Novel Hybrid Conjugates with Dual Suppression of Estrogenic and

Article Cite This: J. Med. Chem. 2018, 61, 8155−8173

pubs.acs.org/jmc

Novel Hybrid Conjugates with Dual Suppression of Estrogenic and Inflammatory Activities Display Significantly Improved Potency against Breast Cancer Wentao Ning,†,∥ Zhiye Hu,†,∥ Chu Tang,‡ Lu Yang,† Silong Zhang,† Chune Dong,† Jian Huang,§ and Hai-Bing Zhou*,† J. Med. Chem. 2018.61:8155-8173. Downloaded from pubs.acs.org by UNIV OF TEXAS SW MEDICAL CTR on 10/02/18. For personal use only.

†

State Key Laboratory of Virology, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China ‡ Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education, School of Life Science and Technology, Xidian University, Xi’an 710126, Shaanxi, China § College of Life Sciences, Wuhan University, Wuhan 430072, China S Supporting Information *

ABSTRACT: In this work, we developed a small library of novel OBHS-RES hybrid compounds with dual inhibition activities targeting both the estrogen receptor α (ERα) and NFκB by incorporating resveratrol (RES), a known inhibitor of NF-κB, into a privileged indirect antagonism structural motif (OBHS, oxabicycloheptene sulfonate) of estrogen receptor (ER). The OBHS-RES conjugates could bind well to ER and showed remarkable ERα antagonistic activity, and they also exhibited excellent NO inhibition in macrophage RAW 264.7 cells. Compared with 4-hydroxytamoxifen, some of them showed better antiproliferative efficacy in MCF-7 cell lines with IC50 up to 3.7 μM. In vivo experiments in a MCF-7 breast cancer model in Balb/c nude mice indicated that compound 26a was more potent than tamoxifen. Exploration of the compliancy of the structure against ER specificity utilizing these types of isomeric three-dimensional ligands indicated that one enantiomer had much better biological activity than the other.

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INTRODUCTION

It mainly includes three groups of antiestrogen drugs, selective estrogen receptor modulators (SERMs), selective estrogen receptor downregulators (SERDs), and aromatase inhibitors (AIs). SERMs, represented by tamoxifen, are effective in some forms of breast cancer, notably ER-positive disease.12 However, in this clinical pathological subtype, de novo resistance to estrogen antagonists is encountered; in addition, endocrine therapy-resistant disease typically develops in patients who are initially responsive.13,14 SERDs are more complete ER antagonists; they can degrade ERs at high doses, providing more effective treatments,15 but resistance is still encountered.16 As an alternative to endocrine therapy, AIs are also effective in this subtype of breast cancer. However, they also encounter treatment resistance with obvious side effects.17 Inflammation is an exacerbating factor in cancers and elicits tumor tissue infiltration and metastases.18 In fact, nearly 150 years ago, the relationship between inflammation and cancers was noticed.19 Patients with chronic inflammatory diseases are at a much higher risk of developing cancer, and inflammation is

Among a variety of cancers, the most commonly diagnosed cancer among females is breast cancer.1,2 Although the overall 5-year relative survival rate has improved in developed countries, 3,4 the incidence rate remains high, with a continuously younger patient population.5 At diagnosis, approximately 70% of patients have ER-positive breast cancer, and ER is well-known to be a favorable prognostic factor in breast cancer.6 The primary function of the ER is to activate gene transcription following binding of estrogen to the receptor.7 As a transcription factor of the nuclear receptor superfamily, ER can be classified into two isoforms, ERα and ERβ.8,9 ERα has a more profound effect not only on the development and function of the mammary gland and uterus but also on maintaining metabolic and skeletal homeostasis, among other effects. By contrast, ERβ has more pronounced effects on the central nervous system.10 Therefore, ERα can serve as an important target for endocrine therapy of breast cancer. Endocrine therapy is a useful treatment for ER-positive breast cancer patients.11 © 2018 American Chemical Society

Received: February 9, 2018 Published: July 27, 2018 8155

DOI: 10.1021/acs.jmedchem.8b00224 J. Med. Chem. 2018, 61, 8155−8173

Journal of Medicinal Chemistry

Article

Figure 1. Structures of resveratrol, resveratrol conjugates, and OBHS and OBHS conjugates.

Figure 2. Design of bifunctional OBHS-RES conjugates.

The natural product resveratrol (3,5,4′-trihydroxystilbene, RES, 1, Figure 1) has numerous biological activities, such as acting as a cardioprotective and neuroprotective agent. In addition, resveratrol is a known inhibitor of NF-κB, which is used as an anti-inflammatory drug.34−36 Although resveratrol is favorable for intervening in some therapeutically interesting pathways, its low potency limits its utility.35,37 To address this issue, Zhu and his colleagues designed and synthesized a novel class of resveratrol-based aspirin prodrugs (2, Figure 1) to enhance anticancer activities and thereby attenuate the side effects caused by aspirin.38 It has also been reported that sulfate-conjugated resveratrol metabolites (3, Figure 1) are less active than resveratrol but retain some degree of antiinflammatory activity.39 Additionally, resveratrol can inhibit hypoxia/reoxygenation-induced NF-κB activation and attenuate hypoxia/reoxygenation-caused inflammatory responses in alveolar epithelial cells. 34 In breast cancer treatment, resveratrol can also enhance the efficacy of sorafenib-mediated apoptosis in MCF-7 cells.40 In recent years, our group has been engaged in the search for breast cancer drug candidates, and some potential SERMs with three-dimensional core scaffolds have been designed and synthesized. OBHS (4, Figure 1), a kind of oxabicycloheptene compound, has been shown to have the greatest potential,

thought to contribute to approximately 25% of all cancer cases worldwide,20−22 suggesting a strong relationship between inflammation and carcinogenesis. Activation of the NF-κB transcription family plays a central role in inflammation.23−25 A large amount of evidence26−28 has shown that ER/NF-κB interactions are implicated in the progression of breast cancer. In addition, NF-κB signaling driving oncogenic transcription programs29,30 may contribute to the development of endocrine therapy resistance.13 Currently, antitumor agents do not uniformly provide optimal antiproliferative as well as anti-inflammatory effects. This consideration is particularly pertinent in ER-positive breast cancer, where antiestrogens, though antiproliferative, generally have poor anti-inflammatory activity, whereas the proproliferative activity of estrogens renders their generally antiinflammatory activity of no benefit in hormone-responsive disease.13 Additionally, estrogen signaling through the ER can also inhibit NF-κB activation to exert anti-inflammatory activity, but it will also promote cell proliferation.26 Antiproliferative agents that also suppress NF-κB signaling could be especially effective as breast cancer endocrine therapeutics.31−33 Therefore, we hoped to develop a type of novel small molecule having dual inhibition that targeted both ER and NF-κB. 8156



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Scheme 1. Synthetic Route of Compound 17aa

a

Reaction conditions: (a) NBS, p-toluenesulfonic acid monohydrate, CHCl3, rt, 12 h; (b) TEA, CH3CN, rt, 12 h; (c) NaH, DMSO, rt, 2 h; (d) BBr3, DCM, 0 °C, 10 h; (e) diisobutylaluminum hydride, THF, −78 °C, 12 h; (f) Ph3P+CH3Br−, NaH, THF, reflux, 2 h; (g) Pd(PPh3)2Cl2, Et3N, DMF, 120 °C, 12 h; (h) Py·HCl, 200 °C, 2 h.

Scheme 2. Synthetic Route of Analogues 20a−e and 22a−ra

Reaction conditions: (a) TEA, DCM, 0 °C, 12 h; (b) BBr3, DCM, 0 °C, 10 h.

a

behaving overall as a partial antagonist,41 and this compound had moderate anti-inflammatory activity.42 In some reports, the introduction of privileged substructure to some molecular scaffolds can increase the binding affinity to receptors and obtain potent derivatives,43 with some successful examples provided in our previous work.44,45 For example, the OBHSHDACi conjugate (5, Figure 1), as a good ER ligand, can efficiently antagonize ERα and inhibit histone deacetylase (HDAC) activity. Another example is the OBHS-ferrocene conjugate (6, Figure 1), which exhibits high binding affinity and good selectivity for the two ER subtypes and provides potent effects against ER-positive and ER-negative breast

cancer cell lines. In addition, when the sulfonate group of OBHS is replaced with sulfonamide, the resulting OBHsulfonamide (OBHSA) exhibits full antagonist activity with efficacy in ERα-positive MCF-7 comparable to that of fulvestrant.46,47 To increase its anti-inflammatory effect, we proposed that the combination of the RES moiety into the OBHS scaffold could afford a new series of OBHS-RES conjugates that would not only have anti-inflammatory activity but also act as a good ER ligand. Herein, two series of OBHS-RES conjugates (Figure 2) were designed via the Diels−Alder reaction. These compounds have an anti-inflammatory unit and 8157



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Scheme 3. Synthetic Route of Target Compounds 24a−d, 25a−r, and 26a−e

1 and the Supporting Information section. We used Nbromosuccinimide as the bromide reagent and p-toluenesulfonic acid as an acid catalyst to react with 4-methoxyacetophenone 7 to provide α-bromo-4-methoxyacetophenone 8, which was treated with 4-bromophenylacetic acid 9 and triethylamine in acetonitrile solution to generate compound 10. Subsequently, an intramolecular aldol reaction of compound 10 under the basic condition provided compound 11. Demethylation of 11 with three equivalents of boron tribromide afforded butenolide 12. Then, compound 12 was reduced at −78 °C in the presence of diisobutylaluminum hydride, after careful acidification at a low temperature, to yield the furan 13. The Heck reaction of 13 and 15a,b, using bis(triphenylphosphine) palladium(II) dichloride as the catalyst, provided the furan derivatives 16a,b. Among them,

adequate three-dimensional topology for binding the receptor. Finally, for comparison, we also synthesized some conjugates that have methyl ether groups on the resveratrol unit, or replacement of the resveratrol moiety with trans-stilbene or 3hydroxypinosylvin, and we evaluated a range of biological activities in vitro and in vivo.

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RESULTS AND DISCUSSION Chemistry. OBHS-RES conjugates were obtained via the Diels−Alder reaction of furan derivative 17a or 23 with different dienophiles (Scheme 3), and the synthetic route of compound 23 has been reported in our previous work.41 Regarding the important intermediate (E)-5-(4-(4-(4Hydroxyphenyl)furan-3-yl)styryl)benzene-1,3-diol 17a in the series I products, the synthetic pathway is presented in Scheme 8158



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Scheme 4. Synthesis and Separation of the Enantiomers of Compound 26aa

a Reaction conditions: (a) benzyl bromide, K2CO3, Cs2CO3, DMF, rt, overnight; (b) TBDPSCl, N-Me-imidazole, DMF, rt, overnight; (c) 20a, 90 °C, neat, 9 h; (d) TBAF, THF, rt, 45 min; (e) N,N-dimethylaniline, AlCl3, DCM, 35 °C, 9 h. The absolute configuration is currently unknown, and enantiomers of 26a are drawn arbitrarily.

compound 15a was obtained by a Wittig reaction with commercially available compound 14a and Ph3P+CH3Br−. Compound 15b was commercially available. Finally, by employing pyridine hydrochloride to demethylate compound 16a, we could acquire the important intermediate 17a. The synthetic route of target dienophiles 20a−e and 22a−r is described in Scheme 2. The synthesis of vinyl sulfonate 20e started with commercially available pterostilbene through a

two-step reaction. First, pterostilbene 18a was allowed to react with 2-chloroethanesulfonyl chloride under basic conditions in an ice bath to generate compound 20a, which was subsequently demethylated with boron tribromide at 0 °C to generate the vinyl sulfonate 20e (Scheme 2A). Vinyl sulfonates 20b−d can be obtained under similar conditions. The synthesis of compounds 18b and 18c is described in the Supporting Information. 8159



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Table 1. Relative Binding Affinity (RBA) of OBHS-RES Conjugates to ERα and ERβa

The RBA values were calculated according to the formula: IC50estradiol/IC50compound × 100 ± the range. We set the RBA value of estradiol as 100%. Ki = (100/RBA) × Kd. For estradiol, the Kd value was 3.49 nM and 4.12 nM for ERα and ERβ, respectively.

a

b

The synthesis of vinyl sulfonates 22a−r is shown in Scheme 2B. In the presence of base, introduction of the vinyl sulfonate functionality was achieved by treatment of different phenols with 2-chloroethanesulfonyl chloride. The target OBHS-RES conjugates were then obtained via the reaction of 17a with the appropriate 22a−r as well as 23 with 20a or 20e (Scheme 3). The Diels−Alder reaction between furans (17a or 23) and vinyl sulfonates went smoothly, and the product yields were generally good. Moreover, for additional SAR investigations, we expanded the diversity of conjugates by appending 16a

onto vinyl sulfonates 22a−c,q (Scheme 3A, series I) or appending 23 onto vinyl sulfonates 20b−d (Scheme 3A, series II). Additionally, starting from 16b, we synthesized a conjugate 25r, in which trans-stilbene replaced the 6-phenol group of OBHS, to investigate the importance of the 3,5-dihydroxyl group. It is worth noting that there was a high stereoselectivity in the cycloaddition reaction, as we have observed previously.41 The thermodynamically favorable exo products were more easily generated through this Diels−Alder reaction, which was 8160



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Figure 3. (A) The RBA values of compounds with substituents at the meta-position of the phenyl sulfonate. (B) The RBA values of compounds with substituents at the para-position of phenyl sulfonate.

cavity very well, thus possibly increasing the binding affinity. It is evident that the position and size of the substituents on the phenyl sulfonate could largely influence the RBA values. Additionally, compounds 24a−c, which have a pterostilbene unit, showed lower ERα binding affinity as well as decreased α/β selectivity, compared to 25a−c, which have a resveratrol unit (Table 1, entries 1−3 vs 5−7). In addition, compound 25r, with trans-stilbene replacing the 6-phenol group of OBHS, exhibited lower ERα binding affinity and reduced subtype selectivity compared with compound 25a (Table 1, entries 22 vs 5). Thus, we conclude that the resveratrol unit makes a positive contribution to the binding affinity of ERα in series I compounds. Taking 25a as an example, in which the phenyl sulfonate moiety lacks the substituent, only moderate ERα binding affinity was found. However, the introduction of an electronwithdrawing group, such as a fluorine, chlorine, and trifluoromethyl group, at the ortho-position or para-position significantly decreased the ERα binding affinity (analogues 25d−f and 25h Table 1, entries 8−10 and 12). Additionally, the ERα binding affinity would increase when we introduced an electron-donating group on the phenyl sulfonate (analogues 25c, 25g, and 25p, Table 1, entries 7, 11 and 20). Substituents at the ortho-position of the phenyl sulfonate unit could hardly affect the RBA value (analogues 25f and 25g, Table 1, entries 10 and 11), except 25h with a trifluoromethyl group, a strong electron-withdrawing group, displayed almost 8-fold and 3-fold reduced affinity compared with 25a for ERα and ERβ, respectively (entries 12 vs 5). Finally, conjugates 26a and 26e in series II possessing the pterostilbene or resveratrol unit in the sulfonate part also demonstrated good RBA values for both ERs and a preferential selectivity for ERα (entries 23 and 27). Compared with conjugates 26c and 26d, conjugates 26a, 26b, and 26e (entries 25 and 26 vs entries 23, 24 and 27) could better bind to ERα, suggesting that the resveratrol unit was preferred to the 3-hydroxypinosylvin unit. Figure 3A shows the effect of the substituents at the metaposition of the phenyl sulfonate on binding affinity. It illustrates that halogen and trifluoromethyl substituents had good RBA values and tended to bind to ERβ, especially compound 25i and 25k, showing 5-fold and 7-fold higher affinity for ERβ than ERα. In addition, compound 25o displayed higher ERα selectivity, which might be attributed to bromine possessing a larger atomic radius. Figure 3B shows the

presumed to be due to the high rate and ready reversibility. Because compound 23 is symmetrical, conjugates 26a−e were studied as single isomers. Because 26a showed the highest cell antiproliferative activity and anti-inflammatory activity, we separated their enantiomers to further investigate their activity (see below, Scheme 4 and Table 5). Synthesis of the target compounds 25a−q was accomplished after 17a reacted with vinyl sulfonates, which interestingly were formed as mainly the one regioisomer; the other regioisomers were hardly found (see Supporting Information for more details of NOESY-NMR of 25b). The major regioisomers were studied as racemates. OBHS-RES Conjugates Exhibit Binding Affinity. We used a competitive fluorometric receptor-binding assay48,49 to examine whether the OBHS-RES conjugates could bind to ER, and the results are reported in Table 1. Overall, the position of the resveratrol group in the OBHS scaffold could significantly influence the binding affinity. According to previous work, we realized that the phenolic group could directly affect whether the compounds could effectively bind to the ER ligand.50 One phenolic ring in OBHS mimics the “A ring” of estrogen, which plays a key role in the binding affinity, while the other can tolerate large substituents and its deletion would reduce the binding affinity to ERα to some extent.41 In further studies, it has been found that replacement of phenolic ring with active groups also sometimes produces very good biological activity.44 In fact, RBA values are lower for ERα in the majority of series I compounds, excluding compound 25p, which possesses a methyl group on the para-position of phenyl sulfonate unit. 25p possesses the highest ERα binding affinity as 33.21 and has the highest α/β subtype selectivity up to 23 among all the conjugates (Table 1, entry 20). The good binding affinity of 25p may derive from its ability to form four hydrogen bonds with ERα (computer model of the complex shown in Supporting Information). The RBA values of OBHS were 8.11 and 3.39 for ERα and ERβ, respectively. However, some of our final products presented even better ERα binding affinity and selectivity than OBHS. These results may be understood based on the observation that resveratrol is capable of modulating the inflammatory response as the pathwayselective ligand for ERα36 and OBHS-RES conjugates could bind to ER by using resveratrol moiety to increase the binding affinity. From the computer model of the ERα compound 25p complex, we observed that OBHS-RES conjugates could fill the 8161



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Table 2. Transcriptional Activity of OBHS-RES Conjugates for ERα and ERβa agonist mode ERα entry

compd

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

24a 24b 24c 24d 25a 25b 25c 25d 25e 25f 25g 25h 25i 25j 25k 25l 25m 25n 25o 25p 25q 25r 26a 26b 26c 26d 26e OBHS

EC50 (μM)

0.146

0.273 1.25

4.93

0.071

0.093

antagonist mode ERβ

Eff (%E2) 10 18 −2 18 29 −9 55 16 −5 79 84 −24 −7 41 16 5 22 12 3 77 −10 13 9 6 13 15 −9 58

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1 3 4 3 7 1 17 12 4 10 2 13 2 11 3 6 1 5 4 21 6 8 2 1 4 3 7 2

EC50 (μM) 5.76 0.941 0.009 0.658 0.003 0.276 0.063 0.242 2.90 1.73 0.043 0.002

0.355 0.780 0.222 2.781 0.004 0.031 0.139 0.749 0.194

ERα Eff (%E2) 57 6 37 20 37 10 52 45 7 25 36 48 29 82 60 17 −3 51 −2 29 65 48 23 25 47 62 51 0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

18 4 2 13 8 3 6 2 1 11 3 2 1 4 24 2 2 4 2 1 13 5 1 6 3 4 8 1

IC50 (μM) 9.27 0.003 0.428 0.061 0.048 0.005 0.062 0.166 0.010 0.072 0.932 6.02 0.947 0.054 2.42 0.002 0.049 1.03 0.007 0.149 0.738 1.41 0.019 0.013

ERβ Eff (%E2) 89 97 32 37 4 15 36 7 11 49 36 3 −10 68 4 17 2 8 21 1 −4 34 48 54 47 29 7 69

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 21 9 5 1 2 5 3 7 16 5 4 8 11 1 3 3 3 5 1 3 2 20 3 5 1 2 10

IC50 (μM)

0.646

2.53

1.02 4.31

0.042

3.11

0.069

0.934

0.585

Eff (%E2) 85 94 39 72 76 43 102 98 33 45 100 82 31 97 91 106 46 88 65 42 71 83 32 74 86 93 99 −15

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5 2 1 4 2 1 7 1 10 3 6 4 8 2 7 3 2 5 3 10 12 1 9 7 5 11 4 2

a

In HEK 293T cells, a luciferase reporter assay was used to test the luciferase activity and normalized to the control as 0% and 10 nM E2 as 100%. Data show the mean ± SD from an average of three experiments. Omitted parts could not be accurately determined. For details, see the Experimental Section.

binding affinities of compounds with substituents at the paraposition of phenyl sulfonate. The RBA value results for 25b, 25d, 25e, 25n, and 25p (Table 1, entries 6, 8, 9, 18 and 20) indicated that a lipophilic character and suitable size were necessary substituents. In addition, compound 25p showed the best binding to ERα, indicating that the para-methyl group on the phenyl sulfonate moiety provided a useful contribution to the binding affinity. On the basis of other changes in the substitutions, we observed that compounds 25j and 25q had a bulkier substituent compared with 25a (Table 1, entries 14 and 21 vs 5), resulting in a decrease in RBA values for ERα and further suggesting the importance of the appropriate size of the substituents. Transcription Activation Assays. We used luciferase reporter gene assays44 to test the ER transcriptional activities of various OBHS-RES conjugates, and the results are described in Table 2. ER agonists are typically classified as normal agonists, partial agonists, or superagonists.51 Overall, most compounds have high efficacy as ERα antagonists and ERβ agonists and show higher efficacy in comparison to OBHS. Compared with compound 24a, compound 25a (Table 2, entries 1 vs 5) with an improved RBA value of ERβ demonstrates an approximately 9-fold higher efficacy but decreased potency as an ERβ agonist; moreover, it displays an antagonistic effect on ERα. From conjugate 25r, which has a trans-stilbene replacing the 6-

phenol group of OBHS, we found that in comparison to 25a, the antagonistic effect on ERα and the agonistic effect on ERβ decreased 2.4-fold and 4.2-fold, respectively, suggesting the importance of the 3,5-dihydroxyl groups for the transcriptional activity of OBHS-RES conjugates (Table 2, entries 22 vs 5). Surprisingly, compared with compounds 24a, 24b, and 24c, which act as partial ERβ agonists or ERα antagonists, compound 24d exhibited good ERβ agonistic and ERα antagonistic effects (Table 2, entries 1−3 vs 4). Compound 25a, an analogue of OBHS with replacement of resveratrol by a 6-phenol group, acted as an ERα antagonist and ERβ agonist. In addition, there was a large change in activity on ERα or ERβ after modifying the phenyl sulfonate unit (compounds 25b−q, Table 2, entries 6−21). The potency of these conjugates as ERα antagonists or ERβ agonists was completely altered after a very slight change in the phenyl sulfonate unit. For example, the introduction of halogens into phenyl sulfonate significantly influenced transcriptional activity. The fluoro-substituted compound 25d and trifluoromethyl-substituted compounds 25h and 25i displayed agonist activity at ERβ; moreover, 25i was a superantagonist on ERα (Table 2, entries 8, 12, and 13). However, the chloro analogues 25e, 25f, and bromo analogue 25b (Table 2, entries 9, 10, and 6) were profiled as ERβ antagonists. In fact, these compounds all showed an antagonistic effect on ERα. Indeed, by comparing the ERα antagonistic activities of 25b, 25d, 25e, 25j, 25n, and 25p 8162



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Table 3. Anti-inflammatory Activity of OBHS-RES Conjugatesa entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

compd NO inhibition IC50 (μM)a 24a 24b 24c 24d 25a 25b 25c 25d 25e 25f 25g 25h 25i 25j 25k 25l

>100 >100 1.26 ± 0.57 0.79 ± 0.04 6.02 ± 0.55 16.5 ± 1.15 11.10 ± 0.41 8.88 ± 0.21 6.50 ± 2.65 5.00 ± 0.13 28.38 ± 0.64 17.22 ± 1.38 14.60 ± 2.21 18.70 ± 0.37 2.14 ± 1.02 >100

NO production % inhibitionb

entry

compd

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

25m 25n 25o 25p 25q 25r 26a 26b 26c 26d 26e RES OBHS 4OHT RES + OBHS

20.44 30.75 60.07 65.39 70.87 22.03 42.29 71.87 62.07 60.86 39.07 38.46 41.65 43.04 62.08 12.29

0.30 2.01 1.79 1.59 0.55 1.15 0.41 0.21 2.65 0.13 0.64 1.38 2.21 0.37 2.67 1.14

NO inhibition IC50 (μM)a NO production % inhibitionb 2.41 ± 0.33 15.50 ± 1.94 2.53 ± 0.33 12.50 ± 0.16 3.29 ± 0.68 9.70 ± 0.20 0.44 ± 0.23 2.23 ± 0.39 6.37 ± 1.18 16.70 ± 0.63 0.93 ± 0.07 13.10 ± 1.11 18.40 ± 2.33 >100 11.10 ± 0.66

68.42 41.03 60.86 42.29 44.38 40.41 95.58 72.44 55.07 24.75 80.97 48.03 41.48 21.51 80.42

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.57 0.04 0.55 0.72 0.41 1.01 0.21 0.72 2.73 2.01 2.65 0.64 1.38 0.39 1.93

Experimental values represent the average of three independent experiments ± standard deviation (mean ± SD). bDetermined at a compound concentration of 10 μM. a

(Table 2, entries 6, 8, 9, 14, 18, and 20), we observed that substitution in the para-position of the phenyl sulfonate unit had remarkable effects, with the preferred substituent exhibiting lipophilic properties and a suitable size. In addition, the results for 25c, 25i, 25k, 25l, and 25m (Table 2, entries 13, 15 vs 7, 16 and 17) suggested that the use of electronwithdrawing substituents (CF3 or Cl) at the meta-position of the phenyl sulfonate unit might convey higher ERα antagonist efficacy than electron-donating (Me, OMe, or OH) groups. Of note, the use of a larger group such as α-naphthyl (25q) to replace the phenyl resulted in stronger ERα antagonistic activity. Compounds in series II also showed impressive activities. Compounds possessing a pterostilbene or resveratrol unit in the sulfonate part were capable of antagonizing ERα and agonizing ERβ efficiently, with compound 26a having EC50 or IC50 values up to 4 and 7 nM, respectively (Table 2, entries 23). Conjugates 26b, 26c, and 26d possessing transstilbene or 3-hydroxypinosylvin in place of the resveratrol moiety were also moderate ERα antagonists and ERβ agonists (Table 2, entries 24−26). Anti-inflammatory Assay for the Inhibition of Nitric Oxide Production in Macrophage RAW 264.7 Cell Lines. NF-κB promotes gene expression for a number of cytokines and enzymes in inflammatory process, including inducible nitric oxide synthase (iNOS), the expression of which is increased in macrophage of patients.24 iNOS, an enzyme catalyzing nitric oxide (NO) production, is an effector located downstream of cytokines and NF-κB, which could link inflammation to cancer.19 NO is a key signaling molecule and a critical component of inflammatory responses;20 furthermore, continuous and excessive production of NO by iNOS causes pathophysiological problems, such as chronic inflammatory diseases and cancer development.39,52,53 Therefore, we chose to inhibit the generation of NO in macrophage RAW 264.7 cell lines as an index to examine the antiinflammatory activity of OBHS-RES conjugates. First, to promote the protein expression of iNOS, we used lipopolysaccharide (LPS) as a stimulant to activate macrophages (the RAW 264.7 cell line). Following the overexpression of iNOS, NO production increased, which can be assayed using an NO assay kit. The results of our compounds

when cotreated with LPS for RAW 264.7 cell lines are displayed in Table 3. We also tested the parent compounds OBHS, resveratrol, and the physical mixture of OBHS and resveratrol for comparison. Interestingly, most OBHS-RES conjugates, such as 24c, 24d, 25a, 25e, 25f, 25k, 25m, 25o, 25q, 26a, 26b, 26c, and 26e, displayed excellent NO inhibition, while 4OHT (4-hydroxytamoxifen), a classic SERM for the treatment of breast cancer, did not show any NO inhibition activity. Compared with their parent compounds and physical mixture group, compound 26a, with a pterostilbene group in the sulfonate moiety, was the best one, with an IC50 up to 0.44 μM and a percent inhibition rate determined at a concentration of 10 μM reaching 95% (Figure 4). In general, the conjugates with the resveratrol unit in the

Figure 4. Dose−response curves of the NO inhibitory activities for 26a, RES, OBHS, and RES+OBHS.

phenyl sulfonate part demonstrated excellent NO inhibition (26a and 26e, Table 3, entries 23 and 27). Although 24c and 24d had moderate binding affinity, their NO inhibition was good, suggesting the absence of a direct correspondence between ER binding affinity and anti-inflammatory activity. Interestingly, we found that the substituents at the ortho- or para-position of the phenyl sulfonate part had to be as small as possible, such as compounds 25b, 25d−h, and 25p (Table 3, entries 6, 8−12, and 20). Unexpectedly, compound 25l exerted 8163



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poor NO inhibition, with an IC50 of more than 100 μM, and compound 25n exhibited only moderate NO inhibition, indicating that the hydrophilic groups did not augment the anti-inflammatory activity. Although compound 25r showed a moderate IC50 for anti-inflammatory activity, the inhibition rate was not appreciable, demonstrating the importance of the resveratrol unit for activity. In addition, compared with compounds 26c and 26d, compounds 26a and 26e (Table 3, entries 25 and 26 vs 23 and 27) displayed much better anti-inflammatory activity, again suggesting the need for the resveratrol unit. Antiproliferative Activity toward Breast Cancer Cells. To test the antiproliferative activity, MCF-7 cells were treated with the target compounds, and the results are shown in Table 4. Overall, compared with OBHS, the antiproliferative activity

JSH-23, which can abrogate NF-κB nuclear translocation, with 26a to cotreat MCF-7 cells for 48 h. On the basis of the results, we found that cell inhibition was significantly decreased in the cotreatment group compared with the 26a only treatment group (Figure 5). These findings suggested that the

Table 4. Cell Anti-proliferative Activity of OBHS-RES Conjugates (IC50, μM)a entry

compd

MCF-7

entry

compd

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

24a 24b 24c 24d 25a 25b 25c 25d 25e 25f 25g 25h 25i 25j 25k 25l

>100 >100 77.1 ± 1.45 49.8 ± 0.72 20.1 ± 0.31 16.9 ± 5.33 10.4 ± 0.64 13.1 ± 0.62 17.9 ± 2.74 40.4 ± 3.22 55.8 ± 0.77 18.3 ± 1.01 11.5 ± 0.41 7.8 ± 2.25 12.9 ± 0.79 23.0 ± 0.58

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

25m 25n 25o 25p 25q 25r 26a 26b 26c 26d 26e RES OBHS 4OHT RES+OBHS

MCF-7 16.3 14.1 19.8 9.2 11.0 25.2 3.7 8.3 11.5 17.8 10.5 22.9 32.0 9.5 25.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Figure 5. A comparison of the antiproliferative activity of 26a and JSH-23 cotreatment with 26a in MCF-7 cells. JSH-23 (10 μM) and compound 26a (5, 10, and 20 μM) cotreatment of MCF-7 cells for 48 h. White columns show the results for only treatment with 26a, and black columns show the results for the cotreatment group.

0.90 3.03 1.84 1.79 0.17 0.39 0.56 0.85 1.08 1.32 1.33 1.84 3.46 1.06 0.41

antiproliferative activity exhibited by 26a was partly due to its anti-inflammatory activity, although its antiproliferative activity was likely mainly derived from its excellent ERα antagonistic activity. In Vivo MCF-7 Breast Cancer Model in Balb/c Nude Mice. Considering the excellent activity of compound 26a, we evaluated the antitumor potency of this compound in vivo in murine MCF-7 breast cancer tumor models in Balb/c nude mice (shown in Figure 6). After the tumor size reached ∼80 mm3, the nude mice were randomly divided into four groups consisting of five animals each and systemically administered vehicle control (consisting of cremophor-EL/DMSO/PBS 1:1:8, total 100 μL), tamoxifen (25 mg/kg), or compound 26a (17.5 mg/kg or 25 mg/kg) once every 2 days. We observed that the group treated with compound 26a at a dose of 17.5 mg/kg had a reduced tumor volume and burden (Figure 6A,C). In addition, the group treated with compound 26a at a dose of 25 mg/kg, a dose equivalent to tamoxifen, had a reduced tumor burden by ∼55% on day 12, whereas tamoxifen was somewhat less efficacious, reducing the tumor burden by ∼48%. Moreover, on day 27, the group treated with compound 26a at 25 mg/kg inhibited tumor growth by up to ∼69%, whereas the group treated with tamoxifen at 25 mg/kg showed only moderate inhibition by ∼33%. It is noteworthy that compound 26a did not significantly reduce body weight (Figure 6B), indicating its low toxicity to normal body tissues. These studies demonstrated that compound 26a was more potent than tamoxifen in a murine tumor model of human breast cancer. OBHS-RES Conjugates Have Anti-inflammatory Effects during Antitumor Progression in Balb/c Nude Mice. Many inflammatory cells and signaling molecules are present in the inflammatory microenvironment, which is beneficial for the malignant progression of transformed cells. The inflammatory microenvironment can facilitate the invasion and migration of tumor cells via breakage of the basement membrane.19 In many studies,18,19,54,55 inhibition of inflammatory factor expression, such as NF-κB, interleukin-6 (IL-6),

Experimental values represent the mean ± SD from an average of three experiments.

a

against MCF-7 cell lines of most OBHS-RES conjugates was much stronger, exceeding even their physical mixture group. Comparison to the approved drug 4-hydroxytamoxifen revealed that compounds 25j, 25p, 26a, and 26b showed higher antiproliferative activity in MCF-7 cell lines (Table 4, entries 14, 20, 23, and 24). In addition, compounds 25c, 25i, 25q, 26c, and 26e were equipotent to 4OHT against MCF-7 cells. Furthermore, the compounds possessing pterostilbene and trans-stilbene substituents instead of the 6-phenol group of OBHS (compounds 24a−d and 25r, Table 4, entries 1−4 and 22) displayed lower antiproliferative activity. These results indicated that the antiproliferative activity of these compounds might mainly derive from the antagonism toward ER, and the anti-inflammatory activity could also improve their potency against MCF-7 cell lines. The Anti-inflammatory Activity of 26a Contributes to Its Increased Antiproliferative Activity. On the basis of the above-described experimental results, we identified the best compound 26a, which displayed excellent antiproliferative activity and anti-inflammatory activity. According to our knowledge, antiproliferative activity may originate from excellent antagonistic activity on ERα. To determine the contribution of the anti-inflammatory to the antiproliferative activity, we selected compound 26a for further study. We used 8164



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Figure 6. Antitumor potency of compound 26a in vivo in a murine MCF-7 breast cancer tumor model in Balb/c nude mice. (A) The changes in tumor volume in different treatment groups measured every 3 days. (B) The changes in body weight in different treatment groups measured every 3 days. (C) Average weight of the tumors excised at the end of treatment. (D) Representative photos of the tumors at the end of treatment.

and tumor necrosis factor α (TNF-α), has been shown to improve the outcome of tumor treatment. Consequently, we further evaluated the anti-inflammatory activity of compound 26a in vivo in murine MCF-7 breast cancer tumor models in Balb/c nude mice. After the tumor size reached ∼80 mm3, the nude mice were randomly divided into four groups consisting of four animals each and systemically administered the vehicle control (consisting of cremophor-EL/ DMSO/PBS 1:1:8, total 100 μL), tamoxifen (25 mg/kg), or compound 26a (17.5 mg/kg or 25 mg/kg) once every 2 days. After 2 weeks, immunohistochemistry was used to evaluate the level of inflammatory parameters in the tissue around the tumor. On the basis of the result, we found that compound 26a exhibited good anti-inflammatory activity in the tissue around the tumor, as assessed by the inhibition of expression of NFκB, IL-6, and TNF-α (Figure 7). We believe that this is an indication that the antitumor activity we observed in the therapeutic model was accompanied by a reduction of inflammation. On the basis of this evidence, we could conclude that the anti-inflammatory activity of 26a might contribute to its antitumor activity. Synthesis, Separation, and Biological Evaluation of the Enantiomers of Compound 26a. As we know, chirality of compounds is very common in medicinal chemistry, and usually the precise structure of a ligand can help to determine whether a receptor for hormones and drugs is activated.56 The relatively rigid endogenous steroid ligands normally bind to steroid hormone receptors, while conformationally flexible lipids have the ability to bind to other nuclear receptor superfamily members and exhibit the plasticity in the ligand-

binding pocket to some extent.57 However, ER can bind and respond to various steroidal or nonsteroidal ligands.56 Given the aforementioned experimental results, we wondered whether one of the two isomers of the best compound 26a, which has a chiral center, might play a main role in this excellent activity. Initially, we attempted to use normal phase Chiralpak columns to separate this compound, but this attempt failed. Fortunately, considering work conducted by Katzenellenbogen and his colleagues,56 we used orthogonal protecting groups and chiral HPLC and successfully separated the two isomers. First, the two phenols of compound 23 were protected as a benzyl ether and a sterically bulky tert-butyldiphenylsilyl (TBDPS) ether. We use the Diels−Alder reaction of this diprotected furan 28 and vinyl sulfonate 20a to obtain a mixture of regioisomers, which could be isolated by normal phase silica chromatography. The structures of these regioisomers were verified by two-dimensional NOESY-NMR (see Supporting Information for details). The next challenge was to explore the appropriate HPLC conditions to separate the enantiomers from one or both regioisomers. After using different chiral columns, we successfully achieved the separation of the two enantiomers 29a and 29b using a normal phase Chiralpak IB column with hexane/2-propanol as the eluent. Then, to complete the synthesis, the orthogonal protecting groups had to be removed. TBDPS could be cleaved by tetrabutylammonium fluoride (TBAF)-promoted deprotection to provide 31 with good yield. However, the final step involving cleavage of the benzyl protecting group seemed to be more challenging than we anticipated. Our first attempt utilized TMSI-mediated 8165



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Figure 7. Anti-inflammatory effect of compound 26a during antitumor progression in a MCF-7 breast cancer model in Balb/c nude mice. (A) IL-6 immunohistochemistry in the tissue around the tumor after 2 weeks of treatment. Scale bar, 50 μm. Quantification of the IL-6 staining signal in the tissue around the tumor (n = 4) is displayed in the column chart. (B) TNF-α immunohistochemistry in the tissue around the tumor after 2 weeks of treatment. Scale bar, 50 μm. Quantification of the TNF-α staining signal in the tissue around the tumor (n = 4) is displayed in the column chart. (C) NF-κB activity monitored by immunohistochemistry of p65 in the tissue around the tumor after 2 weeks of treatment. Scale bar, 50 μm. Quantification of the p65 staining signal in the tissue around the tumor (n = 4) is displayed in the column chart.

Table 5. A Biological Activity Comparison of 26a, Isomers 26a-1 and 26a-2a NO inhibition

MCF-7

compd

ERα

RBA % ERβ

EC50 (μM)

ERβ agonist mode Eff (%E2)

IC50 (μM)

ERα antagonist mode Eff (%E2)

IC50 (μM)

IC50 (μM)

26a-1 26a-2 26a

4.88 ± 0.17 15.11 ± 0.22 10.01 ± 0.81

3.43 ± 0.06 7.91 ± 0.14 5.61 ± 1.28

0.018 0.002 0.004

28 ± 4 17 ± 3 23 ± 1

0.027 0.003 0.007

72 ± 12 11 ± 7 48 ± 20

0.48 ± 0.11 0.30 ± 0.04 0.44 ± 0.23

17.96 ± 2.47 2.31 ± 0.25 3.70 ± 0.56

a

See footnotes in Tables 1−4 for experimental details.

tional activity, and antiproliferative activity in breast cancer cell lines. Computer Models of ERα Compound 26a-1 and 26a2 Complexes. From previous work,59,60 we conclude that either E2 or traditional SERMs and full antagonists directly interact with helix 12 to influence binding transcriptional coactivators. In contrast, the oxabicyclic derivative represented by OBHS can interact with helix 11, indirectly regulating helix 12, which is a new antagonism mechanism. The phenyl sulfonate moiety and the small alkyl ether substituents on the bicyclic core are the key parts.44 Consistent with this model, molecular modeling (Figure 8) showed that the large and nonpolar phenyl sulfonate group of the two conjugates was crucial for the antagonism, which could

debenzylation, but it was unsuccessful. Next, we used the Pd/C hydrogenolysis conditions, which led to debenzylation but a further reduction of olefin. Surprisingly, it also included a reduction of the double bond in the oxabicyclic ring. Another debenzylation condition using AlCl3 allowed us to obtain the desired free phenol derivatives 26a-1 and 26a-2 (Scheme 4).58 We also tested the optical rotation of the two isomers (see Supporting Information for details). Next, we investigated the structure specificity versus the biological activity of these two enantiomers, and the results are summarized in Table 5. The results indicated that except NO inhibition, for which two isomers showed similar activity, isomer 26a-2 showed much better biological activity than isomer 26a-1 in terms of relative binding affinity, transcrip8166



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Therefore, OBHS-RES conjugates exhibit a new strategy to develop the effective antiproliferative and anti-inflammatory agents for the treatment of breast cancer. Most of these novel hybrid compounds act as a good ER ligand with excellent ERα antagonistic activity and potent anti-inflammatory activity. Among them, compound 26a exhibits better activity than tamoxifen in vivo and 4-hydroxytamoxifen in vitro, and the anti-inflammatory activity of this compound is helpful to increase the antitumor activity. In addition, successful separation and testing for the chiral isomers of 26a has laid the foundation for us to find better compounds for breast cancer therapy by using a structure-based strategy. Further mechanistic and druggability studies of the OBHS-RES conjugates against breast cancer are still underway in our group and will be reported at the appropriate time.

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EXPERIMENTAL SECTION

Materials and Methods. If not stated otherwise, the starting materials were purchased commercially and used directly. All solvents involved in the reactions were redistilled and dried to avoid water. The reaction flask was dried in an oven, and then the reaction device was set up with hot and cooled under an argon atmosphere. All reactions proceeded under the protection of an argon stream. The reaction process was monitored by analytical thin layer chromatography and a UV lamp (254 and 365 nm). A Bruker Biospin AV400 (400 MHz, 1H NMR; 100 MHz, 13C NMR) instrument was used to measure the 1H NMR and 13C NMR spectra. Chemical shifts are reported in ppm (parts per million) and are referenced to either tetramethylsilane or the solvent. The purity of all compounds for biological testing was determined using an HPLC (see Supporting Information) and was identified as >95% purity. The Synthesis of intermediate compounds is reported in the Supporting Information. General Procedure for Diels−Alder Reaction (24a−d, 25a− q, and 26a−e). We used the distilled THF (2 mL) as cosolvent and added furans 16 or 17 or 23 (0.6 mmol) and dienophiles 20a−e or 22a−r (0.72 mmol) to the round-bottom flask. The reaction mixture was heated to 90 °C and stirred for 8 h. The crude product was purified by silica gel column chromatography (hexane-EtOAc, 8:1− 2:1). Phenyl-6-(4-((E)-3,5-dimethoxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (24a). Yellow solid, 78% yield, mp 107−109 °C. 1H NMR (400 MHz, acetone-d6) δ 8.78 (s, 1H), 7.58 (m, 2H), 7.43 (m, 1H), 7.41−7.36 (m, 4H), 7.35−7.28 (m, 4H), 7.26 (s, 1H), 7.24 (s, 1H), 6.83 (m, 2H), 6.79 (m, 2H), 6.43 (t, J = 2.2 Hz, 1H), 5.73 (d, J = 1.0 Hz, 1H), 5.52−5.48 (m, 1H), 3.90 (m, 1H), 3.81 (s, 6H), 2.44 (m, 1H), 2.27 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 161.96, 158.61, 150.35, 144.53, 140.09, 137.88, 137.45, 132.46, 130.66, 129.75, 129.02, 128.55, 127.84, 127.61, 126.92, 124.31, 122.97, 116.35, 105.22, 100.67, 84.94, 83.60, 61.64, 55.49, 31.00. HRMS (ESI) calcd for C34H30O7S [M + Na]+ 605.1604, found 605.1605. 4-Bromophenyl-6-(4-((E)-3,5-dimethoxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (24b). Yellow solid, 76% yield, mp 100−102 °C. 1H NMR (400 MHz, acetoned6) δ 8.81 (s, 1H), 7.59−7.54 (m, 4H), 7.36 (m, 3H), 7.30−7.25 (m, 3H), 7.24 (s, 1H), 7.21 (s, 1H), 6.84 (d, J = 8.7 Hz, 2H), 6.79 (d, J = 2.4 Hz, 2H), 6.43 (t, J = 2.2 Hz, 1H), 5.74 (d, J = 1.1 Hz 1H), 5.52− 5.49 (m, 1H), 3.95 (m, 1H), 3.81 (s, 6H), 2.48−2.40 (m, 1H), 2.34− 2.21 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 161.86, 158.52, 149.38, 144.45, 140.00, 137.80, 137.28, 132.31, 130.25, 129.70, 128.94, 128.44, 127.96, 127.53, 126.88, 125.00, 120.57, 116.28, 105.23, 100.59, 84.84, 83.53, 61.75, 55.42, 30.96. HRMS (ESI) calcd for C34H29BrO7S [M + Na]+ 683.0710, found 683.0705. 3-Methoxyphenyl-6-(4-((E)-3,5-dimethoxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (24c). Yellow solid, 63% yield, mp 98−101 °C. 1H NMR (400 MHz, acetoned6) δ 8.76 (s, 1H), 7.59−7.55 (m, 2H), 7.40 (m, 2H), 7.37−7.33 (m,

Figure 8. Docking analysis of OBHS-RES conjugates 26a-1 and 26a-2 bound to ERα (PDB 3ERT). (A) Model of conjugate 26a-1 bound to ERα. The ligand and Cys530 form a hydrogen bond (1.8 Å), and the sulfonate ester moiety collides with helix 11. Another hydrogen bond is formed by the interaction between the hydroxyl group of resveratrol and Tyr537 (2.6 Å), and the phenyl hydroxyl group extends between helices 3 and 6. (B) Model of conjugate 26a-2 bound to ERα. The ligand forms three hydrogen bonds to Cys 530 (2.4 Å), Val 533 (1.9 Å), and Glu 380 (2.1 Å), and the resveratrol group extends between helices 8 and 11.

generate strong steric clashes with helix 11 by engaging in hydrogen bonding with Cys 530 and indirectly regulate helix 12. In addition, we found that the resveratrol group of compound 26a-1 could orient properly toward the hydrogen bond with Tyr 537 on helix 12 and the phenyl hydroxyl group extended between helices 3 and 6. However, compound 26a-2 could form a hydrogen bond with Val 533 to accentuate this clash with helix 11 and a hydrogen bond with Glu 380, thus giving it more potent ERα antagonist activity than compound 26a-1.

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CONCLUSION In the long term treatment of breast cancer, combination therapies can avoid drug resistance to some extent, but there are undeniable side effects. In contrast, conjugates that can simultaneously modulate more targets and overcome pharmacokinetic differences might have fewer side effects and prospects for broader applications. 8167



Article

83.10, 61.09, 30.52. HRMS (ESI) calcd for C32H25FO7S [M + Na]+ 595.1197, found 595.1172. 4-Chlorophenyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25e). Yellow solid, 72% yield, mp 110−112 °C. 1H NMR (400 MHz, acetoned6) δ 8.48 (s, 3H), 7.53 (dd, J = 10.6, 8.4 Hz, 2H), 7.44−7.39 (m, 2H), 7.37−7.34 (m, 2H), 7.33−7.24 (m, 4H), 7.13−7.09 (m, 2H), 6.88−6.82 (m, 2H), 6.62 (m, 2H), 6.34 (t, J = 2.0 Hz, 1H), 5.73 (m, 1H), 5.50 (m, 1H), 3.94 (dd, J = 8.3, 4.4 Hz, 1H), 2.44 (m, 1H), 2.35−2.20 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 159.03, 158.12, 148.44, 144.00, 139.62, 137.53, 136.93, 132.41, 130.18, 129.88, 129.31, 128.06, 127.57, 127.10, 126.96, 124.27, 123.82, 115.93, 105.52, 102.81, 84.48, 83.16, 61.33, 30.59. HRMS (ESI) calcd for C32H25ClO7S [M + Na]+ 611.0902, found 611.0909. 2-Chlorophenyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25f). Yellow solid, 85% yield, mp 117−120 °C. 1H NMR (400 MHz, acetoned6) δ 8.80 (s, 1H), 8.37 (s, 2H), 7.60−7.49 (m, 4H), 7.41 (dd, J = 11.1, 3.2 Hz, 2H), 7.38−7.34 (m, 2H), 7.32−7.28 (m, 2H), 7.12 (m, 2H), 6.87−6.82 (m, 2H), 6.62−6.58 (m, 2H), 6.32 (t, J = 1.9 Hz, 1H), 5.79 (m, 1H), 5.53 (m, 1H), 4.07 (dd, J = 8.3, 4.4 Hz, 1H), 2.56 (m, 1H), 2.39 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 159.52, 158.65, 146.05, 144.67, 140.06, 137.98, 137.44, 132.30, 131.61, 130.23, 129.84, 129.20, 129.00, 128.45, 128.12, 127.53, 127.41, 124.83, 124.28, 116.37, 105.91, 103.22, 85.01, 83.62, 62.96, 31.11. HRMS (ESI) calcd for C32H25ClO7S [M + Na]+ 611.0902, found 611.0895. o-Tolyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25g). Yellow solid, 74% yield, mp 104−107 °C. 1H NMR (400 MHz, acetone-d6) δ 8.50 (m, 3H), 7.54 (m, 2H), 7.40−7.36 (m, 2H), 7.31 (m, 4H), 7.24−7.19 (m, 2H), 7.12 (m, 2H), 6.89−6.82 (m, 2H), 6.61 (m, 2H), 6.34 (t, J = 2.0 Hz, 1H), 5.76 (m, 1H), 5.53 (m, 1H), 4.02 (dd, J = 8.3, 4.4 Hz, 1H), 2.58−2.50 (m, 1H), 2.42 (m, 1H), 2.34 (s, 3H). 13C NMR (100 MHz, acetone-d6) δ 159.27, 158.36, 148.48, 144.32, 140.10, 139.89, 137.72, 137.36, 132.26, 132.05, 130.02, 129.65, 128.20, 127.91, 127.71, 127.51, 127.35, 124.17, 122.72, 116.17, 105.75, 103.01, 84.78, 83.46, 62.08, 31.29, 16.52. HRMS (ESI) calcd for C33H28O7S [M + Na]+ 591.1448, found 591.1446. 2-(Trifluoromethyl)phenyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25h). Yellow solid, 77% yield, mp 110−113 °C. 1H NMR (400 MHz, acetone-d6) δ 8.78 (s, 1H), 8.34 (s, 2H), 7.83−7.76 (m, 2H), 7.59− 7.53 (m, 4H), 7.38 (d, J = 8.4 Hz, 2H), 7.32 (m, 2H), 7.12 (m, 2H), 6.88−6.83 (m, 2H), 6.60 (d, J = 2.1 Hz, 2H), 6.32 (t, J = 2.0 Hz, 1H), 5.82−5.78 (m, 1H), 5.56 (m, 1H), 4.12 (dd, J = 8.3, 4.4 Hz, 1H), 2.56 (m, 1H), 2.33 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 159.58, 158.75, 147.37, 144.87, 140.15, 138.02, 137.44, 135.06, 132.35, 130.46, 130.24, 130.05, 128.85, 128.44, 128.41, 128.32, 127.75, 127.56, 124.28, 123.67, 116.46, 105.99, 103.28, 84.90, 83.70, 63.19, 30.96. HRMS (ESI) calcd for C33H25F3O7S [M + Na]+ 645.1165, found 645.1162. 3-(Trifluoromethyl)phenyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25i). Yellow solid, 76% yield, mp 105−108 °C. 1H NMR (400 MHz, acetone-d6) δ 8.80 (s, 1H), 8.37 (s, 2H), 7.71 (m, 2H), 7.57−7.52 (m, 4H), 7.39−7.36 (m, 2H), 7.30−7.26 (m, 2H), 7.12 (m, 2H), 6.87− 6.82 (m, 2H), 6.59 (m, 2H), 6.31 (t, J = 1.9 Hz, 1H), 5.79 (m, 1H), 5.53 (m, 1H), 4.07 (dd, J = 8.3, 4.4 Hz, 1H), 2.56−2.42 (m, 1H), 2.30 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 159.57, 158.70, 150.38, 144.64, 140.10, 138.05, 137.47, 132.29, 132.01, 130.37, 130.27, 129.87, 128.91, 128.47, 128.15, 127.57, 127.20, 124.68, 124.32, 120.27, 116.42, 105.96, 103.27, 84.94, 83.74, 62.32, 31.10. HRMS (ESI) calcd for C33H25F3O7S [M + Na]+ 645.1165, found 645.1159. [1,1′-Biphenyl]-4-yl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25j). Yellow solid, 79% yield, mp 121−124 °C. 1H NMR (400 MHz, acetone-d6) δ 8.77 (s, 1H), 8.36 (s, 2H), 7.69−7.64 (m, 4H), 7.58 (m, 2H), 7.49−7.45 (m, 2H), 7.41−7.36 (m, 5H), 7.29 (d, J = 8.7 Hz, 2H), 7.14 (m, 2H), 6.84 (m, 2H), 6.61 (m, 2H), 6.34 (t, J = 2.1 Hz, 1H), 5.76 (m, 1H), 5.53 (m, 1H), 3.92 (dd, J = 8.3, 4.4 Hz, 1H),

3H), 7.30 (m, 2H), 7.26−7.22 (m, 3H), 6.86 (m, 2H), 6.79 (m, 2H), 6.43 (t, J = 2.0 Hz, 1H), 5.76 (d, J = 1.2 Hz, 1H), 5.54 (m, 1H), 3.92 (m, 1H), 3.82 (s, 6H), 3.80 (s, 3H), 2.58−2.53 (m, 1H), 2.44 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 162.14, 158.85, 148.33, 142.04, 140.29, 138.08, 137.96, 133.38, 131.00, 130.37, 130.04, 129.25, 128.32, 128.01, 127.64, 127.06, 123.76, 123.02, 116.67, 105.44, 100.81, 85.36, 83.66, 62.21, 55.64, 55.62, 31.64, 23.88. HRMS (ESI) calcd for C35H32O8S [M + Na]+ 635.1710, found 635.1711. 2-Ethylphenyl-6-(4-((E)-3,5-dimethoxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (24d). Yellow solid, 73% yield, mp 110−113 °C. 1H NMR (400 MHz, acetoned6) δ 8.80 (s, 1H), 7.57 (m, 2H), 7.40 (m, 2H), 7.37−7.33 (m, 2H), 7.32−7.28 (m, 2H), 7.27−7.19 (m, 4H), 6.85 (m, 2H), 6.79 (d, J = 2.2 Hz, 2H), 6.43 (t, J = 2.1 Hz, 1H), 5.78−5.76 (m, 1H), 5.55−5.52 (m, 1H), 3.92 (m, 1H), 3.81 (s, 6H), 2.79−2.68 (m, 2H), 2.57 (m, 1H), 2.46−2.28 (m, 1H), 1.23−1.18 (m, 3H). 13C NMR (100 MHz, acetone-d6) δ 161.95, 158.65, 148.14, 144.63, 141.83, 140.09, 137.90, 137.52, 133.18, 132.52, 130.84, 130.19, 129.86, 129.03, 128.40, 127.85, 127.59, 124.30, 122.85, 116.37, 105.27, 100.64, 85.18, 83.65, 62.03, 55.48, 31.46, 23.74, 14.55. HRMS (ESI) calcd for C36H34O7S [M + Na]+ 633.1917, found 633.1918. Phenyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25a). Brown solid, 86% yield, mp 108−110 °C. 1H NMR (400 MHz, acetone-d6) δ 8.77 (s, 1H), 8.35 (s, 2H), 7.55 (m, 2H), 7.40 (m, 2H), 7.36 (m, 3H), 7.33−7.29 (m, 2H), 7.27 (m, 2H), 7.12 (m, 2H), 6.83 (m, 2H), 6.61−6.58 (m, 2H), 6.32 (t, J = 2.2 Hz, 1H), 5.72 (m, 1H), 5.50 (m, 1H), 3.89 (dd, J = 8.3, 4.4 Hz, 1H), 2.43 (m, 1H), 2.27 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 159.55, 158.62, 150.40, 144.49, 140.10, 138.03, 137.54, 132.34, 130.70, 130.36, 129.77, 128.57, 128.35, 127.88, 127.58, 124.39, 123.02, 116.38, 105.93, 103.24, 84.99, 83.64, 61.58, 31.05. HRMS (ESI) calcd for C32H26O7S [M + Na]+ 577.1291, found 577.1284. 4-Bromophenyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25b). Yellow solid, 74% yield, mp 113−115 °C. 1H NMR (400 MHz, acetoned6) δ 8.79 (s, 1H), 8.36 (s, 2H), 7.60−7.51 (m, 4H), 7.35 (d, J = 8.4 Hz, 2H), 7.31−7.21 (m, 4H), 7.12 (m, 2H), 6.83 (m, 2H), 6.61−6.57 (m, 2H), 6.32 (t, J = 1.7 Hz, 1H), 5.72 (m, 1H), 5.51 (m, 1H), 3.95 (dd, J = 8.3, 4.4 Hz, 1H), 2.47−2.39 (m, 1H), 2.36−2.23 (m, 1H). 13 C NMR (100 MHz, acetone-d6) δ 159.57, 158.67, 149.59, 144.53, 140.11, 138.07, 137.48, 133.73, 132.32, 130.37, 129.81, 128.58, 128.09, 127.59, 127.45, 125.15, 124.35, 120.68, 116.39, 105.97, 85.00, 83.67, 61.84, 31.10. HRMS (ESI) calcd for C32H25BrO7S [M + Na]+ 655.0397, found 655.0368. 3-Methoxyphenyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25c). Brown solid, 81% yield, mp 115−117 °C. 1H NMR (400 MHz, acetone-d6) δ 8.77 (s, 1H), 8.40 (s, 2H), 7.54 (m, 2H), 7.35 (m, 2H), 7.30−7.25 (m, 3H), 7.11 (m, 2H), 6.90−6.87 (m, 2H), 6.84 (m, 3H), 6.61−6.59 (m, 2H), 6.33 (t, J = 1.8 Hz, 1H), 5.75−5.71 (m, 1H), 5.52−5.47 (m, 1H), 3.91 (dd, J = 8.3, 4.4 Hz, 1H), 3.76 (s, 3H), 2.45 (m, 1H), 2.27 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 161.34, 159.30, 150.98, 144.30, 139.85, 137.76, 137.25, 132.81, 132.05, 130.81, 130.04, 129.53, 128.28, 127.82, 127.34, 126.65, 124.14, 116.14, 114.55, 108.65, 105.72, 103.02, 84.70, 83.22, 61.38, 55.59, 30.83. HRMS (ESI) calcd for C33H28O8S [M + Na]+ 607.1397, found 607.1392. 4-Fluoropheny-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25d). Yellow solid, 90% yield, mp 100−103 °C. 1H NMR (400 MHz, acetoned6) δ 8.79 (s, 1H), 8.40 (s, 2H), 7.54 (m, 2H), 7.39−7.31 (m, 4H), 7.29−7.25 (m, 2H), 7.19−7.14 (m, 2H), 7.13−7.09 (m, 2H), 6.84 (m, 2H), 6.62 (m, 2H), 6.35 (t, J = 2.0 Hz, 1H), 5.74 (m, 1H), 5.50 (m, 1H), 3.93 (dd, J = 8.3, 4.4 Hz, 1H), 2.45 (m, 1H), 2.27 (m, 1H). 13 C NMR (100 MHz, acetone-d6) δ 161.14 (d, J = 243.0 Hz), 158.96, 145.65, 143.93, 139.62 (d, J = 12.5 Hz), 137.39 (d, J = 14.2 Hz), 136.90, 129.82, 129.25, 128.00, 127.50, 127.04, 126.90, 124.47, 116.80, 116.65 (d, J = 23.9 Hz) 116.56, 116.04, 115.87, 105.46, 84.42, 8168



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2.51−2.43 (m, 1H), 2.32 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 159.61, 158.67, 149.90, 144.51, 140.70, 140.32, 140.15, 138.12, 137.57, 132.42, 130.50, 130.35, 129.77, 129.12, 128.78, 128.51, 128.11, 127.71, 127.67, 124.46, 123.42, 116.43, 106.02, 103.32, 85.14, 83.66, 61.52, 31.13. HRMS (ESI) calcd for C38H30O7S [M + Na]+, 653.1604, found 653.1605. 3-Chlorophenyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25k). Yellow solid, 86% yield, mp 107−110 °C. 1H NMR (400 MHz, acetoned6) δ 8.77 (s, 1H), 8.34 (s, 2H), 7.55 (m, 2H), 7.41 (m, 2H), 7.36 (m, 2H), 7.33−7.23 (m, 4H), 7.11 (m, 2H), 6.84 (m, 2H), 6.59 (m, 2H), 6.31 (t, J = 2.0 Hz, 1H), 5.76 (m, 1H), 5.52 (m, 1H), 3.99 (dd, J = 8.3, 4.4 Hz, 1H), 2.50−2.41 (m, 1H), 2.30 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 159.66, 158.87, 150.87, 144.68, 140.22, 138.17, 137.59, 135.32, 132.38, 132.03, 130.39, 129.92, 128.63, 128.21, 127.71, 127.54, 124.49, 123.51, 121.92, 116.49, 106.05, 103.34, 85.05, 83.80, 62.11, 31.18. HRMS (ESI) calcd for C32H25ClO7S [M + Na]+ 611.0902, found 611.0914. 3-Hydroxyphenyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25l). Taupe solid, 79% yield, mp 123−125 °C. 1H NMR (400 MHz, acetone-d6) δ 8.61 (s, 4H), 7.54 (dd, J = 8.2, 6.2 Hz, 2H), 7.38−7.33 (m, 2H), 7.30−7.25 (m, 2H), 7.18 (m, 1H), 7.13−7.09 (m, 2H), 6.83 (m, 5H), 6.60 (m, 2H), 6.32 (t, J = 2.1 Hz, 1H), 5.71 (m, 1H), 5.50 (m, 1H), 3.90 (dd, J = 8.3, 4.4 Hz, 1H), 2.45 (m, 1H), 2.25 (m, 1H). 13 C NMR (100 MHz, acetone-d6) δ 159.32, 159.18, 158.40, 151.00, 144.27, 139.89, 137.76, 132.13, 130.84, 130.00, 129.62, 128.23, 127.90, 127.36, 124.15, 123.36, 116.17, 114.76, 113.34, 110.06, 105.72, 103.02, 84.73, 83.26, 61.38, 30.86. HRMS (ESI) calcd for C32H26O8S [M + Na]+ 593.1241, found 593.1239. m-Tolyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25m). Brown solid, 78% yield, mp 111−113 °C. 1H NMR (400 MHz, acetone-d6) δ 8.48 (s, 3H), 7.60−7.54 (m, 2H), 7.39 (m, 2H), 7.36−7.32 (m, 2H), 7.30−7.27 (m, 2H), 7.16−7.12 (m, 4H), 6.86 (m, 2H), 6.63 (m, 2H), 6.36 (t, J = 2.1 Hz, 1H), 5.76 (m, 1H), 5.50 (m, 1H), 3.89 (dd, J = 8.3, 4.4 Hz, 1H), 2.49−2.44 (m, 1H), 2.37 (m, 1H), 2.29 (s, 3H). 13C NMR (100 MHz, acetone-d6) δ 159.06, 158.11, 149.85, 144.03, 140.60, 137.58, 137.03, 131.82, 129.95, 129.79, 129.28, 128.16, 128.12, 127.55, 127.16, 126.98, 122.90, 119.49, 116.17, 115.94, 105.55, 102.84, 84.51, 83.17, 60.95, 30.56, 20.65. HRMS (ESI) calcd for C33H28O7S [M + Na]+ 591.1448, found 591.1447. 4-Hydroxyphenyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25n). Brown solid, 66% yield, mp 114−116 °C. 1H NMR (400 MHz, acetone-d6) δ 8.55 (s, 4H), 7.58−7.53 (m, 2H), 7.36 (m, 2H), 7.29− 7.24 (m, 2H), 7.14−7.10 (m, 4H), 6.86−6.83 (m, 2H), 6.82−6.78 (m, 2H), 6.61 (m, 2H), 6.33 (t, J = 2.0 Hz, 1H), 5.70 (m, 1H), 5.49 (m, 1H), 3.83 (dd, J = 8.3, 4.4 Hz, 1H), 2.45−2.40 (m, 1H), 2.24 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 159.23, 158.27, 156.74, 142.59, 139.84, 137.73, 137.28, 132.06, 130.13, 129.95, 129.46, 128.33, 127.74, 127.31, 127.14, 123.77, 116.50, 116.09, 105.70, 102.97, 84.73, 83.34, 60.72, 30.77. HRMS (ESI) calcd for C32H26O8S [M + Na]+ 593.1241, found 593.1235. 3-Bromophenyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25o). Yellow solid, 73% yield, mp 110−112 °C. 1H NMR (400 MHz, acetoned6) δ 8.81 (s, 1H), 8.38 (s, 2H), 7.54 (m, 4H), 7.39−7.35 (m, 3H), 7.29 (m, 3H), 7.11 (m, 2H), 6.83 (m, 2H), 6.60 (m, 2H), 6.33 (t, J = 1.8 Hz, 1H), 5.77−5.76 (m, 1H), 5.51 (m, 1H), 3.99 (dd, J = 8.3, 4.4 Hz, 1H), 2.50−2.42 (m, 1H), 2.29 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 159.39, 158.49, 150.56, 144.41, 141.74, 139.95, 137.89, 137.29, 132.06, 130.91, 130.12, 129.66, 128.36, 127.94, 127.46, 127.29, 126.09, 124.19, 122.07, 116.25, 105.82, 103.12, 84.77, 83.53, 61.85, 30.92. HRMS (ESI) calcd for C32H25BrO7S [M + Na]+ 655.0397, found 655.0372. p-Tolyl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25p). Brown solid, 90% yield, mp 114−116 °C. 1H NMR (400 MHz, acetone-d6) δ 8.43 (s, 3H), 7.57−7.53 (m, 2H), 7.37−7.34 (m, 2H), 7.28−7.24 (m, 2H),

7.16 (m, 4H), 7.13 (m, 2H), 6.83 (m, 2H), 6.61 (m, 2H), 6.33 (t, J = 2.0 Hz, 1H), 5.71−5.70 (m, 1H), 5.50−5.48 (m, 1H), 3.85 (dd, J = 8.3, 4.4 Hz, 1H), 2.44 (m, 1H), 2.39−2.32 (m, 1H), 2.30 (s, 3H). 13C NMR (100 MHz, acetone-d6) δ 159.23, 158.29, 147.91, 144.15, 139.80, 137.71, 137.40, 132.02, 130.76, 130.07, 129.45, 128.27, 128.08, 127.73, 127.28, 124.08, 122.38, 116.08, 105.67, 102.97, 84.68, 83.31, 61.06, 30.75, 20.40. HRMS (ESI) calcd for C33H28O7S [M + Na]+ 591.1448, found 591.1450. Naphthalen-1-yl-6-(4-((E)-3,5-dihydroxystyryl)phenyl)-5-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25q). Earthy yellow solid, 73% yield, mp 127−129 °C. 1H NMR (400 MHz, acetone-d6) δ 8.50 (s, 3H), 8.24−8.19 (m, 1H), 8.00−7.97 (m, 1H), 7.90 (m, 1H), 7.59 (m, 3H), 7.55−7.51 (m, 3H), 7.40−7.35 (m, 2H), 7.30 (m, 2H), 7.12 (m, 2H), 6.84 (m, 2H), 6.60 (m, 2H), 6.32 (t, J = 2.0 Hz, 1H), 5.84 (m, 1H), 5.56 (m, 1H), 4.19 (dd, J = 8.3, 4.4 Hz, 1H), 2.61 (m, 1H), 2.36 (m, 1H). 13C NMR (100 MHz, acetoned6) δ 159.53, 158.65, 146.15, 144.59, 140.11, 137.94, 137.59, 135.72, 132.34, 130.21, 129.88, 128.68, 128.42, 128.16, 127.82, 127.76, 127.56, 127.45, 126.27, 124.36, 122.54, 119.02, 116.54, 116.39, 105.93, 103.22, 85.10, 83.73, 62.46, 31.28. HRMS (ESI) calcd for C36H28O7S [M + Na]+ 627.1448, found 627.1450. Phenyl-5-(4-hydroxyphenyl)-6-(4-((E)-styryl)phenyl)-7oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (25r). Brown solid, 71% yield, mp 104−106 °C. 1H NMR (400 MHz, acetone-d6) δ 8.75 (s, 1H), 7.60 (m, 4H), 7.50 (m, 1H), 7.45−7.41 (m, 2H), 7.38 (m, 5H), 7.35 (m, 1H), 7.30−7.26 (m, 5H), 6.84 (d, J = 8.7 Hz, 2H), 5.74 (m, 1H), 5.51 (m, 1H), 3.90 (dd, J = 8.3, 4.4 Hz, 1H), 2.48−2.41 (m, 1H), 2.27 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 158.66, 150.44, 144.58, 140.43, 138.16, 138.05, 137.56, 132.48, 130.73, 130.52, 129.83, 129.49, 128.93, 128.63, 127.91, 127.65, 127.37, 126.96, 123.05, 116.42, 85.03, 83.69, 61.63, 31.09. HRMS (ESI) calcd for C32H26O5S [M + Na]+ 545.1393, found 545.1371. 4-((E)-3,5-Dimethoxystyryl)phenyl-5,6-bis(4-hydroxyphenyl)-7oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (26a). Yellow solid, 72% yield, mp 108−111 °C. 1H NMR (400 MHz, acetone-d6) δ 8.69 (s, 1H), 8.62 (s, 1H), 7.62−7.57 (m, 2H), 7.26 (m, 4H), 7.24 (d, J = 2.3 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 6.82− 6.79 (m, 2H), 6.78 (d, J = 2.2 Hz, 2H), 6.43 (t, J = 2.2 Hz, 1H), 5.65 (d, J = 1.2 Hz, 1H), 5.44 (dd, J = 4.3, 1.1 Hz, 1H), 3.81 (s, 6H), 3.79 (dd, J = 8.4, 4.6 Hz, 1H), 2.47−2.38 (m, 1H), 2.28 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 161.59, 157.90, 157.78, 149.21, 141.74, 139.54, 137.37, 136.84, 130.16, 129.63, 129.01, 128.21, 127.88, 124.50, 123.76, 122.92, 116.13, 115.94, 105.03, 100.41, 84.78, 83.15, 61.12, 55.18, 30.21. HRMS (ESI) calcd for C34H30O8S [M + Na]+ 621.1554, found 621.1545. 4-((E)-Styryl)phenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (26b). White solid, 75% yield, mp 104−106 °C. 1H NMR (400 MHz, acetone-d6) δ 9.31 (s, 1H), 8.81 (s, 1H), 7.65−7.58 (m, 4H), 7.53 (d, J = 11.0 Hz, 1H), 7.38 (m, 2H), 7.25 (m, 9H), 6.99 (m, 1H), 6.84 (m, 2H), 5.68 (m, 1H), 5.47 (m, 1H), 3.84 (m, 1H), 2.47−2.38 (m, 1H), 2.36−2.26 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 158.69, 158.55, 149.71, 143.72, 140.78, 138.05, 137.44, 133.23, 132.48, 130.55, 130.12, 129.54, 129.49, 128.67, 128.61, 127.90, 127.46, 123.38, 117.51, 116.44, 85.10, 83.59, 61.41, 31.30. HRMS (ESI) calcd for C32H26O6S [M + Na]+ 561.1342, found 561.1336. 3-((E)-3,5-Dimethoxystyryl)phenyl-5,6-bis(4-hydroxyphenyl)-7oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (26c). Yellow solid, 78% yield, mp 107−109 °C. 1H NMR (400 MHz, acetone-d6) δ 8.72 (s, 1H), 8.69 (s, 1H), 7.55 (dd, J = 8.7, 4.9 Hz, 2H), 7.38 (t, J = 7.9 Hz, 1H), 7.28−7.24 (m, 4H), 7.23 (d, J = 4.5 Hz, 2H), 7.16 (dd, J = 8.1, 1.6 Hz, 1H), 6.83 (m, 2H), 6.80 (m, 4H), 6.43 (t, J = 2.1 Hz, 1H), 5.68 (m, 1H), 5.45 (m, 1H), 3.85 (dd, J = 8.3, 4.4 Hz, 1H), 3.81 (s, 6H), 2.45 (m, 1H), 2.29 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 162.02, 158.32, 158.25, 150.86, 142.18, 140.51, 139.79, 137.82, 131.24, 130.88, 129.88, 129.48, 128.31, 125.92, 124.90, 124.16, 121.86, 120.90, 116.48, 116.31, 105.47, 101.05, 85.14, 83.62, 61.76, 55.57, 31.4. HRMS (ESI) calcd for C34H30O8S [M + Na]+ 621.1554, found 621.1552. 8169



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3-((E)-3,5-Dihydroxystyryl)phenyl-5,6-bis(4-hydroxyphenyl)-7oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (26d). Orange solid, 61% yield, mp 122−124 °C. 1H NMR (400 MHz, acetone-d6) δ 8.58 (s, 4H), 7.54 (d, J = 7.9 Hz, 1H), 7.50 (m, 1H), 7.37 (m, 1H), 7.24 (m, 4H), 7.13 (m, 3H), 6.82 (dd, J = 11.6, 8.6 Hz, 4H), 6.60 (m, 2H), 6.33 (t, J = 2.1 Hz, 1H), 5.69 (m, 1H), 5.44 (m, 1H), 3.85 (dd, J = 8.2, 4.4 Hz, 1H), 2.44 (m, 1H), 2.30 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 159.37, 158.09, 158.05, 150.68, 142.04, 140.40, 139.58, 137.64, 131.29, 130.65, 129.73, 129.27, 127.45, 125.65, 124.73, 123.99, 121.57, 120.64, 116.32, 116.13, 105.96, 103.33, 84.95, 83.43, 61.52, 31.23. HRMS (ESI) calcd for C32H26O8S [M + Na]+ 593.1241, found 593.1242. 4-((E)-3,5-Dihydroxystyryl)phenyl-5,6-bis(4-hydroxyphenyl)-7oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (26e). Orange solid, 57% yield, mp 124−126 °C. 1H NMR (400 MHz, acetone-d6) δ 8.74 (s, 1H), 8.67 (s, 1H), 8.36 (s, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.25 (m, 4H), 7.22 (d, J = 6.9 Hz, 2H), 7.09 (m, 2H), 6.84 (d, J = 8.8 Hz, 2H), 6.80 (d, J = 8.8 Hz, 2H), 6.59 (d, J = 2.1 Hz, 2H), 6.33 (t, J = 2.1 Hz, 1H), 5.65 (d, J = 1.2 Hz, 1H), 5.44 (dd, J = 4.3, 1.1 Hz, 1H), 3.78 (dd, J = 8.3, 4.5 Hz, 1H), 2.42 (m, 1H), 2.27 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 159.35, 158.15, 158.01, 149.37, 141.94, 139.77, 137.59, 137.15, 130.61, 129.86, 129.19, 128.33, 127.40, 124.70, 123.95, 123.10, 116.31, 116.10, 105.84, 103.14, 84.98, 83.34, 61.21, 31.19. HRMS (ESI) calcd for C32H26O8S [M + Na]+ 593.1241, found 593.1234. 4-((E)-3,5-Dimethoxystyryl)phenyl(1R,2S,4R)-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (26a-1). Yellow solid, 20% yield, mp 109−111 °C.1H NMR (400 MHz, acetone-d6) δ 8.67 (s, 1H), 8.61 (s, 1H), 7.60 (d, J = 8.7 Hz, 2H), 7.30−7.25 (m, 4H), 7.25−7.23 (m, 2H), 7.21 (d, J = 8.3 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 6.80 (d, J = 8.8 Hz, 2H), 6.78 (d, J = 2.2 Hz, 2H), 6.43 (t, J = 2.2 Hz, 1H), 5.65 (d, J = 1.1 Hz, 1H), 5.44 (dd, J = 4.3, 0.9 Hz, 1H), 3.81 (s, 6H), 3.79 (dd, J = 8.4, 4.6 Hz, 1H), 2.46−2.38 (m, 1H), 2.28 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 162.13, 158.45, 158.32, 149.79, 142.27, 140.08, 137.92, 137.38, 130.67, 130.13, 129.50, 128.68, 128.39, 125.02, 124.28, 123.43, 116.60, 116.39, 105.51, 100.89, 85.29, 83.65, 61.61, 55.65, 31.49. HRMS (ESI) calcd for C34H30O8S [M + Na]+ 621.1554, found 621.1552. Optical rotation was recorded in methanol solution, at 20 °C, sodium light as a light source, [α] = +13.48. 4-((E)-3,5-Dimethoxystyryl)phenyl(1S,2R,4S)-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (26a-2). White solid, 18% yield, mp 108−110 °C. 1H NMR (400 MHz, acetone-d6) δ 8.69 (s, 1H), 8.62 (s, 1H), 7.62−7.57 (m, 2H), 7.26 (m, 4H), 7.24 (d, J = 2.3 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 6.82−6.79 (m, 2H), 6.78 (d, J = 2.2 Hz, 2H), 6.43 (t, J = 2.2 Hz, 1H), 5.65 (d, J = 1.2 Hz, 1H), 5.44 (dd, J = 4.3, 1.1 Hz, 1H), 3.81 (s, 6H), 3.79 (dd, J = 8.4, 4.6 Hz, 1H), 2.47−2.38 (m, 1H), 2.28 (m, 1H). 13C NMR (100 MHz, acetone-d6) δ 162.13, 158.45, 158.32, 149.79, 142.27, 140.08, 137.92, 137.38, 130.67, 130.13, 129.50, 128.68, 128.39, 125.02, 124.28, 123.43, 116.60, 116.39, 105.51, 100.89, 85.29, 83.65, 61.61, 55.65, 31.49. HRMS (ESI) calcd for C34H30O8S [M + Na]+ 621.1554, found 621.1551. Optical rotation was recorded in methanol solution, at 20 °C, sodium light as a light source, [α] = −13.46. Estrogen Receptor Binding Affinity. Relative binding affinities were determined by a competitive fluorometric binding assay. Briefly, 40 nM of a fluorescence tracer and 0.8 μM purified human ERα or ERβ ligand binding domain (LBD) were diluted in 100 mM potassium phosphate buffer (pH 7.4) containing 100 μg/mL bovine gamma globulin (Sigma-Aldrich, MO). Incubations were performed for 2 h at room temperature (25 °C) in the dark. We then used a Cytation 3 microplate reader (BioTek) to measure fluorescence polarization values. The binding affinities are expressed as relative binding affinity (RBA) values with the RBA of 17β-estradiol set to 100%. The values given are the average ± range of two independent determinations. Ki values were calculated according to the following equation: Ki = (100/RBA) × Kd. Gene Transcriptional Activity. The human embryonic kidney cell line HEK 293T was maintained in Dulbecco’s Minimum Essential

Medium (DMEM) (Gibco by Invitrogen Corp., CA) with 10% fetal bovine serum (FBS) (HyClone by Thermo Scientific, UT). Cells were plated in phenol red-free DMEM with 10% FBS. HEK 293T cells were transfected with 25 μL of the mixture per well, containing 300 ng of the 3× ERE-luciferase reporter, 100 ng of the ERα or ERβ expression vector, 125 mM calcium chloride (GuoYao, China), and 12.5 μL of 2× HBS. On the next day, the cells were treated with increasing doses of ER ligands diluted in phenol red-free DMEM with 10% FBS. After 24 h, luciferase activity was measured using a DualLuciferase Reporter Assay System (Promega, MI) according to the manufacturer’s protocol. Anti-Inflammatory Activity Assay. The human macrophage cell line RAW 264.7 was obtained from ATCC and maintained in DMEM with 10% FBS. After RAW 264.7 cells (1 × 105 cells/well) were grown in 96-well microtiter plates (Nest Biotech Co., China) for 24 h, they were treated with samples and 1 μg/mL LPS for 24 h. The Griess reaction was performed to estimate the amount of nitrite. Briefly, 50 μL of Griess reagent I and Griess reagent II was added to 50 μL of supernatant of each well, and the absorbance was measured at 540 nm. Cell Culture and Cell Viability Assay. The MCF-7 human breast cancer cell line was obtained from ATCC. Cells were maintained in DMEM with 10% FBS. For all experiments, the cells were grown in 96-well microtiter plates (Nest Biotech Co., China) with the appropriate ligand in triplicate for 72 h. MTT colorimetric tests (Biosharp, China) were employed to determine cell viability per the manufacturer’s instructions. IC50 values were calculated according to the following equation using Origin 8 software: Y = 100% inhibition + (0% inhibition − 100% inhibition)/(1 + 10 [(logIC50‑X)×Hillslope] ), where Y = fluorescence value, X = log[inhibitor]. Anti-Inflammatory Activity of 26a Contributes to Increased Anti-Proliferative Activity. The MCF-7 human breast cancer cell line was obtained from ATCC. Cells were maintained in DMEM with 10% FBS. For all experiments, cells were grown in 96-well microtiter plates (Nest Biotech Co., China) with the appropriate ligand in triplicate for 48 h. MTT colorimetric tests (Biosharp, China) were employed to determine cell viability per the manufacturer’s instructions. The cell inhibition rate was calculated using Origin 8 software. Mouse Tumor Xenograft Model. Six-week-old female Balb/c nude mice (15−18 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal study procedures were carried out in compliance with the Guide for the Care and Use Committee at Peking University (permit no. 20110039). Mice were orthotopically injected with 1 × 107 MCF-7 cells in 100 μL of 1× PBS into the right axillary mammary fat pad area. When the tumor reached a size of ∼80 mm3, the mice were randomly divided into four groups (five/group) and administered treatment. The mice were treated by gavage with vehicle control (consisting of cremophor-EL/DMSO/PBS 1:1:8, total 100 μL) or the same volume of tamoxifen (25 mg/kg) or compound 26a (17.5 mg/kg or 25 mg/ kg, tamoxifen equivalent dose) once every 2 days. The weight of the mice was measured, and the tumor volume was monitored every 3 days using a digital caliper. Tumor size was calculated using the following formula: volume = 0.5 × (width)2 × (length). At the end of the treatment, the mice were sacrificed and tumor weights recorded. All experiments were performed in accordance with the approved guidelines. OBHS-RES Conjugates Have Anti-Inflammatory Effects during Anti-Tumor Progression in Balb/c Nude Mice. Fiveweek-old female Balb/c nude mice (14−17 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All procedures in animal studies were carried out in compliance with the Guide for the Care and Use Committee at Peking University (permit no. 2011-0039). Mice were orthotopically injected with 1 × 107 MCF-7 cells in 100 μL of 1× PBS into the right axillary mammary fat pad area. When the tumor reached a size of ∼80 mm3, the mice were randomly divided into four groups (four/group) and treatment initiated. The mice were treated by gavage with vehicle control (consisting of cremophor-EL/DMSO/PBS 1:1:8, total 100 8170


Journal of Medicinal Chemistry μL) or the same volume of tamoxifen (25 mg/kg) or compound 26a (17.5 mg/kg or 25 mg/kg, tamoxifen equivalent dose) once every 2 days. After 2 weeks of treatment, immunohistochemistry was used to monitor the activity of IL-6, TNF-α, and NF-κB in the tissue around the tumor. Immunohistochemistry was performed in paraffinembedded mouse tissue sections. The staining signal was quantified by monitoring the average numbers of positively stained cells relative to the total number of cells from six randomly chosen fields. Molecular Modeling. The crystal structure of ERα LBD (PDB 3ERT) was obtained from the PDB, and all water molecules were removed. We used AutoDock software (version 4.2) to dock compounds 26a-1 and 26a-2 into the three-dimensional structure of ERα LBD. The crystallographic coordinates of 26a-1 and 26a-2 were created by Chemoffice. Preparations of all ligands and the protein were performed with AutoDockTools (ADT). A docking cube with edges of 66, 66, and 60 Å in the X, Y, and Z dimensions, respectively (a grid spacing of 0.375 Å). The search parameters were determined using the Genetic Algorithm and the output based on the Lamarckian genetic algorithm (LGA). The figures were prepared using PyMOL.61

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REFERENCES

ER, estrogen receptor; SERMs, selective estrogen receptor modulators; SERDs, selective estrogen receptor downregulators; AIs, aromatase inhibitors; NF-κB, nuclear factor κ B; RES, resveratrol; OBHS, exo-5,6-bis(4-hydroxyphenyl)-7oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl ester; HDACi, histone deacetylase inhibitor; OBHSA, OBHsulfonamide; NBS, N-bromosuccinimide; THF, tetrahydrofuran; RBA, relative binding affinity; LBD, ligand binding domain; E2, 17β-estradiol; iNOS, inducible nitric oxide synthase; NO, nitric oxide; LPS, lipopolysaccharide; 4OHT, 4-hydroxytamoxifen; IL-6, interleukin-6; TNF-α, tumor necrosis factor α; TLC, thin layer chromatography; ERE, estrogen response element; DMEM, Dulbecco’s Minimum Essential Medium; FBS, fetal bovine serum; [α], specific rotation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00224. The synthesis and spectroscopic characterization of intermediate compounds, NOESY-NMR of regioisomers 25b, 29, and 30, the computer model of the ERαcompound 25p complex, the biological curves of the final compounds, 1H NMR and 13C NMR spectra information, HPLC results, and HPLC spectra for the final compounds (DOC) Docking model of ERα±-25P complex (PDB) Docking model of ERα±-26a-1 complex (PDB) Docking model of ERα±-26a-2 complex (PDB) Molecular formula strings and the associated biological data (CSV) Accession Codes

The coordinates and structure factors have been deposited with the RCSB Protein Data Bank under accession code 3ERT for 25p, 26a-1, and 26a-2. The authors will release the atomic coordinates and experimental data upon article publication.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +862768759586. E-mail: [email protected]. ORCID

Hai-Bing Zhou: 0000-0001-8498-063X Author Contributions ∥

W.N. and Z.H. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the NSFC (81773557, 81573279, 81373255), Major Project of Technology Innovation Program of Hubei Province (2016ACA126), NSFHP (2017CFA024), and the Fundamental Research Funds for the Central Universities of China (2015306020201) for support of this research. We thank Professor John A. Katzenellenbogen at UIUC for helpful discussions and critical reading of this manuscript. 8171



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Novel Hybrid Conjugates with Dual Suppression of Estrogenic and

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