Design, microwave-assisted synthesis, biological evaluation and molecular modeling studies of 4-phenylthiazoles as potent fatty acid amide hydrolase inhibitors

Article abstract of DOI:10.1111/cbdd.13670

Endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are endogenous lipids that activate cannabinoid receptors. Activation of these receptors produces anti-inflammatory and analgesic effects. Fatty acid amide hydrolase (FAAH) is a membrane enzyme that hydrolases endocannabinoids; thus, inhibition of FAAH represents an attractive approach to develop new therapeutics for treating inflammation and pain. Previously, potent rat FAAH inhibitors containing 2-naphthyl- and 4-phenylthiazole scaffolds were identified, but up to the present time, very little structure–activity relationship studies have been performed on these moieties. We designed and synthesized several analogs containing these structural motifs and evaluated their inhibition potencies against human FAAH enzyme. In addition, we built and validated a homology model of human FAAH enzyme and performed docking experiments. We identified several inhibitors in the low nanomolar range and calculated their ADME predicted values. These FAAH inhibitors represent promising drug candidates for future preclinical in vivo studies.

Full text of DOI:10.1111/cbdd.13670

MS STEPHANIE R WILT (Orcid ID : 0000-0002-4898-0250)
DR STEVAN PECIC (Orcid ID : 0000-0002-3706-8768)
Article type
: Research Article
Corresponding author mail id : specic@fullerton.edu
Design, Microwave-Assisted Synthesis, Biological Evaluation and Molecular Modeling Studies
of 4-Phenylthiazoles as Potent Fatty Acid Amide Hydrolase Inhibitors
Stephanie R. Wilt, Mark Rodriguez, Thanh NH Le, Emily V. Baltodano, Adrian Salas and Stevan
Pecic*
Department of Chemistry and Biochemistry, California State University-Fullerton, Fullerton,
California 92834, United States
Abstract
Endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are endogenous lipids
that activate cannabinoid receptors. Activation of these receptors produces anti-inflammatory and
analgesic effects. Fatty acid amide hydrolase (FAAH) is a membrane enzyme that hydrolases
endocannabinoids, thus inhibition of FAAH represents an attractive approach to develop new
therapeutics for treating inflammation and pain. Previously, potent rat FAAH inhibitors containing 2-
naphthyl- and 4-phenylthiazole scaffolds were identified, but up to the present time, very little
structure-activity relationship (SAR) studies have been performed on these moieties. We designed and
synthesized several analogs containing these structural motifs and evaluated their inhibition potencies
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differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13670
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against human FAAH enzyme. In addition, we built and validated a homology model of human
FAAH enzyme and performed docking experiments. We identified several inhibitors in the low
nanomolar range and calculated their ADME predicted values. These FAAH inhibitors represent
promising drug candidates for future preclinical in vivo studies.
Introduction
CB1 and CB2 are cannabinoid receptors are present in the central nervous system and the
peripheral nervous system, respectively.(Davis, 2014; Pertwee, 2006) Cannabinoid receptors are
activated by cannabinoid chemical compounds, which play an important role in the regulation of
many physiological processes, including pain and inflammation.(Basavarajappa, 2017) There are
three known classes of cannabinoids: phytocannabinoids, synthetic cannabinoids, and
endocannabinoids. Phytocannabinoids are natural products found in Cannabis sativa, such as 9-
tetrahydrocannabinol (THC) - one of the active ingredients of marijuana.(Thomas & ElSohly, 2016)
To the second group of cannabinoids, synthetic cannabinoids, belong chemically synthesized
cannabinoid receptor agonists. Most synthetic cannabinoids are intended to mimic the natural
cannabinoids, e.g. CP-47497, without containing the typical phytocannabinoid bicyclic
structure.(Hruba & McMahon, 2017; Le Boisselier, Alexandre, Lelong-Boulouard, & Debruyne,
2017) Several recent studies evaluated prolonged exposures of mice to synthetic cannabinoids and
their impact on recognition memory and brain metabolism overall.(F. M. Mouro et al., 2019;
Francisco M. Mouro, Ribeiro, Sebastião, & Dawson, 2018) Endocannabinoids are endogenous lipid
ligands, the non-plant-based neurotransmitters that are naturally produced in the mammalian
body.(Okamoto, Wang, Morishita, & Ueda, 2007; Simon & Cota, 2017) Two main endocannabinoids
are anandamide (AEA) and 2-arachidonoyglycerol (2-AG). By the activation of the CB1 and CB2
receptors, AEA produce a similar pain alleviation effect, such as analgesia, anti-inflammation,
anxiolysis, and anti-depression effects, without giving undesirable side effects as THC or synthetic
cannabinoids, including cognitive and motor impairments.(Ahn, Johnson, & Cravatt, 2009; Scherma
et al., 2019) However, AEA is only synthesized on-demand when there are inflammation and immune
responses, and is rapidly metabolized by the enzyme fatty acid amide hydrolase (FAAH), which is a
mammalian integral membrane enzyme.(Bisogno & Maccarrone, 2013; Morena et al., 2019) FAAH
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enzyme metabolizes AEA by hydrolyzing it to the arachidonic acid and ethanolamine (Figure 1A),
therefore FAAH is mainly responsible for the termination of anandamide signaling in vivo.(Reggio,
2010) The FAAH enzyme belongs to a large group of enzymes called amidase signature (AS), which
is known to share a highly conserved sequence homology structure among diverse species.(Lu &
Anderson, 2017; Ueda, 2002) The X-ray crystallographic structure of the rat FAAH enzyme shows
that FAAH possesses an unusual serine-serine-lysine (Ser241-Ser217-Lys142) catalytic
triad.(McKinney & Cravatt, 2003) The mechanism of the hydrolytic cleavage of the amide bond by
the FAAH enzyme has been proposed by Cravatt et al.(Kay Ahn et al., 2009) In short, once the
substrate AEA enters the active site of FAAH, its amide bond will be in the close proximity of Ser241
residue. In the first step of the postulated mechanism, Lys142 acts as a base and is protonated by
Ser217, followed by the proton transfer from Ser241 to Ser217. Ser241 becomes a nucleophile and
attacks the carbonyl part of the amide bond of the substrate AEA. In the next step, Ser217 acts as an
acid and donates a proton to the nitrogen atom of the amide substrate, giving it a partially positive
charge and making it a good leaving group. An acyl-enzyme intermediate containing Ser241 and the
carbonyl product is formed, with the simultaneous sharing of a proton between Lys142 and Ser217.
The intermediate then reacts with a water molecule, which eventually releases the free fatty acid
product- arachidonic acid. Lys142, Ser217, and Ser241 then return to their initial protonation states
(Figure 1B).
Known FAAH Inhibitors
There are several distinct groups of FAAH inhibitors discovered up to date, including
reversible and irreversible inhibitors (Figure 2). The first investigated group of FAAH inhibitors were
substrate-derived inhibitors, such as the reversible inhibitor trifluoromethyl ketone analog of oleamide
1, with reported Ki of 82 nM.(D. L. Boger et al., 1999) These substrate-based FAAH inhibitors are
highly hydrophobic, have low selectivity, and did not present good lead compounds for future drug
development. The next explored group of FAAH inhibitors were -ketoheterocycles. These inhibitors
were more selective and more potent. One of the very potent inhibitors from this group is OL-135 2,
with Ki of 4.7 nM, which has 60-fold higher selectivity compared to trifluoromethyl ketones.(Dale L.
Boger et al., 2005) However, OL-135 did not show significant inhibition in vivo, probably due to
rapid metabolism.(Lichtman et al., 2004) The third group of FAAH inhibitors discovered were N-
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alkylcarbamates, which are also irreversible inhibitors. The carbamate inhibitor URB597 displays a
high inhibitory potency against rat FAAH, with IC50 of 4.6 nM.(Marco Mor et al., 2004) The research
done on carbamates led to the discovery of the next generation of FAAH inhibitors containing
piperidine-urea moiety. This group also inhibits FAAH irreversibly. PF-3845 3, discovered by Pfizer
is one of the most potent inhibitors from this group with the inhibitory potency Ki of 0.23 nM.(Kay
Ahn et al., 2009) There are many other structurally different FAAH inhibitors that we are not able to
classify into any aforementioned groups, but are known to be potent FAAH inhibitors, such as
boronic acid-based inhibitors, azole derivatives, benzothiazole and others.(K. Ahn et al., 2009;
Lodola, Castelli, Mor, & Rivara, 2015) Wang et al.(Wang et al., 2009) noticed that the binding pocket
of rat FAAH can well tolerate the big and bulky groups located on the left side of the piperidine series
of molecules, while the right side was usually kept small, such as a thiophene group or fluorophenyl
groups. In the separate studies(M. Mor et al., 2008; Tarzia et al., 2003) the synthesis of a series of 2-
naphthyl containing carbamates designed as FAAH inhibitors were reported, showing that this bulky,
hydrophobic moiety is also important for FAAH inhibition (Figure 2). In both of these studies, the rat
FAAH enzyme was used for the biological evaluation of synthesized analogs. Herein we decided to
design, synthesize and biologically evaluate 2-naphthyl- and 4-phenylthiazole- piperidine analogs as
potential inhibitors of human FAAH enzyme. Finally, we performed molecular modeling studies to
further support and analyze our in vitro data. Our results suggest that 4-phenylthiazole piperidine
analogs represent a promising series of FAAH inhibitors with potential therapeutic effects in pain
management.
Materials and Methods:
Reagents and solvents were available from Sigma-Aldrich or Fisher Scientific and used as supplied.
All new compounds were characterized by proton NMR, carbon NMR, and high-resolution mass
spectrometry. NMR spectra were measured on a Bruker 400 magnetic resonance spectrometer. 1H
chemical shifts are reported relative to the residual solvent peak (chloroformꢀ=ꢀ7.26ꢀppm or dimethyl
sulfoxide = 2.50 ppm) as follows: chemical shift (δ), proton ID, multiplicity (sꢀ=ꢀsinglet, bsꢀ=ꢀbroad
singlet, dꢀ=ꢀdoublet, bdꢀ=ꢀbroad doublet, ddꢀ=ꢀdoublet of doublets, tꢀ=ꢀtriplet, qꢀ=ꢀquartet, mꢀ=ꢀmultiplet,
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integration, coupling constant(s) in Hz). 13C chemical shifts are reported relative to the residual
deuterated solvent 13C signals (chloroformꢀ=ꢀ77.2ꢀppm, or dimethyl sulfoxide = 39.5 ppm). High-
resolution mass spectra were obtained using a high-resolution liquid chromatography mass
spectrometer. Microwave reactions were carried out in a CEM Discover SP microwave synthesizer.
Human recombinant fatty acid amide hydrolase (Item No. 100101183, Batch No. 0523867) was
obtained from Cayman Chemical. Molecular modeling studies and docking experiments were
performed using ICM Pro Molsoft software(Abagyan, Totrov, & Kuznetsov, 1994).
Chemistry
The synthesis of 2-naphthyl analogs 8a-c and 4-phenylthiazole analogs 14a-f are illustrated in Scheme
1 and Scheme 2, respectively. In short, 2-naphthylamine 4, and 1-Boc-piperidine-4-carboxylic acid 5,
were coupled under standard EDC-amide coupling conditions to get the amide 6 in 57% yield. The
Boc protecting group was subsequently removed using trifluoroacetic acid (TFA). The obtained
amine was sulfonated with 2-tiophenesulfonyl chloride, benzenesulfonyl chloride and 2-
fluorobenzenesulfonyl chloride to afford analogs 8a, 8b and 8c, respectively.(Pecic, Deng, Morisseau,
Hammock, & Landry, 2012) The synthesis of analogs 14a-f started with condensation of
commercially available 2-bromoacetophenone 9, and 4-aminothiobenzamide 10. The obtained 4-
phenylthiazole aniline 11, was coupled to 1-Boc-piperidine-4-carboxylic 5, using EDC as coupling
reagent and microwave irradiation. The Boc group of the resulting amide 12 was removed with TFA
to afford the amine 13, which was reacted with various different sulfonyl chlorides to yield the target
analogs 14a-f in moderate yields.
Human FAAH Enzyme Inhibition Assay
Measurement of FAAH potency was performed using the substrate N-(6-methoxypyridin-3-yl)
octanamide (OMP) ([S]finalꢀ=ꢀ50ꢀμM) in sodium phosphate buffer (0.1ꢀM, pHꢀ=ꢀ8, 0.1ꢀmg/mL BSA)
and progress of the reaction was measured at λexcitationꢀ=ꢀ303ꢀnm, λemissionꢀ=ꢀ394ꢀnm, 37ꢀ°C. All
experiments were run in triplicate and values reported as averageꢀ± SD.(Pecic et al., 2018) The
substrate OMP was synthesized following synthetic procedure and reaction conditions shown in
Scheme 3.
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Molecular Modeling
Amino acid sequence of human FAAH enzyme was retrieved from the NCBI protein database.
Sequence alignment was carried out with the ICM Pro (based on ZEGA sequence alignment -
Needleman and Wunsch algorithm with zero gap and penalties). To make the homology model, we
used a homology algorithm from ICM Pro. Between several crystal structures available in the PDB
database, the crystal structure of rat FAAH enzyme (PDB code: 3QK5) was selected as a template.
The sequence alignment and the rat FAAH PDB template were converted to the ICM homology
model. Conversion included optimization of hydrogens, several amino acids (H, P, N, C and Q) and
assignment of the secondary structure. In order to validate the model, programs Procheck, Verify3D,
Errat, What check and Prove from SAVES metaserver were used. The small molecule docking
experiments were performed following steps according to the ICM Pro program guidelines. All
synthesized analogs and standard FAAH inhibitor URB-597 were drawn as 2D
structures with ChemDraw Professional version 16, and then energy minimized through Chem3D
version 16/MM2, Job Type: Minimum RMS Gradient of 0.010ꢀkcal/mol and RMS distance of 0.1ꢀÅ,
and saved as MDL MolFiles (*.mol) for purpose of docking with ICM Pro.(Pecic et al., 2010; Pecic,
McAnuff, & Harding, 2011; Pecic et al., 2018) ICM scores were obtained after this procedure. ADME
properties for all synthesized target analogs were calculated using the ICM Chemist program.
Results and Discussion:
Design, synthesis and SAR: In previous studies (Figure 3), Wang et al.(Wang et al., 2009) examined
various benzothiazole and 4-phenylthiazole piperidine moieties identified from high-throughput
screening (HTS) and follow-up structure-activity relationship (SAR) studies. This study mostly
focused on the benzothiazole-piperidine analogs and reported only three analogs with various 4-
phenylthiazole groups on the left side of the piperidine series of analogs. Mor et al.(M. Mor et al.,
2008) reported two potent 2-naphthyl containing FAAH inhibitors. We hypothesized that the FAAH
inhibitory activity may be achieved by introducing the 2-naphthyl ring into piperidine series of
analogs. Our design started with introducing 2-naphthyl ring on the left side of the piperidine moiety
and linking the small thiophene, phenyl or 2-fluorophenyl rings on the right side of the central
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piperidine moiety via sulfonamide bond. Our docking experiments (Table 1) suggested that this series
of bulky, hydrophobic naphthyl analogs fits well in the binding pocket of the human FAAH,
according to the obtained low ICM scores. The first tested 2-naphthyl-piperidine analog 8a showed
only weak submicromolar potency with IC50 of 1700 nM. Replacing the thiophene ring with other
small rings, such as phenyl 8b, or 2-fluorophenyl 8c, did not improve the inhibitory activity of this
series of analogs, as shown in Table 1, with IC50s of 4500 nM and 1200 nM respectively, indicating
that 4-phenylthiazole moiety is the important structural feature on the piperidine series of FAAH
inhibitors. We decided to turn our attention to the 4-phenylthiazole analogs, since this moiety showed
improved potency and was not well explored in previous studies- namely, only three analogs were
synthesized and evaluated in rat FAAH inhibition assay (Figure 3). In addition, 4-phenylthiazole
without any substitution on the phenyl ring was never tested, and our docking experiments suggested
that this moiety will occupy the same hydrophobic pocket as the other bulky, hydrophobic groups
previously reported. In order to explore the structural requirements for this series of analogs, we
prepared several compounds containing 4-phenylthiazole moiety without any substituents on the
phenyl ring on the left side of the piperidine ring, and fluorine and chlorine atoms at various positions
on the aromatic ring on the right side of the molecule and evaluated these analogs for inhibition
against human FAAH enzyme. Biological evaluation of the first 4-phenylthiazole analog 14a,
containing a thiophene ring on the right side of the piperidine moiety, showed very good inhibitory
potency against human FAAH enzyme with IC50 of 23.4 nM. We continued our SAR exploration of
the 4-phenylthiazole analogs with probing the 2-fluorophenyl 14b, 2-chlorophenyl 14c, 4-
fluorophenyl 14d, 4-chlorophenyl 14e, 2,4-difluorophenyl 14f and 2,4-dichlorophenyl 14g groups on
the right side of the 4-phenylthiazole piperidine moiety. All analogs showed improved potencies,
having IC50s in the low-to-medium nanomolar range, and also suggesting that ortho- and para-
substituted and ortho-para disubstituted fluorine atoms are better tolerated than the chlorines at the
same positions on the ring. In addition, the improved potencies in the binding pocket of the human
FAAH, are suggesting potential favorable interactions within the enzyme binding pocket, which were
confirmed by our docking experiments.
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Homology Modeling Studies: In order to better explore the possible binding modes and interactions
for the 4-phenylthiazole analogs, we have conducted molecular modeling studies. Since the crystal
structure of the human FAAH enzyme has not been reported, we decided to build a homology model.
As a template for building the human FAAH enzyme homology model, we selected the crystal
structure of the rat fatty acid amide hydrolase (PDB code: 3QK5)(Gustin et al., 2011) since its crystal
structure is solved at the highest resolution (2.20 Å) that we found deposited. The sequence alignment
of the human FAAH enzyme shows very good sequence identity with rat (and many other species)
FAAH enzyme sequence template, with 80% sequence similarity (See Supplemental Figure S1 and
Figure S2). We also noticed that the alignment of rat and human FAAH sequences contains many
conserved residues (shown by green), especially located in the proximity of the catalytic site of the
FAAH enzyme, that are essential for proper enzyme function. We first built and energy minimized the
homology model using ICM Pro program, and then we assessed validity of the model. In order to
evaluate the constructed human FAAH enzyme homology model for the docking studies, we used
several different programs for evaluation that are available via the server of the UCLA-DOE Institute
for Genomics and Proteomics. Evaluation methods verify whether a model satisfies standard and
geometric criteria, and asses the overall quality of the model. We used PROCHECK(Laskowski,
Rullmann, MacArthur, Kaptein, & Thornton, 1996), ERRAT(Colovos & Yeates, 1993), VERIFY-
3D(Eisenberg, Lüthy, & Bowie, 1997), WHAT-IF(Vriend & Sander, 1993) and PROVE(Pontius,
Richelle, & Wodak, 1996) and assessed the quality of the human FAAH enzyme model we
constructed. We first determined the root-mean-square deviation (RMSD) between the backbone
atoms of the template and the homology model, getting RMSD value of 0.238 A, indicating a close
homology (Figure S3). Therefore, we decided to proceed with the next evaluation steps. We then
evaluated our homology model using the program Procheck. The aim of this program is to assess the
detailed residue-by-residue stereochemical quality of the enzyme structure. Ramachandran plots for
the human FAAH homology model is shown in Figure S4. A simple measure of quality that can be
used from the plot is the percentage of residues in core region, and allowed regions should be very
high (>90% residues). Another important factor in structural assessment is Goodness factor or G-
Factor which shows the quality of dihedral, covalent and overall bond angles. These scores should be
above -0.5 for a reliable model. From observing the Ramachandran plot it can be seen that 89.1% of
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the residues are in the most favorable region. In addition, 10.5% of the residues were found in the
additionally allowed region. To add on, the model was found to have a G-factor of 0.2, meaning the
quality of the bond angles indicates the high quality of our homology model. The results obtained
from Errat are shown in Figure S5. This program evaluates an overall quality factor of nonbonded
atomic interactions. The normally accepted range is above 50% for a high-quality model. The
template structure of rat FAAH enzyme, PDB: 3QK5, has an Errat value of 85.294. From running an
Errat on the FAAH homology model, it was found to have an overall quality factor of 93.585,
suggesting the backbone conformation and nonbonded interactions of the homology model are all
within a normal range. Verify3D evaluates energetic and empirical methods to produce averaged data
points for each residue in order to evaluate the quality of protein structures. Verify3D results shown in
Figure S6 represent the Verify3D average data score of the homology model generated in comparison
with template. Using this scoring function, if more than 80% of the residue has score above 0.2 then
the structure is considered high quality. From the data collected in the table, it was observed that
92.65% of the residues had averaged a 3D score above 0.2. WHAT-IF is used to check the normality
of the local environment of amino acids. The program evaluates: bond lengths, bond angles, omega
angle restraints, side chain planarity, improper dihedral distribution and inside/outside distribution. It
does extensive checking of many stereochemical parameters of the residues in the model and it gives
an overall summary of the quality of the structure as compared with current reliable structures. For a
reliable structure, the WHAT-IF packing scores should be above −5.0. In the case of our homology
model (Table S1), the packing score is -0.794, therefore, the WHAT-IF evaluation also indicates that
the homology model structure is very reasonable.
The Prove results for the human FAAH homology model are shown in Figure S7. Prove provides an
average volume Z-score of all the atoms. High scores have been found to be associated with
uncertainty in the structure. Structures with poor resolution generally have a Zrms greater than 1.2,
while for well-resolved structures, the Zrms is around 1.0.(Pontius et al., 1996) Our model showed
that the average Z score for the model for all resolutions hovered slightly above 0 but below 0.1 with
the exception of one outlier at about 0.8. The Prove score shows that the model is valid, but it contains
an unusual number of buried atoms within it. The buried atoms are most likely hydrophobic residues
within the enzyme. In summary, the geometric quality of the backbone conformation, the residue
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interaction, the residue contacts and the energy profile of the human FAAH homology model are all
well within the limits established for reliable structure and we proceeded with the docking
experiments.
Docking Experiments: In order to better understand the binding modes and interactions of these
inhibitors within the catalytic site of the human FAAH enzyme, we performed docking studies.
Scoring functions from this docking experiment are reported in Table 1. The known irreversible
FAAH inhibitor URB-597 (1) was also docked for comparison. Although the docking scores for the
naphthyl analogs 8a-c had reasonable ICM scores, we were not able to correlate these values with the
obtained IC50 values. On the other hand, the docking scores for URB-597 and for the potent 4-
phenylthiazole analogs 14a-g were in the agreement with our in vitro data, suggesting that our
homology model could be an important tool in the future design for the 4-phenylthiazole series of
FAAH inhibitors. After visual inspections of binding modes of these inhibitors, we observed that
several inhibitors formed a complex with the FAAH enzyme through H-bonding with different amino
acids. All other non-covalent interactions (listed in Table 2) were also located in the proximity of the
catalytic triad of the human FAAH enzyme, suggesting that the low nanomolar potency of these
compounds is probably due to these interactions. In addition, we also observed that the 4-
phenylthiazole moiety was orientated toward the enzyme surface and that the right-side of analogs
14b, 14c, 14e, 14f and 14g is deeply buried within the catalytic site. Figures 4 and 5 show the
observed orientation for the most potent fluoro- and chloro- analogs 14f and 14g, respectively. This
could also rationalize the differences in the observed IC50 values of this series of inhibitors (See
Supplemental Figures S8-S12). According to the list of the non-covalent interactions (Table 2), all
synthesized 4-phenylthiazole analogs are located in the proximity of the catalytic triad residues S217
and S241. We believe that 4-phenylthiazole analogs probably form a non-covalent complex with
Ser241 and S217, similar to the intermediate formed by FAAH and anandamide (Figure 1B) during
the mechanism of hydrolysis. We also observed potential - interactions between F192 and the
phenyl aromatic ring of the analogs 14d, 14f and 14 g which could rationalize the improved potencies
of these analogs.
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Predicting ADME-Tox Properties: Finally, we calculated and performed prediction of the several
pharmacokinetic parameters important for the drug development process, Table 3. All final
compounds showed predicted octanol/water partition coefficient (clogP) in the relatively acceptable
range (2.0 - 6.5 is considered a good logP), moderate predicted aqueous solubility (acceptable range
for logS is between -6.5 and -0.5 moles/liter) and no unwanted or reactive chemical functionalities
(referred as “bad groups” in the table). Other predicted values that we examined were all within
optimum ranges: hydrogen bond donors, hydrogen bond acceptors and overall drug likeness, which
should be within -1 and 1 range. Predicted Caco2 permeability indicates high permeability (all values
above -5 suggest a high permeability) and this series of analogs had excellent predicted values for
hERG inhibition, namely values above 0.5 indicate high probability for drug candidate of being a
hERG inhibitor. All final compounds (except standard URB-597) had values below 0.5. Our further
prediction of plasma half-life showed that these compounds have a moderate predicted half-life of
1.05-3.75 hours. In addition, all newly synthesized 4-phenylthiazole analogs had no unfavorable
substructure/substituents, shown as a tox score of 0.
Conclusion:
We successfully synthesized 10 new compounds using microwave irradiation and evaluated them in
inhibition assay against human FAAH enzyme. Biological evaluation of the two different groups of
inhibitors showed that 4-phenylthiazole moiety is an important structural feature for the potent
inhibition of human FAAH enzyme and we identified several inhibitors in the low nanomolar range.
Using homology modeling and docking studies we further explored the binding modes of the potent
analogs and we noticed several important non-covalent interactions that contribute to the low
nanomolar potency of this series of inhibitors. Our calculation of predicted ADME-Tox properties
suggest that 4-phenylthiazole inhibitors have the potential to be further developed as new lead
candidates and therapeutics in pain management.
Experimental:
General procedure (Scheme 1) for the preparation of naphthyl analogs 8a-c.
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tert-butyl 4-(naphthalen-2-ylcarbamoyl)piperidine-1-carboxylate (6): The mixture of N-Boc-
piperidinecarboxylic acid 5 (1.01 g, 4.4 mmol) and EDC (1.27 g, 6.6 mmol) was dissolved in
anhydrous dichloromethane (45 mL) and was stirred at room temperature. 2-Naphthylamine 4 (756
mg, 5.28 mmol) was slowly added to the reaction mixture and stirred for 48 hours at room
temperature under argon atmosphere. The reaction mixture was transferred to the separatory funnel
and organic layer was washed with 2N HCl (100 mL), an aqueous solution of
saturated NaHCO3 (2x100 mL), and was then dried over anhydrous sodium sulfate. The product was
recrystallized with hexane and collected upon filtration. The product 6 was obtained as an off-white
solid, 891 mg, 57% yield. 1H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 7.77 (t, J = 8.4 Hz, 4H), 7.47 -
7.40 (m, 3H), 4.2 (bs, 1H), 2.78 (bs, 2H), 2.45 - 2.41 (m, 1H), 1.91 (d, J = 14.8 Hz, 2H) 1.81 - 1.77
(m, 2H), 1.49 (s, 9H). 13C NMR (100ꢀMHz, CDCl3): δ 173.0, 154.7, 135.2, 133.8, 130.6, 128.7, 127.6,
127.5, 126.5, 125.0, 119.8, 116.8, 79.8, 44.2, 28.4 ppm.
N-(naphthalen-2-yl)piperidine-4-carboxamide (7): The amide 6 (800 mg, 2.25 mmol) was
dissolved in anhydrous dichloromethane (25 mL) and was then cooled down to 0oC. TFA (4.32 mL,
56.5 mmol) was slowly added and the reaction mixture was stirred at 0oC for additional 30 minutes.
Reaction mixture was stirred at room temperature for 48 hours under argon atmosphere. Next, the
reaction mixture was concentrated. The crude product was dissolved in ethyl acetate (25 mL), and
10% aqueous solution of NaOH (25 mL) was added slowly and vigorously stirred for 15 minutes. The
mixture was transferred to the separatory funnel, organic layer was collected, dried over anhydrous
sodium sulfate, filtered and concentrated. The product 7 was collected as an off-white solid, 351 mg,
1
62% yield. H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.32 (s, 1H), 7.86 – 7.78 (m, 3H), 7.60
(dd, J = 2, 6.8 Hz, 1H), 7.46 (t, J = 6.8 Hz, 1H), 7.39 (t, J = 8 Hz, 1H), 3.92 (bs, 1H), 3.23 (d, J = 12.4
Hz, 2H), 2.80 (t, J = 10 Hz, 2H), 2.66 – 2.59 (m, 1H), 1.89 (d, J = 12 Hz, 2H), 1.78 – 1.70 (m, 2H).
13C NMR (100ꢀMHz, DMSO-d6): δ 173.4, 137.2, 133.8, 130.1, 128.7, 127.8, 127.6, 126.8, 124.9,
120.4, 115.6, 44.0, 41.7, 27.2 ppm.
The amine 7 (50 mg, 0.19 mmol) was dissolved in anhydrous dichloromethane (20 mL), the reaction
mixture was brought to 0oC and N,N-diisopropylethylamine (97 L, 0.59 mmol) was added. The
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reaction mixture was stirred for 30 minutes, followed by the addition of corresponding benzene- (or 2-
thiophene-) sulfonyl chloride (0.30 mmol). The reaction mixture was then stirred at room temperature
under argon atmosphere for 48 hours. Next, the reaction was transferred to the separatory funnel
where the organic layer was washed with an aqueous solution of saturated NaHCO3 (50 mL), then
dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by
column chromatography (ethyl acetate/hexane 1:1).
N-(naphthalen-2-yl)-1-(thiophen-2-ylsulfonyl)piperidine-4-carboxamide (8a) was obtained as an
1
off-white solid in the amount of 30 mg (38% yield): H NMR (400 MHz, DMSO-d6) δ 8.28 (s, 1H),
8.07 (dd, J = 1.2, 4 Hz, 1H), 7.84 – 7.78 (m, 3H), 7.68 (dd, J = 1.2, 2.4 Hz, 1H), 7.55 (dd, J = 2, 6.8
Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.4 Hz, 1H), 7.324 – 7.30 (m, 1H), 3.68 (d, J = 12 Hz,
2H), 2.46 – 2.42 (m, 2H), 1.96 (dd, J = 2.8, 10.4 Hz, 2H), 1.78 – 1.68 (m, 2H). 13C NMR (100ꢀMHz,
DMSO-d6): δ 173.2, 137.1, 135.9, 134.1, 133.8, 133.3, 130.1, 128.7, 127.8, 127.7, 126.8, 124.9,
120.4, 115.6, 45.8, 41.7, 28.0 ppm. HRMS-ESI+: calculated for C20H20N2O3S2 + H: 401.0988; Found:
401.0980.
N-(naphthalen-2-yl)-1-(phenylsulfonyl)piperidine-4-carboxamide (8b) was obtained as an off-
white solid in the amount of 39 mg (50% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.27
(s, 1H), 7.84 – 7.72 (m, 6H), 7.67 (t, J = 8 Hz, 2H), 7.54 (d, J = 6.8 Hz, 1H), 7.45 (t, J = 8 Hz, 1H),
7.38 (t, J = 8.4 Hz, 1H), 3.69 (d, J = 12 Hz, 2H), 2.39 – 2.33 (m, 3H), 1.93 – 1.89 (m, 2H), 1.72 – 1.64
13
(m, 2H). C NMR (100ꢀMHz, DMSO-d6): δ 173.2, 137.1, 136.0, 133.8, 133.6, 130.1, 129.8, 128.7,
127.8, 127.7, 126.8, 124.9, 120.4, 115.6, 45.8, 41.8, 28.1 ppm. HRMS-ESI+: calculated for
C22H22N2O3S + H: 395.1424; Found: 395.1416.
1-((2-fluorophenyl)sulfonyl)-N-(naphthalen-2-yl)piperidine-4-carboxamide (8c) was obtained as a
white solid in the amount of 70 mg (86% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.08 (s, 1H), 8.29
(s, 1H), 7.84 – 7.78 (m, 5H), 7.57 – 7.52 (m, 2H), 7.47 (t, J = 10.6 Hz, 2H), 7.38 (t, J = 6.8 Hz, 1H),
3.76 (d, J = 12 Hz, 2H), 2.66 (t, J = 11.6 Hz, 2H), 1.92 (d, J = 10.4 Hz, 2H), 1.71 – 1.61 (m, 2H). 13C
NMR (100ꢀMHz, DMSO-d6): δ 173.2, 159.9, 157.4, 137.1, 136.4, 136.3, 133.8, 131.2, 130.1, 128.7,
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127.8, 127.7, 126.8, 125.6, 125.3, 125.1, 124.9, 120.4, 118.1, 117.9, 115.7, 45.3, 41.9, 28.2 ppm.
HRMS-ESI+: calculated for C22H21FN2O3S + H: 413.1330; Found: 413.1322.
General procedure (Scheme 2) for the preparation of 4-phenylthaizole analogs 14a-g.
The mixture of 2- bromoacetophenone (1.2 g, 6.02 mmol) and 4-aminothiobenzamide (918 mg, 6.02
mmol) were dissolved in isopropanol (25 mL). The reaction was stirred at 60°C for 2 hours. The
reaction mixture was cooled to 0°C, the crude product was filtered and washed with additional 2 mL
cold isopropanol. The crude product, amine 11 was used for the next step without further purification.
1
11 was obtained as a dark green solid in the amount of 1.370 g (90% yield). H NMR (400 MHz,
DMSO-d6) δ 8.12 (s, 1H), 8.04 – 7.98 (m, 4H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 (d, J = 7.2 Hz, 1H), 7.21
(d, J = 8.8 Hz, 2H), 4.44 (bs, 1H). 13C NMR (100ꢀMHz, DMSO-d6): δ 166.5, 154.9, 133.9, 128.8,
128.2, 127.6, 126.1, 120.3, 114.0 ppm.
N-boc-piperidinecarboxylic acid 5 (750 mg, 3.27 mmol), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) (753 mg, 3.92 mmol), amine 11 (825 mg, 3.27 mmol) and catalytic amount of 4-
dimethylaminopyridine (DMAP) were dissolved in 20 mL anhydrous dichloromethane. The reaction
mixture was subjected to microwave irradiation at 80 °C for 15 min. The mixture was transferred to
a separatory funnel and the organic layer was washed with aqueous solution of 1M HCl (20 mL),
aqueous solution of saturated NaHCO3 (20 mL) and brine (20 mL). The organic layer was then dried
over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by column
chromatography (ethyl acetate/hexane 1:1) and 815 mg, 53% of 12 was obtained as a white solid. 1H
NMR (400 MHz, CDCl3) δ 7.99-7.96 (m, 3H), 7.62 (d, J = 8.8 Hz, 2H), 7.43 (t, J = 8 Hz, 3H), 7.36-
7.32 (m, 1H), 4.18 (bs, 1H), 4.00 (bs, 1H), 2.88 - 2.75 (m, 2H), 2.51 – 2.36 (m, 2H), 1.90 (d, J = 12
Hz, 2H), 1.80 - 1.61 (m, 2H), 1.47 (s, 9H). 13C NMR (100ꢀMHz, CDCl3): δ 179.0, 172.9, 167.3, 156.3,
154.8, 154.8, 139.5, 134.5, 129.9, 128.8, 128.3, 127.5, 126.5, 119.9, 80.0, 44.4, 40.8, 28.5, 27.9 ppm.
The amide 12 (792 mg, 1.71 mmol) was dissolved in anhydrous dichloromethane (20 mL), and
o
reaction mixture was cooled down to 0 C. Trifluoroacetic acid (TFA) (2.62 mL, 34.2 mmol) was
added slowly and the reaction mixture was stirred at room temperature for 24 hours under argon
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atmosphere. Solvents were evaporated, and the crude product was recrystallized from diethyl ether,
and 685 mg (94% yield) was obtained as a TFA salt. A small amount of product was freebased and
used for 1H and 13C NMR analysis. 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.10 (s, 1H), 8.05
(d, J = 7.2 Hz, 2H), 7.97 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.47 (t, J = 7.2 Hz, 2H), 7.37
(t, J = 7.6 Hz, 1H), 3.01 (d, J = 11.6 Hz, 2H), 2.55 – 2.53 (m, 1H), 1.72 (d, J = 10.8 Hz, 2H), 1.60 –
1.50 (m, 2H). 13C NMR (100ꢀMHz, DMSO-d6): δ 173.9, 166.8, 154.9, 141.3, 134.0, 128.7, 128.1,
127.6, 126.8, 126.1, 119.2, 113.8, 45.3, 43.4, 29.0 ppm.
The amine 13 (100 mg, 0.209 mmol) was dissolved in 20 mL of anhydrous dichloromethane and
triethylamine (0.2 mL, 1.045 mmol) was added, followed by addition of corresponding benzene- (or
2-thiophene-) sulfonyl chloride (0.314 mmol) and was subjected to microwave irradiation at 80 °C
for 15 min. The reaction mixture was then transferred to the separatory funnel, the organic layer was
washed with 30 mL of an aqueous solution of saturated sodium bicarbonate, dried over anhydrous
sodium sulfate and then concentrated. The final 4-phenylthiazole analogs were purified by column
chromatography (ethyl acetate/hexane 1:4).
N-(4-(4-phenylthiazol-2-yl)phenyl)-1-(thiophen-2-ylsulfonyl)piperidine-4-carboxamide (14a) was
1
obtained as an white solid in the amount of 54 mg (50% yield): H NMR (400 MHz, DMSO-d6) δ
10.12 (s, 1H), 8.11 (s, 1H), 8.07 – 8.03 (m, 3H), 7.96 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.8 Hz, 2H),
7.68 – 7.67 (m, 1H), 7.47 (t, J = 7.6 Hz, 2H), 7.35 (d, J = 12.8 Hz, 1H), 7.30 (t, J = 3.6 Hz, 1H), 3.68
13
(d, J = 12 Hz, 2H), 2.47 – 2.40 (m, 3H), 1.92 (d, J = 13.6 Hz, 2H), 1.75 – 1.65 (m, 2H). C NMR
(100ꢀMHz, DMSO-d6): δ 173.0, 166.9, 155.1, 141.2, 135.6, 134.2, 133.9, 133.1, 128.9, 128.5, 128.3,
128.0, 127.0, 126.3, 119.5, 114.0, 45.5, 41.5, 27.6 ppm. HRMS-ESI+: calculated for
C25H23N3O3S3 + H: 510.0974; Found: 510.0962.
1-((2-fluorophenyl)sulfonyl)-N-(4-(4-phenylthiazol-2-yl)phenyl)piperidine-4-carboxamide (14b)
was obtained as an white solid in the amount of 44 mg (40% yield). 1H NMR (400 MHz, DMSO-d6) δ
10.14 (s, 1H), 8.10 (s, 1H), 8.03 (d, J = 7.2 Hz, 2H), 7.96 (d, J = 8 Hz, 2H), 7.83 – 7.73 (m, 4H), 7.54
– 7.43 (m, 2H), 7.36 (t, J = 7.2 Hz, 1H), 3.75 (d, J = 12.4 Hz, 2H), 2.65 (t, J = 11.6 Hz, 2H), 1.91
(d, J = 10.4 Hz, 2H), 1.68 – 1.60 (m, 2H). 13C NMR (100ꢀMHz, DMSO-d6): 173.0, 166.9, 155.1,
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141.2, 136.1, 136.0, 134.2, 131.0, 128.9, 128.3, 128.0, 127.0, 126.2, 125.4, 125.3, 125.0, 119.5, 117.8,
117.6, 114.0, 45.0, 41.6, 27.9 δ ppm. HRMS-ESI+: calculated for C27H24FN3O3S2 + H: 522.1316;
Found: 522.1302.
1-((2-chlorophenyl)sulfonyl)-N-(4-(4-phenylthiazol-2-yl)phenyl)piperidine-4-carboxamide (14c)
1
was obtained as an off-white solid in the amount of 62.1 mg (55% yield). H NMR (400 MHz,
DMSO-d6) δ 10.19 (s, 1H), 8.10 (s, 1H), 8.05 – 7.95 (m, 5H), 7.76 – 7.67 (m, 4H), 7.58 (t, J = 8 Hz,
1H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.6 Hz, 1H), 3.78 (d, J = 12.8 Hz, 2H), 2.84 (t, J = 12.4 Hz,
2H), 2.55 – 2.51 (m, 1H), 1.89 (d, J = 10.8 Hz, 2H), 1.62 (dd, J = 11.6, 9.2 Hz, 2H). 13C NMR
(100ꢀMHz, DMSO-d6): δ 172.9, 166.7, 154.9, 141.0, 135.8, 134.4, 134.0, 132.2, 131.5, 130.8, 128.8,
128.7, 128.1, 127.8, 126.8, 126.0, 119.3, 113.8, 44.7, 41.7, 28.0 ppm. HRMS-ESI+: calculated for
C27H24ClN3O3S2 + H: 538.1020; Found: 538.1007.
1-((4-fluorophenyl)sulfonyl)-N-(4-(4-phenylthiazol-2-yl)phenyl)piperidine-4-carboxamide (14d)
1
was obtained as white solid in the amount of 79 mg (72% yield). H NMR (400 MHz, DMSO-d6) δ
10.09 (s, 1H), 8.09 (s, 1H), 8.03 (d, J = 7.2 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H), 7.87 – 7.83 (m, 2H),
7.73 (d, J = 8.8 Hz, 2H), 7.52 – 7.44 (m, 4H), 7.36 (t, J = 7.6 Hz, 1H), 3.68 (d, J = 12 Hz, 2H), 2.36
(t, J = 11.6 Hz, 3H), 1.89 (d, J = 10.4 Hz, 2H), 1.70-1.62 (m, 2H). 13C NMR (100ꢀMHz, DMSO-
d6): δ 172.8, 166.7, 154.9, 141.0, 134.0, 132.1, 132.0, 130.5, 130.4, 128.7, 128.1, 127.8, 126.8, 126.0,
119.3, 116.7, 116.4, 113.8, 45.2, 41.4, 27.5 ppm. HRMS-ESI+: calculated for C27H24FN3O3S2 + H:
522.1316; Found: 522.1302.
1-((4-chlorophenyl)sulfonyl)-N-(4-(4-phenylthiazol-2-yl)phenyl)piperidine-4-carboxamide (14e)
1
was obtained as a white solid, 59 mg (53% yield). H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H),
8.09 (s, 1H), 8.03 (d, J = 7.2 Hz, 2H), 7.95 (d, J = 8.8 Hz, 2H), 7.80-7.77 (m, 2H), 7.73 (d, J = 8.8 Hz,
4H), 7.46 (t, J = 7.2 Hz, 2H), 7.36 (d, J = 7.2 Hz, 1H), 3.68 (d, J = 12 Hz, 2H), 2.42 – 2.35 (m, 3H),
1.89 (d, J = 10.8 Hz, 2H), 1.71 – 1.61 (m, 2H). 13C NMR (100ꢀMHz, DMSO-d6): δ 172.8, 166.7,
154.9, 141.0, 138.1, 134.6, 134.0, 129.5, 129.3, 128.7, 128.1, 127.8, 126.8, 126.1, 119.3, 113.8, 45.2,
41.3, 28.0, 27.5 ppm. HRMS-ESI+: calculated for C27H24ClN3O3S2 + H: 538.1020; Found: 538.1006.
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1-((2,4-difluorophenyl)sulfonyl)-N-(4-(4-phenylthiazol-2-yl)phenyl)piperidine-4-carboxamide
(14f) was obtained as off-white solid, 46 mg (41% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s,
1H), 8.10 (s, 1H), 8.04 (d, J = 7.2 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H), 7.92 – 7.86 (m, 1H), 7.74 (d, J =
9.2 Hz, 2H), 7.66 – 7.61 (m, 1H), 7.47 (t, J = 7.2 Hz, 2H), 7.38 – 7.32 (m, 2H), 3.73 (d, J = 12.4 Hz,
13
2H), 2.66 (t, J = 11.6 Hz, 2H), 1.91 (d, J = 10.4 Hz, 2H), 1.69 – 1.59 (m, 2H). C NMR (100ꢀMHz,
DMSO-d6): δ 173.0, 166.9, 155.1, 141.2, 134.2, 133.1, 128.9, 128.3, 128.0, 127.0, 126.2, 119.5,
114.0, 112.8, 106.5, 44.9, 41.6, 27.9 ppm. HRMS-ESI+: calculated for C27H23F2N3O3S2 + H:
540.1222; Found: 540.1205.
1-((2,4-dichlorophenyl)sulfonyl)-N-(4-(4-phenylthiazol-2-yl)phenyl)piperidine-4-carboxamide
(14g) was obtained as an pale yellow solid in the amount of 93 mg (68 % yield). 1H NMR (400 MHz,
DMSO-d6) δ 10.19 (s, 1H), 8.10 (s, 1H), 8.05 – 7.94 (m, 6H), 7.75 (d, J = 8.8 Hz, 2H), 7.67 (d, J =
10.8 Hz, 1H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.6 Hz, 1H), 3.77 (d, J = 12.8 Hz, 2H), 2.86 (t, J =
13
12 Hz, 2H), 2.55 – 2.51 (m, 1H), 1.89 (d, J = 10.8 Hz, 2H), 1.62 (dd, J = 12, 8.8 Hz, 2H). C NMR
(100ꢀMHz, DMSO-d6): δ 172.8, 166.7, 154.9, 141.0, 138.4, 134.9, 134.0, 132.8, 132.2, 131.7, 128.7,
128.1, 127.9, 127.8, 126.8, 126.0, 119.3, 113.8, 44.7, 41.6, 28.0 ppm. HRMS-ESI+: calculated for
C27H23Cl2N3O3S2 + H: 572.3428; Found: 572.0631.
OMP synthesis (Scheme 3):
A mixture of octanoic acid (350 mg, 2.43 mmol), 5-Amino-2-methoxypyridine (302 mg, 2.43 mmol)
and EDC (700 mg, 3.65 mmol) was dissolved in anhydrous DCM (20 mL) and stirred overnight. The
mixture was transferred to separatory funnel, washed with aqueous solution of 1M HCl (200 mL),
followed by an aqueous solution of saturated sodium bicarbonate, the organic layer was dried over
anhydrous sodium sulfate, filtered and concentrated. The crude OMP was purified by column
chromatography (ethyl acetate/hexane 1:4). The product (301 mg, 49% yield) was obtained as a pale-
yellow solid.
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1
N-(6-methoxypyridin-3-yl) octanamide (OMP): H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), 7.90
(dd, J = 2.8, 6.4 Hz, 1H), 7.51 (s, 1H), 6.71 (d, J = 8.8 Hz, 1H), 3.91 (s, 3H), 2.35 (t, J = 7.2 Hz, 2H),
1.71 (t, J = 6.8 Hz, 2H), 1.29 - 1.28 (m, 8H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (100ꢀMHz,
CDCl3): δ 171.9, 160.9, 138.6, 132.4, 128.6, 110.5, 53.5, 37.3, 31.6, 29.2, 29.0, 25.6, 22.5, 14.0 ppm.
HRMS-ESI+: calculated for C14H23N2O2 + H: 251.1760; Found: 251.1749.
Conflict of Interest:
The authors declare that there is no conflict of interest regarding the publication of this article.
Acknowledgements:
This research was financially supported by startup funds granted by the College of Natural
Sciences and Mathematics at California State University, Fullerton and Project RAISE, U.S.
Department of Education HSI-STEM award number P031C160152. Instrumentation support was
provided by the National Science Foundation MRI (CHE1726903) for acquisition of an UPLC-MS.
Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon
reasonable request.
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Figure Legend
Figure 1A. Inactivation of Endocannabinoid Anandamide and catalytic triad of FAAH.
Figure 1B. Mechanism of the hydrolytic cleavage of anandamide.
Figure 2. Known FAAH inhibitors.
Figure 3. Design of new series of FAAH inhibitors.
Figureꢀ4A.ꢁA docking pose of the inhibitor 14f in the catalytic site of the human FAAH enzyme,
suggesting that 2,4-difluorophenyl part (circled in red) of the inhibitor is orientated within the binding
pocket.
Figure 4B. 2D representation for the lowest energy conformation of inhibitor 14f in the binding
pocket of human FAAH. Green shading represents hydrophobic region; Grey parabolas represents
accessible surface for large areas; Broken thick line around ligand shape indicates accessible surface;
Size of residue ellipse represents the strength of the contact.
Figureꢀ5A.ꢁA docking pose of the inhibitor 14g in the catalytic site of the human FAAH enzyme,
showing that 2,4-dichlorophenyl moiety (circled in red) is located deeply in the binding pocket of the
human FAAH enzyme.
Figure 5B. 2D representation for the lowest energy conformation of inhibitor 14g in the binding
pocket of human FAAH. Green shading represents hydrophobic region; Grey parabolas represent
accessible surface for large areas; Broken thick line around ligand shape indicates accessible surface;
Size of residue ellipse represents the strength of the contact.
Table 1. FAAH inhibitory activity and docking scores of analogs.
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Table 2. The list of non-covalent interactions of 4-phenylthiazol series of analogs
Table 3. Predicted ADME-Tox properties of synthesized analogs
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Compound FAAH IC50 (nM) Docking Score
URB 597
8a
38
1700
4500
1200
23.4
19.5
30.8
9.6
-21.45
-32.73
-29.11
-29.9
8b
8c
14a
14b
14c
14d
14e
14f
-25.87
-22.34
-23.16
-27.78
-21.46
-31.75
-25.01
54
8.4
14g
11.9
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Compound
14a
H-bonds
Hydrophobic interactions
F192, S193, Y194, I238, G239, G240, S241, F244, F381, L404, I407, V422, L429, L433,
M436, T488, V491, W531
Other non-covalent interactions
L401, G485
M191, G402, D403, F432
F192, S193, Y194, G239, G240, S241, F244, F381, D403, L404, I407, V422, L429, L433,
G485, T488, V491, I530, W531
0
M191, G216, S217, L380, L401, M495
M191, G216, S217, L380, L401, G485, M495
M191, G216, G240
14b
14c
14d
14e
14f
F192, S193, Y194, I238, G239, G240, S241, F244, D403, L404, I407, V422, L429, L433,
M436, T488, V491, I530, W531
0
F192, S193, Y194, S217, G239, S241, F244, L380, F381, L404, I407, L429, F432, L433,
M436, T488, V491, I530, W531
0
T488
M191, F192, S193, Y194, I238, G239, D403, L404, I407, V422, L429, L433, M436, I530,
W531
S241, F244, L401, G485, V491
S217, F244
M191, F192, S193, Y194, I238, G239, L380, L404, I407, V422, L429, F432, L433,
M436, T488, V491, I530, W531
G485, S241
0
F192, S193, Y194, G239, G240, S241, F244, L404, I407, V422, L429, F432, L433,
M436, G485, T488, V491, M495, I530, W531
G216, S217, F381, L401, P484
14g
G272, C269 F192, I238, K263, L266, G268, Y271, E274, R277, L278
L154, S190, M191, K267, C269, V270, Q273
URB-597
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Drug
Likeness
1.008
-0.033
-0.081
0.015
0.714
0.666
0.807
1.123
1.223
0.707
0.802
Bad
# of Rot
# of H-B
# of H-B
HalfLife
(hours)
0.85
hERG
Tox
Compound Mol. Wt. cLogP cLogS
CACO2
Groups
Bonds
Acceptors
Donors
Inhibition Score
338.163
400.0915
394.1351
412.1257
509.0901
521.1243
537.0947
521.1243
537.0947
539.1148
571.0557
4.022
4.355
4.424
4.573
5.785
6.003
6.448
6.123
6.568
6.273
-6.168
-5.95
0
0
0
0
0
0
0
0
0
0
0
6
5
5
5
7
7
7
7
7
7
7
5
7
7
7
8
8
8
8
8
8
8
3
1
1
1
1
1
1
1
1
1
1
-4.985
-5.156
-5.053
-5.148
-5.135
-5.177
-5.163
-5.207
-5.168
-5.263
-5.257
0.714
0.149
0.342
0.255
0.149
0.31
2
URB-597
8a
1.531
2.264
3.264
1.083
2.048
2.048
1.839
1.839
3.732
3.732
1.242
-6.359
-6.836
-8.176
-9.062
-9.159
-9.103
-9.543
-9.268
1.242
8b
1.242
8c
0
0
0
0
0
0
0
14a
14b
14c
14d
14e
14f
0.182
0.31
0.182
0.371
0.209
7.163 -10.085
14g
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cbdd_13670_f1a.tiff
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cbdd_13670_f1b.tiff
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cbdd_13670_f2.tiff
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