Resorcinol-sn-glycerol derivatives: Novel 2-Arachidonoylglycerol mimetics endowed with high affinity and selectivity for cannabinoid type 1 receptor

Article abstract of DOI:10.1021/jm200529h

Since the discovery of the endocannabinoid system, evidence has been progressively accumulating to suggest that 2-arachidonoylglycerol (2-AG) rather than anandamide (AEA) is the endogenous ligand for both cannabinoid (CB) receptors. Moreover, other studies have shown that another lipid molecule, 2-arachidonyl-glycerol ether (2-AGE, noladin ether), which acts as a full agonist at cannabinoid receptors, might occur in tissues. Having previously designed a resorcinol-AEA hybrid model, in this paper we have explored the cannabinoid receptor binding properties, the CB₁ functional activity, and the stability to plasma esterases of a novel series of compounds characterized by the conversion of the amide head into the glycerol-ester or glycerol-ether head, typical of 2-AG or the "putative" endocannabinoid 2-AGE, respectively. Glyceryl esters 39 and 41 displayed greater potency for CB₁ (K_i in the nanomolar range) than for CB₂ receptors plus the potential to be exploited as useful hits for the development of novel 2-AG mimetics. (Figure presented)

Full text of DOI:10.1021/jm200529h

Article
pubs.acs.org/jmc
Resorcinol-sn-Glycerol Derivatives: Novel 2-Arachidonoylglycerol
Mimetics Endowed with High Affinity and Selectivity for
Cannabinoid Type 1 Receptor
̂
Antonella Brizzi,*,† Maria Grazia Cascio,‡,⊥ Maria Frosini,§ Alessia Ligresti,‡ Francesca Aiello,∥
Irene Biotti,§ Vittorio Brizzi,† Roger Guy Pertwee,⊥ Federico Corelli,*,† and Vincenzo Di Marzo‡
†Dipartimento Farmaco Chimico Tecnologico, Universita
̀
degli Studi di Siena, Via A. De Gasperi 2, 53100 Siena, Italy
‡Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34,
80078 Pozzuoli, Napoli, Italy
§Dipartimento di Neuroscienze, Sezione di Farmacologia, Universita
̀
degli Studi di Siena, Via A. De Gasperi 2, 53100 Siena, Italy
̂
∥Dipartimento di Scienze Farmaceutiche, Universita
̀
della Calabria, 87036 Arcavacata di Rende, Cosenza, Italy
⊥Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, Scotland, U.K.
S
* Supporting Information
ABSTRACT: Since the discovery of the endocannabinoid
system, evidence has been progressively accumulating to suggest
that 2-arachidonoylglycerol (2-AG) rather than anandamide
(AEA) is the endogenous ligand for both cannabinoid (CB)
receptors. Moreover, other studies have shown that another
lipid molecule, 2-arachidonyl-glycerol ether (2-AGE, noladin
ether), which acts as a full agonist at cannabinoid receptors,
might occur in tissues. Having previously designed a
resorcinol−AEA hybrid model, in this paper we have explored
the cannabinoid receptor binding properties, the CB1 functional
activity, and the stability to plasma esterases of a novel series of
compounds characterized by the conversion of the amide head into the glycerol-ester or glycerol-ether head, typical of 2-AG or
the “putative” endocannabinoid 2-AGE, respectively. Glyceryl esters 39 and 41 displayed greater potency for CB1 (Ki in the
nanomolar range) than for CB2 receptors plus the potential to be exploited as useful hits for the development of novel 2-AG
mimetics.
idonoyl-ethanolamine)4a and 2-AG (2-arachidonoyl-glycer-
INTRODUCTION
■
ol),4b,c whose pharmacological activity and metabolic pathways
Only in the last 20 years have the endogenous counterparts of
(−)-trans-Δ9-tetrahydrocannabinol (THC), the main psycho-
have been carefully investigated; moreover, the brain seems to
produce at least three other chemically similar “endocannabi-
active principle in marijuana, been identified, opening the way
noids”: 2-arachidonyl-glycerol ether (2-AGE, noladin ether),8
for the identification of a rather complex innate signaling
N-arachidonoyl-dopamine (NADA),9 and virodhamine10
system of local lipid mediators, better known as the
endocannabinoid system (ES).1 Interest in investigating and
(Chart 1). More recently, a nonapeptide known as
“hemopressin” has been proposed to be the first endogenous
antagonist/inverse agonist of CB1 receptors, following its
detection in the brain.11
understanding more about the (patho)physiological functions
of this plastic system, characterized by at least two specific G-
protein-coupled receptors, named CB1 and CB2,3 their
2
endogenous ligands,4 and the enzymatic pathways for their
biosynthesis5 and degradation,5a,6 is increasing due to the
multiplicity of endocannabinoid actions and targets and the
complexity of endocannabinoid regulation and signaling.
Indeed, information about this lipid signaling system is still
“evolving” as new bioactive endocannabinoid-related molecules,
non-CB1 non-CB2 receptors for endocannabinoids and
enzymes for endocannabinoid pathways are being recognized
every year.7
2-AG is a unique monoglyceride of arachidonic acid esterified
at the sn-2 position, the levels of which in the brain are much
higher than those of AEA.4c,12 It is able to bind as a full agonist
to both CB1 and CB2 cannabinoid receptors.13 While 2-AG
exhibits a lower CB1 affinity than anandamide, evidence has
progressively accumulated to suggest that it may be the main
endogenous agonist for both cannabinoid receptors, eliciting
biological activities as a signaling molecule, for example in the
Two arachidonoyl-derivatives are still referred to as the
Received: April 29, 2011
“major” endocannabinoids, i.e. anandamide (AEA, N-arach-
Published: November 1, 2011
© 2011 American Chemical Society
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Chart 1. Chemical Structures of AEA, 2-AG, and the other
Proposed Endocannabinoids
Figure 1. General chemical structure of new synthesized compounds.
features of these molecules are: (i) the rigid aromatic backbone,
(ii) the linear or branched aliphatic tail, and (iii) the flexible
alkyloxy chain bearing the ester/ether glyceryl head. These
modifications were carried out to replace the metabolically
unstable polyunsaturated fatty acid,24 typical of endocannabi-
noids, with a stable aromatic scaffold, characteristic of the
phytocannabinoid THC, containing the suitable pharmaco-
phore determinants for cannabinoid receptor recognition.
Moreover, because the available literature on the structure−
activity relationships of arachidonoylglycerol analogues sug-
gested that 1(3)-arachidonoylglycerol may exhibit CB1 and CB2
receptor binding affinities similar to or only slightly lower than
that of 2-AG,25 sn-1(3)-glycerol ester regioisomers were
prepared and tested in cannabinoid receptor binding assays.
In view of the generally high susceptibility of the ester
functionality to plasma esterases, the in vitro stability of one of
the new compounds in the presence of human plasma was also
assessed. Finally, because the cytochrome enzyme superfamily
is involved in the primary oxidation of several endogenous and/
or exogenous compounds,26 with lipophilicity being one of the
major factors in the molecular recognition of different
substrates,27 the metabolic stability of the same compound
was investigated, using an in vitro human recombinant
cytochrome P450 3A4 assay (hCYP3A4) coupled with MS
detection.28
nervous system, i.e. in neuroprotection and neuromodulation,
in the immune system, in cell proliferation, in inflammatory
responses, and in the cardiovascular system.14 Because 2-AG is
a metabolically labile molecule, rapidly hydrolyzed by the
monoglyceride lipase (MAGL),15 it has been somewhat
challenging to assess its biological activity precisely, especially
in vivo; hence, 2-AGE, the ether analogue of 2-AG, was
synthesized4b,16 as a stable chemical tool and tested for its
binding activity at cannabinoid receptors.8a It has also been
postulated to be a novel endocannabinoid.8a However, its
proposed role as an endogenously occurring compound is still
under debate8b,17 and therefore it is better referred to as a
“putative” endocannabinoid, 2-AGE has been shown to act as a
full agonist at both cannabinoid receptor subtypes.8a,18,19
Furthermore, 2-AGE exerts typical cannabimimetic activity in
vivo (i.e., hypothermia, antinociception, decreased locomotor
activity) and has been shown to reduce the intraocular pressure
through the ocular CB1 receptor.20
There has been a growing interest in the development of new
compounds that selectively regulate and/or modify the action
and the levels of endocannabinoids, and many attempts have
been made to obtain stable structural analogues of 2-AG21 and
novel homologues and isomers of 2-AGE22 to investigate the
structure−affinity relationships underlying the biological
activity of alkyl glyceryl ethers. During our research in the
cannabinoid field, we have designed and developed an attractive
medicinal chemistry template23 which turned out to produce
potent cannabimimetic ligands (Chart 2). To validate our
CHEMISTRY
■
As depicted in Scheme 1, the bromoalkyl-glycerol esters 3−6
were obtained in 60−80% yield by reacting commercially
Scheme 1. Synthesis of Protected Compounds 3−6 and 7−
a
10
Chart 2. Proposed Pharmacophore Model for the
Resorcinol-AEA “Hybrids”
a
Reagents: (i) Br(CH2)nCOOH, DCC, DMAP, dry THF, rt,
overnight; (ii) Br(CH2)nBr, NaH, dry THF, 0 °C to reflux, overnight
available cis-1,3-benzylideneglycerol 1 or (±)-solketal 2 with
the appropriate bromoacids in the presence of N,N′-
dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)-
pyridine (DMAP). On the other hand, etherification of various
dibromoalkanes with cis-1,3-benzylideneglycerol 1 in dry THF
in the presence of sodium hydride afforded the bromoalkyl-
glycerol ethers 7−10 in 60−75% yield.
resorcinol−AEA hybrid model, a novel series of 23 compounds
chemically characterized by the conversion of the amide head
into the glycerol-ester or glycerol-ether head (Figure 1), typical
of 2-AG and 2-AGE, respectively, were synthesized and their
cannabimimetic properties evaluated. Important chemical
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a
Scheme 2. Synthesis of Tested Compounds 34−52 and 53−56
a
Reagents: (i) acetone, K2CO3, KF, reflux, 48 h; (ii) EtOH/H2O = 1/1, 37% HCI, 80−90 °C, 3 h; (iii) 60% acetic acid, EtOH, 40−50 °C, 3 h.
The synthetic route for the final products 34−56 is shown in
Scheme 2. Appropriate alkyl-resorcinols, i.e. 5-pentylresorcinol
(olivetol), 4-hexylresorcinol, and 5-(1,1-dimethylheptyl)-
resorcinol, were alkylated with intermediates 3−10 in dry
acetone in the presence of potassium carbonate and potassium
fluoride to furnish the protected compounds 11−33 in
moderate (55%) to good (70%) yields. Although O-alkylation
of various resorcinols usually affords both monoalkylated and
dialkylated products,23 in this case, no dialkylated compound
was isolated.
and a moderate or good selectivity toward CB2. These binding
data suggest that the length of the alkyloxy spacer markedly
influences CB1 receptor affinity, while it does not alter the
interaction with CB2 receptor. In fact, lengthening the chain
from 10 (39 and 41) to 11 (42) or shortening it to seven
carbon atoms (38 and 40) dramatically decreased CB1 Ki values
from 10 (38) to a thousand times (40 and 42) with no change
in CB2 affinity.
Regarding the group of resorcinol-sn-2-glycerol ethers 43−
52, replacement of the carbonyl functionality with a methylene
group proved to be detrimental for cannabinoid receptor
affinity properties. Only compounds 43, 49, and 50 behaved as
quite potent cannabinoid receptor ligands, showing submicro-
molar affinity values for both receptor subtypes. In good
agreement with previous published results for the 2-AGE
series,21b the shorter alkyl chain, characterizing all these
derivatives, seemed to yield the best results, whereas, once
again, the 3-hydroxy-5-(1-methyloctan-2-yl)phenoxy backbone
(49 and 50) possessed the optimal chemical properties for
interacting with both cannabinoid receptors.
Finally, the regioisomeric resorcinol-sn-1(3)-glycerol esters
53−56 possessed a weak ability to interact with cannabinoid
receptors. In fact, the shift of the acyl “head” from position 2 to
position 1(3) of glycerol diminished significantly the
cannabinoid receptor affinities and specifically caused a
complete loss of interaction with the CB1 receptor subtype,
independently of the aromatic scaffold and the alkyloxy chain
length. However, all these derivatives, though not very potent,
yielded ligands selective toward CB2, exhibiting Ki values in the
low micromolar range. In agreement with our previous
results,23c the best receptor interaction occurs when a 5-(2-
methyloctan-2-yl) tail (compound 56, Ki CB2 = 0.31 μM) is
present.
Finally, cleavage of the benzylidene or acetonide protecting
groups by treatment with concentrated HCl29 and 60% acetic
acid,22a respectively, gave the final compounds 34−56 in 75−
90% yield.
RESULTS AND DISCUSSION
■
In Vitro CB1 and CB2 Receptor Binding Assay. All the
newly synthesized compounds were evaluated for their affinity
at recombinant human CB1 and CB2 receptors stably
overexpressed in HEK-293 cells, and the results are
summarized in Table 1.
In the series of resorcinol-sn-2-glycerol esters 34−42, almost
all compounds, with the only exception of 34, 36, and 37,
behaved as potent cannabinoid receptor ligands, exhibiting
affinity values in the submicromolar or nanomolar range of
concentration. In agreement with our previous structure−
affinity relationship observations,23a,c the most potent com-
pounds were those characterized by the presence of a 5-(2-
methyloctan-2-yl) tail (40−42) and/or an alkyloxy flexible
chain of 10 carbon atoms (35, 39, 41): compound 41 proved to
be the most potent CB1 ligand (Ki 9.0 nM) endowed also with
a good CB1 selectivity (SI = 88, calculated as Ki CB2/Ki CB1
ratio). Very interesting was the opposite behavior of the two
regioisomers derived from 4-hexylresorcinol; in fact, while the
4-hexyl-sn-2-glycerol esters (36 and 37) were weak CB1
cannabinoid ligands, the 2-hexyl-sn-2-glycerol ester re-
gioisomers (38 and 39) showed submicromolar or nanomolar
CB1 affinity values (Ki CB1 0.8 μM and 56 nM, respectively)
Functional Activity of 39 and 41 at CB1 Receptors:
MF1 Mouse Brain and hCB1−CHO Cell Membranes. The
CB1 receptor activity of the compounds with the highest affinity
for this receptor subtype, i.e. 39 and 41, was evaluated by
estimating their capacity to stimulate or inhibit [35S]GTPγS
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Table 1. Structure, CB1 and CB2 Receptor Affinity (Ki Values), and Selectivity of Derivatives 34−56 and Reference Compounds
a
AEA, WIN 55,212-2, 2-AG, and 2-AGE
CB1
CB2
CB2/CB1
SI
compd
n
R′
Ki (μM)
Ki (μM)
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
7
5-(CH2)4CH3
5.60 ± 1.43
5.60 ± 2.03
>10
7.87 ± 2.01
0.87 ± 0.06
7.87 ± 1.54
7.87 ± 3.21
1.80 ± 0.22
1.80 ± 0.21
0.79 ± 0.08
0.79 ± 0.07
0.80 ± 0.08
0.91 ± 0.02
1.16 ± 0.09
>10
10
7
5-(CH2)4CH3
4-(CH2)5CH3
10
7
4-(CH2)5CH3
>10
2-(CH2)5CH3
0.80 ± 0.32
0.06 ± 0.01
1.12 ± 0.05
0.01 ± 0.002
2.71 ± 0.78
0.43 ± 0.03
1.86 ± 0.28
>10
10
7
2-(CH2)5CH3
32
88
5-C(CH3)2(CH2)5CH3
5-C(CH3)2(CH2)5CH3
5-C(CH3)2(CH2)5CH3
5-(CH2)4CH3
10
11
5
7
5-(CH2)4CH3
9
5-(CH2)4CH3
11
5
5-(CH2)4CH3
3.78 ± 1.68
>10
>10
4-(CH2)5CH3
1.13 ± 0.96
2.39 ± 1.02
0.19 ± 0.05
0.11 ± 0.01
0.38 ± 0.03
1.67 ± 0.10
7.91 ± 1.13
10.44 ± 0.55
4.82 ± 0.90
0.31 ± 0.06
9
4-(CH2)5CH3
>10
5
C(CH3)2(CH2)5CH3
C(CH3)2(CH2)5CH3
C(CH3)2(CH2)5CH3
C(CH3)2(CH2)5CH3
5-(CH2)4CH3
0.17 ± 0.08
0.27 ± 0.06
2.94 ± 1.01
1.04 ± 0.04
>10
7
9
11
4-(CH2)5CH3
>10
2-(CH2)5CH3
>10
5-C(CH3)2(CH2)5CH3
>10
AEA
0.07
WIN 55,212−2
0.02
0.002
1.4
0.1
3
b
2-AG
0.47
c
2-AGE
0.02
>3
a
Data represent mean value ± SEM for three separate experiments performed in duplicate and are expressed as Ki (μM), for CB1 and CB2 binding
assays. Reference compounds were tested under the same conditions in this study. Anandamide was tested in the presence of PMSF (100 nM). na =
IC50 > 10 in the preliminary screening carried out with rat brain and spleen membranes. Binding affinity constants of the most potent compounds
b
c
(Ki ≤ 1 μM) are highlighted in bold as well as the most selective compounds for CB1 and CB2. Reference 24. Reference 8a.
binding to G proteins using MF1 mouse brain membranes.
Dose−response curves for both compounds and the potent
nonselective CB1/CB2 receptor agonist CP-55940 are shown in
Figure 2.
100 nM (compound 41) and 1 μM (compound 39), these
compounds did not produce any significant rightward shift in
the log concentration−response curve of CP-55940 (Figure 3).
Next, we have evaluated the ability of both compounds to
stimulate or inhibit [35S]GTPγS binding to G proteins using
CHO cells stably transfected with the human CB1 receptor and
the nonselective CB1/CB2 receptor agonist CP-55940 as active
control (data not shown). Experimental results showed that
both compounds did not produce any detectable effect in this
assay (Figure 4), thus suggesting that they are more efficacious
at antagonizing the mouse versus the human receptor, although
behaving overall as very weak CB1 antagonists. This rather
surprising result led us to evaluate the chemical stability of
compound 41 under the experimental conditions by incubating
it in buffer at 30 °C for 90 min and checking by mass analysis
after 60 and 90 min the presence of hydrolysis products in the
sample. The experimental results indicated that 41 was stable
under the conditions routinely used for this biological assay,
thus arguing against the possibility that chemical ester
hydrolysis was the cause of the low efficacy of the compound.
However, the hypothesis of a rapid acyl migration from the
Concentrations ranging from 1 nM up to 10 μM were tested
and both EC50 and Emax values were considered (95%
confidence limits in brackets). Compounds 41 and 39
stimulated [35S]GTPγS binding to mouse whole brain
membranes with high potency (EC50 = 6.3 nM (0.6 and
66.9) and 78.4 nM (9.7 and 635), respectively) (Figure 2).
However, they exhibited very low efficacy, Emax = 14.7% (10.5
and 19.0) and 11.4% (5.5 and 17.3) as compared to the potent
full cannabinoid receptor agonist CP-55,940 [EC50 = 42 nM
(22.4 and 78.8) and Emax = 104% (93.3 and 115.2)]. Of the two
tested compounds, 41 displayed higher potency than
compound 39.
Because both compounds showed a very low efficacy when
tested per se for their effect on [35S]GTPγS binding to mouse
membranes, their ability to antagonize CP-55940-induced
stimulation of [35S]GTPγS binding to mouse brain membranes
was also investigated. Experimental results showed that at
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dependent on the experimental conditions.12,31 Acyl migration
for these compounds would be relevant in the functional assays
because the two regioisomers, i.e. 41 versus 56 and 39 versus
55, showed a significant difference in the relative potencies (see
Table 1).
Stability to Plasma Esterases. The enzymatic stability
study was performed in human plasma and in water as control,
according to the method previously described32 with slight
modifications. Known aliquots of compound 41 from a stock
solution were added to plasma or water (final concentrations of
41 being 1 × 10−4 and 2 × 10−4 M) and incubated at 37 °C for
specified time intervals (0, 15, 30, 60, and 120 min). At the end
of the incubation period, the reaction was stopped by addition
of acetonitrile and each sample, performed in triplicate, was
subjected to mass analysis.
Independently of the test compound concentration, reverse-
phase LC-MS analysis showed no significant hydrolytic activity
of human plasma toward compound 41 compared to the water
control (Figure 5).
Figure 2. Effect of CP-55940 (n = 6), 41 (GL-4, n = 8), and 39 (GL-3,
n = 6) on the level of [35S]GTPγS binding to mouse whole brain
membranes.
Figure 5. Mean (± SEM) changes in 41 concentration determined in
plasma or water solutions incubated at 37 °C for different times (0−
120 min) (A). The compound was tested at two different
concentrations (■ 100 μM in plasma; ▲ 100 μM in water; ▼
200 μM in plasma; ◆ 200 μM in water). Changes of the UV peak area
under the experimental curve (AUC(0−120 min)) of 41 relative to each
concentration was also shown (B). The comparison between
Figure 3. Effect of 1 μM of 39 (GL-3, n = 12) and of 100 nM of 41
(GL-4, n = 4) on the mean log concentration−response curve of CP-
55940 (n = 12, n = 4, respectively) for stimulation of [35S]GTPγS
binding to mouse whole brain membranes.
AUC(0−120
of water vs plasma samples at 100 or 200 μM 41
min)
concentration was performed by using Student’s t test.
Metabolic Stability. A known concentration of compound
41 was incubated with the hCYP3A4 in the presence or
absence of NADPH as cofactor, the final enzyme concentration
corresponding to 0.1−0.2 mg/mL of microsomal protein. After
1 h incubation, the remaining percentage of compound 41 was
24% compared to the typical value of 10% for test compounds.
Therefore, a low substrate conversion was measured under
these experimental conditions, indicating a notably metabolic
stability for compound 41.
CONCLUSIONS
■
Analysis of the binding assay results for this novel series of 23
compounds was multifaceted, and it was not possible to find a
linear and rational trend in the structure−affinity relationships.
In fact, even if all the synthesized compounds shared the
hypothesized key pharmacophoric features for interaction with
cannabinoid receptors, most of them behaving as cannabinoid
receptor ligands, replacement of the amide head with a glycerol-
ester or a glycerol-ether head affected the affinity in a complex
manner, thus leading to compounds expressing very different
Figure 4. Mean log concentration−reponse curves of 39 (GL-3, (A))
and 41 (GL-4, (B)) (n = 6,7). Each symbol represents the mean
percentage change in binding of [35S]GTPγS to hCB1−CHO cell
membranes ± SEM.
2-position to the 1- or 3-position could still be possible. In fact,
this process is characteristic of 2-monoglycerides30 and the
conversion of 2-AG to 1(3)-AG is well-known and highly
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data are within ±0.4% of the theoretical values. All the tested
compounds possessed a purity >95.0%.
affinity values and cannabinoid receptor subtype selectivity. The
carbonyl group seemed to play a crucial role in the cannabinoid
receptor interaction because derivatives having an ester linkage
as a structural feature still behaved as cannabinoid ligands, while
those characterized by an ether bridge showed weak
cannabinoid ligand properties. Interestingly, unlike endocanna-
binoids, in which the metabolically stable glyceryl ether moiety
still endowed the compounds with high cannabinoid receptor
affinity, our hybrids appeared to be substantially more sensitive
to structural changes in the amide moiety, yielding ether
derivatives with low affinities. Moreover, our results underline
the significant influence of the alkyloxy-chain length and the
lipophilic tail on the cannabinoid receptor interaction. The in
vitro functional data presented here suggest that these
compounds exhibit only little efficacy in the [35S]GTPγS
binding to mouse brain membranes and did not show any
detectable effect in the same assay to hCB1−CHO cell
membranes. Last, because it is obvious that any investigation
of the functional activity of these resorcinol-sn-glycerol
derivatives must take into consideration their stability profile,
our findings suggest that these novel compounds are not very
sensitive to chemical and enzymatic hydrolysis and possess
acceptable metabolic stability. These latter data, while
indicating that our novel compounds may represent useful
leads for the future development of novel 2-AG mimetics, also
leave open the question of the reasons underlying their very
weak functional activity, at least in our in vitro assays. It is
possible that, like 2-AG,31 compound 41 undergoes rapid
isomerization to the 1(3)-glycerol ester during the GTPγS
binding assay and that this leads to loss of functional activity.33
General Procedure for the Synthesis of Bromoesters 3−6.
To a mixture of the appropriate bromoacid (1.2 equiv), (±)-solketal
(or cis-1,3-benzylideneglycerol, 1.0 equiv), and DMAP (0.3 equiv) in
dry THF (15 mL), a solution of DCC (1.2 equiv) in the same dry
solvent (5 mL) was added, under stirring and nitrogen atmosphere;
then, the reaction mixture was kept at room temperature overnight.
Afterward, the crude material, obtained from evaporation of solvent,
was taken up in cold chloroform (15 mL) and the resulting cloudy
solution was filtered, washed with water (5 × 8 mL), and dried over
sodium sulfate. Chloroform was evaporated under reduced pressure to
afford a residue, which was purified by silica gel column
chromatography.
Example: cis-2-Phenyl-1,3-dioxan-5-yl 8-bromooctanoate
(3). Eluent: CHCl3. Yield: 70% (transparent oil). 1H NMR
(CDCl3): δ 7.48−7.44 (m, 2H), 7.35−7.30 (m, 3H), 5.46 (s, 1H),
4.60 (s, 1H), 4.18 (d, 2H, J = 12.6 Hz), 4.03 (d, 2H, J = 12.8 Hz), 3.30
(t, 2H, J = 6.9 Hz), 2.36 (t, 2H, J = 7.4 Hz), 1.89−1.72 (m, 2H), 1.69−
1.55 (m, 2H), 1.43−1.20 (mm, 6H). Anal. (C18H25BrO4 (385.29)) C,
H, N.
Example: (±)-(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl 11-
Bromoundecanoate (6). Eluent: CHCl3. Yield: 75% (white pasty
solid); mp <30 °C (K). 1H NMR (CDCl3): δ 4.24−4.21 (m, 1H), 4.05
(t, 2H, J = 4.0 Hz), 3.99 (dd, 1H, J = 6.1 Hz, J = 7.7 Hz), 3.66 (dd, 1H,
J = 6.3 Hz, J = 7.9 Hz), 3.32 (t, 2H, J = 6.9 Hz), 2.26 (t, 2H, J = 7.4
Hz), 1.81−1.71 (m, 2H), 1.55−1.51 (m, 2H), 1.35 (s, 3H), 1.29 (s,
3H), 1.27−1.10 (mm, 12H). Anal. (C17H31BrO4 (379.33)) C, H, N.
General Procedure for the Synthesis of bromoethers 7−10.
A mixture of cis-1,3-benzylidenglycerol (1.0 equiv) and NaH (1.2
equiv) in dry THF (10 mL) was stirred at 0 °C on an ice bath for an
half hour, under nitrogen atmosphere; then, a solution of the
appropriate dibromoalkane (1.0 equiv) in dry THF (10 mL) was
added dropwise and the reaction mixture refluxed overnight.
Afterward, the organic solvent was evaporated under reduced pressure,
the residue diluted with a saturated aqueous solution of ammonium
chloride, and the aqueous layer extracted with chloroform (4 × 15
mL). The collected extracts were washed with water, dried,
evaporated, and the raw material so obtained was purified by silica
gel column chromatography.
Example: cis-5-(6-Bromohexyloxy)-2-phenyl-1,3-dioxane
(7). Eluent: CHCl3. Yield: 72% (transparent oil). 1H NMR
(CDCl3): δ 7.52−7.47 (m, 2H), 7.39−7.31 (m, 3H), 5.54 (s, 1H),
4.32 (d, 2H, J = 11.4 Hz), 4.03 (d, 2H, J = 11.5 Hz), 3.54 (t, 2H, J =
6.5 Hz), 3.39 (t, 2H, J = 6.8 Hz), 3.25−3.23 (m, 1H), 1.92−1.78 (m,
2H), 1.69−1.59 (m, 2H), 1.53−1.42 (m, 4H). MS m/z: 344 [M + H]+
(25), 366 [M + Na]+ (100). Anal. (C16H23BrO3 (343.26)) C, H, N.
General Procedure for the Synthesis of Esters 11−19, 30−
33, and Ethers 20−29. A mixture of the appropriate resorcinol
derivative (olivetol, 4-hexylresorcinol, or 5-(1,1-dimethylheptyl)-
resorcinol, 5.0 mmol), anhydrous potassium carbonate (0.35 g, 2.5
mmol), and potassium fluoride (0.29 g, 5.0 mmol) in dry acetone (30
mL) was heated to reflux with stirring under nitrogen atmosphere for
30 min; then, a solution of the appropriate bromo derivative (3−6 and
7−10, 5.0 mmol) in dry acetone (10 mL) was added, and the reaction
mixture was maintained at reflux temperature for further 48 h.
Afterward, the reaction mixture was concentrated, diluted with
saturated ammonium chloride solution (20 mL), and extracted with
chloroform (3 × 15 mL). The organic layers were collected, dried, and
evaporated under reduced pressure to afford a residue, which was
purified by silica gel column chromatography.
EXPERIMENTAL SECTION
■
Chemicals, Materials, and Methods. All starting materials,
reagents, and solvents were purchased from common commercial
suppliers and were used as received unless otherwise indicated. 5-(1,1-
Dimethylheptyl)resorcinol was obtained with 85% yield by cleavage of
the methoxy groups from commercial 1,3-dimethoxy-5-(2-methyl-
octan-2-yl)benzene using boron tribromide in dry dichloromethane, as
already described in our previous work.23c Organic solutions were
dried over anhydrous sodium sulfate and concentrated with a Buchi
̈
rotary evaporator R-110 equipped with a KNF N 820 FT 18 vacuum
pump. Melting points were determined on a Kofler hot stage apparatus
(K) or using a Mettler FPI apparatus (2 °C/min, M) and are
uncorrected. 1H NMR spectra and 13C NMR spectra were recorded in
the indicated solvent at 25 °C on a Bruker AC200F or on a Bruker
Advance DPX400 employing TMS as internal standard and chemical
shifts are expressed as δ (ppm). The chromatography−mass
spectrometry (LC-MS) system consisted of an Agilent 1100 series
liquid chromatograph system including a 1100 MSD model VL
benchtop mass spectrometer with API-ES interface, a binary high-
pressure gradient pump (0.4 mL/min low flow rate, employing a
binary solvent system of 95/5 methanol/water), and a vacuum solvent
degassing unit. The nebulizer gas, the drying gas, the capillary voltage,
and the vaporizer temperature were set at 40 psi, 9 L/min, 3000 V, and
350 °C, respectively. Nitrogen (purity 99.995%) was used as nebulizer
gas and drying gas. UV detection was monitored at 254 nm. Mass
spectra were acquired in positive mode scanning over the mass range
m/z of 105−1500. The structures of final compounds were
unambiguously assessed by 1H NMR, 13C NMR, and MS. All
compounds were checked for purity by TLC on Merck 60 F254 silica
plates. For column chromatography, Merck 60 silica gel, 230−400
mesh, was used. Final products were purified by a Biotage flash
chromatography system with columns 12.25 mm, packed with KP-Sil,
60A, 32−63 μM. Compound purity was assessed by elemental analysis
on a Perkin-Elmer elemental apparatus model 240 for C, H, N, and the
Example: cis-2-Phenyl-1,3-dioxan-5-yl 11-[3-Hydroxy-5-(2-
methyloctan-2-yl)phenoxy]undecanoate (18). Eluent: CHCl3/
1
MeOH = 50/1. Yield: 65% (yellow oil). H NMR (CDCl3): δ 7.50−
7.48 (m, 2H), 7.40−7.33 (m, 3H), 6.43 (s, 1H), 6.36 (s, 1H), 6.19 (s,
1H), 5.54 (s, 1H), 4.70 (s, 1H), 4.27 (d, 2H, J = 12.6 Hz), 4.14 (d, 2H,
J = 13.0 Hz), 3.87 (t, 2H, J = 6.5 Hz), 2.41 (t, 2H, J = 7.4 Hz), 1.73−
1.31 (mm, 6H), 1.51−1.21 (mm, 26H), 0.84 (t, 3H, J = 6.3 Hz). MS
m/z: 605 [M + Na]+ (100). Anal. (C36H54O6 (582.81)) C, H, N.
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Example: cis-3-(2-Methyloctan-2-yl)-5-[10-(2-phenyl-1,3-di-
oxan-5-yloxy)decyloxy]phenol (28). Eluent: CHCl3/MeOH =
50/0.5. Yield: 58% (pale-yellow oil). 1H NMR (CDCl3): δ 7.56−
7.52 (m, 2H), 7.46−7.33 (m, 3H), 6.41 (s, 1H), 6.36 (s, 1H), 6.20 (s,
1H), 5.53 (s, 1H), 4.32 (d, 2H, J = 12.6 Hz), 4.02 (d, 2H, J = 12.8 Hz),
3.87 (t, 2H, J = 6.5 Hz), 3.38 (t, 2H, J = 6.9 Hz), 3.24 (s, 1H), 1.85−
1.73 (m, 2H), 1.69−1.50 (m, 4H), 1.46−1.20 (mm, 26H), 0.81 (t, 3H,
J = 6.8 Hz). MS m/z: 577 [M + Na]+ (100). Anal. (C35H54O5
(554.80)) C, H, N.
1,3-Dihydroxypropan-2-yl 8-(2-Hexyl-5-hydroxyphenoxy)-
octanoate (38). Eluent: CHCl3/MeOH = 47/3. Yield 85% (yellow
1
oil). H NMR (CDCl3): δ 6.91 (d, 1H, J = 7.9 Hz), 6.35−6.27 (m,
2H), 4.16 (dd, 1H, J = 5.1 Hz, J = 2.2 Hz), 3.91−3.80 (m, 4H), 3.68
(dd, 1H, J = 3.7 Hz, J = 11.3 Hz), 3.58 (dd, 1H, J = 5.8 Hz, J = 11.7
Hz), 2.49 (t, 2H, J = 7.5 Hz), 2.34 (t, 2H, J = 7.3 Hz), 1.76−1.64 (mm,
6H), 1.49−1.28 (mm, 12H), 0.86 (t, 3H, J = 5.0 Hz). 13C NMR
(CDCl3): δ 174.58, 157.80, 154.72, 130.08, 123.42, 106.29, 99.77,
70.40, 67.87, 65.13, 63.09, 34.25, 31.81, 30.22, 29.59, 29.32, 29.11,
29.00, 28.73, 26.01, 24.78, 22.60, 14.10. MS m/z: 433 [M + Na]+
(100). Anal. (C23H38O6 (410.54)) C, H, N.
Example: (±)-(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl 11-[3-
hydroxy-5-(2-methyloctan-2-yl)phenoxy]undecanoate (33). Eluent:
1
1,3-Dihydroxypropan-2-yl 11-(2-Hexyl-5-hydroxyphenoxy)-
CHCl3/MeOH = 50/2. Yield: 63% (yellow oil). H NMR (CDCl3):
undecanoate (39). Eluent: CHCl3/MeOH = 45/5. Yield 87%
δ 6.37 (s, 1H), 6.33 (s, 1H), 6.20−6.16 (m, 1H), 4.29−4.18 (m, 1H),
4.07−3.95 (m, 3H), 3.87−3.78 (m, 2H), 3.66 (dd, 1H, J = 6.2 Hz,
J = 7.9 Hz), 2.26 (t, 2H, J = 7.4 Hz), 1.67−1.55 (m, 2H), 1.51−1.41
(m, 2H), 1.35 (s, 3H), 1.28 (s, 3H), 1.22−1.14 (mm, 28H), 0.76 (t, 3H,
J = 6.1 Hz). MS m/z: 557 [M + Na]+ (100). Anal. (C32H54O6 (534.77))
C, H, N.
General Procedure for the Synthesis of Compounds 34−
52. To a solution of each benzylidene derivative (0.100 g) in ethanol/
water (9 mL, 2/1) was added 37% HCl (0.5 mL) and the reaction
mixture stirred at 80−90 °C. After 3 h, the solvent was evaporated
under reduced pressure and the residue was diluted with ethyl acetate.
The organic phase was washed with brine, dried over sodium sulfate,
filtered, and finally concentrated to dryness, affording a crude material
purified by silica gel column chromatography.
1
(pasty cream solid). mp <30 °C (K). H NMR (CDCl3): δ 6.91 (d,
1H, J = 7.9 Hz), 6.35 (d, 1H, J = 2.3 Hz), 6.29 (dd, 1H, J = 2.6 Hz, J =
7.9 Hz), 4.16 (dd, 1H, J = 5.0 Hz, J = 2.1 Hz), 3.90−3.80 (m, 4H),
3.69 (dd, 1H, J = 3.9 Hz, J = 11.4 Hz), 3.58 (dd, 1H, J = 5.9 Hz, J =
11.7 Hz), 2.49 (t, 2H, J = 7.5 Hz), 2.32 (t, 2H, J = 7.4 Hz), 1.79−1.47
(mm, 6H), 1.44−1.28 (mm, 18H), 0.86 (t, 3H, J = 6.4 Hz). 13C NMR
(CDCl3): δ 174.55, 157.81, 154.64, 130.12, 123.48, 106.23, 99.75,
70.41, 67.84, 65.19, 63.27, 34.14, 31.80, 30.14, 29.52, 29.39, 29.20,
29.18 (×2), 29.09, 28.97 (×2), 26.00, 24.83, 22.71, 14.08. MS m/z:
475 [M + Na]+ (100). Anal. (C26H44O6 (452.62)) C, H, N.
1,3-Dihydroxypropan-2-yl 8-[3-Hydroxy-5-(2-methyloctan-
2-yl)phenoxy]octanoate (40). Eluent: CHCl3/MeOH = 47/3.
Yield 88% (transparent oil). 1H NMR (CDCl3): δ 6.41−6.38 (m,
2H), 6.20−6.19 (m, 1H), 4.14 (d, 1H, J = 5.3 Hz), 4.07−3.81 (m,
4H), 3.69 (dd, 1H, J = 3.7 Hz, J = 11.6 Hz), 3.58 (dd, 1H, J = 6.1 Hz, J
= 11.5 Hz), 2.32 (t, 2H, J = 7.4 Hz), 1.75−1.57 (m, 4H), 1.53−1.34
(mm, 6H), 1.32−1.19 (mm, 16H), 0.81 (t, 3H, J = 6.5 Hz). 13C NMR
(CDCl3): δ 174.15, 160.02, 156.37, 152.98, 105.65, 105.53, 98.60,
70.34, 67.81, 65.20, 63.36, 44.50, 37.83, 34.06, 31.78, 30.02, 29.04
(×2), 28.90, 28.84 (×2), 25.73, 24.72, 24.63, 22.66, 14.06. MS m/z:
453 [M + H]+ (10), 475 [M + Na]+ (100). Anal. (C26H44O6 (452.62))
C, H, N.
1,3-Dihydroxypropan-2-yl 8-(3-Hydroxy-5-pentylphenoxy)-
octanoate (34). Eluent: CHCl3/MeOH = 48/2. Yield 87% (yellow
1
oil). H NMR (CDCl3): δ 6.27−6.20 (m, 3H), 4.14 (d, 1H, J = 5.2
Hz), 3.97−3.78 (m, 4H), 3.72−3.53 (m, 2H), 2.46 (t, 2H, J = 7.7 Hz),
2.31 (t, 2H, J = 7.4 Hz), 1.73−1.55 (mm, 6H), 1.34−1.24 (mm, 10H),
0.86 (t, 3H, J = 6.4 Hz). 13C NMR (CDCl3): δ 174.30, 160.03, 156.66,
145.70, 107.90, 107.18, 99.32, 70.31, 67.81, 65.17, 63.38, 36.03, 34.06,
31.51, 30.83 (×2), 28.99, 28.69, 25.70, 24.70, 22.53, 14.01. MS m/z:
397 [M + H]+ (20), 419 [M + Na]+ (100). Anal. (C22H36O6 (396.52))
C, H, N.
1,3-Dihydroxypropan-2-yl 11-[3-Hydroxy-5-(2-methyloctan-
2-yl)phenoxy]undecanoate (41). Eluent: CHCl3/MeOH = 50/2.
1,3-Dihydroxypropan-2-yl 11-(3-hydroxy-5-pentylphe-
1
Yield 86% (yellow oil). H NMR (CDCl3): δ 6.41 (s, 1H), 6.37 (s,
noxy)undecanoate (35). Eluent: CHCl3/MeOH = 50/2. Yield
1H), 6.21 (d, 1H, J = 1.7 Hz), 4.14 (d, 1H, J = 5.8 Hz), 3.94−3.80 (m,
4H), 3.68 (dd, 1H, J = 3.8 Hz, J = 11.6 Hz), 3.57 (dd, 1H, J = 6.0 Hz, J
= 11.3 Hz), 2.31 (t, 2H, J = 7.4 Hz), 1.76−1.73 (m, 2H), 1.69−1.45
(m, 4H), 1.40−1.26 (mm, 12H), 1.20−1.04 (mm, 14H), 0.81 (t, 3H, J
= 6.5 Hz). 13C NMR (CDCl3): δ 174.33, 160.00, 156.37, 152.91,
105.64, 105.48, 98.62, 70.29, 67.90, 65.18, 63.34, 44.50, 37.86, 34.15,
31.78, 30.02 (×2), 29.30, 29.17 (×2), 29.02 (×2), 28.89 (×2), 25.92,
24.85, 24.6, 22.66, 14.06. MS m/z: 517 [M + Na]+ (100). Anal.
(C29H50O6 (494.70)) C, H, N.
1
85% (yellow oil). H NMR (CDCl3): δ 6.29 (s, 1H), 6.23−6.21 (m,
2H), 4.18−4.14 (m, 1H), 3.95−3.81 (m, 4H), 3.69 (dd, 1H, J = 4.0
Hz, J = 11.6 Hz), 3.58 (dd, 1H, J = 5.8 Hz, J = 11.7 Hz), 2.45 (t, 2H,
J = 7.6 Hz), 2.33 (t, 2H, J = 7.5 Hz), 1.76−1.69 (m, 2H), 1.66−1.52
(m, 4H), 1.19−1.28 (mm, 16H), 0.86 (t, 3H, J = 6.6 Hz). 13C NMR
(CDCl3): δ 174.39, 160.09, 156.71, 145.59, 107.80, 107.12, 99.30,
70.29, 67.75, 65.21, 63.47, 36.01, 34.20, 31.44, 30.82 (×2), 29.32,
29.21, 29.12 (×2), 29.02, 25.78, 24.83, 22.51, 14.03. MS m/z: 439
[M + H]+ (100). Anal. (C25H42O6 (438.60)) C, H, N.
1,3-Dihydroxypropan-2-yl 12-[3-Hydroxy-5-(2-methyloctan-
1,3-Dihydroxypropan-2-yl 8-(4-hexyl-3-hydroxyphenoxy)-
2-yl)phenoxy]dodecanoate (42). Eluent: CHCl3/MeOH = 50/1.
octanoate (36). Eluent: CHCl3/MeOH = 47/3. Yield 89% (yellow
1
Yield 85% (yellow oil). H NMR (CDCl3): δ 6.43 (s, 1H), 6.35 (s,
1
oil). H NMR (CDCl3): δ 6.95 (d, 1H, J = 8.0 Hz), 6.40−6.35 (m,
1H), 6.20 (s, 1H), 4.19−4.14 (m, 1H), 3.92−3.84 (m, 4H), 3.67−3.55
(m, 2H), 2.33 (t, 2H, J = 7.3 Hz), 2.03−1.82 (m, 2H), 1.77−1.52
(mm, 10H), 1.47−1.07 (mm, 22H), 0.82 (t, 3H, J = 6.3 Hz). 13C
NMR (CDCl3): δ 174.25, 160.08, 156.29, 152.90, 105.46, 105.50,
98.56, 70.36, 67.88, 65.20, 63.28, 44.50, 37.90, 34.08, 31.81, 30.09
(×2), 29.50, 29.42, 29.20 (×2), 29.02 (×2), 28.89 (×2), 25.90, 24.85,
24.64, 22.70, 14.08. MS m/z: 509 [M + H]+ (10), 531 [M + Na]+
(100). Anal. (C30H52O6 (508.73)) C, H, N.
2H), 4.15 (d, 1H, J = 4.7 Hz), 4.07−3.79 (m, 4H), 3.69 (dd, 1H, J =
3.9 Hz, J = 11.7 Hz), 3.58 (dd, 1H, J = 5.9 Hz, J = 11.6 Hz), 2.50 (t,
2H, J = 7.5 Hz), 2.33 (t, 2H, J = 7.4 Hz), 1.74−1.51 (mm, 6H), 1.47−
1.28 (mm, 12H), 0.86 (t, 3H, J = 6.5 Hz). 13C NMR (CDCl3): δ
174.32, 158.33, 154.28, 130.40, 123.09, 106.44, 102.31, 70.33, 68.10,
65.22, 63.28, 34.21, 31.82, 30.02, 29.31, 29.08, 28.98, 28.81, 25.87,
24.92, 24.70, 22.58, 14.08. MS m/z: 433 [M + Na]+ (100). Anal.
(C23H38O6 (410.54)) C, H, N.
2-[6-(3-Hydroxy-5-pentylphenoxy)hexyloxy]propane-1,3-
1
1,3-Dihydroxypropan-2-yl 11-(4-Hexyl-3-hydroxyphenoxy)-
undecanoate (37). Eluent: CHCl3/MeOH = 45/5. Yield 90%
(white solid); mp = 68−70 °C (K). 1H NMR (CDCl3): δ 6.46 (d, 1H,
J = 8.2 Hz), 6.42−6.35 (m, 2H), 4.16 (dd, 1H, J = 5.4 Hz, J = 2.4 Hz),
3.95−3.80 (m, 4H), 3.73−3.69 (dd, 1H, J = 3.8 Hz, J = 11.2 Hz), 3.58
(dd, 1H, J = 5.8 Hz, J = 11.4 Hz), 2.50 (t, 2H, J = 7.5 Hz), 2.33 (t, 2H,
J = 7.4 Hz), 1.76−1.52 (mm, 6H), 1.44−1.28 (mm, 18H), 0.87 (t, 3H,
J = 6.5 Hz).
diol (43). Eluent: CHCl3/MeOH = 47/3. Yield 86% (yellow oil). H
NMR (CDCl3): δ 6.27 (d, 1H, J = 1.3 Hz), 6.24 (s, 1H), 6.27 (d, 1H, J
= 1.8 Hz), 3.89 (t, 2H, J = 6.3 Hz), 3.78 (dd, 2H, J = 4.4 Hz, J = 11.7
Hz), 3.68 (dd, 2H, J = 4.7 Hz, J = 11.7 Hz), 3.56 (t, 2H, J = 6.4 Hz),
3.49−3.42 (m, 1H), 2.47 (t, 2H, J = 7.7 Hz), 1.77−1.67 (m, 2H),
1.64−1.52 (m, 4H), 1.48−1.41 (m, 4H), 1.31−1.24 (m, 4H), 0.86 (t,
3H, J = 6.4 Hz). 13C NMR (CDCl3): δ 160.02, 156.60, 145.68, 107.82,
107.21, 99.35, 78.83, 70.37, 68.64, 63.40, 62.28, 36.12, 31.50, 30.72,
30.50, 30.05, 29.62, 26.04, 22.37, 14.06. MS m/z: 355 [M + H]+ (20),
377 [M + Na]+ (100). Anal. (C20H34O5 (354.48)) C, H, N.
13C NMR (CDCl3): δ 174.47, 158.35, 154.24, 130.36, 120.71,
106.35, 102.24, 70.28, 68.00, 65.18, 63.32, 34.14, 31.74, 30.00, 29.26
(×2), 29.18, 29.12 (×2), 29.03 (×2), 25.90, 24.83, 24.68, 22.62, 14.07.
MS m/z: 475 [M + Na]+ (100). Anal. (C26H44O6 (452.62)) C, H, N.
2-[8-(3-Hydroxy-5-pentylphenoxy)octyloxy]propane-1,3-
1
diol (44). Eluent: CHCl3/MeOH = 48/2. Yield 88% (yellow oil). H
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NMR (CDCl3): δ 6.28 (s, 1H), 6.23 (s, 1H), 6.21 (d, 1H, J = 1.9 Hz),
3.89 (t, 2H, J = 6.4 Hz), 3.77 (dd, 2H, J = 4.4 Hz, J = 11.6 Hz), 3.67
(dd, 2H, J = 4.8 Hz, J = 11.7 Hz), 3.55 (t, 2H, J = 6.5 Hz), 3.50−3.42
(m, 1H), 2.48 (t, 2H, J = 7.7 Hz), 1.76−1.69 (m, 2H), 1.66−1.49 (m,
4H), 1.41−1.23 (mm, 12H), 0.86 (t, 3H, J = 6.4 Hz). 13C NMR
(CDCl3): δ 160.18, 156.70, 145.72, 107.89, 107.20, 99.37, 78.75,
70.31, 68.58, 63.50, 62.26, 36.02, 31.53, 30.80, 30.54, 29.80, 29.63
(×2), 29.22, 25.89, 22.50, 14.08. MS m/z: 383 [M + H]+ (20), 405 [M
+ Na]+ (100). Anal. (C22H38O5 (382.53)) C, H, N.
Hz, J = 11.6 Hz), 3.67 (dd, 2H, J = 4.8 Hz, J = 11.6 Hz), 3.56 (t, 2H,
J = 6.7 Hz), 3.50−3.43 (m, 1H), 2.20 (br s, 2H, disappears on
treatment with D2O), 1.77−1.67 (m, 4H), 1.62−1.50 (mm, 6H),
1.48−1.34 (mm, 6H), 1.21 (s, 6H), 1.17−1.03 (mm, 6H), 0.82 (t, 3H,
J = 6.4 Hz). 13C NMR (CDCl3): δ 160.06, 156.47, 152.90, 105.61,
105.49, 98.71, 78.85, 70.52, 68.58, 63.60, 62.21, 44.55, 37.80, 31.66,
30.08, 29.90, 29.83, 29.61 (×2), 29.45, 29.24, 29.02 (×2), 26.00, 22.67,
14.08. MS m/z: 425 [M + H]+ (15), 447 [M + Na]+ (100). Anal.
(C26H46O5 (438.64)) C, H, N.
2-[10-(3-Hydroxy-5-pentylphenoxy]decyloxy)propane-1,3-
2-{10-[3-Hydroxy-5-(2-methyloctan-2-yl)phenoxy]-
decyloxy}propane-1,3-diol (51). Eluent: CHCl3/MeOH = 48/2.
Yield 86% (yellow oil). 1H NMR (CDCl3): δ 6.42 (d, 1H, J = 1.5 Hz),
6.36 (d, 1H, J = 1.5 Hz), 6.20 (t, 1H, J = 2.0 Hz), 3.89 (t, 2H, J = 6.5
Hz), 3.77 (dd, 2H, J = 4.4 Hz, J = 11.8 Hz), 3.67 (dd, 2H, J = 4.9 Hz, J
= 11.8 Hz), 3.55 (t, 2H, J = 6.4 Hz), 3.50−3.43 (m, 1H), 1.81−1.67
(m, 4H), 1.61−1.46 (mm, 6H), 1.41−1.23 (mm, 8H), 1.21 (s, 6H),
1.17−1.02 (mm, 8H), 0.82 (t, 3H, J = 6.5 Hz). 13C NMR (CDCl3): δ
160.10, 156.45, 152.83, 105.56, 105.35, 98.67, 78.70, 70.44, 68.43,
63.61, 62.30, 44.62, 37.88, 31.80, 30.21, 30.04, 29.92 (×2), 29.60 (×2),
29.55, 29.43, 29.34, 29.00 (×2), 26.09, 22.65, 14.10. MS m/z: 467 [M
+ H]+ (20), 489 [M + Na]+ (100). Anal. (C28H50O5 (466.69)) C, H,
N.
2-{12-[3-Hydroxy-5-(2-methyloctan-2-yl)phenoxy]-
dodecyloxy}propane-1,3-diol (52). Eluent: CHCl3/MeOH = 49/
1. Yield 87% (pale-yellow oil). 1H NMR (CDCl3): δ 6.43 (s, 1H), 6.36
(s, 1H), 6.20 (t, 1H, J = 1.7 Hz), 3.89 (t, 2H, J = 6.4 Hz), 3.77 (dd,
2H, J = 4.7 Hz, J = 11.7 Hz), 3.67 (dd, 2H, J = 4.7 Hz, J = 11.5 Hz),
3.55 (t, 2H, J = 6.6 Hz), 3.48−3.43 (m, 1H), 1.78−1.67 (m, 4H),
1.61−1.46 (mm, 6H), 1.44−1.26 (mm, 12H), 1.21 (s, 6H), 1.17−1.02
(mm, 8H), 0.82 (t, 3H, J = 6.4 Hz). 13C NMR (CDCl3): δ 160.15,
156.55, 152.80, 105.61, 105.34, 98.78, 78.90, 70.63, 68.45, 63.50,
62.16, 44.50, 37.85, 31.76, 30.30, 30.08, 30.03, 29.82 (×2), 29.60 (×2),
29.54 (×2), 29.45, 29.17, 29.01 (×2), 26.04, 22.70, 14.06. MS m/z:
495 [M + H]+ (10), 517 [M + Na]+ (100). Anal. (C30H54O5 (494.75))
C, H, N.
1
diol (45). Eluent: CHCl3/MeOH = 48/2. Yield 90% (yellow oil). H
NMR (CDCl3): δ 6.28 (s, 1H), 6.23 (s, 1H), 6.20 (d, 1H, J = 1.7 Hz),
3.88 (t, 2H, J = 6.5 Hz), 3.77 (dd, 2H, J = 4.6 Hz, J = 11.6 Hz), 3.67
(dd, 2H, J = 4.8 Hz, J = 11.7 Hz), 3.54 (t, 2H, J = 6.5 Hz), 3.49−3.42
(m, 1H), 2.47 (t, 2H, J = 7.7 Hz), 1.76−1.66 (m, 2H), 1.56−1.48 (m,
4H), 1.45−1.28 (mm, 16H), 0.86 (t, 3H, J = 6.5 Hz). 13C NMR
(CDCl3): δ 160.12, 156.80, 145.61, 107.78, 107.18, 99.35, 78.77,
70.45, 68.50, 63.45, 62.28, 36.07, 31.54, 30.72, 30.48, 29.91, 29.80
(×2), 29.61 (×2), 29.28, 26.00, 22.58, 14.06. MS m/z: 411 [M + H]+
(15), 433 [M + Na]+ (100). Anal. (C24H42O5 (410.59)) C, H, N.
2-[12-(3-Hydroxy-5-pentylphenoxy)dodecyloxy]propane-
1,3-diol (46). Eluent: CHCl3/MeOH = 50/2. Yield 87% (pale-yellow
oil). 1H NMR (CDCl3): δ 6.28 (s, 1H), 6.22 (s, 1H), 6.19 (d, 1H, J =
1.9 Hz), 3.88 (t, 2H, J = 6.4 Hz), 3.77 (dd, 2H, J = 4.7 Hz, J = 11.7
Hz), 3.67 (dd, 2H, J = 4.8 Hz, J = 11.6 Hz), 3.55 (t, 2H, J = 6.6 Hz),
3.49−3.42 (m, 1H), 2.47 (t, 2H, J = 7.7 Hz), 1.79−1.66 (m, 2H),
1.60−1.49 (m, 4H), 1.48−1.26 (mm, 20H), 0.86 (t, 3H, J = 6.5 Hz).
13C NMR (CDCl3): δ 160.08, 156.75, 145.56, 107.78, 107.16, 99.40,
78.81, 70.69, 68.42, 63.45, 62.20, 36.08, 31.52, 30.81, 30.02 (×2),
29.84, 29.75, 29.60 (×2), 29.53 (×2), 29.38, 29.12, 22.49, 14.10. MS
m/z: 461 [M + Na]+ (100). Anal. (C26H46O5 (438.64)) C, H, N.
2-[6-(4-Hexyl-3-hydroxyphenoxy)hexyloxy]propane-1,3-diol
1
(47). Eluent: CHCl3/MeOH = 47/3. Yield 88% (pale-yellow oil). H
NMR (CDCl3): δ 6.96 (d, 1H, J = 8.0 Hz), 6.41−6.35 (m, 2H), 3.90
(t, 2H, J = 6.3 Hz), 3.78 (dd, 2H, J = 4.3 Hz, J = 11.5 Hz), 3.67 (dd,
2H, J = 4.7 Hz, J = 11.5 Hz), 3.57 (t, 2H, J = 6.3 Hz), 3.49−3.42 (m,
1H), 2.49 (t, 2H, J = 7.6 Hz), 1.77−1.44 (mm, 10H), 1.42−1.23 (mm,
6H), 0.86 (t, 3H, J = 6.3 Hz). 13C NMR (CDCl3): δ 158.20, 154.19,
130.45, 122.90, 106.43, 102.34, 78.75, 70.26, 68.18, 63.35, 62.28,
31.74, 30.12, 29.90, 29.58, 29.35, 29.03, 25.90 (×2), 22.61, 14.10. MS
m/z: 391 [M + Na]+ (100). Anal. (C21H36O5 (368.51)) C, H, N.
2-[10-(4-Hexyl-3-hydroxyphenoxy)decyloxy]propane-1,3-
General Procedure for the Synthesis of Final Compounds
53−56. To a solution of each acetonide (0.100 g) in ethanol (10 mL)
was added 60% aqueous acetic acid (5 mL) and the reaction mixture
mildly heated (40−50 °C). After 3 h, the solvent was concentrated and
the residue partitioned between ethyl acetate and water, the organic
layer dried over sodium sulfate, filtered, and finally concentrated to
dryness, affording a crude material purified by silica gel column
chromatography.
1
diol (48). Eluent: CHCl3/MeOH = 48/2. Yield 86% (yellow oil). H
NMR (CDCl3): δ 6.91 (d, 1H, J = 8.0 Hz), 6.34 (dd, 1H, J = 8.0 Hz, J
= 4.0 Hz), 6.29 (d, 1H, J = 3.7 Hz), 3.83 (t, 2H, J = 7.8 Hz), 3.71 (dd,
2H, J = 4.2 Hz, J = 11.8 Hz), 3.62 (dd, 2H, J = 4.3 Hz, J = 11.7 Hz),
3.51 (t, 2H, J = 7.7 Hz), 3.43−3.40 (m, 1H), 2.45 (t, 2H, J = 6.5 Hz),
1.76−1.64 (m, 4H), 1.57−1.46 (m, 4H), 1.38−1.20 (mm, 16H), 0.81
(t, 3H, J = 6.3 Hz). 13C NMR (CDCl3): δ 158.32, 154.28, 130.55,
123.03, 106.37, 102.20, 78.74, 70.30, 68.08, 63.33, 62.41, 31.80, 30.08,
29.87 (×2), 29.60 (×2), 29.41 (×2), 29.35, 29.22, 29.01, 26.00, 22.62,
14.08. MS m/z: 425 [M + H]+ (15), 447 [M + Na]+ (100). Anal.
(C25H44O5 (424.61)) C, H, N.
2-{6-[3-Hydroxy-5-(2-methyloctan-2-yl)phenoxy]hexyloxy}-
propane-1,3-diol (49). Eluent: CHCl3/MeOH = 47/3. Yield 87%
(yellow oil). 1H NMR (CDCl3): δ 6.42 (s, 1H), 6.37 (s, 1H), 6.21 (d,
1H, J = 1.7 Hz), 3.90 (t, 2H, J = 6.3 Hz), 3.78 (dd, 2H, J = 4.5 Hz, J =
11.6 Hz), 3.68 (dd, 2H, J = 4.6 Hz, J = 11.6 Hz), 3.58 (t, 2H, J = 6.3
Hz), 3.50−3.43 (m, 1H), 1.79−1.69 (m, 2H), 1.66−1.59 (m, 4H),
1.55−1.37 (mm, 6H), 1.21 (s, 6H), 1.18−1.03 (mm, 6H), 0.82 (t, 3H,
J = 6.3 Hz). 13C NMR (CDCl3): δ 160.05, 156.56, 152.92, 105.70,
105.55, 98.71, 78.80, 70.52, 68.60, 63.48, 62.31, 44.60, 37.82, 31.80,
30.15, 30.04, 29.93, 29.62, 29.00 (×2), 25.89, 25.83, 22.70, 14.10. MS
m/z: 425 [M + H]+ (15), 447 [M + Na]+ (100). Anal. (C24H42O5
(410.59)) C, H, N.
2,3-Dihydroxypropyl 11-(3-Hydroxy-5-pentylphenoxy)-
undecanoate (53). Eluent: CHCl3/MeOH = 47/3. Yield 80%
1
(yellow oil). H NMR (CDCl3): δ 6.27−6.20 (m, 3H), 4.14 (d, 1H, J
= 5.1 Hz), 3.96−3.78 (m, 4H), 3.72−3.53 (m, 2H), 2.46 (t, 2H, J = 7.2
Hz), 2.31 (t, 2H, J = 7.4 Hz), 1.75−1.68 (m, 2H), 1.63−1.52 (m, 4H),
1.48−1.26 (mm, 16H), 0.86 (t, 3H, J = 6.5 Hz). 13C NMR (CDCl3): δ
174.64, 160.22, 156.82, 145.59, 107.92, 106.97, 99.37, 70.27, 67.92,
65.11, 63.36, 36.05, 34.13, 31.53, 30.84 (×2), 29.30, 29.17, 29.06 (×2),
29.02, 25.92, 24.83, 22.54, 14.02. MS m/z: 461 [M + Na]+ (100). Anal.
(C25H42O6 (438.60)) C, H, N.
2,3-Dihydroxypropyl 11-(4-Hexyl-3-hydroxyphenoxy)-
undecanoate (54). Eluent: CHCl3/MeOH = 47/3. Yield 78%
(white solid); mp 30−32 °C (K). 1H NMR (CDCl3): δ 6.95 (dd, 1H, J
= 2.0 Hz, J = 7.9 Hz), 6.38−6.34 (m, 2H), 4.14 (d, 1H, J = 5.5 Hz),
3.92−3.77 (m, 4H), 3.71−3.52 (m, 2H), 2.49 (t, 2H, J = 7.5 Hz), 2.32
(t, 2H, J = 7.4 Hz), 1.75−1.64 (m, 4H), 1.60−1.51 (mm, 6H), 1.47−
1.27 (mm, 14H), 0.86 (t, 3H, J = 6.4 Hz). 13C NMR (CDCl3): δ
174.35, 158.26, 154.32, 130.39, 123.05, 106.36, 102.26, 70.08, 68.01,
65.20, 63.35, 34.15, 31.76, 30.02, 29.28 (×2), 29.18, 29.14 (×2), 29.06
(×2), 25.92, 25.13, 24.85, 22.63, 14.10. MS m/z: 453 [M + H]+ (50),
475 [M + Na]+ (100). Anal. (C26H44O6 (452.62)) C, H, N.
2,3-Dihydroxypropyl 11-(2-Hexyl-5-hydroxyphenoxy)-
undecanoate (55). Eluent: CHCl3/MeOH = 47/3. Yield 75%
2-{8-[3-Hydroxy-5-(2-methyloctan-2-yl)phenoxy]octyloxy}-
1
propane-1,3-diol (50). Eluent: CHCl3/MeOH = 48/2. Yield 88%
(pasty cream solid); mp <30 °C (K). H NMR (CDCl3): δ 6.90 (d,
1
(yellow oil). H NMR (CDCl3): δ 6.42 (t, 1H, J = 1.7 Hz), 6.37 (t,
1H, J = 7.9 Hz), 6.34 (s, 1H), 6.29 (dd, 1H, J = 1.9 Hz, J = 8.00 Hz),
4.14 (d, 1H, J = 5.0 Hz), 3.96−3.78 (m, 4H), 3.72−3.53 (m, 2H), 2.48
(t, 2H, J = 7.5 Hz), 2.31 (t, 2H, J = 7.4 Hz), 1.81−1.67 (m, 4H), 1.63−
1H, J = 1.7 Hz), 6.20 (t, 1H, J = 1.8 Hz), 5.54 (br s, 1H, disappears on
treatment with D2O), 3.89 (t, 2H, J = 6.4 Hz), 3.77 (dd, 2H, J = 4.6
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Article
1.46 (mm, 6H), 1.43−1.27 (mm, 14H), 0.86 (t, 3H, J = 6.3 Hz). 13C
NMR (CDCl3): δ 174.60, 157.85, 154.84, 130.03, 123.53, 106.38,
99.69, 70.32, 67.87, 65.12, 63.38, 34.10, 31.85, 30.22 (×2), 29.65,
29.41, 29.20 (×2), 29.12, 28.98 (×2), 26.03, 24.85, 22.61, 14.08. MS
m/z: 475 [M + Na]+ (100). Anal. (C26H44O6 (452.62)) C, H, N.
2,3-Dihydroxypropyl 11-[3-Hydroxy-5-(2-methyloctan-2-yl)-
phenoxy]undecanoate (56). Eluent: CHCl3/MeOH = 48/2. Yield
75% (transparent oil). 1H NMR (CDCl3): δ 6.43 (s, 1H, disappears on
treatment with D2O), 6.37 (s, 1H), 6.25 (s, 1H), 6.21 (s, 1H), 5.27 (br
s, 2H, disappears on treatment with D2O), 4.16−4.14 (m, 1H), 4.07−
3.82 (m, 4H), 3.80−3.52 (m, 2H), 2.32 (t, 2H, J = 7.4 Hz), 1.92−1.83
(m, 2H), 1.76−1.48 (mm, 8H), 1.40−1.06 (mm, 22H), 0.82 (t, 3H, J
= 6.3 Hz). 13C NMR (CDCl3): δ 174.60, 159.98 156.51, 152.87,
105.66, 105.31, 98.70, 70.29, 67.92, 65.13, 62.06, 44.50, 37.81, 34.13,
31.78, 30.02 (×2), 29.32, 29.22 (×2), 29.08 (×2), 29.02 (×2), 25.95,
24.83, 24.64, 22.66, 14.06. MS m/z: 517 [M + Na]+ (100). Anal.
(C29H50O6 (494.70)) C, H, N.
CHO Cells. Chinese hamster ovary cells (CHO) stably transfected
with cDNA encoding human cannabinoid CB1 receptors were
maintained at 37 °C and 5% CO2 Dulbecco’s Modified Eagle’s
Medium nutrient mixture F-12 HAM supplemented with 1 mM L-
glutamine, 10% fetal bovine serum, and 0.6% penicillin−streptomycin
together with G418 (600 μ mL−1). These cells were passaged twice a
week using nonenzymatic cell dissociation solution.
Binding Assay. The preliminary screening was carried out using
three concentrations (5, 10, and 25 μM) of each compound,
membranes from HEK-293 cells stably transfected with either the
human CB1 or CB2 receptors and [3H]-(−)-cis-3-[2-hydroxy-4-(1,1-
dimethylheptyl)-phenyl]-trans-4-(3-hydroxypropyl)-cyclohexanol
([3H]CP-55,940) (Kd = 0.18 nM for CB1 and 0.31 nM for CB2
receptors) as the high affinity ligand as described by the manufacturer
(Perkin-Elmer, Italy).34 Compounds that displaced [3H]CP-55,940 by
more than 50% at 10 μM were further analyzed by carrying out a
complete dose−response curve (using 1, 10, 50, 100, 500, 1000 nM
concentrations). Displacement curves were generated by incubating
drugs with [3H]CP-55,940 (0.14 nM for CB1 and 0.084 μM for CB2
binding assay). In all cases, Ki values were calculated by applying the
Cheng−Prusoff equation35 to the IC50 values (obtained by GraphPad)
for the displacement of the bound radioligand by increasing
concentrations of the test compounds. The concentrations used to
carry out the dose−responses allow us to calculate IC50, and hence Ki,
with at least a 10 nM resolution. Data are reported as mean value ±
SEM of three separate experiments performed in duplicate.
(2.1 mM in DMSO) in a 1 mL volumetric flask with the biological
buffer and incubating for 90 min at 30 °C. At 60 and 90 min time
intervals, 5 μL of the incubated solution were injected in a liquid
chromatography−mass spectrometry system consisted of a Varian
apparatus (Varian Inc.) including a vacuum solvent degassing unit, two
pumps (212-LC), a triple quadrupole MSD (model 320-LC) mass
spectrometer with ES interface and Varian MS Workstation System
Control Vers. 6.9 software. The instrument operated in positive mode
and ESI parameters were: detector 1640 V, drying gas pressure 25.0
psi, desolvation temperature 300.0 °C, nebulizing gas 45.0 psi, needle
5000 V, and shield 600 V. Nitrogen was used as nebulizer gas and
drying gas. The chromatographic separation was performed using a
Lichrospher 100 C18 column (125 mm × 4.0 mm) with 5 μm particle
size. Chromatographic analysis was carried out using gradient elution
with eluent A being acetonitrile and eluent B consisting of an aqueous
solution of formic acid (0.1%); the analysis started with 5% of eluent
A, which was linearly increased up to 95% in 20 min, being the flow
rate 0.4 mL/min.
Human Plasma/Water Stability Assay. A stock solution of
compound 41 (2.2 × 10−2 M) was prepared in DMSO and stored at
−20 °C. Standard solutions for plasma esterase assay were prepared at
a final concentration of 1 × 10−3 M or 2 × 10−3 M by serial dilution of
the stock solution with water immediately prior to use. After being
drawn from the volunteers, human blood samples were placed in a
tube containing a sodium citrate solution as anticoagulant (3.8% p/v,
1:9) and centrifuged immediately to separate the plasma. An aliquot of
225 μL of human plasma was placed in an Eppendorff tube containing
25 μL of 1 × 10−3 or 2 × 10−3 M solution of compound 41, obtaining
a final concentration of 1 × 10−4 and 2 × 10−4 M. After vortex-mixed
for 10 s, samples were incubated at 37 °C for specified time intervals
(0, 15, 30, 60, and 120 min). Control samples were prepared in water
at the same final concentration of the tested compound, and all
experiments were done in triplicates. At the end of incubation time,
Eppendorff tubes were placed in ice and 0.4 mL of acetonitrile was
added to stop the reaction, vortexed for 2 min, and then centrifuged at
17800g for 5 min. The supernatant was aspirated off and stored at
−20 °C until analysis. The chromatographic separation was performed
on a DB-C8 Zorbax Eclipse analytical column (4.6 mm × 150 mm;
5 μm), using a binary mobile phase of 92/8 methanol/water at a flow
rate of 0.6 mL/min. Analysis was carried out following both UV
(254 nm) and MS signals (positive ion mode, Agilent 1100 LC/MSD
VL system G1946C). The sample were injected (20 μL) after
filtration. In these chromatographic conditions, retention times were
4.77 min for compound 41 and 3.52 min for its corresponding acid.
The retention time values were assessed by injecting 20 μL of standard
solutions of both compound 41 and its corresponding acid, prepared
by dissolving each compound in acetonitrile in a volumetric flask.
Data are reported as mean ± SEM of samples run in triplicate. The
area under the experimental curve (AUC, UV signal) was calculated by
a combined linear logarithmic trapezoidal method using Graphpad-
Prism V program (GraphPad Software Inc., San Diego, CA, USA).
The comparison between AUC(0−120 min) of water vs plasma samples
was performed by using Student’s t test. A P value <0.05 was
considered significant.
[35S]GTPγS Assay. The method for measuring agonist-stimulated
[35S]GTPγS binding to cannabinoid CB1 receptors was adapted from
previous described methods.36 The assays were carried out with
GTPγS binding buffer (50 mM Tris-HCl, 50 mM Tris-Base, 5 mM
MgCl2, 1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol, 0.1% BSA)
in the presence of [35S]GTPγS and GDP, in a final volume of 500 μL.
Binding was initiated by the addition of [35S]GTPγS to the wells.
Nonspecific binding was measured in the presence of 30 μM GTPγS.
The drugs were incubated in the assay for 60 min at 30 °C. The
reaction was terminated by addition of ice-cold Tris-binding buffer and
a rapid vacuum filtration. The filters were oven-dried for 60 min and
then placed in 5 mL of scintillation fluid. Radioactivity was quantified
by liquid scintillation spectrometry. In all assays have been used 0.1
nM [35S]GTPγS, 30 μM GDP, and 5 μg of protein per well for mouse
brain membranes and 50 μg per well for CHO−hCB1 cell membranes.
Additionally, mouse brain membranes were preincubated for 30 min at
30 °C with 0.5 U mL−1 adenosine deaminase (200 U mL−1) to remove
endogenous adenosine. Net agonist stimulated [35S]GTPγS binding
values were calculated by subtracting basal binding values (obtained in
absence of agonist) from agonist-stimulated values (obtained in the
presence of agonist) as detailed elsewhere.37 Values for EC50, maximal
effect (Emax) and SEM or 95% confidence limits of these values have
been calculated by nonlinear regression analysis using the equation for
a sigmoid concentration−reponse curve (GraphPad Prism). Agonists
and antagonists were stored at −20 °C as 10 mM stock solutions
dissolved in DMSO. Chemical stability in the binding buffer was
assessed by diluting 200 μL of a standard solution of compound 41
Metabolic Stability Assay. Compound 41 in 10 mM DMSO
solution was added to an incubation mixture in a 96-well microplate
containing 20 pmol/mL of hCYP3A4 (0.1−0.2 mg/mL protein). The
mixture was split in two aliquots: one receiving a NADPH
regenerating system, the other an equal amount of phosphate buffer.
The final substrate concentration was 1 μM along with 0.25% of
organic solvent. Incubation proceeded for 1 h at 37 °C and was
stopped by addition of acetonitrile to precipitate proteins. Metabolic
stability is given as the percent remaining following incubation with
cofactor (NADPH) with reference to the incubation mixture without
NADPH: % remaining = Area NADPH × 100/Area ctrl, where Area
NADPH is the MS peak area of the sample solution with NADPH and
Area ctrl is the MS peak area of the sample solution without NADPH.
The % CV obtained was typically within 10%. Mass analysis was
performed using a Waters UPLC Acquity system with a qTOF LCT
Premiere interface.
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Journal of Medicinal Chemistry
Article
2007, 18, 129−140. (b) Liu, J.; Wang, L.; Harvey-White, J.; Huang, B.
X.; Kim, H. Y.; Luquet, S.; Palmiter, R. D.; Krystal, G.; Rai, R.;
Mahadevan, A.; Razdan, R. K.; Kunos, G. Multiple pathways involved
in the biosynthesis of anandamide. Neuropharmacology 2008, 54, 1−7.
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profile of brain enzymes that hydrolyze the endocannabinoid 2-
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ASSOCIATED CONTENT
* Supporting Information
Physical and spectral data for compounds 4−5, 8−10, 11−17,
19, 20−27, 29, 30−32 and elemental analyses for all final
compounds 34−56. LC-MS chromatogram of 41 incubated in
the biological buffer. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
(7) De Petrocellis, L.; Di Marzo, V. An introduction to the
endocannabinoid system: from the early to the latest concepts. Best
Pract. Res., Clin. Endocrinol. Metab. 2009, 23, 1−15.
AUTHOR INFORMATION
Corresponding Author
*For A.B.: phone, +39 577 234327; fax, +39 577 234333;
E-mail, brizzi3@unisi.it. For F.C.: phone, +39 577 234308; fax,
+39 577 234333; E-mail, corelli@unisi.it.
■
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cerebellar membranes. Br. J. Pharmacol. 2001, 134, 664−672.
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S.; Kodaka, T.; Suhara, Y.; Takayama, H.; Waku, K. Evidence that 2-
Arachidonoylglycerol but Not N-Palmitoylethanolamine or
Anandamide is the Physiological Ligand for the Cannabinoid CB2
Receptor. Comparision of the Agonistic Activities of Various
Cannabinoid Receptor Ligands in HL-60 Cells. J. Biol. Chem. 2000,
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Biochemistry, pharmacology and physiology of 2-arachidonoylglycerol,
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ACKNOWLEDGMENTS
Authors from the University of Siena wish to thank the
■
Ministero dell’Universita
̀
e della Ricerca (PRIN 2006, Prot. no.
2006030948_002) for financial support. We are grateful to
Marco Allara for technical assistance with the binding assays.
̀
Thanks are also due to Dr. Stefania Lamponi for her skillful
support in the human plasma sample preparation.
ABBREVIATIONS USED
■
AG, arachidonoylglycerol; AEA, anandamide; CB, cannabinoid;
AGE, arachidonyl glyceryl ether; THC, (−)-trans-Δ9-tetrahy-
drocannabinol; ES, endocannabinoid system; NADA, N-
arachidonoyl-dopamine; MAGL, monoglyceride lipase; THF,
tetrahydrofuran; DCC, N,N′-dicyclohexylcarbodiimide; DMAP,
4-(dimethylamino)pyridine; hCYP3A4, human recombinant
cytochrome P450 3A4
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