Discovery of heterocyclic carbohydrazide derivatives as novel selective fatty acid amide hydrolase inhibitors: design, synthesis and anti-neuroinflammatory evaluation

Article abstract of DOI:10.1016/j.bmcl.2020.127118

Fatty acid amide hydrolase (FAAH) is a promising target for the development of drugs to treat pain, inflammation, and other central nervous system disorders. Herein, a series of novel heterocyclic carbohydrazide derivatives were firstly designed by the classic scaffold-hopping strategy. Then, multi-steps synthesis and human FAAH enzyme inhibiting activity assays were conducted. Among them, compound 26 showed strong inhibition against human FAAH with IC₅₀ of 2.8 μM. Corresponding docking studies revealed that the acyl hydrazide group of compound 26 well-occupied the acyl-chain binding pocket. It also exhibited high selectivity towards FAAH when comparing with CES2 and MAGL. Additionally, compound 26 effectively suppressed the LPS-induced neuroinflammation of microglial cells (BV2) via the reduction of interleukin-1β and tumor necrosis factor-α. Our results provided significative lead compounds for the further discovery of novel selective and safe FAAH inhibitors with potent anti-neuroinflammation activity.

Full text of DOI:10.1016/j.bmcl.2020.127118

Bioorganic&MedicinalChemistryLetters30(2020)127118
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of heterocyclic carbohydrazide derivatives as novel selective fatty
acid amide hydrolase inhibitors: design, synthesis and anti-
neuroinflammatory evaluation
Yanguo Shang, Qingjing Hao, Kaixuan Jiang, Mengting He, Jinxin Wang⁎
Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009,
China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Fatty acid amide hydrolase (FAAH) is a promising target for the development of drugs to treat pain, in-
flammation, and other central nervous system disorders. Herein, a series of novel heterocyclic carbohydrazide
derivatives were firstly designed by the classic scaffold-hopping strategy. Then, multi-steps synthesis and human
FAAH enzyme inhibiting activity assays were conducted. Among them, compound 26 showed strong inhibition
against human FAAH with IC50 of 2.8 μM. Corresponding docking studies revealed that the acyl hydrazide group
of compound 26 well-occupied the acyl-chain binding pocket. It also exhibited high selectivity towards FAAH
when comparing with CES2 and MAGL. Additionally, compound 26 effectively suppressed the LPS-induced
neuroinflammation of microglial cells (BV2) via the reduction of interleukin-1β and tumor necrosis factor-α. Our
results provided significative lead compounds for the further discovery of novel selective and safe FAAH in-
hibitors with potent anti-neuroinflammation activity.
FAAH inhibitor
AEA
Cannabinoid receptors
Endocannabinoid
Anti-inflammation
The endocannabinoid (eCB) system plays an important neuromo-
dulatory role in the periphery and central nervous system, which can
regulate several physiological processes such as appetite, anxiety, mood
and pain. It contains cannabinoid receptors (CBs, CB1 and CB2) and
their main transmit{Keith, 2014 #55}ters, including endocannabinoids
anandamide (AEA), 2-arachidonoylglycerol (2-AG), and the enzymes
responsible for these biosynthesis and degradation (Fig. 1). AEA is
formed “on-demand” during several physiological and pathophysiolo-
gical conditions and also mediates analgesic and anti-inflammatory
effects by activation of the cannabinoid receptors CB1 and CB2, which
is rapidly hydrolyzed into arachidonic acid (AA) and ethanolamine by
fatty acid amide hydrolase (FAAH). FAAH belongs to the serine hy-
drolase family,3 and characterized by the unusual catalytic triad Ser217
- Lys142 - Ser241. In addition to AEA, FAAH also degrades other
ethanolamides, such as palmitoylethanolamide (PEA) and oleoyletha-
nolamide (OEA), which suppress pain and inflammation through the
peroxisome proliferator-activated receptor (PPAR-α).4
regulation of the AEA signaling through the inhibition of FAAH by the
activation of the cannabinoid (CB) G-protein coupled receptors has
been considered as an attractive therapeutic approach for the treatment
of pain, inflammation7 and other central nervous system disorders.8
In the past years, many potent FAAH inhibitors have been devel-
oped (Fig. 2).9,10,11 The most widely investigated FAAH inhibitor is
URB597, which was reported to display anxiolytic and antidepressant-
like activity in various rodent models.12 It’s worth mentioning that,
several FAAH inhibitors (e.g. BIA 10–2474, JNJ-40355003, ASP 8477)
have entered into clinical trials to assess their potential efficacy in pa-
tients suffering from major depressive disorder (MDD), social anxiety or
post-traumatic stress disorder (PTSD).10 In 2018, PF-04457845 was
demonstrated to be effective on patients suffering from cannabis
withdrawal symptoms in a phase II study.12 Furthermore, FAAH in-
hibitors are useful for the treatment of Parkinson's disease via pre-
venting or reducing the inflammatory process associated with Aβde-
position.13–15
Genetic or chemical inhibition of FAAH produces elevated levels of
AEA in the brain and periphery, as well as potentially benefits in animal
models of anxiety and pain5 without undesirable side effects (such as
hypothermia, catalepsy, and hyperphagia) observed with direct can-
nabinoid receptor agonists.1,5,6 Hence, during recent decades,
However, during the phase I trial of BIA 10-2474, one death and
four mild-to-severe neurological symptoms were reported.16 Sub-
sequent studies had confirmed that the severe adverse effects of BIA 10-
2474 did not result from the inhibition of FAAH, but from the produced
substantial alterations in human cortical neurons (such as CES2 and
⁎ Corresponding author.
E-mail addresses: 1694734664@qq.com (Y. Shang), jinxinwang@cpu.edu.cn (J. Wang).
https://doi.org/10.1016/j.bmcl.2020.127118
Received 2 December 2019; Received in revised form 28 February 2020; Accepted 17 March 2020
Availableonline19March2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

Y. Shang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127118
Fig. 1. FAAH inhibitors mediated biological effects.
Fig. 2. Structures of some known FAAH inhibitors.
PNPLA6),17 which resulted in the neurotoxicity due to disruption of
cellular lipid networks. Thus, the development of novel FAAH in-
hibitors with specificity and safety is an urgent issue to combat central
nervous system disorders.
the carbohydrazide core could form a tetrahedral intermediate with the
Ser241, Ile238 and Gly239, which were essential for inhibitory activity.
In addition, the introduction of different heteroaromatic carbohy-
drazide groups instead of the carbonyl imidazole group was supposed to
optimize physicochemical parameters (such as solubility).
FAAH inhibitors can be broken into the three modular units18: part
tail group, an acyl-chain binding part (urea or carbamate group) and
head group which acts as a leaving group upon covalent binding to Ser
241-OH of FAAH.19,20 As shown Fig. 2, most FAAH inhibitors such as PF-
04457845, ASP8477 and JNJ-40355003 are all urea derivatives with a
long hydrophobic chain as tail group, which had been well tolerated in
phase I study. It was reported that off-target effects of BIA-10–2474
were related to the highly reactive carbonyl imidazole structural ele-
ment.2 Meanwhile, a shorter hydrophobic chain was found to occupy
the hydrophobic cavity in membrane access channel.21 Therefore, em-
ploying BIA-10-2474 as a lead structure, we planned to design and
synthesis of a series of novel highly selective FAAH inhibitors (Fig. 3)
with carbohydrazide cores as acyl-chain binding part (scaffold A).
Moreover, imidazol head group was substituted with either heteroaro-
matic such as indoles, pyrazoles, oxazoles (compounds 1–12) as well
as extended aromatic tail group such as phenyl, naphthalene, benzyloxy
(compounds 12–26). Docking studies indicated that the carbonyl of
Our initial converted the pyridazole moiety of lead compound BIA-
10-2474 to various heteroaromatic groups (compounds 1–6). In ad-
dition, different link chains (cyclohexyl, phenyl) were also investigated
(compounds 7–12). The IC50 results were listed in Table 1. Interest-
ingly, the heteroaromatic compounds such as indolyl, phenylisox-
azoleyl and phenyl-1H-pyrazoleyl, benzimidazolyl exhibited moderate
to good FAAH inhibiting activity, instead of the cyclohexyl with ben-
zene as links the activity had not apparently changed (compounds
7–12). Among them, the head group substituted by benzimidazolely
group resulted in the most active compound 9 (IC50 = 12.4 μM), which
was selected as the new lead compound for further optimization.
Our optimization strategy was replacing the tail groups with various
substitutes (F, Cl) to explore more potent compounds and to establish
the structure–activity relationship. We synthesized the meta-substituted
analogue of 14 (compound 13) and para-substituted analogue of 15
(compound 16). These compounds exhibited a similar trend in the
2

Y. Shang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127118
Fig. 3. Design of new FAAH inhibitors.
Table 1
Table 2
Biological activity of compounds 1–12.
Biological activity of compounds 13–26.
Compd
R1
R2
Inhibition of FAAH IC50(μM)
Compd
R1
Inhibition of FAAHIC50(μM)
1
2-indolyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
phenyl
18.6
> 50
13.0
27.1
41.3
38.7
20.6
46.6
12.4
25.0
38.6
46.1
4.1
2
3-indole ethyl
2-benzimidazolyl
2-benzooxazolyl
5-phenyl-3-pyrazolyl
5-phenyl-3-isoxazolyl
2-indolyl
3
cyclohexyl
13.0
12.4
27.2
17.9
20.1
7.3
3
9
phenyl
4
13
3-flurophenyl
4-fluorophenyl
3-chiorophenyl
4-chlorophenyl
4-methoxyphenyl
4-cyanophenyl
4-hydrophenyl
4-morpholinphenyl
4-biphenyl
5
14
6
15
7
16
8
3-indole ethyl
2-benzimidazolyl
2-benzooxazolyl
5-phenyl-3-pyrazolyl
5-phenyl-3-isoxazolyl
/
phenyl
17
11.5
21.1
24.3
29.8
10.3
7.2
9
phenyl
18
10
phenyl
19
11
phenyl
20
12
phenyl
21
BIA-10–2474
/
22
4-phenoxyphenyl
4-benzyloxyphenyl
2-naphthyl
23
4.2
24
5.7
25
5-indolyl
4.0
inhibitory activities against FAAH, since meta-substitution (compound
13 and 15) decreased the inhibitory activity while para-substitution
(compound 14 and 16) improved it.
26
4-(3-fluorobenzyloxy)phenyl
/
2.8
BIA-10–2474
4.1
As shown in Table 2, compounds synthesized by replacing the tail
groups with a variety of hydrophilic groups (OH, CN, morpholinyl)
were evaluated the FAAH inhibition (e.g 18–20). Substituents in the 4-
position with hydrophilic groups (such as 18, 19, 20) are dis-
advantageous to the activity, while 4-substitution with lipophilic
groups (such 21, 22, 23) tended to improve the potency. After changing
different hydrophobic groups as tail groups, such as naphthalene, in-
dole, benzyloxy and diphenyl, the compound 26 was found to be the
most active one (IC50 = 2.8 μM) among these molecules, which is ap-
proximately 3-fold stronger than compound 9. Those results indicated
that the hydrophobic tail groups (benzyloxy) with appropriate length
and rigidity was quite favorable for the inhibitory activity.
The synthetic routes of the key intermediates 1d/2e/3c were illu-
strated in Scheme 1. Acetophenone 1a was used as the starting mate-
rial. After being treated with diethyl oxalate in the presence of NaOEt,
3

Y. Shang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127118
Scheme 1. Synthesis of intermediates 1d/1e/2e/3c. Reagents and conditions: (a) Diethyl oxalate, NaOEt, 8 h; (b) Hydroxylamine hydrochloride/ hydrazine hydrate,
EtOH, reflux, 2 h; (c) Hydrazine hydrate, EtOH, reflux, 3 h; (d) Glycolic acid, EtOH, reflux; (e) Potassium permanganate, NaOH/H2O, reflux, 5 h; (f) Ethanol, HATU,
THF, rt; (g) Ethyl bromoacetate, pyridine, reflux.
which was claisen condensation, the intermediate 1b was obtained.
Then, 1b was allowed in a ring-closure reaction with hydroxylamine
hydrochloride or hydrazine hydrate to form 1c. Compound 1c was
further treatment with hydrazine hydrate to afford 1d. The 1H-benzi-
midazole-2-carboxylic acid (2c) was synthesized through oxidizing 2-
(hydroxymethyl) - benzimidazole in alkaline conditions with o-pheny-
lenediamine. Synthesize 2e was prepared according to the similar
procedure described above.
inhibitor compound 26, the lipophilic meta-fluorobenzyloxy formed
hydrophobic interactions with the surrounding amino acid residues
Phe381 and Phe432. Whereas compound 9 with a short hydrophobic
chain, which poorly occupied the hydrophobic cavities (Fig. S1).
Moreover, the poor activity of compounds 20 may be attributed to their
increased lipophilicity (the end of the inhibitor is morpholinyl), which
prevented them from interacting with residues Phe381 and Phe432.
These docking results were in accord with their potencies, so this
binding model could explain the SAR observed above.
The target compounds 1–26 were formed according to the reaction
pathways illustrated in Scheme 2. The intermediates hydrazides (1d/
2e/3c) were further refluxed with suitably substituted benzaldehydes
Previous reports have confirmed that many of the off-targets of BIA
10–2474 occurred due to lipolytic enzymes including human carbox-
ylesterase (CES2), which may have a contribution to the neurotoxicity
of BIA 10-2474.18 Therefore, compounds 23, 25 and 26 were tested
against CES2. Meanwhile, the selectivity of these compounds to
monoacylglycerol lipase, which belonged to another major en-
docannabinoid degrading enzyme, was investigated. As shown in
Table 3, these compounds exhibited strong inhibition of FAAH without
inhibition of other serine hydrolase MAGL. Furthermore, compounds
23, 25 and 26 were found low inhibition of CES2 (IC50 > 100 μM). It
was suggested that these novel FAAH inhibitors posed a preferable se-
lectivity, which less likely cause off-targets in the body.
in ethanol for 3 h, gave the target compounds 1–2627
.
For further understanding of the interaction between these in-
hibitors and protein (FAAH) for guiding the structure–activity re-
lationship (SAR), we carried out a docking study of compound 9, 20
and 26 with human FAAH (PDB: 2WJ2) via the program covalent
docking of the Schrödinger suite. The proposed binding mode of com-
pound 26 was illustrated in Fig. 4. It was observed that the central
acylhydrazine module of compound 26 fitted into the oxyanion hole,
which was composed with Ser241, Ile238, Gly239 and Gly240. More-
over, the acylhydrazine groups formed hydrogen bonds with residues
Ser217, Ser193 and Met191, respectively (Fig. 4). At the tail of the
Inflammation was the main causes of neurodegeneration in the
Scheme 2. General synthesis of the target compounds. Reagents and conditions: (h) ethanol, reflux, 1–3 h.
4

Y. Shang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127118
Fig. 4. Docking compounds 26 into the FAAH (PDB: 2WJ2). The protein was shown as ribbons. The key amino acids forming the pocket were represented by stick
with carbon atoms colored in brown. Compound 26 was shown in stick with carbon atoms colored in yellow, oxygen atoms in red and nitrogen atoms in blue,
hydrogen bond was denoted by green dash lines respectively.
promising lead compound for further discovery of new safe and effec-
tive FAAH inhibitors for treatment of inflammation involved the central
nervous system (CNS) disease.
Table 3
Biological activities (IC50 values for FAAH, MAGL and CES2) of compounds 23,
25 and 26.
The toxicity risks (mutagenicity, tumorigenicity, irritation, and re-
production) of partly inhibitors, as well as ADMET prediction experi-
ment, were predicted using Osiris Property explorer and DS4.0.25 All
active compounds reported herein were predicted to be safe and had
good pharmacokinetics and pharmacodynamics properties (Fig. s4). The
additional maximum tolerated dose (MTD) determination showed that
compound 26 had obviously low toxicity with MTD values over 2 g/kg.
In summary, a series of novel heterocyclic carbohydrazide FAAH
inhibitors were firstly reported in this paper. Amongst all the tested
compounds, compound 26 (IC50 = 2.8 μM) was found to be the most
active compound. Docking studies indicated the oxyanion hole channel
was the preferential compound 26 binding sites and the newly devel-
oped compounds possed a long aryl chain in the acylhydrazide group,
which had high inhibition potency on FAAH and strongly reduced their
affinity to MAGL and CES2 respectively. In addition, compound 26
Compd
FAAH IC50 (μM)
MAGL IC50 (μM)
CES2 IC50 (μM)
23
4.2
4.0
2.8
4.1
No active
No active
No active
No active
> 100
> 100
> 100
0.26 μM
25
26
BIA-10-2474
central nervous system (CNS).22,23 The anti-inflammatory activity of
compound 26 was evaluated on LPS-activated microglia inflammatory
cell model.27 As shown in Fig. 5, LPS induced a robust increase in
transcription of IL-1β and TNF-α (p
< 0.0001). Treatment with
compound 26 with 40 μM significantly reduced the unregulated cyto-
kine IL-1β (p < 0.05) and TNF-α (p < 0.001) in a dose-dependent
manner, suggesting that compound 26 could obviously reduce the pa-
thological inflammatory response. Therefore, this compound could be a
5

Y. Shang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127118
Fig. 5. FAAH inhibitor 26 reduced inflammation in cells. Normal BV2 cells were induced by LPS and then treated with different concentrations of 26. Cytokines were
evaluated by ELISA. **p < 0.0001 compared to control group, # #p < 0.05 compared to LPS group, # # #p < 0.001 compared to LPS group.
effectively suppressed the LPS-induced neuroinflammation of mouse-
derived microglial cells (BV2) via the reduction of IL-1β and TNF-α.
These research results displayed novel heterocyclic carbohydrazide 26
is a helpful FAAH inhibitor for the potential treatment of central ner-
vous system disorder. And further structural optimization and biolo-
gical assays are in progress.
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The authors declare that they have no known competing financial
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Acknowledgements
This work was supported by the Double First-Class initiative new
drug development and construction project of China Pharmaceutical
University (CPU2018PZQ07), the Natural Science Foundation of
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