Green synthesis of benzamide-dioxoisoindoline derivatives and assessment of their radical scavenging activity – Experimental and theoretical approach

Article abstract of DOI:10.1016/j.tet.2020.131456

A series of benzamide-dioxoisoindoline derivatives 3 was obtained, starting from phthalic anhydride and different benzoyl hydrazides 2, by ultrasound irradiation in water as solvent and without any catalyst. Five obtained compounds have been reported in this study for the first time and crystal structure of compound 3h was determined. All compounds were subjected to experimental determination of their antioxidative potential. DPPH test revealed that newly synthesized phenolic compounds 3d, 3e, and 3j are the best antioxidants. Additionally, probable radical scavenging pathway was analysed for reactions of the most active compounds and some radicals that can be found in living cells.

Full text of DOI:10.1016/j.tet.2020.131456

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Green synthesis of benzamide-dioxoisoindoline derivatives and assessment of their
radical scavenging activity – Experimental and theoretical approach
Vesna M. Milovanović, Zorica D. Petrović, Slađana Novaković, Goran A. Bogdanović,
Vladimir P. Petrović, Dušica Simijonović
PII:
S0040-4020(20)30628-1
https://doi.org/10.1016/j.tet.2020.131456
TET 131456
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 11 May 2020
Revised Date: 22 June 2020
Accepted Date: 24 July 2020
Please cite this article as: Milovanović VM, Petrović ZD, Novaković Slađ, Bogdanović GA, Petrović VP,
Simijonović Duš, Green synthesis of benzamide-dioxoisoindoline derivatives and assessment of their
radical scavenging activity – Experimental and theoretical approach, Tetrahedron (2020), doi: https://
doi.org/10.1016/j.tet.2020.131456.
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Graphical Abstract
Green synthesis of benzamide-
dioxoisoindoline derivatives and assessment of
their radical scavenging activity -
Leave this area blank for abstract info.
experimental and theoretical approach
Vesna M. Milovanović,a Zorica D. Petrović,a Slađana Novaković,b Goran A. Bogdanović,b Vladimir P. Petrovića
and Dušica Simijonović*a,c
aUniversity of Kragujevac, Faculty of Science, Department of Chemistry, R. Domanovića 12, 34000, Kragujevac,
Serbia
bVinča Institute of Nuclear Sciences, University of Belgrade, P.O. Box 522, 11001, Belgrade, Serbia
cUniversity of Kragujevac, Institute of Information Technologies, Department of Science, Kragujevac, Jovana
Cvijića bb, 34000 Kragujevac, Serbia

1
Tetrahedron
journal homepage: www.elsevier.com
Green synthesis of benzamide-dioxoisoindoline derivatives and assessment of
their radical scavenging activity – experimental and theoretical approach
Vesna M. Milovanović,a Zorica D. Petrović,a Slađana Novaković,b Goran A. Bogdanović,b Vladimir P.
Petrovića and Dušica Simijonović*a,c
aUniversity of Kragujevac, Faculty of Science, Department of Chemistry, R. Domanovića 12, 34000, Kragujevac, Serbia
bVinča Institute of Nuclear Sciences, University of Belgrade, P.O. Box 522, 11001, Belgrade, Serbia
cUniversity of Kragujevac, Institute of Information Technologies, Department of Science, Kragujevac, Jovana Cvijića bb, 34000 Kragujevac, Serbia
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of benzamide-dioxoisoindoline derivatives 3 was obtained, starting from phthalic
anhydride and different benzoyl hydrazides 2, by ultrasound irradiation in water as solvent and
without any catalyst. Five obtained compounds have been reported in this study for the first time
and crystal structure of compound 3h was determined. All compounds were subjected to
experimental determination of their antioxidative potential. DPPH test revealed that newly
Available online
synthesized
phenolic compounds 3d, 3e, and 3j are the best antioxidants. Additionally,
probable radical scavenging pathway was analysed for reactions of the most active
compounds and some radicals that can be found in living cells.
Keywords:
Green synthesis
Ultrasound irradiation
X-ray diffraction
Antioxidants
2009 Elsevier Ltd. All rights reserved
.
Thermodynamics
1. Introduction
All previously mentioned methods for the synthesis of 1,3-
dioxoisoindolines have one or more deficiency, such as high
temperature, a multistep synthesis, low yields, transition metal
catalysts, usage of CO gas, usage of toxic materials such as some
mineral acids, NEt3, MeOH, etc., and the feature of not following
green chemistry principles. Also, the usage of microwave
irradiation for the synthesis of 1,3-dioxoisoindolines, requires the
use of organic solvents and additives.20–22
Heterocyclic compounds containing isoindoline moiety have
been of great interest to the researchers for many years.
Isoindoline nucleus is known to be an integral part of many
compounds that exert wide spectra of biological and
pharmaceutical activities.1 It was shown that this compound class
possesses anticancer,2,3 anti-inflamatory,4 sedative,4 analgesic,5
antihyperglycemic,6 antipsychotic,7 antihypertensive,8 and
cytotoxic9 activity. Commercial drugs, such as lenalidomide,2
apremilast,4 indoprofen,5 (s)-pazinaclone,4 contain isoindoline
scaffold. These compounds exert affinities for dopamine,
serotonine10 and GABA receptors,11 and therefore they have
been known as potential anti-Alzheimer’s agents.12,13 They also
exert similar effect as L-DOPA in Parkinsonism treatment.14 In
addition, isoindolines have found industrial application as a dye,
such as pigment yellow 139, which belongs to the class of highly
resistant dyes.4 The most common method for the synthesis of
1,3-dioxoisoindoline derivatives involves condensation of
phthalic anhydride with different substituted primary amines in
the presence of acids or bases as catalysts.15–17 The literature also
claims a procedure for the synthesis of a range of 1,3-
dioxoisoindoline
derivatives
by
palladium-catalyzed
carbonylation reactions of o-dihaloarenes, o-halobenzoic acids
and their esters, and with primary amines.18,19
Figure 1. Some commercially available drugs with isoindoline moiety.

2
Tetrahedron
Table 1. Optimization of reaction conditions
Entry
Temperature (
)
Time (h)
Yield (%)
Ultrasound irradiation has become significant green and cheap
methodology in the syntheses of various organic compounds.23-33
It is important to emphasize that literature fails with ultrasound
assisted
yl)benzamide derivatives. In addition, ultrasonic methodology
has found application in pharmacy, biotechnology, food industry
and environmental engineering. Compared to the traditional
synthetic methods, ultrasound irradiated reactions have more
advantageous such as: cheap and fast chemical activation which
does not require any catalyst, increasing of reaction rate, product
selectivity, yield and purity of the products, etc.34–37
1
2
3
4
5
6
7
8
rta
10
1
nr
100a
100a
100a
rtb
25
65
82
nr
green
synthesis
of
N-(1,3-dioxoisoindolin-2-
3
5
5
80b
80b
80b
0.5
1
40
65
84
2
In the present study we report green ultrasound assisted
methodology for the synthesis of benzamide-dioxoisoindoline
derivatives in water, in the absence of any catalyst and toxic
organic solvents. This research involved determination of the
structures of reaction products, experimental investigation of
their antioxidative potential using DPPH test and theoretical
investigation of the effects of some medically relevant radicals on
the antioxidative activity and reaction mechanism of the most
active compounds.
Reaction conditions: phthalic anhydride (1 mmol), 2a (1 mmol), 2 mL
H2O; aReaction performed by heating under reflux; bReaction performed
under ultrasound irradiation by heating to 80 ; nr = no reaction.
Therefore, optimal conditions for the synthesis of
benzamide-dioxoisoindoline derivatives
3 are heating to 80
and ultrasound irradiation for 2h in water as solvent
(Table 2). Products 3a and 3b-j (with electron donating
substituents) were simple isolated by precipitation and
filtration, in good to excellent yields (70-89%). However,
when benzoyl hydrazides with electron withdrawing groups
2. Results and Discussion
(
2k-n) were used, the yields of the reactions were lower.
For the synthesis of benzamide-dioxoisoindoline derivatives 3,
differently substituted methyl esters 1 were transformed to
corresponding benzoyl hydrazides 2, which were used for further
reactions. The reactions of 2 and phthalic anhydride were
performed in water and without any catalyst (Scheme 1). In order
to optimize reaction conditions, the reaction between phthalic
anhydride and benzohydrazide (2a) in water was used as a model
reaction for optimization of reaction conditions (Table 1). Firstly,
the reaction was performed at room temperature, but the reaction
did not occur. The product was detected upon reaction mixture
being heated under reflux for 1h, and good yield of product was
obtained after 5h. In order to accelerate the reaction, ultrasound
Prolongation of reaction time to 12h provided good yields of
the reaction products (Table 2), whose structures are
reported on the Fig. 2. It is important to emphasize that five
obtained products have been reported in this study for the
first time (3d-f, 3i and 3j). All products were characterized
by H NMR, 13C NMR, and IR spectroscopy. For newly
1
synthesized compounds elemental analysis was done. In
addition, crystal structure of
3,4,5-trimethoxybenzamide (3h) was determined.
N-(1,3-dioxoisoindolin-2-yl)-
Table 2. Yields of isolated benzamide-dioxoisoindoline
derivatives
Compound
3
irradiation was used and heating to 80
. In that case,
Time (h)
Yield (%)
84
satisfactory yield of benzamide-dioxoisoindoline derivatives 3
was obtained after 2h (Scheme 1, Table 1).
3a
3b
3c
3d
3e
3f
2
2
73
2
85
2
80
2
78
2
74
3g
3h
3i
2
89
Scheme 1. Synthesis of benzamide-dioxoisoindoline
derivatives (
reflux, (ii) phthalic anhydride, benzoyl hydrazides (
water, heating to 80 , ultrasound irradiation.
3
): (
i
) hydrazine monohydrate, esters (
1
),
),
2
74
2
2
72
℃
3j
2
70
3k
3l
12
12
12
12
71
87
3m
3n
70
71

3
The environmental acceptability and efficiency for the used
reaction method were determined. For that purpose, green
chemistry metrics, such as atom economy (AE), atom efficiency
(AEf), carbon efficiency (CE), reaction mass efficiency (RME),
EcoScale, mass intensity (MI), and environmental factor (E-
factor) were determined (Table S1).39–41 The graphical
presentation of the values calculated for the green mass metrics
on the basis of which is estimated incorporation of atoms of
reagents into the product (AE, AEf, CE, RME, and EcoScale)
and green mass metrics for estimating waste minimization (MI
and E-factor) are presented in Fig. 3. The obtained results reveal
that AE is very high and goes in range from 93.7% to 95.6%. The
monitored reactions have high values for AEf and CE (up to 84%
and 89%). The scores of RME in range of 66,2-83,9% label this
method as green synthetic route for preparation of benzamide-
dioxoisoindoline derivatives. The values obtained for EcoScale
(73-83%), indicate that the compounds 3a-n are synthesized in
good to high yield, using low cost chemicals, under safety
conditions and isolated without any purification. Additionally,
the obtained results for MI and E-factor (1.62-1.12 and 0.62-
0.12) convincingly confirm the green features of the method
used.
Figure 2. The structures of isolated benzamide-dioxoisoindoline
derivatives 3.
Mechanism of the reaction for the synthesis of 3 was proposed
and analysed.38 The reaction starts with nucleophilic attack of
nitrogen from hydrazide 2 to the carbonyl group of phthalic
anhydride. In this way, 2-(benzoylhydrazine-carbonyl)benzoic
acid (intermediate I) is formed (Scheme 2). It is important to
emphasize that this intermediate is observed in cases of reactions
with lower yield after 5h, and isolated from the reaction where
electron accepting nitro group was presented. Spectral
characterisation of this intermediate is given in SI. In this acid
medium, oxygen of carbonyl group within carboxylic group of
intermediate I is subjected to protonation to form intermediate II.
This way, carbon atom is more electrophilic, and therefore
subjectable to nucleophilic attack of the nitrogen. This enables
cyclisation and formation of intermediate III. The final step in
this reaction is dehydration and deprotonation of intermediate
III, which yields the corresponding benzamide-dioxoisoindoline
derivatives 3.
Figure 3. Green chemistry metrics calculated for the synthesized
compounds 3a-n.
Scheme 2. Proposed mechanism for the synthesis of
benzamide-dioxoisoindoline derivatives 3.

4
Tetrahedron
bonds. The C2-C7 phenyl ring participates in π…π interaction
2.1. Crystal structure of 3h
with the same ring of neighboring molecule. Two rings are
ideally parallel and form centrosymmetric dimer with
perpendicular distance between the rings of only 3.37 Å
(Distance between ring centroids is 3.71 Å). This π…π
interaction and some additional weak C-H…O
intermolecular hydrogen bonds (Table 4) interconnect to
Molecular structure of 3h, determined by single-crystal X-ray
analysis, is shown in Fig. 4.
form the above described chains of molecules 3h
.
Figure 4. Crystal structure and atom-numbering scheme of 3h
.
Displacement ellipsoids are drawn at the 40% probability level.
All non-H atoms in 3h (except C17 methyl group) are placed
in three planes. The first plane is defined by two fused rings
together with the O1 and O2 atoms (root-mean-square deviation
of all 11 atoms is 0.023 Å). The N2 atom is displaced from this
plane for 0.133 (2) Å. The second plane is defined by the N1-N2-
C9-O3 fragment (rms deviation of four atoms is 0.011 Å).
Dihedral angle between these two planes is 86.56 (7)°. The third
plane is defined by phenyl ring and the atoms bonded to this ring
(rms deviation of all 10 atoms is 0.026 Å). Two methyl groups,
the C16 and C18, are roughly seated in this plane. Dihedral angle
between the last two planes is only 11.56 (9)°, therefore, we can
say overall that the molecule 3h is divided into two halves
approximately orthogonal to each other and connected through
the N1-N2 bond. The N2-C9 bond is somewhat shorter than
corresponding N-C bonds formed by the N1 nitrogen atom but
generally all bonds in 3h molecule have expected values (Table
3).
Figure 5. The molecules of 3h are mutually interconnected into the chains
using hydrogen bonds which are shown in light blue dashed lines. All H
atoms not involved in hydrogen bonding are omitted for clarity.
Table 4 Hydrogen bonding geometry in crystal structure
of 3h
D–H...A
D–H
(Å)
D...A
(Å)
H...A
(Å)
D–
H...A
(°)
Symmetry
codes:
N2–
0.83(2) 2.841(2) 2.05(2)
159(2)
x,-
H1...O1
y+0.5,z+0.5
C3-H3...O2
C4-H4...O4
C5-H5...O3
0.93
0.93
0.93
3.408(2) 2.49
3.509(3) 2.59
3.350(2) 2.50
170
169
153
x,y,z-1
x-1,y,z-1
-x+1,-y,-z+1
Table 3. Selected bond lengths (Å) for 3h
O1-C1
O2-C8
O3-C9
O4-C12
O4-C16
O5-C13
O5-C17
O6-C14
O6-C18
N1-N2
N1-C1
N1-C8
N2-C9
C1-C2
C7-C8
C9-C10
1.208(2)
1.197(2)
1.213(2)
1.365(2)
1.423(2)
1.366(2)
1.393(2)
1.361(2)
1.418(2)
1.375(2)
1.388(2)
1.402(2)
1.361(2)
1.473(2)
1.480(2)
1.489(2)
2.2. Radical scavenging activity
All synthesized benzamide-dioxoisoindoline derivatives
(
3a-
n
) were tested for their in vitro antioxidative activity, Tables
5, S2, and S4. Their scavenging ability was screened using
stable free radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH).
The most probable radical scavenging pathway was analysed
using thermodynamical parameters and reaction enthalpies
for the reactions of the most active compounds with the
some medically relevant radicals (Scheme 3, Tables 5, and
S4).
In this investigation nordihydroguaiaretic acid (NDGA)
and quercetin were used as reference compounds. The
results of this test showed that compounds
dioxoisoindolin-2-yl)-3,4,5-trihydroxybenzamide
N
3j),
-(1,3-
(
N-
(1,3-dioxoisoindolin-2-yl)-3,4-dihydroxybenzamide
and -(1,3-dioxoisoindolin-2-yl)-2,3-dihydroxybenzamide
3d) expressed the best antioxidant activity with IC50 values
(3e),
N
(
of 1.2 µM, 1.9 µM, and 2.6 µM, respectively. Furthermore,
stoichiometric factor has been determined for the most
active compounds.42,43 Taking into account this parameter,
compounds 3d, 3e, and 3j can be considered as significant
The only significant H bond donor in 3h is the N2-H group.
Via the N2-H…O1 hydrogen bonds, it forms a chain along the c
axis of unit cell (Fig. 5). The molecules in the chain are
additionally interconnected through the C3-H…O2 weak H-
radical scavengers, since stoichiometric factor amounts 4.8,
6.6, and 10.4 respectively (Table 5). Based on the fact that
good antioxidants have value for this parameter more than 2,

5
investigated compounds, especially 3j, can be considered as
excellent
antioxidants.42,43
Table 5. Interaction of compounds 3d, 3e, and 3j with the stable radical DPPH, calculated thermodynamical parameters (kJ mol-
1) of antioxidant mechanisms and reaction enthalpies (kJ mol-1) for the reactions of these compounds with the selected radicals in
methanol using B3LYP functional and the 6-311+G(d,p) basis set.
3d
3e
3j
IC50 (μM)
2.6±0.1
1.9±0.1
1.2±0.1
Stoichiometric factor
4.8
6.6
10.4
HAT
SET-PT
SPLET
HAT
SET-PT
SPLET
HAT
SET-PT
IP
SPLET
Thermodynamical parameters (kJ mol-1)
BDE
329
IP
PDE
PA
ETE
375
367
BDE
334
IP
PDE
PA
ETE
368
379
BDE
349
328
348
PDE
1
PA
ETE
389
384
389
4
6
115
125
8
9
127
118
122
106
121
486
488
330
335
510
-20
0
Reaction enthalpies (kJ mol-1)
Radical
•OCH3
ΔHBΔE
-99
ΔHIP
ΔHPΔE
-207
ΔHPA
-97
ΔHETE
-2
ΔHBΔE
-93
ΔHIP
ΔHPΔE
-204
ΔHPA
-84
ΔHETE
ΔHBΔE
-78
-99
-79
-87
-107
-87
-150
-170
-150
-10
-30
-10
-3
ΔHIP
ΔHPΔE
-211
-231
-211
-220
-240
-220
-203
-224
-204
-165
-185
-165
-166
-186
-167
-144
-164
-144
-106
-126
-107
-259
-280
-260
ΔHPA
-90
-106
-90
-99
-114
-99
-83
-98
-83
-44
-60
-44
-45
-61
-46
-23
-39
-24
15
ΔHETE
12
7
-9
2
109
111
-97
-205
-86
-11
-92
-203
-94
133
12
12
7
•OC(CH3)3
-107
-105
-216
-214
-106
-95
-1
-102
-101
-213
-212
-93
-8
2
109
30
111
32
-10
-103
133
54
12
-67
-72
-67
34
29
34
42
37
42
21
16
21
12
7
•OH
-170
-168
-200
-198
-89
-79
-81
-89
-165
-164
-197
-196
-77
-86
-88
-77
•OOH
-30
-28
-161
-159
-51
-40
21
12
-25
-24
-158
-157
-38
-48
14
24
131
139
118
109
298
133
141
120
111
300
155
163
142
132
322
•OOCH3
•OO-CH=CH2
DPPH
-23
-22
-163
-161
-52
-41
29
20
-18
-17
-159
-158
-40
-49
22
32
-23
-4
-22
-21
-140
-139
-30
-19
7
-17
-16
-137
-136
-18
-27
1
-2
-1
11
-23
-3
6
8
-103
-101
8
-2
11
12
-99
-98
20
11
-9
1
26
19
-11
6
-1
26
14
11
34
29
34
•-
O2
42
44
-256
-254
21
32
21
12
47
48
-253
-252
34
24
14
24
62
28
42
12
62
28
It is well known that compounds with several hydroxy
groups bonded to aromatic ring, exert excellent antioxidative
activity,44 and compounds with catechol and pyrogallol
moiety present significant antioxidants.45–49 This can be
explained by stabilisation of formed phenoxy radical (after
dehydrogenation) through intramolecular hydrogen bonding
with the neighbouring hydroxy groups.50 This is in
agreement with obtained results for an excellent antioxidant
activity of 1,3-dioxoisoindolin-benzamide with pyrogallol
moiety (3j), which is even better than the activity of positive
controls quercetin and NDGA. Compounds 3d and 3e have
one catecholic moiety, i.e. one hydroxy group less than 3j,
and therefore they are less active. Lower activity of 3d can
be explained by position of hydroxy groups. Namelly,
hydroxy groups in 3d are ortho and meta positioned relative
to the amide group (-CONH2) with electron-withdrawing
effect. This effect of amide group makes it difficult to build
a radical in ortho hydroxy group. Summarizing all
previously mentioned facts, it can be concluded that the
number of hydroxy groups and their position in aromatic
ring present the most important factor for expressed
antioxidative scavenging activity.

6
Tetrahedron
In addition to in vitro DPPH radical scavenging activity,
compounds 3d 3e, and 3j were subjected to thermodynamic
∆rHBDE = ∆rHIP + ∆rHPDE = ∆rHPA + ∆rHETE
(6)
,
investigation in the absence and in the presence of free
radicals (Schemes 3 and S1, Tables 5, and S4). To get
insight which of the mechanism Hydrogen Atom Transfer
(HAT), Single Electron–Proton Transfer (SET-PT), and
Sequential Proton-Loss Electron-Transfer (SPLET) prevails
in the absence of free radicals, appropriate thermodynamic
parameters (Bond Dissociation Enthalpy (BDE), Ionisation
Potential (IP), and Proton Abstraction (PA) energies,
respectively) were calculated with functionals B3LYP and
M06-2X (Schemes S1, Tables 5 and S4).51–56 It is worth
pointing out that the energies obtained with M06-2X are
generally slightly higher than those with B3LYP functional,
but with the same outcome. Here, the results obtained with
B3LYP functional are presented and discussed, while those
obtained with M06-2X are provided in Table S4 of ESI. For
the examination of the preferred mechanism of radical
scavenging in the presence of free radicals, enthalpies of the
reactions of examined phenolics with selected free radicals
(ΔrH) were calculated using same DFT functionals. Here,
HAT, SET-PT, and SPLET mechanisms are presented with
In both cases (absence and presence of free radicals), the
lowest amount in energy suggests preferred route of radical
scavenging. Thermodynamic parameters and reaction
enthalpies were obtained by optimisation of all relevant
species in methanol, the same solvent which was used in
experimental DPPH assay. Selection of radicals (hydroxy
(•OH), hydroperoxy (•OOH),methylperoxy (CH3–O–O•),
•-
superoxide radical anion (O2 ), methoxy (•OCH3), tert-
butoxy (•OC(CH3)3), vinyl peroxy (CH2=CH–O–O•), and
DPPH) for reaction with the examined compounds was
made based on their appearance and behaviour in the living
cell.47,57,58
Based on the obtained values for BDE, IP, and PA (the
absence of free radicals) one can undoubtedly conclude that
the preferred mechanism of antiradical action is SPLET
Namely, PA is significantly lower than BDE and IP. On the
other hand, obtained reaction enthalpies in the presence of
free radicals imply that the reaction routes are highly
influenced by reacted radical.47,57 Here, SET-PT can be
eliminated as possibility for all examined compounds and
for all radicals, since ΔHIP is considerably higher than
Δ
HBDE, ΔHIP and ΔHPDE, ΔHPA and ΔHETE reaction
enthalpies, respectively (Scheme 3, Tables 5, and S4).51–56
The HAT mechanism is presented with the hydrogen atom
transfer to radical species, and enthalpy of this reaction
Δ
Δ
HBDE and ΔHPA. The only pronounced difference between
HBDE and ΔHPA is in the case of the reaction with OH
•
radical. Here, ΔHBDE values are significantly lower, labelling
HAT as preferred mechanism of •OH radical quenching. Not
so evident difference between ΔHBDE and ΔHPA for the
reactions with alkoxy and peroxy radicals (•OCH3,
(∆rHBDE) can be calculated according to the Eq. 1 (Scheme 3).
The SET-PT mechanism is two-step process, where in the
first step, electron transfer takes place, and enthalpy of this
reaction (∆rHIP) can be determined via Eq. 2. In the second step,
formed radical cation is deprotonated, where enthalpy of this
reaction (∆rHPDE) is calculated according to, Eq. 3. The SPLET
mechanism is two-step process, also. Here, in the first step,
antioxidant is being deprotonated (Eq. 4, ∆rHPA), and in the
second step electron transfer takes place (Eq. 5, ∆rHETE). All
above mentioned mechanisms have the same net thermodynamic
balance, owing to the same reactants and products, Eq. 6.56
•
•OC(CH3)3, OOH, CH3–O–O•, CH2=CH–O–O•) points out
competition between HAT and SPLET mechanisms. The
similar trend is obtained for the reactions with the DPPH
radical. However, ΔHBDE and ΔHPA are presented by low
positive values, which implies slow reactions. Similarly to
previously published results for other phenolic compounds,
•-
enthalpies for the reactions with O2
are the
highest.47,55,56,59,60
3. Conclusions
Catalyst-free and ultrasonic assisted synthesis of
benzamide-dioxoisoindoline derivatives, in water as a solvent,
is reported in this paper. Five of fourteen products have been
presented in this study for the first time (3d-f, 3i, 3j). All
compounds were obtained in high yields and characterized
by melting points, NMR, IR, elemental analysis for new
compounds, and crystal determination for product 3h
Additionally, the mechanism for the synthesis of benzamide-
dioxoisoindoline derivatives was proposed and analysed.
.
3
According to the calculated values of green chemistry
metrics, it can be concluded that this synthesis fulfills the
principles of green chemistry. All products were tested for
their antioxidative potential. The obtained in vitro results
reveal that compounds with catecholic moiety (3d, 3e and
3j) exerted excellent activities comparing to NDGA and
quercetin as reference compounds, and that benzamide-
dioxoisoindoline derivatives with pyrogallolic scaffold (3j
)
Scheme 3. Thermodynamical parameters in the presence of
free radical species
displays the best antioxidant activity with IC50 value 1.2 µM.
In addition, based on thermodynamicall parameters, in the
absence of free radicals, the preferred radical scavenging
pathway is SPLET. On the other hand, enthalpies of the
reactions of investigated compounds with the free radicals
denote competition of HAT and SPLET mechanisms. The
∆rHBDE = [H(AO•) + H(ROH)] – [H(AOH) + H(RO•)] (1)
∆rHIP = [H(AOH•+) + H(RO–)] – [H(AOH) + H(RO•)] (2)
∆rHPDE = [H(AO•) + H(ROH)] – [H(AOH•+) + H(RO–) (3)
∆rHPA = [H(AO−) + H(ROH)] – [H(AOH) + H(RO–)] (4)
•
only difference is in the case of the reaction with OH
∆rHETE = [H(AO•) + H(RO–)] – [H(AO−) + H(RO•)]
(5)
radical, where the HAT reaction pathway is significantly

7
more exergonic, clearly pointing it out as preferred route of
radical quenching.
Beige powder, m.p. 230-232 ; Isolated yield: 81 (78)%; 1H
NMR (200 MHz, DMSO) δ: 6.87 (d, J = 8.4 Hz, 1H), 7.42 – 7.30
(m, 2H), 8.06 – 7.89 (m, 4H), 9.42 (s, 1H), 9.83 (s, 1H), 10.96 (s,
1H), 13C NMR (50 MHz, DMSO) δ: 115.37, 115.51, 120.10,
121.83, 123.89, 129.61, 135.44, 145.32, 149.98, 165.18, 165.64;
IR (cm-1): νmax = 3301-3535 (NH/OH), 1674-1790 (3C=O);
C15H10N2O5 (FW = 298.25): C, 60.41; N, 9.39; H, 3.38%; found:
C, 60.22; N, 8.99; H, 3.71%.
4. Experimental Section
4.1. Materials and Methods
4.2.2.3. N-(1,3-dioxoisoindolin-2-yl)-4-hydroxy-3-
methoxybenzamide (3f)
Chemicals used for the synthesis were acquired from
Sigma Aldrich with purities above 98%. The IR spectra
were obtained on a Perkin-Elmer Spectrum One FT-IR
spectrometer using the KBr disc. The NMR (1H and 13C)
spectra were determined on a Varian Gemini spectrometer
1
White powder, m.p. 233-235 ; Isolated yield: 76 (74)%; H
NMR (200 MHz, DMSO and CDCl3) δ: 3.86 (s, 3H), 6.88 (d, J =
8.1 Hz, 1H), 7.57 – 7.44 (m, 2H), 8.00 – 7.80 (m, 4H), 9.75 (s,
1H), 10.95 (s, 1H), 13C NMR (50 MHz, DMSO and CDCl3) δ:
55.72, 111.57, 115.00, 121.54, 121.79, 123.48, 129.64, 134.81,
147.26, 150.85, 164.84, 165.29; IR (cm-1): νmax = 3395 (NH/OH),
1672-1794 (3C=O), 1219-1277 (OCH3); C16H12N2O5 (FW =
312.28): C, 61.54; N, 8.97; H, 3.87%; found: C, 61.22; N, 9.07;
H, 3.90%.
1
(200 MHz for H and 50 MHz for 13C) using CDCl3 and
DMSO. The UV-Vis measurements were recorded on
Agilent Technologies, Cary 300 Series UV/Vis
Spectrophotometer. Ultrasonication was performed with
ultrasonic bath (BandelinSonorex RK 52 H, Bandelin
electronic GmbH & Co. KG, Berlin, Germany) at 35 kHz
frequency and 150 W power. Melting points were done on a
Mel-Temp capillary melting points apparatus, model 1001.
Elemental (C, H, N) microanalysis were determined at the
University of Belgrade, Faculty of Chemistry.
4.2.2.4. N-(1,3-dioxoisoindolin-2-yl)-2,4-
dihydroxybenzamide (3i)
Brown powder, m.p. 230-232 ; Isolated yield: 75 (72)%; 1H
NMR (200 MHz, DMSO) δ: 6.49 – 6.34 (m, 2H), 7.79 (d, J = 8.6
Hz, 1H), 8.05 – 7.90 (m, 4H), 10.37 (s, 1H), 10.73 (s, 1H), 11.54
(s, 1H), 13C NMR (50 MHz, DMSO) δ: 102.78, 105.86, 108.15,
123.79, 129.53, 131.02, 135.33, 160.80, 163.21, 165.32, 166.62;
IR (cm-1): νmax = 3307 (NH/OH), 1648-1790 (3C=O); C15H10N2O5
(FW = 298.25): C, 60.41; N, 9.39; H, 3.38%; found: C, 60.05; N,
9.70; H, 3.56%.
4.2. Synthetic procedures
4.2.1. General procedure for the synthesis of
hydrazides
2
Hydrazides
2 were obtained in reaction of methyl esters 1
(1 mmol), which synthetized from corresponding acids,61
and hydrazine monohydrate (6 mmol) by heating under
reflux for 4h.62
4.2.2.5. N-(1,3-dioxoisoindolin-2-yl)-3,4,5-
trihydroxybenzamide (3j)
4.2.2. General procedure for the synthesis of
benzamide-dioxoisoindoline derivatives
3
Yellow powder, m.p. 226-228 ; Isolated yield: 72 (70)%; 1H
NMR (200 MHz, DMSO) δ: 6.97 (s, 2H), 8.04 – 7.91 (m, 4H),
9.00 (s, 1H), 9.32 (s, 2H), 10.88 (s, 1H), 13C NMR (50 MHz,
DMSO) δ: 107.38, 120.74, 123.78, 129.52, 135.34, 137.85,
146.71, 165.40, 165.53; IR (cm-1): 3363 (NH/OH), 1652-1786
(3C=O); C15H10N2O6 (FW = 314.25): C, 57.33; N, 8.91; H,
3.21%; found: C, 57.10; N, 9.05; H, 3.26%.
A mixture of phthalic anhydride (1 mmol) and
corresponding hydrazide (1 mmol) in water as a solvent (2
mL), was heated to 80 , using an ultrasound bath. Reaction
progress was monitored using TLC. In all reactions, the
formed precipitation was filtrated and washed with water.
All 1,3-dioxoisoindolin-benzamide products (3a
characterized with melting points, 1HNMR, 13CNMR and IR
spectra. For the newly synthetized compounds 3d 3i and
-n) were
4.3. X-ray crystal structure determination
-f,
3j, purity was confirmed by elemental analysis, too. In
addition, crystal structure of 3h was determined in this study
for the first time. The spectral characterization of new
Single-crystal X-ray diffraction data for compound 3h
were collected at Oxford Gemini S diffractometer, using
monochromatized MoKα radiation (λ = 0.71073 Å). Data
benzamide-dioxoisoindoline derivatives (3d-f, 3i and 3j) is
reduction and empirical absorption correction were
performed with CrysAlisPRO.63 The structure was solved by
direct methods using SHELXS and refined on F2 by full-
matrix least-squares using SHELXL.64 All non-hydrogen
atoms were refined anisotropically. H atoms bonded to C
atoms were placed in geometrically calculated positions and
refined using the riding model with Uiso values constrained
to 1.2Ueq or 1.5Ueq of the parent C atoms. H atom bonded to
N atom was found in a difference Fourier synthesis and
refined isotropically. The PLATON65 software was used to
perform geometrical calculation and the Mercury66 was
employed for molecular graphics. Crystallographic details
are summarized in Table S3. CCDC 1965239 contains the
given in main part of the manuscript, while for other
compounds in Electronic Supplementary Information, as
1
well as copies of H NMR and 13C NMR spectra for all
compounds.
4.2.2.1. N-(1,3-dioxoisoindolin-2-yl)-2,3-
dihydroxybenzamide (3d)
Beige powder, m.p. 220 ; Isolated yield: 85 (80)%; 1H NMR
(200 MHz, DMSO) δ: 6.82 (t, J = 7.9 Hz, 1H), 7.06 (d, J = 7.1
Hz, 1H), 7.39 (d, J = 7.4 Hz, 1H), 8.06-7.90 (m, 4H), 9.95 (s,
1H), 11.04 (s, 1H), 13C NMR (50 MHz, DMSO) δ: 114.60,
118.86, 119.22, 119.96, 123.97, 129.63, 135.49, 146.37, 148.18,
165.22, 167.43; IR (cm-1): νmax = 3289-3503 (NH/OH), 1644-
1787 (3C=O); C15H10N2O5 (FW = 298.25): C, 60.41; N, 9.39; H,
3.38%; found: C, 59.95; N, 9.46; H, 3.63%.
supplementary crystallographic data for compound 3h
.
4.2.2.2. N-(1,3-dioxoisoindolin-2-yl)-3,4-
4.4. DPPH free radical scavenging assay
dihydroxybenzamide (3e)

8
Tetrahedron
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relevant species that participate in the reactions.71,72 To
simulate the influence of methanol (ε = 32.6) as solvents
SMD solvation model was used as implemented in Gaussian
09.68,73 Frequency calculations were done, and the absence of
any imaginary frequency confirmed that all structures are
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Acknowledgements
This work was supported by the Serbian Ministry of
Education, Science and Technological Development
(Agreement No. 451-03-68/2020-14/200122).
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Highlights
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•
•
•
•
N-(1,3-dioxoisoindolin-2-yl)benzamide derivatives were obtained.
“On water” ultrasonic assisted methodology is presented.
Used method presents green approach according to green chemistry parameters.
Five compounds have been reported in this study for the first time.
All obtained compounds were tested for their in vitro antioxidative activity.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: