Welcome to Scribd, the world's digital library. Read, publish, and share books and documents. See more
Download
Standard view
Full view
of .
Look up keyword
Like this
1Activity
0 of .
Results for:
No results containing your search query
P. 1
Design, Synthesis, and Binding Studies of New Potent Ligands of Cannabinoid Receptors

Design, Synthesis, and Binding Studies of New Potent Ligands of Cannabinoid Receptors

Ratings: (0)|Views: 57|Likes:
Published by abazaba151
A study into effectively mapping CB receptors though targeted ligand design.
A study into effectively mapping CB receptors though targeted ligand design.

More info:

Published by: abazaba151 on Apr 16, 2011
Copyright:Attribution Non-commercial

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
download as PDF, TXT or read online from Scribd
See more
See less

02/06/2013

pdf

text

original

 
Design, Synthesis, and Binding Studies of New Potent Ligands of CannabinoidReceptors
 Antonella Brizzi,
†,
* Vittorio Brizzi,
Maria Grazia Cascio,
Tiziana Bisogno,
Rossella Sirianni,
and Vincenzo Di Marzo
 Dipartimento Farmaco Chimico Tecnologico, Universita
`
degli Studi di Siena, Via A. Moro 2, 53100 Siena (Italy), and Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche,Via Campi Flegrei 34, Comprensorio Olivetti, 80078 Pozzuoli, Napoli (Italy) Received February 17, 2005
Despite their different chemical structures,
9
-tetrahydrocannabinol (THC) and anandamide(AEA) have common pharmacological properties. This study was aimed at finding newcannabinoid receptor ligands that overcome the instability of AEA and its analogues. To thisend we planned the synthesis of a series of compounds which retained both a rigid structure,like that of plant cannabinoids, and a flexible portion similar to that of anandamide. Bindingstudies on CB
1
and CB
2
receptors, anandamide membrane transporter (AMT), and fatty acidamide hydrolase (FAAH) showed that some of the newly developed compounds have high affinityand specificity for cannabinoid CB
1
and CB
2
receptors. Compound
25
is a potent CB
1
and CB
2
ligand, with affinity constants significantly lower than AEA and similar to WIN 55-212,compound
52
is a potent CB
2
ligand, although not very selective over CB
1
receptors, andcompound
43
is CB
2
ligand, with at least a 26-fold selectivity over CB
1
receptors. Compound
25
behaved as a inverse agonist at CB
1
receptors as assessed in the cyclic AMP functionalassay.
Introduction
The great popularity of hashish and marihuana, bothderived from the Indian hemp
Cannabis Sativa L.
, andof their major psychoactive component, (
-
)-
trans
-
9
-tetrahydrocannabinol (THC, Chart 1),
1
is due to theirmedicinal as well as their psychotomimetic effects.Cannabinoids have been shown to produce a wide rangeof pharmacological effects
2
mediated by two receptors:CB
1
, very abundant in the brain
3
and cloned in 1990,
4
and CB
2
, expressed predominantly in the periphery,
5
mainly in the spleen and in immune cells. The presenceof these receptors in mammalian cells was indicativeof the existence of an endogenous cannabinoid system.Several years ago the first natural ligand was isolatedfrom porcine brain
6
and identified as an amide relatedto arachidonic acid, the
cis
-5,8,11,14-eicosatetraenoyl-ethanolamide, or anandamide (AEA), showing proper-ties similar to the plant-derived agonist THC. Threeyears later a well-known intermediate in phosphogly-ceride metabolism, 2-arachidonoylglycerol (2-AG), wasfound to act also as a potential ligand at cannabinoidreceptors.
7
-
9
The molecular basis of the biological effects of theseendogenous compounds were elucidated also thanks tothe understanding of their metabolism, including bio-chemical pathways that lead to their synthesis andinactivation. Both AEA and 2-AG are produced from themetabolism of membrane phospholipids and immedi-ately released into extracellular space. Inactivationoccurs in two steps: cellular reuptake, facilitated by aputative transporter protein known as the anandamidemembrane transporter (AMT)
10
and enzymatic hydroly-sis by a fatty acid amide hydrolase (FAAH)
11
or by amonoacylglycerol lipase, in the case of 2-AG. Theproteins of the endocannabinoid system, including CB
1
,CB
2
, AMT, and FAAH, represent excellent targets forthe development of new therapeutic drugs to be em-ployed in many pathologies, like pain, immunodepres-sion, vascular perpherical disorders, increase or lower-ing of appetite, and motor disorders. The therapeutic value of these compounds as analgesic, anti-glaucoma,and antiinflammatory agents, and in the chemotherapy-induced nausea and vomiting and in appetite stimula-tion of patients with AIDS, is nowadays of greatinterest.
12
Since these discoveries several groups have carriedout the synthesis and biological evaluation of AEAanalogues, obtained by modifications of the fatty acylchain or/and of the ethanolamide “head”.
13
Unfortu-nately, arachidonic acid and analogous polyunsaturatedfatty acids are very expensive and/or easily oxidized. AEA itself is very unstable and in the body is rapidlymetabolized by FAAH and/or oxidation. To prevent inpart the degradation of AEA and of its synthetic
* Corresponding author. Fax:
+
39 577 234333; Phone:
+
39 577234327; e-mail: brizzi3@unisi.it.
Universita` degli Studi di Siena.
Consiglio Nazionale delle Ricerche.
Chart 1
7343
 J. Med. Chem.
2005,
48,
7343
-
735010.1021/jm0501533 CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 10/13/2005
   D  o  w  n   l  o  a   d  e   d   b  y   U   N   I   V   O   F   M   I   C   H   I   G   A   N  o  n   O  c   t  o   b  e  r   3   0 ,   2   0   0   9   |   h   t   t  p  :   /   /  p  u   b  s .  a  c  s .  o  r  g    P  u   b   l   i  c  a   t   i  o  n   D  a   t  e   (   W  e   b   )  :   O  c   t  o   b  e  r   1   3 ,   2   0   0   5   |   d  o   i  :   1   0 .   1   0   2   1   /   j  m   0   5   0   1   5   3   3
 
analoguestheiractivityatcannabinoidreceptorsisoftenassessed by conducting receptor binding assays in thepresence of a serine protease inhibitor, phenylmethane-sulfonyl fluoride (PMSF).
14
While AEA is a very flexible molecule, as demon-strated also by NMR studies,
15
and can assume bothlinear and nonlinear conformations, THC, and canna-binoid analogues, including tricyclic and bicyclic can-nabinoids, are rigid compounds. Nevertheless, AEA andTHC have been shown to share many, albeit not all,pharmacological properties. Bearing in mind the phar-macophore requirements
16
of both AEA and THC,necessary for the binding to cannabinoid CB
1
and CB
2
receptors, we have planned the synthesis of a series of stable compounds which consist of both a rigid aromaticportion, as in THC, and a flexible chain, as in AEA.Olivetol, or 5-pentyl-benzene-1,3-diol (5-pentylresor-cinol), is involved in the biogenesis of THC in Cannabis
17
(Figure 1), and it has been selected as the basic aromaticscaffold of the rigid part of our new derivatives; othermodified phenols have also been considered in this workin order to define a structure
-
activity relationship. Asflexible chains we have chosen saturated carboxylicacids with chain lengths of 6 or 11 or 16 carbon atoms,easily converted into the final amides. Finally, we haveevaluated the influence of several factors such as sub-stitutions of the aromatic backbone, the length of thechain carrying the amidic head, and nature of amide. As a result, different series of new compounds weredesigned and synthesized, whose general structure isreported in Figure 2.
Chemistry.
As shown in Scheme 1, the synthesis of the new compounds starts from a substituted phenol[3-pentadecylphenol, olivetol, resorcinol (or benzene-1,3-diol), and 4-hexylresorcinol] which is reacted with abromoalkylmethyl ester (6-bromohexanoic acid methylester or 11-bromoundecanoic acid methyl ester or 16-bromo-hexadecanoic acid methyl ester) in dry acetonein the presence of anhydrous K 
2
CO
3
and KF, givingesters
1
-
14
. The final amides are obtained by either of two simple methods. Method A: esters were refluxedwith methanolic/aqueous sodium hydroxide solution togive the corresponding acids, which were used as suchand reacted with amines in the presence of 1-hydroxy-benzotriazole (HOBt) and 1-cyclohexyl-3-(2-morpho-linoethyl)carbodiimide methyl-
 p
-toluenesulfonate (CMC)in dichloromethane or acetonitrile.
18
Method B: esterswere warmed with ethanolamine as solvent.The O-alkylation of phenols was carried out with orwithout KF; as expected, in both conditions the reactionafforded all possible alkylated products when the aro-matic nucleus had two free hydroxyl groups, althoughaddition of KF improved the yields and specificallyincreased those of monoalkylated over dialkylated prod-ucts. Moreover, O-alkylation of 4-hexylresorcinol af-forded two different monoalkylated regioisomers. Thereaction occurred mainly on the hydroxyl in C-1 irre-spectively of the bromomethyl ester used and indepen-dently from the length of its chain; in fact the C-1position results less hindered when the hexyl chain ispresent as para substituent. The structure of tworegioisomers has been definitively assigned by NOESYexperiments which showed a NOE effect between theOCH
2
and both the ortho aromatic hydrogens (H-2 andH-6) in the isomer with the alkylated hydroxyl in C-1,and a NOE effect between the OCH
2
and the only orthoaromatic hydrogen (H-2) in the isomer with the alkyl-ated hydroxyl in C-3 (Figure 3).
Pharmacological Evaluation. Binding Assays.
For CB
1
and CB
2
receptor binding assays, the newcompounds were first subjected to a preliminary screen-ing carried out using three concentrations (5, 10, and25
µ
M), membranes from rat brain and spleen, and [
3
H]-
 N 
-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophen-yl)-4-methyl-1
 H 
-pyrazole-3-carboxamide hydrochloride(SR141716A, 2 nM) as the high affinity ligand.
19
Com-pounds which displaced [
3
H]-SR141716A by more than50% at 10
µ
M were further analyzed by using P
2
membranes from COS cells transfected with either thehuman CB
1
or CB
2
receptor and [
3
H]-(
-
)-
cis
-3-[2-hy-droxy-4-(1,1-dimethylheptyl)phenyl]-
trans
-4-(3-hydroxy-propyl)cyclohexanol ([
3
H]CP-55,940) (
 K 
d
)
690 pM) asthe high affinity ligand as described by the manufac-turer (Perkin-Elmer, Italy). Displacement curves weregenerated by incubating drugs with 0.5 nM of [
3
H]CP-55,940. In all cases,
i
values were calculated byapplying the Cheng
-
Prusoff equation to the IC
50
values(obtained by GraphPad) for the displacement of thebound radioligand by increasing concentrations of thetest compounds.
Fatty Acid Amide Hydrolase Assays.
The effectof compounds on the enzymatic hydrolysis of [
14
C]-anandamide (6
µ
M) was studied by using membranesprepared from rat brain incubated with increasingconcentrations of compounds in 50 mM Tris-HCl, pH 9,for 30 min at 37 °C.
19
[
14
C]Ethanolamine produced from[
14
C]anandamide hydrolysis was measured by scintilla-
Figure 1.Figure 2.
Scheme 1
a
a
Reagents: (i) Br-(CH
2
)nCOOCH
3
, CH
3
COCH
3
,
2
CO
3
, KF,reflux, 48
-
72 h; (ii) Method A: MeOH/NaOH, reflux, 3 h; amine,HOBt, CMC, r.t., overnight; (iii) Method B: H
2
NCH
2
CH
2
OH, 120
-
130 °C, 5 h.
Figure 3.7344
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 Brizzi et al.
   D  o  w  n   l  o  a   d  e   d   b  y   U   N   I   V   O   F   M   I   C   H   I   G   A   N  o  n   O  c   t  o   b  e  r   3   0 ,   2   0   0   9   |   h   t   t  p  :   /   /  p  u   b  s .  a  c  s .  o  r  g    P  u   b   l   i  c  a   t   i  o  n   D  a   t  e   (   W  e   b   )  :   O  c   t  o   b  e  r   1   3 ,   2   0   0   5   |   d  o   i  :   1   0 .   1   0   2   1   /   j  m   0   5   0   1   5   3   3
 
tion counting of the aqueous phase after extraction of the incubation mixture with 2 volumes of CHCl
3
 /CH
3
-OH (2:1 by volume). In most cases, only the most potentcompounds in the binding assays were subjected to thisassay.
 Anandamide Membrane Transporter (AMT) As-says.
The effect of compounds on the uptake of anan-damide by RBL-2H3 cells was studied as described pre- viously.
19
Cells were incubated with [
14
C]anandamide(4
µ
M) for 5 min at 37 °C, in the presence or absence of  varying concentrations of the inhibitors. Residual [
14
C]-anandamide in the incubation media after extractionwith CHCl
3
 /CH
3
OH (2:1 by volume) was used as a mea-sure of the anandamide that was taken up by cells. Dataare expressed as the concentration exerting 50% inhibi-tion of anandamide uptake (IC
50
) calculated with Graph-Pad. In most cases, only the most potent compounds inthe binding assays were subjected to this assay.
Cyclic AMP Assay.
Cyclic AMP assays were per-formed on intact confluent N18TG2 cells plated in six-well dishes and stimulated for 10 min at 37 °C withforskolin 1
µ
M in 400
µ
L of serum-free Dulbecco’smodified Eagle’s Medium containing 20 mM HEPES,0.1 mg/mL BSA, 0.1 mM 1-methyl-3-isobutylxanthine.
20
Cells were treated with vehicle (methanol, 0.1%) orcompounds, or WIN55,212-2 at various concentrations,or with SR141716A (100 nM). After incubation, 800
µ
Lof ethanol was added, cells were extracted and cyclic AMP was determined by means of a cyclic AMP assaykit (Amersham, UK), as advised by the manufacturer.
Results and Discussion
 All the newly synthesized compounds that exibitedIC
50
e
10
µ
M in the preliminary screening for canna-binoid receptor binding activity were evaluated inradioligand binding assays for affinity at recombinanthuman CB
1
, CB
2
overexpressed in COS cells, or for theirinhibitory actions on FAAH and AMT, and the resultsare summarized in Table 1. Our preliminary goal wasto evaluate the hypothesis that putting together in a
Table 1.
Radioligand Binding Assays and Selectivity over Other Proteins of the Endocannabinoid System of the SynthesizedCompounds
a
compd R R
1
n R
2
CB
1
CB
2
FAAH AMT
15
H 3-CH
2
(CH
2
)
13
CH
3
5 CH
2
CH
2
OH n.t. n.t. n.a.
>
25
16
H 3-CH
2
(CH
2
)
13
CH
3
5
c
.C
3
H
5
n.t. n.t. n.a. n.t.
17
H 3-CH
2
(CH
2
)
13
CH
3
5
p
.OH-C
6
H
4
n.t. n.t. n.a.
>
25
18
H 3-CH
2
(CH
2
)
13
CH
3
10 CH
2
CH
2
OH n.t. n.t. n.t. n.t.
19
H 3-CH
2
(CH
2
)
13
CH
3
10
c
.C
3
H
5
n.t. n.t. n.t. n.t.
20
H 3-CH
2
(CH
2
)
13
CH
3
10
p
.OH-C
6
H
4
n.t. n.t. n.t. n.t.
21
3-OH 5-CH
2
(CH
2
)
3
CH
3
5 CH
2
CH
2
OH n.t. n.a n.a. n.t.
22
3-OH 5-CH
2
(CH
2
)
3
CH
3
5
c
.C
3
H
5
1.3
0.96
n.a n.t.
23
3-OH 5-CH
2
(CH
2
)
3
CH
3
5
p
.OH-C
6
H
4
n.t. n.t. n.a. n.t.
24
3-OH 5-CH
2
(CH
2
)
3
CH
3
10 CH
2
CH
2
OH
0.8 0.16
n.a 25
25
3-OH 5-CH
2
(CH
2
)
3
CH
3
10
c
.C
3
H
5
0.0052 0.013
n.a 17
26
3-OH 5-CH
2
(CH
2
)
3
CH
3
10
p
.OH-C
6
H
4
3 1.4 n.a. n.a.
27
3-OH 5-CH
2
(CH
2
)
3
CH
3
15 CH
2
CH
2
OH 12.5 n.a. n.t. n.t.
28
3-OH 5-CH
2
(CH
2
)
3
CH
3
15
c
.C
3
H
5
>
10 n.a. n.t. n.t.
29
3-OH 5-CH
2
(CH
2
)
3
CH
3
15
p
.OH-C
6
H
4
>
10 n.a. n.t. n.t.
30
3-OH H 5 CH
2
CH
2
OH n.t. n.t. n.a. n.t.
31
3-OH H 5
c
.C
3
H
5
n.t. n.t. n.a. n.t.
32
3-OH H 5
p
.OH-C
6
H
4
n.t. n.t. n.t. n.t.
33
3-OH H 10 CH
2
CH
2
OH
>
10 n.a. n.a. n.t.
34
3-OH H 10
c
.C
3
H
5
>
10 5.4 n.a. n.t.
35
3-OH H 10
p
.OH-C
6
H
4
n.a.
>
10 n.t. n.t.
36
3-OH H 15 CH
2
CH
2
OH
>
10 n.a. n.t. n.t.
37
3-OH H 15
c
.C
3
H
5
4.25 n.a. n.t. n.t.
38
3-OH H 15
p
.OH-C
6
H
4
5 10 n.t. n.t.
39
3-OH 4-CH
2
(CH
2
)
4
CH
3
5 CH
2
CH
2
OH n.t. 6.43 n.a. n.t.
40
3-OH 4-CH
2
(CH
2
)
4
CH
3
5
c
.C
3
H
5
0.18 0.54
n.a. n.t.
41
3-OH 4-CH
2
(CH
2
)
4
CH
3
5
p
.OH-C
6
H
4
n.t. n.t. n.t. n.t.
42
3-OH 4-CH
2
(CH
2
)
4
CH
3
10 CH
2
CH
2
OH
>
10 2.7 n.a. n.a.
43
3-OH 4-CH
2
(CH
2
)
4
CH
3
10
c
.C
3
H
5
>
10
0.35
n.a.
>
25
44
3-OH 4-CH
2
(CH
2
)
4
CH
3
10
p
.OH-C
6
H
4
n.a.
>
10 50
>
25
45
3-OH 4-CH
2
(CH
2
)
4
CH
3
15 CH
2
CH
2
OH n.a. n.a. n.t. n.t.
46
3-OH 4-CH
2
(CH
2
)
4
CH
3
15
c
.C
3
H
5
n.a. n.a. n.t. n.t.
47
3-OH 4-CH
2
(CH
2
)
4
CH
3
15
p
.OH-C
6
H
4
>
10 n.a. n.t. n.t.
48
5-OH 2-CH
2
(CH
2
)
4
CH
3
5 CH
2
CH
2
OH n.t.
>
10 n.a. n.t.
49
5-OH 2-CH
2
(CH
2
)
4
CH
3
5
c
.C
3
H
5
n.t. 3.57 n.a. n.t.
50
5-OH 2-CH
2
(CH
2
)
4
CH
3
5
p
.OH-C
6
H
4
n.t. n.t. n.t. n.t.
51
5-OH 2-CH
2
(CH
2
)
4
CH
3
10 CH
2
CH
2
OH 1.13
0.42
n.a.
>
25
52
5-OH 2-CH
2
(CH
2
)
4
CH
3
10
c
.C
3
H
5
0.21 0.03
>
25 13
53
5-OH 2-CH
2
(CH
2
)
4
CH
3
10
p
.OH-C
6
H
4
3.3 2.11 18.0
>
25
54
5-OH 2-CH
2
(CH
2
)
4
CH
3
15 CH
2
CH
2
OH n.a n.a. n.t. n.t.
55
5-OH 2-CH
2
(CH
2
)
4
CH
3
15
c
.C
3
H
5
5.5 2.49 n.t. n.t.
56
5-OH 2-CH
2
(CH
2
)
4
CH
3
15
p
.OH-C
6
H
4
3 n.a. n.t. n.t.anandamide 72
(
3.1 n M -WIN, 55
-
212 21
(
1.1 nM 2.1
(
0.1 nMHU-210 - 0.15
(
0.03
a
Data are means
(
SEM of 
n
)
3 separate experiments and are expressed as
i
(
 µ
M), for CB
1
and CB
2
binding assays, and in IC
50
(
 µ
M) for FAAH and AMT assays. Reference compounds were tested under the same conditions in this study. Anandamide was tested inthe presence of PMSF (100 nM). n.a.
)
IC
50
>
10 in the preliminary screening carried out with rat brain and spleen membranes; n.t.
)
not tested; insol.
)
insoluble in any of the solvents normally used in binding assays (DMSO, ethanol, or methanol). Binding affinitycostants of the most potent compounds (
 K 
i
e
1
µ
M) are highlighted in bold.
 Potent Ligands of Cannabinoid Receptors Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
7345
   D  o  w  n   l  o  a   d  e   d   b  y   U   N   I   V   O   F   M   I   C   H   I   G   A   N  o  n   O  c   t  o   b  e  r   3   0 ,   2   0   0   9   |   h   t   t  p  :   /   /  p  u   b  s .  a  c  s .  o  r  g    P  u   b   l   i  c  a   t   i  o  n   D  a   t  e   (   W  e   b   )  :   O  c   t  o   b  e  r   1   3 ,   2   0   0   5   |   d  o   i  :   1   0 .   1   0   2   1   /   j  m   0   5   0   1   5   3   3

You're Reading a Free Preview

Download
scribd
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->