Identification of Isoform 2 Acid-Sensing Ion Channel Inhibitors as Tool Compounds for Target Validation Studies in CNS

Article abstract of DOI:10.1021/acsmedchemlett.8b00591

Acid-sensing ion channels (ASICs) are a family of ion channels permeable to cations and largely responsible for the onset of acid-evoked ion currents both in neurons and in different types of cancer cells, thus representing a potential target for drug discovery. Owing to the limited attention ASIC2 has received so far, an exploratory program was initiated to identify ASIC2 inhibitors using diminazene, a known pan-ASIC inhibitor, as a chemical starting point for structural elaboration. The performed exploration enabled the identification of a novel series of ASIC2 inhibitors. In particular, compound 2u is a brain penetrant ASIC2 inhibitor endowed with an optimal pharmacokinetic profile. This compound may represent a useful tool to validate in animal models in vivo the role of ASIC2 in different neurodegenerative central nervous system pathologies.

Full text of DOI:10.1021/acsmedchemlett.8b00591

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Letter
Identification of Isoform 2 Acid-Sensing Ion Channel Inhibitors
as Tool Compounds for Target Validation Studies in CNS
Leda Ivanova Bencheva, Marilenia De Matteo, Luca Ferrante, Marco Ferrara, Adolfo Prandi, Pietro
Randazzo, Silvano Ronzoni, Roberta Sinisi, Pierfausto Seneci, Vincenzo Summa, Mariana Gallo,
Maria Veneziano, Antonella Cellucci, Nausicaa Mazzocchi, Andrea Menegon, and Romano Di Fabio
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00591 • Publication Date (Web): 07 Feb 2019
Downloaded from http://pubs.acs.org on February 8, 2019
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Identification of Isoform 2 Acid-Sensing Ion Channel Inhibitors as Tool
Compounds for Target Validation Studies in CNS
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Leda Ivanova Bencheva,£ Marilenia De Matteo,£ Luca Ferrante,£ Marco Ferrara,£,^ Adolfo Prandi,£ Pietro
Randazzo,£ Silvano Ronzoni,£ Roberta Sinisi,£ Pierfausto Seneci,£,{ Vincenzo Summa,& Mariana Gallo, &
Maria Veneziano,& Antonella Cellucci,& Nausicaa Mazzocchi,$ Andrea Menegon,$,* Romano Di
Fabio£,&,
*
£, Promidis, Via Olgettina 60, 20132, Milan, Italy; $, San Raffaele Scientific Institute, Experimental Imaging Center,
ALEMBIC, Advanced Light and Electron Microscopy BioImaging Center, Via Olgettina 60, 20132, Milan, Italy; &, IRBM
Science Park, Via Pontina Km 30.600, 00070 Pomezia (Rome), Italy; {, Chemistry Department, Università degli Studi di
Milano, Via Golgi 19, I-20133 Milan, Italy. * These two authors share senior authorship.
KEYWORDS: ASICs, ion channels, drug discovery, CNS, PNS, cancer.
ABSTRACT: Acid-sensing ion channels (ASICs) are a family of ion channels permeable to cations and largely responsible for the
onset of acid-evoked ion currents both in neurons and in different types of cancer cells, thus representing a potential target for drug
discovery. Owing to the limited attention ASIC2 has received so far, an exploratory programme was initiated to identify ASIC2
inhibitors using Diminazene, a known pan-ASIC inhibitor, as a chemical starting point for structural elaboration. The performed
exploration enabled the identification of a novel series of ASIC2 inhibitors. In particular, compound 2u is a brain penetrant ASIC2
inhibitor endowed with an optimal pharmacokinetic profile. This compound may represent a useful tool to validate in animal models in vivo
the role of ASIC2 in different neurodegenerative central nervous system pathologies.
The variation of proton concentration in tissues is a tightly
controlled process.1 In particular, a decrease of pH has been
observed both in physiological conditions, i.e. control of neuronal
functions by proton-mediated signalling, and pathological
conditions.2,3 Notably, acidification occurring in pathological
conditions was found to recruit acid-sensing ion channels (ASICs),
a family of proton-activated ion channels4 that are highly expressed
both in central and peripheral neurons5 and in different types of
cancer cells,6 thus representing a potential target for drug
discovery.3,7 ASICs are voltage-insensitive ion channels belonging
to the ENaC/DEG channel super-family, which includes epithelial
Na+ channels (ENaC) and degenerins (DEG). Four ASICs genes
(ASIC1-4) and two specific splice variants for ASIC1 and ASIC2
(a and b) have been described in mammals to date. ASIC1a,
ASIC2a, and ASIC2b are primarily expressed in central nervous
system (CNS) neurons, while all subunits are expressed in the
peripheral nervous system (PNS). ASIC1a, ASIC 2a and ASIC3
subunits assemble to form both homotrimeric and heterotrimeric
channels, whereas ASIC2b and ASIC4 only contribute to forming
heteromeric channels with other ASIC subunits.7,8 In terms of
electrophysiology, while ASIC1a undergoes rapid inactivation, for
ASIC2 and ASIC3 a non-inactivated current was observed,
potentially relevant in chronic pathologies. Thus far, both ASIC1
and ASIC3 have been extensively studied,9 while ASIC2 has
received much less attention. Notably, ASIC2 have recently been
proposed as relevant target in some forms of cancer10,11 whereas, in
combination with ASIC1 subunits, appear to play a key role in
neuronal physiopathology.12 Several natural peptides and synthetic
small molecules, i.e. diminazene 1 (DA, Chart 1), an anti-infective
veterinary drug, have been described as ASICs inhibitor.13,14,15
However, the latter compound shows both poor target and ASIC
isoform specificity, along with negligible blood-brain barrier
(BBB) penetration, hence limiting its usage as therapeutic agent
both for CNS and PNS pathologies.16,17 Hence, an exploratory
projects was initiated, using DA as chemical starting point, to
obtain brain penetrant ASIC2 inhibitors as useful tool compounds
for target validation studies in CNS. To this aim, being ASIC1a and
ASIC2a the most highly expressed ASIC subunits in CNS neurons,
the new chemical entities (NCEs) synthesized were specifically
tested for their effects on murine homotrimeric ASIC1a and
ASIC2a and on the heterotrimeric ASIC1a/2a.18
Chart 1. Structure of diminazene (DA) 1 and early lead 2a
F
H
N
N
N
H2N
NH2
NH
N
NH
N
H2N
1
2a
To identify the most appropriate methodology for in vitro screening
of NCEs, a series of published data19,20,21 on both compound 1 and
Amiloride, a diuretic drug known to interact with ASICs,
specifically drawn our attention and an optical technology based on
membrane potential detection by voltage-sensitive dyes (VSDs)
22,23,24,25
was proposed. This original assay format showed an
adequate throughput performance and ability to efficiently predict
the inhibitory effect of ASICs inhibitors. Moreover, additional
evidence suggested the use of optic based assays, owing to the
membrane potential sensitivity of ASIC1a binding affinity to small
molecules.20
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The preliminary structural elaboration of DA, in three sequential
or heteroaryl substituted boronic acids. The resulting 1-(p-
cyanophenyl) pyrazoles 6a-i were reduced to the corresponding
benzylamines 2a-i with lithium aluminium hydride in moderate to
good overall yields.
1
2
3
4
5
6
7
8
steps, enabled the identification, of the 1,4-diaryl, 3-5-
dimethylpyrazole derivative 2a (Chart 1), an early lead compound
which was fully characterized in terms of ASICs inhibition. In
particular, the linear triazene linker present in DA was initially
replaced by a 1,3-disubstituted 5-membered heterocycle, with the
aim to rigidify the molecular core. Then, to reduce the basic
character of the molecule, with the aim to improve drug-like
character and possibly secure BBB permeability, one of the two
amidine functions was successfully removed, Finally, an initial
exploration was made on the effect of the substitution of the
terminal phenyl ring.
The inhibition of ASICs constructs by compounds 2a-i is reported
in Table 1.
Table 1. Compounds 2a-i: ASICs inhibitiona
Entry
ASIC1a a
ASIC2a a
ASIC1a/2a a
9
1 (DA)
2a
56.9 ± 8.9
>30
169.0 ± 18.1
18.9 ± 6.9
>30
45.2 ± 8.8
10.9 ± 1.6
>30
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As shown in Table 1, compound 2a exhibited greater in vitro
activity than compound 1 and comparable activity on ASIC2a and
ASIC1a/2a (IC50 = 18.9 M and 10.9 M, respectively), while
being inactive on ASIC1a. Based on these preliminary encouraging
results, the rapid “4 points” analoging exploration strategy, as
depicted in Chart 2, focused on the sequential elaboration of the
two aryl moieties (“pink and green”), the heterocycle core
(“fuchsia”) and the suitable rigidification/masking of the
potentially metabolically labile terminal benzyl function (“cyan”),
was envisioned.
2b
2c
>30
>30
8.8 ± 2.0
16.3 ± 4.6
>30b
>30
2d
2e
>30
>30b
>30
>30
2f
>30
>30
>30
2g
>30
4.3 ± 0.5
>30
>30
2h
2i
>30
>30
>30
>30
>30
Chart 2. Structural optimization of early lead 2a
a
IC50 were determined as described in the Supplementary
F
Information; they are expressed in M and are the average value of
at least n=3 independent experiments ± SEM.
- Substitution scan
- Heterocycles
- Benzylamine
substitution and
rigidification
As shown in Table 1, monosubstituted compounds 2c and 2d,
bearing a p-CH3 or p-OCH3 respectively, mostly retained the
activity of the pyrazole lead compound 2a, while isoform-
selectivity for ASIC2a vs ASIC1a/2a was improved. Compound 2g,
the m-F analogue of 2a, was the most potent and selective
compound of this series. Notably, larger and/or charged functions
(2e, p-CF3; 2f, p-CH2NH2), disubstituted aryls (2h, p-CH3, m-F)
and the presence of a pyridine as phenyl replacement (2i, p-CH3,
X=N) led to inactive compounds.
N
N
- Demethylation
- Spacers
H2N
- Heterocycles
Scheme 1. Variations on the 4-aryl substituent: compounds 2a-
ia
Br
Then, the influence of the 3,5-dimethyl substituents present on the
pyrazole core was evaluated by synthesizing the corresponding
“fuchsia” des-methyl analogue 2j (Scheme 2).
OH
B
c
a,b
N
N
OH
N
N
5
NC
NC
NC
Scheme 2. Scaffold hopping: des-methyl compound 2ja
4
3
d
R1
F
F
R1
CH(CHO)2
X
N
X
N
R2
R2
OH
B
2a R1=F, R2=H, X=CH
2b R1=Cl, R2=H, X=CH
2c R1=CH3, R2=H, X=CH
2d R1=OCH3, R2=H, X=CH
2e R1=CF3, R2=H, X=CH
2f R1=CH2NH2, R2=H, X=CH
2g R1=H, R2=F, X=CH
OH
a, b
c
F
e
N
NC
N
N
N
N
N
H2N
H2N
1
NC
NC
2j
2a-i
7
6a-i
2h R1=CH3, R2=F, X=CH
2i R1=CH3, R2=H, X=N
aReagents and conditions: (a) di-tert-butyl diazene-1,2-
.
dicarboxylate, Cu(OAc)2 H2O, MeOH, 65°C , 1h; (b) 2-(4-
aReagents and conditions: (a) di-tert-butyl diazene 1,2-
dicarboxylate, Cu(OAc)2 H2O, MeOH, 65°C, 1h; (b) pentane-2,4-
dione, 4N HCl in dioxane, r.t., 10 min then 80°C, 10 min, 76% (two
steps); (c) NBS, EtOAc, sonication, 25-30°C, 15 min, 80%; (d)
substituted aryl/pyridinyl boronic acid, Pd(PPh3)4, aq. Na2CO3,
DMF, microwave reactor, 140°C, 15-20 min, 52%-70%; (e)
LiAlH4, THF, r.t, 0.5-3 h, 18-75%.
fluorophenyl)propanedial, 4N HCl in dioxane, r.t., 10 min then
80°C, 10 min, 56% two steps; (c) LiAlH4, THF, r.t, 1h, 73%.
.
To this aim, p-cyanophenyl boronic acid 3 was sequentially treated
with di-tert-butyl diazene-1,2-dicarboxylate in presence of
.
Cu(OAc)2 H2O in MeOH at 65°C for 1h, followed by 2-(4-
fluorophenyl)propanedial in 4N HCl in dioxane initially at room
temperature for 10 min, then at 80°C for additional 10 min to give
intermediate 7 in 56% yield. Then, reduction of the cyano group
led to the corresponding benzylamine 2j with lithium aluminium
hydride in 73% yield.
At first, following the synthetic strategy shown in Scheme 1 nine
“pink” analogues 2a-i were synthesized. In particular, p-
cyanophenyl boronic acid 3 was reacted with di-tert-butyl diazene
1,2-dicarboxylate and 2,4-pentanedione, to obtain the 3,5-dimethyl
pyrazole 4. The following bromination reaction with NBS led to
the 4-bromo analogue 5, which was coupled with 9 different aryl
Compound 2j was found inactive in terms of ASICs inhibition (IC50
> 30 M on all ASICs constructs, Table 2), pointing out the
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F
F
relevance of both methyl groups for the recognition of the receptor
1
binding site.
2
3
4
5
6
7
8
To acquire additional SAR information, a methylene spacer was
introduced between the N1 of the pyrazole moiety (“fuchsia”
compound 2k, Scheme 3) by bromination and Suzuki coupling on
3,5-dimethyl pyrazole 8, followed by alkylation at the N1 position
of 3,5-dimethyl pyrazole derivative 10a with p-CN benzyl
bromide, and by final reduction of the nitrile group with lithium
aluminium hydride in poor, unoptimized yields.
b
a
N
2l
NC
N
10a
N
N
H2N
12
F
F
9
Scheme 3. Spacer introduction: compound 2ka
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c
F
N
N
Br
N
N
b
a
H2N
NC
HN
HN
N
8
N
2m
6a
HN
N
10a
9
F
F
c
F
F
d
e
10a
N
N
N
N
d
N
N
CN
N
N
H2N
13
2n
aReagents and conditions: (a) 3-cyanophenylboronic acid,
Cu(OAc)2 H2O, pyridine, DMF, 125°C, 3h, 11%; (b) LiAlH4, THF,
r.t, 3h, 31%; (c) MeMgBr, THF, r.t, 5h, then LiAlH4, THF, 0° to
r.t, 13% (two steps); (d) 4-bromobenzyl cyanide, CuI, L-proline,
K2CO3, DMSO, 140°C, 26h, 33%; (e) NaBH4, CoCl2, MeOH, r.t,
1h, 25%.
11a
2k
CN
.
H2N
aReagents and conditions: (a) NBS, EtOAc, sonication, 25-30°C,
15 min, quantitative; (b) 4-(fluorophenyl)boronic acid or 3-
(fluorophenyl)boronic acid, Pd(PPh3)4, sat. Na2CO3, DMF, 140°C,
20 min, 70%; (c) 4-(bromomethyl)benzonitrile, Cs2CO3, CH3CN,
50°C, 10h, quantitative; (d) LiAlH4, THF, r.t, 1h, 9%.
Compounds 2l, 2m and 2n mostly maintained the activity of the
early pyrazole lead 2a, and showed high ASIC2a isoform-
selectivity (Table 2), whereas the nitrile intermediates 6a was
inactive, pointing out the relevance of the presence of the primary
amine function.
When compound 2k was tested for its ability to inhibit ASICs a
comparable activity and isoform-selectivity was observed for
ASIC2a with respect to compound 2a (Table 2, IC50 = 16.8 M and
18.9 M, respectively).
The “blue” exploration was expanded by constraining the amine
function within a 5-membered ring. To this aim, compounds 2o-r
bearing a more symmetrical (2o, 2q) or unsymmetrical amine (2p,
2r) and a p-F (2o, 2p) or m-F substituent at the 4-phenyl ring (2q,
2r) were prepared. The synthesis of this sub-series of compounds
is reported in Scheme 5. Namely, previously described 4-(p-
fluorophenyl)-3,5-dimethyl pyrazole intermediate 10a, and 4-(m-
fluorophenyl)-3,5-dimethyl pyrazole intermediate 10b (prepared as
10a in Scheme 3, using m-fluorophenyl boronic acid) were N-
arylated with N-Boc-5-bromo isoindoline, using CuI in basic
conditions in a microwave reactor.
Our attention was then focused on the “blue” exploration by
initially moving the p-benzylamine moiety from para to meta
position of the phenyl ring (compound 2l, Scheme 4), by
introducing a methyl group at the benzylic position (compound 2m,
Scheme 4), and by C-1 homologation (compound 2n, Scheme 4).
In particular, as for the synthesis of 2l, 4-(p-fluorophenyl)-3,5-
dimethyl pyrazole 10a was N-arylated with m-cyanophenyl
boronic acid using copper(II) acetate; the resulting 1-(m-
cyanophenyl) pyrazole 12 was reduced to the corresponding
benzylamine 2l with lithium aluminium hydride in unoptimized
poor yields. Compound 2m was synthesized by reacting previously
described 1-(p-cyanophenyl) pyrazole 6a with methylmagnesium
bromide in reducing conditions. Finally, compound 2n was
prepared from 4-(p-fluorophenyl)-3,5-dimethyl pyrazole 10a
which was N-arylated with p-cyanomethyl phenyl bromide,
according to Buchwald-Hartwig reaction experimental protocol26
(microwave reaction, aquous K2CO3 and DMSO, CuI and L-
proline, 140°C, 26 h). The resulting pyrazole intermediate 13 was
then reduced with sodium borohydride in the presence of cobalt (II)
chloride, to obtain the corresponding target benzylamine 2n.
The resulting N-Boc-protected 4-(p-fluorophenyl) pyrazoles 14a
and 15a were obtained in 42% and 15% unoptimized yield,
respectively. Then, removal of the N-Boc protecting group in acidic
conditions afforded the corresponding target compounds 2o and 2q
in 32% and 63% yields, respectively. Intermediates 10a and 10b
underwent the same coupling reaction with 6-bromo indoline,
obtaining the corresponding target compounds 2p and 2r in 44%
and 13% yield, respectively (Scheme 5).
Scheme 4. Variations on the benzylamine: compounds 2l-na
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Scheme 5. Rigidification of the benzylamine: compounds 2o-ra
aReagents and conditions: (a) N-Boc-5-bromo isoindoline, CuI,
homologues 2u and 2v and the N-Me derivative of the latter
compound 2w were prepared. Their synthesis is depicted in
Scheme 6.
1
2
Br
Y
3
4
5
6
7
8
9
Scheme 6. Phenyl substitution: compounds 2s-wa
F
X
F
F
X=NBoc
Y=CH2
X=CH2
Y=NH
F
or
a
b
N
Y
N
Y
N
10a
N
X
c
a
X
N
10a
14a
N
N
a'
N
H2N
N
2o, X=NH, Y=CH2
2p, X=CH2, Y=NH
N
NC
16
2s
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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30
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Br
F
Y
F
F
F
X
X=NBoc
Y=CH2
X=CH2
Y=NH
or
b
c
10a
N
N
N
N
Y
a
Y
N
2t
b
N
N
N
10b
X
X
H2N
N
NC
N
15a
17
a'
2q, X=NH, Y=CH2
2r, X=CH2, Y=NH
F
F
Boc
N
aqueous K2CO3, L-proline, DMSO, microwave, 75°C, 4 h then
140°C, 13 h, 42% (14a) or 15% (15a); (b) 4N HCl in dioxane,
30min, r.t., 32% (2o) or 63% (2q); (a’) 6-bromo indoline, aqueous
K2CO3, CuI, L-proline, DMSO, microwave, 140°C, 6 h, 44% (2p)
or 13% (2r).
Cl
Boc
HN
N
N
N
N
a
N
N
d
N
10a
N
N
N
N
2u
18
Boc
Table 2. Compounds 2k-w and 6a: ASIC inhibitiona
N
Cl
F
F
ASIC1aa
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
ASIC2aa
18.9 ± 6.9
>30
ASIC1a/2aa
10.9 ± 1.6
>30
N
Boc
Boc
Entry
2a
2j
N
N
N
e
f
Cl
N
N
10b
N
N
N
N
N
N
20
Cl
2k
2l
16.8 ± 5.4
15.6 ± 2.9
8.7 ± 2.3
11.7 ± 2.7
8.2 ± 1.5
>30
>30
19
>30
d
2m
2n
2o
2p
2q
2r
>30
F
F
g
>30
HN
N
N
N
>30
N
N
N
N
N
N
>30
2w
2v
9.9 ± 1.1
>30
11.3 ± 1.0
>30
aReagents and conditions: (a) 60% NaH, DMF, 0°C, 30 min then 6-
fluoronicotinonitrile, r.t, 1h (98%, 16) or tert-butyl 4-chloro-5,7-
dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxylate, r.t., 1 h (9%,
18); (b) 5-bromo-2-cyanopyridine, Cs2CO3, CuI, 1,2-
cyclohexanediamine, microwave, 120°C, 13h, 19%; (c) LiAlH4,
THF, r.t, 1h, 4% (2s), 16% (2t); (d) 4N HCl in dioxane, r.t, 1-3h,
8% (2u), 19% (2v); (e) 2/1 THF/DMF, DIPEA, microwave, 150°,
14h, 58%; (f) H2, TEA, MeOH, 10% Pd/C, 45°C, 1 h; (g)
formaldehyde, NaCNBH3, MeOH, r.t, 1 h, 57%.
2s
6.1 ± 1.1
>30
8.5 ± 1.8
>30
2t
2u
2v
2w
6a
17.0 ± 4.7
>30
>30
>30
>30
>30
>30
>30
a
IC50 were determined as described in the Supplementary
Information; they are expressed in M and are the average value of
at least n=3 independent experiments ± SEM.
Previously described 4-(p-fluorophenyl)-3,5-dimethyl pyrazole
intermediate 10a was smoothly N-arylated with p-cyano-2-pyridyl
fluoride in basic conditions (NaH in DMF); the resulting 1-(p-
cyano-2-pyridyl) pyrazole 16 was reduced to the corresponding
benzylamine derivative 2s with lithium aluminium hydride in
unoptimized, poor yields. Instead, the corresponding m-F analogue
2t was synthesized from 10a and 5-bromo-2-cyanopyridine using
Cs2CO3, CuI and 1,2-cyclohexanediamine. This coupling reaction
was run in a microwave reactor for 13 h at 120°C, and afforded
cyano intermediate 17, which was then reduced to amine 2t in a
poor, unoptimized 16% yield. Alternatively, compound 10a was N-
arylated with tert-butyl 4-chloro-5,7-dihydro-6H-pyrrolo[3,4-
d]pyrimidine-6-carboxylate as seen earlier; the resulting N-Boc-
protected pyrazole 18 was deprotected in acid conditions to yield
the target compound 2u in poor, unoptimized yields. Then,
The inhibition of ASICs constructs by “blue” compounds 2o-r is
reported in Table 2. In particular, the isoindole derivative 2o
exhibited good in vitro activity and complete ASIC2a isoform-
selectivity. Conversely, compound 2q inhibited both ASIC2a and
ASIC1a/2a isoforms (IC50 = 9.9 M and 11.3 M, respectively).
Notably, the corresponding indoline derivatives 2p and 2r were
inactive.
Finally, the phenyl ring bearing the benzyl amine function was
replaced (“green” exploration”) by a 2-pyridine (2s) and a 3-
pyridine (2t) moiety, whereas dihydropyrrolo[3,4-d]pyrimidine
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intermediate 10b was transformed into the target compound 2v by
enhancement of the metabolic stability (Cl = 34 ml/min/Kg) with
respect to compound 2a, an appropriate tissue distribution (Vd=1.4
l/Kg), complete absorption after oral administration (F=100%)
along with a significantly higher exposure p.o. with respect to 2a
1
2
3
4
5
6
7
8
N-arylation followed by removal of the Cl atom by hydrogenolysis
to give intermediate 20, which was finally deprotected in acid
conditions to the corresponding NH-free pyrazole 2v. Finally, this
compound was N-methylated to yield compound 2w in good yields
(Scheme 6).
(Cmax 2.6 M), and good brain penetration (B/P = 1.3). The
=
relevant improvement of pharmacokinetic profile of 2u vs 2a was
most likely due to the lack of the basic, metabolically labile primary
benzylamine function present in compound 2a, which was
appropriately masked in 2u within the constrained
dihydropyrrolo[3,4-d]pyrimidine bicyclic moiety.
As shown in Table 2, none of the compounds 2s-w inhibited
ASIC1a. The presence of a 2-pyridyl ring was well tolerated by
ASIC2a (2s). Conversely, the presence of a 3-pyridyl ring led to
complete inactivity (2t). The dihydropyrrolo[3,4-d]pyrimidine
derivative 2u was an ASIC2 inhibitor (IC50 = 17.0 M) selective
against the ASIC1a/2a heterodimer. Surprisingly, its close
congeners 2v and 2w, bearing a 4-(m-fluorophenyl) substitution,
resulted to be completely inactive.
9
In conclusion, the described exploratory strategy enabled the
identification of novel ASIC2 inhibitors. In particular, the “cyano”
optimization approach, focused on the stabilization of the terminal
benzylamine function, allowed to obtain compounds 20 and 2u as
selective ASIC2a the in vivo-compliant lead compound 2u. This
compound, owing to its relevant drug-like character and balanced
pharmacokinetic profile (including brain penetration), may
represent a valuable tool compound to validate p.o. the role of
ASIC2 in vivo in animal models of different type of CNS
pathologies. In addition, 2u can be seen as a foundation molecule
for future optimization studies.
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Compound 2a, 2o and 2u were profiled in terms of early
physicochemical and ADME properties. As to in vivo PK in mice
priority was given to the characterization of 2u over 2o owing to its
more drug-like physicochemical features (cLogP = 2.6 and 4.1;
TPSA = 56 and 29 Å2, for 2u and 2o, respectively). The summary
of both the in vitro and in vivo characterization studies performed
is reported in Table 3.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Table 3. ADME profiling: compounds 2a, 2o and 2u
Compound/Assay
cLogP a
2a
3.9
2u
2.6
2o
4.2
30
TPSA (Å2) a
44
56
Solubility (g/ml)b
PPB (%)c
Experimental procedures for the synthesis and the
analytical characterization of both key intermediates and
final compounds 2a-w, 6a, the in vitro screening of
compounds 2a-w, 6a and in vivo PK studies of
compounds 2a and 2u.
73
32
56
93
94
95
>30
>30
>30
h-ERG (IC50, M)
CYP450 (IC50,
M)d
0.4 [1A2]
0.8 [1A2]
0.2 [1A2]
4.8 [2D6]
NT
Cl (ml/min/Kg)e
224
50.9
0.09
60
34
1.4
2.6
100
1.3
AKNOWLEDGEMENTS
Vd (l/Kg)
Cmax (M)
F (%)
NT
NT
NT
The authors would like to thank Prof. Stefano Maiorana (Promidis),
Dr. Edith Monteagudo (IRBM Science Park), Dr. Daniel Cicero
(IRBM Science Park) and Dr. Annalise Di Marco (IRBM Science
Park) for the useful discussion on the synthesis of the described
chemical series, the in vitro and in vivo ADME studies, the serum
albumin NMR-based analysis and the CYP450 inhibition studies,
respectively. In addition, thanks are due to Dr. Maria Rosaria
Battista (IRBM Science Park) for the in vitro CYP450 inhibition
studies.
B/P ratio f
61
NT
a
b
calculated logP and topological polar surface area; kinetic
c
solubility at pH 7.4; % of bound compound to human serum
albumin measured by NMR-based analysis;
isoforms showing IC50 <10 M are reported; in vivo PK studies
were performed at 1 mg/Kg, i.v. and at 3 mg/Kg, p.o.; brain
d
only CYP450
e
f
AUTHOR INFORMATION
penetration studies were performed at 1 mg/Kg, i.v. and B/P was
calculated from 2h to 8h after dosing.
Corresponding Author
* Phone: +390249491701. E-mail: r.difabio@irbm.it
As to physicochemical descriptors, compound 2u was significantly
less lipophilic than 2a and exhibited a greater TPSA value. Both
compounds showed acceptable kinetic solubility and plasma
protein binding (PPB). No inhibition of hERG was observed up to
30M concentration. In terms of inhibition of CYP450 isoforms,
compound 2a was found to inhibit two different isoforms, i.e. 1A2
and 2D6, although at different extents, whereas 2u inhibited only
the 1A2 isoform. Particularly relevant were the differences
observed in the in vivo pharmacokinetics in mice. Both compounds
were tested at 1 mg/kg, i.v. and at 3 mg/Kg, p.o. Notably, 2a was
highly cleared following i.v. administration (Cl = 224 ml/min/Kg),
but widely distributed in tissues (Vd = 50.9 l/Kg), resulting in a low
Cmax after oral administration (Cmax =0.09 M). However, being
highly brain penetrant (B/P = 61), a relevant total brain
concentration was observed after the administration of 1 mg/Kg
dose, i.v. (Cmax =2.46 M). Conversely, compound 2u showed a
more balanced pharmacokinetic profile, owing to a sizable
Present Addresses
^ Marco Ferrara: Flamma Innovation Srl, Via Cascina Secchi 217,
24040 Isso (BG), Italy.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Funding Sources
The project was in part funded by the COLLEZIONE DEI
COMPOSTI CHIMICI E CENTRO DI SCREENING–CNCCS
scarl
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13) Baron, A.; Lingueglia, E. Pharmacology of acid-sensing ion
Notes
1
channels - Physiological and therapeutical perspectives.
Neuropharmacology. 2015, 94, 19–35.
The authors declare no competing financial interest
2
3
4
5
6
7
8
9
14) Chen, X.; Qiu, L.; Li, M.; Dürrnagel, S.; Orser, B. A.; Xiong,
Z.-G.; MacDonald J. F. Diarylamidines: high potency
inhibitors of acid-sensing ion channels. Neuropharmacology.
2010b, 58, 1045–53.
ABBREVIATIONS
ASICs, acid-sensitive ion channels; DA, diminazene; ENaC,
epithelial Na+ channels; DEG, degenerins; CNS, central nervous
system; PNS, peripheral nervous system; NCE, new chemical
entity; VSD, voltage-sensitive dyes; BBB, blood-brain barrier;
TPSA, topological polar surface area; PK, pharmacokinetic.
15) Lingueglia, E.; Lazdunski, M. Pharmacology of ASIC
channels. Wiley Interdiscip. Rev. Membr. Transp. Signal.
2013, 2, 155–171.
16) Chen, X.; Orser, B. A.; MacDonald J. F. Design and screening
of ASIC inhibitors based on aromatic diamidines for
combating neurological disorders. Eur. J. Pharmacol. 2010a,
648, 15–23.
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F
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H
F
N
N
N
HN
N
H2N
NH2
NH
N
N
N
NH
N
H2N
N
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1 (Diminazene)
2a
2u
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