Synthesis and evaluation of benzothiazole-Based analogues as novel, potent, and selective fatty acid amide hydrolase inhibitors

Article abstract of DOI:10.1021/jm801042a

High-throughput screening (HTS) identified benzothiazole analogue 3 as a potent fatty acid amide hydrolase (FAAH) inhibitor. Structure-activity relationship (SAR) studies indicated that the sulfonyl group, the piperidine ring and benzothiazole were the ke

Full text of DOI:10.1021/jm801042a

170
J. Med. Chem. 2009, 52, 170–180
Synthesis and Evaluation of Benzothiazole-Based Analogues as Novel, Potent, and Selective
Fatty Acid Amide Hydrolase Inhibitors
Xueqing Wang,* Katerina Sarris, Karen Kage, Di Zhang, Scott P. Brown, Teodozyi Kolasa, Carol Surowy, Odile F. El Kouhen,
Steven W. Muchmore, Jorge D. Brioni, and Andrew O. Stewart
Neuroscience Research, Global Pharmaceutical Research and DeVelopment, AP10/303, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, Illinois 60064
ReceiVed August 19, 2008
High-throughput screening (HTS) identified benzothiazole analogue 3 as a potent fatty acid amide hydrolase
(FAAH) inhibitor. Structure-activity relationship (SAR) studies indicated that the sulfonyl group, the
piperidine ring and benzothiazole were the key components to their activity, with 16j being the most potent
analogue in this series. Time-dependent preincubation study of compound 3 was consistent with it being a
reversible inhibitor. Activity-based protein-profiling (ABPP) evaluation of 3 in rat tissues revealed that it
had exceptional selectivity and no off-target activity with respect to other serine hydrolases. Molecular
shape overlay of 3 with a known FAAH inhibitor indicated that these compounds might act as transition-
state analogues, forming putative hydrogen bonds with catalytic residues and mimicking the charge distribution
of the tetrahedral transition state. The modeling study also indicated that hydrophobic interactions of the
benzothiazole ring with the enzyme contributed to its extraordinary potency. These compounds may provide
useful tools for the study of FAAH and the endocannabinoid system.
Introduction
Arachidonyl ethanolamide (AEAa, anandamide, 1), is a
known endogenous cannabinoid ligand and signaling lipid
messenger. It is believed to bind and activate cannabinoid (CB1
and CB2) receptors, as well as vanilloid receptor (TRPV1),1,2
through which it exhibits different biological actions. Its effects
on cell proliferation and apoptosis in various cell lines,3 on
reproduction,4,5 memory processes,6 antinociception,7,8 modula-
tion of anxiety9 and epilepsy10 have been reported. The signaling
function of anandamide, as well as some other related fatty acid
amides such as sleep-inducing agent oleamide,11 anorexigenic
agent N-oleoyl ethanolamide (OEA)12 and anti-inflammatory
agent N-palmitoyl ethanolamide (PEA),13 are all tightly con-
trolled by an intergral membrane enzyme, fatty acid amide
hydrolase (FAAH) through rapid hydrolysis to the corresponding
acids. In the rat brain, neurons that express FAAH are usually
found in the proximity of axon terminals containing CB1
receptors, providing important evidence for a role of FAAH in
anandamide deactivation.14 FAAH knockout studies indicated
that mice lacking FAAH had higher levels of fatty acid amides,
including AEA, and augmented behavioral response to admin-
istered AEA, and displayed CB1 dependent analgesia
phenotypes.15,16 These findings suggest that selective FAAH
inhibitors could enhance the actions of anandamide in brain
regions in which this lipid mediator is physiologically important;
for example, those engaged in the processing of pain8 and
Figure 1. AEA and known FAAH inhibitors.
of this pathway via selective chemical inhibition18 may provide
emotion.17 The increased anandamide tone produced by blocking
FAAH may result in a more restricted spectrum of pharmaco-
an useful therapeutic tool for the treatment of anxiety, epilepsy,
logical effects than those produced by direct-acting CB1 agonists
cancer, eating disorder, pain and sleep disorders.
Despite the therapeutic potential of FAAH in various diseases,
only a few structural classes of FAAH inhibitor have been
and, possibly, in fewer adverse events. Therefore, intervention
* To whom correspondence should be addressed. Phone: 847-936-0362.
identified. Some of the representative examples are shown in
Fax: 847-935-5466. E-mail: xueqing.wang@abbott.com.
a Abbreviations: HTS, high-throughput screening; FAAH, fatty acid
amide hydrolase; AEA, N-arachidonoylethanolamine; OEA, N-oleoyl etha-
nolamide; PEA, N-palmitoyl ethanolamide; TGH, triacylglycerol hydrolase;
MAFP, methyl arachidonyl fluorophosphonate; ABPP, activity-based protein
profiling; TLC, thin layer chromatograpy; HPLC, high pressure liquid
chromatography.
Figure 1. FAAH is the only characterized mammalian enzyme
that is in the amidase signature family that bears an unusual
catalytic Ser-Ser-Lys triad. Most of the recently reported
inhibitors contain functional groups that are capable of forming
a covalent bond with the enzyme catalytic serine, either
10.1021/jm801042a CCC: $40.75
2009 American Chemical Society
Published on Web 12/10/2008

EValuation of Benzothiazole-Based Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 171
Scheme 1a
a Reagents and conditions: (i) DMAP, EDCI, DCM, 12 h; (ii) HCl, dioxane, MeOH, 2 h; (iii) P-BEMP, DMA, 100 °C, microwave, 90 s.
Scheme 2a
Scheme 3a
a Reagents and conditions: (i) Step (i) in Scheme 1; (ii) R3X, Pd(PPh3)4,
K2CO3, THF, reflux.
Scheme 4a
a Reagents and conditions: (i) NaHCO3, acetone, H2O; (ii) Step (i) in
Scheme 1; (iii) pyridine, THF; (iv) NaH, THF, 80 °C.
irreversibly or reversibly. Various reactive carbamate or urea-
containing inhibitors have been identified, including 1a (URB-
597),9 1b (LY2183240),19 1c (BMS-1),20 and 1d (PF-622)21
and are believed to irreversibly acylate active site Ser241,
thereby inactivating the enzyme. Compound 1a had been shown
to exhibit anxiolytic-like,9 antidepressant-like22 and analgesic
activities,23 and has undergone extensive preclinical character-
izations. However, inhibitors with such structures tend to be
nonselective toward off-target serine hydrolases and may result
in unwanted side effects.24,25 Another reported class of FAAH
inhibitors contains the R-keto-heterocylcles such as 2a (OL-
92) and 2b (OL-135).26 It was proposed that the electrophilic
carbonyl in these molecules forms a reversible hemiacetal bond
with the catalytic serine, conferring extraordinary potency.
Extensive structure-activity relationship studies have been
performed and the correlation between the electrophilicity of
the carbonyl and the potency of the inhibitors further supported
the covalent nature of the enzyme-inhibitor interactions.27 This
class of inhibitors was shown to have much improved selectivity
over common off-targets such as triacylglycerol hydrolase
(TGH) and various carboxypeptidases.24,25 Compound 2b was
shown to elevate AEA levels in the brain and spinal cord, exhibit
augmented behavioral response to anandamide,24 and demon-
strate efficacy in various pain models,28 further validating
chemical intervention of FAAH as a viable therapeutic approach.
a Reagents and conditions: (i) i-PrOH, 60 °C; (ii) Step (i) in Scheme 1.
reacting group. The synthesis and SAR studies of these
compounds are presented herein.
Chemistry
Preparations of these benzothiazole analogues are shown in
Scheme 1-4. Different substitutions on the piperidine nitrogen
could be incorporated by sulfonylation or acylation of piperidine
analogue 6b (Scheme 1), which was obtained via acid-catalyzed
deprotection of 6a following coupling of acid 5 with aniline 4.
Analogues with different R2 azacycles (15a-d) were prepared
by acylation of aniline 4 with corresponding sulfonylated acids
7a-d (Scheme 2). Aniline analogue 15e was obtained by
displacement of sulfonate 7e with aniline 4. R3s were introduced
either by acylation of the appropriately substituted anilines 8a-c
or Suzuki coupling of the boronic ester intermediate 10 with
halogen-substituted heterocycles (Scheme 3). Phenylthiazole
analogues 17a-c (Scheme 4) were generated by condensation
of substituted 2-bromoacetophenones with 4-amino thiobenza-
mide, followed by acylation of the resulting aniline with acid
7.
The therapeutic potential of FAAH inhibition clearly calls
for other novel, potent, and selective inhibitors. Toward this
goal, a high-throughput screening (HTS) assay was conducted
on in-house compound selections.29 Benzothiazole analogue 3
was identified as a potent inhibitor with no obvious serine-
Enzymatic Assay
Crude rat FAAH enzyme was isolated from rat cortex. The
human full-length enzyme was isolated from an HEK293 stable

172 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Wang et al.
Table 1. IC50 Values for Compounds of General Structures 13a-h, 14a
To Inhibit Rat FAAH
Table 2. IC50 Values for Compounds 15a-e
compd
X
R1
IC50 (nM)a
3
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
CO
thiophene-2-yl
-CH2CH3
-CH(CH3)2
-N(CH3)2
-C6H5
2-F-C6H5
2-Cl-C6H5
3-Cl-C6H5
4-Cl-C6H5
thiophene-2-yl
18 ( 8
13a
13b
13c
13d
13e
13f
13g
13h
14a
109 ( 37
63 ( 31
48 ( 24
13 ( 7
29 ( 4
263 ( 52
1780 ( 1200
>10000
2370 ( 620
a IC50 ) 50% inhibitory concentration against FAAH prepared from rat
cortex from 9 concentrations (data are expressed as means ( SD from at
least two experiments).
cell line. A tritiated-anandamide carbon filtration assay for
FAAH activity was adapted from Wilson et al.30 Upon incuba-
tion of the enzyme, the compound and tritiated anandamide,
the reaction was quenched with 0.7 M HCl. Hydrolysis of
anandamide by FAAH was assessed by filtering the incubation
mixture through activated carbon and determining the amount
of radiolabeled tritiated ethanolamine product. Eleven different
concentrations (over a range of 4.5 logs) of each compound
were assayed in duplicate and IC50 values were derived.
a IC50 ) 50% inhibitory concentration against FAAH prepared from rat
cortex (data are expressed as means ( SD from at least two experiments).
b Racemic mixtures.
fold loss in potency. Shown in Figure 2a is the postulated
binding mode for an R-keto oxazole analogue 2c, a reversible
inhibitor similar to 2b, proposed by Boger et al.,26 (green
carbons), which is superimposed onto the X-ray crystallographic
structure of the irreversible inhibitor 1e (methyl arachidonyl
fluorophosphonate,32 MAFP; cyan carbons). The R-keto oxazole
forms a reversible hemiketal with a catalytic serine (Ser-241)
in the FAAH active site. In Figure 2b, we show an overlay of
2c (green carbons) and the binding mode proposed for 3 (orange
carbons). It can be seen that the sulfone in 3 mimics the
tetrahedral arrangement at the covalent attachment site that forms
the tetrahedral intermediate in the R-keto oxazole 2c. This
proposition would certainly indicate that the amide carbonyl is
shifted comparing to that of 2c and unable to interact with Ser-
241. The significance of sulfonyl group to the binding potency
and the noncritical role played by the carbonyl group in 3
suggest that the class of compounds presented here may
represent a novel mechanism of inhibition of the FAAH enzyme,
distinct from acting either as an acylating agent or an electro-
philic carbonyl ready for hemiketal formation. This putative
binding mode will be further discussed in the molecular shape
overlay study.
Subsequently, studies were conducted on the bicyclic het-
eroaryl portion of 3. Benzothiazole was much more potent than
the thiazole analogue (16a vs 16d) as shown in Table 3,
suggesting the interactions of this moiety with the enzyme are
mainly hydrophobic in nature. This was confirmed by surveying
several benzo-fused heterocycle analogues. Quinoline 16e, a
bioisostere of benzothiazole, remained as potent. As the
heterocycles shifted to more polar quinoxaline (16f), benzoox-
azole (16b) and benzimidazole (16c), the potency dropped
progressively.
Results and Discussion
Different groups on the piperidine nitrogen were first
investigated (Table 1). While small sulfonyl groups were
tolerated (13a-c), it was the phenyl group, a thiophene
bioisostere (13d), that maintained most of the potency. Small
ortho-fluoro and -chloro substitutions on the phenyl ring were
tolerated (13e,f), while the meta- and para-substituted analogues,
however, saw gradual loss of potencies (13g and h), indicating
potential unfavorable interaction of the para-substituent with
the enzyme. Therefore, 2-thiophenesulfonyl group was kept at
this site for further SAR studies. Carbonyl replacement (14a)
of the sulfonyl led to significant drop in potency, which implies
the possibility of the sulfonyl group mimicking the charge
distribution of the tetrahedral intermediate in, for example, the
reversible hemiketal formed by R-keto-heterocylcle analogue
2b shown in Figure 1.
Next, ring sizes and connecting patterns of the linking
azacycles (Table 2) were evaluated. Compound with 1,3-
substituted piperidine ring (15a) was completely inactive, and
aztidine analogue 15b was much weaker. The 1,3-substituted
pyrrolidine (15c), nevertheless, was only 3 times weaker.
Introduction of an extra methylene linker between piperidine
and the amide group, such as compound 15d, completely
abolished the binding potency, highlighting the importance of
having the right length and shape of the linking group between
two parts of the molecule.
A notable observation was that the des-carbonyl aniline
analogue 15e had almost identical potency as that of 3, indicating
that the carbonyl is not critical for the inhibitory potency and
is in sharp contrast to compounds like 1a or 2a. In those two
cases, the carbonyl was essential for the covalent bond formation
and high inhibitory potency. A recent analysis by Kimball et
al.31 shows that loss of the electrophilic carbonyl in R-keto
oxazole analogues (of which 2b is a member) results in >1000-
The substituents on the benzothiazole ring were also probed
(Table 3). Even though 6-methoxy analogue 16h was slightly
weaker than 3, other substituents such as 6-choro (16g), the
unsubstituted (16a), and 4-methyl (16i) analogues were all
equally potent as 3. This is in qualitative agreement with the

EValuation of Benzothiazole-Based Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 173
Figure 2. Stereo views of inhibitor binding modes for FAAH. (a) Superimposed on the crystallographic bound conformation of fatty acid inhibitor
MAFP (cyan carbons) is the reversible R-keto oxazole inhibitor 2c (green carbons) with a bound conformation analogous to that proposed by Boger
et al.26 (b) The R-keto oxazole inhibitor 2c shown with putative binding mode for compound 3 (orange carbons). The sulfonyl group in 3 mimics
the charge distribution of the R-keto tetrahedral intermediate created by the covalent bond with SER-241. The surface shown in gray indicates the
posterior region of the pocket, which is predominantly hydrophobic in nature.
SAR findings of the insensitivity of substitution on the distal
phenyl ring of 2b by Hardouin et al.,33 where according to our
modeling study, occupies the same hydrophobic pocket as the
benzothiazole of 3. The 4-methoxy analog 16j, however, saw
much boosted potency, resulting in the most active analogue in
this series. The improvement of potency of 16j over 16h may
indicate that larger groups at this site can be accommodated.
This hypothesis was put to test by preparing the 4-phenyl
substituted thiazole analogues. These analogues were also shown
to be very potent FAAH inhibitors (Table 3, 17a-c), in the
same range as their benzothiazole parent compounds. This
indicates that there is probably a large binding pocket in this
region and the potency can potentially be further optimized.
Human FAAH Time-Dependent Preincubation Study. To
determine the likely mechanism of the inhibition, a time-
dependent inhibition study was performed using human FAAH.
The homology between human and rat FAAH is very high and
little species differences26 were observed for active ketone-based
inhibitors. Our data (not shown) also indicated that this class
of compounds exhibited similar potency against human and rat

174 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Wang et al.
Table 3. IC50 Values for Compounds with General Structures 16 and 17
Table 4. IC50 (nM) Values of FAAH Inhibitor at Different
Preincubation Times
compound
0a
5 min
30 min
60 min
1a
2a
3
2187 ( 228b
31 ( 8
11 ( 2
47 ( 7
17 ( 4
20 ( 6
16 ( 2
16 ( 2
34 ( 12
11 ( 2
12 ( 2
19 ( 6
a PreIncubation time of the inhibitor with human FAAH prior to addition
of substrate. b IC50 ) 50% inhibitory concentration against human FAAH
prepared from a HEK-293-based stable cell line expressing recombinant
human FAAH (data are expressed as means ( SD from at least three
experiments).
different between 3 and 2a since, as discussed earlier, ben-
zothiazole compounds are unlikely to form a covalent bond with
the enzyme.
Activity-Based Protein Profiling Study. With the use of a
modified activity-based protein profiling (ABPP) procedure
that was first developed by Leung et al.,36 we have profiled
some known FAAH inhibitors in both rat and human tissues
previously.25 The same screening assay was conducted on
compound 3, along with several known FAAH inhibitors, in
different rat tissues to characterize its selectivity against other
serine hydrolases (Table 5). The color scheme in each box
indicates how selective (red being most nonselective, and green
being most selective) the inhibitor is in this tissue. Number of
off-target protein bands and molecular weights are also shown
in each box. Some of the known irreversible inhibitors such as
1c and 1f (LY2077855),37 a close analogue of 1b, are poorly
selective and multiple protein bands were missing due to the
off-target binding. Compound 1a had significantly improved
selectivity over 1c but still not nearly as selective as the
reversible ketone inhibitor 2b. Compound 3 was exclusively
specific against FAAH in rat brain and had no missing protein
bands in all the other tissues that were tested. No off-targets
such as triacylglycerol hydrolase (TGH), an uncharacterized
membrane-associated hydrolase KIAA 1363, or carboxy es-
terases were observed. The potency determined via this approach
was close to that obtained using the tritiated-anandamide
enzymatic assay (data not shown).
Molecular Shape Overlapping Study. To explore potential
interactions for the class of inhibitors in the present study we
used the X-ray crystallographic bound conformation of MAFP
as a guide in directing placement of plausible bound conforma-
tions for compound 3, which served to constrain the overlays
and maximize the shape and electrostatic similarity with the
bound MAFP conformation.
a IC50 ) 50% inhibitory concentration against FAAH prepared from rat
cortex (data are expressed as means ( SD from at least two experiments).
b n ) 1.
The key features of the resulting pose are shown in Figure
2b (orange carbons), which exhibits a similar position and
orientation as found in other reported docking studies.26,38 The
sulfonyl group of compound 3 (shown in yellow) occupies a
similar location to the acyl moiety of bound MAFP (cyan
carbons in Figure 2a), and can engage in similar hydrogen
bonding to catalytic residues, for example, Ser-217 and Ser-
241. The placement of the benzothiazole heterocycle occupies
a similar region as the hydrocarbon tail in MAFP, which extends
into the predominantly hydrophobic pocket created by residues
such as Phe-381, Ile-491, Tyr-335 and Phe-432. This proposed
binding mode also appears to provide rationalization for the
majority of observed SAR. In particular, it explains the loss of
potency observed with the more polar benzooxazole (16b) and
benzimidazole (16c) analogues, and shows the general tolerance
for the variations present in 16a, 16g-i, and 17a-c. There is
a clear indication for the potential source of the insensitivity to
the loss of carbonyl in 15e, and additionally it gives justification
FAAH. A study in which the preincubation times of the inhibitor
and the enzyme were varied was carried out. In principle, if a
compound inhibits the enzyme via an irreversible mechanism,
upon prolonged preincubation the potency should become
greater; a constant IC50, conversely, supports a reversible
mechanism. This type of time-dependent preincubation study
had been used to characterize mechanism of inhibition of both
FAAH inhibitors34 and other enzyme inhibitors.35
Time-dependent increase of potency was observed for 1a
(Table 4), from micromolar to nanomolar within 10 min of
preincubation. Compound 2a, to the contrary, exhibited no
increase of potency upon prolonged incubation, supporting
literature characterization of it being a reversible inhibitor.26
Benzothiazole compound
3 showed no time-dependent
improvement of potency, suggesting it might also be a reversible
inhibitor. However, the mechanisms of action are most likely

EValuation of Benzothiazole-Based Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 175
Table 5. Selectivity Profiling of FAAH Inhibitors in Representative Rat and Human Tissues
a The number of affected protein bands, if any, with the approximate range of molecular weight (in parentheses) is indicated for each inhibitor in a
particular tissue proteome. b The relative selectivity of FAAH inhibitors is categorized into four classes that are represented by different color schemes:
exceptionally selective (green) with no visible change in band patterns except in rat brain the only protein band with diminished labeling is the 60 kDa
FAAH band, highly selective (yellow) with only 1-2 visible missing or diminished protein bands, moderately selective (orange) with 3-5 visible affected
protein bands, and poorly selective (red) with five or more affected protein bands. c These data were reported previously25 and were included for comparison.
for the loss of potency observed with the carbonyl isostere
analogue 14a.
that these compounds are not acylating agents. The selectivity
of compound 3 against other serine hydrolases is exceptional,
with no off-target proteins identified in multiple rat tissues. The
structural novelty, exquisite potency, exceptional selectivity and
unique mechanism of action for these inhibitors make them
appealing compounds for the study of FAAH-related pharma-
cologies, and the endocannabinoid system.
Conclusions
In the current study, we investigated the structure-activity
relationship of a novel series of benzothiaozle analogues as
potent FAAH inhibitors that sprung from a HTS campaign.
Smaller groups on the sulfonyl are tolerated but thiophene (3)
and phenyl analogues gave the best potency. A piperidine ring
seems to be optimal for activity, while the pyrrolidine analogue
is only 3 times weaker. The interaction between the benzothia-
zole moiety with the enzyme is mainly driven by hydrophobic-
ity, which was manifested by the weaker potency of the thiazole
analogue 16d, as well as the diminished activity of the
corresponding more polar benzoxazole (16b) and benzimidazole
(16c) analogues. Among the substituted benzothiazoles, 4-meth-
oxy analogue 16j provided the best potency (IC50 ) 1.7 nM)
and resulted in 10-fold improvement over HTS hit 3. Similar
substituted phenyl thiazole analogues are also potent FAAH
inhibitors suggesting that this site could allow for further
optimization.
Other two major SAR finding are that the sulfonyl group was
critical and the amide linkage was not necessary, indicating that
these compounds act in a distinctive manner from most of the
previously reported classes of inhibitors. Molecular overlay of
compound 3 with known FAAH inhibitor MAFP suggested
hydrogen bonds between the sulfonyl group and catalytic serine
residues, which form the tetrahedral hemiacetal bonds for the
R-ketoheterocycle inhibitors such as 2a. This putative binding
mode also revealed the hydrophobic interactions of the ben-
zothiazole moiety with the enzyme pocket that is occupied by
the hydrocarbon tail in MAFP and is consistent with major SAR
findings. The potencies of inhibitor 3 at different preincubation
times showed no time-dependent change, was consistent with
a possible reversible mechanism and provided further evidence
Experimental Section
Chemistry. Unless otherwise noted, all solvents, including
anhydrous solvents and chemicals were purchased from Aldrich
Co. and/or Acros Organics, and used without further purification.
Melting points were recorded on a Unimelt apparatus (Arthur H.
Thomas Company, Philadelphia, PA) and are uncorrected. Proton
nuclear magnetic resonance (1H NMR) spectra were recorded at
300 MHz (Varian Mercury 300) using deuterate solvent and TMS
as the internal standard. Splitting patterns are designated as singlet
(s), doublet (d), triplet (t), quartet (q), and multiplet (m). NMR
coupling constants (J) in hertz are indicated parenthetically. Low-
resolution mass spectra were obtained with a Finnigan SSQ7000
single quad mass spectrometer. Elemental analyses were performed
by Quantitative Technologies, Inc., Whitehouse, NJ. Analytical thin
layer chromatography (TLC) was carried out on E. Merck TLC
plates coated with silica gel 60 F254 (0.25 mm layer thickness).
TLC visualization was carried out using either a UV lamp and/or
charring solution as indicated. Flash chromatography was performed
on a Biotage Flash 40 chromatography system (Charlottesville, VA)
using 40, 90, or 120 g cartridges at 32-63 µm, 60 Å silica gel.
Solvent mixtures used for TLC and flash chromatography are
reported in v/v total. Preparative HPLC was performed on a Waters
Symmetry C8 column (25 mm × 100 mm, 7 µm particle size) using
a gradient of 10-100%/0.1% aqueous TFA over 8 min (10 min
run time) at a flow rate of 40 mL/min.
tert-Butyl 4-(4-(6-Methylbenzo[d]thiazol-2-yl)phenylcarbam-
oyl)piperidine-1-carboxylate (6a). To a solution of 1-(tert-
butoxycarbonyl)piperidine-4-carboxylic acid (5, 2.29 g, 10 mmol)
and 4-(6-methylbenzo[d]thiazol-2-yl)aniline (4, 2.40 g, 10 mmol)

176 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Wang et al.
in dichloromethane in a vial was added 4-dimethylamino pyridine
(24.4 mg, 0.2 mmol) followed by addition of ethyl dimethyamino
cabodiimide hydrochloride (2.87 g, 15 mmol) and the mixture was
stirred overnight. The reaction was concentrated and water was
added to the residue. Upon sonication, the solid was collected. It
was triturated with isopropyl alcohol and filtered to give tert-butyl
4-(4-(6-methylbenzo[d]thiazol-2-yl)phenylcarbamoyl)piperidine-1-
carboxylate as off-while solid (6a, 3.9 g, 86%). 1H NMR (300 MHz,
DMSO-d6) δ ppm 1.41 (s, 9 H) 1.45-1.57 (m, 2 H) 1.77-1.85
(m, 2 H) 2.45 (s, 3 H) 2.51-2.61 (m, 1 H) 2.74-2.82 (m, 2 H)
3.99-4.03 (m, 2 H) 7.27-7.40 (m, 1 H) 7.80 (d, J ) 8.82 Hz, 2
H) 7.87-7.93 (m, 2 H) 7.98-8.04 (m, 2 H) 10.23 (s, 1 H); MS
(DCI/NH3) m/z 452 (M + H)+.
N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)piperidine-4-car-
boxamide (6b). To a suspension of 6a (3.61 g, 8 mmol) in ethyl
acetate (20 mL) and methanol (5 mL) was added HCl in dioxane
(4 N, 30 mL) and the reaction was stirred at room temperature
overnight. The solid was filtered to give the HCl salt of 6b (2.68
g, 87%). 1H NMR (300 MHz, DMSO-d6) δ ppm 1.81-1.96 (m, 2
H) 1.97-2.08 (m, 2 H) 2.49 (s, 3 H) 2.69-2.83 (m, 1 H) 2.88-3.02
(m, 2 H) 3.31-3.43 (m, 2 H) 7.36-7.41 (m, 1 H) 7.82-7.90 (m,
2 H) 7.91-7.96 (m, 2 H) 8.02-8.09 (m, 2 H) 8.76 (br, 1 H) 9.13
(br, 1 H) 10.58 (s, 1 H); MS (DCI/NH3) m/z 352 (M + H)+.
1-(Thiophen-2-ylsulfonyl)piperidine-4-carboxylic Acid (7). To
a solution of isonipecotic acid (1.29 g, 10 mmol) and sodium
bicarbonate (920 mg, 11 mmol) in water/acetone (1:1, 15 mL) was
added a solution of thiophene-2-sulfonyl chloride (1.82 g, 10 mmol)
in acetone (8 mL). After 1 h, the solid was collected, dissolved in
dichloromethane and triturated with hexane. The solid was filtered
and dried in vacuo to give the title compound (7, 1.35 g, 50%). 1H
NMR (300 MHz, DMSO-d6) δ ppm 1.52-1.65 (m, 2 H) 1.86-1.97
(m, 2 H) 2.26-2.36 (m, 1 H) 2.43-2.55 (m, 2 H) 3.42-3.50 (m,
2 H) 7.25-7.34 (m, 1 H) 7.62 (dd, J ) 3.73, 1.36 Hz, 1 H)
7.97-8.06 (m, 1 H) 12.28 (br, 1H); MS (DCI/NH3) m/z 276
(M+H)+, 293 (M + NH4)+.
sulfonyl chloride (3.64 g, 20 mmol) in pyridine (15 mL) at -15
°C. The reaction was stirred for 1 h before it was placed in a
refrigerator (-5.5 °C). The cold solution of the reaction mixture
was poured in crushed ice (30 g) and stirred for 1 h. The solid was
filtered and dried to afford the title compound as an off-white solid
(2.75 g, 84%). 1H NMR (300 MHz, DMSO-d6) δ ppm 1.13-1.28
(m, 2 H) 1.62-1.72 (m, 3 H) 2.24-2.33 (m, 2 H) 3.59 (d, J )
11.87 Hz, 2 H) 3.98 (d, J ) 5.76 Hz, 2 H) 7.25 - 7.30 (m, 2 H)
7.60 (dd, J ) 3.73, 1.36 Hz, 1 H) 7.86 (dd, J ) 3.73, 1.36 Hz, 1
H) 8.03 (dd, J ) 5.09, 1.36 Hz, 1 H) 8.16 (dd, J ) 5.09, 1.36 Hz,
1 H); MS (DCI/NH3) m/z 425 (M + NH4)+.
N-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1-
(thiophen-2-ylsulfonyl)piperidine-4-carboxamide (10). Com-
pound 10 was prepared in a similar manner to the synthesis of 6a,
substituting 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
(9) for compound 4 in 55% yield. 1H NMR (300 MHz, DMSO-d6)
δ ppm 1.27 (s, 12 H) 1.61-1.75 (m, 2 H) 1.88-1.94 (m, 2 H)
2.33-2.47 (m, 3 H) 3.60-3.70 (m, 2 H) 7.28-7.32 (m, 1 H) 7.58
(s, 4 H) 7.66 (dd, J ) 3.73, 1.36 Hz, 1 H) 8.06 (dd, J ) 5.09, 1.36
Hz, 1 H) 9.94 (s, 1 H); MS (DCI/NH3) m/z 477 (M + H)+.
N-(4-(4-(3-Methoxyphenyl)thiazol-2-yl)phenyl)-1-(thiophen-
2-ylsulfonyl)piperidine-4-carboxamide (12a). A solution of 4-ami-
nobenzothioamide (268 mg, 1.76 mmol) and 2-bromo-1-(3-
methoxyphenyl)ethanone (404 mg, 1.76 mmol) in isopropyl alcohol
(15 mL) was heated at 60 °C for 2 h. The reaction was cooled to
0 °C and the solid was collected and washed with cold isopropyl
alcohol to obtain the title compound (336 mg, 69%). 1H NMR (300
MHz, DMSO-d6) δ ppm 3.84 (s, 3 H) 6.92-6.96 (m, 3 H) 7.38 (t,
J ) 7.97 Hz, 1 H) 7.54-7.63 (m, 2 H) 7.83-7.87 (m, 2 H) 8.05
(s, 1 H); MS (DCI/NH3) m/z 283 (M + H)+.
N-(4-(4-(4-Methoxyphenyl)thiazol-2-yl)phenyl)-1-(thiophen-
2-ylsulfonyl)piperidine-4-carboxamide (12b). Compound 12b was
prepared in a similar manner to the synthesis of compound 12a,
substituting 2-bromo-1-(4-methoxyphenyl)ethanone for 2-bromo-
1
1-(3-methoxyphenyl)ethanone in 70% yield. H NMR (300 MHz,
1-(Thiophen-2-ylsulfonyl)piperidine-3-carboxylic Acid (7a).
Compound 7a was prepared in a similar manner to the synthesis
of compound 7, substituting nipecotic acid for isonipecotic acid in
50% yield. 1H NMR (300 MHz, DMSO-d6) δ ppm 1.31-1.45 (m,
1 H) 1.51-1.57 (m, 1 H) 1.70-1.86 (m, 2 H) 2.47-2.60 (m, 3 H)
3.29-3.37 (m, 1 H) 3.51-3.53 (m, 1 H) 7.26-7.31 (m, 1 H) 7.64
(dd, J ) 3.73, 1.36 Hz, 1 H) 8.05 (dd, J ) 5.09, 1.36 Hz, 1 H);
MS (DCI/NH3) m/z 276 (M + H)+, 293 (M + NH4)+.
DMSO-d6) δ ppm 3.81 (s, 3 H) 6.95-6.98 (m, 2 H) 6.99-7.05
(m, 2 H) 7.83-7.88 (m, 3 H) 7.93-7.98 (m, 2 H); MS (DCI/NH3)
m/z 283 (M + H)+.
N-(4-(4-(4-Chlorophenyl)thiazol-2-yl)phenyl)-1-(thiophen-2-
ylsulfonyl)piperidine-4-carboxamide (12c). Compound 12c was
prepared in a similar manner to the synthesis of compound 12a,
substituting 2-bromo-1-(4-chlorophenyl)ethanone for 2-bromo-1-
(3-methoxyphenyl)ethanone in 61% yield. 1H NMR (300 MHz,
DMSO-d6) δ ppm 7.00-7.02 (m, 2 H) 7.51-7.56 (m, 2 H)
7.87-7.91 (m, 2 H) 8.03-8.08 (m, 2 H) 8.12 (s, 1 H); MS (DCI/
NH3) m/z 287 (M + H)+.
General Procedure for the Preparation of Sulfonamide
Analogues 13a-13h. A Smith Porcess vial (0-2 mL) was charged
with a stir bar and PS-BEMP resin (5 equiv). To the vessel was
added the sulfonyl chloride monomer (1.2 equiv) in dimethylac-
etamide (1 mL) and the amine 6b (1 equiv) in dimethylacetamide
(0.3 mL). The reaction vessel was sealed and heated to 100 °C for
900 s in an Ermys Optimizer. After cooling, the reaction mixture
was filtered through a prepacked column of celite. The reaction
products were collected and concentrated to dryness. The residues
were dissolved in 1:1 DMSO/MeOH and purified by reverse phase
HPLC.
1-(Thiophen-2-ylsulfonyl)azetidine-3-carboxylic Acid (7b).
Compound 7b was prepared in a similar manner to the synthesis
of compound 7, substituting azitidine-3-carboxylic acid for isonipe-
1
cotic acid in 81% yield. H NMR (300 MHz, DMSO-D6) δ ppm
3.70-3.81 (m, 3 H) 3.96 (t, J ) 8.82 Hz, 2 H) 7.32-7.38 (m,1 H)
7.77 (dd, J ) 3.73, 1.36 Hz, 1 H) 8.14 (dd, J ) 5.09, 1.36 Hz, 1
H)12.68 (s, 1 H); MS (DCI/NH3) m/z 248 (M + H)+, 265 (M +
NH4)+.
1-(Thiophen-2-ylsulfonyl)pyrrolidine-3-carboxylic Acid (7c).
Compound 7c was prepared in a similar manner to the synthesis
of compound 7, substituting pyrrolidine-3-carboxylic acid for
isonipecotic acid in 72% yield. 1H NMR (300 MHz, DMSO-d6) δ
ppm 1.80-2.05 (m, 2 H) 2.91-3.02 (m, 1 H) 3.17-3.30 (m, 2 H)
3.34-3.49 (m, 2 H) 7.28 (dd, J ) 5.09, 3.73 Hz, 1 H) 7.70 (dd, J
) 3.73, 1.36 Hz, 1 H) 8.04 (dd, J ) 5.09, 1.36 Hz, 1 H) 12.54 (s,
1 H); MS (DCI/NH3) m/z 262 (M + H)+, 279 (M + NH4)+.
2-(1-(Thiophen-2-ylsulfonyl)piperidin-4-yl)acetic Acid (7d).
Compound 7d was prepared in a similar manner to the synthesis
of compound 7, substituting piperidin-4-ylacetic acid for isonipe-
1-(Ethylsulfonyl)-N-(4-(6-methylbenzo[d]thiazol-2-yl)phenyl)pi-
peridine-4-carboxamide (13a). The title compound was obtained
1
in 48% yield. H NMR (300 MHz, DMSO-d6) δ ppm 1.18-1.28
(m, 3 H) 1.58-1.73 (m, 2 H) 1.92 (dd, J ) 13.39, 2.88 Hz, 2 H)
2.46 (s, 3 H) 2.52-2.59 (m, 1 H) 2.80-2.94 (m, 2 H) 3.01-3.12
(m, 2 H) 3.59-3.72 (m, 2 H) 7.34 (dd, J ) 8.14, 1.36 Hz, 1 H)
7.80 (d, J ) 8.82 Hz, 2 H) 7.87-7.92 (m, 2 H) 8.01 (d, J ) 8.82
Hz, 2 H) 10.24 (s, 1 H); MS (DCI/NH3) m/z 444 (M + H)+.
1-(Isopropylsulfonyl)-N-(4-(6-methylbenzo[d]thiazol-2-yl)phe-
nyl)piperidine-4-carboxamide (13b). The title compound was
obtained in 54% yield. 1H NMR (300 MHz, DMSO-d6) δ ppm
1.23 (d, J ) 6.78 Hz, 6 H) 1.65 (td, J ) 12.12, 3.56 Hz, 2 H) 1.89
(dd, J ) 13.22, 2.71 Hz, 2 H) 2.46 (s, 3 H) 2.53-2.62 (m, 1 H)
2.94 (td, J ) 12.29, 2.20 Hz, 2 H) 3.32-3.39 (m, 1 H) 3.65-3.69
1
cotic acid in 47% yield. H NMR (300 MHz, DMSO-d6) δ ppm
1.20-1.31 (m, 2 H) 1.58 - 1.66 (m, 1 H) 1.70-1.79 (m, 2 H)
2.14 (d, J ) 6.78 Hz, 2 H) 2.26-2.35 (m, 2 H) 3.58-3.62 (m, 2
H) 7.28 (dd, J ) 5.09, 3.73 Hz, 1 H) 7.61 (dd, J ) 3.73, 1.36 Hz,
1 H) 8.04 (dd, J ) 5.09, 1.36 Hz, 1 H) 12.02 (s, 1 H); MS (DCI/
NH3) m/z 290 (M + H)+, 307 (M + NH4)+.
(1-(Thiophen-2-ylsulfonyl)piperidin-4-yl)methyl Thiophene-
2-sulfonate (7e). To a solution of 4-piperidinemethanol (920 mg,
8 mmol) in pyridine (5 mL) was added a solution of thiophene-2-

EValuation of Benzothiazole-Based Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 177
1
(m, 2 H) 7.34 (dd, J ) 7.97, 1.53 Hz, 1 H) 7.79-7.81 (m, 2 H)
7.87-7.92 (m, 2 H) 7.98-8.04 (m, 2 H) 10.24 (s, 1 H); MS (DCI/
NH3) m/z 458 (M + H)+.
for 5 in 72% yield. H NMR (300 MHz, DMSO-d6) δ ppm 2.45
(s, 3 H) 3.41-3.56 (m, 1 H) 3.87-4.01 (m, 4 H) 7.32-7.41 (m, 2
H) 7.69 (m, 2 H) 7.77-7.81 (m, 1 H) 7.85-7.95 (m, 2 H)
8.00-8.02 (m, 2 H) 8.14-8.18 (m, 1 H) 10.28 (s, 1 H); MS (DCI/
NH3) m/z 470 (M + H)+.
N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-2-yl-
sulfonyl)pyrrolidine-3-carboxamide (15c). The title compound
was prepared in a similar manner to the synthesis of 6a, substituting
7b for 5 in 76% yield. 1H NMR (300 MHz, DMSO-d6) δ ppm
1.85-1.99 (m, 1 H) 2.03-2.15 (m, 1 H) 2.45 (s, 3 H) 3.12 (m, 1
H) 3.25-3.38 (m, 3 H) 3.56 - 3.60 (m, 1 H) 7.27-7.37 (m, 2 H)
7.71-7.75 (m, 3 H) 7.87-7.92 (m, 2 H) 7.98-8.07 (m, 3 H) 10.32
(s, 1 H); MS (DCI/NH3) m/z 484 (M + H)+.
N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-2-(1-(thiophen-2-
ylsulfonyl)piperidin-4-yl)acetamide (15d). The title compound
was prepared in a similar manner to the synthesis of 6a, substituting
7d for 5 in 40% yield. 1H NMR (300 MHz, DMSO-d6) δ ppm
1.23-1.38 (m, 2 H) 1.73-1.85 (m, 3 H) 2.27-2.43 (m, 4 H) 2.45
(s, 3 H) 3.60-3.64 (m, 2 H) 7.26-7.31 (m, 1 H) 7.32-7.35 (m 1
H) 7.62 (dd, J ) 3.73, 1.36 Hz, 1 H) 7.75-7.78 (m, 2 H) 7.87-7.92
(m, 2 H) 7.97-8.06 (m, 3 H) 10.19 (s, 1 H); MS (DCI/NH3) m/z
512 (M + H)+.
1-(N,N-Dimethylsulfamoyl)-N-(4-(6-methylbenzo[d]thiazol-2-
yl)phenyl)piperidine-4-carboxamide (13c). The title compound
was obtained in 43% yield. 1H NMR (300 MHz, DMSO-d6) δ ppm
1.63-1.72 (m, 2 H) 1.84-1.94 (m, 2 H) 2.46 (s, 3 H) 2.52-2.59
(m, 1 H) 2.76 (s, 6 H) 2.83-2.90 (m, 2 H) 3.57-3.67 (m, 2 H)
7.34 (dd, J ) 8.31, 1.86 Hz, 1 H) 7.75-7.84 (m, 2 H) 7.87-7.92
(m, 2 H) 7.99-8.03 (m, 2 H) 10.26 (s, 1 H); MS (DCI/NH3) m/z
459 (M + H)+.
N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(phenylsulfo-
nyl)piperidine-4-carboxamide (13d). The title compound was
1
obtained in 45% yield. H NMR (300 MHz, DMSO-D6) δ ppm
1.59-1.73 (m, 2 H) 1.87-1.93 (m, 2 H) 2.31-2.41 (m, 3 H) 2.45
(s, 3 H) 3.59-3.73 (m, 2 H) 7.34 (dd, J ) 7.97, 1.53 Hz, 1 H)
7.63-7.70 (m, 2 H) 7.71-7.80 (m, 5 H) 7.87-7.92 (m, 2 H)
7.96-8.03 (m, 2 H) 10.13 (s, 1 H); MS (DCI/NH3) m/z 492
(M + H)+.
1-(2-Fluorophenylsulfonyl)-N-(4-(6-methylbenzo[d]thiazol-2-
yl)phenyl)piperidine-4-carboxamide (13e). The title compound
1
was obtained in 35% yield. H NMR (500 MHz, DMSO-d6-D2O)
δ ppm 1.59-1.68 (m, 2 H) 1.87-1.94 (m, 2 H) 2.44-2.51 (m, 4
H) 2.62-2.67 (m, 2 H) 3.74-3.78 (m, 2 H) 7.36 (d, J ) 8.54 Hz,
1 H) 7.45-7.54 (m, 2 H) 7.75-7.84 (m, 4 H) 7.88-7.92 (m, 2 H)
8.00-8.01 (m, 2 H); MS (ESI) m/z 510 (M + H)+.
4-(6-Methylbenzo[d]thiazol-2-yl)-N-((1-(thiophen-2-ylsulfo-
nyl)piperidin-4-yl)methyl)aniline (15e). To a solution of 4-(6-
methylbenzo[d]thiazol-2-yl)aniline (120 mg, 0.5 mmol) and the 7e
(203 mg, 0.5 mmol) in tetrahydrofuran (10 mL) was added sodium
hydride (60%, 60 mg, 1.5 mmol) and heated at 80 °C overnight.
The reaction mixture was concentrated, taken up in water (20 mL)
and extracted with ethyl acetate (3 × 10 mL). The organics were
combined, dried and concentrated and the residue was purified by
flash chromatography (silica gel, 4:1 hexane/ethyl acetate) to obtain
1-(2-Chlorophenylsulfonyl)-N-(4-(6-methylbenzo[d]thiazol-2-
yl)phenyl)piperidine-4-carboxamide (13f). The title compound
1
was obtained in 31% yield. H NMR (500 MHz, DMSO-d6-D2O)
δ ppm 1.57-1.66 (m, 2 H) 1.85-1.92 (m, 2 H) 2.46 (s, 3 H)
2.51-2.57 (m, 1 H) 2.81-2.87 (m, 2 H) 3.76-3.80 (m, 2 H) 7.36
(d, J ) 8.24 Hz, 1 H) 7.57-7.62 (m, 1 H) 7.68-7.78 (m, 4 H)
7.87-7.92 (m, 2 H) 7.99-8.03 (m, 3 H); MS (ESI) m/z 526
(M + H)+.
1
a yellow solid (106 mg, 44%). H NMR (300 MHz, DMSO-d6) δ
ppm 1.21-1.36 (m, 2 H) 1.74-1.79 (m, 1 H) 1.82-1.86 (m, 2 H)
2.25-2.37 (m, 2 H) 2.42 (s, 3 H) 2.95-3.04 (m, 2 H) 3.62-3.71
(m, 2 H) 6.46 (t, J ) 5.59 Hz, 1 H) 6.66 (d, J ) 8.82 Hz, 2 H)
7.24-7.29 (m, 2 H) 7.60-7.65 (m, 1 H) 7.72-7.81 (m, 4 H)
8.01-8.05 (m, 1 H); MS (DCI/NH3) m/z 484 (M + H)+.
N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(thiophen-2-ylsulfonyl)pi-
peridine-4-carboxamide (16a). The title compound was prepared
in a similar manner to the synthesis of 6a, substituting 4-(1,3-
benzothiazol-2-yl)phenylamine for 4 and 7 for 5 in 77% yield. 1H
NMR (300 MHz, DMSO-d6) δ ppm 1.63-1.74 (m, 2 H) 1.95 (m,
2 H) 2.36-2.48 (m, 3 H) 3.64-3.71 (m, 2 H) 7.31 (dd, J ) 5.09,
3.73 Hz, 1 H) 7.41-7.56 (m, 2 H) 7.67 (dd, J ) 3.73, 1.36 Hz, 1
H) 7.78 (d, J ) 8.82 Hz, 2 H) 8.00-8.14 (m, 5 H) 10.17 (s, 1 H);
MS (DCI/NH3) m/z 484 (M + H)+.
1-(3-Chlorophenylsulfonyl)-N-(4-(6-methylbenzo[d]thiazol-2-
yl)phenyl)piperidine-4-carboxamide (13g). The title compound
1
was obtained in 27% yield. H NMR (500 MHz, DMSO-d6-D2O)
δ ppm 1.62-1.71 (m, 2 H) 1.89 (m, 2 H) 2.31-2.39 (m, 3 H) 2.46
(s, 3 H) 3.69 (m, 2 H) 7.36 (d, J ) 8.54 Hz, 1 H) 7.55-7.59 (m,
4 H) 7.73-7.77 (m, 2 H) 7.88-7.91 (m, 2 H) 8.00 (m, J ) 8.85
Hz, 2 H); MS (ESI) m/z 526 (M + H)+.
1-(4-Chlorophenylsulfonyl)-N-(4-(6-methylbenzo[d]thiazol-2-
yl)phenyl)piperidine-4-carboxamide (13h). The title compound
1
was obtained in 45% yield. H NMR (500 MHz, DMSO-d6-D2O)
δ ppm 1.67 (s, 2 H) 1.88 (s, 2 H) 2.29-2.37 (m, 3 H) 2.46 (s, 3 H)
3.70 (m, 2 H) 7.37 (s, 1 H) 7.49 (d, J ) 7.93 Hz, 2 H) 7.66 (d, J
) 8.24 Hz, 2 H) 7.75 (d, J ) 8.85 Hz, 2 H) 7.90 (d, J ) 8.24 Hz,
2 H) 8.00 (d, J ) 8.85 Hz, 2 H); MS (DCI/NH3) m/z 506 (M +
H)+.
N-(4-(6-Methylbenzo[d]oxazol-2-yl)phenyl)-1-(thiophen-2-yl-
sulfonyl)piperidine-4-carboxamide (16b). The title compound was
prepared in a similar manner to the synthesis of 6a, substituting
4-(6-methyl-benzooxazol-2-yl)-phenylamine for 4 and 7 for 5 in
30% yield. 1H NMR (300 MHz, DMSO-d6) δ ppm 1.63-1.78 (m,
2 H) 1.88-2.01 (m, 2 H) 2.37-2.48 (m, 6 H) 3.63-3.71 (m, 2 H)
7.19-7.22 (m, 1 H) 7.29-7.32 (m, 1 H) 7.55-7.70 (m, 3 H)
7.78-7.84 (m, 2 H) 8.05-8.14 (m, 3 H) 10.19 (s, 1 H); MS (DCI/
NH3) m/z 482 (M + H)+.
N-(4-(6-Methyl-1H-benzo[d]imidazol-2-yl)phenyl)-1-(thiophen-
2-ylsulfonyl)piperidine-4-carboxamide (16c). The title compound
was prepared in a similar manner to the synthesis of 6a, substituting
4-(5-methyl-1H-benzoimidazol-2-yl)-phenylamine for 4 and 7 for
5 in 57% yield. 1H NMR (300 MHz, DMSO-d6) δ ppm 1.63-1.78
(m, 2 H) 1.94 (m, 2 H) 2.34-2.47 (m, 6 H) 3.59-3.74 (m, 2 H)
7.00 (t, J ) 7.80 Hz, 1 H) 7.26-7.50 (m, 3 H) 7.66-7.78 (m, 3
H) 8.01-8.11 (m, 3 H) 10.04 (s, 1 H) 12.59 (d, J ) 10.51 Hz, 1
H); MS (DCI/NH3) m/z 481 (M + H)+.
N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(thiophene-2-
carbonyl)piperidine-4-carboxamide (14a). The title compound
was prepared in a similar manner to the synthesis of 6a, substituting
1
thiophene-2-carboxylic acid for 5 and 6b for 4 in 41% yield. H
NMR (300 MHz, DMSO-d6) δ ppm 1.58-1.72 (m, 2 H) 1.86-1.97
(m, 2 H) 2.46 (s, 3 H) 2.64-2.77 (m, 1 H) 3.09 (m, 2 H) 4.30 (m,
2 H) 7.13 (dd, J ) 5.09, 3.73 Hz, 1 H) 7.32-7.37 (m, 1 H) 7.42
(d, J ) 3.73 Hz, 1 H) 7.76 (d, J ) 4.41 Hz, 1 H) 7.80 (d, J ) 8.82
Hz, 2 H) 7.87-7.92 (m, 2 H) 8.02 (d, J ) 8.82 Hz, 2 H) 10.27 (s,
1 H); MS (DCI/NH3) m/z 462 (M + H)+.
N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-2-yl-
sulfonyl)piperidine-3-carboxamide (15a). The title compound was
prepared in a similar manner to the synthesis of 6a, substituting 7a
for 5 in 29% yield. 1H NMR (300 MHz, DMSO-d6) δ ppm 1.36 -
1.63 (m, 2 H) 1.83-1.95 (m, 2 H) 2.28-2.42 (m, 2 H) 2.46 (s, 3
H) 2.68-2.77 (m, 1 H) 3.60-3.64 (m, 1 H) 3.77-3.80 (m, 1 H)
7.31-7.36 (m, 2 H) 7.67-7.68 (m, 1 H) 7.76-7.79 (m, 2 H)
7.88-7.91 (m, 2 H) 8.00-8.06 (m, 3 H) 10.38 (s, 1 H); MS (DCI/
NH3) m/z 498 (M + H)+.
N-(4-(Thiazol-2-yl)phenyl)-1-(thiophen-2-ylsulfonyl)piperidine-
4-carboxamide (16d). To a solution of 10 (144 mg, 0.3 mmol)
and 2-bromothiazole (97 mg, 0.6 mmol) in tetrahydrofuran was
added potassium carbonate (103 mg, 0.75 mmol) and palladium
tetrakistriphenylphosphine (17 mg, 0.015 mmol). The reaction was
heated to reflux overnight, cooled and concentrated. The residue
was partitioned between dichloromethane:isopropyl alcohol (10:1,
N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-2-yl-
sulfonyl)azetidine-3-carboxamide (15b). The title compound was
prepared in a similar manner to the synthesis of 6a, substituting 7c

178 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Wang et al.
3 × 15 mL) and water (10 mL). The organic layers were combined,
dried, concentrated and the residue was purified by HPLC to afford
the title compound in 40% yield. 1H NMR (300 MHz, DMSO-d6)
δ ppm 1.62-1.77 (m, 2 H) 1.93 (m, 2 H) 2.34-2.48 (m, 3 H)
3.62-3.71 (m, 2 H) 7.28-7.33 (m, 1 H) 7.66-7.73 (m, 4 H)
7.85-7.91 (m, 3 H) 8.06 (dd, J ) 5.09, 1.36 Hz, 1 H) 10.08 (s,
1H); MS (DCI/NH3) m/z 434 (M + H)+.
3.73, 1.36 Hz, 1 H) 7.71 - 7.76 (m, 2 H) 7.94 - 7.99 (m, 2 H)
8.07 (dd, J ) 4.75, 1.36 Hz, 1 H) 8.13 (s, 1 H) 10.10 (s, 1 H); MS
(DCI/NH3) m/z 540 (M + H)+.
N-(4-(4-(4-Methoxyphenyl)thiazol-2-yl)phenyl)-1-(thiophen-
2-ylsulfonyl)piperidine-4-carboxamide (17b). The title compound
was prepared in a similar manner to the synthesis of 6a, substituting
1
12b for 5 in 91% yield. H NMR (300 MHz, DMSO-d6) δ ppm
N-(4-(Quinolin-2-yl)phenyl)-1-(thiophen-2-ylsulfonyl)piperi-
dine-4-carboxamide (16e). The title compound was prepared in a
similar manner to the synthesis of 16d, substituting 2-chloroquino-
line for 2-bromothiazole in 40% yield. 1H NMR (300 MHz, DMSO-
d6) δ ppm 1.64-1.79 (m, 2 H) 1.95 (m, 2 H) 2.35-2.48 (m, 3 H)
3.64-3.73 (m, 2 H) 7.31 (dd, J ) 5.09, 3.73 Hz, 1 H) 7.53-7.62
(m, 1 H) 7.65-7.70 (m, 1 H) 7.73-7.79 (m, 3 H) 7.97 (m, 1 H)
8.01-8.13 (m, 3 H) 8.24 (d, J ) 8.82 Hz, 2 H) 8.41 (d, J ) 8.82
Hz, 1 H) 10.05 (s, 1 H); MS (DCI/NH3) m/z 478 (M + H)+.
N-(4-(Quinoxalin-2-yl)phenyl)-1-(thiophen-2-ylsulfonyl)pip-
eridine-4-carboxamide (16f). The title compound was prepared
in a similar manner to the synthesis of 16d, substituting 2-chloro-
quinoxaline for 2-bromothiazole in 58% yield. 1H NMR (300 MHz,
DMSO-d6) δ ppm 1.65-1.79 (m, 2 H) 1.91-2.00 (m, 2 H)
2.37-2.48 (m, 3 H) 3.65-3.73 (m, 2 H) 7.31 (dd, J ) 5.09, 3.73
Hz, 1 H) 7.68 (dd, J ) 3.73, 1.36 Hz, 1 H) 7.78-7.90 (m, 4 H)
8.06-8.12 (m, 3 H) 8.30-8.33 (m, 2 H) 9.55 (s, 1 H) 10.13 (s, 1
H); MS (DCI/NH3) m/z 479 (M + H)+.
N-(4-(6-Chlorobenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-2-yl-
sulfonyl)piperidine-4-carboxamide (16g). The title compound was
prepared in a similar manner to the synthesis of 16d, substituting
2,6-dichlorobenzothiazole for 2-bromothiazole in 25% yield. 1H
NMR (300 MHz, DMSO-d6) δ ppm 1.72 (m, 2 H) 1.90-1.99 (m,
2 H) 2.38-2.49 (m, 3 H) 3.65-3.69 (m, 2 H) 7.28-7.37 (m, 1 H)
7.55 (dd, J ) 8.82, 2.03 Hz, 1 H) 7.65-7.70 (m, 1 H) 7.78 (d, J
) 8.48 Hz, 2 H) 7.98-8.09 (m, 4 H) 8.29 (d, J ) 2.03 Hz, 1 H)
10.19 (s, 1 H); MS (DCI/NH3) m/z 518 (M + H)+.
1.63-1.77 (m, 2 H) 1.92 - 1.97 (m, 2 H) 2.35-2.49 (m, 3 H)
3.63-3.72 (m, 2 H) 3.81 (s, 3 H) 6.99-7.05 (m, 2 H) 7.29-7.32 (m,
1 H) 7.66-7.75 (m, 3 H) 7.92-7.99 (m, 5 H) 8.07 (dd, J ) 5.09,
1.36 Hz, 1 H) 10.10 (s, 1 H); MS (DCI/NH3) m/z 540 (M + H)+.
N-(4-(4-(4-Chlorophenyl)thiazol-2-yl)phenyl)-1-(thiophen-2-
ylsulfonyl)piperidine-4-carboxamide (17c). The title compound
was prepared in a similar manner to the synthesis of 6a, substituting
1
12c for 5 in 88% yield. H NMR (300 MHz, DMSO-d6) δ ppm
1.63-1.77 (m, 2 H) 1.92 - 1.97 (m, 2 H) 2.35-2.47 (m, 3 H)
3.62-3.72 (m, 2 H) 7.31 (dd, J ) 5.09, 3.73 Hz, 1 H) 7.50-7.59
(m, 2 H) 7.66-7.76 (m, 3 H) 7.96 (m, 2 H) 8.04-8.09 (m, 3 H)
8.17 (s, 1 H) 10.10 (s, 1 H); MS (DCI/NH3) m/z 540 (M + H)+.
In Vitro Assay for FAAH Activity. Crude rat FAAH enzyme
was isolated from rat cortex as follows:
Rat cortex (Pel-freeze Catalog number 56006-2) was homog-
enized using a motorized homogenizer in 50 mM Tris, pH 8.0,
buffer containing 320 mM sucrose and the protease inhibitors
benzamidine 1 mM, leupeptin 0.5 µg/mL, pepstatin 0.7 µg/mL, and
aprotinin 2 µg/mL (Sigma) at 1:5 (w/v). The smooth homogenate
was centrifuged at 1100g for 5 min at 4 °C, and the supernatant
carefully taken. This centrifugation was repeated twice, leaving the
pellet each time. The supernatant was then centrifuged at 22 000g
for 30 min at 4 °C, and the resulting P2 pellet containing crude rat
FAAH was resuspended in the same Tris buffer at a 1:0.5 ratio,
original w/v, and stored at -80 °C.
Human FAAH Cloning, Expression and Purification. The
cloning, expression and purification of the human enzyme is
described in Kage et al.29 Briefly, full length human FAAH
(accession number U82535) was amplified from cerebellum
cDNA. The full-length product was subcloned into pcDNA3.1
hygro(-) and used for stable cell line generation by transfection
into HEK293 cells using Lipofectamine 2000 (Invitrogen) as
per the manufacturer’s directions. Crude lysates were made from
individual clones by dislodging adherent cells, spinning down
the cell suspension and resuspending the cell pellet in a small
volume of Tris-EDTA (pH 8) containing protease inhibitors. This
suspension was then sonicated five times for 10 s, aliquoted and
stored at -80 °C. The resulting lysates were screened for FAAH
activity using a [14C]-anandamide assay, as described in Omeir
et al.,39 and a single clone with significant FAAH activity was
selected for scale up.
3H-anandamide carbon filtration assay for FAAH activity was
adapted from Wilson et al.30 Tritiated anandamide was obtained
from American Radiolabeled Chemicals (Catalog number ART626,
60 Ci/mmol, 1 mCi/mL) and unlabeled anandamide was obtained
from Sigma (Catalog number A0580). Prior to the assay, a 10×
stock of substrate was made in ethanol and consisted of 100 µM
anandamide and 0.25 µCi of tritiated anandamide.
A 5× reaction buffer containing 625 mM Tris, pH 8.0 (Invitrogen
15568-025), 5 mM EDTA, and 0.5% BSA, fatty acid free
(Calbiochem 126609) was used for all experiments, and experiments
were carried out in 96 well plates. For preincubation experiments,
20 µL of 5× buffer, 10 µL of enzyme and water were combined
for an 80 µL reaction mix. Ten microliters of 10× compound was
added to the reaction mixture and allowed to preincubate for 15
min, or as indicated. The reaction was started by addition of 10 µL
of 10× substrate and allowed to proceed for 14 min, then stopped
by addition of 10 µL of 0.7 M HCl. For other experiments, 20 µL
of 5× buffer, 10 µL of 10× substrate and water were combined
for an 80 µL reaction mix. Ten microliters of 10× compound was
added to the reaction mix, and the reaction was started by addition
of 10 µL of enzyme. After 14 min, the reaction was stopped by
addition of 10 µL of 0.7 M HCl.
N-(4-(6-Methoxybenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-2-
ylsulfonyl)piperidine-4-carboxamide (16h). The title compound
was prepared in a similar manner to the synthesis of 16d,
substituting 2-chloro-6-methoxybenzothiazole for 2-bromothiazole
1
in 31% yield. H NMR (300 MHz, DMSO-D6) δ ppm 1.63-1.77
(m, 2 H) 1.91 - 1.97 (m, 2 H) 2.36-2.48 (m, 3 H) 3.63-3.72 (m,
2 H) 3.85 (s, 3 H) 7.11 (dd, J ) 8.99, 2.54 Hz, 1 H) 7.31 (dd, J )
5.09, 3.73 Hz, 1 H) 7.66-7.71 (m, 2 H) 7.73 -7.76 (m, 2 H) 7.89
(d, J ) 8.82 Hz, 1 H) 7.91 - 7.98 (m, 2 H) 8.07 (dd, J ) 4.92,
1.19 Hz, 1 H) 10.14 (s, 1 H); MS (DCI/NH3) m/z 514 (M+H)+.
N-(4-(4-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-2-yl-
sulfonyl)piperidine-4-carboxamide (16i). The title compound was
prepared in a similar manner to the synthesis of 16d, substituting
2-chloro-4-methylbenzothiaozole for 2-bromothiazole in 61% yield.
1H NMR (300 MHz, DMSO-d6) δ ppm 1.63-1.78 (m, 2 H)
1.92-1.98 (m, 2 H) 2.36-2.48 (m, 3 H) 2.71 (s, 3 H) 3.64-3.72
(m, 2 H) 7.29-7.35 (m, 3 H) 7.67 (dd, J ) 3.73, 1.36 Hz, 1 H)
7.75-7.80 (m, 2 H) 7.88-7.94 (m, 1 H) 8.01-8.08 (m, 3 H) 10.17
(s, 1 H); MS (DCI/NH3) m/z 498 (M + H)+.
N-(4-(4-Methoxybenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-2-
ylsulfonyl)piperidine-4-carboxamide (16j). The title compound
was prepared in a similar manner to the synthesis of 16d,
substituting 2-chloro-4-methoxybenzothiaozole for 2-bromothiazole
1
in 67% yield. H NMR (300 MHz, DMSO-d6) δ ppm 1.63-1.78
(m, 2 H) 1.92-1.97 (m, 2 H) 2.34-2.48 (m, 3 H) 3.62-3.75 (m,
2 H) 3.98 (s, 3 H) 7.05-7.08 (m, 1 H) 7.28-7.33 (m, 1 H) 7.38 (t,
J ) 7.97 Hz, 1 H) 7.62-7.65 (m, 1 H) 7.67 (dd, J ) 3.73, 1.36
Hz, 1 H) 7.73-7.80 (m, 2 H) 7.97-8.04 (m, 2 H) 8.07 (dd, J )
5.09, 1.36 Hz, 1 H) 10.16 (s, 1 H); MS (DCI/NH3) m/z 514 (M +
H)+.
N-(4-(4-(3-Methoxyphenyl)thiazol-2-yl)phenyl)-1-(thiophen-
2-ylsulfonyl)piperidine-4-carboxamide (17a). The title compound
was prepared in a similar manner to the synthesis of 6a, substituting
1
12a for 5 in 47% yield. H NMR (300 MHz, DMSO-d6) δ ppm
1.63-1.78 (m, 2 H) 1.92-1.97 (m, 2 H) 2.35-2.48 (m, 3 H)
3.63-3.73 (m, 2 H) 3.84 (s, 3 H) 6.95 (m, 1 H) 7.29-7.41 (m, 1
H) 7.38 (t, J ) 7.97 Hz, 1 H) 7.58-7.64 (m, 2 H) 7.67 (dd, J )

EValuation of Benzothiazole-Based Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 179
Isolation of 3H-Ethanolamine Product by Phase Separa-
tion. Twenty-five microliters of activated carbon was loaded into
96 well filter plates (Millipore MultiScreen plates Catalog number
MAFCNOB10). To prewet the carbon, 100 µL of methanol was
pipetted into each well and the plate was then centrifuged into a
“waste” OptiPlate at 755g for 5 min.
Sixty microliters of the above reaction was placed onto the wetted
carbon filtered plate and placed on top of a 96 well OptiPlate
containing 150 µL of MicroScint 40, followed by centrifugation at
755g for 5 min to collect the radiolabeled 3H-ethanolamine product.
The OptiPlate was then read on a Packard TopCount after
incubation for 1 h at room temperature.
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Selectivity Screening. The selectivity screening was conducted
as detailed.25
Molecular Overlay Study. The ligand pose from the cocrys-
tallized X-ray structure of methyl arachidonyl fluorophosphonate
(MAFP) in complex with FAAH (PDB ID 1MT5) was extracted.
An exhaustive set of conformers for 3 was then generated using
OMEGA (OpenEye Scientific Software, Santa Fe, NM; http://
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on similarity to the three-dimensional shape and electrostatics of
the known MAFP bound conformation using the program ROCS
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These poses where then scored by FRED (OpenEye Scientific
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the top of this list was retained and is the pose shown in
Figure 2b.
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Supporting Information Available: Elemental analysis data for
all novel compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
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