Synthesis and structure-activity relationship of the first nonpeptidergic inverse agonists for the human cytomegalovirus encoded chemokine receptor US28

Article abstract of DOI:10.1021/jm050418d

US28 is a human cytomegalovirus (HCMV) encoded G-protein-coupled receptor that signals in a constitutively active manner. Recently, we identified 1 {5-(4-(4-chlorophenyl)-4-hydroxy-piperidin-1-yl)-2,2-diphenylpentanenitrile} as the first reported nonpeptidergic inverse agonist for a viral-encoded chemokine receptor. Interestingly, this compound is able to partially inhibit the viral entry of HIV-1. In this study we describe the synthesis of 1 and several of its analogues and unique structure-activity relationships for this first class of small-molecule ligands for the chemokine receptor US28. Moreover, the compounds have been pharmacologically characterized as inverse agonists on US28. By modification of lead structure 1, it is shown that a 4-phenylpiperidine moiety is essential for affinity and activity. Other structural features of 1 are shown to be of less importance. These compounds define the first SAR of ligands on a viral GPCR (US28) and may therefore serve as important tools to investigate the significance of US28-mediated constitutive activity during viral infection.

Full text of DOI:10.1021/jm050418d

J. Med. Chem. 2005, 48, 6461-6471
6461
Synthesis and Structure-Activity Relationship of the First Nonpeptidergic
Inverse Agonists for the Human Cytomegalovirus Encoded Chemokine
Receptor US28
Janneke W. Hulshof, Paola Casarosa, Wiro M. P. B. Menge, Leena M. S. Kuusisto, Henk van der Goot,
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 May 3, 2005
US28 is a human cytomegalovirus (HCMV) encoded G-protein-coupled receptor that signals
in a constitutively active manner. Recently, we identified 1 {5-(4-(4-chlorophenyl)-4-hydroxy-
piperidin-1-yl)-2,2-diphenylpentanenitrile} as the first reported nonpeptidergic inverse agonist
for a viral-encoded chemokine receptor. Interestingly, this compound is able to partially inhibit
the viral entry of HIV-1. In this study we describe the synthesis of 1 and several of its analogues
and unique structure-activity relationships for this first class of small-molecule ligands for
the chemokine receptor US28. Moreover, the compounds have been pharmacologically
characterized as inverse agonists on US28. By modification of lead structure 1, it is shown
that a 4-phenylpiperidine moiety is essential for affinity and activity. Other structural features
of 1 are shown to be of less importance. These compounds define the first SAR of ligands on a
viral GPCR (US28) and may therefore serve as important tools to investigate the significance
of US28-mediated constitutive activity during viral infection.
Introduction
RANTES, US28 induces migration of vascular smooth
muscle cells, which could provide the molecular basis
of the role of HCMV in vascular diseases. The migration
of smooth muscle cells can also be exploited by HCMV
to enhance dissemination of the virus through the
body.15 Similar to other chemokine receptors, such as
CCR5 and CXCR4, US28 can act as a coreceptor for
HIV-1 entry when coexpressed with CD4.16 Taken
together, US28 is considered as an interesting drug
target.
In previous studies we have shown that in transiently
transfected COS-7 cells as well as in HCMV-infected
cells US28 constitutively activates phospholipase C and
the transcription factor NF-κB in an agonist-indepen-
dent manner.17,18 Interestingly, the CC chemokines
CCL2 and CCL5 do not affect the basal US28 signaling
and act as neutral antagonists, whereas the CX3C
chemokine CX3CL1 partially inhibits the constitutive
signaling and acts as a partial inverse agonist. Consti-
tutive activity is suggested to be a general characteristic
of viral-encoded GPCRs. Besides the HCMV-encoded
receptor US28, other viral-encoded receptors also signal
in the absence of any ligand, namely, the HHV-8 or
Kaposi’s sarcoma-associated herpesvirus (KSHV) en-
coded GPCR ORF7419 and the M33 gene family mem-
bers encoded by MCMV, HCMV, and RCMV.18,20,21
Although the biological relevance of constitutive activity
has not been completely elucidated yet, it is believed to
play an important role in the pathogenesis of virus
infection. This is demonstrated for ORF74, which is a
viral oncogene resulting in the development of Kaposi’s
sarcoma-like lesions in transgenic mice.22,23
Human cytomegalovirus (HCMV) is a widespread
â-herpesvirus that, like other herpes viruses, persists
during the lifetime of the host.1 Infection of individuals
with HCMV is common, reaching a seroprevalence of
50-90% in adults, and is normally without clinical
symptoms.2 However, in immunologically immature or
immunocompromised hosts, like premature neonates,
AIDS patients, and transplant recipients, the virus can
cause serious and even life-threatening disease.1 Infec-
tion with HCMV is furthermore suggested to be associ-
ated with vascular diseases such as arterial restenosis,
atherosclerosis, and chronic allograft rejection.3-5
The genome of human cytomegalovirus encodes four
G-protein-coupled receptors (GPCRs), namely, the open
reading frames (ORFs) UL33, UL78, US27, and US28.6
Two of these GPCRs, namely, UL33 and UL78, have
counterparts in the genome of both rat CMV (R33 and
R78, respectively) and mouse CMV (M33 and M78,
respectively), whereas US27 and US28 are specific for
HCMV.7 US28 shows high homology with â mammalian
chemokine receptors, binds several CC chemokines with
high affinity,8-10 and is able to sequester CC chemokines
from the extracellular environment via endocytosis.11,12
This feature appears to be a putative strategy of the
virus to escape immune surveillance by reducing the
immune response to sites of HCMV infection.13 US28
is able to bind not only CC chemokines but also the
membrane-bound CX3C chemokine CX3CL1/fractalkine,
which has been suggested to play a role in the cell to
cell transfer of HCMV.14 Furthermore, upon binding
with the CC chemokines CCL2/MCP-1 and CCL5/
Inverse agonists are able to inhibit the constitutive
activation of GPCRs. The identification of such mol-
ecules for viral-encoded GPCRs can be an important tool
* To whom correspondence should be addressed. Phone: +31-20-
5987579. Fax: +31-20-5987610. E-mail: r.leurs@few.vu.nl.
10.1021/jm050418d CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/01/2005

6462 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Scheme 1. Synthetic Pathway for 1-16a
Hulshof et al.
a Reagents and conditions: (a) NaH, DMF, 0 °C; (b) 1,ω-dihalopropane; (c) DMF, K2CO3, 90 °C or NaI, Na2CO3, CH3CN, reflux; (d) 6
N HCl, MeOH; (e) R2MgBr, THF, 0 °C or n-BuLi, THF, 0 °C for R2 ) n-butyl; (f) MeI, DCM.
Scheme 2. Synthetic Pathway for 17-20a
a Reagents and conditions: (a) DMF, K2CO3, 90 °C or NaI, Na2CO3, CH3CN, reflux; (b) 2 M NaOH, MeOH, reflux.
to investigate the significance of constitutive activity
and the influence of these receptors in viral infection.
Recently, we identified a small nonpeptidergic molecule
that acts as a full inverse agonist on US28. Further-
more, this compound is able to inhibit 60% of the US28-
mediated HIV entry in cells.18 This molecule has been
previously reported as an antagonist on the human
chemokine receptor CCR124 and was screened on US28
because of the high sequence homology of this viral
receptor with CCR1 (33% identity).10 In this study the
synthesis of VUF2274 and various analogues is reported
and we present the first structure-activity relationships
for the interaction of these ligands with US28.
1,ω-dihalopropanes to yield intermediate 22a or 22b.24,25
Compounds 2, 13, 17, and 18 were synthesized by
stirring intermediate 22a in DMF at 90 °C in the
presence of the corresponding amine and an excess of
K2CO3 (method A).24 The yields after purification were
in the range of 15-50%. The reaction conditions were
optimized by reacting intermediate 22a or 22b with the
corresponding piperidine 23 in the presence of NaI,
Na2CO3, and CH3CN at reflux temperature (method B)26
to give the desired products in yields ranging from 23%
to 79%.
For compounds 5-10 the appropriate piperidines
were not commercially available. Consequently, 5-10
were synthesized according to literature procedures
(Scheme 1, method C).25 Intermediate 22 was reacted
with protected piperidone 24, followed by the deprotec-
tion with HCl to give ketone 25. This was treated with
the appropriate Grignard reagents to yield compounds
5-9, whereas the reaction with n-BuLi afforded com-
Chemistry
For the synthesis of compounds 1-20 the following
synthetic routes were applied (Schemes 1 and 2).
Diphenyl acetonitrile 21 was deprotonated with NaH
at 0 °C followed by a reaction with the appropriate

Nonpeptidergic Inverse Agonist
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6463
Scheme 3. Synthetic Pathway for 26 and 27a
a Reagents and conditions: (a) NaI, Na2CO3, CH3CN, reflux.
Scheme 4. Synthetic Pathway for 31 and 32a
a Reagents and conditions: (a) ethylene glycol, p-toluenesulfonic acid monohydrate, toluene, reflux; (b) 30, DMF, K2CO3, 90 °C or 30,
NaI, Na2CO3, CH3CN, reflux; (c) HCl, MeOH, reflux; (d) NaBH4, EtOH, 0 °C; (e) acetyl chloride, Et3N, Et2O; (f) 10% NaOH, MeOH,
reflux.
pound 10.25 The yields of products 5-10 were moderate
to low, ranging from 5% to 42%, because of low yields
in the recrystallization step.
4-(Diphenylmethylene)piperidine, which was used for
the synthesis of compound 15, was obtained by acid-
catalyzed dehydratation of diphenyl(piperidin-4-yl)-
methanol with TFA.27 The quaternary ammonium salt
16 was synthesized by the methylation of the piperidine
nitrogen atom of 1 with methyl iodide.25
Target compound 19 was synthesized following method
B. The ethyl ester moiety of 19 was hydrolyzed in high
yield with 2 M NaOH, resulting in the carboxylic acid
derivative 20 (Scheme 2).
Compounds 26 and 27 were prepared via the reaction
of intermediates 28 and 29 with 4-chlorophenyl-4-
hydroxypiperidine 30 (Scheme 3).
Compounds 31 and 32 were synthesized as depicted
in Scheme 4. Butyrophenone derivative 31 was synthe-
sized from commercially available reagents as earlier
described.28 The carbonyl group of 33 was ketalized with
ethylene glycol in the presence of p-toluenesulfonic acid
to afford the protected intermediate 34. Alkylation of
piperidine 30 with 34 and deprotection of the carbonyl
group with HCl resulted in product 31. For the synthesis
of compound 32, 4-chloro-1-phenylbutan-1-one 33 was
reduced with NaBH4, resulting in alcohol 36. Subse-
quently, the alcohol group was protected with acetyl
chloride to give intermediate 37, which was reacted with
4-(4-chlorophenyl)-4-hydroxypiperidine 30, resulting in
compound 38. Deprotection with NaOH afforded target
compound 32.
Compounds 39-47 were synthesized according to the
procedure shown in Scheme 5 (method D). Diphenyl-
methanes, which were not commercially available, were
obtained from benzophenones 48 in the presence of
AlCl3 as a Lewis acid and a hydride donor (NaBH4 or
tert-butylamineborane).29,30 Intermediates 49 were depro-
tonated with n-BuLi in THF, and the resulting lithium
salts reacted with 1,3-dibromopropane to afford bro-
mides 50 in low yields.31,32 The conversion of 4-meth-
oxydiphenylmethane (49, R1 ) 4-OCH3, R2 ) H) and
3,4-dichlorodiphenylmethane (49, R1 ) 3,4-Cl2, R2 ) H)
into the corresponding sodium salts or lithium salts
with, respectively, NaNH2 in liquid NH3 or n-BuLi in
THF was not successful. However, deprotonation of 3,4-
dichlorodiphenylmethane was achieved with LDA in
the presence of HMPA. For the deprotonation of 4-meth-
oxydiphenylmethane, the base strength of n-BuLi was
not sufficient and a mixture of n-BuLi and potassium
tert-butoxide in THF was used. Piperidine 30 was
alkylated with intermediate 50 in CH3CN in the pres-
ence of NaI and Na2CO3 to give the desired products
39-47.
Pharmacological Results and Discussion
To identify a potential inverse agonist acting on US28,
several GPCR-directed ligands were screened for their
ability to modulate the basal signaling of this receptor.
This resulted in the identification of compound 1 as the
first nonpeptidergic inverse agonist acting on US28, as
reported in an earlier paper describing US28 pharma-
cology.18 It is noted that compound 1 has previously

6464 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Scheme 5. Synthetic Pathway for 39-47a
Hulshof et al.
a Reagents and conditions: (a) AlCl3, NaBH4, THF, 0 °C or AlCl3 (CH3)3CNH2‚BH3, THF, 0 °C; (b) n-BuLi, THF, -20 °C or LDA,
HMPA, THF, -78 °C or n-BuLi, (CH3)3COK, -100 °C, THF; (c) 1-bromo-3-chloropropane; (d) 30, NaI, Na2CO3, CH3CN, reflux.
Table 1. Chemical Structures and Pharmacological Properties
of Compounds 1-14, 19, and 20 for the HCMV-Encoded
Receptor US28a
been reported as a potent antagonist for the human
chemokine receptor CCR1, but it has no effects on the
human chemokine receptors CCR5, CCR2, and CXCR4.
Additionally, the selectivity of 1 was tested by screening
against a number of human GPCRs and an at least 250-
fold selectivity for the CCR1 chemokine receptor was
observed. As expected, the only cross-reactivity of
compound 1 was demonstrated for several biogenic
amine neurotransmitter receptors.24 Recently, it was
also shown that this molecule binds to the human
chemokine receptor CCR3 with micromolar affinity.33
In all, we consider 1 to be a unique and interesting lead
structure for the development of ligands directed toward
the new class of viral GPCRs and a good tool to study
viral GPCRs in a variety of assays with HCMV-infected
cells. Thus, compound 1 was used as a starting point
for lead optimization, and several analogues were
synthesized in order to investigate the first structure-
activity relationship for inverse agonism on US28. All
the synthesized compounds were evaluated for their
potential to dose-dependently displace [125I]CCL5 bind-
ing in COS-7 cells expressing US28. The effect on the
US28-mediated constitutive inositol phosphate produc-
tion in transiently expressed COS-7 cells was investi-
gated for a selection of compounds. It is evident from
the results in Tables 1-4 that the IC50 and EC50 values
obtained from the binding assay and the functional
assay, respectively, correlate well with each other. To
determine the specificity of action, the selected com-
pounds were also tested on the constitutively active
KSHV-encoded GPCR ORF74. The observed inositol
phosphate production in ORF74 expressing cells was not
affected up to a concentration of 10 µM (data not shown),
indicating that the influence of these compounds on the
inositol phosphate production was selective for US28.
To study the importance of the 4-chlorophenyl-4-
hydroxypiperidine moiety, various substituted piperi-
dine moieties were synthesized (Table 1). The influence
of the substitution pattern of the phenyl ring was
investigated by the substitution of the p-chloro sub-
stituent of 1 for substituents with different electronic
and lipophilic properties in compounds 2-7. Interest-
ingly, all the compounds with variations in the para
position, including the unsubstituted analogue 2, were
a The values are represented as the mean and the interval of
the IC50 and EC50 values of at least three independent experi-
b
ments, unless otherwise indicated.
[
125I]CCL5 displacement.
c Inhibition of [3H]inositol phosphate production. nd ) not deter-
mined. d Result of two independent experiments.
found to be less potent. The introduction of an additional
substituent at the meta position (m-trifluoro or m-chloro
substituents in 3 and 6, respectively) did not result in
compounds with a higher affinity. For the human
chemokine receptor CCR1, there is a 2-fold improve-
ment in Ki value when p-chloro is replaced by a p-bromo
substituent,25 but on the viral-encoded receptor US28
the bromo substitution in 4 causes a 2-fold decrease in
binding affinity.

Nonpeptidergic Inverse Agonist
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6465
Table 2. Chemical Structures and Pharmacological Properties
of Compounds 1 and 15-18 for the HCMV-Encoded Receptor
US28a
Table 3. Chemical Structures and Pharmacological Properties
of Compounds 1, 26, 27, 31, 32, and 39 for the HCMV-Encoded
Receptor US28a
a The values are represented as the mean and the interval of
the IC50 and EC50 values of at least three independent experi-
a The values are represented as the mean and the interval of
b
ments, unless otherwise indicated.
[
125I]CCL5 displacement.
the IC50 and EC50 values of at least three independent experi-
c Inhibition of [3H]inositol phosphate production. nd ) not deter-
b
ments, unless otherwise indicated.
[
125I]CCL5 displacement.
mined. d Result of two experiments.
c Inhibition of [3H]inositol phosphate production.
Methylation of the piperidine nitrogen atom of com-
pound 1 gave quaternary ammonium salt 16. Interest-
ingly, this variation resulted in complete loss of binding
affinity and activity on US28. For the human chemokine
receptor CCR1 this modification resulted in a 6.5-fold
increase in binding affinity,25 clearly illustrating selec-
tivity of compound 16 in favor of the CCR1 chemokine
receptor. This indicates that there is a difference in
structure-activity relationships of these compounds for
both receptors, like that shown earlier in this section
for compound 4. Taken together, several analogues were
synthesized to optimize the piperidine motif in the
molecule, but all the variations in this part of the
molecule resulted in a loss of affinity and potency.
Next, the influence of the diphenylacetonitrile moiety
was investigated (Tables 3 and 4). The influence of the
two phenyl rings was investigated by removal of one of
the phenyl rings in compound 27 or replacement by
more hydrophilic groups, namely, a carbonyl group in
compound 31 and a hydroxy group in compound 32.
Previously, it was suggested that a bulky lipophilic
moiety is important at this position,18 but we observed
that removal of a phenyl ring as in 27 resulted in a
binding affinity comparable to that of lead compound
1. In contrast, the more hydrophilic groups in 31 and
32 resulted in loss of effect. Also, the introduction of an
isosteric oxygen atom in the butyl chain in compound
26 did not improve affinity.
The binding affinities of the unsubstituted analogue
2 and compound 8, with a benzyl group at the 4-position
of the piperidine ring, were comparable, but both
affinities were lower than that of lead compound 1.
Apparently, the introduction of an additional methylene
group has no positive effect on the affinity of the
compound. Removal of the 4-chlorophenyl group in
compound 11 or substitution of this group by a CH3
group in 9 or n-butyl chain in 10 resulted in complete
loss of affinity. This suggests that a phenyl ring at the
4-position is of importance.
Next, the role of the 4-hydroxy group was studied in
compounds 12 and 13. It is noted that the 4-chlorophen-
yl-4-hydroxypiperidine moiety is a structural motif that
is also present in, for example, haloperidol. For this
antipsychotic drug it is known that the 4-hydroxy group
is responsible for the conversion to potentially neuro-
toxic metabolites.34,35 In this respect, it is interesting
to see that the affinity and activity on US28 did not
change significantly after removal of the 4-hydroxy
group in 13. Substitution of the hydroxy group of 2 into
a nitrile group in compound 12 resulted in a complete
loss of both affinity and activity.
The influence of the piperidine ring was investigated
by the synthesis of compounds 15-18 (Table 2). Previ-
ously, we described that the basic nitrogen atom of the
piperidine ring of 1 probably has an important interac-
tion with a glutamic acid residue in transmembrane 7
(Glu277).18 For this reason, the nitrogen atom at this
position was maintained. But substitution of the piper-
idine ring into a piperazine ring in 17 or tetrahydropyr-
idine moiety in 18 abolished activity. The loss of activity
of both compounds might be due to a change in
conformation of the 4-chlorophenyl ring through which
the interaction of the aromatic moiety with the receptor
is lost. Introduction of the bulky 4-(diphenylmethylene)-
piperidine moiety of analogue 15 was detrimental for
both affinity and potency.
Interestingly, the nitrile group of compound 1 is not
essential in the molecule because its removal, resulting
in compound 39, did not affect the activity of the
compound (Table 4). Compounds 40-42 and 44-47
possess a chiral center as a result of the substitution in
one or both of the phenyl rings. The two enantiomers
were not separated, and the activity was thus deter-
mined for the racemic mixtures (Table 4). Compounds
40-42 and 46 were synthesized to determine the effects
of the introduction of para substituents with different

6466 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Hulshof et al.
Table 4. Chemical Structures and Pharmacological Properties
of Compounds 39-47 for the HCMV-Encoded Receptor US28a
These structural insights will be used to develop new
compounds with an improved affinity and potency as
well as a better selectivity for this receptor. These small
molecules may serve as important tools to investigate
the significance of the constitutive activity and the role
of US28 during viral infection.
Experimental Section
Chemistry. General Procedures. 1H NMR spectra were
recorded on a Bruker AC-200 (200 MHz) spectrometer with
tetramethylsilane as internal standard. J. T. Baker silica gel
was used for flash chromatography. Mass spectra were re-
corded on a Finnigan MAT-90 mass spectrometer. Melting
points were measured on an Electrothermal IA9200 apparatus
and were uncorrected. Elemental analyses were performed by
Microanalytisches Labor Pascher, Remagen-Bandorf, Ger-
many, and the results were within (0.4% of the theoretical
values unless otherwise stated. The solvents were dried
according to standard procedures. All reactions were performed
under an atmosphere of dry nitrogen.
General Method A. 5-(4-(4-Chlorophenyl)piperazin-1-
yl)-2,2-diphenypentanenitrile (17). A solution of 22a (0.51
g, 1.88 mmol), which was synthesized according to literature
procedure,24 4-chlorophenylpiperazine (0.45 g, 1.67 mmol), and
K2CO3 (3.53 g, 25.6 mmol) in DMF (25 mL) was stirred
overnight at 90 °C. The solvent was removed in vacuo, and
the residue was dissolved in water (25 mL) followed by an
extraction with EtOAc (3 × 10 mL). The combined organic
layers were washed with water (2 × 10 mL), dried over
anhydrous MgSO4, and filtered. After evaporation in vacuo,
the residue was purified by flash chromatography (0-100%
EtOAc in DCM) to give 107 mg (15%) of 17 as a light-yellow
solid. Mp: 126.2-126.7 °C. 1H NMR (CDCl3): δ 1.52-1.71 (m,
2H), 2.37-2.51 (m, 8H), 3.11 (m, 4H), 6.80 (d, 2H, J ) 9.0 Hz),
7.15-7.40 (m, 12H). MS ESI m/z: 431.0 (M + H)+. Anal.
(C27H28ClN3) C, H, N.
a The values are represented as the mean and the interval of
the IC50 and EC50 values of at least three independent experi-
b
ments, unless otherwise indicated.
[
125I]CCL5 displacement.
c Inhibition of [3H]inositol phosphate production. nd ) not deter-
mined. d Result of two independent experiments.
electronic and lipohilic properties in the two aromatic
rings. The variations in the substitution pattern in the
para position did not significantly influence the activity
of the compounds. Also, the introduction of an additional
p-chloro atom (compound 43) did not increase the
affinity of the compound for US28.
General Method B. 5-(4-(4-Chlorophenyl)-4-hydroxy-
piperidin-1-yl)-2,2-diphenylpentanenitrile (1). A solution
of 22b (0.94 g, 3.00 mmol), which was synthesized according
to literature procedure,25 4-chlorophenyl-4-hydroxypiperidine
30 (0.67 g, 3.15 mmol), NaI (0.50 g, 3.31 mmol), and Na2CO3
(0.64 g, 6.04 mmol) in CH3CN (25 mL) was refluxed overnight.
The solvent was removed in vacuo, and the residue was diluted
with water (50 mL) followed by an extraction with DCM (3 ×
30 mL). The combined organic layers were dried over anhy-
drous MgSO4, filtered, and evaporated in vacuo. Purification
by flash chromatography (0-100% EtOAc in DCM) and
crystallization from EtOAc gave 1.04 g (78%) of 1 as a white
solid. Mp: 133.6-135.2 °C. 1H NMR (CDCl3): δ 1.55-1.70 (m,
4H), 1.93-2.12 (m, 3H), 2.28-2.50 (m, 6H), 2.61-2.78 (m, 2H),
7.18-7.48 (m, 14H). MS ESI m/z: 446.9 (M + H)+. Anal.
(C28H29N2ClO‚0.21EtOAc) C, H, N.
General Method C. 5-(4-(4-Methoxyphenyl)-4-hydroxy-
piperidin-1-yl)-2,2-diphenylpentanenitrile (5). A solution
of (4-methoxyphenyl)magnesium bromide37 (7.7 mL, 0.5 M in
dry Et2O, 3.6 mmol) was cooled to 0 °C, and a solution of 25
(1.00 g, 3.00 mmol), which was synthesized according to
literature procedure,25 dissolved in dry THF (15 mL) was added
in one portion via a dropping funnel. The reaction mixture was
stirred at room temperature for 4 h, and the reaction was
quenched with water (30 mL). This solution was acidified with
1 N HCl, stirred for 15 min and basified with K2CO3. The
product was extracted with EtOAc (3 × 100 mL), and the
combined organic layers were washed with water (2 × 100 mL)
and brine (100 mL), dried over anhydrous MgSO4, filtered, and
evaporated under reduced pressure. Purification by flash
chromatography (EtOAc) afforded 780 mg (59%) of 5. Recrys-
tallization of one batch from Et2O gave 190 mg (14%) of 5 as
white crystals. Mp: 84.7-86.6 °C. 1H NMR (CDCl3): δ 1.69-
1.74 (m, 5H), 2.02-2.18 (m, 2H), 2.35-2.50 (m, 6H), 2.68-
2.79 (m, 2H), 3.78 (s, 3H), 6.86 (d, 2H, J ) 8.9 Hz), 7.24-7.42
(m, 12H). MS ESI m/z: 441.4 (M + H)+. Anal. (C29H32N2O2)
C, H, N.
The differences in binding affinities between the
methyl-substituted analogues 44-46 and the unsubsti-
tuted analogue 39 reveal a slight preference for o-methyl
substitution. This could be caused by the restricted
conformational freedom of the substructure in com-
pound 44. o-Methyl substitution of a similar aromatic
benzhydryl sytem was previously described for a series
of histamine H1 receptor antagonists. It is suggested
that the o-methyl analogue of diphenhydramine has a
markedly reduced antihistamine activity because the
methyl group in the ortho position prevents the molecule
from adopting the conformation necessary for antihis-
tamine activity.36
The influence of a bulky aromatic substituent in the
para position was investigated by the synthesis of
analogue 47. The additional p-phenyl group resulted in
a loss of affinity and potency, and it seems that bulky
substituents are not allowed at this position.
Conclusions
This is the very first study of small molecules acting
as inverse agonists on US28. We described the synthesis
of compound 1 and different analogues, which were
evaluated for their binding affinity by displacement of
[
125I]CCL5. Moreover, a selection of compounds was
evaluated for their functionality by measuring the effect
on the US28-mediated constitutive inositol phosphate
production. This resulted in unique structure-activity
relationships for the first small-molecule US28 receptor
ligands.
With this study we have acquired important new
insights about the first inverse agonists acting on US28.

Nonpeptidergic Inverse Agonist
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6467
General Method D. 4-(4-Chlorophenyl)-1-(4-phenyl-4-
o-tolylbutyl)piperidin-4-ol (50). (i) A solution of 2-methyl-
benzophenone (5.34 g, 27.2 mmol) in dry THF (125 mL) was
cooled to 0 °C, and subsequently AlCl3 (10.06 g, 75.4 mmol)
and NaBH4 (5.26 g, 139 mmol) were added. The reaction
mixture was heated and refluxed for 3 h. Next, the mixture
was cooled to 0 °C and diluted carefully by a dropwise addition
of water (100 mL). After separation of the organic layer, the
water layer was extracted with EtOAc (3 × 100 mL). The
combined organic layers were washed with water (3 × 150 mL)
and brine (150 mL), dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. Purification by flash
chromatography (hexane) gave 3.81 g (77%) of 2-methyldiphen-
ylmethane as a colorless oil. 1H NMR (CDCl3): δ 2.37 (s, 3H),
4.11 (s, 2H), 7.20-7.46 (m, 9H).
(ii) In a dry atmosphere a solution of 2-methyldiphenyl-
methane (1.87 g, 10.3 mmol) in dry THF (20 mL) was cooled
to -20 °C, and n-BuLi (6.40 mL, 1.6 M in hexane, 10.2 mmol)
was added. The solution was stirred for 1 h and subsequently
added slowly to a solution of 1,3-dibromopropane (2.30 mL,
22.7 mmol) in dry THF (35 mL) at -76 °C. The mixture was
allowed to warm to room temperature and was stirred for 30
min. The solvent was evaporated under reduced pressure.
Water (25 mL) was added to the residue, and the water layer
was extracted with EtOAc (3 × 25 mL). The combined organic
layers were washed with water (3 × 50 mL) and brine (50 mL),
dried over anhydrous MgSO4, filtered, and evaporated under
reduced pressure. The residue was purified by flash chroma-
afforded 944 mg of 6. Recrystallization from Et2O gave 212
mg (15%) of 6 as white crystals. Mp: 102.0-103.4 °C. 1H NMR
(CDCl3): δ 1.59-1.68 (m, 5H), 2.01-2.12 (m, 2H), 2.28-2.49
(m, 6H), 2.68-2.79 (m, 2H), 7.24-7.41 (m, 12H), 7.59 (d, 1H,
J ) 2.1 Hz). MS ESI m/z: 479.5 (M + H)+. Anal. (C28H28-
Cl2N2O) C, H, N, Cl.
5-(4-Hydroxy-4-p-tolylpiperidin-1-yl)-2,2-diphenylpen-
tanenitrile Hydrochloride (7). Following method C using
25 (1.00 g, 3.00 mmol) and p-tolylmagnesium bromide37 (3.6
mL, 1.0 M in dry Et2O, 3.6 mmol) gave 280 mg of a thick oil.
The compound was converted to the hydrochloride salt as
described for 3. One batch was recrystallized from Et2O/MeOH
to give 141 mg (10%) of 7 as a white solid. Mp: 225.0-226.3
°C. 1H NMR (CDCl3): δ 1.81-2.03 (m, 5H), 2.30 (s, 3H), 2.62-
2.71 (m, 2H), 2.75-2.92 (m, 2H), 3.01-3.22 (m, 6H), 7.13 (d,
2H, J ) 8.1 Hz), 7.24-7.45 (m, 12H), 12.15 (br s, 1H). MS
ESI m/z: 425.4 (M + H)+. Anal. (C29H33ClN2O) C, H, N, Cl.
5-(4-Benzyl-4-hydroxypiperidin-1-yl)-2,2-diphenylpen-
tanenitrile Hydrochloride (8). Following method C using
25 (1.00 g, 3.00 mmol) and benzylmagnesium bromide37 (3.4
mL, 1.1 M in dry Et2O, 3.7 mmol) gave 0.147 g of thick oil.
The compound was converted to the hydrochloride salt as
described for 3. One batch was recrystallized from Et2O/MeOH
to give 86 mg (5%) of 8 as a white solid. Mp: 121.8-123.8 °C.
1H NMR (CDCl3): δ 1.51-1.65 (m, 3H), 1.90-1.99 (m, 2H),
2.38-2.51 (m, 2H), 2.59-2.65 (m, 2H), 2.80 (s, 2H), 2.89-3.00
(m, 4H), 3.14-3.22 (m, 2H), 7.16-7.38 (m, 15H), 12.02 (br s,
1H). MS ESI m/z: 425.5 (M + H)+. Anal. (C29H33ClN2O‚
1.0H2O) C, H, N, Cl.
5-(4-Hydroxy-4-methylpiperidin-1-yl)-2,2-diphenylpen-
tanenitrile Hydrochloride (9). Following method C using
25 (1.00 g, 4.00 mmol) and methylmagnesium iodide37 (2.8 mL,
1.3 M in dry Et2O, 3.6 mmol) followed by purification by flash
chromatography (0-7% EtOH in DCM) gave 0.50 g of thick
oil. The compound was converted to the hydrochloride salt as
described for 3. Recrystallization from Et2O/MeOH gave 441
mg (42%) of 9 as a white solid. Mp: 192.7-193.5 °C. 1H NMR
(CDCl3): δ 1.32 (s, 3H), 1.63-1.69 (m, 3H), 1.88-2.05 (m, 2H),
2.28-2.44 (m, 2H), 2.62 (t, 2H, J ) 7.9 Hz), 2.90-3.21 (m, 6H),
7.24-7.43 (m, 10H), 11.88 (br s, 1H). MS ESI m/z: 349.4 (M
+ H)+. Anal. (C23H29ClN2O) C, H, N, Cl.
tography (hexane) to give 0.81 g (26%) of bromide 50 (R1
)
2-Me; R2 ) H) as a colorless oil. 1H NMR (CDCl3): δ 1.87-
2.00 (m, 2H), 2.18-2.30 (m, 2H), 2.34 (s, 3H), 3.44 (t, 2H, J )
6.6 Hz), 4.18 (t, 1H, J ) 8.1 Hz), 7.18-7.42 (m, 9H).
(iii) 50 was reacted with 4-chlorophenyl-4-hydroxypiperidine
30 (0.70 g, 3.3 mmol) according to method B to give 834 mg
(72%) of 44 as white crystals after recrystallization from
EtOAc. Mp: 143.1-144.4 °C. 1 H NMR (CDCl3): δ 1.55-1.69
(m, 5H), 2.00-2.12 (m, 4H), 2.25 (s, 3H), 2.33-2.44 (m, 4H),
2.71-2.78 (m, 2H), 4.09 (t, 1H, J ) 7.6 Hz), 7.07-7.33 (m,
11H), 7.41 (d, 2H, J ) 8.8 Hz). MS ESI m/z: 434.4 (M + H)+.
Anal. (C28H32ClNO) C, H, N, Cl.
5-(4-Hydroxy-4-phenylpiperidin-1-yl)-2,2-diphenylpen-
tanenitrile (2). Following method A using 22a (0.51 g, 1.90
mmol) and 4-hydroxy-4-phenylpiperidine (0.34 g, 1.93 mmol)
gave 319 mg (42%) of 2 as a white solid.24 Mp: 73.6-74.7 °C.
1H NMR (CDCl3): δ 1.51-1.80 (m, 4H), 2.06-2.19 (m, 3H),
2.30-2.50 (m, 6H), 2.64-2.78 (m, 2H), 7.12-7.51 (m, 15H).
MS ESI m/z: 411.3 (M + H)+. Anal. (C28H30N2O‚0.36H2O) C,
H, N.
5-(4-(3-Chloro-4-trifluoromethylphenyl)-4-hydroxypi-
peridin-1-yl)-2,2-diphenylpentanenitrile Hydrochloride
(3). Using 22a (0.65 g, 2.40 mmol) and 4-(4-chloro-3-(trifluo-
romethyl)phenyl)-4-piperidinol (0.56 g, 2.01 mmol) gave 0.93
g (90%) of the product as a thick oil. This was dissolved in
MeOH (5 mL), and subsequently 6 N HCl (0.15 mL) and Et2O
(20 mL) were added dropwise while stirring. The hydrochloride
salt was isolated by filtration and recrystallized from Et2O/
MeOH. This gave 666 mg (67%) of 3 as a white solid. Mp:
231.2-234.0 °C (dec). 1H NMR (CDCl3): δ 1.61-1.82 (m, 5H),
2.07-2.26 (m, 2H), 2.31-2.53 (m, 6H), 2.70-2.89 (m, 2H),
7.24-7.46 (m, 13H). MS ESI m/z: 513.4 (M + H)+. Anal.
(C29H29Cl2N2F3O) C, H, N.
5-(4-(4-Bromophenyl)-4-hydroxypiperidin-1-yl)-2,2-
diphenylpentanenitrile (4). Following method B using 22a
(0.65 g, 2.39 mmol) and 4-(4-bromophenyl)-4-hydroxypiperidine
(0.51 g, 2.01 mmol) gave 819 mg (83%) of 4 as a white solid.
Mp: 131.6-132.5 °C. 1H NMR (CDCl3): δ 1.61-1.82 (m, 5H),
2.07-2.26 (m, 2H), 2.31-2.53 (m, 6H), 2.70-2.89 (m, 2H),
7.24-7.46 (m, 14H). MS ESI m/z: 490.8 (M + H)+. Anal.
(C28H29N2BrO‚0.34DCM) C, H, N.
5-(4-Butyl-4-hydroxypiperidin-1-yl)-2,2-diphenylpen-
tanenitrile Hydrochloride (10). This compound was syn-
thesized as described in the literature25 and converted to the
hydrochloride salt as described for 3. One batch was recrystal-
lized from Et2O/MeOH to afford 304 mg (18%) of 10 as a white
1
solid. Mp: 174.2-176.0 °C (dec). H NMR (CDCl3): δ 0.80-
0.93 (t, 3H, J ) 4.6 Hz), 1.18-1.40 (m, 4H), 1.48-1.71 (m, 5H),
1.88-2.08 (m, 2H), 2.28-2.43 (m, 2H), 2.60-70 (m, 2H), 2.89-
3.22 (m, 6H), 7.24-7.44 (m, 10H), 12.02 (br s, 1H). MS ESI
m/z: 391.4 (M + H)+. Anal. (C26H35ClN2O) C, H, N, Cl.
5-(4-Hydroxypiperidin-1-yl)-2,2-diphenylpentaneni-
trile (11). Following method B using 22b (0.95 g, 3.01 mmol)
and 4-hydroxypiperidine (0.38 g, 3.75 mmol) followed by
purification by flash chromatography (10-50% MeOH in
EtOAc) afforded 374 mg (37%) of 11 as a white solid.25 Mp:
114.8-115.8 °C. 1H NMR (CDCl3): δ 1.39-1.68 (m 5H), 1.80-
1.91 (m, 2H), 1.95-2.11 (m, 2H), 2.31-2.44 (m, 4H), 2.59-
2.71 (m, 2H), 3.59-3.72 (m, 1H), 7.22-7.42 (m, 10H). MS ESI
m/z: 335.3 (M + H)+. Anal. (C22H26N2O) C, H, N.
1-(4-Cyano-4,4-diphenylbutyl)-4-phenylpiperidine-4-
carbonitrile (12). Following method B using 22a (0.65 g, 2.40
mmol) and 4-phenylpiperidine-4-carbonitrile (0.45 g, 2.00
mmol) followed by crystallization from DCM/Et2O gave 599
mg (66%) of 12 as a white solid. Mp: 137.5-138.7 °C. 1H NMR
(CDCl3): δ 1.59-1.71 (m, 2H), 2.02-2.08 (m, 4H), 2.33-2.49
(m, 6H), 2.83-2.97 (m, 2H), 7.24-7.50 (m, 15H). MS ESI m/z:
420.7 (M + H)+. Anal. (C29H29N3) C, H, N.
5-(4-(4-Chlorophenyl)piperidin-1-yl)-2,2-diphenylpen-
tanenitrile (13). A solution of 4-(4-chlorophenyl)-1,2,3,6-
tetrahydropyridine hydrochloride (1.00 g, 4.35 mmol) and 5%
Pd/C (0.100 g) in EtOH (25 mL) was stirred for 4 h under
hydrogen. The solution was filtered and evaporated under
reduced pressure. The residue was used without further
5-(4-(3,4-Dichlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-
diphenylpentanenitrile (6). Following method C using 25
(1.00 g, 3.00 mmol) and (3,4-dichlorophenyl)magnesium bro-
mide37 (3.3 mL, 1.1 M in dry Et2O, 3.6 mmol) followed by
purification by flash chromatography (0-100% EtOAc in DCM)

6468 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Hulshof et al.
purification, dissolved in DMF, and reacted following method
A using 22a (0.63 g, 2.34 mmol). This afforded 954 mg (51%)
of 13 as a white solid. Mp: 112.6-113.4 °C. 1H NMR (CDCl3):
δ 1.55-1.83 (m, 6H), 1.89-2.08 (m, 2H), 2.35-2.56 (m, 5H),
2.89-3.01 (m, 2H), 7.09-7.43 (m, 14H). MS ESI m/z: 430.0
(M + H)+. Anal. (C28H29ClN2) C, H, N.
1-(4-Cyano-4,4-diphenylbutyl)piperidine-4-carboxyl-
ic Acid Amide (14). Following method B using 22b (0.94 g,
3.00 mmol) and piperidine-4-carboxylic acid amide (0.39 g, 3.00
mmol) gave 796 mg (73%) of 14 as an orange oil. 1H NMR
(CDCl3): δ 1.61-2.52 (m, 13 H), 2.76-2.99 (m, 2H), 5.57 (br
s, 2H), 7.24-7.45 (m, 10H). MS ESI m/z: 362.3 (M + H)+. Anal.
(C25H27N2O2‚0.44DCM) C, H, N.
4-(4-Chlorophenyl)-1-(4-phenylbutyl)piperidin-4-ol (27).
Following method B using 1-chloro-4-phenylbutane 29 (0.49
mL, 2.98 mmol) and 4-chlorophenyl-4-hydroxypiperidine 30
(0.70 g, 3.30 mmol) gave 310 mg (35%) of 27 as a white solid.33
Mp: 112.5-114.0 °C. 1H NMR (CDCl3): δ 1.50-1.72 (m, 7H),
2.00-2.17 (m, 2H), 2.25-2.43 (m, 4H), 2.62 (t, 2H, J ) 7.1
Hz), 2.72-2.85 (m, 2H), 7.15-7.31 (m, 7H), 7.42 (d, 2H, J )
8.6 Hz). MS ESI m/z: 344.3 (M + H)+. Anal. (C21H26ClNO) C,
H, N, Cl.
4-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-1-phen-
ylbutan-1-one (31). (i) A solution of 33 (6 mL, 37.4 mmol),
ethylene glycol (6 mL, 108 mmol), and p-toluenesulfonic acid
monohydrate (0.50 g, 2.63 mmol) in toluene (400 mL) was
refluxed overnight with azeotropic removal of water. The
organic layer was washed with 5% NaHCO3 (250 mL) and
water (250 mL), dried over anhydrous Na2SO4, and evaporated
in vacuo to give 9.69 g (98%) of 34 as an orange solid.
(ii) Following method B using 34 (0.72 g, 3.19 mmol) and
4-chlorophenyl-4-hydroxypiperidine 30 (0.57 g, 2.69 mmol)
gave 1.281 g of a brown oil. This was dissolved in MeOH (15
mL), and concentrated HCl (1.4 mL) was added. The resulting
reaction mixture was refluxed for 2 h, allowed to cool to room
temperature, and evaporated in vacuo. The resulting brown
oil was dissolved in EtOAc (25 mL) and washed with NH4OH
(5% solution in water, 2 × 15 mL) and water (2 × 15 mL).
The organic layer was dried over anhydrous MgSO4, rinsed
with hexane, and evaporated in vacuo to give 522 mg (53%) of
31 as a white solid. Mp: 130.4-131.8 °C. 1H NMR (CDCl3): δ
1.67-1.79 (m, 2H), 1.92-2.13 (m, 2H), 2.16-2.32 (m, 2H),
2.98-3.54 (m, 8H), 4.18 (br s, OH), 7.03-7.58 (m, 7H), 7.77
(d, 2H, J ) 7.0 Hz). MS ESI m/z: 358.2 (M + H)+. Anal.
(C21H24ClNO2‚0.24CH2(CH2)4CH3) C, H, N.
4-(4-Chlorophenyl)-1-(4-hydroxy-4-phenylbutyl)piper-
idin-4-ol (32). (i) 33 (3.94 g, 21.64 mmol) was dissolved in
EtOH (25 mL), and NaBH4 (0.42 g, 11.10 mmol) was added in
small portions at 0 °C. The reaction mixture was allowed to
warm to room temperature and stirred for 1 h. After the
addition of water (25 mL), the reaction mixture was extracted
with Et2O (3 × 25 mL). The combined organic layers were dried
over anhydrous MgSO4, filtered, and evaporated in vacuo to
give 3.69 g (93%) of 36 as colorless oil, which was used without
further purification.
5-(4-(Diphenylmethylene)piperidin-1-yl)-2,2-diphenyl-
pentanenitrile Hydrochloride (15). Following method B
using 22b (0.92 g, 2.91 mmol) and 4-(diphenylmethylene)-
piperidine27 (0.72 g, 2.86 mmol) followed by purification by
flash chromatography (0-25% EtOAc in DCM) gave a thick
oil. This was dissolved in a solution of MeOH (5 mL), and
subsequently 6 N HCl (0.15 mL) and Et2O (20 mL) were added
dropwise while stirring. The hydrochloride salt was isolated
by filtration and recrystallized from Et2O/MeOH, giving 863
1
mg (58%) of 15 as a white solid. Mp: 106.8-108.5. H NMR
(CH3OH-d4): δ 1.80-1.99 (m, 2H), 2.54-2.75 (m, 6H), 3.20-
3.36 (m, 6H), 7.10-7.47 (m, 20H). MS ESI m/z: 483.9 (M +
H)+. Anal. (C35H36Cl2N2‚1.0H2O) C, H, N.
4-(4-Chlorophenyl)-1-(4-cyano-4,4-diphenylbutyl)-4-
hydroxy-1-methylpiperidinium Iodide (16). Following the
same procedure as described in the literature25 afforded 206
1
mg (70%) of 16 as a white solid. Mp: 215.8-216.9. H NMR
(CDCl3/DMSO): δ 1.70-2.00 (m, 4H), 2.06-2.31 (m, 2H),
2.35-2.60 (m, 2H), 3.18 (s, 3H), 3.30-3.50 (m, 2H), 3.52-3.90
(m, 4H), 4.94 (br s, OH), 7.12-7.43 (m, 14H). MS ESI m/z:
460.0 (M + H)+. Anal. (C27H32ClN2O) C, H, N.
5-(4-(4-Chlorophenyl)-3,6-dihydro-2H-pyridin-1-yl)-2,2-
diphenylpentanenitrile (18). Following method A using 22a
(0.81 g, 3.00 mmol) and 4-(4-chlorophenyl)-1,2,3,6-tetrahydro-
pyridine 27 (0.69 g, 3.00 mmol) afforded 400 mg (31%) of 18
1
as a yellow solid. Mp: 142.1-142.8 °C. H NMR (CDCl3): δ
1.56-1.78 (m, 2H), 2.41-2.62 (m, 8H), 3.06 (d, 2H, J ) 3.1
Hz), 6.01 (m, 1H), 7.22-7.42 (m, 14H). MS ESI m/z: 427.9 (M
+ H)+. Anal. (C28H27ClN2) C, H, N.
(ii) Et3N (8.5 mL, 61.2 mmol) and acetyl chloride (2.6 mL,
30.6 mmol) were added to a solution of 36 in Et2O (50 mL).
The reaction mixture was stirred at room temperature for 1
h, diluted with water (25 mL) and extracted with Et2O (3 ×
25 mL). The combined organic layers were washed with
aqueous K2CO3 (25 mL), dried over anhydrous MgSO4, and
evaporated in vacuo to give 2.51 g (51%) of 37 as brown oil,
which was used without further purification.
1-(4-Cyano-4,4-diphenylbutyl)piperidine-4-carboxyl-
ic Acid Ethyl Ester Hydrochloride (19). Following method
B using 22b (1.26 g, 4.01 mmol) and piperidine-4-carboxylic
acid ethyl ester 24 (0.79 g, 5.03 mmol) gave 1.65 g (84%) of 19
as an orange oil. 1H NMR (CDCl3): δ 1.25 (t, 3H, J ) 8.0 Hz),
1.45-2.00 (m, 8H), 2.15-2.50 (m, 5H), 2.65-2.80 (m, 2H), 4.10
(q, 2H, J ) 7.1), 7.20-7.45 (m, 10H). MS ESI m/z: 391.4 (M
+ H)+. Anal. (C25H30N2O2) C, H, N.
(iii) Following method B using 37 (0.68 g, 3.00 mmol) and
4-chlorophenyl-4-hydroxypiperidine 30 (0.70 g, 3.32 mmol)
gave 1.24 g of 38. This was dissolved in MeOH (25 mL), and
10% NaOH (5 mL) was added. The resulting reaction mixture
was refluxed for 30 min, evaporated in vacuo, and extracted
with DCM (3 × 25 mL). The combined organic layers were
dried over anhydrous MgSO4, filtered, and evaporated in
vacuo. The residue was purified by flash chromatography (0-
10% MeOH in EtOAc) to give 449 mg (41%) of 32 as a white
solid. Mp: 144.4-145.8 °C. 1H NMR (CDCl3): δ 1.51-2.07 (m,
8H), 2.09-2.31 (m, 2H), 2.40-2.69 (m, 4H), 2.74-2.90 (m, 1H),
2.92-3.10 (m, 1H), 4.59-4.71 (m, 1H), 7.10-7.49 (m 9H). MS
ESI m/z: 360.9 (M + H)+. Anal. (C21H26ClN2O2) C, H, N.
4-(4-Chlorophenyl)-1-(4,4-diphenylbutyl)piperidin-4-
ol (39). Following method B using 4-chloro-1,1-diphenylbutane
(0.82 g, 3.16 mmol), which was synthesized according to
literature procedure,31 and 4-chlorophenyl-4-hydroxypiperidine
30 (0.52 g, 2.34 mmol) gave 329 mg (32%) of 39 as a yellow
solid. Mp: 110.1-110.8 °C. 1H NMR (CDCl3): δ 1.39-1.79 (m,
5H), 2.00-2.19 (m, 4H), 2.22-2.49 (m, 4H), 2.67-2.83 (m, 2H),
3.89 (t, 1H, J ) 7.8 Hz), 7.14-7.43 (m, 14H). MS ESI m/z:
421.2 (M + H)+. Anal. (C27H30ClNO) C, H, N.
1-(4-Cyano-4,4-diphenylbutyl)piperidine-4-carboxyl-
ic Acid Hydrochloride (20). A solution of 19 (1.41 g, 3.61
mmol) and 2 M NaOH (5 mL) in MeOH (30 mL) was refluxed
for 3 h. The reaction mixture was allowed to cool to room
temperature, evaporated in vacuo, diluted with water (30 mL),
and extracted with Et2O (1 × 30 mL). The water layer was
acidified with 2 N HCl, and 1.23 g (86%) of 20 was isolated by
filtration as a white solid. Mp: 231.0-233.1 °C. 1H NMR
(DMSO/D2O): δ 1.40-1.80 (m, 4H), 1.75-2.15 (m, 2H), 2.40-
2.65 (m, 3H), 2.70-2.94 (m, 2H), 2.96-3.18 (m, 2H), 3.27-
3.39 (m, 2H), 7.29-7.54 (m, 10H). MS ESI m/z: 363.8 (M +
H)+. Anal. (C23H27ClN2O2‚0.41H2O) C, H, N.
4-(4-Chlorophenyl)-1-(3-phenoxypropyl)piperidin-4-
ol (26). Following method B using 28 (0.65 g, 3.00 mmol) and
4-chlorophenyl-4-hydroxypiperidine 30 (0.42 g, 2.00 mmol)
gave a mixture of 26 and the quaternary product. The
quaternary product was separated from 26 by fractional
crystallization in CHCl3. The filtrate was concentrated under
reduced pressure and recrystallized from EtOAc to give 203
mg (29%) of 26 as white needles. Mp: 125.8-127.3 °C. 1H
NMR (CDCl3/DMSO): δ 1.61-1.82 (m, 3H), 1.88-2.19 (m, 4H),
2.32-2.65 (m, 4H), 2.75-2.90 (m, 2H), 4.03 (t, 2H, J ) 6.0
Hz), 6.80-6.95 (m, 3H), 7.15-7.34 (m, 4H), 7.36-7.45 (m, 2H).
MS ESI m/z: 347.6 (M + H)+. Anal. (C20H24ClNO2) C, H, N.
4-(4-Chlorophenyl)-1-(4-(4-methoxyphenyl)-4-phenyl-
butyl)piperidin-4-ol (40). (i) Following method D (step i)

Nonpeptidergic Inverse Agonist
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6469
starting with 4-methoxybenzophenone (5.75 g, 27.1 mmol) gave
3.18 g (59%) of 4-methoxydiphenylmethane as a colorless oil.
1H NMR (CDCl3): δ 3.87 (s, 3H), 4.06 (s, 2H), 6.97 (d, 2H, J )
8.5 Hz), 7.22-7.41 (m, 7H).
4-(4-Chlorophenyl)-1-(4-(4-chlorophenyl)-4-phenylbu-
tyl)piperidin-4-ol (42). Following method D using 4-chlo-
robenzophenone gave 256 mg (5% overall yield) of 42 as white
crystals. Mp: 126.2-127.0 °C. 1H NMR (CDCl3): δ 1.46-1.53
(m, 2H), 1.64-1.69 (m, 3H), 1.97-2.12 (m, 4H), 2.27-2.43 (m,
4H), 2.68-2.74 (m, 2H), 3.86 (t, 1H, J ) 7.8 Hz), 7.12-7.43
(m, 13H). MS ESI m/z: 454.4 (M + H)+. Anal. (C27H29Cl2NO)
C, H, N, Cl.
1-(4,4-Bis(4-chlorophenyl)butyl)-4-(4-chlorophenyl)pi-
peridin-4-ol Hydrochloride (43). Following method D using
4,4′-dichlorobenzophenone gave 291 mg (5% overall yield) of
thick oil. The compound was converted to the hydrochloride
salt as described for 3. Recrystallization from Et2O/MeOH gave
302 mg (97%) of 43 as a white solid. Mp: 185.0-185.5 °C. 1H
NMR (CDCl3): δ 1.80-1.86 (m, 5H), 2.00-2.11 (m, 2H), 2.70-
2.87 (m, 4H), 3.09-3.22 (m, 4H), 3.85 (t, 1H, J ) 7.5 Hz), 7.09
(d, 4H, J ) 8.47 Hz), 7.20-7.26 (m, 6H), 7.39 (d, J ) 8.6 Hz,
2H), 11.83 (br s, 1H). MS ESI m/z: 488.4 (M + H)+. Anal.
(C27H29Cl4NO) C, H, N, Cl.
4-(4-Chlorophenyl)-1-(4-phenyl-4-m-tolylbutyl)piperi-
din-4-ol Hydrochloride (45). Following method D using
3-methylbenzophenone gave 294 mg (15% overall yield) of
thick oil. The compound was converted to the hydrochloride
salt as described for 3. Recrystallization from Et2O/MeOH gave
299 mg (93%) of 45 as a white solid. Mp: 143.1-144.4 °C. 1H
NMR (CDCl3): δ 1.78-1.84 (m, 4H), 2.01-2.13 (m, 3H), 2.29
(s, 3H), 2.80-2.91 (m, 4H), 3.09-3.29 (m, 4H), 3.88 (t, 1H, J
) 7.9 Hz), 7.00-7.33 (m 11H), 7.41 (d, 2H, J ) 8.5 Hz), 12.15
(br s, 1H). MS ESI m/z: 434.4 (M + H)+. Anal. (C28H33Cl2NO‚
0.17H2O) C, H, N, Cl.
(ii) A solution of potassium tert-butoxide (0.90 g, 8.0 mmol)
in dry THF (20 mL) was added to a solution of n-BuLi (5.00
mL, 1.6 M in hexane, 8.0 mmol) in a dry atmosphere at -100
°C. The reaction mixture was stirred for 5 min, and a solution
of 4-methoxydiphenylmethane (1.58 g, 7.97 mmol) in dry THF
(15 mL) was added slowly in a period of 15 min. The solution
was stirred for 1 h at -95 °C, and 1,3-dibromopropane was
added in one portion (4.0 mL, 39.4 mmol). The reaction mixture
was allowed to warm to room temperature and was stirred
for 1.5 h. The solvent was evaporated under reduced pressure,
and the residue was diluted with water (25 mL) and extracted
with EtOAc (3 × 25 mL). The combined organic layers were
washed with water (3 × 25 mL) and brine (25 mL), dried over
anhydrous MgSO4, filtered, and evaporated under reduced
pressure (1 mmHg) to afford bromide 50 (R1 ) 4-OCH3; R2 )
H). The crude product was used without further purification.
(iii) Bromide 50 (R1 ) 4-OCH3; R2 ) H) was dissolved in
CH3CN, and following method B, purification by flash chro-
matography (50% DCM in EtOAc) and recrystallization from
EtOAc gave 548 mg (15%) of 40 as white crystals. Mp: 119.7-
1
121.0 °C. H NMR (CDCl3): δ 1.47-1.69 (m, 5H), 2.00-2.11
(m, 4H), 2.26-2.43 (m, 4H), 2.69-2.75 (m, 2H), 3.74 (s, 3H),
3.84 (t, 1H, J ) 7.8 Hz), 6.79 (d, 2H, J ) 8.7 Hz), 7.11-7.37
(m, 9H), 7.41 (d, 2H, J ) 8.7 Hz). MS ESI m/z: 450.4 (M +
H)+. Anal. (C28H32ClNO2) C, H, N, Cl.
4-(4-Chlorophenyl)-1-(4-(3,4-dichlorophenyl)-4-phenyl-
butyl)piperidin-4-ol Hydrochloride (41). (i) A solution of
AlCl3 (7.36 g, 55.2 mmol) and tert-butylamineborane (9.95 g,
111 mmol) in DCM (150 mL) was stirred for 10 min at 0 °C,
and a solution of 3,4-dichlorobenzophenone (4.64 g, 18.4 mmol)
in DCM (15 mL) was added. The mixture was stirred for 2 h
at 0 °C and overnight at room temperature. A cooled solution
of 0.1 N HCl (75 mL) at 0 °C was added carefully followed by
extraction with EtOAc (100 mL). The combined organic layers
were washed with 0.1 N HCl (2 × 75 mL) and brine (100 mL),
dried over anhydrous MgSO4, filtered, and evaporated under
reduced pressure. The residue was purified by flash chroma-
tography (hexane) to afford 3.43 g (78%) of 3,4-dichlo-
romethane as a colorless oil. 1H NMR (CDCl3): δ 3.93 (s, 2H),
6.99-7.68 (m, 8H).
(ii) A solution of diisopropylamine (1.22 mL, 8.68 mmol) in
dry THF (5 mL) was added to a solution of n-BuLi (5.43 mL,
1.6 M in hexane, 8.69 mmol) and stirred for 5 min at -10 °C.
The reaction mixture was cooled to -78 °C, and a solution of
3,4-dichlorodiphenylmethane (2.07 g, 8.68 mmol) in dry THF
(10 mL) was added slowly in a period of 15 min. The mixture
was stirred for 1 h at -10 °C followed by the addition of HMPA
(1.5 mL) and 1,3-dibromopropane (4.40 mL, 26.1 mmol) in one
portion at -78 °C. The reaction mixture was allowed to warm
to room temperature and was stirred for 1 h, and the reaction
was quenched with water (25 mL). The solvent was evapo-
rated, and the residue was diluted with water (25 mL) and
extracted with EtOAc (3 × 25 mL). The combined organic
layers were washed with water (3 × 25 mL) and brine (25 mL),
dried over anhydrous MgSO4, filtered, and evaporated under
reduced pressure. The residue was purified by flash chroma-
tography (hexane) to afford 4-bromo-1-(3,4-dichlorophenyl)-1-
phenylbutane 18 (R1 ) 3,4-Cl2; R2 ) H) as a colorless oil (0.15
g, 5%). 1H NMR (CDCl3): δ 1.65-1.79 (m, 2H), 2.03-2.14 (m,
2H), 3.32 (t, 2H, J ) 6.5 Hz), 3.78 (t, 1H, J ) 7.5 Hz), 6.96-
7.26 (m, 8H).
4-(4-Chlorophenyl)-1-(4-phenyl-4-p-tolylbutyl)piperi-
din-4-ol Hydrochloride (46). Following method D using
4-methylbenzophenone gave 468 g (19% overall yield) of thick
oil. The compound was converted to the hydrochloride salt as
described for 3. One batch was recrystallized from Et2O/MeOH
to give 304 mg (60%) of 46 as a white solid. Mp: 174.3-176.0
°C. 1H NMR (CDCl3): δ 1.78-1.84 (m, 4H), 1.99-2.12 (m, 2H),
2.26 (s, 3H), 2.69-2.84 (m, 5H), 3.09-3.18 (m, 4H), 3.84 (t,
1H, J ) 7.9 Hz), 7.07-7.34 (m, 11H), 7.39 (d, 2H, J ) 8.6 Hz),
11.81 (br s, 1H). MS ESI m/z: 434.4 (M + H)+. Anal. (C28H32-
Cl2NO‚0.19H2O) C, H, N, Cl.
1-(4-Biphenyl-4-y)-4-(4-chlorophenyl)butyl]-4-(4-chlo-
rophenyl)piperidin-4-ol (47). Following method D using
4-chloro-4′-phenylbenzophenone gave the final compound as
a white solid. Recrystallization from EtOAc gave 264 mg (9%
overall yield) of 47 as white crystals. Mp: 127.8-128.6 °C. 1H
NMR (CDCl3): δ 1.50-1.70 (m, 5H), 2.00-2.13 (m, 4H), 2.30-
2.46 (m, 4H), 2.72-2.77 (m, 2H), 3.91 (t, 1H, J ) 7.7 Hz), 7.16-
7.55 (m, 17H). MS ESI m/z: 530.5 (M + H)+. Anal. (C33H33-
Cl2NO) C, H, N, Cl.
Pharmacology. Materials. ATP disodium salt, bovine
serum albumin, chloroquine diphosphate, and DEAE-dextran
(chloride form) were obtained from Sigma. Cell culture media,
penicillin, and streptomycin were obtained from Life Technolo-
gies, Inc., and fetal calf serum was purchased from Integro
B.V. (Dieren, The Netherlands). myo-[2-3H]Inositol (17 Ci/
mmol) was obtained from Perkin-Elmer Life Sciences, and
Sephadex G-25 gel filtration columns were purchased from
ICN Pharmaceuticals Inc. (Costa Mesa, CA). The human
chemokine CCL5 (regulated on activation, normal T cell
expressed, and secreted) was obtained from Peprotech (Rocky
Hill, NJ).
Cell Culture and Transfection. COS-7 cells were grown
at 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s medium
supplemented with 5% fetal calf serum, 2 mM L-glutamine,
50 IU/mL penicillin, and 50 µg/mL streptomycin. Transfection
of the COS-7 cells was performed by DEAE-dextran using 2
µg of DNA of each US28 construct pcDEF3-US28 or empty
factor per million cells.17 The total amount of DNA in trans-
fected cells was maintained constant by addition of the empty
vector.
(iii) Following method B gave 132 mg (66%) of thick oil. The
compound was converted to the hydrochloride salt as described
for 3. Recrystallization from Et2O/MeOH gave 124 mg (87%)
of 41 as a white solid. 1H NMR (CDCl3): δ 1.80-1.86 (m, 4H),
2.01-2.16 (m, 3H), 2.80-3.01 (m, 4H), 3.08-3.46 (m, 4H), 3.87
(t, 1H, J ) 7.8 Hz), 7.08-7.46 (m, 12H), 12.23 (br s, 1H). MS
ESI m/z: 488.4 (M + H)+. Anal. (C27H29Cl4NO‚1.0H2O) C, H,
N, Cl.
[
125I]Chemokine Binding Study. Labeling of CCL5 with
125I and binding in COS-7 cells were performed as previously
described.20 Briefly, transfected cells were seeded in 24-well

6470 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Hulshof et al.
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plates; 48 h after transfection, binding was performed on whole
cells for 3 h at 4 °C using 0.3 nM [125I]CCL5 in binding buffer
(50 mM Hepes, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, and 0.5%
bovine serum albumin) in the presence or absence of varying
concentrations of compounds. After incubation, cells were
washed four times at 4 °C with binding buffer supplemented
with 0.5 M NaCl. Nonspecific binding was determined in the
presence of 0.1 µM cold competitor (CCL5).
[3H]Inositol Phoshate Production. Cells were seeded in
24-well plates, and 24 h after transfection they were labeled
overnight in inositol-free medium (modified Eagle’s medium
with Earle’s salts) supplemented with 2 mM L-glutamine,
L-cysteine, L-leucine, L-methionine, L-arginine, glucose, 0.2%
bovine serum albumin, and 2 µCi/ml myo-[2-3H]inositol. Sub-
sequently, the labeling medium was aspirated, cells were
washed for 10 min with Dulbecco’s modified Eagle’s medium
containing 25 mM HEPES (pH 7.4) and 20 mM LiCl and
incubated for 2 h in the same medium in the absence or
presence of varying concentrations of compounds. The incuba-
tion was stopped by aspiration of the medium and addition of
cold 10 mM formic acid. After 90 min of incubation on ice,
inositol phosphates were isolated by anion exchange chroma-
tography (Dowex AG1-X8 columns, Bio-Rad) and counted by
liquid scintillation.
Acknowledgment. J. W. Hulshof was supported by
the Technology Foundation STW. P. Casarosa was
supported by Altana Nederland B.V. (Zwanenburg, The
Netherlands), and M. J. Smit was supported by the
Royal Netherlands Academy of Arts and Sciences.
Supporting Information Available: Results from el-
emental analyses of all target compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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