Novel synthesis of isoindoline/isoindoline-1,3-dione derivatives under solventless conditions and evaluation with the human D2 receptor

Article abstract of DOI:10.1007/s00044-017-1942-6

Isoindolines are the focus of much research because of being an important family of compounds present in a wide array of bioactive molecules. Although many different pathways of synthesis have been described, they do not follow green chemistry principles. The aim of this contribution was to develop a green synthesis technique for isoindolines/dioxoisoindolines. These compounds derived from analogs of important biogenic amines were tested in silico (on the human dopamine receptor D₂) to predict their affinities and some pharmacokinetic parameters. One of them, YaI-01, was evaluated in vivo in a Parkinsonism mouse model. Seven molecules, including three isoindolines and four dioxoisoindolines, were synthesized using simple heating and relatively quick solventless reactions. They were then purified with a methodology as green as possible. Since no published crystal structure exists for dopamine receptor D₂, it was necessary to generate and validate a homology model for molecular docking studies of the seven molecules synthesized presently. Docking was performed to assess affinity in terms of binding energy (?G). Apart from pharmacokinetic parameters, Lipinski’s rule of five and some properties of toxicity from QSAR models were evaluated. Whereas no risk was found for the isoindolines, there was evidence of three kinds of toxicity for the isoindoline-1,3-dione compounds. Overall, the in silico analysis suggests that the three isoindolines herein tested have the best properties as ligands of the dopamine receptor D₂, interacting with the main amino acid residues at its allosteric binding site. YaI-01 (254 μmol/kg) reverted Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Full text of DOI:10.1007/s00044-017-1942-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1942-6
ORIGINAL RESEARCH
Novel synthesis of isoindoline/isoindoline-1,3-dione derivatives
under solventless conditions and evaluation with the human D2
receptor
Erik Andrade-Jorge1 José R. Bahena-Herrera1 Jesus Garcia-Gamez1
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Itzia I. Padilla-Martínez2 José G. Trujillo-Ferrara
1
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Received: 7 March 2017 / Accepted: 2 June 2017
© Springer Science+Business Media, LLC 2017
Abstract Isoindolines are the focus of much research
because of being an important family of compounds present
in a wide array of bioactive molecules. Although many
different pathways of synthesis have been described, they
do not follow green chemistry principles. The aim of this
contribution was to develop a green synthesis technique for
isoindolines/dioxoisoindolines. These compounds derived
from analogs of important biogenic amines were tested in
silico (on the human dopamine receptor D2) to predict their
affinities and some pharmacokinetic parameters. One of
them, YaI-01, was evaluated in vivo in a Parkinsonism
mouse model. Seven molecules, including three iso-
indolines and four dioxoisoindolines, were synthesized
using simple heating and relatively quick solventless reac-
tions. They were then purified with a methodology as green
as possible. Since no published crystal structure exists for
dopamine receptor D2, it was necessary to generate and
validate a homology model for molecular docking studies of
the seven molecules synthesized presently. Docking was
performed to assess affinity in terms of binding energy
(ΔG). Apart from pharmacokinetic parameters, Lipinski’s
rule of five and some properties of toxicity from QSAR
models were evaluated. Whereas no risk was found for the
isoindolines, there was evidence of three kinds of toxicity
for the isoindoline-1,3-dione compounds. Overall, the in
silico analysis suggests that the three isoindolines herein
tested have the best properties as ligands of the dopamine
receptor D2, interacting with the main amino acid residues
at its allosteric binding site. YaI-01 (254 µmol/kg) reverted
Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine.
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Keywords Solventless reactions Dopamine receptor
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Pharmacokinetic parameters 2,3-dihydro-1H-isoindoles
N-substituted phthalimide Docking
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Introduction
Substituted isoindolines are important heterocyclic rings
present in many synthetic compounds, natural products, and
bioactive small molecules, which together display a wide
array of biological activity. They have also proven to be
important intermediates for the synthesis of new drugs with
different applications.(Mancilla et al. 2001; Zang et al.
2012; Lu et al. 2014)
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1942-6) contains supplementary material,
which is available to authorized users.
* José G. Trujillo-Ferrara
jtrujillo@ipn.mx
1
Departamento de Bioquímica y Sección de Graduados, Escuela
Superior de Medicina del Instituto Politécnico Nacional, Plan de
San Luis y Salvador Díaz Mirón s/n, Casco de Santo Tomás,
Mexico City 11340, Mexico
In particular, isoindolines and isoindoline-1,3-dione
modulate the dopamine receptor D3, which suggests a
potential application as antipsychotic agents. In addition,
the inhibition of β-amyloid protein aggregation indicates a
potential capacity in the treatment of Alzheimer’s disease.
Indeed, a wide range of other properties has been
reported for isoindolines, including selective antagonism of
2
Laboratorio de Investigación en Química, Departamento de
Ciencias Básicas, Unidad Profesional Interdisciplinaria de
Biotecnología del Instituto Politécnico Nacional, Av. Acueducto
s/n Barrio la laguna Ticoman, México D.F., Mexico City 07340,
Mexico

Med Chem Res
the ETA receptor (perhaps making them advantageous for a
large number of diseases related to the heart and lungs), a
high affinity for the melanocortin subtype-4 receptor
implicated in body weight regulation (Zang et al. 2012), and
potent and selective inhibition of dipeptidyl peptidase IV,
resulting in in vivo efficacy for antihyperglycemic activity
(Kato et al. 2011). Moreover, N-substituted-isoindoline-1,3-
dione derivatives have shown good anticonvulsant activity
compared to sodium valproate. In fact, one such derivative
is 20 times more active than valproate (Abdei-hafez 2004).
Other isoindoline-1,3-dione derivatives evaluated as acet-
ylcholinesterase inhibitors have demonstrated anti-
Alzheimer effects (Mohammadi-farani et al. 2013; Alia-
badi et al. 2013).
On the other hand, new compounds that incorporate
amino-acetylenic and isoindoline-1,3-dione moieties in their
structure produce anti-inflammatory and analgesic effects
without inducing gastric lesions in the stomach of experi-
mental animals (Shakir et al. 2012; Al-Qaisi et al. 2014).
Some isoindolines have been tested as inhibitors of inte-
grase, which is a key enzyme in the treatment of the human
immunodeficiency virus type 1 (Zhao et al. 2009). These
compounds also possess good antimicrobial activity (Prasad
et al. 2010). They have been evaluated with molecular
docking studies not only as possible inhibitors of lipox-
ygenase (LOX) but also COX-1/COX-2 (Mancilla et al.
2007; Percino et al. 2009). Furthermore, 1-oxo-isoindoline
derivatives are β-adrenergic blocking agents with affinity
for β1 adrenergic receptors. Such affinity is comparable to
that reported for atenolol, but with higher cardioselectivity
(Jindal et al. 2005). Indoprofen is an isoindoline that inhi-
bits both acute and sub-chronic pain, its effect being related
to the inhibition of prostaglandins (Buttinoni and Tomma-
sini 1981). Isoindolines have also been administered for
treating coronary vessel disorders and tested as adrenergic
neuron blocking agents (Mancilla et al. 2001) as well as
L-type Ca+2 channel blockers, the latter carried out through
in silico studies (Mancilla-Percino et al. 2010).
2011; Platania et al. 2012). The alternative splicing of the
D2 receptor mRNA leads to the generation of two isoforms,
the short isoform (D2S) having 414 amino acids and the
long isoform (D2L) 443 amino acids. Both of these are
coupled to a Gαi or Gαo protein (Dal Toso et al. 1989;
Bahena-Trujillo et al. 2000; Platania et al. 2012).
Whereas the D2 receptor has low affinity for dopamine
(Kd 2 × 10−6 M), which is its endogenous ligand, it shows a
high affinity for bromocriptine (agonist) (Kd 5 × 10−9 M).
Although no selective antagonists are known, raclopride
displays selectivity for D2 and D3 receptors, as evidenced
by its Kd values of 0.5 and 5 × 10−9 M, respectively
(Bahena-Trujillo et al. 2000).
Dopamine receptor D2 (D2R) activation triggers the
inhibition of adenylyl cyclase and hence cAMP formation.
D2 receptors may also modulate ion currents, particularly
those activated by voltage, and thus inhibit Ca2+ (an effect
presumably mediated by the Gαo proteins) or facilitate the
opening of K+ channels by Gi3 proteins. Finally, it has also
been reported in cell lines transfected with the D2 receptor
that the stimulation of the same leads to the formation of
IP3 and the mobilization of calcium from intracellular stores
(Bahena-Trujillo et al. 2000; Vangveravong et al. 2006;
Luedtke et al. 2012).
Different disorders affect the dopaminergic system, one
of these being Parkinson’s disease (PD), which is a common
neurodegenerative disease with an etiology that is not
clearly understood. It is likely that PD, first described by
James Parkinson in 1817 (Kitamura et al. 2000; Meredith
et al. 2008a), results from an interaction between genetic
predisposition and the environment. This disease is char-
acterized by the loss of midbrain dopamine neurons of the
nigrostriatal pathway, which results in distinct combinations
of motor symptoms. The clinical features of PD consist of
resting tremor, bradykinesia, rigidity, and postural
instability. In addition, PD includes non-motor disorders,
such as hyposmia, autonomic dysfunction, sleep disorders,
cognitive impairment, and depression. On the other hand,
the main clinical feature in Essential tremor, another neu-
rodegenerative disorder, consists of action (postural and
kinetic) tremor (Meredith et al. 2008b; Kumar et al. 2009;
Wider et al. 2010).
The properties of any given isoindoline depend on the
different substituents. In the current contribution, arylethy-
lamines were used as the R group. These compounds are
structurally related to catecholamines, such as dopamine
(one of the most important neurotransmitters in the human
brain). Dopamine receptors constitute an important group of
G protein-coupled receptors (GPCRs). This large family of
seven transmembrane proteins is the subject of a great deal
of pharmaceutical research and constitutes the main target
of almost 30% of all approved drugs (Sukalovic et al. 2013).
It is well known that dopamine exerts its action through five
different GPCRs (receptors D1–5), grouped into D1-like (D1
and D5) and D2-like (D2, D3, and D4) receptors. These two
categories differ in their signal transductions, binding pro-
file and physiological effects (Caine et al. 2002; Chien et al.
L-DOPA (Levodopa) remains the gold standard for the
treatment of motor symptoms of PD even though it com-
monly leads to the progressive development of involuntary
movements, known as L-DOPA-induced dyskinesia.
Additionally, dopamine can produce reactive oxygen spe-
cies and diketone compounds that cause oxidative stress for
the remaining dopaminergic neurons (Kumar et al. 2009;
Guatteo et al. 2013). These are the main reasons that it is
crucial to develop new compounds having a high affinity
for dopamine and fewer side effects.

Med Chem Res
To facilitate the development of these compounds, in
silico studies can be used to select the best candidates for
in vivo testing. Unfortunately, there are no experimental
structures available in the protein data bank (PDB) for the D2
receptor, nor for most other GPCRs. Consequently, we
herein present molecular modeling of the D2 receptor. Iso-
indolines/dioxoisoindolines are derived from important bio-
genic amines, such as phenylethyl amine and dopamine, the
latter being the endogenous ligand of the dopamine receptor.
In the current contribution, therefore, arylethylamines sub-
stituted with an isoindoline or the isoindoline-1,3-dione were
obtained by green synthesis, and through molecular docking
we predicted the affinity of the synthesized compounds for
the D2 receptor. Finally, we examined the effects of one of
these compounds on 1-methyl-4-phenyl-1,2,3,6-tetra-
hydropyridine (MPTP)-induced Parkinsonism.
performed in an oven-dried flask, while the mixtures were
agitated with a stirring bar and concentrated by means of a
standard rotary evaporator. Melting points were measured by
a Stuart® SMP40 automatic melting point apparatus and are
uncorrected. Infrared spectra (IR) were obtained on a 100
FT-IR spectrometer (Perkin-Elmer) with an universal
attenuated total reflectance (ATR) accessory. Thin layer
chromatography (TLC) was performed on 0.25-mm thick
silica gel 60 F254 plates (Merck, Darmstadt, Germany) and
1
spots were detected under UV light. H and 13C nuclear
magnetic resonance (NMR) spectra were recorded on a
Varian Mercury 300 (1H, 300 MHz; 13C, 75 MHz) spectro-
meter using tetramethylsilane as an internal reference. The
data are reported as follows: chemical shift in ppm (δ),
integration area, multiplicity (s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet), and coupling constant
(Hz). Electrospray ionization high-resolution mass spectro-
It is highly desirable to develop a methodology for the
synthesis of isoindolines using green chemistry principles.
For many decades, chemists have been synthesizing iso-
indoline derivatives with a variety of techniques, such as
alkylation of isoindolines, alkylation and reduction of
phthalimides, and cyclocondensation of α,α′-dibromo-o-
xylene with primary amines (Mancilla et al. 2001). Other
approaches include the borane–tetrahydrofuran reduction of
phthalimide, the reduction of phthalaldehyde by tetra-
carbonylhydridoferrate, the multistep metalationalkylation
of formamidine, catalytic N-heterocyclization by means of a
Cp*Ir complex, co-cyclization of nitrogen-containing acet-
ylenes –NiCl2(PPh3)2, pictet-Spenger cyclization, nickel-
catalyzed cycloaddition (Subbarayappa and Patoliya 2009),
detosylation of N-tosylindoline, the heck-aza-Michael
strategy (Zang et al. 2012), microwave-assisted condensa-
tion of phthalic anhydride with primary amines, continuous-
flow organic synthesis (Ullah et al. 2012), and solid phase
synthesis. These constitute most of the methods described
by different researchers, each with its advantages and dis-
advantages. Unfortunately, many of these methods employ
heavy metals or solvents unfriendly to the environment.
Furthermore, some methods require expensive equipment
(e.g., a microwave-producing apparatus). In order to
develop a green synthesis technique, we proposed a direct
synthesis of 2,3-dihydro-1H-isoindoles and N-substituted
phthalimide in a single step involving short reaction times,
without utilizing any phase transfer, catalysts or solvent
during the reaction.
metry was performed on
instrument.
a Bruker micrOTOf-Q-II
Homology modeling
The modeling of D2R was carried out on the online server I-
TASSER (Roy et al. 2010). The protein sequence of human
D2R (hD2R) was taken from UniProt (The UniProt Con-
sortium 2015) isoform 1 D2 (long, 443 A.A.), identifier:
P14416-1. The model was built by using the dopamine
receptor D3 as a template. The X-ray crystal structure of this
receptor was taken from the PDB (code 3PBL) with a
resolution of 2.89 Å (Chien et al. 2011). The best hD2R
model was selected based on the RMSD and the quality of
the Ramachandran Plot D (Lovell et al. 2003), which dis-
plays the φ and ψ torsion angles for all protein residues. The
model was validated, as done previously (Soriano-Ursúa
et al. 2011), by correlating theoretical and experimental
data, yielding a value of r2 ≥ 0.80 (details not shown).
Docking
All the ligands were modeled with the GaussView
5.0.9 software. As physiological conditions were assumed,
the amino groups of isoindoline were protonated if required
(considering a pH of 7.4). Conformational analysis of the
test compounds was performed by the semi-empirical
molecular orbital calculations method (PM3) with Gaus-
sian 09 software (Frisch et al. 2016).
AutoDock tools 1.5.4 (Morris et al. 2009) and Raccoon
(Forli et al. 2016) were used for docking studies. Before
starting docking simulations, all rotating bonds, torsional
degrees of freedom, atomic partial charges, and non-polar
hydrogens of the ligands were assigned. All rigid docking
simulations were carried out by using the hybrid
Lamarckian genetic algorithm (Morris et al. 1998),
Materials and methods
Synthesis and characterization
All reagents and solvents were used as received from the
commercial supplier Sigma-Aldrich. All reactions were

Med Chem Res
employing an initial population of 100 randomly placed
individuals, adjusting Kollmann partial charges for all
protein atoms, and calculating Gasteiger charges for the
ligands. The grid parameter file was set based on the
orthosteric site reported for D2R at the transmembranal
loops, utilizing a 70 × 70 × 70 Å grid box. The conforma-
tions obtained with an RMSQ of 0.5 A were clustered
together and all calculations were done in Linux-Fedora 21
as the operative system on AutoDock4 software. For each
compound, the lowest energy cluster returned by Auto-
Dock4 was employed for analyzing Gibbs free energy (ΔG),
the dissociation constant (Kd) and the −log(10) dissociation
constant (pKd). Molecules were displayed in the Visual
Molecular Dynamics program (VMD v.1.8.6) (Humphrey
et al. 1996) to describe the principal interactions with hD2R.
(ATR, cm−1) ύ: 3031 (C–H, aromatic), 2908 (C–H, ali-
phatic), 2546 (NH+), 1705 (C=O), 1600 (C=C), 1427
(CH2), 1391 (C–N). MS (m/z): 224.1447 (M + 1).
2-(2-(3,4-dimethoxyphenyl)etyl)-1,3-dihydroisoindoline
hydrobromide (YaI-02): it was obtained as a white solid,
1
80% yield; mp 197–198 °C; H NMR
(DMSO-D6, 300 MHz) δ (PPM) 3.00 (t, H-11), 3.64 (t, H-
10), 3.71 (s, H-18), 3.74 (s, H-19), 4.71 (s, H-1,3), 6.95 (m,
H-13,16,17), 7.40 (m, H-4,5,6,7); 13C NMR (CDCl3, 75
MHz) δ 58.5 (C-1,3), 129.1 (C-4,7), 123.5 (C-5,6), 134.9
(C-8,9), 55.7 (C-10), 31.8 (C-11), 129.8 (C-12), 112.6 (C-
13), 148.4 (C-14), 149.4 (C-15), 113.2 (C-16), 121.3 (C-
17), 56.2 (C-18,19). IR (ATR, cm−1) ύ: 3063 (C–H, aro-
matic), 2990 (C–H, aliphatic), 2842 (O–CH3, aliphatic),
2547 (NH+), 1600 (C=C), 1462 (CH2), 1453 (CH3), 1390
(C–N), 1228 (O–CH3). MS (m/z): 284.1624 (M + 1).
General procedure for the synthesis of 2,3-dihydro-1H-
isoindoles (YaI-01 is a representative)
An amount of 433.80 mg (1.6435 mmol) of α,α′-dibromo-o-
xylene and 199.16 mg (1.6435 mmol) phenylethylamine
were placed into a 50-mL round-bottom flask. The mixture
was stirred and heated to gentle boil 150–250 °C for 5–10
min. The reaction was cooled at room temperature and
monitored by TLC. Afterwards, ethyl acetate (40 mL) was
added and the reaction was sonicated until a white powder
(hydrobromide) formed. The precipitate was filtered and
washed twice with acetone.
(S)-2-(1-phenyl)etyl-1,3-dihydroisoindoline hydrobromide
(YaI-03): it was obtained as a white solid, 97% yield; mp
210–211 °C; H NMR
1
(DMSO-D6, 300 MHz) δ (PPM) 1.90 (d, H-11), 4.28 (dd,
H-1,3), 4.6 (q, H-10), 7.15–7.85 (m, H-4,5,6,7,13,14,15,
16,17); 13C NMR (CDCl3, 75 MHz) δ 58.1 (C-1,3), 123.1
(C-4,7), 128.7 (C-5,6), 133.2 (C-8,9), 67.8 (C-10), 19.6 (C-
11), 136.1 (C-12), 129.8 (C-13,17), 130.0 (C-14,16), 129.2
(C-15). MS (m/z): 224.1359 (M + 1).
General procedure for the synthesis of N-substituted
phthalimide (JaD-01 is representative)
2-(2-phenyletyl) isoindolin-1,3-dione (JaD-01): it was
obtained as a white solid, 85% yield; mp 130–131 °C;
1H NMR
An amount of 327.43 mg (1.1 mmol) of phthalic anhydride
and 243.56 mg (1 mmol) of phenylethylamine were placed
into a 50-mL round-bottom flask. The mixture was stirred
and heated to gentle boil 100–150 °C for 5–10 min. The
reaction was cooled at room temperature and monitored by
TLC. Then the appropriate solvent (40 mL) was added and
the reaction was sonicated until a white powder formed. The
precipitate was filtered, dissolved in acetone and pre-
cipitated again with water (pH 13).
(CDCl3, 300 MHz) δ (PPM) 2.98 (t, H-11), 3.92 (t, H-10),
7.26 (m, H-13,14,15,16,17), 7.70 (m, H-5,6), 7.82 (m, H-
4,7); 13C NMR (CDCl3, 75 MHz) δ 168.1 (C-1,3), 123.2
(C-4,7), 133.9 (C-5,6), 132.0 (C-8,9), 39.2 (C-10), 34.6 (C-
11), 137.9 (C-12), 128.5 (C-13,17), 128.8 (C-14,16), 126.6
(C-15). IR (ATR, cm−1) ύ: 3031 (C–H, aromatic), 2935
(C–H, aliphatic), 1705 (C=O), 1600 (C=C), 1427 (CH2),
1391 (C–N). MS (m/z): 274.0752 (M + Na).
Chemical characterization
2-(2-(3,4-dimethoxyphenyl)etyl) isoindolin-1,3-dione (JaD-
02): it was obtained as a light yellow solid, 90% yield; mp
171–172 °C; H NMR
2-(2-phenyletyl)-1,3-dihydroisoindoline hydrobromide
(YaI-01): it was obtained as a white solid, 90% yield; mp
165-166 °C; H NMR
1
1
(CDCl3, 300 MHz) δ (PPM) 2.93 (t, H-11), 3.90 (t, H-10),
3.80 (s, H-19), 3.83 (s, H-18), 6.78 (m, H-13,16,17), 7.70
(m, H-5,6), 7.82 (m, H-4,7); 13C NMR (CDCl3, 75 MHz) δ
168.2 (C-1,3), 123.19 (C-4,7), 132.0 (C-5,6), 130.4 (C-8,9),
39.3 (C-10), 34.0 (C-11), 133.9 (C-12), 111.1 (C-13), 148.7
(C-14), 147.6 (C-15), 111.8 (C-16), 120.8 (C-17), 55.7 (C-
(DMSO-D6, 300 MHz) δ (PPM) 2.85 (t, H-11), 3.65 (t, H-10),
4.72 (dd, H-1,3), 7.36 (m, H-4,5,6,7,13,14,15,16,17); 13C
NMR (CDCl3, 75 MHz) δ 58.4 (C-1,3), 123.5 (C-4,7),
127.4 (C-5,6), 137.9 (C-8,9), 55.4 (C-10), 33.5 (C-11),
134.9 (C-12), 129.2 (C-13,14,16,17), 127.5 (C-15). IR

Med Chem Res
18,19). IR (ATR, cm−1) ύ: 3063 (C–H, aromatic), 2943
(C–H, aliphatic), 2842 (O–CH3, aliphatic), 1705 (C=O),
1600 (C=C), 1466 (CH2), 1427 (CH3), 1394 (C–N), 1228
(O–CH3). MS (m/z): 334.0956 (M + Na).
determining the total number of movements, distance tra-
veled, and vertical movements during 5 min. The test was
applied 1 h after the treatment and on each of the sub-
sequent 3 days.
(S)-(2-(1-phenyl)etyl)isoindolin-1,3-dione (JaD-03): it was
obtained as a yellow oil, 89% yield; H NMR
Statistical analysis
1
The data from the behavioral test are expressed as the mean
standard error. Each treatment group was compared to the
corresponding control with repeated measures analysis of
variance and Bonferroni post tests. A significant difference
was considered at p < 0.001.
(CDCl3, 300 MHz) δ (PPM) 1.93 (d, H-11), 5.56 (q, H-10),
7.3 (m, H-15), 7.49 (m, H-13,17), 7.50 (m, H-14,16), 7.68
(m, H-5,6), 7.80 (m, H-4,7); 13C NMR (CDCl3, 75 MHz) δ
168.1 (C-1,3), 123.1 (C-4,7), 133.8 (C-5,6), 131.9 (C-8,9),
49.5 (C-10), 17.5 (C-11), 140.2 (C-12), 127.4 (C-13,17),
127.6 (C-14,16), 128.4 (C-15). IR (ATR, cm−1) ύ: 3063
(C–H, aromatic), 2935 (C–H, aliphatic), 1703 (C=O), 1600
(C=C), 1427 (CH2), 1425 (CH3), 1384 (C–N). MS (m/z):
274.0774 (M + Na).
Results and discussion
The synthesis of three isoindolines and four dioxoisoindo-
lines was carried out with a new synthetic strategy based on
the 12 principles of green chemistry (Scheme 1). The fact
that this kind of strategy incorporated almost all materials
used in the reaction without any solvent or catalytic
reagents (and considering some other important points)
allowed it to comply with at least nine principles of green
chemistry. Another important characteristic is the length of
time taken by a chemical reaction. Whereas other reactions
for the synthesis of isoindolines or dioxoisoindolines can
take hours, with this technique the reaction lasted 1–8 min,
synthesizing up to 5 g each time and affording good yield.
To evaluate the affinity for hD2R, docking was carried
out with the seven test ligands (three isoindolines and four
dioxoisoindolines) and the reference compounds. The Gibbs
free energy, dissociation constant and pKd were obtained in
molecular docking assays (Table 1). The data show a
similarity between the two families (isoindolines/dioxoi-
soindolines), although the dioxoisoindolines have a slightly
lower ΔG than the isoindolines.
The amino acid residues involved in the interaction
between hD2R and the isoindolines/dioxoisoindolines were
examined (Table 2). Molecular docking evidence that dur-
ing the recognition of hD2R (Fig. 1a–g), several of the test
compounds interacted with important amino acid residues.
Therefore, these compounds are expected to produce a
biological response, either as agonists or antagonists.
Several interactions were found with hD2R for the YaI-
01 molecule (Fig. 1a), including one electrostatic interaction
(Asp114:O----H:1 at 1.64 Å), four hydrophobic interactions
(Cys118:C----C:1 to 3.52 Å, Val115:C----C:1 to 3.68 Å,
Leu94:C----C:1 to 3.37 Å, Ile183:C----C:1 to 3.66 Å) and
five π–π interactions (Tyr416:O----H:1 at 3.08 Å, Phe110:
C----C:1 to 3.47 Å, Phe389:C----C:1 to 3.48 Å, Tyr416:
C----C:1 to 4.14 Å, Trp386:C----C:1 to 3.46 Å). For the
YaI-02 molecule (Fig. 1b), there were various interactions
with hD2R, including one hydrogen bond (Ser193:O----
2-(2-(3,4-dihydroxyphenyl)etyl) isoindolin-1,3-dione (JaD-
04): it was obtained as a light yellow solid, 90% yield; mp
167–168 °C; H NMR
1
(MeOD, 300 MHz) δ 2.79 (t, H-11), 3.30 (s, OH-14,15),
3.80 (t, H-10), 6.49 (dd, H-17), 6.61 (dd, H-13,16), 7.76 (m,
H-4,5,6,7); 13C NMR (CDCl3, 75 MHz) δ 168.22 (C-1,3),
122.60 (C-4,7), 133.85 (C-5,6), 131.87 (C-8,9), 39.18 (C-
10), 33.29 (C-11), 129.61 (C-12), 114.92 (C-13), 144.85
(C-14), 143.52 (C-15), 115.46 (C-16), 119.68 (C-17). IR
(ATR, cm−1) ύ: 3063 (C–H, aromatic), 2943 (C–H, ali-
phatic), 2842 (O–CH3, aliphatic), 1705 (C=O), 1600
(C=C), 1466 (CH2), 1427 (CH3), 1394 (C–N), 1228
(O–CH3). MS (m/z): 306.0699 (M + Na).
In vivo evaluation of a synthesized isoindoline as the
putative ligand of the D2 receptor
Since theoretical assays suggested that YaI-01 is a potential
D2R-ligand (see below), we examined its effect on parkin-
sonism induced by MPTP. To cause acute motor disruption,
MPTP (18 mg/kg) was administered four times with a 2-h
interval between applications. Briefly, C57BL/6 male mice
weighing 22 2 g were divided into four groups (n ≥ 5): a
negative control group treated with saline solution and three
groups in which parkinsonism was induced by MPTP. Of
the latter groups, one was the positive control, treated with a
saline solution, while the other two were administered L-
DOPA (254 µmol/kg) or YaI-01 (254 µmol/kg). Motor
effects were assessed with the open field test, observing the
spontaneous motor activity, coordinated movement and
avoidance retention capacity of the animals, as reported
elsewhere (Farfán-García et al. 2016). Mice were placed
into cages (50 × 50 cm) for measuring motor activity. These
had detectors every 2.5 cm (Omnialva®, Mexico) for

Med Chem Res
Scheme 1 Structure and route
for the synthesis of isoindoline/
isoindoline-1,3-dione
O
O
R1
N
R3
R5
R3
R5
O
Melting point
100-300 °c
or
H2N
+
(CH2)n
R4
R6
(CH2)n
R4
R6
Br
R2
Br
Molecule
YaI-01
YbI-02
YcI-03
JaD-01
JbD-02
JcD-03
JdD-04
R1
H
H
R2
H
H
R3
H
H
CH3
H
H
R4
H
H
H
H
H
H
H
N
1
1
0
1
1
0
1
R5
H
OCH3
H
H
OCH3
H
OH
R6
H
OCH3
H
H
OCH3
H
H
=O
=O
=O
=O
=O
=O
=O
=O
CH3
H
H
OH
Table 1 Gibbs free energy (ΔG), the dissociation constant (Kd), and
−log(10) dissociation constant (pKd) for the interaction between the
ligands and hD2R
H:217 1.86 Å), one electrostatic interaction (Asp114:O----
H:2 at 1.76 Å) and five hydrophobic interactions (Cys118:
C----C:2 to 3.57 Å, Val115:C----C:2 to 3.33 Å, Trp386:
C----C:2 to 3.05 Å, Thr416:C----C:2 to 3.55 Å, Tyr412:
C----C:2 to 3.30 Å, Ile184:C----C:2 to 3.40 Å) and one π–π
interaction (Phe389:C----C:2 to 3.76 Å). For the YaI-03
molecule (Fig. 1c), diverse interactions were observed with
hD2R, including one electrostatic interaction (Asp114:O----
H:3 at 1.77 Å), four hydrophobic interactions (Cys118:C----
C:3 to 3.28 Å, Val115:C----C:3 to 3.54 Å, Val91:C----C:3
to 3.75 Å, Trp386:C----C:3 to 3.32 Å) and three π–π inter-
actions (Tyr416:O----H:3 at 3.37 Å, Phe110:C----C:3 to
3.55 Å, Phe389:C----C:3 to 3.83 Å). For the JaD-01 mole-
cule (Fig. 1d), several interactions were identified with
hD2R, including one hydrogen bond (Ser193:O----H:1 1.86
Å), six hydrophobic interactions (Cys118:C----C:1 to 3.56
Å, Val115:C----C:1 to 3.59 Å, His393:C----C:1 to 3.12 Å,
Ile394:C----C:1 to 3.04 Å, Ile184:C----C:1 to 3.14 Å,
Val190:C----C:1 to 3.37 Å) and 2 π–π interactions (Phe389:
C----C:1 to 3.12 Å, Phe390:C----C:1 to 3.44 Å). For the
JaD-02 molecule (Fig. 1e), a number of interactions were
found with hD2R, including one hydrogen bond (Ser193:
O----H:2 2.01 Å), five hydrophobic interactions (Cys118:
C----C:2 to 3.49 Å, Val115:C----C:2 to 3.27 Å, Ile184:C----
C:2 to 3.52 Å, Ile183:C----C:2 to 3.50 Å, Val190:C----C:2
to 3.37 Å) and one π–π interaction (Phe389:C----C:2 to
3.47 Å). For the JaD-03 molecule (Fig. 1f), there were
diverse interactions with hD2R, including one hydrogen
bond (Ser193:O----H:3 1.82 Å), five hydrophobic interac-
tions (Cys118:C----C:3 to 3.19 Å, Val115:C----C:3 to 3.56
Å, His393:C----C:3 to 3.20 Å, Ile184:C----C:3 to 3.31 Å,
Ile394:C----C:3 to 3.07 Å) and three π–π interactions
hD2 receptor
Ligand
ΔG (Kcal/mol)
Kd (µM)
pKd
YaI-01
−5.39
−6.06
−5.93
−5.96
−6.17
−6.35
−5.60
−5.01
−7.05
−5.05
−4.84
112.57
35.94
44.65
42.82
30.12
22.25
78.56
213.70
6.84
3.95
4.44
4.35
4.37
4.52
4.65
4.10
3.67
5.16
3.70
3.55
YbI-02
YcI-03
JaD-01
JbD-02
JcD-03
JdD-04
Dopamine
Haloperidol
Quinpirole
Ropinirole
197.27
281.64
(Phe389:C----C:3 to 3.70 Å, Phe390:C----C:3 to 4.72 Å,
Phe386:C----C:3 to 3.71 Å). For molecule JaD-04 (Fig. 1g),
various interactions were detected with hD2R, including
three hydrophobic interactions (Cys118:C----C:1 to 3.24 Å,
Thr412:C----C:1 to 3.59 Å, Val111:C----C:1 to 4.01 Å) and
three π–π interactions (Phe389:C----C:1 to 3.19 Å, Trp386:
C----C:1 to 3.53 Å, Phe110:C----C:1 to 3.26 Å).
We carried out theoretical calculations to examine some
toxicity and physicochemical properties based on Lipinski’s
rule of five (Table 3). The online servers Molinspiration and
OSIRIS Property Explorer were used with the StarDrop
software.

Med Chem Res
Table 2 Amino acid residues
involved in the interaction
between the isoindolines/
Ligand
Residues
YaI-01
YbI-02
YcI-03
JaD-01
JbD-02
JcD-03
JdD-04
Dopamine
Asp114, Cys118, Val115, Leu94, Tyr416, Ile183, Phe110, Trp386, Tyr416, Phe389
Ser193, Asp114, Cys118, Val115, Tyr412, Thr416, Trp386, Ile184, Phe389
Asp114, Cys118, Val115, Trp386, Val91, Tyr416, Phe110, Phe389
Ser193, Cys118, Val115, His393, Ile394, Ile184, Val190, Phe390, Phe389
Ser193, Val115, Ile184, Ile183, Cys118, Phe389, Val190
isoindoline-1,3-dione and hD2R
Ser193, Cys118, Val115, His393, Ile184, Phe389, Ile394, Phe390, Phe386
Cys118, Thr412, Val111, Phe389, Trp386, Phe110
Cys118, Ser121, Asp114, Trp386, Asp80, Val83, Gly415, Asn418, Tyr416, Ser419
Haloperidol Ser194, Val190, Ser193, Ile394, Phe390, His393, Ile184, Val115, Phe389, Ile183, Asp114,
Val111, Tyr408, Cys182, Phe110, Glu99, Leu94
Quinpirole
Ropinirole
Ile184, His393, Phe389, Trp386, Asp114, Thr412, Tyr416
Ile184, Ile183, Asp114, Phe389, Trp386, Cys182, Phe110, Tyr416, Thr412, Tyr416, Leu94
As is well known, LogP is a very important descriptor of
molecular hydrophobicity because it is the logarithm (base
10) of its partition coefficient between n-octanol and water.
To avoid poor absorption and consequently achieve good
bioavailability and hydrophobic drug–receptor interactions,
this value must not be over 5.0. The addition of the iso-
indoline ring improved the logP of the biogenic amines as
well as their absorbance and their ability to cross the
blood–brain barrier. Since the amino group of biogenic
amines is protonated at physiological pH, it is not possible
for these molecules to penetrate the blood–brain barrier and
induce CNS (central nervous system) activity. Such activity
is important for treating neurodegenerative diseases.
LogS or aqueous solubility refers to the solubility mea-
sured in mol/L and has proven to be a good descriptor of
absorption. More than 80 % of the drugs on the market and
all the molecules herein synthesized have a LogS value
greater than − 4.
Another important descriptor is polar surface area,
defined as the sum of the surfaces of polar atoms in a
molecule. It has proven to correlate well with passive
molecular transport through membranes. A value less than
120 is considered desirable, and the values for the iso-
indolines and dioxoisoindolines studied presently are in
accordance with this criterion. Thus, they can probably
cross the blood–brain barrier.
show a maximum number of rotatable bonds of 5, which is
well below the value of 10 considered as a maximum
topological parameter to measure molecular flexibility
(Table 3).
Theoretical analysis is an important tool for drug dis-
covery that should be included as part of green chemistry.
An obligatory study for all drug development is the ADME
profile (absorption, distribution, metabolism and excretion),
and of course, these properties were evaluated in the present
study. Such parameters can be calculated by in silico studies
(Table 4). For all test ligands, the HIA value evidences
absorption from the intestine into the hepatic portal vein,
but without considering the first pass metabolism effects.
All molecules displayed an absorption greater than 30%,
which was primarily related to passive absorption and does
not consider any other kind. A good HIA is necessary but
not sufficient for good oral bioavailability.
Drug efficiency may be affected by the plasma protein
binding profile, because the less bound a drug is, the more
efficiently it can traverse cell membranes. In this regard,
molecules I01–I03 (isoindolines) show plasma protein
binding less than 90%, while molecules D04–D07 (diox-
oisoindolines) exhibit over 90% binding to the same pro-
teins. Therefore, the addition of two carbonyl carbons to the
structure of isoindolines increases binding to plasma pro-
teins and as a result may decrease the drug effect.
Lipinski et al. described some important parameters that
directly affect absorption or permeation, and thus estab-
lished “the rule of 5” (Lipinski et al. 2001). They suggest
that molecules will display poor absorption if they have
more than five H-bond donors or ten H-bond acceptors, a
molecular weight over 500, or a LogP greater than 5.
Regarding the test compounds, these parameters indicate
that they are within the range of good absorption, and
consequently can be considered as potential drugs for the
treatment of PD. Likewise, the ligands included herein
Since metabolism is an important part of any pharma-
cokinetic study, we considered reactions linked to two
isoforms. Firstly, cytochrome P450 2C9 is responsible for
the metabolic clearance of up to 15–20% of all drugs
undergoing phase I metabolism, and it plays a major role in
the oxidation of both xenobiotic and endogenous com-
pounds (Van Booven et al. 2010). Secondly, cytochrome
P450 2D6 is a drug-metabolizing enzyme expressed in the
liver (<2%) and the brain (Mann et al. 2012) that metabo-
lizes 25% of clinical drugs (Wang et al. 2009). The

Med Chem Res
Fig. 1 Binding mode and main amino acid residues interacting with the human D2 receptor obtained by docking simulation for the following
molecules: a YaI-01; b YaI-02; c YaI-03; d JaD-01; e JaD-02; f JaD-03; g JaD-04

Med Chem Res
Table 3 Physicochemical
properties
Ligand LogP <5 LogS (−5 a 1) PSA <120 MW (g/mol) <500 nON <10 nOHNH <5 nrotb <10
YaI-01 3.13
YbI-02 2.30
YcI-03 2.62
JaD-01 2.84
JbD-02 2.70
JcD-03 2.70
JdD-04 2.15
−2.80
−2.84
−2.76
−3.06
−3.10
−3.02
−2.47
3.2400
21.710
3.2400
37.380
55.840
37.380
79.532
223.32
283.37
223.32
251.28
311.33
251.00
283.28
1
3
1
3
5
3
5
0
0
0
0
0
0
2
3
5
2
3
5
2
3
LogP is the octanol/water partition coefficient and LogS the aqueous solubility
PSA polar surface area, MW molecular weight, nON number of hydrogen bond acceptors, nOHNH number of
hydrogen bond donors, nrotb number of rotatable bonds
Table 4 ADME profile
Ligand HIA PPB90 2C9 pKi 2D6 affinity P-gp BBB log([brain]:[blood]) BBB category
YaI-01
YbI-02
YcI-03
JaD-01
JbD-02
JcD-03
JdD-04
+
+
+
+
+
+
+
Low
Low
Low
High
High
High
High
4.552
4.863
4.687
4.760
5.020
4.943
4.613
Medium
Medium
Medium
Medium
Medium
Medium
Medium
No
No
No
No
No
No
No
0.7653
0.6918
0.9554
0.3501
0.1543
0.4137
−0.3118
+
+
+
+
−
+
−
HIA: “+” indicates absorption ≥30% and “−” absorption <30%
PPB90: “Low” denotes <90% and “high” ≥90% of the compound bound to plasma protein
2D6 affinity: “Low” refers to a pKi <5, “medium” a pKi in the range 5–6, “high” in the range 6–7, and “very
high” >7
BBB category: “+” means a ratio ≥−0.5 and “−” a ratio <−0.5
HIA human intestinal absorption, PPB90 plasma protein binding (90% threshold), 2C9 pKi cytochrome P450
CYP2C9 affinity, 2D6 cytochrome P450 CYP2D6 affinity, P-gp P-glycoprotein substrate, BBB log([brain]:
[blood])) blood–brain barrier penetration, BBB category blood–brain barrier penetration category
substrates of this isoform are lipophilic bases with an aro-
matic ring and a nitrogen atom, such as phenylethylamine
derivatives (e.g., catecholamines). They can be protonated
at physiological pH and have been associated with sus-
ceptibility to PD (Wang et al. 2009; Mann et al. 2012; Lu
et al. 2014). Hence, these two isoforms are very important
for the present study because both are involved in the
metabolism of drugs as well as neurotransmitters such as
dopamine. The affinity for both these isoforms is similar in
the case of isoindolines and dioxoisoindolines. The affinity
for P450 2C9 is around 20 µM on the average while being
1–10 µM for P450 2D6.
Another important protein implicated in the absorption
and disposition of a drug is P-glycoprotein, which belongs
to the superfamily of ATP-binding cassette (Lin and
Yamazaki 2003) and acts as a biological barrier by pumping
xenobiotics out of the cells. Accordingly, it is important to
know if the ligands designed herein are a substrate of this
protein. According to the in silico evaluation, none of the
test compounds are a substrate of P-glycoprotein and con-
sequently absorption is probably not affected in this way.
Blood–brain barrier penetration is perhaps the most
important parameter for CNS drug development. With the
in silico study, we estimated two related parameters, BBB
log([brain]:[blood]) and BBB category. In this sense, the
isoindolines proved to be more likely than dioxoisoindo-
lines to cross the BBB and should therefore be considered
as the first option for in vivo testing.
On the other hand, it is possible to predict many prop-
erties of toxicity in silico by using QSAR models with
Stardrop software. Considering both the in vitro and in vivo
evaluations, we predicted a total of 57 toxicity properties
(Table 5). Whereas the data reveal no risk for the three
isoindolines, one of the dioxoisoindolines (D07, a deriva-
tive of dopamine) exhibited some negative properties,
including the following presumptive toxicity: skin sensiti-
zation, hepatotoxicity and chromosome damage. However,
it cannot be assumed that the other test compounds are

Med Chem Res
Table 5 Properties of toxicity evaluated by using the Derek Nexus module with Stardrop software
Carcinogenicity
Irritation of the skin
Phototoxicity
Kidney function-related
toxicity
Photocarcinogenicity
Lachrymation
Anaphylaxis
Blood in urine
Cholinesterase inhibition
Neurotoxicity
Nephrotoxicity
Ocular toxicity
Pulmonary toxicity
Splenotoxicity
Chromosome damage in vitro
Chromosome damage in vivo
Adrenal gland toxicity
Bladder disorders
Photo-induced chromosome damage Cerebral edema
in vitro
Mutagenicity in vitro
Mutagenicity in vivo
Photomutagenicity in vitro
Chloracne
Bladder urothelial hyperplasia
Bone marrow toxicity
Thyroid toxicity
Urolithiasis
Cyanide-type effects
High acute toxicity
Cumulative effect on white cell
count and immunology
Developmental toxicity
Non-specific genotoxicity in vitro
Non-specific genotoxicity in vivo
Methaemoglobinaemia
Bradycardia
Teratogenicity
Mitochondrial dysfunction
Cardiotoxicity
Testicular toxicity
Photo-induced non-specific
genotoxicity in vitro
Uncoupler of oxidative
phosphorylation
HERG channel inhibition in vitro Occupational asthma
Photo-induced non-specific
Oestrogenicity
Hepatotoxicity Respiratory sensitization
genotoxicity in vivo
Eye irritation
Peroxisome proliferation
alpha-2-mu-Globulin nephropathy Photoallergenicity
Kidney disorders Skin sensitization
Irritation of the gastrointestinal tract Phospholipidosis
Irritation of the respiratory tract
completely free of any in vivo toxic effect. Since they are
related to the drug S-thalidomide, a well-known teratogenic
molecule, it is important to evaluate the present compounds
in different animal models to provide conclusive evidence
of their lack of toxicity.
As can be appreciated, theoretical analysis is an impor-
tant tool for drug discovery. Based on the current theoretical
data, we limited in vivo testing to one compound, YaI-01.
In the mouse model employed herein, this compound
reverted motor disruption induced by MPTP on the first day
of testing (Fig. 2), similar to the effect found with L-DOPA.
The fact that no other changes were observed in the sub-
sequent days was expected due to the prompt recovery of
motor behavior of mice, as previously reported (Sedelis
et al. 2000). The motor behavior results strongly suggest
that YaI-01 exerts an effect by dopaminergic pathways, as
does L-DOPA. However, additional studies are needed to
evaluate the biological activity of the synthesized com-
pounds as well as the mechanism involved.
Fig. 2 Effect of YaI-01 compound on the motor activity of male
C57BL/6 mice in the open-field test. The triple-asterisk indicates s
significant difference vs. the group intoxicated and treated with saline
solution (MPTP + S.S.). Data are expressed as the mean SEM (n =
5). Repeated measures analysis of variance and the Bonferroni post
test were used for evaluating statistical significance (p < 0.001)
Indeed, a ‘green’ approach should be considered as a new
model for the development of new drugs in general. We
propose that ‘green design’ (in silico studies) of ligands be
included in the development of potential drugs, adding this
as the 13th point of green chemistry. Currently, most of the
strategies in the industry are based on synthesizing a pool of
candidate molecules, followed by tests of their properties in
different biological models. This not only represents a waste
of economic resources but also unnecessarily pollutes the
environment.
Conclusions
The design of new molecules with potential action on
dopamine receptors is a worthwhile strategy to find alter-
native treatments for PD. This strategy is even more rele-
vant if ‘green conditions’ are involved in the design (in
silico studies) and synthesis (green chemistry) of these
ligands because such a methodology would cause a less
negative impact on the environment.
A better approach would be to generate a pool of
molecules in a virtual environment and carry out molecular

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Acknowledgements This work was supported by SIP of Instituto
Politécnico Nacional, and CONACyT-México.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
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