Fatty Acid Amide Hydrolase (FAAH), Acetylcholinesterase (AChE), and Butyrylcholinesterase (BuChE): Networked Targets for the Development of Carbamates as Potential Anti-Alzheimer's Disease Agents

Article abstract of DOI:10.1021/acs.jmedchem.6b00609

The modulation of the endocannabinoid system is emerging as a viable avenue for the treatment of neurodegeneration, being involved in neuroprotective and anti-inflammatory processes. In particular, indirectly enhancing endocannabinoid signaling to therapeutic levels through FAAH inhibition might be beneficial for neurodegenerative disorders such as Alzheimer's disease, effectively preventing or slowing the progression of the disease. Hence, in the search for a more effective treatment for Alzheimer's disease, in this paper, the multitarget-directed ligand paradigm was applied to the design of carbamates able to simultaneously target the recently proposed endocannabinoid system and the classic cholinesterase system, and achieve effective dual FAAH/cholinesterase inhibitors. Among the two series of synthesized compounds, while some derivatives proved to be extremely potent on a single target, compounds 9 and 19 were identified as effective dual FAAH/ChE inhibitors, with well-balanced nanomolar activities. Thus, 9 and 19 might be considered as new promising candidates for Alzheimer's disease treatment.

Full text of DOI:10.1021/acs.jmedchem.6b00609

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Article
Fatty acid amide hydrolase (FAAH), acethylcholinesterase (AChE)
and butyrylcholinesterase (BuChE): networked targets for the
development of carbamates as potential anti Alzheimer’s Disease agents
Serena Montanari, Laura Scalvini, Manuela Bartolini, Federica Belluti, Silvia Gobbi, Vincenza Andrisano,
Alessia Ligresti, Vincenzo Di Marzo, Silvia Rivara, Marco Mor, Alessandra Bisi, and Angela Rampa
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00609 • Publication Date (Web): 16 Jun 2016
Downloaded from http://pubs.acs.org on June 16, 2016
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Fatty acid amide hydrolase (FAAH), acethylcholinesterase (AChE) and
butyrylcholinesterase (BuChE): networked targets for the development of
carbamates as potential anti Alzheimer’s Disease agents
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Serena Montanari, Laura Scalvini, Manuela Bartolini, Federica Belluti, Silvia Gobbi, Vincenza
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Andrisano, Alessia Ligresti, Vincenzo Di Marzo, Silvia Rivara, Marco Mor, Alessandra Bisi,
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Angela Rampa *
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Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, Via
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Belmeloro 6, 40126 Bologna, Italy; Department of Pharmacy, University of Parma, Parco Area
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delle Scienze 27/A, 43124 Parma, Italy; Department for Life Quality Studies, Alma Mater
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Studiorum University of Bologna, Corso d’Augusto 237, 47921 Rimini, Italy; Endocannabinoid
Research Group, Institute of Biomolecular Chemistry, National Research Council, Via Campi
Flegrei 34, 80078 Pozzuoli, NA, Italy.
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Abstract. The modulation of the endocannabinoid system is emerging as a viable avenue for the
treatment of neurodegeneration, being involved in neuroprotective and antiꢀinflammatory processes.
In particular, indirectly enhancing endocannabinoid signaling to therapeutic levels through FAAH
inhibition might be beneficial for neurodegenerative disorders such as Alzheimer’s disease,
effectively preventing or slowing the progression of the disease. Hence, in the search for a more
effective treatment for Alzheimer’s disease, in this paper, the multiꢀtargetꢀdirected ligand paradigm
was applied to the design of carbamates able to simultaneously target the recently proposed
endocannabinoid system and the classic cholinesterase system, and achieve effective dual
FAAH/cholinesterase inhibitors. Among the two series of synthesized compounds, while some
derivatives proved to be extremely potent on a single target, compounds 9 and 19 were identified as
effective dual FAAH/ChE inhibitors, with wellꢀbalanced nanomolar activities. Thus, 9 and 19 might
be considered as new promising candidates for Alzheimer’s disease treatment.
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Introduction
The progressive aging of the population in western countries undoubtedly reflects the continuous
improvements in living conditions. Consequently, an increase in ageꢀrelated diseases, among which
Alzheimer’s Disease (AD) plays a primary role, is being observed, and a growing trend can be
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anticipated for the coming years.
AD is characterized by progressive mental deterioration, associated with degeneration of brain
regions involved in superior cognitive functions. The neuropathological hallmarks include
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extracellular deposition of amyloidꢀβ peptides to form Aβ plaques, intraneuronal accumulation of
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aberrant forms of hyperphosphorylated tau protein (neurofibrillary tangles), as well as synapse
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dysfunction and neuronal loss. In this scenario, information transfer at synapses begins to fail, the
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number of synapses declines, and neurons eventually die.
The major pathological changes in AD involve cholinergic nerve pathways, from the frontal base to
the cerebral cortex and hippocampus, associated with attention, learning ability, task and memory
related activities, and other cognitive processes. The loss of basal forebrain cholinergic neurons
leads to reduction of the cholinergic tone, and memory and cognitive deficits which are
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characteristic of this disease. Acetylcholinesterase inhibitors (AChEIs) boost cholinergic
neurotransmission in forebrain regions by preventing the breakdown of acetylcholine (ACh), and
temporary ameliorate the clinical condition. Mammal brain contains two major forms of
cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). In healthy
brain, AChE is the major actor in ACh breakdown. Conversely, in AD affected brain, BuChE levels
and activity progressively rise, while AChE activity declines. Therefore, both enzymes are likely to
be involved in the regulation of ACh central levels in AD patients and represent validated
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therapeutic targets to counteract the cholinergic deficit.
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Currently approved AD treatments only provide temporary and modest improvement in cognitive
functions, through cholinergic and antiglutamatergic mechanisms, but they cannot significantly
modify the course and the ultimate outcome of the disease. Thus, there is an urgent need for
diseaseꢀmodifying therapies to fight the upcoming Alzheimer's disease epidemic.
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Over the past two decades, neuroinflammation has emerged as an integral process in the
pathogenesis of AD. Postꢀmortem analysis of the brains of AD patients has revealed an increased
amount of activated microglia and astrocytes as well as significantly higher levels of
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proinflammatory cytokines, namely ILꢀ1, ILꢀ6 and TNFꢀα, and reactive oxygen species (ROS).
Microglia represent brain’s innate immune system, hence the first line of defense against invading
pathogens. However, microglial activation may also be evoked by endogenous proteins and can
significantly contribute to neuronal damage. Indeed, inflammatory changes are observed at the
amyloid deposits, which are rich in activated microglia. Once stimulated, the microglia release a
wide variety of proꢀinflammatory mediators that contribute to neuronal dysfunction and eventually
result in cell death. These mediators create and feed a vicious cycle that may play an important role
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in the pathological progression of AD. When activated, microglia cells may also recruit astrocytes
that actively enhance the inflammatory response to extracellular Aβ deposits. Moreover, reactive
microglia play an important role in governing the expression of Aβ and tau, with a positive
feedback mechanism that drives disease progression.
The modulation of the endogenous cannabinoid system (ECS) is now emerging as a potentially
viable option for the treatment of neurodegeneration. ECS is composed of the endocannabinoid
signaling molecules, including anandamide (Nꢀarachidonoylethanolamine, AEA) and 2ꢀ
arachidonoyl glycerol (2ꢀAG), their G proteinꢀcoupled cannabinoid receptors CB
1 2
and CB , and
enzymes for endocannabinoid biosynthesis and inactivation. CB receptors are highly expressed at
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the terminals of central and peripheral neurons, where they regulate neurotransmitter release. CB
receptor expression is associated with the peripheral immune system and microglia during
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neuroinflammation. Endocannabinoids are lipid compounds of the eicosanoid family, derived
from the processing of membrane phospholipids, and are synthesized onꢀdemand in response to
(
patho)physiological stimuli. Two specific enzymes are mostly responsible for their degradation:
fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), which metabolize AEA
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and 2ꢀAG, respectively. Endocannabinoid signaling has been found to be altered in many
neurodegenerative disorders including AD, in which AEA levels are decreased and are inversely
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correlated with those of Aβ. In AD brain, the expressions of CB receptors in microglia and of
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FAAH in astrocytes are increased, as a response to the inflammatory processes typical of the
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disease. Since AEA is a substrate for FAAH, the higher levels of this enzyme in astrocytes
surrounding neuritic plaques suggest that these cells, via FAAH, might be responsible for the lower
levels of AEA and the decreased neuroprotection promoted by AEA activation of CB and CB
1
2
receptors. FAAH inhibition might therefore be beneficial in preventing or reducing the
inflammatory process associated with Aβ deposition, by indirectly enhancing endocannabinoid
signaling to therapeutic levels, and might be considered an attractive therapeutic avenue, adding a
new piece to the Alzheimer's puzzle.
For the treatment of disorders involving multiple and complex pathways such as AD, a drug
endowed with a singleꢀtarget mechanism of action may not be the most effective agent. In this case,
polypharmacy, that is the synergistic delivery of a “cocktail” of two or more drugs, may give
enhanced beneficial effects and potentially lead to disease modification. However, poor patient
compliance and pharmacokinetic interferences are negative aspects potentially associated with such
an approach. Alternatively, a single therapeutic agent could be purposely designed to interact with
different targets and/or systems involved in the onset or progression of the disease. This multiꢀtarget
approach would allow attacking the disease from many sides, thus greatly enhancing the probability
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of disease modification.
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Taking into account these elements, we tried to tackle AD’s multifaceted nature using the multiꢀ
targetꢀdirected ligand (MTDL) paradigm, thus combining our long lasting experience in the design
of inhibitors of classic AD targets with the attempt to hit the ECS. In this respect, we recently
reported compound 1 (Figure 1) as first dual ChE/FAAH inhibitor, with activity in the nanomolar
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range. In this paper, appropriately designed modifications were performed on lead molecule 1 in
order to increase potency and obtain wellꢀbalanced activities on the selected targets, namely FAAH,
AChE and BuChE. In particular, to explore the chemical space of these enzymes and to determine
the structural features required for high potencies, various carbamic functions were inserted in
selected oxygenated heterocycles, related to natural products and considered as privileged
structures, with spacer chains of different length (Figure 1). As a result, a first small library of 16
compounds (2ꢀ17) was synthesized and tested for activity against AChE, BuChE, and FAAH.
Furthermore, a new series of eight compounds (18ꢀ25, Figure 1) was designed and synthesized by
inserting a triazole ring in the spacer chain, to evaluate the impact of a lower flexibility and the
importance of the basic center for biological activities.
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Figure 1. Structure of lead compound 1 and design strategies for the studied compounds, involving
the introduction of flexible and rigid spacers. Ar represents a variety of substituted heterocycles and
R represents alkyl and phenyl alkyl substituted groups.
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Chemistry
The synthetic procedures to obtain non commercially available hydroxycoumarins (ArꢀOH) are
reported in the supporting information (SI, scheme 1S). The synthesis of compounds 2ꢀ17 was
accomplished as shown in Scheme 1. The hydroxy group of the naturally inspired heterocycles was
2 3
alkylated with 1ꢀbromoꢀ3ꢀchloropropane or 1ꢀbromoꢀ5ꢀchloropentane, in the presence of K CO , to
afford the chloroalkoxy derivatives 26ꢀ37. These intermediates were then reacted with NaI to obtain
the iodinated analogues (38ꢀ49), more reactive species for the following nucleophilic substitution,
18
that was accomplished by refluxing N,Nꢀ(3ꢀhydroxybenzyl)ꢀmethylamine, compounds 38ꢀ49 and
TEA in refluxing toluene to obtain compounds 50ꢀ61. Finally, compounds 50ꢀ61 were treated with
the selected isocyanate, in the presence of NaH, to give the desired compounds 2ꢀ17. The synthetic
procedures to obtain the isocyanate not yet described in literature were reported in the SI (schemes
2
S and 3S).
a
Scheme 1. Synthesis of compounds 2ꢀ17.
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Reagents and conditions: i) K CO , acetone, reflux, 10h; ii) NaI, MeCOEt, reflux; 3h iii) Nꢀ(3ꢀ
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hydroxybenzyl)methylamine, TEA, toluene, reflux, 20h; iv) RNCO, NaH, toluene, rt, 24h.
According to Scheme 2, compounds 18ꢀ25 were synthesized starting from 7ꢀhydroxycoumarin,
which was reacted with propargyl bromide obtaining 62. Then, the 1,2,3ꢀtriazole framework was
introduced via a click chemistry reaction (Huisgen 1,3ꢀdipolar cycloaddition), in which the alkyne
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was reacted with the selected azides, in the presence of CuSO and sodium ascorbate to obtain
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3a,b. The synthetic procedures for achieving the azides are reported in the SI (scheme 4S).
Subsequently, the methoxy group of 63a,b was removed by treatment with BBr (64a,b). When tertꢀ
3
butyl dimethylsilane was employed as protecting group, the free phenolic oxygen (65a,b) was
obtained during purification via flash chromatography. Finally, compounds 64a,b and 65a,b were
combined with the appropriate isocyanate to afford the final carbamates 18ꢀ25.
a
Scheme 2. Synthesis of compounds 18ꢀ25.
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Reagents and conditions: i) K CO , acetone, reflux, 8h; ii) TEA, selected azide, CuSO , sodium
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ascorbate, DMSO, rt, 24h; iii) BBr
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chromatography); v) RNCO, NaH, toluene, rt, 24h.
Results and discussion
Cholinesterase inhibition by compounds 2ꢀ17.
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The activities of the newly synthesized compounds 2ꢀ17 were evaluated against human recombinant
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AChE (hAChE) and BuChE from human serum (hBuChE), using the method of Ellman and the
inhibitory potencies, expressed as IC50 values, are reported in Table 1.
Inhibition values for compound 1 and rivastigmine, the only marketed carbamate drug approved for
AD treatment, are also listed in Table 1 for comparison. Due to the presence of a carbamic
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residue,
all compounds showed a timeꢀdependent ChEs inhibition.
Regarding AChE inhibitory activity, different results were observed maintaining the same carbamic
substituent of 1 and varying the substitution pattern on the coumarin scaffold (2ꢀ8). The
introduction of a methyl group on the 4ꢀposition of the coumarin scaffold (compound 2), and its
extension to a cyclohexane ring (compound 3), did not significantly alter the inhibitory activity with
respect to the lead 1. A lower potency was observed for the corresponding derivative 4, in which an
aromatic ring was introduced on the coumarin lactone ring. The introduction of an aryl moiety in
position 3 of the coumarin was also detrimental (5ꢀ8), especially when a chlorine atom was
introduced in the para position (compound 8). Therefore, the simple coumarin core of the lead 1
was maintained and the phenylpenthyl carbamic function was varied, introducing heptyl (9),
hexylphenyl (12) and phenylhexyl (13) groups. A slight decrease in potency was observed for
compound 13, bearing an additional methylene unit with respect to 1. Compound 9, with a linear
heptyl chain, proved to be the most potent of the series (IC50 = 37.4 nM), being twice as potent as 1.
The presence of a phenyl ring directly bound to the carbamate nitrogen, as in compound 12, led to a
remarkable reduction in potency (two orders of magnitude). Afterwards, in an attempt to improve
potency, the heptyl carbamic function was introduced on the heterocycles that showed a comparable
activity, namely 7,8,9,10ꢀtetrahydroꢀ6Hꢀbenzo [c] chromenꢀ6ꢀone in this series and azaxanthone in
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a previously reported paper. This modification led to an increased inhibitory activity (10 vs 3, IC50
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= 21.6 vs 89.9 nM, and 11 vs the previously reported azaxanthone IC50 = 13.0 vs 89.5 nM).
Finally, for 9ꢀ11, the elongation of the alkoxy chain from three to five methylene units provided a
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remarkable increase in potency, being compounds 15ꢀ17 the most potent of the series, while for
compound 1 chain elongation (14) essentially allowed to maintain the original potency.
All the tested compounds 2ꢀ17 turned out to inhibit hBuChE more effectively than hAChE, with
IC50 values within a close inhibition range (from 0.27 to 3.51 nM), except for derivative 12 (IC50
=
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2.1 nM). In particular, compounds 10ꢀ11 and 15ꢀ17 proved to be the most potent BuChE inhibitors
in the series with inhibitory activities in the subnanomolar range.
Table 1. Inhibitory activity against hAChE, hBuChE and rFAAH, IC50 values of the studied
compounds and of the commercial antiꢀAD drug Rivastigmine (Riva).
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
b
a
IC50 rFAAH (nM) ± SD
Ar
n
R
IC50 (nM) ± SEM
c
d
std protocol
hAChE
hBuChE
preꢀincub prot.
(n≥2)
n≥2)
e
e
7
4.9 ± 2.0
1.57± 0.18
280
50
1
2
1
1
80.5 ± 4.7
89.9 ± 1.8
2.75 ± 0.13
1.44 ± 0.06
459.1 ± 74.2
134.9 ± 5.49
3
1
3484.6 ± 190.5
1385.4 ± 575.6
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5
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7
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9
4
1
390 ± 24
1.19 ± 0.03
1829.9 ± 185.95
312.2 ± 60.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
5
6
1
1
125 ± 15
312 ± 23
2.06 ± 0.08
1.30 ± 0.07
1847.9 ± 340.6
249.5 ± 91.5
322.1 ± 36.2
2333.3 ± 285.9
7
8
9
1
1
1
179 ± 12
900 ± 49
37.4 ± 1.9
1.15 ± 0.06
3.51 ± 0.16
1.36 ± 0.07
2829.0 ± 1675.0
1465.2 ± 101.0
161.4 ± 26.8
210.7 ± 21.6
265.5 ± 112.5
28.5 ± 9.7
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
306.4 ± 66.0
202.1 ± 1.1
1
0
1
1
21.6 ± 0.7
0.27 ± 0.01
0.69 ± 0.02
2487.4 ± 886.5
3134.0 ± 980.9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
O
O
11
13.0 ± 0.6
N
12
13
1
1
3800 ± 540
109 ± 5
32.1 ± 2.9
>50000
>50000
2.85 ± 0.14
1792.2 ± 283.9
529.7 ± 166.5
14
3
3
88.1 ± 4.0
11.3 ± 4.9
1.23 ± 0.10
0.79 ± 0.04
798.1 ± 222.4
1288.7 ± 352.3
120.2 ± 38.5
241.9 ± 38.0
1
5
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9
1
1
6
7
3
3
18.3 ± 0.8
0.28 ± 0.01
0.53 ± 0.03
767.4 ± 110.8
2177.1 ± 419.0
109.1 ± 6.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
6.92 ± 0.58
178.3 ± 15.1
f
f
1535 ± 64
301 ± 14
Riva
a
b
Human recombinant AChE and BuChE from human serum were used. FAAH from rat brain membranes was used. IC50 values represent the
concentration of inhibitor required to decrease enzyme activity by 50% and are the mean of two/three independent measurements, each performed in
c
duplicate. SEM = standard error of the mean. IC50 values of all derivatives but 12 were determined using a 120 minꢀincubation period; for 12 a 60
d
minꢀincubation period was used, according to the inhibition kinetics. IC50 values of all derivatives were determined using 15 or 20 min incubation,
e
17 f
22
according to the inhibition kinetics. Data from reference. Data from reference.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
It is well known that the kinetics of ChE inhibition by carbamate derivatives is strongly influenced
by the substituent at the Nꢀcarbamoyl group. Indeed, while shortꢀchain alkyl carbamates, such as
those bearing a H or a methyl residue, give relatively rapid carbamoylation and decarbamoylation
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(e.g. physostigmine), longꢀchain alkyl carbamates generally provide a more stable carbamoylated
enzyme which is formed slower but also undergoes a slower hydrolysis towards a reactivated
23,24
enzyme.
The kinetics of enzyme inhibition and reactivation influences the duration of action of
a carbamate drug. Indeed, the human pharmacodynamic halfꢀlife (T1/2) of (−)ꢀeptylstigmine, bearing
2
5
3
a hepyl chain at the carbamic nitrogen, is about 10 h, while that of physostigmine (R = CH ) is
26
about 30 min.
Thus, to assess the influence of the different R groups on the kinetics of ChEs carbamoylation,
studies were carried out on the representative compounds 9, bearing a heptyl residue, 12, with a pꢀ
hexylphenyl residue and 13, bearing a phenylhexyl moiety, and data were compared to those
previously obtained for 1, bearing a phenylpentyl group. The four selected derivatives were
characterized by an identical aromatic group (2Hꢀchromenꢀ2ꢀone), an identical length of the spacer
chain (n = 1) and only differed by the R residue. To exclude an influence of the spacer chain length
(n), the inhibition profile of compound 15 (n = 3) was also evaluated. The timeꢀdependent inhibition
of hAChE by an inhibitor concentration close to its IC50 value was monitored. The obtained profiles
are overlaid in Figure 2a. As highlighted in previous studies on 1, the presence of a phenylpentyl
moiety determined a rather slow inactivation of hAChE, and a 120 minꢀincubation period was
17
required to reach the inhibition plateau. To mention, since R is the major determinant on the
carbamoylation kinetics, similar profiles can be assumed for all derivatives, within this work,
bearing a phenylpentyl residue. The introduction of a heptyl chain, as in 9, led to a slightly faster
AChE inactivation (Figure 2a). Neither the substitution of the phenylpentyl residue with a phenylꢀ
hexyl one (as in 13) nor the length of the spacer chain (9, n=1 vs 15, n=3) significantly modified the
kinetics of AChE inactivation (SI, Figure 1S and 2Sa, respectively). Conversely, and quite
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8
9
interestingly, the introduction of a pꢀhexylphenyl residue on the Nꢀcarbamoyl group (compound 12)
determined a much faster AChE inactivation and less than 60 min were required to reach the
maximum inhibition (Figure 2a).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a)
b)
Figure 2. Overlaid carbamoylation profiles of compound 9, 12 and reference compound 1 on a)
hAChE and b) hBuChE.
A similar study was carried out to investigate the kinetics of hBuChE inhibition. On the basis of the
1
7
results previously obtained for 1, which was able to inactivate hBuChE in less than 5 min, the
experiments were performed using incubation times ranging from 2 to 20 min. The inhibition
profiles obtained for derivatives 9 (R =heptyl), 12 (R = pꢀhexylphenyl) and 1, as reference, are
overlaid in Figure 2b.
In opposition to what was observed for hAChE inactivation, hBuChE inactivation by 9 was slightly
slower, although very rapid (maximum inhibition in 5 min), than that observed with 1, while
inactivation by 12 was significantly slower and the maximum inhibition was reached in 20 min.
Finally, similarly to what was observed for AChE, the spacerꢀchain length did not influence the
kinetics of hBuChE carbamoylation (9, n=1 vs 15, n=3, overlaid profiles in SI, Figure 2Sb) and the
hBuChE inhibition by 13, bearing a pꢀphenylhexyl residue, closely matched that of 1, bearing a
phenylpentyl residue.
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Thus, the trend observed for AChE inactivation was pꢀhexylphenyl > heptyl > phenylpentyl
≅
ꢀ
phenylhexyl (from faster to slower), while it was phenylpentyl ≅ꢀphenylhexyl > heptyl > pꢀ
hexylphenyl for BuChE. Consequently, because of the opposite trends, the pꢀhexylphenyl derivative
2 inactivated the two ChEs within much closer timeꢀframes (SI, Figure 3S). kobs (observed pseudoꢀ
firstꢀorder inhibition rate constant) values, calculated at concentrations of 12 close to the IC50
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
ꢀ1
values, were 0.203 min and 0.461 min for AChE and BuChE inhibition, respectively.
The carbamoylation rate constants (k ) for hAChE and hBuChE inhibition by 9, as representative of
derivatives bearing an heptyl substituent, were determined. To this aim, different concentrations of
were added to the enzyme solution and incubated at 37 °C for 15ꢀ120 min. Data were treated
3
9
2
7
according to Feaster and Quinn method. Timeꢀ and concentrationꢀdependent carbamoylation
profiles are shown in Figure 3. The calculated carbamoylation rate constant for AChE inactivation
ꢀ1
ꢀ1
by 9 was 0.238±0.019 min while it was 11.1±4.5 min for BuChE inhibition. A comparison with
the carbamoylation rate constant obtained for 1 confirmed that the presence of the heptyl chain
ꢀ1
ꢀ1
provides a faster inactivation of hAChE (0.238 min vs 0.109 min for 9 and 1, respectively) and a
ꢀ1
ꢀ1
slower inactivation of hBuChE (11.1 min vs 21.4 min for 9 and 1, respectively) with respect to
the phenylpentyl residue (as in 1). However, kinetics for the inactivation of the two cholinesterases
by 9 still differed by an order of magnitude, i.e. 9 carbamoylated hBChE 47 times faster than
hAChE. Thus, on the basis of both inhibitory activity and kinetics, 9 will mainly act as a hBChE
inhibitor. This selectivity profile makes this compound potentially more beneficial in moderate
forms of AD, in which, because of the progressive reduction of AChE levels in the cholinergic
7,8
synapses, BChE becomes the major actor in the regulation of the central cholinergic tone.
a)
b)
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Journal of Medicinal Chemistry
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7
8
9
1
.0
1.0
0.8
0.8
0
.46 nM
1
6.7 nM
0.6
0.4
0.2
0.0
0.6
0.4
0.2
0.0
1
.14 nM
.29 nM
4.58 nM
33.5 nM
7.0 nM
67.5 nM
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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32
33
34
35
36
37
38
39
40
41
42
43
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
1
0
20
40
60
80
100 120
0
5
10
15
20
time (min)
time (min)
Figure 3. Timeꢀ and concentrationꢀdependent carbamoylation of a) hAChE and b) hBuChE by
compound 9. v and v indicate the enzyme activity in the presence and in the absence of inhibitor,
i
0
respectively.
FAAH inhibition
The inhibitory potencies of compounds 2ꢀ17, expressed as IC50 values, against AEA hydrolysis by
FAAH in rat brain membranes are reported in Table 1. All compounds, except for 12, were able to
decrease AEA hydrolysis and, in contrast to what was observed for inhibition of AChE, FAAH
seemed to be more susceptible to modifications of the coumarin core. In particular, the introduction
of a methyl group in position 4 (compound 2) led to a 3ꢀfold decrease in potency with respect to the
lead 1, and a drastic drop in potency emerged when a cyclohexane ring was incorporated in the
coumarin scaffold (compound 3). When the cyclohexane was replaced by a phenyl ring, activity
was partially restored (compound 4), and it was maintained also with the phenylcoumarin core,
independently from the presence of a methoxy or chloro pꢀsubstituent on the phenyl ring
(
compounds 5ꢀ8).
A different behavior was highlighted when the carbamic function was modified: going from the
phenylpentyl carbamic function of compound 1 to the Nꢀheptyl carbamate, an increase in potency
was observed. Remarkably, compound 9, with the coumarin core, showed, after preincubation,
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
activity in the nanomolar range (IC = 28.55 nM) as expected from pseudoirreversible inhibitors,
50
being about twice as potent as the lead compound 1. The same trend of potency was noted for
compound 10 when compared to 3 (IC50 = 306.4 nM vs 1385.4 nM). When the phenyl ring was
directly bound to the carbamic nitrogen, the inhibitory activity was completely lost (compound 12,
with a hexylphenyl carbamate). This detrimental effect had been previously observed for a series of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
28
Oꢀaryl carbamates, and was attributed to the limited hydrolytic stability of O,Nꢀ
2
9
diarylcarbamates. A significant decrease in activity was observed for 13, bearing only an
additional methylene unit, with respect to 1.
Regarding the spacer, the elongation of the alkoxy chain from 3ꢀ to 5ꢀmethylene units allowed to
maintain the inhibitory potency in the nanomolar range, still being 14 twofold less potent than the
corresponding lead 1.
Finally, the modifications performed on the azaxanthone derivatives (compounds 11 and 17) did not
1
7
seem to appreciably affect the activity, unlike what emerged in our previous paper.
FAAH Docking Studies
Docking studies were performed for the lead compound 1 and for other derivatives to explore their
possible binding modes to rat FAAH.
The binding site of FAAH (Figure 4) extends from the Acyl Chain Binding (ACB) channel, which
receives the substrate from the membrane, to the dimerization interface, which includes the
30
hydrophilic region from which ethanolamine is released after substrate cleavage (cytosolic port).
Just beyond the active site, the cytosolic port divides into a minor branch, which is the putative
31
accommodation pocket of the biphenyl group of the reference inhibitor 66 (URB597), and a larger
cavity, lined by the alcoholic side chain of T236. Compound 1, a tertiary amine, could be docked
into FAAH binding site either in its protonatedꢀcationic form or in the neutral one. Additional
biological assays revealed that IC values are affected by pH conditions (Table 2). Specifically, 66
5
0
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9
showed a 6ꢀfold decrease in potency (increase in IC value) when the pH was lowered from 9 to
50
3
2
7
.4. This is consistent with the lower catalytic efficiency of FAAH at lower pH for both amides
33
and esters, which likely reflects a lower carbamoylation rate by the inhibitor. Similarly,
concerning the pHꢀdependence of anandamide hydrolysis by rat brain homogenates, the IC50 values
of oleoyltrifluoromethylketone and arachidonoyl serotonin were increased by 7ꢀ and 3 folds,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
4
respectively. Notably, compound 1, which is characterized by the same Oꢀphenylcarbamic
function as 66, showed a significantly higher (22ꢀfold) loss of potency at pH 7.4. Assuming that the
effect of reduced enzyme activity is the same for both compounds, this difference could be
attributed to different degrees of protonation of the basic chain of 1. This hypothesis, which implies
that the tertiary amine of 1 binds FAAH in its neutral state, was thus followed throughout this work.
Table 2. Values of IC at different pH, with a 10ꢀmin preincubation.
50
Compound
IC50 (nM)
at pH 9.0
n ≥ 2
IC50 (nM)
at pH 7.4
n ≥ 2
6
6
23.5
140.8
1
183.1
4099.1
Docking studies revealed that compound 1, docked in the neutral form, occupies the binding site
almost completely. The carbamate group is placed in close contact with the catalytic serine (S241),
and establishes polar interactions with the oxyanion hole residues (I238, G239 and M191).
While in all docking poses the Nꢀphenylpentyl chain fits the ACB channel, the Oꢀphenyl group
assumes different accommodations within the binding pocket, with different orientations of the
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
methylamine linker and of the coumarin scaffold in the cytosolic port. In the first family of docking
poses, which includes the topꢀranked binding modes, the methylamino group and the coumarin head
occupy the upper portion of the cytosolic port major cleft, with the basic nitrogen involved in a
hydrogen bond with the hydroxyl group of T236. In the second family, which is less populated and
is characterized by lower values of predicted binding affinity, they occupy the minor branch of the
cytosolic port, with the coumarin head laying on the lower edge of the major cleft (Figure 4).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Left panel (A): representation of the FAAH dimer (chain A, light blue ribbons, chain B,
wheat ribbons); compound 1, docked in the neutral form occupies the binding site adopting two
distinct conformations, clustered in family 1 (green carbons) and family 2 (yellow carbons). Right
panel (B): closeꢀup view of the binding modes of compound 1.
The binding mode of the most potent compound 9 in its neutral form to rFAAH (SI, Figure 4S)
strictly reproduced the docking solutions illustrated for compound 1.
Bulkier derivatives characterized by a fiveꢀmethylene chain, including compound 14, tend to adopt
the second binding mode, as a consequence of the longer alkyl spacer (Figure 5).
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9
10
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12
13
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18
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22
23
24
25
26
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30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Binding mode of compound 14 docked in its neutral form.
Following the hypothesis that the tertiary amine nitrogen of 1 could accept a Hꢀbond by T236, we
replaced the amino group with a triazole ring linked to the phenylpentylꢀ or heptylcarbamate group
through a benzyl spacer (compound 18ꢀ21), or directly inserted on the phenyl ring (22ꢀ25). The
inhibitory potencies of compounds 18ꢀ25 are reported in Table 3.
Table 3. Inhibitory activity of compounds 18ꢀ25 and reference compound 1 against rFAAH,
hAChE and hBuChE.
H
N
O
O
O
N ( )
n
N
O
N
R
O
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1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
a
b
Chain
n
R
ꢀ
IC50 rFAAH (nM) ± SD
IC50 (nM) ± SEM
c
d
position
std protocol
preꢀincub prot.
(n≥2)
hAChE
hBuChE
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
(n≥2)
e
e
e
e
1
ꢀ
ꢀ
280
50
74.9 ± 2.0
1.57 ± 0.18
1
8
9
0
1
para
1
112.8 ± 8.6
28.5 ± 7.9
27.9 ± 6.6
55.0 ± 11.2
32.6 ± 8.2
43.3 ± 3.0
44.7 ± 6.1
255 ± 12
1.17 ± 0.12
42.7 ± 1.5
15.9 ±0.5
78.2 ± 4.8
1230 ± 90
15.2 ±1.2
1
2
2
meta
para
meta
para
meta
1
1
1
0
0
157.2 ± 10.5
353.6 ± 35.3
108.6 ± 67.6
236.2 ±24.3
208.7 ± 0.3
922 ± 89
214 ±19
1044± 82
47900 ± 1400
1175 ± 145
22
2
3
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para
0
0
167.4 ± 25.3
61.9 ± 26.9
21.3 ± 6.9
157500 ± 26800
702 ± 18
122000 ± 23000
71.2 ± 2.9
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meta
15.7 ± 1.7
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a
b
FAAH from rat brain membranes was used. Human recombinant AChE and BuChE from human serum were used. SEM= standard error of the
c
d
mean. IC50 values were determined using a 120 minꢀincubation time; IC50 values for phenylpentyl derivatives were determined using a 15ꢀmin
e
17
incubation time, while IC50 values of the heptyl derivatives were determined using a 20ꢀmin incubation time. Data from reference.
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From a first glance at the results, all the tested compounds of the second series (18ꢀ25) turned out to
successfully inhibit FAAH, with IC50 values within a close range (from 15.7 to 55.0 nM), thus
confirming the design hypothesis. It should be noted that, unlike what previously reported (2ꢀ17),
the increase in rigidity of the linker seemed to be the major feature affecting activity. Indeed,
compounds 18ꢀ25 all showed a comparable potency, regardless of the nature and the insertion
position of the carbamate. Remarkably, almost all derivatives in this new series proved to be more
potent than the lead compound 1, and 25, with a triazole ring directly connected to the phenyl ring,
emerged as the most potent compound.
0
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Docking studies performed for compounds 18ꢀ25 revealed the existence of alternative binding
modes, comparable to those obtained for derivatives 1ꢀ17, depending on the position of the
carbamate group and on the flexibility of the triazoleꢀcontaining linker. Compounds 18 and 19,
characterized by a flexible linker, assumed binding modes similar to that of the second family for
compound 1 (Figure 6).
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Figure 6. Details of the binding mode of compounds 18 (left panel, cyan carbons) and 19 (right
panel, purple carbons)
The reduced flexibility of the linker in compounds 22 and 23, due to the direct insertion of the
triazole ring on the phenylcarbamate group, led to solutions similar to the binding modes of
compound 1 family 1 (Figure 7).
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Figure 7. Details of the binding modes of compounds 23 (left panel, orange carbons) and 22 (right
panel, light blue carbons)
Cholinesterase inhibition by compounds 18ꢀ25 and BuChE Docking Studies
1
9
The new series was also tested on human ChEs using the method of Ellman. Biological data,
reported in Table 3, showed a significant loss of potency against hAChE, probably due to the
rigidity of the inhibitor structures, which may hamper a proper accommodation into the enzyme’s
narrow gorge. A general, although less marked, reduction of inhibitory potency against hBuChE
compared to 1 was also found, with IC50 values still in the nanomolar range, except for 22 and 24.
Indeed, a comparison of hBuChE and hAChE gorges shows differences in size, especially in the
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acyl chainꢀbinding pockets. In hBuChE the active site gorge is larger than in hAChE, due to the
presence of a valine and a leucine instead of the bulkier phenylalanines of hAChE, enabling larger
molecules to better fit into the gorge. Compounds 18, 20 and 23 proved to be the most potent
hBuChE inhibitors in this second series.
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Docking studies were performed to investigate the possible binding mode of the triazoleꢀbased
3
6
derivatives within hBuChE active site (PDB ID: 4TPK). Compound 1 gave a binding pose
consistent with enzyme inhibition by carbamoylation of the catalytic serine and in line with the
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accommodation reported for similar compounds in both AChE and BuChE.
In particular,
compound 1 was deeply inserted within the activeꢀsite gorge, assuming a folded conformation, with
the carbamate group directed toward the catalytic triad (S198, H438 and E325), while the coumarin
head and the acyl chain pointed toward the entrance of the gorge (Figure 8A). The carbamate
carbonyl was accommodated close to the catalytic serine (S198, Figure 8B), taking polar
interactions with the oxyanion hole residues (G116 and G117). At the bottom of the binding site,
the phenyl ring attached to the carbamate group pointed toward the cholineꢀbinding pocket,
occluded by W82. This pose was stabilized by a hydrogen bond between the protonated amino
group and the side chain of T120. The coumarin head occupied the wide access channel of the
activeꢀsite gorge, pointing toward the peripheral anionic site (PAS). Here the bicyclic nucleus could
assume different positions in alternative docking solutions: in the pose represented in Figure 8B it
establishes a polar interaction with the side chain of Q289. The protein surface at the entrance of the
gorge is quite flat and hydrophobic. This could be the reason of the good potency of compounds
with tricyclic nuclei, such as 10 (see SI, Figure 5S), 11, 16 and 17. The phenylpentyl chain at the
carbamate nitrogen was accommodated within the putative acyl chainꢀbinding pocket, which is
lined by L286 and V288 in hBuChE. Notably, in hAChE these residues are replaced by F295 and
F297, which reduce the pocket size, hampering the accommodation of bulky lipophilic groups.
Thus, also considering the reduced potency of compounds 18ꢀ25 as hAChE inhibitors, we decided
not to perform docking studies into hAChE models, to avoid overinterpretation of structureꢀactivty
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relationships. Indeed, the different size of the acyl chainꢀbinding pockets is sufficient to explain the
selectivity for hBuChE (approximately 50 fold) of compound 1 and its derivatives, while the Nꢀ
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methyl analog xanthostigmine is 160ꢀfold more potent at hAChE than at hBuChE.
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Figure 8. A. Docking of compound 1 (green carbons) within the catalytic gorge of hBuChE. B.
Detailed picture of the binding mode of compound 1. Hydrogen bonds are marked with yellow
dottedꢀlines.
Docking studies for triazole derivatives provided similar binding modes, and substitution at the
phenyl ring carrying the carbamate group proved to be crucial for structureꢀactivity relationships.
While the paraꢀmethylene spacer of 18 allowed the triazole ring to accept a hydrogen bond from the
hydroxyl group of T120, the metaꢀmethylene spacer of 19 allowed the compound to occupy the
hBuChE gorge, but not to take polar interactions with T120 (Figure 9A). On the other hand, when
the triazole was directly attached to the phenyl ring (compound 22), no docking poses consistent
with carbamoylation of S198 were obtained for the para isomer, due to a steric clash between W82
and the triazole ring, while meta substitution (compound 23) allowed the coumarin head to be
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