Synthesis and Antitumor Evaluation of Novel Hybrids of Phenylsulfonylfuroxan and Estradiol Derivatives

Article abstract of DOI:10.1002/open.201900228

Fifteen novel furoxan-based nitric oxide (NO) releasing hybrids of estradiol derivatives were synthesized and evaluated in vitro anti-proliferative activity in MDA-MB-231, A2780, Hela and HUVEC cell lines. Most of them displayed potent anti-proliferative effects. Among the compounds, 4-bromo-3-((phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-propoxy-estradiol (11 b) exhibited the best activity with IC₅₀ values of 3.58–0.0008 μM. Preliminary pharmacological studies showed that 11 b induced apoptosis and hardly affected the cell cycle of MDA-MB-231 cell line. NO-releasing capacity and inhibition of ERK/MAPK pathway signaling might explain the potent antineoplastic activity of these compounds. The preliminary structure-activity relationship (SAR) showed that steroidal scaffolds with a linker in 3-position were favorable moieties to evidently increase the bioactivities of these hybrids. Overall, these results implied that 11 b merited to be further investigated as a promising anti-cancer candidate.

Full text of DOI:10.1002/open.201900228

DOI: 10.1002/open.201900228
Full Papers
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Synthesis and Antitumor Evaluation of Novel Hybrids of
Phenylsulfonylfuroxan and Estradiol Derivatives
+ [a]
+ [b]
[a]
[a]
[a]
[a]
Qi Wan , Yan Deng , Yaoqing Huang, Zhihui Yu, Chunli Wang, Ke Wang,
[b]
[a]
Jibin Dong,* and Ying Chen*
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Fifteen novel furoxan-based nitric oxide (NO) releasing hybrids
of estradiol derivatives were synthesized and evaluated in vitro
anti-proliferative activity in MDA-MB-231, A2780, Hela and
HUVEC cell lines. Most of them displayed potent anti-prolifer-
ative effects. Among the compounds, 4-bromo-3-((phenylsul-
fonyl)-1,2,5-oxadiazole 2-oxide)-oxy-propoxy-estradiol (11b) ex-
hibited the best activity with IC50 values of 3.58–0.0008 μM.
Preliminary pharmacological studies showed that 11b induced
apoptosis and hardly affected the cell cycle of MDA-MB-231 cell
line. NO-releasing capacity and inhibition of ERK/MAPK pathway
signaling might explain the potent antineoplastic activity of
these compounds. The preliminary structure-activity relation-
ship (SAR) showed that steroidal scaffolds with a linker in 3-
position were favorable moieties to evidently increase the
bioactivities of these hybrids. Overall, these results implied that
11b merited to be further investigated as a promising anti-
cancer candidate.
[
13]
1
. Introduction
One of the most aggressive form of breast cancer, triple-
negative breast cancer (TNBC), which usually lacks expression of
estrogen receptors (ER), progesterone receptors (PgR), and
HER2 protein. Due to consequently unresponsive to both
endocrine and anti-HER2 therapy and limiting the therapeutic
Nitric oxide (NO) that causes vasodilation was found by
[1]
Furchgott in the 1980s. NO, as a key signaling molecule
synthesized naturally from L-arginine by the action of NO
synthase (NOS), plays very important role in a variety of
option, chemotherapy is an alone treatment regimen for these
[2–4]
[14–15]
physiological and pathophysiological processes.
Low levels
patients.
Therefore, developing against triple-negative
of NO are able to promote cancer growth as a signal transducer
by blood flow regulation, smooth muscle relaxation, neuro-
transmission, platelet reactivity and so on, while high levels of
NO can induce cancer cell apoptosis, inhibit metastasis, and
breast cancer drugs is a challenging project and has attracted
many researchers to conduct research.
In our previous research, several furoxan-based NO releasing
hybrids with the coumarin core or steroidal scaffold were
synthesized and exhibited excellent anti-cancer biological
activities through inducing apoptosis, disrupting the phosphor-
ylation of MEK1 and ERK1 and inhibition of angiogenesis.
is noticeable that the hybrids of phenylsulfonylfuroxan and
epiandrosterone/estradiol derivatives show potent anti-prolifer-
[5–7]
sensitize cancer cells to chemotherapy and immunotherapy.
Therefore, NO has a double-edged sword effect on the growth
[8]
[16–19]
of tumors. Furoxan is an important class of NO donors, which
It
[9]
can release high levels of NO in vitro. A series of phenyl-
sulfonylfuroxan and anti-tumor drug coupling compounds,
such as Furoxan/Doxorubicin hybrid, Furoxan/FTS hybrid and
ative activity in MDA-MB-231 (human triple-negative breast
[10–12]
[18–19]
Furoxan/Bifendate hybrid, were reported.
Compared to
cancer cell line).
In particular, compounds 1 and 2 (Figure 1)
their original parent compounds, they have higher anti-tumor
activity, safety and drug resistance reversal activity.
bearing furoxan at 17-position of 3-methoxy estradiol by 2–3 C
linkers showed the strong cytotoxicity for MDA-MB-231 cell line
with IC50 values of 1.8 and 0.7 nM, respectively.
As we known, the endogenous estrogen metabolite 2-
methoxyestradiol (3, 2-ME, Figure 1), formed from estradiol
through the action of catechol-O-methyl transferase, has been
researched for several decades as a potential anticancer agent
+
[
a] Q. Wan, Y. Huang, Z. Yu, C. Wang, K. Wang, Prof. Y. Chen
Department of Medicinal Chemistry
School of Pharmacy
Fudan University
26, Zhangheng Road, Shanghai (China)
8
resulting from its potent inhibition of tumor neovascularization
E-mail: yingchen71@fudan.edu.cn
[20–22]
and cell growth.
The introduction of methoxy group at the
+
[b] Y. Deng, Assoc. Prof. J. Dong
Department of Pharmacology and Biochemistry
School of Pharmacy
2
-position of estradiol leads to miss estrogen activity, while
Fudan University
826, Zhangheng Road, Shanghai (China)
E-mail: jbdong@shmu.edu.cn
+
[
] Q. W. and Y. D. are co-first-authors and contributed equally to this work.
©
2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution Non-Commercial NoDerivs License, which permits use and dis-
tribution in any medium, provided the original work is properly cited, the
use is non-commercial and no modifications or adaptations are made.
Figure 1. The structures of compounds 1, 2 and 3.
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Full Papers
shows a significant anti-cancer action. The result implied that
substituent modification on phenyl ring of estradiol might
pivotally affect its bioactivity including anti-cancer activity. In
consider of our work mentioned above, the interesting anti-
cancer activities of hybrids (1 and 2) of furoxan and estradiol
using different linkers at 17-position prompted us to further
indirectly or directly couple furoxan at 3-hydroxy of 2-ME by
with or without linker, and synthesized three 2-ME derivatives
with furoxan at its 3-position (5a–c). As control, six hybrids (8a–
c, 11a–c) of furoxan and estradiol with 4-bromo or without
other group in phenyl ring were prepared by using similar
linker type. Moreover, six compounds (14a–c, 16, 18 and 20)
including 17-one and 17-alkynyl groups estradiol derivatives
merging furoxan also were developed. Herein, we reported
design, synthesis and biological evaluation of fifteen novel
hybrids of phenylsulfonylfuroxan and estradiol derivatives.
the present of potassium iodide and sodium hydroxide. Finally,
phenylsulfonylfuroxan (21), which was prepared from benzene-
thiol according to the reported method in ref. [23], was
introduced into compounds 4a–b, 7a–b, 10a–b, 13a–b, 15, 17
and 19 in the presence of 8-diazabicyclo[5.4.0]undec-7-ene
(DBU) to synthesize fifteen furoxan and estradiol derivatives
coupling compounds 5a–c, 8a–c, 11a–c, 14a–c, 16, 18 and 20
in 10–70% yields.
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2.2. Biological Evaluations
These fifteen newly synthesized hybrids (5a–c, 8a–c, 11a–c,
14a–c, 16, 18 and 20) were evaluated for their cytotoxic effects
against MDA-MB-231. As shown in Table 1, all target com-
pounds displayed better anti-proliferative activity against MDA-
MB-231 with the IC50 values range from 0.00083 to 0.4995 μM
than that of control compounds phenylsulfonylfuroxan (21,
1.29 μM IC50 value) and 3 (1.27 μM IC50 value). Among them,
anti-cancer activities with 0.00083–0.3107 μM IC50 values of
eight compounds 5a–b, 8a–b, 11a–b and 14a–b containing
furoxan located to 3-position of estradiol and estrone through
2–3 C linkers were higher than that of furoxan group directly
occupied at 3-position four compounds 5c, 8c, 11c and 14c
with the IC50 values of 0.0384–0.4995 μM. The result was
consistent with our previous research mentioned above in
compounds 1 and 2. Moreover, 3 C linker four compounds 8a–
b and 11a–b had more significant anti-proliferative effects with
the IC50 values of 0.00083–0.0046 μM against MDA-MB-231. In
particular, compound 11b (0.00083 μM) bearing 4-bromo and
2. Results and Discussion
2
.1. Chemistry
As depicted in Scheme 1, in the ethanol solution, 4-bromide
estradiol (9) were prepared by the bromination of commercial
available estradiol (6) with NBS in 43% yield. Then, 2-methoxy-
estradiol (3), estradiol (6), estrone (12) and compound 9 were
reacted with 2-bromoethanol or 3-chloropropanol to form
intermediates 4a–b, 7a–b, 10a–b and 13a–b in DMF including
3
C linker with furoxan at 3-position was the most potent
molecule. It was notable that three hybrids of furoxan and 17β-
alkynyl estradiol (16, 18 and 20) also retained the relevant anti-
cancer potency possessing the IC50 values of 0.0132–0.0553 μM
compared
to
2-methoxyl-3-furoxan-oxy-estradiol
5c
(0.0384 μM).
Table 1. Anti-proliferation activities of 5a–c, 8a–c, 11a–c, 14a–c, 16, 18
and 20 in MDA-MB-231.
Compounds
MDA-MB-231
(IC50, μM)
a
2
-ME(3)
1.268
1.293
Furoxan(21)
5
5
5
a
b
c
0.0183
0.0212
0.0384
0.0045
0.0022
0.4995
0.0046
0.00083
0.0780
0.3107
0.0190
0.4228
0.0132
0.0190
0.0553
8a
8
8
1
b
c
1a
11b
1
1
1
1c
4a
4b
14c
1
1
2
6
8
0
Scheme 1. Synthetic routes of target compounds. Reagents and conditions:
(
(
a) furoxan (21), DBU, CH
2
Cl
2
, r.t.; (b) BrCH
2
CH
2
OH, K
2
CO
3
, KI, DMF, 90°C/Cl
[a] The data are the mean of triplicate determinations; IC50 is the
concentration of sample for 50% cell growth inhibitory rate.
CH OH, NaOH, CH COCH
2
)
3
3
3
; (c) NBS, EtOH, r.t.
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According to the data displayed in Table 1, the preliminary
structure-activity relationship (SAR) of the hybrids can be
inferred. First, the anti-proliferative activities of the hybrids
against MDA-MB-231 were improved with the 2–3 C length of
the spacers connecting the NO donors at 3-position of estradiol
and estrone. For example, 8b, 11b and 14b with 3 C linker in
1
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3
-position showed stronger bioactivities than that of 8a, 11a
and 14a containing 2 C linker and without linker compounds
c, 8c, 11c and 14c, respectively. The information suggested
5
that the linker between steroidal skeleton and furoxan was
crucial to preserve the strong anti-cancer activity. Second, 4-
bromo substitution of estradiol was more favorable group for
anti-proliferation activity compared to 2-methoxy derivative.
Thirdly, the replacement of 17-hydroxy in estradiol (5a–c, 8a–c
and 11a–c) with 17-carbonyl group (14a–c) slightly decrease
anti-proliferation action in MDA-MB-231. Fourthly, the introduc-
tion of alkynyl group at 17-position of estradiol can keep the
strong anti-cancer activity. Overall, the best bioactivity hybrid of
furoxan and estradiol with 4-bromo and 3 C linker at 3-position
1
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Figure 2. (A) Apoptosis induced by compound 11b in MDA-MB-231. (B) The
percentage (%) of apoptosis cells induced by compound 11b in MDA-MB-
2
31. Apoptotic cells were detected with annexin V/PI double staining after
incubation with various concentrations of compound 11b or diluent (DMSO)
for 24 h. (C) 11b significantly up-regulated the expression of proapoptotic
protein Bax, but might down-regulate the expression of the antiapoptotic
proteins Bcl-2 after treatment with 80 nM 11b. Meanwhile, 11b could also
up-regulate the expression and the phosphorylation levels of p53, but might
down-regulate the expression of ERK1/2. (D) The profiles showed the
proportions (%) in each phase of MDA-MB-231 cells treated with 11b and
diluent (DMSO). The experiments were repeated three times.
1
1b merited to be further investigated as a promised anti-
cancer candidate.
Furthermore, including 3 C linker at 3-position of estradiol
core three compounds 5b, 8b and 11b were selected to screen
their cytotoxic effects against A2780 (human ovary cancer cell
lines), Hela (human cervical cancer cell lines) and HUVEC
(umbilical vein endothelium cell lines). Interestingly, 5b, 8b and
late apoptotic and necrotic) cells increased from 5.1 to 81.0%
after they were exposed to 20, 40 and 80 nM 11b for 24 h.
We next investigated the signaling pathway involved in
compound 11b induced apoptosis. Western blot analysis
showed that 11b in a dose dependent manner up-regulated
the expression and the phosphorylation levels of p53, an
important tumor suppressor. Meanwhile, 11b significantly up-
regulated the expression of pro-apoptotic protein Bax, and
down-regulated the expression of the anti-apoptotic proteins
Bcl-2 in 80 nM concentration (Figure 2C). The outcome accounts
for that compound 11b induced the expression and the
phosphorylation levels of p53, which subsequently promoted
Bax and reduced Bcl-2 expression. Additionally, as described in
Figure 2C, the expression of ERK1/2 was decreased after treat-
ment with 80 nM of 11b. The result means that 11b also could
disturb the ERK/MAPK signaling pathway in certain degree to
inhibit the growth of tumor. Then, we further tested the effect
of compound 11b on the cell cycle of MDA-MB-231. As
illustrated in Figure 2D, compared to the control cells treated
with DMSO, when MDA-MB-231 cells were treated with
increasing concentrations of 11b (20, 40, and 80 nM), a
significant effect of the cell cycle was not observed.
With the ability to produce high levels of NO in vitro,
furoxan is an important class of NO donor. As shown in
Figure 3A, the intracellular NO production capability of these
furoxan/ hybrids (5a–c, 8a–c, 11a–c, 14a–c, 16, 18 and 20)
were determined and presented as that of nitrite in the cell
lysates using a Griess assay, with phenylsulfonylfuroxan and
compound 3 as positive and negative controls, respectively. As
expected, compound 3 without furoxan group was hardly
detected with nitrite in A2780 cells, whereas the compounds
bearing the furoxan moiety could produce various levels of
1
1b all possessed better bioactivities with the IC50 values of
0
.104–0.725, 0.053–0.418 and 0.0286–0.056 μM than that of 3
(0.59–1.352 μM) and 21 (1.92–1.475 μM) in A2780 and HUVEC
cell lines (Table 2). While in Hela cell lines, activities (3.848 and
3
2
.587 μM) of 8b and 11b were weaker than that of 2-ME and
1 with the IC50 values of 0.993 and 3.051 μM, respectively. The
results implied that inhibiting action of these compounds have
selectivity in different cancer call lines. Then, their pharmaco-
logic action would be further explored in the next work.
Considering that NO induces cellular apoptosis, our steroi-
dal/furoxan hybrids with the ability to release NO might also
have a similar mechanism of inducing apoptosis against tumor
cells. To determine the number and stage of apoptotic cells, the
annexin-V/PI double staining assay was applied to quantitate
1
1b treated MDA-MB-231 cell using flow cytometry. As
depicted in Figure 2A and 2B, the total proportion of annexin
V+/PIÀ (the right lower quadrant representing early apoptotic)
and annexin V+/PI+ (the right upper quadrant representing
Table 2. Anti-proliferation activities of 5b, 8b and 11b in A2780, Hela and
HUVEC.
Compounds
A2780
(IC50, μM)
Hela
(IC50, μM)
HUVEC
(IC50, μM)
a
a
a
2
-ME (3)
0.59
1.92
0.104
0.053
0.029
0.0663
3.051
2.650
3.848
3.587
1.352
1.475
0.725
0.418
0.056
Furoxan (21)
5
8
1
b
b
1b
[a] The data are the mean of triplicate determinations; IC50 is the
concentration of sample for 50% cell growth inhibitory rate.
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the cells were treated with different concentration of tested
compound or DMSO (as negative control) for 48 h. Then the
medium with compound or DMSO was replaced with 200 μL of
fresh medium containing 10% MTT (5 mg/mL in PBS) in each well
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and incubated at 37
°
C for 4 h. Last, the MTT-containing medium
was discarded and 150 μL of DMSO per well was added to dissolve
the formazan crystals newly formed. Absorbance of each well was
determined by a microplate reader (Synergy H4, Bio-Tek) at a
5
70 nm wavelength. The inhibition rates of proliferation were
calculated with the following equation:
Figure 3. (A) Variable levels of NO produced by some steroidal/furoxan
hybrids in A2780 cells. Results are indicated as the mean ꢀ SD (standard
error) of three independent experiments. (B) Effects of hemoglobin on the
anti-proliferative effect of 11b. A2780 was pretreated with the indicated
concentrations of hemoglobin (0, 1.25, 2.5, 5, or 10 μM) for 1 h and treated
with 100 nM 11b for 24 h. The results are expressed as the percentage of
cell growth inhibition relative to control cells. Data are the mean value ꢀ SD
obtained from three determinations.
1
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Inhibition ratio ð%Þ ¼ ðODDMSOÀ ODcompdÞ=ðODDMSOÀ ODblankÞ � 100
The concentrations of the compounds that inhibited cell growth by
5
0% (IC ) were calculated using GraphPad Prism, version 6.0. For
50
the NO scavenge experiment, cells were pretreated with the
indicated concentrations of hemoglobin (Hb) (0, 1.25, 2.5, 5, or
1
0 μM) for 1 h and treated with 100 nM 11b for 24 h. Then the
viability of the cells was determined by MTT reagent as described
above.
nitrite intracellularly with the releasing percentage range from
1
6 to 87 μM, in which the most active compound 11b released
Nitrite measurement in vitro. The levels of NO released by tested
compounds in the cells are presented as that of nitrite and were
determined by the Griess reagent (Beyotime, China), according to
the higher concentration of NO (57 μM) than that of phenyl-
sulfonylfuroxan (25 μM). Furthermore, anti-cancer activity of
[25]
7
the literature with some modifications. Briefly, cells (1×10 per
0 cm dish) were treated with a 100 μM concentration of each
1
1b was diminished by pretreatment with a NO scavenger
1
Hemoglobin in a dose-dependent manner (Figure 3B). The
results indicated that the potent anti-proliferation activities may
be partly attributed to the release of nitric oxide.
compound for 150 min. Subsequently, the cells were harvested and
lysed with 100 μL of RIPA lysis buffer (Beyotime, China) for 30 min
on ice. The cell lysates were mixed with Griess for 30 min in a dark
place, followed by measurement by a microplate reader (Synergy
H4, Bio-Tek) at 540 nm wavelength. The cells treated with diluent
were used to determine the background levels of nitrite produc-
tion, while sodium nitrite at different concentrations was measured
to generate a standard curve.
3. Conclusions
In summary, fifteen novel furoxan-estradiol hybrids (5a–c, 8a–c,
1a–c, 14a–c, 16, 18 and 20) were prepared and tested for
Cell apoptosis analysis. Cell apoptosis was detected by flow
1
[26]
cytometry according to a previously published method. Briefly,
their in vitro anti-cancer activities. Most of them showed
stronger anti-proliferative effects than that of phenylsulfonylfur-
oxan (21) and 2-methoxyestradiol (3) in MDA-MB-231, A2780,
Hela and HUVEC cell lines. And compound 11b had the best
activity against MDA-MB-231 with IC50 value of 0.00083 μM. In
our investigation of preliminary pharmacology, 11b induced
apoptosis and hardly affected the cell cycle of MDA-MB-231. In
addition, higher NO-releasing capacity of compound 11b was
consistent with its better anti-tumor activity, which can be
eliminated by NO scavenger Hemoglobin. The initial SAR
elucidated that the length of 2–3 C linkers between furoxan
group and steroidal scaffold was very important to notably
improve their anti-tumor potency. Moreover, bromo group at 4-
position in aromatic ring of estradiol was much better than
introduction of methoxy to 2-position for improving anti-cancer
activity. Therefore, further investigation of structure-activity
relationship should be conducted in the future. And, all above
results prompted that compound 11b might be a desirable
anti-tumor candidate for further development.
cells were incubated with DMSO or different concentrations of
compound 11b for 24 h. The cells were harvested, washed twice
with cold 1×PBS, and resuspended in 200 μL of binding buffer at a
5
density of 1×10 cells/mL. The cells were then stained with 5 μL of
annexin-V and PI for 15 min in dark conditions at room temperature
and subjected to analysis by flow cytometry (Cytomics FC 500 MPL,
Beckman Coulter). The early apoptosis was evaluated based on the
percentage of cells with annexin V+/PIÀ , while the late apoptosis
was that of annexin V+/PI+. The results were indicated as mean
values from three independent determinations.
Cell cycle analysis. Cell cycle status was detected by flow cytometry
[27]
according to a previously published method and were analysed
by Multicycle AV (for Windows, version 320) software. Briefly, cells
were first treated with DMSO or different concentrations of
compound 11b for 24 h and then harvested, washed twice with 1×
PBS, and resuspended in 200 μL of 1×PBS. The cells were fixed in
4
5
mL of ice-cold 75% ethanol at À 20°C overnight and stained with
00 μL of propidium iodide (50 μg/mL, Sigma) containing 0.1%
RNase (1 mg/mL, Sigma) for 15 min in dark conditions at room
temperature. The cells were then analysed by flow cytometry
(Cytomics FC 500 MPL, Beckman Coulter). The results were
indicated as mean values from three independent determinations.
Western blot analysis. MDA-MB-231 cells were treated with DMSO or
different concentrations of compound 11b for 24 h. Cells were
harvested, washed with cold 1×PBS, and lysed with RIPA lysis
buffer (Beyotime, China) for 30 min on ice, then centrifuged at
12000 g for 15 min at 4°C. The total protein concentration was
determined by BCA protein assay kit (Beyotime, China). Equal
amounts (30 μg per load) of protein samples were subjected to
Experimental Section
In vitro anti-proliferative assay. The in vitro anti-proliferation of the
chemical compounds was measured by the MTT reagent. Briefly, as
[
24]
3
described in the literature. 5×10 cells in 100 μL of medium per
well were plated in 96-well plates. After being incubated for 24 h,
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SDS-PAGE electrophoresis and transferred onto polyvinylidene
fluoride (PVDF) membranes (Millipore) which were then blocked in
2-Methoxy-3-((phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-estradiol
(5c). Compound 5c (0.050 g) was obtained starting from 3 (0.3 g,
1.0 mmol) and furoxan (0.37 g, 1.0 mmol). 5c: yield 10%; Mp 91–
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
1
2
3
4
5
6
7
10% non-fat milk (BD Biosciences) and reacted with primary
+
1
antibodies. The antibodies against Bcl-2, Bax, p53, P-p53 and ERK1/
2 were purchased from Beyotime Biotechnology. The secondary
antibodies conjugated with horseradish peroxidase (HRP) were
from Cell Signaling Technology. The protein bands were developed
by the chemiluminescent reagents (Millipore).
100°C; ESI-MS m/z (%) 527.2 [M+H] ; H NMR (400 MHz, CDCl ) δ
3
7.65–8.16 (5H, m, À SO
C H ), 6.92–6.98 (2H, m, 1-H, 4-H), 4.12 (1H,
2 6 5
m, OH), 3.74 (1H, m, 17-CH), 3.70 (3H, s, 2-OCH ), 0.79 (3H, s, 18-
3
1
3
CH3). C NMR (151 MHz, CDCl ) δ 159.14, 147.62, 140.15, 139.17,
3
138.30, 135.55, 129.69, 129.60, 128.73, 121.66, 110.77, 110.35, 81.82,
7
7.02, 76.81, 55.91, 50.10, 44.49, 43.19, 38.34, 36.70, 30.67, 28.70,
Melting points were measured on a SGW X-4 microscopy melting
+
27.05, 26.37, 23.13, 11.06; ESI-HRMS(m/z) [M+H] Calc.: 527.1846,
1
13
point apparatus without correction. H and C NMR spectral data
were recorded with a Varian 400 MHz spectrometer at 303 K using
TMS as an internal standard. Mass spectra were recorded on Agilent
Technologies 1260 infinity LC/MS instrument, and HRMS spectra
were recorded on an Agilent Technologies LC/MSD TOF instrument.
Analytical and preparative TLC was performed on silica gel HSGF/
UV 254. The chromatograms were conducted on silica gel (100–
Found: 527.1839.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
3-((Phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-ethoxy-estradiol (8a).
Compound 7a (0.35 g) was obtained starting from
6 (1 g,
3.67 mmol), yield 30%. Compound 8a (0.034 g) was obtained
starting from 7a (0.20 g, 0.63 mmol) and furoxan (0.23 g,
0.63 mmol). 8a: yield 10%; Mp 54–56°C; ESI-MS m/z (%) 541.2 [M+
H] ; H NMR (400 MHz, CDCl ) δ 7.52–8.03 (5H, m, À SO C H ), 6.67–
+
1
200 mesh) and visualized under UV light at 254 and 365 nm.
3
2
6
5
7
.23 (3H, m, 1-H, 2-H, 4-H), 4.35–4.75 (4H, m, À OCH CH OÀ ), 3.74
2
2
3-Phenylsulfonyl-4-hydroxylethoxyl-1,2,5-oxadiazole 2-Oxide (21). Syn-
thesis of phenylsulfonylfuroxan from benzenethiol was reported
previously in ref. [23].
13
(1H, t, J=8.36, 8.28, 17-CH), 0.78 (3H, s, 18-CH ). C NMR (151 MHz,
3
CDCl ) δ 158.87, 156.12, 138.31, 138.20, 135.53, 133.70, 129.66,
3
128.59, 126.56, 114.73, 112.31, 110.40, 81.91, 77.02, 76.81, 69.69,
6
2
5
5.30, 50.07, 43.99, 43.28, 38.85, 36.72, 30.63, 29.71, 27.22, 26.36,
3.15, 11.70; ESI-HRMS(m/z) [M+H] Calc.: 541.2003, Found:
41.1997.
General procedure for the preparation of 4a–b, 7a–b, 10a–b and
+
1
3a–b. To a stirred solution of 3, 6, 9 and 12 (1 mmol) in DMF
(5 mL) at room temperature was added corresponding halo alcohol
(
1
2 mmol) and K CO (3 mmol). The mixture was refluxed for 2 to
0 hrs and then poured into water (50 mL). After filtration, the
2
3
3-((Phenylsulfonyl)-1,2,5-oxadiazole
2-oxide)-oxy-propoxy-estradiol
(
4
8b). Compound 7b (0.60 g) was obtained starting from 6 (1.08 g,
mmol), yield 46%. Compound 8b (0.15 g) was obtained starting
residue was washed with water (3×10 mL), yielded yellow or white
solid 4a–b, 7a–b, 10a–b and 13a–b (40–96%).
from 7b (0.20 g, 0.6 mmol) and furoxan (0.22 g, 0.60 mmol). 8b:
+
1
yield 45%; Mp 155–157°C; ESI-MS m/z (%) 555.0 [M+H] ; H NMR
General procedure for the preparation of 5a–c, 8a–c, 11a–c and
(
400 MHz, CDCl ) δ 7.50–8.00 (5H, m, À SO C H ), 6.65–7.23 (3H, m,
1
4a–c. 4a–b, 7a–b, 10a–b and 13a–b (1 equiv.) were added to a
stirred solution of 21 (1 equiv.) in the presence of 8-diazabicyclo
[5.4.0]undec-7-ene (DBU) (3 equiv.) in CH Cl (10 mL). The reaction
3
2
6
5
1-H, 2-H, 4-H), 4.13–4.64 (4H, m, À OCH
CH
OÀ ), 3.74 (1H, t, J=8.36,
2
2
1
3
8
1
1
5
2
.28, 17-CH), 0.78 (3H, s, 18-CH ). C NMR (151 MHz, CDCl ) δ
3
3
2
2
58.94, 156.45, 138.16, 138.11, 135.53, 133.13, 129.64, 128.51,
26.45, 114.56, 112.02, 110.48, 81.93, 77.02, 76.81, 68.20, 63.28,
0.07, 44.00, 43.28, 38.90, 36.73, 30.63, 29.81, 28.69, 27.25, 26.38,
mixture was stirred at room temperature for 3 hrs and then washed
with brine. The organic layer was dried with anhydrous Na SO . The
2
4
solvent was removed under reduced pressure, and the residue was
purified by column chromatography (PE:EtOAc=12:1) to yield 5a–
c, 8a–c, 11a–c and 14a–c (10–64%).
+
3.15, 11.07; ESI-HRMS(m/z) [M+Na] Calc.: 577.1979, Found:
577.1964.
3
-((Phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-estradiol (8c). Com-
2
-Methoxy-3-((phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-ethoxy-es-
tradiol (5a). Compound 4a (0.20 g) was obtained starting from 3
1.0 g, 3.3 mmol), yield 17%. Compound 5a (0.15 g) was obtained
pound 8c (0.35 g) was obtained starting from 6 (0.50 g, 1.8 mmol)
and furoxan (0.80 g, 2.2 mmol). 8c: yield 39%; Mp 104–108 C; ESI-
°
(
+
1
MS m/z (%) 497.2 [M+H] ; H NMR (400 MHz, CDCl
) δ 7.63–8.11
starting from 4a (0.20 g, 0.58 mmol) and furoxan (0.21 g,
3
0.58 mmol). 5a: yield 45%; Mp 154–157°C; ESI-MS m/z (%) 593.2 [M
(
5H, m, À SO C H ), 6.99–7.35 (3H, m, 1-H, 2-H, 4-H), 3.74 (1H, s, 17-
2
6
5
1
3
+
1
CH), 0.79 (3H, s, 18-CH ). C NMR (151 MHz, CDCl ) δ 158.81, 150.36,
+
6
(
(
Na] ; H NMR (400 MHz, CDCl ) δ 7.50–8.03 (5H, m, À SO C H ),
3
3
3
2
6
5
1
1
3
39.18, 139.12, 138.08, 135.74, 129.75, 128.66, 126.94, 119.74,
16.86, 110.80, 81.82, 77.03, 76.82, 50.08, 44.13, 43.21, 38.41, 36.65,
0.60, 29.60, 26.93, 26.16, 23.14, 11.05; ESI-HRMS(m/z) [M+H]
.71–6.84 (2H, m, 1-H, 4-H), 4.40–4.76 (4H, m, À OCH CH OÀ ), 3.80
2
2
13
3H, s, 2-OCH ), 3,73 (1H, m, 17-CH), 0.78 (3H, s, 18-CH ). C NMR
3
3
+
151 MHz, CDCl ) δ 158.95, 147.92, 145.66, 138.18, 135.45, 134.31,
3
Calc.: 497.1741, Found: 497.1731.
129.61, 129.25, 128.61, 116.05, 110.50, 110.09, 81.89, 77.03, 76.82,
69.85, 67.16, 56.18, 50.07, 44.33, 43.27, 38.77, 36.78, 30.68, 29.70,
4-Bromo-3-((phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-ethoxy-es-
+
27.31, 26.55, 23.13, 11.10; ESI-HRMS(m/z) [M+Na] Calc.: 593.1928,
tradiol (11a). Compound 10a (0.45 g) was obtained starting from 9
0.50 g, 1.43 mmol), yield 80%. Compound 11a (0.050 g) was
obtained starting from 10a (0.30 g, 0.76 mmol) and furoxan (0.28 g,
Found: 593.1927.
(
2
-Methoxy-3-((phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-propoxy-
estradiol (5b). Compound 4b (0.30 g) was obtained starting from 3
1.20 g, 4 mmol), yield 21%. Compound 5b (0.12 g) was obtained
starting from 4b (0.20 g, 0.56 mmol) and furoxan (0.203 g,
0
.67 mmol). 11a: yield 10%; Mp 159–163°C; ESI-MS m/z (%) 641.1
+
1
[
6
M+Na] ; H NMR (400 MHz, CDCl ) δ 7.48–8.03 (5H, m, À SO C H ),
(
3
2
6
5
.79–7.27 (2H, m, 1-H, 2-H), 4.40–4.84 (4H, m, À OCH CH OÀ ), 3.74
2
2
13
.56 mmol). 5b: yield 37%; Mp 51–53°C; ESI-MS m/z (%) 585.0 [M+
(
1H, s, 17-CH), 0.78 (3H, s, 18-CH ). C NMR (151 MHz, CDCl ) δ
3
3
0
+
1
1
58.97, 152.65, 138.16, 138.11, 136.10, 135.42, 129.62, 128.54,
H] ; H NMR (400 MHz, CDCl ) δ 7.52–8.02 (5H, m, À SO C H ), 6.66–
3
2
6
5
124.98, 115.87, 111.16, 110.51, 81.83, 77.03, 76.81, 69.44, 67.02,
6.83 (2H, m, 1-H, 4-H), 4.18–4.66 (4H, m, furoxanÀ OCH , 3-OCH ),
2
2
1
3
50.01, 44.23, 43.15, 38.00, 36.64, 31.24, 30.65, 27.41, 26.57, 23.11,
3.77 (3H, s, 2-OCH ), 3.72 (1H, m, 17-CH), 0.79 (3H, s, 18-CH ).
C
3
3
+
1
1.02; ESI-HRMS(m/z) [M+Na] Calc.: 641.0928, Found: 641.0904.
NMR (151 MHz, CDCl
) δ 158.93, 147.60, 146.06, 137.78, 135.52,
3
1
7
2
33.30, 129.64, 129.04, 128.51, 114.63, 110.49, 109.77, 81.91, 77.03,
4
-Bromo-3-((phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-propoxy-es-
6.82, 68.36, 64.96, 56.17, 50.06, 44.30, 43.29, 38.82, 36.78, 30.67,
tradiol (11b). Compound 10b (0.60 g) was obtained starting from 9
1.08 g, 4 mmol), yield 46%. Compound 11b (0.040 g) was obtained
starting from 10b (0.20 g, 0.51 mmol) and furoxan (0.186 g,
+
9.15, 28.67, 27.35, 26.56, 23.13, 11.10; ESI-HRMS(m/z) [M+H]
(
Calc.: 585.2265, Found: 585.2248.
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Full Papers
0
+
6
.51 mmol). 11b: yield 12%; Mp 77–80°C; ESI-MS m/z (%) 633.1 [M
3-((Phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-16,17-acetone ketal-
17-ethynyl- estradiol (18). Compound 18 (0.30 g) was obtained
starting from 17 (0.50 g, 1.42 mmol) and furoxan (0.52 g,
1.42 mmol). 18: yield 37%; Mp 77–83°C; ESI-MS m/z (%) 577.2 [M+
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
1
2
3
4
5
6
7
+
1
H] ; H NMR (400 MHz, CDCl ) δ 7.49–7.99 (5H, m, À SO C H ),
3
2
6
5
.78–7.24 (2H, m, 1-H, 2-H), 4.21–4.73 (4H, m, furoxanÀ OCH , 3-
2
1
3
OCH ), 3.74 (1H, t, J=7.76, 8.60, 17-CH), 0.78 (3H, s, 18-CH ).
C
2
3
+
1
NMR (151 MHz, CDCl
) δ 158.90, 152.87, 138.08, 137.91, 135.49,
H] ; H NMR (400 MHz, CDCl
) δ 7.63–8.11 (5H, m, À SO
C H ), 6.99–
2 6 5
3
3
1
7
2
6
35.41, 129.60, 128.49, 124.89, 115.65, 110.60, 110.50, 81.85, 77.02,
7.35 (3H, m, 1-H, 2-H, 4-H), 4.76 (1H, d, H-16), 2.58 (1H, s, À CCH),
13
6.81, 68.17, 64.73, 50.01, 44.22, 43.15, 38.04, 36.65, 31.20, 30.65,
1.47 (6H, s, À C(CH ) ), 0.93 (3H, s, 18-CH ). C NMR (151 MHz, CDCl )
3
2
3
3
+
8.54, 27.43, 26.59, 23.11, 11.01; ESI-HRMS(m/z) [M+H] Calc.:
δ 158.79, 150.41, 138.98, 138.95, 138.09, 135.75, 129.76, 128.66,
126.90, 119.74, 116.90, 112.14, 110.80, 87.45, 87.35, 84.30, 77.02,
76.81, 74.67, 47.90, 46.51, 43.66, 38.13, 34.22, 30.96, 29.57, 27.73,
33.1265, Found: 633.1259.
4
(
-Bromo-3-((phenylsulfonyl)-1,2,5-oxadiazole
2-oxide)-oxy-estradiol
+
26.98, 25.92, 25.72, 16.93; ESI-HRMS(m/z) [M+H] Calc.: 577.2003,
11c). Compound 11c (0.10 g) was obtained starting from 9 (0.30 g,
Found: 577.1997.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
1
8
.8 mmol) and furoxan (0.80 g, 2.2 mmol). 11c: yield 20%; Mp 81–
+
1
3°C; ESI-MS m/z (%) 597.1 [M+Na] ; H NMR (400 MHz, CDCl ) δ
3-((Phenylsulfonyl)-1,2,5-oxadiazole
2-oxide)-oxy-16-hydroxy-17-
3
7.63–8.19 (5H, m, À SO C H ), 7.17–7.38 (2H, m, 1-H, 2-H), 3.73 (1H, s,
ethynyl-estradiol (20). Compound 20 (0.12 g) was obtained starting
2
6
5
13
17-CH), 0.78 (3H, s, 18-CH ). C NMR (151 MHz, CDCl ) δ 158.46,
3 3
from 19 (0.10 g, 0.32 mmol) and furoxan (0.12 g, 0.32 mmol). 20:
+
1
1
1
3
47.26, 141.77, 139.02, 138.08, 135.78, 129.75, 128.84, 125.68,
18.94, 117.59, 110.68, 81.74, 77.02, 76.81, 50.00, 44.42, 43.08, 37.56,
yield 70%; Mp 120–124°C; ESI-MS m/z (%) 559.0 [M+Na] ; H NMR
(400 MHz, CDCl ) δ 7.63–8.12 (5H, m, À SO C H ), 6.99–7.33 (3H, m,
3
2
6
5
1
3
6.58, 31.06, 30.62, 27.10, 26.35, 23.09, 10.98; ESI-HRMS(m/z) [M+
1-H, 2-H, 4-H), 0.93 (3H, s, 18-CH ). C NMR (151 MHz, CDCl ) δ
3 3
+
Na] Calc.: 597.0665, Found: 597.0643.
158.81, 150.40, 138.99, 138.08, 135.75, 129.76, 128.66, 126.85,
19.75, 116.91, 110.80, 84.50, 78.35, 78.07, 77.03, 76.82, 73.30, 48.87,
1
3
(
(
-((Phenylsulfonyl)-1,2,5-oxadiazole
2-oxide)-oxy-ethoxy-17-estrone
46.63, 43.59, 38.53, 33.95, 29.94, 29.70, 29.58, 27.53, 25.47, 16.33;
ESI-HRMS(m/z) [M+Na] Calc.: 559.1509, Found: 559.1503.
14a). Compound 13a (0.55 g) was obtained starting from 12
1.0 g, 3.7 mmol), yield 47%. Compound 14a (0.33 g) was obtained
+
starting from 13a (0.30 g, 0.95 mmol) and furoxan (0.35 g,
0.95 mmol). 14a: yield 64%; Mp 120–123°C; ESI-MS m/z (%) 539.2
+
1
[M+H] ; H NMR (400 MHz, CDCl
) δ 7.53–8.01 (5H, m, À SO
C H ),
2
3
2 6 5
Acknowledgements
6
0
1
1
3
.67–7.23 (3H, m, 1-H, 2-H, 4-H), 4.13–4.64 (4H, m, À OCH CH OÀ ),
2
13
.91 (3H, s, 18-CH ). C NMR (151 MHz, CDCl ) δ 229.29, 158.86,
3
3
56.28, 138.19, 138.09, 135.54, 133.09, 129.66, 128.59, 126.57,
14.79, 112.45, 110.40, 77.03, 76.81, 69.67, 65.32, 50.44, 48.01, 44.02,
Many thanks to the grant from National Science & Technology
Major Project “Key New Drug Creation and Manufacturing
Program”, China (Number: 2018ZX09711002) for supporting this
study.
8.37, 35.87, 31.60, 29.68, 26.52, 25.97, 21.60, 13.87; ESI-HRMS(m/z)
+
[M+H] Calc.: 539.1846, Found: 539.1834.
3
(
-((Phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-propoxy-17-estrone
14b). Compound 13b (0.62 g) was obtained starting from 12
(2.16 g, 8.0 mmol), yield 23%. Compound 14b (0.12 g) was
obtained starting from 13b (0.30 g, 0.91 mmol) and furoxan (0.33 g,
Conflict of Interest
0
.91 mmol). 14b: yield 24%; Mp 138–141°C; ESI-MS m/z (%) 553.0
+
1
[
M+H] ; H NMR (400 MHz, CDCl ) δ 7.52–8.02 (5H, m, À SO C H ),
The authors declare no conflict of interest.
3
2
6
5
6
.69–7.23 (3H, m, 1-H, 2-H, 4-H), 4.35–4.75 (4H, m, furoxanÀ OCH , 3-
2
13
OCH ), 0.90 (3H, s, 18-CH ). C NMR (151 MHz, CDCl ) δ 229.23,
2
3
3
Keywords: apoptosis
· estradiol · nitric oxide · furoxan
158.93, 156.62, 138.11, 137.94, 135.54, 132.52, 129.64, 128.52,
derivatives · anticancer drugs
126.46, 114.62, 112.16, 110.48, 77.03, 76.82, 68.20, 63.33, 50.44,
4
8.02, 44.03, 38.41, 35.88, 31.61, 29.67, 28.60, 26.55, 25.97, 21.60,
+
13.87; ESI-HRMS(m/z) [M+H] Calc.: 553.2003, Found: 553.1995.
3-((Phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-17-estrone (14c).
Compound 14c (0.10 g) was obtained starting from 12 (0.30 g,
0
.86 mmol) and furoxan (0.31 g, 0.86 mmol). 14c: yield 20%; Mp
[1] R. F. Furchgott, J. V. Zawadzki, Nature 1980, 288, 373–376.
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+
1
1
43–144°C; ESI-MS m/z (%) 495.2 [M+H] ; H NMR (400 MHz,
2
CDCl ) δ 7.33–8.12 (5H, m, À SO C H ), 7.02–7.07 (3H, m, 1-H, 2-H, 4-
3
2
6
5
13
[3] X. Q. Wei, I. G. Charles, A. Smith, J. Ure, G. J. Feng, F. P. Huang, D. Xu, W.
H), 0.92 (3H, s, 18-CH ). C NMR (151 MHz, CDCl ) δ 229.56, 158.79,
3
3
Muller, S. Moncada, F. Y. Liew, Nature 1995, 375, 408–411.
4] L. J. Ignarro, Biochem. Pharmacol. 1991, 41, 485–490.
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1
50.51, 138.89, 138.57, 138.06, 135.77, 129.76, 128.67, 126.96,
[
[
119.85, 117.05, 110.80, 77.03, 76.81, 50.44, 47.91, 44.15, 37.94, 35.83,
+
31.54, 29.47, 26.24, 25.77, 21.60, 13.84; ESI-HRMS(m/z) [M+Na]
Calc.: 517.1404, Found: 517.1387.
[6] R. Sullivan, C. H. Graham, Curr. Pharm. Des. 2008, 14, 1113–1123.
[
[
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Manuscript received: July 9, 2019
Revised manuscript received: October 16, 2019
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