Synthesis and investigations on the oxidative degradation of C3/C5-Alkyl-1,2,4-triarylpyrroles as ligands for the estrogen receptor

Article abstract of DOI:10.1002/cmdc.201000537

In this study, we synthesized 1,2,4-triarylpyrroles as ligands for the estrogen receptor (ER). Two pyrrole series were prepared with either C3-alkyl or C3/C5-dialkyl residues. Compounds from both series were susceptible to oxidative degradation-dialkylated compounds (t_1/2=33-66h) to a higher extent than their monoalkylated congeners (t_1/2=140-211h). Nevertheless, stability was sufficient for determination of in vitro ER binding affinity. The most active agonist in hormone-dependent, ERα-positive MCF-7/2a and U2-OS/α cells was 1,2,4-tris(4-hydroxyphenyl)-3-propyl-1H-pyrrole (6d) (MCF-7/2a: EC₅₀=70nM; U2-OS/α: EC₅₀=1.6nM). A corresponding inactivity in U2-OS/β cells demonstrated the high ERα selectivity. This trend was confirmed in a competition experiment using estradiol (E2) and purified hERα and hERβ proteins (relative binding affinity (RBA) calculated for 6d: RBA(ERα)=1.85%; RBA(ERβ) <0.01%). Generally, C3/C5-dialkyl substitution led to reduction of activity, possibly due to lower stability. Triarylpyrroles with C3-alkyl or C3/C5-dialkyl residues were synthesized as ligands for the estrogen receptor (ER). The compounds exhibited transcription activation selectively for ERα but only marginally displaced estradiol from its binding site. The compounds were susceptible to oxidative degradation-dialkylated compounds to a higher extent than their monoalkylated congeners. The reasons for instability were elucidated; thus, by changing the substitution pattern, it will be possible to generate stable triarylpyrroles.

Full text of DOI:10.1002/cmdc.201000537

MED
DOI: 10.1002/cmdc.201000537
Synthesis and Investigations on the Oxidative Degradation
of C3/C5-Alkyl-1,2,4-triarylpyrroles as Ligands for the
Estrogen Receptor
Anja Schꢀfer,[b] Anja Wellner,[b] and Ronald Gust*[a]
In this study, we synthesized 1,2,4-triarylpyrroles as ligands for
the estrogen receptor (ER). Two pyrrole series were prepared
with either C3-alkyl or C3/C5-dialkyl residues. Compounds from
both series were susceptible to oxidative degradation—dialky-
phenyl)-3-propyl-1H-pyrrole (6d) (MCF-7/2a: EC50 =70 nm; U2-
OS/a: EC50 =1.6 nm). A corresponding inactivity in U2-OS/b
cells demonstrated the high ERa selectivity. This trend was
confirmed in a competition experiment using estradiol (E2)
and purified hERa and hERb proteins (relative binding affinity
(RBA) calculated for 6d: RBA(ERa)=1.85%; RBA(ERb) <0.01%).
Generally, C3/C5-dialkyl substitution led to reduction of activity,
possibly due to lower stability.
1
lated compounds (t = =33–66 h) to a higher extent than their
2
1
monoalkylated congeners (t = =140–211 h). Nevertheless, sta-
2
bility was sufficient for determination of in vitro ER binding af-
finity. The most active agonist in hormone-dependent, ERa-
positive MCF-7/2a and U2-OS/a cells was 1,2,4-tris(4-hydroxy-
Introduction
The estrogen receptors ERa[1] and ERb[2] are ligand-inducible
transcription factors that belong to the nuclear hormone re-
ceptor superfamily.[3] ER activation or inhibition influences the
growth, differentiation, and physiological function of several
organs, such as the reproductive tract,[4] bone,[5] liver,[6] and car-
diovascular system.[7,8] Selective ER modulators (SERM) like ra-
loxifene (RAL) or tamoxifen (TAM) are used as partial ERa an-
tagonists in the therapy of osteoporosis and hormone-depen-
dent ERa-positive breast cancer, respectively.
The binding mode of type I estrogens at ERa is well known
from X-ray studies of estradiol (E2) and diethylstilbestrol (DES),
for example, enabling the determination of a structure–activity
relationship (SAR) for gene activation. In addition to a flat hy-
drophobic skeleton, two hydroxy groups should be present
within an OÀO distance of 10.9–12.5 ꢁ. These moieties interact
with the amino acids Arg394/Glu353, and His524 within the
ligand binding domain (LBD) of ERa. Type II estrogens, such as
4,5-bis(4-hydroxypheny)-2-imidazolines,[11] (Scheme 1a) are an-
gular compounds, preventing the possibility of contact with
His524. Instead, hydrogen bridges to Tyr347 or Asp351 in the
LBD side channel are proposed.[10–12]
Conversion of the 4,5-bis(4-hydroxypheny)-2-imidazolines to
4,5-bis(4-hydroxyphenyl)imidazoles[13] (Scheme 1b) increased
the distance between the hydroxy groups from 5.1 ꢁ to 8.8 ꢁ
and reduced the hormonal activity. Chlorine substituents on
the aromatic rings, as well as N-alkylation, counteract these ef-
fects.[14] However, an attempt to increase the ER activation by
introducing a third phenol ring (Scheme 1c) to enable an E2-
like type I binding mode[15,16] was unsuccessful.
The related 2,4,5-tris(4-hydroxyphenyl)imidazoles were only
weak ER agonists, which may be the consequence of nitrogen
atom arrangement in the heterocycle in relation to the phenol
Despite their unquestionable benefits, SERMs may cause se-
rious side effects, including thromboembolisms and endome-
trial cancer,[9] emphasizing the importance of the search for
new derivatives. Over the past years, several substituted het-
erocycles have been synthesized with the aim of developing
ERa/ERb-selective compounds or substances with tissue-selec-
tive activity. Another approach was the design of drugs (type II
estrogens) with an ER binding mode different from that of es-
tradiol (E2, type I estrogen),[10,11] resulting in the recruitment of
other co-activators or co-repressors.
[a] Prof. Dr. R. Gust
Institut fꢀr Pharmazie, Universitꢁt Innsbruck
Innrain 52, 6020 Innsbruck (Austria)
Fax: (+43)512-507-2940
E-mail: ronald.gust@uibk.ac.at
[b] Dr. A. Schꢁfer, A. Wellner
Institut fꢀr Pharmazie, Freie Universitꢁt Berlin
Kçnigin-Luise-Str. 2+4, 14195 Berlin (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000537.
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1,2,4-Triarylpyrroles as Estrogen Receptor Ligands
the appropriate iodoalkane and potassium tert-butanolate in
THF.[19] Reaction of 1 or 2b–d with 2-aminodiethylmalonate in
boiling acetic acid led to pyrrole ring closure (3a–d) in a modi-
fied Fischer–Fink synthesis.[20,21] Ester hydrolysis and decarboxy-
lation in boiling ethylene glycol with KOH as base yielded pyr-
roles 4a–d which were N-arylated (5a–d) in an Ullman-like re-
action in DMF employing cuprous iodide and l-proline.[22,23]
The final ether cleavage (6a–d) was performed with BBr3 in
CH2Cl2 at 08C (Scheme 2).
1-(4-Methoxyphenyl)pentan-2-one (8), a synthon for type A
pyrroles with various alkyl chains at C3 and a propyl moiety at
C5, was obtained from the reaction of 4-methoxyphenyl acetyl
chloride with N-methyl-N-methoxyammonium hydrochloride
(7) and subsequent reaction with propylmagnesium bromide.
This reaction sequence enables the production of ketones by
Grignard reaction (Scheme 3).[24] It was proposed that a stable
metal chelate is formed which keeps the ketone from reacting
to the corresponding tertiary alcohol. This makes it possible to
synthesize ketones from carboxylic acids.[25]
The a-bromoketones (10a–c) were obtained by Friedel–
Crafts acylation of anisole with the respective acyl chloride and
subsequent bromination. Reaction of 8 with the appropriate a-
bromoketone[26] (10a–c) at À788C resulted in the formation of
1,4-diketones 11 a–c,[27] which underwent ring closure in acetic
acid using p-anisidine (Scheme 3). Ether cleavage was carried
out using boron tribromide.
Scheme 1. Structural formulae for substituted heterocycles as ER ligands.
substituents. Analogously substituted pyrazoles exhibited high
affinity for the ER.[16] 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-
pyrazole PPT (Scheme 1), the
most hormonally active com-
pound of this class, exhibited a
410-fold ERa selectivity.[17]
These findings led us to syn-
thesize 1H-pyrroles and investi-
gate their stability in aqueous
solution. The N-position, in rela-
tion to the aryl ring substituents
on the heterocycle, was selected
from the position in PPT result-
ing in type A or type B pyrroles
(Scheme 1). In this paper, we fo-
cused our attention on type A
pyrroles and attempted to opti-
mize their ER binding affinity
using C3- or C3/C5-dialkyl
chains.
Results and Discussion
Chemistry
The synthesis of type A pyrroles
with
a C3-alkyl chain started
from the reaction of 4-methoxy-
methylbenzoate and 4-methoxy-
phenylethanone in DMSO.[18] The
resulting diketone 1 was C-alky-
Scheme 2. Synthesis of type A pyrroles with a C3-alkyl chain. Reagents and conditions: a) dry DMSO, approx. 88C,
NaH; b) reflux, dry THF, tert-BuOK; c) reflux, acetic acid; d) ethylene glycol, KOH, 1858C, 1 h; e) CuI, dry DMF,
lated (2b–d) with 1.1 equiv of 1408C, 3 days; f) dry CH2Cl2, BBr3, 08C.
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1
Table 1. Half life (t = ) of compounds 6b–d and 13a–c.
2
1
1
Residue
Compd
t = [h]
Compd
t = [h]
2
2
Methyl
Ethyl
Propyl
6b
6c
6d
140
211
169
13a
13b
13c
33
66
56
Figure 1. Diagram of the percentage of original peak area (ÆSD of three
separate experiments) over time for compounds 6b (*) and 13c (&); see
Experimental Section for HPLC method.
The degradation products 6b-1 (tR =3.10 min) and 6b-2
(tR =4.54 min) were able to be isolated using semipreparative
HPLC for characterization by NMR, IR, and mass spectrometry.
Compound 6b-1 showed a molecular peak in the ESI-TOF
spectra at m/z=374.1447, indicating the addition of one
oxygen to 6b (m/z=358.1483). In the IR spectra, a strong
band at 1660 cmÀ1 appeared, characteristic of an amide bond
Scheme 3. Synthesis of type A pyrroles with C3/C5-alkyl chains. Reagents
and conditions: a) dry CH2Cl2, 08C, pyridine, 1 h; b) dry THF, 08C, 3 h; c) dry
CH2Cl2, 08C, AlCl3; d) dry diethyl ether, 08C, 1 h; e) dry THF, À788C, KHMDS
solution; f) acetic acid, reflux, p-anisidine, 3 h; g) dry CH2Cl2, BBr3, 08C. *9a
(4-methoxypropiophenone) was commercially available.
1
carbonyl. H NMR spectra indicated the presence of all AA’BB’
systems of the phenolic rings (Figure 2). The pyrrole proton
C5-H (H1) located at d=6.92 (6b) was shifted to the region of
an alkene bond H (d=5.63). The influence on the CH3 group
was relatively low (shift from 2.06 to 1.83 ppm).
Stability studies
Some pyrroles are known to be sensitive towards oxidation, so
we determined the stability of selected compounds in phos-
phate buffered saline (PBS) using HPLC methodology. Stock
solutions in methanol (10À2 m) were diluted with PBS to a final
concentration of 5ꢃ10À5 m, which were incubated at 378C in
the dark to imitate cell culture conditions.
The ESI-TOF mass chromatogram showed the peak for 6b-2
at m/z=404.1544, signifying an additional m/z of 46 as com-
1
pared to 6b. In the H NMR spectra of 6b-2, the AA’BB’ sys-
tems were arranged similarly to those found for 6b-1. The C5-
H was not detected, while a new singlet (3H) appeared at d=
3.22.
The stability of 6b–d and 13a–c (followed over ~200 h; see
Experimental Section for details) was dependent on substitu-
ents of the heterocycle. Degradation by Diels–Alder reactions
with oxygen or photochemical reactions, for example, is very
complex and follows pseudo first-order kinetics.[28,29,30,31] The
half-life of all compounds (Table 1) was calculated from the
peak area over incubation time correlations as depicted in
Figure 1 for 6b and 13c. Alkyl chains influenced the stability
as well, with dialkylated compounds degraded to a higher
degree than their monoalkylated congeners. Furthermore,
ethyl- and propyl-substituted pyrroles were more stable than
their methyl derivatives (see Table 1).
To the best of our knowledge, only one previous publication
details the decomposition of a pyrrole with both alkyl and aryl
residues.[32] Wasserman and Miller analyzed the stability of 1-
methyl-2,3,5-triphenylpyrrole and identified the cis-dibenzoyl-
styrenoxide as an oxidation product (Scheme 4). When the ni-
trogen was phenyl-substituted, the oxidation product did not
decompose to the 1,4-diketone but rather remained as the
phenylimine intermediate (Scheme 4). Such decomposition
products were not identified in the LC-MS spectra of 6b. Fur-
thermore, NMR and IR spectra of 6b-1 and 6b-2 indicated the
absence of an aldehyde or epoxide functionality, respectively.
Therefore, another mechanism of decomposition is likely.
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1,2,4-Triarylpyrroles as Estrogen Receptor Ligands
Figure 2. 1H NMR spectra of 6b and its decomposition products 6b-1 and 6b-2 (in [D6]DMSO).
(OCH3 group at d=3.22). After nucleophilic attack at C5, the
endoperoxide was transformed into a hydroperoxide, which
subsequently eliminated water to form 6b-2 (Scheme 5). The
related C5-OH derivative of 6b-2 (m/z=390.1400) was detect-
ed in LC-MS studies as well, but isolation of this compound
was unsuccessful.
Another potential mechanism for the building of 6b-1 in-
volves ring opening of the endoperoxide by an intramolecular
electron pair shift, resulting in C2- or C5-hydroperoxides that
are transformed in the following step to the corresponding al-
cohols.[36,37] The resulting aminals are stabilized by a hydride or
aryl shift, accompanied by oxidation of the hydroxy group to a
carbonyl. Of the possible products 6b-1, 6b-1a, and 6b-1b,
only 6b-1 was able to be isolated and spectroscopically char-
acterized.
Scheme 4. a) Decomposition of 1-methyl-2,3,5-triphenylpyrrole.[28] b) Decom-
position of 1,2,3,5-tetraphenylpyrrole, both isomers are formed.
LC–MS analysis (ESI-TOF) of the decomposition products
(peak a–f) of 13c indicated three peaks (a–d) with m/z=
460.2103 and two peaks (e and f) with m/z=426.2050 and
428.2215, respectively. Following the studies of Wasser-
man,[32,38] the decomposition products depicted in Figure 3
could be generated, with m/z values in correlation with those
found in LC–MS experiments. Peak e corresponds to the origi-
nal compound 13c.
Pyrroles are electron-rich heterocycles that can react with
oxygen in a Diels–Alder reaction to form labile endoperox-
ides,[33,34] which can be transformed back to pyrroles and mo-
lecular oxygen or undergo ring opening by solvent molecules
or intramolecular rearrangement.[35] Under the conditions used
to analyze 6b, methanol and water were shown to be present.
Indeed, compound 6b-2 appears to be a methanol adduct
The proposed degradation modes discussed above, begin-
ning in each case with a Diels–Alder reaction of the pyrrole
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R. Gust et al.
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(they have conjugated p-electron systems and are, to
a certain degree, rigid) and thus facilitate their own
decomposition.
Biological activity
Transactivation assays: MCF-7/2a cells
Hormone-dependent, ERa-positive MCF-7 breast
cancer cells were stably transfected with the reporter
plasmid EREwtcluc (MCF-7/2a cells)[39] to study ERa-as-
sociated transcription activation in hormone-compe-
tent cells. U2-OS cells transiently transfected with the
plasmid pSG5-ERa (U2-OS/a), or pSG5-ERb FL (U2-
OS/b) and the reporter plasmid p(ERE)2-luc+, were
used to evaluate ER subtype selectivity.[40] The trans-
activation and intrinsic activity (IA) of type A pyrroles
with only a C3-alkyl chain (6a–d) at 10 mm in MCF-7/
2a cells increased in the order R=H (6a; EC50 >
10000 nm; IA=27%) <methyl (6b; EC50 =440 nm;
IA=68%) <ethyl (6c; EC50 =140 nm; IA=96%) <
propyl (6d; EC50 =70 nm; IA=99%) (Figure 4 and
Table 2). Introduction of a second alkyl chain at C5
(13a–c) led to compounds which exhibited maxi-
mum transcription at 1 mm. This transcription de-
creased at higher concentrations (Figure 4), which is
likely due to reduced cell mass at 10 mm, as these
compounds more strongly inhibited the growth of
MCF-7 cells (see below, “inhibition of cell growth”)
than did their monoalkylated congeners.
Transactivation assays: U2-OS cells
Performing the same experiments with U2-OS/a cells
indicated a higher sensitivity towards hormones. E2
was 25-fold more active in this assay (EC50 (ERa)=
0.004 nm). Again, maximum effect was achieved with
ethyl and propyl derivatives 6c and 6d (EC50 =1.1
and 1.6 nm, respectively). Interestingly, and contrary
to results obtained in MCF-7/2a cells, dialkylated
Scheme 5. Proposed decomposition of 6b.
compounds 13a–c induced gene activation in a
chain length-dependent manner (ERa, Table 2):
methyl (13a; EC50 =45 nm) <ethyl (13b; EC50 =
with singlet oxygen (1O2), generated from triplet oxygen (3O2)
by photo- or heat-activation, was confirmed by incubation of
6b under O2-free conditions. HPLC analysis demonstrated no
degradation over an incubation time of 200 h (data not
shown) after degassing the aqueous solutions in an ultrasound
bath and purging with dry argon for 10 min. Further support is
shown by the lower stability of C3/C5-dialkylated type A pyr-
roles (13a–c). The additional alkyl chain raised the electron
density at the diene and enhanced the kinetic reaction with
1O2. This finding is the subject of a current SAR study using
electron withdrawing groups at pyrrole carbons to stabilize
the pyrrole core. In this context, it should be mentioned that
type A pyrroles fulfill the requirements for photosensitizers
27 nm) <propyl (13c; EC50 =20 nm). Compounds 6a–d were
inactive against U2-OS/b cells and did not induce ERb activa-
tion, indicating high ERa selectivity. An additional alkyl chain
at C5 led to a complete loss of ER subtype selectivity (Table 2).
To explore the potential participation of oxidation products
6b-1 and 6b-2 on the transcriptional activity of 6b, they were
also tested in MCF-7/2a cells. As depicted in Figure 5, 6b-2
was completely inactive, and 6b-1 possessed estrogenic activi-
ty but to a lower extent (EC50 =4700 nm) than 6b. Therefore,
the effect of degradation products on transcriptional activity
cannot be excluded. However, based on the stability studies
mentioned above, application of the compounds was carried
out in such a way that degradation was minimized (freshly pre-
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1,2,4-Triarylpyrroles as Estrogen Receptor Ligands
domain (LBD) of both subtypes.
In contrast to the C3-methyl
(6b) and C3-ethyl (6c) deriva-
tives, C3-H (6a) and C3-propyl
(6d) analogues did not compete
with [3H]E2 for the binding site
in ERb at the concentrations
used. An additional C5-alkyl resi-
due at 6d reduced the relative
binding affinity (RBA) at ERa to
about 0.15–0.30% without in-
creasing affinity for ERb (13a–c).
The higher ERa activity of PPT
compared to 6d, as determined
in cellular assays, was confirmed
in the HAP assay, however with
only a fivefold difference in the
RBA values [RBA(PPT)=10.14%;
RBA(6d)=1.85%].
As the compounds strongly
activated ERa transcription but
only weakly competed with
[3H]E2, it is unclear at this stage
whether the compounds exert
their effects at the receptor pro-
tein. It is possible that the pyr-
roles interact with ERa/b hetero-
dimers, as these are not tested
in our binding assay. Other pos-
sibilities include interaction with
one of the ER coregulators, or
binding at an ER domain other
than the LBD. Additionally, the
compounds may be type II estro-
gens, in which case their differ-
ent binding mode would explain
the very weak binding affinities
in the [3H]E2 displacement assay.
Currently, docking studies are
under way to explore the rea-
sons for the difference in bind-
ing at ERa/b.
Figure 3. Possible decomposition products of 13c.
Table 2. Transactivation of compounds 6a–d and 13a–c as compared to estradiol (E2) in luciferase reporter
assays with MCF-7/2a cells and U2-OSa/b cells.
MCF-7/2a
EC50 [nm]
U2-OS/a
EC50 [nm]
U2-OS/b
EC50 [nm]
Compd
IA [%][a]
IA [%][c]
IA [%]
E2
100
0.1
100
42
107
88
111
85
76
125
119
n.d.
0.004
n.a.
16
1.1
1.6
45
27
20
0.1
n.d.
100
5
5
3
0
53
88
103
5
0.01
–
–
–
–
n.a.
37
20
–
n.d.
6a
6b
6c
6d
13a
13b
13c
PPT
6b-1
27Æ2
68Æ1
96Æ8
99Æ18
34Æ9[b]
58Æ6[b]
61Æ3[b]
97Æ6
68Æ15
>10000
440
140
70
n.a.
n.a.
n.a.
3
4700
n.d.
[a] at 10 mm. [b] at 1 mm. [c] at 1 mm. Estradiol is defined as 100%. –: no estrogenic activity; IA: intrinsic activity;
n.a.: not applicable due to growth inhibitory effects; n.d.: not determined. For MCF-7/2a cells, the mean values
are given ÆSD of three independent determinations performed in quadruplicate. For U2-OS cells transiently
transfected with ERa or Erb, values represent the mean of three independent determinations (SD is <20%).
Inhibition of cell growth
pared oxygen-free stock solutions and concentrations lower
than 10 mm).
Because of the results in MCF-7/2a cells, all compounds were
tested for growth inhibitory effects against hormone-depen-
dent MCF-7 and hormone-independent MDA-MB-231 breast
cancer cells. As expected, dialkylated pyrroles (13a–c, IC50 =6–
10 mm) influenced the growth of MCF-7 cells more effectively
than their monoalkylated congeners (6b–d, IC50 =28–33 mm)
(Table 4). Nearly identical results in hormone-insensitive MDA-
MB-231 cells suggested an ER-independent interference in cell
growth. Interestingly, 6b was nearly three times more toxic in
MDA-MB-231 cells than in MCF-7 cells.
Binding affinity
To gain more insight into the mode of action, competition of
the pyrroles for the [3H]E2 binding site was evaluated in a hy-
droxylapatite (HAP) assay using hERa and hERb (Table 3). Sub-
type selectivity observed in the cellular systems could not be
confirmed with purified ER proteins for 6b and 6c. Both com-
pounds weakly displaced [3H]E2 from the ligand binding
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Table 3. Relative binding affinities (RBA) at hERa and hERb as determined
by HAP assay.[a]
Compd
RBA [%]
ERa
100
ERb
100
<0.01
0.19
E2
6a
6b
6c
6d
13a
13b
13c
PPT
0.56
1.67
0.87
1.85
0.29
0.15
0.14
10.14
0.38
<0.01
<0.01
<0.01
<0.01
0.53
[a] Values represent the mean of two independent determinations in du-
plicate; results are reproducible within Æ20% (see Experimental Section
for conditions).
Table 4. Growth inhibitory effects of type A pyrroles against MCF-7 and
MDA-MB-231 cells.[a]
IC50 [mm]
Residue
Methyl
Ethyl
Cell line
MCF-7
MDA-MB-231
MCF-7
MDA-MB-231
MCF-7
MDA-MB-231
6b–d
13a—c
33.1Æ2.0
12.0Æ1.5
28.3Æ1.9
24.8Æ1.6
29.4Æ2.6
26.4Æ2.6
7.9Æ2.0
12.3Æ0.3
10.0Æ2.8
16.4Æ2.9
6.3Æ1.9
6.1Æ0.3
Propyl
[a] Values represent the mean ÆSD of three independent determinations
(see Experimental Section for details).
Figure 4. Activation–concentration curves for type A pyrroles. a) Type A pyr-
roles with a C3-alkyl residue, with PPT for comparison. Estradiol, &; 6a,
c&c; 6b, *; 6c, ~; 6d, "; PPT, ^; b) Type A pyrroles with C3/C5-
alkyl residues. Estradiol, &; 13a *; 13b, ~; 13c, "; All pyrroles were
tested in a luciferase activation assay using MCF-7/2a cells stably transfected
with plasmid EREwtcluc. The curves are representative examples of one deter-
mination performed in quadruplicate (see Experimental Section for details).
Conclusions
Type A pyrroles were synthesized and analyzed for stability in
aqueous solution (comparable to cell culture conditions) as
well as for gene activation in ERa-positive MCF-7/2a cells and
in U2-OSa/b cells. Alkyl chains at C3/C5 determined the stabili-
ty and the biological effects. C3-alkyl derivatives were more
stable than C3/C5-disubstituted type A pyrroles and activated
the ER protein to a higher extent. Therefore, it is necessary to
increase stability and to exclude possible degradation in cell
culture experiments. This can be attained by introduction of
electron-withdrawing substituents at the phenolic rings and
changing the N-atom position in relation to the substituents of
the heterocycle. These results will be published in forthcoming
papers.
Experimental Section
General procedures: Solvents were purified according to literature
procedures or purchased in pure form. Syntheses of precursors are
described in Supporting Information. IR spectra (KBr pellets) were
obtained using an ATI Mattson Genesis and 1H NMR using an
Avance DPX 400 spectrometer (Bruker, Karlsruhe) at 400 MHz (in-
ternal standard: TMS). All aromatic d are “apparent” doublets. Ele-
mental analysis was performed on a Vario EL (Elementar, Hanau).
Purity of compounds 6a–d and 13a–c was confirmed by elemental
Figure 5. Estrogenic activity of 6b-1 (!) and 6b-2 (^) as compared to 6b
(*), estradiol as control: &.
800
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ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 794 – 803

1,2,4-Triarylpyrroles as Estrogen Receptor Ligands
analysis, with all being >95% (see Supporting Information). Mass
spectrometry (EI) was performed on a CH-7A (Varian MAT Bremen,
Germany), with ESI–TOF for 13c, 6b-1 and 6b-2 on an Agilent
6210 ESI–TOF (Agilent Technologies, Santa Clara, CA, USA). The sol-
vent flow rate was adjusted to 4 mLminÀ1 with the spray voltage
set to 4 kV and the drying gas flow rate set to 15 psi (1 bar). All
other parameters were adjusted for a maximum abundance of the
relative [M+H]+. A Flashscan S12 Microplate Reader (Analytik Jena,
Germany), Wallac Victor2 1420 multilabel counter (Perkin–Elmer,
USA), and Wallac Microbeta TriLux scintillation counter (Perkin–
Elmer, USA) were used.
ArH), 6.85 (s, 1H, CH), 6.76 (d, 2H, J=8.6 Hz, ArH), 6.67 (d, 2H, J=
8.6 Hz, ArH), 6.63 (d, 2H, J=8.8 Hz, ArH), 2.46 (t, 2H, J=8.0 Hz,
CH2CH2CH3), 1.25 (sextet, 2H, J=7.5 Hz, CH2CH2CH3), 0.67 ppm (t,
3H, J=7.3 Hz, CH2CH2CH3); MS (EI, 1708C): m/z (%)=385 [M]+· (83),
356 (100), 330 (16), 193 (7), 131 (7), 77 (3). MS (ESI-TOF): m/z:
386.1740; IR (KBr): n˜ =3370(b,s), 2955/2928(m), 1886(w), 1613(m),
1562 (m), 1449(m), 1388(b,m), 1229(b,s), 1170(m), 1098(w),
835(s) cmÀ1; Anal. calcd for C25H23NO3·0.75H2O: C, H, N.
1,2,4-Tris(4-hydroxyphenyl)-3-methyl-5-propyl-1H-pyrrole (13a):
BBr3 (3.5 mmol, 888 mg) was added to 12a (0.71 mmol, 313 mg)
following Method F to yield 13a as a red solid (0.18 mmol, 0.07 g,
1
25%): mp: 868C; H NMR [(D6)DMSO]: d=9.55 (s, 1H, OH), 9.28 (s,
1H, OH), 9.26 (s, 1H, OH), 7.09 (d, 2H, J=8.4 Hz, ArH), 6.93 (d, 2H,
J=8.6 Hz, ArH), 6.85 (d, 2H, J=8.5 Hz, ArH), 6.78 (d, 2H, J=8.5 Hz,
ArH), 6.68 (d, 2H, J=8.6 Hz, ArH), 6.57 (d, 2H, J=8.5 Hz, ArH), 2.35
(t, 2H, J=7.8 Hz, CH2CH2CH3), 1.87 (s, 3H, CH3), 1.08 (m, 2H,
CH2CH2CH3), 0.53 ppm (t, 3H, J=7.3 Hz, CH2CH2CH3); MS (EI, 508C):
m/z (%)=399 [M]+· (54), 370 (100), 343 (16), 262 (6); IR (KBr): n˜ =
3393(b,s), 2959(m), 1889(w), 1700(s), 1595(m), 1512(s), 1443(s),
1374(s), 1261(b, s), 1171(s), 1101(m), 1044(m), 830(s) cmÀ1; Anal.
calcd for C26H25NO3·2H2O: C, H, N.
Synthesis
General method for ether cleavage (Method F, Schemes 2 and 3):
The respective 1,2,4-tris(4-methoxyphenyl)-1H-pyrrole (5a–d) was
dissolved in 15 mL of dry methylene chloride under inert gas and
cooled to 08C. Boron tribromide (BBr3) dissolved in CH2Cl2 (5 mL)
was added slowly, and the reaction mixture stirred overnight at
room temperature. After evaporation of the solvent, redissolving
several times in MeOH, purification of the residue was performed
on silica gel by column chromatography using CH2Cl2/MeOH (9:1).
3-Ethyl-1,2,4-tris(4-hydroxyphenyl)-5-propyl-1H-pyrrole
(13b):
1,2,4-Tris(4-hydroxyphenyl)-1H-pyrrole (6a): BBr3 (2.2 mmol,
552 mg) was added to 5a (0.44 mmol, 170 mg) following Method F
to produce 6a as a yellow solid (0.4 mmol, 138 mg, 91%): mp:
BBr3 (2.0 mmol, 489 mg) was added to 12b (0.39 mmol, 178 mg)
following Method F to yield 13b as a reddish orange solid
(0.11 mmol, 0.045 g, 28%): mp: 1218C; 1H NMR [(D6)DMSO]: d=
9.52 (s, 1H, OH), 9.28 (s, 1H, OH), 9.26 (s, 1H, OH), 7.09 (d, 2H, J=
8.4 Hz, ArH), 6.93 (d, 2H, J=8.6 Hz, ArH), 6.87 (d, 2H, J=8.4 Hz,
ArH), 6.77 (d, 2H, J=8.4 Hz, ArH), 6.66 (d, 2H, J=8.6 Hz, ArH), 6.57
(d, 2H, J=8.5 Hz, ArH), 2.30 (m, 4H, CH2CH3 and CH2CH2CH3), 1.06
(m, 2H, CH2CH2CH3), 0.73 (t, 3H, J=7.3 Hz, CH2CH2CH3), 0.53 ppm
(t, 3H, J=7.3 Hz, CH2CH2CH3); MS (EI, 1508C): m/z (%)=413 [M]+·
(61), 384 (100), 357 (18), 206 (4). IR (KBr): n˜ =3403(b,m), 2960(m),
1891(w), 1610(m), 1512(s), 1443(m), 1374(m), 1342(w), 1230(b,m),
1
119 8C; H NMR [(D6)DMSO]: d=9.60 (s, 1H, OH), 9.40 (s, 1H, OH),
9.21 (s, 1H, OH), 7.40 (d, 2H, J=8.6 Hz, ArH), 7.17 (d, 1H, J=2.0 Hz,
CH), 7.00 (d, 2H, J=8.7 Hz, ArH), 6.92 (d, 2H, J=8.6 Hz, ArH), 6.74
(d, 2H, J=8.4 Hz, ArH), 6.73 (d, 2H, J=8.4 Hz, ArH), 6.63 (d, 2H, J=
8.6 Hz, ArH), 6.51 ppm (d, 1H, J=2.0 Hz, CH); MS (EI, 2508C): m/z
(%)=343 [M]+· (100), 293(6), 229(6), 224(15), 172(8), 149(25); IR
˜
(KBr): n=3390 (b,s), 1889(w), 1613(m), 1589(m), 1516/1504(s),
1235(b,m), 1171(m), 1102(w), 836(s), 801(m) cmÀ1; Anal. calcd for
C22H17NO3·0.8H2O: C, H, N.
1171(m), 1101(w), 1013(w), 830(m) cmÀ1
C27H27NO3·2.25H2O: C, H, N.
;
Anal. calcd for
1,2,4-Tris(4-hydroxyphenyl)-3-methyl-1H-pyrrole
(6b):
BBr3
(1.9 mmol, 470 mg) was added to 5b (0.38 mmol, 150 mg) follow-
ing Method F to produce 6b as a yellow solid (0.24 mmol, 85 mg,
63%): mp: 1198C; H NMR [(D6)DMSO]: d=9.44 (s (broad), 3H, OH),
7.27 (d, 2H, J=8.5, ArH), 6.89 (m, 5H, 4ArH, 1CH), 6.78 (d, 2H, J=
8.5 Hz, ArH), 6.67 (m, 4H, ArH), 2.06 ppm (s, 3H, CH3); MS (EI,
2308C): m/z (%)=357 [M]+· (100), 238 (8), 223 (18), 179 (6), 107 (4),
1,2,4-Tris(4-hydroxyphenyl)-3,5-dipropyl-1H-pyrrole (13c): BBr3
(1.6 mmol, 400 mg) was added to 12c (0.32 mmol, 150 mg) follow-
ing Method F to yield 13c as a red solid (0.023 mmol, 0.01 g, 7%):
1
1
mp: 1158C; H NMR [(D6)DMSO]: d=9.52 (s, 1H, OH), 9.27 (s, 1H,
OH), 9.25 (s, 1H, OH), 7.07 (d, 2H, J=8.4 Hz, ArH), 6.91 (d, 2H, J=
8.6 Hz, ArH), 6.84 (d, 2H, J=8.5 Hz, ArH), 6.75 (d, 2H, J=8.4 Hz,
ArH), 6.64 (d, 2H, J=8.6 Hz, ArH), 6.55 (d, 2H, J=8.5 Hz, ArH), 2.27
(m, 4H, CH2CH2CH3 und CH2CH2CH3), 1.06 (m, 4H, CH2CH2CH3 and
CH2CH2CH3), 0.57 (t, 3H, J=7.3 Hz, CH2CH2CH3), 0.51 ppm (t, 3H,
J=7.3 Hz, CH2CH2CH3); MS (EI, 1708C): m/z (%)=427 [M]+· (64), 398
(100), 371 (17), 213 (5); IR (KBr): n˜ =3399(b,m), 2957(m), 1889(w),
1611(m), 1512(s), 1444(m), 1376(m), 1342(w), 1229(b,m), 1170(m),
1100(w), 1012(w), 828(m) cmÀ1; Anal. calcd for C28H29NO3·1.5H2O: C,
H, N.
˜
65 (5); MS (ESI-TOF): m/z: 358.1483; IR (KBr): n=3392(b,s), 1887(w),
1613(w), 1565 (w), 1514(s), 1217(b,m), 1171(m), 1101(w),
834,(s) cmÀ1; Anal. calcd for C23H19NO3·H2O: C, H, N.
3-Ethyl-1,2,4-tris(4-hydroxyphenyl)-1H-pyrrole
(6c):
BBr3
(2.4 mmol, 606 mg) was added to 5c (0.48 mmol, 200 mg) follow-
ing Method F to yield 6c as a yellow solid (0.35 mmol, 130 mg,
1
73%): mp: 988C; H NMR [(D6)DMSO]: d=9.46 (s, 1H, OH), 9.42 (s,
1H, OH), 9.25 (s, 1H, OH), 7.26 (d, 2H, J=8.5 Hz, ArH), 6.90 (m, 4H,
ArH), 6.86 (s, 1H, CH), 6.77 (d, 2H, J=8.7 Hz, ArH), 6.67 (d, 2H, J=
8.6 Hz, ArH), 6.64 (d, 2H, J=8.8 Hz, ArH), 2.63 (q, 2H, J=7.5 Hz,
CH2CH3), 0.87 ppm (t, 3H, J=7.4 Hz, CH2CH3); MS (EI, 1708C): m/z
(%)=371 [M]+· (100), 356 (49), 342 (19), 186 (7), 121 (6), 65 (9); IR
Analytical data for 6b-1 and 6b-2: 6b-1: Grey solid; 1H NMR
[(D6)DMSO]: d=9.57 (s, 1H, OH), 9.43 (s, 1H, OH), 9.24 (s, 1H, OH),
7.35 (d, 2H, J=8.6 Hz, ArH), 7.25 (d, 2H, J=8.9 Hz, ArH), 7.01 (d,
2H, J=8.4 Hz, ArH), 6.82 (d, 2H, J=8.6 Hz, ArH), 6.68 (d, 2H, J=
8.5 Hz, ArH), 6.64 (d, 2H, J=8.9 Hz, ArH), 5.63 (s, 1H, CH), 1.83 ppm
(s, 3H, CH3); MS (EI, 2258C): m/z (%)=373 [M]+· (52), 358 (51), 280
(4), 252 (5), 212 (24), 135 (37), 121 (56); MS (ESI-TOF): m/z:
374.1447; IR (KBr): n˜ =3250(b,s), 2880(m), 1870(w), 1720(m),
1640(s), 1590(s), 1500(s), 1430(s), 1390(m), 1360(m), 1210(b, s),
1155(s), 1100(m), 1130(w), 830(s) cmÀ1. 6b-2: Grey solid; 1H NMR
[(D6)DMSO]: d=9.65 (s, 1H, OH), 9.47 (s, 1H, OH), 9.30 (s, 1H, OH),
7.42 (d, 2H, J=8.5 Hz, ArH), 7.12 (d, 2H, J=8.5 Hz, ArH), 7.07 (d,
˜
(KBr): n=3430(b,s), 1889(w), 1613(m), 1512 (m), 1445(m), 1229(b,s),
1172(m), 1101(w), 835(s) cmÀ1; Anal. calcd for C24H21NO3·0.5H2O: C,
H, N.
1,2,4-Tris(4-hydroxyphenyl)-3-propyl-1H-pyrrole
(6d):
BBr3
(1.6 mmol, 410 mg) was added to 5d (0.33 mmol, 140 mg) follow-
ing Method F to yield 6d as a yellow solid (0.14 mmol, 54 mg,
1
43%): mp: 808C; H NMR [(D6)DMSO]: d=9.45 (s, 1H, OH), 9.41 (s,
1H, OH), 9.25 (s, 1H, OH), 7.24 (d, 2H, J=8.5 Hz, ArH), 6.89 (m, 4H,
ChemMedChem 2011, 6, 794 – 803
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www.chemmedchem.org
801

R. Gust et al.
MED
2H, J=8.8 Hz, ArH), 6.84 (d, 2H, J=8.6 Hz, ArH), 6.67 (d, 2H, J=
8.7 Hz, ArH), 6.61 (d, 2H, J=8.9 Hz, ArH), 3.22 (s, 3H, OCH3),
1.72 ppm (s, 3H, CH3); MS (EI, 2508C): m/z (%)=403 [M]+· (1), 373
(38), 358 (39), 284 (6), 252 (4), 212 (22), 135 (71), 121 (32); MS (ESI-
TOF): m/z: 404.1544; IR (KBr): n˜ =3250(b,s), 2900(m), 1870(w),
1730(m), 1660(s), 1600(s), 1500(s), 1430(s), 1390(m), 1360(m),
1330(m), 1210(b, s), 1190(s), 1150(m), 1030(m), 1010(m), 880(w),
pounds). After 18 h incubation, medium was removed and cells
were lysed and luciferase activity assayed as described above.
Estrogen receptor binding: recombinant ERa/ERb (HAP assay): An
ethanol solution (250 fmolmLÀ1) of recombinant human estrogen
receptor (hERa or hERb, Calbiochem) was diluted with Tris-HCl
buffer, pH 8.0 (1:100). These hERa or hERb preparations were ad-
sorbed on hydroxylapatite pellets (prepared in 10 mmolLÀ1 of Tris-
HCl buffer, pH 8.0, containing 1 mgmLÀ1 BSA) which were labeled
840(s) cmÀ1
.
with increasing concentrations of the test compounds (10À10
–
10À5 m) or [3H]E2 (5 nm, Perkin–Elmer) and were incubated for 3 h
at room temperature. Bound [3H]E2 was then successively extract-
ed with EtOH and aliquots of 200 mL of the ethanol extracts were
transferred to scintillation vials containing 3.5 mL of scintillator
Ecoscint H (National Diagnostics, Atlanta) for radioactivity counting.
All measurements were performed in duplicate. The results (rela-
tive radioactivity) were expressed as the percentage of a solvent
treated control sample. Relative concentrations of compounds re-
quired to reduce the binding of [3H]E2 by 50% were determined
as their IC50 values. Relative binding affinities were calculated by di-
viding the IC50 of E2 by the IC50 of the respective compound; there-
fore, binding affinities are expressed relative to estradiol (RBA(E2)=
100%).
Stability testing
For HPLC (Bio-Tek Kontron Instruments, Germany) a C18 reverse
phase column (Eurospher 100–5 C18, 250ꢃ4 mm, Knauer GmbH,
Germany) was used, with water/MeOH (3:7) in an isocratic elution
with a detection wavelength of 265 nm and a flow rate of
0.8 mLminÀ1 over 20 min. Compounds were dissolved in MeOH
(10À2 m) and diluted with PBS to a final concentration of 5ꢃ10À5 m.
Compounds were kept at 378C and were measured once directly
after dissolving and six more times during the following 195 h.
Biological activity
Cell lines and growth conditions: MCF-7/2a cells, MCF-7 cells, and
MDA-MB-231 cells were maintained as monolayer cultures at 378C
in a humidified atmosphere (95% air, 5% CO2) in T-25 flasks. U2-OS
cells were maintained as a monolayer culture at 378C in humidified
atmosphere (92.5% air, 7.5% CO2) in T-25 flasks. Phenol red-free
high glucose DMEM with sodium pyruvate (110 mgLÀ1), 5% dex-
tran charcoal-treated FCS (ct-FCS), and 0.5% geneticin solution was
used as growth media for the MCF-7/2a cells, while l-glutamine
and high glucose DMEM containing sodium pyruvate and 5% FCS
was used for U2-OS, MCF-7, and MDA-MB-231 cells.
Growth inhibition of MCF-7 and MDA-MB-231 human breast cancer
cells: Cells from an 80% confluent monolayer were harvested by
trypsinization and suspended to approximately 104 (MCF-7 cells) or
7.5ꢃ103 cellsmLÀ1 (MDA-MB-231 cells). At the beginning of the ex-
periment, the cell suspension was transferred to 96-well micro-
plates (100 mL per well). After cultivating for 3 days under growth
conditions, the test compounds (dissolved in DMEM) were added
in final concentrations ranging from 3.13 mm–50 mm. Control wells
(16 per plate) contained 0.1% DMSO, which was used for the prep-
aration of stock solutions. Initial cell density was determined by ad-
dition of glutaric dialdehyde to one plate (1% in PBS; 100 mL per
well). After incubation for three (MDA-MB-231 cells) or four (MCF-7
cells) days, the medium was removed, and glutaric dialdehyde (1%
in PBS; 100 mL per well) was added for fixation. After 30 min, the
aldehyde solution was decanted and 150 mL PBS per well was
added. The plates were stored at 48C prior to staining of the cells
by treating them for 30 min with 100 mL of an aqueous crystal
violet solution (0.02%). After decanting, cells were washed several
times with water to remove the adherent dye. After addition of
180 mL EtOH (70%), plates were gently shaken for 3–4 h. Optical
density of each well was measured using a microplate autoreader
at 590 nm.
Transactivation assay with MCF-7/2a cells: Four days before begin-
ning the experiment, MCF-7/2a cells were cultivated in DMEM sup-
plemented with ct-FCS (50 mLLÀ1). Cells from an 80% confluent
monolayer were removed by trypsinization and suspended to ap-
proximately 105 cellsmLÀ1 in the growth medium mentioned
above. The cell suspension was then cultivated in 96-well flat-bot-
tomed plates at growing conditions (see above). After 24 h, test
compounds were added in concentrations ranging from 10À5
–
10À11 m (estradiol 10À7–10À13 m), and the plates were incubated for
50 h. Subsequently, 50 mL of cell culture lysis reagent was added to
each well. After 30 min of lysis at room temperature, 50 mL of the
Promega luciferase assay reagent (Promega, Mannheim, Germany)
were added. Luminescence in relative light units (RLU) was mea-
sured for 10 s using a luminometer. Estrogenic activity was ex-
pressed as the percentage of activation of a 3ꢃ10À9 m estradiol
control (100%).
Acknowledgements
The technical assistance of Silke Bergemann is gratefully ac-
knowledged.
Transactivation assay with U2-OS cells: U2-OS cells were transferred
to DMEM supplemented with ct-FCS 24 h before the start of the
experiment. Cells from a nearly confluent monolayer were split
and seeded in 10 cm Ø Petri dishes at a concentration of 1ꢃ
106 cells per dish at least 24 h prior to transfection. Transient trans-
fection of the cells with 0.05 mg of receptor plasmid pSG5-ERa or
pSG5-ERb FL and 5 mg of reporter plasmid p(ERE)2-luc+ was carried
out after 6 h using 15 mL Fugene 6 (Roche Diagnostics, Mannheim,
Germany) according to the manufacturer’s instructions. After 18 h,
the cells were washed with PBS, harvested by trypsinization, and
seeded in white 96-well plates at a concentration of 104 cells per
well in 100 mL of ct-DMEM. After 3 h, the medium was replaced by
medium containing either E2 or test compounds in final concentra-
tions ranging from 0.1 pm–10 nm (E2) or 0.1 nm–10 mm (test com-
Keywords: autoxidation
·
cytotoxicity
·
drug design
·
hormones · nitrogen heterocycles
[1] G. L. Greene, M. F. Press, J. Steroid Biochem. 1986, 24, 1.
[2] E. Enmark, M. Pelto-Huikko, K. Grandien, S. Lagercrantz, J. Lagercrantz,
G. Fried, M. Nordenskjold, J.-ꢁ. Gustafsson, J. Clin. Endocrinol. Metab.
1997, 82, 4258.
[3] M. G. Parker, Curr. Opin. Cell Biol. 1993, 5, 499.
[4] J. F. Couse, K. S. Korach, Endocr. Rev. 1999, 20, 358.
[5] Q. Qu, M. Perala-Heape, A. Kapanen, J. Dahllund, J. Salo, H. K. Vaananen,
P. Harkonen, Bone 1998, 22, 201.
802
www.chemmedchem.org
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 794 – 803

1,2,4-Triarylpyrroles as Estrogen Receptor Ligands
[6] C. H. Lee, A. M. Edwards, Carcinogenesis 2001, 22, 1473.
[7] P. A. Komesaroff, K. Sudhir, Reprod. Fertil. Dev. 2001, 13, 261.
[8] S. Hulley, D. Grady, T. Bush, C. Furberg, D. Herrington, B. Riggs, E. Vit-
tinghoff, for the Heart and Estrogen/Progestin Replacement Study Re-
search Group, JAMA 1998, 280, 605.
[9] B. Fisher, J. P. Costantino, D. L. Wickerham, C. K. Redmond, M. Kavanah,
W. M. Cronin, V. Vogel, A. Robidoux, N. Dimitrov, J. Atkins, M. Daly, S.
Wieand, E. Tan-Chiu, L. Ford, N. Wolmark, J. Natl. Cancer Inst. 1998, 90,
1371.
[24] U. Ghosh, D. Ganessunker, V. J. Sattigeri, K. E. Carlson, D. J. Mortensen,
B. S. Katzenellenbogen, J. A. Katzenellenbogen, Bioorg. Med. Chem.
2003, 11, 629.
[25] S. Nahm, S. M. Weinreb, Tetrahedron Lett. 1981, 22, 3815.
[26] C. H. Park, R. S. Givens, J. Am. Chem. Soc. 1997, 119, 2453.
[27] D. S. Mortensen, A. L. Rodriguez, K. E. Carlson, J. Sun, B. S. Katzenellen-
bogen, J. A. Katzenellenbogen, J. Med. Chem. 2001, 44, 3838.
[28] R. H. Young, N. Chinh, C. Mallon, Ann. N. Y. Acad. Sci. 1970, 171, 130.
[29] R. H. Young, R. L. Martin, N. Chinh, C. Mallon, R. H. Kayser, Can. J. Chem.
1972, 50, 932.
[10] V. C. Jordan, J. M. Schafer, A. S. Levenson, H. Liu, K. M. Pease, L. A.
Simons, J. W. Zapf, Cancer Res. 2001, 61, 6619.
[30] A. Wassermann, J. Chem. Soc. 1935, 828.
[11] R. Gust, R. Keilitz, K. Schmidt, J. Med. Chem. 2001, 44, 1963.
[12] P. M. Kekenes-Huskey, I. Muegge, M. von Rauch, R. Gust, E.-W. Knapp,
Bioorg. Med. Chem. 2004, 12, 6527.
[13] R. Gust, R. Keilitz, K. Schmidt, M. von Rauch, Arch. Pharm. Chem. Life Sci.
2002, 335, 463.
[14] M. von Rauch, M. Schlenk, R. Gust, J. Med. Chem. 2004, 47, 915.
[15] T. Wiglenda, R. Gust, J. Med. Chem. 2007, 50, 1475.
[16] B. E. Fink, D. S. Mortensen, S. R. Stauffer, Z. D. Aron, J. A. Katzenellenbo-
gen, Chem. Biol. 1999, 6, 205.
[31] L. J. Andrews, R. M. Keefer, J. Am. Chem. Soc. 1955, 77, 6284.
[32] H. H. Wasserman, A. H. Miller, J. Chem. Soc. Chem. Commun. 1969, 199.
[33] D. A. Lightner, C.-S. Pak, J. Org. Chem. 1975, 40, 2724.
[34] K. Gollnick, G. O. Schenck in Organic Chemistry, A series of monographs:
1,4-Cycloaddition reactions Vol. 8 (Ed.: J. Hamer), Academic press, NY
and London, 1967.
[35] G. B. Quistad, D. A. Lightner, Tetrahedron Lett. 1971, 12, 4417.
[36] G. Rio, A. Ranjon, O. Pouchot, C. R. Hebd. Seances Acad. Sci. 1966, 263,
634.
[17] S. R. Stauffer, C. J. Coletta, R. Tedesco, G. Nishiguchi, K. Carlson, J. Sun,
B. S. Katzenellenbogen, J. A. Katzenellenbogen, J. Med. Chem. 2000, 43,
4934.
[37] D. A. Lightner, L. K. Low, J. Heterocycl. Chem. 1972, 9, 167.
[38] H. H. Wasserman, Ann. N. Y. Acad. Sci. 1970, 171, 108.
[39] F. Hafner, E. Holler, E. von Angerer, J. Steroid Biochem. Mol. Biol. 1996,
58, 385.
[18] J. P. Anselme, J. Org. Chem. 1967, 32, 3716.
[19] D. Gibson, I. Binyamin, M. Haj, I. Ringel, A. Ramu, J. Katzhendler, Eur. J.
Med. Chem. 1997, 32, 823.
[40] I. La¨ıos, A. Cleeren, G. Leclercq, D. Nonclercq, G. Laurent, M. Schlenk, A.
Wellner, R. Gust, Biochem. Pharmacol. 2007, 74, 1029.
[20] J. B. Paine, D. Dolphin, J. Org. Chem. 1985, 50, 5598.
[21] H. Fischer, E. Fink, Hoppe-Seyler’s Z. Physiol. Chem. 1944, 280, 123.
[22] S. V. Zaitseva, S. A. Zdanovich, A. S. Semeikin, O. A. Golubchikov, Russ. J.
Gen. Chem. 2003, 73, 467.
Received: December 13, 2010
[23] H. Zhang, Q. Cai, D. Ma, J. Org. Chem. 2005, 70, 5164.
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