N-arylpiperazine-1-carboxamide derivatives: A novel series of orally active nonsteroidal androgen receptor antagonists

Article abstract of DOI:10.1248/cpb.53.402

A novel series of N-arylpiperazine-1-carboxamide derivatives was synthesized and their androgen receptor (AR) antagonist activities and in vivo antiandrogenic properties were evaluated. Reporter assays indicated that trans-2,5-dimethylpiperazine derivativ

Full text of DOI:10.1248/cpb.53.402

402
Chem. Pharm. Bull. 53(4) 402—409 (2005)
Vol. 53, No. 4
N-Arylpiperazine-1-carboxamide Derivatives: a Novel Series of Orally
Active Nonsteroidal Androgen Receptor Antagonists
Isao KINOYAMA,* Nobuaki TANIGUCHI, Eiji KAWAMINAMI, Eisuke NOZAWA, Hiroshi KOUTOKU,
Takashi FURUTANI, Masafumi KUDOH, and Minoru OKADA
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd.; 21 Miyukigaoka, Tsukuba, Ibaraki
305–8585, Japan. Received December 13, 2004; accepted January 18, 2005; published online January 24, 2005
A novel series of N-arylpiperazine-1-carboxamide derivatives was synthesized and their androgen receptor
(AR) antagonist activities and in vivo antiandrogenic properties were evaluated. Reporter assays indicated that
trans-2,5-dimethylpiperazine derivatives are potent AR antagonists, and in this series trans-N-4-[4-cyano-3-(tri-
fluoromethyl)phenyl]-N-(2,4-difluorophenyl)-2,5-dimethylpiperazine-1-carboxamide (18g, YM-175735) exhibited
the most potent antiandrogenic activity. Compared to bicalutamide, YM-175735 is an approximately 4-fold
stronger AR antagonist and has slightly increased antiandrogenic activity, suggesting that YM-175735 may be
useful in the treatment of prostate cancer.
Key words androgen receptor; antagonist; antiandrogen; prostate cancer
Prostate cancer has become the most common cancer antagonists. Among these derivatives, YM-92088 (5) was
among men, and the second leading cause of male cancer shown to be a more potent than bicalutamide as an in vitro
deaths in the United States.1) Testosterone and 5a-dihy- AR antagonist (4). However, the in vivo antiandrogenic activ-
drotestosterone are androgens that are required for the devel- ity of 5 was lower than that of bicalutamide. Hence, to find
opment of both the normal prostate and prostate cancer.2) AR antagonists with greater oral potency, we have conducted
Androgens act through the androgen receptor (AR), which further modification of 5, and in this paper we describe the
belongs to the steroid-receptor superfamily of ligand-depen- results of our studies on the synthesis and pharmacological
dent transcription factors.3,4) Both steroidal and nonsteroidal evaluation of a series of N-arylpiperazine-1-carboxamide
antiandrogens are available, and these molecules are of clini- derivatives as AR antagonists.
cal utility as chemotherapeutic agents for prostate cancer
(Fig. 1).5,6) Cyproterone acetate (CPA: 1) is a typical steroidal Chemistry
AR antagonist.7) It was one of the earliest of these drugs to
Compounds selected for biological evaluation were pre-
be administered orally, but CPA shows agonistic activity and pared as described in Charts 1—4. All synthesized com-
1
overlapping effects with other hormonal systems, leading to a pounds were characterized by H-NMR, mass spectrometry
range of unpleasant side effects. A number of nonsteroidal and elemental analysis.
AR antagonists have been reported in the literature8—17) and
As shown in Chart 1, compounds 7—12 and 19 were pre-
three of these, flutamide (2),18—20) nilutamide (3)21) and bica- pared in good yields by ipso substitution of 4-fluoro-2-(triflu-
lutamide (4)22—25) (Fig. 1), are pure antiandrogens used in the oromethyl)benzonitrile (6) with the corresponding cyclic
treatment of prostate cancer.26) However, these nonsteroidal amines and, in the case of compound 9, by subsequent depro-
AR antagonists exhibit adverse effects such as mastodynia, tection of the Boc group. Treatment of 7—12 with 4-fluo-
gynaecomastia and hepatotoxicity18—25); and therefore potent rophenyl isocyanate afforded the urea derivatives 13—18a.
AR antagonists with fewer adverse effects are highly desir- The amide derivative (20) was obtained from compound 19
able. Moreover, flutamide therapy requires administration by hydrolysis followed by conventional amidation. Com-
three times each day, and bicalutamide is taken once a day. pound 22 was obtained by coupling N-Boc piperidinone with
Therefore, from a quality of life perspective, it would be 4-bromo-2-(trifluoromethyl)benzonitrile, which was prepared
desirable for new generation AR antagonists to have a longer by a Sandmeyer reaction with 21, followed by dehydration
duration of action, at least equal to that of bicalutamide.
using POCl3. Hydrogenation of the dihydropyridine moiety
In a previous paper,27) we reported a new series of of 22 gave the piperidine (23) in good yields. After deprotec-
N-arylpiperazine derivatives as potent nonsteroidal AR tion of the Boc groups of 22 and 23, the piperidines were
treated with 4-fluorophenyl isocyanate to give compounds 24
and 25, respectively (Chart 2). A Pd-C catalyzed Suzuki cou-
pling28,29) between 4-bromo-2-(trifluoromethyl)benzonitrile
and 4-carboxyphenylboronic acid provided the biphenyl 26,
which was then converted to compound 27 in a similar man-
ner to that described for compound 20 (Chart 3). Compound
28 was obtained by ipso substitution of 6 with excess ethyl-
enediamine, followed by reductive amination with benzalde-
hyde using NaBH(OAc)3. The piperazine framework was
constructed in moderate yield by treatment of 28 with gly-
oxal in aqueous conditions,30) and subsequent removal of the
Fig. 1. Structures of Androgen Antagonists
benzyl group by hydrogenolysis gave compound 29. Com-
∗ To whom correspondence should be addressed. e-mail: kinoyama@yamanouchi.co.jp
© 2005 Pharmaceutical Society of Japan

April 2005
403
Chart
1
Chart 2
Chart
3
Chart
4
Chart
5
pound 30 was synthesized by treatment of 29 with 4-fluo- azine framework, such as compound 33. Introduction of the
rophenyl isocyanate (Chart 4). Chart 5 shows the synthesis of alkyl groups on the piperazine framework was achieved by
compounds with bulky groups at the a position on the piper- alkylation of the arylpiperazinone with the corresponding

404
Vol. 53, No. 4
alkyl halides, using lithium diisopropylamide (LDA). Reduc-
tion of the amide with a borane-tetrahydrofuran (THF) com-
plex gave the corresponding amine derivatives (31—33).
Syntheses of compounds 34—36 were similar to that for
compound 30.
Table 1. AR Antagonistic Activities of Arylpiperazine, Arylpiperidine and
Biphenyl Derivatives
Compound
A
IC50 (mM)a)
Results and Discussion
5
13
20
25
24
27
4
0.47
1.6
All the analogues were evaluated for their AR antagonistic
activity using a reporter assay; the resulting IC50 values are
listed in Tables 1—3. As described in the introduction, YM-
92088 (5) shows less potent in vivo antiandrogenic activity
than bicalutamide (4); the reason is unclear, but we speculate
that the piperazine framework of 5 is easily metabolized.
In fact, compound 5 has been found to be metabolically
unstable in human liver microsomes (54% remaining after
1 h), and therefore to find more potent and orally active AR
antagonists, we concentrated our efforts on further modifica-
tion of 5, focusing mainly on the piperazine framework.
Firstly, we converted the piperazine ring of 5 into alterna-
tive cyclic amines, such as homo-piperazine and piperidine
(Table 1). Ring expansion of the piperazine (compound 13)
resulted in an approximately 3-fold decrease in the inhibitory
activity. Replacement of the sp2-like urea nitrogen atom on
the piperazine ring with an sp3 carbon atom in compound 20
led to a substantial reduction in potency, relative to 5. The
piperidine derivative (25) also exhibited a weaker inhibitory
activity, probably due to the change from the sp2-like aniline
nitrogen to an sp3 carbon. However, introduction of the sp2
carbon atom into the piperidine ring in compound 24 pro-
vided a 4-fold improvement in potency, compared to 25.
Moreover, biphenyl derivative (27) was approximately
equipotent with 5. These results suggest that both sp2-like
nitrogen atoms in the piperazine ring were important for
potency, and that the piperazine framework of 5 plays a spa-
tial role as a linker with planar geometry at the N atoms.
Consequently, we selected the N-arylpiperazine-1-carboxam-
ide as an optimal scaffold, and introduced further substituents
onto the piperazine framework of 5.
7.2
4.9
1.2
0.61
0.89
a) Compounds were tested for their ability to inhibit AR mediated transcriptional ac-
tivation using a reporter assay. IC50 values were determined by a single experimental
run in triplicate.
Table 2. AR Antagonistic Activities of the N-Arylpiperazine-1-carboxam-
ide Derivatives
Compound
R
IC50 (mM)a)
5
14
15
34
35
30
36
16
17
18a
4
H
0.47
0.18
0.10
0.14
0.77
17%b)
0.25
8.5
2-Methyl
3-Methyl
3-Ethyl
3-Isopropyl
3-Oxo
3,3-Dimethyl
2,2-Dimethyl
cis-2,6-Dimethyl
trans-2,5-Dimethyl
5.0
0.13
0.89
Next, we introduced an alkyl group onto the piperazine
ring. As shown in Table 2, methyl substitution at the 2-posi-
tion caused an approximately 3-fold increase in the potency
a) Refer to Table 1. b) Percent inhibition at 10 mM.
(IC50ꢀ0.18 and 0.47 mM for 14 and 5, respectively). Since urea bond. Interestingly, the 2,5-trans-dimethyl derivative
addition of a methyl group at the 3-position (15) was pre- (18a) exhibited comparable activity to the monomethyl
ferred over the 2-position, we further introduced another derivatives (14, 15) (IC50ꢀ0.13, 0.18 and 0.10 mM, respec-
alkyl group at the 3-position. Although the ethyl derivative tively). These results suggest that introduction of specific
(34) exhibited comparable inhibitory activity, introduction of methyl group(s) may lead to a preferred conformation of the
an isopropyl group (35) resulted in 8-fold reduction in piperazine ring that increases the AR antagonist activity.
potency, compared to 15, indicating that increased bulkiness
Lastly, we conducted further modification of the 4-fluo-
at this position may be unfavorable for AR antagonism. rophenyl group of the 2,5-trans-dimethyl derivative (18a).
Introduction of an oxo group onto the piperazine ring at the Replacement of the fluorine atom with another halogen, such
3-position resulted in particularly deleterious effects on the as a chlorine or bromine, at the para position resulted in only
inhibitory activity (compound 30). Subsequently, we synthe- a small reduction in potency, and other derivatives (18d—g)
sized di-substituted derivatives for further investigation of exhibited comparable inhibitory activity to 18a (Table 3).
the substituent effects on the piperazine framework. The 3,3-
The 2,5-trans-dimethyl derivatives (18a—g) were also
dimethyl derivative (36) showed a slight decrease in evaluated for in vivo antiandrogenic activity, based on their
inhibitory activity, but the 2,2-dimethyl derivative (16) was inhibition of ventral prostate growth in testosterone propi-
significantly less active relative to the corresponding onate-treated castrated rats, using once daily oral administra-
monomethyl derivative (14). As shown by compound 17, tion for 5 d (Table 3). The 2,5-trans-dimethyl derivative (18a)
introduction of 2,6-cis-dimethyl substituents was also detri- exhibited increased in vivo antiandrogenic activity compared
mental to AR antagonism, probably due to an unfavorable to the unsubstituted derivative (5) (64% and 32% inhibition,
conformation by the interference of free rotation around the respectively, compared to the control), and showed improved

April 2005
405
Table 3. In Vitro and in Vivo Activities of the trans-2,5-Dimethylpiper-
azine Derivatives
carboxamide (18g, YM-175735) exhibited the most potent
antiandrogenic activity. Compared to bicalutamide, YM-
175735 showed an approximately 4-fold stronger activity as
an AR antagonist, and showed a slightly increase in in vivo
antiandrogenic activity, suggesting that YM-175735 may be
useful for the treatment of prostate cancer.
Cpd.
Ar
IC50 (mM)a)
% inhibitionb)
Experimental
In general, all reagents and solvents were commercial quality and were
used without further purification unless otherwise noted. Melting points
were determined on a Yanaco MP-500D micro melting point apparatus with-
out correction. 1H-NMR spectra were measured with a JMN-LA300 or
JMN-EX400 spectrometer; chemical shifts are expressed in d units using
tetramethylsilane as the standard (in NMR description, sꢀsinglet, dꢀdou-
blet, tꢀtriplet, mꢀmultiplet and brꢀbroad peak). MS spectra were deter-
mined with a JEOL JMS-LX2000 spectrometer. Elemental analysis was per-
formed with a Yanaco MT-5 microanalyzer (C, H, N) and Yokogawa IC-
7000S ion chromatographic analyzer (halogens) and were within ꢁ0.4% of
theoretical values.
5
0.47
0.13
32%
18a
64%**
18b
18c
18d
18e
0.24
0.57
0.27
0.11
33%
60%**
27%
17%
4-(1,4-Diazepan-1-yl)-2-(trifluoromethyl)benzonitrile (7) To a solu-
tion of 4-fluoro-2-(trifluoromethyl)benzonitrile (6, 5.0 g, 26.4 mmol) in N,N-
dimethylformamide (DMF, 50 ml) was added 1,4-diazepane (10.6 g,
105.8 mmol) at ambient temperature and stirred at 80 °C for 21 h. The reac-
tion mixture was diluted with H2O and extracted with AcOEt. The organic
layer was washed with H2O, dried over MgSO4 and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(CHCl3/MeOHꢀ10/1) to give the title compound (4.55 g, 64%) as a color-
less solid. 1H-NMR (400 MHz, DMSO-d6) d 1.68—1.80 (2H, m), 2.60—
2.66 (2H m), 2.82—2.90 (2H, m), 3.54—3.70 (4H, m), 7.00—7.06 (2H, m),
7.74 (1H, d, Jꢀ8.4 Hz). FAB-MS m/z: 270 (MꢂHꢂ).
18f
0.11
0.20
62%**
85%**
ED50ꢀ1.1 mg/kg
18g
75%**
ED50ꢀ1.6 mg/kg
4
0.89
a) Refer to Table 1. b) The mean percent changes from the respective control
value of ventral prostate weight after oral administration in testosterone propionate-
treated castrated rats (10 mg/kg/d for 5 d, nꢀ5 or 6). ∗∗ pꢃ0.01 versus control by Dun-
nett’s multiple comparison test.
(ꢀ)-4-(3-Methylpiperazin-1-yl)-2-(trifluoromethyl)benzonitrile
(8)
The title compound was prepared from 2-methylpiperazine in a manner sim-
1
ilar to that described for compound 7 as a colorless solid (quant.). H-NMR
(300 MHz, DMSO-d6) d 1.03 (3H, d, Jꢀ6.3 Hz), 2.36—2.48 (1H, m),
2.62—2.85 (3H, m), 2.90—2.99 (1H, m), 3.79—3.93 (2H, m), 7.16—7.31
(2H, m), 7.79 (1H, d, Jꢀ9.3 Hz). FAB-MS m/z: 270 (MꢂHꢂ).
metabolic stability in human liver microsomes, compared to
compound 5 (72% and 54% remaining, respectively, after
1 h). Although there may be some species difference in meta-
bolic stability, the relative antiandrogenic activities suggest
that the metabolic stability of 18a in rat is also better than
that of 5. Interestingly, different substituents on the phenyl
ring produced various in vivo results. Hence, the activity of
the bromine derivative (18c) was comparable to 18a, but the
chlorine (18b) and methyl (18d) derivatives were less active
than 18a. Although the methoxy derivative (18e) was a po-
tent in vitro AR antagonist, with an IC50 value of 0.11 mM, its
in vivo potency was very weak. Surprisingly, the 2,4-difluoro
(ꢀ)-4-(2-Methylpiperazin-1-yl)-2-(trifluoromethyl)benzonitrile
(9)
tert-Butyl 4-[4-cyano-3-(trifluoromethyl)phenyl]-3-methylpiperazine-1-car-
boxylate was prepared from tert-butyl 3-methylpiperazine-1-carboxylate31)
in a manner similar to that described for compound 7 (65%) as a colorless
powder. A mixture of the intermediate (1.2 g, 3.25 mmol) and TFA (6 ml)
was stirred at 0 °C for 30 min and the solution was concentrated in vacuo.
The residue was diluted with saturated aqueous NaHCO3 and extracted with
AcOEt. The organic layer was dried and concentrated under reduced pres-
sure to give 9 (920 mg, quant.) as a pale yellow oil. 1H-NMR (300 MHz,
DMSO-d6) d 1.11 (3H, d, Jꢀ6.6 Hz), 2.56—2.70 (1H, m), 2.74—2.89
(2H, m), 2.90—3.05 (2H, m), 3.54—3.67 (1H, m), 4.05—4.23 (1H, m),
7.11—7.24 (2H, m), 7.80 (1H, d, Jꢀ9.0 Hz). FAB-MS m/z: 270 (MꢂHꢂ).
4-(3,3-Dimethylpiperazin-1-yl)-2-(trifluoromethyl)benzonitrile
(10)
derivative (18g) strongly inhibited the growth of rat prostate The title compound was prepared from 2,2-dimethylpiperazine32) in a man-
ner similar to that described for compound 7 as a colorless solid (55%).
by 85% at a dose of 10 mg/kg, whereas the 3,4-difluoro de-
rivative (18f) had an effect comparable to that of the 4-fluoro
1H-NMR (300 MHz, DMSO-d6) d 1.05 (6H, s), 2.77—2.90 (2H, m), 3.21
(2H, s), 3.29—3.39 (2H, m), 7.14—7.29 (2H, m), 7.76 (1H, d, Jꢀ8.7 Hz).
derivative (18a). These results indicate that introduction of
an additional fluorine atom at the 2-position on the phenyl
ring may be important for in vivo activity. Compound 18g
showed dose-dependent inhibition of the growth of rat
prostate, and its ED50 value was 1.1 mg/kg, making it more
potent than bicalutamide (ED50ꢀ1.6 mg/kg) and suggesting
that 18g (YM-175735) has potential as a novel nonsteroidal
AR antagonist.
FAB-MS m/z: 284 (MꢂHꢂ).
(ꢀ)-cis-4-(3,5-Dimethylpiperazin-1-yl)-2-(trifluoromethyl)benzonitrile
(11) The title compound was prepared from cis-2,6-dimethylpiperazine in
a manner similar to that described for 7 as a colorless solid (81%). 1H-NMR
(300 MHz, DMSO-d6) d 1.03 (6H, d, Jꢀ6.3 Hz), 2.21—2.41 (2H, m),
2.65—2.83 (2H, m), 3.82—3.94 (2H, m), 7.16—7.32 (2H, m), 7.80 (1H, d,
Jꢀ8.8 Hz). FAB-MS m/z: 284 (MꢂHꢂ).
(ꢀ)-trans-4-(2,5-Dimethylpiperazin-1-yl)-2-(trifluoromethyl)benzoni-
trile (12) The title compound was prepared from trans-2,5-dimethylpiper-
azine in a manner similar to that described for compound 7 as a yellow oil
(quant.). 1H-NMR (300 MHz, CDCl3) d 1.16—1.24 (6H, m), 2.67—2.77
(1H, m), 3.06—3.18 (1H, m), 3.25—3.41 (3H, m), 3.70—3.83 (1H, m), 6.96
(1H, dd, Jꢀ8.7, 2.4 Hz), 7.12 (1H, d, Jꢀ2.4 Hz), 7.62 (1H, d, Jꢀ8.7 Hz).
EI-MS m/z: 283 (Mꢂ).
4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)-1,4-di-
azepane-1-carboxamide (13) To a solution of 4-(1,4-diazepan-1-yl)-2-
(trifluoromethyl)benzonitrile (7, 500 mg, 1.86 mmol) in CH2Cl2 (10 ml) was
added 4-fluorophenyl isocyanate (0.23 ml, 2.04 mmol) at ambient tempera-
ture and stirred for 2 h. The reaction mixture was concentrated in vacuo
and the residue was purified by silica gel column chromatography
Conclusion
A novel series of N-arylpiperazine-1-carboxamide deriva-
tives were synthesized and their androgen receptor (AR)
antagonist activities and in vivo antiandrogenic effects were
evaluated. Reporter assays indicated that trans-2,5-di-
methylpiperazine derivatives were potent AR antagonists,
and in this series, trans-N-4-[4-cyano-3(trifluoromethyl)-
phenyl]-N-(2,4-difluorophenyl)-2,5-dimethylpiperazine-1-

406
Vol. 53, No. 4
benzonitrile (12) and 4-bromophenyl isocyanate in a manner similar to that
described for compound 13 as a colorless solid (84%). mp 192 °C (CH2Cl2).
1H-NMR (300 MHz, DMSO-d6) d 1.09 (3H, d, Jꢀ6.6 Hz), 1.17 (3H, d,
Jꢀ6.6 Hz), 3.30—3.46 (2H, m), 3.67—3.78 (1H, m), 3.82—3.92 (1H, m),
4.29—4.41 (1H, m), 4.43—4.56 (1H, m), 7.20—7.32 (2H, m), 7.37—7.52
(4H, m), 7.84 (1H, d, Jꢀ9.0 Hz), 8.69 (1H, s). FAB-MS m/z: 483, 481
(MꢂHꢂ). Anal. Calcd for C21H20N4OBrF3: C, 52.40; H, 4.19; N, 11.64; Br,
16.60; F, 11.84. Found: C, 52.15; H, 4.24; N, 11.56; Br, 16.31; F, 11.67.
(ꢀ)-trans-4-[4-Cyano-3-(trifluoromethyl)phenyl]-2,5-dimethyl-N-
(4-methylphenyl)piperazine-1-carboxamide (18d) The title compound
was prepared from (ꢁ)-trans-4-(2,5-dimethylpiperazin-1-yl)-2-(trifluo-
romethyl)benzonitrile (12) and p-tolyl isocyanate in a manner similar to that
described for compound 13 as a colorless solid (61%). mp 188 °C (AcOEt).
1H-NMR (300 MHz, DMSO-d6) d 1.10 (3H, d, Jꢀ6.6 Hz), 1.17 (3H, d,
Jꢀ6.6 Hz), 2.23 (3H, s), 3.30—3.45 (2H, m), 3.66—3.77 (1H, m), 3.82—
3.92 (1H, m), 4.28—4.41 (1H, m), 4.43—4.56 (1H, m), 7.05 (2H, d,
Jꢀ8.4 Hz), 7.22—7.39 (4H, m), 7.84 (1H, d, Jꢀ9.0 Hz), 8.46 (1H, s). FAB-
MS m/z: 417 (MꢂHꢂ). Anal. Calcd for C22H23N4OF3: C, 63.45; H, 5.57; N,
13.45; F, 13.69. Found: C, 63.24; H, 5.51; N, 13.42; F, 13.74.
(ꢀ)-trans-4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-methoxy-
phenyl)-2,5-dimethylpiperazine-1-carboxamide (18e) The title com-
pound was prepared from (ꢁ)-trans-4-(2,5-dimethylpiperazin-1-yl)-2-(triflu-
oromethyl)benzonitrile (12) and 4-methoxyphenyl isocyanate in a manner
similar to that described for compound 13 as a colorless solid (61%). mp
195 °C (AcOEt/Et2O). 1H-NMR (300 MHz, DMSO-d6) d 1.10 (3H, d,
Jꢀ6.6 Hz), 1.16 (3H, d, Jꢀ6.6 Hz), 3.31—3.45 (2H, m), 3.66—3.77 (1H,
m), 3.71 (3H, s), 3.81—3.92 (1H, m), 4.28—4.41 (1H, m), 4.42—4.55 (1H,
m), 6.79—6.87 (2H, m), 7.22—7.39 (4H, m), 7.84 (1H, d, Jꢀ9.0 Hz), 8.40
(1H, s). FAB-MS m/z: 433 (MꢂHꢂ). Anal. Calcd for C22H23N4O2F3: C,
61.10; H, 5.36; N, 12.96; F, 13.18. Found: C, 60.84; H, 5.23; N, 13.05; F,
13.05.
(CHCl3/MeOHꢀ50/1). The resulting solid was further purified by recrystal-
lization from AcOEt to give the title compound (550 mg, 73%) as a colorless
crystalline solid. mp 179—180 °C. 1H-NMR (400 MHz, DMSO-d6)
d 1.80—1.95 (2H, m), 3.39—3.50 (2H, m), 3.60—3.85 (6H, m), 6.97—7.15
(4H, m), 7.29—7.37 (2H, m), 7.76 (1H, d, Jꢀ8.8 Hz), 8.34 (1H, s). FAB-MS
m/z: 407 (MꢂHꢂ). Anal. Calcd for C20H18N4OF4: C, 59.11; H, 4.46; N,
13.79; F, 18.70. Found: C, 58.93; H, 4.45; N, 13.82; F, 18.60.
(ꢀ)-4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)-2-
methylpiperazine-1-carboxamide (14) The title compound was prepared
from (ꢁ)-4-(3-methylpiperazin-1-yl)-2-(trifluoromethyl)benzonitrile (8) in a
manner similar to that described for 13 as a colorless solid (84%). mp 211—
216 °C (AcOEt/iPr2O). 1H-NMR (400 MHz, DMSO-d6) d 1.16 (3H, d,
Jꢀ6.9 Hz), 3.06—3.19 (1H, m), 3.29—3.41 (2H, m), 3.84—4.01 (3H, m),
4.35—4.48 (1H, m), 7.03—7.12 (2H, m), 7.25 (1H, dd, Jꢀ8.8, 2.5 Hz), 7.31
(1H, d, Jꢀ2.5 Hz), 7.42—7.52 (2H, m), 7.84 (1H, d, Jꢀ8.8 Hz), 8.55
(1H, s). FAB-MS m/z: 407 (MꢂHꢂ). Anal. Calcd for C20H18N4OF4: C,
59.11; H, 4.46; N, 13.79; F, 18.70. Found: C, 59.38; H, 4.61; N, 13.85; F,
18.59.
(ꢀ)-4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)-3-
methylpiperazine-1-carboxamide (15) The title compound was prepared
from (ꢁ)-4-(2-methylpiperazin-1-yl)-2-(trifluoromethyl)benzonitrile (9) in a
manner similar to that described for compound 13 as a colorless solid
(77%). mp 197—199 °C (AcOEt/iPr2O). 1H-NMR (400 MHz, DMSO-d6)
d 1.10 (3H, d, Jꢀ6.8 Hz), 3.11—3.32 (3H, m), 3.76—3.86 (1H, m), 3.95—
4.14 (2H, m), 4.29—4.40 (1H, m), 7.04—7.12 (2H, m), 7.19—7.30 (2H, m),
7.44—7.51 (2H, m), 7.86 (1H, d, Jꢀ8.7 Hz), 8.61 (1H, s). FAB-MS m/z: 405
(MꢄHꢄ). Anal. Calcd for C20H18N4OF4: C, 59.11; H, 4.46; N, 13.79; F,
18.70. Found: C, 58.94; H, 4.45; N, 13.71; F, 18.97.
4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)-2,2-di-
methylpiperazine-1-carboxamide (16) The title compound was prepared
from 4-(3,3-dimethylpiperazin-1-yl)-2-(trifluoromethyl)benzonitrile (10) in a
manner similar to that described for compound 13 as a colorless solid
(60%). mp 197—201 °C (AcOEt). 1H-NMR (400 MHz, DMSO-d6) d 1.43
(6H, s), 3.54—3.61 (2H, m), 3.67 (2H, s), 3.75—3.86 (2H, m), 7.02—7.09
(2H, m), 7.11 (1H, dd, Jꢀ8.8, 2.5 Hz), 7.17 (1H, d, Jꢀ2.5 Hz), 7.35—7.45
(2H, m), 7.83 (1H, d, Jꢀ8.8 Hz), 8.45 (1H, s). FAB-MS m/z: 421 (MꢂHꢂ).
Anal. Calcd for C21H20N4OF4: C, 60.00; H, 4.80; N, 13.33; F, 18.08. Found:
C, 59.98; H, 4.72; N, 13.34; F, 18.19.
(ꢀ)-trans-4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(3,4-difluoro-
phenyl)-2,5-dimethylpiperazine-1-carboxamide (18f) The title com-
pound was prepared from (ꢁ)-trans-4-(2,5-dimethylpiperazin-1-yl)-2-(triflu-
oromethyl)benzonitrile (12) and 3,4-difluorophenyl isocyanate in a manner
similar to that described for compound 13 as a colorless solid (50%). mp
1
185 °C (MeOH). H-NMR (300 MHz, DMSO-d6) d 1.10 (3H, d, Jꢀ6.6 Hz),
1.53 (3H, d, Jꢀ6.6 Hz), 3.28—3.47 (2H, m), 3.67—3.78 (1H, m), 3.82—
3.92 (1H, m), 4.29—4.55 (2H, m), 7.21—7.37 (4H, m), 7.58—7.70 (1H, m),
7.85 (1H, d, Jꢀ8.7 Hz), 8.76 (1H, s). FAB-MS m/z: 439 (MꢂHꢂ). Anal.
Calcd for C21H19N4OF5: C, 57.53; H, 4.37; N, 12.78; F, 21.67. Found: C,
57.51; H, 4.52; N, 12.74; F, 21.40.
(ꢀ)-cis-4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)-2,6-
dimethylpiperazine-1-carboxamide (17) The title compound was pre-
pared from (ꢁ)-cis-4-(3,5-dimethylpiperazin-1-yl)-2-(trifluoromethyl)ben-
zonitrile (11) in a manner similar to that described for compound 13 as a
1
(ꢀ)-trans-4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(2,4-difluoro-
phenyl)-2,5-dimethylpiperazine-1-carboxamide (18g) The title com-
pound was prepared from (ꢁ)-trans-4-(2,5-dimethylpiperazin-1-yl)-2-(triflu-
oromethyl)benzonitrile (12) and 2,4-difluorophenyl isocyanate in a manner
similar to that described for compound 13 as a colorless solid (82%). mp
169—171 °C (AcOEt/n-hexane). 1H-NMR (300 MHz, DMSO-d6) d 1.11
(3H, d, Jꢀ6.6 Hz), 1.18 (3H, d, Jꢀ6.6 Hz), 3.30—3.48 (2H, m), 3.67—3.88
(2H, m), 4.28—4.51 (2H, m), 6.96—7.07 (1H, m), 7.18—7.43 (4H, m), 7.84
(1H, d, Jꢀ8.7 Hz), 8.39 (1H, s). FAB-MS m/z: 439 (MꢂHꢂ). Anal. Calcd
for C21H19N4OF5: C, 57.53; H, 4.37; N, 12.78; F, 21.67. Found: C, 57.51; H,
4.35; N, 12.99; F, 21.38.
Ethyl 1-[4-Cyano-3-(trifluoromethyl)phenyl]piperidine-4-carboxylate
(19) A mixture of 4-fluoro-2-(trifluoromethyl)benzonitrile (6, 1.0 g, 5.29
mmol), ethyl piperidine-4-carboxylate (0.92 ml, 5.82 mmol) and K2CO3
(1.1 g, 7.94 mmol) in DMF (50 ml) was stirred at ambient temperature for
17 h. The mixture was poured into water and the precipitate was filtered and
washed with water to give the title compound (1.51 g, 88%) as a colorless
solid. 1H-NMR (400 MHz, DMSO-d6) d 1.19 (3H, t, Jꢀ7.1 Hz), 1.52—1.66
(2H, m), 1.85—1.97 (2H, m), 2.59—2.71 (1H, m), 3.01—3.17 (2H, m),
3.92—4.02 (2H, m), 4.08 (2H, q, Jꢀ7.1 Hz), 7.24 (1H, dd, Jꢀ8.8, 2.5 Hz),
7.30 (1H, d, Jꢀ2.5 Hz), 7.81 (1H, d, Jꢀ8.8 Hz). EI-MS m/z: 326 (Mꢂ).
1-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)piperidine-
4-carboxamide (20) A mixture of ethyl 1-[4-cyano-3-(trifluoromethyl)-
phenyl]piperidine-4-carboxylate (19, 1.41 g, 4.32 mmol) and 1 M NaOH
(5.4 ml, 5.40 mmol) in EtOH (50 ml) and THF (20 ml) was stirred at ambient
temperature for 4 d. The reaction mixture was concentrated in vacuo and the
residue was diluted with saturated aqueous NH4Cl and was extracted with
CHCl3. The organic layer was dried over Na2SO4 and concentrated under re-
duced pressure to give 1-[4-cyano-3-(trifluoromethyl)phenyl]piperidine-4-
carboxylic acid (1.23 g, 95%) as a colorless solid. To a solution of 1-
[4-cyano-3-(trifluoromethyl)phenyl]piperidine-4-carboxylic acid (500 mg,
colorless solid (79%). mp 205 °C (AcOEt). H-NMR (400 MHz, DMSO-d6)
d 1.29 (6H, d, Jꢀ6.4 Hz), 3.18—3.31 (2H, m), 3.97—4.14 (2H, m), 4.30—
4.45 (2H, m), 7.02—7.17 (2H, m), 7.30—7.39 (2H, m), 7.44—7.54 (2H, m),
7.84 (1H, d, Jꢀ8.6 Hz), 8.45 (1H, s). FAB-MS m/z: 421 (MꢂHꢂ). Anal.
Calcd for C21H20N4OF4: C, 60.00; H, 4.80; N, 13.33; F, 18.08. Found:
C, 59.91; H, 4.78; N, 13.34; F, 18.34.
(ꢀ)-trans-4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)-
2,5-dimethylpiperazine-1-carboxamide (18a) The title compound was
prepared from (ꢁ)-trans-4-(2,5-dimethylpiperazin-1-yl)-2-(trifluorometh-
yl)benzonitrile (12) and 4-fluoropheny isocyanate in a manner similar to that
described for compound 13 as a colorless solid (74%). mp 200—203 °C
(EtOH). 1H-NMR (400 MHz, DMSO-d6) d 1.11 (3H, d, Jꢀ6.5 Hz), 1.18
(3H, d, Jꢀ6.7 Hz), 3.31—3.48 (2H, m), 3.66—3.79 (1H, m), 3.82—3.95
(1H, m), 4.29—4.42 (1H, m), 4.43—4.57 (1H, m), 7.03—7.13 (2H, m),
7.22—7.33 (2H, m), 7.43—7.54 (2H, m), 7.84 (1H, d, Jꢀ8.9 Hz), 8.60 (1H,
s). FAB-MS m/z: 421 (MꢂHꢂ). Anal. Calcd for C21H20N4OF4: C, 60.00; H,
4.80; N, 13.33; F, 18.08. Found: C, 59.91; H, 4.97; N, 13.26; F, 17.98.
(ꢀ)-trans-N-(4-Chlorophenyl)-4-[4-cyano-3-(trifluoromethyl)phenyl]-
2,5-dimethylpiperazine-1-carboxamide (18b) The title compound was
prepared from (ꢁ)-trans-4-(2,5-dimethylpiperazin-1-yl)-2-(trifluoromethyl)-
benzonitrile (12) and 4-chlorophenyl isocyanate in a manner similar to that
described for compound 13 as a colorless solid (66%). mp 196 °C (EtOH).
1H-NMR (300 MHz, DMSO-d6) d 1.10 (3H, d, Jꢀ6.3 Hz), 1.18 (3H, d,
Jꢀ6.6 Hz), 3.30—3.47 (2H, m), 3.67—3.78 (1H, m), 3.82—3.93 (1H, m),
4.29—4.41 (1H, m), 4.43—4.56 (1H, m), 7.22—7.33 (4H, m), 7.48—7.57
(2H, m), 7.84 (1H, d, Jꢀ9.0 Hz), 8.69 (1H, s). FAB-MS m/z: 437 (MꢂHꢂ).
Anal. Calcd for C21H20N4OClF3: C, 57.74; H, 4.61; N, 12.82; Cl, 8.12; F,
13.05. Found: C, 57.42; H, 4.50; N, 12.79; Cl, 8.42; F, 12.82.
(ꢀ)-trans-N-(4-Bromophenyl)-4-[4-cyano-3-(trifluoromethyl)phenyl]-
2,5-dimethylpiperazine-1-carboxamide (18c) The title compound was
prepared from (ꢁ)-trans-4-(2,5-dimethylpiperazin-1-yl)-2-(trifluoromethyl)-

April 2005
407
1.68 mmol) in CH2Cl2 (25 ml) was added oxalyl chloride (0.3 ml, 3.35
mmol) and DMF (1 drop). After stirring at ambient temperature for 1 h, the
solution was concentrated in vacuo. The residue was dissolved in CH2Cl2
7.93—8.04 (2H, m), 8.16 (1H, d, Jꢀ8.3 Hz), 8.62 (1H, s). FAB-MS m/z: 390
(MꢂHꢂ). Anal. Calcd for C20H15N3OF4: C, 61.70; H, 3.88; N, 10.79; F,
19.52. Found: C, 61.53; H, 3.94; N, 10.76; F, 19.52.
(10 ml) and added to
a cooled solution of 4-fluoroaniline (0.48 ml,
4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)piperidine-
1-carboxamide (25) The title compound was prepared from tert-butyl 4-
[4-cyano-3-(trifluoromethyl)phenyl]piperidine-1-carboxylate (23) in a man-
ner similar to that described for compound 24 as a colorless powder (2 steps
22%). mp 161—162 °C (EtOH/iPr2O). 1H-NMR (400 MHz, DMSO-d6)
d 1.56—1.72 (2H, m), 1.77—1.89 (2H, m), 2.81—2.93 (2H, m), 2.96—3.07
(1H, m), 4.24—4.35 (2H, m), 7.02—7.11 (2H, m), 7.43—7.52 (2H, m),
7.81—7.86 (1H, m), 7.91—7.95 (1H, m), 8.12 (1H, d, Jꢀ7.8 Hz), 8.55 (1H,
s). FAB-MS m/z: 392 (MꢂHꢂ). Anal. Calcd for C20H17N3OF4: C, 61.38; H,
4.38; N, 10.74; F, 19.42. Found: C, 61.26; H, 4.28; N, 10.78; F, 19.75.
4ꢁ-Cyano-3ꢁ-(trifluoromethyl)biphenyl-4-carboxylic Acid (26) Syn-
thesis of 4-bromo-2-(trifluoromethyl)benzonitrile was described as above
5.04 mmol) in CH2Cl2 (10 ml). After stirring at ambient temperature for 1 h,
the precipitate was filtered off and the filtrate was concentrated in vacuo.
The residue was purified by silica gel column chromatography (n-
hexane/AcOEtꢀ1/1). The resulting solid was further purified by recrystal-
lization from AcOEt to give the title compound (371 mg, 56%) as a colorless
crystalline solid. mp 185—187 °C. 1H-NMR (400 MHz, DMSO-d6)
d 1.60—1.76 (2H, m), 1.83—1.96 (2H, m), 2.59—2.70 (1H, m), 2.96—3.13
(2H, m), 4.05—4.19 (2H, m), 7.09—7.18 (2H, m), 7.23—7.29 (1H, m),
7.30—7.35 (1H, m), 7.58—7.67 (2H, m), 7.82 (1H, d, Jꢀ8.8 Hz), 10.01
(1H, s). FAB-MS m/z: 392 (MꢂHꢂ). Anal. Calcd for C20H17N3OF4: C,
61.38; H, 4.38; N, 10.74; F, 19.42. Found: C, 61.42; H, 4.30; N, 10.72; F,
19.60.
(compound 22).
A mixture of 4-bromo-2-(trifluoromethyl)benzonitrile
tert-Butyl 4-[4-Cyano-3-(trifluoromethyl)phenyl]-3,6-dihydropyridine-
1(2H)-carboxylate (22) A suspension of 4-amino-2-(trifluoromethyl)ben-
zonitrile (21, 10.0 g, 53.72 mmol) in 48% HBr (50 ml) was cooled in an ice-
salt bath to 0 °C. A solution of sodium nitrite (3.71 g, 53.72 mmol) in water
(10 ml) was added dropwose at such a rate that the temperature of the reac-
tion mixture was under 5 °C. After stirring for 1 h, the reaction mixture was
poured into a solution of copper(I) bromide (7.71 g, 53.72 mmol) in 48%
HBr (55 ml). The mixture was stirred at ambient temperature for 2 h. The
reaction mixture was poured into ice-water and extracted with AcOEt. The
organic layer was washed with saturated NaHCO3, H2O and concentrated
in vacuo. The residue was purified by silica gel column chromatography
(n-hexane/AcOEtꢀ10/1) to give 4-bromo-2-(trifluoromethyl)benzonitrile
(11.37 g, 85%) as a pale brown oil. A solution of n-BuLi in n-hexane
(2.2 ml, 1.59 M, 3.48 mmol) was added dropwide to a solution of 4-bromo-2-
(trifluoromethyl)benzonitrile (790 mg, 3.16 mmol) in dry THF (30 ml) at
ꢄ78 °C. The reaction mixture was stirred for 15 min, and a solution of tert-
butyl 4-oxopiperidine-1-carboxylate (693 mg, 3.48 mmol) in dry THF (3 ml)
was added to the reaction mixture at ꢄ78 °C. After stirring at ambient tem-
perature for 1 h, the reaction mixture was poured into ice-water and
extracted with AcOEt. The organic layer was dried over Na2SO4 and concen-
trated under reduced pressure. The residue was purified by silica gel column
chromatography (CHCl3/MeOHꢀ50/1) to give tert-butyl 4-[4-cyano-3-(tri-
fluoromethyl)phenyl]-4-hydroxypiperidine-1-carboxylate (380 mg, 32%) as
a pale brown solid. To a cooling solution of tert-butyl 4-[4-cyano-3-(trifluo-
romethyl)phenyl]-4-hydroxypiperidine-1-carboxylate (1.27 g, 3.43 mmol) in
pyridine (25 ml) was added phosphoryl chloride (3.2 ml, 34.3 mmol) at 0 °C.
After stirring at ambient temperature for 1 d, the reaction mixture was
quenched with saturated NaHCO3 and the resultant precipitate was filtered
off and washed with H2O to give the title compound (973 mg, 81%) as a pale
brown solid. 1H-NMR (300 MHz, DMSO-d6) d 1.43 (9H, s), 2.47—2.58
(2H, m), 3.51—3.59 (2H, m), 4.02—4.10 (2H, m), 6.52—6.60 (1H, m),
7.90—8.00 (2H, m), 8.15 (1H, d, Jꢀ8.7 Hz). FAB-MS m/z: 353 (MꢂHꢂ).
tert-Butyl 4-[4-Cyano-3-(trifluoromethyl)phenyl]piperidine-1-carboxy-
late (23) A mixture of tert-butyl 4-[4-cyano-3-(trifluoromethyl)phenyl]-
3,6-dihydropyridine-1(2H)-carboxylate (22, 724 mg, 2.05 mmol) and 10%
Pd-C (36 mg) in MeOH (20 ml) was stirred under H2 at ambient temperature
for 4 h. The precipitate was filtered off and the filtrate was concentrated
in vacuo. The residue was purified by silica gel column chromatography
(n-hexane/AcOEtꢀ1/1) to give the title compound (550 mg, 76%) as a yel-
low oil. 1H-NMR (300 MHz, DMSO-d6) d 1.42 (9H, s), 1.50—1.65 (2H, m),
1.72—1.85 (2H, m), 2.71—3.01 (3H, m), 3.99—4.17 (2H, m), 7.77—7.85
(1H, m), 7.88—7.93 (1H, m), 8.10 (1H, d, Jꢀ8.1 Hz). FAB-MS m/z: 355
(MꢂHꢂ).
(1.88 g, 7.52 mmol), 4-carboxyphenylboronic acid (1.37 g, 8.27 mmol),
Na2CO3 (3.2 g, 30.08 mmol) and 10% Pd-C (320 mg) in EtOH (80 ml) was
refluxed for 2 h. After cooling, the reaction mixture was poured into water.
The precipitate was filtered off, mother liquid was diluted with aqueous cit-
ric acid and extracted with AcOEt. The organic layer was dried and concen-
trated under reduced pressure. The residue was purified by silica gel column
chromatography (CHCl3/MeOHꢀ30/1) to give the title compound (1.80 g,
82%) as a colorless solid. 1H-NMR (300 MHz, DMSO-d6) d 7.95—8.40
(7H, m), 13.16 (1H, br). FAB-MS m/z: 290 (MꢄHꢄ).
4ꢁ-Cyano-N-(4-fluorophenyl)-3ꢁ-(trifluoromethyl)biphenyl-4-carbox-
amide (27) The title compound was prepared from 4ꢅ-cyano-3ꢅ-(trifluo-
romethyl)biphenyl-4-carboxylic acid (26) in a manner similar to that de-
scribed for compound 20 as a colorless powder (46%). mp 191—193 °C
(AcOEt/iPr2O). 1H-NMR (400 MHz, DMSO-d6) d 7.17—7.26 (2H, m),
7.77—7.87 (2H, m), 8.02—8.15 (4H, m), 8.28—8.36 (3H, m), 10.43 (1H,
br). FAB-MS m/z: 385 (MꢂHꢂ). Anal. Calcd for C21H12N2OF4: C, 65.63; H,
3.15; N, 7.29; F, 19.77. Found: C, 65.83; H, 3.04; N, 7.24; F, 19.63.
4-{[2-(Benzylamino)ethyl]amino}-2-(trifluoromethyl)benzonitrile (28)
4-[(2-Aminoethyl)amino]-2-(trifluoromethyl)benzonitrile was prepared from
ethylenediamine in a manner similar to that described for compound 7
(95%). To a solution of 4-[(2-aminoethyl)amino]-2-(trifluoromethyl)ben-
zonitrile (1.15 g, 5.0 mmol) in AcOH (30 ml) was added benzaldehyde
(640 mg, 6.0 mmol) and NaBH(OAc)3 (2.23 g, 10.0 mmol). After stirring at
ambient temperature for 4 h, the solution was concentrated in vacuo. The
residue was diluted with saturated aqueous NaHCO3 and extracted with
AcOEt. The organic layer was concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (AcOEt) to give the title com-
pound (920 mg, 58%) as a yellow oil. 1H-NMR (300 MHz, CDCl3) d 2.89—
2.98 (2H, m), 3.16—3.27 (2H, m), 3.81 (2H, s), 5.07—5.22 (1H, m), 6.60—
6.69 (1H, m), 6.85 (1H, d, Jꢀ2.2 Hz), 7.23—7.38 (5H, m), 7.54 (1H, d,
Jꢀ8.5 Hz). FAB-MS m/z: 320 (MꢂHꢂ).
4-(2-Oxopiperazin-1-yl)-2-(trifluoromethyl)benzonitrile (29) A mix-
ture of 4-{[2-(benzylamino)ethyl]amino}-2-(trifluoromethyl)benzonitrile
(28, 800 mg, 2.5 mmol) and 40% aqueous glyoxal (0.57 ml, 5.0 mmol) in
THF (10 ml) and H2O (5 ml) was stirred at ambient temperature for 14 h.
The solution was diluted with H2O and was extracted with AcOEt. The or-
ganic layer was concentrated in vacuo. The residue was purified by silica gel
column chromatography (n-hexane/AcOEtꢀ3/2) to give 4-(4-benzyl-2-ox-
opiperazin-1-yl)-2-(trifluoromethyl)benzonitrile (590 mg, 66%) as a color-
less powder.
A mixture of 4-(4-benzyl-2-oxopiperazin-1-yl)-2-(trifluo-
romethyl)benzonitrile (580 mg, 1.6 mmol) and 10% Pd-C (59 mg) in AcOH
(30 ml) was stirred under H2 at ambient temperature for 3 h. The precipitate
was filtered off and the filtrate was concentrated in vacuo. The residue was
purified by silica gel column chromatography (CHCl3/MeOHꢀ33/1) to give
the title compound (340 mg, 79%) as a yellow solid. 1H-NMR (300 MHz,
CDCl3) d 3.28 (2H, t, Jꢀ5.5 Hz), 3.76 (2H, s), 3.79 (2H, t, Jꢀ5.5 Hz),
7.69—7.76 (1H, m), 7.81—7.90 (2H, m). FAB-MS m/z: 270 (MꢂHꢂ).
4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)-3-oxopiper-
azine-1-carboxamide (30) The title compound was prepared from 4-(2-
oxopiperazin-1-yl)-2-(trifluoromethyl)benzonitrile (29) in a manner similar
to that described for compound 13 as a colorless powder (83%). mp 174—
4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)-3,6-dihy-
dropyridine-1(2H)-carboxamide (24) To a cooling solution of tert-butyl
4-[4-cyano-3-(trifluoromethyl)phenyl]-3,6-dihydropyridine-1(2H)-carboxy-
late (22, 400 mg, 1.14 mmol) in 1,4-dioxane (10 ml) was added trifluo-
roacetic acid (3 ml). After stirring at ambient temperature for 5 h, the solu-
tion was concentrated in vacuo. The residue was diluted with saturated aque-
ous NaHCO3 and was extracted with AcOEt. The organic layer was washed
with H2O, dried and concentrated to give 4-(1,2,3,6-tetrahydropyridin-4-yl)-
2-(trifluoromethyl)benzonitrile (290 mg, quant.) as a brown solid. The title
compound was prepared from 4-(1,2,3,6-tetrahydropyridin-4-yl)-2-(trifluo-
romethyl)benzonitrile in a manner similar to that described for compound 13
1
175 °C (AcOEt/iPr2O). H-NMR (400 MHz, DMSO-d6) d 3.82—3.89 (2H,
m), 3.90—3.99 (2H, m), 4.34 (2H, s), 7.05—7.14 (2H, m), 7.46—7.54 (2H,
m), 7.94 (1H, dd, Jꢀ8.4, 2.0 Hz), 8.14 (1H, d, Jꢀ2.0 Hz), 8.24 (1H, d,
Jꢀ8.4 Hz), 8.67 (1H, s). FAB-MS m/z: 407 (MꢂHꢂ). Anal. Calcd for
C19H14N4O2F4: C, 56.16; H, 3.47; N, 13.79; F, 18.70. Found: C, 55.98; H,
3.30; N, 13.90; F, 18.91.
as
a
colorless powder (47%). mp 194—196 °C (AcOEt). 1H-NMR
(400 MHz, DMSO-d6) d 2.56—2.65 (2H, m), 3.64—3.73 (2H, m), 4.17—
4.27 (2H, m), 6.62—6.70 (1H, m), 7.04—7.13 (2H, m), 7.45—7.53 (2H, m),

408
(ꢀ)-4-(4-Benzyl-2-ethylpiperazin-1-yl)-2-(trifluoromethyl)benzonitrile
Vol. 53, No. 4
less powder (2 steps 21%). mp 179—180 °C (AcOEt/iPr2O). 1H-NMR
(400 MHz, DMSO-d6) d 1.31 (6H, s), 3.52—3.61 (4H, m), 3.62—3.70 (2H,
m), 7.03—7.12 (2H, m), 7.32—7.42 (2H, m), 7.44—7.53 (2H, m), 7.89 (1H,
d, Jꢀ8.3 Hz), 8.46 (1H, s). FAB-MS m/z: 421 (MꢂHꢂ). Anal. Calcd for
C21H20N4OF4: C, 60.00; H, 4.80; N, 13.33; F, 18.08. Found: C, 59.94; H,
4.91; N, 13.30; F, 18.19.
Evaluation of Transcriptional Activity for Human Androgen Recep-
tor (a) Establishment of CHO Cells Stably Transfected with Human An-
drogen Receptor Gene and MMTV-Luciferase Reporter Gene or SV40-Lu-
ciferase Gene: Chinese hamster ovary (CHO) cells were maintained in
Alpha-modified Eagle’s medium supplemented with 10% Fetal Bovine
(31) 4-(4-Benzyl-3-oxopiperazin-1-yl)-2-(trifluoromethyl)benzonitrile was
prepared from 1-benzylpiperazin-2-one in a manner similar to that described
for compound 7 as a colorless solid (73%). A solution of 4-(4-benzyl-3-ox-
opiperazin-1-yl)-2-(trifluoromethyl)benzonitrile (2.0 g, 5.57 mmol) in dry
THF (20 ml) was added dropwise to a solution of LDA (738 mg, 6.68 mmol)
in dry THF (10 ml) at ꢄ78 °C. The reaction mixture was stirred for 20 min,
and iodoethane (0.67 ml, 8.36 mmol) was added to the reaction mixture at
ꢄ78 °C. The cold bath was removed and the reaction mixture was poured
into saturated NH4Cl at ꢄ10 °C and extracted with AcOEt. The organic
layer was washed with H2O and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography Serum (FBS). The culture medium of neomycin-resistant clone cells was
(n-hexane/AcOEtꢀ2/1) to give 4-(4-benzyl-2-ethyl-3-oxopiperazin-1-yl)-2- supplemented with 10% dextran-coated charcoal-stripped FBS (DCC-FBS)
(trifluoromethyl)benzonitrile (1.50 g, 72%) as a pale yellow form. A solution and 500 mg/ml of neomycin. The CHO cells were transfected at 40—70%
of borane THF complex in THF (6.10 ml, 1.0 M, 6.10 mmol) was added
dropwise to a solution of 4-(4-benzyl-2-ethyl-3-oxopiperazin-1-yl)-2-(triflu- oLUC; MMTV-luciferase reporter plasmid and pSG5-hAR; human androgen
confluence in 10-cm petri dishes with a total of 20 mg DNA (pMAMne-
oromethyl)benzonitrile (1.47 g, 3.80 mmol) in dry THF (30 ml) at 0 °C. After
stirring at 0 °C for 5 h, the reaction mixture was quenched with MeOH
(10 ml) and 1 M HCl (38 ml, 38 mmol) and concentrated in vacuo. The
receptor expression plasmid, or SV40-LUC; SV40-luciferase reporter plas-
mid containing neomycin resistant gene) by calcium phosphate mediated
transfection. The stable transfected cells were selected in the culture medium
residue was neutralized by saturated NaHCO3 and extracted with AcOEt. supplemented with neomycin. The selected clone was designated as
The organic layer was washed with H2O and concentrated in vacuo. AR/CHO#3 (human AR gene and MMTV-luciferase reporter gene inte-
The residue was purified by silica gel column chromatography grated CHO cell) or SV/CHO#10 (SV-40-luciferase reporter gene integrated
(n-hexane/AcOEtꢀ1/1) to give the title compound (670 mg, 47%) as a pale
CHO cell), respectively.
(b) Activities of the Tested Compounds to Inhibit Androgen Receptor
yellow oil. 1H-NMR (300 MHz, DMSO-d6) d 0.67—0.77 (3H, m), 1.40—
1.57 (1H, m), 1.76—1.92 (1H, m), 2.02—2.17 (2H, m), 2.77—2.97 (2H, m), Mediated Transcription Induced by DHT (AR Antagonistic Activity): The
3.08—3.20 (1H, m), 3.40 (1H, d, Jꢀ13.2 Hz), 3.60 (1H, d, Jꢀ13.2 Hz),
stable transfected AR/CHO#3 or SV/CHO#10 cells were plated onto 96 well
3.73—3.86 (1H, m), 3.94—4.08 (1H, m), 7.12—7.42 (7H, m), 7.79 (1H, d, luminoplates (Packard) at a density of 2ꢆ104 cells/well, respectively. Four to
Jꢀ8.7 Hz). FAB-MS m/z: 374 (MꢂHꢂ).
eight hours later, the medium was changed to the medium containing
DMSO, 0.3 nM of DHT, or 0.3 nM of DHT and the tested compound. At the
end of incubation, the medium was removed and then cells were lysed with
20 ml of lysis buffer [25 mM Tris–HCl (pH 7.8), 2 mM dithiothreitol, 2 mM
1,2-cyclohexanediamine-tetraacetic acid, 10% glycerol and 1% TritonX-
(ꢀ)-4-(4-Benzyl-2-isopropylpiperazin-1-yl)-2-(trifluoromethyl)ben-
zonitrile (32) The title compound was prepared from 2-iodopropane in a
manner similar to that described for compound 31 as a colorless oil (3 steps
1
56%). H-NMR (300 MHz, DMSO-d6) d 0.66 (3H, d, Jꢀ6.6 Hz), 0.81 (3H,
d, Jꢀ6.6 Hz), 1.91—2.13 (2H, m), 2.47—2.63 (1H, m), 2.81—2.95 (2H, m),
3.16—3.40 (2H, m), 3.57 (1H, d, Jꢀ13.2 Hz), 3.75—3.93 (2H, m), 7.14—
7.39 (7H, m), 7.73 (1H, d, Jꢀ9.0 Hz). FAB-MS m/z: 388 (MꢂHꢂ).
100]. Luciferase substrate [20 mM Tris–HCl (pH 7.8), 1.07 mM (MgCO3)4Mg-
(OH)2·5H2O, 2.67 mM MgSO4·7H2O, 0.1 mM EDTA, 33.3 mM dithiothreitol,
0.27 mM coenzyme A, 0.47 mM luciferin, 0.53 mM ATP] was added and
luciferase activity was measured with a ML3000 luminometer (Dynatech
4-(4-Benzyl-2,2-dimethylpiperazin-1-yl)-2-(trifluoromethyl)benzoni-
trile (33) The title compound was prepared using 2 equivalents of LDA Laboratories). AR antagonistic activities were calculated by formula below;
and iodomethane in a manner similar to that described for compound 31 as a
AR antagonistic activity (%)ꢀ100(IꢄX)/(IꢄB)
1
colorless oil (3 steps 43%). H-NMR (300 MHz, DMSO-d6) d 1.17 (6H, s),
2.30 (2H, s), 2.47—2.57 (2H, m), 3.23—3.33 (2H, m), 3.51 (2H, s), 7.22—
7.39 (5H, m), 7.46—7.52 (2H, m), 7.95 (1H, d, Jꢀ9.0 Hz). FAB-MS m/z:
374 (MꢂHꢂ).
I: (luciferase activity of AR/CHO#3)/(luciferase activity of SV/CHO#10)
in the presence of 0.3 nM of DHT
B: (luciferase activity of AR/CHO#3)/(luciferase activity of SV/CHO#10)
in the presence of DMSO
X: (luciferase activity of AR/CHO#3)/(luciferase activity of SV/CHO#10)
in the presence of 0.3 nM of DHT and the tested compound
(ꢀ)-4-[4-Cyano-3-(trifluoromethyl)phenyl]-3-ethyl-N-(4-fluoro-
phenyl)piperazine-1-carboxamide (34) A mixture of (ꢁ)-4-(4-benzyl-2-
ethylpiperazin-1-yl)-2-(trifluoromethyl)benzonitrile (31, 650 mg, 1.74 mmol)
and 10% Pd-C (65 mg) in MeOH (20 ml) was stirred under 1 atm H2 gas at
ambient temperature for 7 h. The precipitate was filtered off and the filtrate
was concentrated in vacuo. The residue was purified by silica gel column
chromatography (CHCl3/MeOHꢀ33/1) to give 4-(2-ethylpiperazin-1-yl)-2-
(trifluoromethyl)benzonitrile (460 mg, 93%) as a pale yellow oil. The title
compound was prepared from (ꢁ)-4-(2-ethylpiperazin-1-yl)-2-(trifluo-
romethyl)benzonitrile in a manner similar to that described for compound 13
as a colorless powder (70%). mp 180—182 °C (AcOEt/iPr2O). 1H-NMR
(400 MHz, DMSO-d6) d 0.87 (3H, t, Jꢀ7.3 Hz), 1.42—1.65 (2H, m), 3.13—
3.32 (3H, m), 3.78—3.88 (1H, m), 4.01—4.19 (3H, m), 7.04—7.12 (2H, m),
7.17—7.23 (1H, m), 7.26 (1H, d, Jꢀ2.4 Hz), 7.43—7.51 (2H, m), 7.83 (1H,
d, Jꢀ9.3 Hz), 8.61 (1H, s). FAB-MS m/z: 421 (MꢂHꢂ). Anal. Calcd for
C21H20N4OF4: C, 60.00; H, 4.80; N, 13.33; F, 18.08. Found: C, 59.69; H,
4.71; N, 13.11; F, 17.97.
The concentration of compounds showing 50% of AR antagonistic activ-
ity, IC50 values, were obtained by nonlinear analysis using statistical analysis
system (SAS).
Evaluation of Antiandrogenic Activities in Castrated Immature Rats
Treated with Andorgen Male Wistar rats were supplied by Charles River
Japan Inc. (Atsugi). Prepubertal male rats aged 3 weeks were castrated by
the scrotal route under ether anesthesia. Three days after the castration,
testosterone propionate (TP, 0.5 mg/kg, s.c.) was administered once daily for
5 d alone or in combination with the tested compound (10—30 mg/kg, p.o.).
TP was dissolved in cotton seed oil containing 5% ethanol. The tested com-
pound was suspended with 0.5% methylcellulose. The rats were sacrificed
by excessive chloroform anesthesia 6 h after final dosing, and both ventral
prostates and seminal vesiclesꢂcoagulate glands were removed and
weighed. The antiandrogenic activity was expressed as a percentage of inhi-
bition of the TP effect (TP-treated rats were arbitrarily assigned a value of
0% and vehicle-treated rats a value of 100%).
(ꢀ)-4-[4-Cyano-3-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)-3-iso-
propylpiperazine-1-carboxamide (35) The title compound was prepared
from
(ꢁ)-4-(4-benzyl-2-isopropylpiperazin-1-yl)-2-(trifluoromethyl)ben-
zonitrile (32) in a manner similar to that described for compound 34 as a
colorless amorphous (2 steps 64%). H-NMR (400 MHz, DMSO-d6) d 0.72
Acknowledgements We thank the staff of the Division of Analytical
Science Laboratories for the elemental analysis and spectral measurements.
1
(3H, d, Jꢀ6.8 Hz), 1.02 (3H, d, Jꢀ6.8 Hz), 2.03—2.17 (1H, m), 3.05—3.17
(2H, m), 3.22—3.31 (1H, m), 3.86—3.97 (2H, m), 3.99—4.09 (1H, m),
4.18—4.28 (1H, m), 7.03—7.11 (2H, m), 7.22—7.32 (2H, m), 7.40—7.49
(2H, m), 7.79 (1H, d, Jꢀ8.8 Hz), 8.61 (1H, s). FAB-MS m/z: 435 (MꢂHꢂ).
Anal. Calcd for C22H22N4OF4: C, 60.82; H, 5.10; N, 12.90; F, 17.49. Found:
C, 60.81; H, 5.02; N, 12.79; F, 17.22.
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