Design, Synthesis, and In Silico Evaluation of Methyl 2-(2-(5-Bromo/chloro-2-oxobenzoxazol-3(2H)-yl)-acetamido)-3-phenylpropanoate for TSPO Targeting

Article abstract of DOI:10.1134/S1066362220010142

The high expression of the translocator protein (TSPO) makes it an ideal target for imaging and therapy. The present study is aimed at optimizing acetamidobenzoxazolone-based TSPO ligands by structural modification to overcome the limitations of TSPO ligands of the first two generations such as nonspecificity and polymorphism. Three different TSPO proteins, 2MGY, 4RYQ, and 4UC1, in the native and mutated form were chosen. Acetamidobenzoxazolones modified with phenylalanine methyl ester (methyl 2-(2-(5-bromo/chloro-2-oxobenzooxazol-3(2H)-yl)acetamido)-3-phenylpropanoate, ABPO-Br, ABPO-Cl) through better or comparable docking scores than the known TSPO ligands such as MBMP, FEBMP, FPBMP, and PK11195 are identified as potential TSPO ligands. ABPO-Cl and ABPO-Br were synthesized using phenylalanine methyl ester as a moiety for incorporation of the desired pharmacophoric feature. All the intermediates and final compounds were purified using column chromatography and analytical HPLC (purity > 97%). The purified compounds were characterized by ¹H, ¹³C NMR and mass spectroscopy. Drug likeliness and comparative bioactivity analysis were performed using QikProp through prediction of various properties. Both analogs follow all the five Lipinski rules and three Jogersen’s rules predicting their drug likeliness. The other important aspects related to TSPO ligands such as blood-brain barrier penetration and better contrast have been predicted through lipophilicity (QP log P = 2.76 and 2.74 for ABPO-Br and ABPO-Cl, respectively) and serum binding (QP log K_hsa = ?0.18 and ?0.25 for ABPO-Br and ABPO-Cl, respectively). The selectivity and distribution of these TSPO ligands were confirmed by ^99mTc-ABPO-Br dynamic image in New Zealand rabbit. These results have shown that the ABPO analogs have the potential to act as better ligands as compared to known acetamidobenzoxazolone derivatives and would be of interest as a promising starting point for designing compounds for TSPO targeting.

Full text of DOI:10.1134/S1066362220010142

ISSN 1066-3622, Radiochemistry, 2020, Vol. 62, No. 1, pp. 107–118. © Pleiades Publishing, Inc., 2020.
Design, Synthesis, and In Silico Evaluation
of Methyl 2-(2-(5-Bromo/chloro-2-oxobenzoxazol-3(2H)-yl)-
acetamido)-3-phenylpropanoate for TSPO Targeting
P. Srivastavaa,b,*, P. Kumarb, and Anjani Kumar Tiwaria,c,**
aInstitute of Nuclear Medicine and Allied Sciences, Delhi, 110054 India
bDelhi Technological University, Delhi, 110042 India
cBabasaheb Bhimrao Ambedkar Central University, Lucknow, 226025 India
*e-mail: pushree14@gmail.com,
**e-mail: anjanik2003@gmail.com
Received February 7, 2019; revised April 9, 2019; accepted April 16, 2019
Abstract—The high expression of the translocator protein (TSPO) makes it an ideal target for imaging and therapy.
The present study is aimed at optimizing acetamidobenzoxazolone-based TSPO ligands by structural modification
to overcome the limitations of TSPO ligands of the first two generations such as nonspecificity and polymorphism.
Three different TSPO proteins, 2MGY, 4RYQ, and 4UC1, in the native and mutated form were chosen. Acetami-
dobenzoxazolones modified with phenylalanine methyl ester (methyl 2-(2-(5-bromo/chloro-2-oxobenzooxazol-
3(2H)-yl)acetamido)-3-phenylpropanoate,ABPO-Br,ABPO-Cl) through better or comparable docking scores than
the known TSPO ligands such as MBMP, FEBMP, FPBMP, and PK11195 are identified as potential TSPO ligands.
ABPO-Cl and ABPO-Br were synthesized using phenylalanine methyl ester as a moiety for incorporation of the
desired pharmacophoric feature. All the intermediates and final compounds were purified using column chroma-
tography and analytical HPLC (purity > 97%). The purified compounds were characterized by 1H, 13C NMR and
mass spectroscopy. Drug likeliness and comparative bioactivity analysis were performed using QikProp through
prediction of various properties. Both analogs follow all the five Lipinski rules and three Jogersen’s rules predicting
their drug likeliness. The other important aspects related to TSPO ligands such as blood–brain barrier penetration
and better contrast have been predicted through lipophilicity (QP log P = 2.76 and 2.74 for ABPO-Br and ABPO-
Cl, respectively) and serum binding (QP log Khsa = –0.18 and –0.25 for ABPO-Br and ABPO-Cl, respectively).
The selectivity and distribution of these TSPO ligands were confirmed by 99mTc-ABPO-Br dynamic image in
New Zealand rabbit. These results have shown that the ABPO analogs have the potential to act as better ligands
as compared to known acetamidobenzoxazolone derivatives and would be of interest as a promising starting point
for designing compounds for TSPO targeting.
Keywords: TSPO, acetamidobenzoxazolone, phenylalanine methyl ester, ADME, docking, ABPO-Cl, ABPO-Br
DOI: 10.1134/S1066362220010142
INTRODUCTION
in non mitochondrial site such as nuclear or microsomal
site in some cells [2, 3].
The translocator protein (18 kDa, TSPO), previously
named as the peripheral benzodiazepine receptor (PBR),
was identified as a diazepine peripheral binding site in
1977 [1]. It was distinguished from the central benzo-
diazepine receptor on the basis of the functional role,
unique structure, and cellular location. The TSPO is a
169 amino acid protein arranged in five transmembrane
domains [1]. It is primarily located on the outer mem-
brane of mitochondria and also proposed to be located
TSPO is an important diagnostic and therapeutic
target for inflammation and tumor [4–6]. Since TSPO
was identified by diazepam, benzodiazepines, and
Ro5-4864, several classes of TSPO ligands with di-
verse structures including isoquinoline carboxamides,
benzoazepine, benzothiazepines, indole acetamide,
phenoxyphenylacetamide, imidazopyridine acet-
amides, and pyrazolopyrimidine acetamides have been
developed [1, 7, 8].
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SRIVASTAVA et al.
The isoquinoline carboxamide based ligand PK11195
increasingly important for early screening of potential
drug candidates [24].
is currently considered as the state-of-the-art ligand
for TSPO. Its most widely studied PET ligand, 11C(R)
PK11195, from the first generation has a limitation of
high nonspecific binding and low brain penetration,
resulting in low signal-to-background ratio. Some of the
second-generation TSPO ligands could overcome these
limitations with higher signal-to-noise ratio compared
to PK11195 for improved imaging. Several second-
generation TSPO radioligands used in clinical human
studies are [11C]DAA1106, [18F]DPA714, [11C]PBR28,
and [11C]AC-5216 [9–12].
Around the beginning of 21st century, a simple
thumb rule was reported by Lipinski et al. for in silico
prediction of oral absorption of a compound through
solubility and permeability. As per rule of five, ≈90%
of oral molecules clear three out of following four rules:
MW ≤ 500 Da, calculated log P (clog P) ≤ 5 and ≥ 0,
hydrogen bond acceptors (HBAs) ≤ 10, and hydrogen
bond donors (HBD) ≤ 5 [25, 26]. The bioavailability
of molecules is predicted using Jorgensen’s rule of
3 (log Swat > –5.7, Apparent Caco-2 permeability
>22 nm/s, and number of primary metabolites <7) [27,
28]. Subsequently, a few more in silico properties such
as polar surface area (PSA ≤ 140 Å2) and number of
rotatable bonds (≤ 10–20) have been added to expand
the correlations with ADME parameters [29].
Although
second-generation
TSPO
ligands
have a few advantages over PK11195, there is a
problem of intersubject variability due to single
nucleotide polymorphism [13]. A new skeleton,
acetamidobenzoxazolone (ABO) [14, 15], derived
through opening and reorganizing RoS-4864, has been
explored for PET application (MBMP, FEBMP and
FPBMP) to overcome the problems of intersubject
variability [16–21]. ABO has been modified to ABO-
amino acid (ABO-AA) for SPECT application by
conjugating it with tryptophan methyl ester to maintain
the affinity and biocompatibility [22].
In silico approaches including docking, quantitative
structure–activity relationships, virtual ligand screening,
pharmacophore modeling, etc., are other important tools
in the discovery and optimization of novel molecules
with enhanced affinity and specificity for the selected
therapeutic targets [30–33]. Availability of different
TSPO PDBs from wild type as well as mutants such as
2MGY, 4RYQ, and 4UC1 have enabled TSPO affinity
screening through the docking studies at design level
[34–37].
This encouraged us to try conjugation of
benzoxazolone with other amino acids. Moreover, in a
recent study, preference for amino acid transport system
LAT1 over other LATs, A, and ASC in normal as well
as tumor cells in the uptake of phenylalanine-based
radiotracers has been demonstrated through competitive
uptake inhibition using inhibitors specific for systems
A, ASC, and L [23]. Therefore, phenylalanine can be a
better candidate for improving the cell uptake.
In this study, in continuation of our efforts to develop
effective TSPO ligand, we designed a new skeleton,
methyl
2-(2-(5-bromo/chloro-2-oxobenzooxazol-
3(2H)-yl)acetamido)-3-phenylpropanoate by combining
5-bromo/chlorobenzoxazolone with phenylalanine
methyl ester and evaluated its ADME properties as
well as its affinity for TSPO in silico. Conjugating
TSPO pharmacophore (acetamidobenzoxazolone)
with methyl phenylalanine could be a promising
strategy for drug design, as it may improve the efficacy.
Thereafter, conjugation has been carried out in three
steps. The product was characterized using NMR, mass
spectrometry, and HPLC for further application as TSPO
ligand in animal model.
Before synthesis, it is important to evaluate the
molecule for drug likeliness and TSPO affinity in
silico. The literature of drug discovery gives emphasis
to combinatorial chemistry and the screening of
compounds for drugability involving ADME/toxicity,
as the screening of millions of compounds results in
a limited number of drug-like compounds. Therefore,
it has been advocated that the drugability should be
checked before screening for the receptor activity. In
current scenario, there is a considerable increase in the
application of computer aided drug design methods to
predict potential drugs for the diagnosis/treatment of
several diseases. In silico prediction of pharmacokinetic
parameters, studies of the absorption, distribution,
metabolism, and excretion (ADME) have become
MATERIALS AND METHODS
Chemicals. The chemicals and solvents used in the
reactions were purchased from Sigma–Aldrich and
Merck. MN60 (60–120 mesh) silica was used for column
chromatography, and aluminum plates coated with silica
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DESIGN, SYNTHESIS, AND IN SILICO EVALUATION
109
gel 60 F254 were used for thin layer chromatography
(TLC) for monitoring the reaction.
the structures of the complexes of the TSPO proteins
with its substrate PK11195. The PDB structure files of
the proteins are imported and prepared through protein
preparation wizard of the Schrödinger software by
applying the OPLS_2005 force field. The steps involved
in the process of protein preparation are processing,
optimization, and minimization of proteins.
1
Instrumentation. H and 13C NMR spectra were
recorded on a Bruker Avance III 600 MHz spectrometer
at 600 and 150 MHz, respectively. The mass spectra were
taken with an Agilent 6310 system using electrospray
ionization. High performance liquid chromatography
(HPLC) was carried out on anAgilent 1200 series device
using an analytical T3 Waters column (4.6 × 250 mm).
The docking protocol was validated through docking
back after removing PK11195 from the active site. For
the current study, the protein structures were taken
from RCSB protein data bank (PDB). Namely, we used
2MGY (wild type TSPO), 4RYQ (wild type TSPO,
resolution 1.7 Å), and 4UC1 (mutant TSPO, resolution
1.8 Å).
Gamma camera design. For scintigraphic images of
99mTc-labeled complexes in animal, we used a gamma
camera (Symbia, T2, True point SPECT/CT Siemens,
INMAS, DRDO, Delhi) equipped with a dual-head
low-energy high-resolution parallel-hole collimator
interfaced with a computer network system. For image
acquisition, an animal was fixed on a mechanical support
for standardization of the orientation and position.
Docking. Docking is a computational simulation
process to predict the preferred orientation of protein–
ligand interaction by forming a complex with high
stability. Glide, Schrödinger, and Maestro LLC 9.1 offer
the speedy and accurate high-throughput virtual screening
of several compounds with extremely accurate binding
mode predictions.
Image acquisition parameters. Dorsal ear vein
was used for injection in a rabbit. Images were acquired
at a defined time point in case of static imaging. For
dynamic imaging, acquisition was done for 30 min
immediately after injection. Anterior images were
acquired with 2 s/frame for 2 min, 15 s/frame for 2 min,
and 60 s/frame for 26 min. A 15% window centered on
140 keV (99mTc peak) with a zoom factor of 1 was used.
The matrices used for dynamic imaging were 128 × 128.
Both the newly designed analogs as well as few well-
known TSPO ligands were subjected to docking with the
protein using Glide. The molecules were docked to the
active site of TSPO. The molecules docked with negative
G Score are suitable for further evaluation. Only the
TSPO ligands will have the accurate hydrophobic contact
between the ligand and protein, resulting in favorable
docking scores.
Image analysis. The computational data of
scintigraphy were extracted and analyzed on a Pegasys
workstation (ADAC laboratory). After the image
acquisition, the region of interest (ROI) over the organ
of interest and the total field of view were drawn.
ADME prediction. The ADME properties were
calculated with the aid of QikProp of Schrödinger.
It predicts both physicochemical descriptors and
pharmacokinetic properties. QikProp provides ranges
for all the descriptors and properties of 95% of the
known drugs for comparison purpose. It also evaluates
the suitability of analogs on the basis of Lipinski’s rule
of five, which predicts drug-like pharmacokinetic profile
for rational drug design. In the present study, the QikProp
derived physicochemical properties used for ADME
analysis are molecular mass, number of hydrogen bond
donors, number of hydrogen bond acceptors, partition
coefficients, number of rotatable bonds, molecular
volume, etc., which, in turn, predict the properties, for
assessing drug likeliness, bioavailability, plasma protein
binding, and metabolism. As the molecules are for brain
application, we have considered the following predictive
properties:
Ligand preparation. The skeleton was taken from
our previous work; it proved its potential for TSPO
targeting. Further new analogues were designed and
drawn using Chemdraw Ultra 10.0 and were checked
for drug likeliness using Lipinski’s rule. These analogs
and few known TSPO ligands in .mol format were
used as input structures for processing in LigPrep,
Schrödinger, Maestro LLC 9.1 software. A series of
steps in the LigPrep process are as follows: conversions,
corrections to the structures, generation of variations in
the structures, elimination of unwanted structures, and
finally optimization of the structures.
Protein preparation for docking analysis. Prior to
docking, identification of the binding site in the target
protein is important. This information is taken through
(a) QP log P (octanol/water);
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SRIVASTAVA et al.
(b) QP log S (aqueous solubility);
(c) QP log S (conformation-independent aqueous
solubility);
(d) QP log Khsa (serum protein binding);
(6H). 13C NMR (CDCl3, 150 MHz): 37.8, 42.4, 52.5,
53.4, 127.3, 128.7, 129.2, 135.4, 165.6, 171.3.
Synthesis of methyl 2-(2-(5-chloro-2-oxoben-
zooxazol-3(2H)-yl)acetamido)-3-phenylpropano-
ate, ABPO-Cl (3). To a solution of 5-chlorobenzox-
azol-2(3H)-one (250.0 mg, 1.48 mmol) and K2CO3
(306.0 mg, 2.22 mmol) in DMF (5 mL), methyl
2-(2-chloroacetamido)-3-phenylpropanoate (415.2 mg,
1.63 mmol) was added with cooling on an ice bath.
The reaction mixture was stirred at 60–70°C for 3 h.
Thereafter, the reaction mixture was cooled to room
temperature before adding water. A 1 : 1 mixture of
toluene and ethyl acetate was used as an extractant.
The organic layer was washed with water and brine and
was dried over anhydrous sodium sulfate. The solvent
was removed in vacuo after filtration, and the product
was purified by silica gel column chromatography us-
ing CHCl3/EtOAc (4 : 1, v/v) as eluent. MS (ESI), m/z:
387.1 [M–H]+; calculated m/z: 387.1 [M–H]+. 1H NMR
(CDCl3, 600 MHz): 3.50–3.60 m (2H), 3.86 s (3H),
4.19–4.27 m (2H), 5.07–5.10 m (1H), 6.96–7.33 m
(8H). 13C NMR (CDCl3, 150 MHz): 34.0, 51.2, 53.2,
54.4, 121.3–148.8 (10 peaks), 155.3, 168.1, 168.6.
(e) QP log BB for blood–brain barrier;
(f) number of primary metabolites;
(g) apparent Caco-2 permeability (nm/s);
(h) apparent MDCK permeability (nm/s);
(i) human oral absorption percentage in GI;
(j) qualitative model for human oral absorption.
Synthesis and Characterization
Synthesis of 5-chlorobenzoxazol-2(3H)-one (1).
To a solution of 5-chloro-2-aminophenol (500.0 mg,
2.96 mmol) in 150 mL THF, 1,10-carbonyldiimidazole
(576.0 mg, 3.55 mmol) was added. The mixture was
refluxed with stirring for 2 h. The reaction mixture was
quenched by adding 2 m HCl solution after cooling to
room temperature. After that, the mixture was extracted
with EtOAc. The organic phase was washed with brine,
dried over anhydrous sodium sulfate, and filtered.
Thereafter, the solvent was removed in vacuo to get the
product as a white solid. MS (ESI), m/z: 167.8 [M–H]+;
calculated m/z: 168.0 [M–H]+.
Synthesis of 5-bromobenzoxazol-2(3H)-one (4).
To a solution of 4-bromo-2-aminophenol (500.0 mg,
2.66 mmol) in 150 mL of THF, 1,10-carbonyldiimid-
azole (518.0 mg, 3.19 mmol) was added. The mixture
was refluxed with stirring for 2 h. The reaction mixture
was quenched by adding 2 m HCl solution after cooling
to room temperature. EtOAc was used for the extrac-
tion. The organic phase was washed with brine, dried
over anhydrous sodium sulfate, and filtered. Thereafter,
the solvent was removed in vacuo to get the product
as white solid. MS (ESI), m/z: 211.7 [M–H]+; calcula-
1H NMR (DMSO, 600 MHz): 7.11–7.31 m (3H).
13C NMR (DMSO, 150 MHz):110.3, 111.3, 122.0,
128.2, 132.2, 142.6, 154.7.
Synthesis of methyl 2-(2-chloroacetamido)-
3-phenylpropanoate (2). To
a
solution of
L-phenylalanine methyl ester (500.0 mg, 2.32 mmol)
in deionized water (10 mL), triethylamine (360 μL,
2.52 mmol) was added with stirring under nitrogen at-
mosphere on an ice bath over a period of 5 min. To this
mixture, chloroacetyl chloride (205 μL, 2.52 mmol)
in dichloromethane (10 mL) was added dropwise over
a period of 1 h, and the reaction was carried out for 4
h. Thereafter, the mixture was extracted with dichlo-
romethane. The organic layer was washed twice with
deionized water and brine solution. After washing, the
organic layer was dried over anhydrous sodium sul-
fate. After filtration, the solvent was removed in vacuo,
checked by TLC using CHCl3/MeOH (9 : 1, v/v) as the
mobile phase, and purified by silica gel column chroma-
tography using 100% CHCl3 as eluent. MS (ESI), m/z:
254.0 [M–H]+; calculated m/z: 254.1 [M–H]+. 1H NMR
(CDCl3, 600 MHz): 3.11–3.20 m (2H), 3.74 s (3H),
3.98–4.04 m (2H), 4.86–4.90 m (1H), 7.02–7.33 m
1
ted m/z: 211.9 [M–H]+. H NMR (DMSO, 600 MHz):
7.20–7.28 m (3H). 13C NMR (DMSO, 150 MHz): 111.7,
112.9, 115.8, 124.8, 132.5, 143.0, 154.6.
Synthesis of methyl 2-(2-(5-bromo-2-oxoben-
zooxazol-3(2H)-yl)acetamido)-3-phenylpropano-
ate, ABPO-Br (5). To a solution of 5-bromobenzox-
azol-2(3H)-one (250.0 mg, 1.17 mmol) and K2CO3
(242.2 mg, 1.76 mmol) in DMF (5 mL), methyl
2-(2-chloroacetamido)-3-phenylpropanoate (328.2 mg,
1.29 mmol) was added with cooling on an ice bath.
The reaction mixture was stirred at 60–70°C for 3 h.
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 extractant.
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The organic layer was washed with water and brine and
dried over anhydrous sodium sulfate. The solvent was
removed in vacuo after filtration and was purified by
silica gel column chromatography using CHCl3/EtOAc
(4 : 1, v/v) as an eluent. MS (ESI), m/z: 431.3 [M–
affinity toward TSPO through docking. ABPO consists
of benzoxazolone and phenylalanine methyl ester
moieties coupled by the acetamide group. Two analogs
substituted with chlorine or bromine at the 5-position of
benzoxazolone have been prepared. The choice of the
chloro analog was governed by the fact the presence of
chlorine in several TSPO ligands, whereas the bromo
analog can provide opportunity to further optimize the
property through the substitution reaction. The predicted
affinity for TSPO and the drug likeliness properties of
the ABPO skeleton were found to be appropriate for the
biomedical application in of TSPO overexpression.
1
H]+; calculated m/z: 431.0 [M–H]+. H NMR (CDCl3,
600 MHz): 3.49–3.60 m (2H), 3.86 s (3H), 4.19–4.27 m
(2H), 5.06–5.10 m (1H), 6.91–7.33 m (8H). 13C NMR
(CDCl3, 150 MHz): 34.0, 51.2, 53.2, 54.4, 112.6–149.3
(10 peaks), 155.3, 168.0, 168.5.
Radiolabeling protocol. Radioloabeling was
performed using method mentioned in the literature
[38, 39]. 1 mL of a 1 × 10–2 m SnCl2·2H2O solution
was added to 200 μL of a 3 mM cold ligand solution.
Then, 2 mCi of sodium pertechnetate in 100 μL of saline
was added. 0.1 m NaHCO3 was used for pH adjustment
to 7, and the content was manually shaken and kept at
room temperature for 20 min. An ITLC-SG strip as a
stationary phase and acetone and PAW (pyridine–acetic
acid–water in 3 : 5 : 1.5 ratio) as a mobile phase were
used to determine the complexation with 99mTc and the
purity of the labeled compound. Count measurement was
done by cutting the ITLC strip into 0.1-cm segments.
It is important to know the pharmacokinetic profile of
any molecule to be used in vivo conditions, which can be
studied through ADME properties in silico. Often drugs
fail to enter the market because of poor pharmacokinetic
profile. Thus, it is imperative to design the compound
with easy transportability to desired site, proper
interaction at the desired site, no or minimal metabolism
before the action, and final excretion after the expected
action. The prediction of the desired ADME properties
at early stages of drug discovery using computer-aided
drug design methods reduces the chances of failure of
compound at later stages. This has further advantage of
low cost and less time required for wet screening.
Animal studies. Animals were supplied by the
Experimental Animal Facility (EAF) of the 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 ± 2°C
and 50% humidity under 12 h dark/light cycles.
As we are targeting the receptors/proteins that
are found on the outer mitochondrial surface of glial
cells, our molecules should be able to reach glial
cells of brain. This means that their ability to cross
the blood–brain barrier (BBB) is essential and that
their lipophilicity should be optimum, as lipophilic
substances are required to cross the BBB. During
this process, the transportation/binding pattern with
human serum is also important for consideration.
The ligandsABPO-Cl/Br satisfy the pharmacological
properties of 95% of available drugs available in
Schrödinger database. The drug likeliness or capability
for becoming a good drug candidate for clinical
application was predicted by Lipinski’s rule of 5, which
is normally used in CADD analysis. ABPO-Cl and
ABPO-Br follow all the five rules of Lipinski, reflecting
its drug-like properties. Additionally, hydrogen bond
acceptorsarewellwithintheacceptableproposedlimitof
10 for Lipinski’s rule. However, QP log P, an important
property for molecule to cross the blood–brain barrier,
is followed by both the designed ligands: 2.74 and 2.76
for bromo and chloro analogs, respectively (see table).
Scintigraphy studies. The scintigraphy studies of
99mTc-labeled complexes were carried out with a New
Zealand rabbit. 0.2 mCi of labeled complex was injected
intravenously through the ear vein, and images were
acquired for 30 min post injection of the radiocomplex.
RESULTS AND DISCUSSION
A
new series of acetamidobenzoxazolone–
phenylalanine ligands (ABPO, methyl 2-(2-(5-bromo/
chloro-2-oxobenzoxazol-3(2H)-yl)acetamido)-3-
phenylpropanoate) have been designed, evaluated
for drug likeliness on the basis of predictive ADME
properties in silico, synthesized, and assessed for their
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Bioavailability prediction. The computed param-
eters useful for predicting oral absorption are aqueous
solubility, conformation-independent aqueous solubil-
ity, percentage of human oral absorption in GI, qualita-
tive model for human oral absorption, and compliance
with Jorgensen’s rule of three.
from the first and second generations and the designed
ligands range from –1.09 to 0.362. This range is permis-
sible for crossing the blood–brain barrier (Table 1).
Prediction of binding with plasma protein. The
degree of binding to the blood plasma proteins (like
human serum albumin, glycoprotein, lipoproteins, and
alpha, beta, and gamma globulins) affects the efficiency
of a drug, as it greatly reduces the drug quantity that
passes through traverse cell membranes or decreases the
diffusion efficiency. Therefore, we predicted the ability
of the designed ligands to act on human serum albumin
through QP log Khsa. For the known ligands considered,
this parameter ranges from 0.032 to 1.31. For PK11195,
its value is quite high, i.e., 0.65, resulting in its non-
specific binding, which leads to a poor signal-to-noise
ratio in the case of radiolabeled PET ligands. Among
the ligands chosen for comparison purpose, DPA-713
and DAA1106 are better with QP log Khsa values of 0.03
and 0.35, leading to better signal-to-noise ratio. The de-
signed skeletons ABPO-Br/Cl (–0.18 and –0.26) exhibit
moderate binding, well within the limits (–1.5 to 1.5,
Table 1).
Out of all known ligands taken for comparison
purpose, only two are violating this rule, namely, QP
log S > –5.7 (–7.4 for FGIN-127 and –6.22 for PBR111).
For the designed ligands, the solubilities QP log S range
from –4.06 to –3.72; i.e., the solubility of the ligands is
better than, or comparable to that of the known ligands.
The Caco-2 permeability for the designed ligands
is 248 and 477 nm/s, and for the known ligands it is
between 2290 and 6015 nm/s, i.e., much greater than
25 nm/s, the limit of poor permeability as mentioned by
the software. The other parameter responsible for ab-
sorption, the conformation-independent QP log S val-
ues for the designed molecules (–4.62 and –5.54 for the
chloro and bromo analogs, respectively) are comparable
to those of the known ligands (–7.17 to –4.44). The the
percentage human oral absorption in GI is also reason-
ably good: >85 for the proposed ligands and 100 for the
known ligands. Even qualitative model for the human
oral absorption predicts high oral absorption.
Molecular volume. Another crucial factor for bind-
ing in the active site of the receptor is the molecular vol-
ume. The volume of ligand and that of the active site
should match for perfect interaction. To assess the struc-
tural compatibility of the molecules with an active site,
we analyzed the molecular volume. For RoS4864 and
FGIN-127, it is 941 and 1502 Å3, respectively. However,
the molecular volumes for the other known ligands are
in the range 1129–1307 Å3. Both the designed molecules
have the molecular volume in the range 1144–1172 Å3,
in which the majority the known ligands are lying. Thus,
the proposed ligands have sufficient molecular volume
for the ligand–protein interaction (Table 1).
The molecular flexibility is related to the number
of rotatable bonds, which is associated with the bio-
availability (increases with a decrease in the number
of rotors). Their number in the designed molecule is 6,
which is comparable to the known TSPO ligands such
as DAA1106, PBR06, and PBR28. However, some
known ligands have either high (12 for FGIN-127) or
low (0 for RoS-4864) number of rotatable bonds. There
is certain optimum number of rotatable bonds to bind in
the pocket of the receptor, as there is loss in entropy on
binding because of conformational restriction in case of
flexible molecules. As the molecule is intended for di-
agnostic application, too low number of rotatable bonds
may lead to stickiness to the target receptor. Therefore,
the designed ligands with six rotatable bonds also seem
to be suitable for diagnostic purpose (Table 1).
The ligands were synthesized in three steps as shown
in Scheme 1. The precursors, 5-bromo/chloro benzox-
azol-2(3H)-one and methyl 2-(2-chloroacetamido)-
3-phenylpropanoate, were synthesized starting from
commercially available 4-bromo/chloro-2-aminophenol
and phenylalanine methyl ester using cyclization and
chloroacetylation reactions and following the proce-
dures reported in the literature with some modiifcation
[14, 39]. We used CDI for the cyclization reaction and
chloroacetyl chloride with triethyl amine as a base for
the chloroacetylation reaction. The final compounds
ABPO-Br/Cl were synthesized through alkylation re-
action of 5-bromo/chloro-benzoxazolone with methyl
Prediction of the blood–brain barrier (BBB) pen-
etration. As the molecules proposed have potential ap-
plication for brain, it is mandatory for them to cross the
blood–brain barrier. Too polar molecules may be unable
to cross BBB. To assess the access of drug to the brain,
the blood–brain partition coefficient (QPlog BB) is com-
puted. The values for the known TSPO ligands that are
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SRIVASTAVA et al.
Scheme 1. Synthesis of ABPO-Cl. Reagents: (a) CDI, THF, reflux; (b) chloroacetyl chloride, Et3N, H2O/DCM, 0°C–room
temperature; (c) K2CO3, DMF, 60–70°C, 3 h. APBO-Br was prepared under the same conditions starting from 4-bromo-2-
aminophenol instead of 4-chloro-2-aminophenol.
OH
O
O
H2C
CH
C
Cl
NH2
NH2
(b)
(a)
O
O
C
O
CH
O
NH
Cl
CH2
O
N
Cl
H
1
(c)
2
O
O
O
N
CH
Cl
H
CH2
O
O
3
2-(2-chloroacetamido)-3-phenylpropanoate in the pres-
ence of K2CO3 in DMF. The compounds were purified
using column chromatography and preparative HPLC.
The chemical purity of the compound was determined
using analytical HPLC; the purity was found to be
>97%. All the eight aromatic protons of benzoxazolone
and benzene moieties were observed in the region of
6–8 ppm. The 13C NMR spectra were consistent with
the formula. The C=O carbon signals of benzoxazolone,
amide, and ester were observed in the region of 155–
169 ppm for both analogs, reflecting the synthesis of
ABPO-Cl/Br. The mass spectrometry also confirmed the
2MGY
4RYQ
ABPO-Br
ABPO-Cl
Fig. 1. (Color online) 2D and 3D interaction diagram of ABPO-Br and ABPO-Cl with wild type PDBs 2MGY and 4RYQ.
RADIOCHEMISTRY Vol. 62 No. 1 2020

DESIGN, SYNTHESIS, AND IN SILICO EVALUATION
(b) (c)
115
(a)
Fig. 2. (Color online) 2D interaction diagram of FEBMP, FPBMP with PDBs: (a) 2MGY, (b) 4RYQ, and (c) 4UC1.
Fig. 3. (Color online) 2D and 3D interaction diagram of ABPO-Cl with mutant type PDB 4UC1.
synthesis: The [M–H]+ peaks of ABPO-Cl and ABPO-
Br were observed at m/z 387.1 and 431.3, respectively.
reflect the same. Further studies are required for its vali-
dation (Figs. 2, 3).
The choice of the phenylalanine-based biocompati-
ble ligands was based on their preference for LAT1 over
other LATs, A, and ASC system in the uptake mecha-
nism. Validation for TSPO binding/affinity was carried
out through docking studies to know about interaction
with different amino acid residues of TSPO as compared
to other known TSPO ligands. ABPO-Cl/Br demonstrat-
ed comparable or better affinity for TSPO in terms of
The radiolabeling study was carried out using
the procedure mentioned in the literature with some
modification. Complexation of the synthesized ligands
with 99mTc gave 95.4% stability of 99mTc-ABPO-Br over
24 h. In vitro serum stability clearly demonstrates the
stability of the radiolabeled complexes without much
transchelation to serum protein like albumin, which was
observed to be intact with 92.4% of 99mTc-ABPO-Br.
Gscore, when compared to known ligands like PK11195
The selectivity and distribution of theseTSPO ligands
were confirmed by tracer techniques in a New Zealand
rabbit (dynamic imaging for 30 min, Fig. 4). In brain,
significant uptake was observed; it reduced with time. In
a nut shell, the uptake was majorly in all the TSPO-rich
organs at the initial time point and eventually decreased
with time. In excretory organs, the uptakes were initially
lower but increased with time till the last time point of
the observation.
and MBMP. Various interactions were observed in dock-
ing, including π–π interaction and hydrogen bonding.
The phenylalanine aromatic ring is participating in π–π
interactions, while one hydrogen bond was observed
with the NH group of the acetamidobenzoxalone moiety
(Fig. 1, 2).
In docking studies using mutated PDB 4UC1
(A139T) of RsTSPO, the ligands ABPO-Cl/Br showed
similarity in interactions with FPBMP/FEBMP. The
Gscore values were comparable for FPBMP, FEBMP,
and MBMP. As FEBMP shows no intersubject vari-
ability, it can be predicted that the ABPO-Cl/Br could
After intravenous injection of 99mTc-ABPO-Br, 10-s
frames were taken during dynamic studies. Slight uptake
in lungs, spleen, and brain is seen in these images. As
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SRIVASTAVA et al.
Fig. 4. (Color online) In vivo biodistribution of 99mTc-ABPO-Br in New Zealand rabbit (dynamic image).
the duration of the image capture was 10 s, organs with
relatively low uptake were not clearly visualized. Small
uptake at later time points was seen, which may be due
to fast release kinetics of 99mTc-ABPO-Br (Fig. 4).
mal studies are required to prove its efficiency as TSPO
marker.
ACKNOWLEDGMENTS
In summary, in silico studies of the modified MBMP
skeleton (i.e., ABPO) to evaluate the ADME properties
and interaction with TSPO protein, followed by facile
synthesis, were carried out for overexpressed TSPO
targeting.
The authors are grateful to Dr. Tarun Sekhri, Director of
INMAS, Dehli, Dr. A.K. Mishra, Head of the Division of
Cyclotron and Radiopharmaceutical Science of INMAS, and
Dr. Gaurav Mittal, Head of CCM for providing the facility to
carry out the experiments. The authors are also grateful to Dr.
Himanshu Ojha, Scientist, Dr. Sonia Gandhi, Scientist, Mr.
Sunil Pal, Technical Officer, and Mr. Harish Rawat, INMAS,
for their technical support.
CONCLUSION
The goal of the study was synthesis, characteriza-
tion, and in silico evaluation of acetamidobenzoxazo-
lone–phenylalanine methyl ester, methyl 2-(2-(5-bro-
mo-2-oxobenzooxazol-3(2H)-yl)acetamido)-3-phen-
ylpropanoate, for TSPO targeting. The binding affinity
for TSPO was assessed through ligand–protein docking
and ADME properties by using QikProp modules of
Schrödinger software. The biodistribution of these li-
gands and their selectivity to TSPO were demonstrated
in vivo on a New Zealand rabbit. Further preclinical ani-
FUNDING
The study was supported by INMAS, Delhi, under Task
Project ST/15-16/INM-03.
CONFLICT OF INTEREST
The authors declare that they have no conflict of
interest.
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117
Shimoda, Y., Yamasaki, T., Xie, L., Hatori, A., Maeda, J.,
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