Synthesis, binding studies and molecular modeling of novel cannabinoid receptor ligands

Article abstract of DOI:10.1016/j.bmc.2010.10.050

In the present work, we report upon the design, synthesis and biological evaluation of new anandamide derivatives obtained by modifications of the fatty acyl chain and/or of the ethanolamide 'tail'. The compounds are of the general formula: 6-(substituted

Full text of DOI:10.1016/j.bmc.2010.10.050

Bioorganic & Medicinal Chemistry 18 (2010) 8463–8477
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, binding studies and molecular modeling of novel cannabinoid
receptor ligands
Noha A. Osman a, Amr H. Mahmoud b, Marco Allarà c, Raimund Niess a, Khaled A. Abouzid b,
Vincenzo Di Marzo c, Ashraf H. Abadi a,
⇑
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Al Tagamoa Al Khames, New Cairo City 11835, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo 11566, Egypt
c Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present work, we report upon the design, synthesis and biological evaluation of new anandamide
derivatives obtained by modifications of the fatty acyl chain and/or of the ethanolamide ‘tail’. The com-
pounds are of the general formula: 6-(substituted-phenyl)/naphthyl-4-oxohex-5-enoic acid N-substi-
tuted amide and 7-naphthyl-5-oxohept-6-enoicacid N-substituted amide. The novel compounds had
been evaluated for their binding affinity to CB1/CB2 cannabinoid receptors, binding studies showed that
some of the newly developed compounds have measurable affinity and selectivity for the CB2 receptor.
Compounds XI and XVIII showed the highest binding affinity for CB2 receptor. None of the compounds
exhibited inhibitory activity towards anandamide hydrolysis, thus arguing in favor of their enzymatic
stability. The structure–activity relationship has been extensively studied through a tailor-made homo-
logical model using constrained docking in addition to pharmacophore analysis, both feature and field
based.
Received 30 August 2010
Revised 16 October 2010
Accepted 19 October 2010
Available online 27 October 2010
Keywords:
Endocannabinoids
CB2 selectivity
Anandamide derivatives
Homology modeling
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
compounds are ultimately derivatives of arachidonic acid, and
are biosynthesised ‘on demand’ from membrane phospholipids.
Marijuana or hashish, derived from the Indian hemp Cannabis
sativa have been used for medical purposes in Chinese and Egyp-
tian sources as early as 2700 BC. Apart from its medical use,
marijuana gained great popularity as a recreational drug due to
The finding of endocannabinoids led to the discovery of three addi-
tional proteins, fatty acid amide hydrolase (FAAH), monoacylglyc-
erol lipase (MAGL) and the putative anandamide transporter (AT),
which are involved in their metabolism. Endocannabinoids signal-
ing is terminated by a two-step process: cellular uptake, facilitated
by AT, followed by enzymatic degradation by FAAH, or MAGL in
case of 2-AG.9–11 Hence, the CB1 and CB2 cannabinoid receptors,
their endogenous ligands (endocannabinoids), and enzymes, pro-
teins, and transporters involved in endocannabinoid formation
and inactivation, collectively comprise the endocannabinoid sys-
tem. The components of this system represent excellent targets
for development of therapeutically useful drugs for a range of con-
ditions including pain, inflammation, immunosuppression, loss of
appetite and many others.12
The focus of this work was to design, synthesize and pharmaco-
logically evaluate new anandamide (AEA) derivatives, obtained by
modifications of the fatty acyl chain and/or of the ethanolamide
‘tail’. The synthesis of novel compounds with the general formula:
6-(substituted-phenyl)-4-oxohex-5-enoicacid N-substituted amide
and 7-naphthyl-5-oxohept-6-enoicacid N-substituted amide is
reported in the present work (Fig. 1). All newly synthesized com-
pounds were evaluated for their binding affinity to CB1/CB2
cannabinoid receptors. The SAR was studied extensively using
the presence of the psychotropic component ‘(À)-trans-
hydrocannabinol’ (THC), which has the ability to cause euphoria
and elation.1 It was the identification 9-THC as the major psycho-
D
9-tetra-
D
active ingredient in cannabis, as well as its chemical synthesis that
led to the discovery of the cannabinoid receptors: CB1 and CB2.2
The former is highly expressed in brain and was cloned in 1990,
while the latter is expressed predominantly in the periphery and
appears to be mainly associated with the immune cells and
spleen.3–5 The existence of such specific receptors that recognize
phytocannabinoids stimulated the search for endogenous cannab-
inoid receptor ligands, the ‘endocannabinoids’. By 1992, the first
endocannabinoid, arachidonoyl ethanolamide (AEA or ananda-
mide, Fig. 1), was isolated from porcine brain, and found to mimic
many of the actions of THC.6 Three years later, a second endocan-
nabinoid, 2-arachidonoyl glycerol (2-AG), was discovered.7,8 Both
⇑
Corresponding author. Tel.: +20 2 27590716; fax: +20 2 27581041.
E-mail addresses: ashraf.abadi@guc.edu.eg (A.H. Abadi), ahabadi@yahoo.com
(A.H. Abadi).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.050

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N. A. Osman et al. / Bioorg. Med. Chem. 18 (2010) 8463–8477
O
O
O
R
H
N
R'
N
H
O
R
amide tail
aryl head
linker
H
N
HO
O
Figure 1. Chemical structure of anandamide and the general skeletons of the synthesized compounds.
tailor-made homological model, constrain docking based on site-
directed mutagenesis data and pharmacophore analysis (both field
and feature based).
secondary amides showed IR bands at about 3280 cmÀ1 of the –NH
stretching, and in addition to this, –C@O stretching of the amide
derivativÀe1s showed bands at relatively lower values (about
1650 cm
) than the typical carbonyl stretching at about
1700 cmÀ1. Compounds XIV and XX showed an additional –C@O
2. Results and discussion
2.1. Chemistry
stretchinÀg1 band of the ester at relatively higher values (about
1738 cm
)
.
than the typical carbonyl stretching at about
1700 cmÀ1
The synthesis of (E)-6/7-(substituted-phenyl/naphthyl)-4/5-oxo-
hex-5/hept-6-enoic acids (I–V) (illustrated in Schemes 1 and 2) was
adopted according to the general procedure previously reported in
the literature. This involved condensation of the appropriate alde-
hyde with levulinic acid or 4-acetyl butyric acid using catalytic
amounts of piperidine and acetic acid to give the respective arylidine
keto acid derivatives. Three of the intermediates are reported com-
pounds (I, II and V) while two of them are novel ones (III and IV).13
When 4-acetyl butyric acid was reacted with 2-naphthaldehyde (to
give compound IV) instead of levulinic acid (to give compound III),
a multiplet appeared upfield at d ꢀ 2.05–2.10 ppm characteristic of
the extra –CH2 group added. Moreover, separation of the E-isomer
is evident by the high J value of the olefinic protons, calculated to
be P15 Hz, which is typical for E-isomers rather than the Z ones.
Infrared (IR) spectroscopy showed characteristic broad bands at the
range of about 3300–2400 cmÀ1 for the –OH stretching of the carbox-
ylic acid, bands at about 1690 cmÀ1 for the carboxylic –C@O stretch-
ing and bands at about 1675 cmÀ1 for the ketonic –C@O stretching.
The ketonic –C@O stretching shows relatively lower values than the
2.2. Biology and molecular modeling
All novel compounds were evaluated for their in vitro ability to
bind to human recombinant CB1 and CB2 receptors and their IC50
and Ki values calculated. Results are shown in Table 1. The binding
assay results showed that 5 out of 20 newly synthesized com-
pounds (XI, XIV, XVII, XIX and XXIII) were able to bind selectively
to CB2 receptor with measurable IC50 and Ki values in the low
micromolar range and with selectivity index from 4 to 10.
To explain the SAR profile of these ligands, we developed an
homology model to find the bioactive conformer of a selective
CB2 agonist (GW405833)14 (Fig. 2) and the pharmacophoric fea-
tures of this ligand, and the relation between them in the space.
This homology model was used for three purposes: (1) Alignment
of the synthesized ligands to the bioactive conformer of the selec-
tive CB2 ligand (GW405833) using Field alignment method15 while
applying constraints on the essential field points required for activ-
ity; (2) docking with pharmacophoric restraint based on the idea
that good activity should be linked with good pharmacophore con-
straints together with good interactions in the binding site and ab-
sence of steric clash or high torsional strain; (3) MIF (Molecular
interaction field) analysis of the binding site.
carboxylic acid –C@O due to the presence of an
to the ketonic –C@O.
a, b double bond next
The general synthesis of 6-(substituted-phenyl)-4-oxohex-
5-enoic acid N-substituted amide and 6/7-naphthyl-4/5-oxohex-5-/
hept-6-enoic acid N-substituted amide is described in Schemes 3
and 4, respectively. Synthesis of compounds with biphenyl aryl
heads is illustrated in Schemes 5 and 6. Compounds containing
To develop the CB2 homology model, the strategy of De Graff
and Rognan16 was adopted with some modifications. In short, the
GPCR modeling workflow used in this study followed seven steps:
O
O
O
OH
piperidine/acetic acid
toluene/reflux 6 hrs
H
+
OH
O
O
I, II & V
R
R
Compound
R
I
3, 4-dimethoxy
4-methoxy
4-Ph
II
V
Scheme 1.

N. A. Osman et al. / Bioorg. Med. Chem. 18 (2010) 8463–8477
8465
O
O
O
piperidine/acetic acid
toluene/reflux 6 hrs
OH
H
OH
n
+
n
O
O
III & IV
Compound
n
2
3
III
IV
Scheme 2.
O
O
H
N
OH
O
1. ClCOOEt
R'
O
2. R'NH2
R
I, II & V
VII-IX & XXII
R
Compound
R
R'
VII
VIII
IX
3,4—dimethoxy
3,4—dimethoxy
4-methoxy
4-Ph
-piperidin-1-yl
-CH2-CH2-OH
-4-hydroxyphenyl
-CH2-CH2-OH
XXII
Scheme 3.
O
O
O
O
R'
N
H
1. ClCOOEt
2. R'NH2
OH
n
n
III & IV
X-XXI
Compound
X
n
2
2
2
2
2
2
2
3
3
3
3
3
R’
-piperidin-1-yl
-CH2-CH2-OH
XI
XII
-4-hydroxyphenyl
-cyclopropyl
XIII
XIV
XV
-CH2-COOC2H5
-(CH2-CH2-OH)2
-CH(CH3)-CH2-OH
-piperidin-1-yl
XVI
XVII
XVIII
XIX
XX
-CH2-CH2-OH
-cyclopropyl
-CH2-COOC2H5
-CH(CH3)-CH2-OH
XXI
Scheme 4.

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N. A. Osman et al. / Bioorg. Med. Chem. 18 (2010) 8463–8477
O
Pd/C in MeOH
OH
2 hrs
O
V
O
OH
O
VI
ClCOOC2H5
OH
O
OH
N
H
H2N
O
XXII
Scheme 5.
O
O
O
O
OH
+
O
toluene/
Fenbufen
reflux 6 hrs
O
O
O
O
N
O
i) toluene/
room temperature
ii) morpholine
XXIV
Scheme 6.
(1) Construction of the initial receptor model, consisting of the
seven trans membrane (TM) helices, after identification of the
TM helices (template-independent de novo modeling), amino acid
sequence alignment between target and template receptors and by
rotation/de novo modeling of certain TM helices with putative
alternative helical kinks; (2) construction of a preliminary TM-
ligand complex; (3) energy minimization of the TM-ligand complex;
(4) molecular dynamics simulation refinement of the receptor–
ligand complex; (5) modeling the loops connecting the TM helices;
(6) selection and refinement of the full receptor–ligand complex; (7)
validation of the full receptor–ligand complex was done by docking
of the newly synthesized ligands. Previous studies developed CB2
homology model using Bovine rhodopsin crystal structure,17,18 as
well as the CB2 homological modeling in the inactive state using
b2-adrenergic receptor as a template.19 In this study, we built up
a model to represent the active state using b2-adrenergic receptor
as a template. It is known that CB2 receptors share more structural
features with b2-adrenergic receptor rather than rhodopsin, for in-
stance both CB2 and b2-adrenergic receptors may have small non
covalently bound ligands, and both receptors share the Cys-X-X-X-
Ar motif that is conserved in extracellular loop 2 (EC2) among GPCRs
that bind biogenic amines and peptides20,21 (Fig. 4). The T4-lyso-
zyme inserted into the b2 receptor between Gln231 and Ser262 to
assist crystallization was removed. As the T4-lysozyme replaced
ICL3, which is distant from the binding site, ICL3 was not modeled
in the receptors (Supplementary data, Figs. A-1 and A-2). The CB2
and b2 sequences were aligned based on existing information on
conserved residues within class-A G protein-coupled receptors,

Table 1
Results of radioligand binding assays of the synthesised compounds
Compd Structure
Max tested on CB1 (%
IC50 on CB1
M)
Ki on CB1
M)
Max tested on CB2
IC50 on CB2
M)
Ki on CB2
M)
Selectivity index
(Ki CB1/Ki CB2)
displacement) (lM)
(
l
(
l
(% displacement) (
l
M)
(
l
(
l
O
OH
III
10 (11.84%)
>10
>10
>10
>10
10 (13.72%)
>10
>10
>10
>10
—
—
O
O
O
O
O
IV
10 (21.49%)
10 (<50%)
10 (23.40%)
10 (<50%)
OH
O
N
VII
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
—
—
—
O
O
O
O
H
N
OH
VIII
IX
10 (<50%)
10 (<50%)
10 (<50%)
10 (<50%)
O
O
H
N
O
OH
O
O
O
O
N
X
10 (<50%)
10 (0.00%)
10 (<50%)
>10
>10
>10
>10
>10
>10
10 (<50%)
10 (56.52%)
10 (<50%)
>10
1.00
>10
>10
1.03
>10
—
O
NH
OH
XI
XII
>4.41
O
H
N
—
O
OH
O
O
H
N
XIII
XIV
10 (3.25%)
10 (5.73%)
>10
>10
>10
>10
10 (35.64%)
10 (54.63%)
>10
>10
—
O
O
NH
9.82
2.50
>4.00
O
O
(continued on next page)

Table 1 (continued)
Compd Structure
Max tested on CB1 (%
IC50 on CB1
M)
Ki on CB1
M)
Max tested on CB2
IC50 on CB2
M)
Ki on CB2
M)
Selectivity index
(Ki CB1/Ki CB2)
displacement) (lM)
(
l
(
l
(% displacement) (
l
M)
(
l
(
l
OH
O
O
O
O
XV
10 (1.81%)
>10
>10
10 (27.32%)
>10
>10
—
N
OH
OH
O
O
H
N
XVI
XVII
XVIII
XIX
XX
10 (+2.48%)
10 (34.00%)
10 (4.58%)
10 (15.28%)
10 (41.55%)
10 (0.00%)
10 (5.25%)
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
10 (38.28%)
10 (36.83%)
10 (72.02%)
10 (56.70%)
10 (41.70%)
10 (37.70%)
10 (46.11%)
>10
>10
4.08
8.02
>10
>10
>10
>10
>10
1.03
2.04
>10
>10
>10
—
O
—
N
O
OH
>9.71
>4.90
—
N
H
O
O
N
H
O
O
O
O
O
N
H
O
OH
XXI
XXII
—
N
H
O
O
H
N
—
OH
O
O
H
N
XXIII
XXIV
10 (10.89%)
10 (12.22%)
>10
>10
>10
>10
10 (52.95%)
10 (14.87%)
8.85
>10
2.25
>10
>4.44
—
OH
O
O
N
O

N. A. Osman et al. / Bioorg. Med. Chem. 18 (2010) 8463–8477
8469
During the modeled activation of b2-adrenergic receptor,
Pro288 permits the movement of the intracellular end of TM6
away from TM3 and upwards towards the lipid bilayer, suggesting
that the crucial movements for activation involve flexibility about
the hinge formed by the highly conserved proline in TM6.28
According to Shi et al. there is a rotamer toggle switch which is
able to modulate the TM6 proline kink in the b2-adrenergic recep-
tor and according to this hypothesis Cys285 trans/Trp286 gauche
+/Phe290 gauche + represents the inactive form of the b2-adrener-
gic receptor while Cys285 gauche +/Trp286 trans/Phe290 trans
represents the active state.29 Concerning the CB receptor model
activation, Singh and coworkers study showed that Trp258 and
Phe117 interaction may act as toggle switch for CB2 activation
with Trp258 gauche +/Phe117 trans representing the inactive form
while Trp258 trans/Phe117 gauche + the CB2 active form.(Supple-
mentary data, Fig. A-6).30 Following these results, CB2 modeled
activation was carried out by rotating TM3 and TM6 in counter-
clockwise direction (extracellular point of view) and TM6 was
straightened using Pro260 as a flexible hinge. The suggested switch
Figure 2. GW405833 a selective CB2 agonist.
Table 2.22 The alignment shows consensus with that of CB1 to the b2
adrenergic receptor proposed by Shim et al. For construction of the
helical bundle of the CB2 receptor, the helical boundaries, not TM
boundaries, of the X-ray structure of b2AR were used to extract
the secondary structural information as much as possible. The heli-
cal boundaries were assigned by STRIDE (Supplementary data,
Figs. A-3 and A-4).23 The template structure used in our study was
crystallized in its inactive state20,21,24,25, hence, CB2 receptor was
activated according to the methods mentioned in the literature, to
be able to dock the CB2 selective agonist GW405833 into it. The
modeled activated structure of the GPCR receptor is determined
by the different arrangement of TM3 and TM6. This hypothesis is
based on the fact that the disruption of the interaction between
these two helices produces constitutive modeled receptor activa-
tion. According to Ballesteros et al., the extent of the constitutive
activation is closely correlated with the extent of conformational
rearrangement in TM6.26 Conformational switches in the TM
helices can be generated as a result of the formation of the flexible
molecular hinges by the residue Pro260 in CB2 in the highly
conserved CWXP motif in TM6 (CWXP is CWFP in CB2 represented
by Cys257, Trp258, Phe259 and Pro 260), Supplementary data,
Figure A-5.27
was finally toggled by adjusting the
v1 rotamer of Trp258 and
Phe117 trans the former and gauche + the latter. This complies
with Tuccinardi et al. regarding the CB1 activation.17 The model
of the TM helices was refined using minimization techniques
(steepest descent and conjugate gradient) followed by 100 picosec-
ond of molecular dynamics. No implicit solvent model was applied
here; just a harmonic restraint was set on the backbone. The back-
bone conformation was evaluated using Psi/Phi Ramachandran
plot. In addition Model verification was carried out using Modeler
DOPE score and Verify 3D (Supplementary data, Fig. A-7).31–33
CB2 mutagenesis studies suggested the importance of Ser11234
and Phe19735 in this subtype. We choose the CB2 selective agonist
GW405833 as a reference compound as it shares an aryl moiety at
one end and an amine near the other end as in our compounds. We
docked this ligand manually such that it complies with the above
Table 2
The secondary structure of b2 adrenergic receptor, CB1 receptor and CB2 receptora
a
The secondary structure of was assigned by Stride.23 (2rh1) represents sequence of b2 adrenergic receptor, (P21554) represents the sequence of CB1 receptor and (P34972)
represents the sequence of CB2 receptor. Conservancy of the aligned sequence by CLUSTALW (integrated in Discovery Studio)23,42 is represented by (dark blue)for identical
residues; (blue) for conserved substitutions and (light blue) for semi conserved substitutions. This alignment shows agreement with that done by shim regarding CB1.43

8470
N. A. Osman et al. / Bioorg. Med. Chem. 18 (2010) 8463–8477
sented by the generalized Born solvent accessible surface area im-
plicit membrane model of Spassov et al.36 In this model, the
aqueous solvent comprises a high dielectric region and the lipid
membrane and the interior of the membrane bound protein taken
together comprise a low dielectric region. The initial orientation of
the GPCR agonist model with respect to the bilayer was refined via
a rigid body procedure that systematically rotated and translated
the complex to determine the optimal position of the complex rel-
ative to the implicit membrane. The membrane is presented as a
planar low-dielectric slab and the molecule is treated as a rigid
structure. The optimal orientation corresponds to the minimum
of the solvation energy was calculated as in generalized Born sol-
vent accessible surface area approximation. Minimization of the
complex was carried out using GBIM. Molecular dynamics was car-
ried out as a cascade of heating, equilibration and production to re-
fine the complex and test the ligand stability in the proposed
binding site. It is obvious that the essential interactions for the
selective b2-agonist are as follows: hydrogen bond with Ser112,
aromatic stacking of N1-2,3-dichlorobenzoyl with Phe197 and
Figure 3. The CB2 selective agonist was manually docked such that its hydrophobic
moiety (N1-2,3dichlorobenzoyl) interacts with Phe197 and Trp94 while the
morpholine group is positioned in
a secondary lipophilic pocket. The main
interactions proposed here are: the hydrogen bond with Ser112, the aromatic
stacking of N1-2,3-dichlorobenzoyl with Phe197 and the interaction of N1-2,3-
p–p
dichlorobenzoyl with Trp194.
p–p
interaction of N1-2,3-dichlorobenzoyl with Trp194, Figure 3.
Field Align15 was used to align the synthesized ligands to the bio-
active conformer of the CB2 agonist GW405833. The idea behind
this type of alignment is that two molecules which both bind to
a common active site tend to make similar interactions with the
protein and hence have highly similar field properties. Field align-
ment was used; taking into consideration the excluded volume
provided by the binding site amino acid residues, to align the li-
gands to the bioactive conformer of the CB2 selective agonist,
Figure 5. The main constraints which have been used were the field
points representing the hydrophobic moiety and that representing
the hydrogen bond acceptor.37 The compounds which are top
ranked in similarity index turned to have CB2 selectivity (see Table
3). Investigating the alignment of these compounds show high de-
gree of compliance with the essential features which were con-
strained (hydrogen bond acceptor and hydrophobic aromatic
feature. Investigating the compounds which have no CB2 activity
reveals a high score of excluded volume clash penalty (compounds
X, XVII, XXI, XX and XV) or in general they show no perfect
mapping with the constrained field points (see Supplementary
data, Fig. C1-20). In general the low similarity index scores are
due to the design criteria which stress on two features only in
the CB2 selective agonist (GW405833) and neglect any other
feature.
mentioned essential requirements, this was carried out by insert-
ing the morpholinic group between TM3 and TM5, while the
N1-2,3-dichlorobenzoyl substituent was directed towards the cen-
tral core of TM5 and TM6. This proposal complies with Tuccincardi
et al.17 In this manner the lipophilic core of the ligand was able to
interact with Phe197 and the morpholinic core with Ser112,
Figure 3. The complex was minimized using Steepest descent
method and conjugate gradient with harmonic restraint on the
backbone of the helix. Molecular dynamics (MD) simulation of
the receptor–ligand complex was carried out to refine the model
and test the stability of the proposed interactions of the ligand
and the receptor during the production phase. Modeling the loops
connecting the TM helices loops were added using Modeler,33
where the modified helices were taken as reference template and
b2 adrenergic receptor as the main template. Reference template
co-ordinates were used as it is. EC2 was modeled according to
Ahn et al.,18 where the
a-helix in the EC2 of b2-adrenergic receptor
was introduced into CB2 maintaining the topological orientation
observed for b-2 adrenergic receptor in the X-ray structure, Figure
4 and Supplementary data, Figure A-3. The minimization and MD
protocol was carried out to refine the full receptor-complex using
an implicit solvent model this time. The lipid bilayer was repre-
Figure 4. Sequence comparison of the EC2 loop. The residues which are involved in the disulfide bond of the EC2 loop are in red box. Shown here the sharing of the Cys-X-X-
X-Ar motif among 2rh1 (b2-adrenergic receptor), P21554 (Cb1 receptor) and P34972 (Cb2 receptor).The motif in CB2 is represented by Cys179, Ser180, Glu181, Leu182 and
Phe183. a-Helix in the EC2 of b2-adrenergic receptor is shown below with the disulfide bridge illustrated as yellow bridge.

N. A. Osman et al. / Bioorg. Med. Chem. 18 (2010) 8463–8477
8471
tary data, Figure B-1. It reveals two binding pockets, large lipo-
philic pocket which encompass Phe197 and Trp194 and a small
lipophilic pocket which encompass Ser112. This could explain
the inactivity of ligands III and IV as they have a highly polarizable
carboxylic group acting as hydrogen bond acceptor which is sup-
posed to bind to Ser112 located in the small lipophilic pocket. Fur-
thermore, this can explain the inactivity of ligands which have
slightly polar groups attached to the hydrophobic moiety, for
example, methoxy and dimethoxy derivatives of benzene if com-
pared to naphthalene which is supposed to bind in the large lipo-
philic pocket.
SAR investigation and model support through docking the new-
ly synthesized ligands was done using Gold38 software, as it takes
into consideration the features constraints as interaction filters.
Compounds which have naphthalene moiety showed good activity
due to the ideal interaction with Trp194 through
p–p interaction
together with the stacking interaction with Phe197, Figure 7.
Compounds XI, XIV, XVIII and XIX are good examples for this
case. Only one compound with biaryl ring system showed activity
(XXIII). The activity of this compound was low with respect to the
naphthyl based ligands. Constrained docking gives some rational
for this decrease in activity: The Torsion in case of biaryl system
doesn’t give the ideal orientation in space necessary for the inter-
action. The rotation about the single bond can form steric clash
Figure 5. Ligand XI aligned to the CB2 selective agonist. Two constraints were
applied. The first is the hydrophobic field point in the center of the N1-2,3-
dichlorobenzoyl (yellow sphere) and the second is the negative field point which
represents H-bond donor on the protein (blue sphere). The field points interpre-
tation is: Blue: Negative field points; Red: Positive field points; Yellow: van der
Waals surface field points; Gold/Orange: Hydrophobic field points (describe regions
with high polarisability/hydrophobicity).
with Trp194 which can hinder the
p–p interaction, Figure 8. The
Table 3
interaction can occur but with some torsional strain which give
reason for the lowered activity. Pharmcophoric constraints showed
some important aspects regarding the length of the side chain. It
seems that there is an ideal distance between the centroid of the
Field align similarity score of the newly synthesized ligands to that of the selective
CB2 agonist (GW405833)a
Compd
Similarity
Compd
Similarity
XI
0.514
0.512
0.473
0.457
0.454
0.449
0.443
0.441
0.439
0.433
XVI
XV
XII
XXII
XIII
XX
VII
III
0.427
0.419
0.414
0.414
0.411
0.401
0.364
0.362
0.355
0.322
XVIII
XIV
XXIII
XIX
XXIV
VIII
IX
IV
XXI
XVII
X
a
The top ranked ligands turned to have CB2 selectivity. In general, scores are low
and we focus on two major essential features in GW405833 and neglect others.
Figure 7. Naphthalene moiety is supposed to interact with Trp194 through
p–p
interaction. It seems that the hydrophobic interaction is strong in case of
naphthalene being bicyclic like indole ring of tryptophan. Naphthalene interacts
as well through stacking interaction with Phe197.
Figure 6. Grid analysis show comparative view of the large lipophilic pocket (right)
to that of the small lipophilic pocket (left). Grid analysis was carried out using
C1 = probe.
Figure 8. The biaryl system can form steric clash (shown in the red box) if it has to
comply with the essential interaction with Trp194. This was shown through the
constrained docking carried out by Gold. Otherwise some torsional strain is
The MIF analysis was done to aid in explaining the SAR based on
the nature of binding pocket. Binding site nature was investigated
by Grid39 software using different probes, Figure 6 and Supplemen-
required to form the p–p interaction.

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N. A. Osman et al. / Bioorg. Med. Chem. 18 (2010) 8463–8477
Table 4
Distance between the centroid of the hydrophobic aromatic feature and the hydrogen
bond acceptor of the active compoundsa
Drug
Distance
GW405833
XI
XVIII
XIX
9.808
10.336
11.264
9.903
XXIII
XIV
10.648
10.294
a
Calculated from the manually docked CB2 selective ligand (GW 405833) and
the automatically docked active ligands. The H-bond acceptor in XI, XVIII and XXIII
is hydroxyl oxygen while in XIX and XIV is carbonyl oxygen.
Figure 10. Compound XIII shows obvious non compliance with the desired
distance range (the distance between the hydrophobic aromatic feature centroid
and the hydrogen bond acceptor feature).
glycinate is the hydrogen bond acceptor and remaining part is
small and can be easily accommodated. On the other hand XX
forms hydrogen bond through the carbonyl group marked, Figure
13, leaving the whole ethyl glycinate in the lipophilic pocket
which forms steric clash. Pharmacophore analysis using features
instead of field points, showed results which are in consensus
with the docking results, Figure 14. The compounds which fail
to map completely were those lacking the essential distance be-
tween hydrophobic aromatic feature and hydrogen bond acceptor
feature (III, X, XIII and XXIV) in addition to those which form ste-
ric clash in the small lipophilic pocket (VII, XV and XVII). Both
XXII and XXIII showed very low fitting score (0.15, 0.52, respec-
tively) this emphasis that the flexibility of XXIII gives it higher fit
value. All compounds were also evaluated for their capability of
inhibiting the enzymatic hydrolysis of radiolabelled anandamide
by rat brain membranes, as the finding of such activity would
represent indirect evidence for an amide to act as a potential sub-
strate for FAAH. However, none of the amides exhibited any
Figure 9. Compound X shows the absence of hydrogen bond interaction with
serine 112 in the constrained docking run. The distance between the hydrophobic
feature centroid and the hydrogen bond acceptor feature is 8.878 which is about 1 A
less than desired range. Steric clash is shown (pink line).
in orange.
p–p interaction is shown
hyrdophobic aromatic feature and the hydrogen bond donor fea-
ture (Table 4). This distance ensures that the binding is ideal. Short
distances will lead to failure in satisfaction of one of the essential
interactions. Some compounds failed to attain this distance (X, XIII
and XXIV), Figures 9–11.
inhibitory activity up to a 50 lM concentration, thus ruling out
the possibility of them being substrates for the enzyme and sug-
gesting that they might be enzymatically stable at least as far as
their metabolism by FAAH is concerned.
It is proposed that hydrogen bond acceptor binds to Ser112
which is located in the small hydrophobic pocket. This hypothesis
can be proved by testing the tolerance for different substituents
which binds to this region. It is supposed that large substituents
can make steric clash with the small lipophilic pocket residues.
Docking studies and biological evaluation were used in this anal-
ysis. It is obvious from the docking study that bulky groups like
phenyl (IX), piperidinyl (X, XVII) and diethanolamine (XV) are
not tolerated. Constrained docking here is useful as it try to force
the essential interactions, so any violations during this enforce-
ment can be easily assessed by visual inspection, Figure 12, it is
obvious that small head like cyclopropyl group is tolerated
(XIX). It is important to mention though that (XIII) is not active
though it has cyclopropyl group. This is mainly due to non com-
pliance to the desired distance range mentioned before. Steric
clash and the intolerance of the small lipophilic pocket explains
the difference in activity between XIV and XX, see Figure 13.
Docking showed that the bulky polar head has different roles in
both compounds. In case of XIV the carbonyl group of the ethyl
3. Conclusion
Our compounds represent a new class of CB2 selective ligands
with the general formula: 6-(substituted-phenyl)-4-oxohex-
5-enoic acid N-substituted amide and 6/7-naphthyl-4/5-oxohex-5-/
hept-6-enoic acid N-substituted amide, to be reported for the first
time. The molecular modeling study presented in this work pro-
vides a first study of CB2 in active form based on the structure of
b2-adrenergic receptor. Furthermore, the model was designed
complete with loops and helices and simulated using MD with im-
plicit solvent membrane as a substitute to the resource intensive
explicit models which use real phospholipid membranes. These
combined approaches don’t exist in one model for CB2 previously
made in the literature. The constrained docking proved to be a
valuable method to explain the SAR of the synthesized ligands.
However for the purpose of screening, this model can be further
validated using a library of actives and decoys to find the enrich-
ment capability of the model. Based on the current study, we are

N. A. Osman et al. / Bioorg. Med. Chem. 18 (2010) 8463–8477
8473
Figure 11. Compound XXIV shows noncompliance with the desired distance range (distance between hydrophobic aromatic feature and hydrogen bond acceptor feature).
Figure 12. Diethanolamine in (XV) (A) and piperdinyl group in (XVII) (B) are not tolerated in the binding site. Constrained docking shows obvious steric clash with the small
lipophilic binding site (shown here is Ser112 only) in order to achieve the essential H-bonding interaction with Ser112.
Figure 13. The compound XX inactivity is proposed to be due to the bulkiness of the head (shown in red box), although there is compliance with the distance range. The
compliance of compound XIV with the desired distance range required between hydrophobic pocket and hydrogen bond acceptor together with the presence of small head
which is tolerated in the small lipophilic pocket is the reason why XIV has activity
planning to further direct our research towards the design and
synthesis of even more potent and selective compounds using
the tailor-made homological model.
using silica gel 70–230 mesh. Reaction progress was monitored
by TLC performed on pre-coated silica gel plates (ALUGRAM SIL
G/UV254) and detection of the components was made by UV light
(254 nm).
4. Experimental
4.1. Chemistry
4.2. General procedure for the synthesis of (E)-6/7-(substituted-
phenyl/naphthyl)-4/5-oxohex-5/hept-6-enoic acids (I–V)
Both the respective aldehyde (30 mmol) and levulinic acid
(30 mmol) were dissolved in toluene (100 ml) containing acetic
acid (3 ml) and piperidine (1 ml). The solution was heated under
reflux using Dean-Stark water trap under nitrogen until the theo-
retical amount of water had been collected (ꢁ6 h) and TLC analysis
(CHCl3/CH3OH, 93:7) indicated disappearance of the starting mate-
rial. The solvent was evaporated in vacuo and after cooling the so-
lid product was washed twice with 10 ml of diethyl ether and then
twice with 15 ml of 2 M HCl, dried and recrystallized from the
benzene.13
All starting materials were obtained from Sigma–Aldrich and
were used without further purification and all organic solvents
used were obtained from Al-Goumhoria Company and were of gen-
eral purpose grade. Melting points were determined on Buechi
B-540 Melting Point apparatus and are uncorrected. FTIR spectra
were recorded on Nicolet Avatar 380 spectrometer. 1H NMR spec-
tra were recorded on Varian Mercury VX-300 MHz spectrometer
using CDCl3 or DMSO-d6 as a solvent; chemical shifts (d) were re-
ported in parts per million (ppm) downfield from TMS; multiplic-
ities are abbreviated as: s: singlet; d: doublet; q: quartet; m:
multiplet; dd: doublet of doublet; br: broad. Mass spectra were
made on Hewlett–Packard GC–MS, model 5890, series II at an ion-
ization potential of 70 eV. Elemental analyses were performed by
the Microanalytical Unit, Faculty of Science, Cairo University;
found values were within 0.5% of the theoretical ones, unless
otherwise indicated. Column chromatography was performed
4.2.1. (E)-6-(3,4-Dimethoxyphenyl)-4-oxohex-5-enoic acid (I)13
Yellow crystals; yield: 62%; mp: 88–90 °C; 1H NMR (CDCl3): 1H
NMR (CDCl3): 2.73 (t,, J = 6.6 Hz, 2H), 3.05 (t, J = 6.6 Hz, 2H), 3.92 (s,
6H), 6.68 (d, J = 15 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 7.12 (t, J = 9 Hz,
2H), 7.58 (d, J = 15 Hz, 1H); IR (cmÀ1): 3150–2450 (–OH carboxylic

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N. A. Osman et al. / Bioorg. Med. Chem. 18 (2010) 8463–8477
1684 (–C@O); MS (EI): m/z 280 (M+), 77 (100%). Anal. Calcd for
18H16O3 (m. wt. = 280.3): C, 77.12; H, 5.75. Found: C, 77.30; H,
C
5.79.
4.2.6. (E)-6-Biphenyl-4-yl-4-oxohexanoic acid (VI)
To a suspension of the (E)-6-biphenyl-4-yl-4-oxohex-5-enoic
acid (V) (10.7 mmol) in methanol (50 ml), 300 mg of 10% Pd/C
was added, the resultant mixture was hydrogenated at atmo-
spheric pressure at room temperature for 2 h. The catalyst was fil-
tered off, and the solvent was distilled in vacuum to obtain a white
solid which was recrystallized from CHCl3/hexane.13
4.2.7. (E)-6-Biphenyl-4-yl-4-oxohexanoic acid (VI)13
Fluffy white solid; yield: 70%; mp: 135–136 °C; 1H NMR
(DMSO-d6): 2.40 (t, J = 7.5 Hz, 2H), 2.67 (t, J = 7.5 Hz, 2H), 2.80 (s,
4H), 7.26–7.60 (m, 9H), 12.0 (br s, 1H); IR (cmÀ1): 3300–2400
(–OH carboxylic acid), 1718 (–C@O), 1704 (–C@O); MS (EI): m/z
282 (M+), 167 (100%). Anal. Calcd for C18H18O3 (m. wt. = 282.3):
C, 76.57; H, 6.43. Found: C, 76.58; H, 6.41.
4.3. General procedure for the preparation of 6-(substituted-
phenyl)-4-oxohex-5-enoic acid N-substituted amide (VII, VIII,
IX, XXII, and XXIII)
Figure 14. Pharmacophore manually created based on the bioactive conformer of
GW405833, stressing on the hydrogen bond donor acceptor feature (green arrow),
hydrophobe aromatic and hydrophobic point features. Excluded volume features
were based on the amino acid residues of the binding site.
A mixture of the appropriate acid (0.01 mol) and triethylamine
(0.072 mol) in methylene chloride was cooled in an ice and salt
bath to À10 °C. Ethyl chloroformate (0.05 mol) was added drop-
wise (while stirring) over a period of 10 min and stirring was con-
tinued for 30 min. The amine was added gradually within 10 min
and stirring was continued overnight at room temperature. The
solvent was then distilled under reduced pressure and the residue
was extracted with ethyl acetate. The organic layer was then
washed with saturated solutions of NaHCO3, NH4Cl, and H2O (each
three times with 50 ml, respectively) and dried over anhydrous
NaSO4. The solvent was evaporated under vacuum and the residue
then washed finally with a mixture of diethyl ether/hexane or puri-
fied by column chromatography.40
acid), 1721 (–C@O), 1674 (–C@O); MS (EI): m/z 264 (M+), 64
(100%). Anal. Calcd for C14H16O5 (m. wt. = 264.3): C, 63.63; H,
6.10. Found: C, 63.12; H, 5.80.
4.2.2. (E)-6-(4-Methoxyphenyl)-4-oxohex-5-enoic acid (II)13
Yellow crystals; yield: 60%, mp: 125–127 °C; 1H NMR (CDCl3):
2.75 (t, J = 6.6 Hz, 2H), 3.01 (t, J = 6.6 Hz, 2H), 3.89 (s, 3H), 6.66
(d, J = 15 Hz, 1H), 6.93 (d, J = 6.0 Hz, 2H), 7.49–7.53 (m, 2H), 7.58
(d, J = 15 Hz, 1H); IR (cmÀ1): 3200–2500 (–OH carboxylic acid),
1718 (–C@O), 1672 (–C@O); MS (EI): m/z 235 (M++1), 64 (100%).
Anal. Calcd for C13H14O4 (m. wt. = 234.2): C, 66.66; H, 6.02. Found:
C, 66.21; H, 5.99.
4.4. General procedure for the preparation of 6/7-naphthyl-4/5-
oxohex-5-/hept-6-enoic acid N-substituted amide (X–XXI)
A mixture of the appropriate acid (0.01 mol) and triethylamine
(0.072 mol) in dry chloroform was cooled in an ice and salt bath to
À10 °C. Tertiary butyl chloroformate (0.05 mol) was added drop-
wise (while stirring) over a period of 10 min and stirring was con-
tinued for 30 min. The amine was added gradually within 10 min
and stirring was continued overnight at room temperature. The
solvent was then distilled under reduced pressure and the residue
was extracted with ethyl acetate. The organic layer was then
washed with saturated solutions of NaHCO3, NH4Cl, and H2O (each
three times with 50 ml, respectively) and dried over anhydrous
NaSO4. The solvent was evaporated under vacuum and the residue
then washed finally with a mixture of diethyl ether/n-hexane or
purified by column chromatography.40
4.2.3. (E)-6-Naphthalen-2-yl-4-oxohex-5-enoic acid (III)
Yellow crystals; yield: 67%; mp: 169–171 °C; 1H NMR (CDCl3):
2.79 (t, J = 6.6 Hz, 2H), 3.09 (t, J = 6.6 Hz, 2H), 6.90 (d, J = 15 Hz,
1H), 7.52–7.55 (m, 2H), 7.68–7.88 (m, 5H), 7.98 (s, 1H); IR
(cmÀ1): 3200–2400 (–OH carboxylic acid), 1688 (–C@O), 1679
(–C@O); MS (EI): m/z 254 (M+), 181 (100%). Anal. Calcd for
C16H14O3 (m. wt. = 254.3): C, 75.57; H, 5.55. Found: C, 76.01; H,
5.51.
4.2.4. (E)-7-Naphthalen-2-yl-5-oxohept-6-enoic acid (IV)
Buff crystals; yield: 80%; mp: 172–174 °C; 1H NMR (CDCl3): 2.05–
2.10 (m, 2H), 2.51 (t, J = 6.9 Hz, 2H), 2.84 (t, J = 6.9 Hz, 2H), 6.87 (d,
J = 15 Hz, 1H), 7.52–7.55 (m, 2H), 7.68–7.87 (m, 5H), 7.98 (s, 1H);
IR (cmÀ1): 3200–2400 (–OH carboxylic acid), 1689 (–C@O), 1613
(–C@O); MS (EI): m/z 268 (M+), 181 (100%). Anal. Calcd for
4.4.1. (E)-6-(3,4-Dimethoxyphenyl)4-oxohex-5-enoic acid
piperedinamide (VII)
Dark brown crystals; yield: 35%; mp: 72–73 °C; 1H NMR
(CDCl3): 1.57–1.66 (m, 6H), 2.73 (t, J = 6.9 Hz, 2H), 3.05 (t,
J = 6.9 Hz, 2H), 3.51 (br s, 4H), 3.92 (s, 6H), 6.68 (d, J = 15 Hz, 1H),
6.87 (d, J = 8.1 Hz, 1H), 7.12 (t, J = 9 Hz, 2H), 7.58 (d,
J = 15 Hz,1H); MS (EI): m/z 331 (M+), 86 (100%). Anal. Calcd for
C17H16O3 (m. wt. = 268.3): C, 76.10; H, 6.01. Found: C, 76.10; H, 5.73.
4.2.5. (E)-6-Biphenyl-4-yl-4-oxohex-5-enoic acid (V)13
Yellow crystals; yield: 64%; mp: 188–190 °C; 1H NMR (CDCl3):
2.51 (t, J = 7.2 Hz, 2H), 2.95 (t, J = 7.2 Hz, 2H), 6.94 (d, J = 16.3 Hz,
1H), 7.35–7.50 (m, 3H), 7.67 (d, J = 16.3 Hz, 1H), 7.71–7.81 (m,
6H); IR (cmÀ1): 3300–2400 (–OH carboxylic acid), 1713 (–C@O),
C19H25NO4 (m. wt. = 331.4): C, 68.86; H, 7.60, N, 4.23. Found: C,
69.31; H, 7.74; N, 4.45.

N. A. Osman et al. / Bioorg. Med. Chem. 18 (2010) 8463–8477
8475
4.4.2. (E)-6-(3,4-Dimethoxyphenyl)-4-oxohex-5-enoic acid
4.4.8. (E)-6-Naphthalen-2-yl-4-oxohex-5-enoic acid
ethanolamide (VIII)
glycinamide ethyl ester (XIV)
Brown powder; yield: 35%; mp: 83–85 °C; 1H NMR (CDCl3):
1.26 (t, J = 3.6 Hz, 2H), 2.58 (t, J = 6.6 Hz, 2H), 3.07 (t, J = 6.6 Hz,
2H), 3.72 (t, J = 4.5 Hz, 2H), 3.92 (s, 6H), 6.63 (d, J = 18 Hz, 1H),
6.87 (d, J = 8.1 Hz, 1H), 7.13 (t, J = 6 Hz, 2H), 7.55 (d, J = 18 Hz,
1H); IR (cmÀ1): 3393 (–OH), 2929 (–CH aliphatic), 1641 (–C@O),
1632 (–C@O); MS (EI): m/z 307 (M+), 191 (100%). Anal. Calcd for
Red powder; yield: 46%; mp: 92–95 °C; 1H NMR (CDCl3): 1.31 (t,
J = 7.2 Hz, 3H), 2.67 (t, J = 6 Hz, 2H), 3.10 (t, J = 6 Hz, 2H), 4.03 (d,
J = 8.4 Hz, 2H), 4.23 (q, J = 7.2 Hz, 2H), 6.31 (br s, 1H), 6.88 (d,
J = 18 Hz, 1H), 7.51–7.54 (m, 2H), 7.67–7.86 (m, 5H), 7.96 (s, 1H);
IR (cmÀ1): 3308 (–NH), 3056 (–CH aromatic), 2980 (–CH aliphatic),
1737 (–CO), 1682 (–C@O), 1648 (–C@O); MS (EI): m/z 339 (M+),
152 (100%). Anal. Calcd for C20H21NO4 (m. wt. = 339.4): C, 70.78;
H, 6.24; N, 4.13. Found: C, 70.39; H, 6.56; N, 4.20.
C16H21NO5 (m. wt. = 307.3): C, 62.53; H, 6.89; N, 4.56. Found: C,
62.26; H, 6.95; N, 4.32.
4.4.3. (E)-N-(4-Hydroxyphenyl)-6-(4-methoxyphenyl)-4-
oxohex-5-enamide (IX)
4.4.9. (E)-6-Naphthalen-2-yl-4-oxohex-5-enoic acid
diethanolamide (XV)
Dark brown powder; yield: 32%; mp: 125–127 °C; 1H NMR
(CDCl3): 2.75 (t, J = 6 Hz, 2H), 3.19 (t, J = 6 Hz, 2H), 3.94 (s, 3H),
5.90 (br s, 1H, –NH), 6.66 (d, J = 18 Hz, 1H), 6.88 (d, J = 9 Hz, 4H),
7.13–7.16 (m, 4H, aromatic), 7.57 (d, J = 18 Hz, 1H); IR (cmÀ1):
3319 (–OH), 3048 (–CH aromatic), 2921 (–CH aliphatic), 1658
(–C@O), 1650 (–C@O); MS (EI): m/z 325 (M+), 59 (100%). Anal.
Calcd for C19H19NO4 (m. wt. = 325.4): C, 70.14; H, 5.89; N, 4.31.
Found: C, 70.22; H, 5.83; N, 4.55.
Red solid; yield: 43%; mp: 94–96 °C; 1H NMR (CDCl3): 2.82 (t,
J = 6 Hz, 2H), 3.15 (t, J = 6 Hz, 2H), 3.58–3.65 (m, 4H), 3.85–3.90
(m, 4H), 6.89 (d, J = 15 Hz, 1H), 7.50–7.54 (m, 2H), 7.66–7.88 (m,
5H), 7.96 (s, 1H); IR (cmÀ1): 3307 (–OH), 3056 (–CH aromatic),
2929 (–CH aliphatic), 1660 (–C@O), 1651 (–C@O); MS (EI): m/z
341 (M+), 74 (100%). Anal. Calcd for C20H23NO4 (m. wt. = 341.4):
C, 70.36; H, 6.79; N, 4.10. Found: C, 70.52; H, 6.77; N, 4.23.
4.4.10. (E)-6-Naphthalen-2-yl-4-oxohex-5-enoic acid (2-
hydroxy-1(R)-methylethanolamide (XVI)
4.4.4. (E)-6-Naphthalen-2-yl-4-oxohex-5-enoic acid
piperidinamide (X)
Red powder; yield: 66%; mp: 106–109 °C; 1H NMR (CDCl3): 1.20
(d, J = 6.9 Hz, 3H), 2.58 (t, J = 6.0 Hz, 2H), 3.09 (t, J = 9.0 Hz, 2H),
3.51–3.56 (m, 1H), 3.69 (d, J = 11 Hz, 2H), 4.06 (br s, 1H), 6.05 (br
s, 1H), 6.87 (d, J = 15 Hz, 1H), 7.50–7.53 (m, 2H), 7.66–7.85 (m,
5H), 7.96 (s, 1H); IR (cmÀ1): 3300 (–OH), 3056 (–CH aromatic),
2932 (–CH aliphatic), 1682 (–C@O), 1644 (–C@O); MS (EI): m/z
311 (M+), 152 (100%). Anal. Calcd for C19H21NO3 (m. wt. = 311.4):
C, 73.29; H, 6.80; N, 4.50. Found: C, 73.51; H, 6.42; N, 4.29.
Yellow powder; yield: 42%; mp: 110–112 °C; 1H NMR (CDCl3):
1.55–1.64 (m, 6H), 2.75 (t, J = 6 Hz, 2H), 3.10 (t, J = 6 Hz, 2H), 3.57
(br s, 4H), 6.92 (d, J = 15 Hz, 1H), 7.50–7.53 (m, 2H), 7.68–7.88
(m, 5H), 7.96 (s, 1H); IR (cmÀ1): 3052 (–CH aromatic), 2923 (–CH
aliphatic), 1683 (–C@O), 1635 (–C@O); MS (EI): m/z 321 (M+), 84
(100%). Anal. Calcd for C21H23NO2 (m. wt. = 321.4): C, 78.47; H,
7.21; N, 4.36. Found: C, 78.51; H, 7.42; N, 4.53.
4.4.5. (E)-6-Naphthalen-2-yl-4-oxohex-5-enoic acid
ethanolamide (XI)
4.4.11. (E)-7-Naphthalen-2-yl-5-oxohept-6-enoic
piperidinamide (XVII)
Yellowish-brown powder; yield: 45%; mp: 91–93 °C; 1H NMR
(CDCl3): 2.61 (t, J = 7.5 Hz, 2H,), 3.14 (t, J = 6 Hz, 2H), 3.42–3.47
(m, 2H), 3.74 (t, J = 6 Hz, 2H), 6.30 (br s, 1H), 6.88 (d, J = 15 Hz,
1H), 7.48–7.55 (m, 2H), 7.67–7.86 (m, 5H), 7.97 (s, 1H); IR
(cmÀ1): 3252 (–OH), 3083 (–CH aromatic), 2918 (–CH aliphatic),
1684 (–C@O), 1653 (–C@O); MS (EI): m/z 297 (M+), 151 (100%).
Anal. Calcd for C18H19NO3 (m. wt. = 297.4): C, 72.71; H, 6.44; N,
4.71. Found: C, 72.25; H, 6.28; N, 4.93.
Yellow powder; yield: 55%; mp: 100–103 °C; 1H NMR (CDCl3):
1.57–1.59 (m, 6H), 2.04–2.08 (m, 2H), 2.48 (t, J = 7.2 Hz, 2H), 2.86
(t, J = 7.2 Hz, 2H), 3.52 (br s, 4H), 6.86 (d, J = 18 Hz, 1H), 7.51–
7.54 (m, 2H), 7.73–7.87 (m, 5H), 7.97 (s, 1H); IR (cmÀ1): 3061
(–CH aromatic), 2942 (–CH aliphatic), 1685 (–C@O), 1636
(–C@O); MS (EI): m/z 335 (M+), 152 (100%). Anal. for C22H25NO2
(m. wt. = 335.4): C, 78.77; H, 7.51; N, 4.18. Found: C, 79.15; H,
7.17; N, 4.33.
4.4.6. (E)-6-Naphthalen-2-yl-4-oxohex-5-enoic acid (4-
hydroxyphenyl)-amide (XII)
4.4.12. (E)-7-Naphthalen-2-yl-5-oxohept-6-enoic acid
ethanolamide (XVIII)
Dark brown powder; yield: 53%; mp: 131–133 °C; 1H NMR
(CDCl3): 2.79 (t, J = 7.5 Hz, 2H), 3.20 (t, J = 7.5 Hz, 2H), 6.20 (br s,
1H), 6.66 (d, J = 18 Hz, 1H), 7.10–7.15 (m, 2H), 7.52–7.57 (m, 4H),
7.71–7.86 (m, 5H), 7.97 (s, 1H); IR (cmÀ1): 3317 (–OH), 3060
(–CH aromatic), 2922 (–CH aliphatic), 1658 (–C@O), 1650
(–C@O); MS (EI): m/z 345 (M++2), 237 (100%). Anal. Calcd for
Yellow crystals; yield: 48%; mp: 100–103 °C; 1H NMR (CDCl3):
2.01–2.11 (m, 2H), 2.31 (t, J = 7.5 Hz, 2H), 2.80 (t, J = 6.9 Hz, 2H),
3.41–3.46 (m, 3H), 3.73 (t, J = 5.1 Hz, 2H), 6.20 (br s, 1H), 6.84 (d,
J = 18 Hz, 1H), 7.46–7.55 (m, 2H), 7.65–7.85 (m, 5H), 7.96 (s, 1H);
IR (cmÀ1): 3280 (–OH), 3057 (–CH aromatic), 2935 (–CH aliphatic),
1679 (–C@O), 1637 (–C@O); MS (EI): m/z 311 (M+), 141 (100%).
Anal. Calcd for C19H21NO3 (m. wt. = 311.4): C, 73.29; H, 6.80; N,
4.50. Found: C, 72.85; H, 6.44; N, 4.89.
C22H19NO3 (m. wt. = 345.4): C, 76.50; H, 5.54; N, 4.06. Found: C,
76.88; H, 5.62; N, 3.87.
4.4.7. (E)-6-Naphthalen-2-yl-4-oxohex-5-enoic acid
cyclopropamide (XIII)
4.4.13. (E)-7-Naphthalen-2-yl-5-oxohept-6-enoic acid
cyclopropylamide (XIX)
Red powder; yield: 45%; mp: 154–156 °C; 1H NMR (CDCl3): 0.52
(br s, 2H), 0.74–0.78 (m, 2H), 2.54 (t, J = 6.6 Hz, 2H), 2.71–2.73 (m,
1H), 3.11 (t, J = 6.6 Hz, 2H), 5.80 (br s, 1H), 6.88 (d, J = 15 Hz, 1H),
7.52–7.55 (m, 2H), 7.67–7.89 (m, 5H), 7.97 (s, 1H); IR (cmÀ1):
3243 (–NH), 3051 (–CH aromatic), 2911 (–CH aliphatic), 1682
(–C@O), 1661 (–C@O); MS (EI): m/z 293 (M+), 127 (100%). Anal.
Calcd for C19H19NO2 (m. wt. = 293.4): C, 77.79; H, 6.53; N, 4.77.
Found: C, 77.50; H, 6.67; N, 4.81.
Buff powder; yield: 63%; mp: 125–126 °C; 1H NMR (CDCl3):
0.48–0.52 (m, 2H), 0.75–0.81 (m, 2H), 1.99–2.09 (m, 2H), 2.25 (t,
J = 7.5 Hz, 2H), 2.74–2.86 (m, 3H), 5.78 (br s, 1H), 6.85 (d,
J = 15 Hz, 1H), 7.50–7.56 (m, 2H), 7.67–7.89 (m, 5H), 7.97 (s, 1H);
IR (cmÀ1): 3235 (–NH), 3053 (–CH aromatic), 2968 (–CH aliphatic),
1682 (–C@O), 1661 (–C@O); MS (EI): m/z 307 (M+), 152 (100%).
Anal. Calcd for C20H21NO2 (m. wt. = 307.4): C, 78.15; H, 6.89; N,
4.56. Found: C, 77.67; H, 6.48; N, 4.32.

8476
N. A. Osman et al. / Bioorg. Med. Chem. 18 (2010) 8463–8477
4.4.14. (E)-7-Naphthalen-2-yl-5-oxo-hept-6-enoic acid
glycinamide ethyl ester (XX)
monitored by TLC. Quantitative conversion was observed after
4 h. The suspension was filtered and the residual solid crystallized
from ethanol to yield 43% of a white powder.41
Brown powder; yield: 40%; mp: 78–80 °C; 1H NMR (CDCl3): 1.29
(t, J = 7.2 Hz, 3H), 2.03–2.10 (m, 2H), 2.84 (t, J = 6.9 Hz, 2H), 3.11 (t,
J = 7.2 Hz, 2H), 4.05 (d, J = 5.1 Hz, 2H), 4.21 (q, J = 7.2 Hz, 2H), 6.20
(br s, 1H), 6.86 (d, J = 15 Hz, 1H), 7.51–53 (m, 2H), 7.67–7.85 (m,
5H), 7.97 (s, 1H); IR (cmÀ1): 3291 (–NH), 2943 (–CH aliphatic),
1739 (–CO), 1686 (–C@O), 1652 (–C@O); MS (EI): m/z 353 (M+),
151 (100%). Anal. Calcd for C21H23NO4 (m. wt. = 353.4): C, 71.37;
H, 6.56; N, 3.96. Found: C, 71.74; H, 6.33; N, 4.28.
4.4.21. 1-Biphenyl-4-yl-4-morpholin-4-yl-butane-1,4-dione
(XXIV)
White fluffy powder; yield: 43%; mp: 159–160 °C; 1H NMR
(CDCl3): 2.81 (t, J = 6.6 Hz, 2H), 3.42 (t, J = 6.6 Hz, 2H), 3.63 (br s,
4H), 3.72 (br s, 4H), 7.40–7.51 (m, 3H), 7.62–7.72 (m, 4H), 8.10
(q, J = 4.8 Hz, 2H); IR (cmÀ1): 3050 (–CH aromatic), 2958 (–CH ali-
phatic), 1679 (–C@O), 1652 (–C@O); MS (EI): m/z 323 (M+), 152
(100%). Anal. Calcd for C20H21NO3 (m. wt. = 323.4): C, 74.28; H,
6.55; N, 4.33. Found: C, 73.88; H, 6.72; N, 4.73.
4.4.15. (E)-7-Naphthalen-2-yl-5-oxohept-6-enoic acid (2-
hydroxy-1(R)-methylethanolamide (XXI)
Whitish-brown powder; yield: 65%; mp: 114–116 °C; 1H NMR
(CDCl3): 1.19 (d, J = 6.9 Hz, 3H), 2.03–2.12 (m, 2H), 2.32 (t,
J = 6.9 Hz, 2H), 2.82 (t, J = 6.6 Hz, 2H), 3.51–3.57 (m, 1H), 3.71 (d,
J = 6 Hz, 2H), 4.11 (br s, 1H), 5.82 (br s, 1H), 6.86 (d, J = 18 Hz,
1H), 7.51–7.55 (m, 2H), 7.67–7.89 (m, 5H), 7.97 (s, 1H); IR
(cmÀ1): 3308 (–OH), 3056 (–CH aromatic), 2956 (–CH aliphatic),
1684 (–C@O), 1644 (–C@O); MS (EI): m/z 325 (M+), 152 (100%).
Anal. Calcd for C20H23NO3 (m. wt. = 325.4): C, 73.82; H, 7.12; N,
4.30. Found: C, 74.25; H, 7.39; N, 4.64.
Acknowledgment
The authors are grateful to the Faculty of Postgraduate Studies,
German University in Cairo, for partial financing of this work
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.10.050.
4.4.16. (E)-6-Biphenyl-4-yl-4-oxohex-5-enoic acid
ethanolamide (XXII)
References and notes
Dark brown solid; yield: 65%; mp: 77–79 °C; 1H NMR (CDCl3):
2.58 (t, J = 9 Hz, 2H), 3.11 (t, J = 6 Hz, 2H), 3.22–3.27 (m, 2H), 3.64
(t, J = 6 Hz, 2H), 6.30 (br s, 1H), 6.85 (d, J = 18 Hz, 1H), 7.26–7.96
(m, 10H); IR (cmÀ1): 3254 (–OH), 3030 (–CH aromatic), 2923
(–CH aliphatic), 1656 (–C@O), 1554 (–C@O); MS (EI): m/z 325
(M++2), 73 (100%). Anal. Calcd for C20H21NO3 (m. wt. = 323.4): C,
74.28; H, 6.55; N, 4.33. Found: C, 73.86; H, 6.73; N, 4.75.
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