Development of N -methyl-(2-arylquinolin-4-yl)oxypropanamides as leads to PET radioligands for translocator protein (18 kDa)

Article abstract of DOI:10.1021/jm5007947

Translocator protein (18 kDa), known as TSPO, is a recognized biomarker of neuroinflammation. Radioligands with PET accurately quantify TSPO in neuroinflammatory conditions. However, the existence of three human TSPO genotypes that show differential affinity to almost all useful TSPO PET radioligands hampers such studies. There is an unmet need for genotype-insensitive, high-affinity, and moderately lipophilic TSPO ligands that may serve as leads for PET radioligand development. To address this need, we varied the known high-affinity TSPO ligand (l)-N,N-diethyl-2-methyl-3-(2- phenylquinolin-4-yl)propanamide in its aryl scaffold, side chain tether, and pendant substituted amido group while retaining an N-methyl group as a site for labeling with carbon-11. From this effort, oxygen-tethered N-methyl- aryloxypropanamides emerged as new high-affinity TSPO ligands with attenuated lipophilicity, including one example with attractive properties for PET radioligand development, namely N-methyl-N-phenyl-2-{[2-(pyridin-2-yl)quinolin- 4-yl]oxy}propanamide (22a; rat K_i = 0.10 nM; human TSPO genotypes K_i = 1.4 nM; clogD = 4.18). This article not subject to U.S. Copyright. Published 2014 by the American Chemical Society.

Full text of DOI:10.1021/jm5007947

Article
pubs.acs.org/jmc
Development of N‑Methyl-(2-arylquinolin-4-yl)oxypropanamides as
Leads to PET Radioligands for Translocator Protein (18 kDa)
Chad Brouwer, Kimberly Jenko, Sami S. Zoghbi, Robert B. Innis, and Victor W. Pike*
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Building 10, Room B3 C346A, 10
Center Drive, Bethesda, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: Translocator protein (18 kDa), known as TSPO,
is a recognized biomarker of neuroinflammation. Radioligands
with PET accurately quantify TSPO in neuroinflammatory
conditions. However, the existence of three human TSPO
genotypes that show differential affinity to almost all useful TSPO
PET radioligands hampers such studies. There is an unmet need
for genotype-insensitive, high-affinity, and moderately lipophilic
TSPO ligands that may serve as leads for PET radioligand
development. To address this need, we varied the known high-
affinity TSPO ligand (l)-N,N-diethyl-2-methyl-3-(2-phenylquino-
lin-4-yl)propanamide in its aryl scaffold, side chain tether, and pendant substituted amido group while retaining an N-methyl
group as a site for labeling with carbon-11. From this effort, oxygen-tethered N-methyl-aryloxypropanamides emerged as new
high-affinity TSPO ligands with attenuated lipophilicity, including one example with attractive properties for PET radioligand
development, namely N-methyl-N-phenyl-2-{[2-(pyridin-2-yl)quinolin-4-yl]oxy}propanamide (22a; rat Ki = 0.10 nM; human
TSPO genotypes Ki = 1.4 nM; clogD = 4.18).
been discovered. Thus, [11C]2 fails to image TSPO in some
INTRODUCTION
■
human subjects.27−29 Furthermore, in a study of deceased
Translocator protein 18 kDa (TSPO),1 formerly known as the
peripheral benzodiazepine receptor,2 is located predominantly
individuals diagnosed with multiple sclerosis, 46% had brain
at the mitochondrial membrane3 in association4 with a voltage-
dependent anion channel and an adenine nucleotide trans-
porter. TSPO is present in several major organs, and is
particularly dense in adrenal gland, heart, kidney, and testis.3
Low amounts are present in normal human brain,5 primarily in
microglia where TSPO plays a crucial role in membrane
biogenesis,6 and in steroid7 and heme8 biosynthesis. Activated
microglia upregulate TSPO in instances of neuronal dam-
age9−11 as seen in many neurological disorders including
cerebral ischemia,12 Alzheimer’s disease,13,14 Parkinson’s
disease,15 and multiple sclerosis.16 TSPO can therefore serve
as an important biomarker for neuroinflammation.17
TSPO showing high affinity (Ki ∼ 4 nM) for 2, 23% low affinity
(Ki ∼ 200 nM), and 31% intermediate affinity.30 This
heterogeneity in binding affinity was found to derive from a
genetic polymorphism among individuals of European ancestry,
namely an Ala147Thr31,32 mutation in TSPO. Three
populations exist: those homozygous for Ala147, homozygous
for Thr147, and heterozygous for Ala147/Thr147, now dubbed
high-affinity binders (HABs), low-affinity binders (LABs), and
mixed-affinity (MABs), respectively. Several other second-
generation TSPO ligands are also genotype-sensitive to
different extents.33−36 PET measurements of TSPO density in
studies of neuroinflammation assume that radioligand binding
affinity is the same in all subjects because the output measure in
PET experiments is usually a function of binding potential
(Bmax/KD), the product of TSPO density (Bmax) to radioligand
affinity (1/KD). Therefore, PET radioligands with heteroge-
neous binding affinity may impair data interpretation, especially
comparisons of binding potentials for patient populations with
those of normal subjects, unless all subjects are characterized
for TSPO genotype.37 However, genotyping is resource
demanding and would be unnecessary if a genotype-insensitive
TSPO radioligand could be employed.
[11C]PK 11195 ([11C]1), first as racemate18 and then as the
higher affinity (R)-enantiomer ([11C](R)-1),19 has long been
used to detect human TSPO in vivo with PET.10,11 However,
accurate quantification of TSPO density with [11C](R)-1 is
confounded by limited brain uptake,20 a low ratio of specific to
nonspecific binding,21 and an unfavorable metabolic profile.22
In view of these deficiencies, new TSPO radioligands have been
developed from other structural classes with superior imaging
characteristics (Chart 1).23−25 These include, for example,
[11C]PBR28 ([11C]2), [11C]DAA1106 ([11C]3), [11C]DPA713
([11C]4), [18F]FBR ([18F]5), PBR111 ([11C]6), and
[18F]FEPPA ([18F]7).
With the advent of the more sensitive radioligand, [11C]2,26
heterogeneity in human TSPO binding to PET radioligands has
Received: May 22, 2014
Published: June 20, 2014
This article not subject to U.S. Copyright.
Published 2014 by the American Chemical
Society
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Chart 1. Structures of Some PET TSPO Radioligands
Pharmuka Laboratories, who introduced the prototypical
TSPO ligand 1, later reported N,N-diethyl-2-methyl-3-(2-
phenylquinolin-4-yl)propanamide ( Q; 10a) as a potent
inhibitor of [3H]1 in rat brain cortex (IC50 = 13.7 nM).38
TSPO strongly bound the l-enantiomer (−)-10a (PK 14067;
IC50 = 5.4 nM), and not the d-enantiomer, (+)-10a (PK 14068;
IC50 = 4000 nM). The binding affinity of (−)-10a was also
found to be high in human cerebral cortex (Ki = 44 nM)5
although clearly much lower than in rat. Thus, the (2-
arylquinolinyl-4-yl)propanamide (−)-10a represents a unique
structural class of TSPO ligand that has so far been neglected
for PET radioligand development. In this study, we explored
this structural class for potential to generate high-affinity,
acceptably lipophilic and human genotype-insensitive TSPO
ligands to serve as leads for PET radioligand development.
affinity TSPO ligands emerged from this effort and a few of
these are promising new leads to PET radioligands.
Chemistry. Ligands were synthesized in one of three
general ways, depending on the tether X in the general
structure (Figure 1).
Figure 1. Generalized structure of compounds tested in this study.
For ligands with X = CH2 (10a−w), a propargyl aniline (9a−
t), prepared in situ by copper(I)-catalyzed addition of a
terminal butynamide to an aldimine, was subjected to
intramolecular cyclization to the dihydroquinoline. A second
equivalent of aldimine (or adventitious oxygen) enabled
oxidation of the dihydroquinoline to the desired quinoline,
with the entire sequence conducted in one pot (method C).45
The requisite butynamides were prepared either by PyBroP-
mediated amidation (9a−9k; method A)46 or by α-alkylation of
amides with propargyl bromide (9a, 9l−9t; method B)47
(Scheme 1).
RESULTS AND DISCUSSION
■
Successful PET radioligands for imaging proteins in brain are
required to display a wide array of properties.39−41 Among
these properties are (i) high affinity and selectivity for the target
protein, (ii) low molecular weight and intermediate polar
surface area for blood−brain barrier penetration, (iii) moderate
lipophilicity for adequate brain entry in the absence of excessive
nonspecific binding, and (iv) amenability to labeling with a
positron-emitter. In addition, PET radioligands for imaging
TSPO in humans should ideally be insensitive to genotype.
This study aimed to develop TSPO ligands as leads with a
desirable combination of properties for PET radioligand
development. Ligands were developed by modifying the 2-
(arylquinolinyl-4-yl)propanamide 10a and initially assessed for
binding affinity toward rat TSPO. Lipophilicities (clogD) were
estimated by computation. The lipophilicity cost for high ligand
affinity may be indexed as a lipophilicity efficiency parameter
(LipE), defined42−44 as ligand pIC50 (or pKi) minus clogD. We
sought ligands with improved LipE scores in addition to other
desirable properties. Several ligands that we found to have
appealing properties were also assayed against human HAB and
LAB TSPOs to assess genotype sensitivity. Many new high-
For a ligand analogous to 10m (X = CH2) with X = S (12),
the 2-mercaptoamide (11) was prepared by treatment of
thiolactic acid with N-methylaniline,48 followed by addition of
the product to 4-chloro-2-phenylquinoline in the presence of
base49 (Scheme 2).
A similar reaction with palladium catalysis50 was used to
prepare the analogous ligand with X = NH (14) (Scheme 3).
The required amide 13 was readily prepared from 2-bromo-N-
methyl-N-phenylpropanamide by conversion into the azide
followed by reduction.51
For the analogous ligand with X = O (15a), 2-phenyl-4-
quinolone was alkylated with 2-bromo-N-methyl-N-phenyl-
propanamide in the presence of base52 (Scheme 4). Two
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a
truncated versions of 15a in which either the phenyl group
(15b) or the benzo fusion was absent (15c), were made
similarly (Scheme 4). A series of 1,x-naphthyridine analogues of
15a (x = 5−8; 19a−d) and a quinazoline analogue (x = 3; 19e)
were also prepared similarly (Scheme 5).
Scheme 1. Synthesis of Ligands 10a−w
The o-benzamidoacetopyridines 17a,b,d were made by
acylation of o-benzamidobromopyridines (16a,b,d) according
to Weinreb’s method (Scheme 5),53 whereas 17c was made by
benzoylation of 4-acetyl-3-aminopyridine (Scheme 5). 17a−d
were then subjected to Camps cyclization54,55 to give the
respective 1,x-naphthyridones (18a−d). The quinazoline 18e
was prepared by hydrolysis of 4-chloro-2-phenylquinazoline.
The naphthyridones and quinazolinone (18a−e) were then
alkylated in the same manner (method D) used to make 15a to
give the ligands 19a−e (Scheme 5). The 2-pyridinylquinolines
22a−c were synthesized similarly to round out the series of
regioisomeric nitrogen-substituted ligands (Scheme 5).
a
Reagents and conditions: (i) PyBroP, DIPA, DCM, rt; (ii) propargyl
bromide, LDA, THF, −78 °C; (iii) 10% CuCl/AgOTf, DCA, 100 °C.
Determination of Absolute Configuration of (−)-10a.
Initially, the absolute configuration of (−)-10a was unknown.
We considered that this information could be valuable in
subsequent TSPO ligand design. Therefore, compound 10a was
resolved by chiral HPLC and the optical rotations of the
separate enantiomers were measured. We confirmed that
(−)-10a was the higher affinity enantiomer (Table 1).
(−)-10a is a thick syrup, and attempts to crystallize the picrate
salt failed. This precluded X-ray crystallography for determi-
nation of absolute configuration, and so we resorted to VCD.56
The solvent-corrected IR and VCD spectra for (−)-10a were
obtained (Supporting Information Figure S1). A conforma-
tional search of the R-enantiomer at the molecular mechanics
level was performed on the entire molecule followed by
optimizations using a B3LYP “functional” on a 6-31G(d) basis
set with Gaussian 09. These calculations revealed 12 con-
formers that were all within 1.5 kcal/mol of the lowest-energy
conformer (Supporting Information Figure S2). VCD and IR
spectra were calculated on the optimized geometries of these
conformers. Their Boltzmann summation was compared with
the observed spectra of (−)-10a (Supporting Information
Figure S3). Given the agreement between calculation and
experiment, the absolute configuration of (−)-10a was assigned
to be R.
a
Scheme 2. Synthesis of S-Tethered Ligand 12
a
Reagents and conditions: (i) PhNHMe, 190 °C; (ii) t-BuOK, t-
BuOH, DMF, 135 °C.
a
Scheme 3. Synthesis of N-Tethered Ligand 14
Effect of Structural Changes to 10a on Rat TSPO
Binding Affinity and LipE. The binding affinities (Ki) of all
TSPO ligands, including 10a and its enantiomers, were
determined on rat brain homogenates (Tables 1−5). Except
for 10a, ligands were tested as racemates only. Assuming that
all these ligands bind TSPO enantioselectively, as observed for
10a, the high-affinity enantiomer is expected to have about 2-
fold higher affinity than that recorded for the racemate. Altering
the chiral center of 10a by either removing the methyl group
entirely (10b) or by lengthening this group from methyl to
ethyl (10c) had a substantial detrimental effect on TSPO
affinity. Therefore, a chiral center incorporating a methyl group
appeared necessary for high TSPO affinity. With few
exceptions, we retained this methyl group in all subsequently
prepared ligands.
a
Reagents and conditions: (i) NaN3, DMF, 70 °C; (ii) Ph3P, H2O,
THF, rt; (iii) HCl(g), toluene, rt; (iv) 4% Pd(OAc)2, 8% DPEPhos,
K3PO4, dioxane, 85 °C.
a
Scheme 4. Syntheses of O-Tethered Ligands 15a−c
A tertiary amide is present in almost all high-affinity TSPO
ligands (e.g., see Chart 1). Previous analogues of 1 that have
had rotation about the amide locked or restricted have
significantly reduced affinity for TSPO.57 Nonetheless, we
found that complete restriction of amide bond rotation in the
pyrrolidinyl ligand 10d had almost no adverse effect on affinity
(cf. 10e; Table 1). The ability of the pyrrolidinyl ring to rotate
a
Reagents and conditions: (i) Cs2CO3, acetone, rt.
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a
Scheme 5. Syntheses of Naphthyridine, Quinazoline, and 2-Pyridylquinoline Analogues of 15a
a
Reagents and conditions: (i) PhCOCl, TEA, DCM, DMAP, rt; (ii) BuLi, THF, −78 °C then N-methoxy-N-methylacetamide, −30 °C; (iii) for 17c,
PhCO2H, PyBroP, DIPA, DMAP, DCM, rt; (iv) NaOH, dioxane, 110 °C; (v) for 18e, NaOH, DMSO, 110 °C; (vi) for 19a−e, Cs2CO3, acetone, rt;
(vii) PyBroP, DIPA, DCM, rt; (viii) NaOH, dioxane, 110 °C; (ix) for 22a, K2CO3, MeCN, 50 °C; (x) Cs2CO3, acetone, rt.
Table 1. TSPO Ligands Based on 10a: Dependence of Binding Affinity, Lipophilicity, and Genotype Sensitivity on Side Chain
Alkyl Substituents
a
b
c
ligand
R1
Me
R2
R3
rat Ki (nM)
cLogD
LipE
LAB Ki (nM)
HAB Ki (nM)
(LAB Ki)/(HAB Ki)
1 (PK 11195)
0.5 0.3
2.1 0.6
3.97
4.33
4.33
4.33
4.84
5.26
3.69
4.58
4.3 0.2
4.4 0.1
4.72 0.04
2.9 0.1
2.7 0.2
2.6 0.2
3.6 0.2
2.8 0.1
4
1
4
1
1.0 0.5
10a
Et
Et
Et
Et
Et
Et
(R)-10a (PK 14067)
(R)-Me
(S)-Me
H
Et
0.90 0.09
73 36
743 190
10
2
76 27
(S)-10a (PK 14068)
Et
10b
10c
10d
10e
Et
32 11
Et
CH2-R2
Et
14
53 22
40
6
CH2-R1
Me
Me
Me
Me
8
a
b
c
Mean SD for n = 6, except for 1 (n = 60), and (R)-10a (n = 5). Mean SD for n = 6, except for 1 (n = 12). Mean SD for n = 6, except for 1
(n = 14).
with respect to the isoquinolinyl group perhaps compensates
for absence of amide bond rotation. Creation of this
pyrrolidinyl ring appreciably improved the LipE score (Table
1).
N-pentyl compound (10i) offered no further improvement in
affinity (Table 2). The N-propyl analogue offered the highest
LipE score in this series but still with a quite high clogD value.
Chain branching effects were not as predictable. Thus, TSPO
affinity increased on replacing N-ethyl (10f) with N-isopropyl
(10j) yet decreased on replacing N-propyl (10g) with N-sec-
butyl (10k) or N-iso-butyl (10l). The N-isopropyl analogue
(10j) gave the best LipE score among analogues with branched
N-alkyl groups and the lowest clogD value among 10e−l.
An N-phenyl (10m) or N-benzyl (10n) group was well-
tolerated, with 10m showing subnanomolar TSPO affinity
(Table 2). TSPO showed variable sensitivity to substitution of
Methylation at a secondary amido nitrogen with [11C]methyl
iodide has been a successful strategy for preparing useful PET
radioligands.58 Therefore, we retained an N-methyl group in
subsequently prepared ligands to provide a potential site for
rapid labeling with carbon-11 (t1/2 = 20.4 min). We found that
progressive lengthening of the remaining N-alkyl substituent
from N-methyl to N-butyl (10e−h) dramatically increased
TSPO affinity, yet this effect reached a plateau at N-butyl, as the
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Table 2. TSPO Ligands Based on 10a: Dependence of Binding Affinity, Lipophilicity, and Genotype Sensitivity on Amido
Substituent
a
b
c
ligand
R3
Me
rat Ki (nM)
cLogD
LipE
LAB Ki (nM)
HAB Ki (nM)
(LAB Ki)/(HAB Ki)
10e
10f
10g
10h
10i
10j
40
9
8
2
4.58
4.28
4.74
5.53
6.61
4.22
5.20
5.00
5.57
6.53
5.26
5.35
5.33
5.54
5.64
5.32
2.8 0.1
3.8 0.1
4.2 0.1
3.9 0.2
2.6 0.1
4.3 0.1
2.9 0.2
3.3 0.1
3.5 0.1
1.8 0.2
3.6 0.2
2.7 0.1
1.7 0.1
3.5 0.1
3.5 0.1
4.1 0.2
Et
Pr
1.2 0.4
0.4 0.2
0.6 0.1
2.9 0.9
Bu
89
3
1.0 0.6
89 50
64 28
Pen
i-Pr
973 409
15
2
d
10k
sec-Bu
i-Bu
Ph
5
5
3
2
10l
185 81
53 22
6
2
32 18
27 14
10m
10n
10o
10p
10q
10r
10s
10t
0.9 0.2
1.9 0.5
Bn
5
3
o-Py
m-Py
p-Py
o-F-Ph
m-F-Ph
p-F-Ph
1.6 0.6
10
119 56
4
1
30 18
3
106 24
0.9 0.2
0.8 0.2
0.4 0.1
a
b
c
Mean SD for n = 6, except for 10h,l (n = 9). Mean SD for n = 6, except for 10h,j (n = 3) and 10m (n = 5). Mean SD for n = 6, except for
d
10m,o (n = 5). 10k is a pair of unresolved diastereomers.
the N-phenyl group in 10m with a pyridinyl or fluorophenyl
group. Binding affinity varied with nature and position of the
new heteroatom. Thus, replacement of a phenyl group with an
o-pyridinyl group (10o) left binding affinity unchanged at
subnanomolar. However, there was a 10-fold loss of affinity in
the m-pyridinyl analogue (10p) and a further 10-fold loss in the
p-pyridinyl analogue (10q). In contrast to the pyridinyl
analogues, all three fluoro isomers (10r−t) showed almost
identical subnanomolar affinity. The p-fluorophenyl ligand 10t
had the highest LipE score but still possessed high computed
lipophilicity.
Chlorophenyl groups have featured in examples of high-
affinity TSPO ligands from other structural classes such as the
isoquinoline carboxamide 1. Therefore, we also prepared the
chlorophenyl isomers 10u−w of the N-methyl,N-isopropyl
ligand 10j (Table 3). All three ligands showed lower TSPO
affinity than the phenyl analogue 10j, with the o-isomer (10u)
showing lowest affinity.
Having explored how TSPO affinity was affected by
structural change at the amido nitrogen, we next explored
changes to the methylene group that bridges the amide to the
quinolinyl ring. We replaced the methylene tether of 10m with
an oxygen (15a), sulfur (12), or NH tether (14). In each case,
rat TSPO binding affinity was greatly increased, and particularly
so for the oxygen-tethered compound (15a) which showed a Ki
value of 70 pM (Table 4). Increased affinity may be due to
improved spatial relations of the amido group relative to the
quinolinyl ring via altered bond lengths and dihedral angle.
Further benefits of the heteroatom tethers were decreased
lipophilicities, resulting in higher LipE scores.
We chose to focus on preparing further analogues of the very
high affinity O-tethered lead (15a). We next considered
whether high TSPO affinity might be retained in less lipophilic
analogues in which the phenyl-quinoline scaffold was modified
by removing an aromatic ring or by inserting a nitrogen atom.
Compounds that lacked either the pendant 2-phenyl group
(15b) or the benzo fusion (15c) illustrated that these two rings,
and especially the phenyl group, were quite important for very
high TSPO affinity (Table 5). Nonetheless, 15b and 15c still
exhibited affinity in the low nanomolar range, with radically
reduced clogD values and greatly enhanced LipE scores. All
nitrogen substitutions in the quinoline core resulted in ligands
that retained near- or subnanomolar affinity for TSPO, with
affinities slightly increasing in the following order 19a < 19b <
19c ∼ 19d < 19e. Like the quinazoline (19e), the 2-(o-
pyridyl)quinoline (22a) had very high affinity, approaching that
of 15a, but with much lower computed lipophilicity and hence
a much higher LipE score. The other pyridylquinolines (22b,c)
showed virtually equal subnanomolar affinity.
Table 3. TSPO Ligands Based on 10a: Dependence of Rat
Binding Affinity and Lipophilicity on Pendant Aryl
Substituent
a
ligand
Ar
rat Ki (nM)
cLogD
LipE
10j
Ph
2.9 0.9
4.22
4.50
4.71
4.83
4.3 0.1
3.2 0.1
3.7 0.1
3.6 0.1
10u
10v
10w
o-Cl-Ph
m-Cl-Ph
p-Cl-Ph
22
4
6
1
1
Overall, the presence of a heteroatom tether strikingly
increased LipE score, as may be readily appreciated from a plot
of pKi versus clogD for ligands with each type of tether (Figure
2). In particular, ligands with oxygen tethers clearly cluster into
4
a
Mean SD for n = 6, except for 10u (n = 9).
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Table 4. TSPO Ligands Based on 10a: Dependence of Rat Binding Affinity, Lipophilicity, and Genotype Sensitivity on Tether
for Pendant Alkyl Carboxamido Group
a
b
c
ligand
X
rat Ki (nM)
cLogD
LipE
LAB Ki (nM)
HAB Ki (nM)
(LAB Ki)/(HAB Ki)
10m
12
CH2
S
0.9 0.2
5.57
5.31
4.11
4.73
3.5 0.1
4.10 0.05
5.62 0.03
5.42 0.03
53 22
1.9 0.5
2.5 0.6
0.44 0.08
0.5 0.2
27 14
0.39 0.04
0.19 0.01
26
4
5
1
10
9
3
3
14
NH
O
15a
0.070 0.004
1.3 0.2
2.5 0.9
a
b
c
Mean SD for n = 6, except for 14 (n = 5). Mean SD for n = 6, except for 10m (n = 5). Mean SD for n = 6, except for 10m (n = 5).
Table 5. Oxygen-Tethered TSPO Ligands Based on 15a: Dependence of Binding Affinity, Lipophilicity, and Genotype
Sensitivity on Scaffold Aryl Groups
a
b
c
ligand
Ar
Ph
A
B
C
D
E
rat Ki (nM)
cLogD
LipE
LAB Ki (nM)
HAB Ki (nM) (LAB Ki)/(HAB Ki)
15a
15b
15c
19a
19b
19c
19d
19e
22a
22b
22c
CH
CH
H
CH
CH
CH
CH
CH
CH
H
CH
CH
CH
CH
CH
CH
CH
N
0.070 0.004
5.7 0.5
4.73
2.96
3.52
4.27
4.15
4.15
3.91
5.66
4.18
4.06
4.06
5.42 0.03
5.29 0.04
5.3 0.3
4.6 0.1
5.0 0.1
5.4 0.3
5.63 0.04
4.4 0.5
5.9 0.2
5.6 0.1
5.60 0.04
1.3 0.2
66 22
62 11
0.5 0.2
3.3 0.9
2.0 0.6
1.1 0.3
1.3 0.5
1.4 0.4
0.9 0.2
2.5 0.9
20
31 11
H
9
Ph
3.3 0.6
Ph
N
CH
N
CH
CH
N
CH
CH
CH
N
1.4 0.5
23
25
7
8
21
19
8
9
Ph
CH
CH
CH
CH
CH
CH
CH
0.6 0.1
Ph
CH
CH
CH
CH
CH
CH
0.3 0.2
47 26
33 20
Ph
CH
CH
CH
CH
CH
0.29 0.03
0.13 0.08
0.10 0.05
0.20 0.03
0.22 0.02
4.7 0.7
5
1
2
1
Ph
CH
CH
CH
CH
2
1
2
1
o-Py
m-Py
p-Py
CH
CH
CH
1.4 0.5
1.4 0.2
0.90 0.09
1.0 0.5
1.0 0.4
12
7
3
3
13
7
4
5
a
b
c
Mean SD for n = 6, except for 19d, 22b (n = 5). Mean SD for n = 6, except for 15a,b, 19c (n = 5), and 15c (n = 4). Mean SD for n = 6,
except for 15c (n = 5).
fold lower affinity to HAB TSPO than to rat TSPO (Table 1).
Affinity for LAB TSPO was about 76-fold lower than for HAB
TSPO. All the methylene-tethered ligands had similar or lower
affinity to HAB TSPO than to rat TSPO and had high genotype
sensitivities (Table 2). Thus, overall, variation in amide
substituents had little impact on genotype sensitivity.
For the group of ligands in which only the tether atom
differed (10m, 12, 14, 15a) affinity for HAB TSPO was again
somewhat lower than for rat TSPO, and genotype sensitivity
reduced progressively across the tether series CH2, S, NH, and
O (Table 4). The oxygen-tethered ligand 15a showed very low
sensitivity (2.5) in addition to subnanomolar affinity and high
LipE score. We surmise this improvement may relate to altered
bond lengths and torsional angles at the oxygen atom.
Two truncated versions of 15a (15b,c) showed somewhat
lower affinity for rat TSPO. Remarkably, however, they showed
low nanomolar HAB binding affinities and LAB/HAB Ki ratios
similar to those of 15a (Table 5). The LipE scores of 15b and
15c were similar to those of 15a.
Finally, we looked at the effects of introducing a second
nitrogen into the 2-phenylquinoline scaffold (Table 5). Of the
eight compounds tested, four (19a,b,d, 22b) displayed greater
than 10-fold lower binding affinity to LAB TSPO than to HAB
TSPO. The remaining four compounds were either much less
sensitive (19d, 22c) or insensitive (19e, 22a) to TSPO
Figure 2. Plot of rat pKi versus clogD for ligands having different side
chain tethers.
a separate group to those with methylene tethers. Thus,
introduction of a heteroatom tether was very effective in
mitigating the demands35 of the TSPO binding site for high
ligand lipophilicity.
Assessment of Ligand Sensitivity to Human TSPO
Genotype. Compound 1, the lead compound (R)-10a,
examples of methylene-tethered ligands (10h,j,l,m,o), and the
heteroatom-tethered compounds (12, 14, 15a−c, 19a−e, 22a−
c) were selected to evaluate their human genotype sensitivities
by measurement of their Ki values for binding to leukocytes
from HABs and LABs. Our measurements confirmed that 1 has
low genotype sensitivity (Table 1). (R)-10a showed about 10-
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Champaign using a Micromass Q-Tof Ultima instrument for ESI
(Waters Corp.; Columbia, MD) or a GCT Premier instrument for EI
(Waters Corp.). Preparative HPLC was performed with either a Luna
PFP(2) (5 μm; 100 Å; 30 mm × 250 mm), Gemini C18 (10 μm; 110
Å; 30 mm × 250 mm), or a Lux Amylose-2 (3 μm; 10 mm × 250 mm)
column. HPLC separation conditions are given in parentheses and
refer to column type, flow rate (mL/min), and organic phase ‘O’/aq
phase ‘A’ ratio, as follows: O1 = MeOH, O2 = MeCN, A1 = H2O, A2 =
NH4CO3H (10 mM), A3 = Et2NH (0.1%). The chemical purities of all
compounds were established by HPLC on either a Luna PFP(2) (5
μm; 100 Å; 4.6 mm × 250 mm), Gemini C18 (5 μm; 110 Å; 4.6 mm
× 250 mm), or a Lux Amylose-2 (3 μm; 4.6 mm × 250 mm) column.
Chemical purities were all >95% and typically >99%, as monitored by
absorbance at 220 nm. cLogD was computed with Pallas for Windows
software version 3.8 in default option (CompuDrug; Bal Harbor, FL).
Method A: N,N-Diethyl-2-methylpent-4-ynamide (9a). DIPA
(1.0 mL, 6.0 mmol) was added dropwise to a solution of 2-
methylpent-4-ynoic acid59 (0.33 mL, 3.0 mmol), diethylamine (0.34
mL, 3.3 mmol), and PyBroP (1.4 g, 3.0 mmol) in DCM (3 mL) at rt.
This mixture was stirred for 2 h and then the solvent removed. The
residue was taken up in EtOAc (30 mL) and washed successively with
5% KHSO4 (30 mL × 3), brine (30 mL), 5% NaHCO3 (30 mL × 3),
and brine (30 mL), and finally dried (Na2SO4). FC (hexanes/EtOAc,
1:1) of the residue gave 9a as a colorless oil (0.33 g, 66%). HRMS−
ESI (m/z): [M + H]+ calcd for C10H18NO, 168.1388; found,
genotype. Notably, compounds having the second nitrogen in
nearest proximity to the quinolinyl nitrogen had the least
sensitivity to genotype. Ligand 22a offered the most appealing
combination of properties as a lead for PET radioligand
development, including high HAB TSPO affinity, genotype
insensitivity, and high LipE score for binding to rat and human
TSPO, which are all improved over corresponding values for
10a.
An important consideration in attempts to develop genotype-
insensitive PET radioligands for TSPO, is whether genotype
sensitivity is likely to increase with ligand affinity. Generally, we
observed that genotype sensitivity tended to decrease with
HAB TSPO affinity among the tested ligands (Figure 3).
1
168.1383. H NMR (CDCl3): δ 3.41−3.33 (m, 4H), 2.87 (m, J = 2.4
Hz, 1H), 2.55−2.49 (ddd, J = 17, 7.2, 2.4 Hz, 1H), 2.34−2.28 (ddd, J
= 17, 7.2, 2.4 Hz, 1H), 1.97 (t, J = 2.4 Hz, 1H), 1.21 (t, J = 6.8 Hz,
3H), 1.21 (t, J = 6.8 Hz, 3H), 1.12 (t, J = 6.8 Hz, 3H). 13C NMR
(CDCl3): δ 174.1, 82.7, 69.0, 41.9, 40.4, 35.4, 23.3, 17.7, 14.9, 13.1.
As described in the Supporting Information, this method also gave:
N,2-dimethyl-N-propylpent-4-ynamide (9b), N-butyl-N,2-dimethyl-
pent-4-ynamide (9c), N,2-dimethyl-N-pentylpent-4-ynamide (9d), N-
(butan-2-yl)-N,2-dimethylpent-4-ynamide (9e), N,2-dimethyl-N-(2-
methylpropyl)pent-4-ynamide (9f), N-benzyl-N,2-dimethylpent-4-yna-
mide (9g), N,2-dimethyl-N-(pyridine-2-yl)pent-4-ynamide (9h), N,2-
dimethyl-N-(pyridine-3-yl)pent-4-ynamide (9i), N,2-dimethyl-N-(pyr-
idin-4-yl)pent-4-ynamide (9j), N-(4-fluorophenyl)-N,2-dimethylpent-
4-ynamide (9k), N-(2-fluorophenyl)-N-methylpropanamide (9sa), N-
(3-fluorophenyl)-N-methylpropanamide (9ta), N-(4-acetylpyridin-3-
yl)benzamide (17c), N-(2-acetylphenyl)pyridine-2-carboxamide
(20a), and N-(2-acetylphenyl)pyridine-4-carboxamide (20c).
Method B: N,N-Diethyl-2-methylpent-4-ynamide (9a). n-
Butyl lithium in hexanes (1.6 M, 28 mL, 44 mmol) was added
dropwise to a solution of DIPA (6.1 mL, 43 mmol) in THF (130 mL)
under Ar at −75 °C. This solution was slowly warmed to 0 °C and
then cooled back to −75 °C. N,N-Diethylpropionamide (5.6 mL, 39
mmol) was added dropwise. This solution was slowly warmed to −20
°C and then cooled back to −75 °C. Propargyl bromide (87 mmol) in
toluene (9.4 mL) was added dropwise. This solution slowly was
warmed to rt and stirred for several hours. Brine (200 mL) was added,
the organic layer separated, and the aqueous phase extracted with ether
(100 mL × 2). The combined extracts were washed with brine (100
mL) and dried (MgSO4). Fractional distillation of the residue gave 9a
as a colorless oil (bp 53−54 °C at 5.8 mmHg; 4.8 g, 71%). A small
quantity was further purified by HPLC (Gemini, 30, O1/A1, 55:45); d
0.90 g/mL.
As described in the Supporting Information, this method also gave:
N,N-diethylpent-4-ynamide (9l), N,N,2-triethylpent-4-ynamide (9m),
1-methyl-3-(prop-2-yn-1-yl)pyrrolidin-2-one (9n), N,N,2-trimethyl-
pent-4-ynamide (9o), N-ethyl-N,2-dimethylpent-4-ynamide (9p),
N,2-dimethyl-N-(propan-2-yl)pent-4-ynamide (9q), N,2-dimethyl-N-
phenylpent-4-ynamide (9r), N-(2-fluorophenyl)-N,2-dimethylpent-4-
ynamide (9s), and N-(3-fluorophenyl)-N,2-dimethylpent-4-ynamide
(9t).
Method C: N,N-Diethyl-2-methyl-3-(2-phenylquinolin-4-yl)-
propanamide (10a). Copper(I) chloride (10 mg, 0.10 mmol) and
then silver triflate (26 mg, 0.10 mmol) were added to a solution of N-
benzylideneaniline (362 mg, 2.00 mmol) and 9a (167 mg, 1.00 mmol)
in DCA (1.0 mL) under Ar. The solution was heated to 100 °C for 17
Figure 3. Plot of genotype sensitivity versus HAB TSPO binding
affinity for tested ligands.
CONCLUSIONS
■
Judicious structural modifications to 10a led to several TSPO
ligands with affinity in the nanomolar or subnanomolar range
plus an enhanced LipE score. Truncation of the phenyl-
isoquinoline scaffold as in ligands 15b and 15c was particularly
effective in reducing LipE score, and these ligands may serve as
leads for further PET radioligand development. Introduction of
an oxygen tether in place of the methylene tether was
particularly effective in generating compounds with low
genotype sensitivity as leads for PET radioligand development.
Ligand 22a presents an especially appealing array of properties
for this purpose.
EXPERIMENTAL SECTION
■
Materials and Methods. Reagents and solvents were purchased
unless stated otherwise. Air-sensitive reagents were stored under N2 in
a PureLab HE glovebox (Innovative Technology; Amesbury, MA).
Melting points were determined on an SMP10 apparatus (Stuart;
Staffordshire, UK). Boiling point vacuum pressures were determined
on a DVR-200 apparatus (J-Kem Scientific Inc.; St. Louis, MO).
Optical rotations were determined on a P-1010 instrument (JASCO
Inc.; Easton, MD). IR-VCD spectra were recorded on a Chiral IR-2X
instrument equipped with DualPEM (BioTools, Inc.; Jupiter, FL).
Absolute configuration was determined with ComputeVOA (BioTools
Inc.), employing a B3LYP “functional”, 6-31G(d) basis set on
Gaussian 09 (Gaussian Inc.; Pittsburgh, PA). 1H (400 MHz), 13C
NMR (100 MHz), and 19F NMR (376 MHz) spectra were recorded
on an Avance 400 instrument (Bruker; Billerica, MA). Chemical shifts
for 19F are reported relative to neat TFA in a coaxial insert (δ =
−76.6). HRMS were obtained at the Mass Spectrometry Laboratory,
School of Chemical Sciences, University of Illinois Urbana
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1
h, cooled to rt, and filtered through diatomaceous earth. The mixture
was taken up in EtOAc (10 mL) and washed with aqueous NH4Cl/
NH4OH (pH 8, 10 mL). The aqueous phase was extracted with
EtOAc (10 mL × 2). The combined extracts were washed with brine
(15 mL) and dried (MgSO4). HPLC (PFP, 30, O2/A2, 65:35) of this
material gave 10a as a dark-yellow syrup (0.12 g, 36%). HRMS−ESI
(m/z): [M + H]+ calcd for C23H27N2O, 347.2123; found, 347.2114.
1H NMR (CDCl3): δ 8.20 (bs, 1H), 8.15 (m, 2H), 8.04 (dd, J = 8.4,
0.8 Hz, 1H), 7.74 (s, 1H), 7.70 (dt, J = 7.8, 1.2 Hz, 1H), 7.55 (dt, J =
7.8, 1.2 Hz, 1H), 7.53−7.48 (2H), 7.44 (tt, J = 7.2, 1.6 Hz, 1H), 3.52
(dd, J = 13.6, 9.2 Hz, 1H), 3.36−3.24 (m, 2H), 3.19−3.09 (m, 1H),
2.87 (dq, J = 7.2, 2.4 Hz, 2H), 1.31 (d, J = 6.4 Hz, 3H), 0.88 (t, J = 7.2
Hz, 3H), 0.75 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3): δ 174.4, 156.9,
148.4, 146.7, 139.5, 130.6, 129.3, 128.8, 127.5, 126.5, 126.2, 123.2,
120.0, 41.7, 40.6, 37.0, 36.7, 19.0, 14.5, 13.0.
As described in the Supporting Information, this method also gave:
N,N-diethyl-3-(2-phenylquinolin-4-yl)propanamide (10b), N,N-dieth-
yl-2-[(2-phenylquinolin-4-yl)methyl]butanamide (10c), 1-methyl-3-
[(2-phenylquinolin-4-yl)methyl]pyrrolidin-2-one (10d), N,N,2-tri-
methyl-3-(2-phenylquinolin-4-yl)propanamide (10e), N-ethyl-N,2-di-
methyl-3-(2-phenylquinolin-4-yl)propanamide (10f), N,2-dimethyl-3-
(2-phenylquinolin-4-yl)-N-propylpropanamide (10g), N-butyl-N,2-
dimethyl-3-(2-phenylquinolin-4-yl)propanamide (10h), N,2-dimethyl-
N-pentyl-3-(2-phenylquinolin-4-yl)propanamide (10i), N,2-dimethyl-
3-(2-phenylquinolin-4-yl)-N-(propan-2-yl)propanamide (10j), N-
(butan-2-yl)-N,2-dimethyl-3-(2-phenylquinolin-4-ylpropanamide
(10k), N,2-dimethyl-N-(2-methylpropyl)-3-(2-phenylquinolin-4-yl)-
propanamide (10l), N,2-dimethyl-N-phenyl-3-(2-phenylquinolin-4-
yl)propanamide (10m), N-benzyl-N,2-dimethyl-3-(2-phenylquinolin-
4-yl)propanamide (10n), N,2-dimethyl-3-(2-phenylquinolin-4-yl)-N-
(pyridin-2-yl)propanamide (10o), N,2-dimethyl-3-(2-phenylquinolin-
4-yl)-N-(pyridin-3-yl)propanamide (10p), N,2-dimethyl-3-(2-phenyl-
quinolin-4-yl)-N-(pyridin-4-yl)propanamide (10q), N-(2-fluorophen-
yl)-N,2-dimethyl-3-(2-phenylquinolin-4-yl)propanamide (10r), N-(3-
fluorophenyl)-N,2-dimethyl-3-(2-phenylquinolin-4-yl)propanamide
(10s), N-(4-fluorophenyl)-N,2-dimethyl-3-(2-phenylquinolin-4-yl)-
propanamide (10t), 3-[2-(2-chlorophenyl)quinolin-4-yl)-N,2-dimeth-
yl-N-(propan-2-yl)propanamide (10u), 3-[2-(3-chlorophenyl)-
quinolin-4-yl)-N,2-dimethyl-N-(propan-2-yl)propanamide (10v), and
3-[2-(4-chlorophenyl)quinolin-4-yl)-N,2-dimethyl-N-(propan-2-yl)-
propanamide (10w).
399.1531; found, 399.1532. H NMR (CDCl3): δ 8.15 (dd, J = 8.0,
0.8 Hz, 2H), 7.98 (dd, J = 6.8, 1.6 Hz, 2H), 7.72 (dt, J = 7.4, 1.2 Hz,
1H), 7.54−7.46 (5H), 7.19 (t, J = 2.0 Hz, 3H), 6.94 (2H), 4.12 (q, J =
6.8 Hz, 1H), 3.23 (s, 3H), 1.58 (d, J = 6.8 Hz, 3H). 13C NMR
(CDCl3): δ 171.5, 156.8, 148.3, 144.7, 143.2, 139.5, 130.4, 130.3,
130.0, 129.7, 129.0, 128.3, 127.8, 127.2, 126.8, 124.6, 119.7, 42.8, 38.0,
19.0.
2-Amino-N-methyl-N-phenylpropanamide hydrochloride
(13). NaN3 (2.58 g, 39.8 mmol) and 2-bromo-N-methyl-N-phenyl-
propanamide60 (2.42 g, 10.0 mmol) in DMF (50 mL) were heated to
70 °C under Ar for 2 h. The solution was cooled to rt, diluted with
water (500 mL), and extracted into ether (200 mL × 3). The
combined extracts were washed successively with water (100 mL × 5)
and brine (100 mL) and dried (Na2SO4). The solvent was removed to
yield the azide as a yellow oil (1.98 g, 97%), which was taken up in
THF (100 mL). Ph3P (2.54 g, 9.70 mmol) and water (260 μL, 15
mmol) were added, and the mixture stirred overnight at rt. The solvent
was removed, the residue taken up in benzene (or toluene), and HCl
(g) bubbled through for 10 min. The hydrochloride salt was washed
with ether to give 13 as a cream powder (1.78 g, 83%); mp 230−233
°C dec. HRMS−ESI (m/z): [M + H]+ calcd for C10H15N2O,
179.1184; found, 179.1182. 1H NMR (D2O): δ 7.72−7.63 (3H),
7.55−7.52 (2H), 4.83 (q, J = 7.2 Hz, 1H), 4.21 (q, J = 7.2 Hz, 1H),
3.57 (s, 3H), 3.43 (s, 3H), 1.79 (d, J = 6.8 Hz, 3H), 1.41 (d, J = 6.8
Hz, 3H). 13C NMR (D2O): δ 170.4, 141.0, 130.4, 129.3, 127.2, 47.3,
37.9, 15.8.
N-Methyl-N-phenyl-2-[(2-phenylquinolin-4-yl)amino]-
propanamide (14). A mixture of (13) (322 mg, 1.50 mmol), 4-
chloro-2-phenylquinoline (240 mg, 1.00 mmol), Pd(OAc)2 (9 mg,
0.04 mmol), DPEPhos (43 mg, 0.080 mmol), and K3PO4 (742 mg,
3.50 mmol) in dioxane (4.0 mL) was heated to 85 °C under Ar for 2 d.
The mixture was cooled to rt and filtered through diatomaceous earth,
which was then rinsed with EtOAc (20 mL). The organic phase was
washed with water (20 mL) and the water extracted with EtOAc (20
mL × 2). The combined extracts were washed with brine (20 mL) and
then dried (MgSO4). The product was isolated by FC (CHCl3/
MeOH/TEA, 30:1:1%) and recrystallized (cyclohexane/EtOAc) to
give 14 as pale-yellow chunks (0.13 g, 34%); mp 190−191 °C.
HRMS−ESI (m/z): [M + H]+ calcd for C25H24N3O, 382.1919; found,
382.1917. 1H NMR (CDCl3): δ 8.05 (d, J = 8.0 Hz, 1H), 7.91 (d, J =
6.8 Hz, 2H), 7.85 (dd, J = 8.4, 0.8 Hz, 1H), 7.65 (dt, J = 6.8, 1.2 Hz,
1H), 7.52−7.41 (7H), 7.29−7.27 (2H), 6.41 (s, 1H), 5.92 (d, J = 8.0
Hz, 1H), 4.46 (dquint, J = 6.8, 1.6 Hz, 1H), 3.33 (s, 3H), 1.42 (d, J =
6.8 Hz, 3H). 13C NMR (CDCl3): δ 173.0, 158.3, 148.8, 148.5, 142.7,
141.0, 130.3, 130.2, 129.4, 128.8, 128.7, 128.5, 127.6, 127.4, 124.6,
119.7, 118.1, 96.8, 48.4, 38.0, 18.6.
(R)-N,N-Diethyl-2-methyl-3-(2-phenylquinolin-4-yl)-
propanamide [(R)-10a]. Chiral HPLC (Lux, 12, O2/A3, 50:50) of
10a gave (R)-10a (tR 5.3 min; 99% ee; [α]18 −90° (c 2.86, EtOH).
D
(S)-N,N-Diethyl-2-methyl-3-(2-phenylquinolin-4-yl)-
propanamide [(S)-10a]. Chiral HPLC, as described above, also gave
(S)-10a (tR 6.2 min; 96% ee; [α]18 +95° (c 3.35, EtOH).
Method D: N-Methyl-N-phenyl-2-[(2-phenylquinolin-4-yl)-
oxy]propanamide (15a). 2-Phenylquinolin-4(1H)-one hydrochlor-
ide (144 mg, 0.56 mmol), 2-bromo-N-methyl-N-phenylpropanamide60
(203 mg, 0.84 mmol), and Cs2CO3 (736 mg, 2.26 mmol) were stirred
in acetone (5.0 mL) at rt for 19 h. Solid was filtered off and washed
with a little acetone. Solvent was then removed, and the residue was
recrystallized (DCM/ether) to give 15a as a colorless solid (123 mg,
57%); mp 158−160 °C. HRMS−ESI (m/z): [M + H]+ calcd for
D
N-Methyl-N-phenyl-2-sulfanylpropanamide (11). N-Methyla-
niline (11 mL, 102 mmol) and thiolactic acid (9.0 mL, 102 mmol)
were heated to 190 °C under Ar for 3 d. Water was collected in a
Dean−Stark apparatus. The mixture was cooled to rt, taken up in
CHCl3 (250 mL), washed successively with 10% HCl (100 mL × 2),
saturated NaHCO3 (100 mL × 2), and brine (100 mL), and dried
(MgSO4). The product was distilled (bp = 144 °C at 7.4 mmHg) to
give 11 as a yellow oil (3.8 g, 19%). HRMS−ESI (m/z): [M + H]+
calcd for C10H14NOS, 196.0796; found, 196.0795. 1H NMR (CDCl3):
δ 7.45 (t, J = 7.2 Hz, 2H), 7.38 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 7.2 Hz,
2H), 3.37−3.31 (m, 1H), 3.29 (s, 3H), 2.13 (d, J = 10 Hz, 1H), 1.45
(d, J = 7.2 Hz, 3H). 13C NMR (CDCl3): δ 173.6, 143.5, 123.0, 128.2,
127.3, 37.8, 33.5, 22.9.
N-Methyl-N-phenyl-2-[(2-phenylquinolin-4-yl)sulfanyl]-
propanamide (12). A mixture of 11 (115 mg, 0.59 mmol), 4-chloro-
2-phenylquinoline (120 mg, 0.50 mmol), t-BuOK (61 mg, 0.55
mmol), and t-BuOH (53 μL, 0.55 mmol) in DMF (5.0 mL) was
heated to 135 °C under Ar for 3 d. The mixture was cooled to rt,
diluted with water (50 mL), and extracted into EtOAc (20 mL × 3).
The combined extracts were washed with water (10 mL × 5) and
brine (10 mL) and dried (MgSO4). Product was isolated by HPLC
(Gemini, 43, O1/A2, 80:20) to give 12 as a light-yellow syrup (128 mg,
64%). HRMS−ESI (m/z): [M + H]+ calcd for C25H23N2OS,
1
C25H23N2O2, 383.1760; found, 383.1761. H NMR (CDCl3): δ 8.15
(d, J = 8.4 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 7.2 Hz, 2H),
7.69 (dt, J = 7.8, 1.2 Hz, 1H), 7.53 (tt, J = 6.8, 1.6 Hz, 2H), 7.47 (m,
2H), 7.34−7.31 (3H), 7.21−7.19 (2H), 5.09 (q, J = 6.4 Hz, 1H), 3.31
(s, 3H), 1.68 (d, J = 6.4 Hz, 3H). 13C NMR (CDCl3): δ 169.8, 160.6,
158.5, 149.4, 142.4, 140.4, 130.1, 130.0, 129.2, 129.0, 128.7, 128.5,
127.6, 127.3, 125.4, 122.2, 120.4, 98.9, 71.4, 38.2, 18.1.
As described in the Supporting Information, this method also gave:
N-methyl-N-phenyl-2-(quinolin-4-yloxy)propanamide (15b), N-meth-
yl-N-phenyl-2-[(2-phenylpyridin-4-yl)oxy]propanamide (15c), N-
methyl-N-phenyl-2-[(2-phenyl-1,5-naphthyridin-4-yl)oxy)]-
propanamide (19a), N-methyl-N-phenyl-2-[(2-phenyl-1,6-naphthyri-
din-4-yl)oxy)]propanamide (19b), N-methyl-N-phenyl-2-[(2-phenyl-
1,7-naphthyridin-4-yl)oxy)]propanamide (19c), N-methyl-N-phenyl-
2-[(2-phenyl-1,8-naphthyridin-4-yl)oxy)]propanamide (19d), N-
methyl-N-phenyl-2-[(2-phenylquinazolin-4-yl)oxy)]propanamide
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(19e), N-methyl-N-phenyl-2-{[2-(pyridin-3-yl)quinolin-4-yl]oxy}-
propanamide (22b), and N-methyl-N-phenyl-2-{[2-(pyridin-4-yl)-
quinolin-4-yl]oxy}propanamide (22c).
This method also gave 2-phenyl-1,4-dihydro-1,6-naphthyridin-4-one
(18b), 2-phenyl-1,4-dihydro-1,7-naphthyridin-4-one (18c), and 2-
phenyl-1,4-dihydro-1,8-naphthyridin-4-one (18d) (see Supporting
Information).
N-(3-Bromopyridin-4-yl)benzamide (16b). Benzoyl chloride
(3.7 mL, 32 mmol) was added dropwise to a solution of 4-amino-3-
bromopyridine (5.01 g, 29.0 mmol) and TEA (10 mL) in DCM (25
mL) at rt under Ar. DMAP (325 mg, 2.90 mmol) was then added in
one portion. This mixture was stirred for 30 min and then quenched
by addition of water (250 mL). The organic layer was separated off
and the aqueous phase extracted with DCM (100 mL × 2). The
combined extracts were washed with brine (100 mL) and then dried
(MgSO4). The solvent was removed and the product twice
recrystallized (EtOAc/hexanes) to give 16b as cream needles (1.98
g, 24%); mp 99−101 °C. HRMS−ESI (m/z): [M + H]+ calcd for
2-Phenyl-1,4-dihydroquinazolin-4-one (18e). A slurry of 4-
chloro-2-phenylquinazoline (5.18 g, 21.5 mmol) and NaOH (860 mg,
21.5 mmol) was heated to 110 °C in DMSO (200 mL) for 1 h. This
slurry was then cooled to rt, whereupon fine colorless crystals
appeared. These were collected and washed with water (500 mL × 2)
to give 18e (1.28 g, 27%); mp 238−239 °C (lit. mp62 233−234 °C).
HRMS−EI (m/z): [M + H]+ calcd for C14H11N2O, 222.0793; found,
222.0792. 1H NMR (HFIP−d2): δ 8.40 (d, J = 8.0 Hz, 1H), 8.24 (d, J
= 8.8 Hz, 1H), 8.16 (dt, J = 7.2, 0.8 Hz, 1H), 7.95 (d, J = 7.6 Hz, 2H),
7.83 (t, J = 8.0 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.53 (t, J = 8.0 Hz,
2H), 4.95 (br s, 1H). 13C NMR (HFIP-d2): δ 166.3, 161.8, 151.6,
136.1, 135.6, 130.9, 128.6, 128.3, 127.6, 125.2, 122.7, 114.6. Addition
of water to the mother liquor precipitated unreacted starting material
(1.37 g).
1
C12H10N2OBr, 276.9976; found, 276.9971. H NMR (CDCl3): δ 8.69
(s, 1H), 8.62 (br s, 1H), 8.57 (d, J = 5.6 Hz, 1H), 8.50 (d, J = 5.6 Hz,
1H), 7.95−7.92 (m, 2H), 7.63 (tt, J = 7.6, 1.2 Hz, 1H), 7.56 (tt, J =
7.6, 1.2 Hz, 2H). 13C NMR (CDCl3): δ 165.5, 151.6, 149.9, 142.5,
133.6, 132.9, 129.2, 127.2, 144.8, 111.0.
Method G: N-Methyl-N-phenyl-2-{[2-(pyridin-2-yl)quinolin-
4-yl]oxy}propanamide (22a). 2-(Pyridin-2-yl)-1,4-dihydroquinolin-
4-one55 (222 mg, 1.00 mmol), 2-bromo-N-methyl-N-phenylpropana-
mide60 (270 mg, 1.12 mmol), and K2CO3 (834 mg, 6.03 mmol) in
MeCN (35 mL) were heated to 55 °C (solution temp) under Ar for
16 h. The mixture was then cooled to rt and poured into stirring water
(175 mL). After several min, the precipitate was collected and
recrystallized (cyclohexane/EtOAc) to give 22a as colorless plates
(267 mg, 70%); mp 185−187 °C. HRMS−ESI (m/z): [M + H]+ calcd
N-(3-Bromopyridin-2-yl)benzamide (16d). Benzoyl chloride
(3.9 mL, 33.3 mmol) was added dropwise to a solution of 2-amino-
3-bromopyridine (4.98 g, 28.8 mmol) and TEA (8.0 mL, 57.9 mmol)
in THF (25 mL) at −5 °C under Ar. This mixture was warmed to rt
and stirred overnight. The precipitate was filtered off and washed
several times with THF. Solvent was then removed, MeOH added, and
the precipitate filtered off. This step was repeated with ether. The
ether was removed and the residue twice recrystallized (cyclohexane/
toluene, then cyclohexane/DME) to give 16d as fluffy cream needles
(1.95 g, 24%); mp 109−111 °C. HRMS−ESI (m/z): [M + H]+ calcd
1
for C24H22N3O2, 383.1708; found, 383.1712. H NMR (CDCl3): δ
8.77 (dd, J = 4.8, 0.8 Hz, 1H), 8.69 (d, J = 7.6 Hz, 1H), 8.28 (dd, J =
8.4, 0.8 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.87 (dt, J = 7.6, 1.6 Hz,
1H), 7.86 (s, 1H), 7.69 (dt, J = 7.2, 1.6 Hz, 2H), 7.51−7.45 (3H), 7.37
(dt, J = 4.8, 0.8 Hz, 2H), 5.06 (q, J = 6.8 Hz, 1H), 3.33 (s, 3H), 1.69
(d, J = 6.4 Hz, 3H). 13C NMR (CDCl3): δ 170.2, 160.8, 157.0, 156.3,
149.1, 148.7, 142.5, 136.8, 130.2, 129.9, 129.0, 128.6, 127.9, 125.8,
124.1, 122.4, 121.7, 121.4, 98.7, 71.1, 38.1, 18.5.
This method also gave N-phenyl-2-{[2-(pyridin-2-yl)quinolin-4-
yl]oxy}propanamide (22d) (see Supporting Information).
Determination of Absolute Configuration of Enantiomer of
(−)-10a. The solvent-corrected IR and VCD spectra of a 0.2 M
solution of (−)-10a in CDCl3 in a BaF2 cell (100 μm path length)
were recorded with 4 cm−1 resolution over the span of 22 h. PEM was
optimized at 1400 cm−1.
Determination of Ligand Binding Affinities for Rat Brain
TSPO. Binding assays were performed as previously described,26
except that crude rat brain homogenates were used instead of
mitochondrial fractions. Data were analyzed with nonlinear regression
curve-fitting software (GraphPad Prism 5; GraphPad Prism, San
Diego, CA, USA). Briefly, whole rat brains from Sprague−Dawley rats
were homogenized in cold HEPES buffer (50 mM; pH = 7.4) with a
Teflon pestle and Glas-Col homogenizing system. The homogenates
were centrifuged at 20000g for 15 min at 4 °C. The pellets were then
resuspended, aliquotted into various vials, and stored at −80 °C. A
self-displacement assay on 1 was used as a control along with each
assay of test ligand with [3H]1 as reference radioligand. The
individually calculated control KD values for 1 were compared to the
reported value of 0.707 nM26 as an assurance of the correctness of
results obtained on test ligands. The KD value of 0.707 nM for 1 was
used as the dissociation constant to calculate Ki values for test ligands.
Determination of Ligand Binding Affinity to Human
Leukocyte HAB and LAB TSPO. Assays on human leukocyte
homogenates were performed as described previously32 Data were
analyzed with nonlinear regression curve-fitting software (GraphPad
Prism 5; GraphPad Prism). Ki values for the test TSPO ligands were
measured in triplicate in two HAB and two low LAB tissues whose
genotype had been predetermined. As a control in each assay, a self-
displacement assay on 1 was performed to calculate KD. A mean KD of
4.7 nM29 was used to calculate test ligand Ki. All data were fitted to a
one-site model to determine the ratio of Kis for test ligands between
HABs and LABs.
1
for C12H10N2OBr, 276.9976; found, 276.9974. H NMR (CDCl3): δ
8.54 (br s, 1H), 8.49 (dd, J = 4.8, 1.6 Hz, 1H), 7.97−7.94 (3H), 7.60
(tt, J = 7.2, 1.2 Hz, 1H), 7.52 (tt, J = 7.6, 1.6 Hz, 2H), 7.05 (dd, J = 8.0,
4.8 Hz, 1H). 13C NMR (CDCl3): δ 165.0, 148.8, 147.5, 141.5, 134.2,
132.4, 128.9, 127.5, 121.6, 112.7.
Method E: N-(2-Acetylpyridin-3-yl)benzamide (17a). n-BuLi
in hexanes (24 mL, 38 mmol) was added dropwise to a solution of N-
(2-bromopyridin-3-yl)benzamide61 (4.82 g, 17.4 mmol) in THF (280
mL) at −78 °C under Ar. This solution was stirred for another 30 min
and then warmed to −30 °C for 15 min. N-Methoxy-N-
methylacetamide (4.1 mL, 38 mmol) was added dropwise and the
solution warmed to rt. The mixture was quenched with 0.5 M HCl
(110 mL). Saturated NaHCO3 solution was added to neutralize the
mixture, and the product was extracted into DCM (180 mL × 2). The
combined extracts were washed with brine (180 mL) and then dried
(MgSO4). The product was purified by FC (hexanes/EtOAc/TEA,
10:1:1%) to yield a yellow solid that was further triturated with
hexanes to give 17a as a white powder (1.22 g, 29%); mp 89−91 °C.
HRMS−ESI (m/z): [M + H]+ calcd for C14H13N2O2, 241.0977;
found, 241.0979. 1H NMR (CDCl3): δ 12.53 (s, 1H), 9.32 (dd, J = 8.4,
1.2 Hz, 1H), 8.42 (dd, J = 4.4, 1.6 Hz, 1H), 8.08−8.06 (m, 2H), 7.59−
7.52 (4H), 2.85 (s, 3H). 13C NMR (CDCl3): δ 206.3, 166.5, 143.0,
138.4, 137.4, 134.1, 132.4, 128.9, 128.7, 128.2, 127.5, 27.8.
This method also gave N-(3-acetylpyridin-4-yl)benzamide (17b),
and N-(3-acetylpyridin-2-yl)benzamide (17c) (see Supporting In-
formation).
Method F: 2-Phenyl-1,4-dihydro-1,5-naphthyridin-4-one
(18a). 17a (1.17 g, 4.88 mmol) and NaOH (585 mg, 14.6 mmol)
in dioxane (50 mL) were heated in a pressure vessel to 110 °C under
N2 for 3 h. The mixture was cooled to rt, the solvent removed, and the
residue taken up in water (10 mL) and hexanes (100 mL). The
aqueous phase was acidified with 1 M HCl and then neutralized with
satd NaHCO3. A cream precipitate was collected and dried in vacuo
with BaO for 3 d to give 17a as a hard, mustard-yellow solid (0.93 g,
86%); mp >300 °C. HRMS−ESI (m/z): [M + H]+ calcd for
C14H11N2O, 223.0871; found, 223.0872. 1H NMR (DMSO-d6): δ 8.49
(d, J = 2.8 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 7.2 Hz, 2H),
7.52−7.40 (4H), 6.85 (s, 1H), 3.62 (br s, 1H). 13C NMR (DMSO): δ
173.5, 156.1, 144.7, 143.9, 142.1, 139.7, 135.0, 128.6, 128.3, 126.9,
124.0, 107.6.
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Journal of Medicinal Chemistry
Article
entiation of mouse erythroleukemia cellsa possible involvement of
these receptors in heme biosynthesis. J. Biol. Chem. 1994, 269, 7527−
7531.
(9) Benavides, J.; Fage, D.; Carter, C.; Scatton, B. Peripheral type
benzodiazepine binding sites are a sensitive indirect index of neuronal
damage. Brain Res. 1987, 421, 167−172.
(10) Cagnin, A.; Gerhard, A.; Banati, R. B. In vivo imaging of
neuroinflammation. Eur. Neuropsychopharmacol. 2002, 12, 581−586.
(11) Banati, R. B. Visualising microglial activation in vivo. Glia 2002,
40, 206−217.
(12) Imaizumi, M.; Kim, H.-J.; Zoghbi, S. S.; Briard, E.; Hong, J.;
Musachio, J. L.; Ruetzler, C.; Chuang, D.-M.; Pike, V. W.; Innis, R. B.;
Fujita, M. PET imaging with [11C]PBR28 can localize and quantify
upregulated peripheral benzodiazepine receptors associated with
cerebral ischemia in rat. Neurosci. Lett. 2007, 411, 200−205.
(13) Cagnin, A.; Brooks, D. J.; Kennedy, A. M.; Gunn, R. N.; Myers,
R.; Turkheimer, F. E.; Jones, T.; Banati, R. B. In vivo measurement of
activated microglia in dementia. Lancet 2001, 358, 461−467.
(14) Kreisl, W. C.; Lyoo, C. H.; McGwier, M.; Snow, J.; Jenko, K. J.;
Kimura, N.; Corona, W.; Morse, C. L.; Zoghbi, S. S.; Pike, V. W.;
McMahon, F. J.; Turner, R. S.; Innis, R. B. Biomarkers Consortium
PET Radioligand Project Team. In vivo radioligand binding to
translocator protein correlates with severity of Alzheimer’s disease.
Brain 2013, 136, 2228−2238.
(15) Ouchi, Y.; Yoshikawa, E.; Sekine, Y.; Futatsubashi, M.; Kanno,
T.; Ogusu, T.; Torizuka, T. Microglial activation and dopamine
terminal loss in early Parkinson’s disease. Ann. Neurol. 2005, 57, 168−
175.
(16) Debruyne, J. C.; Versijpt, J.; Van Laere, K. J.; De Vos, F.;
Keppens, J.; Strijckmans, K.; Achten, E.; Slegers, G.; Dierckx, R. A.;
Korf, J.; De Reuck, J. L. PET visualization of microglia in multiple
sclerosis patients using [11C]PK11195. Eur. J. Neurol. 2003, 10, 257−
264.
(17) Owen, D. R. J.; Matthews, P. M. Imaging brain microglial
activation using positron emission tomography and translocator
protein-specific radioligands. In Biomarkers of Neurological and
Psychiatric Disease; Guest, P. C.; Bahn, S., Eds.; Academic Press:
Waltham, MA, 2011; Vol. 101, pp 19−39.
(18) Camsonne, R.; Moulin, M. A.; Crouzel, C.; Syrota, A.; Maziere,
M.; Comar, D. C-11 Labeling of PK11195 and visualization of
peripheral receptors of benzodiazepines by positron-emission
tomography. J. Pharmacol. 1986, 17, 383.
(19) Shah, F.; Hume, S. P.; Pike, V. W.; Ashworth, S.; McDermott, J.
Synthesis of the enantiomers of [N-methyl-11C]PK 11195 and
comparison of their behaviors as radioligands for PK binding sites in
rats. Nucl. Med. Biol. 1994, 21, 573−581.
(20) Debruyne, J. C.; Van Laere, K. J.; Versijpt, J.; De Vos, F.; Eng, J.
K.; Strijckmans, K.; Santens, P.; Achten, E.; Slegers, G.; Korf, J.;
Dierckx, R. A.; De Reuck, J. L. Semiquantification of the peripheral-
type benzodiazepine ligand [11C]PK11195 in normal human brain and
application in multiple sclerosis patients. Acta Neurol. Belg. 2002, 102,
127−135.
ASSOCIATED CONTENT
* Supporting Information
Syntheses of compounds not described in main text and
determination of absolute configuration of (−)-10a. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: 301 594 5986. Fax: 301 480 5112. E-mail: pikev@mail.
nih.gov.
Author Contributions
This paper was written with contributions from all authors. All
authors approved the final version of the manuscript.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by the Intramural Research Program
of the National Institutes of Health (NIH), specifically the
National Institute of Mental Health. We thank the NIH PET
Department for radioisotope production.
ABBREVIATIONS USED
■
DCA, 1,2-dichloroethane; DIPA, diisopropylethylamine; DPE-
Phos, bis-[(2-diphenylphosphino)phenyl]ether; HAB, high-
affinity binder; LAB, low-affinity binder; MAB, mixed-affinity
binder; HFIP, hexafluoroisopropanol; PFP, pentafluorophenyl;
PyBroP, bromotripyrrolidinophosphonium hexafluorophos-
phate; TEA, triethylamine; TSPO, translocator protein (18
kDa)
REFERENCES
■
(1) Anholt, R. R. H.; De Souza, E. B.; Oster-Granite, M. L.; Snyder,
S. H. Peripheral-type benzodiazepine receptors: autoradiographic
localization in whole-body sections of neonatal rats. J. Pharmacol.
Exp. Ther. 1985, 233, 517−526.
(2) Papadopoulos, V.; Baraldi, M.; Guilarte, T. R.; Knudsen, T. B.;
̀
Lacapere, J. J.; Lindemann, P.; Norenberg, M. D.; Nutt, D.; Weizman,
A.; Zhang, M.-R.; Gavish, M. Translocator protein (18 kDa): new
nomenclature for the peripheral-type benzodiazepine receptor based
on its structure and molecular function. Trends Pharmacol. Sci. 2006,
27, 402−409.
(3) Syapin, P. J.; Skolnick, P. Characterization of benzodiazepine
binding sites in cultured cells of neural origin. J. Neurochem. 1979, 32,
1047−1051.
(4) McEnery, M. W.; Snowman, A. M.; Trifiletti, R. R.; Snyder, S. H.
Isolation of the mitochondrial benzodiazepine receptor: Association
with the voltage-dependent anion channel and the adenine nucleotide
carrier. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 3170−3174.
(5) Doble, A.; Malgouris, C.; Daniel, M.; Daniel, N.; Imbault, F.;
(21) Kropholler, M. A.; Boellaard, R.; Schuitemaker, A.; Folkersma,
H.; van Berckel, B. N. M.; Lammertsma, A. A. Evaluation of reference
tissue models for the analysis of [11C](R)-PK11195 studies. J. Cereb.
Blood Flow Metab. 2006, 26, 1431−1441.
́ ́
Basbaum, A.; Uzan, A.; Gueremy, C.; Le Fur, G. Labelling of
(22) Greuter, H. N. J. M.; van Ophemert, P. L. B.; Luurtsema, G.; van
Berckel, B. N. M.; Franssen, E. J. F.; Windhorst, B. D.; Lammertsma,
A. A. Optimizing an online SPE−HPLC method for analysis of (R)-
[11C]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoli-
necarboxamide [(R)-[11C]PK11195] and its metabolites in humans.
Nucl. Med. Biol. 2005, 32, 307−312.
peripheral-type benzodiazepine binding sites in human brain with
[3H]PK 11195: anatomical and subcellular distribution. Brain Res. Bull.
1987, 18, 49−61.
(6) Wright, G.; Reichenbecher, V. The effects of superoxide and the
peripheral benzodiazepine receptor ligands on the mitochondrial
processing of manganese-dependent superoxide dismutase. Exp. Cell
Res. 1999, 246, 443−450.
́
(23) Chauveau, F.; Boutin, H.; Van Camp, N.; Dolle, F.; Tavitian, B.
Nuclear imaging of neuroinflammation: a comprehensive review of
[11C]PK11195 challengers. Eur. J. Nucl. Med. Mol. Imaging 2008, 35,
2304−2319.
(7) Papadopoulos, V.; Lecanu, L.; Brown, R. C.; Han, Z.; Yao, Z.-X.
Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis,
neuropathology and neurological disorders. Neuroscience 2006, 138,
749−756.
(8) Taketani, S.; Kohno, H.; Okuda, M.; Furukawa, T.; Tokunaga, R.
Induction of peripheral-type benzodiazepine receptors during differ-
(24) Schweitzer, P. J.; Fallon, B. A.; Mann, J. J.; Kumar, J. S. D. PET
tracers for the peripheral benzodiazepine receptor and uses thereof.
Drug Discovery Today 2010, 15, 933−942.
6249
dx.doi.org/10.1021/jm5007947 | J. Med. Chem. 2014, 57, 6240−6251

Journal of Medicinal Chemistry
Article
(25) Luus, C.; Hanani, R.; Reynolds, A.; Kassiou, M. The
development of PET radioligands for imaging the translocator protein
(18 kDa): What have we learned? J. Labelled Compd. Radiopharm.
2010, 53, 501−510.
(26) Briard, E.; Zoghbi, S. S.; Imaizumi, M.; Gourley, J. P.; Shetty, H.
U.; Hong, J.; Cropley, V.; Fujita, M.; Innis, R. B.; Pike, V. W. Synthesis
and evaluation in monkey of two sensitive 11C-labeled aryloxyanilide
ligands for imaging brain peripheral benzodiazepine receptors in vivo.
J. Med. Chem. 2008, 51, 17−30.
(27) Brown, A. K.; Fujita, M.; Fujimura, Y.; Liow, J. S.; Stabin, M.;
Ryu, Y. H.; Imaizumi, M.; Hong, J.; Pike, V. W.; Innis, R. B. Radiation
dosimetry and biodistribution in monkey and man of [11C]PBR28: a
PET radioligand to image inflammation. J. Nucl. Med. 2007, 48, 2072−
2079.
(38) Dubroeucq, M.-C.; Ben
́
avides
̀
, J.; Doble, A.; Guilloux, F.; Allam,
́
D.; Vaucher, N.; Bertrand, P.; Gueremy, C.; Renault, C.; Uzan, A.; Le
́
Fur, G. Stereoselective inhibition of the binding of [3H]PK 11195 to
peripheral-type benzodiazepine binding sites by a quinolinepropana-
mide derivative. Eur. J. Pharmacol. 1986, 128, 269−272.
(39) Pike, V. W. Positron-emitting radioligands for studies in vivo
probes for human psychopharmacology. J. Psychopharmacol. 1993, 7,
139−158.
(40) Laruelle, M.; Slifstein, M.; Huang, Y. Relationships between
radiotracer properties and image quality in molecular imaging of the
brain with positron emission tomography. Mol. Imaging Biol. 2003, 5,
363−375.
(41) Patel, S.; Gibson, R. In vivo site-directed radiotracers: a mini-
review. Nucl. Med. Biol. 2008, 35, 805−815.
(28) Fujita, M.; Imaizumi, M.; Zoghbi, S. S.; Fujimura, Y.; Farris, A.
G.; Suhara, T.; Hong, J.; Pike, V. W.; Innis, R. B. Kinetic analysis in
healthy humans of a novel positron emission tomography radioligand
to image the peripheral benzodiazepine receptor, a potential biomarker
for inflammation. Neuroimage 2008, 40, 43−52.
(29) Kreisl, W. C.; Fujita, M.; Fujimura, Y.; Kimura, N.; Jenko, K. J.;
Kannan, P.; Hong, J.; Morse, C. L.; Zoghbi, S. S.; Gladding, R. L.;
Jacobson, S.; Oh, U.; Pike, V. W.; Innis, R. B. Comparison of
[11C](R)-PK 11195 and [11C]PBR28, two radioligands for translocator
protein (18 kDa) in human and monkey: Implications for positron
emission tomographic imaging of this inflammation biomarker.
Neuroimage 2010, 49, 2924−2932.
(30) Owen, D. R.; Howell, O. W.; Tang, S.-P.; Wells, L. A.; Bennacef,
I.; Bergstrom, M.; Gunn, R. N.; Rabiner, E. A.; Wilkins, M. R.;
Reynolds, R.; Matthews, P. M.; Parker, C. A. Two binding sites for
[3H]PBR28 in human brain: implications for TSPO PET imaging of
neuroinflammation. J. Cereb. Blood Flow Metab. 2010, 30, 1608−1618.
(31) Owen, D. R.; Yeo, A. J.; Gunn, R. N.; Song, K.; Wadsworth, G.;
Lewis, A.; Rhodes, C.; Pulford, D. J.; Bennacef, I.; Parker, C. A.; StJean,
P. L.; Cardon, L. R.; Mooser, V. E.; Matthews, P. M.; Rabiner, E. A.;
Rubio, J. P. An 18-kDa Translocator protein (TSPO) polymorphism
explains differences in binding affinity of the PET radioligand PBR28.
J. Cereb. Blood Flow Metab. 2012, 32, 1−5.
(32) Kreisl, W. C.; Jenko, K. J.; Hines, C. S.; Lyoo, C. H.; Corona,
W.; Morse, C. L.; Zoghbi, S. S.; Hyde, T.; Kleinman, J. E.; Pike, V. W.;
McMahon, F. J.; Innis, R. B.; Biomarkers Consortium, PET
Radioligand Project Team. A genetic polymorphism for translocator
protein 18 kDa affects both in vitro and in vivo radioligand binding in
human brain to this putative biomarker of neuroinflammation. J. Cereb.
Blood Flow Metab. 2013, 33, 53−58.
(42) Leeson, P. D.; Springthorpe, B. The influence of drug-like
concepts on decision-making in medicinal chemistry. Nature Rev. Drug
Discovery 2007, 6, 881−890.
(43) Ryckmans, T.; Edwards, M. P.; Horne, V. A.; Correia, A. M.;
Owen, D. R.; Thompson, L. R.; Tran, I.; Tutt, M. F.; Young, T. Rapid
assessment of a novel series of selective CB2 agonists using parallel
synthesis protocols: a lipophilic efficiency (LipE) analysis. Bioorg. Med.
Chem. Lett. 2009, 19, 4406−4409.
(44) Shultz, M. D. Setting expectations in molecular optimizations:
strengths and limitations of commonly used composite parameters.
Bioorg. Med. Chem. Lett. 2013, 23, 5980−5991.
(45) Kuninobu, Y.; Inoue, Y.; Takai, K. Copper(I)- and gold(I)-
catalyzed synthesis of 2,4-disubstituted quinoline derivatives from N-
aryl-2-propynylamines. Chem. Lett. 2007, 36, 1422−1423.
́
(46) Frerot, E.; Coste, J.; Pantaloni, A.; Dufour, M.-N.; Jouin, P.
PyBOP and PyBroP: two reagents for the difficult coupling of the α,α-
dialkyl amino acid, Aib. Tetrahedron 1991, 47, 259−270.
(47) Woodbury, R. P.; Rathke, M. W. Isolation and reactions of α-
lithio N,N-dimethylacetamide. J. Org. Chem. 1977, 42, 1688−1690.
(48) VanAllan, J. A. A new process for the preparation of
thioglycolylamides. J. Am. Chem. Soc. 1947, 69, 2914.
(49) Wolf, C.; Lerebours, R. Use of highly active palladium−
phosphinous acid catalysts in Stille, Heck, amination, and thiation
reactions of chloroquinolines. J. Org. Chem. 2003, 68, 7077−7084.
(50) Margolis, B. J.; Long, K. A.; Laird, D. L. T.; Ruble, J. C.; Pulley,
S. R. Assembly of 4-aminoquinolines via palladium catalysis: a mild
and convenient alternative to SNAr methodology. J. Org. Chem. 2007,
72, 2232−2235.
(51) Knouzi, N.; Vaultier, M.; Carrie,
triphenylphosphine en presence d’eau: une met
chimioselective d’acces aux amines primaires. Bull. Soc. Chim. Fr. 1985,
122, 815−819.
́
R. Red
́
uction d’azides par la
́
́
́
hode generale et
́
́
(33) Owen, D. R. J.; Gunn, R. N.; Rabiner, E. A.; Bennacef, I.; Fujita,
M.; Kreisl, W. C.; Innis, R. B.; Pike, V. W.; Reynolds, R.; Matthews, P.
M.; Parker, C. A. Mixed-affinity binding in humans with 18-kDa
translocator protein ligands. J. Nucl. Med. 2011, 52, 24−32.
(34) Mizrahi, R.; Rusjan, P. M.; Kennedy, J.; Pollock, B.; Mulsant, B.;
Suridjan, I.; De Luca, V.; Wilson, A. A.; Houle, S. Translocator protein
(18 kDa) polymorphism (rs6971) explains in vivo brain binding
affinity of the PET radioligand [18F]-FEPPA. J. Cereb. Blood Flow
Metab. 2012, 32, 968−972.
́
̀
(52) Winters, R. T.; Sercel, A. D.; Showalter, H. D. H. Efficient
synthesis of peri-hydroxylated 9,10-anthracenedione ethers via
alkylation of cesium phenolates. Synthesis 1988, 712−714.
(53) Nahm, S.; Weinreb, S. M. N-Methoxy-N-methylamides as
effective acylating agents. Tetrahedron Lett. 1981, 22, 3815−3818.
(54) Camps, R. Synthese von α- und γ-oxychinolinen. Ber. Dtsch.
Chem. Ges. 1899, 32, 3228−3234.
(35) Pike, V. W.; Taliani, S.; Lohith, T. G.; Owen, D. R. J.; Pugliesi,
I.; Da Pozzo, E.; Hong, J.; Zoghbi, S. S.; Gunn, R. N.; Parker, C. A.;
Rabiner, E. A.; Fujita, M.; Innis, R. B.; Martini, C.; Da Settimo, F.
Evaluation of novel N1-methyl-2-phenylindol-3-ylglyoxylamides as a
new chemotype of 18 kDa translocator protein-selective ligand suitable
for the development of positron emission tomography radioligands. J.
Med. Chem. 2011, 54, 366−373.
(36) Guo, Q.; Colasanti, A.; Owen, D. R.; Onega, M.; Kamalakaran,
A.; Bennacef, I.; Matthews, P. M.; Rabiner, E. A.; Turkheimer, F. E.;
Gunn, R. N. Quantification of the specific translocator protein signal of
18F-PBR111 in healthy humans: a genetic polymorphism effect on in
vivo binding. J. Nucl. Med. 2013, 54, 1915−1923.
(55) Jones, C. P.; Anderson, K. W.; Buchwald, S. L. Sequential Cu-
catalyzed amidation-base-mediated Camps cyclization: a two-step
synthesis of 2-aryl-4-quinolones from o-halophenones. J. Org. Chem.
2007, 72, 7968−7973.
(56) He, Y.; Wang, B.; Dukor, R. K.; Nafie, L. A. Determination of
absolute configuration of chiral molecules using vibrational optical
activity: a review. Appl. Spectrosc. 2011, 65, 699−723.
(57) Cappelli, A.; Anzini, M.; Vomero, S.; De Benedetti, P. G.;
Menziani, M. C.; Giorgi, G.; Manzoni, C. Mapping the peripheral
benzodiazepine receptor binding site by conformationally restrained
derivatives of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-iso-
quinolinecarboxamide (PK11195). J. Med. Chem. 1997, 40, 2910−
2921.
(58) Pike, V. W.; Halldin, C.; Crouzel, C.; Barre, L.; Nutt, D. J.;
́
Osman, S.; Shah, F.; Turton, D. R.; Waters, S. L. Radioligands for PET
studies of central benzodiazepine receptors and PK (peripheral
(37) Guo, Q.; Owen, D. R.; Rabiner, E. A.; Turkheimer, F. E.; Gunn,
R. N. Identifying improved TSPO PET imaging probes through
biomathematics: the impact of multiple TSPO binding sites in vivo.
NeuroImage 2012, 60, 902−910.
6250
dx.doi.org/10.1021/jm5007947 | J. Med. Chem. 2014, 57, 6240−6251

Journal of Medicinal Chemistry
Article
benzodiazepine) binding sitescurrent status. Nucl. Med. Biol. 1993,
20, 503−525.
(59) Pettigrew, J. D.; Freeman, R. P.; Wilson, P. D. Total synthesis of
(−)-xyloketal D and its enantiomerconfirmation of absolute
stereochemistry. Can. J. Chem. 2004, 82, 1640−1648.
(60) Bischoff, C. A. Studien uber Verkettungen. XXVI. Das
̈
Aethylanilin. Ber. Dtsch. Chem. Ges. 1897, 30, 3178−3180.
́
(61) Garnier, E.; Blanchard, S.; Rodriguez, I.; Jarry, C.; Leger, J.-M.;
̀
Caubere, P.; Guillaumet, G. New access to oxazolopyridines via
hydroxyamidine derivatives; application to quinolines. Synthesis 2003,
2033−2040.
(62) Korner, M. Ueber das Benzoyl-o-Amidobenzamid. J. Prakt.
̈
Chem. (Leipzig) 1887, 36, 155−165.
6251
dx.doi.org/10.1021/jm5007947 | J. Med. Chem. 2014, 57, 6240−6251