Structure-activity relationship of a new series of reversible dual monoacylglycerol lipase/fatty acid amide hydrolase inhibitors

Article abstract of DOI:10.1021/jm201327p

The two endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), play independent and nonredundant roles in the body. This makes the development of both selective and dual inhibitors of their inactivation an important priority. In this work we report a new series of inhibitors of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Among them, (±)-oxiran-2-ylmethyl 6-(1,1′-biphenyl-4-yl)hexanoate (8) and (2R)-(-)-oxiran-2-ylmethyl(4-benzylphenyl)acetate (30) stand out as potent inhibitors of human recombinant MAGL (IC₅₀ (8) = 4.1 μM; IC ₅₀ (30) = 2.4 μM), rat brain monoacylglycerol hydrolysis (IC ₅₀ (8) = 1.8 μM; IC₅₀ (30) = 0.68 μM), and rat brain FAAH (IC₅₀ (8) = 5.1 μM; IC₅₀ (30) = 0.29 μM). Importantly, and in contrast to the other previously described MAGL inhibitors, these compounds behave as reversible inhibitors either of competitive (8) or noncompetitive nature (30). Hence, they could be useful to explore the therapeutic potential of reversible MAGL inhibitors.

Full text of DOI:10.1021/jm201327p

Article
pubs.acs.org/jmc
Structure−Activity Relationship of a New Series of Reversible Dual
Monoacylglycerol Lipase/Fatty Acid Amide Hydrolase Inhibitors
†
‡
†
‡
†
́
Jose A. Cisneros, Emmelie Bjorklund, Ines Gonzalez-Gil, Yanling Hu, Angeles Canales,
́
́
́
̈
Francisco J. Medrano,§ Antonio Romero,§ Silvia Ortega-Gutierrez,† Christopher J. Fowler,‡
́
and María L. Lopez-Rodríguez*,†
́
†Departamento de Química Organ
́
ica I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain
‡Department of Pharmacology and Clinical Neuroscience, Umea University, Umea, Sweden
̊
̊
§Departamento de Biología Físico-Química, Centro de Investigaciones Biolog
E-28040 Madrid, Spain
́
icas, Consejo Superior de Investigaciones Científicas,
S
* Supporting Information
ABSTRACT: The two endocannabinoids, anandamide
(AEA) and 2-arachidonoylglycerol (2-AG), play independent
and nonredundant roles in the body. This makes the
development of both selective and dual inhibitors of their
inactivation an important priority. In this work we report a
new series of inhibitors of monoacylglycerol lipase (MAGL)
and fatty acid amide hydrolase (FAAH). Among them,
( )-oxiran-2-ylmethyl 6-(1,1′-biphenyl-4-yl)hexanoate (8)
and (2R)-(−)-oxiran-2-ylmethyl(4-benzylphenyl)acetate (30)
stand out as potent inhibitors of human recombinant MAGL
(IC50 (8) = 4.1 μM; IC50 (30) = 2.4 μM), rat brain
monoacylglycerol hydrolysis (IC50 (8) = 1.8 μM; IC50 (30) = 0.68 μM), and rat brain FAAH (IC50 (8) = 5.1 μM; IC50 (30) =
0.29 μM). Importantly, and in contrast to the other previously described MAGL inhibitors, these compounds behave as
reversible inhibitors either of competitive (8) or noncompetitive nature (30). Hence, they could be useful to explore the
therapeutic potential of reversible MAGL inhibitors.
the pharmaceutical industry.6 MAGL inhibitor pharmacology
INTRODUCTION
■
lagged behind until the discovery of JZL184 (1, Chart 1).7 This
compound, which acts as an irreversible inhibitor, has been
used to explore the therapeutic potential of MAGL, primarily in
pain states.7,8 However, chronic enhancement of 2-AG levels
produced either by repeated treatment with 1 or by genetic
deletion of MAGL induces desensitization of the CB1 receptors
and impairs 2-AG-mediated antinociceptive effects.9 At first
sight, this finding argues against the therapeutic exploitation of
MAGL inhibition. However, both genetic deletion of MAGL
and the use of an irreversible inhibitor of the enzyme represent
a sustained loss of MAGL activity, raising the possibility that
reversible MAGL inhibitors will not produce such desensitiza-
tion. No potent MAGL reversible inhibitors, however, have, to
our knowledge, been characterized. In the present study, we
report the design and synthesis of a new series of biphenyl, 4-
phenylbenzyl, and 4-phenylethylphenyl derivatives and their
ability to inhibit human recombinant MAGL (hrMAGL) as well
as their capacity to interfere with 2-oleoylyglycerol (2-OG, an
alternate MAGL substrate) and AEA hydrolysis in brain
homogenates. Among the synthesized compounds, derivatives
Endocannabinoids (eCBs) constitute a class of lipid messengers
that exert their biological actions through the interaction with
two G protein-coupled cannabinoid receptors, CB1 and CB2.
These components, together with the enzymes responsible for
the biosynthesis and degradation of eCBs, constitute the
endogenous cannabinoid system which regulates a broad
spectrum of physiological processes, such as pain, inflammation,
feeding behavior, and neurodegeneration.1,2 The main eCBs are
N-arachidonoylethanolamine (anandamide, AEA) and 2-arach-
idonoylglycerol (2-AG), and these ligands play separate and
tightly regulated roles in the body, rather than simply acting as
alternate ligands for the same receptors.3
AEA and 2-AG are primarily metabolized by hydrolysis to
arachidonic acid. While the role of FAAH as the key enzyme in
AEA inactivation is well established,4 several enzymes are
responsible for 2-AG inactivation, of which monoacylglycerol
lipase (MAGL) is the key hydrolytic enzyme. About 85% of the
total 2-AG hydrolysis in the brain has been ascribed to MAGL,
while the remaining activity is mainly due to the activities of α/
β hydrolase-6 (ABHD-6) and α/β hydrolase-12 (ABHD-12).5
The central roles of FAAH and MAGL in gating eCB levels
make them promising targets for drug development. A wide
variety of FAAH inhibitors have been described, not least by
Received: October 4, 2011
Published: December 21, 2011
© 2011 American Chemical Society
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Chart 1. Structure of the Monoacylglcyerol Lipase (MAGL) Inhibitors JZL184 (1), UCM162 (2), UCM505 (3), and SAR629
(34)
8 and 30 show good potency toward MAGL and do not
produce the sort of time-dependent inhibition seen with
irreversible inhibitors. These compounds could open new
perspectives for modulating MAGL activity and to assess
whether reversible inhibition is a strategy with which to exploit
the therapeutic potential of this enzyme.
carbonyl group at the adequate distance and orientation to
interact with the catalytic serine.
Glide docking of the derivative with five methylenic units
between the carbonyl group and the biphenyl system
(compound 8) into the active site of MAGL afforded three
binding poses. In all of them, the catalytic serine (Ser 132) is
located in the vicinity of the carbonyl of 8 and the methylenic
chain attached to the biphenyl group points toward the exit of
the channel where the hydrophobic residues that interact with
the arachidonoyl moiety of 2-AG are located. The refined
model of the less energy binding pose obtained with Prime16
(Figure 1A) shows a hydrogen bond between the oxygen atom
of the ester carbonyl and the backbone NH of Ala 61. A similar
hydrogen bond was found between the carbonyl oxygen of the
MAGL covalent inhibitor SAR629 (34, Chart 1) and Ala 61 in
the crystal structure of the complex, and it was also proposed in
the docking models described for the natural substrates (2-AG
and 1(3)-AG).13 In our case, the biphenyl group interacts
mainly with the hydrophobic residues Leu 158, Ala 174, Leu
186, Leu 215, Ile 221, Leu 223, and Leu 224, which are also
involved in van der Waals contacts with the fluorophenyl rings
of inhibitor 34 in the crystal structure.13 Therefore, according
to docking calculations, compound 8 could be recognized by
MAGL occupying the channel where the natural substrate
should be accommodated.
Docking calculations carried out with the derivative with one
methylene between the two benzene rings and a spacer of three
methylenes between the carbonyl group and the first phenyl
ring (compound 22) yielded a similar binding mode to the
docking pose obtained for 8, with the carbonyl group of 22
located near the nucleophilic oxygen atom of the catalytic
serine (Figure 1B). The main difference with 8 is the location
of the oxirane ring of the molecule, which is more buried into
the polar pocket of the enzyme in the case of compound 22
than in compound 8. Due to this disposition and the shorter
length of the spacer, it is possible that this compound would
establish fewer interactions with the hydrophobic residues of
the enzyme channel than derivative 8, since residues Ala 174,
Leu 186, Ile 221, and Leu 215 are not directly involved in
ligand recognition.
RESULTS AND DISCUSSION
■
Design. In a previous study10 we identified oxiran-2-
ylmethyl (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate (2) and
tetrahydro-2H-pyran-2-ylmethyl (5Z,8Z,11Z,14Z)-icosa-
5,8,11,14-tetraenoate (3) as inhibitors of monoacylglycerol
hydrolysis (Chart 1). However, these compounds were based
on the structure of 2-AG, and thereby, they retain the
arachidonic acid chain in their structure. This moiety could
be prone to oxidation in biological media, and it may limit the
selectivity of the compounds, given that arachidonic acid is a
key metabolite in several pathways and therefore can be
recognized by different enzymes, including an assortment of
lipases and cyclooxygenases. Hence, we sought to replace the
arachidonic acid moiety with a suitable bioisostere. Optimally,
the selected group should be stable in biological settings and
chemically versatile to enable structure exploration. Since the
biphenyl group has been suggested as an isostere of arachidonic
acid,11 we envisioned a series of esters that contained a
biphenyl core and the heterocyclic moieties previously
optimized. Since our data indicate that derivative 2 behaves
as a competitive inhibitor (unpublished observations), it is
conceivable that its carbonyl group is placed close to the
catalytic serine (Ser 132) of the enzyme. On the basis of the
recently reported 3D structure of MAGL,12,13 an important site
of interaction in addition to the active site is the hydrophobic
pocket defined by the side chains of residues Leu 158, Ala 174,
Leu 186, Ile 189, Leu 215, Val 217, Ile 221, Leu 223, and Leu
224. Therefore, we carried out docking studies in order to
obtain some clues about whether the introduction of a suitable
spacer between the biphenyl core and the oxygenated ring or
between both benzene rings of the biphenyl group could
facilitate the simultaneous interaction of the compound with
the catalytic serine and the hydrophobic pocket.
According to these computational models, we designed two
different series of derivatives 4−33 (Figure 1C) where the
lipophilic moiety has been substituted by biphenyl, 4-
benzylphenyl (Y = CH2), and 4-phenylethylphenyl (Y =
CH2CH2) groups.
Chemistry, Biological Evaluation, and Structure−
Activity Relationship (SAR) Study. The synthesis of the
esters 4−31 is indicated in Scheme 1. In general, appropriate
commercially available or previously synthesized carboxylic
Docking calculations have been performed by using Glide
software14,15 as described in the Experimental Section. Docking
results suggest that the incorporation of a spacer with five
methylenic units between the carbonyl group and the biphenyl
system (Figure 1A) or of one methylene between the two
benzene rings and a spacer of three methylenes between the
carbonyl group and the first phenyl ring (Figure 1B) allows for
the favorable hydrophobic interactions of the aromatic rings of
the compounds and the hydrophobic pocket while keeping the
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Figure 1. Computational models of the complexes between compounds (A) 8 and (B) 22 and MAGL (PDB code 3JW8). Residues of the catalytic
triad have been labeled in blue. The hydroxyl group of Ser 132 and the carbonyl group of the compounds are marked with an arrow (in green).
Shown models correspond to the R enantiomer for compound 8 and to the S enantiomer for compound 22. (C) Design of compounds 4−33.
acids were condensed with the corresponding alcohol in the
presence of dicyclohexylcarbodiimide (DCC) and catalytic
amounts of N,N-dimethyl-4-aminopyridine (DMAP), except
compound 19, which has been prepared by Wittig reaction
b e t w e e n t e t r a h y d r o - 2 H - p y r a n - 2 - y l m e t h y l
(triphenylphosphoranylidene)acetate and (1,1′-biphenyl-4-yl)-
acetaldehyde.
Final compounds 4−31 were tested not only for their ability
to inhibit MAGL activity in vitro but also for their capacity to
block 2-OG hydrolysis in brain homogenates, since the most
interesting compounds are those that can inhibit both hrMAGL
in vitro and also monoacylglycerol hydrolysis in rat brain, since
this would allow the compound to be used in biologically more
relevant studies. Furthermore, in view of our previous finding
that some compounds (such as troglitazone) show assay
dependency in their ability to inhibit MAGL,17 it is important
to demonstrate that active compounds are active regardless of
the MAGL assay used. The hrMAGL inhibition assay was
carried out as previously reported18 with some modifications17
using commercially available his-tagged hrMAGL. The capacity
of the compounds to interfere with the hydrolysis of 2-AG in
cytosolic fractions was determined using 2-oleoylglycerol (2-
OG) as a surrogate. Effects upon the FAAH-catalyzed
hydrolysis of AEA by the membrane fractions were also
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a
Scheme 1. Synthesis of Derivatives 4-31
a
Reagents and conditions: (a) X-OH, DCC, DMAP, CH2Cl2, Ar, rt; (b) toluene, Δ.
determined to characterize the selectivity of the compounds for
MAGL vs FAAH. 2-OG and AEA hydrolytic activities were
measured using a substrate concentration of 0.5 μM and the
assay procedures described previously using membrane and
cytosolic fractions of rat cerebella.19 These results are shown in
Tables 1−4. For consistency of notation, throughout the tables,
IC50 and pI50 values for hrMAGL, 2-OG, or AEA hydrolysis
inhibition are denoted as hrMAGL, 2-OG, and AEA,
respectively. In some cases, 100% inhibition was not seen at
the highest dose tested. This type of situation is indicated
throughout the tables by adding the percentage of maximum
inhibition value obtained for the highest concentration tested
(10 μM for hrMAGL or 100 μM for 2-OG or AEA hydrolysis).
Influence of the Spacer. First, we analyzed the effect of
the length of the spacer in the biphenyl derivatives 4−15
(Table 1) on the endocannabinoid hydrolysis inhibition. In
general, for oxirane esters 4−9, increasing the length of the
methylenic spacer enhances the inhibitory activity of the
compounds at hrMAGL, from no or low inhibition (for
compounds 4 and 5) to IC50 values between 1.8 and 6.6 μM
(compounds 6−8, with spacers of 3 to 5 methylenic units,
respectively). Further elongation decreases inhibition of
hrMAGL, as shown by derivative 9 (11% inhibition at 10
μM). Given the importance of the fact that compounds which
behave as in vitro hrMAGL inhibitors also show the capacity to
block monoacylglycerol hydrolysis in the brain, the results
obtained in brain cytosolic fractions using 2-OG as a 2-AG
surrogate highlight the importance of compounds 7 [IC50
(hrMAGL) = 6.6 μM; IC50 (2-OG) = 2.6 μM] and 8 [(IC50
(hrMAGL) = 4.1 μM; IC50 (2-OG) = 1.8 μM]. The effect of
the length of the spacer of oxirane derivatives 4−9 on FAAH
inhibition shows, however, an opposite trend, with the shortest
spacers (n = 1−3, compounds 4−6) being the ones that yielded
the lowest IC50 values (0.59, 0.87, and 0.71 μM, respectively).
When the heterocyclic subunit is a tetrahydropyran ring
(derivatives 10−15), no significant simultaneous inhibition
for hrMAGL and for 2-OG hydrolysis inhibition was observed
for any of them [IC50 (2-OG) > 100 μM]. These compounds
showed, nonetheless, a moderate potency as FAAH inhibitors,
with IC50 values ranging from 3.9 μM (compound 15) to 20
μM (compound 13). Next, we studied whether the presence of
one unsaturation of trans geometry in the spacer could be
favorable for the inhibition of MAGL activity and AEA
hydrolysis (Table 2). None of the synthesized compounds
(16−19), regardless of the presence of oxirane or tetrahy-
dropyran as heterocyclic subunit, showed any significant
capacity to block concomitantly hrMAGL activity and 2-OG
hydrolysis. It is interesting to note, however, the good capacity
of derivatives 16 and 17 to inhibit hrMAGL (IC50 values of 5.1
and 0.76 μM, respectively). With respect to FAAH, only 16
showed a moderate activity toward FAAH inhibition with an
IC50 value of 3.8 μM. In sum, among all these biphenyl
derivatives 4−19, compounds 7 and 8 are the most potent
inhibitors of the series. Furthermore, derivative 8, which is
more potent than initial hits 2 and 3 to inhibit 2-OG hydrolysis
[IC50 (8) = 1.8 μM; IC50 (2) = 4.5 μM; IC50 (3) = 5.6 μM)],
acts directly at MAGL [IC50 (8, hrMAGL) = 4.1 μM], and lacks
the arachidonic acid chain in its structure.
Modifications in the Biphenyl Moiety. Taking into
account the docking models (Figure 1B) and the previous
results, we also studied the possibility of replacing the biphenyl
moiety for two benzene rings separated by different spacers
(one or two methylenic units) and decreasing the length chain
that separates the hydrophobic moiety and the heterocyclic
subunit (Table 3) in compounds 20−27. Only the oxirane
derivative 20, with one methylenic unit between the two
benzene rings and one methylenic unit in the spacer (m, n = 1),
showed a significant capacity to inhibit hrMAGL (IC50 value of
16 μM) and 2-OG hydrolysis (IC50 value of 10 μM).
Nonetheless, some of the oxirane esters with one methylenic
unit between the two benzene rings (m = 1) exhibited some
capacity to block 2-OG hydrolysis [IC50 (21) = 19 μM; IC50
(22) = 8 μM)], a result that suggests that these compounds
may be able to distinguish between MAGL activities in different
species or between distinct 2-OG hydrolyzing activities. The
increase of the length of the linker between the two benzene
rings to two methylenic units (m = 2) abolished all activity, as
shown by compound 23, where only 18% inhibition at the
maximal concentration tested was observed for 2-OG
hydrolysis and resulted inactive at hrMAGL. Regarding the
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Table 1. Influence of the Length of the Spacer
Table 2. Influence of Unsaturation in the Spacer
a
For an explanation of the data, see the footnotes for Table 1.
Table 3. Influence of the 4-Benzylphenyl and 4-
Phenylethylphenyl Moieties
a
Throughout the tables, the pI50 values (−log10 [IC50]) are expressed
as mean s.e.m., and they are calculated from data obtained in 3−10
independent experiments. The IC50 values derived from the mean pI50
values are given in brackets. n.i. indicates <10% inhibition at the
b
highest concentration tested (10 μM for hrMAGL or 100 μM for 2-
c
OG or AEA hydrolysis). When the inhibitable component was less
than 50%, when the data could not be fitted to a curve due to a
marginal degree of inhibition, or when the pI50 value was lower (and
hence the IC50 value higher) than the highest concentration tested (10
μM for hrMAGL or 100 μM for 2-OG or AEA hydrolysis), the pI50
values have been indicated as <5 or <4 (i.e., IC50 value >10 μM or IC50
value >100 μM, respectively), and the percentage of inhibition attained
at 10 or 100 μM has been indicated between parentheses as mean
d
s.e.m. When the data was better fitted to an inhibition curve with a
a
For an explanation of the data, see the footnotes for Table 1.
residual activity (i.e., the “bottom” value) >0, the percentage of
inhibitable component (100-“bottom” value) is given in the table as
maximum inhibition and the data expressed as pI50 [IC50, percentage
of maximum inhibition].
stereogenic center does not exert a great influence in the
inhibitory ability of compounds 28 and 29, with close IC50
values for 2-OG hydrolysis of 1.8 μM (racemic 8), 4.9 μM (R-
enantiomer 28), and 5.1 μM (S-enantiomer 29). Both
enantiomers inhibited hrMAGL, but <50% inhibition was
seen at the highest concentration tested (10 μM), precluding
determination of their IC50 values. In contrast, the config-
uration of the sterogenic center plays an important role in the
case of the 4-benzylphenyl derivative 20, where a remarkable
difference between the activities of both enantiomers can be
observed. While S-enantiomer 31 showed a lack of activity at
hrMAGL or a low inhibitory capacity in cytosolic brain
fractions [IC50(2-OG) = 70 μM] compared with the racemic 20
[IC50(hrMAGL) = 16 μM; IC50(2-OG) = 10 μM], its R-
counterpart 30 behaves as a potent inhibitor of hrMAGL (IC50
= 2.4 μM) and of 2-OG hydrolysis with an IC50 value in the
submicromolar range (0.68 μM). No such stereoselectivity was
seen for the inhibition of AEA hydrolysis by FAAH.
tetrahydropyran derivatives 24−27, none of the modifications
yielded significant inhibition values for either 2-OG hydrolysis
or hrMAGL activity (Table 3). With respect to FAAH
inhibition, in general, all these compounds were able to inhibit
AEA hydrolysis, with moderate IC50 values between 0.28 and
50 μM.
Influence of the Stereogenic Center. In summary, from
all synthesized compounds 4−27, the ones with the best
inhibitory profile toward 2-AG hydrolysis were the biphenyl
derivative 8 [IC50 (hrMAGL) = 4.1 μM; IC50 (2-OG) = 1.8
μM] and the 4-benzylphenyl derivative 20 [IC50 (hrMAGL) =
16 μM; IC50 (2-OG) = 10 μM]. Since both of them contain a
stereogenic center, we next studied its influence by synthesizing
the two enantiomers of 8 (derivatives 28 and 29) and 20 (30
and 31). The results compiled in Table 4 indicated that the
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Table 4. Influence of the Stereogenic Center and the Glycerol Moiety in Derivatives 8 and 20
a
For an explanation of the data, see the footnotes for Table 1.
Replacement of the Oxirane Group with the 2-
Glycerol Moiety. Finally, and in an attempt to further
optimize the potency of 8 and 20 to inhibit 2-OG hydrolysis,
we have replaced their oxirane ring with the 2-glycerol moiety,
present in the molecule of 2-AG. Derivatives 32 and 33 were
obtained through esterification of the corresponding carboxylic
acids with 1,3-dibenzyloxypropan-2-ol followed by deprotection
of the hydroxyl groups by catalytic hydrogenation in the
presence of palladium hydroxide (Scheme 2). The activity
assays (Table 4) showed that this modification did not
significantly affect the inhibitory ability of 32 when compared
with its analogue 8, with IC50 values for hrMAGL inhibition of
7.5 and 4.1 μM, respectively, and 2-OG hydrolysis inhibition of
1.5 and 1.8 μM, respectively (Table 4). On the contrary, the
same modification basically abolished the activity of the parent
compound 20 in both assays. Derivative 33 did not inhibit
hrMAGL whereas 20 had an IC50 value of 16 μM and similarly,
for 2-OG hydrolysis, 33 showed only a 49% inhibition at 100
μM, whereas 20 had an IC50 value of 10 μM.
a
In summary, from the SAR study, compounds 8 and 30
exhibited the best inhibitory profiles (see Figure 2 for
representative curves) and were therefore selected for a more
detailed study of their inhibition mechanism.
Scheme 2. Synthesis of Derivatives 32 and 33
Study of the Inhibition Mechanism. Given the finding
that sustained increase of 2-AG levels resulting either from
repeated administration of the irreversible MAGL inhibitor 1 or
by genetic deletion of the enzyme desensitizes CB1 in the
central nervous system and impairs antinociceptive effects of
MAGL inhibitors,9 it is important to identify reversible
inhibitors in order to determine whether such compounds
a
Reagents and conditions: (a) DCC, DMAP, CH2Cl2, Ar, rt; (b)
Pd(OH)2, CH2Cl2/EtOH, rt.
○
▼
▲
Figure 2. Concentration−response curves for compounds 8 (A) and 30 (B) ( , AEA hydrolysis; , rat cytosolic 2-OG hydrolysis; , hrMAGL
NPA hydrolysis). Means and sem (when not enclosed by the symbol), n = 3−7.
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avoid this problem. Therefore, we next studied the inhibition
mechanism of the optimal compounds identified in these series
8 and 30. To assess the reversible character of inhibition, we
studied the effect of preincubation and dilution (Figure 3) in
If the compound is a freely reversible inhibitor, then the
inhibition should drop upon dilution, whereas, if it is a tight-
binding inhibitor, the inhibition should remain. Thus, for a fully
reversible compound, the inhibition produced by preincubation
with a 10 μM concentration of the compound (p10), for
example, should be the same after dilution as that produced by
0.5 μM of the inhibitor added after the dilution stage.
Conversely, for a tight-binding compound, the inhibition
produced by p10 should be similar to that produced by 10
μM of the inhibitor added after the dilution stage, given that the
compounds tested do not show time-dependent inhibition.
Accordingly, derivative 8 behaves as a freely reversible inhibitor
(the inhibition caused by 10 μM of the compound significantly
decreases compared with the inhibition observed upon the 20-
fold dilution (p10 in Figure 3B, p < 0.01), and no significant
difference between p10 and 0.5 μM is observed. On the other
hand, 30 is something in between freely reversible and tight-
binding (Figure 3B), since a drop in activity is observed
between a concentration of the compound of 3 μM and p3
(compatible with freely reversible) but the inhibition of p3 is
significantly higher than that produced by 0.15 μM (compatible
with tight binding). This latter behavior has been previously
observed in other MAGL inhibitors, such as troglitazone.17
Similar results were found for AEA hydrolysis by 8 and 30 (data
not shown).
Having confirmed the reversibility of both 8 and 30, we then
determined the mode of inhibition using hrMAGL expressed
and purified in our laboratories (see Supporting Information for
details). Our results indicate that compound 8 acts as a
competitive inhibitor (Figure 4A) with a Ki value of 8 2 μM
whereas inhibitor 30 is noncompetitive (Figure 4B) with a Ki
value of 40 3 μM. Furthermore, additional NMR experiments
in the presence of hrMAGL indicated that, in fact, 8 was
hydrolyzed by MAGL over time whereas 30 was unaffected
during the same time interval (Figure S1 of the Supporting
Information). These results are in agreement with the fact that
8 could inhibit the enzyme by acting as a competing substrate,
and they also support the noncompetitive nature of 30, which is
not hydrolyzed by hrMAGL under the same experimental
conditions. In order to check whether the initially proposed
model was indeed compatible with these experimental results,
we confirmed that both enantiomers of 8 (compounds 28 and
29) fitted in a similar manner in the binding pocket of MAGL
(Figure S2). In addition, the distance and relative orientation of
the hydroxyl group of the catalytic serine and the carbonyl
group of enantiomers 28 and 29 makes possible the
nucleophilic attack of the serine and the subsequent hydrolysis
of the substrate. Hence, this model is in agreement with the
lack of stereoselectivity and with the competitive mechanism
observed. On the contrary, the facts that compound 30 is not
hydrolyzed under the same conditions as those for 8 (Figure
S1) and that 30 behaves in a noncompetitive manner suggest
that these molecules have a different mechanism of action. A
reasonable explanation could be that while enantiomers 28 and
29 bind in the same pocket used by the endogenous substrate,
derivative 30 inhibits MAGL activity by binding in a different
site of the enzyme. Alternatively, it is also possible that 30 can
bind simultaneously to 2-AG, given that the hydrophobic
channel leading to the catalytic serine is quite wide. Any of
these two possibilities is compatible with the fact that 30 is not
hydrolyzed in the presence of enzyme as observed by NMR
(Figure S1) and with the noncompetitive inhibition mechanism
experimentally observed (Figure 4B) but cannot be unequiv-
Figure 3. (A) Effect of preincubation for 0 min (white bars) or 60 min
(dashed bars) of compounds 8, 30, and, for comparative purposes, 1 at
37 °C upon the inhibition of the hydrolysis of 2-OG. Shown are means
and s.e.m., n = 3−4. ***p < 0.01 vs the corresponding non-
preincubation values, two-tailed paired t test; otherwise, p > 0.05 (note
that the significance level for 0.1 μM 30 was p = 0.06). (B) Assessment
of the reversibility of the inhibition by 8 and 30 of rat cerebellar
cytosolic 2-OG hydrolysis. Samples were preincubated for 60 min with
either vehicle or the concentration shown (”p10” = preincubated with
10 μM) and then diluted 20-fold, after which substrate was added and
the samples were incubated for 10 min. Following the preincubation
phase, vehicle samples were either treated with vehicle or the
concentrations of 8 or 30 (1 is also included for comparison). Shown
are means and s.e.m., n = 3, of the hydrolysis rates as percent of the
vehicle controls. *, <0.05; **, p < 0.01; NS, not significant, for the
comparisons between the values for the inhibitor added before dilution
vs after dilution, Tukey’s multiple comparison test following significant
one way ANOVA for repeated measures.
the inhibitory capacity of the compounds. An irreversible
inhibitor will increase its capacity to block the enzyme if
preincubated previously in the presence of enzyme prior to
addition of substrate and assay of activity. This is indeed the
case with compound 1 (Figure 3A), a known irreversible
inhibitor of MAGL.7 On the contrary, neither 8 nor 30 showed
any significant increase in their ability to block MAGL activity
after 60 min (Figure 3A). In order to further support this
finding, we have carried out dilution experiments (Figure 3B).
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Figure 4. Inhibition of the activity of the hrMAGL: (A) competitive nature of compound 8 and (B) noncompetitive nature of derivative 30. Shown
are means sem (n = 2−3, error bars for each point are smaller than the symbols).
were within 0.5% of the theoretical values, confirming a purity of at
least 95% of all tested compounds. In the case of HPLC-MS,
satisfactory chromatograms (purity >95%) were obtained. HPLC-MS
analysis was performed using an Agilent 1200LC-MSD VL instrument.
LC separation was achieved with an Eclipse XDB-C18 column (5 μm,
4.6 mm × 150 mm) together with a guard column (5 μm, 4.6 mm ×
12.5 mm). The gradient mobile phases consisted of A (95:5 water/
acetonitrile or water/methanol) and B (5:95 water/acetonitrile or
water/methanol) with 0.1% ammonium hydroxide and 0.1% formic
acid as the solvent modifiers. The gradient started at 0% B (for 5 min)
and increased linearly to 100% B over the course of 20 min, with a
flow rate of 0.5 mL/min, and it was followed by an isocratic gradient of
100% B for 5 min before equilibrating for 5 min at 0% B. MS analysis
was performed with an electrospray ionization (ESI) source. The
capillary voltage was set to 3.0 kV, and the fragmentor voltage was set
at 70 eV. The drying gas temperature was 350 °C, the drying gas flow
was 10 L/min, and the nebulizer pressure was 20 psi. HRMS was
carried out on a FTMS Bruker APEX Q IV spectrometer in ESI mode
at UCM’s mass spectrometry core facility. Thin-layer chromatography
(TLC) was run on Merck silica gel type 60 F-254 plates. For normal
pressure chromatography, Merck silica gel type (size 70−230) was
used. Unless stated otherwise, the starting materials used were high-
grade commercial products from Sigma-Aldrich, Acros, Fluka, Merck,
or Panreac. Methylene chloride was used freshly distilled over CaH2.
Elemental or HPLC-MS purity analyses of final compounds 4−33 and
synthesis of all intermediates are described in the Supporting
Information.
ocally demonstrated with the current available structural
models.
In conclusion, in this work we have developed new dual
MAGL and FAAH inhibitors. Among the synthesized
compounds we identified two reversible inhibitors of hrMAGL,
compounds 8 and 30, that block not only hrMAGL activity
[IC50(8) = 4.1 μM; IC50(30) = 2.4 μM] but also brain
monoacylglycerol hydrolysis [IC50(8) = 1.8 μM; IC50(30) =
0.68 μM]. Derivative 8 was found to inhibit MAGL by acting as
a competing substrate, whereas 30 behaved as a noncompetitive
inhibitor. Such compounds could open new perspectives for
assessing the therapeutic potential of the time-controlled and
fine-tuned modulation of 2-AG levels by means of reversible
inhibition of MAGL activity.
EXPERIMENTAL SECTION
■
Molecular Modeling. Docking calculations have been carried out
using Glide.14,15 Protein coordinates were downloaded from the
Protein Data Bank, accession code 3JW8. Chain B, 2-methyl-pentane-
2,4-diol, and water molecules (except water molecules 446 and 529)
were removed. The box was centered on the centroid of residues Ala
61 and Leu 215, as in the previous work of Bertrand et al.13 Docking
solutions were refined using MM-GBSA (molecular mechanics with
generalized Born surface area), as implemented in Prime.16 Residues at
a distance of 12 Å from the ligand were considered as a flexible region
in the refinement.
General Procedure for the Synthesis of Final Compounds
4−18 and 20−31. To a stirred solution of 1 equiv (100 mg) of
carboxylic acid in dry dichloromethane (0.82 mL/mmol) and the
appropriate alcohol (5 equiv) in dry dichloromethane (0.27 mL/
mmol) in an ice bath under argon was added dropwise a solution of
DCC (1 equiv) and DMAP (0.068 equiv) in dry dichloromethane (1.9
mL/mmol). The mixture was stirred for 5 min at this temperature and
then removed from the cooling bath and stirred at rt (3−6 h) until no
further evolution was observed by TLC. The dicyclohexylurea was
filtered off, and the filtrate was washed with saturated NaHCO3. The
organic extracts were dried over anhydrous Na2SO4. Then, the solvent
was evaporated under reduced pressure and the product purified by
column chromatography on silica gel using the appropriate eluent.
( )-Tetrahydro-2H-pyran-2-ylmethyl (2E)-4-(1,1′-Biphenyl-
4-yl)but-2-enoate (19). To a solution of (1,1′-biphenyl-4-yl)-
acetaldehyde (1.0 mmol, 1 equiv) in anhydrous toluene (12 mL/
mmol) was added tetrahydro-2H-pyran-2-ylmethyl
(triphenylphosphoranylidene)acetate (1 equiv). The reaction mixture
was stirred at reflux for 30 min. The solvent was evaporated under
reduced pressure and the product purified by column chromatography
on silica gel.
Chemistry. Melting points (mp, uncorrected) were determined on
a Stuart Scientific electrothermal apparatus for all solid compounds.
Those compounds for which mp was not determined were oils.
Infrared (IR) spectra were measured on a Perkin-Elmer 781,
Shimadzu-8300 infrared spectrophotometer, or a Bruker Tensor 27
instrument equipped with a Specac ATR accessory of 5200−650 cm−1
transmission range; frequencies (ν) are expressed in cm−1. Optical
1
rotation [α] was measured using a Perkin-Elmer 781 polarimeter. H
and 13C NMR spectra have been obtained at the UCM’s NMR core
facility and were recorded on a Varian VXR-300S, Bruker Avance 300
a.m. or Bruker 200-AC instrument at room temperature (rt) unless
stated otherwise. Chemical shifts (δ) are expressed in parts per million
relative to internal tetramethylsilane; coupling constants (J) are in
hertz (Hz). The following abbreviations are used to describe peak
patterns when appropriate: s (singlet), d (doublet), t (triplet), q
(quartet), qt (quintuplet), sept (septuplet), m (multiplet), br (broad),
app (apparent). For all final compounds, purity was determined either
by elemental analyses or high-performance liquid chromatography
coupled to mass spectrometry (HPLC-MS) and high-resolution mass
spectrometry (HRMS). Elemental analyses (C, H, N) were performed
on a LECO CHNS-932 apparatus at the UCM’s analysis services and
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13C NMR (CDCl3, δ): 24.8, 28.8, 31.1 (3CH2), 34.0, 35.4 (ArCH2,
CH2CO), 44.7 (CH2 oxirane), 49.4 (CH oxirane), 64.8 (CH2-ox),
127.0 (3CHAr), 127.1 (2CHAr), 128.7 (2CHAr), 128.8 (2CHAr), 138.7,
141.1, 141.6 (3CAr), 173.5 (CO). Chromatography: hexane/dichloro-
methane, 1:9. HRMS (ESI): calcd for (M + Na)+: 347.1623. Found:
347.1621. Anal.: (C21H24O3) C, H, N.
( )-Oxiran-2-ylmethyl 7-(1,1′-Biphenyl-4-yl)heptanoate (9).
Yield: 27%. Rf: 0.3 (dichloromethane). mp: 67 °C. IR (CH2Cl2, cm−1):
3026, 2929, 2856, 1737, 1519, 1255, 1174, 846, 760. 1H NMR (CDCl3,
δ): 1.30 (qt, J = 3.6 Hz, 4H, (CH2)2−(CH2)2−CO); 1.53−1.65 (m,
4H, 2CH2); 2.28 (t, J = 7.5 Hz, 2H, CH2CO); 2.54−2.60 (m, 3H,
ArCH2, 1H oxirane); 2.76 (app t, J = 4.5 Hz, 1H, 1H oxirane); 3.10−
3.15 (m, 1H, 1H oxirane); 3.83 (dd, J = 12.3; 6.3 Hz, 1H, CH2a-ox);
4.34 (dd, J = 12.3; 3.0 Hz, 1H, CH2b-ox); 7.16−7.18 (m, 2H, 2HAr);
7.21−7.27 (m, 1H, 1HAr); 7.32−7.37 (m, 2H, 2HAr); 7.42−7.45 (m,
2H, 2HAr); 7.48−7.53 (m, 2H, 2HAr). 13C NMR (CDCl3, δ): 24.8,
28.9, 29.0, 31.3 (4CH2), 34.1, 35.5 (ArCH2, CH2CO), 44.7 (CH2
oxirane), 49.4 (CH oxirane), 64.8 (CH2-ox), 127.0 (3CHAr), 127.1
(2CHAr), 128.7 (2CHAr), 128.8 (2CHAr), 138.6, 141.2, 141.8 (3CAr),
173.5 (CO). Chromatography: dichloromethane. HRMS (ESI): calcd
for (M + Na)+: 361.1780. Found: 361.1778. Anal.: (C22H26O3) C, H,
N.
General Procedure for the Synthesis of Final Compounds 32
and 33. To a solution of 1.0 mmol of the corresponding benzylated
intermediates in a mixture of dry dichloromethane/absolute ethanol
(3:1; 115 mL/mmol) was added Pd(OH)2 (327 mg/mmol), and the
mixture was hydrogenated in a Parr hydrogenator (hydrogen initial
pressure 50 psi) during 3 h at rt. The catalyst was filtered off over
Celite, and the solvent was evaporated under reduced pressure. The
resulting final compounds were purified by column chromatography
on silica gel.
( )-Oxiran-2-ylmethyl (1,1′-Biphenyl-4-yl)acetate (4). Yield:
55%. Rf: 0.3 (dichloromethane). mp: 55−57 °C. IR (CH2Cl2, cm−1):
2922, 2854, 1740, 1462, 1248, 1152, 1009, 856. 1H NMR (CDCl3, δ):
2.63 (dd, J = 4.9; 2.6 Hz, 1H, 1H oxirane); 2.82−2.86 (m, 1H, 1H
oxirane); 3.19−3.27 (m, 1H, 1H oxirane); 3.72 (s, 2H, CH2CO); 3.96
(dd, J = 12.3 Hz, 6.2 Hz, 1H, CH2a-ox); 4.47 (dd, J = 12.3; 3.0 Hz, 1H,
CH2b-ox); 7.31−7.48 (m, 5H, 5HAr); 7.54−7.61 (m, 4H, 4HAr). 13C
NMR (CDCl3, δ): 40.5 (CH2CO), 44.4 (CH2 oxirane), 49.1 (CH
oxirane), 65.1 (CH2-ox), 126.9 (2CHAr), 127.1 (CHAr), 127.2
(2CHAr), 128.5 (2CHAr), 129.5 (2CHAr), 132.5, 140.3, 140.8 (3CAr),
171.3 (CO). Chromatography: dichloromethane. HRMS (ESI): calcd
for (M + Na)+: 291.0997. Found: 291.0986. Anal.: (C17H16O3) C, H,
N.
( )-Oxiran-2-ylmethyl 3-(1,1′-Biphenyl-4-yl)propanoate (5).
(
)-Tetrahydro-2H-pyran-2-ylmethyl (1,1′-Biphenyl-4-yl)-
Yield: 59%. Rf: 0.4 (dichloromethane). IR (CH2Cl2, cm−1): 2925,
acetate (10). Yield: 58%. Rf: 0.3 (chloroform). mp: 37 °C. IR
1
(CH2Cl2, cm−1): 2935, 2853, 1736, 1152, 1088, 1048, 1007, 752. H
1
2856, 1737, 1520, 1289, 1158, 840, 763. H NMR (CDCl3, δ): 2.54
(dd, J = 4.9; 2.6 Hz, 1H, 1H oxirane); 2.65 (t, J = 7.4 Hz, 2H,
CH2CO); 2.75 (app t, J = 4.5 Hz, 1H, 1H oxirane); 2.94 (t, J = 7.7 Hz,
2H, ArCH2); 3.07−3.15 (m, 1H, 1H oxirane); 3.86 (dd, J = 12.3; 6.3
Hz, 1H, CH2a-ox); 4.35 (dd, J = 12.3; 3.0 Hz, 1H, CH2b-ox); 7.19−
7.53 (m, 9H, 9HAr). 13C NMR (CDCl3, δ): 30.4, 35.5 (ArCH2,
CH2CO), 44.5 (CH2 oxirane), 49.2 (CH oxirane), 64.9 (CH2-ox),
126.9 (2CHAr), 127.0 (CHAr), 127.1 (2CHAr), 128.6 (4CHAr), 139.2,
139.3, 140.8 (3CAr), 172.4 (CO). Chromatography: dichloromethane.
HRMS (ESI): calcd for (M + Na)+: 305.1154. Found: 305.1149.
HPLC-MS: >95%.
NMR (CDCl3, δ): 1.49−1.53 (m, 5H, 5H tetrahydropyran); 1.75−
1.82 (m, 1H, 1H tetrahydropyran); 3.31−3.54 (m, 2H, 2H
tetrahydropyran); 3.64 (s, 2H, CH2CO); 3.91−4.04 (m, 3H, 1H
tetrahydropyran, CH2-tet); 7.29−7.48 (m, 5H, 5HAr); 7.52−7.61 (m,
4H, 4HAr). 13C NMR (CDCl3, δ): 22.8, 25.5, 27.6 (3CH2
tetrahydropyran), 40.6 (CH2CO), 67.7 (CH2-tet), 68.2 (OCH2
tetrahydropyran), 75.2 (OCH tetrahydropyran), 126.8 (2CHAr),
127.0 (CHAr), 127.1 (2CHAr), 128.5 (2CHAr), 129.5 (2CHAr),
132.9, 139.8, 140.7 (3CAr), 171.4 (CO). Chromatography: chloroform.
HRMS (ESI): calcd for (M + Na)+: 333.1467. Found: 333.1461. Anal.:
(C20H22O3) C, H, N.
( )-Oxiran-2-ylmethyl 4-(1,1′-Biphenyl-4-yl)butanoate (6).
Yield: 41%. Rf: 0.3 (chloroform). IR (CH2Cl2, cm−1): 2928, 2855,
( )-Tetrahydro-2H-pyran-2-ylmethyl 3-(1,1′-Biphenyl-4-yl)-
propanoate (11). Yield: 49%. Rf: 0.4 (dichloromethane). mp: 45
°C. IR (CH2Cl2, cm−1): 2926, 2851, 1735, 1175, 1089, 1049, 835, 698.
1H NMR (CDCl3, δ): 1.40−1.50 (m, 5H, 5H tetrahydropyran); 1.73−
1
2118, 1737, 1524, 1246, 1178, 1144, 846, 759. H NMR (CDCl3, δ):
1.94 (qt, J = 7.4 Hz, 2H, CH2CH2CH2); 2.35 (t, J = 7.4 Hz, 2H,
CH2CO); 2.56−2.67 (m, 3H, ArCH2, 1H oxirane); 2.77 (app t, J = 4.5
Hz, 1H, 1H oxirane); 3.09−3.17 (m, 1H, 1H oxirane); 3.84 (dd, J =
12.3; 6.4 Hz, 1H, CH2a-ox); 4.36 (dd, J = 12.3; 3.0 Hz, 1H, CH2b-ox);
7.16−7.53 (m, 9H, 9HAr). 13C NMR (CDCl3, δ): 26.4 (CH2CH2CH2),
33.4, 34.7 (ArCH2, CH2CO), 44.7 (CH2 oxirane), 49.4 (CH oxirane),
64.9 (CH2-ox), 127.0 (2CHAr), 127.1 (CHAr), 127.2 (2CHAr), 128.8
(2CHAr), 129.0 (2CHAr), 139.1, 140.4, 141.0 (3CAr), 173.2 (CO).
Chromatography: dichloromethane. HRMS (ESI): calcd for (M +
Na)+: 319.1310. Found: 319.1305. HPLC-MS: >95%.
( )-Oxiran-2-ylmethyl 5-(1,1′-Biphenyl-4-yl)pentanoate (7).
Yield: 56%. Rf: 0.4 (chloroform). IR (CH2Cl2, cm−1): 2921, 2853,
1737, 1484, 1256, 1175, 758. 1H NMR (CDCl3, δ): 1.57−1.71 (m, 4H,
(CH2)2CH2CO); 2.33 (t, J = 7.1 Hz, 2H, CH2CO); 2.49−2.63 (m,
3H, ArCH2, 1H oxirane); 2.76 (app t, J = 4.5 Hz, 1H, 1H oxirane);
3.10−3.16 (m, 1H, 1H oxirane); 3.84 (dd, J = 12.3; 6.3 Hz, 1H, CH2a-
ox); 4.35 (dd, J = 12.3; 3.0 Hz, 1H, CH2b-ox); 7.16−7.19 (m, 2H,
2HAr); 7.22−7.28 (m, 1H, 1HAr); 7.33−7.38 (m, 2H, 2HAr); 7.42−
7.45 (m, 2H, 2HAr); 7.48−7.52 (m, 2H, 2HAr). 13C NMR (CDCl3, δ):
24.5, 30.9 (2CH2), 34.0, 35.2 (ArCH2, CH2CO), 44.7 (CH2 oxirane),
49.4 (CH oxirane), 64.9 (CH2-ox), 127.0 (2CHAr), 127.1 (CHAr),
127.3 (2CHAr), 128.7 (2CHAr), 128.8 (2CHAr), 138.8, 141.1, 141.2
(3CAr), 173.3 (CO). Chromatography: chloroform. HRMS (ESI):
calcd for (M + Na)+: 333.1467. Found: 333.1466. HPLC-MS: >95%.
( )-Oxiran-2-ylmethyl 6-(1,1′-Biphenyl-4-yl)hexanoate (8).
Yield: 53%. Rf: 0.2 (hexane/dichloromethane, 1:9). mp: 64−65 °C. IR
(CH2Cl2, cm−1): 2923, 2854, 2119, 1737, 1485, 1257, 1174, 1013, 759.
1H NMR (CDCl3, δ): 1.26−1.38 (m, 2H, CH2(CH2)2CO); 1.56−1.68
(m, 4H, 2CH2); 2.30 (t, J = 7.5 Hz, 2H, CH2CO); 2.56−2.61 (m, 3H,
ArCH2, 1H oxirane); 2.76 (app t, J = 4.5 Hz, 1H, 1H oxirane); 3.10−
3.15 (m, 1H, 1H oxirane); 3.83 (dd, J = 12.3; 6.3 Hz, 1H, CH2a-ox);
4.35 (dd, J = 12.3; 3.0 Hz, 1H, CH2b-ox); 7.16−7.53 (m, 9H, 9HAr).
1.78 (m, 1H, 1H tetrahydropyran); 2.64 (t, J = 7.9 Hz, 2H, CH2CO);
2.93 (t, J = 7.6 Hz, 2H, ArCH2); 3.30−3.45 (m, 2H, 2H
tetrahydropyran); 3.90−4.07 (m, 3H, 1H tetrahydropyran, CH2-tet);
7.30−7.60 (m, 7H, 7HAr); 7.54−7.60 (m, 2H, 2HAr). 13C NMR
(CDCl3, δ): 22.9, 25.7, 27.8 (3CH2 tetrahydropyran), 30.5, 35.6
(CH2CO, ArCH2), 67.5 (CH2-tet), 68.4 (OCH2 tetrahydropyran),
75.4 (OCH tetrahydropyran), 126.9 (2CHAr), 127.1 (CHAr), 127.2
(2CHAr), 128.7 (2CHAr), 128.8 (2CHAr), 139.2, 139.6, 140.9 (3CAr),
172.9 (CO). Chromatography: dichloromethane. HRMS (ESI): calcd
for (M + Na)+: 347.1623. Found: 347.1632. Anal.: (C21H24O3) C, H,
N.
( )-Tetrahydro-2H-pyran-2-ylmethyl 4-(1,1′-Biphenyl-4-yl)-
butanoate (12). Yield: 71%. Rf: 0.2 (hexane/chloroform, 1:9). mp:
51 °C. IR (CH2Cl2, cm−1): 2938, 2852, 1734, 1179, 1089, 844, 756,
1
699. H NMR (CDCl3, δ): 1.40−1.49 (m, 5H, 5H tetrahydropyran);
1.78−1.82 (m, 1H, 1H tetrahydropyran); 1.92 (qt, J = 7.5 Hz, 2H,
CH2CH2CH2); 2.34 (t, J = 7.4 Hz, 2H, CH2CO); 2.63 (t, J = 7.6 Hz,
2H, ArCH2); 3.31−3.49 (m, 2H, 2H tetrahydropyran); 3.90−4.07 (m,
3H, 1H tetrahydropyran, CH2-tet); 7.16−7.18 (m, 2H, 2HAr); 7.21−
7.27 (m, 1H, 1HAr); 7.32−7.38 (m, 2H, 2HAr); 7.42−7.45 (m, 2H,
2HAr); 7.47−7.51 (m, 2H, 2HAr). 13C NMR (CDCl3, δ): 23.0 (CH2
tetrahydropyran), 25.8 (CH2CH2CH2), 26.5, 28.0 (2CH2 tetrahy-
dropyran), 33.6, 34.8 (ArCH2, CH2CO), 67.4 (CH2-tet), 68.4 (OCH2
tetrahydropyran), 75.6 (OCH tetrahydropyran), 127.0 (2CHAr), 127.1
(CHAr), 127.2 (2CHAr), 128.8 (2CHAr), 129.0 (2CHAr), 139.0, 140.6,
141.1 (3CAr), 173.5 (CO). Chromatography: hexane/chloroform, 1:9.
HRMS (ESI): calcd for (M + Na)+: 361.1780. Found: 361.1777. Anal.:
(C22H26O3) C, H, N.
( )-Tetrahydro-2H-pyran-2-ylmethyl 5-(1,1′-Biphenyl-4-yl)-
pentanoate (13). Yield: 54%. Rf: 0.3 (dichloromethane). IR
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(CH2Cl2, cm−1): 3028, 2933, 2853, 1735, 1519, 1487, 1177, 1088,
1050, 760, 698. 1H NMR (CDCl3, δ): 1.35−1.50 (m, 5H, 5H
tetrahydropyran); 1.61−1.68 (m, 4H, (CH2)2CH2CO); 1.76−1.81 (m,
1H, 1H tetrahydropyran); 2.33 (t, J = 7.1 Hz, 2H, CH2CO); 2.60 (t, J
= 7.1 Hz, 2H, ArCH2); 3.32−3.50 (m, 2H, 2H tetrahydropyran);
3.91−4.05 (m, 3H, 1H tetrahydropyran, CH2-tet); 7.16−7.19 (m, 2H,
2HAr); 7.23−7.28 (m, 1H, 1HAr); 7.32−7.40 (m, 2H, 2HAr); 7.42−
7.45 (m, 2H, 2HAr); 7.48−7.53 (m, 2H, 2HAr). 13C NMR (CDCl3, δ):
23.0 (CH2 tetrahydropyran), 24.6 (CH2), 25.8, 27.9 (2CH2
tetrahydropyran), 30.9 (CH2), 34.1, 35.2 (ArCH2, CH2CO), 67.4
(CH2-tet), 68.5 (OCH2 tetrahydropyran), 75.5 (OCH tetrahydropyr-
an), 127.0 (3CHAr), 127.1 (2CHAr), 128.7 (2CHAr), 128.8 (2CHAr),
138.9, 141.3, 141.7 (3CAr), 173.7 (CO). Chromatography: dichloro-
methane. HRMS (ESI): calcd for (M + Na)+: 375.1936. Found:
375.1941. HPLC-MS: >95%.
( )-Tetrahydro-2H-pyran-2-ylmethyl 6-(1,1′-Biphenyl-4-yl)-
hexanoate (14). Yield: 29%. Rf: 0.2 (dichloromethane). IR
(CH2Cl2, cm−1): 3029, 2929, 2854, 1733, 1453, 1272, 1180, 1089,
756, 698. 1H NMR (CDCl3, δ): 1.20−1.66 (m, 11H, 3CH2, 5H
tetrahydropyran); 1.77−1.80 (m, 1H, 1H tetrahydropyran); 2.29 (t, J
= 7.5 Hz, 2H, CH2CO); 2.58 (t, J = 7.7 Hz, 2H, ArCH2); 3.32−3.48
(m, 2H, 2H tetrahydropyran); 3.90−4.04 (m, 3H, CH2-tet, 1H
tetrahydropyran); 7.15−7.18 (m, 2H, 2HAr); 7.22−7.27 (m, 1H,
1HAr); 7.32−7.40 (m, 2H, 2HAr); 7.42−7.45 (m, 2H, 2HAr); 7.48−
7.60 (m, 2H, 2HAr). 13C NMR (CDCl3, δ): 23.0 (CH2 tetrahydropyr-
an), 24.8 (CH2), 25.8, 27.9 (2CH2 tetrahydropyran), 28.8, 31.1
(2CH2), 34.1, 35.4 (ArCH2, CH2CO), 67.4 (CH2-tet), 68.5 (OCH2
tetrahydropyran), 75.5 (OCH tetrahydropyran), 127.0 (3CHAr), 127.1
(2CHAr), 128.7 (2CHAr), 128.8 (2CHAr), 138.7, 141.2, 141.7 (3CAr),
173.9 (CO). Chromatography: dichloromethane. HRMS (ESI): calcd
for (M + Na)+: 389.2093. Found: 389.2091. HPLC-MS: >95%.
( )-Tetrahydro-2H-pyran-2-ylmethyl 7-(1,1′-Biphenyl-4-yl)-
heptanoate (15). Yield: 32%. Rf: 0.2 (hexane/ethyl acetate, 9:1).
IR (CH2Cl2, cm−1): 3028, 2930, 2853, 1735, 1519, 1177, 1088, 760,
733. 1H NMR (CDCl3, δ): 1.18−1.41 (m, 6H, 3CH2); 1.43−1.66 (m,
7H, CH2, 5H tetrahydropyran); 1.75−1.80 (m, 1H, 1H tetrahy-
dropyran); 2.28 (t, J = 7.5 Hz, 2H, CH2CO); 2.56 (t, J = 7.7 Hz, 2H,
ArCH2); 3.32−3.49 (m, 2H, 2H tetrahydropyran); 3.90−4.04 (m, 3H,
CH2-tet, 1H tetrahydropyran); 7.15−7.18 (m, 2H, 2HAr); 7.21−7.27
(m, 1H, 1HAr); 7.32−7.39 (m, 2H, 2HAr); 7.41−7.45 (m, 2H, 2HAr);
7.47−7.53 (m, 2H, 2HAr). 13C NMR (CDCl3, δ): 23.0 (CH2
tetrahydropyran), 24.9 (CH2), 25.8, 27.8 (2CH2 tetrahydropyran),
28.9, 29.0, 31.3 (3CH2), 34.2, 35.5 (ArCH2, CH2CO), 67.3 (CH2-tet),
68.5 (OCH2 tetrahydropyran), 75.5 (OCH tetrahydropyran), 126.9
(2CHAr), 127.0 (3CHAr), 128.7 (2CHAr), 128.8 (2CHAr), 138.6, 141.2,
141.9 (3CAr), 173.9 (CO). Chromatography: hexane/ethyl acetate,
9:1. HRMS (ESI): calcd for (M + Na)+: 403.2249. Found: 403.2253.
HPLC-MS: >95%.
7.57 (m, 6H, 6HAr); 7.68 (d, J = 16.0 Hz, 1H, ArCH). 13C NMR
(CDCl3, δ): 23.0, 25.8, 27.9 (3CH2 tetrahydropyran), 67.6 (CH2-tet),
68.5 (OCH2 tetrahydropyran), 75.6 (OCH tetrahydropyran), 117.8
(CHCO), 127.0 (2CHAr), 127.5 (2CHAr), 127.8 (CHAr), 128.6
(2CHAr), 128.9 (2CHAr), 133.4, 140.2, 143.1 (3CAr), 144.6 (ArCH),
167.1 (CO). Chromatography: dichloromethane. HRMS (ESI): calcd
for (M + Na)+: 345.1467. Found: 345.1469. Anal.: (C21H22O3) C, H,
N.
( )-Tetrahydro-2H-pyran-2-ylmethyl (3E)-4-(1,1′-Biphenyl-
4-yl)but-3-enoate (18). Yield: 69%. Rf: 0.2 (hexane/dichloro-
methane, 2:8). mp: 92−94 °C. IR (CH2Cl2, cm−1): 3032, 2938,
1
2852, 1734, 1262, 1162, 1090, 759. H NMR (CDCl3, δ): 1.22−1.56
(m, 5H, 5H tetrahydropyran); 1.77−1.82 (m, 1H, 1H tetrahydropyr-
an); 3.25 (dd, J = 6.9; 1.1 Hz, 2H, CH2CO); 3.34−3.47 (m, 2H, 2H
tetrahydropyran), 3.98−4.09 (m, 3H, CH2-tet, 1H tetrahydropyran);
6.28 (dt, J = 15.9; 7.0 Hz, 1H, CHCH2); 6.46 (d, J = 15.9 Hz, 1H,
ArCH), 7.26−7.38 (m, 5H, 5HAr), 7.46−7.54 (m, 4H, 4HAr). 13C
NMR (CDCl3, δ): 23.0, 25.8, 27.9 (3CH2 tetrahydropyran), 38.3
(CH2CO), 67.8 (CH2-tet), 68.5 (OCH2 tetrahydropyran), 75.4 (OCH
tetrahydropyran), 121.9 (CHCH2), 126.7 (2CHAr), 126.9 (2CHAr),
127.2 (2CHAr), 127.3 (CHAr), 128.8 (2CHAr), 133.0 (ArCH), 136.3,
140.3, 140.7 (3CAr), 171.6 (CO). Chromatography: hexane/dichloro-
methane, 2:8. HRMS (ESI): calcd for (M + Na)+: 359.1623. Found:
359.1622. Anal.: (C22H24O3) C, H, N.
( )-Tetrahydro-2H-pyran-2-ylmethyl (2E)-4-(1,1′-Biphenyl-
4-yl)but-2-enoate (19). Yield: 67%. Rf: 0.2 (hexane/dichloro-
methane, 2:8). mp: 71 °C. IR (CH2Cl2, cm−1): 3029, 2936, 2850,
1718, 1653, 1269, 1087, 763. 1H NMR (CDCl3, δ): 1.18−1.55 (m, 5H,
5H tetrahydropyran); 1.76−1.81 (m, 1H, 1H tetrahydropyran); 3.32−
3.53 (m, 4H, ArCH2, 2H tetrahydropyran); 3.91−4.10 (m, 3H, CH2-
tet, 1H tetrahydropyran); 5.83 (dt, J = 15.6; 1.6 Hz, 1H, CH2CH
CH); 7.10 (dt, J = 15.6; 6.7 Hz, 1H, CHCO); 7.19−7.39 (m, 5H,
5HAr); 7.46−7.53 (m, 4H, 4HAr). 13C NMR (CDCl3, δ): 23.0, 25.8,
27.9 (3CH2 tetrahydropyran), 38.1 (ArCH2), 67.4 (CH2-tet), 68.4
(OCH2 tetrahydropyran), 75.5 (OCH tetrahydropyran), 122.2
(CH2CHCH), 127.1 (2CHAr), 127.2 (CHAr), 127.5 (2CHAr),
128.8 (2CHAr), 129.3 (2CHAr), 136.7, 139.7, 140.9 (3CAr), 147.7
(CHCO), 166.5 (CO). Chromatography: hexane/dichloromethane,
2:8. HRMS (ESI): calcd for (M + Na)+: 359.1623. Found: 359.1616.
Anal.: (C22H24O3) C, H, N.
( )-Oxiran-2-ylmethyl (4-Benzylphenyl)acetate (20). Yield:
71%. Rf: 0.3 (dichloromethane). IR (CH2Cl2, cm−1): 2922, 2853, 1738,
1
1147, 1011, 854. H NMR (CDCl3, δ): 2.61 (dd, J = 4.8; 2.6 Hz, 1H,
1H oxirane), 2.80−2.84 (m, 1H, 1H oxirane), 3.16−3.24 (m, 1H, 1H
oxirane), 3.64 (s, 2H, ArCH2CO), 3.89−3.97 (m, 3H, ArCH2Ar,
CH2a-ox), 4.43 (dd, J = 12.3; 3.0 Hz, 1H, CH2b-ox), 7.05−7.25 (m,
9H, 9HAr). 13C NMR (CDCl3, δ): 40.6 (ArCH2CO), 41.5 (ArCH2Ar),
44.6 (OCH2 oxirane), 49.3 (OCH oxirane), 65.2 (CH2-ox), 126.1
(CHAr), 128.4 (2CHAr), 128.9 (2CHAr), 129.2 (2CHAr), 129.4
(2CHAr), 131.5, 140.1, 141.5 (3CAr), 171.5 (CO). Chromatography:
dichloromethane. HRMS (ESI): calcd for (M + Na)+: 305.1154.
Found: 305.1150. HPLC-MS: >95%.
(
)-Oxiran-2-ylmethyl (2E)-3-(1,1′-Biphenyl-4-yl)acrilate
(16). Yield: 41%. Rf: 0.3 (dichloromethane). mp: 99 °C. IR
(CH2Cl2, cm−1): 2925, 2852, 1713, 1636, 1450, 1349, 1266, 1187,
1177, 985, 852, 768. H NMR (CDCl3, δ): 2.72 (dd, J = 4.9; 2.6 Hz,
1
( )-Oxiran-2-ylmethyl 3-(4-Benzylphenyl)propanoate (21).
Yield: 75%. Rf: 0.2 (hexane/dichloromethane, 3:7). IR (CH2Cl2,
cm−1): 3020, 2926, 2855, 1737, 1156, 907, 848. 1H NMR (CDCl3, δ):
2.58−2.70 (m, 3H, 1H oxirane, CH2CO); 2.79−2.83 (m, 1H, 1H
oxirane); 2.90−2.98 (m, 2H, ArCH2CH2); 3.13−3.21 (m, 1H, 1H
oxirane); 3.84−3.95 (m, 3H, ArCH2Ar, CH2a-ox); 4.40 (dd, J = 12.3;
3.1 Hz, 1H, CH2b-ox); 7.07−7.35 (m, 9H, 9HAr). 13C NMR (CDCl3,
δ): 30.5, 35.7 (CH2CO, ArCH2CH2), 41.6 (ArCH2Ar), 44.7 (CH2
oxirane), 49.3 (CH oxirane), 65.0 (CH2-ox), 126.1 (CHAr), 128.4
(2CHAr), 128.5 (2CHAr), 128.9 (2CHAr), 129.1 (2CHAr), 138.0, 139.2,
141.2 (3CAr), 172.6 (CO). Chromatography: hexane/dichlorome-
thane, 3:7. HRMS (ESI): calcd for (M + Na)+: 319.1310. Found:
319.1302. HPLC-MS: >95%.
1H, 1H oxirane); 2.88−2.92 (m, 1H, 1H oxirane); 3.27−3.35 (m, 1H,
1H oxirane); 4.06 (dd, J = 12.3; 6.3 Hz, 1H, CH2a-ox); 4.57 (dd, J =
12.3; 3.0 Hz, 1H, CH2b-ox); 6.51 (d, J = 16.0 Hz, 1H, CH−CO);
7.34−7.51 (m, 3H, 3HAr); 7.59−7.67 (m, 6H, 6HAr); 7.77 (d, J = 16.0
Hz, 1H, ArCH). 13C NMR (CDCl3, δ): 44.8 (CH2 oxirane), 49.5 (CH
oxirane), 65.1 (CH2-ox), 117.2 (CHCO), 127.1 (2CHAr), 127.6
(CHAr), 127.9 (2CHAr), 128.7 (2CHAr), 128.9 (2CHAr), 133.2, 140.1,
143.3 (3CAr), 145.2 (ArCH), 166.7 (CO). Chromatography: dichloro-
methane. HRMS (ESI): calcd for (M + Na)+: 303.0997. Found:
303.0998. Anal.: (C18H16O3) C, H, N.
( )-Tetrahydro-2H-pyran-2-ylmethyl (2E)-3-(1,1′-Biphenyl-
4-yl)acrilate (17). Yield: 60%. Rf: 0.2 (dichloromethane). mp:
113−115 °C. IR (CH2Cl2, cm−1): 2940, 2850, 1712, 1636, 1317,
1175, 1085, 1048, 867, 801. 1H NMR (CDCl3, δ): 1.30−1.59 (m, 5H,
5H tetrahydropyran); 1.79−1.90 (m, 1H, 1H tetrahydropyran); 3.35−
3.46 (m, 1H, 1H tetrahydropyran); 3.49−3.57 (m, 1H, 1H
tetrahydropyran); 4.09−4.22 (m, 3H, 1H tetrahydropyran, CH2-tet);
6.48 (d, J = 16.0 Hz, 1H, CHCO); 7.26−7.43 (m, 3H, 3HAr), 7.52−
( )-Oxiran-2-ylmethyl 4-(4-Benzylphenyl)butanoate (22).
Yield: 76%. Rf: 0.3 (dichloromethane). IR (CH2Cl2, cm−1): 3019,
2924, 2854, 1737, 1504, 1449, 1177, 1143, 850. 1H NMR (CDCl3, δ):
1.85 (qt, J = 7.3 Hz, 2H, CH2CH2CH2); 2.27 (t, J = 7.5 Hz, 2H,
CH2CO); 2.49−2.56 (m, 3H, ArCH2CH2, 1H oxirane); 2.74 (app t, J
833
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Article
= 6.0 Hz, 1H, 1H oxirane); 3.05−3.13 (m, 1H, 1H oxirane); 3.74−
3.89 (m, 3H, ArCH2Ar, CH2a-ox); 4.30 (dd, J = 12.3; 3.0 Hz, 1H,
CH2b-ox); 7.07−7.23 (m, 9H, 9HAr). 13C NMR (CDCl3, δ): 26.4
(CH2CH2CH2), 33.4, 34.7 (CH2CO, ArCH2), 41.5 (ArCH2Ar), 44.6
(CH2 oxirane), 49.3 (CH oxirane), 64.8 (CH2-ox), 126.0 (CHAr),
128.4 (2CHAr), 128.6 (2CHAr), 128.8 (2CHAr), 128.9 (2CHAr), 138.9,
139.0, 141.3 (3CAr), 173.2 (CO). Chromatography: dichloromethane.
HRMS (ESI): calcd for (M + Na)+: 333.1467. Found: 333.1461.
HPLC-MS: >95%.
( )-Oxiran-2-ylmethyl 4-(2-Phenylethyl)benzoate (23). Yield:
75%. Rf: 0.3 (dichloromethane). IR (CH2Cl2, cm−1): 2921, 2853, 1720,
1458, 1179, 1106, 843. 1H NMR (CDCl3, δ): 2.65 (dd, J = 4.8; 2.6 Hz,
1H, 1H oxirane); 2.79−2.94 (m, 5H, 1H oxirane, Ar(CH2)2Ar); 3.22−
3.30 (m, 1H, 1H oxirane); 4.08 (dd, J = 12.3; 6.2 Hz, 1H, CH2a-ox);
4.57 (dd, J = 12.3; 3.0 Hz, 1H, CH2b-ox); 7.05−7.23 (m, 7H, 7HAr);
7.89 (d, J = 8.2 Hz, 2H, 2HAr). 13C NMR (CDCl3, δ): 37.3, 37.7
(2CH2Ar), 44.6 (CH2 oxirane), 49.4 (CH oxirane), 65.1 (CH2-ox),
125.9 (CHAr), 127.3 (CAr), 128.2 (2CHAr), 128.3 (2CHAr), 128.5
(2CHAr), 129.7 (2CHAr), 140.9, 147.4 (2CAr), 166.1 (CO).
Chromatography: dichloromethane. HRMS (ESI): calcd for (M +
Na)+: 305.1154. Found: 305.1146. HPLC-MS: >95%.
tetrahydropyran); 3.55−3.66 (m, 1H, 1H tetrahydropyran); 3.93−4.13
(m, 1H, 1H tetrahydropyran); 4.19−4.28 (m, 2H, CH2-tet,); 7.05−
7.25 (m, 7H, 7HAr); 7.90 (dd, J = 6.6; 1.7 Hz, 2H, 2HAr). 13C NMR
(CDCl3, δ): 22.9, 25.7, 28.0 (3CH2 tetrahydropyran), 37.3, 37.7
(2CH2Ar), 67.6 (CH2-tet), 68.3 (OCH2 tetrahydropyran), 75.4 (OCH
tetrahydropyran), 125.9 (CHAr), 127.8 (CAr), 128.2 (2CHAr), 128.3
(2CHAr), 128.4 (2CHAr), 129.7 (2CHAr), 141.0, 147.0 (2CAr), 166.4
(CO). Chromatography: dichloromethane. HRMS (ESI): calcd for (M
+ Na)+: 347.1623. Found: 347.1617. HPLC-MS: >95%.
́
(2R)-(−)-Oxiran-2-ylmethyl 6-(1,1-Biphenyl-4-yl)hexanoate
(28). Data of 28 were identical to those recorded for the racemic
20
material 8 except for the optical rotation. 28: [α]D −15.5 (c = 1.9,
ethanol). Anal.: (C21H24O3) C, H, N.
(2S)-(+)-Oxiran-2-ylmethyl 6-(1,1-Biphenyl-4-yl)hexanoate
(29). Data of 29 were identical to those recorded for the racemic
́
20
material 8 except for the optical rotation. 29: [α]D +16.3 (c = 1.9,
ethanol). Anal.: (C21H24O3) C, H, N.
(2R)-(−)-Oxiran-2-ylmethyl(4-benzylphenyl)acetate (30).
Data of 30 were identical to those recorded for the racemic material
20
20 except for the optical rotation. 30: [α]D −9.1 (c = 1.5, ethanol).
HPLC-MS: >95%.
(2S)-(+)-Oxiran-2-ylmethyl(4-benzylphenyl)acetate (31).
(
)-Tetrahydro-2H-pyran-2-ylmethyl (4-Benzylphenyl)-
Data of 31 were identical to those recorded for the racemic material
acetate (24). Yield: 69%. Rf: 0.2 (chloroform). mp: 65 °C. IR
(CH2Cl2, cm−1): 3026, 2937, 2850, 1736, 1147, 1089, 1012. 1H NMR
(CDCl3, δ): 1.21−1.56 (m, 5H, 5H tetrahydropyran); 1.81−1.86 (m,
1H, 1H tetrahydropyran); 3.36−3.58 (m, 2H, 2H tetrahydropyran);
3.64 (s, 2H, ArCH2CO); 3.96 (s, 2H, ArCH2Ar); 3.98−4.09 (m, 3H,
CH2-tet, 1H tetrahydropyran); 7.12−7.33 (m, 9H, 9HAr). 13C NMR
(CDCl3, δ): 23.0, 25.8, 27.8 (3CH2 tetrahydropyran), 40.8
(ArCH2CO), 41.6 (ArCH2Ar), 67.8 (CH2-tet), 68.4 (OCH2
tetrahydropyran), 75.4 (OCH tetrahydropyran), 126.1 (CHAr), 128.5
(2CHAr), 128.9 (2CHAr), 129.1 (2CHAr), 129.4 (2CHAr), 131.8, 139.9,
141.1 (3CAr), 171.8 (CO). Chromatography: chloroform. HRMS
(ESI): calcd for (M + Na)+: 347.1623. Found: 347.1626. Anal.:
(C21H24O3) C, H, N.
20
20 except for the optical rotation. 31: [α]D +9.0 (c = 1.5, ethanol).
HPLC-MS: >95%.
2-Hydroxy-1-(hydroxymethyl)ethyl 6-(1,1′-biphenyl-4-yl)-
hexanoate (32). Yield: 28%. Rf: 0.1 (dichloromethane/ethanol,
1
9.6:0.4). IR (CH2Cl2, cm−1): 3369, 2924, 2855, 1735, 1176, 744. H
NMR (CDCl3, δ): 1.35−1.45 (m, 2H, CH2(CH2)2CO); 1.62−1.74
(m, 4H, 2CH2); 2.36 (t, J = 7.5 Hz, 2H, CH2CO); 2.65 (t, J = 7.6 Hz,
2H, ArCH2); 3.54−3.70 (m, 2H, 2CH2aOH); 3.80−3.94 (m, 1H,
CHO); 4.11−4.22 (m, 2H, 2CH2bOH); 7.22−7.59 (m, 9H, 9HAr). 13
C
NMR (CDCl3, δ): 24.8, 28.7, 31.0 (3CH2), 34.1, 35.3 (ArCH2,
CH2CO), 63.4, 65.2 (2CH2OH), 70.3 (CH), 127.0 (2CHAr), 127.1
(3CHAr), 128.7 (2CHAr), 128.8 (2CHAr), 138.7, 141.1, 141.6 (3CAr),
174.2 (CO). Chromatography: dichloromethane/ethanol (9.6:0.4).
HRMS (ESI): calcd for (M + Na)+: 365.1729. Found: 365.1709.
HPLC-MS: > 95%.
(
)-Tetrahydro-2H-pyran-2-ylmethyl 3-(4-Benzylphenyl)-
propanoate (25). Yield: 76%. Rf: 0.2 (hexane/dichloromethane,
2:8). IR (CH2Cl2, cm−1): 3023, 2925, 2852, 1735, 1447, 1174, 1090,
855. H NMR (CDCl3, δ): 1.26−1.61 (m, 5H, 5H tetrahydropyran);
1
2-Hydroxy-1-(hydroxymethyl)ethyl (4-Benzylphenyl)acetate
(33). Yield: 70%. Rf: 0.3 (dichloromethane/ethanol, 9.7:0.3). mp: 82
1.81−1.86 (m, 1H, 1H tetrahydropyran); 2.62−2.70 (m, 2H,
CH2CO); 2.89−2.96 (m, 2H, ArCH2CH2); 3.36−3.53 (m, 2H, 2H
tetrahydropyran); 3.95−4.13 (m, 5H, ArCH2Ar, 1H tetrahydropyran,
CH2-tet); 7.12−7.35 (m, 9H, 9HAr). 13C NMR (CDCl3, δ): 23.0, 25.8,
27.8 (3CH2 tetrahydropyran), 30.5, 35.8 (CH2CO, ArCH2CH2), 41.6
(ArCH2Ar), 67.5 (CH2-tet), 68.4 (OCH2 tetrahydropyran), 75.5
(OCH tetrahydropyran), 126.1 (CHAr), 128.4 (2CHAr), 128.5
(2CHAr), 128.9 (2CHAr), 129.0 (2CHAr), 138.2, 139.1, 141.2 (3CAr),
173.0 (CO). Chromatography: hexane/dichloromethane, 2:8. HRMS
(ESI): calcd for (M + Na)+: 361.1780. Found: 361.1769. HPLC-MS:
>95%.
1
°C. IR (CH2Cl2, cm−1): 3404, 2924, 1732, 1157, 726, 699. H NMR
(CDCl3, δ): 2.15 (br s, 2H, 2OH), 3.51−3.93 (m, 5H, 2CH2aOH,
CH2CO, CH), 3.98 (s, 2H, ArCH2Ar), 4.13−4.24 (m, 2H,
2CH2bOH), 7.15−7.28 (m, 9H, 9HAr). 13C NMR (CDCl3, δ): 41.2
(CH2CO), 41.9 (ArCH2Ar), 63.6, 66.1 (2CH2OH), 70.5 (CH), 126.5
(CHAr), 128.9 (2CHAr), 129.3 (2CHAr), 129.6 (2CHAr), 129.7
(2CHAr), 131.7, 140.7, 141.3 (3CAr), 172.5 (CO). Chromatography:
dichloromethane/ethanol, 9.8:0.2. HRMS (ESI): calcd for (M + Na)+:
300.1362. Found: 300.1365. Anal.: (C18H20O4) C, H, N.
Enzyme Inhibition Assays in Brain Fractions. All final
compounds were tested for their ability to inhibit 2-OG and AEA
hydrolysis using a substrate concentration of 0.5 μM and the assay
procedures described previously.19 FAAH and MAGL assays were
undertaken using membrane and cytosolic fractions of rat cerebella.
Briefly, cerebella that had been obtained previously and stored frozen
were thawed and homogenized in 0.32 M sucrose containing 50 mM
sodium phosphate, pH 8.0. Following homogenization, the samples
were centrifuged at 100000g for 60 min at 4 °C, and the supernatants
(“cytosolic fractions”) were collected. The pellets were resuspended in
50 mM sodium phosphate, pH 8.0 (“membrane fractions”), and the
fractions were stored frozen in aliquots until they were used for assay.
Protein concentrations for the assays were set so that the initial
velocities were always measured, with the fractions being diluted with
10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH
7.2. Each assay consisted of the fraction to be tested, test compound,
and substrate ([3H]-2-OG or [3H]-AEA labeled in its glycerol or
ethanolamine moiety, respectively, final concentration 0.5 μM). The
radiolabeled substrates were obtained from American Radiolabeled
Chemicals, St. Louis, MO, USA. The substrate solution contained fatty
acid-free bovine serum albumin (BSA), to give an assay concentration
(
)-Tetrahydro-2H-pyran-2-ylmethyl 4-(4-Benzylphenyl)-
butanoate (26). Yield: 75%. Rf: 0.2 (hexane/ethyl acetate, 8:2). IR
(CH2Cl2, cm−1): 3023, 2938, 2851, 1734, 1447, 1089, 772. H NMR
1
(CDCl3, δ): 1.23−1.59 (m, 5H, 5H tetrahydropyran); 1.81−1.96 (m,
3H, 1H tetrahydropyran, CH2CH2CH2); 2.34 (t, J = 7.4 Hz, 2H,
CH2CO); 2.58 (t, J = 7.6 Hz, 2H, ArCH2CH2); 3.36−3.54 (m, 2H, 2H
tetrahydropyran); 3.89−4.08 (m, 5H, 1H tetrahydropyran, CH2-tet,
ArCH2Ar); 7.07−7.28 (m, 9H, 9HAr). 13C NMR (CDCl3, δ): 23.0
(CH2 tetrahydropyran), 25.8 (CH2), 26.6, 27.9 (2CH2 tetrahydropyr-
an), 33.6, 34.7 (CH2CO, ArCH2CH2), 41.6 (ArCH2Ar), 67.4 (CH2-
tet), 68.4 (OCH2 tetrahydropyran), 75.5 (OCH tetrahydropyran),
126.0 (CHAr), 128.5 (2CHAr), 128.6 (2CHAr), 128.9 (4CHAr), 138.8,
139.2, 141.3 (3CAr), 173.6 (CO). Chromatography: hexane/ethyl
acetate, 8:2. HRMS (ESI): calcd for (M + Na)+: 375.1936. Found:
375.1925. HPLC-MS: >95%.
(
)-Tetrahydro-2H-pyran-2-ylmethyl 4-(2-Phenylethyl)-
benzoate (27). Yield: 74%. Rf: 0.2 (dichloromethane). IR (CH2Cl2,
cm−1): 3029, 2933, 2853, 1718, 1091. 1H NMR (CDCl3, δ): 1.37−1.61
(m, 5H, 5H tetrahydropyran); 1.81−1.84 (m, 1H, 1H tetrahydropyr-
an); 2.79−2.96 (m, 4H, Ar(CH2)2Ar); 3.33−3.46 (m, 1H, 1H
834
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of 0.125% w/v. After incubation, usually for 10 min at 37 °C, reactions
were stopped by the addition of 400 μL of active charcoal mixture (80
μL of charcoal + 320 μL of 0.5 M HCl). After vortex mixing and phase
separation, aliquots (200 μL) of the aqueous phase were taken and
tritium content was determined by liquid scintillation spectrometry
with quench collection. Blanks were prepared in the same manner but
without an enzyme source. Results were expressed as percent of the
activity of controls containing the same volume of solvent carrier.
hrMAGL Inhibition Assay. The method of Muccioli et al.18 was
modified17 using a 96-well microtiter plate (100 mL total volume).
Clear lysates of human recombinant his tagged MAGL (Cayman
Chemical Co., Ann Arbor, MI, USA) in 10 mM Tris−HCl, 1 mM
EDTA, pH 7.4 (added volume 70 μL) were added to wells containing
test compounds (3 μL in vehicle) or vehicle alone. Buffer (7 μL) was
added to each well to make up the volume. To start the hydrolysis
reactions, 4-nitrophenyl acetate (NPA, 20 μL, final concentration of
0.25 mM, unless otherwise shown) was added rapidly. The samples
were incubated at rt, and the absorbance was measured at 405 nm after
0 min (to rule out effects of the compounds per se on the absorbance)
and thereafter at two 20 min intervals using a Thermomax Microplate
Reader (ThermoMax Kinetic Microplate Reader, Molecular Devices,
Sunnyvale, CA, USA). The 20 min time point was used here and the
data expressed as percent of vehicle control after subtraction of blank
values (wells with buffer in place of enzyme).
Enzymatic Activity for Kinetic Studies. Enzymatic activity was
assayed as previously described20 using umbelliferyl arachidonate from
Cayman Chemical Co. (Ann Arbor, MI) as substrate dissolved in
dimethylsulfoxide. The fluorescence of the product 7-hydroxycoumar-
in released by the hydrolysis was measured with excitation and
emission wavelengths of 355 and 460 nm, respectively. Fluorescence
measurements were carried out in a Perkin-Elmer luminescence
spectrophotometer LS50B. Excitation and emission bandwidths were
2.5 and 10 nm, respectively. The fluorescence cell (1 cm × 0.5 cm)
was mounted on a thermostatted holder. All fluorescence measure-
ments were collected at a constant temperature of 37 °C. The reaction
mixture was prepared as follows50 μg of MAGL protein expressed
and purified as described in the Supporting Informationand the
desired amount of substrate was added to 1.0 mL of buffer (50 mM
Na-HEPES, pH 8.2, and 1 mM EDTA), and the reaction was followed
for 2.5 min. Initial velocities were used to calculate the kinetic
constants Km and kcat. The inhibition constants were calculated by
performing the kinetic measurements in the presence of different
amounts of the inhibitors. All constants (Km, kcat, and Ki) were
calculated with the program GraphPad Prism (version 5) using the
global nonlinear regression option.
(MICINN, SAF-2010/22198-C02-01, M.L.L.-R.) and from the
́
Comunidad Autonoma de Madrid (S2010/BMD-2353, M.L.L.-
R.), the Swedish Science Research Council (Grant 12158,
medicine, to C.J.F.) and by the Research Funds of the Medical
Faculty, Umea University (C.J.F.). The authors thank MICINN
̊
and European Social Fund for predoctoral (J.A.C.) and Ramon
́
y Cajal grants (S.O.-G. and A.C.).
ABBREVIATIONS USED
■
ABHD-6, α/β hydrolase-6; ABHD-12, α/β hydrolase-12; AEA,
N-arachidonoylethanolamine, anandamide; 2-AG, 2-arachido-
noylglycerol; (e)CB, (endo)cannabinoid; FAAH, fatty acid
amide hydrolase; hrMAGL, human recombinant MAGL;
MAGL, monoacylglycerol lipase; NPA, 4-nitrophenyl acetate;
2-OG, 2-oleoylglycerol; SAR, structure−activity relationship
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ASSOCIATED CONTENT
* Supporting Information
■
S
Full synthetic procedures; analytical and spectral character-
ization data for all intermediates; HPLC-MS and elemental
analyses for all final compounds; molecular biology methods for
MAGL cloning; expression and purification; NMR spectra of
compounds 8 and 30 in the presence of hrMAGL over time;
and computational models of the complex between the
enantiomers of compound 8 (derivatives 28 and 29). This
material is available free of charge via the Internet at http://
pubs.acs.org.
(10) Cisneros, J. A.; Vandevoorde, S.; Ortega-Gutier
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rez, S.; Paris, C.;
Fowler, C. J.; Lopez-Rodríguez, M. L. Structure-activity relationship of
́
a series of inhibitors of monoacylglycerol hydrolysiscomparison
with effects upon fatty acid amide hydrolase. J. Med. Chem. 2007, 50,
5012−5023.
AUTHOR INFORMATION
Corresponding Author
*Phone: +34 913944239. Fax: +34 913944103. E-mail:
mluzlr@quim.ucm.es.
■
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synthesis, and structure−activity relationships of alkylcarbamic acid
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ACKNOWLEDGMENTS
The authors are indebted to Eva Hallin and Britt Jacobsson for
excellent technical assistance. This work has been supported by
■
grants from the Spanish Ministerio de Ciencia e Innovacion
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