Synthesis and binding study of certain 6-arylalkanamides as molecular probes for cannabinoid receptor subtypes

Article abstract of DOI:10.3109/14756366.2011.645241

Tetrahydrocannabinol and other mixed cannabinoid (CB) receptors CB1/CB2 receptor agonists are well established to elicit antinociceptive effects and psychomimetic actions, however, their potential for abuse have dampened enthusiasm for their therapeutic d

Full text of DOI:10.3109/14756366.2011.645241

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 436–439
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.645241
RESEARCH ARTICLE
Synthesis and binding study of certain 6-arylalkanamides as
molecular probes for cannabinoid receptor subtypes
Azza T. Taher1, Hanan H. Kadry1, Marco Allarà2, Vincenzo Di Marzo2, Ashraf H. Abadi3, and
Khaled A. Abouzid4
1Department of Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt, 2Endocannabinoid Research
Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Napoli, Italy, 3Department of
Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, New Cairo City, Egypt,
and 4Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt
Abstract
Tetrahydrocannabinol and other mixed cannabinoid (CB) receptors CB /CB2 receptor agonists are well established
to elicit antinociceptive effects and psychomimetic actions, however,1 their potential for abuse have dampened
enthusiasm for their therapeutic development. In an effort to refine a semi-rigid structural framework for CB2
receptors binding, we designed novel compounds based on aromatic moiety and flexible linker with various amides
mimicking the outlook of the endogenous anandamide which could provide as CB2 receptor ligand. In this direction,
we developed and synthesized new aryl or arylidene hexanoic acid amides and aryl alkanoic acid diamide carrying
different head groups. These new compounds were tested for their affinities for human recombinant CB receptors
CB1 and CB and fatty acid amide hydrolase. Although, the preliminary screening of these compounds demonstrated
weak bindi2ng activity towards CB receptor subtypes at 10 µmole, yet this template still could serve up as probes for
further optimization and development of affinity ligand for CB receptors.
Keywords: Cannabinoid, selective CB2 ligands, synthesis
Introduction
the psychomimetic effects of CBs, as well as the poten-
tial for abuse and dependence of Tetrahydrocannabinol
(THC). Although CB1 is also expressed in lung, liver, and
kidney, its role in peripheral tissue is not well understood.
On the other hand, CB2 receptors are sparsely expressed
in the CNS and are predominately expressed on activated
immune cells, including natural killer cells, at higher levels
Since the discovery of the endogenous cannabimimetic
(anandamide), many structural ligands were developed
for cannabinoid (CB) receptor subtypes. During the last
decade, numerous selective ligands for each of the canna-
binergic proteins were designed and synthesized1. Many
of these agents serve as important molecular probes, pro-
viding structural information about receptor binding sites,
as well as serving as pharmacological tools for obtaining
information about the role of each of these targets in phys-
iological and disease states. e endocannabinoid system
is represented by CB1 and CB2, two well characterized G
protein-coupled receptors2–4. Furthermore, there is evi-
dence for the presence of additional CB receptors5,6, how-
ever, their full characterizations are still incomplete. CB1
receptors in the central nervous system mediate (CNS)
6
than CB1 . us, the CB2 receptor represents a viable target
for the development of anti-inflammatory and analgesic
agents that lack overt behavioral effects, and has therefore
gained much recent attention, as evidenced by a rapidly
growing body of research7.
Recently, ethyl sulfonamide THC analogue, O-3223
displayed excellent selectivity and efficacy for the CB2
receptor when evaluated in a variety of murine models of
pain and inflammation Figure 18.
Address for Correspondence: Prof. Khaled A. Abouzid, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams
University, Cairo 1156, Egypt. E-mail: abouzid@yahoo.com
(Received 21 October 2011; revised 20 November 2011; accepted 21 November 2011)
436

Synthesis and binding study of certain 6-arylalkanamides 437
In the same direction, in our previous work, we
designed and synthesized a series of arylidene oxoalka-
noic acid amides as CB2 selective ligands. e ethanol-
amide candidate Figure 1 displayed promising affinity to
CB2 receptor at 1 µmol9. is finding motivated us to high-
light essential pharmacophoric features necessary for
CB2 binding affinity of this scaffold. In continuous effort
to optimize this model, we decided to explore and impart
certain modifications for this framework through replac-
ing the amide tail with different hydrophilic moieties.
To achieve this goal, we report here the synthesis
of several candidates such as 6-arylalkanamide and
diamide derivatives. In these compounds, we replaced
the aromatic portion of the selective CB2 ligand O-3223
with a variety of oxygenated and non oxygenated aryl
moieties and a tail of 6 carbon atoms and different
amidic groups.
4-(naphthalene-1-ylamino)-4-oxo-butanoic acid (2e13)
were prepared as previously reported.
General procedure for the preparation of 6-(substituted-
phenyl)-4-oxohex-N-substituted-5-enamide (3a–m)
A mixture of the appropriate acid 2a–f (0.01 mol) and
triethylamine (0.072 mol) in dry methylene chloride
(10 mL) was cooled in an ice and salt bath to −10°C. Ethyl
chloroformate (0.05 mol) was added dropwise, while stir-
ring over a period of 10 min and stirring was continued for
30 min. e amine (0.01 mol) was added gradually within
10 min and stirring was continued overnight at room
temperature. e solvent was evaporated under vacuum
and after cooling, the residue was extracted twice with
(20 mL) ethyl acetate. e ethyl acetate layer was washed
with 10% hydrochloric acid twice with (15mL), then
washed with 5% sodium hydroxide twice with (15mL).
e organic layer dried over anhydrous sodium sulphate,
filtered and the filtrate was evaporated under reduced
pressure and cooled. e crystalline solid was separated,
collected and crystallized from chloroform.
ese compounds were tested for their affinity to CB1/
CB2 hoping to obtain new CB2 selective receptor ligands
with semi-rigid components.
e synthesized compounds were tested for their
binding affinities for CB receptors CB1 and CB2 receptors
adopting reported methods14,15 and Ki values were calcu-
lated by applying the Cheng–Prusoff equation to the IC50
values (obtained by GraghPad) for the displacement of
the bound radioligand by increasing concentrations of
the test compound. Data are means SEM of at least n= 3
experiments and presented in Table S1.
On the other hand, the effect of increasing concen-
trations of the synthetic compounds on the enzymatic
hydrolysis of anandamide was studied using membranes
prepared from rat brain16. e results of anadamide
hydrolysis assay is tabulated in Table S2.
Material and methods
Chemistry
General procedure for the synthesis of (E)-6-(Substituted-
phenyl)-4-oxohex-5-enoic acids (2a–f)
Both the respective aldehyde (30 mmol) and levulinic
acid (30 mmol) were dissolved in dry toluene (100mL)
containing acetic acid (3 mL) and piperidine (1 mL). e
solution was heated under reflux using Dean–Stark water
trap under nitrogen until the theoretical amount of water
had been collected (~6–8 h) and TLC analysis (CHCl3:
CH3OH 93:7) indicated disappearance of the starting
material. e solvent was evaporated under vacuum,
cooled and ice cold water (30 mL) was added. e solid
formed was collected, washed twice with (10mL) of
diethyl ether and then twice with 2 M HCl (15 mL), dried
and crystallized from the benzene/ methanol mixture
(10:5 mL).
Discussion
(E)-6-(Substituted-phenyl)-4-oxohex-6-enoic acids (2a–f)
were synthesized through condensation of the appropri-
ate aldehyde with levulinic acid using catalytic amounts of
piperidine and acetic acid to give the respective arylidene
keto acid derivatives according to the general procedure
previously reported in the literature (Scheme 19). e E
6-Phenyl-4-oxohex-5-enoic
6-(4-methylphenyl)-4-oxohex-5-enoic 2b and 6-(N,N-
dimethylaminophenyl)-4-oxohex-5-enoic
(2c11)
6-(3,4-dimethoxylphenyl)-4-oxohex-5-enoic (2d12) and
2a10,
Figure 1. Structures of CB1/CB2 agents.
© 2013 Informa UK, Ltd.

438 A. T. Taher et al.
isomers were obtained as the major products through
crystallization of the crude products and this was con-
firmed with the high coupling constant values of the
olefinic protons, which is about 15 Hz, typical for E rather
than the Z isomers. Infrared (IR) spectroscopy showed
characteristic broad bands at the range of 3300–2400cm−1
for the O-H stretching of the carboxylic group, and bands
at 1690cm−1 for the carboxylic C=O stretching in addition
to bands at 1730–1665cm−1 for the ketonic C=O stretching.
Scheme 1 also describes the synthesis of 6-(substituted-
phenyl)-4-oxohex-5-enoic acid N-substituted amides (3a–
m and 5a–c) through mixed anhydride method using ethyl
chloroformate, triethylamine and the appropriate amine17.
Secondary amides showed IR bands at 3280cm−1 of the
−NH stretching, and in addition to −C=O stretching of the
amide derivatives which showed bands at relatively lower
values 1650cm−1 than the typical carbonyl stretching at
1700cm−1. e ester amide containing compounds showed
an additional −C=O stretching of the ester at 1738cm−1.
Another series of compounds containing diamides or
amide ester was obtained in two step reaction as described
in Scheme 2. e first step involved the reaction of 1-ami-
nonaphthalene with succinic anhydride in acetic acid at
ambient temperature to afford the amic acid derivative 4.
e formed amic acid was subsequently reacted in a similar
manner with amines as previously described in Scheme 1 to
give the diamide 5a–b or amide ester 5c derivatives.
On the other hand, the results of the binding assay
revealed that most of the tested compounds did not
exhibit appreciable binding affinity for CB2 at 10 µM
except compound 3f which exhibited weak CB2 affinity at
15.9 % and compound 3c which could be weak allosteric
modulator (+11.4 %). On the other hand, compounds 5c,
3g, 3m, 5b exhibited maximum displacement percentage
of 30.3, 25, 21.8 % and 21.21, respectively, on CB1 at 10 µM.
It is worth to mention that, although, the results of anan-
damide hydrolysis assay for most of tested compounds
did not demonstrate appreciable activity at 10 µM, yet,
compounds 3b and 3e which possess trihydroxyalkyl
amide tail demonstrated stimulation of fatty acid amide
hydrolase enzyme rather than inhibition. is could be
related to allosteric activation of the enzyme.
Scheme 1. Reagents and conditions: (A) piperidine/acetic acid, toluene/reflux, (B) ethyl chloroformate /triethylamine, H-ZR (where Z refer
to nitrogen or oxygen and R alkyl or aryl), stirring at rt.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and binding study of certain 6-arylalkanamides 439
Scheme 2. Reagents and conditions: (A) Acetic acid, stirring at rt, (B) ethyl chloroformate/triethylamine, at 10 °C then H-ZR and stirring at rt.
6. Joong Y S. Understanding functional residues of the cannabinoid
CB1 receptor for drug discovery. Current Topics in Med Chem
Conclusion
2010;10:779–798.
ese findings could verify that the proposed com-
pounds may lack proper features and/or dimensions of
the tail necessary for optimum binding with the target
CB2 receptor. Although these compounds demonstrated
7. Pasquini L, Botta T, Semeraro C, Mugnaini A, Ligresti E, Palazzo
S, Maione V, Di Marzo F. ree-dimensional quantitative structure
selectivity relationships analysis guided rational design of a highly
selective ligand for the cannabinoid receptor CB2. J. Med Chem
weak binding affinity yet these finding will be useful as
starting points for further structure refinement and for
exploring further structural requirements of the CB1/CB2
receptor binding site. is will also serve for further opti-
mization of this proposed template for the development
of new ligands by changing the rigid aryl moiety and/or
the spacer or the head amide group.
2008;51:5075–5084.
8. Steven G, Kinsey a, Anu Mahadevan b, Bingjun Z, Hang S, Pattipati
S, Naidu R, Razdan K, Dana E, Selley M, Imad D, Aron L. e CB2
cannabinoid receptor-selective agonist O-3223 reduces pain and
inflammation without apparent cannabinoid behavioral effects.
Neuropharm 2011;60:244–251.
9. Noha A, Osman A, Mahmoud H, Marco A, Raimund N, Khaled A,
Abouzid, Vincenzo M, Ashraf A. Synthesis, binding studies and
molecular modeling of novel cannabinoid receptor ligands. Bioorg
Med Chem 2010;18:8463–8477.
10. OlppT. Productclass2:2-oxoaldehydesandheteroatomanalogues.
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Acknowledgment
11. Tran Q, Nguyen D. Synthesis and structures of some 6-(2-
arylvinyl)-4,5-dihydropyridazin-3(2H)-ones. Tap Chi Hoa Hoc
2004;42:325–328. rough CA 143:347118.
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e authors are grateful to the staff members micro-an-
alytical unit, Cairo University for performing the spectra
data for the synthesized compounds.
Declaration of interest
13. Gluzman M, Mil’ner S. Investigation of the acylation process of
amines by solid succinic and benzoic anhydrides under isothermal
conditions. Khim Khimich Tekhno 1960;3:684–690.
e authors report no conflict of interest.
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