E-ring modified steroids as novel potent inhibitors of 17β- hydroxysteroid dehydrogenase type 1

J. Med. Chem. 2005, 48, 5749-5770

5749

E-Ring Modified Steroids as Novel Potent Inhibitors of 17â-Hydroxysteroid

Dehydrogenase Type 1

Delphine S. Fischer,^†Gillian M. Allan,^†Christian Bubert,^†Nigel Vicker,^†Andrew Smith,^†Helena J. Tutill,^‡

Atul Purohit,^‡Lynn Wood,^#Graham Packham,^#Mary F. Mahon,^§Michael J. Reed,^‡and Barry V. L. Potter*^,†

Medicinal Chemistry, Department of Pharmacy and Pharmacology and Sterix Ltd., University of Bath, Claverton Down BA2

7AY, U.K., Endocrinology and Metabolic Medicine and Sterix Ltd., Faculty of Medicine, Imperial College, St. Mary’s Hospital,

London W2 1NY, U.K., Cancer Research UK Oncology Unit, Cancer Sciences Division, University of Southampton School of

Medicine, Southampton General Hospital, Southampton SO16 6YD, U.K., and Department of Chemistry, University of Bath,

Bath BA2 7AY, U.K.

Received April 14, 2005

17â-Hydroxysteroid dehydrogenases (17â-HSDs) are an important class of steroidogenic

enzymes that regulate the bioavailability of active estrogens and androgens and are as yet a

relatively unexploited therapeutic target. Based on our investigations and those of others, E-ring

modified steroids were identified as a useful template for the design of inhibitors of 17â-HSD

type 1, an enzyme involved in the conversion of estrone into estradiol. The synthesis and

biological evaluation of a new series of N- and C-substituted 1,3,5(10)-estratrien-[17,16-c]-

pyrazoles and the corresponding SAR are discussed. Among the N-alkylated analogues, the

most potent inhibitor was the 1′-methoxyethyl derivative, 41, with an IC₅₀of 530 nM in T47-D

human breast cancer cells. The X-ray crystal structure of the 1′-isobutyl derivative, 37, was

determined. Further optimization of the template using parallel synthesis resulted in a library

of C5′-linked amides from which 73 emerged. This pyridylethyl amide had an IC₅₀of 300 nM

and its activity, with that of 41, suggests the importance of hydrogen bond acceptor groups in

the pyrazole side chain. Both 41 and 73 displayed selectivity over 17â-HSD type 2, and

preliminary investigations showed 41 to be nonestrogenic in vitro in a luciferase reporter gene

assay in contrast to the parent pyrazole 25. Molecular modeling studies, which support these

findings, and a QSAR, the predictive power of which was demonstrated, are also presented.

Introduction

synthesis of estrogens where the successive action of

steroid sulfatase (STS) and 17â-hydroxysteroid dehy-

drogenase (17â-HSD) type 1 convert estrone-3-O-sulfate

(E1S) into the biologically active estradiol (E2). Both of

these enzymes therefore represent attractive targets for

estrogen suppression strategies. While STS inhibitors

are in clinical trials,⁸we decided to explore the inhibi-

tion of 17â-HSD type 1 as a therapeutic target.

Hormones play a major role in the promotion and

development of breast cancer because between one-third

and two-thirds of all breast carcinomas rely on estrogens

for their sustained growth. Steroidogenic enzyme in-

hibitors can potentially deplete circulating and tissue

levels of active estrogens by blocking their biosynthetic

pathways. Such compounds therefore represent an

attractive treatment option for patients with hormone-

dependent breast cancer (HDBC).^1-5

In postmenopausal women, who account for nearly

80% of all cases of HDBC, almost all the estrogens are

synthesized extraglandularly from inactive precursors.

Aromatase inhibitors, which prevent the conversion of

androgens into estrogens, have proven therapeutic and

are currently used as adjuvant therapy to treat HDBC.⁴

It was subsequently shown however that steroid sulfa-

tase is likely to be the primary enzyme responsible for

estrone production in hormone-dependent breast tu-

mors: estrone production in such tumors is approxi-

mately 10-fold higher per gram of protein for the

sulfatase (estrone-3-O-sulfate to estrone) pathway than

for aromatase.^6,7Estrone sulfate precursor may thus act

as a reservoir for the peripheral and intratumoral

The 17â-HSD type 1 enzyme is a 34.9 kDa protein

with 327 amino acid residues that belongs to the class

of 17â-hydroxysteroid dehydrogenases.⁹These enzymes

catalyze the interconversion of the oxidized and reduced

form of estrogenic and androgenic steroids at the 17-

position. The conversion of E1 and E2 involving the

17â-HSDs is outlined in Figure 1. Although they are

reversible, their activity is mainly unidirectional and

thus can be classified as reductive or oxidative. Of

the thirteen isoforms of the enzyme that have been

identified to date,¹⁰eleven exist in humans where they

regulate the bioavailability of active estrogens and

androgens.¹¹While they all require NAD(P)H or NAD-

(P) as a cofactor, each type has a selective substrate af-

finity, a directional activity, and a particular tissue dis-

tribution. The 17â-HSD type 1 isoform, which converts

E1 to E2 and to a minor extent dehydroepiandrosterone

(DHEA) to 5-androsten-3â,17â-diol (∆⁵-diol), is found in

the ovaries, endometrium, placenta, and in normal and

cancerous breast tissue.¹²Since it preferentially reduces

its substrate,¹³the activity of 17â-HSD type 1 directly

* Author to whom correspondence should be addressed. E-mail:

B.V.L.Potter@Bath.ac.uk. Phone: +44 1225 386639. Fax: +44 1225

386114.

^†University of Bath, Department of Pharmacy & Pharmacology.

^‡Imperial College London.

^§University of Bath, Department of Chemistry.

^#University of Southampton.

Published on Web 08/17/2005

5750 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18

Fischer et al.

Chemistry

The syntheses of the various 17â-HSD type 1 inhibi-

tors discovered are depicted in Schemes 1-8. Estrone

was initially protected as a 3-tert-butyldimethylsilyl

ether, 1, and subsequently formylated at C16 as de-

scribed previously (Scheme 1).^23-26The reaction of 1

with sodium methoxide and ethyl formate afforded 2 in

98% yield. Conversion of the enol functionality of 2 into

a cyano group was performed according to a literature

procedure,²⁷using O,N-bis-(trifluoroacetyl)-hydroxy-

lamine in a mixture of toluene and pyridine at reflux.

This gave 3 in 71% yield, and subsequent deprotection

using a solution of tetrabutylammonium fluoride (TBAF)

in THF afforded 4 in a good yield.

Figure 1. Conversion of E1 and E2 by 17â-HSD subtypes.

The final product 4 was found to exist as a mixture

of diastereoisomers in solution in deuterated DMSO

with a ratio of 1.6:1 for the two components. Structural

assignment of the isomers was made on the basis of ¹H

NMR-NOE difference experiments involving the sig-

nals for the proton at C16 in each isomer (δ 3.84 and δ

4.38) and C18 (δ 0.91 and δ 0.93). Significant enhance-

ment of the C16-H signal at δ 4.38 was observed upon

irradiation of a sample of 4 at the resonance frequency

of the C18 methyl peak of the minor isomer (δ 0.91).

The same experiment on the methyl peak of the major

isomer (δ 0.93) did not produce any signal enhancement

in the region of C16-H, suggesting a cis (18-methyl,-

16-cyano) 16â-configuration for this product. The recip-

rocal experiments (irradiation at the resonance fre-

quency of the C16-H signals) confirmed these observa-

tions. These findings were in agreement with the

reported stereochemical ratio of isomers for the 3-methyl

ether analogue of 4, where the â product was identified

as the major isomer formed.²⁸

Further functionalization at C16 was performed using

3-O-benzyl precursors. The benzyl ether 5 was prepared

by an adaptation of a reported procedure²⁹by reacting

E1 with sodium hydride and benzyl bromide (Scheme

1). Further treatment of 5 with sodium hydride and

methyl iodide in DMSO afforded 6 in 49% yield. Depro-

tection of the latter by hydrogenolysis gave 7. Compound

9 was then accessed following a similar route to that

applied for the preparation of 3, via the 16-hydroxy-

methylene intermediate 8. The isomeric mixture of 9

was then subjected to alkylation with methyl iodide to

yield 10 as a single isomer. Since we observed correla-

tion with similar 3-methyl ether derivatives of E1, the

orientation of the cyano group of 10 was assigned â in

accordance with the report of Schaub and co-workers.²⁸

Subsequent cleavage of the benzyl protecting group of

10 afforded 11.

Figure 2. Inhibitors of 17â-HSD type 1 (I-III) and novel

E-ring modified targets (IV).

supports the growth and development of estrogen-

dependent tumors.^14-16

Although the therapeutic value of inhibiting 17â-HSD

type 1 has been known for many years, relatively few

potent and selective inhibitors have been reported in

the literature. Early work on affinity ligands identified

3-O-methyl-16-bromoacetoxyestradiol,¹⁷and acetylenic

16-seco-estradiol as alkylating agents.¹⁸More recently,

Poirier et al. reported 16-(bromoalkyl)-estradiols to be

active against human placental 17â-HSD type 1.¹⁹The

best inhibitor in this series, 16R-bromopropyl-estradiol,

I (Figure 2), had an IC₅₀of 0.46 µM and was shown to

act via a competitive irreversible mechanism. It was a

pure agonist on the estrogen-sensitive human breast

tumor cell line ZR-75-1, which is undesirable in endo-

crine therapy. Attempts to suppress the intrinsic estro-

genicity of such compounds using alkylamide side

chains led to loss of activity.²⁰

Interest in the inhibition of 17â-HSD type 1 was

initiated after the identification of various 16-substi-

tuted steroids as moderately potent inhibitors of this

enzyme. Prompted by these results, we decided to

explore the potential of modification of the 16 position

on the E1 nucleus, a strategy that has resulted in a

recent patent application by our group.²¹Recently, we

reported the discovery of the highly potent 17â-HSD

type 1 inhibitors II and III (Figure 2), which showed

selectivity over 17â-HSD type 2.²²As an extension of

this work, the 16 position was further explored alone

and in combination with 17-substituents. This culmi-

nated in the discovery of novel E-ring pyrazole steroids

(general formula IV, Figure 2) as potent, nonestrogenic

inhibitors of 17â-HSD type 1 that did not inhibit 17â-

HSD type 2. Herein, we report the chemical synthesis

and the biological activity of these inhibitors and

describe the SAR studies that led to the identification

of active novel compounds. Furthermore, molecular

modeling studies and QSAR give encouraging in silico

results that support this work.

The investigation of compounds devoid of chirality at

C16 was then initiated with the 16-hydroxymethylene

derivative 12, which was obtained after deprotection of

the synthetic intermediate 2 using TBAF in THF

(Scheme 1). Further homologation at the C1′ position

was possible by reacting the precursors 1 or 5 with a

series of ethyl esters (Scheme 2). Literature precedent

for the synthesis of C1′-substituted 16-hydroxymethyl-

ene compounds include 16-acetylation and 16-trifluo-

roacetylation of 3-methoxyestrone.^30,31The correspond-

ing 3-hydroxy analogues were prepared, along with a

pyridyl and an ethyl ester derivative, as part of our SAR

investigation. To this end, a solution of 1 in toluene was

E-Ring Modified Steroids as Inhibitors of 17â-HSD

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5751

Scheme 1^a

^aReagents and conditions: (a) imidazole, tert-butyldimethylsilyl chloride (TBDMSCl), DMF; (b) NaOMe/toluene, HCO₂Et; (c) O,N-bis-

(trifluoroacetyl)-hydroxylamine, toluene/pyridine, reflux; (d) TBAF/THF, (e) NaH/DMF, BnBr; (f) NaH/DMSO, MeI; (g) Pd/C, H₂, MeOH/

t

THF; (h) BuOK/toluene, HCO₂Et; (i) NaH/DMF, MeI.

Scheme 2^a

Scheme 3^a

t

^aReagents and conditions: (a) BuOK, (CH₃)₂CH(CH₂)₂ONO;

(b) H₂NOH.HCl, NaOAc, MeOH/H₂O; (c) H₂NOCH₂CHCH₂.HCl,

NaOAc, MeOH/H₂O; (d) 230 °C.

toluene to give 16 in 80% yield. Subsequent deprotection

by hydrogenolysis under standard conditions afforded

20.

For some of these C1′-substituted derivatives, enol-

ketone tautomerism was observed by ¹H NMR. Interest-

ingly, while the methyl analogues 16 and 20 were a

mixture of tautomers in a solution of CDCl₃, the trifluoro

derivatives 13 and 17 were found to exist predominantly

in the enol form. This is in accordance with previous

reports, where it is believed that the electronegative

character of the trifluoromethyl group in addition to the

electronic properties of fluorine atoms are involved in

the stabilization of the enolic form.^31,32The pyridyl

derivatives 14 and 18, as well as their ethyl ester

analogues 15 and 19, with extended conjugated systems,

were also found to be totally enolized in CDCl₃.

^aReagents and conditions: (a) ^tBuOK or NaOEt/toluene,

R¹CO₂Et; (b) TBAF/THF; (c) Pd/C, H₂, MeOH/THF.

treated with potassium tert-butoxide and ethyl trifluo-

roacetate or ethyl nicotinate. The corresponding prod-

ucts 13 and 14, obtained respectively in 100% and 77%

yield, were then deprotected to afford 17 and 18.

Reaction of 1 with diethyl oxalate in the presence of

sodium ethoxide gave 15, which after deprotection

afforded 19 in high yield. Similarly, 5 was reacted with

potassium tert-butoxide and ethyl acetate at reflux in

5752 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18

Fischer et al.

Scheme 4^a

^aReagents and conditions: (a) H₂NNH₂‚H₂O, EtOH, reflux; (b) NaH/DMF, R¹X; (c) Pd/C, H₂, MeOH/THF.

Scheme 5^a

^aReagents and conditions: (a) H₂NNH₂‚H₂O, EtOH, reflux; (b) ^tBuOK/toluene, CH₂CHCN; (c) TBAF/THF; (d) H₂NNH(CH₂)₂CN, EtOH.

Scheme 6^a

Scheme 7^a

^aReagents and conditions: (a) H₂NNH₂‚H₂O, EtOH, reflux; (b)

TBAF/THF.

^aReagents and conditions: (a) BuOK, (CO₂Et)₂, toluene; (b)

H₂NNH₂‚H₂O, EtOH/DCM, reflux, then rt; (c) NaOH/EtOH, reflux;

(d) DMAP, EDC, NEt₃, DCM, R¹NHR²; (e) H₂, Pd/C, EtOH/THF.

t

Preparation of the 16-oxime derivative 21 was per-

formed as reported previously (Scheme 3).³³This iso-

steric analogue of 12 was further reacted with hydroxy-

lamine hydrochloride to give the bis-oximino derivative

22 in moderate yield. Because of the enolic character of

12 and that of some of its C1′ analogues, it was

envisaged that internal hydrogen bonding might occur

between the carbonyl at C17 and the C1′-OH group to

create a pseudo-E-ring system on the steroidal backbone

(Figure 3). Similarly, this was conceivable in the oximes

21 and 22, which prompted us to investigate the

synthesis of E-ring five- and six-membered heterocyclic

derivatives of E1 as bioisoteric replacements for this

intramolecular hydrogen bond network.

A [17,16-b]-pyridine derivative of E1 was initially

synthesized as a putative six-membered ring mimic

where the CdN bond is an isostere of the carbonyl at

E-Ring Modified Steroids as Inhibitors of 17â-HSD

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5753

Table 1. 1′- and 2′-Alkylated Pyrazole Derivatives of E1

Scheme 8^a

R

R¹

Bn

TBDMS

H

Bn

TBDMS

H

CH₃

27

29

31

33

35

37

39

41

46

28

30

32

34

36

38

40

42

47

CH₂CH(CH₃)₂

CH₂CO₂CH₃

(CH₂)₂OCH₃

(CH₂)₂CN

^aReagents and conditions: (a) H₂, Pd/C, EtOH/THF; (b) LiAlH₄/

THF.

44

45

derivatives 27-34 in yields ranging from 19% to 48%.

Debenzylation by hydrogenolysis yielded the final prod-

ucts 35-42 (Table 1).

Structural assignment of the regioisomers was ini-

1

tially made on the basis of H NMR-NOE difference

Figure 3. Intramolecular hydrogen bonding in C1′ derivatives

experiments between compounds 27 and 28. Irradiation

of a sample of 27 (the less polar isomer) at the resonance

frequency of the C18-methyl signal only yielded back-

ground noise. The same experiment performed on 28

(the more polar isomer) yielded a significant peak at δ

3.81, corresponding to the enhancement of the signal

of the N-methyl group. Given that the NOE effect is only

observed over short distances (2-4 Å), the N-methyl

pyrazole derivative 28 was assigned the structure of a

2′-alkylated compound. Structural assignment of the

other members of this series was made by analogy. For

each alkyl motif, we also noticed that the chemical shift

of the heterocyclic proton (C5′-H) showed a downfield

shift of ca. 0.2 ppm in all of the more polar isomers. This

can be used to predict the position of the N-substitution

by comparing the ¹H NMR spectra of two regioisomers.

of E1 and E-ring fused systems proposed as bioisosters.

C17. The synthesis was accomplished via the thermal

rearrangement of an O-allyl oxime precursor, a method

widely used to generate pyridine heterocycles,^34-37

which has however, to the best of our knowledge, not

been applied to steroidal systems. Following the adapta-

tion of a literature procedure,³⁸the O-allyl oxime

derivative 23 was easily obtained in 94% yield after

treatment of E1 with O-allylhydroxylamine hydrochlo-

ride and sodium acetate in a mixture of MeOH and H₂O.

The latter was then heated neat at 230 °C for 46 h to

give a crude dark material, from which 24 was isolated

in spectroscopically pure form after repeated column

chromatography. The harsh conditions required for this

reaction, which resulted in loss of material through

decomposition and a consequent tedious separation of

24 from the crude material explains the poor yield of

<10% for this final product. This is also consistent with

moderate to low yields generally reported for this

rearrangement.^34-37

To probe the SAR for five-membered E-ring ana-

logues, a series of N- and C-substituted 1,3,5(10)-

estratrien-[17,16-c]-pyrazoles was synthesized (Schemes

4-8). Besides retaining the carbonyl isostere CdN at

C17, the fused pyrazole ring system is also a convenient

scaffold with three different attachment points for the

rapid exploration of the three-dimensional space. Early

reports describe the preparation of steroidal E-ring

pyrazoles by reaction of hydrazine with an appropriate

hydroxymethylene precursor.^39-41This strategy was

applied to the synthesis of our target compounds start-

ing from 8, while 3-hydroxy-1,3,5(10)-estratrien-[17,16-

c]-pyrazole, 25, was prepared from 12 as previously

reported.⁴²Condensation of 8 with hydrazine monohy-

drate in EtOH at reflux rapidly yielded the versatile

precursor 26 in high yield. N-Alkylation was then

performed under standard conditions, using sodium

hydride in DMF, followed by treatment with the desired

alkyl halide. As expected, each alkylation yielded two

regioisomers corresponding to 1′- and 2′-alkylated prod-

ucts. Their difference in polarity allowed partial or total

separation by flash chromatography, yielding the benzyl

The conclusive evidence for assigning the structures

of the regioisomers was provided by the X-ray crystal

structure of the N-isobutyl derivative 37 (Figure 4). This

compound was obtained after deprotection of the less

polar regioisomer isolated after N-alkylation of 26 with

isobutyl bromide. The experiment was performed on a

crystal (approximate dimensions 0.50 × 0.50 × 0.30

mm³) obtained by recrystallization from MeOH. As

1

predicted from the H NMR experiments on 27/28, 37

was found to correspond to a 1′-alkylated product. The

ORTEX⁴³plot shows all four steroidal rings with the

additional heterocyclic system fused to the 16 and 17

positions. The presence of a molecule of MeOH in the

cell indicates that 37 cocrystallized with the solvent, to

which it hydrogen bonds via O1 of the hydroxyl group

at C3. Moreover, N2′ exhibits a hydrogen bond to the

methanolic hydrogen, H2, of its closest lattice neighbor,

affording hydrogen-bonded ribbons in the supramolecu-

lar array [H1‚‚‚O2 1.847 Å, H2‚‚‚N2′ 1.911(1) Å; O1-

H1-O2 171.1(1)°, O2-H2-N2′ 172.6(1)°]. This is the

first report of a crystal structure of a substituted

pyrazole fused E-ring steroidal derivative.

While the N-methyl, N-isobutyl, N-methyl acetate and

N-(2-methoxyethyl) pyrazole derivatives could easily be

accessed following the reaction sequence described

above, the synthesis of the N-cyanoethyl analogues

5754 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18

Fischer et al.

Figure 4. ORTEX⁴³plot of the X-ray crystal structure of 37. Ellipsoids are shown at the 30% probability level.

proved to be problematic. The use of bromopropionitrile

to alkylate 26 under a variety of conditions only yielded

mixtures in which the starting material was predomi-

nant. A 1,4-addition of the pyrazole anion on acrylo-

nitrile was then envisaged to access the N-cyanoethyl

derivative. For this pathway, the precursor 2 was

reacted with hydrazine monohydrate to give 43 in 81%

yield. Subsequent reaction with acrylonitrile in toluene

yielded the regioisomers 44 and 45 in low yields.

Deprotection using TBAF in THF afforded the phenolic

derivatives 46 and 47.

pling reactions were carried out using a Radleys Green-

house synthesizer followed by purification by flash

chromatography, affording 56-62 in moderate to low

yields. A diminished reactivity of the carboxylic acid at

C5′ might explain the difficulties encountered in cou-

pling 55 with amines in high yields. Subsequent depro-

tection of the benzyl precursors under a hydrogen

blanket with Pd/C yielded the amides 63-69 (Table 2)

in a high average purity of 96%. It is noteworthy that

the tetrahydrofuran derivative 66 was obtained upon

hydrogenation of the corresponding furan precursor 59

during the deprotection step.

A variation of this method was necessary for the

preparation of 71, since the pyridyl amide side chain

was shown to be unstable under hydrogenation condi-

tions. The key precursor 55 was therefore deprotected

prior to coupling (method B) to give 70 in 98% yield.

Reaction of the latter with 3-picolylamine afforded 71

in a poor 16% yield. The pyridyl-containing amides 72

and 73 were similarly prepared; however, as in the case

of 71, poor reactivity and solubility of the phenolic

precursor 70 may have contributed to the low yields

obtained.

In an attempt to improve on the yields for the

synthesis of N-cyanoethyl derivatives, 2 was directly

reacted with N-cyanoethylhydrazine (Scheme 5). The

partially condensed product 48 was obtained in 71%

yield as the sole product of the reaction. Its structure

1

was assigned on the basis of H and ¹³C NMR data.

Reaction of 48 with TBAF afforded the aromatized and

deprotected product 47 in good yield.

To further explore the SAR around the pyrazole

nucleus, the synthesis of C5′-substituted pyrazole de-

rivatives of E1 was also envisaged, starting from the

C1′-substituted 16-hydroxymethylene derivatives previ-

ously prepared (Scheme 6). Both 17 and 20 were reacted

with hydrazine monohydrate, yielding 49 and 50 in 60%

and 35% yield, respectively. Because of the poor solubil-

ity of the 3-pyridyl precursor 18, annulation to give the

pyrazole was performed on the protected derivative 14.

The product of this reaction was however not the

aromatized heterocyclic derivative but the hydrated

analogue 51, which was obtained in a yield of 89% as a

single diastereoisomer. Removal of the TBDMS group

using TBAF in THF afforded the dehydrated product

52 in good yield.

Additional C5′-linked pyrazole derivatives of E1 were

prepared using solution-phase parallel synthesis (Scheme

7). The intermediate 55, easily accessible in three steps

from 5, was chosen as the common intermediate for the

preparation of a library of amides. Reaction of 5 with

diethyloxalate gave 53, which upon condensation with

hydrazine monohydrate, afforded the pyrazole 54. Hy-

drolysis of the ethyl ester released the free acid 55,

which was obtained in an overall yield of 77% from 5.

Diversity was then introduced by amide coupling to 55

(method A), after activation of the carboxylic acid moiety

with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

(EDC) in dichloromethane (DCM), followed by treatment

with the required amine in the presence of 4-dimethyl-

aminopyridine (DMAP) and triethylamine. These cou-

Compound 54 was used for the synthesis of additional

final C5′-linked derivatives (Scheme 8). Debenzylation

of 54 yielded 74 in 82% yield, while reduction of its ester

moiety with LiAlH₄followed by deprotection afforded

76 in good yield.

Results and Discussion

Inhibition of 17â-HSD Type 1 and Type 2. The

compounds synthesized were tested for their ability to

inhibit 17â-HSD type 1 activity in T47-D cells. As a

measure of selectivity, inhibition of type 2 activity was

also measured for each compound in MDA-MB-231 cells.

The evaluation of the ability to inhibit these enzymes

was performed by measuring the amount of labeled E1

or E2 formed from the labeled natural substrate. The

percentage of inhibition achieved for a 10 µM concentra-

tion of the inhibitor, as well as the IC₅₀for some of the

most potent compounds, is given in Tables 3-6.

Preliminary investigations around the 16-position of

the E1 skeleton demonstrated that the active site of the

enzyme was tolerant of small groups with varied steric

and electronic properties. This was exemplified by the

activity of compounds 4, 7, and 11, where enzymatic

activity was inhibited in excess of 77% for a 10 µM

concentration of compound. Interestingly, the 16-cyano

E-Ring Modified Steroids as Inhibitors of 17â-HSD

Table 2. C5′-Linked Pyrazole Amide Derivatives^a

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5755

^a- - - indicates the point of attachment to the amide nitrogen.

derivative 4, which is a mixture of diastereoisomers (R/â

1:1.6), was a moderately potent inhibitor of 17â-HSD

type 1 with an IC₅₀of 10 µM. Compound 11, where the

cyano group is fixed in â orientation, was found to be

equipotent to 4 at 10 µM.

of 22 when compared to 21. This prompted us to probe

the activity of compounds containing smaller ring

systems, and the choice of a pyrazole E-ring system was

driven by several considerations. First, 3-hydroxy-1,3,5-

(10)-estratrien-[17,16-c]-pyrazole 25 had been reported

as a 17â-HSD type 1 inhibitor by Sweet et al.,⁴²having

a K_iof 4.1 µM on human placental enzyme. Second, five-

membered ring systems and in particular pyrazole

heterocycles are synthetically accessible, the corre-

sponding steroid derivatives being obtained from 16-

hydroxymethylene precursors. In addition, the pyrazole

heterocycle is amenable to rapid SAR exploration with

three potential sites of diversification, namely N1′, N2′,

and C5′, and the CdN bond in the heterocycle is a

carbonyl mimic.

In the N-alkylated pyrazole series, all the 1′-substi-

tuted compounds significantly inhibited 17â-HSD type

1 activity; however, selectivity over the type 2 isozyme

was modest for most of these derivatives. An inhibition

of 94% or higher was observed at 10 µM for compounds

bearing a side chain with directional electronics, 39, 41,

and 46 showing IC₅₀values between 530 and 920 nM.

A bulky hydrophobic group was not as well tolerated,

as suggested by 37, which was the weakest inhibitor of

the series. The methoxyethyl derivative 41 was the most

potent analogue (IC₅₀of 530 nM), suggesting the

importance of hydrogen bond acceptor interactions of

the ether oxygen atom in the side chain.

Removing the chirality at C16 led to the discovery of

highly potent and selective inhibitors of 17â-HSD type

1. This was exemplified in the series of 16-hydroxy-

methylene analogues by the unsubstituted compound

12. With an IC₅₀of 110 nM, it is among the most potent

17â-HSD type 1 inhibitors reported to date and is also

selective over 17â-HSD type 2. Measurement of the

selectivity of 12 and other compounds reported herein

for other steroidogenic enzymes such as 17â-HSD type

3, 3â-HSD, and the 11â-HSDs is part of a planned

program of work in this area. Probing for hydrophobic

interactions, hydrogen bonding capabilities, or both in

the active site gave the moderate inhibitors 17 and 19.

Although 10 times less potent than 12, the isosteric 16-

oximino-estrone 21 (IC₅₀) 1 µM) remained of interest

for further investigations. Replacing the C17 carbonyl

by an oxime moiety unfortunately further depleted the

activity, 22 achieving only 55% inhibition of 17â-HSD

type 1 at 10 µM.

Given the possibility of a hydroxy-ketone hydrogen

bond network existing between the D-ring moieties in

compounds such as 12 or 21, we investigated E-ring

heterocyclic derivatives of E1. The six-membered ring

analogue 24 was moderately active against 17â-HSD

type 1, which was in accordance with the lower activity

Interestingly, the activity of the 2′-alkylated deriva-

tives was significantly lower than that of the corre-

5756 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18

Fischer et al.

Table 3. Inhibition of 17â-HSD Type 1 and Type 2 by C16 Derivatives of E1 and 24

^aMean of at least two measurements with typically a SD of (5%. ^bNot determined.

Table 4. Inhibition of 17â-HSD Type 1 and Type 2 by

N-Alkylated Pyrazole Derivatives of E1

Table 5. Inhibition of 17â-HSD Type 1 and Type 2 by

C5′-substituted Pyrazole Derivatives of E1

% inhibition

at 10 µM^a

IC₅₀

(µM)

R

compd

type 1 type 2

type 1

% inhibition

IC₅₀

% inhibition

at 10 µM^a

(µM)

at 10 µM^a

CF₃

CH₃

49

50

52

74

70

76

75

b

36

b

R

compd type 1 type 2 type 1 compd type 1 type 2

3-pyridyl

CH₃CO₂Et

CO₂H

b

H

25^b

35

97

94

77

95

32

24

61

19

48

44

0.18

2.75

c

0.92

0.53

0.73

94

32

94

7

1.85

b

CH₃

36

38

40

42

47

43

70

79

83

c

32

44

30

55

c

-6

-1

CH₂CH(CH₃)₂37

CH₂OH

0.95

CH₂CO₂CH₃

(CH₂)₂OCH₃

(CH₂)₂CN

39

41

46

^aMean of at least two measurements with typically a SD of

(5%. ^bNot determined.

^aMean of at least two measurements with typically a SD of

the highest drop of activity was provoked by introducing

a methyl side chain, 35 being 15 times less potent than

25. The most potent novel N-alkylated derivative 41 was

only 3 times less active than 25, which indicates the

importance of the interactions the side chain may have

with the amino acid residues in the active site.

Most of the C5′-substituted pyrazoles prepared dis-

played activity against 17â-HSD type 1 with a high de-

gree of selectivity over 17â-HSD type 2. In the first

series of compounds (Table 5), the ethyl ester 74 and

the hydroxymethyl analogue 76 inhibited enzymatic

activity by 94% at a 10 µM concentration and had res-

pective IC₅₀values of 1.85 and 0.95 µM. This may

indicate the importance of hydrogen bond donor/acceptor

interactions in the corresponding region of the active

site. The carboxylic acid derivative 70 was however

found inactive and probably has detrimental electronic

effects in this region. The moderate activity observed

(5%. ^bMay exist as a 1′-H or 2′-H tautomer. ^cNot determined.

sponding 1′-alkylated compounds with inhibitory ac-

tivities of 83% or below at 10 µM. The influence of the

position of the substitution was especially noticeable in

the case of 36. While its N1′ analogue 35 achieved a

level of inhibition close to 95%, compound 36 showed

moderate inhibition of type 1 activity at 10 µM. How-

ever, for bulkier or longer side chains, the difference

between 1′- and 2′-substitution was less marked. The

selectivity for 17â-HSD type 1 over 17â-HSD type 2 was

also poor for the 2′-alkylated compounds. These results

clearly indicate that the active site has a limited

tolerance for certain substituents in this area of space,

where such moieties may prevent the steroid from

binding in a high-affinity orientation.

In our assay, the unsubstituted analogue 25 was

highly potent with an IC₅₀of 180 nM. Unexpectedly,

E-Ring Modified Steroids as Inhibitors of 17â-HSD

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5757

Table 6. Inhibition of 17â-HSD Type 1 and Type 2 by C5′-Amide Pyrazole Derivatives of E1^b

^aMean of at least two measurements with typically a SD of (5%. ^b- - - indicates the point of attachment to the amide nitrogen. ^cNot

determined.

for the trifluoromethyl derivative 49 suggested that

hydrophobic groups at C5′ were not well tolerated.

These observations were also confirmed when the activ-

ity of the series of amides 63-69 and 71-73 (Table 6)

was examined.

observation indicates that there may be an interaction

between the nicotinamide amide moiety and the amide

carbonyl of the 16â side chain. In addition, a 16â side

chain with sp³substitution may hinder the Pro(S)

hydrogen being transferred to the 17 carbonyl 2.21 Å

away. One of the hydrogen atoms in the methylene

linker attached to the 16 position is only 2.63 Å from

the Pro(S) hydrogen, and the methine hydrogen at the

16 position is 1.81 Å away. The pyridyl nitrogen of the

16â side chain is 3.16 Å from a phosphate oxygen and

could form an interaction similar to Ile 14 with an

alternative phosphate oxygen at the same phosphorus

center. These additional interactions proposed for com-

pound II with the cofactor may in part explain the high

potency of this novel inhibitor and that of compounds

such as 73. Moreover, the fact that compounds bearing

polar side chains display overall better activity indicates

that they might interact with the cofactor binding

domain, resulting in an enhanced affinity similar to II.

In this series, the methyl and isopropyl analogues 63

and 64 were among the least potent compounds with

moderate inhibition (62% or below) achieved at a 10 µM

concentration. In contrast, compounds possessing a

heterocyclic group in the side chain displayed overall

high activity against the enzyme (>80% inhibition at

10 µM), while remaining selective over 17â-HSD type

2. Weak activity was however observed for 68, possibly

due to the hydrophilic character and the bulk of the side

chain. The best inhibitors were obtained with com-

pounds containing a pyridyl side chain, namely, 71-

73. These analogues inhibited the enzymatic activity in

excess of 92% at 10 µM. The position of the heteroatom

was found not to be crucial for the activity, although

the 3-pyridyl analogue 71 was slightly more potent than

72 (2-pyridyl). Homologation of the side chain of 71 by

one carbon atom gave 73, the most potent and selective

inhibitor of this series, with an IC₅₀of 300 nM.

Such results are consistent with our previous findings

that II (Figure 1) is a highly potent inhibitor of 17â-

HSD type 1.²²In the crystal structure of human 17â-

HSD type 1 co-crystallized with E2 and cofactor, the

nicotinamide carbonyl and the amide nitrogen form

hydrogen bonds to the NH of Val 188 and the oxygen of

Thr 140, respectively. The docked conformation of II

shows that the carbonyl oxygen of the amide in the side

chain is 3.16 Å from the closest nicotinamide NH. This

Molecular Modeling

Compounds 41 and 73 were identified as the most

potent and selective inhibitors of 17â-HSD type 1 from

our pyrazole library with IC₅₀values of 530 and 300 nM,

respectively. The crystal structure of 17â-HSD1 in

complex with E2 and NADP (PBD code 1FDT)⁴⁴has

previously been used by our group in docking studies

of potent 17â-HSD1 inhibitors.²²To compare the pro-

posed binding modes of the pyrazoles 41 and 73 with

II, they were docked in a similar manner into 1FDT

with E2 removed using the flexible docking program

GOLD (Figure 5).⁴⁵

5758 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18

Fischer et al.

relation of observed versus predicted activity with an

r²value of 0.9, so the QSAR can be used in a predictive

fashion to calculate activities in silico. This QSAR will

be further refined as more data becomes available from

other series with the same steroid template.

Compound 41 was docked into the crystal structure

(PDB code 3ERD)⁵⁰of the ligand binding domain of

human estrogen receptor R (ERR) using GOLD. Poor

docking scores indicated that 41 did not fit well in

ERR: the maximum score observed was 28. This gives

an in silico prediction that 41 would not be strongly

estrogenic and similar docking studies examining the

orientation of binding in ERR have previously proven

predictive in identifying ERR selective agonists using

the same crystal structure.⁵¹

Estrogenicity Measurements in Vitro. The N-

alkylated compound 41 was evaluated for its estrogenic

activity in vitro in a luciferase reporter gene assay. For

the purpose of comparison, the nonalkylated pyrazole

25, for which no estrogenicity data are available, was

also tested in this assay, and the results are presented

in Figure 8.

Figure 5. Compounds 41 (green) and 73 (brown) docked in

1FDT.

The top-ranked conformations of 41 and 73 gave high

fitness scores of 64.0 and 78.6, respectively, and are

depicted in Figure 5. The steroid backbones of 41 and

73 are essentially overlaid when docked into 1FDT

forming the same hydrophobic interactions as the

steroid substrate. The main common hydrophobic in-

teractions involving the steroid backbone of both 41 and

73 are with amino acids Leu 149, Val 225, Phe 226, and

Phe 259 (residues in gray); these hydrophobic interac-

tions are also observed in the crystal structures reported

wherein the steroid nucleus is cocrystallized.⁴⁴The

hydrogen bond acceptor groups in the side chains of 41

and 73 are in close proximity to the primary amide

group of the nicotinamide group of the cofactor and may

form favorable electrostatic interactions with this func-

tionality. The ether oxygen atom in the side chain of

41 is 4.0 Å from the closest nicotinamide NH and the

amide carbonyl of 73 is a similar distance away (3.8 Å)

from the other nicotinamide NH. The side chain sub-

stitution distal to the hydrogen bond acceptor groups

may form further interactions with the cofactor and

protein backbone. These additional interactions of com-

pounds 41 and 73 with the cofactor may in part explain

the high potency of these inhibitors.

The novel 17â-HSD type 1 inhibitors described herein

for which an IC₅₀had been determined were used to

develop a QSAR using the three-dimensional method

of comparative molecular field analysis (CoMFA), part

of the SYBYL suite, where a set of aligned molecules is

used in QSAR generation.⁴⁶The set of molecules was

aligned using FlexS;^47,48CoMFA was then used to

calculate the steric and electrostatic interaction energy

of a probe atom with each molecule at points on a grid

surrounding the molecules. The aligned molecules with

the favored and disfavored steric interactions and elec-

trostatic interaction regions are depicted in Figure 6a,b.

Using the IC₅₀values as continuous response data,

the statistical tool in QSAR for CoMFA utilizes partial

least squares (PLS) for regression analysis.⁴⁹The results

of the QSAR are shown in Figure 7, where the negative

log of the observed IC₅₀is plotted against the negative

log of the predicted IC₅₀. Compounds 41, 67, and 71

were used as the validation set and compounds 4, 12,

21, 35, 39, 46, 72-74, and 76 as the training set to

predict activity. The graph in Figure 7 shows good cor-

From the data collected, it is clear that the presence

of a side chain at N1′ contributes to a reduced estroge-

nicity of the pyrazole nucleus. While 25 is estrogenic at

the lowest concentration tested (100 pM), the estrogenic

activity of the substituted derivative remains similar

to that of the control over the range 100 pM to 100 nM.

These results suggest that the side chain at the N1′

position of the pyrazole template may prevent binding

of 41 at the estrogen receptor. Alternatively, 41 may

act as an antagonist, and this possibility needs to be

further investigated since a compound displaying both

inhibition of 17â-HSD type 1 and antiestrogenic proper-

ties would be a potential drug for the treatment of

HDBC. A previous attempt to design “dual action”

inhibitors by Poirier and co-workers had some success

and the derivatives that emerged from such investiga-

tions were moderately potent against 17â-HSD type 1

but not fully antagonistic.²⁰

Conclusions

The inhibition of 17â-HSD type 1 represents an

important option in the search for novel endocrine

agents to treat HDBC, yet this is a relatively unexplored

area. There is a considerable need to identify lead

structures and pharmacophores. Not only should the

ideal 17â-HSD type 1 inhibitor be highly potent against

its target, it also needs to be selective over other 17â-

HSD isoforms and devoid of estrogenic activity. All these

requirements have resulted in only a few candidates

being identified, none of which has entered the clinic

or even been shown to be efficacious in vivo. In an effort

to develop new potent inhibitors of 17â-HSD type 1, we

synthesized E-ring pyrazole derivatives of E1.

Preliminary work in our laboratory has identified the

16,17-fused pyrazole derivatives of E1 as useful tem-

plates for the design of novel 17â-HSD type 1 inhibitors.

Compound 25 was functionalized at the 1′, 2′, and 5′

positions to build a comprehensive SAR which resulted

in the discovery of several active compounds as new 17â-

HSD type 1 inhibitors with selectivity over 17â-HSD

type 2. The N1′-methoxyethyl analogue 41 and C5′-

pyridylethyl amide derivative 73 emerged as novel and

E-Ring Modified Steroids as Inhibitors of 17â-HSD

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5759

Figure 6. CoMFA alignment (a) showing regions of steric interactions with green indicating favored regions and yellow disfavored

steric areas and (b) showing electrostatic interactions with blue areas indicating regions where hydrogen bond acceptor interactions

are favored and red showing regions where hydrogen bond donor interactions are favored.

highly potent inhibitors of 17â-HSD type 1 with IC₅₀

values of 530 and 300 nM, respectively. This has sub-

stantially extended the understanding of potential

binding interactions in this area and suggests the

importance of hydrogen bond acceptor interactions in

the side chain at the N1′ position of the pyrazole

template with the amino acid residues in the active site.

This also strengthens the hypothesis that compounds

with the appropriate side chain at C16 or at an

equivalent position may also interact with the cofactor

binding site.²²Preliminary in vitro evaluation showed

that N-alkylated compounds such as 41 are devoid of

estrogenicity, which is in agreement with predictive in

silico methods. New compounds of this type could be of

importance to develop proof of concept models for

evaluation of the therapeutic potential of in vivo inhibi-

tion of 17â-HSD1 in oncology.

Experimental Section

Chemistry. All chemicals were purchased from Aldrich

Chemical Co. (Gillingham, U.K.) or Lancaster Synthesis

(Morecambe, U.K.). All organic solvents of A. R. grade were

supplied by Fisher Scientific (Loughborough, U.K.). E1 was

purchased from Sequoia Research Products (Oxford, U.K.).

Thin-layer chromatography (TLC) was performed on pre-

coated plates (Merck TLC aluminum sheets silica gel 60 F₂₅₄).

Product(s) and starting material(s) were detected by either

viewing under UV light or treating with an ethanolic solution

of phosphomolybdic acid followed by heating, or both. Flash

column chromatography was performed on silica gel (Sorbsil/

Matrex C60) or using Argonaut prepacked columns with a

Flashmaster. IR spectra were recorded on a Perkin-Elmer

Spectrum RXI FT-IR as KBr disks, and peak positions are

expressed in cm^-1. ¹H NMR (270 or 400 MHz) and distortion-

less enhancement by polarization transfer (DEPT)-edited ¹³

NMR (100.4 MHz) spectra were recorded with a JEOL Delta

270 or a Varian Mercury VX 400 NMR spectrometer, and

C

5760 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18

Fischer et al.

Figure 7. QSAR showing activity against predicted activity

(units are -log IC₅₀values). Points in blue are the training set,

and compounds in pink are the validation set.

chemical shifts are reported in parts per million (ppm, δ).

HPLC analyses were performed on a Waters Millenium 32

instrument equipped with a Waters 996 PDA detector. A

Waters Radialpack C18 reversed phase column (8 × 100 mm)

was eluted with a MeOH/H₂O gradient at 2 mL/min. Fast atom

bombardment (FAB) low- and high-resolution mass spectra

were recorded at the Mass Spectrometry Service Center,

University of Bath, using m-nitrobenzyl alcohol (NBA) as the

matrix. Electrospray (ES) and atmospheric pressure chemical

ionization (APCI) low-resolution mass spectra were obtained

on a Waters Micromass ZQ. Elemental analyses were per-

formed by the Microanalysis Service, University of Bath.

Melting points were determined using a Reichert-Jung Ther-

mo Galen Kofler block and are uncorrected. The X-ray crystal-

lographic study of 37 was carried out by Dr. M. F. Mahon in

the Department of Chemistry, University of Bath, on a κ CCD

diffractometer with area detector.

Compounds 1,⁵²5,²⁹12,²⁵and 21^33,53have been described

in the literature.

Molecular Modeling. For the docking studies of com-

pounds 41 and 73, the starting conformations used for receptor

docking were generated from a random conformational search

performed using the MMFF94S force field as implemented in

Sybyl 7.0. The resulting lowest energy conformer was then

used for docking studies. Charge calculations were determined

using the MMFF94S method, and GOLD, version 2.1, was then

used with default parameters to perform the docking studies.

The active site was defined as a 12 Å radius around the C R

atom of serine 142, and 30 attempts were computed for each

compound and scored using Gold Score.

Figure 8. Estrogenic activity of 17â-estradiol (E2), 25, and

41. The results are luciferase activity (y-axis) relative to control

cells (no added compound) in the presence of E2 and com-

pounds 25 and 41 at different concentrations (x-axis). Data

are means ( SD of triplicate determinations. Figure shows

results from two separate experiments each with E2 as a

positive control.

To develop a QSAR using CoMFA, the ligands were initially

minimized using the MMFF94S force field as implemented

within the Sybyl 7.0 package. The molecules were aligned

using FlexS with a common core elucidated by DISTILL, which

corresponded to the steroid backbone. Gastegier-Huckel

charges were used for the charge descriptors in FlexS, and

CoMFA was performed using the aligned compounds and the

standard Sybyl 7.0 CoMFA fields.

mixture was achieved using TLC (DCM/EtOAc, 4:1 v/v), and

the mass of ³H-E2 produced was calculated from the ³H counts

detected and recovery of ¹⁴C-E2.

Biology. Radiolabeled E1 and E2 (³H and ¹⁴C) were

purchased from New England Nuclear (Boston, MA) or Am-

ersham Biosciences UK Limited (Amersham, U.K.).

Inhibition of 17â-HSD Type 2 Activity. Briefly MDA-

3

MB-231 human breast cancer cells were incubated with H-

E2 at a concentration of 2 nM per flask in the absence or

presence of the inhibitor (0.1 nM to 10 µM). After incubation

of the substrate with or without inhibitor for 3 h at 37 °C, the

products were isolated from the mixture by extraction with

Et₂O (4 mL) using ¹⁴C-E1 (5000 dpm) to monitor procedural

losses. Separation of ³H-E1 from the mixture was achieved

using TLC (DCM/EtOAc, 4:1 v/v), and the mass of ³H-E1

produced was calculated from the ³H counts detected and

recovery of ¹⁴C-E1.

T47-D and MDA-MB-231 cells have previously been shown

to possess predominantly reductive or oxidative 17â-HSD

activity, respectively.⁵⁴

Inhibition of 17â-HSD Type 1 Activity. Briefly T47-D

human breast cancer cells were incubated with ³H-E1 at a

concentration of 2 nM per well in a 24-well tissue culture plate

in the absence or presence of the inhibitor (0.1 nM to 10 µM).

After incubation of the substrate with or without inhibitor for

30 min at 37 °C, the products were isolated from the mixture

by extraction with Et₂O (4 mL) using ¹⁴C-E2 (5000 dpm) to

monitor procedural losses. Separation of ³H-E2 from the

Estrogenicity Assay. The ability of compounds to activate

ERR was determined by transfecting cells with plasmid

E-Ring Modified Steroids as Inhibitors of 17â-HSD

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5761

pERET81luc, which contains ER binding sites cloned upstream

of a minimal thymidine kinase (TK) promoter and the lu-

ciferase reporter gene. Cells were cotransfected with an ERR

expression plasmid. Addition of estrogenic compounds stimu-

lated the ER and increased the expression of luciferase. Cells

were also transfected with a â-galactosidase expression plas-

mid to control for well-to-well variation in sample handling

and potential nonspecific effects of compounds.

organics were extracted with EtOAc (2 × 50 mL), washed with

H₂O (2 × 30 mL) and brine (2 × 30 mL), dried (Na₂SO₄),

filtered, and concentrated under reduced pressure. The crude

product was purified by flash chromatography.

Method 4. Amide Coupling in a Radleys Greenhouse

Synthesizer: Synthesis of 56-62. To a solution of 55 (107

mg, 250 µmol) in anhydrous DCM (2 mL) and NEt₃(25 µL,

180 µmol) under N₂was added 1 mL of a stock solution

containing EDC (58 mg, 300 µmol), NEt₃(17 µL, 160 µmol),

and DMAP (catalytic) in anhydrous DCM. The reaction was

stirred at room temperature for 30 min before addition of

amine (1.1 equiv), and stirring was continued for 24 h. The

mixture was then washed with saturated aqueous NaHCO₃

(2 mL) before separation and concentration of the organic

layer. The product was semipurified by flash chromatography

using EtOAc as eluent, except in the case of 62 for which 5%

MeOH in DCM was used. Yields of products ranged from 9%

to 35%. Products were then debenzylated without further

purification.

Method 5. Hydrogenolysis in a Radleys Greenhouse

Synthesizer: Synthesis of 63-69. A solution/suspension of

the starting material in EtOH/THF (1:1, 2 mL) was degassed

by bubbling N₂through for 3 min before Pd-C 5% (catalytic)

was added. Degassing was continued for 5 min more before

hydrogen gas (balloon) was passed over the reaction, and

stirring under a hydrogen blanket was continued until TLC

indicated completion (up to 48 h). The mixture was then

filtered through Celite, and the filtrate was concentrated under

reduced pressure. The product was purified by flash chroma-

tography using an elution gradient of hexane to EtOAc with

the exception of 68 for which 10% MeOH in DCM was used.

16-Cyano-estrone (4). Following method 2, a solution of

3 (350 mg, 856 µmol) in anhydrous THF (20 mL) was treated

with a 1.0 M solution of TBAF in anhydrous THF (1.0 mL, 1.0

mmol) for 30 min. The crude product was triturated in EtOAc/

hexane to give 4 as a pale yellow solid (R/â ) 1:1.6, 223 mg,

88%): δ_H(DMSO-d₆, 400 MHz) 0.91 (s, C-18-H₃, minor isomer),

0.93 (s, C-18-H₃, major isomer), 1.25-2.82 (13H, m), 3.84 (dd,

J ) 9.9 Hz, J ) 8.8 Hz, C-16-H, major isomer), 4.38 (dd, J )

10.4 Hz, J ) 2.0 Hz, C-16-H, minor isomer), 6.45 (1H, d, J )

2.4 Hz, C-4-H), 6.51 (1H, dd, J ) 8.4 Hz, J ) 2.4 Hz, C-2-H),

7.04 (1H, d, J ) 8.4 Hz, C-1-H), 9.05 (s, OH, minor isomer),

9.06 (s, OH, major isomer); MS m/z (FAB+) 295.2 [100, (M +

H)⁺]; Acc MS m/z (FAB+) 295.1572, C₁₉H₂₁NO₂requires

295.1572. Anal. (C₁₉H₂₁NO₂) C, H, N.

Human 293 HEK cells (ERR negative) were cultured for 3

days in growth medium depleted of estrogenic compounds

(phenol-red free Dulbecco’s modified Eagle’s Medium (PRF-

DMEM; Invitrogen) supplemented with 10% (v/v) charcoal-

stripped serum, glutamine, and antibiotics). Cells (5 × 10⁶)

were then plated in a 150 mm tissue culture dish in the same

medium. The following day, DNA for transfection was prepared

by combining 6 µg of pERET81luc, 6 µg of an ERR expression

plasmid (pSG5-HEGO), and 3 µg of a â-galactosidase expres-

sion plasmid (CMV-â-gal) in a total volume of 0.1 mL of sterile

water. The DNA was added to a mixture of 995 µL of PRF-

DMEM and 45 µL of Fugene 6 transfection reagent (Roche),

and the solution was gently mixed and incubated at room

temperature for 30 min. The DNA/Fugene mix was then added

dropwise to the cells and incubated overnight. The following

day, cells were recovered by trypsinization, and equal cell

numbers were plated in individual wells of a 96-well plate

(typically 5000-10 000 cells/well in a total volume of 50 µL).

After 2 h, various concentrations of test compounds or 17â-

estradiol as a positive control were added (each 50 µL, diluted

at 2× final concentration in complete estrogen-depleted growth

medium). For the negative control, 50 µL of complete estrogen-

depleted growth medium lacking compound was added. Each

determination was made in triplicate. The following day, 100

µL of SteadyLite luciferase reagent (Packard/Perkin-Elmer

Life Sciences) was added to each well. Cell lysate (100 µL) was

transferred to a white 96-well plate, and luciferase activity

was determined using a TopCount scinitillation counter (Pack-

ard) in single photon counting mode. To measure â-galactosi-

dase activity, 50 µL of cell lysate was transferred to a clear

96-well plate, and 50 µL of O-nitrophenyl-â-^D-galactopyrano-

side (ONPG) reagent (Sigma) was added. The plate was

incubated at 37 °C for approximately 2 h before measuring

absorbance at 405 nm using a Dynatech MR-500 plate reader.

Relative luciferase activity was calculated by dividing mean

luciferase activity (of triplicate determinations) by mean

â-galacotosidase activity, relative to untreated cells (value set

at 1).

3-O-Benzyl-16,16-dimethyl-estrone (6). Methyl iodide

(155 µL, 2.50 mmol) and NaH (60% dispersion in mineral oil,

146 mg, 3.66 mmol) were added to a stirred solution of 5 (300

mg, 832 µmol) in anhydrous DMSO (15 mL) at room temper-

ature under an atmosphere of N₂. The resulting mixture was

stirred at room temperature for 12 h, after which it was heated

to 80 °C for 1 h. After the mixture cooled, H₂O (200 mL) was

added, and the organics were extracted with EtOAc (2 × 200

mL), washed with H₂O (2 × 150 mL) and then brine (2 × 150

mL), dried (Na₂SO₄), filtered, and concentrated under reduced

pressure. The crude product was recrystallized from EtOH to

give 6 as off-white crystals (157 mg, 49%): IR (KBr) 2955-

2855, 1735, 1605, 1500, 1235 cm^-1; δ_H(CDCl₃, 400 MHz) 0.93

(3H, s, C-16-H₃), 1.08 (3H, s, C-16-H₃), 1.21 (3H, s, C-18-H₃),

1.18-2.91 (13H, m), 5.02 (2H, s, OCH₂Ar), 6.71 (1H, d, J )

2.7 Hz C-2-H), 6.77 (1H, dd, J ) 8.6 Hz, J ) 2.7 Hz, C-2-H),

7.18 (1H, d, J ) 8.6 Hz, C-1-H), 7.27-7.42 (5H, m, C₆H₅); MS

m/z (FAB+) 388.1 [47, M⁺], 91.0 [100]; Acc MS m/z (FAB+)

388.2383, C₂₇H₃₂O₂requires 388.2402.

16,16-Dimethyl-estrone (7). Following method 1, a sus-

pension of 6 (90 mg, 0.225 mmol) and Pd-C 10% (40 mg) in

MeOH/THF 2:1 (30 mL) was hydrogenated overnight to give

7 as a white solid (74 mg, 96%). This was recrystallized from

MeOH/H₂O to give white crystals (42 mg, 54%): IR (KBr) 3300,

2960-2815, 1720, 1615, 1505-1450, 1235 cm^-1; δ_H(CDCl₃, 400

MHz) 0.95 (3H, s, C-16-H₃), 1.10 (3H, s, C-16-H₃), 1.22 (3H, s,

C-18-H₃), 1.37-2.89 (13H, m), 4.75 (1H, s, OH), 6.58 (1H, d, J

) 2.7 Hz, C-4-H), 6.64 (1H, dd, J ) 8.2 Hz, J ) 2.7 Hz, C-2-

H), 7.14 (1H, d, J ) 8.2 Hz, C-1-H); MS m/z (FAB+) 597.2 [61,

General Methods. Method 1. Hydrogenolysis. A sus-

pension of Pd-C, 5% or 10% (catalytic), in THF (2 mL) was

added to a solution of the starting material in MeOH/THF or

EtOH/THF. The resulting suspension was hydrogenated at

room temperature using a hydrogen-filled balloon until TLC

indicated completion. After removal of the supported catalyst

by filtration through Celite and concentration of the filtrate

under reduced pressure, the product obtained was purified by

flash chromatography, recrystallized, or both.

Method 2. Deprotection using TBAF. A 1.0 M solution

of TBAF in anhydrous THF (1.5 or 2 equiv) was added to a

stirred solution of the starting material in anhydrous THF

under an atmosphere of N₂. The resulting solution was stirred

at room temperature until TLC indicated completion, after

which the solvent was removed under reduced pressure and

H₂O added. For compounds 4, 18, and 19, the final mixture

was acidified with AcOH (0.5 mL). The organics were extracted

with EtOAc, washed with H₂O and then brine, dried (Na₂SO₄),

filtered, and concentrated under reduced pressure. The crude

product was purified by flash chromatography or recrystallized

or triturated.

Method 3. Synthesis of Alkylated Derivatives 27-34.

NaH (60% dispersion in mineral oil, 1.5 equiv) was added to a

stirred solution of 26 in anhydrous DMF at 0 °C under an

atmosphere of N₂. After 20 min of stirring, the parent alky-

lating agent (2 equiv) was added, and the resulting mixture

was stirred at room temperature. The reaction was monitored

by TLC and quenched with H₂O (50 mL) at completion. The

5762 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18

Fischer et al.

(2M + H)⁺], 452.1 [39, (M + H + NBA)⁺], 298.2 [100, M⁺]; Acc

MS m/z (FAB+) 298.1934, C₂₀H₂₆O₂requires 298.1933. HPLC

(MeOH/H₂O, 90:10) R_t) 3.26 min, 100%.

309.1276, C₂₀H₂₃NO₂requires 309.1279. HPLC (MeOH/H₂O,

80:20) R_t) 2.53 min, 98%.

3-O-tert-Butyl-dimethylsilyl-16-(1′-hydroxy-2′,2′,2′-tri-

fluoro-ethylidene)-estrone (13). ^tBuOK (875 mg, 7.80 mmol)

was added to a stirred solution of 1 (1.0 g, 2.60 mmol) in

anhydrous toluene (20 mL) at 0 °C under an atmosphere of

N₂. After 20 min of stirring, ethyl trifluoroacetate (2.16 mL,

18.2 mmol) was added, and the mixture was heated to reflux

for 2 h. After the mixture cooled, the resulting dark orange

suspension was poured into H₂O (50 mL) and acidified with 5

M HCl. The organics were extracted with EtOAc (2 × 50 mL),

washed with H₂O (2 × 30 mL) and then brine (2 × 30 mL),

dried (Na₂SO₄), filtered, and concentrated under reduced

pressure. The crude orange oil that crystallized on standing

was used without further purification (1.4 g, 100%). An

analytical sample was recrystallized from EtOH to give 13 as

light yellow crystals: mp 145-146 °C; IR (KBr) 2930-2860,

1690, 1640-1495 cm^-1; δ_H(CDCl₃, 400 MHz) 0.19 (6H, s, Si-

(CH₃)₂), 0.98 (9H, s, C(CH₃)₃), 0.99 (3H, s, C-18-H₃), 1.42-2.90

(13H, m), 6.57 (1H, d, J ) 2.6 Hz, C-4-H), 6.63 (1H, dd, J )

8.4 Hz, J ) 2.6 Hz, C-2-H), 7.11 (1H, d, J ) 8.4 Hz, C-1-H),

C-1′-OH not seen; δ_C(CDCl₃, 100.4 MHz) -4.4 (2×q, Si(CH₃)₂),

14.8 (q, C-18), 18.1 (s, C(CH₃)₃), 25.2 (t), 25.6 (t), 25.7 (3×q,

C(CH₃)₃), 26.7 (t), 29.3 (t), 31.0 (t), 37.5 (d), 43.9 (d), 48.5 (s,

C-13), 49.5 (d), 110.9 (s, C-16), 117.4 (d), ∼119 (app d, J )

272 Hz, CF₃), 120.0 (d), 126.0 (d), 132.0 (s), 137.4 (s), 153.6 (s,

C-3), ∼154 (app d, J ) 38 Hz, C-1′), 216.0 (s, CdO); MS m/z

(FAB+) 480.1 [47, M⁺], 423.0 [38, (M - C₄H₉)⁺], 73.0 [100];

Acc MS m/z (FAB+) 480.2296, C₂₆H₃₅F₃O₃Si requires 480.2308.

Anal. (C₂₆H₃₅F₃O₃Si) C, H.

t

3-O-Benzyl-16-hydroxymethylene-estrone (8). BuOK

(9.4 g, 84.1 mmol) was added portionwise to a stirred solution

of 5 (10 g, 27.7 mmol) in anhydrous toluene (250 mL) at room

temperature under an atmosphere of N₂. After it was stirred

for 20 min, ethyl formate (14.8 mL, 194 mmol) was added, and

the resulting suspension was stirred for 2.5 h. The final

mixture was poured into H₂O (300 mL) and acidified with 5

M HCl. The organics were extracted with EtOAc (2 × 200 mL),

washed with H₂O (3 × 100 mL) and then brine (2 × 100 mL),

dried (Na₂SO₄), filtered, and concentrated under reduced

pressure. The crude product was triturated with boiling EtOAc

to give 8 as an off-white solid (9.25 g, 86%): mp 149-151°C;

IR (KBr) 3235, 2930-2855, 1700, 1675-1500 cm^-1; δ_H(DMSO-

d₆, 400 MHz) 0.81 (3H, s, C-18-H₃), 1.31-2.85 (13H, m), 5.05

(2H, s, OCH₂Ar), 6.71 (1H, d, J ) 2.7 Hz, C-4-H), 6.75 (1H,

dd, J ) 8.4 Hz, J ) 2.7 Hz, C-2-H), 7.16 (1H, d, J ) 8.4 Hz,

C-1-H), 7.28-7.33 (1H, m, dCHOH), 7.35-7.43 (5H, m, C₆H₅),

10.6-10.9 (1H, br s, exchanged with D₂O, dCHOH); δ_C

(DMSO-d₆, 100.4 MHz) 15.3 (q, C-18), 24.9 (t), 26.5 (t), 27.1

(t), 30.0 (t), 32.3 (t), 38.2 (d), 44.3 (d), 48.6 (s, C-13), 49.1 (d),

69.7 (t, OCH₂Ar), 112.9 (d), 113.9 (s, C-16), 115.2 (d), 126.7

(d), 128.1 (2 × d), 128.3 (d), 129.0 (2 × d), 132.7 (s), 138.0 (2 ×

s), 150.8 (d, C-1′), 156.7 (s, C-3), 209.1 (s, CdO); MS m/z

(FAB+) 389.3 [31, (M + H)⁺], 91.1 [100, (CH₂Ar)⁺]; Acc MS

m/z (FAB+) 389.2099, C₂₆H₂₉O₃requires 389.2117.

3-O-Benzyl-16-cyano-estrone (9). O,N-Bis-(trifluoroacetyl)-

hydroxylamine²⁷(486 mg, 2.16 mmol) was added to a solution

of 8 (300 mg, 772 µmol) and pyridine (460 µL, 5.70 mmol) in

toluene (10 mL), and the resulting mixture was heated to

reflux for 3 h. After the mixture cooled, H₂O (20 mL) was

added, and the organics were separated, washed with H₂O (10

mL) and then brine (10 mL), dried (Na₂SO₄), filtered, and

concentrated under reduced pressure. The crude product was

purified by flash chromatography (DCM) to give an off-white

solid (200 mg, 67%). This was triturated in EtOAc/hexane to

give 9 as light yellow crystals (R/â ) 1:1.2, 84 mg, 28%): IR

3-O-tert-Butyl-dimethylsilyl-16-(1′-hydroxy-1′′-pyridin-

t

3′′-ylmethylene)-estrone (14). BuOK (875 mg, 7.80 mmol)

was added to a stirred solution of 1 (1.0 g, 2.60 mmol) in

anhydrous toluene (30 mL) at 0 °C under an atmosphere of

N₂. After 20 min of stirring, ethyl nicotinate (2.48 mL, 18.2

mmol) was added, and the mixture was heated to reflux for

1.5 h. After it had cooled, the resulting dark red suspension

was poured into H₂O (200 mL) and acidified with 5 M HCl.

The organics were extracted with EtOAc (2 × 100 mL), washed

with H₂O (100 mL) and then brine (2 × 100 mL), dried (Na₂-

SO₄), filtered, and concentrated under reduced pressure. The

crude yellow solid was recrystallized from EtOH to give 14 as

white flaky crystals (985 mg, 77%): mp 169-170 °C; IR (KBr)

2930-2855, 1660, 1605, 1495 cm^-1; δ_H(CDCl₃, 400 MHz) 0.21

(6H, s, Si(CH₃)₂), 0.99 (9H, s, C(CH₃)₃), 1.11 (3H, s, C-18-H₃),

1.38-2.95 (13H, m), 6.58 (1H, d, J ) 2.6 Hz, C-4-H), 6.63 (1H,

dd, J ) 8.5 Hz, J ) 2.6 Hz, C-2-H), 7.12 (1H, d, J ) 8.5 Hz,

C-1-H), 7.41 (1H, ddd, J ) 8.1 Hz, J ) 4.9 Hz, J ) 0.8 Hz,

C-5′′-H), 8.09 (1H, app dt, J ) 8.1 Hz, J ) 2.0 Hz, C-4′′-H),

8.69 (1H, dd, J ) 4.9 Hz, J ) 1.9 Hz, C-6′′-H), 8.97 (1H, app

dd, J ) 2.1 Hz, J ) 0.8 Hz, C-2′′-H), 13.63 (1H, s, exchanged

with D₂O, C-1′-OH); MS m/z (FAB+) 490.1 [100, (M + H)⁺];

Acc MS m/z (FAB+) 490.2775, C₃₀H₄₀NO₃Si requires 490.2777.

(KBr) 2930-2870, 2240, 1750, 1615, 1605, 1500, 1230 cm^-1

;

δ_H(CDCl₃, 400 MHz) 0.93 (s, C-18-H₃, minor isomer), 0.95 (s,

C-18-H₃, major isomer), 1.32-2.85 (13H, m), 3.87 (dd, J ) 10.1

Hz, J ) 8.6 Hz, C-16-H, major isomer), 4.40 (dd, J ) 10.1 Hz,

J ) 1.9 Hz, C-16-H, minor isomer), 5.07 (2H, s, OCH₂Ar), 6.74

(1H, d, J ) 2.7 Hz, C-4-H), 6.78 (1H, dd, J ) 8.4 Hz, J ) 2.7

Hz, C-2-H), 7.18 (1H, d, J ) 8.4 Hz, C-1-H), 7.31-7.45 (5H,

m, C₆H₅); MS m/z (FAB+) 385.1 [40, M⁺], 91.0 [100, (CH₂Ar)⁺];

Acc MS m/z (FAB+) 385.2053, C₂₆H₂₇NO₂requires 385.2042.

3-O-Benzyl-16-cyano,16-methyl-estrone (10). NaH (60%

dispersion in mineral oil, 1.2 equiv, 0.248 mmol, 10 mg) was

added to a stirred solution of 9 (0.207 mmol, 80 mg) in

anhydrous DMF (5 mL) at 0 °C under an atmosphere of N₂.

After 30 min of stirring, methyl iodide (0.310 mmol, 19 µL)

was added to the white suspension, and the mixture was

stirred for 3 h, in which time it was allowed to warm to room

temperature. The resulting light yellow solution was then

poured into H₂O (50 mL), and the organics were extracted with

EtOAc (3 × 20 mL), washed with brine (2 × 20 mL), dried

(Na₂SO₄), filtered, and evaporated under reduced pressure.

The crude product was purified by flash chromatography

(DCM/hexane, 3:2) to give 10 as a crystalline light yellow solid

(60 mg, 72%): δ_H(DMSO-d₆, 270 MHz) 1.02 (3H, s, C-18-H₃),

1.25-2.83 (16H, m), 5.06 (2H, s, OCH₂Ar), 6.73-6.80 (2H, m,

C-4-H and C-2-H), 7.18 (1H, d, J ) 8.4 Hz, C-1-H), 7.29-7.46

(5H, m, C₆H₅); Anal. (C₂₇H₂₉NO₂) C, H, N.

16-Cyano,16-methyl-estrone (11). Following method 1, a

suspension of 10 (90 mg, 0.225 mmol) and Pd-C 10% (40 mg)

in MeOH/THF 1:1 (40 mL) was hydrogenated overnight to give

a white foam (46 mg, 66%). A sample was then triturated in

hexane to give 11 as a creamy powder: δ_H(CDCl₃, 270 MHz)

1.12 (3H, s, C-18-H₃), 1.32-2.92 (16H, m), 4.55 (1H, s, C-3-

OH), 6.73-6.58 (1H, app d, C-4-H), 6.63 (1H, dd, J ) 8.5 Hz,

J ) 2.5 Hz, C-2-H), 7.13 (1H, d, J ) 8.5 Hz, C-1-H); MS m/z

(FAB+) 309.1 [60, M⁺], 97.1 [50], 83.0 [65]; Acc MS m/z (FAB+)

3-O-tert-Butyl-dimethylsilyl-16-(1′-hydroxy-2′-carboxy-

lic acid ethyl ester-ethylidene)-estrone (15). Sodium

ethoxide (340 mg, 5.00 mmol) was added to a stirred solution

of 1 (700 mg, 1.82 mmol) and diethyl oxalate (1.0 mL, 7.36

mmol) in toluene (20 mL). The resulting yellow solution was

stirred for 2 h at room temperature after which it was poured

into 6 M HCl (20 mL). The organics were extracted with EtOAc

(50 mL), dried (Na₂SO₄), filtered, and concentrated under

reduced pressure. The crude product was recrystallized from

EtOH to give 15 as colorless crystals (737 mg, 83%): mp 162-

163 °C; δ_H(CDCl₃, 400 MHz) 0.19 (6H, s, Si(CH₃)₂), 0.98 (9H,

s, C(CH₃)₃), 1.00 (3H, s, C-18-H₃), 1.40 (3H, t, J ) 7.2 Hz),

1.43-3.08 (13H, m), 4.36 (2H, q, J ) 7.2 Hz), 6.57 (1H, d, J )

2.3 Hz, C-4-H), 6.63 (1H, dd, J ) 8.6 Hz, J ) 2.3 Hz, C-2-H),

7.11 (1H, d, J ) 8.6 Hz, C-1-H), 12.47 (1H, br s, C-1′-OH); δ_C

(CDCl₃, 100.4 MHz) -4.2 (2×q, Si(CH₃)₂), 14.3 (q), 15.0 (q),

18.3 (t), 25.8 (q), 25.9 (t), 26.8 (t), 27.5 (t), 29.5 (t), 31.2 (t),

37.7 (d), 44.0 (d), 48.8 (s), 49.5 (d), 62.0 (t), 116.5 (s), 117.2 (d),

119.9 (d), 125.8 (d), 132.1 (s), 137.4 (s), 152.4 (s), 153.3 (s),

162.5 (s), 216.6 (s); MS m/z (FAB+) 485.3 [100, (M + H)⁺],

E-Ring Modified Steroids as Inhibitors of 17â-HSD

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5763

73.1 [61]; Acc MS m/z (FAB+) 484.2638 C₂₈H₄₀O₅Si requires

16-(1′-Hydroxy-ethylidene)-estrone (20). Following

method 1, a suspension of 16 (200 mg, 497 µmol) and Pd-C

10% (80 mg) in MeOH/THF 2:1 (30 mL) was hydrogenated for

2 h to give 20 as a white solid (98 mg, 63%). This product was

recrystallized from acetone/hexane to give white crystals (72

mg, 46%): mp 211-214 °C; IR (KBr) 3450, 2915-2860, 1705,

1655-1510 cm^-1; δ_H(CDCl₃, 400 MHz) 0.83, 0.96 (3H, 2 × s,

C-18-H₃), 1.39-2.89 (16H, m), 3.43 (dd, J ) 9.4 Hz, J ) 8.2

Hz, C-16-H, ketone tautomer), 4.70 (1H, br s, exchanged with

D₂O, C-3-OH), 6.55-6.57 (1H, m, C-4-H), 6.62 (1H, dd, J )

8.0 Hz, J ) 2.7 Hz, C-2-H), 7.11 (1H, d, J ) 8.0 Hz, C-1-H),

C-1′-OH (enol tautomer) not seen; MS m/z (FAB+) 313.1 [100,

(M + H)⁺], 73.0 [19]; Acc MS m/z (FAB+) 313.1799, C₂₀H₂₅O₃

requires 313.1804. Anal. (C₂₀H₂₄O₃) C, H.

3-Hydroxy-16,17-bis-oximino-estra-1,3,5(10)-triene (22).

NaOAc (556 mg, 6.79 mmol) followed by hydroxylamine

hydrochloride (520 mg, 7.48 mmol) was added to a solution of

21 (200 mg, 668 µmol) in a mixture of MeOH/H₂O 5:1 (36 mL).

The resulting pale yellow solution was stirred at room-

temperature overnight, after which the solvent was removed

under reduced pressure and brine was added (100 mL). The

organics were extracted with EtOAc (100 mL), washed with

brine (2 × 50 mL), dried (Na₂SO₄), filtered, and concentrated

under reduced pressure. The crude product was recrystallized

from acetone to give 22 as off-white crystals (109 mg, 52%):

mp 245-247 °C; IR (KBr) 3420-3200, 3020, 2935-2870, 1705,

1620, 1585-1500 cm^-1; δ_H(DMSO-d₆, 400 MHz) 0.97 (3H, s,

C-18-H₃), 1.28-2.83 (13H, m), 6.44 (1H, d, J ) 2.4 Hz, C-4-

H), 6.51 (1H, dd, J ) 8.5 Hz, J ) 2.4 Hz, C-2-H), 7.04 (1H, d,

J ) 8.5 Hz, C-1-H), 9.02 (1H, s, exchanged with D₂O, OH),

10.81 (1H, s, exchanged with D₂O, NOH), 11.19 (1H, s,

exchanged with D₂O, NOH); δ_C(DMSO-d₆, 100.4 MHz) 14.5

(q, C-18), 26.5 (t), 27.1 (t), 27.3 (t), 29.5 (t), 35.5 (t), 37.4 (d),

43.4 (d), 46.2 (s, C-13), 49.0 (d), 113.2 (d), 115.4 (d), 126.4 (d),

130.5 (s), 137.4 (s), 155.5 (s), 155.7 (s), 160.2 (s); MS m/z

(FAB+) 315.2 [100, (M + H)⁺]; MS m/z (FAB-) 466.2 [66, (M

- H + NBA)^-], 313.2 [100, (M - H)^-], 276.1 [80]; Acc MS m/z

(FAB+) 315.1711, C₁₈H₂₃N₂O₃requires 315.1709. HPLC (MeOH/

H₂O, 70:30) R_t) 2.71 min, 99%. Anal. [C₁₈H₂₂N₂O₃.(CH₃)₂O]

C, H, N; C: calcd, 67.72; found 67.30.

484.2645. Anal. (C₂₈H₄₀O₅Si) C, H.

3-Benzyloxy-16-(1′-hydroxy-ethylidene)-estra-1,3,5(10)-

t

triene-17-one (16). BuOK (438 mg, 4.14 mmol) was added

to a stirred solution of 5 (500 mg, 1.38 mmol) in anhydrous

toluene (6 mL) and DMSO (1.5 mL) at 0 °C under an

atmosphere of N₂. EtOAc (1.27 mL, 17.94 mmol) was then

added, and the mixture was heated to reflux for 1 h. After

cooling, the resulting brown solution was poured into H₂O (200

mL) and acidified with 5 M HCl. The organics were extracted

with EtOAc (2 × 150 mL), washed with H₂O (2 × 100 mL)

and then brine (2 × 100 mL), dried (Na₂SO₄), filtered, and

concentrated under reduced pressure. The crude product was

purified by flash chromatography (DCM) to yield 16 as a light

yellow crystalline solid (447 mg, 80%). This compound was

recrystallized from EtOH to give yellow crystals (351 mg,

63%): mp 126-128 °C; IR (KBr) 2930-2860, 1660, 1615-1455

cm^-1; δ_H(CDCl₃, 400 MHz) 0.83, 0.95 (3H, 2 × s, C-18-H₃),

1.38-2.92 (16H, m), 3.43 (app dd, J ) 9.4 Hz, J ) 8.2 Hz,

C-16-H, ketone tautomer), 5.02 (2H, s, OCH₂Ar), 6.71 (1H, d,

J ) 2.8 Hz, C-4-H), 6.77 (1H, dd, J ) 8.3 Hz, J ) 2.8 Hz, C-2-

H), 7.17 (1H, d, J ) 8.3 Hz, C-1-H), 7.28-7.42 (5H, m, C₆H₅),

12.91 (br s, exchanged with D₂O, C-1′-OH, enol tautomer); MS

m/z (FAB+) 403.1 [68, (M + H)⁺], 91.0 [100, (CH₂Ar)⁺]; Acc

MS m/z (FAB+) 403.2246, C₂₇H₃₁O₃requires 403.2273. Anal.

(C₂₇H₃₀O₃) C, H.

16-(1′-Hydroxy-2′,2′,2′-trifluoro-ethylidene)-estrone (17).

Following method 2, a solution of 13 (1.18 g, 2.42 mmol) in

anhydrous THF (50 mL) was treated with a 1.0 M solution of

TBAF in anhydrous THF (4.90 mL, 4.90 mmol) for 4 h. The

crude product was purified by flash chromatography (DCM)

to give 17 as a light brown oil (535 mg, 60%). This compound

was crystallized from cold DCM to give white crystals (249

mg, 28%): mp 91-93 °C; IR (KBr) 3430, 2930-2860, 1685,

1610, 1500 cm^-1; δ_H(CDCl₃, 400 MHz) 1.02 (3H, s, C-18-H₃),

1.43-2.91 (13H, m), 6.58 (1H, d, J ) 2.7 Hz, C-4-H), 6.64 (1H,

dd, J ) 8.4 Hz, J ) 2.7 Hz, C-2-H), 7.14 (1H, d, J ) 8.4 Hz,

C-1-H), C-3-OH and C-1′-OH not seen; MS m/z (FAB+) 366.1

[100, M⁺]; Acc MS m/z (FAB+) 366.1476, C₂₀H₂₁F₃O₃requires

366.1443. HPLC (MeOH/H₂O, 96:4) R_t) 1.66 min, 100%. Anal.

[C₂₀H₂₁F₃O₃.(CH₂Cl₂)_3/4] C, H.

3-Hydroxy-17-O-allyl-oximino-estra-1,3,5(10)-triene (23).

NaOAc (3 g, 10.17 mmol) followed by O-allyl-hydroxylamine

hydrochloride (4.5 g, 41.44 mmol) was added to a solution of

E1 (1.0 g, 3.70 mmol) in a mixture of MeOH/H₂O 5:1 (180 mL).

The resulting solution was stirred at room-temperature over-

night, after which the solvent was removed under reduced

pressure and H₂O was added (200 mL). The organics were

extracted with EtOAc (300 mL), washed with H₂O (2 × 100

mL) and then brine (2 × 100 mL), dried (Na₂SO₄), filtered,

and concentrated under reduced pressure to give a white

product (1.37 g). The crude material was recrystallized from

MeOH/H₂O to give 23 as white crystals (1.13 g, 94%): mp 81-

83 °C; IR (KBr) 3455, 3185, 2955-2845, 1640-1505, 1240

cm^-1; δ_H(CDCl₃, 400 MHz) 0.94 (3H, s, C-18-H₃), 1.35-2.87

(15H, m), 4.50-4.60 (2H, m, C-1′-H₂), 4.74 (1H, s, exchanged

with D₂O, OH), 5.19 (1H, app dq, C-3′-H), 5.28 (1H, app dq,

C-3′-H), 5.59-6.01 (1H, m, C-2′-H), 6.56 (1H, d, J ) 2.7 Hz,

C-4-H), 6.63 (1H, dd, J ) 8.2 Hz, J ) 2.7 Hz, C-2-H), 7.15

(1H, d, J ) 8.2 Hz, C-1-H); δ_C(CDCl₃, 100.4 MHz) 17.7 (q,

C-18), 23.4 (t), 26.5 (t), 26.6 (t), 27.6 (t), 30.0 (t), 34.5 (t), 38.5

(d), 44.3 (d), 44.8 (s, C-13), 53.3 (d), 74.6 (t, C-1′), 113.0 (d),

115.5 (d), 117.3 (t, C-3′), 126.7 (d), 132.4 (s), 134.7 (d, C-2′),

138.2 (s), 153.7 (s, C-3), 171.3 (s, CdN); MS m/z (FAB+) 326.1

[100, (M + H)⁺], 268.1 [30]; Acc MS m/z (FAB+) 326.2116,

C₂₁H₂₈NO₂requires 326.2120. HPLC (MeOH/H₂O, 90:10) R_t

) 3.01 min, 99%. Anal. (C₂₁H₂₇NO₂) C, H, N.

16-(1′-Hydroxy-1′′-pyridin-3′′-ylmethylene)-estrone (18).

Following method 2, a solution of 14 (350 mg, 716 µmol) in

anhydrous THF (10 mL) was treated with a 1.0 M solution of

TBAF in anhydrous THF (1.43 mL, 1.43 mmol) for 3 h. The

crude product was triturated with boiling EtOH to give 18 as

a pale yellow solid (125 mg, 50%): mp 275-278 °C; IR (KBr)

2950-2855, 1645, 1610-1500 cm^-1; δ_H(CDCl₃, 400 MHz) 1.09

(3H, s, C-18-H₃), 1.39-2.91 (13H, m), 5.48 (1H, s, exchanged

with D₂O, C-3-OH), 6.59 (1H, d, J ) 2.7 Hz, C-4-H), 6.65 (1H,

dd, J ) 8.4 Hz, J ) 2.7 Hz, C-2-H), 7.15 (1H, d, J ) 8.4 Hz,

C-1-H), 7.41 (1H, ddd, J ) 8.1 Hz, J ) 4.9 Hz, J ) 0.8 Hz,

C-5′′-H), 8.09 (1H, dt, J ) 8.1 Hz, J ) 1.8 Hz, C-4′′-H), 8.67

(1H, dd, J ) 4.9 Hz, J ) 1.8 Hz, C-6′′-H), 8.94 (1H, app d, J )

1.8 Hz, C-2′′-H), 13.58 (1H, s, exchanged with D₂O, C-1′-OH);

MS m/z (FAB+) 376.0 [100, (M + H)⁺], 242.1 [54]; Acc MS

m/z (FAB+) 376.1903, C₂₄H₂₆NO₃requires 376.1913. Anal.

[C₂₄H₂₅NO₃.(H₂O)_1/2] C, H, N.

16-(1′-Hydroxy-2′-carboxylic acid ethyl ester-ethyl-

idene)-estrone (19). Following method 2, a solution of 15 (485

mg, 1.00 mmol) in anhydrous THF (25 mL) was treated with

a 1.0 M solution of TBAF in anhydrous THF (1.50 mL, 1.50

mmol) for 1 h. The crude product was recrystallized from

EtOAc/hexane to give 19 as colorless crystals (303 mg, 82%):

mp 200-202 °C; δ_H(CDCl₃, 400 MHz) 1.00 (3H, s, C-18-H₃),

1.40 (3H, t, J ) 7.2 Hz), 1.42-3.09 (13H, m), 4.36 (2H, q, J )

7.2 Hz), 4.61 (1H, br s, OH), 6.59 (1H, d, J ) 2.3 Hz, C-4-H),

6.64 (1H, dd, J ) 8.2 Hz, J ) 2.3 Hz, C-2-H), 7.14 (1H, d, J )

8.2 Hz, C-1-H), 12.47 (1H, br s, OH); δ_C(DMSO-d₆, 100.4 MHz)

14.2 (q), 15.0 (q), 25.9 (t), 26.7 (t), 27.5 (t), 29.4 (t), 31.1 (t),

37.7 (d), 43.9 (d), 48.8 (s), 49.4 (d), 62.2 (t), 112.8 (d), 115.2

(d), 116.5 (s), 126.2 (d), 131.6 (d), 137.7 (s), 152.4 (s), 153.4 (s),

162.6 (s), 216.7 (s); MS m/z (FAB+) 370.2 [100, (M + H)⁺].

Anal. (C₂₂H₂₆O₅) C, H.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-b]-pyridine (24).

Compound 23 (2.45 g, 7.53 mmol) was stirred and heated to

230 °C (using a sand bath) for 46 h. The resulting dark brown

solid was allowed to cool, and EtOH was added until most of

the solid was dissolved. The remaining undissolved material

was crushed to a fine powder, silica was added, and the solvent

was removed under reduced pressure. This was purified by

flash chromatography (CHCl₃/EtOAc, 9:1) to give 24 as a dark

5764 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18

Fischer et al.

orange solid (200 mg, 9%). A second flash chromatography

(CHCl₃/EtOAc, 95:5) yielded a light orange powder (146 mg,

6%). For analysis, a sample was triturated with CHCl₃to give

an off-white powder: mp 279-284 °C (dec); IR (KBr) 3415,

3115-3025, 2985-2855, 1615-1500 cm^-1; δ_H(DMSO-d₆, 400

MHz) 0.92 (3H, s, C-18-H₃), 1.36-2.83 (13H, m), 6.47 (1H, d,

J ) 2.4 Hz, C-4-H), 6.54 (1H, dd, J ) 8.4 Hz, J ) 2.4 Hz, C-2-

H), 7.05-7.09 (2H, m, C-1-H and C-5′-H), 7.60 (1H, d, J )

7.4 Hz, C-4′-H), 8.25 (1H, app d, J ) 4.3 Hz, C-6′-H), 9.05

(1H, s, exchanged with D₂O, OH); δ_H(CDCl₃, 400 MHz) 1.01

(3H, s, C-18-H₃), 1.43-2.96 (13H, m), 6.08 (1H, br s, OH), 6.61

(1H, d, J ) 2.6 Hz, C-4-H), 6.67 (1H, dd, J ) 8.4 Hz, J ) 2.6

Hz, C-2-H), 7.04 (1H, dd, J ) 7.4 Hz, J ) 4.8 Hz, C-5′-H),

7.18 (1H, d, J ) 8.4 Hz, C-1-H), 7.54 (1H, d, J ) 7.4 Hz, C-4′-

H), 8.33 (1H, d, J ) 4.8 Hz, C-6′-H); δ_C(DMSO-d₆, 100.4 MHz)

18.5 (q, C-18), 26.9 (t), 27.9 (t), 29.9 (t), 30.4 (t), 34.4 (t), 38.2

(d), 44.7 (d), 46.3 (s, C-13), 55.4 (d), 113.5 (d), 115.7 (d), 121.7

(d), 126.5 (d), 130.9 (s), 133.2 (d), 136.4 (s), 137.7 (s), 147.2

(d), 155.6 (s, C-3), 173.1 (s, CdN); MS m/z (FAB+) 306.1 [85,

(M + H)⁺], 207.0 [95], 114.9 [100]; Acc MS m/z (FAB+)

306.1864, C₂₁H₂₄NO requires 306.1858. HPLC (MeOH/H₂O, 80:

20) R_t) 2.90 min, 98%. Anal. [C₂₁H₂₃NO.(CHCl₃)_1/5] C, H, N.

3-Benzyloxy-estra-1,3,5(10)-triene-[17,16-c]-pyrazole

(26). Hydrazine hydrate (751 µL, 15.44 mmol) was added to a

suspension of 8 (4 g, 10.3 mmol) in EtOH (200 mL) at room

temperature under an atmosphere of N₂. The resulting yellow

suspension was heated to reflux for 45 min and allowed to cool.

After acidification with 5 M HCl, the solvent was removed

under reduced pressure until precipitation of the product. H₂O

(20 mL) was then added, and the precipitate was filtered and

dried to give 26 as an off-white powder (3.73 g, 94%): mp 102-

105°C; IR (KBr) 3410, 2930-2860, 1610-1500, 1255 cm^-1; δ_H

(DMSO-d₆, 400 MHz) 0.91 (3H, s, C-18-H₃), 1.36-2.87 (13H,

m), 5.05 (2H, s, OCH₂Ar), 6.72 (1H, d, J ) 2.7 Hz, C-4-H), 6.76

(1H, dd, J ) 8.6 Hz, J ) 2.7 Hz, C-2-H), 7.18 (1H, d, J ) 8.6

Hz, C-1-H), 7.27 (1H, s, C-5′-H), 7.29-7.44 (5H, m, C₆H₅),

12.00 (1H, s, exchanged with D₂O, NH); MS m/z (FAB+) 385.3

[75, (M + H)⁺], 91.1 [100, (CH₂Ar)⁺]; Acc MS m/z (FAB+)

385.2280, C₂₆H₂₉N₂O requires 385.2280.

3-Benzyloxy-estra-1,3,5(10)-triene-[17,16-c]-(1′-methyl)-

pyrazole (27) and 3-Benzyloxy-estra-1,3,5(10)-triene-[17,-

16-c]-(2′-methyl)-pyrazole (28). Following method 3, 26 (150

mg, 390 µmol) was treated with NaH (19 mg, 468 µmol) in

DMF (6 mL), and the subsequent reaction with methyl iodide

(57 µL, 780 µmol) was complete within 50 min. Purification of

the crude mixture by flash chromatography (DCM/EtOAc, 98:2

to 95:5, gradient) gave two products.

The less polar fraction gave 27 as a light yellow oil that

crystallized on standing (52 mg, 33%): mp 133-135 °C; IR

(KBr) 2930-2860, 1606-1500, 1245 cm^-1; δ_H(CDCl₃, 400

MHz) 1.01 (3H, s, C-18-H₃), 1.42-2.99 (13H, m), 3.84 (3H, s,

N-CH₃), 5.03 (2H, s, OCH₂Ar), 6.73 (1H, d, J ) 2.5 Hz, C-4-

H), 6.79 (1H, dd, J ) 8.5 Hz, J ) 2.5 Hz, C-2-H), 6.97 (1H, s,

C-5′-H), 7.22 (1H, d, J ) 8.5 Hz, C-1-H), 7.29-7.45 (5H, m,

C₆H₅); δ_C(CDCl₃, 100.4 MHz) 18.4 (q, C-18), 24.0 (t), 26.2 (t),

27.4 (t), 29.6 (t), 34.1 (t), 37.5 (d), 38.5 (q, N-CH₃), 40.8 (s,

C-13), 44.4 (d), 61.1 (d), 69.8 (t, OCH₂Ar), 111.9 (d), 114.6 (d),

121.5 (s), 124.0 (d), 125.9 (d), 127.1 (2 × d), 127.5 (d), 128.2 (2

× d), 132.6 (s), 136.9 (s), 137.5 (s), 156.4 (s), 168.6 (s); MS m/z

(FAB+) 399.3 [74, (M + H)⁺], 91.1 [100, (CH₂Ar)⁺], 73.0 [24];

Acc MS m/z (FAB+) 399.2447, C₂₇H₃₁N₂O requires 399.2436.

Anal. (C₂₇H₃₀N₂O) C, H, N.

+ H)⁺], 91.1 [100, (CH₂Ar)⁺]; Acc MS m/z (FAB+) 399.2434,

C₂₇H₃₁N₂O requires 399.2436. Anal. (C₂₇H₃₀N₂O) C, H, N.

3-Benzyloxy-estra-1,3,5(10)-triene-[17,16-c]-(1′-isobutyl)-

pyrazole (29) and 3-Benzyloxy-estra-1,3,5(10)-triene-[17,-

16-c]-(2′-isobutyl)-pyrazole (30). Following method 3, 26

(250 mg, 650 µmol) was treated with NaH (31 mg, 780 µmol)

in DMF (10 mL) and the subsequent reaction with 1-bromo-

2-methyl-propane (124 µL, 1.30 mmol) was complete within 2

h. Purification of the crude mixture by flash chromatography

(DCM/EtOAc, 95:5) gave two products.

The less polar fraction gave 29 as a pale yellow oil (121 mg,

42%): IR (KBr) 2960-2870, 1705, 1640-1495 cm^-1; δ_H(CDCl₃,

400 MHz) 0.87 (3H, d, J ) 6.6 Hz, C-3′′-H₃), 0.89 (3H, d, J )

6.6 Hz, C-4′′-H₃), 1.01 (3H, s, C-18-H₃), 1.42-2.98 (14H, m),

3.81 (1H, dd, J_BA) 13.8 Hz, J ) 7.5 Hz, N-CH_AH_B), 3.86 (1H,

dd, J_AB) 13.8 Hz, J ) 7.5 Hz, N-CH_AH_B), 5.04 (2H, s, OCH₂-

Ar), 6.71-6.75 (1H, m, C-4-H), 6.79 (1H, dd, J ) 8.6 Hz, J )

2.7 Hz, C-2-H), 6.94-6.99 (1H, m, C-5′-H), 7.22 (1H, d, J )

8.6 Hz, C-1-H), 7.31-7.46 (5H, m, C₆H₅); MS m/z (FAB+) 441.2

[100, (M + H)⁺], 91.1 [93, (CH₂Ar)⁺]; Acc MS m/z (FAB+)

441.2894, C₃₀H₃₇N₂O requires 441.2906.

The more polar fraction gave 30 as a white crystalline solid

(56 mg, 19%): mp 126-128 °C; IR (KBr) 2960-2870, 1635-

1455, 1255 cm^-1; δ_H(CDCl₃, 400 MHz) 0.92 (3H, d, J ) 6.6

Hz, C-3′′-H₃), 0.95 (3H, d, J ) 6.6 Hz, C-4′′-H₃), 1.02 (3H, s,

C-18-H₃), 1.46-2.94 (14H, m), 3.74 (1H, dd, J_BA) 13.3 Hz, J

) 7.8 Hz, N-CH_AH_B), 3.85 (1H, dd, J_AB) 13.3 Hz, J ) 7.4

Hz, N-CH_AH_B), 5.04 (2H, s, OCH₂Ar), 6.74 (1H, d, J ) 2.6

Hz, C-4-H), 6.79 (1H, dd, J ) 8.6 Hz, J ) 2.6 Hz, C-2-H), 7.17-

7.21 (2H, m, C-1-H and C-5′-H), 7.31-7.45 (5H, m, C₆H₅); MS

m/z (FAB+) 441.1 [100, (M + H)⁺], 91.1 [60, (CH₂Ar)⁺]; Acc

MS m/z (FAB+) 441.2898, C₃₀H₃₇N₂O requires 441.2906.

3-Benzyloxy-estra-1,3,5(10)-triene-[17,16-c]-(1′-meth-

yl acetate)-pyrazole (31) and 3-Benzyloxy-estra-1,3,5(10)-

triene-[17,16-c]-(2′-methyl acetate)-pyrazole (32). Follow-

ing method 3, 26 (300 mg, 78 µmol) was treated with NaH (47

mg, 1.17 mmol) in DMF (10 mL) and the subsequent reaction

with methyl chloroacetate (136 µL, 1.56 mmol) was complete

within 2.5 h. Purification of the crude mixture by flash

chromatography (DCM to DCM/EtOAc, 8:2, gradient, Flash-

master) gave two products.

The less polar fraction gave 31 as a white crystalline solid

(173 mg, 48%): mp 133-135°C; IR (KBr) 2960-2850, 1750,

1740, 1615-1500, 1265, 1255 cm^-1; δ_H(CDCl₃, 400 MHz) 1.03

(3H, s, C-18-H₃), 1.44-2.99 (13H, m), 3.76 (3H, s, OCH₃), 4.83

(1H, d, J_BA) 17.8 Hz, N-CH_ACH_B), 4.88 (1H, d, J_AB) 17.8

Hz, N-CH_ACH_B), 5.04 (2H, s, OCH₂Ar), 6.74 (1H, d, J ) 2.6

Hz, C-4-H), 6.79 (1H, dd, J ) 8.6 Hz, J ) 2.6 Hz, C-2-H), 7.07

(1H, s, C-5′-H), 7.22 (1H, d, J ) 8.6 Hz, C-1-H), 7.29-7.44

(5H, m, C₆H₅); δ_C(CDCl₃, 100.4 MHz) 18.9 (q, C-18), 24.5 (t),

26.7 (t), 27.9 (t), 30.1 (t), 34.4 (t), 38.0 (d), 41.4 (s, C-13), 44.8

(d), 52.9 (d or q), 53.2 (t, N-CH₂), 61.3 (d or q), 70.3 (t, OCH₂-

Ar), 112.5 (d), 115.1 (d), 123.2 (s), 125.0 (d), 126.4 (d), 127.7 (2

× d), 128.1 (d), 128.7 (2 × d), 133.1 (s), 137.5 (s), 138.0 (s),

156.9 (s), 169.1 (s), 169.8 (s); MS m/z (FAB+) 457.3 [55, (M +

H)⁺], 91.1 [100, (CH₂Ar)⁺], 73.0 [72]; Acc MS m/z (FAB+)

457.2495, C₂₉H₃₃N₂O₃requires 457.2491.

The more polar fraction gave 32 as a pale yellow solid (70

mg, 20%): mp 78-82 °C; IR (KBr) 2930-2850, 1760, 1740,

1635-1500, 1260, 1210 cm^-1; δ_H(CDCl₃, 400 MHz) 1.02 (3H,

s, C-18-H₃), 1.42-2.98 (13H, m), 3.78 (3H, s, OCH₃), 4.80 (1H,

d, J_BA) 17.2 Hz, N-CH_ACH_B), 4.86 (1H, d, J_AB) 17.2 Hz,

N-CH_ACH_B), 5.03 (2H, s, OCH₂Ar), 6.74 (1H, d, J ) 2.6 Hz,

C-4-H), 6.78 (1H, dd, J ) 8.6 Hz, J ) 2.6 Hz, C-2-H), 7.18

(1H, d, J ) 8.6 Hz, C-1-H), 7.25 (1H, s, C-5′-H), 7.31-7.44

(5H, m, C₆H₅); MS m/z (FAB+) 457.3 [64, (M + H)⁺], 91.1 [100,

(CH₂Ar)⁺], 73.0 [27]; Acc MS m/z (FAB+) 457.2507, C₂₉H₃₃N₂O₃

requires 457.2491.

3-Benzyloxy-estra-1,3,5(10)-triene-[17,16-c]-(1′-meth-

oxyethyl)-pyrazole (33) and 3-Benzyloxy-estra-1,3,5(10)-

triene-[17,16-c]-(2′-methoxyethyl)-pyrazole (34). Following

method 3, 26 (300 mg, 780 µmol) was treated with NaH (47

mg, 1.17 mmol) in DMF (10 mL), and the subsequent reaction

with 1-chloro-2-methoxy-ethane (142 µL, 1.56 mmol) was

The more polar fraction gave 28 as an off-white solid (55

mg, 35%): mp 168-171°C; IR (KBr) 2930-2870, 1640-1495,

1255 cm^-1; δ_H(CDCl₃, 400 MHz) 1.01 (3H, s, C-18-H₃), 1.44-

2.98 (13H, m), 3.82 (3H, s, N-CH₃), 5.04 (2H, s, OCH₂Ar), 6.74

(1H, d, J ) 2.8 Hz, C-4-H), 6.80 (1H, dd, J ) 8.6 Hz, J ) 2.8

Hz, C-2-H), 7.15 (1H, s, C-5′-H), 7.20 (1H, d, J ) 8.6 Hz, C-1-

H), 7.30-7.45 (5H, m, C₆H₅); δ_C(CDCl₃, 100.4 MHz) 17.9 (q,

C-18), 24.6 (t), 26.5 (t), 27.7 (t), 30.1 (t), 34.5 (t), 37.3 (q,

N-CH₃), 37.8 (d), 42.0 (s, C-13), 44.6 (d), 62.3 (d), 70.3 (t,

OCH₂Ar), 112.6 (d), 115.1 (d), 123.9 (s), 126.2 (d), 127.6 (2 ×

d), 128.1 (d), 128.7 (2 × d), 132.7 (s), 133.4 (d), 137.4 (s), 138.1

(s), 157.0 (s), 1 singlet not seen; MS m/z (FAB+) 399.3 [55, (M

E-Ring Modified Steroids as Inhibitors of 17â-HSD

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5765

complete within 4 h. Purification of the crude mixture by flash

chromatography (DCM/EtOAc, 9:1) gave two products.

mL) was hydrogenated overnight to give a light yellow solid

(103 mg). The crude product was recrystallized from EtOAc/

hexane to yield 38 as off-white crystals (33 mg, 92%): mp 194-

196 °C (dec); IR (KBr) 3195, 2960-2845, 1620, 1585, 1500

cm^-1; δ_H(CDCl₃, 400 MHz) 0.92 (3H, d, J ) 6.6 Hz, C-3′′-H₃),

0.95 (3H, d, J ) 6.6 Hz, C-4′′-H₃), 1.03 (3H, s, C-18-H₃), 1.41-

2.91 (14H, m), 3.77 (1H, dd, J_BA) 13.4 Hz, J ) 8.2 Hz,

N-CH_AH_B), 3.89 (1H, dd, J_AB) 13.4 Hz, J ) 7.6 Hz,

N-CH_AH_B), ∼5.50 (∼1H, br s, exchanged with D₂O, OH), 6.60

(1H, d, J ) 2.6 Hz, C-4-H), 6.66 (1H, dd, J ) 8.4 Hz, J ) 2.6

Hz, C-2-H), 7.14 (1H, d, J ) 8.4 Hz, C-1-H), 7.22 (1H, s, C-5′-

H); MS m/z (FAB+) 351.3 [100, (M + H)⁺]; Acc MS m/z (FAB+)

351.2444, C₂₃H₃₁N₂O requires 351.2436. HPLC (MeOH/H₂O,

80:20) R_t) 3.59 min, 100%.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(1′-methyl ac-

etate)-pyrazole (39). Following method 1, a suspension of

31 (90 mg, 197 µmol) and Pd-C 10% (40 mg) in MeOH/THF

2:1 (30 mL) was hydrogenated for 6 h to give a white solid (66

mg). The crude product was recrystallized from MeOH to yield

39 as colorless crystals (49 mg, 68%): mp 236-239 °C; IR

(KBr) 3210, 2955-2855, 1755, 1610-1455, 1215 cm^-1; δ_H

(CDCl₃, 400 MHz) 1.03 (3H, s, C-18-H₃), 1.41-2.96 (13H, m),

3.75 (3H, s, OCH₃), 4.83 (1H, d, J_BA) 17.6 Hz, N-CH_AH_B),

4.88 (1H, d, J_AB) 17.6 Hz, N-CH_AH_B), 5.04 (1H, s, OH), 6.58

(1H, d, J ) 2.7 Hz, C-4-H), 6.64 (1H, dd, J ) 8.4 Hz, J ) 2.7

Hz, C-2-H), 7.07 (1H, s, C-5′-H), 7.16 (1H, d, J ) 8.4 Hz, C-1-

H); δ_C(CDCl₃, 100.4 MHz) 18.8 (q, C-18), 24.5 (t), 26.7 (t), 27.9

(t), 29.9 (t), 34.3 (t), 38.0 (d), 41.4 (s, C-13), 44.8 (d), 52.9 (d or

q), 53.1 (t, N-CH₂), 61.3 (d or q), 113.0 (d), 115.5 (d), 123.3

(s), 125.2 (d), 126.5 (d), 132.5 (s), 138.1 (s), 153.9 (s), 169.1 (s),

169.8 (s); MS m/z (FAB+) 367.3 [100, (M + H)⁺], 73.1 [29];

Acc MS m/z (FAB+) 367.2038, C₂₂H₂₇N₂O₃requires 367.2022.

Anal. (C₂₂H₂₆N₂O₃.MeOH) C, H, N.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(2′-methyl ac-

etate)-pyrazole (40). Following method 1, a suspension of

32 (60 mg, 131 µmol) and Pd-C 10% (25 mg) in MeOH/THF

2:1 (30 mL) was hydrogenated overnight to give a light brown

foam (34 mg). The crude product was purified by flash

chromatography (DCM/EtOAc, 8:2) to yield 40 as a light yellow

solid (18 mg, 37%): mp 92-95°C; IR (KBr) 3250, 2925-2855,

1760, 1745, 1615-1455 cm^-1; δ_H(CDCl₃, 400 MHz) 1.02 (3H,

s, C-18-H₃), 1.42-2.91 (14H, m), 3.77 (3H, s, OCH₃), 4.81 (1H,

d, J_BA) 17.4 Hz, N-CH_AH_B), 4.87 (1H, d, J_AB) 17.4 Hz,

N-CH_AH_B), 6.59 (1H, d, J ) 2.7 Hz, C-4-H), 6.64 (1H, dd, J )

8.6 Hz, J ) 2.7 Hz, C-2-H), 7.12 (1H, d, J ) 8.6 Hz, C-1-H),

∼7.25 (∼1H, s, C-5′-H, under solvent peak); MS m/z (FAB+)

367.1 [37, (M + H)⁺], 97.0 [39], 73.0 [55], 57.0 [100]; Acc MS

m/z (FAB+) 367.2029, C₂₂H₂₇N₂O₃requires 367.2022.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(1′-methoxy-

ethyl)-pyrazole (41). Following method 1, a suspension of

33 (80 mg, 181 µmol) and Pd-C 10% (35 mg) in MeOH/THF

2:1 (30 mL) was hydrogenated for 6 h to give a white

crystalline solid (58 mg). The crude product was recrystallized

from MeOH to yield 41 as white crystals (32 mg, 50%): mp

123-125°C; IR (KBr) 3235, 2930-2850, 1640-1505 cm^-1; δ_H

(CDCl₃, 400 MHz) 1.02 (3H, s, C-18-H₃), 1.43-2.93 (13H, m),

3.33 (3H, s, OCH₃), 3.67-3.75 (2H, m, C-1′′-H₂or C-2′′-H₂),

4.17-4.28 (2H, m, C-1′′-H₂or C-2′′-H₂), 5.57 (1H, br s,

exchanged with D₂O, OH), 6.59 (1H, d, J ) 2.7 Hz, C-4-H),

6.64 (1H, dd, J ) 8.4 Hz, J ) 2.7 Hz, C-2-H), 7.09 (1H, s, C-5′-

H), 7.16 (1H, d, J ) 8.4 Hz, C-1-H); MS m/z (FAB+) 353.3

[100, (M + H)⁺]; Acc MS m/z (FAB+) 353.2232, C₂₂H₂₉N₂O₂

requires 353.2229. HPLC (MeOH/H₂O, 80:20) R_t) 2.47 min,

98%. Anal. [C₂₂H₂₈N₂O₂.(MeOH)_1/2] C, H, N.

The less polar fraction gave 33 as a pale yellow solid (92

mg, 27%): mp 97-98 °C; IR (KBr) 2930-2870, 1615, 1500,

1255, 1105 cm^-1; δ_H(CDCl₃, 400 MHz) 1.01 (3H, s, C-18-H₃),

1.46-2.99 (13H, m), 3.33 (3H, s, OCH₃), 3.67-3.76 (2H, m,

C-1′′-H₂or C-2′′-H₂), 4.17-4.28 (2H, m, C-1′′-H₂or C-2′′-H₂),

5.04 (2H, s, OCH₂Ar), 6.74 (1H, d, J ) 2.8 Hz, C-4-H), 6.80

(1H, dd, J ) 8.3 Hz, J ) 2.8 Hz, C-2-H), 7.09 (1H, s, C-5′-H),

7.23 (1H, d, J ) 8.3 Hz, C-1-H), 7.31-7.45 (5H, m, C₆H₅); MS

m/z (FAB+) 443.3 [100, (M + H)⁺], 91.1 [80, (CH₂Ar)⁺]; Acc

MS m/z (FAB+) 443.2696, C₂₉H₃₅N₂O₂requires 443.2698.

The more polar fraction gave 34 as a pale yellow solid (80

mg, 23%): mp 86-88 °C; IR (KBr) 2930-2850, 1635-1495

cm^-1; δ_H(CDCl₃, 400 MHz) 1.04 (3H, s, C-18-H₃), 1.42-2.99

(13H, m), 3.32 (3H, s, OCH₃), 3.73-3.83 (2H, m, C-1′′-H₂or

C-2′′-H₂), 4.14-4.22 (2H, m, C-1′′-H₂or C-2′′-H₂), 5.04 (2H, s,

OCH₂Ar), 6.74 (1H, d, J ) 2.7 Hz, C-4-H), 6.79 (1H, dd, J )

8.6 Hz, J ) 2.7 Hz, C-2-H), 7.19-7.21 (2H, m, C-1-H and C-5′-

H), 7.30-7.44 (5H, m, C₆H₅); MS m/z (FAB+) 443.3 [100, (M

+ H)⁺], 91.1 [95, (CH₂Ar)⁺], 73.1 [44]; Acc MS m/z (FAB+)

443.2712, C₂₉H₃₅N₂O₂requires 443.2698.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(1′-methyl)-

pyrazole (35). Following method 1, a suspension of 27 (50

mg, 125 µmol) and Pd-C 10% (20 mg) in MeOH/THF 2:1 (30

mL) was hydrogenated overnight to give a pale yellow solid

(31 mg). The crude product was recrystallized from EtOH/H₂O

to yield 35 as off-white crystals (26 mg, 68%): mp 287-289°C;

IR (KBr) 3115, 2975-2855, 1605-1500 cm^-1; δ_H(DMSO-d₆,

400 MHz) 0.89 (3H, s, C-18-H₃), 1.32-2.83 (13H, m), 3.73 (3H,

s, N-CH₃), 6.45 (1H, d, J ) 2.4 Hz, C-4-H), 6.52 (1H, dd, J )

8.5 Hz, J ) 2.4 Hz, C-2-H), 7.06 (1H, d, J ) 8.5 Hz, C-1-H),

7.24 (1H, s, C-5′-H), 9.02 (1H, s, OH); MS m/z (FAB+) 309.2

[100, (M + H)⁺], 219.2 [52]; Acc MS m/z (FAB+) 309.1975,

C₂₀H₂₅N₂O requires 309.1967. HPLC (MeOH/H₂O, 96:4) R_t)

1.90 min, 98%. Anal. (C₂₀H₂₄N₂O) C, H, N.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(2′-methyl)-

pyrazole (36). Following method 1, a suspension of 28 (50

mg, 125 µmol) and Pd-C 10% (20 mg) in MeOH/THF 2:1 (30

mL) was hydrogenated overnight to give a white solid (40 mg).

The crude product was recrystallized from EtOH to yield 36

as white crystals (20 mg, 53%): mp 321-323 °C (dec); IR (KBr)

3105-3010, 2935-2860, 1610-1500; δ_H(DMSO-d₆, 400 MHz)

0.95 (3H, s, C-18-H₃), 1.32-2.83 (13H, m), 3.73 (3H, s, N-CH₃),

6.45 (1H, d, J ) 2.6 Hz, C-4-H), 6.52 (1H, dd, J ) 8.4 Hz, J )

2.6 Hz, C-2-H), 7.04 (1H, s, C-5′-H), 7.06 (1H, d, J ) 8.4 Hz,

C-1-H), 9.03 (1H, s, OH); MS m/z (FAB+) 391.3 [28], 309.2

[100, (M + H)⁺]; Acc MS m/z (FAB+) 309.1974, C₂₀H₂₅N₂O

requires 309.1967. HPLC (MeOH/H₂O, 96:4) R_t) 2.41 min,

99%.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(1′-isobutyl)-

pyrazole (37). Following method 1, a suspension of 29 (110

mg, 250 µmol) and Pd-C 10% (40 mg) in MeOH/THF 2:1 (30

mL) was hydrogenated overnight to give a white solid (78 mg).

The crude product was recrystallized from MeOH to yield 37

as colorless crystals (42 mg, 48%): mp 122-123 °C; IR (KBr)

3235, 2955-2853, 1635, 1610, 1575-1455, 1230 cm^-1; δ_H

(CDCl₃, 400 MHz) 0.87 (3H, d, J ) 6.4 Hz, C-3′′-H₃), 0.89 (3H,

d, J ) 6.4 Hz, C-4′′-H₃), 1.01 (3H, s, C-18-H₃), 1.42-2.95 (14H,

m), 3.81 (1H, dd, J_BA) 13.7 Hz, J ) 7.4 Hz, N-CH_AH_B), 3.86

(1H, dd, J_AB) 13.7 Hz, J ) 7.4 Hz, N-CH_AH_B), 5.61 (1H, s,

exchanged with D₂O, OH), 6.59 (1H, d, J ) 2.6 Hz, C-4-H),

6.65 (1H, dd, J ) 8.4 Hz, J ) 2.6 Hz, C-2-H), 6.97 (1H, s, C-5′-

H), 7.16 (1H, d, J ) 8.4 Hz, C-1-H); δ_C(CDCl₃, 100.4 MHz)

18.5 (q, C-18), 20.0 (2 × t, C-3′′ and C-4′′), 24.1 (t), 26.4 (t),

27.5 (t), 29.6 (t), 30.0 (d), 34.2 (t), 37.6 (d), 41.0 (s, C-13), 44.5

(d), 59.6 (t, N-CH₂), 61.2 (d), 112.8 (d), 115.3 (d), 121.2 (s),

123.9 (d), 126.4 (d), 132.5 (s), 138.1 (s), 153.8 (s), 168.7 (s);

MS m/z (FAB+) 351.3 [100, (M + H)⁺]; Acc MS m/z (FAB+)

351.2448, C₂₃H₃₁N₂O requires 351.2436. HPLC (MeOH/H₂O,

80:20) R_t) 3.27 min, 99%. Anal. (C₂₃H₃₀N₂O.MeOH) C, H, N.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(2′-methoxy-

ethyl)-pyrazole (42). Following method 1, a suspension of

34 (70 mg, 158 µmol) and Pd-C 10% (30 mg) in MeOH/THF

2:1 (30 mL) was hydrogenated overnight to give an off-white

foam (50 mg). The crude product was purified by flash

chromatography (DCM/EtOAc, 8:2) to yield 42 as an off-white

oil (25 mg, 45%): IR (KBr) 2920-2855, 1610-1500, 1255 cm^-1

;

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(2′-isobutyl)-

pyrazole (38). Following method 1, a suspension of 30 (45

mg, 102 µmol) and Pd-C 10% (20 mg) in MeOH/THF 2:1 (30

δ_H(CDCl₃, 400 MHz) 1.05 (3H, s, C-18-H₃), 1.42-2.95 (13H,

m), 3.31 (3H, s, OCH₃), 3.74-3.82 (2H, m, C-1′′-H₂or C-2′′-

H₂), 4.12-4.23 (2H, m, C-1′′-H₂or C-2′′-H₂), 6.60 (1H, d, J )

5766 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18

Fischer et al.

2.6 Hz, C-4-H), 6.66 (1H, dd, J ) 8.6 Hz, J ) 2.6 Hz, C-2-H),

7.14 (1H, d, J ) 8.6 Hz, C-1-H), 7.23 (1H, s, C-5′-H), OH not

seen; MS m/z (FAB+) 353.3 [100, (M + H)⁺]; Acc MS m/z

(FAB+) 353.2236, C₂₂H₂₉N₂O₂requires 353.2229.

8.4 Hz, J ) 2.7 Hz, C-2-H), 7.02 (1H, s, C-5′-H), 7.08 (1H, d,

J ) 8.4 Hz, C-1-H); MS m/z (FAB+) 348.1 [90, (M + H)⁺], 147.1

[64], 73.0 [100]; Acc MS m/z (FAB+) 348.2072, C₂₂H₂₆N₃O

requires 348.2076. HPLC (MeOH/H₂O, 90:10) R_t) 2.27 min,

96%.

3-O-tert-Butyl-dimethylsilyl-estra-1,3,5(10)-triene-[17,-

16-c]-pyrazole (43). Hydrazine hydrate (440 µL, 9.1 mmol)

was added to a suspension of 2 (2.5 g, 6.1 mmol) in EtOH (125

mL) at room temperature under an atmosphere of N₂. The

resulting mixture was heated to reflux for 45 min. After the

mixture cooled, the solvent was removed under reduced

pressure, H₂O (200 mL) was added, and the mixture was

acidified with 5 M HCl. The organics were extracted with

EtOAc (2 × 10 mL), washed with H₂O (2 × 50 mL) and then

brine (2 × 50 mL), dried (Na₂SO₄), filtered, and concentrated

under reduced pressure to give a yellow foam (2.9 g). The crude

product was recrystallized from EtOH/H₂O to give 43 as yellow

crystals (2.0 g, 81%): mp 122-124 °C; IR (KBr) 3190, 2930-

2855, 1605-1495 cm^-1; δ_H(DMSO-d₆, 400 MHz) 0.18 (6H, s,

Si(CH₃)₂), 0.95 (3H, s, C-18-H₃), 0.96 (9H, s, C(CH₃)₃), 1.36-

2.87 (13H, m), 6.55 (1H, d, J ) 2.3 Hz, C-4-H), 6.62 (1H, dd,

J ) 8.5 Hz, J ) 2.3 Hz, C-2-H), 7.16 (1H, d, J ) 8.5 Hz, C-1-

H), 7.28 (1H, s, C-5′-H), 12.02 (1H, s, exchanged with D₂O,

NH); δ_C(CDCl₃, 100.4 MHz) -3.8 (2×q, Si(CH₃)₂), 18.6 (s,

C(CH₃)₃), 18.8 (q, C-18), 24.3 (t), 26.2 (3×q, C(CH₃)₃), 26.6 (t),

27.9 (t), 30.0 (t), 34.5 (t), 38.0 (d), 41.1 (s, C-13), 44.9 (d), 61.9

(d), 117.4 (d), 120.2 (d), 121.8 (s), 123.2 (s), 126.2 (d), 133.2

(s), 137.8 (s), 153.5 (s), 168.6 (s); MS m/z (FAB+) 409.2 [100,

(M + H)⁺], 73.0 [33]; Acc MS m/z (FAB+) 409.2662, C₂₅H₃₇N₂-

OSi requires 409.2675. Anal. [C₂₅H₃₆N₂OSi.(H₂O)_2/3] C, H, N.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(2′-propioni-

trile)-pyrazole (47). Method A. Following method 2, a

solution of 45 (35 mg, 76 µmol) in anhydrous THF (5 mL) was

treated with a 1.0 M solution of TBAF in anhydrous THF (150

µL, 150 µmol) for 3 h. The crude brown oil was purified by

flash chromatography (DCM/EtOAc, 8:2) to give 47 as a light

brown oil (10 mg, 38%).

Method B. Following method 2, a solution of 48 (220 mg,

458 µmol) in anhydrous THF (20 mL) was treated overnight

with a 1.0 M solution of TBAF in anhydrous THF (916 µL,

916 µmol). The crude product was purified by flash chroma-

tography (EtOAc/CHCl₃, 9:1) to give 47 as a pale pink foam

(136 mg, 85%). This compound was then triturated with

EtOAc/hexane to give white crystals (99 mg, 62%): mp 198-

200 °C; IR (KBr) 3160, 2925-2860, 2250, 1610-1500 cm^-1

;

δ_H(CDCl₃, 400 MHz) 1.06 (3H, s, C-18-H₃), 1.41-3.05 (15H,

m), 4.22-4.34 (2H, m, N-CH₂), 5.42 (1H, br s, exchanged with

D₂O, OH), 6.57 (1H, d, J ) 2.4 Hz, C-4-H), 6.64 (1H, dd, J )

8.3 Hz, J ) 2.4 Hz, C-2-H), 7.12 (1H, d, J ) 8.3 Hz, C-1-H),

7.25 (1H, s, C-5′-H); δ_C(CDCl₃, 100.4 MHz) 18.6 (q, C-18),

19.7 (t, CH₂CN), 24.5 (t), 26.5 (t), 27.6 (t), 29.8 (t), 34.8 (t),

37.7 (d), 42.4 (s, C-13), 44.4 (d), 45.9 (t, N-CH₂), 62.2 (d), 113.1

(d), 115.7 (d), 117.4 (s), 124.3 (s), 126.4 (d), 132.0 (s), 135.2

(d), 138.2 (s), 154.1 (s), 157.98 (s); MS m/z (FAB+) 348.1 [100,

(M + H)⁺], 147.0 [50], 85.1 [75], 73.0 [94]; Acc MS m/z (FAB+)

348.2060, C₂₂H₂₆N₃O requires 348.2076. Anal. (C₂₂H₂₅N₃O) C,

H, N.

3-O-tert-Butyl-dimethylsilyl-estra-1,3,5(10)-triene-[17,-

16-c]-(1′-propionitrile)-pyrazole (44) and 3-O-tert-Butyl-

dimethylsilyl-estra-1,3,5(10)-triene-[17,16-c]-(2′-propion-

t

itrile)-pyrazole (45). BuOK (91 mg, 807 µmol) was added

3-O-tert-Butyl-dimethylsilyl-estra-1,3,5(10)-triene-[17,-

16-c]-(2′-propionitrile-3′,4′-dihydro)-pyrazol-3′-ol (48). Cy-

anoethylhydrazine (177 µL, 2.18 mmol) was added to a stirred

solution of 2 (600 mg, 1.45 mmol) in EtOH (50 mL) under an

atmosphere of N₂. After 4 h of stirring at room temperature,

the resulting orange mixture was concentrated under reduced

pressure, and H₂O (100 mL) was added, followed by 5 M HCl.

The organics were extracted with EtOAc (2 × 100 mL), washed

with H₂O (50 mL) and then brine (50 mL), dried (Na₂SO₄),

filtered, and concentrated under reduced pressure. The crude

product was purified by flash chromatography (CHCl₃/EtOAc,

7:3) to give 48 as a pale yellow solid (510 mg, 73%). This

compound was recrystallized from EtOH to give white needles

(240 mg, 34%): mp 145-147 °C; IR (KBr) 3380, 2950-2860,

2260, 1610, 1495 cm^-1; δ_H(DMSO-d₆, 400 MHz) 0.15 (6H, s,

Si(CH₃)₂), 0.88 (3H, s, C-18-H₃), 0.93 (9H, s, C(CH₃)₃), 1.19-

3.16 (18H, m), 5.97 (1H, s, exchanged with D₂O, OH), 6.48 (1H,

d, J ) 2.7 Hz, C-4-H), 6.56 (1H, dd, J ) 8.2 Hz, J ) 2.7 Hz,

C-2-H), 6.74 (1H, d, J ) 1.9 Hz, C-5′-H), 7.10 (1H, d, J ) 8.2

Hz, C-1-H); δ_C(CDCl₃, 100.4 MHz) -4.4 (2×q, Si(CH₃)₂), 15.6

(q, C-18), 17.1 (t, CH₂CN), 18.0 (s, C(CH₃)₃), 25.6 (3×q,

C(CH₃)₃), 25.8 (t), 27.0 (t), 29.1 (t), 29.4 (t), 31.4 (t), 38.6 (d),

43.2 (d), 43.9 (t, N-CH₂), 47.1 (s, C-13), 49.5 (d), 56.7 (d), 104.2

(s), 116.8 (d), 119.4 (d), 119.9 (s, CN), 126.0 (d), 132.5 (s), 137.3

(s), 144.6 (s), 152.4 (s); MS m/z (FAB+) 480.1 [100, (M + H)⁺],

462.1 [63, (M + H - H₂O)⁺], 72.9 [77]; MS m/z (FAB-) 632.3

[32, (M + NBA)^-]; Acc MS m/z (FAB+) 480.3038 C₂₈H₄₂N₃O₂-

Si requires 480.3046.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(5′-methyl)-

pyrazole (49). Hydrazine hydrate (56 µL, 1.15 mmol) was

added to a refluxing solution of 17 (240 mg, 768 µmol) in EtOH

(15 mL) under an atmosphere of N₂. The resulting pale yellow

solution was heated to reflux for 45 min, after which the

solvent was removed under reduced pressure, H₂O (50 mL)

was added, and the mixture was acidified with 5 M HCl. The

organics were extracted with EtOAc (2 × 50 mL), washed with

H₂O (30 mL) and then brine (2 × 30 mL), dried (Na₂SO₄),

filtered, and concentrated under reduced pressure. The crude

product was purified by flash chromatography (DCM/EtOAc,

8:2) to give 49 as a pale brown solid (84 mg, 35%). This was

triturated with acetone to give an off-white solid (32 mg,

13%): mp 234-236 °C; IR (KBr) 3295, 2930-2860, 1620-1500

to a stirred solution of 43 (300 mg, 734 µmol) in anhydrous

THF (10 mL) at 0 °C under an atmosphere of N₂. After 20 min

of stirring, acrylonitrile (58 µL, 881 µmol) was added, and the

resulting bright orange solution was stirred for 5 h at room

temperature. The resulting dark orange mixture was then

concentrated under reduced pressure, and H₂O (50 mL) was

added. The organics were extracted with EtOAc (2 × 50 mL),

washed with H₂O (2 × 30 mL) and then brine (2 × 30 mL),

dried (Na₂SO₄), filtered, and concentrated under reduced

pressure. The crude product was purified by flash chromatog-

raphy (DCM/EtOAc, 9:1) to give two products.

The less polar fraction gave 44 as a light yellow oil (29 mg,

8%): IR (KBr) 2930-2860, 2250, 1640-1495 cm^-1; δ_H(CDCl₃,

400 MHz) 0.20 (6H, s, Si(CH₃)₂), 0.98 (9H, s, C(CH₃)₃), 1.02

(3H, s, C-18-H₃), 1.42-3.01 (15H, m), 4.28-4.40 (2H, m,

N-CH₂), 6.57 (1H, d, J ) 2.6 Hz, C-4-H), 6.63 (1H, dd, J )

8.5 Hz, J ) 2.6 Hz, C-2-H), 7.10 (1H, s, C-5′-H), 7.14 (1H, d,

J ) 8.5 Hz, C-1-H); MS m/z (FAB+) 462.2 [100, (M + H)⁺],

73.0 [55]; Acc MS m/z (FAB+) 462.2914, C₂₈H₄₀N₃OSi requires

462.2941.

The more polar fraction gave 45 as a light brown oil that

crystallized on standing (42 mg, 12%): mp 80-82 °C; IR (KBr)

2930-2855, 2255, 1705-1445 cm^-1; δ_H(CDCl₃, 400 MHz) 0.20

(6H, s, Si(CH₃)₂), 0.98 (9H, s, C(CH₃)₃), 1.07 (3H, s, C-18-H₃),

1.41-3.06 (15H, m), 4.21-4.38 (2H, m, N-CH₂), 6.57 (1H, d,

J ) 2.4 Hz, C-4-H), 6.63 (1H, dd, J ) 8.7 Hz, J ) 2.4 Hz, C-2-

H), 7.11 (1H, d, J ) 8.7 Hz, C-1-H), 7.25 (1H, s, C-5′-H); MS

m/z (FAB+) 462.1 [100, (M + H)⁺], 73.0 [79]; Acc MS m/z

(FAB+) 462.2922, C₂₈H₄₀N₃OSi requires 462.2941.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(1′-propioni-

trile)-pyrazole (46). Following method 2, a solution of 44 (25

mg, 50 µmol) in anhydrous THF (5 mL) was treated with a

1.0 M solution of TBAF in anhydrous THF (110 µL, 110 µmol)

for 3 h. The crude yellow oil was purified by flash chromatog-

raphy (DCM/EtOAc, 8:2) to give 46 as a light yellow oil (12

mg, 63%). This product was then triturated with EtOAc/

hexane to give a light yellow powder (8 mg): mp 92-94 °C;

IR (KBr) 3230, 3095, 2925-2860, 2265, 1665-1500, 1245 cm^-1

;

δ_H(CDCl₃, 400 MHz) 0.94 (3H, s, C-18-H₃), 1.34-2.94 (15H,

m), 4.19-4.31 (2H, m, N-CH₂), 4.92 (1H, br s, exchanged with

D₂O, OH), 6.50 (1H, d, J ) 2.7 Hz, C-4-H), 6.55 (1H, dd, J )

E-Ring Modified Steroids as Inhibitors of 17â-HSD

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5767

cm^-1; δ_H(DMSO-d₆, 400 MHz) 1.01 (3H, s, C-18-H₃), 1.34-

2.85 (13H, m), 2.28 (3H, s, C-1′′-H₃), 6.45 (1H, d, J ) 2.6 Hz,

C-4-H), 6.52 (1H, dd, J ) 8.6 Hz, J ) 2.6 Hz, C-2-H), 7.04

(1H, d, J ) 8.6 Hz, C-1-H), 9.04 (1H, s, exchanged with D₂O,

NH or OH), NH or OH not seen; MS m/z (FAB+) 309.2 [100,

(M + H)⁺], 95.1 [33], 69.0 [39]; Acc MS m/z (FAB+) 309.1978,

C₂₀H₂₅N₂O requires 309.1967. HPLC (MeOH/H₂O, 96:4) R_t)

1.98 min, 99%.

after which AcOH (5 mL) and H₂O (100 mL) were added. The

organics were extracted with EtOAc (50 mL), washed with H₂O

(100 mL) and then brine (50 mL), dried (Na₂SO₄), filtered, and

concentrated under reduced pressure. The light yellow crude

was recrystallized from EtOH to give 53 as a colorless

crystalline solid (7.28 g, 99%): mp 176-178 °C; δ_H(CDCl₃,

400 MHz) 1.01 (3H, s, C-18-H₃), 1.41 (3H, t, J ) 7.0 Hz), 1.44-

3.10 (13 H, m), 4.37 (2H, q, J ) 7.0 Hz), 5.05 (2H, s, OCH₂Ar),

6.75 (1H, d, J ) 2.7 Hz, C-4-H), 6.80 (1H, dd, J ) 8.6 Hz, J )

2.7 Hz, C-2-H), 7.20 (1H, d, J ) 8.6 Hz, C-1-H), 7.30-7.46 (5H,

m, C₆H₅), 12.48 (1H, s, OH); δ_C(CDCl₃, 100.4 MHz) 14.7 (q),

15.4 (q), 26.2 (t), 27.2 (t), 27.9 (t), 30.0 (t), 31.5 (t), 38.1 (d),

44.4 (d), 49.1 (s), 49.8 (d), 62.4 (t), 70.3 (t), 112.7 (d), 115.1 (d),

116.8 (s), 126.4 (d), 127.7 (d), 128.1 (d), 128.8 (d), 132.3 (s),

137.4 (s), 137.9 (s), 152.7 (s), 157.0 (s), 162.8 (s), 217.0 (s); MS

m/z (FAB+) 461.2 [100, (M + H)⁺]. Anal. (C₂₉H₃₂O₅) C, H.

3-O-Benzyl-estra-1,3,5(10)-triene-[17,16-c]-(5′-carboxy-

lic acid ethyl ester)-pyrazole (54). Hydrazine hydrate (0.75

mL, 15 mmol) was added to a suspension of 53 (6.91 g, 15.0

mmol) in EtOH (100 mL) and DCM (20 mL) at room temper-

ature. The resulting mixture was heated to reflux for 15 min

and stirred overnight at room temperature. The solvents were

removed under reduced pressure to give a white solid, which

was dissolved in EtOH (50 mL). p-Toluenesulfonic acid mono-

hydrate (200 mg, 1.05 mmol) was then added, and the mixture

was heated to reflux for 5 min. After the mixture cooled, H₂O

(50 mL) was added, and the organics were extracted with

EtOAc (120 mL), washed with saturated aqueous NaHCO₃(30

mL) and then brine (30 mL), dried (Na₂SO₄), filtered, and

concentrated under reduced pressure. The crude product was

recrystallized from EtOAc/hexane to give 54 as fine colorless

crystals (5.43 g, 79%): mp 208-211 °C; δ_H(CDCl₃, 400 MHz)

1.05 (3H, s, C-18-H₃), 1.39 (3H, t, J ) 7.0 Hz), 1.44-3.00 (13

H, m), 4.37 (2H, q, J ) 7.0 Hz), 5.05 (2H, s, OCH₂Ar), 6.73

(1H, d, J ) 2.3 Hz, C-4-H), 6.81 (1H, dd, J ) 8.6 Hz, J ) 2.3

Hz, C-2-H), 7.23 (1H, d, J ) 8.6 Hz, C-1-H), 7.30-7.46 (5H,

m, C₆H₅), NH not seen; δ_C(CDCl₃, 100.4 MHz) MS m/z (FAB+)

457.4 [100, (M + H)⁺]; Acc MS m/z (FAB+) 457.2488,

C₂₉H₃₃N₂O₃requires 457.2491. Anal. (C₂₉H₃₂N₂O₃) C, H, N.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(5′-trifluoro-

methyl)-pyrazole (50). Hydrazine hydrate (32 µL, 655 µmol)

was added to a refluxing solution of 20 (160 mg, 437 µmol) in

EtOH (15 mL) under an atmosphere of N₂. The resulting pale

yellow solution was heated to reflux for 3 h, after which

p-toluenesulfonic acid monohydrate (∼10 mg) was added. The

mixture was then heated to reflux overnight, and after the

mixture cooled, the solvent was removed under reduced

pressure, and H₂O (50 mL) was added, followed by 5 M HCl.

The organics were extracted with EtOAc (2 × 50 mL), washed

with H₂O (30 mL) and then brine (2 × 30 mL), dried (Na₂-

SO₄), filtered, and concentrated under reduced pressure. The

crude product was purified by flash chromatography (DCM/

EtOAc, 8:2) to give 50 as a pale yellow solid (95 mg, 60%).

This compound was precipitated from DCM/hexane (1:5) to

give an off-white solid (51 mg, 32%): mp 152-155°C; IR (KBr)

3220, 2930-2860, 1610-1500 cm^-1; δ_H(CDCl₃, 400 MHz) 1.01

(3H, s, C-18-H₃), 1.40-2.89 (13H, m), 4.63 (1H, s, exchanged

with D₂O, C-3-OH), 6.53 (1H, d, J ) 2.7 Hz, C-4-H), 6.59 (1H,

dd, J ) 8.2 Hz, J ) 2.7 Hz, C-2-H), 7.08 (1H, d, J ) 8.2 Hz,

C-1-H), NH not seen; MS m/z (FAB+) 363.1 [100, (M + H)⁺];

Acc MS m/z (FAB+) 363.1689, C₂₀H₂₂N₂F₃O requires 363.1684.

HPLC (MeOH/H₂O, 85:15) R_t) 2.43 min, 98%.

3-O-tert-Butyl-dimethylsilyl-estra-1,3,5(10)-triene-[17,-

16-c]-[5′-(1′′-pyridin-3′′-yl)-2′,3′-dihydro)]-pyrazol-3′-ol (51).

Hydrazine hydrate (75 µL, 1.53 mmol) was added to a refluxing

solution of 14 (500 mg, 1.02 mmol) in EtOH (20 mL) under an

atmosphere of N₂. The resulting pale yellow solution was

heated to reflux for 1 h, after which the solvent was partially

removed under reduced pressure and H₂O (100 mL) was added.

The resulting white precipitate was filtered and dried (460 mg,

89%), and an analytical sample was recrystallized from EtOH/

H₂O to give 51 as white crystals: mp 144-145 °C; IR (KBr)

3480, 3330, 3190, 2940-2860, 1610-1495 cm^-1; δ_H(DMSO-

d₆, 400 MHz) 0.14 (6H, s, Si(CH₃)₂), 0.92 (9H, s, C(CH₃)₃), 0.94

(3H, s, C-18-H₃), 1.12-2.71 (13H, m), 3.43 (1H, app d, J ) 9.0

Hz, C-16-H), 5.97 (1H, s, exchanged with D₂O, C-17-OH), 6.46

(1H, d, J ) 2.5 Hz, C-4-H), 6.55 (1H, dd, J ) 8.4 Hz, J ) 2.5

Hz, C-2-H), 7.10 (1H, d, J ) 8.4 Hz, C-1-H), 7.26 (1H, s,

exchanged with D₂O, NH), 7.35 (1H, app ddd, J ) 8.1 Hz, J )

4.6 Hz, J ) 0.8 Hz, C-5′′-H), 7.91 (1H, app dt, J ) 8.1 Hz, J )

1.7 Hz, C-4′′-H), 8.43 (1H, dd, J ) 4.6 Hz, J ) 1.7 Hz, C-6′′-

H), 8.74 (1H, app d, J ) 1.7 Hz, C-2′′-H); MS m/z (FAB+) 504.1

[100, (M + H)⁺], 486.1 [27, (M + H - H₂O)⁺]; Acc MS m/z

(FAB+) 504.3045, C₃₀H₄₂N₃O₂Si requires 504.3046.

3-O-Benzyl-estra-1,3,5(10)-triene-[17,16-c]-(5′-carboxy-

lic acid)-pyrazole (55). A 2 M NaOH solution (7.5 mL, 15

mmol) was added to a suspension of 54 (2.28 g, 5.0 mmol) in

EtOH (40 mL). The resulting mixture was heated to reflux

for 30 min and allowed to cool. AcOH (5 mL) was then added,

and the mixture was stirred for an additional 2 h, while the

product precipitated. This was filtered off, washed with H₂O

(50 mL) and EtOH (20 mL), and dried under high vacuum for

2 days to give 55 as a white powder (2.13 g, 99%): mp > 290

°C (dec); δ_H(DMSO-d₆, 400 MHz) 0.95 (3H, s, C-18-H₃), 1.40-

2.94 (13 H, m), 5.06 (2H, s, OCH₂Ar), 6.73 (1H, d, J ) 2.3 Hz,

C-4-H), 6.78 (1H, dd, J ) 8.6 Hz, J ) 2.3 Hz, C-2-H), 7.20

(1H, d, J ) 8.6 Hz, C-1-H), 7.30-7.45 (5H, m, C₆H₅), 12.90

(2H, br s, OH, NH); MS m/z (FAB+) 429.2 [100, (M + H)⁺].

Data for a Representative Benzylated Intermediate,

3-O-Benzyl-estra-1,3,5(10)-triene-[17,16-c]-[5′-(furan-2′′-

ylmethyl)carbamoyl]-pyrazole (59). Prepared according to

method 4. 18%; δ_H(CDCl₃, 270 MHz) 1.02 (3H, s, C-18-H₃),

1.35-1.94 (5H, m), 2.10-2.48 (5H, m), 2.70-2.90 (3H, m), 4.61

(2H, d, J ) 5.7 Hz), 5.03 (2H, s), 6.28-6.33 (2H, m), 6.72 (1H,

d, J ) 2.6 Hz, C-4-H), 6.78 (1H, dd, J ) 8.4 Hz, J ) 2.6 Hz,

C-2-H), 7.20 (1H, d, J ) 8.4 Hz, C-1-H), 7.24-7.44 (6H); MS

m/z (ES+) 508.3 [M + H]⁺. HPLC (MeOH/H₂O, 96:4) R_t) 2.77

min, 93%.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(5′-methyl-

carbamoyl)-pyrazole (63). Prepared according to method 5.

57%; δ_H(CD₃OD, 270 MHz) 1.03 (3H, s, C-18-H₃), 1.39-2.88

(13H, m), 2.89 (3H, s), 6.50 (1H, d, J ) 2.5 Hz, C-4-H), 6.54

(1H, app dd, C-2-H), 7.09 (1H, d, J ) 8.2 Hz, C-1-H); MS m/z

(APCI+) 350.21 [M - H]⁺. Acc MS m/z (FAB+) 353.2110,

C₂₁H₂₇N₃O₂requires 353.2103. HPLC (MeOH/H₂O, 96:4) R_t)

1.66 min, 99%.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-[5′-(1′′-pyri-

din-3′′-yl)]-pyrazole (52). Following method 2, a solution of

51 (110 mg, 218 µmol) in anhydrous THF (10 mL) was treated

overnight with a 1.0 M solution of TBAF in anhydrous THF

(436 µL, 436 µmol) for 3 h. The crude foam was triturated with

boiling EtOAc/CHCl₃(1:1) to give 52 as a white powder (61

mg, 75%): mp 298-300 °C (dec); IR (KBr) 2980-2840, 1610-

1495 cm^-1; δ_H(DMSO-d₆, 400 MHz) 0.98 (3H, s, C-18-H₃),

1.37-2.87 (13H, m), 6.45 (1H, d, J ) 2.6 Hz, C-4-H), 6.51 (1H,

dd, J ) 8.5 Hz, J ) 2.6 Hz, C-2-H), 7.05 (1H, d, J ) 8.5 Hz,

C-1-H), 7.37-7.48 (1H, m, C-4′′-H or C-5′′-H), 7.97-8.06 (1H,

m, C-4′′-H or C-5′′-H), 8.42-8.49 (1H, m, C-2′′-H or C-6′′-H),

8.84-8.89 (1H, m, C-2′′-H or C-6′′-H), 9.01 (1H, s, exchanged

with D₂O, OH), 12.65 and 12.72 (1H, 2 × s, exchanged with

D₂O, NH); MS m/z (FAB+) 372.0 [100, (M + H)⁺]; Acc MS m/z

(FAB+) 372.2080, C₂₄H₂₆N₃O requires 372.2076.

3-O-Benzyl-16-(1′-hydroxy-2′-carboxylic acid ethyl ester-

t

ethylidene)-estrone (53). BuOK (2.24 g, 20.0.mmol) was

added to a solution of 5 (5.77 g, 16.0 mmol) and diethyl oxalate

(5 mL, 36.8 mmol) in toluene (100 mL). The resulting clear

yellow solution was stirred overnight at room temperature

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(5′-isopropy-

lcarbamoyl)-pyrazole (64). Prepared according to method

5. 63%; δ_H(CDCl₃, 270 MHz) 0.97 (3H, s, C-18-H₃), 1.25 (3H,

5768 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18

Fischer et al.

s), 1.27 (3H, s), 1.47-2.94 (13H, m), 4.19-4.30 (1H, m), 5.88

(1H, d, J ) 8.1 Hz), 6.60 (1H, d, J ) 2.3 Hz, C-4-H), 6.65 (1H,

dd, J ) 8.3 Hz, J ) 2.9 Hz, C-2-H), 7.16 (1H, d, J ) 8.3 Hz,

C-1-H); MS m/z (AP+) 378.35 [M - H]⁺; Acc MS m/z (FAB+)

380.2324, C₂₃H₃₀N₃O₂requires 380.2338. HPLC (MeOH/H₂O,

96:4) R_t) 1.72 min, 95%.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-(5′-ethylme-

thylcarbamoyl)-pyrazole (65). Prepared according to method

5. 76%; δ_H(CD₃OD, 270 MHz) 0.96 (3H, s, C-18-H₃), 1.12 (3H,

t, J ) 7.2 Hz), 1.25-2.79 (13H, m), 2.95 and 3.09 (total 3H, 2

× s), 3.44-3.54 (2H, m), 6.40 (1H, d, J ) 2.6 Hz, C-4-H), 6.46

(1H, dd, J ) 8.4 Hz, J ) 2.6 Hz, C-2-H), 7.00 (1H, d, J ) 8.4

Hz, C-1-H); MS m/z (FAB+) 380.0 [M + H]⁺. Acc MS m/z

(FAB+) 380.2330, C₂₃H₃₀N₃O₂requires 380.2338. HPLC (MeOH/

H₂O, 96:4) R_t) 1.69 min, 93%.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-[5′-(tetrahy-

drofuran-2′′-ylmethyl)carbamoyl]-pyrazole (66). Prepared

according to method 5. 30%; δ_H(CD₃OD, 270 MHz) 0.92 (3H,

s, C-18-H₃), 1.25-2.82 (17H, m), 3.25-3.45 (2H, m), 3.59-3.71

(1H, m), 3.74-3.85 (1H, m), 3.89-4.00 (1H, m), 6.40 (1H, d, J

) 2.6 Hz, C-4-H), 6.45 (1H, dd, J ) 8.4 Hz, J ) 2.6 Hz, C-2-

H), 6.99 (1H, d, J ) 8.4 Hz, C-1-H); MS m/z (APCI+) 422.3 [M

+ H]⁺. Acc MS m/z (FAB+) 422.2433, C₂₅H₃₂N₃O₃requires

422.2443. HPLC (MeOH/H₂O, 96:4) R_t) 1.70 min, 100%.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-[5′-(1′′-meth-

ylpyrrol-2′′-ylmethyl)carbamoyl]-pyrazole (67). Prepared

according to method 5. 36%; δ_H(CD₃OD, 270 MHz) 0.94 (3H,

s, C-18-H₃), 1.05-2.79 (13H, m), 3.52 (3H, s), 4.43 (2H, s),

5.85-5.86 (1H, m), 5.96-5.98 (1H, m), 6.40 (1H, d, J ) 2.7

Hz, C-4-H), 6.45 (1H, dd, J ) 8.4 Hz, J ) 2.7 Hz, C-2-H), 6.51-

6.53 (1H, m), 7.01 (1H, d, J ) 8.4 Hz, C-1-H); MS m/z (FAB+)

431.0 [M + H]⁺. Acc MS m/z (FAB+) 431.2442, C₂₆H₃₁N₄O₂

requires 431.2447. HPLC (MeOH/H₂O, 96:4) R_t) 2.11 min,

100%.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-[5′-(1′′-meth-

ylpiperazin-4′′-ylmethyl)carbamoyl]-pyrazole (68). Pre-

pared according to method 5. 96%; δ_H(CD₃OD, 270 MHz) 1.05

(3H, s, C-18-H₃), 1.28-2.50 (14H, m), 2.33 (3H, s), 2.67-2.94

(3H, m), 3.52-3.90 (4H, m), 6.50 (1H, d, J ) 2.6 Hz, C-4-H),

6.55 (1H, dd, J ) 8.5 Hz, J ) 2.6 Hz, C-2-H), 7.10 (1H, d, J )

8.5 Hz, C-1-H); MS m/z (APCI+) 421.4 [M + H]⁺. Acc MS m/z

(FAB+) 421.2598, C₂₅H₃₃N₄O₂requires 421.2603. HPLC (MeOH/

H₂O, 96:4) R_t) 1.68 min, 94%.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-[5′-(5′′-meth-

ylpyrazin-2′′-ylmethyl)carbamoyl]-pyrazole (69). Pre-

pared according to method 5. 82%; δ_H(CD₃OD, 400 MHz) 1.04

(3H, s, C-18-H₃), 1.06-2.50 (10H, m), 2.54 (3H, s), 2.78-2.90

(3H, m), 4.66 (2H, s), 6.50 (1H, d, J ) 2.5 Hz, C-4-H), 6.55

(1H, dd, J ) 8.4 Hz, J ) 2.5 Hz, C-2-H), 7.09 (1H, d, J ) 8.4

Hz, C-1-H), 8.48 (1H, s), 8.49 (1H, s); MS m/z (APCI+) 442.3

[M - H]⁺. Acc MS m/z (FAB+) 444.2385, C₂₆H₃₀N₅O₂requires

444.2400. HPLC (MeOH/H₂O, 96:4) R_t) 1.67 min, 89%.

3-Hydroxy-1,3,5(10)-triene-[17,16-c]-(5′-carboxylic acid)-

pyrazole (70). Following method 1, a suspension of 55 (214

mg, 0.50 mmol) and Pd-C 5% (50 mg) in EtOH/THF 1:1 (30

mL) was hydrogenated for 24 h to give 70 as a light gray solid

(165 mg, 98%): mp > 300 °C; δ_H(DMSO-d₆, 400 MHz) 0.92

(3H, s, C-18-H₃), 1.30-2.86 (13H, m), 6.44 (1H, d, J ) 2.3 Hz,

C-4-H), 6.52 (1H, dd, J ) 8.6 Hz, J ) 2.3 Hz, C-2-H), 7.05

(1H, d, J ) 8.6 Hz, C-1-H), 9.02 (1H, s, OH), 12.90 (2H, br s,

OH, NH); MS m/z (FAB+) 329.1 [45, (M + H)⁺], 176.0 [100];

Acc MS m/z (FAB+) 339.1711 C₂₀H₂₃N₂O₃requires 339.1709.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-[5′-(pyridin-

3′′-ylmethyl)carbamoyl]-pyrazole (71). DMAP (catalytic),

EDC (116 mg, 0.6 mmol), and NEt₃(77 µL, 0.55 mmol) were

added to a suspension of 70 (170 mg, 0.5 mmol) in anhydrous

DCM (10 mL) under N₂. After the suspension was stirred at

room temperature for 20 min, 3-aminomethylpyridine (60 µL,

0.59 mmol) and anhydrous DMF (10 mL) were added, and the

resulting solution was stirred at room temperature for 24 h.

After the solution was washed with saturated aqueous NaH-

CO₃, the organic layer was separated and concentrated under

reduced pressure. The crude product was precipitated from

DCM/hexane to give an off-white precipitate, which was

further purified by flash chromatography (DCM to DCM/

MeOH, 90:10, gradient) to give 71 as a white crystalline solid

(340 mg, 16%): mp > 240 °C (dec); δ_H(DMSO-d₆, 400 MHz)

0.92 (3H, s, C-18-H₃), 1.37-2.89 (13H, m), 4.41 (2H, app d),

6.44 (1H, d, J ) 2.3 Hz, C-4-H), 6.51 (1H, dd, J ) 8.2 Hz, J )

2.3 Hz, C-2-H), 7.04 (1H, d, J ) 8.2 Hz, C-1-H), 7.33 (1H, app

s), 7.69 (1H, app s), 8.29-8.63 (3H, m), 9.02 (1H, s), 12.65 and

12.92 (total 1H, 2 × s, NH); MS m/z (ES+) 429.2 [M + H]⁺;

Acc MS m/z (FAB+) 429.2279 C₂₆H₂₉N₄O₂requires 429.2291.

HPLC (MeOH/H₂O, 90:10) R_t) 2.04 min, 98%.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-[5′-(pyridin-

2′′-ylmethyl)carbamoyl]-pyrazole (72). DMAP (catalytic),

EDC (65 mg, 0.3 mmol), and NEt₃(52 µL, 0.37 mmol) were

added to a solution of 70 (950 mg, 0.28 mmol) in anhydrous

DCM (6 mL) and anhydrous DMF (2 mL) under N₂. After the

mixture was stirred at room temperature for 15 min, 2-ami-

nomethylpyridine (40 µL, 0.39 mmol) was added, and the

resulting solution was stirred at room temperature for 4 days.

After the solution was washed with saturated aqueous NaH-

CO₃, the organic layer was separated and concentrated under

reduced pressure. The crude product was purified by flash

chromatography (DCM to DCM/MeOH, 90:10) followed by

precipitation from hexane/DCM to give 72 as a white solid (10

mg, 8%): mp 187-190 °C; δ_H(DMSO-d₆, 270 MHz) 0.94 (3H,

s, C-18-H₃), 1.72-2.97 (13H, m), 4.45-4.55 (2H, m), 6.45 (1H,

d, J ) 2.6 Hz, C-4-H), 6.51 (1H, dd, J ) 8.6 Hz, J ) 2.6 Hz,

C-2-H), 7.05 (1H, d, J ) 8.6 Hz, C-1-H), 7.22-7.33 (1H, m),

7.74 (1H, m), 8.28 (1H, m), 8.52 (1H, app s), 9.03 (1H, s); MS

m/z (FAB+) 429.3 [M + H]⁺; Acc MS m/z (FAB+) 429.2279

C₂₆H₂₉N₄O₂requires 429.2291. HPLC (MeCN/H₂O, 96:4) R_t)

1.67 min, 100%.

3-Hydroxy-estra-1,3,5(10)-triene-[17,16-c]-[5′-(pyridin-

3′′-ylethyl)carbamoyl]-pyrazole (73). Following the same

procedure as for the preparation of 72, reaction of 70 (95 mg,

0.28 mmol) with 3-aminoethylpyridine (34 µL, 0.33 mmol) gave

73 as an off-white solid (12 mg, 10%): mp > 190 °C (dec); δ_H

(DMSO-d₆, 270 MHz) 0.93 (3H, s, C-18-H₃), 1.36-3.50 (17H,

m), 6.47 (1H, app d, C-4-H), 6.52 (1H, app dd, C-2-H), 7.07

(1H, d, J ) 8.2 Hz, C-1-H), 7.31-7.36 (1H, m), 7.67 (1H, app

d), 8.45 (2H, m), 9.07 (1H, s); MS m/z (APCI+) 441.5 [M -

H]⁺; Acc MS m/z (FAB+) 443.2442 C₂₇H₃₁N₄O₂requires

443.2447. HPLC (MeCN/H₂O, 80:20) R_t) 2.15 min, 88%.

3-Hydroxy-1,3,5(10)-triene-[17,16-c]-[5′-(carboxylic acid

ethyl ester)]-pyrazole (74). Following method 1, a suspen-

sion of 54 (913 mg, 2.00 mmol) and Pd-C 5% (100 mg) in

EtOH/THF 1:1 (60 mL) was hydrogenated for 72 h. The crude

product was crystallized from CHCl₃to give 74 as pale yellow

crystals (602 mg, 82%): mp 166-170 °C; δ_H(DMSO-d₆, 400

MHz) 0.92 (3H, s, C-18-H₃), 1.12-2.84 (16H, m), 4.14-4.40

(2H, m), 6.45 (1H, d, J ) 2.3 Hz, C-4-H), 6.50 (1H, dd, J ) 8.6

Hz, J ) 2.3 Hz, C-2-H), 7.05 (1H, d, J ) 8.6 Hz, C-1-H), 9.01

(1H, s, OH), 13.07 (1H, s, NH); MS m/z (FAB+) 367.2 [100,

(M + H)⁺]; Acc MS m/z (FAB+) 367.2038, C₂₂H₂₇N₂O₃requires

367.2022.

3-O-Benzyl-1,3,5(10)-triene-[17,16-c]-(5′-hydroxymethyl)-

pyrazole (75). Compound 54 (457 mg, 1.0 mmol) was added

to a suspension of LiAlH₄(100 mg, 2.64 mmol) in anhydrous

THF (20 mL) under an atmosphere of N₂. The resulting

mixture was stirred for 30 min at room temperature, after

which H₂O (1 mL) was carefully added. Stirring was continued

for 1 h, and AcOH (0.5 mL) was added. The suspension was

filtered through a layer of Celite, and the resulting clear

solution was diluted with EtOAc (50 mL), washed with brine

(50 mL), dried (Na₂SO₄), and concentrated under reduced

pressure. The residue was crystallized from EtOAc/Et₂O to give

75 as colorless crystals (215 mg, 52%): mp 210-213 °C; δ_H

(CDCl₃, 400 MHz) 1.01 (3H, s, C-18-H₃), 1.40-3.00 (13 H, m),

4.69 (2H, s), 5.04 (2H, s, OCH₂Ar), 6.28 (2H, br s, OH, NH),

6.73 (1H, d, J ) 2.3 Hz, C-4-H), 6.80 (1H, dd, J ) 8.6 Hz, J )

2.3 Hz, C-2-H), 7.21 (1H, d, J ) 8.6 Hz, C-1-H), 7.30-7.45 (5H,

m, C₆H₅); δ_C(CDCl₃, 100.4 MHz) 18.8 (q), 23.9 (t), 26.6 (t),

27.9 (t), 30.1 (t), 34.3 (t), 38.0 (d), 41.5 (s), 44.8 (d), 56.5 (t),

62.0 (d), 70.3 (t), 109.3 (s), 112.5 (d), 115.1 (d), 119.5 (s), 126.4

(d), 127.7 (d), 128.1 (d), 128.7 (d), 132.9 (s), 137.4 (s), 138.0

E-Ring Modified Steroids as Inhibitors of 17â-HSD

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5769

(s), 156.9 (s), 168.0 (s); MS m/z (FAB+) 415.3 [60, (M + H)⁺],

91.1 [100]; Acc MS m/z (FAB+) 415.2384, C₂₇H₃₁N₂O₂requires

415.2385.

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17HSD/KSR enzymes. J. Mol. Endocrinol. 1999, 23, 1-11.

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beta-hydroxysteroid dehydrogenase type 1 and type 2. Mol. Cell.

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dehydrogenase: Predominance of estrone reduction and its

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physiology and disease. J. Mol. Endocrinol. 2000, 25, 1-16.

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17 beta-hydroxysteroid dehydrogenases in sex steroid formation

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3-Hydroxy-1,3,5(10)-triene-[17,16-c]-(5′-hydroxymethyl)-

pyrazole (76). Following method 1, a suspension of 75 (120

mg, 0.289 mmol) and Pd-C 5% (50 mg) in EtOH/THF 1:1 (40

mL) was hydrogenated for 24 h to give 76 as a white solid (93

mg, 99%): mp > 213 °C (dec); δ_H(DMSO-d₆, 270 MHz) 0.82

(3H, s, C-18-H₃), 1.20-2.80 (13H, m), 4.31 (2H, s), 4.95 (1H,

br s, OH), 6.37 (1H, s), 6.44 (1H, d, J ) 8.5 Hz), 6.97 (1H, d,

J ) 8.5 Hz), 8.92 (1H, s, OH), 11.78 (1H, br s, NH); MS m/z

(FAB+) 325.2 [100, M⁺]; Acc MS m/z (FAB+) 325.1925,

C₂₀H₂₅N₂O₂requires 325.1916.

X-ray Crystallography. Crystal Data for 37. C₂₄H₃₄N₂O₂,

M ) 382.53, λ ) 0.710 73 Å, orthorhombic, space group

P2₁2₁2₁, a ) 8.3600(1), b ) 13.2660(2), c ) 19.1680(4) Å, U )

2125.80(6) Å³, Z ) 4, D_c) 1.195 mg/m³, µ ) 0.076 mm^-1

,

F(000) ) 832, crystal size 0.50 × 0.50 × 0.30 mm³, 4846 unique

reflections [R(int) ) 0.0594], observed I > 2σ(I) ) 4002, data/

restraints/parameters ) 4846/0/261, R1 ) 0.0414, wR2 )

0.0879 (obs. data), R1 ) 0.0567, wR2 ) 0.0939 (all data), max

peak/hole 0.199 and -0.176 e Å^-3, software used, SHELXS,⁵⁵

SHELXL,⁵⁶and ORTEX.⁴³H1, H2 located in the penultimate

difference Fourier map, but included at calculated positions

in final least squares cycles.

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Acknowledgment. We acknowledge Sterix Ltd, as

a member of the Ipsen group for a studentship to D.S.F.

We thank Dr. S. J. Black for useful discussions and Ms.

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Supporting Information Available: Experimental and

spectroscopic data for compounds 2, 3, and 25, microanalysis

data on selected compounds, and crystallographic data for

compound 37 (which have also been deposited with the

Cambridge Crystallographic Data Centre as CCDC263244;

copies of the data can be obtained free of charge on application

to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax (+44)

1223 336033, email: deposit@ccdc.cam.ac.uk]). This material

is available free of charge via the Internet at http://pubs.ac-

s.org.

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JM050348A

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E-ring modified steroids as novel potent inhibitors of 17β- hydroxysteroid dehydrogenase type 1

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