyl-
amino group of 11 was done with 3-bromopropionic acid and 6-
bromohexanoic that afforded the mifepristone derivatives 12 and
13 with 51% and 39% yield, respectively.
O
O CH3
O CH3
H
N
NH3Cl
NH2
OH
NH2
OH
a
b
c
O2N
O CH3
O2N
O2N
O
O
O
O
1
2
3
4
d
O
H
H
H
BnOOC
COOBn
COOBn
H
H
H
N
N
N
N
N
N
N
N
N
N
f
e
NO2
N
H
NO2
O2N
N
H
O
BnOOC
7
6
5
g
OOC
OOC
COOH
COO
HOOC
HOOC
COOH
COOH
N
N
N
N
N
h
Gd3
H2N
N
N
H2N
N
9
8
Scheme 1. Synthesis of p-NH2-Bn-DOTA and its Gd-complex. Reagents and conditions: (a) concd H2SO4/concd HNO3; (b) CH3OH/HCl gas; (c) DMF/TEA/BrCH2COOCH3; (d)
CH3OH/diethylenetriamine/reflux for 11 days; (e) THF/BH3 in THF/concd HCl; (f) CH3CN/BrCH2CO2Bn/Na2CO3/70 °C; (g) 5% Pd/C/H2 gas at 30 psi/CH3OH/H2O; (h) GdCl3ꢁ6H2O/
100 mM Na2CO3 solution/pH 8/80 °C.

P. Saha et al. / Bioorg. Med. Chem. 18 (2010) 1891–1898
1893
CH3
N
CH3
N
CH3
N
HO
H
H3C
OH
OH
OH
H3C
H3C
H3C
n
O
C
C CH3
C
C CH3
C C CH3
i
j
O
O
O
10
11
12: n = 1
13: n = 4
Scheme 2. Attaching linker groups to desmethylmifepristone. Reagents: (i) CaO/I2; (j) Br(CH2)n+1COOH/NaHCO3 (n = 1 for 12 and 4 for 13).
O
O
O
CH3
N
CH3
N
O
HO
OH
OH
H3C
H3C
n
n
O
C C CH3
C C CH3
k
O
O
12: n = 1
13: n = 4
14: n = 1
15: n = 4
l
CH3
H
N
OOC
COO
COO
N
N
N
Gd3
N
n
OH
H3C
O
C C CH3
N
HOOC
O
16: n = 1
17: n = 4
Scheme 3. Syntheses of bioconjugates 16 and 17. Reagents: (k) THF/TEA/isobutylchloroformate; (l) DMF/compound 9.
For conjugate 17, the spectrum in positive mode showed abun-
dant signals corresponding to the [M+2Na+H]2+ ion of the proposed
structure, as well as corresponding to the [M+Na+H]+ ion of the
same compound. In negative mode, only a single dominant signal
was visible corresponding to the [M]ꢀ ion of the proposed struc-
ture. All other signals were below 2% in relative intensity and re-
sulted from the addition of Na or NaCl.
In TLC every conjugate showed two very close spots [for 16 Rf
value = 0.46 and 0.39 in MeCN/H2O (20:7); for 17 Rf = 0.39 and
0.32 in MeCN/H2O (10:3)] and the two spots were separated by
TLC from each conjugate. MS displayed almost identical spectra
for the two separated spots from 16 and 17 and in both cases the
most abundant signals were corresponded to [M]ꢀ ion of the ex-
pected conjugates. As the mass spectra for both fractions are prac-
tically identical we conclude that the two main compounds
represent stereo-isomers, most probably related to the non-com-
plexing molecular part. The isomerisation (epimerisation) might
also be taken place at the condensation reaction conditions at
which the gadolinium ion is already present. An induction of isom-
erisation due to the presence of a metal ion or caused by the reac-
tion conditions need to be considered. However, further thorough
investigation is suggested in this regard. For all biological experi-
ments we used the mixture of these two isomers.
It should be noted that the highest yield of 16 and 17 was ob-
tained if the coupling reaction was done after preparation of the
gadolinium complex 9. Furthermore Gd complex formation dimin-
ishes the risk of contamination from trace metals during purifica-
tion of the conjugates. Hüber et al.30 reported that they were not
able to make the conjugate complex (rhoda-DOTA) unless Gd+3
was introduced into p-NH2-DOTA prior to the addition of tetra-
methyl rhodamine (TRITC) due to sterically shielding the binding
cavity of the macrocycle that prevents Gd+3 coordination.
2.2. Antiprogestagenic activity
Antagonistic activity of conjugates 16, 17 and intermediates 11,
12 and 13 was determined in cell culture using the AP (alkaline
phosphatase) assay in human T47-D tumour cells. Inhibition of
progesterone-induced AP activity was found for all compounds.
IC50 values were estimated from dose–response curves and the rel-
ative potencies (defined as the ratio of IC50 value of the reference
antagonist 10 to the IC50 value of the individual test compound
and multiplied by 100) are summarised in Table 1. Antiprogesta-
genic activity of the intermediates 11 and 13 was 3–9 whereas
12 was more than 100-fold lower than that of the reference
antagonist mifepristone (10). Conjugates 16 and 17 exhibited

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P. Saha et al. / Bioorg. Med. Chem. 18 (2010) 1891–1898
Table 1
Table 2 shows the results. At a ‘physiological’ concentration of 10ꢀ7
mol/L the number R of binding sites at increasing incubation time
increases from 6.2 ꢂ 103 at 0 h to 25.8 ꢂ 103 at 48 h. These numbers
are in good agreement with the number of binding sites per cell
which was determined for T47D breast cancer cells by radioligand
assay as reported by Gunnet et al.34 The number given in this paper
was 32,947 14,517 per cell, resp. 3296 947 fmol/mg protein. To
see if mifepristone (10) interferes with the binding of compound
17 we have added 10 in a concentration of 10ꢀ6 mol/L during a per-
iod of 48 h and again determined R. The result is a considerable
drop of R from 25,800 to 10,800. From this we conclude that com-
pound 17 selectively binds to the PR binding sites in T47D tumour
cells. If the incubation concentration is enhanced to 10ꢀ6 mol/L, the
calculated number of binding sites is greatly enhanced in the region
from 8100 at 0 h to 259,000 at 48 h, probably because at this con-
centration compound 17 is not only PR bound, but also free dis-
solved in the cytoplasm.
Antiprogestinic activity of the new conjugates, intermediates and mifepristone from
alkaline phosphatase assaya
Compound
IC50 valuesb
Relative potencyc
10
11
12
13
16
17
0.05
0.17
34
0.44
45
100
29
0.15
11
0.11
0.11
47
a
Inhibition of progesterone-induced (10ꢀ9 mol/L) alkaline phosphatase activity
in T 47-D cells.
b
IC50 values were determined from dose–response curve and are given in nmol/L.
Relative potency is defined as the ratio of IC50 (10): IC50 (x) and multiplied by
c
100 (x = 11, 12, 13, 16, 17).
considerable antiprogestagenic activity with IC50 values in the
nano molar range although the relative potencies of both conju-
gates were approximately 900-fold lower compared with the par-
ent compound 10. Conjugates showed no antagonistic activity at
10 nM but displayed almost complete antagonistic activity at
100 nM concentration.
3. Conclusion
The newly synthesised conjugates bind selectively to the pro-
gesterone binding sites of T47D human breast cancer cells. How-
ever the concentration one can achieve in the cells by the
application of physiological useful incubation concentration is
not high enough to use the conjugates as a diagnostic tool in
MRI. It however can be used to determine the number of progester-
one binding sites in these breast tumour cells. In addition the
present conjugates might be the lead compounds to design
receptor-targeted MRI contrast agents as well as a series of steroid
conjugates holding therapeutic or diagnostic agents with high
affinities for their cognate receptors for potential use for therapeu-
tic, mechanistic and diagnostic purposes. It is also predictable that
PR-binding specificity can be enhanced by using highly selective
antiprogestins thus minimising binding to other steroid hormone
receptors.15,16,35,36
To assess ligand-receptor interaction of conjugates 16, 17, inter-
mediates 11, 12 and 13 in cell culture, antiprogestagenic activity of
all compounds was determined using AP Assay which was com-
pared with that of parent compound 10, mifepristone, as reference
antagonist. Nevertheless after attachment of Gd-chelate significant
antiprogestagenic activity was observed. Since functional activity
is approximately in agreement with receptor binding affinity33
hence considerable affinity of conjugates 16 and 17 to PR is
assumed.
2.3. Proton relaxation experiments
T47D human breast cancer tumour cells have been incubated
with different concentrations (10ꢀ7, 10ꢀ8 and 10ꢀ9 M) of com-
pound 17. After washing the cells and transferring the same num-
ber of cells into NMR tubes, no influence on the relaxation
behaviour could be observed. Therefore, we washed the cells and
investigated the relaxation of the water molecules of the cell ly-
sate. The cell lysate with the same number of cells was dispersed
in DMSO and the T1-relaxation rate of the water molecules was
determined. However, the T1 values (about 1.4 s) obtained for dif-
ferent incubation times did not show any dependency on incuba-
tion time. We therefore concluded that the Gd-concentration in
the cells was too small to reduce the relaxation time of water con-
siderably and decided to determine the mean Gd concentration in
the tumour cells to gain information about the number of proges-
terone binding sites per cell.
4. Experimental
All reagents and solvents for syntheses were purchased from
Sigma–Aldrich, Fluka or Merck and used without further purifica-
tion. Reagent-grade solvents were purified and dried using stan-
dard methods. DMF was purified by azeotropic distillation and
dried over 4 Å molecular sieve. Trietylamine and diethylenetria-
mine were dried over KOH. For preparation of absolute methanol,
98% methanol was first treated with fresh calcinated CaO and dis-
tilled; subsequently, it was refluxed with magnesium splinters and
iodine and distilled again. THF and acetonitrile were dried over
2.4. Determination of progesterone binding sites
Table 2
Determination of progesterone binding sites using conjugate 17 in T47D cells
A sample of 10 Mio. T47D cells was incubated with 17 at differ-
ent time periods and after washing, the cells were destroyed and
Gd concentration was determined with inductively coupled plas-
ma mass spectrometry. As there is usually almost no Gd present
in tissues (ultra)traces of Gd or Gd-containing molecules are easily
quantifiable with ICPMS. The precision is <5% at a concentration of
1.0 ng Gd/L. From the amount of pg Gd and the number of cells one
can calculate the mean value of Gd atoms in a cell, thus giving an
estimate of progesterone binding sites per cell.
Incubation time in hour
Number of binding site
Rb
Ra
Rc
0
3
6
24
48
8.1 ꢂ 103
6.2 ꢂ 103
5.87 ꢂ 104
1.44 ꢂ 104
1.77 ꢂ 104
2.58 ꢂ 104
8.8 ꢂ 103
6.22 ꢂ 104
9.18 ꢂ 104
2.59 ꢂ 105
1.08 ꢂ 105
Number of binding sites estimated using the concentration 10ꢀ6 mol/L of con-
a
jugate 17.
Equation for calculation of binding sites:
b
Number of binding sites estimated using the concentration 10ꢀ7 mol/L of con-
x ¼ pg Gd; y ¼ Mio: cells; R ¼ number of binding sites
R ¼ ðx ꢂ 10ꢀ12 ꢂ 6:02 ꢂ 1023=y ꢂ 106 ꢂ 157Þ ¼ ðx=yÞ ꢂ 3834
jugate 17.
c
Number of binding sites estimated using the concentration 10ꢀ7 mol/L of con-
jugate 17 plus 10ꢀ6 mol/L of mifepristone during an incubation period of 48 h.

P. Saha et al. / Bioorg. Med. Chem. 18 (2010) 1891–1898
1895
sodium wire and P2O5 (0.5–1% w/v) respectively. Solvents of
analytical and spectroscopic grade as well as deuterated NMR sol-
vents were purchased from Merck and Chemische Fabrik Uetikon,
respectively.
46.08; H, 5.03; N, 10.75; Cl, 13.60. Found: C, 45.93; H, 4.82; N,
10.66; Cl, 13.33.
4.1.3. N-[(Methoxycarbonyl)methyl]-(S)-p-nitrophenylalanine
methyl ester (4)
UV spectra were recorded on a Shimadzu UV-160 Å spectrome-
ter and absorption maxima kmax are given in nm. IR spectra were
taken on a Perkin–Elmer FT-IR 2000 spectrometer. Absorption
maxima mmax are given in cmꢀ1 and referred to as s (strong), m
(medium), w (weak) and br (broad). NMR spectra were recorded
on a Varian Unity Inova 400/600 NMR spectrometer equipped with
a tuneable broadband probe. Inverse detected gradient selected 2D
experiments (gCOSY, HSQC, HMBC) were done using standard
pulse sequence programs. HMBC experiments were optimised for
a long range-coupling constant of 8 Hz. Tetramethylsilane (TMS)
was used as internal reference standard. Chemical shifts d are given
in ppm, coupling constants (J values) are expressed in Hz and mul-
tiplicities are referred to as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet) and br (broad).
Prepared as described in the literature26 26.1 g (0.10 mol) 3 was
reacted with methyl bromoacetate (45.9 g, 0.30 mol) and triethyl-
amine under nitrogen yielding 4 as a pale yellow oil (24.9 g, 84%).
TLC (CHCl3/CH3OH = 50:1): Rf = 0.53 and (10% aqueous solution of
ammonium acetate/CH3OH = 1:1): Rf = 0.85. UV/vis (MeOH):
207.0, 232.0, 270.5. IR (KBr): 3443 (m, NH), 1739 (s, C@O), 1519
(s, NO2). 1H NMR (400 MHz, CD3OD): d = 3.10 (dd, 1H, J = 14.8
and 6.9, ArCH2CH), 3.14 (dd, 1H, J = 14.8 and 6.9, ArCH2CH), 3.40
(d, 1H, J = 17.7, NHCH2CO), 3.42 (d, 1H, J = 17.7, NHCH2CO), 3.66
(s, 3H, CO2CH3), 3.70 (s, 3H, CO2CH3), 3.71 (t, 1H, J = 6.9, ArCH2CH),
7.48 (d, 2H, ArH), 8.16 (d, 2H, ArH). 13C NMR (100 MHz, CD3OD):
d = 39.4 (ArCH2), 49.1 (HNCH2), 51.97 (CH3), 52.11 (CH3), 62.37
(CH), 124.09 (Ar), 131.25 (Ar), 146.46 (Ar), 147.99 (Ar), 173.35
(C@O), 174.61 (C@O). GC–MS (m/z): 237 (42), 177 (19), 160
(100), 100 (49), 72 (11), 59 (5); calcd for C13H16N2O6: 296.28.
The T1-relaxation time was determined by inversion recovery
experiments with standard experiments provided in the NMR soft-
ware VNMR (Varian). For these experiments, lysates of the cells
(1.7 million cells) were dissolved in 690
H2O.
lL DMSO-d6 and 30 lL
4.1.4. 3-(S)-(4-Nitrobenzyl)-2,6-dioxo-1,4,7,10-tetraazacyclodo-
decane (5)
Column chromatography was carried out on Silica Gel 60
(0.063–0.200 mm). Flash chromatography was done by Flash Mas-
ter Personal, Jones Chromatography. Column width 27 mm, length
150 mm, silica gel Merck 60 (0.063–0.200 mm; 70–230 mesh
ATSM). Preparative HPLC was done using a Labomatic HD-200
pump equipped with a Labocord-700 UV/vis detector (absorption
recorded at k = 254 nm); column 1: YMC-Pack™ ODS-A™ (C18),
Following the procedure described in the literature26 a solution
of compound 4 (26.75 g, 0.09 mol) and dry diethylenetriamine
(9.29 g, 0.09 mol) in absolute CH3OH was refluxed for 11 days un-
der nitrogen yielding 5 as a pale yellow solid (4.73 g, 16%). TLC
(10% w/v aqueous ammonium acetate/CH3OH = 1:1): Rf = 0.60.
UV/vis (MeOH): 233.0, 272.0. IR (KBr): 3270 (s, NH), 1655 (C@O),
1512 (NO2). 1H NMR (400 MHz, DMSO-d6): d = 1.72 (NHCH2),
2.49–3.34 (m, 13 H, ArCH2, ArCH2CH, NCH2), 4.03 (NHCH2), 7.49
(d, 2H, ArH), 7.74 (NHCO), 7.78 (NHCO), 8.14 (d, 2H, ArH). 13CNMR
(100 MHz, DMSO-d6): d = 37.14 (CH2), 37.26 (CH2), 38.45 (CH2),
45.01 (CH2), 45.35 (CH2), 51.95 (CH2), 64.60 (CH), 123.23 (Ar),
130.50 (Ar), 146.23 (Ar), 147.12 (Ar), 171.72 (C@O), 172.84
(C@O). Elemental Anal. Calcd for C15H21N5O4: C, 53.72; H, 6.31;
N, 20.88. Found: C, 53.16; H, 6.19; N, 20.38.
10
l
m, 250 ꢂ 20 mm and column. Analytical HPLC was done using
a HP 1050 autosampler and pump equipped with an Agilent 1100.
G1314A VWD UV/vis detector; column 2: YMC-Pack™ ODS-A™
(C18), 5
l
m, 250 ꢂ 4.6 mm. Elemental analyses were performed
at the Institute of Organic Chemistry, University of Graz.
EI-mass spectra were measured with a Finnigan MAT 212 or
Varian MAT 312 spectrometer at 70 eV ionising voltage. GC–MS
were measured on a HP-GC 6890-HP-MSD 7890. Intensities are gi-
ven in % of the base peak. High resolution mass spectra were ob-
tained in the positive and negative mode on an Agilent 1100
Trap SL ion trap in the range of 100–1000 m/z or 100–2000 m/z
using electrospray ionisation.
4.1.5. 2-(S)-(4-Nitrobenzyl)-1,4,7,10-tetraazacyclododecane
trihydrochloride (6)
Prepared as described in the literature.26,27
4.1.5.1. Caution: explosion hazard. All glass wares were dried in
an oven. In a three necked 1-L flask a suspension of compound 5
(3.0 g, 9 mmol) in 300 mL of dry THF under nitrogen, 200 mL of
1 mol/L BH3/THF solution was added drop wise at 0 °C during
3 h. This reaction mixture was stirred for 1 h at 0 °C. Temperature
was increased gradually and the mixture refluxed for 24 h. After
that 90 mL water was added slowly during 1 h to destroy excess
BH3 and thereafter the mixture was cooled to 0 °C and concd HCl
was added slowly during 1 h. The resulting mixture was refluxed
for 14 h. Two thirds of the solvent was removed in vacuo and the
resulting precipitate was filtered off and recrystallised from abso-
lute CH3OH and dried in vacuum. Compound 6 was obtained as
white solid (1.09 g, 28%). TLC (CH3OH/10% aq ammonium acetate
w/v = 1:1): Rf = 0.18 and (CHCl3/MeOH/25% aq NH3 = 100:30:6):
Rf = 0.11 as free base. UV/vis (MeOH): 205.0, 233.0, 271.0. IR
(KBr): 3446 (w, br, NH), 1515 (s, NO2). 1H NMR (400 MHz, D2O):
d = 2.4–3.25 (m, 16 H, NCH2, ArCH2), 3.4 (m, 1H, NCHC), 7.49 (d,
2H, ArH), 8.14 (d, 2H, ArH). 13C NMR (100 MHz, D2O): d = 39.34
(ArCH2), 43.22 (CH2), 45.00 (CH2), 45.52 (CH2), 46.48 (CH2), 46.77
(CH2), 47.05 (CH2), 50.13 (CH2), 56.55 (CH), 126.65 (Ar), 132.90
(Ar), 147.39 (Ar), 149.32 (Ar). Elemental Anal. Calcd for
C15H25N5O2ꢁ3HClꢁH2O: C, 41.44; H, 6.95; N, 16.11; Cl, 24.46. Found:
C, 41.92; H, 6.80; N, 16.01; Cl, 24.25.
4.1. Synthesis of bifunctional chelating agent (p-NH2-Bn-DOTA)
and its gadolinium complex
4.1.1. (S)-p-Nitrophenylalanine (2)
Following the procedure described in the literature21 33 g
(0.20 mol) of (S)-phenylalanine (1) was dissolved in concd H2SO4
and reacted with concd HNO3, yielding 27.46 g (65%) 2 as a white
crystalline solid. TLC (CHCl3/CH3OH = 3:1): Rf = 0.07 and (10% w/v
aq ammonium acetate/CH3OH = 1:1): Rf = 0.83. UV/vis (MeOH):
210.0, 229.5, 261.5. IR (KBr): 3433 (br, OH), 1741 (C@O), 1514 (s,
NO2). 1H NMR (400 MHz, CD3OD): d = 3.36 (dd, 1H, J = 15.3 and
7.2, CH2), 3.46 (dd, 1H, J = 15.3 and 6.4, CH2), 4.38 (dd, 1H, J = 6.4
and 7.2), 7.60 (d, 2H, ArH), 8.27 (d, 2H, ArH).
4.1.2. (S)-p-Nitrophenylalanine methyl ester hydrochloride (3)
Prepared as described in the literature25 compound 2 (10.0 g,
47.6 mmol) was treated with CH3OH, saturated with HCl (g), to
give the compound 3 as a white solid (7.4 g, 60%). TLC (CHCl3/
CH3OH = 4:1): Rf = 0.75 and (10% aqueous solution of ammonium
acetate/CH3OH = 1:1): Rf = 0.83. UV/vis (MeOH): 207.0, 228.5,
264.0. IR (KBr): 1745 (s, C@O). 1H NMR (400 MHz, CD3OD):
d = 3.27–3.42 (m, 2H), 3.83 (s, 3H), 4.41 (t, 1H), 7.52 (d, 2H, ArH),
8.23 (d, 2H, ArH). Elemental Anal. Calcd for C10H12N2O4ꢁHCl: C,

1896
P. Saha et al. / Bioorg. Med. Chem. 18 (2010) 1891–1898
4.1.6. 2-(S)-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclododecane-
N,N0,N00,N000-tetraacetic acid tetrabenzyl ester (7)
d = 0.50 (s, 3H, C-18 H), 1.40 (m, 1H, C-15 H), 1.42 (m, 1H, C-7
H), 1.68 (m, 2H, C-14 H, C-15 H), 1.84 (s, 3H, C-21 H), 1.89 (td,
J = 11.4, J = 3.2, 1H, C-16 H), 1.98 (m, 1H, C-7 H), 2.16 (m, 1H, C-
16 H), 2.21 (m, 1H, C-12 H), 2.29 (m, 4H, C-1 H, 2 ꢂ C-2 H, C-12
H), 2.42 (dd, J = 10.4, J = 0.8, 1H, C-8 H), 2.52 (m, 2H, C-6 H), 2.73
(m, 1H, C-1 H), 2.86 (s, 3H, NCH3), 4.31 (d, J = 6.4, 1H, C-11 H),
5.52 (s, 1H, C-4 H), 6.61 (m, 2H, C-30/50H), 6.97 (m, 2H, C-20/60H).
13C NMR (100 MHz, CDCl3): d = 3.7 (q, C-21), 13.6 (q, C-18), 23.2
(t, C-15), 25.6 (t, C-1), 27.2 (t, C-7), 31.0 (t, C-6), 36.7 (t, C-2),
38.8 (t, C-12, C-16), 39.0 (d, C-8), 39.4 (d, C-11), 40.5 (q, NCH3),
46.7 (s, C-13), 49.7 (d, C-14), 79.9 (s, C-17), 82.0 (s, C-20), 82.4 (s,
C-19), 112.7 (d, C-30, C-50), 122.5 (d, C-4), 127.3 (d, C-20, C-60),
128.8 (s, C-10), 132.0 (s, C-10), 146.8 (s, C-9), 148.4 (s, C-40),
156.9 (s, C-5), 199.5 (s, C-3). MS: (m/z): 415 (M+, 100), 397 (7),
331 (11), 266 (71), 213 (23), 120 (47), 107 (76), 91 (54); calcd for
C28H33NO2: 415.25.
Prepared according to the literature.13,28 Free base of 2-(p-nitro-
benzyl)-1,4,7,10-tetraazacyclododecane (4-NO2-Bn-cyclen) was
prepared by dissolving the HCl salt (6, 434.8 mg, 1 mmol) in
2 mL of H2O. The pH was raised to about 13 by addition of solid
NaOH and the free base was extracted with CHCl3 (3 ꢂ 25 mL).
After drying the CHCl3 solution with K2CO3, the solvent was re-
moved and the residue was dried in vacuo. The free base was dis-
solved in 10 mL of anhydrous MeCN, benzyl bromoacetate (2.29 g,
10 mmol) and anhydrous Na2CO3 (1.06 g, 10 mmol) were added.
The mixture was stirred at 70 °C under argon for 96 h. The reaction
mixture was filtered to remove excess Na2CO3 and the solvent was
evaporated. The resulting yellow oil was purified by flash chroma-
tography using first CHCl3 and finally CHCl3/MeOH (15:4) as eluent
to give 7 (766 mg, 85%). 7 was used for the next step without fur-
ther purification. TLC (CHCl3/CH3OH = 15:2): Rf = 0.46. UV/vis
(MeOH): 206.5, 283.0. IR (KBr): 1732 (s, C@O), 1515 (s, NO2). 1H
NMR (400 MHz, CD3OD): d = 2.10–3.80 (m, 25H, ArCH2C, NCH2,
NCH), 5.00–5.40 (m, 8H, ArCH2O), 7.20–7.40 (m, 20H, ArH), 7.48
(d, 2H, ArH), 8.05 (d, 2H, ArH). 13C NMR (100 MHz, CDCl3):
d = 44.05–67.13 (ArCH2, NCH2, CH), 123.64–135.20 (Ar), 146.33
(Ar), 146.64 (Ar), 147.75 (Ar), 173.45 (C@O), 173.81 (C@O),
173.91 (C@O), 173.99 (C@O).
4.2.2. 3-N-Methyl-N-[4-(11b,17b)-17-hydroxy-3-oxo-17a-(1-
propynyl)estra-4,9-dien-11-yl]-phenylaminopropionic acid (12)
Prepared according to the literature.31,32 3-Bromopropionic acid
(367 mg, 2.4 mmol) was dissolved in 4 mL of water and the pH of
the solution was brought to 7.7 by the addition of NaHCO3. Com-
pound 11 (200 mg, 0.48 mmol) was dissolved in 4 mL EtOH and
was added to the previous aqueous mixture, the pH was kept at
9 during the reaction by the addition of NaHCO3. This mixture
was stirred for 20 h at 70 °C, cooled to room temperature and pH
was adjusted to 6 by adding HCl/EtOH. Solvent was evaporated
and the product was extracted with ethyl acetate three times
and the extract was dried over sodium sulfate. After evaporation
of the solvent the product (12) was purified by HPLC column 1
(flow rate 23 mL/min, k = 254 nm, acetonitrile/water 6:4, retention
time for = 5.22 min). After lyophilisation, 12 was isolated as yellow
solid (120 mg, 51%). TLC (ethyl acetate): Rf = 0.16, (chloroform/
MeOH/25% aqueous ammonia solution = 100:30:6): Rf = 0.41. Ana-
lytical HPLC (column 2; acetonitrile/H2O (90:10, vol/vol); flow:
1 mL/min): tR = 4.161 min. UV/vis (MeOH): 207.5, 263.5, 303.0.
UV/vis (CH2Cl2): 300.0. IR (KBr): 3424 (m, br, O–H), 2939 (s, C–
H), 2870 (m, C-H), 2242 (w, C„C), 1728 (s, C@O), 1656 (s, C@O),
1615(s, C@C). 1H NMR (400 MHz, CDCl3): d = 0.53 (s, 3H, C-18 H),
1.35 (m, 1H, C-15 H), 1.46 (m, 1H, C-7 H), 1.72 (m, 1H, C-14 H),
1.73 (m, 1H, C-15 H), 1.89 (s, 3H, CH3, C-21 H), 1.94 (t, 1H, C-16
H), 2.02 (m, 1H, C-7 H), 2.22 (m, 1H, C-16 H), 2.25 (m, 1H, C-12
H), 2.32 (m, 1H, C-1 H), 2.35 (m, 1H, C-12 H), 2.37 (m, 1H, C-2
H), 2.44 (m, 1H, C-2 H), 2.46 (m, 1H, C-8 H), 2.60 (m, 4H, C-6 H,
C-200H), 2.75 (m, 1H, C-1 H), 2.93 (s, 3H, ꢀNCH3), 3.63 (m, 2H, C-
100 H), 4.36 (d, J = 6.6, 1H, 11-H), 5.78(s, 1H, C-4 H), 6.81 (d,
4.1.7. 2-(S)-(p-Aminobenzyl)-1,4,7,10-tetraazacyclododecane-
N,N0,N00,N000-tetraacetic acid (8)
Following the procedure described in the literature29 7 (1.58 g,
1.75 mmol) was hydrogenated to compound 8 (802.5 mg, 90%)
white crystal. TLC (10% w/v aqueous ammonium acetate/
CH3OH = 1:1): Rf = 0.44. UV/vis (MeOH): 204.0, 242.0. IR (KBr):
3430 (s, br, NH2), 1700 (s, C@O). 1H NMR (400 MHz, D2O):
d = 2.5–4.0 (m, 25H, NCH, ArCH2, NCH2, NCH2CO), 7.2–7.4 (m, 4H,
ArH). 13C NMR (100 MHz, D2O): d = 34.30–63.66 (ArCH2, NCH2,
NCH2CO, NCH), 126.45 (Ar), 132.36 (Ar), 133.84 (Ar), 140.67 (Ar),
173.48 (C@O). MS (m/z): [M+H]+: 510.1, [M+Na]+: 532.1,
[M+NaCl+H]+: 568.0, [M+NaCl+Na]+: 590.0, [MꢀH]ꢀ: 508.7,
[M+Naꢀ2H]ꢀ: 530.1, [M+NaClꢀH]ꢀ: 566.0, [M+NaCl+Naꢀ2H]ꢀ:
588.2; calcd for C23H35N5O8: 509.25.
4.1.8. [Gd-(p-NH2-Bn-DOTA)] (9)
Prepared according to the literature.30 GdCl3ꢁ6H2O (74.34 mg,
0.2 mmol) was added to
a stirred solution of compound 8
(101.91 mg, 0.2 mmol) in 100 mM aqueous Na2CO3 solution (pH
ꢃ8). This mixture was stirred at 80 °C for 12 h. The solvent was
evaporated, yielding 9. TLC (MeCN/H2O = 10:3): Rf = 0.22 and
(MeOH/10% aq w/v CH3COONH4 solution = 1:1): Rf = 0.51. UV/vis
(H2O): 217.0, 236.0. IR (KBr): 3416 (s, br, NH2), 1604 (s, C@O).
MS (m/z): [M+2Na]+: 708.9, [M+NaCl+2Na]+: 766.9, [MꢀH]ꢀ:
661.3; calcd for C23H31GdN5O8ꢀ: 662.77.
J = 7.2, 2H, C-3 /5 H), 7.06 (d, 2H, C-2 /6H). 13C NMR (100 MHz,
CDCl3): d = 3.84 (q, C-21), 13.81 (q, C-18), 23.34 (t, C-15), 25.81
(t, C-1), 27.33 (t, C-7), 31.14 (t, C-6), 31.22 (t, C-200), 36.76 (t, C-
2), 38.90 (t, 2C, C-12, C-16), 39.18 (d, C-8), 39.45(q, NCH3), 39.69
(d, C-11), 46.88 (s, C-13), 49.66 (t, C-100), 49.78 (d, C-14), 80.18 (s,
C-17), 82.30 (s, C-19), 82.54 (s, C-20), 114.59 (d, C-30, C-50),
122.74 (d, C-4), 127.95 (d, C-20, C-60), 129.25 (s, C-10), 135.10 (s,
C-10), 145.60 (s, C-40), 146.66 (s, C-9), 157.28 (s, C-5), 175.85 (s,
C-300), 200.01 (s, C-3). MS (m/z): [M+Na]+ 510.4; calcd for
C31H37NO4: 487.27.
0
0
0
´
4.2. Synthesis of mifepristone derivatives by attaching linkers
4.2.1. Desmethylmifepristone (DMM) [17b-hydroxy-17a-propy-
nyl-11b-(4-N-methylaminophenyl)-estra-4,9-dien-3-one (11)
Following the procedure described in the literature1,31 500 mg
(1.16 mmol) of mifepristone (10) was converted to 135 mg of com-
pound 11 (28%) as a fluffy pale yellow solid. Purification: HPLC:
column 1, acetonitrile: H2O, (84:16, vol/vol); flow: 23 mL/min;
tR = 3.4 min. Adduct 10 was recovered (130 mg, 26%), tR = 4.3 min.
Analytical HPLC: column 2; acetonitrile: H2O (90:10, vol/vol); flow:
1 mL/min): tR = 4.822. UV/vis: (MeOH): 209.0, 250.0, 303.5. TLC
(cyclohexane/EtOAc = 4:6): Rf = 0.51 and (CH2Cl2/MeOH, 9:1):
Rf = 0.71. UV/vis (MeOH): 209.0, 250.0, 303.5. IR (KBr): 3387 [m
(br), N–H, O–H], 2929 (s, C–H), 2870 (m, C–H), 2250 (w, C„C),
1654 [s (br),C@O], 1615 (s, C@C). 1H NMR (400 MHz, CDCl3):
4.2.3. 6-N-Methyl-N-[40-[17b-hydroxy-3-oxo-17
a-(1-propynyl)-
estra-4,9-dien-11b-yl]-phenylaminohexanoicacid (13)
Following the procedure described for the synthesis of 12, reac-
tion of 6-bromohexanoic acid (468 mg, 2.4 mmol) and compound
11 (200 mg, 0.48 mmol) gave after purifying by flash-chromatogra-
phy with EtOAc and MeOH and preparative HPLC (column 2;
acetonitrile/H2O (56:44, vol/vol), flow: 23 mL/min; tR = 8 min
30 s) 100 mg (39%) of compound 13 as a fluffy yellow solid.
Analytical HPLC (column 3; acetonitrile/H2O (90:10, vol/vol); flow:

P. Saha et al. / Bioorg. Med. Chem. 18 (2010) 1891–1898
1897
1 mL/min): tR = 4.668 min. TLC (chloroform/MeOH/25% aqueous
ammonia solution = 100:30:6): Rf = 0.47 and (EtOAc): Rf = 0.36.
UV/vis (MeOH): 207.0, 262.5, 303.0. IR (KBr): 3420 (m, br, OH),
2937 (s, CH), 2866 (m, CH), 2242 (w, C„C), 1730 (s, C@O), 1659
(s, C@O), 1612 (s, C@C). 1H NMR (400 MHz, CDCl3): d = 0.55 (s,
3H, C-18 H), 1.36 (m, 1H, C-15 H), 1.38 (m, 2H, C-300 H), 1.46 (m,
1H, C-7 H), 1.56 (m, 2H, C-200 H), 1.68 (m, 2H, C-400 H), 1.72 (m,
1H, C-14 H), 1.73 (m, 1H, C-15 H), 1.89 (s, 3H, CH3, C-21 H), 1.94
(t, 1H, C-16 H), 2.02 (m, 1H, C-7 H), 2.22 (m, 1H, C-16 H), 2.28 (t,
J = 7.0, 2H, C-500 H), 2.32 (m, 1H, C-1 H), 2.35 (m, 1H, C-12 H),
2.40 (m, 1H, C-2 H), 2.44 (m, 1H, C-2 H), 2.46 (m, 1H, C-8 H),
2.57 (m, 2H, C-6 H), 2.73 (m, 1H, C-1 H), 2.88 (s, 3H, -NCH3), 3.26
viously1,37,38 with some minor modifications. Cells were washed
with 0.9% saline and stored at ꢀ80 °C for at least 30 min after
48 h incubation. After defrosting cells were incubated with 50-lL
p-nitrophenyl phosphate (5 mM, Fluka) dissolved in aqueous
diethanolamine (1 M, supplemented with 0.5 mM magnesium
chloride and 20 lM zinc sulfate and adjusted to pH 9.8) for 2–5 h
in the dark at room temperature. Absorbance of p-nitrophenolate
was measured in a Lucy 1 multimode plate reader (anthos labtec
instruments) at 405 nm (vs 690 nm as reference). For each com-
pound, at least four independent experiments (with six data points
each) were carried out. After background subtraction (absorbance
of cells without hormonal treatment), absorbance was normalised
to the absorbance resulting from the progesterone incubation.
Median (central tendency) median absolute deviation (variabil-
ity) was calculated. IC50 values were determined from dose–re-
sponse curves and are given in nmol/L.
(t, J = 8.0, 2H, C-100 H), 4.34 (d, J = 6.8, 1H, 11
a-H), 5.76 (s, 1H, C-4
H), 6.59 (m, 2H, C-30/50 H), 6.98 (m, 2H, C-20/60 H). 13C NMR
(100 MHz, CDCl3): d = 3.80 (q, C-21), 13.68 (q, C-18), 23.27 (t, C-
15), 24.67 (t, C-400), 25.80 (t, C-1), 26.39 (t, C-200), 26.66 (t, C-300),
27.32 (t, C-7), 31.09 (t, C-6), 34.34 (t, C-500), 36.85 (t, C-2), 38.19
(q, NCH3), 38.74 (t, C-12), 38.83 (t, C-15), 39.07 (d, C-8), 39.46 (d,
C-11), 46.82 (s, C-13), 49.83 (d, C-14), 52.71 (t, C-100), 80.13 (s, C-
17), 82.35 (s, C-20), 82.37 (s, C-19), 112.20 (d, C-30, C-50), 122.62
(d, C-4), 127.54 (d, C-20, C-60), 128.98 (s, C-10), 131.27 (s, C-10),
146.86 (s, C-9), 147.24 (s, C-40), 156.97 (s, C-5), 173.60 (s, C-600),
199.64 (s, C-3). MS (m/z): [M+Na]+ 552, [M]ꢀ 529; calcd for
C34H43NO4: 529.32.
4.5. Determination of Gd concentration with ICPMS (inductively
coupled plasma mass spectrometry)
Lysed cell pellets were quantitatively transferred into 15 mL
polyethylene using 20% (v/v) nitric acid. The Gd-concentration
was directly determined in these solutions with an Agilent
7500ce inductively coupled plasma mass spectrometer (Agilent,
Waldbronn, Germany) at m/z 157. The calibration standards were
4.3. Synthesis of desired conjugates
prepared from a 10.0 0.5 lg Gd/mL stock standard solution (P/N
4400-010031, CPI International, Amsterdam, Netherlands) in a
concentration range of 1.0–100 ng Gd/L. Throughout the measure-
ments Indium at m/z 115 was used as internal standard (P/N
4400-1000241, CPI International, Amsterdam, Netherlands) to
compensate for instrumental drifts.
4.3.1. 3-N-Methyl-N-[40-[17b-hydroxy-3-oxo-17
a-(1-propynyl)-
estra-4,9-dien-11b-yl]-phenyl-amino-propanoyl-[Gd-(p-NH2-
Bn-DOTA)] conjugate (16)
Extremely dried 9 (27 mg, 0.04 mmol) was dissolved in 8 mL of
dry DMF in a 10 mL flask under N2 using a sonicator (3–4 min).
In another flask compound 12 (19.5 mg, 0.04 mmol) was dissolved
Acknowledgements
in 0.8 mL of dry THF with 14
lL of triethylamine by stirring
5 min in an N2 atmosphere. Isobutyl chloroformate (5.76
l
L,
The present study was financially supported by Austrian Ex-
change Service (OÄD), Austrian Cancer Aid of Styria and Federal
Ministry of Education & Research Germany.
0.044 mmol) was added and this mixture was stirred about
2 min under N2 atmosphere. The solution of 9 was then added
and the reaction mixture was stirred for 5 h under N2 atmosphere
at room temperature. Solvent was evaporated and the residue was
purified by silica gel column chromatography using MeCN/H2O
(20:7) as eluent yielding 16 after evaporation as a pale yellow so-
References and notes
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lid. C54H67GdN6O11
; (MW: 1133.40). TLC (MeCN/H2O = 20:7):
Rf = 0.39 and (MeOH: 10% aq w/v CH3COONH4 solution = 1:1):
Rf = 0.58. UV/vis (DMSO): 253.0, 265.0. IR(KBr): 3421 (s, br, NH),
1609 (s, C@O). MS (m/z): [M]ꢀ: 1132.2.
4.3.2. 6-N-methyl-N-[40-[17b-hydroxy-3-oxo-17
a-(1-propynyl)-
estra-4,9-dien-11b-yl]-phenyl-amino-hexanoyl-[Gd-(p-NH2-Bn-
DOTA)] conjugate (17)
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Following the procedure described for the synthesis of conju-
gate 16. Product 17 was synthesised by the coupling reaction
between 9 and 13 and purified by silica gel column chromatogra-
phy using MeCN/H2O (10:3) yielding 17 as a pale yellow solid.
C57H73GdN6O11; (MW: 1175.49) calcd C, 58.24; H, 6.26; N, 7.15;
found +1/3 H2O: C, 57.92; H, 6.10; N, 7.08. TLC (MeCN/H2O =
10:3): Rf = 0.32 and (MeOH: 10% aq w/v CH3COONH4 solu-
tion = 1:1): Rf = 0.52. UV/vis (DMSO): 252.0, 264.0, 281.5, 303.0.
IR (KBr): 3422 (s, br, NH), 1605 (s, C@O). MS (m/z): [M+2Na+
H]2+: 610.6, [M+Na+H]+: 1198.2, [M]ꢀ: 1174.2.
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