Synthesis and structure-activity relationship studies of O-biphenyl-3-yl carbamates as peripherally restricted fatty acid amide hydrolase inhibitors

Article abstract of DOI:10.1021/jm4007017

The peripherally restricted fatty acid amide hydrolase (FAAH) inhibitor URB937 (3, cyclohexylcarbamic acid 3′-carbamoyl-6-hydroxybiphenyl-3-yl ester) is extruded from the brain and spinal cord by the Abcg2 efflux transporter. Despite its inability to enter the central nervous system (CNS), 3 exerts profound antinociceptive effects in mice and rats, which result from the inhibition of FAAH in peripheral tissues and the consequent enhancement of anandamide signaling at CB₁ cannabinoid receptors localized on sensory nerve endings. In the present study, we examined the structure-activity relationships (SAR) for the biphenyl region of compound 3, focusing on the carbamoyl and hydroxyl groups in the distal and proximal phenyl rings. Our SAR studies generated a new series of peripherally restricted FAAH inhibitors and identified compound 35 (cyclohexylcarbamic acid 3′-carbamoyl-5- hydroxybiphenyl-3-yl ester) as the most potent brain-impermeant FAAH inhibitor disclosed to date.

Full text of DOI:10.1021/jm4007017

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationship Studies of O‑Biphenyl-
3-yl Carbamates as Peripherally Restricted Fatty Acid Amide
Hydrolase Inhibitors
Guillermo Moreno-Sanz,†,‡,⊥ Andrea Duranti,§,⊥ Laurin Melzig,† Claudio Fiorelli,† Gian Filippo Ruda,†
Giampiero Colombano,† Paola Mestichelli,§ Silvano Sanchini,§ Andrea Tontini,§ Marco Mor,∥
Tiziano Bandiera,† Rita Scarpelli,† Giorgio Tarzia,§ and Daniele Piomelli*,†,‡
†Drug Discovery and Development, Fondazione Istituto Italiano di Tecnologia, via Morego 30, I-16163 Genova, Italy
‡Departments of Anatomy and Neurobiology, Pharmacology and Biological Chemistry, University of California, Irvine, Irvine,
California 92697-4621, United States
§Dipartimento di Scienze Biomolecolari, University of Urbino “Carlo Bo”, Piazza del Rinascimento 6, I-61029 Urbino, Italy
∥Dipartimento Farmaceutico, University of Parma, Viale G. P. Usberti 27/A, I-43124 Parma, Italy
ABSTRACT: The peripherally restricted fatty acid amide hydrolase
(FAAH) inhibitor URB937 (3, cyclohexylcarbamic acid 3′-
carbamoyl-6-hydroxybiphenyl-3-yl ester) is extruded from the brain
and spinal cord by the Abcg2 efflux transporter. Despite its inability
to enter the central nervous system (CNS), 3 exerts profound
antinociceptive effects in mice and rats, which result from the
inhibition of FAAH in peripheral tissues and the consequent
enhancement of anandamide signaling at CB1 cannabinoid receptors
localized on sensory nerve endings. In the present study, we examined the structure−activity relationships (SAR) for the biphenyl
region of compound 3, focusing on the carbamoyl and hydroxyl groups in the distal and proximal phenyl rings. Our SAR studies
generated a new series of peripherally restricted FAAH inhibitors and identified compound 35 (cyclohexylcarbamic acid 3′-
carbamoyl-5-hydroxybiphenyl-3-yl ester) as the most potent brain-impermeant FAAH inhibitor disclosed to date.
to limit access of compounds of interest to the CNS were either
to increase the compounds’ topological polar surface area
(TPSA)11 or to exploit the recognition by drug transporters
present in the blood−brain barrier (BBB).12
INTRODUCTION
■
The therapeutic exploitation of the endocannabinoid system
with exogenous agonists is limited by the undesired side effects
caused by indiscriminate activation of cannabinoid type-1 (CB1)
receptors, particularly in the brain.1 An alternative strategy to
direct CB1 receptor targeting is to upregulate the signaling
activity of the endogenous cannabinoid ligands, arachidonoyle-
thanolamide (anandamide)2 and 2-arachidonoyl-sn-glycerol (2-
AG),3 by blocking their intracellular degradation. Anandamide is
released on demand by stimulated neurons2 and inhibitors of the
enzyme responsible for its hydrolytic cleavage, fatty acid amide
hydrolase (FAAH), have been shown to increase anandamide
levels and activate central and peripheral CB1 receptors without
causing signs of cannabinoid intoxication.4,5
Outside the central nervous system (CNS), CB1 receptors are
found in organs such as liver, kidney, and intestine, as well as in
peripheral sensory terminals and dorsal root ganglia (DRG)
neurons.6 Evidence suggests that therapeutic gain devoid of
central liability can be achieved in conditions such as pain and
metabolic syndrome by targeting these peripheral receptors.7,8
Thus, developing pharmacological agents that do not cross the
blood−brain barrier (BBB) provides a potential approach to
identify endocannabinoid-based therapies that are both safe and
effective. Indeed, synthetic efforts aimed at creating CB1 receptor
agonists and antagonists with restricted access to the CNS have
been reported.9−12 In those studies, the main strategies adopted
Selective activation of peripheral CB1 receptors by endoge-
nously produced anandamide was first achieved with compound
URB937 (3, cyclohexylcarbamic acid 3′-carbamoyl-6-hydrox-
ybiphenyl-3-yl ester, Figure 1), which blocks FAAH activity only
outside the CNS through an irreversible mechanism.13
Compound 3 inhibits visceral and inflammatory pain responses
in rodents by reducing nociceptive inputs to the spinal cord.13
Genetic and pharmacological studies have shown that this
compound is extruded from the CNS by the ATP-binding
cassette transporter Abcg2 (ABCG2 in humans).14 Furthermore,
Abcg2 limits the access of 3 not only to the CNS but also to the
fetoplacental unit of pregnant rodents.15 Recognition by Abcg2
may be exploited, therefore, as a novel strategy to develop
therapeutic agents devoid of CNS-mediated effects and, possibly,
safe to be used during pregnancy.16
We have previously shown that introduction of a hydroxyl
group in the para position of the proximal phenyl ring of
cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester (1,
URB597, Figure 1) generates the peripherally restricted
Received: May 10, 2013
© XXXX American Chemical Society
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Figure 1. Design of peripherally restricted FAAH inhibitors.
derivative 3 (Figure 1).13 Conversely, removing the carbamoyl
moiety from the distal phenyl ring yields cyclohexylcarbamic acid
6-hydroxybiphenyl-3-yl ester, a compound that readily enters the
brain (2, URB694, Figure 1).17,18 These observations suggest
that the R1 and R2 regions (Figure 1) are essential to limit the
penetration of O-biphenyl-3-yl carbamate FAAH inhibitors into
the CNS. To elucidate the substitutions in R1 and R2 that best
combine inhibitory potency and lack of brain penetration, we
progressively modified the R1 and R2 regions in the scaffold of 3
and tested the new compounds for their ability to inhibit FAAH
activity in vitro and in vivo. Dose−response exploration studies
allowed us to establish a hierarchy among the different
substituents based on their access to the brain. Furthermore,
the involvement of Abcg2 in the peripheral distribution of the
new compounds was tested using the selective Abcg2 inhibitor,
Ko-143.19 These studies allowed us to identify a series of novel
peripherally restricted FAAH inhibitors, including the highly
potent compound 35, and obtain new insights on the structural
requirements underpinning the impaired access of these agents
to the CNS.
a
Scheme 1
a
Reagents and conditions: (a′) BBr3, DCM, −78 °C, 2 h, 97%; (b′)
ArB(OH)2, CsOAc, PdCl2dppf, dioxane, 80 °C, 5−12 h, 16−62%;
(a″) ArB(OR)2 (5d,e) or ArB(OH)2 (5f,g), CsOAc, PdCl2dppf,
dioxane, 80 °C, 3−12 h, 19−77%; (b″) cyclohexene, 10% Pd/C,
EtOH, 60 °C, 2 h (7d,f,g) or BBr3, DCM, −78 °C to rt, 2 h (7e), 56−
90%.
CHEMISTRY
■
Different synthetic approaches were utilized for the preparation
of compounds 7a−g and 11a−c, bearing structural modifications
on the distal phenyl ring (Schemes 1 and 2).
Compounds 7a−c were prepared by the introduction of the
targeted structural variations at the last step of the synthesis via a
Suzuki cross-coupling reaction directly on the common
intermediate 6, obtained from 420 through an O-debenzylation
by using boron tribromide in dichloromethane (Scheme 1).
Alternatively, compounds 7d−g were prepared using a synthetic
methodology recently reported for the preparation of multigram
scale of 3.20 The intermediates 5d−g were obtained from 4
through a Suzuki cross-coupling reaction and converted into the
final compounds via a O-debenzylation reaction employing
either cyclohexene and Pd/C in dioxane or boron tribromide in
dichloromethane (Scheme 1).
1824 via a nucleophilic aromatic substitution with benzyl alcohol
in the presence of potassium tert-butoxide, followed by
hydrolysis of the acetal group under acidic condition. The
intermediate 18 was efficiently converted into the biphenyl
aldehyde 19 under ligand-free Suzuki cross-coupling condi-
tions25 by reacting with 3-carbamoylphenylboronic acid in
aqueous potassium carbonate in the presence of palladium
acetate. Compound 19 was then reduced under standard
conditions to the corresponding alcohol 20 that was O-
debenzylated and selectively converted with cyclohexyl iso-
cyanate into the final compound 21.
The synthetic procedure for the preparation of compound 29
is described in Scheme 5. Starting from 22, a Friedel−Crafts
acylation produced compound 23 which was oxidized to
carboxylic acid 24 by a two-step sequence consisting in a Claisen
condensation with diethyl oxalate followed by an oxidative
cleavage with potassium peroxymonosulfate.26 The conversion
of 24 into 25 was carried out using boron tribromide in
dichloromethane. Compound 25 was then chemoselectively
transformed into the O-benzylester 2627 that, after a Suzuki
cross-coupling reaction, gave 27. Conversion of the phenol
derivative 27 into the carboxylic acid 29 was carried out through a
carbamoylation reaction by using cyclohexyl isocyanate in
Compounds 11a−c were obtained in three steps starting from
813,21 through a Suzuki cross-coupling reaction followed by
carbamoylation and Pd/C catalyzed hydrogenative deprotection
(Scheme 2).
Compound 1513 was obtained in a three-step synthetic
procedure starting from the commercially available aldehyde 12,
which was converted into phenol derivative 13 through a Dakin
oxidation,22 followed by a Suzuki cross-coupling reaction to give
14 that was then treated with cyclohexyl isocyanate in
acetonitrile to afford the carbamate 15 (Scheme 3).
The synthetic procedure for the preparation of compound 21
is reported in Scheme 4. Starting from the commercially available
aldehyde 16, which was protected as acetal 17,23 a two-step
sequence was employed to obtain the O-benzylated bromide
B
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a
Scheme 2
a
Reagents and conditions: (a) ArB(OH)2, Na2CO3, Pd(PPh3)4, toluene/H2O, reflux, 12 h, 64−78%; (b) c-C6H11NCO, Et3N, CH3CN, reflux, 5 h,
55−61%; (c) H2 (4 atm), 10% Pd/C, EtOH/EtOAc (11b) or EtOH (11a,c), 50 °C, 4 h, 19−50%.
a
Scheme 3
a
Reagents and conditions: (a) (i) m-CPBA, DCM, 40 °C, 72 h, (ii) NaOCH3, EtOH, rt, 1 h, 60%; (b) 3-carbamoylphenylboronic acid, Na2CO3,
Pd(PPh3)4, toluene/H2O, reflux, 12 h, 81%; (d) c-C6H11NCO, Et3N, CH3CN, reflux, 2 h, 77%.
a
Scheme 4
a
Reagents and conditions: (a) ethylene glycol, p-TSA, toluene, reflux, 12h, 93%; (b) t-BuOK, BnOH, dry dioxane, 85 °C, 1 h; (c) 2 N HCl, rt, 2 h,
75% (over 2 steps); (d) ArB(OH)2, K2CO3, Pd(OAc)2, EGME/H2O (3:1), rt, 40 min, 95%; (e) NaBH4, EtOH, 0 °C to rt, 2 h, 81%; (f) 10% Pd/C,
γ-terpinene, dioxane, reflux, 1 h; (g) c-C6H11NCO, Et3N, CH3CN/EtOH, rt, 12h, 44% (over 2 steps).
a
Scheme 5
a
Reagents and conditions: (a) AcCl, ZrCl4, DCM, 0 °C to rt, 1 h, 55%; (b) diethyl oxalate, t-BuONa, THF, rt, 30 min, 77%; (c) Oxone, acetone, 0
°C, 2 h, 61%; (d) BBr3, DCM, 0 °C to rt, 12 h, 61%; (e) BnBr, KHCO3, DMF, rt, 12 h; (f) 3-carbamoylphenylboronic acid, PdCl2dppf, K2CO3,
dioxane/H2O, 90 °C, 1 h, 57%; (g) c-C6H11NCO, Et3N, CH3CN/EtOH, 45 °C, 12 h, 76%; (h) 10% Pd/C, cyclohexene, dioxane, 80 °C, 2 h, 44%.
acetonitrile/ethanol followed by Pd/C catalyzed hydrogenolysis
with cyclohexene in dioxane.
Scheme 6 reports the synthesis of compound 35 from 31 that
was converted by a nucleophilic aromatic substitution in 32.28
A
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a
a
Scheme 6
Scheme 7
a
Reagents and conditions: (a) t-BuONa, BnOH, dry DMF, 90 °C, 3 h,
a
Reagents and conditions: (a) (CF3SO2)2O, DMAP, DCM, 0 °C to rt,
15 min; (b) 3-carbamoylphenylboronic acid, Na2CO3, PdCl2, EtOH,
reflux, 12 h, 45%; (c) BBr3, DCM, 0 °C to rt, 30 min, 40%; (d) c-
C6H11NCO, Et3N, CH3CN, reflux, 2 h, 65%.
72%; (b) 3-carbamoylphenylboronic acid, K2CO3, Pd(OAc)2, EGME/
H2O (3:1), 60 °C, 20 min, 70%; (c) 10% Pd/C, cyclohexene, dioxane,
80 °C, 2 h, 100%; (d) c-C6H11NCO, CuCl, DMF, rt, 30 min, 29%.
ligand-free Suzuki cross coupling reaction was utilized29 to afford
the corresponding biphenyl derivative 33 that was quantitatively
debenzylated to obtain compound 34. Attempts to selectively
monocarbamoylate 34 in the desired carbamate 35 were
troublesome. Compound 34 was treated with cyclohexyl
isocyanate (1.1 equiv) in the presence of triethylamine or 4-
(dimethylamino)-pyridine (DMAP) in acetonitrile at room
temperature for 12 h to afford mixtures containing the
dicarbamoylated derivative 36 as the major side product. In the
specific, we observed the formation of (0.6:1.3:1.0) and
(1.3:1.0:2.3) mixtures of compounds (34: 35: 36) by using
triethylamine (1.1 equiv) and DMAP (0.1 equiv), respectively.
Eventually, the use of cyclohexyl isocyanate in N,N-dimethyl-
formamide at room temperature in the presence of copper
chloride as promoter30 yielded a (1:2:1) mixture of compounds
(34:35:36), which were separated by chromatographic purifica-
tion. Alternatively, attempts to carry out selective monopro-
tection of compound 34 with tert-butyldimethylsilyl chloride
(TBSCl) or triisopropylsilyl chloride (TIPSCl) also failed.
Compound 41 was obtained in a four-step synthetic procedure
starting from the commercially available phenol 37, which was
converted in the corresponding triflate 3831 and directly used in
the Suzuki cross coupling reaction to afford 39. Compound 39
was selectively monodemethylated to 40 using boron tribromide
in dichloromethane and then converted to the carbamate 41
under standard conditions (Scheme 7).
carbamates inhibit FAAH through a covalent, irreversible
mechanism,32 the degree of FAAH inhibition measured ex vivo
provides a useful estimate of the amount of compound reaching
that tissue.14,15
Analogues of 3 Bearing Different Substituents on the
Meta- Position of the Distal Phenyl Ring. The results of
explorative chemistry targeting the meta position of the distal
phenyl ring (R1 region) of 3 are summarized in Table 1.
Substituting the carbamoyl group with a methyl (11a), 1-
hydroxyethyl (11b), or hydroxymethyl (11c) group yielded
compounds that retained FAAH inhibitory activity in vitro but
readily accessed the CNS in vivo: 11a−c produced similar
inhibitory effects on FAAH activity in brain and liver. Likewise,
the methylketone derivative 7a displayed good brain penetration
in vivo, along with increased in vitro potency on FAAH (Table
1). These results confirm that the carbamoyl functionality in the
R1 region of 3 plays a key role in the peripheral distribution of this
compound. This idea was further tested by preparing new
compounds in which such functionality was progressively
alkylated to a secondary (7d) or tertiary (7e) amide. When
administered in vivo (1 mg/kg, ip), both 7d and 7e displayed
impaired access to the brain but maintained the ability to block
FAAH activity in the liver (Table 1). A dose−exploration study
revealed that 7d and 7e gain access to the brain at higher dosages,
similarly to what previously found for 3,13 possibly by saturating
the mechanism that mediates their extrusion from the brain
(Figure 2a). Interestingly, the ED50 values of these compounds
for brain FAAH inhibition ex vivo (3, 40 mg/kg; 7d, 15 mg/kg;
7e, 3.5 mg/kg) progressively decreased as the number of methyl
substituents linked to the carbamoyl moiety was increased
(Figure 2a). The reverse amide 7c displayed significantly higher
brain penetration relative to 3, 7d, and 7e at all doses tested
(Figure 2a). The involvement of the Abcg2 transporter in
restricting the access of 7d and 7e to the brain was tested by
preadministration of the selective inhibitor Ko-143 (15 mg/kg,
i.p.). Pharmacological blockade of Abcg2 allowed subeffective
doses of 3 and 7d to inhibit FAAH activity in the brain but failed
to do so with 7e (Figure 2c). We interpret these findings to
indicate that a primary (3) or secondary (7d) carbamoyl moiety
is a key determinant for the interaction of O-biphenyl-3-yl
carbamate FAAH inhibitors with Abcg2 in vivo. By contrast, the
RESULTS
■
Previous studies have shown that compound 3 inhibits FAAH
activity in liver and other peripheral tissues of mice with a median
effective dose (ED50) of 0.2 mg/kg (intraperitoneal, ip), which is
200 times lower than the ED50 for FAAH inhibition in the brain
(40 mg/kg).13 The present study evaluated the effects that
structural modifications at the meta position of the distal phenyl
ring (R1 region) and the para or meta positions of the proximal
phenyl ring (R2 region) exert on the inhibitory potency and
systemic distribution of 3. Median concentrations to inhibit
FAAH activity (IC50) were determined in vitro using rat brain
homogenates. FAAH inhibition was also measured ex vivo in
liver and brain tissue of mice 1 h after systemic administration of
test compounds (1 mg/kg, ip). Because O-biphenyl-3-yl
D
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Table 1. Inhibitory Potency (IC50) and Systemic Distribution of 3′-Substituted O-Biphenyl-3-yl Carbamates
a
b
b
d
R
in vitro IC50 (nM)
FAAH inhibition in liver (%)
FAAH inhibition in brain (%)
PSA (Å2)
3
CONH2
2.0
0.3
32
91.7 0.7
88.1 0.8
−3.0 8.0
89.2 0.6
83
62
77
72
73
65
99
78
49
65
66
7a
COCH3
c
c
7b
7c
COOH
30.0 2.6 66.4 4.1
2.8 1.3 −6.8 2.7
NHCOCH3
CONHCH3
CON(CH3)2
SO2NH2
15
76.0 1.6
87.2 2.4
84.2 2.5
81.1 1.3
73.9 4.6
72.1 4.9
80.9 0.9
86.6 0.6
31.6 1.5
0.9 3.7
22.6 7.8
7.0 1.7
6.6 2.3
85.4 0.5
89.6 0.6
88.6 0.7
7d
7e
6.0
1.5
2.7
6.3
2.5
2.0
1.6
7f
7g
11a
11b
11c
SO2CH3
CH3
CH(OH)CH3
CH2OH
a
b
IC50 measured in membrane preparations of Wistar rat brain. FAAH inhibition measured ex vivo 1 h after injection in Swiss Webster mice (1 mg/
c
d
kg, intraperitoneal, n = 3). FAAH inhibition measured ex vivo 1 h after injection in Swiss Webster mice (3 mg/kg, intraperitoneal, n = 3). PSA
values were calculated using ICM version 3.7 (Molsoft LLC, San Diego, CA).
asymmetric tissue distribution of 7e at low doses appears to be
independent of Abcg2.
restricted when administered at the dosage of 1 mg/kg (Table 2).
Compound 21 behaved similarly to 3 and 7d, in that it inhibited
brain FAAH when given at high doses (ED50 = 15 mg/kg, Figure
3a) and gained access to the brain when coadministered with the
Abcg2 inhibitor Ko-143 (Figure 3b). By contrast, 29 failed to
enter the brain even at the highest dose tested (75 mg/kg, Figure
3a). A similar behavior was displayed by the sulfate derivative 30
(Table 2, Figure 3a). Neither 29 nor 30 entered the brain despite
pretreatment with Ko-143 (Figure 3b), indicating that Abcg2 is
not involved in restricting their access to the CNS. It is likely that
the presence of absolute charges on the surface of 29 and 30
hinders their diffusion across the BBB.
Lastly, we asked whether relocation of the hydroxyl group of 3
from the para- to the meta- position on the proximal phenyl ring
affects FAAH activity and brain penetration. The m-hydroxy
derivative 35 retained both a strong inhibitory potency toward
FAAH in vitro (IC50 = 0.5 mg/kg) and a marked peripheral
distribution (Table 2). A dose exploration study revealed that 35
had a markedly restricted access to the brain (ED50 = 75 mg/kg,
Figure 3a). For comparison, compound 3 inhibited brain FAAH
with an ED50 of 40 mg/kg. The inability of 35 to access the brain
appears to require Abcg2 because pharmacological blockade of
this transporter allowed a high dose of 35 (40 mg/kg) to inhibit
central FAAH activity (Figure 3b). Similarly to what found for
15, the asymmetric tissue distribution of 35 was lost in the
corresponding m-methoxy derivative 41, which inhibited FAAH
in liver and brain to a similar extent after systemic administration
(1 mg/kg, Table 2).
To further test the relevance of the carbamoyl group in the
distal ring of 3 and probe its possible role in the interaction with
Abcg2 acting as a H-bond donor, we synthesized and tested the
carboxylic acid derivative 7b (Table 1). The compound displayed
impaired access to the brain but failed to fully inhibit liver FAAH
activity at 1 mg/kg (30% inhibition, Table 1). By increasing the
dosage to 3 mg/kg, we were able to improve the blockade of liver
FAAH activity (66% inhibition, Table 1), still without affecting
brain FAAH activity. These findings suggest that 7b has
restricted access to the CNS. However, we did not further
pursue the characterization of this compound due to its relatively
low potency both in vitro and in vivo. On the other hand, the
corresponding bioisosteric sulphonamide (7f) and methylsul-
fone (7g) derivatives were potent, single-digit nanomolar FAAH
inhibitors in vitro and were effective at inhibiting liver FAAH
activity in vivo (1 mg/kg, ip) while having significantly reduced
brain penetration (Table 1). In agreement with the results
obtained with the primary and secondary carbamoyl derivatives 3
and 7d, we found that 7f displayed a more strongly restricted
access to the CNS compared to 7g, with brain ED50 values of 75
and 3 mg/kg, respectively (Figure 2b). However, pharmaco-
logical blockade of Abcg2 with Ko-143 did not increase the
access of a subeffective dose of 7f (40 mg/kg) or 7g (1 mg/kg) to
the brain (Figure 2c), indicating that these compounds are
excluded from the CNS by a mechanism that is independent of
Abcg2.
Analogues of 3 with Different Substituents on the
Meta- or Para- Position of the Proximal Phenyl Ring. Next,
we turned our attention to the SAR exploration of the R2 region
of compound 3. The results are summarized in Table 2. We
hypothesized that the hydroxyl group in the para position of the
proximal phenyl ring, which differentiates 3 from the globally
active inhibitor 1 (Figure 1), might be a key element in the
peripheral distribution of 3. Supporting this idea, we previously
showed that the p-methoxy derivative 15 readily accesses the
brain following systemic administration.13 In agreement with this
finding, hydroxyl-containing substituents such as the hydrox-
ymethyl 21 and the carboxylic acid 29 were also peripherally
CONCLUSIONS
■
Previous studies have identified compound 3 as a potent and
selective FAAH inhibitor, whose passage through the blood−
tissue barriers of the CNS and fetoplacental unit is restricted at by
the Abcg2 transporter.14,15 Despite this restricted systemic
distribution, 3 exhibits marked antihyperalgesic and antiallodynic
properties in mice and rats,13 suggesting that peripherally
restricted FAAH inhibitors might represent a novel class of
clinically relevant analgesics.21 The primary objective of the
present study was to identify new brain-impermeant FAAH
inhibitors, which could be used both as tools to understand the
E
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Among them, the m-hydroxyl derivative 35 showed the greatest
inhibitory potency in vitro (IC50 = 0.5 nM) and the lowest brain
penetration in vivo (ED50 = 75 mg/kg). This compound thus
provides a valuable addition to our still limited armamentarium
of peripherally restricted FAAH inhibitors.
The present study also offered several insights on the
molecular mechanism responsible for the peripheral distribution
of O-biphenyl-3-yl carbamate FAAH inhibitors. By showing that
progressive alkylation of the carboxamide group in the distal
phenyl ring of 3 (R1 region) leads to a higher degree of brain
penetration, our results confirm a key role for this moiety in
determining the peripheral distribution of 3. Contrary to what
was found for the primary and secondary amides (3 and 7d), the
restricted access to the brain of the tertiary amide 7e was not
mediated by Abcg2. A plausible interpretation of this finding is
that the carboxamide hydrogen bond donors may be necessary
for 3 and 7d to interact with Abcg2, possibly through H-bonding.
We were able to confirm the key role played by the hydroxyl
substituent of the proximal ring of 3 in the peripheral distribution
of O-biphenyl-3-yl carbamate FAAH inhibitors. Some flexibility
is allowed around this motif, however, because both the p- and m-
hydroxy (3 and 35), as well as the p-hydroxymethyl derivative 21
behaved as substrates for Abcg2 in vivo, gaining access to the
brain after pharmacological blockade of the transporter by Ko-
143. By contrast, compounds 29 and 30 did not enter the brain,
even at the highest dosage tested, and their distribution was not
influenced by Ko-143 treatment. The case of the sulfate
derivative 30 is particularly significant. This compound was
initially synthesized to test the putative role of phenolsulfo-
transferases (PSTs) in the peripheralization of 3. Sulfate
conjugation is known to increase the affinity of Abcg2 for its
substrates33 and PSTs to colocalize with the transporter in brain,
intestinal epithelium, and other tissues.34 However, the transfer
of the sulfate group is expected to occur in the cytosol of
epithelial cells, where PSTs and the substrate-binding site of
Abcg2 are located, once the compound has diffused through the
apical side of the membrane. The present findings suggest that
the higher PSA of these two compounds might preclude their
diffusion through cellular membranes and therefore their access
to the CNS.
Figure 2. Inhibition of brain FAAH activity by analogues of 3 bearing
different substituents on the meta- position of the distal phenyl ring. (a)
Dose-dependent effects of secondary (7c), tertiary (7d), or reverse (7e)
amide derivatives of compound 3 in Swiss Webster mice; doses were
0.3−40 mg/kg (subcutaneous); FAAH activity was measured ex vivo 1 h
after injection. (b) Dose-dependent effects of sulfonamide (7f) and
methylsulfone (7g) derivatives of compound 3 in Swiss Webster mice;
doses were 0.3−75 mg/kg (sc). (c) Effects of pharmacological blockade
of the Abcg2 transporter (Ko-143, 15 mg/kg, ip, closed bars) on brain
inhibition of FAAH activity by a subeffective dose (selected from the
dose−response study: 3 (25); 7d (10); 7e (1); 7f (40); 7g (1) in mg/kg,
sc, open bars) of analogues of compound 3 bearing different
functionalities on the meta- position of the distal phenyl ring. Results
are expressed as mean SEM (n = 3−4). *** P < 0.001 vs non-Ko-143
treated group.
Even though further experiments are required to fully
characterize the mechanisms that preclude O-biphenyl-3-yl
carbamates from entering the brain, our results may be relevant
to the design of new pharmacological agents with restricted
access to the CNS. In the case of the Abcg2/ABCG2 transporter,
the structure of which has not been yet resolved, the information
gleamed from the present SAR studies may be useful to
understand the interaction between the pharmacophoric sites of
35 and amino acid residues of the transporter involved in
substrate recognition.
EXPERIMENTAL SECTION
role of anandamide in peripheral tissues and as potential
candidates for preclinical development. The identification of
brain-impermeant drugs is often based on the use of cellular
efflux transport assays. However, these in vitro systems do not
realistically capture the complexity of the BBB. For this reason,
we opted for testing the newly synthesized compounds in vivo,
using inhibition of FAAH activity in brain and liver tissues as a
measure of central and peripheral exposure to the drug. The
contribution of Abcg2 was examined using Ko-143, a selective
inhibitor of this transporter. Our SAR exploration of two
pharmacophoric regions in the scaffold of 3 allowed us to identify
several novel FAAH inhibitors with restricted access to the brain.
■
Animals. Adult male Swiss-Webster mice (25−30 g) were kept in a
temperature-controlled environment with a 12 h light/12 h dark cycle
receiving standard chow and water ad libitum. All procedures met the
National Institutes of Health guidelines for the care and use of
laboratory animals and were approved by the Institutional Animal Care
and Use Committee of the University of California, Irvine.
Drug Administration. FAAH inhibitors were dissolved in warm
saline/PEG400/Tween80 (18:1:1) under sonication and were admin-
istered by ip or subcutaneous injection between the shoulder blades. Ko-
143 (Tocris, Ellisville, MO) was dissolved in the same vehicle containing
30% DMSO (Sigma, St. Louis, MO) and administered by ip injection 20
min prior to FAAH inhibitors.
F
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Table 2. Inhibitory Potency (IC50) and Systemic Distribution of 5-(or 6-)Substituted 3′-Carbamoyl-O-biphenyl-3-yl Carbamates
a
b
b
d
R1
R2
in vitro IC50 (nM)
FAAH inhibition in liver (%)
FAAH inhibition in brain (%)
PSA (Å2)
3
OH
H
H
H
H
H
2.0
0.5
91.7 0.7
94.6 0.7
91.5 1.1
86.3 1.3
84.0 1.2
89.5 1.1
85.6 2.4
−3.0 8.0
86.4 2.1
10.5 1.5
−2.1 0.5
−11.7 2.6
−4.2 2.5
82.6 0.4
83
74
15
21
29
30
35
41
OCH3
CH2OH
COOH
OSO3NH4
H
1.2
83
2100
34
94
c
117
84
OH
0.5
H
OCH3
2.0
74
a
b
IC50 measured in membrane preparations of Wistar rat brain. FAAH inhibition measured ex vivo 1 h after injection in Swiss Webster mice (1 mg/
c
kg, intraperitoneal, n = 3). 30 was obtained from 3 upon treatment with SO3−DMF complex in dry DCM (see Experimental Section for further
details). PSA values were calculated using ICM version 3.7 (Molsoft LLC, San Diego, CA).
d
protein concentration using a bicinchoninic acid (BCA) assay kit
(Pierce, Rockford, IL).
Ex Vivo FAAH Activity Assay. FAAH activity was measured at 37
°C for 30 min in 0.5 mL of Tris buffer (50 mM, pH 7.5) containing fatty
acid-free bovine serum albumin (BSA) (0.05%, w/v), protein from
tissue homogenates (50 μg from rat brain, 10 μg from liver),
nonradioactive anandamide (10 μM), and anandamide-
[ethanolamine-3H] (10000 cpm, specific activity 60 Ci/mmol, ARC,
St. Louis, MO) as substrate. Reactions were stopped with chloroform/
methanol (1:1, 1 mL), and radioactivity was measured in the aqueous
layer by liquid scintillation counting. For in vitro IC50 determination,
homogenates (50 μg from rat brain) were preincubated with inhibitors
for 20 min at 37 °C prior to substrate addition.
Chemicals, Materials, and Methods. Solvents and reagents were
obtained from commercial suppliers and were used without further
purification. NMR experiments were run on a Bruker AC 200
spectrometer (200.07 MHz for 1H, and 50.31 MHz for 13C) and on a
Bruker Avance III 400 system (400.13 MHz for 1H, and 100.62 MHz for
13C), equipped with a BBI probe and Z-gradients. Spectra were acquired
at 300 K, using deuterated dimethylsulfoxide (DMSO-d6) or deuterated
chloroform (chloroform-d) as solvents. Chemical shifts (d) for 1H and
13C spectra are reported in parts per million (ppm) using the residual
nondeuterated solvent resonance as the internal standard (for
chloroform-d: 7.26 ppm, 1H and 77.16 ppm, 13C; For DMSO-d6: 2.50
ppm, 1H; 39.52 ppm, 13C). Data are reported as follows: chemical shift
(sorted in descending order), multiplicity (indicated as: s, singlet; d,
doublet; t, triplet; q, quartet; p, pentet; m, multiplet and combinations
thereof), coupling constants (J) in Hertz (Hz) and integration. UPLC/
MS analyses were run on a Waters ACQUITY UPLC/MS system
consisting of a single quadropole detector (SQD) mass spectrometer
(MS) equipped with an electrospray ionization (ESI) interface and a
photodiode array (PDA) detector. PDA range was 210−400 nm. ESI in
positive and negative mode was applied. Mobile phases: (A) 10 mM
NH4OAc in H2O, pH 5; (B) 10 mM NH4OAc in CH3CN/H2O (95:5)
pH 5. Analyses were performed with either methods A, B, or C. Method
A: Gradient 5− 95% B over 3 min; flow rate 0.5 mL/min; temperature
40 °C. Pre column: Vanguard BEH C18 (1.7 μm 2.1 mm × 5 mm).
Figure 3. Inhibition of brain FAAH activity by analogues of compound 3
bearing different substituents on the meta- or para- position of the
proximal phenyl ring. (a) Dose-dependent inhibition of brain FAAH
activity by p-hydroxymethyl (21), p-carboxyl (29), p-sulfate (30), and
m-hydroxy (35) derivatives of compound 3 in Swiss Webster mice;
doses were 0.3−75 mg/kg (sc). (b) Effects of pharmacological blockade
of the Abcg2 transporter (Ko-143, 15 mg/kg, ip, closed bars) on brain
inhibition of FAAH activity caused by a subeffective dose (selected from
the dose−response study: 3 (25); 21 (10); 29 (40); 30 (75); 35 (40) in
mg/kg, sc, open bars) of analogues of compound 3 bearing different
functionalities on the meta- position of the distal phenyl ring. Results are
expressed as mean SEM (n = 3−4). *** P < 0.001, ** P < 0.01 vs non-
Ko-143 treated group.
Column: BEH C18 (1.7 μm 2.1 mm × 50 mm). Method B: Gradient 0−
50% B over 3 min; flow rate 0.5 mL/min; temperature 40 °C. Pre
column: VanGuard HSS T3 C18 (1.7 μm 2.1 mm × 5 mm). Column
HSS T3 (1.8 μm 2.1 mm × 50 mm). Method C: Gradient: 50−100% B
over 3 min, flow rate 0.5 mL/min; temperature 40 °C. Pre column:
Vanguard BEH C18 (1.7 μm 2.1 mm × 5 mm). Column: BEH C18 (1.7
μm 2.1 mm × 50 mm). Flash column chromatography was performed
automatically on Teledyne ISCO apparatus (CombiFlash Rf) with
prepacked silica gel columns of different sizes (Redisep) or manually on
silica gel (Kieselgel 60, 0.040−0.063 mm, Merck). TLC analyses were
Tissue Processing. Mice were slightly anesthetized with isofluorane
and killed by decapitation 1 h after drug injections. Brain and liver were
immediately removed and frozen in liquid N2. Samples were weighed
and homogenized in 10 volumes of ice-cold Tris-HCl (50 mM, 5−9 vol,
pH 7.5) containing 0.32 M sucrose. Homogenates were centrifuged at
1000g for 10 min at 4 °C, and supernatants were collected and tested for
G
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performed on precoated silica gel on aluminum sheets (Kieselgel 60
F254, Merck). Purifications by preparative HPLC/MS were run on a
Waters Autopurification system consisting of a 3100 single quadropole
detector (SQD) mass spectrometer (MS) equipped with an electrospray
ionization (ESI) interface and a 2998 photodiode array (PDA) detector.
HPLC system included a 2747 sample manager, 2545 binary gradient
nodule, System Fluidic Organizer, and 515 HPLC pump. PDA range
was 210−400 nm. Purifications were performed on a XBridge Prep C18
OBD column (100 mm × 19 mmID, particle size 5 μm) with a XBridge
Prep C18 (10 mm × 19 mmID, particle size 5 μm) guard cartridge.
Mobile phase was 10 mM NH4OAc in H2O at pH 5 adjusted with AcOH
(A) and 10 mM NH4OAc in CH3CN−H2O (95:5) at pH 5 (B). ESI in
positive and negative mode was used. All final compounds displayed
≥95% purity as determined by UPLC analysis.
General Procedure for the Synthesis of Carbamates 5d−g and
7a−c (Procedure A). A mixture of compound 4 (or 6) (1.0 equiv), the
appropriate aryl boronic acid (or aryl boronic ester) (1.5 equiv), and
CsOAc (2.0 equiv) in dioxane (0.1 M) was degassed with a stream of N2
for 30 min. PdCl2dppf (0.05 equiv) was added, and the reaction mixture
was heated at 80 °C until UPLC-MS analysis revealed completion of the
reaction. The reaction mixture was cooled down to room temperature,
and a saturated aqueous NH4Cl solution was added (3 mL). The
aqueous phase was separated and extracted with EtOAc (2 × 15 mL).
The combined organic phases were washed with brine and dried
(Na2SO4). After evaporation of the solvent, the residue was purified by
flash chromatography (SiO2) eluting with a gradient of EtOAc/
cyclohexane or MeOH/DCM.
General Procedure for the Synthesis of Carbamates 7d−g
(Procedure B). Compound 5d (5e, 5f, or 5g) (1.0 equiv) was heated
in a 1:5 mixture of cyclohexene/EtOH (0.2 M) at 60 °C in the presence
of 10% Pd/C (catalyst loading: 2.5% w/w) until UPLC-MS analysis
revealed completion of the reaction. The reaction mixture was filtered
through a pad of Celite and concentrated in vacuo. The residue was
purified by flash chromatography (SiO2) eluting with a gradient of
cyclohexane/EtOAc or MeOH/DCM.
General Procedure for the Synthesis of Phenols 9a,b and 14
(Procedure C). A mixture of 8 (or 13) (1.0 equiv), the appropriate
boronic acid (1.2 equiv), and Na2CO3 (5 equiv, 10% aqueous solution)
in toluene (0.2 M) was degassed with a stream of N2 for 30 min.
Pd(PPh3)4 (0.05 equiv) was added, and the reaction mixture was stirred
at reflux for 12 h, cooled down to room temperature, and filtered
through a pad of Celite. 2N HCl (5 mL) was added, and the mixture was
extracted with EtOAc. The combined organic layers were dried
(Na2SO4). After evaporation of the solvent, the residue was purified
by flash chromatography (SiO2) eluting with cyclohexane/EtOAc or
MeOH/DCM.
General Procedure for the Synthesis of Carbamates 10a,b and 15
(Procedure D). To a solution of 9a (or 9b, or 14) (1.0 equiv) in CH3CN
(0.4 M), c-hexyl-isocyanate (1.1 equiv) and Et3N (1.1 equiv) were
added. The mixture was stirred for 5 h at reflux, cooled down to room
temperature, and concentrated in vacuo. The residue was purified by
flash chromatography (SiO2) eluting with cyclohexane/EtOAc.
General Procedure for the Synthesis of Carbamates 11a−c
(Procedure E). Compound 10a (or 10b) (1.0 equiv) was heated in
EtOAc/EtOH (1:1, 0.1 M) (for 10a) or EtOH (0.1 M) (for 10b) at 50
°C under H2 atmosphere (4 atm) in the presence of 10% Pd/C (catalyst
loading: 10% w/w) for 4 h. The mixture was then cooled down to room
temperature and filtered through a pad of Celite. After evaporation of
the solvent, the residue was purified by flash chromatography (SiO2)
eluting with cyclohexane//EtOAc.
Cyclohexylcarbamic Acid 3′-Acetamido-6-benzyloxybiphenyl-3-yl
Ester (5d). The title compound 5d was prepared according to general
procedure A using compound 4 (0.202 g, 0.50 mmol), PdCl2dppf (18.3
mg, 0.025 mmol), CsOAc (192 mg, 1.00 mmol), and N-methyl-3-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (195 mg, 0.75
mmol); reaction time, 2 h. The residue was purified by flash
chromatography (0−50% EtOAc in cyclohexane) to afford 5d as a
white solid; 43 mg, 19%. 1H NMR (400 MHz, chloroform-d) δ 7.85 (s,
1H), 7.77−7.72 (m, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.41 (t, J = 7.7 Hz,
1H), 7.34−7.26 (m, 5H), 7.12 (d, J = 2.7 Hz, 1H), 7.05 (dd, J = 2.7, 8.8
Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 6.19 (d, J = 4.8 Hz, 1H), 5.04−4.99
(m, 1H), 4.99 (s, 2H), 3.59−3.51 (m, 1H), 2.90 (d, J = 4.8 Hz, 3H),
2.03−1.96 (m, 2H), 1.77−1.69 (m, 2H), 1.65−1.58 (m, 1H), 1.41−1.30
(m, 2H), 1.28−1.14 (m, 3H). MS (ES) C28H30N2O4 requires m/z 458,
found 459 [M + H]+.
Cyclohexylcarbamic Acid 6-Benzyloxy-3′-dimethylcarbamoylbi-
phenyl-3-yl Ester (5e). The title compound 5e was prepared according
to general procedure A using compound 4 (0.404 g, 1.0 mmol),
PdCl2dppf (36.6 mg, 0.05 mmol), CsOAc (0.384 mg, 2.0 mmol), and
N,N-dimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
benzamide (0.413 g, 1.5 mmol); reaction time, 6 h. The residue was
purified by flash chromatography (0−50% EtOAc in cyclohexane) to
1
afford 5e as a colorless solid; 229 mg, 48%. H NMR (400 MHz,
chloroform-d) δ 7.65−7.55 (m, 2H), 7.47−7.35 (m, 2H), 7.35−7.27 (m,
5H), 7.17−7.10 (m, 1H), 7.07 (d, J = 8.81 Hz, 1H), 7.00 (d, J = 8.82 Hz,
1H), 5.03 (s, 2H), 4.94−4.85 (m, 1H), 3.64−3.51 (m, 1H), 2.95 (s, 6H),
2.09−1.95 (m, 2H), 1.80−1.67 (m, 2H), 1.67−1.56 (m, 1H), 1.46−1.30
(m, 2H), 1.30−1.12 (m, 3H). MS (ES) C29H32N2O4 requires m/z 472,
found 473 [M + H]+.
Cyclohexylcarbamic Acid 6-Benzyloxy-3′-sulfamoylbiphenyl-3-yl
Ester (5f). The title compound 5f was prepared according to general
procedure A using compound 4 (162 mg, 0.4 mmol), PdCl2dppf (14.6
mg, 0.02 mmol), CsOAc (154 mg, 0.80 mmol), and (3-
sulfamoylphenyl)boronic acid (201 mg, 0.60 mmol); reaction time, 8
h. The residue was purified by flash chromatography (0−30% EtOAc in
cyclohexane) to afford 5f as a white solid; 105 mg, 55%. 1H NMR (400
MHz, chloroform-d) δ 8.17 (s, 1H), 7.93 (d, J = 7.4 Hz, 1H), 7.83 (d, J =
7.9 Hz, 1H), 7.75 (dd, J = 7.4, 7.9 Hz, 1H), 7.37−7.28 (m, 5H), 7.16 (d, J
= 2.7 Hz, 1H), 7.11 (dd, J = 2.7, 8.8 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H),
5.04 (s, 2H), 4.93 (d, J = 7.7 Hz, 1H), 4.61 (s, 2H), 3.61−3.52 (m, 1H),
2.05−1.98 (m, 2H), 1.78−1.70 (m, 2H), 1.66−1.60 (m, 1H), 1.41−1.31
(m, 2H), 1.31−1.15 (m, 3H). MS (ESI): C26H28N2O5S requires m/z
480, found 481 [M + H]+.
Cyclohexylcarbamic Acid 6-Benzyloxy-3′-methylsulfonylbiphen-
yl-3-yl Ester (5g). The title compound 5g was prepared according to
general procedure A using compound 4 (162 mg, 0.4 mmol), PdCl2dppf
(14.6 mg, 0.02 mmol), CsOAc (154 mg, 0.80 mmol), and (3-
methylsulfonylphenyl)boronic acid (200 mg, 0.60 mmol); reaction
time, 4 h. The residue was purified by flash chromatography (0−30%
EtOAc in cyclohexane) to afford 5g as a colorless solid; 148 mg, 77%. 1H
NMR (400 MHz, chloroform-d) δ 8.19 (s, 1H), 7.86 (d, J = 7.9 Hz, 1H),
7.82 (d, J = 7.7 Hz, 1H), 7.56 (dd, J = 7.7, 7.9 Hz, 1H), 7.35−7.27 (m,
5H), 7.16 (d, J = 2.7 Hz, 1H), 7.12 (dd, J = 2.7, 8.8 Hz, 1H), 7.04 (d, J =
8.8 Hz, 1H), 5.05 (s, 2H), 4.95 (d, J = 7.86 Hz, 1H), 3.61−3.52 (m, 1H),
2.89 (s, 3H), 2.06−1.98 (m, 2H), 1.78−1.70 (m, 2H), 1.66−1.60 (m,
1H), 1.44−1.32 (m, 2H), 1.23 (m, 3H). MS (ESI) C27H29NO5S requires
m/z 479, found 480 [M + H]+.
Cyclohexylcarbamic Acid 3-Bromo-4-hydroxyphenyl Ester (6). To
a solution of 4 (4.04 g, 10.0 mmol) in dry DCM (50 mL) at −78 °C,
BBr3 (20.0 mL, 1.0 M solution in DCM) was slowly added under Ar
atmosphere and the reaction mixture stirred at −78 °C for 2 h and then
quenched with saturated aqueous NH4Cl solution. The aqueous layer
was extracted with DCM (3 × 50 mL), and the combined organic phases
were washed with brine and dried (Na2SO4). After evaporation of the
solvent, the residue was purified by flash chromatography (0−20%
EtOAc in cyclohexane) to afford 6 as a white solid; 3.06 g, 97%. 1H NMR
(400 MHz, chloroform-d) δ 7.27 (d, J = 2.5 Hz, 1H), 6.97 (dd, J = 2.5,
8.8 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 5.59 (s, 1H), 4.89 (d, J = 6.9 Hz,
1H), 3.59−3.50 (m, 1H), 2.04−1.96 (m, 2H), 1.78−1.70 (m, 2H),
1.66−1.59 (m, 1H), 1.43−1.31 (m, 2H), 1.27−1.14 (m, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 154.1, 151.8, 143.9, 126.4, 122.5, 116.4, 108.8,
50.2, 33.0, 25.6, 25.0. MS (ESI) C13H16BrNO3 requires m/z 313, 315,
found 314, 316 [M + H]+.
Cyclohexylcarbamic Acid 3′-Acetyl-6-hydroxybiphenyl-3-yl Ester
(7a). The title compound 7a was prepared according to general
procedure A using compound 6 (0.157 g, 0.5 mmol), PdCl2dppf (18.3
mg, 0.025 mmol), CsOAc (192 mg, 1.00 mmol), and 3-methox-
yphenylboronic acid (114 mg, 0.75 mmol); reaction time, 5 h. The crude
was purified by flash chromatography (0−40% EtOAc in cyclohexane)
1
to afford 7a as a colorless solid; 109 mg, 62%. H NMR (400 MHz,
H
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1
DMSO-d6) δ 9.63 (s, 1H), 8.11 (s, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.82 (d,
J = 7.8 Hz, 1H), 7.62−7.52 (m, 2H), 7.04 (s, 1H), 6.96−6.91 (m, 2H),
3.35−3.28 (m, 1H), 2.62 (s, 3H), 1.86−1.79 (m, 2H), 1.74−1.66 (m,
2H), 1.60−1.52 (m, 1H), 1.33−1.19 (m, 4H), 1.15−1.07 (m, 1H). 13C
NMR (101 MHz, DMSO-d6) δ 198.4, 154.4, 151.8, 144.1, 138.6, 137.2,
134.2, 129.1, 128.9, 127.4, 127.1, 123.6, 122.6, 116.8, 50.2, 33.0, 27.3,
25.6, 25.0. MS (ES) C21H23NO4 requires m/z 353, found 354 [M + H]+.
Cyclohexylcarbamic Acid 3′-Carboxy-6-hydroxybiphenyl-3-yl
Ester (7b). The title compound 7b was prepared according to general
procedure A using compound 6 (0.157 g, 0.5 mmol), PdCl2dppf (18.3
mg, 0.025 mmol), CsOAc (192 mg, 1.00 mmol), and 3-phenylboronic
acid (124 mg, 0.75 mmol); reaction time, 12 h. The crude was purified
by flash chromatography (0−10% MeOH in DCM) to afford 7b as a
yellow solid; 28 mg, 16%. 1H NMR (400 MHz, DMSO-d6) δ 12.90 (s,
1H), 9.62 (s, 1H), 8.12 (s, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 7.8
Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 6.99 (s, 1H),
6.92 (s, 2H), 3.34−3.30 (m, 1H), 1.83−1.79 (m, 2H), 1.72−1.67 (m,
2H), 1.57−1.53 (m, 1H), 1.30−1.18 (m, 4H), 1.16−1.05 (m, 1H). MS
(ESI) C20H21NO5 requires m/z 355, found 356 [M + H]+.
Cyclohexylcarbamic Acid 3′-Acetamido-6-hydroxybiphenyl-3-yl
Ester (7c). The title compound 7c was prepared according to general
procedure A using compound 6 (157 mg, 0.50 mmol), PdCl2dppf (18.3
mg, 0.025 mmol), CsOAc (192 mg, 1.00 mmol), and (3-
acetamidophenyl)boronic acid (179 mg, 0.75 mmol); reaction time, 6
h. The crude was purified by flash chromatography (0−50% EtOAc in
cyclohexane) to afford 7c as an off-white solid; 110 mg, 60%. 1H NMR
(400 MHz, DMSO-d6) δ 9.94 (s, 1H), 9.47 (s, 1H), 7.71 (s, 1H), 7.57
(d, J = 7.9 Hz, 2H), 7.30 (t, J = 7.9 Hz, 1H), 7.20 (d, J = 7.8 Hz, 1H), 6.89
(s, 2H), 3.29 (s, 1H), 2.04 (s, 3H), 1.81 (d, J = 9.5 Hz, 2H), 1.75−1.65
(m, 3H), 1.55 (d, J = 12.5 Hz, 1H), 1.33−1.17 (m, 4H), 1.18−1.04 (m,
1H). 13C NMR (101 MHz, DMSO-d6) δ 168.7, 154.4, 151.7, 144.0,
139.5, 138.7, 128.7, 128.2, 124.3, 123.5, 122.0, 120.2, 118.0, 116.7, 50.2,
33.0, 25.6, 25.0, 24.5. MS (ES) C21H24N2O4 requires m/z 368, found
369 [M + H]+.
Cyclohexylcarbamic Acid 6-Hydroxy-3′-methylcarbamoylbiphen-
yl-3-yl Ester (7d). The title compound 7d was prepared according to
general procedure B using compound 5d (41.3 mg, 0.09 mmol) and 10%
Pd/C (16.5 mg); reaction time, 2 h. The residue was purified by flash
chromatography (0−2% MeOH in DCM) to afford 7d as a white solid;
23 mg, 56%. 1H NMR (400 MHz, chloroform-d) δ 7.65 (d, J = 7.7 Hz,
1H), 7.58 (s, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.35 (dd, J = 7.7, 7.7 Hz, 1H),
6.90−6.83 (m, 3H), 6.73 (d, J = 8.7 Hz, 1H), 6.67 (d, J = 4.8 Hz, 1H),
5.08 (bd, J = 8.1 Hz, 1H), 3.56−3.51 (m, 1H), 2.90 (d, J = 4.8 Hz, 3H),
2.01−1.96 (m, 2H), 1.76−1.70 (m, 2H), 1.65−1.59 (m, 1H), 1.41−1.30
(m, 2H), 1.27−1.16 (m, 3H). 13C NMR (101 MHz, chloroform-d) δ
171.3, 168.7, 155.0, 150.9, 144.2, 137.7, 134.8, 132.2, 128.8, 127.4, 126.4,
123.5, 122.2, 117.2, 60.5, 50.4, 33.3, 25.6, 24.9. MS (ES) C21H24N2O4
requires m/z 368, found 369 [M + H]+.
Cyclohexylcarbamic Acid 3′-Dimethylcarbamoyl-6-hydroxybi-
phenyl-3-yl Ester (7e). To a solution of 5e (227 mg, 0.48 mmol) in
dry DCM (5 mL) at −78 °C, BBr3 (0.96 mL, 1.0 M solution in DCM)
was slowly added under Ar atmosphere. The reaction was warmed to
room temperature, stirred for 1 h, and then quenched with saturated
aqueous NH4Cl solution. The aqueous solution was extracted with
EtOAc (3 × 20 mL) and the organic phase dried (Na2SO4). After
evaporation of solvent, the residue was purified by flash chromatography
(0−4% MeOH in DCM) to afford 5e as a colorless solid; 133 mg, 72%.
1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 7.64−7.54 (m, 3H),
solid; 75 mg, 87%. H NMR (400 MHz, DMSO-d6) δ 9.72−9.68 (m,
1H), 8.01 (s, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.77−7.73 (m, 1H), 7.62−
7.56 (m, 2H), 7.35 (s, 2H), 7.01 (s, 1H), 6.96−6.93 (m, 2H), 3.39−3.26
(m, 1H), 1.85−1.77 (m, 2H), 1.74−1.66 (m, 2H), 1.59−1.52 (m, 1H),
1.32−1.19 (m, 4H), 1.18−1.08 (m, 1H). 13C NMR (101 MHz, DMSO-
d6) δ 154.4, 151.8, 144.4, 144.1, 138.9, 132.7, 129.07, 126.8, 126.5, 124.3,
123.6, 122.9, 116.9, 50.2, 33.0, 25.6, 25.0. MS (ESI) C19H22N2O5S
requires m/z 390, found 391 [M + H]+.
Cyclohexylcarbamic Acid 6-Hydroxy-3′-methylsulfonylbiphenyl-
3-yl Ester (7g). The title compound 7g was prepared according to
general procedure B using compound 5g (149 mg, 0.31 mmol) and 10%
Pd/C (59.6 mg); reaction time, 2 h. The residue was purified by flash
chromatography (0−50% EtOAc in cyclohexane) to afford 7g as a
colorless solid; 109 mg, 90%. 1H NMR (400 MHz, DMSO-d6) δ 9.75 (s,
1H), 8.09−8.07 (m, 1H), 7.94−7.90 (m, 1H), 7.88−7.84 (m, 1H),
7.71−7.65 (m, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.11−7.08 (m, 1H), 6.98−
6.92 (m, 2H), 3.34−3.27 (m, 1H), 3.25 (s, 3H), 1.85−1.77 (m, 2H),
1.74−1.66 (m, 2H), 1.59−1.52 (m, 1H), 1.32−1.16 (m, 4H), 1.16−1.06
(m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 153.9, 151.3, 143.7, 140.7,
138.8, 134.0, 129.1, 127.2, 125.9, 125.2, 123.2, 122.7, 116.4, 49.7, 43.6,
32.6, 25.1, 24.6. MS (ESI) C20H23NO5S requires m/z 389, found 390 [M
+ H]+.
3-(2-Benzyloxy-5-hydroxyphenyl)benzaldehyde (9a). The title
compound 9a was prepared according to general procedure C using
compound 8 (278 mg, 1.0 mmol), 3-formylphenylboronic acid (0.174 g,
1.2 mmol), Na2CO3 (0.53 g, 5 mmol), and Pd(PPh3)4 (0.058 g, 0.05
mmol). The crude was purified by flash chromatography (cyclohexane/
EtOAc 85:15) to afford 9a as amber oil; 0.194 g, 64%. 1H NMR (200
MHz, DMSO-d6) δ 10.04 (s, 1H), 9.14 (s, 1H), 8.05 (t, J = 1.5 Hz, 1H),
7.88−7.81 (m, 2H), 7.62 (t, J = 7.6 Hz 1H), 7.33−7.22 (m, 5H), 7.06 (d,
J = 9.0 Hz 1H), 6.81−6.74 (m, 2H), 5.00 (s, 2H). MS (ESI) C20H16O3
requires m/z 304, found 303 [M − H]−.
1-[3-(2-Benzyloxy-5-hydroxyphenyl)phenyl]ethanone (9b). The
title compound 9b was prepared according to general procedure C
using compound 8 (0.278 g, 1.0 mmol), 3-acetylphenylboronic acid
(0.196 g, 1.2 mmol), Na2CO3 (0.53 g, 5 mmol), and Pd(PPh3)4 (0.058 g,
0.05 mmol). The crude was purified by flash chromatography
(cyclohexane/EtOAc 75:25 and then DCM/MeOH 99:1) to afford
9b as white solid after crystallization from EtOH. 0.248 g, 78%. 1H NMR
(200 MHz, DMSO-d6) δ 9.12 (br, 1H), 8.09 (t, J = 1.6 Hz, 1H), 7.89 (dt,
J = 7.7 Hz, J = 1.4 Hz, 1H), 7.75 (dt, 7.7 Hz, J = 1.4 Hz, 1H), 7.54 (t, J =
7.7 Hz, J = 1.4 Hz, 1H), 7.33−7.28 (m, 5H), 7.06 (d, 8.8 Hz, 1H), 6.80−
6.73 (m, 2H), 4.99 (s, 2H), 2.55 (s, 3H). MS (ESI) C21H18O3 requires
m/z 318, found 319 [M + H]+, 317 [M − H]−.
Cyclohexylcarbamic Acid 6-Benzyloxy-3′-formylbiphenyl-3-yl
Ester (10a). The title compound 10a was prepared according to general
procedure D using compound 9a (0.304 g, 1.0 mmol), c-C6H11NCO
(0.137 mg, 1.1 mmol), and Et3N (0.11 g, 0.154 mL, 1.1 mmol). The
crude was purified by flash chromatography (cyclohexane/EtOAc
75:25) to afford 10a as amber oil; 0.236 g, 55%. 1H NMR (200 MHz,
DMSO-d6) δ 10.04 (s, 1H), 8.10 (t, J = 1.5 Hz 1H), 7.91−7.85 (m, 2H),
7.67−7.59 (m, 2H), 7.39−7.08 (m, 8H), 5.14 (s, 2H), 3.37−3.33 (m,
1H), 1.83−1.54 (m, 5H), 1.24−1.19 (m, 5H). MS (ESI) C27H27NO4
requires m/z 429, found 430 [M + H]+.
Cyclohexylcarbamic Acid 3′-Acetyl-6-benzyloxybiphenyl-3-yl
Ester (10b). The title compound 10b was prepared according to
general procedure D using compound 9b (0.318 g, 1.0 mmol), c-
C6H11NCO (0.137 mg, 1.1 mmol), and Et3N (0.11 g, 0.154 mL, 1.1
mmol). The crude was purified by flash chromatography (cyclohexane/
EtOAc 70:30) to afford 10b as white solid, after crystallization from
EtOH; 0.270 g, 61%. 1H NMR (200 MHz, DMSO-d6) δ 8.12 (t, J = 1.5
Hz, 1H), 7.90 (dt, J = 8.0 Hz, J = 1.5 Hz, 1H), 7.79 (dt, J = 8.0 Hz, J = 1.5
Hz 1H), 7.64−7.52 (m, 2H), 7.40−7.20 (m, 6H), 7.12−7.07 (m, 2H),
5.13 (s, 2H), 3.38−3.31 (m, 1H), 2.55 (s, 3H), 1.83−1.53 (m, 5H),
1.23−1.06 (m, 5H). MS (ESI) C28H29NO4 requires m/z 443, found 444
[M + H]+.
7.50−7.41 (m, 1H), 7.33 (d, J = 7.5 Hz, 1H), 7.00 (s, 1H), 6.97−6.89
(m, 2H), 3.33−3.28 (m, 1H), 3.01−2.93 (m, 6H), 1.85−1.78 (m, 2H),
1.71−1.66 (m, 2H), 1.59−1.51 (m, 1H), 1.33−1.17 (m, 4H), 1.16−1.04
(m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 170.6, 154.4, 151.8, 144.1,
138.2, 136.6, 130.3, 128.5, 127.9, 127.5, 125.9, 123.6, 122.4, 116.8, 55.3,
50.2, 33.0, 25.6, 25.0. MS (ES) C22H26N2O4 requires m/z 382, found
383 [M + H]+.
Cyclohexylcarbamic Acid 6-Hydroxy-3′-sulfamoylbiphenyl-3-yl
Ester (7f). The title compound 7f was prepared according to general
procedure B using compound 5f (106 mg, 0.22 mmol) and 10% Pd/C
(42.4 mg); reaction time, 2 h. The residue was purified by flash
chromatography (0−50% EtOAc in cyclohexane) to afford 7f as a white
Cyclohexylcarbamic Acid 6-Hydroxy-3′-methylbiphenyl-3-yl Ester
(11a). The title compound 11a was prepared according to general
procedure E using compound 10a (0.429 mg, 1 mmol) and 10% Pd/C
(0.044 g). The crude was purified by flash chromatography (cyclo-
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hexane/EtOAc 40:60) to afford 11a as a white solid, after crystallization
from Et2O; 0.061 g, 19%. 1H NMR (200 MHz, chloroform-d) δ 7.39−
7.18 (m, 4H), 7.01−6.84 (m, 3H), 5.60 (s, 1H), 4.95 (d, J = 8.0 Hz, 1H),
3.68−3.48 (m, 1H), 2.40 (s, 3H), 2.04−1.99 (d, J = 10.4 Hz, 2H), 1.78−
1.61 (m, 3H), 1.48−1.11 (m, 5H). 13C NMR (50 MHz, chloroform-d) δ
154.2, 149.9, 144.3, 139.0, 136.47, 129.7, 129.1, 128.7, 128.6, 126.0,
123.0, 122.0, 116.3, 50.1, 33.3, 25.4, 24.7, 21.5. MS (ESI) C20H23NO3
requires m/z 325, found 326 (M + H]+.
Cyclohexylcarbamic Acid 6-Hydroxy-3′-(1-hydroxyethyl)-
biphenyl-3-yl Ester (11b). The title compound 11b was prepared
according to general procedure E using compound 10b (0.443 g, 1.0
mmol) and 10% Pd/C (0.044 g). The crude was purified by flash
chromatography (cyclohexane/EtOAc 40:60) to afford 11b as a white
amorphous solid; 0.176 g, 50%. 1H NMR (200 MHz, chloroform-d) δ
7.37−7.27 (m, 4H), 6.96−6.61 (m, 3H), 5.11 (d, J = 8.0 Hz, 1H), 4.81−
4.71 (m, 1H), 3.55−3.51 (m, 1H), 2.82 (br, 1H), 2.05−1.96 (m, 2H),
1.69−1.59 (m, 3H), 1.42 (d, J = 6.4 Hz, 3H), 1.33−1.15 (m, 6H). 13C
NMR (50 MHz, chloroform-d) δ 181.4, 154.7, 150.4, 146.1, 144.0,
137.2, 128.7, 128.1, 126.3, 124.6, 123.2, 121.8, 116.8, 70.2, 50.2, 33.2,
25.4, 25.0, 24.7. MS (ESI) C21H25NO4 requires m/z 355, found 354 [M
− H]−, 356 [M + H]+.
Cyclohexylcarbamic Acid 6-Hydroxy-3′-hydroxymethylbiphenyl-
3-yl Ester (11c). The title compound 11c was prepared according to
general procedure E using compound 10a (0.429 g, 1 mmol) and 10%
Pd/C (0.044 g). The crude was purified by flash chromatography
(cyclohexane/EtOAc 40:60) to afford 11c as a white amorphous solid;
0.112 g, 30%. 1H NMR (200 MHz, chloroform-d) δ 7.44−7.34 (m, 4H),
7.00−6.83 (m, 3H), 5.60 (s, 1H), 4.93 (d, J = 8.2 Hz,1H), 4.69 (s, 2H),
3.57−3.43 (m, 1H), 2.05−1.97 (m, 3H), 1.77−1.70 (m, 3H), 1.42−1.06
(m, 4H). 13C NMR (50 MHz, chloroform-d) δ 154.5, 150.2, 144.1,
141.5, 137.1, 129.0, 128.6, 128.2, 127.7, 126.3, 123.2, 121.9, 116.7, 66.0,
50.2, 33.2, 25.4, 24.7. MS (ESI) C20H23NO4 requires m/z 341, found
342 [M + H]+.
2-(2-Bromo-4-fluorophenyl)-1,3-dioxolane (17).23 To a solution of
16 (4.0 g, 19.7 mmol) in dry toluene (30 mL), ethylene glycol (5.56 mL,
98.5 mmol), and p-TSA (187 mg, 1 mmol) were added and the reaction
mixture was heated at reflux for 12 h. The mixture was cooled down to
room temperature and then poured into saturated aqueous NH4Cl
solution (50 mL). The two phases were separated, and the organic
solution was washed with brine and dried (Na2SO4). Evaporation of the
solvent gave 17 as light-yellow oil that was used in the next step without
1
further purification; 4.5 g. H NMR (400 MHz, chloroform-d) δ 7.62
(dd, J = 8.3, 6.1 Hz, 1H), 7.34 (dd, J = 8.3, 2.6 Hz, 1H), 7.08 (td, J = 8.3,
2.6 Hz, 1H), 6.07 (s, 1H), 4.46−3.85 (m, 4H).
4-Benzyloxy-2-bromobenzaldehyde (18). To a solution of 17 (4.0 g,
16.19 mmol) in dry dioxane (60 mL), BnOH (6.27 mL, 64.78 mmol),
and t-BuOK (7.27 g, 64.78 mmol) were added and then the mixture was
heated at 85 °C for 1 h. The mixture was cooled down to room
temperature and then poured into H2O (150 mL) and EtOAc (200 mL),
and the two phases were separated. To the organic solution 2 N HCl
(150 mL) was added, and stirring was continued for 2 h at room
temperature. The two phases were then separated, and the organic layer
was dried (Na2SO4). After evaporation of the solvent, the residue was
purified by flash chromatography (0−10% EtOAc in cyclohexane) to
yield 18 as a white solid. 3.53 g, 75%. 1H NMR (400 MHz, chloroform-
d) δ 10.25 (d, J = 0.8 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.48−7.34 (m,
5H), 7.25 (d, J = 2.5 Hz, 1H), 7.03 (dd, J = 8.8, 2.5 Hz, 1H), 5.16 (s, 2H).
MS (ESI): no ionization.
3-(5-Benzyloxy-2-formylphenyl)benzamide (19). To a solution of
18 (1.41g, 4.85 mmol) in ethyleneglycol monomethyl ether (EGME)
(30 mL), H2O was slowly added (8 mL), followed by the addition of
K2CO3 (1.34 g, 9.69 mmol), 3-carbamoylbenzeneboronic acid (1.2 g,
7.27 mmol), and Pd(OAc)2 (10.8 mg, 0.049 mmol). The mixture was
stirred at room temperature for 40 min until the mixture became dark,
and a precipitate was formed. H2O (40 mL) was then added, and the
solid was filtered and washed with H2O (15 mL) to afford 19 as a whitish
solid; 1.53 g, 95%. 1H NMR (400 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.08
(bs, 1H), 8.00−7.92 (m, 3H), 7.65−7.55 (m, 2H), 7.55−7.33 (m, 6H),
7.25 (dd, J = 8.6, 2.5 Hz, 1H), 7.15 (d, J = 2.5 Hz, 1H), 5.30 (s, 2H). MS
(ESI) C21H17NO3 requires m/z 331, found 332 [M + H]+
3-[5-Benzyloxy-2-hydroxymethylphenyl]benzamide (20). To a
suspension of 19 (1.6 g, 4.83 mmol) in EtOH (20 mL), NaBH4 (365
mg, 9.67 mmol) was added slowly at 0 °C and the mixture was stirred for
2 h. The reaction was diluted with DCM (50 mL) and quenched by the
addition of saturated aqueous Na2CO3 solution (20 mL) and H2O (20
mL) to afford a precipitate that was filtered to give 20 as a grayish solid;
1.3 g, 81%. 1H NMR (400 MHz, DMSO-d6) δ 8.00 (bs, 1H), 7.95−7.83
(m, 2H), 7.67−7.27 (m, 9H), 7.06 (dd, J = 8.5, 2.7 Hz, 1H), 6.92 (d, J =
2.7 Hz, 1H), 5.16 (s, 2H), 5.02 (t, J = 5.2 Hz, 1H), 4.32 (d, J = 5.2 Hz,
2H). MS (ESI) C21H19NO3 requires m/z 333, found 316 [M − H2O +
H]+
Cyclohexylcarbamic Acid 3′-Carbamoyl-6-hydroxymethylbiphen-
yl-3-yl Ester (21). A suspension of 20 (1.3 g, 3.89 mmol) in MeOH (80
mL) under N2 atmosphere was heated at reflux until complete
dissolution, and then 10% Pd/C (700 mg) were rapidly added followed
by the addition of γ-terpinene (6.2 mL, 38.9 mmol). The mixture was
heated at reflux for additional 1 h then was cooled down to room
temperature, filtered through Celite, and washed with MeOH (15 mL).
The filtrate was concentrated to dryness, affording a colorless solid,
which was dissolved in a 1:1 mixture of CH3CN/EtOH (20 mL). To this
solution, Et3N (0.32 mL, 2.35 mmol) and c-C6H11NCO (0.5 mL, 3.89
mmol) were added, and the mixture was stirred at room temperature for
12 h. The reaction was quenched by the addition of EtOAc (80 mL) and
2 N HCl (80 mL) that, after vigorously stirring, were then separated.
The aqueous solution was extracted with EtOAc (40 mL), and then the
combined organic layers were dried (Na2SO4). After evaporation of the
solvent, the residue was purified by flash chromatography (100%
EtOAc) and then crystallized from EtOH/H2O to afford 21 as a white
solid; 630 mg, 44%. 1H NMR (400 MHz, DMSO-d6) δ 8.03 (bs, 1H),
7.94−7.80 (m, 2H), 7.71 (d, J = 7.9 Hz, 1H), 7.64−7.47 (m, 3H), 7.39
(bs, 1H), 7.13 (dd, J = 8.4, 2.5 Hz, 1H), 6.99 (d, J = 2.5 Hz, 1H), 5.15 (t, J
= 5.1 Hz, 1H), 4.38 (d, J = 5.1 Hz, 2H), 3.42−3.22 (m, 1H), 2.03−1.49
(m, 5H), 1.37−0.99 (m, 5H). 13C NMR (101 MHz, DMSO-d6) δ 168.2,
3-Bromo-4-methoxyphenol (13). To a solution of 12 (214 mg, 1
mmol) in DCM (5 mL), m-CPBA (0.173 g, 1 mmol) was added. The
mixture was stirred for 72 h at 40 °C and then washed with a saturated
aqueous Na2S2O3 solution (5 mL) and with a saturated aqueous
NaHCO3 solution (5 mL). The combined organic layers were dried
(Na2SO4). After evaporation of the solvent, the residue was dissolved in
EtOH (5 mL), NaOCH3 (0.108 g, 2 mmol) was added, and the mixture
was stirred for 1 h at room temperature, concentrated in vacuo, and
acidified with 2 N HCl (5 mL) and extracted with DCM (5 × 3 mL).
The combined organic layers were dried (Na2SO4). After evaporation of
the solvent, the residue was purified by flash chromatography
1
(cyclohexane/DCM = 20:80) to afford 13; 0.121 g, 60%. MS and H
NMR are according to the literature.35
3-(5-Hydroxy-2-methoxyphenyl)benzamide (14). The title com-
pound 14 was prepared according to general procedure C using
compound 13 (0.202 g, 1 mmol), 3′-carbamoylphenylboronic acid
(0.198 g, 1.2 mmol), Na2CO3 (0.52 g, 5 mmol), and Pd(PPh3)4 (0.020
mg). The crude was purified by flash chromatography (DCM/MeOH =
94:6) to afford 14 as a white solid; 0.197 g, 81%. 1H NMR (200 MHz,
DMSO-d6) δ 9.10 (s, 1H), 8.03 (s, 1H), 7.93−7.91 (m, 1H), 7.82−7.78
(m, 1H), 7.61−7.57 (m, 1H), 7.49−7.45 (m, 1H), 7.41−7.39 (m, 1 H),
6.96−9.91 (m, 1H), 6.77−6.71 (m, 2H), 3.65 (s, 3H). MS (ESI)
C14H13NO3 requires m/z 243, found 242 [M − H]−.
Cyclohexylcarbamic Acid 3′-Carbamoyl-6-methoxybiphenyl-3-yl
Ester (15). The title compound 15 was prepared according to general
procedure D using compound 14 (0.243 g, 1.0 mmol), c-C6H11NCO
(0.137 mg, 1.1 mmol), and Et3N (0.11 g, 0.154 mL, 1.1 mmol). The
crude was purified by flash chromatography (cyclohexane/EtOAc
30:70) to afford 15 as a white solid; 0.284 g, 77%. 1H NMR (200 MHz,
chloroform-d) δ 7.93−7.91 (m, 1H), 7.82−7.77 (m, 1H), 7.72−7.68 (m,
1H), 7.52−7.44 (m, 1H), 7.14−7.07 (m, 2H), 6.97−6.92 (m, 1H), 6.26
−5.73 (m, 2H), 4.98−4.82 (m, 1H), 3.80 (s, 3H), 3.58−3.54 (m, 1H),
1.13−2.04 (m, 10H). 13C NMR (50 MHz, chloroform-d) δ 169.4, 154.1,
153.7, 144.6, 138.2, 133.2, 133.1, 130.1, 128.3, 126.3, 124.0, 121.9, 111.8,
56.0, 50.2, 33.3, 25.4, 24.7. MS (ESI) C21H24N2O4 requires m/z 368,
found 369 [M + H]+.
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153.9, 150.3, 140.9, 140.0, 136.3, 134.9, 132.2, 129.7, 128.6, 128.3, 127.0,
122.9, 121.3, 60.8, 50.3, 33.0, 25.6, 25.0. MS (ESI) m/z C21H24N2O4
requires m/z 368, found 386 [M + NH4]+, 737 [2M + H]+.
mL) was added followed by the addition of Na2CO3 (2.9 g, 27.36 mmol)
and 3-carbamoylphenylboronic acid (3.4 g, 20.52 mmol). To this
solution, PdCl2dppf (500 mg, 0.034 mmol) was added and then the
mixture was heated at 90 °C for 1.5 h under N2 atmosphere. The mixture
was cooled down to room temperature and then poured into 1 N HCl
(200 mL) and EtOAc (200 mL) under stirring. After 30 min, the two
phases were separated and the aqueous phase was extracted with EtOAc
(100 mL). The combined organic layers were dried (Na2SO4). After
evaporation of the solvent, the residue was purified by flash
chromatography (40−100% EtOAc in DCM) to afford 27 as a whitish
solid; 2.73 g, 57%. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (bs, 1H),
8.02 (bs, 1H), 7.92−7.76 (m, 3H), 7.51−7.33 (m, 3H), 7.30−7.23 (m,
3H), 7.11−7.01 (m, 2H), 6.89 (dd, J = 8.6, 2.5 Hz, 1H), 6.75 (d, J = 2.5
Hz, 1H), 5.02 (s, 2H). MS (ESI) C21H17NO4 requires m/z 347, found
348 [M + H]+.
Cyclohexylcarbamic Acid 6-Benzyloxycarbonyl-3′-carbamoylbi-
phenyl-3-yl Ester (28). A suspension of 27 (2.7g, 7.78 mmol) in dioxane
(100 mL) was heated at 50 °C until a yellow solution was formed. The
reaction mixture was cooled down to room temperature and DMAP
(250 mg, 2.04 mmol) and c-C6H11CNO (1.2 mL, 9.33 mmol) were
added and the mixture was heated at 45 °C for 12 h then cooled down to
room temperature and poured into 1 N HCl (200 mL) and EtOAc (200
mL) under stirring. The two phases were separated, and the aqueous
layer was extracted with EtOAc (100 mL). The combined organic layers
were dried (Na2SO4). After evaporation of the solvent, the residue was
purified by flash chromatography (20−50% EtOAc in DCM) to afford
28 as a white fluffy solid; 2.8 g, 76%. 1H NMR (400 MHz, DMSO-d6) δ
8.05 (bs, 1H), 7.95−7.82 (m, 4H), 7.52−7.36 (m, 2H), 7.33−7.24 (m,
4H), 7.21 (d, J = 2.4 Hz, 1H), 7.13−7.00 (m, 2H), 5.08 (s, 2H), 3.32 (m,
1H), 1.91−1.48 (m, 5H), 1.38−1.01 (m, 6H). MS (ESI) C28H28N2O5
requires m/z 472, found 473 [M + H]+.
Cyclohexylcarbamic Acid 3′-Carbamoyl-6-carboxybiphenyl-3-yl
Ester (29). To a solution of 28 (2.7 g, 5.72 mmol) in dioxane (200 mL),
cyclohexene (50 mL) and 10% Pd/C (2 g) were added. The mixture was
heated at 85 °C for 2 h, then was cooled down to room temperature,
activated carbon (2 g) added, and filtered through a pad of Celite.
Evaporation of solvent gave 29 as white solid; 960 mg, 44%. 1H NMR
(400 MHz, DMSO-d6) δ 12.61 (bs, 1H), 8.04 (bs, 1H), 7.95−7.73 (m,
4H), 7.55−7.43 (m, 2H), 7.38 (s, 1H), 7.23 (dd, J = 8.5, 2.4 Hz, 1H),
7.15 (d, J = 2.4 Hz, 1H), 3.33 (m, 1H), 2.00−1.48 (m, 5H), 1.39−0.99
(m, 5H). 13C NMR (101 MHz, DMSO-d6) δ 169.0, 168.6, 153.3, 153.3,
142.9, 140.7, 134.6, 131.6, 131.5, 128.7, 128.4, 127.8, 127.0, 124.0, 121.1,
50.4, 32.9, 25.6, 25.0. MS (ESI) C21H22N2O5 requires m/z 382, found
383 [M + H]+.
Ammonium Cyclohexylcarbamic Acid 3′-Carbamoyl-6-sulfatebi-
phenyl-3-yl Ester (30). To a suspension of 3 (200 mg, 0.62 mmol) in dry
DCM (5 mL), SO3−DMF complex (593 mg, 3.73 mmol) was added.
After stirring at room temperature for 1 h, pyridine (2 mL) was added
and the reaction mixture was concentrated in vacuo to give a colorless oil
that was purified by preparative HPLC (column, C18), using the
following eluent conditions: 20% B for 0.5 min then 20% to 60% B in 7
min; Rt, 4.5 min to afford 30 as a white solid; 125 mg, 44%. 1H NMR
(400 MHz, DMSO-d6) δ 8.08−8.01 (m, 1H), 7.91 (bs, 1H), 7.87−7.76
(m, 2H), 7.69 (d, J = 7.9 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.48 (t, J = 7.7
Hz, 1H), 7.36 (bs, 1H), 7.11 (d, J = 2.9 Hz, 1H), 7.05 (dd, J = 8.9, 2.9 Hz,
1H), 3.50 (bs, 4H), 3.41−3.22 (m, 1H), 1.89−1.78 (m, 2H), 1.78−1.66
(m, 2H), 1.63−1.49 (m, 1H), 1.43−0.95 (m, 5H). 13C NMR (101 MHz,
DMSO-d6) δ 168.6, 154.1, 147.8, 147.1, 137.7, 134.7, 133.2, 132.7,
128.6, 128.3, 126.7, 123.4, 122.3, 121.9, 50.2, 33.0, 25.6, 25.0. MS (ESI)
C20H22N2O7S requires m/z 434, found 433 [M − H]− .
1-(2-Bromo-4-methoxyphenyl)ethanone (23). To a suspension of
ZrCl4 (37.63 g, 0.16 mol) in dry DCM (500 mL), 22 (16.78 mL, 0.17
mol) was added at −10 °C under N2 atmosphere followed by the
addition of AcCl in 15 mL of DCM dropwise. The orange turbid
solution was stirred at −10 °C for 1 h, then the reaction mixture was
carefully poured into a 3 L flask containing of 2 N HCl (500 mL) and
DCM (150 mL) and stirred for 40 min. The phases were separated and
the milky organic phase dried (Na2SO4). The residue was dissolved in
MTBE (300 mL), and the mixture was filtered through a pad of Celite to
give a clear colorless solution. After evaporation of the solvent, the
residue was purified by flash chromatography (0−20% EtOAc in
cyclohexane) to afford 23 as a light-yellow oil; 17 g, 55%. 1H NMR (400
MHz, chloroform-d) δ 7.61 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 2.5 Hz, 1H),
6.90 (dd, J = 8.7, 2.5 Hz, 1H), 3.87 (s, 3H), 2.65 (s, 3H). MS (ESI)
C9H9BrO2 requires m/z 228, 230, found 229, 231 [M + H]+.
2-Bromo-4-methoxybenzoic Acid (24). To a suspension of t-BuONa
(2.77 g, 28.8 mmol) in dry THF (50 mL), diethyl oxalate (6.06 mL, 39.3
mmol) was carefully added under N2 atmosphere and the yellow
reaction mixture was stirred at room temperature for 30 min. A solution
of 23 (3.0 g, 13.10 mmol) in dry THF (15 mL) was then added
dropwise, and the mixture was stirred at room temperature for
additional 30 min. The reaction mixture was carefully poured into a
mixture of 1 N HCl (200 mL) and EtOAc (200 mL), then the phases
were separated and the organic layer was concentrated in vacuo to give a
yellow oil. It was dissolved in a 5:3 mixture of acetone/H2O (160 mL),
then NaHCO3 was added (11.0 g, 131.0 mmol) and cooled down to 0
°C. To this mixture, Oxone (20.1 g, 32.8 mmol) was added very carefully
and an evolution of gas was immediately observed. The mixture was
stirred at 0 °C for 2 h then the solids were filtered off. To the filtrate,
solid Na2S2O3 was added under stirring until disappearance of oxidant
(KI solution test). The mixture was concentrated to remove residual
acetone, and then 2N HCl (20 mL) was added until acidic pH while a
white precipitate immediately was formed which was filtered and
washed with H2O (20 mL) to afford 24 as a white solid; 2.3 g, 77%. 1H
NMR (400 MHz, DMSO-d6) δ 13.00 (bs, 1H), 7.82 (d, J = 8.7 Hz, 1H),
7.27 (d, J = 2.5 Hz, 1H), 7.04 (dd, J = 8.7, 2.5 Hz, 1H), 3.84 (s, 3H). MS
(ESI) C8H7BrO3 requires m/z 230, 232, found 231, 233 [M + H]+.
2-Bromo-4-hydroxybenzoic Acid (25). To a suspension of 24 (3.0 g,
13.0 mmol) in dry DCM (50 mL) BBr3 (39 mL, 1 M solution in DCM)
was added dropwise at 0 °C under N2 atmosphere for 30 min until
complete dissolution and then left under stirring at room temperature
for 12 h while a precipitate was formed. The reaction mixture was
quenched at 0 °C by a careful addition of 5 N NaOH (10 mL) until pH
>11. H2O (50 mL) and DCM (50 mL) were then added, and the
mixture was stirred for additional 2 h. The two phases were separated,
and the organic layer was washed with H2O (30 mL). The aqueous
phase was carefully acidified with 37% HCl until pH 1. NaCl (10 g) was
added portionwise, and a white precipitate immediately was formed.
The mixture was stirred at 0 °C for 1.5 h and then filtered to give 25 as a
1
white solid; 1.7 g, 61%. H NMR (400 MHz, DMSO-d6) δ 12.78 (bs,
1H), 10.54 (bs, 1H), 7.75 (d, J = 8.6 Hz, 1H), 7.08 (d, J = 2.4 Hz, 1H),
6.84 (dd, J = 8.6, 2.4 Hz, 1H). MS (ESI) C7H5BrO3 requires m/z 216,
217, found 215, 217 [M − H]−.
Benzyl 2-Bromo-4-hydroxybenzoate (26). To a solution of 25 (3.0
g, 13.8 mmol) in DMF (30 mL), KHCO3 (2.1 g, 20.7 mmol) was added
under vigorous stirring followed by the addition of BnBr (1.47 mL, 12.44
mmol). The yellow mixture was stirred for 12 h at room temperature
and then poured into a mixture of 1 N HCl (200 mL) and MTBE (200
mL) under stirring. The two phases were separated, and the organic
layer was dried (Na2SO4). Evaporation of solvent gave 26 as a yellow oil
1,3-Dibenzyloxy-5-bromobenzene (32). To a solution of t-BuONa
(19.9 g, 207.3 mmol) and BnOH (21.3 mL, 207.3 mmol) in dry DMF
(200 mL), 1-bromo-3,5-difluorobenzene 31 (4.8 mL, 41.5 mmol) was
added under N2 atmosphere. The reaction mixture was stirred at 90 °C
for 3 h. The dark-yellow mixture was cooled down to room temperature
and, under stirring, slowly transferred in a 3 L flask containing H2O (600
mL) and of MTBE (500 mL). After 30 min, the organic phase was
separated, washed with H2O (400 mL), and dried (Na2SO4).
Evaporation of the solvent gave 32 as yellow oil that crystallized after
12 h upon cooling at −19 °C. The solid was treated with 180 mL of
1
that was used in the next step without further purification; 4.1 g. H
NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 7.80 (d, J = 8.7 Hz, 1H),
7.51−7.45 (m, 2H), 7.45−7.33 (m, 3H), 7.12 (d, J = 2.4 Hz, 1H), 6.87
(dd, J = 8.7, 2.4 Hz, 1H), 5.30 (s, 2H). MS (ESI) C14H11BrO3 requires
m/z 306, 308, found 307, 309 [M + H]+.
Benzyl 2-(3-Carbamoylphenyl)-4-hydroxybenzoate (27). To a
solution of 26 (4.2 g, 13.68 mmol) in dioxane (100 mL), H2O (80
K
dx.doi.org/10.1021/jm4007017 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
MeOH then filtered and washed with cold MeOH (30 mL); 11 g, 72%.
1H NMR (400 MHz, chloroform-d) δ 7.52−7.31 (m, 10H), 6.80 (d, J =
J = 2.4 Hz, 2H), 6.53 (t, J = 2.2 Hz, 1H), 3.82 (s, 6H). MS (ESI)
C15H15NO3 requires m/z 257, found 258 [M + H]+.
3-(3-Hydroxy-5-methoxyphenyl)benzamide (40). To a solution of
39 (0.257 g, 1.0 mmol) in dry DCM (9 mL), BBr3 (5.0 mL, 1.0 M
solution in DCM, 5 mmol) was added at 0 °C. The mixture was stirred
for 30 min at room temperature, then H2O (5 mL) was added and the
mixture was extracted with DCM (5 mL) and EtOAc (5 × 2 mL). The
combined organic layers were dried (Na2SO4). After evaporation of the
solvent, the residue was purified by flash chromatography (DCM/
MeOH 95:5) to afford 40 as a white amorphous solid; 97 mg, 40%. 1H
NMR (200 MHz, DMSO-d6) δ 7.87−7.81 (m, 3H), 7.59−7.42 (m, 4H),
5.80 (br, 3H), 1.57 (s, 3H). MS (ESI) C14H13NO3 requires m/z 243,
found 244 [M + H]+.
2.2 Hz, 2H), 6.57 (t, J = 2.2 Hz, 1H), 5.03 (s, 4H). MS (ESI)
C20H17BrO2 requires m/z 368, found 367 (M − H)−.
3-(3,5-Dibenzyloxyphenyl)benzamide (33). To a solution of 32
(11.0 g, 29.8 mmol) in EGME (152 mL), H2O (54 mL) was added
dropwise, followed by the addition of K2CO3 (8.2 g, 59.6 mmol), 3-
carbamoylphenylboronic acid (7.4 g, 44.7 mmol), and Pd(OAc)2 (80.3
mg 0.36 mmol). The reaction mixture was stirred at 60 °C for 20 min.
Then H2O (100 mL) were added, and a precipitate was formed which
was filtered and washed with cold H2O (50 mL). The solid was
recrystallized from MeOH/THF (2.5:1, 350 mL) to give 33 as a light-
gray solid; 8.5 g, 70%. 1H NMR (400 MHz, DMSO-d6) δ 8.15 (t, J = 1.8
Hz, 1H), 8.12 (bs, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H),
7.61−7.30 (m, 12H), 7.00 (d, J = 2.2 Hz, 2H), 6.73 (t, J = 2.2 Hz, 1H),
5.19 (s, 4H). MS (ESI) C27H23NO3 requires m/z 409, found 410 (M +
H)+.
Cyclohexylcarbamic Acid 3′-Carbamoyl-5-metoxybiphenyl-3-yl
Ester (41). To a solution of 40 (0.243 g, 1 mmol) in CH3CN (25
mL), c-C6H11NCO (0.137 mg, 1.1 mmol), and Et3N (0.11 g, 0.154 mL,
1.1 mmol) were added. The mixture was refluxed for 2 h and
concentrated. The residue was purified by flash chromatography
(DCM/EtOAc 70:30) to afford 41 as a white solid after crystallization
3-(3,5-Dihydroxyphenyl)benzamide (34). To a suspension of 33
(8.5 g, 20.8 mmol) in dioxane (260 mL), cyclohexene (80 mL) was
added and the mixture was heated at 50 °C for 15 min until complete
dissolution, cooled down to room temperature, and 10% Pd/C (2 g)
added. The reaction mixture was heated at 80 °C for 2 h, and an
additional amount of 10% Pd/C (2 g) was then added. After additional 2
h, the mixture was cooled down to room temperature and filtered
through a pad of Celite and washed with dioxane (100 mL) and absolute
EtOH (100 mL). The clear solution was concentrated in vacuo to afford
34 as a light-yellow solid; 4.8 g, 100%. 1H NMR (400 MHz, DMSO-d6)
δ 9.38 (s, 2H), 8.10 (bs, 1H), 8.07−8.03 (m, 1H), 7.83 (d, J = 7.8 Hz,
1H), 7.68 (d, J = 7.8 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.38 (bs, 1H), 6.55
(d, J = 2.1 Hz, 2H), 6.27 (t, J = 2.1 Hz, 1H). MS (ESI) C13H11NO3
requires m/z 229, found 230 (M + H)+.
1
from EtOAc/petroleum ether; 0.240 g, 65%. H NMR (200 MHz,
DMSO-d6) δ 8.14−8.09 (m, 2H), 7.80−7.79 (m, 2H), 7.70 (d, J = 8.0
Hz, 1H), 7.53 (t, J = 7.7 Hz,1H), 7.39 (s, 1H), 7.13 (m, 1H), 7.05 (t, J =
1.7 Hz, 1H), 6.71 (t, J = 2.1 Hz, 1H), 3.84 (s, 3H), 3.41−3.30 (m, 1H),
1.85−1.55 (m, 5H), 1.25−1.21 (m, 5H). 13C NMR (50 MHz, DMSO-
d6) δ 168.2, 160.9, 153.7, 153.1, 141.8, 139.7, 135.4, 129.9, 129.4, 127.6,
126.1, 113.1, 109.6, 107.7, 56.0, 50.3, 33.0, 25.6, 25.0. MS (ESI)
C21H24N2O4 requires m/z 368, found 369 [M + H]+.
AUTHOR INFORMATION
■
Corresponding Author
Cyclohexylcarbamic Acid 3′-Carbamoyl-5-hydroxybiphenyl-3-yl
Ester (35). To a solution of 34 (2.6 g, 11.4 mmol) in dry DMF (30 mL),
CuCl (1.1 g, 11.4 mmol) was added and the reaction mixture turned
rapidly to a brown color. c-C6H11CNO (1.45 mL, 11.4 mmol) was then
added, and the mixture was stirred at room temperature for 30 min. To
this solution, a mixture of 3% aqueous citric acid solution (200 mL) and
EtOAc (100 mL) were then added. The organic phase was separated and
dried (Na2SO4). After evaporation of the solvent, the residue was
purified by flash chromatography (50−100% EtOAc in cyclohexane) to
afford 35 as a white solid. The solid was dissolved in a 6.5:2.0:1.5 mixture
of H2O:acetone:EtOH (75 mL). To this solution, H2O (30 mL) were
then added and a precipitate was formed which was filtered to afford 35
as a white solid; 1.17 g, 29%. 1H NMR (400 MHz, DMSO-d6) δ 9.86 (s,
1H), 8.13 (bs, 1H), 8.11−8.09 (m, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.75 (d,
J = 7.7 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.41
(bs, 1H), 6.95 (t, J = 1.9 Hz, 1H), 6.89 (t, J = 1.9 Hz, 1H), 6.53 (d, J = 1.9
Hz, 1H), 3.46−3.32 (m, 1H), 1.99−1.46 (m, 6H), 1.46−0.99 (m, 4H).
13C NMR (101 MHz, DMSO-d6) δ 168.2, 158.9, 153.8, 153.0, 141.8,
139.9, 135.4, 129.7, 129.4, 127.4, 126.0, 111.4, 110.8, 108.8, 50.2, 33.0,
25.6, 25.0. MS (ESI) C20H22N2O4 requires m/z 354, found 355 (M +
H)+.
3,5-Dimethoxyphenyl Trifluoromethanesulfonate (38). To a
solution of 37 (0.154 g, 1.0 mmol) in DCM (3 mL), DMAP (0.183 g,
1.5 mmol) and (CF3SO2)2O (0.366 g, 0.22 mL, 1.3 mmol) were added
at 0 °C. The mixture was stirred for 15 min at room temperature and
concentrated. The residue was purified by flash chromatography
(cyclohexane/EtOAc 3:7) to afford 38, which was used directly in the
next step without characterization.
3-(3,5-Dimethoxyphenyl)benzamide (39). To a solution of (n-
BuN)4Br (0.332 g, 1.0 mmol) and Na2CO3 (0.265 g, 2.5 mmol) in
EtOH (5 mL), 38 (0.286, 1.0 mmol), 3-carbamoylphenylboronic acid
(0.165 g, 1.0 mmol) and PdCl2 (18 mg, 0.1 mmol) were added. The
mixture was refluxed for 12 h, filtered on a plug of Celite, acidified with 2
N HCl (5 mL), and extracted with EtOAc (5 × 3 mL). The combined
organic layers were dried (Na2SO4). After evaporation of the solvent, the
residue was purified by flash chromatography (DCM/MeOH 95:5 and
then cyclohexane/EtOAc 30:70) gave 39 as a white solid; 0.115 g, 45%.
1H NMR (200 MHz, DMSO-d6) δ 8.12 (t, J = 1.6 Hz, 1H), 8.08 (br,
1H), 7.88−7.80 (m, 2H), 7.52 (t, J = 7.7 Hz, 1H), 7.39 (br, 1H), 6.85 (d,
*Phone: +3901071781509. Fax: +3901071781228. E-mail:
daniele.piomelli@iit.it.
Author Contributions
⊥These authors contributed equally to design and perform the
research.
Notes
The authors declare the following competing financial
interest(s): Patents protecting the class of compounds disclosed
in this paper were filed by the University of California Irvine,
Istituto Italiano di Tecnologia, Universit degli Studi di Urbino
Carlo Bo, and Universit degli Studi di Parma on behalf of
following individuals: Piomelli, Daniele; Moreno-Sanz, Guiller-
mo; Bandiera, Tiziano; Mor, Marco, and Tarzia, Giorgio.
ACKNOWLEDGMENTS
■
We thank Sine Mandrup Bertozzi for reverse phase HPLC
purifications, Luca Goldoni for NMR technical support, Silvia
Venzano for compounds handling, and Masih A. Babagoli for
experimental assistance. Funding by NIH grant DA-012423 (to
D.P.) is gratefully acknowledged. Universities of Parma and
Urbino “Carlo Bo” also supported the work.
ABBREVIATIONS USED
■
FAAH, fatty-acid amide hydrolase; ABCG2, ATP-binding
cassette transporter G2; BBB, blood−brain barrier; CB1,
cannabinoid type-1 receptor; CNS, central nervous system;
ED50, median effective dose; IC50, median inhibitory dose;
Oxone, potassium peroxymonosulfate; PSA, polar surface area;
PST, phenol sulfotransferase; SAR, structure−activity relation-
ship; dppf, 1,1′-bis(diphenylphosphino)ferrocene; EGME, eth-
yleneglycol monomethyl ether; MTBE, methyl tert-butyl ether;
SiO2, silica gel; TBSCI, tert-butyldimethylchlorosilane; TIPSCI,
triisopropylchlorosilane
L
dx.doi.org/10.1021/jm4007017 | J. Med. Chem. XXXX, XXX, XXX−XXX

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