Modified benzoxazolone (ABO-AA) based single photon emission computed tomography (SPECT) probes for 18 kDa translocator protein

Article abstract of DOI:10.1002/ddr.21547

Acetamidobenzoxazolone (ABO) has been modified to ABO-AA, 2-(2-(5-bromo/chloro benzoxazolone)acetamide)-3-(1H-indol-3-yl)propionate to improve pharmacokinetics and lipophilicity (log p = 2.04). The final compound was synthesized in better yield and in few

Full text of DOI:10.1002/ddr.21547

Received: 3 February 2019
DOI: 10.1002/ddr.21547
Revised: 9 April 2019
Accepted: 11 May 2019
R E S E A R C H A R T I C L E
Modified benzoxazolone (ABO-AA) based single photon
emission computed tomography (SPECT) probes for 18 kDa
translocator protein
Pooja Srivastava1,2 | Dipti Kakkar1 | Pravir Kumar2 | Anjani Kumar Tiwari1,3
1Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Delhi, India
2Molecular Neuroscience and Functional Genomic Laboratory, Department of Biotechnology, Delhi Technological University, Delhi, India
3Department of Chemistry, School of Physical & Decision Sciences (SPDS), Babasaheb Bhimrao Ambedkar Central University, Lucknow, UP, India
Abstract
Correspondence
Anjani K. Tiwari, Department of Chemistry,
Acetamidobenzoxazolone (ABO) has been modified to ABO-AA, 2-(2-(5-bromo/chloro
School of Physical & Decision Sciences (SPDS),
benzoxazolone)acetamide)-3-(1H-indol-3-yl)propionate to improve pharmacokinetics
and lipophilicity (log p = 2.04). The final compound was synthesized in better yield and in
fewer steps than previously reported MBIP-Br (70% vs. 62%). Computational docking
confirmed binding of MBIP-Cl with translocator protein (TSPO) as well as with mutant
TSPO (−8.99 for PDB: 4RYQ and −9.30 for PDB: 4UC1, respectively). Ex-vivo bio-
distribution and scintigraphy showed that 99mTc-MBIP-Cl is better than 99mTc-MBIP-Br
in terms of uptake in TSPO-rich organs and release kinetics 0–120 min postinjection. At
15 min, uptake was 2.75-fold (12.91%ID/g vs. 4.69%ID/g) in lung and seven-fold (5.16%
ID/g vs. 0.72%ID/g) in heart for 99mTc-MBIP-Cl compared to that of 99mTc-MBIP-Br
which gives warrant to utilize this single photon emission computed tomography agent
in higher animals.
Babasaheb Bhimrao Ambedkar Central
University, Vidya Vihar, Raebareli Road,
Lucknow 226025, UP, India & Division of
Cyclotron and Radiopharmaceutical Sciences,
Institute of Nuclear Medicine and Allied
Sciences, Brig. S. K. Mazumdar Road, Delhi
110 054, India.
Email: anjanik2003@gmail.com
Pooja Srivastava, Division of Cyclotron and
Radiopharmaceutical Sciences, Institute of
Nuclear Medicine and Allied Sciences, Brig.
S. K. Mazumdar Road, Delhi 110054, India.
Email: pushree14@gmail.com
Funding information
INMAS
K E Y W O R D S
acetamidobenzoxazolone, inflammation, SPECT, tryptophan methyl ester, TSPO
respectively. All three of them showed compatibility to tetrapyrrole pro-
toporphyrin IX (PPIX) structure which is a well-known endogenous
1
| INTRODUCTION
Translocator protein (TSPO) first described as peripheral benzodiaze-
pine receptor (PBR) is a 18 kDa protein and initially identified in 1977
as peripheral binding site for the benzodiazepine. The name PBR
described its high affinity binding to benzodiazepine in peripheral tissues
(Chen & Guilarte, 2008; Fan, Lindemann, Feuilloley, & Papadopoulos,
2012; Milenkovic, Rupprecht, & Wetzel, 2015; Veenman, Vainshtein, &
Gavish, 2015). It is an intracellular protein which forms a complex with
voltage dependent anion channel (VCAD, 32 kDa) and adenine nucleo-
tide translocator (ANT, 30 kDa) in mitochondria. There are few classical
ligands available such as PK11195, RoS-4864, and FGIN-127 which are
isoquinoline carboxamide, benzodiazepine and indole derivatives,
TSPO ligand (Best, Ghadery, Pavese, Tai, & Strafella, 2019; Veenman,
Vainshtein, Yasin, Azrad, & Gavish, 2016). TSPO has been investigated
as biomarker for inflammatory conditions of lung, liver, kidney, and
brain, etc. Its role has also been studied in other disorders such as
depression, anxiety, and neurodegenerative disorders (Hatori et al.,
2014; Jean et al., 2015; Jones, Marino, Shakur, & Morrell, 2003; Kraus,
Kadriu, Lanzenberger, Zarate, & Kasper, 2019; Lagarde, Sarazin, &
Bottlaender, 2018; Veenman & Gavish, 2006; Vicidomini et al., 2015).
Therefore, high affinity TSPO ligands can find application for noninva-
sive diagnosis of pathologically affected tissues. This led to the develop-
ment of TSPO ligands as diagnostic ligands (Arlicot et al., 2012; Boutin
Drug Dev Res. 2019;1–9.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

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SRIVASTAVA ET AL.
et al., 2007; Dolle, Luus, Reynolds, & Kassiou, 2009; Hatori et al., 2014;
Kreisl et al., 2010; Trapani, Palazzo, de Candia, Lasorsa, & Trapani, 2013;
Veenman et al., 2016).
10 weeks old, male) and New Zealand rabbit (2.3 kg, 1 year 5 months,
male) were used for biodistribution studies and imaging.
Animals were supplied by experimental animal facility (EAF) of
Institute of Nuclear Medicine and Allied Sciences (INMAS), Delhi. The
animals were maintained as per the regulations of the Committee for
the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA). All the animals were maintained on standard diet
(Hindustan Lever Ltd., Mumbai, India) and water. They were kept in
the animal house of the institute which was maintained at 22 2ꢀC
and 50% humidity under a 12 hr dark/light cycles.
11C(R) PK11195 ligand is most widely studied PET TSPO ligand from
first generation which has limitation of high lipophilicity and low in vivo
specific binding. These limitations were overcome by second generation
TSPO ligands for improved imaging. Some of the TSPO ligands used in
clinical human studies are [18F]FEDAA, [11C]DAA, [11C]PBR28, [18F]
DPA714, and [11C]AC-5216 (Arlicot et al., 2012; Boutin et al., 2007;
Dolle et al., 2009; Hatori et al., 2014; Kreisl et al., 2010; Trapani et al.,
2013; Veenman et al., 2016). These second-generation ligands have the
problem of intersubject variability (Owen, Yeo, & Gunn, 2012). A new
skeleton acetamidobenzoxazolone (ABO) which has been derived from
RoS-4,864 by opening the diazepine ring and reorganizing it (Fukaya
et al., 2012; Fukaya et al., 2013) to overcome the problems of inter-
subject variability and nonspecific binding (Tiwari, Yui, et al., 2015). In
recent past, our group has explored this skeleton for diagnostic
application using positron emission tomography (PET) with 11C and 18F
radionuclides such as 2-[5-(4-[11C]Methoxyphenyl)-2-oxo-1,3-benzoxazol-
3(2H)-yl]-N-methyl-N-phenylacetamide (11C-MBMP), 2-[5-(4-[18F]
fluoroethoxy/propoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-
N-phenylacetamide (18F-FEBMP and 18F-FPBMP) and using single photon
emission computed tomography (SPECT) with 99mTc such as 99mTc-MBIP
(Kumari et al., 2017; Srivastava, Kaul, Ojha, Kumar, & Tiwari, 2016; Tiwari,
Fujinaga, et al., 2014; Tiwari, Yui, et al., 2014; Tiwari, Ji, et al., 2015).
Here we compare two ligands of similar category acetamido-
benzaoxazolone, 99mTc-MBIP-Br, and 99mTc-MBIP-Cl, in terms of
ex vivo biodistribution and in vivo radioactivity uptake in TSPO enriched
organs. The release kinetics of these tracers has been compared to see
their retention in different organs under in vivo conditions. Besides that,
computational ligand–protein interaction, synthetic yield and lipophilicity
have also been compared to get more insight about the complete phar-
macokinetics of ABO-AA skeleton for SPECT application.
A lung inflammation model was prepared in balb/c mice by intra-
tracheal delivery of lipopolysaccharide (LPS). For this, 6–8 weeks old
balb/c (22–25 g, male) mice were anesthetized by isoflurane sedation.
While anesthetized, an incision was made around the thoracic region
in mice and 100 μL of 0.5 mg/mL of LPS solution was delivered intra-
tracheally into the mice. After 24 hr, the mice were used for experi-
mental purpose to validate the uptake of radioligand in inflamed
lungs, overexpressing TSPO receptors. The blocking studies were per-
formed in these models by preadministering 10 mg/kg unlabeled
PK11195, 10 min prior to 100 μCi 99mTc-MBIP-Cl injection and was
imaged 40 min postinjection of the radioligand. The lung model was
assessed by tracer technique by comparing biodistribution of radio-
complex in lung-modeled mice with and without blocking.
2.3 | In vitro cell uptake of 99mTc-MBIP-cl in A549
cells
Human lung carcinoma A549 cells were cultured as per protocol in lit-
erature (Dumoga et al., 2016). A549 cells were maintained in RPM1
medium supplemented with 10% (v/v) heat inactivated fetal bovine
serum, 2 mM L-glutamine and 100 U ml−1 penicillin–streptomycin in a
humidified incubator at 37ꢀC in 5% CO2. The A549 cells (1 × 106 per
well) were incubated with 99mTc-MBIP-Cl/Br (in 10% ethanol, 5%
tween 20 and water) for 10, 30, 60, and 120 min, respectively. In an
inhibition study for specificity, 300 mM solution of PK11195 was
added to the cells prior to incubation and the cells were washed with
PBS after 1 hr. Thereafter radiocomplex (99mTc-MBIP-Cl/Br) was
added and its cell uptake was studied at 10, 30, 60, and 120 min with
the help of a gamma counter.
2
| MATERIALS AND METHODS
2.1 | Instrumentation
Spectroscopic techniques used for the characterization were 1H and
13C NMR spectroscopy (Bruker Avance III 600 MHz system) at
600 MHz and 150 MHz, respectively, and mass spectroscopy (Agilent
6310 system) using ESI. HRMS was recorded on Agilent 6520 Q-TOF
(ESI-HRMS) mass spectrometer having ESI source in positive mode.
Radioligand related studies were performed on Siemens Symbia Cam-
era for imaging and γ-scintillation counter for radioactivity counting.
The lipophilicities of the compounds were calculated using the
ChemDraw software.
2.4 | Synthesis
2.4.1 | Synthesis of methyl 2-(2-chloroacetamido)-
3-(1H-indol-3-yl)propanoate
To a solution of L-tryptophan methyl ester (500 mg, 2.32 mmol) and
deionized water (10 mL), triethyl amine (355 μL, 2.55 mmol) was
added with constant stirring under nitrogen atmosphere on ice bath
for 5 min. To the mixture, chloroacetyl chloride (203 μL, 2.55 mmol) in
dichloromethane (10 mL) was added drop wise over 1 hr and the reac-
tion was carried out for 4 hr. Thereafter, the mixture was extracted
with dichloromethane (3×). The organic layer was washed twice by
deionized water and brine solution. After washing, the organic layer
2.2 | Animal models
Institutional Animal Ethics Committee (INM/DASQA/IAEC/09/015)
approved protocols were used for studies. BALB/c mice (22–28 g,

SRIVASTAVA ET AL.
3
was dried over anhydrous sodium sulfate. After filtration, the solvent
was removed in vacuo, and purified by silica gel column chromatogra-
phy using 100% CHCl3 as eluent (Yield = 75%).
1H-NMR (DMSO, 400 MHz): δ (ppm) 3.1–3.2 (m, 2H), 3.6 (s, 3H),
4.4–4.6 (m, 3H), 6.9–7.5 (m, 8H)
13C-NMR (DMSO, 100 MHz): δ (ppm) 28.7, 44.4, 52.4, 53.9,
1H-NMR (CDCl3, 600 MHz): δ (ppm) 3.3–3.4 (d, 2H), 3.7 (s, 3H),
4.0 (s, 2H), 4.9–5.0 (m, 1H), 7.0–7.6 (m, 5H), 8.5 (br s, 1H)
13C-NMR (CDCl3, 150 MHz): δ (ppm) 27.5, 42.5, 52.6, 53.3,
109.3–136.3 (8 peaks), 166.0, 171.8
111.0–141.5 (14 C), 154.2, 166.3, 172.3
MS (ESI) m/z: 470.2 [M−H+], calculated: 470.0 [M−H+]
HPLC: T3 column, 4.6 × 250 mm; MeCN/H2O, 6/4–8/2 (v/v);
flow rate: 0.8 mL/min; λuv = 254 nm; tR = 8.751 min
HRMS: m/z 474.0496 ([C21H18BrN3O5] + H)+.
MS (ESI) m/z: 293.1 [M−H+], calculated m/z: 293.1 [M−H+].
2.5 | 99mTc radiolabeling
2.4.2 | Synthesis of 5-chloro benzoxazol-2(3H)-one
To a solution of 4-chloro-2-aminophenol (5 g, 34.83 mmol) in 150 mL
THF, 1,10-carbonyldiimidazole (5.65 g, 34.83 mmol) was added. The
mixture was refluxed with stirring for 2 hr. The reaction mixture was
quenched by adding 2 M HCl solution after cooling down to room
temperature. EtOAc was used for extraction which was washed with
brine, dried over anhydrous sodium sulfate and filtered. Thereafter,
solvent was removed in vacuo to get the product as a white solid
(yield = 91%).
To evaluate the stability, specificity, and biodistribution profile of
MBIP-Cl and MBIP-Br compounds, they were labeled with 99mTc
using procedure from literature with slight modifications (Kakkar
et al., 2012; Srivastava et al, 2013). Briefly, 100 μL of sterile sodium
pertechnetate (approximately 74–110 MBq of 99mTcO4) was reduced
with 50 μL of stannous chloride solution (1 × 10−2 M in 10% glacial
acetic acid) and pH was adjusted to 6.5 using 0.5 M sodium bicarbon-
ate. A solution of MBIP-Cl and MBIP-Br were added to this mixture
separately and after thorough mixing, incubated for 15 min at room
temperature (25ꢀC). ITLC-SG strips as stationary phase and acetone
as mobile phase were used to determine complexation of MBIP-Cl
and MBIP-Br with 99mTc and purity of the labeled compound.
1H-NMR (DMSO, 600 MHz): δ (ppm) 7.1–7.4 (m, 3H)
13C-NMR (DMSO, 150 MHz): δ (ppm) 110.3, 111.3, 122.0, 128.2,
132.2, 142.6, 154.7
MS (ESI) m/z: 167.8 [M−H+], Calculated m/z: 168.0 [M−H+].
2.4.3 | Synthesis of 2-(2-(5-chloro benzoxazolone)
acetamido)-3-(1H-indol-3-yl) propanoate (MBIP-Cl)
2.6 | In vitro human serum stability assay
In vitro human serum stability of the radiolabeled compounds was
evaluated using our previous approach with some modifications
(Kumar et al., 2010). Blood collected from healthy volunteers was
clotted in humidified incubator for 1 hr under 5% CO2 and 95% air
and used to prepare serum. Sample was centrifuged at 400 rpm and
filtered through 0.22 μm filter into sterile plastic culture tubes.
99mTc-MBIP-Cl and 99mTc-MBIP-Br were incubated with freshly
prepared serum at 37ꢀC. Samples were analyzed at different time
points using ITLC-SG strips. Acetone was used as mobile phase for
measurement to find out percentage dissociation of the complex in
serum with a function of time.
To a solution of 5-chloro benzoxazol-2(3H)-one (250 mg, 1.48 mmol)
and K2CO3 (306 mg, 2.22 mmol) in DMF (5 mL), methyl-2-(2-
chloroacetamido)-3-(1H-indol-3-yl) propionate (435 mg, 1.48 mmol)
was added with cooling on ice bath. The reaction mixture was stirred
at 60–70ꢀC for 3 hr. Thereafter, the reaction mixture was cooled to
room temperature before adding water. A mixture of toluene and
ethyl acetate, in 1:1 ratio, was used as extraction solvent. Water and
brine solution were used for washing the organic layer and anhydrous
sodium sulfate for drying. Solvent was removed in vacuo after filtra-
tion and was purified by silica gel column chromatography using
CHCl3/EtOAc (4:1, v/v) as eluent (yield = 70%).
1H-NMR (CDCl3, 600 MHz): δ (ppm) 3.5–3.7 (m, 2H), 3.8 (s, 3H),
3.9–4.0 (m, 2H), 5.0–5.1 (m, 1H), 6.7–7.5 (m, 8H), 8.1 (br s, 1H)
13C-NMR (CDCl3, 150 MHz): 24.1, 51.2, 53.1, 54.3, 110.4–148.8
(14 peaks), 155.5, 168.2, 168.8
MS (ESI) m/z: 426.3 [M−H+], Calculated m/z: 426.1 [M−H+]
HPLC: T3 column, 4.6 × 250 mm; MeCN/H2O, 6/4–8/2 (v/v);
flow rate: 0.8 mL/min; λuv = 254 nm; tR = 7.214 min.
2.7 | Biodistribution studies
Balb/c mice were used for the initial in vivo assessment of 99mTc-
MBIP-Cl and 99mTc-MBIP-Br (Srivastava et al., 2016). Radioactivity
uptake was measured in specific organs of the mice at different time
points, that is, 15 min, 30 min, 60 min, and 120 min postinjection of
100 μCi of both radioligands via a tail vein and results were expressed
as percentage of injected dose per gram (%ID/g) of tissue. The experi-
ments were performed in triplicate at each time point. Radioactivities
in organs were measured with the help of a gamma counter. Radioac-
tivity injected in each mouse was found out by subtracting the activity
left in tail from the activity injected. Total blood volume was calcu-
lated as 7% of the total body weight.
HRMS: m/z 428.1000 ([C21H18ClN3O5] + H)+.
2.4.4 | Synthesis of 2-(2-(5-bromo benzoxazolone)
acetamido)-3-(1H-indol-3-yl) propanoate (MBIP-Br)
MBIP-Br was synthesized by the procedure mentioned in our previous
paper (Srivastava et al., 2016).

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SRIVASTAVA ET AL.
3
| RESULTS AND DISCUSSION
Encouraged by the results of our recently developed TSPO ligands
having TSPO affinity in nanomolar range (Tiwari, Fujinaga, et al.,
2014; Tiwari, Yui, et al., 2014; Tiwari, Yui, et al., 2015), we directed
our efforts towards development of analogs of ABO for 99mTc labeling
to target 18 kDa TSPO. The basis of our research was the successful
outcome of radioligands [11C]MBMP and [18F]FE/FPBMP showing
promise for TSPO visualization and quantification. The new ligands
conjugate acetamidobenzoxazolone to tryptophan methyl ester. Fur-
ther, the 99mTc-MBIP-Cl showed fast clearance from the requisite
organs as well as from circulating blood. The 99mTc-MBIP-Cl exhibited
reasonable uptake in heart, lungs, kidney, and spleen, which are
known to express TSPO.
ADME analysis of both the analogs, MBIP-Cl and MBIP-Br, were
focused mainly on the issue of blood brain permeability and serum pro-
tein binding which were found to be in the range of 95% of well-
established drugs in computational studies using QikProp module of
Schrodinger. They do not violate any of the five Lipinski rules and three
Jorgensen rules. Both the ligands have lower lipophilicity (2.04 for
chloro and 2.31 for bromo analogs) than known TSPO ligands. Analogs
of ABO, [11C]MBMP and [18F]FEBMP, demonstrated high specific bind-
ing with TSPO in ischemic rat brain but they have moderate brain kinet-
ics in higher animals. These PET ligands have been modified to other
ABO ligand 18F-2-(5-(6-Fluoropyridin-3-yl)-2-oxobenzo[d]oxazol-3(2H)-
yl)-N-methyl-N-phenylacetamide for reducing the lipophilicity for faster
brain kinetics.29 Thereafter, TSPO binding/affinity of MBIP-Cl was
found out through docking methods with TSPO proteins (Figure 1).
Score of MBIP-Cl with 4RYQ and 4UC1 were −8.99 and −9.30, respec-
tively. The interaction of MBIP-Cl in terms of Gscore was found compara-
ble with other known TSPO ligands for 4RYQ and with TSPO ligands
not significantly affected by single nucleotide polymorphism such as
FEBMP and PK11195 for 4UC1 (Tables 1 and 2).
FIGURE 1 2D interaction diagram of MBIP-Cl on docking with
TSPO [PDB ID: 4RYQ (a) and 4UC1 (b)] MBIP-Cl formed pi–pi
interactions of aromatic rings with Phe90 and H-bond interactions
with Try51 in 4RYQ interaction and Try50 is involved in H bond
interaction with 4UC1
The final ligand was synthesized by the procedure mentioned in
Scheme 1 in three steps with modification in earlier used procedure
having four steps for MBIP-Br (Srivastava et al., 2016). The synthetic
procedure involved synthesis of methyl-2-(2-chloroacetamido)-3-(1H-
indol-3-yl)propanoate and 5-chloro benzoxazol-2(3H)-one, as interme-
diates which were combined in the final step. The precursor molecule
5-chloro benzoxazolone was prepared by cyclization reaction of
4-chloro-2-amino phenol in presence of CDI. Simultaneously,
chloroacetylation was performed on L-tryptophan methyl ester.
Thereafter, both the above-mentioned products were combined
through alkylation reaction in presence of K2CO3 in DMF. The synthe-
sized MBIP-Cl had good yield (70%). Column chromatography was
used for purification of the synthesized products and spectroscopic
methods were used for characterization of the intermediates as well
as the final products. The synthesis of final product was confirmed by
m/z = 426.3 [M−H+] peak present in mass spectroscopic data. The
synthetic scheme followed for MBIP-Cl is better than reported
scheme for its bromo analog in terms of yield and duration of overall
synthesis (Srivastava et al., 2016).
TABLE 1 Gscorea of TSPO ligands with PDB: 4RYQb
Ligand
Gscore
MBIP-Cl
MBIP-Br
DAA1106
PK11195
PBR28
−8.99
−6.92
−7.06
−7.17
−7.14
−7.035
DPA-713
aGlide score is an empirical scoring function that approximates the ligand
binding free energy.
b4RYO is wild TSPO X-ray crystal structure with resolution 1.7 Å taken
from RCSB protein data bank.
Subsequently, radiochemistry of MBIP-Cl was optimized with
99mTc for maximum complexation stability (Srivastava et al., 2016;
Tiwari, Ji, et al., 2015). The stability of 99mTc-MBIP-Cl was found
>94% in saline solution and >91% in serum at 4 hr (Figure 2) indicating

SRIVASTAVA ET AL.
5
that both 99mTc-MBIP-Cl and 99mTc-MBIP-Br are suitable for SPECT
application in terms of stability, the latter being relatively better (91%
vs. 93% in serum at 4 hr). This technetium-99 m labeled MBIP-Cl was
then used to evaluate its binding affinity to TSPO expressing A549
human lung carcinoma cells by an in vitro assay. The results of the
in vitro uptake of the radiocomplex are shown in Figure 3. They demon-
strate that the uptake is time dependent and increases until last time
point of study, that is, 60 min for both bromo and chloro analogs. The
blocking studies were also performed in the presence of PK11195 as
blocking agent. The compound was incubated for 1 hr before adding
radiolabeled compound. The maximum reduction was 41.1% and 43.6%
for chloro and bromo analogs, respectively. Although there was an
increase in the uptake of blocked cells with time implying that the cells
are not fully blocked, still it gives preliminary idea about the specificity
of radiolabeled MBIP-Cl towards TSPO.
Biodistribution studies were performed to determine how 99mTc-
MBIP-Cl was distributed in TSPO enriched organs as well as in excre-
tory organs in terms of %ID/g of 99mTc-MBIP-Cl at four different time
points as shown in Figure 4 and was compared with biodistribution
studies of 99mTc-MBIP-Br. After 15 min, there was high uptake of
radioactivity into organs known to have TSPO sites, such as kidney
TABLE 2 G score of TSPO ligands with PDB: 4UC1a
Ligand
Gscore
MBIP-Cl
MBIP-Br
FEBMP
−9.30
−9.61
−8.07
−8.25
PK11195
FIGURE 2 % intact 99mTc-MBIP-cl as a function of time
demonstrating stability in saline and human serum at 370 C over 24 h
a4UC1 is mutant TSPO X-ray crystal structure with resolution 1.8 Å taken
from RCSB protein data bank.
SCHEME 1 Synthesis of MBIP-Cl
reagents: (a) CDI, THF, reflux;
(b) chloroacetyl chloride, Et3N,
H2O/DCM, 0ꢀC-rt; and (c) K2CO3, DMF,
reflux

6
SRIVASTAVA ET AL.
(29.57%ID/g), heart (5.16%ID/g), spleen (2.87%ID/g), and lungs
(12.91%ID/g). The uptake of 99mTc-MBIP-Cl in lungs and heart was
approximately 2.75 and 7 folds higher to that of 99mTc-MBIP-Br
uptake at 15 min showing better distribution pattern for TSPO
targeting (Figure 5) which, in turn, would result in a better SPECT
ligand. Uptake of 99mTc-MBIP-Cl in liver was relatively lower than that
of 99mTc-MBIP-Br (19.74 vs. 23.12%ID/g).24 Rate of reduction was
high in heart and lungs, which was reduced to 14.7% (5.16%ID/g to
0.76%ID/g) and 8.9% (12.91%ID/g to 1.12%ID/g) between 15 and
120 min, respectively. Over the same period, uptake in kidney, liver,
spleen, and blood was reduced to 54.5%, 34.0%, 33.8%, and 45.0%,
respectively. The amount of radiotracer in the blood and brain
decreases with time. Uptake in kidney and liver reflected that the
excretion occurred through renal and hepatobiliary tract as expected
for a compound with moderate lipophilicity.
be higher in heart (2.96%ID/g vs. negligible uptake) and lung (6.16%
ID/g vs. 1.19 %ID/g) and comparable in spleen (2.18%ID/g vs. 2.07%
ID/g) at 15 min as reported in CD-1 mice.31 Uptake of [123I]-CLINME,
an iodine based SPECT ligand, in heart, lungs, and spleen of SD rat
were 3.53%ID/g, 3.53%ID/g, 2.65%ID/g at 15 min. This was low-
er/comparable to the uptake of 99mTc-MBIP-Cl in respective organs.32
The liver uptake was lower for 99mTc-MBIP-Cl in comparison with
99mTc-labeled 2-quinolinecarboxamide and 99mTc-MBIP-Br (Cappelli
et al., 2008; Cumming et al., 2018; Srivastava et al., 2016). This
accounts for better image visualization (target organ to liver ratio) for
99mTc-MBIP-Cl.
Comparison of release rate among SPECT agents between 30 and
60 min for 99mTc-MBIP-Cl, [123I]-CLINME (Mattner et al., 2015), 99mTc-
labeled 2-quinolinecarboxamide and 99mTc-MBIP-Br reflected better
release rate of 99mTc-MBIP-Cl in lung, liver, heart, and spleen (Cappelli
et al., 2008; Srivastava et al., 2016). Further, it was also compared with
PET and SPECT ligands of ABO category. The release kinetic rates
between 15 and 60 min for 18F-FEBMP (Tiwari, Ji, et al., 2015), 11C-
MBMP (Tiwari, Fujinaga, et al., 2014), 18F-2-(5-(6-Fluoropyridin-3-yl)-
2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (Fujinaga
et al., 2017), and 99mTc-MBIP-Cl are 0.175%ID/g/min, 0.150%ID/g/min,
0.070%ID/g/min, and 0.085%ID/g/min in heart, this ratio became more
comparable as 0.382%ID/g/min, 0.375%ID/g/min, 0.155%ID/g/min and
0.231%ID/g/min in lungs. In liver and kidney, release kinetic rates were
approximately 3–30 folds faster in 99mTc-MBIP-Cl which makes it inter-
esting for further evaluation. These evaluations demonstrated release
rate of 99mTc-MBIP-Cl from different organs are comparable to known
TSPO ligands, therefore it can be further evaluated for TSPO targeting.
In vivo specificity of MBIP-Cl was evaluated in lung inflammation
models of balb/c mice using PK11195. The preadministration of cold
PK11195 in these inflammation models demonstrated significant
reduction of uptake in the lung region as expected. This was clearly
evident in the whole-body SPECT image taken 40 min postinjection
of 99mTc-MBIP-Cl (Figure 6).
Further, 99mTc-MBIP-Cl was also compared with the SPECT
ligands reported in literature. As compared to 99mTc-labeled
2-quinolinecarboxamide, the uptake of 99mTc-MBIP-Cl, was found to
(a)
(b)
In order to have better clarity about organs uptake, in vivo imaging
was performed in New Zealand rabbit. Scintigraphy study of 99mTc-
MBIP-Cl was carried out by intravenous injection of 300 μCi in
200 μL of radiolabeled complex through ear vein. The acquisition was
performed 4 min postinjection for 1 min. The biodistribution pattern
shown in the image (figure 7) is similar to ex vivo biodistribution
FIGURE 3 Uptake kinetics of (a) 99mTc-MBIP-Cl and (b) 99mTc-
MBIP-Br in A549 cells in the presence (blue) and absence (red) of
PK11195. Data are expressed as counts per well (mean with SD n = 3)
FIGURE 4 Biodistribution (%
ID/g, mean SD) data for 99mTc-
MBIP-Cl. Data are derived from
tissues taken from normal
15 min 30 min 60 min 120 min
30.00
20.00
10.00
0.00
BALB/c mice (n = 3) at different
time points after intravenous
injection of 100 Ci of 99mTc-
MBIP-Cl via tail vein in each
mouse. Radioactivity uptake in
different organs were displayed
blood
heart
lungs
liver
spleen kidney stomach intestine brain
Organ
as mean of % injected dose per
gram SD

SRIVASTAVA ET AL.
7
FIGURE 5 Time-activity curve
of 99mTc-MBIP-Cl and 99mTc-MBIP-
Br in organs expressed as %ID/g
(y axis) versus time in min (x axis)
FIGURE 6 In vivo SPECT image showing activity distribution of
99mTc-MBIP-Cl in (a) normal and (b) inflamed mouse without and with
blocking at 40 min postinjection. Blockage was done with PK11195
(10 mg/kg), administered 10 min before radioligand injection
FIGURE 7 In vivo biodistribution of 99mTc-MBIP-cl in
New Zealand rabbit
observed for the radioligand. Also small uptakes in brains were
observed demonstrating its blood brain barrier crossing ability.
As per these findings, 99mTc-MBIP-Cl was found to be better than
99mTc-MBIP-Br in terms of in vivo uptake of the compound in tissues
expressing TSPO. This ligand therefore has the potential to specifically tar-
get TSPO. Further, the introduction of the chloro group in place of bromo
in ligand shows improved pharmacokinetic properties which in turn will
improve the imaging resolution in mice using the SPECT modality. These
data clearly indicated that MBIP-Cl should be considered as a viable SPECT
ligand for further investigation in pathological conditions expressing TSPO.

8
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Cappelli, A., Mancini, A., Sudati, F., Valenti, S., Anzini, M., Belloli, S., …
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4
| CONCLUSION
The goal of this study was to optimize acetamidobenzoxazolone-
tryptophan methyl ester based radiotracer for imaging inflammation in
TSPO enriched organs. In this work, a new modified SPECT ligand
99mTc-MBIP-Cl has been evaluated for TSPO expression imaging. This
ligand is able to form stable coordinated complex with 99mTc and
remains stable (>91%) in serum up to 4 hr (in vitro). It also has favor-
able pharmacokinetic properties especially in terms of lipophilicity and
biodistribution. The specificity of 99mTc-MBIP-Cl for TSPO was vali-
dated with the known TSPO ligand PK11195 in lung inflammation
models. The current ligand for SPECT, 99mTc-MBIP-Cl, on comparison
with 99mTc-MBIP-Br, has improved %ID uptake in peripheral organs,
better release rate and increased synthetic yield for ligand. High
uptake at early time point and fast kinetics suggests that 99mTc-MBIP-
Cl may have potential as SPECT ligand for evaluation of TSPO-over
expression. in vivo studies on rabbit demonstrated selectivity of
99mTc-MBIP-Cl for TSPO as only TSPO rich organs are showing signif-
icant uptake. Further studies in comparison with other known TSPO
ligands are the next step to establish it as a promising TSPO ligand
for TSPO.
Fan, J., Lindemann, P., Feuilloley, M. G., & Papadopoulos, V. (2012). Struc-
tural and functional evolution of the translocator protein (18 kDa).
Current Molecular Medicine, 12, 369–386.
Fujinaga, M., Luo, R., Kumata, K., Zhang, Y., Hatori, A., Yamasaki, T., …
Zhang, M. R. (2017). Development of a 18F-labeled radiotracer with
improved brain kinetics for positron emission tomography imaging of
translocator protein (18 kDa) in ischemic brain and glioma. Journal of
Medicinal Chemistry, 60, 4047–4061.
Fukaya, T., Kodo, T., Ishiyama, T., Kakuyama, H., Nishikawa, H., Baba, S., &
Masumoto, S. (2012). Design, synthesis and structure–activity relation-
ships of novel benzoxazolone derivatives as 18 kDa translocator pro-
tein (TSPO) ligands. Bioorganic & Medicinal Chemistry, 22, 5568–5582.
Fukaya, T., Kodo, T., Ishiyama, T., Nishikawa, H., Baba, S., & Masumoto, S.
(2013). Design, synthesis and structure–activity relationship of novel
tricyclic benzimidazolonederivatives as potent 18 kDa translocator pro-
tein (TSPO) ligands. Bioorganic & Medicinal Chemistry, 21, 1257–1267.
Hatori, A., Yui, J., Xie, L., Yamasaki, T., Kumata, K., Fujinaga, M., …
Zhang, M. R. (2014). Visualization of acute liver damage induced by
cycloheximide in rats using PET with [18F]FEDAC, a radiotracer for
translocator protein (18 kDa). PLoS One, 9, e86625.
ACKNOWLEDGMENTS
We extend special thanks to Dr Tarun Sekhri, Director INMAS and Dr
A. K. Mishra, Head, Division of Cyclotron and Radiopharmaceutical
Science, INMAS, Delhi, for providing us the facility to carry out experi-
ments. I also thank Dr Sonia Gandhi, Scientist and Mr Sunil Pal, Tech-
nical Officer and Dr Ankur Kaul, Technical Officer, INMAS for their
technical support. This work was supported by INMAS, Delhi under
Task Project ST/15-16/INM-03.
Jean, R., Bribe, E., Knabe, L., Petit, A. F., Vachier, I., & Bourdin, A. (2015).
TSPO is a new anti-inflammatory target in the airway of COPD. Revue
des Maladies Respiratoires, 32, 320.
CONFLICT OF INTEREST
There is no conflict of interest among authors of this manuscript.
Jones, H. A., Marino, P. S., Shakur, B. H., & Morrell, N. W. (2003). In vivo
assessment of lung inflammatory cell activity in patients with COPD
and asthma. European Respiratory Journal, 21, 567–573.
ORCID
Kakkar, D., Tiwari, A. K., Chuttani, K., Khanna, A., Datta, A., Singh, H., &
Mishra, A. K. (2012). Design, synthesis, and antimycobacterial property
of PEG–bis (INH) conjugates. Chemical Biology & Drug Design, 80,
245–253.
Anjani Kumar Tiwari
https://orcid.org/0000-0001-6271-3055
Kakkar, D., Tiwari, A. K., Chuttani, K., Kumar, R., Mishra, K., Singh, H., &
Mishra, A. K. (2011). Polyethylene-glycolylated isoniazid conjugate for
reduced toxicity and sustained release. Therapeutic Delivery, 2(2),
205–212.
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