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

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.

Introduction

Nitric oxide (NO) that causes vasodilation was found by Furchgott in the 1980s.1 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 physiological and pathophysiological processes.2, 3, 4 Low levels of NO are able to promote cancer growth as a signal transducer by blood flow regulation, smooth muscle relaxation, neurotransmission, platelet reactivity and so on, while high levels of NO can induce cancer cell apoptosis, inhibit metastasis, and sensitize cancer cells to chemotherapy and immunotherapy.5, 6, 7 Therefore, NO has a double‐edged sword effect on the growth of tumors.8 Furoxan is an important class of NO donors, which can release high levels of NO in vitro.9 A series of phenylsulfonylfuroxan and anti‐tumor drug coupling compounds, such as Furoxan/Doxorubicin hybrid, Furoxan/FTS hybrid and Furoxan/Bifendate hybrid, were reported.10, 11, 12 Compared to their original parent compounds, they have higher anti‐tumor activity, safety and drug resistance reversal activity.

One of the most aggressive form of breast cancer,13 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 option, chemotherapy is an alone treatment regimen for these patients.14, 15 Therefore, developing against triple‐negative 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 phosphorylation of MEK1 and ERK1 and inhibition of angiogenesis.16, 17, 18, 19 It is noticeable that the hybrids of phenylsulfonylfuroxan and epiandrosterone/estradiol derivatives show potent anti‐proliferative activity in MDA‐MB‐231 (human triple‐negative breast cancer cell line).18, 19 In particular, compounds 1 and 2 (Figure 1) 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.

Figure 1

The structures of compounds 1, 2 and 3.

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 resulting from its potent inhibition of tumor neovascularization and cell growth.20, 21, 22 The introduction of methoxy group at the 2‐position of estradiol leads to miss estrogen activity, while 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 (5 a–c). As control, six hybrids (8 a–c, 11 a–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 (14 a–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.

Results and Discussion

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 4 a–b, 7 a–b, 10 a–b and 13 a–b in DMF including the present of potassium iodide and sodium hydroxide. Finally, phenylsulfonylfuroxan (21), which was prepared from benzenethiol according to the reported method in ref. [23], was introduced into compounds 4 a–b, 7 a–b, 10 a–b, 13 a–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 5 a–c, 8 a–c, 11 a–c, 14 a–c, 16, 18 and 20 in 10–70 % yields.

Scheme 1

Synthetic routes of target compounds. Reagents and conditions: (a) furoxan (21), DBU, CH2Cl2, r.t.; (b) BrCH2CH2OH, K2CO3, KI, DMF, 90 °C/Cl(CH2)3OH, NaOH, CH3COCH3; (c) NBS, EtOH, r.t.

Biological Evaluations

These fifteen newly synthesized hybrids (5 a–c, 8 a–c, 11 a–c, 14 a–c, 16, 18 and 20) were evaluated for their cytotoxic effects against MDA‐MB‐231. As shown in Table 1, all target compounds 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 5 a–b, 8 a–b, 11 a–b and 14 a–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 5 c, 8 c, 11 c and 14 c 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 8 a–b and 11 a–b had more significant anti‐proliferative effects with the IC50 values of 0.00083–0.0046 μM against MDA‐MB‐231. In particular, compound 11 b (0.00083 μM) bearing 4‐bromo and 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 5 c (0.0384 μM).

Table 1

Anti‐proliferation activities of 5 a–c, 8 a–c, 11 a–c, 14 a–c, 16, 18 and 20 in MDA‐MB‐231.

Compounds	MDA‐MB‐231 (IC50, μM)a
2‐ME(3)	1.268
Furoxan(21)	1.293
5 a	0.0183
5 b	0.0212
5 c	0.0384
8 a	0.0045
8 b	0.0022
8 c	0.4995
11 a	0.0046
11 b	0.00083
11 c	0.0780
14 a	0.3107
14 b	0.0190
14 c	0.4228
16	0.0132
18	0.0190
20	0.0553

[a] The data are the mean of triplicate determinations; IC50 is the concentration of sample for 50 % cell growth inhibitory rate.

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, 8 b, 11 b and 14 b with 3 C linker in 3‐position showed stronger bioactivities than that of 8 a, 11 a and 14 a containing 2 C linker and without linker compounds 5 c, 8 c, 11 c and 14 c, respectively. The information suggested 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 (5 a–c, 8 a–c and 11 a–c) with 17‐carbonyl group (14 a–c) slightly decrease anti‐proliferation action in MDA‐MB‐231. Fourthly, the introduction 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 11 b merited to be further investigated as a promised anti‐cancer candidate.

Furthermore, including 3 C linker at 3‐position of estradiol core three compounds 5 b, 8 b and 11 b 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, 5 b, 8 b and 11 b 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 3.587 μM) of 8 b and 11 b were weaker than that of 2‐ME and 21 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 pharmacologic action would be further explored in the next work.

Table 2

Anti‐proliferation activities of 5 b, 8 b and 11 b in A2780, Hela and HUVEC.

Compounds	A2780 (IC50, μM)a	Hela (IC50, μM)a	HUVEC (IC50, μM)a
2‐ME (3)	0.59	0.0663	1.352
Furoxan (21)	1.92	3.051	1.475
5 b	0.104	2.650	0.725
8 b	0.053	3.848	0.418
11 b	0.029	3.587	0.056

[a] The data are the mean of triplicate determinations; IC50 is the concentration of sample for 50 % cell growth inhibitory rate.

Considering that NO induces cellular apoptosis, our steroidal/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 11 b treated MDA‐MB‐231 cell using flow cytometry. As depicted in Figure 2A and 2B, the total proportion of annexin V+/PI− (the right lower quadrant representing early apoptotic) and annexin V+/PI+ (the right upper quadrant representing late apoptotic and necrotic) cells increased from 5.1 to 81.0 % after they were exposed to 20, 40 and 80 nM 11 b for 24 h.

Figure 2

(A) Apoptosis induced by compound 11 b in MDA‐MB‐231. (B) The percentage (%) of apoptosis cells induced by compound 11 b in MDA‐MB‐231. Apoptotic cells were detected with annexin V/PI double staining after incubation with various concentrations of compound 11 b or diluent (DMSO) for 24 h. (C) 11 b 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 11 b. Meanwhile, 11 b 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 11 b and diluent (DMSO). The experiments were repeated three times.

We next investigated the signaling pathway involved in compound 11 b induced apoptosis. Western blot analysis showed that 11 b in a dose dependent manner up‐regulated the expression and the phosphorylation levels of p53, an important tumor suppressor. Meanwhile, 11 b 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 11 b 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 treatment with 80 nM of 11 b. The result means that 11 b 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 11 b 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 11 b (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 (5 a–c, 8 a–c, 11 a–c, 14 a–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 nitrite intracellularly with the releasing percentage range from 16 to 87 μM, in which the most active compound 11 b released the higher concentration of NO (57 μM) than that of phenylsulfonylfuroxan (25 μM). Furthermore, anti‐cancer activity of 11 b was diminished by pretreatment with a NO scavenger 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.

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 11 b. 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 11 b 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.

Conclusions

In summary, fifteen novel furoxan‐estradiol hybrids (5 a–c, 8 a–c, 11 a–c, 14 a–c, 16, 18 and 20) were prepared and tested for their in vitro anti‐cancer activities. Most of them showed stronger anti‐proliferative effects than that of phenylsulfonylfuroxan (21) and 2‐methoxyestradiol (3) in MDA‐MB‐231, A2780, Hela and HUVEC cell lines. And compound 11 b had the best activity against MDA‐MB‐231 with IC50 value of 0.00083 μM. In our investigation of preliminary pharmacology, 11 b induced apoptosis and hardly affected the cell cycle of MDA‐MB‐231. In addition, higher NO‐releasing capacity of compound 11 b 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 11 b might be a desirable anti‐tumor candidate for further development.

Experimental Section

In vitro anti‐proliferative assay. The in vitro anti‐proliferation of the chemical compounds was measured by the MTT reagent. Briefly, as described in the literature.24 5×103 cells in 100 μL of medium per well were plated in 96‐well plates. After being incubated for 24 h, 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 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 570 nm wavelength. The inhibition rates of proliferation were calculated with the following equation:

The concentrations of the compounds that inhibited cell growth by 50 % (IC50) were calculated using GraphPad Prism, version 6.0. For the NO scavenge experiment, cells were pretreated with the indicated concentrations of hemoglobin (Hb) (0, 1.25, 2.5, 5, or 10 μM) for 1 h and treated with 100 nM 11 b for 24 h. Then the viability of the cells was determined by MTT reagent as described above.

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 literature with some modifications.25 Briefly, cells (1×107 per 10 cm dish) were treated with a 100 μM concentration of each 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 production, while sodium nitrite at different concentrations was measured to generate a standard curve.

Cell apoptosis analysis. Cell apoptosis was detected by flow cytometry according to a previously published method.26 Briefly, cells were incubated with DMSO or different concentrations of compound 11 b for 24 h. The cells were harvested, washed twice with cold 1×PBS, and resuspended in 200 μL of binding buffer at a density of 1×105 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+/PI−, 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 according to a previously published method27 and were analysed by Multicycle AV (for Windows, version 320) software. Briefly, cells were first treated with DMSO or different concentrations of compound 11 b 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 mL of ice‐cold 75 % ethanol at −20 °C overnight and stained with 500 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 11 b 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 SDS‐PAGE electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore) which were then blocked in 10 % non‐fat milk (BD Biosciences) and reacted with primary 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).

Melting points were measured on a SGW X‐4 microscopy melting point apparatus without correction. 1H and 13C 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 (100–200 mesh) and visualized under UV light at 254 and 365 nm.

3‐Phenylsulfonyl‐4‐hydroxylethoxyl‐1,2,5‐oxadiazole 2‐Oxide (21). Synthesis of phenylsulfonylfuroxan from benzenethiol was reported previously in ref. [23].

General procedure for the preparation of . To a stirred solution of 3, 6, 9 and 12 (1 mmol) in DMF (5 mL) at room temperature was added corresponding halo alcohol (2 mmol) and K2CO3 (3 mmol). The mixture was refluxed for 2 to 10 hrs and then poured into water (50 mL). After filtration, the residue was washed with water (3×10 mL), yielded yellow or white solid 4 a–b, 7 a–b, 10 a–b and 13 a–b (40–96 %).

General procedure for the preparation of . 4 a–b, 7 a–b, 10 a–b and 13 a–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 CH2Cl2 (10 mL). The reaction mixture was stirred at room temperature for 3 hrs and then washed with brine. The organic layer was dried with anhydrous Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (PE : EtOAc=12 : 1) to yield 5 a–c, 8 a–c, 11 a–c and 14 a–c (10–64 %).

2‐Methoxy‐3‐((phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐ethoxy‐estradiol (5 a). Compound 4 a (0.20 g) was obtained starting from 3 (1.0 g, 3.3 mmol), yield 17 %. Compound 5 a (0.15 g) was obtained starting from 4 a (0.20 g, 0.58 mmol) and furoxan (0.21 g, 0.58 mmol). 5 a: yield 45 %; Mp 154–157 °C; ESI‐MS m/z (%) 593.2 [M+Na]+; 1H NMR (400 MHz, CDCl3) δ 7.50–8.03 (5H, m, −SO2C6H5), 6.71–6.84 (2H, m, 1‐H, 4‐H), 4.40–4.76 (4H, m, −OCH2CH2O−), 3.80 (3H, s, 2‐OCH3), 3,73 (1H, m, 17‐CH), 0.78 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 158.95, 147.92, 145.66, 138.18, 135.45, 134.31, 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, 27.31, 26.55, 23.13, 11.10; ESI‐HRMS(m/z) [M+Na]+ Calc.: 593.1928, Found: 593.1927.

2‐Methoxy‐3‐((phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐propoxy‐estradiol (5 b). Compound 4 b (0.30 g) was obtained starting from 3 (1.20 g, 4 mmol), yield 21 %. Compound 5 b (0.12 g) was obtained starting from 4b (0.20 g, 0.56 mmol) and furoxan (0.203 g, 0.56 mmol). 5 b: yield 37 %; Mp 51–53 °C; ESI‐MS m/z (%) 585.0 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 7.52–8.02 (5H, m, −SO2C6H5), 6.66–6.83 (2H, m, 1‐H, 4‐H), 4.18–4.66 (4H, m, furoxan−OCH2, 3‐OCH2 ), 3.77 (3H, s, 2‐OCH3), 3.72 (1H, m, 17‐CH), 0.79 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 158.93, 147.60, 146.06, 137.78, 135.52, 133.30, 129.64, 129.04, 128.51, 114.63, 110.49, 109.77, 81.91, 77.03, 76.82, 68.36, 64.96, 56.17, 50.06, 44.30, 43.29, 38.82, 36.78, 30.67, 29.15, 28.67, 27.35, 26.56, 23.13, 11.10; ESI‐HRMS(m/z) [M+H]+ Calc.: 585.2265, Found: 585.2248.

2‐Methoxy‐3‐((phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐estradiol (5 c). Compound 5 c (0.050 g) was obtained starting from 3 (0.3 g, 1.0 mmol) and furoxan (0.37 g, 1.0 mmol). 5 c: yield 10 %; Mp 91–100 °C; ESI‐MS m/z (%) 527.2 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 7.65–8.16 (5H, m, −SO2C6H5), 6.92–6.98 (2H, m, 1‐H, 4‐H), 4.12 (1H, m, OH), 3.74 (1H, m, 17‐CH), 3.70 (3H, s, 2‐OCH3), 0.79 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 159.14, 147.62, 140.15, 139.17, 138.30, 135.55, 129.69, 129.60, 128.73, 121.66, 110.77, 110.35, 81.82, 77.02, 76.81, 55.91, 50.10, 44.49, 43.19, 38.34, 36.70, 30.67, 28.70, 27.05, 26.37, 23.13, 11.06; ESI‐HRMS(m/z) [M+H]+ Calc.: 527.1846, Found: 527.1839.

3‐((Phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐ethoxy‐estradiol (8 a). Compound 7 a (0.35 g) was obtained starting from 6 (1 g, 3.67 mmol), yield 30 %. Compound 8 a (0.034 g) was obtained starting from 7 a (0.20 g, 0.63 mmol) and furoxan (0.23 g, 0.63 mmol). 8 a: yield 10 %; Mp 54–56 °C; ESI‐MS m/z (%) 541.2 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 7.52–8.03 (5H, m, −SO2C6H5), 6.67–7.23 (3H, m, 1‐H, 2‐H, 4‐H), 4.35–4.75 (4H, m, −OCH2CH2O−), 3.74 (1H, t, J=8.36, 8.28, 17‐CH), 0.78 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 158.87, 156.12, 138.31, 138.20, 135.53, 133.70, 129.66, 128.59, 126.56, 114.73, 112.31, 110.40, 81.91, 77.02, 76.81, 69.69, 65.30, 50.07, 43.99, 43.28, 38.85, 36.72, 30.63, 29.71, 27.22, 26.36, 23.15, 11.70; ESI‐HRMS(m/z) [M+H]+ Calc.: 541.2003, Found: 541.1997.

3‐((Phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐propoxy‐estradiol (8 b). Compound 7 b (0.60 g) was obtained starting from 6 (1.08 g, 4 mmol), yield 46 %. Compound 8 b (0.15 g) was obtained starting from 7 b (0.20 g, 0.6 mmol) and furoxan (0.22 g, 0.60 mmol). 8 b: yield 45 %; Mp 155–157 °C; ESI‐MS m/z (%) 555.0 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 7.50–8.00 (5H, m, −SO2C6H5), 6.65–7.23 (3H, m, 1‐H, 2‐H, 4‐H), 4.13–4.64 (4H, m, −OCH2CH2O−), 3.74 (1H, t, J=8.36, 8.28, 17‐CH), 0.78 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 158.94, 156.45, 138.16, 138.11, 135.53, 133.13, 129.64, 128.51, 126.45, 114.56, 112.02, 110.48, 81.93, 77.02, 76.81, 68.20, 63.28, 50.07, 44.00, 43.28, 38.90, 36.73, 30.63, 29.81, 28.69, 27.25, 26.38, 23.15, 11.07; ESI‐HRMS(m/z) [M+Na]+ Calc.: 577.1979, Found: 577.1964.

3‐((Phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐estradiol (8 c). Compound 8 c (0.35 g) was obtained starting from 6 (0.50 g, 1.8 mmol) and furoxan (0.80 g, 2.2 mmol). 8 c: yield 39 %; Mp 104–108 °C; ESI‐MS m/z (%) 497.2 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 7.63–8.11 (5H, m, −SO2C6H5), 6.99–7.35 (3H, m, 1‐H, 2‐H, 4‐H), 3.74 (1H, s, 17‐CH), 0.79 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 158.81, 150.36, 139.18, 139.12, 138.08, 135.74, 129.75, 128.66, 126.94, 119.74, 116.86, 110.80, 81.82, 77.03, 76.82, 50.08, 44.13, 43.21, 38.41, 36.65, 30.60, 29.60, 26.93, 26.16, 23.14, 11.05; ESI‐HRMS(m/z) [M+H]+ Calc.: 497.1741, Found: 497.1731.

4‐Bromo‐3‐((phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐ethoxy‐estradiol (11 a). Compound 10 a (0.45 g) was obtained starting from 9 (0.50 g, 1.43 mmol), yield 80 %. Compound 11 a (0.050 g) was obtained starting from 10 a (0.30 g, 0.76 mmol) and furoxan (0.28 g, 0.67 mmol). 11 a: yield 10 %; Mp 159–163 °C; ESI‐MS m/z (%) 641.1 [M+Na]+; 1H NMR (400 MHz, CDCl3) δ 7.48–8.03 (5H, m, −SO2C6H5), 6.79–7.27 (2H, m, 1‐H, 2‐H), 4.40–4.84 (4H, m, −OCH2CH2O−), 3.74 (1H, s, 17‐CH), 0.78 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 158.97, 152.65, 138.16, 138.11, 136.10, 135.42, 129.62, 128.54, 124.98, 115.87, 111.16, 110.51, 81.83, 77.03, 76.81, 69.44, 67.02, 50.01, 44.23, 43.15, 38.00, 36.64, 31.24, 30.65, 27.41, 26.57, 23.11, 11.02; ESI‐HRMS(m/z) [M+Na]+ Calc.: 641.0928, Found: 641.0904.

4‐Bromo‐3‐((phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐propoxy‐estradiol (11 b). Compound 10 b (0.60 g) was obtained starting from 9 (1.08 g, 4 mmol), yield 46 %. Compound 11 b (0.040 g) was obtained starting from 10 b (0.20 g, 0.51 mmol) and furoxan (0.186 g, 0.51 mmol). 11 b: yield 12 %; Mp 77–80 °C; ESI‐MS m/z (%) 633.1 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 7.49–7.99 (5H, m, −SO2C6H5), 6.78–7.24 (2H, m, 1‐H, 2‐H), 4.21–4.73 (4H, m, furoxan−OCH2, 3‐OCH2 ), 3.74 (1H, t, J=7.76, 8.60, 17‐CH), 0.78 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 158.90, 152.87, 138.08, 137.91, 135.49, 135.41, 129.60, 128.49, 124.89, 115.65, 110.60, 110.50, 81.85, 77.02, 76.81, 68.17, 64.73, 50.01, 44.22, 43.15, 38.04, 36.65, 31.20, 30.65, 28.54, 27.43, 26.59, 23.11, 11.01; ESI‐HRMS(m/z) [M+H]+ Calc.: 633.1265, Found: 633.1259.

4‐Bromo‐3‐((phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐estradiol (11 c). Compound 11 c (0.10 g) was obtained starting from 9 (0.30 g, 1.8 mmol) and furoxan (0.80 g, 2.2 mmol). 11 c: yield 20 %; Mp 81–83 °C; ESI‐MS m/z (%) 597.1 [M+Na]+; 1H NMR (400 MHz, CDCl3) δ 7.63–8.19 (5H, m, −SO2C6H5), 7.17–7.38 (2H, m, 1‐H, 2‐H), 3.73 (1H, s, 17‐CH), 0.78 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 158.46, 147.26, 141.77, 139.02, 138.08, 135.78, 129.75, 128.84, 125.68, 118.94, 117.59, 110.68, 81.74, 77.02, 76.81, 50.00, 44.42, 43.08, 37.56, 36.58, 31.06, 30.62, 27.10, 26.35, 23.09, 10.98; ESI‐HRMS(m/z) [M+Na]+ Calc.: 597.0665, Found: 597.0643.

3‐((Phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐ethoxy‐17‐estrone (14 a). Compound 13 a (0.55 g) was obtained starting from 12 (1.0 g, 3.7 mmol), yield 47 %. Compound 14 a (0.33 g) was obtained starting from 13 a (0.30 g, 0.95 mmol) and furoxan (0.35 g, 0.95 mmol). 14 a: yield 64 %; Mp 120–123 °C; ESI‐MS m/z (%) 539.2 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 7.53–8.01 (5H, m, −SO2C6H5), 6.67–7.23 (3H, m, 1‐H, 2‐H, 4‐H), 4.13–4.64 (4H, m, −OCH2CH2O−), 0.91 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 229.29, 158.86, 156.28, 138.19, 138.09, 135.54, 133.09, 129.66, 128.59, 126.57, 114.79, 112.45, 110.40, 77.03, 76.81, 69.67, 65.32, 50.44, 48.01, 44.02, 38.37, 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 (14 b). Compound 13 b (0.62 g) was obtained starting from 12 (2.16 g, 8.0 mmol), yield 23 %. Compound 14 b (0.12 g) was obtained starting from 13 b (0.30 g, 0.91 mmol) and furoxan (0.33 g, 0.91 mmol). 14 b: yield 24 %; Mp 138–141 °C; ESI‐MS m/z (%) 553.0 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 7.52–8.02 (5H, m, −SO2C6H5), 6.69–7.23 (3H, m, 1‐H, 2‐H, 4‐H), 4.35–4.75 (4H, m, furoxan−OCH2, 3‐OCH2), 0.90 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 229.23, 158.93, 156.62, 138.11, 137.94, 135.54, 132.52, 129.64, 128.52, 126.46, 114.62, 112.16, 110.48, 77.03, 76.82, 68.20, 63.33, 50.44, 48.02, 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 (14 c). Compound 14 c (0.10 g) was obtained starting from 12 (0.30 g, 0.86 mmol) and furoxan (0.31 g, 0.86 mmol). 14 c: yield 20 %; Mp 143–144 °C; ESI‐MS m/z (%) 495.2 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 7.33–8.12 (5H, m, −SO2C6H5), 7.02–7.07 (3H, m, 1‐H, 2‐H, 4‐H), 0.92 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 229.56, 158.79, 150.51, 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.

3‐((Phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐17‐ethynyl‐estradiol (16). Compound 16 (0.10 g) was obtained starting from 15 (0.10 g, 0.34 mmol) and furoxan (0.13 g, 0.34 mmol). 16: yield 57 %; Mp 84–87 °C; ESI‐MS m/z (%) 521.2 [M+H]+; 1H NMR (400 MHz, CDCl3) δ 7.63–8.12 (5H, m, −SO2C6H5), 6.99–7.33 (3H, m, 1‐H, 2‐H, 4‐H), 0.89 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 158.80, 150.39, 139.10, 139.06, 138.09, 135.75, 129.75, 128.66, 126.97, 119.74, 116.89, 110.80, 87.41, 79.82, 77.03, 76.81, 74.17, 49.48, 47.05, 43.72, 38.97, 38.97, 32.70, 29.61, 26.93, 26.24, 22.81, 12.66; ESI‐HRMS(m/z) [M+Na]+ Calc.: 543.1560, Found: 543.1559.

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+H]+; 1H NMR (400 MHz, CDCl3) δ 7.63–8.11 (5H, m, −SO2C6H5), 6.99–7.35 (3H, m, 1‐H, 2‐H, 4‐H), 4.76 (1H, d, H‐16), 2.58 (1H, s, −CCH), 1.47 (6H, s, −C(CH3)2), 0.93 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) 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, 26.98, 25.92, 25.72, 16.93; ESI‐HRMS(m/z) [M+H]+ Calc.: 577.2003, Found: 577.1997.

3‐((Phenylsulfonyl)‐1,2,5‐oxadiazole 2‐oxide)‐oxy‐16‐hydroxy‐17‐ethynyl‐estradiol (20). Compound 20 (0.12 g) was obtained starting from 19 (0.10 g, 0.32 mmol) and furoxan (0.12 g, 0.32 mmol). 20: yield 70 %; Mp 120–124 °C; ESI‐MS m/z (%) 559.0 [M+Na]+; 1H NMR (400 MHz, CDCl3) δ 7.63–8.12 (5H, m, −SO2C6H5), 6.99–7.33 (3H, m, 1‐H, 2‐H, 4‐H), 0.93 (3H, s, 18‐CH3). 13C NMR (151 MHz, CDCl3) δ 158.81, 150.40, 138.99, 138.08, 135.75, 129.76, 128.66, 126.85, 119.75, 116.91, 110.80, 84.50, 78.35, 78.07, 77.03, 76.82, 73.30, 48.87, 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.

Conflict of interest

The authors declare no conflict of interest.