Hemisynthesis and preliminary evaluation of novel endocannabinoid analogues

Article abstract of DOI:10.1016/S0960-894X(03)00348-2

Three new endocannabinoid analogues in which amide moiety was replaced either by oxomethylene group or ester moiety with simultaneous substitution of both α-hydrogens with methyl groups were synthesized and their abilities to interact with CB1-receptor and FAAH were investigated.

Full text of DOI:10.1016/S0960-894X(03)00348-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1977–1980
Hemisynthesis and Preliminary Evaluation of Novel
Endocannabinoid Analogues
Siham El Fangour,a Laurence Balas,a,* Jean-Claude Rossi,a Andrey Fedenyuk,b
Natalia Gretskaya,b Mikhail Bobrov,b Vladimir Bezuglov,b Cecilia J. Hillardc
and Thierry Duranda
a
´
UMR CNRS 5074, Faculte de Pharmacie, 15 av. C. Flahault, BP 14491, F-34093 Montpellier Cedex 5, France
bShemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10 Miklukho-Maklaya str., 117437 Moscow, Russia
cDepartment of Pharmacology and Toxicology, Medicinal College of Wisconsin, Milwaukee, WI 53226, USA
Received 13 January 2003; revised 18 March 2003; accepted 2 April 2003
Abstract—Three new endocannabinoid analogues in which amide moiety was replaced either by oxomethylene group or ester
moiety with simultaneous substitution of both a-hydrogens with methyl groups were synthesized and their abilities to interact with
CB1-receptor and FAAH were investigated.
# 2003 Elsevier Science Ltd. All rights reserved.
Endocannabinoids1 are endogenous ligands of cannabi-
noid receptors which, interestingly are less psychoactive
than Á9-tetrahydrocannabinol (Á9-THC), the well-
known active component of marijuana. Thus, since the
discovery in 1992 of the first endocannabinoid, named
anandamide2 1, research on cannabimimetic drugs took
an important revival of interest.
Acting on cannabinoid (CB1/CB2) receptors, endo-
cannabinoids have been shown to influence the activity
of nervous, vascular and immune systems. Thus, interest
in endocannabinoids as therapeutic agents10,11 has led
us to design novel anandamide analogues that might be
resistant to hydrolases degradation.
Various structure–affinity and structure–activities rela-
tionship studies for the CB1 receptor,12 the anandamide
transporter13,14 and the fatty acid amide hydrolase
(FAAH) have been reported.15,16
To date, several new families of endocannabinoids were
identified in central and peripherical nervous systems:
while two of them (2-AG3 2 and virodhamine4) are
arachidonate esters of glycerol and ethanolamine,
respectively, an ether linkage confers a higher metabolic
stability to noladin ether 3.5 In addition, among the
amide family containing anandamide 1 as a leader,
oleamide (amide of oleic acid) that accumulates in the
cerebrospinal fluid under conditions of sleep depriva-
tion and induces physiological sleep in animals,6 could
be referred as a prototypical member of this family.
Several aminoacid derivatives: AA-Gly, AA-GABA and
AA-Ala with analgesic activity were recently found in
bovine and rat brains.7 Besides, cannabimimetic arachi-
donoyldopamine,8 which was recently proposed to be
endogenous activator of vanilloid receptor,9 sig-
nificantly expands the physiological role of the men-
tioned amide family.
Thus, numerous endocannabinoid analogues were syn-
thesized as tools to probe the influence of the lipid
chain, the importance of the carbonyl function and the
tolerance to steric hindrance on the polar head moiety.
Modifications of the amide linkage were also investi-
gated: esters,17,14 carbamates,18 ureas,18 ethers,18 meth-
ylene-linked, ketone19 and amine20 derivatives as either
anandamide or 2-AGanalogues.
Keeping in mind all the above data, we based our
investigations on template of arachidonoyl-ethylenegly-
col 4 (AA-EG). Thus, we report herein the synthesis of
the novel arachidonyl derivatives 6 and 5 together with
its linoleyl derivative 9.
AA-EG 4, firstly prepared21 as 2-AGanalogue, pos-
sesses true cannabimimetic properties22 and can be
regarded as ‘promiscuous’ analogue of both ananda-
*Corresponding author. Tel.:+33-4-67-54-86-24; fax:+33-4-67-54-86-
25; e-mail: balas@pharma.univ-montp1.fr
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00348-2

1978
S. El Fangour et al. / Bioorg. Med. Chem. Lett. 13(2003) 1977–1980
mide 1, in which amide moiety has been replaced with
ester functionality, or 2-AG 2, simplified by replacement
of one hydroxymethylene group with hydrogen.
Second, we envisioned to prevent the analogues from
hydrolase degradation by replacement of the CO–NH
bond of anandamide 1 by a CO–CH2 linkage. Sugiura
et al. have already published19 the total synthesis of
keto-analogue 8 of 2-AG. We thought that hemisynth-
esis would offer a more concise route to ketone analo-
gues 5 than the one presented by Sugiura et al.19 (10
steps) for preparation of compound 8.
Investigations towards the synthesis of the ketone deri-
vatives were performed using linoleic acid 13 prior to
apply our best results to arachidonic acid 10, much
more expensive.
Two approaches were considered for the carbon-carbon
bond formation (Scheme 2): Pathway A relies upon
organozinc cross-coupling to acid chloride 14 while
pathway B is based on carbanion condensation to the
Weinreb amide 15. Thus, both strategies required the
same commercially available starting material that is,
3-bromo-1-propanol 16 and the desired free poly-
unsaturated fatty acid. The choice of the TBDPS pro-
tection was guided by its readily UV detection to
monitor both organozinc and carbanion intermediates
formation.
First, we decided to protect the ester bond of AA-EG 4
by steric hindrance at the a-position to the carbonyl
functionality created by two methyl groups as in analo-
gue 6. Such a strategy was proved to be successfully
efficient on anandamide 1 leading to structure 7.23
The synthesis of analogue 624 was afforded, in 62%
yield,25 by coupling a,a-dimethyl arachidonic acid 11 with
ethylene glycol via intermediate acyl fluoride 12 according
to our published procedure.22 Acid 11 was synthesized
from arachidonic acid 10 by esterification, followed by
two runs of known23 condensation of methyliodide to
a-lithium derivative and then saponification (Scheme 1).
Pathway A is highlighted in Scheme 3. Thus, O-silyl-
ation of bromopropanol 16, followed by Finkelstein
halogen-exchange reaction produced 3-iodopropyl tert-
butyldiphenylsilyl ether 17 in 98% yield.
Unfortunately, all our initial efforts to carry out palla-
dium cross-coupling reaction between the crude lino-
leoyl chloride 14 (0.7 mmol) and organozinc
intermediate 18 (according to Tamaru et al. proce-
dure26) afforded ketone 19 in less than 30% yield. Sur-
prisingly, such bad yields were also obtained with
freshly purified methyl 5-chloro 5-oxovalerate and
methyl 4-chloro-4-oxobutyrate as models. It was subse-
quently found that efficiency in the preparation of this
ketone could be nicely improved when carrying out the
chemistry on larger scale. Effectively, the synthesis of
ketone 19 reached 65% yield using 5.0 mmol of linoleic
acid 13.
Scheme 1. Synthesis of the dimethyl ester analogue 6: (a) CH2N2,
MeOH, 0 ꢀC, 20 min, 100%; (b) LDA, THF, ꢁ80 ꢀC, 1 h then MeI,
ꢁ70 to ꢁ30 ꢀC, 1.5 h followed by LDA, THF, ꢁ80 ꢀC, 1 h then MeI,
ꢁ70 to ꢁ30 ꢀC, 1.5 h, 77%; (c) NaOH, THF, MeOH, 50 ꢀC, 7.5 h,
98%; (d) cyanuric fluoride, Py, MeCN, 23 ꢀC, 1 h; (e) ethylene glycol,
DMAP, MeCN, 23 ꢀC, 18 h, 62% from 11.
In our hands, such a molar size dependence is therefore
financially cumbersome for arachidonic acid.
Scheme 2. Retrosynthetic analysis.

S. El Fangour et al. / Bioorg. Med. Chem. Lett. 13(2003) 1977–1980
1979
Scheme 3. Synthesis of linoleyl-ketone 9.
Abilities of novel compounds 5, 6, and 9 to interact
with CB1-receptor and FAAH were then investigated
using rat forebrain membranes according to Jarrahian
et al. procedure.13 The results are summarized in
Table 1.
Scheme 4. Synthesis of arachidonyl-ketone 5: (a) iBuOCOCl, MeCN,
NMM, 0 ꢀC, 40 min then HCl. HN(OMe)Me, ꢁ15 ꢀC, 82%; (b) gen-
densation: Et2O, ꢁ78–0 ꢀC, 15 min; (c) TBAF, THF, 96%.
eration of carbanion 20: BuLi, 17, Et2O, ꢁ80 to ꢁ60 ꢀC, then con-
t
Arachidonyl-ketone 5 exhibits a moderate affinity for
the CB1 receptor with a Ki value 4-fold higher than that
of anandamide 1, while linoleyl ketone 9 and both esters
4 and 6 were inactive.
Thus, the difficulties encountered with pathway A
prompted us to examine pathway B. This later strategy
is outlined in Scheme 3.
Moreover, compound 6 displays an interesting inhibi-
tory effect on the FAAH activity (very close to ananda-
mide 1), while the presence of the keto-compounds 5
and 9 does not influence it.
Linoleic acid 13 was readily converted to the Weinreb
amide 15 using the mixed anhydride method.8 Thus,
treatement of the fatty acid with isobutyl chloroformate
followed by N,O-dimethylamine and N-methylmorpho-
line furnished the corresponding amide 15 in excellent
yield. Condensation of the lithiated carbanion 20 to the
N-methoxy-N-methylamide 15 provided the expected
ketone 19 in 85% yield. The TBDPS protecting group
of ketone 19 was removed by treatement with TBAF to
produce the expected linoleyl keto-alcohol 9 in 94%
yield.27
As a conclusion, three novel anandamide analogues 6,
5, and 9 were synthesized. The evaluation of their affi-
nity for the CB1 cannabinnoid receptor and FAAH
inhibition was presented as preliminary biological
results. Ability of these three analogues to bind to the
CB2 receptor and the anandamide carrier (ANT) toge-
ther with their pharmacological behavior in the tetrad
test in mice are currently under progress. To argue a
SAR study, the synthesis of other keto-analogues, espe-
cially virodhamine and AA-Gly analogues, is under
investigation too.
Application of pathway B to arachidonic acid 10 pro-
vided target ketone 528 (Scheme 4) in only 3 steps in
overall 27% yield (non optimized).
Table 1. Evaluation of the cannabinoid properties of analogues 5, 6 and 9 in comparison with anandamide 1 and arachidonoylethylene glycol 4
Compd
(1 mM)
% CB1 affinityb
(PMSF)
CB1 affinity
(PMSF) Kia,b
FAAH activityc
FAAH activity
at 10 mM
FAAH
activitya Ki
at 1 mM
1
4
5
6
9
19
83
38
100
72
90 nM (ꢂ32)
>10 mMd
3 mM (ꢂ0.5)
100
91
90
100
70
78
12
100
360 (ꢂ10 nM)
>10 mM
2.1 (ꢂ0.1 mM)
2.1 (ꢂ1 mM)
aValues are means of three experiments, standard deviation is given in parentheses.
bCompetition with [3H]-CP55940 agonist.
cPercentage of anandamide hydrolysis in the presence of tested compounds.
dPMSF is not an effective inhibitor of mono-acyl glycerol lipase and other lipases, possibly responsible for AA-EGhydrolysis.

1980
S. El Fangour et al. / Bioorg. Med. Chem. Lett. 13(2003) 1977–1980
19. Suhara, Y.; Nakane, S.; Arai, S.; Takayama, H.; Waku, K.;
Acknowledgements
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Financial support from CNRS (PICS no. 1582), Rus-
sian Foundation for Basic Research (grant #02-04-
22002) and NIH (grant DA09155) to C.J.H. is gratefully
acknowledged.
22. Bezuglov, V. V.; Bobrov, M.Yu.; Gretskaya, N. M.;
Archakov, A. V.; Serkov, I. V.; Fedenuyk, A. P.; Ver-
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24. Colorless oil; TLC Rf 0.44 (benzene/ethylacetate 4/1); H
1
NMR (CDCl3, 600 MHz) d(ppm): 0.89 (t; 3H, J=7 Hz; H2O);
1.22 (s, 6H, a-CH3); 1.15–1.4 (m, 6H, H17, H18, H19); 1.55–
1.64 (m, 2H, H3); 1.97–2.09 (m, 4H, H4, H16); 2.75–2.87 (m,
6H, H7, H10, H13); 3.82 (br s, 2H, H20); 4.21 (t, 2H, J=5 Hz,
H10); 5.29–5.43 (m, 8H, H5, H6, H8, H9, H11, H12, H14,
H15). 13C NMR (CDCl3, 150 MHz) d (ppm): 14.1 (C20); 22.6
(C4); 23.0 (C19); 25.1 (2 a-Me); 25.5–25.6–27.2–29.3–31.5 (C3,
C7, C10, C13, C16, C17, C18); 40.5 (C2); 61.5 (C20); 66.1
(C10); 127.5–127.9–128.2–128.3–128.6–129.5–130.5 (C5, C6,
C8, C9, C11, C12, C14, C15); 178.2 (C1).
IR n (cmꢁ1): 711; 914; 1137; 1176; 1730 (C¼O); 2857–3012
(–C–H); 3465 (OH). FAB+ MS (NBA): m/z=399 (M+Na)+;
377 (M+H)+; 359 (MH+ꢁH2O).
25. The yield of coupling with free ethyleneglycol never
exceeded 65% regardless acyl fluoride or diisopropyl carbo-
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27. Colorless oil; TLC Rf 0.13 (heptane/ethylacetate: 8/2). H
1
NMR (CDCl3, 400 MHz) d (ppm): 0.89 (t, 3H, H18, JH18-
H17=6.9 Hz); 1.23–1.41 (m, 14H, H4, H5, H6, H7, H15, H16,
H17); 1.53–1.64 (m, 2H, H3); 1.79–1.93 (m, 3H, H20 and
OH); 2.00–2.10 (m, 4H, H8, H14); 2.42 (t, 2H, H2, JH2-
H3=7.30 Hz); 2.55 (t, 2H, H30, JH3 ,H2 =6.9 Hz); 2.77 (t, 2H,
H11, JH11ꢁH10=JH11ꢁH12=6.5 Hz); 3.65 (t, 2H, H10,
0
0
0
0
JH1 ꢁH2 =6.1 Hz); 5.29–5.43 (m, 4H, H9, H10, H12, H13).
13C NMR (CDCl3, 100 MHz) d (ppm): 14.0 (C18); 22.5
(C17); 23.9 (C3); 25.6 (C11); 26.5 (C20); 27.2 (C8, C14); 29.1
(CH2-C); 29.2 (CH2-C); 29.3 (CH2-C); 29.6 (C15); 31.5 (C16);
39.5 (C30); 42.9 (C2); 62.3 (C10); 127.9 (C10 or C12); 128.0
(C12 or C10); 130.0 (C9 or C13); 130.2 (C13 or C9); 211.8
(C¼O).
IR n (cmꢁ1): 722; 914; 1058; 1458; 1711 (C¼O); 2857–3000
(–C–H); 3416 (C–OH). FAB+ MS (NBA): m/z=345
(M+Na)+; 323 (M+H)+; 305 (MH+ꢁH2O).
28. Pale yellow oil; TLC Rf 0.20 (heptane/ethylacetate: 7/3).
1H NMR (CDCl3, 400 MHz) d (ppm): 0.89 (s, 3H, CH3); 1.21–
1.51 (m, 6H, H17, H18, H19); 1.63–1.73 (m, 2H, H3); 1.80–
2.04 (m, 3H, H20, OH); 2,09–2,19 (m, 4H, H4, H16); 2.42 (t,
2H, H2, JH2ꢁH3=7.5 Hz); 2.55 (t, 2H, H30, JH3 ꢁH2 =6.9 Hz);
0
0
2.74–3,.8 (m, 6H, H7, H10, H13); 3.4 (t, 2H, H10,
0
0
JH1 ꢁH2 =6,09 Hz); 5.27–5.53 (m, 8H, H5, H6, H8, H9, H11,
H12, H14, H15).13C NMR (CDCl3, 100 MHz) d (ppm): 14.2
(C20); 22.5 (C19); 23.6 (C3); 25.6 (C7, C10, C13); 27.2 (C4,
C16); 29.3 (C17); 31.2 (C18); 39.5 (C30); 42.2 (C2); 62.3 (C10);
127.5 (CH¼CH); 127.9 (CH¼CH); 128.1 (CH¼CH); 128.2
(CH¼CH); 128.6 (CH¼CH); 128.8 (CH¼CH); 129.2
(CH¼CH); 130.5(CH¼CH); 211.4 (C¼O). IR n (cmꢁ1): 915;
1059; 1111; 1267; 1373; 1455; 1650; 1711 (C¼O); 2857–3000
(C–H); 3012 (¼C–H); 3418 (O–H). FAB+MS (NBA): m/z
=369 (M+Na)+; 347 (M+H)+; 329 (MH+ꢁH2O).