Novel nonsteroidal ligands with high binding affinity and potent functional activity for the androgen receptor

Eur. J. Med. Chem. 37 (2002) 619–634

www.elsevier.com/locate/ejmech

Original article

Novel nonsteroidal ligands with high binding afﬁnity and potent

functional activity for the androgen receptor

Yali He, Donghua Yin ¹, Minoli Perera ¹, Leonid Kirkovsky, Nina Stourman, Wei Li,

James T. Dalton ¹, Duane D. Miller *

Department of Pharmaceutical Sciences, College of Pharmacy, Uni6ersity of Tennessee-Memphis, 847 Monroe A6enue, Faculty Building,

Room 327, Memphis, TN 38163, USA

Received 31 May 2000; received in revised form 3 January 2002; accepted 7 January 2002

Abstract

While nonsteroidal androgen receptor (AR) antagonists have been known for many years, and used in the clinic for the

treatment of hormone dependent prostate cancer, very little is known about nonsteroidal AR agonists. We designed and

synthesized a series of chiral bicalutamide analogs, which bear electron-withdrawing groups (either a cyano or a nitro group at

the 4-position and a triﬂuoromethyl group at the 3-position) in the aromatic A ring, and different substituents at the para position

in the aromatic B ring of the parent molecule. We also synthesized a series of racemic bicalutamide analogs, which have a

triﬂuoromethyl group instead of a methyl group at the R₂position. We examined AR binding afﬁnities of our compounds in a

competitive binding assay with a radiolabeled high afﬁnity AR ligand, ³H-mibolerone, and also measured their abilities to

stimulate AR-mediated transcriptional activation in a cotransfection assay. These studies demonstrated that (1) nonsteroidal

ligands can be structurally modiﬁed from known nonsteroidal antiandrogens to generate ligands capable of activating AR-medi-

ated transcriptional activation. (2) R-isomer analogs exhibit higher AR binding afﬁnity and more potent functional activity than

their corresponding S-isomers in all cases. (3) All sulphide analogs show higher AR binding afﬁnity and more potent functional

activity than their corresponding sulphone analogs, with the exception of ligand R-8. Those ligands which exhibit high AR binding

afﬁnity and potent functional activity for human AR may provide effective clinical uses for male fertility, male contraception, and

´

Keywords: Nonsteroidal ligands; Androgen receptor agonists; Bicalutamide derivatives

Nonsteroidal antiandrogens were extensively re-

1. Introduction

ported. Some of these (e.g. ﬂutamide 3 [3,4] and bicalu-

tamide 4 [5,6] in Fig. 1) are successfully used in the

clinic for the treatment of AR dependent prostate can-

cer. We reported on the ﬁrst nonsteroidal agonists for

the human AR [7]. Recently, others have reported on

new chemical classes of AR agonists [8]. Based on these

previous reports and the known properties of non-

steroidal antiandrogens [9,10], nonsteroidal androgens

can be designed and synthesized that will mimic the

pharmacological effects of testosterone 1, and would

likely avoid many of the undesired physicochemical and

pharmacokinetic properties of their steroidal counter-

parts, including poor oral bioavailability, rapid hepatic

metabolism, and activation of other steroid receptors.

Nonsteroidal AR agonists would be potentially useful

for the treatment of a number of hormone dependent

The androgen receptor (AR) is an important cellular

regulatory protein and plays a critical role in numerous

physiological processes, including the development and

maintenance of male secondary sexual characteristics

such as muscle, hair and bone mass, prostate growth

and spermatogenesis [1]. The AR is also thought to be

involved in prostate carcinogenesis [2]. The natural

steroids, testosterone 1 and 5a-dihydrotestosterone (5a-

DHT) 2 are the natural ligands for the AR.

* Corresponding author. Tel.: +1-901-4487529; fax: +1-901-

4486828

E-mail address: dmiller@utmem.edu (D.D. Miller).

¹Present address: College of Pharmacy, The Ohio State University,

Columbus, OH 43210, USA.

´

PII: S0223-5234(02)01335-1

620

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

Fig. 1). To optimise our parent molecule (5, Fig. 1)

with the ultimate aim of designing a new series of

human AR agonists, we designed and synthesized a

series of chiral bicalutamide analogs bearing electron-

withdrawing groups, either a cyano or a nitro group at

the 4-position and a triﬂuoromethyl group at the 3-po-

sition in the aromatic A ring, and different substituents

at the para position in the aromatic B ring of the parent

molecule (5, Fig. 1). We also synthesized a series of

racemic bicalutamide analogs, which have a triﬂ-

uoromethyl group instead of a methyl group at the R₂

position. Substitution at the meta position in the aro-

matic B ring resulted in a dramatic decrease in both

binding and functional activities for the AR (data were

not included). Modiﬁcation of the aromatic B ring only

focused on the para position in this paper.

conditions, ranging from aging or hypogonadal men

requiring hormone replacement therapy [11], to male

fertility, male contraception [12] and performance en-

hancement [13,14].

We discovered that nonsteroidal ligands with andro-

genic activity can be generated by means of structural

modiﬁcations of known nonsteroidal antiandrogens

and ﬁrst reported these drugs as nonsteroidal agonists

for the human AR [7]. Based on Structure–activity

relationship (SAR) studies of nonsteroidal AR ligands

[15–17], electron-withdrawing groups together with a

branched alkyl group a to the amidic carbonyl in the

aromatic A ring were required for binding and func-

tional activity (5, Fig. 1). There may be a distinctive

role for this electron deﬁcient aromatic A ring with

respect to direct AR receptor interaction. It is also

known that the hydroxyl group (5, Fig. 1) plays an

important role in the interaction between the AR recep-

tor and ligand [15]. Electron-attracting substituents in

the aromatic A ring can enhance the proton donor

capability of the hydroxyl group by increasing the

acidity of the amide moiety [15]. Chemically, the R₂

group could affect the proton donating ability of the

hydroxyl group via electronic effect. We assumed that

the AR binding afﬁnity and functional activity might be

increased by introducing a strong electron-withdrawing

functional substituent (for example, a NO₂group ins-

tead of a CN group) at the 4-position in the aromatic A

ring, and/or by introducing an electron-attracting

group (for example, a CF₃group) at the R₂position (5,

In summary, the present study examined the struc-

tural requirements of the aromatic A ring (R₁), aro-

matic B ring (R₃), functional group X-position (sulfonyl

or thio), and/or a triﬂuoromethyl group instead of a

methyl group at the R₂position in the parent molecule

(5, Fig. 1) for optimum activity on the human AR.

2. Chemistry

The synthesis of chiral ligands R-3–R-17 is outlined

in Fig. 2 (with R-isomers as an example). For a series

of cyano ligands (cyano group at the 4-position in

aromatic A ring), the starting material R-2 (N-[4-cy-

Fig. 1. Molecular structures of testosterone (T), 5a-dihydrotestosteroen (5a-DHT), ﬂutamide, R-bicalutamide, and parent molecule.

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

621

Fig. 2. (a) 1.25 equiv. NaH, 1.25 equiv. 4-aminothiophenol, anhydrous THF, 25 °C for 24 h, 75% (R-3), 73% (R-4). (b) Excess acetic anhydride,

25 °C for 10 min, 74% (R-5), 98% (R-9); 1.1 equiv. chloroacetyl chloride, 10 equiv. CaCO₃, anhydrous CH₂Cl₂, 25 °C for 24 h, 72% (R-6), 77%

(R-11); 5 equiv. triﬂuoroacetic anhydride, 5 equiv. Et₃N, anhydrous THF, 0–25 °C for 24 h, 95% (R-7); 1 equiv. methanesulfonyl chloride, 2

equiv. Et₃N, anhydrous THF, 0–25 °C for 24 h, 60% (R-8); 1.5 equiv. propionyl chloride, 5 equiv. CaCO₃, anhydrous CH₂Cl₂, 25 °C for 72 h,

20% (R-10). (c) Excess peracetic acid, EtOAc, 25 °C for 1 h, nearly quantitative yield.

The preparation of racemic ligands 24–28 is outlined

in Fig. 3. Commercially available starting material 18

(3-bromo-1,1,1-triﬂuoro acetone) was coupled with

para-nitrothiophenol sodium salt [19] to afford 19,

which reacted with potassium cyanide and 25% sulfuric

acid to provide the cyano precursor 20. Hydrolysis of

the cyano group of compound 20 in a reﬂuxing mixture

of concentrated HCl and glacial acetic acid afforded

compound 21 as the precursor for the preparation of

compounds 24–28. After the reduction of the nitro

group of 21 with tin(II) chloride, compounds 22 and 23

were prepared by acylation of the amino intermediate

with acetyl chloride or chloroacetyl chloride. Coupling

of 22 or 23 with commercially available 4-nitro-3-(tri-

ﬂuoromethyl)aniline yielded the corresponding amides,

24 and 25, respectively. Ligand 25 was oxidized to its

corresponding sulfonyl derivative 26 by peracetic acid.

Coupling of 21 with commercially available 4-nitro-3-

(triﬂuoromethyl)aniline or 4-amino-2-triﬂuoromethyl

benzonitrile afforded compounds 27 and 28,

respectively.

ano - 3 - (triﬂuoromethyl)phenyl] - (2R) - 3 - bromo - 2-

hydroxy-2-methylpropanamide) was synthesized from

commercially available 4-amino-2-triﬂuoromethyl ben-

zonitrile and R-proline (L-proline for S-isomers) in four

steps [16,18]. In the same manner as the series of cyano

ligands, the starting material R-1 (N-[4-nitro-3-(tri-

ﬂuoromethyl)phenyl] - (2R) - 3 - bromo - 2 - hydroxy - 2-

methyl propanamide) for a series of nitro ligands (a

nitro group at the 4-position in the aromatic A ring)

was prepared from commercially available 4-nitro-3-

(triﬂuoromethyl) aniline and R-proline (also L-proline

for S-isomers). The starting material (R-1 or R-2) was

coupled with 4-aminothiophenol to afford R-3 and R-4,

respectively. The acetylamino compounds (R-5 and R-

9) were obtained by acylation of the corresponding

aniline R-3 or R-4 with acetyl anhydride at room

temperature for 10 min, while the propionylamino

analog R-10 was obtained by acylation of R-4 with

propionyl chloride in anhydrous methylene chloride at

room temperature for 3 days. In the same manner as

acetylamino derivatives, chloroacetylamino compounds

R-6 and R-11 were prepared by acylation of R-3 and

R-4 with chloroacetyl chloride, respectively. The tri-

ﬂuoroacetylamino analog R-7 was prepared by reaction

of R-3 with triﬂuoroacetyl anhydride in anhydrous

THF. The methanesulfonylamino analog R-8 was ob-

tained by reaction of R-3 with methanesulfonyl chloride

in anhydrous THF. Sulphide derivatives (R-5–R-11)

were oxidized to their corresponding sulfonyl deriva-

tives (R-12–R-17) by peracetic acid or chloroperbenzoic

acid.

3. Biological results and discussion

3

A competitive binding assay with H-mibolerone, a

high afﬁnity AR binding ligand [7] was employed to

examine AR binding afﬁnities of the new analogs pre-

pared in this study. CV-1 cells cotransfected with a

human AR expression vector, an androgen-sensitive

luciferase reporter vector and a control b-galactosidase

vector were employed to evaluate pharmacological

622

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

conﬁguration. In previous studies on hydroxy ﬂutamide

analogs [7], we found that structural modiﬁcation by

replacing the para-cyano group with a nitro functional

group in the aromatic A ring signiﬁcantly improved the

binding and functional activity. However, this SAR was

not obvious in bicalutamide derivatives tested here

(compare R-5 vs. R-9, R-6 vs. R-11, R-12 vs. R-16, and

R-13 vs. R-17). This suggested that the mode of binding

for bicalutamide analogs differs from that for hydro-

xyﬂutamide analogs.

To optimise the substitution at the para position of

the aromatic B ring, various substituents were intro-

duced. Comparing to R-3 and R-4 which bear an amino

group at the para position in the aromatic B ring, and

produced only minimal efﬁcacies, 2.590.3 and 1.49

0.1% (mean9S.D.), respectively, introduction of a

short para acylamino group (for example, R-5–R-7,

R-9–R-14) or para sulfonylamino group (R-8 and R-15)

in the aromatic B ring resulted in a signiﬁcant increase

in AR binding afﬁnity, and in the degree of efﬁcacy.

Structural modiﬁcations at the para position in the

aromatic B ring resulted in the most potent agonist

activities of these ligands. For potent ligands, AR-media-

ted transcriptional activation increased with increasing

ligand concentrations, then reached a plateau at high

concentration. Only the R-isomer analogs and racemic

analogs with high binding afﬁnity were evaluated for

their ability to stimulate AR-mediated transcriptional

activation (Table 1). Control studies showed that the

efﬁcacy of transcriptional activation induced by 500

nM of each testing compound was below 1% in the

absence of androgen receptor expression construct,

which demonstrated that the observed increases in luci-

ferase expression were the result of speciﬁc AR-media-

ted effects.

Both the 4-nitro-3-triﬂuoromethyl and 4-cyano-3-

triﬂuoromethyl analogs exhibited similar AR binding

afﬁnity and functional activity. The R-isomer analogs

exhibited higher AR binding afﬁnity and more potent

functional activity than their corresponding S-isomers

in all cases (Table 1). These results suggested that the

structural elements required for the AR binding afﬁnity

in the aromatic A ring were an electron deﬁcient aro-

matic ring, and the most active isomers have the R-

Fig. 3. (a) 1 equiv. para-nitrothiophenol sodium salt, anhydrous THF, 0–25 °C for 3 h, 50%. (b) 1.15 equiv. KCN, 25% (v/v) sulphuric acid

aqueous solution, 0–25 °C for 20 h, 67%. (c) Concentrated HCl and glacial acetic acid (17:3, v/v), reﬂux for 24 h, 47%. (d) 3 equiv. tin(II)

chloride, methanol and concentrated HCl (1:1), 0–25 °C for 24 h. (e) 1.5 equiv. acetyl chloride (22), or 1.2 equiv. chloroacetyl chloride (23), 10

equiv. CaCO₃, anhydrous MeCN, 25 °C for 48 h, 20% (22), 62% (23). (f) 1 equiv. 4-nitro-3-(triﬂuoromethyl) aniline, 1.25 equiv. thionyl chloride,

anhydrous DMA, −15 to −10 °C then 25 °C for 3 days, 13% (24), 14% (25). g. 3 equiv. meta-chloro-perbenzoic acid, CH₂Cl₂, 25 °C for 1 h,

25%. (h) 1 equiv. 5-amino-2-cyanobenzotriﬂuoride (27), 1 equiv. 4-Nitro-3-(triﬂuoromethyl) aniline (28), 1.25 equiv. thionyl chloride, anhydrous

DMA, −15 to −10 °C then 25 °C for 3 days, 20% (27), 20% (28).

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

623

Table 1

Structures, binding afﬁnity (K_i) and functional activity of DHT, bicalutamide and synthesized nonsteroidal ligands

Efﬁcacy ^b(% of DHT)

Potency ^c(nM)

a

R₁

R₂

X

R₃

Ligands

K_i(nM)

DHT

R ^d-Bical. ^e

S ^f-Bical. ^e

R ^d-3

S ^f-3

R-4

S-4

R-5

S-5

R-6

0.2890.02

11.091.5

365910

57.291.5

528926

91.0916.0

832950

2.9490.23

123927

3.3290.36

97.097.0

2.5890.39

49.891.4

4.8890.18

137930

26.692.25

1.6590.10

410949

9.3290.81

5899127

6.9191.20

233974

6.2391.13

15.491.7

15.990.93

7.0490.88

2.5390.14

4.9790.56

5.0490.66

18.894.6

20.3491.88

100

8.2892.66

1

1000

CN

NO₂

CN

Me

CF₃

SO₂

S

SO₂

S

F

NH₂

2.5090.3

1.4090.1

79.295.7

90.097.2

500

100

500

CN

NO₂

CN

NO₂

CN

NHCOMe

NHCOCH₂Cl

NHCOCF₃

NHSO₂Me

NHCOMe

NHCOEt

NHCOCH₂Cl

NHCOMe

NHCOCH₂Cl

NHCOCF₃

NHSO₂Me

NHCOMe

NHCOCH₂Cl

NHCOMe

NHCOCH₂Cl

NO₂

S-6

R-7

R-8

R-9

127.2917

ND ^g

500

ND ^g

S-9

R-10

R-11 ^h

S-11 ^h

R-12

S-12

R-13

S-13

R-14

R-15

R-16

R-17

24 ⁱ

ND ^g

95.5921.6

ND ^g

100

45.195.2

41.392.9

500

100

9.894.4

3.390.4

30.1917.8

65.496.4

136.3932.9

106.3912.9

97.899.9

ND ^g

500

100

500

CN

NO₂

CN

S

SO₂

S

25 ⁱ

26 ⁱ

500

27 ⁱ

ND ^g

NO₂

S

NO₂

28 ⁱ

ND ^g

Data are expressed as means9SEM (standard error).

^aThe radioligand used in the competitive binding assay was ³H-mibolerone.

^bMaximal percentage of transcriptional activation observed for each ligand.

^cThe lowest concentration of the ligand capable of maximally stimulating AR-mediated transcription during transfection experiments.

^dR-isomer analog.

^eBical., bicalutamide.

^fS-isomer analog.

^gND, not determined.

^hRef. [7].

ⁱRacemic mixture (compounds 24–28).

activity. Among these substituents, the acetamido,

chloroacetamido, or triﬂuoroacetamido sulphide

derivatives (ligands R-5–R-7, R-9, R-11, 24, and 25)

demonstrated a mean efﬁcacy of 80–136% at 100–500

nM concentrations. However, the potency of these li-

gands was ca. 100–500 fold lower than that of DHT.

Replacement of an acetylamino group (R-9) with a

propionylamino group (R-10) or methanesulfonylamino

group (R-8) decreased the AR binding afﬁnity. These

observations suggest that the nature of the substituent

in the para-position in the aromatic B ring play an

important role in drug (compare R-9 vs. R-10).

To investigate the effects on the AR binding afﬁnity

and functional activity by introducing either a sulphide

or a sulfonyl group at the X-position (5, Fig. 1), we

designed and synthesized both sulphide and sulphone

derivatives of some ligands. The results show that all

sulphide analogs (R-5–R-7, R-9, R-11, and 25) show

higher AR binding afﬁnity and more potent functional

activity than their corresponding sulphone analogs

624

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

(R-12–R-14, R-16, R-17, and 26), with the exception of

R-8 which exhibited lower binding afﬁnity than its

corresponding sulfonyl analog (R-15), and ligand 25

whose AR binding afﬁnity and functional activity re-

mained practically unchanged comparing to ligand 26.

The effect of introducing a triﬂuoromethyl group

instead of a methyl group at the R₂position on AR

binding afﬁnity and functional activity was also exami-

ned (5, Fig. 1). A series of racemic bicalutamide

analogs (24–28) was synthesized and biologically evalua-

ted (Table 1). While the AR binding afﬁnities of the

CF₃series of ligands were on the same order magnitude

as that of the CH₃series of ligands. Ligand 24 demons-

trated the most potent functional activity, mean efﬁcacy

of 136% at concentration as low as 100 nM. These

results suggested that the replacement of a methyl

group by a triﬂuoromethyl group at the R₂position

might be helpful for the AR binding afﬁnity. However,

it has still been argued that the electronic property of

R₂and the electron-withdrawing groups on the aro-

matic ring A group were a crucial factor for the proton

donating capability of a hydroxyl group in the interac-

tion between the AR receptor and ligand [15]. To

further attempt to optimise our analogs, we also syn-

thesized ligands 27 and 28 in the CF₃series of ligands.

The AR binding afﬁnity of 27 was essentially equipo-

tent with that of 28, which further implied that a nitro

group at the 4-position in the aromatic A ring has a

similar effect as the cyano group for AR binding

afﬁnity in the AR competitive binding assay. It is clear

that additional structural modiﬁcations of these ligands

to improve the efﬁcacy and potency of androgenic

activity are needed, and will be the subject of future

research in our laboratory.

mately equal to that of ligand 26. These new AR

agonist compounds are considerably different than the

known steroidal AR agonists and in vivo studies need

to be carried out to see if some of the undesired

properties such as the lack of good oral bioavailability

and metabolic breakdown of the steroidal androgen

agonists are overcome, steroidal counterparts are the

subjects for continuing investigation. The development

of new nonsteroidal human AR agonists should

provide effective clinical opportunities for therapeutical

uses.

5. Experimental protocols

5.1. Chemistry

Melting points were determined on a Thomas–

Hoover capillary melting point apparatus and are un-

corrected. Infrared spectra were recorded on

a

Perkin–Elmer System 2000 FTIR. Optical rotations

were determined on a Autopol^®III Automatic Pola-

rimeter (Rudolph Research Model III-589-10, Fairﬁeld,

New Jersey). Proton and carbon-13 magnetic resonance

spectra were obtained on a Bruker AX 300 spectrome-

1

ter (300 and 75 MHz for H- and ¹³C-NMR, respec-

tively). Chemical shift values were reported as parts per

million (l) relative to tetramethylsilane (TMS). Spectral

data were consistent with assigned structures. Mass

spectra were determined on a Bruker-HP Esquire LC

System. Elemental analyses were performed by Atlantic

Microlab Inc. (Norcross, GA), and found values were

within 0.4% of the theoretical values. Routine thin-

layer chromatography (TLC) was performed on silica

gel on aluminium plates (silica gel 60 F 254, 20×20

cm, Aldrich Chemical Company Inc., Milwaukee, WI).

Flash chromatography was performed on silica gel

4. Conclusions

,

(Merck, grade 60, 230–400 mesh, 60 A). Tetrahydro-

Nonsteroidal human AR ligands with high binding

afﬁnity and potent functional activity were obtained by

means of structural modiﬁcations of bicalutamide, a

known nonsteroidal antiandrogen used in the treatment

of hormone dependent prostate cancer by using co-

transfection and binding assays as guides. R-isomer

analogs exhibit higher binding afﬁnity and more potent

functional activity for the human AR than their corres-

ponding S-isomers in all cases. The ligands bearing an

acetamido, chloroacetamido, or triﬂuoroacetamido

functional group at the para position in the aromatic B

ring represented the most active analogs in this series of

ligands. All sulphide analogs showed higher AR bin-

ding afﬁnity and more potent functional activity than

their corresponding sulphone analogs, with the excep-

tion of sulphide R-8 which showed lower AR binding

afﬁnity than sulphone R-15 and ligand 25 whose AR

binding afﬁnity and functional activity were approxi-

furan (THF) was dried by distillation from sodium

metal. Acetonitrile (MeCN) and methylene chloride

(CH₂Cl₂) were dried by distillation from P₂O₅.

(17a-Methyl-³H)-mibolerone

(³H-MIB,

83.5

Ci mmol⁻¹) and unlabeled MIB were purchased from

DuPont Research NEN Products (Boston, MA). Tri-

amcinolone acetonide, phenylmethylsulfonyl ﬂuoride

(PMSF), TRIS base, sodium molybdate and dithiothrei-

tol were purchased from Sigma Chemical Company (St.

Louis, MO). Hydroxyapatite (HAP) was purchased

from Bio-Rad Laboratories (Hercules, CA). EcoLite

Plus™ scintillation cocktail was purchased from ICN

Research Products Division (Costa Mesa, CA). Dulbec-

co’s modiﬁed essential medium (DMEM) and Lipofec-

tamine™ transfection reagent were purchased from Life

Technologies (Gaithersburg, MA). Fetal bovine serum

(FBS) was purchased from Atlanta Biologicals, Inc.

(Norcross, GA).

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

625

5.1.1. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2R)-3-[(4-

aminophenyl)sulfanyl]-2-hydroxy-2-methylpropanamide

(R-3)

IR (KBr, cm⁻¹): 3370 (NH and OH), 1685 (CꢀO,

amide), 1599 (CꢀC, Ar), 1521 (CꢀC, Ar), 1421, 1350,

1153; ESIMS; m/z: 438.2 [M+Na]⁺; [h]_D²⁶−23.7°

(c=0.53, acetone); Anal. C₁₇H₁₆F₃N₃O₄S·0.25C₄H₈O₂·

(C, H, N).

A solution of 4-aminothio phenol (7.51 g, 0.06 mol)

in 60 ml of anhydrous THF was added to a suspension

of previously washed 60% oil dispersion of NaH (2.30

g, 0.06 mol) in 60 ml of anhydrous THF under argon

atmosphere. The resulting mixture was stirred for 22 h

until a white suspension was formed. A solution of R-1

[7] (17.84 g, 48.0 mmol) in 60 ml of anhydrous THF

was added dropwise to the above suspension, and

stirred for 24 h. The reaction mixture was ﬁltered,

washed with THF, and the ﬁltrate was evaporated in

vacuo to give an oil. Puriﬁcation by ﬂash column

chromatography on silica gel (hexanes–chloroform,

1:1–1:2 with 0.1% ammonium as eluent) yielded 15 g

(75%) of the desired compound (R-3) as a yellow

5.1.3. N-[4-cyano-3-(triﬂuoromethy)phenyl]-(2R)-3-[(4-

aminophenyl)sulfanyl]-2-hydroxy-2-methylpropanamide

(R-4)

The title compound was prepared from compound

R-2 [7] (2.55 g, 7.40 mmol) and 4-aminothiophenol

(1.38 g, 11.0 mmol) by the same procedure used for

compound R-3. The crude oily product was solidiﬁed

after addition of a small amount of MeOH. Recrystalli-

sation from MeOH afforded 2.53 g (73%) of the desired

compound as white crystals: m.p. 166–167 °C; ¹H-

NMR (300 MHz, DMSO-d₆): l 10.44 (s, 1H, NH), 8.48

(d, J=1.9 Hz, 1H, ArH), 8.23 (dd, J=8.6, 1.9 Hz, 1H,

ArH), 8.06 (d, J=8.6 Hz, 1H, ArH), 7.05–7.01 (m, 2H,

ArH), 6.48–6.43 (m, 2H, ArH), 6.08 (s, 1H, OH), 5.14

(br s, 2H, NH₂), 3.17 (d, J=13.0 Hz, 1H, CHH_a), 3.07

(d, J=13.0 Hz, 1H, CHH_b), 1.40 (s, 3H, Me); ¹³C-

NMR (75 MHz, DMSO-d₆): l 175.0, 148.2, 143.2,

136.1, 133.4, 131.4 (q, J=31.7 Hz), 122.5 (q, J=273.6

Hz), 122.6, 119.8, 117.4 (q, J=5.0 Hz), 115.8, 114.2,

101.7 (q, J=2 Hz), 75.3, 47.3, 25.6; IR (KBr, cm⁻¹):

3486, 3447, 3386, 3363, (NH, NH₂, OH), 2234 (CN),

1681 (CO), 1623, 1596, 1583, 1513, 1331; [h]²_D⁷+33.0°

(c=2, acetone); Anal. C₁₈H₁₆F₃N₃O₂S (C, H, N).

1

powder: m.p. 94–95 °C; H-NMR (300 MHz, DMSO-

d₆): l 10.36 (s, 1H, CONH), 8.41 (d, J=2.1 Hz, 1H,

ArH), 8.19 (dd, J=2.1, 8.9 Hz, 1H, ArH), 8.07 (d,

J=8.9 Hz, 1H, ArH), 7.00–6.96 (m, 2H, ArH), 6.38–

6.34 (m, 2H, ArH), 6.01 (s, 1H, OH), 5.09 (s, 2H, NH₂),

3.17 (d, J=12.8 Hz, 1H, CHH_a), 3.07 (d, J=12.8 Hz,

1H, CHH_b), 1.43 (s, 3H, CH₃); ¹³C-NMR (75 MHz,

DMSO-d₆): l 175.0 (CꢀO), 148.2 (CꢁN, Ar), 143.2

(CꢁNO₂, Ar), 141.5 (CꢁN, Ar), 133.4, 127.2, 123.0,

122.6 (q, J=32.2 Hz), 122.1 (q, J=271.5 Hz, CF₃),

119.9, 118.3 (q, J=6.0 Hz), 114.2, 75.3 (COꢁCꢁO),

47.3 (CH₂ꢁS), 25.6 (CH₃); IR (KBr, cm⁻¹): 3496, 3397,

3357 (NH and OH), 1678 (CꢀO, amide), 1624 (CꢀC,

Ar), 1598 (CꢀC, Ar), 1513 (CꢀC, Ar), 1417, 1360, 1153;

ESIMS; m/z: 438.2 (M+Na)⁺; [h]_D²⁶+22.8° (c=0.54,

acetone); Anal. C₁₇H₁₆F₃N₃O₄S·0.25C₄H₈O₂·0.2C₆H₁₄

(C, H, N).

5.1.4. N-[4-cyano-3-(triﬂuoromethy)phenyl]-(2S)-3-[(4-

aminophenyl)sulfanyl]-2-hydroxy-2-methylpropanamide

(S-4)

The title compound was prepared from compound

S-2 (2.55 g, 7.40 mmol) and 4-aminothiophenol (1.38 g,

11.0 mmol) in the same manner as compound R-4.

Recrystallization from MeOH gave 2.46 g (71%) of the

desired compound as white crystals: m.p. 166–167 °C;

¹H-NMR (300 MHz, DMSO-d₆): l 10.41 (s, 1H, NH),

8.47 (d, J=1.9 Hz, ArH), 8.22 (dd, J=8.6, 2.0 Hz, 1H,

ArH), 8.05 (d, J=8.6 Hz, 1H, ArH), 7.06–7.01 (m, 2H,

ArH), 6.48–6.44 (m, 2H, ArH), 6.05 (s, 1H, OH), 5.22

(br s, 2H, NH₂), 3.17 (d, J=13.0 Hz, 1H, CHH_a), 3.07

(d, J=13.0 Hz, 1H, CHH_b), 1.39 (s, 3H, Me); ¹³C-

NMR (75 MHz, DMSO-d₆): l 175.0, 147.8, 143.2,

136.1, 133.3, 131.4 (q, J=31.7 Hz), 122.6, 122.5 (q,

J=273.5 Hz), 120.2, 117.4 (q, J=5.0 Hz), 115.8, 114.4,

101.7 (q, J=2.0 Hz), 75.3, 47.2, 25.6; IR (KBr, cm⁻¹):

3485, 3447, 3385, 3365 (NH, NH₂, OH), 2234 (CN),

1681 (CO), 1623, 1596, 1583, 1517, 1330; [h]²_D⁷−33.7°

(c=2, acetone); Anal. C₁₈H₁₆F₃N₃O₂S (C, H, N).

5.1.2. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2S)-3-[(4-

aminophenyl)sulfanyl]-2-hydroxy-2-methylpropanamide

(S-3)

The title compound was prepared from compound

S-1 (0.85 g, 2.29 mmol) and 4-aminothiophenol (0.36 g,

2.86 mmol) in the same manner as compound R-3.

Puriﬁcation by ﬂash column chromatography on silica

gel (hexanes–chloroform, 1:1–1:2 with 0.1% ammo-

nium as eluent) yielded 0.5 g (54%) of the desired

compound as a yellow powder: m.p. 92–93 °C; ¹H-

NMR (300 MHz, DMSO-d₆): l 10.51 (s, 1H, CONH),

8.50 (d, J=2.1 Hz, 1H, ArH), 8.29 (dd, J=2.1 Hz,

J=9.0 Hz, 1H, ArH), 8.16 (d, J=9.0 Hz, 1H, ArH),

7.07–7.03 (m, 2H, ArH), 6.44–6.40 (m, 2H, ArH), 6.10

(s, 1H, OH), 5.16 (s, 2H, NH₂), 3.18 (d, J=12.9 Hz,

1H, CHH_a), 3.08 (d, J=12.9 Hz, 1H, CHH_b), 1.41 (s,

3H, CH₃); ¹³C-NMR (75 MHz, DMSO-d₆): l 175.0

(CꢀO), 148.2 (CꢁN, Ar), 143.3 (CꢁNO₂, Ar), 141.5

(CꢁN, Ar), 133.4, 127.3, 123.9, 122.7 (q, J=33.0 Hz),

122.1 (q, J=272.3 Hz, CF3), 119.9, 118.3 (q, J=6.0

Hz), 114.3, 75.4 (COꢁCꢁO), 47.3 (CH₂ꢁS), 25.7 (CH₃);

5.1.5. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2R)-3-{[4-

[acetylamino)phenyl]sulfanyl}-2-hydroxy-2-methylpro-

panamide (R-5)

Compound R-3 (0.70 g, 1.69 mol) was completely

626

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

[h]²_D⁶−24.0° (c=0.56, acetone); Anal. C₁₉H₁₈F₃N₃O₅S·

0.4C₄H₈O₂(C, H, N).

dissolved in 10 ml of acetic anhydride, and the reaction

solution was stirred for 10 min at room temperature.

After the end of the reaction was established by TLC,

the resulting solution was poured into 50 ml of satu-

rated NaHCO₃solution (more solid NaHCO₃was

added), stirred, and extracted with EtOAc (20 ml×3).

The EtOAc extract was dried over anhydrous Na₂SO₄,

ﬁltered through Celite, and evaporated in vacuo to give

an oil. Puriﬁcation by ﬂash column chromatography on

silica gel (hexanes–ethyl acetate, 2:1–1:1) yielded 0.56 g

(74%) of the desired compound as a yellow powder:

5.1.7. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2R)-3-

({4-[(2-chloroacetyl)amino]phenyl}sulfanyl)-2-

hydroxy-2-methylpropanamide (R-6)

Chloro acetyl chloride (0.21 g, 1.85 mmol) was added

dropwise to a suspension of compound R-3 (0.70 g,

1.69 mmol) and CaCO₃(1.84 g, 16.85 mmol) in 60 ml

of anhydrous CH₂Cl₂under argon atmosphere. The

resulting suspension was stirred overnight, ﬁltered

through Celite, washed with CH₂Cl₂, and the ﬁltrate

was evaporated completely in vacuo to furnish an oil.

Puriﬁcation by ﬂash column chromatography on silica

gel (hexanes–ethyl acetate, 2:1–1:1) yielded 0.6 g (72%)

of the desired compound as a yellow powder: m.p.

1

m.p. 104–107 °C, softened then melted; H-NMR (300

MHz, DMSO-d₆): l 10.47 (s, 1H, CONH), 9.84 (s, 1H,

CONH), 8.43 (d, J=2.1 Hz, 1H, ArH), 8.24 (dd,

J=2.1 Hz, J=9.0 Hz, 1H, ArH), 8.13 (d, J=9.0 Hz,

1H, ArH), 7.42–7.39 (m, 2H, ArH), 7.28–7.23 (m, 2H,

ArH), 6.17 (s, 1H, OH), 3.37 (d, J=13.2 Hz, 1H,

CHH_a), 3.20 (d, J=13.2 Hz, 1H, CHH_b), 1.99 (s, 3H,

COCH₃), 1.44 (s, 3H, CH₃); ¹³C-NMR (75 MHz,

DMSO-d₆): l 174.7 (CꢀO), 168.0 (CꢀO), 143.1 (CꢁNO₂,

Ar), 141.5 (CꢁN, Ar), 137.7 (CꢁS, Ar), 130.3, 129.4

(CꢁN, Ar), 127.1, 122.9, 122.5 (q, J=33.0 Hz), 122.0

(q, J=271.5 Hz, CF₃), 119.2, 118.2 (q, J=6.0 Hz),

75.1 (COꢁCꢁO), 44.7 (CH₂ꢁS), 25.6 (COCH₃), 23.8

(CH₃); IR (KBr, cm⁻¹): 3340 (NH and OH), 1677

(CꢀO, amide), 1596 (CꢀC, Ar), 1523 (CꢀC, Ar), 1397

(CH3), 1321, 1153; ESIMS; m/z: 480.2 [M+Na]⁺;

[h]²_D⁶+24.2° (c=0.40, acetone); Anal. C₁₉H₁₈F₃N₃O₅S·

0.5C₄H₈O₂·0.2C₆H₁₄(C, H, N).

1

58–60 °C, softened then melted; H-NMR (300 MHz,

DMSO-d₆): l 10.49 (s, 1H, CONH), 10.22 (s, 1H,

CONH), 8.45 (d, J=2.1 Hz, 1H, ArH), 8.25 (dd,

J=2.1, 9.0 Hz, 1H, ArH), 8.14 (d, J=9.0 Hz, 1H,

ArH), 7.46–7.42 (m, 2H, ArH), 7.33–7.28 (m, 2H,

ArH), 6.20 (s, 1H, OH), 4.20 (s, 2H, COCH₂Cl), 3.38

(d, J=13.7 Hz, 1H, CHH_a), 3.34 (d, J=13.7 Hz, 1H,

CHH_b), 1.45 (s, 3H, CH₃); ¹³C-NMR (75 MHz,

DMSO-d₆): l 174.7 (CꢀO), 164.4 (CꢀO), 143.1 (CꢁNO₂,

Ar), 141.5 (CꢁN, Ar), 136.7 (CꢁS, Ar), 130.8 (CꢁN,

Ar), 130.1, 127.1, 122.8, 122.5 (q, J=33.0 Hz), 122.0

(q, J=270.8 Hz, CF₃), 119.7, 118.2 (q, J=6.0 Hz),

75.1 (COꢁCꢁO), 44.5 (COCH₂Cl), 43.4 (CH₂ꢁS), 25.6

(CH₃); IR (KBr, cm⁻¹): 3355 (NH and OH), 1679

(CꢀO, amide), 1596 (CꢀC, Ar), 1524 (CꢀC, Ar), 1400,

1322, 1153; [h]²_D⁶+17.4° (c=0.46, acetone); Anal.

C₁₉H₁₇ClF₃N₃O₅S (C, H, N).

5.1.6. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2S)-3-{[4-

[acetylamino)phenyl]sulfanyl}-2-hydroxy-2-methylpro-

panamide (S-5)

The title compound was prepared from compound

S-3 (0.70 g, 1.69 mmol) and acetic anhydride (10 ml) in

the same manner as compound R-5. Puriﬁcation by

ﬂash column chromatography on silica gel (hexanes–

ethyl acetate, 2:1–1:1) yielded 0.70 g (91%) of the

desired compound as a light yellow powder: m.p. 105–

108 °C, softened then melted; ¹H-NMR (300 MHz,

DMSO-d₆): l 10.35 (s, 1H, CONH), 9.72 (s, 1H,

CONH), 8.34 (d, J=2.1 Hz, 1H, ArH), 8.15 (dd,

J=2.1, 8.7 Hz, 1H, ArH), 8.04 (d, J=8.7 Hz, 1H,

ArH), 7.35–7.32 (m, 2H, ArH), 7.21–7.16 (m, 2H,

ArH), 6.11 (s, 1H, OH), 3.35 (d, J=13.0 Hz, 1H,

CHH_a), 3.2 (d, J=13.0 Hz, 1H, CHH_b), 1.99 (s, 3H,

COCH₃), 1.46 (s, 3H, CH₃); ¹³C-NMR (75 MHz,

DMSO-d₆): l 174.7 (CꢀO), 168.1 (CꢀO), 143.1 (CꢁNO₂,

Ar), 141.5 (CꢁN, Ar), 137.7 (CꢁS, Ar), 130.3, 129.4

(CꢁN, Ar), 127.2, 122,9, 122.6 (q, J=33.0 Hz), 122.1

(q, J=270.8 Hz, CF₃), 119.2, 118.2 (q, J=6.0 Hz),

75.1 (COꢁCꢁO), 44.7 (CH₂ꢁS), 25.7 (COCH₃), 23.8

(CH₃); IR (KBr, cm⁻¹): 3348 (NH and OH), 1677

(CꢀO, amide), 1596 (CꢀC, Ar), 1523 (CꢀC, Ar), 1397

(CH3), 1321, 1154; ESIMS; m/z: 480.2 [M+Na]⁺;

5.1.8. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2S)-3-

({4-[(2-chloroacetyl)amino]phenyl} sulfanyl)-2-

hydroxy-2-methylpropanamide (S-6)

The title compound was prepared from compound

S-3 (0.70 g, 1.69 mmol) and chloroacetyl chloride (0.21

g, 1.85 mmol) in the same manner as compound R-6.

Puriﬁcation by ﬂash column chromatography on silica

gel (hexanes–ethyl acetate, 2:1–1:1) yielded 0.68 g

(82%) of the desired compound as a light yellow pow-

der: m.p. 60–63 °C, softened then melted; ¹H-NMR

(300 MHz, DMSO-d₆): l 10.50 (s, 1H, CONH), 10.32

(s, 1H, CONH), 8.45 (d, J=2.1 Hz, 1H, ArH), 8.25

(dd, J=2.1, 9.0 Hz, 1H, ArH), 8.14 (d, J=9.0 Hz, 1H,

ArH), 7.45–7.42 (m, 2H, ArH), 7.32–7.29 (m, 2H,

ArH), 6.21 (s, 1H, OH), 4.20 (s, 2H, COCH₂Cl), 3.39

(d, J=13.1 Hz, 1H, CHH_a), 3.23 (d, J=13.1 Hz, 1H,

CHH_b), 1.45 (s, 3H, CH₃); ¹³C-NMR (75 MHz,

DMSO-d₆): l 174.7 (CꢀO), 164.4 (CꢀO), 143.1 (CꢁNO₂,

Ar), 141.6 (CꢁN, Ar), 136.7 (CꢁS, Ar), 130.8 (CꢁN,

Ar), 130.1, 127.2, 122.9, 122.6 (q, J=33.0 Hz), 122.1

(q, J=271.5 Hz, CF₃), 119.7, 118.2 (q, J=6.0 Hz),

75.2 (COꢁCꢁO), 44.5 (COCH₂Cl), 43.4 (CH₂ꢁS), 25.7

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

627

(CH₃); IR (KBr, cm⁻¹): 3349 (NH and OH), 1680

(CꢀO, amide), 1596 (CꢀC, Ar), 1525 (CꢀC, Ar), 1400,

1322, 1153; ESIMS; m/z: 514.2 (M+Na)⁺; [h]_D²⁶

−18.1° (c=0.50, acetone); Anal. C₁₉H₁₇ClF₃N₃O₅S·

0.4C₄H₈O₂(C, H, N).

chromatography on silica gel (ethyl acetate–hexanes,

1:1–2:1) yielded 0.28 g (60%) of the desired compound

as a light yellow powder: m.p. 57–60 °C, softened then

1

melted; H-NMR (300 MHz, CDCl₃): l 9.09 (s, 1H,

NH), 7.97 (d, J=2.2 Hz, 1H, ArH), 7.95 (d, J=8.9

Hz, 1H, ArH), 7.83 (dd, J=8.9, 2.2 Hz, 1H, ArH),

7.41–7.37 (m, 2H, ArH), 7.06–7.04 (m, 2H, ArH), 6.70

(s, 1H, OH), 3.76 (d, J=14.1 Hz, 1H, CHH_a), 3.15 (d,

J=14.1 Hz, 1H, CHH_b), 2.98 (s, 3H, CH₃SO₂), 1.56 (s,

3H, CH₃); ¹³C-NMR (75 MHz, CDCl₃): l 173.0 (CꢀO),

143.2 (CꢁNO₂, Ar), 141.3 (CꢁN, Ar), 136.3, 132.7,

130.1, 127.0, 125.2 (q, J=33.8 Hz), 122.0, 121.7 (q,

J=272.3 Hz, CF₃), 120.8, 118.1 (q, J=6.0 Hz), 75.5,

45.2 (CH₂S), 39.7 (CH₃SO₂), 26.2 (CH₃); IR (KBr,

cm⁻¹): 3350 (OH and NH), 1703 (CꢀO, amide), 1598,

1523, 1495 (CꢀC, Ar), 1421, 1324, 1152; ESIMS; m/z:

516.2 [M+Na]⁺; [h]_D²⁶+12.0° (c=0.68, acetone);

Anal. C₁₈H₁₈F₃N₃O₆S₂·0.5C₄H₈O₂(C, H, N).

5.1.9. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2R)-2-

hydroxy-2-methyl-3-({4-[(2,2,2-triﬂuoroacetyl)amino]-

phenyl}sulfanyl)propanamide (R-7)

To a solution of compound R-3 (0.50 g, 1.20 mmol)

in 15 ml of anhydrous THF was added 0.85 ml of Et₃N

(6.0 mmol) at room temperature, and the resulting

solution was cooled to 0 °C in an ice bath. Tri-

ﬂuoroacetic anhydride (0.85 ml, 6.0 mmol) was added

dropwise to the above solution. The reaction mixture

was warmed to room temperature, stirred overnight

and then evaporated in vacuo to afford a residue. A

solution of the residue in 20 ml of CH₂Cl₂was washed

with brine (20 ml×2), 1N HCl solution (20 ml×2),

dried over anhydrous Na₂SO₄, ﬁltered through Celite,

and evaporated in vacuo to dryness. Puriﬁcation by

ﬂash column chromatography on silica gel (CHCl₃–

EtOAc, 6:1) yielded 0.58 g (95%) of the desired com-

pound as a yellow amorphic powder: m.p. 50–53 °C,

5.1.11. N-[4-cyano-3-(triﬂuoromethyl)phenyl]-(2R)-3-

{[4-[acetylamino)phenyl]sulfanyl}-2-hydroxy-2-

methylpropanamide (R-9)

The title compound was prepared from compound

R-4 (0.38 g, 0.97 mmol) and acetic anhydride (2 ml) by

the same procedure used for compound R-5. Puriﬁca-

tion by ﬂash column chromatography on silica gel

(ethyl acetate–hexanes, 1:1) yielded 0.41 g (98%) of the

desired compound as a white solid: m.p. 121–123 °C;

¹H-NMR (300 MHz, DMSO-d₆): l 10.41 (s, 1H, NH),

9.85 (s, 1H, NH), 8.41 (d, J=1.9 Hz, 1H, ArH), 8.18

(dd, J=8.6, 1.9 Hz, 1H, ArH), 8.02 (d, J=8.6 Hz, 1H,

ArH), 7.42–7.39 (m, 2H, ArH), 7.27–7.23 (m, 2H,

ArH), 6.17 (s, 1H, OH), 3.36 (d, J=13.1 Hz, 1H,

CHH_a), 3.20 (d, J=13.1 Hz, 1H, CHH_b), 1.70 (s, 3H,

Me), 1.44 (s, 3H, Me); ¹³C-NMR (75 MHz, DMSO-d₆):

l 174.8, 168.1, 143.1, 137.7, 136.1, 131.4 (q, J=31.5

Hz), 130.3, 129.4, 122.6, 122.5 (q, J=271.5 Hz), 119.2,

117.3 (q, J=5.25 Hz), 115.8, 101.8 (q, J=2.3 Hz),

75.1, 44.7, 25.7, 23.9; IR (KBr, cm⁻¹): 3442, 3329 (NH,

OH), 2231 (CN), 1683 (CO), 1590, 1524 (CꢀC, Ar),

1431, 1320, 1181, 1130; [h]²_D⁷+27.1° (c=0.60, acetone);

Anal. C₂₀H₁₈F₃N₃O₃S (C, H, N).

1

softened then melted; H-NMR (300 MHz, DMSO-d₆):

l 11.61 (s, 1H, NH), 10.51 (s, 1H, NH), 8.41 (d, J=2.0

Hz, 1H, ArH), 8.22 (dd, J=2.0, 9.0 Hz, 1H, ArH), 8.13

(d, J=9.0 Hz, 1H, ArH), 7.51–7.48 (m, 2H, ArH),

7.36–7.34 (m, 2H, ArH), 6.25 (s, 1H, OH), 3.44 (d,

J=13.3 Hz, 1H, CHH_a), 3.25 (d, J=13.3 Hz, 1H,

CHH_b), 1.46 (s, 3H, CH₃); ¹³C-NMR (75 MHz,

DMSO-d₆): l 174.7 (CꢀO), 154.2 (q, J=36.8 Hz,

COCF₃), 143.1 (CꢁN, Ar), 141.5 (CꢁNO₂, Ar), 134.3,

133.1, 129.6, 127.2, 123.0, 122.5 (q, J=33.0 Hz), 122.1

(q, J=271.5 Hz, CF₃), 121.1, 118.1 (q, J=5.3 Hz),

115.7 (q, J=286.5 Hz, COCF₃), 75.1, 43.8, 25.7 (CH₃);

IR (KBr, cm⁻¹): 3353 (OH and NH), 1712 (CON),

1599, 1543 (CꢀC, Ar), 1422, 1352; ESIMS; m/z: 534.1

[M+Na]⁺; [h]²_D⁶+7.2° (c=0.36, acetone); Anal.

C₁₉H₁₅F₆N₃O₅S·0.5C₄H₈O₂(C, H, N).

5.1.10. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2R)-2-

hydroxy-2-methyl-3-({4-[(methylsulfonyl)amino]-

phenyl}sulfanyl)propanamide (R-8)

5.1.12. N-[4-cyano-3-(triﬂuoromethyl)phenyl]-(2S)-3-

{[4-[acetylamino)phenyl]sulfanyl}-2-hydroxy-2-

methylpropanamide (S-9)

To a solution of compound R-3 (0.40 g, 0.96 mmol)

in 15 ml of anhydrous THF was added 0.28 ml of Et₃N

(2.0 mmol) at room temperature, and the resulting

solution was cooled to 0 °C in an ice bath. Methanesul-

fonyl chloride (0.11 g, 0.96 mmol) was added dropwise

to the above mixture. The reaction mixture was

warmed to room temperature, stirred overnight, and

evaporated in vacuo to afford a residue. A solution of

the residue in 20 ml of CH₂Cl₂was washed with brine

(20 ml×2), 1N HCl solution (20 ml×2), dried over

anhydrous Na₂SO₄, ﬁltered through Celite, and evapo-

rated in vacuo to dryness. Puriﬁcation by ﬂash column

The title compound was prepared from compound

S-4 (0.30 g, 0.76 mmol) and acetic anhydride (2 ml) in

the same manner as compound R-9. Recrystallization

from ethyl acetate–hexanes afforded 0.26 g (78%) of

the desired compound as white solid: m.p. 122–124 °C;

¹H-NMR (300 MHz, DMSO-d₆): l 10.41 (s, 1H, NH),

9.85 (s, 1H, NH), 8.41 (d, J=1.9 Hz, 1H, ArH), 8.18

(dd, J=8.6, 1.9 Hz, 1H, ArH), 8.02 (d, J=8.6 Hz, 1H,

ArH), 7.42–7.39 (m, 2H, ArH), 7.27–7.23 (m, 2H,

ArH), 6.17 (s, 1H, OH), 3.36 (d, J=13.1 Hz, 1H,

628

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

CHH_a), 3.20 (d, J=13.1 Hz, 1H, CHH_b), 1.70 (s, 3H,

Me), 1.44 (s, 3H, Me); ¹³C-NMR (75 MHz, DMSO-d₆):

l 174.8, 168.1, 143.1, 137.7, 136.1, 131.4 (q, J=31.5

Hz), 130.3, 129.4, 122.6, 122.5 (q, J=271.5 Hz), 119.2,

117.3 (q, J=5.25 Hz), 115.8, 101.8 (q, J=2.3 Hz),

75.1, 44.7, 25.7, 23.9; IR (KBr, cm⁻¹): 3442, 3329 (NH,

OH), 2231 (CN), 1683 (CO), 1590, 1524 (CꢀC, Ar),

1431, 1320, 1181, 1130; [h]²_D⁷−27.2° (c=0.58, ace-

tone); Anal. C₂₀H₁₈F₃N₃O₃S (C, H, N).

(dd, J=8.6, 1.9 Hz, 1H, ArH), 8.03 (d, J=8.6 Hz, 1H,

ArH), 7.45–7.43 (m, 2H, ArH), 7.31–7.28 (m, 2H,

ArH), 6.20 (s, 1H, OH), 4.20 (s, 2H, CH₂Cl), 3.38 (d,

J=13.1 Hz, 1H, CHH_a), 3.23 (d, J=13.1 Hz, 1H,

CHH_b), 1.45 (s, 3H, Me); ¹³C-NMR (75 MHz, DMSO-

d₆): l 174.8, 164.4, 143.1, 136.7, 136.1, 131.4 (q, J=

31.7 Hz), 130.9, 130.1, 122.6, 122.5 (q, J=273.7 Hz),

119.7, 117.3 (q, J=5.0 Hz), 115.8, 101.9 (q, J=2.0

Hz), 75.2, 44.5, 43.4, 25.7; IR (KBr, cm⁻¹) 3464, 3321,

3265 (NH, OH), 2238 (CN), 1679 (CO), 1609, 1597,

1522, 1429, 1327, 1174; [h]²_D⁷+23.4° (c=2, acetone);

Anal. C₂₀H₁₇ClF₃N₃O₃S (C, H, N).

5.1.13. N-[4-cyano-3-(triﬂuoromethyl)phenyl]-(2R)-3-

{[4-[propionylamino)phenyl]sulfanyl}-2-hydroxy-2-

methylpropanamide (R-10)

Propionyl bromide (0.27 g, 1.89 mmol) was added

dropwise to a mixture of compound R-4 (0.5 g, 1.26

mmol) and CaCO₃(0.63 g, 6.32 mmol) in anhydrous

CH₂Cl₂(50 ml) at room temperature under argon at-

mosphere. The reaction mixture was stirred for 3 days.

After the end of the reaction was established by TLC,

the resulting mixture was ﬁltered through Celite,

washed with EtOAc, and the ﬁltrate was evaporated

completely in vacuo to dryness. Puriﬁcation by ﬂash

column chromatography on silica gel (ethyl acetate–

hexanes, 1:1) yielded 0.15 g (20%) of the desired com-

5.1.15. N-[4-cyano-3-(triﬂuoromethyl)phenyl]-(2S)-3-

({4-[(2-chloroacetyl)amino]phenyl}sulfanyl)-2-

hydroxy-2-methylpropanamide (S-11)

The title compound was prepared from compound

S-4 (0.41 g, 1.04 mmol) and chloroacetyl chloride (0.1

ml, d=1.418, 1.20 mmol) in the same manner as com-

pound R-11. Recrystallization from ethyl acetate–he-

xanes gave 0.35 g (56%) of the desired compound as

1

white crystals: m.p. 132–134 °C; H-NMR (300 MHz,

DMSO-d₆): l 10.44 (s, 1H, NH), 10.23 (s, 1H, NH),

8.43 (d, J=1.7 Hz, 1H, ArH), 8.20 (dd, J=8.6, 1.9 Hz,

1H, ArH), 8.04 (d, J=8.6 Hz, 1H, ArH), 7.45–7.42 (m,

2H, ArH), 7.31–7.28 (m, 2H, ArH), 6.20 (s, 1H, OH),

4.20 (s, 2H, CH₂Cl), 3.38 (d, J=13.2 Hz, 1H, CHH_a),

3.23 (d, J=13.1 Hz, 1H, CHH_b), 1.44 (s, 3H, Me);

¹³C-NMR (75 MHz, DMSO-d₆): l 174.8, 164.5, 143.1,

136.7, 136.1, 131.4 (q, J=31.7 Hz), 130.9, 130.1, 122.7,

122.5 (q, J=273.8 Hz), 119.7, 117.3 (q, J=5.3 Hz),

115.8, 101.9 (q, J=1.8 Hz), 75.2, 44.5, 43.5, 25.8; IR

(KBr, cm⁻¹) 3464, 3320, 3268 (NH, OH), 2239 (CN),

1679 (CO), 1609, 1598, 1521, 1429, 1327, 1174 1135;

[h]²_D⁷−23.4° (c=2, acetone); Anal. C₂₀H₁₇ClF₃N₃O₃S

(C, H, N).

1

pound as a white solid: m.p. 125–127 °C; H-NMR

(300 MHz, DMSO-d₆): l 10.43 (NH), 9.78 (NH), 8.41

(d, J=1.6 Hz, 1H, ArH), 8.18 (dd, J=1.7, 8.6 Hz, 1H,

ArH), 8.03 (d, J=8.6 Hz, 1H, ArH), 7.43–7.40 (m, 2H,

ArH), 7.26–7.23 (m, 2H, ArH), 6.19 (s, 1H, OH), 3.34

(d, J=13.2 Hz, 1H, CHH_a), 3.19 (d, J=13.2 Hz, 1H,

CHH_b), 3.27 (q, J=7.5 Hz, 2H, CH₂), 1.43 (s, 3H,

Me), 1.05 (t, J=7.5 Hz, 3H, Me); ¹³C-NMR (75 MHz,

DMSO-d₆): l 174.8, 171.8, 143.1, 137.8, 136.1, 131.4 (q,

J=31.58 Hz), 130.3, 129.3, 122.6, 122.5 (q, J=271.73

Hz, CF₃), 119.2, 117.3 (q, J=5.3 Hz), 115.8, 102.0 (q,

J=1.9 Hz), 75.1, 44.7, 29.4, 25.7, 9.5; IR (KBr, cm⁻¹):

3415, 3352 (NH, OH), 2231 (CN), 1680 (CꢀO), 1589,

1522 (CꢀC, Ar), 1431, 1400, 1329, 1183, 1136; [h]_D²⁷

+28.3° (c=0.53, acetone); Anal. C₂₁H₂₀F₃N₃O₃S (C,

H, N).

5.1.16. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2R)-3-

{[4-[acetylamino)phenyl]sulfonyl}-2-hydroxy-2-

methylpropanamide (R-12)

Peracetic acid (1 ml) was added to a solution of

compound R-5 (0.40 g, 0.87 mmol) in 2 ml of EtOAc

and the resulting mixture was stirred for 1 h at room

temperature. After the end of the reaction was esta-

blished by TLC, the reaction solution was diluted with

EtOAc (150 ml), washed with saturated Na₂SO₃solu-

tion (30 ml×2), brine (30 ml×2), dried over anhy-

drous Na₂SO₄, ﬁltered through Celite, and evaporated

in vacuo to give an oil. The oily crude product was

puriﬁed by ﬂash column chromatography on silica gel

(hexanes–ethyl acetate, 3:1) to yield 0.43 g (99%) of the

desired compound as a yellow powder: m.p. 101–

5.1.14. N-[4-cyano-3-(triﬂuoromethyl)phenyl]-(2R)-3-

({4-[(2-chloroacetyl)amino]phenyl}sulfanyl)-2-

hydroxy-2-methylpropanamide (R-11)

Chloro acetyl chloride (0.1 ml, d=1.418, 1.2 mmol)

was added dropwise to a vigorously stirred suspension

of compound R-4 (0.41 g, 1.04 mmol) and CaCO₃(0.56

g, 5.6 mmol) in anhydrous CH₂Cl₂(50 ml) over a

period of 1–2 min under argon atmosphere. The reac-

tion mixture was stirred overnight, ﬁltered through

Celite, and the ﬁltrate was completely evaporated in

vacuo to give a white solid. Recrystallization from ethyl

acetate–hexanes yielded 0.48 g (77%) of the desired

compound as ball-shape crystals: m.p. 134.5–135 °C;

¹H-NMR (300 MHz, DMSO-d₆): l 10.44 (s, 1H, NH),

10.24 (s, 1H, NH), 8.43 (d, J=1.8 Hz, 1H, ArH), 8.20

1

103 °C; H-NMR (300 MHz, DMSO-d₆): l 10.32 (s,

1H, CONH), 10.19 (s, 1H, CONH), 8.39 (d, J=2.0 Hz,

1H, ArH), 8.21 (dd, J=2.0, 9.0 Hz, 1H, ArH), 8.12 (d,

J=9.0 Hz, 1H, ArH), 7.76–7.74 (m, 2H, ArH), 7.67–

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

629

7.64 (m, 2H, ArH), 6.36 (s, 1H, OH), 3.89 (d, J=14.6

Hz, 1H, CHH_a), 3.61 (d, J=14.6 Hz, 1H, CHH_b), 2.02

(s, 3H, COCH₃), 1.40 (s, 3H, CH₃); ¹³C-NMR (75

MHz, DMSO-d₆): l 173.5 (CꢀO), 168.9 (CꢀO), 143.8

(CꢁNO₂, Ar), 143.2 (CꢁS, Ar), 141.5 (CꢁN, Ar), 133.6

(CꢁN, Ar), 129.4, 127.1, 123.0, 122.6 (q, J=33.0 Hz),

122.1 (q, J=271.5 Hz, CF₃), 118.2 (q, J=6.0 Hz),

118.1, 73.0 (COꢁCꢁO), 63.6 (CH₂ꢁSO₂), 27.3 (COCH₃),

23.9 (CH₃); IR (KBr, cm⁻¹): 3343 (NH and OH), 1691

(CꢀO, amide), 1594 (CꢀC, Ar), 1528 (CꢀC, Ar), 1403,

1323, 1142; ESIMS; m/z: 512.2 [M+Na]⁺; [h]_D²⁶

−41.5° (c=0.42, acetone); Anal. C₁₉H₁₈F₃N₃O₇S·

0.5C₄H₈O₂·0.2C₆H₁₄(C, H, N).

J=14.7 Hz, 1H, CHH_b), 1.41 (s, 3H, CH₃); ¹³C-NMR

(75 MHz, DMSO-d₆): l 173.7 (CꢀO), 165.2 (CꢀO),

143.2 (CꢁNO₂, Ar), 142.9 (CꢁSO₂, Ar), 141.6 (CꢁN,

Ar), 134.8 (CꢁN, Ar), 129.5, 127.2, 123.1, 122.6 (q,

J=33.0 Hz), 122.1 (q, J=271.5 Hz, CF₃), 118.6, 118.3

(q, J=6.0 Hz), 73.1 (COꢁCꢁO), 63.6 (CH₂ꢁSO₂), 43.5

(COCH₂Cl), 27.2 (CH₂); IR (KBr, cm⁻¹): 3454, 3337

(NH and OH), 1690 (CꢀO, amide), 1593 (CꢀC, Ar),

1521 (CꢀC, Ar), 1403, 1356, 1143; [h]²_D⁶−47.4° (c=

0.56, acetone); Anal. C₁₉H₁₇ClF₃N₃O₇S (C, H, N).

5.1.19. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2S)-3-

({4-[(2-chloroacetyl)amino]phenyl}sulfonyl)-2-hydroxy-

2-methylpropanamide (S-13)

5.1.17. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2S)-3-{[4-

[acetylamino)phenyl]sulfonyl}-2-hydroxy-2-methyl-

propanamide (S-12)

The title compound was prepared from compound

S-6 (0.44 g, 0.89 mmol) and peracetic acid (2 ml) in the

same manner as compound R-13. Puriﬁcation by ﬂash

column chromatography on silica gel (hexanes–ethyl

acetate, 4:1) yielded 0.47 g (99%) of the desired com-

pound as a light yellow powder: m.p. 155–157 °C;

¹H-NMR (300 MHz, DMSO-d₆): l 10.59 (s, 1H,

CONH), 10.37 (s, 1H, CONH), 8.42 (d, J=2.1 Hz, 1H,

ArH), 8.22 (dd, J=2.1, 9.0 Hz, 1H, ArH), 8.13 (d,

J=9.0 Hz, 1H, ArH), 7.83–7.80 (m, 2H, ArH), 7.71–

7.68 (m, 2H, ArH), 6.37 (s, 1H, OH), 4.24 (s, 2H,

COCH₂Cl), 3.93 (d, J=14.7 Hz, 1H, CHH_a), 3.63 (d,

J=14.7 Hz, 1H, CHH_b), 1.41 (s, 3H, CH₃); ¹³C-NMR

(75 MHz, DMSO-d₆) d 173.6 (CꢀO), 165.2 (CꢀO), 143.2

(CꢁNO₂, Ar), 142.9 (CꢁSO₂, Ar), 141.6 (CꢁN, Ar),

134.8 (CꢁN, Ar), 129.5, 127.1, 123.1, 122.6 (q, J=33.0

Hz), 122.1 (q, J=271.5 Hz, CF₃), 118.7, 118.3 (q,

J=6.0 Hz), 73.1 (COꢁCꢁO), 63.6 (CH₂ꢁSO₂), 43.4

(COCH₂Cl), 27.9 (CH₃); IR (KBr, cm⁻¹): 3337 (NH

and OH), 1690 (CꢀO, amide), 1593 (CꢀC, Ar), 1521

(CꢀC, Ar), 1403, 1356, 1143; ESIMS; m/z: 546.3 [M

+Na]⁺; [h]²_D⁶+47.7° (c=0.53, acetone); Anal.

C₁₉H₁₇ClF₃N₃O₇S·0.35C₄H₈O₂(C, H, N).

The title compound was prepared from compound

S-5 (0.30 g, 0.66 mmol) and peracetic acid (1 ml) in the

same manner as compound R-12. Puriﬁcation by ﬂash

column chromatography on silica gel (hexanes–ethyl

acetate, 3:1) yielded 0.32 g (99%) of the desired com-

pound as a light yellow powder: m.p. 100–103 °C;

¹H-NMR (300 MHz, DMSO-d₆): l 10.32 (s, 1H,

CONH), 10.19 (s, 1H, CONH), 8.39 (d, J=2.0 Hz, 1H,

ArH), 8.20 (dd, J=2.0, 9.0 Hz, 1H, ArH), 8.12 (d,

J=9.0 Hz, 1H, ArH), 7.77–7.74 (m, 2H, ArH), 7.67–

7.64 (m, 2H, ArH), 6.37 (s, 1H, OH), 3.90 (d, J=14.6

Hz, 1H, CHH_a), 3.61 (d, J=14.6 Hz, 1H, CHH_b), 2.02

(s, 3H, COCH₃), 1.41 (s, 3H, CH₃); ¹³C-NMR (75

MHz, DMSO-d₆): l 173.5 (CꢀO), 168.9 (CꢀO), 143.8

(CꢁNO₂, Ar), 143.2 (CꢁS, Ar), 141.6 (CꢁN, Ar), 133.6

(CꢁN, Ar), 129.4, 127.1, 123.0, 122.6 (q, J=33.0 Hz),

122.1 (q, J=270.8 Hz, CF₃), 118.3 (q, J=6.0 Hz),

118.1, 73.0 (COꢁCꢁO), 63.6 (CH₂ꢁSO₂), 27.3 (COCH₃),

24.0 (CH₃); IR (KBr, cm⁻¹) 3352 (NH and OH), 1692

(CꢀO, amide), 1594 (CꢀC, Ar), 1527 (CꢀC, Ar), 1403,

1323, 1142; ESIMS; m/z: 512.2 [M+Na]⁺; [h]_D²⁶

+

41.7° (c=0.52, acetone); Anal. C₁₉H₁₈F₃N₃O₇S·

0.6C₄H₈O₂(C, H, N).

5.1.20. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2R)-2-

hydroxy-2-methyl-3-({4-[(2,2,2-triﬂuoroacetyl)amino]-

phenyl}sulfonyl)propanamide (R-14)

5.1.18. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2R)-3-

({4-[(2-chloroacetyl)amino]phenyl}sulfonyl)-2-hydroxy-

2-methylpropanamide (R-13)

The title compound was prepared from compound

R-7 (0.10 g, 0.2 mmol) and peracetic acid (0.5 ml) by

the same procedure used for compound R-12. Puriﬁca-

tion by ﬂash column chromatography on silica gel

(hexanes–ethyl acetate, 4:1) yielded 0.1 g (98%) of the

desired compound as a yellow powder: m.p. 70–73 °C,

The title compound was prepared from compound

R-6 (0.38 g, 0.77 mmol) and peracetic acid (1 ml) by the

same procedure used for compound R-12. Puriﬁcation

by ﬂash column chromatography on silica gel (hexa-

nes–ethyl acetate, 4:1) yielded 0.40 g (99%) of the

desired compound as a yellow powder: m.p. 154–

1

softened then melted; H-NMR (300 MHz, DMSO-d₆):

l 11.51 (s, 1H, NH), 10.3 (s, 1H, NH), 8.38 (d, J=1.7

Hz, 1H, ArH), 8.20 (dd, J=1.7 Hz, J=9.0 Hz, 1H,

ArH), 8.13 (d, J=9.0 Hz, 1H, ArH), 7.89–7.86 (m, 2H,

ArH), 7.80–7.77 (m, 2H, ArH), 6.45 (s, 1H, OH), 3.95

(d, J=14.8 Hz, 1H, CHH_a), 3.67 (d, J=14.8 Hz, 1H,

CHH_b), 1.40 (s, 3H, CH₃); ¹³C-NMR (75 MHz,

DMSO-d₆): l 173.5 (CꢀO), 154.3 (q, J=37.5 Hz,

COCF₃), 143.2 (CꢁN, Ar), 141.6 (CꢁNO₂, Ar), 140.9

1

156 °C; H-NMR (300 MHz, DMSO-d₆): l 10.58 (s,

1H, CONH), 10.36 (s, 1H, CONH), 8.41 (d, J=2.1 Hz,

1H, ArH), 8.22 (dd, J=2.1, 9.0 Hz, 1H, ArH), 8.14 (d,

J=9.0 Hz, 1H, ArH), 7.82–7.79 (m, 2H, ArH), 7.72–

7.69 (m, 2H, ArH), 6.36 (s, 1H, OH), 4.24 (s, 2H,

COCH₂Cl), 3.90 (d, J=14.7 Hz, 1H, CHH_a), 3.64 (d,

630

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

(CꢁN, Ar), 136.5, 129.7, 127.2, 123.1, 122.6 (q, J=33.0

Hz), 122.1 (q, J=267.0 Hz, CF₃), 120.3, 118.3 (q,

J=6.0 Hz), 115.5 (q, J=287.3 Hz, CF₃), 73.0, 63.5,

27.4 (CH₃); IR (KBr, cm⁻¹): 3344 (NH and OH), 1718

(CON), 1598, 1546 (CꢀC, Ar), 1410, 1353, 1321, 1151;

[h]²_D⁶−53.8° (c=0.58, acetone); Anal. C₁₉H₁₅F₆N₃O₇S

(C, H, N).

5.1.23. N-[4-cyano-3-(triﬂuoromethyl)phenyl]-(2R)-

3-({4-[(2-chloroacetyl)amino]phenyl}sulfonyl)-2-

hydroxy-2-methylpropanamide (R-17)

The title compound was prepared from compound

R-11 (0.22 g, 0.47 mmol) and peracetic acid (4 ml) in

the same manner as compound R-12. Recrystallization

from ethyl acetate–hexanes gave compound R-17 as a

white solid with quantitative yield: m.p. 174–175 °C;

¹H-NMR (300 MHz, DMSO-d₆): l 10.61 (s, 1H, NH),

10.33 (s, 1H, NH), 8.40 (d, J=1.8 Hz, 1H, ArH), 8.17

(dd, J=8.6, 1.9 Hz, 1H, ArH), 8.05 (d, J=8.6 Hz, 1H,

ArH), 7.81–7.78 (m, 2H, ArH), 7.70–7.67 (m, 2H,

ArH), 6.38 (s, 1H, OH), 4.25 (s, 2H, CH₂Cl), 3.89 (d,

J=14.7 Hz, 1H, CHH_a), 3.62 (d, J=14.7 Hz, 1H,

CHH_b), 1.39 (s, 3H, Me); ¹³C-NMR (75 MHz, DMSO-

d₆): l 173.7, 165.2, 143.1, 136.1, 133.8, 131.4 (q, J=

31.6 Hz), 129.5, 122.8, 122.5 (q, J=273.6 Hz), 118.7,

117.4 (q, J=5.0 Hz), 115.9, 101.9 (q, J=2.0 Hz), 73.1,

63.7, 43.5, 27.3; IR (KBr, cm⁻¹): 3477, 3357, 3325

(NH, OH), 2235 (CN), 1692 (CO), 1592, 1527, 1422,

1403, 1329, 1301, 1186, 1144; [h]²_D⁷−49.4° (c=2,

acetone); Anal. C₂₀H₁₇ClF₃N₃O₅S (C, H, N).

5.1.21. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-(2R)-2-

hydroxy-2-methyl-3-({4-[(methyl sulfonyl)amino]-

phenyl}sulfonyl)propanamide (R-15)

The title compound was prepared from compound

R-8 (0.19 g, 0.38 mmol) and peracetic acid (0.5 ml) in

the same manner as compound R-12. Puriﬁcation by

ﬂash column chromatography on silica gel (hexanes–

ethyl acetate, 1:1–1:2) yielded 0.19 g (98%) of the

desired compound as a yellow powder: m.p. 75–78 °C,

1

softened then melted; H-NMR (300 MHz, DMSO-d₆):

l 10.44 (s, 1H, NH), 10.41 (s, 1H, NH), 8.47 (d, J=2.1

Hz, 1H, ArH), 8.27 (dd, J=2.1, 9.0 Hz, 1H, ArH), 8.18

(d, J=9.0 Hz, 1H, ArH), 7.82–7.79 (m, 2H, ArH),

7.30–7.27 (m, 2H, ArH), 6.36 (s, 1H, OH), 3.88 (d,

J=14.7 Hz, 1H, CHH_a), 3.64 (d, J=14.7 Hz, 1H,

CHH_b), 3.09 (s, 3H, CH₃SO₂), 1.41 (s, 3H, CH₃);

¹³C-NMR (75 MHz, DMSO-d₆): l 173.9 (CꢀO), 143.3

(CꢁNO₂, Ar), 143.2 (CꢁN, Ar), 141.7, 134.6, 130.0,

127.3, 123.2, 122.7 (q, J=33.0 Hz), 122.1 (q, J=271.5

Hz, CF₃), 118.5 (q, J=6.0 Hz), 117.4, 73.2, 63.6

(CH₂SO₂), 40.0 (CH₃SO₂), 27.2 (CH₃); IR (KBr,

cm⁻¹): 3438 (OH and NH), 1701 (CꢀO, amide), 1598,

1524 (CꢀC, Ar), 1323, 1142; ESIMS; m/z: 548.1 [M+

Na]⁺; [h]²_D⁶−49.2° (c=0.56, acetone); Anal.

C₁₈H₁₈F₃N₃O₈S₂·0.6C₄H₈O₂(C, H, N).

5.1.24. 1,1,1-Triﬂuoro-3-[(4-nitrophenyl)sulfanyl]acetone

(19)

3-Bromo-1,1,1-triﬂuoroacetone (5.0 g, 26.0 mmol)

was added dropwise to a solution of para-nitrothiophe-

nol sodium salt (4.6 g, 26.0 mmol) [19] in anhydrous

THF (30 ml) at 0–5 °C in an ice bath. The reaction

mixture was warmed to room temperature, and stirred

for 3 h. After the end of the reaction was established by

TLC, the mixture was ﬁltered through Celite, washed

with THF, and the ﬁltrate was evaporated in vacuo to

dryness. The crude product was separated by ﬂash

column chromatography on silica gel (CHCl₃–MeOH,

19:1) to yield 3.57 g (50%) of the desired compound as

5.1.22. N-[4-cyano-3-(triﬂuoromethyl)phenyl]-(2R)-3-

{[4-[acetylamino)phenyl]sulfonyl}-2-hydroxy-2-

methylpropanamide (R-16)

1

yellow powder: m.p. 82–84 °C; H-NMR (300 MHz,

The title compound was prepared from compound

R-9 (0.10 g, 0.23 mmol) and peracetic acid (0.5 ml) in

the same manner as compound R-12. Recrystallization

from ethyl acetate–hexanes afforded 0.11 g (93%) of

DMSO-d₆): l 8.11–8.14 (m, 2H, ArH), 7.55–7.58 (m,

2H, ArH), 3.42 (s, 2H, CH₂S); ¹³C-NMR (75 MHz,

DMSO-d₆): l 147.7, 144.5, 126.6, 125.6 (q, J=270.8

Hz, CF₃), 123.7, 92.1 (q, J=30.6 Hz, CCF₃), 37.2

(CH₂S); IR (KBr, cm⁻¹): 3418 (broad), 1768 (CꢀO),

1582, 1517 (CꢀC, Ar), 1344, 1205, 1170, 1108, 1022,

958, 855, 740, 699; ESIMS; m/z: 264.4 [M]⁻; Anal.

C₉H₆F₃NO₃S·H₂O (C, H, N).

1

compound R-16 as white solid: m.p. 198–199 °C; H-

NMR (300 MHz, DMSO-d₆): l 10.27 (s, 1H, NH),

10.20 (s, 1H, NH), 8.36 (d, J=1.6 Hz, 1H, ArH), 8.15

(dd, J=8.6, 1.6 Hz, 1H, ArH), 8.03 (d, J=8.6 Hz, 1H,

ArH), 7.75–7.72 (m, 2H, ArH), 7.65–7.62 (m, 2H,

ArH), 3.88 (d, J=14.6 Hz, 1H, CHH_a), 3.60 (d, J=

14.6 Hz, 1H, CHH_b), 2.04 (s, 3H, Me), 1.39 (s, 3H,

Me); ¹³C-NMR (75 MHz, DMSO-d₆): l 173.5, 168.9,

143.7, 143.1, 136.1, 133.6, 131.3 (q, J=31.5 Hz), 129.4,

122.7, 122.5 (q, J=272.3 Hz), 118.0, 117.3 (q, J=5.25

Hz), 115.6, 101.8 (q, J=2.3 Hz), 73.0, 63.6, 27.3, 24.0;

IR (KBr, cm⁻¹): 3435, 3380, 3305 (NH, OH), 2243

(CN), 1686 (CꢀO), 1593, 1528 (CꢀC, Ar), 1430, 1404,

1327, 1303, 1174, 1144; [h]²_D⁷−36.2° (c=0.54, ace-

tone); Anal. C₂₀H₁₈F₃N₃O₅S (C, H, N).

5.1.25. 2,2,2-Triﬂuoro-1-hydroxy-1-{[(4-nitrophenyl)-

sulfanyl]methyl}ethyl cyanide (20)

An aqueous solution (25% v/v) of sulfuric acid (3.4

ml) was added dropwise to a mixture of compound 19

(3.57 g, 13.0 mmol) and KCN (1.0 g, 15.0 mmol) in 5

ml of water at 0–5 °C in an ice bath. The reaction

mixture was warmed to room temperature, stirred for

20 h, diluted with water (50 ml), and extracted with

Et₂O (3×150 ml). The Et₂O extracts were washed

with saturated NaHCO₃solution, brine, dried over

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

631

1

anhydrous Na₂SO₄, ﬁltered through Celite, and evapo-

rated completely in vacuo to afford 2.74 g (67%) of the

desired compound as a light yellow solid: m.p. 62–

the desired compound as an ivory solid: H-NMR (300

MHz, DMSO-d₆): l 10.01 (s, 1H, NH), 9.97 (s, 1H,

OH), 7.50–7.53 (m, 2H, ArH), 7.32–7.36 (m, 2H,

ArH), 3.37 (d, J=13.3 Hz, 1H, CHH_a), 3.33 (d, J=

13.2 Hz, 1H, CHH_b), 2.01 (s, 3H, Me).

1

64 °C; H-NMR (300 MHz, DMSO-d₆): l 8.15–8.10

(m, 2H, ArH), 7.59–7.54 (m, 2H, ArH), 7.40 (s, 1H,

OH), 3.42 (s, 2H, CH₂S); ¹³C-NMR (75 MHz, DMSO-

d₆): l 147.7 (CꢁN, Ar), 144.5 (CꢁS, Ar), 126.6, 124.2,

123.7, 123.0 (q, J=379.12 Hz, CF₃), 92.1 (q, J=30.75

Hz, CCF₃), 37.2 (CH₂S); IR (KBr, cm⁻¹): 3488, 3418

(OH), 1585, 1583, 1517 (CꢀC, Ar), 1480, 1344, 1280,

1204, 1186, 1170, 1108, 1085.

5.1.28. 2-[({4-[(2-Chloroacetyl)amino]phenyl}sulfanyl)-

methyl]-3,3,3-triﬂuoro-2-hydroxy propanoic acid (23)

The title compound was prepared from compound 21

(0.31 g, 1.0 mmol), tin(II) chloride dihydrate (0.68 g,

3.0 mmol), CaCO₃(1.0 g, 10.0 mmol), and chloroacetyl

chloride (0.1 ml, 1.2 mmol) by the same procedure used

for compound 22,. Puriﬁcation by ﬂash column chro-

matography on silica gel (CHCl₃ꢁMeOHꢁAcOH,

19:1:5) yielded 0.2 g (62%) of the desired compound:

¹H-NMR (300 MHz, DMSO-d₆): l 10.37 (s, 1H, NH),

10.33 (s, 1H, OH), 7.52–7.45 (m, 2H, ArH), 7.34–7.30

(m, 2H, ArH), 4.23 (s, 2H, CH₂Cl), 3.46 (d, J=13.8

Hz, CHH_a), 3.35 (d, J=13.8 Hz, 1H, CHH_b).

5.1.26. 3,3,3-Triﬂuoro-2-hydroxy-2-{[(4-nitrophenyl)-

sulfanyl]methyl}propanoic acid (21)

A mixture of compound 20 (1.5 g, 5 mmol), concen-

trated HCl (17 ml), and AcOH (3 ml) was heated to

reﬂux overnight with vigorous stirring. After the end of

the reaction was established by TLC, the mixture was

diluted with water (100 ml) and extracted with Et₂O

(4×100 m) which was in turn washed with saturated

NaHCO₃solution (4×100 ml). The NaHCO₃solution

was acidiﬁed with concentrated HCl to pH 1 and

extracted with Et₂O (4×150 ml). The Et₂O extracts

were dried over anhydrous MgSO₄, ﬁltered through

Celite, and evaporated completely in vacuo to dryness.

Trituration in ligroin afforded 0.75 g (47%) of the

desired compound as yellow powder: m.p. 92–93 °C;

¹H-NMR (300 MHz, DMSO-d₆): l 8.10–8.15 (m, 2H,

ArH), 7.58–7.63 (m, 2H, ArH), 3.61 (s, 2H, CH₂S);

¹³C-NMR (75 MHz, DMSO-d₆): l 168.2, 146.3, 144.9,

127.3, 123.7, 123.8 (q, J=286.5 Hz, CF₃), 77.4 (q,

J=27.2 Hz, CCF₃), 34.9 (CH₂S); IR (KBr, cm⁻¹):

3510 (broad), 3192 (broad), 1753, 1718, 1584, 1519,

1340, 1256, 1199, 1184, 1125, 1045, 982, 853, 741; Anal.

C₁₀H₈F₃NO₅S (C, H, N).

5.1.29. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-2-({[4-(2-

acetylamino)phenyl]sulfanyl}methyl-3,3,3-triﬂuoro-

2-hydroxypropanamide (24)

Thionyl chloride (0.1 ml, 1.5 mmol) was added drop-

wise to a solution of compound 22 (0.40 g, 1.2 mmol) in

anhydrous DMA (10 ml) at −15 to −10 °C under

argon atmosphere. The reaction mixture was stirred for

1 h, and a solution of 5-amino-2-nitrobenzotriﬂuoride

(0.25 g, 1.2 mmol) in anhydrous DMA (3 ml) was

added dropwise to the above reaction solution. The

resulting mixture was warmed to room temperature and

stirred for 72 h. After the end of the reaction was

established by TLC, the mixture was evaporated in

vacuo, diluted with saturated NaHCO₃solution (50

ml), and extracted with Et₂O (3×50 ml). The Et₂O

extracts were ﬁltered through Celite, dried over anhy-

drous Na₂SO₄, and evaporated in vacuo to dryness.

The crude product was separated by ﬂash column

chromatography on silica gel (CHCl₃–MeOH, 19:1) to

yield 80 mg (13%) of the desired compound as a light

5.1.27. 2-({[4-(Acetylamino)phenyl]sulfanyl}methyl)-

3,3,3-triﬂuoro-2-hydroxypropanoic acid (22)

A solution of compound 21 (2.02 g, 6.5 mmol) in

MeOH (10 ml) was added dropwise to a solution of

tin(II) chloride dihydrate (4.40 g, 19.5 mmol) in concen-

trated HCl (10 ml) cooled in an ice bath. The reaction

mixture was stirred overnight, ﬁltered through Celite,

and evaporated in vacuo to give a residue. The identity

was conformed by ¹H-NMR to be 2-({[4-(amino)-

phenyl]sulfanyl}methyl)-3,3,3-triﬂuoro-2-hydroxypro-

panoic acid without further puriﬁcation.

Acetyl chloride (0.7 ml, 9.8 mmol) was added drop-

wise to a mixture of above residue and CaCO₃(6.50 g,

65 mmol) in anhydrous CH₃CN (30 ml). The resulting

mixture was stirred for 48 h under argon atmosphere.

After the end of the reaction was established by TLC,

the mixture was ﬁltered through Celite, washed with

CH₃CN, and the ﬁltrate was evaporated in vacuo to

dryness. Puriﬁcation by ﬂash column chromatography

on silica gel (CHCl₃–MeOH, 3:1) yielded 0.4 g (20%) of

1

yellow powder: m.p. 203–204 °C; H-NMR (300 MHz,

DMSO-d₆): l 10.78 (s, 1H, NH), 9.84 (s, 1H, NH), 8.32

(d, J=1.7 Hz, 1H, ArH), 8.17 (dd, J=2.0, 9.0 Hz, 1H,

ArH), 8.12 (d, J=9.0 Hz, 1H, ArH), 8.00 (s, 1H, OH),

7.36–7.33 (m, 2H, ArH), 7.27–7.25 (m, 2H, ArH), 3.71

(d, J=13.6 Hz, 1H, CHH_a), 3.36 (d, J=13.6 Hz, 1H,

CHH_b), 1.97 (s, 3H, Me); ¹³C-NMR (75 MHz, DMSO-

d₆): l 168.1, 166.3, 142.1, 138.6, 131.8, 127.1, 126.8,

125.9, 123.7, 122.5 (q, J=33.8 Hz), 122.1, 122.0 (q,

J=270.8 Hz, CF₃), 119.0, 118.7 (q, J=6.0 Hz), 77.2

(q, J=26.0 Hz, CCF₃), 38.0 (CH₂S), 23.9 (Me); IR

(KBr, cm⁻¹): 3382 (broad), 1701, 1663, 1595, 1522,

1398, 1353, 1323, 1178, 1155, 1046, 840; Anal.

C₁₉H₁₅F₆N₃O₅S (C, H, N).

632

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

5.1.30. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-2-[({4-

[(2-chloroacetyl)amino]phenyl}sulfanyl)methyl]-3,3,3-

triﬂuoro-2-hydroxypropanamide (25)

(0.31 g, 1.0 mmol) and 5-amino-2-cyanobenzotriﬂuoride

(0.19 g, 1.0 mmol) by the procedure used for compound

24. Puriﬁcation by ﬂash column chromatography on

silica gel (CHCl₃–MeOH, 19:1) yielded 95 mg (20%) of

the desired compound as a yellow solid: m.p. 172–

The title compound was prepared from compound 23

(0.33 g, 0.9 mmol) and 5-amino-2-nitrobenzotriﬂuoride

(0.21 g, 1.0 mmol) by the same procedure used for

compound 24. Puriﬁcation by ﬂash column chromatog-

raphy on silica gel (CHCl₃–MeOH, 19:1) yielded 70 mg

(14%) of the desired compound as pale-yellow powder:

1

174 °C; H-NMR (300 MHz, DMSO-d₆): l 10.84 (s,

1H, NH), 8.26 (d, J=1.6 Hz, 1H, ArH), 8.22 (s, 1H,

OH), 8.13 (dd, J=1.8, 8.6 Hz, 1H, ArH), 8.04 (d,

J=8.6 Hz, 1H, ArH), 8.01–7.97 (m, 2H, ArH), 7.61–

7.57 (m, 2H, ArH), 4.00 (d, J=13.7 Hz, 1H, CHH_a),

3.57 (d, J=13.7 Hz, 1H, CHH_b); ¹³C-NMR (75 MHz,

DMSO-d₆): l 166.3, 145.1, 144.6, 141.9, 136.2, 131.3 (q,

J=27.0 Hz), 128.4, 124.3 (q, J=205.5 Hz, CF₃), 122.3

(q, J=270.0 Hz, CF₃), 123.5, 123.3, 117.7 (q, J=4.5

Hz), 115.5, 102.9, 77.2 (q, J=26.6 Hz, CCF₃), 35.4

(CH₂S); IR (KBr, cm⁻¹) 3357 (broad), 2236, 1709,

1616, 1589, 1530, 1434, 1345, 1329, 1281, 1186, 1137,

1052, 855, 744; Anal. C₁₈H₁₁F₆N₃O₄S (C, H, N).

1

m.p. 119–120 °C; H-NMR (300 MHz, DMSO-d₆): l

10.79 (s, 1H, NH), 10.22 (s, 1H, NH), 8.33 (d, J=2.0

Hz, 1H, ArH), 8.17 (dd, J=2.1, 9.0 Hz, 1H, ArH), 8.12

(d, J=9.0 Hz, 1H, ArH), 8.02 (s, 1H, OH), 7.33–7.30

(m, 2H, ArH), 7.40–7.36 (m, 2H, ArH), 4.18 (s, 2H,

CH₂Cl), 3.72 (d, J=13.3 Hz, 1H, CHH_a), 3.39 (d,

J=13.3 Hz, 1H, CHH_b); ¹³C-NMR (75 MHz, DMSO-

d₆): l 166.3, 164.5, 142.2, 142.0, 137.6, 131.6, 128.3,

127.1, 123.9 (q, J=289.5 Hz, CF₃), 123.7, 122.0 (q,

J=274.5 Hz, CF₃), 122.5 (q, J=33.0 Hz), 119.5, 118.8

(q, J=6.0 Hz), 77.3 (q, J=25.5 Hz, CCF₃), 43.4

(CH₂Cl), 37.8 (CH₂S); IR (KBr, cm⁻¹): 3372 (broad),

1701, 1595, 1530, 1405, 1324, 1255, 1186, 1155, 1046,

843, 547; Anal. C₁₉H₁₄ClF₆N₃O₅S (C, H, N).

5.1.33. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-3,3,3-

triﬂuoro-2-hydroxy-2-{[(4-nitrophenyl)sulfanyl]methyl}-

propanamide (28)

The title compound was prepared from compound 21

(0.31 g, 1.0 mmol) and 4-nitro-3-(triﬂuoromethyl) ani-

line (0.21 g, 1.0 mmol) by the same procedure used for

compound 24,. Puriﬁcation by ﬂash column chromato-

graphy on silica gel (CH₂Cl₂–EtOAc, 19:1) yielded 0.11

g (20%) of the desired compound as a yellow solid: m.p.

5.1.31. N-[4-nitro-3-(triﬂuoromethyl)phenyl]-2-[({4-[(2-

chloroacetyl)amino]phenyl}sulfonyl)methyl]-3,3,3-

triﬂuoro-2-hydroxypropanamide (26)

A solution of meta-chloro-perbenzoic acid (0.20 g,

1.1 mmol) in CH₂Cl₂(5 ml) was added dropwise to a

solution of compound 25 (0.20 g, 0.4 mmol) in CH₂Cl₂

(10 ml) at room temperature. The reaction mixture was

stirred for 1 h. After the end of the reaction was

established by TLC, the mixture was diluted with

CH₂Cl₂(50 ml), washed successively with saturated

Na₂SO₃solution (3×30 ml), saturated Na₂HCO₃solu-

tion (30 ml), brine (30 ml), ﬁltered through Celite, and

evaporated completely in vacuo to afford 50 mg (25%)

of the desired compound as a pale yellow solid: m.p.

1

143–144 °C; H-NMR (300 MHz, DMSO-d₆): l 10.89

(s, 1H, NH), 8.29 (d, J=1.9 Hz, 1H, ArH), 8.23 (s, 1H,

OH), 8.18 (dd, J=2.0, 9.0 Hz, 1H, ArH), 8.13 (d,

J=9.0 Hz, 1H, ArH), 8.02–7.97 (m, 2H, ArH), 7.61–

7.56 (m, 2H, ArH), 4.00 (d, J=13.6 Hz, 1H, CHH_a),

3.59 (d, J=13.6 Hz, 1H, CHH_b); ¹³C-NMR (75 MHz,

DMSO-d₆): l 166.3, 145.1, 144.7, 142.3, 141.9, 128.3,

127.1, 124.3 (q, J=222.8 Hz, CF₃), 123.9, 123.6, 121.9

(q, J=271.5 Hz, CF₃), 118.7 (q, J=6.0 Hz), 77.2 (q,

J=27.0 Hz, CCF₃), 35.4 (CH₂S); IR (KBr, cm⁻¹):

3482, 3352, 1710, 1617, 1599, 1543, 1520, 1346, 1323,

1279, 1181, 1044, 847, 744; Anal. C₁₇H₁₁F₆N₃O₆S (C,

H, N).

1

160–162 °C; H-NMR (300 MHz, DMSO-d₆): l 10.80

(s, 1H, NH), 10.64 (s, 1H, NH), 8.43 (s, 1H, OH), 8.39

(d, J=1.8 Hz, 1H, ArH), 8.23 (dd, J=2.0, 9.0 Hz, 1H,

ArH), 8.17 (d, J=9.0 Hz, 1H, ArH), 7.83–7.80 (m, 2H,

ArH), 7.71–7.67 (m, 2H, ArH), 4.25 (s, 2H, CH₂Cl),

4.17 (d, J=14.6 Hz, 1H, CHH_a), 3.90 (d, J=14.6 Hz,

1H, CHH_b); ¹³C-NMR (75 MHz, DMSO-d₆): l 165.3,

165.1, 143.5, 142.2, 142.2, 133.7, 129.7, 127.2, 123.8,

122.5 (q, J=32.2 Hz), 122.0 (q, J=272.3 Hz, CF₃),

118.8 (q, J=6.0 Hz), 118.7, 75.5 (q, J=28.0 Hz, CCF₃),

56.7 (CH₂SO₂), 43.4 (CH₂Cl); IR (KBr, cm⁻¹): 3378

(broad), 1716, 1594, 1530, 1405, 1323, 1255, 1184, 1163,

1047, 844, 546; Anal. C₁₉H₁₄ClF₆N₃O₇S (C, H, N).

5.2. Biological studies

5.2.1. Preparation of cytosolic androgen receptor

Male Sprague–Dawley rats (Harlan Sprague–Daw-

ley, Indianapolis, IN), weighing ca. 250 g were castrated

24 h prior to the removal of prostates. Ventral prostates

were surgically removed and immersed immediately in

ice-cold homogenisation buffer consisting of 10 mM

Tris, 1.5 mM disodium EDTA, 0.25 M sucrose, 10 mM

sodium molybdate, and 1 mM PMSF adjusted to pH

7.4 [20]. The prostate tissue (about 0.4 g per rat) was

minced, weighed, and homogenized (Model PRO 200

homogeniser, Pro Scientiﬁc, Monroe, CT) with 1

5.1.32. N-[4-cyano-3-(triﬂuoromethyl)phenyl]-3,3,3-

triﬂuoro-2-hydroxy-2-{[(4-nitrophenyl)sulfanyl]methyl}-

propanamide (27)

The title compound was prepared from compound 21

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

633

ml of the homogenisation buffer per 500 mg of prostate

tissue. The homogenate was then centrifuged at 114 000

g for 1 h at 0 °C in an ultracentrifuge (Model L8-M,

Beckman Instruments Inc., Palo Alto, CA) [21]. The

supernatant (cytosol) containing AR protein was re-

moved and stored at −80 °C until use.

librium dissociation constant (K_i). The K_iof each ligand

was calculated using the following equation:

K_i=(IC₅₀×K_d)/(L+K_d)

where K_dwas the equilibrium dissociation constant of

3

³H-MIB, L was the concentration of H-MIB in the

incubate, and IC₅₀was as deﬁned above.

5.2.2. AR competiti6e binding afﬁnity assay

AR binding afﬁnities of the synthesized ligands were

determined by competitive binding in the presence of

the high afﬁnity AR ligand, ³H-MIB. AR binding

studies were performed by incubating increasing con-

centrations (10⁻²–10⁴nM) of each ligand with cytosol

5.2.4. Transcriptional acti6ation assay

To examine the AR agonistic activity of synthesized

compounds, AR-mediated transcriptional activation as-

says were performed in transfected CV-1 cells (Ameri-

can Type Culture Collection, Rockville, MD) as

previously described [7] with the exception of certain

modiﬁcations. One day before transfection, CV-1 cells

were seeded into DMEM supplemented with 10% FBS

at a density of 2×10⁵cells/well in 12-well tissue culture

plates. For the following steps, DMEM without phenol

red was used. Transient transfection of plated cells was

carried out in serum-free medium using Lipofectamine

according to the manufacturer’s instructions. Cells in

each well were transfected with 50 ng of a human AR

expression construct (pCMVhAR; generously provided

by Dr Donald J. Tindall, Mayo Clinic and Mayo

Foundation, Rochester, MN), 1 mg of a luciferase re-

porter construct (pMMTV-Luc; generously provided by

Dr Ronald Evans at The Salk Institute, San Diego,

CA), and 1 mg of a control b-galactosidase expression

construct (pSV-b-galactosidase; Promega Corporation,

Madison, WI) using 6 ml of Lipofectamine. After 10 h

of transfection, cells were washed once with DMEM,

and recovered in fresh DMEM supplemented with 0.2%

FBS for 10–20 h. After recovery, the cells were treated

with various concentrations of testing compound, 1 nM

DHT, or vehicle for 48 h. All drugs were initially

dissolved in 100% ethanol, and then serially diluted to

the desired concentration using DMEM containing

0.2% FBS. The volumes of ethanol in ﬁnal solutions

were less than 0.5%. The ﬁnal concentrations of testing

compound were 1, 10, 100, and 500 nM. The drug

containing solutions were replaced with freshly pre-

pared ones at 24 h during the incubation. Following the

treatment, the cells were washed twice with 1×PBS,

and lysed with 200 ml well⁻¹of 1×Reporter Lysis

Buffer (Promega Corporation, Madison, WI) at room

temperature for 30 min. Cell lysates were then collected

in Eppendorf tubes, vortex brieﬂy, and centrifuge at

12 000 g for 2 min. Fifty microliter of supernatant from

each well was used for measurement of b-galactosidase

activity using a 96 well plate reader (Titertek Multiskan

MCC/340, Labsystems Inc., Franklin, MA) set at 414

nm. A separate 100 ml of supernatant from each well

was used for measurement of luciferase activity (Luci-

ferase Assay System, Promega Corporation, Madison,

WI) using an automated luminometer (Model Au-

toLumat LB953, Wallac Inc., Gaithersberg, MD).

3

and a saturating concentration of H-MIB (1 nM) at

4 °C for 18 h. In preliminary experiments, the equi-

librium dissociation constant (Kd) of MIB was deter-

mined under the same procedure by incubating

increasing concentrations of ³H-MIB (0.01–10 nM)

with cytosol. We found that the minimum concentra-

3

tion of H-MIB required to saturate AR sites in the

cytosol preparation was 1 nM. The incubates also

contained 1000 nM triamcinolone acetonide to prevent

interaction of MIB with progesterone receptors [22].

For the determination of non-speciﬁc binding, separate

experiments were conducted by adding 1000 nM unla-

beled MIB to the incubate. Separation of bound and

free radioactivity at the end of incubation were

achieved by the hydroxyapatite (HAP) method, as des-

cribed previously [23], and 0.8 ml of the ethanolic

supernant was added to 5 ml of scintillation cocktail.

Radioactivity was counted in a Beckman LS6500 liquid

scintillation counter (Beckman Instruments Inc., Irvine,

CA).

5.2.3. Data analysis of AR binding afﬁnity

For competitive binding experiments, speciﬁc binding

3

was calculated by subtracting the binding of H-MIB

observed in the presence of excess unlabeled MIB (non-

3

speciﬁc binding) from the binding of H-MIB observed

in the absence of unlabeled MIB (total binding). Com-

petitive displacement curves were constructed for each

ligand with percent speciﬁc binding (speciﬁc binding of

³H-MIB at a particular ligand concentration expressed

as a percentage of the speciﬁc binding of ³H-MIB in the

absence of ligand) on the vertical axis and ligand

concentration on the horizontal axis. The ligand con-

centration that reduced the percentage of speciﬁc bin-

ding by 50% (IC₅₀) was determined by computer-ﬁtting

the data for each ligand to the following equation:

B=B_o[1−C/(IC₅₀+C)]

where B was the speciﬁc binding of ³H-MIB in the

presence of a particular concentration of ligand, B_owas

the speciﬁc binding of ³H-MIB in the absence of ligand,

and C was the ligand concentration. Binding afﬁnities

of the ligands were then compared using their equi-

634

Y. He et al. / European Journal of Medicinal Chemistry 37 (2002) 619–634

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[7] J.T. Dalton, A. Mukherjee, Z. Zhu, L. Kirkovsky, D.D. Miller,

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Acknowledgements

This research was supported by grants to J.T.D. from

the National Institute of Child Health and Human

Development (c R15 HD-35329), the National Cancer

Institute (c1 R29 CA68090), and the St. Francis of

Assisi Foundation of Memphis.

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