Synthesis and evaluation of B-, C-, and D-ring-substituted estradiol carboxylic acid esters as locally active estrogens

1886

J . Med. Chem. 2003, 46, 1886-1904

Syn th esis a n d Eva lu a tion of B-, C-, a n d D-Rin g-Su bstitu ted Estr a d iol Ca r boxylic

Acid Ester s a s Loca lly Active Estr ogen s

David C. Labaree,^†,‡J ing-xin Zhang,^†,‡Heather A. Harris,^§Craig O’Connor,^†Toni Y. Reynolds,^†and

Richard B. Hochberg*^,†

Department of Obstetrics and Gynecology, and Comprehensive Cancer Center, Yale University School of Medicine,

New Haven, Connecticut 06520, and Women’s Health Research Institute, Wyeth Research, 500 Arcola Road,

Collegeville, Pennsylvania 19426

Received October 1, 2002

We have synthesized derivatives of estradiol that are structurally modified to serve as “soft”

estrogens and act within a geographically limited area of the body; estrogens without systemic

action. We have previously shown with 16R-substituted analogues of estradiol that carboxylates

proximal to the steroid ring neither bind to the estrogen receptor nor activate estrogen-

responsive genes. However, when the carboxylic acid is masked as an ester, they bind to the

receptor and stimulate estrogenic responses. Enzymatic hydrolysis through nonspecific esterases

can inactivate these estrogens and thereby limit their area of action. Here, we describe our

continued studies to design “soft” estrogens by synthesizing carboxylic acid esters of estradiol

at the 7R-, 11â-, and 15R-positions in the steroid nucleus at which bulky substituents are

accommodated by the estrogen receptor. These compounds were tested for estrogen receptor

binding (estrogen receptors R and â), stimulation of an estrogen sensitive gene in Ishikawa

cells in culture, and as substrates for enzymatic hydrolysis. Likely candidates were tested in

in vivo assays for systemic and local estrogenic action. The biological studies showed that

regardless of the point of attachment, all of the short-chain carboxylic acids, C-1 to C-3, were

devoid of hormonal action, while many of the esters were estrogenic. The site on the steroid

nucleus had great influence on hormonal activity and esterase hydrolysis. Formate esters at

7R and 15R were good estrogens, but lengthening the chain to acetate dramatically decreased

hormonal activity. However, the 7R-formate esters were not enzymatically hydrolyzed. At 11â,

the acetate (methyl ester) was an effective estrogen, but increasing the chain length to

propionate dramatically reduced hormonal activity. In general, the length of the alcohol from

methyl to butyl had only a small effect on receptor binding, and as the size of the alcohol

increased, so did esterase hydrolysis. One exception was the 11â-acetate esters where increasing

the alcohol moiety from methyl to ethyl eliminated estrogenic activity (Ishikawa cells) without

affecting estrogen receptor binding. Several of the esters were tested in vivo, and two, the

methyl and ethyl esters of estradiol-15R-formate, appeared to have the requisite properties

(high local and low systemic activity) of superior “soft” estrogens.

In tr od u ction

most common structural feature of these “soft” drugs

are ester groups that can be hydrolyzed to inactive

carboxylic acids through the action of nonspecific and

ubiquitous esterases.⁷

Estrogen replacement therapy for menopausal women

is one of the most common therapeutic regimens. It is

prescribed for the relief of a number of menopausal

symptoms, including hot flashes, vaginal dyspareunia,

and the prevention of osteoporosis as well as heart

disease. However, it has been shown that estrogen

therapy and/or estrogen-progestin therapy is not with-

out its risks^1-4(see editorial⁵). It has been our goal to

synthesize a locally active estrogen that could be used

to treat vaginal dyspareunia without the risks involved

in systemic treatment. Therapeutic agents whose bio-

logical actions are limited to the regions to which they

are applied have been termed “soft drugs”.⁶These drugs

have geographically limited actions through rapid me-

tabolism into inactive products in tissues and blood. The

Recently, we described a series of compounds in which

analogues of estradiol (E₂) were modified at 16R with a

series of carboxylic esters to produce a locally active

estrogen that could be applied directly into and act

solely within the vagina, without producing systemic

effects.⁸While various preparations of estrogens are

currently available for direct vaginal application, they

are adsorbed from the vagina into the blood stream, and

thus, they act at sites throughout the body.^9-12Our

design for a locally active estrogen is based upon the

synthesis of analogues of estradiol containing carboxy-

lates in proximity to the steroid nucleus. These organic

acids have poor affinity for the estrogen receptor while

esters of these same carboxylic acids bind very well to

this receptor.⁸To be restricted to local action, these

potent estrogens would be rapidly inactivated by es-

terases. The 16R-carboxylates of E₂that we described

were designed and tested for the following character-

* To whom correspondence and reprint requests should be ad-

dressed. Tel: (203) 785-4001. Fax: (203) 737-4391. E-mail:

richard.hochberg@yale.edu.

^†Yale University School of Medicine.

^‡Both contributed equally to these studies.

^§Women’s Health Research Institute.

Published on Web 04/05/2003

Estradiol Carboxylic Acid Esters as Locally Active Estrogens

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1887

F igu r e 1. Abbreviation key for the E₂-carboxy esters. Ex-(m + 1),(n + 1): where x is the position in the steroid nucleus from

which the ester chain originates and the quantity (m + 1) is the number of carbon atoms in the acid and (n + 1), the alcohol

portion of the chain containing the ester. For example, E11-2,2, lower right in the figure, steroid position (x) is 11, m and n ) 1.

In the case of C-7, both epimers were prepared and their stereochemistry is provided within the abbreviation.

istics: affinity for the estrogen receptor; biological

activity in an in vitro estrogen sensitive model (endome-

trial cells in culture); as substrates for esterase hydroly-

sis; local action through in vivo estrogenic stimulation

of the vagina; systemic activity (uterotrophic action).

Two of these E₂analogues, the ethyl and 2′-monofluo-

rethyl esters of E₂-16R-formate, showed the requisite

properties with significant differences in their systemic

and local actions.

Our aim in the present study was to design a second

generation of local estrogens with increased estrogenic

potency leading to greater local action and yet decreased

systemic action. To this end, we synthesized carboxylic

esters of estradiol at positions in the steroid nucleus,

7R, 11â, and 15R (Figure 1), at which substituents are

known to be tolerated by the estrogen receptor.¹³In

addition, several 7â analogues were isolated during the

synthesis of the 7R analogues. These esters and car-

boxylic acids were tested for estrogenic potential (bind-

ing to the estrogen receptor; in vitro bioassay; in vivo,

vaginal and uterotrophic assays) and as substrates for

esterase enzyme(s).

duces a 1:5 mixture of the acid E7R-1,0 7 and E7â-1,0

13 and can be separated by reversed-phase HPLC

(Scheme 1). Esterification of 13 as above gave the esters

14 and 15 whose H NMR spectra have a ddd signal at

δ 2.62 ppm with J ) 6, 8.5, and 9 Hz for H-7R consistent

with its axial orientation.

1

The synthesis of the 7R-carboxymethyl analogues of

estradiol 19-21 is shown in Scheme 2. Aldehyde 5 was

cleanly reduced to the alcohol 16 using NaBH₄in

ethanol at 0 °C. That the stereochemistry of C-7 was

unchanged during the NaBH₄reduction of 5 to 16 is

indicated by the fact that reduction of 5 and 16 with

LiAlH₄in ether, conditions known not to affect epimer-

izable asymmetric centers,^18,19gives the same product

1

by inspection of the H NMR (data not shown). Tosy-

lation of 16 followed by cyanation gave 18, which was

hydrolyzed to the acid E7R-2,0 19. Esterification as

above gave E7R-2,1 20 and E7R-2,2 21.

The synthesis of the 11â-carboxymethyl analogues of

estradiol 32-34 is shown in Scheme 3. Estradiol was

protected with 3- and 17-benzyl ether groups and

oxidized with DDQ in methanol and dioxane to give 24.

Hydroboration and oxidation followed by further oxida-

tion with PCC with methodology used previously^18,20

gave the 11-ketone 26, which was converted to the 11-

methylene compound 27 by Peterson olefination.^21,22

Hydroboration/oxidation of 11-methylene-substituted

steroids occurs with delivery of the reagent to the less

stearically hindered R-face giving the 11â-hydroxym-

ethyl substituent.^23-25Olefin 27 was converted to 28 in

this manner. Tosylation of the hydroxyl group followed

by displacement with cyanide gave the nitrile 30, which

was hydrolyzed to the acid 31. Removal of the benzyl

groups produced E11-2,0 32. Esterification as above

gave E11-2,1 33 and E11-2,2 34.

Synthesis of the E11-1 formyl ester series was at-

tempted by oxidation of hydroxymethyl steroid 28 with

J ones reagent. However, oxidation went no further than

the aldehyde, which was stable to a variety of oxidants

(TPAP/NMO,²⁶H₂O₂, NaClO₂,²⁷m-CPBA, Ag₂O), and

only nonpolar degradation products were obtained with

KMnO₄, Cr₂O₃, and NaOCl.²⁸

Ch em istr y

The synthesis of the 7R-formyl ester anologues of

estradiol 8-12 is shown in Scheme 1. A cyano group is

introduced stereoselectively at the 7R-position of di-

enone 1 with diethylaluminum cyanide in THF.^14-16The

1

signal for H-7 in the H NMR of 2 appears as a ddd at

δ 3.01 with J ) 2, 4, and 5.6 Hz consistent with that of

an equatorial hydrogen. Aromatization of the A-ring of

2 with CuBr₂-LiBr in acetonitrile at reflux^15,17followed

by reduction of the cyano group of 3 with DIBAL-H in

toluene gave the R-aldehyde 4¹⁴along with removal of

the protecting groups. Reprotection with Ac₂O in pyri-

dine followed by J ones oxidation¹⁶of the aldehyde 5 gave

the carboxylic acid 6. Deprotection with MeOH/HCl (1:

1) at room temperature produced the acid E7R-1,0 7.

The esters 8-12 were prepared by reacting 7 with the

appropriate alcohols in the presence of SOCl₂or H₂SO₄.

1

The signal for H-7â in the H NMR spectrum of these

esters appears as a poorly resolved dd at 2.88 ppm with

J ) 3.6, 5.6 Hz consistent with that of an equatorial

hydrogen.

For the synthesis of the 7â-formyl analogues, hydroly-

sis of 7R-cyanoestradiol 3 in KOH/ethylene glycol pro-

The synthesis of the 11â-carboxyethyl analogues of

estradiol 39-41 is shown in Scheme 4 and uses an

intermediate 36 prepared with methodology used to

prepare similar 11â-alkyl-substituted steroids.²⁰Addi-

1888 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10

Labaree et al.

Sch em e 1^a

a

Key: (a) Et₂AlCN, THF (1 f 2); (b) CuBr₂, LiBr (2 f 3); (c) DIBAL, toluene (3 f 4); (d) Ac₂O, pyridine (4 f 5); (e) CrO₃, H₂SO₄,

acetone (5 f 6); (f) MeOH, aq HCl (6 f 7); (g) ROH, SOCl₂or H₂SO₄; (h) KOH, ethylene glycol, 120 °C (3 f 13).

Sch em e 2^a

a

Key: (a) NaBH₄, EtOH 0 °C (5 f 16); (b) pTsCl, pyridine (16 f 17); (c) NaCN, DMSO, 90 °C (17 f 18); (d) KOH, ethylene glycol, 150

°C (18 f 19); (e) ROH, SOCl₂(19 f 20, 21).

tion of allylmagnesium bromide to ketone 26 from the

less stearically hindered R-face yields the 11R-allylhy-

droxy steroid 35. Reduction with triethylsilane and BF₃‚

OEt₂occurs with inversion of C-11 giving the 11â-allyl

steroid 36. Hydroboration/oxidation of the terminal

olefin followed by J ones’ oxidation produced the acid 38

which was deprotected to yield E11-3,0 39. Esterifica-

tion as above gave E11-3,1 40 and E11-3,2 41.

The synthesis of the 15R-formyl ester analogues of

estradiol 49-54 is shown in Scheme 5 and employs

methodology used previously by Bernstein to prepare

15R-carboxyl substituted estradiol derivatives.^29,30Ketal

42³¹was protected as the 3-benzyl ether 43 and care-

fully deketalized with pTsOH in aqueous acetone at

room temperature to give enone 44. Conjugate addition

of NaCN in aqueous THF at reflux gave the 15â-cyano

steroid 45. Ketone reduction followed by nitrile hydroly-

sis gave the 15R-carboxylic acid 47 via epimerization of

the intermediate carboxamide. Hydrogenolysis of 47

with 5% Pd-C/H₂gave the acid 48, E151,0. The methyl,

ethyl, trifluoroethyl, n-propyl, isopropyl, and n-butyl

esters [E15-1,1 (49), E15-1,2 (50), E15-1,2 F₁(51), E15-

1,3 (52), E15-1,3i (53), and E15-1,4 (54)] were prepared

by reacting 48 with the appropriate alcohol in the

presence of a catalytic amount of H₂SO₄.

To support the assignment of stereochemistry at C-15,

the ester function of E15-1,2 (50) was converted to a

methyl group giving the known 3-methoxy-15R-methyl-

1,3,5(10)-estratriene-17â-ol 60³²as follows (Scheme 5).

Methylation of the phenolic hydroxyl group followed by

Estradiol Carboxylic Acid Esters as Locally Active Estrogens

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1889

Sch em e 3^a

a

Key: (a) (i) NaH, 22, DMF, THF, (ii) BnBr (22 f 23); (b) DDQ, MeOH, dioxane (23 f 24); (c) (i) catecholborane, LiBH₄, THF, (ii)

NaOH, H₂O₂; (d) PCC, CH₂Cl₂(25 f 26); (e) (i) Me₃SiCH₂MgCl, Et₂O, (ii) HCl, acetone (26 f 27); (f) pTsCl, pyridine (28 f 29); (g) NaCN,

DMSO, 90 °C (29 f 30); (h) KOH, ethylene glycol, 140 °C (30 f 31); (i) BCl₃, CH₂Cl₂, 0 °C (31 f 32); (j) BCl₃, ROH (32 f 33, 34).

Sch em e 4^a

a

Key: (a) allylmagnesium bromide, THF (26 f 35); (b) HSiEt₃, BF₃‚Et₂O, 0 °C (35 f 36); (c) (i) catecholborane, LiBH₄, THF, (ii)

NaOH, H₂O₂; (36 f 37); (d) CrO₃, H₂SO₄, acetone (37 f 38); (e) BCl₃, CH₂Cl₂, 0 °C (38 f 39); (f) BCl₃, ROH (39 f 40, 41).

protection of the 17â-hydroxyl group as the MOM-ether

gave 56. Reduction of the 15-ester with LiAlH₄, a

reagent known not to affect epimerizable asymmetric

centers^18,19gave the hydroxymethyl steroid 57, which

was tosylated giving 58. Reductive removal of the tosyl

group followed by deprotection of the 17-hydroxyl group

yielded 60 whose ¹H NMR was identical with that

reported in the literature.³²In addition, there is a triplet

procedure employed by Kojima to prepare 15R-car-

boxymethyltestosterone derivatives.³⁴Sodium diethyl-

malonate was added to enone 44 in a Michael reaction

to give mainly the R-epimer of 61. Ester hydrolysis

followed by decarboxylation produced only the 15R-

carboxymethyl steroid 63. Ketone reduction with NaBH₄

followed by deprotection with 5% Pd-C/H₂gave the acid

E15-2,0, 65. The esters E15-2,1 66 and E15-2,2 67 were

prepared by reacting 65 with the appropriate alcohol

in the presence of SOCl₂. Confirmation of the stereo-

chemistry at C-15 was provided by conversion of 67 to

the known 15R-allylestradiol 69³⁵(Scheme 6) by DIBAL

reduction to 68 followed by methyleneation with Nys-

tead reagent to 69. The ¹H NMR spectrum of 69 is

identical to that reported for 15R-allylestradiol. In

1

signal (J ) 10.2 Hz) for H-14R at δ 0.90 ppm in the H

NMR spectrum of 60, indicating a trans-diaxial rela-

tionship with both H-8 and H-15, and is in accord with

that seen with other 15R-substituted estradiol com-

pounds.³³

The synthesis of the 15R-carboxymethyl analogues of

estradiol 65-67 is shown in Scheme 6 and uses the

1890 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10

Labaree et al.

Sch em e 5^a

a

i

Key: (a) BnBr, K₂CO₃, Pr₂NEt, acetone (42 f 43); (b) pTsOH, acetone (43 f 44); (c) NaCN, THF, 75 °C (44 f 45); (d) NaBH₄,

MeOH, THF (45 f 46); (e) KOH, ethylene glycol 160 °C (46 f 47); (f) 5% Pd-C/H₂, EtOH (47 f 48); (g) ROH, H₂SO₄(48 f 49-54); (h)

i

CH₃I, K₂CO₃, acetone 60 °C (50 f 55); (i) MOMCl, PrEt₂N, toluene (55 f 56); (j) LiAlH₄, Et₂O (56 f 57); (k) pTsCl, pyridine, 4 °C (57

f 58); (l) LiEt₃BH, THF, 65 °C (58 f 59); (m) HCl-MeOH (59 f 60).

particular the signal for H-14R of 15R-allylestradiol 69

appears as a distinct triplet at δ 1.00 ppm with J ) 10.0

Hz, whereas the signal for H-14R of the â-allyl epimer

would be contained in a region of overlapping signals δ

1.1-1.5 ppm.³⁵In addition, this triplet signal at δ 1.00

structure/activity relationships.⁸Increasing the length

of the carboxyl chain from C-1 to C-3 (formate, acetate,

and propionate) progressively decreased binding to the

estrogen receptor. Accordingly, as the carboxylate chain

length increased the biological activity (Ishikawa cells)

decreased, whereas the length of the alcohol group in

the esters had a negligible affect on estrogen receptor

binding. Thus, within the limits of the small-chain

esters that were synthesized, the size of the alcohol was

not as important as the length of the carboxyl moiety.

Consequently, esters of exactly the same size but with

different carboxyl and alcohol groups had very different

affinities for the estrogen receptor, for example, E16-

1,3 and E16-2,2 or E16-3,1. Increasing the length of

either the alcohol or the carboxylic substituents in-

creased the rate of enzymatic hydrolysis. In studying

the potent 16R-formate esters, we noted that while the

effect of the length of the alcohol substituent on estrogen

receptor binding was minor, the biological effectiveness

of the various esters decreased markedly as the alcohol

was lengthened greater than ethyl. Since longer alcohol

moieties do not appreciably affect estrogen receptor

binding but are hydrolyzed faster, it is likely that the

decrease in biological estrogenic activity is due to the

increase in esterase hydrolysis. Therefore, esterase

susceptibility of these compounds is an important

1

ppm is present in the H NMR spectrum of each of the

15R-carboxymethyl steroids 65-67.

The synthesis of the 15R-carboxyethyl analogues of

estradiol 77-79 is shown in Scheme 7 and is based on

methodology used previously by Bojack et al.³³and

Dionne et al.³⁵to prepare 15R-allylestradiol. The 1,

2-addition of allylmagnesium chloride to enone 44 in

THF at 0 °C gave 70 as the only isomer. Anionic oxy-

Cope rearrangement with KH and 18-crown-6 in THF

produced exclusively the 15R-allylestrone 71. Reduction

of the 17-ketone followed by acetylation of the resulting

17â-alcohol gave 73. Hydroboration of 73 followed by

oxidation with trimethylamine N-oxide in diglyme

produced the alcohol 74, which was oxidized with CrO₃-

H₂SO₄to give the acid 75. Saponification of the acetate

group followed by hydrogenolysis of the benzyl group

gave E15-3,0 77. Esterification of 77 with the appropri-

ate alcohol and SOCl₂gave E15-3,1 78 and E15-3,2 79.

Resu lts a n d Discu ssion

In our previous study of 16R-carboxylate esters of E₂,

several general observations could be made about their

Estradiol Carboxylic Acid Esters as Locally Active Estrogens

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1891

Sch em e 6^a

a

Key: (a) (i) NaH, diethyl malonate, THF, (ii) 44 (44 f 61R,â); (b) NaOH, EtOH, H₂O (61 f 62); (c) diglyme, 162 °C (62 f 63); (d)

NaBH₄, EtOH (63 f 64); (e) 5% Pd-C/H₂, EtOH (64 f 65); (f) SOCl₂, ROH, 40 °C (65 f 66,67); (g) DIBAL, toluene, -60 °C (67 f 68);

(h) Nystead reagent, BF₃‚OEt₂, THF (68 f 69).

Sch em e 7^a

a

Key: (a) allylmagnesium chloride, THF, 0 °C (44 f 70); (b) KH, THF, 18-crown-6 (70 f 71); (c) NaBH₄THF (71 f 72); (d) Ac₂O,

pyridine (72 f 73); (e) (i) BH₃‚THF, THF, (ii) Me₃NO, 150 °C (73 f 74); (f) CrO₃-H₂SO₄, acetone, 0 °C (74 f 75); (g) KOH, MeOH, 55

°C (75 f 76); (h) 5% Pd-C/H₂, EtOH (76 f 77); (i) SOCl₂, ROH (77 f 78, 79).

determinant in their estrogenic action, and therefore,

in their ability to act as “soft” estrogens.

7r- a n d 7â- Su bstitu tion . As can be seen in Table

1, the 7R-formate derivatives appear to have the req-

uisite estrogenic characteristics that are desired for a

“soft” estrogen. The carboxylate E7R-1,0 has very poor

affinity for the estrogen receptor and shows almost no

activity in the estrogen bioassay with cultured Ishikawa

cells. As desired, the methyl and ethyl esters bind to

the estrogen receptor with affinities equal to or greater

In this study, we synthesized 4 different families of

carboxylic acid analogues of E₂at 7R, 7â, 11â, 15R and

their esters and evaluated them as “soft” estrogens in

several different types of assays designed to measure

their inherent estrogenic potency as well as to dif-

ferentiate their systemic and local actions.

1892 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10

Ta ble 1. Estrogenic Properties of E₂-alkyl Esters

Labaree et al.

dramatically: E7R-2,1 and E7R-2,2 were hydrolyzed at

rates almost comparable to E16-1,2 (30% and 70%

respectively). However, like the 16R- analogues, their

affinity for the estrogen receptor and their biological

activity is exceedingly low. The 7â-formate esters E7â-

1,1 and E7â-1,2 are also not hydrolyzed, and more

importantly, they are very poor estrogens.

estrogen

receptor

(RBA^b)

Ishikawa

cell

esterase

(RHA^d)

compd^a

AlkP(RSA^c)

E₂

E₁

E16-1,2

100

9 ( 6

100

4 ( 2

14 ( 4

<0.1

9 ( 3

100

E7R-1,0 (7)

E7R-1,1 (8)

E7R-1,2 (9)

E7R-1,2F₁(12)

E7R-1,3 (10)

E7R-1,4 (11)

E7R-2,0 (19)

E7R-2,1 (20)

E7R-2,2 (21)

E7â-1,1 (14)

E7â-1,2 (15)

E11-2,0 (32)

E11-2,1 (33)

E11-2,2 (34)

E11-3,0 (39)

E11-3,1 (40)

E11-3,2 (41)

E15-1,0 (48)

E15-1,1 (49)

E15-1,2 (50)

E15-1,2F₁(51)

E15-1,3 (52)

E15-1,3i (53)

E15-1,4 (54)

E15-2,0 (65)

E15-2,1 (66)

E15-2,2 (67)

E15-3,0 (77)

E15-3,1 (78)

E15-3,2 (79)

0.3 ( 0.3

40 ( 25

15 ( 12

2 ( 1

1 ( 0.7

0.6 ( 0.3

0

0.8 (0.3

0.2 ( 0.03

0.4 ( 0.1

0.4 ( 0.3

0.3 ( 0.3

16 ( 10

e

11â-Su bstitu tion . We synthesized and tested the

11â-acetate (E11-2) and propionate (E11-3) analogues

of E₂. As can be seen in Table 1, the acetate-substituted

esters (E11-2,1 and E11-2,2) are hydrolyzed by the

esterase preparation at about the same rate as E16-

1,2. The propionate analogue E11-3,1 is also cleaved at

about the same rate (RHA ) 64%), while E11-3,2 is

hydrolyzed very rapidly (RHA ) 542%). As we antici-

pated, both E11-2,0 and E11-3,0 are poor ligands for

the estrogen receptor, and consequently, they both have

low activity in the Ishikawa cell assay. While this

activity is low it is nevertheless significant. This re-

sidual activity in both assays is higher than any of the

other parent carboxylates, formic acid derivatives at 7R,

15R, and 16R. E11-2,1 is a good ligand for the ER (RBA

of 24%) with high biological activity in the Ishikawa

assay (RSA 16%). However, the results of the Ishikawa

assay with E11-2,2 were unexpected. This ester has a

very high binding affinity in the estrogen receptor assay,

RBA ) 45%, which is much higher than all of the other

compounds that we tested in this study and yet it

demonstrates only very little estrogenic activity in the

Ishikawa assay. The esters of the propionate analogue,

E11-3,1 and E11-3,2, also have relatively good affinity

for the estrogen receptor, RBA ) 18% and 25%, respec-

tively, and they too show low activity in the Ishikawa

cell assay. This discrepancy between receptor binding

and estrogenic activity appears not to be related to rapid

hydrolysis to the parent carboxylates since similar

esters at 16R with lower affinities for the estrogen

receptor and similar or much more rapid rates of

enzymatic hydrolysis, have relatively high activity in

the Ishikawa cell assay.⁸Preliminary studies indicate

that these compounds are strong antiestrogens, and

experiments are currently underway in this laboratory

to uncover the underlying mechanism.

12 ( 4

10 ( 4

3 ( 0.3

4 ( 1

0

9 ( 2

0.2 ( 0.2

3 ( 0.8

1 ( 0.2

0.1 ( 0.1

0.4 ( 0.4

0.3 ( 0.2

24 ( 8

45 ( 19

0.2 ( 0.2

18 ( 3

25 ( 6

0

30 ( 1

70 ( 5

0

106 ( 17

152 ( 15

e

0

64 ( 5

542 ( 84

20 ( 10

25 ( 12

8 ( 3

11 ( 5

18 ( 10

3 ( 0.2

2 ( 1

1.7 ( 0.1

2.3 ( 0.1

4.5 ( 0.1

7.8 ( 0.4

0.4 ( 0.1

7 ( 0.5

17 ( 5

5 ( 4

0.3 ( 0.06

0.7 ( 0.4

0.2 ( 0.2

1 ( 1

8 ( 2

<0.1

5 ( 3

31 ( 6

71 ( 7

2 ( 0

0.4 ( 0.5

0

0.8 ( 0.1

<0.1

248 ( 26

674 ( 14

a

Abbreviations are in Figure 1, with examples as follows. E15-

2,0 is the 15R-propionic acid analogue of E₂. E15-2,1 is the methyl

ester, 3i, the propyl ester, etc. The stereochemistry is not assigned

in the abbreviations C-11, C-15, and C16; they are 11â, 15R, and

b

16R. RBA is the relative binding affinity in the ER assay, where

E₂) 100. ^cRSA is the relative stimulatory activity in the induction

of alkaline phosphatase (AlkP) activity in the Ishikawa estrogen

d

bioassay, where E₂) 100. RHA is the relative hydrolytic activity

in the esterase assay with hepatic microsomes in comparison to

E16-1,2. ^eThese compounds produced a small estrogenic response

at high concentrations, reaching a plateau at a value of 10-20%

of that produced by E₂. The dash (-) indicates not done. All values

are ( SD.

than estrone (E₁), and for these types of E₂-esters, they

have very high activity in the Ishikawa cell assay.

However, they are not hydrolyzed by the nonspecific

esterase from rat hepatic microsomes. They are com-

pletely stable to enzymatic hydrolysis even after incuba-

tion with esterase preparations for 4 h, conditions under

which the standard E16-1,2 was hydrolyzed completely

in 2 min (not shown). In an attempt to increase the rate

of esterase hydrolysis, we synthesized 3 other esters of

E7R-1; lengthening the alcohol moiety, E7R-1,3 and

E7R-1,4; or employing fluorine in the alcohol E7R-1,-

2F₁. In our previous study, both strategies increased the

rate of E16-1 ester hydrolysis without having deleteri-

ous effects on their estrogenic potency.⁸However, as can

be seen in Table 1, neither the insertion of fluorine nor

the extension of the alcohol substituent to C-3 or C-4

produced compounds that were hydrolyzed appreciably,

and in contrast to the 16R-formate esters their estro-

genic potency decreased dramatically. In the case of the

16R-esters, lengthening the carboxylate group from

formate to acetate increased esterase hydrolysis. This

tactic did increase the hydrolysis of the 7R-2 esters

15r-Su bstitu tion . In addition to the above B- and

C-ring-substituted esters, we also synthesized a series

of D-ring carboxylates and esters at C-15R. Like the E7

and E11 series, the E15-3 (propionate) esters are very

poor ligands for the estrogen receptor, and consequently,

they are devoid of estrogenic activity in the Ishikawa

cell assay. While the C15-2 (acetate) analogues were

somewhat better ligands for the estrogen receptor than

the E15-3 esters, with RBAs for E15-2,1 and E15-2,2 of

5% and 2%, respectively, their biological activity in the

Ishikawa bioassay is low. The E15-1 compounds (for-

mate) have very good estrogenic potential. The formate

analogue E15-1,0 does not bind to the estrogen receptor,

nor does it have estrogenic activity in the Ishikawa cell

assay. However, as would be desired for “soft” hormones,

the esters E15-1,1 and E15-1,2 are very potent ligands

for estrogen receptor (RBA ) 20% and 25%) with very

good activity in the Ishikawa cell assay (RSA ) 11%

and 18%). However, the rate of enzymatic hydrolysis is

relatively slow for both (RHA: E15-1,1 ) 1.7% and E15-

1,2 ) 2.3%). As with the 7R-analogues, we attempted

Estradiol Carboxylic Acid Esters as Locally Active Estrogens

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1893

Ta ble 2. Binding of E₂-Alkyl Esters to the LBD of Human

ERR and ERâ

Ta ble 3. In Vivo Estrogenic Action: Systemic (Uterotrophic)

and Local (Vaginal) Activity^a

uterotrophic

effect

vaginal

effect

compd

ERR^a

ERâ^a

ERR/ERâ

E₂

100

9 ( 2

1

100 µg dose uterotrophic 50 ng dose

vaginal

compd

(ng E₂equiv) R.A. × 10^{3 b}(pg E₂equiv) R.A. × 10^{3 b}

E7R-1,2 (9)

E11-2,2 (34)

E11-3,2 (41)

E15-1,2 (50)

E16-1,2

11 ( 2

66 ( 11

66 ( 3

22 ( 5

27 ( 6

1 ( 0.1

1 ( 0.04

1 ( 0.4

3 ( 0.6

95 ( 47

70 ( 14

64 ( 7

7 ( 2

E16-1,2F₁

2^c

0.02

0.11

0.04

0.07

n.s.

21^c

50^c

0.42

1.0

0.44

0.42

0.38

0.42

E7R-1,1 (8) 10.5 (10-11)

E7R-1,2 (9)

E11-2,1 (33)

4 (3.5-4.4)

22^c

0.3 ( 0.1

6.5^c

21^c

E15-1,1 (49) n.s. (0-1.3)

E15-1,2 (50) n.s. (0.7-3)

19 (15-23)

21 (18-25)

a

RBA of the indicated ester compared to E₂. Values are ( SD.

The inhibition of the binding of [³H]E₂in lysates of E. coli in which

the LBD of human ERR and ERâ were separately expressed.

Abbreviations are in Table 1. LBD is the ligand binding domain.

n.s.

a

In the uterotrophic assay, the results are compared to the

effect of 5 ng (total dose administered over 3 days) of E₂injected

subcutaneously in immature rats; in the vaginal assay the results

are compared to 50 pg of E₂administered vaginally to ovariecto-

mized adult mice. In the uterotrophic assays n ) 5, and in the

vaginal assays n ) 5-6. Values in parentheses show the range.

to increase the hydrolytic rate by introducing fluorine

(E15-1,2F₁) or by lengthening the alcohol moiety of the

ester (E15-1,3 and E15-1,4). While the hydrolytic rates

do increase, the estrogenic activity of these three

compounds decreases precipitously when compared to

the methyl or ethyl esters. Steric factors are important

determinants in the rate of enzymatic hydrolysis: the

isopropyl ester E15-1,3i has an RHA ) 0.3%, which is

much slower than the propyl ester E15-1,3, RHA ) 7.

Again, lengthening the carboxylic acid chain increases

the rate of hydrolysis; compare the RHA of E15-3 >

E15-2 > E15-1 esters containing the same alcohols.

However, as discussed above, the esters in the E15-2

and E15-3 series have low affinity for the estrogen

receptor and poor estrogenic action.

Bin d in g to ERr a n d ERâ. In addition to the

classical estrogen receptor, now called estrogen receptor

R (ERR), there is another subtype of estrogen receptor,

termed ERâ. These estrogen-activated transcription

factors are expressed differently in various tissues, and

although they both bind E₂avidly, they have a some-

what different affinity for other estrogens.^36,37Various

substitutions at 16R of E₂have a profound differential

effect on the binding of the two estrogen receptor

subtypes, with preferential binding to ERR.^8,38In this

study, we investigated the relative binding of represen-

tative ethyl esters of the analogues at 7R, 11â, and 15R

(E7R-1,2, E11-2,2, and E15-1,2) to the ligand binding

domain (LBD) of ERR and ERâ. For comparison, E16-

1,2, which is ERR specific,⁸was also included in these

experiments. As can be seen in Table 2, of the five

estrogens, E7R-1,2, E11-2,2, and E11-3,2 bound about

equally to ERR and ERâ, while E15-1,2 showed a small

selectivity, about 3-fold for ERR. Again, E16-1,2 was a

highly selective ligand with about a 95-fold preference

for ERR.

In Vivo Stu d ies. Several of the analogues were

tested for estrogenic potency in in vivo assays for

systemic (uterotrophic assay) and local (vaginal assay)

action. In both of these assays, the esters were compared

to E₂; 50 pg of E₂in the vaginal assay (within the dose

range that produces a linear response) and 5 ng of E₂

in the uterotrophic assay (the minimum dose that we

found reproducibly produces a statistically significant

uterine stimulation). The objective of this study was to

determine which compounds show the greatest dif-

ferential between local (high) and systemic (low) action.

The results are in Table 3. E7R-1,1 and E7R-1,2, have

relatively high activity in the vaginal assay. However,

both are also very active in the uterotrophic assay.

Likewise, E11-2,1, which is estrogenically active in the

b

R.A. activity relative to E₂. n.s. ) not significantly different from

the control. Except where noted, each compound was assayed in

three different experiments. ^cData are from a single experiment.

Ishikawa assay, is also estrogenic in the vaginal assay,

and it too is uterotrophic with an activity even greater

than E7R-1,2. The E15-1,1 and E15-1,2 analogues are

estrogenic in vitro and in the vaginal assay (50 ng,

equivalent to approximately 20 pg of E₂sabout the same

potency as E7R-1,2 and E11-2,1). However, unlike the

other three analogues tested in vivo, neither of these

15R-substituted estrogens produced a statistically sig-

nificant uterotrophic response at the 100 µg dose. Both

were slightly estrogenic at a dose of 300 µg, producing

a statistically significant stimulation (P < 0.05), E15-

1,1 and E15-1,2, equivalent to 4 and 5 ng of E₂,

respectively. For comparative purposes, in one of the

uterotrophic assays with E15-1,1 and E15-1,2 we in-

cluded a 100 µg dose of the E16-1,2F₁group. This

compound showed the best differential in comparing

local vs systemic estrogenic effects in our previous

study.⁸In this assay, 100 µg of E16-1,2F₁produces a

statistically significant (P < 0.01) uterotrophic stimula-

tion equivalent to 2 ng of E₂. This is approximately what

was observed previously, although in those experiments

the standard deviations were higher and the stimulation

was not statistically significant.

The relatively high estrogenic potency of the 7R-

carboxy esters in both types of in vivo assays is easily

explained because these esters are very active in the

estrogen receptor binding assay and the Ishikawa cell

bioassay and because they cannot be enzymatically

hydrolyzed. The esterase enzyme apparently cannot

attack the formate esters at 7R (or 7â) due to steric

constraints of the proximal steroid ring system. Length-

ening the carboxylate by one methylene group to E7R-2

allows esterase access to the ester function, but the E7R-

2,1 and E7R-2,2 have very low activity in estrogen

receptor and Ishikawa assays. It is evident that while

the methyl and ethyl formate esters (E7R-1,1 and E7R-

1,2) have the requisite estrogenic action, their resistance

to enzymatic hydrolysis made it predictable that they

would be estrogenic in the systemic assay. Conse-

quently, they cannot act as local estrogens since they

are not inactivated by hydrolysis to E7R-1,0.

Our previous studies showed that esters of carboxylic

analogues of E₂at C-16R that have high estrogenic

potential (estrogen receptor binding and Ishikawa cell

stimulation) and rapid esterase hydrolysis (E16-1,2 and

E16-1,2F₁) generate a large differential between local

1894 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10

Labaree et al.

and systemic estrogenic activity in vivo.⁸In this view,

analogues such as E7R-1 that have high estrogenic

potential but are not enzymatically hydrolyzed do not

provide this divergence since they have both high local

activity as well as high systemic action. Thus, the

results of the studies with the E7R and E16 analogues

support the rationale behind the design of a “soft”

estrogen.

low, not measurable, but as discussed above the hy-

drolysis of the 15-esters by esterase is relatively slow.

However, this rate of hydrolysis is evidently sufficient

to hydrolytically deactivate these E15-1 esters. Ad-

ditionally, the rate of esterase hydrolysis of the E₂-

analogues measures only one of the potential catabolic

routes of metabolism, albeit the one designed to play

the major role. It is well-known that the major secretory

estrogen, E₂, is metabolized by a large number of

catabolic enzymes. While in the case of the E₂analogues

the esterase enzyme probably plays the most important

role, there are other enzymatic routes that also inacti-

vate these steroids. Substituents on E₂analogues are

known to affect these enzymes and play an important

role in their metabolic clearance. For example, substit-

uents at C-11 protect steroidal estrogens from metabo-

lism, specifically from the metabolically important

2-hydroxylase⁴⁰and thus, such substituents at C-11

have a major impact, decreasing metabolic clearance

and increasing potency.⁴¹In this view, substitution at

11â as in E11-2,1 protects the estrogen from other

catabolic enzymes, thereby extending the estrogenic

effect of the parent E11-2,0. This would result in a more

systemically potent estrogen. Conversely, substitution

at 15R could increase metabolism and therefore decrease

the biological t_1/2of the E15-1 analogues. Thus, in-

creased metabolism in concert with enzymatic hydroly-

sis would eliminate systemic activity.

Nevertheless, regardless of the reason, the 15R-alkyl

esters E15-1,1 and E15-1,2 have the characteristics of

a “soft” estrogen. Interestingly, E15-1,1 and E15-1,2 are

as potent in stimulating a vaginal response as E16-1,-

2F₁, the best compound in our previous study.⁸However,

as discussed above, E15-1,1 and E15-1,2 were inactive

in the uterotrophic assay, in contrast to E16-1,2F₁,

which produced a small but statistically significant

response. Thus, the E15-esters have an improved local

to systemic estrogenic profile. E15-1,1 and E15-1,2 have

excellent potential for being useful “soft” therapeutic

agents for the local treatment of estrogen deprivation.

However, the analogues E11-2,1, E15-1,1, and E15-

1,2 do not appear to behave according to this model.

E11-2,1 is a good ligand for the ER with an RBA

considerably higher than estrone; it is also more than

four times as active as estrone in the Ishikawa assay,

and therefore, as expected, it is a relatively potent

estrogen in the vaginal assay. As this ester is cleaved

fairly rapidly (RHA ) 106%) in the esterase assay, it

might be surprising that it is active in the systemic

uterotrophic assay. It is even more active than E7R-1,2,

which is not hydrolyzed by esterase. Additionally, the

results with E15-1,1 and E15-1,2 are unexpected. Both

are highly potent estrogens in receptor binding and in

the Ishikawa assay, more potent than either E₁or E16-

1,2. Consequently, they are very active in the vaginal

assay. Since they are cleaved by esterase(s) at a

relatively low rate, RHAs of about 2%, our model

would predict them to have a relatively high systemic

(uterotrophic) action. However, neither of these 15R-

alkyl esters produce a statistically significant estrogenic

response in the uterus at the 100 µg dose. As described

above, the systemic effect of the 15R-analogues is lower

than that produced by E16-1,2F₁, which we previously

found shows the greatest differential action. Thus, the

uterotrophic activity of E15-1,1 and E15-1,2 was unusu-

ally low for all of these E₂-alkyl esters.

The fact that esters such as E11-2,1, E15-1,1, and

E15-1,2 show such divergence between the Ishikawa

assay (where they are almost as potent as E₂) and the

uterotrophic assay (where they are either inactive or

almost inactive) demonstrates their unusual suscepti-

bility to catabolism, likely through esterase hydrolysis.

This discrepancy between the uterotropic and Ishikawa

assays is strong evidence that the esters of the E₂-

carboxylates are acting as labile estrogens, since gener-

ally the potency of estrogens in the Ishikawa assay

closely mirrors in vivo activity.³⁹The high systemic

activity of E11-2,1 even with its high esterase hydrolysis

and (conversely) the low systemic activity of E15-1,1 and

E15-1,2, despite their low enzymatic hydrolysis, appear

to contradict the “soft” estrogen model. How can estro-

gens that are enzymatically hydrolyzed at low rates

(E15-1,1 and E15-1,2) seem to have the qualities of local

estrogens, while an estrogen that is rapidly cleaved

(E11-2,1) has such high systemic action? The answer

for the high residual activity of E11-2,1 results from the

relatively high estrogen receptor binding and Ishikawa

bioactivity of the parent carboxylate, E11-2,0. As dis-

cussed above both E11-2,0 and E11-3,0 showed measur-

able estrogen receptor binding and Ishikawa bioactivity.

Although the potency of E11-2,0 in both systems is less

than 1% of estradiol, it is considerably higher than all

of the other carboxylates at C-7, -15, and -16. Appar-

ently, E11-2,0 is of sufficient potency to produce a

systemic estrogenic response at high dose. In contrast,

the RBA and RSA of the carboxylate, E15-1,0, is very

Exp er im en ta l Section

1

Gen er a l Meth od s. H NMR spectra were recorded with a

Bruker AM500 and chemical shifts are reported relative to

residual CHCl₃(7.27 ppm) or DMSO (2.5 ppm). Purification

by flash chromatography was performed according to the

procedure of Still⁴²using 230-400 mesh silica gel (EM Science,

Darmstadt Germany). High-resolution mass spectra were

obtained by electrospray ionization on a Micromass Q-Tof

spectrometer by Dr. Walter J . McMurray at the Yale Univer-

sity Comprehensive Cancer Center using either PEG as an

internal standard with NH₄OAc or NaI as an internal stan-

dard. Elemental analyses were performed by Schwarzkopf

Micro Analytical Laboratory, Woodside, NY. The computer

program Prism was purchased from GraphPad Software, Inc.

(San Diego, CA). The cell culture reagents were obtained from

Gibco-BRL (Grand Island, NH). Unless otherwise indicated,

solvents (analytical or HPLC grade) and reagents were used

as supplied, and all reactions were carried out under nitrogen.

Ch r om a togr a p h ic System s. 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, hexanes/EtOAc (2:1); T-2,

hexanes/EtOAc (1:1); T-3, CHCl₃/MeOH (5:1); T-4, hexanes/

EtOAc (4:1). Analytical high-performance liquid chromatog-

raphy (HPLC) was performed on a Waters 600E system

(Waters Co. Milford MA) equipped with

a 484 variable

wavelength detector set at 280 nm using the following columns

Estradiol Carboxylic Acid Esters as Locally Active Estrogens

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1895

and systems. Ultrasphere ODS column (5 µm, 10 mm × 25

cm, Altex Scientific Operations Co.) with the following solvent

systems at 3 mL/min: H-1, HOAc/CH₃CN/H₂O (0.15:25:74.85);

H-2, CH₃OH/H₂O (60:40); H-3, CH₃CN/H₂O (40:60); H-4,

HOAc/CH₃CN/H₂O (0.13:35:64.87); H-5, CH₃CN/H₂O (50:50);

H-6, HOAc/CH₃CN/H₂O (0.14:30:69.86); H-7, HOAc/CH₃CN/

H₂O (0.12:40:59.88); H-8, CH₃CN/H₂O (45:55). LiChrospher

100 Diol column (5 µm, 4.6 mm × 25 cm, EM Science) with

the following solvent systems at 1 mL/min: H-9, CH₂Cl₂/ⁱPrOH

(90:10); H-10, CH₂Cl₂/ⁱPrOH (99:1); H-11, CH₂Cl₂/ⁱPrOH (98:

2); H -12, CH₂Cl₂/ⁱPrOH (95:5); H -13, HOAc/CH₂Cl₂/ⁱPrOH

(0.094:94.25:5.65); H-14, HOAc/CH₂Cl₂/ⁱPrOH (0.1:6:93.9); H-15,

CH₂Cl₂; H-16, CH₂Cl₂/isooctane (80:20); H-17, HOAc/CH₂Cl₂/

ⁱPrOH (0.1:3:96.9); H-18, CH₂Cl₂/isooctane (90:10). Protein I-60

column (7.8 mm × 30 cm, Waters Co.) with the following

solvent systems at 3 mL/min: H-19, HOAc/ⁱPrOH/CH₂Cl₂(0.1:

6:93.9); H-20, HOAc/ⁱPrOH/CH₂Cl₂(0.1:5.99:93.91); H-21, CH₂-

Cl₂; H-22, HOAc/ⁱPrOH/CH₂Cl₂(0.1:3:96.9). Beckman System

Gold HPLC system (Beckman Coulter, Inc. Fullerton, CA)

consisting of a model 126 solvent module and a model 168

diode array detector set at 280 nm using a Microsorb-MV C18

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

ments) in the following solvent systems at 1 mL/min: H-23,

HOAc/CH₃CN/H₂O (0.15:25:74.85); H-24, CH₃CN/H₂O (35:65);

H-25, CH₃CN/H₂O (40:60); H-26, CH₃CN/H₂O (50:50); H-27,

HOAc/CH₃CN/H₂O (0.13:35:64.87); H-28, CH₃CN/H₂O (45:55);

H-29, HOAc/CH₃CN/H₂O (0.14:30:69.86); H-30, HOAc/CH₃CN/

H₂O (0.13:33:66.87).

17â-Acetoxy-7r-cya n oestr -4-en e-3-on e (2). To a solution

of 1.96 g of 6-dehydro-19-nortestosterone acetate 1 (6.2 mmol)

in THF (100 mL) was added 23 mL of diethylaluminum

cyanide (1.0 M solution in toluene, Aldrich). The reaction

mixture was stirred at room temperature for 1 h, poured into

cold aqueous NaOH solution (0.5 N, 300 mL), and extracted

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

washed with H₂O, dried over Na₂SO₄, and evaporated. Puri-

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

silica gel using 1:1 hexanes/EtOAc as eluent gave 1.56 g (74%)

of 2.¹⁴Data for 2: ¹H NMR (400 MHz, CDCl₃) δ 0.88 (s, 3H,

H-18), 2.06 (s, 3H, OAc), 3.01 (ddd, 1H, J ) 2.4, 3.6, 5.6 Hz,

H-7â), 4.70 (dd, 1H, J ) 8.8, 7 Hz, H-17R), 5.98 (s, 1H, H-4).

(s, 3H, OAc), 2.29 (s, 3H, OAc), 2.76 (m, 1H, H-7â), 3.07 (m,

2H, H-6), 4.73 (t, 1H, J ) 8.4 Hz, H-17R), 6.89 (m, 2H, H-2 &

4), 7.29 (d, 1H, J ) 9.2 Hz, H-1), 9.85 (d, 1H, J ) 1.9 Hz, CHO).

3,17â-Diacetoxyestr a-1,3,5(10)-tr ien e-7r-car boxylic Acid

(6). J ones’ reagent⁴³solution (8 N CrO₃in aqueous H₂SO₄, 0.1

mL) was added to a solution of 240 mg of 5 (0.6 mmol) in

acetone (13 mL) that was cooled to 0 °C. After the mixture

was stirred for 30 min, MeOH (50% in H₂O, 5 mL) was added,

and the mixture was extracted with EtOAc (3×, 50 mL). The

combined extracts were washed with H₂O, dried over Na₂SO₄,

and evaporated. Purification by flash chromatography on a

column of silica gel using hexanes/EtOAc (1.5:1) as eluent gave

0.12 g (48%) of 6.

3,17â-Dih yd r oxyest r a -1,3,5(10)-t r ien e-7r-ca r b oxylic

Acid (7, E7r-1,0). To a mixture of MeOH (5 mL) and

concentrated HCl (5 mL) was added 120 mg of 6 (0.3 mmol).

The solution was stirred at room temperature for 4 h and then

evaporated under a N₂stream. Purification by preparative

reversed-phase HPLC using system H-1 (t_R) 15 min) as eluent

gave 52 mg (54%) of 7. Data for 7: ¹H NMR (400 MHz, DMSO-

d₆) δ 0.65 (s, 3H, H-18); 2.80 (m, 2H, H-6), 3.48 (m, 1H, H-17R),

6.48 (d, 1H, J ) 2.6 Hz, H-4), 6.54 (dd, 1H, J ) 2.4, 8 Hz,

H-2), 7.00 (d, 1H, J ) 8.2 Hz, H-1); HRMS (ES⁺) calcd for

C

₁₉H₂₄O₄Na (M + Na⁺) m/e 339.1572, found m/e 339.1580.

HPLC system H-9, t_R) 7.1 min, and system H-23, t_R) 14.8

min, >99% pure.

Meth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -7r-yl)for -

m a te (8, E7r-1,1). To a solution of 2 mg of 7 (0.006 mmol) in

MeOH (1 mL) was added SOCl₂(10 µL). The mixture was

stirred at 50 °C for 17 h, evaporated under a N₂stream diluted

with EtOAc (5 mL), washed with saturated aqueous NaHCO₃

(2 mL) and H₂O, dried over Na₂SO₄, and evaporated. Purifica-

tion by preparative reversed-phase HPLC using system H-2

as eluent gave 1.5 mg (75%) of 8. Data for 8: ¹H NMR (400

MHz, CDCl₃) δ 0.79 (s, 3H, H-18), 2.89 (dd, 1H, J ) 3.6, 5.6

Hz, H-7â), 3.00 (m, 2H, H-6), 3.60 (s, 3H, OCH₃), 3.76 (t, 1H,

J ) 8.2 Hz, H-17R), 4.70 (s, 1H, OH), 6.57 (d, 1H, J ) 2.6 Hz,

H-4), 6.63 (dd, 1H, J ) 2.6, 8.4 Hz, H-2), 7.15 (d, 1H, J ) 8.4

Hz, H-1); HRMS (ES⁺) calcd for C₂₀H₂₆O₄Na (M + Na⁺) m/e

353.1729, found m/e 353.1718. HPLC system H-10, t_R) 9.7

min, and system H-24, t_R) 10.7 min, >99% pure.

7r-Cya n o-3-h yd r oxyestr a -1,3,5(10)-tr ien -17â-yl Aceta te

(3). Compound 3 was prepared by aromatization of 2 (1.56 g,

4.6 mmol) with CuBr₂(2 g, 9 mmol) and LiBr (0.38 g, 4.4 mmol)

as described in the literature.¹⁴Purification by flash chroma-

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

EtOAc as eluent gave 0.94 g (60%) of 3. Data for 3: ¹H NMR

(400 MHz, CDCl₃) δ 0.85 (s, 3H, H-18), 2.07 (s, 3H, OAc), 3.10

(m, 2H, H-6), 4.76 (t, 1H, J ) 8.1 Hz, H-17R), 4.88 (s, 1H,

3-OH), 6.57 (d, 1H, J ) 2.7 Hz, H-4), 6.70 (dd, 1H, J ) 2.5 Hz,

8.3 Hz, H-2), 7.20 (d, 1H, J ) 8.4 Hz, H-1).

3,17â-Dih yd r oxyest r a -1,3,5(10)-t r ien e-7r-ca r b oxa ld e-

h yd e (4). To a solution of 0.25 mL of DIBAL-H (25% in

toluene) in toluene (1 mL) was added 50 mg of 3 (0.15 mmol).

The reaction mixture was shaken vigorously at room temper-

ature for 3 h. A solution of MeOH (0.2 mL) and 2 N HCl (0.35

mL) was added carefully. The mixture was stirred for 15 min

and extracted with EtOAc (3×, 1 mL). The combined extracts

were washed with H₂O, dried over Na₂SO₄, and evaporated.

Purification by flash chromatography on a 1 × 15 cm column

of silica gel using 1:1 hexanes/EtOAc as eluent gave 27 mg

(61%) of 4. Data for 4: ¹H NMR (400 MHz, CDCl₃) δ 0.67 (s,

3H, H-18), 3.54 (t, 1H, J ) 8.4 Hz, H-17R), 6.50 (m, 2H, H-2 &

4), 7.05 (d, 1H, J ) 8.5 Hz, H-1), 9.76 (s, 1H, CHO).

7r-For m ylestr a-1,3,5(10)-tr ien e-3,17â-diyl Diacetate (5).

A solution of 25 mg of 4 (0.08 mmol) in Ac₂O (0.25 mL) and

pyridine (0.5 mL) was stirred at room temperature for 17 h.

The mixture was poured into H₂O (5 mL), acidified with

concentrated HCl (0.45 mL), and extracted with EtOAc (1 ×

25 mL). The organic extract was washed with 1 N HCl (2 × 5

mL) and H₂O, dried over Na₂SO₄, and evaporated. Purification

by flash chromatography on a 1 × 15 cm column of silica gel

using 3:1 hexanes/EtOAc as eluent gave 18 mg (56%) of 5. Data

for 5: ¹H NMR (400 MHz, CDCl₃) δ 0.84 (s, 3H, H-18), 2.07

Eth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -7r-yl)for -

m a te (9, E7r-1,2). Compound 9 was prepared by esterification

of the acid 7 (8 mg, 0.025 mmol) with EtOH as described for

the preparation of 8. Purification of this material by prepara-

tive reversed-phase HPLC using system H-3 (t_R) 12 min) gave

5 mg (57%) of 9. Data for 9: ¹H NMR (400 MHz, CDCl₃) δ 0.71

(s, 3H, H-18), 1.12 (t, 3H, J ) 7.1 Hz, OCH₂CH₃), 2.79 (m, 1H,

H-7â), 2.94 (m, 2H, H-6), 3.67 (m, 1H, H-17R), 4.00 (m, 2H,

OCH₂CH₃), 4.94 (s, 1H, OH), 6.53 (m, 2H, H-2 & 4), 7.07 (d,

1H, J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for C₂₁H₂₈O₄Na (M +

Na⁺) m/e 367.1885, found m/e 367.1895. HPLC system H-11,

t_R) 7.3 min, and system H-25, t_R) 10.4 min, >99% pure.

n -P r op yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -7r-yl)-

for m a te (10, E7r-1,3). Compound 10 was prepared by es-

terification of the acid 7 (6 mg, 0.019 mmol) with n-PrOH as

described for the preparation of 8. Purification of this material

by preparative reversed-phase HPLC using system H-3 (t_R

)

17 min) gave 2 mg (29%) of 10. Data for 10: ¹H NMR (400

MHz, CDCl₃): δ 0.80 (s, 3H, H-18), 0.84 (t, 3H, J ) 7.2 Hz,

OCH₂CH₂CH₃), 2.88 (m, 1H, H-7â), 3.05 (m, 2H, H-6), 3.78

(m, 1H, H-17R), 4.00 (m, 2H, OCH₂CH₂CH₃), 4.49 (s, 1H, OH),

6.59 (d, 1H, J ) 2.7 Hz, H-4), 6.64 (dd, 1H, J ) 2.4, 8.3 Hz,

H-2), 7.16 (d, 1H, J ) 8.6 Hz, H-1), HRMS (ES⁺) calcd for

C

₂₂H₃₀O₄Na (M + Na⁺) m/e 381.2042, found m/e 381.2050.

HPLC system H-12, t_R) 9 min, and system H-25, t_R) 15.4

min, >99% pure.

n -Bu t yl (3,17â-Dih yd r oxyest r a -1,3,5(10)-t r ien -7r-yl)-

for m a te (11, E7r-1,4). Compound 11 was prepared by es-

terification of the acid 7 (6 mg, 0.019 mmol) with nBuOH as

described for the preparation of 8. Purification of this material

by preparative reversed-phase HPLC using system H-3 (t_R

23 min) gave 1.2 mg (17%) of 11. Data for 11: H NMR (400

MHz, CDCl₃) δ 0.80 (s, 3H, H-18), 2.88 (m, 1H, H-7â), 3.04

)

1

1896 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10

Labaree et al.

(m, 2H, H-6), 3.78 (m, 1H, H-17R), 4.03 (m, 2H, OCH₂CH₃),

4.48 (s, 1H, OH), 6.59 (d, 1H, J ) 2.6 Hz, H-4), 6.64 (dd, 1H,

J ) 2.5, 8.5 Hz, H-2), 7.16 (d, 1H, J ) 8.5 Hz, H-1); HRMS

(ES⁺) calcd for C₂₃H₃₂O₄Na (M + Na⁺) m/e 395.2198, found

m/e 395.2201. HPLC system H-11, t_R) 6.4 min, and system

H-26, t_R) 9.8 min, >99% pure.

h. The solution was poured into saturated aqueous NaHCO₃

(200 mL) and extracted with EtOAc (3×, 60 mL). The combined

extracts were washed with H₂O, dried over Na₂SO_4,and

evaporated. Purification by flash chromatography on a column

of silica gel using 3:1 hexanes/EtOAc as eluent gave 15 mg

(46%) of 17. Data for 17: ¹H NMR (400 MHz, CDCl₃) δ 0.78

(s, 3H, H-18), 2.05 (s, 3H, OAc), 2.28 (s, 3H, OAc), 2.43 (s, 3H,

ArCH₃), 3.80 (t, 1H, J ) 9.4 Hz, H-17R), 4.12 & 4.62 (m, 2H,

CH₂OTs), 6.65 (d, 1H, J ) 2.3 Hz, H-4), 6.84 (dd, 1H, J ) 2.3,

8.5 Hz, H-2), 7.21 (d, 1H, J ) 8.9 Hz, H-1), 7.30 (d, 2H, J )

8.5 Hz, ArH), 7.70 (d, 2H, J ) 8.3 Hz, ArH).

7r-Cya n om eth ylestr a -1,3,5(10)-tr ien e-3,17â-d iyl Dia c-

eta te (18). A mixture of 15 mg of 17 (0.028 mmol), NaCN (20

mg), and DMSO (1 mL) was stirred at 90 °C for 2 h. The

solution was cooled to room temperature and poured into

saturated aqueous NH₄Cl (50 mL). The mixture was extracted

with CH₂Cl₂(3×, 100 mL). The combined extracts were washed

with H₂O, dried over Na₂SO₄, and evaporated. Purification by

flash chromatography on a 1 × 15 cm column of silica gel using

6:1 hexanes/EtOAc as eluent gave 10 mg (89%) of 18.

2′-F lu or oeth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -

7r-yl)for m a te (12, E7r-1,2F ₁). Compound 12 was prepared

by esterification of the acid 7 (5 mg, 0.014 mmol) with

fluoroethanol as described for the preparation of 8. Purification

of this material by preparative reversed-phase HPLC using

system H-3 (t_R) 10.9 min) gave 0.7 mg (14%) of 12. Data for

1

12: H NMR (400 MHz, CDCl₃) δ 0.71 (s, 3H, H-18), 2.87 (m,

1H, H-7â), 2.96 (m, 2H, H-6), 3.67 (m, 1H, H-17R), 4.30 (m,

4H, OCH₂CH₂F), 6.50 (d, 1H, J ) 2.5 Hz, H-4), 6.57 (dd, 1H,

J ) 2.7, 8.4 Hz, H-2), 7.07 (d, 1H, J ) 8.4 Hz, H-1); HRMS

(ES⁺) calcd for C₂₁H₂₇FO₄Na (M + Na⁺) m/e 385.1791, found

m/e 385.1788. HPLC system H-10, t_R) 9.8 min, and system

H-25, t_R) 8.3 min, >99% pure.

3,17â-Dih ydr oxyestr a-1,3,5(10)-tr ien -7â-car boxylic Acid

(13, E7â-1,0). A solution of 40 mg of 3 (0.12 mmol), 200 mg

KOH (3.6 mmol), and ethylene glycol (2 mL) was heated at

120 °C overnight. The reaction mixture was diluted with H₂O

(50 mL) and extracted with ether (3×, 50 mL). The combined

organic extracts were coevaporated with toluene and purified

by preparative reversed-phase HPLC using system H-1 to give

3 mg (8%) of 7 (t_R) 15 min) and 15 mg (41%) of 13 (t_R) 17

min). Data for 13: ¹H NMR (400 MHz, DMSO-d₆) δ 0.65 (s,

3H, H-18), 2.77 (m, 2H, H-6), 3.47 (m, 1H, H-17R), 6.50 (m,

2H, H-2 & 4), 6.98 (d, 1H, J ) 8.3 Hz, H-1); HRMS (ES⁺) calcd

for C₁₉H₂₄O₄Na (M + Na⁺) m/e 339.1572, found m/e 339.1587.

HPLC system H-9, t_R) 7.2 min, and system H-23, t_R) 15.1

min, >96% pure.

(3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -7r-yl)a cetic Acid

(19, E7r-2,0). A solution of 18 (10 mg, 0.025 mmol), KOH (0.1

g), and ethylene glycol (1 mL) was heated at 150 °C for 5 days,

poured into H₂O, and extracted with EtOAc. Purification by

preparative reversed-phase HPLC with system H-6 as eluent

gave 6 mg (71%) of 19. Data for 19: ¹H NMR (400 MHz, DMSO-

d₆) δ 0.66 (s, 3H, H-18), 3.52 (t, 1H, J ) 8 Hz, H-17R), 6.40 (d,

1H, J ) 2.4 Hz, H-4), 6.52 (dd, 1H, J ) 2.6, 8.4 Hz, H-2), 7.07

(d, 1H, J ) 8.6 Hz, H-1); HRMS (ES⁺) calcd for C₂₀H₂₆O₄Na

(M + Na⁺) m/e 353.1729, found m/e 353.1731. HPLC system

H-13, t_R) 12.2 min, and system H-29, t_R) 8.6 min >99%

pure.

Meth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -7r-yl)a c-

eta te (20, E7r-2,1). A solution of 4 mg of 19 (0.012 mmol),

MeOH (1 mL), and SOCl₂(20 µL) was stirred at 55 °C for 15

h. The solvent was evaporated under a N₂stream, and the

residue dissolved in EtOAc (50 mL). The solution was washed

with saturated aqueous NaHCO, dried over MgSO₄, and

evaporated. Purification by preparative reversed-phase HPLC

using system H-3 gave 2 mg (48%) of 20. Data for 20: ¹H NMR

(400 MHz, CDCl₃) δ 0.80 (s, 3H, H-18), 3.59 (s, 3H, OCH₃),

3.67 (m, 1H, H-17R), 4.46 (s, 1H, OH), 6.48 (d, 1H, J ) 2.5 Hz,

H-4), 6.58 (dd, 1H, J ) 2.6, 8.3 Hz, H-2), 7.12 (d, 1H, J ) 8.4

Hz, H-1); HRMS (ES⁺) calcd for C₂₁H₂₈O₄Na (M + Na⁺) m/e

367.1885, found m/e 367.1892. HPLC system H-11, t_R) 7.2

min, and system H-24, t_R) 14.2 min >99% pure.

Eth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -7r-yl)a c-

eta te (21, E7r-2,2). Compound 21 was prepared by esterifi-

cation of 19 (4 mg, 0.012 mmol) with EtOH as described for

the preparation of 20. Purification with preparative reversed-

phase HPLC using system H-5 as eluent gave 1.4 mg (32%) of

21. Data for 21: ¹H NMR (400 MHz, CDCl₃) δ 0.72 (s, 3H,

H-18), 1.15 (t, 3H, J ) 7.2 Hz, OCH₂CH₃), 3.66 (m, 1H, H-17R),

4.04 (q, 2H, J ) 7.2 Hz, OCH₂CH₃), 4.51 (s, 1H, OH), 6.47 (d,

1H, J ) 2.6 Hz, H-4), 6.57 (dd, 1H, J ) 2.6, 8.4 Hz, H-2), 7.05

(d, 1H, J ) 8.5 Hz, H-1); HRMS (ES⁺) calcd for C₂₂H₃₀O₄Na

(M + Na⁺) m/e 381.2042, found m/e 381.2035. HPLC system

H-10, t_R) 8.7 min, and system H-28, t_R) 8.7 min >99% pure.

3,17â-Diben zyloxyestr a -1,3,5(10)-tr ien e (23). A suspen-

sion of 20 g of NaH (0.8 mmol) in DMF (100 mL) was cooled

to 0 °C with ice bath. Then 25 g of estradiol 22 (92 mmol) in

THF (100 mL) was added dropwise over 40 min followed by

the addition of BnBr (27 mL) dropwise over 30 min. The

reaction was stirred at room temperature for 20 h, diluted with

H₂O, and extracted with EtOAc (2×, 250 mL). The combined

organic extracts were washed with H₂O, dried over Na₂SO₄,

and evaporated. The residue was diluted with methanol (150

mL), and the resulting solid was filtered and washed with

MeOH giving 40 g (96%) of 23: mp 68-70 °C.

Meth yl 3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -7â-yl)for -

m a te (14, E7â-1,1). Compound 14 was prepared by esterifi-

cation of the acid 13 (2 mg, 0.006 mmole) with MeOH as

described for the preparation of 8 gave 1 mg (50%). Data for

1

14: H NMR (400 MHz, CDCl₃) δ 0.80 (s, 3H, H-18), 2.23 (s,

3H, OCH₃), 2.62 (ddd, 1H, J ) 8.5, 5.8, 9.2 Hz, H-7R), 2.97

(m, 1H, H-6), 3.70 (t, 1H, J ) 8.2 Hz, H-17R), 4.71 (s, 1H, OH),

6.57 (d, 1H, J ) 2.7 Hz, H-4), 6.67 (dd, 1H, J ) 2.6, 8.3 Hz,

H-2), 7.15 (d, 1H, J ) 8.4 Hz, H-1); HRMS (ES⁺) calcd for

C

₂₀H₂₆O₄Na (M + Na⁺) m/e 353.1729, found m/e 353.1734.

HPLC system H-10, t_R) 11 min, and system H-24, t_R) 11.7

min. >99% pure.

E t h yl 3,17â-Dih yd r oxyest r a -1,3,5(10)-t r ien -7â-yl)for -

m a te (15, E7â-1,2). Compound 15 was prepared by esterifi-

cation of the acid 13 (7 mg, 0.022 mmol) with EtOH as

described for the preparation of 8. Purification of this material

by preparative reversed-phase HPLC using system H-3 (t_R

)

10 min) gave 3 mg (39%) of 15. Data for 15: ¹H NMR (400

MHz, CDCl₃) δ 0.82 (s, 3H, H-18), 1.29 (t, 3H, J ) 7.3 Hz,

OCH₂CH₃), 2.62 (m, 1H, H-7R), 2.90(m, 1H, H-6), 3.68 (m, 1H,

H-17R), 4.15 (m, 2H, OCH₂CH₃), 4.50 (s, 1H, OH), 6.58 (d, 1H,

J ) 2.7 Hz, H-4), 6.68 (dd, 1H, J ) 2.8, 8.3 Hz, H-2), 7.16 (d,

1H, J ) 8.6 Hz, H-1); HRMS (ES⁺) calcd for C₂₁H₂₈O₄Na (M +

Na⁺) m/e 367.1885, found m/e 367.1888. HPLC system H-10,

t_R) 10.2 min, and system H-25, t_R) 11.1 min, >99% pure.

7r-Hyd r oxym eth ylestr a -1,3,5(10)-tr ien -3,17â-d iyl Di-

a ceta te (16). To a solution of 5 (5 mg, 0.013 mmol) in ethanol

(0.5 mL) at 0 °C was added 2 mg of NaBH₄. After being stirred

at 0 °C for 50 min, the mixture was extracted with EtOAc (10

mL), washed with H₂O, dried over Na₂SO₄, and evaporated.

Purification by flash chromatography on a 1 × 15 cm column

of silica gel using 2.5:1 hexanes/EtOAc as eluent gave 2 mg

(40%) of 16. Data for 16: ¹H NMR (400 MHz, CDCl₃) δ 0.82

(s, 3H, H-18), 2.06 (s, 3H, 17-AcO), 2.28 (s, 3H, 3-AcO),

3.47&3.76 (m, 2H, OCH₂), 4.70 (dd, 1H, J ) 7.6, 8.4 Hz,

H-17R), 6.84 (m, 2H, H-2 & 4), 7.28 (d, 1H, J ) 8.4 Hz, H-1).

3,17â-Dia cetoxy-(7r-tolu en esu lfon yloxym eth yl)estr a -

1,3,5(10)-tr ien (17). To a solution of 24 mg of 16 (0.06 mmol)

in pyridine (2 mL) was added TsCl (40 mg). After the mixture

was stirred for 17 h at room temperature, another portion of

TsCl (40 mg) was added and the stirring was continued for 3

3,17â-Diben zyloxyestr a -1,3,5(10),9(11)-tetr a en e (24). To

a solution of 1 g of 23 (2.2 mmol) in MeOH (50 mL) and dioxane

(10 mL) was added 0.7 g of DDQ (3 mmol). The reaction was

stirred at room temperature for 22 h. The solvent was

evaporated at 50 °C, and the residue was dissolved in CH₂Cl₂

and filtered. The filtrate was evaporated and diluted with

Estradiol Carboxylic Acid Esters as Locally Active Estrogens

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1897

MeOH. The resulting solid was collected and washed with

using 6:1 hexanes-EtOAc as eluent gave 140 mg (93%) of 30.

Data for 30: ¹H NMR (400 MHz, CDCl₃): δ 1.10 (s, 3H, H-18),

3.57 (t, 1H, J ) 7.4 Hz, H-17R), 4.64 (s, 2H, benzylic-H), 5.09

(s, 2H, benzylic-H), 6.77 (m, 2H, H-2 & 4), 7.09 (d, 1H, J ) 8.6

Hz, H-1), 7.48 (m, 10H, ArH).

MeOH giving 0.44 g (44%) of 24:^20,44mp 105-110 °C.

3,17â-Diben zyloxyestr a -1,3,5(10)-tr ien -11r-ol (25). The

compound 25 was prepared by hydroxylation of 24 (3 g, 6.6

mmol) with LiBH₄(0.2 g, 10 mmol) and catecholborane (20

mL, 1.0 M in THF, Aldrich) as described in the literature.²⁰

The solution was stirred at room temperature for 18 h and

added dropwise over 30 min to an ice-cold mixture of NaOH

(3 g), water (10 mL), EtOH (30 mL), and H₂O₂(35%, 20 mL).

The solution was stirred at room temperature for 5 h and

extracted with EtOAc (3×, 100 mL). The combined organic

extracts were washed with H₂O, dried over Na₂SO₄, and

evaporated. Purification by flash chromatography on a 3 × 21

cm column of silica gel using 20:1 CH₂Cl₂/EtOAc as eluent gave

2.4 g (78%) of 25.

3,17â-Diben zyloxyest r a -1,3,5(10)-t r ien -11-on e (26) To

the solution of 1.0 g of 25 (2.1 mmol) in CH₂Cl₂(20 mL) was

added 1.0 g of PCC (4.8 mmol). The reaction was stirred at

room temperature for 3 h, diluted with Et₂O (20 mL), filtered

through Florisil, and washed through with CH₂Cl₂/Et₂O (1:

1). The filtrate was washed with H₂O, dried over Na₂SO₄, and

evaporated. Purification by flash chromatography on a column

of silica gel using hexanes-EtOAc (5:1) as eluent gave 680

mg (68%) of 26.

3,17â-Dib en zyloxy-11-m et h ylen est r a -1,3,5(10)-t r ien e

(27). To a solution of 26 (0.52 g, 1.1 mmol) in Et₂O (15 mL)

was added trimethylsilylmethylmagnesium chloride (15 mL,

1.0 M in ether). The solution was stirred at room temperature

for 17 h, quenched with saturated aqueous NH₄Cl (250 mL),

and extracted with CHCl₃(500 mL). The organic extract was

washed with H₂O, dried over MgSO₄, and evaporated. The

resulting white solid (0.7 g) was dissolved in a mixture of

acetone (10 mL) and concentrated HCl (30µL) and stirred at

room temperature for 17 h. The mixture was evaporated to

dryness with a N₂stream, dissolved in EtOAc (50 mL), washed

with saturated aqueous NaHCO₃and H₂O, dried over Na₂SO₄,

and evaporated. Purification by flash chromatography on a 1

× 15 cm column of silica gel using 15:1 hexanes/EtOAc as

eluent gave 0.37 g (72%) of 27. Data for 27: ¹H NMR (400 MHz,

CDCl₃): δ 0.84 (s, 3H, H-18), 2.90 (m, 1H, H-6), 3.58 (t, 1H, J

) 8.2 Hz, H-17R), 4.57 (s, 2H, benzylic-H), 4.85 (d, 2H, J )

11.2 Hz, dCH₂), 5.03 (s, 2H, benzylic-H), 6.71 (d, 1H, J ) 2.4

Hz, H-4), 6.78 (dd, 1H, J ) 2.5, 8.6 Hz, H-2), 7.35 (m, 11 H,

ArH).

(3,17â-Dib en zyloxyest r a -1,3,5(10)-t r ien -11â-yl)a cet ic

Acid (31). A mixture of 140 mg of 30 (0.29 mmol), 700 mg of

KOH (13 mmol), and ethylene glycol (5 mL) was heated at 140

°C for 5 days. The mixture was extracted with EtOAc (3×, 10

mL). The combined extracts were washed with H₂O, dried over

Na₂SO₄, and evaporated. Purification by flash chromatography

on a 1 × 15 cm column of silica gel using 1:2 hexanes/EtOAc

1

as eluent gave 34 mg (23%) of 31. Data for 31: H NMR (400

MHz, CDCl₃) δ 1.05 (s, 3H, H-18), 3.49 (t, 1H, J ) 8.6 Hz,

H-17R), 4.56 (s, 2H, benzylic-H), 5.03 (s, 2H, benzylic-H), 6.70

(d, 1H, J ) 2.6 Hz, H-4), 6.80 (dd, 1H, J ) 2.7, 8.3 Hz, H-2),

7.12 (d, 1H, J ) 8.3 Hz, H-1), 7.40 (m, 10H, ArH).

(3,17â-Dih ydr oxyestr a-1,3,5(10)-tr ien -11â-yl)acetic Acid

(32, E11-2,0). To a solution of 15 mg of 31 (0.03 mmol) in

CH₂Cl₂(1.5 mL) at 0 °C was added BCl₃(0.6 mL, 1 M in CH₂-

Cl₂, Aldrich). The reaction was stirred at room temperature

for 40 min, quenched with 10 mL of H₂O, and extracted with

EtOAc (3×, 20 mL). The combined organic extracts were

washed with H₂O, dried over Na₂SO₄, and evaporated. Puri-

fication by preparative reversed-phase HPLC using system H-4

(t_R) 14 min) as eluent gave 5 mg (51.5%) of 32. Data for 32:

¹H NMR (400 MHz, DMSO-d₆) δ 0.78 (s, 3H, H-18), 3.49 (t,

1H, J ) 8 Hz, H-17R), 6.43 (d, 1H, J ) 2.8 Hz, H-4), 6.54 (dd,

1H, J ) 2.8, 8.4 Hz, H-2), 6.90 (d, 1H, J )8.4 Hz, H-1); HRMS

(ES⁺) calcd for C₂₀H₂₆O₄Na (M + Na⁺) m/e 353.1729, found

m/e 353.1738. HPLC system H-13, t_R) 18.5 min, and system

H-27, t_R) 9.6 min, >99% pure.

Meth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -11â-yl)-

a ceta te (33, E11-2,1). At 0 °C, BCl₃(0.4 mL, 1.0 M in CH₂-

Cl₂) was added to a solution of 7 mg of 31 (0.014 mmol) in

CH₂Cl₂(0.6 mL). The reaction was stirred at 0 °C for 40 min,

and then MeOH (0.5 mL) was added. The mixture was stirred

for 2 h, diluted with 0.5 mL of saturated aqueous NaHCO₃,

and extracted with CH₂Cl₂(3×, 20 mL). Combined extracts

were evaporated under N₂stream, and the residue was

purified by preparative reversed-phase HPLC with system H-3

(t_R) 15 min) gave 3 mg (64%) of 33. Data for 33: ¹H NMR

(400 MHz, CDCl₃) δ 1.07 (s, 3H, H-18), 3.79 (s, 3H, OCH₃),

3.87 (t, 1H, J ) 7.8 Hz, H-17R), 6.73 (d, 1H, J ) 2 Hz, H-4),

6.80 (dd, 1H, J ) 2, 8.6 Hz, H-2), 7.20 (d, 1H, J ) 8.6 Hz,

H-1); HRMS (ES⁺) calcd for C₂₁H₂₈O₄Na (M + Na⁺) m/e

367.1885, found m/e 367.1885. System H-11, t_R) 7.4 min, and

system H-25, t_R) 11.2 min, >99% pure.

3,17â-Diben zyloxy-11â-h yd r oxym eth ylestr a -1,3,5(10)-

tr ien e (28). Compound 28 was prepared by hydroxylation of

the olefin 27 (0.15 g, 0.3 mmol) with catecholborane as

described for the preparation of 25. Purification by flash

chromatography on a 1 × 15 cm column of silica gel using 3:1

hexanes/EtOAc as eluent gave 0.11 g (76%) of 28. Data for

Eth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -11â-yl)a c-

eta te (34, E11-2,2). The compound 34 was prepared by

deprotection of 31 (9 mg, 0.018 mmol) and esterification with

EtOH as described for the preparation of 33. Purification with

preparative reversed-phase HPLC with system H-5 (t_R) 8

1

28: H NMR (400 MHz, CDCl₃): δ 1.01 (s, 3H, H-18), 3.52 (t,

1H, J ) 8.5 Hz, H-17R), 3.59&3.72 (m, 2H, CH₂OH), 4.61 (s,

2H, benzylic-H), 5.04 (s, 2H, benzylic-H), 6.71 (d, 1H, J ) 2.8

Hz, H-4), 6.80 (dd, 1H, J ) 2.7, 8.4 Hz, H-2), 7.24 (d, 1H, J )

8.4 Hz, H-1), 7.38 (m, 10 H, ArH).

1

min) gave 2 mg (31%) of 34. Data for 34: H NMR (400 MHz,

CDCl₃) δ 0.93 (s, 3H, H-18), 1.23 (t, 3H, J ) 7 Hz, OCH₂CH₃),

3.72 (t, 1H, J ) 8.1 Hz, H-17R), 4.10 (m, 2H, OCH₂CH₃), 4.56

(s, 1H, OH), 6.54 (d, 1H, J ) 2.8 Hz, H-4), 6.64 (dd, 1H, J )

2.9, 8.8 Hz, H-2), 7.09 (d, 1H, J ) 8.7 Hz, H-1); HRMS (ES⁺)

calcd for C₂₂H₃₀O₄Na (M + Na⁺) m/e 381.2042, found m/e

381.2038. HPLC system H-11, t_R) 6.5 min, and system H-28,

t_R) 10.5 min >99% pure.

11r-Allyl-3,17â-d ib en zyloxyest r a -1,3,5(10)-t r ien -11â-

ol (35). Allylmagnesium bromide (2.5 mL, 1.0 M in ether,

Aldrich) was added to a solution of 26 (0.12 g, 0.26 mmol) in

THF (2.5 mL) under N₂. The reaction was stirred at room

temperature for 1.5 h, quenched with saturated aqueous NH₄-

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

combined extracts were washed with H₂O, dried over Na₂SO₄,

and evaporated. Purification by flash chromatography on a 1

× 15 cm column of silica gel using 4:1 hexanes/EtOAc as eluent

gave 98 mg (74%) of 35.

3,17â-Diben zyloxy(11â-tolu en esu lfon yloxym eth yl)estr a-

1,3,5(10)-tr ien e (29). To a solution of 200 mg of 28 (0.4 mmol)

in pyridine (5 mL) was added 400 mg of pTsCl (2 mmol). The

reaction was stirred for 28 h, poured into saturated aqueous

NaHCO₃(200 mL), and extracted with EtOAc (3×, 100 mL).

Combined organic extracts were washed with H₂O, dried over

Na₂SO₄, and evaporated. Purification by flash chromatography

on a 1 × 15 cm column of silica gel using 4:1 hexanes/EtOAc

as eluent gave 0.2 g (76%) of 29. Data for 29: ¹H NMR (400

MHz, CDCl₃) δ 0.82 (s, 3H, H-18), 2.43 (s, 3H, ArCH₃), 3.48 (t,

1H, J ) 8 Hz, H-17R), 4.00&4.06 (m, 2H, TsOCH₂), 4.57 (d,

2H, J ) 3 Hz, benzylic-H), 5.06 (s, 2H, benzylic-H), 6.68 (d,

1H, J ) 2.6 Hz, H-4), 6.72 (dd, 1H, J ) 2.5, 8.4 Hz, H-2), 6.97

(d, 1H, J ) 8.7 Hz, H-1), 7.43 (m, 12H, ArH), 7.71 (d, 2H, J )

8.4 Hz, ArH).

3,17â-Diben zyloxy-11â-cya n om eth ylestr a -1,3,5(10)-tr i-

en e (30). The compound 30 was prepared by cyanation of 29

(200 mg, 0.3 mmol) as described for the preparation of 18.

Purification by flash chromatography on a column of silica gel

11â-Allyl-3,17â-d iben zyloxyestr a -1,3,5(10)-tr ien e (36).

To a solution of 98 mg of 35 (0.19 mmol) in CH₂Cl₂(10 mL)

was added HSiEt₃(1 mL). The mixture was cooled to 0 °C,

1898 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10

Labaree et al.

and BF₃‚Et₂O (2 mL) was added. The reaction was stirred at

0 °C for 40 min, washed with saturated aqueous NaHCO₃

followed by H₂O, dried over Na₂SO₄, and evaporated. Purifica-

tion by flash chromatography on a 1 × 15 cm column of silica

gel using 20:1 hexanes/EtOAc as eluent gave 90 mg (95%) of

36. Data for 36: ¹H NMR (400 MHz, CDCl₃) δ 1.03 (s, 3H,

H-18), 3.48 (t, 1H, J ) 8.5 Hz, H-17R), 4.57 (s, 2H, benzylic-

H), 5.00 (m, 2H, CdCH₂), 5.02 (s, 2H, benzylic-H), 5.80 (m,

1H, C-CHdC), 6.69 (d, 1H, J ) 2.8 Hz, H-4), 6.78 (dd, 1H, J

) 2.9, 8.6 Hz, H-2), 7.09 (d, 1H, J ) 8.6 Hz, H-1), 7.36 (m,

10H, ArH).

3,17â-Dib en zyloxy-11â-(3′-h yd r oxyp r op yl)est r a -1,3,5-

(10)-tr ien e (37). Compound 37 was prepared by hydroxylation

of the olefin 36 (20 mg, 0.04 mmol) with LiBH₄(1.5 mg) and

catecholborane (0.2 mL) as described for the preparation of

25. Purification by flash chromatography on a 1 × 15 cm

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

16 mg (78%) of 37. Data for 37: ¹H NMR (400 MHz, CDCl₃) δ

0.98 (s, 3H, H-18), 2.75 (m, 2H, H-6), 3.50 (t, 1H, J ) 7.2 Hz,

H-17R), 3.59 (m, 2H, CH₂O), 4.60 (s, 2H, benzylic-H), 5.04 (s,

2H, benzylic-H), 6.71 (d, 1H, J ) 2.4 Hz, H-4), 6.80 (dd, 1H, J

) 2.7, 8.7 Hz, H-2), 7.08 (d, 1H, J ) 8.6 Hz, H-1), 7.38 (m, 10

H, ArH).

3-(3,17â-Dib en zyloxyest r a -1,3,5(10)-t r ien -11â-yl)p r o-

p a n oic a cid (38). Compound 38 was prepared by CrO₃

oxidation of 37 (40 mg, 0.08 mmol) as described for the

preparation of 6. Purification by flash chromatography on a

column of silica gel using 4:1 hexanes/EtOAc as eluent gave

15 mg (36%) of 38. Data for 38: ¹H NMR (400 MHz, CDCl₃) δ

1.03 (s, 3H, H-18), 3.49 (t, 1H, J ) 8.5 Hz, H-17R), 4.59 (d,

2H, J ) 4.6 Hz, benzylic-H), 5.04 (s, 2H, benzylic-H), 6.70 (d,

1H, J ) 2.5 Hz, H-4), 6.81 (dd, 1H, J ) 2.6, 8.5 Hz, H-2), 7.09

(d, 1H, J ) 8.6 Hz, H-1), 7.38 (m, 10 H, ArH), 8.4 (broad, 1H,

COOH).

3-(3,17â-Dih ydr oxyestr a-1,3,5(10)-tr ien -11â-yl)pr opan o-

ic Acid (39), Meth yl Ester (40), a n d Eth yl Ester (41). BCl₃

(0.6 mL) was added to a solution of 38 (15 mg, 0.029 mmol) in

CH₂Cl₂(1.5 mL) at 0 °C and the mixture stirred for 40 min.

The solution was divided into three aliquots, H₂O (0.5 mL),

MeOH (0.5 mL), or EtOH (0.5 mL), respectively, was added,

and the reactions were stirred at room temperature for 3 h.

The three mixtures were separately extracted with EtOAc (3×,

10 mL). The organic extracts from each were washed with H₂O,

dried over Na₂SO₄and evaporated. The residues were purified

by preparative reversed-phase HPLC.

1.06 mL (6.07 mmol) of diisopropylethylamine in 50 mL

acetone was stirred at room temperature for 3 days. The

reaction mixture was poured into H₂O (150 mL) and extracted

with EtOAc (3×, 100 mL). Combined organic extracts were

dried over Na₂SO₄and concentrated in vacuo giving a yellow

oil. Purification by flash chromatography on a 3 × 21 cm

column of silica gel using 3:1 hexanes/EtOAc as eluent gave

2.37 g (97%) of 43. Data for 43: TLC, T-1, R_f0.54.

3-Ben zyloxyestr a -1,3,5(10),15-tetr a en -17-on e (44). A so-

lution of 1.04 g (2.57 mmol) of 43, 117 mg (0.617 mmol) of

pTsOH in acetone (70.3 mL), and H₂O (11.4 mL) was stirred

at room temperature for 1.5 h. The reaction mixture was

adjusted to pH 7 with 5% NaHCO₃and concentrated by

rotovap to about 30 mL without heating. The solution was

poured into H₂O (50 mL) and extracted with EtOAc (3×, 70

mL). Combined organic extracts were dried over Na₂SO₄and

concentrated in vacuo giving a white solid. Crystallization of

the residue from CH₂Cl₂/hexanes gave 477 g (52%) of 44 as

fine white needles. Data for 44: TLC, T-1, R_f0.5; ¹H NMR

(400 MHz, CDCl₃) δ 1.12 (s, 3H, H-18), 5.05 (s, 2H, benzylic-

H), 6.10 (dd, 1H, J ) 5.9, 3.2 Hz, H-15), 6.76 (d, 1H, J ) 2.7

Hz, H-4), 6.81 (dd, 1H, J ) 8.6, 2.7 Hz, H-2), 7.22 (d, 1H, J )

8.6 Hz, H-1), 7.31-7.45 (m, 5H, Ar-H), 7.64 (dd, 1H, J ) 5.9,

1.1 Hz, H-16).

3-Ben zyloxy-15â-cya n oestr a -1,3,5(10)-tr ien -17-on e (45).

This procedure is based on the literature method.^29,30

A

solution of 1.03 g (2.88 mmol) of 44 and 2.03 g (41.5 mmol) of

NaCN in THF (30 mL) with 21 drops of H₂O was stirred and

heated at 75 °C under reflux for 2 h. The reaction mixture

was poured into ice-water (300 mL) and extracted with CH₂-

Cl₂(3×, 100 mL). Combined organic extracts were washed with

H₂O (100 mL), dried over Na₂SO₄, and concentrated in vacuo

giving a brown oil. Purification by flash chromatography on a

3 × 17 cm column of silica gel using 2:1 hexanes/EtOAc as

eluent gave 557 mg (50%) of 45 as a white solid. Data for 45:

TLC, T-1, R_f0.3; ¹H NMR (400 MHz, CDCl₃) δ 1.24 (s, 3H,

H-18), 5.06 (s, 2H, benzylic-H), 6.76 (d, 1H, J ) 2.7 Hz, H-4),

6.81 (dd, 1H, J ) 8.6, 2.7 Hz, H-2), 7.20 (d, 1H, J ) 8.6 Hz,

H-1), 7.33-7.45 (m, 5H, Ar-H).

3-Ben zyloxy-15â-cya n oestr a -1,3,5(10)-tr ien -17â-ol (46).

A solution of 558 mg (1.45 mmol) of 45 in THF (6.6 mL) and

MeOH (34 mL) was stirred at room temperature as 400 mg

(10.6 mmol) of NaBH₄was added in small portions over 10

min. The reaction was stirred at room temperature under N₂

for 3.5 h, the solvent was evaporated, and the residue was

dissolved in EtOAc (100 mL) and H₂O (50 mL). The phases

were separated, and the aqueous phase was extracted with

EtOAc (2×, 70 mL). Combined organic extracts were dried over

Na₂SO₄and concentrated in vacuo giving a yellow foam which

was used without purification in the next step. Data for 46:

3-(3,17â-Dih ydr oxyestr a-1,3,5(10)-tr ien -11â-yl)pr opan o-

ic a cid (39, E11-3,0) using system H-7, t_R) 11 min, gave 1.2

mg (36%). Data for 39: ¹H NMR (400 MHz, DMSO-d₆) δ 0.77

(s, 3H, H-18), 3.47 (t, 1H, J ) 8 Hz, H-17R), 6.40 (d, 1H, J )

2.1 Hz, H-4), 6.52 (dd, 1H, J ) 2.1, 8.1 Hz, H-2), 6.95 (d, 1H,

J ) 8.7 Hz, H-1); HRMS (ES⁺) calcd. for C₂₁H₂₈O₄Na (M +

Na⁺) m/e 367.1885, found m/e 367.1878. HPLC system H-9, t_R

) 7 min, and system H-27, t_R) 11.8 min >99% pure.

Meth yl 3-(3,17â-d ih yd r oxyestr a -1,3,5(10)-tr ien -11â-yl)-

p r op a n oa te (40, E11-3,1) using system H-8, t_R) 11 min,

1

TLC, T-2, R_f0.22; H NMR (400 MHz, CDCl₃) δ 1.07 (s, 3H,

H-18), 3.75 (t, 1H, J ) 8.7 Hz, H-17R), 5.05 (s, 2H, benzylic-

H), 6.75 (d, 1H, J ) 2.7 Hz, H-4), 6.80 (dd, 1H, J ) 8.5, 2.7

Hz, H-2), 7.21 (d, 1H, J ) 8.5 Hz, H-1), 7.33-7.45 (m, 5H,

Ar-H).

1

gave 2 mg (59%). Data for 40: H NMR (400 MHz, CDCl₃): δ

3-Ben zyloxy-17â-h ydr oxyestr a-1,3,5(10)-tr ien e-15r-car -

boxylic Acid (47). A solution of 571 mg (1.47 mmol) of 46

crude, 2.54 g (45.3 mmol) of KOH in H₂O (6 mL), and ethylene

glycol (34 mL) was stirred and heated at 160 °C for 112 h

without a reflux condenser to allow H₂O to evaporate. The

reaction mixture was poured into H₂O (700 mL) and washed

with Et₂O (2×, 100 mL). The aqueous phase was adjusted to

pH 2 with concentrated HCl and extracted with Et₂O (3×, 100

mL). Combined organic extracts were washed with 10% sodium

metabisulfite (50 mL) and H₂O (50 mL), dried over Na₂SO₄,

and concentrated in vacuo giving 535 mg (89%, two steps) of

0.94 (s, 3H, H-18), 3.62 (s, 3H, OCH₃), 3.73 (t, 1H, J ) 8 Hz,

H-17R), 4.52 (s, 1H, OH), 6.55 (d, 1H, J ) 2.6 Hz, H-4), 6.66

(dd, 1H, J ) 2.7, 8.5 Hz, H-2), 7.05 (d, 1H, J ) 8.3 Hz, H-1);

HRMS (ES⁺) calcd. for C₂₂H₃₀O₄Na (M + Na⁺) m/e 381.2042,

found m/e 381.2045. HPLC system H-10, t_R) 8.6 min, and

system H-28, t_R) 9.6 min >99% pure.

Eth yl 3-(3,17â-d ih yd r oxyestr a -1,3,5(10)-tr ien -11â-yl)-

p r op a n oa te (41, E11-3,2) using system H-5, t_R) 11 min,

1

gave 2 mg, (56%). Data for 41: H NMR (400 MHz, CDCl₃) δ

0.94 (s, 3H, H-18), 3.73 (m, 1H, H-17R), 4.12 (q, 2H, J ) 7.2

Hz, OCH₂), 4.53 (s, 1H, OH), 6.55 (d, 1H, J ) 2.5 Hz, H-4),

6.67 (dd, 1H, J ) 2.6, 8.6 Hz, H-2), 7.06 (d, 1H, J ) 8.7 Hz,

H-1); HRMS (ES⁺) calcd for C₂₃H₃₂O₄Na (M + Na⁺) m/e

395.2198, found m/e 395.2208. HPLC system H-10, t_R) 8 min,

and system H-28, t_R) 12.9 min >99% pure.

1

47 as a tan solid. Data for 47: TLC, T-3, R_f0.575; H NMR

(400 MHz, DMSO-d₆) δ 0.69 (s, 3H, H-18), 3.61 (ddd, 1H, J )

8.7, 8.7, 5.0 Hz, H-17R), 4.73 (d, 1H, J ) 5.0 Hz, 17-OH), 5.04

(s, 2H, benzylic-H), 6.67 (d, 1H, J ) 2.6 Hz, H-4), 6.74 (dd,

1H, J ) 8.7, 2.6 Hz, H-2), 7.17 (d, 1H, J ) 8.7 Hz, H-1), 7.31-

7.42 (m, 5H, Ar-H), 12.07 (s, 1H, OH).

3-Ben zyloxy-17,17-eth ylen ed ioxyestr a -1,3,5(10),15-tet-

r a en e (43). A solution of 1.90 g (6.07 mmol) of 42,³¹940 µL

(7.89 mmol) of benzyl bromide, 1.09 (7.89 mmol) of K₂CO₃, and

3,17â-Dih yd r oxyest r a -1,3,5(10)-t r ien e-15r-ca r b oxylic

Acid (48, E15-1,0). A solution of 479 mg (1.18 mmol) of 47 in

Estradiol Carboxylic Acid Esters as Locally Active Estrogens

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1899

EtOH (10 mL) was warmed to dissolve solid, cooled to room

temperature, and added to a suspension of 50 mg 5% Pd on

carbon in EtOH (5 mL) and stirred at room temperature under

an atmosphere of H₂for 20 h. The reaction mixture was filtered

through a 1 in. plug of Celite and washed through with EtOH

(50 mL). The solvent was evaporated giving 368 mg (99%) of

48 as a white solid. Purification of 25.5 mg of this material by

HPLC in system H-20 followed by acid/base extraction gave

a 2 × 17 cm column of silica gel using 1:1 hexanes/EtOAc as

eluent gave 21.9 mg of 52. Further purification by HPLC with

system H-21 in 3 portions gave 22.3 mg (58%) of 52 as a white

solid. Crystallization from acetone-petroleum ether gave 12

mg (31%) of 52 as white needles. Data for 52: TLC, T-3, R_f

0.64; ¹H NMR (400 MHz, CDCl₃) δ 0.83 (s, 3H, H-18), 0.98 (t,

3H, J ) 7.5 Hz, OCH₂CH₂CH₃), 3.93 (t, 1H, J ) 8.6 Hz, H-17R),

4.05-4.08 (m, 2H, OCH₂CH₂CH₃), 6.54 (d, 1H, J ) 2.7 Hz,

H-4), 6.63 (dd, 1H, J ) 8.2, 2.7 Hz, H-2), 7.20 (d, 1H, J ) 8.2

Hz, H-1); HRMS (ES⁺) calcd for C₂₂H₃₀O₄Na (M + Na⁺) m/e

381.2042, found m/e 381.2034. HPLC system H-15, t_R) 11.97

min, and system H-25, t_R) 19.35 min, >99% pure.

1

11.9 mg 48 for bioassay. Data for 48: TLC, T-3, R_f0.475; H

NMR (400 MHz, DMSO-d₆) δ 0.69 (s, 3H, H-18), 3.60 (ddd,

1H, J ) 8.7, 8.7, 5.0 Hz, H-17R), 4.72 (d, 1H, J ) 5.0 Hz, OH),

6.40 (d, 1H, J ) 2.3 Hz, H-4), 6.49 (dd, 1H, J ) 8.5, 2.3 Hz,

H-2), 7.04 (d, 1H, J ) 8.5 Hz, H-1), 9.00 (s, 1H, OH), 12.05 (br

s, 1H, OH); HRMS (ES^-) calcd for C₁₉H₂₃O₄(M-H) m/e

315.1597, found m/e 315.1603. HPLC system H-14, t_R) 12.85

min, and system H-23, t_R) 12.08 min, >99% pure.

Meth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-estr a tr ien -15r-

yl)for m a te (49, E15-1,1). A solution of 41.8 mg (0.132 mmol)

of 48 and 1 drop of concentrated H₂SO₄in MeOH (2 mL) was

heated at 60 °C in a sealed vial for 75 h. The reaction mixture

was poured into saturated aqueous NaHCO₃(50 mL) and

extracted with EtOAc (3×, 70 mL). Combined organic extracts

were dried over Na₂SO₄and concentrated in vacuo giving a

yellow oil. Purification of the residue by flash chromatography

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

as eluent gave 21.2 mg of 49 as a white solid. Further

Isopr opyl (3,17â-Dih ydr oxyestr a-1,3,5(10)-tr ien -15r-yl)-

for m a te (53, E15-1,3i). Compound 53 was prepared by

esterification of 48 (34.6 mg, 0.109 mmol) with ⁱPrOH as

described for 49. Purification of the residue by flash chroma-

tography on a 2 × 17 cm column of silical gel using 1:1

hexanes/EtOAc gave 21.3 mg of 53. Further purification by

HPLC with system H-21 in 4 portions gave 16 mg (41%) of 53

as a white solid. Crystallization from Et₂O-petroleum ether

gave 13.6 mg (35%) of 53 as white needles. Data for 53: TLC,

1

T-3, R_f0.74; H NMR (400 MHz, CDCl₃) δ 0.82 (s, 3H, H-18),

1.26 (d, 6H, J ) 6.3 Hz, -OCH(CH₃)₂), 3.91 (t, 1H, J ) 8.5

Hz, H-17R), 5.04 (septet, 1H, J ) 6.3 Hz, -OCH(CH₃)₂), 6.55

(d, 1H, J ) 2.6 Hz, H-4), 6.63 (dd, 1H, J ) 8.5, 2.6 Hz, H-2),

7.16 (d, 1H, J ) 8.5 Hz, H-1); HRMS (ES⁺) calcd for C₂₂H₃₀O₄-

Na (M + Na⁺) m/e 381.2042, found m/e 381.2038. HPLC system

H-16, t_R) 11.3 min, and system H-28, t_R) 11.4 min, >99%

pure.

purification of this material by HPLC with system H-21 (t_R

)

12-14 min) gave 19.1 mg (44%) of 49. Crystallization from

acetone-petroleum ether gave 16.9 mg (39%) of 49 as white

needles for bioassay. Data for 49: TLC, T-2, R_f0.45; ¹H NMR

(400 MHz, CDCl₃) δ 0.83 (s, 3H, H-18), 3.71 (s, 3H, OCH₃),

3.93 (t, 1H, J ) 8.8 Hz, H-17R), 4.56 (br s, 1H, OH), 6.55 (d,

1H, J ) 2.8 Hz, H-4), 6.63 (dd, 1H, J ) 8.6, 2.8 Hz, H-2), 7.16

(d, 1H, J ) 8.6 Hz, H-1); HRMS (ES⁺) calcd for C₂₀H₂₆O₄Na

(M + Na⁺) m/e 353.1729, found m/e 353.1737. HPLC system

H-15, t_R) 13.21 min, and system H-25, t_R) 8.15 min, >99%

pure.

Bu tyl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -15r-yl)for -

m a te (54, E15-1,4). Compound 54 was prepared by esterifi-

cation of 48 (29.1 mg, 0.0920 mmol) with ⁿBuOH as described

for 49. Purification of the residue by flash chromatography on

a 3 × 21 cm column of silica gel using 1:1 hexanes/EtOAc as

eluent gave 16.2 mg of 54. Further purification by HPLC with

system H-21 in 4 portions gave 10.5 mg (31%) of 54 as a white

solid. Crystallization from Et₂O-petroleum ether gave 9.6 mg

(28%) of 54 as white needles. Data for 54: TLC, T-3, R_f0.85;

¹H NMR (400 MHz, CDCl₃) δ 0.83 (s, 3H, H-18), 0.96 (t, 3H, J

) 7.3 Hz, -OCH₂CH₂CH₂CH₃), 3.92 (t, 1H, J ) 8.8 Hz, H-17R),

4.11 (t, 2H, J ) 6.6 Hz, -OCH₂-), 6.55 (d, 1H, J ) 2.5 Hz,

H-4), 6.63 (dd, 1H, J ) 8.6, 2.5 Hz, H-2), 7.16 (d, 1H, J ) 8.6

Hz, H-1); HRMS (ES⁺) calcd for C₂₃H₃₂ONa (M + Na⁺) m/e

395.2198, found m/e 395.2193. HPLC system H-15, t_R) 14.78

min, and system H-25, t_R)32.53 min, >99% pure.

E t h yl (17â-H yd r oxy-3-m et h oxyest r a -1,3,5(10)-t r ien -

15r-yl)for m a te (55). A solution of 53.7 mg (0.144 mmol) of

50, 400 µL (6.40 mmol) of CH₃I, and 96 mg (0.695 mmol) of

K₂CO₃in acetone (5 mL) was stirred at room temperature for

19 h and then heated at 60 °C for 6 h. The reaction mixture

was allowed to cool to room temperature, poured into H₂O (70

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

extracts were dried over Na₂SO₄and concentrated in vacuo.

Purification by flash chromatography on a 2 × 15 cm column

of silica gel using 2:1 hexanes/EtOAc as eluent gave 55.7 mg

(100%) of 55 TLC, T-2, R_f0.51.

Eth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -15r-yl)for -

m a te (50, E15-1,2). Compound 50 was prepared by esterifi-

cation of 48 (43.1 mg, 0.136 mmol) with EtOH as described

for 49. Purification of the residue by flash chromatography on

a 2 × 17 cm column of silica gel using 1:1 hexanes/EtOAc as

eluent followed by HPLC with system H-21 gave 23.3 mg (50%)

of 50. Crystallization from acetone-petroleum ether gave 18.6

mg (40%) of 50 as white needles for bioassay. Data for 50:

1

TLC, T-2, R_f0.375; H NMR (400 MHz, CDCl₃) δ 0.83 (s, 3H,

H-18), 1.29 (t, 3H, J ) 7.1 Hz, -OCH₂CH₃), 3.93 (t, 1H, J )

8.9 Hz, H-17R), 4.17 (q, 2H, J ) 7.2 Hz, -OCH₂CH₃), 4.59 (br

s, 1H, OH), 6.55 (d, 1H, J ) 2.7 Hz, H-4), 6.63 (dd, 1H, J )

8.6, 2.7 Hz, H-2), 7.16 (d, 1H, J ) 8.6 Hz, H-1); HRMS (ES⁺)

calcd for C₂₁H₂₈O₄Na (M + Na⁺) m/e 367.1885, found m/e

367.1882. HPLC system H-15, t_R) 12.55 min, and system

H-25, t_R) 11.77 min, >99% pure.

2′-F lu or oeth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -

15r-yl)for m a te (51, E15-1,2F ₁). Compound 51 was prepared

by esterification of 48 (83.9 mg, 0.265 mmol) with 2′-fluoro-

ethanol as described for 49. Purification of the residue by flash

chromatography on a 3 × 21 cm column of silica gel using 1:1

hexanes/EtOAc as eluent gave 33.6 mg of 51. Further purifica-

tion by HPLC with system H-21 in 3 portions gave 29.1 mg

(29%) of 51 as a white solid. Crystallization from acetone-

petroleum ether gave 24.9 mg (25%) of 51 as white needles

for bioassay. Data for 51: TLC, T-3, R_f0.65; ¹H NMR (400

MHz, CDCl₃) δ 0.84 (s, 3H, H-18), 3.93 (t, 1H, J ) 8.8 Hz,

H-17R), 4.36 (m, 2H, -OCH₂CH₂F), 4.63 (dt, 2H, J ) 47.5,

4.1, 4.1 Hz, -OCH₂CH₂F), 6.55 (d, 1H, J ) 2.7 Hz, H-4), 6.63

(dd, 1H, J ) 8.3, 2.7 Hz, H-2), 7.16 (d, 1H, J ) 8.3 Hz, H-1);

HRMS (ES⁺) calcd for C₂₁H₂₇FO₄Na (M + Na⁺) m/e 385.1791,

found m/e 385.1791. HPLC system H-15, t_R) 12.69 min, and

system H-25, t_R) 9.37 min, >99% pure.

E t h yl (3-Met h oxy-17â-(m et h oxym et h oxy)est r a -1,3,5-

(10)-tr ien -15r-yl)for m a te (56). A solution of 59.9 mg (0.155

i

mmol) of 55, 269 µL (1.55 mmol) of PrEt₂N, 118 µL (1.55

mmol) of MOMCl in anhydrous toluene (2 mL) was stirred at

room temperature for 22 h. The reaction mixture was poured

into H₂O (70 mL) and extracted with CH₂Cl₂(3x, 50 mL).

Combined organic extracts were dried over Na₂SO₄and

concentrated in vacuo giving a yellow oil. Purification by flash

chromatography on a 2 × 17 cm column of silica gel using 3:1

hexanes/EtOAc as eluent gave 53.7 mg (80%) of 56. Data for

56: TLC, T-2, R_f0.8; ¹H NMR (500 MHz, CDCl₃) δ 0.85 (s,

3H, H-18), 1.29 (t, 3H, J ) 7.2 Hz, CH₂CH₃), 3.37 (s, 3H,

-CH₂OCH₃), 3.77 (t, 1H, J ) 8.7 Hz, H-17R), 3.78 (s, 3H,

ArOCH₃), 4.20-4.14 (m, 2H, -CH₂CH₃), 4.66 (s, 2H, OCH₂O),

6.61 (d, 1H, J ) 2.8 Hz, H-4), 6.71 (dd, 1H, J ) 8.8, 2.8 Hz,

H-2), 7.21 (d, 1H, J ) 8.8 Hz, H-1).

P r op yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -15r-yl)-

for m a te (52, E15-1,3). Compound 52 was prepared by esteri-

fication of 48 (33.9 mg, 0.107 mmol) with ⁿPrOH as described

for 49. Purification of the residue by flash chromatography on

15r-Hydr oxym eth yl-3-m eth oxy-17â-(m eth oxym eth oxy)-

est r a -1,3,5(10)-t r ien e (57). A solution of 53.7 mg (0.125

1900 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10

Labaree et al.

mmol) of 56 in anhydrous Et₂O (2 mL) was stirred at room

temperature as 47.3 mg (1.25 mmol) of LiAlH₄was added and

reaction was stirred at room temperature for 2 h. The reaction

mixture was poured into saturated Na-K tartrate (100 mL)

and extracted with EtOAc (3×, 70 mL). Combined organic

extracts were dried over Na₂SO₄and concentrated in vacuo

giving 47.1 mg (87%) of 57 as a clear colorless oil which was

a single isomer as judged by ¹H NMR and used without further

purification in the next step. Data for 57: TLC, T-2, R_f0.31;

¹H NMR (400 MHz, CDCl₃) δ 0.88 (s, 3H, H-18), 1.05 (t, 1H, J

) 10.6 Hz, H-14), 3.38 (s, 3H, -CH₂OCH₃), 3.42 (dd, 1H, J )

10.3, 8.2 Hz, CH₂OH), 3.60 (t, 1H, J ) 8.9 Hz, H-17R), 3.78 (s,

3H, ArOCH₃), 3.89 (dd, 1H, J ) 10.3, 3.2 Hz, -CH₂OH), 4.66

& 4.69 (AB quartet, 2H, J _AB) 6.5 Hz, OCH₂O), 6.62 (d, 1H, J

) 2.6 Hz, H-4), 6.72 (dd, 1H, J ) 8.7, 2.6 Hz, H-2), 7.22 (d,

1H, J ) 8.7 Hz, H-1).

3-Meth oxy-17â-m eth oxym eth oxy-15r-tolu en esu lfon yl-

oxym eth ylestr a -1,3,5(10)-tr ien e (58). A solution of 47.1 mg

(0.109 mmol) of 57 and 457 mg (2.39 mmol) of pTsCl in

pyridine (7 mL) was allowed to stand in a sealed vial at 4 °C

for 24 h. The reaction mixture was poured into H₂O (100 mL)

and extracted with CH₂Cl₂(3×, 70 mL). Combined organic

extracts were dried over Na₂SO₄and concentrated in vacuo

giving a yellow oil. Purification by flash chromatography on a

2 × 17 cm column of silica gel using 3:1 hexanes/EtOAc as

eluent gave 60 mg (94%) of 58. Data for 58: TLC, T-2, R_f0.73;

¹H NMR (400 MHz, CDCl₃) δ 0.82 (s, 3H, H-18), 0.99 (t, 1H, J

) 10.9 Hz, H-14), 2.45 (s, 3H, ArCH₃), 3.35 (s, 3H, CH₂OCH₃),

3.52 (t, 1H, J ) 8.8 Hz, H-17R), 3.78 (s, 3H, ArOCH₃), 3.80

(dd, 1H, J ) 9.5, 8.0 Hz, CH₂OH), 4.26 (dd, 1H, J ) 9.5, 3.1

Hz, CH₂OH), 4.61 (s, 2H, OCH₂O), 6.60 (d, 1H, J ) 2.7 Hz,

H-4), 6.71 (dd, 1H, 8.6, 2.7 Hz, H-2), 7.19 (d, 1H, J ) 8.6 Hz,

H-1), 7.37 (d, 2H, J ) 8.1 Hz, Ar-H), 7.82 (d, 2H, J ) 8.1 Hz,

Ar-H).

1,3,5(10)-tr ien -15â-yl)m a lon a te (61â). A suspension of 46.1

mg of NaH (76.8 mg of 60% dispersion in oil, washed with

hexanes, 1.92 mmol) in anhydrous THF (1 mL) was stirred at

room temperature as 264 µL (1.74 mmol) of diethyl malonate

was added, and the reaction mixture was stirred at room

temperature for 30 min. To this was added a solution of 312.5

mg (0.872 mmol) of 44 in THF (2 mL), and the reaction was

stirred at room temperature for 1.5 h, poured into saturated

aqueous NH₄Cl, and extracted with CH₂Cl₂(3×, 30 mL).

Combined organic extracts were dried over Na₂SO₄and

concentrated in vacuo giving a slightly yellow oil. Purification

by flash chromatography on a 3 × 21 cm column of silica gel

using 3:1 hexanes/EtOAc as eluent gave 392 mg (87%) of 61

as an inseparable 5:1 mixture of 15R and 15â epimers. Data

for 61R: TLC, T-1, R_f0.45; ¹H NMR (400 MHz, CDCl₃) δ 1.01

(s, 3H, H-18), 1.27 (t, 3H, J ) 7.1 Hz, OCH₂CH₃), 1.29 (t, 3H,

J ) 7.1 Hz, OCH₂CH₃), 4.01 (d, 1H, J ) 4.0 Hz, HC(CO₂Et)₂),

4.22 (m, 4H, OCH₂CH₃), 5.04 (s, 2H, benzylic-H), 6.72 (d, 1H,

J ) 2.6 Hz, H-4), 6.80 (dd, 1H, J ) 8.7, 2.6 Hz, H-2), 7.21 (d,

1H, J ) 8.7 Hz, H-1), 7.33-7.44 (m, 5H, Ar-H). Data attributed

to 61â: ¹H NMR (400 MHz, CDCl₃) δ 1.21 (s, 3H, H-18), 3.72

(d, 1H, J ) 3.5 Hz, HC(CO₂Et)₂).

(3-Ben zyloxy-17-oxoestr a -1,3,5(10)-tr ien -15r-yl)m a lon -

ic Acid (62). A solution of 392 mg (0.756 mmol) of 61 and 2 g

NaOH in 30 mL of EtOH and 10 mL of H₂O was stirred at

room temperature for 17 h. The reaction mixture was concen-

trated to about 15 mL and poured into 100 mL of H₂O and

washed with Et₂O (1×, 20 mL). The aqueous phase was

adjusted to pH 2 with concentrated HCl and extracted with

EtOAc (3×, 70 mL). Combined organic extracts were washed

with saturated aqueous NaCl (1×, 50 mL), dried over Na₂SO₄,

and concentrated in vacuo giving 297 mg (85%) of 62 as a white

solid. It was one isomer by inspection of the ¹H NMR and it

was used without purification in the next step. Data for 62:

1

TLC, T-3, R_f0.08; H NMR (400 MHz, CDCl₃) δ 1.03 (s, 3H,

3-Meth oxy-17â-m eth oxym eth oxy-15r-m eth ylestr a-1,3,5-

(10)-tr ien e (59). A solution of 15.8 mg (0.0269 mmol) of 58 in

anhydrous THF (500 µL) was stirred at room temperature as

108 µL of a 1 M solution of LiEt₃BH in THF (0.108 mmol) was

added, and the reaction was stirred and heated at 65 °C for 5

h and then quenched at room temperature with EtOH (1 mL).

To the reaction mixture were added diglyme (2 mL) and 48

mg (0.431 mmol) Et₃NO, and the reaction was stirred and

heated at 150 °C for 1 h allowing the THF to evaporate. The

reaction mixture was allowed to cool to room temperature,

poured into H₂O (50 mL), and extracted with EtOAc (3×, 50

mL). Combined organic extracts were washed with 10%

Na₂S₂O₅(30 mL), H₂O (30 mL), dried over Na₂SO₄, and

concentrated in vacuo giving a colorless oil. Purification by

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

4:1 hexanes/EtOAc as eluent gave 9 mg (97%) of 59. Data for

H-18), 4.14 (d, 1H, J ) 3.7 Hz, HC(CO₂H), 5.04 (s, 2H, benzylic-

H), 6.72 (s, 1H, J ) 2.7 Hz, H-4), 6.80 (dd, 1H, J ) 8.7, 2.7

Hz, H-2), 7.20 (d, 1H, J ) 8.7 Hz, H-1), 7.33-7.45 (m, 5H,

Ar-H).

(3-Ben zyloxy-17-oxoest r a -1,3,5(10)-t r ien -15r-yl)a ce-

tic Acid (63). A solution of 297 mg (0.642 mmol) of 62 crude

in 10 mL of 2-methoxyethyl ether was stirred and heated at

162 °C for 15 min. The reaction mixture was allowed to cool

to room temperature, poured into H₂O (100 mL), and extracted

with EtOAc (3x, 70 mL). Combined organic extracts were

washed with 10% Na₂S₂O₅and concentrated in vacuo giving

a yellow oil which was used without purification in the next

step. Data for 63: TLC, T-3, R_f0.58; ¹H NMR (400 MHz,

CDCl₃) δ 1.01 (s, 3H, H-18), 5.04 (s, 2H, benzylic-H), 6.72 (d,

1H, J ) 2.6 Hz, H-4), 6.81 (dd, 1H, J ) 8.5, 2.6 Hz, H-2), 7.22

(d, 1H, J ) 8.5 Hz, H-1), 7.30-7.45 (m, 5H, Ar-H).

1

59: TLC, T-1, R_f0.73; H NMR (400 MHz, CDCl₃) δ 0.86 (s,

3H, H-18), 0.90 (t, 1H, J ) 10.2 Hz, H-14), 1.12 (d, 3H, J )

6.2 Hz, 15R-CH₃), 3.38 (s, 3H, CH₂OCH₃), 3.64 (t, 1H, J ) 8.6

Hz, H-17R), 3.79 (s, 3H, ArOCH₃), 4.65-4.67 (AB quartet, 2H,

J _AB) 6.6 Hz, OCH₂O), 6.62 (d, 1H, J ) 2.8 Hz, H-4), 6.72 (dd,

1H, J ) 8.6, 2.8 Hz, H-2), 7.22 (d, 1H, J ) 8.6 Hz, H-1).

(3-Ben zyloxy-17â-h yd r oxyestr a -1,3,5(10)-tr ien -15r-yl)-

a cetic Acid (64). A solution of crude 63 and 97 mg (2.57

mmol) of NaBH₄in EtOH (20 mL) was stirred at room

temperature for 22 h. The reaction mixture was poured into

saturated Na₂CO₃(70 mL) and washed with Et₂O (1×, 50 mL).

The aqueous phase was adjusted to pH 1 and extracted with

EtOAc (2×, 70 mL). Combined organic extracts were washed

with H₂O (2×, 50 mL), dried over Na₂SO₄, and concentrated

in vacuo giving 220 mg (82%, 2 steps) of 64 as a white solid.

3-Meth oxy-15r-m eth ylestr a -1,3,5(10)-tr ien -17â-ol (60).

A solution of 9 mg (0.026 mmol) of 59 and 5 µL of concentrated

HCl in MeOH (2 mL) was stirred at room temperature for 15

min then heated at 60 °C for 30 min. Another 25 mL of

concentrated HCl was added, and the reaction was stirred at

60 °C for 1 h. The reaction mixture was poured into saturated

aqueous NaHCO₃(5 mL) and extracted with EtOAc (3×, 5

mL). Combined organic extracts were dried over Na₂SO₄and

concentrated in vacuo giving a clear yellow oil. Purification

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

using 2:1 hexanes/EtOAc as eluent gave 7.3 mg (93%) of 60.³²

1

Data for 64: TLC, T-3, R_f0.46; H NMR (400 MHz, CDCl₃) δ

0.85 (s, 3H, H-18), 1.03 (t, 1H, J ) 10.5 Hz, H-14), 3.77 (t, 1H,

J ) 8.9 Hz, H-17R), 5.04 (s, 2H, benzylic-H), 6.71 (d, 1H, J )

2.9 Hz, H-4), 6.80 (dd, 1H, J ) 8.3, 2.9 Hz, H-2), 7.22 (d, 1H,

J ) 8.3 Hz, H-1), 7.33-7.45 (m, 5H, Ar-H).

(3,17â-Dih ydr oxyestr a-1,3,5(10)-tr ien -15r-yl)acetic Acid

(65, E15-2,0). Compound 65 was prepared from 64 (220 mg,

0.523 mmol) as described for 48 giving a yellow oil which

crystallized on standing. Further purification of 33.9 mg of this

material by HPLC with system H-20 gave 15.8 mg of 65 for

bioassay. Data for 65: TLC, T-3, R_f0.33; ¹H NMR (400 MHz,

DMSO-d₆) δ 0.70 (s, 3H, H-18), 0.91 (t, 1H, J ) 9.9 Hz, H-14),

3.51 (t, 1H, J ) 8.9 Hz, H-17R), 6.41 (d, 1H, J ) 2.7 Hz, H-4),

6.50 (dd, 1H, J )8.6, 2.7 Hz, H-2), 7.04 (d, 1H, J ) 8.6 Hz,

1

Data for 60: TLC, T-1, R_f0.31; H NMR (400 MHz, CDCl₃) δ

0.82 (s, 3H, H-18), 0.90 (t, 1H, J ) 10.2 Hz, H-14) 1.13 (d, 3H,

J ) 6.3 Hz, 15R-CH₃), 3.76 (t, 1H, J ) 8.7 Hz, H-17R), 3.79 (s,

3H, ArOCH₃), 6.63 (d, 1H, J ) 2.7 Hz, H-4), 6.72 (dd, 1H, J )

8.6, 2.7 Hz, H-2), 7.22 (d, 1H, J ) 8.6 Hz, H-1).

Dieth yl (3-Ben zyloxy-17-oxoestr a -1,3,5(10)-tr ien -15r-

yl)m a lon a te (61r) a n d Dieth yl (3-Ben zyloxy-17-oxoestr a -

Estradiol Carboxylic Acid Esters as Locally Active Estrogens

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1901

H-1); HRMS (ES⁺) calcd for C₂₀H₂₆O₄Na (M + Na⁺) m/e

353.1729, found m/e 353.1731. HPLC system H-20, t_R) 13.57

min, and system H-23, t_R) 21.28 min, >99% pure.

solid. Purification by flash chromatography on a 3 × 21 cm

column of silica gel using 3:1 hexanes/EtOAc as eluent gave

389 mg (73%) of 70 as a white solid. Data for 70: TLC, T-1, R_f

0.61; ¹H NMR (400 MHz, CDCl₃) δ 0.96 (s, 3H, H-18), 5.05 (s,

2H, benzylic-H), 5.16-5.20 (m, 2H, dCH₂), 5.67 (dd, 1H, J )

6.0, 3.2 Hz, H-15), 5.90-5.98 (m, 1H, -HCd), 5.99 (dd, 1H, J

) 6.0, 1.6 Hz, H-16), 6.74 (d, 1H, J ) 2.7 Hz, H-4), 6.80 (dd,

1H, J ) 8.6, 2.7 Hz, H-2), 7.21 (d, 1H, J ) 8.5 Hz, H-1), 7.31-

7.45 (m, 5H, ArH).

15r-Allyl-3-ben zyloxyestr a -1,3,5(10)-tr ien -17-on e (71).

A 556 mg portion (4.85 mmol) of a 35% oil dispersion of KH

was washed with hexanes and suspended in anhydrous THF

(2 mL). To this was added a solution of 389 mg (0.971 mmol)

of 70 and 1.28 g (4.85 mmol) of 18-crown-6 in THF (6 mL).

The reaction mixture was stirred at room temperature for 3

h, transferred dropwise to EtOH (10 mL), diluted with

saturated aqueous NH₄Cl (70 mL), and extracted with CH₂-

Cl₂(3×, 70 mL). Combined organic extracts were washed with

10% Na₂S₂O₅(30 mL) and H₂O (30 mL), dried over Na₂SO₄,

and concentrated in vacuo giving an orange oil. Purification

by flash chromatography on a 3 × 21 cm column of silica gel

using 4:1 hexanes/EtOAc as eluent gave 311 mg (80%) of 71

as a white solid. Data for 71: TLC, T-1, R_f0.37; ¹H NMR (400

MHz, CDCl₃) δ 0.99 (s, 3H, H-18), 1.36 (t, 1H, J ) 10.7 Hz,

H-14), 5.01-5.05 (m, 2H, dCH₂), 5.05 (s, 2H, benzylic-H),

5.74-5.85 (m, 1H, -HCd), 6.73 (d, 1H, J ) 2.6 Hz, H-4), 6.80

(dd, 1H, J ) 8.7, 2.8 Hz, H-2), 7.22 (d, 1H, J ) 8.7 Hz, H-1),

7.33-7.45 (m, 5H, ArH).

15r-Allyl-3-ben zyloxyestr a -1,3,5(10)-tr ien -17â-ol (72). A

solution of 341 mg (0.850 mmol) of 71 and 129 mg (3.40 mmol)

of NaBH₄in THF (9 mL) with 37 drops of H₂O was stirred at

room temperature for 3 h, poured into saturated aqueous NH₄-

Cl (50 mL), and extracted with EtOAc (3x, 50 mL). Combined

organic extracts were washed with 10% Na₂S₂O₅(30 mL) and

H₂O (30 mL), dried over Na₂SO₄, and concentrated in vacuo

giving a yellow oil. Purification by flash chromatography on a

3 × 21 cm column of silica gel using 2:1 hexanes/EtOAc as

eluent gave 311 mg (91%) of 72. Data for 72: TLC, T-1, R_f

0.37; ¹H NMR (500 MHz, CDCl₃) δ 0.83 (s, 3H, H-18), 1.02 (t,

1H, J ) 10.3 Hz, H-14), 3.70 (t, 1H, J ) 9.2 Hz, H-17R), 5.01-

5.06 (m, 2H, dCH₂), 5.04 (s, 2H, benzylic-H), 5.78-5.86 (m,

1H, -CHd), 6.72 (d, 1H, J ) 2.7 Hz, H-4), 6.79 (dd, 1H, J )

8.6, 2.7 Hz, H-2), 7.22 (d, 1H, J ) 8.6 Hz, H-1), 7.31-7.44 (m,

5H, ArH).

(15r-Allyl-3-ben zyloxyestr a -1,3,5(10)-tr ien -17â-yl) Ac-

eta te (73). A solution of 311 mg (0.770 mmol) of 72 and 2 mL

of Ac₂O in pyridine (4 mL) was stirred at room temperature

for 17 h. The reaction mixture was poured into H₂O (70 mL)

and extracted with CH₂Cl₂(3×, 50 mL). Combined organic

extracts were dried over Na₂SO₄and concentrated in vacuo

giving a yellow oil. Purification by flash chromatography on a

3 × 21 cm column of silica gel using 5:1 hexanes/EtOAc as

eluent gave 323 mg (94%) of 73. Data for 73: TLC, T-4, R_f

0.45; ¹H NMR (400 MHz, CDCl₃) δ 0.89 (s, 3H, H-18), 1.11 (t,

1H, J ) 10.2 Hz, H-14), 2.07 (s, 3H, OAc), 4.69 (t, 1H, J ) 8.8

Hz, H-17R), 5.01-5.07 (m, 2H, dCH₂), 5.04 (s, 2H, benzylic-

H), 5.76-5.87 (m, 1H, -HCd), 6.71 (d, 1H, J ) 2.7 Hz, H-4),

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

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

Meth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -15r-yl)-

a ceta te (66, E15-2,1). Compound 66 was prepared from 65

(50.6 mg, 0.153 mmol) as described for 8. Purification by flash

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

EtOAc/hexanes as eluent gave 38.5 mg of 66. Further purifica-

tion by HPLC with system H-21 gave 27.9 mg of 66. Crystal-

lization from Et₂O-petroleum ether gave 22.4 mg (43%) of 66

as white needles. Data for 66: TLC, T-3, R_f0.61; ¹H NMR (400

MHz, CDCl₃) δ 0.84 (s, 3H, H-18), 1.00 (t, 1H, J ) 10.5 Hz,

H-14), 3.69 (s, 3H, OCH₃), 3.74 (t, 1H, J ) 8.4 Hz, H-17R),

6.55 (d, 1H, J ) 2.6 Hz, H-4), 6.64 (dd, 1H, J ) 8.5, 2.6 Hz,

H-2), 7.17 (d, 1H, J ) 8.5 Hz, H-1); HRMS (ES⁺) calcd for

C

₂₁H₂₈O₄Na (M + Na⁺) m/e 367.1885, found m/e 367.1872.

HPLC system H-21, t_R) 16.5 min, and system H-25, t_R) 10.78

min, >99% pure.

Eth yl (3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -15r-yl)a c-

eta te (67, E15-2,2). Compound 67 was prepared from 65 (41.9

mg, 0.127 mmol) with EtOH as described for 8. Purification

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

using 2:1 EtOAc/hexanes as eluent gave 36.7 mg of 67. Further

purification by HPLC with system H-21 gave 36 mg of 67.

Crystallization from Et₂O-petroleum ether gave 26.6 mg (58%)

of 67 as fine needles. Data for 67: TLC, T-3, R_f0.67; ¹H NMR

(400 MHz, CDCl₃) δ 0.84 (s, 3H, H-18), 1.01 (t, 1H, J ) 10.5

Hz, H-14), 1.28 (t, 3H, J ) 7.0 Hz, CH₂CH₃), 3.74 (br t, 1H, J

) 8.5 Hz, H-17R), 4.15 (q, 1H, J ) 7.0 Hz, CH₂CH₃), 6.56 (d,

1H, J ) 2.7 Hz, H-4), 6.64 (dd, 1H, J ) 8.5, 2.7 Hz, H-2), 7.17

(d, 1H, J ) 8.5 Hz, H-1); HRMS (ES⁺) calcd for C₂₂H₃₀O₄Na

(M + Na⁺) m/e 381.2042, found m/e 381.2028. HPLC system

H-21, t_R) 15.70 min, and system H-25, t_R) 16.38 min, >99%

pure.

3-Ben zyloxy-17â-h yd r oxyestsr a -1,3,5(10)-tr ien -15r-yl)-

a ceta ld eh yd e (68). A solution of 10 mg (0.0279 mmol) of 67

in anhydrous toluene (500 µL)was stirred at -60 °C as 200

µL (0.3 mmol) of a 1.5 M solution of DIBAL in toluene was

added. The reaction mixture was stirred at -60 °C for 1.5 h,

poured into H₂O (3 mL), and extracted with EtOAc (2×, 3 mL).

Combined organic extracts were dried over Na₂SO₄and

concentrated in vacuo giving a clear colorless oil. Purification

by flash chromatography on a 1 × 17 cm column of silica gel

using 2:1 EtOAc/hexanes gave 1.8 mg (20%) of 68. Data for

68: TLC, T-2, R_f0.2; ¹H NMR (500 MHz, CDCl₃) δ 0.85 (s,

3H, H-18), 1.05 (t, 1H, J ) 10.5 Hz, H-14), 3.75 (br t, 1H, J )

9.7 Hz, H-17R), 4.53 (s, 1H, OH), 6.56 (d, 1H, J ) 2.8 Hz, H-4),

6.64 (dd, 1H, J ) 8.5, 2.8 Hz, H-2), 7.17 (d, 1H, J ) 8.5 Hz,

H-1), 9.79 (d, 1H, J ) 1.6 Hz, CHO).

15r-Allylestr a -1,3,5(10)-tr ien e-3,17â-d iol (69). A solution

of 1.8 mg (0.0057 mmol) of 68 in anhydrous THF (500 µL) was

stirred at 0 °C as 110 µL (0.048 mmol) of Nystead reagent and

1 µL of BF₃‚OEt₂was added. The reaction mixture was allowed

to warm to room temperature, stirred for 2 h, transferred to 1

N

HCl (3 mL), and extracted with EtOAc (3×, 2 mL).

Combined organic extracts were dried over Na₂SO₄and

concentrated in vacuo giving a clear colorless oil. Purification

by flash chromatography on a 1 × 12 cm column of silica gel

using 2:1 hexanes/EtOAc as eluent gave 1 mg (55%) of 69.³⁵

1

Data for 69: TLC, T-2, R_f0.54; H NMR (500 MHz, acetone-

(3-Ben zyloxy-15r-(3′-h yd r oxyp r op yl)est r a -1,3,5(10)-

tr ien -17â-yl) Aceta te (74). A solution of 304 mg (0.685 mmol)

of 73 in anhydrous THF (4.9 mL) was stirred at 0 °C as 890

µL (0.890 mmol) of a 1 M solution of BH₃-THF in THF was

added dropwise. The reaction mixture was stirred at room

temperature for 2 h and diluted with EtOH (2 mL) and diglyme

(10 mL). To this was slowly added 396 mg (3.56 mmol)

trimethylamine N-oxide dihydrate and the reaction was heated

at 150 °C for 1 h allowing the THF to evaporate. The reaction

was cooled to room temperature, poured into H₂O (100 mL)

and extracted with EtOAc (3×, 70 mL). Combined organic

extracts were washed with 10% Na₂S₂O₅(60 mL), H₂O (60 mL),

dried over Na₂SO₄, and concentrated in vacuo giving a yellow

oil. Purification by flash chromatography on a 3 × 21 cm

column of silica gel using 1.5:1 hexanes/EtOAc as eluent gave

d₆) δ 0.83 (s, 3H, H-18), 1.02 (t, 1H, J ) 10.3 Hz, H-14), 3.63

(t, 1H, J ) 8.9 Hz, H-17R), 4.97 (d, 1H, J ) 10.5 Hz, dCH₂),

5.03 (d, 1H, J ) 17.1 Hz, dCH₂), 5.81-5.90 (m, 1H, -CH)CH₂),

6.51 (d, 1H, J ) 2.6 Hz, H-4), 6.59 (dd, 1H, J ) 8.5, 2.6 Hz,

H-2), 7.10 (d, 1H, J ) 8.5 Hz, H-1).

17r-Allyl-3-ben zyloxyestr a -1,3,5(10),15-tetr a en -17â-ol

(70). A solution of 477 mg (1.33 mmol) of 44 in anhydrous THF

(5.15 mL) was stirred at 0 °C as 1.99 mL (3.99 mmol) of a 2 M

solution of allylmagnesium chloride in THF was added drop-

wise slowly over 10 min. The reaction mixture was stirred at

0 °C for 3 h, poured into saturated aqueous NH₄Cl (70 mL)

and extracted with EtOAc (3×, 70 mL). Combined organic

extracts were washed with 10% Na₂S₂O₅(30 mL), H₂O (50 mL),

dried over Na₂SO₄, and concentrated in vacuo giving a white

1902 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10

Labaree et al.

1

261 mg (82%) of 74. Data for 74: TLC, T-3, R_f0.59; H NMR

1H, J ) 8.4 Hz, H-17R), 4.12-4.18 (m, 2H, OCH₂CH₃), 6.56

(d, 1H, J ) 2.7 Hz, H-4), 6.63 (dd, 1H, J ) 8.3, 2.7 Hz, H-2),

7.17 (d, 1H, J ) 8.3 Hz, H-1); HRMS (ES⁺) calcd for C₂₃H₃₂O₄-

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

H-18, t_R) 8.2 min, and system H-25, t_R) 21.98 min, >99%

pure.

(400 MHz, CDCl₃) δ 0.88 (s, 3H, H-18), 1.06 (t, 1H, J ) 10.2

Hz, H-19), 2.07 (s, 3H, OAc), 3.66 (t, 2H, J ) 6.6 Hz, CH₂OH),

4.69 (t, 1H, J ) 8.6 Hz, H-17R), 5.04 (s, 2H, benzylic-H), 6.71

(d, 1H, J ) 2.9 Hz, H-4), 6.79 (dd, 1H, J ) 8.6, 2.9 Hz, H-2),

7.21 (d, 1H, J ) 8.6 Hz, H-1), 7.33-7.45 (m, 5H, Ar-H).

3-(17â-Acetoxy-3-ben zyloxyestr a-1,3,5(10)-tr ien -15r-yl)-

p r op a n ioc Acid (75). A solution of 261 mg (0.564 mmol) 74in

acetone (35 mL) was stirred at 0 °C as 211 µL (0.564 mmol) of

2.64 M CrO₃-H₂SO₄⁴³in H₂O was added. The reaction mixture

was stirred at 0 °C for 20 min, poured into 1:1 MeOH/H₂O

(120 mL), concentrated to about 60 mL, and extracted with

EtOAc (3×, 80 mL). Combined organic extracts were washed

with H₂O (20 mL), dried over Na₂SO₄, and concentrated in

vacuo giving 213 mg of 75 as a yellow foam which was used

in the next step without purification. Data for 75: TLC, T-3,

R_f0.54; ¹H NMR (400 MHz, CDCl₃) δ 0.88 (s, 3H, H-18), 1.07

(t, 1H, J ) 10.2 Hz, H-14), 2.06 (s, 3H, OAc), 4.68 (t, 1H, J )

8.5 Hz, H-17R), 5.04 (s, 2H, benzylic-H), 6.71 (d, 1H, J ) 2.7

Hz, H-4), 6.79 (dd, 1H, J ) 8.6, 2.7 Hz, H-2), 7.21 (d, 1H, J )

8.6 Hz, H-1), 7.31-7.45 (m, 5H, Ar-H).

Com p etitive Bin d in g to th e Estr ogen Recep tor ERr

a n d ERâ. Binding affinities relative to E₂were performed in

incubations with the ER (ERR³⁶) in uterine cytosol prepared

from Sprague Dawley rats that had been castrated and 24 h

prior to sacrifice. For assay, the cytosol was incubated with 1

nM [³H]E₂in the presence and absence of nonradioactive E₂,

estrone (E₁), E16-1,2 and the E₂-carboxy analogues over a

range of concentrations 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.⁴⁵The details of the assay are as

we previously described.⁸Binding affinity (RBA) was deter-

mined by analysis of the displacement curves by the curve-

fitting program Prism. The results shown in Table 1 are from

at least three separate experiments performed in duplicate.⁵²

A subset of the E₂-alkyl esters was also compared for binding

3-(3-Ben zyloxy-17â-h yd r oxyest r a -1,3,5(10)-t r ien -15r-

yl)p r op a n oic Acid (76). A solution of 213 mg (0.446 mmol)

of 75 in 5% aqueous KOH (20 mL) and MeOH (20 mL) was

stirred and heated at 55 °C for 3 h. The reaction mixture was

allowed to cool to room temperature, poured into H₂O (70 mL),

adjusted to pH 2 with 10% HCl, and extracted with Et₂O (3x,

40 mL). Combined organic extracts were washed with 10%

Na₂S₂O₅(30 mL), H₂O (30 mL), dried over Na₂SO₄, and

concentrated in vacuo giving a white foam. Purification by

flash chromatography on a 2 × 15 cm column of silica gel using

0.5:5 EtOH/CHCl₃gave 134 mg (55%, two steps) of 76. Data

to the ligand binding domain (LBD) of human ERR (M₂₅₀

-

46

V₅₉₅

)

and human ERâ (M₂₁₄-Q₅₃₀).⁴⁷The assay was per-

formed in competition with [³H]E₂in lysates of Escherichia

coli in which the LBDs are expressed as described, with the

exception that the incubation was performed overnight at 0-2

°C.⁴⁸The results, the average of 3 experiments, each performed

in duplicate, as RBAs compared to E₂and the ratio, RBA of

ERR/ERâ, are shown in Table 2.

Estr ogen ic P oten cy in Ish ik a w a Cells. The estrogenic

potency of the E₂-analogues was determined in an estrogen

bioassay, the induction of alkaline phosphatase (AlkP) in

human endometrial adenocarcinoma cells (Ishikawa) grown

in 96-well microtiter plates as we have previously described.³⁹

In short, the 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. E₂, E₁, and

E16-1,2 were included for comparison. 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. The relative stimulatory activity (RSA)

represents the ratio of EC₅₀of E₂to that of the steroid analogue

× 100, using the curve fitting program Prism to determine

the EC₅₀. Each compound was analyzed in at least three

separate experiments performed in duplicate.

1

for 76: TLC, T-3, R_f0.46; H NMR (500 MHz, CDCl₃) δ 0.82

(s, 3H, H-18), 1.00 (t, 1H, J ) 10.0 Hz, H-14), 3.73 (t, 1H, J )

8.6 Hz, H-17R), 5.04 (s, 2H, benzylic-H), 6.72 (d, 1H, J ) 2.8

Hz, H-4), 6.79 (dd, 1H, J ) 8.5, 2.8 Hz, H-2), 7.22 (d, 1H, J )

8.5 Hz, H-1), 7.21-7.44 (m, 5H, Ar-H).

3-(3,17â-Dih yd r oxyest r a -1,3,5(10)-t r ien -15r-yl)p r op i-

on ic Acid (77, E15-3,0). Compound 77 was prepared from

76 (104 mg, 0.239 mmol) as described for 48. The filtrate was

concentrated in vacuo giving 82 mg (100%) of 77 as a yellow

oil. HPLC purification of a 43.3 mg portion of this material

using system H-22 gave 36.2 mg of 77 for bioassay. Data for

1

77: TLC, T-3, R_f0.44; H NMR (400 MHz, DMSO-d₆) δ 0.68

(s, 3H, H-18), 3.48 (t, 1H, J ) 8.7 Hz, H-17R), 6.41 (d, 1H, J )

2.6 Hz, H-4), 6.50 (dd, 1H, J ) 8.6, 2.6 Hz, H-2), 7.04 (d, 1H,

J ) 8.6 Hz, H-1); HRMS (ES⁺) calcd for C₂₁H₂₈O₄Na (M + Na⁺)

m/e 367.1885, found m/e 367.1882. HPLC system H-17, t_R

11.2 min, and system H-23, t_R) 32.9 min, >99% pure.

)

Meth yl 3-(3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -15r-yl)-

p r op ion a te (78, E15-3,1). Compound 78 was prepared from

77 (51.6 mg, 0.150 mmol) as described for 8. Purification by

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

1:1 hexanes/EtOAc as eluent gave 48.5 mg of 78. Further

purification by HPLC with system H-21 gave 42.3 mg. Crys-

tallization from Et₂O-petroleum ether gave 31.2 mg (58%) of

78 as white needles. Data for 78: TLC, T-3, R_f0.62; ¹H NMR

(400 MHz, CDCl₃) δ 0.83 (s, 2H, H-18), 0.98 (t, 1H, J ) 10.2

Hz, H-14), 3.71 (s, 3H, OCH₃), 3.73 (t, 1H, J ) 8.5 Hz, H-17R),

6.57 (d, 1H, J ) 2.7 Hz, H-4), 6.65 (dd, 1H, J ) 8.3, 2.7 Hz,

H-2), 7.18 (d, 1H, J ) 8.3 Hz, H-1); HRMS (ES⁺) calcd for

In Vivo Estr ogen Bioa ssa ys: Uter in e Weigh t. Systemic

estrogenic potency was determined by an uterotrophic assay

in immature rats as described.⁴⁹Female Sprague-Dawley

rats, 22 days old, were injected subcutaneously daily for 3 days

with a solution of 0.1 mL of the various steroids in sesame oil.

Control animals received sesame oil. On the fourth day, the

animals were killed, the uteri were removed, dissected, blotted,

and weighed. Except where noted, each compound was assayed

in 2 separate experiments with n ) 5. The results comparing

100 µg (total dose) of the E₂-analogues to 5 ng of E₂run

concurrently are presented in Table 3.

C

₂₂H₃₀O₄Na (M + Na⁺) m/e 381.2042, found m/e 381.2032.

In Vivo Estr ogen Bioa ssa ys: Va gin a l Red u cta ses. The

estrogenic action of locally applied estrogens on the vagina was

determined by measuring the induction of vaginal reductases.⁵⁰

Female CD-1 mice were ovariectomized, and 1 week later the

E₂-alkyl esters or E₂were instilled into the vagina in 10 µL of

sesame oil. (The details of the assay and the use of sesame oil

to increase the t_1/2of the steroid has been previously dis-

cussed.⁸) Briefly, the next morning 2,3,5-triphenyltetrazolium

chloride is injected into the vagina and 30 min later the

animals are euthanized and the vaginas removed, washed, and

extracted with ethanol/tetrachloroethylene (3:1). The formazan

product in the organic extract is quantified at 500 nm. Except

where noted, each compound was assayed on at least two

HPLC system H-18, t_R) 8.5 min, and system H-25, t_R) 14.45

min, >99% pure.

Eth yl 3-(3,17â-Dih yd r oxyestr a -1,3,5(10)-tr ien -15r-yl)-

p r op ion a te (79, E15-3,2). Compound 79 was prepared from

77 (51.65 mg,0.150 mmol) with EtOH as described for 8.

Purification by flash chromatography on a 2 × 17 cm column

of silica gel using 1:1 hexanes/EtOAc as eluent gave 48.7 mg.

Further purification by HPLC with system H-21 gave 41.3 mg.

Crystallization from Et₂O-petroleum ether gave 30.9 mg

(56%) of 79 as white needles. Data for 79: TLC, T-3, R_f0.71;

¹H NMR (400 MHz, CDCl₃) δ 0.81 (s, 3H, H-18), 0.98 (t, 1H, J

) 10.1 Hz, H-14), 1.28 (t, 3H, J ) 7.1 Hz, OCH₂CH₃), 3.72 (t,

Estradiol Carboxylic Acid Esters as Locally Active Estrogens

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Ester a se. Esterase activity was measured in rat hepatic

microsomes essentially using the conditions described⁵¹with

some minor modifications. Briefly, rat hepatic microsomes

were incubated with 50 µM E2-alkyl ester. Since the rates of

reaction are widely different for the various esters, the

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cordingly to obtain linear kinetics. In every experiment, E16-

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E7R-2,0 by HPLC with system H-29 and E11-2,0 and E11-3,0

by HPLC with system H-30. Peaks for the hydrolysis product

(E16-1,0 t_R) 10.7 min; E15-1,0 t_R) 6.9 min; E15-2,0 t_R) 9.9

min; E15-3,0 t_R) 14 min; E11-2,0 t_R) 11.9 min; E11-3,0 t_R

)

14.8 min; E7R-2,0 t_R) 8.6 min) and the internal standard (t_R

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Ack n ow led gm en t. This work was supported in part

by NIH Grant Nos. CA37799 and HL61432 and by the

Yale Cancer Center.

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J M0204340

Article Doi

Synthesis and evaluation of B-, C-, and D-ring-substituted estradiol carboxylic acid esters as locally active estrogens

DOI: 10.1021/jm0204340

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Full text of DOI:10.1021/jm0204340

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