Identification of novel allosteric nonpeptidergic inhibitors of the human cytomegalovirus-encoded chemokine receptor US28

Article abstract of DOI:10.1016/j.bmc.2009.11.060

Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory properties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated

Full text of DOI:10.1016/j.bmc.2009.11.060

Bioorganic & Medicinal Chemistry 18 (2010) 675–688
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of novel allosteric nonpeptidergic inhibitors of the human
cytomegalovirus-encoded chemokine receptor US28
,
*
Henry F. Vischer , Janneke W. Hulshof , Saskia Hulscher, Silvina A. Fratantoni, Mark H. P. Verheij,
Jane Victorina, Martine J. Smit, Iwan J. P. de Esch, Rob Leurs
Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV, Amsterdam,
The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory proper-
ties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad
spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic
chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit
chemokine binding to US28.
Received 21 September 2009
Revised 25 November 2009
Accepted 28 November 2009
Available online 6 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
HCMV
Viral GPCR
Nonpeptidergic
Constitutive activity
Chemokine
Inverse agonist
Herpesvirus
1. Introduction
signaling induces a transformed and pro-angiogenic phenotype in
NIH-3T3 cells, and promotes tumorigenesis in a nude mice model.8
Human cytomegalovirus (HCMV) is an infectious b-herpesvirus
that establishes a lifelong latent infection in the majority of the
human population and (ab)uses human cells for survival and prop-
agation. Occasional reactivation and shedding of new infectious
virions allow viral dissemination to other individuals. In order to
establish viral latency and widespread propagation, HCMV has to
hijack infected host cells and evade anti-viral immune responses.
To this end, HCMV encodes important regulatory proteins that
are expressed by infected host cells. One of these virally-encoded
genes is the G protein-coupled receptor US28. US28 binds a broad
variety of inflammatory chemokines allowing cell migration along
chemokine gradients. In addition, recruitment of leukocytes to the
microenvironment of HCMV-infected cells is impaired as inflam-
matory chemokines are depleted by constitutive and rapid US28-
mediated internalization.1–3 Although latent infection is generally
asymptomatic, HCMV has been associated with several chronic
inflammatory diseases and cancer.4 Importantly, US28 activates
in a ligand-independent manner proliferative and inflammatory
signal transduction pathways.5–7 This US28-mediated constitutive
Identification of small nonpeptidergic ligands is essential to
investigate the role of US28 in evasion of the immune system and
HCMV-associated cancers. Moreover, such molecules might be con-
sidered as promising therapeutics for anti-viral intervention. Previ-
ously, we identified VUF2274 (1) [5-(4-(4-chlorophenyl)-4-
hydroxy-piperidin-1-yl)-2,2-diphenylpentanenitrile] as a nonpept-
idergic allosteric inverse agonist for US28 (Fig. 1a).9 Compound 1
inhibited US28-mediated constitutive signaling, chemokine binding,
and US28-mediated human immunodeficiency virus (HIV) co-recep-
tor activity with a low l
M potency (EC50 and IC50, respectively).9 In
subsequent lead optimization programs, approximately 100 new
analogues were synthesized and pharmacologically character-
ized.10,11 These structure–activity relationship (SAR) studies re-
vealed the importance of the 4-phenylpiperidine moiety. However,
thepotencyofthisclassofcompoundscouldnotbeimprovedbeyond
the low lM range. In addition to these 4-substituted piperidine der-
ivates (e.g., 1 and 2), series of piperazinyldibenzothiepine (e.g., 3, 4),
cinchonidine derivates (e.g., 5) and benzamides like 6 have been de-
scribed in the patent literature as inhibitors of chemokine binding to
US28 with IC50 values in the 0.3–1 l
M range (Fig. 1a).12–14
In order to identify novel chemotypes that can inhibit constitu-
tive US28-mediated signaling and/or chemokine binding that
might be used as new starting point for lead optimization, we
screened a selection of compounds from our in-house collection.
* Corresponding author. Tel.: +31 0 205987588; fax: +31 0 205987610.
E-mail address: vischer@few.vu.nl (H.F. Vischer).
Both authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.060

676
H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
Figure 1. (A) Chemical structures of previously described nonpeptidergic ligands for US28. (B) Screening results using 10 lM nonpeptidergic ligand. Data are represented as
percentage CCL5 binding (X-axis) and constitutive US28-mediated InsP production (Y-axis) in the absence of small ligands. The average of at least three independent
experiments are shown. For clarity no error bars (<5%) are shown. Previously described compounds 1–6 are indicated and depicted in black, whereas most significant hits of
this screen are indicated 7, 36, and 41.
O
O
a
Br
b
N
OH
OH
OH
HN
Cl
Cl
48
49
50
Scheme 1. Synthetic pathway for the synthesis of compound 50. Reagents and conditions: (a) p-TSA, 2-bromoethanol, toluene, reflux; (b) NaI, Na2CO3, CH3CN, microwave
(15 min, 160 °C).
Compounds (7–47) were selected based on (sub)structural similar-
ity to known ligands acting on US28 (Fig. 1a). Subsequently, the
structure–activity relationships (SAR) around three promising hits
(i.e., (7), (36), (41)) were explored by synthesizing structural ana-
logues and subsequent pharmacological characterization.
3.2. Synthesis of analogues of hit 36
Reduction of 11,12-dihydrodibenzo[b,f]azocin-6(5H)-one 52
with LiAlH4 resulted in 5,6,11,12-tetrahydrodibenzo[b,f]azocine
53,16 which was alkylated with 1-bromo-3-chloropropane in the
microwave (15 min, 200 °C) to yield intermediate 54. N-alkylation
of piperidine moieties 55, 49 and 56 with chloride 54 afforded inter-
2. Compound selection criteria
Table 1
For this screening campaign, compounds 7–47 were selected
from our in-house compound collection that share (sub)structural
similarity to reported US28 ligands. To this end, substructure sim-
ilarity searches were performed using ChemFinder Std. 7.0 (Cam-
bridgeSoft Corporation). In addition, compounds were selected
based on subjective structural similarity criteria.
Chemical structures and pharmacological properties on US28 of compounds 1, 7 and
50–51
R2
R1
X
N
OH
3. Chemistry
Cl
3.1. Synthesis of analogues of hit 7
R2
Bromide intermediate 48, which was synthesized in a quantita-
tive yield by an acid-catalysed condensation of diphenylmethanol
with 2-bromoethanol with azeotropic removal of water,15 was re-
acted with 4-(4-chlorophenyl)piperidin-4-ol 49 in the microwave
in the presence of NaI, Na2CO3 and CH3CN to give compound 50
(Scheme 1). Compound 51 (Table 1) was synthesized in an analo-
gous manner by the alkylation of the commercially available 4,40-
(4-chlorobutane-1,1-diyl)bis(fluorobenzene) with piperidine 49.
No.
R1
R2
X
IC50
(
l
M)
EC50 (lM)
1
7
50
51
CN
H
H
H
F
H
F
CH2
O
O
4.9 (4.4–5.5)
2.3 (1.5–3.2)
2.1 (1.8–2.4)
3.0 (2.5–3.5)
6.3 (5.9–6.8)
4.6 (2.5–4.0)
3.5 (1.7–5.4)
5.2 (3.2–7.2)
H
CH2
The IC50 and EC50 values (mean and interval) were generated in radioligand binding
experiments using 125I-CCL5 and InsP accumulation experiments, respectively. All
experiments were performed in triplicate and repeated at least three times.

H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
677
O
Cl
a
NH
NH
N
b
52
53
54
R
HN
Cl
c
c
HN
49 R = OH
55 R = CN
Cl
OH
56
Cl
OH
N
N
N
N
R
Cl
60
57 R = OH
58 R = CN
d
59 R = CH2NH2
Scheme 2. Synthetic pathway for the synthesis of 57, 59 and 60. Reagents and conditions: (a) LiAlH4, THF, reflux; (b) 1-bromo-3-chloropropane, Na2CO3, microwave (15 min,
200 °C); (c) NaI, Na2CO3, CH3CN, microwave (15 min, 160 °C) or NaI, Na2CO3, CH3CN, reflux; (d) AlCl3, LiAlH4, THF.
mediate 58, and target compounds 57 and 60, respectively (Scheme
2). Piperidine moiety 55 was synthesized starting from bis(2-chloro-
ethyl)amine hydrochloride as previously described in the litera-
ture.11,17 The 3-substituted piperidine moiety 56 was synthesized
by a Grignard reaction of BOC-protected piperidin-3-one with 4-
chlorophenylmagnesiumbromidefollowedbyadeprotectionunder
acidicconditions.18 Thenitrilegroupof intermediate58was reduced
in the presence of AlCl3 and LiAlH4 to yield target compound 59.
5) to give the substituted 2-(phenylmethylene)-2,3-dihydro-1H-
inden-1-ones 82.22 Reduction of both the carbonyl group as well
as the double bond in one pot with lithium aluminium hydride
was not successful. Therefore, hydrogenation of the double bond
and subsequent reduction of the carbonyl group with sodium boro-
hydride afforded the substituted 2-benzyl-2,3-dihydro-1H-inden-
1-ols 83 as 1:1 mixture of both cis/trans isomers in moderate to
high yields. Intermediates 83 were converted to the corresponding
1-azido-2-benzyl-2,3-dihydro-1H-indenes 84 in the presence of di-
phenyl phosphorazidate (DPPA) and 2,3,4,6,7,8,9,10-octahydropy-
rimidol[1,2-a]azepine (DBU) in THF.23,24 For some of the azide
intermediates it was possible to separate the two diastereomers
via column purification. Hydrogenation of the azide group with
palladium on activated carbon afforded target compounds 41 and
85–91. The separation of the two diastereomers could not be
accomplished after the last step of the synthesis route. Treatment
of 2-benzyl-2,3-dihydro-1H-inden-1-ol derivatives 83 with thionyl
chloride gave a 1:1 mixture of the two diastereomers of 1-chloro-
2-(2,4-dichlorobenzyl)-2,3-dihydro-1H-indenes 92 in a quantita-
tive yield. Alkylation of chloro derivatives 92 with commercially
available amines in the presence of NaI, Na2CO3, and CH3CN at re-
flux temperature or in the microwave, resulted in mainly elimina-
tion of HCl.11 Therefore, the synthesis was performed in a different
manner by heating the corresponding amine in CH3CN to reflux
temperature in the presence of Na2CO3 and NaI and subsequent
addition of the appropriate chloride 92 when reflux temperature
was reached. This resulted in the successful synthesis of target
compounds 93–98 in moderate to low yields. This synthetic route
resulted in the formation of only one diastereomer of the desired
compounds.
3.3. Synthesis of analogues of compound 57
Unsubstituted amine 65 was commercially available, and amines
66 and 67 were synthesized in a quantitative yield via a reductive
amination reaction with the corresponding anilines 61–62 and
benzaldehydes 63–64 in the presence of sodium triacetoxyborohy-
dride and acetic acid in DCE. The amines 65–67 were deprotonated
with NaNH2 in toluene at reflux temperature followed by an alkyl-
ation with 1-bromo-3-chloropropane to give intermediates 68–70,
which were reacted with piperidine 49 in the presence of NaI,
Na2CO3, and CH3CN (Scheme 3) to give compounds 71, 72 and 73.
3.4. Synthesis of compound 80
Rigid tricyclic analogue 80 was synthesized as depicted in
Scheme 4. The reaction of 2-(2-iodophenyl)acetic acid 74 with 4-
(methylthio)benzenethiol 75 in the presence of KOH, Cu and water
gave intermediate 76.19 A subsequent ring closure to the tricyclic
system of 77 was performed by a Friedel–Crafts acylation with
PPA in toluene.20 Reduction of the carbonyl group of 77 with
NaBH4 in MeOH resulted in alcohol 78, which was reacted with
BF3ꢀ(Et)2O and 2-bromoethanol in toluene to give bromide 79.21
N-Alkylation of piperidine 49 with 79 yielded target compound 80.
4. Results and discussion
3.5. Synthesis of analogues of hit 41
To identify new chemotypes that inhibit chemokine binding
to US28 and/or display inverse agonism, 198 compounds were
selected on the merit of (sub)structural similarities to known
Indanones 81 were treated with the appropriate substituted
benzaldehydes in a saturated solution of K2CO3 in EtOH (Scheme
US28 ligands. Efficacy of these compounds (10 lM) was deter-

678
H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
R1
R1
N
Cl
NH
R2
a
b, c
R1
NH2
+
O
R1 = Cl
R
1 = H
1 = Cl
61
62
63
64
R2
R2
R
1 = H
R
R1 = H; R2 = H
R1 = Cl; R2 = H
68
69
70
R
1 = H; R2 = H
1 = Cl; R2 = H
65
66
67
R
R
1 = H; R2 = Cl
R1 = H; R2 = Cl
d
R1
N
N
OH
R2
Cl
R
1 = H; R2 = H
71
72
73
R
1 = Cl; R2 = H
R1 = H; R2 = Cl
Scheme 3. Synthetic pathway for the synthesis of 71–73. Reagents and conditions: (a) NaBH(OAc)3, CH3COOH, DCE; (b) NaNH2, toluene, reflux; (c) 1-bromo-3-chloropropane,
reflux; (d) 49, NaI, Na2CO3, CH3CN, microwave (15 min, 160 °C).
S
COOH
S
S
a
b
+
HOOC
S
S
I
O
SH
74
77
75
76
c
S
S
S
S
S
O
N
O
OH
e
d
S
OH
78
Br
Cl
79
80
Scheme 4. Synthetic pathway for the synthesis of 80. Reagents and conditions: (a) Cu, KOH, water, reflux; (b) PPA, toluene, reflux; (c) NaBH4, MeOH; (d) BF3ꢀ(Et)2O, 2-
bromoethanol, toluene; (e) 49, NaI, Na2CO3, CH3CN, reflux.
mined by measuring their effect on the US28-mediated constitu-
tive production of inositol phosphates. Their potency to alloster-
ically inhibit chemokine binding to US28 was analyzed by means
oligand competition curves and dose–response curves,
respectively.
of 125I-CCL5 radioligand binding assays in the presence of 10
lM
4.1. Structural analogues of VUF2274 (1)
of these compounds (Fig. 1b). Compounds that were identified as
allosteric inhibitors of 125I-CCL5 binding acted either as antago-
nists or inverse agonists when considering US28-mediated con-
stitutive signaling. On the other hand, inverse agonistic
compounds inhibited 125I-CCL5 binding as well. Typically, no in-
verse agonists were identified that did not affect 125I-CCL5 bind-
ing (i.e., lower left half of Fig. 1b). Several compounds were
identified as novel allosteric inhibitors of US28, and those that
inhibited CCL5 binding more than 50% were analyzed more
extensively by determining their IC50 and EC50 values from radi-
Screening our in-house compound collection yielded the
VUF2274 (1)-like compound (7) that displays a slightly higher po-
tency to inhibit constitutive US28 signaling and CCL5 binding as
compared with compound 1 (Fig. 1b and Table 1). To evaluate
the importance of the fluoro atoms in the phenyl rings and the
ether linkage in the alkyl chain between the diphenyl group and
the piperidine moiety of 7, compounds 50 and 51 were synthesized
(Scheme 1). Removal of both fluoro atoms (i.e., 50) or substitution
of the oxygen atom with CH2 (i.e., 51) did not significantly affect

H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
679
R3
R4
N
Cl
Cl
Cl
Cl
Cl
g
93-98
f
O
O
OH
b, c
a
R1
R1
R1
R2
O
R2
81
R2
82
d
NH2
N1
R1
e
R1
R2
84
R2
41, 85-91
Scheme 5. Synthetic pathway for the synthesis of 41, 85–91 and 93–98 (R1–R4 are listed in Tables 8 and 9). Reagents and conditions: (a) K2CO3, EtOH; (b) 10% Pd/C, H2,
MeOH; (c) NaBH4, MeOH; (d) DPPA, DBU, THF; (e) 10% Pd/C, H2, MeOH; (f) SOCfe, DCM; (g) amine, Nal, CH3CN, 90 °C.
the potency to inhibit chemokine binding and US28-mediated sig-
naling as compared to 7 (Table 1).
phenylbutyl group (i.e., compound 17) restores the potency to in-
hibit CCL5 binding to US28 (Table 2). Subsequent replacement of
the phenyl group of 17 with an alcohol moiety (i.e., 18) diminishes
potency again. Compounds 16 and 17, but not 18, display weak in-
verse agonistic efficacy.
4.2. Structural analogues of methiothepin (3) and
octoclothepin (4)
The piperazinyldibenzothiepine derivates methiothepin (3) and
octoclothepin (4) inhibit CCL5 binding to US28 with a comparable
potency as previously described for the binding of CX3CL1 to US28
(Fig. 1b and Table 2).13 Compound 3 has a higher potency than the
previously described 4-phenylpiperidine scaffold-based com-
pounds (e.g., 1).9–11 Replacement of the thiomethylene group of
compound 3 by a chloro atom in 4 resulted in a reduced potency
to inhibit CCL5 binding which is comparable to that of 1. On the
other hand, both 3 and 4 act as partial inverse agonists on US28
(Fig. 1b) with slightly reduced potency as compared to 1 (Tables
1 and 2). The piperazinyldibenzodiazepine derivate clozapine (8),
an atypical antipsychotic drug that binds to a variety of bioaminer-
gic receptors, also diminished (ꢁ50%) CCL5 binding to US28 at a
4.3. Tropine derivates
Our screening campaign also identified tropine derivatives as a
new hits for US28. Compound B83 (19) inhibited CCL5 binding and
constitutive US28 activity with IC50 and EC50 values, respectively,
in the low
lM range (Table 3). Replacement of the octyl group at
the R position of 19 by a phenetyl group (i.e., compound 20), and
subsequent shortening of the alkyl linker between the basic nitro-
gen atom of the tropine ring and the phenyl ring (i.e., compound
21) did not significantly affect the inhibitory properties on CCL5
binding or constitutive signaling (Table 3). On the other hand,
small methyl and ethyl chains (i.e., compound 22 and 23, respec-
tively), N-hydroxypropionimidamide or ethyl-guanidine groups
(i.e., compound 24 and 25, respectively) are detrimental for their
interaction with US28. In compounds 26–28 an unsaturated bridge
between the two phenyl rings in the tricyclic moiety is introduced,
which in combination with short branched alkyl chains at the R po-
sition (i.e., compounds 26 and 27) possess no inhibitory properties
on US28. However, placing a cyclohexyl group at this R position
yielded an inhibitor (i.e., compound 28) with IC50 and EC50 values
concentration of 10 lM (Table 2). Elongation of the R2 group of 8
from methyl into ethyl (9), or butyl (10) resulted in IC50 values in
the low micromolar range (Table 2). However, compounds 8–10
displayed only weak inverse agonistic activities on US28-mediated
constitutive signaling. Substitution of the diazepine with an oxaze-
pine moiety decreases the potency to block CCL5 binding to US28,
whereas inverse agonistic activities were similar (11) or abolished
(12) (Table 2). Changing the chloro substituent from the 8- (11) to
the 2-position (13) of the dibenzo[b,f][1,4]oxazepine moiety atten-
uates both potency to block CCL5 binding and inverse agonistic
activity (Table 2). Substitution of the oxazepine moiety of com-
pounds 13 and 11 with a thiazepine moiety (compounds 14 and
15, respectively) resulted in neutral antagonists with an increased
potency to inhibit CCL5 binding (Table 2). Compounds 16–18 have
a high flexibility due to their open structure as compared with the
tricyclic moieties of compounds 3–4 and 8–15. Compound 16 dis-
plays a lower potency to block CCL5 binding compared to the dis-
tinct tricyclic compounds (i.e., 3, 4, 8, 11, 13, 14, 15). Interestingly,
in the low lM range (Table 3).
4.4. 1-Phenylpropan-2-amine derivates
Various N-substituted analogues of 1-phenylpropan-2-amine
(i.e., compounds 29–35) display low lM potencies to inhibit CCL5
binding and constitutive activity, even though their tricyclic moie-
ties are quite different with respect to rigidity and the conformation
of the two aromatic phenyl rings (Table 4).The two aromatic rings of
the tricyclic moiety of compound 29 are tilted out of plane due to the
presence of three carbon atoms between the two phenyl rings,
whereas the tricyclic ring system of 30 is nearly planar, as deter-
substitution of the methyl group at R2 position of 16 with a
x-

680
H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
Table 2
Chemical structures and pharmacological properties on US28 of compounds 3–4 and 8–18
R1
N
N
R2
Cl
Cl
N
N
O
S
S
HN
Cl
S
Cl
A
E
B
C
D
Cl
N
N
N
O
S
S
Cl
Cl
F
G
H
No.
R1
R2
IC50
(l
M)
EC50 (lM)
3
4
8
A
B
C
C
C
D
D
E
–CH3
–CH3
–CH3
–CH2CH3
–(CH2)3CH3
–CH3
–CH2CH3
–CH3
–CH3
–CH3
–CH3
–(CH2)4Ph
–(CH2)4OH
0.41 (0.37–0.45)
4.9 (1.6–8.1)
47%a
7.6 (4.5–10.7)
12.1 (5.6–18.6)
11%a
16%a
19%a
22%a
0%a
9
3.8 (2.6–5.1)
4.7 (3.2–6.2)
19%a
10
11
12
13
14
15
16
17
18
38%a
13%a
0%a
F
28%a
0%a
G
H
H
H
47%a
0%a
13%a
20%a
18%a
0%a
47%a
13%a
The IC50 and EC50 values (mean and interval) were generated in radioligand binding experiments using 125I-CCL5 and InsP accumulation experiments, respectively. All
experiments were performed in triplicate and repeated at least three times.
a
Inhibition of CCL5 binding and constitutive US28 signaling at a 10 lM compound concentration.
mined by conformational analysis using the molecular modeling
program MOE (molecular operating environment, version
2004.03). Saturation of the double bond of compound 30 (i.e., com-
pound 31), or introduction of an unsaturated bridge between the
two phenyl rings in compound 32 did not significantly affect their
potency to inhibit CCL5 binding or constitutive signaling (Table 4).
Interestingly, a thioxanthene tricyclic moiety in combination with
ing the amine nitrogen atom in the tricyclic moiety to an amide
nitrogen atom (i.e., compounds 38 and 39) resulted in a significant
reduced inhibition of CCL5 binding to US28 (Table 5). Hence, a ba-
sic nitrogen in the tricyclic moiety seemed to be essential for inter-
action of these antagonists with US28.
Previous SAR studies revealed that a 4-phenylpiperidine moiety
of lead compound 1 is essential for inverse efficacy at US28.10
Anticipating that this moiety would confer inverse efficacy to neu-
tral antagonist 36, we synthesized an hybrid compound 57 in
which the N-methylpiperazine group of 36 was substituted with
4-(4-chlorophenyl)piperidin-4-ol (Fig. 2 and Scheme 2). Indeed,
this hybrid compound 57 displayed inverse efficacy on US28-med-
a p-chloro substituent on the phenethyl group confers low lM po-
tency to block CCL5 binding, but weak inverse agonistic efficacy to
compound 33 (Table 4). Similar inhibitory potencies were observed
for compound 34 which consists of an acridine tricyclic ring system
in combination with a m-trifluoromethyl group on the phenethyl
group (Table 4). Replacing the tricyclic ring system in 34 with a
4,40-difluorophenyl group (i.e., compound 35) reduces the potency
to block CCL5 binding to US28 (Table 4).
iated constitutive signaling with an EC50 of 4.7 lM (Table 5). More-
over, compound 57 was sevenfold more potent in inhibiting CCL5
to binding than the two compounds (i.e., 1 and 36), from which
it originated (Table 5). In contrast to the previously described
VUF2274-like compound 2,11 substitution of the 4-hydroxy group
of compound 57 with a methylamine group (i.e., compound 59),
did not yield the anticipated increase in inhibitory potencies (Table
5). Introduction of a chiral center by moving the 4-hydroxy group
and the 4-chlorophenyl substituent to the 3-position of the piper-
idine ring (i.e., compound 60) did not change the pharmacological
properties as compared to compound 57 (Table 5).
4.5. Identification and conversion of US28 antagonists into
inverse agonists
The tricyclic imipramine analogue A194 (36) was identified in
this screen as the first allosteric neutral antagonist for US28 as it
blocks CCL5 binding to US28 with an IC50 value of 4.9 lM, but dis-
played no inverse efficacy (Fig. 1b and Table 5). Replacing the N-
methyl group in 36 with a 2-ethanol group in compound 37 re-
sulted in a slightly lower IC50 value (Table 5). In contrast, convert-
Next, flexible analogues of compound 57 were synthesized to
explore the significance of rigidity in the tricyclic moiety for the

H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
681
Table 3
Chemical structures and pharmacological properties on US28 of compounds 19–28
Table 4
Chemical structures and pharmacological properties on US28 of compounds 29–35
No.
Structure
IC50
(lM)
EC50
(lM)
X
O
R
N
29
4.2 (3.6–4.8)
39%a
NH
No.
X
R
IC50
(l
M)
EC50 (lM)
19
20
21
22
23
CH2–CH2
CH2–CH2
CH2–CH2
CH2–CH2
CH2–CH2
(CH2)7CH3
(CH2)2Ph
CH2Ph
CH3
CH2CH3
5.6 (3.4–7.8)
11.9 (8.7–15.1)
10.5 (8.3–12.6)
15%a
4.9 (3.7–6.2)
4.3 (3.5–5.1)
8.8 (5.9–11.7)
0%a
30
31
7.0 (5.9–8.1)
5.2 (4.4–6.0)
42%a
NH
7%a
0%a
N OH
NH2
NH
24
CH2–CH2
0%a
0%a
0%a
10.9 (6.0–15.8)
5.2 (3.2–7.1)
34%a
NH
25
CH2–CH2
NH
NH
NH
H2N
26
27
28
CH@CH
CH@CH
CH@CH
CH(CH3)2
(CH2)2CH3
Cyclohexyl
13%a
3%a
18%a
0%a
8.3 (6.8–9.8)
5.8 (3.3–8.3)
The IC50 and EC50 values (mean and interval) were generated in radioligand binding
experiments using 125I-CCL5 and InsP accumulation experiments, respectively. All
experiments were performed in triplicate and repeated at least three times.
Inhibition of CCL5 binding and constitutive US28 signaling at a 10
pound concentration.
32
3.7 (3.2–4.2)
a
lM com-
S
interaction with US28 (Scheme 3). Exclusion of the ethylene bridge
between the two phenyl rings as in open analogue 71 decreased
the potency to block CCL5 binding (Table 6). Subsequent introduc-
tion of a chloro substituent at the 4-position of one of the phenyl
rings revealed an interesting regioselectivity with compound 72
having a slightly higher potency to inhibit CCL5 binding than com-
pound 73. Not surprisingly, these modifications in the tricyclic
moiety did not significantly affect the inverse agonistic potencies
of compounds 71, 72, and 73 as compared to 57 (Table 6).
Challenged by the observed increase in potency of compound
57 to block CCL5 binding to US28 as compared to compounds 1
and 36, we decided to design and synthesize another hybrid com-
pound by fusing the 4-(4-chlorophenyl)piperidin-4-ol scaffold to
the tricyclic moiety of compound 3 (Scheme 4). Compound 3 had
the lowest IC50 value so far observed and we anticipated to gener-
ate a full inverse agonist with an increased potency to inhibit CCL5
binding. Unfortunately, this hybrid compound (80) displayed de-
creased potency to block CCL5 binding to US28 (Table 6).
33
34
2.6 (2.0–3.2)
5.5 (4.5–6.5)
21%a
Cl
34%a
N
F
NH
NH
CF3
35
39%a
38%a
CF3
F
The IC50 and EC50 values (mean and interval) were generated in radioligand binding
experiments using 125I-CCL5 and InsP accumulation experiments, respectively. All
experiments were performed in triplicate and repeated at least three times.
4.6. Structural analogues of S(ꢂ)-IBZM (6)
a
Inhibition of CCL5 binding and constitutive US28 signaling at a 10
pound concentration.
lM com-
Automated similarity searches of our in-house compound col-
lection using ChemFinder Std. 7.0 software did not yield any ana-
logues. However, inspired by the substructure of S(ꢂ)-IBZM (6)
several analogues were selected from our library by subjective vi-
sual inspection. Pharmacological analysis of these S(ꢂ)-IBZM-like
analogues revealed some interesting preliminary SAR. Compound
41 [2-(2,4-dichlorobenzyl)-2,3-dihydro-1H-inden-1-amine] was
seems to be essential as indicated by compound 45. Interestingly,
compounds 46 and 47, in which the amine substituent is replaced
with a bulky 2-methoxy-2-phenylethanamine group, displayed
similar IC50 and EC50 values as the smaller analogues 41 and 42
(Table 7). Replacement of the dichloro substituents (46) in the phe-
nyl ring with a t-butyl group (47) at position 4 did not alter the
pharmacological properties.
The structure of compound 41 provided some interesting op-
tions for further lead optimization. Firstly, compounds 85–91 with
various modifications in the 2,4-dichlorobenzyl moiety were de-
signed and synthesized to optimize the substitution pattern in
the benzyl ring (Scheme 5 and Table 8). Substitution of the electron
withdrawing chloro groups with electron donating methyl groups
in compound 85 resulted in a small decrease in the potency to in-
hibit CCL5 binding to US28, without affecting inverse efficacy. Sub-
sequent introduction of two methoxy groups at the ortho and para
identified as
a novel scaffold for inverse agonism on US28
(Fig. 1b and Table 7). Conversion of the cyclopentyl group of 41
into cyclohexyl (i.e., compound 42) or cycloheptyl (i.e., compound
43) did not affect the potency to inhibit CCL5 binding (Table 7).
However, compound 43 displayed significantly less inverse agonis-
tic potency as compared to compounds 41 and 42. Interestingly,
increasing the conformational freedom by replacing the rigid ring
structure with a more flexible propyl chain as in compound 44
did not significantly affect the pharmacological properties as com-
pared to compounds 41 and 42 (Table 7). However, the presence of
3,4-dichloro substituents on (at least one of) the phenyl rings

682
H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
Table 5
Table 6
Chemical structures and pharmacological properties on US28 of compounds 36–39
and 57, 59 and 60
Chemical structures and pharmacological properties on US28 of compounds 57, 71–
73 and 80
X
N
R
R
1
R2
N
Cl
No.
R1
R2
IC50
(
lM)
EC50 (lM)
No.
X
R
IC50
(l
M)
EC50 (lM)
N
36
CH2
4.9 (2.8–7.1)
1.6 (1.3–1.9)
>100
3% 15%
0%a
N
N
N
N
57
OH
0.7 (0.6–0.9)
4.1 (2.8–5.4)
4.7 (2.8–6.6)
37
38
39
57
CH2
OH
OH
C(@O)
C(@O)
CH2
0%a
N
N
N
N
71
72
OH
OH
3.0 (2.8–6.0)
0%a
N
>100
OH
0.7 (0.6–0.9)
4.7 (2.8–6.6)
N
Cl
Cl
H2N
1.8 (1.4–2.1)
5.0 (3.9–6.2)
59
60
CH2
CH2
1.6 (1.0–2.2)
0.9 (0.7–1.0)
7.6 (4.8–10.5)
3.9 (3.6–4.2)
N
N
Cl
N
Cl
Cl
OH
The IC50 and EC50 values (mean and interval) were generated in radioligand binding
experiments using 125I-CCL5 and InsP accumulation experiments, respectively. All
73
OH
5.7 (4.3–7.1)
5.5 (5.0–6.0)
experiments were performed in triplicate and repeated at least three times.
N
a
Inhibition of CCL5 binding and constitutive US28 signaling at a 10
pound concentration.
lM com-
S
O
position of the benzyl ring, significantly attenuated the potency of
compound 86 to inhibit CCL5 binding as well as constitutive US28
signaling. Although a 2-methoxy substitution is allowed at the
benzyl moiety (compound 87), the presence of a 4-methoxy group
in compounds 86 and 88 seemed to be detrimental for interaction
with US28. Interestingly, the potency to inhibit CCL5 binding and
inverse agonism was restored in compound 89 by introducing an
additional 3-methyl group in 4-methoxyphenyl ring (Table 8).
80
OH
12.4 (11.2–13.5)
7.1 (4.9–9.3)
S
The IC50 and EC50 values (mean and interval) were generated in radioligand binding
experiments using 125I-CCL5 and InsP accumulation experiments, respectively. All
experiments were performed in triplicate and repeated at least three times.
CN
N
OH
N
N
N
Cl
1
36
N
OH
Cl
N
57
Figure 2. Chemical structures of inverse agonist 1, neutral antagonist 36 and hybrid compound 57.

H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
683
Table 7
binding to US28, as well as US28-mediated constitutive signaling
(Table 9).
Chemical structures and pharmacological properties on US28 of compounds 40–47
No. Structure
IC50
(l
M)
EC50
(lM)
5. Conclusions
N
40
12.9 (5.4–20.4)
10%a
To identify novel nonpeptidergic ligands acting on the constitu-
tively active HCMV-encoded receptor US28, a selection of chemo-
types extracted from our in-house compound collection was
screened on their potency to inhibit CCL5 binding to US28 and con-
stitutive receptor signaling. Compounds 7 and 41 were identified
as novel inverse agonists acting on US28, whereas compound 36
was identified as a neutral antagonist. Combining the tricyclic
group of this neutral antagonist with the 4-(4-chlorophenyl)piper-
idin-4-ol moiety of lead inverse agonist 1, resulted in a hybrid
compound 57 displaying inverse efficacy and an increased potency
to block CCL5 binding. Hence, inverse efficacy can indeed be attrib-
uted to the 4-(4-chlorophenyl)piperidin-4-ol moiety as previously
indicated.10,11 SAR of compounds 7, 57 and 41 were further ex-
plored by generating series of structural analogues, however none
of the modifications resulted in improved inhibitory activities on
US28. The identification of potent nonpeptidergic inverse agonists
and antagonists will provide us with a tool to validate the signifi-
cance of US28 in HCMV-associated pathologies.
O
Cl
41
9.5 (6.8–12.3)
8.5 (6.8–10.2)
11.1 (8.3–13.8)
Cl
Cl
NH2
Cl
12.6 (10.0–
15.1)
42
43
NH2
Cl
11.8 (11.5–
12.0)
26%a
NH2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
44
45
7.5 (5.0 -10.0)
11.7 (9.3–14.1)
34%a
NH2
6. Experimental
5%a
6.1. General procedures
NH2
O
THF and DCM were freshly distilled from lithium aluminium hy-
dride. All reactions were performed under an atmosphere of dry
nitrogen. Microwave reactions were performed in a CEM Explorer
single mode MW reactor equipped with auto sampler. 1H NMR and
13C NMR spectra were recorded on a Bruker AC-200 (200 MHz) spec-
trometer unless otherwise stated. J.T. Baker silica gel was used for
Cl
46
9.2 (6.3–12.0)
10.2 (7.2–9.5)
NH2
flash chromatography. Melting points were measured on
a
MPA100 OptiMelt automated melting point system apparatus and
were uncorrected. HRMS mass spectra were recorded on a Finnigan
MAT 900 mass spectrometer. 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 chromatograph mass spectrometer. The analyses
were performed using the following conditions; condition I: a Xbrid-
ge(C18)5um column (100 mm ꢃ 4.9 mm) with 70% MeOH–30%
H2O–0.1%formicacid(MethodIa); 60% MeOH–40% H2O–0.1%formic
acid (Method Ib); 50% MeOH–50% H2O–0.1% formic acid (Method
Ic); 40% MeOH–60% H2O–0.1% formic acid (Method Id) or 30%
MeOH–70% H2O–0.1% formic acid (Method Ie). Flow rate = 1.0 mL/
min. Total run time 15 min unless otherwise stated. Condition II: a
Xbridge(C18)5um column (100 mm ꢃ 4.9 mm) with 50% CH3CN–
50% H2O–0.1% formic acid (Method IIa); 40% CH3CN–60% H2O–
0.1%formicacid(MethodIIb), 35%CH3CN–65%H2O–0.1%formicacid
(Method IIc), 30% CH3CN–70% H2O–0.1% formic acid (Method IId);
25% CH3CN–75% H2O–0.1% formic acid (Method IIe) or 20%
CH3CN–80% H2O–0.1% formic acid (Method IIf). Flow rate = 1.0 mL/
min. Total run time 20 min. Condition III: a Xbridge(C18)5um col-
umn (100 mm ꢃ 4.9 mm) with 60% CH3CN–40% H2O with 10%
NH4HCO3/NH4OH buffer pH 8 (Method IIIa); 50% CH3CN–50% H2O
with 10% NH4HCO3/NH4OH buffer pH 8 (Method IIIb). Flow
rate = 1.0 mL/min. Total run time 20 min. Compounds that were iso-
latedasfumaricacidsaltsall showedan extrapeakaround2 min. Fu-
maric acid blancs were used to determine the tR of fumaric acid.
Purities calculated are based on RP.
47
8.2 (6.3–10.0)
8.4 (7.2–9.5)
O
NH2
The IC50 and EC50 values (mean and interval) were generated in radioligand binding
experiments using 125I-CCL5 and InsP accumulation experiments, respectively. All
experiments were performed in triplicate and repeated at least three times.
Inhibition of CCL5 binding and constitutive US28 signaling at a 10
pound concentration.
a
lM com-
Introductionofanacetamidesubstituentattheparapositionofthe
phenyl ring (i.e., compound 90) or removal of all substituents (i.e.,
compound 91) impaired all inhibitory activities at US28 (Table 8).
To evaluate the importance of the amine group of lead com-
pound 41, several analogues were designed and synthesized in
which the nitrogen atom was incorporated into different ring
systems (Scheme 5 and Table 9). Substitution of the amine group
with a 4-(4-chlorophenyl)piperidin-4-ol group (i.e., compound
93), a piperidine ring (i.e., compound 94), or an N-methylpiper-
azine moiety (i.e., compound 95) are tolerable and did not have
a major impact on the pharmacological properties as compared
to lead compound 41 (Table 9). On the other hand, introduction
of an N-phenylpiperazine group (i.e., compound 96) or unsatu-
rated tetrahydropyridine moiety (i.e., compound 97), or attach-
ment of a benzyl group to the nitrogen atom (i.e., compound
98) are not allowed and impaired the potency to inhibit CCL5
Compounds 1, 2, 11 and 12 were previously described.9,11,25
Compounds 3, 4, 6, 8, and 13 were purchased from Sigma–Aldrich

684
H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
Table 8
15 min in the microwave at a temperature of 160 °C (settings: ramp
time 5 min, hold time 15 min, power 200 watt, 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 ꢃ 15 mL). The
combined organic layers were washed with water (3 ꢃ 40 mL) and
brine (40 mL), dried over anhydrous MgSO4, filtered, and evaporated
in vacuo. Purification by flash chromatography (0–50% EtOAc in
DCM) gave 480 mg (57%) of the free base as an oil. This was dissolved
in EtOAc and acidified by the addition of a saturated solution of fu-
maric acid in Et2O. The fumaric salt was isolated by filtration and
recrystallized from MeOH/Et2O to give 281 mg (52%) of 50 as a white
solid. 1H NMR (CDCl3): d 1.60–1.81 (m, 2H), 2.08–2.43 (m, 2H), 2.84–
3.25 (m, 6H), 3.65–3.78 (m, 2H), 5.28 (s, 1H), 6.63 (s, 2H), 7.11–7.36
(m, 14H). 13C (CDCl3): d 36.29, 49.42, 56.91, 64.68, 69.54, 84.64,
126.57, 127.30, 128.09, 128.89, 134.06, 135.30, 141.78, 148.74,
169.53. Anal. RP-HPLC Ib: tR = 8.60 min (purity 100%), IId:
tR = 12.13 min (purity 100%). HRMS (ESI) m/z calcd for C26H28ClNO2:
421.1808; found: 421.1803.
Chemical structures and pharmacological properties on US28 of compounds 41, and
85–91
NH2
R
No.
R
IC50
(l
M)
EC50 (lM)
41
85
86
87
88
89
90
91
2,4-Cl2
9.5 (6.8–12.3)
17.7 (14.5–20.9)
>100
37.3 (28.8–45.7)
>100
59.7 (50.1–69.2)
>100
80.6 (32.4–128.8)
11.1 (8.3–13.8)
13.7 (9.1–18.2)
>100
42.7 (41.7–43.7)
>100
16.4 (14.1–18.6)
>100
>100
2,4-Me2
2,4-(OMe)2
2-OMe
4-OMe
3-Me-4-OMe
4-NH(C@O)CH3
H
The IC50 and EC50 values (mean and interval) were generated in radioligand binding
experiments using 125I-CCL5 and InsP accumulation experiments, respectively. All
experiments were performed in triplicate and repeated at least three times.
6.1.2. General method B: 1-(4,4-bis(4-fluorophenyl)butyl)-4-(4-
chlorophenyl)piperidin-4-ol (51)
A solution of 4,40-(4-chlorobutane-1,1-diyl)bis(fluorobenzene)
(0.56 g, 1.99 mmol), 4-(4-chlorophenyl)piperidin-4-ol 49 (0.51 g,
2.41 mmol), NaI (0.30 g, 2.00 mmol) and Na2CO3 (0.43 g,
4.06 mmol) in CH3CN (20 mL) was refluxed overnight. The solvent
was removed in vacuo, the residue was diluted with water (50 mL)
and extracted with DCM (3 ꢃ 30 mL). The combined organic layers
were washed with water (3 ꢃ 50 mL) and brine (50 mL), dried over
anhydrous MgSO4, filtered and evaporated in vacuo. Purification by
flash chromatography (0–100% EtOAc in DCM) and recrystalliza-
tion from EtOAc gave 853 mg (94%) of 51 as a white solid. 1H
NMR (CDCl3): d 1.48–1.72 (m, 5H), 1.92–2.24 (m, 4H), 2.30–2.55
(m, 4H), 2.69–2.85 (m, 2H), 3.86 (t, J = 7.8 Hz, 1H), 6.90–7.43 (m,
12H). 13C (CDCl3): d 25.76, 34.27, 38.71, 49.83, 50.14, 58.92,
71.41, 115.57, 115.78, 126.47, 128.82, 129.43, 129.51, 133.22,
140.86, 140.89, 147.16, 160.54, 162.97. Anal. RP-HPLC Ib:
tR = 8.78 min (purity 100%), IIb: tR = 13.99 min (purity 100%). HRMS
(EI) m/z calcd for C27H28ClF2NO: 455.1827; found: 455.1826.
Table 9
Chemical structures and pharmacological properties on US28 of compounds 41, and
93–98
R
Cl
Cl
No.
R
IC50
(
l
M)
EC50 (lM)
41
9.5 (6.8–12.3)
11.1 (8.3–13.8)
6.0 (4.0–7.9)
NH2
N
OH
93
94
95
96
97
11.0 (10.0–12.0)
Cl
13.4 (11.2–15.5)
8.1 (6.6–9.5)
>100
2.6 (2.1–3.0)
12.1 (9.1–15.1)
>100
N
N
N
N
N
N
6.1.3. General method C: 1-(3-((4-chlorobenzyl)(phenyl)amino)-
propyl)-4-(4-chlorophenyl)-piperidin-4-ol fumarate (73)
(i) A solution of aniline 62 (1.82 mL, 20.0 mmol), 4-chlorobenz-
aldehyde 64 (2.35 mL, 20.0 mmol) and CH3COOH (1.14 mL,
19.9 mmol) in DCE (100 mL) was stirred for 24 h at room temper-
ature and NaBH(OAc)3 (6.36 g, 30.0 mmol) was added. The reaction
mixture was stirred for another 18 h, quenched with 5% Na2CO3
(100 mL) and the water layer was extracted with DCM
(3 ꢃ 100 mL). The combined organic layers were washed with
brine (150 mL), dried over anhydrous Na2SO4, filtered and evapo-
rated in vacuo to give 4.42 g (100%) of 67 as a brown solid. 1H
NMR (CDCl3): d 4.06 (br s, 1H), 4.29 (s. 2H), 6.51–6.58 (m, 2H),
7.07–7.14 (m, 2H), 7.24–7.36 (m, 5H).
>100
>100
Cl
98
>100
>100
NH
The IC50 and EC50 values (mean and interval) were generated in radioligand binding
experiments using 125I-CCL5 and InsP accumulation experiments, respectively. All
experiments were performed in triplicate and repeated at least three times.
(ii) NaNH2 (0.39 g, 10.0 mmol) was added to a solution of 67
(1.09 g, 5.01 mmol) in toluene (15 mL) and this was refluxed for
18 h. 1-Bromo-3-chloropropane (2.5 mL, 25.3 mmol) was added
and the reaction mixture was refluxed for another 18 h. Water
(25 mL) was added and the water layer was extracted with EtOAc
(3 ꢃ 20 mL). The combined organic layers were washed with water
(3 ꢃ 30 mL) and brine (40 mL), dried over anhydrous Na2SO and fil-
tered. After evaporation under reduced pressure, the residue was
purified by flash chromatography (5% DCM in hexane) to give
142 mg (10%) of 70 as a light yellow oil. 1H NMR (CDCl3): d 1.98–
2.18 (m, 2H), 3.45–3.68 (m, 4H), 4.50 (s, 2H), 6.51–6.75 (m, 3H),
7.03–7.28 (m, 6H).
Co. (USA). Compounds 14 and 15 were kindly donated by Dr. Aebi-
scher from the Sandoz Research Institute in Bern. Compounds 5, 7,
9, 10 and 16–47 were selected from our in-house screening library.
Purity of all compounds was P95% as determined by LC–MS
measurements.
6.1.1. General method A: 1-(2-(benzhydryloxy)ethyl)-4-(4-chloro-
phenyl)piperidin-4-ol fumarate (50)
Bromide intermediate 48 (0.58 g, 1.99 mmol), which was synthe-
sized following a method previously described,15 4-(4-chloro-
phenyl)piperidin-4-ol 49 (0.51 g, 2.41 mmol), NaI (0.30 g,
2.00 mmol), Na2CO3 (0.42 g, 3.96 mmol) and 3 mL CH3CN were
added in a 10 mL microwave vessel and this was reacted during
(iii) Following method B using 70 gave 142 mg of the free base
as an oil. This was converted to the fumaric salt as described for 50

H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
685
to give 178 mg (63%) of 73 as a white solid. 1H NMR (MeOH-d4): d
1.81–1.93 (m, 2H), 1.97–2.17 (m, 2H), 2.19–2.39 (m, 2H), 2.98–3.39
(m, 6H), 3.50 (t, J = 7.2 Hz, 2H), 4.54 (s, 2H), 6.59–6.30 (m, 4H),
7.02–7.51 (m, 11H).13C (MeOH-d4): d 23.81, 36.88, 49.57, 50.09,
55.21, 55.84, 69.75, 114.39, 118.29, 127.48, 129.44, 129.58,
129.61, 130.30, 133.52, 134.07, 137.00, 139.26, 147.47, 149.37,
173.62. Anal. RP-HPLC Ib: tR = 4.87 min (purity 100%), IIb:
tR = 3.89 min (purity 100%). HRMS (EI) m/z calcd for C27H30Cl2N2O:
468.1735; found: 468.1721.
time 20 min): tR = 14.35 min (purity 100%), IIe (total runtime
30 min): tR = 21.45 min (purity 96%). HRMS (EI) m/z calcd for
C30H36ClN3: 473.2598; found: 473.2579.
6.1.7. 3-(4-Chlorophenyl)-1-(3-(11,12-dihydrodibenzo[b,f]azocin-
5(6H)-yl)propyl)piperidin-3-ol fumarate (60)
Following method A using 54 (0.18 g, 0.63 mmol) and 3-(4-
chlorophenyl)piperidin-3-ol 56 (0.11 g, 0.52 mmol), which was
synthesized as described in the literature,18 gave 77 mg of the free
base as an oil. This was converted to the fumaric salt as described
for 50 to give 50 mg (17%) of 60 as a white solid. 1H NMR (MeOH-
d4): d 1.76–2.29 (m, 6H), 2.79–3.10 (m, 5H), 2.91 (s, 2H), 3.13–3.39
(m, 6H), 4.13 (s, 2H), 6.69 (s, 2H), 6.79–7.12 (m, 8H), 7.37–7.49 (m,
4H). 13C NMR (MeOH-d4): d 20.44, 23.57, 34.56, 34.75, 35.80, 52.18,
53.72, 56.81, 61.36, 62.93, 71.47, 121.23, 124.28, 127.22, 127.77,
128.10, 128.49, 129.67, 129.94, 130.90, 132.24, 134.91, 136.16,
138.21, 139.02, 143.20, 144.30, 151.23, 171.08. Anal. RP-HPLC Ib
(total run time 20 min): tR = 6.78 min (purity 99%), IIa (total run
time 30 min): tR = 14.64 min (purity 98%). HRMS (EI) m/z calcd
for C29H33ClN2O: 460.2281; found: 460.2283.
6.1.4. 5-(3-Chloropropyl)-5,6,11,12-tetrahydrodibenzo[b,f]azo-
cine (54)
5,6,11,12-Tetrahydrodibenzo[b,f]azocine 53 (0.42 g, 2.01 mmol),
which was synthesized as previously described in the literature,16
and Na2CO3 (0.42 g, 3.96 mmol) were added in a 10 mL microwave
vessel and 1-bromo-3-chloropropane (3 mL) was added. This solu-
tion was reacted in the microwave for 15 min at a temperature of
200 °C (settings: ramp time 5 min, hold time 15 min, power 200
watt, pressure 17.2 bar) and filtered. The solvent was evaporated
in vacuo and the residue was purified by flash chromatography
(15% DCM in hexane) to give 228 mg (40%) of 54 as a colorless oil.
1H NMR (CDCl3): d 1.85–1.98 (m, 2H), 3.04–3.48 (m, 8H), 4.15 (s,
2H), 6.80–7.24 (m, 8H).
6.1.8. 1-(3-(Benzyl(phenyl)amino)propyl)-4-(4-chlorophenyl)pi-
peridin-4-ol hemifumarate (71)
Following method C starting with commercially available N-
benzylaniline gave 331 mg of the free base as an oil. The final
product was converted to the fumaric salt as described for 50
and recrystallized from MeOH/Et2O to give 261 mg (24% over
two steps) of 71 as white crystals. 1H NMR (CDCl3/DMSO-d6):
d 1.53–1.72 (m, 2H), 1.74–1.97 (m, 2H), 2.00–2.21 (m, 2H),
2.41–2.70 (m, 4H), 2.82–3.01 (m, 2H), 3.26–3.48 (t, J = 7.4 Hz,
2H), 4.42 (s, 2H), 6.49–6.88 (m, 5H), 6.98–7.42 (m, 11H). 13C
6.1.5. 4-(4-Chlorophenyl)-1-(3-(11,12-dihydrodibenzo[b,f]azocin-
5(6H)-yl)propyl)piperidine-4-ol fumarate (57)
Following method A using 54 (0.29 g, 1.01 mmol) gave 289 mg
of the free base as an oil. This was converted to the fumaric salt
as described for 50 and recrystallized from MeOH/Et2O to give
199 mg (38%) of 57 as white crystals. 1H NMR (CDCl3): d 1.50–
1.92 (m, 5H), 2.09–2.36 (m, 2H), 2.41–2.70 (m, 4H), 2.78–3.34
(m, 8H), 4.11 (s, 2H), 6.69 (s, 2H), 6.72–7.35 (m, 12H). 13C (CDCl3):
d 23.15, 33.28, 34.22, 35.92, 48.11, 51.14, 55.08, 60.96, 69.57,
119.32, 122.17, 125.91, 126.82, 127.15, 128.39, 128.79, 129.73,
131.08, 132.94, 135.45, 136.06, 137.65, 141.83, 145.37, 150.02,
171.33. Anal. RP-HPLC Ib (total run time 20 min): tR = 14.43 min
(purity 100%), IIc: tR = 11.35 min (purity 97%). HRMS (EI) m/z calcd
for C29H33ClN2O: 460.2281; found: 460.2284.
NMR (CDCl3/DMSO-d6):
d 22.90, 36,45, 48.43, 54.32, 54.76,
69.23, 112.30, 116.32, 126.13, 126.43, 126.61, 128.02, 128.34,
129.04, 135.15, 138.49, 146.62, 148.14, 170.21. Anal. RP-HPLC
Ib: tR = 10.01 min (purity 100%), IIb (total run time 30 min):
tR = 8.05 min (purity 100%), IIb (total run time 30 min):
tR = 18.22 min (purity 100%). HRMS (EI) m/z calcd for
C27H31ClN2O: 434.2125; found: 434.2113.
6.1.6. (4-(4-Chlorophenyl)-1-(3-(11,12-dihydrodibenzo[b,f]azo
cin-5(6H)-yl)propyl)piperidin-4-yl)methanamine fumarate (59)
(i) Following method B using 54 and 4-(4-chlorophenyl)-piper-
idine-4-carbonitrile hydrochloride 55, which was synthesized as
previously described in the literature,11 afforded 675 mg of 58 as
a light yellow oil. The crude product was used without further
purification.
6.1.9. 1-(3-(Benzyl(4-chlorophenyl)amino)propyl)-4-(4-chloro-
phenyl)piperidin-4-ol hemifumarate (72)
Following the analogous route (including the reductive amina-
tion and alkylation of the piperidine nitrogen atom) as described
in method C starting with benzaldehyde and 4-chloroaniline. Con-
version of the final product to the fumaric salt as described for 50
and recrystallized from Et2O/MeOH resulted in the isolation of
112 mg of 72 as white crystals (6% over three steps) 1H NMR
(CDCl3/DMSO-d6): d 1.61–2.05 (m, 4H), 2.13–2.37 (m, 2H), 2.53–
2.86 (m, 2H), 2.99–3.12 (m, 2H), 3.37 (t, J = 7.1 Hz, 2H), 4.44 (s,
2H), 6.46–6.68 (m, 2H), 6.72 (s, 2H), 7.02–7.39 (m, 11H). 13C
NMR (CDCl3/DMSO-d6): d 22.64, 36.16, 48.38, 48.70, 54.54, 69.37,
113.62, 121.22, 126.02, 126.40, 126.86, 128.26, 128.49, 128.88,
132.73, 135.35, 137.92, 145.84, 146.72, 170.93. Anal. RP-HPLC Ib
(ii) AlCl3 (0.37 g, 2.78 mmol) was added portion wise to a suspen-
sion of LiAlH4 (0.11 g, 2.90 mmol) in THF (10 mL) at 0 °C and this was
stirred for 5 min followed by the drop wise addition of a solution of
58 (675 mg, 1.44 mmol) in THF (5 mL). The reaction mixture was al-
lowed to warm to room temperature, 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; the filtrate was dried over anhydrous MgSO4, filtered
againandevaporatedinvacuo. Purificationbyflashchromatography
(5% Et3N in EtOAc) afforded 183 mg (39%) of the free base as an oil.
This was converted to the fumaric salt as described for 50 to give
199 mg (24%) of 59 as a light brown solid. 1H NMR (CDCl3/DMSO-
d6): d 1.54–1.75 (m, 2H), 1.82–1.93 (m, 2H), 2.05–2.18 (m, 4H),
2.27–2.39 (m, 2H), 2.59–2.68 (m, 2H), 2.86 (s, 2H),2.92–2.99 (m,
2H), 3.03–3.19 (m, 6H), 4.11 (s, 2H),6.60 (s, 2H), 6.73 (t, J = 7.3 Hz,
1H), 6.89–7.04 (m, 6H), 7.11–7.39 (m, 5H). 13C NMR (CDCl3/DMSO-
d6): d 24.06, 31.36, 32.79, 33.75, 38.67, 48.42, 50.68, 54.91, 60.22,
119.50, 121.68, 125.47, 126.33, 126.68, 128.51, 128.59, 128.79,
129.20, 130.57, 131.95, 134.13, 135.82, 137.06, 141.06, 146.64,
149.77. Anal. RP-HPLC Ib: tR = 4.43 min (purity 99%), Ic (total run
:
tR = 5.03 min (purity 100%), IIa (total run time 30 min):
tR = 14.46 min (purity 98%). HRMS (EI) m/z calcd for C27H30Cl2N2O:
468.1735; found: 468.1718.
6.1.10. 4-(4-Chlorophenyl)-1-(2-(8-(methylthio)-10,11-dihydro-
dibenzo[b,f]thiepin-10-yloxy)-ethyl)piperidin-4-ol fumarate (80)
(i) 4-(Methylthio)benzenethiol 75 (5.24 g, 20.0 mmol) was
added to a solution of 2-(2-iodophenyl)acetic acid 74 (3.12 g,
20.0 mmol) and KOH (4.49 g, 80.0 mmol) in water (60 mL). After
the addition of Cu (205 mg), the reaction mixture was refluxed
for 48 h, cooled to room temperature and filtered. The filtrate
was acidified with 1.0 M HCl, filtered and the residue was dried un-
der vacuum to yield 4.83 g (83%) of 2-(2-(4-(methylthio)phenyl-

686
H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
thio)phenyl)acetic acid 76 as a pink solid. 1H NMR (CDCl3): d 2.43
(s, 3H), 7.12–7.35 (m, 8H).
quantitative yield. 1H NMR (CDCl3): d 2.28 (s, 3H), 2.30 (s, 3H),
2.41–2.59 (m, 1H), 2,72–3.05 (m, 2H), 3.10–3.22 (m, 1H), 3.37
(dd, J = 10.4 Hz and 3.9 Hz, 1H), 6.81–7.01 (m, 3H), 7.25–7.41 (m,
2H), 7.54 (d, J = 7.5 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H).
(ii) PPA (50.0 g) was added to
a solution of 76 (4.83 g,
16.6 mmol) in toluene (30 mL) and the reaction mixture was
heated and refluxed for 18 h. Water (50 mL) was added, the organic
layer was separated and the water layer was extracted with EtOAc
(3 ꢃ 30 mL). The combined organic extracts were washed with 5%
NaOH (50 mL) and brine (50 mL), dried over anhydrous Na2SO4, fil-
tered and evaporated in vacuo to give 3.50 g (77%) of 8-(methyl-
thio)dibenzo[b,f]thiepin-10(11H)-one 77 as a brown oil. 1H NMR
(CDCl3): d 2.46 (s, 3H), 4.36 (s, 2H), 7.13–7.64 (m, 6H), 8.02 (d,
J = 2.3 Hz, 1H).
(iii) A solution of 77 (3.50 g, 12.8 mmol) and NaBH4 (1.01 g,
26.7 mmol) in MeOH (100 mL) was stirred overnight at room tem-
perature. The reaction mixture was evaporated in vacuo, water was
added (50 mL) and the water layer was extracted with Et2O
(3 ꢃ 25 mL). The combined organic extracts were washed with
water (3 ꢃ 50 mL) and brine (50 mL), dried over anhydrous
Na2SO4, filtered and evaporated in vacuo to give 3.14 g (89%) of
(iii) A solution of 2-(2,4-dimethylbenzyl)-2,3-dihydro-1H-in-
den-1-one (1.15 g, 4.59 mmol) and NaBH4 (0.17 g, 4.59 mmol) in
MeOH (10 mL) was stirred overnight at room temperature. The
reaction mixture was evaporated in vacuo, water was added
(25 mL) and the water layer was extracted with EtOAc
(3 ꢃ 20 mL). The combined organic extracts were washed with
water (3 ꢃ 25 mL) and brine (25 mL), dried over anhydrous
Na2SO4, filtered and evaporated in vacuo to give a quantitative
yield of 2-(2,4-dimethylbenzyl)-2,3-dihydro-1H-inden-1-ol as a
1:1 mixture of two diastereomers. 1H NMR (CDCl3): d 1.70 (br s,
1H), 2.30 (s, 6H), 2.35–2.88 (m, 3H), 2.98–3.12 (m, 2H), 4.83–5.01
(m, 1H), 6.89–7.01 (m, 2H), 7.04–7.41 (m, 6H).
(iv) A solution of 2-(2,4-dimethylbenzyl)-2,3-dihydro-1H-in-
den-1-ol (1.15 g, 4.59 mmol) and DPPA (1.39 mL, 6.41 mmol) in
THF (10 mL) was stirred for 10 min, cooled to 0 °C and DBU
(0.98 mL, 6.42 mmol) was added in a drop wise manner. The reac-
tion mixture was allowed to warm to room temperature and stir-
red for 18 h. The solvent was evaporated in vacuo, water (25 mL)
was added and the water layer was extracted with Et2O
(3 ꢃ 15 mL). The combined organic extracts were washed with
brine (25 mL), dried over anhydrous Na2SO4, filtered and evapo-
rated in vacuo. Purification by column chromatography (hexane)
afforded 763 mg (60%) of 1-azido-2-(2,4-dimethylbenzyl)-2,3-
dihydro-1H-indene as a 1:1 mixture of two diastereomers. 1H
NMR (CDCl3): d 2.29 (s, 3H), 2.30 (s, 3H), 2.51–2.84 (m, 4H),
2.90–3.11 (m, 1H), 4.52 and 4.70 (d, J = 4.7 and 5.0 Hz, 1H), 6.87–
7.03 (m, 3H), 7.11–7.39 (m, 4H).
8-(methylthio)-10,11-dihydrodibenzo[b,f]thiepin-10-ol 78 as
a
brown solid. 1H NMR (CDCl3): d 2.44 (s, 3H), 3.31–3.49 (m, 1H),
3.69–3.78 (m, 1H), 5.49 (br s, 1H), 6.95–7.49 (m, 7H).
(iv) BF3ꢀ(Et)2O (1.60 mL, 12.6 mmol) was added drop wise to a
solution of 78 (3.14 g, 11.4 mmol) and 2-bromoethanol (1.22 mL,
17.2 mmol) in toluene (30 mL) and the reaction mixture was stir-
red for 1 h. Water (25 mL) was added and the water layer was ex-
tracted with EtOAc (3 ꢃ 20 mL). The combined organic extracts
were washed with water (25 mL), 5% NaOH (2 ꢃ 25 mL) and brine
(25 mL), dried over anhydrous Na2SO4, filtered and evaporated in
vacuo to give 3.37 g of 11-(2-bromoethoxy)-2-(methylthio)-
10,11-dihydrodibenzo[b,f]thiepine 79 as a brown oil. The crude
product was used without further purification.
(v) Following method B using crude 79 (0.57 g, 1.49 mmol) and
4-(4-chlorophenyl)piperidin-4-ol 49 (0.21 g, 1.00 mmol) gave
88 mg of a light yellow oil. This was converted to the fumaric salt
as described for 50 to give 101 mg (16%) of 80 as a white solid. 1H
NMR (CDCl3/DMSO-d6): d 1.67–1.80 (m, 2H), 2.21–2.57 (m, 5H),
2.78–3.31 (m, 6H), 3.42–3.62 (m, 2H), 3.78–3.99 (m, 2H), 5.22–
5.33 (m, 1H), 6.73 (s, 2H), 7.00–7.41 (m, 12H). 13C NMR (CDCl3/
DMSO-d6): d 15.53, 35.91, 38.84, 48.92, 49.19, 56.61, 64.57,
68.79, 77.22, 125.02, 125.61, 126.28, 127.43, 128.16, 129.64,
130.97, 131.39, 131.82, 132.58, 133.66, 134.94, 137.86, 138.80,
142.22, 146.10, 169.42. Anal. RP-HPLC Ia: tR = 7.76 min (purity
99%), IIa (total run time 30 min): tR = 19.48 min (purity 97%). HRMS
(EI) m/z calcd for C28H30ClNO2S2: 511.1406; found: 511.1405.
(v) A solution of 1-azido-2-(2,4-dimethylbenzyl)-2,3-dihydro-
1H-indene (0.76 g, 2.74 mmol) and 10% Pd/C (3 mg) in MeOH
(10 mL) was hydrogenated with H2 (atmospheric pressure) for
3 days. The reaction mixture was filtrated over Hyflo, the residue
was washed with EtOH and the filtrate was evaporated in vacuo.
Purification by flash chromatography (2% Et3N in hexane) gave
646 mg of the free base. This was dissolved in a solution of HCl
gas in EtOH and the solvent was evaporated in vacuo. The hydro-
chloride salt was isolated by filtration and recrystallized from
Et2O/MeOH.
6.1.12. 2-(2,4-Dimethylbenzyl)-2,3-dihydro-1H-inden-1-amine
hydrochloride (85)
Yield: 665 mg (84%) of 85 as a 1:1 mixture of two diastereomers.
1H NMR (MeOH-d4): d 2.30 (s, 3H), 2.33 (s, 3H), 2.61–3.18 (m, 5H),
4.51 and 4.83 (d, J = 3.5 and 6.0 Hz, 1H), 6.92–7.15 (m, 3H), 7.21–
7.40 (m, 3H), 7.49–7.63 (m, 1H). 13C NMR (MeOH-d4): d 18.64,
20.02, 31.51, 35.25, 35.87, 43.08, 58.19, 125.44, 125.73, 126.78,
127.33, 129.29, 129.96, 131.25, 134.43, 136.07, 136.21, 138.73,
143.83 (one diastereomer). 13C NMR (MeOH-d4): d 17.38, 18.59,
31.51, 35.25, 35.87, 45.21, 60.52, 125.18, 125.31, 127.48, 129.66,
131.27, 134.49, 136.27, 136.30, 137.94, 143.58 (other diastereomer).
Anal. RP-HPLC Ib (total run time 30 min): tR = 12.89 min (purity
100%), IIb: tR = 11.79, 12.79 min (purity 100%). HRMS (EI) m/z calcd
for C18H21N: 251.1674; found: 251.1665.
6.1.11. General method for the synthesis of target compounds
41, 85–91
(i) 2,4-Dimethylbenzaldehyde (1.67 g, 10.0 mmol) and 1-inda-
none(77, R1 = H);1.33 g, 10.0 mmol) were added toa saturatedsolu-
tion of anhydrous K2CO3 in EtOH (25 mL).The resulting reaction
mixture was stirred vigorously for 24 h, 37% HCl (2 mL) was added
and the solvent was evaporated in vacuo. Water (50 mL) was added
and the water layer was extracted with DCM (3 ꢃ 25 mL). The com-
bined organic extracts were washed with brine (50 mL), dried over
anhydrous Na2SO4, filtered and evaporated in vacuo. The residue
was recrystallized from EtOH to give 1.95 g (79%) of 2-(2,4-dimeth-
ylbenzylidene)-2,3-dihydro-1H-inden-1-one as white crystals. 1H
NMR (CDCl3): d 2.33 (s, 3H), 2.43 (s, 3H), 3.95 (s, 2H), 6.98–7.11 (m,
2H), 7.31–7.60 (m, 3H), 7.80–7.95 (m, 2H).
(ii) A suspension of 2-(2,4-dimethylbenzylidene)-2,3-dihydro-
1H-inden-1-one (1.14 g, 5.49 mmol) and 10% Pd/C (5 mg) in MeOH
(10 mL) was hydrogenated with H2 (atmospheric pressure) for
18 h. The reaction mixture was filtrated over Hyflo and the residue
was washed with EtOH. The filtrate was evaporated in vacuo to
6.1.13. 2-(2,4-Dimethoxybenzyl)-2,3-dihydro-1H-inden-1-amine
hydrochloride (86)
Yield: 539 g (17% yield over five steps) of 86 as a 1:1 mixture of
two diastereomers. 1H NMR (MeOH-d4): d 2.58–3.14 (m, 5H), 3.78
and 3.79 (s, 3H), 3.81 and 3.82 (s, 3H), 4.43 and 4.69 (d, J = 4.1 and
5.8 Hz, 1H), 6.45–6.57 (m, 2H), 7.02–7.52 (m, 5H). 13C NMR
(MeOH-d4): d 35.44, 35.79, 45.35, 54.82, 54.84, 54.91, 57.98,
98.55, 104.89, 119.62, 125.36, 125.41, 127.24, 129.95, 131.31,
138.83, 144.09, 158.89, 160.45 (one diastereomer). 13C NMR
give 2-(2,4-dimethylbenzyl)-2,3-dihydro-1H-inden-1-one in
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H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
687
(MeOH-d4): d 28.70, 32.75, 43.45, 54.82, 54.84, 54.91, 60.26, 98.50,
104.61, 119.77, 124.92, 125.59, 127.32, 129.80, 130.80, 138.12,
143.62, 158.54, 160.52 (other diastereomer). Anal. RP-HPLC Ia :
tR = 8.47 and 9.81 min (purity 100%), IIIb: tR = 5.29 and 5.61 min
(purity 100%). HRMS (EI) m/z calcd for C18H21NO2: 283.1572;
found: 283.1582.
(m, 9H). 13C NMR (MeOH-d4): d 35.03, 35.29, 44.29, 58.13, 125.29,
125.47, 125.61, 127.37, 128.73, 128.80, 130.02, 138.68, 139.59,
143.77. RP-HPLC Ic: tR = 5.52 min (purity 100%), IId: tR = 5.94 min
(purity 97%), IIIa: tR = 3.37 min (purity 99%). HRMS (EI) m/z calcd
for C16H17N: 223.1361; found: 223.1363.
6.1.19. General method for the synthesis of target compounds
93–98
6.1.14. 2-(2-Methoxybenzyl)-2,3-dihydro-1H-inden-1-amine
hydrochloride (87)
(i) 2,4-Dichlorobenzaldehyde (17.5 g, 100 mmol) and 1-inda-
none 81 (13.2 g, 100 mmol) were added to a saturated solution of
anhydrous K2CO3 in EtOH (250 mL). The resulting reaction mixture
was stirred vigorously for 18 h and 37% HCl (20 mL) was added.
The precipitate was isolated by filtration, washed with water and
dried under vacuum to give 25.8 g (89%) of 2-(2,4-dichlorobenzylid-
ene)-2,3-dihydro-1H-inden-1-one as white crystals. 1H NMR
(CDCl3): d 3.92 (s, 2H), 7.12–7.66 (m, 5H), 7.80–7.99 (m, 2H).
(ii) A suspension of 2-(2,4-dichlorobenzylidene)-2,3-dihydro-
1H-inden-1-one (11.6 g, 40.1 mmol) and 10% Pd/C (40 mg) in MeOH
(100 mL) was hydrogenated with H2 (atmospheric pressure) for 6 h
at a temperature of 50 °C. The reaction mixture was filtrated over
Hyflo and the filtrate was evaporated in vacuo. Recrystallization
from EtOH afforded 4.6 g (39%) of 2-(2,4-dichlorobenzyl)-2,3-dihy-
dro-1H-inden-1-one as white crystals. 1H NMR (CDCl3): d 2.65–
2.88 (m, 2H), 2.95–3.20 (m, 2H), 3.27–3.40 (m, 1H), 7.06–7.20 (m,
2H), 7.26–7.40 (m, 3H), 7.56 (t, J = 7.4 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H).
(iii) A solution of 2-(2,4-dichorolbenzyl)-2,3-dihydro-1H-inden-
1-one (1.86 g, 6.40 mmol) and NaBH4 (0.17 g, 4.59 mmol) in MeOH
(40 mL) was stirred for 3 h at room temperature. The reaction mix-
ture was evaporated in vacuo, water was added (20 mL) and the
waterlayer was extractedwith EtOAc(3 ꢃ 15 mL). Thecombinedor-
ganic extracts were washed with water (3 ꢃ 25 mL) and brine
(25 mL), driedover anhydrousNa2SO4, filtered and evaporatedin va-
cuo to give 1.84 (98%) of 2-(2,4-dichlorobenzyl)-2,3-dihydro-1H-in-
den-1-ol as a 1:1 mixture of two diastereomers. 1H NMR (CDCl3): d
1.72 (br s, 1H), 2.41–3.02 (m, 5H), 4.92 (br s, 1H), 7.16–7.38 (m, 7H).
(iv) Thionyl chloride (0.75 mL, 10.3 mmol) was added to a solu-
tion of 2-(2,4-dichlorobenzyl)-2,3-dihydro-1H-inden-1-ol (1.84 g,
6.28 mmol) in toluene (30 mL) at 0 °C. The reaction mixture was
stirred at room temperature for 30 min, heated to 55 °C and stirred
for 1 h. The solution was cooled to room temperature, washed with
ice-water (2 ꢃ 15 mL), dried over anhydrous Na2SO4, filtered and
evaporated in vacuo to give 1.88 g (96%) of 1-chloro-2-(2,4-dichlo-
robenzyl)-2,3-dihydro-1H-indene 92 as a 1:1 mixture of two dia-
stereomers. 1H NMR (CDCl3): d 2.55–3.29 (m, 5H), 5.10 and 5.27
(d, J = 5.3 and 4.8 Hz, 1H), 7.08–7.41 (m, 7H).
Yield: 439 mg (17% yield over five steps) of one diastereomer of
87 as a white solid. 1H NMR (MeOH-d4): d 2.52–2.80 (m, 2H), 2.85–
3.15 (m, 3H), 3.85 (s, 3H), 4.71 (d, J = 5.9 Hz, 1H), 6.89–6.97 (m,
2H), 7.01–7.36 (m, 5H), 7.50 (d, J = 6.6 Hz, 1H). 13C NMR (MeOH-
d4): d 30.37, 36.41, 44.21, 55.82, 59.00, 111.76, 121.82, 126.30,
126.43, 128.28, 128.54, 129.17, 131.01, 131.43, 139.80, 145.96,
158.71. RP-HPLC Ic: tR = 4.19 min (purity 100%), IIb: tR = 3.95 min
(purity 100%). HRMS (EI) m/z calcd for C17H19NO: 253.1467; found:
253.1476.
6.1.15. 2-(4-Methoxybenzyl)-2,3-dihydro-1H-inden-1-amine
hydrochloride (88)
Yield: 439 mg (19% yield over five steps) of 88 as a 1:1 mixture
of two diastereomers. 1H NMR (MeOH-d4): d 2.51–3.29 (m, 5H),
3.77 (s, 3H), 3.79 (s, 3H), 4.49 and 4.77 (d, J = 3.9 and 6.2 Hz, 1H),
6.80–6.94 (m, 2H), 7.08–7.52 (m, 6H). 13C NMR (MeOH-d4): d
35.89, 38.28, 46.58, 54.73, 60.39, 114.14, 125.03, 125.67, 127.44,
129.75, 130.06, 131.55, 137.93, 143.47, 158.92 (one diastereomer).
13C NMR (MeOH-d4): d 34.12, 35.33, 44.47, 54.73, 58.08, 114.11,
125.35, 125.46, 127.34, 129.92, 129.98, 131.49, 138.73, 143.82,
158.92 (other diastereomer). RP-HPLC Ic: tR = 5.73 min (purity
100%), IId: tR = 5.48 min (purity 96%). HRMS (EI) m/z calcd for
C17H19NO: 253.1467; found: 253.1459.
6.1.16. 2-(4-Methoxy-3-methylbenzyl)-2,3-dihydro-1H-inden-
1-amine hydrochloride (89)
Yield: 470 mg (20% yield over five steps) of one diastereomer of
89 as a white solid. 1H NMR (MeOH-d4): d 2.42 (s, 3H), 2.42–3.09
(m, 5H), 3.80 (s, 3H), 4.77 (d, J = 6.0 Hz, 1H) 6.82–7.54 (m, 7H).
13C NMR (MeOH-d4): d 15.35, 34.14, 35.37, 44.48, 54.84, 58.09,
110.21, 125.30, 125.46, 126.72, 131.04, 127.05, 127.31, 129.96,
130.89, 138.78, 143.86, 156.94. Anal. RP-HPLC Ib: tR = 4.56 min
(purity 100%), IIa: tR = 4.33 min (purity 99%). HRMS (EI) m/z calcd
for C18H21NO: 267.1623; found: 267.1623.
6.1.17. N-(4-((1-Amino-2,3-dihydro-1H-inden-2-yl)methyl)phen-
yl)acetamide (90)
(v) A solution of piperidine (70 lL, 0.71 mmol), NaI (75 mg,
Yield: 581 mg (23% yield over five steps) of 90 as a 1:5 mixture
of two diastereomers. (MeOH-d4): d 2.10 (s, 3H), 2.20–2.34 (m, 1H),
2.41–77 (m, 2H), 2.81–2.92 (m, 1H), 3.00–3.15 (m, 1H), 3.98 and
4.28 (d, J = 7.6 and 7.8 Hz, 1H), 6.98–7.51 (m, 8H). 13C NMR
(MeOH-d4): d 22.73, 36.38, 38.92, 52.76, 61.76, 120.45, 123.50,
124.48, 126.56, 127.41, 129.28, 136.94, 137.22, 141.91, 146.11,
0.50 mmol), and Na2CO3 (106 mg, 1.0 mmol) in CH3CN (10 mL)
was heated to reflux temperature and 1-chloro-2-(2,4-dichloroben-
zyl)-2,3-dihydro-1H-indene 92 (0.16 g, 0.51 mmol) in CH3CN (1 mL)
was added. The reaction mixture was refluxed overnight, the solvent
was removed in vacuo and the residue was diluted with water
(15 mL) followed by an extraction with DCM (3 ꢃ 10 mL). The com-
bined organic layers were dried over anhydrous NaSO4, filtered,
evaporated in vacuo, and subsequently purified by flash chromatog-
raphy (25% EtOAc in DCM) and recrystallized from EtOAc.
170.55 (major diastereomer). 13C NMR (MeOH-d4):
d 22.73,
34.10, 35.19, 46.63, 58.46, 120.32, 124.23, 124.65, 124.77, 127.63,
129.17, 136.82, 137.34, 141.91, 146.11, 170.55 (minor diastereo-
mer). RP-HPLC Ie (total run time 30 min): tR = 10.42 min (minor
diastereomer), 13.62 min (major diastereomer) (purity 100%), IIf:
tR = 5.21 min (minor diastereomer), 6.22 min (major diastereomer)
(purity 100%). HRMS (EI) m/z calcd for C18H20N2O: 280.1576;
found: 280.1571.
6.1.20. 1-(2-(2,4-Dichlorobenzyl)-2,3-dihydro-1H-inden-1-yl)pi-
peridine (94)
Yield: 90 mg (49%) of 94 as white crystals. 1H NMR (CDCl3): d
1.29–1.51 (m, 6H), 2.29–2.70 (m, 6H), 2.76–2.99 (m, 3H), 3.95 (d,
J = 3.4 Hz, 1H), 7.03–7.22 (m, 5H), 7.29–7.41 (m, 3H). 13C (CDCl3): d
24.61, 26.41, 37.00, 38.18, 38.80, 50.19, 76.04, 124.50, 125.88,
126.09, 126.65, 127.38, 129.12, 131.78, 132.15, 134.78, 137.23,
142.58. Anal. RP-HPLCIc(totalruntime 30 min):tR = 19.88 min(pur-
ity 100%), IIb (total run time 30 min): tR = 13.77 min (purity 100%).
HRMS (EI) m/z calcd for C21H23Cl2N: 359.1208; found: 359.1199.
6.1.18. 2-Benzyl-2,3-dihydro-1H-inden-1-amine hydrochloride
(91)
Yield: 350 mg (14% yield over five steps) of one diastereomer of
91 as a white solid. 1H NMR (MeOH-d4): d 2.52–2.66 (m, 1H), 2.70–
3.05 (m, 3H), 3.09–3.20 (m, 1H), 4.79 (d, J = 6.4 Hz, 1H), 7.25–7.55

688
H. F. Vischer et al. / Bioorg. Med. Chem. 18 (2010) 675–688
6.1.21. 4-(4-Chlorophenyl)-1-(2-(2,4-dichlorobenzyl)-2,3-dihydro-
1H-inden-1-yl)-1,2,3,6-tetrahydropyridine hydrochloride (97)
Yield: 50 mg (19%) of one diastereomer of 97 as a light brown
solid. 1H NMR (MeOH-d4): d 2.61–2.92 (m, 6H), 3.15–3.39 (m,
3H), 3.51–3.81 (m, 2H), 4.61–4.69 (m, 1H), 6.01–6.07 (m, 1H),
7.31–7.54 (m, 10H), 7.67 (d, J = 7.1 Hz, 1H). 13C (MeOH-d4): d
26.22, 37.74, 38.29, 40.69, 47.72, 47.83, 75.74, 118.08, 127.30,
127.70, 128.68, 128.96, 129.74, 130.48, 132.14, 134.00, 134.59,
135.07, 135.83, 136.27, 136.78, 138.51, 146.52. Anal. RP-HPLC Ib
(total run time 20 min): tR = 13.04 min (purity 99%), IIb:
tR = 6.78 min (purity 96%). HRMS (EI) m/z calcd for C27H24Cl3N:
469.0945 (second isotope); found: 469.0931.
J = 6.1 Hz, 1H), 6.68 (s, 2H), 7.27–7.53 (m, 12H). 13C NMR (MeOH-
d4): d 32.41, 36.00, 44.64, 51.56, 65.52, 126.53, 126.60, 127.91,
128.49, 129.71, 130.01, 130.30, 130.33, 130.51, 133.42, 134.08,
135.48, 135.89, 136.00, 137.77, 144.58, 170.70. Anal. RP-HPLC Ib:
tR = 8.22 min (purity 100%), IIa: tR = 12.37 min (purity 97%). HRMS
(ESI) m/z calcd for C27H25Cl2NO4: 381.1051; found: 381.1046.
6.2. Pharmacology
Pharmacological characterization of nonpeptidergic ligands was
performed as previously described.11
Acknowledgments
6.1.22. 4-(4-Chlorophenyl)-1-(2-(2,4-dichlorobenzyl)-2,3-
dihydro-1H-inden-1-yl)piperidin-4-ol hydrochloride (93)
Yield: 61 mg (23%) of one diastereomer of 93 as a white solid 1H
NMR (MeOH-d4): d 1.81–1.92 (m, 4H), 2.50 (br s, 1H), 2.70–3.12
(m, 5H), 3.15–3.36 (4H), 4.41–4.47 (m, 1H), 7.15–7.48 (m, 10H),
8.00 (s, 1H), 12.08 (br s, 1H). 13C (MeOH-d4): d 35.03, 35.26,
37.25, 37.94, 38.73, 44.31, 45.84, 46.37, 69.58, 75.31, 125.81,
126.00, 127.64, 127.97, 128.67, 128.76, 129.60, 131.01, 132.58,
132.80, 133.54, 133.63, 134.80, 134.83, 144.42, 144.51. Anal. RP-
HPLC Ib : tR = 6.19 min (purity 100%), IIa (total run time 30 min):
tR = 13.45 min (purity 100%). HRMS (EI) m/z calcd for C27H26Cl3NO:
485.1080; found: 485.1073.
H. F. Vischer, J. W. Hulshof, 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.
References and notes
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6.1.23. 1-(2-(2,4-Dichlorobenzyl)-2,3-dihydro-1H-inden-1-yl)-
4-phenylpiperazine dihydrochloride (96)
Yield: 48 mg (22%) of one diastereomer of 96 as a white solid.
1H NMR (MeOH-d4): d 2.72–2.97 (m, 3H), 3.05–3.55 (m, 8H),
3.69–3.88 (m 2H), 4.64–4.72 (m, 1H), 6.88–7.08 (m, 2H), 7.19–
7.54 9 m, 9H), 7.74 (d, J = 7.2 Hz, 1H). 13C (MeOH-d4): d 37.56,
38.05, 40.90, 50.58, 76.79, 118.22, 122.92, 127.46, 128.73, 128.78,
129.44, 130.41, 130.51, 132.52, 133.79, 134.00, 134.66, 136.28,
136.55, 146.84, 150.63. Anal. RP-HPLC Ib: tR = 7.07 min (purity
100%), IIa: tR = 4.13 min (purity 98%), IIb (total run time 30 min):
tR = 19.11 min (purity 98%). HRMS (EI) m/z calcd for C26H26Cl2N2:
436.1473; found: 436.1471.
13. Schall, T. J.; McMaster, B. E.; Dairaghi, D. J. World (PTC) Patent WO0217900,
2002.
14. McMaster, B. E.; Schall, T. J.; Penfold, M. E.; Wright, J. J.; Dairaghi, D. J. World
(PTC) Patent WO03018549, 2003.
6.1.24. 1-(2-(2,4-Dichlorobenzyl)-2,3-dihydro-1H-inden-1-yl)-
4-methylpiperazine dihydrochloride (95)
Yield: 105 mg (46%) of one diastereomer of 95 as a light yellow
solid. 1H NMR (MeOH-d4): d 2.61–3.03 (m, 4H), 2.93 (s, 5H), 3.12–
3.87 (m, 8H), 4.55–4.64 (m, 1H), 7.15–7.49 (m, 6H), 7.72 (d,
J = 7.1 Hz, 1H). 13C (MeOH-d4): d 37.59, 38.05, 40.99, 43.36, 47.18,
51.93, 77.02, 127.37, 128.72, 129.35, 130.46, 132.33, 133.98,
134.55, 136.27, 136.76, 146.61. Anal. RP-HPLC Ib (total run time
20 min): tR = 12.99 min (purity 95%), IId: tR = 12.70 min (purity
96%). HRMS (EI) m/z calcd for C21H24Cl2N2: 374.1317; found:
374.1311.
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6.1.25. N-Benzyl-2-(2,4-dichlorobenzyl)-2,3-dihydro-1H-inden-
1-amine fumarate (98)
Yield: 20 mg (8%) of one diastereomer of 98 as a white solid. 1H
NMR (MeOH-d4): d 2.69–2.79 (m, 2H), 2.89–2.95 (m, 1H), 3.02–
3.08 (m, 1H), 3.17–3.25 (m, 1H), 4.14–4.21 (m, 2H), 4.62 (d,