Synthesis and evaluation of technetium-99m labelled 1-(2-methoxyphenyl)piperazine derivative for single photon emission computed tomography imaging for targeting 5-HT1A

Article abstract of DOI:10.1016/j.bioorg.2021.104972

Quantitative changes in expression level of 5HT_1A are somewhere related to common neurological disorders such as anxiety, major depression and schizophrenia. We have designed EDTA conjugated SPECT imaging probe for localization of 5HT_1A

Full text of DOI:10.1016/j.bioorg.2021.104972

Bioorganic Chemistry 111 (2021) 104972
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and evaluation of technetium-99m labelled 1-(2-methoxyphenyl)
piperazine derivative for single photon emission computed tomography
imaging for targeting 5-HT1A
Neelam Kumaria c, Ankur Kaulb, Raunak Varshneyb, Vinay Kumar Singhd,
,b,
,b,*
Krishna Srivastavae, Sunita Bhagatc, Anil Kumar Mishrab, Anjani Kumar Tiwaria
a Department of Chemistry, School of Physical & Decision Sciences (SPDS), Babasaheb Bhimrao Ambedkar University (A Central University), Lucknow 226025, UP,
India
b Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Road, Delhi 110054, India
c Department of Chemistry, University of Delhi, Delhi 110054, India
d Department of Chemistry, Dr. S.M.N.R University, Lucknow 226018, India
e Faculty of Chemical Sciences, Shri Ramswaroop Memorial University, Lucknow 225003, UP, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Quantitative changes in expression level of 5HT1A are somewhere related to common neurological disorders such
as anxiety, major depression and schizophrenia. We have designed EDTA conjugated SPECT imaging probe for
localization of 5HT1A receptor in brain. For designing SPECT probe we have employed the concept of bivalent
approach and a homodimeric system with desirable pharmacokinetics of 5HT1A imaging. 99mTc-EDHT was also
evaluated for its stability through serum stability assay and glutathione challenge experiment. Biodistribution
study showed the highest accumulation of radioactivity in kidney which depicted the renal mode of excretion
from the body. However in brain the uptake of 1.21% ID per gram was observed in initial 5 min of drug
administration. On blocking the receptor this percent get decreased to 0.97% ID per gram. The regional distri-
bution in brain was also performed which showed the accumulation of drug in cerebellum, cortex and hippo-
campus part, which are already known for 5HT1A expression. Dynamic study in rabbit is also in support of results
derived from biodistribution and blood kinetics experiment. These finding suggest that 99mTc-EDHT holds
promising place for further optimization before nuclear medicine applications in different animal species.
Serotonin
Molecular docking
Acetamidobenzoxazolone
5-HT1A
SPECT/PET and Inflammation
1. Introduction
of neurotransmitter receptors and transporters associated with disease
onset and progression. Only a handful of established imaging agents
Enormous development in the field of molecular imaging has enabled
neuroscientists to gain a deeper insight into the functioning of the
central nervous system during the last few decades.[1–3] The discovery
of high resolution imaging techniques having the capability to explore
physiological, structural, and chemical properties in brain has really
opened up the field of in-vivo brain mapping. New imaging techniques
which are non-invasive in the nature can explore changes in receptor
availability during neurological diseases and disorders involving the
central nervous system including Parkinson’s disease, schizophrenia,
Alzheimer’s disease, epilepsy and addiction.[4–6] These molecular im-
aging helps in understanding the changes in concentration and function
exist for the imaging of CNS targets despite the fact that development of
molecular tracers for in-vivo imaging of specific targets in the CNS began
more than 25 years ago.[7–18] So, it’s a great interest in molecular
imaging field to develop ligands of high affinity and selectivity for the
receptor to perform brain imaging (see Table 1).
In patients of psychiatric disorders including depression, anxiety and
schizophrenia, there is reduction in serotonin receptor levels.[19–27]
Only 1–2% of total body serotonin is found in central nervous system.
5HT1 family of receptors have a role in inhibiting cell firing. Out of all
5HT1 receptor family, 5HT1A is the highest investigated serotonin re-
ceptor. It has also been found to have more control over the anxiety/
* Corresponding author at: Department of Chemistry, School of Physical & Decision Sciences (SPDS), abasaheb Bhimrao Ambedkar University (A Central Uni-
versity), Lucknow 226025, UP, India.
E-mail address: anjani7797@rediffmail.com (A.K. Tiwari).
https://doi.org/10.1016/j.bioorg.2021.104972
Received 26 November 2020; Received in revised form 17 April 2021; Accepted 4 May 2021
Available online 13 May 2021
0045-2068/© 2021 Published by Elsevier Inc.

N. Kumari et al.
Bioorganic Chemistry 111 (2021) 104972
the synthesis of neutral, lipophilic, small size oxotechnetium complexes.
This approach uses the simultaneous action of a dianionic tridentate
ligand (having SSS, SOS or SN(R)S donor atom set) and a monodentate
thiol as co-ligand on a suitable oxotechnetium precursor.[54–56,58,62]
The best part of these complexes are their appropriate capability to cross
blood–brain barrier due to its neutrality and diverse aspects of conju-
gation with pharmacologically relevant groups.
Table 1
Hematological and Biochemistry Values data of animals treated with single dose
of EDHT.
Control
7 day
14 day
Haemoglobin(Hb)/ gm/dl
Neutrophil / %
12.50
12.80
13.05
32.16 ± 1.1
33.17 ±
0.12
36.15 ±
1.20
Lymphocyte / %
92.35 ±
2.18
93.67 ± 2.5
95.52 ±
1.20
Fig. 1 represents the representative compounds of all category.
Compound 1 and 2 shows 3 + 1 approach based mixed ligand complex
in which the requisite pharmacophore moiety has been placed in two
opposite direction. Compound 3 and 4 represents 4 + 1 and 2 + 1 + 1
complexes. The other two approaches tetradentate N2S2 chelates and the
Tc-tricarbonyl approaches have been shown by compound 5 and 6.
Compound 7 represent nonmetallic PET ligand labelled with carbon-11
isotope having 1-(2-methoxyphenyl) piperazine moiety.
Eosinophil / %
2.15 ± 0.51
3.61 ± 0.11
0 ± 0
2.82 ± 1.16
1.98 ± 1.17
0 ± 0
2.51 ± 1.50
2.12 ± 0.19
0 ± 0
Monocyte/ %
ESR (Westegren’s Method) /mm/
1st hr.
RBC Count / Millions/cmm
P.C.V/Haematocrit / %
8.33 ± 0.51
57.21 ±
0.95
7.51 ± 0.3.5
55.27 ±
0.951
7.85 ± 0.8
55.56 ±
2.50
M C V / fl.
61.53 ±
3.10
61.25 ±
1.76
62.52 ±
5.20
The presented compound 8 of this work comprises benzoxazolone
conjugated with MPP system having EDTA chelator to evaluate the
5HT1A expression in brain. Idea behind using EDTA in place of other
vehicle, is to improve the lipophilicity of the ligand towards brain
regions.
M C H / Picogram
M C H C / gm/dl
21.15 ±
2.50
18.98 ±
1.61
17.26 ±
3.15
32.13 ±
1.10
33.25 ±
2.15
35.53 ±
3.15
Platelet Count / Lakh/cmm
AST (IU/L)
7.25 ± 3.50
75.2 ±
8.87 ± 2.90
68.2 ±
6.96 ± 1.30
68.3 ± 9.30
16.10
19.30
2. Experimental
ALT (IU/L)
25.7 ± 6.17
5.30 ± 1.53
3.60 ± 1.30
22.2 ± 5.30
5.02 ± 1.21
3.65 ±
24.7 ± 5.50
5.22 ± 1.32
5.52 ± 1.32
Protein (g/dL)
Albumin (g/dL)
2.1. Synthesis (Chemistry)
1.165
2.1.1. Material and methods
All chemicals and solvents required for the synthesis have been
purchased from Sigma Aldrich, Alfa Aesar and Spectrochem Pvt. Ltd.
Buchi Mꢀ 560 instrument is used for determining the melting point and
IR spectra has been drawn from Perkin-Elmer FT-IR spectrometer. For
some compounds KBr disc was prepared and some compounds were
analyzed in form of thin film. NMR spectra were recorded at Jeol alpha-
400 spectrometer. Chemical shift values in case of 1H NMR are reported
on δscale and coupling constants (J) are reported in Hz unit. Mass
spectra’s were obtained on a 6530 Q-TOF instrument from Agilent
Technologies.
anxiolytic action in comparison to the other members of the 5HT1
family. The hyperpolarisation of the neuron is caused because of 5HT1A
activation by triggering voltage gated K + channels. The most common
location of postsynaptic 5HT1A receptors is on GABAergic and gluta-
minergic neurons, which has high concentrations in several cortical and
subcortical areas mainly in limbic system.[28–29]
Molecular imaging techniques using PET or SPECT are the tools used
in the investigation of changes in 5HT1A receptor expression. During
past decades, there is a rise of interest of medicinal chemist for providing
integrating molecular medicine solutions using new radiolabeled li-
gands with dedicated and optimized imaging systems to investigate in-
vivo changes of 5HT1A receptors.[30–32]
3. Animals
Metal complexes are not generally found suitable for normal brain
imaging probes due to their low BBB permeability but it can be utilized
in diseased state. Despite the exponential growth in the number of nu-
clear medicine hospitals equipped with SPECT, 99mTc is one of the most
common radionuclide owing to its good imaging characteristics and
easy availability of 99mTc/99Mo generators. There are many reviews
which have been written for the development of metal coordinated
imaging probes in CNS applications.[33–36]
Young healthy male Sprague Dawley Rats matured for 2–3 months,
weight 150–200 g were acquired from the Experimental animal facility
(EAF) of Institute of Nuclear Medicine and Allied Sciences (INMAS),
Delhi. The take care of animals were regulated as per the Committee for
the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) and the study protocol was approved by the Institutional
Animal Ethics Committee (IAEC) of the institute (INM/DASQA/IAEC/
09/015). All the animals were administered food (Hindustan Lever Ltd,
Mumbai, India) and water ad libitum. Management and husbandry
conditions were identical in all groups of experimental animals with 12/
In general, imaging addresses two main things: structure and func-
tion. With the aid of different imaging modalities we can image anatomy
or examine physiological functions of the brain. Arylpiperazines is one
of the most promising classes to design 5HT1A receptor ligands. The most
common skeletons are gepirone, buspirone, WAY-100135/ WAY-
100635 and others.[37–44] WAY-100635 (N-(2-(4-(2-methoxyphenyl)-
1-piperazinyl) ethyl)-N-(2-pyridyl)-cyclohexane carboxamide) was the
first successful compound (selective antagonists) in this category. The
most important pharmacophore present in WAY-100635 is 1-(2-
methoxyphenyl)piperazine which have high affinity for the 5HT1A
subreceptors.[45] The amalgamation of aromatic ring and basic nitro-
gen containing piperazine is the primary requisite scaffold for 5HT1A
receptor binding. The basic nitrogen in the piperazine can further
exploited for functionalization with a long chain alkyl linker.
◦
12 h light/dark cycle at 22 ± 2 C. The animals were acclimatized to
laboratory conditions for at least one week before experimentation. The
animals were kept under observation during the entire experimental
period.
3.0.1. General procedure for synthesis of 2,2′-(ethane-1,2-diylbis((2-((3-
(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-2-oxo-2,3-dihydrobenzo
[d]oxazol-5-yl)amino)-2-oxoethyl)azanediyl))diacetic acid (EDHT)
• Synthesis of 5-nitro-1,3-benzoxazol-2(3H)-one (2)
To a solution of 2-amino-4-nitrophenol derivative 1 (0.8 g, 5.19
mmol) in THF (30 mL) was added 1,10-carbonyldiimidaziole (1.42 g,
6.23 mmol) at room temperature. The mixture was stirred at reflux for 3
hrs and cooled to room temperature. The reaction was then quenched by
adding 2 M HCl solution, and the mixture was extracted with EtOAc. The
organic layer was washed with brine and dried over anhydrous sodium
Three different mixed ligand complex approaches (3 + 1, 4 + 1, 2 +
1 + 1) were used to attach the technetium to the pharmacophore moiety
of WAY 100635.[46–57]Besides that tetradentate N2S2 chelates and the
Tc-tricarbonyls were also used to attach this pharmacophore.[58–61]
Out of these approaches, the most successful was the 3 + 1 concept for
2

N. Kumari et al.
Bioorganic Chemistry 111 (2021) 104972
Fig. 1. Representative systems having 1-(2-methoxyphenyl) piperazine moiety for 5HT1A receptor imaging.
sulphate. After filtration, the solvent was removed in vacuo to give 2
Physical state and yield: Yellow solid, 99% yields.
Rf = 0.7 (40% ethyl acetate in hexane)
• Synthesis of 3-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-5-
nitrobenzo[d]oxazol-2(3H)-one (4)
M. Pt.: 98–99 ◦C
To an ice-cool solution of 3 (0.4 g, 1.2 mmol) in DMF (20 mL)
anhydrous K2CO3 (0.35 g, 2.5 mmol) was added followed by the addi-
tion of 1-(2-methoxyphenyl)piperazine (0.227 g, 1.18 mmol). The
mixture was stirred at 0 ◦C for 3 hrs. Reaction mixture was cooled and
poured into ice water which results into formation of precipitate. The
collected precipitate was further purified by silica gel column chroma-
tography using ethyl acetate in hexane as gradient solvent system to
afford 4.
IR (thin film) νmax: 3127, 1785, 1678, 1522, 1473 and 1344 cmꢀ 1
1H NMR (DMSO‑d6, 400 MHz): δ 7.92 (1H, dd, J = 2.4 and 8.8 Hz),
7.76 (1H, d, J = 2.8 Hz) and 7.36 (1H, d, J = 9.2 Hz)
13C NMR (DMSO‑d6, 100.6 MHz): δ 154.7, 148.2, 144.2, 131.5,
119.2, 110.35 and 105.6;
HR-ESI-TOF-MS: m/z 179.0083 ([Mꢀ H]+), calcd. for [C7H4N2O4-
H]+ 179.0087.
Physical state and Yield: pale yellow solid, 92%
• Synthesis of 3-(4-bromobutyl)-5-nitrobenzo[d]oxazol-2(3H)-one (3)
Rf = 0.3 (10% MeOH in CHCl3)
IR (KBr) νmax: 2942, 2816, 1787, 1673, 1492, 1342, 1238, 1141,
1023 and 749 cmꢀ 1
1, 4-dibromobutane (2.4 g, 11.1 mmol) in acetone (10 mL) was
added dropwise to the solution of 5-nitrobenzo[d]oxazol-2(3H)-one
(0.5 g, 2.7 mmol) and potassium carbonate (0.76 g, 5.5 mmol) in
acetone (30 mL) at room temperature followed by the refluxing for 4 h.
The mixture was filtered for eliminating the inorganic material and the
solvent was removed through rotary evaporator to obtain a residue. The
solution was extracted with dichloromethane (3 × 50 mL) and the
resultant extract were washed, dried and evaporated. The solution was
dried over anhydrous sodium sulphate, filtered and concentrated under
reduced pressure. The residue was purified by flash chromatography on
silica gel by using ethyl acetate in hexane as gradient solvent system to
afford 3 as white solid.
1H NMR (CDCl3, 400 MHz): δ 8.26, (1H, s), 8.09 (1H, d, J = 9.2 Hz),
7.58 (1H, d, J = 9.2 Hz), 6.90–6.83 (4H, m), 3.94 (2H, t, J = 6.3 Hz),
3.75 (3H, s), 2.88 (4H, m), 2.50–2.49 (4H, m), 2.34 (2H, t, J = 6.3 Hz),
1.78 (2H, s) and 1.52 (2H, s).
13C NMR (DMSO‑d6, 100.6 MHz): δ 153.6, 151.9, 146.5, 144.0,
141.2, 132.0, 122.3, 120.8, 118.9, 117.8, 111.8, 110.5, 104.8, 57.1,
55.3, 53.0, 50.0, 42.1, 24.9 and 23.0.
HR-ESI-TOF-MS:
m/z
427.1968
([M+H]+),
calcd.
for
[C22H26N4O5+H]+ 427.20.
• Synthesis of 5-amino-3-(4-(4-(2-methoxyphenyl)piperazin-1-yl)
butyl)benzo[d]oxazol-2(3H)-one (5)
Physical state and Yield: yellow oil, 88%
Rf = 0.7 (20% EtOAc in Hexane)
IR (KBr) νmax: 3744, 2876, 1787, 1617, 1529, 1344, 1264 and 750
cmꢀ 1
To a solution of purified compound 4 (0.3 g, 0.75 mmol) in methanol
(20 mL) was added 10% palladium on charcoal (97 mg). The flask was
supplied with hydrogen balloon and the mixture was stirred for 2 hrs at
room temperature. At finishing of the reactant, the catalyst was celite
filtered, the filtrate was evaporated under reduced pressure to afford
amine derivative 5
1H NMR (DMSO‑d6, 400 MHz): δ 8.27 (1H, d, J = 9.6 Hz), 8.068
(1H, d, J = 8.0 Hz), 7.57 (1H, d, J = 8.4 Hz), 3.95–3.89 (2H, m),
3.58–3.53 (2H, m) and 1.87–1.71 (4H, m)
13C NMR (DMSO‑d6, 100.6 MHz): δ 153.6, 146.5, 144.1, 132.0,
118.9, 110.1, 104.9, 60.20, 34.6, 29.4 and 26.0.
Physical state and Yield: White solid, 74%
HR-ESI-TOF-MS:
m/z
315.004
([M+H]+),
calcd.
for
Rf = 0.1 (20% MeOH in CHCl3)
[C11H11BrN2O4+H]+ 315.00.
IR (KBr) νmax: 3744, 3356, 2946, 2880, 2823, 1760, 1628, 1497,
1366, 1240, 1023 and 754 cmꢀ 1
1H NMR (DMSO‑d6, 400 MHz): δ 6.97–6.85 (5H, m), 6.44 (1H, s),
3

N. Kumari et al.
Bioorganic Chemistry 111 (2021) 104972
6.28 (1H, s), 5.07 (2H, brs), 3.78–3.71 (5H, m), 3.37–3.36 (2H, m), 2.97
(4H, s), 2.60 (4H, s), 1.72–1.65 (2H, m) and 1.51 (2H, s).
13C NMR (DMSO‑d6, 100.6 MHz): δ 154.5, 151.9, 145.9, 133.2,
131.41, 122.6, 120.8, 117.9, 111.9, 109.9, 107.0, 94.9, 55.3, 52.6, 41.0
and 24.9.
The characterization of 99mTc-EDHT was accomplished by
comparative studies after co-injection of the Re complexes and corre-
sponding 99mTc complexes at tracer level with HPLC analysis with
UV–vis and gamma detection. HPLC analysis was performed with a
reverse phase C18 column. The isocratic solvent CH3CN:H2O were used
in the ratio of 70:30 for 99mTc- EDHT.
HR-ESI-TOF-MS:
m/z
397.2230
([M+H]+),
calcd.
for
[C22H28N4O3+H]+ 297.22.
3.3. Glutathione challenge
• Synthesis of 2,2′-(ethane-1,2-diylbis((2-((3-(4-(4-(2-methoxyphenyl)
piperazin-1-yl)butyl)-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)
amino)-2-oxoethyl)azanediyl))diacetic acid (EDHT)
To test the 99mTc-EDHT stability, it’s challenged with Glutathione
within concentration range of 25 mM- 100 mM. Labelled compound was
incubated with solution mixture of glutathione in saline and percentage
of transcomplexation and labelled compound was determined at
different time interval by instant thin layer chromatography (ITLC).
A solution of compound 5 (0.35 g, 0.89 mmol) and Et3N (0.19 g, 1.9
mmol) in anhydrous DMF (10 mL) was stirred at room temperature.
EDTA dianhydride (0.10 g, 0.39 mmol) was added slowly to the reaction
mixture and was continued to stir at 50 ◦C for next 20 h. After finishing
of the starting material in reaction mixture, solvent was removed and
residue was redissolved in methanol and diethyl ether to get the pre-
cipitate. Achieved crude product was further dried under reduce vac-
uum to afford compound EDHT.
3.4. In-Vitro serum stability of 99mTc-EDHT
The radiolabeled drug 99mTc-EDHT was tested for its in-vitro serum
stability by ascending instant thin layer chromatography. For in-vitro
stability in physiological saline and serum, 100 µL of the radiolabel
was mixed with 2 mL each of 0.9% saline and serum. Instant thin layer
chromatography was carried out to assess the labeling efficiency after
incubating at 37 ◦C for 30 min. For in-vitro protein binding, 37 MBq of
99mTc-EDHT in 0.1 mL was mixed with 2 mL of plasma (n = 6). Aliquots
were taken at various time intervals and proteins were precipitated by
adding equal volumes of 12.5% trichloroacetic acid (TCA) and plasma.
The radioactivity in the precipitate and supernatant was measured in a
well type gamma counter.
Physical state and Yield: white solid, 80%
IR (KBr) νmax: 3394, 2361, 1593, 1435, 769 cmꢀ 1
1H NMR (DMSO‑d6, 400 MHz): δ 10.48 (2H, brs), 7.61 (2H, s),
7.30–6.79 (12H, m), 4.24 (25H, m), 3.73(12H, s), 3.41–3.35 (10H, m),
2.96–2.49 (6H, m) and 1.67–1.54 (8H, m).
13C NMR (DMSO‑d6, 100.6 MHz): δ 174.5, 170.0, 1154.5, 153.2,
141.2, 138.3, 136.1, 132.7, 134.4, 122.4, 122.6, 119.2, 110.3, 112.2,
109.7, 101.22, 66.20, 58.2, 55.3, 53.2, 49.6, 40.2, 25.6 and 23.2
HR-ESI-TOF-MS: m/z 1071.4904 ([M+Na]+), calcd. for
[C54H68N10O12+Na]+ 1071.49.
3.5. Blood kinetics
For in-vivo studies, 74 MBq of radiotracer was administered to the
rabbit and blood samples were withdrawn at various time intervals.
Drug was injected through the ear vein and blood samples were
collected at different time intervals post administration (n = 3). The
radioactivity in blood samples was measured in a well type gamma
counter and was calculated as percentage of the injected dose.
Formula used for Blood-clearance:
Reaction conditions: (i) CDI, THF, reflux, 3hrs; (ii) 1,4-dibromobu-
tane, K2CO3, acetone, reflux, 4hrs; (iii) arylpiperazine, K2CO3, acetoni-
trile, stirred at 0 ◦C, 3hrs (iv) Pd/C, MeOH, rt; (v) 4,4′-(ethane-1,2-diyl)
bis(morpholine-2,6-dione), Et3N, DMF, 50 ◦C
3.1. In vitro receptor binding assay
%Radioactivity in blood = (Counts *100 * Volume of blood)
(Weight of blood * Total counts injected)
In vitro receptor binding assay was performed through competitive
binding experiments by using [3H]-8-OH-DPAT as radioligand in
duplicate. Aliquots of volume 50
μ
L of rat hippocampal homogenates
3.6. Biodistribution
were mixed with 50
μ
L [3H]-8-OH-DPAT (0.180 nM) tris-HCl buffer (50
The animals were used from the experimental animal facility at the
Institute. The animals were fed laboratory chow pellets, in ad libitum
with free access to food and water. The animals were in the room with
daytime light and no light after 1900 h until morning with the tem-
perature of approx. 25 ◦C.
mM tris-HCl, 0.1% ascorbic acid, 2 mM CaCl2 at pH7.5) and 50 μL of
increasing concentrations of competing EDHT and its Re complex.
Nonspecific binding was defined with 10 μM 8-OH-DPAT. The mixture
was incubated for 20 min at 37 ◦C and then bound and free radioligand
was separated by filtration and resoaked with 1% bovine serum albumin
(BSA). It was washed three times with 3 mL of ice cold tris-HCl buffer
(50 mM tris-HCl, 150 mM NaCl) and dried over for 10 min, then placed
in a 2 mL scintillation cocktail. The results of competitive experiments
were analyzed to obtain the IC50 values.
In-vivo distribution of 99mTc-EDHT was studied in 2 months old
BALB/c mice (n = 3; each weighing approximately 22 g). An aliquot of
100 µL of 99mTc-EDHT (3.7 MBq) was administered intravenously to
each mouse, weighing about 25 g, through the tail vein. The animals
were sacrificed at different time intervals and different organs were
removed, collected into preweighed tubes. The radioactivity in each
organ was counted using well-type gamma spectrometer and Counts per
minute (CPM) values were decay-corrected and results were calculated
as % ID per gram of wet tissue. All animal studies were approved by the
institutional Experimental Animal Ethical Committee and performed in
accordance with their guidelines.
3.2. Radiolabeling of EDHT
The radiolabelling of EDHT is performed by using sodium pertech-
nate as labeling reagent and SnCl2 as a reducing agent. In summary,
EDHT was dissolved in water, then 1 N HCl solution of 50 mg
SnCl2⋅2H2O was added to the solution and the pH was adjusted to 7.0.
After filtration through a 0.22
μm millipore filter and lyophilized. An
Formula for biodistribution studies:
eluate of 1 mL Na99mTcO4 was added to a lyophilized kit. The labelling
efficiency was more than 98%. The radiochemical purity of 99mTc-EDHT
was determined by ascending instant thin layer chromatography (ITLC)
using silica gel coated fibre glass sheets (Pall Corporation) and dual
solvent systems, namely (a) 100% acetone and (b) a solvent mixture of
Ethanol, Ammonia and Water (2:1:5 v/v) as mobile phases. The radio-
active contaminants were identified as reduced/hydrolyzed (R/H) 99mTc
(Rf = 0.0) and free 99mTc-pertechnetate (Rf = 1.0) and the labeled
product remained at the point of application when 100% acetone alone
was used as mobile phase.
% Injected Dose per Gram Organ = (Counts * 100)
(Wt of organ* Total counts injected)
3.7. SPECT imaging of 99mTc-EDHT
SPECT whole body scintigraphic scan was carried out after intrave-
nous administration of 40 MBq of radiolabeled EDHT in normal New
Zealand rabbit through the dorsal ear vein. The animal was anaes-
thetized by intramuscular injection of diazepam 10 min before imaging.
The animal was fixed on a board in supine position and imaging was
performed using Single Photon Emission Computerized Tomography
4

N. Kumari et al.
Bioorganic Chemistry 111 (2021) 104972
(Symbia® Siemens,USA).
agent. Now this amine group can be utilized to conjugate with EDTA
dianhydride to afford EDHT (6) and can be conjugated to isocynate
group of FITC to form FIHT (7). The structures of all synthesized in-
termediates and compounds were unambiguously established on the
basis of their spectral data (1H NMR, 13C NMR spectra, IR spectra and
HRMS) analysis (Scheme 1).
3.8. In-vivo safety evaluation
For safety evaluation, the animals were injected with a single dose of
0.5 mg/ kg body weight of tested compound, intravenously and left for
seven days and fourteen days. After the experimental period, animals
were sacrificed and blood sample was collected through cardiac punc-
ture into tubes containing EDTA for hematological estimations. The
hematological parameters were evaluated using hematological auto-
matic analyzer. The parameters determined were as follows:White blood
count, WBC (103/µl), Red blood cell, RBC count (106/µl), Hemoglobin,
Hb (g/dL), Hematocrit HCT (%), Mean Corpuscular Volume, MCV (fL),
Mean Cell Hemoglobin, MCH (pg), Mean Cell Hemoglobin Concentra-
tion, MCHC (g/dL), Platelet (103/µl), Clotting time (s), Lymphocyte (%),
Neutrophil (%), Monocyte (%), Basophil (%), Eosinophil (%).
4.2. Radiolabeling of EDHT
The radiolabeling efficiency of the compounds and stability are
controlled by several factors including temperature, type of complexing
ligand, pH, reducing agent and the incubation time. Prior to in-vivo
studies, by keeping all parameters constant and the labeling efficiency
was measured through ITLC. It was observed that the radiolabeling ef-
ficiency was considerably high for the complex at a pH 7. So, it qualifies
the complexing agent for in-vivo administration and evaluation. The
labeling efficiency in solvent system (b) is shown in Fig. 2.
4. Result and discussion
4.1. Synthesis (Chemistry)
4.3. Glutathione challenge and in vitro binding assay
In first step 2-amino-4-nitrophenol was cyclised by using CDI reagent
in THF solvent at refluxing for 3 h to afforded 5-nitrobenzo[d]oxazol-2
(3H)-one (2) in 90% yield. In next step 1, 4 dibromobutane is conjugated
to compound 2 in K2CO3 and acetone on refluxing for 4 h to produce 3-
(4-bromobutyl)-5-nitrobenzo[d]oxazol-2(3H)-one (3). The bromo group
in compound 3 is further utilized for 1-(2-methoxyphenyl)piperazine
conjugation to afford compound 4 with 92% yield. Subsequently nitro
group of the compound is reduced to amine group (5) as a reducing
99mTc-EDHT showed only 0.72% of transcomplexation of 99mTc on
glutathione concentration of 0.25 mM and 95.31% of the radiiolabelled
compound remain associated at 1 mM concentration of 37 ◦C even after
2 h of glutathione challenge (Fig. 3).
The analogous Re compounds were synthesized as a surrogate for the
99mTc complexes for use in receptor binding assays. During in vitro
binding assays, the Re analogues were tested with a competition assay
against [3H]-8-OH-DPAT. The ligand and its metal complex showed
Scheme 1. Synthesis of 2,2′-(ethane-1,2-diylbis((2-((3-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)amino)-2-oxoethyl)
azanediyl))diacetic acid (EDHT).
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Bioorganic Chemistry 111 (2021) 104972
4.5. Blood kinetics
New Zealand albino rabbits weighing 2.5 to 3.0 kg were used for
blood clearance studies. Blood Kinetics of 99mTc-EDHT in whole blood
after intravenous administration in rabbits exhibited bi-exponential
clearance pattern.
The % radioactivity associated with blood at 1 h was found to be
8.97% and this value decreased to 2.70% at 24 h post administration.
Fig. 5 shows that radiolabelled EDHT showed initial fast elimination
followed by slow clearance from systemic circulation.
4.6. Biodistribution
Fig. 2. Radiochromatogram of 99mTc-EDHT using ITLC-SG in Ethanol,
Biodistribution of 99mTc-EDHT in various organs in mice at various
time intervals post administration is shown in Fig. 6.
Ammonia and Water (2:1:5 v/v) at room temperature (25 ◦C).
Among all the organs studied, kidneys showed maximum uptake of
9.21% of injected dose per whole organ at 5 min and liver showed the
uptake of 7.28% of injected dose per whole organ at 5 min. Significant
radioactivity in kidney and liver suggest its excretion through both the
hepatobiliary and renal routes. Low uptake by blood was observed and
low protein binding justifies for its low activity in blood pool. Further-
more, absence of radioactivity in the stomach confirms the in-vivo sta-
bility of the radiolabel throughout the duration of the study. The
targeted organ in the studies i.e. brain showed the uptake of 1.21%
injected dose per gram in initial 5 min of drug administration followed
by the slow wash out in next 2hr. Biodistribution in blocking case was
also performed to analyze the specificity. In Fig. 7 the radiolabelled
EDHT uptake is shown when the receptor is already pre-blocked by the
cold substitute. The reduction of % ID in brain after blocking, in com-
parison to the control case is expected due to the specificity of the ligand
toward serotonin receptor. Preblocked brain showed the highest uptake
of 0.97% ID at 5 min and at the same time point in control case the
uptake is 1.21% ID.
Fig. 3. Transcomplexation of 99mTc-EDHT by glutathione challenge.
Biodistribution pattern is further observed in various region of brain
also to determine the accumulation of radioactivity in 5HT1A rich re-
gion. In control case at 5 min, 10 min, 30 min, 60 min and 120 min of
time point the % ID of drug is determined for cerebellum, cortex,
striatum, and hippocampus. The highest accumulation in the hippo-
campus and cortex was because of expression of 5HT1A receptor. One of
the way to see the regional brain uptake in Hippocampus (HP) Cere-
bellum region (CB). The ratio of HP/CB increased till 10 min but de-
creases in between 30 and 60 min and further increases 60–120 min.
With the time the decline in radioactivity is observed (Fig. 8). On
comparing the activity we found that the radioactive accumulation is
reduced in case of cerebellum, cortex and hippocampus but remain
unaffected in case of stratium. It indicates that the uptake of radioac-
tivity in hippocampus, cerebellum and cortex is specifically because of
5HT1A receptor.
nanomolar affinities to the target receptor. The binding affinity of the
EDHT displayed appropriate affinity for the 5-HT1A receptor (IC50
=
11.3 nM) in a competition assay against [3H]-8-OH-DPAT. No significant
impact was found after introduction of a Re metal into EDHT (IC50 =
38.7 nM for Re-EDHT).
4.4. In-Vitro serum stability of 99mTc-EDHT
In-vitro studies like Serum-stability, glutathione challenge test are
done to confirm the strength of the radio conjugation when adminis-
tered into systemic circulation. In presence of serum, the radiolabeled
EDHT showed good stability upto 24 h post labelling which is important
for further in vivo studies (Fig. 4).
During blocking experiment the upatake of radioactivity in the
Fig. 4. In-vitro serum stability studies of 99mTc-EDHT.
Fig. 5. Blood clearance of 99mTc-EDHT in rabbits.
6

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Bioorganic Chemistry 111 (2021) 104972
Fig. 6. Bio-distribution-study of radiolabeled EDHT in BALB/c mice.
Fig. 7. Bio-distribution-study of 99mTc-EDHT in blocked (PK11195) BALB/c mice.
blocked and non blocked mice as been dipected in form of graph in
Fig. 9.
urinary bladder which indicates its renal mode of exrection. This scan
facilitated in explaining the in-vivo delivery of radiocomplex to different
organs and correlated with the findings of the tissue distribution. A
representative static image at 30 min has been shown in Fig. 11.
In conclusion for biodistribution of 99mTc-EDHT was not homoge-
neous in the brain. 5-HT1A rich regions like hippocampus, showed low
concentrations of radioactivity and somehow in other regions does not
bind specifically to 5-HT1A receptors in vivo.
4.8. Hematology and biochemistry evaluation
4.7. SPECT imaging of 99mTc-EDHT
No mortality was observed in experimental animal models till 14
days.
Dynamic imaging was performed on rabbit and the images were
acquired on gamma camera for 60 min (Fig. 10). The early phase showed
the high uptake in the brain and the wash out was observed in 30 min.
The highest accumulation in brain is seen at till 15 min of post injection
followed by slow wash out. The uptake is also observable in kidney and
5. of controlled and treated mice was recorded and no deviation
was found till 14 day.
The variation in the clinical biochemistry parameters in the control
7

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Bioorganic Chemistry 111 (2021) 104972
Fig. 8. Regional brain uptake of 99mTc-EDHT in BALB/c mice.
Fig. 9. Regional brain uptake of 99mTc-EDHT in PK11195 pre-treated (blocking) condition.
and test groups were less than 10%. There was no uniformity in in-
crease/decrease pattern of parameters in the studied animals in the same
or different groups. Any increase and decrease in particular group could
be considered as incidental.
further development of complexes with improved biological profiles of
ligands.
Declaration of Competing Interest
6. Conclusion
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
In this work, we have designed EDTA conjugated SPECT imaging
probe for localization of 5HT1A receptor in brain. Our results demon-
strate the potential of 99mTc-EDHT with the ability to bind 5HT1A re-
ceptor. However, substantial imaging of 5-HT1A with 99mTc requires
8

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Bioorganic Chemistry 111 (2021) 104972
Fig. 10. Dynamic SPECT imaging in New Zealand rabbit (0–30 min).
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