Synthesis of diphenylmethyl analogues and their affinity for the melanocortin-4 receptor and the serotonin transporter

Article abstract of DOI:10.1248/cpb.55.1044

While examining antagonists of the melanocortin-4 receptor (MC4 receptor), we found that compound 12b, containing a diphenylmethyl moiety, had a relatively high affinity for the MC4 receptor. When diphenylmethyl analogues were further examined, compounds

Full text of DOI:10.1248/cpb.55.1044

1044
Chem. Pharm. Bull. 55(7) 1044—1050 (2007)
Vol. 55, No. 7
Synthesis of Diphenylmethyl Analogues and Their Affinity for the
Melanocortin-4 Receptor and the Serotonin Transporter
Dai NOZAWA,* Taketoshi OKUBO, Takaaki ISHII, Shigeyuki CHAKI, Shigeru OKUYAMA, and
Atsuro NAKAZATO
Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd.; 1–403 Yoshino-cho, Kita-ku, Saitama, Saitama
331–9530, Japan. Received March 12, 2007; accepted April 11, 2007; published online April 12, 2007
While examining antagonists of the melanocortin-4 receptor (MC4 receptor), we found that compound 12b,
containing a diphenylmethyl moiety, had a relatively high affinity for the MC4 receptor. When diphenylmethyl
analogues were further examined, compounds 12c and 18 were also found to exhibit a high affinity for the MC4
receptor (IC50ꢀ46.7 nM and 33.2 nM, respectively). Furthermore, compound 12c was also found to show a high
affinity for the serotonin transporter (IC50ꢀ10.7 nM). Here, we describe the synthesis and biological evaluation of
various diphenylmethyl analogues in relation to their actions on the MC4 receptor and the serotonin transporter.
Key words melanocortin-4 receptor; serotonin transporter; depression; anxiety
Adrenocorticotropic hormone (ACTH) and melanocyte- serotonin reuptake inhibitors (SSRIs) have since become
stimulating hormones (a-MSH, b-MSH and g-MSH) are de- first-line drugs for the treatment of not only depression, but
rived by enzymatic processing from proopiomelanocortin also anxiety disorders.21) Although most patients are success-
(POMC) and are collectively referred to as the melano- fully treated with SSRIs, the existence of non-responders and
cortins. Five melanocortin receptors (MC1—MC5 receptors) of patients who respond slowly to the drug remains a draw-
belonging to the seven-transmembrane G-protein-coupled re- back of SSRIs therapy. Therefore, much effort is being ex-
ceptor family have been reported to date.1—3) The MC1 re- pended on the development of new antidepressants with
ceptor, bound mainly by a-MSH, is prominently expressed mono-aminergic mechanisms of action. Previously, we re-
in the skin and melanoma cells and plays a major role in reg- ported that 2 (MCL0042; Fig. 1) exhibited relatively high
ulating skin pigmentation. The MC2 receptor, bound by only affinities for both the MC4 receptor and the serotonin trans-
ACTH, is prominently expressed in the adrenal cortex and is porter. This compound, with its unique activity for both the
involved in steroidogenesis. The MC3 receptor, bound by MC4 receptor and the serotonin transporter, was also shown
both a- and g-MSH with equal affinity, is widely expressed to exhibit antidepressant and anxiolytic-like effects.18) Thus,
in the central nervous system as well as the placenta and drugs exhibiting a combination of MC4 antagonism and
plays a role in fat metabolism and energy homeostasis to- serotonin transporter inhibition might represent a new class
gether with the MC4 receptor. The MC4 receptor, which ex- of antidepressants.
hibits higher affinities for a-MSH and b-MSH than for g-
In this paper, we report the synthesis and biological evalu-
MSH, is primarily expressed in the brain. The MC5 receptor, ation of various diphenylmethyl analogues in relation to their
bound by a-MSH, is expressed in various peripheral tissues actions on the MC4 receptor. Moreover, selected compounds
and plays a role in exocrine gland function.
with a high affinity for the MC4 receptor were also examined
Numerous studies have suggested that the MC4 receptor is to determine their affinity for the serotonin transporter.
involved in the regulation of feeding behavior, energy home- Chemistry Charts 1—3 show the synthesis of com-
ostasis, sexual functions and protection against tumor-in- pounds 12a—p and 18—20. Compounds 12a—p were pre-
duced reductions in body weight.4—13) In addition, this recep- pared using two synthetic routes (Methods A and B) from
tor has been the focus of interest for its possible relationship
to stress and the regulation of emotional behavior14—16) in re-
lation to conditions such as depression and anxiety.17—20) The
findings to date indicate that the MC4 receptor may be a
promising target for the development of drugs for the above-
mentioned conditions, and numerous agonists25—29) and an-
tagonists22—24,30—40) of the MC4 receptor have been devel-
oped. We previously reported that the MC4 receptor antago-
nist 3 (MCL0129; Fig. 1) exhibited antidepressant, anxi-
olytic, and anti-stress effects in a variety of animal models.17)
These findings suggest that MC4 receptor blockade may be a
useful approach for treating subjects with depressive and
anxiety disorders. This hypothesis is supported by the
demonstration in various rodent models of depression and
anxiety that other MC4 receptor antagonists also exert anti-
depressant and anxiolytic effects.19)
Serotonin transporter blockade has been recognized as
Fig. 1. Bis-Piperazine Compounds as MC4 Receptor Antagonists
being effective for the treatment of depression, and selective
∗ To whom correspondence should be addressed. e-mail: dai.nozawa@po.rd.taisho.co.jp © 2007 Pharmaceutical Society of Japan

July 2007
1045
Compounds 18—20 were also synthesized from com-
pound 11 (Chart 3). The reaction of 11 with alkyl halide 21
i
in the presence of Pr2NEt (yielding 18) and followed by hy-
drolysis yielded 19. Compound 20 was prepared from 19 by
treatment with thionyl chloride, followed by treatment with
aqueous NH3 (Method C; Chart 3).
Results and Discussion
The affinities of all the compounds were evaluated by as-
sessing their binding to the membranes of COS-1 cells ex-
pressing the human MC4 receptor and estimating the result-
ing affinities using the inhibition curve for [125I]Nle4-D-Phe7-
a-MSH binding17); the respective IC50 values are shown in
Tables 1 and 2. In addition, selected compounds were tested
for their affinity to the serotonin transporter (SERT) by as-
sessing [3H]Paroxetine binding to the rat cortical membrane
Reagents and conditions: (a) CHCl3, reflux; (b) NaBH4, EtOH, 50 °C and then 4 M
HCl/EtOAc; (c) 9, SOCl2, benzene, 50 °C and then N-R1-piperazine (15), iPr2NEt, ben-
i
zene, 60 °C (78% from 7); (d) KOH, EtOH, reflux (85%); (e) Pr2NEt, DMF, 60 °C (Table 3).
(58%)
We previously reported that compound 1 (Fig. 1), a bis-
Chart 1. Synthesis of Compound 12c; General Synthetic Method for
12b, 12c, 12e and 12h—o (Method A)
piperazine compound with a carbonyl group that was identi-
fied in a high-throughput screening against an internal com-
pounds library, exhibited moderate affinity for the MC4 re-
ceptor (IC50ꢀ399 nM).23) Bis-piperazine analogues with a
carbonyl group (including 1), antagonists of the MC4 recep-
tor, have also been reported by Arasasingham et al.22) They
also reported that bis-piperazine compound 6 (Table 1), with-
out the carbonyl group, exhibited almost no affinity for the
MC4 receptor. These findings indicate that the carbonyl
group is crucial for the binding of bis-piperazine compounds
to the MC4 receptor. However, compound 1 has a beta amino
ketone moiety in its structure, possibly resulting in the beta-
elimination of the amine part. Therefore, we attempted to de-
sign non-carbonyl compounds with a higher affinity for the
MC4 receptor.
Reagents and conditions: (f) EDC, CHCl3, rt; (g) LiAlH4, THF, reflux (73% from 11)
Chart 2. Synthesis of Compound 12a; General Synthetic Method for
12a, 12f, 12g and 12p (Method B)
At first, to increase the binding affinity of non-carbonyl
compound 6 for the MC4 receptor, we investigated a non-
carbonyl compound with a longer length of an alkylene
linker moiety between the piperazine ring and the benzene
ring, designed based on the results of previously reported in-
formation regarding the structure–activity relationships
(SARs) on the bis-piperazine compounds.23) As expected, the
resultant non-carbonyl compound 12a, with a four-carbon
linker, exhibited a higher affinity for the MC4 receptor (12a:
IC50ꢀ1050 nM) than compound 6 with a three-carbon linker.
We next focused attention on p-electron of carbonyl
group, and anticipated that a phenyl group having p-electron
i
Reagents and conditions: (h) Pr2NEt, DMF, 70 °C (89%); (i) HCl, reflux (72%); (j)
SOCl2, benzene, 80 °C and then aq. NH3, rt (quant.)
Chart 3. Synthesis of Compounds 18—20 (Method C)
fluoroacetophenone 7 via compound 1123) (Charts 1, 2). The could be used as an alternative to the carbonyl group. We
reaction of 7 with N-CO2Et-piperazine 14 followed by the re- prepared compound 12b, in which a phenyl group was intro-
duction of the carbonyl group with NaBH4 yielded 9. Chlori- duced into the benzyl position of compound 12a, and evalu-
nation of the hydroxyl group of 9 with thionyl chloride fol- ated the affinity of the resultant compound for the MC4 re-
lowed by coupling with N-R1-piperazine 15 in the presence ceptor. Fortunately, compound 12b showed a much higher
i
of Pr2NEt yielded 10. Removal of the ethoxycarbonyl group affinity for the MC4 receptor (IC50ꢀ153 nM) than the lead
of 10 using KOH yielded 11. Compounds 11 were converted compound 12a (IC50ꢀ1050 nM) and also had a higher affinity
to 12b, 12c, 12e and 12h—o by treatment with alkyl halides than compound 1 (IC50ꢀ399 nM). Therefore, we started to
(16 for 12c) in the presence of iPr2NEt (Method A; Chart 1). examine diphenylmethyl analogues with a higher affinity for
Compound 12d was obtained by the hydrogenation of 12j the MC4 receptor.
using Pd–C as a catalyst. Furthermore, compound 12a was
Next, to increase the binding affinity for the MC4 receptor,
prepared by the reduction of 13, which had been prepared we investigated an iso-propyl group at R1, because the iso-
from 11 by condensation with carboxylic acid 17, using propyl group was previously reported to be an optimized
LiAlH4. Compounds 12f, 12g and 12p were also prepared group in bis-piperazine analogues.23) Moreover, we explored
from 11 by the same synthetic method as that for 12a the effects of introducing fluorine atoms at the benzene ring
(Method B; Chart 2).
on the binding affinity for the receptor. The resultant 12c,

1046
Vol. 55, No. 7
Table 1. Binding Affinity for the MC4 Receptor
Table 2. Binding Affinity for the MC4 Receptor
Compound
IC50 (nM)
18
19
20
33.2
2960
618
Compound
R1
R2
IC50 (nM)
6
Me
Me
3829
1050
Table 3. Binding Affinities for the MC4 Receptor and SERT
12a
IC50 (nM)
Compound
12b
12c
12d
Me
153
MC4
SERT
2
3
4
5
12c
12i
118
8.13
11.2
33.0
46.7
64.5
42.3
383
3130
1370
10.7
444
isoPr
isoPr
46.7
43.8
with the iso-propyl group and fluorine atoms at the benzene
ring, showed a high affinity (IC50ꢀ46.7 nM) for the MC4 re-
ceptor, and the iso-propyl group was employed in subsequent
research efforts to explore diphenylmethyl analogues further.
We then explored the effects of benzene ring substituents
in 12c on the binding affinity for the MC4 receptor. Substitu-
tion with a methyl (12f) or methoxy (12g) group at position 4
of the benzene ring resulted in a decrease in affinity for the
MC4 receptor (12f: IC50ꢀ97.7 nM and 12g: IC50ꢀ224 nM),
compared to that of a fluorine-substituted compound, 12c
(IC50ꢀ46.7 nM). Compound 12d substituted with a fluorine
atom at position 3 of the benzene ring exhibited almost the
same affinity for the MC4 receptor (IC50ꢀ43.8 nM) as that of
12c with fluorine substitution at position 4, whereas substitu-
tion with a fluorine atom at position 2 resulted in a 2-fold de-
crease in the binding affinity (12e: IC50ꢀ90.8 nM). These re-
sults suggest that substitution at positions 3 or 4 of the ben-
zene ring might be more favorable for obtaining an increased
binding affinity for the MC4 receptor than substitution at po-
sition 2.
12e
12f
isoPr
isoPr
90.8
97.7
12g
12h
isoPr
Me
224
92.0
12i
12j
isoPr
isoPr
64.5
45.1
Next, we examined diphenylmethyl analogues with other
alkylene linkers between the piperazine ring and the
diphenylmethyl moiety. At first, we focused on the confor-
mational rigidity of the carbonyl group of compound 1 and
introduced a carbon–carbon double bond to 12b. The result-
ant 12h, containing a carbon–carbon double bond, was tested
for its affinity for the MC4 receptor. The result revealed that
12h exhibited a slightly higher affinity for the MC4 receptor
(IC50ꢀ92.0 nM) than 12b, without a carbon–carbon double
bond. This finding encouraged us to optimize the binding
affinity of this series of compounds containing a carbon–car-
bon double bond. Compounds 12i, 12j and 12k, substituted
with a fluorine atom at positions 4, 3 or 2 of the benzene
ring, respectively, exhibited similar affinities (IC50ꢀ64.5,
45.1 and 68.2 nM, respectively) to those of compounds with-
out a carbon–carbon double bond (12i vs. 12c, 12j vs. 12d,
12k vs. 12e). We next prepared compounds 12l—o substi-
tuted with a chlorine atom (12l), methoxy (12m), trifluoro-
methyl (12n) or trifluoromethoxy (12o) group at position 4
of the benzene ring and tested the binding affinities of these
compounds for the MC4 receptor. The results showed that
12k
12l
isoPr
isoPr
68.2
83.6
12m
isoPr
148
12n
12o
isoPr
isoPr
365
87.5
12p
isoPr
52.2

July 2007
1047
Experimental
none of these compounds exhibited a higher affinity for the
receptor than 12i (12l: IC50ꢀ83.6 nM, 12m: IC50ꢀ148 nM,
12n: IC50ꢀ365 nM, 12o: IC50ꢀ87.5 nM). These results sug-
gest that a steric bulk at this position may not be well toler-
Melting points were determined on a Yanaco MP-500D melting point ap-
paratus and are uncorrected. Proton nuclear magnetic resonance (1H-NMR)
spectra were obtained using a Varian Gemini 2000 (200 MHz) or Varian
Unity Inova 300 (300 MHz). Chemical shifts are reported in parts per mil-
ated. We then investigated diphenylmethyl analogues with lion relative to tetramethylsilane (TMS) as an internal standard. IR spectra
were recorded on a Perkin–Elmer Spectrum One FT-IR spectrometer. Mass
spectra (MS) were obtained on Micromass Platform LC (ES). High resolu-
tion spectra were recorded on a Micromass Q-TOF2 instrument. Elemental
analyses were performed by a Perkin–Elmer 2400 or a Yanaco MT-6. Silica
longer alkylene linkers between the piperazine ring and the
diphenylmethyl moiety. The results showed that 12p, with a
four-carbon linker, exhibited a similar affinity for the MC4
receptor (IC50ꢀ52.2 nM) to 12i, with a three-carbon linker.
Since the introduction of the carbon–carbon double bond
had no significant decrease of the binding affinity for MC4
receptor, we attempted to explore the effects of introducing
RA (Chart 3) on the binding affinity for the receptor (Table
2). The introduction of a methoxycarbonyl group at RA re-
sulted in a slight increase in affinity for the MC4 receptor
(18: IC50ꢀ33.2 nM), compared with that of a non-substituted
compound, 12c. Thus, a methoxycarbonyl group at RA had a
favorable effect on the binding affinity for the MC4 receptor:
however, a carboxyl or carbamoyl group at RA proved to be
unfavorable (19: IC50ꢀ2960 nM and 20: IC50ꢀ618 nM).
gel C-200 (100—200 mesh, Wako Pure Chemical) and Chromatorex NH
(100—200 mesh, Fuji Silysia Chemical Ltd.) were used for column chro-
matography, using the solvent systems (volume ratios) indicated below.
General Methods for the Synthesis of (ꢁ)-12b—e and (ꢁ)-12h—o
(Method A). (ꢁ)-1-[4,4-Bis(4-fluorophenyl)butyl]-4-[2-(4-fluorophenyl)-
2-(4-isopropylpiperazin-1-yl)ethyl]piperazine 3 Maleate ((ꢁ)-12c)
A
mixture of (ꢁ)-1-[1-(4-fluorophenyl)-2-piperazin-1-ylethyl]-4-isopropyl-
piperazine (ꢁ)-1123) (0.20 g, 0.6 mmol), 1,1ꢂ-(4-chlorobutane-1,1-diyl)bis(4-
i
fluorobenzene) 16 (0.42 g, 1.5 mmol) and Pr2NEt (0.26 ml, 1.5 mmol) in
DMF (5 ml) was heated at 60 °C for 6 h. The mixture was partitioned be-
tween EtOAc and saturated aqueous NaHCO3, and separated organic phase
was washed with brine. The organic phase was dried over Na2SO4, filtered
and concentrated in vacuo. The residue was chromatographed on
Chromatorex NH (hexane/EtOAc 9 : 1) to obtain (ꢁ)-1-[4,4-bis(4-fluo-
rophenyl)butyl]-4-[2-(4-fluorophenyl)-2-(4-isopropylpiperazin-1-
yl)ethyl]piperazine (0.20 g, 58%) as an oily product. 1H-NMR (300 MHz,
CDCl3) d: 1.01 (6H, d, Jꢀ6.5 Hz), 1.39—1.44 (2H, m), 1.90—2.02 (2H, m),
2.18—2.67 (20H, m), 2.82 (1H, dd, Jꢀ6.2, 12.8 Hz), 3.51 (1H, t, Jꢀ6.8 Hz),
Selected compounds with a high affinity for the MC4 re-
ceptor were also examined for their affinity to SERT (Table
3). Although 3, 4 and 5 (Fig. 1), antagonists of the MC4 re-
ceptor previously reported by us,23) and 12i had a higher 3.83 (1H, t, Jꢀ7.6 Hz), 6.88—7.01 (6H, m), 7.09—7.21 (6H, m). MS (ESI,
Pos) m/z: 579 (MꢃH)ꢃ. The above free base (0.18 g, 0.31 mmol) was dis-
solved in EtOH (2 ml), and to the solution was added a solution of maleic
acid (0.11 g, 0.95 mmol) in EtOH (1 ml). After stirred at room temperature
for 1 h, the resulting precipitate was collected by filtration to obtain (ꢁ)-1-
affinity for the MC4 receptor than for SERT, 12c exhibited a
high affinity (IC50ꢀ10.7 nM) for SERT. Interestingly, the only
difference between 12c and 12i was the presence of a car-
bon–carbon double bond, yet 12c exhibited a high affinity for
SERT (IC50ꢀ10.7 nM) while 12i had a low affinity for SERT
(IC50ꢀ444 nM). These results suggest that, in the case of
these analogues, the carbon–carbon double bond might sig-
nificantly impact the affinity for SERT. Furthermore, the
finding that the affinities of 12c for both the MC4 receptor
and SERT were greater than those of 2, which exhibited anti-
depressant and anxiolytic-like effects,18) suggests that 12c
may represent a promising tool for investigating the patho-
genesis of stress-related disorders, such as depression and
anxiety.
[4,4-bis(4-fluorophenyl)butyl]-4-[2-(4-fluorophenyl)-2-(4-isopropylpiper-
azin-1-yl)ethyl]piperazine 3 maleate (ꢁ)-12c (0.20 g, 70%) as a crystal. H-
1
NMR (300 MHz, DMSO-d6) d: 1.20 (6H, d, Jꢀ6.5 Hz), 1.41—1.52 (2H, m),
1.88—2.12 (2H, m), 2.55—3.65 (22H, m), 3.95—4.03 (1H, m), 6.12 (6H,
s), 7.11—7.32 (12H, m). MS (ESI, Pos) m/z: 579 (MꢃH)ꢃ. Anal. Calcd for
C35H45F3N4·3C4H4O4·1.0H2O: C, 59.74; H, 6.29; N, 5.93. Found: C, 59.80;
H, 6.11; N, 5.66. mp 148—150 °C.
(ꢁ)-12b, 12e and (ꢁ)-12h—o were prepared by using method of (ꢁ)-
12c.
(ꢁ)-1-(4,4-Diphenylbutyl)-4-[2-(4-fluorophenyl)-2-(4-methylpiper-
azin-1-yl)ethyl]piperazine 3 Maleate ((ꢁ)-12b) (ꢁ)-12b was obtained as
a crystal. 1H-NMR (300 MHz, DMSO-d6) d: 1.41—1.55 (2H, m), 1.98—
2.08 (2H, m), 2.72 (3H, s), 2.74—3.46 (20H, m), 3.85—3.96 (2H, m), 6.10
(6H, s), 7.13—7.33 (14H, m). MS (ESI, Pos) m/z: 515 (MꢃH)ꢃ. Anal. Calcd
for C33H43FN4·3C4H4O4: C, 62.63; H, 6.42; N, 6.49. Found: C, 62.36; H,
6.42; N, 6.53. mp 181—182 °C (crystallized from EtOH).
Conclusion
We have reported the synthesis and evaluation of a new se-
ries of MC4 receptor ligands, diphenylmethyl analogues. Re-
moval of a carbonyl and methyl group from compound 1 pro- 12e was obtained as a crystal. H-NMR (300 MHz, DMSO-d6) d: 1.20 (6H,
vided our lead compound 12a. Structural optimization based
on the SAR study around the piperazine side chain moiety
(ꢁ)-1-[4,4-Bis(2-fluorophenyl)butyl]-4-[2-(4-fluorophenyl)-2-(4-iso-
propylpiperazin-1-yl)ethyl]piperazine 4 Hydrochloride ((ꢁ)-12e) (ꢁ)-
1
d, Jꢀ6.5 Hz), 1.61—1.76 (2H, m), 2.02—2.21 (2H, m), 2.51—2.70 (1H, m),
3.09—3.75 (21H, m), 4.03—4.21 (1H, m), 4.42—4.57 (2H, m), 7.07—7.43
(12H, m), 9.80 (1H, br s), 11.3—11.5 (1H, m). MS (ESI, Pos) m/z: 579
led to the identification of diphenylmethyl analogues with
high affinities for the MC4 receptor, namely, 12c and 18,
which showed an over 20-fold higher affinity for the receptor
than our lead compound 12a. Among these compounds, the
introduction of a carbon–carbon double bond, such as in 12i,
resulted in a higher selectivity for the MC4 receptor over
SERT. In contrast, compound 12c without a carbon–carbon
double bond exhibited high affinities for both the MC4 re-
ceptor and SERT. Considering that SSRIs are widely pre-
scribed for the treatment of both depressive and anxiety dis-
orders in clinical settings, this new class of compounds, such
as 12c, displaying dual high affinities for both the MC4 re-
ceptor and SERT might also be useful for the treatment of
these disorders.
(MꢃH)ꢃ. Anal. Calcd for C35H45F3N4·4HCl·1.2H2O: C, 56.33; H, 6.94; N,
7.51. Found: C, 56.08; H, 7.08; N, 7.50. mp 170—173 °C (crystallized from
a mixture of EtOAc and MeOH).
(ꢁ)-1-(4,4-Diphenylbut-3-en-1-yl)-4-[2-(4-fluorophenyl)-2-(4-meth-
ylpiperazin-1-yl)ethyl]piperazine 3 Maleate ((ꢁ)-12h) (ꢁ)-12h was ob-
1
tained as a crystal. H-NMR (300 MHz, DMSO-d6) d: 2.33—2.41 (2H, m),
2.72 (3H, s), 2.62—3.59 (20H, m), 3.91—3.99 (1H, m), 6.05 (1H, t,
Jꢀ7.2 Hz), 6.10 (6H, s), 7.14—7.47 (14H, m). MS (ESI, Pos) m/z: 513
(MꢃH)ꢃ. Anal. Calcd for C33H41FN4·3C4H4O4: C, 62.78; H, 6.21; N, 6.51.
Found: C, 62.50; H, 6.15; N, 6.57. mp 182—184 °C (crystallized from
EtOH).
(ꢁ)-1-[4,4-Bis(4-fluorophenyl)but-3-en-1-yl]-4-[2-(4-fluorophenyl)-2-
(4-isopropylpiperazin-1-yl)ethyl]piperazine 3 Maleate ((ꢁ)-12i) (ꢁ)-
1
12i was obtained as a crystal. H-NMR (300 MHz, DMSO-d6) d: 1.20 (6H,
d, Jꢀ6.5 Hz), 2.18—3.82 (23H, m), 3.96—4.08 (1H, m), 6.04 (1H, t,
Jꢀ7.2 Hz), 6.12 (6H, s), 7.09—7.32 (12H, m). MS (ESI, Pos) m/z: 577
(MꢃH)ꢃ. Anal. Calcd for C35H43F3N4·3C4H4O4·1.5H2O: C, 59.30; H, 6.14;
N, 5.89. Found: C, 59.31; H, 5.90; N, 5.62. mp 161—164 °C (crystallized
from EtOH).

1048
(ꢁ)-1-[4,4-Bis(3-fluorophenyl)but-3-en-1-yl]-4-[2-(4-fluorophenyl)-2-
Vol. 55, No. 7
0.092 mmol) was dissolved in EtOH (1 ml), treated with 4 M HCl in AcOEt
(0.10 ml) at room temperature, and the mixture was concentrated in vacuo.
The residue was crystallized in a mixture of AcOEt and MeOH, and the pre-
cipitate was collected by filtration to obtain (ꢁ)-1-[4,4-bis(3-fluoro-
phenyl)butyl]-4-[2-(4-fluorophenyl)-2-(4-isopropylpiperazin-1-yl)ethyl]-
piperazine 4 hydrochloride (ꢁ)-12d (45 mg, 66%) as a crystal. 1H-NMR
(300 MHz, DMSO-d6) d: 1.20 (6H, d, Jꢀ6.5 Hz), 1.56—1.69 (2H, m),
2.03—2.22 (2H, m), 2.52—2.69 (1H, m), 3.10—3.79 (22H, m), 3.99—4.21
(1H, m), 4.45—4.57 (1H, m), 6.98—7.05 (2H, m), 7.17—7.41 (10H, m),
9.90 (1H, br s), 11.5 (1H, br s). MS (ESI, Pos) m/z: 579 (MꢃH)ꢃ. Anal.
Calcd for C35H45F3N4·4HCl·0.5H2O: C, 57.30; H, 6.87; N, 7.64. Found: C,
57.34; H, 7.08; N, 7.60. mp 196—198 °C.
General Methods for the Synthesis of 12a, 12f, 12g and 12p (Method
B). (ꢁ)-1-[2-(4-Fluorophenyl)-2-(4-methylpiperazin-1-yl)ethyl]-4-(4-
phenylbutyl)piperazine 3 Maleate ((ꢁ)-12a) To a mixture of (ꢁ)-1-[1-
(4-fluorophenyl)-2-piperazin-1-ylethyl]-4-methylpiperazine (ꢁ)-11 (0.385 g,
1.26 mmol) and 4-phenylbutanoic acid 17 (0.217 g, 1.32 mmol) in CHCl3
(5 ml) was added EDC·HCl (0.253 g, 1.32 mmol) and the mixture was
stirred at room temperature for 15 h. The mixture was partitioned between
EtOAc and saturated aqueous NaHCO3, and separated organic phase was
washed with brine. The organic phase was concentrated in vacuo to ob-
tain crude (ꢁ)-1-[2-(4-fluorophenyl)-2-(4-methylpiperazin-1-yl)ethyl]-4-(4-
phenylbutanoyl)piperazine (ꢁ)-13 (0.555 g, quantitative) as an oily product.
A mixture of the above crude (ꢁ)-13 (0.555 g, 1.26 mmol) and LiAlH4
(48 mg, 1.3 mmol) in THF (5 ml) was refluxed for 1 h. To the mixture were
added Et2O (5 ml) and 25% aqueous NH3 (1 ml) at room temperature. After
stirred for 1 h, the mixture was filtered with Celite and the filtrate was con-
centrated in vacuo. The residue was chromatographed on Silica gel C-200
(CHCl3/MeOH 4 : 1) to obtain (ꢁ)-1-[2-(4-fluorophenyl)-2-(4-methylpiper-
azin-1-yl)ethyl]-4-(4-phenylbutyl)piperazine (0.40 g, 73%) as an oily prod-
(4-isopropylpiperazin-1-yl)ethyl]piperazine 4 Hydrochloride ((ꢁ)-12j)
1
(ꢁ)-12j was obtained as a crystal. H-NMR (300 MHz, DMSO-d6) d: 1.20
(6H, d, Jꢀ6.5 Hz), 2.17—2.30 (2H, m), 2.62—2.77 (2H, m), 3.10—3.78
(18H, m), 4.09—4.22 (1H, m), 4.49—4.61 (1H, m), 6.32 (1H, t, Jꢀ7.1 Hz),
6.70—6.94 (2H, m), 6.99—7.07 (5H, m), 7.20—7.54 (7H, m), 10.2 (1H,
br s), 11.9 (1H, br s). MS (ESI, Pos) m/z: 577 (MꢃH)ꢃ. Anal. Calcd for
C35H43F3N4·4HCl·0.5H2O: C, 57.46; H, 6.61; N, 7.66. Found: C, 57.51; H,
6.72; N, 7.62. mp 198—202 °C (crystallized from a mixture of EtOAc and
MeOH).
(ꢁ)-1-[4,4-Bis(2-fluorophenyl)but-3-en-1-yl]-4-[2-(4-fluorophenyl)-2-
(4-isopropylpiperazin-1-yl)ethyl]piperazine 3.8 Hydrochloride ((ꢁ)-12k)
1
(ꢁ)-12k was obtained as a crystal. H-NMR (300 MHz, DMSO-d6) d: 1.20
(6H, d, Jꢀ6.5 Hz), 2.08—2.26 (1H, m), 2.51—2.59 (1H, m), 3.12—3.77
(22H, m), 4.01—4.20 (1H, m), 4.42—4.58 (1H, m), 6.12 (1H, t, Jꢀ7.2 Hz),
7.05—7.41 (12H, m), 9.90 (1H, br s), 11.7 (0.8H, br s). MS (ESI, Pos) m/z:
577 (MꢃH)ꢃ. Anal. Calcd for C35H43F3N4·3.8HCl·0.3H2O: C, 58.33; H,
6.63; N, 7.77. Found: C, 58.56; H, 6.89; N, 7.71. mp 200—203 °C (crystal-
lized from a mixture of EtOAc and MeOH).
(ꢁ)-1-[4-(4-Chlorophenyl)-4-(4-fluorophenyl)but-3-en-1-yl]-4-[2-(4-
fluorophenyl)-2-(4-isopropylpiperazin-1-yl)ethyl]piperazine 3.8 Hydro-
chloride ((ꢁ)-12l) (a Mixture of E and Z) (ꢁ)-12l was obtained as a
1
crystal. H-NMR (300 MHz, DMSO-d6) d: 1.21 (6H, d, Jꢀ6.5 Hz), 2.12—
2.31 (1H, m), 2.45—2.59 (1H, m), 2.60—2.77 (1H, m), 3.18—3.41 (9H, m),
3.43—3.77 (10H, m), 4.12 (1H, t, Jꢀ7.2 Hz), 4.38—4.95 (3H, m), 6.09—
6.22 (1H, m), 7.09—7.53 (12H, m), 10.2 (1H, br s), 11.8 (0.8H, br s). MS
(ESI, Pos) m/z: 593 (MꢃH)ꢃ. Anal. Calcd for C35H43F2ClN4·3.8HCl·
0.5H2O: C, 56.90; H, 6.62; N, 7.58. Found: C, 56.71; H, 6.76; N, 7.49. mp
187—190 °C (crystallized from a mixture of EtOAc and MeOH).
(ꢁ)-1-(1-(4-Fluorophenyl)-2-{4-[4-(4-fluorophenyl)-4-(4-me-
thoxyphenyl)but-3-en-1-yl]piperazin-1-yl}ethyl)-4-isopropylpiperazine
3.8 Hydrochloride ((ꢁ)-12m) (a Mixture of E and Z) (ꢁ)-12m was ob-
tained as a crystal. 1H-NMR (300 MHz, DMSO-d6) d: 1.21 (6H, d,
Jꢀ6.5 Hz), 2.09—2.22 (1H, m), 3.19—3.77 (24H, m), 4.06—4.20 (1H, m),
4.30—4.60 (3H, m), 6.09—6.22 (1H, m), 7.09—7.49 (12H, m), 9.80 (1H,
br s), 11.5—11.7 (0.8H, m). MS (ESI, Pos) m/z: 589 (MꢃH)ꢃ. Anal. Calcd
for C36H46F2N4O·3.8HCl·0.9H2O: C, 58.15; H, 6.99; N, 7.54. Found: C,
58.34; H, 7.28; N, 7.53. mp 172—174 °C (crystallized from a mixture of
EtOAc and MeOH).
1
uct. H-NMR (200 MHz, CDCl3) d: 1.60—1.72 (4H, m), 2.22—2.69 (24H,
m), 2.84 (1H, dd, Jꢀ6.2, 12.8 Hz), 3.56 (1H, d, Jꢁ6.5 Hz), 6.91—7.05 (2H,
m) 7.10—7.35 (7H, m). MS (ESI, Pos) m/z: 439 (MꢃH)ꢃ. The above free
base (0.31 g, 0.71 mmol) was dissolved in EtOH (2 ml), and to the solution
was added a solution of maleic acid (0.25 g, 2.1 mmol) in EtOH (1 ml). After
stirred at room temperature for 1 h, the resulting precipitate was collected
by filtration to obtain (ꢁ)-1-[2-(4-fluorophenyl)-2-(4-methylpiperazin-1-
yl)ethyl]-4-(4-phenylbutyl)piperazine 3 maleate (ꢁ)-12a (0.45 g, 90%) as a
1
crystal. H-NMR (200 MHz, DMSO-d6) d: 1.50—1.70 (4H, m), 2.55—3.98
(26H, m), 6.10 (6H, s), 7.16—7.40 (9H, m). MS (ESI, Pos) m/z: 439
(MꢃH)ꢃ. Anal. Calcd for C27H39FN4·3C4H4O4: C, 59.53; H, 6.53; N, 7.12.
Found: C, 59.41; H, 6.55; N, 7.06. mp 185—187 °C.
(ꢁ)-1-[1-(4-Fluorophenyl)-2-(4-{4-(4-fluorophenyl)-4-[4-(trifluo-
romethyl)phenyl]but-3-en-1-yl}piperazin-1-yl)ethyl]-4-isopropylpiper-
azine 3.8 Hydrochloride ((ꢁ)-12n) (a Mixture of E and Z) (ꢁ)-12n was
obtained as a crystal. 1H-NMR (300 MHz, DMSO-d6) d: 1.21 (6H, d,
Jꢀ6.5 Hz), 2.05—2.22 (1H, m), 3.18—3.80 (21H, m), 4.00—4.55 (4H, m),
6.18—6.32 (1H, m), 7.10—7.49 (10H, m), 7.64—7.83 (2H, m), 9.80 (1H,
br s), 11.6—11.7 (0.8H, m). MS (ESI, Pos) m/z: 527 (MꢃH)ꢃ. Anal. Calcd
for C36H43F5N4·3.8HCl·2.0H2O: C, 53.96; H, 6.39; N, 6.99. Found: C,
54.23; H, 6.66; N, 7.15. mp 173—175 °C (crystallized from a mixture of
EtOAc and MeOH).
(ꢁ)-1-[1-(4-Fluorophenyl)-2-(4-4-(4-fluorophenyl)-4-[4-(trifluo-
romethoxy)phenyl]but-3-en-1-yl}piperazin-1-yl)ethyl]-4-isopropylpiper-
azine 4 Hydrochloride ((ꢁ)-12o) (a Mixture of E and Z) (ꢁ)-12o was
obtained as a crystal. 1H-NMR (300 MHz, DMSO-d6) d: 1.21 (6H, d,
Jꢀ6.5 Hz), 2.09—2.21 (1H, m), 3.18—4.23 (25H, m), 6.11—6.22 (1H, m),
7.10—7.44 (12H, m), 9.85 (1H, br s), 11.6—11.8 (1H, m). MS (ESI, Pos)
m/z: 643 (MꢃH)ꢃ. Anal. Calcd for C36H43FN4O·4HCl·0.1H2O: C, 54.71;
H, 6.02; N, 7.09. Found: C, 54.47; H, 5.97; N, 6.92. mp 212—215 °C (crys-
tallized from a mixture of EtOAc and MeOH).
12f, 12g and 12p were prepared by using method of (ꢁ)-12a.
(ꢁ)-1-[4,4-Bis(4-methylphenyl)butyl]-4-[2-(4-fluorophenyl)-2-(4-iso-
propylpiperazin-1-yl)ethyl]piperazine 4 Hydrochloride ((ꢁ)-12f) (ꢁ)-
1
12f was obtained as a crystal. H-NMR (300 MHz, DMSO-d6) d: 1.22 (6H,
d, Jꢀ6.5 Hz), 1.52—1.69 (2H, m), 1.97—2.10 (2H, m), 2.13 (6H, s), 2.55—
2.72 (1H, m), 3.10—3.73 (19H, m), 3.83 (1H, t, Jꢀ7.6 Hz), 4.09—4.22 (1H,
m), 4.35—4.60 (3H, m), 7.07 (4H, d, Jꢀ8.1 Hz), 7.17 (4H, d, Jꢀ8.2 Hz),
7.25 (2H, t, Jꢀ8.6 Hz), 7.34—7.43 (2H, m), 10.1 (1H, br s), 11.6 (1H, br s).
MS (ESI, Pos) m/z: 571 (MꢃH)ꢃ. Anal. Calcd for C37H51FN4·4HCl·
1.2H2O: C, 60.19; H, 7.84; N, 7.59. Found: C, 60.18; H, 7.91; N, 7.54. mp
174—176 °C (crystallized from a mixture of EtOAc and MeOH).
(ꢁ)-1-[4,4-Bis(4-methoxyphenyl)butyl]-4-[2-(4-fluorophenyl)-2-(4-iso-
propylpiperazin-1-yl)ethyl]piperazine 4 Hydrochloride ((ꢁ)-12g) (ꢁ)-
1
12g was obtained as a crystal. H-NMR (300 MHz, DMSO-d6) d: 1.22 (6H,
d, Jꢀ6.5 Hz), 1.53—1.70 (2H, m), 1.96—2.09 (2H, m), 2.11—2.28 (1H, m),
2.57—2.73 (1H, m), 3.09—3.42 (9H, m), 3.44—3.78 (9H, m), 3.72 (6H, s),
3.82 (1H, t, Jꢀ7.8 Hz), 4.17 (1H, t, Jꢀ12.4 Hz), 4.49—4.59 (1H, m), 5.15—
5.55 (2H, m), 6.82 (4H, d, Jꢀ8.7 Hz), 7.20 (4H, d, Jꢀ8.7 Hz), 7.25 (2H, t,
Jꢀ8.7 Hz), 7.35—7.44 (2H, m), 10.2 (1H, br s), 11.1 (1H, br s). MS (ESI,
Pos) m/z: 603 (MꢃH)ꢃ. Anal. Calcd for C37H51FN4O2·4HCl·1.5H2O: C,
57.29; H, 7.54; N, 7.22. Found: C, 57.20; H, 7.57; N, 7.18. mp 169—172 °C
(crystallized from a mixture of EtOAc and MeOH).
(ꢁ)-1-[4,4-Bis(3-fluorophenyl)butyl]-4-[2-(4-fluorophenyl)-2-(4-iso-
propylpiperazin-1-yl)ethyl]piperazine
4
Hydrochloride ((ꢁ)-12d)
A
mixture of (ꢁ)-1-[4,4-bis(3-fluorophenyl)but-3-en-1-yl]-4-[2-(4-fluoro-
phenyl)-2-(4-isopropylpiperazin-1-yl)ethyl]piperazine 4 hydrochloride (ꢁ)-
12j (0.25 g, 0.34 mmol), 5% Pd–C (25 mg) in MeOH (2.5 ml) was stirred at
room temperature under H2 atmosphere for 12 h. The mixture was parti-
tioned between EtOAc and saturated aqueous NaHCO3, and separated or-
ganic phase was washed with brine. The organic phase was dried over
Na2SO4, filtered and concentrated in vacuo. The residue was chro-
matographed on Chromatorex NH (hexane/EtOAc 9 : 1) to obtain (ꢁ)-1-
[4,4-bis(3-fluorophenyl)butyl]-4-[2-(4-fluorophenyl)-2-(4-isopropylpiper-
azin-1-yl)ethyl]piperazine (53 mg, 27%) as an oily product. 1H-NMR
(300 MHz, CDCl3) d: 1.01 (6H, d, Jꢀ6.5 Hz), 1.31—1.44 (2H, m), 1.92—
2.04 (2H, m), 2.10—2.66 (20H, m), 2.82 (1H, dd, Jꢀ5.8, 12.7 Hz), 3.53
(1H, t, Jꢀ7.0 Hz), 3.84 (1H, t, Jꢀ8.0 Hz), 6.80—7.02 (8H, m), 7.10—7.25
(4H, m). MS (ESI, Pos) m/z: 579 (MꢃH)ꢃ. The above free base (53 mg,
(ꢁ)-1-[5,5-Bis(4-fluorophenyl)pent-4-en-1-yl]-4-[2-(4-fluorophenyl)-2-
(4-isopropylpiperazin-1-yl)ethyl]piperazine 4 Hydrochloride ((ꢁ)-12p)
1
(ꢁ)-12p was obtained as a crystal. H-NMR (300 MHz, DMSO-d6) d: 1.20
(6H, d, Jꢀ6.5 Hz), 1.82—1.98 (2H, m), 2.03—2.22 (3H, m), 2.58—2.76
(1H, m), 3.02—3.16 (2H, m), 3.18—3.43 (8H, m), 3.45—3.79 (8H, m), 4.14
(1H, t, Jꢀ8.7 Hz), 4.30—4.90 (3H, m), 6.12 (1H, t, Jꢀ7.2 Hz), 7.06—7.43
(12H, m), 10.0 (1H, br s), 11.8 (1H, br s). MS (ESI, Pos) m/z: 591 (MꢃH)ꢃ.
Anal. Calcd for C36H45F3N4·4HCl·0.5H2O: C, 55.96; H, 6.91; N, 7.25.
Found: C, 55.91; H, 6.95; N, 7.25. mp 203—207 °C (crystallized from a
mixture of EtOAc and MeOH).

July 2007
Synthesis of 18—20 (Method C). (ꢁ)-Methyl 2,2-bis(4-fluoro-
1049
Binding Test. Material
[
125I][Nle4,D-Phe7]a-Melanocyte stimulating
hormone ([Nle4,D-Phe7]a-MSH) (specific radioactivity: 81.4 TBq/mmol)
was purchased from Amersham International (Buckinghamshire, England).
COS-1 cells were purchased from American Type Culture Collection
(Rocksville, MD, U.S.A.). [Nle4,D-Phe7]a-MSH was purchased from Penin-
sula Laboratories (Belmont, CA, U.S.A.). All other chemicals used in this
study were obtained commercially, and all were of the highest purity avail-
able.
phenyl)-5-{4-[2-(4-fluorophenyl)-2-(4-isopropylpiperazin-1-yl)ethyl]
piperazin-1-yl}pentanoate 3.5 Maleate ((ꢁ)-18) A mixture of (ꢁ)-1-[1-
(4-fluorophenyl)-2-piperazin-1-ylethyl]-4-isopropylpiperazine (ꢁ)-11 (1.0 g,
3.0 mmol), methyl 5-bromo-2,2-bis(4-fluorophenyl)pentanoate 21 (1.2 g,
i
3.1 mmol) and Pr2NEt (0.55 ml, 3.1 mmol) in DMF (10 ml) was heated at
70 °C for 5 h. The mixture was partitioned between EtOAc and saturated
aqueous NaHCO3, and separated organic phase was washed with brine. The
organic phase was dried over Na2SO4, filtered and concentrated in vacuo.
The residue was chromatographed on Chromatorex NH (hexane/EtOAc 4 : 1)
to obtain (ꢁ)-methyl 2,2-bis(4-fluorophenyl)-5-{4-[2-(4-fluorophenyl)-2-(4-
isopropylpiperazin-1-yl)ethyl]piperazin-1-yl}pentanoate (1.7 g, 89%) as an
oily product. 1H-NMR (300 MHz, CDCl3) d: 1.00 (6H, d, Jꢀ6.5 Hz), 1.11—
1.29 (2H, m), 1.58—1.70 (2H, m), 2.15—2.64 (20H, m), 2.82 (1H, dd,
Jꢀ6.0, 12.8 Hz), 3.53 (1H, t, Jꢀ6.8 Hz), 3.74 (3H, s), 6.97 (2H, t,
Jꢀ8.8 Hz), 7.12—7.23 (10H, m). MS (ESI, Pos) m/z: 637 (MꢃH)ꢃ. The
above free base (1.2 g, 1.9 mmol) was dissolved in EtOH (10 ml), and to the
solution was added a solution of maleic acid (0.66 g, 5.7 mmol) in EtOH
(5 ml). After stirred at room temperature for 1 h, the resulting precipitate was
collected by filtration to obtain (ꢁ)-methyl 2,2-bis(4-fluorophenyl)-5-{4-[2-
(4-fluorophenyl)-2-(4-isopropylpiperazin-1-yl)ethyl]piperazin-1-yl}pen-
tanoate 3.5 maleate (ꢁ)-18 (0.46 g, 18%) as a crystal. IR (KBr) cmꢄ1: 1729,
1623, 1605, 1576, 1511, 1480, 1386, 1356, 1228, 1165. 1H-NMR (300 MHz,
DMSO-d6) d: 1.20 (6H, d, Jꢀ6.5 Hz), 1.22—1.36 (2H, m), 1.91—2.10 (1H,
m), 2.22—2.37 (2H, m), 2.65—3.15 (16H, m), 3.22—3.45 (4H, m), 3.63
(3H, s), 3.96—4.03 (1H, m), 6.13 (7H, s), 7.17—7.29 (9H, m), 7.30—7.39
(3H, m). MS (ESI, Pos) m/z: 637 (MꢃH)ꢃ. Anal. Calcd for
C37H47F3O2N4·3.5C4H4O4·0.5H2O: C, 58.22; H, 5.94; N, 5.33. Found: C,
57.92; H, 6.02; N, 5.13. mp 104—106 °C.
[
125I][Nle4,D-Phe7]a-MSH Binding to Recombinant MC4 Receptor
COS-1 cells expressing the MC4 receptor, prepared according to the method
reported previously (Chaki et al. 2003),17) were washed with phosphate
buffered saline, scraped and pelleted by centrifugation. Cell pellets were ho-
mogenized with 50 mM Tris–HCl buffer (pH 7.4) containing 2 mM EDTA,
10 mM CaCl2, and 100 mM phenylmethylsulfonylfluoride, and centrifuged at
48000ꢅg for 20 min at 4 °C. The pellet was washed twice with the buffer,
and the final pellet was suspended in an assay buffer (50 mM Tris–HCl buffer
(pH 7.4) containing 2 mM EDTA, 10 mM CaCl2, 100 mM phenylmethylsul-
fonylfluoride and 0.1% bovine serum albumin (BSA)), and served as crude
membrane preparation for binding studies. Binding assays of [125I][Nle4,D-
Phe7]a-MSH were performed according to Chaki et al.17) Membranes were
incubated with [125I][Nle4,D-Phe7]a-MSH (0.2 nM) for 120 min at 25 °C, and
the reaction was terminated by rapid filtration over a GF/C filter presoaked
with 0.5% BSA, after which the filters were washed three times with the
buffer. Radioactivity was quantified in a g-counter. Nonspecific binding was
determined in the presence of 1 mM [Nle4,D-Phe7]a-MSH. Specific binding
was determined by subtracting nonspecific from total binding. In the compe-
tition assay, concentration of the test compound that caused 50% inhibition
of the specific binding (IC50 value) was determined from each concentra-
tion–response curve.
(ꢁ)-2,2-Bis(4-fluorophenyl)-5-{4-[2-(4-fluorophenyl)-2-(4-isopro-
pylpiperazin-1-yl)ethyl]piperazin-1-yl}pentanoic Acid 4 Hydrochloride
((ꢁ)-19) A solution of (ꢁ)-methyl 2,2-bis(4-fluorophenyl)-5-{4-[2-(4-flu-
orophenyl)-2-(4-isopropylpiperazin-1-yl)ethyl]piperazin-1-yl}pentanoate
(ꢁ)-18 (0.50 g, 0.79 mmol) in conc. HCl (20 ml) was refluxed for 12 h. After
the mixture was concentrated in vacuo, the residue was stirred in a mixture
of Et2O and EtOH. The resulting precipitate was collected by filtration to
obtain (ꢁ)-2,2-bis(4-fluorophenyl)-5-{4-[2-(4-fluorophenyl)-2-(4-isopropyl-
piperazin-1-yl)ethyl]piperazin-1-yl}pentanoic acid 4 hydrochloride (ꢁ)-19
(0.44 g, 72%) as an amorphous. IR (KBr) cmꢄ1: 1718, 1606, 1510, 1469,
1448, 1230, 1165. 1H-NMR (300 MHz, DMSO-d6) d: 1.22 (6H, d,
Jꢀ6.5 Hz), 1.41—1.56 (2H, m), 2.15—2.32 (1H, m), 2.34—2.42 (2H, m),
2.61—2.78 (1H, m), 3.08—3.79 (21H, m), 4.09—4.23 (1H, m), 4.50—4.72
(1H, m), 7.17 (4H, t, Jꢀ8.8 Hz), 7.22—7.32 (5H, m), 7.35—7.43 (3H, m),
10.3 (1H, br s), 11.7 (1H, br s). MS (ESI, Pos) m/z: 623 (MꢃH)ꢃ. Anal.
Calcd for C36H45F3N4O2·4HCl·1.5H2O: C, 54.35; H, 6.59; N, 7.04. Found:
C, 54.14; H, 6.70; N, 6.90.
References
1) Chaki S., Okuyama S., Peptides, 26, 1952—1964 (2005).
2) Chaki S., Nakazato A., Drugs Future, 29, 1065—1074 (2004).
3) Wikberg J. E. S., Eur. J. Pharmacol., 375, 295—310 (1999).
4) Fan W., Boston B. A., Kesterson R. A., Hruby V. J., Cone R. D., Nature
(London), 385, 165—168 (1997).
5) Forbes S., Bui S., Robinson B. R., Hochgeschwender U., Brenna M.
B., Proc. Natl. Acad. Sci. U.S.A., 98, 4233—4237 (2001).
6) Huszar D., Lynch C. A., Fairchild-Huntress V., Dunmore J. H., Fang
Q., Berkemeier L. R., Gu W., Kesterson R. A., Boston B. A., Cone R.
D., Smith F. J., Campfield L. A., Burn P., Lee F., Cell, 88, 131—141
(1997).
7) Kask A., Rago L., Wikberg J. E. S., Schiotch H. B., Eur. J.
Pharmacol., 360, 15—19 (1998).
8) Murphy B., Nunes C. N., Ronan J. J., Harper C. M., Beall M. J., Han-
away M., Fairhurst A. M., van der Ploeg L. H. T., MacIntyre D. E.,
Mellin T. N., Neuropeptides, 32, 491—497 (1998).
(ꢁ)-2,2-Bis(4-fluorophenyl)-5-{4-[2-(4-fluorophenyl)-2-(4-isopropyl-
piperazin-1-yl)ethyl]piperazin-1-yl}pentanamide 4 Hydrochloride ((ꢁ)-
20) A mixture of (ꢁ)-2,2-bis(4-fluorophenyl)-5-{4-[2-(4-fluorophenyl)-2-
(4-isopropylpiperazin-1-yl)ethyl]piperazin-1-yl}pentanoic acid 4 hydrochlo-
ride (ꢁ)-19 (0.20 g, 0.26 mmol) and one drop of DMF in SOCl2 (5 ml) was
stirred at 80 °C for 2 h, and concentrated in vacuo to obtain crude product as
an amorphous. A mixture of the above crude product in a mixture of 25%
aqueous NH3 (15 ml) and THF (15 ml) was stirred at room temperature for
1 h. The mixture was partitioned between EtOAc and brine. The separated
organic phase was dried over Na2SO4, filtered and concentrated in vacuo.
The residue was chromatographed on Chromatorex NH (hexane/EtOAc 1 : 1)
to obtained (ꢁ)-2,2-bis(4-fluorophenyl)-5-{4-[2-(4-fluorophenyl)-2-(4-iso-
propylpiperazin-1-yl)ethyl]piperazin-1-yl}pentanamide (80 mg, 50%) as an
9) Giraudo S. Q., Billington C. J., Levine A. S., Brain Res., 809, 302—
306 (1998).
10) Kask A., Rago L., Mutulis F., Pahkla R., Wikberg J. E. S., Schiotch H.
B., Biochem. Biophys. Res. Commun., 245, 90—93 (1998).
11) van der Ploeg L. H. T., Martin W. J., Howard A. D., Nargund R. P.,
Austin C. P., Guan X., Drisko J., Cashen D., Sebhat I., Patchett A. A.,
Figueroa D. J., DiLella A. G., Connolly B. M., Weinberg D. H., Tan C.
T., Palyha O. C., Pong S., MacNeil T., Rosenblum C., Vongs A., Tang
R., Yu H., Sailer A. W., Fong T. M., Huang C., Tota M., Chang R. S.,
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1
oily product. H-NMR (300 MHz, CDCl3) d: 1.01 (6H, d, Jꢀ6.5 Hz), 1.62
(4H, br s), 2.20—2.68 (20H, m), 2.81 (1H, dd, Jꢀ5.8, 12.8 Hz), 3.52 (1H, t,
Jꢀ7.5 Hz), 5.38 (1H, br s), 5.99 (1H, br s), 6.93—7.04 (5H, m), 7.12—7.21
(2H, m), 7.23—7.31 (5H, m). MS (ESI, Pos) m/z: 622 (MꢃH)ꢃ. The above
free base (80 mg, 0.13 mmol) was dissolved in EtOH (1 ml), treated with 4 M
HCl in 1,4-dioxane (0.20 ml) at room temperature, and the mixture was con-
centrated in vacuo to give (ꢁ)-2,2-bis(4-fluorophenyl)-5-{4-[2-(4-fluo-
rophenyl)-2-(4-isopropylpiperazin-1-yl)ethyl]piperazin-1-yl}pentanoic acid
4 hydrochloride (ꢁ)-20 (90 mg, quantitative) as an amorphous. IR (KBr)
cmꢄ1: 1668, 1605, 1510, 1469, 1454, 1229, 1165. 1H-NMR (300 MHz,
DMSO-d6) d: 1.21 (6H, d, Jꢀ6.5 Hz), 1.31—1.56 (2H, m), 2.12—2.42 (3H,
m), 2.60—2.79 (1H, m), 3.00—4.23 (23H, m), 4.41—4.58 (1H, m), 7.01—
7.20 (4H, m), 7.22—7.43 (8H, m), 10.2 (1H, br s), 11.5 (1H, br s). MS (ESI,
Pos) m/z: 622 (MꢃH)ꢃ. HR-MS m/z: 622.3745 (Calcd for C36H46F3N5O:
622.3733).

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Vol. 55, No. 7
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