Novel indole and azaindole (pyrrolopyridine) cannabinoid (CB) receptor agonists: Design, synthesis, structure-activity relationships, physicochemical properties and biological activity

Article abstract of DOI:10.1016/j.ejmech.2011.08.021

The discovery, synthesis and structure-activity relationship (SAR) of a novel series of cannabinoid 1 (CB₁) and cannabinoid 2 (CB ₂) receptor ligands are reported. Based on the aminoalkylindole class of cannabinoid receptor agonists, a biphenyl moiety was introduced as novel lipophilic indole 3-acyl substituent in 11-16. Furthermore, the 3-carbonyl tether was replaced with a carboxamide linker in 17-20 and the azaindole (pyrrolopyridine) nucleus was designed as indole bioisostere with improved physicochemical properties in 21-25. Through these SAR efforts, several high affinity CB₁/CB₂ dual cannabinoid receptor ligands were identified. Indole-3-carboxamide 17 displayed single-digit nanomolar affinity and ～80 fold selectivity for CB₁ over the CB₂ receptor. The azaindoles displayed substantially improved physicochemical properties (lipophilicity; aqueous solubility). Azaindole 21 elicited potent cannabinoid activity. Cannabinoid receptor agonists 17 and 21 potently modulated excitatory synaptic transmission in an acute rat brain slice model of cannabinoid receptor-modulated neurotransmission.

Full text of DOI:10.1016/j.ejmech.2011.08.021

European Journal of Medicinal Chemistry 46 (2011) 5086e5098
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel indole and azaindole (pyrrolopyridine) cannabinoid (CB) receptor agonists:
Design, synthesis, structureeactivity relationships, physicochemical properties
and biological activity
,
c
,
a
b
a
c
Antoni R. Blaazer a , Jos H.M. Lange , Martina A.W. van der Neut , Arie Mulder , Femke S. den Boon ,
*
Taco R. Werkman c, Chris G. Kruse a c, Wytse J. Wadman c
,
a Chemical Design & Synthesis Unit, Abbott Healthcare Products B.V., C. J. van Houtenlaan 36, 1381 CP Weesp, The Netherlands
b Preclinical Candidate Selection Unit, Abbott Healthcare Products B.V., C. J. van Houtenlaan 36, 1381 CP Weesp, The Netherlands
c Center for NeuroScience, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery, synthesis and structure-activity relationship (SAR) of a novel series of cannabinoid 1 (CB1)
and cannabinoid 2 (CB2) receptor ligands are reported. Based on the aminoalkylindole class of canna-
binoid receptor agonists, a biphenyl moiety was introduced as novel lipophilic indole 3-acyl substituent
in 11ꢀ16. Furthermore, the 3-carbonyl tether was replaced with a carboxamide linker in 17ꢀ20 and the
azaindole (pyrrolopyridine) nucleus was designed as indole bioisostere with improved physicochemical
properties in 21ꢀ25. Through these SAR efforts, several high affinity CB1/CB2 dual cannabinoid receptor
ligands were identified. Indole-3-carboxamide 17 displayed single-digit nanomolar affinity and w80 fold
selectivity for CB1 over the CB2 receptor. The azaindoles displayed substantially improved physico-
chemical properties (lipophilicity; aqueous solubility). Azaindole 21 elicited potent cannabinoid activity.
Cannabinoid receptor agonists 17 and 21 potently modulated excitatory synaptic transmission in an
acute rat brain slice model of cannabinoid receptor-modulated neurotransmission.
Received 2 May 2011
Received in revised form
11 August 2011
Accepted 16 August 2011
Available online 23 August 2011
Keywords:
Azaindole
Cannabinoid receptor agonist
Indole
Physicochemical properties
Pyrrolopyridine
Ó 2011 Elsevier Masson SAS. All rights reserved.
Structure-activity relationships
1. Introduction
Recently, therapeutic application in various disorders such as
osteoporosis [9,10], multiple sclerosis [11,12], Alzheimer’s disease
The endocannabinoid system mediates wide-ranging physio-
logical processes through at least two distinct G protein-coupled
receptors (GPCR) and their endogenous ligands which are
controlled by several metabolic pathways [1e3]. The cannabinoid 1
receptor (CB1) subtype is expressed abundantly in the central
nervous system (CNS) and is also found in the periphery [4e6]. In
contrast, the cannabinoid 2 receptor (CB2) is principally expressed
in the immune system and periphery, although recently it has also
been found in the CNS [7,8]. There is 44% overall sequence
homology between CB1 and CB2 receptors, with 68% amino acid
identity in the putative transmembrane regions.
[13,14] as well as other neurodegenerative diseases [15,16], allergy
[17], gastro-intestinal conditions [18], diabetes [19,20], and cancer
[21e24] has attracted considerable attention. Furthermore,
cannabinoid ligands are useful in the management of emesis and
anorexia in cancer and AIDS chemotherapy and wasting syndrome
[25,26]. Although to different extents, activation of CB1 as well as
CB2 receptors results in analgesic activity in models of neuropathic,
inflammatory, chronic and post-operative pain as well as central
hypersensitization and pain initiation [27e36]. Moreover, there is
evidence for cannabinoid-mediated neuroprotection [37,38] and
neurogenesis [39,40].
Activation of the endocannabinoid system results in psychoac-
tive effects and consequently has been the basis of drug abuse.
The herb Cannabis sativa L. contains several active ingredients
which mediate their medicinal and psychoactive effects through
the endocannabinoid system [41]. After the structure of the prin-
cipal active ingredient
D D
9-tetrahydrocannabinol ( 9-THC, 1, Fig. 1)
was elucidated [42], various analogues such as nabilone (2) have
been prepared [43e45]. Simplification of the classical tricyclic
terpenoid structure led to the design of potent non-classical
cannabinoids such as CP-55,940 (3) [45,46]. The aminoalkylindole
* Corresponding author. Chemical Design & Synthesis Unit, Abbott Healthcare
Products B.V., C. J. van Houtenlaan 36, 1381 CP Weesp, The Netherlands. Tel.: þ31
610 05 95 48.
E-mail address: ton@blaazer.nl (A.R. Blaazer).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.021

A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
5087
O
N
OH
O
OH
OH
OH
O
O
O
N
O
HO
1 (Δ9-THC)
2 (Nabilone)
3 (CP-55,940)
4 (WIN-55,212-2)
Fig. 1. Prototypical cannabinoid receptor agonists.
(AAI) class of cannabinoid receptor ligands was conceived by the
disclosure of WIN-55,212-2 (4) [47e49]. Next-generation canna-
binoid receptor agonists based on AAI structure-activity relation-
ship (SAR) studies are exemplified by novel compounds such as
L-768,242 (GW405833, 5, Fig. 2) [50], CRA13 (6) [51], MDA19 (7)
[52], A-796260 (8) [53] and GSK554418A (9) [54]. The rapidly
expanding diversity of cannabinoid receptor agonists has recently
been captured in two excellent reviews [55,56].
Currently, the AAI class of cannabimimetic indoles comprises
numerous compounds [57e61]. It was revealed that the hetero-
cyclic aminoalkyl group attached to the indole nitrogen could be
replaced by a straight alkyl chain, while retaining cannabinoid
activity [62,63]. The n-pentyl analogue JWH-018 (10, Fig. 3) was
found to have high affinity for both cannabinoid receptors with
good in vivo analgesic activity [64,65]. Recently, the SAR of
a large set of indole N-1 side chain modifications has been
reported and the n-pentyl analogue of A-796260 (8) displayed
a potent full CB2 receptor agonist profile [66]. Furthermore, the
Although the indole nucleus is present in many cannabinoid
receptor agonists, various other heterocycles such as benzimid-
azoles, benzoxazoles, indolinones, quinazolines and imidazopyr-
idines
have
also
been
reported
[55,56].
Azaindoles
(pyrrolopyridines), such as GSK554418A (9) [54], have recently
been disclosed as cannabinoid receptor ligands [79e81]. Resulting
from the combination of an electron-deficient pyridine with
a pyrrole ring, azaindoles possess unique physicochemical prop-
erties hypothetically leading to advantageous solubility and
bioavailability [82,83].
Cannabinoid receptors are characterized by lipophilic binding
pockets delimited by amino acid residues which principally interact
with AAI analogues through aromatic stacking [84]. This is reflected
by the physicochemical properties of cannabinoid receptor ligands,
which are inherently lipophilic in nature. It is apparent from
a
survey of oral drug candidates that the most lipophilic
compounds are discontinued from development [85]. Therefore,
optimization of the physicochemical properties of cannabinoid
receptor ligands is of critical importance. To this aim, several
modifications (Fig. 3) have been introduced using dual CB1/CB2
receptor agonist JWH-018 (10) as starting point. The main lipophilic
elements of JWH-018 (10) are the naphthoyl substituent, the alkyl
side chain and the indole nucleus. These considerations motivated
the design of target compounds 11ꢀ25, with the aim of reducing
lipophilicity and increasing aqueous solubility while retaining
cannabinoid activity. To this end, the 3-carbonyl tether of AAI
analogues was replaced by a carboxamide linker. Furthermore, the
bioisosteric replacement of the indole nucleus with azaindole
heterocycles is reported. To broaden the SAR, biphenyl moieties
were explored as novel lipophilic 3-acyl substituents. Also, the n-
pentyl side chain at N-1 was varied with a 2-methoxyethyl side
chain throughout the series.
introduction of
a N-(2-methoxyethyl) side chain resulted in
substantial CB2 selectivity over the CB1 receptor, while retaining
full agonism.
The orientation of the 3-acyl substituent of AAI cannabinoid
agonists has been examined by docking WIN-55,212-2 (4) in CB1
and CB2 homology models, suggesting an out-of-plane orientation
of the naphthoyl group relative to the indole nucleus [67e69].
Various substituted naphthoyl [70,71] and phenylacetyl [72] groups
at indole C-3 are tolerated and thought to interact with the
cannabinoid receptors through aromatic stacking. It was reported
that some of the 1-pentyl-3-phenylacetylindoles combined a full
CB1 agonist character with partial agonism at the CB2 receptor.
The majority of cannabinoids from the AAI class have
a carbonyl tether at C-3 linking the indole nucleus with aromatic
groups. The hydrogen bonding capability of the carbonyl group is
not essential for binding at CB1, since a C-3 methylene tether is
allowed as well [70]. Polar tethers other than carbonyl are also
accepted by the cannabinoid receptors, as evidenced by the SAR of
potent cannabinoid receptor agonists with a C-3 carboxamide
linker [73e78].
2. Chemistry
Biphenyl analogues 11ꢀ16 were synthesized according to
Scheme 1. The unsubstituted indole nucleus was N-alkylated with
the appropriate alkyl halide using NaH in DMF. The 3-acyl
O
O
O
NH
N
N
O
HN
Cl
O
O
N
N
N
N
N
N
O
N
O
O
O
Cl
O
Cl
5 (L-768,242 (GW405833))
6 (CRA13)
7 (MDA19)
8 (A-796260)
9 (GSK554418A)
Fig. 2. Recently reported cannabinoid receptor agonists.

5088
A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
Scheme 4. For the 6-azaindole, intermediate 43 was prepared in
heterocyclic
nucleus
lipophilic
substituent
a Bartoli-type cyclization by reacting 2-chloro-3-nitropyridine 42
with excess vinylmagnesium bromide and quenching with satu-
rated aqueous NH4Cl [93,94]. Remarkably, a novel procedure for the
hydrodehalogenation of halopyridines and quinolines, using cata-
lytic PdCl2(dppf)$CH2Cl2 with TMEDA and NaBH4 in THF [95], was
found applicable for the dehalogenation of 43 leading to key
intermediate 37 under mild conditions. The 5-azaindole nucleus
was prepared following a slightly adapted literature procedure [96].
The 4-amino-3-bromopyridine 44 was converted to acetamide 45
in a straightforward amidation. Stille coupling with (Z)-1-ethoxy-2-
tributylstannylethene and subsequent cyclization with hydro-
chloric acid in methanol afforded 5-azaindole 38.
O
O
4
7
3
5
6
2
N
N
1
linker
side
chain
10 (JWH-018)
Fig. 3. Structure of JWH-018 (10) and the sites of modification.
substituents were introduced by reacting the appropriate acyl
chloride with diethylaluminum chloride in an adapted Friedel-
Crafts acylation [86]. Appropriate biphenyl acyl chlorides were
prepared in high yields by treatment of the corresponding
carboxylic acids with oxalyl chloride and a catalytic amount of DMF.
The indole-3-carboxamides 17ꢀ20 were prepared as depicted in
Scheme 2. Unsubstituted 3-indolecarboxylate methyl ester 33 was
treated with the appropriate alkyl halide affording N-alkylated
intermediates 34 and 35. Base-catalyzed ester hydrolysis of 34 with
KOH, followed by acid chloride formation using oxalyl chloride with
catalytic DMF provided the indole-3-carbonyl chloride interme-
diate in high yield. Subsequent amidation with 2-naphthylamine
produced target compound 18 in unexpected low yield. Alterna-
tively, naphthylamines were treated with trimethylaluminum and
reacted with N-alkylated indolecarboxylate methyl esters 34 and
35, following a Weinreb amidation procedure [87], affording car-
boxamides 17, 19, 20 in high yields.
Reviews of various routes to the synthetically challenging
azaindoles and their reactivity have recently been published
[88e91]. The azaindoles 21ꢀ25 were prepared according to
Scheme 3. The azaindole nucleus (36ꢀ38) underwent Friedel-Crafts
acylation with 1-naphthoyl chloride in the presence of excess
aluminum chloride [92], giving intermediates 39ꢀ41 in low to
moderate yields. It was noted that using excess 1-naphthoyl chlo-
ride resulted in an additional Friedel-Crafts acylation on the
naphthyl ring, hence stoichiometric quantities of 1-naphthoyl
chloride are required. Target compounds 21ꢀ25 were obtained
after N-alkylation with the appropriate alkyl halides.
3. Results and discussion
The CB1 and CB2 receptor binding activities of biphenyl
analogues 11ꢀ16 are reported in Table 1. The ortho-substituted
analogue 13 was found to have the highest affinity for both
cannabinoid receptors, while para-substitution (compound 11)
decreased binding. The meta-substituted biphenyl analogue 12
elicited a high CB1 affinity with w9-fold selectivity over the CB2
receptor. Replacing the n-pentyl side chain with a 2-methoxyethyl
side chain at the indole nitrogen was hypothesized to lead to
compounds with increased CB2 selectivity (vide supra), however
this resulted in compounds (14ꢀ16) with reduced affinity for CB2
as well as CB1 receptors. As is apparent from the data, none of the
compounds with a 2-methoxyethyl side chain has appreciable
affinity for the CB2 receptor. Although in the low submicromolar
range, the meta and ortho-substituted biphenyl analogues (15 and
16) possess a higher CB1 receptor binding affinity than their
para-substituted congener, a trend also seen in the n-pentyl
derivatives.
Replacement of the 3-carbonyl tether by an amide group led to
indole-3-carboxamides 17ꢀ20, reported in Table 2. Surprisingly,
amide 17 displayed a very high affinity for CB1 (which equals the
pKi value of the potent reference compound CP-55,940), with 79-
fold selectivity over the CB2 receptor. Although some CB1-selec-
tive agonists are known [55], substantial selectivity for CB1 over the
CB2 receptor is a feature generally lacking from the AAI class of
cannabinoid receptor ligands. Changing the naphthylamine orien-
tation of 17 to compound 18 resulted in a 50-fold decrease in
affinity for the CB1 receptor, although some selectivity was
retained. Introduction of the 2-methoxyethyl side chain in this
The 7-azaindole nucleus 36 was commercially available.
The synthesis of 6-azaindole 37 and 5-azaindole 38 is shown in
(a)
O
O
HO
Cl
26: m-biphenyl
27: o-biphenyl
28: m-biphenyl
29: o-biphenyl
11: R1 = n-pentyl, R2 = p-biphenyl
12: R1 = n-pentyl, R2 = m-biphenyl
13: R1 = n-pentyl, R2 = o-biphenyl
14: R1 = 2-MeOEt, R2 = p-biphenyl
15: R1 = 2-MeOEt, R2 = m-biphenyl
16: R1 = 2-MeOEt, R2 = o-biphenyl
O
R2
(b)
(c)
N
H
N
N
28, 29
R1
R1
31: R1 = n-pentyl
30
32: R1 = 2-MeOEt
Scheme 1. Reagents and conditions: (a) oxalyl chloride, DMF (cat), CH2Cl2, 0 ꢁC to rt, 16 h, 95ꢀ99%; (b) alkyl halide, NaH, DMF, 0 ꢁC to rt, 16 h, 75ꢀ95%; (c) Et2AlCl,
CH2Cl2, ꢀ10 ꢁCe0 ꢁC, 4 h, 59ꢀ87%.

A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
5089
O
NH
N
(b), (c), (d)
18
O
O
O
O
(a)
N
N
17: R1 = n-pentyl, R2 = 1-naphthyl
H
R1
(e)
18: R1 = n-pentyl, R2 = 2-naphthyl
19: R1 = 2-MeOEt, R2 = 1-naphthyl
20: R1 = 2-MeOEt, R2 = 2-naphthyl
R2
NH
34: R1 = n-pentyl
35: R1 = 2-MeOEt
O
33
N
R1
17, 19, 20
Scheme 2. Reagents and conditions: (a) alkyl halide, NaH, DMF, 0 ꢁC to rt, 24 h, 57ꢀ95%; (b) KOH, H2O, EtOH, reflux, 20 h, 91%; (c) oxalyl chloride, DMF (cat), CH2Cl2, ꢀ10 ꢁC to rt,
16 h, 94%; (d) R2NH2, DIPEA, CH2Cl2, rt, 62 h, 24%; (e) R2NH2, Al(CH3)3, 1,2-dichloroethane, reflux, 16 h, 85ꢀ92%.
series (compounds 19 and 20) resulted in a considerable reduction
in affinity for the CB1 receptor. Compound 19 displayed some
selectivity for CB2 and binds with submicromolar affinity to this
receptor.
affinity for both cannabinoid receptors and was non-selective. The
n-pentyl substituted 6-azaindole 23 and 5-azaindole 25 elicited
reduced CB1 receptor binding affinities but retained agonist activ-
ities. Compound 25 displayed w5-fold binding preference for the
CB2 receptor and demonstrated 98% stimulation in the CB2 receptor
functional assay. Both 6-azaindoles 23 and 24 displayed 97ꢀ100%
stimulation in the CB2 functional assay, furthermore 2-
methoxyethyl analogue 24 was at least 35-fold selective for the
CB2 receptor. When compared with its indole parent JWH-018 (10),
it can be seen that the affinity as well as the activity of azaindoles
21ꢀ25 is decreased for both cannabinoid receptors. Nevertheless,
the CB1 potencies of 7-azaindole 21 as well as the CB2 receptor
preference of 5-azaindole 25 demonstrate that the azaindole
nucleus can be used as effective indole bioisostere in this class of
cannabinoid receptor ligands.
The predicted and experimental physicochemical properties of
prototypical cannabinoid receptor agonists nabilone (2), CP-55,940
(3), WIN-55,212-2 (4), JWH-018 (10) and target compounds 11ꢀ25
are reported in Table 3. The biphenyl analogues with n-pentyl side
chain (11ꢀ13) were predicted to be more lipophilic than their
naphthyl parent JWH-018 (10). The effect of replacing the 3-
carbonyl tether of JWH-018 (10) with a carboxamide linker was
found to result in the slightly improved predicted lipophilicity of
amides 17 and 18. From the experimental logDpH7.4 of biphenyl
In analogues of A-796260 (8), the observed selectivity for CB2
over the CB1 receptor in the 2-methoxyethyl (w230-fold) versus
the n-pentyl (w80-fold) side chain analogue is considerable [66].
This trend is also seen with 17 compared to 19. Moreover,
throughout the indole series, the 2-methoxyethyl side chain was
found to lead to a consistent overall reduction in affinity. This
effect might be attributed to a decrease of van der Waals inter-
actions from the more polar 2-methoxyethyl chain compared to
the n-pentyl side chain with the cannabinoid receptors. In addi-
tion, the degree of entropy gain by hydrophobic binding is
expected to be lower. However, with respect to carboxamides 19
and 20, the 2-methoxyethyl side chain was found to suppress CB1
receptor binding affinity. Despite its modest CB2 receptor affinity
(pKi ¼ 6.6), amide 19 was a potent full agonist at the CB2 receptor
(pEC50 ¼ 8.2).
The azaindole nucleus was designed as indole bioisostere,
leading to compounds 21ꢀ25 (Table 2). The 7-azaindole analogue
21 displayed potent binding to CB1, maintained full agonist activity
and demonstrated a minor 4-fold binding preference over the CB2
receptor. Its 2-methoxyethyl counterpart 22 exhibited reduced
O
O
(a)
(b)
N
N
N
N
H
N
H
N
R
21: 7-azaindole, R1 = n-pentyl
22: 7-azaindole, R1 = 2-MeOEt
23: 6-azaindole, R1 = n-pentyl
24: 6-azaindole, R1 = 2-MeOEt
25: 5-azaindole, R1 = n-pentyl
36: 7-azaindole
37: 6-azaindole
38: 5-azaindole
39: 7-azaindole
40: 6-azaindole
41: 5-azaindole
Scheme 3. Reagents and conditions: (a) 1-naphthoyl chloride, AlCl3, CH2Cl2, rt, 6ꢀ16 h, 11ꢀ60%; (b) alkyl halide, NaH, DMF, 0 ꢁC to rt, 6ꢀ24 h; 16ꢀ84%.

5090
A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
(a)
(b)
N
N
N
N
N
N
N
H
N
H
NO2
Cl
42
Cl
43
37
Br
Br
(c)
(d), (e)
O
N
H
N
H
NH2
44
45
38
Scheme 4. Reagents and conditions: (a) vinylmagnesium bromide, THF, ꢀ78 ꢁC to ꢀ20 ꢁC, 16 h; followed by quench with NH4Cl (aq), 0 ꢁC, 38%; (b) PdCl2(dppf)$CH2Cl2, TMEDA,
NaBH4, THF, rt, 24 h, 71%; (c) acetyl chloride, DIPEA, CH2Cl2, 5 ꢁC to rt, 16 h, 89%; (d) (Z)-(EtO-HC ¼ CH)SnBu3, Pd(PPh3)2Cl2, TBAC, MeCN, reflux, 2.5 h, 68%; (e) HCl, MeOH, reflux,
5 h, 67%.
analogues 14ꢀ16 as well as amides 19 and 20, it can be appreciated
that replacing the n-pentyl at N-1 with a 2-methoxyethyl side chain
leads to a substantially lower lipophilicity, this aspect was further
improved by replacing the carbonyl tether with a carboxamide
linker.
a slightly improved solubility when compared with its indole
parent JWH-018 (10). The solubility of the other azaindoles was
found to be improved, culminating with the good solubility of 5-
azaindole 25. These findings are in agreement with the kinetic
(turbidimetric) aqueous solubility of selected compounds.
The predicted lipophilicity of 7-azaindole 21 (clogD7.4 ¼ 4.99)
was well below its indole parent JWH-018 (10, clogD7.4 ¼ 6.86), the
experimental logDpH7.4 of 21 was >5. The other azaindoles (22ꢀ25)
had greatly improved, that is, decreased experimental logDpH7.4
values when compared with JWH-018 (10). The location of the
fused pyridine nitrogen seems to have a substantial effect on the
lipophilicity of the azaindoles, since the experimental logDpH7.4
value of each n-pentyl variant (23, 25) and 2-methoxyethyl variant
(22, 24) is lower with the more exposed pyridyl nitrogen atom.
All target compounds (11ꢀ25) had a calculated topological polar
surface area (TPSA <60 Å2) suitable for CNS penetration [97]. The
thermodynamic aqueous solubility of cannabinoid receptor
agonists 2ꢀ4, JWH-018 (10), substituted indoles 11ꢀ20 and azain-
doles 21ꢀ25 has been determined experimentally at pH 7.4 and pH
4 (Table 3). Nabilone (2), marketed for its antiemetic and analgesic
properties, was found to have a low thermodynamic solubility
The incorporation of heteroatoms in the aromatic moiety of the
central indole nucleus was hypothesized to lead to a decrease in
lipophilicity as well as improving the aqueous solubility of the
resulting compounds. Azaindoles provide an additional site for
potential polar interactions within the receptor and the pyridyl
nitrogen has hydrogen bonding capabilities [82,83]. Furthermore,
azaindoles are moderately basic, unsubstituted 6-azaindole (37)
is reported to have a pKa value of 8 [98], while the substituted
5-azaindole GSK554418A (9) has a pKa value of 6.1 [54]. The
predicted and experimental pKa of the reference compounds 2ꢀ4
and azaindoles 21ꢀ25 are reported in Table 3. Overall, the
experimental pKa of the azaindoles are somewhat below the
predicted values. The 7-azaindoles 21 and 22 displayed the lowest
experimental pKa, which was found to be increased in the
6-azaindoles 23 and 24. Compound 25 was the most basic
azaindole from the series, with an experimental pKa value of 6.5,
slightly surpassing the 5-azaindole cannabinoid CB2 receptor
agonist GSK554418A (9).
Membrane permeability data for cannabinoid agonists CP-
55,940 (3), WIN-55,212-2 (4) as well as carboxamide 19 and
selected azaindoles (22, 23, 25) are summarized in Table 3. All
tested compounds demonstrated good permeability in Caco-2 cell
lines, a model for gastrointestinal absorption [99], with no indica-
tion of active efflux transport. Furthermore, no P-glycoprotein
transport of CP-55,940 (3) (AI < 2) was found in our in-house PK1
LLC MDR assay [100].
(<1 mM). CP-55,940 (3) was found to have a slightly better solu-
bility, whereas the prototypical AAI agonist WIN-55,212-2 (4)
showed the highest solubility of the reference compounds. The
thermodynamic solubility of JWH-018 (10), biphenyl analogues
(11ꢀ16) as well as amides 17, 18 and 20 was low (<1
mM), with
amide 19 being a notable exception. The 7-azaindole 21 displayed
Table 1
Cannabinoid receptor binding data of JWH-018 (10) and biphenyl analogues 11ꢀ16.
The preferred sites of metabolism by cytochrome P450 (CYP)
enzymes has been assessed for compounds 11ꢀ25 with MetaSite
software [101,102]. The predicted primary site of metabolism was
the N-1 side chain, followed by multiple sites at the aromatic
naphthyl and phenyl portions, as well as the indole C-6 position.
The 2-methoxyethyl side chain was predicted to be slightly more
resistant to metabolism than its n-pentyl counterpart. All sites
predicted by MetaSite are in accordance with the recently reported
metabolites and proposed metabolic pathways of JWH-018 (10)
[103], and its 2-methyl-1-propyl analogue JWH-015 [104].
The properties of the most potent compounds, carboxamide 17
and 7-azaindole 21 prompted further characterization of these
compounds in an ex vivo model of cannabinoid receptor-
modulated neurotransmission. Activation of presynaptic CB1
Compound
CB1 bindinga
CB2 bindingb
Selectivityc
pKi ꢂ SEM
pKi ꢂ SEM
CB2/CB1
10 (JWH-018)
8.7 ꢂ 0.1
7.5 ꢂ 0.3
8.1 ꢂ 0.1
8.2 ꢂ 0.1
<6
8.2 ꢂ 0.1
7.4 ꢂ 0.1
7.2 ꢂ 0.1
8.1 ꢂ 0.1
<6
<6
6.2 ꢂ 0.1
3
1
9
1
11
12
13
14
15
16
6.6 ꢂ 0.4
>4
3
6.6 ꢂ 0.3
a
Binding assay for human CB1 receptors stably transfected in CHO cells using [3H]
CP-55,940 as radioligand. All values represent the average of at least three inde-
pendent determinations.
b
Binding assay for human CB2 receptors stably transfected in CHO cells using [3H]
CP-55,940 as radioligand. All values represent the average of at least three inde-
pendent determinations.
c
Receptor subtype selectivity based on CB2 (Ki)/CB1 (Ki).
receptors leads to a decrease in neurotransmitter release

A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
5091
Table 2
In vitro pharmacological evaluation of compounds 2ꢀ4, JWH-018 (10), and target compounds 17ꢀ25.
Compound
CB1 bindinga
CB1 functionalb
CB2 bindingc
CB2 functionald
Selectivitye
pKi ꢂ SEM
pEC50 ꢂ SEM
pKi ꢂ SEM
pEC50 ꢂ SEM
CB2/CB1
2 (Nabilone)
3 (CP-55,940)
4 (WIN-55,212-2)
10 (JWH-018)
17
18
19
20
21
22
23
24
25
8.4 ꢂ 0.1
8.9 ꢂ 0.1
7.1 ꢂ 0.1
8.7 ꢂ 0.1
8.9 ꢂ 0.1
7.2 ꢂ 0.2
<6
8.4 ꢂ 0.1
9.2 ꢂ 0.3
7.5 ꢂ 0.4
8.3 ꢂ 0.2
8.0 ꢂ 0.1
<6
8.2 ꢂ 0.2
9.3 ꢂ 0.1
8.2 ꢂ 0.1
8.2 ꢂ 0.1
7.0 ꢂ 0.1
6.2 ꢂ 0.3
6.6 ꢂ 0.1
6.5 ꢂ 0.1
7.3 ꢂ 0.2
6.7 ꢂ 0.1
6.3 ꢂ 0.1
7.6 ꢂ 0.2
7.3 ꢂ 0.1
7.8 ꢂ 0.4
9.1 ꢂ 0.4
8.8 ꢂ 0.1
8.3 ꢂ 0.3
8.1 ꢂ 0.2
5.3 ꢂ 0.1
8.2 ꢂ 0.1g
NDf
1.7
0.4
0.07
3
79
10
<0.3
<0.3
4
0.4
3
NDf
<6
NDf
7.9 ꢂ 0.2
6.3 ꢂ 0.1
6.8 ꢂ 0.2
<6
7.5 ꢂ 0.1
6.8 ꢂ 0.2g
<6
<6
7.0 ꢂ 0.1
<6
7.3 ꢂ 0.1
NDf
NDf
<0.03
0.2
6.5 ꢂ 0.2
NDf
a
Binding assay for human CB1 receptors stably transfected in CHO cells using [3H]CP-55,940 as radioligand. All values represent the average of at least three independent
determinations.
b
c
Functional human CB1 receptor [3H]-arachidonic acid (AA) release assay in CHO cells. All values represent the average of at least three independent determinations.
Binding assay for human CB2 receptors stably transfected in CHO cells using [3H]CP-55,940 as radioligand. All values represent the average of at least three independent
determinations.
d
Functional human CB2 receptor cAMP accumulation assay in CHO cells. Values represent the average of at least three independent determinations, unless otherwise noted.
e
f
Receptor subtype selectivity based on CB2 (Ki)/CB1 (Ki).
ND ¼ not determined.
g
Result of two independent determinations.
probability in various cortical regions [105,106]. The prefrontal
cortex (PFC) is critically implicated in higher cognitive functions
[107e109], and plays a role in the cannabinoid hypothesis of
schizophrenia [110,111]. An extracellular electrophysiological
method was used to study the effects of CB1/CB2 receptor agonist
JWH-018 (10) and target compounds 17 and 21 on cannabinoid
receptor-modulated neurotransmission. Field potentials were
recorded in an acute brain slice preparation of the rat prelimbic PFC.
Reference compound JWH-018 (10) was revealed to reduce the field
excitatory postsynaptic potential (fEPSP) amplitude in a concen-
tration-dependent fashion to 27% of baseline, with an IC50 of
0.28
(IC50 ¼ 0.52
m
M (Fig. 4). Target compounds 17 (IC50 ¼ 0.45
mM) and 21
m
M) were found to reduce the fEPSP amplitude to
22ꢀ33% of baseline, in a manner similar to JWH-018 (10). The
current results are in line with the recently reported effects of JWH-
018 (10) in cultured hippocampal neurons and the notion that CB1
receptor activation by 10 leads to a potent inhibition of excitatory
synaptic transmission in certain brain areas [112].
Table 3
Predicted and experimental physicochemical properties of compounds 2ꢀ4, JWH-018 (10), substituted indoles 11ꢀ20 and azaindoles 21ꢀ25.
Compound
ACD/
Thermodynamic solubility
Kinetic
ACD/
pKa
Permeability
LogDa
solubilityd
e
AIh
b
f
g
logDpH7.4
pH 7.4c
pH 4c
pKa
Papp
2 (Nabilone)
3 (CP-55,940)
4 (WIN-55,212-2)
10 (JWH-018)
7.25
6.20
5.05
6.86
7.61
7.73
7.24
5.49
5.61
5.13
5.97
5.97
3.85
3.85
4.99
2.91
5.39
3.32
5.02
>5
>5
<1
2
5
<1
2
8
9.67
10.25
6.66
10.60 ꢂ 0.05
11.16 ꢂ 0.07
4.14 ꢂ 0.03
30/100
3/20
5.7
12.1
1.92
0.74
4.08 ꢂ 0.01
>5
<1
<1
<1
<1
<1
<1
<1
<1
<1
3
<1
<1
NDi
NDi
<1
<1
<1
<1
<1
3
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
>5
>5
>5
4.5 ꢂ 0.1
4.4 ꢂ 0.1
3.31 ꢂ 0.01
>5
>5
2.94 ꢂ 0.01
3.69 ꢂ 0.01
>5
3.57 ꢂ 0.01
4.72 ꢂ 0.06
3.13 ꢂ 0.01
4.44 ꢂ 0.08
30/100
27
0.35
<1
1
3
<1
1
3.60
3.32
6.96
6.72
7.75
<2
<2
5.83 ꢂ 0.07
5.80 ꢂ 0.06
6.49 ꢂ 0.07
3
10/65
3/10
26
13
0.37
0.4
4
NDi
NDi
133
NDi
5
10/30
10
0.7
a
Predicted lipophilicity clogD (pH 7.4) with ACD/LogD v12.
b
c
d
e
f
Experimental log D determined at pH 7.4 with measureable range logDpH7.4 1.0e5.0.
Thermodynamic aqueous solubility ( M), determined at pH 7.4 and at pH 4.
Kinetic (turbidimetric) aqueous solubility ( M), expressed as minimal/maximal value.
m
m
Predicted acid dissociation constant (pKa) with ACD/pKa v12.
Experimental acid dissociation constant (pKa).
g
h
i
Membrane permeability in Caco-2 cells, apical to basolateral (AB), expressed in ꢃ 10ꢀ6 cm/s.
Asymmetry index (AI) of Caco-2 cell membrane permeability, ratio of BA/AB transfer rate.
ND ¼ not determined.

5092
A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
silica (SigmaeAldrich). Proton NMR spectra were recorded on
a Bruker AV400 instrument (400 MHz) with chemical shifts ( in
d
ppm) reported relative to trimethylsilane as internal standard.
Exact mass measurement (HRMS) was performed on a Waters
Micromass QTOF-2 equipped with ESI spray and APCI spray ioni-
zation and Waters Micromass GCT (CA049) equipped with EI
source. LC/MS analysis was performed using a Waters SunFire C18
HPLC column, 2.5
HPLC Pump 1525
m
m
m, 4.6 ꢃ 30 mm, equipped with a Waters Binary
and a Waters 2777 Sample Manager, coupled to
a Waters Photodiode Array Detector 2996 (wavelength (l) range
240e320 nm), Waters ELS Detector 2424 and a Waters ZQ MM1
mass detector. The reported yields refer to isolated pure products,
yields were not optimized. The purity of all final compounds was
established as ꢄ95% based on LC/MS/UV/ELSD.
Fig. 4. Effects of JWH-018 (10) and target compounds 17 and 21 on field potentials
recorded in layer II of the rat prelimbic PFC. All compounds reduced the amplitude of
5.1.1. [1,10-Biphenyl]-3-carbonyl chloride (28)
the fEPSP in
intervals: 10 (0.28
0.28e0.97
a
concentration-dependent manner. Mean IC50 and 95% confidence
To a
solution of [1,10-biphenyl]-3-carboxylic acid (2.00 g,
10.09 mmol) in 30 mL CH2Cl2 at ꢀ5 ꢁC, was added a catalytic
amount of anhydrous DMF (0.01 mL, 0.10 mmol). A solution of
oxalyl chloride (1.28 mL, 15.13 mmol) in 10 mL CH2Cl2 was added
dropwise, the release of gasses was noted. The reaction was allowed
to increase to ambient temperature, continuing overnight (16 h).
The reaction mixture was concentrated in vacuo, affording 2.37 g of
acid chloride 28 (98% yield), which was used immediately without
further characterization.
m
M; 0.12e0.66 M); 17 (0.45 M; 0.33e0.62 M); 21 (0.52 M;
m
m
m
m
m
M). Mean Emax ꢂ SEM: 10 (73.5% ꢂ 10.8); 17 (78.1% ꢂ 4.1); 21 (67.1% ꢂ 6.6).
Data points are normalized to baseline and represent mean values ꢂ SEM; for each
data point n > 3 (range 3e24) determinations.
4. Conclusions
In conclusion, the discovery, synthesis and physicochemical
properties of a novel series of cannabinoid receptor ligands are
described. It is apparent from biphenyl analogues 11ꢀ16 that the
position of the terminal phenyl substituent plays an important role
in binding to cannabinoid receptors. The carboxamide linked
compounds 17ꢀ20 highlight the complex SAR of cannabinoid
receptor ligands. Replacing the C-3 carbonyl linker with a carbox-
amide tether provided access to 17, which displayed a very high CB1
receptor potency and subtype selectivity. Furthermore, the bio-
isosteric replacement of the indole moiety with the azaindole
nucleus provided compounds 21ꢀ25 with substantially improved
physicochemical properties (lipophilicity and aqueous solubility).
The 7-azaindole derivative 21 elicited potent CB1 receptor activity,
whereas the 6-azaindole congener 24 showed distinct CB2 receptor
subtype selectivity. Compounds 17 and 21 were found to potently
inhibit excitatory synaptic transmission in an electrophysiological
brain slice model of cannabinoid receptor-modulated neurotrans-
mission. Overall, novel series of cannabinoid receptor ligands have
been disclosed which provide viable starting points for further
optimization of physicochemical properties as well as cannabinoid
receptor subtype selectivity.
5.1.2. [1,10-Biphenyl]-2-carbonyl chloride (29)
To
a
solution of [1,10-biphenyl]-2-carboxylic acid (2.00 g,
10.09 mmol) in 20 mL CH2Cl2 at ꢀ5 ꢁC, was added a catalytic
amount of anhydrous DMF (0.01 mL, 0.10 mmol). This was followed
by the dropwise addition of oxalyl chloride (1.28 mL,15.13 mmol) in
5 mL CH2Cl2, the reaction expelled gasses and was allowed to
increase to ambient temperature, continuing overnight (16 h). The
reaction mixture was concentrated in vacuo, yielding 2.24 g of acid
chloride 29 (97% yield), which was used immediately without
further characterization.
5.1.3. 1-Pentyl-1H-indole (31)
1H-indole (2.50 g, 21.36 mmol) in 20 mL DMF was added to
a solution of NaH (60% dispersion in mineral oil,1.03 g, 25.63 mmol)
in 15 mL DMF, at 5 ꢁC. The mixture was allowed to warm to ambient
temperature and was stirred for 30 min. The mixture was cooled to
5 ꢁC and 1-bromopentane (3.17 mL, 25.63 mmol) in 5 mL DMF was
added dropwise to the solution, after addition was complete the
mixture was allowed to warm to ambient temperature. After 16 h,
the mixture was cooled to 5 ꢁC, quenched with 40 mL H2O and
extracted with 40 mL Et2O. The aqueous fraction was extracted with
Et2O (2 ꢃ 20 mL) and the combined organic fractions were
concentrated in vacuo. Automated flash chromatography (Et2O/
petroleum ether 1:19) was used to separate the reaction products,
yielding 31 (4.01 g, 95% yield) as yellow oil. 1H NMR (400 MHz,
5. Experimental section
5.1. Chemistry
All solvents and reagents were obtained from commercial
sources and were used as received. All reactions were carried out
under inert N2 atmosphere and magnetically stirred. Reaction
progress was followed with thin-layer chromatography (TLC silica
gel 60 glass-backed plates with F254 indicator, Merck) and LC/MS
analysis. TLC-MS (CAMAG TLC-MS Interface coupled to PE SCIEX API
150EX LC/MS system) was used to identify reaction products by
mass detection on TLC plates. Flash chromatography was per-
formed using silica gel 60 (0.040e0.063 mm, Merck). Column
chromatography was performed using silica gel 60
(0.063e0.200 mm, Merck). Automated flash chromatography was
performed on a Sepacore system (Büchi AG) consisting of a C-660
fraction collector equipped with C-630 UV Monitor, C-615 Pump
Manager and two C-605 Pump Modules, using Versapak disposable
CDCl3)
d
7.63 (d, J ¼ 7.9 Hz, 1H), 7.34 (d, J ¼ 8.2 Hz, 1H), 7.20
(t, J ¼ 7.6 Hz, 1H), 7.13e7.04 (m, 2H), 6.48 (d, J ¼ 3.0 Hz, 1H), 4.11
(t, J ¼ 7.2 Hz, 2H), 1.90e1.76 (m, 2H), 1.42e1.02 (m, 4H), 0.88
(t, J ¼ 6.9 Hz, 3H).
5.1.4. 1-(2-Methoxyethyl)-1H-indole (32)
1H-indole (3.00 g, 25.61 mmol) in 20 mL DMF was added to
a solution of NaH (60% dispersion in mineral oil,1.23 g, 30.73 mmol)
in 35 mL DMF at 0 ꢁC. The mixture was stirred for 30 min at ambient
temperature, cooled to
0
ꢁC and 1-bromo-2-methoxyethane
(2.89 ml, 30.73 mmol) was slowly added to the solution. The
reaction mixture was allowed to warm to ambient temperature and
the solution was stirred for 16 h. The reaction was quenched by
slowly pouring the reaction mixture in iced HCl (1 M), followed by
cartridges prepacked with 20e45 mm, matrix active group spherical

A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
5093
the addition of Et2O. The organic fraction was washed with 1 M HCl
( ꢃ 2) and concentrated in vacuo. Automated flash chromatography
(EtOAc/petroleum ether 1:6) was used to furnish pure 32 (3.56 g,
in hexanes, 8.13 mL, 8.13 mmol) in 5 mL CH2Cl2. The mixture was
stirred for 45 min at ꢀ5 ꢁC whereafter [1,10-biphenyl]-4-carbonyl
chloride (1.76 g, 8.13 mmol) in 25 mL CH2Cl2 was added drop-
wise. The orange acylation reaction was stirred at 0 ꢁC for 4 h,
carefully poured in iced HCl (1 M), extracted with CH2Cl2 ( ꢃ 2) and
concentrated in vacuo. The crude product was dissolved in CH2Cl2
and washed twice with a solution of K2CO3 (15 g in 100 mL H2O)
and purified by automated flash chromatography (EtOAc/petro-
leum ether, 2:5 and then CH2Cl2) to provide 14 (1.54 g, 76% yield) as
75% yield). 1H NMR (400 MHz, CDCl3)
d
7.62 (d, J ¼ 7.9 Hz, 1H), 7.33
(dd, J ¼ 8.2, 0.7 Hz, 1H), 7.24e7.05 (m, 3H), 6.48 (dd, J ¼ 3.2, 0.8 Hz,
1H), 4.24 (t, J ¼ 5.6 Hz, 2H), 3.66 (t, J ¼ 5.6 Hz, 2H), 3.28 (s, 3H).
5.1.5. [1,10-Biphenyl]-4-yl(1-pentyl-1H-indol-3-yl)methanone (11)
To a solution of 31 (0.76 g, 4.02 mmol) in 20 mL CH2Cl2 at ꢀ15 ꢁC
was added dropwise diethylaluminum chloride (1 M in hexanes,
6.03 mL, 6.03 mmol). After 30 min, [1,10-biphenyl]-4-carbonyl
chloride (1.31 g, 6.03 mmol) in 35 mL CH2Cl2 was added dropwise.
The temperature was allowed to increase to 0 ꢁC and the resulting
mixture was stirred for 4 h. The reaction was quenched by slowly
pouring the mixture in iced HCl (1 M) at 5 ꢁC, followed by extraction
with CH2Cl2. The aqueous layer was extracted with CH2Cl2 ( ꢃ 2) and
the combined organic fractions were concentrated in vacuo. The
obtained crude material was dissolved in Et2O and washed twice
with a solution of K2CO3 (10 g in 100 mL H2O), concentrated in vacuo
and purified by automated flash chromatography (Et2O/petroleum
ether, 1:4) to afford 11 (1.12 g, 72% yield) as a gum. 1H NMR
yellow flakes. 1H NMR (400 MHz, CDCl3)
d 8.52e8.42 (m, 1H), 7.92
(d, J ¼ 8.3 Hz, 2H), 7.77e7.63 (m, 5H), 7.54e7.29 (m, 6H), 4.34
(t, J ¼ 5.3 Hz, 2H), 3.74 (t, J ¼ 5.2 Hz, 2H), 3.31 (s, 3H); HRMS
(C24H22NO2) [M þ H]þ m/z calcd 356.1651, found 356.1673.
5.1.9. [1,10-Biphenyl]-3-yl(1-(2-methoxyethyl)-1H-indol-3-yl)
methanone (15)
To a solution of 32 (1.00 g, 5.42 mmol) in 25 mL CH2Cl2 at ꢀ5 ꢁC
was added diethylaluminum chloride (1 M in hexanes, 8.13 mL,
8.13 mmol) in 5 mL CH2Cl2. The mixture was stirred for 45 min,
freshly prepared 28 (1.96 g, 8.13 mmol) in 15 mL CH2Cl2 was added
at ꢀ5 ꢁC and the reaction was allowed to reach ambient tempera-
ture overnight (16 h). The reaction was quenched by slowly pouring
the solution in iced HCl (1 M, 0 ꢁC) and extracted with CH2Cl2 ( ꢃ 3).
The combined organic fractions were washed twice with K2CO3
(15 g in 100 mL H2O) and concentrated in vacuo. The mixture was
purified by automated flash chromatography (EtOAc/petroleum
ether, 1:2 and then CH2Cl2), excess solvents were removed from the
sample in a vacuum oven (85 ꢁC, 50ꢀ100 mbar, 4 h), to give target
(400 MHz, CDCl3) d 8.50e8.40 (m,1H), 7.95e7.86 (m, 2H), 7.75e7.60
(m, 5H), 7.54e7.25 (m, 6H), 4.17 (t, J ¼ 7.2 Hz, 2H),1.97e1.79 (m, 2H),
1.52e1.28 (m, 4H), 0.89 (t, J ¼ 6.9 Hz, 3H); HRMS (C26H26NO)
[M þ H]þ m/z calcd 368.2014, found 368.2038.
5.1.6. [1,10-Biphenyl]-3-yl(1-pentyl-1H-indol-3-yl)methanone (12)
To a solution of 31 (1.25 g, 6.61 mmol) in 20 mL CH2Cl2 at ꢀ10 ꢁC
was added dropwise diethylaluminum chloride (1 M in hexanes,
9.91 mL, 9.91 mmol). After 45 min, all the freshly prepared 28 in
25 mL CH2Cl2 was added dropwise and the resulting mixture was
stirred for 4 h at 0 ꢁC. The reaction mixture was successively poured
into iced HCl (1 M), extracted with CH2Cl2, the aqueous layer was
extracted with CH2Cl2 ( ꢃ 3) and the combined organic fractions
were concentrated in vacuo. The obtained crude material was dis-
solved in Et2O and washed three times with a solution of K2CO3
(15 g in 100 mL H2O), concentrated in vacuo and purified using
automated flash chromatography (Et2O/petroleum ether, 1:4) to
give 12 (2.12 g, 87% yield) as gray oil. 1H NMR (400 MHz, CDCl3)
compound 15 (1.46 g, 72%). 1H NMR (400 MHz, CDCl3)
d 8.52e8.43
(m, 1H), 8.06 (t, J ¼ 1.6 Hz, 1H), 7.85e7.75 (m, 2H), 7.73e7.62 (m,
3H), 7.57 (t, J ¼ 7.7 Hz,1H), 7.51e7.30 (m, 6H), 4.33 (t, J ¼ 5.2 Hz, 2H),
3.72 (t, J ¼ 5.2 Hz, 2H), 3.27 (s, 3H); HRMS (C24H22NO2) [M þ H]þ
m/z calcd 356.1651, found 356.1656.
5.1.10. [1,10-Biphenyl]-2-yl(1-(2-methoxyethyl)-1H-indol-3-yl)
methanone (16)
To a solution of 32 (1.00 g, 5.42 mmol) in 20 mL CH2Cl2 at ꢀ5 ꢁC
was added dropwise a solution of diethylaluminum chloride (1 M
in hexanes, 8.13 mL, 8.13 mmol) in 10 mL CH2Cl2. The mixture was
stirred for 45 min at ꢀ5 ꢁC whereafter a solution of freshly prepared
29 (1.76 g, 8.13 mmol) in 10 mL CH2Cl2 was added. The acylation
reaction was allowed to continue for 3.5 h at 5 ꢁC, and was
quenched by slowly pouring the solution in iced HCl (1 M), fol-
lowed by extraction with CH2Cl2 ( ꢃ 3). The combined organic
fractions were concentrated in vacuo. The crude product was
purified by automated flash chromatography (CH2Cl2), to yield 16
d
8.50e8.41 (m, 1H), 8.05 (t, J ¼ 1.6 Hz, 1H), 7.78 (ddt, J ¼ 6.8, 3.0,
1.2 Hz, 2H), 7.68e7.51 (m, 4H), 7.50e7.29 (m, 6H), 4.14 (t, J ¼ 7.2 Hz,
2H), 1.94e1.76 (m, 2H), 1.34e1.28 (m, 4H), 0.87e0.85 (m, 3H);
HRMS (C26H26NO) [M þ H]þ m/z calcd 368.2014, found 368.2035.
5.1.7. [1,10-Biphenyl]-2-yl(1-pentyl-1H-indol-3-yl)methanone (13)
To a solution of 31 (1.25 g, 6.61 mmol) in 20 mL CH2Cl2 at ꢀ10 ꢁC
was added dropwise diethylaluminum chloride (1 M in hexanes,
9.91 mL, 9.91 mmol). After 45 min, freshly prepared 29 (2.15 g,
9.91 mmol) in 25 mL CH2Cl2 was added dropwise and the resulting
mixture was stirred for 4 h at 0 ꢁC. The reaction mixture was poured
in iced HCl (1 M) and extracted with CH2Cl2, the aqueous layer was
extracted with CH2Cl2 ( ꢃ 4) and the combined organic fractions
were concentrated in vacuo. The crude material was dissolved in
Et2O andwashedthreetimes with asolution ofK2CO3 (15g in 100 mL
H2O) and concentrated in vacuo. Automated flash chromatography
(Et2O/petroleum ether, 1:4 and then CH2Cl2) gave 13 (2.15 g, 88%
(1.21 g, 60% yield). 1H NMR (400 MHz, CDCl3)
d 8.32e8.16 (m, 1H),
7.58e7.37 (m, 6H), 7.34e7.26 (m, 4H), 7.25e7.12 (m, 3H), 4.17
(t, J ¼ 5.4 Hz, 2H), 3.60 (t, J ¼ 5.4 Hz, 2H), 3.24 (s, 3H); HRMS
(C24H22NO2) [M þ H]þ m/z calcd 356.1651, found 356.1647.
5.1.11. Methyl 1-pentyl-1H-indole-3-carboxylate (34)
Methyl 1H-indole-3-carboxylate (3.00 g, 17.12 mmol) in 25 mL
DMF, was added to a solution of NaH (60% dispersion in mineral oil,
0.82 g, 20.55 mmol) in 35 mL DMF at 0 ꢁC. After stirring for 30 min,
1-bromopentane (2.54 mL, 20.55 mmol) was slowly added to the
solution. The mixture was allowed to warm to ambient tempera-
ture and the solution was stirred for 20 h. The reaction was
quenched by slowly pouring the solution in iced HCl (1 M), fol-
lowed by the addition of Et2O. The organic fraction was washed
twice with HCl (1 M) and concentrated in vacuo. Automated flash
chromatography (Et2O/petroleum ether 1:4) gave pure 34 (2.55 g,
yield) as a gum. 1H NMR (400 MHz, CDCl3)
d 8.31e8.14 (m, 1H),
7.59e7.35 (m, 6H), 7.35e7.08 (m, 7H), 3.97 (t, J ¼ 7.2 Hz, 2H),1.71 (dt,
J ¼ 15.0, 7.4 Hz, 2H),1.38e1.09 (m, 4H), 0.87 (t, J ¼ 7.3 Hz, 3H); HRMS
(C26H26NO) [M þ H]þ m/z calcd 368.2014, found 368.2036.
5.1.8. [1,10-Biphenyl]-4-yl(1-(2-methoxyethyl)-1H-indol-3-yl)
methanone (14)
58% yield). 1H NMR (400 MHz, CDCl3)
d 8.23e8.13 (m, 1H), 7.81
To a solution of 32 (1.00 g, 5.42 mmol) in 20 mL CH2Cl2 at ꢀ10 ꢁC
was added dropwise a solution of diethylaluminum chloride (1 M
(s, 1H), 7.40e7.19 (m, 3H), 4.11 (t, J ¼ 7.2 Hz, 2H), 3.91 (s, 3H),
1.92e1.79 (m, 2H), 1.36e1.25 (m, 4H), 0.88 (t, J ¼ 7.0 Hz, 3H).

5094
A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
5.1.12. Methyl 1-(2-methoxyethyl)-1H-indole-3-carboxylate (35)
To a solution of NaH (60% dispersion in mineral oil, 0.82 g,
20.55 mmol) in 15 mL DMF was added dropwise a solution of
methyl 1H-indole-3-carboxylate (3.00 g,17.12 mmol) in 20 mL DMF,
at 0 ꢁC. The reaction mixture was allowed to warm to ambient
temperature. After 30 min the mixture was cooled to 0 ꢁC and
a solution of 1-bromo-2-methoxyethane (1.93 ml, 20.55 mmol) in
5 mL DMF was added dropwise. The temperature was allowed to
increase to ambient temperature and the mixture was stirred for
90 h. The reaction was quenched by slowly pouring the solution in
iced HCl (1 M), extraction with EtOAc and purification by auto-
mated flash chromatography (EtOAc/petroleum ether 1:3) afforded
recrystallization from Et2O ( ꢃ 2) to give 18 (0.34 g; 24% yield) as
a fine white powder. 1H NMR (400 MHz, CDCl3)
1H), 8.15e8.08 (m, 1H), 7.90e7.76 (m, 5H), 7.65 (dd, J ¼ 8.8, 2.1 Hz,
1H), 7.50e7.37 (m, 3H), 7.36e7.30 (m, 2H), 4.16 (t, J ¼ 7.2 Hz, 2H),
1.95e1.83 (m, 2H), 1.39e1.29 (m, 4H), 0.90 (t, J ¼ 6.9 Hz, 3H); HRMS
(C24H25N2O) [M þ H]þ m/z calcd 357.1967, found 357.1990.
d
8.35 (d, J ¼ 1.8 Hz,
5.1.15. 1-(2-Methoxyethyl)-N-(naphthalen-1-yl)-1H-indole-3-
carboxamide (19)
To a solution of naphthalen-1-amine (1.05 g,7.33 mmol) in
20 mL 1,2-dichloroethane at 15 ꢁC was added trimethylaluminum
(2 M in toluene, 3.67 mL, 7.33 mmol). The mixture was stirred for
30 min at ambient temperature whereafter 35 (1.20 g, 4.89 mmol)
in 15 mL 1,2-dichloroethane was added dropwise. The reaction
mixture was refluxed for 16 h, CH2Cl2 (30 mL) was added and the
reaction mixture was poured slowly in iced HCl (1 M), extracted
with CH2Cl2 ( ꢃ 2) and the combined organic fractions were
concentrated in vacuo. Automated flash chromatography (Et2O)
and subsequent recrystallization from Et2O ( ꢃ 2) yielded pure 19
pure 35 (3.95 g, 94% yield). 1H NMR (400 MHz, CDCl3)
d 8.24e8.14
(m, 1H), 7.89 (s, 1H), 7.40e7.34 (m, 1H), 7.29e7.25 (m, 2H), 4.31
(t, J ¼ 5.3 Hz, 2H), 3.91 (s, 3H), 3.72 (t, J ¼ 5.3 Hz, 2H), 3.31 (s, 3H).
5.1.13. N-(Naphthalen-1-yl)-1-pentyl-1H-indole-3-carboxamide
(17)
To a solution of naphthalen-1-amine (0.42 g, 2.90 mmol) in
10 mL 1,2-dichloroethane was added trimethylaluminum (2 M in
toluene,1.45 mL, 2.90 mmol), at ambient temperature. After 10 min,
intermediate 34 (0.50 g, 1.94 mmol) dissolved in 10 mL 1,2-
dichloroethane was added dropwise. The mixture was heated to
reflux temperature and allowed to react for 24 h. The reaction was
quenched by slowly pouring the solution in iced HCl (1 M) and the
resulting mixture was twice extracted with CH2Cl2. The combined
organic fractions were concentrated in vacuo and recrystallization
from Et2O ( ꢃ 3) to provide pure 17 (0.62 g, 85% yield). 1H NMR
(1.13 g, 64% yield). 1H NMR (400 MHz, CDCl3)
d 8.20e8.14 (m, 1H),
8.12e8.04 (m, 2H), 8.00 (dd, J ¼ 7.9, 5.7 Hz, 1H), 7.94e7.87 (m, 2H),
7.73 (d, J ¼ 8.2 Hz, 1H), 7.57e7.41 (m, 4H), 7.38e7.30 (m, 2H), 4.35 (t,
J ¼ 5.3 Hz, 2H), 3.75 (t, J ¼ 5.3 Hz, 2H), 3.33 (s, 3H); HRMS
(C22H21N2O2) [M þ H]þ m/z calcd 345.1603, found 345.1611.
5.1.16. 1-(2-Methoxyethyl)-N-(naphthalen-2-yl)-1H-indole-3-
carboxamide (20)
To a solution of naphthalen-2-amine (0.87 g, 6.11 mmol) in 25 mL
1,2-dichloroethane was added trimethylaluminum (2 M in toluene,
3.05 mL, 6.11 mmol). The solution was stirred for 30 min at ambient
temperature, intermediate 35 (1.00 g, 4.07 mmol) in 5 mL 1,2-
dichloroethane was added dropwise and the mixture was refluxed
for 16 h. Hereafter, CH2Cl2 (30 mL) was added and the reaction
mixture was poured slowly in iced HCl (1 M). After extraction with
CH2Cl2 ( ꢃ 2), the combined organic fractions were concentrated in
vacuo. Purification by automated flash chromatography (CH2Cl2/
MeOH 98:2) and recrystallization from Et2O provided 19 (1.35 g, 91%
(400 MHz, CDCl3)
d
8.18e8.05 (m, 3H), 8.01 (dd, J ¼ 7.8, 6.1 Hz, 1H),
7.94e7.84 (m, 2H), 7.73 (d, J ¼ 8.2 Hz, 1H), 7.59e7.40 (m, 4H),
7.39e7.29 (m, 2H), 4.19 (t, J ¼ 7.2 Hz, 2H), 1.99e1.80 (m, 2H),
1.54e1.28 (m, 4H), 0.91 (t, J ¼ 6.9 Hz, 3H); HRMS (C24H25N2O)
[M þ H]þ m/z calcd 357.1967, found 357.1982.
5.1.14. N-(Naphthalen-2-yl)-1-pentyl-1H-indole-3-carboxamide
(18)
Step 1. To a solution of 34 (2.34 g, 9.44 mmol) in 45 mL EtOH was
added dropwise a solution of KOH (85%,1.87 g, 28.33 mmol) in 5 mL
H2O, and the resulting mixture was stirred overnight at ambient
temperature. Hereafter, an additional portion of KOH (85%, 1 g) was
added and the mixture was successively heated to 75 ꢁC for 4 h,
acidified with 1 M HCl (2 ꢃ 25 mL) and extracted with Et2O ( ꢃ 3).
Residual water and EtOH was removed in vacuo with CH3CN as
azeotrope ( ꢃ 4), additional concentration in vacuo gave 1-pentyl-
1H-indole-3-carboxylic acid (2.11 g, 92% yield). 1H NMR (400 MHz,
yield).1H NMR (400 MHz, DMSO-d6)
d
9.98 (s,1H), 8.47 (d, J ¼ 1.5 Hz,
1H), 8.38 (s, 1H), 8.27 (d, J ¼ 7.3 Hz, 1H), 7.93e7.78 (m, 4H), 7.62
(d, J ¼ 8.0 Hz, 1H), 7.52e7.35 (m, 2H), 7.31e7.14 (m, 2H), 4.45
(t, J ¼ 5.1 Hz, 2H), 3.74 (t, J ¼ 5.1 Hz, 2H), 3.26 (s, 3H); HRMS
(C22H21N2O2) [M þ H]þ m/z calcd 345.1603, found 345.1626.
5.1.17. Naphthalen-1-yl(1H-pyrrolo[2,3-b]pyridin-3-yl)methanone
(39)
CDCl3)
d
8.27e8.20 (m, 1H), 7.93 (s, 1H), 7.42e7.37 (m, 1H),
1H-Pyrrolo[2,3-b]pyridine (1 g, 8.46 mmol) in 20 mL CH2Cl2 was
added to a solution of aluminum chloride (3.5 g, 26.24 mmol) in
20 mL CH2Cl2 at ambient temperature. After 1 h, a solution of 1-
naphthoyl chloride (1.77 g, 9.31 mmol) in 20 mL CH2Cl2 was added
dropwise and the mixture was stirred for 8 h. An ice bath was applied
and the reaction was quenched by the cautious dropwise addition of
MeOH (25 mL) at 0 ꢁC. The resulting solution was concentrated in
vacuo. Crude products were dissolved in EtOAc and NaOH (2 N) was
added. Theaqueouslayerwasquicklyextracted withEtOAc( ꢃ 2) and
the combined organic fractions were concentrated in vacuo. Auto-
matedflashchromatography(EtOAc/MeOH, 97.5:2.5)gave 39(1.46g,
60% yield) as yellow crystalline needles. 1H NMR (400 MHz, CDCl3)
7.33e7.28 (m, 2H), 4.17 (t, J ¼ 7.2 Hz, 2H), 1.94e1.85 (m, 2H),
1.38e1.30 (m, 4H), 0.90 (t, J ¼ 6.9 Hz, 3H).
Step 2. To a solution of 1-pentyl-1H-indole-3-carboxylic acid
(2.11 g, 9.12 mmol) and CH2Cl2 at ꢀ10 ꢁC was successively added
a catalytic amount of DMF (0.04 mL, 0.46 mmol) and a solution of
oxalyl chloride (1.16 mL, 13.68 mmol) in CH2Cl2. The mixture was
stirred at ambient temperature for 16 h and concentrated in vacuo
to give 2.27 g (95%) of the corresponding acyl chloride, which was
immediately used in the next step.
Step 3. To a solution of freshly prepared 1-pentyl-1H-indole-3-
carbonyl chloride (1.14 g, 3.51 mmol) in 65 mL CH2Cl2 was added
DIPEA (0.73 mL, 4.22 mmol) which formed a white fog upon
addition. Naphthalen-2-amine (0.50 g, 3.51 mmol) in 15 mL CH2Cl2
was added to the mixture at ambient temperature. After 4 h,
evaluation of the reaction progress indicated the reaction was not
completed and was allowed to continue for 62 h. Hereafter, the
mixture was washed twice with 1 M HCl and once with 5 M HCl,
the organic fraction was concentrated in vacuo, subjected to auto-
mated flash chromatography (CH2Cl2/petroleum ether 5:1) and
d
12.79 (s,1H), 8.82 (dd, J ¼ 7.9,1.6 Hz,1H), 8.40 (dd, J ¼ 4.9,1.6 Hz,1H),
8.18 (d, J ¼ 8.2 Hz, 1H), 8.00 (d, J ¼ 8.3 Hz, 1H), 7.94e7.89 (m, 1H),
7.76e7.65 (m, 2H), 7.59e7.44 (m, 3H), 7.33 (dd, J ¼ 7.9, 4.9 Hz, 1H).
5.1.18. Naphthalen-1-yl(1-pentyl-1H-pyrrolo[2,3-b]pyridin-3-yl)
methanone (21)
To a solution of NaH (60% in mineral oil, 0.05 g, 1.33 mmol) in
5 mL DMF at 0 ꢁC was added a solution of 39 (0.31 g, 1.02 mmol) in

A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
5095
10 mL DMF. The mixture was allowed to increase to ambient
temperature and was stirred for 30 min. An ice bath was applied
and a solution of 1-bromopentane (0.14 mL, 1.13 mmol) in 10 mL
DMF at 0 ꢁC was added dropwise. After 90 h at ambient tempera-
ture, the reaction was quenched by slowly pouring the solution into
iced HCl (1 M) and extracted with Et2O ( ꢃ 2). Residual water was
azeotropically removed from the combined organic fractions in
vacuo with CH3CN. Automated flash chromatography (EtOAc/
MeOH, 99.5:0.5) was used to purify title compound 21 (0.31 g, 84%
multiple times using batches of 43 (0.25e2.5 g), giving consistent
65ꢀ75% yields at reaction times of 14e60 h 1H NMR (400 MHz,
CDCl3)
d
8.75 (s, 1H), 8.04 (d, J ¼ 5.5 Hz, 1H), 7.53e7.46 (m, 1H), 7.43
(t, J ¼ 2.8 Hz, 1H), 6.64 (dd, J ¼ 3.0, 2.1 Hz, 1H).
5.1.22. Naphthalen-1-yl(1H-pyrrolo[2,3-c]pyridin-3-yl)methanone
(40)
To a stirred solution of aluminum chloride (4.15 g,31.16 mmol) in
25 mL CH2Cl2 was added 37 (1.25 g, 10.05 mmol) in 25 mL CH2Cl2.
After 1 h, 1-naphthoyl chloride (2.11 g, 11.06 mmol) in 10 mL CH2Cl2
was added dropwise. The reaction was quenched after 9 h, a total of
100 mL MeOH was added to dissolve the black sticky tar which had
formed in the flask. The resulting bright yellow solution was
concentrated in vacuo, dissolved in EtOAc and washed with K2CO3
(15 g in 75 mL H2O). To the aqueous fractionwas added 100 mL NaOH
(1 N), which was quickly extracted with EtOAc ( ꢃ 2). The combined
organic fractions were concentrated in vacuo and purified using
automated flash chromatography (CH2Cl2/MeOH, 90:10 and then
EtOAc/acetone 1:1) to give 40 (0.39 g, 13.54%). 1H NMR (400 MHz,
yield). 1H NMR (400 MHz, CDCl3)
d
8.68 (dd, J ¼ 7.9, 1.6 Hz, 1H), 8.44
(dd, J ¼ 4.7, 1.6 Hz, 1H), 8.18 (d, J ¼ 8.3 Hz, 1H), 8.00 (d, J ¼ 8.2 Hz,
1H), 7.96e7.89 (m, 1H), 7.68 (dd, J ¼ 7.0, 1.1 Hz, 1H), 7.59e7.46
(m, 4H), 7.30 (dd, J ¼ 7.9, 4.8 Hz, 1H), 4.30e4.22 (m, 2H), 1.90e1.78
(m, 2H), 1.33e1.27 (m, 4H), 0.85 (t, J ¼ 7.0 Hz, 3H); HRMS
(C23H23N2O) [M þ H]þ m/z calcd 343.1810, found 343.1794.
5.1.19. (1-(2-Methoxyethyl)-1H-pyrrolo[2,3-b]pyridin-3-
yl)(naphthalen-1-yl)methanone (22)
To a solution of NaH (60% in mineral oil, 0.13 g, 3.14 mmol) in
10 mL DMF at 0 ꢁC was added a solution of 39 (0.31 g, 1.02 mmol) in
15 mL DMF. The temperature was allowed to increase to ambient
temperature and the mixture was stirred for 45 min. An ice bath
was applied and a solution of 1-bromo-2-methoxyethane (0.30 mL,
3.14 mmol) in 10 mL DMF at 0 ꢁC was added dropwise. Hereafter,
the reaction temperature was allowed to increase to ambient
temperature. After 30 h, the reaction was quenched by slowly
pouring the solution into iced H2O and extracted with Et2O ( ꢃ 2).
Residual water was azeotropically removed from the organic frac-
tion with CH3CN under reduced pressure. Automated flash chro-
matography (EtOAc/MeOH, 99.5:0.5 and then Et2O) was used to
DMSO-d6)
d
12.52(s,1H), 8.88(s,1H), 8.36(d, J¼ 5.4Hz,1H), 8.15e7.98
(m, 4H), 7.95 (s, 1H), 7.74 (dd, J ¼ 6.9, 0.8 Hz, 1H), 7.67e7.50 (m, 3H).
5.1.23. Naphthalen-1-yl(1-pentyl-1H-pyrrolo[2,3-c]pyridin-3-yl)
methanone (23)
To a solution of 40 (150.0 mg, 0.52 mmol) in 15 mL anhydrous
DMF was added NaH (60% dispersion in mineral oil, 23.02 mg,
0.58 mmol). The resulting mixture was stirred for 45 min at
ambient temperature. Thereafter, 1-bromopentane (0.07 mL,
0.58 mmol, in 5 mL DMF) was added. After 6 h, the reaction was
quenched by pouring the mixture in 15 mL H2O and extracted with
Et2O ( ꢃ 4). Flash column chromatography (EtOAc) was used to give
yield pure 22 (0.64 g, 70% yield). 1H NMR (400 MHz, CDCl3)
d 8.70
(dd, J ¼ 7.9, 1.6 Hz, 1H), 8.43 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.21 (dd, J ¼ 8.2,
0.9 Hz, 1H), 7.99 (d, J ¼ 8.2 Hz, 1H), 7.95e7.88 (m, 1H), 7.69 (dd,
J ¼ 7.0,1.1 Hz,1H), 7.64 (s, 1H), 7.59e7.45 (m, 3H), 7.35e7.27 (m,1H),
4.45 (t, J ¼ 5.1 Hz, 2H), 3.74e3.68 (m, 2H), 3.24 (s, 3H); HRMS
(C21H19N2O2) [M þ H]þ m/z calcd 331.1447, found 331.1443.
pure 23 (103.00 mg, 54.60%). 1H NMR (400 MHz, CDCl3)
d 8.86
(s, 1H), 8.48 (d, J ¼ 5.4 Hz, 1H), 8.24e8.14 (m, 2H), 8.01 (d, J ¼ 8.2 Hz,
1H), 7.93 (d, J ¼ 7.7 Hz, 1H), 7.66 (dd, J ¼ 7.0, 1.1 Hz, 1H), 7.58e7.47
(m, 4H), 4.18 (t, J ¼ 7.2 Hz, 2H), 1.87 (dt, J ¼ 14.6, 7.4 Hz, 2H),
1.39e1.23 (m, 4H), 0.86 (t, J ¼ 6.8 Hz, 3H); HRMS (C23H23N2O)
[M þ H]þ m/z calcd 343.1810, found 343.1831.
5.1.20. 7-Chloro-1H-pyrrolo[2,3-c]pyridine (43)
2-Chloro-3-nitropyridine (10.0 g, 63.07 mmol) was dissolved in
500 mL anydrous THF and magnetically stirred at ꢀ78 ꢁC. Excess
vinylmagnesium bromide (1 M in THF, 200 mL, 200.0 mmol) was
added dropwise, after the addition was complete, the solution was
allowed to warm to ꢀ20 ꢁC. After 16 h, an aqueous solution of
saturated ammonium chloride (300 g/L, 150 mL, 841.25 mmol) was
added dropwise under vigorous stirring. The resulting suspension
was filtered over Hyflo Super Cel medium (calcined) and extracted
with EtOAc ( ꢃ 3). The combined organic fractions were concen-
trated and automated flash chromatography (CH2Cl2/MeOH, 97:3
and then CH2Cl2/MeOH/NH4OH, 980:18.75:1.25) was used to obtain
5.1.24. (1-(2-Methoxyethyl)-1H-pyrrolo[2,3-c]pyridin-3-
yl)(naphthalen-1-yl)methanone (24)
To a solution of 40 (150 mg, 0.52 mmol) in 15 mL anhydrous
DMF was added NaH (60% dispersion in minderal oil, 23.02 mg,
0.58 mmol). The resulting mixture was stirred magnetically at
ambient temperature. After 45 min, 1-bromo-2-methoxyethane
(0.05 mL, 0.58 mmol) in 5 mL DMF was added. After 6 h, the
reaction was quenched by pouring the mixture in 15 mL H2O, fol-
lowed by extraction with Et2O ( ꢃ 4). Flash column chromatography
(CH2Cl2/MeOH/NH4OH, 960:37.5:2.5) was used to give 24 (30 mg,
16% yield). 1H NMR (400 MHz, CDCl3)
d 8.86 (s, 1H), 8.48 (d,
pure 43 (3.87 g, 38% yield). 1H NMR (400 MHz, CDCl3)
d
8.73 (s, 1H),
J ¼ 5.3 Hz, 1H), 8.20 (dd, J ¼ 19.2, 6.8 Hz, 2H), 7.99 (d, J ¼ 8.2 Hz, 1H),
7.96e7.88 (m, 1H), 7.67 (dd, J ¼ 7.0, 1.1 Hz, 1H), 7.62e7.44 (m, 4H),
4.33 (t, J ¼ 5.1 Hz, 2H), 3.70 (t, J ¼ 5.1 Hz, 2H), 3.25 (s, 3H); HRMS
(C21H19N2O2) [M þ H]þ m/z calcd 331.1447, found 331.1427.
8.04 (d, J ¼ 5.5 Hz, 1H), 7.53e7.46 (m, 1H), 7.43 (dd, J ¼ 6.2, 3.4 Hz,
1H), 6.64 (dd, J ¼ 3.1, 2.1 Hz, 1H).
5.1.21. 1H-Pyrrolo[2,3-c]pyridine (37)
To a solution of anhydrous THF (35.50 mL) was added 43 (0.71 g,
4.42 mmol). The resulting solution was degassed for 2 h using N2.
Hereafter, PdCl2(dppf)$CH2Cl2 (180.50 mg, 0.22 mmol), TMEDA
(2.25 mL, 15.03 mmol) and NaBH4 (0.57 g, 15.03 mmol) were added
in sequence. The reaction was allowed to continue for 60 h at
ambient temperature. The reactionwas quenched by slowly pouring
the solution in brine. After extraction with EtOAc ( ꢃ 3), the
combined organic fractions were filtered over Hyflo Super Cel
medium (calcined), concentrated in vacuo and purified using auto-
mated flash chromatography (EtOAc/MeOH/NH4OH, 960:37.5:2.5)
to give 37 (0.39 g; 70.95% yield). Note: This procedure was repeated
5.1.25. N-(3-Bromopyridin-4-yl)acetamide (45)
3-Bromopyridin-4-amine (5.10 g, 29.48 mmol) and DIPEA
(5.66 mL, 32.43 mmol) were dissolved in 6 mL CH2Cl2. Acetyl chloride
(2.31 mL, 32.43 mmol) in 10 mL CH2Cl2 was added dropwise at 5 ꢁC.
The solution was stirred overnight, washed with H2O and concen-
tratedinvacuotogive45(5.65g, 89%yield).1HNMR(400MHz,CDCl3)
d
8.64 (s,1H), 8.40 (dd, J ¼ 17.5, 5.6 Hz, 2H), 7.79 (br s,1H), 2.29 (s, 3H).
5.1.26. 1H-Pyrrolo[3,2-c]pyridine (38)
Step 1. A solution of 45 (5.18 g, 24.09 mmol), (Z)-tribu-
tyl(2-ethoxyvinyl)stannane (9.65 mL, 28.90 mmol) and TBAC

5096
A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
(6.69 g, 24.09 mmol) were degassed in CH3CN (100 mL) for 2 h.
Hereafter, Pd(PPh3)2Cl2 (0.51 g, 0.72 mmol) was added and the
mixture was heated to reflux temperature. After 2.5 h, the crude
mixture was concentrated in vacuo, dissolved in CH2Cl2 and
magnetically stirred for 2 h in the presence of a 10% KF solution. The
resulting suspension was successively filtered over Hyflo Super Cel
medium (calcined), extracted with CH2Cl2, concentrated in vacuo
and purified using column chromatography (CH2Cl2/MeOH/
NH4OH, 960:37.5:2.5) giving 6.20 g (w80% purity) of intermediate
UV diode array and it is assumed that the largest peak in the
chromatogram is the compound of interest.
5.2.2. Thermodynamic solubility
The thermodynamic solubility (in
mM) of samples was deter-
mined at pH 7.4 and pH 4.0. A 200 M mixture of the compound was
m
prepared in phosphate buffer (10 mM) at pH 7.4 or pH 4.0 and
magnetically stirred for 24 h at room temperature. Before analysis,
each solution was centrifuged at 4700 tr/min for 5 min. The super-
natant was then collected and analyzed by HPLC with and without
dilution. Analytical column: Chromolith RP-18-e, 50 ꢃ 4.6 mm;
mobile phase: gradient of water þ 0.05% TFA and MeOH þ 0.05%
TFA; flow rate: 2 mL/min; UV detection wavelength: 255 ꢂ 25 nm.
(Z)-N-(3-(2-ethoxyvinyl)pyridin-4-yl)acetamide,
which
was
immediately used in the next step.
Step 2. Intermediate (Z)-N-(3-(2-ethoxyvinyl)pyridin-4-yl)acet-
amide (6.20 g, 24.05 mmol) was dissolved in MeOH (50 mL), 5 mL
concentrated HCl was added and the resulting mixture was heated to
reflux temperature for 5 h. Hereafter, the crude mixture was
concentrated in vacuo, basified with K2CO3 (15 g in 100 mL H2O) and
extracted with CH2Cl2. Column chromatography (CH2Cl2/MeOH/
NH4OH, 920:75.5:5) afforded 38(1.90g, 67%yield).1HNMR(400MHz,
5.2.3. Kinetic (turbidimetric) solubility
The experiments were conducted by Cyprotex Discovery Ltd.
Four dilutions of a 10 mM test compound solution were prepared in
DMSO. Each dilution was then further diluted 1 in 100 in the
appropriatebuffer (0.01Mphosphate-buffered saline, pH 7.4)togive
a final DMSO concentration of 1% in each well. After an incubation
period of 2 h at 37 ꢁC, absorbance was measured at 620 nm and the
solubility wasestimated fromthe concentration of test compound(7
replicates per concentration) that produced an increase in absor-
bance above the vehicle control (1% DMSO in buffer, pH 7.4).
CDCl3)
d
11.07 (br s,1H), 8.99 (d, J ¼ 0.8 Hz,1H), 8.30 (d, J ¼ 5.8 Hz,1H),
7.35 (dd, J ¼ 9.2, 4.5 Hz, 2H), 6.67 (dd, J ¼ 3.2, 0.8 Hz, 1H).
5.1.27. Naphthalen-1-yl(1H-pyrrolo[3,2-c]pyridin-3-yl)methanone
(41)
To a solution of aluminum chloride (0.79 g, 5.93 mmol) in 50 mL
CH2Cl2 was added 1-naphthoyl chloride (1.13 g, 5.93 mmol) in
10 mL CH2Cl2 at ambient temperature. After 10 min, intermediate
38 (0.70 g, 5.93 mmol) was added and the mixture was stirred for
16 h at ambient temperature. Hereafter, MeOH was added drop-
wise, followed by NaOH (2 N). The resulting mixture was extracted
with CH2Cl2 ( ꢃ 2). The combined organic fractions were concen-
trated in vacuo and purified by flash column chromatography
(CH2Cl2/MeOH/NH4OH, 960:37.5:2.5), giving pure 41 (0.17 g, 11%
5.2.4. Acid dissociation constant (pKa)
The pKa of samples was investigated using the fast UV-metric
method, by Sirius Analytical Ltd. Each sample was titrated in triple
titrations between pH 1.6 and pH 12.4 in 50%, 40% and 30% MeOH.
Sample concentrations were typically in the range 17e32 mM. All
titrations were carried out at 25 ꢁC. The pKa values of the samples
were determined by mathematical analysis of the obtained spec-
troscopic data and were extrapolated to aqueous conditions by the
Yasuda-Shedlovsky method.
yield). 1H NMR (400 MHz, CDCl3)
d 11.15 (br s, 1H), 9.81
(d, J ¼ 0.9 Hz, 1H), 8.45 (d, J ¼ 5.8 Hz, 1H), 8.24e8.18 (m, 1H), 7.96
(d, J ¼ 8.3 Hz, 1H), 7.89 (dd, J ¼ 7.2, 2.1 Hz, 1H), 7.72 (dd, J ¼ 7.0,
1.0 Hz, 1H), 7.64 (s, 1H), 7.55e7.40 (m, 4H).
5.2.5. Cell membrane permeability
Cell membrane permeability experiments were performed
using Caco-2 cell lines, by Cyprotex Discovery Ltd. Test compound
was added to either the apical or basolateral side of a confluent
monolayer of human colon carcinoma cells (bi-directional; pH 7.4/
pH 7.4: HBSS medium) and permeability was measured by moni-
toring the appearance of the test compound on the opposite site of
the membrane using an LC-MS/MS read-out. The apparent
permeability (Papp) coefficient, efflux ratio for the test compound
(10ꢀ5 M) and recovery values were the determined parameters.
5.1.28. Naphthalen-1-yl(1-pentyl-1H-pyrrolo[3,2-c]pyridin-3-yl)
methanone (25)
To a solution of NaH (60% dispersion in minderal oil, 0.03 g,
0.69 mmol) in 10 mL DMF was added 41 (0.17 g, 0.62 mmol), at
ambient temperature. After
1 h, 1-bromopentane (0.10 g,
0.69 mmol) in 4 mL DMF was added and the mixture was stirred for
16 h. The reaction was quenched by the addition of H2O, extracted
with CH2Cl2, concentrated in vacuo and purified by column chro-
matography (CH2Cl2/MeOH/NH4OH, 960:37.5:2.5) to afford 25
(0.10 g, 44% yield). 1H NMR (400 MHz, CDCl3)
d
9.65 (s, 1H), 8.49
5.3. Biology
(d, J ¼ 5.8 Hz, 1H), 8.19 (d, J ¼ 8.2 Hz, 1H), 7.99 (d, J ¼ 8.2 Hz, 1H),
7.94e7.88 (m, 1H), 7.67 (dd, J ¼ 7.0, 1.1 Hz, 1H), 7.58e7.44 (m, 3H),
7.39 (s, 1H), 7.33e7.28 (m, 1H), 4.07 (t, J ¼ 7.3 Hz, 2H), 1.86e1.73 (m,
2H), 1.30 (ddd, J ¼ 9.4, 8.4, 4.0 Hz, 4H), 0.85 (t, J ¼ 7.1 Hz, 3H); HRMS
(C23H23N2O) [M þ H]þ m/z calcd 343.1810, found 343.1802.
5.3.1. Receptor binding assays
Cannabinoid CB1 and CB2 receptor binding affinities were
determined using membrane preparations of Chinese hamster
ovary (CHO) cells in which the hCB1 receptor or hCB2 receptor is
stably transfected, using [3H]CP-55,940 as radioligand. In the pres-
ence or absence of test compound, the radioligand [3H]CP-55,940
was incubated with freshly prepared membrane preparations,
separation of bound and free radioligand was performed by filtra-
tion over glassfiber filters. Radioactivity on the filter was measured
by liquid scintillation counting. The IC50 values from at least three
independent measurements was determined and converted to
binding affinity Ki (nM) using the ChengePrusoff equation.
5.2. Physicochemical properties
5.2.1. Lipophilicity
The Log D of each sample at pH 7.4 was investigated using the
liquideliquid distribution chromatography (LDA) method, by Sirius
Analytical Ltd. The LDA is an isocratic chromatography system,
which uses an octanol-coated column with an octanol saturated
mobile phase adjusted to pH 7.4. A set of standard compounds with
well known Log D octanol values are run through the column in
order to generate a calibration curve from their measured retention
times. The calibration curve is subsequently used to convert the
sample retention time to a Log D value. Detection is performed by
5.3.2. Functional assay for arachidonic acid release
Cannabinoid CB1 receptor agonist functional activity was
determined using CHO cells stably expressing hCB1 receptors, using
an arachidonic acid release assay, as described previously [100].

A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
5097
Briefly, cell cultures were incubated with [3H]-arachidonic acid in
Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, UK), for 18 h at
37 ꢁC, in 95% air ꢀ 5% CO2. The incubation medium was aspirated
and the cells were washed (3 ꢃ ) with 0.5 mL DMEM containing
0.2% bovine serum albumin (BSA). Stimulation of the CB1 receptor
by test compounds leads to activation of PLA2 and subsequent [3H]-
arachidonic acid release in the medium. Maximal stimulation was
(1ꢀ2 mL/min) with aCSF (containing <0.1% DMSO). Test
compounds were dissolved in DMSO and diluted with aCSF per-
fusion medium to obtain the desired concentration. Two baseline
control recordings were performed at the start of each experiment
before control perfusion medium was switched to drug-containing
medium. The effects on the fEPSP amplitude were determined after
20 min of perfusion with the drug-containing medium. The IC50
determined using reference agonist WIN-55,212-2 (1
m
M) and basal
(mM) and Emax (%) values were obtained by nonlinear regression
[3H]-arachidonic acid release was used as baseline. Compounds
were tested in a logarithmic concentration range, in quadruplicate,
agonist pEC50, Emax (% stimulation compared to maximal effect of
WIN-55,212-2) were calculated.
analysis using a variable slope four-parameter curve fit (GraphPad
Prism 5.0) for a sigmoid concentration-response curve; Emax is
defined as the maximal ligand-evoked effect (% reduction of the
fEPSP amplitude).
5.3.3. Functional assay for cAMP accumulation
Acknowledgements
The functional activity of test compounds at the cannabinoid
CB2 receptor was determined using CHO cells stably expressing
hCB2 receptors, according to a cAMP accumulation protocol as
previously described [113,114]. Briefly, cell cultures were grown in
CHO-S-SFM-II medium (Gibco, UK), supplemented with 2 mM L-
Jord van Schaik and Mark Kuil are gratefully acknowledged for
practical support, Alice Borst and Hugo Morren for analytical
support, Maria van Dongen, Isabelle Robert, Veronique Lucas and
Pieter Spaans for the supply of physicochemical data.
glutamine; 500
mg/mL Geneticin-G418 (Gibco, UK); 400 mg/mL
Hygromycine B (Gibco, UK); 10% heat-inactivated Fetal Bovine
Serum (FBS); in 93% air ꢀ 7% CO2. For the cAMP accumulation assay,
CHO-hCB2 cells (105 cells/mL) were transferred to 96-well plates
and grown for 48 h to produce a 100% confluent monolayer. Test
compounds were diluted using assay medium (DMEM (Gibco, UK),
References
[1] V. Di Marzo, M. Bifulco, L. De Petrocellis, Nat. Rev. Drug Discov. 3 (2004)
771e784.
[2] V. Di Marzo, Nat. Rev. Drug Discov. 7 (2008) 438e455.
[3] J.M. McPartland, Brain Res. Rev. 45 (2004) 18e29.
[4] L.A. Matsuda, S.J. Lolait, M.J. Brownstein, A.C. Young, T.I. Bonner, Nature 346
(1990) 561e564.
[5] W.A. Devane, F.A. Dysarz 3rd, M.R. Johnson, L.S. Melvin, A.C. Howlett, Mol.
Pharmacol. 34 (1988) 605e613.
[6] A.C. Howlett, F. Barth, T.I. Bonner, G. Cabral, P. Casellas, W.A. Devane,
C.C. Felder, M. Herkenham, K. Mackie, B.R. Martin, R. Mechoulam,
R.G. Pertwee, Pharmacol. Rev. 54 (2002) 161e202.
[7] S. Munro, K.L. Thomas, M. Abushaar, Nature 365 (1993) 61e65.
[8] M.D. Van Sickle, M. Duncan, P.J. Kingsley, A. Mouihate, P. Urbani, K. Mackie,
N. Stella, A. Makriyannis, D. Piomelli, J.S. Davison, L.J. Marnett, V. Di Marzo,
Q.J. Pittman, K.D. Patel, K.A. Sharkey, Science 310 (2005) 329e332.
[9] O. Ofek, M. Karsak, N. Leclerc, M. Fogel, B. Frenkel, K. Wright, J. Tam, M. Attar-
Namdar, V. Kram, E. Shohami, R. Mechoulam, A. Zimmer, I. Bab, Proc. Natl.
Acad. Sci. USA 103 (2006) 696e701.
containing 1 mM IBMX; 0.3
ated by addition of 100 L assay medium in the presence or absence
of test compound, at 37 ꢁC for 20 min. The plates were washed with
water (HPLC grade) and nucleotides were extracted with 100
mM forskolin). Incubations were initi-
m
mL
lysis buffer (63% CH3CN in HPLC grade water, with 10 mM
NH4HCO3, adjusted to pH 9.4 with 25% NH4OH solution) and plates
were sealed, centrifuged and stored at 4 ꢁC until analysis. Levels of
cAMP and ATP were analyzed using HPLC-MS/MS. Data was
expressed as percentage of control values (forskolin-stimulated
cAMP accumulation). Maximal response was determined by refer-
ence agonist JWH-133 (1 mM). Compounds were tested in a loga-
rithmic concentration range, in duplicate, agonist pEC50, Emax were
calculated using SigmaPlot (SYSTAT 9).
[10] I. Bab, A. Zimmer, E. Melamed, Ann. Med. 41 (2009) 560e567.
[11] D. Baker, G. Pryce, J.L. Croxford, P. Brown, R.G. Pertwee, J.W. Huffman,
L. Layward, Nature 404 (2000) 84e87.
[12] D. Baker, G. Pryce, Curr. Pharm. Des. 14 (2008) 2326e2336.
[13] L.M. Eubanks, C.J. Rogers, A.E.t. Beuscher, G.F. Koob, A.J. Olson, T.J. Dickerson,
K.D. Janda, Mol. Pharm. 3 (2006) 773e777.
5.3.4. Ex vivo extracellular field potential recordings
Coronal prelimbic prefrontal cortex (PFC) slices (400 mm) were
[14] N.E. Campillo, J.A. Paez, Mini Rev. Med. Chem. 9 (2009) 539e559.
[15] J. Romero, J.M. Orgado, CNS Neurol. Disord. Drug Targets 8 (2009) 440e450.
[16] L.G. Bilsland, L. Greensmith, Curr. Pharm. Des. 14 (2008) 2306e2316.
[17] M. Karsak, E. Gaffal, R. Date, L. Wang-Eckhardt, J. Rehnelt, S. Petrosino,
K. Starowicz, R. Steuder, E. Schlicker, B. Cravatt, R. Mechoulam, R. Buettner,
S. Werner, V. Di Marzo, T. Tuting, A. Zimmer, Science 316 (2007) 1494e1497.
[18] A.A. Izzo, K.A. Sharkey, Pharmacol. Ther. 126 (2010) 21e38.
[19] L. Weiss, M. Zeira, S. Reich, M. Har-Noy, R. Mechoulam, S. Slavin, R. Gallily,
Autoimmunity 39 (2006) 143e151.
[20] L. Weiss, M. Zeira, S. Reich, S. Slavin, I. Raz, R. Mechoulam, R. Gallily,
Neuropharmacology 54 (2008) 244e249.
[21] L.D. Recht, R. Salmonsen, R. Rosetti, T. Jang, G. Pipia, T. Kubiatowski, P. Karim,
A.H. Ross, R. Zurier, N.S. Litofsky, S. Burstein, Biochem. Pharmacol. 62 (2001)
755e763.
[22] J. Flygare, K. Gustafsson, E. Kimby, B. Christensson, B. Sander, FEBS Lett. 579
(2005) 6885e6889.
[23] B. Herrera, A. Carracedo, M. Diez-Zaera, M. Guzman, G. Velasco, FEBS Lett.
579 (2005) 5084e5088.
[24] S. Sarfaraz, V.M. Adhami, D.N. Syed, F. Afaq, H. Mukhtar, Cancer Res. 68
(2008) 339e342.
[25] J.L. Croxford, CNS Drugs 17 (2003) 179e202.
obtained from the brains of 6e8 week-old male Wistar rats. The
rats were killed by decapitation, the brain was quickly removed and
placed in cold (4 ꢁC) artificial cerebrospinal fluid (aCSF, containing
(in mM): 120 NaCl; 3.5 KCl; 25 NaHCO3; 10 D-glucose; 2.5 CaCl2; 1.3
MgSO4; 1.25 NaH2PO4, gassed with a mixture of 95% O2 ꢀ 5% CO2).
The brain slices were cut at 4 ꢁC using a vibrating blade microtome
(Leica VT1000S), subsequently placed in a CSF (32 ꢁC) for 15 min
and thereafter kept at ambient temperature for at least 1 h before
recording. Glass recording electrodes were pulled from
0.86 ꢃ 1.50 ꢃ 80 mm (GB150-8P, Science Products) borosilicate
glass tubes on a Flaming/Brown micropipette puller (model P-87,
Sutter Instrument), filled with aCSF and positioned in layer II of the
PFC. A concentric tungsten microelectrode (TM33CCNON, World
Precision Instruments) was placed in layer V of the PFC. Field
potentials were evoked by applying biphasic 200 ms stimulation
pulses with an interpulse interval of 8 s. At higher stimulation
intensities large field excitatory postsynaptic potentials (fEPSPs)
could be recorded in layer II. Input-output relationships were
determined to establish the stimulation intensities (range
[26] R.G. Pertwee, Br. J. Pharmacol. 156 (2009) 397e411.
[27] D.R. Haubrich, S.J. Ward, E. Baizman, M.R. Bell, J. Bradford, R. Ferrari,
M. Miller, M. Perrone, A.K. Pierson, J.K. Saelens, et al., J. Pharmacol. Exp. Ther.
255 (1990) 511e522.
[28] A. Calignano, G. La Rana, A. Giuffrida, D. Piomelli, Nature 394 (1998)
50e400 mA) at which maximal field potential amplitudes could be
277e281.
[29] N. Agarwal, P. Pacher, I. Tegeder, F. Amaya, C.E. Constantin, G.J. Brenner,
T. Rubino, C.W. Michalski, G. Marsicano, K. Monory, K. Mackie, C. Marian,
S. Batkai, D. Parolaro, M.J. Fischer, P. Reeh, G. Kunos, M. Kress, B. Lutz,
C.J. Woolf, R. Kuner, Nat. Neurosci. 10 (2007) 870e879.
evoked. The effects of the test compounds on the fEPSP were
determined at maximal stimulation intensities. For recordings,
a slice was transferred to the recording chamber (SliceMate, Sci-
entifica) which was kept at 31 ꢁC and continuously perfused
[30] R.G. Pertwee, Prog. Neurobiol. 63 (2001) 569e611.

5098
A.R. Blaazer et al. / European Journal of Medicinal Chemistry 46 (2011) 5086e5098
[31] P. Goya, N. Jagerovic, L. Hernandez-Folgado, M.I. Martin, Mini Rev. Med.
Chem. 3 (2003) 765e772.
[32] M.M. Ibrahim, M.L. Rude, N.J. Stagg, H.P. Mata, J. Lai, T.W. Vanderah,
F. Porreca, N.E. Buckley, A. Makriyannis, T.P. Malan Jr., Pain 122 (2006)
36e42.
[33] M.P. Davis, Expert Opin. Investig. Drugs 17 (2008) 85e95.
[34] E.J. Rahn, A.M. Zvonok, G.A. Thakur, A.D. Khanolkar, A. Makriyannis,
A.G. Hohmann, J. Pharmacol. Exp. Ther. 327 (2008) 584e591.
[35] P. Anand, G. Whiteside, C.J. Fowler, A.G. Hohmann, Brain Res. Rev. 60 (2009)
255e266.
[36] Y. Cheng, S.A. Hitchcock, Expert Opin. Investig. Drugs 16 (2007) 951e965.
[37] D. Panikashvili, C. Simeonidou, S. Ben-Shabat, L. Hanus, A. Breuer,
R. Mechoulam, E. Shohami, Nature 413 (2001) 527e531.
[72] J.W. Huffman, P.V. Szklennik, A. Almond, K. Bushell, D.E. Selley, H. He,
M.P. Cassidy, J.L. Wiley, B.R. Martin, Bioorg. Med. Chem. Lett. 15 (2005)
4110e4113.
[73] J. Hynes, K. Leftheris, H. Wu, C. Pandit, P. Chen, D.J. Norris, B.-C. Chen, R. Zhao,
P.A. Kiener, X. Chen, L.A. Turk, V. Patil-Koota, K.M. Gillooly, D.J. Shuster,
K.W. McIntyre, Bioorg. Med. Chem. Lett. 12 (2002) 2399e2402.
[74] K. Leftheris, R. Zhao, B.-C. Chen, P. Kiener, H. Wu, C.R. Pandit, S. Wrobleski, P.
Chen, J. Hynes, Jr., M. Longphre, D.J. Norris, S. Spergel, Tokarski, J. PCT Int.
Appl. WO 2001/58869, 2001.
[75] K. Leftheris, H. Wu, S. Wrobleski, P. Chen, J. Hynes, Tokarski, J. U.S. Patent
6,653,304, 2003.
[76] T. Kiyoi, M. York, S. Francis, D. Edwards, G. Walker, A.K. Houghton, J.E. Cottney,
J. Baker, J.M. Adam, Bioorg. Med. Chem. Lett. 20 (2010) 4918e4921.
[77] E.M. Moir, K. Yoshiizumi, J. Cairns, P. Cowley, M. Ferguson, F. Jeremiah,
T. Kiyoi, R. Morphy, J. Tierney, G. Wishart, M. York, J. Baker, J.E. Cottney,
A.K. Houghton, P. McPhail, A. Osprey, G. Walker, J.M. Adam, Bioorg. Med.
Chem. Lett. 20 (2010) 7327e7330.
[38] I. Galve-Roperh, T. Aguado, J. Palazuelos, M. Guzman, Curr. Pharm. Des. 14
(2008) 2279e2288.
[39] Y. Marchalant, H.M. Brothers, G.L. Wenk, Mol. Psychiatry 14 (2009)
1068e1069.
[40] Y. Marchalant, H.M. Brothers, G.J. Norman, K. Karelina, A.C. DeVries,
G.L. Wenk, Neurobiol. Dis. 34 (2009) 300e307.
[41] Y. Gaoni, R. Mechoulam, J. Am. Chem. Soc. 93 (1971) 217e224.
[42] Y. Gaoni, R. Mechoulam, J. Am. Chem. Soc. 86 (1964) 1646e1647.
[43] R.K. Razdan, Pharmacol. Rev. 38 (1986) 75e149.
[44] R. Mechoulam, N. Lander, M. Srebnik, A. Breuer, M. Segal, J.J. Feigenbaum,
T.U. Jarbe, P. Consroe, NIDA Res. Monogr. 79 (1987) 15e30.
[45] L.S. Melvin, M.R. Johnson, NIDA Res. Monogr. 79 (1987) 31e47.
[46] P.J. Little, D.R. Compton, M.R. Johnson, L.S. Melvin, B.R. Martin, J. Pharmacol.
Exp. Ther. 247 (1988) 1046e1051.
[78] J.M. Adam, J. Cairns, W. Caulfield, P. Cowley, I. Cumming, M. Easson,
D. Edwards, M. Ferguson, R. Goodwin, F. Jeremiah, T. Kiyoi, A. Mistry, E. Moir,
R. Morphy, J. Tierney, M. York, J. Baker, J.E. Cottney, A.K. Houghton,
P.J. Westwood, G. Walker, Med. Chem. Commun. 1 (2010) 54e60.
[79] Z. Wei, R. Dolaine, C. Walpole, Yang, H. PCT Int. Appl. WO 2004/087704,
2004.
[80] A.J. Eatherton, G.M.P. Giblin, M.R. Johnson, W.L. Mitchell, A. Perboni, Slingsby,
B.P. PCT Int. Appl. WO 2005/121140, 2005.
[81] A.J. Eatherton, G.M.P. Giblin, K.S. Jandu, M.R. Johnson, W.L. Mitchell, A.
Naylor, Sweeting, J.A. PCT Int. Appl. WO 2007/017264, 2007.
[82] P.-W. Wu, W.-T. Hsieh, Y.-M. Cheng, C.-Y. Wei, P.-T. Chou, J. Am. Chem. Soc.
128 (2006) 14426e14427.
[47] M.R. Bell, T.E. D0Ambra, V. Kumar, M.A. Eissenstat, J.L. Herrmann, J.R. Wetzel,
D. Rosi, R.E. Philion, S.J. Daum, J. Med. Chem. 34 (1991) 1099e1110.
[48] T.E. D0Ambra, K.G. Estep, M.R. Bell, M.A. Eissenstat, K.A. Josef, S.J. Ward,
D.A. Haycock, E.R. Baizman, F.M. Casiano, J. Med. Chem. 35 (1992) 124e135.
[49] M.A. Eissenstat, M.R. Bell, T.E. D0Ambra, E.J. Alexander, S.J. Daum,
J.H. Ackerman, M.D. Gruett, V. Kumar, K.G. Estep, J. Med. Chem. 38 (1995)
3094e3105.
[50] M. Gallant, C. Dufresne, Y. Gareau, D. Guay, Y. Leblanc, P. Prasit, C. Rochette,
N. Sawyer, D.M. Slipetz, N. Tremblay, K.M. Metters, M. Labelle, Bioorg. Med.
Chem. Lett. 6 (1996) 2263e2268.
[83] M.T. Cash, P.R. Schreiner, R.S. Phillips, Org. Biomol. Chem.
3 (2005)
3701e3706.
[84] T. Tuccinardi, P.L. Ferrarini, C. Manera, G. Ortore, G. Saccomanni,
A. Martinelli, J. Med. Chem. 49 (2006) 984e994.
[85] M.C. Wenlock, R.P. Austin, P. Barton, A.M. Davis, P.D. Leeson, J. Med. Chem. 46
(2003) 1250e1256.
[86] T. Okauchi, M. Itonaga, T. Minami, T. Owa, K. Kitoh, H. Yoshino, Org. Lett. 2
(2000) 1485e1487.
[51] E.K. Dziadulewicz, S.J. Bevan, C.T. Brain, P.R. Coote, A.J. Culshaw, A.J. Davis,
L.J. Edwards, A.J. Fisher, A.J. Fox, C. Gentry, A. Groarke, T.W. Hart, W. Huber,
I.F. James, A. Kesingland, L. La Vecchia, Y. Loong, I. Lyothier, K. McNair,
C. O’Farrell, M. Peacock, R. Portmann, U. Schopfer, M. Yaqoob, J. Zadrobilek,
J. Med, Chem 50 (2007) 3851e3856.
[87] J.I. Levin, E. Turos, S.M. Weinreb, Synth. Commun. 12 (1982) 989e993.
[88] J.-Y. Mérour, B. Joseph, Curr. Org. Chem. 5 (2001) 471e506.
[89] F. Popowycz, S. Routier, B. Joseph, J.-Y. Mérour, Tetrahedron 63 (2007)
1031e1064.
[90] F. Popowycz, J.-Y. Mérour, B. Joseph, Tetrahedron 63 (2007) 8689e8707.
[91] J.J. Song, J.T. Reeves, F. Gallou, Z. Tan, N.K. Yee, C.H. Senanayake, Chem. Soc.
Rev. 36 (2007) 1120e1132.
[52] P. Diaz, J. Xu, F. Astruc-Diaz, H.M. Pan, D.L. Brown, M. Naguib, J. Med. Chem.
51 (2008) 4932e4947.
[53] J.M. Frost, M.J. Dart, K.R. Tietje, T.R. Garrison, G.K. Grayson, A.V. Daza, O.F. El-
Kouhen, L.N. Miller, L. Li, B.B. Yao, G.C. Hsieh, M. Pai, C.Z. Zhu, P. Chandran,
M.D. Meyer, J. Med. Chem. 51 (2008) 1904e1912.
[54] G.M. Giblin, A. Billinton, M. Briggs, A.J. Brown, I.P. Chessell, N.M. Clayton,
A.J. Eatherton, P. Goldsmith, C. Haslam, M.R. Johnson, W.L. Mitchell,
A. Naylor, A. Perboni, B.P. Slingsby, A.W. Wilson, J. Med. Chem. 52 (2009)
5785e5788.
[92] Z. Zhang, Z. Yang, H. Wong, J. Zhu, N.A. Meanwell, J.F. Kadow, T. Wang, J. Org.
Chem. 67 (2002) 6226e6227.
[93] G. Bartoli, G. Palmieri, M. Bosco, R. Dalpozzo, Tetrahedron Lett. 30 (1989)
2129e2132.
[94] Z. Zhang, Z. Yang, N.A. Meanwell, J.F. Kadow, T. Wang, J. Org. Chem. 67 (2002)
2345e2347.
[95] G. Chelucci, Tetrahedron Lett. 51 (2010) 1562e1565.
[96] T. Sakamoto, C. Satoh, Y. Kondo, H. Yamanaka, Heterocycles 34 (1992)
2379e2384.
[55] G.A. Thakur, R. Tichkule, S. Bajaj, A. Makriyannis, Expert Opin. Ther. Pat. 19
(2009) 1647e1673.
[56] K. Worm, R.E. Dolle, Curr. Pharm. Des. 15 (2009) 3345e3366.
[57] T.E. D’Ambra, M.A. Eissenstat, J. Abt, J.H. Ackerman, E.R. Bacon, M.R. Bell,
P.M. Carabateas, K.A. Josef, V. Kumar, J.D. Weaver, R. Arnold, F.M. Casiano,
S.M. Chippari, D.A. Haycock, J.E. Kuster, D.A. Luttinger, J.I. Stevenson,
S.J. Ward, W.A. Hill, A. Khanolkar, A. Makriyannis, Bioorg. Med. Chem. Lett. 6
(1996) 17e22.
[97] H. van de Waterbeemd, G. Camenisch, G. Folkers, J.R. Chretien, O.A. Raevsky,
J. Drug Target. 6 (1998) 151e165.
[98] S.M. Twine, L. Murphy, R.S. Phillips, P. Callis, M.T. Cash, A.G. Szabo, J. Phys.
Chem. B. 107 (2002) 637e645.
[99] H. van De Waterbeemd, D.A. Smith, K. Beaumont, D.K. Walker, J. Med. Chem.
44 (2001) 1313e1333.
[58] J.W. Huffman, L.W. Padgett, Curr. Med. Chem. 12 (2005) 1395e1411.
[59] H. Deng, PhD thesis, University of Connecticut (USA), 2000.
[60] A. Makriyannis, Deng, H. U.S. Patent 6,900,236, 2005.
[61] A. Makriyannis, Deng, H. U.S. Patent 7,173,027, 2007.
[62] J.W. Huffman, D. Dai, B.R. Martin, D.R. Compton, Bioorg. Med. Chem. Lett. 4
(1994) 563e566.
[63] J.W. Huffman, Cannabimimetic indoles, pyrroles, and indenes: structure-
activity relationsphips and receptor interactions. in: P.H. Reggio (Ed.), The
Cannabinoid Receptors. Humana Press, New York, 2009, pp. 49e94.
[64] J.L. Wiley, D.R. Compton, D. Dai, J.A. Lainton, M. Phillips, J.W. Huffman,
B.R. Martin, J. Pharmacol, Exp. Ther. 285 (1998) 995e1004.
[65] M.M. Aung, G. Griffin, J.W. Huffman, M. Wu, C. Keel, B. Yang, V.M. Showalter,
M.E. Abood, B.R. Martin, Drug Alcohol Depend. 60 (2000) 133e140.
[66] J.M. Frost, M.J. Dart, K.R. Tietje, T.R. Garrison, G.K. Grayson, A.V. Daza, O.F. El-
Kouhen, B.B. Yao, G.C. Hsieh, M. Pai, C.Z. Zhu, P. Chandran, M.D. Meyer,
J. Med. Chem. 53 (2010) 295e315.
[67] X.Q. Xie, X.W. Han, J.Z. Chen, M. Eissenstat, A. Makriyannis, J. Med. Chem. 42
(1999) 4021e4027.
[68] O.M. Salo, M. Lahtela-Kakkonen, J. Gynther, T. Jarvinen, A. Poso, J. Med. Chem.
47 (2004) 3048e3057.
[69] J.Y. Shim, A.C. Howlett, J. Chem. Inf. Model. 46 (2006) 1286e1300.
[70] J.W. Huffman, R. Mabon, M.J. Wu, J. Lu, R. Hart, D.P. Hurst, P.H. Reggio,
J.L. Wiley, B.R. Martin, Bioorg. Med. Chem. 11 (2003) 539e549.
[71] J.W. Huffman, G. Zengin, M.J. Wu, J. Lu, G. Hynd, K. Bushell, A.L. Thompson,
S. Bushell, C. Tartal, D.P. Hurst, P.H. Reggio, D.E. Selley, M.P. Cassidy,
J.L. Wiley, B.R. Martin, Bioorg. Med. Chem. 13 (2005) 89e112.
[100] J.H.M. Lange, H.K.A.C. Coolen, H.H. van Stuivenberg, J.A.R. Dijksman,
A.H.J. Herremans, E. Ronken, H.G. Keizer, K. Tipker, A.C. McCreary,
W. Veerman, H.C. Wals, B. Stork, P.C. Verveer, A.P. den Hartog, N.M.J. de Jong,
T.J.P. Adolfs, J. Hoogendoorn, C.G. Kruse, J. Med. Chem. 47 (2004) 627e643.
[101] MetaSite v3.0.6, Molecular Discovery Ltd., 2009.
[102] G. Cruciani, E. Carosati, B. De Boeck, K. Ethirajulu, C. Mackie, T. Howe,
R. Vianello, J. Med. Chem. 48 (2005) 6970e6979.
[103] A. Wintermeyer, I. Moller, M. Thevis, M. Jubner, J. Beike, M.A. Rothschild,
K. Bender, Anal. Bioanal. Chem. 398 (2010) 2141e2153.
[104] Q. Zhang, P. Ma, R.B. Cole, G. Wang, Anal. Bioanal. Chem. 386 (2006)
1345e1355.
[105] R.I. Wilson, R.A. Nicoll, Science 296 (2002) 678e682.
[106] M. Kano, T. Ohno-Shosaku, Y. Hashimotodani, M. Uchigashima, M. Watanabe,
Physiol. Rev. 89 (2009) 309e380.
[107] E.K. Miller, J.D. Cohen, Annu. Rev. Neurosci. 24 (2001) 167e202.
[108] N. Ramnani, A.M. Owen, Nat. Rev. Neurosci. 5 (2004) 184e194.
[109] A.F. Arnsten, Nat. Rev. Neurosci. 10 (2009) 410e422.
[110] H. Ujike, Y. Morita, J. Pharmacol. Sci. 96 (2004) 376e381.
[111] D.T. Malone, M.N. Hill, T. Rubino, Br. J. Pharmacol. 160 (2010) 511e522.
[112] B.K. Atwood, J. Huffman, A. Straiker, K. Mackie, Br. J. Pharmacol. 160 (2010)
585e593.
[113] W. Goutier, P.A. Spaans, M.A.W. van der Neut, A.C. McCreary, J.H. Reinders,
J. Neurosci. Methods 188 (2010) 24e31.
[114] J.H.M. Lange, M.A.W. van der Neut, H.C. Wals, G.D. Kuil, A.J. Borst, A. Mulder,
A.P. den Hartog, H. Zilaout, W. Goutier, H.H. van Stuivenberg, B.J. van Vliet,
Bioorg. Med. Chem. Lett. 20 (2010) 1084e1089.