Arylisothiocyanato selective androgen receptor modulators (SARMs) for prostate cancer

Article abstract of DOI:10.1016/j.bmc.2006.06.019

A new series of androgen receptor targeted agents (ARTA) was prepared and tested in androgen-dependent and -independent prostate cancer cell lines. These agents were bicalutamide analogs with isothiocyanato substituted B-rings. Also, the linker sulfone of R-bicalutamide was maintained or replaced with several alternative linkages including ether, amine, N-methylamine, thioether, and methylene (in this case the product was a racemic mixture) functional groups at the X-position. To expand the structure-activity relationship (SAR) of these arylisothiocyanato AR ligands, B-ring halogenated arylisothiocyanato ligands were also prepared and tested. The arylisothiocyanato AR ligands showed strong binding affinities to AR ranging from 0.6 to 54 nM. Among them, thioether and ether linkages demonstrated high binding affinities (0.6 and 4.6 nM, respectively) and selective cell growth inhibition (approximately 3- to 6-fold) for LNCaP, an androgen-dependent prostate cancer cell line, when compared to the androgen independent prostate cell lines (DU145, PC-3, and PPC-1) and a bladder cell line (TSU-Pr1). However, the ligands were inactive (IC₅₀>100 mM) in a normal monkey kidney cell line (CV-1) that was used as the control for non-specific toxicity.

Full text of DOI:10.1016/j.bmc.2006.06.019

Bioorganic & Medicinal Chemistry 14 (2006) 6525–6538
Arylisothiocyanato selective androgen receptor
modulators (SARMs) for prostate cancer
Dong Jin Hwang,a Jun Yang,b Huiping Xu,b Igor M. Rakov,a
Michael L. Mohler,a James T. Daltonb and Duane D. Millera,*
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center,
Memphis, TN 38163, USA
bDivision of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
Received 3 January 2006; revised 6 June 2006; accepted 8 June 2006
Available online 7 July 2006
Abstract—A new series of androgen receptor targeted agents (ARTA) was prepared and tested in androgen-dependent and -inde-
pendent prostate cancer cell lines. These agents were bicalutamide analogs with isothiocyanato substituted B-rings. Also, the linker
sulfone of R-bicalutamide was maintained or replaced with several alternative linkages including ether, amine, N-methylamine, thio-
ether, and methylene (in this case the product was a racemic mixture) functional groups at the X-position. To expand the structure–
activity relationship (SAR) of these arylisothiocyanato AR ligands, B-ring halogenated arylisothiocyanato ligands were also pre-
pared and tested. The arylisothiocyanato AR ligands showed strong binding affinities to AR ranging from 0.6 to 54 nM. Among
them, thioether and ether linkages demonstrated high binding affinities (0.6 and 4.6 nM, respectively) and selective cell growth inhi-
bition (approximately 3- to 6-fold) for LNCaP, an androgen-dependent prostate cancer cell line, when compared to the androgen
independent prostate cell lines (DU145, PC-3, and PPC-1) and a bladder cell line (TSU-Pr1). However, the ligands were inactive
(IC50>100 mM) in a normal monkey kidney cell line (CV-1) that was used as the control for non-specific toxicity.
ꢀ 2006 Published by Elsevier Ltd.
1. Introduction
non-steroidal AR inhibitors that are effective even in
hormone refractory prostate cancer.5 Until now, the
blockage of the pharmacological effects of androgens
by non-steroidal AR ligands was achieved through the
use of nilutamide, flutamide, and bicalutamide in
advanced CaP patients.6–8
Prostate cancer (CaP) is a major health hazard for men
living in United States1 and developed countries.
Approximately 90% of prostate tumors are androgen-
dependent.2 Androgens, primarily testosterone (T) and
dihydrotestosterone (DHT), bind to the androgen recep-
tor (AR) and support the development and maintenance
of normal prostate tissue but more importantly can have
a pathologic role in prostate cancer patients by promot-
ing the growth of the tumor.3 Antiandrogen therapy to
block these pathological effects of endogenous andro-
gens has been the golden standard for unrespectable
prostate cancer (disease spread beyond prostate). How-
ever, despite the high initial response rate to hormonal
therapy, almost all patients relapse and become hor-
mone refractory.4 Our effort is directed at finding potent
All current AR antagonists are reversible ligands that
interact with the AR via non-covalent bonds such as
hydrophobic, electrostatic, and hydrogen bond interac-
tions. Unlike a reversible ligand, an irreversible ligand
can bind to the receptor via a covalent bond that perma-
nently attaches the ligand to the receptor. Our laborato-
ry has a long interest in the discovery of novel anticancer
drugs that act via the AR. Recently, we reported that an
isothiocyanate substituent at the para-position of the
B-ring of thioether and sulfonyl-linked derivatives of
bicalutamide bound tightly and may form a covalent
bond to the AR.9 In this study, we expand our previous
efforts10,11 regarding non-steroidal AR ligands to in-
clude bicalutamide analogs with the potential to bind
irreversibly to the AR. In pursuit of this objective, chiral
arylisothiocyanato derivatives were synthesized and
tested in the prostate cancer cell lines LNCaP, DU145,
Keywords: Prostate cancer (CaP); Selective androgen receptor modu-
lators (SARMs); Isothiocyanate; Bicalutamide; Androgen receptor
target agents (ARTA); Non-steroidal; Irreversible; Androgen receptor
(AR).
*
Corresponding author. Tel.: +1 901 448 6026; fax: +1 901 448
3446; e-mail: dmiller@utmem.edu
0968-0896/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.06.019

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D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
PC-3, PPC-1, and a bladder cancer cell line (TSU-Pr1)
as well as a normal monkey kidney cell line (CV-1). In
this report, our previous ligands S-23 and R-35 (Table
1)10,11 were tested on CaP cell lines as selective androgen
receptor modulators (SARMs) and diversified at the X-
position and halogenated on the B-ring. This work was
carried out to find suitable ligands, which are selective
AR-dependent CaP inhibitors with strong AR binding
affinity and no cytotoxicity on normal cell lines, and
to address the key issues in enhancing AR binding affin-
ities of bicalutamide derivatives and their use in prostate
cancer therapy. First, we focused on which linker was
most suitable for AR potential prostate cancer therapy.
With this objective, we prepared and tested several ana-
logs with ether, methylene, thioether, sulfone, amino,
and methylamino groups as outlined in general structure
S/R-1 (Fig. 1). The synthetic methods for these linker
variants are shown in Schemes 1–5. Our second goal
was the determination of the pharmacological effects
of halogenation at the 2- or 3-position of the 4-isothio-
cyanato substituted B-ring derivatives as outlined in
general structure S-2 (Fig. 1). Kim et al.12 reported the
para B-ring substituent is a major structural determi-
nant of in vivo disposition and activity of non-steroidal
AR agonists (a.k.a. selective androgen receptor modula-
tors or SARMs). SARMs are a new set of non-steroidal
AR ligands with potential therapeutic activity similar to
testosterone. They may be used orally with androgenic
and anabolic activity and have fewer side effects than
testosterone. Chen et al.13 reported that halogenation
at the 2- or 3-position of the B-ring increased their bind-
ing affinities to AR. To further probe the SAR of 2- or
3-halogenated SARMs, we tested the AR binding affin-
ity and cancer cell growth inhibition of the 2- or 3-halo-
genated isothiocyanato ligands. Our last goal was the
characterization of SAR A-ring para-position regarding
AR affinity and cell growth inhibition. We compared the
NO2 or CN group on the A-ring of our analogs. The
in vitro prostate cancer directed cell growth inhibitory
properties of the chiral and racemic AR ligands in
Figure 1 were characterized. The AR binding affinity
and the concentration of the ligand that inhibited cell
growth by 50% (IC50) were determined by radioligand
competitive binding assay and sulforhodamine
B
(SRB) assay, respectively. The AR binding affinities
and cell growth inhibitory effect of these compounds
(S and/or R)-23 ꢀ (S and/or R)-37 with diverse substitu-
ents on the aromatic A and B rings are reported.
2. Chemistry
A total of 17 arylisothiocyanato derivatives (S and/or
R)-23 to (S and/or R)-37 (Table 1) with several linkages
(X = O, CH2, NH, NCH3, S, and SO2) were prepared as
discussed in Schemes 1–4, out of which 14 novel SARMs
were newly synthesized. The target arylisothiocyanato
derivatives were mainly synthesized by two known
Table 1. Structures and pharmacological activities of arylisothiocyanato SARMs against prostate cancer cell lines (LNCaP, DU145, PC-3, and
PPC-1), bladder cancer cell line (TSU-Pr1), and normal monkey kidney cell line (CV-1) (IC50 in lM)
O
*
R1
F3C
A
N
X
NCS
B
H
HO CH3
R2
R3
R1
X
R2 R3
IC50 (lM)
Ki (nM) on hAR
LNCaP
DU145
PC-3
PPC-1
TSU-Pr1
CV-1
DHT
R-bicalutamidea
S-23 NO2
R-23b NO2
0.43 0.01
11.0 2.0
4.6 0.310
45.9 8.8
17.4 4.9
24.9 1.7
17.8 12.8
13.9 2.9
66.3 14.9
30.6 11.0
52.0 15.3
23.8 2.0
20.3 1.4
20.6 3.8
21.5 0.7
13.8 0.4
N.D.
74.7 12.2
74.7 5.9
28.7 1.7
76.8 8.2
50.8 6.0
76.0 6.9
72.1 3.3
55.0 6.1
47.9 3.9
29.4 2.9
23.4 1.3
29.4 2.9
N.D.
71.6 6.7
39.3 1.8
74.7 5.9
22.3 0.1
43.2 14.4
34.3 5.1
56.9 5.1
43.5 7.4
53.0 10.7
31.8 1.3
22.2 1.2
20.7 0.9
26.5 1.3
21.9 1.2
N.D.
8.3 1.6 38.1 9.2
57.1 2.3 >100
O
O
O
O
O
O
O
O
O
H
H
H
H
H
H
H
F
H
H
H
F
59.1 3.3
15.7 1.4
69.5 4.8
47.2 2.9
62.1 12.2
41.2 8.2
48.6 2.5
39.5 1.4
26.8 1.6
21.1 2.7
32.4 2.1
27.8 1.8
N.D.
28.7 1.7 44.3 1.1 36.2 2.8
S-24
S-25
S-26
S-27
S-28
S-29
S-30
31c
CN
54.8 3.1 N.D.
45.3 6.1 N.D.
54.8 3.9 N.D.
39.8 7.3 N.D.
51.1 6.7 N.D.
49.0 1.0 N.D.
26.9 1.1 N.D.
23.8 3.0 N.D.
38.3 1.0 N.D.
14.7 0.3 N.D.
8.1 0.3
12.6 1.1
6.3 1.0
NO2
CN
F
NO2
CN
Cl
Cl
H
H
H
H
H
H
H
H
H
H
11.9 2.7
8.6 0.9
NO2
NO2
34.6 6.5
31.1 1.8
31.8 1.8
21.4 7.0
41.2 5.9
54.8 3.0
0.6 0.111
430 13
Cl
H
H
H
H
H
H
H
H
NO2 CH2
NO2 NH
NO2 NCH3
S-32
S-33
R-34
R-35
S-35b
R-36
R-37
NO2
CN
CN
S
S
S
N.D.
88.3 8.2
>100
N.D.
65.6 4.2 >100
>100 >100
N.D.
10.6 7.1
64.0 5.9
29.4 1.6
42.7 5.3
91.1 6.3
>100
86.9 4.1
>100
NO2 SO2
CN SO2
59.3 1.9
81.5 28.7
58.9 4.1
70.1 7.6
43.9 3.1
69.4 1.8
43.3 3.7 N.D.
59.2 6.7 >100
14.4 4.4
41.0 2.011
N.D., not determined.
a Reversible ligand; tested on above cell lines and AR binding affinity determined.
b Opposite enantiomeric (inactive) isomers synthesized using the chiral auxiliary starting from L-proline (vs D-proline for the other compounds via the
same synthetic procedure as S-23 and R-35).
c Racemic mixture.

D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
6527
O
O
R
A
N
O
B
N C S
R
N
H
X
N
C
S
H
HO CH3
HO CH3
F3C
X1
X2
F3C
X = CH2*, O, S, SO2, NH, NCH3
R = NO2, CN
R = NO2, CN
X1, X2 = H, F, Cl
*: racemic mixture
S -2
S/R -1
Figure 1. General structures of putative irreversible SARMs.
O
Route B
O
R
N
Br
HO CH3
R
N
H
O
NHt-Boc
a
H
HO CH3
F3C
F3C
X1
X2
R-3, R = NO2
R-4, R = CN
9
5 , X = X = H
1
2
HO
NHt-Boc
6, X =H, X = F
1
2
7, X =F, X = H
1
2
X1 X2
8, X =Cl, X = H
1
2
10
S-9
R = NO , X =X =H from 5
2 1 2
S-10 R = NO , X =H, X =F from 6
2
1
2
a
Route A
b
S-11 R = CN, X =X =H from 5
1
2
S-12 R = CN, X =H, X =F from 6
1
2
HO
NCS
S-13 R = NO , X =F, X =H from 7
2
1
2
21, X = X = H
1
2
S-14 R = NO , X =Cl, X =H from 8
2
1
2
22, X =H, X = Cl
X1
X2
1
2
O
O
R
N
O
NCS
R
N
O
NH2
H
H
HO CH3
HO CH3
F3C
X1
X2
F3C
X1
X2
c
10
S-23
R = NO , X =X =H (Route A and B)
2
1
2
S-15 R = NO , X =X =H from S-9
2
1
2
S-24 R = CN, X =X =H (Route B from S-17)
S-16 R = NO , X =H, X =F from S-10
1
2
2
1
2
S-17 R = CN, X =X =H from S-11
1
2
S-25 R = NO , X =H, X =F (Route B from S-16)
2
1
2
S-18 R = CN, X =H, X =F from S-12
1
2
S-26 R = CN, X =H, X =F (Route B from S-18)
1
2
S-19 R = NO , X =F, X =H from S-13
2
1
2
S-27 R = NO , X =H, X =Cl (Route A from 22)
2
1
2
S-20 R = NO , X =Cl, X =H from S-14
2 1 2
S-28 R = CN, X =H, X =Cl (Route A from 22)
1
2
S-29 R = NO , X =F, X =H (Route B from S-19)
2
1
2
S-30 R = NO , X =Cl, X =H (Route B from S-20)
2
1
2
Scheme 1. Reagents and conditions: (a) i—K2CO3, acetone, reflux, ii—substituted phenols (5, 6, 7, 8, 21 or 22), K2CO3, methylethylketone, reflux;
(b) EtOH, AcCl, 0 ꢁC; (c) CSCl2, aq NaHCO3, CHCl3.
O
NO2
a
HO
NO2
O
HO CH3
38
39
O
c
b
HO
NH2
HO CH3
40
O
O
O2N
F3C
N
H
NH2
d
O2N
F3C
N
H
NCS
HO CH3
HO CH3
42
31
Scheme 2. Reagents and conditions: (a) i—Me3SiCN, ZnI2 (cat.), CH2Cl2, ii—HCl, AcOH, reflux; (b) H2, Pd/C, MeOH, 30 psi; (c) i—SOCl2, THF,
DMF (cat.), ii—4-nitro-3-(trifluoromethyl)aniline (41), Et3N; (d) CSCl2, aq NaHCO3, CHCl3.
methods (Leclerc14 and Marhefka10). Scheme 1 de-
scribes the synthesis of ether-linked analogs. The pro-
tected arylamines (S-9 to S-14) were converted to free
arylamines (S-15 to S-20) by deprotection under acidic
conditions using AcCl and absolute EtOH.15 The result-
ing free amines (S-15 to S-20) were then converted to

6528
D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
O
R
a or a'
O2N
F3C
N
Br
HO CH3
HN
NHt-Boc
H
+
R-3
47 R = H
48 R = CH3
O
R
N
O
R
N
O2N
F3C
N
NCS
b
O2N
F3C
N
NHt-Boc
H
HO CH3
H
HO CH3
S-32, R = H
S-33, R = CH3
S-49 R = H
S-50 R = CH3
Scheme 3. Reagents and conditions: (a) Et3N, THF, reflux, for S-49 from 47. (a0) NaH, THF, 0 ꢁC, for S-50 from 48; (b) i—EtOH, AcCl, 0 ꢁC,
ii—CSCl2, aq NaHCO3, CHCl3.
O
O
R
N
S
NH2
R
N
Br
HO CH3
a
H
+
HS
NH2
HO CH3
H
F3C
F3C
R-52, R = NO2
R-5311, R =CN
51
R-3, R = NO2
R-4, R = CN
O
c
R
N
H
S
HO CH3
NCS
F3C
R-34, R = NO2
R-3511, R =CN
O
O
O
O
b
R
N
S
NCS
R
N
S
NH2
H
H
HO CH3
O
HO CH3
O
F3C
F3C
R-36, R = NO2
R-3711, R = CN
R-54, R = NO2
R-5530, R =CN
Scheme 4. Reagents and conditions: (a) NaH, THF, 0 ꢁC; (b) CSCl2, aq NaHCO3, CHCl3; (c) 3-chloroperoxy-benzoic acid, CH2Cl2.
Derivatives having CH2, NH, N-CH3, S, and SO2 link-
a
HO
NHt-Boc
HO
NH2
ages (31, S-32, S-33, and R-34 ꢀ R-37) were prepared
from corresponding precursors (42, S-49, S-50, and R-
52 ꢀ R-55) as shown in Schemes 2–4. The synthetic
route outlined in Scheme 2 provided the optimized
method for the methylene linkage (–CH2–) 31, which
is a racemic mixture.
X1
X2
X1
X2
5810 X1 = X2 = H
510 X1 = X2 = H from 58
6 X1 = H, X2 = F from 59
7 X1 = F, X2 = H from 60
8 X1 = Cl, X2 = H from 61
59 X1 = H, X2 = F from 56
60 X1 = F, X2 = H from 57
61 X1 = Cl, X2 = H
Nitrophenyl butanone 38 was prepared by refluxing
1-bromomethyl-4-nitrobenzene and pentane-2,4-dione
with K2CO3 in ethanolic medium.17 Trimethylsilyl
cyanide (Me3SiCN) with catalytic zinc iodide (ZnI2)
provided the cyanohydrin intermediates,18 which on
subsequent hydrolysis under acidic conditions afforded
the racemic butyric acid 39 in 91% yield. The nitro
group of compound 3916 was converted to correspond-
ing amine 40 by catalytic reduction. The aniline precur-
sor 42 was prepared by coupling of hydroxybutyric acid
40 with 4-nitro-3-trifluoromethylaniline (41). In order to
prevent acid chloride of 40 from undergoing self con-
densation, the nitroaniline 41 was rapidly added to the
solution under ice-cold conditions, followed by triethyl-
amine (Et3N) to gain desired anilino product 42 in rea-
sonable yield of 68%.
Scheme 5. Reagents and conditions: (a) Et3N, di-tert-butyl dicarbon-
ate, THF.
arylisothiocyanato derivatives (S-23 to S-26, S-29, and
S-30) by reaction with thiophosgene in heterogeneous
(chloroform/water) co-solvents under basic condition.14
Arylisothiocyanato S-23 was obtained by a straightfor-
ward procedure adopted by Marhefka et al.10 through
the epoxide intermediate, by using 4-isothiocyanatophe-
nol (21). With 3-chloro-4-isothiocyanatophenol (22),
compounds S-27 and S-28 were prepared by a similar pro-
cedure from the amidobromide R-3 and R-4. The propi-
onamide derivatives R-3 and R-4 were prepared from R-
3-bromo-2-hydroxy-2-methylpropionic acid and 4-nitro
or 4-cyano-3-trifluoromethylaniline as reported.11

D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
6529
The S-32 and S-33 were synthesized from N-protected
S-49 and S-50, respectively (Scheme 3) by deprotection
under acidic condition to produce the corresponding
aniline precursors (not isolated), and then to target ami-
no-linked S-32, S-33 in 80% and 67% overall yields. The
coupling of R-bromoanilide R-3 with N-protected
diamine 47/48 was carried out under the following two
basic conditions. The first method employed sodium
hydride (NaH) in THF for N-methyl di-amino com-
pound 48 to undergo coupling with R-bromoanilide R-
3 in 78% yield. However, the protected diamino deriva-
tive 47 under sodium hydride condition failed to yield
the desired product S-49. A solution of 47 and R-3 in
THF, on refluxing with Et3N, afforded amino-linked
precursor S-49 in 63% yield. The amino-protected
diamine 4719 was prepared from 4-nitroaniline (43)
through amino-protection in presence of Et3N to yield
(4-nitrophenyl)-carbamic acid tert-butyl ester (44),20
which was reduced catalytically in EtOAc at 30 psi to
give the amino-protected diamine 47. The same
reduction of nitro group of methyl-(4-nitrophenyl)amine
(45) gave N-methylbenzene-1,4-diamine (46)21 quantita-
tively, and this was used for the preparation of
compound 48 by adding di-tert-butyl dicarbonate (Boc
anhydride).
form a covalent bond with the AR and thus irreversibly
block its pathological role in prostate cancer.5,24
The SARMs were tested for AR binding affinities as well
as cell growth inhibition on prostate cancer cell lines
(LNCaP, DU145, PC-3, and PPC-1), bladder cancer cell
line (TSU-Pr1), and a normal renal cell line (CV-1),
which was included as the control for non-specific cell
growth inhibition (Table 1). We tested the pharmacolog-
ical activities of the isothiocyanato AR ligands against
prostate/bladder cancer cell lines and normal cell line
(IC50 in lM) as shown in Table 1. Generally, the isomers
that are analogs to R-bicalutamide (R- for X = S and
SO2, S- for X = O) have higher AR binding affinity as
described in our previous report.11 The cell growth inhi-
bition results for the ether-linked S-23 showed an IC50
value on LNCaP of S-23 (18.8 lM) which is 1.5 times
higher than R-23 (28.8 lM). In this study, the in vitro
cell proliferation data also indicated that S-23 was more
selective in inhibiting cell growth by 3- to 4-fold in the
AR-dependent cell line LNCaP compared to the AR-in-
dependent prostate/bladder cancer cell lines (DU145,
PC-3, PPC-1, and TSU-Pr1). This suggests that the S-
isomer of ether-linked isothiocyanates generally demon-
strated higher binding affinity and better selectivity for
the AR-positive prostate tumors. The A-ring substitu-
tion of the isothiocyanato ligands also has an effect on
the AR binding affinity. The presence of nitro group
in para-position of S-23 demonstrated slightly stronger
AR binding affinity than the corresponding cyano ana-
log as in case of S-24. The methylene (CH2) linked 31
was a racemic mixture that inhibited the proliferation
of various prostate cancer cell lines (i.e. had no selectiv-
ity for AR-dependent versus independent cell growth
inhibition) despite intermediate levels of the AR binding
affinity (Ki). Amino-linked ligands (S-32 and S-33) also
showed mild inhibitions of CaP cell lines
(13.8 ꢀ 32.4 lM) with intermediate Ki values (21.4 nM
for S-32, 41.2 nM for S-33). The N-methyl ligand S-33
showed more activity against LNCaP (13.8 lM) and
TSU-Pr1 (14.7 lM) cell lines but the binding affinity
to AR was less than S-32 (21.5 lM for LNCaP,
38.3 lM for TSU-Pr1). We saw a trend of S-33 being
more active in cancer cell lines but binding less to AR
than S-32. Thus, N-methyl ligand S-32 lowered AR
binding but not anticancer activity. The thioether-
linked, nitro group containing R-34, ad a mild binding
affinity, but was not tested in the CaP cell lines. Howev-
er, the cyano A-ring variant, R-35, had selective andro-
gen-dependent (10.6 lM on LNCaP) inhibition and no
significant cell growth inhibition on the normal cell line
(>100 lM on CV-1). Compound R-35 also had the
strongest AR binding affinity (0.6 nM) as previously
reported.11 The thioether-linked enantiomeric isomer,
S-35, bound very weakly to AR (Ki = 430 nM), showed
sixfold less activity against the androgen-dependent
prostate cancer cell line (LNCaP), and no cell growth
inhibition on normal cells (>100 lM, CV-1). However,
ether-linked enantiomeric isomer, R-23 (28.8 lM for
LNCaP), had similar LNCaP inhibition as its isomer
S-23 (18.8 lM) despite much weaker AR binding affinity
(141 vs 4.6 nM). The sulfone ligands R-36 and R-37 re-
tained the selectivity for AR-dependent cell growth inhi-
The S- and SO2-linked ligands (R-34 to R-37) were pre-
pared from corresponding amines (R-52 to R-55) by
Leclerc’s method14 as shown in Scheme 4. The thio-
amine R-52 and R-53, precursors for the R-36 and R-
37 respectively, were oxidized to produce sulfone R-54
and R-55 following a literature procedure.11,17 The R-
bromoanilide R-3 and R-4 were synthesized from D-pro-
line as the chiral auxiliary.11 We also tested the other
isomeric arylisothiocyanato compounds, R-23 and S-
35, synthesized from L-proline in order to optimize the
ligand for inhibition of prostate cancer cell lines.
The selective amino-protection for aminophenol (5–8),
which used the synthesis of ether linkage (X = O) S-9
to S-14, is depicted in Scheme 5. The reduction of the
nitrophenol22 was carried out using Pd/C with H2, fol-
lowed by amino-protection with Boc anhydride, to ob-
tain the desired products 5, 6, 7, and 8.
3. Results and discussion
A novel series of arylisothiocyanato 1,3-disubstituted-2-
hydroxy-2-methylpropionamide AR ligands were pre-
pared (Table 1) with several variations of the linkers
(O, CH2, NH, N-CH3, S, and SO2; S/R–23, S-24, 31,
S-32, S-33, R-34, S/R-35, R-36, and R-37) and modifica-
tions in position and degree of halogenation (2-, or 3-po-
sitions with F/Cl substitution on B-ring; S-25 to S-30).
Irreversibly binding ligands have been proven to be very
useful pharmacological tools in a number of systems.23
The reaction of isothiocyanato compounds may provide
a unique mechanism for suppressing cell proliferation of
prostate cancer cell growth. In this study, the isothiocy-
anate groups are capable of reacting with nucleophilic
side chains that contain thiol (cysteine), hydroxyl (thre-
onine or serine), or amino (lysine) functionalities to

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D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
bition in LNCaP cells. R-36 showed selectivity for AR-
dependent (29.4 lM for LNCaP), greater than AR-inde-
pendent prostate carcinomas (43.9 lM for PPC-1). And,
R-37 showed 42.7 lM for AR-dependent LNCaP cell
line, greater than 69.3 lM for the AR-independent
PPC-1 cell line. Comparison of the irreversibly binding
isothiocyanate analog R-37 with R-bicalutamide re-
vealed that there was no significant cell growth inhibi-
tion on normal cell lines (CV-1; >100 lM for R-37 vs
38.1 lM for R-bicalutamide), despite weak inhibition
on bladder cancer cell lines (TSU-Pr1; 59.2 lM for R-
37 vs 8.3 lM for R-bicalutamide) and its weak binding
affinity to the AR (Ki; 41.0 nM for R-37 vs 11.0 nM
for R-bicalutamide). Earlier studies disclosed that
dihalogenation of the B-ring increased the binding affin-
ity of 4-halogenated compounds on AR.13 The 2- or 3-
halogenated 4-isothiocyanato (NCS) compounds S-25
to S-30 were synthesized. Unfortunately the addition
of halogens on the B-ring did not consistently enhance
activity. The 3-fluoro B-ring S-25 and the 3-chloro B-
ring S-27 had approximate 2- to 3-fold lower binding
affinities (12.6 and 11.9 nM, respectively) than the
non-halogenated variant S-23. As a result, only relative-
ly modest to no LNCaP-selective cell growth inhibition
was observed (13.9 and 30.6 lM on LNCaP, respective-
ly, vs greater than or equal to 34.3 and 39.8 lM on other
CaP cell lines). The cyano A-ring variants S-26 and S-
28, and the 2-halogenated, NO2 group-containing S-29
and S-30 compounds had no selectivity on AR-depen-
dent line (LNCaP) in spite of stronger Ki values in some
cases (6.3 and 8.6 nM for S-26 and S-28, respectively).
In conclusion, ether-linked S-23 and thioether-linked
R-35 arylisothiocyanato linkage ligands are effective on
androgen-dependent prostate cancer cells (LNCaP)
and have no effect on androgen-independent cell lines
(DU145, PC-3, PPC-1, and TSU-Pr1). Also, S-23 and
R-35 demonstrated high binding affinities to hAR (4.6
and 0.6 nM, respectively). The current effort was an at-
tempt to improve our binding affinity and selectivity in
AR-dependent prostate cancer cell growth inhibition
by the synthesis and testing of 2- and 3-halogenated
ether linkages and the variants at X-position. We found
in this study that CaP activities of SARMs are depen-
dent on their linkers and substituents on B-ring. Unfor-
tunately, the 2-, or 3- halogenation on the 4-
isothiocyanato B-ring derivatives reported here did not
produce an improved CaP cell growth inhibition profile.
Replacing the NO2 group with CN on the A-ring of
isothiocyanato SARMs produced several compounds
that had significant AR binding affinity and/or favorable
(AR-dependent) CaP cell growth inhibition profiles. De-
spite our SAR exploration around S-23 and R-35, these
compounds still remain the best compounds to date for
irreversible AR-targeted therapies for hormone-resistant
prostate cancer as exhibited by their strong selectivity
for CaP inhibition. We do not see a good correlation be-
tween AR binding and CaP inhibition (e.g. S-23 vs R-
23). In order to provide a rationale for the observations,
we will continue to investigate the requirements for cell
growth inhibition in prostate cancer cell lines with com-
pounds structurally related to S-23 and R-35, and we
plan to examine selected compounds for in vivo activity
in prostate cancer animal models.
4. Experimental
4.1. Biological tests
4.1.1. Cell culture. Four prostate cancer cell lines
(LNCaP, DU145, PC-3, and PPC-1), a bladder cancer
cell line (TSU-Pr1), and a normal monkey kidney cell
line (CV-1) were obtained from ATCC. Prostate cancer
cells and bladder cancer cell were grown in RPMI-1640
medium and CV-1 cells were grown in Dulbecco’s mod-
ified Eagle’s medium (DMEM) containing 2 mM L-glu-
tamine supplemented with 10% fetal bovine serum
(FBS) and maintained in a 5% CO2/95% air humidified
atmosphere at 37 ꢁC, respectively.
4.1.2. Assay for cell growth inhibition (sulforhodamine B
assay). Viable cells were quantified using the sulforhoda-
mine B (SRB) assay. Cells were seeded for one day prior to
the addition of different drugs at a range of concentrations
(0–100 lM). After 96 h incubation, cells were fixed by the
addition of 50 lL of 50% cold trichloroacetic acid and
incubated at 4 ꢁC for 1 h. The plate was washed five times
with tap water and allowed to air-dry. The cells were then
stained with 0.4% SRB dissolved in 1% acetic acid for
20 min at room temperature. Unbound SRB was washed
away with five washes of 1% acetic acid. The plate was al-
lowed to air-dry, bound SRB stain representing surviving
cells was dissolved in 200 lL of Tris base (10 mM). The
optical density was determined at 540 nm on a microplate
reader (Dynex Technologies, Chantilly, VA). Mean val-
ues were obtained from at least five wells per treatment
condition. Plots of percent inhibition of cell growth versus
drug concentration were constructed, and the concentra-
tion that inhibited cell growth by 50% relative to the
untreated control (IC50) was determined by non-linear
least squares regression using WinNonlin software
(version 4.1).
4.1.3. Assay for androgen receptor binding affinity. The AR
binding affinity of tested compounds was determined by a
radioligand competitive binding assay with cytosolic AR
prepared from rat ventral prostates. The AR preparation
was incubated with 1 nM of [3H]mibolerone (MIB), 1 lM
triamcinolone acetonide at 4 ꢁC for 18 h, in the presence
of increasing concentrations of the test compound (10 dif-
ferent concentrations ranging from 10ꢁ2 to 104 nM) or in
the absence of the compound. After incubation, the pro-
tein-bound radioactivity was separated from free radioac-
tivity by hydroxyapatite (HAP) precipitation. The bound
radioactivity was then extracted from HAP by ethanol,
and counted in a Beckman LS6000 liquid scintillation
counter (Beckman Instruments Inc., Palo Alto, CA).
The specific binding of [3H]MIB at each concentration
of the compound of interest (B) was obtained after
subtracting the non-specific binding of [3H]MIB, and ex-
pressed as the percentage of the specific binding in the
absence of the compound of interest (B0). The concentra-
tion of compound that reduced the specific binding of
[3H]MIB by 50% (IC50) was determined by computer-
fitting the data to the following equation using Win-
Nonlin (Pharsight Corporation, Mountain View, CA):
B = B0 · [1 ꢁ C/(IC50 + C)], where C is the concentration
of the compound of interest. The apparent equilibrium

D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
6531
binding constant (Ki) of the compound of interest was cal-
culatedbyKi = Kd · IC50/(Kd + L), whereKd isthe equilib-
rium dissociation constant of [3H]MIB (Kd = 0.19 nM;
determined in preliminary experiments) and L is the con-
centration of [3H]MIB used in the experiment (L = 1 nM).
mediate R-3 or R-4 with protected phenols (5, 6, 7 or 8
in Scheme 5; yield 57 to 77% for ether linkages). Acetyl
chloride (10 mL) was added dropwise to a solution of
the protected amine (2 mmol) in EtOH (30 mL) under
ice-cold conditions. The reaction mixture was raised to
room temperature and stirred for 3 h. The completion
of the reaction was determined by the disappearance
of starting material (compounds S-9 to S-14), as moni-
tored by TLC. Under reduced pressure, the solvent
was removed to give crude white or brown foam of ani-
line S-15 to S-20. A solution of the amines in CHCl3
(30 mL) and NaHCO3 (841 mg, 10 mmol) in 30 mL
H2O was cooled in an ice-bath and stirred vigorously.
Thiophosgene (0.31 mL, 4 mmol) was slowly added to
the solution and the mixture was stirred at room temper-
ature for 5 h. The reaction mixture in CHCl3 was
washed with water (50 mL 3·), dried with MgSO4 and
purified using flash column chromatography using either
CH2Cl2/hexane or EtOAc/hexane as an eluent to obtain
S-23 to S-30, R-23 and S-35 as a yellowish solid. Yield
38–85%. The protected 4-amino phenol 5, 6, 7, and 8
were prepared from corresponding 58, 59,25 60,26 and
6127 by using Boc anhydride with Et3N in THF solvent
(Scheme 5).28 Amino compound 59 was obtained from
nitro phenol 56 by reduction with H2 on Pd/C on the
Parr hydrogenation apparatus (mp 136.5–137.5 ꢁC;
lit.29 mp 138–139 ꢁC).
4.2. Synthesis and physical data for SARMs
Melting points were determined using a Thomas-Hoover
capillary apparatus and are uncorrected. The NMR spec-
tra were recorded on Brucker ARX 300 (300 MHz for 1H
NMR and 75 Hz for 13C NMR) or Varian Inova FT spec-
trometers (500 MHz for 1H NMR and 125 MHz for 13
C
NMR). The chemical shifts are reported in ppm downfield
relative to tetramethylsilane. Spectral data were consis-
tent with assigned structures. Elemental analyses were
performed by Atlantic Microlab Inc., Norcross, GA,
and found values were within 0.4% of the theoretical val-
ues. Analytical thin-layer chromatography was carried
out on pre-coated silica gel (Merck Kieselgel 60 F254 layer
thickness 0.25 mm). Flash column chromatography was
carried out using Silica gel (230–400 mesh, Merck).
4.3. General procedure for ether-linked compounds S-23
to S-30
The target isothiocyanate compounds S-23 to S-30 were
prepared by two synthetic methods (Route A and/or B)
as outlined below in Scheme 1.
4.4. (2-Fluoro-4-hydroxyphenyl)carbamic acid tert-butyl
ester (6)
4.3.1. Route A. Compounds S-23, S-27, and S-28 were
synthesized by coupling of chiral intermediate R-3 or R-
4 with appropriate phenols (21 or 22). The synthesis of
target compounds was achieved by a previously reported
method.10 Briefly, the mixture of bromide R-3 or R-4
(0.81 mmol) and potassium carbonate (2.4 mmol) in ace-
tone medium (30 mL) was heated to reflux for 30 min.
After complete conversion of starting bromide R-3 or
R-4 to corresponding epoxides as monitored by TLC,
the solvent was evaporated under reduced pressure to give
a yellowish residue. To the residue, suspended in methyl-
ethylketone or isopropanol (30 mL), substituted phenol
(0.81 mmol) and an additional amount of potassium car-
bonate (1.6 mmol) were added. The reaction mixture was
refluxed for 2–4 h. The solvent in the mixture was re-
moved under reduced pressure. The residue was dissolved
in EtOAc (50 mL), washed with brine, aqueous ammoni-
um chloride, and water. The ethyl acetate extract was
dried by MgSO4, concentrated and purified by flash col-
umn chromatography using EtOAc/hexane as an eluent
to give target compounds. The overall reaction was car-
ried out in one-pot two-step process as shown in Scheme
1 and the yields ranged from 23% to 48%.
Yield 27% (from 59); brown oil; Rf 10.17 (EtOAc/hexane
1:5); ESI-MS; m/z: 226.0 [MꢁH]ꢁ; H NMR (CDCl3) d
7.54 (t, J = 8.1 Hz, 1H, OH), 7.01 (br s, 1H, NH), 6.59–
6.46 (m, 3H, ArH), 1.55 (s, 9H, CH3); 13C NMR
(CDCl3) d 154.4 (d, JC–F = 155.1 Hz), 153.0 (d, JC–F
=
10.9 Hz), 152.2, 123.3, 117.7 (d, JC–F = 11.5 Hz), 110.8
(d, JC–F = 3.2 Hz), 103.0 (d, JC–F = 22.4 Hz), 80.7,
27.8; Anal. Calcd for C11H14FNO3: C, 58.14; H, 6.21;
N, 6.16. Found: C, 58.19; H, 6.26; N, 6.28.
4.5. (3-Fluoro-4-hydroxyphenyl)carbamic acid tert-butyl
ester (7)
Yield 56% (from 60); white solid; Rf 0.43 (EtOAc/hexane
1
1:2); mp 122–123 ꢁC; ESI-MS; m/z: 226.0 [MꢁH]ꢁ; H
NMR (CDCl3) d 7.31 (d, JH–F = 12.0 Hz, 1H, ArH),
6.91–6.80 (m, 2H, ArH), 6.47 (br s, 1H, OH), 5.54 (br s,
1H, NH), 1.52 (s, 9H, CH2); 13C NMR: (CDCl3) d
151.9, 150.4 (d, JC–F = 254.3 Hz), 139.0 (d, JC–F
=
14.3 Hz), 130.9, 116.7, 114.9, 107.3 (d, JC–F = 23.8 Hz),
80.3, 27.8; Anal. Calcd for C11H14FNO3: C, 58.14; H,
6.21; N, 6.16. Found: C, 58.20; H, 6.15; N, 6.15.
4.3.2. Route B. Compounds S-23 to S-26 and S-29 to S-
30 were synthesized by coupling of chiral intermediate
R-3 or R-4 with appropriate protected phenols (5, 6, 7
or 8) to get S-9 to S-14. Removal of the Boc group
under acidic condition afforded free amine intermediates
S-15 to S-20. Treating with thiophosgene under basic
condition afforded the target compounds S-23 to S-26
and S-29 to S-30. Compounds S-9 to S-14 were pre-
pared by synthetic method A by coupling of chiral inter-
4.6. (3-Chloro-4-hydroxyphenyl)carbamic acid tert-butyl
ester (8)
Yield 65% (from 61); white solid; Rf 0.48 (EtOAc/hexane
1:2); mp 77–79 ꢁC; ESI-MS; m/z: 241.9 [MꢁH]ꢁ; 1H
NMR (CDCl3) d 7.55 (s, 1H, ArH), 7.03 (dd, J = 8.9,
2.4 Hz, 1H, ArH), 6.92 (d, J = 8.9 Hz, 1H, ArH), 6.38
(br s, 1H, NH), 5.44 (br s, 1H, OH), 1.51 (s, 9H,
CH3); 13C NMR (CDCl3) d 151.5, 147.8, 132.2, 120.3,

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D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
1
120.1, 119.8, 116.5, 81.1, 28.7; Anal. Calcd for
C11H14ClNO3: C, 54.22; H, 5.79; N, 5.75. Found: C,
54.15; H, 5.74; N, 5.72.
mp: 56–59 ꢁC; ESI-MS; m/z: 496.2 [MꢁH]ꢁ; H NMR
(CDCl3) d 9.20 (br s, 1H, NH), 8.13 (d, J = 2.1 Hz,
1H, ArH), 7.98 (dd, J = 8.7, 2.1 Hz, 1H, ArH), 7.89
(br s, 1H, NH), 7.82 (d, J = 8.7 Hz, 1H, ArH), 7.68
(m, 2H, ArH), 6.53 (m, 1H, ArH), 4.42 (d, J = 9.0 Hz,
1H, CH2), 3.96 (d, J = 9.0 Hz, 1H, CH2), 3.63 (br s,
1H, OH), 1.58 (s, 3H, CH3), 1.52 (s, 9H, CH3); 13C
NMR (CDCl3) d 172.1, 153.1 (d, JC–F = 10.1 Hz),
152.3 (d, JC–F = 242.6 Hz), 152.2, 140.9, 135.3, 133.7
(q, JC–F = 32.6 Hz), 121.6 (q, JC–F = 272.4 Hz), 121.3,
4.7. R-{4-[2-Hydroxy-2-(4-nitro-3-trifluoromethylphe-
nylcarbamoyl)propoxy]phenyl} carbamic acid tert-butyl
ester (R-9)
Prepared by Route B; yield 75% (from S-3 with 5, S-3 is
opposite isomer of R-3 synthesized from L-proline);
yellowish solid; Rf 0.22 (EtOAc/hexane 1:2); ESI-MS;
121.2, 120.5 (d, JC–F = 10.7 Hz), 116.8 (q, JC–F
=
1
m/z: 498.0 [MꢁH]ꢁ; mp 145–147 ꢁC; H NMR (CDCl3)
4.9 Hz), 114.9, 109.8 (d, JC–F = 3.2 Hz), 104.1 (d,
JC–F = 2.0 Hz), 102.3 (d, JC–F = 23.0 Hz), 80.5, 75.2,
72.6, 27.7, 22.4; Anal. Calcd for C23H23F4N3O5: C,
55.53; H, 4.68; N, 8.45. Found: C, 55.26; H, 4.70; N, 8.29.
d 9.20 (br s, 1H, NH), 8.10 (s, 1H, ArH), 8.01 (m, 2H,
ArH), 7.27 (d, J = 8.6 Hz, 2H, ArH), 6.84 (d,
J = 8.6 Hz, 2H, ArH), 6.40 (br s, 1H, NH), 4.42 (d,
J = 9.0 Hz, 1H, CH2), 3.95 (d, J = 9.0 Hz, 1H, CH2),
3.54 (br s, 1H, OH), 1.58 (s, 3H, CH3), 1.51 (s, 9H,
CH3); Anal. Calcd for C22H24F3N3O7: C, 52.91; H,
4.84; N, 8.41. Found: C, 52.77; H, 4.91; N, 8.42.
4.11. S-{3-Fluoro-4-[2-hydroxy-2-(4-nitro-3-trifluoro-
methylphenylcarbamoyl)propoxy]phenyl}carbamic acid
tert-butyl ester (S-13)
4.8. S-{2-Fluoro-4-[2-hydroxy-2-(4-nitro-3-trifluorometh-
ylphenylcarbamoyl)propoxy] phenyl}carbamic acid tert-
butyl ester (S-10)
Prepared by Route B; yield 31% (two steps from R-3
with 7); brown solid; Rf 0.28 (EtOAc/hexane 2:3); mp:
69–72 ꢁC; ESI-MS; m/z: 516.1 [MꢁH]ꢁ; 1H NMR
(CDCl3) d 9.39 (br s, 1H, NH), 8.11 (s, 1H, ArH),
7.94 (m, 2H, ArH), 7.28 (br s, 1H, NH), 6.71–6.77 (m,
3H, ArH), 4.41 (d, J = 9.0 Hz, 1H, CH2), 4.23 (br s,
1H, OH), 3.97 (d, J = 9.0 Hz, 1H, CH2), 1.55 (s, 3H,
CH3), 1.44 (s, 9H, CH3); 13C NMR: (CDCl3) d 172.6,
152.3, 152.2 (d, JC–F = 242.8 Hz), 143.7, 141.4, 140.8
(d, JC–F = 11.2 Hz), 133.2 (d, JC–F = 11.2 Hz), 126.7,
Prepared by Route B; yield 59% (two steps from R-3 with
6); yellowish solid; Rf 0.22 (EtOAc/hexane 1:2); ESI-MS;
1
m/z: 516.0 [MꢁH]ꢁ; mp 54–57 ꢁC; H NMR (CDCl3) d
9.23 (br s, 1H, NH), 8.11 (s, 1H, ArH), 8.03 (m, 2H,
ArH), 7.89 (br s, 1H, NH), 8.68 (m, 2H, ArH), 6.54 (m,
1H, ArH), 4.43 (d, J = 9.0 Hz, 1H, CH2), 3.96 (d,
J = 9.0 Hz, 1H, CH2), 3.59 (br s, 1H, OH), 1.59 (s, 3H,
CH3), 1.53 (s, 9H, CH3); 13C NMR (CDCl3) d 172.1,
153.1 (d, JC–F = 10.2 Hz), 152.3 (d, JC–F = 242.8 Hz),
152.2, 142.7, 140.9, 126.5, 125.0 (q, JC–F = 34.0 Hz),
121.6, 121.3 (q, JC–F = 272.0 Hz), 121.2, 120.5 (d,
JC–F = 10.7 Hz), 117.7 (q, JC–F = 5.9 Hz), 109.8 (d,
JC–F = 3.3 Hz), 102.3 (d, JC–F = 23.0 Hz), 80.5, 75.3,
72.6, 27.6, 22.4; Anal. Calcd for C22H23F4N3O7: C,
51.07; H, 4.48; N, 8.12. Found: C, 51.27; H, 4.67; N, 8.17.
124.9 (q, JC–F = 33.8 Hz), 121.8, 121.2 (q, JC–F
272.0 Hz), 117.9 (q, JC–F = 5.8 Hz), 116.5 (d, JC–F
=
=
1.8 Hz), 113.9, 107.4 (d, JC–F = 23.3 Hz), 75.0, 74.7,
65.2, 27.9, 22.2; Anal. Calcd for C22H23F4N3O7: C,
51.07; H, 4.48; N, 8.12. Found: C, 51.06; H, 4.63; N,
7.99.
4.11.1. S-{3-Chloro-4-[2-hydroxy-2-(4-nitro-3-trifluoro-
methylphenylcarbamoyl)propoxy]phenyl}carbamic acid
tert-butyl ester (S-14). Prepared by Route B; yield 44%
(two steps from R-3 with 8); brown solid; Rf 0.28
(EtOAc/hexane 2:3); mp 71–74 ꢁC; ESI-MS; m/z: 532.0
[MꢁH]ꢁ; 1H NMR (CDCl3) d 9.39 (br s, 1H, NH), 8.12
(s, 1H, ArH), 7.92 (m, 2H, ArH), 7.43 (br s, 1H, NH),
7.06 (m, 1H, ArH), 6.87 (s, 1H, ArH), 6.77 (d,
J = 8.7 Hz, 1H, ArH), 4.34 (d, J = 9.0 Hz, 1H, CH2),
4.21 (br s, 1 H, OH), 3.96 (d, J = 9.0 Hz, 1H, CH2), 1.63
(s, 3H, CH3), 1.47 (s, 9H, CH3); 13C NMR (CDCl3) d
173.3, 153.0, 149.2, 143.4, 141.8, 133.8, 127.2, 125.3 (q,
4.9. S-{4-[2-(4-Cyano-3-trifluoromethylphenylcarba-
moyl)-2-hydroxypropoxy]phenyl} carbamic acid
tert-butyl ester (S-11)
Prepared by Route B; yield 57% (two steps from R-4
with 5); white solid; Rf 0.27 (EtOAc/hexane 2:3); mp:
156–158 ꢁC; ESI-MS; m/z: 478.1 [MꢁH]ꢁ; 1H NMR
(CDCl3) d 9.17 (br s, 1H, NH), 8.10 (s, 1H, ArH),
7.96 (d, J = 8.4 Hz, 1H, ArH), 7.80 (d, J = 8.4 Hz, 1H,
ArH), 7.27 (d, J = 8.1 Hz, 2H, ArH), 6.84 (d,
J = 8.1 Hz, 2H, ArH), 6.43 (br s, 1H, NH), 4.42 (d,
J = 9.0 Hz, 1H, CH2), 3.95 (d, J = 9.0 Hz, 1H, CH2),
3.58 (br s, 1H, OH), 1.57 (s, 3H, CH3), 1.50 (s, 9H,
CH3); Anal. (C23H24F3N3O5 Æ H2O) C, H, N. Anal.
Calcd for C23H24F3N3O5: C, 57.62; H, 5.05; N, 8.76.
Found: C, 57.41; H, 5.10; N, 8.66.
JC–F = 34.5 Hz), 123.8, 122.5, 121.9 (q, JC–F
=
241.9 Hz), 121.2, 118.7 (q, JC–F = 5.9 Hz), 118.6, 115.6,
75.7, 74.7, 66.0, 28.4, 23.0; Anal. Calcd for C22H23ClF3
N3O7: C, 49.88; H, 4.71; N, 7.27. Found: C, 49.96; H,
4.70; N, 7.30.
4.12. S-3-(4-Aminophenoxy)-2-hydroxy-2-methyl-N-(4-
nitro-3-trifluoromethylphenyl) propionamide (S-15)
4.10. S-{4-[2-(4-Cyano-3-trifluoromethylphenylcarba-
moyl)-2-hydroxypropoxy]-2-fluoro phenyl}carbamic acid
tert-butyl ester (S-12)
Prepared from S-9 by Route B; brown solid; Rf 0.29
(CHCl3/MeOH 20:1); mp 138–140 ꢁC; ESI-MS; m/z:
398.0 [MꢁH]ꢁ; 1H NMR (CD3OD)
d 8.38 (d,
J = 1.8 Hz, 1H, ArH), 8.17 (dd, J = 8.7, 1.8 Hz, 1H,
ArH), 8.04 (d, J = 8.7 Hz, 1H, ArH), 7.11 (d,
Prepared by Route B; yield 65% (two steps from R-4
with 6); yellowish solid; Rf 0.12 (EtOAc/hexane 1:2);

D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
6533
J = 9.0 Hz, 2H, ArH), 6,98 (d, J = 9.0 Hz, 2H, ArH),
4.32 (d, J = 9.6 Hz, 1H, CH2), 4.03 (d, J = 9.6 Hz, 1H,
CH2), 1.52 (s, 3H, CH3); 13C NMR (CDCl3) d 172.6,
4.16. S-3-(4-Amino-2-chlorophenoxy)-2-hydroxy-2-meth-
yl-N-(4-nitro-3-trifluoromethyl phenyl)propionamide
(S-20)
150.3, 142.6, 141.1, 140.6, 126.5, 124.6 (q, JC–F
=
33.9 Hz), 121.5, 121.1 (q, JC–F = 272.0 Hz), 117.7 (q,
JC–F = 5.9 Hz), 115.9 (2C), 115.7 (2C), 75.3, 73.0, 22.4;
Anal. Calcd for C17H16F3N3O5: C, 51.13; H, 4.04; N,
14.27. Found: C, 51.35; H, 4.06; N, 14.19.
Prepared from S-14 by Route B; yellowish solid; Rf 0.15
(EtOAc/hexane 1:1); mp 108–110 ꢁC; ESI-MS; m/z:
432.1 [MꢁH]ꢁ; 1H NMR (DMSO-d6) d 10.71 (br s, 1H,
NH), 10.27 (br s, 2H, NH2), 8.57 (s, 1H, ArH), 8.34 (d,
J = 7.8 Hz, 1H, ArH), 8.18 (d, J = 7.8 Hz, 1H, ArH),
7.42 (s, 1H, ArH), 7.30 (br s, 2H, ArH), 4.25 (d,
J = 9.0 Hz, 1H, CH2), 4.19 (d, J = 9.0 Hz, 1H, CH2),
1.48 (s, 3H, CH3); 13C NMR (DMSO-d6) d 174.2, 153.2,
143.2, 141.6, 127.2, 125.8, 124.3, 123.2, 122.9, 122.2 (q,
JC–F = 32.9 Hz), 122.0 (q, JC–F = 271.4 Hz), 121.8, 118.3
(q, JC–F = 5.8 Hz), 114.9, 75.1, 74.9, 22.6.
4.13. S-3-(4-Aminophenoxy)-N-(4-cyano-3-trifluorometh-
ylphenyl)-2-hydroxy-2-methylpropionamide (S-17)
Prepared from S-11 by Route B; brown solid; Rf 0.24
(EtOAc/hexane 2:1); mp 177–179 ꢁC; ESI-MS; m/z:
378.1 [MꢁH]ꢁ; 1H NMR (DMSO-d6) d 10.54 (br s,
1H, NH), 8.57 (d, J = 1.8 Hz 1H, ArH), 8.32 (dd,
J = 8.7, 1.8 Hz, 1H, ArH), 8.11 (d, J = 8.7 Hz, 1H,
ArH), 6.63 (d, J = 9.0 Hz, 2H, ArH), 6.48 (d,
J = 9.0 Hz, 2H, ArH), 6.17 (br s, 1H, OH), 4.61 (br s,
2H, NH2), 4.08 (d, J = 9.6 Hz, 1H, CH2), 3.85 (d,
J = 9.6 Hz, 1H, CH2), 1.18 (s, 3H, CH3); 13C NMR
(DMSO-d6) d 174.7, 149.8, 143.2, 142.7, 136.2, 131.6
(q, JC–F = 33.8 Hz), 122.6, 122.4 (q, JC–F = 264.1 Hz),
117.3 (q, JC–F = 4.9 Hz), 115.8, 115.6 (2C), 114.8 (2C),
101.8, 75.0, 74.5, 22.9; Anal. Calcd for C18H16F3N3O3
0.2H2O: C, 56.46; H, 4.32; N, 10.97. Found: C, 56.35;
H, 4.26; N, 11.09.
4.17. S-2-Hydroxy-3-(4-isothiocyanatophenoxy)-2-meth-
yl-N-(4-nitro-3-trifluoromethyl phenyl)propionamide
(S-23)10
Prepared by Route B; (from S-9 through S-15, S-9
synthesized starting from D-proline); ESI-MS; m/z:
439.8 [MꢁH]ꢁ; Anal. Calcd for C18H14F3N3O5S: C,
48.98; H, 3.20; N, 9.52. Found: C, 49.10; H, 3.31; N,
9.44.
4.18. R-2-Hydroxy-3-(4-isothiocyanatophenoxy)-2-
methyl-N-(4-nitro-3-trifluoromethyl phenyl)propionamide
(R-23)
4.14. S-3-(4-Amino-3-fluorophenoxy)-N-(4-cyano-3-tri-
fluoromethylphenyl)-2-hydroxy-2-methylpropionamide
(S-18)
Prepared by Route B (R-23 is opposite isomer of S-23);
yield 81% (two steps from R-9 through R-15, R-9 syn-
thesized starting from L-proline); yellowish solid; Rf
0.21 (EtOAc/hexane 3:2); ESI-MS; m/z: 439.8 [MꢁH]ꢁ;
1H NMR (CDCl3) d 9.14 (br s, 1H, NH), 8.04 (s, 1H,
ArH), 7.92 (m, 2H, ArH), 7.08 (d, J = 8.4 Hz, 2H,
ArH), 6,80 (d, J = 8.4 Hz, 2H, ArH), 4.40 (d,
J = 9.0 Hz, 1H, CH2), 3.93 (d, J = 9.0 Hz, 1H, CH2),
3.39 (br s, 1H, OH), 1.53 (s, 3H, CH3); 13C NMR
(DMSO-d6) d 172.7, 156.8, 143.5, 141.7, 135.2, 127.2
(2C), 125.4 (q, JC–F = 34.1 Hz), 125.3, 122.4, 122.6 (q,
JC–F = 272.0 Hz), 118.5 (q, JC–F = 5.8 Hz), 116.0 (2C),
76.0, 73.0, 23.1; Anal. Calcd for C18H14F3N3O5S: C,
48.98; H, 3.20; N, 9.52. Found: C, 48.71; H, 3.25; N,
9.57.
Prepared from S-12 by Route B; brown solid; Rf 0.33
(EtOAc/hexane 1:1); mp 179–181 ꢁC; ESI-MS; m/z:
395.9 [MꢁH]ꢁ; 1H NMR (DMSO-d6) d 10.55 (br s,
1H, NH), 8.56 (d, J = 1.8 Hz, 1H, ArH), 8.30 (dd,
J = 8.7, 1.8 Hz, 1H, ArH), 8.10 (d, J = 8.7 Hz, 1H,
ArH), 6.68 (m, 2H, ArH), 6.50 (dd, J = 8.7, 2.1 Hz,
1H, ArH), 6.21 (br s, 1H, OH), 4.63 (br s, 2H, NH2),
4.10 (d, J = 9.6 Hz, 1 H, CH2), 3.86 (d, J = 9.6 Hz, 1H,
CH2), 1.40 (s, 3H, CH3); 13C NMR (DMSO-d6) d 174.6,
150.9 (d, JC–F = 180.9 Hz), 149.4 (d, JC–F = 64.5 Hz),
143.2, 136.2, 131.5 (q, JC–F = 31.4 Hz), 130.1, 122.6,
122.5 (q, JC–F = 271.7 Hz), 117.4, 116.6 (q, JC–F
5.9 Hz), 115.8, 111.1, 103.0, 101.9, 74.9, 74.6, 22.9.
=
4.15. S-3-(4-Amino-2-fluorophenoxy)-2-hydroxy-2-meth-
yl-N-(4-nitro-3-trifluoromethyl phenyl)propionamide
(S-19)
4.19. S-N-(4-Cyano-3-trifluoromethylphenyl)-2-
hydroxy-3-(4-isothiocyanatophenoxy)-2-methyl-
propionamide (S-24)
Prepared from S-13 by Route B; brown solid; Rf 0.17
(EtOAc/hexane 1:1); mp 103–105 ꢁC; ESI-MS; m/z:
416.1 [MꢁH]ꢁ; 1H NMR (DMSO-d6) d 10.71 (br s, 1H,
NH), 10.25 (br s, 2H, NH2), 8.57 (s, 1H, ArH), 8.35 (d,
J = 8.1 Hz, 1H, ArH), 8.19 (d, J = 8.1 Hz, 1H, ArH),
7.35–7.24 (m, 2H, ArH), 7.15 (m, 1H, ArH), 4.31 (d,
J = 9.3 Hz, 1H, CH2), 4.13 (d, J = 9.3 Hz, 1H, CH2),
1.46 (s, 3H, CH3); 13C NMR (DMSO-d6) d 174.3, 151.2
(d, JC–F = 244.9 Hz), 145.8 (d, JC–F = 10.3 Hz), 143.2,
141.7, 127.3, 125.5, 123.1, 122.2 (q, JC–F = 33.0 Hz),
122.1 (q, JC–F = 271.4 Hz), 119.3 (d, JC–F = 3.2 Hz),
118.3 (q, JC–F = 5.9 Hz), 116.3 (d, JC–F = 1.8 Hz), 111.4
(d, JC–F = 21.7 Hz), 75.0, 74.9, 22.8.
Prepared by Route B; yield 84% (two steps from S-11
through S-17); brown solid; Rf 0.46 (EtOAc/hexane
1
1:1); mp: 41–43 ꢁC; ESI-MS; m/z: 420.1 [MꢁH]ꢁ; H
NMR (CDCl3) d 9.14 (br s, 1H, NH), 8.13 (d,
J = 1.8 Hz, 1H, ArH), 7.99 (dd, J = 8.4, 1.8 Hz, 1H,
ArH), 7.84 (d, J = 8.4 Hz, 1H, ArH), 7.20 (d,
J = 9.0 Hz, 2H, ArH), 6.91 (d, J = 9.0 Hz, 2H, ArH),
4.49 (d, J = 9.0 Hz, 1H, CH2), 4.02 (d, J = 9.0 Hz, 1H,
CH2), 3.39 (br s, 1H, OH), 1.62 (s, 3H, CH3); 13C
NMR (CDCl3) d 171.9, 156.0, 140.8, 135.4, 134.2,
126.6, 126.3 (q, JC–F = 33.8 Hz), 124.7, 122.0 (q,
JC–F = 270.2 Hz), 121.3, (2 C), 116.7 (q, JC–F
4.9 Hz), 115.2 (2C), 114.8, 104.1, 75.3, 72.2, 22.4; Anal.
=

6534
D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
Calcd for C19H14F3N3O3S: C, 54.15; H, 3.35; N, 9.97.
Found: C, 54.07; H, 3.44; N, 9.61.
4.23. S-3-(3-Chloro-4-isothiocyanatophenoxy)-N-(4-cya-
no-3-trifluoromethylphenyl)-2-hydroxy-2-methylpropi-
onamide (S-28)
4.20. S-3-(3-Fluoro-4-isothiocyanatophenoxy)-2-hydroxy-
2-methyl-N-(4-nitro-3-trifluoromethylphenyl)propion-
amide (S-25)
Prepared by Route A; yield 48% (two steps from R-4
with 22); white solid; Rf 0.24 (EtOAc/hexane 2:3); mp
50–53 ꢁC; ESI-MS; m/z: 454.4 [MꢁH]ꢁ; 1H NMR
(CDCl3) d 9.21 (br s, 1H, NH), 8.14 (d, J = 1.2 Hz,
1H, ArH), 7.98 (dd, J = 8.7, 1.2 Hz, 1H, ArH), 7.81
(d, J = 8.7 Hz, 1H, ArH), 7.16 (d, J = 9.0 Hz, 1H,
ArH), 6.99 (d, J = 2.7 Hz, 1H, ArH), 6.80 (dd, J = 9.0,
2.7 Hz, 1H, ArH), 4.46 (d, J = 9.0 Hz, 1H, CH2), 4.02
(d, J = 9.0 Hz, 1H, CH2), 3.65 (br s, 1H, OH), 1.61 (s,
3H,CH3); 13CNMR (CDCl3) d 171.9, 156.2, 140.9,
135.7, 135.3, 133.5 (q, JC–F = 32.7 Hz), 132.1, 126.8,
123.1, 121.6 (q, JC–F = 272.4 Hz), 121.3, 116.8 (q,
JC–F = 4.9 Hz), 116.0, 114.9, 113.6, 104.1, 75.2, 72.6,
22.4; Anal. Calcd for C19H13ClF3N3O3S: C, 50.06; H,
2.87; N, 9.22. Found: C, 50.13; H, 3.04; N, 9.08.
Prepared by Route B; yield 85% (two steps from S-10
through S-16); yellowish solid; Rf 0.24 (EtOAc/hexane
2:3); mp 42–45 ꢁC; ESI-MS; m/z: 458.0 [MꢁH]ꢁ; 1H
NMR (CDCl3) d 9.24 (br s, 1H, NH), 8.15 (br s, 1H,
ArH), 8.08–8.00 (m, 2H, ArH), 7.14 (t, J = 8.7 Hz, 1H,
ArH), 6.78–6.68 (m, 2H, ArH), 4.50 (d, J = 9.0 Hz,
1H, CH2), 4.05 (d, J = 9.0 Hz, 1H, CH2), 3.44 (br s,
1H, OH), 1.64 (s, 3H, CH3); 13C NMR (CDCl3) d
174.5, 156.0, 154.1 (q, JC–F = 144.9 Hz), 143.2, 136.2,
131.4 (q, JC–F = 126.9 Hz), 129.4, 122.6, 122.5, 122.4
(q, JC–F = 272.0 Hz), 117.3 (q, JC–F = 5.8 Hz), 115.8 (q,
JC–F = 287.3 Hz), 103.3, 103.0, 102.4 (q, JC–F
=
82.4 Hz), 74.8, 74.7, 22.9; Anal. Calcd for C18H13F4
N3O5S: C, 47.06; H, 2.85; N, 9.15. Found: C, 46.98;
H, 2.87; N, 9.10.
4.24. S-3-(2-Fluoro-4-isothiocyanatophenoxy)-2-hydroxy-
2-methyl-N-(4-nitro-3-trifluoro methylphenyl)propion-
amide (S-29)
4.21. S-N-(4-Cyano-3-trifluoromethylphenyl)-3-(3-fluoro-
4- isothiocyanatophenoxy)-2-hydroxy-2-methylpropion-
amide (S-26)
Prepared by Route B; yield 73% (two steps from S-13
through S-19); yellowish solid; Rf 0.14 (EtOAc/hexane
1:2); mp 51–53 ꢁC; ESI-MS; m/z: 457.9 [MꢁH]ꢁ; 1H
NMR (CDCl3) d 9.19 (br s, 1H, NH), 8.12 (s, 1H,
ArH), 8.01 (m, 2H, ArH), 6.99 (m, 3H, ArH), 4.51 (d,
J = 9.0 Hz, 1H, CH2), 4.07 (d, J = 9.0 Hz, 1H, CH2),
3.60 (br s, 1 H, OH), 1.63 (s, 3H, CH3); 13C NMR:
(CDCl3) d 172.6, 152.5 (d, JC–F = 247.7 Hz), 145.3 (d,
JC–F = 10.9 Hz), 143.6, 141.6, 137.1, 127.4, 126.3,
126.1, 125.5 (q, JC–F = 34.1 Hz), 122.4, 121.9 (q,
JC–F = 272.0 Hz), 118.6 (d, JC–F = 5.9 Hz), 116.6, 114.5
(d, JC–F = 21.3 Hz), 75.9, 74.6, 23.1; Anal. Calcd for
C18H13F4N3O5S: C, 47.95; H, 3.60; N, 8.06. Found: C,
48.08; H, 3.57; N, 8.09.
Prepared by Route B; yield 79% (two steps from S-12
through S-18); white solid; Rf 0.23 (EtOAc/hexane
2:3); mp 43–46 ꢁC; ESI-MS; m/z: 438.1 [MꢁH]ꢁ; 1H
NMR (CDCl3) d 9.17 (br s, 1H, NH), 8.14 (d,
J = 1.2 Hz, 1H, ArH), 7.99 (dd, J = 8.4, 1.2 Hz, 1H,
ArH), 7.82 (d, J = 8.4 Hz, 1H, ArH), 7.15 (t,
J = 8.4 Hz, 1H, ArH), 6.76–6.66 (m, 2H, ArH), 4.47
(d, J = 9.0 Hz, 1H, CH2), 4.02 (d, J = 9.0 Hz, 1H,
CH2), 3.49 (br s, 1H, OH), 1.62 (s, 3H, CH3); 13C
NMR (CDCl3) d 171.8, 158.4 (d, JC–F = 158.4 Hz),
156.9, 140.9, 139.9, 135.5, 135.3, 133.6 (q, JC–F
32.7 Hz), 126.4, 121.4 (q, JC–F = 272.4 Hz), 121.3,
116.8 (q, JC–F = 4.9 Hz), 114.9, 110.3 (d, JC–F
=
=
4.25. S-3-(2-Chloro-4-isothiocyanatophenoxy)-2-hy-
droxy-2-methyl-N-(4-nitro-3-trifluoromethylphenyl)-
propionamide (S-30)
3.4 Hz), 104.1, 103.3 (d, JC–F = 22.2 Hz), 75.2, 72.6,
22.4; Anal. Calcd for C19H13F4N3O3S: C, 51.94; H,
2.98; N, 9.56. Found: C, 51.71; H, 3.13; N, 9.41.
Prepared by Route B; yield 77% (two steps from S-14
through S-20); yellowish solid; Rf 0.14 (EtOAc/hexane
1:2); mp 57–59 ꢁC; ESI-MS; m/z: 473.9 [MꢁH]ꢁ; 1H
NMR (CDCl3) d 9.18 (br s, 1H, NH), 8.13 (s, 1H,
ArH), 8.02 (m, 2H, ArH), 7.29 (d, J = 2.4 Hz, 1H ArH),
7.14 (dd, J = 8.7, 2.4 Hz, 1H ArH), 6.95 (d, J = 8.7 Hz,
1H ArH), 4.49 (d, J = 9.0 Hz, 1H, CH2), 4.09 (d,
J = 9.0 Hz, 1H, CH2), 3.62 (br s, 1H, OH), 1.66 (s, 3H,
CH3); 13C NMR: (CDCl3) d 172.7, 152.5, 143.2, 141.6,
4.22. S-3-(3-Chloro-4-isothiocyanatophenoxy)-2-hy-
droxy-2-methyl-N-(4-nitro-3-trifluoromethylphenyl)-
propionamide (S-27)
Prepared by Route A; yield 38% (two steps from R-3
with 22); yellowish solid; Rf 0.25 (EtOAc/hexane 2:3);
1
mp 44–46 ꢁC; ESI-MS; m/z: 474.0 [MꢁH]ꢁ; H NMR
(CDCl3) d 9.23 (br s, 1H, NH), 8.14 (m, 1H, ArH),
8.01 (m, 2H, ArH), 7.18 (d, J = 9.0 Hz, 1H, ArH),
6.99 (d, J = 2.7 Hz, 1H, ArH), 6.81 (dd, J = 9.0,
2.7 Hz, 1H, ArH), 4.48 (d, J = 9.0 Hz, 1H, CH2),
4.02 (d, J = 9.0 Hz, 1H, CH2), 3.47 (br s, 1H, OH),
1.62 (s, 3H, CH3); 13C NMR (CDCl3) d 171.8,
156.1, 142.8, 140.9, 137.8, 132.2, 128.4, 126.8, 126.5,
137.2, 127.7, 127.3, 126.3, 125.6, 125.4 (q, JC–F
=
34.0 Hz), 124.1, 122.5, 121.9 (q, JC–F = 272.1 Hz), 118.7
(q, JC–F = 5.9 Hz), 115.0, 75.7, 74.3, 23.2; Anal. Calcd
for C18H13ClF3N3O5S Æ 0. 2C4H8O2: C, 45.76; H, 2.98;
N, 8.52. Found: C, 45.97; H, 3.02; N, 8.46.
124.8 (q, JC–F = 34.1 Hz), 121.6, 121.8 (q, JC–F
=
4.26. Preparation for methylene-linked compound 31
272.0 Hz), 117.8 (q, JC–F = 5.9 Hz), 116.0, 113.6,
75.3, 72.5, 22.4; Anal. Calcd for C18H13ClF3N3O5S:
C, 45.44; H, 2.75; N, 8.83. Found: C, 45.63; H,
2.92; N, 8.74.
4.26.1. 2-Hydroxy-2-methyl-4-(4-nitrophenyl)butyric acid
(39). To the solution of the nitro ketone 38 (710 mg,
3.7 mmol) in anhydrous CH2Cl2 (20 mL) were added

D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
6535
dropwise trimethylsilyl cyanide (0.54 mL, 4.0 mmol) and
catalytic amounts of zinc iodide (ZnI2) at ice-bath under
argon atmosphere. The reaction mixture was stirred for
1 h at room temperature. The completion of the reaction
was determined by the disappearance of starting materi-
al (compound 38), as monitored by TLC. After remov-
ing the solvent under reduced pressure, concd
hydrochloric acid (5 mL) and acetic acid (5 mL) were
added to the mixture and heated to reflux for 30 min.
The solution was diluted with water (100 mL), extracted
with EtOAc (2· 30 mL), and washed with satd NaHCO3
solution. The aqueous layer was acidified with 3 N
hydrochloric acid, extracted with Et2O, concentrated
under reduced pressure, and crystallized from CH2Cl2/
hexane to obtain compound 39 (805 mg, 91% yield) as
white solid; mp 110–111 ꢁC; ESI-MS; m/z: 237.9
[MꢁH]ꢁ; 1H NMR (DMSO-d6) d 12.6 (br s, 1H,
CO2H), 8.16 (d, J = 8.7 Hz, 2H ArH), 7.46 (d,
J = 8.7 Hz, 2H ArH), 3.33 (br s, 1H, OH), 2.84 (m,
1H CH2), 2.64 (m, 1H CH2), 1.96 (m, 1H CH2), 1.86
(m, 1H CH2), 1.32 (s, 3H, CH3); 13C NMR: (DMSO-
d6) d 177.2, 150.5, 145.8, 129.5 (2C), 123.4 (2C), 73.0,
41.1, 29.7, 25.8; Anal. Calcd for C11H13NO5: C, 55.23;
H, 5.48; N, 5.86. Found: C, 55.34; H, 5.52; N, 5.87.
272.0 Hz), 118.4 (q, JC–F = 5.9 Hz), 115.7, 77.1, 42.0,
29.4, 27.3; Anal. Calcd for C18H18F3N3O4: C, 54.41;
H, 4.57; N, 10.58. Found: C, 54.44; H, 5.52; N,
10.67.
4.26.4. 2-Hydroxy-4-(4-isothiocyanatophenyl)-2-methyl-
N-(4-nitro-3-trifluoromethyl phenyl)butyramide (31). Pre-
pared by Route B (followed by Method c in Scheme 1);
yellowish oil; Yield 81% (from 42); Rf 0.24 (EtOAc/
hexane 1:2); ESI-MS; m/z: 438.1 [MꢁH]ꢁ; 1H NMR
(CDCl3) d 9.27 (br s, 1H, NH), 8.14 (s, 1H, ArH),
7.98 (m, 2H, ArH), 7.14 (d, J = 8.4 Hz, 2H, ArH),
7.07 (d, J = 8.4 Hz, 2H, ArH), 2.90 (br s, 1H, OH),
2.81 (m, 1H CH2), 2.63 (m, 1H CH2), 2.33 (m, 1H,
CH2), 1.95 (m, 1H, CH2), 1.62 (s, 3H, CH3); 13C
NMR: (CDCl3) d 174.5, 143.1, 141.9, 140,7, 135.1,
129.7 (2C), 129.2, 127.2, 125.9 (2C), 125.4 (q, JC–F
=
33.9 Hz), 122.3, 121.9 (q, JC–F = 272.0 Hz), 118.4 (q,
JC–F = 5.9 Hz), 76.6, 41.9, 29.8, 27.2; Anal. Calcd for
C19H16F3N3O4S: C, 51.93; H, 3.67; N, 9.56. Found: C,
52.04; H, 3.52; N, 9.67.
4.27. Preparation for amine- and N-methylamine-linked
compounds S-32 and S-33
4.26.2.
4-(4-Aminophenyl)-2-hydroxy-2-methylbutyric
The amino linkages of S-32 and S-33 were directly intro-
duced by two synthetic methods (a and a0) from protect-
ed S-49 and S-50 through the deprotection without
isolating corresponding aniline moieties.
acid (40). The solution of compound 39 (635 mg,
2.7 mmol) and Pd/C (64 mg) in MeOH (10 mL) was re-
duced with H2 gas on Parr apparatus under 30 psi pres-
sure for 3 h. The mixture was filtered through Celite 521
and concentrated on reduced pressure, and then, crystal-
lized from CH2Cl2/hexane to obtain compound 40 (quan-
titative yield) as white solid; mp 158–161 ꢁC; ESI-MS; m/
z: 208.4 [MꢁH]ꢁ; 1H NMR (DMSO-d6) d 6.79 (d,
J = 8.1 Hz, 2H, ArH), 6.47 (d, J = 8.1 Hz, 2H, ArH),
3.33 (br s, 1H, OH), 2.55 (m, 1H CH2), 2.30 (m, 1H
CH2), 1.83 (m, 1H CH2), 1.65 (m, 1H CH2), 1.28 (s, 3H,
CH3); 13C NMR: (DMSO-d6) d 177.5, 146.2, 129.1,
128.4 (2C), 114.1 (2C), 73.2, 42.6, 28.8, 25.9.
4.27.1. S-{4-[2-Hydroxy-2-(4-nitro-3-trifluoromethylphe-
nylcarbamoyl)propylamino]phenyl}carbamic acid tert-bu-
tyl ester (S-49). Method (a) in Scheme 3: To a solution
of 47 (300 mg, 1.44 mmol) and R-3 (490 mg, 1.44 mmol)
in THF (30 mL) was added triethylamine (0.37 mL,
2.6 mmol). The mixture was refluxed overnight. After
cooling, the mixture was concentrated under reduced
pressure, poured into EtOAc (30 mL), washed with
water and dried with MgSO4, concentrated under re-
duced pressure, and purified by flash column chroma-
tography to gain compound S-49 (412 mg, 63%) as
yellowish solid. mp 78–81 ꢁC; ESI-MS; m/z: 497.2
[MꢁH]ꢁ; 1H NMR (CDCl3) d 9.26 (br s, 1H, NH),
8.04 (s, 1H, ArH), 7.90 (m, 2H, ArH), 7.07 (d,
J = 7.5 Hz, 2H, ArH), 6.57 (d, J = 7.5 Hz, 2H, ArH),
6.43 (br s, 1H, NH), 4.13 (br s, 2H, OH & NH), 3.70 (d,
J = 12.9 Hz, 1H, CH2), 3.07 (d, J = 12.9 Hz, 1H, CH2),
1.51 (s, 3H, CH3); 13C NMR: (CDCl3) d 174.0, 153.5,
4.26.3. 4-(Aminophenyl)-2-hydroxy-2-methyl-N-(4-nitro-
3-trifluoromethylphenyl) butyramide (42). To the solution
of compound 40 (230 mg, 1.1 mmol) in THF (10 mL)
were added thionyl chloride in dropwise manner
(0.14 mL, 1.8 mmol) and catalytic amounts of DMF at
ice-water bath. The mixture was stirred for 1 h at room
temperature. Nitroaniline 41 (189 mg, 0.9 mmol) was
added, followed by dropwise addition of triethylamine
(0.19 mL, 1.4 mmol), to the solution. The mixture was
stirred for 3 h at room temperature, concentrated under
reduced pressure, poured into EtOAc (20 mL), washed
with satd NaHCO3, brine, dried with MgSO4, and puri-
fied with flash column chromatography using EtOAc/
hexane (1:1) as an eluent to gain compound 42
(246 mg, yield 68%) as brown solid; Rf 0.25 (EtOAc/hex-
ane 1:1); mp 52–55 ꢁC; ESI-MS; m/z: 396.0 [MꢁH]ꢁ; 1H
NMR (CDCl3) d 9.15 (br s, 1H, NH), 8.07 (s, 1H, ArH),
7.99 (m, 2H, ArH), 6.97 (d, J = 8.1 Hz, 2H, ArH), 6.60
(d, J = 8.1 Hz, 2H, ArH), 3.15 (br s, 3H, NH2 & OH),
2.69 (m, 1H, CH2), 2.58 (m, 1H, CH2), 2.32 (m, 1H,
CH2), 1.92 (m, 1H, CH2), 1.56 (s, 3H, CH3); 13C NMR:
(CDCl3) d 174.6, 144.8, 143.2, 141.9, 130.8, 129.4, 127.2,
143.8, 142.3, 141.2, 128.7, 126.3 (2C), 124.6 (q, JC–F
=
33.9 Hz), 121.7 (2C), 121.4, 121.3 (q, JC–F = 272.0 Hz),
117.8 (q, JC–F = 5.9 Hz), 113.8, 79.9, 75.4, 52.3, 27.2,
23.6; Anal. Calcd for C22H25F3N4O6: C, 53.01; H, 5.06;
N, 11.24. Found: C, 52.92; H, 5.16; N, 11.29.
4.27.2. S-(4-{[2-Hydroxy-2-(4-nitro-3-trifluoromethylphe-
nylcarbamoyl)propyl]methylamino}phenyl)carbamic acid
tert-butyl ester (S-50). Method (a0) in Scheme 3: To a
suspension of sodium hydride (1.2 mmol) in THF
(10 mL) was added protected aniline (48, 1.1 mmol) at
ice-bath under argon and raised room temperature.
After 30 min, a solution of R-3 (1.1 mmol) in THF
(10 mL) was added to the suspension at ice-bath. The
mixture was stirred overnight under argon. Water
125.5 (q, JC–F = 34.1 Hz), 122.2, 122.0 (q, JC–F
=

6536
D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
(10 mL) was added carefully, and the mixture was
extracted with EtOAc (3· 20 mL). The combined organ-
ic extracts were dried with MgSO4, concentrated under
reduced pressure and purified with flash column chro-
matography to gain compound S-50 (390 mg, yield
78%) as brown solid; Rf 0.19 (EtOAc/hexane 1:2); mp
84–85 ꢁC; ESI-MS; m/z: 511.0 [MꢁH]ꢁ; 1H NMR
(CDCl3) d 9.36 (br s, 1H, NH), 8.07 (s, 1H, ArH),
7.97 (m, 2H, ArH), 7.19 (d, J = 8.6 Hz, 2H, ArH),
7.82 (d, J = 8.6 Hz, 2H, ArH), 6.48 (br s, 1H, NH),
4.13 (br s, 1H, OH), 3.85 (d, J = 14.6 Hz, 1 H, CH2),
3.33 (d, J = 14.6 Hz, 1H, CH2), 2.82 (s, 3H, NCH3),
1.52 (s, 3H, CH3), 1.50 (s, 9H, CH3); 13C NMR:
(CDCl3) d 174.5, 152.8, 146.4, 142.5, 141.2, 130.3,
126.4 (2C), 124.6, (q, JC–F = 33.9 Hz), 121.6, 121.2 (q,
JC–F = 271.9 Hz), 120.1, 117.8 (q, JC–F = 5.9 Hz), 115.8
(2C), 79.8, 74.9, 62.0, 40.6, 27.8, 24.3; Anal. Calcd for
C23H27F3N4O6: C, 53.90; H, 5.31; N, 10.93. Found: C,
53.78; H, 5.30; N, 10.67.
Synthesis of R-52 and R-53: To a suspension of sodium
hydride (3 mmol) in THF (10 mL) was added 4-amino-
thiophenol (51, 2.7 mmol) at ice-bath under argon and
later raised to room temperature. After 30 min, a solu-
tion of R-3 or R-4 (2.7 mmol) in THF (30 mL) was add-
ed to the suspension at ice-bath. The mixture was stirred
under argon for 20 h. Water (30 mL) was added careful-
ly, and the mixture was extracted with EtOAc (3·
30 mL). The combined organic extracts were dried with
MgSO4, concentrated under reduced pressure, and puri-
fied with flash column chromatography using EtOAc/
hexane (2:3) as an eluent to gain compound R-52 or
R-53 as below.
Synthesis of R-54 and R-55: To a solution of R-52 or R-
53 (1.8 mmol) in methylene chloride (20 mL) was added
3-chloroperoxybenzoic acid (935 mg, 5.4 mmol) at room
temperature. The mixture was stirred for 2 h, and then
washed with sodium sulfite solution and water, dried
with MgSO4, concentrated under reduced pressure,
and purified with flash column chromatography as an
eluent of EtOAc/hexane (2:3) to gain compound R-54
or R-55 as below.
4.27.3. S-2-Hydroxy-3-(4-isothiocyanatophenylamino)-2-
methyl-N-(4-nitro-3-trifluoromethylphenyl)propionamide
(S-32). Prepared by S-49 (Route B in Scheme 1, fol-
lowed by Method (c); yellowish solid; yield 80% (two
steps from S-49); Rf 0.29 (EtOAc/hexane 2:3); mp 63–
4.28.1. R-3-(4-Aminophenylsulfanyl)-2-hydroxy-2-meth-
yl-N-(4-nitro-3-trifluoromethylphenyl)propionamide (R-
52). Yellowish oil; yield 85% (from R-3 with 51); Rf
0.26 (EtOAc/hexane 2:3); ESI-MS; m/z: 414.1 [MꢁH]ꢁ;
1H NMR (CDCl3) d 9.04 (br s, 1H, NH), 7.92 (m, 2H,
ArH), 7.74 (m, 1H, ArH), 7.26 (d, J = 8.4 Hz, 2H,
ArH), 6.60 (d, J = 8.4 Hz, 2H, ArH), 3.78 (d,
J = 14.4 Hz, 1H, CH2), 3.68 (br s, 3H, NH2 & OH),
2.93 (d, J = 14.4 Hz, 1H, CH2), 1.51 (s, 3H, CH3); 13C
NMR: (CDCl3) d 172.9, 146.6, 146.4, 142.4, 141.0,
133.3 (2C), 126.2, 124.5 (q, JC–F = 33.8 Hz), 121.4,
121.3 (q, JC–F = 272.0 Hz), 117.6 (q, JC–F = 5.9 Hz),
115.2 (2C), 74.3, 45.8, 24.1; Anal. Calcd for
C17H16F3N3O4SÆ0.1H2O: C, 48.94; H, 3.91; N, 10.07.
Found: C, 49.02; H, 3.87; N, 10.17.
1
66 ꢁC; ESI-MS; m/z: 439.2 [MꢁH]ꢁ; H NMR (CDCl3)
d 9.20 (br s, 1H, NH), 8.13 (s, 1H, ArH), 8.00 (m, 2H,
ArH), 7.17 (d, J = 8.4 Hz, 2H, ArH), 6.98 (d,
J = 8.4 Hz, 2H, ArH), 4.47 (d, J = 9.0 Hz, 1H, CH2),
4.01 (d, J = 9.0 Hz, 1H, CH2), 3.42 (br s, 1H, OH),
1.62 (s, 3H, CH3); 13C NMR (CDCl3) d 172.7, 156.8,
143.5, 141.6, 135.2, 127.3 (2C), 126.0 (q, JC–F
=
34.0 Hz), 125.8, 122.3, 121.9 (q, JC–F = 272.0 Hz),
118.5 (q, JC–F = 5.9 Hz), 117.7, 116.0 (2C), 76.0, 73.0,
22.9; Anal. Calcd for C18H15F3N4O4S: C, 49.09; H,
3.43; N, 12.72. Found: C, 48.92; H, 3.26; N, 12.53.
4.27.4. S-2-Hydroxy-3-[(4-isothiocyanatophenyl)methyl-
amino]-2-methyl-N-(4-nitro-3-trifluoromethylphenyl)pro-
pionamide (S-33). Prepared by S-50 (Route B in Scheme
1, followed by Method (c); brown oil; yield 67% (two
steps from S-50); Rf 0.28 (EtOAc/hexane 2:3); ESI-MS;
m/z: 453.1 [MꢁH]ꢁ; 1H NMR (CDCl3) d 9.27 (br s, 1H,
NH), 8.09 (s, 1H, ArH), 7.98 (m, 2H, ArH), 7.08 (d,
J = 8.4 Hz, 2H, ArH), 7.81 (d, J = 8.4 Hz, 2 H, ArH),
3.94 (d, J = 15.0 Hz, 1H, CH2), 3.57 (d, J = 15.0 Hz,
1H, CH2), 3.28 (br s, 1H, OH), 2.97 (s, 3H, NCH3),
1.60 (s, 3H, CH3); 13C NMR: (CDCl3) d 173.6, 148.9,
4.28.2. R-3-(4-Aminophenylsulfanyl)-N-(4-cyano-3-triflu-
oromethylphenyl)-2-hydroxy-2-methylpropionamide (R-
53)11. Prepared from R-4 and 51; white solid; ESI-MS;
1
m/z: 394.1 [MꢁH]ꢁ; H NMR (CDCl3) d 9.01 (br s,
1H, NH), 7.93 (d, J = 1.7 Hz, 1H ArH), 7.69 (d,
J = 8.4 Hz, 1H ArH), 7.62 (dd, J = 8.1, 1.7 Hz, 1H
ArH), 7.17 (d, J = 8.4 Hz, 2H, ArH), 6.38 (d,
J = 8.4 Hz, 2H ArH), 3.72 (br s, 3 H, NH2 & OH),
3.65 (d, J = 14.1 Hz, 1H, CH2), 2.92 (d, J = 14.1 Hz,
1H, CH2), 1.47 (s, 3H, CH3); 13C NMR: (CDCl3) d
173.0, 146.4, 141.0, 135.1, 134.0, 133.8, 133.2 (q,
142.7, 141.0, 132.7, 126.5, 126.2 (2C), 124.7 (q, JC–F
=
34.2 Hz), 121.6, 121.2 (q, JC–F = 272.0 Hz), 120.4, 117.8
(q, JC–F = 5.9 Hz), 113.4 (2C), 76.9, 60.6, 39.7, 24.2; Anal.
Calcd for C19H17F3N4O4S: C, 50.22; H, 3.77; N, 12.33.
Found: C, 50.25; H, 3.85; N, 12.18.
JC–F = 32.8 Hz), 130.2, 125.1, 121.4 (q, JC–F
=
275.4 Hz), 116.7 (q, JC–F = 5.0 Hz), 115.1, 103.8, 74.5,
45.9, 25.8; Anal. Calcd for C18H16F3N3O2S: C, 54.68;
H, 4.08; N, 10.63. Found: C, 54.44; H, 4.02; N, 10.60.
4.28. Preparation for thioether- and sulfone-linked
compounds, R-34 to R-37
4.28.3. R-3-(4-Aminobenzenesulfonyl)-2-hydroxy-2-meth-
yl-N-(4-nitro-3-trifluoromethyl phenyl)propionamide (R-
54). Yellowish solid; yield 71% (from R-52); Rf 0.12
(EtOAc/hexane 2:3); mp 90–93 ꢁC; ESI-MS; m/z: 445.9
[MꢁH]ꢁ; 1H NMR (CDCl3) d 9.30 (br s, 1H, NH),
8.03 (s, 1H, ArH), 7.92 (d, J = 8.7 Hz, 1H, ArH), 7.82
(d, J = 8.7 Hz, 1H, ArH), 7.56 (d, J = 8.4 Hz, 2H,
Compounds R-34 to R-37 were prepared by coupling the
propanamide derivatives (R-3, R-4) with 4-aminothi-
ophenol (51) and subsequent conversion of the amine
(R-52 to R-55) to isothiocyanate (R-34 to R-37) by a
procedure similar to Leclerc’s method14 as shown in
Scheme 4.

D. J. Hwang et al. / Bioorg. Med. Chem. 14 (2006) 6525–6538
6537
ArH), 6.54 (d, J = 8.4 Hz, 2H, ArH), 5.35 (br s, 1H,
OH), 4.34 (br s, 2H, NH2), 4.02 (d, J = 14.4 Hz, 1H,
CH2), 3.43 (d, J = 14.4 Hz, 1H, CH2), 1.57 (s, 3H,
CH3); 13C NMR: (CDCl3) d 171.4, 151.7, 142.6, 141.0,
130.4, 129.7 (2C), 126.2, 124.3 (q, JC–F = 34.0 Hz),
4.28.7.
R-2-Hydroxy-3-(4-isothiocyanatobenzenesulfo-
nyl)-2-methyl-N-(4-nitro-3-trifluoromethylphenyl)propion-
amide (R-36). Prepared from R-54; yellowish oil; yield
81%; Rf 0.22 (EtOAc/hexane 1:1); ESI-MS; m/z: 487.9
[MꢁH]ꢁ; 1H NMR (CDCl3) d 9.29 (br s, 1H, NH),
8.03 (s, 1H, ArH), 7.94 (m, 4H, ArH), 7.32 (d,
J = 8.1 Hz, 2H, ArH), 4.99 (br s, 1H, OH), 4.06 (d,
J = 14.4 Hz, 1H, CH2), 3.56 (d, J = 14.4 Hz, 1H, CH2),
1.63 (s, 3H, CH3); 13C NMR: (CDCl3) d 171.2, 142.8,
140.7, 140.0, 137.4, 136.6, 129.1 (2C), 126.4, 125.9
121.8, 121.1 (q, JC–F = 272.0 Hz), 117.8 (q, JC–F
=
5.9 Hz), 113.3 (2C), 73.7, 61.2, 22.4; Anal. Calcd for
C17H16 F3N3O6S: C, 45.64; H, 3.60; N, 9.39. Found:
C, 45.82; H, 3.71; N, 9.40.
4.28.4. R-3-(4-Aminophenylsulfonyl)-N-(4-cyano-3-triflu-
oromethylphenyl)-2-hydroxy-2-methylpropionamide (R-
55).30 White solid; mp 157.5–159 ꢁC; ESI-MS; m/z:
426.0 [MꢁH]ꢁ; 1H NMR (DMSO-d6) d 10.35 (br s, 1H,
NH), 8.44 (d, J = 1.2 Hz, 1H, ArH), 8.21 (dd, J = 8.4,
1.2 Hz, 1H, ArH), 8.06 (d, J = 8.4 Hz, 1H, ArH), 7.43
(d, J = 8.7 Hz, 2H, ArH), 6.52 (d, J = 8.7 Hz, 2H, ArH),
6.29 (br s, 1 H, OH), 6.02 (br s, 2H, NH2), 3.76 (d,
J = 14.4 Hz, 1H, CH2), 3.47 (d, J = 14.4 Hz, 1H, CH2),
1.39 (s, 3H, CH3); 13C NMR: (CDCl3) d 173.8, 153.5,
143.2, 136.0, 131.3 (q, JC–F = 31.5 Hz), 129.8, 125.4,
(2C), 124.6 (q, JC–F = 34.0 Hz), 121.7, 121.2 (q, JC–F =
272.0 Hz), 117.9 (q, JC–F = 5.9 Hz), 74.0, 61.5, 20.5;
Anal. Calcd for C18H14F3N3O6S2: C, 44.17; H, 2.88;
N, 8.59. Found: C, 43.99; H, 2.76; N, 8.53.
4.28.8.
R-N-(4-Cyano-3-trifluoromethylphenyl)-2-hy-
droxy-3-(4-isothiocyanatophenyl sulfonyl)-2-methylpropi-
onamide (R-37).11 Prepared from R-55; white solid; m/z:
467.7 [MꢁH]ꢁ; 1H NMR (DMSO-d6) d 10.33 (br s, 1H,
NH), 8.41 (s, 1H, ArH), 8.19 (d, J = 8.4, 1H, ArH), 8.08
(d, J = 8.4 Hz, 1H, ArH), 7.90 (d, J = 8.4 Hz, 2H, ArH),
7.51 (d, J = 8.4 Hz, 2H, ArH), 6.46 (br s, 1H, OH), 3.98
(d, J = 14.7 Hz, 1H, CH2), 3.74 (d, J = 14.7 Hz, 1H,
CH2), 1.41 (s, 3H, CH3); 13C NMR: (CDCl3) d 173.5,
122.8, 122.2 (q, JC–F = 272.1 Hz), 117.4 (q, JC–F
=
5.0 Hz), 115.8, 112.3, 101.8, 73.2, 63.9, 27.1; Anal. Calcd
for C18H16F3N3O4S: C, 50.58; H, 3.77; N, 9.83. Found: C,
50.40; H, 3.88; N, 9.63.
143.0, 139.0, 136.5, 136.1, 134.8, 131.4 (q, JC–F
=
31.4 Hz), 130.1, 126.2, 122.7, 122.4 (q, JC–F
=
The thioether-linked isothiocyanate compounds (R-34
and R-35) and sulfone-linked compounds (R-36 and
R-37) were prepared as shown in Scheme 4. The synthet-
ic methods of the following compounds R-34, R-35, R-
36, and R-37 were same as method c of Route B in
Scheme 1.
272.0 Hz), 117.4 (q, JC–F = 5.1 Hz), 115.7, 102.0, 73.0,
63.3, 27.2; Anal. Calcd for C19H14F3N3O4S2: C, 48.61;
H, 3.01; N, 8.95. Found: C, 48.71; H, 2.95; N, 8.79.
Acknowledgments
4.28.5. R-2-Hydroxy-3-(4-isothiocyanatophenylsulfanyl)-
2-methyl-N-(4-nitro-3-trifluoromethylphenyl)propionamide
(R-34). Prepared from R-52; brown oil; yield 76%; Rf
0.36 (EtOAc/hexane 2:3); ESI-MS; m/z: 455.9 [MꢁH]ꢁ;
1H NMR (CDCl3) d 9.09 (br s, 1H, NH), 7.98 (br s,
2H, ArH), 7.86 (m, 1H, ArH), 7.37 (d, J = 7.8 Hz, 2H,
ArH), 7.06 (d, J = 7.8 Hz, 2H, ArH), 3.74 (d,
J = 13.8 Hz, 1H, CH2), 3.48 (br s, 1H, OH), 3.22 (d,
J = 13.8 Hz, 1H, CH2), 1.59 (s, 3H, CH3); 13C NMR:
(CDCl3) d 172.3, 142.7, 140.8, 136.4, 133.0, 131.0, 129.9
(2C), 126.5, 125.9 (2C), 124.7 (q, JC–F = 34.0 Hz),
This work was supported by grants from National Insti-
tutes of Health (NIH-5R01DK065227-02 and -03) and
GTx-Inc.
References and notes
1. Greenlee, R. T.; Hill-Harmon, M. B.; Murray, T.; Thun,
M. CA-Cancer J. Clin. 2001, 51, 15.
2. Clement, O. O.; Freeman, C. M.; Hartmann, R. W.;
Handratta, V. D.; Vasaitis, T. S.; Brodie, A. M. H.; Njar,
V. C. O. J. Med. Chem. 2003, 46, 2345.
3. McConnell, J. D. Urol. Clin. North Am. 1991, 18, 1.
4. Carter, B. S.; Carter, H. B.; Isaacs, J. T. Prostate 1990, 16,
187.
121.5, 121.2 (q, JC–F = 272.0 Hz), 117.7 (q, JC–F
=
5.9 Hz), 75.1, 44.1, 25.6; Anal. Calcd for C18H14F3
N4O2S2: C, 47.26; H, 3.08; N, 9.19. Found: C, 47.36;
H, 3.03; N, 9.01.
5. (a) Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.;
Dalton, J. T. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
6201; (b) Reddy, V. K.; Sarkar, A.; Valasinas, A.; Marton,
L. J.; Basu, H. S.; Frydman, B. J. Med. Chem. 2001, 44,
404; (c) Valasinas, A.; Sarkar, A.; Reddy, V. K.; Marton,
L. J.; Basu, H. S.; Frydman, B. J. Med. Chem. 2001, 44,
390; (d) Frydman, B.; Bhattacharya, S.; Sarkar, A.;
Drandarov, K.; Chesnov, S.; Guggisberg, A.; Popaj, K.;
Sergeyev, S.; Yurdakul, A.; Hesse, M.; Basu, H. S.;
Marton, L. J. J. Med. Chem. 2004, 47, 1051; (e) Le, H. T.;
Schaldach, C. M.; Firestone, G. L.; Bjeldanes, L. F.
J. Biol. Chem. 2003, 278, 21136; (f) Ohtsu, H.; Xiao, Z.;
Ishida, J.; Nagai, M.; Wang, H.-K.; Itokawa, H.;
Su, C.-Y.; Shih, C.; Chiang, T.; Chang, E.; Lee, Y.; Tsai,
M.-Y.; Chang, C.; Lee, K.-H. J. Med. Chem. 2002, 45,
5037; (g) Matias, P. M.; Carrondo, M. A.; Coelho, R.;
Thomaz, M.; Zhao, X.-Y.; Wegg, A.; Crusius, K.; Egner,
4.28.6.
R-N-(4-Cyano-3-trifluoromethylphenyl)-2-hy-
droxy-3-(4-isothiocyanatophenyl sulfanyl)-2-methylpropi-
onamide (R-35).11 Prepared from R-53; white solid; ESI-
MS; m/z: 336.1 [MꢁH]ꢁ; 1H NMR (CDCl3) d 9.05 (br s,
1H, NH), 7.97 (s, 1H, ArH), 7.79 (m, 2H, ArH), 7.36 (d,
J = 7.5 Hz, 2H, ArH), 7.04 (d, J = 7.5 Hz, 2H, ArH),
3.73 (d, J = 13.8 Hz, 1H, CH2), 3.51 (br s, 1H, OH),
3.20 (d, J = 13.8 Hz, 1H, CH2), 1.58 (s, 3H, CH3). 13C
NMR: (CDCl3) d 173.2, 141.5, 137.1, 136.0, 134.1 (q,
JC–F = 32.6 Hz), 133.8, 131.7, 130.5, 126.3, 122.3 (q,
JC–F = 272.4 Hz), 122.0, 117.4 (q, JC–F = 4.8 Hz),
115.7, 104.7, 75.8, 44.8, 26.4; Anal. Calcd for
C19H14F3N3O2S2: C, 52.17; H, 3.23; N, 9.61. Found:
C, 52.09; H, 3.16; N, 9.53.

6538
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