Synthesis and in vitro evaluation of iodinated derivatives of piperazine as a new ligand for sigma receptor imaging by single photon emission computed tomography

Article abstract of DOI:10.1248/cpb.54.470

A new series of radioiodinated analogues of 1-[2-(3,4-dimethoxyphenyl) ethyl]-4-(3-phenylpropyl)piperazine (SA4503) was synthesized and evaluated as a potential brain sigma-1 receptor imaging ligands by single photon emission computed tomography (SPECT).

Full text of DOI:10.1248/cpb.54.470

470
Chem. Pharm. Bull. 54(4) 470—475 (2006)
Vol. 54, No. 4
Synthesis and in Vitro Evaluation of Iodinated Derivatives of Piperazine as
a New Ligand for Sigma Receptor Imaging by Single Photon Emission
Computed Tomography
Masahiko HIRATA, Tetsuya MORI, Seigo SOGA, Takuya UMEDA, and Yoshiro OHMOMO*
Osaka University of Pharmaceutical Sciences; 4–20–1 Nasahara, Takatsuki, Osaka 569–1094, Japan.
Received September 20, 2005; accepted December 15, 2005
A new series of radioiodinated analogues of 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine
(SA4503) was synthesized and evaluated as a potential brain sigma-1 receptor imaging ligands by single photon
emission computed tomography (SPECT). Iodinated analogues of SA4503 (4a—c) were prepared from piper-
azine in a high yield. The in vitro competition binding studies using [3H] DTG (sigma-1, 2), [3H] (ꢀ)-pentazocine
(sigma-1), and [3H] DTG in the presence of carbetapentane (sigma-2) as sigma receptor selective radioligands
were revealed that iodinated analogues 4a—c possess high affinities to sigma receptors (IC50: 4aꢁ7.1, 4bꢁ31.0,
and 4cꢁ77.3 nM). In particular, the affinity of 4a, bearing iodine at ortho position on the phenyl ring, was 4.4
times greater than SA4503, and 3 times greater than that of haloperidol. The meta-iodo analogue 4b was the
same to SA4503, the lead compound. The radioiodinated derivatives, [125I] 4a, 4b were synthesized no-carrier-
added from the corresponding tributyltin precursors by the iododestannylation reaction with high yields. The
binding of [125I] 4a, 4b have been characterized in the rat brain membranes. These compounds were indicated
single population binding to sigma receptor with high affinity (4a: Kdꢁ1.86ꢂ0.34 nM, Bmaxꢁ205ꢂ28.9 fmol/mg
protein, 4b: Kdꢁ3.30ꢂ0.51 nM, Bmaxꢁ231.5ꢂ13.8 fmol/mg protein). In vitro blocking studies were confirmed that
the high specificity of 4a, 4b. These results suggest that radioiodinated 4a and 4b are promising sigma receptors
imaging ligand for pursuing further in vivo studies.
Key words sigma receptor; radiopharmaceutical; single photon emission computed tomography (SPECT); SA4503; piperazine;
radioiodine
Sigma receptors were suggested as subtypes of the opiate (SA4503), have been reported that a potential drug candidate
receptors in 1976 by Martin et al.,1) however, the later studies with high binding affinity to sigma receptors.30,31) The bind-
of the pharmacological binding and behavior studies had to ing affinity of SA4503 to sigma receptors indicates over 100
be modified that they were non-opiate and non-phencyclidine times higher than that of other receptors. Therefore, SA4503
(PCP) binding site.2) From binding studies, sigma receptors has a potential to become lead compound of useful radio-
have been classified into at least two subtypes, termed sigma- pharmaceutical for sigma receptors studies in the brain. In
1 and sigma-2, for their pharmacological profiles and differ- fact, 11C and 18F labeled SA4503 have been reported and
ent molecular weights.2,3) They are widely distributed in the [11C] SA4503 has been successfully used for PET studies of
central nervous system (CNS), endocrine, immune and other sigma receptors in cat, monkey, and human brain.32—36) De-
peripheral organs.4—6) Furthermore, they have also been ex- spite attractive features associated with PET techniques, PET
pressed in a variety of human and rodent tumor cell lines.7,8) studies are still limited, since they usually require on-site cy-
Recently, cDNAs of sigma-1 were cloned, and their amino clotrons. On the other hand, SPECT studies are more com-
acid sequences were deduced.9—12) However, sigma-2 has not monly used in nuclear medicine clinics. We have explored
been cloned yet. It has been demonstrated that sigma recep- the feasibility of 123I radioiodinated sigma ligands as an alter-
tors are involved in a variety of physiological functions and native to [11C] SA4503 for functional sigma receptor studies
that sigma ligands have diversified pharmacological ef- in the brain with SPECT.
fects.13—18) In addition, sigma receptors may be associated
with such as Alzheimer’s disease,19) ischemia,20) schizophre- sigma receptor had been reported.30,31) It is also known that
nia,21) depression,22) and certain psychiatric disorders.23) allyl iodides are more stable than alkyl iodides.37,38) Thus, io-
Structure–activity relationships studies of SA4503 to
A
wide variety of structurally and pharmacologically diverse dinated analogue of SA4503 were designed on the basis of
compounds such as haloperidol, NE-100, DTG, carbetapen- the structure activity relationships to sigma receptors and in
tane, (ꢀ)-3-PPP, (ꢁ)-SKF10047, have been reported to bind vivo stability.
toward sigma receptors.
We reported here the synthesis of three iodinated ana-
Positron emission tomography (PET) and single photon logues amenable to radiolabeling 125I. In vitro studies on the
emission computed tomography (SPECT) have been success- inhibitory potency and selectivity toward sigma receptors
fully employed for non-invasive studies of the biochemical were also performed.
transformation and physiological processes in the living
human brain, utilizing organic molecules labeled with a Results and Discussion
positron emitter or a single photon emitter. For imaging
sigma receptors by SPECT,24—26) and PET,27—29) several radi- compound has carried out already by Fujimura et al.,39) how-
olabeled ligands have been developed. ever, the yield of this procedure was low. Therefore, iodi-
Chemical Synthesis The synthesis of SA4503 as lead
Recently, new high selective sigma-1 agonist, 1-[2(3,4- nated derivatives of SA4503 and SA4503 were synthesized
dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine by the reaction outlined in Chart 1, using a modified method
∗ To whom correspondence should be addressed. e-mail: Ohmomo@gly.oups,ac.jp
© 2006 Pharmaceutical Society of Japan

April 2006
471
Chart 1
of Moore et al. which introduced substituent into piperazine
directly.40)
1-(3,4-Dimethoxyphenylacetyl)piperazine (2) was synthe-
sized directly without the protection group by condensation
of 3,4-dimethoxyphenylacetic chloride. The reaction was
controlled acid conditions in the presence of some bromo-
phenol blue as the indicator (pH 3.0—4.6). This method was
superior in giving the mono-substitute piperazine analogue in
a high yield (78%) and less side reactions. Compounds 3a—
c were prepared by condensation of the corresponding
iodophenylpropionyl chloride in high yields (77—87%). The
desired compounds 4a—c were obtained by reduction of
the corresponding derivatives 3a—c using 1.0 M diborane-
tetrahydrofuran solution under argon gas in yields of 70—
93%. In a synthesis of SA4503, SA4503 was prepared in a
high yield (total yield 51.1%) using our method compared
with method of Fujimura et al. (lit.39) 26.2%). This yield was
about 2 times higher than that of Fujimura’s method, and our
method was successful in improvement of yield drastically.
In Vitro Competitive Binding Assay The binding affin-
ity of the synthesized compounds 4a—c to sigma receptors
in rat brain membranes were evaluated by competitive bind-
ing assay using [3H] DTG (sigma-1, 2), [3H] (ꢀ)-pentazocine
(sigma-1), and [3H] DTG with carbetapentane (sigma-2).41,42)
Several typical sigma ligands, haloperidol (sigma-1, 2 and
dopamine), DTG (sigma-1, 2), carbetapentane (sigma-1),
(ꢀ)-3-PPP (sigma-1), (ꢁ)-SKF10047 (sigma-1) and SA4503
(sigma-1), were also assessed under the same conditions.
Figure 1 displayed competitive binding curves of these com-
pounds, and 50% inhibition concentration (IC50) values were
summarized in Table 1. These results show that the new se-
ries of iodinated analogues 4a—c were indicated to possess
high affinity to brain sigma receptors (IC50: 4aꢂ7.1 nM,
4bꢂ31.0 nM, 4cꢂ77.3 nM). In particular, the IC50 value of 4a
was 4.4 times greater than that of the lead compound,
SA4503, and the IC50 value of 4b was the same as the IC50
Fig. 1. Inhibition of [3H] DTG Binding to Sigma-1, 2 Receptors (A),
[3H] (ꢀ)-Pentazocine Binding to Sgma-1 Receptors (B), and [3H] DTG
Binding to Sigma-2 Receptors in the Presence of Carbetapentane to Mask
Sigma-1 Site (C) in Rat Brain Membranes by, 4a (ꢀ), 4b (ꢁ), 4c (ꢂ),
SA4503 (ꢃ), Haloperidol (ꢄ), DTG (ꢅ), (ꢀ)-3-PPP (∗), Carbetapentane
(ꢆ), and (ꢁ)-SKF10047 (ꢃ)
Each point is the mean value of triplicate determinations.
Table 1. Inhibitory Effects of Sigma Ligands against Sigma Receptors
IC50 (nM)
Compound
[3H] DTG
(Sigma non-
selective)
[3H] (ꢀ)-
pentazocine
(Sigma-1)
[3H] DTGꢀ
carbetapentane
(Sigma-2)
4a
4b
4c
7.1ꢁ1.2
31.0ꢁ6.2
77.3ꢁ11.0
31.0ꢁ1.4
22.0ꢁ4.1
45.5ꢁ6.4
76.3ꢁ16.9
307.5ꢁ17.7
1650ꢁ70
6.4ꢁ0.7
8.9ꢁ2.8
19.2ꢁ2.8
4.0ꢁ1.9
10.2ꢁ1.9
54.5ꢁ6.6
160.0ꢁ7.8
55.7ꢁ4.7
65.3ꢁ4.1
27.7ꢁ4.0
145.0ꢁ7.1
530.0ꢁ11.0
3700ꢁ141
SA4503
Haloperidol
DTG
(ꢀ)-3-PPP
Carbetapentane
(ꢁ)-SKF10047
2.4ꢁ0.5
75.0ꢁ7.1
29.5ꢁ6.4
16.5ꢁ0.7
151.7ꢁ14.4
Data are mean valueꢁS.D. of triplicate determinations. DTG: 1,3-di-o-tolylguani-
dine, (ꢀ)-3-PPP: (ꢀ)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, SKF10047: N-allyl-
value of SA4503 (IC50ꢂ31.0 nM). The IC50 value of 4a was normetazocine.
3-times greater than that of haloperidol (IC50ꢂ22.0 nM),
known a potent sigma ligand,43) and the IC50 value of 4b was
comparable with that of haloperidol. The compound 4c was dicated high binding affinity to sigma receptors. In particular,
weakest affinity among three new analogues, but it was rank 4a indicated good characteristic for binding to sigma recep-
along with (ꢀ)-3-PPP which was one of the traditional sigma tors. In general, the compound against the receptor as a target
ligand (IC50ꢂ76.3 nM).44) Among them, compound 4a, 4b in- was attenuated by the introduction of a heavy heteroatom as

472
Vol. 54, No. 4
Chart 2
iodine. However, novel compounds 4a and 4b, which were
introduced iodine to ortho- and meta-position, increased
binding affinity to sigma receptors. These in vitro examina-
tions were confirmed the validity of the compound design of
new series of iodinated analogues for sigma receptors imag-
ing. The potent compound 4a and 4b, among the iodinated
derivatives tested in vitro, were chosen for further evalua-
tions.
Radioiodination The electrophilic iododestannylation
reaction offers several advantages for radioiodination, since
it is performed in short times under very mild conditions and
with very high regional selectivity, and also affords a high
specific radioactivity. Thus, the radiolabeled ligands,
Fig. 2. Blocking Effect of Various Drugs on [125I] 4a Binding (Top Panel)
and [125I] 4b Binding (Bottom Panel) in Rat Brain Membranes
[
125I] 4a, and [125I] 4b were performed using an iodo-
destannylation reaction with the corresponding tributyltin HT2), naloxone (opiate), atropine (muscarinic), (ꢄ)-nicotine
precursor by the reaction outlined in Chart 2.45) The tri-n- (nicotinic), (ꢄ)-propranolol (b, 5-HT1) and phentolamine
butylstannyl precursor 5a, 5b was prepared from its corre- (a), failed to block the [125I] 4a or 4b binding. Haloperidol
sponding 4a, 4b using bis(tri-n-butyltin) in the presence of a (sigma-1, -2, dopamine) was observed a significant reduction
catalytic amount of tetrakis-(triphenylphosphine)palladium. of radioactivity also binds dopamine receptor. But each of
The radioiodination of 5a, 5b was achieved using hydrogen
[
125I] 4a and 4b binding were not reduced by pretreated (ꢀ)-
peroxide as an oxidant and sodium [125I] iodide (specific ac- SCH23390, D1 ligand, or (ꢄ)-sulpiride, D2 ligand. These
tivity 74 GBq/mmol) in 0.1 M HCl/ethanol solution at room data were indicated the two radioiodinated ligands bound to
temperature, followed by HPLC purification. The radiochem- only sigma receptors.
ical yields of product, [125I] 4a, and [125I] 4b, were 90—94%
Saturation Binding of [125I] 4a and [125I] 4b to Rat
based on sodium [125I] iodide activity. The radiochemical pu- Brain Membranes For comparison with pharmacological
rity of [125I] 4a, and [125I] 4b were higher than 99% as as- characterization, the crude membrane fraction from rat brain
sessed by HPLC analysis, with specific radioactivity of ap- was used for further binding experiments. The saturation
proximately 74 GBq/mmol, respectively. This method should binding of [125I] 4a and [125I] 4b was studied with the use of
be a applicable for labeling 123I a suitable radioisotope (half- Scatchard analysis (Figs. 3, 4).
life 13 h and gamma ray energy of 159 keV) for in vivo imag-
The resulting linear Scatchard plot was consistent with the
ing with SPECT. Importantly, the tin precursors 5a, 5b were theory that [125I] 4a and [125I] 4b bind to single population of
stable over 6 months when stored at ꢄ20 °C and [125I] 4a, binding sites with high affinity and kinetic parameter, 4a:
and [125I] 4b can be readily generated.
Kdꢂ1.86ꢁ0.34 nM and Bmaxꢂ205ꢁ28.9 fmol/mg protein, 4b:
In Vitro Blocking Studies In vitro pharmacological Kdꢂ3.30ꢁ0.51 nM and Bmaxꢂ231.5ꢁ13.8 fmol/mg protein,
blocking study was performed to examine the degree of spe- respectively. These data confirm the possibility that similar
cific binding of [125I] 4a and [125I] 4b in rat brain membranes. parameter with [3H] (ꢀ) pentazocine and [3H] DTG as well
Figure 2 shows that the total amount of [125I] 4a (top panel) known sigma ligand, has indicated single class of binding
or [125I] 4b binding (bottom panel) were indicated significant site in rat brain membranes, ([3H] (ꢀ) pentazocine: Kdꢂ
reduction by the pretreatment with sigma ligands, such 6.90 nM, Bmaxꢂ280 fmol/mg protein, [3H] DTG: Kdꢂ15.8 nM,
as SA4503, haloperidol, DTG, (ꢁ)-pentazocine, (ꢀ)-3-PPP,
carbetapentane, rimcazole, and (ꢁ)-SKF10047. In addition,
B
maxꢂ291 fmol/mg protein).43,44) These results suggested that
[
125I] 4a and [125I] 4b bound to same binding site of [3H] (ꢀ)
the reduction rate of the radioactivity by treatment of these pentazocine and [3H] DTG, in rat brain membranes with high
ligands, was correlated with the avidity of each sigma ligand. affinity.
On the other hand, the non-sigma receptor ligands, such as
(ꢀ)-SCH23390 (D1), (ꢄ)-sulpiride (D2), spiperone (D2, 5-

April 2006
473
Fig. 3. Binding of [125I] 4a in Rat Brain Membranes
Left: binding curves of [125I] 4a; total binding (ꢂ), specific binding (ꢀ), non-specific binding (ꢁ). Right: scatchard plot of the binding. Data represent meanꢁS.D. of triplicate
determinations.
Fig. 4. Binding of [125I] 4b in Rat Brain Membranes
Left: binding curves of [125I] 4b; total binding (ꢂ), specific binding (ꢀ), non-specific binding (ꢁ). Right: scatchard plot of the binding. Data represent meanꢁS.D. of triplicate
determinations.
To the solution of 3,4-dimethoxyphenylacetic acid (2.00 g, 10.2 mmol) in
20 ml tetrahydrofuran was added thionyl chloride (20 ml) and heated at re-
flux for 2 h. The volatiles were removed in vacuo to give yellow oil as acid
chloride. Then, 4 M HCl was added to a solution of piperazine·6H2O (7.92 g,
Conclusion
This work demonstrated that a new series of iodinated
compound have been synthesized and characterized in vitro
receptor binding assay. Among the prepared compounds, 4a
and 4b were found to possess high inhibitory potency against
sigma receptors. Each of [125I] 4a and [125I] 4b were synthe-
sized by iododestannylation reaction with no-carrier-condi-
tions which can apply to label with 123I using for SPECT. The
binding and the blocking study data suggest that [125I] 4a and
40.8 mmol) in water (10 ml) containing some bromophenol blue until the
neutral tint of the indicator appeared. The acid chlorides were dissolved in
dry CHCl3 (2 ml), and 1 M NaOH were added in the piperazine solution at
intervals with vigorous stirring at 0 °C. The reaction mixture was stirred at
room temperature for 30 min, followed by, 0.5 N NaOH was added until pH
9. The mixture was extracted with CHCl3 (30 mlꢃ3). The combined organic
extracts were washed with water (30 ml), dried over sodium sulfate and the
solvent was removed in vacuo to give an oil. This oil was purified by silica
gel column chromatography (MeOH/CHCl3ꢂ5/1) to provide the pure prod-
uct.
[
125I] 4b were considered to be a candidate for further studies
as a SPECT radiopharmaceutical for imaging sigma recep-
tors. Further studies of these new agents are now in progress.
1
Yield 77.9%, IR (CHCl3): 2938, 1631, 1516, 1463, 1262 cmꢄ1. H-NMR
(CDCl3): 1.94 (1H, br, NH), 2.67 (2H, t, Jꢂ5.1 Hz, piperazine), 2.81 (2H, t,
Jꢂ5.1 Hz, piperazine), 3.43 (2H, t, Jꢂ5.1 Hz, piperazine), 3.62 (2H, t,
Jꢂ5.1 Hz, piperazine), 3.68 (2H, s, phCH2CO), 3.87 (6H, s, OCH3ꢃ2),
Experimental
All melting points are uncorrected. Proton nuclear magnetic resonance
(1H-NMR) spectra were recorded on a Varian Gemini-200 (200 MHz) spec- 6.74—6.83 (3H, m, aromatics). CI-HR-MS Calcd for C14H20N2O3 (MHꢀ)
trometer and the chemical shifts are reported in ppm downfield from an in-
ternal tetramethylsilane standard. Infrared (IR) spectra were recorded on a
m/z: 264.1474, Found: 264.1470.
Substituted 1-(3,4-Dimethoxyphenylacetyl)-4-(3-phenylpropionyl)-
JASCO IR-700 spectrometer. High resolution mass spectra (HR-MS) were piperazine (3a—c) 3-(Iodophenyl)propionyl acid (0.79 g, 3.0 mmol) was
obtained on a Hitachi M-80 instrument. The radio HPLC system used in-
cluded a Waters M-600 pump, a Lambda-Max 481 UV detector (254 nm), a
added to thionyl chloride (5 ml) and the solution was refluxed for 2 h. The
volatiles were removed in vacuo to give a light yellow oil. This acid chloride
Beckman 170 NaI radioactivity detector, and a Cosmosil 5C18-AR column was dissolved in CHCl3 (2 ml) and added dropwise to another flask contain-
3
(10ꢃ250 mm, Nacalai Tesque). The H radioactivity was determined by a ing compound 2 (0.50 g, 3.0 mmol) and triethylamine (0.30 g, 3.0 mmol) and
Packard TRI-CARB1600CA liquid scintillation spectrometer. The 125I ra- CHCl3 (20 ml). The reaction mixture was stirred for 15 h at room tempera-
dioactivity was measured using an Aloka ARC-300 NaI (Tl) gamma scintil-
lation counter.
[
ture and then washed with water (25 ml). The organic layer was dried with
K2CO3 and the solvent was evaporated in vacuo to afford the crude product
125I] NaI, [3H] DTG, and [3H] pentazocine were purchased from Amer- as yellow oil. This oil was purified by silica gel column chromatography
sham Japan. All other chemicals used were of reagent grade and were pur- (MeOH/CHCl3ꢂ5/1) to provide the pure product.
chased commercially. Wistar rats were obtained from Japan SLC Co., Ltd.
1-(3,4-Dimethoxyphenylacetyl)-4-[3-(2-iodophenyl)propionyl]piperazine
These animals were kept at least one week before the experiments. Animals (3a): Yield 87.6%, IR (CHCl3): 2962, 1639, 1515, 1459, 1261 cmꢄ1
.
1H-
chow and drinking water were allowed ad libitum. Animals were housed and NMR (CDCl3): 2.59 (2H, t, Jꢂ7.5 Hz, phCH2CH2), 2.90 (2H, t, Jꢂ7.5 Hz,
experiments were performed according to guidelines stipulated by the Osaka
University of Pharmaceutical Sciences Animal Care and Use Committee.
phCH2CH2), 3.29—3.58 (8H, m, piperazine), 3.68 (2H, s, phCH2CO), 3.86
(6H, s, OCH3ꢃ2), 6.76—6.83 (3H, m, aromatics), 6.99—7.55 (4H, m, aro-
Chemical Synthesis. 1-(3,4-Dimethoxyphenylacetyl)piperazine (2) matics). CI-HR-MS Calcd for C23H27IN2O4 (MHꢀ) m/z: 522.1017. Found:

474
Vol. 54, No. 4
522.1010.
man GF/B glass fiber filters presoaked in 0.5% polyethylenimine for at least
1-(3,4-Dimethoxyphenylacetyl)-4-[3-(3-iodophenyl)propionyl]piperazine 30 min at room temperature prior to use, followed by a rapid washing 3
(3b): Yield 90.5%, IR (CHCl3): 2962, 1639, 1515, 1459, 1261 cmꢄ1
NMR (CDCl3): 2.59 (2H, t, Jꢂ7.5 Hz, phCH2CH2), 2.90 (2H, t, Jꢂ7.5 Hz,
.
1H-
times with 4 ml of ice-cold 10 mM Tris–HCl buffer (pH 8.0). The radioactiv-
ity trapped on the filters was counted by scintillation spectrometer after an
phCH2CH2), 3.29—3.58 (8H, m, piperazine), 3.68 (2H, s, phCH2CO), 3.86 overnight extraction of counts in 10 ml scintillation fluid. These IC50 values
(6H, s, OCH3ꢃ2), 6.76—6.83 (3H, m, aromatics), 6.99—7.55 (4H, m, aro- were determined from displacement curves of the percent inhibition of
matics). CI-HR-MS Calcd for C23H27IN2O4 (MHꢀ) m/z: 522.1017. Found: [3H] DTG binding.
522.1014. Sigma-1 and sigma-2 receptor binding assays were examined the same
1-(3,4-Dimethoxyphenylacetyl)-4-[3-(4-iodophenyl)propionyl]piperazine manner described above except using [3H]-(ꢀ)-pentazocine (0.1 nmol,
(3c): Yield 86.7%, IR (CHCl3): 2930, 1640, 1515, 1431, 1261 cmꢄ1
NMR (CDCl3): 2.59 (2H, t, Jꢂ7.8 Hz, phCH2CH2), 2.90 (2H, t, Jꢂ7.8 Hz,
phCH2CH2), 3.33—3.58 (8H, m, piperazine), 3.69 (2H, s, phCH2CO), 3.87
(6H, s, OCH3ꢃ2), 6.76—6.83 (3H, m, aromatics), 6.96 (2H, d, Jꢂ8.4 Hz,
.
1H-
0.46 kBq) and [3H] DTG (0.1 nmol, 0.46 kBq) in the presence of carbetapen-
tane (0.1 mM) to mask sigma-1 binding site, respectively.
Synthesis of Precursors. Substitute 1-[2-(3,4-Dimethoxyphenyl)-
ethyl]-4-[3-(tributylstannylphenyl)propyl]piperazine (5a—c) The mix-
aromatics), 7.60 (2H, d, Jꢂ8.4 Hz, aromatics). CI-HR-MS Calcd for ture of compound 4a—c (0.20 g, 0.40 mmol) and bistributyltin (0.64 g,
C23H27IN2O4 (MHꢀ) m/z: 522.1017. Found: 522.1007.
Substitute 1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piper- anhydrous toluene (7 ml) was refluxed under argon for 17 h. After cooling,
azine (4a—c) The compounds 3a—c (0.91 g, 2.28 mmol) were dissolved the reaction mixture was filtered through celite. The filtrate was concentrated
1.12 mmol) and tetrakis(triphenylphosphine)palladium (30 mg, 26 mmol) in
in anhydrous tetrahydrofuran (5 ml), followed by, 1.0 M diborane-tetrahydro-
in vacuo. The oily residue was purified by silica gel column chromatography
furan solution were dropwised (17.0 ml) at room temperature under argon (MeOH/CHCl3ꢂ1/20) affording 4a—c.
gas. The reaction mixture was heated at reflux for 18 h. The resulting solu-
tion was added 1 M HCl (25 ml) at 0 °C, and stirred for 15 min at room tem-
perature. After refluxed for 1 h, the solution was cooled, and evaporated in
vacuo to reduce tetrahydrofran. The residue was added 4 M NaOH until pH
1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-[3-(2-tributylstannylphenyl)propyl]-
piperazine (5a): Yield 47.5%, IR (CHCl3): 3018, 2398, 1518, 1422,
1225 cmꢄ1 1H-NMR (CDCl3): 0.86—1.57 (27H, m, Bu3), 1.83 (2H, q,
Jꢂ7.5 Hz, phCH2CH2CH2), 2.41 (4H, t, Jꢂ7.5 Hz, CH2N), 2.56—2.64
.
11 and the mixture was extracted with CHCl3 (20 mlꢃ3). The organic layer (10H, m, phCH2CH2CH2 and piperazine), 2.76 (2H, t, Jꢂ7.5 Hz,
was dried over K2CO3 and the solvent was evaporated in vacuo to afford the phCH2CH2N), 3.87 (6H, s, OCH3ꢃ2), 6.73—6.81 (3H, m, aromatics),
crude product as yellow oil. This oil was purified by silica gel column chro- 7.10—7.35 (4H, m, aromatics). CI-HR-MS Calcd for C35H58N2O2Sn (MHꢀ)
matography (MeOH/CHCl3ꢂ1/20), the oil product was converted to its hy- m/z: 658.3520. Found: 658.3514.
drobromide salt. Recrystallized from methanol afforded 4a—c.
1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-[3-(3-tributylstannylphenyl)propyl]-
1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-[3-(2-iodophenyl)propyl]piperazine piperazine (5b): Yield 45.0%, IR (CHCl3): 3018, 2398, 1518, 1422,
1
(4a): Yield 80.8%, mp 246—248 °C, IR (KBr): 2910, 2439, 1521, 1462, 1225 cmꢄ1. H-NMR (CDCl3): 0.86—1.57 (27H, m, Bu3), 1.83 (2H, q,
1267 cmꢄ1
.
1H-NMR (Free Base, CDCl3): 1.80 (2H, q, Jꢂ7.8 Hz,
Jꢂ7.5 Hz, phCH2CH2CH2), 2.41 (4H, t, Jꢂ7.5 Hz, CH2N), 2.56—2.64
phCH2CH2CH2), 2.37 (4H, t, Jꢂ7.8 Hz, CH2N), 2.56—2.62 (10H, m, (10H, m, phCH2CH2CH2 and piperazine), 2.76 (2H, t, Jꢂ7.5 Hz,
phCH2CH2CH2 and piperazine), 2.77 (2H, t, Jꢂ7.8 Hz, phCH2CH2N), 3.87 phCH2CH2N), 3.87 (6H, s, OCH3ꢃ2), 6.73—6.81 (3H, m, aromatics),
(6H, s, OCH3ꢃ2), 6.73—6.81 (3H, m, aromatics), 6.98—7.56 (4H, m, aro-
7.10—7.35 (4H, m, aromatics). CI-HR-MS Calcd for C35H58N2O2Sn (MHꢀ)
matics). CI-HR-MS (Free Base) Calcd for C23H31IN2O2 (MHꢀ) m/z: m/z: 658.3520. Found: 658.3517.
494.1432. Found: 494.1423. Anal. Calcd for C23H31IN2O2·2HBr: C, 42.10;
H, 5.07; N, 4.27. Found: C, 41.93; H, 5.26; N, 4.24.
Radiolabeling Aqueous hydrogen peroxide (10 ml, 30%, w/v) was
added to a mixture of [125I] NaI (10 ml, 37.0 MBq, 74 TBq/mmol), 0.1 N HCl
1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-[3-(3-iodophenyl)propyl]piperazine (25 ml) and tributylstannyl precursor 5a, b (0.01 mg in 10 ml ethanol) in a
(4b): Yield 92.6%, mp 247—249 °C, IR (KBr): 2910, 2439, 1521, 1462, sealed vial. After stirring for 10 min at room temperature, the reaction mix-
1267 cmꢄ1
.
1H-NMR (Free Base, CDCl3): 1.80 (2H, q, Jꢂ7.8 Hz,
ture was quenched with aqueous sodium bisulfite (0.1 mg in 10 ml). The
phCH2CH2CH2), 2.37 (4H, t, Jꢂ7.8 Hz, CH2N), 2.56—2.62 (10H, m, mixture was isolated by HPLC using NaH2PO4 solution (10 mM)/MeOH
phCH2CH2CH2 and piperazine), 2.77 (2H, t, Jꢂ7.8 Hz, phCH2CH2N), 3.87 (15/85) as an eluent at a flow rate of 3.0 ml/min. The fraction corresponding
(6H, s, OCH3ꢃ2), 6.73—6.81 (3H, m, aromatics), 6.98—7.56 (4H, m, aro-
of 4a, b was collected and the solvent was removed in vacuo. The retention
matics). CI-HR-MS (Free Base) Calcd for C23H31IN2O2 (MHꢀ) m/z: times were 13.8 min for [125I] 4a and 14.5 min for [125I] 4b. The radiochemi-
494.1432. Found: 494.1414. Anal. Calcd for C23H31IN2O2·2HBr: C, 42.10; cal yields based on [125I] NaI were 90.0 and 94.0%, respectively. The radio-
H, 5.07; N, 4.27. Found: C, 42.16; H, 5.04; N, 4.09.
1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-[3-(4-iodophenyl)propyl]piperazine than 99% and approximately 74 TBq/mmol.
(4c): Yield 91.4%, mp 247—249 °C. IR (KBr): 2938, 2434, 1517, 1451, In Vitro Binding Studies Crude P2 membrane fractions of rat brain
chemical purity and the specific activities of all three tracers were greater
1264 cmꢄ1
.
1H-NMR (Free Base, CDCl3): 1.75 (2H, q, Jꢂ7.8 Hz,
(2.0 mg of protein) in Tris–HCl (50 mM, pH 8.0) were incubated for 90 min
phCH2CH2CH2), 2.33 (4H, t, Jꢂ7.8 Hz, CH2N), 2.49—2.58 (10H, m, at 25 °C with [125I] 4a or [125I] 4b (1—300 nM, 0.74 kBq, respectively). The
phCH2CH2CH2 and piperazine), 2.71 (2H, t, Jꢂ7.8 Hz, phCH2CH2N), 3.80 total incubation volume was 1.0 ml. Nonspecific binding was determined in
(6H, s, OCH3ꢃ2), 6.67—6.74 (3H, m, aromatics), 6.87 (2H, d, Jꢂ8.4 Hz,
the presence of haloperidol (10 mM). Specific binding was defined as the dif-
aromatics), 7.52 (2H, d, Jꢂ8.4 Hz, aromatics). CI-HR-MS (free base) Calcd ference between the total and the nonspecific binding. Assays were termi-
for C23H31IN2O2 (MHꢀ) m/z: 494.1432. Found: 494.1424. Anal. Calcd for nated by rapid vacuum filtration through Whatman GF/B glass fiber filters
C23H31IN2O2·2HBr: C, 42.10; H, 5.07; N, 4.27. Found: C, 42.00; H, 5.00; N, presoaked in 0.5% polyethylenimine for at least 30 min at room temperature
4.05.
Preparation of Rat Brain Membranes Receptor binding assays were
prior to use, and assayed filters were washed 6 times with 4 ml ice-cold
10 mM Tris–HCl buffer (pH 7.4). The filter-bound radioactivity was counted
performed using the crude synaptosomal (P2) membrane fraction of rat in an auto-g-counter. The saturation binding data were analyzed by
brain, according to a modified the method of Leitner et al.44) Male Wistar Scatchard plot analysis for determination of the equilibrium dissociation
rats (250—300 g) were decapitated and the brain, minus cerebellum, were constant (Kd), and the maximal number of binding site (Bmax).
homogenized in 10 volumes of ice-cold 10 mM Tris–HCl/0.32 M sucrose
buffer (pH 7.4), using a Polytorone homogenizer at 24000 rpm for 20 s. The
In Vitro Blocking Studies Crude P2 membrane fractions of rat brain
(2.0 mg of protein) were incubated in Tris–HCl (50 mM, pH 8.0) for 90 min
homogenates were centrifuged at 1000 g for 10 min at 4 °C to remove cell at 25 °C with [125I] 4a or [125I] 4b (0.74 kBq) and various receptor ligand
debris. The supernatants were centrifuged at 45000 g for 15 min at 4 °C. The
final pellets were taken and adjusted to a protein concentration of 2 mg/ml in DTG, (ꢀ)-3-PPP, (ꢁ)-SKF10047, (ꢀ)-SCH23390, (ꢄ)-sulpiride, (ꢄ)-nico-
(1 mM, 100 ml), 4a—c, SA4503, haloperidol, rimcazole, carbetapentane,
ice-cold 10 mM Tris–HCl buffer (pH 7.4). Protein concentration was deter-
mined according to the Lowly method.46)
tine, phentolamine, atropine, and naloxone. The total incubation volume was
1.0 ml. Assays were terminated by rapid vacuum filtration through Whatman
In Vitro Competitive Binding Assays Sigma receptor binding assay GF/B glass fiber filters, presoaked in 0.5% polyethylenimine for at least
was carried out under the follow conditions. Crude P2 membrane fractions 30 min at room temperature prior to use. These filters were washed 6 times
of rat brain (2.0 mg of protein) were incubated with 0.1 nmol [3H] DTG
(0.46 kBq) and the test compound (3.3ꢃ10ꢄ9—1.0ꢃ10ꢄ5 M) in 50 mM
Tris–HCl (pH 8.0) at 25 °C for 90 min. The total incubation volume was
1.0 ml. Nonspecific binding were determined in the presence of haloperidol
(10 mM). Assays were terminated by rapid vacuum filtration through What-
with 4 ml ice-cold Tris–HCl buffer (10 mM, pH 7.4). The filter-bound ra-
dioactivity was counted in an auto-g-counter.
Acknowledgements This work was supported by Grant-in-Aid for Sci-
entific Research (C) No. 13670972, 17591298 and High Technology Re-

April 2006
475
search from Ministry of Education, Culture, Sports, Science and Technol-
ogy, Japan.
23, 761—766 (1996).
25) Waterhouse R. N., Chapman J., Izard B., Donald A., Belbin K.,
O’Brien J. C., Collier T. L., Nucl. Med. Biol., 24, 587—593 (1997).
26) Waterhouse R. N., Mardon K., Brien J. C. O., Nucl. Med. Biol., 24,
45—51 (1997).
27) Waterhouse R. N., Collier T. L., Nucl. Med. Biol., 24, 127—134
(1997).
28) Dence C. S., John C. S., Bowen W. D., Welch M. J., Nucl. Med. Biol.,
24, 333—340 (1997).
29) Ishiwata K., Noguchi J., Ishii S., Hatano K., Ito K., Nabeshima T.,
Senda M., Nucl. Med. Biol., 25, 195—202 (1998).
30) Kobayashi T., Matsuno K., Nakata K., Mita S., J. Pharmacol. Exp.
Ther., 279, 106—113 (1996).
31) Matsuno K., Nakazawa M., Okamoto K., Kawashima Y., Mita S., Eur.
J. Pharmacol., 306, 271—279 (1996).
32) Kawamura K., Ishiwata K., Tajima H., Ishii S., Matsuno K., Homma
Y., Senda M., Nucl. Med. Biol., 27, 255—261 (2000).
33) Kawamura K., Ishiwata K., Shimada Y., Kimura Y., Kobayashi T.,
Matsuno K., Ann. Nucl. Med., 14, 285—292 (2000).
References
1) Martin W. R., Eades C. E., Thompson J. A., Huppler R. E., Gilbert P.
E., J. Pharmacol. Exp. Ther., 197, 517—532 (1976).
2) Walker J. M., Bowen W. D., Walker F. O., Matsumoto R. R., De Costa
B. R., Rice K. C., Pharmacol. Rev., 42, 355—402 (1990).
3) Quirion R., Bowen W. D., Itzhak Y., Junien J. L., Rothman R. B., Su T.
P. , Tam S. W., Taylor D. P., Trends Neurosci., 10, 444—446 (1992).
4) Hellewell S. B., Bruce A., Feinstein G., Orringer J., Williams W.,
Bowen W. D., Eur. J. Pharmcol., 268, 9—18 (1994).
5) Wolfe S. A., Jr., Clup S. G., De Souza E. B., Endocrinology, 124,
1160—1172 (1988).
6) Su T. P., London E. D., Jaffe J. H., Science, 240, 219—221 (1988).
7) Brent P. J., Pang G. T., Eur. J. Pharmcol., 278, 151—160 (1995).
8) Vilner B. J., John C. S., Bowen W. D., Cancer Res., 55, 408—413
(1995).
9) Honner M., Moebius F. F., Flandorfer A., Knaus H.-G., Striessnig J.,
Kempner E., Glossmann H., Proc. Natl. Acad. Sci. U.S.A., 93, 8072— 34) Kawamura K., Elsinga P. H., Kobayashi T., Ishii S., Wang W. F., Ma-
8077 (1996).
tsuno K., Vaalburg W., Ishiwata K., Nucl. Med. Biol., 30, 273—284
(2003).
35) Ishiwata K., Tsukada H., Kawamura K., Kimura Y., Nishiyama S.,
Kobayashi T., Synapse, 40, 235—237 (2001).
10) Pan Y.-X., Mei J., Wan B.-L., Zukermann A., Pasternak G. W., J. Neu-
rochem., 70, 2279—2285 (1998).
11) Prasad P. D., Li H. W., Fey Y.-J., Ganpathy M. E., Fujita T., Plumley L.
H., Yang-Feng T. L., Leibach F. H., Ganapathy V., J. Neurochem., 70, 36) Kawamura K., Kimura Y., Tsukada H., Kobayashi T., Nishiyama S.,
443—451 (1998).
Kakiuchi T., Ohba H., Harada N., Matsuno K., Ishii K., Ishiwata K.,
Neurobiol. Aging, 24, 745—752 (2003).
37) Magata Y., Saji H., Horiuchi K., Torizuka K., Yokoyama A., Nucl.
Med. Biol., 14, 7—13 (1987).
12) Seth P., Fei Y.-J., Li H. W., Huang W., Leibach F. H., Ganapathy V., J.
Neurochem., 78, 922—931 (1998).
13) Su T. P., Eur. J. Biochem., 200, 6336—6342 (1991).
14) Junien J. L., Gue M., Bueno L., Neurophamacol., 30, 1119—1124 38) Eckelman W. C., “Radiopharmaceuticals,” ed. by Nunn A. D., Marcel
(1991). Dekker, New York, 1992, pp. 167—219.
15) Kamei J., Iwamoto Y., Kawashima N., Hitosugi H., Misawa M., Ka- 39) Fujimura K., Matsumoto J., Niwa M., Kobayashi T., Kawashima Y.,
suya Y., Eur. J. Pharmacol., 224, 39—43 (1992). Bioorg. Med. Chem., 5, 1675—1683 (1997).
16) Riviere P. J. M., Rao R. K., Pascaud X., Junien J. L., Porreca F., J. 40) Moore T. S., Boyle M., Thorn V. M., J. Chem. Soc., Part 1, 39—51
Pharmacol. Exp. Ther., 264, 1268—1274 (1993).
17) Maurice T., Su T. P., Privat A., Neuroscience, 83, 739—748 (1998).
18) Nakazawa M., Matsuno K., Mita S., Neurochem. Int., 32, 337—343
(1998).
19) Matsuno K., Kobayashi T., Tanaka M. K., Mita S., Eur. J. Pharmacol.,
312, 267—271 (1996).
20) Maurice T., Lockhart A. A., Prog. Neuro-Psycopharmacol. Biol. Psy-
chiat., 21, 69—102 (1997).
21) Nabeshima T., Okuyama S., Jpn. J. Psychopharmacol., 14, 51—76
(1994).
(1929).
41) De Costa B. R., Radesaca L., Paolo L. D., Bowen W. D., J. Med.
Chem., 35, 38—47 (1992).
42) Weber W., Sonders M., Quarum M., Mclean S., Pou S., Keana J. F. W.,
Proc. Natl. Acad. Sci. U.S.A., 83, 8784—8788 (1986).
43) Cagnotto A., Bastone A., Mennini T., Eur. J. Pharmacol., 266, 131—
138 (1994).
44) Leitner M. L., Hohmann A. G., Patrick S. L., Walker J. M., Eur. J.
Pharmacol., 259, 65—69 (1994).
45) Blaszczac L. C., Halligan N. G., Seitz D. E., J. Labelled. Compd., 27,
401—406 (1989).
22) Amano M., Yamada K., Matsuno K., Nabeshima T., Jpn. J. Psy-
chopharmacol., 16, 73—84 (1996).
23) Hayashi T., Su T. P., CNS Drugs, 18, 269—284 (2004).
24) John C. S., Gulden M. E., Vilner B. J., Bowen W. D., Nucl. Med. Biol.,
46) Gornal A. G., Bradwill C. J., David M. M., J. Biol. Chem., 177, 751—
766 (1948).