Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model

Article abstract of DOI:10.1016/j.bmcl.2018.11.048

Conceptual design and modification of urea moiety in chemotype PF-3845/04457845, the bench marking irreversible inhibitor of fatty acid amide hydrolase (FAAH), led to discovery of a novel nicotinamide-based lead 12a having reversible mechanism of action.

Full text of DOI:10.1016/j.bmcl.2018.11.048

Accepted Manuscript
Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model
Debnath Bhuniya, Rajendra K. Kharul, Atul Hajare, Nadim Shaikh, Sandeep
Bhosale, Sandip Balwe, Fouzia Begum, Siddhartha De, Sonalee Athawankar,
Dhananjay Joshi, Vamsi Madgula, Kaushal Joshi, Amol A. Raje, Ashwinkumar
V. Meru, Amol Magdum, Kasim A. Mookhtiar, Rashmi Barbhaiya
PII:
S0960-894X(18)30924-7
https://doi.org/10.1016/j.bmcl.2018.11.048
BMCL 26159
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 October 2018
20 November 2018
22 November 2018
Please cite this article as: Bhuniya, D., Kharul, R.K., Hajare, A., Shaikh, N., Bhosale, S., Balwe, S., Begum, F., De,
S., Athawankar, S., Joshi, D., Madgula, V., Joshi, K., Raje, A.A., Meru, A.V., Magdum, A., Mookhtiar, K.A.,
Barbhaiya, R., Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.048
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Discovery and evaluation of novel FAAH
inhibitors in neuropathic pain model
Leave this area blank for abstract info.
Debnath Bhuniya,* Rajendra K. Kharul, Atul Hajare, Nadim Shaikh, Sandeep Bhosale, Sandip Balwe, Fouzia
Begum, Siddhartha De, Sonalee Athawankar, Dhananjay Joshi, Vamsi Madgula, Kaushal Joshi, Amol A. Raje,
Ashwinkumar V. Meru, Amol Magdum, Kasim A. Mookhtiar, Rashmi Barbhaiya

1
Bioorganic & Medicinal Chemistry Letters
Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model
Debnath Bhuniya,* Rajendra K. Kharul, Atul Hajare, Nadim Shaikh, Sandeep Bhosale, Sandip Balwe,
Fouzia Begum, Siddhartha De, Sonalee Athawankar, Dhananjay Joshi, Vamsi Madgula, Kaushal Joshi,
Amol A. Raje, Ashwinkumar V. Meru, Amol Magdum, Kasim A. Mookhtiar, Rashmi Barbhaiya
Drug Discovery Facility – Pune, Advinus Therapeutics Limited. Head Office: Block No. 21 & 22, Phase II, Peenya Industrial Area, Bangalore 560058, India
*Corresponding author: Current address: A2/202, PRISM, Sr No. 6&7, Aundh, Pune 411007, India; Tel.: +91 989 064 6615. E-mail address:
dbhuniya@yahoo.com (D. Bhuniya)
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Conceptual design and modification of urea moiety in chemotype PF-3845/ 04457845, the
bench marking irreversible inhibitor of fatty acid amide hydrolase (FAAH), led to
discovery of a novel nicotinamide-based lead 12a having reversible mechanism of action.
Focused SAR around the pyridine heterocycle (Ar) in 12a (Table 1 & 2) resulted into four
shortlisted compounds, (-)-12a, (-)-12i, (-)-12l-m. The required (-)-enantiomers were
obtained via diastereomeric resolution of a novel chiral dissymmetric intermediate 15.
Based on comparative profile of FAAH potency, metabolic stability in liver microsome,
liability of inhibiting major hCYP450 isoforms, rat PK, and brain penetration ability, two
SAR optimized compounds, (-)-12l and (-)-12m, were selected for efficacy study in rat
model of chemotherapy-induced peripheral neuropathy (CIPN). Both the compounds
exhibited dose related antihyperalgesic effects, when treated with 3 to 30 mg/kg po for 7
days. The effects at 30 mg/kg are comparable to that of PF-04457845 (10 mg/kg) and
Tramadol (40 mg/kg).
Keywords:
Fatty Acid Amide Hydrolase (FAAH) inhibitors
Chemotherapy-Induced Peripheral Neuropathy
Reversible Mechanism of Action
Diastereomeric Resolution
2009 Elsevier Ltd. All rights reserved.
Neuropathic pain (NP) is a chronic pain caused by lesion or
disease affecting any part of the nervous system leading to
clinical conditions ranging from painful neuropathy to poststroke
central pain.1 Anticonvulsant drugs, Gabapentin and Pregabalin,
acting on α2δ subunit-containing voltage-dependent calcium
channels (VDCCs), and antidepressant agents such as serotonin-
norepinephrine reuptake inhibitor (SNRI) Duloxetine are the
first-line treatment options for NP.2-3 However, their efficacies
are mediocre, and also associated with several side effects
including dizziness, vertigo, nausea, dry mouth and dairrhea.3c-f
Hence, there is an unmet medical need to discover a new
pharmacological class for the treatment of neuropathic pain that
can also be safely combined with the current medication. We are
particularly interested in condition like pain due to cancer
chemotherapy-induced peripheral neuropathy (CIPN) for its
growing importance in connection with the cancer
chemotherapy.4
reversible as well as irreversible mechanism of action.6-7 Fig. 1
exemplifies some of the well characterized FAAH inhibitors 1-11
including those tested in early phase of clinical trials.8-19 In
general, pharmacological inhibition as well as genetic ablation20
of FAAH has been shown to be associated with increased levels
of endogenous fatty acid amides (FAAs) including AEA,
eliciting analgesic effects in various animal models of
inflammation and chronic pain primarily via activation of
endocannabinoid CB receptors. Therefore, it has been also
hypothesized that a FAAH inhibitor may avoid side effects as
well as abuse of cannabis and exocannabinoids.21
In its first wave, clinical trials with FAAH inhibitors targeted
several neuropsychiatric conditions such as schizophrenia,
Tourette syndrome, symptoms associated with cannabis
withdrawal, and inflammatory pain due to osteoarthritis (OA). In
its careful second wave, NP and major depressive disorder
(MDD) are being considered as more suitable indications,
evidenced by advancement of 7a (V-158866),14 11 (ASP-8477),19
6 (SSR-411298)13 and 10b (JNJ-42165279)18b-c in Phase-2
clinical trials.
Fatty acid amide hydrolase (FAAH) is an intracellular
membrane-bound enzyme that utilizes unusual catalytic triad Ser-
Ser-Lys to degrade and inactivate fatty acid amide family of
signaling lipids, including the endogenous cannabinoid ligands
N-arachidonoylethanolamine (Anandamide, AEA) and 2-
arachidonoyl glycerol (2-AG).5 Since the discovery of FAAH as
molecular target in 1996, several chemotypes have been reported
and reviewed in the literature as FAAH inhibitors having
Interaction of Ser-241 residue of FAAH catalytic site with its
inhibitors have been particularly demonstrated by co-crystal
structures,22 initially with
a
substrate analog methoxy
arachidonoyl fluorophosphonate (MAFP), and subsequently with

2
non-FA based synthetic inhibitors such as, (i) OL-1358 (forms
reversible oxyanion intermediate), (ii) URB-59716 and (iii) PF-
750/384517 (ii and iii form covalently bound irreversible
carbamates). We conceptualized that replacing the urea moiety in
PF-3845 with an amide functional group, as depicted in newly
designed analog 12a (Fig. 2), might lead to a reversible
inhibition, via formation of the tetrahedral oxyanion
intermediate.
Fig 2. Design of a new reversible inhibitor (R)-12a.
Such an intermediate would neither have the option of permanent
covalent bonding with the Ser-241 nor a hydrolysis, as the
aliphatic cyclohexylamine moiety is not a good leaving group.
Additionally, the newly designed 12a scaffold would have the
potential of retaining well established selectivity profile of PF-
3845/04457845.17 Suitability of replacing the urea moiety with an
amide FG was verified by qualitative docking 12a to the co-
crystal structure of PF-3845 (PDB:3LJ6) (Fig.
2
and
supplementary Fig. S1). Based on this in silico exercise, we have
concluded that (R)-configuration of 12a could only be docked to
the catalytic site with the desired pose as compared to its (S)-
enantiomer.
Accordingly, a new heteroaryl amide series was invented as
FAAH inhibitors broadly claiming the scaffold 12, and the
intellectual property has been protected by PCT application.23
Objective of this preliminary communication is to disclose a
focused set of SAR around 12 which has also yielded two
optimized leads suitable for further development.
Data of initial analogs (±)-12a-h have been shown in Table 1
wherein SAR was observed for the intended change of the right-
hand side pyridin-3-yl ring of 12a to various other N-containing
heteroaryl ring (Ar) disposing the ring-N at different directions.
Based on consistent human and rat FAAH potency as well as
intrinsic metabolic stability in HLM and RLM, the analogs 12a,
12e and 12g were qualified for the next round of SAR and
optimization. Further focus was made on pyridine-3-yl (12a) and
pyrimidin-5-yl (12e) analogs wherein the ring substitution SAR,
particularly with additional -N atom, was studied as in 12i-12p.
Whereas, all other analogs maintained the FAAH potencies, 12j-
k lost the rat FAAH potency in a commonly observed
phenomena in this series that, any electron withdrawing group
which could potentially reduce the basicity of the 3-pyridyl-N,
resulted in particular loss of rat potency. Subsequently, 12n-p
were also disqualified due to relatively less solubility which were
reflected in <10% oral bioavailability in rats (supplementary
Table S5a).
Fig 1. Representative FAAH inhibitors from literature.
Based on above SAR inputs, compounds 12a, 12i, 12l-m were
resolved using chiral prep. HPLC and both the enantiomers (with
>96% ee) were screened against FAAH inhibition assay. In all
the cases, (-)-enantiomers were found to be >100 fold more
potent than (+)-enantiomers irrespective of the human or rat
FAAH enzyme (supplementary Table S1). Based on the
comparative FAAH potency of the enantiomers, and supported

3
by the docking analysis, an (R)- stereochemistry has been
tentatively assigned to the more potent (-)-enantiomers.
As was expected, all the four (-)-enantiomers were near 1 and
10 nM potent in h- and r-FAAH respectively and possessed
adequate metabolic stability in HLM and RLM. The observed
low intrinsic MR (0.06-0.04 nmol/min/mg) reflected well into the
rat PK profile having low plasma clearance (CL), required longer
Profile of the shortlisted (-)-12a, (-)-12i, and (-)-12l-m have
been presented in Table 2 which included additional two
plasm
a
half-
Compound
12a
12b
12c
12d
12e
12f
12g
12h
life
(t1/2)
and
good
oral
hFAAH IC50 (nM)a
2.2±0.04
(n = 8)
508
14
9.0±1
(n = 6)
2.6 ±0.2
(n = 3)
10
2.6±0.4
(n = 6)
6
27.8±1.9
(n=8)
NDb
--
15%c
--
194±35.2
(n = 3)
100±13.1
(n = 3)
266±64.1
(n = 5)
64.6±11.3
(n = 7)
145±53.6
(n = 3)
rFAAH IC50 (nM)a
--
<0.04; 0.07
--
--
HLM; RLM
0.06; 0.06
0.04; <0.04
MR (nmol/min/mg)d
BA
(%F).
In
additi
on,
all
the
four
comp
ounds
Compound
hFAAH IC50 (nM)a
12i
12j
12k
12l
12m
2
12n
12o
12p
5.0±0.1
(n = 5)
5.1±0.7
(n = 3)
4
3±0.9
(n = 3)
6.7±1.4
(n = 5)
3
2.2±0.2
(n = 5)
rFAAH IC50 (nM)a
31.3±5.5
(n = 5)
0.07; 0.05
80.7±6.9
(n = 3)
0.06; 0.04
52±8.5
(n = 3)
0.06; <0.04
29±4.2
(n = 3)
0.09; 0.04
16
6.6±0.5
(n = 4)
0.11; 0.09
17.2±0.2
(n = 7)
0.08; 0.04
9
0.03; 0.02
0.09; 0.04
HLM; RLM
MR (nmol/min/mg)d
ADMET evaluation: (a) CYP inhibition; (b) rat PK and brain
penetration. Selectivity against inhibition of major human
CYP450 isoforms is an important aspect of modern-day drug
optimization as the drug otherwise may lead to potential drug-
drug-interactions (DDI). This is particularly important in the area
of CIPN where multiple drugs are co-administered. CNS or brain
penetration was another important requirement due the adequate
therapeutic benefits in NP.
were found to be available in brain, indicating CNS penetration
of the drugs. Compound (-)-12a, however, encountered with
unacceptable hCYP3A4 inhibition (IC50 0.4 µM) in addition to a
border line
liability for
CYP
2C9
and
2C19.
Table 1. FAAH inhibition and metabolic stability SAR of analogs around (±)-
12.
Compound
(-)-12a
(-)-12i
(-)-12l
(-)-12m
hFAAH IC50
(nM)b
0.7±0.06
(n=3)
1.2±0.03
(n=3)
1.3±0.1
(n=7)
0.6 ±0.03
(n = 3)
rFAAH IC50
(nM)b
13±1.9
(n=3)
11.6±0.9
(n=3)
9.2±0.8
(n=6)
6.2±1.0
(n = 4)
a) Each IC50 value corresponds to an average of at least two experiments (n≥2) each in
triplicate data set and obtained as per the experimental procedure described in
supplementary section. Values are presented as average±SEM when n>2.
b) Not Determined.
HLM; RLM MR
(nmol/min/mg)c
0.04; <0.04
<0.04; 0.06
0.06; 0.04
<0.04; 0.04
c) % inhibition at 100 nM concentration of the inhibitor.
hCYP IC50 (nM)d
1A2, 2C9,
2C19, 2D6,
3A4
d) Intrinsic metabolic rate (MR) in Human or Rat Liver Microsome (HLM or RLM) in the
presence of NADPH cofactor (in nmol/min/mg of the LM protein). MR value of <0.1
approx correlates to >70% unmetabolized after 30 min. of incubation, and it indicates that
the compound is suitable for progression to PK study.
>10, 2.7,
4.6, >10,
0.4 (2.4)
>12.5, 3.4,
9.1, >12.5,
>12.5(>12.5)
>12.5, 9.2,
>12.5, 6.5,
>12.5,>12.5,
>12.5(>12.5)
>12.5,>12.5,
>12.5(>12.5)
Rat PK:e
CL(mL/min/kg)
Vss (L/kg)
t1/2 (h)
2.4±0.2
0.9±0.1
5.1±1.2
35
0.7±0.03
0.6±0.04
9.2±2.6
42
0.8±0.07
1.2±0.1
17 ±2.3
46
0.6±0.1
0.8±0.2
17.2±1.7
46
%F
Brain : plasma
1.5±0.2
0.6±0.01
0.5±0.04
0.8±0.02
Table 2. Profile of the shortlisted (-)-enantiomers.a
a)Chiral prep. HPLC method used for the resolution into the enantiomers (>96% ee).
FAAH IC50 for the less potent (+)-isomers are given in supplementary Table S1.
b) Ref to footnote a) in Table 1.
c) Ref to footnote d) in Table 1.
d)Ref to supplementary section for protocol. For hCYP3A4, midazolam as well as
testosterone (value in parenthesis) were used as substrates due to different binding sites.
e)Male SD rats (fasted overnight) were administered compounds at 3 mg/kg iv (solution
formulation) and 10 mg/kg po (NaCMC suspension). PK values presented as mean ± SD.
Ref to supplementary data for the protocol and formulation details.
Fig 3. Reversible mechanism of action of (-)-12a. Briefly, hFAAH (10x of
regular assay conc) and the inhibitor of choice (at 10xIC50 conc) were

4
preincubated for 1 hour followed by addition of arachidonyl-7-amino-4-
methyl coumarin amide (AAMCA) as substrate (Lane 1, hatched bar) or a 10-
fold dilution of the preincubated enzyme-inhibitor complex into a reaction
buffer containing the substrate (Lane 2, crossed grid bar). A reference
condition was created without the preincubation period but doing 10-fold
dilution process to mimic the IC50 situation (Lane 3, solid bar). The resultant
% FAAH enzyme activities have been plotted for the three conditions. PF-
3845: FAAH activity could not be recovered upon dilution after the
preincubation (crossed grid bar not detectable), as the inhibitor formed an
irreversible complex with the enzyme. However, near 20% FAAH activity
40
was recovered without the preincubation (solid bar). OL-135 and (-)-12a:
Near 30% FAAH activity were recovered after the preincubation followed by
dilution (crossed grid bars) as the effective conc. of the inhibitors decreased
by a factor of 10 (close to its IC50 conc), indicating the formation of reversible
enzyme-inhibitor complex.
30
*
*
*
*
*
*
*
*
*
*
*
*
20
10
0
*
* *
*
*
*
*
*
*
*
*
*
*
*
*
*
Basal
240 min
Basal
240 min
Basal
240 min
DAY 3
DAY 7
DAY 1
Vehicle, 3 ml/kg, PO
(-)-12l, 3mg/kg, PO
PF-04457845, 10mg/kg, PO
Tramadol, 40mg/kg, PO
Naive
(-)-12l, 30mg/kg, PO
(-)-12m, 3mg/kg, PO
(-)-12m, 30mg/kg, PO
Date is shown as Mean ± S.E.M. (n=3-7); Significantaly different as compared to
Vehicle,*P < 0.05; One-way Annova followed by Dunnett Multiple Comparison test
Fig 4. Effect of (-)-12l and (-)-12m (synthesized by diastereomeric resolution method; >96% ee) on thermal hyperalgesia in a rat model of CIPN. The model was
developed in SD rats by administration of cisplatin (Cytoplatin Injection, Cipla) intraperitoneally once a week at a dose of 3 mg/kg for 4 weeks (cumulative dose,
12 mg/kg). After that, thermal hyperalgesia was assessed using plantar test apparatus at 25 IR intensity and cut off time of 45 seconds. Testing comprised
alternate presentations of radiant heat from below to hindpaw, focused on the mid-plantar surface. The heat source cut off automatically with paw withdrawal and
the reaction time (Paw Withdrawal Latency) was recorded. Paw withdrawal latency was defined as the mean of three trials applied to paw. All the treated and the
vehicle groups were assessed for the thermal hyperalgesia on day 1, 3, 7 at the time of 0 h (before dosing, basal) and 4 h (240 min) after once daily (qd) oral (po)
treatment. Vehicle control: Tween 80® - 1% (v/v); 0.5% (w/v) NaCMC in water q.s.; dose vol. 3 mL/kg. Reversible FAAH inhibitors: (-)-12l, (-)-12m at 3 and
30 mg/kg po qd dose. Irreversible FAAH inhibitor: PF-04457845 at 10 mg/kg po qd dose. A standard positive control: Tramadol at 40 mg/kg po qd dose. Dosing
formulation for the test compounds: oral suspension in Tween 80® - 1% (v/v); 0.5% (w/v) NaCMC in water q.s; dose vol. 3 mL/kg. Ref to supplementary data
for the detailed protocol.
The desired optimized lead like properties were achieved in
compounds (-)-12l and (-)-12m with a balanced profile of FAAH
potency (h-IC50: 1.3 & 0.6 nM; r-IC50: 9.2 & 6.2 nM
respectively), selectivity against five major hCYP450 isoforms
(>5000 fold), and rat PK properties (46% BA, 50-80% brain
penetration). Finally, for the development of FAAH inhibitors,
selectivity against a related enzyme, monoacylglycerol lipase
(MAGL),6b,6f is very important as the latter is primarily
responsible for hydrolysis of another endocannabinoid 2-AG,
essential for the brain and CNS functions. We did not see any
inhibition of MAGL tested in vitro up to 10 μM of (-)-12l and (-)-
12m (>10000 fold selectivity).
(quantified by mass spectroscopic method) over a period of 1 h
incubation with hFAAH enzyme. In contrast, there was a gradual
loss of PF-3845 observed under the similar condition
(supplementary Table S2).
Finally, the optimized leads (-)-12l and (-)-12m were tested in
rat model of CIPN (3 and 30 mg/kg, po, qd, administered for 7
days) and the results have been shown in Fig. 4. Under acute set
up, after single oral administration at 30 mg/kg, both the
compounds exerted significant improvement of the NP condition
and thermal hyperalgesia by exhibiting increased Paw
Withdrawal Latency and reaction time compared to vehicle
(measured at 4 h post dosing on day 1). The pain relieving effect
was maintained in
A reversible mechanism of action by this chemotype was
established based on a principle of qualitative recovery of FAAH
enzyme activity upon dilution of a preincubated enzyme-inhibitor
complex. As briefed in Fig. 3, recovery of hFAAH activity, for
hydrolysis of its substrate AAMCA, was possible in case of
preincubation with the known reversible inhibitors OL-135, and
(-)-12a. On the contrary, the enzyme activity could not be
recovered when preincubated with the covalent and irreversible
inhibitor PF-3845. Reversible as well a non-substrate like MoA
for the FAAH inhibition by (-)-12a was further confirmed in a
separately designed experiment by near 100% recovery of (-)-12a

5
o
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 100 C, 18 h, 95%;
(b) NaBH4, MeOH, 0 oC, 1 h, 99%; (c) SOCl2, CH2Cl2, 0 oC to RT, 1 h, 99%;
(d) P(OEt)3, 140 oC, 18 h, 85%; (e) 1,4-dioxaspiro[4.5]decan-8-one, NaH,
o
o
THF, 0 C to RT, 18 h, 63%; (f) 1(M) HCl, acetone, 60 C, 2-4 h, 100%; (g)
(i) 2(M) NH3 in EtOH, Ti(O-i-Pr)4, RT, 6 h; (ii) NaBH4, RT, 18 h; (iii)
NH4OH, 60% combined; (h) ArCO2H, EDCI.HCl, HOBt, NMM, DMF, RT,
14-18 h, 40-50% (for 12a-h, 12n-p); (i) ArCOCl, Et3N, DCM, 0 oC to 26 oC,
2 h, 79% (for 12a); (j) ArCO2H, PyBOP, DIPEA, CH2Cl2, RT, 18 h, 60-72%
(for 12i, 12k-m); (k) RCOOH, oxalyl chloride, Et3N, CH2Cl2, 0 to 26 oC, 2 h,
o
82% (for 12j); (l) (i) (+)-O,O'-di-p-toluoyl-D-tartaric acid, EtOH, 70 C, 2 h,
then cool to RT, ppt collected; (ii) Crystallization using MeOH:EtOH (3:1)
followed by recrystallization using MeOH:EtOH (1:1); (iii) 5% NaOH,
AcOEt (pH 10), 10-15 oC, 30 min. >98% chiral purity by HPLC.
antibody upon chronic administration. The current FAAH
inhibitors are expected not to encounter with such problems.
a subacute condition upon repeat dosing for 6 days and measured
after 24h of post dosing (same as basal reading on day 7).
Though the antihyperalgesic effect of the compounds at 3 mg/kg
po could not be seen upon single administration, it indeed
established the effect on day 7 in a dose dependent manner
(measured at 4 h post dosing on day 7). Overall, the reversible
FAAH inhibitors (-)-12l and (-)-12m at 30 mg/kg po manifested
the therapeutic effects comparable to the tested irreversible
FAAH inhibitor PF-04457845 (at 10 mg/kg po dose) and a
standard positive control Tramadol (at 40 mg/kg po dose).
Acknowledgments
Authors would like to acknowledge the larger FAAH team
members for various contributions. Dr. Jagadeesh N. Mavinahalli
is being acknowledged for molecular docking. Advinus
publication number: ADV-A-032.
References and notes
Synthetic outline of the FAAH inhibitors 12a-p has been
shown in Scheme-1 (ref. to supplementary section for
experimental). A novel dissymmetric amine 15 acted as a
common intermediate which was synthesized from commercially
available synthons, 2-chloro-5-(trifluoromethyl) pyridine and 3-
hydroxybenzaldehyde in seven steps via the formation of
pyridyloxybenzylchloride intermediate 13 (steps a, b, c) followed
by the spiro-olefinic intermediate 14 (steps d, e). Deprotection of
ketal in 14 and subsequent reductive amination using ammonia
and Ti(O-i-Pr)4 (steps f, g) furnished the racemic amine 15,24 which
upon amide coupling with appropriate carboxylic acids or acid
chlorides (steps h/i/j/k) yielded the racemic compounds 12a-p.
Enantiomers of 12a,25 12i,26 12l-m27-28 were initially separated
using chiral preparative HPLC and the required enantiomers were
used for in vitro and PK studies as mentioned in Table 2.
Subsequently, for multigram requirement of (-)-12l-m (CIPN
efficacy study and toxicology experiments), the racemic
intermediate 15 was resolved into (-)-(R)-15 via diastereomeric
salt formation using (+)-O,O'-di-p-toluoyl-D-tartaric acid (step l)
and was used for the intended amide coupling reactions (step j).
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In conclusion, a novel scaffold 12 has been discovered as
FAAH inhibitor with reversible mechanism of action. Design
strategy was to maintain the overall structural features of PF-
3845/04457845 to take advantage of the well-established
selectivity profile of PF-chemotype. The reversible and non-
substrate like mechanism of action has been ascertained based on
a qualitative recovery of the active enzyme and near quantitative
recovery of (-)-12 upon preincubation with hFAAH. After a
systematic screening, two compounds (-)-12l and (-)-12m have
been identified as optimized leads and therefore nominated for
preclinical candidate profiling towards an indication of
neuropathic pain (NP). Irreversible inhibitors particularly with
longer plasma half-life can potentially generate circulating
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6
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Pabst, B.; Bhattacharya, K.; Nugent, R. A.; Kamtekar, S.; Cravatt,
B. F.; Ahn, K.; Stevens, R. C. Proc. Natl. Acad. Sci. U.S.A. 2008,
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400, 743–754.
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L.; Chambers, A.; Pierce, J. M.; Seierstad, M. et al. Bioorg. Med.
Chem. Lett. 2014, 24, 1280–1284.
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Lanza, T.J.; Chang, L.; Bakshi, R.K. et al. ACS Med. Chem.
Lett. 2014, 5, 717-721.
13. For 6 (SSR-411298): Griebel1, G.; Jeanne Stemmelin, J.; Lopez-
Grancha, M.; Fauchey, V.; Slowinski, F.; Pichat, P.; Dargazanli,
G.; Abouabdellah, A.; Caroline Cohen, C.; Bergis, O. E. Nature
Sci. Rep. 2018, 8, 2416.
23. Kharul, R. K.; Bhuniya, D.; Mookhtiar, K. A.; Singh, U.; Hazare,
A.; Patil, S.; Datrange, L.; Thakkar, M. International PCT
publication No. WO 2013/042139 A1.
14. For 7a (V158866, structure unknown): (a) Pawsey, S.;
Browne, M. W. H.; Donaldson, K.; Christie, M.; Warrington, S.
Drugs R. D. 2016, 16, 181–191. (b) Roughley, S.; Walls, S.; Hart,
T. et al. Azetidine derivatives. International PCT publication No.
WO 2009/109743.
24. 4-[[3-[[5-(Trifluoromethyl)-2-
pyridyl]oxy]phenyl]methylene]cyclohexanamine (15): Thick
liquid. 1H NMR (400 MHz, CDCl3) δ: 1.13-1.23 (m, 1H, -CH2-),
1.25-1.34 (m, 1H, -CH2-), 1.87-1.94 (m, 1H, -CH2-), 1.95-2.04 (m,
2H, -CH2-), 2.25 (dt, J = 12.9, 3.4 Hz, 1H, >CH-NH2), 2.38 (d, J =
13.7 Hz, 1H, -CH2-), 2.84-2.95 (m, 2H, -CH2-), 3.48 (s, 2H, -
NH2), 6.26 (s, 1H, olefinic), 6.97-7.01 (m, 3H, Py-3H & Phenoxy-
ortho-H), 7.08 (d, J = 7.6 Hz, 1H, Phenoxy-para-H), 7.37 (t, J =
7.8 Hz, 1H, Phenoxy-meta-H), 7.89 (dd, J = 8.5, 2.2 Hz, 1H, Py-
4H), 8.45 (s, 1H, Py-6H). 13C NMR (100 MHz, CDCl3) δ: 27.1,
35.3, 36.9, 37.6, 50.1, 111.3, 118.9, 121.5 (q, J = 33.3 Hz, 1C),
121.8, 122.1, 123.7 (q, J = 270 Hz, 1C), 126.1, 129.4, 136.7 (q, J
= 3.1 Hz, 1C), 140.2, 142.4, 145.6 (q, J = 4.1 Hz, 1C), 153.0,
165.9. LC-MS (ES) m/z 349.2 (M+1). HRMS (ESI) m/z calcd for
C19H20F3N2O [M+H]+ 349.1522, found 349.1523. (-)-15: Chiral
purity: >98 % (by HPLC). [α]20D -28.8° (c 0.5, MeOH).
25. N-[4-[[3-[[5-(Trifluoromethyl)-2-
15. For 7b (BIA-10-2474): (a) Kiss, L. E.; Learmonth, D. A.; Rosa,
C. P. et al. International PCT Publication No. WO
2010/074588A2. (b) Clinical Study Protocol N° BIA-102474-101
(Version 1.2, 01 July 2015) submitted to the French ethics
committee and regulatory authorities (CPP and ANSM) by BIAL-
Portela & C, S.A. (c) Kiss, L. E.; Beliaev, A.; Ferreira, H.
S.; Rosa, C. P.; Bonifácio, M. J.; Loureiro, A. I.; Pires, N.
M.; Palma, P. N.; Soares-da-Silva, P. ChemMedChem. 2018, 13,
2177-2188.
16. For 8 (URB-597/937): (a) Russo, R.; Loverme, J.; La Rana, G.;
Compton, T. R.; Parrott, J.; Duranti, A.; Tontini, A.; Mor, M.;
Tarzia, G.; Calignano, A.; Piomelli, D. J Pharmacol. Exp.
Ther. 2007, 322, 236–242. (b) Clapper, J. R.; Moreno-Sanz, G.;
Russo, R.; Guijarro, A.; Vacondio, F.; Duranti, A.; Tontini, A.;
Sanchini, S.; Sciolino, N. R.; Spradley, J. M.; Hohmann, A. G.;
Calignano, A.; Mor, M.; Tarzia, G.; Piomelli, D. Nature Neurosci.
2010, 13, 1265-1270. (c) Moreno-Sanz, G.; Duranti, A.; Melzig,
L.; Fiorelli, C.; Ruda, G. F.; Colombano, G.; Mestichelli, P.;
Sanchini, S.; Tontini, A.; Mor, M.; Bandiera, T.; Scarpelli, R.;
Tarzia, G.; Piomelli, D. J Med. Chem., 2013, 56, 5917-5930 and
the ref. cited herein.
pyridyl]oxy]phenyl]methylene]cyclohexyl]pyridine-3-
carboxamide (12a): Mp 140-142 oC. 1H NMR (400 MHz, CDCl3)
δ: 1.37 (dq, J = 11.0, 5.6 Hz, 1H, -CH2-), 1.48 (dq, J = 11.3, 5.1
Hz, 1H, -CH2-), 2.15-2.59 (m, 3H, -CH2-), 2.37-2.49 (m, 2H, -
CH2-), 2.88-2.94 (m, 1H, -CH2-), 4.18-4.28 (m, 1H, >CH-NH2),
6.00 (d, J = 7.4 Hz, 1H, amide-NH), 6.32 (s, 1H, olefinic), 6.99-
7.03 (m, 3H, Py-3H & Phenoxy- ortho-H), 7.00 (d, J = 7.9 Hz,
1H, Phenoxy-para-H), 7.37-7.41 (m, 2H, Phenoxy-meta-H &
RHS-Py-5H), 7.90 (dd, J = 8.8, 2.4 Hz, 1H, Py-4H), 8.10 (dt, J =
7.8, 1.7 Hz, 1H, RHS-Py-4H), 8.45 (s, 1H, Py-6H), 8.72 (dd, J =
4.9, 1.7 Hz, 1H, RHS-Py-6H), 8.94 (d, J = 1.7 Hz 1H, RHS-Py-
2H). LC-MS (ES) m/z 454.1 (M+1). HRMS (ESI) m/z calcd for
C25H22F3N3O2 [M+H]+ 454.1737, found 454.1742. (-)-12a: Mp
135-136 °C. [α]20D -22.8° (c 0.2, CHCl3).
17. For 9 (PF-3845/04457845): (a) Ahn, K.; Johnson, D. S.; Mileni,
M.; Beidler, D.; Long, J. Z.; McKinney, M. K.;Weerapana, E.;
Sadagopan, N.; Liimatta,M.; Smith,S.E.; Lazerwith,
S.;Stiff,C.;Kamtekar,S.;Bhattacharya,K.; Zhang, Y.; Swaney, S.;
Van Becelaere, K.; Stevens, R. C.; Cravatt, B. F. Chem. Biol.
2009, 16, 411–420. (b) Johnson, D. S.; Stiff, C.; Lazerwith, S. E.;
Kesten, S. R.; Fay, L. K.; Morris, M.; Beidler, D.; Liimatta, M. B.;
Smith, S. E.; Dudley, D. T.; Sadagopan, N.; Bhattachar, S. N.;
Kesten, S. J.; Nomanbhoy, T. K.; Cravatt, B. J.; Ahn, K. ACS
Med. Chem. Lett, 2011, 2, 91-96. (c) Ahn, K.; Smith, S. E.;
Liimatta, M. B.; Beidler, D.; Sadagopan, N.; Dudley, D. T.;
Young, T.; Wren, P.; Zhang Y.; Swaney, S.; Becelaere, K. V.;
Blankman, J. L.; Nomura, D. K.; Bhattachar, S. N.; Stiff, C.;
Nomanbhoy, T. K.; Weerapana, E.; Johnson, D. S.; Cravatt, B. F.
J Pharmacol. Exp. Ther. 2011, 338, 114-124.
26. 6-Amino-N-[4-[[3-[[5-(trifluoromethyl)-2-pyridyl]oxy]phenyl]
methylene]cyclohexyl]pyridine-3-carboxamide (12i). Mp 156-157
oC. 1H NMR (400 MHz, CDCl3) δ: 1.28-1.39 (m, 1H, -CH2-),
1.41-1.50 (m, 1H, -CH2-), 2.09-2.20 (m, 3H, -CH2-), 2.35-2.45 (m,
2H, -CH2-), 2.90 (d, J = 14.2 Hz, 1H, -CH2-), 4.15-4.23 (m, 1H,
1H, >CH-NH2), 4.79 (brs, 2H, RHS-Py-6-NH2), 5.80 (d, J = 7.6
Hz, 1H, amide-NH), 6.31 (s, 1H, olefinic), 6.49 (d, J = 8.5 Hz, 1H,
RHS-Py-5H), 6.99-7.02 (m, 3H, Py-3H & Phenoxy- ortho-H),
7.10 (d, J = 7.6 Hz, 1H, Phenoxy-para-H), 7.38 (t, J = 7.8 Hz, 1H,
Phenoxy-meta-H), 7.85-7.91 (m, 2H, Py-4H & RHS-Py-4H ), 8.44

7
(brs, 2H, Py-6H & RHS-Py-2H). LC-MS (ESI) m/z 469.3 (M+1).
HRMS (ESI) m/z calcd for C25H24F3N4O2 [M+H]+ 469.1846, found
469.1849. (-)-12i: Mp 145-147 °C. [α]20D -56.3° (c 0.2, CHCl3).
27. 6-Amino-5-methyl-N-[4-[[3-[[5-(trifluoromethyl)-2-
pyridyl]oxy]phenyl] methylene]cyclohexyl]pyridine-3-
(d, J = 13.6 Hz, 1H, -CH2-), 2.80 (d, J = 13.6 Hz, 1H, -CH2-),
3.93-4.02 (m, 1H, >CH-NH2), 6.29 (s, 1H. olefinic), 6.99 (s, 1H,
amide-NH), 7.03 (d, J = 8.1 Hz, 1H, Phenoxy-ortho-H), 7.10-7.17
(m, 3H, Phenoxy-ortho-H, pyrimidine-3 & 5-H), 7.22 (d, J = 8.6
Hz, 1H Py-3H), 7.40 (d, J = 7.8 Hz, 1H, Phenoxy-para-H), 8.02 (t,
J = 7.9 Hz, 1H, Phenoxy-meta-H), 8.22 (dd, J = 8.6, 2.0 Hz, 1H,
carboxamide (12l). Mp 165-168 oC. 1H NMR (400 MHz, CDCl3) δ
1.25-1.38 (m, 1H, -CH2-), 1.41-1.51 (m, 1H, -CH2-), 2.10-2.21 (m,
6H, -CH2-, RHS-Py-5-CH3), 2.35-2.45 (m, 2H), 2.87-2.92 (m,
1H), 4.15-4.25 (m, 1H, >CH-NH2), 4.74 (brs, 2H, RHS-Py-6-
NH2), 5.79 (d, J = 7.3 Hz, 1H, amide-NH), 6.31 (s, 1H, olefinic),
6.99-7.02 (m, 3H, Py-3H & Phenoxy- ortho-H), 7.10 (d, J = 7.5
Hz, 1H, Phenoxy-para-H), 7.38 (t, J = 7.6 Hz, 1H, Phenoxy-meta-
H), 7.72 (s, 1H, RHS-Py-4H), 7.90 (dd, J = 8.6, 2.4 Hz, 1H, Py-
4H), 8.30 (s, 1H, RHS-Py-2H), 8.45 (s, 1H, Py-6H). 13C NMR
(100 MHz, CDCl3) δ 16.9, 27.1, 33.5, 34.2, 35.3, 48.3, 111.4,
115.9, 119.1, 121.1, 121.5 (q, J = 33.3 Hz, 1C), 121.8, 123.7 (q, J
= 269.4 Hz, 1C), 122.7, 126.1, 129.5, 136.7 (q, J = 3.1 Hz, 1C),
140.0, 141.5, 145.0, 145.5 (q, J = 3.8 Hz, 1C), 153.0, 159.3, 165.5,
165.8, 165.9. LC-MS (ESI) m/z 483.1 (M+1). HRMS (ESI) m/z
calcd for C26H26F3N4O2 [M+H]+ 483.2002, found 483.2004. (-)-
12l: Mp 142-144 °C. [α]20D -60.4° (c 0.2, CHCl3).
Py-4H), 8.56 (s, 1H, Py-6H), 8.63 (s, 2H, pyrimidine-NH2). 13
NMR (100MHz, CDCl3) δ 27.1, 33.5, 34.2, 35.2, 48.5, 111.5,
C
118.6, 119.2, 121.6 (q, J = 33.3 Hz, 1C), 121.9, 123.0, 123.8 (q, J
= 269.4 Hz, 1C), 126.2, 129.6, 136.8 (q, J = 3.1 Hz, 1C), 139.9,
141.1, 145.6 (q, J = 3.9 Hz, 1C), 153.1, 157.9 (2C), 163.5, 164.0,
165.9. LC-MS (ESI) m/z 470.2 [M+H]+. (-)-12m: Mp 173-175 °C.
[α]20D -72° (c 0.5, MeOH).
Supplementary Data
Molecular docking study; synthetic details of compounds 12;
brief protocols of pharmacological and DMPK experiments;
additional FAAH MoA data associated with the article.
28. 2-Amino-N-[4-[[3-[[5-(trifluoromethyl)-2-pyridyl]oxy]phenyl]
methylene]cyclohexyl]pyrimidine-5-carboxamide (12m). Mp 187-
190 oC. 1H NMR (400 MHz, DMSO-d6) δ 1.27-1.37 (m, 1H, -
CH2-), 1.40-1.49 (m, 1H, -CH2-), 1.80-1.96 (m, 2H, -CH2-), 2.06
(t, J = 12.8 Hz, 1H, -CH2-), 2.27 (t, J = 13.2 Hz, 1H, -CH2-), 2.38
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Highlights:





Novel scaffold as FAAH inhibitor with
reversible mechanism of action
Proof of MoA by recovery of active enzyme
upon preincubation with inhibitor
Synthesis via diastereomeric resolution of novel
chiral dissymmetric intermediate
Drug-like DMPK properties of Opt. Leads (-)-
12l-m suitable for preclinical development
Antihyperalgesic effects, in rat CIPN model at 3
to 30 mg/kg po dose.