Sulfonyl fluoride inhibitors of fatty acid amide hydrolase

Article abstract of DOI:10.1021/jm301205j

Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl

Full text of DOI:10.1021/jm301205j

Article
pubs.acs.org/jmc
Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase
Shakiru O. Alapafuja,⊥ Spyros P. Nikas,*,† Indu T. Bharathan,† Vidyanand G. Shukla,† Mahmoud L. Nasr,†
Anna L. Bowman,† Nikolai Zvonok,† Jing Li,† Xiaomeng Shi,‡ John R. Engen,‡
and Alexandros Makriyannis*,†
†Center for Drug Discovery and Departments of Chemistry and Chemical Biology and Pharmaceutical Sciences, Northeastern
University, Boston, Massachusetts 02115, United States
‡Department of Chemistry and Chemical Biology and Barnett Institute of Chemical and Biological Analysis, Northeastern University,
Boston, Massachusetts 02115, United States
⊥MAK Scientific LLC, 801 Albany Street Suite 107/108, Boston, Massachusetts 02119, United States
S
* Supporting Information
ABSTRACT: Sulfonyl fluorides are known to inhibit esterases. Early work from our
laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo
inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl
fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat
and human FAAH, we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506)
has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution
assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an
irreversible manner. Our SAR results are highlighted by molecular docking of key analogs.
INTRODUCTION
■
Fatty acid amide hydrolase (FAAH) is an intracellular
membrane-bound enzyme that degrades and inactivates
members of the fatty acid amide (FAA) family of endogenous
signaling lipids, including anandamide (1, Figure 1) and
oleamide (2).1,2 Anandamide3 binds and activates the CB1
and CB2 cannabinoid receptors,4 the molecular targets of plant-
derived (−)-Δ9-terahydrocannabinol ((−)-Δ9-THC), while
oleamide induces physiological sleep5 and modulates seroto-
nergic systems6 and GABAergic transmission.7 Fatty acid amide
hydrolase is currently the only characterized mammalian
enzyme that is in the amidase signature (AS) family bearing
the unusual Ser-Ser-Lys catalytic triad, as confirmed by the
crystal structure of the enzyme after reaction with methyl
arachidonoyl fluorophosphonate (MAFP).1,2
The pharmacological effects of FAAH inhibition have been
demonstrated in FAAH knockout mice8 as well as by chemical
inhibition.9,10 Increased central and peripheral neuronal levels
of anandamide and other FAAs produce physiological effects
including analgesia,10,11 apoptosis in various cancer cells,12−14
modulation of memory processes,15,16 neuroprotection,9,17−19
epilepsy,20 feeding,21 and prevention of neurotoxicity of the
human amyloid-β peptide in Alzheimer’s disease.22 In addition,
antidepressant, anxiolytic, anti-inflammatory, antihypertensive,
Figure 1. Representative substrates (1, 2) and inhibitors (3−7) of
gastrointestinal, and sleep-inducing effects have been ob-
served.10,23−26 These pharmacological effects are devoid of
unwanted central “cannabinoid effects”, such as hypomotility,
hypothermia, catalepsy, and weight gain, which accompany
directly acting exogenous cannabinoid agonists such as (−)-Δ9-
THC.27 Thus, there is significant therapeutic potential for
FAAH inhibitors as analgesic, neuroprotective, anti-inflamma-
fatty acid amide hydrolase (FAAH).
Received: August 17, 2012
Published: October 19, 2012
© 2012 American Chemical Society
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a
Table 1. Compound Inhibition Data Results for Rat FAAH and Human MGL
a
Inhibition data for rFAAH and hMGL were determined using medium throughput fluorescent assays as described in the Experimental Section.
IC50 values were determined from three independent experiments and are expressed as the mean of three values. N.D.: not determined.
b
tory, and antianxiety drugs, and as agents for the treatment of
metabolic and sleep disorders.
ester derivatives of azetidinone, (thio)hydantoin analogs, as
well as boronic acids have been reported to inhibit
FAAH.28,37,38
Work from our and other laboratories had provided evidence
that the catalytic serine in FAAH is a more reactive nucleophile
compared to the serine residues in other esterases. This has
served as a basis for the development of more selective FAAH
inhibitors. In the course of our program,9,17−19,25,26,29,39−46
aimed at developing potent and selective inhibitors for the
endocannabinoid deactivating enzymes, we have examined the
Over the last 13 years an increasing number of irreversible
and reversible FAAH inhibitors were disclosed.10,28 Irreversible
inhibitors include sulfonyl fluorides29 (e.g., 3 and 4) as well as
aryl carbamates and ureas24,30−32 (e.g., 7). Reversible inhibitors
include a number of synthetic agents bearing electrophilic
carbonyl groups such as trifluoromethyl ketones (e.g., 5), α-
keto-esters and amides, aldehydes, α-halo-ketones, and the α-
keto-heterocyclic type of inhibitors (e.g., 6).33−36 Additionally,
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abilities of a series of second generation sulfonyl fluorides
(Table 1) to inhibit FAAH. The structural features of the
irreversible inhibitors hexadecyl sulfonylfluoride 3 (AM374),29
an early generation FAAH inhibitor developed in our
laboratory, and phenylmethane sulfonyl fluoride 4 (PMSF), a
generic esterase inhibitor, were incorporated into a phenylalkyl
template (analogs 11a−11f, Table 1). Furthermore, a hydro-
philic hydroxyl group was added to the phenyl ring (analogs
21a−21d) and the benzylic methylene group was replaced by
the polar oxygen atom (analog 26). Extension of our structure
activity relationship (SAR) study to include synthetic
intermediates (analogs 20a−20d) shows that addition of the
bulky benzyloxy group on the phenyl ring successfully modifies
the phenylalkyl template, resulting in potent FAAH inhibitors.
All analogs synthesized were tested for their inhibitory activity
on fatty acid amide hydrolase. In addition, initial testing for
selectivity was carried out by also comparing the FAAH
activities of the most potent compounds against three
endocannabinoid targets, namely, CB1 and CB2 receptors as
well as the other major endocannabinoid inactivating enzyme
monoacylglycerol lipase (MGL).
One of the most successful analogs identified in this study, 5-
(4-hydroxyphenyl)pentane sulfonyl fluoride (21d,
AM3506),25,41 has served as a valuable pharmacological tool
to explore the cardiovascular, gastrointestinal, and amygdala-
mediated fear extinction effects related to FAAH inhib-
ition.25,26,40 Additionally, as reported earlier,25 21d exhibited
low “off target” effects when tested against a large number of
serine hydrolases using activity-based proteomic methods.
Chemistry. Synthesis of phenylalkyl sulfonyl fluorides 11a−
11f was accomplished by the reaction sequence shown in
Scheme 1. Commercially available phenylalkyl alcohols 8b−8f
(19−23%). Treatment of these intermediates with NH4F in
refluxing acetone gave phenylalkyl sulfonyl fluorides 11a−11f
in excellent yields (91−93%).
The preparation of the benzyloxy and hydroxyl substituted
phenylalkyl sulfonyl fluorides 20a−20d and 21a−21d is
summarized in Scheme 2. Commercially available phenoxyalkyl
bromides 12a and 12b were reacted with triphenylphosphine in
refluxing benzene47 to give the respective (phenoxyalkyl)-
triphenylphosphonium bromides 13a and 13b in very good
yields (84−85%). These were treated with potassium bis-
(trimethylsilyl)amide, and the generated phosphoranes were
coupled with 4- or 3- or 2-anisaldehyde in an olefination
reaction to furnish intermediate alkenes 14a−14d in 91−93%
yields. This Wittig reaction afforded isomeric mixtures of
1
alkenes favoring the cis isomer (cis/trans = 92−94:8−6 by H
NMR).
Catalytic hydrogenation of 14a−14d led to the correspond-
ing phenolic methyl ethers 15a−15d in 94−96% yields.
Exposure of these compounds to boron tribromide in
methylene chloride47,50 cleaved the two ether groups and
introduced the bromo group at the terminal carbon atom of the
alkyl moiety, producing bromides 16a−16d in very good yields
(90−93%). Protection of the phenolic hydroxyl in 16a−16d led
to benzyl ethers 17a−17d (77−81%) that upon reaction with
sodium sulfite in refluxing EtOH/H2O for 24 h afforded
sodium sulfonates 18a−18d. Subsequent treatment with
thionyl chloride in the presence of catalytic amounts of DMF
gave sulfonyl chlorides 19a−19d in 37−40% overall yields for
the combined steps. During the course of this work, we found
that use of microwave irradiation remarkably decreases the time
required for the conversion of bromides to sodium sulfonates
and enhances the yields of the respective sulfonyl chlorides.
Optimal conditions involve microwave heating (160 °C) of the
bromides and sodium sulfite in a mixture of THF/EtOH/H2O
(1:2:2) for 15 min. For example, treatment of 17d with sodium
sulfite under our optimized conditions and exposure of the
respective sulfonate 18d to thionyl chloride in the presence of
catalytic amounts of DMF gave sulfonyl chloride 19d in 50%
yield. Details of this work were published separately.39
a
Scheme 1.
Intermediate sulfonyl chlorides 19a−19d were treated with
NH4F to produce the respective sulfonyl fluorides 20a−20d in
90−93% yields. However, debenzylation of the phenolic
hydroxyl group in 20a−20d using palladium on active carbon
was unsuccessful. The problem was solved by using a mixture of
HS(CH2)2SH and BF3.Et2O for cleavage of the benzyl group,51
to give sulfonyl fluorides 21a−21d in 66−70% yields.
The arylalkyl ether analog 26 and ester 27 were synthesized
as shown in Scheme 3. In a similar fashion, etherification of
commercially available 4-benzyloxyphenol (22) with 1,4-
dibromobutane gave bromide 23 in 50% yield. Microwave
heating of 23 with sodium sulfite and exposure of the resulting
sodium sulfate 24 to SOCl2/DMF led to 25 in 51% overall
yield. Subsequent treatment with NH4F gave the required
sulfonyl fluoride 26 (93% yield). Sulfonic ester 27 was
produced by reacting sulfonyl chloride 19a with methanol.
a
Reagents and conditions: (a) PPh3, imidazole, I2, MeCN/Et2O, 0 °C
to rt, 72−85%; (b) (i) t-BuLi, Et2O/pentane, −78 °C, (ii) SO2Cl2,
−78 °C, 19−23%; (c) NH4F, acetone, reflux, 91−93%.
RESULTS AND DISCUSSION
were converted to the respective iodides 9b−9f in very good
yields (72−85%) using the triphenylphosphine, iodine,
imidazole method.47 Low temperature lithium−iodine ex-
change48 between the primary alkyl iodides 9a−9f and t-BuLi
afforded the corresponding primary alkyllithium reagents that
were treated49 in situ with sulfuryl chloride to produce
phenylalkyl sulfonyl chlorides 10a−10f in moderate yields
■
Structure−Activity Relationships. Inhibition data (Table
1) for FAAH and MGL were determined using fluorescent
assay protocols as described in the Experimental Section. The
principle of these protocols is based on monitoring the
fluorescence produced by enzyme catalyzed hydrolysis of a
fluorogenic substrate to give a highly fluorescent compound. A
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a
Scheme 2.
a
Reagents and conditions: (a) Ph3P, benzene, reflux, 2 days, 84−85%; (b) (Me3Si)2N−K+, THF, 0 °C, 5 min, then 4- or 3- or 2-anisaldehyde, 10 min,
91−93%; (c) H2, Pd/C, AcOEt, 30 psi, rt, 6 h, 94−96%; (d) BBr3, CH2Cl2, −30 °C to rt, 2 h, 90−93%; (e) K2CO3, acetone, BnBr, reflux, 6 h, 77−
81%; (f) Na2SO3, EtOH/H2O, reflux, 24 h; or Na2SO3, THF/EtOH/H2O, M.W., 160 °C, 15 min; (g) SOCl2, benzene/cat. DMF, 50 °C, 3 h, 37−
50% from 17a−17d; (h) NH4F, acetone, reflux, 2 h, 90−93%; (i) BF3·OEt2, HS(CH2)2SH, rt, 1 h, 66−70%.
a
Scheme 3.
a
Reagents and conditions: (a) K2CO3, 18-crown-6, 1,4-dibromobutane, acetone, reflux, 3 h, 50%; (b) Na2SO3, THF/EtOH/H2O, M.W., 160 °C, 15
min; (c) SOCl2, benzene/cat. DMF, 50 °C, 3 h, 51% from 23; (d) NH4F, acetone, reflux, 2 h, 93%; (3) MeOH, rt, overnight, 84%.
fluorescent assay protocol to monitor FAAH activity has
already been described.52 This methodology uses human FAAH
expressed in Chinese Hamster Ovary (CHO) cell lines and
arachidonyl-7-amino-4-methylcoumarin amide (AAMCA) as
the fluorogenic substrate. As detailed in the Experimental
Section, our modified protocol involves transmembrane
domain-deleted rat FAAH (ΔTM rFAAH) expressed in E.
coli53 and AAMCA as the substrate. MGL activity was
monitored with a simple, sensitive fluorometric assay recently
developed in our laboratory that was amenable to high-
throughput screening.43,45,46 This methodology involves
recombinant hexa-histidine-tagged human MGL (hMGL)
overexpressed in E. coli and the novel fluorogenic substrate
arachidonoyl 7-hydroxy-6-methoxy-4-methylcoumarin ester
(AHMMCE).43,45,46 Each compound’s inhibition is presented
in Table 1 as an IC50 value from a full eight point curve
performed in triplicate.
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Table 2. Affinities (Ki) of Selected Analogs for Rat CB1 and Mouse CB2 Cannabinoid Receptors (95% Confidence Limits)
a
Affinities for CB1 and CB2 were determined using rat brain (CB1) or membranes from HEK293 cells expressing mouse CB2, and [3H]CP-55,940
as the radioligand following previously described procedures.50,56 Data were analyzed using nonlinear regression analysis. Ki values were obtained
from three independent experiments run in duplicate and are expressed as the mean of the three values.
The present study involves sulfonyl fluoride derivatives
where the sulfonyl fluoride group is connected to a substituted
or nonsubstituted phenyl ring through a linear alkyl linker.
Substituents of the phenyl ring comprise the hydrophilic
hydroxyl and the hydrophobic and bulky benzyloxy groups.
Examination of the IC50 values of the analogs carrying a
nonsubstituted phenyl ring (11a−11f, Table 1) reveals that a
five to seven carbon long linker is optimal for FAAH inhibition.
It can be postulated that, within the FAAH’s catalytic
channel, this linking methylene chain places the phenyl ring in a
position that mimics the π-unsaturations of the endogenous
substrates anandamide (Δ8,9 double bond) and oleamide (Δ9,10
double bond). It should be noted that similar trends were
observed earlier with the α-keto-heterocyclic type of inhibitors
where the phenyl-binding region appears to constitute a special
site at the intersection of the FAAH’s membrane access channel
and the chain-binding pocket.34,54,55 Thus, the phenylpentyl
(11c), phenylhexyl (11d), and phenylheptyl (11e) analogs
block FAAH activity with IC50’s of 37−50 nM. Of these three
compounds, the analog with the lowest IC50 value (11e) was
selected as the main template for further studies aimed at
exploring the effect of phenyl ring substitution on FAAH
inactivation. As can be seen from the inhibition data depicted in
Table 1, the presence of a hydroxyl or benzyloxy group at the
phenyl ring of 11e can result in significant enhancement of the
compound’s ability to inhibit FAAH. More specifically, addition
of ortho-, meta-, or para-hydroxy groups (21c, 21b, and 21a)
leads to 15- to 31-fold increases in the compounds’ potencies.
However, the ortho-hydroxy analog (21c) exhibits the lowest
selectivity for FAAH (47-fold) over MGL. Thus, in terms of
inhibitory activity and selectivity for FAAH, the order for the
hydroxyl substituted phenylheptyl analogs is as follows: 21a >
21b > 21c. Interestingly, the successful addition of a para-
hydroxy group on the phenylheptyl template (compound 11e)
seems to work equally well for the phenylpentyl prototype
(compound 11c). Thus, the less lipophilic analog 21d, with the
pentyl linker, exhibits a similar inhibitory activity and selectivity
profile as 21a.
Extension of our SAR study shows that the presence of the
bulky benzyloxy group at the ortho-, meta-, or para-position of
the phenylheptyl template (20c, 20b, and 20a) enhances
FAAH inhibitory activity by 9−10-fold. This suggests additional
favorable binding contacts within FAAH’s active site. Again, the
ortho-substituted analog (20c) shows the lowest selectivity for
FAAH (27-fold) over MGL. The order of inhibitory activity/
selectivity for the benzyloxy substituted phenylheptyl analogs is
as follows: 20b > 20a > 20c. A comparison of the IC50 values of
20a and 20d indicates that modification of the length of the
linker from seven carbon atoms to five carbon atoms retains the
inhibitory activity for FAAH and slightly improves selectivity
over MGL. Furthermore, as seen in analog 26, replacement of
the benzylic methylene group with the polar oxygen atom does
not affect the compound’s potency and selectivity for FAAH.
Conversely, replacement of the sulfonyl fluoride group with a
sulfonyl ester moiety (compound 27) leads to a significant
decrease (73-fold) in the compound’s inhibitory activity.
Our SAR suggests that, within the FAAH’s catalytic channel
and around the phenyl ring binding region, there is suitable
space and flexibility to accommodate the hydrophilic hydroxyl
and the bulky benzyloxy groups. Presumably, the benzyloxy
groups, that represent conformationally defined π-unsaturated
systems, are capable of mimicking the length, the conforma-
tional properties, and the π-characteristics of the terminal
double bonds (e.g., Δ11,12 and Δ14,15 in anandamide) in the
endogenous lipids that are recognized by FAAH.
Overall, this systematic SAR study suggests that (1), for a
phenylalkyl template, a five to seven carbon long linker is
optimal for FAAH inhibition; (2) addition of a hydroxyl or
benzyloxyl group in the phenyl ring enhances the inhibitory
activity by 9- to 31-fold; and (3) the most successful
compounds in terms of inhibitory activity and selectivity for
FAAH over MGL are the hydroxyl substituted analogs 21a, 21d
and the benzyloxy substituted analogs 20b, 20d, and 26.
Subsequently, the most successful sulfonyl fluorides, 21a,
21d, 20b, 20d, and 26, were tested for their CB1 and CB2
receptor binding affinities50,56 to determine selectivity over the
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other two endocannabinoid proteins, and the results are
depicted in Table 2. We observe that the tested compounds
had moderate to low affinities for binding to rat CB1 and
mouse CB2, with the benzyloxy substituted analogs (20b, 20d,
26) exhibiting slightly higher CB receptor binding affinities
when compared to the hydroxyl substituted ones (21a and
21d). These receptor binding affinity data reported here were
carried out using a radioligand displacement assay indicating
that our FAAH inhibitors interact with CB1 and CB2 at their
respective orthosteric sites. We have no information on
whether these ligand−receptor interactions are reversible or
of a covalent nature. It should be pointed out that unlike their
sulfonyl chloride counterparts, sulfonyl fluorides exhibit
relatively low to moderate electrophilic properties and are,
thus, capable of exhibiting remarkable selectivities for their
targets. This is clearly demonstrated in our recently published
results for 21d.25 Activity based protein profiling experiments in
mouse brain and liver demonstrate that this compound has
selectivity for FAAH over a large number of serine hydrolases.
Of the two most successful compounds (21a and 21d)
identified here, we chose the less lipophilic 21d to probe the
interaction of FAAH with sulfonyl fluorides in both in vitro and
in vivo models. Experiments with the recombinant human
enzyme, studies on the mode of inhibition, and molecular
modeling work follow, while assessment of target selectivity and
in vivo studies with 21d were published recently.25,26,40
Figure 2. Irreversible inhibition of purified ΔTM rFAAH with 21d.
Ordinate, enzymatic activity after rapid dilution of untreated (control)
and 21d treated FAAH. A negligible enzymatic activity (8%) was
observed with 21d.
To further explore the inhibition mode of the sulfonyl
fluorides reported here, we next carried out liquid chromatog-
raphy-time-of-flight mass spectrometry (LC-QTOF) experi-
ments to directly test whether 21d covalently modified ΔTM
rFAAH. It should be noted that mass spectrometric character-
ization of the FAAH−inhibitor complex with sulfonyl fluorides
is reported here for the first time while similar work with
carbamate and urea based inhibitors was reported earlier.59,60
As detailed in the Experimental Section, samples of purified
ΔTM rFAAH were incubated for 60 min with a 2-fold molar
excess of 21d in DMSO or with DMSO vehicle only, after
which incubation samples were desalted and analyzed by MS to
determine the intact mass of each. The spectrum of the DMSO
treated sample showed a prominent peak with an average mass
of 64390.7 Da for the ΔTM rFAAH. The average intact mass of
21d-treated ΔTM rFAAH was 226.6 Da greater than that of the
unmodified enzyme (Figure 3), a result consistent with enzyme
sulfonylation by 21d and the consequent calculated mass
increase of 226.2 Da. Together, our experiments indicate that
compound 21d acts as an irreversible inhibitor that covalently
modifies the enzyme. Most probably, this involves an SN2
reaction resulting in sulfonylation of ΔTM rFAAH at the
catalytic Ser241 (Figure 4). It should be noted that this
potential pathway is congruent with the reported mechanism of
inactivation of serine esterases by sulfonyl fluorides.61,62
Molecular Modeling. To gain insight into the features
required for potent FAAH inhibition, three sulfonyl fluoride
analogs, 11c, 20d, and 21d, were covalently docked into
rFAAH as detailed in the Experimental Section. Our docking
experiments are based on enzyme sulfonylation at the catalytic
Ser241 (Figure 4). In each docked pose (Figure 5), the ligand is
found in the acyl binding channel of rFAAH, with one oxygen
of the sulfonyl moiety involved in extensive hydrogen bonding
with the oxyanion hole (formed by Ile238, Gly239, Gly240, and
Ser241). Addition of a hydroxyl group in the phenyl ring allows
for the formation of a hydrogen bond between the ligand and
Thr488, possibly increasing the potency of 21d. Similarly,
introduction of a benzyloxy substituent produces analog 20d,
whose enhanced potency is probably due to an increase in
hydrophobic contacts within the enzyme’s binding channel.
Inhibition of Recombinant Human FAAH. Rat FAAH
exhibits 84% sequence identity with the human enzyme.57 This
divergent nature of rat and human FAAH could possibly result
in species-based differences in inhibitory potency. For this
reason, the key inhibitor 21d was examined against the human
enzyme and the results are shown in Table 3. We observe that
the tested compound exhibits similar inhibitory activity for both
rat and human FAAH.
a
Table 3. Inhibition of Rat and Human FAAH by 21d
b
b
compd
rFAAH (IC50, nM)
2.8 0.3
hFAAH (IC50, nM)
5.1 0.5
21d
a
Inhibition for hFAAH was determined using a medium through-put
b
fluorescent assay as described for rFAAH. IC50 values were
determined from three independent experiments and are expressed
as the mean of three values.
Enzyme Inhibition Studies. We utilized the rapid dilution
assay58 to assess whether 21d inhibits FAAH through a
reversible or irreversible mechanism. Under the conditions of
the rapid dilution assay, incubation of the enzyme with a
reversible inhibitor leads to an equilibrated system. Rapid
dilution of this system perturbs the equilibrium between the
inhibitor and the enzyme and results in virtually complete
recovery of enzymatic activity. In contrast, incubation of the
enzyme with an irreversible inhibitor results in the formation of
an irreversible enzyme−inhibitor complex, with very little or no
enzymatic activity being recovered after dilution of the assay
mixture. As detailed in the Experimental Section, two purified
FAAH enzyme aliquots were preincubated for 1 h with equal
volumes of DMSO without inhibitor (control) or excess (40-
fold higher than its IC50 value) of 21d. As shown in Figure 2,
after rapid dilution, a negligible recovery of FAAH activity (8%)
was observed for 21d after 80 min and did not essentially
change after 24 h incubation (data not shown), suggesting that
21d inhibits FAAH through an irreversible mechanism.
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EXPERIMENTAL SECTION
■
Materials. All reagents and solvents were purchased from Aldrich
Chemical Co., unless otherwise specified, and used without further
purification. All anhydrous reactions were performed under a static
argon atmosphere in flame-dried glassware using scrupulously dry
solvents. Flash column chromatography employed silica gel 60 (230−
400 mesh). All compounds were demonstrated to be homogeneous by
analytical TLC on precoated silica gel TLC plates (Merck, 60 F245 on
glass, layer thickness 250 μm), and chromatograms were visualized by
phosphomolybdic acid staining. Melting points were determined on a
micromelting point apparatus and are uncorrected. IR spectra were
recorded on a Perkin-Elmer Spectrum One FT-IR spectrometer. NMR
spectra were recorded in CDCl3, unless otherwise stated, on a Bruker
AC 300 (1H at 300 MHz, 13C at 75 MHz) or on a Bruker Ultra Shield
400 WB plus (1H at 400 MHz, 13C at 100 MHz) or on a Varian
INOVA-500 (1H at 500 MHz, 13C at 125 MHz) spectrometer, and
chemical shifts are reported in units of δ relative to internal TMS.
Multiplicities are indicated as br (broadened), s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet), and coupling constants (J) are
reported in hertz (Hz). Low- and high-resolution mass spectra were
performed at the School of Chemical Sciences, University of Illinois at
Urbana−Champaign. Mass spectral data are reported in the form of
m/z (intensity relative to base =100). Elemental analyses were
obtained at Baron Consulting Co, Milford, CT, and were within
0.4% of the theoretical values (see Supporting Information). Purities
of the tested compounds were determined by elemental analysis and
were >95%.
General Procedure for the Synthesis of Phenylalkyl Iodides
(9b−9f). A round-bottom flask was charged with phenylalkyl alcohol 8
(1 equiv), acetonitrile/diethyl ether mixture (1:2), triphenyl
phosphine (1.3 equiv), imidazole (1.3 equiv), and iodine (1.3
equiv). The solution was blanketed with argon and capped, and the
reaction mixture was stirred for 4−5 h at room temperature. The
resulting mixture diluted with diethyl ether, washed with water,
aqueous sodium thiosulfate, and brine, dried (MgSO4), and
evaporated. Purification by flash column chromatography on silica
gel (10% diethyl ether/hexane) gave phenylalkyl iodide 9 in 72−85%
yield.
Figure 3. MS analysis of ΔTM rFAAH enzyme masses. Intact masses
of unmodified ΔTM rFAAH (A) and 21d-modified ΔTM rFAAH (B).
The increase in mass is consistent with enzyme sulfonylation by 21d.
CONCLUSIONS
■
4-Phenylbutyl Iodide63 (9b). Colorless oil, yield: 83%. H NMR
1
In summary, our SAR study includes sulfonyl fluoride
derivatives where the sulfonyl fluoride group is connected to
a substituted or nonsubstituted phenyl ring through a linear
alkyl linker. Biodata suggest the following: (1) a five to seven
carbon long linker is optimal for FAAH deactivation; (2)
addition of a hydroxy or benzyloxy group in the phenyl ring
enhances inhibitory activity; (3) replacement of the linker’s
benzylic methylene group with an oxygen atom is well
tolerated; and (4) all new analogs show a significant degree
of selectivity for FAAH over MGL. Studies with recombinant
human FAAH revealed that 21d has similar inhibitory potency
for both the rat and the human enzyme while experiments
using rapid dilution assays and mass spectrometric analysis
suggested that the compound is a covalent modifier for the
enzyme and inhibits its action in an irreversible manner.
Cannabinoid receptor binding studies of the most successful
analogs indicate that they had low to moderate binding
affinities for CB1 and CB2. Docking experiments have
identified key pharmacophoric features for potent FAAH
inhibition.
(300 MHz, CDCl3) δ 7.40−7.11 (m, 5H, ArH), 3.20 (t, J = 7.2 Hz,
2H, −CH2I), 2.61 (t, J = 7.0 Hz, 2H, −CH2−Ph), 1.93−1.59 (m, 4H).
5-Phenylpentyl Iodide64 (9c). Colorless oil, yield: 85%. H NMR
1
(300 MHz, CDCl3) δ 7.49−7.11 (m, 5H, ArH), 3.18 (t, J = 7.3 Hz,
2H, −CH2I), 2.62 (t, J = 7.1 Hz, 2H, −CH2−Ph), 1.98−1.32 (m, 6H).
6-Phenylhexyl Iodide64 (9d). Colorless oil, yield: 81%. H NMR
1
(300 MHz, CDCl3) δ 7.44−7.08 (m, 5H, ArH), 3.17 (t, J = 7.2 Hz,
2H, −CH2I), 2.60 (t, J = 7.0 Hz, 2H, −CH2-Ph), 1.81 (m, 2H), 1.68−
1.06 (m, 6H); mass spectrum m/z (relative intensity) 288 (M+, 5),
117 (7), 105 (13), 91 (100), 77 (18), 65 (32).
7-Phenylheptyl Iodide65 (9e). Colorless oil, yield: 76%. H NMR
1
(300 MHz, CDCl3) δ 7.40−7.05 (m, 5H, ArH), 3.19 (t, J = 7.3 Hz,
2H, −CH2I), 2.60 (t, J = 7.1 Hz, 2H, −CH2-Ph), 1.80 (m, 2H), 1.71−
1.02 (m, 8H).
8-Phenyloctyl Iodide64 (9f). Colorless oil, yield: 72%. H NMR
1
(300 MHz, CDCl3) δ 7.41−7.01 (m, 5H, ArH), 3.18 (t, J = 7.3 Hz,
2H, −CH2I), 2.59 (t, J = 7.1 Hz, 2H, −CH2−Ph), 1.81 (m, 2H), 1.70−
1.10 (m, 10H).
General Procedure for the Synthesis of Phenylalkylsulfonyl
Chlorides (10a−10f). A solution of phenylalkyl iodide 9 (1 equiv) in
a mixture of dry n-pentane/diethyl ether (3:2) was cooled to −78 °C
under argon, and t-BuLi (2.2 equiv, using a 1.7 M solution of t-BuLi in
Figure 4. Probable mechanism of ΔTM rFAAH sulfonylation by 21d.
10080
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Figure 5. 11c (cyan carbons), 21d (green carbons), and 20d (magenta carbons) covalently docked to the catalytic Ser241 of rFAAH in the acyl
chain binding channel. There is significant hydrogen bonding of all ligands with the oxyanion hole (formed by the backbone of Ile238, Gly239,
Gly240, and Ser241). 21d also forms a hydrogen bond with the backbone carbonyl of Thr488. Hydrogen bonds are denoted by a blue dashed line,
and the surface of the binding channels is shown in gray.
hexane) was added dropwise over a 2 min period. The mixture was
stirred for 10 min at −78 °C and then was transferred by cannula to a
cooled (−78 °C) and dry solution of SO2Cl2 in n-pentane over a 20
min period. Following the addition, the reaction mixture was stirred
for 1 h at −78 °C and then allowed to warm to room temperature over
a 3 h period. The reaction mixture was quenched by dropwise addition
of water and then diluted with diethyl ether, and the organic phase was
separated. The aqueous phase was extracted with diethyl ether, the
combined organic layer was dried (MgSO4), and the solvent was
evaporated. Purification by flash column chromatography on silica gel
gave phenylalkylsulfonyl chloride 10 in 19−23% yield.
Phenylalkylsulfonyl Fluorides (11). The synthesis was carried
out as described for 20a (see text below), using phenylalkylsulfonyl
chlorides 10a−10f (1 equiv) and NH4F (2 equiv) in dry acetone. The
crude products obtained after workup were purified by flash column
chromatography on silica gel (diethyl ether in hexane) to give 11a−
11f as colorless viscous liquids in 91−93% yields.
1
3-Phenylpropane-1-sulfonyl Fluoride (11a). Yield: 91%. H NMR
(300 MHz, CDCl3) δ 7.46−7.15 (m, 5H, ArH), 3.33 (m as dt, J = 10.5
Hz, J = 4.4 Hz, 2H, −CH2SO2F), 2.82 (t, J = 7.3 Hz, 2H, −CH2−Ph),
2.29 (qt, J = 7.5 Hz, 2H); mass spectrum m/z (relative intensity) 202
(M+, 19), 117 (30), 91 (100). Exact mass calculated for C9H11FO2S,
202.0464; found, 202.0465.
3-Phenylpropane-1-sulfonyl Chloride66 (10a). Colorless viscous
1
1
4-Phenylbutane-1-sulfonyl Fluoride (11b). Yield: 92%. H NMR
oil, yield: 19%. H NMR (300 MHz, CDCl3) δ 7.50−7.03 (m, 5H,
(300 MHz, CDCl3) δ 7.45−7.08 (m, 5H, ArH), 3.35 (m as dt, J = 10.5
Hz, J = 4.3 Hz, 2H, −CH2SO2F), 2.63 (t, J = 7.2 Hz, 2H, −CH2−Ph),
2.10−1.52 (m, 4H); mass spectrum m/z (relative intensity) 216 (M+,
39), 104 (7), 91 (100). Exact mass calculated for C10H13FO2S,
216.0620; found, 216.0621.
ArH), 3.66 (m as t, J = 7.7 Hz, 2H, −CH2SO2Cl), 2.63 (t, J = 7.2 Hz,
2H, −CH2−Ph), 2.22 (qt, J = 7.4 Hz, 2H); mass spectrum m/z
(relative intensity) 220 (M+ + 2, 3), 218 (M+, 9), 183 (5), 117 (69),
91 (100). Exact mass calculated for C9H11ClO2S, 218.0168; found,
218.0165.
1
4-Phenylbutane-1-sulfonyl Chloride67 (10b). Colorless viscous oil
(lit.67 mp = 41−41.5 °C), yield: 22%. 1H NMR (300 MHz, CDCl3) δ
7.40−7.08 (m, 5H, ArH), 3.65 (m as t, J = 7.6 Hz, 2H, −CH2SO2Cl),
2.70 (t, J = 7.1 Hz, 2H, −CH2−Ph), 2.12 (qt, J = 7.2 Hz, 2H), 1.82
(qt, J = 6.9 Hz, 2H).
5-Phenylpentane-1-sulfonyl Fluoride (11c). Yield: 93%. H NMR
(300 MHz, CDCl3) δ 7.41−7.08 (m, 5H, ArH), 3.34 (m as dt, J = 10.3
Hz, J = 4.2 Hz, 2H, −CH2SO2F), 2.63 (t, J = 7.3 Hz, 2H, −CH2−Ph),
2.05 (qt, J = 7.0 Hz, 2H), 1.80−1.39 (m, 4H); mass spectrum m/z
(relative intensity) 230 (M+, 20), 117 (4), 105 (8), 91 (100). Exact
mass calculated for C11H15FO2S, 230.07768; found, 230.07763.
5-Phenylpentane-1-sulfonyl Chloride68 (10c). Colorless viscous
1
6-Phenylhexane-1-sulfonyl Fluoride (11d). Yield: 93%. H NMR
1
oil, yield: 23%. H NMR (300 MHz, CDCl3) δ 7.41−7.09 (m, 5H,
(300 MHz, CDCl3) δ 7.35−7.09 (m, 5H, ArH), 3.33 (m as dt, J = 10.5
Hz, J = 4.3 Hz, 2H, −CH2SO2F), 2.62 (t, J = 7.3 Hz, 2H, −CH2−Ph),
1.94 (qt, J = 7.6 Hz, 2H), 1.75−1.22 (m, 6H); mass spectrum m/z
(relative intensity) 244 (M+, 17), 117 (2), 105 (9), 91 (100). Exact
mass calculated for C12H17FO2S, 244.0933; found, 244.0936.
ArH), 3.65 (m as t, J = 7.7 Hz, 2H, −CH2SO2Cl), 2.65 (t, J = 7.3 Hz,
2H, −CH2−Ph), 2.07 (qt, J = 7.2 Hz, 2H), 1.82−1.40 (m, 4H); mass
spectrum m/z (relative intensity) 248 (M+ + 2, 2), 246 (M+, 6), 117
(55), 91 (100).
6-Phenylhexane-1-sulfonyl Chloride (10d). Colorless viscous oil,
yield: 22%. 1H NMR (300 MHz, CDCl3) δ 7.39−6.98 (m, 5H, ArH),
3.63 (m as t, J = 7.6 Hz, 2H, −CH2SO2Cl), 2.62 (t, J = 7.2 Hz, 2H,
−CH2−Ph), 1.97 (qt, J = 7.6 Hz, 2H), 1.72−1.19 (m, 6H); mass
spectrum m/z (relative intensity) 262 (M+ + 2, 3), 260 (M+, 9), 91
(100). Exact mass calculated for C12H17ClO2S, 260.0638; found,
260.0639.
1
7-Phenylheptane-1-sulfonyl Fluoride (11e). Yield: 91%. H NMR
(300 MHz, CDCl3) δ 7.39−7.08 (m, 5H, ArH), 3.33 (m as dt, J = 10.5
Hz, J = 4.3 Hz, 2H, −CH2SO2F), 2.62 (t, J = 7.2 Hz, 2H, −CH2−Ph),
2.09−1.74 (m, 2H), 1.71−1.12 (m, 8H); mass spectrum m/z (relative
intensity) 258 (M+, 18), 105 (11), 91 (100). Exact mass calculated for
C13H19FO2S, 258.1090; found, 258.1087.
1
8-Phenyloctane-1-sulfonyl Fluoride (11f). H NMR (300 MHz,
7-Phenylheptane-1-sulfonyl Chloride (10e). Colorless viscous oil,
yield: 20%. 1H NMR (300 MHz, CDCl3) δ 7.39−7.07 (m, 5H, ArH),
3.64 (m as t, J = 7.8 Hz, 2H, −CH2SO2Cl), 2.62 (t, J = 7.2 Hz, 2H,
−CH2−Ph), 2.11−1.75 (m, 2H), 1.72−1.13 (m, 8H); mass spectrum
m/z (relative intensity) 276 (M+ + 2, 2), 274 (M+, 6), 91 (100). Exact
mass calculated for C13H19ClO2S, 274.0794; found, 274.0790.
8-Phenyloctane-1-sulfonyl Chloride (10f). Colorless viscous oil,
yield: 21%. 1H NMR (300 MHz, CDCl3) δ 7.42−7.08 (m, 5H, ArH),
3.64 (m as t, J = 7.8 Hz, 2H, −CH2SO2Cl), 2.61 (t, J = 7.1 Hz, 2H,
−CH2−Ph), 2.04 (m, 2H), 1.68−1.05 (m, 10H); mass spectrum m/z
(relative intensity) 290 (M+ + 2, 3), 288 (M+, 9), 91 (100). Exact mass
calculated for C14H21ClO2S, 288.0951; found, 288.0954.
CDCl3) δ 7.45−7.05 (m, 5H, ArH), 3.32 (m as dt, J = 10.3 Hz, J = 4.2
Hz, 2H, −CH2SO2F), 2.59 (t, J = 7.1 Hz, 2H, −CH2−Ph), 2.10−1.72
(m, 2H), 1.70−1.13 (m, 10H). mass spectrum m/z (relative intensity)
272 (M+, 19), 117 (2), 105 (13), 91 (100). Exact mass calculated for
C14H21FO2S, 272.1246; found, 272.1244.
(6-Phenoxyhexyl)triphenylphosphonium Bromide (13a).
The synthesis was carried out as described for 13b (see text below),
using 6-phenoxyhexyl bromide (12a) (2.8 g, 10.9 mmol) and
triphenylphosphine (3.14 g, 12 mmol) in anhydrous benzene (11
mL), and gave 13a as a white solid in 84% yield (4.75 g). mp 143−145
1
°C; H NMR (500 MHz, CDCl3) δ 7.87 (dd, J = 12 Hz, J = 8.0 Hz,
6H, -PPh3), 7.78 (td, J = 8.0 Hz, J = 1.5 Hz, 3H, -PPh3), 7.69 (td, J =
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8.0 Hz, J = 3.5 Hz, 6H, -PPh3), 7.25 (t, J = 7.7 Hz, 2H, 3-H, 5-H,
-OPh), 6.91 (t, J = 7.7 Hz, 1H, 4-H, -OPh), 6.84 (d, J = 7.7 Hz, 2H, 2-
H, 6-H, -OPh), 3.95−3.87 (m and t overlapping, 4H, −CH2OPh and
−CH2PPh3, especially 3.90, t, J = 6.3 Hz, −CH2OPh), 1.80−1.60 (m,
6H), 1.49 (quintet, J = 7.7 Hz, 2H).
3.85 mmol), potassium bis(trimethylsilyl)amide (0.762 g, 3.83 mmol)
in dry THF (30 mL), and a solution of 2-methoxybenzaldehyde (0.20
g, 1.47 mmol) in dry THF (5 mL). The crude product obtained after
workup was purified by flash column chromatography on silica gel (5%
diethyl ether in hexane) to give 14c as a colorless liquid in 91% yield
(396 mg). On the basis of 1H NMR analysis, the product is a mixture
(4-Phenoxybutyl)triphenylphosphonium Bromide (13b). A
mixture of 4-phenoxybutyl bromide (12b) (22.0 g, 96.1 mmol) and
triphenylphosphine (27.6 g, 105.3 mmol) in anhydrous benzene (96
mL) was refluxed for 2 days under argon. The reaction mixture was
cooled to room temperature, and the precipitating product (13b) was
isolated by filtration under reduced pressure and washed with
anhydrous diethyl ether. Yield: 85% (40.0 g); white solid, mp 185−
1
of cis and trans isomers in the ratio 93:7, respectively. cis isomer: H
NMR (500 MHz, CDCl3) δ 7.29−7.21 (m, 4H, 3-H, 5-H, -OPh and 4-
H, 6-H, -Ph−OMe, overlapping), 6.95−6.86 (m, 5H, 2-H, 4-H, 6-H,
-OPh and 3-H, 5-H, -Ph−OMe, overlapping), 6.52 (d, J = 11.5 Hz, 1H,
1′-H), 5.73 (dt, J = 11.5 Hz, J = 7.5 Hz, 1H, 2′-H), 3.93 (t, J = 6.7 Hz,
2H, -CH2−OPh), 3.83 (s, 3H, -OMe), 2.28 (m as q, J = 7.2 Hz, 2H,
3′-H), 1.76 (qt, J = 7.3 Hz, 2H), 1.53−1.46 (m, 4H); mass spectrum
m/z (relative intensity) 296 (M+, 4), 203 (6), 173 (4), 159 (7), 147
(34), 134 (7), 121 (100), 107 (8), 91 (32), 77 (11); Exact mass
1
186 °C; H NMR (500 MHz, CDCl3) δ 7.96 (dd, J = 12 Hz, J = 8.0
Hz, 6H, -PPh3), 7.88 (td, J = 8.0 Hz, J = 1.5 Hz, 3H, -PPh3), 7.80 (td, J
= 8.0 Hz, J = 3.5 Hz, 6H, -PPh3), 7.25 (t, J = 7.9 Hz, 2H, 3-H, 5-H,
-OPh), 6.92 (t, J = 7.9 Hz, 1H, 4-H, -OPh), 6.82 (d, J = 7.9 Hz, 2H, 2-
H, 6-H, -OPh), 4.09 (t, J = 5.5 Hz, 2H, −CH2OPh), 4.04−3.98 (m,
2H, −CH2PPh3), 2.25 (qt, J = 6.4 Hz, 2H), 1.93−1.85 (m, 2H).
1-(4-Methoxyphenyl)-7-phenoxyhept-1-ene (14a). To a
stirred suspension of (6-phenoxyhexyl)triphenylphosphonium bro-
mide (13a) (4.60 g, 8.86 mmol) in dry THF (80 mL) at 0 °C, under
an argon atmosphere, was added potassium bis(trimethylsilyl)amide
(1.76 g, 8.84 mmol). The resulting slurry was stirred for 5 min, and
then a solution of 4-methoxybenzaldehyde (0.61 g, 4.49 mmol) in dry
THF (10 mL) was added dropwise. The reaction was stirred for an
additional 10 min and upon completion was quenched by the addition
of saturated aqueous NH4Cl solution. The mixture was warmed to
room temperature, the organic layer was separated, and the aqueous
phase was extracted with diethyl ether. The combined organic layer
was washed with brine and dried over MgSO4, and the solvent was
evaporated under reduced pressure. The residue was purified through
a short column of silica gel (5% diethyl ether in hexane) to give 14a as
1
calculated for C20H24O2, 296.1776; found, 296.1775; trans isomer: H
NMR (500 MHz, CDCl3) δ 6.49 (d, J = 15.6 Hz, 1H, 1′-H), 6.22 (dt, J
= 15.6 Hz, J = 7.4 Hz, 1H, 2′-H).
1-(4-Methoxyphenyl)-5-phenoxypent-1-ene (14d). The syn-
thesis was carried out as described for 14a, starting from 13b (29.0 g,
59.1 mmol), potassium bis(trimethylsilyl)amide (11.7 g, 58.8 mmol)
in dry THF (200 mL), and a solution of 4-methoxybenzaldehyde (2.9
g, 21.3 mmol) in dry THF (10 mL). The crude product obtained after
workup was purified by flash column chromatography on silica gel (5%
diethyl ether in hexane) to give 14d as a colorless liquid in 93% yield
(5.31 g). On the basis of 1H NMR analysis, the product is a mixture of
1
cis and trans isomers in the ratio 95:5, respectively. cis isomer: H
NMR (500 MHz, CDCl3) δ 7.26 (t, J = 7.3 Hz, 2H, 3-H, 5-H, -OPh),
7.22 (d, J = 8.7 Hz, 2H, 2-H, 6-H, -Ph−OMe), 6.92 (t, J = 7.3 Hz, 1H,
4-H, -OPh), 6.87 (d, J = 7.3 Hz, 2H, 2-H, 6-H, -OPh), 6.85 (d, J = 8.7
Hz, 2H, 3-H, 5-H, -Ph−OMe), 6.39 (d, J = 11.5 Hz, 1H, 1′-H), 5.60
(dt, J = 11.5 Hz, J = 7.5 Hz, 1H, 2′-H), 3.98 (t, J = 6.5 Hz, 2H,
−CH2−OPh), 3.80 (s, 3H, -OMe), 2.51 (m as qd, J = 7.5 Hz, J = 2.1
Hz, 2H, 3′-H), 1.94 (qt, J = 7.2 Hz, 2H, 4′-H); mass spectrum m/z
(relative intensity) 268 (M+, 2), 149 (16), 107 (52), 91 (100); Exact
mass calculated for C18H20O2, 268.1463; found, 268.1466. trans
1
a colorless liquid in 91% yield (1.21 g). On the basis of H NMR
analysis, the product is a mixture of cis and trans isomers in a ratio
94:6, respectively. cis isomer: 1H NMR (500 MHz, CDCl3) δ 7.27 (t, J
= 7.2 Hz, 2H, 3-H, 5-H, -OPh), 7.21 (d, J = 8.7 Hz, 2H, 2-H, 6-H, -Ph-
OMe), 6.92 (t, J = 7.2 Hz, 1H, 4-H, -OPh), 6.88 (d, J = 7.2 Hz, 2H, 2-
H, 6-H, -OPh), 6.86 (d, J = 8.7 Hz, 2H, 3-H, 5-H, -Ph-OMe), 6.35 (d,
J = 11.5 Hz, 1H, 1′-H), 5.57 (dt, J = 11.5 Hz, J = 7.5 Hz, 1H, 2′-H),
3.94 (t, J = 7.3 Hz, 2H, −CH2−OPh), 3.81 (s, 3H, -OMe), 2.41−2.20
(m as q, J = 7.5 Hz, 2H, 3′-H), 1.78 (qt, J = 7.2 Hz, 2H), 1.58− 1.48
(m, 4H); mass spectrum m/z (relative intensity) 296 (M+, 12), 203
(17), 173 (8), 159 (8), 147 (78), 134 (18), 121 (100), 107 (6), 91
(19), 77 (8); Exact mass calculated for C20H24O2, 296.1776; found,
1
isomer: H NMR (500 MHz, CDCl3) δ 6.37 (d, J = 15.6 Hz, 1H, 1′-
H), 6.10 (dt, J = 15.6 Hz, J = 7.4 Hz, 1H, 2′-H).
1-(4-Methoxyphenyl)-7-phenoxyheptane (15a). A mixture of
14a (1.19 g, 4.02 mmol) and 10% Pd/C (0.18 g, 15% w/w) in ethyl
acetate (40 mL) was placed in a Parr apparatus (Parr Instrument Co,
Moline, IL) and treated with hydrogen at 30 psi for 6 h. The catalyst
was removed by filtration through a pad of Celite, and the filtrate was
evaporated under reduced pressure to give 15a as a white solid (1.14 g,
95% yield) which was used in the next step without further
1
296.1774; trans isomer: H NMR (500 MHz, CDCl3) δ 6.33 (d, J =
1
15.6 Hz, 1H, 1′-H), 6.08 (dt, J = 15.6 Hz, J = 7.4 Hz, 1H, 2′-H).
1-(3-Methoxyphenyl)-7-phenoxyhept-1-ene (14b). The syn-
thesis was carried out as described for 14a, starting from 13a (3.20 g,
6.17 mmol), potassium bis(trimethylsilyl)amide (1.22 g, 6.15 mmol)
in dry THF (55 mL), and a solution of 3-methoxybenzaldehyde (0.28
g, 2.06 mmol) in dry THF (5 mL). The crude product obtained after
workup was purified by flash column chromatography on silica gel (5%
diethyl ether in hexane) to give 14b as a colorless liquid in 92% yield
(564 mg). On the basis of 1H NMR analysis, the product is a mixture
purification. mp 32−34 °C; H NMR (500 MHz, CDCl3) δ 7.30 (t,
J = 8.5 Hz, 2H, 3-H, 5-H, -OPh), 7.11 (d, J = 8.2 Hz, 2H, 2-H, 6-H,
-Ph−OMe), 6.95 (t, J = 8.5 Hz, 1H, 4-H, -OPh), 6.92 (d, J = 8.5 Hz,
2H, 2-H, 6-H, -OPh), 6.84 (d, J = 8.2 Hz, 2H, 3-H, 5-H, -Ph−OMe),
3.97 (t, J = 6.7 Hz, 2H, −CH2−OPh), 3.81,(s, 3H, OMe), 2.57 (t, J =
7.5 Hz, 2H, −CH2−Ph−OMe), 1.78 (qt, J = 6.7 Hz, 2H of the 7-
phenoxyheptyl group), 1.62 (qt, J = 7.5 Hz, 2H of the 7-
phenoxyheptyl group), 1.48 (qt, J = 7.5 Hz, 2H of the 7-
phenoxyheptyl group), 1.44−1.34 (m, 4H of the 7-phenoxyheptyl
group); mass spectrum m/z (relative intensity) 298 (M+, 17), 204 (5),
147 (11), 134 (7), 121 (100), 107 (3), 94 (7), 77 (8); Exact mass
calculated for C20H26O2, 298.1933; found, 298.1933.
1
of cis and trans isomers in the ratio 92:8, respectively. cis isomer: H
NMR (500 MHz, CDCl3) δ 7.27−7.21 (t and t overlapping, 3H, 3-H,
5-H, -OPh and 5-H, -Ph−OMe), 6.92 (t, J = 7.3 Hz, 1H, 4-H, -OPh),
6.88 (d, J = 7.3 Hz, 2H, 2-H, 6-H, -OPh), 6.87 (d, J = 8.5 Hz, 1H, 6-H,
-Ph-OMe), 6.82 (d, J = 2.7 Hz, 1H, 2-H, -Ph-OMe), 6.77 (dd, J = 8.5
Hz, J = 2.7 Hz, 1H, 4-H, -Ph−OMe), 6.39 (d, J = 11.7 Hz, 1H, 1′-H),
5.67 (dt, J = 11.7 Hz, J = 7.5 Hz, 1H, 2′-H), 3.94 (t, J = 6.5 Hz, 2H,
-CH2OPh), 3.80 (s, 3H, OMe), 2.38 (q, J = 6.5, 2H, 3′-H), 1.78 (qt, J
= 6.5 Hz, 2H), 1.56−1.46 (m, 4H); mass spectrum m/z (relative
intensity) 296 (M+, 43), 203 (39), 173 (9), 159 (20), 147 (42), 134
(28), 121 (100), 107 (8), 91 (23), 77 (16); Exact mass calculated for
1-(3-Methoxyphenyl)-7-phenoxyheptane (15b). The synthesis
was carried out as described for 15a, using 14b (0.55 g, 1.86 mmol),
and 10% Pd/C (0.080 g, 15% w/w) in AcOEt (20 mL), and gave 15b
1
as a colorless viscous liquid in 96% yield (530 mg). H NMR (500
MHz, CDCl3) δ 7.27 (t, J = 7.7 Hz, 2H, 3-H, 5-H, -OPh), 7.19 (t, J =
8.0 Hz, 1H, 5-H, -Ph−OMe), 6.92 (t, J = 7.7 Hz, 1H, 4-H, -OPh), 6.89
(d, J = 7.7 Hz, 2H, 2-H, 6-H, -OPh), 6.77 (d, J = 8.0 Hz, 1H, 6-H,
-Ph−OMe), 6.74−6.70 (d and d overlapping, 2H, 2-H, 4-H, -Ph−
OMe), 3.94 (t, J = 6.5 Hz, 2H, −CH2OPh), 3.79 (s, 3H, OMe), 2.58
(t, J = 7.5 Hz, 2H, −CH2−Ph−OMe), 1.77 (qt, J = 7.0 Hz, 2H of the
7-phenoxyheptyl group), 1.62 (qt, J = 7.0 Hz, 2H of the 7-
phenoxyheptyl group), 1.50−1.42 (m, 2H of the 7-phenoxyheptyl
group), 1.42−1.34 (m, 4H of the 7-phenoxyheptyl group); mass
1
C20H24O2, 296.1776; found, 296.1776; trans isomer: H NMR (500
MHz, CDCl3) δ 6.36 (d, J = 15.6 Hz, 1H, 1′-H), 6.22 (dt, J = 15.6 Hz,
J = 7.4 Hz, 1H, 2′-H).
1-(2-Methoxyphenyl)-7-phenoxyhept-1-ene (14c). The syn-
thesis was carried out as described for 14a, starting from 13a (2.0 g,
10082
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spectrum m/z (relative intensity) 298 (M+, 45), 204 (6), 147 (7), 134
(9), 121 (100), 107 (6), 94 (14), 77 (9); Exact mass calculated for
C20H26O2, 298.1933; found, 298.1934.
7-Bromo-1-(2-hydroxyphenyl)heptane (16c). The synthesis
was carried out as described for 16a, using 15c (300 mg, 1.01
mmol) and BBr3 (1 M solution in CH2Cl2, 2.2 mL, 2.2 mmol) in
anhydrous CH2Cl2 (10 mL). The crude product obtained after workup
was purified by flash column chromatography on silica gel (20%
diethyl ether in hexane) to give 16c as a viscous liquid in 90% yield
(247 mg). 1H NMR (500 MHz, CDCl3) δ 7.11 (dd, J = 7.5 Hz, J = 2.0
Hz, 1H, 6-H, -Ph−OH), 7.07 (td, J = 7.5 Hz, J = 2.0 Hz, 1H, 4-H,
-Ph−OH), 6.87 (td, J = 7.5 Hz, J = 1.5 Hz, 1H, 5-H, -Ph−OH), 6.75
(dd, J = 7.5 Hz, J = 1.5 Hz, 1H, 3-H, -Ph−OH), 4.62 (br s, 1H, OH),
3.40 (t, J = 7.0 Hz, 2H, −CH2Br), 2.60 (t, J = 8.0 Hz, 2H, -CH2−Ph−
OH), 1.85 (qt, J = 6.7 Hz, 2H of the 7-bromoheptyl group), 1.62 (qt, J
= 7.2 Hz, 2H of the 7-bromoheptyl group), 1.4 (qt, J = 7.5 Hz, 2H of
the 7-bromoheptyl group), 1.40−1.35 (m, 4H of the 7-bromoheptyl
group); mass spectrum m/z (relative intensity) 272 (M+ + 2, 9), 270
(M+, 9), 147 (2), 133 (4), 120 (5), 107 (100), 91 (4), 77 (9); Exact
mass calculated for C13H19BrO, 270.0619; found, 270.0621.
5-Bromo-1-(4-hydroxyphenyl)pentane (16d). The synthesis
was carried out as described for 16a, using 15d (3.43 g, 12.7 mmol)
and BBr3 (1 M solution in CH2Cl2, 32 mL, 32 mmol) in anhydrous
CH2Cl2 (120 mL). The crude product obtained after workup was
purified by flash column chromatography on silica gel (20% diethyl
ether in hexane) to give 16d as a viscous liquid in 92% yield (2.84 g).
1H NMR (500 MHz, CDCl3) δ 7.04 (d, J = 8.5 Hz, 2H, 2-H, 6-H,
-Ph−OH), 6.75 (d, J = 8.5 Hz, 2H, 3-H, 5-H, -Ph−OH), 4.55 (br s,
1H, OH), 3.34 (t, J = 6.7 Hz, 2H, -CH2Br), 2.55 (t, J = 7.7 Hz, 2H,
-CH2−Ph−OH), 1.88 (qt, J = 7.5 Hz, 2H of the 5-bromopentyl
group), 1.60 (qt, J = 7.5 Hz, 2H of the 5-bromopentyl group), 1.46
(qt, J = 7.4 Hz, 2H of the 5-bromopentyl group); mass spectrum m/z
(relative intensity) 244 (M+ + 2, 9), 242 (M+, 9), 120 (2), 107 (100),
91 (4), 77 (7); Exact mass calculated for C11H15BrO, 242.0306; found,
242.0303.
1-(2-Methoxyphenyl)-7-phenoxyheptane (15c). The synthesis
was carried out as described for 15a, using 14c (0.35 g, 1.18 mmol)
and 10% Pd/C (0.050 g, 14% w/w) in AcOEt (20 mL), and it gave
15c as a colorless viscous liquid in 94% yield (330 mg). 1H NMR (500
MHz, CDCl3) δ 7.27 (t, J = 7.5 Hz, 2H, 3-H, 5-H, -OPh), 7.16 (t, J =
7.5 Hz, 1H, 4-H, -Ph−OMe), 7.12 (d, J = 7.5 Hz, 1H, 6-H, -Ph−
OMe), 6.95−6.81 (m, 5H, 2-H, 4-H, 6-H, -OPh and 3-H, 5-H, -Ph−
OMe, overlapping), 3.95 (t, J = 6.7 Hz, 2H, −CH2−OPh), 3.81 (s, 3H,
OMe), 2.60 (t, J = 7.7, 2H, −CH2−Ph−OMe), 1.78 (qt, J = 7.0 Hz,
2H of the 7-phenoxyheptyl group), 1.59 (qt, J = 6.9 Hz, 2H of the 7-
phenoxyheptyl group), 1.48−1.43 (m, 2H of the 7-phenoxyheptyl
group), 1.42−1.35 (m, 4H of the 7-phenoxyheptyl group); mass
spectrum m/z (relative intensity) 298 (M+, 19), 204 (7), 147 (13),
134 (11), 121 (100), 107 (8), 94 (43), 77 (10); Exact mass calculated
for C20H26O2, 298.1933; found, 298.1935.
1-(4-Methoxyphenyl)-5-phenoxypentane (15d). The synthesis
was carried out as described for 15a, using 14d (3.67 g, 13.59 mmol)
and 10% Pd/C (0.55 g, 15% w/w) in AcOEt (100 mL), and gave 15d
1
as a white solid in 95% yield (3.52 g). mp 32−34 °C; H NMR (500
MHz, CDCl3) δ 7.27 (t, J = 7.5 Hz, 2H, 3-H, 5-H, -OPh), 7.09 (d, J =
8.5 Hz, 2H, 2-H, 6-H, -Ph−OMe), 6.92 (t, J = 7.5 Hz, 1H, 4-H, -OPh),
6.88 (d, J = 7.5 Hz, 2H, 2-H, 6-H, -OPh), 6.82 (d, J = 8.5 Hz, 2H, 3-H,
5-H, -Ph−OMe), 3.94 (t, J = 6.5 Hz, 2H, -CH2−OPh), 3.78 (s, 3H,
OMe), 2.58 (t, J = 7.5 Hz, 2H, -CH2−Ph−OMe), 1.80 (qt, J = 6.7 Hz,
2H of the 5-phenoxypentyl group), 1.66 (qt, J = 7.0 Hz, 2H of the 5-
phenoxypentyl group), 1.49 (qt, J = 7.5 Hz, 2H of the 5-
phenoxypentyl group); mass spectrum m/z (relative intensity) 270
(M+, 18), 177 (7), 147 (18), 134 (6), 121 (100), 94 (6), 77 (8); Exact
mass calculated for C18H22O2, 270.1620; found, 270.1620.
7-Bromo-1-(4-hydroxyphenyl)heptane (16a). To a stirred
solution of 15a (1.1 g, 3.69 mmol) in anhydrous CH2Cl2 (40 mL),
at −30 °C, under an argon atmosphere, was added boron tribromide
(8 mL, 1 M solution in CH2Cl2). The mixture was gradually warmed
to room temperature and stirred for an additional 2 h. Unreacted
boron tribromide was destroyed by the addition of aqueous saturated
NaHCO3 solution at 0 °C. The resulting mixture was warmed to room
temperature, and volatiles were removed in vacuo. The residue was
diluted with ethyl acetate and washed with saturated NaHCO3 and
brine. The organic layer was dried over MgSO4 and the solvent
evaporated under reduced pressure. Purification by flash column
chromatography on silica gel (20% diethyl ether in petroleum ether)
7-Bromo-1-(4-benzyloxyphenyl)heptane (17a). To a stirred
solution of 16a (900 mg, 3.32 mmol) in anhydrous acetone (40 mL)
was added anhydrous K2CO3 (1.38 g, 10 mmol) and benzyl bromide
(624 mg, 3.65 mmol), and the mixture was refluxed for 6 h under
argon. The reaction mixture was cooled to room temperature and
diluted with acetone, and insoluble materials were filtered off. The
filtrate was evaporated under reduced pressure, and the residue was
dissolved in diethyl ether (50 mL). The ethereal solution was washed
with water and brine, dried (MgSO4), and evaporated. Purification by
flash column chromatography on silica gel (5% diethyl ether in
hexane) afforded 17a as a white solid in 78% yield (938 mg). mp 32−
34 °C; 1H NMR (500 MHz, CDCl3) δ 7.43 (d, J = 7.0 Hz, 2H, 2-H, 6-
H, -OCH2Ph), 7.38 (t, J = 7.0 Hz, 2H, 3-H, 5-H, -OCH2Ph), 7.32 (t, J
= 7.0 Hz, 1H, -OCH2Ph), 7.08 (d, J = 8.5 Hz, 2H, 2-H, 6-H, -Ph-
OBn), 6.90 (d, J = 8.5 Hz, 2H, 3-H, 5-H, -Ph-OBn), 5.04 (s, 2H,
-OCH2Ph), 3.34 (t, J = 7.2 Hz, 2H, -CH2Br), 2.54 (t, J = 7.5 Hz, 2H,
-CH2−Ph−OBn), 1.85 (qt, J = 7.7 Hz, 2H of the 7-bromoheptyl
group), 1.58 (qt, J = 7.5 Hz, 2H of the 7-bromoheptyl group), 1.46−
1.38 (m, 2H of the 7-bromoheptyl group), 1.37−1.30 (m, 4H of the 7-
bromoheptyl group); mass spectrum m/z (relative intensity) 362 (M+
+ 2, 6), 360 (M+, 6), 184 (7), 91 (100), 65 (9); Exact mass calculated
for C20H25BrO, 360.1089; found, 360.1088.
1
gave 16a as a viscous liquid in 93% yield (930 mg). H NMR (500
MHz, CDCl3) δ 7.03 (d, J = 8.5 Hz, 2H, 2-H, 6-H, -Ph−OH), 6.74 (d,
J = 8.5 Hz, 2H, 3-H, 5-H, -Ph−OH), 4.59 (br s, 1H, OH), 3.40 (t, J =
6.7 Hz, 2H, −CH2Br), 2.53 (t, J = 7.7 Hz, 2H, -CH2−Ph−OH), 1.84
(qt, J = 7.0 Hz, 2H of the 7-bromoheptyl group), 1.57 (qt, J = 7.5 Hz,
2H of the 7-bromoheptyl group), 1.46−1.38 (m, 2H of the 7-
bromoheptyl group), 1.36−1.31 (m, 4H of the 7-bromoheptyl group);
mass spectrum m/z (relative intensity) 272 (M+ + 2, 7), 270 (M+, 7),
147 (2), 133 (4), 120 (3), 107 (100), 91 (2), 77 (5); Exact mass
calculated for C13H19BrO, 270.0619; found, 270.0620.
7-Bromo-1-(3-hydroxyphenyl)heptane (16b). The synthesis
was carried out as described for 16a, using 15b (500 mg, 1.68 mmol)
and BBr3 (1 M solution in CH2Cl2, 3.7 mL, 3.7 mmol) in anhydrous
CH2Cl2 (16 mL). The crude product obtained after workup was
purified by flash column chromatography on silica gel (20% diethyl
ether in hexane) to give 16b as a viscous liquid in 92% yield (420 mg).
1H NMR (500 MHz, CDCl3) δ 7.14 (t, J = 7.7 Hz, 1H, 5-H, -Ph−
OH), 6.75 (d, J = 7.7 Hz, 1H, 6-H, -Ph−OH), 6.68−6.63 (d and d
overlapping, 2H, 2-H, 4-H, -Ph−OH), 4.70 (br s, 1H, OH), 3.40 (t, J =
6.7 Hz, 2H, -CH2Br), 2.56 (t, J = 7.7 Hz, 2H, -CH2-Ph−OH), 1.85 (qt,
J = 7.0 Hz, 2H of the 7-bromoheptyl group), 1.60 (qt, J = 7.3 Hz, 2H
of the 7-bromoheptyl group), 1.46−1.38 (m, 2H of the 7-bromoheptyl
group), 1.36−1.30 (m, 4H of the 7-bromoheptyl group); mass
spectrum m/z (relative intensity) 272 (M+ + 2, 8), 270 (M+, 8), 149
(4), 147 (3), 133 (3), 121 (13), 108 (100), 91 (4), 77 (10); Exact
mass calculated for C13H19BrO, 270.0619; found, 270.0618.
7-Bromo-1-(3-benzyloxyphenyl)heptane (17b). The synthesis
was carried out as described for 17a, using 16b (400 mg, 1.48 mmol),
K2CO3 (612 mg, 4.44 mmol), and benzyl bromide (278 mg, 1.63
mmol) in anhydrous acetone. The crude product obtained after
workup was purified by flash column chromatography on silica gel
(10% diethyl ether in hexane) to give 17b as a viscous liquid in 77%
1
yield (411 mg). H NMR (500 MHz, CDCl3) δ 7.44 (d, J = 7.7 Hz,
2H, 2-H, 6-H, -OCH2Ph), 7.39 (t, J = 7.7 Hz, 2H, 3-H, 5-H,
-OCH2Ph), 7.32 (t, J = 7.7 Hz, 1H, 1H, 4-H, -OCH2Ph), 7.19 (t, J =
7.9 Hz, 1H, 5-H, -Ph−OBn), 6.83−6.76 (m, 3H, 2-H, 4-H, 6-H, -Ph−
OBn), 5.05 (s, 2H, -OCH2Ph), 3.40 (t, J = 6.6 Hz, 2H, −CH2Br), 2.58
(t, J = 7.7 Hz, 2H, −CH2−Ph−OBn), 1.84 (qt, J = 7.1 Hz, 2H of the
7-bromoheptyl group), 1.61 (qt, J = 7.7 Hz, 2H of the 7-bromoheptyl
group), 1.42 (qt, J = 7.0 Hz, 2H of the 7-bromoheptyl group), 1.35−
1.31 (m, 4H of the 7-bromoheptyl group); mass spectrum m/z
(relative intensity) 362 (M+ + 2, 13), 360 (M+, 13), 183 (3), 91 (100),
10083
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65 (6); Exact mass calculated for C20H25BrO, 360.1089; found,
360.1090.
mL) was added thionyl chloride (890 mg, 7.49 mmol), and the
mixture was heated at 50 °C for 3 h under argon. The reaction mixture
was quenched by dropwise addition of water at room temperature and
extracted with diethyl ether. The organic layer was washed with brine
and dried (MgSO4), and the solvent was evaporated under reduced
pressure. Purification by flash column chromatography on silica gel
(20% diethyl ether in hexane) gave 19a as a white solid in 40% yield
from 17a (380 mg). mp 33−35 °C; IR (neat) 3033, 2945, 2923, 1610,
7-Bromo-1-(2-benzyloxyphenyl)heptane (17c). The synthesis
was carried out as described for 17a, using 16c (230 mg, 0.85 mmol),
K2CO3 (352 mg, 2.55 mmol), and benzyl bromide (160 mg, 0.936
mmol) in anhydrous acetone. The crude product obtained after
workup was purified by flash column chromatography on silica gel
(10% diethyl ether in hexane) to give 17c as a viscous liquid in 78%
1
1
1513, 1371 (s), 1244, 1164 (s); H NMR (500 MHz, CDCl3) δ 7.44
yield (240 mg). H NMR (500 MHz, CDCl3) δ 7.44 (d, J = 7.5 Hz,
(d, J = 7.5 Hz, 2H, 2-H, 6-H, -OCH2Ph), 7.38 (t, J = 7.5 Hz, 2H, 3-H,
5-H, -OCH2Ph), 7.32 (t, J = 7.5 Hz, 1H, 4-H, -OCH2Ph), 7.08 (d, J =
8.5 Hz, 2H, 2-H, 6-H, -Ph-OBn), 6.90 (d, J = 8.5 Hz, 2H, 3-H, 5-H,
-Ph-OBn), 5.04 (s, 2H, -OCH2Ph), 3.64 (m as t, J = 8.0 Hz, half of an
AA′XX′ system, 2H, −CH2SO2Cl), 2.55 (t, J = 7.5 Hz, 2H, -CH2−
Ph−OBn), 2.03 (m as qt, J = 7.7 Hz, 2H, -CH2CH2SO2Cl), 1.62−1.54
(m, 2H), 1.52−1.46 (m, 2H), 1.40−1.30 (m, 4H); mass spectrum m/z
(relative intensity) 382 (M+ + 2, 2), 380 (M+, 6), 282 (7), 149 (8),
107 (7), 91 (100); Exact mass calculated for C20H25ClO3S, 380.1213;
found, 380.1211.
2H, 2-H, 6-H, -OCH2Ph), 7.39 (t, J = 7.5 Hz, 2H, 3-H, 5-H,
-OCH2Ph), 7.32 (t, J = 7.5 Hz, 1H, 4-H, -OCH2Ph), 7.18−7.13 (m,
2H, 4-H, 6-H, -Ph-OBn), 6.92−6.88 (m, 2H, 3-H, 5-H, -Ph−OBn),
5.08 (s, 2H, -OCH2Ph), 3.37 (t, J = 7.0 Hz, 2H, −CH2Br), 2.67 (t, J =
7.5 Hz, 2H, −CH2−Ph−OBn), 1.82 (qt, J = 7.7 Hz, 2H of the 7-
bromoheptyl group), 1.62 (qt, J = 7.5 Hz, 2H of the 7-bromoheptyl
group), 1.39 (qt, J = 7.2 Hz, 2H of the 7-bromoheptyl group), 1.36−
1.31 (m, 4H of the 7-bromoheptyl group); mass spectrum m/z
(relative intensity) 362 (M+ + 2, 10), 360 (M+, 10), 190 (4), 91 (100),
77 (3), 65 (6); Exact mass calculated for C20H25BrO, 360.1089; found,
360.1089.
5-Bromo-1-(4-benzyloxyphenyl)pentane (17d). The synthesis
was carried out as described for 17a, using 16d (2.79 g, 11.5 mmol),
K2CO3 (4.24 g, 30.75 mmol), and benzyl bromide (2.31 g, 13.51
mmol) in anhydrous acetone. The crude product obtained after
workup was purified by flash column chromatography on silica gel (5%
diethyl ether in hexane) to give 17d as a semisolid material in 81%
yield (3.11 g). 1H NMR (500 MHz, CDCl3) δ 7.42 (d, J = 7.5 Hz, 2H,
2-H, 6-H, -OCH2Ph), 7.37 (t, J = 7.5 Hz, 2H, 3-H, 5-H, -OCH2Ph),
7.31 (t, J = 7.5 Hz, 1H, -OCH2Ph), 7.08 (d, J = 8.5 Hz, 2H, 2-H, 6-H,
-Ph−OBn), 6.90 (d, J = 8.5 Hz, 2H, 3-H, 5-H, -Ph−OBn), 5.03 (s, 2H,
-OCH2Ph), 3.39 (t, J = 6.7 Hz, 2H, −CH2Br), 2.56 (t, J = 7.7 Hz, 2H,
−CH2−Ph−OBn), 1.87 (qt, J = 6.7 Hz, 2H of the 5-bromopentyl
group), 1.61 (qt J = 7.7 Hz, 2H of the 5-bromopentyl group), 1.47 (qt
J = 6.7 Hz, 2H of the 5-bromopentyl group); mass spectrum m/z
(relative intensity) 334 (M+ + 2, 15), 332 (M+, 15), 197 (2), 91 (100),
65 (7); Exact mass calculated for C18H21BrO, 332.0776; found,
332.0776.
7-(4-Benzyloxyphenyl)heptanesulfonic Acid Sodium Salt
(18a). A stirred mixture of 17a (900 mg, 2.49 mmol) and Na2SO3
(423 mg, 3.36 mmol) in EtOH (20 mL)/H2O (10 mL) was heated
under reflux for 24 h. The reaction mixture was cooled to room
temperature and the solvent evaporated under reduced pressure. The
residue was scrupulously dried under high vacuum, and the crude
product 18a (pale yellow solid) was used in the next step without
further purification.
7-(3-Benzyloxyphenyl)heptanesulfonic Acid Sodium Salt
(18b). The synthesis was carried out as described for 18a, using
17b (400 mg, 1.11 mmol), Na2SO3 (190 mg, 1.5 mmol), and an EtOH
(8 mL)/H2O (4 mL) mixture. The crude product 18b was used in the
next step without further purification.
7-(3-Benzyloxyphenyl)heptanesulfonyl Chloride (19b). The
synthesis was carried out as described for 19a, using 18b and thionyl
chloride (360 mg, 3.03 mmol) in benzene (9 mL)/DMF (0.1 mL).
The crude product obtained after workup was purified by flash column
chromatography on silica gel (20% diethyl ether in hexane) to give
19b as a viscous liquid in 39% yield from 17b (163 mg). IR (neat)
1
3033, 2945, 2920, 1610, 1513, 1371 (s), 1242, 1160 (s); H NMR
(500 MHz, CDCl3) δ 7.44 (d, J = 7.5 Hz, 2H, 2-H. 6-H, -OCH2Ph),
7.39 (t, J = 7.5 Hz, 2H, 3-H, 5-H, -OCH2Ph), 7.32 (t, J = 7.5 Hz, 1H,
4-H, -OCH2Ph), 7.20 (t, J = 7.6 Hz, 1H, 5-H, -Ph-OBn), 6.83−6.76
(m, 3H, 2-H, 4-H, 6-H, -Ph-OBn), 5.05 (s, 2H, -OCH2Ph), 3.64 (m as
t, J = 8.0 Hz, half of an AA′XX′ system, 2H, -CH2SO2Cl), 2.58 (t, J =
7.6 Hz, 2H, -CH2−Ph−OBn), 2.03 (m as qt, J = 7.7 Hz, half of an
AA′XX′ system, 2H, -CH2CH2SO2Cl), 1.62 (qt, J = 7.5 Hz, 2H), 1.48
(qt, J = 7.5 Hz, 2H), 1.42−1.30 (m, 4H); mass spectrum m/z (relative
intensity) 382 (M+ + 2, 1), 380 (M+, 3), 149 (8), 107 (7), 91 (100);
Exact mass calculated for C20H25ClO3S, 380.1213; found, 380.1215.
7-(2-Benzyloxyphenyl)heptanesulfonyl Chloride (19c). The
synthesis was carried out as described for 19a, using 18c and thionyl
chloride (228 mg, 1.92 mmol) in benzene (9 mL)/DMF (0.1 mL).
The crude product obtained after workup was purified by flash column
chromatography on silica gel (20% diethyl ether in hexane) to give 19c
as a viscous liquid in 38% yield from 17c (92 mg). IR (neat) 3028,
1
2941, 2921, 1611, 1517, 1371 (s), 1244, 1164 (s); H NMR (500
MHz, CDCl3) δ 7.44 (d, J = 8.0 Hz, 2H, 2-H, 6-H, -OCH2Ph), 7.39 (t,
J = 8.0 Hz, 2H, 3-H, 5-H, -OCH2Ph), 7.33 (t, J = 8.0 Hz, 1H, 4-H,
-OCH2Ph), 7.18−7.13 (t and d overlapping, 2H, 4-H, 6-H, -Ph−OBn),
6.91 (d, J = 8.0 Hz, 1H, 3-H, -Ph−OBn), 6.90 (t, J = 8.0 Hz, 1H, 5-H,
-Ph−OBn), 5.08 (s, 2H, -OCH2Ph), 3.58 (m as t, J = 8.0 Hz, half of an
AA′XX′ system, 2H, −CH2SO2Cl), 2.67 (t, J = 7.7 Hz, 2H, -CH2−
Ph−OBn), 1.99 (m as qt, J = 7.7 Hz, half of an AA′XX′ system, 2H,
-CH2CH2SO2Cl), 1.62 (qt, J = 7.5 Hz, 2H), 1.46−1.40 (m, 2H),
1.39−1.33 (m, 4H); mass spectrum m/z (relative intensity) 382 (M+ +
2, 1), 380 (M+, 3), 149 (21), 107 (9), 91 (100); Exact mass calculated
for C20H25ClO3S, 380.1213; found, 380.1211.
5-(4-Benzyloxyphenyl)pentanesulfonyl Chloride (19d). The
synthesis was carried out as described for 19a, using 18d and thionyl
chloride (1.0 g, 8.40 mmol) in benzene (27 mL)/DMF (0.3 mL). The
crude product obtained after workup was purified by flash column
chromatography on silica gel (20% diethyl ether in hexane) to give
19d as a white solid in 37% yield from 17d (372 mg).
7-(2-Benzyloxyphenyl)heptanesulfonic Acid Sodium Salt
(18c). The synthesis was carried out as described for 18a, using 17c
(0.231 g, 0.64 mmol), Na2SO3 (0.11 g, 0.89 mmol), and an EtOH (8
mL)/H2O (4 mL) mixture. The crude product 18c was used in the
next step without further purification.
5-(4-Benzyloxyphenyl)pentanesulfonic Acid Sodium Salt
(18d). Method A: The procedure described for 18a was followed,
using 17d (0.95 g, 2.85 mmol), Na2SO3 (0.5 g, 4.0 mmol), and an
EtOH (20 mL)/H2O (10 mL) mixture. The crude product 18d was
used in the next step without further purification. Method B: A
mixture of 17d (0.95 g, 2.85 mmol) and Na2SO3 (0.5 g, 4.0 mmol) in
THF/EtOH/H2O (1:2:2 mixture, 15 mL) was heated for 15 min at
160 °C under microwave irradiation (300 W) using a CEM Discover
system. The reaction mixture was cooled to room temperature, and
volatiles were removed under reduced pressure. The residue was
scrupulously dried under high vacuo, and the crude product 18d (a
pale yellow solid) was used in the next step without further
purification.
Using 18d derived from Method B and thionyl chloride (1.0 g, 7.41
mmol) in benzene (27 mL)/DMF (0.3 mL), the title compound was
obtained in 50% yield from 17d (501 mg). mp 58−60 °C; IR (neat)
1
3031, 2944, 2923, 1610, 1512, 1372 (s), 1244, 1164 (s); H NMR
(500 MHz, CDCl3) δ 7.43 (d, J = 7.5 Hz, 2H, 2-H, 6-H, -OCH2Ph),
7.38 (t, J = 7.5 Hz, 2H, 3-H, 5-H, -OCH2Ph), 7.32 (t, J = 7.5 Hz, 1H,
4-H, -OCH2Ph), 7.07 (d, J = 8.5 Hz, 2H, 2-H, 6-H, -Ph−OBn), 6.90
(d, J = 8.5 Hz, 2H, 3-H, 5-H, -Ph−OBn), 5.03 (s, 2H, -OCH2Ph), 3.63
(m as t, J = 8.0 Hz, half of an AA′XX′ system, 2H, -CH2SO2Cl), 2.59
(t, J = 7.5 Hz, 2H, -CH2−Ph−OBn), 2.06 (m as qt, J = 7.7 Hz, 2H,
7-(4-Benzyloxyphenyl)heptanesulfonyl Chloride (19a). To a
stirred suspension of 18a in anhydrous benzene (20 mL)/DMF (0.2
10084
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-CH2CH2SO2Cl), 1.66 (qt, J = 7.5 Hz, 2H), 1.51 (qt, J = 7.4 Hz, 2H);
mass spectrum m/z (relative intensity) 354 (M+ + 2, 4), 352 (M+, 15),
256 (39), 238 (37), 196 (41), 168 (33), 107 (38), 91 (100), 77 (79);
Exact mass calculated for C18H21ClO3S, 352.0900; found, 352.0900.
7-(4-Benzyloxyphenyl)heptanesulfonyl Fluoride (20a). To a
stirred solution of 19a (300 mg, 0.79 mmol) in dry acetone (20 mL)
was added anhydrous NH4F (66 mg, 1.8 mmol), and the mixture was
refluxed for 2 h under argon. The reaction mixture was cooled to room
temperature, and the solvent was evaporated under reduced pressure.
The residue was dissolved in diethyl ether, and the ethereal solution
was successively washed with water and brine, dried (MgSO4), and
evaporated in vacuo. Purification by flash column chromatography on
silica gel (20% diethyl ether in hexane) gave 20a as a white solid in
93% yield (267 mg). mp 35−38 °C; IR (neat) 3043, 2955, 1512, 1397
(qt, J = 7.5 Hz, 2H), 1.50 (qt, J = 7.5 Hz, 2H); 13C NMR (100 MHz,
CDCl3) δ 157.14 (4-C, -CH2−Ph−OBn), 137.17 (1-C, -O−CH2−Ph),
134.01 (1-C, -CH2−Ph−OBn), 129.25, 128.57, 127.92, 127.47, 114.82
(3-C, 5-C, -CH2−Ph−OBn), 70.08 (-O−CH2−Ph), 50.89 (d, J = 16.1
Hz, -CH2SO2F), 34.47 (-CH2−Ph−OBn), 30.77, 27.33, 23.32; mass
spectrum m/z (relative intensity) 336 (M+, 17), 260 (3), 224 (14),
196 (9), 149 (3), 121 (9), 107 (4), 91 (100), 65 (9); Exact mass
calculated for C18H21FO3S, 336.1195; found, 336.1198.
7-(4-Hydroxyphenyl)heptanesulfonyl Fluoride (21a). To a
solution of 20a (182 mg, 0.5 mmol) in ethanedithiol (4 mL), at room
temperature, under an argon atmosphere was added BF3·Et2O (282
mg, 2.0 mmol). The reaction mixture was stirred for 1 h and then
diluted with diethyl ether and water. The organic layer was separated
and the aqueous phase extracted with diethyl ether. The combined
organic layer was washed with brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (50% diethyl ether in
hexane) to give 21a as a white solid in 70% yield (96 mg). mp 47−51
°C; IR (neat) 3396 (br, OH), 2923, 2851, 1611, 1513, 1394 (s), 1237,
1
(s), 1235, 1194 (s); H NMR (500 MHz, CDCl3) δ 7.43 (d, J = 7.5
Hz, 2H, 2-H, 6-H, -OCH2Ph), 7.38 (t, J = 7.5 Hz, 2H, 3-H, 5-H,
-OCH2Ph), 7.32 (t, J = 7.5 Hz, 1H, 4-H, -OCH2Ph), 7.08 (d, J = 8.7
Hz, 2H, 2-H, 6-H, -Ph−OBn), 6.90 (d, J = 8.7 Hz, 2H, 3-H, 5-H, -Ph−
OBn), 5.04 (s, 2H, -OCH2Ph), 3.34 (m as dt, J = 11.0 Hz, J = 4.5 Hz,
2H, -CH2SO2F), 2.55 (t, J = 7.5 Hz, 2H, -CH2−Ph−OBn), 1.94 (m as
qt, J = 8.0 Hz, 2H, -CH2CH2SO2F), 1.59 (qt, J = 7.0 Hz, 2H), 1.47 (qt,
J = 7.4 Hz, 2H), 1.40−1.31 (m, 4H); mass spectrum m/z (relative
intensity) 364 (M+, 10), 149 (2), 107 (6), 91 (100), 65 (6); Exact
mass calculated for C20H25FO3S, 364.1508; found, 364.1512.
1
1198 (s); H NMR (500 MHz, CDCl3) δ 7.03 (d, J = 8.5 Hz, 2H,
ArH), 6.75 (d, J = 8.5 Hz, 2H, ArH), 4.62 (br s, 1H, OH), 3.33 (m as
dt, J = 11.0 Hz, J = 4.5 Hz, 2H, -CH2SO2F), 2.53 (t, J = 7.3 Hz, 2H,
-CH2−Ph−OH), 1.93 (m as qt, J = 8.0 Hz, 2H, -CH2CH2SO2F), 1.58
(qt, J = 6.9 Hz, 2H), 1.47 (qt, J = 7.4 Hz, 2H), 1.40−1.30 (m, 4H);
mass spectrum m/z (relative intensity) 274 (M+, 18), 120 (3), 107
(100), 91 (2), 77 (6); Exact mass calculated for C13H19FO3S,
274.1039; found, 274.1037.
7-(3-Benzyloxyphenyl)heptanesulfonyl Fluoride (20b). The
synthesis was carried out as described for 20a, using 19b (149 mg, 0.39
mmol) and NH4F (29 mg, 0.78 mmol) in dry acetone (10 mL). The
crude product obtained after workup was purified by flash column
chromatography on silica gel (20% diethyl ether in hexane) to give
20b as a viscous liquid in 90% yield (129 mg). IR (neat) 3051, 2952,
7-(3-Hydroxyphenyl)heptanesulfonyl Fluoride (21b). The
synthesis was carried out as described for 21a, using 20b (100 mg,
0.27 mmol) and BF3.Et2O (140 mg, 1.0 mmol) in ethanedithiol (4
mL). The residue obtained after work up was purified by flash column
chromatography on silica gel (50% diethyl ether in hexane) to give
21b as a viscous liquid in 68% yield (51 mg). IR (neat) 3380 (br, OH),
1
1512, 1397 (s), 1234, 1194 (s); H NMR (500 MHz, CDCl3) δ 7.43
(d, J = 7.5 Hz, 2H, 2-H, 6-H, -OCH2Ph), 7.39 (t, J = 7.5 Hz, 2H, 3-H,
5-H, -OCH2Ph), 7.32 (t, J = 7.5 Hz, 1H, 4-H, -OCH2Ph), 7.19 (t, J =
7.7 Hz, 1H, 5-H, -Ph-OBn), 6.82−6.76 (m, 3H, 2-H, 4-H, 6-H, -Ph-
OBn), 5.05 (s, 2H, -OCH2Ph), 3.34 (m as dt, J = 11.0 Hz, J = 4.5 Hz,
2H, -CH2SO2F), 2.58 (t, J = 7.5 Hz, 2H, -CH2−Ph−OBn), 1.93 (m as
qt, J = 7.8 Hz, 2H, -CH2CH2SO2F), 1.61 (qt, J = 7.5 Hz, 2H), 1.47 (qt,
J = 7.2 Hz, 2H), 1.40−1.29 (m, 4H); mass spectrum m/z (relative
intensity) 364 (M+, 13), 149 (3), 107 (2), 91 (100), 77 (3), 65 (6);
Exact mass calculated for C20H25FO3S, 364.1508; found, 364.1507.
7-(2-Benzyloxyphenyl)heptanesulfonyl Fluoride (20c). The
synthesis was carried out as described for 20a, using 19c (85 mg, 0.22
mmol) and NH4F (18 mg, 0.486 mmol) in dry acetone (10 mL). The
crude product obtained after workup was purified by flash column
chromatography on silica gel (20% diethyl ether in hexane) to give 20c
as a viscous liquid in 90% yield (73 mg). IR (neat) 3037, 2940, 1513,
1398 (s), 1235, 1194 (s); 1H NMR (500 MHz, CDCl3) δ 7.43 (d, J =
7.5 Hz, 2H, 2-H. 6-H, -OCH2Ph), 7.39 (t, J = 7.5 Hz, 2H, 3-H, 5-H,
-OCH2Ph), 7.34 (t, J = 7.5 Hz, 1H, 4-H, -OCH2Ph), 7.18−7.12 (t and
d overlapping, 2H, 4-H, 6-H, -Ph−OBn), 6.91 (d, J = 8.0 Hz, 1H, 3-H,
-Ph−OBn), 6.90 (t, J = 8.0 Hz, 1H, 5-H, -Ph−OBn), 5.07 (s, 2H,
-OCH2Ph), 3.28 (m as dt, J = 11.0 Hz, J = 4.5 Hz, 2H, -CH2SO2F),
2.67 (t, J = 7.5 Hz, 2H, −CH2−Ph−OBn), 1.89 (m as qt, J = 7.8 Hz,
2H, -CH2CH2SO2F), 1.62 (qt, J = 7.5 Hz, 2H), 1.46−1.40 (m, 2H),
1.39−1.33 (m, 4H); mass spectrum m/z (relative intensity) 364 (M+,
12), 107 (7), 91 (100), 65 (6); Exact mass calculated for C20H25FO3S,
364.1508; found, 364.1506.
1
2935, 2854, 1612, 1513, 1394 (s), 1238, 1198 (s); H NMR (500
MHz, CDCl3) δ 7.14 (t, J = 7.5 Hz, 1H, 5-H, -Ph−OH), 6.74 (d, J =
7.5 Hz, 1H, 6-H, -Ph−OH), 6.66−6.63 (m, 2H, 2-H, 4-H, -Ph−OH),
4.74 (br s, 1H, OH), 3.35 (m as dt, J = 11.0 Hz, J = 4.5 Hz, 2H,
-CH2SO2F), 2.56 (t, J = 7.7 Hz, 2H, -CH2−Ph−OH), 1.94 (m as qt, J
= 7.7 Hz, 2H, -CH2CH2SO2F), 1.61 (qt, J = 7.5 Hz, 2H), 1.48 (qt, J =
7.2 Hz, 2H), 1.40−1.31 (m, 4H); mass spectrum m/z (relative
intensity) 274 (M+, 25), 121 (21), 108 (100), 91 (23), 77 (17); Exact
mass calculated for C13H19FO3S, 274.1039; found, 274.1039.
7-(2-Hydroxyphenyl)heptanesulfonyl Fluoride (21c). The
synthesis was carried out as described for 21a, using 20c (65 mg,
0.18 mmol) and BF3·Et2O (92 mg, 0.65 mmol) in ethanedithiol (2
mL). The residue obtained after work up was purified by flash column
chromatography on silica gel (50% diethyl ether in hexane) to give 21c
as a viscous liquid in 69% yield (34 mg). IR (neat) 3395 (br, OH),
1
2927, 2854, 1612, 1513, 1395 (s), 1238, 1198 (s); H NMR (500
MHz, CDCl3) δ 7.10 (dd, J = 7.5 Hz, J = 1.6 Hz, 1H, 6-H, -Ph−OH),
7.07 (td, J = 7.5 Hz, J = 1.6 Hz, 1H, 4-H, -Ph−OH), 6.87 (td, J = 7.5
Hz, J = 1.2 Hz, 1H, 5-H, -Ph−OH), 6.75 (dd, J = 7.5 Hz, J = 1.2 Hz,
1H, 3-H, -Ph−OH), 4.73 (br s, 1H, OH), 3.35 (m as dt, J = 11.0 Hz, J
= 4.5 Hz, 2H, -CH2SO2F), 2.61 (t, J = 7.5 Hz, 2H, -CH2−Ph−OH),
1.94 (m as qt, J = 7.8 Hz, 2H, -CH2CH2SO2F), 1.62 (m, 2H), 1.52−
1.44 (m, 2H), 1.42−1.34 (m, 4H); mass spectrum m/z (relative
intensity) 274 (M+, 16), 120 (3), 107 (100), 91 (7), 77 (8); Exact
mass calculated for C13H19FO3S, 274.1039; found, 274.1042.
5-(4-Hydroxyphenyl)pentanesulfonyl Fluoride (21d). The
synthesis was carried out as described for 21a, using 19d (0.24 g,
0.71 mmol) and BF3·Et2O (0.47 g, 3.32 mmol) in ethanedithiol (7
mL). The residue obtained after workup was purified by flash column
chromatography on silica gel (50% diethyl ether in hexane) to give
21d as a white solid in 66% yield (117 mg). mp 32−35 °C; IR (neat)
3387 (br, OH), 2920, 2851, 1611, 1514, 1394 (s), 1238, 1196 (s); 1H
NMR (500 MHz, CDCl3) δ 7.02 (d, J = 8.2 Hz, 2H, ArH), 6.76 (d, J =
8.2 Hz, 2H, ArH), 4.65 (br s, 1H, OH), 3.34 (m as dt, J = 11.0 Hz, J =
4.5 Hz, 2H, -CH2SO2F), 2.57 (t, J = 7.0 Hz, 2H, -CH2−Ph−OH), 1.96
(m as qt, J = 7.7 Hz, 2H, -CH2CH2SO2F), 1.64 (qt, J = 7.5 Hz, 2H),
1.50 (qt, J = 7.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 156.45 (4-
5-(4-Benzyloxyphenyl)pentanesulfonyl Fluoride (20d). The
synthesis was carried out as described for 20a, using 19d (300 mg, 0.85
mmol) and NH4F (60 mg, 1.64 mmol) in dry acetone (20 mL). The
crude product obtained after workup was purified by flash column
chromatography on silica gel (20% diethyl ether in hexane) to give
20d as a white solid in 92% yield (263 mg). mp 66−68 °C; IR (neat)
1
3042, 2961, 1511, 1397 (s), 1235, 1195 (s); H NMR (500 MHz,
CDCl3) δ 7.43 (d, J = 7.5 Hz, 2H, 2-H, 6-H, -OCH2Ph), 7.38 (t, J =
7.5 Hz, 2H, 3-H, 5-H, -OCH2Ph), 7.32 (t, J = 7.5 Hz, 1H, 4-H,
-OCH2Ph), 7.08 (d, J = 8.5 Hz, 2H, 2-H, 6-H, -Ph−OBn), 6.90 (d, J =
8.5 Hz, 2H, 3-H, 5-H, -Ph−OBn), 5.04 (s, 2H, -OCH2Ph), 3.34 (m as
dt, J = 11.0 Hz, J = 4.5 Hz, 2H, -CH2SO2F), 2.58 (t, J = 7.3 Hz, 2H,
-CH2−Ph−OBn), 1.97 (m as qt, J = 7.7 Hz, 2H, -CH2CH2SO2F), 1.65
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C, -CH2−Ph−OH), 135.63 (1-C, -CH2−Ph−OH), 131.22 (2-C, 6-C,
-CH2−Ph−OH), 116.35 (3-C, 5-C, -CH2−Ph−OH), 50.92 (d, J =
16.1 Hz, -CH2SO2F), 35.72 (-CH2−Ph−OH), 30.91, 27.43, 22.37;
mass spectrum m/z (relative intensity) 246 (M+, 14), 120 (3), 107
(100), 91 (4), 77 (7); Exact mass calculated for C11H15FO3S,
246.0726; found, 246.0725.
The ethereal solution was washed with water and brine, dried
(MgSO4), and evaporated under reduced pressure. Purification by
flash column chromatography on silica gel (20% diethyl ether in
hexane) gave 27 as a white solid in 84% yield (41 mg). mp 57−59 °C;
1H NMR (500 MHz, CDCl3) δ 7.43 (d, J = 7.5 Hz, 2H, 2-H, 6-H,
-OCH2Ph), 7.38 (t, J = 7.5 Hz, 2H, 3-H, 5-H, -OCH2Ph), 7.32 (t, J =
7.5 Hz, 1H, 4-H, -OCH2Ph), 7.08 (d, J = 8.0 Hz, 2H, 2-H, 6-H, -Ph−
OBn), 6.90 (d, J = 8.0 Hz, 2H, 3-H, 5-H, -Ph−OBn), 5.04 (s, 2H,
-OCH2Ph), 3.88 (s, 3H, OMe), 3.08 (m as t, J = 7.8 Hz, half of an
AA′XX′ system, 2H, -CH2SO2OMe), 2.54 (t, J = 7.2 Hz, 2H, -CH2−
Ph−OBn), 1.85 (m as qt, J = 8.0 Hz, 2H, -CH2CH2SO2OMe), 1.58
(qt, J = 7.0 Hz, 2H), 1.43 (qt, J = 7.9 Hz, 2H), 1.38−1.30 (m, 4H).
Preparation of Transmembrane Domain-Deleted Rat FAAH
(ΔTM rFAAH). Rat ΔTM FAAH was expressed in E. coli cells and
purified using the procedure disclosed by Patricelli et al.53
4-Bromo-1-(4-benzyloxyphenoxy)butane (23). A stirred mix-
ture of 4-benzyloxyphenol (22, 400 mg, 2 mmol), anhydrous
potassium carbonate (331 mg, 2.4 mmol), 18-crown-6 (580 mg, 2.2
mmol), and 1,4-dibromobutane (518 mg, 2.4 mmol) in anhydrous
acetone (20 mL) was heated under reflux for 3 h. The reaction mixture
was cooled to room temperature, the solid materials were filtered off,
and the solvent was removed in vacuo. The residue was dissolved in
diethyl ether, and the ethereal solution was washed sequentially with
water and brine, dried (MgSO4), and evaporated under reduced
pressure. Purification by flash column chromatography on silica gel
(20% diethyl ether in hexane) gave 23 as a white solid in 50% yield
Preparation of Human FAAH in Fusion with N-Terminal
Maltose Binding Tag (MBPΔTMhFAAH). Human FAAH without
putative transmembrane domain following maltose binding protein
was expressed in E. coli cells using pMALcE4 vector (New England
Biolabs) (unpublished data).
1
(337 mg). mp 68−69 °C; H NMR (300 MHz, CDCl3) δ 7.46−7.28
(m, 5H, 2-H, 3-H, 4-H, 5-H, 6-H, -O−CH2−Ph), 6.90 (m as d, J = 8.7
Hz, 2H, -O−Ph−OBn), 6.81 (m as d, J = 8.7 Hz, 2H, -O−Ph−OBn),
5.01 (s, 2H, -OCH2−Ph), 3.94 (t, J = 6.0 Hz, 2H, -CH2−O−Ph-), 3.48
(t, J = 6.6 Hz, 2H, -CH2Br), 2.58 (qt, J = 6.5 Hz, 2H of the 4-
bromobutyl group), 1.91 (qt, J = 6.6 Hz, 2H of the 4-bromobutyl
group).
Fluorescent Assay Protocol for ΔTM Rat FAAH. The
compounds N-arachidonoyl, 7-amino-4-methylcoumarin amide
(AAMCA),52 and arachidonoylmethylcoumarin (AMC) were dis-
solved in DMSO (10 mM) and kept as stock solutions at −20 °C
before the assay was performed. The compounds were diluted in 50:50
DMSO/assay buffer (50 mM HEPES, 1 mM EDTA, 0.1% BSA, pH
7.4) so as to have a final DMSO concentration below 8% in each
reaction. For the initial screening assay, 3 concentrations (1 μM, 10
μM, and 100 μM) of test compounds, 15 μg of total protein in E. coli
lysate containing ΔTM rFAAH, and assay buffer were preincubated for
15 min at 25 °C. AAMCA (20 μM) was added prior to incubation at
25 °C and kinetic fluorescence reading every 20 min (λex = 360/λem=
460) for 4 h on a BioTek Synergy HT Microplate Reader (BioTek
Instruments, Winooski, VT). The fluorescence reading at the 3 h time
point (linear enzyme kinetics) was used to calculate percent inhibition
based on control assays without inhibitor present. For the full dose
response curves, 8 concentrations of the test compounds were used
and IC50 values were determined using Prizm software (GraphPad
Software, Inc.).
Preparation of Human MGL (hMGL). Recombinant hexa-
histidine-tagged human MGL (hMGL) was expressed in E. coli cells
and purified following our recently reported procedures.45,46
Fluorescent Assay Protocol for hMGL. Compound inhibition of
hMGL activity was assessed by a fluorometric assay recently developed
in our laboratory.43,45,46 This medium throughput assay involved a 96-
well plate format in which hMGL activity was monitored by the
hydrolysis of the substrate 7-hydroxy-6-methoxy-4-methylcoumarin
ester (AHMMCE)43,45,46 to form the fluorescent product, coumarin.
In brief, various concentrations of each compound were preincubated
with hMGL (175 ng of total protein in E. coli lysate containing hMGL)
for 15 min at room temperature. Upon the addition of AHMMCE, the
reaction was incubated at 25 °C for 120 min; fluorescence readings
were taken every 15 min at 360 nm/460 nm (λexcitation/λemission) using a
Synergy HT Plate Reader (Bio-Tek, Winooski, VT). Under these
incubation conditions, negligible spontaneous AHMMCE hydrolysis
was observed. External standards were used to convert relative
fluorescence units to the amount of 4-methylcoumarin formed. All
MGL assays were performed in triplicate for each inhibitor
concentration, and IC50 values were calculated using Prizm software
(GraphPad Software, Inc., San Diego, CA).
4-(4-Benzyloxyphenoxy)butanesulfonic Acid Sodium Salt
(24). The synthesis was carried out as described for 18d (Method B)
using 4-bromo-1-(4-benzyloxyphenoxy)butane (23) (250 mg, 0.75
mmol) and Na2SO3 (121 mg, 1.2 mmol) in THF/EtOH/H2O (1:2:2,
5 mL). The crude product 24 (a pale yellow solid) was used in the
next step without further purification.
4-(4-Benzyloxyphenoxy)butanesulfonyl Chloride (25). The
synthesis was carried out as described for 19a. Using 24 derived from
Method B and thionyl chloride (180 mg, 1.51 mmol) in benzene (9
mL)/DMF (0.1 mL), the title compound was obtained in 51% yield
(from 23, 136 mg) as a viscous liquid, after purification by flash
column chromatography on silica gel (20% diethyl ether in hexane). IR
1
(neat) 3033, 2945, 2868, 1610, 1512, 1371 (s), 1164 (s); H NMR
(500 MHz, CDCl3) δ 7.44 (d, J = 7.8 Hz, 2H, 2-H, 6-H, -OCH2Ph),
7.40 (t, J = 7.8 Hz, 2H, 3-H, 5-H, -OCH2Ph), 7.34 (t, J = 7.9 Hz, 1H,
4-H, -OCH2Ph), 6.93 (d, J = 8.5 Hz, 2H, -O−Ph−OBn), 6.84 (d, J =
8.5 Hz, 2H, -O−Ph−OBn), 5.04 (s, 2H, -OCH2Ph), 4.00 (t, J = 6.0
Hz, 2H, -CH2−O−Ph−OBn), 3.81 (m as t, J = 7.2 Hz, half of an
AA′XX′ system, 2H, -CH2SO2Cl), 2.29 (m as qt, J = 7.1 Hz, 2H,
-CH2CH2SO2Cl), 2.00 (qt, J = 7.3 Hz, 2H, -CH2CH2CH2SO2Cl);
mass spectrum m/z (relative intensity) 356 (M+ + 2, 3), 354 (M+, 9),
246 (9), 200 (4), 139 (4), 128 (3), 107 (23), 91 (100), 65 (9); Exact
mass calculated for C17H19ClO4S, 354.0693; found, 354,0692.
4-(4-Benzyloxyphenoxy)butanesulfonyl Fluoride (26). The
title compound was synthesized as described for 20a, using 25 (100
mg, 0.3 mmol) and NH4F (52 mg, 1.4 mmol) in dry acetone (4 mL).
Purification by flash column chromatography on silica gel (20% diethyl
ether in hexane) gave 26 as a white solid (mp 108−110 °C) in 93%
yield (88 mg). IR (neat) 3062, 2961, 2874, 1512, 1397 (s), 1235, 1194
(s); 1H NMR (500 MHz, CDCl3) δ 7.44 (d, J = 8.0 Hz, 2H, 2-H, 6-H,
-OCH2Ph), 7.40 (t, J = 8.0 Hz, 2H, 3-H, 5-H, -OCH2Ph), 7.34 (t, J =
8.0 Hz, 1H, 4-H, -OCH2Ph), 6.93 (d, J = 9.0 Hz, 2H, -O−Ph−OBn),
6.83 (d, J = 9.0 Hz, 2H, -O−Ph−OBn), 5.04 (s, 2H, -OCH2Ph), 4.00
(t, J = 6.2 Hz, 2H, -CH2−O−Ph−OBn), 3.51 (m as dt, J = 11.0 Hz, J =
4.5 Hz, 2H, -CH2SO2F), 2.20 (m as qt, J = 7.5 Hz, 2H,
-CH2CH2SO2F), 1.98 (qt, J = 7.5 Hz, 2H, -CH2CH2CH2SO2F); 13C
NMR (100 MHz, CDCl3) δ 153.75 (4-C, -O−Ph−OBn), 152.21 (1-C,
-O−Ph−OBn), 137.22 (1-C, -O−CH2−Ph), 129.27, 128.21, 127.73,
115.87, 115.23, 70.27 (-O−CH2−Ph), 66.57 (-CH2−O−Ph-), 50.82
(d, J = 16.2 Hz, -CH2SO2F), 27.48, 21.17; mass spectrum m/z (relative
intensity) 338 (M+, 15), 196 (3), 139 (6), 107 (8), 91 (100), 65 (7);
Exact mass calculated for C17H19FO4S, 338.0988; found, 338.0985.
7-(4-Benzyloxyphenyl)heptane-1-sulfonic Acid Methyl Ester
(27). A solution of 19a (50 mg, 0.13 mmol) in MeOH (5 mL) was
stirred at room temperature overnight. The solvent was evaporated
under reduced pressure, and the residue was dissolved in diethyl ether.
Radioligand Binding Assays. Rat brain CB1 and mouse CB2
assays. Compounds were tested for their affinities for the rat CB1 and
mouse CB2 receptors using membrane preparations from rat brain or
HEK 293 cells overexpressing mouse CB2 and [3H]CP-55,940 as
previously described.50,56,69
Rapid Dilution Assay. The assay was performed by following
procedures similar to those described earlier.58 Briefly, DMSO (2.5
μL; positive control of enzyme activity) and inhibitor 21d in DMSO
(2.5 μL, 2.35 μM; enzyme inhibition at a concentration 40 times
higher than its IC50 value) were added to two samples of 50 μL assay
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buffer (50 mM HEPES, 1 mM EDTA 0.1% BSA, pH 7.4) containing
0.2 μg (3.1 pmol) of purified rat ΔTM FAAH, respectively, and
incubated at room temperature for 1 h. A substrate self-hydrolysis
control sample, containing 2.5 μL of DMSO in 50 μL of assay buffer
(50 mM HEPES, 1 mM EDTA 0.1% BSA, pH 7.4) without enzyme
was incubated at room temperature for 1 h. All samples were diluted
100 times in assay buffer, and aliquots of the resulting solutions (198
μL) were added in a 96-well assay plate (costar 3650) containing 2 μL
of a 1 mM DMSO solution of the fluorogenic substrate arachidonoyl
7-amino-4-methylcoumarin amide (AAMCA). Accumulation of the
fluorescent product 7-amino-4-methylcoumarin (AMC) was measured
at 360 nm/460 nm (λexcitation/λemission) each 20 min up to 24 h using an
EnVision Multilabel Reader at 25 °C (PerkinElmer Inc., Shelton, CT).
To account for self-hydrolysis of the fluorogenic substrate, the average
readings values of control without enzyme were systematically
subtracted from samples with enzyme.
LC/MS Intact Mass Analysis of 21d-Treated rat ΔTM FAAH.
Two samples of purified rat ΔTM FAAH in 100 mM NaCl, 50 mM
Tris, pH 8.0 buffer (50 μL, 40 μM) were incubated with 2.5 μL of
DMSO and 2.5 μL of 1.68 mM 21d (final inhibitor concentration was
80 μM), respectively, at room temperature for 1 h. To evaluate
enzyme inhibition, an aliquot of each mixture was analyzed in the
fluorescent-based assay as previously described. The DMSO and
inhibitor excess were removed from the samples on a Bio-Spin 6
column equilibrated with the same buffer. Samples were then taken for
intact mass determination using a LCT-PremierXE mass spectrometer
(Waters). The instrument was calibrated with horse myoglobin
(Sigma-Aldrich) and operated under the following conditions: source
temperature, 80 °C; desolvation, 175 °C; a capillary voltage of 3200 V;
and a cone voltage of 35 V. Samples (200 pmol) were injected onto a
self-packed POROS 20 R2 protein trap column (Applied Biosystems)
and desalted with 1.0 mL of Buffer A (0.05% TFA in H2O). The
desalted protein was eluted from the trap column with a linear 15−
75% acetonitrile B (acetonitrile, 0.05% TFA) gradient over 4 min at 50
μL/min.
Molecular Modeling Methods. The sulfonyl fluorides were
covalently docked to the catalytic Ser241 of rFAAH (PDB ID:
1MT5)2 with a covalent docking module in Prime.70 This protocol
forms the specified covalent bond and exhaustively samples the
rotatable bonds of the ligand, producing a large number of potential
poses. After clustering, the enzyme-ligand complexes are minimized
and ranked by prime energy.
To validate the covalent docking protocol for FAAH, the
methodology was tested on two covalent complexes: methyl
arachidonyl fluorophosphonate (MAFP) bound to rFAAH (PDB
ID: 1MT5)2 and PF3845, a urea-based inhibitor, bound to the
humanized variant of rFAAH (PDB ID: 3LJ6).71 The ligands were
extracted from the catalytic site and redocked in a covalent binding
mode. The root mean-square deviation (RMSD) between the lowest
prime energy complexes and the crystal structures for MAFP and
PF3845 were 1.15 and 0.31 Å, respectively. These results indicate that
the covalent docking module in Prime can provide a reliable ligand
pose for FAAH, in terms of reproducing the experimental observed
binding mode.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Institute
on Drug Abuse: DA009158, DA003801, and DA007215.
ABBREVIATIONS
■
AEA, N-arachidonoylethanolamine; 2-AG, 2-arachidonoyl
glycerol; FAA, fatty acid amide; CB1, cannabinoid receptor 1;
CB2, cannabinoid receptor 2; FAAH, fatty acid amide
hydrolase; MGL, monoacylglycerol lipase; AS, amidase
signature; (−)-Δ9-THC, (−)-Δ9-tetrahydrocannabinol; PMSF,
phenylmethanesulfonyl fluoride; SAR, structure−activity rela-
tionship; DMF, dimethyl formamide; CHO, Chinese hamster
ovary; AAMCA, arachidonyl-7-amino-4-methylcoumarin
amide; ΔTM-rFAAH, transmembrane-deleted rat FAAH;
AHMMCE, arachidonoyl 7-hydroxy-6-methoxy-4-methylcou-
marin ester; HEK293, human embryonic kidney cell line;
NMR, nuclear magnetic resonance; QTOF, quadrupole time-
of-flight
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ASSOCIATED CONTENT
* Supporting Information
■
S
Elemental analysis results for compounds 11a−11f, 20a−20d,
21a−21d, 26, and 27. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*S.P.N.: Center for Drug Discovery; tel, +1-617-373-7620; fax,
+1-617-373-7493; e-mail, s.nikas@neu.edu. A.M.: Center for
Drug Discovery and Departments of Chemistry and Chemical
Biology and Pharmaceutical Sciences; tel, +1-617-373-4200;
fax, +1-617-373-7493; e-mail, a.makriyannis@neu.edu.
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