Synthesis, fluorine-18 radiolabeling, and in vitro characterization of 1-iodophenyl-N-methyl-N-fluoroalkyl-3-isoquinoline carboxamide derivatives as potential PET radioligands for imaging peripheral benzodiazepine receptor

Article abstract of DOI:10.1016/j.bmc.2008.04.046

The isoquinoline carboxamide derivative 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (PK11195) has been shown to bind strongly and selectively to the peripheral benzodiazepine receptor (PBR) binding sites. A series of PK11195

Full text of DOI:10.1016/j.bmc.2008.04.046

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 6145–6155
Synthesis, fluorine-18 radiolabeling, and in vitro characterization
of 1-iodophenyl-N-methyl-N-fluoroalkyl-3-isoquinoline
carboxamide derivatives as potential PET radioligands for
imaging peripheral benzodiazepine receptor
Weiping Yu,a Eric Wang,a Ronald J. Voll,a Andrew H. Millerb and Mark M. Goodmana,*
aDepartment of Radiology, School of Medicine, Emory University, Atlanta, GA 30322, USA
bDepartment of Psychiatry and Behavioral Sciences, School of Medicine, Emory University, Atlanta, GA 30322, USA
Received 17 March 2008; revised 15 April 2008; accepted 16 April 2008
Available online 25 April 2008
Abstract—The isoquinoline carboxamide derivative 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide
(PK11195) has been shown to bind strongly and selectively to the peripheral benzodiazepine receptor (PBR) binding sites. A series
of PK11195 analogues have been synthesized and biologically characterized. The affinities of the analogues for the PBR were deter-
mined using in vitro competitive binding assays with [3H]PK11195 in rat kidney mitochondrial membranes. The results showed that
the 1-(2-iodophenyl)-N-methyl-N-(3-fluoropropyl)-3-isoquinoline carboxamide (9a) was the most potent compound (Ki = 0.26 nM)
of this series and is an excellent lead ligand for additional studies for labeling with fluorine-18 to determine whether it possesses the
desired in vivo performance in non-human primates by PET imaging. Thus, radiolabeling of 9a with fluorine-18 was developed.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
pies, and for monitoring patient response to therapy.
These advances have also helped to identify targets for
molecular radiotherapy, making it possible to target
radiation at the cellular and molecular level. The under-
standing of the structure and the function of PBR is not
as advanced as that of CBR. Nonetheless, its concentra-
tion at brain lesions, tumors, and inflammatory sites
now engenders much clinical interest.
The development and biological evaluation of radioli-
gands for positron emission tomography (PET) studies
of central benzodiazepine receptors (CBRs) and periph-
eral benzodiazepine receptors (PBRs) are of great value
as they have been associated with a variety of disease
processes. Although CBR and PBR are pharmacologi-
cally and structurally distinct from each other,1,2 despite
their historical connection through the benzodiazepines,
they both hold interest for present and future PET stud-
ies on, for example, epilepsy, Alzheimer’s disease, alco-
holism, and anxiety with respect to CBR, and for
stroke, neuro-protection, inflammation, and tumors
with respect to PBR.
PBR has been implicated in the regulation of various
cellular processes, including regulation of steroidogene-
sis, immunomodulation, porphyrin transport, heme syn-
thesis, cell apoptosis and proliferation.1,3–5 PBR
expression levels were shown to be increased in a variety
of cancers indicating its important role in cancer devel-
opment.6–12 PBR has been reported to be a sensitive
marker for various pathologies of glial activation in
brain injuries.13–22 Increased and decreased PBR levels
have been demonstrated as biological indicators of
stressful conditions23 in several psychiatric disorders as
it is closely associated with personality traits for anxiety
tolerance.24
Advances in our understanding of the molecular biology
of cancer and other diseases have identified molecules
and signaling pathways that we can visualize, in vivo,
for diagnosis and staging, for identifying optimal thera-
Keywords: PK11195; Fluorine-18; Positron emission tomography; In-
flammation; Peripheral benzodiazepine receptor.
PBR is a ubiquitous high affinity drug ligand and choles-
terol binding protein. Functional PBR is described as a
*
Corresponding author. Tel.: +1 404 727 9366; fax: +1 404 727
3488; e-mail: mgoodma@emory.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.046

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W. Yu et al. / Bioorg. Med. Chem. 16 (2008) 6145–6155
multimeric complex composed of evolutionarily con-
served, highly hydrophobic 18 kDa receptor protein,25
the 32 kDa voltage-dependent anion channel (VDAC)
protein required for benzodiazepine binding and the
30 kDa adenine nucleotide carrier (ANC) of an as yet
unclear function in the complex.26–29 PBR is mainly lo-
cated and spanned on the outer mitochondrial mem-
brane, but its localization has also been detected on
plasma membranes,30,31 the Golgi apparatus, lysosomes,
rough endoplasmic reticular microsomes, peroxi-
somes,30 and nuclear membranes.9,32
analogues labeled with fluorine-18 to image PBR with
PET. In this paper, we describe the synthesis and the
preliminary in vitro evaluation of a series of 1-iodo-
phenyl-N-methyl-N-fluoroalkyl-3-isoquinoline carbox-
amides (9a–d). The best PBR affinity ligand 9a was
chosen to explore the fluorine-18 labeling methodology.
Although [11C]PK11195 has been used as a valuable
PET reagent to image PBR, the development of 1-iodo-
phenyl-N-methyl-N-[18F]fluoroalkyl-3-isoquinoline car-
boxamides as new PBR raioligands has at least three
advantages over [11C]PK11195. Firstly, [18F] has a
half-life of 110 min while [11C] has a 20 min half-life.
The longer half-life of [18F] allows for longer imaging
time which gives rise to effective clearance of non-spe-
cific binding and thus allows for imaging of more subtle
specific PBR binding. Secondly, the new radioligands
[18F]9a–d do not possess a chiral center whereas [11C]-
Specific PBR ligands are divided into two classes, endog-
enous and synthetic ligands. Endogenous PBR ligands
include the human protein sequences of diazepam bind-
ing inhibitor (DBI)33 and its derived fragments (TTN,
triakontatetraneuropeptide, DBI 17–50; EPN, eicosa-
neuropeptide, DBI 26–50; and ODN, octadecaneuropep-
tide, DBI 33–50),34 porphyrines35 and cholesterol.36,37
The representative synthetic PBR ligands include 40-chlo-
PK11195 does, including its analogues
[
125I]-
PK11195,52 [11C]PK11211,53,54 and [18F]PK14105.55,56
It is well known that in general individual enantiomers
may behave differently than racemic mixtures in vivo
with respect to specific receptor binding, protein bind-
ing,57 pharmacokinetics,57,58 and metabolism.59 Where
such differences exist the mathematical interpretation
of kinetic data from the use of the racemic radioligands
may lead to more complicated kinetic modeling.60 It has
also been reported that the binding of PK11195 to PBR
binding sites may be inhibited stereoselectively.61 These
factors have not been evaluated for PK11195.2 By using
non-chiral radiotracers for imaging PBR with PET, it
not only eliminates all the above mentioned complica-
tions caused by racemic ligands, but also significantly
simplifies the synthesis procedures, since synthesis of
>99% enantiomerically enriched compounds poses sig-
nificant challenges for chemists. An additional advan-
tage of [18F] ligands is that it can be produced off-site
whereas [11C] requires production via an on-site cyclo-
tron. Results of these new tracers’ in vivo biodistribu-
tion and displacement experiments, in vivo metabolic
stability, and comparative microPET studies with
PK11195 in non-human primates are currently in pro-
gress and will be reported in a separate publication.
rodiazepam
Ro54864
[7-chloro-5-(4-chlorophenyl)-
1-methyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one],38,39
isoquinoline carboxamide PK11195 [1-(2-chlorophenyl)-
N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxam-
ide],40,41 indolacetamide derivative FGIN-1-27 [N,N-di-
n-hexyl-2-(4-fluorophenyl) indole-3-acetamide]40,42–44
,
and pyridazinoindole derivative SSR180575 (7-chloro-
N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridoz-
ino[4,5-b]indole-1-acetamide)45–47 (Fig. 1). These com-
pounds’ affinity (Kd) for human PBR is selective in the
nanomolar range: Ro54864, 6 nM; PK11195, 2 nM;
FGIN-1-27, 4 nM and SSR180575, 3.5 nM.37
The development of specific potent PBR ligands may be
of great value for the management of a broad range of
different pathological applications such as cancer, auto-
immune diseases, viral infection, neurodegeneration,
stress, and brain injury. PK11195 was the first non-ben-
zodiazepine high affinity PBR ligand and is the most
widely used pharmacological tool for the study of the
expression and the function of PBR. The isoquinolines
that bind specifically to PBR interact with the 18 kDa
subunit of the receptor complex. PK11195 does not
show the variations in affinity that the majority of ben-
zodiazepines do, and it has a consistent high affinity
(Kd < 20 nM) in all species40,41,43,48,49 whereas the affin-
ity for Ro54864 is highly species-dependent50,51 and
temperature sensitive.40
2. Results and discussion
2.1. Synthesis of reference compounds
The syntheses of 1-iodophenyl-3-isoquinoline carboxylic
acids 7a–c were shown in Scheme 1. Iodobenzoyl chlo-
rides 2a–c, which were prepared from their correspond-
Based on but not limited to the above mentioned facts,
we decided to develop new isoquinoline carboxamide
O
O
O
N
H
N
N
N
F
N
N
Cl
N
N
Cl
Cl
O
N
O
N(n-Hex)2
Cl
PK11195
Ro5-4864
FGIN-1-27
SSR180575
Figure 1. Representative synthetic PBR ligands.

W. Yu et al. / Bioorg. Med. Chem. 16 (2008) 6145–6155
6147
OH
OH
COCl
a
X
Y
NH
b
N
+
.
O
NH2 HCl
X
Y
X
Y
Z
Z
Z
(4a)
(4b)
X=I, Y=Z=H
(1)
(2a)
(2b)
(3a) X=I, Y=Z=H
(3b) X=Z=H, Y=I
(3c) X=Y=H, Z=I
X=I, Y=Z=H
X=Z=H, Y=I
X=Z=H, Y=I
(4c) X=Y=H, Z=I
(2c) X=Y=H, Z=I
CH2Br
CHO
CO2H
N
X
c
N
X
e
N
d
X
Y
Y
Y
Z
Z
Z
(5a)
X=I, Y=Z=H
X=Z=H, Y=I
(6a)
(6b)
X=I, Y=Z=H
X=Z=H, Y=I
(7a) X=I, Y=Z=H
(7b) X=Z=H, Y=I
(7c) X=Y=H, Z=I
(5b)
(5c) X=Y=H, Z=I
(6c) X=Y=H, Z=I
Scheme 1. Syntheses of 1-iodophenyl-3-isoquinoline carboxylic acids 7a–c. Reagents and conditions: (a) 5% NaOH, CH2Cl2, 0 ꢁC to rt, overnight;
(b) P2O5, o-dichlorobenzene, reflux, overnight; (c) NBS, AIBN, CCl4, reflux, overnight; (d) NaIO4, DMF, reflux, overnight; (e) AgNO3, NaOH,
EtOH/H2O, rt, 2 h.
ing acids by treatment with phosphorous (V) chloride,
were reacted with norephedrine hydrochloride (1),
respectively, to afford benzamides 3a–c. The benzamides
were cyclized by a modified Pictet–Gams reaction62 to
provide isoquinoline derivatives 4a–c. The bromides
5a–c were obtained by free radical-promoted bromina-
tion of 4a–c, respectively, using N-bromosuccinimide
(NBS)/2,2-azobisisobutyro-nitrile (AIBN). Oxidative
hydrolysis of the bromides by treatment of 5a–c with so-
dium periodate gave the aldehydes 6a–c, respectively.
Compounds 5b–c and 6b–c were used for the next reac-
tion without further purification. For characterization,
compounds 5a and 6a were isolated and analyzed to
confirm their identities. 1-Iodophenyl-3-isoquinoline
carboxylic acids 7a–c were synthesized by further oxida-
tion of aldehydes 6a–c with silver nitrate, respectively.
to give secondary amides 8a–c, respectively, which were
then N-alkylated with 3-fluoropropyl bromide to provide
the N-fluoropropylated PK11195 analogues 9a–c, respec-
tively. Amide 8a was reacted with 2-fluoroethyl bromide
and afforded 1-(2-iodophenyl)-N-methyl-N-(2-fluoroeth-
yl)-3-isoquinoline carboxamide 9d.
2.2. Synthesis of labeling precursor and radiosynthesis
The precursor for fluorine-18 radiolabeling of the most
potent isoquinoline carboxamide 9a (see Section 2.3)
was prepared as shown in Scheme 3. First amide 8a
was reacted with 3-triphenylmethoxypropylbromide
10, which was prepared by tritylation of 3-bromopro-
pyl alcohol, in the presence of sodium hydride in
DMF to give amide 11. The trityl group of 11 was re-
moved by treatment with trifluoroacetic acid (TFA) to
afford the alcohol 12, followed by using p-toluenesulfo-
nyl chloride to produce the tosylate 13 as F-18 labeling
precursor.
Preparations of fluorine-19 reference compounds 9a–d
are displayed in Scheme 2. 1-Iodophenyl-3-isoquinoline
carboxylic acids 7a–c were first coupled with methylamine
O
O
CO2H
( )n
NH
N
F
N
N
N
a
b
X
Y
X
Y
X
Y
Z
Z
Z
(7a)
(7b)
(7c)
(9a)
(9b)
(9c)
(9d)
X=I, Y=Z=H
X=Z=H, Y=I
X=Y=H, Z=I
(8a) X=I, Y=Z=H
(8b) X=Z=H, Y=I
(8c) X=Y=H, Z=I
X=I, Y=Z=H, n=1
X=Z=H, Y=I, n=1
X=Y=H, Z=I, n=1
X=I, Y=Z=H, n=0
Scheme 2. Syntheses of F-19 references of isoquinoline carboxamides 9a–d. Reagents and conditions: (a) 1—SOCl2, CH2Cl2, 50 ꢁC, 1 h; 2—
CH3NH2, THF, ꢀ70 ꢁC to rt, overnight; (b) 1—NaH, DMF, rt, 40 min; 2—BrCH2CH2CH2F (for 9a–c) or BrCH2CH2F (for 9d), rt, 24 h.

6148
W. Yu et al. / Bioorg. Med. Chem. 16 (2008) 6145–6155
a
Br
OH
Br
OTr
(10) Tr=CPh3
O
I
O
I
O
I
NH
N
O Tr
N
OH
N
b
N
c
N
(11)
(12)
Tr=CPh3
O
(8a)
O
I
O
I
N
O
S
N
18F
e
d
N
O
N
([18F]9a)
(13)
Scheme 3. Synthesis of [18F] labeling precursor 13 and radiosynthesis of iosquinoline carboxamide [18F]9a. Reagents and conditions: (a) TrCl, Py,
CH2Cl2, 60 ꢁC, 5 h then rt, overnight; (b) (1) NaH, DMF, rt, 30 min. (2) 10, rt, overnight; (c) TFA, CH2Cl2, rt, 40 min; (d) p-TsCl, Et3N, CH2Cl2,
0 ꢁC, 4 h; (e) [18F]KF, K2CO3, K222, DMSO, 120 ꢁC, 10 min.
Radiofluorination of [18F]9a was achieved through no-
carrier-added (NCA) nucleophilic substitution with dried
[18F]KF, potassium carbonate, and Kryptofix in DMSO
from the tosylate 13 (Scheme 3). The exchange between
[18F]fluoride and the leaving group occurred in 10 min
at 120 ꢁC. Unreacted [18F]fluoride and any radiolabel-
charged by-products were eliminated by passing the reac-
tion mixture through a silica SepPak. Purification by
semipreparative, reverse-phase HPLC afforded [18F]9a
in anaverage (n = 4) radiochemical yield of8%, decaycor-
rected from the end of bombardment (EOB). The identity
of the final [18F] radioligand was confirmed by co-injec-
tion with the authentic sample of non-radioactive 9a on
analytical HPLC. Analytical HPLC demonstrated that
the radiochemical purity of [18F]9a was over 99%. The to-
tal synthesis time including HPLC purification and radio-
has been demonstrated that in kidney68 and adrenal,69
these receptors are primarily associated with the mito-
chondrial compartment stably expressing PBR. In this
study, the binding affinity experiments were performed
by NovaScreen (Hanover, MD) under the NIMH Psy-
chotherapeutic Drug Discovery & Development Pro-
gram. The compounds 9a–d were tested for their ability
to inhibit the binding of [3H]PK11195, and their affinities
are given as Ki and IC50 in Table 1. The Ki values of
PK11195, Ro54864, and diazepam (CBR ligand)2 were
also screened under the same assay system for references.
The data in Table 1 indicate that compounds 9a and 9b
displayed high affinities for PBR, with Ki values of
0.26 nM and 1.30 nM, respectively, approximately 110
and 22 times more potent compared to the Ki value of
PK11195, respectively, and 130 and 26 times to
Ro54864, respectively, in this in vitro assay model.
Changing the iodo group on the benzene ring of the iso-
quinoline carboxamide from ortho (9a) to meta (9b) po-
sition resulted in 5-fold decreased affinity for PBR.
Further decreased affinity occurred when the iodo group
is on para (9c) position, over 240-fold decrease com-
pared to compound 9a. Replacing the N-fluoropropyl
group of 9a to N-fluoroethyl group afforded 9d, which
led to even more dramatic decrease in PBR affinity,
exhibited over 1600-fold less Ki value relative to 9a.
Thus the order of PBR affinities in this series (Ki in
nM) was 9a > 9b > 9c > 9d. This structure–affinity rela-
tionship may be of value to help predicting new PBR li-
gands as it may apply on other halogens (fluoro, chloro
and bromo), or other groups (nitro, alkoxy, alkyl, etc.)
instead of iodo on the benzene ring in the similar mole-
cules. Also the best PBR ligand 9a in this study provided
a suitable candidate to label this compound with [11C]
(N-methyl), [18F] (N-fluoropropyl), and [123I] (iodo-
pharmaceutical
administration was approximately 90 min from EOB.
formulation
for
intravenous
The lipophilicity of radioligand [18F]9a was measured
according to Wilson et al.’s procedure,63 by extracting
the test compound between octanol and pH 7.4, phos-
phate buffer. The logP7.4 value of [18F]9a is 3.0 0.05
(n = 3). It has been reported that the logP7.4 value of
radiotracers in the range of 1.0–3.0 was considered opti-
mal for brain penetrance thus, the compound [18F]9a
should be expected to enter the brain readily.64
2.3. Binding affinity assays
New isoquinoline carboxamide derivatives 9a–d were
screened for binding affinities to PBR using in vitro com-
petition binding assays with a known PBR antagonist
[3H]PK11195 in rat kidney mitochondrial membranes
according to previously reported procedures.65–67 We
chose rat renal mitochondria as a tissue source since it

W. Yu et al. / Bioorg. Med. Chem. 16 (2008) 6145–6155
6149
Table 1. In vitro competition assays of 1-iodophenyl-N-methyl-N-
fluoroalkyl-3-isoquinoline carboxamides 9a–d with [3H]PK11195 in rat
kidney mitochondrial membranes
Merck Kieselgel silica gel 60 (230–400 mesh). Silica and
C-18 SepPaks were purchased from Waters, Inc. (Mil-
ford, MA, USA). The Gelman teflon filters were pur-
chased from VWR.
O
Melting points were measured in capillary tubes using a
Mel-Temp II apparatus (Laboratory Devices, Inc., Holl-
iston, MA, USA) and are uncorrected. H NMR and
( )n
N
F
N
1
9a
X=I, Y=Z=H, n=1
9b X=Z=H, Y=I, n=1
9c X=Y=H, Z=I, n=1
13C spectra were recorded on Varian 400 MHz,
300 MHz or GE 300 MHz spectrometers at NMR Cen-
ter in Emory University, and chemical shifts (d values)
were reported as parts per million (ppm) downfield from
tetramethylsilane (TMS). Elemental analyses were per-
formed by Atlantic Microlabs, Inc. (Norcross, GA,
USA) and were within 0.4% of the theoretical values.
Mass Spectra were performed on JEOL JMS-SX102/
SX102A/E or VG 70-S double focusing mass spectrom-
eters at Mass Spectroscopy Center at Emory University
using high-resolution electrospray ionization (ESI), or
electron impact ionization (EI). Semipreparative HPLC
column (Waters XTerra Prep RP18 5 lm, 19· 100 mm)
and analytical HPLC column (Waters Nova-Pak C18,
3.9· 150 mm) were purchased from Waters Inc.
X
Y
9d
X=I, Y=Z=H, n=0
Z
Compound Ki (nM) IC50 (nM) Ki(PK11195) Ki(Ro54864)
9a
9b
0.26
1.3
0.28
1.4
111
22.2
0.46
0.07
131
26.2
0.54
0.08
9c
9d
63.1
421
66.5
445
PK11195
Ro54864
Diazepam
28.8
34
574
phenyl) to imaging PBR with PET and SPECT. Further
work is in progress in this laboratory.
The [18F]fluoride was produced at Emory University with
a 11 MeV Siemens RDS 112 negative-ion cyclotron
(Knoxville, TN, USA) by the 18O (p,n) 18F reaction using
[18O]H2O (95%). The isolated radiochemical yields were
determined using a dose-calibrator (Capintec CRC-
712M). Thin-layer chromatograms of the radiolabeled
compounds were analyzed with a Raytest System (model:
Rita Star, Germany) using the same type of silica TLC
plates from Whatman.
3. Conclusions
A series of 1-iodophenyl-N-methyl-N-fluoroalkyl-3-iso-
quinoline carboxamide derivatives 9a–d have been pre-
pared and the in vitro affinities of these compounds to
PBR in rat kidney mitochondrial membranes have been
conducted. Competition binding assays showed that the
ability of these compounds to inhibit the standard PBR li-
gand PK11195 decreased in the order of 9a, 9b, 9c, and 9d.
The most potent compound 9a was radiolabeled with
[18F] in moderate radiochemical yield with high radio-
chemical purity from its tosylated precursor 13. The lipo-
philicity of [18F]9a was measured as logP7.4, which
indicates that this tracer is suitable to image PBR in or-
gans in the body, as well as in the brain. This tracer model
labeled with [11C], [18F], or [123I] may be used as a tool to
evaluate the potential of isoquinoline carboxamide deriv-
atives as PET and SPECT radioligands for imaging PBR.
4.2. 2-Iodo-N-(2-hydroxy-1-methyl-2-phenylethyl)benz-
amide (3a)
To an ice-cooled mixture of L,D-norephedrine hydrochlo-
ride (1, 5.28 g, 28.1 mmol) in CH2Cl2 (100 mL) and 5% so-
dium hydroxide aqueous solution (40 mL) was added
dropwise a solution of 2-iodobenzoyl chloride (2a, 7.5 g,
28.1 mmol) in CH2Cl2 (15 mL). The resulting mixture
was stirred at 0 ꢁC for 4 h and at room temperature over-
night. The solvent was removed in vacuo. The residue was
washed with water, dried, and recrystallized from ethanol
to afford a white solid (9.07 g, 84.7%). Mp 129–130 ꢁC. 1H
NMR (400 MHz, CDCl3) d 1.12, 1.13 (3H, d, J = 6.8 Hz, -
CH3), 4.51 (1H, m, -CHCH3), 5.05 (1H, d, J = 2.8 Hz, -
CHOH), 5.96 (1H, d, J = 8 Hz, -NH), 7.08–7.12 (1H, m,
aromatic), 7.26–7.42 (m, 7H, aromatic), 7.84, 7.86 (1H,
d, J = 8.0 Hz, aromatic). 13C NMR (75 MHz, CDCl3) d
13.74, 51.55, 75.64, 92.44, 126.02, 139.70, 140.66,
141.85, 169.57. Anal. Calcd for C16H16INO2: C, 50.41;
H, 4.23; N, 3.67. Found: C 50.25; H, 4.18; N, 3.58.
4. Experimental
4.1. Materials and instrumentation
All chemicals used were obtained from commercially
available sources (Aldrich Chemicals Co., Milwaulkee,
WI, USA; Sigma Chemical Co. St. Louis, MO, USA),
were of analytical or higher grade, and were used without
further purification. Solvents used in reactions and chro-
matographic purification were purchased from Aldrich
Chemicals Co. and from VWR Scientific Products (West
Chester, PA, USA). Thin-layer chromatography (TLC)
analyses were performed with 250 lm thick layers of fluo-
rescence UV254 silica gel backing on aluminum plates
purchased from Whatman Ltd (Maidstone, Kent, Eng-
land). Flash chromatography was carried out using
4.3. 3-Iodo-N-(2-hydroxy-1-methyl-2-phenylethyl)benz-
amide (3b)
The title compound 3b was obtained from L,D-norephe-
drine hydrochloride (1, 7.02 g, 37.5 mmol) and 3-iodo-
benzoyl chloride (2b, 10 g, 37.5 mmol) following the
procedure described for preparation of compound 3a.
Compound 3b, white solid (12.84 g, 90%). Mp 139–

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W. Yu et al. / Bioorg. Med. Chem. 16 (2008) 6145–6155
141 ꢁC. 1H NMR (300 MHz, CDCl3) d 1.10 (3H, d,
J = 6.9 Hz, CH3), 3.51 (1H, d, J = 3.3 Hz, –CHCH3),
4.42–4.52 (1H, m, –OH), 4.97 (1H, bs, CHOH), 6.42
(1H, d, J = 7.8 Hz, NH), 7.14 (1H, t, J = 7.8 Hz, aro-
matic), 7.25–7.42(5H, m, aromatic), 7.69 (1H, d,
J = 7.8 Hz, aromatic), 7.82 (1H, d, J = 8.1 Hz, aromatic),
8.08 (1H, s, aromatic). 13C NMR (75 MHz, CDCl3) d
14.20, 51.45, 76.18, 94.24, 126.14, 127.63, 128.26,
130.19, 136.00, 136.25, 140.41, 140.55, 166.26. Anal.
Calcd for C16H16INO2: C, 50.41; H, 4.23; N, 3.67. Found:
C, 50.48; H, 4.28; N, 3.62.
lid (6.5 g, 62%). Mp 77–78 ꢁC. 1H NMR (300 MHz,
CDCl3) d 2.72 (3H, s, CH3), 7.23 (1H, t, J = 7.8 Hz, aro-
matic), 7.40–7.52 (2H, m, aromatic), 7.57–7.66 (2H, m,
aromatic), 7.71–7.85 (2H, m, aromatic), 7.92 (1H, d,
J = 8.7 Hz, aromatic), 8.02 (1H, s, aromatic). 13C NMR
(75 MHz, CDCl3) d 24.31, 94.31, 118.36, 126.39, 126.92,
129.08, 129.83, 129.99, 137.34, 137.47, 138.53, 141.70,
150.80, 158.36. Anal. Calcd for C16H12IN: C, 55.67; H,
3.50; N, 4.06. Found: C, 55.53; H, 3.56; N, 4.05.
4.7. 1-(4-Iodophenyl)-3-methyl-isoquinoline (4c)
4.4. 4-Iodo-N-(2-hydroxy-1-methyl-2-phenylethyl)benz-
amide (3c)
The title compound 4c was obtained from amide (3c,
8.54 g, 22.4 mmol) following the procedure described
for preparation of compound 4a. Compound 4c, white so-
lid (6.1 g, 79%). Mp 147–148 ꢁC. H NMR (300 MHz,
CDCl3) d 2.72 (3H, s, CH3), 7.38–7.51 (4H, m, aromatic),
7.61 (1H, t, J = 6.9 Hz, aromatic), 7.75 (1H, d, J = 8.1 Hz,
aromatic), 7.84 (1H, d, J = 8.1 Hz, aromatic), 7.94 (1H, d,
J = 8.7 Hz, aromatic). 13C NMR (75 MHz, CDCl3) d
24.34, 94.58, 118.20, 124.56, 126.31, 126.42, 126.94,
129.96, 131.68, 137.41, 137.51, 139.12, 150.88, 158.97.
Anal. Calcd for C16H12IN: C, 55.67; H, 3.50; N, 4.06.
Found: C, 55.63; H, 3.55; N, 4.07.
1
The title compound 3c was obtained from L,D-norephe-
drine hydrochloride (1, 7.02 g, 37.5 mmol) and 4-iodo-
benzoyl chloride (2c, 10.1 g, 37.9 mmol) following the
procedure described for preparation of compound 3a.
Compound 3c, white solid (12.8 g, 90%). Mp 209–
1
210 ꢁC. H NMR (300 MHz, DMSO-d6) d 1.03 (3H, d,
J = 6.6 Hz, CH3), 4.05–4.12 (1H, m, –CHCH3), 4.65
(1H, s, –CHOH), 5.46 (1H, d, J = 3.6 Hz, NH), 7.14
(1H, t, J = 7.2 Hz, aromatic), 7.23 (2H, t, J = 735 Hz,
aromatic), 7.32 (2H, d, J = 7.2 Hz, aromatic), 7.53
(2H, d, J = 8.4 Hz, aromatic), 7.75 (2H, d, J = 8.1 Hz,
aromatic), 8.30 (1H, d, J = 8.4 Hz, aromatic). 13C
NMR (75 MHz, DMSO-d6) d 14.70, 51.30, 74.41,
98.48, 126.23, 126.73, 127.75, 129.29, 134.19, 137.03,
143.59, 164.70. Anal. Calcd for C16H16INO2: C, 50341;
H, 4.23; N, 3.67. Found: C, 50.35; H, 4.24; N, 3.65.
4.8. 1-(2-Iodophenyl)-3-bromomethyl-isoquinoline (5a)
A mixture of 1-(2-iodophenyl)-3-methyl-isoquinoline (4a,
112 mg, 0.33 mmol), N-bromosuccinimide (NBS)
(104 mg, 0.59 mmol), and 2,2-azobisisobutyro-nitrile
(AIBN) (12 mg, 0.07 mmol) in CCl4 (6 mL) was heated
to reflux for 20 h. The reaction mixture was cooled to room
temperature and filtered. The filtrate was washed with sat-
urated NaHCO3 aqueous solution (1· 5 mL), dried
(Na2SO4), and filtered. The filtrate was concentrated in va-
cuo, thereby affording crude 1-(2-iodophenyl)-3-bromo-
methyl-isoquinoline 5a as yellow oil, which was purified
by flash chromatography (20% ethyl acetate/hexane) to
4.5. 1-(2-Iodophenyl)-3-methyl-isoquinoline (4a)
A mixture of amide (3a, 820 mg, 2.15 mmol) and P2O5
(3.8 g) in o-dichlorobenzene (15 mL) was refluxed over-
night. After it was cooled to room temperature, it was fur-
ther cooled to 0 ꢁC by application of an external ice-water
bath. To this cooled mixture was cautiously added 30 mL
of water. After the vigorous reaction had subsided, the
dark solution was washed with toluene (2· 10 mL). The
aqueous layer was cooled to 0 ꢁC and was adjusted to
pH > 11 with 50% aqueous sodium hydroxide. The result-
ing mixture was extracted with toluene (4· 10 mL). The or-
ganic layers were dried over sodium sulfate and filtered.
The filtrate was concentrated in vacuo. The residue was
recrystallizedfrom benzene, thereby affording a white solid
1
give a clear oil (90.2 mg, 64.6%). H NMR (400 MHz,
CDCl3) d 4.80 (2H, s, –CH2Br), 7.17–8.01 (9H, m, aro-
matic). HRMS (ESI), calcd for C16H1279Br127IN
[M+H]+, 423.91924; found 423.91898 (85.7%).
4.9. 1-(2-Iodophenyl)-3-formyl-isoquinoline (6a)
The mixture of bromide (5a, 216 mg, 0.51 mmol) and so-
dium periodate (110 mg, 0.51 mmol) in DMF (10 mL)
was refluxed for 19 h. Water (20 mL) was added and
the product was extracted into ether (2· 10 mL). The
combined organic layers were washed with water (1·
10 mL), dried over sodium sulfate, filtered, and concen-
trated under reduced pressure. The crude product was
subjected to flash chromatography (20% ethyl acetate/
hexane) to give 6a as white semisolid (116 mg, 63.4%).
1H NMR (400 MHz, CDCl3) d 7.21–8.11 (8H, m, aro-
matic), 8.47 (1H, s, aromatic), 10.31 (1H, s, CHO).
HRMS (ESI), calcd for C16H11127INO [M+H]+,
359.98854; found 359.98798 (100%).
1
(366 mg, 49.4%). Mp 111–112 ꢁC. H NMR (400 MHz,
CDCl3) d 2.77 (3H, s, -CH3), 7.16–7.19 (1H, m, aromatic),
7.39–7.65 (6H, m, aromatic), 7.80 (1H, d, J = 8.0 Hz, aro-
matic), 7.98 (1H, d, J = 8.0 Hz, aromatic). 13C NMR
(75 MHz, CDCl3) d 24.05, 97.82, 118.26, 124.45, 125.98,
126.77, 127.70, 129.43, 129.79, 129.92, 136.73, 138.78,
143.74, 150.24, 161.34. HRMS (ESI), calcd for
C16H13127IN [M+H]+, 346.00873; found 346.00949
(100%). Anal. Calcd for C16H12IN: C, 55.67; H, 3.50; N,
4.06. Found: C, 55.77; H, 3.51; N, 3.96.
4.6. 1-(3-Iodophenyl)-3-methyl-isoquinoline (4b)
4.10. 1-(2-Iodophenyl)-3-isoquinoline carboxylic acid (7a)
The title compound 4b was obtained from amide (3b,
11.54 g, 30.3 mmol) following the procedure described
for preparation of compound 4a. Compound 4b, white so-
To a solution of aldehyde (6a, 274 mg, 0.76 mmol) in
ethanol (10 mL) was slowly added a solution of silver ni-

W. Yu et al. / Bioorg. Med. Chem. 16 (2008) 6145–6155
6151
trate (320 mg, 1.9 mmol) in water (5 mL). To this stirred
4.13. 1-(2-Iodophenyl)-N-methyl-3-isoquinoline carbox-
amide (8a)
solution was added dropwise a solution of sodium
hydroxide (240 mg, 6.1 mmol) in water (5 mL). The
resulting black slurry was stirred at room temperature
for 2 h. Then the mixture was filtered through a small
Celite column. The filter cake was washed with ether.
The ether was evaporated and the aqueous solution
was adjusted to pH 5 with concentrated hydrochloric
acid. The precipitate was collected by filtration, and
washed with water. It was recrystallized from CH3CN,
thereby affording a pale yellow crystalline (246 mg,
To a solution of 1-(2-iodophenyl)-3-isoquinoline car-
boxylic acid (7a, 189 mg, 0.5 mmol) in 5 mL of dichloro-
methane was added dropwise thionyl chloride (180 mg,
1.5 mmol). The mixture was heated to 50 ꢁC for 1 h
and the solvent was removed under reduced pressure.
The resulting residue was re-dissolved in 5 mL of THF
and the mixture was cooled to ꢀ70 ꢁC followed by the
addition of methylamine (78 mg, 2.5 mmol) in THF
(2 mL). The reaction was allowed to warm up to room
temperature overnight. Water (5 mL) was added and
the compound 8a was extracted with dichloromethane
(3· 5 mL). The combined organic layers were washed
with water (1· 5 mL), dried (Na2SO4), and filtered.
The filtrate was concentrated in vacuo. The residue
was purified on column (SiO2, 50% ethyl acetate/hexane,
0.1% Et3N), thereby giving a white foam (189.2 mg,
1
86.3%). Mp 174–176 ꢁC. H NMR (400 MHz, CDCl3)
d 7.25–8.12 (8H, m, aromatic), 8.73 (1H, s, aromatic).
13C NMR (75 MHz, CDCl3) d 24.05, 97.82, 118.26,
124.45, 125.98, 126.77, 127.70, 129.43, 129.79, 129.92,
136.73, 138.78, 143.74, 150.24, 161.34. HRMS (ESI),
calcd for C16H11127INO2 [M+H]+, 375.98291; found,
375.98203 (100%).
1
4.11. 1-(3-Iodophenyl)-3-isoquinoline carboxylic acid (7b)
97.5%). Mp 167–168 ꢁC. H NMR (400 MHz, CDCl3)
d 3.05, 3.06 (3H, two s, N-CH3), 7.21–8.07 (8H, m, aro-
matic), 8.21 (1H, br s, NH), 8.67 (1H, s, aromatic). 13C
NMR (75 MHz, CDCl3) d 26.13, 97.79, 120.02, 127.29,
127.75, 127.98, 128.53, 128.76, 130.13, 130.31, 130.81,
136.81, 139.42, 142.59, 143.30, 160.79, 165.32. HRMS
(ESI), calcd for C17H14127IN2O [M+H]+, 389.01454;
found 389.01309 (100%).
The crude compound 5b (530 mg, yellow oil) was ob-
tained from1-(3-iodophenyl)-3-methyl-isoquinoline (4b,
573 mg, 1.66 mmol) following the procedure described
for preparation of compound 5a, which was used di-
rectly in next step without further purification. The
crude product 1-(3-iodophenyl)-3-formyl-isoquinoline
6b was obtained as dark yellow oil (315 mg) following
the procedure described for preparation of compound
6a. This oil was used without further purification. The
title compound 7b was obtained following the procedure
described for preparation of compound 7a. Compound
7b, yellow crystalline (222 mg, 36%). Mp 145–146 ꢁC.
1H NMR (300 MHz, DMAO-d6) d 7.37 (1H, t,
J = 6.0 Hz, aromatic), 7.65–7.71 (2H, m, aromatic),
7.80 (1H, t, J = 4.8 Hz, aromatic), 7.91 (1H, d,
J = 6.6 Hz, aromatic), 7.95 (1H, d, J = 6.3 Hz, aro-
matic), 8.01 (1H, s, aromatic), 8.14 (1H, d, J = 6.0 Hz,
aromatic), and 8.44 (1H, s, aromatic). HRMS (EI) calcd
for C16H10INO2, 374.9756; found: 374.9756.
4.14. 1-(3-Iodophenyl)-N-methyl-3-isoquinoline carbox-
amide (8b)
The title compound 8b was obtained from 1-(3-iodo-
phenyl)-3-isoquinoline carboxylic acid (7b, 119 mg,
0.32 mmol) following the procedure described for prep-
aration of compound 8a. Compound 8b, white solid
1
(59.2 mg, 48%). Mp 172–174 ꢁC. H NMR (300 MHz,
CDCl3) d 3.03, 3.04 (3H, two s, N-CH3), 7.24 (1H, t,
J = 8.7 Hz, aromatic), 7.55–7.65 (2H, m, aromatic),
7.71 (1H, t, J = 8.1 Hz, aromatic), 7.82 (1H, d,
J = 8.7 Hz, aromatic), 7.95–8.05 (3H, m, aromatic),
and 8.19 (1H, broad s, CONH), 8.57 (1H, s, aromatic).
13C NMR (75 MHz, CDCl3) d 26.16, 94.24, 119.86,
127.15, 128.70, 128.96, 129.13, 129.91, 130.77, 137.14,
137.85, 138.57, 140.99, 142.67, 157.62, 165.25. HRMS
(EI) calcd for C17H13IN2O, 388.0073; found: 388.0071.
4.12. 1-(4-Iodophenyl)-3-isoquinoline carboxylic acid (7c)
The crude compound 5c (240 mg, yellow oil) was ob-
tained from1-(4-iodophenyl)-3-methyl-isoquinoline (4c,
259 mg, 0.75 mmol) following the procedure described
for preparation of compound 5a, which was used di-
rectly in next step without further purification. The
crude product 1-(4-iodophenyl)-3-formyl-isoquinoline
6c was obtained as dark yellow oil (140 mg) following
the procedure described for preparation of compound
6a. This oil was used without further purification. The
title compound 7c was obtained following the procedure
described for preparation of compound 7a. Compound
7c, a pale yellow solid (101 mg, 37%). Mp 243–245 ꢁC.
1H NMR (300 MHz, DMSO-d6) d 7.44 (1H, d,
J = 8.1 Hz, aromatic), 7.59 (1H, d, J = 8.1 Hz, aro-
matic), 7.68–7.78 (2H, m, aromatic), 7.84 (1H, t,
J = 7.8 Hz, aromatic), 7.90 (1H, d, J = 8.4 Hz, aro-
matic), 7.97 (1H, d, J = 8.4 Hz, aromatic), 8.20 (1H, d,
J = 8.1 Hz, aromatic), and 8.56 (1H, s, aromatic).
HRMS (EI) calcd for C16H10INO2, 374.9756; found:
374.9761.
4.15. 1-(4-Iodophenyl)-N-methyl-3-isoquinoline carbox-
amide (8c)
The title compound 8c was obtained from 1-(4-iodo-
phenyl)-3-isoquinoline carboxylic acid (7c, 207 mg,
0.55 mmol) following the procedure described for prep-
aration of compound 8a. Compound 8c, white solid
1
(172 mg, 81%). Mp 163–164 ꢁC. H NMR (300 MHz,
CDCl3) d 3.07, 3.09 (3H, two s, N-CH3), 7.46 (2H, d,
J = 8.4 Hz, aromatic), 7.64 (1H, t, J = 7.2 Hz, aromatic),
7.77 (1H, t, J = 6.9 Hz, aromatic), 7.92 (2H, d,
J = 8.4 Hz, aromatic), 8.07 (2H, t, J = 7.8 Hz, aro-
matic), 8.24 (1H, br s, NH), 8.02 (1H, s, aromatic).
13C NMR (75 MHz, CDCl3) d 26.05, 95.14, 119.54,
127.02, 128.58, 128.75, 130.60, 131.43, 131.56, 137.02,
137.40, 138.28, 142.58, 158.09, 165.12. HRMS (EI) calcd
for C17H13IN2O, 388.0073; found: 388.0077.

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W. Yu et al. / Bioorg. Med. Chem. 16 (2008) 6145–6155
4.16. 1-(2-Iodophenyl)-N-methyl-N-(3-fluoropropyl)-3-
isoquinoline carboxamide (9a)
104 mg, 0.27 mmol) and 2-fluoroethylbromide (274 mg,
2.16 mmol) following the procedure described for prep-
aration of compound 9a. Compound 9d, white solid
(48 mg, 41%) was obtained. Mp 142–143 ꢁC. H NMR
(300 MHz, CDCl3) d 3.22, 3.27 (3H, two s, N-CH3),
3.67–4.15 (2H, m, N-CH2), 4.42–4.92 (2H, m, CH2F),
7.15–7.25 (1H, m, aromatic), 7.27–7.37(1H, m, aro-
matic), 7.45–7.64 (3H, m, aromatic), 7.67–7.77 (1H, m,
aromatic), 7.97 (2H, t, J = 7.5 Hz, aromatic), 8.11,
8.21 (1H, two s, aromatic). Anal. Calcd for C19H16FI-
N2O: C, 52.55; H, 3.71; N, 6.45. Found: C, 52.64; H,
3.78; N, 6.38.
1
To a solution of 1-(2-iodophenyl)-N-methyl-3-isoquino-
line carboxamide (8a, 20 mg, 0.05 mmol) in DMF
(2 mL) was added sodium hydride (12 mg, 0.5 mmol)
in one portion. The mixture was stirred under argon at
room temperature for 40 min and 3-fluropropyl bromide
(36 mg, 0.25 mmol) was added. After stirring under ar-
gon at room temperature for 24 h, the mixture was
quenched by adding water (5 mL). The mixture was ex-
tracted with dichloromethane (3· 15 mL). The com-
bined organic layers were dried (Na2SO4) and filtered.
The filtrate was concentrated in vacuo. The residue
was purified by flash chromatography (SiO2, 30% ethyl
acetate/hexane, 0.1% Et3N), thereby affording clear oil
(7.9 mg, 35.3%) and the starting material (8a,
12.2 mg). 1H NMR (400 MHz, CDCl3) d 1.96–2.18
(2H, m, CH2), 3.16, 3.19 (3H, two s, N-CH3), 3.58–
3.82 (2H, m, N-CH2), 4.27, 4.39, 4.54, 4.66 (2H, qt,
J = 79.8, 47.1, 6 Hz, CH2F), 7.20–7.98 (8H, m, aro-
matic), 8.10, 8.17 (1H, two s, aromatic). 19F NMR
(376 MHz, CDCl3) d ꢀ221.17 to ꢀ220.31 (1F, m,
FCH2). HRMS (ESI), calcd for C20H19F127IN2O
[M+H]+, 449.05207; found 449.05186 (100%).
4.20. 3-Triphenylmethoxypropyl bromide (10)
To a mixture of 3-bromo-1-propanol (1.39 g, 10 mmol),
pyridine (4 mL, 50 mmol) in CH2Cl2 (10 mL) at 0 ꢁC
was added dropwise a solution of triphenylmethyl chlo-
ride (3.13 g, 11 mmol) in CH2Cl2 (5 mL). The resulting
mixture was heated at 60 ꢁC for 5 h and then kept at
room temperature overnight. It was poured into ice-
water and extracted with dichloromethane (3· 20 mL).
The combined organic layers were dried over sodium
sulfate, filtered and concentrated in vacuo. The residue
was purified by column chromatography (silica gel,
10% ethyl acetate/hexane), thereby affording a colorless
1
4.17. 1-(3-Iodophenyl)-N-methyl-N-(3-fluoropropyl)-3-
isoquinoline carboxamide (9b)
oil (1.44 g, 37.8%). H NMR (CDCl3) d 2.09–2.13 (2H,
m, CH2), 3.20 (2H, t, J = 6 Hz, CH2O), 3.56 (2H, t,
J = 6.8 Hz, CH2Br), and 7.21–7.31 (15H, m, aromatic).
The title compound 9b was obtained from 1-(3-iodo-
phenyl)-N-methyl-3-isoquinoline carboxamide (8b,
22 mg, 0.06 mmol) following the procedure described
for preparation of compound 9a. Compound 9b, semi-
solid (4.4 mg, 37%). 1H NMR (300 MHz, CDCl3) d
2.04–2.24 (2H, m, CH2), 3.16,3.18(3H, two s, N-CH3),
3.60–3.75 (2H, m, N-CH2), 4.25–4.70(2H, qt, J = 67.8,
47.1, 5.7 Hz, CH2F), 7.20–7.30 (1H, m, aromatic),
7.55–7.65 (2H, m, aromatic), 7.73 (1H, t, J = 7.2 Hz,
aromatic), 7.83 (1H, d, J = 7.8 Hz, aromatic), 7.94
(1H, d, J = 8.4 Hz, aromatic), 7.98–8.09 (2H, m, aro-
matic), and 8.12 (1H, s, aromatic). HRMS (EI) calcd
for C20H18FIN2O, 448.0448; found: 448.0461.
4.21. 1-(2-Iodophenyl)-N-methyl-N-(3-triphenylmethoxy-
propyl)-3-isoquinoline carboxamide (11)
A
mixture 1-(2-iodophenyl)-N-methyl-3-isoquinoline
carboxamide (8a, 65 mg, 0.17 mmol) and sodium hy-
dride (20.4 mg, 0.84 mmol) in DMF (4 mL) was stirred
at room temperature for 30 min. To this mixture was
added dropwise a solution of 3-triphenylmethoxypropyl
bromide (10, 128 mg, 0.34 mmol) in DMF (2 mL). The
resulting mixture was stirred under argon at room tem-
perature overnight and then quenched by addition of
5 mL water. The mixture was extracted with dichloro-
methane (3· 15 mL). The combined organic layers were
dried (Na2SO4) and filtered. The filtrate was concen-
trated in vacuo. The residue was purified by column
chromatography (silical gel, 50% ethyl acetate/hexane,
0.1% Et3N), thereby affording clear oil (56 mg, 48.5%).
1H NMR (400 MHz, CDCl3) d 1.82–1.98 (2H, m,
CH2), 2.95–3.88 (4H, m, CH2O, CH2N), 3.05, 3.06
(3H, two s, N-CH3), 7.14–8.07 (23H, m, aromatic),
8.67 (1H, s, aromatic). HRMS (ESI), calcd for
4.18. 1-(4-Iodophenyl)-N-methyl-N-(3-fluoropropyl)-3-
isoquinoline carboxamide (9c)
The title compound 9c was obtained from 1-(4-iodo-
phenyl)-N-methyl-3-isoquinoline carboxamide (8c,
25 mg, 0.06 mmol) following the procedure described
for preparation of compound 9a. Compound 9c, semi-
solid (11 mg, 40%). 1H NMR (300 MHz, CDCl3) d
2.01–2.22 (2H, m, CH2), 3.15, 3.17 (3H, two s, N-
CH3), 3.60–3.76 (2H, m, N-CH2), 4.22–4.76 (2H, qt,
J = 77.7, 47.1, 5.7 Hz, CH2F), 7.36–7.47 (1H, m, aro-
matic), 7.5–7.81 (4H, m, aromatic), 7.82–7.99 (2H, m,
aromatic), 7.83–8.16 (2H, m, aromatic). HRMS (EI),
calcd for C20H18FIN2O, 448.0448; found: 448.0435.
C39H34F127IN2O2
689.16620 (47.9%).
[M+H]+,
689.16596;
found
4.22. 1-(2-Iodophenyl)-N-methyl-N-(3-hydroxypropyl)-3-
isoquinoline carboxamide (12)
To a solution of 1-(2-iodophenyl)-N-methyl-N-(3-triphe-
nylmethoxypropyl)-3-isoquinoline carboxamide (11,
187 mg, 0.27 mmol) in dichloromethane (5 mL) was
added dropwise trifluoroacetic acid (TFA) (1 mL). The
mixture was stirred at room temperature for 40 min.
Then it was transferred into a separatory funnel and
4.19. 1-(2-Iodophenyl)-N-methyl-N-(2-fluoroethyl)-3-iso-
quinoline carboxamide (9d)
The title compound 9d was obtained from 1-(2-iodo-
phenyl)-N-methyl-3-isoquinoline carboxamide (8a,

W. Yu et al. / Bioorg. Med. Chem. 16 (2008) 6145–6155
6153
washed with saturated sodium bicarbonate solution (2·
10 mL), water (1· 10 mL), and brine (1· 10 mL), then
dried (Na2SO4), and filtered. After removing the solvent
in vacuo, the residue was purified on column (silica gel,
2% MeOH/CH2Cl2, 0.1% Et3N), thereby affording start-
ing material (32 mg) and the title compound as clear oil
0.2 lm Gelman teflon filters into a dose vial, which was
ready for use in in vitro and in vivo studies.
4.25. Quality control of purified radioligand [18F]9a
The identity of the radiolabeled product [18F]9a was
confirmed by co-injection with the authentic sample of
non-radioactive 9a on an analytical HPLC, equipped
with UV and radio detectors, eluted with 70:30
MeOH/H2O (v/v) at a flow rate of 1 mL per minute.
The retention time of [18F]9a was 4.48 min. Its radio-
chemical purity was also determined by analytical
HPLC.
1
(50 mg, 41%). H NMR (CDCl3) d 1.85–1.87 (2H, m,
CH2), 3.11, 3.18 (3H, two s, N-CH3), 3.52–3.95 (4H,
m, CH2N, CH2O), 7.20–8.02 (8H, m, aromatic), 8.18,
8.26 (1H, two s, aromatic). HRMS (ESI), calcd for
C20H20127IN2O2 [M+H]+, 447.05641; found: 447.05692
(100%).
4.23. 1-(2-Iodophenyl)-N-methyl-N-(3-p-toluenesulfonyl-
oxypropyl)-3-isoquinoline carboxamide (13)
4.26. Lipophilicity measurement
To
hydroxypropyl)-3-isoquinoline
a
solution of 1-(2-iodophenyl)-N-methyl-N-(3-
carboxamide (12,
Measurement of distribution coefficient of radiolabeled
[18F]9a was performed according to a previously re-
ported procedure.63 Briefly, the test tubes containing
2 mL of 1-octanol, 2 mL of 0.02 M, pH 7.4, sodium
phosphate buffer, and approximately 185–370 MBq por-
tion of the radiotracer were mixed for 10 min at room
temperature and then centrifuged at 75g for 5 min. A
0.5 mL portion of samples from the 1-octanol and the
buffer layers, respectively, were counted for radioactivity
on a Packard Cobra II automated gamma-counter.
Each assay condition was performed in triplicate. The
logP7.4 values were calculated with decay corrected for
each replicate, which were averaged to give the logP7.4
value for the radiotracer.
42 mg, 0.09 mmol) and Et3N (0.02 mL, 0.14 mmol) in
CH2Cl2 (2 mL) at 0 ꢁC was added dropwise p-toluene-
sulfonyl chloride (69 mg, 0.36 mmol) in CH2Cl2
(1 mL). The resulting mixture was stirred at 0 ꢁC and
monitored by TLC. After stirring at 0 ꢁC for 4 h, TLC
showed that no starting material was left. Then it was
transferred into a separatory funnel and washed with
water (1· 10 mL). The organic layer was dried (Na2SO4)
and filtered. The filtrate was concentrated in vacuo. The
residue was purified on column (silica gel, 2% MeOH/
CH2Cl2, 0.1% Et3N), thereby affording an oil (19 mg,
1
35%). H NMR (400 MHz, CDCl3) d 1.97–2.08 (2H,
m, CH2) 2.39, 2.41 (3H, two s, –PhCH3), 3.05, 3.11
(3H, two s, N-CH3), 3.45–3.72 (2H, m, CH2N), 3.89,
4.15 (2H, two m, CH2O), 7.17–7.99 (12H, m, aromatic),
8.06, 8.15 (1H, two s, aromatic). HRMS (ESI), calcd for
4.27. Binding assays
PBR binding affinity assays were performed by Nova-
Screen using a previously reported procedure which
was modified.65–67 Briefly, tissue preparations of rat kid-
ney homogenate were incubated with [3H]PK11195 and
competitors (i.e. 9a–d, at 12 concentrations ranging
from 10ꢀ11 to 10ꢀ6 nM) in an assay buffer. The assay
mixture was incubated for 60 min at room temperature
with stirring and the resulting samples were rapidly fil-
tered through Whatman GF/B glass-fiber filters pre-
treated with 0.2% protamine base and washed with
cold buffer, pH 7.4. The filters were counted in a liquid
scintillation counter at an efficiency of 65% for tritium
saturation binding, scatchard and competition experi-
ments were analyzed with the iterative nonlinear least
square curve-fitting program.
C27H26127IN2O432S
601.06537 (100%).
[M+H]+,
601.06526;
found:
4.24. Radiosynthesis of [18F]9a
[18F]HF was produced with a Siemens 11 MeV RDS 112
cyclotron by the 18O (p,n) 18F reaction using [18O]H2O.
A fully automated synthesis program developed for the
Siemens computer programmable chemistry process
control unit (CPCU) was used for the preparation of
[18F]9a. The CPCU is a valve-and-tubing system which
is designed to accommodate one or two glass reaction
vessels and a number of reagent and solvent reservoir
containers. Briefly, the automated production of
[18F]9a was performed by reacting 3–5 mg (5–8 lmol)
of tosylate precursor 13 in 0.3 mL of DMSO with 3.7–
5.6 · 107 MBq NCA [18F]fluoride in the presence of
Kryptofix 222 (5 mg, 13.3 lmol) and potassium carbon-
ate (3 mg, 21.7 lmol) at 120 ꢁC for 10 min. After passing
a silica SepPak, the product [18F]9a was purified by
semipreparative reverse-phase HPLC, using mobile
phase consisting of 60:40 MeOH/H2O (v/v) at a flow rate
of 6 mL per minute. The desired fractions were com-
bined, diluted 1:2 (v/v) with sterile water, and loaded
onto a C-18 SepPak. After washing with 40 mL of saline
(0.9% NaCl aq), the radioactive product was eluted
from C-18 SepPak with 1 mL of ethanol into a sealed
sterile vial containing 9 mL of saline. The resulting solu-
tion was passed successively through a 1 lm and then a
Acknowledgments
We are grateful to Dr. Bing Wang, NMR Center of Em-
ory University, for the help with NMR instrumentation,
sample treatment, and data analysis. We acknowledge
the use of Shared Instrumentation for the mass spectros-
copy data.
References and notes
1. Beurdeley-Thomas, A.; Miccoli, L.; Oudard, S.; Dutrill-
aux, B.; Poupon, M. F. J. Neurooncol. 2000, 46, 45.

6154
W. Yu et al. / Bioorg. Med. Chem. 16 (2008) 6145–6155
2. Pike, V. W.; Halldin, C.; Crouzel, C.; Barre, L.; Nutt, D.
J.; Osman, S.; Shah, F.; Turton, D. R.; Waters, S. L. Nucl.
Med. Biol. 1993, 20, 503.
3. Casellas, P.; Galiegue, S.; Basile, A. S. Neurochem. Int.
2002, 40, 475.
4. Papadopoulos, V. Ann. Pharm. Fr. 2003, 61, 30.
5. Bribes, E.; Bourrie, B.; Casellas, P. Immunol. Lett. 2003,
88, 241.
6. Amitani, M.; Zhang, M.-R.; Noguchi, J.; Kumata, K.; Ito,
T.; Takai, N.; Suzuki, K.; Hosoi, R.; Inoue, O. . Nucl.
Med. Biol. 2006, 33, 971.
31. Olson, J. M. M.; Ciliax, B. J.; Mancini, W. R.; Young, A.
B. Eur. J. Pharmacol. 1988, 152, 47.
32. Decaudin, D. Anti-Cancer Drugs 2004, 15, 737.
33. Guidotti, A.; Forchetti, C. M.; Corda, M. G.; Konkel, D.;
Bennett, C. D.; Costa, E. Proc. Natl. Acad. Sci. U.S.A.
1983, 80, 3531.
34. Costa, E.; Guidotti, A. Life Sci. 1991, 49, 325.
35. Verma, A.; Nye, J. S.; Snyder, S. H.Proc. Natl. Acad. Sci.
U.S.A. 1987, 84, 2256.
36. Li, H.; Papadopoulos, V. Endocrinology 1998, 139, 4991.
37. Galiegue, S.; Tinel, N.; Casellas, P. Curr. Med. Chem.
2003, 10, 1563.
7. Black, K. L.; Ikezaki, K.; Santori, E.; Becker, D. P.;
Vinters, H. V. Cancer 1990, 65, 93.
38. Basile, A. S.; Klein, D. C.; Skolnick, P. Mol. Brain Res.
1986, 1, 127.
39. Rao, V. L. R.; Butterworth, R. F. . Eur. J. Pharmacol.
1997, 340, 89.
8. Cornu, P.; Benavides, J.; Scatton, B.; Hauw, J. J.;
Philippon, J. Acta Neurochir. 1992, 19, 146.
9. Hardwick, M.; Fertikh, D.; Culty, M.; Li, H.; Vidic, B.;
Papadopoulos, V. Cancer Res. 1999, 59, 831.
10. Katz, Y.; Eitan, A.; Gavish, M. Oncology 1990, 47, 139.
11. Miettinen, H.; Kononen, J.; Haapasalo, H.; Helen, P.;
Sallinen, P.; Harjuntausta, T.; Helin, H.; Alho, H. Cancer
Res. 1995, 55, 2691.
12. Venturini, I.; Zeneroli, M. L.; Corsi, L.; Avallone, R.;
Farina, F.; Alho, H.; Baraldi, C.; Ferrarese, C.; Pecora,
N.; Frigo, M.; Ardizzone, G.; Arrigo, A.; Pellicci, R.;
Baraldi, M. Life Sci. 1998, 63, 1269.
40. Le Fur, G.; Perrier, M. L.; Vaucher, N.; Imbault, F.;
Flamier, A.; Benavides, J.; Uzan, A.; Renault, C.; Dub-
roeucq, M. C.; Gueremy, C. Life Sci. 1983, 32, 1839.
41. Le Fur, G.; Guilloux, F.; Rufat, P.; Benavides, J.; Uzan,
A.; Renault, C.; Dubroeucq, M. C.; Gueremy, C. Life Sci.
1983, 32, 1849.
42. Farges, R.; Joseph-Liauzun, E.; Shire, D.; Caput, D.; Le
Fur, G.; Ferrara, P. Mol. Pharmacol. 1994, 46, 1160.
43. Le Fur, G.; Vaucher, N.; Perrier, M. L.; Flamier, A.;
Benavides, J.; Renault, C.; Dubroeucq, M. C.; Gueremy,
C.; Uzan, A. Life Sci. 1983, 33, 449.
13. Banati, R. B.; Myers, R.; Kreutzberg, G. W. . G. W. J.
Neurocytol. 1997, 26, 77.
14. Benavides, J.; Fage, D.; Carter, C.; Scatton, B. Brain Res.
1987, 421, 167.
15. Bourdiol, F.; Toulmond, S.; Serrano, A.; Benavides, J.;
Scatton, B. Brain Res. 1991, 543, 194.
16. Chen, M.-K.; Baidoo, K.; Verina, T.; Guilarte, T. R.
Brain: J. Neurol. 2004, 127, 1379.
17. Dubois, A.; Benavides, J.; Peny, B.; Duverger, D.; Fage,
D.; Gotti, B.; MacKenzie, E. T.; Scatton, B. Brain Res.
1988, 445, 77.
18. Guilarte, T. R.; Kuhlmann, A. C.; O’Callaghan, J. P.;
Miceli, R. C. Neurotoxicology 1995, 16, 441.
19. Maeda, J.; Higuchi, M.; Inaji, M.; Ji, B.; Haneda, E.;
Okauchi, T.; Zhang, M.-R.; Suzuki, K.; Suhara, T. Brain
Res. 2007, 1157, 100.
20. Myers, R.; Manjil, L. G.; Cullen, B. M.; Price, G. W.;
Frackowiak, R. S. J.; Cremer, J. E. J. Cereb. Blood Flow
Metab. 1991, 11, 314.
44. Romeo, E.; Auta, J.; Kozikowski, A. P.; Ma, D.;
Papadopoulos, V.; Puia, G.; Costa, E.; Guidotti, A. J.
Pharmacol. Exp. Ther. 1992, 262, 971.
45. Bribes, E.; Bourrie, B.; Esclangon, M.; Galiegue, S.; Vidal,
H.; Casellas, P. Eur. J. Pharmacol. 2002, 452, 111.
46. Ferzaz, B.; Brault, E.; Bourliaud, G.; Robert, J.-P.;
Poughon, G.; Claustre, Y.; Marguet, F.; Liere, P.;
Schumacher, M.; Nowicki, J.-P.; Fournier, J.; Marabout,
B.; Sevrin, M.; George, P.; Soubrie, P.; Benavides, J.;
Scatton, B. J. Pharmacol. Exp. Ther. 2002, 301, 1067.
47. Vin, V.; Leducq, N.; Bono, F.; Herbert, J. M. Biochem.
Biophys. Res. Commun. 2003, 310, 785.
48. Awad, M.; Gavish, M. . Life Sci. 1988, 43, 167.
49. Wang, J. K. T.; Morgan, J. I.; Spector, S. Proc. Natl.
Acad. Sci. U.S.A. 1984, 81, 753.
50. Gavish, M.; Katz, Y.; Bar-Ami, S.; Weizman, R. J.
Neurochem. 1992, 58, 1589.
21. Raghavendra Rao, V. L.; Dogan, A.; Bowen, K. K.;
Dempsey, R. J. Exp. Neurol. 2000, 161, 102.
22. Stephenson, D. T.; Schober, D. A.; Smalstig, E. B.; Mincy,
R. E.; Gehlert, D. R.; Clemens, J. A. J. Neurosci. 1995, 15,
5263.
51. Krueger, K. E. Biochim. Biophys. Acta, Rev. Biomembr.
1995, 1241, 453.
52. Gildersleeve, D. L.; Lin, T. Y.; Wieland, D. M.; Ciliax, B.
J.; Olson, J. M. M.; Young, A. B. Nucl. Med. Biol. 1989,
16, 423.
23. Nakamura, K.; Fukunishi, I.; Nakamoto, Y.; Iwah-
ashi, K.; Yoshii, M. Psychopharmacology 2002, 162,
301.
24. Nakamura, K.; Yamada, K.; Iwayama, Y.; Toyota, T.;
Furukawa, A.; Takimoto, T.; Terayama, H.; Iwahashi, K.;
Takei, N.; Minabe, Y.; Sekine, Y.; Suzuki, K.; Iwata, Y.;
Pillai, A.; Nakamoto, Y.; Ikeda, K.; Yoshii, M.; Fukuni-
shi, I.; Yoshikawa, T.; Mori, N. Am. J. Med. Genet., B:
Neuropsychiatr. Genet. 2006, 141B, 222.
25. Bernassau, J. M.; Reversat, J. L.; Ferrara, P.; Caput, D.;
Lefur, G. J. Mol. Graph. 1993, 11, 236.
26. Papadopoulos, V. Endocr. Res. 2004, 30, 677.
27. Casellas, P.; Galiegue, S.; Basile, A. S. . Neurochem. Int.
2002, 40, 475.
53. Dubroeucq, M.-C.; Renault, C.; Le Fur, G. French
Patent, 1982.
54. Shah, F.; Pike, V. W.; Turton, D. R. J. Labelled Cmpd.
Radiopharm. 1993, 32, 166.
55. Doble, A.; Ferris, O.; Burgevin, M. C.; Menager, J.; Uzan,
A.; Dubroeucq, M. C.; Renault, C.; Gueremy, C.; Le Fur,
G. Mol. Pharmacol. 1987, 31, 42.
56. Pascali, C.; Luthra, S. K.; Pike, V. W.; Price, G. W.;
Ahier, R. G.; Hume, S. P.; Myers, R.; Manjil, L.; Cremer,
J. E. Appl. Radiat. Isot. 1990, 41, 477.
57. Simonyi, M.; Fitos, I.; Visy, J. Trends Pharmacol. Sci.
1986, 7, 112.
58. Walle, T.; Walle, U. K. Trends Pharmacol. Sci. 1986, 7,
155.
28. Gavish, M.; Bachman, I.; Shoukrun, R.; Katz, Y.;
Veenman, L.; Weisinger, G.; Weizman, A. Pharmacol.
Rev. 1999, 51, 629.
29. Papadopoulos, V. Endocr. Rev. 1993, 14, 222.
30. O’Beirne, G. B.; Woods, M. J.; Williams, D. C. Eur. J.
Biochem. 1990, 188, 131.
59. Testa, B. Trends Pharmacol. Sci. 1986, 7, 60.
60. Ariens, E. J. Trends Pharmacol. Sci. 1986, 7, 200.
61. Dubroeucq, M. C.; Benavides, J.; Doble, A.; Guilloux, F.;
Allam, D.; Vaucher, N.; Bertrand, P.; Gueremy, C.;
Renault, C.; Uzan, A.; Le Fur, G. Eur. J. Pharmacol.
1986, 128, 269.

W. Yu et al. / Bioorg. Med. Chem. 16 (2008) 6145–6155
6155
62. Pictet, A.; Gams, A. Berichte der Deutschen Chemischen
Gesellschaft 1909, 42, 2943.
66. Cheng, Y.-C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.
63. Wilson, A. A.; Houle, S. J. Labelled Cmpd. Radiopharm.
1999, 42, 1277.
67. Rao, V. L. R.; Audet, R.; Therrien, G.; Butterworth, R. F.
. Dig. Dis. Sci. 1994, 39, 1055.
64. Dischino, D. D.; Welch, M. J.; Kilbourn, M. R.; Raichle,
M. E. J. Nucl. Med. 1983, 24, 1030.
68. Lukeman, D. S. F.; Darrell, D., et al. J. Pharmacol. Exp.
Ther. 1987, 241, 950.
65. Skowronski, R.; Fanestil, D. D.; Beaumont, K. . K. Eur.
J. Pharmacol. 1988, 148, 187.
69. Anholt, R. R.; Pedersen, P. L.; DeSouza, E. B.; Snyder, S.
H. J. Biol. Chem. 1986, 261, 576.