Synthesis and biological evaluation of novel 4-benzylpiperazine ligands for sigma-1 receptor imaging

Article abstract of DOI:10.1016/j.bmc.2011.03.037

We report the synthesis and evaluation of 4-benzylpiperazine ligands (BP-CH₃, BP-F, BP-Br, BP-I, and BP-NO₂) as potential σ₁ receptor ligands. The X-ray crystal structure of BP-Br, which crystallized with monoclinic space

Full text of DOI:10.1016/j.bmc.2011.03.037

Bioorganic & Medicinal Chemistry 19 (2011) 2911–2917
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel 4-benzylpiperazine ligands
for sigma-1 receptor imaging
Zi-Jing Li a, Hui-Ying Ren a, Meng-Chao Cui a, Winnie Deuther-Conrad b, Rui-Kun Tang a, Jörg Steinbach b,
Peter Brust b, Bo-Li Liu a, Hong-Mei Jia a,
⇑
a Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China
b Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmacy, Neuroradiopharmaceutical Division, 04318 Leipzig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and evaluation of 4-benzylpiperazine ligands (BP-CH3, BP-F, BP-Br, BP-I, and BP-
Received 5 February 2011
Revised 17 March 2011
Accepted 17 March 2011
Available online 23 March 2011
NO2) as potential
r1 receptor ligands. The X-ray crystal structure of BP-Br, which crystallized with mono-
clinic space group P21/c, has been determined. In vitro competition binding assays showed that all the
five ligands exhibit low nanomolar affinity for r1 receptors (Ki = 0.43–0.91 nM) and high subtype selec-
tivity (r2 receptor: Ki = 40–61 nM; Kir2/Kir
1 = 52–94). [125I]BP-I (1-(1,3-benzodioxol-5-ylmethyl)-4-(4-
iodobenzyl)piperazine) was prepared in 53 10% isolated radiochemical yield, with radiochemical purity
of >99% by HPLC analysis after purification, via iododestannylation of the corresponding tributyltin pre-
cursor. The log D value of [125I]BP-I was found to be 2.98 0.17, which is within the range expected to
give high brain uptake. Biodistribution studies in mice demonstrated relatively high concentration of
Keywords:
r1 receptor
4-Benzylpiperazine
Iodine-125
radiolabeled substances in organs known to contain
r1 receptors, including the brain, lung, kidney, heart,
Imaging agent
and spleen. Administration of haloperidol 5 min prior to injection of [125I]BP-I significantly reduced the
concentration of radioactivity in the above-mentioned organs. The accumulation of radiolabeled sub-
stance in the thyroid was quite low suggesting that [125I]BP-I is relatively stable to in vivo deiodination.
These findings suggest that the binding of [125I]BP-I to r1 receptors in vivo is specific.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
was also eliminated by r1 receptor blockers, and the r1 receptor
agonist carbetapentane citrate was found to mimic the effect of
Sigma receptors represent a distinct class of proteins, with two
subtypes (r1 and
2) known.1 They are expressed in the central
nervous system (CNS), endocrine, immune and certain peripheral
tissues, and are believed to play an important role in regulating
and integrating nervous, endocrine, and immune responses.2–4 It
DHEAS. The main functional consequence of this DHEAS effect is
presumably to protect neurons under ischemia.13 More and more
evidence suggests that the r1 receptors represent potential thera-
peutic targets in many human diseases.14 Development of r1
receptor ligands could have wide therapeutic applications, while
development of specific radiotracers for in vivo imaging of r1
receptors may provide the necessary diagnostic tools in combating
the above-mentioned diseases.
r
is noteworthy that the human
mosome 9, band p13, which is known to be associated with differ-
ent psychiatric disorders.5 Accordingly, the
1 receptors are linked
r1 receptor gene is located on chro-
r
to a number of brain disorders, including schizophrenia, depres-
sion, Alzheimer’s disease, as well as ischemia.3,4,6–8 They regulate
the activity of various ion channels and possess protein chaperone
It is well known that many compound classes with diverse
structures possess high affinity for r1 receptors. In the past few
years, many potential radiotracers for imaging r1 receptor
expression with PET and SPECT have also been reported. But until
function.3 Recently, it has been reported that
r
-receptor ligands
inhibit the cardiac voltage-gated Na+ channel Nav1.5, which may
now, only a few tracers such as [11C]SA4503,15,16 18F]FPS,17 and
[
explain some of the actions of
r
-receptor ligands on the cardiovas-
[
123I]TPCNE18 have been evaluated in human studies. Moreover,
cular system.9 Moreover, dehydroepiandrosterone sulfate (DHEAS),
an agonist of the r1 receptors,10,11 could inhibit the persistent so-
dium currents, which is involved in the pathological process of
brain disease.12 The effect of DHEAS on persistent sodium currents
few of the r1 tracers reported to date have ideal properties for
imaging. [11C]SA4503 was found to have about 70% specific
binding rate,19 but has high affinity also to the emopamil binding
protein (Ki = 1.72 nM)20 and to the vesicular acetylcholine
transporter (Ki = 50.2 nM).21 On the other hand, [18F]FPS22 and
[
123I]TPCNE18 were found to have irreversible kinetics. Recently,
⇑
Corresponding author. Tel./fax: +86 10 58808891.
E-mail address: hmjia@bnu.edu.cn (H.-M. Jia).
a new promising 18F-labeled spirobenzofuran, named fluspidine,
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.037

2912
Zi-Jing Li et al. / Bioorg. Med. Chem. 19 (2011) 2911–2917
was developed and evaluated in mice. [18F]Fluspidine demon-
strated favorable target affinity and specificity as well as meta-
bolic stability both in vitro and in animal experiments. The
in vivo properties of [18F]fluspidine offer a high potential of this
radiotracer for neuroimaging and quantitation of r1 receptors
in vivo.23
reacted with bis(tributyltin) for 6 h in toluene at reflux under
nitrogen in the presence of palladium catalyst, followed by purifi-
cation with chromatography (ethyl acetate/petroleum ether = 3:2
v/v) to afford yellow-light oil 3 in 18% yield. The yields of 2a–2e
were ranging from 60.4% to 81.5%. The synthesized compounds
were characterized by NMR, MS, and EA.
In 2008 1-(1,3-benzodioxol-5-ylmethyl)-4-(4-bromobenzyl)-
piperazine (BP-Br) was reported to be used for the treatment of so-
dium channel mediated disease.24 Based on the evidence that r1
receptors regulate the activity of sodium ion channels,9,12 the pos-
sible mechanisms of the proposed treatment effect of BP-Br could
be related to r1 receptors. Therefore, we have synthesized five
4-benzylpiperazine ligands (BP-CH3, BP-F, BP-Br, BP-I, and BP-
NO2), determined the X-ray crystal structure of BP-Br, and mea-
sured their affinity to r1 receptors by in vitro radioligand binding
assays. Moreover, we synthesized [125I]BP-I (1-(1,3-benzodioxol-
5-ylmethyl)-4-(4-iodobenzyl)piperazine), determined its log D
value, and evaluated its potential as a putative SPECT tracer for
imaging of r1 receptors by biodistribution studies in mice.
2.2. Crystal data of BP-Br compound
From the general point of view, 4-benzylpiperazine derivatives
consist of two amine sites (basic nitrogen atom) flanked by two
hydrophobic regions. According to the pharmacophore model pro-
posed by Glennon,25 a structural feature common to high-affinity
r1 receptor ligands is C–N(R)–X–Ph. N is a basic nitrogen atom,
representing the amine site. Ph and C represent the primary hydro-
phobic region and secondary hydrophobic region, respectively.
Both C and Ph are associated with regions of bulk tolerance. We as-
sume that N1 (Fig. 1) is the amine binding site, and the phenyl ring
with dioxol group (aromatic A) is the primary binding site, while
the phenyl ring with Br substitute (aromatic B) (Scheme 1) is the
secondary binding site. In order to examine the binding model of
4-benzylpiperazine derivatives, the single X-ray crystal structure
analysis of BP-Br was performed. The crystal structure with the
atomic numbering scheme of the BP-Br compound is shown in
Figure 1. Crystal data together with details of the determinations
are summarized in the experimental part. Atomic coordinates
(ꢀ104) and equivalent isotropic displacement parameters
(Å2 ꢀ 103) are listed in Table 1.
2. Results and discussion
2.1. Chemistry
The synthetic route of BP derivatives is shown in Scheme 1.
Alkylation of compound 1 with the corresponding benzyl bromide
or benzyl chloride provided compound 2a–2e. Compound 2b
Based on the obtained X-ray crystal structure of BP-Br, the dis-
tances between N1 and the aromatic centroid (A) as well as the
aromatic centroid (B) are 6.410 and 3.787 Å, respectively. The dis-
tance between the centroid of the aromatic A and that of B is
10.144 Å. Based on Glennon’s pharmacophore model, the distance
between N and the primary hydrophobic region (Ph) is 6–10 Å,
while the distance between N and the secondary region (C) is
2.5–3.9 Å. Therefore, the above structural features of BP-Br are in
good agreement with the general structural features found optimal
for r1 receptor binding. BP derivatives may show high affinity for
r1 receptors.
2.3. Radiolabeling
[
125I]BP-I was prepared via an iododestannylation reaction
using H2O2 as the oxidizing agent. The reaction was quenched with
saturated NaHSO3 solution, and the resulting mixture was purified
using a C18 Sep-Pak cartridge. After removing inorganic salts
including [125I]NaI by washing with water, [125I]BP-I was eluted
with ethanol. The product was further purified by radio-HPLC
using a reverse-phase column and mobile phase consisting of ace-
tonitrile with a flow rate of 2 mL/min.
In order to identify the radiotracer, the non-radioactive BP-I was
co-injected and co-eluted with the radioactive product. Their HPLC
profiles using acetonitrile and water (85:15 v/v) as mobile phase
at a flow rate of 1 mL/min are present in Figure 2. From Figure 2,
the retention times of BP-I and [125I]BP-I were observed to be 4.18
and 4.60 min, respectively. The difference in retention time was in
good agreement with the time lag which corresponds with the vol-
ume and flow rate within the distance between the UV and radioac-
tivity detector of our HPLC system. After purification by HPLC, no
significant chemicalimpurities were detected by analytical RP-HPLC
(UV, 254 nm). The isolated radiochemical yield of [125I]BP-I was 55–
65% (n = 5). The radiochemical purity was higher than 99%. The spe-
cific activity of the n.c.a. [125I]NaI was >2200 Ci/mmol (>80 GBq/
l
mol) at time of delivery. The specific activity of the product was
not determined. To prepare a suitable solution of [125I]BP-I for
in vivo use, the eluted radioactive substance (peak corresponding
Scheme 1. Reagents and conditions: (a) CH2Cl2, rt, 24 h; (b) (Bu3Sn)2, (Ph3P)4Pd,
toluene, reflux, 6 h; (c) [125I]NaI, H2O2, HCl.

Zi-Jing Li et al. / Bioorg. Med. Chem. 19 (2011) 2911–2917
2913
Figure 1. Crystal structure of BP-Br compound.
Table 1
Atomic coordinates (ꢀ104) and equivalent isotropic displacement parameters
(Å2 ꢀ 103) for BP-Br compound
x
y
z
U (equiv)
Br(1)
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
9729(1)
9263(2)
8940(2)
8619(2)
8616(1)
8947(2)
9266(2)
8307(2)
7780(2)
7422(2)
7153(2)
7509(2)
6615(2)
6315(1)
6326(2)
6023(2)
5708(1)
5697(1)
5992(2)
5146(2)
7965(1)
6964(1)
5358(1)
5376(1)
13,543(1)
11,224(7)
10,687(8)
8961(7)
7737(7)
8353(7)
10,087(8)
5729(8)
3329(6)
3068(6)
6608(6)
6871(6)
4214(7)
2182(6)
943(7)
ꢁ868(7)
ꢁ1388(6)
ꢁ212(6)
1571(6)
ꢁ2962(7)
5488(5)
4453(5)
ꢁ1130(5)
ꢁ3098(4)
8232(1)
8220(5)
7150(4)
7126(4)
8140(4)
9211(4)
9262(5)
8055(4)
8995(4)
9917(4)
9635(4)
8712(4)
10,546(4)
10,469(4)
11,491(4)
11,481(4)
10,408(4)
9380(4)
9366(4)
8953(4)
8969(3)
9651(3)
8423(2)
10,150(3)
105(1)
59(1)
63(1)
58(1)
51(1)
61(1)
66(1)
67(1)
59(1)
53(1)
54(1)
53(1)
57(1)
46(1)
49(1)
50(1)
45(1)
45(1)
48(1)
56(1)
49(1)
43(1)
63(1)
60(1)
C(9)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
C(17)
C(18)
C(19)
N(1)
N(2)
O(1)
O(2)
to [125I]BP-I) was collected. After the solvent was removed in vacuo,
the product was re-dissolved in less than 0.1 mL ethanol and diluted
with sterile saline to provide approximately 1
active substance per 0.1 mL of solution.
lCi (37 kBq) of radio-
The apparent distribution coefficient of [125I]BP-I was deter-
mined between 1-octanol and 0.05 mol Lꢁ1 sodium phosphate buf-
fer at pH 7.4 as previously reported.26 At pH 7.4, the log D value of
Figure 2. HPLC co-elution profiles of BP-I and [125I]BP-I, with retention times of
[
125I]BP-I was determined to be 2.98 0.17 (n = 3), which is within
4.18 and 4.60 min, respectively.
the range expected to give high brain uptake.
2.4. Biological studies
Table 2
Binding affinities of the 4-benzylpiperazine compounds towards
r
1 and
Ki(
r
2 receptors
2.4.1. In vitro radioligand competition studies
r
1 and r2 receptor competition binding assays were performed
Ki ( 1) (nM) Ki ( 2) (nM)
r
r
r
2)/Ki(r1
)
as previously reported.27 The r1 and r2 receptor affinities of BP
derivatives were determined in competition experiments with the
radioligands (+)-[3H]pentazocine and [3H]ditolylguanidine using
rat brain and rat liver preparations, respectively. In vitro competi-
tion binding assays showed that all five ligands exhibit low nanomo-
lar affinity for r1 receptors (Ki = 0.43–0.91 nM, Table 2) and high
BP-I
BP-F
BP-CH3
BP-NO2
BP-Br
0.80 0.14
0.85 0.91
0.91 0.81
0.43 0.19
0.78 0.26
61.1 20.1
52.2 13.3
49.8 8.89
40.5 11.7
40.5 9.23
76.4
61.4
54.7
94.2
51.9
Values are means SD of three experiments performed in triplicate.

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Zi-Jing Li et al. / Bioorg. Med. Chem. 19 (2011) 2911–2917
Table 4
subtype selectivity (r2 receptors: Ki = 40–61 nM; Kir2/Kir1 = 52–
Effects of preinjection of haloperidol (0.1 mL, 1.0 mg kgꢁ1) 5 min prior to the injection
of [125I]BP-I on the biodistribution of radioactivity in female ICR micea
94, Table 2). Substitution with electron-withdrawing group, such
as NO2, in p-position of the benzyl moiety led to a slight increase
of r1 receptor affinity. The high affinities of the BP derivatives are
in good agreement with the structural features of BP-Br found opti-
Organ
60 min (control)
60 min (blocking)
% Blocking
pb
Blood
Brain
Heart
Liver
Spleen
Lung
1.32 0.41
3.64 0.66
1.72 0.31
5.73 0.95
4.35 0.66
3.84 0.69
8.18 0.13
1.08 0.28
1.36 0.19
1.17 0.15
6.85 1.17
2.30 0.39
2.37 0.44
5.48 0.31
ꢁ18
ꢁ63
ꢁ32
20
ꢁ47
ꢁ38
ꢁ33
0.311
<0.001
0.007
0.136
<0.001
0.011
mal for the
r1 receptors binding.
2.4.2. Biodistribution and blocking studies in mice
In vivo biodistribution studies of [125I]BP-I were performed in
female ICR mice. The uptake of radiolabeled substance in the or-
gan of interest at 15, 30, 60, 120, and 240 min after intravenous
Kidney
<0.001
a
Data are means of %ID/g of tissue SD, n = 4–5.
p values for the control vs blocking group at 60 min p.i. calculated by Student’s t
administration of (0.10 mL) 1 l
Ci [125I]BP-I is summarized in
b
Table 3. [125I]BP-I showed a high initial brain uptake, with slow
clearance and high brain-to-blood ratios. The radioactivity con-
centration determined in the brain was highest at 15 min
post-injection (p.i.) with 5.03 0.60 %ID/g, and slowly cleared
thereafter with 4.96 0.16 %ID/g at 30 min, 4.63 0.41 %ID/g at
60 min, 3.75 0.20 %ID/g at 120 min, and 2.09 0.21 %ID/g at
240 min p.i. The values are higher than that of [11C]SA450319
and comparable to those determined for [18F]fluspidine.23 In con-
trast to the high uptake and retention in the brain, the blood
radioactivity levels were low with 1.34 0.37 %ID/g at 15 min
and 0.72 0.03 %ID/g at 240 min p.i., resulting in high brain-
to-blood ratios (3.75, 5.64, 5.72, and 4.87 at 15, 30, 60 and
120 min p.i., respectively). Similar to what was found for
test (independent, two-tailed).
imaging in the above brain diseases. However,
[
11C]SA4503
needs an on-site cyclotron. Longer half-life 18F- and 123I- labeled
compounds are more suitable for r1 receptors imaging. Unfortu-
nately, there is lack of suitable 18F-labeled PET radiotracer and
123I-labeled SPECT radiotracer for the neuroimaging of r1 recep-
tors until now. The major problems of the currently available r1
receptor radiotracers such as [18F]FPS22 and [123I]TPCNE,18 are
the irreversible kinetics and lack of a suitable reference region.
Since r1 receptors regulate the activity of sodium ion chan-
nels,9,12 we proposed that the possible mechanisms of the treat-
ment effect of sodium channel mediated disease of BP-Br could
be related to r1 receptors. BP-Br could possess affinity for r1
receptors. Based on the obtained X-ray crystal structure of BP-
Br, the structural features of BP-Br are in good agreement with
the Glennon’s pharmacophore model for r1 receptor binding.
Moreover, the five synthesized 4-benzylpiperazine ligands exhi-
bit low nanomolar affinity for r1 receptors and high subtype
selectivity. It is reasonable to synthesize [18F]BP-F and [123I]BP-
I radioligands for further evaluation. For the routine clinical
applications, imaging with SPECT have some advantages over
PET such as more widespread availability, no need for an on-site
cyclotron and lower cost. So [125I]BP-I was synthesized first for
[
18F]fluspidine high uptakes (4.0–12.7 %ID/g at 15 min p.i.) were
observed in organs known to contain receptors, including
r
the lungs, kidneys, heart, spleen and liver, followed by slow
clearance over time. Third, the accumulation of radioactivity in
the thyroid at 240 min p.i. was quite low suggesting that
[
125I]BP-I is relatively stable to in vivo deiodination.
In order to verify the specific binding of [125I]BP-I to
r
receptors
in vivo, the effects of preinjection of haloperidol (0.1 mL,
1.0 mg kgꢁ1) on the biodistribution of radioactivity in various
organs of female ICR mice were examined. Either saline or haloper-
idol was injected 5 min prior to the radiotracer injection. The
results of blocking studies at 60 min p.i. are given in Table 4. Of
particular interest was a significant reduction (63%, p <0.001) in
the brain accumulation of radioactivity at 60 min p.i. which corre-
sponds to values obtained with [18F]fluspidine.23 Moreover, the
in vivo evaluation. In biodistribution studies,
[
125I]BP-I was
found to have higher brain uptake than that of [11C]SA4503, high
specific binding to r1 receptors comparable to that determined
for [11C]SA4503, as well as limited in vivo deiodination. It is
noteworthy that [125I]BP-I showed clearance from the brain after
the peak uptake at 15 min. This is an advantage compared to
concentration of radioactivity in organs known to possess
r recep-
tors was significantly reduced with heart by 32%, spleen by 47%,
and lung by 38%. These data suggest that the binding of [125I]BP-I
[
123I]TPCNE. The brain uptake of [123I]TPCNE remained constant
to
r
receptors was specific in vivo.
Currently, in vivo [11C]SA4503 imaging studies have shown
after initial uptake over the whole investigation time of
180 min.18 Our results encourage the synthesis and evaluation
of iodine-123 labeled BP-I as a putative tracer for imaging r1
receptors with SPECT.
that the density of r1 receptors was decreased in Parkinson’s
disease (PD) and Alzheimer’s disease (AD) patients.15,16 This tra-
cer could be used as a probe in clinical studies of r1 receptors
Table 3
Biodistribution of [125I]BP-I in female ICR micea
Organ
15 min
30 min
60 min
120 min
240 min
Blood
Brain
Heart
Liver
Spleen
Lung
Kidney
Thyroid
Stomachb
Muscle
Small intestine
1.34 0.37
5.03 0.60
3.98 0.33
9.00 0.90
6.68 0.67
12.49 2.18
12.72 1.43
6.55 0.66
2.32 0.11
2.35 0.11
5.77 1.33
0.88 0.16
4.96 0.16
2.79 0.29
6.39 0.20
6.53 0.21
8.70 0.23
9.65 0.72
2.30 0.54
2.12 0.14
1.78 0.29
10.21 1.29
0.81 0.12
4.63 0.41
1.71 0.17
5.68 0.42
5.70 0.37
6.29 0.83
8.73 1.37
1.53 0.16
1.78 0.26
1.50 0.32
7.45 1.32
0.77 0.19
3.75 0.20
1.24 0.18
5.08 0.88
4.79 0.60
4.91 1.36
7.82 0.76
1.40 0.16
2.54 0.17
1.06 0.29
10.73 4.12
0.72 0.03
2.09 0.21
0.75 0.03
3.95 0.27
2.83 0.24
2.69 0.34
6.89 0.51
1.22 0.20
1.68 0.35
0.72 0.09
10.95 1.31
a
Data are means of %ID/g of tissue SD, n = 5.
%ID/organ.
b

Zi-Jing Li et al. / Bioorg. Med. Chem. 19 (2011) 2911–2917
2915
4.4. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-bromobenzyl)pipera-
zine (2b)
3. Conclusions
4-Benzylpiperazine derivatives have been synthesized and eval-
Mp 106–108 °C. Yield 63%. 1H NMR (400 MHz, CDCl3)
(ppm): 7.42 (d, J = 8.3 Hz, 2H), 7.18 (d, J = 8.2 Hz, 2H), 6.84 (s,
1H), 6.73 (s, 2H), 5.93 (s, 2H), 3.44 (s, 2H), 3.41 (s, 2H), 2.45
(br s, 8H). ESI+-MS([M+H]+): 389.4. Anal. Calcd for C19H21BrN2O2:
C, 58.62; N, 7.20; H, 5.44; O, 8.22; Br, 20.53. Found: C, 58.57; N,
6.89; H, 5.99.
d
uated as high-affinity
r1 receptor ligands with relatively high sub-
type selectivity. [125I]BP-I has been prepared in good radiochemical
yield and high radiochemical purity. The log D value of [125I]BP-I
was within the range expected to give excellent brain uptake. In
biodistribution studies [125I]BP-I was found to possess very high
initial brain uptake followed by slow clearance. The in vivo binding
pattern of the tracer was in good agreement with the known distri-
bution of r1 receptors, and blocking studies confirmed high spe-
cific binding. These findings suggest further synthesis and
evaluation of [123I]BP-I as a suitable radiotracer for imaging r1
receptors with SPECT in vivo.
4.5. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-iodobenzyl)piperazine
(2c)
Mp 104–106 °C. Yield 59%. 1H NMR (400 MHz, CDCl3)
d
(ppm): 7.62 (d, J = 7.4 Hz, 2H), 7.06 (d, J = 7.5 Hz, 2H), 6.84 (s,
1H), 6.73 (s, 2H), 5.93 (s, 2H), 3.42 (d, J = 9.4 Hz, 4H), 2.44 (br
s, 8H). ESI+-MS([M+H]+): 437.1. Anal. Calcd for C19H21IN2O2: C,
52.31; N, 6.42; H, 4.85; I, 29.09; O, 7.33. Found: C, 52.39; N,
6.37; H, 4.85.
4. Experimental section
4.1. General information
[
125I]NaI (specific activity as I: 17.4 Ci/mg, >80 GBq/
lmol, sol-
4.6. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-nitrobenzyl)pipera-
zine (2d)
vent: 1.0 E–05 mol/L, pH 8–11) was bought from Perkin–Elmer Life
and Analytical Sciences. All other reagents and chemicals were pur-
chased from commercial sources and used without further purifica-
tion unless otherwise indicated. 1H NMR spectra were recorded on a
Bruker Avance III (400 MHz) NMR spectrometer. Chemical shift (d)
are reported in ppm downfield from tetramethylsilane and cou-
pling constants (J) are reported in Hertz (Hz). MS spectra were ob-
tained by Quattro micro API ESI/MS (Waters, USA). Elemental
analyses were obtained on an Elementar 240C (Perkin–Elmer,
USA). Melting point was recorded on an X-6 micro melting point
apparatus (Beijing Taike Co., Ltd, China) and was uncorrected.
HPLC analyses were performed on a Shimadzu SCL-10AVP sys-
tem (SHIMAZU Corporation, Japan) which consisted of a binary
pump with on-line degasser, a model SPD-10AVP UV–vis detector
operating at a wavelength of 254 nm, and a Packard 500TR series
flow scintillation analyzer (Packard BioScience Co., USA). The sam-
ples were analyzed on a Agilent TC-C18(2) column (150 ꢀ 4.6 mm,
Mp 104–105 °C. Yield 56%. 1H NMR (400 MHz, CDCl3) d (ppm):
8.16 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H), 6.84 (s, 1H), 6.73
(s, 2H), 5.93 (s, 2H), 3.59 (s, 2H), 3.43 (s, 2H), 2.47 (br s, 8H).
ESI+-MS([M+H]+): 356.4. Anal. Calcd for C19H21N3O4: C, 64.21; N,
11.82; H, 5.96; O, 18.01. Found: C, 64.46; N, 11.65; H, 6.08.
4.7. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-methylbenzyl)-
piperazine (2e)
Mp 102–103 °C. Yield 78%. 1H NMR (400 MHz, CDCl3) d (ppm):
7.18 (d, J = 7.8 Hz, 2H), 7.10 (d, J = 7.8 Hz, 2H), 6.83 (s, 1H), 6.72 (s,
2H), 5.92 (s, 2H), 3.46 (s, 2H), 3.40 (s, 2H) 2.45 (br s, 8H), 2.32 (s,
3H). ESI+-MS([M+H]+): 325.5. Anal. Calcd for C20H24N2O2: C,
74.04; N, 8.64; H, 7.46; O, 9.86. Found: C, 74.32; N, 8.69; H, 6.92.
5 lm) using acetonitrile and water (85:15 v/v) as mobile phase at a
4.8. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-(tributylstannyl)-
benzyl)piperazine (3)
flow rate of 1 mL/min. HPLC separation was carried out on a
Shimadzu LC-20AT HPLC system with a UV–vis detector operating
at a wavelength of 254 nm, and a SPD-M20A flow scintillation
analyzer. The sample was separated on an Alltech Alltima RPC-18
Compound 2b (100 mg, 0.24 mmol) was dissolved in anhydrous
toluene (5 mL) under nitrogen. Bis(tributyltin) (0.36 mL,
0.71 mmol), tetrakis(triphenylphosphine) palladium(0) (11 mg,
0.01 mmol) were added. The mixture was refluxed for 6 h. After
cooling the inorganic salts were filtered off and the solvent was re-
moved under reduced pressure. The residue was purified by chro-
matography (ethyl acetate/petroleum ether = 3:2 v/v) to afford
25 mg white solid, yield 18%. 1H NMR (400 MHz, CDCl3) d (ppm):
7.39 (d, J = 8.5 Hz, 2H), 7.25 (d, J = 8.5 Hz, 2H), 6.84 (s, 1H), 6.73
(s, 2H), 5.92 (s, 2H), 3.49 (s, 2H), 3.41 (s, 2H), 2.46 (br s, 8H),
1.60–1.54 (m, 6H), 1.39–1.34 (m, 6H), 1.10–1.06 (m, 6H),
0.93–0.90 (m, 9H). 13C NMR (400 MHz, CDCl3) d 146.57, 145.51,
139.23, 136.79, 135.30, 131.17, 127.84, 121.21, 108.54, 106.78,
99.81, 62.10, 61.78, 52.04, 29.62, 28.07, 26.46, 26.35, 25.76,
16.30, 12.60, 8.97, 8.55. ESI+-MS([M+H]+): 600.9.
column (250 ꢀ 4.6 mm, 5
lm) using acetonitrile as mobile phase
at a flow rate of 2 mL/min.
4.2. General procedure for synthesis of BP derivatives
To produce compounds 2a, 2b, 2c, 2d, and 2e, 1-(1,3-ben-
zodioxol-5-ylmethyl)piperazine (220 mg, 1.0 mmol) was dissolved
in anhydrous dichloromethane (5 mL), the corresponding benzyl
bromide or benzyl chloride (1.0 mmol) were added. The mixture
was stirred at room temperature for 24 h. After filtration, the sol-
vent was removed under reduced pressure. The residue was puri-
fied by chromatography (ethyl acetate/petroleum ether = 1:1 v/v)
to afford white solid. The solid was crystallized from ethyl acetate
as colorless crystal.
4.9. Crystal data for BP-Br compound
4.3. 1-(1,3-Benzodioxol-5-ylmethyl)-4-(4-fluorobenzyl)piper-
azine (2a)
Data were collected for a colorless crystal on a Bruker Smart APEX
II diffractometer (Bruker Co., Germany). Crystal data together with
Mp 101–103 °C. Yield 71%. 1H NMR (400 MHz, CDCl3) d (ppm):
7.26 (t, J = 8.6 Hz, 2H), 6.98 (t, J = 8.7 Hz, 2H), 6.84 (s, 1H), 6.73
(s, 2H), 5.92 (s, 2H), 3.46 (s, 2H), 3.41 (s, 2H), 2.45 (br s, 8H).
ESI+-MS([M+H]+): 329.4. Anal. Calcd for C19H21FN2O2: C, 69.49; N,
8.53; H, 6.45; F, 5.79. Found: C, 69.61; N, 8.48; H, 6.48.
details of the determinations are summarized as follows. C19H21
-
BrN2O2,0 Mr = 389.29, monocyclic, space group P21/c, a = 25.48
0
0
50(19) ÅA, b = 6.3928(5) ÅA, c = 11.1585(9) ÅA,
c
a
= 90°, b = 100.247(2)°,
= 90°, V = 1789.0(2) ÅA3; Z = 4; calculated density = 1.445 Mg mꢁ3
Absorption coefficient 2.311 mmꢁ1; F(0 0 0) = 800; crystal size:
0
;

2916
Zi-Jing Li et al. / Bioorg. Med. Chem. 19 (2011) 2911–2917
0.24 ꢀ 0.20 ꢀ 0.04 mm. Reflections collected/unique: 6493/3135
[R(int) = 0.0275]; absorption correction: semi-empirical from
equivalents. Max. and min. transmission: 0.9132 and 0.6070; refine-
ment method: full-matrix least-squares on F2; data/restraints/
parameters: 3135/0/217; Goodness-of-fit on F2: 1.026; final R indi-
values were calculated according to Cheng and Prusoff and rep-
resent data from at least three independent experiments, each
performed in triplicate. The results are given as mean standard
deviation (SD).
ces [I > 2r(I)]: R1 = 0.0499, wR2 = 0.1178; R indices (all data):
4.13. Biodistribution and blocking studies in mice
R1 = 0.0950, wR2 = 0.1399; largest diff. peak and hole: 0.839 and
ꢁ0.826 e.Aꢁ3
.
Experiments in female ICR mice (n = 5, 18–22 g) were carried
out in compliance with the national laws related to the care and
experiments on laboratory animals. The HPLC-purified [125I]BP-I
4.10. Preparation of [125I]BP-I
was injected via tail vein (0.1 mL, 1 lCi). The mice were sacrificed
by cervical fracture at various time points. Samples of blood and
organs of interest were removed, weighed and counted in an auto-
matic c-counter (Wallac 1470 Wizard, USA). The results were ex-
pressed in terms of the percentage of the injected dose per gram
(%ID/g) of blood or organs.
For blocking studies, mice were injected via tail vein with either
saline (0.1 mL) or haloperidol (0.1 mL, 1.0 mg kgꢁ1) 5 min prior to
radiotracer injection. The animals were sacrificed by cervical
fracture at 60 min p.i. Blood or organs were isolated and analyzed
as described above for the biodistribution study. Significant
differences between control and test groups were determined by
Student’s t test (independent, two-tailed). The criterion for signifi-
cance was p 6 0.05.
To a solution of the tributyltin precursor (BP-Sn, 3) in eth-
anol (1 mg/mL, 100
cific activity >2200 Ci/mmol), 100
prepared) and 100
vial. The reaction mixture was kept at room temperature for
15 min. The reaction was quenched with 50 L saturated NaH-
l
L), 2
l
L Na125I (795
lCi, carrier free, spe-
lL
H2O2 (3%, freshly
l
L HCl (1 mol Lꢁ1) were added to a sealed
l
SO3 solution. The pH value of the mixture was adjusted to 7.5
with NaOH solution (1 mol Lꢁ1). The mixture was then passed
across a C18 Sep-Pak cartridge. After elution with water, inor-
ganic salts including [125I]NaI was separated from the product.
The crude product was recovered into a sample vial by slowly
flushing the cartridge with 10 mL of absolute ethanol. The sol-
vent was evaporated under reduced pressure and the residue
was purified by radio-HPLC. As analyzed by HPLC, the final
product was obtained with a radiochemical purity of >99%.
The isolated radiochemical yield of
[
125I]BP-I was 55–65%
Acknowledgment
(n = 5). In order to identify the radioactive product, the stable
BP-I was co-injected and co-eluted with the radioactive
product.
This work was supported by the National Natural Science Foun-
dation of China (No. 21071023).
4.11. Determination of log D value
Supplementary data
The log D value of [125I]BP-I was determined by measuring
the distribution of the radiotracer between 1-octanol and
0.05 mol Lꢁ1 sodium phosphate buffer at pH 7.4. The two
phases were pre-saturated with each other. 1-Octanol (3 mL)
and phosphate buffer (3 mL) were pipetted into a 10 mL plastic
centrifuge tube. Two microliters of a solution of HPLC-purified
Supplementary data (CIF file of BP-Br) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmc.2011.03.037.
References and notes
[
125I]BP-I (2
texed for 5 min, followed by centrifugation for 15 min
(3500 rpm, Anke TDL80-2B, China). About 50 L of the 1-octanol
layer was weighed in a tared tube. The 1-octanol layer was re-
moved and about 500 L of the buffer layer was weighed in a
lCi) in ethanol was then added. The tube was vor-
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haloperidol. Ki

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