Synthesis and CB1 receptor activities of novel arachidonyl alcohol derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.03.093

Novel derivatives of arachidonyl alcohol were synthesized and evaluated for their CB1 receptor activity by [³⁵S]GTP_γS assay using rat cerebellar membranes.

Full text of DOI:10.1016/j.bmcl.2004.03.093

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3231–3234
Synthesis and CB1 receptor activities of novel arachidonyl alcohol
derivatives
Teija Parkkari,a,* Juha R. Savinainen,b Anu L. Rauhala,a Tiina L. Tolonen,a
a
Tapio Nevalainen, Jarmo T. Laitinen, Jukka Gynther and Tomi Jarvinen
b
a
a
€
aDepartment of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland
bDepartment of Physiology, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland
Received 8September 2003; revised 29 March 2004; accepted 30 March 2004
Abstract—Novel derivatives of arachidonyl alcohol were synthesized and evaluated for their CB1 receptor activity by [35S]GTPcS
assay using rat cerebellar membranes.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
the linkage between the polar head and the lipophilic
tail. With AEA some progress has been made by
methylating postitions C-102;3 and C-2,4 and using
reversed amide linkage.2 The replacement of an ester
linkage of 2-AG with an ether linkage (HU-310, here-
after defined as third endogenous cannabinoid,5
although this has been recently disputed6), or with a
ketogroup,7 has improved stability, but efficacy
decreases considerably compared to 2-AG.1
Since the discovery of D9-THC, remarkable progress has
been made in the area of CB1 receptor ligands. CB1
receptor agonists are endogenous, exogenous or syn-
thetic compounds which are traditionally divided into
four major groups according to their chemical structure:
(1) classical cannabinoids, like D9-THC, (2) nonclassical
cannabinoids developed by Pfizer (e.g., CP 55,940), (3)
compounds similar to endogenous cannabinoids called
eicosanoids and (4) finally aminoalkylindoles developed
by the Sterling Winthrop research team (e.g., WIN55,
212-2).
The aim of the study was to synthesize ester, carbonate
and carbamate derivatives of arachidonyl alcohol in an
effort to achieve three main goals; firstly, the CB1
receptor activities for derivatives of arachidonyl alcohol
have not been previously investigated, secondly, carba-
mate, carbonate and reversed ester linkages were
developed for possible improved enzymatic stability,
and finally, to achieve the liberation of arachidonyl
alcohol instead of arachidonic acid after enzymatic or
chemical hydrolysis of the derivative. In the present
study we describe the syntheses of these compounds and
their CB1 receptor activities.
The most potent CB1 receptor ligands are synthetic and
belong to the group of classical cannabinois. The
endogenous cannabinoid, 2-arachidonoyl glycerol (2-
AG), is not as potent, but according to the [35S]GTPcS
activation studies, it is the most efficient CB1 agonist so
far.1 The high efficacy of 2-AG provides interest in the
molecular structure of eicosanoids, which was also the
starting point of this study.
The endogenous cannabinoids, arachidonoyl ethanol
amide (AEA) and 2-AG, are metabolically labile mole-
cules, and several attempts have been made to stabilize
2. Synthesis
The syntheses of the arachidonyl alcohol esters (Fig. 1,
compounds 1–7) are outlined in Scheme 1. Preparation
of the esters of arachidonyl alcohol turned out to be
difficult due to self-degradation and polymerization.
Compounds 1–5 were synthesized as follows (Scheme 1).
Keywords: Cannabinoid; CB1 receptor agonist; Synthesis; [35S]GTPcS
activation.
* Corresponding author. Tel.: +358-17-162431; fax: +358-17-162456;
e-mail: teija.parkkari@uku.fi
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.093

3232
T. Parkkari et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3231–3234
O
O
O
O
OH
OH
OH
O
O
O
O
O
OH
OH
O
OH
7
8
10
11
1
4a
4b
O
O
O
O
OH
O
O
O
OH
O
N
H
OH
2
O
O
O
O
OH
OH
O
O
H
O
O
OH
O
O
N
H
3a
9a
9b
5
6
O
O
OH
OH
N
H
O
OH
3b
Figure 1. Chemical structures of synthesized molecules.
O
O
O
HO
HO
O
O
O
O
OH
OH
O
O
OH
HO
O
O
O
OH
Cl
TBDMS
TBDPS
HO
O
a, b
c
26
l, m
16
25
15
1
l
O
O
OH
O
O
TBDPS
O
O
HO
O
O
O
OH
d, e, f
j, k
HO
O
4a, b
HO
O
O
27
18
17
+
28
O
O
O
OH
TBDPS
O
OH
O
OH
HO
O
g, h
R
j, k
R
R
k, n
k, n
2
R=H
19
R=H
21
R=H
3a, b R=Me
20a, b R=Me
22a, b R=Me
O
O
OH
OH
OH
OH
O
O
O
O
O
TBDMS
OH
O
HO
OH
HO
O
i
j, k
6
7
5
23
24
Scheme 1. (a) TBDMSCl, imidazole, DMF, 63%; (b) 2 M oxalyl chloride, DCM, 84%; (c) arachidonyl alcohol, Et3N, DCM, 84%; (d) 1. NaOH, 2.
HCl, 3. BnBr, 60%; (e) TBDPSCl, imidazole, THF, 100%; (f) Pd/C, H2, 88%; (g) TBDPSCl, THF, 76%; (h) LiOH, 83%; (i) 1. TBDMSCl, imidazole,
THF, 71%, 2. THF/H2O, 48%; (j) arachidonyl alcohol, DCC, DMAP, 64–95%; (k) Bu4NþFÀ, 30–66%; (l) benzaldehyde, toluene, p-TSA, 51–82%;
(m) 1. KOH, 2. HCl, 3. Et3N, D, 31%; (n) HCl/MeOH, 74%.
Compound 15 was first treated with t-butyldimethylsilyl
chloride (TBDMSCl), followed by acid chloride for-
mation using oxalyl chloride.8 Activated and protected
16 was attached to arachidonyl alcohol under basic
conditions.9 Preparation of 4a and 4b began with
enantiomerically pure starting materials.10–12 Compound
17 was first unprotected under basic conditions, fol-
lowed by protection of carboxylic acid group with
benzyl (Bn)13 and then hydroxyl groups with t-butyl-
diphenylsilyl group (TBDPS).14 Benzyl group was
removed by catalytic hydrogenation,13 and compound
18 was coupled with arachidonyl alcohol. The syntheses
of compounds 3a and 3b also began from enantiomeri-
cally pure compounds. Compounds 19 and 20 were
protected with TBDPS and then hydrolyzed under basic
conditions.14
centrated HCl/MeOH (1:1) solution, leading to the final
products 6 and 7.
The syntheses of 8–11 are outlined in Scheme 2. Car-
bamates 8, 9a and 9b were prepared by attaching p-
nitrophenylchlorofomate activated arachidonyl alcohol
to aminoalcohol under basic condition.17;18 The p-nitro-
phenylchloroformate activation was also used in and the
preparation of carbonates 10 and 11. However, in this
case the carboxylic acid residue was activated and at-
tached to arachidonyl alcohol.
3. In vitro studies
Compounds 21 and 22 were coupled with arachidonyl
alcohol using N,N0-dicyclohexylcarbodiimide (DCC)
coupling. All silyl protected intermediates were depro-
tected using Bu4NþFÀ in THF, leading to the final
products 1–5.15 The synthetic pathways for compounds
6 and 7 are presented in Scheme 1. The hydroxyl groups
of 25 and 26 were protected with benzylidene acetal.
After protection, 26 was decarboxylated, leading to 28.16
Compounds 27 and 28 were attached to arachidonyl
alcohol with DCC coupling and deprotected with con-
Maximal responses (Emax, % basal) to all compounds, as
well as their CB1 receptor-dependent activity, were
determined by [35S]GTPcS-binding studies. These stud-
ies were conducted using four-week-old male Wistar
rats. All animal experiments were approved by the local
ethics committee. The animals lived in a 12-h light/12-h
dark cycle (lights on at 07:00 h) with water and food
available ad libitum. The rats were decapitated 8h after
lights on (15:00 h), whole brains were removed, dipped
in isopentane on dry ice and stored at )80 ꢁC. Rat

T. Parkkari et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3231–3234
3233
O
O
R
c
+
NO2
OH
OH
O
O
OH
+
O2N
O2N
O
Cl
H2N
30
d
a
b
O
R
O
OH
O
N
H
+
O
R
8, 9a, 9b
R = Me or H
31 R=OPr
32 R=OEt
O
R
O
O
10, 11
R = Et or Pr
Scheme 2. (a) Ethanol/propanol, pyridine, THF, 77–88%; (b) DMAP, DCM, 77–91%; (c) pyridine, THF, 87%; (d) Et3N, THF, 80–100%.
cerebellar membranes were prepared as previously
described.1 Incubations using PMSF-pretreated (1 mM)
rat cerebellar membranes were carried out essentially as
previously described.1 For agonist dose–response and
antagonist experiments, results are presented as mean-
SEM of at least three independent experiments, per-
formed in duplicate. Data analysis for dose–response
curves were calculated as nonlinear regressions by
GraphPad Prism 3.0 for Windows.
[35S]GTPcS binding, or the activity achieved was not
attenuated by 1 lM AM251 (data not shown).
Acknowledgements
The authors are grateful to Mrs Miia Reponen and Mrs
Tiina Koivunen for their skilful technical help. The
study was supported by a grant from the National
Technology Agency of Finland.
4. Results and discussion
Among all the ligands synthesized, only compounds 4a
and 4b showed dose-dependent CB1 activity (Table 1
and Fig. 2). For both of these compounds, responses at
5 · 10À5 M were reversed by CB1 receptor antagonist,
AM251 (1 lM), to 109 2 and 115 6 (% basal SEM,
n ¼ 2), respectively. However, both efficacy and potency
values of 4a and 4b were weaker than those of AEA
(Table 1). Other compounds did not stimulate
References and notes
€
1. Savinainen, J. R.; Jarvinen, T.; Laine, K.; Laitinen, J. T.
Br. J. Pharmacol. 2001, 134, 664.
2. Abadji, V.; Lin, S.; Taha, G.; Griffin, G.; Stevenson, L. A.;
Pertwee, R. G.; Makriyannis, A. J. Med. Chem. 1994, 37,
1889.
3. Lin, S.; Khanolkar, A. D.; Fan, P.; Goutopoulos, A.; Qin,
C.; Papahadjis, D.; Makriyannis, A. J. Med. Chem. 1998,
41, 5353.
Table 1. Comparison of efficacy (Emax) and potency (À log EC50) of 4a
and 4b and AEA
4. Adams, I. B.; Ryan, W.; Singer, M.; Razdan, R. K.;
Compton, D. R.; Martin, B. R. Life Sci. 1995, 56, 2041.
^
5. Hanus, L.; Abu-Lafi, S.; Fride, E.; Breuer, A.; Vogel, Z.;
Compound
CB1 activation
Shalev, D. E.; Kustanovich, I.; Mechoulam, R. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 3662.
6. Oka, S.; Tsuchie, A.; Tokumura, A.; Muramatsu, M.;
Suhara, Y.; Takayama, H.; Waku, K.; Sugiura, T.
J. Neurochem. 2003, 85, 1374.
Emax (% basal SE)
ÀLogEC50 ꢀ SE
4.4 0.2
4a
4b
278 21
301 84.6 0.1
AEA
380
6
4.8 0.0
7. Suhara, Y.; Nakane, S.; Arai, S.; Takayama, H.; Waku,
K.; Ishima, Y.; Sugiura, T. Bioorg. Med. Chem. Lett. 2001,
11, 1985.
8. Glabe, A. R.; Sturgeon, K. L.; Ghizzoni, S. B.; Musker,
W. K.; Takahashi, J. N. J. Org. Chem. 1996, 61, 7212.
9. Gu, L.; Dunn, J.; Dvorak, C. Drug Dev. Ind. Pharm. 1989,
15, 209.
4a
4b
AEA
10. (R)-2,3-Di-[(t-butyldiphenylsilyl)oxy] propionic acid (18).
Methyl-(R)-2,2-dimethyl-1,3-dioxalane-4-carboxylate (17)
(47 mg, 2.93 mmol) was dissolved in THF/H2O (150 mL/
85 mL), and the solution was cooled on an ice bath. 2 M
NaOH (2.6 mL) was added slowly. After the addition, the
cooling bath was removed and stirring was continued for
1 h. The reaction mixture was acidified with 2 M HCl and
stirred at rt for 2 h. The solvent was evaporated. The
residue was dissolved in MeOH/H2O (5 mL/0.5 mL), and
the solution was neutralized with 20% NaHCO3. The
solvent was evaporated. The residue was dissolved in dry
DMF, and benzylbromide (0.38mL, 3.22 mmol) was
added. The reaction mixture was stirred under Ar at rt
for 2 days. The solvent was evaporated, the residue was
100
-7
-6
-5
-4
log[ligand]
Figure 2. Dose–response curves for 4a and 4b and AEA for G-protein
binding in rat cerebellar membranes (mean SEM, n ¼ 3).

3234
T. Parkkari et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3231–3234
dissolved in EtOAc and washed with brine. The organic
solution of (R)-2,3-di-[(t-butyldiphenyl-silyl)oxy] propi-
onic acid icosa-5,8,11,14-tetraenyl ester (170 mg,
0.20 mmol) and 1 M Bu4NþFÀ (300 lL) in dry THF
(3 mL) was stirred under Ar at rt for 2 h. THF was
evaporated, and the crude product was purified by flash
chromatography, eluting with petroleum ether/EtOAC
3:1. Evaporation of solvents yielded 59 mg of an oily
layer was dried over Na2SO4, filtered and evaporated. The
crude product was purified by flash chromatography,
eluting with EtOAc/petroleum ether 3:1. Yield of the
colourless oil was 340 mg (60%). Rf ¼ 0:46 (EtOAc/petro-
leum ether 4:1). 1H NMR (CDCl3): 7.40–7.33 (m, 5H),
5.29–5.24 (m, 2H), 4.31 (t, J ¼ 3:5 Hz, 1H), 3.93–3.86 (m,
2H). The mixture of benzyl-((R)-2,3-dihydroxy) prop-
ionate (340 mg, 1.73 mmol), TBDMSCl (0.98mL,
3.81 mmol) and imidazole (366 mg, 5.37 mmol) in DMF
(7 mL) was stirred under Ar at rt for 5 h. The solvent was
evaporated, and the residue was dried in vacuo. The
reaction proceeded quantitively. Rf ¼ 0:70 (EtOAc/petro-
leum ether 1:4). 1H NMR (CDCl3): 7.76–7.74 (m, 1H),
7.66–7.57 (m, 8H), 7.41–7.27 (m, 14H), 7.18–7.16 (m, 2H),
4.94 (d, J ¼ 12:3 Hz, 1H), 4.90 (d, J ¼ 12:3 Hz, 1H), 4.37
(t, J ¼ 4:3 Hz, 1H), 3.93 (dd, J ¼ 4:5 Hz, 10.2 Hz, 1H),
3.86 (dd, J ¼ 4:4 Hz, 10.3 Hz, 1H), 1.07 (s, 9H), 1.00 (s,
9H). Benzyl-{(R)-2,3-di-[(t-butyldiphenylsilyl)oxy]} prop-
ionate (1.16 g, 1.73 mmol) was dissolved in dry THF
(10 mL) and Pd/C (941 mg) was added. The reaction
mixture was stirred under H2-balloon for 3 days. The
catalyst was filtered, and the solvent was evaporated. The
crude product was purified with flash chromatography,
eluting with petroleum ether/EtOAc 8:1. Evaporation of
solvents yielded colourless oil (890 mg, 88%). Rf ¼ 0:29
(petroleum ether/EtOAc 4:1). 1H NMR (CDCl3): 7.74–
7.72 (m, 1H), 7.65–7.58(m, 8H), 7.45–7.28(m, 11H), 4.32
(t, J ¼ 4:3 Hz, 1H), 3.88 (dd, J ¼ 3:1 Hz, 10.7 Hz, 1H),
3.64 (dd, J ¼ 3:1 Hz, 10.7 Hz, 1H), 1.14 (s, 9H), 1.06 (s,
9H).
1
product (78%). H NMR (CDCl3): d 5.43–5.31 (m, 8H),
4.26 (br, 1H), 4.23 (t, J ¼ 6:6 Hz, 2H), 3.87 (dd, 2H), 3.23
(br, 1H), 2.85–2.80 (m, 6H), 2.25 (br, 1H), 2.11 (q,
J ¼ 7:1 Hz, 2H), 2.06 (q, J ¼ 7:1 Hz, 2H), 1.70 (qui,
J ¼ 6:8Hz, 2H), 1.44 (qui, J ¼ 7:7 Hz, 2H), 1.39–1.26 (m,
6H), 0.89 (t, J ¼ 6:8Hz, 3H). 13C NMR (CDCl3): d 173.1,
130.5, 129.4, 128.6, 128.5, 128.2, 128.2, 127.9, 127.5, 71.5,
66.1, 64.1, 31.5, 29.3, 28.1, 27.2, 26.7, 26.6, 25.7 (3C), 22.6,
14.1. Rf ¼ 0:11 (petroleum ether/EtOAc 3:1). Elemental
analysis. Calculated for C23H38O4*1/10H2O: C 72.63%; H
10.12%. Found C 72.49%, H 10.18%.
12. (S)-2,3-Dihydroxy-propionic acid icosa-5,8,11,14-tetrae-
nyl ester (4b). The synthesis was similar to the synthesis
of compound 4a. 1H NMR (CDCl3): d 5.43–5.31 (m, 8H),
4.26 (br, 1H), 4.23 (t, J ¼ 6:6 Hz, 2H), 3.86 (dd, 2H), 2.85–
2.80 (m, 6H), 2.25 (br, 1H), 2.11 (q, J ¼ 7:2 Hz, 2H), 2.06
(q, J ¼ 7:2 Hz, 2H), 1.70 (qui, J ¼ 6:7 Hz, 2H), 1.44 (qui,
J ¼ 7:6 Hz, 2H), 1.39–1.26 (m, 6H), 0.89 (t, J ¼ 6:9 Hz,
3H). 13C NMR (CDCl3): d 173.1, 130.5, 129.4, 128.6,
128.5, 128.2, 128.2, 127.9, 127.5, 71.6, 66.1, 64.1, 31.5,
29.3, 28.1, 27.2, 26.7, 26.6, 25.8, 25.7 (2C), 22.6, 14.1.
Rf ¼ 0:11 (petroleum ether/EtOAc 3:1). ESI-MS 400.3
(M+Na). Elemental analysis. Calculated for C23H38O4*1/
10H2O: C 72.63%; H 10.12%. Found C 72.30%, H
10.30%.
13. Shin, I.; Lee, M.-R.; Lee, J.; Jung, M.; Lee, W.; Yoon, J.
J. Org. Chem. 2000, 65, 7667.
14. Oliver, J. E.; Doss, R. P.; Williamson, T. R.; Carney, J. R.;
DeVilbiss, E. D. Tetrahedron 2000, 56, 7633.
15. Lal, B.; Gangopadhyay, A. K.; Rajagopalan, R.; Ghate,
A. V. Bioorg. Med. Chem. 1998, 6, 2061.
16. Scriba, G. K. E. Arch. Pharm. 1993, 326, 477.
17. Andrews, D. M.; Cherry, P. C.; Humber, D. C.; Jones,
P. S.; Keeling, S. P.; Martin, P. F.; Shaw, C. D.; Swanson,
S. Eur. J. Med. Chem. 1999, 34, 563.
11. (R)-2,3-Dihydroxy-propionic acid icosa-5,8,11,14-tetrae-
nyl ester (4a). (R)-2,3-Di-[(t-butyldiphenyl-silyl)oxy] pro-
pionic acid (18) (408mg, 0.71 mmol), was dissolved in dry
DCM (10 mL) and arachidonyl alcohol (172 mg,
0.59 mmol), DCC (244 mg, 1.18mmol) and DMAP
(43 mg, 0.35 mmol) were added. The reaction mixture
was stirred under Ar at rt over night. The white precipitate
was filtered, and the filtrate was washed with 10% citric
acid, 5% NaHCO3 and water. The combined organic
layers were dried over Na2SO4, filtered and evaporated.
The crude product was purified by flash chromatography,
eluting with 0–4% EtOAc in petroleum ether. Evaporation
of solvents yielded a colourless oil (320 mg, 57%). The
18. Hay, M. P.; Wilson, W. R.; Denny, W. A. Tetrahedron
2000, 56, 645.