A pyridone analogue of traditional cannabinoids. A new class of selective ligands for the CB2 receptor

Article abstract of DOI:10.1016/S0968-0896(01)00155-9

A pyridone analogue (5) of the potent bicyclic cannabinoid CP 47,497 (6) has been synthesized as a model for one conformational isomer of anandamide and to test the hypothesis that an amide carbonyl may serve as a hydrogen bond acceptor in interactions with the CB1 cannabinoid receptor. Pyridone 5 was synthesized from 6-bromo-2-methoxypyridine (10) by palladium catalyzed coupling with 1-pentyne to provide 11. Catalytic hydrogenation of 11 and hydrolysis to pyridone 13 followed by N-alkylation gave 1-propyl-6-pentyl-2-pyridone (15). Bromination of 15 gave dibromide 18, which underwent Heck coupling with cyclohex-2-en-1-one to give enone 19. Catalytic hydrogenation of 19 gave ketone 20 which was reduced using NaBH₄ to alcohol 5. Reduction of 20 with K-Selectride gave the axial epimer of 5 (21). Neither alcohol 5 nor 21 have significant affinity for the CB₁ receptor (K_i > 970 nM), but both have moderately high affinity for the CB₂ receptor (K_i < 60 nM). All rights reserved. Copyright

Full text of DOI:10.1016/S0968-0896(01)00155-9

Bioorganic & Medicinal Chemistry 9 (2001) 2863–2870
A Pyridone Analogue of Traditional Cannabinoids.
A New Class of Selective Ligands for the CB2 Receptor
John W. Huffman,a,* Jianzhong Lu,a George Hynd,a Jenny L. Wileyb
and Billy R. Martinb
aHoward L. Hunter Laboratory, Clemson University, Clemson, SC 29634-1905, USA
bDepartment of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University,
Richmond, VA 23298-0613, USA
Received 28February 2001; accepted 3 May 2001
Abstract—A pyridone analogue (5) of the potent bicyclic cannabinoid CP 47,497 (6) has been synthesized as a model for one con-
formational isomer of anandamide and to test the hypothesis that an amide carbonyl may serve as a hydrogen bond acceptor in
interactions with the CB1 cannabinoid receptor. Pyridone 5 was synthesized from 6-bromo-2-methoxypyridine (10) by palladium
catalyzed coupling with 1-pentyne to provide 11. Catalytic hydrogenation of 11 and hydrolysis to pyridone 13 followed by N-
alkylation gave 1-propyl-6-pentyl-2-pyridone (15). Bromination of 15 gave dibromide 18, which underwent Heck coupling with
cyclohex-2-en-1-one to give enone 19. Catalytic hydrogenation of 19 gave ketone 20 which was reduced using NaBH4 to alcohol 5.
Reduction of 20 with K-Selectride gave the axial epimer of 5 (21). Neither alcohol 5 nor 21 have significant affinity for the CB1
receptor (Ki > 970 nM), but both have moderately high affinity for the CB2 receptor (Ki < 60 nM). # 2001 Elsevier Science Ltd.
All rights reserved.
Introduction
At the present time, several structurally diverse classes
of compounds have been identified which bind to the
cannabinoid CB1 and CB2 receptors and which elicit the
behavioral effects characteristic of cannabinoids.1,2
These compounds include inter alia classical cannabi-
noids, such as Á9-tetrahydrocannabinol (Á9-THC, 1),
the principal psychoactive constituent of marijuana,
cannabimimetic indoles, pyrroles and indenes, of which
WIN-55,212-2 (2) is the prototypical example3 and can-
nabimimetic fatty acid derivatives, of which the endo-
genous cannabinoid anandamide (3) is typical.4 These
various types of cannabinoids bind to both receptors,
and structure–activity relationships (SAR) for each class
of compound have been devised.1À5
A number of attempts have been made to develop a
pharmacophore to accommodate both the cannabimi-
metic fatty acid derivatives such as anandamide (3) and
classical cannabinoids related to Á9-THC (1).6À8 Tho-
mas et al. described a model in which the amide car-
bonyl oxygen of anandamide is superimposed on the
pyran oxygen of Á9-THC (1), the terminal hydroxyl of 3
is aligned with the C-1 hydroxyl of the traditional can-
nabinoid and the saturated aliphatic chain of ananda-
mide is aligned with the cannabinoid C-3 side chain.6
*Corresponding author. Tel.: +1-864-656-3133; fax: +1-864-656-
3133; e-mail: huffman@clemson.edu
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00155-9

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J. W. Huffman et al. / Bioorg. Med. Chem. 9 (2001) 2863–2870
An alternative alignment was described by Tong et al.,
who employed constrained conformational searching
and a CoMFA study which aligned anandamide with a
traditional cannabinoid, 9-nor-9b-hydroxyhexahydro-
cannabinol (HHC, 4).7 In this alignment, the five term-
inal carbons of anandamide correspond to the aliphatic
side chain of HHC, the ethanol hydroxyl of anandamide
was superimposed on the 9-hydroxyl of 4, and the oxy-
gen of the amide carbonyl was aligned with the C-1
hydroxyl of HHC. Very recently, Fischera et al. carried
out a 3-D-QSAR study to develop a pharmacophore
which would accommodate traditional cannabinoids,
cannabimimetic indoles and anandamide.8 The align-
ment of anandamide and its analogues with Á9-THC (1)
was that suggested by Thomas6 and the alignment of the
indole based cannabinoids with traditional cannnabi-
noids was similar to that suggested by Huffman and co-
workers.9 All three of these approaches provided results
which were consistent with the pharmacology of the
compounds included in the studies. In an alternative
approach to the interaction of the anandamide and its
analogues with the CB1 receptor, Barnett-Norris et al.
employed the conformational memories technique to
investigate biologically relevant conformations of anan-
damide and then carried out docking studies with a
computer generated model of the receptor.10
indicated congruence of the salient structural features of
the two molecules. In the alignment of traditional can-
nabinoids and cannabimimetic indoles suggested by
Huffman et al. it was considered that the ketonic car-
bonyl of the indoles corresponded to the C-1 hydroxyl
of traditional cannabinoids.3,9 In this model, the car-
bonyl group of the indole serves as a hydrogen bond
acceptor, and the amide carbonyl of pyridone 5 pro-
vides a possible model for the feasibility of this hypo-
thetical alignment of traditional cannabinoids and
cannabimimetic indoles.
Results
The initial retrosynthetic analysis of pyridone 5 is out-
lined in Scheme 1. This relatively simple and direct
approach employed commercially available 2,5-dibro-
mopyridine as starting material and took advantage of
the chemoselectivity of the 2-bromo substituent toward
palladium catalyzed coupling with terminal alkynes.17
Reaction of 2,5-dibromopyridine with 1-pentyne would
afford 2-(1-pentynyl)-5-bromopyridine (7), selective
hydrogenation of which would provide 2-(1-pentyl)-5-
bromopyridine (8). Halogen metal interconversion of 8,
followed by reaction with 3-ethoxycyclohex-2-en-1-one
and mild acid hydrolysis would afford ketone 9. Con-
version of 9 to the appropriate quaternary salt followed
by oxidation with potassium ferricyanide would give the
target pyridone.18
By employing a combination of Dreiding molecular
models and the program PCModel, an alternative
alignment of anandamide and Á9-THC (1) has been
developed.11 In this alignment, which is similar to that
suggested by Tong et al.,7 the amide carbonyl of anan-
damide is superimposed upon the phenolic hydroxyl of
Á9-THC, and the terminal carbons of anandamide are
aligned with the alkyl side chain of Á9-THC. The amide
nitrogen of anandamide is aligned with C-2 of Á9-THC
(1) and the N-substituent of anandamide is considered
to correspond to a C-2 substituent on the cannabinoid
nucleus. A C-2 substituent should have a relatively
minor impact on cannabinoid activity based upon
observations made some years ago by Edery,12 and the
more recent observation that 2-iodo-Á8-THC has mod-
erate affinity for the CB1 receptor (Ki=89 nM) and
shows typical cannabinoid pharmacology in vivo.13
Also, a conformationally constrained analogue of Á8-
THC which has an alkyl substituent at C-2 has good
affinity for the CB1 receptor (Ki=22.3 nM).14
In practice, the palladium catalyzed coupling of 2,5-
dibromopyridine with 1-pentyne proceeded smoothly
and in good yield to provide 2-(1-pentynyl)-5-bromo-
pyridine (7), however the selective hydrogenation of the
alkyne in the presence of the aromatic halogen
failed.17,19 Repeated attempts gave only 2-pentylpyr-
idine. In order to circumvent this problem, 2-(1-penty-
nyl)-5-bromopyridine (7) was subjected to halogen
metal interconversion and coupling with 3-ethoxy-
cyclohex-3-en-1-one. Catalytic hydrogenation provided
ketone 9 and a variety of methods were explored to
convert ketone 9 to the corresponding N-alkyl 2-pyr-
idone (5). However, the result of these efforts were
either recovered starting material or products of gross
decomposition. A number of alternative approaches
On the basis of the alignment described above, a pyr-
idone based cannabinoid (5) was designed to test this
model. Pyridone 5 replaces the aromatic benzenoid ring
of Á9-THC with a heterocylic ring containing an amide
carbonyl and a bicyclic structure designed in analogy to
the potent non-traditional cannabinoid CP 47,497 (6).15
The n-pentyl side-chain characteristic of Á9-THC is
appended to the 6-position of the pyridone. Based on
the observation that the analogue of anandamide in
which the nitrogen substituent was propyl rather than
hydroxyethyl had greater affinity for the CB1 receptor,
and greater hydrolytic stability than anandamide,16
a
propyl substituent was employed as the nitrogen sub-
stituent. Comparison, employing PCModel, of a mini-
mized structure of pyridone 5 with that of CP 47,497 (6)
Scheme 1.

J. W. Huffman et al. / Bioorg. Med. Chem. 9 (2001) 2863–2870
2865
similar in concept to those described above, based upon 2-
(1-pentynyl)-5-bromopyridine (7) were also unsuccessful.19
Scheme 3). However, reaction of 15 with one equivalent
of bromine in acetic acid gave a mixture from which
only 16% yield of a monobromo compound could be
isolated, in addition to recovered starting material (15)
and 38% of dibromopyridone 18. The structure of the
monobromo compound was established on the basis of
It seemed probable that the difficulties encountered in
converting ketone 9 to the corresponding pyridone were
due to steric crowding. An alternative approach was
devised in which the oxygen of the pyridone would be
introduced early in the synthesis and the alicyclic ring
would be introduced subsequently. Accordingly, 2-
methoxy-6-bromopyridine (10, Scheme 2)20 was sub-
jected to palladium catalyzed coupling with 1-pentyne
to give 2-methoxy-6-(1-pentynyl)pyridine (11) in 90%
yield. Catalytic hydrogenation of 11 afforded 2-meth-
oxy-6-pentylpyridine (12) in excellent (95%) yield.
However, attempted directed metalation adjacent to the
methoxyl employing n-butyllithium was unsuccessful
due to the fact that the a-proton of the pentyl group was
removed in preference to the aromatic hydrogen.19
1
the H NMR spectrum in which the aromatic protons
appeared as a doublet, J=9.6 Hz, indicating that they
were on adjacent carbons. The conclusion that this
compound was the undesired 5-bromo isomer (17) was
based on the observation that there was no NOE
enhancement of either aromatic proton when the a-
proton of the side chain was irradiated. Acting on the
assumption that it would be possible to selectively carry
out reaction at one of the two bromine substituents of
dibromopyridone 18, reaction of 15 with excess bromine
in acetic acid was carried out to give 18 in 86% yield.
Reaction of dibromopyridone 18 with one equivalent of
n-butyllithium, at À78 ꢀC followed by quenching with
water gave a 16% yield of 1-propyl-6-pentyl-2-pyridone
(15) and a 32% yield of 5-bromo-1-propyl-6-pentyl-2-
pyridone (17). Since it was apparent that the 3-bromo
substituent was more reactive than the 5-bromo group,
the reaction was repeated, however the intermediate
organolithium was treated with 3-ethoxycyclohex-2-en-
1-one to give a product in 13% yield (Scheme 3), the
spectroscopic properties (see Experimental) of which
were consistent with those of the desired enone (19),
however an alternative structure resulting from halo-
gen–metal interconversion at C-5 could not be excluded.
The assigned structure (19) was confirmed by X-ray
crystallography. No attempt was made to optimize the
yield for this reaction since it was found nearly simul-
taneously that palladium catalyzed reaction of dibro-
mopyridone 18 with cyclohex-2-en-1-one under Heck
reaction conditions also gave enone 19, although in only
11% yield.
In the successful approach to pyridone 5, 2-methoxy-6-
pentylpyridine (12) was hydrolyzed to 1(H)-6-pentyl-2-
pyridone (13) in 95% yield using hot 48% hydrogen
bromide. A number of methods for effecting the
N-alkylation of pyridone 13 were investigated, all of
which afforded O-alkylation as the major reaction path.
Reaction of 13 with 1-iodopropane in the presence of
sodium or potassium carbonate in DMF gave 2-pro-
poxy-6-pentylpyridine (14) as the major product, with
less than 10% of the desired product, 1-propyl-6-pentyl-
2-pyridone (15). Similar results were obtained with
LDA in THF. A number of years ago, Adams and
Schrecker reported that 6-methyl-2-pyridone underwent
N-alkylation in the presence of sodium ethoxide in ben-
zene.21 Similar conditions applied to the reaction of 13
with 1-iodopropane afforded 14 and 15 in a ratio of 84
to 13. However, when sodium hydroxide and lithium
bromide were used in a one to one ratio in DMF, the
ratio of 14 to 15 improved to 73 to 27. It was eventually
found that 13 and 1-iodopropane with sodium hydrox-
ide and lithium bromide in a ratio of four to one in
DMF gave 1-propyl-6-pentyl-2-pyridone (15) in a mod-
est, but acceptable yield of 52% accompanied by 32%
of 2-propoxy-6-pentylpyridine (14).
It was envisioned that electrophilic bromination of pyr-
idone 15 would provide a mixture of the desired 3-bro-
mopyridone (16) and the undesired regioisomer (17,
Scheme 3. (a) Br2/HOAc, 25 ꢀC; (b) n-BuLi/THF, À78ꢀ C, then 3-
ethoxycyclohex-2-en-1-one followed by NH4Cl, or cyclohex-2-en-1-
one, Na2CO3/Pd(OAc)2/tri-o-tolylphosphine,/DMF; (c) H2(g), 10%
Pd(C)/EtOH/40psig; (d) NaBH4/EtOH, 25 ꢀC; (e) K(sec-butyl)3BH/
THF, À78 ꢀC to 25 ꢀC, then NaOH/H2O2/EtOH/H2O.
Scheme 2. (a) 1-Pentyne, (PPh3)2Cl2Pd/Et3N/CuI, 25 ꢀC; (b) H2(g),
10% Pd(C)/EtOH/20psig; (c) 48% HBr, reflux; (d) 1-iodopropane,
NaOH/LiBr/DMSO, 70 ꢀC.

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J. W. Huffman et al. / Bioorg. Med. Chem. 9 (2001) 2863–2870
This result was surprising since it is known that the
Heck reaction usually proceeds with the syn addition of
the aromatic substrate, to the alkene, followed by syn
elimination of hydrogen and palladium(II) as the final
step.22 Although this path is not possible with cyclo-
hexenone as a reactant, there is precedent for intra-
molecular Heck additions to cyclohexenones similar to
the intermolecular reaction of 18 with cyclohexenone.23
It has been noted that in the addition to a conjugated
enone, the palladium adduct is actually an enolate
which may equilibrate prior to elimination of palladium
(II) and hydrogen. A number of experiments were
carried out to investigate the potential utility of this
reaction and to optimize the yield. It was ultimately
found that the yield of enone 19 could be improved to
50% using palladium acetate, tri-o-tolylphosphine and
sodium carbonate in DMF at 120 ꢀC.
models for the active conformational isomer of ana-
ndamide. The observation that alcohol 21 has little affi-
nity for the CB1 receptor is in itself not surprising in
view of the fact that cannabinoids with a-substituents at
C-9 are known to have attenuated affinity for the CB1
receptor.25
Comparison of the structures of CP 47,497 (6) and pyr-
idone 5 using PCModel11 indicate that with the excep-
tion of the substitution of an amide carbonyl in 5 for the
phenolic hydroxyl of 6 and the presence of a propyl
substituent at a position corresponding to C-2 of CP
47,497, there is excellent overlap between the structures
of 5 and 6. However, the affinity of pyridone 5 for the
CB1 receptor (973Æ105 nM) is many fold less than that
of CP 47,497 (6, 2.20Æ0.47 nM),15 which provides
experimental evidence that an amide carbonyl does not
substitute for the phenolic hydroxyl of 6 and traditional
cannabinoids as a hydrogen bond acceptor. The sub-
stitution of a pentyl side chain in 5 for the dimethyl-
heptyl side chain of 6 may also contribute somewhat to
the diminished affinity of 5 for the CB1 receptor. The
failure of pyridone 5 to have appreciable affinity for the
CB1 receptor implies that the amide carbonyl of canna-
bimimetic indoles may also not serve as a hydrogen
bond acceptor as suggested previously.3,9 The fact that
several indene derivatives structurally related to the
cannabimimetic indoles and which also lack this car-
bonyl group have high affinity for the CB1 receptor
provides additional evidence that a carbonyl group is
not essential for interaction of cannabimimetic indoles
and related compounds with the CB1 receptor.26
The synthesis of pyridone 5 was completed by hydro-
genation of the enone double bond (Scheme 3), which
was accompanied by simultaneous hydrogenolysis of
the bromine to provide ketone 20. Sodium borohydride
reduction of ketone 20 gave the target pyridone (5). The
1H NMR spectrum of this reduction product was con-
sistent with the assigned stereochemistry which showed
the carbinol proton as a multiplet at d 3.73–3.81, con-
sistent with an equatorial hydroxyl group. The axial
isomer of alcohol 5 (21) was prepared by reduction of
ketone 20 with K-Selectride in THF. The 1H NMR
spectrum of alcohol 21 showed a carbinol proton as a
broadened singlet at d 4.20, which is consistent with the
assigned stereochemistry.
As shown in Table 1, neither alcohol 5 nor 21 have
appreciable affinity for the CB1 receptor, however both
alcohols have significant affinity, in the 50 nM range,
for the CB2 receptor. Alcohol 5 was designed as a model
compound for one conformation of anandamide (3),
and also to test the hypothesis that an amide carbonyl
group could serve as a surrogate for the phenolic
hydroxyl of traditional cannabinoids.3,9 The distance
between the terminal methyl of the pentyl side chain of
5 and the pyridone carbonyl can be brought to 5.74 A
by allowing 5 to assume a minimum energy conforma-
tion (although not the global minimum) as determined
using PCModel. This distance is somewhat greater than
that between the terminal methyl group of anandamide
(3) and the carbonyl carbon of a conformational isomer
of anandamide which has been suggested to be biologi-
cally important (4.89 A).24 The very weak affinity of
alcohols 5 and 21 for the CB1 receptor suggests that
either this conformation of anandamide is not that
which is responsible for interaction with the CB1 recep-
tor or that pyridones 5 and 21 are not appropriate
Based on studies employing a mutant CB1 receptor,
Song and Bonner suggested that a hydrogen bonding
interaction between the phenolic hydroxyl of traditional
cannabinoids such as Á9-THC (1) and the Pfizer non-
traditional cannabinoids such as CP 47,497 (6) is
important for binding to the CB1 receptor.27 This inter-
action is not important for binding to the CB2 receptor,
inasmuch as methyl ethers derived from analogues of
Á9-THC (1) and several 1-deoxy traditional cannabi-
noids have high affinity for the CB2 receptor, but little
affinity for the CB1 receptor.28 The affinities of pyr-
idones 5 and 21 for the CB2 receptor (Ki=56Æ11 and
50Æ4 nM, respectively) are similar to that of 1-deoxy-
Á8-THC (22, Ki=32Æ9 nM).28 As noted above, pyr-
idones 5 and 21 share a general molecular template with
CP 47,497 (6), which in turn can be related structurally
to the traditional cannabinoids, hexahydrocannabinol
(HHC, 4) and Á9-THC (1). Although pyridones 5 and
21 can be related structurally to cannabinoids 1, 4 and
6, their affinities for the CB1 and CB2 receptors correlate
well with those of 1-deoxy-Á8-THC (22). Deoxy canna-
binoid 22 is closely related structurally to traditional
Table 1. Receptor affinities of pyridones 5 and 21 and Á9-THC (1)
Compound
Ki (nM)
CB1
CB2
5
21
1
973Æ105
1054Æ6850
41Æ232
56Æ11
Æ4
36Æ1033

J. W. Huffman et al. / Bioorg. Med. Chem. 9 (2001) 2863–2870
2867
cannabinoids, such as 1 and 4, however 22 lacks the
phenolic hydroxyl characteristic of traditional cannabi-
noids with high affinity for the CB1 receptor. On the
basis of these structural relationships and the simila-
rities in affinities for the CB2 receptor, pyridones 5 and
21 may be considered to be analogues of 1-deoxy-Á8-
THC (22).
80 mL of dry EtOH was added 0.5 g of 10% Pd on
carbon. The mixture was shaken for 18h under H 2 at 20
psig. The catalyst was removed by filtration through
Celite and the solvent was removed in vacuo to give 4.37 g
(95%) of 2-methoxy-6-pentylpyridine (12) as a pale yel-
low oil which was used in the subsequent step without
further purification: 1H NMR (300 MHz, CDCl3) d 0.82
(t, J=6.9 Hz, 3H), 1.20–1.28(m, 4H), 1.60–1.69 (m,
2H), 2.59 (t, J=7.8Hz, 2H), 3.38 (s, 3H), 6.52 (d,
J=8.2 Hz, 1H), 6.69 (d, J=7.2 Hz, 1H), 7.45 (dd,
J=7.2, 8.0 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d
14.0, 22.5, 29.0, 31.5, 37.8, 53.1, 107.0, 115.0, 138.5, 160.4,
163.6; MS (EI) m/z 179 (5), 150 (14), 136 (17), 123 (100).
Although pyridones 5 and 21 do not constitute viable
rigid models for anandamide, they do provide evidence
that an amide carbonyl, and by extension the acyl car-
bonyl of cannabimimetic indoles, does not serve as a
surrogate for the phenolic hydroxyl group of traditional
cannabinoids in interactions with the CB1 receptor.
However, these pyridones constitute a new class of CB2
receptor ligands, with affinities similar to that of 1-
deoxy-Á8-THC (22).
1(H)-6-Pentyl-2-pyridone (13). A suspension of 0.48g
(2.68mmol) of 2-methoxy-6-pentylpyridine ( 12) in 12
mL of 48% HBr was heated at reflux for 4 h. The reac-
tion mixture was neutralized with saturated aqueous
NaHCO3 and extracted with ether. The ethereal extracts
were washed with water, dried (MgSO4) and the solvent
was removed in vacuo to give 0.42 g (95%) of 1(H)-6-
pentyl-2-pyridone (13) as a white solid: 1H NMR
(300 MHz, CDCl3) d 0.88 (t, J=6.9 Hz, 3H), 1.30–1.35
(m, 4H), 1.64–1.72 (m, 2H), 2.61 (t, J=7.5 Hz, 2H), 6.06
(d, J=6.9 Hz, 1H), 6.41 (d, J=9.0 Hz, 1H), 7.37 (dd,
J=6.9, 9.0 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d
13.9, 22.3, 28.1, 31.0, 32.9, 105.0, 116.5, 141.7, 150.4,
165.9; MS (EI) m/z 165 (9), 122 (15), 109 (100).
Experimental
General
IR spectra were obtained using Nicolet 5DX or Magna
1
spectrometers; H and 13C NMR spectra were recorded
on Bruker AC300 or JEOL 500 MHz spectrometers.
Mass spectral analyses were performed on a Hewlett-
Packard 5890A gas chromatograph with a mass sensi-
tive detector and HRMS data were obtained in the
Mass Spectrometry Laboratory, School of Chemical
Sciences, University of Illinois. Ether and THF were
distilled from Na-benzophenone ketyl immediately
before use, and other solvents were purified using stan-
dard procedures. Column chromatography was carried
out on Universal silica gel (32–63 m) using the indicated
solvents as eluents. All new compounds were homo-
geneous to TLC and 13C NMR.
6-Pentyl-2-propoxypyridine (14) and 6-pentyl-1-propyl-2-
pyridone (15). To a solution of 0.40 g (2.42 mmol) of
1(H)-6-pentyl-2-pyridone (13) in 2 mL of DMSO was
added 0.75 g (8.64 mmol) of LiBr, 0.16 g (4.0 mmol) of
NaOH and 0.3 mL (3.1 mmol) of 1-iodopropane. The
reaction mixture was heated to 70 ꢀC and stirred for 12 h.
The mixture was poured into water, extracted with
ether, washed with water and brine, dried (MgSO4) and
the solvent removed in vacuo. Chromatography (petro-
leum ether/ethyl acetate, 2.5:1) gave 0.16 g (32%) of 6-
pentyl-2-propoxypyridine (14): 1H NMR (300 MHz,
CDCl3) d 0.89 (t, J=6.4 Hz, 3H), 1.01 (t, J=7.2 Hz,
3H), 1.32–1.35 (m, 4H), 1.70–1.84 (m, 4H), 2.64 (t,
J=7.2 Hz, 2H), 4.22 (t, J=7.2 Hz, 2H), 6.51 (d, J=8.2
Hz, 1H), 6.67 ( d, J=7.2 Hz, 1H), 7.44 (t, J=7.3 Hz,
1H); 13C NMR (75.5 MHz, CDCl3) d 10.6, 14.0, 22.4,
22.5, 29.1, 31.5, 37.9, 67.3, 107.1, 114.8, 138.5, 160.4,
163.6; MS (EI) m/z 207 (8), 178 (29), 151 (78), 109 (100).
Further elution with the same solvent system gave 0.26 g
(52%) of 6-pentyl-1-propyl-pyridone (15): 1H NMR
(300 MHz, CDCl3) d 0.93 (t, J=6.9 Hz, 3H), 0.99 (t,
J=7.5 Hz, 3H), 1.36–1.41 (m, 4H), 1.57–1.78(m, 4H),
2.59 (t, J=7.8Hz, 2H), 3.96 (t, J=7.8Hz, 2H), 5.99 (d,
J=6.9 Hz, 1H), 6.42 (d, J=9.0 Hz, 1H), 7.20 (dd,
J=6.9, 9.1 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d
11.3, 13.8, 22.0, 22.3, 28.5, 31.4, 32.9, 45.3, 105.5, 117.6,
138.4, 149.8, 163.6; MS (EI) m/z 207 (62), 164 (75), 136
(50), 122 (54), 109 (100); HRMS calcd for C13H21NO:
207.1623, found 207.1629.
2-Methoxy-6-(1-pentynyl)pyridine (11). To a stirred
solution of 0.40 g (2.13 mmol) of 2-bromo-6-methoxy-
pyridine in 8mL of triethylamine at ambient tempera-
ture, 0.019 g (0.10 mmol) of CuI was added and the
mixture was degassed by passing a stream of argon
through the solution for 15 min. After cooling to 0 ꢀC,
0.3 mL (2.24 mmol) of 1-pentyne was added, followed
by 0.035 g (0.050 mmol) of dichlorobis(triphenylphos-
phine)palladium (II). The reaction was stirred for 1 h at
0 ꢀC, allowed to warm to ambient temperature and stir-
red for 8h. The mixture was filtered through Celite, and
the solvent was removed in vacuo. Chromatography
(petroleum ether/ethyl acetate, 30:1) gave 0.34 g (91%)
of 2-methoxy-6-(1-pentynyl)pyridine (11): 1H NMR
(300 MHz, CDCl3) d 1.05 (t, J=7.2 Hz, 3H), 1.65 (sex-
tet, J=7.2 Hz, 2H), 2.42 (t, J=7.0 Hz, 2H), 3.94 (s,
3H), 6.65 (d, J=7.9 Hz, 1H), 6.98(d, J=7.5 Hz, 1H),
7.47 (t, J=7.5 Hz, 1H); 13C NMR (75.5 MHz, CDCl3)
d?13.6, 21.4, 21.9, 53.4, 80.5, 90.2, 110.4, 120.2, 138.3,
140.8, 163.7; MS (EI) m/z 174 (100), 146 (35), 130 (12),
117 (17); HRMS calcd for C11H13NO: 175.0997, found
175.0998.
5-Bromo-6-pentyl-1-propyl-2-pyridone (17) and 3,5-Di-
bromo-6-pentyl-1-propyl-2-pyridone (18). To a stirred
solution of 0.11 g (0.53 mmol) of pyridone 15 in 3.0 mL
of glacial acetic acid at ambient temperature was added
2-Methoxy-6-pentylpyridine (12). To a solution of 4.50 g
(25.7 mmol) of 2-methoxy-6-(1-pentynyl)pyridine (11) in

2868
J. W. Huffman et al. / Bioorg. Med. Chem. 9 (2001) 2863–2870
dropwise a solution of 0.093 g (0.58mmol) of bromine
in 0.28mL of acetic acid. The reaction mixture was
stirred at ambient temperature for 10 h, poured into
saturated aqueous NaHCO3 and extracted with ether.
The ether extract was washed successively with water
and brine, dried (MgSO4) and the solvent removed in
vacuo. The residue was chromatographed (petroleum
ether/ethyl acetate, 3:1) to give 0.026 g (17%) of 16 as a
C19H26BrNO2: C, 60.00; H, 6.89; N, 3.68; found: C,
60.00; H, 6.81; N, 3.68.
B. To a solution of 0.73 g (2.0 mmol) of dibromopyr-
idone 18 in 4 mL of dry THF at À78 ꢀC was added
dropwise 0.38mL (0.75 mmol) of 1.9M8
n-butyl-
lithium. The solution was stirred at À78 ꢀC for 1 h,
warmed to ambient temperature and stirred for an
additional 2 h. After cooling to À78 ꢀC, a solution of
0.44 g (3.1 mmol) of 3-ethoxycyclohex-2-en-1-one in 1.5
mL of THF was added dropwise and the reaction was
stirred at À78 ꢀC for 4 h, quenched with saturated aqu-
eous NH4Cl and extracted with ether. The ether extract
was washed with water, dried (MgSO4) and the solvent
was removed in vacuo. Chromatography (petroleum
ether/ethyl acetate 5:1) gave 0.10 g (13%) of 19 as a
green solid, identical in all respects to the material
described in part A, above.
1
pale yellow oil: H NMR (300 MHz, CDCl3) d 0.94 (t,
J=6.9 Hz, 3H), 1.00 (t, J=7.2 Hz, 3H), 1.37–1.48(m,
4H), 1.55–1.64 (m, 2H), 1.75–1.86 (m, 2H), 2.79 (t,
J=8.1 Hz, 2H), 4.00 (t, J=7.8Hz, 2H), 6.35 (d, J=9.6
Hz, 1H), 7.35 (d, 9.6 1H); 13C NMR (75.5 MHz, CDCl3)
d 11.2, 13.8, 22.1, 22.4, 27.5, 31.6, 32.8, 47.0, 99.7, 118.8,
142.6, 147.8, 162.2; MS (EI) m/z 287 (33), 285 (31), 244
(10), 242 (10), 189 (100); HRMS calcd for C13H20BrNO:
285.0728, found 285.0734. Further elution with petro-
leum ether/ethyl acetate, 2:1 gave 0.073 g (38%) of
dibromopyridone 18 as a pale yellow oil: 1H NMR
(300 MHz, CDCl3) d 0.94 (t, J=6.9 Hz, 3H), 1.00 (t,
J=7.5 Hz, 3H), 1.35–1.48(m, 4H), 1.54–1.62 (m, 2H),
1.72 (sextet, J=7.5 Hz, 2H), 2.78(t, J=7.4 Hz, 2H),
3-(3-Oxocyclohexyl)-6-pentyl-1-propyl-2-pyridone (20).
To a solution of 0.10 g (0.26 mmol) of 19 in 30 mL of
dry ethanol was added 0.02 g of 10% Pd/C followed by
0.5 mL of triethylamine. The mixture was shaken under
an atmosphere of H2 at 40 psig for 16 h. The solvent
was removed in vacuo and the residue was chromato-
graphed (petroleum ether/ethyl acetate 2.5:1) to give
4.04 (t, J=7.8Hz, 2H), 7.08 (s, 1H);
13C NMR
(75.5 MHz, CDCl3) d 11.2, 13.8, 22.1, 22.2, 27.4, 31.6,
32.9, 48.6, 98.5, 113.7, 143.8, 147.6, 158.6; MS (EI) m/z
365 (25), 294 (31), 280 (28), 267 (100); HRMS calcd for
C13H19Br2NO: 362.9834, found 362.9826.
1
0.035 g (44%) of ketone 20 as a colorless oil: H NMR
(300 MHz, CDCl3) d 0.90 (t, J=7.5 Hz, 3H), 0.99 (t,
J=7.5 Hz, 3H), 1.36–1.43 (m, 4H), 1.60–1.74 (m, 4H),
1.78–1.84 (m, 2H), 2.03–2.07 (m, 2H), 2.38–2.67 (m,
6H), 3.20–3.25 (m, 1H), 3.96 (t, J=7.8Hz, 2H), 5.98(d,
J=7.2 Hz, 1H), 7.04 (d, J=7.2 Hz, 1H); 13C NMR
(75.5 MHz, CDCl3) d 11.4, 13.9, 22.1, 22.4, 25.1, 28.5,
30.0, 31.4, 32.8, 39.6, 41.3, 45.7, 45.9, 104.9, 131.3,
133.8, 147.7, 162.4, 211.6; MS (EI) m/z 303 (76), 275
(55), 260 (100), 234 (80); HRMS calcd for C19H29NO2:
303.2198, found 303.2199.
3,5-Dibromo-6-pentyl-1-propyl-2-pyridone (18). To
a
stirred solution of 0.33 g (1.59 mmol) of 6-pentyl-3-
propyl-2-pyridone (15) in 2.5 mL of glacial acetic acid at
ambient temperature was added dropwise 0.59 g (3.5
mmol) of bromine in 3 mL of acetic acid. The reaction
mixture was stirred at ambient temperature for 10 h,
poured into saturated aqueous NaHCO3 and extracted
with ether. The ether extract was washed with successive
portions of water and brine, dried (MgSO4) and the
solvent removed in vacuo. The residue was chromato-
graphed (petroleum ether/ethyl acetate, 10:1) to give
0.50 g (86%) of 18, which was identical to the material
described above.
cis-3-(3-Hydroxycyclohexyl)-6-pentyl-1-propyl-2-pyridone
(5). To a solution of 0.028g (0.092 mmol) of ketone 20
in 1 mL of dry ethanol at 0 ꢀC was added 0.03 g (0.79
mmol) of NaBH4, the reaction mixture was allowed to
warm to ambient temperature and stirred for 18h. The
reaction was quenched with 10% aqueous HCl, extrac-
ted with ether, dried (MgSO4) and the solvent was
removed in vacuo. Chromatography (petroleum ether/
ethyl acetate, 1:1) gave 0.019 g (68%) of 5 as a colorless
5-Bromo-3-(3-oxocyclohex-1-enyl)-6-pentyl-1-propyl-2-
pyridone (19), A. To a solution of 0.66 g (1.8mmol) of
dibromopyridone 18 in 4 mL of DMF at ambient tem-
perature was added 0.26 g (2.7 mmol) of cyclohex-2-en-
1-one, 0.29 g (2.7 mmol) of anhydrous Na2CO3, 0.020 g
(0.09 mmol) of Pd(OAc)2 and 0.055 g (0.18mmol) of tri-
o-tolylphosphine. The mixture was heated at 120 ꢀC for
12 h under an atmosphere of argon, cooled poured into
brine and extracted with ether. The ether extracts were
washed with three portions of brine, dried (MgSO4) and
the solvent was removed in vacuo. The residue was
purified by chromatography (petroleum ether/ethyl ace-
tate 30:1) to give 0.35 g (51%) of 19 as a green solid: mp
83–84 ꢀC; 1H NMR (300 MHz, CDCl3) d 0.95 (t, J=6.9
Hz, 3H), 1.02 (t, J=7.2 Hz, 3H), 1.40–1.45 (m, 4H),
1.62–1.76 (m, 4H), 2.05–2.13 (m, 2H), 2.46 (t, J=6.4
Hz, 2H), 2.75 (t, J=5.7 Hz, 2H), 2.82 (t, J=7.8Hz,
2H), 4.02 (t, J=7.8Hz, 2H), 6.48(s, 1H), 7.50 (s, 1H);
13C NMR (75.5 MHz, CDCl3) d 11.3, 13.8, 22.1, 22.3,
22.9, 27.5, 28.2, 31.7, 33.2, 37.5, 47.5, 99.6, 127.8, 128.4,
140.6, 149.2, 157.8, 159.8, 199.9. Anal. calcd for
1
oil: H NMR (300 MHz, CDCl3) d 0.95 (t, J=7.0 Hz,
3H), 1.01 (t, J=7.2 Hz, 3H), 1.22–1.30 (m, 4H), 1.36–
1.49 (m, 4H), 1.62–1.74 (m, 4H), 1.83–1.87 (m, 2H),
2.00–2.04 (m, 2H), 2.14 (d, J=10.7 Hz, 1H), 2.57 (t,
J=7.9 Hz, 2H), 2.91 (t, J=12.1 Hz, 1H), 3.73–3.81 (m,
1H), 3.97 (t, J=7.8Hz, 2H), 5.99 (d, J=7.2 Hz, 1H),
7.06 (d, J=7.3 Hz, 1H); 13C NMR (75.5 MHz, CDCl3)
d 11.4, 13.9, 22.1, 22.4, 24.3, 28.5, 31.1, 31.5, 32.8, 35.4,
35.9, 41.2, 45.8, 70.8, 105.1, 132.9, 133.4, 146.7, 162.8;
MS (EI) m/z 305 (35), 287 (100), 244 (65), 189 (80);
HRMS calcd for C19H31NO2: 305.2355, found
305.2355.
trans-3-(3-Hydroxycyclohexyl)-6-pentyl-1-propyl-2-pyri-
done (21). To a solution of 0.044 g (0.15 mmol) of
ketone 20 in 1 mL of dry THF at À78 ꢀC was added

J. W. Huffman et al. / Bioorg. Med. Chem. 9 (2001) 2863–2870
2869
0.31 mL (0.31 mmol) of 1.0 M potassium tri-sec-butyl-
borohydride (K-Selectride) in THF. The reaction mix-
ture was stirred at À78 ꢀC for 2 h, allowed to warm to
ambient temperature and stirred for 1 h. The reaction
was quenched with 1 mL of water and 5 mL of ethanol
followed by 2 mL of 15% aqueous NaOH and 2 mL of
30% H2O2. After extraction with three portions of
ether, the combined extracts were washed with brine
and dried (MgSO4). The solvent was removed in vacuo
and the crude material was chromatographed (petro-
leum ether/ethyl acetate, 1:1) to give 0.027 g (61%) of
was resuspended in 3 mL of buffer B (50 mM Tris–HCl,
1 mM EDTA, 3 mM MgCl2, pH 7.4) to yield a protein
concentration of approximately 1 mg/mL. The tissue
preparation was divided into equal aliquots, frozen on
dry ice, and stored at À70 ꢀC. Binding was initiated by
the addition of 40–50 mg membrane protein to silanized
tubes containing [3H]CP-55,940 (102.9 Ci/mmol) and a
sufficient volume of buffer C (50 mM Tris–HCl, 1 mM
EDTA, 3 mM MgCl2, and 5 mg/mL fatty acid free
BSA, pH 7.4) to bring the total volume to 0.5 mL. The
addition of 1 mM unlabelled CP-55,940 was used to
assess nonspecific binding. Following incubation (30 ꢀC
for 1 h), binding was terminated by the addition of 2
mL of ice cold buffer D (50 mM Tris–HCl, pH 7.4, plus
1 mg/mL BSA) and rapid vacuum filtration through
Whatman GF/C filters (pretreated with poly-
ethyleneimine (0.1%) for at least 2 h). Tubes were rinsed
with 2 mL of ice cold buffer D, which was also filtered,
and the filters subsequently rinsed twice with 4 mL of
ice cold buffer D. Before radioactivity was quantitated
by liquid scintillation spectrometry, filters were shaken
for 1 h in 5 mL of scintillation fluid.
1
alcohol 21 as a pale yellow oil: H NMR (500 MHz,
CDCl3) d 0.91 (t, J=6.9 Hz, 3H), 0.97 (t, J=7.3 Hz,
3H), 1.21 (t, J=10.8Hz, 1H), 1.32–1.51 (m, 7H), 1.56–
1.65 (m, 3H), 1.69 (sextet, J=7.4 Hz, 2H), 1.78–1.95 (m,
3H), 2.04 (br d, J=13.8Hz, 1H), 2.56 (t, J=7.8Hz,
2H), 3.25 (br t, J=9.2 Hz, 1H), 3.94 (d, J=5.5 Hz, 1H),
3.96 (d, J=5.5 Hz, 1H), 4.20 (s, 1H), 5.99 (d, J=7.3 Hz,
1H), 7.06 (d, J=6.9 Hz, 1H); 13C NMR (125.8MHz,
CDCl3) d 11.4, 13.9, 20.5, 22.1, 22.4, 28.6, 31.2, 31.7,
32.1, 32.2, 32.8, 38.6, 45.8, 66.7, 105.2, 133.2, 134.1,
146.4, 162.9; MS (EI) m/z 305 (35), 287 (15), 262 (45),
244 (63), 234 (100); HRMS calcd for C19H31NO2:
305.2355, found 305.2354.
CP-55,940 and all cannabinoid analogues were prepared
by suspension in assay buffer from a 1 mg/mL ethanolic
stock without evaporation of the ethanol (final con-
centration of no more than 0.4%). When anandamide
was used as a displacing ligand, experiments were per-
formed in the presence of phenylmethylsulfonyl fluoride
(50 mM). Competition assays were conducted with 1 nM
[3H]CP-55,940 or 1 nM [3H]SR141716A and six con-
centrations (0.1 nM to 10 mM displacing ligands). Dis-
placement IC50 values were originally determined by
unweighted least-squares linear regression of log con-
centration-percent displacement data and then con-
verted to Ki values using the method of Cheng and
Prusoff.31
Receptor binding assays. CB1 assay. [3H]CP-55,940
(KD=690 nM) binding to P2 membranes was conducted
as described elsewhere,29 except whole brain (rather than
cortex only) was used. Displacement curves were gener-
ated by incubating drugs with 1 nM of [3H]CP-55,940. The
assays were performed in triplicate, and the results repre-
sent the combined data from three individual experiments.
CB2 assay. Human embryonic kidney 293 cells were
maintained in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% fetal clone II (HyClone, Logan,
UT, USA) and 5% CO2 at 37 ꢀC in a Forma incubator.
Cell lines were created by transfection of CB2pcDNA3
into 293 cells by the Lipofectamine reagent (Life Tech-
nologies, Gaithersburg, MD, USA). The human CB2
cDNA was provided by Dr. Sean Munro (MRC, Cam-
bridge, UK). Stable transformants were selected in
growth medium containing geneticin (1 mg/mL,
reagent, Life Technologies, Gaithersburg, MD, USA).
Colonies of about 500 cells were picked (about 2 weeks
post-transfection) and allowed to expand, then tested
for expression of receptor mRNA by northern blot
analysis. Cell lines containing moderate to high levels of
receptor mRNA were tested for receptor binding prop-
erties. Transfected cell lines were maintained in DMEM
with 10% fetal clone II plus 0.3–0.5 mg/mL geneticin
and 5% CO2 at 37 ꢀC in a Forma incubator.
Acknowledgements
The work at Clemson University was supported by
grant DA03590, and that at The Medical College of
Virginia, Virginia Commonwealth University by grant
DA03672, both from the National Institute on Drug
Abuse. High resolution mass spectral data were deter-
mined by the Mass Spectrometry Laboratory, School of
Chemical Sciences, University of Illinois. We thank Drs.
William T. Pennington and Mariusz Krawiec for carry-
ing out the X-ray structure determination. The purchase
of the JEOL 500 MHz NMR instrument was supported
by grant CHE-9700278from the NSF Chemical Instru-
mentation Program.
The current assay is a modification of Compton et al.30
Cells were harvested in phosphate-buffered saline con-
taining 1 mM EDTA and centrifuged at 500g. The cell
pellet was homogenized in 10 mL of solution A (50 mM
Tris–HCl, 320 mM sucrose, 2 mM EDTA, 5 mM
MgCl2, pH 7.4). The homogenate was centrifuged at
1600g (10 min), the supernatant saved, and the pellet
washed three times in solution A with subsequent cen-
trifugation. The combined supernatants were cen-
trifuged at 100,000g (60 min). The (P2 membrane) pellet
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