Synthesis and pharmacological evaluation of coumarin derivatives as cannabinoid receptor antagonists and inverse agonists

Article abstract of DOI:10.1016/j.bmc.2009.02.027

In the present study we synthesized 36 coumarin and 2H-chromene derivatives applying a recently developed umpoled domino reaction using substituted salicylaldehyde and α,β-unsaturated aldehyde derivatives as starting compounds. In radioligand binding stud

Full text of DOI:10.1016/j.bmc.2009.02.027

Bioorganic & Medicinal Chemistry 17 (2009) 2842–2851
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological evaluation of coumarin derivatives
as cannabinoid receptor antagonists and inverse agonists
a,
Andrea Behrenswerth a, Nicole Volz b, Jakob Toräng b, Sonja Hinz a, Stefan Bräse b, , Christa E. Müller
*
*
a PharmaCenter Bonn, University of Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, An der Immenburg 4, D-53121 Bonn, Germany
b Institute of Organic Chemistry, University of Karlsruhe, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, D-76131 Karlsruhe, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study we synthesized 36 coumarin and 2H-chromene derivatives applying a recently
developed umpoled domino reaction using substituted salicylaldehyde and ,b-unsaturated aldehyde
derivatives as starting compounds. In radioligand binding studies 5-substituted 3-benzylcoumarin deriv-
Received 2 November 2008
Revised 7 February 2009
Accepted 15 February 2009
Available online 21 February 2009
a
atives showed affinity to cannabinoid CB1 and CB2 receptors and were identified as new lead structures.
In further GTP
c
S binding studies selected compounds were shown to be antagonists or inverse agonists.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Coumarins
Cannabinoid receptors
Antagonists
Inverse agonists
1. Introduction
the relief of pain in case of multiple sclerosis and neuropathic
pain.1,13 It is further approved for the treatment of nausea and
Cannabinoid receptors are part of the endocannabinoid system,
which consists of cannabinoid receptors, endogenous cannabinoids
(endocannabinoids), and the enzymes that synthesize and degrade
endocannabinoids.1 The cloning of the cannabinoid CB1 and CB2
receptor subtypes2,3 in the early 1990s followed by the discovery
and characterization of their endogenous ligands—that is, ananda-
mide (3)4 and 2-arachidonoylglycerol5—offered new areas for ther-
apeutic interventions.6 Recently, the orphan receptor GPR55 has
been described as a putative third cannabinoid receptor subtype,
which appears to be also activated by other lipid metabolites
including lysophosphatidic acid and sphingosine 1-phosphate.7–10
Clinical trials involving either cannabinoid receptor ligands or Can-
nabis sativa extracts demonstrated pharmacological activities.11
vomiting associated with chemotherapy and as an appetite stimu-
lant for AIDS patients.1 In animal models, cannabinoids have
shown to reduce allergic contact dermatitis.14
Antagonistsat cannabinoidCB1 receptorsalso exhibittherapeutic
potential while being devoid of severe psychotropic side effects such
as addiction.15,16 The CB1 antagonist rimonabant (SR141716A, 5)
was approved as a novel therapeutic for the treatment of obesity17
but meanwhile withdrawn from the market due to potential side ef-
fects; it may also be helpful for smoking cessation.18 Another prom-
ising application for such drugs is the treatment of drug dependence
(from alcohol, opioids, or cannabis) as indicated by both animal and
human studies.19 Furthermore, there is preliminary evidence for the
usefulness of CB1 antagonists in the treatment of Parkinson’s and
Huntington’s diseases20 probably due to a direct, physical, as well
as an indirect, functional interaction between adenosine A2A and
cannabinoid CB1 receptors in the brain striatum.21
For instance,
D D
9-tetrahydrocannabinol ( 9-THC, 1) was shown to
be effective in the treatment of Tourette’s syndrome tics12 and in
The physiological role of CB2 receptors has not been fully ana-
lyzed yet. CB2 agonists are effective in chronic pain models1 and
possess potent peripheral analgesic activity.22
Abbreviations: AIDS, acquired immune deficiency syndrome; BSA, bovine serum
albumin; CB1, cannabinoid receptor type 1; CB2, cannabinoid receptor type 2;
DABCO, 1,4-diazobicyclo[2.2.2]octane; DIC, diisopropylcarbodiimide; DMAP, 4-
(dimethylamino)pyridine; DTT, dithiothreitol; EtOAc, ethylacetate; GPCR, G pro-
Both CB1 and CB2 cannabinoid receptors belong to the superfam-
ily of G protein-coupled receptors (GPCRs). Both CB1 and CB2 recep-
tors are coupled via Gi/o proteins to inhibition of adenylate cyclase
and activation of mitogen-activated protein (MAP) kinase.23 Gs
coupling of the CB1 receptor has been described under certain con-
tein-coupled receptor; GPR55, G protein-coupled receptor 55; [35S]GTP
c
S, guano-
sine 50-( 35S)-triphosphate; HEK, human embryonic kidney; HR-EIMS, high
c-
resolution mass spectrometry; IR, infrared; mp, melting point; MS, mass spec-
trometry; NMR, nuclear magnetic resonance; n-/c-Hex, n-/cyclo-hexane; Rf, ratio of
fronts; THC, tetrahydrocannabinol.
* Corresponding authors. Tel.: +49 721 608 2902; fax: +49 721 608 8581 (S.B.);
tel.: +49 228 73 2301; fax: +49 228 73 2567 (C.E.M.).
ditions.24 In contrast, GPR55 is coupled to G 13 mediating activation
a
of rhoA, cdc42, and rac1.7,9 The highest density of the CB1 cannab-
inoid receptor is found in the cerebellum, hippocampus, cerebral
E-mail addresses: braese@ioc.uka.de (S. Bräse), christa.mueller@uni-bonn.de
(C.E. Müller).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.027

A. Behrenswerth et al. / Bioorg. Med. Chem. 17 (2009) 2842–2851
2843
cortex, basal ganglia, substantia nigra pars compacta, and in some
regions of the globus pallidus.25 It is also present in peripheral or-
gans such as the adrenal glands, bone marrow, lungs, testis, and
uterus.26 In contrast to the CB1 cannabinoid receptor, the CB2
receptor is limited essentially to cells associated with the immune
system, like leukocytes, spleen, thymus, and tonsils.27
possess a variety of pharmacological activities dependent on the
substitution pattern.40,41 Suitably substituted coumarin derivatives
that show structural similarity to cannabinol, such as compounds 7
and 8, have been described as highly selective CB2 receptor
agonists.22,42
In the present study, we synthesized a novel series of coumarin
and related 2H-chromene derivatives and explored their potential
as cannabinoid receptor ligands. The new compounds were inves-
tigated in radioligand binding studies to determine their affinity
for cannabinoid CB1 and CB2 receptor subtypes. Furthermore,
Due to the immense therapeutic potential of cannabinoid recep-
tor ligands, considerable efforts have been undertaken to identify
and optimize selective CB1 and CB2 receptor agonists and antago-
nists.28,29 The cannabinoid receptor agonists are classified into four
different groups. The classical cannabinoids consist of a tricyclic
[
35S]GTP
cS binding studies were performed in order to investigate
benzopyran-containing ring system like
D
9-THC (1). A dihydro-
the intrinsic activity (agonistic/antagonistic/inverse agonistic) of
benzopyran-type structure with a hydroxyl group at the C-1 aro-
matic position and an alkyl group on the C-3 aromatic position
seems to be a requirement for high cannabinoid receptor affinity.30
The length of the chain at C-3 is of major importance, but an all car-
bon side chain is not an absolute requirement, it may contain an
ethereal oxygen.31 The methyl group on C-9 and the double bond
in the terpene ring is not required for activity.32 The terpene ring
may be exchanged for some heterocyclic systems.33 Changes in
the stereochemistry at various carbons of THC-type molecules
may cause significant changes in pharmacological activity. Non-
the new compounds.
2. Results and discussion
2.1. Chemistry
3-Alkylcoumarins 9–40 were synthesized from salicylaldehydes
and a,b-unsaturated aldehydes utilizing nucleophilic carbenes in a
recently developed umpoled domino reaction (Scheme 1).43 While
compounds 9, 10, 12–15, 17, 20–24, 30, 32, and 37–40 were re-
ported earlier without any biological data43–50 all other com-
pounds are new derivatives.
classical cannabinoids lacking the dihydropyran ring of
D
9-THC
are represented by the bicyclic analogue CP55,940 (2).2 Amino-
alkylindoles possessing cannabimimetic properties, such as
WIN55,212-2 (4), have also been described.34 The prototypic mem-
ber of the eicosanoid group of cannabinoid receptor agonists is
anandamide (3), the first of five known endogenous cannabinoid
receptor agonists. In addition to the biarylpyrazole derivatives,
for example, rimonabant (5), several other heterocyclic classes of
compounds such as triazoles,35,36 thiazoles,36 pyrazolines,37 imida-
zoles36,38, and pyridines39 with antagonistic or inverse agonistic
properties have been described.
The chromene 41 was prepared as described earlier while the
chromenes 42–44 were obtained according to procedures previ-
ously published by our group (see Schemes 2–4).43 The starting
compound for the preparation of 44 was obtained as described.51
2.2. Biological evaluation
The new compounds were investigated in radioligand binding
studies at CB1 receptors using rat brain cortical membrane prepa-
rations, and at CB2 receptors using commercially available mem-
brane preparations of human embryonic kidney (HEK293) cells
Coumarins (chromene-2-ones, benzopyran-2-ones) are a natu-
rally occurring class of compounds, which have been shown to
H3C
R4
R4
N
Me2PO4
N
CH3
R3
R2
CHO
OH
R3
R2
R5
1 equiv.
R5
+
O
1 equiv. K2CO3
O
O
toluene, 100 °C, 18 h
R1
R1
9−40
Scheme 1. One-pot synthesis of 3-alkylcoumarins.
R2
R2
O
O
CHO
OH
R4
0.5 equiv. Na2CO3
R4
dioxane/H2O 3:1, 55 °C, 48 h
R3
+
R3
O
R1
R1
41, 42
Scheme 2. One-pot synthesis of 2H-chromenes 41 and 42 (for full structures see Table 1).
O
O
CHO
OH
2 equiv. DABCO
+
dioxane:H2O 1:2
sonification
15%
O
HO
H
43
Scheme 3. Synthesis of tetrahydroxanthone 43.

2844
A. Behrenswerth et al. / Bioorg. Med. Chem. 17 (2009) 2842–2851
1) HSCH2CH2SH, BF3 • Et2O,
EtO2C
CH2Cl2, r.t., 18 h
Ph
OH
2) EtO2CCH2CCO2H, DIC, DMAP,
CH2Cl2, r.t., 24 h
OHC
S
O
O
O
3) PhCH=CHCHO, piperidine,
acetic acid, benzene,
mol sieves, reflux, 4 h
35%
S
O
44
Scheme 4. Synthesis of 2H-chromene 44.
recombinantly expressing the human CB2 receptor. [3H](ꢀ)-cis-3-
[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxy-
propyl)cyclohexanol (CP55,940, 2) was used as CB1 and CB2 radio-
ligand. The assays were performed in a 96-well plate format to
allow fast screening, and the assay conditions were optimized for
that format (see Experimental). The evaluation of standard com-
pounds—unlabeled CP55,940; WIN55,212-2; and AM281—con-
firmed that our assays produced results which were in
accordance with published data52,53 (see Table 1). Initially the
at various positions of the 3-benzyl residue (compounds 24–32
and 34). These modifications were well tolerated by both receptor
subtypes and yielded compounds with relatively high affinity and
no or only weak selectivity. In general, substituents in the ortho-
position were best tolerated; the rank order of affinity was as fol-
lows: o-methyl (25) > p-methyl (26); o-chloro (27) > p-chloro (28);
o-methoxy (30) > m-methoxy (31) > p-methoxy (32) P o,p-dime-
thoxy (34). The ortho-substituted derivatives had similarly high
affinity for CB1 and CB2 receptors and showed Ki values around
compounds were tested a concentration of 10
l
M. In cases where
1
l
M or even in the submicromolar range. The compound with
the highest affinity was 3-o-methoxybenzyl-5-methoxy-7-methyl-
coumarin (30) with Ki values of 0.738 M at CB1 and 1.03 M at
inhibition of radioligand binding was greater than 60%, full concen-
tration–inhibition curves were determined using seven concentra-
tions spanning three orders of magnitude in order to determine
IC50 and to calculate Ki values. For all investigated compounds sig-
moidal concentration–inhibition curves were obtained. Selected
compounds of the present series were investigated for their func-
l
l
CB2 receptors. The corresponding 8-methyl isomer (3-o-methoxy-
benzyl-5-methoxy-8-methylcoumarin, 35) showed a 17-fold lower
Ki value at CB1 and a fourfold lower affinity for CB2 receptors. Also,
combination of 3-benzyl-5-methoxycoumarin (13) with a bromine
atom in the 8-position was unfavorable (compound 37). Another
trisubstituted coumarin derivative, 3-benzyl-5-isopropyl-8-meth-
ylcoumarin (38), showed micromolar affinity for both receptor
tional properties in [35S]GTP
cS binding at rat brain cortical mem-
branes and at HEK cells expressing the human CB1 receptor.
2.3. Structure–activity relationships
subtypes (Ki CB1 2.76 lM, CB2 3.93 lM) indicating that an isopro-
pyl residue in the 5-position of 3-benzylcoumarin may be favor-
able. However, in this case, o-methoxy-substitution of the 3-
benzyl residue did not enhance affinity (compound 36) possibly
due to a different binding mode of the trisubstituted coumarin
derivative. In the 7-position, a methyl group improved affinity by
several-fold in comparison with a hydrogen atom as shown in a
comparison of the lead compound 30 and its 7-unmethylated ana-
logue 33.
A series of 32 coumarin derivatives (9–40) with variations in
the 3-, 5-, 6-, 7-, and 8-position, as well as four 2H-chromene deriv-
atives (41–44) were investigated in order to assess their potential
to bind to cannabinoid CB1 and/or CB2 receptors and to study their
structure–activity relationships. Selected concentration–inhibition
curves from radioligand binding studies are shown in Figure 3.
In the 3-position of the coumarin scaffold, a methyl, benzyl, o-
hydroxybenzyl, methoxybenzyl (o, p, m), o,p-dimethoxybenzyl, or
chlorobenzyl (o, p) residue was present. None of the compounds
with a methyl residue in the 3-position displayed significant binding
neither towards CB1 nor CB2 receptors (12, 15, 17, 18, 21, 23, 39 and
40). In contrast, active compounds were identified among the ben-
zyl-substituted derivatives (e.g., 10, 24–36 and 38). However, a 3-
benzyl residue alone was not sufficient, since the 3-benzyl coumarin
9 was virtually inactive. Introduction of an additional, 5,6-annelated
benzene ring into 3-benzylcoumarin (yielding the tricyclic com-
pound 10) was favorable, particularly with respect to CB1 affinity
Two of the four investigated 2H-chromene derivatives, com-
pounds 43 and 44, also showed good affinity for CB1, but not CB2
receptors, with Ki values of 4.90 lM (43) and 1.37 lM (44), respec-
tively. Thus it appears that the pyranone structure of coumarins is
not required for interaction with the receptors, a partially hydroge-
nated benzopyran structure is sufficient. Again, a large, bulky, aro-
matic substituent is required in the 3-position of the benzopyran
ring system (like in compound 43) or nearby (44), while small sub-
stituents as in 41 and 42 result in virtually inactive or only slightly
active compounds. Thus a bulky, aromatic group in that position
appears to be essential.
(Ki 4.19
selective (Ki 2.06
5-position of 3-benzylcoumarin (9) resulting in compound 13 also
led to an increase in CB1 affinity (Ki 19.1 M), but not CB2 affinity.
lM), while the o-methoxybenzyl derivative 11 was CB2-
lM). The introduction of a methoxy group in the
2.4. Species differences
l
If a methoxy group was introduced into the 6-, 7-, or 8-position in-
stead (compounds 14, 20 and 22) weakly active or inactive com-
pounds were obtained. Residues other than methoxy in the 6-
position (iodo, nitro) also resulted in only weakly active or inactive
compounds (16 and 19). Careful analysis of the data appeared to
showthata 6-methoxygroupor a 6-iodoresiduewas somewhatbet-
ter tolerated by the CB2 receptor than by the CB1 receptor.
Additional introduction of a 7-methyl group into the best CB1 li-
gand so far, 3-benzyl-5-methoxycoumarin (13), increased affinity
for CB1 receptors by almost sixfold (24, Ki 3.46 lM) without much
The CB1 cannabinoid receptor has been cloned from rat,
mouse, and human tissues and it exhibits 97–99% amino acid se-
quence identity across species.55–58 The CB2 cannabinoid receptor
shows ca. 48% homology with the CB1 cannabinoid receptor.3 The
mouse CB2 receptor has an 82% sequence identity to the human
CB2 receptor.59 Sequence analysis of the CB2 genomic clone indi-
cates 93% amino acid identity between rat and mouse and 81%
amino acid identity between rat and human.42,60 Affinities and
efficacies of several reference cannabinoid ligands were investi-
gated at CB1 and CB2 receptors in three different species (rat,
mouse, and human). The ligands follow the same ranking of affin-
ity for the cannabinoid receptors in the mouse, rat, and human. In
own competition experiments at the rat cannabinoid CB1 recep-
effect on the CB2 affinity thus yielding a CB1-selective compound.
This derivative was selected as a lead compound for further varia-
tion: a methyl, chloro, hydroxy, or methoxy group was introduced

A. Behrenswerth et al. / Bioorg. Med. Chem. 17 (2009) 2842–2851
2845
Table 1
Affinities of coumarin and 2H-chromene derivatives for cannabinoid receptor subtypes
EtO2C
O
Ph
R5
OMe
O
O
R6
R7
R3
O
S
O
O
O
S
O
O
O
O
HO
H
R8
9−40
Ph
Br
41
42
43
44
Compound
R3
R5
R6
R7
R8
Ki SEM (
lM) (% inhibition SEM)
Rat CB1 versus [3H]CP55,940
0.00124 0.00058 (0.00137)39
Human CB2 versus [3H]CP55,940
0.000439 0.000021 (0.00069)38
a
a
2 CP55,940 (agonist)
4 WIN55,212-2
(agonist)
6 AM281
(inverse agonist)
9
—
—
—
—
—
—
—
—
—
—
0.00606 0.00062 (0.0044,54 0.00994)39 0.00536 0.00303 (0.0012)40
0.0124 0.0038 0.01244
>10 (18 7%)c
4.87 0.63 (n = 2) 4.2 [44]b
Benzyl
Benzyl
H
H
H
H
H
H
>10 (28 3%)c
10
11
12
4.19 2.00
P10 (59 9%)c
o-Methoxybenzyl
H
H
H
H
>10 (41 3%)c
>10 (10 4%)c
2.06 1.19
Methyl
ꢁ10 (2 1%)c
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Benzyl
Benzyl
Methyl
Benzyl
Methyl
Methyl
Benzyl
Benzyl
Methyl
Benzyl
Methyl
Benzyl
o-Methylbenzyl
p-Methylbenzyl
o-Chlorobenzyl
p-Chlorobenzyl
o-Hydroxybenzyl
o-Methoxybenzyl
m-Methoxybenzyl
p-Methoxybenzyl
o-Methoxybenzyl
Methoxy
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
19.1 4.0
>10 (28 7%)c
>10 (37 1%)c
ꢁ10 (7 8%)c
P10 (44 2%)c
>10 (10 7%)c
ꢁ10 (3 10%)c
>10 (15 5%)c
>10 (33 4%)c
ꢁ10 (2 3%e)c
>10 (25 3%)c
>10 (4 2%)c
P10 (45 3%)c
2.16 0.80
4.76 0.54
1.54 0.39
9.39 2.23
5.24 0.87
1.03 0.22
3.37 0.90
Methoxy
Methoxy
I
I
ꢁ10 (8 2%)c
>10 (15 10%)c
>10 (31 7%)c
>10 (12 10%)c
ꢁ10 (8 5%)c
>10 (32 15%)c
>10 (31 13%)c
ꢁ10 (1 6%)c
Cl
Nitro
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
—
—
—
—
Methoxy
Methoxy
H
H
H
H
H
Methoxy ꢁ10 (6 9%)c
Methoxy ꢁ10 (6 3%)c
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methoxy
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
H
Methyl
H
H
H
H
H
H
H
H
H
H
H
H
3.46 1.14
1.17 0.32
9.14 1.87
0.978 0.264
2.08 0.40
1.10 0.12
0.738 0.414
4.11 1.38
6.14 0.69
9.57 2.00
6.23 1.43
12.3 3.2
H
H
H
5.81 2.39
3.49 0.33d
2.78 1.22
o,p-Dimethoxybenzyl Methoxy
o-Methoxy-benzyl
o-Methoxy-benzyl
Benzyl
Benzyl
Methyl
Methyl
—
Methoxy
Isopropyl
Methoxy
Isopropyl
Methyl
Methyl
Br
3.65 1.20
6.18 0.96
>10 (39 6%)c
2.76 0.66
>10 (26 11%)c
5.67 1.10
>10 (29 2%)c
4.27 0.10
Methyl
Methyl
Isopropyl
H
H
—
—
—
—
ꢁ10 (0 2%)c
ꢁ10 (9 7%)c
ꢁ10 (3 1%)c
ꢁ10 (7 7%)c
>10 (37 5%)c
>10 (22 3%)c
H
—
—
—
—
Methoxy >10 (17 6%)c
—
—
—
—
P10 (46 7%)c
>10 (34 8%)c
4.90 1.11
—
—
—
1.37 0.27
a
Membrane preparations.
Binding to rat cannabinoid CB2 receptors on spleen tissue.
Percent inhibition of radioligand binding at 10 lM.
n = 2.
b
c
d
tors only very minor species differences were observed for the
CB1 receptors, for example, the determined Ki values for
CP55,940 (2) at rat and human receptors were quite similar
(1.24 nM at rat CB1 and 0.71 nM at human CB1 receptors; K. At-
zler and C.E. Müller, unpublished data). Therefore, Ki values deter-
mined at rat CB1 receptors should be highly predictive of Ki
values at human CB1 receptors.
2.5. Functional properties
The compounds of the present series that had shown the
highest affinities were investigated in [35S]GTP
S binding assays
at rat brain CB1 receptors and at human recombinant CB1 recep-
tors in order to study their intrinsic activity (see Fig. 4 and Table
2). GDP–GTP exchange is an early event in the signal transduc-
c

2846
A. Behrenswerth et al. / Bioorg. Med. Chem. 17 (2009) 2842–2851
tion mechanism of a G protein-coupled receptor. Measuring the
binding of [35S]GTP
S—a radiolabeled non-hydrolyzable analogue
3. Conclusions
c
of GTP—provides direct quantitative information on the interac-
tion between the receptor and the G protein. Binding of an ago-
nist to the GPCR will increase binding of the guanine nucleotide,
while a neutral antagonist does not have any effects on the
nucleotide binding, and an inverse agonist will decrease the
In conclusion, we have synthesized 32 coumarin (9–40) deriva-
tives, 22 of which are new compounds not previously described in
the literature, as well as four new 2H-chromene derivatives (41–
44). The compounds were evaluated for their potency to bind to
and activate cannabinoid receptors. 5-Substituted 3-benzylcouma-
rin derivatives were identified as new lead structures for the devel-
opment of cannabinoid CB1 and CB2 receptor antagonists/inverse
agonists, and initial structure activity relationships were analyzed.
3-o-Methoxybenzyl-5-methoxy-7-methylcoumarin (30) was the
compound with the highest affinity of the present series exhibiting
[
35S]GTP
at a high concentration of 30
cS binding to the G protein. The compounds were tested
l
M, and the determined effects
were compared to those elicited by the full agonist CP55,940
(2) and the inverse agonist AM281 (6). As expected CP55,940 in-
duced an increase in [35S]GTP
cS binding from a basal level of
100% to 169 9% at CB1 receptors natively expressed in rat brain
cortical membranes, and to 228 12% in membranes prepared
from HEK293 cells expressing the human CB1 receptor, respec-
a Ki value of 0.738 lM at CB1 and 0.944 lM at CB2 receptors. Some
of the compounds showed a certain degree of selectivity for CB1 or
CB2 receptors. The new compounds will be used as novel lead
structures for the development of more potent CB1 or CB2 antago-
nists/inverse agonists. In particular, the introduction of more bulky
substituents in the 7-position in order to fill the lipophilic pocket
tively (Fig. 4). AM281 had no apparent effect on [35S]GTP
cS
binding to CB1 receptors in rat cortex, but induced a decrease
in binding of the nucleotide to human recombinant CB1 recep-
tors expressed in HEK293 cells. AM281, previously described as
which is, for example, occupied by the pentyl chain of
may further increase affinity.
D
9-THC
full inverse agonist,61 led to a reduction of GTP
cS binding from
a basal level of 100% to 73 13% at CB1 receptors in HEK cell
membranes. The discrepant effect of the inverse agonist
AM281 at native CB1 receptors (neutral antagonistic effect) as
compared to CB1 receptors expressed in HEK293 cells (inverse
agonistic effect) can be explained by the high density of CB1
receptors in the recombinant cell system as compared to the
lower density in cortex. Similar effects have been observed be-
fore, for example, for the antagonist/inverse agonist
rimonabant.62,63
4. Experimental
4.1. Chemical synthesis
NMR spectra were recorded on a Varian XL-300 (1H: 300 MHz,
13C: 75 MHz) or a Bruker DRX 500 (1H: 500 MHz, 13C: 125 MHz).
Deuterated CDCl3 was used as a solvent unless otherwise noted.
The chemical shifts of the remaining protons of the solvent were
used as internal standard: 1H, 7.26 ppm; 13C, 77.0 ppm. All chemi-
cal shifts (d) were expressed in ppm. Coupling constants (J) are gi-
ven in Hertz (Hz). The reactions were monitored by thin layer
Figure 4 shows the effects of the coumarin derivatives 10, 24,
30, 32, and 38 on the binding of [35S]GTP
c
S to HEK293 cell mem-
branes expressing the human CB1 receptor. In Table 2 the results of
all [35S]GTP
S assays are collected.
None of the investigated compounds led to an increase in
35S]GTP
S binding at rat brain CB1 cannabinoid receptors thus
c
[
c
OH
showing that they were all antagonists. In HEK293 cells, which
were stably transfected with the human CB1 receptor, the
9
OH
coumarin derivatives induced a decrease in [35S]GTP
cS binding
OH
H
from a basal level of 100% down to between 72% and 90%. Com-
pound 10 was as efficacious as the full inverse agonist AM281
(72 6%). Thus, a condensed phenyl ring at the 5- and 6-position
led to selective CB1 receptor ligands with full inverse agonistic
activity. The most potent compound of the present series, cou-
H
H
O
9
HO
1
CP55,940
(−)-Δ -Tetrahydrocannabinol (
)
(2)
O
marin 30, led to a reduction in [35S]GTP
c
S binding from a basal
level of 100% to 79 2% indicating that it is also an efficacious
inverse agonist. Compounds 24, 32, and 38 showed less
pronounced inhibitory effect on [35S]GTP
S binding. This indi-
O
OH
a
N
H
N
c
O
cates that minor structural modifications may have a significant
influence on the inverse agonistic efficacy of coumarin
derivatives.
N
O
Anandamide (3)
WIN55212
(4)
N
2.6. Structural considerations
HN
O
A comparison of the coumarin-based agonists 7 and 8 (Fig. 2)
with the 3-benzylcoumarin derivatives identified in the present
study to be agonist/inverse agonists at CB1 receptors reveals
important differences. The agonists 7 and 8 feature a phenolic
OH group and a long lipophilic alkyl side chain on the same phenyl
N
N
R
Cl
ring. Comparing the structures of 7 and 8 with
D
9-THC (1, Fig. 1)
Cl
R = Cl Rimonabant(5)
R = I
shows that this partial structure is found in both agonistic classes
of compounds; however, it is lacking in the corresponding posi-
tions of the coumarin derivatives investigated in the present study
(position 5 and position 7, for OH and the long alkyl chain, respec-
tively). This may explain why the new compounds are CB receptor
antagonists rather than agonists.
AM281(6)
Figure 1. Structures of standard cannabinoid receptor ligands:
D
9-THC (1, agonist),
CP55,940 (2, agonist), anandamide (3, endogenous agonist), WIN55,212-2
(4, agonist), rimonabant (SR141716A, inverse agonist) (5), and AM281 (6, inverse
agonist).

A. Behrenswerth et al. / Bioorg. Med. Chem. 17 (2009) 2842–2851
2847
The combined organic phases were dried over magnesium sulfate
and the solvent was removed under reduced pressure. The product
was purified by flash column chromatography.
R
OH
4.1.2. 2-(2-Methoxybenzyl)-3H-benzo[f]chromen-3-one (11)
54.3 mg, 15% (Scale: 1.21 mmol). Rf (n-Hex/EtOAc 20:1) = 0.10.
mp: 142–145 °C. 1H NMR (400 MHz, CDCl3): d = 3.84 (s, 3H, OCH3),
4.00 (s, 2H, CH2), 6.92–6.96 (m, 1H, Har), 6.97–7.01 (m, 1H, Har),
7.29–7.36 (m, 2H, Har), 7.45 (d, 3J = 9.0 Hz, 1H, Har), 7.52–7.54 (m,
1H, Har), 7.58–7.62 (m, 1H, Har), 7.86–7.91 (m, 2H, Har), 8.02 (d,
3J = 8.3 Hz, 1H, Har), 8.08 (s, 1H, CH). 13C NMR (100 MHz, CDCl3):
d = 31.3 (s, CH2), 55.4 (p, OCH3), 110.6 (t, CarH), 113.6 (q, Car–CH),
116.8 (t, CarH), 120.8 (t, CarH), 121.4 (t, CarH), 125.7 (t, CarH), 126.0
(q, C–CH), 127.7 (q, Car–CH2), 127.8 (t, CarH), 127.9 (q, Car), 128.4 (t,
CarH), 128.9 (t, CarH), 130.2 (q, Car), 131.3 (t, CarH), 131.6 (t, CH),
134.7 (t, CarH), 152.3 (q, Car–OCO), 157.6 (q, Car–OCH3), 161.9 (q,
O
O
R = OCH3 AM1710 (7)
R = OH AM1714 (8)
Figure 2. Structure of coumarin derivatives AM-1710 (7) and AM-1714 (8), two CB2
receptor agonists (AM-1710: Ki 360 nM (rCB1), 6.7 nM (mCB2); AM-1714: Ki 400 nM
(rCB1), 0.82 nM (mCB2)).22
chromatography (TLC) using aluminum sheets with silica gel 60
F254 (Merck). The melting points were determined on a Laboratory
Devices Inc., model Mel-Temp II melting point apparatus, and are
uncorrected. Elemental analyses were performed in the Institute
of Organic Chemistry, University of Karlsruhe. Mass spectra were
collected on an MS-50 A.E.I. (Manchester) mass spectrometer with
an ionization energy of 70 eV. The coumarins 11, 25–29, 31, and
33–36 were synthesized as described.42
C@O). IR (KBr): 1713 (s,
m
C@O) cmꢀ1. MS (EI): m/z (%) = 316 (100,
M+), 304 (31), 285 (15, (MꢀCH3)+). HR-EIMS (C10H8O2): calcd
316.1099, found 316.1102. C21H16O3 (316 g/mol): Calcd C, 79.73*;
H, 5.10. Found: C, 78.45*; H, 5.14 (*deviation > 0.4%).
4.1.3. 3-Benzyl-6-iodochromen-2-one (16)
4.1.1. General procedure
Under an atmosphere of argon, 0.602 mmol of substituted sali-
cylaldehyde, 0.602 mmol of potassium carbonate, 1.51 mmol of
53.1 mg, 17% (Scale: 0.876 mmol). Rf (c-Hex/EtOAc 5:1) = 0.40.
mp: 147–150 °C. 1H NMR (400 MHz, CDCl3): d = 3.86 (br s, 2H,
CH2), 7.04 (d, 1H, 3J = 8.6 Hz, C8–H), 7.11 (br s, 1H, C4–H), 7.24–
7.35 (m, 8H, Har), 7.65 (d, 1H, 4J = 2.0 Hz, C5–H), 7.68 (dd, 1H,
4J = 2.0 Hz, 3J = 8.7 Hz, C7–H). 13C NMR (100 MHz, CDCl3): d = 36.6
(s, CH2), 87.1 (q, C-6), 118.4 (t, C-7), 121.5 (q, C-4a), 127.1 (t, CarH),
128.9 (+, 2 ꢂ CarH), 129.5 (t, 2 ꢂ CarH), 130.7 (q, C-3), 135.8 (t, C-5),
137.2 (q, Car), 137.7 (t, C-7), 139.3 (t, C-4), 152.6 (q, C-8a), 161.0 (q,
a,b-unsaturated aldehyde and 0.602 mmol of 1,3-dimethylimi-
dazolium dimethylphosphate were suspended in 2.00 ml of tolu-
ene. The reaction mixture was stirred at 100 °C for 24 h, after
which it was allowed to cool to room temperature. The reaction
was quenched by addition of 10 ml of water. The product was ex-
tracted with 2 ꢂ 10 ml of EtOAc and 1 ꢂ 10 ml of dichloromethane.
C-2). IR (KBr): 1726 (s,
m
C@O) cmꢀ1. MS (EI): m/z (%) = 362 (100,
120
100
80
60
40
20
0
CP55,940 (2)
WIN55212 (4)
24
30
32
38
13
10
10-1210-1110-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2
conc. (M)
120
100
80
60
40
20
0
CP55,940 (2)
30
32
38
10-1210-1110-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2
conc. (M)
Figure 3. Competition curves of selected compounds versus 0.5 nM [3H]CP55,940 at membrane preparations of rat brain cortical membranes (A), and at human embryonic
kidney (HEK293) cells expressing the human CB2 receptor (B). Data points represent means SEM of three independent experiments performed in triplicate.

2848
A. Behrenswerth et al. / Bioorg. Med. Chem. 17 (2009) 2842–2851
250
200
150
100
50
0
-50
-100
-150
Figure 4. [35S]GTP
cS binding assay at human CB1 cannabinoid receptors expressed in HEK293 cells. Data are expressed as means SEM of at least three separate experiments
performed in triplicate. Obtained data were normalized with respect to the maximal effect of AM281 (set at ꢀ100%).
M+), 333 (16). HR-EIMS (C17H14O3): calcd 361.9804, found
361.9804. C17H14O3 (266 g/mol): Calcd C, 53.06; H, 3.06. Found:
C, 53.46; H, 3.16.
(16), 178 (12). HR-EIMS (C16H11NO4): calcd 281.0688, found
281.0689. C16H11NO4 (281 g/mol): Calcd C, 68.32; H, 3.94; N,
4.98. Found: C, 68.54; H, 4.11; N, 4.70.
4.1.4. 8-Bromo-6-chloro-3-methylchromen-2-one (18)
4.1.6. 5-Methoxy-7-methyl-3-(2-methylbenzyl)-2H-chromen-2-
one (25)
28.1 mg, 17% (Scale: 0.602 mmol). Rf (c-Hex/EtOAc 5:1) = 0.29.
mp: 147–150 °C. 1H NMR (400 MHz, CDCl3): d = 2.24 (d, 3H,
4J = 1.3 Hz, CH3), 7.34 (d, 1H, 4J = 2.3 Hz, C5-H), 7.40 (qt, 1H,
4J = 1.4 Hz, C4-H), 7.66 (d, 1H, 4J = 2.4 Hz, C7-H). 13C NMR
(100 MHz, CDCl3): d = 17.2 (p, CH3), 110.7 (q, C-8), 121.3 (q,
C-4a), 125.5 (t, C-5), 128.2 (q, C-3), 129.7 (q, C-6), 133.4 (t, C-4),
71.8 mg, 41% (Scale: 0.602 mmol). Rf (c-Hex/EtOAc 30:1) = 0.04.
mp: 132–135 °C. 1H NMR (400 MHz, CDCl3): d = 2.28 (s, 3H, CH3),
2.40 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 3.86 (d, 4J = 1.1 Hz, 2H, CH2),
6.47 (s, 1H, Har), 6.73 (s, 1H, Har), 7.19–7.22 (m, 4H, Har), 7.71 (d,
4J = 1.1 Hz, 1H, CH). 13C NMR (100 MHz, CDCl3): d = 17.0 19.5 (p,
CH3), 22.3 (p, CH3), 33.9 (s, CH2), 55.7 (p, OCH3), 106.2 (t, CarH),
107.7 (q, Car–CH), 109.0 (t, CarH), 125.0 (q, C–CH2), 126.2 (t, CarH),
126.9 (t, CarH), 130.2 (t, CarH), 130.5 (t, CarH), 134.0 (t, CH), 136.1
(q, Car–CH2), 136.8 (q, Car–CH3), 142.5 (q, Car–CH3), 154.0 (q,
Car–O–CO), 155.3 (q, Car–OCH3), 162.1 (q, C@O). IR (KBr): 1714 (s,
137.6 (t, C-7), 148.7 (q, C-8a), 160.6 (q, C-2). IR (KBr): 1718 (s,
m
C@O) cmꢀ1. MS (EI): m/z (%) = 272/274 (100/98, M+), 243/245
(40/38, M+ꢀCO). HR-EIMS (C10H6BrClO2): calcd 271.9240, found
271.9246. C10H6BrClO2 (272 g/mol): Calcd C, 43.91*; H, 2.21.
Found: C, 44.55*; H, 2.59 (*deviation > 0.4%).
m
C@O) cmꢀ1 MS (EI): m/z (%) = 294 (100, M+), 276 (18), 203 (31,
4.1.5. 3-Benzyl-6-nitrochromen-2-one (19)
(C12H11O3)+). HR-EIMS (C10H8O2): calcd 294.1256, found
294.1258. C19H18O3 (294 g/mol): Calcd C, 77.53*; H, 6.16. Found
C, 76.89*; H, 6.17 (*deviation > 0.4%).
23.8 mg, 5% (Scale: 1.64 mmol). Rf (cHex/EtOAc 5:1) = 0.35. mp:
167–171 °C. 1H NMR (300 MHz, CDCl3): d = 3.92 (s, 2H, CH2), 7.27–
7.45 (m, 7H, CarH, C4–H), 8.29 (d, 1H, 4J = 2.3 Hz, C5–H), 8.32 (dd,
1H, 4J = 2.6 Hz, 3J = 8.9 Hz, C7–H). 13C NMR (75 MHz, CDCl3):
d = 36.7 (s, CH2), 117.6 (t, C-8), 119.5 (q, C-4a), 123.3 (t, C-5),
125.6 (t, C-7), 127.3 (t, CarH), 129.0 (t, 2 ꢂ CarH), 129.5 (t, 2 ꢂ CarH),
132.2 (q, C-3), 136.6 (q, Car), 137.8 (t, C-4), 144.0 (q, C-6), 156.5 (q,
4.1.7. 5-Methoxy-7-methyl-3-(4-methylbenzyl)-2H-chromen-2-
one (26)
70.2 mg, 35% (Scale:0.602 mmol). Rf (c-Hex/EtOAc 20:1) = 0.12.
mp: 120–123 °C. 1H NMR (400 MHz, CDCl3): d = 2.33 (s, 3H, CH3),
2.40 (s, 3H, CH3), 3.82 (s, 2H, CH2), 3.86 (s, 3H, OCH3), 6.48 (s, 1H,
Har), 6.71 (s, 1H, Har), 7.13 (d, 3J = 7.9 Hz, 2H, Har), 7.20 (d,
C-8a), 160.1 (q, C-2). IR (KBr): 1735 (s,
m C@O), 1529 (s, mas NO2),
1342 (s, msym NO2) cmꢀ1. MS (EI): m/z (%) = 281 (100, M+), 252
Table 2
Results from [35S]GTP
c
S binding studies of selected compounds at native rat CB1 receptors and human CB1 receptors expressed in HEK cells in comparison with Ki values from
competition binding studies
Compd.
CB1 rat brain cortical
Effect on [35S]GTP
c
S binding at rat brain cortical
Effect on [35S]GTP
cS binding at membranes from HEK cells
membranes versus
membranes in relation to the full agonist
CP55,940 set at 100% ( SEM)
expressing the human CB1 receptor in relation to the full
agonist CP55,940 set at 100% ( SEM)
[3H]CP55,940 Ki (
lM)
CP55,940
AM281
10
24
30
0.00124
0.01264
4.19
3.46
0.738
6.14
100 0a
2
100 0a
11b
3
ꢀ19 9c
15
3
ꢀ21
ꢀ8
4
9
7
ꢀ2
7
4
ꢀ16
ꢀ10
ꢀ9
1
7
32
38
13
2.76
19 12
8
a
The full agonist CP55,940 led to a maximal stimulation of 169 6% (n = 3) at native rat CB1 receptors in brain cortical membranes, and 228 12% at human CB1 receptors
expressed in HEK cells (n = 2) over basal (=100%).
b
The full inverse agonist AM281 had no apparent effect on [35S]GTP
c
S binding in rat cortical membranes: 102 7% (n = 3), basal (=100%).
c
The full inverse agonist AM281 reduced [35S]GTP
cS binding in CB1-transfected HEK293 cells from 100% basal to 73 13% (n = 2).

A. Behrenswerth et al. / Bioorg. Med. Chem. 17 (2009) 2842–2851
2849
3J = 7.9 Hz,, 2H, Har), 7.72 (s, 1H, CH). 13C NMR (100 MHz, CDCl3):
d = 21.0 (p, CH3), 22.3 (p, CH3), 36.3 (s, CH2), 55.8 (p, OCH3), 106.2
(t, CarH), 107.8 (q, Car), 109.1 (t, CarH), 126.1 (q, C–H), 129.0 (t,
2 ꢂ CarH), 129.3 (t, 2 ꢂ CarH), 134.4 (t, CH), 135.3 (q, Car–CH2),
136.0 (q, Car–CH3), 142.4 (q, Car–CH3), 154.2 (q, Car–O–CO), 155.4
Har), 6.85–6.87 (m, 1H, Har), 6.89 (d, 3J = 7.6 Hz, 1H, Har), 7.22–
7.26 (m, 1H, Har), 7.72 (s, 1H, CH). 13C NMR (100 MHz, CDCl3):
d = 22.9 (p, CH3), 36.7 (s, CH2), 55.1 (p, OCH3), 55.7 (p, OCH3),
106.2 (t, CarH), 107.7 (q, Car––CH), 109.0 (t, CarH), 111.9 (t, CarH),
114.8 (t, CarH), 121.5 (t, CarH), 125.6 (q, C–CH2), 129.5 (t, CarH),
134.7 (t, CH), 139.9 (q, Car–CH2), 142.5 (q, Car–CH3), 154.1 (q, Car–
OCH3), 155.3 (q, Car–OCH3), 159.7 (q, Car–O–CO), 162.0 (q, C@O).
(q, Car–OCH3), 162.0 (q, C@O). IR (KBr): 1711 (s,
m
C@O) cmꢀ1. MS
(EI): m/z (%) = 294 (100, M+), 279 (24, (MꢀCH3)+), 265 (11). HR-
EIMS (C10H8O2): calcd 294.1256, found 294.1254. C19H18O3
(294 g/mol): Calcd C, 77.53; H, 6.16. Found: C, 77.51; H, 6.12.
IR (KBr): 1701 (s,
m
C@O) cmꢀ1. MS (EI): m/z (%) = 310 (100, M+),
279 (10, (MꢀOCH3)+), 203 (13). HR-EIMS (C10H8O2): calcd
310.1205, found 310.1208. C19H18O4 (310 g/mol): Calcd C, 73.53*;
H, 5.85. Found: C, 73.03*; H, 5.93 (*deviation > 0.4%).
4.1.8. 3-(2-Chlorobenzyl)-5-methoxy-7-methyl-2H-chromen-2-
one (27)
39.7 mg, 21% (Scale: 0.602 mmol). Rf (c-Hex/EtOAc 40:1) = 0.03.
mp: 138–144 °C. 1H NMR (400 MHz, CDCl3): d = 2.40 (s, 3H, CH3),
3.83 (s, 3H, OCH3), 3.99 (s, 2H, CH2), 6.48 (s, 1H, Har),6.72 (s, 1H,
Har), 7.19–7.25 (m, 2H, Har), 7.36–7.41 (m, 2H, Har), 7.64 (s, 1H,
CH). 13C NMR (100 MHz, CDCl3): d = 22.3 (p, CH3), 34.2 (s, CH2),
55.8 (p, OCH3), 106.2 (t, CarH), 107.7 (q, Car–CH), 109.0 (t, CarH),
123.8 (q, C–CH2), 127.0 (t, CarH), 128.2 (t, CarH), 129.6 (t, CarH),
131.6 (t, CarH), 134.4 (q, Car–Cl), 134.8 (t, CH), 135.8 (q, Car–CH2),
142.7 (q, Car–CH3), 154.1 (q, Car–O–CO), 155.4 (q, Car–OCH3),
4.1.12. 5-Methoxy-3-(2-methoxybenzyl)-2H-chromen-2-one
(33)
57.8 mg, 30% (Scale: 0.789 mmol). Rf (c-Hex/EtOAc 40:1) = 0.05.
mp: 147–150 °C. 1H NMR (400 MHz, CDCl3): d = 3.81 (s, 3H, OCH3),
3.85 (s, 3H, OCH3), 3.88 (s, 2H, CH2), 6.65 (d, 3J = 8.1 Hz, 1H, Har),
6.88–6.91 (m, 2H, Har), 6.92–6.96 (m, 1H, Har), 7.24–7.28 (m, 2H,
Har), 7.32–7.36 (m, 1H, Har), 7.69 (s, 1H, CH). 13C NMR (100 MHz,
CDCl3): d = 31.0 (s, CH2), 55.3 (p, OCH3), 55.8 (p, OCH3), 104.8 (t,
CarH), 108.8 (t, CarH), 110.2 (q, Car–CH), 110.5 (t, CarH), 120.6 (t,
CarH), 126.3 (q, Car–CH2), 126.5 (q, Car–CH2), 128.0 (t, CarH), 130.8
(t, CarH), 131.0 (t, CarH), 134.0 (t, CH), 154.0 (q, Car–O–CO), 155.6
(q, Car–OCH3), 157.6 (q, Car–OCH3), 161.9 (q, C@O). IR (KBr): 1723
161.9 (q, C@O). IR (KBr): 1707 (s,
m
C@O) cmꢀ1. MS (EI): m/z
(%) = 314 (8, M+), 279 (100, (MꢀCl)+). HR-EIMS (C10H8O2): calcd
314.0710, found 314.0706. C18H15ClO3 (314 g/mol): Calcd C,
68.68; H, 4.80. Found: C, 68.43; H, 4.85.
(s,
m
C@O) cmꢀ1. MS (EI): m/z (%) = 296 (15, M+), 189 (11). HR-EIMS
(C10H8O2): calcd 296.1049, found 296.1050. C18H16O4 (296 g/mol):
4.1.9. 3-(4-Chlorobenzyl)-5-methoxy-7-methyl-2H-chromen-2-
one (28)
Calcd C, 72.96; H, 5.44. Found: C, 72.99; H, 5.54.
50.3 mg, 26% (Scale: 0.602 mmol). Rf (c-Hex/EtOAc 20:1) = 0.04.
mp: 143–147 °C. 1H NMR (400 MHz, CDCl3): d = 2.40 (s, 3H, CH3),
3.82 (s, 2H, CH2), 3.86 (s, 3H, OCH3), 6.49 (s, 1H, Har), 6.70 (s, 1H,
Har), 7.22–7.29 (m, 4H, Har), 7.71 (s, 1H, CH). 13C NMR (100 MHz,
CDCl3): d = 22.3 (p, CH3), 36.2 (s, CH2), 55.8 (p, OCH3), 106.2 (t,
CarH), 107.6 (q, Car), 109.1 (t, CarH), 125.2 (q, C–CH), 128.7 (t,
2 ꢂ CarH), 130.5 (t, 2 ꢂ CarH), 132.3 (q, Car–CH2), 134.7 (t, CH),
136.8 (q, Car–CH3), 142.8 (q, Car–CH3), 154.2 (q, Car–O–CO), 155.4
4.1.13. 3-(2,4-Dimethoxybenzyl)-5-methoxy-7-methyl-2H-
chromen-2-one (34)
27.3 mg, 21% (Scale: 0.602 mmol) Rf (c-Hex/EtOAc 5:1) = 0.12.
mp: 161–165 °C. 1H NMR (400 MHz, CDCl3): d = 2.39 (s, 3H, CH3),
3.78 (s, 3H, OCH3), 3.79 (s, 2H, CH2), 3.81 (s, 3H, OCH3), 3.84 (s,
3H, OCH3), 6.45–6.48 (m, 3H, Har), 6.71 (s, 1H, Har), 7.16 (d,
3J = 8.2 Hz, 1H, Har), 7.61 (s, 1H, CH). 13C NMR (100 MHz, CDCl3):
d = 22.3 (p, CH3), 30.3 (s, CH2), 55.3 (p, OCH3), 55.4 (p, OCH3),
55.8 (p, OCH3), 98.7 (t, CarH), 104.1 (t, CarH), 106.1 (t, CarH), 108.0
(q, Car–CH), 109.0 (t, CarH), 118.9 (q, Car–CH2), 125.6 (q, C–CH2),
131.3 (t, CarH), 133.9 (t, CH), 142.0 (q, Car–CH3), 154.0 (q, Car–O–
CO), 155.3 (q, Car–OCH3), 158.5 (q, Car–OCH3), 159.8 (q, Car–
(q, Car–OCl), 161.8 (q, C@O). IR (KBr): 1711 (s,
m
C@O) cmꢀ1. MS
(EI): m/z (%) = 314 (100, M+), 279 (80, (MꢀCl)+), 125 (18). HR-EIMS
(C10H8O2): calcd 314.0710, found 314.0707. C18H15ClO3 (314 g/
mol): Calcd C, 68.68; H, 4.80. Found: C, 68.58; H, 4.83.
OCH3), 162.2 (q, C@O). IR (KBr): 1724 (s,
m
C@O) cmꢀ1. MS (EI):
4.1.10. 3-(2-Hydroxybenzyl)-5-methoxy-7-methyl-2H-
chromen-2-one (29)
m/z (%) = 340 (100, M+), 325 (17, (MꢀCH3)+), 309 (18, (MꢀOCH3)+).
HR-EIMS (C10H8O2): calcd 340.1311, found 340.1314. C20H20O5
(340 g/mol): Calcd C, 70.57; H, 5.92. Found: C, 70.38; H, 6.05.
187 mg, 42% (Scale: 0.602 mmol). Rf (c-Hex/EtOAc 20:1) = 0.18.
mp: 173–177 °C. 1H NMR (400 MHz, CDCl3): d = 2.41 (s, 3H, CH3),
3.83 (s, 2H, CH2), 3.92 (s, 3H, OCH3), 6.53 (s, 1H, Har), 6.72 (s, 1H,
Har), 6.87 (ddd, 3J = 7.5 Hz, 4J = 1.2 Hz, 1H, Har), 6.96 (dd,
3J = 8.1 Hz, 4J = 1.2 Hz, 1H, Har), 7.14 (ddd, 3J = 8.1 Hz, 4J = 1.7 Hz,
1H, Har), 7.22 (dd, 3J = 7.5 Hz, 4J = 1.7 Hz, 1H, Har), 8.08 (s, 1H,
CH), 8.19 (s, 1H, OH). 13C NMR (100 MHz, CDCl3): d = 22.4 (p,
CH3), 32.3 (s, CH2), 55.9 (p, OCH3), 106.7 (t, CarH), 108.0 (q, Car),
109.1 (t, CarH), 118.2 (t, CarH), 120.8 (t, CarH), 125.0 (q, C–CH2),
125.3 (q, Car–CH2), 128.6 (t, CarH), 130.5 (t, CarH), 136.0 (t, CH),
143.4 (q, Car–CH3), 153.9 (q, Car–O–CO), 154.7 (q, Car–OH), 155.4
4.1.14. 5-Methoxy-3-(2-methoxybenzyl)-8-methyl-2H-
chromen-2-one (35)
50.4 mg, 26% (Scale: 1.50 mmol). Rf (c-Hex/EtOAc 10:1) = 0.11.
mp: 124–126 °C. 1H NMR (400 MHz, CDCl3): d = 2.35 (s, 3H, CH3),
3.81 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.89 (s, 2H, CH2), 6.56 (d,
3J = 8.3 Hz, 1H, Har), 6.90 (d, 3J = 8.3 Hz, 1H, Har), 6.92–6.96 (m, 1H,
Har), 7.18 (d, 3J = 8.4 Hz, 1H, Har), 7.23–7.28 (m, 2H, Har), 7.70 (s,
1H, CH). 13C NMR (100 MHz, CDCl3): d = 14.8 (p, CH3), 30.9 (s, CH2),
55.3 (p, OCH3), 55.7 (p, OCH3), 104.3 (t, CarH), 110.0 (q, Car–CH),
110.5 (t, CarH), 117.5 (q, Car–CH3), 120.6 (t, CarH), 126.0 (q, Car–
CH2), 126.4 (q, C–CH), 128.0 (t, CarH), 131.0 (t, CarH), 131.7 (t, CarH),
134.4 (t, CH), 152.0 (q, Car–O–CO), 153.8 (q, Car–OCH3), 157.6 (q,
(q, Car–OCH3), 165.0 (q, C@O). IR (KBr): 1725 (s,
m
C@O) cmꢀ1. MS
(EI): m/z (%) = 296 (100, M+), 190 (35), 159 (19). HR-EIMS
(C10H8O2): calcd 296.1049, found 296.1046. C18H16O4 (296 g/
mol): Calcd C, 72.96*; H, 5.44. Found: C, 72.49*; H, 5.48
(*deviation > 0.4%).
Car–OCH3), 162.0 (q, C@O). IR (KBr): 1711 (s,
m
C@O) cmꢀ1. MS (EI):
m/z (%) = 310 (100, M+), 279 (10, (MꢀOCH3)+), 203 (13). HR-EIMS
(C10H8O2): calcd 310.1205, found 310.1208. C19H18O4 (310 g/mol):
Calcd C, 73.53; H, 5.85. Found: C, 73.13; H, 5.81.
4.1.11. 5-Methoxy-3-(3-methoxybenzyl)-7-methyl-2H-
chromen-2-one (31)
84.1 mg, 45% (Scale: 0.602 mmol). Rf (c-Hex/EtOAc 20:1) = 0.04.
mp: 108–112 °C. 1H NMR (400 MHz, CDCl3): d = 2.39 (s, 3H, CH3),
3.80 (s, 3H, OCH3), 3.84 (s, 2H, CH2), 3.85 (s, 3H, OCH3), 6.48 (s,
1H, Har), 6.70 (s, 1H, Har), 6.79 (dd, 3J = 8.2 Hz, 4J = 2.5 Hz, 1H,
4.1.15. 5-Isopropyl-3-(2-methoxybenzyl)-7-methyl-2H-
chromen-2-one (36)
130 mg, 55% (Scale: 0.729 mmol). Rf (c-Hex/EtOAc 80:1) = 0.15.
mp: 107–111 °C. 1H NMR (400 MHz, CDCl3): d = 1.21 (d, 3J = 6.8 Hz,

2850
A. Behrenswerth et al. / Bioorg. Med. Chem. 17 (2009) 2842–2851
i
6 H, 2 ꢂ Pr–CH3), 2.41 (s, 3H, CH3), 3.17 (sept, 3J = 6.8 Hz, 1H, Pr–
CH), 3.81 (s, 3H, OCH3), 3.91 (s, 2H, CH2), 6.89–6.97 (m, 2H, Har),
7.02–7.04 (m, 1H, Har), 7.23–7.33 (m, 3H, Har), 7.64 (s, 1H, CH).
13C NMR (100 MHz, CDCl3): d = 15.4 (p, CH3), 23.4 (t, iPr–CH),
cates. Binding studies at human CB2 receptors were performed in
duplicates and at least three separate experiments were performed
unless otherwise noted. The incubation was started by adding the
protein, and terminated after 2 h by rapid filtration using a Brandel
96-channel cell harvester (Brandel, Gaithersburgh, Maryland, USA)
through GF/C glass fiber filters (Perkin Elmer, Boston, USA). Filters
were rinsed three times with 2 ml of ice-cold washing buffer each,
containing 50 mM Tris–HCl, pH 7.4, and 0.1% bovine serum albu-
min (BSA). Radioactivity of filter plates, which had been dried for
90 min at 50 °C, was measured in a TopcountÒ microplate scintilla-
tion and luminescence counter after 10 h of preincubation with
0.05 ml of MicroscintTM20 scintillation cocktail (Perkin Elmer, Bos-
ton, USA).
i
i
28.4 (p, 2 ꢂ Pr–CH3), 31.4 (s, CH2), 55.2 (p, OCH3), 110.3 (t, CarH),
116.9 (q, Car–CH), 119.9 (t, CarH), 120.7 (t, CarH), 123.2 (q,
Car–CH2), 126.2 (q, C–CH), 126.9 (q, Car–CH3), 128.2 (t, CarH),
131.2 (t, CarH), 131.7 (t, CarH), 135.9 (t, CH), 143.2 (q, Car-iPr),
151.9 (q, Car–O–CO), 157.5 (q, Car–OCH3), 161.7 (q, C@O). IR
(KBr): 1718 (s,
m
C@O) cmꢀ1. MS (EI): m/z (%) = 322 (6, M+), 310
(100), 91 (20). HR-EIMS (C10H8O2): calcd 322.1569, found
322.1570. C21H22O3 (322 g/mol): Calcd C, 78.23; H, 6.88. Found:
C, 78.13; H, 6.89.
4.2. Biological assays
4.2.1. Materials
4.2.4. [35S]GTP
cS binding assays
Assays were performed essentially as previously described.68,69
Membrane preparations of rat brain cortical membranes, or com-
mercially available membrane preparations expressing the human
GTPcS, EDTA, GDP, AM281, and cell culture materials were ob-
tained from Sigma–Aldrich (Steinheim, Germany). Tris and BSA
were from Carl Roth GmbH (Karlsruhe, Germany), HCl and DTT
from Applichem (Darmstadt, Germany), MgCl2 and sucrose from
Fluka (Buchs, Switzerland), NaCl from KMF Optichem (Lohmar,
Germany), DMSO from Riedel-de Haën (Seelze, Germany), and
CP55,940 and WIN55,212-2 from Tocris (Ellisville, MO, USA).
[3H]CP55,940 was obtained from PerkinElmer Life Sciences, Rod-
gau-Jügesheim, Germany (158 Ci/mmol; 5.85 TBq/mmol).
CB1 receptor, respectively, 5
0.1 nM [35S]GTP
S (46.3 TBq/mmol, PerkinElmer Life Sciences,
Roddgau-Jügesheim, Germany) in a total volume of 200 l of
Tris–HCl buffer, 50 mM, pH 7.4, containing 1 mM EDTA, 5 mM
MgCl2, 1 mM dithiothreitol (DTT), 30 M GDP, 100 mM NaCl,
0.5% BSA, and test compound dissolved in DMSO. The final DMSO
concentration in the assay was 1%. Nonspecific binding was deter-
mined in the presence of 30 lM of unlabeled GTPcS. Incubations
lg per tube, were incubated with
c
l
l
were terminated after 60 min of incubation at rt by the addition
of 2 ml of ice-cold buffer containing 50 mM Tris–HCl, pH 7.4, and
5 mM MgCl2, and rapid filtration through GF/B glass fiber filter
(Schleicher and Schuell, Germany) on a BrandelÒ 48-channel har-
vester, followed by two washing steps with ice-cold buffer, 2 ml
each. Radioactivity on the wet filters was measured by liquid scin-
tillation counting after punching out the filters and incubating
them for 9 h in 2.5 ml of scintillation cocktail (Ready SafeTM, Beck-
man Coulter, USA).
4.2.2. Membrane preparations
Frozen rat brains were purchased from Pel FreezÒ, Rogers, AR,
USA. Rat brain cortical membrane preparations were obtained as
previously described65 with minor modifications. Brains were
thawed and then kept on ice while the cortex was dissected. The
cortex was subsequently homogenized in ice-cold 0.32 M sucrose
solution using an Ultra-TurraxÒ T25 basic homogenizer, IKA Labor-
technik for 10 s at setting 3. The suspension was centrifuged at
1000g for 5 min (4 °C), the pellet was discarded and the superna-
tant was subsequently centrifuged at 37,000g for 60 min (4 °C).
The supernatant was discarded and the pellet was resuspended
in ice-cold water, homogenized with the Ultra TurraxÒ and recen-
trifuged at 37,000g for 60 min (4 °C). The resulting membrane pel-
let was resuspended in Tris–HCl buffer, 50 mM, pH 7.4, and stored
at ꢀ80 °C. Protein concentration was determined by the method of
Lowry.66 Membrane preparations of human embryonic kidney
(HEK) 293 EBNA cells expressing the human CB1 or the human
CB2 receptor, respectively, were obtained from Perkin Elmer, Bos-
ton, USA.
4.2.5. Data analysis
Data were analyzed using Graph Pad PRISM Version 3.0 (San
Diego, CA, USA). For the calculation of Ki values by nonlinear
regression analysis, the Cheng Prusoff equation and KD values of
0.58 nM (rCB1) and 0.7 (hCB2) for [3H]CP55,940 were used.
Acknowledgments
This study was supported by the Deutsche Forschungsgemeins-
chaft (GRK-804, scholarships for A.B. and J.T.). We thank Alexander
Zielinski for performing some of the radioligand binding assays.
4.2.3. Radioligand binding assays
Competition experiments were performed versus the cannabi-
noid receptor agonist radioligand [3H]CP55,940 as described be-
fore67 with some minor modifications. Stock solutions of
compounds were prepared in DMSO. The final DMSO concentration
in the assays was 2.5%. Inhibition curves were determined using
seven different concentrations of test compound spanning three
orders of magnitude. Experiments were performed using 0.5 nM
[3H]CP55,940 at rt in 96-well microtest plates (0.5 ml, CostarÒ
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.02.027.
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