Identification and structure-activity relationships of a novel series of estrogen receptor ligands based on 7-thiabicyclo[2.2.1]hept-2-ene-7-oxide

Article

pubs.acs.org/jmc

Identification and Structure−Activity Relationships of a Novel Series

of Estrogen Receptor Ligands Based on 7-Thiabicyclo[2.2.1]hept-2-

ene-7-oxide

Pengcheng Wang,^†Jian Min,^†Jerome C. Nwachukwu,^§Valerie Cavett,^§Kathryn E. Carlson,^‡Pu Guo,^†

Manghong Zhu,^†Yangfan Zheng,^†Chune Dong,^†John A. Katzenellenbogen,*^,‡Kendall W. Nettles,^§

and Hai-Bing Zhou*^,†

^†State Key Laboratory of Virology, Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University, Ministry of

Education, Wuhan University School of Pharmaceutical Sciences, Wuhan 430072, China

^‡Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, United States

^§Department of Cancer Biology, The Scripps Research InstituteFlorida, 130 Scripps Way, Jupiter, Florida 33458, United States

S

* Supporting Information

ABSTRACT: To develop estrogen receptor (ER) ligands having novel

structures and activities, we have explored compounds in which the

central hydrophobic core has a more three-dimensional topology than

typically found in estrogen ligands and thus exploits the unfilled space

in the ligand-binding pocket. Here, we build upon our previous

investigations of 7-oxabicyclo[2.2.1]heptene core ligands, by replacing

the oxygen bridge with a sulfoxide. These new 7-thiabicyclo[2.2.1]hept-

2-ene-7-oxides were conveniently prepared by a Diels−Alder reaction of

3,4-diarylthiophenes with dienophiles in the presence of an oxidant and give cycloadducts with endo stereochemistry. Several

new compounds demonstrated high binding affinities with excellent ERα selectivity, but unlike oxabicyclic compounds, which are

transcriptional antagonists, most thiabicyclic compounds are potent, ERα-selective agonists. Modeling suggests that the gain in

activity of the thiabicyclic compounds arises from their endo stereochemistry that stabilizes an active ER conformation. Further,

the disposition of methyl substituents in the phenyl groups attached to the bicyclic core unit contributes to their binding affinity

and subtype selectivity.

three-dimensional topology than is commonly found in both

steroidal and nonsteroidal ER ligands. This design strategy was

based on structural studies of the ligand binding pockets of

both ERα and ERβ: In addition to the obvious flexibility and

deformability of the ligand binding pocket,¹⁶it was notable that

the cavity of ERα has a probe-accessible size of ca. 450 Å³,

whereas estradiol (E₂) has a molecular volume of only 245 Å³;

though somewhat smaller, the ligand pocket in ERβ is also

considerably larger than that of E₂.¹⁷As a result of these

marked pocket vs ligand volume differences, there is substantial

unoccupied space on the α face of the B-ring and the β face of

the C-ring.¹⁸By incorporating a more three-dimensional

hydrophobic bicyclic unit as the core structure of a ligand, we

hoped to exploit this unfilled, opportunity space, thereby

enhancing their binding affinity and/or ER subtype selectivity,

and potentially uncovering novel patterns of estrogen responses

through the ERs. We and a number of other investigators have

prepared some ER ligands having more 3-dimensional

character, such as those with ferrocene,¹⁹⁻²¹carborane,^22,23

INTRODUCTION

■

Estrogens are known to play important roles in the develop-

ment and maintenance of both reproductive and non-

reproductive tissues in both women and men.^1,2While

estrogens are required and can provide some health benefits

in some tissues, such as those of the reproductive,³skeletal,⁴

cardiovascular,⁵and central nervous systems,⁶the pro-

proliferative effect of estrogens can be pathological and

promote cancer in the breast and uterus.⁷⁻⁹The multiple

actions of estrogens are mediated by two estrogen receptors

(ERα and ERβ) that, although similar, are distinct gene

products with nonoverlapping and even opposing functions.¹

These different functions, combined with the distinct tissue

distribution patterns of these two receptors, result in the

remarkable tissue-selective effects of estrogens²and, thus, have

heightened interest in searching for selective estrogen receptor

modulators (SERMs) that are also subtype selective and thus

best able to support estrogen health benefits and minimize the

risk of cancer.¹⁰⁻¹⁵

As part of our long-term interest in the development of

ligands for the ERs having novel structures and activities, we

have undertaken exploratory studies by preparing new

compounds having a central core that has, overall, a more

Received: November 18, 2011

Published: January 26, 2012

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Scheme 1. Described Three-Dimensional, Thiophene, or Sulfur Containing ER Ligands and the Title Compounds

a

Scheme 2. Synthesis of Thiophenes 1a−f

a

Reagents and conditions: a) n-BuLi, −78 °C, 1 h; B(OMe)₃, −78 °C-rt; b) [Pd]/PPh₃, Na₂CO₃, toluene, reflux 48 h; c) BBr₃, CH₂Cl₂, 0 °C-rt, 24

h.

polycyclic,^23,24and cyclopentadienyl metal tricarbonyl core

Beyond the oxygen-containing furan and pyran-type hetero-

cycles, sulfur-containing heterocycles also frequently constitute

the cores of ER ligands, as the benzothiophene core of

raloxifene and the benzoxathiin core of other ER ligands

(Scheme 1).³⁴Some simple aryl-substituted thiophenes can

also be ER ligands, as well as inhibitors of certain steroid

dehydrogenases.^35,36It is noteworthy that although some aryl-

substituted thiophenes exhibit good ER binding affinities, as

thus far reported, they have limited selectivity and bioactivity.

In light of these recent reports and in continuation of our

interest in nonsteroidal estrogens, we extended our previous

study of OBHS by replacing the oxabicyclic core with a 7-

thiabicyclic core.

structures.²⁵

In previous studies, we prepared a series of 7-

oxabicyclo[2.2.1]hept-5-ene compounds as ER ligands

(Scheme 1).²⁶The best compound, exo-5,6-bis-(4-hydroxy-

phenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl

ester (which we named OBHS), exhibited modest ER subtype

selectivity, with the relative binding affinity (RBA) values 9.3%

and 1.7% for ERα and ERβ, respectively (RBA[estradiol] =

100%), and was profiled as an antagonist on both ER subtypes,

with a modest potency preference for ERβ.²⁶Bearing some

structural relationship to other bicyclic ER ligands, such as

bicyclo[3.3.1]nonanes^27,28and oxabicyclo[3.3.1]nonenes,²⁹⁻³¹

the 7-oxabicyclo[2.2.1]hept-5-enes mimic an element of the

core of high affinity furan-based ER ligands that we have

studied,^32,33and they also embody a 1,2-diarylethylene unit, a

motif found in many high-affinity nonsteroidal estrogens.

Unlike furan, thiophene is not a good diene for the Diels−

Alder reaction because of its higher aromaticity.³⁷In addition,

the sulfur or sulfone bridge is not very stable, and such Diels−

Alder adducts can spontaneously lose sulfur or sulfur dioxide,

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a

Scheme 3. Synthesis of Dienophiles 8

a

Reagents and conditions: a) NaOH, CH₂Cl₂, 0 °C; b) BBr₃, CH₂Cl₂, 0 °C-rt, 24 h.

respectively, leading to benzene ring formation.³⁸⁻⁴¹Con-

sequently, after a brief survey, we chose a 7-thiabicyclo[2.2.1]-

hept-2-ene-7-oxide as the core structure of these novel ER

ligands, because of its greater stability and ease of preparation

(Scheme 1).

Scheme 4. Diels−Alder Reaction of Thiophene 1 with

Dienophiles 8 to Give SOBHS Adducts 9

In this report, we describe novel sulfoxide-bridged OBHS

analogues constituted of a 7-thiabicyclo[2.2.1]hept-2-ene 7-

oxide core that can be prepared conveniently by a Diels−Alder

reaction of thiophene with an appropriate dienophile in the

presence of an oxidant.⁴²This bicyclic core system, which we

term SOBHS, expands our exploration of ER ligands having an

overall three-dimensional topology, and it introduces some new

characteristics as well. Therefore, this structure potentially

could be further investigated and developed as the basis for new

estrogen pharmacological agents. We also evaluate the effect of

SOBHS analogues on ER binding affinity and estrogen

responsive element (ERE)-driven transcriptional activity.

(Scheme 4).^42,43A wide range of dienophiles were examined to

obtain a broad structure−activity relationship of this series. On

the other hand, the dienophiles were restricted to mostly arene

esters of vinylsulfonic acid, because earlier work in the OBHS

series had indicated that the Diels−Alder products from

vinylsulfones and various maleic acid derivatives generally gave

products with low affinity for the ERs,²⁶although we did

prepare some of these analogues for comparison purposes.

Unlike the high yields obtained in the Diels−Alder reactions

with furans, the Diels−Alder reaction with the thiophenes was

very sluggish; conversions were typically around 60%, and the

yields of the Diels−Alder adducts were moderate. Also, while

the exo products predominated in the Diels−Alder reaction

with furans, presumably because, as we described previously,²⁶

this very facile cycloaddition is reversible under the conditions

used, we observed high endo stereoselectivity in the Diels−

Alder reaction with phenolic thiophenes. This endo stereo-

chemistry for one compound (13) (Scheme 5) was verified by

the two-dimensional ROESY-NMR (Figure 1, see legend). This

observation is in accord with other studies documenting that

the Diels−Alder reaction with these systems takes place

exclusively in an endo-mode with 100% π-face selectivity, in

which dienophiles add in an endo fashion to the thiophene S-

monoxide on the syn-π-face relating to the SO bond, which is

the combined result of secondary orbital energy interaction and

steric factors.^44,45It should be noted that all compounds were

studied as racemates, and in the one case where an

unsymmetrical thiophene was used as a diene, we were unable

to separate the regioisomeric products (compound 15), despite

our best efforts, although the very low affinity of this compound

makes this less of an issue.

RESULTS AND DISCUSSION

■

Chemical Synthesis. The preparation of the 7-thiabicyclic

oxide-bridged compounds involves a Diels−Alder reaction of

aryl-substituted thiophenes with various dienophiles. The 3,4-

bis(4-hydroxyphenyl)-substituted thiophenes (1a−c) can be

conveniently prepared from 3,4-dibromothiophene by a Suzuki

coupling sequence that, together with a boronic acid synthesis

and a phenol demethylation, proceeds in three steps (Scheme

2A). 3,4-Diphenylthiophene (1d) and 3,4-di-p-tolylthiophene

(1e) can be prepared in one step from commercially available

boronic acids (Scheme 2B). The unsymmetrical thiophene 1f

was obtained by demethylation of 7, which was prepared by

two successive cross-coupling reactions (Scheme 2C). In the

first step, 1 equiv of 3,4-dibromothiophene reacted with 1.2

equiv of phenyl boronic acid using standard conditions. In the

second step, the resulting monosubstituted thiophene 6 was

subsequently submitted to a second cross-coupling reaction

with 1.2 equiv of aryl boronic acid 2c to yield the intermediate

7, with ether cleavage with boron tribromide giving the final

compound 1f.

The synthesis of vinyl sulfonates 8a−k was accomplished by

the reaction of 2-chloroethanesulfonyl chloride with substituted

phenols under basic conditions, as shown below (Scheme 3).

The synthesis of 7-thiabicyclic oxide bridged compounds was

achieved by a Diels−Alder reaction of thiophene 1 with various

dienophiles 8 (2 equiv) (Scheme 4) in the presence of an in situ

oxidant (m-CPBA) and a Lewis acid (BF₃); the results are

summarized in Table 1. This transformation is presumed to

proceed via two steps: in situ oxidation of the thiophene to the

thiophene S-monoxide, followed by Diels−Alder reaction to

produce the 7-thiabicyclic[2.2.1]hept-2-ene-7-oxide structure

In our previous work on 7-oxabicyclic[2.2.1]hept-5-ene

(OBHS)-core ER ligands,²⁶we found that compounds bearing

a p-hydroxyphenyl group in both the C-5 and C-6 positions and

a phenyl sulfonate in the C-2 position of the bicyclic core

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Table 1. Diels−Alder Reaction of Thiophene 1 and Dienophiles 8

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Table 1. continued

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Table 1. continued

a

b

The conversion was calculated accounting for the recovered thiophene. Isolated yield by column chromatography purification based on the

thiophene consumed.

1

Scheme 5. H NMR Assignments of endo 13

series.²⁶Using molecular modeling as a guiding tool, we

designed and synthesized a small array of 29 SOBHS analogues.

Despite the generally moderate yields (30−50%) that we

obtained with the diphenolic thiophenes 1a−c and dienophiles,

we found that the reaction of 1a−c with the ethenesulfonic acid

4-hydroxyphenyl ester 8k and diethyl maleate 8m gave the

corresponding products in lower yields (15−32%) (Table 1,

entries 3, 5, 16, and 24). Part of the reduced yield appears to be

the sensitivity of the products to purification by silica gel

chromatography. In comparison, compounds 1d,e, which have

no hydroxyl group on the phenyl ring, reacted well with 8k,

giving products 13 and 14 in 55% and 49% yields, respectively

(Table 1, entries 27 and 28).

Binding Affinity for Estrogen Receptors ERα and ERβ.

The binding affinities of the SOBHS compounds for both ERα

and ERβ were determined using a competitive radiometric

assay and are reported in Table 2.^46,47These affinities are

presented as relative binding affinity (RBA) values, where

estradiol has an affinity of 100%. At the start, it should be noted

that comparisons between compounds in the SOBHS series,

presented here, and the OBHS compounds, prepared earlier,²⁶

are between SOBHS endo stereoisomers and OBHS exo

stereoisomers, although, in the one case we investigated

previously, there was relatively little difference in the affinity

between exo and endo OBHS isomers.²⁶

always had the highest ER binding affinities (see below).

Therefore, we wondered whether the replacement of the

oxygen atom on the bridge with a sulfoxide group might lead to

ligands in the SOBHS series with increased binding affinity.

Thus, we started our investigation with compound 10a, as

shown in Table 1 (entry 1). In addition, we explored the

potential binding affinities and estrogenic properties of the

SOBHS analogues by varying the substituents on phenol

groups in the 5,6-positions, and the phenyl group of sulfonate,

while keeping the 7-thiabicyclic[2.2.1]hept-2-ene-7-oxide skel-

eton intact. Meanwhile, the adducts of diaryl thiophenes with

other dienophiles, e.g., naphthyl vinyl sulfonate as well as

diethyl maleate and N-phenylmaleimide, were also prepared for

comparison with compounds prepared previously in the OBHS

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Figure 1. ROESY-NMR of endo 13. The peaks at δ 4.86 and δ 4.49 are the hydrogen atoms on the bridgehead carbons (H₁and H₄), and the peak

between them (at δ 4.59) is the hydrogen attached to the carbon bearing the sulfonate group (H₂). It is evident that the H₂interacts with the

bridgehead hydrogen H₁. Since the bridgehead hydrogen is necessarily at an exo position, this interaction indicates that H₂is also at the exo position,

and, as a result, the sulfonate group is disposed in an endo configuration.

As a global observation, it is notable first that members of the

SOBHS class bind with somewhat lower affinity than the

corresponding members of the OBHS class.²⁶Second, addition

of a methyl group in the 5,6-substituted phenol rings, as well as

the substituents at the 2- and 3-positions of the 7-

thiabicycloheptane 7-oxide core, has very significant effects on

the binding affinity of the ligands. The series of compounds 11

that have an o-methyl group in both of the core phenyl

substituents (o means adjacent to the attachment site to the

bicyclic system; see Scheme 1) demonstrate a better binding

affinity than the other two series (10 and 12). The compound

that has the highest binding affinity for ERα is endo-phenyl-5,6-

bis(4-hydroxy-2-methylphenyl)-7-thiabicyclo[2.2.1]hept-5-ene-

2-sulfonate-7-oxide (11a), a compound that possesses a p-

hydroxyl group and an o-methyl group in both of the core

phenyl substituents and a phenyl sulfonate moiety at the 2-

position of the bicyclic unit. The RBA values of this compound

are 8.11 and 0.348 for ERα and ERβ, respectively (Table 2,

entry 6), which are comparable to those of the best compounds

we have reported in the original OBHS series.²⁶However, the

compounds in the 12 series, which possesses a m-methyl group

instead of an o-methyl group as in the 11 series, show the

highest ER subtype selective binding affinity. For example,

compounds 12a and 12c, which possess a p-hydroxyl group and

a m-methyl group in both of the core phenyl substituents, have

an ERα/ERβ selectivity as high as 249 and 248, respectively,

which are the highest selectivity values among the 29

compounds, being more than 10 times greater than 11a and

11c (Table 2, entries 18 vs 6, and 20 vs 8). In fact, the ERα

binding preference for these compounds approaches that of the

most ERα selective ligand of which we are aware, propyl

pyrazole triol (PPT), a compound on which we reported some

time ago, though the absolute affinity of the SOBHS

compounds for ERα is less than that of PPT.⁴⁸

The position of the hydroxyl group is also of great

importance. The hydroxyl group in the 5, 6-substituted phenyl

rings is more important than that in the phenyl sulfonate

moiety at the 2-position, as can be surmised, to some extent,

from a comparison of compounds 15 and 12a (Table 2, entries

29 and 18). As is widely known, the presence of a phenolic ring

in ER ligands is crucial to their binding affinity, as this ring is

needed to mimic the steroidal “A ring” present in natural

estrogens.⁴⁹This phenol forms important hydrogen bonds with

residues Glu353 and Arg394 and with a structured water

molecule in ERα or the corresponding residues in ERβ.¹⁸

Therefore, this dependence of the RBA value on the position of

the hydroxyl group suggests that the hydroxyl group in the 5,6-

substituted phenyl rings is better positioned to establish these

critical hydrogen bonds with the ERs. Consistent with this

required phenolic ring feature, compounds 13 and 14, which

lack phenolic hydroxyl groups on the core phenyls, show low

affinities (Table 2, entries 27 and 28).

While one might imagine that a single core phenol group, as

in compound 15 (Table 2, entry 29) might prove sufficient to

engender good binding to the ERs, this is clearly not the case,

nor was it the case in the OBHS series studied earlier.²⁶In the

crystal structure of OBHS-like compounds in ERα, one of the

core phenols is in the steroidal A-ring position, engaged in the

crucial hydrogen bonds, but the second core phenol projects

upward in the ligand pocket, roughly in a direction that

corresponds to a steroidal 11β substituent.⁵⁰This places the

second phenolic OH close to Thr347, which, aside from

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a

Table 2. Relative Binding Affinity (RBA) of 7-Thiabicyclic-7-oxide Analogues for ERα and ERβ

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Table 2. continued

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Table 2. continued

a

compound

Relative binding affinity (RBA) values are determined by competitive radiometric binding assays and are expressed as 100IC₅₀^estradiol/IC₅₀

the range or standard deviation (RBA, estradiol = 100%). In these assays, the K_dfor estradiol is 0.2 nM on ERα and 0.5 nM on ERβ. For details, see

the Experimental Section.

Glu353 and Arg394, is the only other polar residue in the

ligand-binding pocket. This is very likely an energetically

productive interaction, as the second phenol in the bisphenolic

ligands of the cyclofenil class, which also greatly enhances the

affinity of these ligands, is thought to play the same role.²⁸

In the ligands studied here, the substituents on the C-2 or C-

3 position of the bridged core have a significant effect on

binding affinity and selectivity. Because many nonsteroidal

estrogens are triphenols, the introduction of an additional

hydroxyl group is also investigated; however, as was the case in

the OBHS series,²⁶the placement of a methoxyl or hydroxyl

group at the para position of the phenyl sulfonate ring caused a

decrease in affinity for ERα (the trend is not clear for ERβ) and

for ER subtype selectivity (Table 2, entries 2, 3, 7, 16, 19, and

24). In fact, the introduction of a third hydroxyl group results in

a remarkable drop in affinity for ERα; only compound 11k still

shows a moderate binding affinity for ERα; however, its affinity

for ERβ increases (RBA ERα 2.21 and ERβ 0.812) (Table 2,

entry 16). It is not clear why ERα and ERβ show different

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responses to these substituent alterations, and this phenomen-

on is different from that of the OBHS-core ligands.²⁶

Table 3. Transcriptional Activities of 7-Thiabicyclic-7-oxide

Analogues through ERα and ERβ

Compounds bearing halogens on the sulfonate phenyl group

were also evaluated; however, all of them have decreased

binding affinity for both ERα and ERβ. With the chlorinated

compounds 11d, 11g, and 11i (Table 2, entries 9, 12, and 14),

the position of the substituent has little effect on binding

affinity. For the para halogenated compounds 11f−h (Table 2,

entries 11−13), the bromo compound (11h) seems to be

superior to the other two. By contrast, for the o-halogenated

compounds 11c−e and 12c−e (Table 2, entries 8−10 and 20−

22), the fluorine-substituted compounds (11c and 12c) are the

best. Other changes to the sulfonate moiety, such as replacing

the phenyl with a naphthyl group, as in compound 11j and 12f

(Table 2, entries 15 and 23), lower both binding affinity and

subtype selectivity.

ERα EC

ERα (%

ERβ EC₅₀

ERβ (%

a⁵⁰

entry cmpd

(nM)

E₂)

(nM)

E₂)

E₂

2.2

100 16

85 20

48 0.2

11

100

3.4

19

10

24

27

66

32

54

46

21

6

1

10a

10b

10c

10d

10e

11a

11b

11c

11d

11e

11f

11g

11h

11i

11j

11k

11l

670

2

9

3

1100

7100

48

31

55

62

76

56

73

67

4

8

7

4

5

7

3

9

3

7

2

3

2

4

5

6

0.14

210

2.2

4400

160

7

8

9

2.8

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

14

200

110

n.d.

74 10

45 11

50 10

100 10

n.d.

When other dienophiles, such as diethyl maleate and N-

phenylmaleimide, were used, the Diels−Alder adducts all gave

very poor binding affinity and selectivity (compounds 10e and

12i, Table 2, entries 5 and 26, and compounds 10d, 11l, and

12h, Table 2, entries 4, 17, and 25). The products from these

dienophiles in the furan series also showed very low affinity.²⁶

We also measured the ER binding affinities of the three 3,4-

bisphenolic thiophenes (1a−c) used for the preparation of the

three series of SOBHS compounds in this report (Table 2,

entries 30−32). Comparison of the affinities of the three parent

thiophenes with members of the three series of SOBHS

compounds derived from them is interesting. First, incorpo-

ration of a thiophene into the 7-thiabicyclo[2.2.1]heptane 7-

oxide phenyl sulfonate system in each case raises ERα binding

affinity but lowers ERβ binding affinity (Table 2, entry 30 vs 1;

entry 31 vs 6; entry 32 vs 18). Very likely, this has to do with

the smaller volume of the ligand-binding pocket in ERβ.¹⁷

Second, the highest affinity thiophene (compound 1b, Table 2,

entry 31), which has two o-methyl groups, gives rise to, overall,

the highest affinity SOBHS series (compounds 11, Table 2,

entries 6−17); however, the lowest affinity thiophene

(compound 1c, Table 2, entry 32), having two meta methyl

groups, gives a series of SOBHS compounds that overall have

higher affinity (compounds 12, Table 2, entries 18−26) than

those derived from the unsubstituted thiophene (compounds

10, Table 2, entries 1−5). Thus, the sulfoxide bridge structure

and other elements of the three-dimensional SOBHS ligand

core design make strong contributions to the binding affinity

and selectivity of the parent thiophene precursors. Further

studies on many other members of the parent thiophene class

will be described in a subsequent publication. Overall then, the

disposition of the methyl group in the appended phenol

substituents in the C-5 and C-6 positions of the bicyclic core

unit, and the electron withdrawing group derived from the

dienophiles, all prove to be factors in determining the binding

affinity and selectivity of these novel SOBHS-core ligands for

ERs.

67

71

n.d.

59

58

65

57

55

72

51

42

n.d.

29

22

24

32

5

9

760

n.d.

27

7

6

5

6

4

3

7

6

2

83

82

3

6

9.2

14000

800

150

12

82 20

12a

12b

12c

12d

12e

12f

12g

12h

12i

13

3.8

9.8

0

1

2

11000

1300

2000

1200

770

n.d.

0

10

n.d.

3.4

3.6

0

2

n.d.

3

2

6

2

1

14

5500

340

10

38

4

15

3

a

Luciferase activity was measured in HepG2 cells transfected with 3X-

ERE-driven luciferase reporter and expression vectors encoding ERα

or ERβ and treated in triplicate with increasing doses (up to 10⁻⁵M)

of the compounds. EC₅₀and average efficacy (mean S.E.M.), shown

as a percentage of 10⁻⁵M 17β-estradiol (E₂), were determined. Effects

of 11i and 12g were not determined (n.d.). Omitted EC₅₀values were

too high, while omitted %E₂values were too low to be determined

accurately.

Activation of ERα and ERβ Mediated Transcription.

Various SOBHS compounds were tested by luciferase reporter

gene assays for their ability to stimulate the transcriptional

activities of ERα and ERβ compared to 17β-estradiol (E₂).

Luciferase assays were conducted in human liver cancer

(HepG2) cells transfected with full-length human ERα or

ERβ, and a widely used estrogen-responsive element (ERE)-

driven luciferase reporter.⁵¹These results are summarized in

Table 3, and dose−response curves for a few examples are

shown in Figure 2.

Figure 2. Illustrative dose−response curves for estradiol and two

sulfoxide-bridged SOBHS compounds in ERα and ERβ reporter gene

assays in HepG2 cells. For details, see the Experimental Section.

Compound 10a, which showed approximately 100-fold

weaker binding affinity for ERα than E₂(Table 2, entry 1),

stimulated ERα activity with about 300-fold weaker potency

than E₂, but near full efficacy (Table 3). Within this compound

10 scaffold, modifications of the phenyl sulfonate moiety (i.e.,

compounds 10a−e, entries 1−5) further reduced the potency

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Figure 3. Structure of ODE and models of OBHS and SOBHS binding within the ERα LBD. (A) Crystal structure of transcriptionally active ERα

LBD in complex with ODE (PDB ID: 2QH6)⁵⁰showing ligand orientation relative to helices 3, 6, 8, 11, and 12 (purple). The ODE hydroxyphenyl

groups form hydrogen bonds with Thr347, Glu353, and Arg394, while the exo ethyl ester moiety displaces His524 (shown in bold), compared to its

position in the estradiol-bound LBD structure (PDB ID: 1ERE) (gray). (B) Model of OBHS binding within the ERα LBD. Like the ethyl ester

moiety of ODE, the exo phenyl sulfonate moiety of OBHS clashes with helix-11 residues including His524. (C) Model of SOBHS binding within the

ERα LBD. The endo phenyl sulfonate moiety of SOBHS is accommodated in a different region of the pocket, avoiding the clash with helix-11.

and efficacy with which the compounds stimulate ERα activity

(Table 3), and the binding affinity for ERα (Table 2).

Compounds having a p-hydroxyl group on the phenyl

sulfonate moiety or lacking p-hydroxyl groups on both phenyl

substituents, which decreased their binding affinity (Table 2,

10c and 13−15, entries 3 and 27−29), also show decreased

potency and efficacy as ERα agonists (Table 3). Interestingly,

introducing p-hydroxyl and m-methyl groups to one of the

phenyl substituents (i.e., compound 15) improved binding

affinity (Table 2, entry 29), as well as potency as ER agonists

(Table 3).

Compound 11a, which has an o-methyl group in both of the

core hydroxyphenyl substituents and about 8-fold improved

binding affinity for ERα (Table 2, entry 6), demonstrated about

5,000-fold higher potency but reduced efficacy as an ERα

agonist (Figure 2), compared to compound 10a (entry 1)

(Table 3). Modifications of the phenyl sulfonate moiety within

this compound 11 scaffold (i.e., compounds 11a−l) further

reduced potency (Table 3) and affinity (Table 2, entries 6−17)

as ERα agonists.

ERα (Table 2), as well as potency as ERα agonists to various

extents (Table 3).

Structure−Activity Relationships. Crystal structures of

ERα LBD in complex with oxabicyclic compounds (PBD ID:

2QH6 and 2QR9)⁵⁰show that one p-hydroxyphenyl group

attached to the oxabicyclic core engages in hydrogen bonding

with Glu353 and Arg394, while the other p-hydroxyphenyl

group forms a hydrogen bond with Thr347. The ethyl ester

moieties of oxabicyclic diethyl ester (ODE, i.e., diethyl 5,6-

bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicar-

boxylate), which are attached to the oxabicyclic core in the exo

position, are accommodated in the pocket at least in part by

displacing helix-11 residues, including His524, which engages in

hydrogen bonding with 17β-estradiol (E₂) (PDB ID: 1ERE)

(see the two indicated positions for His524 in Figure 3A).¹⁸

Displacement of helix-11 toward the dimer interface is

associated with reduced ERα transcriptional activity, a

mechanism sometimes termed “passive antagonism”, suggesting

a suboptimal ERα LBD conformation for full agonist

activity.^50,52

Crystal structures of the ER LBD in complex with OBHS or

SOBHS have not yet been reported; therefore, the exact

orientation of their phenyl sulfonate moieties in the ER ligand-

binding pocket is unclear. The molecular model of OBHS

bound to the ERα LBD suggests that its exo phenyl sulfonate

moiety will displace helix-11 residues such as His524, at least as

severely as the exo ethyl ester moiety of ODE (Figure 3B) and

consistent with its greater antagonistic activity. In contrast, the

endo phenyl sulfonate moiety of SOBHS is likely accom-

modated in a different space within the pocket and, therefore, is

not expected to displace helix-11 as extensively (Figure 3C).

Consistent with these models, SOBHS compound 10a, which

lacks ERα antagonist activity, is an effective ERα agonist (Table

3), compared to OBHS, which is a potent ERα antagonist.²⁶

An examination of the ODE structure suggests that the o-

versus m-methyl substitutions differentially impact how the

ligand interacts with the receptor (Figure 3A). The m-methyl

groups of compound 12a cannot be accommodated in the

pocket without shifting the ligand further away from the

hydrogen bonding partners, Glu353, Arg394, and/or Thr347,

consistent with the lower affinity and transactivation potency of

these compounds. This shift in the ligand is transmitted to the

sulfonate phenyl substitution, where it could further shift helix

11 out of the position required for agonist activity. This is most

apparent with the ERβ compound 12 series, which display very

little agonist activity. These differences in the ER subtypes are

consistent with our previous findings that ERβ has a smaller

Unlike OBHS or compound 10a (entry 1), both of which do

not activate ERβ, compound 11a (entry 6) showed about 3-fold

improved binding affinity for ERβ compared to 10a (Table 2),

and stimulated ERβ activity, albeit with about 30,000-fold less

potency than ERα (Table 3). As ERβ agonists, a 1-naphthyl

modification of the phenyl sulfonate moiety (i.e., compound

11j, entry 15) improved the efficacy of the compound-11

scaffold (Table 3). Furthermore, introduction of a p-bromine

atom to the phenyl sulfonate moiety (i.e., compound 11h)

improved ERβ binding affinity as effectively as 11a (Table 2,

entries 13 and 6) and increased ERβ agonist efficacy with about

6-fold more potency than 11a (Table 3). In addition, the N-

phenylmaleimide adduct, compound 11l, which showed about

24-fold weaker affinity for ERβ (Table 2, entry 17) was more

efficacious and at least 5-fold more potent as an ERβ agonist

than compound 11a. Therefore, the ability of compounds

bearing the compound 11 scaffold to stimulate ERβ activity

does not correlate with their relative binding affinities for ERβ.

By contrast, compound 12a, which has a m-methyl group in

each of the core hydroxyphenyl substituents and shows

improved binding affinity for ERα but not ERβ (Table 2,

entry 18), exhibits the typical ERα-selective agonist properties

of the SOBHS scaffold, with 56-fold higher potency than

compound 10a (entry 1) (Table 3). Modifying the phenyl

sulfonate moiety within the compound 12 scaffold (i.e.,

compounds 12a-I, entry 18−26) reduced binding affinity for

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uncorrected. The purity of all compounds for biological testing was

determined by HPLC (see Supporting Information), confirming >95%

purity.

pocket and is more sensitive to ligand-induced shifts in helix

11.⁵³In contrast, the o-methyl substitutions are positioned to

make additional hydrophobic contacts, consistent with the

higher affinity of these compounds in series 11.

General Procedure for Boronic Acid Synthesis. n-BuLi (2.5 M,

2 equiv) was added dropwise to a solution of bromobenzene derivative

in THF at −78 °C, resulting in a white suspension. The reaction

mixture was stirred for 30 min at −78 °C, and then B(OMe)₃(4

equiv) was added. The resulting mixture was stirred at −78 °C for a

further 30 min and then allowed to warm to room temperature. The

reaction mixture was acidified with 10% aqueous HCl solution and

extracted with EtOAc (3 × 30 mL). The organic layer was then dried

over anhydrous MgSO₄and concentrated under vacuum. The crude

product was purified by chromatography to afford an off-white

crystalline material.

General Procedure for Suzuki Coupling Reaction. A solution

of deoxygenated toluene was added to a mixture of Pd(OAc)₂(5%

mol) (Pd₂(dba)₃is used in the synthesis of 5b) and PPh₃(25% mol)

and stirred at an atmosphere of argon for 15 min at room temperature.

Then arylboronic acid (4 equiv) was added to the reaction mixture and

stirred for 5 min. A deoxygenated 3,4-dibromothiophene (1 equiv) was

added to the mixture and stirred for a further 5 min. A deoxygenated 2

M Na₂CO₃solution was added to the reaction mixture, which was

stirred at room temperature for a further 5 min before being heated at

reflux for 40 h. The mixture was cooled to room temperature and

quenched with H₂O, after which the organic material was extracted

with EtOAc (3 × 30 mL) and dried over anhydrous MgSO₄and the

solvent was evaporated under vacuum. The crude product was

subjected to column chromatography and recrystallized in ether to

afford the target product.

Another factor could be contributing to the increased affinity

of the series 11 compounds compared to their unsubstituted

analogues (series 10). In an indene system that we studied

earlier, we noted that addition of an o-methyl group in a cis-

stilbene core structure also raised binding affinity to a

considerable degree; we presumed that this was due to an

increased twist of the aryl upon methyl substitution, which

would increase the molecular surface area.⁵⁴Here, we note that

according to simple MM2 energy minimization, addition of an

o-methyl group to the two hydroxyphenyl groups increases the

aryl dihedral angles from ca. 18° in 10a to 30° in 11a, which

likely again is responsible for the increased affinity.

CONCLUSION

■

To further explore the consequences of expanding ligands for

the ERs in the third dimension in terms of ER binding affinity

and selectivity, and cellular activity, we have prepared a series of

novel ligands for these receptors based on an inherently three-

dimensional 7-thiabicyclo[2.2.1]hept-5-ene-7-oxide (SOBHS)

core, analogues of the oxa-bridged (7-oxabicyclo[2.2.1]hept-5-

ene) OBHS compounds. Ligands in this sulfoxide-bridged

series can be readily prepared by an in situ oxidative Diels−

Alder reaction between a 3,4-disubstituted thiophene and

various dienophiles, which produces exclusively the endo

stereoisomers. Careful SAR analysis of ER binding affinities

and transcriptional output showed that these novel ligands are

largely ERα-selective, and the disposition of methyl groups in

the appended phenol substituents has a marked effect on their

ER binding affinity and subtype selectivity. The compounds

with o-substituted methyl groups show increased binding

affinity for ERα, while those in the m-substituted methyl series

show significantly enhanced ERα subtype binding-selectivity.

The compounds with o-substituted methyl groups also exhibit

partial ERβ agonist activity in transcription assays.

3,4-Bis(4-methoxyphenyl)thiophene (5a). Obtained as a white

1

solid (76% yield); H NMR (400 MHz, CDCl₃) δ 7.23 (s, 2H), 7.12

(d, J = 8.8 Hz, 4H), 6.80 (d, J = 8.8 Hz, 4H), 3.80 (s, 6H); ¹³C NMR

(100 MHz, CDCl₃) δ 158.57, 141.32, 130.10, 129.22, 123.08, 113.57,

55.22. MS (ESI) m/z: 297 (M + 1)⁺.

3,4-Bis(4-methoxy-2-methylphenyl)thiophene (5b). Obtained as a

white solid (69% yield); ¹H NMR (400 MHz, CDCl₃) δ 7.15 (s, 2H),

6.94 (d, J = 8.4 Hz, 2H), 6.64−6.58 (m, 4H), 3.75 (s, 6H), 2.03 (s,

6H); ¹³C NMR (100 MHz, CDCl₃) δ 158.46, 141.98, 137.67, 131.61,

128.98, 123.26, 115.26, 110.62, 55.08, 20.52. MS (ESI) m/z: 325 (M +

1)⁺.

3,4-Bis(4-methoxy-3-methylphenyl)thiophene (5c). Obtained as a

white solid (81% yield); ¹H NMR (400 MHz, CDCl₃) δ 7.21 (s, 2H),

7.05 (s, 2H), 6.93 (dd, J = 8.4, 2.1 Hz, 2H), 6.69 (d, J = 8.4 Hz, 2H),

3.81 (s, 6H), 2.16 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 154.56,

139.23, 130.98, 129.02, 125.14, 124.46, 120.58, 107.17, 53.00, 13.98.

MS (ESI) m/z: 325 (M + 1)⁺.

Lastly, ER remains an important pharmaceutical target, and

the diversity associated with ER ligands provides a strategic

platform to improve our understanding of how biological

information is encoded within ligand structure and transmitted

through ER. Generation of this new series of ER ligands

provides key insight into the diversity of structures that can

function as ER ligands and specifically as SERMs. Further

cellular and in vivo studies on members of this new class of ER

ligands, as well as X-ray crystallographic analyses, which are

underway, will be reported in due course.

3,4-Diphenylthiophene (1d). Obtained as a white solid (89% yield)

(mp 105−106 °C); ¹H NMR (400 MHz, CDCl₃) δ 7.25 (dd, J = 5.0,

1.6 Hz, 6H), 7.20−7.17 (m, 4H), as previously reported.⁵⁵MS (ESI)

m/z: 237 (M + 1)⁺.

3,4-Di-p-tolylthiophene (1e). Obtained as a white solid (84% yield)

1

(mp 68−69 °C); H NMR (400 MHz, CDCl₃) δ 7.26 (s, 2H), 7.08

(dd, J = 12.1, 8.0 Hz, 8H), 2.33 (s, 6H). MS (ESI) m/z: 265 (M + 1)⁺.

3-(4-Methoxy-3-methylphenyl)-4-phenylthiophene (7). Obtained

1

as a white solid (73% yield); H NMR (400 MHz, CDCl₃) δ 7.32−

EXPERIMENTAL SECTION

7.23 (m, 7H), 7.05 (d, J = 1.9 Hz, 1H), 6.95 (q, J = 2.2 Hz, 1H), 6.72

(d, J = 8.4 Hz, 1H), 3.84 (s, 3H), 2.18 (s, 3H); ¹³C NMR δ 156.85,

141.74, 141.61, 136.85, 131.37, 129.06, 128.61, 128.13, 127.45, 126.82,

126.25, 123.88, 123.08, 109.52, 55.30, 16.25. MS (ESI) m/z: 281 (M +

1)⁺.

■

Materials and Methods. Unless otherwise noted, reagents and

materials were obtained from commercial suppliers and were used

without further purification. Tetrahydrofuran and toluene were dried

over Na and distilled prior to use. Dichloromethane was dried over

CaH₂and distilled prior to use. Glassware was oven-dried, assembled

while hot, and cooled under an inert atmosphere. Unless otherwise

noted, all reactions were conducted in an inert atmosphere. Reaction

progress was monitored using analytical thin-layer chromatography

General Procedure for Demethylation. To a solution of 3,4-

diarylthiophene (1 equiv) in dry CH₂Cl₂at 0 °C was added dropwise

BBr₃in CH₂Cl₂(1 M, 3 equiv per methoxyl function). The reaction

mixture was stirred for 24 h at room temperature under argon. Water

was added to quench the reaction, and the aqueous layer was extracted

with EtOAc. The combined organic layers were washed with brine,

dried over Na₂SO₄, evaporated to dryness under vacuum, and purified

by column chromatography.

1

(TLC). Visualization was achieved by UV light (254 nm). H NMR

and ¹³C NMR spectra were obtain on a Bruker Biospin AV400 (400

MHz) instrument. The chemical shifts are reported in ppm and are

referenced to either tetramethylsilane or the solvent. Mass spectra

were recorded under electron impact conditions at 70 eV. Melting

points were obtained on a SGW X-4 melting point apparatus and are

3,4-Bis(4-hydroxyphenyl)thiophene (1a). Obtained as a white solid

1

(92% yield) (mp 198−201 °C); H NMR (400 MHz, DMSO-d₆) δ

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9.41 (s, 2H), 7.40 (s, 2H), 6.93 (d, J = 8.5 Hz, 4H), 6.65 (d, J = 8.5

Hz, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 156.14, 140.94, 129.69,

126.98, 123.11, 114.87. MS (ESI) m/z: 269 (M + 1)⁺.

N-Phenyl-5,6-bis(4-hydroxyphenyl)-7-thiabicyclo[2.2.1]hept-5-

ene-2,3-dicarboxamide-7-oxide (10d). Obtained as a white solid

1

(36% yield) (mp 264−265 °C); H NMR (400 MHz, DMSO-d₆) δ

9.74 (s, 2H), 7.52−7.33 (m, 3H), 7.02 (d, J = 8.7 Hz, 5H), 6.64 (d, J =

8.8 Hz, 4H), 4.76 (dd, J = 2.8, 1.7 Hz, 2H), 4.21 (dd, J = 2.7, 1.7 Hz,

2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.56, 167.43, 131.97,

129.11, 126.42, 99.98, 69.45, 65.50, 61.01, 56.46. HRMS (ESI) calcd

for C₂₆H₁₉NNaO₅S [M + Na]⁺480.0882; found 480.0893.

3,4-Bis(4-hydroxy-2-methylphenyl)thiophene (1b). Obtained as a

1

white solid (95% yield) (mp 214−215 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.28 (s, 2H), 7.41 (s, 2H), 6.80 (d, J = 8.2 Hz, 2H), 6.57

(s, 2H), 6.50 (d, J = 8.2 Hz, 2H), 1.98 (s, 6H); ¹³C NMR (100 MHz,

DMSO-d₆) δ 156.09, 141.68, 136.75, 131.19, 126.97, 123.24, 116.38,

112.20, 20.00. MS (ESI) m/z: 319 (M + Na)⁺.

Diethyl-5,6-bis(4-hydroxyphenyl)-7-thiabicyclo[2.2.1]hept-5-ene-

2,3-dicarboxylate-7-oxide (10e). Obtained as a yellow solid (21%

yield) (mp 193−194 °C); ¹H NMR (400 MHz, DMSO-d₆) δ 9.60 (s,

2H), 7.07 (d, J = 8.8 Hz, 4H), 6.62 (d, J = 8.8 Hz, 4H), 4.46 (t, J = 1.6

Hz, 2H), 3.94−3.86 (m, 6H), 0.96 (t, J = 7.1 Hz, 6H); ¹³C NMR (100

MHz, DMSO-d₆) δ 158.60, 142.36, 130.00, 129.56, 129.11, 125.32,

123.70, 115.49, 71.17, 65.52, 56.00, 18.95. HRMS (ESI): calcd for

C₂₄H₂₄NaO₇S [M + Na]⁺479.1140, found 479.1156.

3,4-Bis(4-hydroxy-3-methylphenyl)thiophene (1c). Obtained as a

1

white solid (93% yield) (mp 183−184 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.32 (s, 2H), 7.38 (s, 2H), 6.95 (s, 2H), 6.67 (dd, J =

21.5, 8.2 Hz, 4H), 2.06 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ

154.66, 141.57, 131.36, 127.44, 123.81, 114.48, 16.47. MS (ESI) m/z:

318 (M + Na)⁺.

3-(4-Hydroxy-3-methylphenyl)-4-phenylthiophene (1f). Obtained

as a white solid (90% yield) (mp 114−115 °C); ¹H NMR (400 MHz,

DMSO-d₆) δ 7.29−7.19 (m, 7H), 7.01 (d, J = 1.6 Hz, 1H), 6.83 (dd, J

= 8.2, 2.1 Hz, 1H), 6.63 (d, J = 8.2 Hz, 1H), 4.66 (s, 1H), 2.18 (s, 3H);

¹³C NMR (100 MHz, CDCl₃) δ 152.86, 141.70, 141.47 136.74,

Phenyl-5,6-bis(4-hydroxy-2-methylphenyl)-7-thiabicyclo[2.2.1]-

hept-5-ene-2-sulfonate-7-oxide (11a). Obtained as a white solid

1

(41% yield) (mp 275−276 °C); H NMR (400 MHz, DMSO-d₆) δ

9.36 (s, 1H), 9.31 (s, 1H), 7.43 (t, J = 7.6 Hz, 2H), 7.34 (t, J = 7.3 Hz,

1H), 7.26 (d, J = 7.8 Hz, 2H), 6.90 (d, J = 8.3 Hz, 1H), 6.64 (d, J = 8.4

Hz, 1H), 6.44−6.36 (m, 4H), 4.59 (s, 1H), 4.53 (dd, 1H), 4.19 (s,

1H), 2.95 (ddd, 1H), 2.51 (dd, J = 13.5, 4.6 Hz, 1H), 1.89 (s, 3H),

1.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 157.02, 156.74,

148.61, 137.22, 136.84, 132.70, 132.19, 130.84, 130.64, 130.41, 130.25,

128.78, 127.88, 125.76, 124.95, 122.09, 116.84, 112.67, 68.26, 68.02,

58.34, 26.73, 20.15. HRMS (ESI) calcd for C₂₆H₂₄NaO₆S₂[M + H]⁺

497.1103; found 497.10865.

131.67, 129.21, 129.01, 127.83, 126.81, 123.83, 123.42, 123.07, 114.60,

15.70. MS (ESI) m/z: 267 (M + 1)⁺.

General Procedure for Diels−Alder Reaction. To a solution of

3,4-diarylthiophene and dienophile (2 equiv) in dry CH₂Cl₂(10 mL)

was added slowly BF₃·Et₂O (10 equiv) under an inert atmosphere and

at −20 °C. The reaction mixture was stirred for 10 min at −20 °C, and

then a solution of m-CPBA (2 equiv) in dry CH₂Cl₂(5 mL) was

added slowly. The reaction mixture was stirred for 4 h at −20 °C.

Then the suspension was poured into a mixture of concentrated

aqueous NaHCO₃solution (25 mL) and CH₂Cl₂(25 mL) and stirred

at room temperature for 20 min. The organic phase was separated, and

the aqueous phase was extracted with EtOAc (3 × 30 mL). The

combined organic phase was washed with water and brine and dried

over anhydrous MgSO₄. After removal of the solvent under vacuum,

the residue was chromatographed on silica gel to give the target

compound.

4-Methoxyphenyl-5,6-bis(4-hydroxy-2-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (11b). Obtained as

1

a white solid (26% yield) (mp 224−226 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.48 (s, 1H), 9.43 (s, 1H), 7.25 (d, J = 9.1 Hz, 2H), 7.00

(d, J = 9.1 Hz, 2H), 6.96 (d, J = 8.3 Hz, 1H), 6.71 (d, J = 8.2 Hz, 1H),

6.48−6.41 (m, 4H), 4.62 (s, 1H), 4.55 (dd, J = 7.2, 6.2 Hz, 1H), 4.24

(s, 1H), 3.77 (s, 3H), 3.00 (ddd, 1H), 2.56 (dd, J = 13.9, 4.7 Hz, 1H),

1.95 (s, 3H), 1.90 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 158.13,

157.01, 156.72, 141.93, 137.22, 136.83, 136.76, 132.27, 130.86, 130.40,

125.77, 124.98, 123.11, 116.86, 116.78, 115.02, 112.69, 68.31, 68.12,

58.03, 56.03, 55.54, 26.74, 20.11. HRMS (ESI) calcd for

C₂₇H₂₆NaO₇S₂[M + Na]⁺549.1018; found 549.1021.

Phenyl-5,6-bis(4-hydroxyphenyl)-7-thiabicyclo[2.2.1]hept-5-ene-

2-sulfonate-7-oxide (10a). Obtained as a white solid (36% yield) (mp

1

206−207 °C); H NMR (400 MHz, DMSO-d₆) δ 9.70 (s, 1H), 9.65

(s, 1H), 7.50 (t, J = 7.8 Hz, 2H), 7.41 (t, J = 7.3 Hz, 1H), 7.33 (d, J =

7.8 Hz, 2H), 7.08 (dd, J = 8.4, 5.5 Hz, 4H), 6.65 (dd, J = 20.1, 8.6 Hz,

4H), 4.74 (s, 1H), 4.59−4.55 (m, 1H), 4.39 (s, 1H), 2.96 (ddd, J =

13.2, 9.6, 3.5 Hz, 1H), 2.36 (dd, J = 13.5, 4.3 Hz, 1H); ¹³C NMR (100

MHz, DMSO-d₆) δ 157.39, 157.12, 148.58, 132.97, 130.26, 129.82,

129.50, 129.05, 127.64, 125.09, 124.83, 122.13, 115.33, 114.93, 67.43,

67.34, 58.50, 26.67. HRMS (ESI) calcd for C₂₄H₂₀O₆S₂H [M + H]⁺

469.0780; found 469.0777.

2-Fluorophenyl-5,6-bis(4-hydroxy-2-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (11c). Obtained as

1

a white solid (40% yield) (mp 210−212 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.46 (s, 1H), 9.40 (s, 1H), 7.51−7.41 (m, 3H), 7.30 (t, J

= 7.6 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H),

6.53−6.40 (m, 4H), 4.76−4.62 (m, 2H), 4.29 (s, 1H), 3.09 (t, J = 10.6

Hz, 1H), 2.63 (dd, J = 13.7, 3.6 Hz, 1H), 1.95 (s, 3H), 1.91 (s, 3H);

¹³C NMR (100 MHz, DMSO-d₆) δ 157.06, 156.62, 155.05, 152.58,

4-Methoxyphenyl-5,6-bis(4-hydroxyphenyl)-7-thiabicyclo[2.2.1]-

137.42, 136.76, 135.66, 132.14, 130.39, 129.18, 125.72, 125.56, 124.85,

124.77, 117.48, 117.30, 116.88, 116.81, 112.71, 112.46, 68.35, 59.56,

56.01, 26.84, 20.10, 18.44. HRMS (ESI) calcd for C₂₆H₂₃FO₆S₂H [M

+ H]⁺515.0995; found 515.0998.

hept-5-ene-2-sulfonate-7-oxide (10b). Obtained as a white solid

1

(36% yield) (mp 225−226 °C); H NMR (400 MHz, DMSO-d₆) δ

9.70 (s, 1H), 9.65 (s, 1H), 7.27 (d, J = 9.1, 2H), 7.08 (dd, J = 8.6, 5.5

Hz, 4H), 7.02 (d, J = 9.2 Hz, 2H), 6.65 (dd, J = 20.0, 8.7 Hz, 4H), 4.72

(s, 1H), 4.54 (ddd, J = 9.6, 4.3, 3.8 Hz, 1H), 4.38 (s, 1H), 2.94 (ddd, J

= 13.0, 9.4, 3.5 Hz, 1H), 2.33 (dd, J = 13.4, 4.7 Hz, 1H); ¹³C NMR

(100 MHz, DMSO-d₆) δ 175.34, 132.37, 132.15, 131.98, 131.19,

129.99, 129.50, 129.11, 128.98, 128.79, 127.03, 115.80, 99.98, 68.87,

65.50, 56.48, 46.62, 30.46. HRMS (ESI) calcd for C₂₅H₂₂NaO₇S₂[M +

Na]⁺521.0705; found 521.0679.

2-Chlorophenyl-5,6-bis(4-hydroxy-2-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (11d). Obtained as

1

a white solid (31% yield) (mp 248−249 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.47 (s, 1H), 9.41 (s, 1H), 7.68 (dd, J = 7.5, 1.8 Hz, 1H),

7.57−7.35 (m, 3H), 6.94 (d, J = 8.3 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H),

6.53−6.39 (m, 4H), 4.76−4.69 (m, 2H), 4.28 (s, 1H), 3.10 (ddd, J =

13.1, 9.7, 3.7 Hz, 1H), 2.68 (dd, J = 13.4, 3.8 Hz, 1H), 1.95 (s, 3H),

1.91 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 157.03, 156.91,

156.61, 144.40, 137.44, 136.81, 136.77, 132.16, 130.79, 130.40, 128.91,

128.87, 126.13, 125.74, 124.87, 124.27, 116.87, 116.72, 112.70, 112.55,

68.36, 68.15, 60.14, 27.00, 20.13, 19.82. HRMS (ESI) calcd for

C₂₆H₂₃ClO₆S₂H [M + H]⁺531.0703; found 531.0695.

4-Hydroxyphenyl-5,6-bis(4-hydroxyphenyl)-7-thiabicyclo[2.2.1]-

hept-5-ene-2-sulfonate-7-oxide (10c). Obtained as a white solid

1

(32% yield) (mp 235−236 °C); H NMR (400 MHz, DMSO-d₆) δ

9.81 (s, 1H), 9.68 (s, 1H), 9.63 (s, 1H), 7.13 (d, J = 9.0 Hz, 2H),

7.09−7.06 (m, 4H), 6.81 (d, J = 9.0 Hz, 2H), 6.64 (dd, J = 20.5, 8.6

Hz, 4H), 6.70 (s, 1H), 4.50 (t, J = 3.8 Hz, 1H), 4.38 (s, 1H), 2.92

(ddd, J = 13.3, 9.8, 3.6 Hz, 1H), 2.32 (dd, J = 13.4, 4.5 Hz). ¹³C NMR

(100 MHz, DMSO-d₆) δ 167.45, 141.26, 133.48, 132.17, 131.99,

130.33, 129.12, 125.69, 123.61, 116.58, 115.75, 68.00, 65.50, 58.40,

56.43, 30.46, 19.10. HRMS (ESI) calcd for C₂₄H₂₀NaO₇S₂[M + Na]⁺

507.0548; found 507.0564.

2-Bromophenyl-5,6-bis(4-hydroxy-2-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (11e). Obtained as

1

a white solid (35% yield) (mp 246−247 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.47 (s, 1H), 9.40 (s, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.48

(q, J = 8.0 Hz, 2H), 7.38−7.29 (m, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.71

(d, J = 8.3 Hz, 1H), 6.55−6.39 (m, 4H), 4.80−4.68 (m, 2H), 4.28 (s,

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1H), 3.10 (ddd, J = 12.9, 9.4, 3.4 Hz, 1H), 2.70 (dd, J = 13.5, 4.1 Hz,

1H), 1.95 (s, 3H), 1.91 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ

157.06, 156.76, 156.61, 137.45, 136.77, 134.01, 132.17, 130.40, 129.45,

129.11, 125.74, 124.88, 124.00, 116.79, 115.46, 112.71, 112.56, 68.39,

68.19, 60.29, 27.08, 20.70, 19.82. HRMS (ESI) calcd for

C₂₆H₂₃BrO₆S₂H [M + H]⁺575.0198; found 575.0199.

Hz, 2H), 7.03 (d, J = 8.1 Hz, 1H), 6.87 (d, J = 6.9 Hz, 2H), 6.76 (d, J

= 8.3 Hz, 1H), 6.56−6.48 (m, 4H), 4.67 (s, 1H), 4.59−4.66 (m, 1H),

4.29 (s, 1H), 3.03 (ddd, J = 13.3, 9.8, 3.6 Hz, 1H), 2.57−2.61 (m, 1H),

1.95 (s, 3H), 1.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.84,

167.44, 156.42, 141.24, 133.34, 132.17, 131.98, 131.32, 130.88, 129.48,

129.12, 127.70, 127.34, 125.66, 124.55, 123.60, 116.59, 114.88, 68.04,

67.90, 65.50, 30.46, 19.10, 18.94. HRMS (ESI) calcd for

C₂₆H₂₄NaO₇S₂[M + Na]⁺535.0861; found 535.0868.

4-Fluorophenyl-5,6-bis(4-hydroxy-2-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (11f). Obtained as

1

a white solid (28% yield) (mp 238−240 °C); H NMR (400 MHz,

N-Phenyl-5,6-bis(4-hydroxy-2-methylphenyl)-7-thiabicyclo-

DMSO-d₆) δ 9.46 (s, 1H), 9.41 (s, 1H), 7.43−7.26 (m, 4H), 6.95 (d, J

= 8.2 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H), 6.55−6.39 (m, 4H), 4.69−

4.59 (m, 1H), 4.26 (s, 1H), 3.03 (ddd, 1H), 2.57 (dd, J = 13.6, 4.6 Hz,

1H), 1.94 (s, 1H), 1.91 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ

162.27, 159.84, 157.52, 157.24, 145.07, 137.33, 137.27, 131.35, 125.42,

124.60, 117.47, 117.24, 113.19, 68.85, 68.58, 60.24, 59.07, 27.27,

20.61, 20.32, 14.52. HRMS (ESI) calcd for C₂₆H₂₃FNaO₆S₂[M +

Na]⁺537.0818; found 537.0818.

[2.2.1]hept-5-ene-2,3-dicarboxamide-7-oxide (11l). Obtained as a

1

white solid (34% yield) (mp 274−276 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.53 (s, 2H), 7.53 (t, J = 7.6 Hz, 2H), 7.44 (t, J = 7.4 Hz,

1H), 7.21 (d, J = 7.5 Hz, 2H), 6.77 (d, J = 8.1 Hz, 2H), 6.48 (d, J = 9.2

Hz, 4H), 4.64 (s, 2H), 4.25 (s, 2H), 1.77 (s, 6H); ¹³C NMR (100

MHz, DMSO-d₆) δ 174.97, 157.05, 136.63, 129.95, 128.99, 128.43,

126.43, 125.19, 117.13, 113.02, 69.35, 46.25, 19.89. HRMS (ESI) calcd

for C₂₈H₂₃NNaO₅S [M + Na]⁺508.1195; found 508.1168.

4-Chlorophenyl-5,6-bis(4-hydroxy-2-methylphenyl)-7-

Phenyl-5,6-bis(4-hydroxy-3-methylphenyl)-7-thiabicyclo[2.2.1]-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (11g). Obtained as

hept-5-ene-2-sulfonate-7-oxide (12a). Obtained as a white solid

1

a white solid (28% yield) (mp 218−220 °C); H NMR (400 MHz,

(50% yield) (mp 215−216 °C); H NMR (400 MHz, DMSO-d₆) δ

DMSO-d₆) δ 9.47 (s, 1H), 9.41 (s, 1H), 7.55 (d, J = 8.9 Hz, 2H), 7.36

(d, J = 8.9 Hz, 2H), 6.94 (d, J = 8.3 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H),

6.51−6.41 (m, 4H), 4.67−4.60 (m, 2H), 4.26 (s, 1H), 3.03 (ddd, J =

13.3, 9.9, 3.5 Hz, 1H), 2.58 (dd, J = 13.5, 4.0 Hz, 1H), 1.94 (s, 3H),

1.91 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 155.55, 153.08,

136.29, 136.16, 133.55, 131.25, 129.73, 129.26, 127.65, 127.33, 126.06,

125.52, 125.28, 124.36, 123.87, 117.99, 114.98, 114.66, 68.04, 67.93,

60.23, 27.28, 16.41, 16.29. HRMS (ESI) calcd for C₂₆H₂₃ClNaO₆S₂

[M + Na]⁺533.0522; found 553.0526.

9.55 (s, 1H), 9.50 (s, 1H), 7.50 (t, J = 7.5 Hz, 2H), 7.40 (t, J = 7.4 Hz,

1H), 7.34 (d, J = 3.2 Hz, 1H), 7.04 (d, J = 6.7 Hz, 2H), 6.86 (t, J = 8.7

Hz, 2H), 6.62 (dd, J = 19.0, 8.4 Hz), 4.72 (s, 1H), 4.56 (dd, 1H), 4.37

(s, 1H), 2.95 (ddd, 1H), 2.36 (dd, J = 13.4, 4.6 Hz, 1H), 2.02 (s, 3H),

1.99 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 155.44, 155.17,

148.55, 132.82, 130.79, 130.38, 130.25, 128.86, 127.62, 127.20, 126.85,

125.05, 124.77, 123.90, 123.32, 122.12, 114.43, 114.09, 67.46, 67.39,

58.55, 30.66, 15.95, 15.85. HRMS (ESI) calcd for C₂₆H₂₄O₆S₂H [M +

H]⁺497.1093; found 497.1103.

4-Bromophenyl-5,6-bis(4-hydroxy-2-methylphenyl)-7-

Methoxyphenyl-5,6-bis(4-hydroxy-3-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (11h). Obtained as

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (12b). Obtained as

1

a white solid (32% yield) (mp 209−210 °C); H NMR (400 MHz,

a white solid (27% yield) (mp 221−223 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.46 (s, 1H), 9.41 (s, 1H), 7.68 (d, J = 8.9 Hz, 2H), 7.30

(d, J = 8.9 Hz, 2H), 6.93 (d, J = 8.4 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H),

6.52−6.40 (m, 4H), 4.68−4.61 (m, 2H), 4.26 (s, 1H), 3.03 (ddd, J =

13.8, 9.9, 4.1 Hz, 1H), 2.58 (dd, J = 13.5, 4.2 Hz, 1H), 1.94 (s, 3H),

1.91 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 157.53, 157.26,

148.24, 137.78, 137.33, 137.26, 133.57, 132.68, 130.89, 128.37, 126.25,

125.44, 124.84, 120.65, 117.57, 113.04, 68.85, 68.57, 59.33, 27.28,

20.61, 20.32. HRMS (ESI) calcd for C₂₆H₂₃BrNaO₆S₂[M + Na]⁺

597.0017; found 597.0008.

DMSO-d₆) δ 9.58 (s, 1H), 9.53 (s, 1H), 7.27 (d, J = 7.0 Hz, 2H), 7.03

(t, J = 6.8 Hz, 4H), 6.86 (t, J = 10.0 Hz, 2H), 6.62 (dd, J = 18.6, 8.4

Hz, 2H), 4.69 (s, 1H), 4.52 (dd, 1H), 4.36 (s, 1H), 2.94 (ddd, 1H),

2.33 (dd, J = 11.6, 4.5 Hz, 1H), 2.02 (s, 1H), 1.99 (s, 1H); ¹³C NMR

(100 MHz, DMSO-d₆) δ 158.60, 155.93, 155.64, 142.38, 138.80,

133.33, 131.29, 125.59, 125.30, 123.39, 123.81, 123.66, 115.50, 114.95,

114.61, 81.17, 68.03, 67.89, 56.50, 18.99, 16.46, 16.33. HRMS (ESI)

calcd for C₂₇H₂₆NaO₇S₂[M + Na]⁺549.1018; found 549.0998.

2-Fluorophenyl-5,6-bis(4-hydroxy-3-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (12c). Obtained as

3-Chlorophenyl-5,6-bis(4-hydroxy-2-methylphenyl)-7-

1

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (11i). Obtained as

a white solid (32% yield) (mp 203−204 °C); H NMR (400 MHz,

1

a white solid (28% yield) (mp 217−219 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.58 (s, 1H), 9.53 (s, 1H), 7.53−7.40 (m, 3H), 7.30 (t, J

= 7.6 Hz, 1H), 7.03 (s, 2H), 6.86 (t, J = 8.2 Hz, 2H), 6.62 (dd, J =

19.7, 8.4 Hz, 2H), 4.68−4.63 (m, 1H), 4.39 (s, 1H), 3.01 (ddd, J =

13.2, 9.7, 3.3 Hz, 1H), 2.40 (dd, J = 13.4, 4.4 Hz, 1H), 2.02 (s, 3H),

1.98 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 157.54, 157.26,

157.12, 147.75, 137.78, 137.32, 137.25, 132.69, 132.42, 131.34, 130.90,

130.61, 126.28, 125.43, 124.49, 117.28, 113.20, 113.11, 113.03, 112.95,

68.85, 68.58, 59.32, 27.29, 20.62, 20.33. HRMS (ESI) calcd for

C₂₆H₂₃FNaO₆S₂[M + Na]⁺537.0818; found 537.0809.

DMSO-d₆) δ 9.46 (s, 1H), 9.40 (s, 1H), 7.56−7.45 (m, 2H), 7.41 (s,

1H), 7.32 (d, J = 7.6 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 8.2

Hz, 1H), 6.56−6.38 (m, 4H), 4.76−4.62 (m, 1H), 4.27 (s, 1H), 3.05

(ddd, J = 12.5, 9.4, 3.2 Hz, 1H), 2.60 (dd, J = 13.7, 3.8 Hz, 1H), 1.94

(s, 3H), 1.92 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 156.89,

156.79, 148.83, 136.81, 133.91, 132.15, 131.54, 130.84, 130.41, 127.79,

122.46, 121.08, 116.84, 116.74, 112.69, 112.59, 112.54, 68.33, 68.04,

58.96, 26.72, 20.14, 19.85. HRMS (ESI) calcd for C₂₆H₂₃ClNaO₆S₂

[M + Na]⁺553.0522; found 553.0536.

2-Chlorophenyl-5,6-bis(4-hydroxy-3-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (12d). Obtained as

Naphthalen-1-yl-5,6-bis(4-hydroxy-2-methylphenyl)-7-

1

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (11j). Obtained as

a white solid (34% yield) (mp 219−220 °C); H NMR (400 MHz,

1

a white solid (33% yield) (mp 273−275 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.58 (s, 1H), 9.53 (s, 1H), 7.67 (d, J = 7.7 Hz, 1H),

7.56−7.37 (m, 3H), 7.04 (s, 2H), 6.87 (t, J = 8.2 Hz, 2H), 6.62 (dd, J

= 20.7, 7.9 Hz, 2H), 4.77 (s, 1H), 4.74−4.68 (m, 1H), 4.39 (s, 1H),

3.03 (ddd, J = 13.0, 9.5, 3.4 Hz, 1H), 2.47 (dd, 1H), 2.03 (s, 3H), 1.98

(s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 155.47, 155.18, 144.38,

133.04, 130.94, 130.76, 130.39, 128.87, 128.72, 127.17, 126.85, 126.13,

124.99, 124.95, 124.80, 124.76, 124.24, 123.91, 123.35, 123.28, 67.58,

67.45, 60.33, 26.90, 15.96, 15.83. HRMS (ESI) calcd for

C₂₆H₂₃ClNaO₆S₂H [M + Na]⁺553.0522; found 553.0513.

DMSO-d₆) δ 9.60 (s, 1H), 9.55 (s, 1H), 8.06−8.00 (m, 2H), 7.96 (d, J

= 8.0 Hz, 1H), 7.65−7.60 (m, 2H), 7.54 (dt, J = 15.4, 7.7 Hz, 2H),

7.06 (d, J = 15.9 Hz, 2H), 6.88 (ddd, J = 13.2, 8.3, 2.0 Hz, 2H), 6.61

(dd, J = 18.6, 8.4 Hz, 2H), 4.88−4.82 (m, 1H), 4.81 (s, 1H), 4.39 (s,

1H), 3.02 (ddd, J = 13.2, 9.6, 3.5 Hz, 1H), 2.01 (s, 3H), 1.95 (s, 3H);

¹³C NMR (100 MHz, DMSO-d₆) δ 155.44, 155.17, 144.21, 134.36,

132.87, 130.80, 130.42, 128.87, 127.92, 127.37, 127.29, 127.22, 127.11,

126.85, 126.62, 125.67, 123.92, 123.37, 121.35, 118.70, 114.44, 114.12,

80.68, 67.68, 59.57, 26.84, 15.93, 15.81. HRMS (ESI) calcd for

C₃₀H₂₆O₆S₂H [M + H]⁺547.1249; found 547.1260.

2-Bromophenyl-5,6-bis(4-hydroxy-3-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (12e). Obtained as

1

a white solid (32% yield) (mp 216−217 °C); H NMR (400 MHz,

4-Hydroxyphenyl-5,6-bis(4-hydroxy-2-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (11k). Obtained as

DMSO-d₆) δ 9.58 (s, 1H), 9.52 (s, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.48

(q, J = 8.0 Hz, 2H), 7.37−7.27 (m, 1H), 7.04 (s, 2H), 6.86 (t, J = 7.3

Hz, 2H), 6.64 (dd, J = 8.3, 1.8 Hz, 1H), 6.59 (dd, J = 8.4, 1.7 Hz, 1H),

1

a white solid (24% yield) (mp 240−241 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.86 (s, 1H), 9.49 (s, 1H), 9.43 (s, 1H), 7.17 (d, J = 6.8

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4.77 (s, 1H), 4.75−4.67 (m, 1H), 4.39 (s, 1H), 3.03 (ddd, J = 13.0, 9.5,

3.3 Hz, 1H), 2.48 (dd, 1H), 2.02 (s, 3H), 1.98 (s, 3H); ¹³C NMR (100

MHz, DMSO-d₆) δ 155.31, 155.18, 145.64, 134.02, 133.04, 130.76,

130.40, 129.46, 129.08, 128.74, 127.17, 126.86, 124.99, 124.77, 123.91,

123.34, 123.27, 67.59, 67.47, 60.45, 26.95, 15.96, 15.81. HRMS (ESI)

calcd for C₂₆H₂₃BrNaO₆S₂H [M + Na]⁺579.0017; found 579.0028.

Naphthalen-1-yl-5,6-bis(4-hydroxy-3-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (12f). Obtained as

MHz, DMSO-d₆) δ 9.84 (s, 1H), 9.65 (s, 0.5H), 9.60 (s, 0.5H), 7.72

(d, J = 7.8 Hz, 1H), 7.56 (t, J = 8.1 Hz, 1H), 7.33−7.21 (m, 4H), 7.14

(t, J = 8.6 Hz, 2H), 7.02 (s, 1H), 6.88−6.78 (m, 2H), 6.62 (dd, J =

20.2, 8.2 Hz, 1H), 4.78 (s, 1H), 4.54 (dd, J = 7.6, 2.0 Hz, 1H), 4.43 (s,

1H), 2.96 (t, J = 11.7 Hz, 1H), 2.42−2.29 (m, 1H), 2.01 (s, 1.5H),

1.97 (s, 1.5H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.83, 167.44,

157.37, 157.09, 156.85, 141.30, 137.71, 137.34, 137.26, 133.20, 132.77,

131.98, 131.36, 131.13, 130.90, 129.28, 129.12, 128.38, 126.32, 125.55,

123.57, 117.26, 116.66, 113.09, 68.79, 68.62, 65.50, 60.23, 58.25,

30.47, 27.20, 20.63, 20.34, 19.11, 18.93. HRMS (ESI) calcd for

C₂₅H₂₂NaO₆S₂[M + Na]⁺505.0755; found 505.0763.

Estrogen Receptor Binding Affinity. Relative binding affinities were

determined by a competitive radiometric binding assay, as previously

described,^46,47using 2 nM [³H]estradiol as tracer ([2,4,6,7-³H]estra-

1,3,5(10)-triene-3,17-β-diol, 70−115 Ci/mmol, Perkin-Elmer, Wal-

tham, MA), and purified full-length human ERα and ERβ, purchased

from PanVera/Invitrogen (Carlsbad, CA). Incubations were for 18−24

h at 0 °C. Hydroxyapatite (BioRad, Hercules, CA) was used to absorb

the receptor−ligand complexes, and free ligand was washed away. The

binding affinities are expressed as relative binding affinity (RBA) values

with the RBA of estradiol set to 100%. The values given are the

average range or SD of two to three independent determinations.

Estradiol binds to ERα with a K_dof 0.2 nM and to ERβ with a K_dof

0.5 nM.

Luciferase Assay. Assays were performed as previously described

with a few modifications.^51,56HepG2 cells were cultured in growth

media containing Dulbecco’s minimum essential medium (DMEM)

(Cellgro by Mediatech, Inc., Manassas, VA) supplemented with 10%

fetal bovine serum (FBS) (Hyclone by Thermo Scientific, South

Logan, UT) and 1% nonessential amino acids (Cellgro), penicillin−

streptomycin−neomycin antibiotic mixture, and Glutamax (Gibco by

Invitrogen Corp. Carlsbad, CA), and maintained at 37 °C and 5%

CO₂. The cells were transfected with 10.0 μg of 3XERE-luciferase

reporter plus 1.6 μg of ER expression vector per 10 cm dish using

FugeneHD reagent (Roche Applied Sciences, Indianapolis IN). The

next day, the cells were resuspended in phenol red-free growth media

containing 10% charcoal−dextran sulfate-treated FBS, transferred to

384-well plates at a density of 20,000 cells/well, incubated overnight at

37 °C and 5% CO₂, and treated in triplicate with increasing doses of

ER ligands. After 24 h, luciferase activity was measured using BriteLite

reagent (PerkingElmer Inc., Shelton, CT) according to the

manufacturer’s protocol.

1

a white solid (37% yield) (mp 235−237 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.58 (s, 1H), 9.53 (s, 1H), 8.03 (t, J = 6.3 Hz, 2H), 7.97

(d, J = 7.7 Hz, 1H), 7.63 (t, J = 6.7 Hz, 2H), 7.58−7.51 (m, 2H), 7.06

(d, J = 13.7 Hz, 2H), 6.93−6.83 (m, 2H), 6.62 (dd, J = 18.5, 8.4 Hz,

2H), 4.89−4.83 (m, 1H), 4.82 (s, 1H), 4.40 (s, 1H), 3.02 (ddd, J =

13.1, 9.5, 3.3 Hz, 1H), 2.55 (d, J = 3.5 Hz, 1H), 2.01 (s, 3H), 1.96 (s,

3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 155.87, 155.60, 144.59,

138.80, 134.81, 133.35, 131.26, 131.16, 130.88, 129.30, 129.23, 128.41,

127.80, 127.63, 127.31, 127.06, 126.14, 125.47, 125.27, 124.49, 123.95,

121.24, 119.19, 114.85, 68.11, 67.98, 60.03, 23.55, 16.41, 16.28. HRMS

(ESI) calcd for C₃₀H₂₆O₆S₂H [M + H]⁺547.1249; found 547.1273.

4-Hydroxyphenyl-5,6-bis(4-hydroxy-3-methylphenyl)-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (12g). Obtained as

1

a white solid (15% yield) (mp 211−212 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.80 (s, 1H), 9.55 (s, 1H), 9.50 (s, 1H), 7.1 3(d, J = 8.8

Hz, 2H), 7.03 (d, J = 5.4 Hz, 2H), 6.89−6.80 (m, 4H), 6.62 (dd, J =

18.9, 8.2 Hz, 2H), 4.68 (s, 1H), 4.48 (dd, 1H), 4.35 (s, 1H), 2.93 (ddd,

1H), 2.34 (dd, 1H), 2.02 (s, 3H), 1.99 (s, 3H); ¹³C NMR (100 MHz,

DMSO-d₆) δ 157.37, 157.09, 156.85, 141.30, 137.71, 137.26, 133.20,

131.98, 130.90, 129.28, 129.12, 128.38, 126.32, 125.55, 123.57, 117.26,

116.66, 113.09, 68.79, 68.62, 65.50, 30.47, 20.63, 20.34. HRMS (ESI)

calcd for C₂₆H₂₄NaO₇S₂[M + Na]⁺535.0861; found 535.0837.

N-Phenyl-5,6-bis(4-hydroxy-3-methylphenyl)-7-thiabicyclo-

[2.2.1]hept-5-ene-2,3-dicarboxamide-7-oxide (12h). Obtained as a

1

white solid (39% yield) (mp 260−261 °C); H NMR (400 MHz,

DMSO-d₆) δ 9.62 (s, 2H), 7.47−7.38 (m, 3H), 7.03−6.97 (m, 4H),

6.84 (dd, J = 8.3, 2.0 Hz, 2H), 6.62 (d, J = 8.4 Hz, 2H), 4.74 (s, 2H),

4.19 (s, 1H), 1.98 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ 175.35,

156.02, 132.40, 131.05, 130.92, 129.47, 128.95, 127.35, 124.99, 124.29,

114.93, 68.90, 46.65, 19.00. HRMS (ESI) calcd for C₂₈H₂₃NNaO₅S

[M + Na]⁺508.1195; found 508.1179.

Diethyl-5,6-bis(4-hydroxy-3-methylphenyl)-7-thiabicyclo[2.2.1]-

hept-5-ene-2,3-dicarboxylate-7-oxide (12i). Obtained as a white

solid (18% yield) (mp 200−201 °C); ¹H NMR (400 MHz, DMSO-d₆)

δ 9.48 (s, 2H), 7.00 (s, 2H), 6.85 (dd, J = 8.3, 2.0 Hz, 2H), 6.59 (d, J =

8.4 Hz, 2H), 4.43 (s, 2H), 3.92−3.87 (m, 6H), 2.00 (s, 6H), 0.97 (t, J

= 7.1 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.56, 155.47,

138.80, 131.62, 126.38, 123.75, 114.57, 81.17, 69.48, 60.98, 44.93,

18.99, 14.04. HRMS (ESI) calcd for C₂₆H₂₈NaO₇S [M + Na]⁺

507.1453; found 407.1464.

Molecular Modeling. Crystal structures of ER LBD in complex with

E₂and ODE were downloaded from the protein data bank (PDB IDs:

1ERE and 2QH6).⁵⁷OBHS or SOBHS was docked into the electron

density of ODE using the molecular graphic program, COOT.⁵⁸The

models were transferred to CCP4MG and superposed for

presentation.⁵⁹

4-Hydroxyphenyl-5,6-diphenyl-7-thiabicyclo[2.2.1]hept-5-ene-2-

sulfonate-7-oxide (13). Obtained as a white solid (55% yield) (mp

194−195 °C); ¹H NMR (400 MHz, DMSO-d₆) δ 9.86 (s, 1H), 7.33−

7.21 (m, 9H), 7.14 (d, J = 9.6 Hz, 2H), 6.83 (d, J = 8.9 Hz, 2H), 4.86

(s, 1H), 4.59 (dd, 1H), 4.49 (s, 1H), 3.01 (ddd, J = 13.3, 9.7, 3.6 Hz,

1H), 2.43 (dd, J = 13.5, 4.4 Hz, 1H); ¹³C NMR (100 MHz, DMSO-

d₆) δ 156.54, 140.67, 135.49, 134.17, 134.12, 131.53, 128.60, 128.55,

128.39, 128.16, 128.13, 128.07, 123.11, 116.15, 67.55, 67.40, 57.62,

26.50. HRMS (ESI): calcd for C₂₄H₂₀NaO₅S₂[M + Na]⁺475.0650,

found 475.0652.

4-Hydroxyphenyl-5,6-di-p-tolyl-7-thiabicyclo[2.2.1]hept-5-ene-2-

sulfonate-7-oxide (14). Obtained as a yellow solid (49% yield) (mp

164−165 °C); ¹H NMR (400 MHz, DMSO-d₆) δ 9.81 (s, 1H), 7.16−

7.12 (m, 9H), 7.05 (d, J = 8.1 Hz, 2H), 6.81 (d, J = 8.9 Hz, 2H), 4.80

(s, 1H), 4.43−4.56 (m, 1H), 4.44 (s, 1H), 2.95 (ddd, J = 13.2, 9.8, 3.6

Hz, 1H), 2.39 (dd, J = 13.5, 4.4 Hz, 1H), 2.26 (dd, J = 6.6 Hz, 6H);

¹³C NMR (100 MHz, DMSO-d₆) δ 165.98, 140.73, 137.85, 137.51,

ASSOCIATED CONTENT

* Supporting Information

HPLC results and HPLC spectra for compounds 10−14. This

material is available free of charge via the Internet at http://

pubs.acs.org.

■

S

AUTHOR INFORMATION

Corresponding Author

*J.A.K.: telephone, 1 217 333 6310; fax, 217-333-7325; e-mail,

jkatzene@uiuc.edu. H.-B.Z.: telephone, 86 27 68759586; fax, 86

27 68759850; e-mail, zhouhb@whu.edu.cn.

■

Notes

The authors declare no competing financial interest.

134.66, 133.33, 132.72, 131.33, 130.64, 129.16, 128.41, 127.88, 123.11,

116.10, 67.52, 57.72, 26.53, 20.72. HRMS (ESI) calcd for

C₂₆H₂₄NaO₅S₂[M + Na]⁺503.0963; found 503.0963.

4-Hydroxyphenyl-6-(4-hydroxy-3-methylphenyl)-5-phenyl-7-

thiabicyclo[2.2.1]hept-5-ene-2-sulfonate-7-oxide (15, Mixture of 1:1

Isomers). Obtained as a brown solid (35% yield); ¹H NMR (400

ACKNOWLEDGMENTS

■

We are grateful to the NSFC (20872116, 20972121,

91017005), the Program for New Century Excellent Talents

in University (NCET-10-0625), the National Mega Project on

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Major Drug Development (2009ZX09301-014-1), and the

Research Fund for the Doctoral Program of Higher Education

of China (20100141110021) for support of this research.

Research support from the National Institutes of Health (PHS

R37 DK015556 to J.A.K. and R01 DK077085 to K.W.N.) is

gratefully acknowledged.

ABBREVIATIONS USED

■

E₂, estradiol; ER, estrogen receptor; HepG2, human liver

cancer cells; RBA, relative binding affinity; OBHS, 7-

oxabicyclo[2.2.1]hept-5-ene; ODE, diethyl 5,6-bis(4-hydroxy-

phenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboxylate; SAR,

structure−activity relationship; SERMs, selective estrogen

receptor modulators; SOBHS, 7-thiabicyclo[2.2.1]hept-5-ene-

7-oxide; THF, tetrahydrofuran; m-CPBA, m-chloroperbenzoic

acid

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Article Doi

Identification and structure-activity relationships of a novel series of estrogen receptor ligands based on 7-thiabicyclo[2.2.1]hept-2-ene-7-oxide

DOI: 10.1021/jm201556r

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