Synthesis and pharmacological characterization of novel inverse agonists acting on the viral-encoded chemokine receptor US28

Article abstract of DOI:10.1016/j.bmc.2006.06.054

G-protein coupled receptors encoded by viruses represent an unexplored class of potential drug targets. In this study, we describe the synthesis and pharmacological characterization of the first class of inverse agonists acting on the HCMV-encoded receptor US28. It is shown that replacement of the 4-hydroxy group of lead compound 1 with a methylamine group results in a significant 6-fold increase in affinity. Interestingly, increasing the rigidity of the spacer by the introduction of a double bond also leads to a significant increase in binding affinity compared to 1. These novel inverse agonists serve as valuable tools to elucidate the role of constitutive signaling in the pathogenesis of viral infection and may have therapeutic potential as leads for new antiviral drugs.

Full text of DOI:10.1016/j.bmc.2006.06.054

Bioorganic & Medicinal Chemistry 14 (2006) 7213–7230
Synthesis and pharmacological characterization of novel inverse
agonists acting on the viral-encoded chemokine receptor US28
Janneke W. Hulshof, Henry F. Vischer, Mark H. P. Verheij, Silvina A. Fratantoni,
Martine J. Smit, Iwan J. P. de Esch and Rob Leurs*
Leiden/Amsterdam Center for Drug Research (LACDR), Division of Medicinal Chemistry,
Faculty of Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Received 19 April 2006; revised 8 June 2006; accepted 23 June 2006
Available online 14 July 2006
Abstract—G-protein coupled receptors encoded by viruses represent an unexplored class of potential drug targets. In this study, we
describe the synthesis and pharmacological characterization of the first class of inverse agonists acting on the HCMV-encoded
receptor US28. It is shown that replacement of the 4-hydroxy group of lead compound 1 with a methylamine group results in a
significant 6-fold increase in affinity. Interestingly, increasing the rigidity of the spacer by the introduction of a double bond also
leads to a significant increase in binding affinity compared to 1. These novel inverse agonists serve as valuable tools to elucidate
the role of constitutive signaling in the pathogenesis of viral infection and may have therapeutic potential as leads for new antiviral
drugs.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
tors can only be activated by CC chemokines, while
CXC chemokines only bind to CXC chemokine recep-
tors.2 Two exceptions are the promiscuous chemokine
binding protein DARC (Duffy antigen receptor for che-
mokines) and the human cytomegalovirus (HCMV)-en-
coded receptor US28. DARC binds chemokines of both
the CC and CXC subclasses with high affinity,5 while the
viral-encoded receptor US28 binds several CC-chemo-
kines, including CCL2/MCP-1, CCL3/MIP-1a, CCL4/
MIP-1b and CCL5/RANTES as well as the only
member of the CX3C chemokine subclass, namely
CX3CL1/fractalkine.6–9
Chemokines are a group of small (8–14 kDa) soluble
proteins that belong to a large family of chemotactic
cytokines.1 They play an important role in the migration
and activation of leukocytes in a wide variety of im-
mune-mediated disorders. These proteins are subdivided
by structure into four major groups, namely CC, CXC,
CX3C and XC chemokines, based on the number and
position of conserved cysteine residues in their amino-
terminus.2
Chemokines mediate their effects by binding to chemo-
kine receptors, which belong to the family of G-protein
coupled receptors. These cell surface proteins are major
targets for therapeutic intervention and are targeted by
more than 40% of all marketed drugs.3 Binding of che-
mokines to their cognate receptors appears promiscuous
and redundant, as most chemokine receptors can bind
more than one chemokine and most chemokines can
activate several chemokine receptor subtypes.4 Howev-
er, binding to chemokine receptors is often restricted
to a single subclass of chemokines; CC chemokine recep-
HCMV is a species-specific b-herpesvirus that persists
lifelong in the host without any clinical symptoms in
immunocompetent individuals, but the virus can cause
severe illness in immunocompromised individuals, like
premature neonates, transplant recipients, and HIV-
infected people.10,11 After primary infection, the viral gen-
ome establishes a lifelong latent infection within the
host. The virus has developed several strategies to evade
the immune system, such as the expression of genes that
mimic host genes that are involved in the immune sys-
tem.12–14 One of these viral genes encodes a G-protein
coupled receptor, namely US28, with significant homol-
ogy to mammalian chemokine receptors.6,8 US28 shows
a 30% amino acid sequence homology with the human
CCR1 chemokine receptor,6 suggesting that HCMV
Keywords: US28; HCMV; Chemokines; Inverse agonists.
*
Corresponding author. Tel.: +31 20 5987600; fax: +31 20
5987610; e-mail: r.leurs@few.vu.nl
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.054

7214
J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
exploits chemokine signaling pathways to interfere with
the host immune system through chemokine mimicry.15
The homology of US28 compared to the CC chemokine
receptors is even higher within the N-terminus, crucial
for chemokine binding, with an amino acid sequence
homology of 70% and 52% with the human CCR1 and
CCR2 chemokine receptors, respectively.6
tive signaling of viral-encoded GPCRs could be valuable
tools to elucidate the role of constitutive signaling in the
pathogenesis of viral infection and may have therapeutic
potential as new antiviral drugs acting against patholo-
gies caused by HCMV infection. Screening of a variety
of GPCR-directed ligands for their ability to modulate
the basal signaling of US28 resulted in the identification
of the small nonpeptidergic molecule VUF2274 (1) as an
inverse agonist.28 This molecule is not only able to block
the basal signaling of US28, but also inhibits 60% of the
US28-mediated HIV entry in cells.
Currently, the role of US28 is still unknown, but due to
its promiscuous chemokine binding profile it is suggest-
ed that the receptor acts as a chemokine scavenger by
sequestering CC chemokines from the extracellular envi-
ronment.16,17 By this means the virus would elude im-
mune surveillance, as chemokines play an important
role in the regulation of the immune response. Further-
more, US28 induces the migration of vascular smooth
muscle cells upon binding with the chemokines CCL2
and CCL5, which could be exploited by HCMV to dis-
seminate the virus through the human body.18 This
could also provide a possible link between HCMV and
the development of vascular diseases, such as arterial
restenosis,19 atherosclerosis,20 and chronic allograft
rejection.21 Moreover, CCL2 and CCL5 play an impor-
tant role in the pathogenesis of vascular disease.22 Like
many mammalian chemokine receptors, of which CCR5
and CXCR4 are the primary HIV-1 co-receptors in vivo,
US28 can also act as a co-receptor for HIV-1 entry into
cells in vitro.23
Recently, a limited series of analogues of 1 was synthe-
sized to study the very first structure–activity relation-
ships for inverse agonism on a viral-encoded chemokine
receptor.29 To our knowledge, these molecules are cur-
rently the only nonpeptidergic inverse agonists acting
on US28. In this study, we describe a new series of mole-
cules, in which the rigidity is increased by the introduction
of conformationally restrained tricyclic ring systems or
rigid fused and nonfused piperidine ring systems. More-
over, we changed the spacer length between the diphenyl
acetonitrile group and the piperidine moiety, and intro-
duced rigidity in this part of the molecule. These novel in-
verse agonists acting on US28 give us more knowledge
about the SAR of this class of compounds.
2. Chemistry
Chemokine receptors have been shown to be involved in
the pathophysiology of different diseases.1,2 The identifi-
cation of small nonpeptidergic chemokine receptor
antagonists that are able to block these receptors pro-
ceeds rapidly, with some compounds in clinical trials
at the moment.
Target compounds 1–12 were synthesized via an N-
alkylation of the appropriate bromide or chloride inter-
mediates with commercially available 4-(4-chlorophe-
nyl)piperidine-4-ol 13 as outlined in Scheme 1.29–31
Compounds 14–21 were synthesized following an ana-
logue synthetic route. Microwave chemistry was used
for the N-alkylations to shorten the reaction times.
The tricyclic precursors for compounds 2–6 were synthe-
sized following methods previously described in the
literature.31–33 9H-carbazole, 10H-phenothiazine, 2-
chloro-10H-phenothiazine, and 1-(10H-phenothiazin-2-
yl)ethanone were easily deprotonated with NaH at room
temperature followed by an alkylation with 1-bromo-3-
chloropropane and a reaction with 13 to give target
compounds 7 and 10–12.34,35 However, the deprotona-
tion of 10,11-dihydro-5H-dibenzo[b,f]azepine and
diphenylamine, which were used for the synthesis of 8
In contrast to mammalian chemokine receptors, we pre-
viously showed that US28 signals in a constitutively
active manner and that the receptor affects different sig-
naling pathways, such as phospholipase C and NF-jB,24
and the transcription factors nuclear factor of activated
T-cells (NFAT) and cAMP response element binding
protein (CREB).25–27 The putative role of constitutive
activity in viral pathogenesis is not elucidated yet, but
it could be a way of the virus to alter the normal homeo-
stasis of a host cell for its own benefit.24,27 Potent
inverse agonists that are able to influence the constitu-
Scheme 1. Synthetic pathway for the synthesis of 1–12. Reagents and conditions: (a) NaH in DMF or di-n-butylether; (b) 1-bromo-3-chloropropane;
(c) NaI, Na2CO3, CH3CN, reflux or NaI, Na2CO3, CH3CN, microwave (15 min, 160 ꢁC).

J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
7215
and 9, could not be accomplished under these reaction
conditions and was therefore achieved with NaH as a
base in di-n-butylether at reflux temperature.36 Cyclo-
hexyl analogue 14 was synthesized starting from cyclo-
hexylphenylacetonitrile.31 1-Benzyl-4-tert-butylbenzene,
which was used for the synthesis of 15, was synthesized
from the corresponding benzophenone,29 and 4-(2-ben-
zothiazolyl)propylamine, an intermediate for the synthe-
sis of 16, was synthesized using o-aminothiophenol and
4-chlorobutyryl chloride.37 The unsaturated compounds
22 and 23 were synthesized according to the procedure
shown in Scheme 2. Cyclopropyldiphenylmethanol 24
was reacted with MgI2, formed in situ from Mg and
I2, to give 4-iodo-1,1-diphenylbut-1-ene 25, which was
line-2-one compound 38 was obtained by an intramolec-
ular cyclization reaction of 42 by treatment with p-
toluenesulfonic acid.43
Compounds 43 and 44 were synthesized following a
method previously described in the literature.31 The ace-
tate group of compound 45 was formed by reacting
compound 1 with acetylchloride in the presence of tri-
ethylamine. Reduction of the methyl ester group of com-
pound 43 with LiAlH4 resulted in alcohol 46.31 The
synthetic route for the preparation of 47–52 is outlined
in Schemes 4 and 5. Target compounds 47 and 50 were
synthesized by a reaction of 4-chloro-1,1-diphenylbu-
tane44 with 53 or 54 to give intermediates 55 or 56, fol-
lowed by a reduction of the nitrile group in the presence
of AlCl3 and LiAlH4. Compound 53, which was used for
the synthesis of 47, was commercially available as the
hydrochloride salt, but 54 needed to be synthesized.
Thus, 2-(4-chlorophenyl)acetonitrile was deprotonated
by NaNH2 and treated with Boc-protected bis-(2-chlo-
ro-ethyl)amine 57 resulting in intermediate 58, which
was deprotected under acidic conditions resulting in
the desired piperidine moiety 54.45 Substituents were
easily introduced on the amine group of 47, so 48 was
synthesized in a high yield by reacting 47 with formic
acid in the microwave. The monomethyl-substituted
amine 49 was synthesized by refluxing 48 in THF in
the presence of LiAlH4. The hydroxy group of 59 was
converted to an acetamide group in 51 via a Ritter reac-
tion.46 In strongly acidic media, a highly electrophilic
tertiary carbocation is formed from the tertiary hydroxy
group of 59, and this is followed by an acid-induced
nucleophilic addition of the nitrile group of acetonitrile
and a hydrolysis resulting in the desired compound 51.
The acetamide group of 51 was hydrolyzed under acidic
conditions to the corresponding amine group in 52.47
reacted with piperidine 13 to give compound 22.38,39
A
reduction of the carbonyl group of 26 with NaBH4 gave
alcohol 27 in a quantitative yield.40 This alcohol was
dehydrated under acidic conditions to give intermediate
28, which was used for the alkylation of 13 to afford tar-
get compound 23.
Compounds 29–31 were synthesized following the same
method as described in Scheme 1. Reduction of the car-
bonyl group of 31 with NaBH4 resulted in target com-
pound 32. The different fused and non-fused piperidine
moieties of compounds 33–36 were reacted with the
appropriate chloride intermediates in a manner similar
to that described for target compounds 1–12. The fused
and nonfused ring systems of 33, 34 and 36 were com-
mercially available, and the six-membered spiropiperi-
dine moiety of 35 was synthesized following
a
literature procedure.41 1-Benzyl-2-methylbenzene, which
was used for the synthesis of 34, was synthesized by the
reduction of 2-methylbenzophenone.29 The synthesis of
compounds 37 and 38 is outlined in Scheme 3. Com-
pound 37 was synthesized by reacting o-bromobenzoic
acid with 2 equiv of n-BuLi,42 followed by a reaction
with ketone 39. Reductive amination of 40 with di-
tert-butyl 2-(2-aminophenyl)malonate 41, which was
synthesized from the reaction of 2-fluoronitrobenzene
with di-tert-butyl malonate and subsequent reduction
of the nitro group, yielded intermediate 42. The indo-
3. Results and discussion
Starting from lead compound 1 we synthesized a
new series of compounds, which were evaluated for
Scheme 2. Synthetic pathway for the synthesis of 22 and 23. Reagents and conditions: (a) MgI2, Et2O, reflux; (b) 13, Na2CO3, CH3CN, reflux;
(c) NaHCO3, NaBH4, EtOH; (d) HCl, reflux; (e) 13, NaI, Na2CO3, CH3CN, microwave (15 min, 160 ꢁC).

7216
J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
Scheme 3. Synthetic pathway for the synthesis of 37 and 38. Reagents and conditions: (a) n-BuLi, THF, ꢀ78 ꢁC; (b) di-tert-butylmalonate, NaH,
DMF, 90 ꢁC; (c) Pd/C, H2, EtOAc; (d) AcOH, NaBH(OAc)3, 1,2-dichloroethane; (e) PTSA, toluene, reflux.
Scheme 4. Synthetic pathway for the synthesis of 47–50. Reagents and conditions: (a) di-tert-butyl dicarbonate, Et3N, DCM; (b) NaNH2, 2-(4-
chlorophenyl)acetonitrile, toluene, 70 ꢁC; (c) EtOH/HCl; (d) 4-chloro-1,1-diphenylbutane, NaI, Na2CO3, CH3CN, reflux; (e) AlCl3, LiAlH4, THF; (f)
formic acid, microwave (5 min, 200 ꢁC) for 48 or formic acid, microwave (5 min, 200 ꢁC) followed by LiAlH4, THF, reflux for 49.
their
125I]CCL5 binding to US28. The inverse agonistic
properties of a selection of compounds were investi-
potential
to
dose-dependently
displace
gated by testing their potential to inhibit the US28-
mediated constitutive inositol phosphate production
in SVEC4-10 cells.
[

J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
7217
Scheme 5. Synthetic pathway for the synthesis of 51 and 52. Reagents and conditions: (a) H2SO4, CH3CN for 51 followed by HCl, reflux for 52.
In our previous study,29 it was shown that a piperidine
ring was important for activity and therefore this struc-
tural motif was maintained. It was also revealed that the
nitrile group in the structure was not essential for affin-
ity and efficacy, so this group could be omitted.29 In this
study, we focussed our chemistry program on other
parts of the structure, namely on the diphenyl group
(Table 1) and the propyl linker (Table 2). Furthermore,
we introduced different substituents at the 4-position of
the piperidine ring (Tables 3 and 4).
bulky tert-butyl group in compound 15 is permitted at
this position. Interestingly, replacement of the diphenyl-
acetonitrile group by a benzothiazole ring in compound
16 caused a 3.5-fold drop in binding affinity to the recep-
tor. Taken together, the structural modifications indi-
cate that changes in this part of the molecule are well
tolerated and that the conformation of the two phenyl
rings does not seem to be important.
Furthermore, the importance of the propyl linker be-
tween the diphenylacetonitrile group and the piperidine
moiety was investigated by the introduction of different
structural modifications in this part of the structure (Ta-
ble 2). First, we studied the effect of varying the chain
length of the linker. Compound 1 has been previously
reported as a potent antagonist acting on the human
CCR1 chemokine receptor.34 Interestingly, we recently
demonstrated that the SAR of compound 1 and its ana-
logues is completely different on the viral-encoded recep-
tor US28 compared to that of the human chemokine
receptor CCR1.29 Now, the shortening of the propyl
spacer with one methylene group in 17 caused a small
decrease in affinity on US28, while the addition of one
methylene group in the structure of 1, resulting in com-
pound 18, did not change the affinity or efficacy. In con-
trast, for the human CCR1 chemokine receptor it was
shown that shortening of the propyl chain with one
methylene group caused a large decrease in Ki value,
while addition of one or two methylene groups in the
structure of 1, resulting in, respectively, a butyl and a
pentyl chain, did not change the affinity on the CCR1
chemokine receptor.31
First, more rigidity was introduced by incorporation of
the two phenyl rings in different tricyclic moieties (Table
1). The rotation of the two aromatic phenyl rings is
restricted by incorporation of an ethylene group be-
tween the two phenyl rings in 3, a bioisosteric thiometh-
ylene or oxomethylene bridge in 4 and 5 or an
unsaturated bridge as in 6. All these changes were toler-
ated and did not influence the affinity and potency of
the compounds. Interestingly, introduction of a nearly
planar tricyclic ring system in 2 also resulted in a
compound with a comparable affinity.
The introduction of a nitrogen atom in the tricyclic sys-
tem could provide an additional position for hydrogen
bonding, but this structural modification did not influ-
ence the affinity of the compound. For tricyclic antipsy-
chotics and antidepressants it is known that there is a
relationship between the folding of the tricyclic moiety
and biological activity. If the sulfur atom of neuroleptic
phenothiazine drugs is replaced by an ethylene bridge it
results in dibenzazepine derivatives, which have an anti-
depressant activity.48 In contrast, replacement of the sul-
fur atom of phenothiazine analogue 10 into an ethylene
bridge, resulting in the tricyclic moiety of dibenzazepine
analogue 8, did not result in any change in affinity or
potency on US28. Additionally, introduction of the
more rigid and planar carbazole ring system in analogue
7 did not change the affinity for the receptor, as was also
shown for compound 2. The introduction of a chloro or
acetyl group in one of the aromatic rings in phenothia-
zine analogues 11 and 12 resulted in a decrease in bind-
ing affinity, so these substitutions are not preferred in
the phenothiazine ring.
In compounds 19–21 one of the phenyl rings was re-
moved and this series of compounds showed a similar
trend as demonstrated for compounds 1, 17, and 18,
namely that removal of one methylene group in 19
resulted in a decrease of binding affinity, while com-
pounds 20 and 21 have binding affinities comparable
to compounds 1 and 18. Interestingly, the introduction
of a more rigid chain in unsaturated analogue 22 result-
ed in a 3-fold increase in binding affinity compared to
lead compound 1. However, in the functional assay both
compounds were equipotent. In our previous study it
was shown that removal of a phenyl ring as in 20 result-
ed in a binding affinity comparable to compound 1, but
this is not true for the more rigid and unsaturated ana-
logues 22 and 23. Compound 23 has a binding affinity
that is more than 10-fold reduced compared to com-
pound 22.
Replacement of one of the phenyl rings by a bulky sat-
urated cyclohexyl ring in 14 is allowed. Previously, we
found that introduction of a bulky phenyl substituent
at the 4-position of one of the phenyl rings was not
allowed to maintain binding affinity.29 In contrast, the

7218
J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
Table 1. Chemical structures and pharmacological properties of compounds 1–12 and 14–16 for the HCMV-encoded receptor US28
OH
R
N
Cl
R
a
b
Compound
VUF
R
IC50 (lM)
EC50 (lM)
CN
1
2274
4.9 (4.4–5.5)
6.0 (5.5–6.5)
6.6 (4.8–8.3)
6.1 (4.6–7.6)
3.2 (2.5–4.0)
5.7 (4.3–7.1)
5.7 (3.0–8.5)
5.4 (4.0–6.9)
2
3
4
10004
5713
5727
CN
CN
S
CN
O
5
10007
7.7 (5.9–9.3)
6.2 (3.4–8.9)
CN
CN
6
7
8
10003
5932
5982
6.0 (4.7–7.4)
8.4 (6.2–10.7)
6.4 (4.6–8.3)
5.1 (2.5–7.8)
4.5 (2.8–6.2)
7.1 (3.0–11.2)
N
N
9
5983
6.9 (3.5–10.2)
6.4 (5.2–7.6)
4.4 (2.4–6.3)
6.8 (5.2–8.3)
N
S
10
10005
N
S
N
S
N
11
12
10006
6902
10.5 (6.5–14.5)
11.0 (5.5–16.6)
n.d.
Cl
10.7 (6.9–14.5)
O
14
15
5892
7.7 (5.5–10.0)
5.9 (4.6–7.2)
5.2 (3.2–7.1)
7.6 (3.2–12.0)
CN
( ) 5937
16
5933
18.0 (13.2–22.9)
n.d.
N
S
The values are represented as means and the interval of the IC50 and EC50 values of at least three independent experiments.
a
[
125I]CCL5 displacement.
b Inhibition of [3H]inositol phosphate production. n.d., not determined.

J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
7219
Table 2. Chemical structures and pharmacological properties of compounds 1 and 17–23 for the HCMV-encoded receptor US28
OH
N
Cl
a
b
Compound
VUF
5742
R
IC50 (lM)
EC50 (lM)
Ph CN
17
9.2 (7.9–10.5)
4.9 (4.4–5.5)
4.6 (3.6–5.5)
21.1 (15.8–26.3)
7.8 (6.8–8.9)
6.6 (4.6–8.7)
1.7 (1.1–2.3)
20.0 (19.5–20.4)
4.1 (1.5–6.6)
Ph
Ph
CN
Ph
1
18
19
20
21
22
23
2274
3.2 (2.5–4.0)
5.5 (1.9–9.1)
n.d.
Ph CN
Ph
5743
Ph
5745
Ph
Ph
5746
n.d.
5752
n.d.
Ph
Ph
Ph
6869
4.8 (1.3–8.3)
n.d.
(E/Z) 6870
The values are represented as means and the interval of the IC50 and EC50 values of at least three independent experiments.
a
[
125I]CCL5 displacement.
b Inhibition of [3H]inositol phosphate production. n.d., not determined.
Next, the influence of the substitution pattern at the
4-position of the piperidine ring was investigated to
further define the structure–activity relationships on
US28 (Tables 3 and 4). We previously showed that
a phenyl ring at this position is important,29 so this
was maintained in the structure. In compounds 33–
38 the aromatic ring is incorporated in a heterocyclic
system, while in compounds 30–32 an additional car-
bon atom is present between the piperidine ring and
the phenyl ring. Both the replacement of the 4-chloro
atom in the phenyl ring by a 3-trifluoromethyl group
in 29 as well as the introduction of a bulky diphenyl-
methanol group in compound 30 did not influence the
potency of the compounds. In compound 31 a 4-fluoro-
phenylmethanone group was introduced at the 4-posi-
tion of the piperidine ring and reduction of the
carbonyl group resulted in analogue 32. The introduc-
tion of both the 4-fluorophenylmethanone group in
compound 31 as well as the 4-fluorophenylmethanol
group in analogue 32 did not influence the affinity
for US28.
functional groups, which can act as hydrogen bond
acceptor or donor groups. Both compounds 33 and 34
contain a spiropiperidine amide group, but compound
34 was not synthesized with a diphenylacetonitrile group
as in compound 33, but with a ortho methyl-substituted
diphenyl group, because recently it was demonstrated
that there was a slight preference for this structural mo-
tif.29 However, in this series of compounds the binding
affinities of 33 and 34 were comparable and in the same
order as lead compound 1. Additionally, the introduc-
tion of the spiropiperidine moieties in both compounds
35 and 37 was allowed to maintain affinity for US28.
The benzimidazolone piperidine moiety of 36 is often
seen in ligands acting on various G-protein coupled
receptors, such as serotonergic and dopaminergic recep-
tors.43 Interestingly, the introduction of an indolin-2-
one group in compound 38 resulted in a compound with
an activity compared to 1, while the introduction of the
benzimidazolone piperidine moiety of 36 made the affin-
ity drop slightly.
Moreover, the 4-hydroxy group was replaced by other
substituents (Table 4), because a hydroxy group at this
position of a piperidine ring is suggested to be a site of
potential metabolic toxicity.49 The introduction of an es-
ter group in 43, an acetyl group in 44, an acetate group
in 45 or a hydroxy methyl group in 46 did not result in
compounds with a higher affinity.
Different fused and nonfused ring systems (heterocyclic-
substituted piperidine analogues or bicyclic heterocyclic
groups) were introduced at the 4-position of the piperi-
dine ring, because these structural motifs appear
frequently in CC chemokine receptor antagonists.
Additionally, the heterocyclic groups contain different

7220
J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
Table 3. Chemical structures and pharmacological properties of compounds 1 and 29–38 for the HCMV-encoded receptor US28
R2
R3
R1
a
b
Compound
VUF
2274
R1
H
R2
R3
IC50 (lM)
EC50 (lM)
OH
OH
1
CN
4.9 (4.4–5.5)
3.2 (2.5–4.0)
4.3 (2.5–6.2)
Cl
N
N
N
29
30
6984
5729
H
H
CN
CN
5.2 (4.2–6.3)
4.8 (4.3–5.4)
CF3
6.1 (2.3–10.0)
OH
F
F
31
32
6868
H
H
H
H
4.8 (3.9–5.6)
6.4 (4.6–8.3)
n.d.
N
O
( ) 10010
3.0 (2.8–3.2)
N
N
OH
NH
O
33
5893
H
CN
3.7 (3.5–4.0)
2.0 (1.3–2.8)
N
O
NH
O
N
34
35
( ) 5997
Me
H
2.6 (1.2–3.9)
7.6 (6.0–9.1)
4.2 (1.1–7.4)
N
O
N
N
NH
6967
H
CN
n.d.
O
NH
N
36
6985
H
CN
7.1 (6.3–7.9)
n.d.
Cl
O
O
N
N
37
38
6047
6048
H
H
CN
H
11.9 (10.0–13.8)
5.5 (4.9–6.2)
n.d.
O
N
7.3 (2.2–12.3)
The values are represented as means and the interval of the IC50 and EC50 values of at least three independent experiments.
125I]CCL5 displacement.
a
[
b Inhibition of [3H]inositol phosphate production. n.d., not determined.

J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
7221
Table 4. Chemical structures and pharmacological properties of compounds 1 and 43–52 for the HCMV-encoded receptor US28
R1
R2
N
X
a
b
Compound
VUF
R1
R2
X
IC50 (lM)
EC50 (lM)
1
43
44
45
46
47
48
49
50
51
52
2274
5934
5984
5936
6881
6046
6987
6989
6966
6981
6993
CN
CN
CN
CN
H
OH
Cl
H
H
Cl
H
H
H
H
Cl
Cl
Cl
4.9 (4.4–5.5)
8.0 (6.0–10.0)
7.9 (6.3–9.5)
8.6 (7.4–9.8)
7.5 (5.6–9.3)
1.6 (1.1–2.0)
2.5 (2.0–3.0)
1.3 (1.1–1.5)
0.8 (0.7–1.0)
2.2 (2.0–2.5)
1.4 (1.3–1.4)
3.2 (2.5–4.0)
n.d.
n.d.
(C@O)OCH3
(C@O)CH3
O(C@O)CH3
–CH2OH
n.d.
n.d.
H
–CH2NH2
3.1 (1.7–4.6)
n.d.
H
–CH2NHC(@O)H
–CH2NHCH3
–CH2NH2
H
3.1 (1.2–4.9)
3.6 (2.6–4.6)
4.1 (1.0–7.1)
5.7 (2.5–8.9)
H
H
–NHC(@O)CH3
–NH2
H
The values are represented as means and the interval of the IC50 and EC50 values of at least three independent experiments.
a
[
125I]CCL5 displacement.
b Inhibition of [3H]inositol phosphate production. n.d., not determined.
Furthermore, amine analogue 47 was synthesized to
investigate the influence of a primary amine group at
the 4-position of the piperidine ring. It was demonstrat-
ed earlier that a nitrile group at the 4-position of the
piperidine ring was detrimental for both affinity and
activity.29 However, the reduction of this group into a
methylamine group of derivative 47 resulted in a 3-fold
increase in binding affinity compared to lead compound
1, while the efficacy of both compounds was compara-
ble. From previous studies28,29 it was known that
removal of the p-chloro substituent resulted in a de-
crease in binding affinity. Thus, a chloro atom was intro-
duced at the 4-position of the phenyl ring in compound
50, which resulted in a further 2-fold increase of binding
affinity compared to unsubstituted analogue 47. The
amine group of compound 47 was substituted, resulting
in compounds 48 and 49. Unfortunately, these substitu-
tions did not cause an increase in the binding affinity on
US28. Compound 51 was synthesized to investigate the
importance of the position of the amine group and this
amine was synthesized starting from compound 52, in
which the amine group is substituted with an acetyl
group. Both compounds have an affinity higher than
lead compound 1, but slightly lower than that of our
novel lead compound 50.
between the diphenyl group and the piperidine moiety
in compound 22 caused a significant increase in binding
affinity to US28. Currently, these molecules are being
used as tools to further investigate the role of constitu-
tive signaling of US28 in the pathogenesis of viral infec-
tion. In the future, potent and selective inverse agonists
acting on constitutively active viral GPCRs may have
therapeutical potential in the treatment of pathologies
caused by viral infections.
5. Materials and methods
5.1. General procedures
The solvents were dried according to standard proce-
dures. All reactions were performed under an atmo-
sphere of dry nitrogen. Microwave reactions were
performed in a CEM Explorer single mode MW reactor
1
equipped with auto sampler. H NMR and 13C NMR
spectra were recorded on a Bruker AC-200 (200 MHz)
spectrometer. J. T. Baker silica gel was used for flash
chromatography. HRMS mass spectra were recorded
on a Finnigan MAT 900 mass spectrometer. Melting
points were measured on a MPA100 OptiMelt Auto-
mated Melting Point System apparatus and are uncor-
rected. Analytical HPLC-MS analyses were conducted
using a Shimadzu LC-8A preparative liquid chromato-
graph pump system with a Shimadzu SPD-10AV UV–
VIS detector set at 254 nm, with the MS detection
performed with a Shimadzu LCMS-2010 liquid chro-
matograph mass spectrometer. The analyses were per-
formed using the following conditions; condition I: an
Alltima(C18)5u column (150 mm · 4.6 mm) with 70%
MeOH–30% H2O–0.1% formic acid (method Ia); 60%
MeOH–40% H2O–0.1% formic acid (method Ib) or
50% MeOH–50% H2O–0.1% formic acid (method Ic).
Flow rate = 1.0 mL/min. Total run time 15 min unless
otherwise stated. Condition II: an Alltima(C18)5u
4. Conclusion
In summary, we described the synthesis and structure–
activity relationships of inverse agonists acting on the
viral-encoded GPCR US28. These molecules are consid-
ered as valuable tools to investigate the (patho)physio-
logical role of US28 during viral infection.
Replacement of the 4-hydroxy group of lead compound
1 into a methylamine group as in compound 50 resulted
in the, to our knowledge, highest affinity inverse agonist
acting on US28 currently known. Interestingly, the
introduction of a double bond in the propyl linker

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J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
column (150 mm · 4.6 mm) with 50% CH3CN–50%
H2O–0.1% formic acid (method IIa); 40% CH3CN–
60% H2O–0.1% formic acid (method IIb) or 30%
CH3CN–70% H2O–0.1% formic acid (method IIc). Flow
rate = 1.0 mL/min. Total run time 20 min. Compounds
that were isolated as fumaric acid salts all showed a
fumaric acid peak around 2 min. Purities calculated
are based on RP HPLC–UV peak surface area using
the condition that showed the highest level of impurities
(disregarding the fumaric acid peak). Reference com-
pounds 1 and 20 have been described previously and
taken from stock.29 Compounds 3–6, 14, 17, 18, 43,
and 46 were synthesized as previously described in the
literature31 and the characterization data confirmed that
the desired compounds had been formed. Compounds
15, 32, and 34 were tested as racemic mixtures.
uo. Purification by flash chromatography (EtOAc) and
recrystallization from EtOAc gave 293 mg (59%) of 30
as a white solid. Mp: 77.9–78.5 ꢁC. H NMR (CDCl3):
d 1.34–1.69 (m, 5H), 1.85–2.20 (m, 4H) 2.30–2.50 (m,
5H), 2.79–2.99 (m, 2H), 7.11–7.47 (m, 20H). 13C NMR
(CDCl3): 22.80, 26.03, 37.25, 43.85, 51.47, 53.71,
57.60, 79.32, 122.22, 125.57, 126.39, 126.69, 127.69,
128.04, 128.71, 129.23, 139.94, 145.70. Anal. RP-HPLC
Ib: tR = 9.48 min (purity 98%), IIa: tR = 6.11 min (purity
98%). HRMS (EI) m/z calcd for C35H36N2O: 500.2828;
found: 500.28170.
1
5.4. General method C. 1-(3-(9H-Carbazol-9-yl)propyl)-
4-(4-chlorophenyl)piperidin-4-ol (7)
(i) A solution of 9H-carbazole (0.84 g, 5.01 mmol) in
DMF (20 mL) was cooled to 0 ꢁC and NaH (0.22 g,
5.58 mmol) was added in small portions. After stirring
for 1 h at room temperature, the reaction mixture was
cooled to 0 ꢁC and 1-bromo-3-chloropropane (0.5 mL,
5.06 mmol) was added. The reaction mixture was al-
lowed to warm to room temperature and stirred over-
night. Water (50 mL) was added and the water layer
was extracted with EtOAc (3· 25 mL). The combined
organic extracts were washed with water (3· 25 mL)
and brine (25 mL), dried over anhydrous Na2SO4, and
filtered. After evaporation under reduced pressure, the
residue was purified by flash chromatography (0–15%
DCM in hexane) to give 853 mg (70%) of 9-(3-chloro-
5.2. General method A. 5-(4-Hydroxy-4-(3-(trifluoro-
methyl)phenyl)piperidin-1-yl)-2,2-diphenylpentanenitrile
fumarate (29)
4-(3-(Trifluoromethyl)phenyl)piperidine-4-ol (0.51 g, 2.07
mmol), 5-chloro-2,2-diphenylpentanenitrile34 (0.54 g,
2.00 mmol), NaI (0.30 g, 2.00 mmol), Na2CO3 (0.42 g,
2.00 mmol) and 3 mL CH3CN were added in a 10 mL
microwave and this mixture was reacted during
15 min in the microwave at a temperature of 160 ꢁC
(settings: ramp time 5 min, hold time 15 min, power
200 W, pressure 17.2 bar). The solvent was removed
in vacuo and the residue was diluted with water
(20 mL), followed by an extraction with DCM (3·
15 mL). The combined organic layers were washed with
water (3· 40 mL) and brine (40 mL), dried over anhy-
drous MgSO4, filtered, and evaporated in vacuo. Puri-
fication by flash chromatography (0–50% EtOAc in
DCM) gave 772 mg (81%) of oil. This was dissolved
in EtOH and converted to the corresponding fumaric
salt by the addition of fumaric acid (0.19 g, 1.6 mmol).
The fumaric salt was isolated by evaporation in vacuo
and recrystallized from EtOH/Et2O to give 803 mg
(68%) of 29 as a white solid. Mp: 123.7–125.3 ꢁC
(dec). 1H NMR (DMSO-d6): d 1.47–1.78 (m, 5H),
1.98–2.20 (m, 2H), 2.35–2.91 (m, 8H), 6.57 (s, 2H),
7.33–7.81 (m, 14H). 13C NMR (CDCl3): d 30.74,
35.31, 36.14, 47.91, 51.18, 55.68, 68.78, 121.49,
121.85, 123.64, 126.44, 127.89, 128.20, 128.69, 128.84,
134.72, 134.91, 139.22, 148.43, 169.59. Anal. RP-HPLC
Ib: tR = 6.64 min (purity 100%), IIb: tR = 11.69 min
(purity 100%). HRMS (EI) m/z calcd for
C29H29F3N2O: 478.2232; found: 478.22249.
1
propyl)-9H-carbazole. H NMR (CDCl3): d 2.28–2.49
(m, 2H), 3.51 (t, J = 6.0 Hz, 2H), 4.50 (t, J = 6.4 Hz,
2H), 7.21–7.31 (m, 2H), 7.40–7.48 (m, 4H), 8.11 (d,
J = 7.7 Hz, 2H).
(ii) Following method B using 9-(3-chloropropyl)-9H-
carbazole (0.57 g, 2.34 mmol) gave 727 mg (75%) of 7
as a light yellow solid after recrystallization in EtOAc.
Mp: 130.1–131.4 ꢁC. 1H NMR (CDCl3): d 1.54–1.72
(m, 3H), 2.01–2.14 (m, 4H), 2.28–2.41 (m, 4H), 2.68–
2.73 (m, 2H), 4.41 (t, J = 6.6 Hz, 2H), 7.17–7.49 (m,
10H), 8.09 (d, J = 7.7 Hz, 2H). 13C NMR (CDCl3): d
25.91, 38.24, 40.54, 49.17, 55.20, 70.92, 108.62, 118.64,
120.17, 122.67, 125.40, 125.97, 128.27, 132.66, 140.33,
146.60. Anal. RP-HPLC Ib: tR = 9.37 min (purity
100%), IIb: tR = 10.33 min (purity 100%). HRMS (EI)
m/z calcd for C26H27ClN2O: 418.1812; found:
418.18153.
5.5. 9-(3-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-
propyl)- 9H-fluorene-9-carbonitrile hydrochloride (2)
5.3. General method B. 5-(4-(Hydroxydiphenylmeth-
yl)piperidin-1-yl)-2,2-diphenylpentanenitrile (30)
(i) 9H-Fluorene-9-carbonitrile33 (1.03 g, 5.39 mmol) was
dissolved in DMF (25 mL) and NaH (0.22 g, 5.58 mmol)
was added in small portions. The reaction mixture was
heated to 70 ꢁC and after 2 h 1-bromo-3-chloropropane
(2.75 mL, 27.8 mmol) was added in one portion. The
reaction mixture was allowed to cool to room tempera-
ture and stirred overnight. Water (50 mL) was added
and the water layer was extracted with Et2O (3·
50 mL). The combined organic extracts were washed
with water (3· 50 mL) and brine (50 mL), dried over
anhydrous MgSO4, filtered, and evaporated in vacuo
to give 1.06 g of 9-(3-chloropropyl)-9H-fluorene-9-car-
A solution of 4-bromo-2,2-diphenylbutanitrile31 (0.31 g,
1.00 mmol), diphenyl(piperidin-4-yl)methanol (0.36 g,
1.20 mmol), NaI (0.45 g, 3.00 mmol), and Na2CO3
(0.64 g, 6.00 mmol) in CH3CN (30 mL) was refluxed
overnight. The solvent was removed in vacuo, the resi-
due was diluted with water (50 mL) and extracted with
DCM (3· 30 mL). The combined organic layers were
washed with water (3· 50 mL) and brine (50 mL), dried
over anhydrous MgSO4, filtered, and evaporated in vac-

J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
7223
bonitrile as a yellow solid. The crude product was used
without further purification.
2.32–2.47 (m, 4H), 2.69–2.87 (m, 2H), 3.77 (t,
J = 7.3 Hz, 2H), 6.88–7.03 (m, 4H), 7.21–7.45 (m,
10H). 13C NMR (CDCl3): d 25.33, 38.84, 49.86, 50.54,
56.20, 71.47, 121.38, 121.60, 126.49, 128.83, 129.64,
133.23, 136.18, 148.43. Anal. RP-HPLC Ib:
tR = 8.62 min (purity 100%), IIb: tR = 11.11 min (purity
99%). HRMS (EI) m/z calcd for C26H29ClN2O:
420.1968; found: 420.19699.
(ii) Following method A using crude 9-(3-chloropropyl)-
9H-fluorene-9-carbonitrile (0.49 g, 2.16 mmol) gave
445 mg of oil. This was dissolved in Et2O and dry hydro-
chloride gas was bubbled through the solution. Isolation
of the hydrochloride salt by filtration and recrystalliza-
tion from MeOH/Et2O gave 201 mg (17%) of 2 as a
1
white solid. Mp: 156.2–158.1 ꢁC. H NMR (CDCl3): d
5.8. 1-(3-(10H-Phenothiazin-10-yl)propyl)-4-(4-chloro-
phenyl)piperidine-4-ol (10)
1.55–1.94 (m, 5H), 2.33–2.54 (m, 2H), 2.77–3.18 (m,
6H), 3.26–3.40 (m, 2H), 7.35–7.60 (m, 8H), 7.66–7.88
(m, 4H). 13C NMR (CDCl3/DMSO-d6): d 18.52, 34.88,
36.00, 47.29, 48.64, 56.16, 68.26, 120.45, 120.68,
124.08, 126.17, 128.22, 128.57, 129.67, 132.92, 139.93,
141.57, 145.12. Anal. RP-HPLC Ib: tR = 5.10 min (puri-
ty 99%), IIb: tR = 8.39 min (purity 99%). HRMS (EI)
m/z calcd for C28H27ClN2O: 442.1812; found: 442.18072.
Following method C starting with 10H-phenothiazine
gave 462 mg (34% over two steps) of 10 as a light yellow
solid. Mp: 59.0–60.6 ꢁC. 1H NMR (CDCl3): d 1.51–2.11
(m, 7H), 2.21–2.56 (m, 4H), 2.63–2.89 (m, 2H), 3.93 (t,
J = 6.7 Hz, 2H), 6.87–7.41 (m, 12H). 13C NMR
(CDCl3): d 25.34, 38.86, 45.61, 49.84, 55.99, 71.28,
115.97, 117.14, 122.90, 125.64, 126.47, 127.63, 127.89,
128.82, 133.26, 145.58. Anal. RP-HPLC Ia:
tR = 3.45 min (purity 97%), Ib (total run time 20 min):
tR = 13.93 min (purity 98%), IIb: tR = 13.46 min (purity
99%). HRMS (EI) m/z calcd for C26H27ClN2OS:
450.1533; found: 450.15394.
5.6. 4-(4-Chlorophenyl)-1-(3-(10,11-dihydro-5H-
dibenzo[b,f]azepin-5-yl)propyl)piperidin-4-ol (8)
(i) NaH (0.40 g, 10.03 mmol) was added portionwise to
a
solution of 10,11-dihydro-5H-dibenzo[b,f]azepine
(1.95 g, 9.99 mmol) in di-n-butylether (30 mL). The reac-
tion mixture was heated, refluxed for 3.5 h, and 1-bro-
mo-3-chloropropane (4.00 mL, 40.5 mmol) was added
at 100 ꢁC. The reaction mixture was refluxed overnight,
water was added (50 mL) and the water layer was
extracted with toluene (3· 75 mL). The combined organ-
ic extracts were washed with water (3· 100 mL) and
brine (100 mL), dried over anhydrous Na2SO4, and fil-
tered. After evaporation in vacuo, the residue was puri-
fied by flash chromatography (5% DCM in hexane) to
give 737 mg (27%) of 5-(3-chloropropyl)-10,11-dihy-
5.9. 1-(3-(2-Chloro-10H-phenothiazin-10-yl)propyl)-4-(4-
chlorophenyl)piperidin-4-ol (11)
Following method C starting with 2-chloro-10H-pheno-
thiazine gave 376 mg (24% over two steps) of 11 as a
light yellow solid. Mp: 66.1–66.7 ꢁC. 1H NMR (CDCl3):
d 1.49–1.80 (m, 3H), 1.87–2.18 (m, 4H), 2.30–2.58 (m,
4H), 2.68–2.85 (m, 2H), 3.91 (t, J = 6.8 Hz, 2H), 6.84–
7.42 (m, 11H). 13C NMR (CDCl3): d 24.12, 38.33,
45.62, 49.84, 55.71, 71.01, 116.31, 116.33, 122.76,
123.42, 124.08, 125.35, 126.48, 127.87, 127.97, 128.34,
128.83, 133.30, 133.68, 144.77, 146.89. Anal. RP-HPLC
Ib: tR = 4.27 min (purity 97%), IIa: tR = 6.45 min (purity
98%). HRMS (EI) m/z calcd for C26H26Cl2N2OS:
484.1143; found: 484.11331.
1
dro-5H-dibenzo[b,f]azepine as a colorless oil. H NMR
(CDCl3): d 1.99–2.16 (m, 2H), 3.18 (s, 4H), 3.57 (t,
J = 6.4 Hz, 2H), 3.91 (t, J = 6.5 Hz, 2H), 6.91–6.99 (m,
2H), 7.08–7.24 (m, 6H).
(ii) Following method A using 5-(3-chloropropyl)-10,11-
dihydro-5H-dibenzo[b,f]azepine (0.42 g, 1.55 mmol)
gave 494 mg (71%) of 8 as white crystals after recrystal-
lization from EtOAc. Mp: 116.0–117.6 ꢁC. 1H NMR
(CDCl3): d 1.61–1.84 (m, 5 H), 1.96–2.11 (m, 2H),
2.26–2.47 (m, 4H), 2.62–2.78 (m, 2H), 3.15 (s, 4H), 3.76
(t, J = 6.8 Hz, 2H), 6.85–6.93 (m, 2H), 7.06–7.15 (m,
6H), 7.24–7.41 (m, 4H). 13C NMR (CDCl3): d 25.74,
32.63, 38.67, 49.33, 49.83, 56.95, 71.40, 120.35, 122.86,
126.48, 126.77, 128.79, 130.22, 133.18, 134.64, 147.17,
148.65. Anal. RP-HPLC Ib (total run time 20 min):
tR = 12.71 min (purity 99%), IIa: tR = 4.76 min (purity
100%), IIb: tR = 15.55 min (purity 99%). HRMS (EI)
m/z calcd for C28H31ClN2O: 446.2125; found: 446.21311.
5.10. 1-(10-(3-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-
yl)propyl)-10H-phenothiazin-2-yl)ethanone (12)
Following method C starting with 1-(10H-phenothiazin-
2-yl)ethanone gave 410 mg (22% over two steps) of 12 as
a light yellow solid. Mp: 108.9–110.6 ꢁC. 1H NMR
(CDCl3): d 1.39–1.68 (m, 5H), 1.81–2.12 (m, 4H),
2.22–2.81 (m, 4H), 2.50 (s, 3H), 3.96 (t, J = 6.7 Hz,
2H), 6.79–6.92 (m, 2H), 7.01–7.42 (m, 9H). 13C NMR
(CDCl3): d 23.71, 26.47, 37.90, 45.11, 49.22, 55.29,
70.70, 113.81, 115.76, 122.75, 123.00, 123.71, 125.92,
126.89, 127.33, 127.54, 128.24, 132.13, 132.65, 136.10,
144.19, 145.33, 197.31. Anal. RP-HPLC Ib:
tR = 10.46 min (purity 100%), IIb: tR = 10.90 min (puri-
ty 100%). HRMS (EI) m/z calcd for C28H29ClN2O2S:
492.1638; found: 492.16307.
5.7. 4-(4-Chlorophenyl)-1-(3-(diphenylamino)propyl)pip-
eridin-4-ol (9)
(i) This was synthesized as described for 8 starting with
diphenylamine (1.69 g, 10.00 mmol) to give 683 mg (16%
over two steps) of 9 as a white solid after recrystalliza-
tion from EtOAc. Mp: 109.9–111.9 ꢁC. 1H NMR
(CDCl3): d 1.50–1.95 (m, 5H), 2.03–2.22 (m, 2H),
5.11. 1-(4-(4-tert-Butylphenyl)-4-phenylbutyl)-4-(4-chloro-
phenyl)piperidin-4-ol (15)
This was synthesized following a method previously
described29 starting with 4-tert-butylbenzophenone

7224
J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
(1.43 g, 6.01 mmol) to give 801 mg (28% over three
steps) of 15 as white crystals after recrystallization from
hexane/EtOAc. Mp: 104.0–105.3 ꢁC. 1H NMR (CDCl3):
d 1.26 (s, 9H), 1.48–1.72 (m, 5H), 2.02–2.14 (m, 4H),
2.28–2.44 (m, 4H), 2.64–2.79 (m, 2H), 3.85 (t,
J = 7.8 Hz, 1H), 7.12–7.43 (m, 13H). 13C NMR
(CDCl3): d 27.14, 31.21, 33.15, 34.17, 37.31, 49.08,
50.62, 58.24, 70.45, 125.19, 125.89, 125.99, 127.13,
127.66, 128.30, 128.33, 132.91, 135.46, 141.59, 144.64,
148.71. Anal. RP-HPLC Ib: tR = 5.24 min (purity
100%), IIa: tR = 9.46 min (purity 99%). HRMS (EI) m/
z calcd for C31H38ClN2O: 475.2642; found: 475.26301.
(ii) This was synthesized according to method B
using 1-bromo-5-phenylpentane (0.55 g, 2.44 mmol)
to give 666 mg (77%) of 21 as white crystals after
recrystallization from EtOAc. Mp: 103.4–105.2 ꢁC.
1H NMR (CDCl3): d 1.30–1.42 (m, 2H), 1.50–1.73
(m, 7H), 2.02–2.19 (m, 2H), 2.34–2.42 (m, 4H),
2.60 (t, J = 7.7 Hz, 2H), 2.78–2.84 (m, 2H), 7.15–
7.45 (m, 9H). 13C NMR (CDCl3): d 27.07, 27.66,
31.72, 36.25, 38.66, 49.82, 59.10, 71.41, 126.05,
126.50, 128.65, 128.78, 128.82, 133.22, 142.98,
147.15. Anal. RP-HPLC Ib: tR = 4.57 min (purity
100%), IIb: tR = 6.49 min (purity 100%). HRMS
(EI) m/z calcd for C22H28ClNO: 357.1859; found:
357.18617.
5.12. (1-(3-Benzo[d]thiazol-2-yl)propyl)-4-(4-chlorophe-
nyl)piperidin-4-ol (16)
5.15. 4-(4-Chlorophenyl)-1-(4,4-diphenylbut-3-enyl)pip-
eridin-4-ol (22)
Following method B using 2-(3-chloropropyl)benzo-
[d]thiazole (0.636 g, 3.00 mmol), which was synthesized
according to literature procedure,37 gave 306 mg
(26 %) of 16 as a light yellow solid after recrystallization
from EtOAc. Mp: 109.3–110.8 ꢁC. 1H NMR (CDCl3): d
1.61–1.76 (m, 3H), 2.21–2.39 (m, 4H), 2.50–2.79
(m, 4H), 2.93–3.08 (m, 2H), 3.18 (t, J = 7.3 Hz, 2H),
7.24–7.48 (m, 6H), 7.82–7.96 (m, 2H). 13C NMR
(CDCl3): d 25.76, 31.85, 37.48, 49.11, 57.13, 70.51,
121.41, 122.36, 124.68, 125.86, 125.90, 128.34, 132.83,
134.98, 146.01, 152.97, 170.99. Anal. RP-HPLC Ic:
tR = 8.42 min (purity 99%), IIc: tR = 10.70 min (purity
99%). HRMS (EI) m/z calcd for C21H23ClN2OS:
386.1220; found: 386.12301.
Following method B using 25 (1.00 g, 3.00 mmol),
which was synthesized according to literature proce-
dure,38,39 followed by recrystallization from EtOAc/
hexane gave 611 mg (49%) of 22 as a white solid. Mp
117.6–118.6 ꢁC. 1H NMR (CDCl3): d 1.48–1.79 (m,
3H), 1.98–2.20 (m, 2H), 2.29–2.62 (m, 6H), 2.67–2.89
(m, 2H), 6.07 (t, J = 7.2 Hz, 1H), 7.15–7.44 (m, 14H).
13C NMR (CDCl3): d 27.44, 38.27, 49.13, 58.43,
70.88, 125.97, 126.06, 126.83, 126.89, 127.03, 127.08,
127.96, 128.09, 128.25, 129.66, 132.58, 139.86, 142.40,
142.55, 146.80. Anal. RP-HPLC Ib: tR = 10.43 min
(purity 100%), IIb: tR = 13.29 min (purity 100%).
HRMS (EI) m/z calcd for C27H28ClNO: 417.1859;
found: 417.18446.
5.13. 4-(4-Chlorophenyl)-1-(3-phenylpropyl)piperidin-4-ol
(19)
5.16. 4-(4-Chlorophenyl)-1-(4-phenylbut-3-enyl)piperidin-
4-ol (23)
Following method B using 1-bromo-3-phenylpropane
(0.60 g, 3.03 mmol) afforded 729 mg (73%) of 19 as a
light yellow solid. Mp: 95.7–96.8 ꢁC. 1H NMR (CDCl3):
d 1.60–1.95 (m, 5H), 2.03–2.21 (m, 2H), 2.36–2.49 (m,
4H), 2.64 (t, J = 7.7 Hz, 2H), 2.80–2.86 (m, 2H), 7.16–
7.31 (m, 7H), 7.42 (d, J = 8.7 Hz, 2H). 13C NMR
(CDCl3): d 28.80, 34.12, 38.61, 49.76, 58.44, 71.39,
126.25, 126.49, 128.75, 128.77, 128.83, 133.26, 142.27,
147.06. Anal. RP-HPLC Ic: tR = 6.17 min (purity
100%), IIc: tR = 10.14 min (purity 100%). HRMS (EI)
m/z calcd for C20H24ClNO: 329.1546; found: 329.15489.
(i)
A solution of HCl (6 mL) and 27 (0.55 g,
2.98 mmol), which was synthesized as previously
described,40 was heated and refluxed for 4.5 h. The
reaction mixture was allowed to cool to room temper-
ature, slowly basified with a saturated solution of
Na2CO3, and the water layer was extracted with
EtOAc (3· 25 mL). The combined organic extracts
were washed with water (3· 50 mL) and brine
(50 mL), and dried over anhydrous MgSO4. Evapora-
tion in vacuo and purification by flash chromatogra-
phy (hexane) gave 99 mg (20%) of 28 as a colorless
5.14. 4-(4-Chlorophenyl)-1-(5-phenylpentyl)piperidin-4-ol
(21)
1
oil. H NMR (CDCl3): d 2.61–2.72 (m, 2H), 3.61 (t,
J = 6.9 Hz, 2H), 6.12–6.26 (dt, J = 6.9 Hz, 1H), 6.48
(d, J = 15.9 Hz, 1H), 7.19–7.38 (m, 5H).
(i) PBr3 (0.22 mL, 2.34 mmol) was added to a solution
of 5-phenyl-1-pentanol (0.82 g, 5.02 mmol) in Et2O
(25 mL) and this was stirred for 24 h at room tempera-
ture. Water (50 mL) was added and the solution was
basified with K2CO3 followed by an extraction with
Et2O (3· 30 mL). The combined organic extracts were
washed with water (100 mL) and brine (100 mL), dried
over anhydrous MgSO4 and filtered. Evaporation in
vacuo and purification by flash chromatography (hex-
ane) gave 555 mg (49%) of 1-bromo-5-phenylpentane
(ii) Following method A using 28 (0.10 g, 0.59 mmol)
gave 101 mg (50%) of 23 as white needles after
recrystallization from EtOAc. Mp: 136.3–137.2 ꢁC.
1H NMR (CDCl3): d 1.51–1.82 (m, 3H), 2.02–2.30
(m, 2H), 2.38–2.70 (m, 6H), 2.79–3.00 (m, 2H),
6.12–6.29 (dt, J = 6.6 Hz, 1H), 6.44 (d, J = 16.0 Hz,
1H), 7.18–7.46 (m, 9H). 13C NMR (CDCl3):
d
30.66, 38.30, 49.21, 58.21, 70.96, 125.83, 125.95,
126.89, 128.27, 128.36, 130.89, 133.01, 137.79. Anal.
RP-HPLC Ic: tR = 8.94 min (purity 100%), IIb:
tR = 4.79 min (purity 99%). HRMS (EI) m/z calcd
for C21H24ClNO: 341.1546; found: 341.15600.
1
as a colorless oil. H NMR (CDCl3): d 1.36–1.54 (m,
2H), 1.58–1.75 (m, 2H), 1.82–1.98 (m, 2H), 2.61 (t,
J = 7.5 Hz, 2H), 3.38 (t, J = 6.8 Hz, 2H), 7.13–7.38 (m,
5H).

J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
7225
5.17. [1-(4,4-Diphenylbutyl)-piperidin-4-yl]-(4-fluorophe-
nyl)methanone (31)
5.20. 1-Phenyl-8-(4-phenyl-4-o-tolylbutyl)-1,3,8-triaza-
spiro[4.5]decan-4-one (34)
This was synthesized according to method B using 4-
chloro-1,1-diphenylbutane44 (1.59 g, 6.50 mmol) and 4-
(4-fluorobenzoyl)piperidine toluenesulfonate (2.28 g,
6.00 mmol) to give 1.25 g (50%) of 31 as white crystals
after recrystallization from EtOAc. Mp: 88.9–90.6 ꢁC.
1H NMR (CDCl3): d 1.38–1.53 (m, 2H), 1.69–2.12
(m, 8H), 2.36 (t, J = 7.5 Hz, 2H), 2.80–2.99 (m, 2H),
3.06–3.23 (m, 1H), 3.88 (t, J = 7.8 Hz, 1H), 6.98–7.34
(m, 12H), 7.80–8.00 (m, 2H). 13C NMR (CDCl3): d
25.34, 28.59, 33.48, 43.64, 51.17, 53.12, 58.61,
115.36, 115.80, 125.94, 127.68, 128.27, 130.61,
130.79, 144.89, 162.90, 167.96, 200.92. Anal. RP-
HPLC Ib: tR = 8.30 min (purity 100%), IIa:
tR = 5.13 min (purity 100%), IIb: tR = 16.06 min (puri-
ty 100%). HRMS (EI) m/z calcd for C28H30FNO:
415.2311; found: 415.23053.
Following method A using 1-(4-chloro-1-phenylbutyl)-
2-methylbenzene29 (0.16 g, 0.52 mmol) and 1-phenyl-
1,3,8-triaza-spiro[4.5]decan-4-one (0.14 g, 0.62 mmol)
gave 188 mg (80%) of 34 as white crystals after recrystal-
lization from hexane/CHCl3. Mp: 184.3–186.0 ꢁC. 1H
NMR (CDCl3): d 1.39–1.83 (m, 4H), 1.93–2.11 (m,
2H), 2.19 (s, 3H), 2.33–2.81 (m, 8H), 4.11 (t,
J = 7.7 Hz, 1H), 4.80 (s, 2H), 6.61 (br s, 1H), 6.86–
6.92 (m, 2H), 7.00–7.38 (m, 12H). 13C NMR (CDCl3):
d 19.78, 29.00, 33.97, 41.28, 46.59, 49.57, 58.24, 58.89,
115.36, 118.89, 125.76, 125.85, 126.41, 128.04, 128.15,
129.12, 130.34, 136.14, 177.56. Anal. RP-HPLC Ib:
tR = 8.12 min (purity 100%), IIb: tR = 11.45 min (purity
100%). HRMS (EI) m/z calcd for C30H35N3O: 453.2780;
found: 453.27945.
5.21. 5-(2-Oxo-1,2-dihydrospiro[benzo[d][1,3]oxazine-
4,40-piperidine]-10-yl)-2,2-diphenylpentanenitrile (35)
5.18. (1-(4,4-Diphenylbutyl)piperidin-4-yl)(4-fluorophe-
nyl)methanol fumarate (32)
Following method A using spiro[benzo[d][1,3]oxazine-
4,40-piperidin]-2(1H)one (0.41 g, 1.62 mmol), which
was synthesized according to literature procedure,41
afforded 416 mg (57%) of 35 as a white solid. Mp:
172.7–174.3 ꢁC. 1H NMR (CDCl3): d 1.56–1.78 (m,
2H), 1.96–2.21 (m, 4H), 2.34–2.83 (m, 8H), 6.83 (d,
J = 9.0 Hz, 2H), 6.98–7.56 (m, 12H), 8.89 (br s, 1H).
13C NMR (CDCl3): d 23.65, 35.76, 37.83, 48.59, 52.04,
58.08, 81.91, 114.97, 122.71, 123.66, 124.08, 127.27,
128.28, 129.28, 129.44, 124.71, 140.57, 152.52. Anal.
RP-HPLC Ic: tR = 9.84 min (purity 100%), IIb:
tR = 4.58 min (purity 99%). HRMS (EI) m/z calcd for
C29H29N3O2: 451.2260; found: 451.22625.
A solution of 31 (0.16 g, 0.38 mmol) and NaBH4
(0.05 g, 1.32 mmol) in MeOH (20 mL) was stirred
overnight at room temperature. The reaction mixture
was evaporated in vacuo, water was added (15 mL),
and the water layer was extracted with DCM (3·
10 mL). The combined organic extracts were washed
with water (3· 25 mL) and brine (25 mL), dried over
anhydrous MgSO4, filtered, and evaporated in vacuo
to give 149 mg of oil. This was dissolved in EtOAc
and acidified by the addition of a saturated solution
of fumaric acid in Et2O. The fumaric salt was isolated
by filtration and recrystallized from IPA/Et2O to give
161 mg (79%) of 32 as a white solid. Mp: 77.4–
79.3 ꢁC. 1H NMR (CDCl3/DMSO-d6): d 1.29–1.79
(m, 7H), 1.91–2.40 (m, 5H), 2.61–2.83 (m, 2H), 3.08–
3.40 (m, 2H), 3.77 (t, J = 7.9 Hz, 1H), 4.12–4.30 (m,
1H), 6.64 (s, 2H), 6.89 (t, J = 8.7 Hz, 2H), 7.05–7.24
(m, 12H). 13C NMR (CDCl3): d 22.03, 25.75, 32.32,
41.46, 50.48, 56.29, 77.08, 114.81, 115.23, 126.25,
127.51, 127.85, 128.00, 128.45, 135.12, 138.56, 143.89,
159.51, 170.43. Anal. RP-HPLC Ib: tR = 6.38 min
(purity 100%), IIb: tR = 10.26 min (purity 96%).
HRMS (EI) m/z calcd for C28H32FNO: 417.2468;
found: 417.24489.
5.22. 5-(4-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imi-
dazol-1-yl)piperidin-1-yl)-2,2-diphenylpentanenitrile (36)
Following method A using 5-chloro-1-(piperidin-4-yl)-
1H-benzo[d]imidazol-2(3H)-one (0.50 g, 2.00 mmol)
gave 734 mg (76%) of 36 as a white solid. Mp: 203.1–
1
204.6 ꢁC. H NMR (DMSO-d6): d 1.35–1.68 (m, 4H),
1.82–2.09 (m, 2H), 2.20–2.59 (m, 6H), 2.77–2.98 (m,
2H), 4.02–4.23 (m, 1H), 6.98–7.04 (m, 2H), 7.20–7.45
(m, 11H), 11.05 (br s, 1H). 13C NMR (CDCl3): d
23.23, 29.02, 37.27, 50.85, 51.49, 52.98, 57.51, 110.21,
120.87, 122.21, 126.66, 126.72, 127.52, 127.77, 128.61,
128.75, 128.82, 139.99, 155.03. Anal. RP-HPLC Ib:
tR = 5.81 min (purity 100%), IIb: tR = 8.83 min (purity
100%). HRMS (EI) m/z calcd for C29H29ClN4O:
484.2030; found: 484.20548.
5.19. 5-(4-Oxo-1-phenyl-1,3,8-triazaspiro[4.5]decan-8-
yl)-2,2-diphenylpentanenitrile (33)
Following method B using 1-phenyl-1,3,8-triaza-spi-
ro[4.5]decan-4-one (0.83 g, 3.61 mmol) afforded 842 mg
(60%) of 33 after recrystallization from CHCl3. Mp:
204.1–206.0 ꢁC. 1H NMR (CDCl3): d 1.60–1.76 (m,
4H), 2.38–2.68 (m, 10H), 4.70 (s, 2H), 6.45 (s, 1H),
6.80–6.92 (m, 3H), 7.24–7.42 (m, 12H). 13C NMR
(CDCl3): d 23.22, 28.84, 37.34, 49.58, 51.49, 57.38,
59.14, 115.11, 118.75, 122.23, 126.70, 127.71, 128.74,
129.11, 140.07, 142.92, 177.93. Anal. RP-HPLC Ib:
tR = 5.41 min (purity 100%), IIb: tR = 8.17 min (purity
100%). HRMS (EI) m/z calcd for C30H32N4O:
464.2576; found: 464.25859.
5.23. 5-(3-Oxo)-3H-spiro[isobenzofuran-1,40-piperidine]-
10-yl-2,2-diphenylpentanenitrile hydrochloride (37)
A solution of o-bromobenzoic acid (0.76 g, 3.76 mmol)
in THF (20 mL) was cooled to ꢀ78 ꢁC and n-BuLi
(4.7 mL, 1.6 M in hexane, 7.52 mmol) was added slowly
in a period of 20 min. The solution was stirred for 2 h at
ꢀ78 ꢁC and 39 (1.5 g, 4.51 mmol), which was synthe-
sized according to literature procedure,31 was added
slowly in a period of 30 min. The reaction mixture was

7226
J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
allowed to warm to room temperature and water
(30 mL) was added. The water layer was extracted with
Et2O (5· 10 mL) and acidified with HCl until a pH of 2.
The solution was heated to reflux temperature for 1 h,
cooled to room temperature, and stirred overnight. A
10% NaOH solution was added to a pH of 10, and the
water layer was rapidly extracted with CHCl3 (5·
20 mL). The combined organic extracts were washed
with water (3· 10 mL), brine (20 mL), dried over anhy-
drous MgSO4, filtered and evaporated in vacuo to afford
1.01 g of a white solid. This was converted to the hydro-
chloride salt as described for 2 to give 516 mg (31%) of
37 after recrystallization from MeOH/Et2O. Mp: 129.8–
131.5 ꢁC. 1H NMR (CDCl3): d 1.68–1.92 (m, 4H), 2.48–
2.91 (m, 8H), 2.98–3.21 (m, 2H), 7.18–7.60 (m, 12H),
7.66 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 9.0 Hz, 1H). 13C
NMR (CDCl3): d 21.60, 34.28, 36.80, 49.27, 51.40,
57.08, 82.78, 121.26, 121.94, 125.00, 125.88, 126.56,
127.86, 128.91, 129.63, 134.49, 139.51, 152.25, 169.01.
Anal. RP-HPLC Ic: tR = 9.86 min (purity 98%), IIb:
tR = 5.64 min (purity 95%). HRMS (EI) m/z calcd for
C29H28N2O2: 436.2151; found: 436.21533.
solid. The crude product was used without further
purification.
(iv) This was dissolved in toluene (20 mL) and p-toluene
sulfonic acid (0.19 g, 9.99 mmol) was added. The reac-
tion mixture was refluxed for 3 h, quenched with a satu-
rated solution of NaHCO3 (25 mL), and the water layer
was extracted with Et2O (3· 15 mL). The combined
organic extracts were washed with water (3· 20 mL)
and brine (20 mL), dried over anhydrous MgSO4, and
filtered. After evaporation in vacuo, the residue was
purified by flash chromatography (0–50% EtOAc in
DCM) and recrystallized from Et2O to give 112 mg
1
(26%) of 38 as white crystals. Mp: 139.2–140.8 ꢁC. H
NMR (CDCl3): d 1.31–1.78 (m, 4H), 1.91–2.19 (m,
4H), 2.22–2.50 (m, 4H), 2.95 (m, 2H), 3.48 (s, 2H),
3.89 (t, J = 7.7 Hz, 1H), 4.19-4.35 (m, 1H), 7.02–7.31
(m, 14H). 13C NMR (CDCl3): d 26.04, 28.45, 31.30,
34.01, 36.25, 50.27, 51.73, 53.69, 58.81, 110.91, 122.19,
124.89, 125.19, 126.52, 127.92, 128.24, 128.83, 144.07,
145.42, 175.25. Anal. RP-HPLC Ib: tR = 5.58 min (puri-
ty 99%), IIb: tR = 9.11 min (purity 99%). HRMS (EI)
m/z calcd for C29H32N2O: 424.2515; found: 424.25005.
5.24. 1-(1-(4,4-Diphenylbutyl)piperidin-4-yl)indolin-2-one
(38)
5.25. 5-(4-Acetyl-4-phenylpiperidin-1-yl)-2,2-diphenyl-
pentanenitrile hydrochloride (44)
(i) NaH (1.96 g, 49.00 mmol) was added in 5 min to a
solution of di-t-butyl malonate (10 mL, 44.61 mmol) in
DMF (250 mL) and this was stirred for 15 min. Subse-
quently, 2-fluoro-nitrobenzene (4.95 mL, 46.83 mmol)
was added in one portion and the reaction mixture
was stirred for 8 h at 90 ꢁC and two days at room tem-
perature. Water (300 mL) was added and the water layer
was extracted with Et2O (3· 200 mL). The combined
organic extracts were washed with water (4· 150 mL)
and brine (150 mL), dried over Na2SO4 and filtered.
After evaporation in vacuo, the residue was purified
by flash chromatography (20% EtOAc in hexane) to give
3.0 g (20%) of di-t-butyl 2-(2-nitrophenyl)malonate as a
Following method A using 1-(4-phenylpiperidin-4-
yl)ethanone (0.48 g, 2.00 mmol) gave 728 mg of an oil.
This was converted to the hydrochloride salt as de-
scribed for 2 and recrystallized from MeOH/Et2O to
give 552 mg (54 %) of 44 as a white solid. Mp: 247.1–
1
249.0 ꢁC. H NMR (CDCl3): d 1.75–2.02 (m, 2H), 1.91
(s, 3H), 2.42–3.08 (m, 10H), 3.29–3.51 (m, 2H), 7.11–
7.56 (m, 15H), 12.44 (br s, 1H). 13C NMR (CDCl3):
d 20.11, 25.13, 27.23, 28.92, 29.61, 34.87, 48.43,
49.59, 51.17, 121.72, 125.25, 125.53, 126.19, 127.39,
127.85, 139.05, 139.42, 207.72. Anal. RP-HPLC Ib:
tR = 4.84 min (purity 100%), IIb: tR = 10.56 min (purity
100%). HRMS (EI) m/z calcd for C30H32N2O: 436.2515;
found: 436.25253.
1
light brown solid. H NMR (CDCl3): d 1.48 (s, 18H),
5.09 (s, 1H), 7.39–7.78 (m, 3H), 8.01 (d, J = 9.1 Hz, 1H).
(ii) This was dissolved in EtOAc (100 mL) and 10% Pd/
C (0.35 g) was added. The reaction mixture was hydro-
genated with H2 (5 bar) overnight, filtrated over Hyflo,
and washed with EtOAc. After evaporation in vacuo,
the residue was purified by recrystallization (Et2O/hex-
ane) to give 915 mg (34%) of 41 as purple crystals. H
NMR (CDCl3): d 1.43 (s, 18H), 4.48 (s, 1H), 6.62–6.78
(m, 2H), 7.03–7.19 (m, 2H).
5.26. 4-(4-Chlorophenyl)-1-(4-cyano-4,4-diphenyl-
butyl)piperidin-4- yl acetate fumarate (45)
Et3N (0.12 mL, 0.9 mmol) and acetyl chloride (0.02 mL,
0.28 mmol) were added to a solution of 1 (0.13 g,
0.30 mmol) in DCM (5 mL) at 0 ꢁC. The reaction mix-
ture was stirred at 0 ꢁC for 30 min and 1 h at room tem-
perature. Water (10 mL) was added and the water layer
was extracted with DCM (3· 10 mL). The combined
organic extracts were washed with water (3· 15 mL)
and brine (15 mL), dried over anhydrous MgSO4, and
filtered. After evaporation in vacuo, the residue was
purified by flash chromatography (0–25% EtOAc in
DCM) to afford 89 mg of a thick oil. This was converted
to the fumaric salt as described for 32 and recrystallized
from MeOH/Et2O to give 75 mg (41%) of 45 as white
crystals. Mp: 176.4–177.3 ꢁC. 1H NMR (CDCl3/
DMSO-d6): d 1.42–1.69 (m, 2H), 1.81–2.07 (m, 5H),
2.16–2.56 (m, 8H), 2.65–2.83 (m, 2H), 6.59 (s, 2H),
7.11–7.24 (m, 14H). 13C NMR (DMSO-d6): d 21.46,
34.51, 35.57, 48.27, 51.19, 56.18, 64.70, 78.96, 122.21,
1
(iii) Both 41 (0.31 g, 1.00 mmol) and 40 (0.31 g,
1.00 mmol), which was synthesized following literature
procedures,29,31 were dissolved in 1,2-dichloroethane
˚
(10 mL). Next, molsieves (4 A) and AcOH (0.06 mL,
1.00 mmol) were added. The reaction mixture was stir-
red for 30 min, NaBH(OAc)3 (0.30 g, 1.42 mmol) was
added, and this was stirred for another 24 h. A saturated
solution of NaHCO3 (15 mL) was added and the water
layer was extracted with Et2O (3· 20 mL). The com-
bined organic extracts were washed with water (3·
25 mL) and brine (25 mL), dried over MgSO4, filtered,
and evaporated in vacuo to give 520 mg of 42 as a white

J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
7227
126.21, 126.38, 127.68, 127.94, 128.82, 131.37, 133.92,
139.79, 143.16, 166.01, 168.76. Anal. RP-HPLC Ib:
tR = 6.42 min (purity 100%), IIa: tR = 5.39 min (purity
100%), IIb: tR = 17.41 min (purity 100%). HRMS (EI)
m/z calcd for C30H31ClNO2: 486.2074; found:
486.20619.
tR = 5.20 min (purity 100%), IIb: tR = 6.08 min (purity
93%). HRMS (EI) m/z calcd for C29H34N2O: 426.2671;
found: 426.26775.
5.29. 1-(1-(4,4-Diphenylbutyl)-4-phenylpiperidin-4-yl-
methyl)-N- methylmethanamine dihydrochloride (49)
5.27. (1-(4,4-Diphenylbutyl)-4-phenylpiperidin-4-yl)meth-
anamine (47)
LiAlH4 (0.75 g, 19.76 mmol) was added to a solution of
48 (0.20 g, 0.47 mmol) in THF (10 mL) at 0 ꢁC. The
reaction mixture was refluxed for 6 h, quenched with a
saturated solution of Na2CO3 and the water layer was
extracted with DCM (3· 10 mL). The combined organic
extracts were dried over anhydrous MgSO4 and evapo-
rated in vacuo. The residue was purified by flash chro-
matography (5% Et3N in EtOAc) to give 150 mg of a
white solid. This was converted to the corresponding
hydrochloride salt as described for 2 to give 104 mg
(46%) of 49 as a white solid. Mp: 50.9–52.5 ꢁC. 1H
NMR (MeOH-d4): d 1.57–1.79 (m, 2H), 1.98–2.27 (m,
4H), 2.59 (s, 3H), 2.60–2.82 (m, 4H), 2.92–3.06 (m,
2H), 3.18–3.30 (m, 2H), 3.40–3.57 (m, 2H), 3.88–4.09
(m, 1H), 7.10–7.33 (m, 10H), 7.36–7.61 (m, 5H). 13C
NMR (CDCl3): d 22.66, 30.45, 35.42, 40.00, 49.34,
51.01, 55.53, 57.59, 61.17, 127.33, 128.08, 128.27,
128.98, 129.03, 130.66, 137.48, 144.28. Anal. RP-HPLC
Ic: tR = 3.52 min (purity 100%), IIc: tR = 4.63 min (puri-
ty 100%). HRMS (EI) m/z calcd for C29H36N2:
412.2878; found: 412.28610.
(i) Following method B using 4-chloro-1,1-diphenylbu-
tane44 (1.22 g, 4.98 mmol) and 53 (0.92 g, 4.15 mmol)
gave 1.59 g (81%) of 55 as a white solid. 1H NMR
(CDCl3): d 1.36–1.71 (m, 2H), 1.99–2.21 (m, 6H),
2.30–2.56 (m, 4H), 2.88–3.05 (m, 2H), 3.88 (t, 1H,
J = 7.8 Hz), 7.09–7.51 (m, 15H).
(ii) A suspension of AlCl3 (0.93 g, 6.98 mmol) in THF
(10 mL) was added to a suspension of LiAlH4 (0.26 g,
6.85 mmol) in THF (10 mL) at 0 ꢁC and this was stirred
for 5 min followed by the dropwise addition of a solu-
tion of 55 (1.36 g, 3.45 mmol) in THF (15 mL). The
reaction mixture was allowed to warm to room temper-
ature, stirred overnight, cooled with an ice bath, and
quenched with a saturated solution of Na2CO3 in water
until the foaming stopped. Subsequently, the suspension
was filtered, and the filtrate was dried over anhydrous
MgSO4. Filtration, evaporation in vacuo, and purifica-
tion by flash chromatography (5% Et3N in EtOAc)
afforded 863 mg (63%) of 47 as a white solid. Mp:
5.30. (4-(4-Chlorophenyl)-1-(4,4-diphenylbutyl)piperidin-
4-yl)methanamine dihydrochloride (50)
1
228.2–230.2 ꢁC. H NMR (MeOH-d4): d 1.53–1.72 (m,
2H), 1.98–2.21 (m, 4H), 2.42–2.99 (m, 6H), 3.15 (s,
2H), 3.21–3.40 (m, 2H), 3.94 (t, J = 7.9 Hz, 1H), 7.02–
7.54 (m, 15H). 13C NMR (DMSO-d6): d 22.45, 29.81,
31.80, 47.69, 49.87, 55.33, 64.70, 125.87, 126.70,
126.98, 127.31, 127.48, 128.21, 128.79, 144.60. Anal.
RP-HPLC Ic: tR = 3.91 min (purity 100%), IIc:
tR = 4.44 min (purity 100%). HRMS (EI) m/z calcd for
C28H34N2: 398.2722; found: 398.27414.
(i) Et3N (20 mL) was added to a solution of bis-(2-chlo-
roethyl)amine hydrochloride (8.17 g, 45.77 mmol) in
DCM (75 mL) and this was stirred for 15 min. After
the addition of di-tert-butyl dicarbonate (10.00 g,
45.82 mmol) in DCM (50 mL), the reaction mixture
was stirred for 20 h and quenched with water
(200 mL). The water layer was extracted with DCM
(4· 100 mL) and the combined organic extracts were
dried over anhydrous Na2SO4 and filtered. After evapo-
ration in vacuo, the residue was purified by flash chro-
matography (30% hexane in DCM) to give 3.90 g
5.28. N-((1-(4,4-Diphenylbutyl)-4-phenylpiperidin-4-
yl)methyl)formamide hydrochloride (48)
1
Compound 47 (0.11 g, 0.23 mmol) and formic acid
(2 mL) were added in a 10 mL microwave vessel and this
was reacted during 5 min in the microwave at a temper-
ature of 200 ꢁC (settings: ramp time 5 min, hold time
5 min, power 200 watt, pressure 17.2 bar). The reaction
mixture was quenched with a saturated solution of
Na2CO3 (2 mL), and the water layer was extracted with
DCM (3 · 5 mL). The combined organic extracts were
dried over MgSO4 and filtered. After evaporation in
vacuo, the residue was purified by flash chromatography
(5% Et3N in EtOAc) and the free base was converted to
the hydrochloride salt as described for 2 to give 100 mg
(94%) of 48 as a white solid. Mp: 52.2–52.8 ꢁC. 1H
NMR (CDCl3): d 1.42–1.74 (m, 2H), 1.86–2.42 (m,
8H), 2.50–2.89 (m, 2H), 3.10–3.29 (m, 2H), 3.39
(d, J = 7.6 Hz, 2H), 3.87 (t, J = 7.8 Hz, 1H), 5.05
(br s, 1H), 7.01–7.61 (m, 15H), 8.03 (s 1H). 13C
NMR (CDCl3): d 24.62, 31.69, 32.24, 33.72, 49.83,
51.45, 58.51, 126.65, 127.42, 128.14, 128.87, 129.00,
129.60, 144.51, 144.96, 161.78. Anal. RP-HPLC Ib:
(35%) of 57 as a light yellow oil. H NMR (CDCl3): d
1.48 (s, 9H), 3.49–3.70 (m, 8H).
(ii) A suspension of NaNH2 (0.80 g, 20.51 mmol) in tolu-
ene (10 mL) was added to a solution of 2-(4-chlorophe-
nyl)acetonitrile in toluene (10 mL) at 0 ꢁC. The reaction
mixture was stirred for 30 min at 0 ꢁC and added drop
wise to a solution of 57 (2.78 g, 10.01 mmol) in toluene
(50 mL) at 0 ꢁC. This was heated to 70 ꢁC, stirred over-
night, and quenched with water (100 mL). The organic
layer was separated and the water layer was extracted
with DCM (3· 50 mL). The combined organic extracts
were dried over anhydrous Na2SO4 and filtered. Evapora-
tion in vacuo and purification by flash chromatography
(DCM) gave 600 mg (19%) of 58 as a light yellow oil.
1H NMR (CDCl3): d 1.44 (s, 9H), 1.72–2.11 (m, 4H),
3.04–3.29 (m, 2H), 4.13–4.35 (m, 2H), 7.31–7.49 (m, 4H).
(iii) This was dissolved in EtOH (30 mL), which was
saturated with hydrochloride gas, and the resulting

7228
J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
reaction mixture was stirred for 5 min. Evaporation in
vacuo gave a quantitative yield of 54 as a white solid.
1H NMR (DMSO-d6): d 2.31–2.44 (m, 4H), 2.99–3.20
(m, 2H), 3.39–3.48 (m, 2H), 7.49–7.62 (m, 4H).
white solid. This was converted to the fumaric salt as
described for 29 to give 110 mg (19%) of 52 as white
crystals after recrystallization from MeOH/Et2O. Mp:
189.9–190.7 ꢁC. H NMR (MeOH-d4): d 1.57–1.68 (m,
1
2H), 1.98–2.08 (m, 2H), 2.08–2.18 (m, 2H), 2.13–2.18
(s, 2H), 2.31–2.42 (m, 2H), 1.82–2.95 (m, 4H), 3.15–
3.24 (m, 2H), 3.96 (t, J = 7.8 Hz, 1H), 7.11–7.19 (m,
2H) 7.23–7.32 (m, 8H) 7.44 (d, J = 8.7 Hz, 2H), 7.52
(d, J = 8.8 Hz, 2H). 13C NMR (DMSO-d6): d 23.88,
35.02, 48.17, 50.04, 52.48, 56.42, 125.78, 127.32,
127.79, 127.90, 128.16, 131.43, 134.70, 144.85, 167.23.
Anal. RP-HPLC Ic: tR = 5.85 min (purity 100%), IIc:
tR = 6.63 min (purity 100%). HRMS (EI) m/z calcd for
C27H31ClN2: 418.2176; found: 418.21760.
(iv) Following method B using 4-chloro-1,1-diphenylbu-
tane44 (0.56 g, 2.28 mmol) and 54 (0.49 g, 1.90 mmol)
afforded 500 mg of 56 as a white solid (62%). 1H
NMR (CDCl3): 1.32–1.72 (m, 2H), 1.90–2.08 (m, 6H),
2.23–2.59 (m, 4H), 2.72–3.02 (m, 2H), 3.91 (t,
J = 7.8 Hz, 1H), 6.99–7.52 (m, 14H).
(v) This was synthesized as described for 47 starting
from 56 (0.30 g, 0.47 mmol) to give 130 mg of oil. This
was converted to the hydrochloride salt as described
for 2 to give 100 mg (42%) of 50 as a white solid. Mp:
257.3–258.9 ꢁC (dec). 1H NMR (MeOH-d4): d 1.57–
1.80 (m, 2H), 2.01–2.29 (m, 6H), 2.56–2.81 (m, 2H),
2.92–3.20 (m, 4H), 3.32–3.54 (m, 2H), 3.82–4.00 (m,
1H), 7.09–7.59 (m, 14H). 13C NMR (DMSO-d6): d
21.80, 29.23, 30.48, 31.64, 47.21, 49.80, 54.98, 60.16,
125.92, 127.30, 128.23, 128.80, 131.97, 135.28, 144.46.
Anal. RP-HPLC Ic: tR = 6.07 min (purity 100%), IIc:
tR = 7.57 min (purity 100%). HRMS (EI) m/z calcd for
C28H33ClN2: 432.2332; found: 432.23193.
5.33. Pharmacology
5.33.1. Transient and stable expression of US28. COS-7
cells were grown as described previously.50 Transient
transfection of COS-7 cells was performed by DEAE–
dextran, using 2 lg of US28-pcDEF3 DNA per million
cells.50 SVEC4-10 (ATCC CRL2181) is a cell line de-
rived by SV40 transformation of endothelial cells from
the murine axillary lymph node vessels. SVEC4-10 cells
were grown as described previously.50 SVEC4-10 cells
were transfected with US28-pTJE8 using 25 kDa linear
polyethylenimine (PEI; Polysciences, Inc.). ‘Empty’ vec-
tor was used for mock transfections. Briefly, 10 lg DNA
and 50 lg PEI were separately diluted into 250 lL of
150 mM NaCl solution. The PEI solution was added
to the DNA solution, vortexed and incubated for
15 min at room temperature. One million SVEC4-10
cells were seeded in a 10-cm dish the day before transfec-
tion. Culture medium was replaced by 6 mL DMEM
and the DNA/PEI solution was added dropwise to the
cells. The solution was mixed by gently shaking and
the dish was incubated at 37 ꢁC for 5 h. The transfection
solution was replaced by culture medium and incubated
O/N. SVEC4-10 cells stably expressing US28 were
selected in culture medium containing 500 lg/mL neo-
mycin G418. Clones were selected based on US28-med-
iated constitutive IPx accumulation and chemokine
binding analysis.
5.31. N-(4-(4-Chlorophenyl)-1-(4,4-diphenylbutyl)-piperi-
din-4-yl)acetamide (51)
A solution of 95% H2SO4 (3.3 mL, 12.4 mmol) was add-
ed dropwise to a solution of 59 (1.37 g, 3.26 mmol) in
CH3CN (16 mL), while the temperature was maintained
between 25 ꢁC and 30 ꢁC. The reaction mixture was stir-
red overnight at room temperature, poured into ice, and
neutralized with a 30% solution of NaOH. Water was
added (20 mL) and the water layer was extracted with
DCM (3· 30 mL). The combined organic layers were
washed with water (3· 30 mL) and brine (20 mL), dried
over anhydrous Na2SO4, and filtered. After evaporation
in vacuo, the residue was purified by flash chromatogra-
phy (0–50% MeOH in EtOAc) and recrystallized from
EtOH/Et2O to give 655 mg (75%) of 51 as a white solid.
Mp: 189.1–190.4 ꢁC. 1H NMR (CDCl3): d 1.39–1.83 (m,
6H), 1.89–2.50 (m, 6H), 1.98 (s, 3H), 2.67–2.88 (m, 2H),
3.88 (t, J = 7.8 Hz, 1H), 5.52 (br s, 1H), 7.13–7.27 (m,
14H). 13C NMR (CDCl3): d 24.02, 24.52, 33.14, 34.32,
49.21, 50.97, 55.54, 57.82, 126.14, 126.47, 127.30,
127.59, 128.38, 132.51, 143.40, 144.45, 169.58. Anal.
RP-HPLC Ib: tR = 5.66 min (purity 100%), IIb:
tR = 8.20 min (purity 100%). HRMS (EI) m/z calcd for
C29H33ClN2O: 460.2281; found: 460.22991.
5.33.2. [125I]CCL5 binding experiments. Radioiodination
[
125I] of CCL5 (Peprotech, Rocky Hill, NJ) was per-
formed using iodogen (Pierce Chemical Co., Rockford,
IL) as described previously.51 Displacement binding
experiments were performed on US28-expressing COS-
7 cell membranes. Two days after transfection, COS-7
cells were homogenized in ice-cold buffer A (i.e.,
15 mM Tris–HCl, pH 7.5, 2 mM MgCl2, 0.3 mM
EDTA, and 1 mM EGTA) and centrifuged at 200g for
10 min. Supernatant was collected and centrifuged at
48,000g for 30 min. The pellet was resuspended in buffer
B (i.e., 7.5 mM Tris–HCl, pH 7.5, 12,5 mM MgCl2,
0.3 mM EDTA, 1 mM EGTA, and 250 mM sucrose),
aliquoted, and stored at ꢀ80 ꢁC until use. Cell mem-
branes (approximately 0.2 lg/sample) were incubated
with 0.3 nM [125I]CCL5 in binding buffer (i.e., 50 mM
Hepes, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 100 mM
NaCl, and 0.2% bovine serum albumin) in the absence
or presence of various concentrations of nonpeptidergic
5.32. 4-(4-Chlorophenyl)-1-(4,4-diphenylbutyl)piperidin-
4-amine fumarate (52)
A solution of 51 (0.66 g, 1.42 mmol) in 8% HCl
(240 mL) was refluxed for three days. Water was added
(40 mL), the water layer was basified with a 20% solu-
tion of NaOH and extracted with DCM (3· 30 mL).
The combined organic layers were dried over anhydrous
Na2SO4 and filtered. After evaporation in vacuo, the
residue was purified by flash chromatography (5%
MeOH and 1% Et3N in EtOAc) to give 290 mg of a

J. W. Hulshof et al. / Bioorg. Med. Chem. 14 (2006) 7213–7230
7229
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ligands at 37 ꢁC for 1 h. Incubations were terminated by
filtration through a UniFilter-96 GF/C filter plate (Per-
kin-Elmer Life Sciences, Wellesley, MA) presoaked in
0.3% PEI, followed by three rapid washes with ice-cold
binding washing buffer (i.e., 50 mM Hepes, pH 7.4,
1 mM CaCl2, 5 mM MgCl2, and 500 mM NaCl) using
a MicroBeta FilterMate-96 Harvester (PerkinElmer).
Non-specific binding was determined on membranes
from mock-transfected cells. Radioactivity was quanti-
fied with a liquid scintillation using a Wallac MicroBeta
TriLux (PerkinElmer).
5.33.3. [3H]inositol phosphate accumulation. SVEC4-10
cells (approximately 4 · 105 cells/well) were seeded into
poly-L-lysine-coated 96-well plates in 200 lL culture
medium. After approximately 24 h, medium was re-
placed with 100 lL/well inositol-free Dulbecco’s modi-
fied Eagle’s medium supplemented with 10 lCi/mL
myo-[2-3H]-inositol (PerkinElmer) and the cells were
further incubated. The next day, cells swere washed
and preincubated for 10 min with assay buffer (i.e., Dul-
becco’s modified Eagle’s medium containing 10 mM
LiCl). Next, cells were incubated in assay buffer in the
absence or presence of various concentrations of nonp-
eptidergic ligands at 37 ꢁC for 2 h. Inositol phosphates
were extracted using 10 mM formic acid and quantified
using 0.5 mg/well Ysi-RNA-binding SPA beads (Amer-
sham Biosciences), in white clear-bottomed 96-well
isoplates (PerkinElmer) using a Wallac MicroBeta
TriLux, essentially as previously described.52
Acknowledgments
29. Hulshof, J. W.; Casarosa, P.; Menge, W. M. P. B.;
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J. W. Hulshof, H. F. Vischer, and M. H. P. Verheij were
supported by the Technology Foundation STW and
M. J. Smit was supported by the Royal Netherlands
Academy of Arts and Sciences.
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