Synthesis and in vivo biodistribution of F-18 labeled 3-cis-, 3-trans-, 4-cis-, and 4-trans-fluorocyclohexane derivatives of WAY 100635

Article abstract of DOI:10.1016/j.bmc.2006.01.064

Radioligands that are specific for the serotonin 5-HT_1A receptor will be useful in characterizing the physiological action of this receptor subtype. With radioligands of varying pharmacokinetic properties, investigators can measure not only rec

Full text of DOI:10.1016/j.bmc.2006.01.064

Bioorganic & Medicinal Chemistry 14 (2006) 3737–3748
Synthesis and in vivo biodistribution of F-18 labeled 3-cis-,
3-trans-, 4-cis-, and 4-trans-fluorocyclohexane
derivatives of WAY 100635
Lixin Lang,* Elaine Jagoda, Ying Ma, Mark B. Sassaman and William C. Eckelman
PET Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD, USA
Received 18 July 2005; accepted 13 January 2006
Available online 20 February 2006
Abstract—Radioligands that are specific for the serotonin 5-HT1A receptor will be useful in characterizing the physiological action
of this receptor subtype. With radioligands of varying pharmacokinetic properties, investigators can measure not only receptor den-
sity, but also the effect of endogenous serotonin concentration. To this end, three additional fluorinated analogs of WAY 100635
were prepared and evaluated as 5-HT1A receptor ligands of varyin|%1e[OK]g pharmacokinetic properties based on our previous
studies. These four compounds are cis-4-fluoro-, trans-4-fluoro-, cis-3-fluoro-, and trans-3-fluoro-N-{2-[4-(2-methoxyphenyl)pipera-
zin-1-yl]ethyl}-N-(pyridin-2-yl)cyclohexanecarboxamides (FCWAYs). All four compounds were characterized and radiolabeled
with fluorine-18, a 109.7 min half-life radionuclide used in positron emission tomography. We then determined in vitro inhibition
constants at the 5-HT1A receptor; in vitro metabolic profile, using rat hepatocytes and liquid chromatography/mass spectroscopy
(LC/MS); and the rate of defluorination and hippocampus to cerebellum ratio ex vivo. This led to the conclusion that high affinity
4-trans-F-18 FCWAY had the best properties for measuring receptor density given its high hippocampus to cerebellum ratio and 3-
cis-F-18 FCWAY had the best properties for measuring dynamic change in receptors, with lower affinity and faster
pharmacokinetics.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
elucidated,2 and show the value, if not the necessity, of
adhering to this ideal.
The serotonin 5-HT1A receptor was identified in 1981,1
but silent antagonists (compounds possessing no intrin-
sic agonist activity) were not identified until 12 years lat-
er.2 This delay prevented the clear definition of the
action of this receptor due, in part, to the complexities
of the 5-HT system, for which at least 15 separate recep-
tor subtypes have been identified,3 and in the difficulty
of characterizing the agonist and antagonist properties
of potential ligands using physiological models alone.2
In general, radiolabeled silent antagonists with high
affinity for a specific receptor subtype represent an ‘ide-
al’ ligand for studying that subtype in vivo by biomedi-
cal imaging techniques such as single photon emission
tomography (SPET) and positron emission tomography
(PET). The intricacies and problems associated with the
5-HT1A receptor in particular have recently been
The first 5-HT1A silent antagonist, 4-phenylethyl-1-(2-
methoxyphenyl)piperazine, possessed high binding affin-
ity for that subtype, but also bound to a1 adrenoceptors.
An added N-t-butyl carboxamide group produced (S)-
N-t-butyl-3-[4-[(2-methoxyphenyl)piperazin-1-yl]-2-phe-
nylpropanamide [(S)WAY-100135], a 5-HT1A silent
antagonist having the ‘ideal’ properties of both high
specificity and high affinity.4 Despite this decidedly
advantageous combination of attributes, (S)WAY-
100135, with an IC50 of 15 nM for displacement of
[3H]8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetra-
lin] binding to rat hippocampal 5-HT1A receptors,5 was
still considered to be an order of magnitude too weak to
be useful as an external imaging agent.
The introduction of WAY 100635 (1, Scheme 1),6 with
an IC50 of 1.35 nM against [3H]8-OH-DPAT binding,7
was a significant breakthrough in the development of
external imaging agents. Mathis et al. radiolabeled
this compound with carbon-11, a positron emitting
radionuclide with a 20.4 min half-life, and studied its
Keywords: 5-HT1A receptors; Fluorine-18; WAY 100635; In vitro
metabolites.
*
Corresponding author. Tel.: +1 301 451 3532; fax: +1 301 402
3521; e-mail: llang@mail.cc.nih.gov
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.064

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L. Lang et al. / Bioorg. Med. Chem. 14 (2006) 3737–3748
selected for development and radiolabeled with iodine-
123 and 125 for in vivo evaluation in rats.14,13 Subse-
quently, the 4-fluorophenylcarboxamide derivative,
p-MPPF (4),13 was radiolabeled, first with tritium,15
and followed shortly thereafter with fluorine-18
(t1/2 = 109.7 min).16,17
At this point in the development of compounds that
could be used specifically for PET imaging, studies of
phenyl- and cyclohexanecarboxamides further diverged
into two groups: those labeled with fluorine-18 and
those labeled with carbon-11. The research group at
NIH, concentrating on fluorine-18, prepared a series
of derivatives of WAY 100635 from the following acids:
4-fluorobenzoic (4), 4-fluoro-3-methylbenzoic (5), trans-
4-fluorocyclohexanecarboxylic (7), 4-fluoromethylben-
zoic (8), and 4-fluoromethyl-3-nitrobenzoic (9).18 The
biological properties of the F-18 labeled compounds
were compared to those of [carbonyl-11C] WAY
100635 in rats. The two benzylic fluorides (8 and 9) were
eliminated from further study due to rapid defluorina-
tion in vivo. Unlike other benzylic halides, fluorides
are generally stable to nucleophilic attack even under
fairly strenuous conditions. This anomalous behavior
prompted further study, which confirmed that in vivo
defluorination was not due to nucleophilic displace-
ment,19 however, the mechanism, presumed to be oxida-
tion–elimination, is yet to be elucidated.
Of the remaining compounds, the two phenylcarboxa-
mides p-MPPF, which was identified as FBWAY (4)
in our early publication18 and the 4-fluoro-3-methylphe-
nyl derivative (5) showed (definitively in the first case
and presumptively in the second) brain uptake and
biphasic clearance in rats, the uptake phase peaking at
approximately 5 min postinjection. The normalized
uptake correlating with the beginning of the second
phase for both compounds was considerably lower than
for WAY 100635 [carbonyl-11C], which was run concur-
rently as a control. The last compound, the trans-4-flu-
orocyclohexyl derivative (7), referred to as 4-trans-
FCWAY, had a high affinity for the 5-HT1A receptor
and a high hippocampus to cerebellum [(H/Cb) target/
nontarget] ratio.18 By comparison, the [O-methyl-11C]cis
4-methoxycyclohexanecarboxylic acid derivative (10)
did not bind in vivo and the trans-4-methoxy (11) had
a high affinity in vitro, but had a relatively low H/Cb
ratio.20 Two reviews, covering both historical and
current trends in 5-HT1A radioligand development
pertinent to positron emission tomography, have
recently been published.2,21
Scheme 1. Structures based on the N-{2-[4-(2-methoxyphenyl)pipera-
zin-1-yl]ethyl}-N-(pyridin-2-yl)amide platform.
biodistribution in monkeys.8 A detailed analytical study
of the major metabolic pathways (in vitro) for 1, by li-
quid chromatography/mass spectroscopy (LC/MS),
showed oxidation in the methoxyphenyl moiety as the
primary mode in rat hepatocytes, while amide hydrolysis
predominated in human hepatocytes.9 Metabolism in
monkeys (in vivo) paralleled the hydrolysis mode ob-
served in human hepatocytes, when blood samples were
analyzed by TLC.10 When 1 was labeled with carbon-11
in the methoxyphenyl moiety, this predominant amide
hydrolysis (in human and nonhuman primates) gave
the radiolabeled metabolite corresponding to WAY-
100634 (2), which easily crossed the BBB and bound
weakly to both 5-HT1A and a1 receptors.11 This problem
was partially circumvented by labeling the carboxyl
group with carbon-11, the acid liberated upon hydroly-
sis having markedly less BBB permeability.12
We have now synthesized additional F-18 labeled deriv-
atives in an attempt to produce a range of pharmacoki-
netic properties with a longer half-life radionuclide that
will facilitate the determination of the metabolic-cor-
rected plasma input function.22 Our hypothesis is that
the use of subtype selective ligands with different recep-
tor affinities will provide sensitivity to a range of seroto-
nergic processes. Using a high affinity ligand should
provide a more accurate measure of serotonin receptor
density, suitable for comparison among patient groups.
Lower affinity ligands may be useful for measuring
The next series of compounds developed were based on
replacing the cyclohexanecarboxamide moiety of 1 with
(o-, m-, and p-) substituted phenylcarboxamides.13 Inhibi-
tion constants against the binding of [125I]8-OH-PIPAT in
rat hippocampal homogenates for all of the compounds
tested (with the exception of the o-iodo-derivative)
were comparable to 1. The 4-iodophenylcarboxamide
derivative, 4-iodo-N-{2-[4-(2-methoxyphenyl)piperazin-
1-yl]ethyl}-N-(pyridin-2-yl)benzamide (p-MPPI, 3), was

L. Lang et al. / Bioorg. Med. Chem. 14 (2006) 3737–3748
3739
dynamic changes in neurotransmitter concentration.
2.2. Synthesis of labeling precursors
Recently, Rice et al.23 showed that a high affinity ligand,
such as WAY 100635, was unaffected by the 5-HT
releasers, p-chloroamphetamine and fenfluramine.
However, a recent publication showed that the binding
of p-MPPF can be modulated by release of endogenous
serotonin using fenfluramine.24 The combination of
these data supports our hypothesis that a lower affinity
compound may be more susceptible to blocking by
endogenous serotonin.
Compounds 21 and 22 were prepared by hydrogenating
3-hydroxybenzoic acid using rhodium as the catalyst.
The resulting mixture of 3-cis and 3-trans hydroxycyc-
lohexanecarboxylic acid was converted to the pentam-
ethylbenzyl ester and separated by silica gel flash
chromatography (Scheme 3). Unlike the 4-hydroxy
counterpart (19 and 20),18 nosylate derivatives of 21
and 22 are unstable, and the mesylate derivatives were
prepared instead. The reactivity of mesylate at the 3 po-
sition is similar to that of the nosylate at 4 position.
The first lower affinity compound, MeFBWAY (5),
has a low H/Cb ratio in rats and, therefore, a low
contrast between receptor-rich and receptor-poor
regions, which limits the sensitivity of the radiophar-
maceutical. We have previously reported on trans-4-
FCWAY (7).18 In this report, we compare this high
affinity compound with cis-4-FCWAY (12), trans-3-
FCWAY (13), and cis-3-FCWAY (14) in search of a
less tightly bound radiotracer. The criteria are the ease
of synthesis of the precursor, the radiochemical yield,
the in vitro inhibition constant, metabolic profile, the
rate of defluorination in vivo, and the target gray
matter to cerebellum ratio.
2.3. Synthesis of F-18 labeled WAY 100635 analogs
The four radiolabeled compounds were prepared by
generating the radiolabeled acid first starting from either
the 4-nosylate derivatives or 3-mesylate derivatives with
F-18 and then combining this acid with the amine,
WAY 100634, after conversion of the acid to the acid
chloride. All substitution reactions undergo conversion
of the configuration. The 4-cis-nosylate and the
4-trans-nosylate gave similar radiolabeling yields of
25–40% in acetonitrile and 35–50% in acetone. The
3-trans-mesylate gave 25–35% incorporation of F-18
and 3-cis-mesylate gave 15-25% incorporation of F-18
in acetonitrile. The reactions in acetone for both 3-trans-
and 3-cis-mesylate gave only ꢀ5% labeling yield. The
3-trans-mesylate had higher yields than the 3-cis-mesy-
late, but the radiochemical yield for substitution at the
3 position was lower than at the 4 position.
2. Results and discussion
2.1. Synthesis of WAY 100635 analogs
Three new fluorinated WAY 100635 analogs (12, 13,
and 14) were prepared using a similar procedure used
for preparing (7) starting from corresponding Pentam-
ethylbenzyl fluoroxycyclohexanecarboxylates (15, 17,
and 18). The major difficulty in making these analogs
is the synthesis of the fluorinated cyclohexanecarboxy-
lates. Previously, when DAST was used as the fluorinat-
ing agent, the yield of fluorinated products was very low
(ꢀ5%). The major side product was the unsaturated
cyclohexenecarboxylate. The current procedure (Scheme
2) of using the mixture of DAST and HF/pyridine as the
fluorinating agent gives a much higher yield (ꢀ70%).
However, the products are the mixtures of four fluori-
nated isomers, which require extensive HPLC proce-
dures to separate these isomers. The configuration of
these fluorocyclohexanecarboxylates was established by
proton NMR. A simple force field calculation of the
possible 3- and 4-fluorocyclohexanecarboxylic methyl
esters shows that in all cases, an axial carbomethoxy
group is 7–8 kcal molꢁ1 higher in energy than an equa-
torial one, while there is no energetic preference for fluo-
rine being either axial or equatorial (inhouse molecular
mechanics calculation using software PC Model). Hav-
ing established the basis for the carbomethoxy (or large)
group’s disposition toward remaining equatorial, the
configuration at fluorine can be determined by a
comparison of its geminal proton by 1H NMR, an
equatorial proton always appearing downfield of the
corresponding axial proton in substituted cyclohexanes.
This proton is easily discernible from the remaining ring
protons, since it is strongly shifted downfield by its prox-
imity to fluorine and additionally distinguishable by the
large proton-fluorine coupling constant (doublet).
2.4. In vitro assays
We have developed three classes of compounds: those
based on cyclohexanecarboxylic acid, those based on
benzoic acid, and those based on benzoic acid with
replacement of the pyridine with other aromatic ring sys-
tems.22 None of the compounds tested showed agonist
properties in the GTP-c-S binding (data not shown).
Among the compounds in the first class of compounds,
the trans-4-FCWAY has a higher affinity than the
comparable cis compound (Table 1); the trans- and cis-
3-FCWAY show the same trend (Table 1). trans-4-
FCWAY has a higher affinity compared with the benzoic
acid series and this was reflected in the pharmacokinetics
of these two groups of compounds.
Of the compounds in the third class (i.e., those contain-
ing other types of aromatic rings in place of the parent’s
pyridine) showed a lower range of inhibition constants,
which did not lead to a clear structure–activity
relationship.22
The study in rat hepatocytes showed that trans-4-
FCWAY produced eight metabolites. The major metab-
olite was the oxidation product of metabolism of the
aromatic ring. cis-4-FCWAY had a nearly identical
metabolite profile.25 The cis-3-FCWAY also had a sim-
ilar profile, but the trans-3-FCWAY had a measurable
amount of an oxidation product of the fluorocyclohex-
ane ring (Figs. 1 and 2). cis-3-FCWAY had a slower rate
of defluorination than that for the trans-3-FCWAY.

3740
L. Lang et al. / Bioorg. Med. Chem. 14 (2006) 3737–3748
O
CH3CH2
CH3CH2
O
C
O
C
F
DAST/HF/Pyridine
CH3CH2
O
O
C
F
OH
O
Cis and Trans mixture
Cis and Trans mixture
CH2Cl
O
O
O
NaOH
C
O H2C
C
O CH2CH3
C
OH
F
F
F
3- and 4- Fluoro Mixture
HPLC
O
C
O
C
F
CH2
O
CH2
O
O
F
16
17
O
O
C
CH2
O C
CH2
F
F
15
1) TFA
18
O
O
C
WAY-100634
CH2
O C
Cl
F
2) Cl2CHOCH3
F
15
OCH3
N
N
N
N
C
O
F
12
Scheme 2. Synthesis of FCWAYs.
2.5. In vivo studies in rats
WAY 100635 was needed to reduce FBWAY hippocam-
pal binding to that of the cerebellum.18 The low net
retention of cis-4-FCWAY at 30 min was due to rapid
efflux, because the distribution of cis-4-FCWAY at
5 min was within a factor of 2 to that obtained with
trans-4-FCWAY (Table 3). The uptake of trans-4-
FCWAY did not seem to be related to a1 receptor bind-
ing. Co-injection of 50 nmol of prazosin, an a1 receptor
antagonist, did not show a statistically significant
change in the distribution of trans-4-FCWAY (Table
4). However, in the case of cis-4-FCWAY, binding in
the cerebellum was blocked to some extent. This pre-
cluded a meaningful calculation of a target to non-target
tissue ratio, where the cerebellum is (normally) used as
the non-target tissue. A detectable decrease after prazo-
sin co-injection occurred in both the thalamus and
The biodistribution of trans-4-FCWAY and cis-4-
FCWAY showed the profound effects of the stereo-
chemistry of the fluorine. The uptake of the 4-cis isomer
at 30 min was roughly an order of magnitude lower than
the 4-trans isomer in the rat hippocampus and cortex.
The 4-cis-FCWAY had properties similar to those of
the 4-fluorophenylcarboxamide derivatives (FBWAY
and MeFBWAY) in that the expression for specific
binding (H/Cb ꢁ 1) was 2.6, compared to values of 4.7
and 4.9 for the two 4-fluorophenylcarboxamides (Table
2). The blood clearance of the cis- and trans-4-fluoro
compounds was similar. These compounds were blocked
by co-injection of 50 nmol of WAY 100635 indicating
saturable binding. As previously shown, 500 nmol of

L. Lang et al. / Bioorg. Med. Chem. 14 (2006) 3737–3748
3741
CH2Cl
HO
OH
O
C
H2 /Rh
O
C
OH
HO
COOH
HO
+
OH
O
C
O
C
OH
CH2
O
CH2 O
+
22
21
O
MsCl
O
C
OH
OMs
CH2
O C
CH2
O
26
22
18F
18F
18F- / K2CO3 / K-222
1) TFA
O
C
O
C
Cl
CH2
O
2) Cl2CHOCH3
WAY-100634
[F-18]17
OCH3
N
N
18F
N
N
C
O
[F-18]13
Scheme 3. Synthesis F-18 labeled FCWAY.
Table 1. Inhibition constants (Ki, nM) obtained by in vitro assay
Trans-3
90
80
70
60
50
40
30
20
10
Compound
Ki versus [3H]8-OH-DPAT human clone
MeFBWAY
FBWAY
0.79 0.11
1.1
0.25 0.08
trans-4-FCWAY
cis-4-FCWAY
trans-3-FCWAY
cis-3-FCWAY
FPWAY
1.45
<1
1.2
<1
Cis-3
0
0
90
80
70
60
50
40
30
20
10
0
20
40
60
80
100
120
Incubation Time(mins)
Figure 2. F-18 labeled trans-3-FCWAY and metabolites present after
incubation with rat hepatocytes. trans-3-FCWAY (diamond), fluoride
(triangle), aromatic oxidation (circle), and fluorocyclohexane oxida-
tion (square).
cortex, but none in the hippocampus, most likely be-
cause of the relatively high concentration of 5 HT1A
receptors in that tissue. From these data, the cis-4-
FCWAY appears to be inferior to the FBWAY and
MeFBWAY as the target to nontarget ratio is lower
and binding to the a1 receptor is evident.
0
20
40
60
80
100
120
Incubation Time (mins)
Figure 1. F-18 labeled cis-3-FCWAY and metabolites present after
incubation with rat hepatocytes. cis-3-FCWAY (diamond), fluoride
(triangle), and aromatic oxidation (circle).

3742
L. Lang et al. / Bioorg. Med. Chem. 14 (2006) 3737–3748
Table 2. Biodistribution (DUR) of radiolabeled WAY derivatives at 30 min in rats with and without 50 nmol of co-injected WAY 100635 (n = 4)
Compound
Cortex (Ctx)
Hippocampus (H)
Hypothalamus
Cerebellum
(Ctx ꢁ Cb)/Cb
(H ꢁ Cb)/Cb
4.7
FBWAY
+50 nmol
0.17 0.02
0.09 0.01
0.13 0.02
0.05 0.02
2.01 0.36
0.56 0.03
0.14 0.02
0.07 0.009
0.38 0.11
0.14 0.01
0.32 0.05
0.04 0.01
3.05 0.42
1.48 0.14
0.26 0.05
0.07 0.007
0.13 0.02
0.03 0.007
ND
0.066 0.008
0.05 0.01
0.05 0.01
0.04 0.006
0.16 0.03
0.10 0.006
0.07 0.008
0.07 0.007
1.5
1.0
1.4
0.1
11.6
4.6
1.0
0.0
2.1
4.9
MeFBWAY
+50 nmol
ND
0.0
4-trans-FCWAY
+50 nmol
1.35 0.29
0.37 0.04
0.19 0.04
0.11 0.02
18.1
13.8
2.6
4-cis-FCWAY
+50 nmol
0.0
Table 3. Biodistribution (DUR) of radiolabeled WAY derivatives at 5 min and 30 min after injection of cis-4-FCWAY and trans-4-FCWAY in rats
(n P 4)
FCWAY
Blood
Caudate
Thalamus
Hippocampus
Brain stem
Cerebellum
Cortex
50-cis
0.85 0.12
0.52 0.06
1.05 0.09
0.64 0.04
0.44 0.05
0.08 0.02
0.89 0.18
0.28 0.05
0.71 0.13
0.11 0.03
1.68 0.24
0.88 0.12
1.30 0.32
0.23 0.03
4.31 0.20
3.06 0.20
0.74 0.13
0.14 0.04
1.87 0.04
ND
0.39 0.07
0.07 0.02
0.59 0.02
0.18 0.04
0.95 0.21
0.13 0.03
3.82 0.11
1.97 0.16
300-cis
50-trans
300-trans
(DUR) differential uptake ratio: [(% ID/g) · body weight (g)/100].
Table 4. Percent reduction of cis-4-FCWAY and trans-4-FCWAY in
rats 30 min after co-injection of 50 nmol 4-FCWAY, 200 nmol WAY
100635, or 50 nmol of prazosin
Table 6. Biodistribution (DUR) of F-18 3-trans FCWAY in rats after
30 min with and without coinjection of 200 nmol of prazosin (n P 4)
NCA
+200 nmol prazosin % reduction
% reductiona
Blood
Caudate
Thalamus
0.44 0.03 0.54 0.07
0.36 0.05 0.25 0.04
1.56 0.25 1.12 0.34
ꢁ22.3
31.8a
28.3
15.0
17.3
11.8
22.9
50 nmol of 4-
FCWAY
200 nmol of
WAY 100635
50 nmol of
prazosin
Hippocampus 3.66 0.60 3.11 0.41
4-trans 4-cis
4-trans 4-cis 4-trans 4-cis
Brain stem
Cortex
Cerebellum
1.35 0.15 1.12 0.14
2.81 0.28 2.48 0.31
0.18 0.03 0.14 0.04
Blood
Caudate
Thalamus
ꢁ2.42 ꢁ3.66 ꢁ0.23 17.77 ꢁ8.47 ꢁ2.70
26.52 16.81b 27.23 22.67 9.08 20.13b
76.80b 26.16b 77.38b 27.99 17.14 26.83b
a Values in bold represent reduction which is significantly different
Hippocampus 88.77b 66.96b 92.93b 70.82b 15.67
2.66
from the control tissue (p < 0.05; n P 4).
Cerebellum
Cortex
13.74 10.33 18.51 11.33 ꢁ6.41 26.76b
88.95b 48.28b 91.58b 46.69b 11.43 30.62b
a % reduction = [(tissue uptake ꢁ tissue uptake with inhibitor)/tissue
uptake] · 100.
these experiments. These ratios are higher than those
obtained for MeBWAY (Table 2) and thus meet our cri-
terion for a compound with an effective specific binding
between that of MeBWAY and trans-4-FCWAY. The
3-trans derivative had H/Cb and Ctx/Cb ratios as high
as those for trans-4-FCWAY but gave lower radiochem-
ical yields (Table 5). To determine if a1 adrenoceptor
binding was involved in the 3-trans compound’s biodis-
tribution, 200 nmol of prasozin was co-injected, and
blocking was observed (Table 6). As a result of these
findings, this compound was not pursued.
b Values in bold represent reduction which is significantly different
from the control tissue (p < 0.05; n P 4).
The second group of radiopharmaceuticals studied
included the two 3-FCWAY analogs, cis-3-FCWAY
and trans-3-FCWAY. The cis analog has an H/Cb ratio
between 9 and 10, and a cortex/cerebellum (Ctx/Cb) ra-
tio between 3 and 4 at 30 min (Table 5). Data obtained
on two separate days show that reproducibility is high in
Table 5. Biodistribution (DUR) of F-18 3 cis-FCWAY in rats after 30 min with and without co-injection of 200 nmol of WAY 100635 (n P 6) and
biodistribution (DUR) of F-18 3-trans-FCWAY in rats after 30 min with and without coinjection of 200 nmol of WAY 100635 (n P 4)
F-18 3 cis-FCWAY
+200 nmol WAY
F-18 3-trans FCWAY
NCA
% reduction
NCA
+200 nmol WAY
% reduction
Blood
Caudate
0.31 0.05
0.17 0.04
0.24 0.06
1.09 0.06
0.26 0.02
0.12 0.01
0.41 0.06
0.32 0.04
0.33 0.05
0.11 0.03
0.13 0.04
0.12 0.02
0.11 0.03
0.09 0.02
0.12 0.03
0.34 0.03
ꢁ3.88
33.30a
48.44a
89.12a
56.20a
18.39
0.52 0.03
0.44 0.06
1.07 0.10
3.73 0.27
1.23 0.07
0.16 0.01
2.91 0.36
0.83 0.05
0.54 0.07
0.24 0.03
0.23 0.02
0.31 0.04
0.18 0.02
0.16 0.01
0.24 0.01
1.11 0.03
ꢁ5.14
45.39
Thalamus
Hippocampus
Brain stem
Cerebellum
Cortex
78.82a
91.62a
85.64a
1.59
71.88a
91.68a
Femur
ꢁ5.54
ꢁ34.56
a Values in bold represent reduction which is significantly different from the control tissue (p < 0.05; n P 6). Average of two different days.

L. Lang et al. / Bioorg. Med. Chem. 14 (2006) 3737–3748
3743
The final criterion is the rate of defluorination in vivo. It
is clear from these data at 30 min in rats that the uptake
in bone is low compared to fluoride itself, which gave a
DUR of 5.1 whereas FBWAY gave a DUR at 0.25. The
3-cis is clearly the best of the four FCWAY compounds
in this regard with a femur uptake at 30 min in rat of 0.3
DUR compared to 1.4 for 4-trans-FCWAY, 1.3 for
4-cis-FCWAY, 0.8 for 3-trans and 5.1 for fluoride. Rat
metabolite study showed that all these radioligands were
rapidly metabolized in the blood with ꢀ70% parent
activity at 5 min decreasing to 30% at 30 min. However,
the metabolites did not get into the brain, after 30 min
the brain was >97% parent for all the F-18 FCWAYs.
are given in Hertz. Mass spectra were recorded on a
Hewlett Packard 5989B Mass Spectrometer linked to a
Hewlett Packard 5890 Series II Plus Gas Chromato-
graph. Elemental analyses were performed by Galbraith
Laboratories (Knoxville, TN). Thin-layer chromatogra-
phy of the radioactive products was performed on
Whatman LK6DF silica gel glass-backed plates
(5 · 20 cm, 250 lm). TLC radio chromatograms were
obtained using Fuji Bio-imaging Analysis System 1500.
Semi-prep HPLC was carried out using a Perkin-Elmer
series 200 LC pump and UV absorbance was monitored
with a Waters 486 UV detector and radioactivity was
monitored using a Beckman Model 170 radioisotope
detector. The purification of radiolabeled compounds
was performed using a reversed-phase semi-prep Rainin
Microsorb, 5 lm (10 · 250 mm) C-18 column.
The position and configuration of the fluorine substitu-
tion can affect both affinity and stability of F-18 labeled
WAY 100635 derivatives. The 4-cis isomer has the
weakest affinity of the four isomers and the lowest hip-
pocampus to cerebellum ratio. Although the 3-trans
had a useable radiochemical yield, the H/Cb ratio was
similar to that obtained with trans-4-FCWAY and we
observed a1 adrenoceptor binding. The 3-cis isomer
has the lowest defluorination, nanomolar affinity, and
an intermediate H/Cb ratio. The closest competitor is
the previously reported compound with the 1,3-pyrimi-
dine substituted for the pyridine in the FBWAY com-
pound (FPWAY).22 It has slightly poorer properties
when compared to cis-3-FCWAY with an H/Cb ratio
of 6.5 compared to an H/Cb ratio of 10 for cis-3-
FCWAY. Neither of these compounds has yet to be
shown to be sensitive to changes in serotonin concentra-
tion in vivo and, until that has been shown, trans-4-
FCWAY is the compound of choice. However, two
compounds, cis-3-FCWAY and FPWAY, are now
available to test whether serotonin ligands can be used
to measure endogenous serotonin.
3.2. Mixture of trans-, cis-ethyl 4-fluorocyclohexane-
carboxylates and trans-, cis-ethyl 3-fluorocyclo-
hexanecarboxylates
To an ice cold solution of 100 g of 70% hydrogen fluo-
ride in pyridine containing 2.52 g of sodium fluoride
(60.0 mmol) was added 6.89 g of commercially available
ethyl 4-hydroxycyclohexanecarboxylate (40.0 mmol,
mixture of cis and trans isomers) and 5.28 ml (diethyla-
mino)sulfur trifluoride (40.0 mmol). The mixture was
stirred at room temperature for 4 h and then treated
with 300 ml of 25% sodium carbonate solution and more
solid sodium carbonate was added slowly until the solu-
tion was neutral. The aqueous solution was extracted
with 2· 200 ml of dichloromethane and treated with
2 ml of bromine to add across the double bond of the
ethyl cyclohex-3-enecarboxylate formed during the reac-
tion. The solution was dried over anhydrous sodium sul-
fate and solvent evaporated at reduced pressure. The
residue was distilled at 100 ꢁC under the vacuum
(0.1 mm) to give a mixture of 4.81 g of the four cis
and trans fluorinated products in 70% yield.
In conclusion, cis-3-FCWAY has a factor of 2 higher H/
Cb ratio compared to FPWAY and MeBWAY. cis
3-FCWAY has a low rate of defluorination in this series
of four alkyl fluoride containing compounds, but not as
low as that obtained for FPWAY. A series of radiofluo-
rinated compounds that bind to the 5-HT1A receptor are
now available to study a range of serotonergic processes.
High affinity 4-trans-FCWAY had the best properties
for measuring receptor density given its high hippocam-
pus to cerebellum ratio and 3-cis-FCWAY had the best
properties of the analogs with lower affinity and faster
pharmacokinetics to be responsive to endogenous
serotonin concentrations.
3.3. Mixture of trans-, cis-4-fluorocyclohexanecarboxylic
acids and trans-, cis-3-fluorocyclohexanecarboxylic acids
To an aqueous solution of 10% sodium hydroxide
(40 ml) was added 4.01 g of above fluorinated mixture
(23.0 mmol). The solution was stirred at room tempera-
ture overnight and then acidified with 12 N HCl drop-
wise until the solution was tested acidic by pH paper.
Upon standing at room temperature, the hydrolyzed
products precipitated out of the solution as the mixture
of four fluorinated cyclohexanecarboxylic acids (2.61 g,
yield 78%).
3. Experimental
3.4. Mixture of trans-, cis-pentamethylbenzyl
4-fluorocyclohexanecarboxylates and trans-,
cis-pentamethylbenzyl 3-fluorocyclohexanecarboxylates
3.1. General chemistry
WAY 100634 (2) was obtained from Med-Life Systems,
Inc. Upper Darby, PA. All other chemicals were
purchased from Aldrich Chemical Company,
Milwaukee, WI. The 1H and 13C NMR spectra were ob-
tained on a Varian Gemini-2000 at 200 and 50 MHz,
respectively. Chemical shifts are reported in ppm (d)
downfield of tetramethylsilane and coupling constants
To a solution of the above mixture of the fluorinated
cyclohexanecarboxylic acids (2.00 g, 13.7 mmol) in
20 ml N,N-dimethylformamide were added pentameth-
ylbenzyl chloride (2.69 g, 13.7 mmol) and triethylamine
(1.39 g, 13.7 mmol). The mixture was stirred at room
temperature overnight. The reaction mixture was

3744
L. Lang et al. / Bioorg. Med. Chem. 14 (2006) 3737–3748
poured into a 200 ml 10% sodium bicarbonate solution
to give a white precipitate. The solid products were col-
lected by filtration and dried under vacuum to give
3.62 g of mixture of four pentamethylbenzyl esters of
fluorocyclohexanecarboxylic acid (86%).
JCF = 20.5 Hz), 41.4 (d, JCF = 11.2 Hz), 62.6, 91.4 (d,
JCF = 173.3 Hz), 129.4, 133.2, 134.1, 136.3, 174.9 (d,
JCF = 2.4 Hz). MS (EI) 306 (M+), 160, 145, 131, 105.
3.7. cis- and trans-Pentamethylbenzyl 4-hydroxycycloh-
exanecarboxylates (19, 20)
3.5. cis-Pentamethylbenzyl 4-fluorocyclohexanecarboxylate
(15)
Preparation of pure cis-pentamethylbenzyl 4-hydroxy-
cyclohexanecarboxylate (19) has been described previ-
ously starting from the mixture of cis- and trans-ethyl
4-hydroxycyclohexanecarboxylates.18 The pure cis iso-
mer was obtained by fractional recrystallization from
the mixture of cis and trans isomers of pentamethylben-
zyl 4-hydroxycyclohexanecarboxylate. After recrystalli-
zation and collection of cis isomer, the mother liquor
was evaporated to obtain a mixture of isomers, enriched
in trans (20). This could be carried through to obtain
pure trans-nosylate (23) in the next step.
Part of the mixture (1.98 g) was dissolved in 10% ethyl
acetate/hexane and about 60 mg of mixture in 0.5 ml
solution was injected each time onto a normal phase
prep-HPLC column running with 5% ethyl acetate/hex-
ane at 10 ml/min. The pure cis-pentamethylbenzyl 4-flu-
oroxycyclohexanecarboxylate came out at about 11 min
and remaining mixture came out about 13 min. After 30
injections, the fractions containing the product were
combined to yield 250 mg of cis-pentamethylbenzyl
4-fluorocyclohexanecarboxylate as white crystals, mp
132–133 ꢁC. 1H NMR (CDCl3) d 1.28–1.62 (m, 2H),
1.72–2.12 (m, 6H), 2.17–2.48 (m, 16H), 4.76 (dm,
JHF = 47.9 Hz, 1H), 5.24 (s, 2H). 13C NMR (CDCl3) d
16.4, 16.8, 17.2, 23.4 (d, JCF = 2.7 Hz), 30.0 (d,
JCF = 18.7 Hz), 41.9, 62.4, 88.4 (d, JCF = 168.9 Hz),
129.5, 133.1, 134.1, 136.2, 175.6. MS (EI) 306 (M+),
160, 145,131, 105.
3.8. trans- and cis-Pentamethylbenzyl 3-hydroxycycloh-
exanecarboxylates (21, 22)
To a solution of 2.00 g of 3-hydroxybenzoic acid in
100 ml ethyl acetate in a 300 ml pressure bottle were
added 300 mg of rhodium (5 wt. % on alumina) and
1.0 ml of acetic acid. The mixture was hydrogenated at
60 psi overnight. Analysis by GC/MS indicated a mix-
ture of unreduced 3-hydroxybenzoic acid, fully reduced
cyclohexanecarboxylic acid, and 3-cis-and 3-trans-
hydroxycyclohexanecarboxylic acid. The catalyst was
then filtered and the solvent evaporated to give 1.95 g
of solid mixture. The mixture was redissolved in 20 ml
of N,N-dimethylformamide and 2.85 g of pentamethylb-
enzyl chloride, 2.0 ml of triethylamine were added to the
solution. After stirring at room temperature overnight,
the solution was poured into a 200 ml 10% sodium
bicarbonate solution to give a white precipitate.
3.6. trans-Pentamethylbenzyl 4-fluoroxycyclohexane-
carboxylate, trans- and cis-pentamethylbenzyl 3-fluor-
oxycyclohexanecarboxylate (16, 17, and 18)
The remaining of the above mixture was further separated
with reversed-phase semi-prep HPLC with 60%
acetonitrile running at 6 ml/min to give 410 mg of trans-
pentamethylbenzyl
4-fluorocyclohexanecarboxylate
(16), 320 mg of trans pentamethylbenzyl 3-fluorocycloh-
exanecarboxylate (17), and 210 mg of cis-pentamethylb-
enzyl 3-fluorocyclohexanecarboxylate (18).
The solid was collected by filtration and air-dried to give
3.40 g of a mixture of cis- and trans-pentamethylbenzyl
3-hydroxycyclohexanecarboxylate. This mixture was
dissolved in minimal amount of dichloromethane, load-
ed onto a 5.5 · 20 cm flash chromatography silica gel
column, and eluted with 10% ethyl acetate/hexanes (v/
v). The fractions containing the product were combined
and solvent evaporated to give 0.74 g of trans-pentam-
ethylbenzyl 3-hydroxycyclohexanecarboxylate (21)
(17%) and 1.38 g of cis-pentamethylbenzyl 3-hydroxy-
cyclohexanecarboxylate (22) (32%).
Compound 16: mp 104–105 ꢁC. 1H NMR (CDCl3) d
1.40–1.67 (m, 4H), 1.92–2.18 (m, 4H), 2.20–2.40 (m,
16H), 4.50 (dm, JHF = 48.4 Hz, 1H), 5.25 (s, 2H). 13C
NMR (CDCl3) d 16.4, 16.8, 17.2 26.1 (d, JCF = 10.8 Hz),
31.8 (d, JCF = 19.0 Hz), 41.7 (d, JCF = 1.5 Hz), 62.5,
91.2 (d, JCF = 172.4 Hz), 129.4, 133.2, 134.1, 136.3,
175.6 (d, JCF = 2.4 Hz). MS (EI) 306 (M+), 160, 145,
131, 105.
Compound 17: mp 105–106 ꢁC. 1H NMR (CDCl3) d
1.22–1.78 (m, 5H), 1.82–2.02 (m, 3H), 2.10–2.38 (m,
15H), 2.75 (tt, 1H), 4.90 (dm, JH = 48.4 Hz, 1H), 5.24
(s, 2H). 13C NMR (CDCl3) d 16.4, 16.8, 17.2, 19.7 (d,
JCF = 2.0 Hz), 28.3, 30.3, (d, JCF = 21.0 Hz), 33.2 (d,
JCF = 21.0 Hz), 38.0, (d, JCF = 1.5 Hz), 62.3, 88.6 (d,
JCF = 168.9 Hz), 129.4, 133.1, 134.0, 136.2, 176.0. MS
(EI) 306 (M+), 160, 145, 131, 105.
Compound 21: mp 139.5–141.0 ꢁC. 1H NMR (CDCl3) d
1.4–1.7 (m, 6H), 1.7–1.9 (m, 2H), 2.2–2.4 (m, 15H), 2.8
(m, 1H), 4.0 (m, 1H), 5.2 (s, 2H). 13C NMR (CDCl3) d
16.5, 16.9, 17.3, 20.0, 28.4, 33.0, 35.7, 38.2, 62.4, 66.3,
129.5, 133.0, 134.0, 136.1, 176.3. MS (EI) 304 (M+),
160, 145, 131 105.
Compound 18: mp 99–100 ꢁC. 1H NMR (CDCl3) d
1.22–1.57 (m, 3H), 1.59–1.69 (m, 2H), 1.80–2.00 (m,
2H), 2.00–2.14 (m, 1H), 2.18–2.37 (m, 16H), 4.46 (dm,
JHF = 48.2 Hz, 1H) 5.25 (s, 2H). 13C NMR (CDCl3) d
16.4, 16.8, 17.2, 22.6 (d, JCF = 11.2 Hz), 27.9 (d,
JCF = 2.0 Hz), 32.2 (d, JCF = 18.6 Hz), 35.0 (d,
Compound 22: mp 134–135 ꢁC. 1H NMR (CDCl3) d
1.2–1.6 (m, 4H), 1.8–2.1 (m, 4H), 2.2–2.4 (m, 16H), 3.7
(m, 1H), 5.3 (s, 2H). 13C NMR (CDCl3) d 16.5, 16.9,
17.3, 23.3, 28.2, 35.1 37.8, 42.1, 62.6, 69.9, 129.3,
133.1, 134.0, 136.2, 175.5. MS (EI) 304 (M+), 160, 145,
131 105.

L. Lang et al. / Bioorg. Med. Chem. 14 (2006) 3737–3748
3745
3.9. trans- and cis-Pentamethylbenzyl 4-(4-nitro-
benzenesulfonyl)-oxycyclohexanecarboxylates (23, 24)
32.6, 35.1, 39.1, 42.0, 62.8, 80.1, 129.2, 133.1, 134.0,
136.3, 174.2. MS (EI) 382 (M+), 338, 308, 286, 256,
205, 175, 160, 145, 131, 108, 81.
To a solution of 0.61 g (2.0 mmol) mixture of 19 and 20
obtained from the mother liquor as described earlier in
10 ml methylene chloride were added 4-nitro-
benzenesulfonyl chloride (0.44 g, 2.0 mmol) and triethyl-
amine (0.28 ml, 2.0 mmol). The mixture was stirred at
room temperature overnight. At the end of the reaction,
the solid was filtered and the solution was loaded on a
5.5 · 20 cm silica gel and eluted with 20% ethyl ace-
tate/hexane to give 0.41 g 23 (42%) and 0.15 g of 24
(16%) as a white solid.
3.12. cis-4-Fluoro-N-{2-[4-(2-methoxyphenyl)piperazin-1-
yl]ethyl}-N-(2- pyridyl)cyclohexanecarboxamide (12)
To a 5 ml V-vial were added 150 mg of 15, 0.3 ml ani-
sole, and 2 ml of trifluoroacetic acid. The mixture was
stirred at room temperature for 5 min. Trifluoroacetic
acid was evaporated and 0.20 ml of dichloromethyl
methylether was added and heated at 90 C for 5 min.
The vial was cooled and evaporated with argon flow.
To the above residue, 0.20 g of WAY-100634 (2) and
85 lL of TEA in 3.0 ml of methylene chloride were add-
ed and stirred at room temperature for 2 h. At the end of
the reaction, the solution was loaded onto a flash chro-
matography silica gel column and eluted with ethyl
acetate containing 0.1% TEA to give 140 mg of product
(65%). 1H NMR (CDCl3) d 1.16–1.44 (m, 2H), 1.58–1.63
(m, 2H), 1.86–2.06 (m, 4H), 2.27–2.33 (m, 1H),
2.59–2.66 (m, 6H) 2.99 (s, broad, 4H), 3.84 (s, 3H),
3.99 (t, 2H), 4.71 (dm, JHF = 48.8 Hz, 1H), 6.82–7.03
(m, 4H), 7.21–7.34 (m, 2H), 7.76 (td, 1H), 8.51 (dd,
1H). 13C NMR (CDCl3) d 23.6, 30.0 (d, JCF = 21.5 Hz),
Compound 23: mp 131 ꢁC (d). 1H NMR (CDCl3) d
1.4–1.6 (m, 4H), 1.9–2.1 (m, 4H), 2.2–2.4 (m, 16H), 4.6
(m, 1H), 5.22 (s, 2H), 8.1 (d, 2H), 8.4 (d, 2H). 13C
NMR (CDCl3) d 16.4, 16.8, 17.2, 26.5, 31.3, 41.3, 62.6,
82.4, 124.7, 129.2, 129.2, 133.2, 134.0, 136.4, 143.5,
150.9, 175.0. MS (EI) 446, 412, 363, 322, 308, 286,
271, 225, 203, 160, 147, 131, 108, 81.
Compound 24: mp 137 ꢁC (d). 1H NMR (CDCl3) d
1.4–2.0 (m, 8H), 2.2–2.3 (m, 15H), 2.4 (m, 1H) 4.9 (m,
1H), 5.22 (s, 2H), 8.1 (d, 2H), 8.4 (d, 2H). 13C NMR
(CDCl3) d 16.6, 16.9, 17.3, 23.5, 30.2, 41.1, 62.6, 80.6,
124.6, 129.1, 129.2, 133.1, 134.0, 136.3, 143.5, 150.8,
174.9. MS (EI) 488 (M+), 446, 442, 362, 354, 308, 286,
243, 225, 203, 160, 147, 131, 81.
41.2, 45.4, 50.7, 53.5, 55.4, 56.3, 87.8 (d, JCF
=
168.4 Hz), 111.4, 118.3, 121.1, 122.4, 122.5, 123.0,
138.5, 141.6, 149.5, 152.5, 156.2, 176.6. MS (EI) 440
(M+), 425, 311, 278, 249, 218, 205, 190, 162, 149. Anal.
Calcd (C25H33FN4O2F): C, 68.16; H, 7.55; N, 12.72.
Found, C, 67.85; H, 7.80, N, 12.50. Compounds 13
and 14 are prepared using a similar procedure.
3.10. trans-Pentamethylbenzyl 3-(methanesulfonyl)oxy-
cyclohexanecarboxylate (25)
To a solution of 21 (81 mg, 0.27 mmol) in 2 ml methy-
lene chloride were added methanesulfonyl chloride
(22 ll, 0.28 mmol) and diisopropylethylamine (51 ll,
0.29 mmol). The mixture was stirred at room tempera-
ture overnight. At the end of the reaction, the solution
was loaded on to a 3.8 · 20 cm flash chromatography
silica gel column and eluted with 3% ethyl acetate in
methylene chloride. The fractions containing the prod-
uct were combined and the solvent evaporated to give
3.13. trans-3-Fluoro-N-{2-[4-(2-methoxyphenyl)pipera-
zin-1-yl]ethyl}-N-(2-pyridyl)cyclohexanecarboxamide
(13)
1H NMR (CDCl3) d 1.22–2.22 (m, 9H), 2.58–2.81 (m,
6H), 2.99 (s, broad, 4H), 3.84 (s, 3H), 4.00 (t, 2H),
4.89 (dm, JHF = 48.2 Hz, 1H), 6.83–7.04 (m, 4H),
7.22–7.33 (m, 2H), 7.77 (dt, 1H), 8.53 (dd, 1H). 13C
NMR (CDCl3) d 19.6, 28.7, 30.1 (d, JCF = 21.5 Hz),
33.7 (d, JCF = 20.0 Hz), 36.6, 45.4, 50.8, 53.6, 55.5,
56.4, 89.1 (d, JCF = 167.0 Hz), 111.5, 118.4, 121.2,
122.5, 122.7, 123.1, 138.5, 141.7, 149.6, 152.6, 155.9,
175.9. MS (EI) 440 (M+), 425,311, 218, 205, 190, 162,
149. Purity: 95% by HPLC.
1
55 mg (54%) of white solid. Mp 120–121 ꢁC. H NMR
(CDCl3) d 1.4–1.8 (m, 4H), 1.8–2.0 (m, 4H), 2.1–2.4
(m, 15H), 2.8 (m, 1H), 3.0 (s, 3H), 5.0 (m, 1H), 5.25
(s, 2H). 13C NMR (CDCl3) d 16.6, 16.9, 17.3, 20.0,
28.1, 31.0, 33.4, 38.3, 38.7, 62.7, 78.3, 129.2, 133.1,
134.0, 136.3, 175.2. MS (EI) 382 (M+), 338, 308, 286,
256, 205, 175, 160, 145, 131,108, 81.
3.14. cis-3-Fluoro-N-{2-[4-(2-methoxyphenyl)piperazin-1-
yl]ethyl}-N-(2- pyridyl)cyclohexanecarboxamide (14)
3.11. cis-Pentamethylbenzyl 3-(methanesulfonyl)oxycyc-
lohexanecarboxylate (26)
1H NMR (CDCl3) d 1.01–1.14 (m, 1H), 1.26–1.56 (m,
2H), 1.76–1.85 (m, 3H), 1.99–2.38 (m, 3H), 2.59–2.66
(m, 6H), 2.99 (s, broad, 4H), 3.84 (s, 3H), 3.98 (t, 2H),
4.24 (dm, JHF = 48.8 Hz, 1H), 6.82–7.02 (m, 4H),
7.22–7.34 (m, 4H), 7.78 (td, 1H), 8.53 (dd, 1H). 13C
NMR (CDCl3) d 23.0 (d, JCF = 12.2 Hz), 28.4, 32.3 (d,
JCF = 18.1 Hz), 35.6 (d, JCF = 20.0 Hz), 40.4 (d,
JCF = 11.22 Hz), 45.7, 50.8, 53.6, 55.5, 56.3, 91.7 (d,
JCF = 173.3 Hz), 111.5, 118.4, 121.2, 122.3, 122.7,
123.1, 138.6, 141.6, 149.7, 152.6, 156.0, 174.6. MS (EI)
440 (M+), 425,311, 218, 205, 190, 162, 149. Purity:
96% by HPLC.
To a solution of 22 (200 mg, 0.66 mmol) in 4 ml methy-
lene chloride were added methanesulfonyl chloride
(54 ll, 0.68 mmol) and diisopropylethylamine (126 ll,
0.71 mmol). The mixture was stirred at room tempera-
ture overnight. At the end of the reaction, the product
was purified by flash chromatography on silica gel as de-
scribed above to give 146 mg white solid (58%). mp
1
120.0–121.5 ꢁC. H NMR (CDCl3) d 1.2–2.0 (m, 6H),
2.1–2.5 (m, 17H), 3.0 (s, 3H), 4.6 (m, 1H), 5.25 (s,
2H). 13C NMR (CDCl3) d 16.5, 16.9,17.3, 23.4, 27.8,

3746
L. Lang et al. / Bioorg. Med. Chem. 14 (2006) 3737–3748
3.15. Synthesis of [18F] labeled WAY 100635 analogs
In addition, a complete analysis (Ki SEM) was
carried out for MeFBWAY (0.791 0.113) and
trans-4-FCWAY (0.247 0.085 nM). The biogenic
amine profile for trans-4-FCWAY gave the following
Ki (nM) values: adrenergic a1 40.9 4.3, adrenergic
a2 3600 400, dopamine D2 152 15.2, dopamine
D3 79 23, and serotonin 5-HT2A 514 143. The bio-
genic amine profile for MeFBWAY gave the following
Ki (nM) values: adrenergic a1 12.5 2, adrenergic a2
342 14, dopamine D2 119 7, dopamine D3
26 10, serotonin 5-HT2A 133 32, r1 527 162,
and r2 5300 1000. Testing was performed by MDS
Panlabs, Seattle, WA.
To a 1 ml V-vial containing 3 lmol of potassium car-
bonate in 15 ll of water and 6 lmol of Kryptofix-
2.2.2. in 30 ll of acetonitrile, 300–500 ll of aqueous
F-18 fluoride activity (30–50 mCi) was added. The
water was removed with argon flow three times using
anhydrous acetonitrile on a 105 ꢁC heating block. To
the above vial containing the anhydrous [18F]fluoride
ion, 3 mg of substrate (23, 24, 25, or 26,) in 0.1 ml
of acetonitrile or acetone was added. The vial was
sealed and heated on the heating block for 10 min.
The reaction mixture was cooled to room temperature
and diluted with 1 ml ether. The ether solution was
passed through a small silica gel column (0.5 ml) to
a 5 ml V-vial and rinsed with another 1 ml ether.
The solvent was evaporated with argon flow and
the pentamethylbenzyl ester was hydrolyzed with
0.1 ml trifluoroacetic acid in 2 min at room tempera-
ture. The trifluoroacetic acid was removed with argon
flow and 40 lL of a,a-dichloromethyl methylether was
added to the reaction vial. The vial was sealed using
a cap with Teflon seal, heated at 90 ꢁC for 5 min, and
cooled in ice water for 2 min. The vial cap was
opened and the remaining a,a-dichloromethyl methy-
lether was evaporated with argon flow. The vial con-
taining the acid chloride was added 5 mg of substrate
(2) in 0.2 ml acetonitrile containing 5 lL triethyl-
amine. The vial was sealed and heated at 90 ꢁC for
5 min. The vial was cooled in ice water and the
reaction mixture diluted with 0.3 ml HPLC solvent
(40–50% acetonitrile in 5 mM phosphate buffer con-
taining triethylamine at a flow rate of 5 ml/min) and
injected onto the reversed-phase HPLC column. The
fraction containing the product was collected, diluted
with 2 volumes of water, and passed through a 1 ml
C-18 Bond Elute column. The column was washed
with 10 ml of water and the product trapped on the
column was eluted with 0.5 ml ethanol. The over
radiochemical yields are 10–15% for 4-cis and 4-trans
compounds and 5–10% for 3-cis and 3-trans
compounds. The radiochemical purities were >98%
for all four compounds. The specific activity for these
compounds was between 2000 and 8000 Ci/mmol
EOB.
3.17. Rat biodistribution studies
In the biodistribution studies, the rats were injected
intravenously with 50 lCi of 18F radioligand or coinject-
ed with 50 lCi of 18F radioligand and 50 nmol of nonra-
dioactive WAY 100635. The rats were sacrificed at
30 min and the brain was immediately placed in 0.3 M
sucrose on ice. The blood and other tissues were excised
from each animal and weighed. The brain was dissected
on ice and the various brain regions weighed. The radio-
active content of the blood and various tissues was as-
sessed by gamma counting.
3.18. Rat metabolite studies
The rats were injected intravenously with 100 lCi of
the 18F WAY 100635 analogs and sacrificed at 5,
10, 15, and 30 min. Blood and brain were removed,
weighed, and counted to determine the total activity.
After the blood was centrifuged, the serum was re-
moved, mixed with an equal volume of acetonitrile,
and centrifuged. The brain was placed in an equal vol-
ume of acetonitrile and homogenized for 15–30 s
bursts. Following centrifugation, the radioactive con-
tent of the supernatant and pellets was determined.
The supernatants were applied to TLC plates, devel-
oped, and placed on a phosphorimaging plate over-
night. The plates were scanned for the WAY analog
and its metabolites the next day using a Fuji Bio-im-
aging Analysis System 1500.
3.19. Incubation of cold and F-18 labeled FCWAYs with
hepatocytes
3.16. In vitro assays (Table 1)
Receptor binding studies were carried out using
1.5 nM H-3 8-OH-DPAT with human recombinant
CHO-K1 Cells containing serotonin 5-HT1A receptors.
Five concentrations of each compound were used to
determine an IC50, which was converted to a Ki. Bind-
ing to adrenergic alpha1 and dopamine D2 receptors
was also determined. All compounds showed inhibi-
tion of binding to the adrenergic a1 and dopamine
D2 receptors at a concentration of 10 lM. The inhibi-
tion at the adrenergic a1 receptor was greater than
95% for all compounds at this concentration, whereas
the inhibition at the dopamine D2 receptor was much
lower and always less than 50% inhibition. Testing
was performed at MDS Panlabs, Seattle, WA, on
two separate occasions.
The cryopreserved hepatocytes from male Sprague–
Dawley rats (In Vitro Technologies, Inc., Baltimore,
MD) were used for in vitro metabolism studies of
FCWAYs. The cells, which were stored in liquid nitro-
gen, were thawed rapidly at 37 ꢁC in a water bath and
gradually diluted with cell culture medium (RPMI
Medium 164O media, Life Technologies, Rockville,
MD). After washing the cells with the medium, the via-
ble cell concentration was adjusted to 1.0 million per ml
and the resulting cell suspension was incubated at 37 ꢁC
for 15 min prior to the introduction of test compound.
From stock solution of FCWAY (2.0 mg/ml in 10%
EtOH in water), 10 ll was added into 1 ml of cell sus-
pension, to give a final concentration of 20 lg/ml. The
suspension was maintained at 37 ꢁC, 100 ll of cell

L. Lang et al. / Bioorg. Med. Chem. 14 (2006) 3737–3748
3747
receptor antagonist WAY100635. Eur. J. Pharmacol.
1994, 264, 95–97.
suspension was removed and added to 100 ll acetoni-
trile at 10, 30, 60, and 120 min. Each suspension was
centrifuged at 5000 rpm for 5 min. The metabolites in
20 ll supernatant were analyzed by LC/MS.
7. Fletcher, A.; Forster, E. A.; Bill, D. J.; Brown, G.; Cliffe,
I. A., et al. Electrophysiological, biochemical, neurohor-
monal and behavioural studies with WAY-100635, a
potent, selective and silent 5-HT1A receptor antagonist.
Behav. Brain Res. 1996, 73, 337–353.
8. Mathis, C. A.; Simpson, N. R.; Mahmood, K.; Kinahan,
P. E.; Mintun, M. A. [11C]WAY 100635: A radioligand for
imaging 5-HT1A receptors with positron emission tomog-
raphy. Life Science 1994, PL403–PL407.
9. Ma, Y.; Lang, L.; Kiesewetter, D. O.; Jagoda, E.;
Sassaman, M., et al. Metabolism of fluorine-18 labeled
5-HT1A antagonist FCWAY by human and rat hepato-
cytes. J. Chromatogr. A 2004, 1034, 149–153.
10. Carson, R. E.; Lang, L.; Watabe, H.; Der, M. G.; Adams,
H. R., et al. PET evaluation of [(18)F]FCWAY, an
analog of the 5-HT(1A) receptor antagonist, WAY-
100635. Nucl. Med. Biol. 2000, 27, 493–497.
For the analysis of radiolabeled compounds, 0.5 mCi of
[18F]FCWAY in 20 ll ethanol was added to 1.0 ml sus-
pension of cells and corresponding unlabeled com-
pound, prepared as described in the preceding
paragraph. Metabolites at 10, 30, 60, and 120 min were
chosen for analysis, which was performed in the same
manner as before. Supernatant (20 ll) was injected into
the HPLC with a radioactivity detector and analyzed by
LC/MS.
3.20. LC/MS
All experiments were performed with a Finnigan LCQ
MS (Finnigan Corporation, San Jose, CA, USA) cou-
pled with HP series 1100 HPLC system (Agilent Tech-
nologies Company, Palo Alto, CA, USA). HPLC
utilized a YMC-pack ProC-18 reversed-phase column
(150 · 4.6 mm ID, YMC, Inc c/o Waters, Milford,
MA, USA), eluting with 50 mM ammonium acetate
and acetonitrile at 0.5 ml/min flow rate and a gradient
of 0–65% acetonitrile over 10 min followed by iso-
cratic elution at 65% for an additional 10 min. The
entire column eluent was introduced into the ESI
MS source with a standard high flow tune method.
Ion detection was achieved with the Finnigan ESI
using a spray voltage of +4200 V, capillary heater
temperature of 200 ꢁC, sheath gas flow of 80 ml/min
(N2), and an auxiliary gas flow of 20 ml/min (N2).
By using the entire column eluent, FCWAYs could
be determined with the detection limit of 1 pmol on-
column.
11. Pike, V. W.; McCarron, J. A.; Lammertsma, A. A.; Hume,
S. P.; Poole, K., et al. First delineation of 5-HT1A
receptors in human brain with PET and [11C]WAY-
100635. Eur. J. Pharmacol. 1995, 283, R1–R3.
12. Pike, V. W.; McCarron, J. A.; Lammertsma, A. A.;
Osman, S.; Hume, S. P., et al. Exquisite delineation of 5-
HT1A receptors in human brain with PET and [carbon-
yl-11C]WAY-100635. Eur. J. Pharmacol. 1996, 301,
R5–R7.
13. Zhuang, Z. P.; Kung, M. P.; Kung, H. F. Synthesis and
evaluation of 4-(20-methoxyphenyl)-1-[20-[N-(200-pyridi-
nyl)-p-iodobenzamido]ethyl]piperazine
(p-MPPI):
a
new iodinated 5-HT1A ligand. J. Med. Chem. 1994, 37,
1406–1407.
14. Kung, M. P.; Zhuang, Z. P.; Frederick, D.; Kung, H. F. In
vivo binding of [123I] 4-(20-methoxyphenyl)-1-[20-(N-200-
pyridinyl)-p-iodobenzamido-]ethylpiperazine, p-MPPI, to
5-HT1A receptors in rat brain. Synapse 1994, 359–366.
15. Kung, H. F.; Stevenson, D. A.; Zhuang, Z. P.; Kung, M.
P.; Frederick, D., et al. New 5-HT1A receptor antagonist:
[3H]p-MPPF. Synapse 1996, 23, 344–346.
16. Shiue, C. Y.; Shiue, G. G.; Mozley, P. D.; Kung, M. P.;
Zhuang, Z. P., et al. P-[18F]-MPPF: a potential radioli-
gand for PET studies of 5-HT1A receptors in humans.
Synapse 1997, 25, 147–154.
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