Nonpolar and short side chain groups at C-11β of estradiol result in antiestrogens

1428

J. Med. Chem. 2005, 48, 1428-1447

Nonpolar and Short Side Chain Groups at C-11â of Estradiol Result in

Antiestrogens

Jing-xin Zhang,^†,‡David C. Labaree,^‡and Richard B. Hochberg*

Department of Obstetrics/Gynecology and Reproductive Sciences, and the Comprehensive Cancer Center,

Yale University School of Medicine, New Haven, Connecticut 06520

Received August 6, 2004

We have previously found that esters of 11â-estradiol carboxylates are transformed from an

estrogen into an antiestrogen when the 11â-side chain is increased in length from four to five

non-hydrogen atoms (n g 5). To understand the structural requirements for this transformation

and obtain metabolically stable analogues that are not susceptible to esterase cleavage, we

have synthesized other compounds having an 11â-side chain composed of other functional

groups: ketones, amides, ethers, and thiono esters. With the exception of amides, which bind

poorly to the estrogen receptor (ER), all of these compounds exhibit antiestrogenic action when

the side chain length is n g 5. Ethers (n g 5), studied in more detail, inhibit the action of

estradiol with either ERR or ERâ. In rat uteri they are estrogen antagonists/weak agonists

and decrease the concentration of cholesterol in blood (an hepatic estrogenic action). Thus,

these short chain and nonpolar 11â-analogues of estradiol have tissue specific antiestrogenic/

estrogenic actions, characteristics of selective estrogen receptor modulators.

Introduction

In the course of studies to produce locally active “soft”

estrogens for the treatment of menopausal dyspareunia

(vaginal dryness), we synthesized various derivatives

of estradiol (E₂) in which alkyl esters of carboxylates

were appended to the steroid nucleus.^1,2These groups

were attached at C-7R, C-11â, 15R, and 16R, positions

in the nucleus of E₂that are known to interfere

minimally with binding to the estrogen receptor (ER).³

Biological studies of these esters of E₂-carboxylates

showed unusual activities in which esters at the same

positions and with the same chain length but different

alkoxy groups had disparate activities. However, the

Figure 1. Examples of E₂-11â-ester, -ketone, and -ether,

where the side chain has a length of five non-hydrogen atoms.

most unusual estrogenic response was observed with the

methyl and ethyl esters of E₂-11â-methylcarboxylate

(E11-2,1 and E11-2,2, respectively) (Figure 1). Both of

these esters of the E₂-11â-carboxylate bound with high

affinity to ER, with E11-2,2 the better ligand. However,

Thus, we showed that E11-2,2 is an antiestrogen in the

uterus and an estrogen in liver and bone.

Compounds that have tissue-selective estrogenic/

while E11-2,1 caused an appropriate estrogenic simula-

antiestrogenic actions are called selective estrogen

tion in the estrogen-responsive Ishikawa cell, E11-2,2

receptor modulators, SERMs, well-known therapeutic

was almost inactive.⁴Subsequently, we found that E11-

agents, exemplified by raloxifene and tamoxifen. Their

2,2 is an antiestrogen: it inhibits estrogen stimulation

action is different from pure antiestrogens, such as ICI

of an endogenous gene (alkaline phosphatase) in Ish-

164,384, that act as antiestrogens in estrogen-respon-

sive tissues.⁷Both pure antiestrogens and SERMs,

whether nonsteroidal or steroidal, share a common

structural feature of a long side chain with a polar or

charged substituent. This side chain interferes with the

agonist-induced conformation of helix 12 in ER,^8-10

which in turn interferes with coactivator binding re-

quired for transcriptional activation of estrogen-respon-

sive genes.^11,12E11-2,2 has a relatively short and

nonpolar side chain at the 11â-position of E₂. This side

chain is dramatically different from that of all other

antiestrogens or SERMs, and consequently, it appears

that E11-2,2 may act with ER by another mechanism.

E11-2,1 and E11-2,2 are esters that were originally

intended to act locally within the tissue (vagina) to

ikawa cells and in cells transfected with a construct of

either ER subtypes, ERR or ERâ, and an estrogen

response element (ERE) reporter gene construct.⁵When

tested in vivo in immature rats, E11-2,2 inhibited

uterotrophic stimulation with E₂. When administered

alone to ovariectomized rats, E11-2,2 had minimal

uterotrophic action, but it had the estrogenic effect of

decreasing plasma cholesterol (upregulation of hepatic

lipoprotein receptors⁶) and stimulating bone growth.

* Corresponding author. Phone: (203)785-4001. Fax: (203) 737-

4391. E-mail: richard.hochberg@yale.edu.

^†Current address: Sackler Institute of Graduate Biomedical Sci-

ences, New York University School of Medicine, New York, NY 10016.

^‡Both contributed equally to these studies.

Published on Web 02/10/2005

C-11â-Analogues of Estradiols Result in SERMs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1429

Scheme 1^a

^a(a) ROH, SOCl₂; (b) DMAP, DCC, neopentyl alcohol, CH₃CN; (c) BCl₃, CH₂Cl₂, 0 °C.

Scheme 2^a

which they are applied. They were designed to be

metabolically labile, e.g. substrates for esterases that

would convert them to their parent carboxylates which

are inactive, and thus, they would lack systemic activity.

This current study was undertaken to probe what

structural features of the 11â-side chain esters produce

this changeover of E11-2,1 from an estrogen to E11-2,2,

an antiestrogen. This transformation of an agonist into

an antagonist occurs with the addition of a single

methylene unit, replacing a methyl ester with an ethyl

ester, thereby increasing the length of the C-11â side-

chain of E₂from four to five non-hydrogen atoms

(termed the n g 5 rule). We now ask, do other analogues

containing portions of the ester group (ketones and

ethers) or substitutions (amides or thiono esters) also

exhibit this property? We expect that antagonists

resulting from this study would be considerably more

metabolically stable than esters and therefore poten-

tially therapeutically useful.

^a(a) ROH, SOCl₂; (b) Lawessons reagent, o-xylene, 140 °C; (c)

BCl₃, CH₂Cl₂, 0 °C.

esterification of 12 followed by aminolysis of the result-

ing ethyl ester 15 with 33% ethanolic methylamine

catalyzed by NaCN^15,16to give 16. Removal of the benzyl

groups with BCl₃as above gave E11-3,1_amide(17).

Chemistry

The isopropyl and propyl esters [E11-2,iPr (2) and

E11-2,3 (3)] were prepared by reacting 1²with the

appropriate alcohol in the presence of SOCl₂(Scheme

1); the neopentyl ester, E11-2,5 neo 6, was prepared by

reacting the previously prepared benzyl-protected com-

pound 4²with neopentyl alcohol in the presence of

DMAP and 1,3-dicyclohexylcarbodiimide. Deprotection

with BCl₃in CH₂Cl₂at 0 °C gave the ester 6.

For the synthesis of the thiono ester 9, E11-2S,2

(Scheme 2), the protected acid 4 was first esterified with

ethanol in the presence of SOCl₂and then thiated with

Lawesson’s reagent¹³in o-xylene at 140 °C in a sealed

vial to give 8. Deprotection as above with BCl₃gave E11-

2S,2 (9).

The syntheses of the E11-2 and E11-3 amide deriva-

tives are shown in Scheme 3. The protected acid 4 or

12 was coupled¹⁴with ethylamine in the presence of

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC)

and DMAP in CH₂Cl₂to give 10 and 13, respectively.

These compounds were then deprotected with BCl₃as

above, giving E11-2,2_amide(11) and E11-3,2_amide(14). The

methyl amide in the E11-3 series was prepared by

The syntheses of the ketone analogues are shown in

Scheme 4. Addition of ethylmagnesium bromide or

propylmagnesium chloride to the previously obtained

nitrile 18²followed by acidic workup¹⁷gave the ketones

19 and 21, respectively. Deprotection with BCl₃as above

gave E11-2K,2 (20) and E11-2K,3 (22). For the E11-3K

series, hydroboration/oxidation of the 11â-vinyl steroid

23¹⁸gave the terminal alcohol 24. Tosylation of the

alcohol followed by displacement with NaCN gave the

nitrile 26. Reaction of 26 with the appropriate Grignard

reagent as above followed by deprotection with BCl₃

gave E11-3K,1 (28), E11-3K,2 (30), and E11-3K,3 (32).

For the synthesis of E11-0,4_ether(40) (Scheme 5), the

protecting group on 11â-hydroxyestrone 3-acetate¹⁹was

replaced with a benzyl group and the 17-ketone was

protected as a ketal giving 36. Deprotonation of 36 with

KH, followed by reaction with butyl p-toluenesulfonate²⁰

in toluene at 80 °C, gave the ether 37. Removal of the

ketone protecting group and stereoselective reduction

of the ketone with LiAlH₄in THF at -78 °C gave 39.

Hydrogenolysis of the benzyl group using 5% palladium

1430 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5

Zhang et al.

Scheme 3^a

^a(a) EDC, DMAP, EtNH₂, CH₂Cl₂; (b) BCl₃, CH₂Cl₂, 0 °C; (c) EtOH, SOCl₂, 45 °C; (d) NaCN, EtOH, CH₃NH₂.

Scheme 4^a

^a(a) (i) RMgX, Et₂O, (ii) 1 M HCl; (b) BCl₃, CH₂Cl₂, 0 °C; (c) (i) catechol borane, LiBH₄, THF, rt (ii) NaOH, H₂O₂, EtOH-H₂O; (d) TsCl,

Pyr; 0 °C, (e) NaCN, DMSO, 90 °C.

on carbon in EtOH under an atmosphere of H₂produced

E11-0,4_ether(40).

respective ether. Hydrogenolysis of the benzyl groups

using 5% palladium on carbon under an atmosphere of

H₂gave E11-2,iPr_ether(62), E11-2,tBu_ether(64), and E11-

2,2F_ether(66).

For the preparation of the E11-3 ethers (Scheme 8)

the procedure for the E11-2 ethers was used with alcohol

67², giving E11-3,1_ether(69), E11-3,2_ether(71), E11-

3,iPr_ether(74), E11-3,tBu_ether(76), and E11-3,2F_ether(78).

The synthesis of the E11-1 ethers is shown in Scheme

6. 11â-hydroxymethyl steroid 41²was deprotonated with

KH and reacted with the appropriate alkyl iodide

followed by deprotection with BCl₃in CH₂Cl₂or by

hydrogenolysis giving E11-1,1_ether(43), E11-1,2_ether(45),

E11-1,3_ether(47), and E11-1,4_ether(49). To construct the

2-fluoroethyl ether, the anion of 41, generated in the

presence of 18-crown-6, was reacted with (2-bromo-

ethoxy)-tert-butyldimethylsilane, giving 50. Desilylation,

mesylation of the resulting alcohol, and displacement

with fluoride gave the 2-fluoroethyl ether 53. Hydro-

genolysis of the benzyl groups with 5% palladium on

carbon in EtOH-EtOAc under an atmosphere of H₂

gave E11-1,2F_ether(54).

The synthesis of the E11-2 ethers is shown in Scheme

7. The anion of alcohol 24 was reacted with the ap-

propriate alkyl iodide followed by deprotection with BCl₃

in CH₂Cl₂, giving E11-2,1_ether(56), E11-2,2_ether(58), and

E11-2,3_ether(60). For the bulky or substituted ethers,

alcohol 24 was tosylated and then reacted with the

anion of the appropriate alcohol (generated in the

presence of 18-crown-6) in toluene at 80 °C, giving the

Results

These studies were designed to determine whether

the transformation from estrogen agonist to estrogen

antagonist occurs when the length of the side chain is

increased from four to five (non-hydrogen) atoms in 11â-

substituted compounds containing functional groups

other than esters (n g 5 rule). We synthesized various

E₂-11â-side chain analogues: ethers, ketones, amides,

and thiono esters (Tables 1 and 2). In addition, we

synthesized three esters in the E11-2 series that are

larger than E11-2,2 in order to determine whether they

retain the antagonist property. These compounds were

tested for their affinity to the ER by displacement

assays (inhibition of the binding of [³H]E₂to ER in

ovariectomized rat uterine cytosol) and for their estro-

C-11â-Analogues of Estradiols Result in SERMs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1431

Scheme 5^a

E11-2,3 (3), E11-2,iPr (2), E11-2,diMePr (6) (Table 1).

As expected (n g 5), all of these compounds are estrogen

antagonists. E11-2,3 has an RBA for the ER of 38% and

a K_iin the Ishikawa assay of 1 nM compared to E11-

2,2, which has an RBA of 45% and a K_iof 3.9 ( 1.4 nM.⁵

While E11-2,iPr, a relative of E11-2,2 with a branched

methyl group, has a lower RBA of 17%, it is a better

antagonist than E11-2,2, with a K_iof 2 nM. E11-

2,diMePr, a bulky relative of E11-2,3, has comparatively

poor binding and inhibition with respect to E11-2,3, 10%

and 5 nM, respectively. It appears in this case that the

bulky 2,2-dimethylpropyl group hinders binding to the

ER to an extent that causes lower inhibition.

Ketones. We synthesized a set of ketones, compounds

in which the ester oxygen in the E₂-ester analogues is

replaced by a methylene unit (Table 1). For example,

E11-2K,2 (20) related in this manner to the ester E11-

2,1 has a comparable RBA 18% vs 24% ( 8% for the

ester.²Like the ester E11-2,1, which is an agonist with

a relative stimulatory activity (RSA) of 16% ( 10%,²

E11-2K,2 is also an agonist, albeit a weak one with an

RSA of 1.9% (E11-2K,2 never reaches maximal stimula-

tion and plateaus at about 50% of the maximal value

produced by E₂. An example of this behavior is shown

with E11-1,2_etherin Figure 2a. E11-3K,1 (28) (n ) 4) has

very low binding with an RBA of 1% and is inactive in

the Ishikawa assay. E11-2K,3 (22), analogous to the

ester E11-2,2, is an antagonist with an RBA of 13% and

a K_iof 3.1 nM. E11-3K,2 (30), an antagonist related to

the ester E11-3,1, has an RBA of 3.7% and a K_iof 16

nM as compared with ester E11-3,1 with an RBA of 18

( 3%²and a K_iof 10 ( 2 nM (unpublished data). E11-

3K,3 (32), an antagonist analogous to the ester E11-

3,2, has an RBA of 7.7% and a K_iof 13 nM as compared

with ester E11-3,2 with an RBA of 25% ( 6%²and a K_i

of 7 ( 4 nM (unpublished data).

These ketones fit the pattern where compounds with

side chains of four non-hydrogen atoms or less exhibit

agonist activity and side chains of five non-hydrogen

atoms or greater exhibit antagonist activity (n g 5 rule).

In general, for ketones of the same length, compounds

with the ketone group at the 2′-position have better

binding than compounds with the ketone group at the

3′-position. Most of these ketones have lower ER binding

affinity and the same biological activity, albeit not

necessarily the same potency, as their corresponding

ester.

Ethers. We prepared a series of ethers, compounds

in which the carbonyl unit of the ester group in the E₂-

ester analogues is replaced by a methylene unit (Table

2). For example, E11-2,1_ether(56), related in this manner

to the ester E11-2,1, has an RBA of 25%, almost

identical to that of the ester E11-2,1 (discussed above).

However, it is a mixed agonist/antagonist in the Ish-

ikawa cell. E11-2,2_ether(58), an antagonist analogous to

the ester E11-2,2, has an RBA of 31%, not significantly

different from that of the ester, and a K_iof 0.3 nM,

which is about 10 times the potency of the ester. E11-

3,1_ether(69), an antagonist analogous to the ester E11-

3,1, has an RBA of 34% and a K_iof 0.3 nM; it is a better

ligand for ER and a much more potent antagonist

compared to the corresponding ester. E11-3,2_ether(71),

an antagonist analogous to the ester E11-3,2, has an

RBA of 48% and a K_iof 0.3 nM, which is again a much

^a(a) 1% KOH-MeOH, 50 °C; (b) BnBr, CHCl₃, MeOH, K₂CO₃;

(c) ethylene glycol, pTsOH, benzene, reflux; (d) (i) KH, toluene,

80 °C, (ii) butyl toluenesulfonate, toluene; (e) pTsOH, acetone, H₂O;

(f) LiAlH₄, THF, -78 °C; (g) 5% Pd-C, H₂, EtOH, rt.

genic and antiestrogenic actions in the Ishikawa cell

bioassay [the induction of alkaline phosphatase (AlkP)].⁴

The E₂-11â-ether analogues were also tested for their

binding affinity to the ligand-binding domain (LBD) of

both human ERR and ERâ. A representative ether, E11-

2,2_ether(58), was tested for its antiestrogenic affect in

cells that were transfected separately with ERR or ERâ

with an ERE-luciferase reporter gene construct;⁵in

addition, it was tested in vivo in rats to assesses its

estrogenic/antiestrogenic action on the uterus (uterine

weight) and liver (plasma cholesterol concentration).⁵

Amides. We synthesized the amides corresponding

to the esters E11-2,2, E11-3,1, and E11-3,2: E11-2,2_amide

(11), E11-3,1_amide(17), and E11-3,2_amide(14), respectively

(Table 1). Unlike the esters, the amides bind poorly to

ER, and therefore, as would be expected, all of them are

only weakly active in the Ishikawa estrogen bioassay.

Thiono Esters. We synthesized E₂analogues in

which the carbonyl oxygen of the ester was replaced

with sulfur, resulting in thiono esters, compounds that

are known to be resistant to hydrolysis by esterase.²¹

We attempted the synthesis of two thiono esters, E11-

2S,2 (9) and E11-3S,1. E11-3S,1, the methyl ester of 11â-

(2-thiocarboxyethyl)-E₂, proved to be unstable, as it

decomposed by hydrolysis to the corresponding ester

11â-(2-carboxyethyl)-E₂, E11-3,1. E11-2S,2 has a rela-

tive binding affinity (RBA) of 10% (Table 1), which is

significantly lower than that of the ester E11-2,2 (45%

( 19%).²In the Ishikawa assay E11-2S,2 is an estrogen

antagonist, inhibiting the E₂stimulation of alkaline

phosphatase (AlkP) with a K_iof ∼3 nM, which is about

the same as previously found for the ester E11-2,2.⁵The

decrease in binding activity without an accompanying

decrease in antagonism may best be explained by an

increase in metabolic stability of the thiono ester.

Esters. We synthesized three esters in the E11-2

series with increasing chain length and/or steric bulk,

1432 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5

Zhang et al.

Scheme 6^a

^a(a) (i) KH, toluene, rt, (ii) R-I, rt; (b) BCl₃, CH₂Cl₂, 0 °C; (c) (i) KH, toluene, rt, (ii) (2-bromoethoxy)-tert-butyldimethylsilane, 60 °C;

(d) ⁿBu₄NF, THF, (e) MsCl, pyr, 4 °C; (f) 5% Pd-C, H₂, EtOAc-EtOH, rt.

Scheme 7^a

^a(a) (i) KH, toluene, rt (ii) R-I, rt; (b) BCl₃, CH₂Cl₂, 0 °C; (c) TsCl, pyr, 4 °C; (d) (i) KH, 18-crown-6, ROH, toluene, rt (ii) 25, 80 °C (e)

5% Pd-C, H₂, EtOAc-EtOH, rt.

more potent ER ligand and antagonist than its corre-

sponding ester.

(n ) 5), E11-1,4_ether(49), and E11-2,3_ether(60) (n ) 6)

are all highly potent antagonists with K_i) 0.1-0.3 nM.

We also synthesized 11â-butoxy-E₂, E11-0,4_ether(40) (n

) 5), which binds very weakly to the ER with an RBA

of 2.1% and almost no biological activity.

We tested for the effect of fluorine incorporation at

the 11â-ether side chain terminus, synthesizing E11-

1,2F_ether(54), E11-2,2F_ether(66), and E11-3,2F_ether(78).

The compounds E11-1,2F_etherand E11-2,2F_ethershow

modest ER binding of 10% and 16%, respectively, and

We synthesized a number of additional ethers to

examine the effect of ether chain length in terms of the

n g 5 rule, and the position of the ether oxygen on ER

binding and estrogen antagonistic activity (Figure 2,

Table 2). All of these ethers adhere to the rule. E11-

1,1_ether(43) (n ) 3) is a very potent agonist with an RSA

of 125%. Like E11-2,1_ether, E11-1,2_ether(45) (n ) 4) is

an agonist/antagonist. The compounds E11-1,3_ether(47)

C-11â-Analogues of Estradiols Result in SERMs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1433

Scheme 8^a

^a(a) (i) KH, toluene, rt (ii) R-I, rt; (b) BCl₃, CH₂Cl₂, 0 °C; (c) TsCl, pyr, 4 °C; (d) (i) KH, 18-crown-6, ROH, toluene, rt (ii) 72, 80 °C (e)

5% Pd-C, H₂, EtOAc-EtOH, rt.

show poor biological activity. While E11-3,2F_ethershows

good ER binding (47%), it is only a modest antagonist

with a K_iof 1.1 nM.

trophic stimulation of E₂in the immature rat. This

experiment was performed four times with similar

results. One such experiment is shown in Figure 4. It

can be seen that E11-2,2_etherat 100 ng has only a small

stimulatory effect on uterine weight (P < 0.01) and this

dose partially inhibits the uterotrophic stimulation of

20 ng of E₂(P < 0.001). At 1 µg, E11-2,2_etheris again

slightly estrogenic (P < 0.001), and it also inhibits the

uterotrophic stimulation of 20 ng of E₂(P < 0.001).

There were slight differences in the other experiments:

in two of them, 100 ng of E11-2,2_etherhad no uterotrophic

effect but still partially inhibited the action of E₂; in two

experiments, the 1 µg dose of E11-2,2_etherwas again

slightly uterotrophic but completely abolished the uter-

ine response to E₂. In all experiments, the low dose of

E11-2,2_etherinhibits E₂stimulation of the uterus, and

the high dose inhibits even more (in some of the

experiments completely). While the low dose was slightly

estrogenic in some experiments, it was not in others

(likely due to a relatively high SD). The high dose was

slightly estrogenic in all experiments. Thus, these

experiments clearly show that E11-2,2_etheris an antago-

nist with weak agonist activity, a mixed agonist/

antagonist, in the uterus of the rat.

We compared the action of E11-2,2_etheron the liver

and uterus by measuring its effect on plasma cholesterol

level and uterine weight in the ovariectomized rat. The

rats were injected with two separate doses of E11-

2,2_ether: (1) 36 ng/day and (2) 180 ng/day and E₂20 ng/

day. E₂produced both a significant increase in uterine

weight and a decrease in plasma cholesterol concentra-

tion (P < 0.01) (Figure 5a,b). In contrast, each dose of

E11-2,2_etherhad no statistical effect on uterine weight

and yet both produced a dose-dependent decrease in

plasma cholesterol level (P < 0.01). The lack of

uterotrophic activity in this experiment compared to the

previous one in immature rats is most probably due to

the lower dose employed in this experiment with the

We prepared a series of compounds in which we

incorporated branched methyl groups at the end of the

11â-ether side chain to investigate the possibility that

a bulky side chain terminus might enhance antiestro-

genic activity in compounds with good ER binding

(Table 2). The compounds are analogues of E11-2,2_ether

and E11-3,2_ether. In the case of E11-2,iPr_ether(62), ER

binding, RBA, is decreased by half, but the antiestro-

genic activity is unaffected as compared to that of E11-

2,2_ether. However, for E11-2,tBu_ether(64), estrogen re-

ceptor binding is dramatically decreased as is anti-

estrogenic activity. Interestingly, there is a dramatic

improvement in both the ER binding and in estrogen

antagonism as the same modifications are made in the

E11-3 ether series. Here E11-3,iPr_ether(74) (RBA )

114%) has a K_iof 0.2 nM and E11-3,tBu_ether(76) (RBA

) 81%) has a K_iof 0.09 nM.

To determine the selectivity of the binding of the ether

analogues to ERâ, we measured the binding to the LBD

of human ERâ (M₂₁₄-Q₅₃₀)

²²in comparison to the LBD

of human ERR (M₂₅₀-V₅₉₅).²³As can be seen in Table

2, the 11â-ethers of E₂have little binding selectivity for

ERR or ERâ. In addition, we investigated the action of

a representative ether, E11-2,2_ether(the ether analogue

of the ester E11-2,2 characterized previously^2,5), in JAR

cells that had been separately transfected with ERR or

ERâ with a consensus ERE linked to a luciferase

reporter gene. Like the ester E11-2,2, the ether E11-

2,2_etheris an estrogen antagonist with both ER subtypes;

it inhibits E₂stimulated transcription of the reporter

gene with both ERR and ERâ (Figure 3)

We investigated the possibility of a differential in vivo

action of E11-2,2_etheron the uterus and liver. Accord-

ingly, E11-2,2_etherwas studied in vivo in the classical

estrogen bioassay, determining its effect on the utero-

1434 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5

Table 1. Estrogenic Properties of 11â-Analogues of E₂

Zhang et al.

^aPartial structure showing the side chain at the 11â-position of the E₂-analogue. ^bRBA of the indicated compound (compared to E₂)

100%) to the ER in rat uterine cytosol. Values are (SD. ^cK_iwas determined by the inhibition of the stimulatory effect of 1 nM E₂on

alkaline phosphatase activity in the Ishikawa cells. ^dWeak mixed agonist/antagonist; weak activity that does not reach 50% of maximal

and is reflected by low RBA in ER. ^ew.ag., weak agonist with low activity which does not approach 50% of maximal. ^fValues in parentheses

are the relative stimulatory activity (RSA) in Ishikawa cells with respect to E₂.

C-11â-Analogues of Estradiols Result in SERMs

Table 2. Estrogenic Properties of E₂-11â-Ethers

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1435

^aPartial structure showing the side chain at the 11â-position of the E₂-analogue. ^bRBA (compared to E₂) 100%) of the indicated

E₂-11â-ether determined by inhibition of the binding of [³H]E₂to ER in rat uterine cytosol or lysates of E. coli in which the ligand binding

domain (LBD) of human ERR and ERâ were separately expressed. ^cK_iwas determined in Ishikawa cells as described in the legend to

Table 1. ^dRSA with respect to E₂. ^eMixed agonist/antagonist moderate activity that plateaus significantly below 100% of maximal activity

(see Figure 2). ^fWeak mixed agonist/antagonist; weak activity that does not reach 50% of maximal activity and is reflected by low RBA

in ER binding. Values are (SD.

1436 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5

Zhang et al.

Figure 2. Estrogenic (a) and antiestrogenic (b) action of the E11-1 ethers (11â-side chain length of n ) 3, E11-1,1_ether, through

n ) 6, E11-1,4_ether) on the expression of AlkP in Ishikawa cells. The cells were grown for 3 days with the indicated amounts of

each E11-1 ether alone (a) or with 1 nM E₂(b). In both panels 100% is the AlkP response normalized to 1 nM E₂alone. E11-

1,1_etheris a full agonist (a), without significant effect on the action of E₂(b). E11-1,2_etheris defined as a mixed agonist/antagonist

(ag/ant.) that alone stimulated AlkP (a) and inhibited E₂action (b), but in neither case did the effect approach 100% activity.

E11-1,3_etherand E11-1,4_etherare estrogen antagonists; inactive alone (a) but completely inhibit the stimulatory effect of E₂(b). In

both panels 100% is the AlkP response to 1 nM E₂. Error bars are SEM.

Figure 3. Antiestrogenic action of E11-2,2_etherin JAR cells transfected with a consensus ERE-Luc and ERR (a) or ERâ (b). The

cells were grown for 12 h in the presence of 1 nM E₂and the indicated concentrations of E11-2,2_ether. In both panels, 100% is the

luciferase response normalized to 1 nM E₂alone. Error bars are SEM.

ovariectomized rats. However, these low doses produced

striking effects on lowering plasma cholesterol. In a

separate study the pure antiestrogen ICI 182,780⁷was

coadministered with the higher dose (2) of E11-2,2_ether

(Figure 5, inset panel c). The antiestrogen had a small

but statistically significant effect (P < 0.001) in blocking

the agonist action of E11-2,2_etheron plasma cholesterol.

that both the carbonyl group and the ester oxygen

function composing the ester side chain are not required

to be present together for antiestrogenic activity. In fact,

those compounds lacking the carbonyl group, ethers,

exhibit much higher antagonist activity than their

corresponding esters.

For ethers of the same length (Table 2), the RBA for

the ER appears to be uninfluenced by the position of

the ether oxygen along the 11â-chain, with the exception

of E11-0,4_ether, which is a poor ER ligand. Given the

weak activity of this E₂-11â-ether, it is noteworthy that

Discussion

From the results of the ketone (Table 1) and ether

analogue studies (Table 2) where n g 5, it is apparent

C-11â-Analogues of Estradiols Result in SERMs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1437

Figure 4. Antiestrogenic and estrogenic action of E11-2,2_etheron the uterus of the immature rat. Twenty-one day old Sprague-

Dawley rats were injected with a total dose of 20 ng of E₂, 100 ng of E11-2,2_ether(1) or 1 µg of E11-2,2_ether(2) separately or together

with 20 ng of E₂(+ E₂) in 0.1 mL of sesame oil. The control group was injected with sesame oil alone. n ) 6 animals/group. Error

bars are SEM. Relevant statistical comparisons are provided in the text.

Figure 5. Tissue-specific effect of E11-2,2_etheron uterine weight (panel a) and liver (plasma cholesterol) (panel b) of the

ovariectomized rat. Ovariectomized rats (∼250 g) were treated sc daily for 7 days with 33 ng of E₂, 36 ng of E11-2,2_ether(1) or 180

ng E11-2,2_ether(2) in 0.1 mL of sesame oil. The control group was injected with sesame oil alone. n ) 6 animals/group. (inset panel

c) In a separate experiment ovariectomized animals were injected with 180 ng of E11-2,2_ether(2) or 180 ng of E11-2,2_ether(2) + ICI

182,780 in 0.1 mL of sesame oil. Error bars are SEM. Relevant statistical comparisons are provided in the text.

moving the ether oxygen anywhere else in the chain

leads to compounds with good ER binding and, when n

g 5, strong estrogen antagonism.

fluorine substitution as compared to E11-3,2_ether. Clearly,

fluorine addition at the side chain terminus offers no

advantage in the biological activity of these estradiol

analogues.

We prepared a series of compounds analogous to E11-

2,2_etherand E11-3,2_etherin which we incorporated

branched methyl groups at the end of the 11â-ether side

chain to investigate the possibility that a bulky side

chain terminus might enhance antiestrogenic activity

in compounds with good ER binding (Table 2). However,

compounds having the side chain branch point too close

For steroidal esters of E₂at 16R, estrogenic action is

enhanced with fluorine at the terminus of the substitu-

ent.¹We tested for a similar effect in compounds with

fluorine incorporation at the 11â-ether side chain ter-

minus, synthesizing E11-1,2F_ether, E11-2,2F_ether, and

E11-3,2F_ether. However, the fluorine compounds E11-

1,2F_etherand E11-2,2F_ethershow, instead, a significant

decrease in ER binding as compared to their unfluori-

nated counterparts E11-1,2_etherand E11-2,2_ether, respec-

tively. In both fluorine compounds, the low RBAs were

reflected in poor biological activity: E11-2,2F_etherhas a

much decreased antiestrogenic action when compared

to its unfluorinated counterpart E11-2,2_ether, and E11-

1,2F_etheris characterized as a weak agonist/antagonist

(a compound that has a relatively large EC₅₀or K_i,

respectively, and does not exceed 50% of maximal

activity in each case) With E11-3,2F_ether, ER binding is

unaffected, but estrogen antagonism is decreased by

to the steroid nucleus (E11-2,iPr_ether, E11-2,tBu_ether

)

exhibit lower binding to the ER. On the other hand,

moving the branch one methylene unit out (E11-

3,iPr_ether, E11-3,tBu_ether) enhances both ER binding and

estrogen antagonism.

We have previously found in the 11â-ester series that

E11-2,2 and E11-3,2 bound equally to the LBD of

human ERR and human ERâ. ERR is the classical

estrogen receptor found in most estrogen target organs,

while ERâ is an ER isoform that is distributed in a more

1438 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5

Zhang et al.

restricted manner.²⁴Both ERR and ERâ bind estrogens,

but they have somewhat different affinity for particular

estrogens. For example, C-16R-analogues of E₂bind with

a 20-90-fold ERR selectivity compared to ERâ.^1,25

However, most of the E₂-ethers in this study appear to

bind about equally to each ER isoform, although in some

cases there is modest selectivity (Table 2). Of note is

the larger RBA observed for most of the ether analogues

for both human ER LBDs compared to the ER in rat

uterine cytosol. This is not unexpected, as it probably

represents the temperature difference in the two assays.

The uterine assay is run at 0-2 °C while the LBD

assays are run at room temperature. It is well-known

that this elevated temperature increases the RBA for

11â-analogues of E₂.^26-28Furthermore, this difference

in RBA could result from the source (rat and human)

as well as a disparity between the intact ER and the

ER receptor fragment (LBD). Regardless, the compari-

son of the potency of the analogues to each other in the

various binding assays is the important point.

As we showed (Table 2), the 11â-ethers of E₂have

little binding selectivity for ERR or ERâ, and thus, it

might be presumed that they would act similarly as

antiestrogens with both ER isoforms. Indeed, we previ-

ously found that the antiestrogenic effect of the ester

E11-2,2 is exhibited through both ERR and ERâ.⁵In the

present study, we investigated the action of E11-2,2_ether

in JAR cells that had been separately transfected with

ERR and ERâ and a consensus ERE linked to a

luciferase reporter gene. Like the ester E11-2,2, the

ether E11-2,2_etheris an estrogen antagonist with both

ER subtypes; it inhibits E₂-stimulated transcription of

the reporter gene with both ERR and ERâ (Figure 3).

The experiment shown in Figure 3 with E11-2,2_etheris

representative of other experiments we performed with

the other n g 5 11â-ethers. Prior to these studies, it has

been reported that other 11â-analogues of E₂, substi-

tuted with pure alkanes and alkenes from C₅to C₉in

length show differential behavior; in cells transfected

separately with the two estrogen receptor subtypes and

a reporter gene, the alkanes and alkenes are ERâ

antagonists, but to the contrary, they are ERR ago-

nists.²⁹This selective behavior between the two ER

subtypes is not unprecedented. The R,R-enantiomer of

tetrahydrochrysene (R,R-THC), which also has short

alkyl side chains (two ethyl groups), is likewise an ERâ

antagonist and an ERR agonist.³⁰It is not clear what

makes the E₂-11â-alkanes/alkenes ERR-agonists while

all of the compounds described in this study are ERR-

antagonists, but it seems that hydrogen binding to the

ether oxygen must play a role. Since the E₂-11â substi-

tuted alkanes and alkenes are ERR agonists,²⁹then

E11-2,2_ether, which is a ERR antagonist, must be acting

through a markedly different mechanism with ERR.

in the uterus in vivo (Figure 4). As might be expected,

E11-2,2_etherwas considerably more potent in this assay

than the labile ester E11-2,2.⁵

While the SERM raloxifene exhibits an antiestrogenic

effects in specific tissues, such as the uterus, in other

tissues, however, including the liver, it is estrogenic.³⁴

This is manifested by a decrease in plasma cholesterol,

a well-known estrogenic action on the liver, which in

the rat produces an increase in hepatic LDL receptors,

resulting in a concomitant clearance of LDL and HDL

cholesterol.⁶As with the antiestrogenic action on the

uterus (above), the effect of E11-2,2_etheron plasma

cholesterol is much more potent than that which we

previously found for the labile ester E11-2,2.⁵This

follows from the fact that the ethers such as E11-2,2_ether

,

in contrast to the esters, are not susceptible to esterase

hydrolysis. It is noteworthy that E11-2,2_etherproduces

an agonist effect on plasma cholesterol at a dose that

has no effect on the uterus (Figure 5). As would be

expected, the pure antiestrogen ICI 187,780 inhibited

the agonist action of E11-2,2_etheron plasma cholesterol

(Figure 5, inset panel c). While the agonist activity of

E11-2,2_etherwas significantly blocked (P < 0.001), the

effect was comparatively small as the pure antagonist

ICI 187,780 increased the cholesterol levels of the E11-

2,2_ether-treated rats only slightly. This is not unexpected,

as it has been observed previously that the antiestrogen

is remarkably less potent in inhibiting estrogenic stimu-

lation of the liver (plasma cholesterol) compared to the

uterus.³⁵

Like the ester E11-2,2, these studies demonstrate that

E11-2,2_etheris a SERM: it is antiestrogenic in the

Ishikawa cell and in JAR cells transfected with ERR or

ERâ, in vivo in the uterus it is an antagonist with weak

agonist activity, and in the liver it is estrogen agonist.

These nonpolar short 11â-side chain analogues of E₂are

different from the classical antiestrogens and SERMs

that uniformly contain a long and polar or charged side

chain.³⁶This side chain is essential for the antiestro-

genic action, as it interferes with the agonist conforma-

tion of helix 12 in the ER and consequently binding of

required coactivators.^8,9,11This suggests the possibility

that these ethers act on ER by a different mechanism

(perhaps similar to the mechanism by which the short-

chain ERâ-antagonist THC acts on ERâ³⁷), and thus,

they could be unique therapeutic agents. The increased

antiestrogenic action of the analogues with bulky side

chains, E11-3,iPr_etherand E11-3,tBu_ether, indicates that

other sterically constrained analogues might have even

greater activity, and studies investigating this possibil-

ity are underway in this laboratory.

Experimental Section

General Methods. ¹H NMR spectra were recorded with a

Bruker Avance 400 or Avance 500 spectrometer, and chemical

shifts are reported relative to residual CHCl₃(7.27 ppm) or

acetone-d₆(2.05 ppm). Purification by flash chromatography

was performed according to the procedure of Still³⁸using 230-

400 mesh silica gel (EM Science, Darmstadt Germany). High-

resolution mass spectra were obtained by electrospray ioniza-

tion on a Micromass Q-Tof spectrometer by Dr. Walter J.

McMurray at the Yale University Comprehensive Cancer

Center/WM Keck Biotechnology Resource Center using NaI

as an internal standard. The computer program Prism was

purchased from GraphPad Software, Inc. (San Diego, CA). The

cell culture reagents were obtained form Gibco-BRL (Grand

The antiestrogenic action of E11-2,2_etherwas investi-

gated further in vivo in the classical estrogen bioassay,

determining its effect on the uterotrophic stimulation

of E₂in the immature rat. The rat uterus has a

preponderance of ERR compared to ERâ,³¹and the

uterotrophic response to estrogens has been shown to

be ERR-ligand selective;³²furthermore, the uterus of the

ERR-knockout mouse does not respond to E₂.³³These

experiments confirm that E11-2,2_etheracts through ERR

and demonstrate its mixed agonist/antiestrogenic action

C-11â-Analogues of Estradiols Result in SERMs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1439

Island, NH). The synthesis of compounds 1, 4, 12, 18, 41, and

67 were previously described.²Unless otherwise noted, sol-

vents (analytical or HPLC) and reagents were used as sup-

plied, and all reactions were carried out under nitrogen. ICI

182,780 was obtained from Tocris Cookson Inc (Ellisville, MO).

of silica gel using 7:1 hexanes/EtOAc as eluent gave 14 mg

(61%) of 5 as a white solid.

2,2-Dimethylpropyl (3,17â-Dihydroxyestra-1,3,5(10)-

trien-11â-yl)acetate (6, E11-2,diMePr). At 0 °C, BCl₃(0.4

mL, 1.0 M solution in CH₂Cl₂) was added to a solution of

compound 5 (10 mg, 0.017 mmol) in CH₂Cl₂(1.5 mL). The

reaction was stirred at 0 °C for 30 min, quenched with H₂O

(10 mL), and extracted with EtOAc (3×, 20 mL). Combined

organic extracts were washed with brine, dried over Na₂SO₄,

and concentrated in vacuo. Purification by HPLC using system

H-2 (t_R) 7.5 min) gave 3 mg (46%) of 6 as a white solid. Data

for 6: ¹H NMR (400 MHz, CDCl₃) δ 0.94 (s, 9H, -C(CH₃)₃),

0.96 (s, 3H, H-18), 3.74 (t, 2H, J ) 7.4 Hz, H-17R), 3.73 &

3.79 (AB quartet, 2H, J_AB) 10.6 Hz, -OCH₂-), 6.57 (d, 1H,

J ) 2.7 Hz, H-4), 6.66 (dd, 1H, J ) 8.3, 2.7 Hz, H-2), 7.10 (d,

1H, J ) 8.3 Hz, H-1); HRMS (ES⁺) calcd for C₂₅H₃₆O₄Na (M +

Na⁺) m/e 423.2511, found m/e 423.2525; HPLC system H-20,

t_R) 16.4 min, and system H-12, t_R) 10.9 min, >99% pure.

Ethyl (3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-yl)-

acetate (7). Compound 7 was prepared from 4 (140 mg, 0.274

mmol) and SOCl₂(700 µL, 9.5 mmol) in EtOH (25 mL) as

described for 2. Purification by flash chromatography on a 2

× 17 cm column of silica gel using 6:1 hexanes/EtOAc as eluent

gave 147 mg (100%) of 7: TLC, T-2, R_f0.68.

O-Ethyl (3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-

yl)thioacetate (8). A solution of 10 mg (0.018 mmol) of 7 and

20 mg (0.049 mmol) of Lawesson’s reagent¹³in o-xylene (1 mL)

was stirred and heated at 140 °C in a sealed vial for 24 h. The

reaction mixture was allowed to cool to room temperature and

subjected to flash chromatography on a 2 × 17 cm column of

silica gel using 25:1 hexanes/EtOAc as eluent, giving 3 mg

(28%) of 8 (TLC system T-2, R_f0.52) and 5.6 mg of recovered

7 (TLC system T-2, R_f0.34). Data for 8: ¹H NMR (400 MHz,

CDCl₃) δ 1.06 (s, 3H, H-18), 1.40 (t, 3H, J ) 7.0 Hz, -CH₂CH₃),

3.50 (t, 1H, J ) 7.9 Hz, H-17R), 4.47-4.57 (m, 2H, -OCH₂-

CH₃), 4.57 (s, 2H, OBn), 5.04 (s, 2H, OBn), 6.71 (d, 1H, J )

2.5 Hz, H-4), 6.82 (dd, 1H, J ) 8.7, 2.5 Hz, H-2), 7.22 (d, 1H,

J ) 8.7 Hz, H-1), 7.30-7.46 (m, 10H, Ar-H).

Chromatographic Systems. Thin-layer chromatography

(TLC) was performed using Merck silica gel plates (F254) (EM

Science) and visualized using phosphomolybdic acid or UV

illumination. TLC systems: T-1, EtOAc/hexanes (2:1); T-2,

hexanes/EtOAc (6:1); T-3, hexanes/EtOAc (2:1); T-4, hexanes/

EtOAc (4:1); T-5, hexanes/EtOAc (1:1); T-6, hexanes/EtOAc (3:

1); T-7, hexanes/EtOAc (1:3). Analytical high-performance

liquid chromatography (HPLC) was performed on a Beckman

System Gold HPLC system (Beckman Coulter, Inc. Fullerton,

CA) consisting of a model 126 solvent module and a model 168

diode array detector at 210 and 280 nm using the following

columns and systems. With an Ultrasphere ODS column (5

µm, 10 mm × 25 cm, Altex Scientific Operations Co.), the

following solvent systems at 3 mL/min were used, unless

otherwise noted: H-1, CH₃CN/H₂O (1:1); H-2, CH₃CN/H₂O (65:

35); H-3, CH₃CN/H₂O (55:45); H-4, CH₃CN/H₂O (45:55); H-5,

CH₃CN/H₂O (40:60); H-6, CH₃CN/H₂O/HOAc (42:57.884:0.116);

H-7, CH₃CN/H₂O/HOAc (40:59.88:0.12); H-8, CH₃CN/H₂O (60:

40); H-9, CH₃OH/H₂O (60:40), 2 mL/min; H-10, CH₃OH/H₂O

(70:30), 1.5 mL/min; H-11, CH₃OH/H₂O (75:25), 1.2 mL/min.

With a LiChrospher 100 Diol column (5 µm, 4.6 mm × 25 cm,

EM Science) the following solvent systems at 1 mL/min were

used: H-12, CH₂Cl₂; H-13, CH₂Cl₂/iPrOH (92:8); H-14, CH₂-

Cl₂/iPrOH (90:10); H-15, CH₂Cl₂/iPrOH (99:1); H-16, CH₂Cl₂/

isooctane (90:10); H-17, CH₂Cl₂/iPrOH (97:3); H-18, CH₂Cl₂/

iPrOH (98:2). With a Protein I-60 column (7.8 mm × 30 cm,

Waters Co.) the following solvent system at 3 mL/min was

used: H-19, CH₂Cl₂at 2 mL/min. With a Microsorb-MV C18

column (5 µm, 4.6 mm × 25 cm, Varian Analytical Instru-

ments) the following solvent systems were used at 1 mL/min:

H-20, CH₃CN/H₂O (1:1); H-21, CH₃CN/H₂O (40:60).

Isopropyl (3,17â-Dihydroxyestra-1,3,5(10)-trien-11â-

yl)acetate (2, E11-2,iPr). A solution of 10.0 mg (0.030 mmol)

of 1 and 100 µL (163 mg, 1.37 mmol) of SOCl₂in 2-propanol

(1 mL) was stirred and heated at 60 °C in a sealed vial for 24

h. The reaction mixture was poured into saturated aqueous

NaHCO₃(50 mL) and extracted with EtOAc (3×, 50 mL).

Combined organic extracts were washed with H₂O (20 mL),

dried over Na₂SO₄, and concentrated in vacuo. Purification of

the residue by HPLC using system H-1 gave 5.6 mg (49%) of

2 as a white solid. Data for 2: TLC, T-1, R_f0.63; ¹H NMR

(400 MHz, CDCl₃) δ 0.91 (s, 3H, H-18), 1.17 (d, 3H, J ) 6.2

Hz, OCH(CH₃)₂), 1.22 (d, 3H, J ) 6.2 Hz, OCH(CH₃)₂), 3.70

(m, 1H, H-17R), 4.98 (septet, 1H, J ) 6.2 Hz, OCH(CH₃)₂), 6.53

(d, 1H, J ) 2.2 Hz, H-4), 6.64 (dd, 1H, J ) 8.4, 2.6 Hz, H-2),

7.08 (d, 1H, J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for C₂₃H₃₂O₄-

Na (M + Na⁺) m/e 395.2198, found m/e 395.2197; HPLC system

H-20, t_R) 10.2 min, and system H-12, t_R) 12.5 min, >99%

pure.

O-Ethyl (3,17â-Dihydroxyestra-1,3,5(10)-trien-11â-yl)-

thioacetate (9, E11-2S,2). At 0 °C, BCl₃(1.0 mL, 1.0 M

solution in CH₂Cl₂) was added to a solution of compound 8

(25 mg, 0.045 mmol) in CH₂Cl₂(1.5 mL). The reaction was

stirred at 0 °C for 30 min, quenched with saturated aqueous

NaHCO₃(10 mL), and extracted with EtOAc (3×, 20 mL).

Combined organic extracts were washed with brine, dried over

Na₂SO₄, and concentrated in vacuo. Purification by HPLC

using system H-1 gave 5 mg (31%) of 9 as a white solid. Data

1

for 9: TLC, T-3, R_f0.4; H NMR (400 MHz, CDCl₃) δ 0.98 (s,

3H, H-18), 1.40 (t, 3H, J ) 7.1 Hz, -OCH₂CH₃), 3.73 (m,1H,

H-17R), 4.49-4.55 (m, 2H, -OCH₂CH₃), 6.56 (d, 1H, J ) 2.7

Hz, H-4), 6.67 (dd, 1H, J ) 8.5, 2.7 Hz, H-2), 7.17 (d, 1H, J )

8.5 Hz, H-1); HRMS (ES⁺) calcd for C₂₂H₃₀O₃SNa (M + Na⁺)

m/e 397.1813, found m/e 397.1813; HPLC system H-20, t_R

14.6 min, and system H-12, t_R) 9.4 min, >99% pure.

)

Propyl (3,17â-Dihydroxyestra-1,3,5(10)-trien-11â-yl)-

acetate (3, E11-2,3). Compound 3 was prepared by esterifi-

cation of 1 (8.5 mg, 0.026 mmol) with n-propanol as described

for 2. Purification by HPLC using system H-1 gave 6.3 mg

(66%) of 3 as a white solid. Data for 3: ¹H NMR (400 MHz,

CDCl₃) δ 0.92 (t, 3H, J ) 7.5 Hz, -CH₃), 0.93 (s, 3H, H-18),

3.73 (m, 1H, H-17R), 4.00 (m, 2H, -OCH₂-), 6.55 (d, 1H, J )

2.5 Hz, H-4), 6.64 (dd, 1H, J ) 8.4, 2.5 Hz, H-2), 7.08 (d, 1H,

J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for C₂₃H₃₂O₄Na (M + Na⁺)

N-Ethyl(3,17â-dibenzyloxyestra-1,3,5(10)-trien-11â-yl)-

acetamide (10). The synthesis of 10 is based on the literature

procedure.¹⁴A mixture of 32 mg (0.063 mmol) of 4, 24 mg (0.15

mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 13

mg (0.11 mmol) of DMAP, and 29 mg (51 µL, 0.64 mmol of a

70% aqueous solution) of ethylamine in CH₂Cl₂(2.5 mL) was

stirred at room temperature for 24 h. The reaction mixture

was poured into CH₂Cl₂(50 mL) and washed with saturated

aqueous NH₄Cl (2×, 50 mL). The organic layer was dried over

Na₂SO₄and concentrated in vacuo. Purification of the residue

by flash chromatography on a 2 × 17 cm column of flash silica

gel using 2:1 hexanes/EtOAc as eluent gave 12 mg (36%) of

10. Data for 10: TLC, T-5, R_f0.66; ¹H NMR (400 MHz, CDCl₃)

δ 1.02 (s, 3H, H-18), 1.10 (t, 3H, J ) 7.1 Hz, -CH₂CH₃), 2.29

(d, 1H, J ) 13.6 Hz, H-12â), 2.60 (dd, 1H, J ) 10.7, 5.2 Hz,

H-9), 2.72-2.86 (m, 2H, H-6), 3.16-3.36 (m, 3H, H-11, -CH₂-

CH₃), 3.51 (t, 1H, J ) 8.9 Hz, H-17R), 4.56 & 4.59 (AB quartet,

2H, J_AB) 12.3 Hz, OBn), 5.03 (s, 2H, OBn), 5.17 (br s, 1H,

m/e 395.2198, found m/e 395.2197; HPLC system H-20, t_R

10.6 min, and system H-12, t_R) 12.9 min, >99% pure.

)

2,2-Dimethylpropyl (3,17â-Dibenzyloxyestra-1,3,5(10)-

trien-11â-yl)acetate (5). A solution of 20.0 mg (0.039 mmol)

of 4, 15 mg (0.170 mmol) of neopentyl alcohol, 1 mg (0.008

mmol) of DMAP, and 8 mg (0.04 mmol) of 1,3-dicyclohexyl-

carbodiimide in CH₃CN (1 mL) was stirred and heated at 50

°C for 24 h. The reaction mixture was cooled to room temper-

ature, filtered, and washed through with CH₃CN. The filtrate

was dried over Na₂SO₄and concentrated in vacuo. Purification

of the residue by flash chromatography on a 2 × 17 cm column

1440 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5

Zhang et al.

NH), 6.70 (d, 1H, J ) 2.6 Hz, H-4), 6.80 (dd, 1H, J ) 8.9, 2.6

Hz, H-2), 7.19 (d, 1H, J ) 8.9 Hz, H-1), 7.30-7.45 (m, 10H,

Ar-H).

procedure.¹⁷To a solution of 30 mg (0.061 mmol) of 18 in 1

mL of Et₂O was added 60 µL (0.18 mmol) of a 3 M solution of

ethylmagnesium bromide in Et₂O and reaction was stirred at

room temperature for 24 h. To the reaction mixture was added

Et₂O (1 mL) and 1 M HCl (1 mL) and stirring was continued

at room temperature for 2 h. The reaction was poured into

H₂O (50 mL) and extracted with EtOAc (3×, 30 mL). Combined

organic extracts were dried over Na₂SO₄and concentrated in

vacuo. Purification by flash chromatography on a 2 × 17 cm

column of silica gel using 6:1 hexanes/EtOAc as eluent gave

23 mg (72%) of 19. TLC, T-4, R_f0.62.

1-(3,17â-Dihydroxyestra-1,3,5(10)-trien-11â-yl)butan-2-

one (20, E11-2K,2). Compound 20 was prepared from 19 (20

mg, 0.038 mmol) as described for 6. Purification by HPLC

using system H-1 gave 9.5 mg (72%) of 20 as a white solid.

Data for 20: ¹H NMR (400 MHz, CDCl₃) δ 0.91 (s, 3H, H-18),

0.98 (t, 3H, J ) 7.3 Hz, -CH₂CH₃), 2.70-2.86 (m, 2H, H-6),

3.21-3.24 (m, 1H, H-11), 3.74 (dd, 1H, J ) 9.0, 7.4 Hz, H-17R),

6.57 (d, 1H, J ) 2.6 Hz, H-4), 6.61 (dd, 1H, J ) 8.4, 2.6 Hz,

H-2), 6.97 (d, 1H, J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for

C₂₂H₃₀O₃Na (M + Na⁺) m/e 365.2093, found m/e 365.2082;

HPLC system H-20, t_R) 8.1 min, and system H-19, t_R) 9.4

min, >99% pure.

N-Ethyl(3,17â-dihydroxyestra-1,3,5(10)-trien-11â-yl)-

acetamide (11, E11-2,2_amide). Compound 11 was prepared

from 10 (12 mg, 0.022 mmol) as described for 6. Purification

by HPLC using system H-5 gave 2.4 mg (30%) of 11 as a white

solid. Data for 11: TLC, T-7, R_f0.125; ¹H NMR (400 MHz,

acetone-d₆) δ 0.93 (s, 3H, H-18), 1.04 (t, 3H, J ) 7.3 Hz,

-CH₂CH₃), 2.53 (dd, 1H, J ) 10.8, 4.8 Hz, H-9), 2.63-2.79

(m, 2H, H-6), 3.08-3.22 (m, 3H, H-11, -CH₂CH₃), 3.65 (t, 1H,

J ) 8.2 Hz, H-17R), 6.52 (d, 1H, J ) 2.5 Hz, H-4), 6.62 (dd,

1H, J ) 8.3, 2.5 Hz, H-2), 6.79 (br s, 1H, NH), 7.06 (d, 1H, J

) 8.3 Hz, H-1); HRMS (ES⁺) calcd for C₂₂H₃₁NO₃Na (M + Na⁺)

m/e 380.2202, found m/e 380.2202; HPLC system H-21, t_R

7.9 min, and system H-13, t_R) 7.25 min, >99% pure.

)

N-Ethyl-3-(3,17â-dibenzyloxyestra-1,3,5(10)-trien-11â-

yl)propionamide (13). Compound 13 was prepared from 12

(30 mg, 0.057 mmol) as described for 10. Purification by flash

chromatography on a 2 × 17 cm column of silica gel using 1.5:1

hexanes/EtOAc as eluent gave 18 mg (57%) of 13. Data for

1

13: TLC, T-5, R_f0.58; H NMR (400 MHz, CDCl₃) δ 1.03 (s,

3H, H-18), 1.14 (t, 3H, J ) 7.2 Hz, -CH₂CH₃), 2.28 (d, 1H, J

) 13.6 Hz, H-12â), 2.58 (dd, 1H, J ) 10.6, 4.3 Hz, H-9), 2.69-

2.86 (m, 2H, -CH₂CH₃), 3.24-3.31 (m, 2H, -CH₂CH₃), 3.49

(t, 1H, J ) 8.0 Hz, H-17R), 4.57 & 4.61 (AB quartet, 2H, J_AB

) 12.2 Hz, OBn), 5.04 (s, 2H, OBn), 5.31 (br s, 1H, NH), 6.69

(d, 1H, J ) 2.5 Hz, H-4), 6.81 (dd, 1H, J ) 8.5, 2.5 Hz, H-2),

7.11 (d, 1H, J ) 8.5 Hz, H-1), 7.30-7.46 (m, 10H, Ar-H).

N-Ethyl-3-(3,17â-dihydroxyestra-1,3,5(10)-trien-11â-yl)-

propionamide (14, E11-3,2_amide). Compound 14 was pre-

pared from 13 (9 mg, 0.016 mmol) as described for 6.

Purification by HPLC using system H-1 gave 3 mg (50%) of

14 as a white solid. Data for 14: ¹H NMR (400 MHz, acetone-

d₆) δ 0.93 (s, 3H, H-18), 1.05 (t, 3H, J ) 7.3 Hz, -CH₂CH₃),

3.13-3.20 (m, 2H, -CH₂CH₃), 3.64 (t, 1H, J ) 8.3 Hz, H-17R),

6.52 (dd, 1H, J ) 2.6 Hz, H-4), 6.62 (dd, 1H, J ) 8.4, 2.6 Hz,

H-2), 6.95 (br s, 1H, NH), 7.05 (d, 1H, J ) 8.4 Hz, H-1); HRMS

(ES⁺) calcd for C₂₃H₃₃NO₃Na (M + Na⁺) m/e 394.2358, found

m/e 394.2356; HPLC system H-20, t_R) 6.3 min, and system

H-14, t_R) 5.8 min, >99% pure.

1-(3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-yl)pentan-

2-one (21). Compound 21 was prepared from 18 (420 mg,

0.854 mmol) and propylmagnesium chloride (2.8 mL of a 2.0

M solution in Et₂O, 5.6 mmol) as described for 19. Purification

by flash chromatography on a 2 × 17 cm column of silica gel

using 6:1 hexanes/EtOAc as eluent gave 400 mg (87%) of 21.

1

Data for 21: TLC, T-4, R_f0.56; H NMR (400 MHz, CDCl₃) δ

0.87 (t, 3H, J ) 7.3 Hz, -CH₂CH₃), 0.99 (s, 3H, H-18), 2.73-

2.85 (m, 2H, H-6), 3.19-3.25 (m, 1H, H-11), 3.49 (t, 1H, J )

7.9 Hz, H-17R), 4.56 (s, 2H, OBn), 5.03 (s, 2H, OBn), 6.71 (d,

1H, J ) 2.5 Hz, H-4), 6.75 (dd, 1H, J ) 8.6, 2.5 Hz, H-2), 7.00

(d, 1H, J ) 8.6 Hz, H-1), 7.29-7.45 (m, 10H, Ar-H).

1-(3,17â-Dihydroxyestra-1,3,5(10)-trien-11â-yl)pentan-

2-one (22, E11-2K,3). Compound 22 was prepared from 21

(30 mg, 0.056 mmol) as described for 6. Purification by HPLC

using system H-3 gave 10 mg (50%) of 22. Data for 22: ¹H

NMR (400 MHz, CDCl₃) δ 0.86 (t, 3H, J ) 7.4 Hz, -CH₂CH₃),

0.91 (s, 3H, H-18), 2.70-2.85 (m, 2H, H-6), 3.19-3.24 (m, 1H,

H-11), 3.74 (t, J ) 6.6 Hz, H-17R), 6.57 (d, 1H, J ) 2.5 Hz,

H-4), 6.60 (dd, 1H, J ) 8.5, 2.5 Hz, H-2), 6.97 (d, 1H, J ) 8.5

Hz, H-1); HRMS (ES⁺) calcd for C₂₃H₃₂O₃Na m/e 379.2249,

found m/e 379.2241; HPLC system H-20, t_R) 11.2 min, and

system H-19, t_R) 8.4 min, > 99% pure.

Ethyl (3,17â-dibenzyloxyestra-1,3,5(10)-trien-11â-yl)-

propionate (15). Compound 15 was prepared from 12 (110

mg, 0.210 mmol) and SOCl₂(0.2 mL, 2.74 mmol) in EtOH (3

mL) at 45 °C as described for 2. Purification by flash chroma-

tography on a 2 × 17 cm column of silica gel using 7:1 hexanes/

EtOAc as eluent gave 85 mg (73%) of 15.

2-(3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-yl)etha-

nol (24). A solution of 605 mg (1.26 mmol) of 23¹⁸and 43.0

mg (1.97 mmol) of LiBH₄in a 1 M solution of catecholborane

in THF was stirred at room temperature for 21 h. The reaction

mixture was poured into a 0 °C solution of NaOH (0.57 g),

EtOH (6 mL), H₂O₂(6 mL), and H₂O (1.4 mL) and allowed to

stir at room temperature for 6 h. The reaction mixture was

then poured into H₂O (70 mL) and extracted with EtOAc (3×,

70 mL). Combined organic extracts were dried over Na₂SO₄

and concentrated in vacuo, giving a slightly yellow oil.

Purification by flash chromatography on a 3 × 21 cm column

of silica gel using 2:1 hexanes/EtOAc as eluent gave a colorless

oil which was dissolved in EtOAc (100 mL) and washed with

15% aqueous NaOH (50 mL) and H₂O (until the aqueous phase

was colorless), giving 0.5 g (80%) of 24. Data for 24: TLC, T-6,

R_f0.18; ¹H NMR (400 MHz, CDCl₃) δ 1.02 (s, 3H, H-18), 2.25

(dd, 1H, J ) 13.8, 1.5 Hz, H-12â), 2.54-2.86 (m, 4H, H-6, 9,

11), 3.48 (dd, 1H, J ) 8.7, 7.3 Hz, H-17R), 3.64-3.80 (m, 2H,

-CH₂O), 4.57 & 4.58 (AB quartet, 2H, J_AB) 12.3 Hz, OBn),

5.03 (s, 2H, OBn), 6.70 (d, 1H, J ) 2.8 Hz, H-4), 6.80 (dd, 1H,

J ) 8.5, 2.8 Hz, H-2), 7.10 (d, 1H, J ) 8.5 Hz, H-1), 7.29-7.49

(m, 10H, Ar-H).

N-Methyl-3-(3,17â-dibenzyloxyestra-1,3,5(10)-trien-11â-

yl)propionamide (16). The synthesis of 16 is based on the

literature procedure.^15,16A solution of 20 mg (0.036 mmol) of

15 and 1.3 mg (0.026 mmol) of NaCN in 33% ethanolic CH₃-

NH₂(2 mL) was stirred and heated at 60 °C for 20 h. The

solvent was evaporated under a N₂stream and the residue

was purified by flash chromatography using 2:1 hexanes/

EtOAc as eluent to give 15 mg (77%) of 16. Data for 16: TLC,

1

T-3, R_f0.27; H NMR (400 MHz, CDCl₃) δ 1.03 (s, 3H, H-18),

2.69-2.86 (m, 2H, H-6), 2.78 (d, 3H, J ) 4.9 Hz, NCH₃), 3.49

(t, 1H, J ) 8.1 Hz, H-17R), 4.57 & 4.60 (AB quartet, 2H, J_AB

) 12.3 Hz, OBn), 5.04 (s, 2H, OBn), 5.33 (br s, 1H, NH), 6.70

(d, 1H, J ) 2.7 Hz, H-4), 6.81 (dd, 1H, J ) 8.6, 2.7 Hz, H-2),

7.10 (d, 1H, J ) 8.6 Hz, H-1), 7.30-7.46 (m, 10H, Ar-H).

N-Methyl-3-(3,17â-dihydroxyestra-1,3,5(10)-trien-11â-

yl)propionamide (17, E11-3,1_amide). Compound 17 was

prepared from 16 (15 mg, 0.028 mmol) as described for 6.

Purification by HPLC using system H-5 gave 3.5 mg (35%) of

17 as a white solid. Data for 17: ¹H NMR (400 MHz, acetone-

d₆) δ 0.93 (s, 3H, H-18), 2.65 (d, 3H, J ) 4.9 Hz, NCH₃), 3.64

(dd, 1H, J ) 8.8, 7.4 Hz, H-17R), 6.52 (d, 1H, J ) 2.3 Hz, H-4),

6.62 (dd, 1H, J ) 8.3, 2.3 Hz, H-2), 6.93 (br s, 1H, NH), 7.05

(d, 1H, J ) 8.3 Hz, H-1); HRMS (ES⁺) calcd for C₂₂H₃₁NO₃Na

(M + Na⁺) m/e 380.2202, found m/e 380.2203; HPLC system

H-21, t_R) 8.5 min, and system H-13, t_R) 10 min, >99% pure.

1-(3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-yl)butan-

2-one (19). The synthesis of 19 is based on the literature

2-(3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-yl)ethyl

Toluenesulfone (25). A solution of 68 mg (0.14 mmol) of 24

and 390 mg (2.01 mmol) of pTsCl in pyridine (5 mL) was

allowed to stand at 4 °C for 6 days. Reaction was poured into

H₂O (70 mL) and extracted with CH₂Cl₂(3×, 70 mL). Com-

bined organic extracts were dried over Na₂SO₄and concen-

C-11â-Analogues of Estradiols Result in SERMs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1441

using system H-1 gave 3.6 mg (27%) of 32 as a white solid.

trated in vacuo. Purification by flash chromatography on a 2

1

× 21 cm column of silica gel using 4:1 hexanes/EtOAc as eluent

Data for 32: TLC, T-1, R_f0.55; H NMR (400 MHz, CDCl₃) δ

1

gave 60 mg (68%) of 25. Data for 25: TLC, T-6, R_f0.78; H

0.91 (t, 3H, J ) 7.6 Hz, -CH₃), 0.93 (s, 3H, H-18), 2.67-2.85

(m, 2H, H-6), 3.73 (dd, 1H, J ) 8.8, 7.4 Hz, H-17R), 6.55 (d,

1H, J ) 2.7 Hz, H-4), 6.66 (dd, 1H, J ) 8.4, 2.7 Hz, H-2), 7.07

(d, 1H, J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for C₂₄H₃₄O₃Na

(M + Na⁺) m/e 393.2406, found m/e 393.2402; HPLC system

H-11, t_R) 29 min, and system H-15, t_R) 8.2 min, >99% pure.

3,11â-Dihydroxyestra-1,3,5(10)-trien-17-one (34). A so-

lution of 1.13 g (3.4 mmol) of 33¹⁹in 1% KOH-MeOH (25 mL)

was stirred and heated at 50 °C for 1 h. The reaction mixture

was poured into H₂O (200 mL) adjusted to pH 2 with

concentrated HCl. The resulting white solid was collected by

vacuum filtration and used without further purification in the

next step. Data for 34: TLC, T-5, R_f0.31; ¹H NMR (400 MHz,

CDCl₃) δ 1.17 (s, 3H, H-18), 2.81-2.96 (m, 2H, H-6), 4.79 (dd,

1H, J ) 5.6, 2.4 Hz, H-11), 6.64 (d, 1H, J ) 2.6 Hz, H-4), 6.70

(dd, 1H, J ) 8.3, 2.6 Hz, H-2), 7.17 (d, 1H, J ) 8.3 Hz, H-1).

3-Benzyloxy-11â-hydroxyestra-1,3,5(10)-trien-17-one

(35). A solution of 2.74 g (19.8 mmol) of K₂CO₃in CHCl₃(60

mL) and MeOH (30 mL) was stirred and heated at 70 °C for

20 min. To this was added a solution of crude 34 in CHCl₃(40

mL) and MeOH (20 mL) followed by 600 µL (5.06 mmol) of

benzyl bromide. The reaction mixture was stirred at reflux for

6 h then allowed to stir at room temperature overnight. The

reaction mixture was poured into H₂O (300 mL) and extracted

with EtOAc (2×, 250 mL). Combined organic extracts were

dried over Na₂SO₄and concentrated in vacuo. Purification by

flash chromatography on a 3 × 17 cm column of silica gel using

1:1 hexanes/EtOAc as eluent gave 990 mg (77%, two steps) of

35. Data for 35: TLC, T-5, R_f0.64; ¹H NMR (400 MHz, CDCl₃)

δ 1.17 (s, 3H, H-18), 2.84-2.98 (m, 2H, H-6), 4.79 (dd, 1H, J

) 5.5, 2.6 Hz, H-11), 5.06 (s, 2H, OBn), 6.78 (d, 1H, J ) 2.7

Hz, H-4), 6.85 (dd, 1H, J ) 8.6, 2.7 Hz, H-2), 7.21 (d, 1H, J )

8.6 Hz, H-1), 7.32-7.45 (m, 5H, Ar-H).

NMR (400 MHz, CDCl₃) δ 0.90 (s, 3H, H-18), 2.41 (s, 3H,

-CH₃), 3.45 (t, 1H, J ) 7.9 Hz, H-17R), 4.08-4.17 (m, 2H,

-CH₂O), 4.54 (s, 2H OBn), 5.04 (s, 2H, OBn), 6.69 (d, 1H, J )

2.6 Hz, H-4), 6.77 (dd, 1H, J ) 8.5, 2.6 Hz, H-2), 7.00 (d, 1H,

J ) 8.5 Hz, H-1), 7.30-7.47 (m, 12H, Ar-H), 7.79 (d, 2H, J )

8.1 Hz, Ar-H).

3-(3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-yl)pro-

panenitrile (26). A solution of 220 mg (0.34 mmol) of 25 and

100 mg (2.0 mmol) of NaCN in DMSO (8 mL) was stirred and

heated at 90 °C for 1.5 h. The reaction was allowed to cool to

room temperature, poured into saturated aqueous NH₄Cl (50

mL), and extracted with CH₂Cl₂(2×, 50 mL). Combined

organic extracts were washed with H₂O (20 mL), dried over

Na₂SO₄, and concentrated in vacuo. Purification by flash

chromatography on a 2 × 17 cm column of silica gel using 5:1

hexanes/EtOAc gave 150 mg (88%) of 26. Data for 26: TLC,

T-4, R_f) 0.51; ¹H NMR (400 MHz, CDCl₃) δ 0.99 (s, 3H, H-18),

3.51 (t, 1H, J ) 8.0 Hz, H-17R), 4.63 & 4.56 (AB quartet, 2H,

J_AB) 12.4 Hz, OBn), 5.06 (s, 2H, OBn), 6.73 (d, 1H, J ) 2.5

Hz, H-4), 6.85 (dd, 1H, J ) 8.6, 2.5 Hz, H-2), 7.12 (d, 1H, J )

8.6 Hz, H-1), 7.30-7.48 (m, 10H, Ar-H).

4-(3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-yl)butan-

2-one (27). Compound 27 was prepared from 26 (27 mg, 0.053

mmol) and methylmagnesium bromide (120 µL of a 3.0 M

solution in Et₂O, 0.36 mmol) in Et₂O (1.5 mL) as described for

19. Purification by flash chromatography on a 2 × 17 cm

column of silica gel using 8:1 hexanes/EtOAc gave 14 mg (50%)

of 27.

4-(3,17â-Dihydroxyestra-1,3,5(10)-trien-11â-yl)butan-2-

one (28, E11-3K,1). Compound 28 was prepared from 27 (13

mg, 0.25 mmol) as described for 6. Purification by HPLC using

system H-1 gave 2 mg (23%) of 28 as a white solid. Data for

1

28: TLC, T-5, R_f0.35; H NMR (400 MHz, CDCl₃) δ 0.93 (s,

3-Benzyloxy-17,17-ethylenedioxyestra-1,3,5(10)-trien-

11â-ol (36). A solution of 990 mg (2.63 mmol) of 35, ethylene

glycol (17.4 mL), and pTsOH (100 mg) in benzene (150 mL)

was stirred and heated at reflux for 4 h in a 250 mL flask

equipped with a reflux condenser and a Dean-Stark trap. The

trap was emptied and 4A sieves were added to it and heating

was continued for additional 1 h. The reaction mixture was

allowed to cool to room temperature, poured into saturated

aqueous NaHCO₃(200 mL), and extracted with EtOAc (2×,

200 mL). Combined organic extracts were dried over Na₂SO₄

and concentrated in vacuo. Purification by flash chromatog-

raphy on a 3 × 17 cm column of silica gel using 2:1 hexanes/

EtOAc as eluent gave 985 mg (89%) of 36.

3H, H-18), 2.12 (s, 3H, -CH₃), 2.67-2.82 (m, 2H, H-6), 3.73

(dd, 1H, J ) 8.9, 7.3 Hz, H-17R), 6.55 (d, 1H, J ) 2.5 Hz, H-4),

6.65 (dd, 1H, J ) 8.5, 2.5 Hz, H-2), 7.06 (d, 1H, J ) 8.5 Hz,

H-1); HRMS (ES⁺) calcd for C₂₂H₃₀O₃Na (M + Na⁺) m/e

365.2093, found m/e 365.2096; HPLC system H-9, t_R) 26 min,

and system H-15, t_R) 11.0 min, >99% pure.

1-(3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-yl)pentan-

3-one (29). Compound 29 was prepared from 26 (32 mg, 0.063

mmol) and ethylmagnesium bromide (130 µL of a 3 M solution

in Et₂O, 0.39 mmol) as described for 19. Purification by flash

chromatography using 8:1 hexanes/EtOAc as eluent gave 22

mg (65%) of 29: TLC, T-4, R_f0.61.

1-(3,17â-Dihydroxyestra-1,3,5(10)-trien-11â-yl)pentan-

3-one (30, E11-3K2). Compound 30 was prepared from 29 (20

mg, 0.037 mmol) as described for 6. Purification by HPLC

using system H-1 gave 3.7 mg (28%) of 30 as a white solid.

3-Benzyloxy-11â-butoxy-17,17-ethylenedioxyestra-1,3,5-

(10)-triene (37). A suspension of 47 mg (16 mg, 0.41 mmol)

of a 35% dispersion of KH in mineral oil was washed in a 10

mL pear flask with hexanes (3 mL) and resuspended in

anhydrous toluene (1 mL). To this was added a solution of 115

mg (0.273 mmol) of 36 in 2 mL of toluene, and the reaction

mixture was stirred and heated at 80 °C for 30 min. To this

was added a solution of 312 mg (1.36 mmol) of butyl toluene-

sulfonate²⁰in toluene (1 mL) and stirring was continued at

80 °C for 20 h. The reaction mixture was allowed to cool to

room temperature, poured into saturated aqueous NH₄Cl (200

mL), and extracted with CH₂Cl₂(2×, 150 mL). Combined

organic extracts were dried over Na₂SO₄and concentrated in

vacuo. Purification by flash chromatography on a 2 × 21 cm

column of silica gel using 5:1 hexanes/EtOAc as eluent gave

264 mg of product containing material which was used in the

1

Data for 30: TLC, T-5, R_f0.45; H NMR (400 MHz, CDCl₃) δ

0.94 (s, 3H, H-18), 1.04 (t, 3H, J ) 7.3 Hz, -CH₃), 2.67-2.85

(m, 2H, H-6), 3.73 (t, 1H, J ) 8.0 Hz, H-17R), 6.55 (d, 1H, J )

2.7 Hz, H-4), 6.66 (dd, 1H, J ) 8.4, 2.7 Hz, H-2), 7.07 (d, 1H,

J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for C₂₃H₃₂O₃Na (M + Na⁺)

m/e 379.2249, found m/e 379.2260; HPLC system H-10, t_R

43 min, and system H-15, t_R) 8.2 min, >99% pure.

)

1-(3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-yl)hexan-

3-one (31). Compound 31 was prepared from 26 (30 mg, 0.059

mmol) and propylmagnesium chloride (200 µL of a 2 M solution

in Et₂O, 0.4 mmol) as described for 19. Purification by flash

chromatography on a 2 × 17 cm column of silica gel using 10:1

hexanes/EtOAc as eluent gave 24 mg (73%) of 31. Data for

1

31: TLC, T-4, R_f0.71; H NMR (400 MHz, CDCl₃) δ 0.92 (t,

next step without further purification. Integration of the H

3H, J ) 7.4 Hz, -CH₃), 1.02 (s, 3H, H-18), 2.70-2.87 (m, 2H,

H-6), 3.49 (t, 1H, J ) 7.9 Hz, H-17R), 4.58 & 4.61 (AB quartet,

2H, J_AB) 12.3 Hz, OBn), 5.04 (s, 2H, OBn), 6.70 (d, 1H, J )

2.4 Hz, H-4), 6.81 (dd, 1H, J ) 8.6, 2.4 Hz, H-2), 7.11 (d, 1H,

J ) 8.6 Hz, H-1), 7.29-7.47 (m, 10H, Ar-H).

1-(3,17â-Dihydroxyestra-1,3,5(10)-trien-11â-yl)hexan-

3-one (32, E11-3K,3). Compound 32 was prepared from 31

(20 mg, 0.36 mmol) as described for 6. Purification by HPLC

NMR signals at δ 0.87 and 0.81 ppm indicates that 45 mg of

this material is 37 (45% yield) and 218 mg is recovered butyl

toluenesulfonate. In addition, 55 mg of 36 was recovered. Data

for 37: TLC, T-3, R_f0.7; ¹H NMR (400 MHz, CDCl₃) δ 0.81 (t,

3H, J ) 7.4 Hz, -CH₃), 1.09 (s, 3H, H-18), 2.74-2.93 (m, 2H,

H-6), 3.16-3.21 (m, 1H, OCH₂), 3.57-3.63 (m, 1H, OCH₂),

3.87-3.97 (m, 4H, 17-ketal), 4.25 (dd, 1H, J ) 5.7, 2.7 Hz,

H-11), 5.03 (s, 2H, OBn), 6.69 (d, 1H, J ) 2.7 Hz, H-4), 6.76

1442 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5

Zhang et al.

(dd, 1H, J ) 8.6, 2.7 Hz, H-2), 7.06 (d, 1H, J ) 8.6 Hz, H-1),

7.31-7.45 (m, 5H, Ar-H).

339.1936, found m/e 339.1934; HPLC system H-5, t_R) 12 min,

and system H-17, t_R) 7.9 min, >99% pure.

3-Benzyloxy-11â-butoxyestra-1,3,5(10)-trien-17-one (38).

A solution of impure 37 from the previous step, 200 mg (1.05

mmol) of pTsOH in acetone (15 mL), and 20 drops of H₂O was

stirred and heated at 70 °C for 1 h. The reaction mixture was

allowed to cool to room temperature, poured into saturated

NaHCO₃(200 mL), and extracted with CH₂Cl₂(2×, 150 mL).

Combined organic extracts were dried over Na₂SO₄and

concentrated in vacuo, giving 217 mg of crude 38 which was

used without purification in the following step.

3,17â-Dibenzyloxy-11â-ethoxymethylestra-1,3,5(10)-

triene (44). Compound 44 was prepared from 41²(85 mg, 0.18

mmol) and EtI (50 µL, 0.6 mmol) as described for 42. Purifica-

tion by flash chromatography on a 2 × 17 cm column of silica

gel using 17:1 hexanes/EtOAc as eluent gave 30 mg (33%) of

44 and 22 mg of recovered 41. Data for 44: TLC, T-3, R_f0.84;

¹H NMR (400 MHz, CDCl₃) δ 1.02 (s, 3H, H-18), 1.19 (t, 3H, J

) 6.9 Hz, -CH₃), 2.58-2.64 (m, 2H, H-9, 12â), 2.69-2.88 (m,

3H, H-6, 11), 3.30-3.42 (m, 4H, OCH₂), 3.51 (t, 1H, J ) 7.9

Hz, H-17R), 4.60 & 4.65 (AB quartet, 2H, J_AB) 12.5 Hz, OBn),

5.05 (s, 2H, OBn), 6.70 (d, 1H, J ) 2.6 Hz, H-4), 6.81 (dd, 1H,

J ) 8.7, 2.6 Hz, H-2), 7.23 (d, 1H, J ) 2.6 Hz, H-1), 7.28-7.47

(m, 10H, Ar-H).

11â-Ethoxymethylestra-1,3,5(10)-triene-3,17â-diol (45,

E11-1,2_ether). Compound 45 was prepared from 44 (15 mg,

0.029 mmol) as described for 6. Purification by flash chroma-

tography on a 2 × 17 cm column of silica gel using 1.5:1 EtOAc/

hexanes as eluent gave 7 mg of 45. Further purification by

HPLC using system H-1 gave 5.3 mg (55%) of 45 as a white

solid. Data for 45: TLC, T-1, R_f0.51; ¹H NMR (400 MHz,

CDCl₃) δ 0.92 (s, 3H, H-18), 1.16 (t, 3H, J ) 7.0 Hz, -CH₃),

2.46 (dd, 1H, J ) 13.2, 1.8 Hz, H-12â), 2.58 (dd, 1H, J ) 10.6,

4.9 Hz, H-9), 2.68-2.86 (m, 3H, H-6, 11), 3.25-3.40 (m, 4H,

OCH₂), 3.74 (dd, 1H, J ) 9.0, 7.4 Hz, H-17R), 6.54 (d, 1H, J )

2.8 Hz, H-4), 6.64 (dd, 1H, J ) 8.4, 2.8 Hz, H-2), 7.17 (d, 1H,

J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for C₂₁H₃₀O₃Na (M + Na⁺)

3-Benzyloxy-11â-butoxyestra-1,3,5(10)-trien-17â-ol (39).

A solution of the crude 38 from the previous step in anhydrous

THF (5 mL) was stirred at -78 °C as 104 mg (2.73 mmol) of

LiAlH₄was added. The reaction mixture was stirred at -78

°C for 1 h, quenched with EtOAc (5 mL), poured into saturated

aqueous sodium-potassium tartrate (50 mL), and extracted

with CH₂Cl₂(3×, 70 mL). The combined organic extracts were

dried over Na₂SO₄and concentrated in vacuo. Purification by

flash chromatography on a 2 × 21 cm column of silica gel using

2:1 hexanes/EtOAc as eluent gave 31 mg (26%, 3 steps) of 39.

1

Data for 39: TLC, T-4, R_f0.13; H NMR (400 MHz, CDCl₃) δ

0.81 (t, 3H, J ) 7.9 Hz, -CH₃), 0.99 (s, 3H, H-18), 3.13-3.23

(m, 1H, OCH₂), 3.58-3.64 (m, 1H, OCH₂), 3.70 (t, 1H, J ) 7.9

Hz, H-17R), 4.22 (dd, 1H, J ) 5.5, 2.6 Hz, H-11), 5.04 (s, 2H,

OBn), 6.69 (d, 1H, J ) 3.0 Hz, H-4), 6.77 (dd, 1H, J ) 8.7, 3.0

Hz, H-2), 7.06 (d, 1H, J ) 8.7 Hz, H-1), 7.31-7.49 (m, 5H,

Ar-H).

11â-Butoxyestra-1,3,5(10)-triene-3,17â-diol (40, E11-

0,4_ether). A suspension of 31 mg (0.071 mmol) of 39 and 5 mg

of 5% Pd-C in EtOH (5 mL) was stirred at room temperature

under an atmosphere of H₂for 24 h. The reaction mixture was

filtered through a 1 in. pad of Celite and washed through with

EtOAc (50 mL). The filtrate was concentrated in vacuo and

purified by flash chromatography on a 2 × 21 cm column of

silica gel using 2:1 hexanes/EtOAc as eluent, giving 23 mg of

40. Further purification by HPLC using system H-6 gave 11

mg (45%) of 40 as a white solid. Data for 40: TLC, T-3, R_f

0.27; ¹H NMR (400 MHz, CDCl₃) δ 0.82 (t, 3H, J ) 7.4 Hz,

-CH₃), 0.99 (s, 3H, H-18), 2.71-2.91 (m, 2H, H-6), 3.17-3.23

(m, 1H, OCH₂), 3.58-3.64 (m, 1H, OCH₂), 3.70 (t, 1H, J ) 8.1

Hz, H-17R), 4.20 (dd, 1H, J ) 5.6, 2.7 Hz, H-11), 6.54 (d, 1H,

J ) 2.6 Hz, H-4), 6.62 (dd, 1H, J ) 8.4, 2.6 Hz, H-2), 7.02 (d,

1H, J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for C₂₂H₃₂O₃Na (M +

Na⁺) m/e 367.2249, found m/e 367.2241; HPLC system H-7, t_R

) 25 min, and system H-16, t_R) 11 min, >99% pure.

3,17â-Dibenzyloxy-11â-methoxymethylestra-1,3,5(10)-

triene (42). A suspension of 150 mg (52 mg, 1.31 mmol) of a

35% dispersion of KH in mineral oil was washed with hexanes

(2 mL) and suspended in 1 mL of anhydrous toluene. To this

was added a solution of 100 mg (0.21 mmol) of 41²in 1 mL of

toluene and the reaction was stirred at room temperature for

30 min. To this was added 80 µL (1.25 mmol) of CH₃I, and

stirring was continued for 24 h. Another 200 µL (3.2 mmol) of

CH₃I was added, and the reaction was stirred for another 24

h, poured into saturated aqueous NH₄Cl (20 mL), and ex-

tracted with CH₂Cl₂(3×, 30 mL). Combined organic extracts

were dried over Na₂SO₄and concentrated in vacuo. Purifica-

tion by flash chromatography on a 2 × 17 cm column of silica

gel using 8:1 hexanes/EtOAc as eluent gave 26 mg (25%) of

42. TLC, T-6, R_f0.59.

m/e 353.2093, found m/e 353.2097; HPLC system H-20, t_R

8.5 min, and system H-12, t_R) 18.3 min, >99% pure.

)

3,17â-Dibenzyloxy-11â-propoxymethylestra-1,3,5(10)-

triene (46). Compound 46 was prepared from 41²(42 mg,

0.087 mmol) and 1-iodopropane (50 µL, 0.52 mmol) as de-

scribed for 42. Purification by flash chromatography on a 2 ×

17 cm column of silica gel using 12:1 hexanes/EtOAc as eluent

1

gave 17 mg (37%) of 46. Data for 46: TLC, T-2, R_f0.69; H

NMR (400 MHz, CDCl₃) δ 0.92 (t, 3H, -CH₃), 1.00 (s, 2H,

H-18), 2.57-2.65 (m, 2H, H-9,12â), 2.69-2.87 (m, 3H, H-6,11),

3.19-3.38 (m, 4H, OCH₂), 3.51 (t, 1H, J ) 8.1 Hz, H-17R), 4.57

& 4.64 (AB quartet, 2H, J_AB) 12.0 Hz, OBn), 5.03 (s, 2H,

OBn), 6.69 (d, 1H, J ) 2.7 Hz, H-4), 6.79 (dd, 1H, J ) 8.7, 2.7

Hz, H-2), 7.22 (d, 1H, J ) 8.7 Hz, H-1), 7.29-7.46 (m, 10H,

Ar-H).

11â-Propoxymethylestra-1,3,5(10)-triene-3,17â-diol (47,

E11-1,3_ether). Compound 47 was prepared from 46 (15 mg, 0.29

mmol) as described for 6. Purification by HPLC using system

H-1 gave 7 mg (71%) of 47 as a white solid. Data for 47: ¹H

NMR (400 MHz, CDCl₃) δ 0.91 (t, 3H, J ) 7.4 Hz, -CH₃), 0.92

(s, 3H, H-18), 2.58-2.87 (m, 4H, H-6, 9, 11), 3.19-3.37 (m,

4H, OCH₂), 3.71-3.77 (m, 1H, H-17R), 6.55 (d, 1H, J ) 2.6

Hz, H-4), 6.65 (dd, 1H, J ) 8.3, 2.6 Hz, H-2), 7.19 (d, 1H, J )

8.3 Hz, H-1); HRMS (ES⁺) calcd for C₂₂H₃₂O₃Na (M + Na⁺)

m/e 367.2249, found m/e 367.2241; HPLC system H-20, t_R

13.1 min, and system H-18, t_R) 6.8 min, >99% pure.

)

3,17â-Dibenzyloxy-11â-butoxymethylestra-1,3,5(10)-

triene (48). Compound 48 was prepared from 41²(100 mg,

0.21 mmol) and iodobutane (142 µL, 1.25 mmol) as described

for 42. Purification by flash chromatography on a 2 × 17 cm

column of silica gel using 8:1 hexanes/EtOAc as eluent gave

95 mg (85%) of 48: TLC, T-6, R_f0.66.

11â-Butoxymethylestra-1,3,5(10)-triene-3,17â-diol (49,

E11-1,4_ether). Compound 49 was prepared from 48 (95 mg, 0.18

mmol) as described for 40. Purification by flash chromatog-

raphy on a 2 × 27 cm column of silica gel using 1:1 hexanes/

EtOAc as eluent gave 41 mg of 49. Further purification by

HPLC using system H-1 gave 32 mg (50%) of 49 as a white

solid. Data for 49: TLC, T-3, R_f0.27; ¹H NMR (500 MHz,

CDCl₃) δ 0.90 (t, 3H, J ) 7.3 Hz, -CH₃), 0.91 (s, 3H, H-18),

2.45 (dd, 1H, J ) 13.2, 2.0 Hz, H-12â), 2.58 (dd, 1H, J ) 10.5,

4.9 Hz, H-9), 2.68-2.86 (m, 3H, H-6, 11), 3.21-3.35 (m, 4H,

OCH₂), 3.73 (dd, 1H, J ) 8.9, 7.5 Hz, H-17R), 6.54 (d, 1H, J )

2.5 Hz, H-4), 6.64 (dd, 1H, J ) 8.5, 2.5 Hz, H-2), 7.17 (d, 1H,

J ) 8.5 Hz, H-1); HRMS (ES⁺) calcd for C₂₃H₂₄O₃Na (M + Na⁺)

11â-Methoxymethylestra-1,3,5(10)-triene-3,17â-diol (43,

E11-1,1_ether). Compound 43 was prepared from 42 (26 mg,

0.052 mmol) in 5:1 EtOH/EtOAc as described for 40. Purifica-

tion by flash chromatography on a 2 × 17 cm column of silica

gel using 1:1 hexanes/EtOAc gave 13.3 mg of 43. Further

purification by HPLC using system H-5 gave 10.5 mg (63%)

of 43 as a white solid. Data for 43: TLC, T-3, R_f0.13; ¹H NMR

(500 MHz, CDCl₃) δ 0.92 (s, 3H, H-18), 2.42 (dd, 1H, J ) 13.2,

1.9 Hz, H-12â), 2.57-2.60 (m, 1H, H-9), 2.67-2.86 (m, 3H, H-6,

11), 3.22-3.32 (m, 2H, OCH₂), 3.24 (s, 3H, OCH₃), 3.74 (dd,

1H, J ) 8.8, 7.6 Hz, H-17R), 6.54 (d, 1H, J ) 2.6 Hz, H-4),

6.65 (dd, 1H, J ) 8.8, 2.6 Hz, H-2), 7.16 (d, 1H, J ) 8.8 Hz,

H-1); HRMS (ES⁺) calcd for C₂₀H₂₈O₃Na (M + Na⁺) m/e

C-11â-Analogues of Estradiols Result in SERMs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1443

m/e 381.2406, found m/e 381.2410; HPLC system H-1, t_R) 15

min, and system H-12, t_R) 12.4 min, >99% pure.

Na⁺) m/e 371.1998, found m/e 371.1988; HPLC system H-5, t_R

) 14.5 min, and system H-12, t_R) 14.5 min, >99% pure.

3,17â-Dibenzyloxy-11â-(2-methoxyethyl)estra-1,3,5(10)-

triene (55). Compound 55 was prepared from 24 (50 mg, 0.10

mmol) and CH₃I (50 µL, 0.80 mmol) as described for 42.

Purification by flash chromatography using 8:1 hexanes/EtOAc

as eluent gave 51 mg (100%) of 55: TLC, T-6, R_f0.73.

11â-(2-Methoxyethyl)estra-1,3,5(10)-triene-3,17â-diol (56,

E11-2,1_ether). Compound 56 was prepared from 55 (20 mg,

0.039 mmol) as described for 6. Purification by HPLC using

system H-5 gave 7.2 mg (56%) of 56 as a white solid. Data for

56: ¹H NMR (400 MHz, CDCl₃) δ 0.95 (s, 3H, H-18), 2.55-

2.85 (m, 4H, H-6, 9, 11), 3.32 (s, 3H, OCH₃), 3.45-3.46 (m,

2H, OCH₂), 3.74 (t, 1H, J ) 8.8, 7.5 Hz, H-17R), 6.55 (d, 1H,

J ) 2.7 Hz, H-4), 6.65 (d, 1H, J ) 8.4, 2.7 Hz, H-2), 7.07 (d,

1H, J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for C₂₁H₃₀O₃Na (M +

Na⁺) m/e 353.2093, found m/e 353.2094; HPLC system H-21,

t_R) 12.9 min, and system H-12, t_R) 22.5 min, >99% pure.

3,17â-Dibenzyloxy-11â-(2-ethoxyethyl)estra-1,3,5(10)-

triene (57). Compound 57 was prepared from 24 (32 mg, 0.064

mmol) and EtI (50 µL, 0.62 mmol) as described for 42.

Purification by flash chromatography on a 2 × 17 cm column

of silica gel using 20:1 hexanes/EtOAc gave 10 mg (29%) of

57. Data for 57: TLC, T-6, R_f0.79; ¹H NMR (400 MHz, CDCl₃)

δ 1.05 (s, 3H, H-18), 1.20 (t, 3H, J ) 7.0 Hz, -CH₃), 2.27 (d,

1H, J ) 13.2 Hz, H-12â), 2.54-2.62 (m, 2H, H-9, 11), 2.69-

2.87 (m, 2H, H-6), 3.39-3.55 (m, 5H, H-17R, OCH₂), 4.60 (s,

2H, OBn), 5.04 (s, 2H, OBn), 6.69 (d, 1H, J ) 2.7 Hz, H-4),

6.80 (dd, 1H, J ) 8.5, 2.7 Hz, H-2), 7.12 (d, 1H, J ) 8.5 Hz,

H-1), 7.28-7.46 (m, 10H, Ar-H).

11â-(2-Ethoxyethyl)estra-1,3,5(10)-triene-3,17â-diol (58,

E11-2,2_ether). Compound 58 was prepared from 57 (10 mg,

0.019 mmol) as described for 6. Purification by HPLC using

system H-1 gave 4.6 mg (70%) of 58. Data for 58: ¹H NMR

(400 MHz, CDCl₃) δ 0.96 (s, 3H, H-18), 1.20 (t, 3H, J ) 6.9

Hz, -CH₃), 2.52-2.62 (m, 2H, H-9, 11), 2.66-2.84 (m, 2H,

H-6), 3.39-3.55 (m, 4H, OCH₂), 3.73 (dd, 1H, J ) 8.9, 7.2 Hz,

H-17R), 6.54 (d, 1H, J ) 2.7 Hz, H-4), 6.63 (dd, 1H, J ) 8.5,

2.7 Hz, H-2), 7.07 (d, 1H, J ) 8.5 Hz, H-1); HRMS (ES⁺) calcd

for C₂₂H₃₂O₃Na (M + Na⁺) m/e 367.2249, found m/e 367.2242;

HPLC system H-20, t_R) 9.2 min, and system H-12, t_R) 16.6

min, >99% pure.

3,17â-Dibenzyloxy-11â-(2-propoxyethyl)estra-1,3,5(10)-

triene (59). Compound 59 was prepared from 24 (60 mg, 0.12

mmol) and 1-iodopropane (50 µL, 0.51 mmol) as described for

42. Purification by flash chromatography on a 2 × 17 cm

column of silica gel using 12:1 hexanes/EtOAc as eluent gave

29 mg (44%) of 59: TLC, T-2, R_f0.71.

11â-(2-Propoxyethyl)estra-1,3,5(10)-triene-3,17â-diol (60,

E11-2,3_ether). Compound 60 was prepared from 59 (15 mg,

0.028 mmol) as described for 6. Purification by HPLC using

system H-1 gave 5 mg (50%) of 60 as a white solid. Data for

60: ¹H NMR (400 MHz, CDCl₃) δ 0.93 (t, 3H, J ) 7.5 Hz,

-CH₃), 0.96 (s, 3H, H-18), 2.52-2.83 (m, 4H, H-6, 9, 11), 3.30-

3.59 (m, 4H, OCH₂), 3.73 (dd, 1H, J ) 9.0, 7.1 Hz, H-17R),

6.53 (d, 1H, J ) 2.8 Hz, H-4), 6.62 (dd, 1H, J ) 8.4, 2.8 Hz,

H-2), 7.08 (d, 1H, J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for

C₂₃H₃₄O₃Na (M + Na⁺) m/e 381.2406, found m/e 381.2408;

HPLC system H-20, t_R) 11.2 min, and system H-12, t_R) 12.2

min, >99% pure.

11â-(tert-Butyldimethylsiloxyethoxy)methyl-3,17â-

dibenzyloxyestra-1,3,5(10)-triene (50). Compound 50 was

prepared from 41²(57 mg, 0.12 mmol) and (2-bromoethoxy)-

tert-butyldimethylsilane (500 µL, 2.4 mmol) at 60 °C as

described for 42, except 374 mg (1.42 mmol) of 18-crown-6 was

added along with the KH. Purification by flash chromatogra-

phy on a 2 × 17 cm column of silica gel using 12:1 hexanes/

EtOAc as eluent gave 48 mg (63%) of 50: TLC, T-2, R_f0.4.

2-((3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-yl)-

methoxy)ethanol (51). A solution of 48 mg (0.074 mmol) of

50 was stirred at room temperature in a 1 M solution of ⁿBu₄-

NF in THF (1 mL) for 30 min. The reaction mixture was

poured into H₂O (50 mL) and extracted with EtOAc (3×, 50

mL). The combined organic extracts were dried over Na₂SO₄

and concentrated in vacuo. Purification by flash chromatog-

raphy on a 2 × 17 cm column of silica gel using 2:1 hexanes/

EtOAc as eluent gave 22 mg (55%) of 51. Data for 51: ¹H NMR

(400 MHz, CDCl₃) δ 0.98 (s, 3H, H-18), 2.52 (d, 1H, J ) 13.0

Hz, H-12â), 2.60 (dd, 1H, J ) 10.8, 4.3 Hz, H-9), 2.70-2.86

(m, 3H, H-6, 11), 3.38-3.42 (m, 4H, OCH₂), 3.50 (t, 1H, J )

8.0 Hz, H-17R), 3.65-3.68 (m, 2H, OCH₂), 4.59 (s, 2H, OBn),

5.03 (s, 2H, OBn), 6.70 (d, 1H, J ) 2.3 Hz, H-4), 6.80 (dd, 1H,

J ) 8.6, 2.3 Hz, H-2), 7.22 (d, 1H, J ) 8.6 Hz, H-1), 7.29-7.45

(m, 10H, Ar-H).

3,17â-Dibenzyloxy-11â-(methanesulfonyloxyethoxymeth-

yl)estra-1,3,5(10)-triene (52). A solution of 22 mg (0.041

mmol) of 51 and 32 µL (0.41 mmol) of MsCl in pyridine (2 mL)

was allowed to stand at 4 °C overnight. The reaction mixture

was poured into H₂O (30 mL) and extracted with CH₂Cl₂(3×,

30 mL). Combined organic extracts were dried over Na₂SO₄

and concentrated in vacuo. Purification by flash chromatog-

raphy on a 2 × 17 cm column of silica gel using 2:1 hexanes/

EtOAc gave 24 mg (96%) of 52. Data for 52: TLC, T-3, R_f0.40;

¹H NMR (400 MHz, CDCl₃) δ 0.99 (s, 3H, H-18), 2.55 (dd, 1H,

J ) 13.1, 1.6 Hz, H-12â), 2.60 (dd, 1H, J ) 11.0, 4.8 Hz, H-9),

2.71-2.85 (m, 3H, H-6, 11), 2.95 (s, 3H, -CH₃), 3.36-3.45 (m,

2H, OCH₂), 3.51 (t, 1H, J ) 7.9 Hz, H-17R), 3.55-3.58 (m, 2H,

OCH₂), 4.30-4.33 (m, 2H, OCH₂), 4.57 (s, 2H, OBn), 5.04 (s,

2H, OBn), 6.70 (d, 1H, J ) 2.7 Hz, H-4), 6.80 (dd, 1H, J ) 8.7,

2.7 Hz, H-2), 7.19 (d, 1H, J ) 8.7 Hz, H-1), 7.29-7.45 (m, 10H,

Ar-H).

3,17â-Dibenzyloxy-11â-(2-fluoroethoxymethyl)estra-

1,3,5(10)-triene (53). A solution of 24 mg (0.039 mmol) of 52

n

was stirred and heated at 70 °C in a 1 M solution of Bu₄NF

in THF (1 mL) for 30 min. The reaction mixture was cooled to

room temperature, poured into H₂O (30 mL), and extracted

with CH₂Cl₂(3×, 30 mL). The combined organic extracts were

dried over Na₂SO₄and concentrated in vacuo. Purification by

flash chromatography on a 2 × 17 cm column of silica gel using

4:1 hexanes/EtOAc as eluent gave 15 mg of 53. Data for 53:

1

TLC, T-3, R_f0.69; H NMR (400 MHz, CDCl₃) δ 1.00 (s, 3H,

H-18), 2.57-2.86 (m, 5H, H-6, 9, 11, 12â), 3.40-3.43 (m, 2H,

OCH₂), 3.48-3.53 (m, 1H, H-17R, OCH₂), 3.57-3.61 (m, 1H,

OCH₂), 4.45-4.65 (m, 2H, FCH₂-), 4.58 & 4.63 (AB quartet,

2H, J_AB) 12.2 Hz, OBn), 5.03 (s, 2H, OBn), 6.69 (d, 1H, J )

2.9 Hz, H-4), 6.80 (dd, 1H, J ) 8.6, 2.9 Hz, H-2), 7.22 (d, 1H,

J ) 2.9 Hz, H-1), 7.29-7.45 (m, 10H, Ar-H).

11â-(2-Fluoroethoxymethyl)estra-1,3,5(10)-triene-3,17â-

diol (54, E11-1,2F1_ether). Compound 54 was prepared from

53 (15 mg, 0.028 mmol) in EtOH (5 mL) and EtOAc (1 mL) as

described for 40. Purification by flash chromatography on a 2

× 17 cm column of silica gel using 1:1 hexanes/EtOAc gave

7.5 mg of 54. Further purification by HPLC using system H-5

gave 5.4 mg (54%) of 54 as a white solid. Data for 54: TLC,

3,17â-Dibenzyloxy-11â-(2-isopropoxyethyl)estra-1,3,5-

(10)-triene (61). A suspension of 570 mg (200 mg, 5.0 mmol)

of a 35% dispersion of KH in mineral oil was washed in a 25

mL pear-shaped flask with hexanes (2 mL). To this was added

a solution of 1.39 g (5.25 mmol) of 18-crown-6 and 380 µL (4.96

mmol) of anhydrous iPrOH in anhydrous toluene (4 mL). The

reaction was stirred at room temperature for 10 min and then

a solution of 162 mg (0.250 mmol) of 25 in toluene (4 mL) was

added. The reaction was stirred and heated at 80 °C for 2 h,

allowed to cool to room temperature, poured into H₂O (100

mL), and extracted with EtOAc (3×, 50 mL). The combined

organic extracts were dried over Na₂SO₄and concentrated in

vacuo. Purification by flash chromatography on a 2 × 17 cm

column of silica gel using 10:1 hexanes/EtOAc as eluent gave

1

T-3, R_f0.15; H NMR (400 MHz, CDCl₃) δ 0.91 (s, 3H, H-18),

2.46 (dd, 1H, J ) 13.5, 1.7 Hz, H-12â), 2.59 (dd, 1H, J ) 10.7,

4.7 Hz, H-9), 2.68-2.82 (m, 2H, H-6), 2.84-2.91 (m, 1H, H-11),

3.35-3.44 (m, 2H, OCH₂), 3.74 (dd, 1H, J ) 8.8, 7.6 Hz, H-17R),

4.50 (dt, 2H, J ) 47.5, 4.2, 4.2 Hz, -CH₂F), 6.54 (d, 1H, J )

2.7 Hz, H-4), 6.64 (dd, 1H, J ) 8.4, 2.7 Hz, H-2), 7.17 (d, 1H,

J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for C₂₁H₂₉FO₃Na (M +

1444 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5

Zhang et al.

1

130 mg (96%) of 61. Data for 61: TLC, T-6, R_f0.66; H NMR

J ) 8.3 Hz, H-1); HRMS (ES⁺) calcd for C₂₂H₃₁FO₃Na (M +

Na⁺) m/e 385.2155, found m/e 385.2152; HPLC system H-5, t_R

) 16.5 min, and system H-12, t_R) 13.1 min, >99% pure.

(400 MHz, CDCl₃) δ 1.03 (s, 3H, H-18), 1.13 (d, 3H, J ) 5.7

Hz, -CH₃), 1.14 (d, 3H, J ) 5.7 Hz, -CH₃), 2.25 (dd, 1H, J )

13.5, 1.1 Hz, H-12â), 2.52-2.58 (m, 2H,H-9, 11), 2.68-2.84 (m,

2H, H-6), 3.39-3.57 (m, 4H, H-11, 17R, OCH₂), 4.58 (s, 2H,

OBn), 5.03 (s, 2H, OBn), 6.67 (d, 1H, J ) 2.5 Hz, H-4), 6.78

(dd, 1H, J ) 8.6, 2.5 Hz, H-2), 7.10 (d, 1H, J ) 8.6 Hz, H-1),

7.28-7.45 (m, 10H, Ar-H).

11â-(2-Isopropoxyethyl)estra-1,3,5(10)-triene-3,17â-di-

ol (62, E11-2,iPr_ether). Compound 62 was prepared from 61

(17 mg, 0.032 mmol) in EtOAc (4 mL) as described for 40.

Purification by flash chromatography on a 2 × 17 cm column

of silica gel using 1:1 hexanes/EtOAc as eluent gave 5.8 mg of

62. Further purification by HPLC using system H-4 gave 4.4

mg (38%) of 62 as a white solid. Data for 62: ¹H NMR (500

MHz, CDCl₃) δ 0.95 (s, 3H, H-18), 1.12 (d, 3H, J ) 6.1 Hz,

-CH₃), 1.14 (d, 3H, J ) 6.1 Hz, -CH₃), 2.13 (dd, 1H, J ) 13.5,

1.8 Hz, H-12â), 2.52-2.61 (m, 2H, H-9, 11), 2.66-2.80 (m, 2H,

H-6), 3.37-3.42 (m, 1H, OCH₂), 3.47-3.56 (m, 2H, OCH₂,

-CH(CH₃)₂), 3.72 (t, 1H, J ) 8.2 Hz, H-17R), 6.52 (d, 1H, J )

2.8 Hz, H-4), 6.63 (dd, 1H, J ) 8.5, 2.8 Hz, H-2), 7.07 (d, 1H,

J ) 8.5 Hz, H-1); HRMS (ES⁺) calcd for C₂₃H₃₄O₃Na (M + Na⁺)

m/e 381.2406, found m/e 381.2413; HPLC system H-4, t_R) 15.5

min, and system H-12, t_R) 12.3 min, >99% pure.

3,17â-Dibenzyloxy-11â-(3-methoxypropyl)estra-1,3,5-

(10)-triene (68). Compound 68 was prepared from 67²(42 mg,

0.082 mmol) and CH₃I (50 µL, 0.8 mmol) as described for 42.

Purification by flash chromatography on a 2 × 17 cm column

of silica gel using 12:1 hexanes/EtOAc as eluent gave 17 mg

(39%) of 68: ¹H NMR (400 MHz, CDCl₃) δ 1.03 (s, 3H, H-18),

2.60-2.87 (m, 2H, H-6), 3.29-3.33 (m, 2H, OCH₂) 3.31 (s, 3H,

OCH₃), 3.49 (t, 1H, J ) 7.9 Hz, H-17R), 4.59 (s, 2H, OBn), 5.04

(s, 2H, OBn), 6.70 (d, 1H, J ) 2.8 Hz, H-4), 6.80 (dd, 1H, J )

8.5, 2.8 Hz, H-2), 7.07 (d, 1H, J ) 8.5 Hz, H-1), 7.29-7.47 (m,

10H, Ar-H).

11â-(3-Methoxypropyl)estra-1,3,5(10)-triene-3,17â-di-

ol (69, E11-3,1_ether). Compound 69 was prepared from 68 (15

mg, 0.028 mmol) as described for 6. Purification by HPLC

using system H-1 gave 6 mg (61%) of 69 as a white solid. Data

for 69: ¹H NMR (400 MHz, CDCl₃) δ 0.94 (s, 3H, H-18), 2.67-

2.83 (m, 2H, H-6), 3.28-3.33 (m, 2H, OCH₂), 3.30 (s, 3H,

OCH₃), 3.72 (t, 1H, J ) 7.7 Hz, H-17R), 6.55 (d, 1H, J ) 2.7

Hz, H-4), 6.65 (dd, 1H, J ) 8.5, 2.7 Hz, H-2), 7.04 (d, 1H, J )

8.5 Hz, H-1); HRMS (ES⁺) calcd for C₂₂H₃₂O₃Na (M + Na⁺)

m/e 367.2249, found m/e 367.2254; HPLC system H-20, t_R) 9

min, and system H-15, t_R) 10 min, >99% pure.

3,17â-Dibenzyloxy-11â-(2-tert-butoxyethyl)estra-1,3,5-

(10)-triene (63). A mixture of 11 mg (0.017 mmol) of 25, 44

mg (0.16 mmol) of 18-crown-6, and 18 mg (0.16 mmol) of

KOtBu in anhydrous toluene (1 mL) was stirred and heated

at 80 °C for 4 h. The reaction mixture was cooled to room

temperature, poured into H₂O (30 mL), and extracted with

CH₂Cl₂(3×, 30 mL). The combined organic extracts were dried

over Na₂SO₄and concentrated in vacuo. Purification by flash

chromatography on a 2 × 21 cm column of silica gel using 8:1

hexanes/EtOAc as eluent gave 3.2 mg (35%) of 63. Data for

3,17â-Dibenzyloxy-11â-(3-ethoxypropyl)estra-1,3,5(10)-

triene (70). Compound 70 was prepared from 67 (50 mg, 0.098

mmol) and EtI (100 µL, 1.2 mmol) as described for 42.

Purification by flash chromatography on a 2 × 17 cm column

of silica gel using 12:1 hexanes/EtOAc as eluent gave 28 mg

1

(53%) of 70. H NMR (400 MHz, CDCl₃) δ 1.03 (s, 3H, H-18),

1.19 (t, 3H, J ) 6.9 Hz, -CH₃), 2.65-2.84 (m, 2H, H-6), 3.33-

3.37 (m, 2H, OCH₂), 3.42-3.51 (m, 3H, H-17R, OCH₂), 4.60

(s, 2H, OBn), 5.04 (s, 2H, OBn), 6.71 (d, 1H, J ) 2.5 Hz, H-4),

6.80 (dd, 1H, J ) 8.6, 2.5 Hz, H-2), 7.08 (d, 1H, J ) 8.6 Hz,

H-1), 7.28-7.47 (m, 10H, Ar-H).

1

63: TLC, T-4, R_f0.62; H NMR (400 MHz, CDCl₃) δ 1.04 (s,

3H, H-18), 1.17 (s, 9H, tBu), 2.55 (dd, 1H, J ) 13.5, 1.2 Hz,

H-12â), 2.51-2.58 (m, 2H, H-9, 11), 2.68-2.82 (m, 2H, H-6),

3.30-3.48 (m, 2H, OCH₂), 3.48 (t, 1H, J ) 7.9 Hz, H-17R), 4.59

(s, 2H, OBn), 5.03 (s, 2H, OBn), 6.66 (d, 1H, J ) 2.6 Hz, H-4),

6.78 (dd, 1H, J ) 8.4, 2.6 Hz, H-2), 7.11 (d, 1H, J ) 8.4 Hz,

H-1), 7.27-7.44 (m, 10H, Ar-H).

11â-(3-Ethoxypropyl)estra-1,3,5(10)-triene-3,17â-diol (71,

E11-3,2_ether). Compound 71 was prepared from 70 (15 mg, 0.28

mmol) as described for 6. Purification by HPLC using system

H-1 gave 5 mg (50%) of 71 as a white solid. Data for 71: ¹H

NMR (400 MHz, CDCl₃) δ 0.94 (s, 3H, H-18), 1.19 (t, 3H, J )

6.9 Hz, -CH₃), 2.67-2.85 (m, 2H, H-6), 3.34-3.37 (m, 2H,

OCH₂), 3.42-3.47 (m, 2H, OCH₂), 3.72 (dd, 1H, J ) 8.9, 7.0

Hz, H-17R), 6.55 (d, 1H, J ) 2.8 Hz, H-4), 6.65 (dd, 1H, J )

8.4, 2.8 Hz, H-2), 7.04 (d, 1H, J ) 8.4 Hz, H-1); HRMS (ES⁺)

calcd for C₂₃H₃₄O₃Na (M + Na⁺) m/e 381.2406, found m/e

381.2403; HPLC system H-20, t_R) 10.2 min, and system H-15,

t_R) 8.8 min, >99% pure.

11â-(2-t-Butoxyethyl)estra-1,3,5(10)-triene-3,17â-diol (64,

E11-2,tBu_ether). Compound 64 was prepared from 63 (3.2 mg,

0.0058 mmol) as described for 40. Purification by HPLC using

system H-1 gave 1 mg (48%) of 64 after crystallization from

1

acetone-petroleum ether. Data for 64: TLC, T-5, R_f0.7; H

NMR (400 MHz, CDCl₃) δ 0.95 (s, 3H, H-18), 1.16 (s, 9H, tBu),

2.13 (dd, 1H, J ) 13.4, 1.7 Hz, H-12â), 2.51-2.59 (m, 2H, H-9,

11), 2.65-2.80 (m, 2H, H-6), 3.38 (m, 2H, OCH₂), 3.71 (dd, 1H,

J ) 9.0, 7.1 Hz, H-17R), 6.51 (d, 1H, J ) 2.7 Hz, H-4), 6.63

(dd, 1H, J ) 8.4, 2.7 Hz, H-2), 7.07 (d, 1H, J ) 8.4 Hz, H-1);

HRMS (ES⁺) calcd for C₂₄H₃₆O₃Na (M + Na⁺) m/e 395.2562,

found m/e 395.2563; HPLC system H-1, t_R) 15 min, and

system H-12, t_R) 11.8 min, >99% pure.

3-(3,17â-Dibenzyloxyestra-1,3,5(10)-trien-11â-yl)pro-

pyl Toluenesulfone (72). Compound 72 was prepared from

67 (242 mg, 0.473 mmol) as described for 25. Purification by

flash chromatography on a 3 × 17 cm column of silica gel using

4:1 hexanes/EtOAc as eluent gave 132 mg (42%) of 72: TLC,

1

T-6, R_f0.4; H NMR (500 MHz, CDCl₃) δ 0.93 (s, 3H, H-18),

2.20 (d, 1H, J ) 13.9 Hz, H-12â), 2.36-2.41 (m, 1H, H-11),

2.41 (s, 3H, -CH₃), 2.52 (dd, 1H, J ) 10.7, 4.3 Hz, H-9), 2.69-

2.82 (m, 2H, H-6), 3.45 (t, 1H, J ) 7.9 Hz, H-17R), 3.95-3.97

(m, 2H, -OCH₂), 4.57 & 4.54 (AB quartet, 2H, J_AB) 12.2 Hz,

OBn), 5.04 (s, 2H, OBn), 6.69 (d, 1H, J ) 2.5 Hz, H-4), 6.77

(dd, 1H, J ) 8.4, 2.5 Hz, H-2), 6.99 (d, 1H, J ) 8.4 Hz, H-1),

7.29-7.45 (m, 12H, Ar-H), 7.72 (d, 2H, J ) 8.3 Hz, Ar-H).

3,17â-Dibenzyloxy-11â-(3-isopropoxypropyl)estra-1,3,5-

(10)-triene (73). Compound 73 was prepared from 72 (30 mg,

0.045 mmol) and iPrOH (69 µL, 0.90 mmol) as described for

61. Purification by flash chromatography on a 2 × 17 cm

column of silica gel using 10:1 hexanes/EtOAc as eluent gave

12 mg (50%) of 73: TLC, T-6, R_f0.71;

11â-(3-Isopropoxypropyl)estra-1,3,5(10)-triene-3,17â-

diol (74, E11-3,iPr_ether). Compound 74 was prepared from 73

(12 mg, 0.023 mmol) in EtOH (4 mL) and EtOAc (2 mL) as

described for 40. Purification by flash chromatography on a 2

× 17 cm column of silica gel using 1:1 hexanes/EtOAc as eluent

gave 6.8 mg of 74. Further purification by HPLC using system

H-1 gave 6.1 mg (73%) of 74 as a white solid. Data for 74:

3,17â-Dibenzyloxy-11â-(2-(2-fluoroethoxy)ethyl)estra-

1,3,5(10)-triene (65). Compound 65 was prepared from 25 (38

mg, 0.59 mmol) and 2-fluoroethanol (69 µL, 2.3 mmol) as

described for 61. Purification by flash chromatography on a 2

× 17 cm column of silica gel using 6:1 hexanes/EtOAc gave 30

mg (94%) of 65: TLC, T-4, R_f0.5.

11â-(2-(2-Fluoroethoxy)ethyl)estra-1,3,5(10)-triene-3,17â-

diol (66, E11-2,2F_1ether). Compound 66 was prepared from 65

(30 mg, 0.056 mmol) in EtOH (4 mL) and EtOAc (4 mL) as

described for 40. Purification by flash chromatography on a 2

× 17 cm column of silica gel using 1:1 hexanes/EtOAc as eluent

gave 11 mg of 66. Further purification by HPLC using system

H-5 gave 8.2 mg (41%) of 66 as a white solid. Data for 66: ¹H

NMR (400 MHz, CDCl₃) δ 0.95 (s, 3H, H-18), 2.14 (dd, 1H, J

) 13.4, 1.7 Hz, H-12â), 2.55 (dd, 1H, J ) 11.1, 4.6 Hz, H-9),

2.59-2.64 (m, 1H, H-11), 2.66-2.84 (m, 2H, H-6), 3.53-3.57

(m, 2H, OCH₂), 3.58-3.62 & 3.65-3.69 (m, 2H, J_HF) 29.9

Hz, OCH₂), 3.72 (dd, 1H, J ) 8.9, 7.5 Hz, H-17R), 4.54 (dt,

2H, J ) 4.1, 4.1 Hz, J_HF) 47.8 Hz, CH₂F), 6.54 (d, 1H, J )

2.7 Hz, H-4), 6.64 (dd, 1H, J ) 8.3, 2.7 Hz, H-2), 7.07 (d, 1H,

C-11â-Analogues of Estradiols Result in SERMs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1445

1

TLC, T-3, R_f0.23; H NMR (400 MHz, CDCl₃) δ 0.93 (s, 3H,

Competitive Binding to Rat Cytosolic ER, Human

LBD-ERr, and Human LBD-ERr ERâ. Binding affinities

relative to E₂were performed in incubations with the ER

(ERR²⁴) in rat uterine cytosol. Female Sprague-Dawley rats

were castrated and sacrificed 24 h later. The uterus was

removed, homogenized in ice-cold TEGDMo buffer (10 mM

Tris, 1.5 mM Na₂EDTA, 10% (v/v) glycerol, 1.0 mM dithio-

threitol, 25 mM sodium molybdate, pH 7.4 at 4 °C), and

centrifuged at 105 000g for 45 min at 4 °C. The supernatant

(cytosol) was frozen on dry ice and stored at -80 °C until assay.

For assay, the cytosol was defrosted, diluted, and incubated

with 1 nM [³H]E₂in the presence and absence of nonradioac-

tive E₂, estrone, or the E₂-analogues over a range of concentra-

tions from 10^-12to 10^-6M. Incubations were carried out on

ice overnight, and bound radioactivity was separated from free

by adsorption with dextran-coated charcoal and quantified by

counting.³⁹Relative binding affinity (RBA) was determined by

analysis of the displacement curves by the curve-fitting

program Prism. For comparison, we measured the binding of

H-18), 1.11 (d, 3H, J ) 6.1 Hz, -CH₃), 1.12 (d, 3H, J ) 6.1

Hz, -CH₃), 2.23 (dd, 1H, J ) 13.5, 1.7 Hz, H-12â), 2.42-2.48

(m, 1H, H-11), 2.54 (dd, 1H, J ) 10.4, 4.2 Hz, H-9), 2.67-2.83

(m, 2H, H-6), 3.27-3.36 (m, 2H, OCH₂), 3.51 (septet, 1H, J )

6.1 Hz, CH(CH₃)₂), 3.71 (dd, 1H, J ) 9.0, 7.5 Hz, H-17R), 6.54

(d, 1H, J ) 2.5 Hz, H-4), 6.63 (dd, 1H, J ) 8.5, 2.5 Hz, H-2),

7.04 (d, 1H, J ) 8.5 Hz, H-1); HRMS (ES⁺) calcd for C₂₄H₃₆O₃-

Na (M + Na⁺) m/e 395.2562, found m/e 395.2554; HPLC system

H-1, t_R) 13.2 min, and system H-12, t_R) 12.7 min, >99%

pure.

3,17â-Dibenzyloxy-11â-(3-tert-butoxypropyl)estra-1,3,5-

(10)-triene (75). Compound 75 was prepared from 72 (77 mg,

0.116 mmol) and tBuOH (218 µL, 2.31 mmol) as described for

61. Purification by flash chromatography on a 2 × 17 cm

column of silica gel using 12:1 hexanes/EtOAc gave 35 mg

(53%) of 75. Data for 75: TLC, T-4, R_f0.62; ¹H NMR (400 MHz,

CDCl₃) δ 1.02 (s, 3H, H-18), 1.17 (s, 9H, tBu), 2.39 (dd, 1H, J

) 13.7, 1.3 Hz, H-12â), 2.42-2.48 (m, 1H, H-11), 2.54 (dd, 1H,

J ) 10.6, 4.1 Hz, H-9), 2.69-2.86 (m, 2H, H-6), 3.20-3.28 (m,

2H, OCH₂), 3.48 (t, 1H, J ) 7.9 Hz, H-17R), 4.57 & 4.60 (AB

quartet, 2H, J_AB) 12.2 Hz, OBn), 5.03 (s, 2H, OBn), 6.70 (d,

1H, J ) 2.8 Hz, H-4), 6.80 (dd, 1H, J ) 8.6, 2.8 Hz, H-2), 7.09

(d, 1H, J ) 8.6 Hz, H-1), 7.28-7.46 (m, 10H, Ar-H).

11â-(3-tert-Butoxypropyl)estra-1,3,5(10)-triene-3,17â-

diol (76, E11-3,tBu_ether). Compound 76 was prepared from

75 (10 mg, 0.019 mmol) as described for 40. Purification by

flash chromatography on a 1 × 10 cm column of silica gel using

1:1 hexanes/EtOAc as eluent gave 7.1 mg of 76. Further

purification by HPLC using system H-1 gave 4.5 mg (61%) of

76 as a white solid. Data for 76: TLC, T-4, 0.06; ¹H NMR (500

MHz, CDCl₃) δ 0.93 (s, 3H, H-18), 1.16 (s, 9H, tBu), 2.24 (dd,

1H, J ) 13.6, 1.8 Hz, H-12â), 2.42-2.48 (m, 1H, H-11), 2.54

(dd, 1H, J ) 10.3, 4.6 Hz, H-9), 2.67-2.82 (m, 2H, H-6), 3.20-

3.28 (m, 2H, OCH₂), 3.71 (t, 1H, J ) 8.4 Hz, H-17R), 6.54 (d,

1H, J ) 2.8 Hz, H-4), 6.64 (dd, 1H, J ) 8.5, 2.8 Hz, H-2), 7.04

(d, 1H, J ) 8.5 Hz, H-1); HRMS (ES⁺) calcd for C₂₅H₃₈O₃Na

(M + Na⁺) m/e 409.2719, found m/e 409.2719; HPLC system

H-1, t_R) 16.2 min, and system H-12, t_R) 12.9 min, >99%

pure.

all E₂-11â-ethers to the LBD of human ERR (M₂₅₀-V₅₉₅)

²³and

human ERâ (M₂₁₄-Q₅₃₀).²²The assay was performed overnight

at room temperature in competition with [³H]E₂in lysates of

Escherichia coli in which the LBDs are expressed as de-

scribed.⁴⁰The results, as RBAs compared to E₂, of all receptor

studies shown in Table 2, are from at least three separate

experiments performed in duplicate. RBAs represent the ratio

of the EC₅₀of E₂to that of the steroid analogue × 100 using

the curve-fitting program Prism to determine the EC₅₀.

Estrogenic Potency in Ishikawa Cells. The estrogenic

potency of the E₂-analogues was determined in an estrogen

bioassay, the induction of AlkP in human endometrial adeno-

carcinoma cells (Ishikawa) grown in 96-well microtiter plates,

as we have previously described.⁴The cells are grown in phenol

red free medium with estrogen-depleted (charcoal stripped)

bovine serum in the presence or absence of varying amounts

of the steroids, across a dose range of at least 6 orders of

magnitude. After 3 days, the cells are washed, frozen, thawed,

and then incubated with 5 mM p-nitrophenyl phosphate, a

chromogenic substrate for the AlkP enzyme, at pH 9.8. To

ensure linear enzymatic analysis, the plates are monitored

kinetically for the production of p-nitrophenol at 405 nm. For

antagonists, the effect (K_i) of each compound tested at a range

from 10^-6to 10^-12M was measured for the inhibition of the

action of 10^-9M E₂(EC₅₀∼ 0.2 nM). Each compound was

analyzed in at least three separate experiments performed in

duplicate. The K_iand RSA (RSA ) ratio of 1/EC₅₀of the steroid

analogue to that of E₂× 100) were determined using the curve-

fitting program Prism.

ERr-, ERâ-, and ERE-Transfected JAR Cells. The

specificity of the antiestrogenic activity relative to the ER

subtypes, ERR and ERâ, was determined in the human

choriocarcinoma JAR cell line transfected with plasmids

containing a consensus estrogen response element (ERE) fused

to a firefly luciferase reporter gene and separately with either

the expression vectors for human ERR or human ERâ exactly

as we described.⁵After transfection, the medium was changed

to phenol red free RPMI containing 10% dextran-coated

charcoal-treated calf serum. Twenty-four hours later, E₂or

E11-2,2_ether(10^-12-10^-6M) + E₂(1 nM) were added and the

cells were incubated for 12 h at 37 °C in 5% CO₂. The cells

were harvested in 10 mM Tris-HCl/10 mM EDTA/150 mM

NaCl and centrifuged for 4 min at 4000 rpm, supernatant was

removed, and cell pellets were lysed in lysis buffer 2 (Bio-Orbit,

Turku, Finland). Luciferase activity was measured using the

GenGlow system (Promega, Madison, WI).

Uterotrophic Stimulation in Immature Rats. All ani-

mal procedures were approved by the Yale University Insti-

tutional Animal Care and Use Committee. The uterotrophic

assay was performed in immature rats as described.⁴¹Female

Sprague-Dawley rats (n ) 6/group), 21-days-old, were injected

subcutaneously daily for 3 days with (total dose) E₂(20 ng),

E11-2,2_ether(100 ng and 1 µg), or a combination of the 2 doses

of E11-2,2_ether+ E₂in 0.1 mL of sesame oil. Control animals

3,17â-Dibenzyloxy-11â-(3-(2-fluoroethoxy)propyl)estra-

1,3,5(10)-triene (77). Compound 77 was prepared from 72 (69

mg, 0.10 mmol) and 2-fluoroethanol (61 µL, 1.0 mmol) as

described for 61. Purification by flash chromatography on a 2

× 17 cm column of silica gel using 6:1 hexanes/EtOAc as eluent

1

gave 24 mg (42%) of 77. Data for 77: TLC, T-2, R_f0.28; H

NMR (400 MHz, CDCl₃) δ 1.01 (s, 3H, H-18), 2.34 (dd, 1H, J

) 13.5, 1.4 Hz, H-12â), 2.42-2.47 (m, 1H, H-11), 2.54 (dd, 1H,

J ) 10.6, 4.0 Hz, H-9), 2.69-2.86 (m, 2H, H-6), 3.40-3.44 (m,

2H, OCH₂), 3.48 (dd, 1H, J ) 8.5, 7.3 Hz, H-17R), 3.59-3.61

& 3.66-3.68 (m, 2H, J_HF) 29.5, OCH₂), 4.59-4.48 & 4.58-

4.60 (m, 2H, J_HF) 47.7 Hz, CH₂F), 4.58 (s, 2H, OBn), 5.03 (s,

2H, OBn), 6.69 (d, 1H, J ) 2.7 Hz, H-4), 6.79 (dd, 1H, J ) 8.5,

2.7 Hz, H-2), 7.07 (d, 1H, J ) 8.5 Hz, H-1), 7.28-7.45 (m, 10H,

Ar-H).

11â-(3-(2-Fluoroethoxy)propyl)estra-1,3,5(10)-triene-

3,17â-diol (78, E11-3,2F_1ether). Compound 78 was prepared

from 77 (24 mg, 0.044 mmol) in EtOH (2 mL) and EtOAc (2

mL) as described for 40. Purification by flash chromatography

on a 2 × 17 cm column of silica gel using 1.5:1 hexanes/EtOAc

as eluent gave 13 mg of 78. Further purification by HPLC

using system H-4 gave 11 mg (66%) of 78 as a white solid.

1

Data for 78: TLC, T-5, R_f0.42; H NMR (400 MHz, CDCl₃) δ

0.95 (s, 3H, H-18), 2.24 (dd, 1H, J ) 13.7, 1.4 Hz, H-12â), 2.44-

2.50 (m, 1H, H-11), 2.56 (dd, 1H, J ) 11.0, 5.5 Hz, H-9), 2.68-

2.85 (m, 2H, H-6), 3.42-3.45 (m, 2H, OCH₂), 3.60-3.62 &

3.66-3.69 (m, 2H, J_HF) 29.4 Hz, OCH₂), 3.73 (dd, 1H, J )

9.0, 7.1 Hz, H-17R), 4.47-4.49 & 4.59-4.61 (m, 2H, J_HF) 47.7

Hz, CH₂F), 6.56 (d, 1H, J ) 2.8 Hz, H-4), 6.65 (dd, 1H, J )

8.6, 2.8 Hz, H-2), 7.04 (d, 1H, J ) 8.6 Hz, H-1); HRMS (ES⁺)

calcd for C₂₃H₃₃FO₃Na (M + Na⁺) m/e 399.2311, found m/e

399.2322; HPLC system H-4, t_R) 14 min, and system H-12,

t_R) 12.1 min, >99% pure.

1446 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5

Zhang et al.

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received the vehicle. On the fourth day, the animals were

killed, and the uteri were removed, dissected, blotted, and

weighed.

Tissue-Selective Effects in Ovariectomized Rats.

Sprague-Dawley rats (∼250 g) were ovariectomized and

starting that evening were injected daily with either 33 ng/kg

of E₂or E11-2,2_ether(36 or 180 ng) or vehicle alone (0.1 mL of

sesame oil) subcutaneously for 7 days. The following day the

animals were killed by exsanguination while under ether

anesthesia. Serum was obtained and total cholesterol concen-

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(Roche Diagnostics, Indianapolis IN). The uteri were dissected

and weighed. In a separate study, the ovariectomized rats were

injected with vehicle alone (control), E11-2,2_ether(180 ng) or

E11-2,2_ether(180 ng) + ICI 182,780 (250 µg) as above, and

serum cholesterol was determined.

Acknowledgment. We gratefully acknowledge the

excellent technical assistance of Toni Yeasky and Chris-

topher Forestiere. This work was supported in part by

NIH grants CA-37799 and HL-61342.

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JM049352X

Article Doi

Nonpolar and short side chain groups at C-11β of estradiol result in antiestrogens

DOI: 10.1021/jm049352x

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Article abstract of DOI:10.1021/jm049352x

Full text of DOI:10.1021/jm049352x

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