Nitric Oxide-Releasing Selective Estrogen Receptor Modulators: A Bifunctional Approach to Improve the Therapeutic Index

Article abstract of DOI:10.1021/acs.jmedchem.9b00171

When using selective estrogen receptor modulators (SERMs) in cancer therapy, adverse effects such as endothelial dysfunction have to be considered. Estrogens and, consequently, SERMs regulate the synthesis of vasoactive nitric oxide (^?NO). We h

Full text of DOI:10.1021/acs.jmedchem.9b00171

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Article
Nitric oxide-releasing selective estrogen receptor modulators (NO-
SERMs): a bifunctional approach to improve the therapeutic index
Nicole Bechmann, Torsten Kniess, and Jens Pietzsch
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00171 • Publication Date (Web): 17 May 2019
Downloaded from http://pubs.acs.org on May 18, 2019
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Nitric oxide-releasing selective estrogen receptor modulators
(NO-SERMs): a bifunctional approach to improve the
therapeutic index
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a,#
a
a,b,*
Nicole Bechmann , Torsten Kniess , Jens Pietzsch
a
Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research,
Department of Radiopharmaceutical and Chemical Biology, 01328 Dresden, Germany
b
Technische Universität Dresden, School of Science, Faculty of Chemistry and Food Chemistry,
01069 Dresden, Germany
#
Present address: Institute for Clinical Chemistry and Laboratory Medicine, University Hospital
Carl Gustav Carus at Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden,
Germany
*Corresponding
author:
Helmholtz-Zentrum
Dresden-Rossendorf,
Institute
of
Radiopharmaceutical Cancer Research, Department Radiopharmaceutical and Chemical
Biology, Bautzner Landstrasse 400, 01328 Dresden, Germany; Tel: +49 351 2602622; e-mail
address: j.pietzsch@hzdr.de
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Abstract
When using selective estrogen receptor modulators (SERMs) in cancer therapy adverse effects
such as endothelial dysfunction have to be considered. Estrogens and, consequently, SERMs
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
regulate the synthesis of vasoactive nitric oxide ( NO). We hypothesized that a bifunctional
approach combining the antagonistic action of SERMs with a targeted NO-release could diminish
vascular side effects. We synthesized a series of NO-releasing SERMs (NO-SERMs) and the
corresponding SERMs (after NO-release) derived from a triaryl olefin lead. Compounds showed
antagonistic activity for ERβ (IC (ERβ)=0.2–2.7µM), but no interaction with ERα. Growth of ERβ-
50
positive breast cancer and melanoma cells was significantly decreased by treatment with SERM

5d. This anti-proliferative effect was diminished by the additional release of NO by the

corresponding NO-SERM 4d. Moreover, targeted release of NO by 4d counteracted the anti-
proliferative effect of 5d in normal vascular tissue cells. Summarizing, the therapeutic index of
SERMs might be improved by this bifunctional approach.
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Introduction
Estrogen signaling pathways regulate diverse physiological functions, but are also key players in
osteoporosis, neurodegenerative and cardiovascular diseases, and in the progression of certain
cancers, like breast, ovarian, colorectal, prostate, and endometrial cancer.1-4 Estrogen receptors
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(ERs) are ligand-dependent transcription factors that can be modulated by selective estrogen
receptor modulators (SERMs), like tamoxifen (TAM) and raloxifene (Fig. 1A).5-6 SERMs are able
to interact with both subtypes of the receptor, ERα and/or ERβ, acting as estrogen agonists or
7
antagonists, in a tissue-specific manner. For example, on the one hand, tamoxifen acts as an
estrogen antagonist, which is currently be used for prevention and treatment of breast cancer, on
the other hand, agonistic action of tamoxifen increases the incidence of endometrial cancer.8-9
Raloxifene is currently used for the treatment of osteoporosis in postmenopausal women,
10
because of its property to preserve bone mineral density without uterotropic side effects. Daily
treatment with lasofoxifene reduced the risk of nonvertebral and vertebral fractures, ER-positive
breast cancer, coronary heart disease, and stroke in postmenopausal women with osteoporosis,
but enhanced the risk of venous thromboembolic events.11 In contrast to SERMs that modulate
receptor conformation and regulate coactivators and corepressors for recruitment of the receptor,
12
selective ER degraders (SERDs), like fulvestrant (Fig. 1A) or RAD1901 , act via binding and
13
degrading the receptor. A third strategy to block the estrogen action, e.g., in tumor cells, is the
inhibition of estrogen biosynthesis by aromatase inhibitors, like letrozole and exemestane.14-15 All
three estrogen blocking strategies are associated with postmenopausal symptoms. For example,
the treatment with SERMs is accompanied by hot flushes, but more important, with long-term
effects, such as endothelial dysfunction, thromboembolic and cardiovascular disease. 17β-
estradiol (E2) exerts direct and indirect effects on the cardiovascular system via mechanisms
involving genomic and non-genomic pathways (Fig. 1B), whereby, e.g., the vascular homeostasis
is maintained.16 Antagonistic action of SERMs is responsible for the vascular side effects,
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especially by inhibition of the PI3-K/Akt/endothelial NO-synthase (eNOS)-signaling (Fig. 1C) and

17
the resulting imbalance in nitric oxide ( NO) homeostasis. Hayashi et al. could demonstrate that
treatment of human umbilical vein endothelial cells (HUVECs) with E2 enhanced the release of

the potent vasodilator NO via an increased expression of eNOS. Furthermore, the associated
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cardioprotective effect of E2 is repealed via the antagonistic activity of SERMs.18-19
Major challenge in the development of novel SERMs is the improvement of the therapeutic index
to prevent vascular side effects in particular. In contrast to the design of novel SERMs with higher
selectivity towards ERα or ERβ, we describe another potential approach to improve the
therapeutic index of SERMs, combining the selectivity of SERM action with a targeted release of
NO through the introduction of a NO-releasing moiety. Our strategy aims to compensate the
reduced endogenous NO-release by the SERM-mediated inhibition of the PI3-K/Akt/eNOS
signaling cascade. Therapeutic agents with an additional NO-releasing moiety gain increasing

attention with regard to the different actions of NO as vasorelaxant, neurotransmitter and free
radical.20 This approach is currently being intensively investigated, for example in the
development of novel selective cyclooxygenase-2 inhibitors (coxibs). The origin of this study was
the development of such NO-releasing coxibs (NO-coxibs), which also are considered as potent
bifunctional radiosensitizers.21-22 By using two lead structures suggested for coxib/NO-coxib
development, (pyrazolyl)benzenesulfonamides21, 23 and acyclic triaryl olefins 24-28, soon it became
obvious that the selected triaryl olefins do not fulfil the requirements for potent COX-2 inhibition
(supporting information). However, the structural similarity of the novel triaryl olefins to tamoxifen
prompted us to investigate the functional activity (e.g., antagonistic and/or agonistic) of the
compounds on human ERα and ERβ. Furthermore, we indroduced a NO-releasing moiety to the
triaryl olefine lead structure. First studies on the potential benefit of this bifunctional approach
were performed on ERα/β-positive human melanoma and breast cancer cell lines. Investigations
on human umbilical vein endothelial cells allowed us to draw first conclusions regarding the impact
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of an additional NO-release on the side effect profile of SERMs on normal tissue cells of the
vascular system.
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Fig. 1: Structure and modulation of the estrogen signaling by selective estrogen receptor modulators
(SERMs). (A) Chemical structure of the SERMs tamoxifen, raloxifene, and fulvestrant. (B) Showed the most
important intracellular signaling pathways of 17β-estradiol (E2) that could be influenced by SERMs. Signaling
pathway 1 (yellow star): canonical estrogen pathway, the ligand-activated estrogen receptor (ER) binds
specifically to the estrogen responsive element (ERE) in the promoter region of the targeted gene. Signaling
pathway 2: non-ERE-depending estrogen pathway: the ligand-activated estrogen receptor interacts with other
transcription factors (TF), like NF-κB, and, activates gene expression in ERE-independent manner. Signaling
pathway 3: non-genomic estrogen pathway, ERs that are localized in cell membrane activate PI3-K/MAPK-

signaling and regulate for example vasodilation by an activation of the endothelial nitric oxide ( NO) synthase
(eNOS). Moreover, a ligand-independent mechanism by the activation of different growth factors was described
(not shown). (C) In particular, the influence of SERMs on the NO-signaling is coupled with several side effects,
like endothelial dysfunction. PI3-K: Phosphatidylinositol-4,5-bisphosphate 3-kinase; Akt: Protein kinase B; MAPK:
mitogen-activated protein kinase; NF-κB: Nuclear factor kappa B; BCL2: B-cell lymphoma 2; ERK: extracellular-
signal-regulated kinase; JNK: c-Jun N-terminal kinase.
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Results
Synthesis of NO-releasing triaryl olefins. Starting from a triaryl olefin lead structure (3)
a group of 5-(4-(methylsulfonyl)phenyl)-6-6-diphenylalkenyls (4a,c, 5a,c) and 6,6´-bis(4-
fluorophenyl)-5-(4-(methylsulfonyl)phenyl) alkenyls (4b,d, 5b,d) with and without a NO-
releasing moiety were synthesized by applying the reaction sequence illustrated in Figure
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2. Building of the triaryl olefin lead structure started with a Friedel-Crafts acylation of
thioanisole with 4-chlorobutanolychloride or 5-chloropentanoylchloride to yield 4-chloro-1-
(4-(methylthio)phenyl)butan-1-one
(1a,
86%)
and
5-chloro-1-(4-
(methylthio)phenyl)pentan-1-one (1b, 87%), respectively. A similar synthesis strategy was
29
applied for 5-chloro-1-phenylpentan-1-one. Subsequent oxidation of the ketones 1a-b
with
oxone®
(potassium
peroxymonosulfate)
(2a, 76%)
generated
and
4-chloro-1-(4-
5-chloro-1-(4-
(
(
methylsulfonyl)phenyl)butan-1-one
methylsulfonyl)phenyl)pentan-1-one (2b, 92%) for subsequent McMurry olefination.
Reductive cross-coupling reaction of 2a-b with benzophenone or 4,4´-
difluorobenzophenone formed the chloroalkyl substituted triaryl olefin derivatives 3a-d in
40-75% yield. The desired NO-SERMs (4a-d) were obtained by nucleophilic substitution
of the chloroalkyl derivatives 3a-d with silver nitrate in acetonitrile (S 2). Due to the
N
moderate nucleophilic power of the nitrate that resulted from the delocalization of the
negative charge in the anion and the comparatively poor chloro-leaving group, the reaction
mixture had to be heated at 80°C for 24 h. Introduction of the nitroester functionality was
confirmed by high-resolution mass spectrometry (HRMS) and infrared spectroscopy
-1
(Supporting information). Additionally, asymmetric (1640 – 1620 cm ) and symmetric
-1
(1285 – 1270 cm ) stretching vibration for the introduced nitroester group could be
observed.
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O
R
S
O
R
R
O
O
S
S
O
a
b
S
c
O
R= H, F
Cl
n=3,4
nCl
Cl
nCl
O
n
Cl
O
1
1
a, n=3
b, n=4
2a, n=3
2b, n=4
R
3
a, n=3, R=H
b, n=3, R=F
c, n=4, R=H
d, n=4, R=F
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e
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OH
R
5
5
a, n=3, R=H
b, n=3, R=F
5c, n=4, R=H
d, n=4, R=F
n
O
5
O2N
4a, n=3, R=H
4b, n=3, R=F
4
4
c, n=4, R=H
d, n=4, R=F
Fig. 2: Synthetic pathway of NO-releasing SERMs (4) and reference SERMs (5). Reagents and conditions:
®
(
a) AlCl
3
, CHCl
3
, argon, 2.5 h rt, (b) oxone (potassium peroxymonosulphate), MeOH/THF (1/1, v/v), 31 h, (c)
Zn, TiCl
4
, reflux 4.5 h, (d) AgNO
3
, MeCN, 24 h, 80°C, (e) CuSO
4
x 5 H
2
O, DMSO/H
2
O (1/3, v/v), 130°C, 48 h.
Synthesis of corresponding hydroxyalkyl triaryl olefins. From literature, it’s well

known that organic nitrates release NO after a 3-electron-transmission to leave a hydroxyl
rest via an enzymatic or thiol dependent mechanism.30 The corresponding hydroxyl
substituted triaryl olefin derivatives 5a-d were synthesized as reference compounds. To
introduce the hydroxyl group on the chlorinated side chain of the triaryl olefin lead, a
synthesis strategy previously described by Abdellatif et al. was used.27 Each of the
chloroalkyl derivatives 3a-d was heated (130°C) in presence of CuSO x 5 H O in a
4
2
mixture of DMSO/water (1/3, v/v) for 48 h and the reference compounds after NO-release
5a-d were obtained in 52-74% yield. In further experiments, it could be demonstrated that
the reaction follows a nucleophilic substitution (S 2) mechanism with the hydroxyl ions
N
from water, whereby the presence of sulfate or copper rather disturbs the reaction (Fig.
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S1). A direct synthesis of the hydroxyalkyl compounds by using the hydroxy-1-(4-
(methylsulfonyl)phenyl)alkan-1-ones was not feasible (intensively described in the
supporting information, Fig. S2-S4). To compare the impact of the fluorine substituents on
the benzene rings and the nitroester- and the hydroxyl-substituents on the alkenyl chain,
a fluorinated reference compound 8 was synthesized by following the reaction sequence
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illustrated in Figure 3, previously described by Uddin et al. Ketone 6 was oxidized to 1-
(4-(methylsulfonyl)phenyl)pentan-1-one (7) and after McMurry olefination with 4,4´-
difluorobenzophenone,
4,4'-(2-(4-(methylsulfonyl)phenyl)hex-1-ene-1,1-
diyl)bis(fluorobenzene) (8) was obtained in 32% yield.
O
S
O
F
O
S
O
S
O
a
b
c
Cl
O
O
6
7
F
8
Fig. 3: Synthetic pathway of SERM 8. Reagents and conditions: (a) thioanisole, AlCl
3 3
, CHCl , 1.5 h rt (b),
oxone® (potassium peroxymonosulphate), MeOH/THF (1/1, v/v), 15 h, (c) 4,4´-difluorobenzophenone, Zn,
TiCl , reflux 4.5 h, reflux.
4
ER interactions of compounds 4 and 5. The estrogen receptor binding affinity of the
novel NO-SERMs 4 and the corresponding SERMs 5 after NO-release was evaluated by
using human receptor reporter assays. The cell-based luminescence assay allows the
screening of test compounds to quantify the influence of the functional activity of the
receptors both in agonist and antagonist manner. However, preliminary experiments
showed that the agonist assay mode is not applicable for triaryl olefin derivatives. This is
also the case for the partial agonist TAM as it was confirmed by the manufacturer. In vivo,
TAM is easily converted by the liver cytochrome P450 into 4-hydroxytamoxifen (4OHT)
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that binds to ERs within the equal binding pocket like E2.31-32 The resulting nuclear
complex decreases DNA synthesis and inhibits the action of E2.32 Furthermore, the
complex also recruits co-repressor proteins, that contribute to the antagonistic action of
33
4
OHT. It could be demonstrated that the agonistic effect of tamoxifen depends on the
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cell type, ER subtype, and ERE-promoter context.34 Thus, the cell-based ER reporter
assay is not useable for the analysis of the agonistic action of triaryl olefins.
ERα activity was not affected by the novel compounds up to a concentration of 5 µM (Tab.
1
). In contrast, all compounds, except 4b, showed promising antagonistic activity on ERβ
IC50(NO-SERMs 4): 0.2 - 2.7 µM; IC50(SERMs 5): 0.18 - 3.0 µM). The fluorinated derivatives with a
butyl side chain 4d (IC (ERβ): 0.20 µM) and 5d (IC (ERβ): 0.18 µM) were the most
(
50
50
potent, selective ERβ antagonists (4d: S.I.(ERβ) > 25.00; 5d: S.I.(ERβ) > 27.78). The
introduction of the nitroester group seems to have only a slight influence on ERβ affinity.
These data and the findings from Chen et al. indicate that the methylsulfonyl group is
responsible
for
ERβ
selectivity.35
2-(4-(methylsulfonyl)phenyl)hex-1-ene-1,1-
diyl)dibenzene, previously described by Uddin et al., also showed a high ERβ selectivity
whereas the corresponding NH-sulfoximine and N-cyano sulfoximine derivatives displayed
an affinity for both estrogen receptors.26, 35 Because of the ERβ selectivity of the novel
compounds the additional NO-release is more important to reduce cardiovascular events.
Studies on knockdown mice have demonstrated that ERβ is required for E2-mediated
vasodilatation and protection from age-related hypertension.36-37 Hence, the diminished
vasodilatation that might be associated with antagonistic activity on ERβ of the novel

compounds, might possibly be compensated by the release of the vasodilator NO.
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Tab. 1: Estrogen receptor antagonistic activity and NO-release of NO-releasing SERMs and the corresponding
SERMs.
IC50 [µM]
NO-release [%]c
plasma
SI
L-cysteine
compound
ERαa
ERβa
ERβb
PBS
oxidant
S-nitroso-
S-nitroso-
thiol
1.3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
nitritefree
NOtotal
nitritefree
NOtotal
thiol
3.6
2.1
3.1
1.3
4.0
2.5
4a
> 5
> 5
> 5
> 5
-
2.7
> 5
1.3
0.20
-
> 1.85
1.00
> 3.85
> 25.00
-
n.d
2.2±0.2
0.1±0.1
n.d.
1.5±0.5
0.1±0.1
1.1±0.7
2.5±1.4
48.3±4.2
0.2±0.1
0.2±0.2
0.7±0.2
0.2±0.2
0.2±0.1
0.3±0.2
1.4±0.3
3.8±0.5
2.8±1.5
3.3±1.7
1.5±1.5
4.3±1.4
3.9±1.5
0.7±0.3
0.1±0.1
0.9±0.5
0.4±0.4
0.3±0.3
0.8±0.1
2.0±1.3
2.0±1.3
0.5±0.6
3.8±2.6
3.6±1.3
4.4±1.5
4
b
c
1.9
4
0.4
4d
PA
3.4
33.5±1.3
n.d.
3.3
NO-aspirin
-
-
-
3.6
5
a
b
> 5
> 5
> 5
> 5
1.5
3.0
> 3.34
> 1.67
> 3.57
> 27.78
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5
5c
1.4
5d
0.18
tamoxifen
citrat
1
.7d
0.005d
377.8
-
-
-
-
-
-
-
-
n.d. not detectable.
PA Piloty´ s acid (N-hydroxybenzenesulfonamide).
a
Values are means of two determinations using a cell based human estrogen receptor alpha or beta reporter assay (Human Estrogen
Receptor Beta Reporter Assay System, #IB00411 or Human Estrogen Receptor Alpha Reporter Assay System, #IB00401; Indigo
Biosciences; PA; USA).
The IC50 value was calculated by non- linear fitting (Software: Origin, logistic fit).
b
c
ERβ selectivity index in vitro (IC50(ERα)/IC50(ERβ)).
NO measured by nitrite using the reaction of nitrite with 2,3-diaminonaphthalen (DAN) to the high fluorescence 2,3-naphthotriazol (NAT) under
acid conditions. Percent of NO-released based on the theoretical maximum release of 1 mol of NO/mol of the test compound. Three to four
independent experiments were performed, each in duplicate (n = 6–8). Mean ± SEM.
d
analyzed by the manufacturer of the cell based human estrogen receptor alpha and beta reporter assay (Indigo Bioscience, Technical Manual).
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In vitro stability of 4d and 5d. Exemplarily, in vitro stability of the most potent NO-SERM
4d and the corresponding SERM 5d was analyzed after incubation in human whole blood
(Fig. 4). SERM 5d is stable over a period of > 6 h, no metabolites could be detected by
HPLC after 6 h incubation time. The measurable decrease of 5d between 0 min and 1 h
incubation, suggests a binding of 5d (logPcalculated: 4.85) on lipophilic components of the
blood. In opposite, a 6 h incubation of NO-SERM 4d in whole blood resulted in the release
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
of NO and formation of the corresponding SERM 5d, as expected. The low ratio of 5d
indicates a slow NO-release by 4d. Other metabolites could not be detected, but there is
also a loss of 4d (logPcalculated: 6.21) engendered with the high lipophilicity after 1 h
incubation time. These results suggest that the novel NO-SERMs and the corresponding
SERMs are stable under physiological conditions, as human whole blood. This underlines
suitability for further applications in vitro and in vivo.
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Fig. 4: Stability of SERM 5d and NO-SERM 4d in human blood. Stability was analyzed by HPLC after zero
up to six hours incubation in human whole blood at 37°C. After incubation, proteins were separated by using
supersol-precipitating agent (1:2, v/v) and the clear solution were analyzed using the following conditions.
Agilent 1100; Zorbrax 300SB-C18 (250 x 9.4 mm; 4 µm); Zorbrax 300SB-C18 (4.6 x 12.5 mm, 5 µm); 254 nm;
A: H
2
O + 0.1% TFA B: MeCN + 0.1% TFA, 1-5 min 85% A, 10 min 70% B, 25-26 min 95% B, 29-30 min 85%
A; flow rate 3.0 mL/min at 50°C.
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Evaluation of the NO-release by NO-SERMs. In presence of oxygen, the free radical
nitric oxide is immediately oxidized to nitrite and further to nitrate. The exogenous NO-
release by the novel NO-SERMs in vitro was determined by a fluorescence-based nitrite
21
assay. For quantification the reaction of nitrite with 2,3-diaminonaphthalene (DAN) to the
highly fluorescent 2,3-naphthotriazole (NAT) under acidic conditions was utilized.
Mercury(II)chloride was added to the samples to detect nitric oxide that was stabilized as
bioactive S-nitrosothiols in presence of free thiol groups. The NO-release by the novel NO-
SERMs was determined after incubation at 37°C for 90 min in (a) phosphate-buffered-
saline (PBS, pH 7.5) to analyze the spontaneous NO-release, (b) PBS containing human
plasma to measure enzyme-mediated mechanisms, (c) PBS with 50 µM K [Fe(CN) ], as
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oxidizing agent, or (d) PBS in presence of 5 mM L-cysteine to detect the release by free
thiol groups. The results were summarized in table 1. The NO-release by the novel organic
nitrates 4a-d is moderate, as expected. Interestingly, 4b showed a comparatively high

spontaneous release of NO (2.2 ± 0.2%), whereby for the other compounds no NO-
release after incubation in PBS could be observed. After incubation under oxidizing
conditions, an inverse effect was found. For 4b no NO-release was detected, whereas 4a
and 4c-d showed a NO-release of 1.1 - 2.5%. For all compounds a moderate NO-release
under enzymatic and thiol dependent conditions could be observed (0.5 - 3.8%). A similar
NO-release was verifiable for the organic nitrate, NO-aspirin. A moderate NO-release was
aimed to ensure a good manageability of the NO-SERMs, and to prevent side effects

associated with high exogenous NO concentration.
Anti-tumorigenic activity of the novel compounds in dependence of their NO-
release. For analyzing the influence of the novel compounds on tumor cell proliferation,
different breast adenocarcinoma (MCF-7 and MDA-MD-231) and human melanoma
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(
A2058 and Mel-Juso) cell lines were selected as model. Their dissimilar basal COX-2
22
protein expression was the initial intent for using the melanoma cell lines. The melanoma
model was retained in this investigation because it allows a direct comparison of both NO-
SERMs and NO-coxibs, particularly, with regard to the additional NO-release. However,
the stronger emphasis on SERM development led to the introduction of a suitable breast
adenocarcinoma model. Beside the ERα/β-positive MCF-7 cells, we used MDA-MB-231
with no detectable expression of ERα. ER protein expression of the melanoma cell lines
was analyzed by Western blot analysis in comparison to the MCF-7 cells (Fig. 5A). All cell
lines showed a comparable ERβ protein synthesis. Protein synthesis of ERα was lower in
A2058 and Mel-Juso cells than in MCF7 cells. Blocking of estrogen-dependent cell
proliferation is utilized as a common treatment option for several types of ER-positive
breast cancer. Helguero and coworkers suggested that application of ERβ agonists could
be a useful addition in the treatment regime of breast cancer that at the moment only aimed
at ERα inhibition, with routinely use of TAM (ER antagonist).38
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Fig. 5: ERα and ERβ expression of two melanoma cell lines and the influence of NO-SERMs 4 or SERMs
5 on cell viability and proliferation of both cell lines. (A) ERα and ERβ expression in A2058 and Mel-Juso
human melanoma cells was determined by Western blotting. Human breast cancer cells (MCF-7) here were
used as positive control. Representative sections of the immunochemical Western blot analysis of ERα, ERβ
and β-actin used as housekeeping protein are shown (three independent experiments). (B) The influence of
NO-SERMs 4 and SERMs 5 on cell viability was investigated. Interestingly, the additional NO-release
preserved the cell viability. Three to five independent experiments were performed (n = 9 - 15). Mean ± SEM,
ANOVA and Bonferroni post hoc test comparison vs. DMSO control, * p < 0.05 or, ** p < 0.01. Moreover, the
influence of the corresponding compounds 4d and 5d on the number of proliferating cells on (C) A2058 and
(D) Mel-Juso cells was analyzed. The proliferation assays confirmed the results of the viability assays that the
additional release of NO reduces the anti-tumorigenic activity of SERMs in melanoma cells. Three
independent experiments were performed (n = –5-6). Mean ± SEM, ANOVA and Bonferroni post hoc test
comparison vs. DMSO control, * p < 0.05 or, ** p < 0.01.
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In order to investigate the anti-proliferative activity of our novel ERβ antagonists on breast
cancer cells, we performed cell viability and proliferation assays for the most potent NO-
SERM 4d and the corresponding SERM 5d. Viability of the ERα/β-positive MCF-7 cells
was reduced significantly after 24 h treatment with 50 µM 4d or 5d (Fig. 6A). The additional
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release of NO diminished this effect and preserved the viability of the cells. In comparison,
viability of MDA-MB-231 cells, naturally lacking ERα, was not affected by the treatment
with 4d and 5d. In both breast cancer cell lines the number of proliferating cells
(proliferation assay) was significantly decreased after 48 h treatment with SERM 5d in a

concentration dependent manner. Interestingly, the additional release of NO maintained
the number of proliferating cells of both cell lines. The high ERβ selectivity resulted in a
comparable response behavior of both breast cancer cell lines independent of their
different ERα expression pattern. However, the concentration of SERM 5d needed to
reduce the number of proliferating cells significantly is relative high.
The impact of the additional NO-release was evaluated more in detail by the use of two
ERα/β-positive melanoma cell lines to obtain results that could be compared with our
previous study on NO-coxibs.21-22 Viability of both ER-positive melanoma cell lines was
significantly diminished by treatment with the novel SERMs 5a-d (Fig. 5B). The half-
maximal effective concentration (EC ) of SERMs 5 was in a micromolar range (25.7 -
50
38.5 µM). 5c was the only compound that showed no significant influence on cell viability.
Remarkably, the additional NO-release resulted in a preservation of cell viability.
Treatment with the NO-SERMs 4a-d up to a concentration of 50 µM showed no detectable
influence on cell viability. Exemplary, for the most potent NO-SERM 4d and the
corresponding SERM 5d the impact on the number of proliferating cells was analyzed (Fig.
5C-D). A concentration of 25 µM SERM 5d significantly decreased the number of
proliferating cells in both cell lines. In contrast, 50 µM NO-SERM 4d showed no influence
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on the number of proliferating cells. In summary, the additional NO-release reduces the
influence on cell proliferation on both ER-positive melanoma cells. These preliminary
results indicate that by the additional NO-release the tolerability of SERMs could be
enhanced but their anti-tumorigenic activity is decreased.
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Fig. 6: Influence of NO-SERMs 4 or SERMs 5 on cell viability and proliferation of breast cancer cell
lines. (A) Influence of the NO-SERMs 4 and SERMs 5 on cell viability was investigated. Additional release of
NO preserved viability of MCF-7 cells. Three independent experiments were performed (n =9). Mean ± SEM,
ANOVA and Bonferroni post hoc test comparison vs. DMSO control, * p < 0.05 or, ** p < 0.01. Furthermore,
the influence of the corresponding compounds 4d and 5d on the number of proliferating cells on (B) MCF-7
and (C) MDA-MB-231 cells was analyzed. In both breast cancer cell lines, the number of proliferating cells

was preserved by the additional release of NO. Four independent experiments were performed (n = 8). Mean
±
SEM, ANOVA and Bonferroni post hoc test comparison vs. DMSO control, * p < 0.05 or, ** p < 0.01.
Impact of the additional NO-release on normal vascular tissue cells. Taking into
account our working hypothesis, we evaluated the benefit of the additional NO-release on
vascular cells. Therefore, human umbilical vein endothelial cells, HUVECs, were used. ER
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protein expression of HUVECs was analyzed by Western blotting (Fig. 7A). ERβ protein
synthesis was comparable with the expression of the MCF-7 cells, used as control.
Moreover, HUVECs also showed a comparatively high ERα protein expression that is only
slightly lower than in the MCF-7 cells. To confirm the protective influence of the additional
NO-release on normal vascular tissue cells viability assays were performed (Fig. 7B). 24
h treatment with SERM 5d resulted in a significant decrease of HUVECs viability.
Moreover, treatment with 50 µM 5d led to the complete loss of cell viability. As expected,
HUVECs are more sensitive for the treatment with SERM 5d than the breast cancer and
the melanoma cell lines. In contrast, treatment with NO-SERM 4d had no measurable
influence on the cell viability up to a concentration of 50 µM. These data indicate that
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additional release of NO does protect endothelial cell viability. Caulin-Glaser et al. showed
that the 17β-estradiol dependent regulation of the basal NO-release of HUVECs is a non-
genomic effect of E2 39 via the activation of Akt signaling40.
Fig. 7: ERα and ERβ expression and the influence of NO-SERM 4d or SERM 5d on viability of HUVEC.
(A) ERα and ERβ expression in HUVECs as determined by Western blotting. Representative sections of the
immunochemical Western blot analysis of ERα, ERβ and β-actin used as housekeeping protein are shown
(three independent experiments). (B) The influence of NO-SERMs 4d and SERM 5d on cell viability was
investigated. The additional NO-release resulted in a preservation of the cell viability in comparison to the
strong decrease of cell viability by the treatment with SERM 5d. Three to five independent experiments were
performed (n = 3 - 15). n.E.d.: no EC50-value could be detected; b.l.d.: below limit of detection.
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The influence of E2 in comparison to the treatment with NO-SERM 4d on bioavailability of
NO in HUVECs was analyzed (Fig. 8A) via a fluorescence-based assay. Treatment with
200 nM E2 showed no detectable impact on NO-bioavailability. Furthermore, the influence
of the NO-SERM 4d and SERM 5d on COX-2 protein expression was investigated as
marker for inflammatory stress. Since the ER/PI3K pathway is also associated with a
regulation of the transcription factor nuclear factor-kappa B (NF-κB), the activation of NF-
κB resulted in an increase in COX-2 transcription.41 Twenty-four hours treatment with
200 nM E2 showed no influence on COX-2 expression (Fig. 8B). TAM (up to 1 µM) slightly
affected the COX-2 expression level, while treatment with the ERβ selective SERM 5d
resulted in a significant up-regulation of COX-2 in a concentration dependent manner.
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0

Importantly, this effect was diminished by the additional release of NO caused by NO-
SERM 4d. Tamura et al. demonstrated on primary human uterine microvascular
endothelial cells (HUMEC) that various levels of E2 significantly increased COX-2
expression via estrogen receptors.42 This effect could not be observed in the utilized
HUVECs. A tissue-specific regulation of COX-2 expression via estrogens and anti-
estrogens was previously described and document the molecular complexity of estrogen
receptor signaling.43
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Fig. 8: Influence of NO-SERM 4d or SERM 5d on (A) NO-bioavailability and (B) the inflammatory marker

COX-2 of HUVECs. (A) The influence of SERMs and the novel NO-SERM 4d on the bioavailable NO was
measured as sum of nitrite and S-nitrosothiols in cell culture supernatants. Therefore, cells were treated with
different concentrations of SERM 5d, NO-SERM 4d, tamoxifen (TAM) or 17β-estradiol and the amount of nitrite
and S-nitrosothiol after 24 h treatment was analyzed in HUVECs. Three independent experiments were
performed (n = 6). Mean ± SEM, ANOVA and Bonferroni post hoc test comparison vs. DMSO control were
performed. (B) The influence of the ER-modulation on the inflammatory marker COX-2 was determined by
Western blotting. Representative sections of the four independent experiments were shown.
Discussion and Conclusion
Starting from the hypothesis, that a bifunctional approach combining the antagonistic action of
SERMs with a targeted exogenous release of NO could diminish vascular side effects associated
with treatment with this commonly used drugs, we synthesized a series of novel NO-SERMs
basing on a triaryl olefin lead bearing a nitroester functionality. NO-SERMs as well as the
corresponding SERMs showed an antagonistic activity towards ERβ, whereby the methylsulfonyl
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group of the compounds seems to be responsible for the high ERβ selectivity. The novel
SERMs with a NO-releasing moiety underlie a moderate NO-release, this was aimed to

prevent side effects associated with high NO concentration, and to ensure a good
manageability of the novel compounds. Similar results were obtained for NO-coxibs basing
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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0
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21
on a (pyrazolyl)benzenesulfonamide lead. Using for example a sulfohydroxamic acid
(
SO NHOH) substituent on a triaryl olefin lead resulted in an undesirable increased
2
spontaneous NO-release (6 - 14%) that was enhanced by the addition of oxidizing agents
(4 - 26%).44
For the first time, the present study could demonstrate that the targeted release of NO
diminished the anti-proliferative activity of SERMs on normal vascular tissue cells as well as on
different ERβ-positive breast cancer and melanoma cells. In order to evaluate these in vitro

findings, several facts have to be taken into account. After the release of NO in the
vascular system or in the tumor, the corresponding SERM with its increased anti-tumor
activity becomes available, and would logically affect the ERβ-positive tumor. Further, it
can be assumed that the NO-release occurs faster in vivo than under in vitro conditions.
On the other hand, NO-release can be considered as therapeutically useful, since in
contrast to the effect as gasotransmitter and vasodilator in the musculature of the vascular
system, here the damaging effect of the free radical is the major impact. In this context,
NO-releasing compounds are considered as potential radiosensitizers or
chemosensitizers.22, 45-46 By following this concept, coxibs and NO-coxibs can be used for

a comparative analysis. Data achieved by us demonstrate that the effect of NO is
22
dependent on the underlying inhibitory mechanism of the lead structure. The additional

release of NO by coxibs resulted in an increase of the anti-tumorigenic activity of these
compounds, while the effect is reduced in SERMs. We could demonstrate that the
bifunctional NO-coxibs are more potent radiosensitizers than the corresponding
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conventional coxibs. The radiation response of different tumor entities is regulated by
several factors. In this context, the role of ERβ is largely unknown. For further studies, we
hypothesized that a manipulation of the ER-signaling, by SERMs in particular, provides a
promising strategy to sensitize radioresistant ER-positive tumor cells for radiotherapy.47
0
1
2
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9
0
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0

Perhaps, this effect is enhanced by the additional release of NO. The negligible
cytotoxicity of the novel NO-SERMs in this regard is a positive aspect, because an ideal
radiosensitizer shows a significant impact on tumor cells without cytotoxic side effects.48-
49
However, confirmation of this concept requires further investigations.
In conclusion, a group of novel triaryl olefins with or without a NO-releasing moiety was
synthesized, via McMurry olefination and identified as potential ERβ selective SERMs. To

the best of our knowledge, our data showed for the first time that a targeted release of NO
could diminish the anti-proliferative and pro-inflammatory effect of SERMs in normal
vascular tissue and thereby could possibly improve the therapeutic index of this
extensively used drug class. The high affinity and selectivity towards ER expressing cells
should also contribute to reinforced effect on ER-positive tumor cells. The role of ERβ in
tumor progression, metastasis and therapy resistance, in particular towards radiotherapy,
is largely unknown. By our novel ERβ-selective SERMs with or without a NO-releasing
moiety, useful tools to investigate these therapy relevant questions are available.
Experimental Section
Synthetic procedures. If not indicated otherwise, all reagents and solvents were of highest
purity available from VWR International GmbH (Darmstadt, Germany), Sigma-Aldrich
GmbH (Taufkirchen, Germany), Merck KGaA (Darmstadt, Germany), Thermo Fisher
Scientific Inc. (Bonn, Germany) or Carl Roth GmbH & Co. KG (Karlsruhe, Germany).
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Column chromatography was performed using silica gel with a mesh size of 40-63 µm.
Semi-preparative HPLC by using the HPLC system ProStar from Varian was performed
for further purification. The purity of the nitroester and hydroxyalkyl reference compounds
was determined by analytical HPLC (Varian ProStar, microsorb 60, C18, 250 x 21.4 mm;
acetonitrile/water + 0.1% TFA, 5/5, v/v). The purity of the novel compounds were ≥ 95%.
1
1
1
1
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0
For determination of R -values by thin-layer chromatography (TLC) silica gel F-254
f
aluminum plates (Merck KGaA, Darmstadt, Germany) were used. Melting points were
recorded by a Galen III (Cambridge Instruments, Cambridge, UK) melting point apparatus
1
13
19
and are uncorrected. H NMR, C NMR and F NMR were recorded on Agilent DD2-400
OneNMR probe (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were
1
13
19
referenced to the residual solvent shifts for H and C, for F CFCl as internal standard
3
was used. Infrared (IR) spectroscopy was performed by FTIR spectrometer iS5 (Thermo
Fisher Scientific, Rockfort, USA) and the spectra were recorded by ATR (attenuated total
reflection). Low-resolution mass spectra (MS) were recorded by MS/MS Xevo TQ-S
(Water GmbH, Eschborn, Germany) using electrospray (ES) ionization. High-resolution
mass spectra were obtained by ESI-TOF-MS (Impact II, Bruker Daltonics) also using ES
ionization. Elemental analysis was performed for C, H, N and S with the elemental analyzer
EA3000 from EuroVector (Italia) and was within ± 0.5% of the theoretical values. All
spectra are shown in the supporting information.
General procedure for synthesis of compound (1a-b). To a stirring suspension of AlCl (1.3
3
eq.) in CHCl , 1.2 eq. chloroalkanolychloride and 1 eq. thioanisole were added under argon
3
atmosphere at 0°C. After 2.5 h stirring at room temperature the reaction mixture was added
to water. The organic layer was separated and washed three times with water. The
combined aqueous solution was extracted three times with CHCl , the combined organic
3
layer was dried over Na SO and the solvent was removed under vacuum.
2
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4
-chloro-1-(4-(methylthio)phenyl)butan-1-one (1a). 4 ml 4-chlorobutanolychloride (35.7 mmol)
was stirred with 3.4 mL thioanisole (28.9 mmol) in presence of 5.28 g AlCl (39.6 mmol). After
3
50
reconditioning 1a was obtained as white solid (86.1% yield). mp 53-58°C (lit. 73-75°C ); Rf 0.60
1
(ethyl acetate/petroleum ether; 1/1; v/v); H NMR (400 MHz, CD CN) δ (ppm) 2.17–2.08 (m, 2H,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
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3
3
CH ), 2.53 (s, 3H, CH ), 3.12 (t, J=7.1 Hz, 2H, CH ), 3.68 (t, J=6.6 Hz, 2H, CH ), 7.33 (d,
2-β
3
2-α
2-γ
3J=8.7 Hz, 2H, CHaromatic), 7.89 (d, J=8.8 Hz, 2H, CH
3
); C NMR (101 MHz, CD CN) δ (ppm)
3
13
aromatic
14.82 (CH ), 27.98 (CH ), 36.05 (CH ), 45.75 (CH ), 125.87, 129.36 (2 x CHaromatic), 134.22,
3
2-β
2-α
2-γ
146.89 (2 x Caromatic), 199.02 (Cketone); C H ClOS [Mmonoisotopic] requires 228, ESI-MS (ES+) m/z:
11
13
35
+
37
+
35
-
229 [M( Cl)+H] , 231 [M( Cl)+H] ; (ES-) m/z: 227 [M( Cl)-H] ; HRMS (ESI+) m/z calcd for
+ 35
C H ClOS [M+Na , Cl] 251.02733, found 251.02694.
11
13
5-chloro-1-(4-(methylthio)phenyl)pentan-1-one (1b). 5.58 mL 5-chloropentanoylchloride (49.9
mmol) and 4.14 mL thioanisole (35.2 mmol) were stirred in presence of 5.29 g AlCl (39.7 mmol).
3
1
yellow solid; yield: 86.7%; mp 76-80°C, Rf 0.62 (ethyl acetate/petroleum ether; 1/1; v/v); H NMR
(400 MHz, CD CN) δ (ppm) 1.87–1.74 (m, 4H, CH , CH ), 2.53 (s, 3H, CH ), 3.03–2.96 (m, 2H,
3 2-β 2-γ 3
3
3
3
CH ), 3.63 (t, J=6.4 Hz, 2H, CH ), 7.33 (d, J=8.7 Hz, 2H, CH ), 7.88 (d, J=8.7 Hz, 2H,
aromatic
2-α
2-δ
13
CHaromatic); C NMR (101 MHz, CD CN) δ (ppm) 14.83 (CH ), 22.30 (CH ), 32.84 (CH ), 38.06
3
3
2-γ
2-β
(CH ), 46.04 (CH2- δ), 125.87, 129.38, (2 x CHaromatic), 134.38, 146.68, (2 x Caromatic), 198.76
2-α
35
+
(Cketone); C H ClOS [M
] requires 242, ESI-MS (ES+) m/z: 243 [M( Cl)+H] , 245
12 15
monoisotopic
37
+
+ 35
[M( Cl)+H] , HRMS (ESI+) m/z calcd for C H ClOS [M+Na , Cl] 265.04298, found 265.04226.
12
15
General procedure for synthesis of compound (2a-b). To an ice cooled solution of 1 (1 eq.)
®
in THF/MeOH (1/1; v/v) an aqueous solution of oxone (4 eq.) was added. The reaction
mixture was stirred at room temperature for 31 h. After removal of the solvent EtOAc and
water were added. The aqueous fraction was extracted three times with EtOAc and the
combined organic fraction was washed with water, followed by drying with Na SO . The
2
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vacuum dried row product was purified by column chromatography (ethyl acetate /
petroleum ether, 1/1, v/v).
4-chloro-1-(4-(methylsulfonyl)phenyl)butan-1-one
(2a).
2.56
g
4-chloro-1-(4-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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0
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8
9
0
(methylthio)phenyl)butan-1-one (1a, 11.2 mmol) an aqueous solution of oxone (14.45 g, 43.8
mmol) was added. After purification 2a was obtained as white solid (75.8% yield). mp 88-93°C;
1
Rf 0.37 (ethyl acetate/petroleum ether; 1/1; v/v); H NMR (400 MHz, CD CN) δ (ppm) 2.21–2.12
3
3
3
(
m, 2H, CH ), 3.10 (s, 3H, CH ), 3.22 (t, J=7.0 Hz, 2H, CH ), 3.70 (t, J=6.6 Hz, 2H, CH ),
2-β 3 2-α 2-γ
3
3
13
8
.03 (d, J=8.7 Hz, 2H, CH
), 8.16 (d, J=8.7 Hz, 2H, CH
); C NMR (101 MHz, CD CN)
aromatic
aromatic 3
δ (ppm) 27.64 (CH ), 36.88 (CH ), 44.48 (CH ), 45.55 (CH ), 128.60, 129.76 (2 x CHaromatic),
2-β
2-α
2-γ
3
141.74, 145.43 (2 x Caromatic), 199.44 (Cketone). C H ClO S [Mmonoisotopic] requires 260, ESI-MS
11
13
3
35
+
37
+
35
+
35
+
(ESI+) m/z: 261 [M( Cl)+H] , 263 [M( Cl)+H] , 283 [M( Cl)+Na] , 285 [M( Cl)+H] ; (ES-) m/z:
35
-
+ 35
2
60 [M( Cl)-H] ; HRMS (ESI+) m/z calcd for C H ClO S [M+Na , Cl] 283.01716, found
11 13 3
2
83.01656; Anal. Calcd for C H ClO S: C: 50.67, H: 5.03, S: 12.30. Found: C: 50.80, H: 5.01,
11 13 3
S: 11.90.
5-chloro-1-(4-(methylsulfonyl)phenyl)pentan-1-one
(2b).
9.89
g
5-chloro-1-(4-
®
(
methylthio)phenyl)pentan-1-one (1b, 40.7 mmol) and 50.11 g oxone (163.0 mmol) were stirred
as previously described. After purification 2b was obtained as white solid in 92.3% yield. mp 99-
1
1
02°C; Rf 0.46 (ethyl acetate/petroleum ether; 1/1; v/v); H NMR (400 MHz, CDCl ) δ (ppm) 2.02–
3
3
1
.83 (m, 4H, CH , CH ), 3.16–3.00 (m, 5H, CH , CH ), 3.60 (t, J=6.1 Hz, 2H, CH ), 8.06 (d,
2-β
2-γ
3
2-α
2-δ
3J=8.5 Hz, 2H, CHaromatic), 8.13 (d, J=8.5 Hz, 2H, CH
3
); C NMR (101 MHz, CDCl ) δ (ppm)
3
13
aromatic
2
1.38 (CH ), 31.99 (CH ), 38.25 (CH ), 44.48 (CH ), 44.72 (CH ), 128.01, 129.01
2-γ 2-β 2-α 2-δ 3
(2 x CHaromatic), 140.88, 144.33 (2 x Caromatic), 198.38 (Cketone). C H ClO S [Mmonoisotopic] requires
12
15
3
35
+
37
35
+
274, ESI-MS (ES+) m/z: 275 [M( Cl)+H] , 277 [M( Cl)+H]+, 297 [M( Cl)+Na] , 299
37
+
35
-
+ 35
[M( Cl)+Na] , (ES-) m/z: 274 [M( Cl)-H] ; HRMS (ESI+) m/z calcd for C H ClO S [M+Na , Cl]
12
15
3
+
35
2
97.03281, found 297.03241, calcd for [2M+Na , Cl] 571.07585, found 571.07531.
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General procedure for synthesis of compound (3a-d). To a stirred suspension of zinc
powder (8 eq.) in dry THF under argon atmosphere at -10°C titan tetrachloride (4 eq.) was
added dropwise. The suspension was headed under reflux for 2 h and after cooling the
suspension to 0°C a solution of 2 (1 eq.) and 4,4´-difluoro-benzophenone or
benzophenone (1 eq.) in dry THF was added. After 2.5 h heating under reflux the reaction
mixture was tempered to 25°C and poured into a 10% aqueous potassium carbonate
solution. The mixture was stirred for 5 min and the insoluble material was removed by
vacuum filtration. After separation of the organic fraction the aqueous fraction was
extracted with EtOAc three times. The combined organic fraction was dried over Na SO
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
and the solvent was removed under vacuum. The raw product was purified by column
chromatography (EtOAc/petroleum ether).
5-chloro-2-(4-(methylsulfonyl)phenyl)pent-1-en-1,1-diyl)dibenzene (3a). To the stirring
complex of 1.06 mL TiCl (1.8 mmol) and 0.99 g Zinc powder (15.1 mmol) in 1.1 mL THF a
4
suspension of 4-chloro-1-(4-(methylsulfonyl)phenyl)butan-1-one (2a, 0.50 g, 1.9 mmol) and
benzophenone (0.35 g, 1.9 mmol) in 38 mL THF was added. Upon purification by column
chromatography (ethyl acetate/petroleum ether; 2/3; v/v) 3a was obtained as white solid with
1
6
3.3% yield. mp: 129-133°C; Rf 0.55 (EtOAc/petroleum ether, 1/1, v/v); H NMR (400 MHz,
CD CN) δ (ppm) 1.80–1.68 (m, 2H, CH ), 2.62–2.55 (m, 2H, CH ), 3.00 (s, 3H, CH ), 3.46 (t,
3
2-β
2-α
3
3
J=6.6 Hz, 2H, CH ), 6.97–6.91 (m, 2H, CHaromatic), 7.10–7.03 (m, 3H, CHaromatic), 7.35–7.28 (m,
2-γ
3
13
3H, CHaromatic), 7.44–7.38 (m, 4H, CHaromatic), 7.71 (d, J=8.6 Hz, 2H, H-2,6 CH SO Phe). C NMR
3
2
(
101 MHz, CD CN) δ (ppm) 32.29 (CH ), 33.69 (CH ), 44.42 (CH ), 45.83 (CH ), 127.37,
3 2-α 2-β 3 2-γ
127.66, 128.08, 128.68, 129.40, 129.89, 131.15, 131.53, 138.92, 139.77, 142.90, 143.27, 143.47,
35
+
37
+
1
48.90. C H ClO S [M
] 410, MS (ES+) m/z: 411 [M( Cl)+H] , 413 [M( Cl)+Na] , 433
24
23
2
monoisotopic
35
+
37
+
+ 35
[
M( Cl)+Na] , 435 [M( Cl)+Na] ; HRMS (ESI+) m/z calcd for C H ClO S [M+Na , Cl]
24 23 2
+
35
4
33.10050, found 433.09975, calcd for [2M+Na , Cl] 843.21123, found 843.21068; IR (ATR): ѵ
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-1
=
1589 (m, olefin), 1149 (s, SO ) cm ; Anal. calcd for C H ClO S: C 70.14, H 5.64, S: 7.80,
2
24 23
2
found: C 70.33, H 5.71, S 7.34.
4
,4´-(5-chloro-2-(4-(methylsulfonyl)phenyl)pent-1-en-1,1-diyl)bis(fluorobenzene) (3b). To
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
3
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5
5
5
5
5
6
0
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0
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0
the stirring complex of 2.2 mL TiCl (19.7 mmol) and 2.00 g Zinc powder (30.6 mmol) in 36 mL
4
THF a suspension of 1.00 g 4-chloro-1-(4-(methylsulfonyl)phenyl)butan-1-one (2a, 3.8 mmol) and
0
.85 g 4,4´-difluorobenzophenone (3.9 mmol) in 76 mL THF was added. After purification by
column chromatography (ethyl acetate/petroleum ether; 1/3; v/v) 3b was obtained as white solid
with 56.3% yield. mp: 140-142°C; Rf 0.49 (EtOAc/petroleum ether, 1/1, v/v); purity: 99.0%
1
(
analyzed: H O/MECN + 0.1% TFA; 1/1; v/v); H-NMR (400 MHz, CD CN) δ (ppm) 1.78–1.69 (m,
2
3
3
2H, CH ), 2.60–2.55 (m, 2H, CH ), 3.01 (s, 3H, CH ), 3.46 (t, J=6.4 Hz, 2H, CH ), 6.81 (t,
2-β
2-α
3
2-γ
3J=3JH,F=9.0 Hz, 2H, H–3,5 FPhe), 6.93 (dd, J=8.9 Hz, JH,F=5.6 Hz, 2H, H–2,6 FPhe), 7.15 (t,
3
4
3J=3JH,F= 8.9 Hz, 2H, H–3,5 FPhe), 7.30 (dd, J=8.8 Hz, JH,F=5.5 Hz, 2H, H–2,6 FPhe), 7.38 (d,
3
4
3
J=8.6 Hz, 2H, H–3,5 CH SO Phe), 7.73 (d, J=8.6 Hz, 2H, H–2,6 CH SO Phe). 13C NMR (101
3
3
2
3
2
MHz, CD CN) δ (ppm) 32.19 (CH ), 33.71 (CH ), 44.50 (CH ), 45.82 (CH ), 115.48 (d,
3
2-α
2-β
3
2-γ
2JC,F=21.6 Hz, C–3,5 FPhe), 116.21 (d, 2JC,F=21.6 Hz, C–3,5 FPhe), 127.83, 131.50, 131.95 (d,
3JC,F=8.1 Hz, C–2,6 FPhe), 133.11 (d, 3JC,F=8.1 Hz, C–2,6 FPhe), 139.38 (d, 4JC,F=3.3 Hz, C–4
FPhe), 139.46 (d, 4JC,F=3.4 Hz, C–4 FPhe), 139.94, 139.97, 140.70, 148.62, 162.25 (d,
1JC,F=244.4 Hz, C–1 FPhe), 162.88 (d, 1JC,F=244.2 Hz, C–1 FPhe). F NMR (376 MHz, CD CN)
19
3
δ (ppm) -114.40 – -114.51 (m), -113.97 – -114.09 (m). C H ClF O S [Mmonoisotopic] requires 446,
24
21
2
2
35
+
37
+
35
+
37
+
ESI-MS (ES+) m/z: 447 [M( Cl)+H] , 449 [M( Cl)+H] , 469 [M( Cl)+Na] , 471 [M( Cl)+Na] ;
+
35
HRMS (ESI+) m/z calcd for C H ClF O S [M+Na , Cl] 469.08165, found 469.08061, calcd for
24
21
2
2
+ 35
[
2M+Na , Cl] 915.17353, found 915.17299.
6-chloro-2-(4-(methylsulfonyl)phenyl)hex-1-en-1,1-diyl)dibenzene (3c). To the stirring
complex of 3.9 mL TiCl (35.6 mmol) and 3.50 g Zinc powder (53.5 mmol) in 63 mL THF a
4
suspension of 1.85 g 5-chloro-1-(4-(methylsulfonyl)phenyl)pentan-1-one (2b, 6.7 mmol) and 1.25
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g benzophenone (6.8 mmol) in 133 mL THF was added. Upon purification by column
chromatography (ethyl acetate/petroleum ether; 1/4; v/v) 3c was obtained as white solid with
1
3
9.4% yield. mp: 96-100°C; Rf 0.60 (EtOAc/petroleum ether, 1/1, v/v); H NMR (400 MHz, CD CN)
3
δ (ppm) 1.47–1.37 (m, 2H, CH ), 1.71–1.59 (m, 2H, CH ), 2.50–2.43 (m, 2H, CH ), 3.00 (s,
2-β
2-γ
2-α
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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8
9
0
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9
0
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0
3
3
H, CH ), 3.44 (t, J=6.6 Hz, 2H, CH ), 6.97–6.92 (m, 2H, CHaromatic), 7.10–7.03 (m, 3H,
3 2-δ
3
CHaromatic), 7.34–7.27 (m, 3H, CHaromatic), 7.44–7.36 (m, 4H, CHaromatic), 7.70 (d, J=8.2 Hz, 2H, H-
13
2,6 CH SO Phe). C NMR (101 MHz, CD CN) δ (ppm) 26.40 (CH ), 33.03 (CH ), 35.30 (CH
2-
3
2
3
2-γ
2-β
), 44.44 (CH ), 45.72 (CH ), 127.32, 127.59, 128.03, 128.67, 129.38, 129.92, 131.20, 131.53,
α
3
2-δ
1
39.67, 139.94, 142.35, 143.37, 143.64, 149.18. C H ClO S [Mmonoisotopic] requires 424, ESI-MS
25 25 2
35
+
37
+
(
ES+) m/z: 447 [M( Cl)+Na] , 449 [M( Cl)+Na] , HRMS (ESI+) m/z calcd for C H ClO S
25 25 2
+ 35
+ 35
[
M+NH , Cl] 442.16498, found 442.16020, calcd for [M+Na , Cl] 447.11615, found 447.11560,
4
+
35
calcd for [2M+Na , Cl] 871.24253, found 871.24198; IR (ATR): ѵ = 1591 (m, olefin), 1147 (s,
-1
SO ) cm ; Anal. calcd for C H ClO S: C 70.65, H 5.93, S: 7.55, found: C 70.76, H 5.99, S 7.22.
2
25 25
2
4,4´-(6-chloro-2-(4-(methylsulfonyl)phenyl)hex-1-en-1,1-diyl)bis(fluorobenzene) (3d). To a
stirring suspension of 1.12 mL TiCl4 (8.0 mmol) and 1.05 g Zinc powder (16.3 mmol) in 19 mL
THF a suspension of 0.56 g 5-chloro-1-(4-(methylsulfonyl)phenyl)pentan-1-one (2b, 2.0 mmol)
and 0.44 g 4,4´-difluorobenzophenone (2.0 mmol) in 40 mL THF was added. Upon purification by
column chromatography (ethyl acetate/petroleum ether; 1/1; v/v) 3d was obtained as colorless oil
1
with 75.2% yield. mp: 99-105°C; Rf 0.56 (EtOAc/petroleum ether, 1/1, v/v); H NMR (400 MHz,
CD CN) δ (ppm) 1.47–1.35 (m, 2H, CH ), 1.70–1.58 (m, 2H, CH ), 2.52–2.42 (m, 2H, CH ),
3
2-β
2-γ
2-α
3
3
3
.01 (s, 3H, CH ), 3.48–3.41 (m, 2H, CH ), 6.81 (t, J= JH,F=9.0 Hz, 2H, H–3,5 FPhe), 6.92 (dd,
3 2-δ
3J=8.9 Hz, 4JH,F=5.6 Hz, 2H, H–2,6 FPhe), 7.15 (t, J= JH,F=8.9 Hz, 2H, H–3,5 FPhe), 7.29 (dd,
3
3
3J=8.8 Hz, 4JH,F=5.5 Hz, 2H, H–2,6 FPhe), 7.38 (d, J=8.6 Hz, 2H, H–3,5 CH SO Phe), 7.72 (d,
3
3
2
3
13
J=8.6 Hz, 2H, H–2,6 CH SO Phe). C NMR (101 MHz, CD CN) δ (ppm) 26.32 (CH ), 33.02
3
2
3
2-γ
2
(
CH ), 35.34 (CH ), 44.50 (CH ), 45.72 (CH ), 115.45 (d, JC,F=21.6 Hz, C–3,5 FPhe), 116.03
2-β 2-α 3 2-δ
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(
d, 2JC,F=21.5 Hz, C–3,5 FPhe), 127.75, 131.49, 131.94 (d, 3JC,F=8.2 Hz, C–2,6 FPhe), 133.15 (d,
3JC,F=8.1 Hz, C–2,6 FPhe), 139.47 (d, 4JC,F=3.2 Hz, C–4 FPhe), 139.61 (d, 4JC,F=3.5 Hz, C–4
FPhe), 139.85, 140.11, 140.95, 148.90, 162.21 (d, 1JC,F=244.6 Hz, C–1 FPhe), 162.85 (d,
1JC,F=244.2 Hz, C–1 FPhe). C H ClF O S [Mmonoisotopic] requires 460, ESI-MS (ES+) m/z: 483
25
23
2
2
1
1
1
1
1
1
1
1
1
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5
5
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0
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0
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9
0
35
+
37
+
35
-
[
M( Cl)+Na] ; 485 [M( Cl)+Na] , (ES-) m/z: 459 [M( Cl)-H] .
General procedure for synthesis of compound (4a-d). To a solution of 3 (1 eq.) in
acetonitrile silver nitrate (3 eq.) was added and the reaction mixture was heated for 24
hours at 80°C. The resulting silver salt was filtered off and the solvent was evaporated at
reduced pressure. The dry reaction mixture was suspended in EtOAc, washed three times
with water and dried with Na SO . The raw product was purified by column
2
4
chromatography (EtOAc/ petroleum ether).
-(4-(methylsulfonyl)phenyl)-5,5-diphenylpent-4-en-1-yl nitrate (4a). 21 g 5-chloro-2-(4-
4
(methylsulfonyl)phenyl)pent-1-en-1,1-diyl)dibenzene (3a, 0.5 mmol) and 0.27 g silver nitrate (1.6
mmol) were heated in 3.5 mL acetonitrile. After purification with column chromatography (ethyl
acetate/petroleum ether; 2/3; v/v) and semi-preparative HPLC (acetonitrile/water + 0.1% TFA;
7
/3; v/v; 5 mL/min) 4a was obtained as white solid. yield: 32.6%; purity: 98.6% (water/acetonitrile
1
+
0.1% TFA; 1/1; v/v); mp: 109-111°C; Rf 0.61 (EtOAc/petroleum ether, 1/1, v/v); H NMR (400
MHz, CD CN) δ (ppm) 1.75–1.65 (m, 2H, CH ), 2.59–2.52 (m, 2H, CH ), 3.00 (s, 3H, CH ),
3
2-β
2-α
3
3
4
.35 (t, J=6.5 Hz, 2H, CH ), 6.97–6.93 (m, 2H, CHaromatic), 7.10–7.04 (m, 3H, CHaromatic), 7.36–
2-γ
3
7
.26 (m, 3H, CHaromatic), 7.44–7.37 (m, 4H, CHaromatic), 7.74 (d, J=8.2 Hz, 2H, H–2,6 CH SO Phe).
3
2
1
3
C NMR (101 MHz, CD CN) δ (ppm) 26.33 (CH ), 32.31 (CH ), 44.44 (CH ), 74.11 (CH ),
3
2-β
2-α
3
2-γ
127.44, 127.70, 128.15, 128.71, 129.47, 129.75, 131.11, 131.57, 138.70, 139.86, 142.13, 143.15,
+
143.44, 148.66. C H NO S [M
] requires 437, ESI-MS (ES+) m/z: 460 [M+Na] , 476
24
23
5
monoisotopic
+
+
[M+K] ; HRMS (ESI+) m/z calcd for C H NO S [M+Na] 460.11946, found 460.11830, calcd for
24
23
5
+
[
2M+Na] 897.24915, found 897.24861; IR (ATR): ѵ = 1618, 1283 (s, O-NO ), 1599 (m, olefin),
2
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