Modified benzoxazolone derivative as 18-kDa TSPO ligand

Article abstract of DOI:10.1111/cbdd.12971

We have synthesized six new congeners of acetamidobenzoxazolone for Translocator Protein [18 kDa, TSPO] imaging. The best in vitro binding affinity (10.8?±?1.2?nm) for TSPO was found for N-methyl-2-(5-(naphthalen-1-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-phen

Full text of DOI:10.1111/cbdd.12971

DR. ANJANI K TIWARI (Orcid ID : 0000-0001-6271-3055)
Received Date : 16-Jun-2016
Revised Date : 20-Jan-2017
Accepted Date : 13-Feb-2017
Article type : Research Article
Modified benzoxazolone derivative as 18 kDa TSPO Ligand
a,b
a
a
c
Neelam Kumari, Nidhi Chadha , Pooja Srivastava , Lokesh Chandra Mishra ,
b
a
a
Sunita Bhagat , Anil K Mishra and Anjani K. Tiwari *
a
Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and
Allied Sciences, Defence Research & Development Organization, Brig S K Mazumdar Road, Delhi-
110054, India.
b
Organic Synthesis Research Laboratory, Department of Chemistry, A.R.S.D. College, University of
Delhi, New Delhi-110021, India.
Department of biological Sciences, Hans Raj College, North Campus, University of Delhi-110054,
c
Delhi
*Corresponding Author
Anjani K. Tiwari: Division of Cyclotron and Radiopharmaceutical Sciences, Institute of
Nuclear Medicine and Allied Sciences, Defence Research & Development Organization, Brig
S
K
Mazumdar Road, Delhi-110054, India; Phone: +91-11-23905387; E-mail:
anjanik2003@rediffmail.com
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12971
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*Co-Corresponding Author
Anil K Mishra: Division of Cyclotron and Radiopharmaceutical Sciences, Institute of
Nuclear Medicine and Allied Sciences, Defence Research & Development Organization, Brig
S
K
Mazumdar Road, Delhi-110054, India; Phone: +91-11-23905117; E-mail:
akmishra63@gmail.com
Sunita Bhagat: Organic Synthesis Research Laboratory, Department of Chemistry, A.R.S.D.
College, University of Delhi, New Delhi-110021, India
sunitabhagat28@rediffmail.com
Abstract
We have synthesized six new congeners of acetamido-benzoxazolone for Translocator
Protein [18 kDa, TSPO] imaging. The best in vitro binding affinity (10.8 ± 1.2 nM) for TSPO
was
phenylacetamide, (NBMP). NBMP was synthesised by suzuki coupling reaction between 2-
5-bromo-2-oxo-1,3-benzoxazol-3(2H)-yl)-N-phenylacetamide and napthalene-1-boronic
found
for
N-methyl-2-(5-(naphthalen-1-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-
(
acid. Computational docking and simulation studies showed not much impact of intersubject
variability on binding which is one of the major drawbacks of several TSPO ligands. These
findings suggested that NBMP may become a promising marker for visualization of
neuroinflammation via TSPO targeting.
Keywords: Translocator Protein (18kDa, TSPO), Neuroinflammation, Microglia activation
and Benzoxazolone
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Introduction
Development of ligand for Translocator Protein [18 kDa, TSPO] to study its role in
activation of glial cells in the injured brain as well as in neurodegenerative diseases is one of
the critical issues of biomedical imaging [1-3]. Most of PET radioligands bind TSPO with
high affinity in small animals; however, their specific binding is reduced in human brain.
Moreover, some ligands such as PBR28 exhibited mixed affinity with TSPO in “binder” and
“
non-binder” human subjects [4-6]. Therefore a new candidate as TSPO ligand with different
structural skeleton is desirable. Recently, we have synthesized a new series of PET ligands
-[5-(4- Methoxyphenyl)- 2-oxo-1,3-benzoxazol-3(2H)-yl] -N-methyl-N- phenyl acetamide
2
11
labeled with carbon-11 ([ C]MBMP) and two new 18F-labelled molecules, 2-[5-(4-
18
18
18
18
[
F]fluoroethoxy- ([ F]FEBMP) and 2-[5-(4-[ F]fluoropropyloxy- ([ F]FPBMP) -2-oxo-
1
,3-benzoxazol-3(2H)-yl)-N-methyl-N-phenyl acetamide (Figure 1) and evaluated in vitro
and in vivo for imaging of TSPO expression in brain [7-9]. The in vitro autoradiography and
in vivo PET study demonstrated appropriate specific binding and higher binding potential of
1
1
[
C]MBMP toward TSPO in the ischemic rat brain but moderate kinetics in primates
unpublished data).
(
Although these second-generation TSPO radioligands have improved signal-to-noise ratios,
and minimal intersubject variability for binding. A rs6971 polymorphism in the gene that
encodes TSPO is fully responsible for such difference in affinity. This polymorphism leads to
an amino acid substitution, from alanine to threonine (Ala147Thr), and resulted into three
distinct binding affinity classes; high-affinity binder (HAB, Ala/Ala amino acid at 147
position of TSPO protein, major genotype) and low-affinity binder (LAB, Thr/Thr amino
acid at 147 position of TSPO protein, minor genotype) express a single binding site for TSPO
high and low affinity, respectively, whereas the mixed-affinity binder (MAB, Ala/Thr amino
acid) expresses both binding sites[10-11].
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In our previous studies post-mortem human brains (Two HABs and two LABs) were used for
in vitro autoradiographic studies for a correlation analysis [9].Autoradiography with known
11
11
ligands [ C]AC-5216 and [ C]DAA1106 showed decreased binding in LAB-1 compared to
1
1
(
R)[ C]PK11195, and this is also consistent with previous publications. Moreover, there was
an excellent correlation (R = 0.98, p < 0.01) between the ratio of binding of TSPO ligands for
HAB-1 to LAB-1 (RB(L/H)). The presence of unlabelled PK11195 caused significant decrease
18
18
in the binding of [ F]FEBMP in both human brains, and the R
value of [ F]FEBMP
Ki(L/H)
were estimated to be 0.9, suggesting that binding of MBMP and their derivatives have very
little affect by TSPO rs6971 polymorphism [9].
Here we report synthesis and evaluation of modified benzoxazolone derivatives which may
be either equivalent or better than MBMP in terms of binding affinity towards TSPO. The
intersubject variability issue was also assessed for the best compound through molecular
docking and MD simulation with TSPO, along with natural polymorph (Ala147→Thr
polymorphism, rs6971) TSPO protein.
Results
All the six compounds were synthesised as per schemes 1-3. Cyclization of commercially
o
available 2-amino phenol derivatives (1 and 9) with CDI in THF at 80 C for 4 - 6 hrs
afforded benzo[d]oxazol-2(3H)-one derivatives (2 and 10) in 91 - 99 % yields. To attain
convergent methodology in scheme at second phase we had coupled 2-bromoacetyl bromide
with N-methyl aniline by using triethylamine in DCM to afford 2-bromo-N-methyl-N-
phenylacetamide (4) in 66 % yield. Subsequently benzoxazolone derivatives (2 or 10) and 4
were coupled together by using potassium carbonate in N,N-dimethylformamide (DMF)
solvent at room temperature to give N-methyl-2-(2-oxobenzo[d]oxazol-3(2H)-yl)-N-
phenylacetamide (5 and 13) in 53 - 69 % yields. Further, reduction of nitro group in
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compound 5 and 13 was achieved by Pd-C under hydrogen atmosphere in methanol solvent
to afford amine compound 6 and 14 in 70 - 73 % yields. Compound 6 and 14 was further
2 3
alkylated with 2-(bromomethyl)naphthalene by using K CO in DMF which ultimately leads
to the mixture of mono and disubstituted product. Desired monosubstituted product 7 and 15
was successfully isolated and purified by flash column chromatography with 50 - 55 %
yields. The ORTEP diagram of compound 8 is shown as crystallographically independent
unit. The detailed crystallographic data of compound 7 has been deposited in the Cambridge
Crystallographic Data Centre with supplementary publication number 1051947. Compound 8
was also synthesized from precursor 6 by refluxing it with 5,6-dimethoxy-2-oxo-2H-
chromene-4-carbaldehyde in ethanol solvent and catalytic amount of acetic acid.
In scheme 3 synthesis of N-methyl-2-(5-(naphthalen-1-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-
phenylacetamide has been shown by employing Suzuki coupling between 2-(5-bromo-2-
oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide 16 and 4,4,5,5-tetramethyl-2-
(naphthalen-1-yl)-1,3,2-dioxaborolane.
In vitro binding assay
i
We used rat brain homogenates to determine the inhibition constants (K ) of synthesised
congeners for TSPO and central benzodiazepine receptor (CBR). As shown in Table 1, 17 or
NBMP bound TSPO with highest (nanomolar) affinity, confirming the results of the
computer modelling study. The computed lipophilicities at pH = 7.4 (cLogD) is also
calculated, and the corresponding experimental values (LogD) were determined using the
shake flask method.
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Computational studies
Recently reported three dimensional structure of mitochondrial TSPO in complex with the
ligand PK11195 (gold standard of TSPO binding) has opened the new avenues to direct the
design and development process for effective TSPO ligands and targeting its variable binding
profiles. TSPO has many diagnostic ligands of which PK11195 is the best characterized
ligand and was found to bind TSPO in all species with equal affinity. Recently the solution
structure of PK11195 has been elucidated to understand its interactions with hydrophobic
sites of TSPO [13-15].
In present work, we have designed the computational protocol to identify the potential
preferred binding sites for TSPO known ligands which can be further differentiated as high
affinity binding site and low binding site on monomer and dimer TSPO. 2MGY, which is
three dimensional high-resolution structure of mammalian TSPO, is reconstituted in micelles
in complex with its high-affinity ligand PK11195. In this PK11195 is bound by a tight bundle
of five transmembrane helices that form a hydrophobic pocket. Hence, firstly we have taken
the monomeric identified crystal structure of TSPO, 2MGY and explored the binding site of
PK11195 and NBMP to understand their prospective interactions with TSPO.
In the monomeric TSPO, we have explored other top-binding sites on the basis of various
parameters such as size, site score, drug binding score, hydrophobic/hydrophilic contents and
donor/acceptor balance (supplementary data).
The docking studies were performed for PK11195, MBMP and NBMP at all the five sites of
2MGY and mutant 2MGY (Ala147Thr mutation) on the basis of above mentioned parameters
as shown in table 2.
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Discussion
T. Fukaya and group were the first to introduce benzoxazolone class of TSPO ligand [12].
Further in the process of optimization in pharmacophoric model structure they made some
important conclusions like - 1) Tertiary acetamide is a necessary structural moiety required
for TSPO binding, 2) Alkyl chain elongation between amide group and benzoxazolone
skeleton will reduce the TSPO affinity and 3) Hydrophobic substitution at C-5 position is the
preferred substitution on benzoxazolone ring. By keeping all these essential perspective in
common we have designed six new congeners of 2-oxobenzo[d]oxazol-3(2H)-yl)-N-
phenylacetamide as novel candidates which belong to the pre-existing benzoxazolone class of
TSPO ligand. The structures of all synthesized intermediates and compounds were
1
13
unambiguously established on the basis of their spectral data ( H-, C- NMR spectra, IR
spectra and HRMS) analysis.
The biological in vitro data confirmed that MBMP and compound no 17, NBMP both have
similar in vitro binding efficacy (below 10 nM). For MBMP we cannot proceed in higher
animals due to kinetic issues. Here NBMP has shown a promising aspect through
computational analysis in terms of TSPO rs6971 polymorphism. Initially the pharmacokinetic
property of NBMP has been found better than the native compound with improved
bioavailability and plasma clearance. The metabolic stability of this ligand was found much
suitable in human liver and rat liver S-9 fraction (buffered solution containing the fraction
from a pool of livers).
The ligands, NBMP showed higher Gscore of -11.566 at site 1 and -10.950 at site 2 in
comparison to PK11195 and MBMP for 2MGY binding (table 2). In similar terms, the known
Ala147Thr mutation has been introduced and mutated TSPO structure (2MGY mutant) was
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modelled for studying the binding patterns of ligands in which NBMP showed higher Gscore in
comparison to PK11195 and MBMP. The binding site residues were found to be conserved
among the mutated 2MGY and thus showed the impact of Ala147Thr mutation.
Although it has been reported that TSPO binds ligands monomers, it also forms dimer and
oligomers. Therefore from the literature information dimer of TSPO was modeled on the
basis of crystalized 2MGY 3D structure. The dimeric 3D structure of TSPO was determined
for Rhodobacter sphaeroides and reconstituted it as helical crystal by using electron
cryomicroscopy. The structure at the 10Å resolution showed the arrangement of ten helixes
as bundles within transmembrane region [16-18] and large dimeric interface between them
with the monomers aligned as antiparallel and parallel orientations where TSPO might be
functional. The protein-protein docking module of Schrödinger has been used for designing
dimeric conformations of TSPO; parallel TSPO dimer (abbreviated as P) and antiparallel
(marked as AP) TSPO. For AP dimer, NBMP showed higher Gscore of -11.293 at site 1 and -
7.286 at site 2 in comparison to PK11195 and MBMP for 2MGY (table 3). Similarly for AP
dimer mutant Gscore was -10.880 at site 1 and it was -9.536 at site 2. The other binding sites
were not taken into consideration as the score was only good in case of NBMP but not for the
known standard PK11195.
In 2D and 3D interaction diagram for 2MGY (AP-dimer), NBMP as well as the references
ligand PK11195. NBMP was involved in the π-π stacking interactions with Trp143 (figure 2).
The similar trend was observed at site 2 of the Ala147Thr AP-TSPO mutant with the slight
decrease in the binding score due to presence of either no interactions or only hydrogen
bonding or π-π stacking with Trp93 in all the ligands. This has been also confirmed in our
experimental studies for PK11195 and MBMP analogues [9].
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Similarly in case of parallel TSPO dimer ( P), the NBMP shows -11.714 and -11.697 at site 1
and site 2, respectively (table 4). In case of site 1 of P-TSPO dimer, the conformation of
molecule is different as in case of monomer and AP-dimer TSPO. Although the binding
pocket is still conserved with residues Ala23, Val26, Leu49, Ala50, Ile52, Trp107, Ala110,
Leu114, Ala147 and Leu150 but NBMP makes the cation-π interaction with Arg27 and
hydrogen bond with the side chain of Trp53 which was not found in earlier binding poses
(figure 3). At site 2 of P-TSPO dimer, the binding pose at this conformation of NBMP
enhances the binding score as compared to previous conformation due to π-π stacking
interactions with Phe99 which was not found earlier.
Conclusion A new derivative from acetamidobezoxazolone, NBMP has been synthesised
and evaluated for its in vitro affinity. Computational analysis showed its low possibility of
variation in the affinity with respect to different subjects. Further, primate’s study is required
to evaluate the usefulness of this compound. Subsequently, with these data we can
characterize this ligand in human binder and non- binder brain, specifically for the high
specific binding in non-binder brains.
Experimental Section
Melting points were determined on Buchi M-560 instrument and were uncorrected. The IR
spectra were recorded on a Perkin-Elmer model 2000 FT-IR spectrometer by making KBr
1
13
disc for solid samples and thin film for oils. The H- and C- NMR spectra were recorded at
Jeol alpha-400 spectrometer at 400 and 100.6 MHz, respectively using TMS as internal
standard. The chemical shift values are on δ scale and the coupling constants (J) are in Hz.
The mass spectra analyses were done on a 6530 Q-TOF instrument from Agilent
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Technologies. Analytical TLCs were performed on precoated Merck silica-gel 60F254 plates;
the spots were detected under UV light. Silica gel (100-200 mesh) was used for column
chromatography.
General procedure for the synthesis of Nitro-benzoxazol-2(3H)-one derivatives (2 and
10). To a solution of 2-amino phenol derivatives 1 (0.8 g, 5.19 mmol) or 9 (0.8 g, 4.26 mmol)
in THF (30 mL) was added 1,10-carbonyldiimidaziole (1.42 g, 6.23 mmol) at room
temperature. The mixture was stirred at reflux for 2 hrs and cooled to room temperature. The
reaction was then quenched by adding 2 M HCl solution, and the mixture was extracted with
EtOAc. The organic layer was washed with brine and dried over anhydrous sodium sulphate.
After filtration, the solvent was removed in vacuo to give 2 and 10 in 91 - 99 % yields.
5
0
1
-nitro-1,3-benzoxazol-2(3H)-one (2). It was obtained as yellow solid (0.928 g, 99 %). R =
f
o
.7 (40 % ethyl acetate in hexane); M. Pt.: 98-99 C; IR (thin film) νmax: 3127, 1785, 1678,
-1 1
522, 1473 and 1344 cm ; H NMR (DMSO-d , 400 MHz): δ 7.92 (1H, dd, J = 2.4 and 8.8
6
13
Hz), 7.76 (1H, d, J = 2.8 Hz) and 7.36 (1H, d, J = 9.2 Hz); C NMR (DMSO-d
6
, 100.6
MHz): δ 154.76, 148.27, 144.20, 131.52, 119.27, 110.35 and 105.67; HR-ESI-TOF-MS: m/z
+
+
1
5
9
1
7 4 2 4
79.0083 ([M-H] ), calcd. for [C H N O -H] 179.0087.
-chloro-7-nitrobenzo[d]oxazol-2(3H)-one (10). It was obtained as yellow solid (0.828 g,
o
1 %). R
f
= 0.4 (40 % ethyl acetate in hexane); M. Pt.: 239-241 C; IR (thin film) νmax: 3127,
-1 1
968, 1763, 1527 and 1341 cm ; H NMR (DMSO-d
6
, 400 MHz): δ 7.85 (1H, d, J = 2.4 Hz)
1
3
and 7.58 (1H, d, J = 2.0 Hz); C NMR (DMSO-d
6
, 100.6 MHz): δ 153.48, 136.77, 134.57,
+
1
31.16, 127.53, 116.04 and 115.47; HR-ESI-TOF-MS: m/z 212.9706 ([M-H] ), calcd. for
+
7 3 2 4
[C H ClN O -H] 212.9709.
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2
-Bromo-N-methyl-N-phenylacetamide (4). Bromoacetyl bromide (1.95 mL, 22.39 mmol)
in dichloromethane (10 mL) was added dropwise to the solution of N-methylaniline (2.03
mL, 18.66 mmol) and triethylamine (3.9 mL, 27.99 mmol) in dichloromethane (30 mL) at 0
o
C. The solution was allowed to warm to ambient temperature and was stirred for 2 hrs, then
was poured into a separating funnel and extracted with DCM. The solution was dried over
anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The residue
was purified by flash chromatography on silica gel by using ethyl acetate in hexane as
f
gradient solvent system to afford 4 (2.8 g, 66 %) as white crystalline solid. R = 0.3 (20 %
o
ethyl acetate in petroleum ether); M. Pt.: 46-47 C; IR (thin film) νmax: 1667, 1495, 1379 and
-1 1
6
99 cm ; H NMR (CDCl , 400 MHz): δ 7.48-7.40 (3H, m), 7.29 (2H, d, J = 7.6 Hz), 3.67
3
1
3
(
2H, s) and 3.31 (3H, s); C NMR (CDCl
3
, 100.6 MHz): δ 166.50, 143.05, 129.97, 128.49,
+
1
26.98, 38.05 and 26.72; HR-ESI-TOF-MS: m/z 228.0048 ([M+H] ), calcd. for
+
9
[C H10BrNO+H] 228.0019.
General procedure for the synthesis of N-methyl-2-(2-oxobenzo[d]oxazol-3(2H)-yl)-N-
phenylacetamide nitro-derivatives (5 and 13). To a well-stirred and ice-cool solution of
benzoxazol-2(3H)-one derivatives 2 (0.4 g, 2.22 mmol) or 10 (0.4 g, 1.87 mmol) in DMF (20
mL) anhydrous K
2
CO
3
(0.607 g, 6.66 mmol) was added followed by the addition of 4 (0.713
o
g, 2.66 mmol). The mixture was stirred at 0 C for 30 min and then at room temperature for 2
hrs. Reaction mixture was cooled and poured into ice water which results into formation of
yellow colour precipitate. The precipitate was then collected by vacuum filtration, washed
and dried in oven. The residue was purified by silica gel column chromatography using ethyl
acetate in hexane as gradient solvent system to afford 5 and 13 in 53 - 69 % yields.
N-methyl-2-(5-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-phenylacetamide (5). It was
f
obtained as pale yellow solid (0.726 g, 69 %). R = 0.4 (30 % ethyl acetate in hexane); M. Pt.:
o
-1
1
1
67–168 C; IR (thin film) νmax: 1798, 1673, 1530, 1493, 1344 and 1262 cm ; H NMR
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6
(DMSO-d , 400 MHz): δ 8.33 (1H, s), 8.11 (1H, dd, J = 2.8 and 8.8 Hz),7.62-7.46 (6H, m),
13
4
.51 (2H, s) and 3.23 (3H, s); C NMR (DMSO-d
6
, 100.6 MHz): δ 164.70, 153.58, 146.12,
1
44.03, 141.78, 132.45, 129.97, 128.34, 127.31, 118.94, 110.10, 105.76, 44.11 and 37.16;
+
+
HR-ESI-TOF-MS: m/z 328.0925 ([M+H] ), calcd. for [C16
-(5-chloro-7-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (13). It
was obtained as yellow solid (0.359 g, 53 %). R = 0.5 (30 % ethyl acetate in hexane); M. Pt.:
13 3 5
H N O +H] 328.0928.
2
f
o
-1 1
1
6
72-174 C; IR (thin film) νmax: 1801, 1670, 1542,1373 and 1339 cm ; H NMR (DMSO-d ,
1
3
4
00 MHz): δ 7.98-7.93 (2H, m), 7.58-7.32 (5H, m), 4.45 (2H, s) and 3.23 (3H, s); C NMR
(
DMSO-d
6
, 100.6 MHz): δ 164.23, 152.89, 141.64, 135.26, 135.01, 131.49, 130.03, 128.45,
+
1
27.97, 127.39, 116.75, 115.96, 44.38 and 37.21; HR-ESI-TOF-MS: m/z 362.0520 ([M+H] ),
+
calcd. for [C16
H
12ClN
3
O
5
+H] 362.0538.
General procedure for the synthesis of N-methyl-2-(2-oxobenzo[d]oxazol-3(2H)-yl)-N-
phenylacetamide amino-derivatives (6 and 14). To a solution of 5 (0.3 g, 0.917 mmol) and
13 (0.3 g, 0.829 mmol) in MeOH (20 mL) was added half mole equivalent 10 % palladium
on charcoal (97 mg). The reaction flask was flushed with hydrogen and the mixture was
stirred for 2 hrs at room temperature. At completion of reaction, the catalyst was filtered off
through celite, the filtrate was evaporated under reduced pressure to afford 6 and 14 in 70-73
%
yields.
-(5-amino-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (6). It was
obtained as white solid (0.191 g, 70 %). R = 0.3 (5 % methanol in dichloromethane); M. Pt.:
2
f
o
-1 1
2
68-270 C; IR (thin film) νmax: 3358, 1752, 1661 and 1220 cm ; H NMR (DMSO-d
6
, 400
MHz): δ 7.52-7.27 (5H, m), 6.94 (1H, d, J = 8.4 Hz), 6.29-6.27 (2H, m), 5.13 (2H, brs), 4.21
1
3
(
2H, s) and 3.20 (3H, s); C NMR (DMSO-d
6
, 100.6 MHz): δ 165.20, 154.53, 145.69,
1
41.93, 133.20, 131.66, 130.09, 128.21, 127.35, 109.68, 107.12, 95.16, 43.21 and 37.22; HR-
+
+
15 3 3
ESI-TOF-MS: m/z 298.1186 ([M+H] ), calcd. for [C16H N O +H] 298.1186.
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2-(7-amino-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (14). It was
obtained as white solid (0.18 g, 73 %). R = 0.5 (5 % methanol in dichloromethane); M. Pt.:
f
o
-1 1
2
66-268 C; IR (thin film) νmax: 3347, 1773, 1656 and 1123 cm ; H NMR (DMSO-d
6
, 400
MHz): δ 7.54-7.43 (5H, m), 6.95 (1H, t, J = 8.4 Hz), 6.61-6.52 (2H, m), 4.28 (4H, brs) and
1
3
3
1
2
.18 (3H, s); C NMR (DMSO-d
6
, 100.6 MHz): δ 164.94, 142.01, 131.98, 130.00, 129.80,
28.90, 128.29, 127.28, 124.09, 110.84, 99.40, 43.48 and 37.14; HR-ESI-TOF-MS: m/z
+
+
15 3 3
98.1187 ([M+H] ), calcd. for [C16H N O +H] 298.1186.
General procedure for the synthesis of N-methyl-2-(5-((naphthalen-2-ylmethyl)amino)-2-
oxobenzo[d]oxazol-3(2H)-yl)-N-phenylacetamide (7) and N-methyl-2-(7-((naphthalen-2-
ylmethyl)amino)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-phenylacetamide (15). To a solution
of 2-(5-amino-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (6) (0.2 g, 0.67
mmol) and 2-(7-amino-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (14)
2 3
(0.2 g, 0.67 mmol) in DMF (15 mL) were added anhydrous K CO (0.28 g, 2.06 mmol) and
2-(bromomethyl)naphthalene (0.152 g, 0.68 mmol) and reaction mixture was continued to
stirred at room temperature for 3 hrs. The progress of reaction is monitored by TLC. On
completion of reaction the contents were poured over ice cold water and extracted with ethyl
acetate (3 x 50 mL). The combined organic layer was washed with brine (2 x 50 mL) and was
2 4
dried over anhydrous Na SO . The excess of solvent was removed under reduced pressure
and the residue thus obtained was purified by silica gel column chromatography using ethyl
acetate in hexane as gradient solvent system to afford 7 and 15 in 50-55 % yields.
N-methyl-2-(5-((naphthalen-2-ylmethyl)amino)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-
f
phenylacetamide (7). It was obtained as white crystalline solid (0.147 g, 50 %). R = 0.2 (30
o
%
ethyl acetate in hexane); M. Pt.: 180-181 C; IR (thin film) νmax: 3382, 1761, 1633 and
-1 1
1
224 cm ; H NMR (DMSO-d
6
, 400 MHz): δ 7.89-7.83 (4H, m), 7.55-7.41 (7H, m), 6.99
(1H, d, J = 9.2Hz), 6.35-6.33 (3H, m), 4.43 (2H, d, J = 6.0Hz), 4.18 (2H, s) and 3.10 (3H, s);
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13
C NMR (DMSO-d , 100.6 MHz): δ 164.85, 154.52, 146.09, 141.97, 137.68, 133.30,
6
1
1
4
32.96, 132.17, 131.92, 130.01, 128.29, 127.87, 127.50, 127.47, 127.80, 126.07, 125.84,
25.50, 125.25, 109.77, 105.69, 93.55, 47.31, 43.13 and 37.07; HR-ESI-TOF-MS: m/z
+
+
23 3 3
38.1818 ([M+H] ), calcd. for [C27H N O +H] 438.1812.
N-methyl-2-(7-((naphthalen-2-ylmethyl)amino)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-
phenylacetamide (15). It was obtained as white crystalline solid (0.162 g, 55 %). R = 0.4
f
o
(
30 % ethyl acetate in petroleum ether); M. Pt.: 179–181 C; IR (thin film) νmax: 3382, 1761,
-1 1
1
633 and 1224 cm ; H NMR (DMSO-d , 400 MHz): δ 7.87-7.84 (5H, m), 7.56-7.43 (6H,
6
m), 6.84 (1H, t, J = 8.4 Hz), 6.62 (1H, t, J = 6.4 Hz), 6.35 (2H, d, J = 8.4 Hz), 4.56 (2H, d, J
1
3
=
6
6.4 Hz), 4.26 (2H, s) and 3.20 (3H, s); C NMR (DMSO-d , 100.6 MHz): δ 166.42,
1
1
42.79, 141.85, 137.70, 132.84, 132.32, 132.04, 131.55, 129.93, 128.64, 128.20, 127.81,
27.34, 127.23, 125.97, 125.47, 125.40, 124.87, 124.14, 122.86, 106.16, 97.45, 46.48, 43.51
+
+
and 37.09; HR-ESI-TOF-MS: m/z 438.1821 ([M+H] ), calcd. for [C27
23 3 3
H N O +H]
438.1812.
Synthesis of (Z)-2-(5-(((5,6-dimethoxy-2-oxo-2H-chromen-4-yl)methylene)amino)-2-
oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (8)
2-(5-amino-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide (0.10 g, 0.34
mmol) and 5,6-dimethoxy-2-oxo-2H-chromene-4-carbaldehyde (0.079 g, 0.34 mmol) were
dissolved together in ethanol (25 mL) followed by the addition of acetic acid (0.05 mL) as a
catalyst. The reaction mixture was stirred at reflux for 5 hrs and completion of reaction was
confirmed by TLC. The reaction mixture was cooled and extracted with ethyl acetate (3 x 50
2 4
mL). After drying the ethyl acetate over anhydrous Na SO the organic layer were
concentrated at reduced pressure. The residue thus obtained was purified by silica gel column
chromatography using ethyl acetate in hexane as gradient solvent system to furnish 8 (0.115
o
g, 67 %) as yellow solid. R = 0.5 (2 % methanol in chloroform); M. Pt.: 270-271 C; IR (thin
f
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-1 1
film) νmax: 1787, 1715, 1672 and 1294 cm ; H NMR (DMSO-d
6
, 400 MHz): δ 8.91 (1H, s),
8.59 (1H, d, J = 8.8 Hz), 7.58-7.44 (7H, m), 7.24 (1H, dd, J = 2.0 and 8.0Hz), 7.18 (1H, d, J
13
=
5.2Hz), 6.85 (1H, s), 4.41 (2H, s), 3.95 (3H, s), 3.86 (3H, s) and 3.23 (3H, s); C NMR
(
DMSO-d , 100.6 MHz): δ 164.76, 159.80, 157.57, 155.30, 153.99, 147.86, 146.30, 145.48,
6
1
1
41.31, 135.25, 132.37, 129.99, 128.33, 127.32, 122.05, 155.59, 111.01, 110.67, 108.98,
+
03.55, 60.71, 56.31, 43.64, 37.16 and 28.90;HR-ESI-TOF-MS: m/z 514.1601 ([M+H] ),
+
calcd. for [C28
H
23
N
3
O
7
+H] 514.1609.
N-methyl-2-(5-(naphthalen-1-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-phenylacetamide,
NBMP or (17)
A mixture of 2-(5-bromo-2-oxo-1,3-benzoxazol-3(2H)-yl)-N-phenylacetamide (1.44 g, 4.0
mmol) and napthalene-1-boronic acid (826 mg, 4.8 mmol), K CO (829 mg, 6.0 mmol) and
2
3
tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4 (462 mg, 0.4 mmol) in 1,4-
dioxane/water (3/1, 10.0 mL) was heated at 90°C under nitrogen atmosphere. The reaction
mixture was stirred for 6 h and cooled to room temperature. Subsequently, the mixture was
extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous
sodium sulphate. After filtration, the solvent was removed in vacuo, and the residue was
purified by silica gel column chromatography using hexane/EtOAc (2/1, v/v) as the eluent to
give NBMP (72.5% yield) as white solid. MP 155–160°C. Chemical purity: >99% by HPLC:
Capcell Pack UG80 C18 column, 4.6 mm i.d. × 250 mm; MeCN/H
2
O/Et
3
N, 65/35/0.01
), 10.4 min. H NMR (400 MHz,
CDCl ) d 7.56–7.49 (6H, m), 7.48–7.41 (3H, m), 7.39–7.32 (3H, m), 7.30 (1H, dd, J = 8.3,
1
(v/v/v); flow rate, 1.0 mL/min; λuv, 254 nm; retention time (t
R
3
1.7 Hz), 7.24 (1H, d, J = 8.3 Hz),7.04 (1H, d, J = 1.2 Hz), 4.36 (2H, s), 3.58 (3H, s)
HRMS (ESI) m/z calcd for [C26 ] 396.14; found 396.8.
20 2 3
H N O
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Supplementary data: Site map data of 2MGY in both monomer and dimer (parallel and
anti-parallel) are shown in Table S1-S3 while RMSD/RMSF deviation along with fractions
of interactions in MBNP are shown in figure S1-S6.
The crystal data of compound 8 is mentioned in table S4 and figure S7.
Acknowledgements
This work was supported by INMAS, Delhi under Task Project ST/15-16/INM-03.The first
author also would like to thanks University Grants Commission, India for senior research
fellowship.
This is certified that, there is no issue of conflict between the authors of this manuscript.
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Figure legends
Figure 1. Structures of known acetamidobenzoxazolone TSPO ligands
th
(
Benzoxazolone ring has been substituted at 5 position by phenoxy methyl/ phenoxy fluoro ethyl / phenoxy
fluoro propyl)
Figure2: Two-Dimensional (2D) and Three-Dimensional (3D) ligand interaction diagram of
NBMP with site 1 and site 2 of AP dimer TSPO
Figure 3: Two-Dimensional (2D) and Three-Dimensional(3D) ligand-protein interaction
diagram of NBNP at site 1 and site 2 of P dimer TSPO.
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Scheme 1. Synthetic route
oxobenzo[d]oxazol-3(2H)-yl)-N-phenylacetamide (7) and (Z)-2-(5-(((5,6-dimethoxy-2-oxo-
H-chromen-4-yl)methylene)amino)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-
of N-methyl-2-(5-((naphthalen-2-ylmethyl)amino)-2-
2
phenylacetamide (8).
Scheme 2. Synthetic route of N-methyl-2-(7-((naphthalen-2-ylmethyl)amino)-2-
oxobenzo[d]oxazol-3(2H)-yl)-N-phenylacetamide (13).
Scheme 3. Synthetic route of N-methyl-2-(5-(naphthalen-1-yl)-2-oxobenzo[d]oxazol-3(2H)-
yl)-N-phenylacetamide (17)
Table legends
Table-1 In vitro binding affinity for TSPO and lipophilicity of compounds
a) Binding affinities represent the mean ± standard error of the mean (SE) of triplicate samples of brain
homogenates.
3
b) Concentration of [ H]-flumazenil was taken at 10 µM.
c) LogD values were measured in octanol/phosphate buffer (pH = 7.4) using the shake flask method (n =
3; maximum range ± 5%).
d) cLogD values were calculated using Pallas 3.4 software.
Table 2: Glide XP docking results (Gscore) of PK11195, MBMP and NBMP with five binding
sites of 2MGY (monomer)
Gscore = 0.05*vdW + 0.15*Coul + Lipo + Hbond + Metal + Rewards + BuryP + RotB + Site
(Where, vdw is the Van der Waals energy, Coul is coulomb energy, Lipo means lipophilic term, Hbond means
Hydrogen-bonding term, BuryP is penalty for buried polar groups, RotB is penalty for freezing rotatable bonds and
site polar interactions in the active site.)
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Table 3: Glide XP docking results of PK11195, MBMP and NBMP with five binding sites of
MGY (antiparallel-dimer) and its mutant
2
(Glide extra-precision (XP) mode combines a scoring function without unfavorable energies during Glide XP
docking, rigid receptor is considered where Van der Waal scaling is performed for nonpolar atoms of receptor or
ligand or both, and reorients the specified hydroxyl groups optimized for hydrogen bonding.)
Table 4: Glide XP docking results of PK1195, MBMP and NBMP with five binding sites of
2MGY (parallel-dimer)
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Lipophilicity
Binding affinity
Binding affinity Experimental
Calculated
(
K ) nM
i
(Ki) nM
Compound
a
b
c
d
TSPO
CBR
LogD
3.4
cLogD
>
>
>
200
100
100
MBMP
3.9 ± 0.6
4.4 ± 0.4
15.2 ± 1.1
18.2 ± 15
18.9 ± 1.5
8.1 ± 0.8
3.3
5.1
3.8
3.2
3.8
3.7
PK11195
3.7
7
8
3.8
>100
3.3
>
100
100
15
3.9
>
NMBP
3.6
Table-1 In vitro binding affinity for TSPO and lipophilicity of compounds
e) Binding affinities represent the mean ± standard error of the mean (SE) of triplicate
samples of brain homogenates.
3
f) Concentration of [ H]-flumazenil was taken at 10 µM.
g) LogD values were measured in octanol/phosphate buffer (pH = 7.4) using the shake
flask method (n = 3; maximum range ± 5%).
h) cLogD values were calculated using Pallas 3.4 software.
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2
MGY
PK11195
-3.966
NBMP
-11.566
-10.950
MBMP
-6.789
-9.658
SITE 1
SITE 2
-9.231
SITE 3
SITE 4
-2.607
-2.878
-3.672
-2.957
-1.176
-9.018
NBMP
-5.117
-5.762
-3.265
-2.764
-2.057
-5.405
MBMP
-5.593
-4.485
-4.707
-3.325
SITE 5
-3.551
PK11195
-4.769
2
MGY Mutant
SITE 1
SITE 2
-
5.05
-5.175
1.975
SITE 3
SITE 4
-
SITE 5
-1.525
-2.899
-4.031
Table 2: Glide XP docking results (Gscore) of PK11195, MBMP and NBMP with five binding
sites of 2MGY (monomer)
Gscore = 0.05*vdW + 0.15*Coul + Lipo + Hbond + Metal + Rewards + BuryP + RotB + Site
(Where, vdw is the Van der Waals energy, Coul is coulomb energy, Lipo means lipophilic term, Hbond means
Hydrogen-bonding term, BuryP is penalty for buried polar groups, RotB is penalty for freezing rotatable bonds and
site polar interactions in the active site.)
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AP Dimer
SITE 1
PK11195
-8.681
NBMP
-11.293
MBMP
-11.371
SITE 2
-6.067
-7.286
-7.058
SITE 3
SITE 4
SITE 5
-5.175
-3.518
-5.428
-3.975
-3.451
-3.898
-3.856
-8.999
-11.252
AP Dimer Mutant
SITE 1
PK11195
-8.871
NBMP
-10.880
MBMP
-10.896
SITE 2
-8.275
-9.536
-10.470
SITE 3
SITE 4
SITE 5
-4.413
-3.968
-5.428
-2.854
-6.562
-4.163
-3.974
-3.975
-6.556
Table 3: Glide XP docking results of PK11195, MBMP and NBMP with five binding sites of
MGY (antiparallel-dimer) and its mutant
2
(Glide extra-precision (XP) mode combines a scoring function without unfavorable energies during Glide XP
docking, rigid receptor is considered where Van der Waal scaling is performed for nonpolar atoms of receptor or
ligand or both, and reorients the specified hydroxyl groups optimized for hydrogen bonding.)
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P Dimer
SITE 1
SITE 2
PK11195
-8.93
NBMP
-11.714
MBMP
-10.659
-
9.655
3.949
-11.697
-5.091
-10.659
-5.495
SITE 3
-
SITE 4
SITE 5
-5.962
-3.415
-4.364
-9.069
NBMP
-6.648
-8.570
-5.495
P Dimer Mutant PK111952
MBMP
-10.404
SITE 1
SITE 2
-9.334
-
9.257
5.316
-9.798
-7.84
-10.204
-7.416
SITE 3
-
SITE 4
SITE 5
-4.398
-4.193
-4.316
-4.529
-5.265
-5.793
Table 4: Glide XP docking results of PK1195, MBMP and NBMP with five binding sites of
MGY (parallel-dimer)
2
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