Conformationally constrained analogues of 2-arachidonoylglycerol

Article abstract of DOI:10.1016/j.bmcl.2007.07.064

Novel monocyclic analogues of 2-arachidonoylglycerol (2-AG) were designed in order to explore the pharmacophoric conformations of this endocannabinoid ligand at the key cannabinergic proteins. All 2-arachidonoyl esters of 1,2,3-cyclohexanetriol [meso-7 (A

Full text of DOI:10.1016/j.bmcl.2007.07.064

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5959–5963
Conformationally constrained analogues of 2-arachidonoylglycerol
Subramanian K. Vadivel, Sundararaman Vardarajan, Richard I. Duclos, Jr.,
JodiAnne T. Wood, Jianxin Guo and Alexandros Makriyannis*
Center for Drug Discovery, Northeastern University, 116 Mugar Hall, 360 Huntington Avenue, Boston, MA 02115, USA
Received 15 June 2007; revised 18 July 2007; accepted 19 July 2007
Available online 21 August 2007
Abstract—Novel monocyclic analogues of 2-arachidonoylglycerol (2-AG) were designed in order to explore the pharmacophoric
conformations of this endocannabinoid ligand at the key cannabinergic proteins. All 2-arachidonoyl esters of 1,2,3-cyclohexanetriol
[meso-7 (AM5504), ( )-8 (AM5503), and meso-9 (AM5505)] were synthesized by regioselective acylation of 2,3-dihydroxycyclohexa-
none followed by selective reductions. The optically active isomers (+)-8 (AM4434) and (À)-8 (AM4435) were synthesized from
(2S,3S)- and (2R,3R)-2,3-dihydroxycyclohexanone, respectively, via a chemoenzymatic route. These head group constrained and
conformationally restricted analogues of 2-AG as well as the 1-keto precursors were evaluated as substrates for the endocannabinoid
deactivating hydrolytic enzymes monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH), and also were tested for
their affinities for CB1 and CB2 cannabinoid receptors. The observed biochemical differences between these ligands can help define
the conformational requirements for 2-AG activity at each of the above endocannabinoid protein targets.
Ó 2007 Elsevier Ltd. All rights reserved.
2-Arachidonoylglycerol (2-AG, 1) (Fig. 1) is a monoac-
ylglycerol identified as an endogenous ligand which
binds to both CB1 and CB2 cannabinoid receptors.1,2
The other key endocannabinoid is N-arachidonoyletha-
nolamine (AEA, 2), although it has been postulated that
2-AG is the primary endocannabinoid agonist ligand for
CB13,4 as well as CB25 receptors. For example, 2-AG
binds to the CB1 cannabinoid receptors on presynaptic
axons during the duration of its existence in the extracel-
lular space and functions as a retrograde synaptic neuro-
transmitter,6,7 where it elicits a variety of cannabinergic
effects in vitro and in vivo.8 2-AG is primarily inacti-
vated by an efficient transporter system-mediated cellu-
lar uptake followed by intracellular enzymatic
hydrolysis to arachidonic acid and glycerol by monoac-
ylglycerol lipase (MGL).9–12 In addition to the hydroly-
sis of this metabolically labile molecule by MGL,
hydrolysis by fatty acid amide hydrolase (FAAH),10,12
phosphorylation,13 as well as metabolism by lipoxogen-
ases (LOX)14 and cyclooxygenase 2 (COX 2)15 also oc-
cur, although the biological relevance of these other
mechanisms for 2-AG deactivation has not yet been
fully established. Thus, 2-AG interacts not only with
the CB receptors, but with a transporter system, intra-
cellular MGL and FAAH, as well as other enzymes.
We postulated that the conformations of 2-AG required
for interactions with each of these targets may be differ-
ent and that probing its bioactive conformation in each
target could lead to information useful in the design of
selective inhibitors which have the potential to be thera-
peutic drugs.16–18
Our approach for the design of conformationally de-
fined 2-AG analogues involved constraining the confor-
mation of the glycerol moiety by incorporation of its key
pharmacophoric features into a six-membered carbocy-
clic ring system. This series of analogues includes all
the 2-arachidonoyl esters of 1,2,3-cyclohexanetriol as
well as the corresponding keto analogues. These ligands
were synthesized and assayed for their affinities for the
two known cannabinoid receptors and evaluated as sub-
strates for MGL and FAAH, as we have been particu-
larly interested in identifying the structural features of
the 2-AG molecule which can discriminate between
these two endocannabinoid deactivating enzymes.
As shown in Scheme 1, 2,3-dihydroxycyclohexanone (4)
was an intermediate in the synthesis of all possible iso-
meric 2-AG analogues. Commercially available
cyclohexenone (3) was treated with osmium tetroxide
and N-methylmorpholine N-oxide (NMO) to give the
Keywords: 2-Arachidonoylglycerol, 2-AG; Monoacylglycerol lipase,
MGL; Fatty acid amide hydrolase, FAAH; Cannabinoid,
Endocannabinoid.
*
Corresponding author. Tel.: +1 617 373 4200; fax: +1 617 373
7493; e-mail: a.makriyannis@neu.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.064

5960
S. K. Vadivel et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5959–5963
OH
O
O
OH
OH
O
N
H
2-AG
AEA
2
1
Figure 1. Endocannabinoids 2-arachidonoylglycerol (2-AG, 1) and N-arachidonoylethanolamine (AEA, 2).
O
O
O
O
O
O
O
O
arachidonic acid
EDCI, DMAP
OH
OH
OsO4
NMO
+
OH
OH
AM5501
( )-5
AM5502
( )-6
( )-4
3
NaBH4
NaBH4
OH
OH
O
OH
O
O
O
O
O
OH
OH
OH
AM5503
( )-8
AM5505
meso-9
AM5504
meso-7
Scheme 1. Syntheses of 1,2,3-cyclohexanetriol ester analogues of 2-AG.
racemic ketodiol ( )-4, which was acylated at the more
acidic a-hydroxyl group using arachidonic acid in the
presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide (EDCI) and 4-dimethylaminopyridine (DMAP)
at À20 °C to give ( )-5 (AM5501) and a minor amount
of the epimerized byproduct ( )-6 (AM5502). Both ( )-
5 and ( )-6 were reduced with sodium borohydride in
methanol at 0 °C to give the corresponding 2-AG ana-
logues meso-7 (AM5504), ( )-8 (AM5503), and meso-9
(AM5505). These 2-arachidonoyl esters of 1,2,3-cyclo-
hexanetriol were free of acyl migration byproducts usu-
ally observed for 2-acyl glycerols such as 2-AG4,19–21
and have been characterized.22
ported from an enzymatic method which utilized lipase
from Pseudomonas sp. (SAM-II, Amano).23 We utilized
a different lipase from Candida rugosa (lipase L1754,
Sigma) to selectively hydrolyze one enantiomer of ester
( )-11 and give the corresponding alcohol (+)-12 which
was readily chromatographically separable from the
unreactive enantiomeric ester (À)-11. Both ester (À)-11
([a]D À48.1° (c 1.00, CH3OH); Lit.23 [a]D À41.2°
(c 0.7, CHCl3)) and alcohol (+)-12 ([a]D +10.8° (c 1.00,
CH3OH); Lit.23 [a]D +12.3° (c 0.6, CHCl3)) were ob-
tained in good yields and high optical purities. Ketodiol
(À)-(2R,3R)-4 was then prepared from (À)-11 and con-
verted to (À)-8 (AM4435, [a]D À16.4° (c 1.2, CHCl3)),
while ketodiol (+)-(2S,3S)-4 was prepared from (+)-12
and converted to (+)-8 (AM4434, [a]D +17.0° (c 0.9,
CHCl3)).
Racemic compound ( )-8 (AM5503) was not resolvable
by chiral HPLC (CHIRALPAK AD). Therefore, enzy-
matic resolution of the chiral ketodiol ( )-4 was carried
out (Scheme 2) to give the required crucial intermediates
(À)-4 and (+)-4 in optically active forms. The synthetic
intermediates (À)-11 and (+)-12 were previously re-
Compounds were tested for their affinities for the CB1
and CB2 receptors using membrane preparations from
rat brain or mouse spleen, respectively, as previously de-
O
O
OH
O
O
OH
MeO OMe
lipase from
OH
OH
, H+
1.
O
O
Candida rugosa
O
O
O
O
+
2. CH3CH2CH2COCl,
Et3N, DMAP
phosphate
buffer
meso-10
( )-11
(-)-11
(+)-12
pH 7.2
1. LiOH, THF/H2O
2. Dess-Martin periodinane
3. CH3OH, H+
1. Dess-Martin periodinane
2. CH3OH, H+
(+)-4
(-)-4
Scheme 2. Chemoenzymatic syntheses of optically active starting 2,3-dihydroxycyclohexanones (À)-4 and (+)-4.

S. K. Vadivel et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5959–5963
5961
Table 1. Substrate hydrolysis by FAAH and MGL enzymes with
standard deviations
when the CB1-containing rat brain membrane prepara-
tions were pretreated27 with phenylmethylsulfonyl fluo-
ride (PMSF). All other compounds exhibited CB1 and
CB2 Ki values larger than 1000 nM without substantial
differences except for ( )-5 (AM5501, CB2 Ki 410 nM).
Compound
Name
Substrate assay
(% hydrolysis)
FAAH
MGL
1
2-AG
100
91
0
1
0
3
0
2
3
6
74
4
FAAH and MGL enzymes were partially purified from
adult Sprague–Dawley rat brains purchased from Pel-
Freeze Biologicals according to a previously reported
procedure.31,32 The pellet from the last centrifugation
step (microsomal fraction) was resuspended in 25 mM
Tris–HCl, 5 mM MgCl2, 1 mM EDTA, and pH 7.4
(TME) buffer for the FAAH assay (plus 0.1% BSA),
and the supernatant from the last centrifugation step
(cytosol fraction without BSA) was used for the MGL
assay. All compound stock solutions used were 10 mM
in DMSO, and the chemical stabilities of the compounds
were first checked under the assay conditions (100 lM
substrate) without any added FAAH or MGL enzymes.
To screen compounds as substrates for FAAH, assays
were carried out according to our previously reported
procedures.32,33 Samples (100 lL) were taken at the start
of the assay to obtain the background concentration of
arachidonic acid in the biological sample, and then after
15 min of FAAH hydrolysis. Samples were diluted 1:5
with acetonitrile and centrifuged (20,000g, 5 min, room
temperature) to precipitate the proteins. The resulting
supernatant was analyzed by HPLC to determine the
percentage of compound hydrolyzed in the reaction
time. The assays with cytosolic MGL were carried out
in similar fashion to the FAAH assay described above,
except that TME buffer was used without BSA, the
MGL enzyme preparation used 30 lg of protein, and
the reaction time was 20 min.
( )-5
( )-6
meso-7
( )-8
(+)-8
(À)-8
meso-9
AM5501
AM5502
AM5504
AM5503
AM4434
AM4435
AM5505
48
8
3
3
2
1
3
2
4
100
2
2
100
88
1
10
13
19
88
78
scribed24–27 via competition-equilibrium binding with
[3H]CP55940 as the radioligand. The results were ana-
lyzed using nonlinear regression to determine the actual
IC50 of the ligand (Prizm by GraphPad Software, Inc.)
and the Ki values were calculated from the IC50.28 All
data were in duplicate with IC50 and Ki values deter-
mined from single experiments. This series of rigid 2-
AG analogues had affinities for the CB receptors that
were comparable to the endogenous cannabinoid 2-
AG (Ki CB1 472,1 2400,2 100,20 53829; Ki CB2 1400,1
100,20 110030; review8) even in the presence of the bulky
–(CH2)3– methylenes which were used to constrain the
glycerol headgroup portion of the ligands. For example,
the Ki’s for (+)-8 (AM4434, CB1 970 nM, CB2 370 nM)
and (À)-8 (AM4435, CB1 4200 nM, CB2 1200 nM) were
determined, though some ester hydrolysis did occur dur-
ing the course of the binding assays, as evidenced by the
examples of the CB1 Ki’s for (+)-8 (AM4434) and (À)-8
(AM4435), which were 360 and 770 nM, respectively,
Figure 2. Molecular models for ( )-5 (AM5501), ( )-6 (AM5502), meso-7 (AM5504), the enantiomers (À)-8 (AM4435) and (+)-8 (AM4434), and
meso-9 (AM5505) were obtained on a Silicon Graphics Fuel workstation using Insight II (2000). The O-arachidonoyl groups were modeled in
extended conformations34,35 but are not displayed. All structures were subject to molecular mechanics calculations using the steepest descent method
for the first 1000 iterations, then the conjugate gradient method until the maximum derivative was less than 0.001 kcal/mol. Only for meso-7
(AM5504) was the 2-O-arachidonoyl group found to be axial in the lowest energy conformation (see Fig. 3).

5962
S. K. Vadivel et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5959–5963
These raw data in Table 1 from the enzyme susceptibil-
ity screenings have not been adjusted to reflect the sta-
bility of the substrates in the absence of enzyme,
where (+)-5 (AM5501) showed the most chemical insta-
bility (12% and 11% hydrolyses) and meso-7 (AM5504)
showed the least instability (2% and 1% hydrolyses) un-
der control conditions for FAAH and MGL screenings,
respectively. However, the data clearly indicated that the
stereochemical features of the triol ester headgroup of
some analogues could be used to distinguish the active
sites for ester hydrolysis at the MGL and FAAH en-
zyme active sites. The preferred conformations of the
individual analogues are represented in Figure 2. In gen-
eral, FAAH was very effective in hydrolyzing all equato-
rial arachidonoyl esters. However, with the energy
difference of 2.0 kcal/mol between the axial and equato-
rial conformations (see Fig. 3), meso-7 (cis,cis-7,
AM5504) exists primarily (97% at 25 °C) in a conforma-
tion where the arachidonoyl ester is axial, and it was not
a good substrate for FAAH. The arachidonoyl ester of
meso-9 (trans,trans-9, AM5505) was only somewhat less
susceptible to FAAH hydrolysis than ( )-8 (AM5503),
which represents a conformation of 2-AG that is readily
hydrolyzable by FAAH. Interestingly, no difference was
seen between the two enantiomers (+)-8 (AM4434) and
(À)-8 (AM4435) for this observed excellent FAAH spec-
ificity, but it should be noted that both endocannabinoid
substrates (AEA and 2-AG) are achiral. FAAH also
readily hydrolyzes ketone ( )-6, which, like 8, has one
hydroxyl group adjacent and in the plane of the arachi-
donoyl ester.
The best access to the ester linkage by MGL was in the
cases of ketone ( )-5, and to a much lesser extent, meso-
9 (trans,trans-9, AM5505). Thus, the combination of a
carbonyl which is adjacent and in the plane of the
arachidonoyl ester with a hydroxyl group axial as in
( )-5 resulted in the highest MGL activity for 2-O-
arachidonoylglycerol analogue hydrolysis.
This screening of conformationally restricted 2-AG ana-
logues has identified key structural features which distin-
guish the endocannabinoid hydrolytic enzymes MGL
and FAAH, and the Km values for these compounds
are being determined. We are also interested in evaluat-
ing this series of 2-AG analogues as competitive inhibi-
tors of MGL and FAAH, and for their effects on signal
transduction via the cannabinoid receptors in both the
forskolin-stimulated cyclic AMP accumulation assay
and the [35S]GTPcS binding assay. Future work on con-
strained analogues of 2-AG will involve, not only mod-
ifying the triol ester headgroups through the use of
Figure 3. Molecular models of both possible chair conformations of meso-7 (AM5504) were obtained on a Silicon Graphics Fuel workstation using
Insight II (2000). The O-arachidonoyl groups were modeled in extended conformations as reported by Reggio et al.34 for 2-AG and were constrained
between C1 and C15. A restraint file for the cyclohexyl ring was also incorporated during the dynamics run in order to prevent possible isomerization
and/or racemization at high temperature. The energy-minimized structures underwent constrained molecular dynamics performed by heating it to
1200 °K and recording 100 atomic coordinate trajectories every 10,000 iterations (1 fs per iteration). Next, each trajectory was subjected to simulated
annealing followed by energy minimization using the steepest descent method for 100 iterations followed by conjugate gradient method until the
maximum derivative was less than 0.001 kcal/mol. Two families of low-energy conformers were identified, and the conformer with the axial 2-O-
arachidonoyl group (top) was found to be 2.0 kcal/mol lower in energy than the corresponding equatorial 2-O-arachidonoyl conformer (bottom).

S. K. Vadivel et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5959–5963
5963
18. Pertwee, R. G. AAPS J. 2005, 7, E625.
different-sized rings, but also modifications of the 2-
arachidonoyl group. These conformationally well-de-
fined 2-AG analogues can now be used to develop lead
compounds which are selective inhibitors of MGL or
FAAH.
19. Rouzer, C. A.; Ghebreselasie, K.; Marnett, L. J. Chem.
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22. ( )-5 (CDCl3, 500 MHz) d 5.35–5.44 (m, 8H), 5.25 (d,
J = 2.9 Hz, 1H), 4.48 (br s, 1H), 2.82 (m, 6H), 2.41–2.55
(m, 4H), 2.14–2.25 (m, 4H), 2.09–2.10 (m, 2H), 1.96–2.09
(m, 2H), 1.65–1.83 (m, 2H), 1.22–1.42 (m, 6H), 0.91 (t,
J = 6.6 Hz, 3H); ( )-6 (CDCl3, 500 MHz) d 5.28–5.33 (m,
8H), 4.98 (d, J = 9.9 Hz, 1H), 3.85–3.92 (dt, J = 10.4,
4.7 Hz, 1H), 2.82 (m, 6H), 2.47–2.55 (m, 4H), 2.14–2.25
(m, 4H), 2.09–2.10 (m, 2H), 1.96–2.09 (m, 2H), 1.78–1.83
(m, 2H), 1.22–1.42 (m, 6H), 0.91 (t, J = 6.6 Hz, 3H); meso-
7 (CDCl3, 400 MHz) d 5.30–5.43 (m, 8H), 4.95 (br s, 1H),
3.95 (br s, 2H), 2.79–2.85 (m, 6H), 2.43 (t, J = 7.6 Hz, 2H),
2.14–2.15 (m, 2H), 2.04–2.06 (m, 2H), 1.73–1.77 (m, 4H),
1.60–1.67 (m, 2H), 1.27–1.37 (m, 8H), 0.89 (t, J = 7.0 Hz,
3H); ( )-8 (CDCl3, 400 MHz) d 5.30–5.43 (m, 8H), 4.72–
4.82 (m, 1H), 4.04–4.05 (m, 1H), 3.68–3.71 (m, 1H), 2.84–
2.88 (m, 6H), 2.38 (t, J = 7.6 Hz, 2H), 2.06–2.20 (m, 4H),
1.67–1.78 (m, 8H), 1.28–1.39 (m, 6H), 0.89 (t, J = 7.0 Hz,
3H); meso-9 (CDCl3, 400 MHz) d 5.30–5.43 (m, 8H), 4.63–
4.65 (m, 1H), 3.48–3.53 (m, 1H), 3.34–3.40 (dt, J = 3.4,
9.1 Hz, 1H), 2.79–2.85 (m, 6H), 2.35 (t, J = 7.4 Hz, 2H),
2.00–2.17 (m, 4H), 1.68–1.75 (m, 4H), 1.19–1.39 (m, 10H),
0.88 (t, J = 6.9 Hz, 3H).
23. Dumortier, L.; Van der Eycken, J.; Vandewalle, M.
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Acknowledgments
The authors are grateful to Ying Pei, Yan Peng, Alexan-
der A. (Sasha) Zvonok, and Pusheng Fan for the bio-
chemical assays of these compounds and to
Lakshmipathi Pandarinathan for helpful discussions.
This work was supported by grants from the National
Institute on Drug Abuse DA03801, DA09158, and
DA07215.
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