Development of novel tail-modified anandamide analogs

Article abstract of DOI:10.1016/j.bmcl.2008.07.110

To explore the hydrophobic groove subsite within the CB1 cannabinoid receptor we have designed and synthesized a group of tail-substituted anandamide analogs. Our design involves the introduction of aryl or heterocyclic ring as terminal substituents that

Full text of DOI:10.1016/j.bmcl.2008.07.110

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5912–5915
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of novel tail-modified anandamide analogs
*
Fenmei Yao, Chen Li, Subramanian K. Vadivel, Anna L. Bowman, Alexandros Makriyannis
Center for Drug Discovery, Northeastern University, 116 Mugar Life Sciences Building, 360 Huntington Avenue, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
To explore the hydrophobic groove subsite within the CB1 cannabinoid receptor we have designed and
synthesized a group of tail-substituted anandamide analogs. Our design involves the introduction of aryl
or heterocyclic ring as terminal substituents that are connected to the last cis-arachidonyl double bond
through aliphatic chains of variable lengths. Our results indicate that there are strict stereochemical
requirements for the interaction of such analogs with the CB1 receptor. The optimal pharmacophore
includes the phenyl, p-substituted phenyl, or 3-furyl substituents attached to the cis-double bond
through a four methylene chain.
Received 15 June 2008
Revised 23 July 2008
Accepted 28 July 2008
Available online 31 July 2008
Keywords:
Anandamide
Tail modification
CB1 receptor
Cannabinoid
Endocannabinoid
Published by Elsevier Ltd.
Anandamide,1 a key endogenous cannabinoid ligand, is a partial
CB1 cannabinoid receptor agonist and is known to produce a con-
centration-dependent inhibition of the electrically evoked twitch
response of the mouse vas deferens,2,3 as well as antinociception,
hypothermia, hypomotility, and catalepsy in mice.4–8 In brain
and liver, anandamide is hydrolyzed enzymatically to yield arachi-
donic acid and ethanolamine. The reaction is catalyzed by a mem-
brane-bound amidohydrolase9–12 (anandamide amidohydrolase or
fatty acid amide hydrolase, FAAH) which has been cloned and fully
characterized.13 This enzyme was shown to be co-localized in the
same brain regions as the CB1 cannabinoid receptor.12 The hydro-
lytic breakdown of anandamide can be prevented in vitro by phen-
ylmethanesulfonyl fluoride (PMSF), a general serine protease
inhibitor.9,10 PMSF can be included in the competitive binding as-
says of anandamide analogs, where amidase-catalyzed ananda-
mide hydrolysis might be a complicating factor.14 There is also
evidence pointing to the existence of carrier-mediated anandamide
transport,15 which is essential for the termination of the biological
effects of anandamide. Thus, anandamide interacts not only with
the CB receptors but also possibly with endocannabinoid trans-
porter system and FAAH.
chain is also very sensitive to structural modifications.18,22,23 Com-
plete saturation, or replacement of the olefins with alkynes, results
in the total loss of receptor affinity. Substitution of the arachidonoyl
chain with other fatty acid chains with
x-olefinic bonds, with a trans
double bond or analogs with non -6 structure leads to a major
x
reduction in affinity for CB1. Earlier studies on the hydrophobic tail
of anandamide suggested that the pentyl chain may mimic the five
carbon side chain of (À)-
D
9-tetrahydrocannabinol, the principal
active ingredient of cannabis.16–18,28 Substitution of the terminal
pentyl group of anandamide with a 1,1-dimethyl moiety leads to
significant enhancement of its affinity for CB1, as has also been
observed with (À)-
D
9-THC.17 The present communication further
explores this terrain through the design and synthesis of novel
chain-modified anandamide analogs.
In earlier work, we have shown that introduction of
x-isothio-
cyanato and -azido groups within the anandamide structure
x
leads to substantial enhancement of CB1 affinity and potency.29
The present work describes the synthesis and evaluation of a series
of anandamide analogs of variable chain lengths in which the ter-
minal carbon is functionalized with a phenyl, substituted phenyl,
or heterocyclic rings. All the compounds carry the R-methyletha-
nolamine moiety, a headgroup which was earlier demonstrated
to provide analogs (e.g. methanandamide, AM356) with optimized
affinity for the CB1 receptor as well as robust metabolic stability.14
Our results confirm that, as with anandamide, all analogs exhibit
CB1 selectivity over CB2. Additionally, our data reveal that the ste-
reochemical features of the anandamide tail may have a major im-
pact on ligand’s affinity for CB1.
Anandamide exhibits selectivity toward the CB1 cannabinoid
receptor. Additionally, structure–activity relationship (SAR) studies
of anandamide analogs have provided insight into the stereoelec-
tronic requirements for interaction with the CB1 receptor.14,16–27
Although much of the reported work has addressed the head group,
SAR of the arachidonoyl side chain has revealed that this hydrophobic
The synthesis of the anandamide analogs is illustrated in
Scheme 1. Methyl 5-hexynoate 1 was coupled with 4-chloro-bu-
tyn-1-ol in the presence of copper (I) iodide,30 and the alcohol
* Corresponding author. Tel.: +1 617 373 4200; fax: +1 617 373 7493.
E-mail address: a.makriyannis@neu.edu (A. Makriyannis).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.07.110

F. Yao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5912–5915
5913
Scheme 1. Reagents and conditions: (i) 4-chloro-butyn-1-ol, CuI, NaI, K2CO3, DMF, rt, overnight, 84%; (ii) PPh3, CBr4, rt, 95%; (iii) 3-butyn-1-ol, CuI, NaI, K2CO3, DMF, rt,
overnight, 80%; (iv) H2, Lindlar catalyst, quinoline, ether, 0–10 °C, 86%; (v) Dess–Martin reagent, CH2Cl2, rt, 30 min, 86%; (vi) Wittig reagent from the corresponding
phosphonium salt (see Scheme 2) with n-BuLi in THF at À78 °C, 60–80%; (vii) R-(À)-2-amino-propanol, NaCN (cat), Methanol, 55 °C, sealed vial, 74%; (viii) bis-tributyltin,
Pd(PPh3)4, toluene, reflux, 40%; (ix) I2, CH2Cl2, rt, 95%.
intermediate was converted to the corresponding bromide using
CBr4/PPh3. Bromide 2 was coupled in an analogous procedure with
3-butyn-1-ol to afford alcohol 3, which underwent hydrogenation
with Lindlar’s catalyst in the presence of quinoline to afford triene
4 in high yield.31,32 Dess–Martin periodinane33 oxidation of alcohol
4 resulted in the formation of the unstable aldehyde 5, which was
immediately subjected to Wittig olefination with various phospho-
nium salts to furnish cis-alkenes 6. The methanandamide analogs
were obtained by direct amidation of the corresponding esters
with R-(À)-alaninol catalyzed by sodium cyanide.34 The iodo-
substituted compound 7j was prepared from the corresponding
bromo compound 9 via the tin intermediate.
tionalized iodobenzene 8 and hex-5-yn-1-ol 9 catalyzed by tetraki-
striphenylphosphinepalladium(0),35 the resulting alcohols were
hydrogenated to give
x-phenyl alcohols 11. Iodination of alcohol
11 by I2/PPh3 followed by heating with PPh3 gave the desired phos-
phonium salts 13 in high yields (85–95%). All the other phospho-
nium salts were obtained from commercially available iodide and
triphenylphosphine, and were used for the Wittig reaction without
further purification. The furyl derivatives 7o–7s were synthesized
by utilizing an approach published earlier by our research group.29
This involves the coupling of furyl-substituted terminal alkynes
(14 or 15) with 13-bromo-trideca-5,8,11-triynoic acid methyl ester
16 followed by partial hydrogenation with Lindlar’s catalyst to give
esters 17 (Scheme 3), which were converted to the corresponding
amides using the same method described in Scheme 1.
Scheme 2 illustrates the preparation of the phosphonium salts
utilized in Scheme 1. Starting with the coupling of properly func-
Scheme 2. Reagents and conditions: (i) Pd(PPh3)4, CuI, piperidine, DMF, 0 °C–rt, 50–82%; (ii) H2, 5% Pd-C, methanol, rt, 85–90%; (iii) I2, PPh3, imidazole, 0 °C, CH2Cl2, rt, 80–
92%; (iv) PPh3, neat, 110 °C, 80–92%.
Scheme 3. Reagents and conditions: (i) CuI, NaI, K2CO3, DMF, rt, overnight, 60%; (ii) H2, Lindlar catalyst, quinoline, ether, 0–10 °C, 94%.

5914
F. Yao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5912–5915
Binding affinities for the CB1 and CB2 receptors were deter-
(Ki = 17.9 nM in the presence of PMSF). Decreasing the number of
methylene carbons between C15 cis-double bond and the phenyl
group gave analogs 7a–7c and showed progressively weaker affin-
ities for the CB1 receptor with decreasing length of the alkyl chain.
Conversely, compound 7e which differs from 7d by one extra
methylene group displayed approximately over fivefold lower
affinity compared to 7d. These results clearly indicate that a four
methylene linker is optimal between the distal arachidonyl double
bond and the pendant phenyl moiety. In an effort to enhance the
polar features of anandamide analogs, we have substituted the
phenyl ring with the more polar furan moiety. A similar trend
was observed with the 3-furyl derivative (7o). However, surpris-
ingly the corresponding 2-furyl derivative (7q) showed a substan-
mined according to previously reported procedures.36,21,23 For the
CB1 receptor, binding data were obtained using a rat brain mem-
brane in the presence of phenylmethanesulfonyl fluoride (PMSF),
a general serine protease inhibitor to protect the analogs from
the hydrolytic activity of fatty acid amide hydrolase. CB2 data were
obtained using a membrane preparation from mouse spleen
known to be rich in CB2 receptor. [3H]CP-55,940, a most widely
used radioligand, was chosen as a competing ligand for the assays
as it has high affinity for both CB1 and CB2 receptors and is
nonselective.37
The binding affinities of novel anandamide analogs are summa-
rized in Table 1, where (R)-methanandamide is included for com-
parison. It is apparent from the CB2 affinities reported here that
these novel anandamide analogs show selectivity for the CB1
receptor. Analogs 7a–7e were synthesized to assess the optimal
length for the phenyl group at the hydrophobic tail for receptor
affinity. The binding results show that 7d with four methylene car-
bons separating C15 from the phenyl group exhibited comparable
activity (Ki = 13.4 nM) for the CB1 receptor as (R)-methanandamide
tially weaker affinity. Introduction of terminal
a-naphthyl group
(7f) resulted in the loss of CB1 receptor affinity.
Earlier data have suggested the presence of a hydrophobic
groove subsite capable of accommodating the side chain of classi-
cal cannabinoids and possibly the terminal five carbon alkyl group
of anandamide.38 Our present results are congruent with the exis-
tence of the hydrophobic subsite for the anandamide tail within
the CB1 receptor capable of accommodating a terminal phenyl
group. It can be argued that a short chain linking the phenyl ring
with the last double bond as seen in 7a (n = 1) and 7b (n = 2)
may not occupy a sufficient portion of this groove, resulting in
low binding affinity. Conversely, a linker that is too long, as seen
in 7e (n = 5), may be too large to be fully encompassed by the sub-
site. The ideal length, given by 7d, completely fills the groove and
yet does not incur any steric penalties. The groove also accommo-
dates the 3-furyl moiety, but not the 2-furyl isomer, suggesting
that the relative position of the furyl oxygen within the receptor
subsite may affect the overall ability of the ligand for an optimal
interaction with the receptor. Substitution of the phenyl ring with
Table 1
Affinity (Ki)a of new anandamide analogs for CB1 and CB2 receptors
O
OH
N
H
R
Analogs
R
CB1 Ki (nM) (PMSF)
CB2 Ki (nM)
(R)-Methanandamide
(CH2)4CH3
CH2Ph
(CH2)2Ph
(CH2)3Ph
(CH2)4Ph
(CH2)5Ph
17.9
348.5
241.2
69.2
13.3
76.6
678.3
30.4
89.4
868
826.3
335
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7s
533.7
1117
240.6
3996
987.3
605.1
1054
1564
2040
839.7
N.R.
250.4
1027
869
749.8
3255
an a-naphthyl moiety results in severe loss in affinity in CB1, an ef-
fect which is probably due to its large size.
(CH2)4-
a-naphthyl
To further probe this CB1 receptor subsite, different substitu-
ents were introduced on the pendent phenyl ring. Our results indi-
cate that, while p-phenyl substituents are tolerated in the receptor
subsite, meta or ortho substitution leads to a substantial reduction
in affinity. The most potent analog is that with the p-bromophenyl
group (7n). Substitution at the meta and ortho position is not toler-
ated. However, the introduction of bromine at the para position re-
sults in an increase in CB1 receptor affinity. The above
stereochemical postulate for the anandamide distal tail pharmaco-
phore is pictorially summarized in Figure 1.
In conclusion, new anandamide analogs with structural modifi-
cations at the hydrophobic tail were synthesized and evaluated for
their affinities for the cannabinoid receptors. Introduction of a pen-
dant phenyl, aryl, or furyl group separated from the last cis double
bond of arachidonic acid by methylene chains of variable lengths
(CH2)4Ph-m-F
(CH2)4Ph-m-Cl
(CH2)4Ph-m-Br
(CH2)4Ph-m-I
(CH2)4Ph-m-CF3
(CH2)4Ph-m-Me
(CH2)4Ph-o-Br
(CH2)4Ph-p-Br
(CH2)4(3-furyl)
(CH2)5(3-furyl)
(CH2)4(2-furyl)
(CH2)5(2-furyl)
95.3
533.3
284.1
344.3
170.0
8.9
12.0
170
325
561.2
CB1 affinities were determined using rat brain membranes and 0.8 nM [3H]
CP-55,940 as the radioligand. Mouse spleen was used as source of CB2 receptor.
Data were analyzed using nonlinear regression analysis. Ki values were obtained
from a minimum of two independent experiments run in duplicate.
a
Figure 1. Pictorial representation of the CB1 binding groove for the tail of (R)-methanandamide analogs. Compound 7a (green), 7b (cyan), 7c (magenta), 7d (yellow), 7e
(pink), 7i (gray), 7m (blue), 7n (orange). Structures were minimized with the OPLS_2005 force field in Macromodel.39

F. Yao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5912–5915
5915
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