Synthesis, preclinical evaluation and molecular modelling of macrocyclic appended 1-(2-methoxyphenyl)piperazine for 5-HT1A neuroreceptor imaging

Article abstract of DOI:10.1039/c5ra13432c

5-HT_1A receptors are known to be implicit in a number of neuropsychiatric fluctuations related to mood and anxiety. Their visualization in the human brain using PET, SPECT or MRI is of great importance in the management and treatment of neurolo

Full text of DOI:10.1039/c5ra13432c

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PAPER
Synthesis, preclinical evaluation and molecular
modelling of macrocyclic appended 1-(2-
methoxyphenyl)piperazine for 5-HT1A
neuroreceptor imaging†
Cite this: RSC Adv., 2016, 6, 7288
Puja Panwar Hazari,a Surbhi Prakash,ab Virendra Kumar Meena,ab Niraj Singh,a
Krishna Chuttani,a Nidhi Chadha,a Pooja Singh,ab Shrikant Kukretib
a
*
and Anil Kumar Mishra
5-HT1A receptors are known to be implicit in a number of neuropsychiatric fluctuations related to mood and
anxiety. Their visualization in the human brain using PET, SPECT or MRI is of great importance in the
management and treatment of neurological disorders. The present work focuses on the metal
, ) of DO3A-butyl-MPP to be used as brain (cerebral cortex,
complexes (Gd3+ Eu3+ and Ga3+
hippocampus, and amygdala) imaging agents using different modalities. Synthesis of 2,20,200-(10-(2-(4-(4-
(2-methoxyphenyl)piperazin-1-yl)butylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)
triacetic acid (DO3A-butyl-MPP) was achieved by conjugating the chloroacetylated derivative of 1-(2-
methoxyphenyl)piperazine-butylamine with trisubstituted cyclen with subsequent cleavage with
trifluoroacetic acid (TFA). The resulting compound was then labeled with GdCl3 and 68GaCl3 to perform
MRI (relaxivity studies) and PET respectively. The longitudinal relaxivity (r1), and transverse relaxivity (r2),
were determined to be 6.66 and 11.486 mMꢀ1 sꢀ1 respectively on 7 T at 21 ꢁC. The SD (Sprague Dawley)
rat brain uptake was 3.91% ID per g (percentage of the injected dose per gram) at 30 min post injection.
Homology modeling and docking studies at the shallow antagonist binding pocket of 5-HT1A show a high
G score of ꢀ12.132 that confers high binding of the ligand at the target receptor.
Received 9th July 2015
Accepted 6th January 2016
DOI: 10.1039/c5ra13432c
www.rsc.org/advances
whose residue, 1-(2-methoxyphenyl)piperazine (MPP), is known
to bind with high affinity to the serotonin receptors.4 Many 11C
and 18F radiolabeled derivatives of WAY-100635 have been
synthesized and evaluated for use in positron emission tomog-
raphy.5 The radiolabeling of p-MPPF with 18F, yielding a uoro-
analogue of WAY-100635 has provided encouraging results in
vivo by autoradiography in cats, monkeys and human beings.6
Despite the success of these tracers as PET imaging agents,
Introduction
Radiopharmaceuticals that bind to CNS receptors in vivo are
potentially useful for understanding the pathophysiology of
a number of neurological and psychiatric disorders, their
diagnosis and treatment.1 The neurotransmitter serotonin
(5-hydroxytryptamine, 5-HT) is involved in various central
nervous system functions and psychiatric disorders.2 The
receptors that are activated by 5-HT have been classied into
different classes, of which the 5-HT1A receptors have been
implicated in the pathogenesis of depression, anxiety, schizo-
phrenia, epilepsy, and eating disorders.3 For the visualization of
these receptors, several ligands have been evaluated, starting
from the lead structure of WAY-100635, a potent antagonist
the dependency on
a nearby cyclotron for radioisotope
production and high cost of production has redirected the
interest in development of a WAY-100635 analogue incorpo-
rating radionuclide that is easily available commercially and has
low cost per dose.7 Moreover, complexes of macrocyclic poly-
dentate ligands based on 1,4,7,10-tetraazacyclododecanetetra-
acetic acid (DOTA) with radioactive metals are used in potential
diagnostic/therapeutic applications.8 Additionally, DOTA is
known to form stable complexes with many metal ions, such as
Ga(III), Gd(III), Eu(III), Tb(III) ions.9 The set of coordinating donor
atoms (four nitrogens and four oxygens) wraps around a met-
al(III) ion in a very efficient way to yield complexes endowed with
a very high thermodynamic and kinetic stability.10
aDivision of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear
Medicine and Allied Sciences, Brig SK Mazumdar Road, Delhi-110054, India. E-mail:
akmishra63@gmail.com
bDepartment of Chemistry, University of Delhi, Delhi-110007, India
† Electronic supplementary information (ESI) available: Fig. S1–S25 NMR and
mass spectra (ESI and high resolution mass spectra) characterization details of
all synthesized compounds, cytotoxicity graph, human serum stability,
radioligand binding assays, radio HPLC and radio TLC, molecular modeling
studies, calibration curves and specic activity. See DOI: 10.1039/c5ra13432c
Thus, in this current study we have designed and synthesized
macrocyclic chelating agent covalently linked to 1-(2-
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methoxyphenyl) piperazine and radiolabeled with 68Ga, which do enhances the affinity by interacting with the receptor, which is
not require the presence of a nearby cyclotron for its production. suggestive of its hydrophobic nature of interaction.16
68Ga is a positron emitting isotope which is produced from
The binding affinity of 5-HT1A receptor ligands towards the
generator with half-life of 67.71 min. Gallium-based radiophar- aryl substituent group (R ¼ o-CH3, o-OBu, o-OCH3, o-NHCOPr,
maceutical preparation is easy and fast and an alternative to the m-NHCOPr, o-CN, o-COOPr, m-NH2, m-Br) were also evaluated
preparation of 18F- or 11C-labeled PET agents, leading to and reported by various groups, suggesting that the o-OCH3
a minimum loss of activity.11 The present molecule has also been group have the highest binding affinity to the 5-HT1A recep-
complexed with Gd(III) to show its properties as a MR contrast tors.17 Moreover, PET radioligands for 5-HT1A with o-OCH3
agent as MRI provides unmatched and/or unparalleled so tissue group have been used extensively to track the 5-HT1A neuro-
details alongside giving plethora of functional information.
receptor uctuations in the brain.
Considering no single modality is perfect and sufficient to
We hereby present an efficient approach in the design and
gain all the necessary information, the combination of multiple synthesis of a multi-functional chelating agent based on 1-(2-
molecular imaging techniques can offer synergistic advantages methoxyphenyl) piperazine for targeting 5-HT1A receptors in
over any modality alone.12 Therefore, we have attempted to brain.
synthesize a 5-HT1A antagonist which can be utilized for neu-
roimaging using PET as well as MRI.13 We hereby report an
efficient scalable strategy for the synthesis of 1-(2-methoxy-
phenyl)piperazine functionalized macrocyclic chelating agent
Results and discussion
Chemistry
DO3A-butyl-MPP in good yield (80–85%).
1-(2-Methoxyphenyl)piperazine was alkylated with N-(4-bromo-
The macrocyclic ligand has been designed on the basis of butyl)phthalimide and cleaved with hydrazine hydrate to give 4-
structure–activity relationship (SAR) studies reported in the liter- (4-(2-methoxyphenyl)piperazine-1-yl)butan-1-amine (2).18 N-(4-
ature for enhanced affinities towards the target receptors.14 In Bromobutyl)phthalimide was used to increase the yield of
order to achieve optimal selectivity and affinity the macrocyclic compound (2). Compound (2) was then chloroacetylated in
ligand was constructed keeping in consideration the following dichloromethane : water (1 : 1) to yield 2-chloro-N-(4-(4-(2-
aspects; rstly methoxyphenylpiperazine moiety has basic methoxyphenyl)piperazin-1-yl)butyl)acetamide (3). Chloroacety-
nitrogen which can be functionalized easily. Secondly, function- lated butyl-MPP (3) was conjugated to trisubstituted cyclen (4)
alization with butyl linker increases affinity towards the 5-HT1A to give tert-butyl-2,20,200-(10-(2-(4-(4-(2-methoxyphenyl)piperazin-
receptor. Attaching methoxyphenylpiperazine moiety to macro- 1-yl)butylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-
cycle ligand provides an excellent agent for imaging purpose. It is 1,4,7-triyl)triacetate (5) which was further reacted with tri-
well documented in literature4h that (CH2)n is a spacer attached to uoroacetic acid (TFA) to cleave the boc groups and obtain
–NH of the arylpiperazine, with respect to n two to four methylene the nal compound 2,20,200-(10-(2-(4-(4-(2-methoxyphenyl)
groups appear optimal. This chain length (n) can inuence piperazin-1-yl)butylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclo-
affinity and selectivity of the molecule.15 It is known from the dodecane-1,4,7-triyl) triacetic acid (6) with overall yield of 80–
previous reports, that the linker length signicantly enhances the 85% starting from 1-(2-methoxyphenyl)piperazine (Scheme 1).
binding affinity towards 5-HT1A receptors and reduce the affinity
Conjugation of butyl-methoxyphenylpiperazine moiety to
for a1-adrenergic as well. The most optimal condition for high macrocycle ligand provides an excellent agent for the
binding affinity is provided by linker length of (CH2)n, n ¼ 4 with complexation of trivalent metal ion for imaging application.
subnanomolar Ki value for 5-HT1A receptor ligands.16 When there The introduction of the bifunctional butyl spacer and conju-
is a change in value of ‘n’ [i.e. n ¼ 2, 3, 5, 6] it subsequently reduces gation of the DOTA group with MPP accomplished the synthetic
the affinity of ligands and disrupts the most optimal condition for design aiming to ensure simplistic and scalable approach with
high binding to the 5-HT1A receptor. The alkyl amide bond further high yield. This was achieved using N-(4-bromobutyl)phthali-
mide which gave the product 4-(4-(2-methoxyphenyl)piperazin-
1-yl)butan-1-amine in more than 70% yield aer subsequent
cleavage of phthalimide group whereas using unprotected
bromobutylamine, less than 40% yield was obtained. Direct
functionalization of –NH group of methoxyphenylpiperazine
with DOTA gave product with less than 20% yield.
For the development of PET imaging agent, DO3A-butyl-MPP
was complexed with 68Gallium. Gallium-based radiopharma-
ceutical preparation is easy and fast, contrary to the preparation
of covalently 18F- or 11C-labeled PET agents, leading to
a minimum loss of activity. The most important requirements
for a Ga(III)-based radiopharmaceutical agent are thermody-
namic stability and kinetic inertness during the period of clin-
ical use in order to avoid ligand exchange with the blood serum
proteins, such as transferrin. DOTA is also advantageous
considering that it can more than saturate Gallium's common
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Scheme 1 (a) Synthesis of 2-chloro-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)acetamide (3). Reagents and conditions: (i) ACN, K2CO3, 4-
bromobutylphthalimide, 70 ꢁC (ii) hydrazine hydrate, ethanol, reflux, 2 h (iii) ClCH2COCl, DCM/H2O, 0 ꢁC. (b) Synthesis of 2,20,200-(10-(2-(4-(4-(2-
methoxyphenyl)piperazin-1-yl)butylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (6). Reagents and conditions:
(iv) ACN, NaHCO3, 0 ꢁC, tert-butylbromoacetate (v) ACN, 3, 70 ꢁC (vi) 20% TFA in DCM, 0 ꢁC.
six coordination sphere, offering free carboxymethyl arms for concentration of the complex. At low concentration, an increase
conjugation to targeting molecules. On achieving encouraging in contrast results to an increase in signal intensity due to effect
results from PET studies, we extended our approach of devel- on T1 concentration of the Gd(III) complex.
oping multifunctional contrast agent towards the prospect of
A plot of relaxivities as a function of the concentration of this
utilization of the ligand DO3A-butyl-MPP as magnetic reso- complex was plotted (Fig. 1B and C) where the values of r1 and r2
nance imaging agent. The coordination chemistry of lanthanide were calculated from the slope of 1/T values obtained from
complexes of polyaminocarboxylates is very important because samples at different concentrations. The measured relaxivities
of their application in biomedical sciences as MR contrast arise from the exchange between the coordinated water mole-
agent. It is well known that gadolinium complexes are cules and the surrounding water molecules in solution. The
T1-weighted MRI contrast agents, and thus the T1 relaxivity rate
is an important parameter. With the aim to synthesize MR
imaging agent possessing potential of targeting brain region,
DO3A-butyl-MPP was complexed with gadolinium and its
relaxation properties were investigated. To assess the hydration
state of complex, the luminescence lifetime measurements on
Eu(III) complex of DO3A-butyl-MPP were conducted.
Relaxometric studies
The enhancement of the longitudinal relaxation rate of water
protons (1/T1) occurs due to the presence of paramagnetic
Gd(III) in complexes. The prociency of the Gd(III) complexes is
expressed in terms of relaxivity (r1, mMꢀ1 sꢀ1) values, which is
the increase in the longitudinal proton relaxation rates induced
by 1 mM. Longitudinal and transverse proton spin relaxation
rates were measured for Gd complex of DO3A-butyl-MPP in an
aqueous solution (pH-7) at various concentrations (0.625, 1.25,
Fig. 1 (A) T1 weighted image of Gd-DO3A-butyl-MPP at different
concentrations varying from 0.625 mM to 40 mM. Concentration
2.5, 5, 10, 20, 40, 80 mM). A contrast enhancement in range of 0.625–40 mM of Gd-DO3A-butyl-MPP exhibited in MR
studies, the effect being more marked at lower concentrations. At
concentration as low as 0.625 mM, enhanced contrast was obtained
whereas darker image intensity was observed at higher concentration
of the complex. (B & C) In vitro MR measurement of the longitudinal
T1-weighted images in the concentration range of 0.625-20 mM
of Gd-DO3A-butyl-MPP exhibited in MR studies (Fig. 1A), the
effect being more marked at lower concentrations. At concen-
tration as low as 0.625 mM, enhanced contrast was obtained
and transverse relaxivity, r1 and r2 of Gd-DO3A-butyl-MPP with varying
whereas darker image intensity was observed at higher concentrations.
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longitudinal relaxivity r1, and transverse relaxivity r2, was between the Gd3+ complexes and the metal ions Cu2+, Zn2+ or
determined to be 6.66 ꢂ 0.11 mMꢀ1 sꢀ1 and 11.49 ꢂ 0.39 Eu3+ were studied (Table 3).
mMꢀ1 sꢀ1 respectively. Thus the r1 value and stability constant
The determination of the protonation constants of the
with the M(III) metal ion promises its potential as an MR ligand and the properties of its metal complexes are useful
contrast agent for various ex vivo imaging purposes of 5-HT1A for determining stable rigid complexes with M(III) metal ions.
neuroreceptor in brain.
Since the ligand, DO3A-butyl-MPP is tridentate as compared
to DOTA and DTPA the effect of protonation could be seen by
lower values of pKa1 and pKa2. The stability constant of DO3A-
butyl-MPP was slightly lower than DOTA and DTPA but good
enough for high binding with M(III) complexes. The amide
bond of DO3A-butyl-MPP participates in slightly weaker
coordination with Gd3+ which lowers the stability constant as
compared to DOTA and DTPA.19 Even though there is reduced
affinity of DO3A-butyl-MPP towards Gd3+ when compared to
DOTA and DTPA, DO3A-butyl-MPP also have lesser tendency
to bind with endogenous Ca2+, Zn2+ and Cu2+ increasing the
binding selectivity of DO3A-butyl-MPP for Gd3+ over Zn2+ and
Cu2+. A sufficiently high thermodynamic stability of the
Gd(III) complex and good selectivity of the chelating ligand
Assessment of number of coordinated water molecules (q)
Assessment of the number of coordinated water molecules (q)
was carried out using luminescence lifetime measurements.
The values of luminescence lifetimes calculated for Eu-DO3A-
butyl-MPP in H2O and D2O are depicted in Table 1. The
hydration number “q” calculated from equation of Supkowski
and Horricks comes out to be 1.43. It indicates that more than
one water molecule is bound in inner sphere.
The lifetime increases substantially in D2O solution due to
the known sensitivity of Eu(III) luminescence towards deuter-
ated solvent. Since Eu(III) neighbour Gd(III) in the periodic table
and has similar ionic radii, the results of luminescence tech-
nique gives a good estimate for the hydration number of the
corresponding Gd(III) chelate. Thus the hydration number “q”
indicated that more than one water molecule probably bound
to the inner sphere of Gd(III). Attempts have been made to
increase the low hydration number of Gd(III) chelates but the
toxicity concerns of the Gd(III) chelates hamper this way of
increasing the proton relaxivity. The thermodynamic and
kinetic stabilities of the complex decreases considerably when
more than one coordination site is occupied by water.19
However as seen through potentiometric titrations, the ligand
is found to form stable complex with Gd(III) which eliminates
this risk of toxicity.
for Gd(III) over other endogenous metal ions such as Cu2+
,
Zn2+ are needed as prior condition for all biomedical
applications.
Stability constant of DO3A-butyl-MPP complexes with Eu(III)
and Gd(III) showed similar log b. The lower log b values for Cu2+
and Zn2+ conrmed that DO3A-butyl-MPP forms more stable
complex with lanthanide ions, thus it will not undergo trans-
metallation with endogenous metal ions.
68Ga radio-complexation of DO3A-butyl-MPP, serum stability
and log P
The preparation of 68Ga-DO3A-butyl-MPP was simple and effi-
cient by using sodium acetate. The radio-TLC was performed on
silica RP18 F256S plate with sodium citrate (0.15 M) as the
mobile phase. Radio TLC showed peak at 5.73 for free
68Ga-acetate (<2%) and 3.21 for 68Ga-DO3A-butyl-MPP.
Radiochemical yield was calculated from decay-corrected
68Ga at the end of synthesis (EOS) including SPE purica-
tion (Fig. S19 ESI†). The decay corrected radiochemical yield
was found to be 86.42 ꢂ 3.9%, n ¼ 40 runs with specic
activity of 529.08 GBq mmolꢀ1 (decay corrected and SPE
purication for 15 min, EOS). Aer tc18 cartridge purication
of 68Ga-DO3A-butyl-MPP, it showed no evidence of presence
of free 68Ga. Radio HPLC analysis of the puried product
68Ga-DO3A-butyl-MPP showed 99% radiochemical purity and
the presence of only one peak (RT ¼ 7.8 min) conrms the
formation of only one species of the complex which quanti-
tatively reected the radiochemical purity of the sample
Determination of protonation and stability constant
The protonation constants of DO3A-butyl-MPP and stability
constant of the complex was calculated by pH potentiometric
titrations. Solutions of Gd(III), Eu(III), Zn(II) and Cu(II) in MilliQ
water were prepared by dissolving the appropriate amounts of
their salts. For titrations of complexes, an appropriate volume
of the previously prepared metal solution was added via
micropipette to reach the 1 : 1 ligand to metal ratio. The
protonation constants of ligand DO3A-butyl-MPP in compar-
ison with DOTA and DTPA are given in Table 2.
The pKa1 and pKa2 of DO3A-butyl-MPP was found to be lower
than DOTA and DTPA indicating better ionization and disso-
ciation at any given pH. pKa3 was found to be comparable for all
the chelating systems.
For the determination of kinetic stabilities of the complexes,
the rates of exchange (trans-metallation) reactions taking place
Table 2 pKa values of ligand DOTA, DTPA and DO3A-butyl-MPP
Table 1 Luminescence lifetime measurements of Eu-DO3A-butyl-
MPP
pKa
DOTA
DTPA
DO3A-butyl-MPP
pKa1
pKa2
1.43 pKa3
11.14
9.69
4.85
10.49
8.37
4.09
9.58
7.13
4.78
Complex
1/sH O (ms)
1/sD O (ms)
q
2
2
Eu-DO3A-butyl-MPP
2.617
1.257
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Table 3 Stability constant of metal complexes of DO3A-butyl-MPP
revealed that the labeled compound exhibited high affinity on
hippocampal neuronal cultures with a Kd value of 0.64 nM and
0.27 nM in competitive binding assay with serotonin and WAY-
1000635 respectively.
with Gd3+, Zn2+, Eu3+, Cu2+
Gd-DO3A-
butyl-MPP
Eu-DO3A-
butyl-MPP
Zn-DO3A-
butyl-MPP
Cu-DO3A-
butyl-MPP
The binding affinity of the compound, DO3A-butyl-MPP on
the targeted receptors was determined by harvesting different
parts of brain expressing serotonin receptors and transporters.
5-HT1A receptor binding assay exhibited lower affinity of
68Ga-DO3A-butyl-MPP towards 5-HT2A receptors (Kd ¼ 136 ꢂ
3.4) than 5-HT1A receptors (Kd ¼ 0.39 ꢂ 0.08) in brain homog-
enates (Table 4). Scatchard plot of the radioligand binding
assays of 5-HT1A, 5HTT and 5-HT2A brain homogenate are given
in ESI† (Fig. S24).
A subnanomolar affinity for the 5-HT1A receptor was ob-
tained for the above derivative in the in vitro binding assay
which was sufficiently high to carry out imaging. The selectivity
of the complex for 5-HT1A receptors was observed to be 1000
times as high as for 5-HT2A receptors (Table 4). 5-HTT also
showed high affinity with a value of 56 ꢂ 0.7 nM.
log b
17.6969
17.4256
7.0897
6.0173
(Fig. S20, ESI†). The optimisation of radiolabeling with
respect to pH, temperature, duration of heating and the
amount of precursor used in labeling is described in detail in
ESI (Fig. S21 ESI†).
The rate of decomplexation of the radiolabeled complex was
studied in human serum under physiological conditions at
different time intervals of 5, 10, 15, 30, 60, 120 and 180 min. No
measurable loss of metal ion from the macrocyclic core and
high stability of the radiolabeled DO3A-butyl-MPP were
observed (see Fig. S22 ESI†). Thus high specic activity, stability
was achieved and log P was found to be 2.59. The log P of the
radiolabeled DO3A-butyl-MPP was found to be lipophilic in
nature, appropriate to cross the blood brain barrier.
PET image of rat whole brain
The results found in the micro PET imaging of SD (Sprague
Dawley) rat brain indicated that the radiotracer showed
remarkable accumulation in the rat brain striatum over 30
min compared with surrounding normal tissue (Fig. 2). The
decline in activity was observed aer 30 min. This could be
well supported by the fact that terminal elimination half life of
antagonist WAY 100635 is also fast with a value of 33 min.22
The radioactivity distribution was in concordance with the
known 5-HT1A receptor distribution, with high uptake of
radioactivity in hippocampus, caudate putamen and amygdala
(Fig. 2A). The time activity curves revealed accumulation of the
radioligand in whole brain over the rst few minutes which
were followed by a clearance of the compound from hippo-
campus (Fig. 2).
The pre-treatment experiment with WAY-100635 (known
5-HT1A antagonist) markedly reduced the level of radioactivity
in all examined regions. Cerebellum was used as a reference
region for the calculation of binding potential due to the low
5-HT1A receptor density. Rat MRI atlas was superimposed on
the micro PET for anatomical conrmation of the brain
regions. VOI analysis was done using PMOD, data is deter-
mined as VOI (CPS cmꢀ3) brain region/VOI (CPS cmꢀ3) cere-
bellum. VOI analysis showed highest uptake in hippocampus
followed by hippocampus and amygdala (Fig. 3). VOI of 5.606
in hippocampus region and 2.59 in caudate putamen was
observed. VOI of 3.2 and 2.48 were in amygdala and
Cell viability studies of DO3A-butyl-MPP
The in vitro cytotoxicity of the ligand, DO3A-butyl-MPP was
studied by incubating human embryonic kidney (HEK) cells
with varying concentrations of the compound at time intervals
of 24, 48 and 72 h. Using 100 mL of HBSS (Hank Balance Saline
Solution) as control, experiment was conducted. Signicant
percentage of viable cells were obtained when HEK cells were
treated with low concentrations (1–100 mM) of the compound.
Aer 24 and 48 h incubation of 10 mM concentration of the
compound cell survival (percentage relative to untreated
controls) in the range of 70–75% was observed (Fig. S23, ESI†).
The cytotoxicity studies assessed in normal HEK cells showed
high IC50 indicating its safe and favorable applications to be
used in nuclear medicine imaging techniques.
Radioligand binding assay
The specicity of the 1-(2-methoxyphenyl)piperazine function-
alized compound to bind to the cell surface receptors on
primary hippocampal culture cells was examined by radioligand
receptor binding assays using 68Ga-DO3A-butyl-MPP as the
labeled ligand. Primary hippocampal cells were cultured as re-
ported in literature.20 It has been demonstrated previously that
moderate to heavy signal were detected for 5-HT1A mRNA
transcripts in hippocampal neurons and glial cells.21 The
specicity of the 1-(2-methoxyphenyl)piperazine functionalized
compound to bind to the cell surface receptors on primary
hippocampal culture cells was examined by radioligand
receptor binding assays using 68Ga-DO3A-butyl-MPP as the
labeled ligand.
Table
4 Dissociation constants of DO3A-butyl-MPP for 5-HT1A,
5-HT2A, and 5-HTT receptorsa
Kd (nM)
5-HT1A
0.39 ꢂ 0.09
5-HT2A
5-HTT
56 ꢂ 0.7
Non-specic binding was obtained by using 100-fold excess
of unlabeled serotonin and WAY-100635. Analysis of the
binding curve exhibited saturable binding of the radio-
conjugate in subnanomolar range. Scatchard plot analysis
68Ga-DO3A-butyl-MPP
136 ꢂ 3.4
a
Nonspecic binding was determined by serotonin, ketanserin and
paroxetine.
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Fig. 2 (A) 0–30 minutes summation images of rat brain coronal microPET scan depicting activity in different section (slices) of the brain. (B) Time
activity curve (TAC) of different regions for 30 minutes and (C) TAC for 60 minutes post 68Ga-DO3A-butyl-MPP injection in rat (n ¼ 5).
hypothalamus respectively as compared to the cerebellum with
a VOI of 1.1.
Regional uptake in rat brain
Regional binding indices were calculated by % ID per g of the
different regions of the brain by their respective weights (Fig. 4).
In regional biodistribution experiments, the hippocampus
showed the highest uptake of radiolabeled compound, followed
by the caudate putamen which showed a binding of 1.499% ID
per g and cerebellum showed least uptake with a value of 0.08%
ID per g. Whole brain uptake at 30 min increased to 3.724% ID
per g and 73% fall of activity at 60 min. The values were
concordant with the VOI obtained in PET brain scan. The 5-HT
receptor-rich regions showed signicantly higher binding index
than the cerebellum.
Plasma clearance and biodistribution
The plasma clearance and in vivo biodistribution of 68Ga-DO3A-
Fig. 3 MicroPET images of rat brain showing coronal, transverse and
butyl-MPP was studied in SD rats at different time intervals.
sagittal sections respectively after the intravenous injection of 37 MBq
Plasma clearance indicates the clearance rate from the body.
The biological half life t1/2 (fast) results in the increase in target
to non target ratio. Biodistribution studies were performed to
study the accumulation pattern of tracer in the whole body. It
was observed that the clearance of complex was through the
renal and hepatobiliary routes.
of 68Ga-DO3A-butyl-MPP. (A) SD rat depicting high accumulation of
radioactivity in hippocampus, amygdala and caudate putamen. (B)
Coregisteration of rat MRI atlas on the microPET scan for anatomical
confirmation of the brain regions was done using PMOD and (C)
Blocking studies with WAY-100635.
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VOIs generated from the PET studies. Cardiac uptake was seen
at early time points (Fig. 6).
MR imaging
The preliminary in vivo imaging was done on rat brain with and
without injection of the Gd(III)-complex, which shows that the
ligand Gd-DO3A-butyl-MPP at 2 mM was showing enhanced
images as compared to the control brains. T1-weighted signal
enhancement was observed on an in vivo MRI, notably within
hippocampus, which further supports the specicity of the
complexed ligand towards 5-HT1A receptors.
The T1 were statistically decreased by Gd-DO3A-butyl-MPP at
2 mM administration (0.176 mmol kgꢀ1) for hippocampus
compared to baseline scan (Fig. 7A). The enhancement of in vivo
MRI in the control brains was statistically signicant with
a positive signal at 7 T. It is worthwhile to note that the short-
ening of T1 relaxation was observed in treated rats at 30 min
post injection at a concentration of 0.176 mmol kgꢀ1 of
Gd-DO3A-butyl-MPP, as compared to control rats. There was
increase in T1 relaxation on different regions of brain when
WAY100635 (0.2 mmol kgꢀ1) and Gd-DO3A-butyl-MPP (0.176
mmol kgꢀ1) were co administered at 30 min pi (Fig. 7B).
It was also observed that the cerebral blood ow was
increased when the Gd(III)-DO3A-buyl-MPP was given alone
which is an indication of antagonistic property of the
compound. It can be hypothesized from the study conducted on
rats that in pathological state when there is an up regulation of
5-HT1A receptors, this contrast agent could be useful in visual-
izing the uptake at high density and high concentration. It
could be useful as a surrogate tracer of 68Ga-DO3A-buyl-MPP for
MR imaging.
Fig. 4 Regional distribution of 68Ga-DO3A-butyl-MPP in SD rat brain.
WBS ¼ whole brain section Hip ¼ hippocampus, Th ¼ thalamus, Ctx ¼
frontal and parietal cortex, CPu ¼ caudate putamen, Cb ¼ cerebellum
at 10, 30 and 45 min post injection.
The plasma clearance followed a biphasic trend with
a rapidly clearing initial phase followed by slow phase. Plasma
clearance rate (CR) was found to be 37.39 ꢂ 0.31 mg mLꢀ1 minꢀ1
(Fig. 5).
In biodistribution studies, major accumulation of the
labeled compound was observed in kidneys (14.62 ꢂ 0.52% ID
per g) followed by liver (6.19 ꢂ 0.61% ID per g) at 2 h showing
that the complex is excreted by renal and hepatobiliary routes.
Retention of radioactivity in the non-target organs (lungs,
spleen, intestine etc.) was less than 2.0% ID per g at 4 h post
injection. Accumulation in lungs attributed to the lipophilic
nature of complex. The retention of activity follows a declining
trend in the all the organs for the later time points except for the
kidneys at 4 h, wherein the clearance was observed at 8-10 h.
In vivo biodistribution of 68Ga-DO3A-butyl-MPP in SD rats
showed 3.52 ꢂ 0.39% ID per g activity in brain at 10 min and
3.91 ꢂ 0.54% ID per g activity at 30 min which drastically
decreased at 2 h with a value of 0.48 ꢂ 0.03% ID per g activity.
The selective high brain uptake could be seen in regional bio-
distribution studies which were found concomitant with the
Theoretical evidence
Molecular modeling and docking studies with monomeric 5-
HT1A receptor models. Homology modeling and exible dock-
ing studies has been performed for DO3A-butyl-MPP with
human 5-HT1A homology modeled receptor. The 5-HT1A model
was optimized; rened and Ramachandran plot validates the
models where residues are present in allowed region without
any steric clashes (Fig. 8A). The reliable modeling of TM region
Fig.
5
Plasma clearance of 68Ga-DO3A-butyl-MPP (inset: initial Fig. 6 Biodistribution of 68Ga-DO3A-butyl-MPP in SD rats following
clearance up to 20 min).
intravenous injection of 8.14 MBq activity.
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Fig. 8 (A) Model of the human 5-HT1A receptor and Ramachandran
plot showing the transmembrane helices in different colours. (B) Two
dimensional view of DO3A-butyl-MPP with 5-HT1A receptor where
green colored are hydrophobic, light blue are polar, red are negative
and purple are positive charged residues. Pink lines are hydrogen
bonds, red lines are p–cation interactions. (C) Three dimensional view
of DO3A-butyl-MPP (pink carbon layout) at the binding pocket of
5-HT1A homology modeled receptor. Yellow dashed lines are showing
the hydrogen bonds.
Fig. 7 (A) In vivo MR imaging in control and treated SD rats depicting
T1 shortening in 30 min images post Gd-DO3A-butyl-MPP (0.176
mmol kgꢀ1) injection. (B) Representation of relaxivity with respect to
the different regions in the brain.
DO3A-butyl-MPP, the carboxylate groups and carbonyl moiety of
DOTA is involved in hydrogen bonding with side chain residues,
His376, Gly382, Ile189 and Lys191. In addition, p–cation
interactions were observed for protonated –NH of piperazine
moiety with Phe112. This interaction is an example of non
covalent bonding between a monopole (cation) and a quadru-
pole (p system). Here, the cation is nitrogen which is protonated
and p-electrons are in residue Phe112.
In addition to the above interactions, the aromatic ring of
piperazine moiety of DO3A-butyl-MPP was involved in p–p
stacking with Phe361, Phe362 and hydrogen bond interactions
with the side chain of Asp116. The resident binding pocket of
both uncomplexed and complexed DO3A-butyl-MPP was found
to be conserved and comparable to our pervious results24 with
known antagonists in which docking was performed in exible
mode showing appreciable G-score.
The Asp116 interactions with azapirones, buspirone and
sunepitron are well known with piperazine amine25 as in case of
DO3A-butyl-MPP. This binding mode is in general agreement
with putative binding modes suggested in a published ligand-
supported homology model of 5-HT1A receptor.26 Also the
binding pose is quite consistent with the available experimental
studies25 and importance of these residues is evidenced by site-
directed mutagenesis. From docking pose, the antagonist
binding pocket was found to be relatively shallow and involved
in various interactions at the site.
is important since ligand binding pockets lies in TM region of
the 5-HT1A monomer (Fig. 8A).
Three antagonist; p-MPPF, BMMP and WAY-100635 were
used in the docking studies to understand the binding pose of
our ligand with 5-HT1A receptor.23
Our molecular modelling results showed a promising results
for thermodynamic parameters obtained by docking of the
DO3A-butyl-MPP and interactive residue present in the active
site of the 5-HT1A receptor. DO3A-butyl-MPP was aligned in
hydrophobic pocket conserved in all three studied antagonists,
p-MPPF, BMMP and WAY-100635, the Glide XP scoring mode in
exible docking has predicted the minimized energy complexes
in terms of XP score (Table 5). The antagonist p-MPPF is
involved in p–p interactions with Phe362 and hydrogen
bonding with backbone of Ile189 whereas MefWAY is involved
in p–p interactions Arg181 and hydrogen bonding with back-
bone of Ile189. But in case of WAY-100635, one additional p–p
interactions with Phe361 was obtained (see Fig. S25 ESI†).
The docking pose of DO3A-butyl-MPP at the shallow antag-
onist binding pocket of 5-HT1A was found to have high G score
of ꢀ12.132.
The high G score signies the contribution due to important
hydrophobic residues surrounding the ligands. In the ligand,
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Table IFD Glide score and ligand interactions of antagonists
Paper
5
uptake of radiolabeled compound in brain in PET images. The
desired molecule also shows antidepressant actions in the
behavioral tests conducted on CMS model of rats, and
completely reverses the depression levels. The action of the
DO3A-butyl-MPP can be speculated by acting on 5-HT1A via SSRI
effect, which further supports its potency in treating depres-
sion. So the preliminary results and preclinical evaluations of
the given compound DO3A-butyl-MPP show it as a favorable
molecule and promises good potential as a CNS imaging system
which can be used as a MR contrast, and PET imaging system.
This will also encourage the development of brain imaging
compounds for different applications.
MefWAY, WAY-100635 and p-MPPF and DO3A-butylMPP
G score
monomeric
Residual interactions
Ligand
Non-polar residues
Polar residues
IFD
DO3A-butyl- Ile189, Asp116, Phe112, Lys191, Thr200, ꢀ12.132
MPP
Phe362, Gly382
Asn386
p-MPPF
Val117, Cys120, Ile189, Lys191, Thr200
la203, Phe361, Phe362,
Val364, Ala365, Leu368
ꢀ9.11
ꢀ9.81
ꢀ8.81
MefWAY
Ile189, Phe361, Leu368, Thr196, Ser190,
Gly382, Arg181
Lys191, Ser199,
Thr379
WAY-100635 Ile189, Tyr195, Phe361, Thr196, Thr200,
Experimental
General
Leu368, Gly382
Ser190, Lys191,
Ser199, Thr379
1-(2-Methoxyphenyl)piperazine, N-(4-bromobutyl)phthalimide,
hydrazine
hydrate,
tert-butylbromoacetate,
potassium
carbonate, 1,4,7,10-tetraazacyclododecane (cyclen), triuoro-
acetic acid, chloroacetyl chloride, sodium bicarbonate, sero-
tonin hydrochloride, WAY-100635 maleate were purchased
from Sigma-Aldrich. Acetonitrile (HPLC grade), N,N-dime-
thylformamide (HPLC grade), dichloromethane (HPLC grade),
water (HPLC grade), MTT and triuoroacetic acid were obtained
from E. Merck Ltd., India. Metal salts (GaCl3, EuCl3 and GdCl3),
DMEM F12, EDTA, nystatin, formaldehyde, D-glucose, agarose
and sodium chloride were purchased from Aldrich (99.9%).
Penicillin, streptomycin sulfate, FBS and trypsin were
purchased from GIBCO (Invitrogen). The reactions demanding
anhydrous conditions or involving moisture sensitive reactants
were carried out under an atmosphere of dry nitrogen using
oven dried (80 ꢁC) glassware. All reaction temperatures reported
indicate the temperature of the bath in contact with the reaction
vessel. Sep-Pak tC18 Plus short cartridges were purchased from
Waters. TLC was run on the silica gel coated aluminum sheets
(silica gel 60 F254, Merck, Germany). Instant thin layer chro-
matography was used to detect radiocomplexation and radio-
chemical purity. Radio TLC was analyzed on Peak Simple 3.29.
Gallium-68 complex radio HPLC was performed on semi auto-
mated GE FXFN TracerLab integrated with radioactivity detec-
tion and UV detection at 254 nm (xed wavelength detector
K-2001, Knauer, Germany), using isocratic pump SykamS-
1021, elution with methanol and water (25/80, v/v) mobile
Although promising, PET compounds, particularly 11C
compounds, are generally limited to those facilities with
a cyclotron and the high cost hinders commercialization of
viable agents which is driving the need to develop 68Ga-based
agents that can target 5-HT1A. Progress in receptor pharma-
cology with PET radiotracers has led to the design of numerous
candidate for CNS imaging. Fragments of WAY100635, (i.e.
arylpiperazine), have been combined with various bifunctional
chelates consisting of a ligand that binds to technetium-99m
and a linker that joins to the targeting vector. Johannsen et al.
prepared a WAY-based complex using an N2S2 chelate to bind to
99mTc including similar derivatives of arylpiperazine linked to
other technetium(V) chelates (NxSy or NxPy, x ¼ 1–4, y ¼ 4 ꢀ x).27
Although their IC50 and Ki values appear promising, these
agents have low or negligible brain uptake. To date, no 68Ga
labeled compound has been developed which can image the
5-HT1A receptor in vivo. DO3A-butyl-MPP provides efficient
macrocyclic framework for complexation of M3+ metal ions for
multiple biomedical applications. Its improved affinity and
selectivity with high brain uptake is evident through PET
imaging in rat brain which could plausibly be translated
clinically.
Conclusions
phase and ow rate of 2 mL minꢀ1 on C-18 reversed phase
1
We have efficiently synthesized a chelating system containing Nucleosil column (250 mm ꢃ 10 mm, 5 mm). H and 13C NMR
1-(2-methoxyphenyl)piperazine unit as a vector for evaluation of spectra were recorded on a Bruker Avance II 400 MHz system
5-HT1A receptors. Keeping in mind the concept of multiple (Ultra shield). Chemical shis ‘d’ were reported in ppm relative
imaging modalities, the compound has been labeled with to TMS. Mass spectra (ESI-MS in positive and negative ion
Gd(III), Eu(III), and 68Ga so that it can be exploited as MRI and mode) were performed on an in-house Agilent 6310 system ion
PET imaging agent. Encouraging results have been obtained trap. High resolution mass spectroscopy was done using
with all three modalities. The compound (Gd-DO3A-butyl-MPP) accurate-mass Quadrupole time-of-ight (Q-TOF) LC/MS at
has shown longitudinal relaxivity, r1 of 6.66 mMꢀ1 sꢀ1 and Delhi University. All the computational studies were performed
transverse relaxivity, r2 of 11.49 mMꢀ1 sꢀ1 on 500 MHz at 21 ꢁC. with Schrodinger, LLC, New York, NY, 2014 maestro 9.7. MR
28
¨
Hydration number, ‘q’ was found to be 1.43 from luminescence studies were done using an inhouse 7 T Bruker Biospec USR
lifetime studies. Compound (68Ga-DO3A-butyl-MPP) shows high 70/30, (AVANCE III) horizontal bore animal MRI scanner tuned
affinity for hippocampal cells found in brain, proving its rele- to 20 MHz. In vivo imaging studies were performed using micro
vance as a CNS imaging system, shown also by the appreciable PET imaging as non-invasive technology. The acquisitions were
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performed using a GE FLEX Triumph (TriFoil Imaging) micro- The organic layer was dried over anhydrous sodium sulfate and
PET/SPECT/CT scanner. This scanner consists of a micro-PET evaporated to product 3 (820 mg, 80%). The product was used as
module (LabPET4) with 202010 mm3 LYSO/LGSO scintillators in such without any further purication.1H NMR (400 MHz, 25 ꢁC,
an 8-pixel, quad-APD detector module arrangement. Potentio- CDCl3), d (ppm): 1.5 (s, 4H, CH2), 2.3 (s, 2H, CH2), 2.5 (s, 4H,
metric measurements were carried out with an automatic titration CH2), 3.1 (s, 4H, CH2), 3.3 (t, 2H, J ¼ 6 Hz, CH2), 3.8 (s, 3H, CH3),
system consisting of metrohm 713 pH meter equipped with 4.0 (s, 2H, CH2), 6.7–6.9 (m, 4H, CH); 13C NMR (100 MHz, 25 ꢁC,
Metrohm A.60262.100 electrode, 800 Dosino autoburet. The image CDCl3), d (ppm): 24.2, 27.3, 39.7, 42.7, 50.6, 53.5, 55.3, 58.3,
processing was done using PMOD 3.2 and Amide 1.0.4 soware. 111.1, 118.2, 120.9, 122.9, 141.2, 152.2, 165.8; MS (ESI+) m/z
calculated for C17H26ClN3O6 339.8, found [M + H]+ 340.5.
Cell culture
Synthesis of 1,4,7-tri(tert-butoxymethane)-1,4,7,10-
tetraazacyclododecane (t-Bu-DO3A) (4)
Monolayer cultures of normal embryonic kidney cells, HEK, in
DMEM (Sigma, USA) supplemented with 10% fetal bovine
serum (GIBCO), 50 U mLꢀ1 penicillin, 50 mg mLꢀ1 streptomycin Under an atmosphere of nitrogen, 1,4,7,10-tetraazacyclodode-
sulfate, and 2 mg mLꢀ1 nystatin were maintained at 37 ꢁC in cane (2.906 mmol, 500 mg) was dissolved in dry acetonitrile
a humidied CO2 incubator (5% CO2, 95% air). Cells were (10 mL) at 0 ꢁC. To the reaction ask NaHCO3 (8.72 mmol, 732
routinely subcultured using 0.05% trypsin (Sigma, USA) in mg) was added and stirred for 30 min. tert-Butyl bromoacetate
0.02% EDTA in humidied atmosphere of 95% air and 5% CO2 (8.72 mmol, 1.28 mL) was added slowly from a dropping funnel
ꢁ
at 37 C twice a week.
in 2 h at 0 ꢁC. The reaction was stirred for additional 1 h at 0 ꢁC
and then at room temperature for 40 h. The progress of
the reaction was checked by TLC (9 : 1; dichlor-
omethane : methanol). Aer the completion of the reaction, the
reaction mixture was ltered and the ltrate was evaporated to
dryness. The crude compound was puried by column chro-
matography (silica gel, 10% methanol in dichloromethane, Rf
0.67) to give nal compound t-Bu-DO3A (1120 mg, 75%) as white
powder. 1H NMR (400 MHz, 25 ꢁC, CDCl3); 1.4 (27H, s, C(CH3)3),
2.8–3.1 (16H, m, CH2), 3.2–3.3 (6H, m, CH2); 13C NMR (400 MHz,
25 ꢁC, CDCl3): 28.0, 47.5, 49.1, 51.1, 55.6, 58.2, 81.7, 170.5, 172.9;
m/z calculated for C26H50N4O6 514.7, found 515.8 [M + H]+.
Ethics statement
The human serum study was approved by the institutional
ethical committee. Blood samples for human serum study were
collected from healthy volunteers who were well informed about
the study. Written consent from the volunteers above 18 years of
age was obtained.
Animal models
All animal experiments were conducted in accordance with the
guidelines of INMAS animal ethics committee (CPCSEA Regn
no. 8/GO/a/99). Blood clearance, imaging, and biodistribution
were carried on SD rats. Rats were housed under conditions of
controlled temperature of 22 ꢂ 2 ꢁC and normal diet ad libitum.
Synthesis of tert-butyl 2,20,200-(10-(2-(4-(4-(2-methoxyphenyl)
piperazin-1-yl)butylamino)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetate (5)
Under nitrogen atmosphere, to a stirring solution of 1,4,7-
tris(carbobutoxymethyl)-1,4,7,10-tetraaza-cyclododecane (500 mg,
0.97 mmol) in dry acetonitrile (20 mL), K2CO3 (669 mg,
4.85 mmol) was added. Aer 30 min, (3) (396 mg, 1.17 mmol)
dissolved in acetonitrile was added drop wise and heated to 70 ꢁC.
Aer 24 h, the reaction mixture was cooled to room temperature,
ltered, and evaporated under reduced pressure to give crude oily
residue. The compound was puried by column chromatography
(silica gel, 5% methanol in dichloromethane) to give the ligand
Data analysis
The competition curve of the receptor binding experiments was
analyzed by nonlinear regression using algorithms in GraphPad
PRISM 5.0 (San Diego, CA). Biodistribution data is reported as
mean ꢂ standard deviation (S.D.). VOI analyses were carried out
on PMOD 3.2.
Synthetic approach
1
ꢁ
Synthesis of compounds 2-(4-(4-(2-methoxyphenyl)piperazin-1- (5) as a brown solid (600 mg, 75%). H NMR (400 MHz, 25 C,
yl)butyl)isoindoline-1,3-dione (1) and 4-(4-(2-methoxyphenyl) CDCl3), d (ppm): 1.4 (s, 27H, C(CH3)3), 1.6–3.3 (m, 40H, CH2), 3.8
piperazin-1-yl)butan-1-amine (2) have been done as reported (s, 3H, CH3), 6.8–6.9 (m, 4H, CH), 8.4 (br, 1H, NH); 13C NMR
previously by our group.18
(100 MHz, 25 ꢁC, CDCl3), d (ppm): 22.9, 26.6, 27.9, 38.5, 49.2, 52.8,
55.7, 56.1, 57.6, 81.8, 111.2, 118.4, 120.9, 133.2, 140.5, 152.2,
171.5, 172.3. HRMS (ESI+) m/z calculated for C43H75N7O8
817.5677, found [M + H]+ 818.5756, [M + Na]+ 840.5572 (see ESI†
Fig. S9).
Synthesis of 2-chloro-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)
butyl)acetamide (3)
2-Chloroacetylchloride (410 mg, 3.636 mmol in 10 mL of chlo-
roform) and potassium carbonate (627 mg, 4.5 mmol in 10 mL of
water) were added simultaneously through dropping funnels to
a stirred solution of 2 (800 mg, 3.03 mmol) in 10 mL of chloro-
form at 0 ꢁC over a period of 1 h. The reaction mixture was
Synthesis of 2,20,200-(10-(2-(4-(4-(2-methoxyphenyl)piperazin-
1yl)butylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-
1,4,7-triyl)triacetic acid (6)
stirred for an additional 4 h at room temperature. The chloro- The ligand 5 (500 mg, 0.595 mmol) was dissolved in dichloro-
form layer was separated and washed with water (50 mL) twice. methane (4 mL) and 1 mL of triuoroacetic acid was added at
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ꢁC and stirred for 4 h. The reaction mixture was stirred at
Paper
0
Spectroscopic measurement
room temperature for additional 10 h. The solvent was evapo-
rated and residue was dissolved in 1 mL of methanol, followed
by addition of 30 mL of diethyl ether drop wise at 0–5 C and
The luminescence lifetime was obtained by measuring the
decay at the maximum of emission spectra and the signals were
analyzed as single exponential decays. The instrument settings
were: gate-time of 10 ms, integration time of 1 s, ash count – 8
and proper slit widths were used. The excitation wavelength
used for Eu-DO3A-butyl-MPP was 365 nm and luminescence
lifetimes are the average of ve independent experiments. The
slit widths for both excitation and emission monochromators
were kept open. The number of coordinated water molecules (q)
present in the inner sphere of the complex is calculated by using
equation of Supkowski and Horricks for Eu(III).
ꢁ
stirring for 1 h at room temperature. The solvent was decanted
and the precipitated compound dried under reduced pressure.
(370 mg, 96%). 1H NMR (400 MHz, 25 ꢁC, D2O), d (ppm): 1.4 (m,
2H, CH2), 1.6 (m, 2H, CH2), 2.6–3.9 (m, 39H, CH2 and CH3 of
–OCH3), 6.8–6.9 (m, 2H, CH), 7.0–7.1 (m, 2H, CH); 13C NMR
ꢁ
(100 MHz, 25 C, CDCl3), d (ppm): 13.9, 20.6, 25.3, 38.3, 48.1,
48.7, 50.1, 53.8, 54.1, 55.2, 56.2, 65.9, 111.9, 114.8, 116.2, 117.7,
120.6, 121.2, 126.7, 135.9, 151.8, 162.3, 163.4, 171.5 HRMS
(ESI+) m/z calculated for C31H51N7O8 649.3799, found [M + H]+
650.3999 (see ESI† Fig. S11).
q ¼ AEu(1/sH O ꢀ 1/sD O ꢀ aEu
)
2
2
Complexation of DO3A-butyl-MPP with EuCl3
Solutions of Eu(III) complexed DO3A-butyl-MPP complex
were prepared by mixing its appropriate volume in MilliQ water
or in D2O and ligand in Tris buffer 0.1 M (pH ¼ 7.4) or in D2O
followed by addition of NaOD until pH ¼ 7. Measurement of the
Ln(III) luminescence lifetimes in solutions of H2O and D2O gave
the estimation of the number of coordinated water molecules
present in complex by using above equation.
To a solution of DO3A-butyl-MPP (200 mg; 0.3076 mmol) in
water (10 mL) was added EuCl3$6H2O (112.5 mg; 0.3076 mmol)
and the pH of the reaction mixture was adjusted to 5.5. The
ꢁ
reaction was stirred at 60 C for 6 h. The reaction mixture was
ltered and the solvent was evaporated to obtain the
Eu-complex as a pale green solid. HR-ESI-MS calculated for
C
31H48EuN7O8 800.2855, found [M + H]+ 800.2840.
Potentiometric titrations
Complexation of DO3A-butyl-MPP with GdCl3
Potentiometric titrations were carried out at 25 ꢁC and ionic
strength “I” ¼ 1 M tert-butylammoniumchloride (TBACl). The
protonation constant of DO3A-butyl-MPP were determined
potentiometrically by titrating 2 mM of HCl (2 mL) in the
presence of 0.1 mM DO3A-butyl-MPP (15 mL) with 0.01 mM
(3 mL) tert-butylammoniumhydroxide (TBAOH). Titrations were
performed in the pH range of 1.0–10.0 for protonation
constants. The protonation constant of ligand is expressed by
eqn (1) and stability constant of the metal complex is given
by eqn.
To a solution of DO3A-butyl-MPP (200 mg; 0.3076 mmol) in
water (10 mL) was added GdCl3$6H2O (114 mg; 0.3076 mmol)
and the pH of the reaction mixture was adjusted to 5.5. The
reaction was stirred at 60 ꢁC for 12 h. The reaction mixture was
ltered and the solvent was evaporated to obtain the Gd
complex as a pale white solid. HR-ESI-MS C31H48GdN7O8
calculated for 804.2805, found [M + H]+ 805.2896.
Complexation of DO3A-butyl-MPP with GaCl3
½HiLꢄ
10 mg (0.0153 mmol) DO3A-butyl-MPP was dissolved in water
and pH was adjusted to 4.5 with the help of 1 M sodium acetate
solution. The resulting solution was perched with nitrogen to
maintain inert atmosphere. Then GaCl3 (3.2 mg) was added.
The temperature of reaction mixture was then raised to 80 ꢁC for
8 h. Aer that the solution was brought to room temperature
and chelex 100 was added to it which was then stirred for
30 min. Then the solution was ltered and evaporated under
reduced pressure to give a white colored compound. HRMS
(ESI+) calculated for C31H48GaN7O8: 715.2820, found [M]+
713.0644.
Ki ¼
(1)
½Hiꢀ1Lꢄ½Hþꢄ
½MHiLꢄ
KMH L
¼
where i ¼ 0; 1; 2; 3
(2)
i
½MHiꢀ1Lꢄ½Hþꢄ
Radiolabeling of DO3A-butyl-MPP with 68Ga
68Ga was eluted from 68Ge/68Ga generator (Eckert and Ziegler)
with 0.1 N HCl (3 mL) to obtain 480 MBq of 68Ga. Sodium
acetate was added to adjust pH of the elute 4. Then compound
(200 mg) was added from 1 mg mLꢀ1 stock solution of the
compound and the mixture incubated at 90 ꢁC for 10 min. It was
Relaxation measurements
MR imaging was done using an inhouse 7 T Bruker Biospec USR then cooled and passed through C-18 cartridge (preconditioned
70/30, (AVANCE III) horizontal bore animal MRI scanner. For all with ethanol and water). The compound was eluted with 1 mL
experiments, RARE T1 + T2 sequence was used to acquire ethanol. Then ITLC was run in 0.15 M sodium acetate. The
relaxometry images. The scan was acquired at 5 TE times (11, radiochemical purity was assessed by omniscan EZ-TLC scanner
33, 55, 77, 99 ms) and at different TR's (6000, 5000, 4500, 4000, and then the radiolabeled conjugate was puried using a C-18
3500, 3000, 2500 and 1981 ms). Relaxation data were analyzed reversed phase extraction cartridge which was preconditioned
with Topspin 2.0 (Bruker) soware.
with 20 mL methanol and subsequently activated with 30%
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methanol. The cartridge was successively rinsed with 5 mL was measured (reference lter: 630 nm). Surviving fraction at
distilled water and radiolabeled conjugate was eluted in 5 mL of (0.001–10 mM) concentration range was plotted against
40% ethanol. The 68Ga-DO3A-butyl-MPP was reconstituted in concentration for DO3A-butyl-MPP.
saline, and ltered through a sterile 0.22 mm Millipore (Milford,
Measurement of 68Ga-DO3A-butyl-MPP uptake in hippo-
MA) Millex-GV® disposable syringe lter. Radiotracer purity campal primary cultures. Primary cultured cortical neuronal
and stability were monitored using radio-HPLC. In each HPLC cells were obtained from the hippocampus of fetal SD rats at
analysis, the eluent was collected and counted along with 17–20 days of gestation. Single cell suspension from the
a standard prepared from the injectate.
hippocampus of fetal rats was seeded on the poly-L-lysine coated
Determination of log P and stability in blood serum. The culture slide at 3 ꢃ 105 cells per well. Culture was incubated in
partition coefficient of the complex was determined by DMEM (F-12) medium supplemented with 10% heat inactivated
measuring the activity that partitioned between the 1-octanol horse serum and 5% fetal bovine serum for 1–7 days aer
and aqueous phosphate buffer (0.025 mol Lꢀ1, pH 7.4) under plating together with glutamine 2.5 mM, glucose 17.5 mM, and
strict equilibrium conditions. 2 mL 1-octanol and 2 mL of NaHCO3 14.3 mM. The cultures were maintained at 37 ꢁC in
68Ga-DO3A-butyl-MPP phosphate buffer were mixed in a centri- a humidied 5% CO2 atmosphere. Aer 3 days non-neuronal
fuge tube. The mixture was vortexed at room temperature for cells were removed by addition of cytosine arabinoside
5 min and then centrifuged at 5000 rpm minꢀ1 for 5 min. The (10 mM). Only mature cultures surviving 15–18 days in vitro were
counts in 0.1 mL samples of both organic and inorganic layers used for the experiments. Aer 18 days of culture in vitro,
were determined by a well type gamma counter. The measure- successful cultured neurons were selected for binding studies
ment was repeated three times. The partition coefficient (P) was which were carried out as described previously.20 The amount of
calculated as a ratio of the counts in the octanol fraction to the radioactivity (CPM) in cell lysates was determined by gamma
counts in the water fraction (average of n ¼ 5).
scintillation counting. Uptake was then calculated and
Human serum stability. Fresh human serum was prepared expressed in units of pmoles per mg protein per min. Compet-
by allowing blood collected from healthy volunteers to clot for itive binding of 68Ga-DO3A-butyl-MPP with unlabeled serotonin
1 h at 37 ꢁC in a humidied incubator maintained at 5% carbon and WAY-100635 were assessed to calculate dissociation
dioxide/95% air. Then, the samples were centrifuged at 400g, constant (Kd). Final concentrations in the well were 10 pM to
and the serum was ltered through 0.22 mm syringe lter into 10 mM. Hippocampal cultures were washed with HBSS and were
sterile tubes. The radiolabeled DO3A-butyl-MPP was immedi- incubated in HBSS at 37 ꢁC for 20 min prior to the experiment.
ately placed in a CO2 chamber incubated at 37 ꢁC and then Binding experiments were performed at 37 ꢁC. Cells were
analyzed to check for any dissociation of the complex. The incubated for 30 min with increasing concentrations (10 pM to
mixture was stored in the same environment conditions, and 10 mM) of 68Ga-DO3A-butyl-MPP in the absence and presence of
aliquots of 100 mL (in triplicate) were taken at appropriate the 100 folds excess unlabeled serotonin/WAY100635 to esti-
periods of time (0 min, 30 min, 1 h and 3 h). The aliquots were mate the total binding and non-specic binding respectively.
ꢁ
treated with 200 mL of ethanol, cooled (4 C), and centrifuged Specic binding was obtained by subtracting non-specic
ꢁ
during 15 min at 4000 rpm, at 4 C, in order to precipitate the binding from total binding. The cells were washed with cold
serum proteins. A 200 mL aliquot of supernatant was collected PBS and 0.9% saline four times at the end of each experiment.
for activity counting in a g well-counter. The sediment was Gamma scintillation counting was used to determine the cell-
washed twice with 1 mL of ethanol and its activity was counted. associated radioactivity.
The activity of the supernatant was compared to that of the
5-HT1A receptor binding assay. The hippocampus of rat
sediment in order to determine the percentage of the chelate brain was homogenized in 10 volumes of ice-cold buffer (50 mM
associated to the proteins. The activity of the supernatant at 3 h Tris–HCl pH-7.6) using an Ultra Turrax T10 (IKA). The homog-
was evaluated by TLC in order to check whether the chelate enate was centrifuged for 10 min at 20 000g. The resulting pellet
remained intact.
was resuspended with the Ultra-Turrax and centrifuged again at
20 000g for 10 min. The same procedure was repeated again.
Aer that the pellet was resuspended in 10 volumes of buffer
Biological studies
ꢁ
and stored at ꢀ80 C until used in binding studies.
Cell viability assay of DO3A-butyl-MPP. To test the cytotoxic
The binding assay was carried out in a nal volume of 2.5 mL
effect of DO3A-butyl-MPP exposure on cells, MTT assays were Tris–HCl buffer (50 mM, pH 7.4, 0.1% ascorbic acid, 2 mM
conducted. Exponentially growing cells were plated in a 96 well CaCl2) containing varied concentrations (1 nM to 1 mM) of the
microtitre plate at a cell density of 4000 cells per well for 24 h 68Ga-DO3A-butyl-MPP complex and membrane homogenate
before treatment. Cells were treated with the varying concen- (about 0.5 mg per mL protein). Nonspecic binding was deter-
trations of the drug (0.001–10 mM) at different time intervals mined as the amount of 68Ga-DO3A-butyl-MPP bound in the
24 h, 48 h, 72 h. At the end of treatment, both the treated cells presence of 10 mM serotonin. The binding assay was conducted
ꢁ
and negative control were incubated with MTT at a nal in triplicates at 20 C for 120 min. The incubation was termi-
concentration of 0.05 mg mLꢀ1 for 2 h at 37 ꢁC and followed by nated by rapid ltration through GF/B glass ber lters. The
removal of the medium. Triplicate wells from each treatment lters were rapidly washed with 4 mL portions of ice cold buffer,
were lysed and the formazan crystals were dissolved using transferred into 4 mL scintillation cocktail and analyzed for
150 mL of DMSO. Optical density of 150 mL of extracts at 570 nm radioactivity by scintillation counting.
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per gram of the tissue (% ID per g). Animal protocols have been
approved by Institutional Animal Ethics Committee and in
accordance with the guidelines of INMAS animal ethics
committee (CPCSEA Regn no. 8/GO/a/99).
5-HT2A receptor binding assay. The cortex of rat brain was
homogenized and prepared analogously to the procedure
described above and stored at ꢀ80 ꢁC until used in binding
studies. The assay was performed in a volume of 5.0 mL Tris–
HCl buffer (pH 7.6) containing 100 folds excess of ketanserin
tartarate, membrane homogenate (about 0.9 mg per mL
protein) and various concentrations (1 nM to 1 mM) of
68Ga-DO3A-butyl-MPP. Triplicates of the samples were incu-
bated at 20 ꢁC for 60 min. Filtration and counting of the
samples were the same as described above.
Brain biodistribution experiment. Three adult SD rats
weighing between 300 and 350 g were used in the procedure.
Intravenous injection of 0.5 mL of labeled 68Ga-DO3A-butyl-
MPP solution in 5% ethanol with an activity of 6.1 MBq was
injected through the tail vein to each rat. Aer 10, 30 and
45 min, the rats were dissected and the brains were removed
and placed in ice, washed with physiological saline, their weight
measured on an analytical balance and their activity counted.
The brains were then dissected, and the weights of the following
regions were calculated: hippocampus, caudate putamen,
cortex and cerebellum. The radioactivity in each section was
counted and regional uptake of the ligand was assessed.
5-HT transporter binding assay. The caudate nucleus of rat
brain was homogenized and prepared analogously to the
ꢁ
procedure described above and stored at ꢀ80 C until used in
binding studies. The nal volume of the binding assay was 5.0
Tris–HCl buffer (pH 7.4) containing 120 mM NaCl, 5 mM KCl,
membrane homogenate (about 1.2 mg per mL protein) and
various concentrations (1 nM to 1 mM) of the 68Ga-DO3A-butyl-
MPP complex in presence and absence of 100 fold excess of
paroxetine. Filtration and counting of the samples were the
same as described above.
Computational methodology
The homology model for human 5-HT1A receptor was prepared
as per our previously reported study24 by using prime homology
modeling.25 The 5-HT1A model was built on crystal structure of
PET acquisition. Animals were anesthetized by inhalation of
2% isourane in 2 L minꢀ1 oxygen in the prone position.
Animals were injected with 37 MBq activity of 68Ga-DO3A-butyl-
MPPimmediately prior to their micro PET scan on the camera
bed at the start of a dynamic acquisition. Frames of 1 ꢃ 1 and
5 ꢃ 5 min were accordingly sequentially recorded for 60 min.
The resulting PET was reconstructed using 50 iterations of the
maximum likelihood expectation maximization algorithm.
In vivo MR imaging. The animals were anaesthetized and
injected with the Gd-DO3A-butyl-MPP at 2 mM concentration
through tail vein. MR images were acquired using loop coil on
brain. T1 measurements were performed using a fast spin echo
sequence with TRs from 140–6000 ms with TE ¼ 8 ms. The slice
thickness, matrix and pixel size were same for all treated and
control animals.
Plasma clearance and biodistribution. Plasma clearance is
a quantitative study and reveals about the clearance kinetics of
the drug. Radiolabeled conjugate 68Ga-DO3A-butyl-MPP 200 mL
(10 mmol kgꢀ1, 14.8 MBq activity) was administered intrave-
nously through the tail vein of the SD rats (n ¼ 3). Blood
samples were withdrawn at different time intervals (0, 5, 10, 15,
20, 30, 60, 120 and 240 min). Decay corrected radioactivity is
expressed as % injected dose assuming theoretical blood
volume to be approximately 7% of the body weight. Plasma
clearance was plotted as mg mLꢀ1 minꢀ1 against time.
For biodistribution, twelve SD rats (weighing 110–140 g) were
divided into 4 groups (n ¼ 3) for time intervals-10 min, 30 min,
1 h and 4 h. Intravenous injection of 68Ga-DO3A-butyl-MPP
conjugate in a volume of 100 mL (5 mmol kgꢀ1, 8.14 MBq
activity) was injected through the tail vein of each rat. Rats were
dissected at 10 min, 30 min, 1 h and 4 h post injection. Various
organs were removed, made free of adhering tissue, rinsed with
chilled saline, blotted to remove excess liquid, weighed, and
radioactivity measured in each organ in a gamma counter
calibrated for 68Ga energy. Uptake of the radiotracer in each
tissue was evaluated and expressed as percentage injected dose
the b2-adrenergic receptor as template. The model was rened
28
¨
by using protein preparation wizard of Schrodinger and opti-
mized for further docking studies. Also, the ligand, ‘DO3A-
butyl-MPP’ was prepared using the Ligprep v2.9 module.23 All
the possible protonation states at pH ¼ 7.0 ꢂ 2.0 were generated
using ionization tool. Specied chiralities of these ligands were
retained during ligand preparation. Low energy conformers
were obtained in the force eld OPLS2005 without any
constraints. These conformers were retained for docking
studies. We have performed docking studies of all structured
5-HT1A models on the known binding sites and corresponding
grids were generated. Flexible (IFD) docking studies were per-
formed to predict the binding mode of the ligand with high
accuracy where the Glide docking under extra precision (XP)
mode was employed. Post docking minimizations were per-
formed by including ve poses per ligand.
Acknowledgements
We are thankful to Dr R. P. Tripathi, Director of Institute of
Nuclear Medicine and Allied Sciences, Defence Research and
Development Organization and Department of Chemistry,
University of Delhi for providing excellent research facilities.
We are also thankful Dr Poonam Rana for MR imaging at NMR
Research Centre, INMAS. This work was supported by INMAS
Project INM-311(3.1).
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