Deep eutectic solvents as convenient media for synthesis of novel coumarinyl schiff bases and their QSAR studies

Article abstract of DOI:10.3390/molecules22091482

Deep eutectic solvents, as green and environmentally friendly media, were utilized in the synthesis of novel coumarinyl Schiff bases. Novel derivatives were synthesized from 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide and corresponding aldehyde

Full text of DOI:10.3390/molecules22091482

molecules
Article
Deep Eutectic Solvents as Convenient Media for
Synthesis of Novel Coumarinyl Schiff Bases and
Their QSAR Studies
1
1
Maja Molnar 1, Mario Komar 1, Harshad Brahmbhatt 1, Jurislav Babic´ , Stela Jokic´ and
Vesna Rastija 2,
*
ID
1
Faculty of Food Technology Osijek, Josip Juraj Strossmayer University of Osijek, Franje Kuhaca 20,
Osijek 31000, Croatia; mmolnar@ptfos.hr (M.M.); mario.komar@ptfos.hr (M.K.);
brahmbhattharshad@hotmail.com (H.B.); jurislav.babic@ptfos.hr (J.B.); stela.jokic@ptfos.hr (S.J.)
Faculty of Agriculture in Osijek, Josip Juraj Strossmayer University of Osijek, Vladimira Preloga 1,
Osijek 31000, Croatia
2
*
Correspondence: vrastija@pfos.hr; Tel.: +385-31-554-903
Received: 8 August 2017; Accepted: 2 September 2017; Published: 5 September 2017
Abstract: Deep eutectic solvents, as green and environmentally friendly media, were utilized
in the synthesis of novel coumarinyl Schiff bases. Novel derivatives were synthesized from
2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide and corresponding aldehyde in choline
chloride:malonic acid (1:1) based deep eutectic solvent. In these reactions, deep eutectic solvent acted
as a solvent and catalyst as well. Novel Schiff bases were synthesized in high yields (65–75%) with
no need for further purification, and their structures were confirmed by mass spectra, 1H and 13
C
NMR. Furthermore, their antioxidant activity was determined and compared to antioxidant activity
of previously synthesized derivatives, thus investigating their structure–activity relationship utilizing
quantitative structure-activity relationship QSAR studies. Calculation of molecular descriptors
has been performed by DRAGON software. The best QSAR model (Rtr = 0.636; Rext = 0.709)
obtained with three descriptors (MATS3m, Mor22u, Hy) implies that the pairs of atoms higher
−1
mass at the path length 3, three-dimensional arrangement of atoms at scattering parameter s = 21 Å
,
and higher number of hydrophilic groups (-OH, -NH) enhanced antioxidant activity. Electrostatic
potential surface of the most active compounds showed possible regions for donation of electrons to
1,1-diphenyl-2-picryhydrazyl (DPPH) radicals.
Keywords: coumarin; schiff base; deep eutectic solvents; antioxidant activity; QSAR
1. Introduction
Coumarins are a class of compounds widely distributed in the plant kingdom [1], but lots of
synthetic studies have been done on them in the last few decades. Most of the synthetic modifications
aim at the synthesis of biologically active derivatives with more potent specific biological activity and
their potential application in pharmacy, cosmetic industry or medicine. Different antioxidants have
been synthesized and investigated by many researchers in this regard. We have been investigating the
antioxidant activity of different synthetic coumarin derivatives for years and showed that different
synthetic modifications on the basic coumarin core can increase its antioxidant activity [
2–5]. For this
purpose, a series of Schiff bases were synthesized [ ] conventionally; however, we have noticed
2
their formation when deep eutectic solvents (DESs) are applied as well. DESs have proven to
be a convenient media for many synthetic routes and are often characterized as environmentally
friendly [6–8]. Their application in organic synthesis and extraction, as well as an extensive analysis of
their properties, were described in some good reviews published in the last few years [6–11].
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As the emphasis is put on green chemistry approaches on a daily basis, “green solvents” are
the focus of many researchers, since the most waste that is produced in the synthesis of different
compounds are actually waste solvents [12]. DESs are an emerging class of new, non-toxic, biodegradable
solvents, which have already found their application in organic synthesis, and they are becoming
more prominent every day, since they have many advantages compared to conventional solvents.
Capua et al. [12] synthesized 2-aminoimidazoles in one-pot protocol in high yields and with very simple
post synthetic workup, in shorter times than with conventional organic solvents like tetrahydrofuran (THF),
and this procedure resulted in no by-products, while DES was recovered and recycled. Massolo et al. [13]
employed DESs in enantioselective transformations, also obtaining products at high yields with the
possibility of DESs being recycled and reused, and the same advantages of DESs were noticed in the
synthesis of imines and hydrobenzamides [14] and enantioselective aldol reaction [15]. DESs have
proven to be effective in umpolung sulfenylation reactions [16], applicable in material science in epoxy
resin polymerization [17], and as catalysts in CO2 fixation epichlorohydrin [18].
Due to DESs’ numerous advantages compared to organic solvents, the aim of this study was to
synthesize novel coumarinyl Schiff bases utilizing choline chloride based DES as reaction media and
catalyst as well. The fact that there was no need for a specific catalyst, like acetic acid, which is often
used in these kinds of reactions, was noticed. Not only did we get a novel compounds, but we also
applied an environmentally friendly approach, utilizing a solvent made of biodegradable components,
with low toxicity and vapor pressure, which can be easily recycled and reused [19]. The purpose of
the study was also to determine their antioxidant activity and compare it to antioxidant activities of
previously synthesized similar Schiff bases [2,5].
Many theoretical studies have showed that biological activity of natural and synthetic antioxidants
is related to their structure [20]. Different quantitative structure–activity relationship (QSAR)
techniques have been employed to correlate the antioxidant capacity with various structural features
of coumarin derivatives that affect the antioxidant activity and predict the same for the untested and
future molecules, such as multiple linear regression [21,22], genetic function approximation genetic
partial least squares [23 24], and artificial neural networks [25]. Wide ranges of molecular descriptors,
,
mathematical representations of a molecule, have been used for QSAR modeling of antioxidant
activities of a series of coumarin derivatives. Thus, physicochemical properties such as hydrophilic
factor [24
two-dimensional (2D) descriptors Moran autocorrelation and BCUT (Burden - CAS - University of
Texas eigen values) descriptors and three-dimensional (3D) descriptors GATEWAY descriptors [27
,25] and lipophilicity [21]; energy based descriptors, such as bond dissociation enthalpy [26];
]
showed the most significant influences on the QSAR modeling of antioxidant activity of coumarines.
Accurate QSAR models have been generated using quantum-chemistry descriptors, such as HOMO
(highest occupied molecular orbital), LUMO (lowest unoccupied molecular orbital), the difference of
the energies HOMO-LUMO, and hardness [24,25,28,29].
QSAR models obtained for series of coumarin derivatives that were modeled for their antioxidant
activity based on their ability to inhibit DPPH free radical, revealed that compounds with a higher
degree of branching, oxygen atom-bearing fragment and tertiary carbon as substituent lie in the higher
activity range [23]. QSAR study of 3-carboxycoumarin derivatives has implied an importance of the
presence of hydroxyl groups on the ring structure and a higher number of hydrophilic groups for
enhanced DPPH radical scavenging activity [25]. In this work, QSAR study was performed on the
set of synthesized coumarin derivatives in order to signify the importance of structural and chemical
attributes for their antioxidant activity.
2. Results
2.1. Synthesis
Synthesis of Schiff bases is often performed by a reaction of amine with aldehyde, usually with
the addition of catalysts such as acetic acid. Here, we describe a synthesis of Schiff bases from

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2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide and different aldehydes utilizing choline
chloride:malonic acid DES. DES was easily prepared from choline chloride and malonic acid (1:1),
simply by heating their mixture at 80 ◦C until clear liquid was formed and used, as such, without
any further purification, in synthesis of desired Schiff bases. First, we performed reactions with some
commercial aldehydes, 4-methoxybenzaldehyde and 4-dimethylaminobenzaldehyde, to obtain Schiff
bases 10 and 29, which have already been synthesized conventionally in our previous research [
The yields of compounds obtained conventionally (60% for compound 10 and 86% for compound
29 ]) compared to those obtained in DES (44% for compound 10 and 70% for compound 29) are
2,5].
[
5
not much higher, which justified the use of this environmentally acceptable approach in further
synthesis of novel Schiff bases. Therefore, further synthesis was performed with some new aldehydes
to obtain compounds 30–36 (Scheme 1). Pyrazole based aldehydes utilized for a synthesis of Schiff
bases 30 35 were synthesized in a reaction of substituted acetophenones with different hydrazines and
–
subsequent formylation to yield 1H-pyrazole-4-carbaldehyde. For synthesis of Schiff bases, choline
chloride:malonic acid (1:1) DES was used as reaction media. The synthetic approach (Scheme 1) in the
synthesis of mentioned Schiff bases in DES is rather simple. When DES is prepared, an equimolar ratio
of 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide and aldehyde was added to the solvent,
◦
stirred at 70 C and, upon completion of the reaction, water was added and a solid product separated.
DESs were found to be very effective in this kind of synthesis, both, as solvents and catalysts, since
reaction times were not longer than four hours, and purity of final compounds was satisfying as well
as the yields. The mechanism of their action in accordance with Yadav et al. [30] is shown in Scheme 2.
Compound
R
Compound
R
Compound
R
4
5
6
7
8
H
2-OH
3-OH
17
18
19
20
21
22
23
24
25
26
27
28
29
3-O-Ph
2-OH; 5-NO2
3,4,5-OCH3
2-Cl
30
31
32
33
34
35
R1 = Cl; R2 = F
R1 = Br; R2 = CH3
R1 = I; R2 = CH3
R1 = OCH3; R2 = CH3
R1 = Cl; R2 = CH3
R1 = NO2; R2 = Cl
4-OH
2-OCH3
3-OCH3
4-OCH3
2,3-OH
2,4-OH
2,5-OH
3,4-OH
3,5-OH
3-OCH3; 4-OH
3-Cl
2-Br
3-Br
4-Br
2-F
3-F
4-F
9
10
11
12
13
14
15
16
styryl
4-N(CH3)2
Scheme 1. Synthesis of novel coumarinyl Schiff bases in deep eutectic solvent (DES).

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Scheme 2. Proposed mechanism for synthesis of Schiff bases in choline chloride:malonic acid (1:1) DES.
Schiff bases were obtained in high yields (44–95%) and characterized by 1H- and 13C-NMR, as
well as mass spectrometry. Typical shifts for coumarin C3 proton (6.20–6.22 ppm) and C4-methyl group
(2.40 ppm) were noticed. The synthesized coumarin derivatives show characteristic peaks for aromatic
protons, coumarin aromatic protons, protons of aromatic rings derived from corresponding aldehyde
and pyrazole proton. All molecular masses were also in accordance with molecular ions obtained by
mass spectrometry. The structures of synthesized compounds (30–
36) are presented in Table 1. The 1H-
and 13C-NMR spectra of synthesized compounds are presented in supplementary file 1 (File S1).
2.2. Antioxidant Activity
Novel compounds (30
were investigated for their antioxidant activity expressed as % DPPH scavenging activity.
Data presented in Table 1 show that substituents on the phenyl ring have a great influence
on antioxidant activity. Compounds were used as basic structures in antioxidant activity
determination to demonstrate how modification of a starting compound can have a great influence on
antioxidant activity. Compared to coumarin’s basic structure ( ), substitution in position 4 with methyl
and in position 7 with hydroxy group enhances antioxidant activity ( ). Substitution of 7-OH group
–36), together with Schiff bases synthesized in our previous work [2,5],
1–3
1
2
with -CO-NH-NH2 group in 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide (
3
) additionally
increased antioxidant activity.
Novel compounds, together with Schiff bases synthesized in our previous work [
investigated for their antioxidant activity expressed as % DPPH scavenging activity. Structural details
of all studied molecules and % DPPH are shown in Table 1 Data presented in Table 1 show that
substituents on the phenyl ring have a great influence on antioxidant activity. Compounds with two
hydroxyl groups in ortho position (11 14) exhibited the best antioxidant activity, although hydroxyl
2,5] were
.
,
groups at the 2,3-position (11) compared to the 3,4-position (14) showed enhanced antioxidant activity.
Catehol structure or 3,4-diOH substitution of phenyl ring allows oxygen atoms to cause electron
delocalization and stabilisation of phenoxyl radical [2,5,31]. Novel compounds (30–35) did not show
significant antioxidant activity. Replacement of aromatic ring by pyrazole ring in these compounds
reduced antioxidant power compared to compounds (4–29).

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Table 1. Structures of analysed compounds with their antioxidant activities (%DPPH) *.
Mol. ID
1
R1/R2
% DPPH
Mol. ID
3
R
% DPPH
H-/H-
1.8 ± 0.22
H2N-
14.4 ± 0.81
1.6 ± 0.00
2
-CH3/-OH
2.4 ± 0.12
28
36
14.1 ± 0.10
Mol. ID
Supstituents
% DPPH
Mol. ID
Supstituents
% DPPH
4
5
6
7
8
H
2-OH
3-OH
4-OH
2-OCH3
3-OCH3
4-OCH3
2,3-OH
2,4-OH
2,5-OH
3,4-OH
3,5-OH
22.9 ± 0.50
16.6 ± 0.56
3.6 ± 0.24
4.4 ± 0.37
3.1 ± 0.86
0.2 ± 0.08
3.7 ± 0.23
75.4 ± 2.62
8.4 ± 0.12
33.4 ± 3.74
42.6 ± 4.10
4.6 ± 2.17
16
17
18
19
20
21
22
23
24
25
26
27
29
3-OCH3; 4-OH
3-O-Ph
2-OH; 5-NO2
3,4,5-OCH3
2-Cl
32.0 ± 0.25
2.2 ± 0.68
2.9 ± 0.99
3.5 ± 0.42
11.1 ± 0.87
7.5 ± 0.37
6.6 ± 0.62
3.0 ± 0.32
5.0 ± 0.27
3.0 ± 0.76
2.3 ± 0.68
7.4 ± 0.12
5.9 ± 0.62
9
3-Cl
2-Br
3-Br
4-Br
2-F
3-F
4-F
10
11
12
13
14
15
4-N(CH3)2
Mol. ID
R
% DPPH
Mol. ID
R
% DPPH
30
31
32
R1 = Cl; R2 = F
R1 = Br; R2 = CH3
R1 = I; R2 = CH3
1.6 ± 0.23
0.5 ± 0.01
1.6 ± 0.18
33
34
35
R1 = OCH3; R2 = CH3
R1 = Cl; R2 = CH3
R1 = NO2; R2 = Cl
0.8 ± 0.09
1.4 ± 0.19
1.6 ± 0.31
* data are means ± standard deviation of three replicates.
2.3. QSAR Models
The best QSAR model for antioxidant activity of 36 coumarinyl Schiff bases was obtained by
DRAGON descriptors:
log % DPPH = 0.696 + 1.474 (0.488) Mor22u + 1.948 (0.398) MATS3m + 0.387 (0.398) Hy
(1)
N(training) = 29; N(test) = 7 (6,12,19,24,28,33,34)
Statistical parameters of obtained models are given in Table 2. The variables in Equation (1) are
listed in order of relative importance by their standardized regression coefficients (β, in brackets).

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Table 3 presents a correlation matrix of descriptors included in model (1) and proves that descriptors
are not mutually correlated (correlation coefficient, R 0.7). Low collinearity is also verified by the
low values of Kxx and K ( 0.05) [32] (Table 2). The molecular descriptor values; experimental and
≤
∆
≥
calculated log %DPPH by model (1) have been tabulated in supplementary file 2 (Table S2). A scatter
plot of experimentally obtained antioxidant activity versus calculated by model (1) is shown in Figure 1.
Table 2. The model statistical results for the QSAR for antioxidant activity.
Model (1)
Fittinig criteria
Model (2)
R2
R2
s
F
Kxx
∆K
RMSEtr
MAEtr
CCCtr
0.636
0.592
0.369
14.559
0.242
0.170
0.342
0.271
0.778
0.673
0.632
0.315
16.467
0.241
0.178
0.292
0.241
0.805
adj
Internal validation criteria
Q2
0.512
0.544
0.345
0.285
3.326
0.733
0.112
−0.221
LOO
RMSEcv
MAEcv
PRESScv
CCCcv
R2 scr
0.397
0.314
4.560
0.710
0.108
−0.214
Q2Y scr
Y
External validation criteria
RMSEext
0.299
0.271
0.627
0.709
0.523
0.169
0.722
0.732
0.311
0.283
0.677
0.712
0.558
0.103
0.629
0.701
MAEext
PRESSext
R2
Q2ext
Q2 F1
Q2F 2
F3
CCCext
rm2
0.619
0.050
0.600
0.197
∆r2m
Applicability domain (h* = 0.4138)
N compounds outlier
N compounds out of app.dom.
1 (9)
0
0
0
LOO (the leave-one out); R2 (coefficient of determination); R2 (adjusted coefficient of determination); s (standard
adj
deviation of regression); F (Fisher ratio); Kxx (global correlation among descriptors);
descriptors); RMSEtr (root-mean-square error of the training set); MAEtr (mean absolute error of the training set);
CCCtr (concordance correlation coefficient of the training set); Q2
(cross-validated explained variance); RMSEcv
∆K (global correlation among
LOO
(root-mean-square error of the training set determined through the cross validated method; MAEcv (mean absolute
error of the internal validation set); PRESScv (predictive residual sum of squares determined through cross-validated
method); CCCcv (concordance correlation coefficient test set using cross validation); R2Yscr (Y-scramble correlation
coefficients); Q2
(Y-scramble cross-validation coefficients); RMSEex (root-mean-square error of the external
Yscr
validation set); MAEex (mean absolute error of the external validation set); PRESSext (predictive residual sum
of squares determined through cross-validated LOO method in the external prediction set; R2 (coefficient of
ext
determination of validation set); Q2F1, Q2F2, Q2 (predictive squared correlation coefficients); CCCext (concordance
F3
correlation coefficient of the test set); r2m (average value of squared correlation coefficients between the observed and
(leave-one-out) predicted values of the compounds with and without intercept); ∆r2m (absolute difference between
the observed and leave-one-out predicted values of the compounds with and without intercept); h* (warning
leverage for the applicability domain of the model).

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Figure 1. Observed versus predicted log % DPPH for the coumarinyl Schiff bases calculated by
model (1).
Table 3. Correlation matrix for the descriptors included in model 1.
MATS3m
Mor22u
Hy
MATS3m
Mor22u
Hy
1
−0.260
1
0.194
0.309
1
According the statistical results presented in Table 2, model (1) satisfactoriness threshold for
fitting validation parameters: R2 > 0.60 and value of R2 is close to the value of R2. The leave-one out
adj
(LOO) validation highlights that the model is stable, not obtained by chance, since Q2
> 0.5; R2yscr,
LOO
and Q2yscr < 0.2, as R2yscr > Q2yscr [33
the training and validation sets are similar. The chosen models demonstrate a satisfactory stability in
external validation: R2
0.60; small difference between CCCtr and CCCext; small difference between
,34]. Moreover, the root-mean-square error (RMSE) values for
≥
ext
RMSEtr and RMSEex, and between mean absolute error of the training set (MAEtr) and mean absolute
error of the external validation set (MAEex) [35]. Parameter r2m considers the actual difference between
the experimental and the predicted values, serving as more accurate measures for rating of model
predictivity. Value of rm2
> 0.6 confirms high prediction ability for both internal and external validation
sets [36]. Williams plot (Figure 2) was used in order to define the chemical domain of applicability for
which a given QSAR model makes reliable predictions [37]. Inspection of the Williams plot revealed
no compounds out of hat value of leverage or warning leverage (HAT, h* = 0.414), which means that
all predicted data for compounds belonging to the chemical domain and have reliable prediction.
Williams plot identified one outlier (compound
than 2.5 standard deviation units. Outlying behaviour of compound
demonstrated lowest antioxidant activity (Table 1). The methoxy group in phenyl ring, especially in
position 3 (compound ), inactivates the ring, which negatively influences the ability of the compound
to form a stable radical upon scavenging DPPH radicals [38].
9
), which has standardized residual predictions greater
9
was expected since it has
9
After removal of the compound
following improved QSAR model:
9 from the training set, subsequent re-analysis produced a
log % DPPH = 0.733 + 1.331 (0.495) Mor22u + 1.744 (0.401) MATS3m + 0.375 (0.375) Hy
N(training) = 28; N(test) = 7 (6,12,19,24,28,33,34) (β in brackets)
(2)
Calculated values of log %DPPH by model (2) have been tabulated in Table S2. Since compound
9
belonged to the training set, the obtained model gives the better statistics in terms of improvement in

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fitting and internal criteria. Exclusion of outliers generated the model with higher values of R2, R2
,
adj
F, CCCtr, CCCcv, Q2
and lower values of s, RMSEtr, MAEtr RMSEcv, and MAEcv. The obtained
LOO
model in external validation showed only improvement in R2ext, which demonstrated that the removal
of outliers can improve the fitting, but not the predictivity of a model.
The largest values of standardized regression coefficients in Equations (1) and (2) has 3D-MoRSE
(Molecular Representation of Structures based on Electronic diffraction) descriptor Mor22u. According
the positive coefficients of Mor22u in Equations (1) and (2), compounds with significant antioxidant
ability have enhanced positive values of this descriptor (Table S2). Descriptor Mor22u denotes
unweighted descriptors with scattering parameter s = 21 Å−1. Since it is unweighted, the descriptor has
no discriminative ability precisely and treats each atom equally. Though each 3D-MoRSE descriptor
reflects the three-dimensional arrangement of the atoms in molecules, their final values are derived
mostly from short distances [39]. Since mainly QSAR study covers structurally similar sets of
compounds, 3D-MoRSE descriptors in a model can be interpreted using just several pairs of neighbor
atoms. Specifically, descriptor Mor22u has the possibility to distinguish the difference between bond
lengths of any kinds of atoms at least 0.03 Å [39]. The relative discriminative power of descriptor
Mor22u can be observed at molecules 11–15, which have two hydroxyl groups on phenyl rings (Table 1
and Table S2). This descriptor is extremely sensitive to the difference in di-OH substitution of the
phenyl ring. The value of Mor22u for the most active compound 11 (log %DPPH = 1.817), which
has two hydroxyl groups in position 2,3 is 0.3. Substitution of two OH groups in compound 12 at
the position 2,4 decreased values of Mor22u (0.216) and antioxidant activity (log %DPPH = 0.924).
Our recent study has also evinced sensitivity of the 3D-MoRSE descriptor Mor19e to the changes of
substituents and their position on the 3,4-ethylenedioxythiophene central unit [40].
Figure 2. Williams plot of the applicability domain of the the QSAR model for antioxidant activity
calculated by model (1).
The 2D autocorrelation molecular descriptor, MATS3m, corresponds to the Moran autocorrelation
descriptor –lag 3/weighted by atomic masses. The given descriptor describes how atomic mass is
distributed along a topological molecular structure, precisely indicating dependence of one atom on
value of mass through a topological structure of compounds [41]. Its positive regression coefficient
in models (1) and (2) suggests that the increased number of pairs of atoms higher atomic mass at
the path length 3 characterized compounds with enhanced antioxidant activity. The descriptor is
sensitive to kind and position of substituents on the phenyl ring (Table 1 and Table S2). The best
differences can be observed between compounds 11 and 14, as well as 12 and 15. Compounds

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bearing 2,3-dihydroxyphenyl (11) and 3,4-dihydroxyphenyl moieties (14) on benzene rings have two
oxygen atoms at path length 3. Consequently, these compounds have higher values of MATS3m
than compounds with 2,4-dihydroxyphenyl (12) and 3,5-dihydroxyphenyl (15) moieties, and exhibit
higher antioxidant activity. Hence, descriptor MATS3m has ability to distinguish the structure
with two hydroxyl groups in ortho position at the phenolic ring from structure with two hydroxyl
groups in meta position, the structural requirements relevant for the effective radical scavenging of
phenolic compounds.
The third variable in models (1) and (2) is hydrophilic index (Hy). Hydrophilic index takes
into account a total number of hydrogen atoms attached to oxygen, sulfur and nitrogen atoms, and
the number of carbon atoms in relation of number of non-hydrogen atoms [42]. Positive regression
coefficients of Hy in both models indicate that higher number of -OH and -NH groups are favorable
for antioxidant activity. This is expected since DPPH radicals become stable molecules by accepting
hydrogen radicals from organic substances [43]. Since the hydrophilic factor can predict the ability
of molecules to donate a hydrogen atom, it is an important descriptor in DPPH radical scavenging.
Hydrophilic index counts only the number of hydrogen donating groups, and it is independent
from their positions in molecules; therefore, its relative importance in models (1) and (2) is minimal.
Hydrophilic index has been found in previous QSAR studies of the DPPH radical scavenging activity
of coumarin derivatives. Results of those studies showed that incorporation of hydroxyl groups
in coumarin molecules enhanced hydrophilic index, which increased DPPH radical scavenging
effects [24,25].
2.4. Electrostatic Potential (ESP) Surface
Electrostatic potential (ESP) surface provides a visualization of total charge distribution of the
molecule and relative polarity of the molecule [44]. Figure 3 presents an ESP mapped density surface
of the most active molecule (11) and the least active molecule (9), for comparison. The red region
shows the greatest increase in electron density or region of negative ESP. The light blue shows about
zero, while the white color positive electron density. The negative regions (red color) correspond to the
aggregation of electron density. The positive regions, which increase in order blue < violet < white
color, indicate positive ESP that corresponds to the repulsion of the proton by the atomic nuclei or
nucleophilic reactivity [45].
(11) most active
(9) least active
(11) ESP
(9) ESP
ESP color map
Figure 3. Three-dimensional optimized structure (MM+, AM1) and electrostatic potential (ESP) surface
maps of the most active compound (11) and the least active compound (9).
ESP maps of compounds (
located mainly over the oxygen and nitrogen from imino groups (>C=N-) with minimum value of
0.0409 au. A weakly positive region (from 0.0045 to 0.0318 au) is localized mostly on the carbon
9) and (11) shows that the greatest negative electrostatic potential is
−

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atoms of phenyl rings. Highly positive electron density is located on the hydrogen atoms. Observing
ESP maps can help in the explanation of the role of substitution in antioxidant activity. Comparing the
ESP maps of molecules, it could be observed that positive charge is more spread over the phenyl ring
of inactive molecule (9). Negative charge of oxygen atoms from the 3-OCH3 group stabilizes positive
charge, decreasing the ability for electron transfer to the DPPH radical. On the contrary, a smaller
positive region in the phenyl ring of the most active molecule (11) allows easy electron transfer from
negative charges located on two oxygen atoms from 2,3-OH groups.
3. Materials and Methods
3.1. Chemistry
All the chemicals were of p.a. purity and purchased from commercial suppliers. Melting points
were determined on a capillary melting point apparatus (Electrothermal Engineering Ltd., Rochford,
UK) and are uncorrected. Thin-layer chromatography was performed with fluorescent silica gel plates
F254 (Merck, Darmstadt, Germany), under UV (254 and 365 nm) light, with benzene–acetone–acetic
acid (8:1:1, v/v) as a solvent. The mass spectra were recorded on liquid chromatography tandem
mass spectrometry (LC/MS/MS) API 2000 (Applied Biosystems/MDS SCIEX, Foster City, CA, USA).
NMR spectra were recorded on a Bruker Avance 600 MHz NMR Spectrometer (Bruker Biospin GmbH,
Rheinstetten, Germany) at 293 K in dimethylsulfoxide-d6 (DMSO-d6). The absorbance was measured
on UV visible spectrophotometer Helios γ (ThermoSpectronic, Cambridge, UK).
3.2. Preparation of DES
DES was prepared as described previously [7]. Briefly, choline chloride and malonic acid were
mixed together in molar ratio 1:1 and heated up to 80 ◦C until a clear liquid was obtained. This DES
was used as such in synthesis of desired compounds as described in Section 3.3.
3.3. Synthesis of Schiff Bases in DES Choline Chloride:Malonic Acid (1:1)
An equimolar ratio of 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide and corresponding
◦
aldehyde was mixed in DES and stirred at 70 C until the completion of reaction, which was monitored
by thin-layer chromatography (TLC). Upon addition of water, a crude product was precipitated and
washed with ethanol to obtain a pure Schiff base.
3.4. Characterization of Compounds
(E)-N0-(4-Methoxybenzylidene)-2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide (10). Yield 44%;
◦
m.p. = 263 C; 1H-NMR (ppm): 2.40 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 4.79–5.27 (s, 2H, CH2), 6.22 (s, H,
CH), 6.95–7.70 (7H, arom), 7.98 (s, H, CH), 11.53 (s, H, NH); 13C-NMR (ppm): 18.1, 55.3, 65.2, 101.5,
111.2, 112.3, 113.3, 114.2, 114.5, 126.3, 128.6, 128.8, 143.8, 147.9, 153.3, 154.5, 160.1, 160.8, 161.3, 163.3,
168.2; MS m/z: 365.20 [M − H]+, (Mr = 366.37).
(E)-N0-(4-(Dimethylamino)benzylidene)-2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide (29). Yield
70%; m.p. = 262 ◦C; Rf = 0.4; 1H-NMR (ppm): 2.40 (s, 3H, CH3), 2.97 (s, 6H, 2(CH3)), 4.76–5.25 (s, 2H,
CH2), 6.22 (s, H, CH), 6.72–7.54 (7H, arom), 7.89 (s, H, CH), 11.44 (s, H, NH); 13C-NMR (ppm): 18.6,
65.7, 101.9, 102.1, 111.7, 112.2, 112.8, 113.8, 121.7, 126.8, 127.0, 128.8, 145.3, 149.3, 151.9, 153.8, 155.0,
160.6, 161.9, 163.3, 168.2; MS m/z: 378.20 [M − H]+, (Mr = 379.41).
(E)-N0-((3-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyra◦zol-4-yl)methylene)-2-((4-methyl-2-oxo-2H-chromen-
1
7-yl)oxy)acetohydrazide (30). Yield 75%; m.p. = 200 C; Rf = 0.5; H-NMR (ppm): 2.40 (s, 3H, CH3),
4.82–5.20 (s, 2H, CH2), 6.22 (s, H, CH), 6.93–7.86 (12H, arom), 8.49 (s, H, CH), 11.82 (s, H, NH);
13C-NMR (ppm): 18.6, 65.5, 102.2, 111.7,113.9, 123.4,123.8, 126.8, 128.6, 129.3, 135.6, 142.9, 146.5, 153.8,
154.9, 160.6, 161.7, 164.3, 168.8; MS m/z: 529.30 [M + H]+, (Mr = 530.94).

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(E)-N0-((3-(4-Bromophenyl)-1-(p-tolyl)-1H-pyrazol-4-yl)methylene)-2-((4-methyl-2-oxo-2H-chromen-7-yl)
◦
oxy)acetohydrazide (31). Yield 83%; m.p. = 248–251 C; Rf = 0.47; 1H-NMR (ppm): 2.35 (s, 3H, CH3), 2.49
(s, 3H, CH3), 4.76–5.11 (s, 2H, CH2), 6.22 (s, H, CH), 6.88–7.87 (12H, arom), 8.97 (s, H, CH), 11.47 (s, H,
NH); 13C-NMR (ppm): 18.1, 20.4, 65.2, 101,7, 111.4,118.4, 121.8, 126.4, 129.9, 130.4, 131.4, 131.7, 136.5,
153.3, 154.5, 160.1; MS m/z: 570.80 [M − H]+, (Mr = 571.43).
(E)-N0-((3-(4-iodophenyl)-1-(p-tolyl)-1H-pyrazol-4-yl)methylene)-2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)
1
acetohydrazide (32). Yield 73%; m.p. = 265 ◦C; Rf = 0.45; H-NMR (ppm): 2.26 (s, 3H, CH3), 2.41 (s,
3H, CH3), 5.32 (s, 2H, CH2), 6.21 (s, H, CH), 6.95–7.79 (12H, arom), 8.97 (s, H, CH), 10.91 (s, H, NH);
13C-NMR (ppm): 18.1, 65.2, 95.8, 101.6, 111.2,112.3, 113.3, 126.3, 128.3, 129.5, 130.4, 137.5,147.8, 153.2,
154.6, 160.0, 161.4; MS m/z: 617.20 [M − H]+, (Mr = 618.08)
(E)-N0-((3-(4-methoxyphenyl)-1-(p-tolyl)-1H-pyrazol-4-yl)methylene)-2-((4-methyl-2-oxo-2H-chromen-7-yl)
oxy)acetohydrazide (33). Yield 93%; m.p. = 238 ◦C; Rf = 0.41; 1H-NMR (ppm): 2.36 (s, 3H, CH3), 2.40 (s,
3H, CH3), 3.79-3.83 (s, 3H, OCH3), 4.78–5.18 (s, 2H, CH2), 6.22 (s, H, CH), 7.05-8.12 (12H, arom), 8.94 (s,
H, CH), 11.48 (s, H, NH); 13C-NMR (ppm): 18.6, 20.9, 55.7, 101.9, 111.7,112.7, 114.4, 118.9, 126.9, 128.4,
130.2, 130.4, 136.7, 137.3, 147.8, 153.8, 168.3; MS m/z: 521.40 [M − H]+, (Mr = 522.2).
(E)-N0-((3-(4-Chlorophenyl)-1-(p-tolyl)-1H-pyrazol-4-yl)methylene)-2-((4-methyl-2-oxo-2H-chromen-7-yl)
oxy)acetohydrazide (34). Yield 65%; m.p. = 255 ◦C; Rf = 0.47; 1H-NMR (ppm): 2.36 (s, 3H, CH3), 2.40 (s,
3H, CH3), 4.79–5.13 (s, 2H, CH2), 6.23 (s, H, CH), 6.90–8.12 (12H, arom), 8.98 (s, H, CH), 11.50 (s, H,
NH); 13C-NMR (ppm): 18.6, 20.9, 101.9, 102.2, 111.7, 112.7, 119.1, 119.3, 126.9, 129.0, 129.2, 130.5, 130.6,
153.8; MS m/z: 525.30 [M − H]+, (Mr = 526.98).
(E)-N0-((1-(4-Chlorophenyl)-3-(4-nitrophenyl)-1H-pyra◦zol-4-yl)methylene)-2-((4-methyl-2-oxo-2H-chromen-7-
1
yl)oxy)acetohydrazide (35). Yield 69%; m.p. = 281 C; Rf = 0.42; H-NMR (ppm): 2.40 (s, 3H, CH3),
4.81–5.09 (s, 2H, CH2), 6.23 (s, H, CH), 6.86–8.47 (12H, arom), 9.11 (s, H, CH), 11.59 (s, H, NH);
13C-NMR (ppm): 18.6, 101.9, 111.8, 112.6, 121.5, 124.1, 124.2, 126.8, 130.0, 130.2, 168.4; MS m/z: 556.30
[M − H]+, (Mr = 557.94).
0
(E)-N -((4-Chloro-2-oxo-2H-chromen-3-yl)methylene)-2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide
(
36). Yield 78%; m.p. = 233 ◦C; Rf = 0.28; 1H-NMR (ppm): 2.40 (s, 3H, CH3), 4.78–5.25 (s, 2H, CH2),
6.21 (s, H, CH), 6.91–8.03 (12H, arom), 8.21 (s, H, CH), 11.98 (s, H, NH); 13C-NMR (ppm): 18.1, 65.2,
101.4, 111.3, 112.1, 113.4, 116.5, 118.4, 125.3, 126.1, 126.4, 133.6, 1370.5, 153.3, 154.5; MS m/z: 439.20
[M + H]+, (Mr = 438.82).
3.5. Determination of DPPH Scavenging Activity
DPPH scavenging activity for previously synthesized compounds [
2,
5] and new compounds
(30–
36) was performed according to the procedure described in our previous work [4
]. For each sample,
antioxidant activity determination was performed in triplicate and expressed as % scavenging activity
(% DPPH).
3.6. QSAR Studies
3.6.1. Data Set
The dataset used for building QSAR models consists of 36 molecules whose antioxidant activities
were measured and described in the present study. Antioxidant activity, expressed as % DPPH), were
converted in the form of the logarithm (log % DPPH) and presented in Table 1 together with structures
of molecules (While transformation of the experimental data to both logit % DPPH and log %DPPH
afforded a normal distribution, the latter approach afforded a QSAR model, which appears to provide
a better relationship between the structures of the molecules and their activities).

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3.6.2. Descriptor Calculation and Selection
The 3D structures of 36 molecules were optimized applying the Avogadro 1.2.0. (University
of Pittsburgh, Pittsburgh, PA, USA) [46] using the molecular mechanics force field (MM+) [47].
Subsequently, all structures were submitted to geometry optimization using the semiempirical AM1
method [48]. Two sets of descriptors were generated using Parameter Client (Virtual Computational
Chemistry Laboratory, an electronic remote version of the Dragon program [49]). In order to reduce the
huge number of calculated descriptors (about 1260), firstly, zero value descriptors were excluded from
the initial pool. Further exclusion was performed using QSARINS-Chem 2.2.1 (University of Insubria,
Varese, Italy) [50]: constant and semi-constant descriptors, i.e., those having chemical compounds
with a constant value for more than 80%, and descriptors that are too inter-correlated (>85%) were
rejected. Data sets were randomly divided into training (80%, Ntrain = 29) and test (20%, Ntest = 7) set
using QSARINS.
3.6.3. Regression Analysis and Validation of Models
The best QSAR models were obtained by Genetic Algorithm (GA) using QSARINS. The number
of descriptors (I) in the multiple regression equation was limited to three. The models have been
assessed by: fitting criteria; internal cross-validation using the leave-one out (LOO) method and
Y-scrambling; and external validation. Fitting criteria included: the coefficient of determination
(R2), adjusted (R2adj), cross-validate R2 using the leave-one-out method (Q2LOO), global correlation
among descriptors (Kxx), the difference between global correlation between molecular descriptors
and y the response variable, and global correlation among descriptors (
∆K), standard deviation of
regression (s), and Fisher ratio (F) [32 51 52]. Internal and external validations also included the
,
,
following parameters: root-mean-square error of the training set (RMSEtr); root-mean-square error
of the training set determined through the cross validated LOO method (RMSEcv), root-mean-square
error of the external validation set (RMSEex), squared correlation coefficients between the observed
and (leave-one-out) predicted values of the compounds with and without intercept (r2m ); concordance
correlation coefficient of the training set (CCCtr), test set using LOO cross validation (CCCcv), and of the
external validation set (CCCex), mean absolute error of the training set (MAEtr), mean absolute error
of the internal validation set (MAEcv) and mean absolute error of the external validation set (MAEex)
predictive residual sum of squares determined through cross-validated LOO method (PRESScv) in
the training set and in the external prediction set (PRESSex) [23,51]. The analysed external validation
parameters also include the coefficient of determination (R2ex). Robustness of QSAR models was tested
by a Y-randomisation test.
Investigation of the applicability domain of a prediction model was performed by leverage plot
(plotting residuals vs. leverage of training compounds). The warning leverage h* is defined as 3p0/n,
0
where n is the number of training compounds and p is the number of model adjustable parameters [37].
Tools of regression diagnostics as residual plots and Williams plots were used to check the quality of
the best models and define their applicability domain using QSARINS.
3.6.4. Visualization of Electrostatic Potential Surface
Electrostatic potential surface has been generated from optimized structures by ArgusLab 4.0.1
(Mark A. Thompson, Planaria Software LLC, Seattle, WA, USA). In an ESP-mapped density surface,
the electron density surface gives the shape of the surface, while the value of the ESP on that surface
gives the colors. Maximum mapped surface was set to 0.05, while the minimum to −0.05 a.u.
4. Conclusions
A series of novel coumarinyl Schiff bases in DES have been synthesized and evaluated for
antioxidant activity. Compared to previously synthesized coumarin derivatives, novel compounds
have not showed an improvement in antioxidant effect. QSAR study has clarified the importance of

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two hydroxyl groups in ortho position on phenyl ring and hydrophilicity for enhanced antioxidant
activity coumarin derivatives. Electrostatic potential surface provides a visualization of possible
regions in molecules that allow easy electron transfer to DPPH radicals.
Supplementary Materials: Supplementary materials are available online. File S1: 1H- and 13C-NMR spectra
of synthesized compounds; Table S2: Values of the descriptors included in models (1–2), as experimental and
calculated log %DPPH.
Acknowledgments: The research leading to these results has received funding from the European Union Seventh
Framework Programme (FP7 2007-2013) under Grant No. 291823 Marie Curie FP7-PEOPLE-2011-COFUND
(the new International Fellowship Mobility Programme for Experienced Researchers in Croatia—NEWFELPRO).
This presentation has been prepared as a part of a project “Synthesis and characterization of some chalcone based
heterocyclic compounds and their biological screening as potential in vitro antioxidant agent”, which has received
funding through the NEWFELPRO project under Grant No. 84.
Author Contributions: Maja Molnar, Mario Komar and Harshad Brahmbhatt conceived, designed the experiments
and performed the experiments; Vesna Rastija performed QSAR analysis and wrote the paper together with
Maja Molnar, while Jurislav Babic´ and Stela Jokic´ helped with experiment design and feasibility.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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Sample Availability: Samples of the compounds 3–36 are available from the authors.
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