Synthesis and pharmacological evaluation of functionalized isoindolinones on GABA-activated chloride currents in rat cerebellum granule cells in culture

Article abstract of DOI:10.1016/j.bmcl.2016.09.042

A focused N-substituted 3-(2-piperazin-1-yl-2-oxoethyl)-2-(pyridin-2-yl)iso-indolin-1-ones small library was synthesized for modulation of GABA-A receptor function and compared to Zopiclone for the ability to increase GABA-activated chloride currents. All

Full text of DOI:10.1016/j.bmcl.2016.09.042

Accepted Manuscript
Synthesis and Pharmacological Evaluation of Functionalized Isoindolinones on
GABA-Activated Chloride Currents in Rat Cerebellum Granule Cells in Culture
Antonia Di Mola, Elena Gatta, Carmen Petronzi, Aroldo Cupello, Paolo De
Caprariis, Mauro Robello, Antonio Massa, Rosanna Filosa
PII:
S0960-894X(16)30984-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.09.042
BMCL 24266
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 August 2016
14 September 2016
15 September 2016
Please cite this article as: Mola, A.D., Gatta, E., Petronzi, C., Cupello, A., Caprariis, P.D., Robello, M., Massa, A.,
Filosa, R., Synthesis and Pharmacological Evaluation of Functionalized Isoindolinones on GABA-Activated
Chloride Currents in Rat Cerebellum Granule Cells in Culture, Bioorganic & Medicinal Chemistry Letters (2016),
doi: http://dx.doi.org/10.1016/j.bmcl.2016.09.042
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Synthesis and Pharmacological Evaluation of
Functionalized Isoindolinones on GABA-Activated
Chloride Currents in Rat Cerebellum Granule Cells in
Culture
Antonia Di Molaa#, Elena Gattab#, Carmen Petronzid,c, Aroldo Cupellob,
Paolo De Caprariisa, Mauro Robellob, Antonio Massa d*, Rosanna Filosaa,c*
a Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 84084 Fisciano, SA, Italy
bDipartimento di Fisica, Università di Genova, Via Dodecaneso 33, 16146 Genova, Italy
cDepartment of Experimental Medicine, Second University of Naples, Via Costantinopoli, 16, 80138 Naples, Italy
dDepartment of Chemistry and Biology, University of Salerno, Via Giovanni Paolo II, 84084 Fisciano, SA, Italy
# these author contribute equally
Corresponding authors at: Rosanna Filosa, Department of Experimental Medicine, Second
University of Naples, Via Costantinopoli, 16 - 80138 Naples, Italy; Phone:0815667663; e-
mail: rosanna.filosa@unina2.it
1

Abstract
A focused N-substituted 3-(2-piperazin-1-yl-2-oxoethyl)-2-(pyridin-2-yl)iso-indolin-1-ones small
library was synthesized for modulation of GABA-A receptor function and compared to Zopiclone
for the ability to increase GABA-activated chloride currents. All compounds were tested for their
effects on GABA-activated chloride currents in rat cerebellar granule cells by use of the whole-cell
patch clamp technique. Electrophysiological studies on cultured cerebellar granule cells revealed 3-
[2-(4-Methylpiperazin-1-yl)-2-oxoethyl]-2-(5-nitropyridin-2-yl)iso-indolin-1-one (Id) as a partial
agonist displaying 34% increase of the 10 μM GABA evoked peak chloride currents, antagonized
by flumazenil. Moreover, a second group of compounds, with bulky functional groups at N-4
position of piperazine, have shown inverse agonist effects.
The simple synthetic procedure and the possibility of modulating the efficacy of this class of
ligands through additional structural modifications pave the way for further development of new
molecules as a novel class of compounds able to interfere with benzodiazepine receptors.
2

GABA-A receptor complexes are associated with, and interact with, membrane channels for
chloride ion transport, these complexes are located both within the central nervous system and
peripherally.1,2 There is little doubt regarding the therapeutic possibilities of modulation of GABA-
A receptor function.3,4 These receptors are indeed targets of important drugs such as
benzodiazepines, barbiturates, neuroactive steroids and anaesthetics.5,6 The emerging understanding
of the role of different GABA-A receptor subtypes in mediating different physiological functions
and pathological processes continues to offer opportunities for novel therapeutics. The
benzodiazepine (Bzd) site is an important player in the pharmacology of GABA-A receptors.
Clinically used classical 1,4-benzodiazepines such as diazepam have a large spectrum of
pharmacological effects such as anxiolysis, sedation/hypnosis, myorelaxation and antiepileptic
effects. The 1,5-benzodiazepine clobazam shares the anxiolytic and antiepileptic actions, with less
adverse effects. Non benzodiazepine drugs such as zolpidem share some but not all the
pharmacological actions. In fact, the hypnotic zolpidem, like zaleplon and indiplon, interacts
preferentially with the GABA-A receptors containing, in addition to the β2/3 γ2, the α1 subunit.3
This receptor subtype mediates the sedative hypnotic action of classical benzodiazepines. Receptors
containing α2 or α3 subunit are responsible for the anxiolytic effect.3 Classical 1,4-
benzodiazepines are non selective for the α subunit ( a part from the absence of interaction with
receptors containing the α6 one) and this explains the presence of a cohort of adverse side effects,
when they are used as anxiolytics or antiepileptics.3
Zopiclone and its active dextrorotatory stereoisomer eszopiclone, belongs to the class of drugs known
as cyclopyrrolones, sedative and anxiolytic drugs widely used in the treatment of insomnia.
They belong to the group of the nonbenzodiazepine drugs which however act as positive allosteric
modulators at the benzodiazepine site or a site closely associated with it, within GABA-A
receptors.7-10 Upon binding to the benzodiazepine site, Zopiclone is believed to allosterically
3

modulate the activity of GABA-A receptors by increasing transmembrane conductance of chloride
ions,10,11 thus stabilizing neuronal membrane potentials and dampening excitatory input.
Much of the interest about Zopiclone is due to the fact that, at difference from classical 1,4-
benzodiazepine, it has an anxiolytic and hypnotic effect without negative side effects such as
addiction12,13. Also in this case, GABA-A receptor subtype selectivity is thought to underlie such
characteristics. In general, studies in this respect only partially confirm this concept.10,11
However, the study by Petroski et al.14 indicates a greater potency of Zopiclone at α1β2γ2 and
α5β2γ2 in the respect of α2β2γ2 and α3β3γ2 receptor subtypes. Moreover, in contrast to
Zopiclone, pagoclone15 and pazinaclone produce anxiolytic effects with little or no sedative or
amnestic actions at low doses. This is because these compounds are subtype-selective drugs which
bind primarily to the α2/α3 subtypes of the GABA-A receptor which are responsible for the anti-
anxiety effects of drugs of this kind, but have relatively little efficacy at the α1 subtype which
produces the sedative and memory loss effects.3
In the present article, we describe the synthesis and the pharmacological evaluation of a focused
library with isoindolinone backbone and the evaluation of their effects on GABA-activated chloride
currents by use of the whole-cell patch clamp technique activity in rat cerebellar granule cells, a
useful model for studying new benzodiazepine site ligands.16
In these cells in culture we previously evaluated the following distribution of GABA-A receptors
expressed on the plasma membrane and contributing to the rapidly desensitizing (peak) chloride
current: 25 % α6 β2/3 incomplete receptors, 45 % α1 β2/3 γ2, and 30 % α6 β2/3 γ2 + α1 α6 β2/3
γ2 subtypes.17 Tandem reactions of 2-formyl benzonitriles 4 (2-cyano benzaldehydes) are among
the most powerful strategies for the synthesis of 3-substituted isoindolinones.18 Taking advantage
from these very convenient and operational simple one-pot methodologies, we envisioned effective
routes to the designed derivatives Ia-e and IIa-e object of the present investigation (Scheme 1). In
an early approach, the N-methyl piperazine scaffold was introduced reacting the mono-
malonoamide derivative 3 with 4, followed by Krapcho decarboxylation (pathway A).19 Then, Cu(I)
4

NH-arylation of the resulting lactam20 6 with 5-substituted 2-iodopyridines led to the desired
products Ia-e. Hydrogenation of nitro group in Ie gave in almost quantitative yield compound If,
with amino group on the pyridine ring. However, the described approach allows a linear synthesis
of the target compounds, not particularly convenient to build up a diversified library to effectively
tackle the major challenges in medicinal chemistry. For these reasons, recently, we focused on the
obtaining of a common key intermediate to be employed in a divergent approach to diverse
functionalized isoindolinones (pathway B). In this respect, tandem reaction of dimethyl malonate
with 2-cyanobenzaldehyde21 and the following HCl 6M decarboxylation furnished the isoindolinone
acetic acid 8 in very high yields, a synthetic sequence also applicable for large scale synthesis. This
compound was, then, subjected to the amide coupling with different 4-substituted arylpiperazines
9a-e, in the presence of EDC.HCl (N-(3-dimethylaminopropyl)-N-ethylcarbodiimidehydrochloride)
and HOBT (1-hydroxybenzotriazole hydrate).22 The obtained products 10a-e were arylated in an
improved version of the Cu(I) NH arylation, giving the target compounds in very good overall
yields.
5

pathway A
O
O
+
HN
N
O
O
1
2
pathway B
a
O
O
O
O
N
N
O
O
+
O
1
CHO
3
CHO
CN
b
CN
b
4
4
O
O
NH
NH
O
O
O
7
O
O
O
N
O
N
d
H
N
5
c
O
N
O
O
NH
N
O
9a-e
f
X
N
O
NH
R
O
e
OH
8
N
N
N
N
R
R
Ia-e
6 R= CH3
10a-e
IIa-e
X
R
Compound
Ia
Ib
H
CH3
CH3
X = NO2,
R= CH3
g
Br
Ic
Id
Ie
CH3
NO2
Cl
CH3
CH3
CH3
O
N
NH2
N
O
IIa
IIb
NO2
NO2
Ph
4Cl-Ph
N
IIc
IId
IIe
NO2
NO2
NO2
4CH3-Ph
N
4OCH3-Ph
4F-Ph
If
Scheme 1
Reagents and conditions. a) Toluene, reflux 48h; b) K2CO3, CH3CN, r.t; c) LiCl, DMF/H2O, MW; d) HCl 6M, 100 °C
1h; e) EDC.HCl, 5-HOBt, 4-substituted phenylpiperazines, Et3N, THF, r.t, 16h.; f) 5-substituted 2-iodopiridines, CuI,
K3PO4, N,N1 dimethyl-ethylen-diamine, dry toluene, 100°C (for Ia-e) or dry dioxane 80 °C (for IIa-e) , 24h; g) H2,
Pd/C 5%, MeOH, 24h.
6

We synthesized isoindolin-1-one derivatives in racemic form, different substituents, chloro,
bromine, methyl, and nitro groups to the p-position in pyridine core were introduced, and in order to
increase the water solubility of the compounds as hydrochloride salts, we introduced a substituent at
the 3-position in the form of a 2-(4-methyl-1-piperazinyl)-2-oxoethyl group also present in the lead
compound. A part from compounds If and IIc which show limited solubility in DMSO, all the
compounds were fully soluble in this solvent, slightly soluble in ethanol and insoluble in water.
Solubility in phosphate buffer (pH 3.2) was very high due to salt formation by protonation of
piperazine ring.
First of all, the effect of the lead compound Zopiclone on the peak chloride current elicited by 10
µM GABA (a GABA concentration close to the EC50 for the peak current activation in this model)
23 was determined at the concentration of 1 µM. This concentration is already in the saturation range
of zopiclone positive allosteric effect on recombinant α1 β3 γ2 receptor subtype11 and in our model
the α1 β2/3 γ2 subtype is the only one bearing a benzodiazepine site.16An augmentation of the peak
current by 58 ± 7 % resulted (Table I). In these experiments all the zopiclone analogue tested were
at 1 µM concentration, as an index of their efficacy (only If and IIc were at respectively 0.47 and
0.58 µM, as the maximal concentrations possible due to limited solubility in the media used). As
shown in Table I the structural simplification of the lead demonstrated a decrease in activity (Ia vs
Zopiclone: pz <0.01) and the introduction of electron-releasing and electron-withdrawing groups in
p-position (Ib and Ie) demonstrated no potentiation of the current in the respect of Ia.
Besides, compound Ic exerted an increment in efficacy versus Ie (p <0.05) and furthermore, the 4-
nitro analog Id demonstrated (Fig. 2) maximum potentiation 34% above control.
Interestingly, reduction of NO2 group led to an inverse agonist If (Table I).
Modification of the terminal methyl group on piperazine substituent of Id into a bulky substituent
was next examined. (Table II).
7

Compounds
X
E%
Statistical significance
(Student’s t-test)
O
N
N
N
N
Cl
O
O
N
N
pa <0.01 pb<0.01
58 % ± 7
Zopiclone
O
N
N
X
O
N
N
H
20% ± 3
pa <0.01 pb<0.01 pz <0.01
Ia
CH3
Br
21% ± 3
26 % ± 3
34% ± 4
18% ± 2
-23% ± 2
pa <0.01 pb<0.05
pa <0.01 n.s.b
Ib
Ic
Id
Ie
If
pa <0.01
NO2
Cl
pa <0.01 pb<0.01
NH2
pa <0.01
Table I
8

Compounds
R
X
E%
Statistical significance
(Student’s t-test)
O
N
N
X
O
N
N
R
H
NO2
NO2
NO2
NO2
NO2
-27% ± 2
-36% ± 4
pa <0.01
pa <0.01
IIa
IIb
IIc
IId
IIe
4-chloro
4-methyl
4-methoxy
4-fluoro
n.sa
pa<0.01
-21% ± 6
n.sa
Table II
9

Here, it is interesting to underline that in compound IIa the replacement of methyl with phenyl in
the piperazine group caused a transition from agonistic to inverse agonistic activity. This change is
displayed by compounds IIb (Fig.3) and IId as well and indicates that most probably the 
interaction with receptor protein is highly required to display an inverse agonist profile. As shown
in Fig.’s 1-3, both the agonistic (first series of compounds) and the inverse agonist activities
(second series of compounds) were reversed by the Bzd site antagonist flumazenil. These reversions
were repeated on three different experiments. Zopiclone is a non-benzodiazepine hypnotic agent
able to bind at or near benzodiazepine sites on GABA-A macromolecular receptor complexes8,9. It
is part of a relatively new generation of drugs able to produce comparable anxiolytic effects with
less sedation, muscle relaxation, or addictive potential24,25
.
Fig.1
10

Fig.2
Fig.3
11

Racemic Zopiclone and its more active stereoisomer S-Zopiclone augment chloride currents due to
α1β2γ2, α2β2γ2 and α3β3γ2 GABA-A receptor subtypes10. According to Petrovski et al. (2006)14,
its maximal potency is versus α1β2γ2 and α5β2γ2 receptor subtypes. This might explain its
specificity as an hypnotic, since α1βxγ2 receptor subtypes are those mediating sedation by Bzd
agonists, whereas anxiolysis involves receptors containing α2 or α3 subunits4,26
.
Along this line, it seems interesting to find new drugs which may have an even greater specificity in
terms of interaction with GABA-A receptor subtypes and with improvement in metabolic stability.
The metabolism of Zopiclone, in fact is rapid, complex, and differs among species; of the more than
ten metabolites of the compound that have been identified, however, only two reportedly exhibit
pharmacological activity in humans: N-desmethyl zopiclone and zopiclone-N-oxide. It is interesting
to note that approximately 50% of the administered dose is decarboxylated to inactive metabolites.
Moreover, Zopiclone has also an affinity for acetylcholine muscarinic receptors27, consequently,
administration results in adverse effects such as, but are not limited to, drymouth, thirst, slowing
and acceleration of the heart, dilated pupils, blurred vision, restlessness, fatigue, headache,
hallucinations and delirium.
In our research interested in synaptic transmission in the central nervous system, and particularly
amino acid neurotransmission 28-30, we synthesized and tested new compounds with isoindolinone
skeleton and with 2-oxoethyl group instead of ester group. It is interesting to note that the major
metabolic routes, affecting more than 50% of dose of the parent compound Zopiclone, involve
the transformation to the inactive decarboxylated metabolite, taking in mind that, in general,
carbamate is more susceptible to hydrolysis than amides31, we replaced it to the relatively
metabolically stable amidic group.
In the present study and model, the two series of zopiclone related new compounds were tested for
their effects on the GABA activated rapidly desensitizing (peak) current component. In previous
studies it was determined that this component is due to GABA-A receptors of the α6 β2/3
incomplete receptors, α1 β2/3 γ2, and α6 β2/3 γ2 + α1 α6 β2/3 γ2 subtypes17. Since the effects (
12

both positive and negative allosteric modulation) by the new compounds tested are reversed by the
benzodiazepine antagonist flumazenil, only the α1β2/3γ2 GABA-A receptor subtype is involved. In
fact, the α6 containing receptors are not affected by benzodiazepines.32,33
Within the first group of compounds Ia-e, the most active was found to be Id, with a very
interesting 34% increment of chloride peak current activated by 10 µM GABA.
Thus, since in our model the α1β2/3γ2 receptor subtype represents 45 % of the total of the GABA-
A receptors generating the peak current, the actual effect on the affected receptors can be estimated
as 2.20 times the one evaluated on the basis of the difference in the peaks with versus without the
drugs studied. This is exactly what we experimentally found for diazepam and clobazam erasing by
furosemide the contribution of α6 containing receptors to the peak current.34
This enhancement of chloride current was antagonized by flumazenil, thus confirms the
involvement of the benzodiazepine site. The lower activity vs Zopiclone is probably due to the
absence in Id of one of the two nitrogens present in the Zopiclone pyrazine group that acts as an
electron donor for the H1 hydrogen bond within the receptor7,9. The absence of the N atom in
compound Id may decrease the binding to the receptor and the pharmacological effect.
It is remarkable that passing from Id to If the only change is a -NH2 group instead of -NO2,
however the pharmacological activity changes completely from agonist (+ 34 %) to inverse agonist
(-23 %).
Referring to the second group of compounds IIa-e, they share a nitro group on the pyridine ring
with bulky groups at the N in position 4 of the piperazine ring and behave as inverse agonists. An
explanation of the drastic change of activity may be found, as described by Borea et al. (1987)7, in
the interaction between agonists/antagonists/inverse agonists and the Bzd site.
According to this, there are two lipophilic interaction points whose engagement determines the
positive allosteric (agonistic) activity: AG1 and AG2. Within this scheme, Zopiclone interacts with
AG1 with the 3-Cl-piridine ring and with AG2 with the piperazine ring, this results in an agonistic
activity. The introduction of bulky groups on the nitrogen in position 4 in the piperazine ring may
13

push piperazine and its substituent out of the AG2 interaction point and laterally towards a
different interaction point, which Borea et al.7 defined as IAG, critical for the inverse agonist
activity like that of β-carboline structure derivatives.
This possibility can be further tested by studying the effects of different substituents on the
piperazine ring, As an example, introducing a cyclohexane would permit to investigate whether a
bulky but more flexible and non aromatic structure maintains or not the transition to negative
allosteric modulation in the resulting molecule.
In conclusion, a novel series of agonists at the Bzd binding site of the GABA-A receptor based on
modifications of a known template were described. The optimization work surprisingly yielded both
agonists and inverse agonists depending on the substitution patterns.
Currently, further work is ongoing to evaluate compound Id in several different in vivo models with
the aim of obtaining a safer pharmacokinetic profile.
Acknowledgements
This work was supported by the grant PON 03 PE_00060_07 (MIUR) Sviluppo pre-clinico di
nuove terapie e di strategie innovative per la produzione di molecole ad azione farmacologica.
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16

Legends to Schemes, Tables and Figures
Scheme 1: Pathway of synthesis of compounds Ia-f and IIa-e
Table I: Structures of the first series of compounds, Ia-f and the lead compound Zopiclone and
percent stimulations of the chloride current elicited by 10 μM GABA. The data are reported as
mean E % (see main text) ± SEM. Student’s t-test. pa: compound plus GABA vs GABA alone; pb:
compound vs Id; n.s.b: not significant, compound vs Id; pz: compound vs Zopiclone. The compounds were
tested at 1 M, except If, which for solubility limit reason was tested at 0.47 M. The numbers of samples
were: Zopiclone (N=5); Ia (N=7); Ib (N=6); Ic (N=6) Id (N=8); Ie (N=8); If (N=15).
Table II: Structures of the second series of compounds, IIa-e and percent changes of the chloride
current elicited by 10 μM GABA. The data are reported as mean E % (see main text) ± SEM.
Student’s t-test. pa: Compound plus GABA vs GABA alone; n.s.a: not significant, compound plus GABA vs
GABA alone. The compounds were tested at 1 M, except IIc which for solubility limit reason was tested at
0.58 M. The numbers of samples were: IIa (N=19); IIb (N=14); IIc (N=19); IId (N=6); IIe (N=24).
Figure 1: Chloride currents evoked by (from left to right): 10 μM GABA alone, Zopiclone 1 μM
plus 10 μM GABA, 10 μM plain flumazenil and Zopiclone 1 μM plus 10 μM GABA in the
presence of 10 μM flumazenil.
Figure 2: Chloride currents evoked by (from left to right): 10 μM GABA alone, Id 1 μM plus 10
μM GABA, 10 μM plain flumazenil and Id 1 μM plus 10 μM GABA in the presence of 10 μM
flumazenil.
Figure 3: Chloride currents evoked by (from left to right): 10 μM GABA alone, IIb 1 μM plus 10
μM GABA, 10 μM plain flumazenil and IIb 1 μM plus 10 μM GABA in the presence of 10 μM
flumazenil.
17

Graphical Abstract
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Synthesis and Pharmacological Evaluation
of Functionalized Isoindolinones
on GABA-Activated Chloride Currents
in Rat Cerebellum Granule Cells in Culture
Antonia Di Molaa#, Elena Gattab#, Carmen Petronzi d,c, Aroldo Cupellob,
Paolo De Caprariisa, Mauro Robellob, Antonio Massa d*, Rosanna Filosaa,c*
O
O
N
O
N
O
NO2
N
NO2
N
N
N
N
N
Id
IIb
Cl
18