Design, synthesis and biological evaluation of methyl-2-(2-(5-bromo benzoxazolone)acetamido)-3-(1H-indol-3-yl)propanoate: TSPO ligand for SPECT

Article abstract of DOI:10.1039/C6RA19514H

The translator protein (TSPO, 18 kDa), a transmembrane mitochondrial protein, has been explored as an important biomarker by researchers because of its involvement in inflammation, immune modulation and cell proliferation. Recently, our group has explored a modified benzoxazolone derivative for diagnostic applications that has overcome few problems of first and second generation TSPO PET ligands. In this study, a new skeleton acetamidobenzoxazolone-indole, a conjugation of two TSPO pharmacophoric moieties benzoxazolone and indole, has been designed, synthesized and evaluated for TSPO targeting for SPECT. The methyl-2-(2-(5-bromo benzoxazolone)acetamido)-3-(1H-indol-3-yl)propanoate (MBIP) ligand was designed on the basis of pharmacophore modeling done on benzoxazolone based TSPO ligands which was then validated computationally for TSPO binding through docking studies (PDB ID: 4RYO, 4RYQ, and 4UC1) which showed a comparable Glide G_score as compared to known ligands like PK11195, PBR28, and FGIN-127. MBIP was synthesized by amidation reaction of 2-(5-bromo-benzoxazolone)acetic acid with tryptophan methyl ester hydrochloride (yield 62%). The compound was synthesized and characterized using spectroscopic techniques like ¹H-NMR, ¹³C-NMR, and mass spectroscopy. Purification was carried out by column chromatography and analytical HPLC (purity > 97%). The purified compound was labelled with ^99mTc (radiochemical yield > 96%). The radiolabelled compound showed >94% stability in solution and >91% stability in serum after 24 h indicating the stable nature of the radio complex. A biodistribution study on BALB/c mice showed uptake of ^99mTc-MBIP in TSPO rich organs and appropriate pharmacokinetics of excretion and release for a SPECT agent. Further evaluation of the ^99mTc-MBIP may prove it as a potential candidate for TSPO targeting using SPECT.

Full text of DOI:10.1039/C6RA19514H

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Design, synthesis and biological evaluation of
methyl-2-(2-(5-bromo benzoxazolone)acetamido)-
3-(1H-indol-3-yl)propanoate: TSPO ligand for
SPECT†
Cite this: RSC Adv., 2016, 6, 114491
Received 2nd August 2016
Accepted 22nd November 2016
ab
a
a
b
a
*
*
Ankur Kaul, Himanshu Ojha, Pravir Kumar and Anjani K. Tiwari
Pooja Srivastava,
DOI: 10.1039/c6ra19514h
www.rsc.org/advances
The translator protein (TSPO, 18 kDa), a transmembrane mitochondrial
protein, has been explored as an important biomarker by researchers
because of its involvement in inflammation, immune modulation and
cell proliferation. Recently, our group has explored a modified ben-
zoxazolone derivative for diagnostic applications that has overcome
few problems of first and second generation TSPO PET ligands. In this
study, a new skeleton acetamidobenzoxazolone–indole, a conjuga-
tion of two TSPO pharmacophoric moieties benzoxazolone and
indole, has been designed, synthesized and evaluated for TSPO tar-
geting for SPECT. The methyl-2-(2-(5-bromo benzoxazolone)acet-
amido)-3-(1H-indol-3-yl)propanoate (MBIP) ligand was designed on
the basis of pharmacophore modeling done on benzoxazolone based
TSPO ligands which was then validated computationally for TSPO
binding through docking studies (PDB ID: 4RYO, 4RYQ, and 4UC1)
which showed a comparable Glide Gscore as compared to known
ligands like PK11195, PBR28, and FGIN-127. MBIP was synthesized by
amidation reaction of 2-(5-bromo-benzoxazolone)acetic acid with
tryptophan methyl ester hydrochloride (yield 62%). The compound
was synthesized and characterized using spectroscopic techniques
like 1H-NMR, 13C-NMR, and mass spectroscopy. Purification was
carried out by column chromatography and analytical HPLC (purity >
97%). The purified compound was labelled with 99mTc (radiochemical
yield > 96%). The radiolabelled compound showed >94% stability in
solution and >91% stability in serum after 24 h indicating the stable
nature of the radio complex. A biodistribution study on BALB/c mice
showed uptake of 99mTc-MBIP in TSPO rich organs and appropriate
pharmacokinetics of excretion and release for a SPECT agent. Further
evaluation of the 99mTc-MBIP may prove it as a potential candidate for
TSPO targeting using SPECT.
1. Introduction
Inammation is linked to the preliminary stage of many
complications of diseases associated with lung inammatory
diseases, chronic obstructive pulmonary disease, acute liver
damage, immune response to heart, brain injury, neurodegen-
erative diseases, psychiatric disorders and cancer.1–8 Peripheral
benzodiazepine receptor (PBR), renamed as translocator
protein (TSPO, 18 kDa),9 has been found to be an important
biomarker for inammation in the TSPO enriched organs.
Recent investigations of TSPO in various biological processes
and its importance in certain pathological situations due to
altered concentration has led to development of various ligands
for diagnostic purposes.10–14
Various radionuclides have been tried for diagnostic appli-
cations by targeting TSPO such as 11C and 18F for positron
emission tomography (PET),15–21 123I and 99mTc for single
photon emission computed tomography (SPECT).22–27 Other
nuclides such as Eu3+ and Gd3+ have also been tried for optical
and magnetic resonance imaging (MRI), respectively.28,29
Different classes of TSPO ligands, for example, isoquinoline
carboxamides, indoleacetamides, pyrazolopyrimidines, imida-
zolepyridines, benzodiazepines and benzoxazolone analogs
have been developed.30,31 Till date no ligand has been found
ideal for human application throughout the globe. Few rst
generation ligands, such as PK11195 showed poor specicity
and poor signal-to-noise ratio. Although second generation
ligands, such as PBR28, DAA1106, DPA713, and PBR111
demonstrated improved signal-to-noise ratio but suffered from
inter subject variability. This inter subject variability was
attributed to rs6971 polymorphism in TSPO gene which results
into single amino acid substitution i.e. Ala147Thr.32 This
amino acid substitution constitute three classes in terms of
binding affinity: high affinity binder (HAB) having alanine at
147, low affinity binder (LAB) having threonine at 147 and
mixed affinity binder (MAB) having alanine and threonine
both. Low-binding affinity TSPO is present in 30% of Cauca-
sians and 25% of Africans.20
aDivision of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear
Medicine and Allied Sciences, Brig. S. K. Mazumdar Road, Delhi-110054, India.
E-mail: pshree_14@yahoo.co.in; anjani7797@rediffmail.com; Fax: +91-11-
23919509; Tel: +91-11-23905362; +91-11-23905387
bMolecular Neuroscience and Functional Genomics Laboratory, Department of
Biotechnology, Delhi Technological University, Delhi 110042, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra19514h
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11C and 18F labeling have their own limitations as they chromatography (TLC) on aluminium plates coated with silica
require on site cyclotron to produce the radioisotopes. 99mTc, an gel 60 F254 and instant thin layer chromatography silica gel
ideal single photon of 140 keV emitting radionuclide with half (ITLC-SG) was used for identication of labeling, purity and Rf
life of 6 h, has lesser radiation burden as compared to PET of radiocomplex.
radionuclide and is generated through 99Mo/99mTc generator.
2.1.2. Instrumentation. 1H and 13C NMR spectra were
Although number of TSPO ligands have been synthesized but recorded on Bruker Avance II 400 MHz system at 400 MHz and
few of them have been modied for diagnostic purposes using 100 MHz, respectively. Mass spectroscopy was done on 6310
metals for example 99mTc, 188Re, Gd3+ and Eu3+ etc.24–29 As these system of Agilent using ESI positive mode. g-Scintigraphic
metal based ligands have been used for receptor targeting, this studies were done on Hawkeye Camera and a g-scintillation
aspect can be further explored for TSPO. Some modication on counter was used for counting radioactivity. High performance
quinoline carboxamide25 and imidazolepyridines24,26 have been liquid chromatography (HPLC) was carried out on 1200 series of
reported with 99mTc for SPECT application which are the basis of Agilent using analytical T3 Waters column (4.6 ꢀ 250 mm) for
TSPO ligands like analogues of PK11195 and CB86, respectively. analysis and C-18 column (21.2 ꢀ 150 mm) for purication.
Recently, acetamidobenzoxazolone based skeleton, devel- SPECT images were taken on GE Triumph system.
oped from rst generation ligand RoS-4864 has been used by
2.1.3. Animal models. Animal protocols were approved by
our group for the development of PET ligand [11C]MBMP and the Institutional Animal Ethics Committee (INM/DASQA/IAEC/
[18F]FEBMP with improved TSPO targeting properties over (R) 09/015). BALB/c mice (22–28 g) were used for animal
[11C]PK11195.18–21 MBMP/FEBMP skeleton has shown favour- experiments.
able biodistribution pattern, high in vitro and in vivo specic
2.1.4. Pharmacophore hypothesis generation. The phar-
binding for TSPO and has overcome the problem of intersubject macophore hypothesis was developed by aligning pharmaco-
variability. In our previous studies human postmortem brains phoric features of 51 TSPO ligands retrieved from the
of HABs and LABs were used for in vitro autoradiographic literature33,34 using PHASE, Schrodinger, Maestro LLC 9.1 so-
¨
studies of MBMP/FEBMP. The binding ratios of (R)[11C] ware. Generation of conformers was carried out with the help of
PK11195, [11C]DAA1106, [11C]AC-5216, and [11C]PBR28, for HAB MacromodelConfGen using OPLS2005 force eld with rapid
to that of LAB were approximately 0.45, 1.19, 4.60, and 11.1, torsional search. Geometric alignment of site points in the
respectively, showing excellent correlation with the Ki ratios of active molecules to the site points in the hypothesis was the
these ligands for LAB to that of HAB. The compound [18F] basis of hypothesis score generation. Each active molecule
FEBMP has shown binding ratio of LAB to that of HAB as 0.9. conformer generates hypothesis. Scores of the compounds were
This study presents [18F]FEBMP as a promising TSPO ligand determined by 3D-QSAR model developed from compounds by
and skeleton for new ligands with negligible effect of rs6971 aligning on the hypothesis. PLS analysis was carried out to
polymorphism on binding towards TSPO.20
evaluate the pharmacophore hypothesis using atom-based
Taking lead from this work, we have developed a new SPECT PHASE QSAR models generation with a maximum of three
ligand using 99mTc for TSPO imaging. We have introduced PLS factors for each pharmacophore type. Best hypothesis was
a new skeleton acetamidobenzoxazolone indole propanoate as further used for designing the TSPO ligand.
a TSPO ligand by conjugating acetamidobenzoxazolone moiety
2.1.5. Docking studies. To design modied TSPO ligand,
present in MBMP with indole moiety present in TSPO ligand, docking studies for ligand–protein interaction were carried out
FGIN-127.30 MBMP and FGIN-127 both display TSPO affinity in with extra precision (XP) mode of GLIDE, Schrodinger, Maestro
¨
nanomolar range. The affinity of MBIP towards TSPO has been LLC 9.1 soware. Structures of ligands were drawn using
validated through docking studies using wild type as well as ChemBioDraw. We have chosen wild type (4RYO and 4RYQ) as
mutant PDBs of TSPO. Thereaer, newly designed potential well as mutant (4UC1) PDBs for docking studies from different
TSPO ligand was synthesized through a simple and efficient sources such as BcTSPO and RsTSPO. The rs6971 poly-
four steps synthetic procedure which was characterized by morphism in human resulting in A147T single amino acid
NMR, mass and puried by HPLC. The puried compound was change is equivalent to A139T in RsTSPO. Ultra 10.0.LigPrep
radiolabelled with 99mTc for biological evaluation. Stability of and protein preparation modules were used for ligands and
complex for in vivo application was evaluated in serum and protein preparation, respectively. Protein preparation module
biodistribution study was performed to evaluate its % uptake consists of a series of steps that perform preprocess, optimi-
with time in TSPO enriched organs.
zation and minimization of protein structure. Receptor grids
generation were required for docking with ligands. OPLS2005
forceeld has been used for the receptor grid as well as ligand
grid generation. Docking simulation was performed in implicit
solvent.
2. Materials and methods
2.1. Chemistry
2.1.1. Chemicals. All the chemicals and solvents were
The ligand strain energy is the difference between the energy
purchased from Sigma-Aldrich and Merck. 99mTc was procured of the ligand as it is in the complex and the energy of the
from Regional Centre for Radiopharmaceuticals, Board of extracted ligand, minimized, starting from the geometry in the
Radiation and Isotope Technology (BRIT), Department of rened complex. This local strain energy is used for comparison
Atomic Energy, India. Column chromatography was carried out of different poses of the same ligand. Calculations to assess the
using silica MN60 having mesh 60–120, thin layer strain were performed in implicit solvent.
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141.52 (14 carbon of aromatic rings), 154.15 (O–C]O–N),
166.28 (NH–C]O), 172.35 (O–C]O).
2.1.6. Synthesis
2.1.6.1 Synthesis of 2-(2-(5-bromo benzoxazolone)acetamido)-3-
(1H-indol-3-yl)propanoate, 5 (MBIP). 2-(5-Bromo-benzoxazolone)
acetic acid was synthesized as per Scheme 1 by following the
procedure reported in the literature.33 To a solution of 2-(5-bromo-
benzoxazolone)acetic acid (250 mg, 0.92 mmol) in DMF (5 ml),
tryptophan methyl ester hydrochloride (351.2 mg, 1.38 mmol),
HOBt (124.2 mg, 0.92 mmol) and EDCl (264.3 mg, 1.38 mmol)
were added at room temperature and stirred for 7 h. Subse-
quently, water was added and the mixture was extracted using
a toluene and ethyl acetate (1 : 1). Water and brine were used for
washing of organic layer and subsequently anhydrous Na2SO4 was
added for drying. Aer ltration, the solvent was removed in
vacuo and purication of residue was carried out on silica gel
column chromatography using CHCl3/EtOAc (4 : 1 v/v) as eluent
to give amide as a white solid (MBIP: yield 62%). Chemical purity
> 97% by HPLC: T3 column, 4.6 ꢀ 250 mm; MeCN/H2O, 6/4–8/2
(v/v); ow rate: 0.8 ml minꢁ1; luv ¼ 254 nm; tR ¼ 8.75 min.
1H-NMR (DMSO, 400 MHz): d (ppm) 3.10–3.16 (2H, m), 3.59
(3H), 4.47–4.53 (3H, s), 7.00–7.50 (8H, m), 8.85–8.87 (1H, d),
10.90 (1H, s).
MS (ESI) m/z: 496.3 [M + Na]+; calculated mass 496.38
[M + Na]+.
2.1.6.2 Radiosynthesis. 200 ml solution of the compound
(0.03 nM) was added in SnCl2$2H2O (1 ꢀ 10ꢁ2 M) (purged
nitrogen 10% acetic acid (1 ml). Thereaer, 75 MBq, 100 ml
saline solution of sodium pertechnetate (freshly eluted NaTcO4)
was added. The pH was adjusted to 7.0 by addition of 0.1 M
NaHCO3 solution to the reaction mixture and the contents were
shaken manually. Vial was kept at room temperature for 20–
30 min. 99mTc complex's labeling, chemical purity and Rf were
determined by ITLC-SG strips by using developing solvents such
as acetone and PAW (pyridine, acetic acid and water in 3 : 5 : 1.5
ratio) simultaneously. 0.1 cm segments of ITLC were taken for
count measurement to nd out the percentage of free and
labeled ligand i.e. radiochemical purity.
2.1.7. In vitro serum stability assay. Blood was collected
from healthy volunteers and was made to clot in a humidied
incubator maintained at 5% carbon dioxide and 95% air at
37 ꢂC for 1 h and was used to prepare serum. Centrifugation of
sample at 400 rpm was followed by ltration through 0.22 mm
syringe and ltrate was kept into sterile plastic culture tubes.
13C-NMR (DMSO, 100 MHz): d (ppm) 28.72 (CH2–CH), 44.41
(CH3–O), 52.44 (NH–CH–C]O), 53.91 (N–CH–C]O), 111–
t
Scheme 1 Chemical synthesis. Reagents and conditions: (i) CDI, THF, rt; (ii) butylbromoacetate, K2CO3, DMF, 70 ꢂC; (iii) HCl, dioxane, AcOH,
50 ꢂC; (iv) tryptophan methyl ester hydrochloride, EDCl, HOBt, DMF, rt.
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Incubation of the freshly prepared radiocomplex in fresh designing new ligands. Pharmacophore model was developed
human serum was done at physiological conditions i.e. at 37 ꢂC with TSPO ligands having benzoxazolone to understand the key
(concentration 100 nM mlꢁ1) and then it was analyzed by ITLC- features responsible for affinity. Ki values taken for all the
SG to detect any dissociation of the complex at different time ligands were studied against TSPO of the kidney membrane.
intervals. Percentage dissociation of the complex was deter-
As per our previous ndings about acetamidobenzoxazolone
mined by calculating percentage of free pertechnetate at pharmacophore based skeleton was found suitable for new
a particular time by using saline and acetone as mobile phases. TSPO ligand which is not affected by intersubject variability.
This represents percentage dissociation of the complex at that Besides that Fukaya and others33 have also conrmed that
particular time point in serum.
appropriate hydrophobic/acceptor groups attached with ben-
2.1.8. Biodistribution studies. Tissue distribution studies zoxazolone and acetamide have potential to bind TSPO selec-
were performed on BALB/c mice. All the guidelines of the tively in nM concentration.
Institutional Animal Ethics Committee were followed during
Different pharmacophoric features like hydrogen bond
animal handling and experimentation. Radiolabelled donor (D), acceptor (A), hydrophobic group (H), and aromatic
compound was injected in mice through tail vein (10 mCi). Mice ring (R) were chosen to generate the hypothesis. The 51 TSPO
were sacriced at six time intervals (5 min, 15 min, 30 min, ligands were divided into test set and training set with 13 and 38
60 min, 120 min, and 1440 min), blood was collected and ligands, respectively. Ligands of these sets were aligned with
different organs (brain, heart, lung, liver, spleen, kidney, each other to nd the pharmacophore and thereaer PLS
stomach and intestine) were dissected for analysis. Radioac- analysis was performed. Hypothesis AAAR showed best result in
tivity was measured in the gamma counter. Radioactivity terms of predictive ability and internal validation (Fig. 1) with R2
injected in each mouse was found out by subtracting the activity ¼ 0.95 and standard deviation in the range of 0.237 to 0.760.
le in tail from the activity injected. Radioactivity found in each This hypothesis was further utilized for designing of new
organ was expressed as percentage administered dose per gram ligand.
(% ID per g) of tissue. Blood volume was calculated as 7% of the
total body weight. Decay correction was applied to radioactivity ligand. A substituent on the benzoxazolone derivative speci-
measurements. cally at C-5 position of phenyl ring plays an important role in
Based on these concepts we have designed MBIP as TSPO
2.1.9. Preparation of mice model. The inammatory model TSPO binding. Substitution by halogen in place of aromatic ring
was prepared as per previous literature.35–39 10 weeks old male system at this position provides sub-nanomolar binding
BALB/c mice of 22–25 g weight were taken to develop inam- affinity.33 This is the rationale behind selecting Br for C-5
mation model. 40 ml of E. coli lipopolysaccharide (5 mg kgꢁ1) in position for designing the benzoxazolone ring. This has also
PBS was administered intranasally in each mouse. Mice were been mentioned in literature that compound suffered from
housed under a 12 h dark–light cycle and were allowed free poor solubility due to phenyl substituent at acetamida side. We
access of food pellets and water. Aer 24 h the mice were used have tried biocompatible amino acid analogue at this position
for animal studies.
For ex vivo biodistribution blocking evaluation, three
different concentration of TSPO ligand (PK11195) 2.5 mg kgꢁ1
,
5 mg kgꢁ1, and 10 mg kgꢁ1 were injected 10 min prior to 99mTc-
MBIP injection (100 ml of 1 mg kgꢁ1). All the experiments were
performed in triplicate set (n ¼ 3). During this study 5 mg kgꢁ1
blocking was found most appropriate that is why it was taken
for in vivo imaging at 40 min post radioligand intravenous
injection.
3. Result and discussion
MBIP, acetamidobenzoxazolone–indole based ligand designed
on the basis of pharmacophore modeling and docking studies,
has been synthesized as per Scheme 1 and radiolabelled with
99mTc for its evaluation as a diagnostic agent for TSPO targeting
whose concentration is perturbed during inammation in TSPO
enriched organs. The stability of 99mTc complex, biodistribution
and serum stability studies of the ligand were found appro-
priate for SPECT application.
Ligand based drug design makes use of the knowledge of
other known ligands for same target. These ligands could
provide information about minimum structural characteristics
necessary for binding with biological target in terms of phar-
Fig. 1 Pharmacophore model AAAR used for designing TSPO ligand
derived from the benzoxazolone derivatives. Pharmacophoric features
with colour code: A-hydrogen bond acceptor (pink) and R-aromatic
macophoric model. This model/hypothesis could be useful in moiety (orange).
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to solve solubility issue for better prospect of clinical applica-
tion in future. The R contained in MBIP is also present in TSPO
ligands-FGIN-127 which has nanomolar binding affinity for
TSPO. Its limited in vivo use is due to its high lipophilicity. The
designed ligand MBIP showed decreased computational log P
(2.31) value as compared to FGIN-127 (computational log P ¼
6.94).30
The intersubject variability issue for MBIP has been studied
computationally by taking 2 wild type PDBs (4RYO and 4RYQ)
and one mutant PDB (4UC1) representing required SNP for
docking studies. MBIP has shown good interactive score (Gscore
)
for wild type and mutant PDBs similar to PK11195 and FEBMP
which are successful ligands against intersubject variability
(ESI†). The validation of docking protocol was done before
docking the compounds with PDB. The ligand present in co-
crystallized PDB was removed and docked with binding site.
The root mean square deviation between predicted and
observed X-ray crystallographic conformers was found to be
ꢀ
around 0.5 A predicting the reproduction of binding mode of
docked compound using this docking protocol.
Aer designing, the next step is validation for TSPO binding/
affinity which was carried out through docking studies to gain
better insight of its interaction with TSPO as compared to other
known TSPO ligands. MBIP demonstrated comparable or better
affinity with TSPO in terms of Gscore (ꢁ10.448 for MBIP > ꢁ7.451
to ꢁ10.15 for other known ligands) (Table 1). Both the parent
ligands FEBMP and FGIN-127 have Gscore ꢁ10.151 and ꢁ9.319,
respectively on docking with TSPO. The MBIP interaction with
4RYO has been reected in their interactions shown in 2D and
3D interaction diagram (Fig. 2). Various interactions were
observed in docking including pi–pi interaction with Phe90 and
Phe95, hydrogen bonding with Gly44 and Ser22. Both the
aromatic rings were participating in pi–pi interactions. One
hydrogen bond was observed due to NH of indole moiety in the
ligand. These interactions were different from interactions of
MBMP and its uoro analog with TSPO (Try27, Tyr28, Phe19
and Try99).19
Table 1 Glide Gscore of MBIP and other known TSPO ligands on
docking with TSPO (PDB ID: 4RYO). Docking was performed using
extra precision mode of GLIDE, Schrodinger, Maestro LLC 9.1 software
¨
Fig. 2 3D (A) and 2D (B) interaction diagram of MBIP on docking with
TSPO (PDB ID: 4RYO). Docking was performed using extra precision
Ligand
G-Score
mode of GLIDE, Schrodinger, Maestro LLC 9.1 software. MBIP formed
¨
pi–pi interactions of aromatic rings with Phe90 and Phe95 and
hydrogen bond interactions with Gly44 and Ser22.
MBIP
ꢁ10.448
ꢁ9.493
ꢁ7.451
ꢁ6.180
ꢁ10.151
ꢁ9.809
ꢁ9.319
ꢁ9.633
ꢁ9.658
ꢁ7.475
ꢁ9.881
ꢁ9.034
ꢁ7.992
ꢁ9.406
DAA1106
DIAZEPAM
DPA713
FEBMP
BcTSPO-PK11195 model showed interaction between oxygen
of carbonyl group of PK11195 with indole groups of Try51 and
Try138, van der Waal interaction with Ser22, Tyr32, Pro42, Ile47,
Phe55, Phe90, Ser91, GIn94, Cys107, Ala142 and Leu145. Few of
the interaction of PK11195 as on Ser22 and Phe90 were similar
to MBIP interactions with TSPO protein.40 MBIP and FEBMP
both the compounds have shown similar binding modes. The
C]O of benzoxazolone forms H-bond with the Try51 in 4RYQ.
This is in agreement with the interaction of PK11195 with
FEDAA1106
FGIN-127
FPBMP
PBR28
PBR111
PK11195
RO5-4864
SSR180575
XBD173
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4RYQ. Few more interaction sites are common to MBIP in 4RYO benzoxazolone C]O, respectively. MS run in the positive mode
and 4RYQ such as Ser22, Phe55, Phe90, Ser91, Gln94, Cys107, has peak at 496.3 [M + Na]+ which was in accordance with the
Ala142. Their glide Gscore and their contribution from different proposed structural formula. Aer synthesis, biological evalu-
sub interactions were found comparable (ESI†).
ation was performed by tracer method using 99mTc. The radio-
The precursor 2-(5-bromo-benzoxazolone)acetic acid of labelling study was carried out with the procedure reported in
compound MBIP was synthesized in three steps starting from 4- our previous work.41 Stability of the complex is one of the most
bromo-2-amino phenol by the procedure reported in the liter- important criteria for use of the ligand in vivo as it avoids
ature.33 In the last step, the ligand 2-(2-(5-bromo benzoxazolone) metabolite formation and transchelation which changes the
acetamido)-3-(1H-indol-3-yl)propanoate
was
synthesized desired property for SPECT application. Complexation of the
through amidation reaction of 5-bromo-benzoxazolone acetic synthesized ligand with 99mTc gave sufficient stability over 24 h
acid and tryptophan methyl ester hydrochloride in the presence (Fig. 3). In vitro serum stability clearly indicates the stable
of HOBt and EDCl. The yield of the nal product was 62% and nature of radio complex without any transchelation to serum
was puried using column chromatography and preparative protein like albumin which was observed to be >91% intact aer
HPLC. The retention time of nal product MBIP was found to be 24 h (Fig. 4). Stability studies showed requisite stability of the
8.75 min. The chemical purity of the compound was determined complex to act as a SPECT agent.
using analytical HPLC and purity was found to be >97%. In the
Biodistribution using radiocomplex was carries out to have
1H-NMR, two singlet NH peaks of same intensity were observed an idea whether the radioligand is reaching the organs of
at 8.8 and 10.9 ppm which correspond to NH of amide and NH interest which are rich in TSPO and its excretion from the body.
of indole, respectively. This indicates the successful synthesis of The distribution in different organs is represented as
the proposed structure. All the eight aromatic protons of ben- percentage of injected dose per organ at different time points.
zoxazolone and indole moieties were observed in the region 6– Table 2 showed the distribution of radioactive compound at six
8 ppm. 13C-NMR reected appropriate peaks as per its formula. different time points post injection into the mice (n ¼ 3). At
Three C]O peaks were observed at 172.3, 166.2 and 154.1 ppm 5 min, peak value of activity was recorded in all the organs.
which correspond to ester C]O, amide C]O and Highest % ID per g was observed in liver i.e. 28.87 followed by
Fig. 3 Labeling efficiency (% LE) of MBIP with 99mTc. NaTcO4 at pH 7 was used for labeling. 0.1 cm segment of ITLC were used after developing it
in solvents acetone and PAW (pyridine, acetic acid and water in 3 : 5 : 1.5 ratio) for count measurement.
Fig. 4 Serum stability study in terms of labeling efficiency (% LE) of 99mTc-MBIP over 24 h. Studies were performed in fresh human serum from
healthy volunteers at physiological conditions i.e. 37 ꢂC.
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Table 2 Biodistribution of 99mTc-MBIP in normal BALB/c mice. Mice (n ¼ 3) received 10 mCi of 99mTc-MBIP by tail vein injection. Radioactivity
uptake in different organs were measured by gamma counting and expressed as % ID per g, mean ꢃ standard deviation
Organ/tissue
5 min
15 min
30 min
60 min
120 min
1440 min
Blood
Brain
Heart
Lungs
Liver
Spleen
Kidney
Stomach
Intestine
2.23 ꢃ 0.11
0.06 ꢃ 0.01
0.72 ꢃ 0.04
5.20 ꢃ 0.26
28.87 ꢃ 1.44
10.25 ꢃ 0.51
11.19 ꢃ 0.56
0.55 ꢃ 0.03
0.53 ꢃ 0.03
1.62 ꢃ 0.08
0.04 ꢃ 0.01
0.72 ꢃ 0.04
4.70 ꢃ 0.23
23.12 ꢃ 1.16
6.88 ꢃ 0.34
4.57 ꢃ 0.23
0.29 ꢃ 0.01
0.45 ꢃ 0.02
1.84 ꢃ 0.09
0.04 ꢃ 0.01
0.49 ꢃ 0.02
3.15 ꢃ 0.16
21.50 ꢃ 1.07
5.18 ꢃ 0.26
3.50 ꢃ 0.17
0.46 ꢃ 0.02
0.45 ꢃ 0.02
1.02 ꢃ 0.09
0.03 ꢃ 0.02
0.37 ꢃ 0.02
1.33 ꢃ 0.16
18.21 ꢃ 1.07
4.97 ꢃ 0.26
2.27 ꢃ 0.17
0.30 ꢃ 0.02
0.30 ꢃ 0.02
0.98 ꢃ 0.05
0.03 ꢃ 0.01
0.02 ꢃ 0.02
0.94 ꢃ 0.07
12.07 ꢃ 0.91
3.42 ꢃ 0.25
1.09 ꢃ 0.11
0.04 ꢃ 0.02
0.02 ꢃ 0.02
0.65 ꢃ 0.05
0.01 ꢃ 0.01
0.16 ꢃ 0.01
0.29 ꢃ 0.05
9.62 ꢃ 0.60
1.72 ꢃ 0.17
0.90 ꢃ 0.05
0.23 ꢃ 0.01
0.08 ꢃ 0.01
Fig. 5 99mTc-MBIP ex vivo biodistribution in lung, liver, spleen and kidney at 40 min post i.v. injection with 10 min preadministration of 2.5/5/
10 mg kgꢁ1 PK11195 as blocking agent.
kidney, spleen and lung which were 11.19, 10.25, and 5.20, mice model. 99mTc-MBIP showed uptake in all the TSPO
respectively. 15 min onwards spleen contained highest radio- enriched organs. Blocking percentage in lungs and other
activity aer liver i.e. 6.88, 5.18, 4.97, 3.42, and 1.72% ID per g at peripheral organs was found in the range of 23–60% as per ROI
5 min, 15 min, 30 min, 60 min, 120 min, and 1440 min, derived data (ESI†) which is similar to the ndings of previous
respectively. Lung and kidney also have signicant amount of ligands used in nuclear medicine. Literature reported 99mTc-
radioligand present till 30 min i.e. 3.15 and 3.50% ID per g, labelled 2-quinoline carboxamide ligands showed only signi-
respectively. Thereaer, radioactivity decreased sharply in lungs cant effect in uptake of lung but not in remaining tissues on pre-
with 25% to 5.5% of uptake at 5 min from 60 min to 1440 min. administration of PK11195.25 PET TSPO ligands [18F]-DPA-714 17
Similar trend was observed in kidney i.e. 20% of the uptake at and [18F]VC701 42 showed blocking in the range of 46–72% and
5 min in 60 min and subsequent reduction to 8% in 1440 min. 36–88%, respectively.
The biodistribution pattern revealed that the radioactivity is
TSPO enriched peripheral organs have shown blocking but it
mainly localized in TSPO-expressing organs such as lung, liver, was not concentration dependent for all (Fig. 5), which could be
spleen, and kidney. The good uptake of radioactivity in requisite attributed to small sample size or staging of inamed condi-
organs proves it as TPSO targeting ligand. In literature, highest tion. Further studies with different animal models may clarify
uptake in liver, aer intestine, has been reported for 99mTc the saturation trend once blocked with cold reference like
labelled 2-quinoline carboxamide analogs.25 99mTc-MBIP PK11195.
demonstrated negligible uptake in intestine which is desir-
able for TSPO ligand.
In summary, in silico studies on modied MBMP skeleton
followed by facile synthesis and biological evaluation were
In brain, small uptake of 0.06% ID per g at 5 min was carried out for TSPO target for SPECT application.
observed which decreased to 0.01% at the last time point of
observation. The small % ID in brain could be attributed to
moderate expression of TSPO in the healthy brain. Maximum
4. Conclusion
quantity of radioactivity in liver showed that the clearance of the This work resulted in the synthesis of a new TSPO ligand 2-(2-(5-
radioligand is through hepatobiliary route.
bromo benzoxazolone)acetamido)-3-(1H-indol-3-yl)propanoate
The saturation aspect of TSPO binding site by 99mTc-MBIP (MBIP) for diagnostic application using SPECT. The ligand
has been studied by blocking with PK11195 on lung inamed binding interaction with TSPO was calculated through MM
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analysis and subsequently synthesized in four steps. MBIP 15 W. C. Kreisl, M. Fujita, Y. Fujimura, N. Kimura, K. J. Jenko,
formed a stable complexation with 99mTc which remained >91%
stable at 24 h in serum alongwith favorable ex vivo bio-
distribution in TSPO expressing organs. Stable nature of
P. Kannan, J. Hong, C. L. Morse, S. S. Zoghbi,
R. L. Gladding, S. Jacobson, U. Oh, V. W. Pike and
R. B. Innis, NeuroImage, 2010, 49, 2924–2932.
complex, high uptake at early time points in desired organs with 16 T. Yamasaki, K. Kumata, K. Yamamoto, A. Hatori, M. Takei,
fast kinetics proved MBIP as potential candidate for SPECT.
Further studies are required to prove its efficiency as TSPO
ligands for human clinical application.
Y. Nakamura, S. Koike, K. Ando, K. Suzuki and M. R. Zhang,
Nucl. Med. Biol., 2009, 36, 801–809.
17 C. Vicidomini, M. Panico, A. Greco, S. Gargiulo,
A. R. D. Coda, A. Zannetti, M. Graman zini, G. N. Roviello,
M. Quarantelli, B. Alfano, B. Tavitian, F. Dolle,
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Acknowledgements
We extend special thanks to Dr A. K. Mishra, Head, Division of
Cyclotron and Radiopharmaceutical Science, INMAS, Delhi, for 18 A. K. Tiwari, J. Yui, M. Fujinaga, K. Kumata, Y. Shimoda,
providing us the facility to carry out experiments. We thank Dr
Anshoo Gautam, Scientist D, INMAS for her support to develop
T. Yamasaki, L. Xie, A. Hatori, J. Maeda, N. Nengaki and
M. R. Zhang, J. Neurochem., 2014, 129(4), 712–720.
animal model. This work was supported by INMAS, Delhi under 19 A. K. Tiwari, M. Fujinaga, J. Yui, T. Yamasaki, L. Xie,
Task Project ST/15-16/INM-03. There is no conict of interest
among authors of this manuscript.
K. Kumata, A. K. Mishra, Y. Shimoda, A. Hatori, B. Ji,
M. Ogawa, K. Kawamura, F. Wang and M. R. Zhang, Org.
Biomol. Chem., 2014, 12(47), 9621–9630.
20 A. K. Tiwari, B. Ji, M. Fujinaga, T. Yamasaki, L. Xie, R. Luo,
Y. Shimoda, K. Kumata, Y. Zhang, A. Hatori, J. Maeda,
M. Higuchi, F. Wang and M. R. Zhang, Theranostics, 2015,
5(9), 961–969.
21 A. K. Tiwari, J. Yui, Y. Zhang, M. Fujinaga, T. Yamasaki,
L. Xie, Y. Shimoda, K. Kumata, A. Hatori and M. R. Zhang,
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