Syntheses and antigestagenic activity of mifepristone derivatives

Article abstract of DOI:10.1021/jm800985z

A series of mifepristone derivatives with different "linker groups" in position 4' of the phenyl ring in the 11β- position of the steroid scaffold (2-41) have been synthesized. Their antigestagenic activites were determined in a cell-based assay (alkali p

Full text of DOI:10.1021/jm800985z

1268
J. Med. Chem. 2009, 52, 1268–1274
Syntheses and Antigestagenic Activity of Mifepristone Derivatives
Claudia Ho¨dl,† Katrin Raunegger,† Rainer Strommer,† Gerhard F. Ecker,‡ Olaf Kunert,† Sonja Sturm,§ Christoph Seger,§
Ernst Haslinger,† Rudolf Steiner,| Wolfgang S. L. Strauss,*,| and Hans-Wolfgang Schramm*,†
Department of Pharmaceutical ChemistrysMedicinal Chemistry, Institute of Pharmaceutical Sciences, UniVersity of Graz, UniVersita¨tsplatz 1,
A-8010 Graz, Austria, Department of Medicinal Chemistry, UniVersity of Vienna, Althanstrasse 14, A-1090 Vienna, Austria, Institute of
Pharmacy, Department of Pharmakognosy, UniVersity of Innsbruck, Innrain 52, A-6020 Innsbruck, Austria, Institute for Lasertechnology in
Medicine and Metrology at the UniVersity of Ulm, Helmholtzstrasse 12, D-89081 Ulm, Germany
ReceiVed August 4, 2008
A series of mifepristone derivatives with different “linker groups” in position 4′ of the phenyl ring in the 11ꢀ-
position of the steroid scaffold (2-41) have been synthesized. Their antigestagenic activites were determined in
a cell-based assay (alkali phosphatase assay in T47-D breast cancer cells) and compared with that of the parent
compound mifepristone. SAR and QSAR studies reveal the influence of both lipophilicity and partial charge
based van der Waals surface area descriptors on biological activity. Within the series of compounds described in
this study, three mifepristone derivatives are identified with considerably high antigestagenic activity. These
compounds are regarded as useful starting materials for the synthesis of either physiologically stable or cleavable
progesterone receptor-binding conjugates for therapeutic or diagnostic purposes.
Scheme 1. Synthesis of O-Analogues of Mifepristone Starting
from I
Introduction
Worldwide, breast cancer is the most common cancer among
women and the number of incidences has significantly increased
since 1970, especially in Western Europe and in North
America.1 Mifepristone, or RU 486 (1), is a synthetic steroid
compound with a p-(dimethylamino)phenyl group in 11ꢀ-
position of the steroid scaffold and acts as a progesterone
receptor (PRa) antagonist.2-4 Its binding affinity is more than
five times that of progesterone.5 Therefore, it can be used as
abortifacient and has been considered to be effective in treating
progesterone-dependent breast cancer.6,7
In a preceding study, we have shown that a conjugate
containing a mifepristone residue attached via an ω-thioure-
thano-C6-alkyl linker to a fluorescein residue is selectively
accumulated in PR receptor positive T47D breast cancer cells.8
This work has also demonstrated that such a conjugate is even
translocated into the cell nuclei. Thus, the PR might serve for
a site-directed delivery of pharmaceutically active agents. In a
continuous effort to develop conjugates, which may be useful
for a selective enrichment of anticancer agents, e.g., taxodiol
or doxorubicin, into these tumor cells, we focused our attention
on the nature of the linker group. In particular, the linker group
used for attachment of the bioactive molecule to the mifepristone
residue should impair the biological activity of 1 as low as
possible. In addition, it is assumed that the linker must provide
a certain distance between both molecular moieties to retain
biological activity of conjugates at least partly. Therefore, linker
groups of different chain length bearing additionally different
groups at the ω-terminus, e.g., carboxy, ester and amide groups,
were synthesized and evaluated in cell culture. Because detach-
ment of the pharmaceutically active agent from the conjugate
is required in certain cases, e.g., for the anticancer agents
mentioned above, linker groups that will be cleaved in cellular
environment are also considered in this study.
Derivatization of 1 at the aromatic region is necessary to retain
high PR-binding affinity.9 Therefore, the position 4′ was chosen
for derivatization and attachment of the various linker groups.
Mifepristone (1) was demethylated to 2 as reported previously,10
which was the common starting material for compounds 6-41.
The different linker groups have been attached to the nitrogen
atom of the secondary amino group yielding the variety of
compounds summarized in Scheme 3.
* To whom correspondence should be addressed. For H-W.S.: phone,
+43/316/380-5381; fax, +43/316/380-9846; E-mail, wolfgang.schramm@uni-
graz.at. For W.S.L.S.: phone, +49/731/1429-21; fax, +49/731/1429-42;
E-mail: wolfgang.strauss@ilm.uni-ulm.de.
The present paper describes the synthesis of a series of mostly
novel mifepristone analogues as well as SAR and QSAR studies
on their antigestagenic activity.
†
Department of Pharmaceutical ChemistrysMedicinal Chemistry, In-
stitute of Pharmaceutical Sciences, University of Graz.
‡
Department of Medicinal Chemistry, University of Vienna.
Institute of Pharmacy, Department of Pharmakognosy, University of
§
Innsbruck.
Chemistry and Structural Assignment
|
Institute for Lasertechnology in Medicine and Metrology at the
All compounds described in this study have been obtained
University of Ulm.
a Abbreviations: PR, progesterone receptor; AP, alkaline phosphatase.
by partial synthesis9 either starting from 3,3-dimethoxy-5R,10R-
10.1021/jm800985z CCC: $40.75
2009 American Chemical Society
Published on Web 02/13/2009

Syntheses and Antigestagenic ActiVity of Mifepristones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1269
Scheme 2. Synthesis of N-Derivatives via Demethylation of Mifepristone
Scheme 3. Structures of compounds 1-42
O-Analogues of Mifepristone. Compound 3 was obtained
as described in literature6-8 and converted to 4 with diazo-
methane. Alkylation of 3 with 1,9-dibromo nonane yielded
compound 5.
N-Derivatives of Mifepristone. Starting material for all these
compounds was 2, which was prepared as described previously.8
To introduce an alkyl residue at the nitrogen atom, 2 was
alkylated with the 1-bromo alkanes, yielding compounds 6-8.
Alkylation with ω-bromo 1-alcohols resulted in compounds 9
and 10 (42 was obtained as byproduct together with 9). Reacting
2 with 6-bromohexanal ethylene glycol acetal led to compound
11, which gave 12 after acid hydrolysis. Introduction of an
ω-bromoalkyl carboxylic acid residue gave compounds 13-16.
Ester 17 was obtained by reaction of methyl 3-bromopropionate
with 2; conversion of compounds 14-16 by diazomethane
resulted in the esters 18-20. After activation of acid 14 by
isobutyl chloroformate, amides 21 and 22 were obtained by
reaction of the intermediate with ammonia or n-butylamine,
respectively. Acid 13 was activated in the same manner and
converted with 4-amino-2-hydroxybenzamide to compound 36.
Reaction of 2 with 4-(3′-bromopropoxymethyl)-2-hydroxyben-
zamide yielded compound 37. Acylation of 9 and 10 with
succinic acid anhydride formed acid 38 and 39, respectively,
which yielded esters 40 and 41 after treatment with diazo-
methane. Reaction of 2 with hexanoyl chloride or cyclic acid
anhydrides led to compounds 23-25; again, esterification with
diazomethane yielded esters 26 and 27. Reaction of 2 with
different ω-iso(thio)cyanates gave the corresponding (thio)urea
compounds 28-31 and 33. Saponification of ester 33 resulted
in acid 32, which was activated by isobutyl chloroformate and
further reacted with L-leucine t-butylester, resulting in ester 34,
which was further hydrolyzed to 35.
Structural Assignment. Structures of all new compounds
were confirmed by IR, MS, and NMR spectroscopic methods.
In most cases complete and unambiguous assignments for all
1H and 13C resonances could be achieved on the basis of 1D
and 2D experiments.
Antigestagenic Activity
To demonstrate ligand-receptor interaction of the new
compounds in cell culture, antigestagenic activity was deter-
mined using two independent cell-based assays, and biological
epoxyestra-9(11)-en-17-one (I) or mifepristone (1), as shown
in Schemes 1 and 2, respectively.

1270 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Ho¨dl et al.
Table 1. Calculated logP Values and Antigestagenic Activity of
Compounds 1-41
and a MOE-database was generated. PEOE charges were
calculated, and all compounds were subjected to energetic
minimization using the MMFF94x force field (gradient 0.01)
implemented in MOE.
alkaline phosphatase assaya
transactivation assayb
calcd
compd logP
IC50
relative
IC50
relative
valuec
potencyd
valuec
potencyd
Hansch Analyses. For multiple linear regression analyses, a
set of descriptors provided by MOE was calculated. These
include physicochemical parameters such as logP, polar surface
area, hydrophobic van der Waals surface area, molar refractivity,
number of H-bond donors and acceptors, and atomic polariz-
ability, as well as connectivity indices and a set of fractional
van der Waals surface area descriptors (PEOE_VSA, SLOG-
P_VSA, SMR_VSA). For generation of a global model, the data
matrix was subjected to PLS analysis using a svl-script provided
by the Chemical Computing Group exchange program (auto-
QSAR). For local models, regression analyses were performed.
1
2
3
4
5
6
7
8
4.57
4.30
4.35
4.61
7.14
5.53
6.85
8.18
4.32
5.65
6.02
5.74
4.17
5.49
6.38
7.70
4.43
5.76
6.64
7.97
4.76
6.51
5.95
4.14
4.58
4.41
4.85
5.19
5.78
6.04
6.63
4.71
4.97
7.19
5.71
4.11
5.39
4.46
5.79
4.73
6.05
0.045
0.17
1.3
0.095
1.0
0.27
0.66
1.4
0.12
0.06
0.17
0.15
100
26
3
47
4.5
17
7
0.021
0.073
100
29
(0.025)
0.18
0.38
0.105
0.071
0.073
(84)
12
6
20
30
29
3
9
37
75
26
30
0.13
10
17
10
23
11
10
7
8
5
7
0.04
0.06
3
2.5
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
34
0.44
0.27
0.46
0.16
0.40
0.47
0.63
0.55
0.9
Results and Discussion
A variety of different chemical structures have been attached
to the p-position of the aromatic ring in 11ꢀ-position of the
steroid scaffold. In total a series of 41 compounds have been
synthesized and tested with respect to their antigestagenic
activity in cell culture. Structural variations comprise different
substitution patterns on the aniline nitrogen atom as well as
exchanging the nitrogen atom by oxygen. Substituents on the
nitrogen atom include alkyl and alkanoyl chains of different
length bearing additionally different groups at the ω-terminus,
e.g., carboxy, ester, and amide groups.
O-Analogues of Mifepristone. Compound 4 exhibited con-
siderably antigestagenic activity. The relative potency was only
2-fold lower than that of 1. However, if the methoxy group is
replaced by the more polar hydroxyl function (3), biological
activity decreases by one decade. The same is true for compound
5, which has an alkyl bromide side chain attached to the oxygen
atom. Although 5 (calcd logP ) 7.14) is more lipophilic than
the N-derivative 10 (R ) 6-hydroxyhexyl, calcd logP ) 5.65),
its biological activity is about 15-fold lower than that of 10
(see Table 1, Scheme 3). Therefore, we concluded that O-
analogues of mifepristone with the phenyloxy group at position
11ꢀ are less favorable for the development of bioactive
conjugates.
N-Derivatives of Mifepristone. Some of these compounds
have retained considerable biological activity. Most IC50 values
are still in the subnanomolar range, and a relative potency
between 10% and 25% was calculated for some of the test
compounds (Table 1).
Comparison of 6, 7, and 8 with n-propyl, n-hexyl, and n-nonyl
residues at the nitrogen atom reveals that the relative potency
decreases from 17% over 7% to 3% as compared to 1 with
increasing chain length. If, however, the alkyl residue bears an
ω-hydroxyl group (9, 10) the relative potency is considerable
higher, exhibiting relative potencies of 37% and 75%, respec-
tively. In this case, a longer alkyl chain seem to enhance
biological activity.
0.69
∼ 100
78
1.55
1.8
0.79
0.73
0.27
0.16
72
2.2
1.7
6
17
28
0.06
2
2.6
0.06
10
10
2.1
18
3.8
26
72
0.44
0.46
2.1
0.25
1.2
0.17
a Inhibition of progesterone-induced (10-9 mol/L) alkaline phosphatase
b
activity in T45-D cells. Inhibition of progesterone-induced (10-9 mol/L)
transcriptional activity in T47-D-C124 cells. c IC50 values were determined
from dose-response curves and are given in nmol/L. d Relative potency is
defined as the ratio of IC50 (1): IC50 (X) multiplied by 100 (X ) 2-41).
activity of each test compound was compared with that of the
parent compound 1, mifepristone, as reference antagonist. In
all cases, antigestagenic activity was assessed using the alkaline
phosphatase assay in T47-D breast cancer cells; in some cases
also a transactivation assay in T47-D-C124, i.e., T47-D cells
stably transfected with the luciferase gene linked to the steroid
hormone responsive MMTV promoter, was applied. Dose-
dependent inhibition of progesterone-induced increase of either
alkaline phosphatase or luciferase activity was found for most
test compounds. IC50 values determined from dose-response
curves and the relative potencies (rp defined as the ratio of the
IC50 value of the reference antagonist 1 to the IC50 value of the
individual test compound) are summarized in Table 1. Assess-
ment of biological activity in a cell-based assay display both
receptor binding affinity as well as intracellular availability of
the test compound.
In contrast, a carboxyl group at the ω-terminus of the alkyl
chain resulted again in a considerable reduction of antigestagenic
activity (e.g.: 13: rp ) 0.13%). In this case, length of the alkyl
chain does not obviously affect biological activity as deduced
from the comparison of 14, 15, and 16. However, compound
13 exhibits an almost negligible antigestagenic activity for
reasons unknown so far. Most likely the polar, and under
physiological pH also negatively charged, carboxyl group
impairs the transport of the compound through the cell
membrane; consistently, esterification of the carboxyl group
partly restores biological activity (17: rp ) 23%). However,
SAR and QSAR Studies
Molecular Modeling QSAR Studies. Structures were built
within MOE (Chemical Computing Group, Montreal, Canada),

Syntheses and Antigestagenic ActiVity of Mifepristones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1271
Table 4. Relative Potencies of Compounds 38, 39, 40, and 41
Table 2. Antigestagenic Activity (AP-assay) of Compounds 17-20
compd
side chain
IC50 value
relative potency
compd
side chain
IC50 value relative potency
17
18
19
20
-(CH2)2COOCH3
-(CH2)5COOCH3
-(CH2)7COOCH3
-(CH2)10COOCH3
0.16
0.40
0.47
0.63
23
11
10
7
38
39
40
41
-(CH2)3OCO(CH2)2COOH
-(CH2)6OCO(CH2)2COOH
-(CH2)3OCO(CH2)2COOCH3
-(CH2)6OCO(CH2)2COOCH3
2.10
0.25
1.20
0.17
2.1
18
3.8
26
for the compounds with longer alkyl chains esterification does
not affect antigestagenic activity (18-20 as compared to 14-16,
Table 2).
Compound 35 bearing an enzymatic cleavable linker group
exhibit almost negligible antigestagenic activity (rp ) 0.06%).
Even after esterification (34), biological activity remains quite
moderate (rp ) 2.6%). However, comparison of 34 vs 33 and
35 vs 32 reveal that attachment of the amino acid L-leucine at
the ω-terminus does not significantly affect biological activity.
Compound 39 with a hydrolytic cleavable linker group show
considerable antigestagenic activity (rp ) 18%), which is again
increased after esterification (41, rp ) 26%). In contrast,
biological activities of 38 and 40 are 6-8-fold lower as
compared to 39 and 41, respectively. This indicates that in the
case of a hydrolytic cleavable linker group chain length might
be an important determinant for biological activity (Table 4).
Because the aniline derivative 7 and the anilide 23 exhibit
the same biological activity, electron density at the nitrogen atom
seem to be not important for ligand receptor interaction.
Therefore, it is concluded that no hydrogen bonding is involved
in the N-region of mifepristone analogues, which is in agreement
with the present knowledge on the interaction of mifepristone
with steroid hormone receptors at the structural level.11,12 This
conclusion is strengthened by comparing biological activities
of the urea and thiourea derivatives (28 vs 29 and 30 vs 31). If,
however, the anilide 23 is additionally combined with a carboxyl
group at the ω-terminus of the side chain (25), antigestagenic
activity is vanished (25, rp ) 0.06%). Again, esterification result
in a significant increase in biological activity (27, rp ) 2.5%),
which is probably due to a higher intracellular availability of
the less polar compound; however, its antigestagenic activity
is considerably lower than that of the corresponding N-alkyl
derivative 18 (rp ) 11%). Comparing mifepristone derivatives
bearing a C6-alkyl chain with different oxygen functions at the
ω-terminus shows that the alcohol has the highest relative
potency (10, rp ) 75%). An acetal (11) aldehyde (12), or
carboxyl group (14) decreases the antigestagenic activity of the
compounds; esterification (18) does not have much influence
(see above). Converting the carboxyl function to an amide group
(21 and 22) further reduces biological activity (Table 3).
Alkyl amides show only moderate antigestagenic activity (21,
rp ) 8% and 22, rp ) 5%), whereas the aromatic amide 36
exhibit a somewhat higher relative potency of 10%. Comparable
results are found in the case of the derivatives with an urea or
thiourea bond at the nitrogen atom of the mifepristone residue
(28, R ) n-butyl, rp ) 6% vs 30, R ) p-chlorophenyl, rp )
17% and 29 (rp ) 6%) vs 31 (rp ) 28%). However, although
compounds 30 and 31 have considerable high biological activity,
they offer only limited possibilities to attach linker groups. If
an urea bond at the aromatic nitrogen atom is additionally
combined with a carboxyl group at the ω-terminus of the side
chain (32), antigestagenic activity is vanished (rp ) 0.06%),
which is again partly restored after esterification (33, rp ) 2%).
QSAR Studies and Regression Analysis. The activity pattern
of alkyl derivatives 6-8 indicate that a given logP value is
needed to achieve sufficient intracellular concentrations. The
general influence of lipophilicity within this series of compounds
is further strengthened by a regression analysis using alkyl
derivatives 6-8, alcohols 9 and 10, the acetal 11, the aldehyde
12, and esters 17-20 (r2 ) 0.64; Figure 1).
Interestingly, a negative correlation is observed. This indicates
that mifepristone itself might exhibit already an optimal logP
value. However, it should be stressed that increasing lipophilicity
is likely to result in (nonspecific) binding of test compound to
cellular membranes competing with the specific binding to PR.
Compound 10 (R ) 6-hydroxyhexyl) is a positive outlier
showing higher biological activity than expected from its logP
value. This might be due to an additional interaction mediated
by the OH group. This is further strengthened by including either
the topological polar surface area (TPSA) or the atom polariz-
ability (apol) further enhancing the regression coefficient (r2 )
0.68, Q2 ) 0.56), whereby logP shows a negative contribution
and apol/TPSA exhibit positive coefficients in the equation.
However, adding amides 22 and 23 to the data set dramatically
decreases the predictive power of the model (r2 ) 0.45). Also
within the series of anilides 23, 26, 27, and urea/thiourea
derivatives 28, 29, 30, 31, and 33, a good correlation between
logP(o/w) and the log(1/IC50) value is observed (r2 ) 0.81). In
Table 3. Relative Potencies of Compounds 10, 11, 12, 14, 18, 21, and 21

1272 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Ho¨dl et al.
Figure 1. Plot of log potency vs calculated logP values for alkyl
derivatives 6-8, alcohols 9 and 10, the acetal 11, the aldehyde 12,
and esters 17-20 (r2 ) 0.64); excluding compound 10 increases the r2
value to 0.79.
Figure 3. Plot of observed vs calculated log(1/IC50) values for
compounds 1-41 (r2 ) 0.73, r2 ) 0.62); excluding compound 13
cv
increases the r2 value to 0.78.
Table 5. Descriptors Present in the Final Equation
PEOE_VSA+0 sum of accessible van der Waals surface area (in Å2)
for all atoms with a partial charge qi in the range of
[0.00, 0.05)
PEOE_VSA-5 sum of accessible van der Waals surface area (in Å2)
for all atoms with a partial charge qi in the range of
[-0.30, -0.25)
Q_VSA_POS
total positive van der Waals surface area; this is the
sum of the accessible van der Waals surface area of
all atoms with positive partial charge.
pmiX
x component of the principal moment of inertia
(external coordinates).
SlogP_VSA7
sum of accessible van der Waals surface area (in Å2)
for all atoms with an logP(o/w) contribution between
(0.25, 0.30]
SlogP_VSA9
SMR_VSA7
ASA
sum of accessible van der Waals surface area (in Å2)
for all atoms with an logP(o/w) contribution >0.40
sum of accessible van der Waals surface area (in Å2)
for all atoms with an SMR value >0.56
water accessible surface area calculated using a radius
of 1.4 Å for the water molecule; a polyhedral
representation is used for each atom in calculating
the surface area
Figure 2. Plot of log potency vs calculated logP values for anilides
23, 26, and 27, and ureas/thioureas 28-31, and 33 (r2 ) 0.81).
vdw_area
vdw_vol
van der Waals volume (Å3) calculated using a
connection table approximation
area of van der Waals surface (Å2) calculated using a
connection table approximation
this case, the coefficient of logP shows a positive value,
indicating that increasing the value enhances biological activity
(Figure 2).
screening of small virtual libraries for further improvement of
the linker group.
These local differences in the influence of lipophilicity might
be the reason why attempts to create a global model with
reasonable predictivity just on basis of a set of simple ADME-
type descriptors (logP, TPSA, number of H-bond donors and
acceptors, molar refractivity, number of rotable bonds) failed.
The best model obtained showed an r2 value of 0.50 and a cross-
validated r2 value of 0.34 (leave one out). Therefore we used
an svl-script (autoQSAR), which automatically selects out of a
large set of descriptors the most important ones. This led to a
Conclusions
Within this paper we describe the synthesis, biological
activity, and structure-activity relationships of a series of
mifepriston analogues bearing different linker groups at the
nitrogen atom of the phenyl ring in position 11ꢀ of the steroid
scaffold. SAR and QSAR studies reveal the influence of both
lipophilicity and partial charge based van der Waals surface
area descriptors on biological activity. Some of the new
compounds retain considerable antigestagenic activity (with IC50
values in the subnanomolar range and relative potencies between
26% and 75% as compared to the parent compound mifepris-
tone), indicating high receptor binding affinity as well as
sufficient intracellular availability. Within the series of com-
pounds described in this study, 9 (R ) 3-hydroxypropyl) and
10 (R ) 6-hydroxyhexyl) are regarded as good starting materials
for the synthesis of stable conjugates (without degradation within
cellular environment), which will accumulate in PR-positive
final equation that shows reasonable predictivity (r2 ) 0.73, r2
cv
) 0.62) comprising a final set of 10 descriptors (8 components).
The plot of observed vs predicted (leave one out) log(1/IC50)
values showed only the propanoic acid derivative 13 as a clear
outlier (Figure 3). Important descriptors present in the final
equation include, among others, PEOE-VSA descriptors, Slog-
P_VSA descriptors, and van der Waals area and volume. A full
list with a short comment on the nature of the descriptors is
given in Table 5. As this model covers already a relatively broad
chemical space, it might serve as a useful tool for in silico

Syntheses and Antigestagenic ActiVity of Mifepristones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1273
General Procedure III. After dissolving 20 mg of 14-16, 24,
or 25 (0.038-0.034 mmol) in 2 mL of THF, a 10-fold excess of
diazomethane in 2 mL of diethyl ether was added and the mixture
stirred for 1 h at room temperature. Solvent and excess of
diazomethane solution were removed in vacuo and the residue
dissolved in acetonitrile/water and lyophilized. Compounds 18-20,
26, and 27 were obtained in about 80% yield.
General Procedure IV. Compound 2 (116 mg, 0.26 mmol) and
an excess of the iso(thio)cyanate were dissolved in 3 mL of dry
DMF and stirred for 3 h at 70 °C for. The mixture was poured into
3 mL of ice water; pH of the mixture was adjusted to 2 with 2
mol/L HCl. After extraction with diethyl ether, the organic layer
was washed with water and dried over Na2SO4. The solvent was
removed in vacuo and the residue purified by preparative HPLC.
After lyophilization, colorless solid compounds 28-31 and 33 were
obtained in typically 33% yield.
tumor cells. A promising starting material for the synthesis of
cleavable conjugates seem to be compound 39, which is obtained
by reacting 10 with succinic anhydride. Besides intracellular
accumulation, cleavable conjugates enables additionally the
detachment of the pharmacologically active agent, which seem
to be essential in some cases, e.g., for anticancer agents like
doxorubicin13 or taxol.14 Thus, it is concluded that compounds
9, 10, and 39 are promising and versatile starting materials for
the development of stable or cleavable conjugates for diagnostic
or therapeutic purposes in PR expressing tumors, i.e., breast,
ovarian, and endometrial cancer. Specific targeting of PR-
positive cancer cells may contribute to further improve clas-
sification, diagnosis, stratification, and treatment of cancer
patients.
17ꢀ-Hydroxy-11ꢀ-{4-[1,14,14-trimethyl-11-(2-methylpropyl)-
2,9,12-trioxo-1,3,10-triaza-13-oxa-pentadecyl]-phenyl}-17r-(1-
propinyl)-estra-4,9-dien-3-one (34). Compound 32 (115 mg, 0.2
mmol) and an equimolar amount of triethylamine (20.2 mg, 0.2
mmol) were dissolved in 3 mL of THF under argon at 0 °C. Isobutyl
chloroformate (27.3 mg, 0.2 mmol) was added, and the mixture
was stirred for 40 min at 0 °C. Subsequently, a solution of leucine-
t-butyrate in 0.2 mL THF [the free base was extracted from leucine-
t-butyrate·HCl (37 mg, 0.2 mmol) with NaOH (2 mol/L)] was
added and the mixture stirred for additional 1.5 h. The product was
purified by flash chromatography with EtOAc and by preparative
HPLC (Prontosil 120-5-C 18H; acetonitrile: H2O (67:33, vol/vol).
Yield: 56%. Purity HPLC 100%, tR ) 4.105 min.
17ꢀ-Hydroxy-11ꢀ-[4-(12-hydroxy-1-methyl-9,12-dioxo-1-aza-
8-oxa-dodecyl)-phenyl]-17r-(1-propinyl)-estra-4,9-dien-3-one
(39). Compound 10 (51.6 mg, 0.1 mmol), succinic anhydride (10
mg, 0.1 mmol), 4-(dimethylamino)pyridine16 (7.3 mg, 0.06 mmol),
and an excess of triethylamine (20.2 mg, 0.2 mmol) were dissolved
in 4 mL of CH2Cl2 and refluxed for 5 h. The solvent was removed
in vacuo, the residue suspended in EtOAc, and the reaction mixture
adjusted to pH ) 4.0 by adding HCl (2 mol/L) in MeOH; the
residue was purified by flash chromatography with EtOAc as eluent.
Yield: 45%. Purity HPLC 95.4%, tR ) 5.087 min.
Experimental Section
General. All reagents and solvents for syntheses were purchased
from Sigma-Aldrich, Fluka, or Merck and used without further
purification. Reagent-grade solvents were purified and dried using
standard methods. Solvents of analytical and spectroscopic grade
as well as deuterated NMR solvents were purchased from Merck
and Chemische Fabrik Uetikon, respectively. NMR spectra were
recorded on a Varian Unity Inova 400/600 NMR spectrometer
equipped with a tuneable broadband probe. Purity was determined
by elemental analyses (Mikroanalytisches Laboratorium of the
Institute of Physical Chemistry, University of Vienna, Austria) and/
or HPLC; purity of key target compounds was g95% except
otherwise noted.
N-Alkylation of 2 with 1-Bromo Alkanes and ω-Bromo-1-
alkanols: General Procedure I. Compound 2 (100 mg, 0.24 mmol)
and 3-fold excess of freshly distilled 1-bromo alkane (0.72 mmol)
or ω-bromo-1-alkanol (0.72 mmol) were dissolved in anhydrous
DMF (2.0 mL). The mixture was stirred in the presence of dry
K2CO3 (33.3 mg, 0.24 mmol) and in the absence of moisture for
23 h at 70 °C. The solvent was removed in vacuo and the residue
purified by flash-chromatography (EtOAc:cyclohexane, (3:2, vol/
vol)) and by preparative HPLC. After lyophilization, colorless or
light-yellow solid compounds 6-10 were obtained in 20-55%
yield.
17ꢀ-Hydroxy-11ꢀ-[4-(6-hydroxy-N-methylhexylamino)-phen-
yl]-17r-(1-propinyl)-estra-4,9-dien-3-one (10). Compound 10 was
synthesized according to general procedure I reacting 2 (100 mg,
0.24 mmol) with 6-bromo-1-hexanol (0.72 mmol); the product was
purified by flash-chromatography (EtOAc: cyclohexane, 3:2, vol/
vol)) and preparative HPLC (YMC-Pack ODS-A (C18); acetonitrile:
H2O, 2:3, vol/vol). Yield: 54%. Anal. (C34H45NO3) calcd C 79.18,
H 8.79, N 2.72; found C 78.51, H 8.80, N 3.25.
N-Alkylation of 2 with ω-Bromo Carboxylic Acids: General
Procedure II.15 Compound 2 (150 mg, 0.361 mmol) was dissolved
in 3.6 mL of ethanol and added to a solution of 1.80 mmol ω-bromo
carboxylic acid (dissolved in 3.6 mL water and adjusted to pH 9.25
with 4.3 mmol NaHCO3). The mixture was stirred for 20 h at 70
°C. Thereafter, the pH of the reaction mixture was adjusted to 6.1
by adding 0.7 mL of HCl (2 mol/L) in 10 mL ethanol. The solvent
was removed in vacuo, the residue suspended in a small amount
of water, extracted with EtOAc, and washed with brine. The organic
layer was dried over Na2SO4. The solvent was removed in vacuo,
and the residue purified by flash chromatography with EtOAc and
MeOH and by preparative HPLC. After lyophilization, yellow solid
compounds 13-16 were obtained in 30-55% yield.
Acknowledgment. This work was supported by the Austrian
Cancer Aid/Styria (grant no. 03/2001) and the Federal Ministry
of Education and Research of Germany (grants nos. 01 KS
9605/2 and 13 N 8490). In particular, we are grateful to Bayer
Schering Pharma AG (Germany) for a gift of compound I. We
want also to especially thank Eva Winkler and Petra Kruse,
Institute for Lasertechnology in Medicine and Metrology at the
University of Ulm, for perfect technical assistance.
Supporting Information Available: Preparation, purification,
spectroscopic (1H NMR, 13C NMR, IR, and UV/vis), and chro-
matographic data (TLC, HPLC) of compounds 3-42 as well as a
description of the biological assays. This material is available free
of charge via the Internet at http://pubs.acs.org.
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