Synthesis, antioxidant activity and SAR study of novel spiro-isatin-based Schiff bases

Article abstract of DOI:10.1007/s11030-018-09910-7

Abstract: A new series of 21 Schiff bases of spiro-isatin was synthesized, and their DPPH, CUPRAC and ABTS cation radical scavenging abilities were investigated for antioxidant activity. The results showed that all the synthesized compounds exhibited anti

Full text of DOI:10.1007/s11030-018-09910-7

Molecular Diversity
https://doi.org/10.1007/s11030-018-09910-7
ORIGINAL ARTICLE
Synthesis, antioxidant activity and SAR study of novel
spiro‑isatin‑based Schif bases
Fatih Sonmez1 · Zuhal Gunesli1 · Belma Zengin Kurt2 · Isil Gazioglu3 · Davut Avci4 · Mustafa Kucukislamoglu5
Received: 22 September 2018 / Accepted: 13 December 2018
© Springer Nature Switzerland AG 2019
Abstract
A new series of 21 Schif bases of spiro-isatin was synthesized, and their DPPH, CUPRAC and ABTS cation radical scav-
enging abilities were investigated for antioxidant activity. The results showed that all the synthesized compounds exhib-
ited antioxidant activity for each assay. 5̍-(2,3-Dihydroxybenzylideneamino)spiro[[1,3] dioxolane-2,3̍-indoline]-2̍-on (5c)
(IC50 =4.49 µM, for DPPH; IC50 =0.39 µM, for ABTS.+; and A0.50 =0.42 µM, for CUPRAC) showed signifcantly better
ABTS, CUPRAC and DPPH radical scavenging ability than quercetin (IC50 =8.69 µM, for DPPH; IC50 =15.49 µM, for
ABTS.+; and A0.50 =18.47 µM, for CUPRAC), which is used as a standard. SAR study showed that the synthesized com-
pounds had higher ABTS.+ activity than DPPH and CUPRAC activities. Moreover, the compounds (5c and 5d), containing
two hydroxyl groups, exhibited the highest antioxidant activities for all assays. Quantum chemical calculations were also
carried out to support SAR results.
Graphical abstract
Keywords ABTS · Antioxidant activity · CUPRAC · DPPH · Isatin · Schif base
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-018-09910-7) contains
supplementary material, which is available to authorized users.
3
* Fatih Sonmez
Department of Analytical Chemistry, Faculty of Pharmacy,
Bezmialem Vakif University, 34093 Istanbul, Turkey
fsonmez@sakarya.edu.tr
4
5
Department of Physics, Sakarya University, 54100 Sakarya,
Turkey
1
Pamukova Vocational High School, Sakarya University
of Applied Sciences, 54900 Sakarya, Turkey
Department of Chemistry, Sakarya University,
54100 Sakarya, Turkey
2
Department of Pharmaceutical Chemistry, Faculty
of Pharmacy, Bezmialem Vakif University, 34093 Istanbul,
Turkey
Vol.:(0123456789)
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Molecular Diversity
mammals at different concentrations [12]. Most of the
isatin derivatives have shown a variety biological activi-
ties, such as antimicrobial [13], antioxidant [14], anticancer
[15], MAO inhibitor [16], antibacterial [17], α-glucosidase
inhibitor [18], and antitubulin agent [19]. The presence of
the indole ring in the structure of anticancer drugs such as
sunitinib causes this skeletal structure to be subject to many
anticancer-related investigations [20–22]. The classic phe-
nolic compounds, aromatic and heterocyclic amines having
N–H bond functions showed antioxidant properties. It has
also been reported that most of the isatin derivatives have
potential antioxidant activity [23, 24], and the lactam ring
of isatin is responsible for having free radical scavenging
activity due to its N–H and C=O moieties [25, 26].
Introduction
Reactive oxygen species (ROS) play an important role in
the formation of various serious diseases, such as cancer,
heart diseases, diabetes, arteriosclerosis and cataracts. The
harmful efects of free radicals that cause potential biologi-
cal damage are called oxidative stress [1]. Free radicals in
the human body play a pathogenic role in the formation of
many chronic degenerative diseases such as cancer, auto-
immune, infammatory and cardiovascular neurodegenera-
tive diseases [2–4]. Free radicals are one or more unpaired
electron-donating molecules, and they have the short half-
life, low stability and high chemical reactivity [5, 6]. These
radicals arise naturally or due to some biological functions
related to phagocytosis, regulation of cell proliferation,
synthesis of substances and signalling between cells. Radi-
cals damage lipids, proteins or DNA [7]. In order to protect
the tissue from these damages, it is important that the free
radicals are put into an inefective state [8]. For this reason,
antioxidants have been shown to play an important role in
protecting people against many fatal diseases [9, 10]. Anti-
oxidants (Fig. 1) are defned as substances that delay, inhibit
or eliminate oxidative damage to a target molecule when
present in low concentrations in food or in the body. The
human body has developed various mechanisms against the
oxidative stress by naturally producing antioxidants in situ
(endogenous antioxidants) as well as by providing antioxi-
dants through food (exogenous antioxidants) [11].
Schif bases are another important class of organic com-
pounds. It is also known that Schif bases have various phar-
macological activities, such as antibacterial, antifungal, anti-
malarial, anti-infammatory and antipyretic as well as their
use as dyes, pigments, starting materials, organic synthesis
intermediates, catalysts and polymer stabilizers. The imine
group present in the Schif bases is responsible for biological
activities, and these activities can be modulated in general
by modifying the substituent groups in the molecules. In
addition, Schif bases are known as excellent ligands because
the imine groups, forming chelates with metal ions, show
strong afnity to transition metal ions [27, 28].
In this study, on the basis of the reported evidences, new
Schif bases, containing spiro-isatin derivatives, which can
show strong antioxidant activity, taking into account the
known properties of isatin and Schif bases, were synthe-
sized. The antioxidant activities of the synthesized com-
pounds were determined using three different methods
(ABTS, CUPRAC and DPPH), and structure–activity rela-
tionships were examined in detail. The frontier molecular
Isatin (1H-indole-2,3-dione) is an important chemical
building block. In humans, it is also found as a metabolic
by-product of adrenaline and also in tissues and fuids of
Fig. 1 The structures of some well-known antioxidant compounds
1 3

Molecular Diversity
orbitals (FMOs) energies of the selected compounds were
obtained by using the B3LYP/6-311G(d,p) level.
It is known that protecting by ethylene glycol is sensi-
tive to pH value. Thus, in order to exhibit the diversity of
spiro, the selected compound 5u was mixed under the test
condition (pH = 6.3 for ABTS and pH = 8.6 for DPPH).
After that, 1H and 13C NMR of the products were acquired
(Fig. 2). The presence of the signals of spiro moiety (at
~ 4.25 ppm for 1H NMR and at ~ 66.3 ppm for 13C NMR)
confrms the structure is spiro-isatin derivatives not the
isatin derivatives under the test conditions for ABTS and
DPPH. It is not needed for CUPRAC assays due to carry
out in neutral media (using NH4CH3COO bufer, pH = 7).
Results and discussion
Chemistry
The synthetic procedures employed to obtain the target
compounds 5a–u are depicted in Scheme 1. 5-Nitroisatin
(2) was synthesized from isatin according to the literature
[29]. Due to having high reactivity, the carbonyl group of
isatin was protected with ethylene glycol [30]. The nitro
moiety of 5-nitrospiro-isatin (3) was reduced with Pd/C,
and 5-aminospiro-isatin (4) was reacted with diferent
aldehyde derivatives to obtain Schif base (5a–u) [31].
All the new compounds were characterized by 1H NMR,
Antioxidant activity assay
It has been previously reported that antioxidant capacity
determined by in vitro assays may difer from each other.
Diferences between DPPH and ABTS radical scavenging
activities can be ascribed to reaction media. The DPPH
assay is conventionally conducted under 50% ethanol/water,
while the ABTS assay is carried out in aqueous conditions.
Besides, compounds solubility in both media should be
taken in consideration. Certain bioactive compounds may
not soluble into reaction media and cannot express radical
scavenging activities. Otherwise, the antioxidant capacities
of the compounds depend on the mechanism of the assay.
While the reaction runs on the cation radical in the ABTS
assay, the reaction runs over free radical in the DPPH assay.
In the CUPRAC assay, the antioxidant capacity depends on
copper ion reduction power of the compound [32]. Conse-
quently, in this study, these three diferent methods have
been used for the determination of antioxidant capacity.
1
13C NMR, IR, MS and elemental analysis. H NMR, 13C
NMR, IR, MS of the 5a–t are given in the Supplemen-
tary Materials. In the infrared spectra of the synthesized
compounds, it was possible to observe the absorptions
between 3140 and 3300 cm−1 relating to NH stretch
for indole group, between 3350 and 3440 cm−1 relat-
ing to OH stretch for hydroxyl group, about 1520 cm−1
relating to C=N stretch for imine, absorptions in about
1700-1746 cm−1 from isatin carbonyl moiety stretch and
absorptions between 1190 and 1230 cm−1 from acetal
1
C–O moiety stretching. From the H NMR spectra, the
signal of proton NH at indole ring was between 10.50
and 10.65 ppm. The signals for aromatic hydrogens were
observed between 6.70 and 7.90 ppm, and the signal of
proton at imine group was detected about 8.60 ppm. The
signals for aliphatic hydrogens were observed between
4.25 and 4.40 ppm. From the 13C NMR spectra, the sig-
nals can be seen at 175 ppm, relating to isatin carbonyl
moiety. This is followed by the sign about 159 ppm for
imine carbon and about 90-150 ppm for aromatic carbons.
DPPH free radical scavenging assay
DPPH method is commonly used to measure the abil-
ity of antioxidants to sweep free radicals. In this spec-
trophotometric method, a stable free radical, DPPH
Scheme 1 Synthesis of new
Schif base-substituted spiro-
isatin derivatives. Reaction
conditions; (i) KNO3, H2SO4,
2 h, rt; (ii) ethylene glycol,
PTSA, benzene, 24 h, refux;
(iii) cyclohexene, 10% Pd–C,
EtOH, 2 h, refux; (iv) aldehyde
derivatives, Et3N, EtOH, 8 h,
refux
1 3

Molecular Diversity
Fig. 2 a 1H NMR of 5u before antioxidant assay; b 1H NMR of 5u after mixing pH 6.3; c 1H NMR of 5u after mixing pH 8.6; d 13C NMR of 5u
before antioxidant assay; e 13C NMR of 5u after mixing pH 6.3
(2,2-diphenyl-1-picrylhydrazyl), is used [33]. Antioxidants
are based on the ability to degrade the DPPH radical, and
when it interacts with radical hydrogen donors, hydrazine
is reduced. According to this method, compounds must
have strong hydrogen donor groups to exhibit good anti-
oxidant properties. The IC50 values of the synthesized
compounds were between 4.49 µM and 204.90 µM for
DPPH activity. Among them, only 5c (IC50 = 4.49 µM)
showed stronger antioxidant activity than quercetin
(IC50 = 8.69 µM). Other compounds showed lower DPPH
activity than quercetin (Table 1).
1 3

Molecular Diversity
Table 1 IC50 and A0.50 values (µM) of synthesized imine derivatives for antioxidant activities
Compound
R
DPPH
ABTS·+
CUPRAC
A
0.50 (µM)b
IC50 (µM)a
IC50 (µM)a
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
H
3-OH
171.74 1.24
94.81 0.83
4.49 0.45
18.45 1.31
1.93 0.33
0.39 0.02
0.86 0.03
1.57 0.05
6.83 0.84
1.45 0.28
0.95 0.01
1.56 0.23
1.25 0.14
1.06 0.20
0.98 0.22
2.43 0.11
1.48 0.32
1.28 0.11
1.85 0.23
1.45 0.45
1.24 0.14
19.2 0.57
1.56 0.46
1.38 0.56
15.49 2.33
75.87 0.04
18.76 0.69
0.42 0.02
2,3-di-OH
2,5-di-OH
2,4,6-tri-OH
4-OCH3
3,4-di-OCH3
2,5-di-OCH3
4-N(CH3)2
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-NO2
3-NO2
4-NO2
–
18.65 0.03
64.03 0.39
145.00 0.25
130.00 0.20
81.78 0.38
112.33 2.49
125.4 0.63
137.75 2.67
90.15 0.59
129.40 0.46
178.91 0.37
110,95 0,72
143.90 1.85
204.90 3.52
127.16 0.97
162.91 0.98
126.62 0.21
118.86 0.68
8.69 0.24
1.35 0.01
12.99 1.13
28.74 0.03
28.06 0.05
17.59 0.99
15.38 0.38
29.38 0.02
24.76 0.38
16.99 1.47
26.31 0.05
18.13 1.56
16.67 0.40
20.92 0.42
16.90 0.68
16.15 0.56
15.12 0.23
19.23 0.50
26.03 0.04
18.47 0.03
5s
5t
5u
Quercetin
aIC50 values represent the mean SEM of three parallel measurements (p<0.05)
bA0.50 values represent the mean SEM of three parallel measurements (p<0.05)
ABTS cation radical scavenging assay
CUPRAC assay
The ABTS method is based on the ability of hydrogen or
electron-donating antioxidants to decolorize the performed
radical monocation of 2,2′-azino-bis(3-ethylbenzothiazo-
line-6-sulphonic acid) generated due to oxidation of ABTS
with potassium persulfate [34]. The results indicated that
the synthesized compounds exhibited good radical scaveng-
ing ability (Table 1.). IC50 values of the synthesized com-
pounds ranged from 0.39 µM to18.45 µM for ABTS·+ activ-
ity. They (except 5a and 5s) showed stronger antioxidant
properties than the standard quercetin (IC50 = 15.49 µM).
Among them, 5c, 5d, 5h and 5l (IC50 =0.39, 0.86, 0.95 and
0.98 µM, respectively) showed signifcantly better activity
than quercetin.
As a distinct advantage over other electron-transfer-based
assays (e.g. Folin, FRAP, ABTS, DPPH), CUPRAC is supe-
rior in regard to its realistic pH close to the physiological
pH, favourable redox potential, accessibility and stability of
reagents, and applicability to lipophilic antioxidants as well
as hydrophilic ones. The cupric reducing antioxidant capaci-
ties of the synthesized compounds (5a–u) were determined
according to the literature method [35] using quercetin as
the standard compound. A0.50 values of the synthesized com-
pounds were between 0.42 µM and 75.87 µM for CUPRAC
activity. 5c, 5d and 5e (A0.50 = 0.42, 1.35 and 12.99 µM,
respectively) showed better cupric reducing antioxidant
activity than quercetin (A0.50 =18.47 µM). The others have
1 3

Molecular Diversity
Fig. 3 The proposed mechanism of the synthesized compounds for antioxidant assays; (i) DPPH, (ii) ABTS, (iii) CUPRAC
Fig. 4 The formation of quinone by oxidizing 5c
1 3

Molecular Diversity
Fig. 5 The formation of quinone by oxidizing 5d
so much less or the similar cupric reducing antioxidant
capacity than quercetin (Table 1).
shown in Fig. 3. According to this proposed mechanism,
the possible products of the best antioxidant compounds (5c
and 5d) in this study are given in Figs. 4 and 5, respectively.
These two compounds have phenolic moiety, so it is pos-
sible they can be converted to the corresponding quinones
after the antioxidant assays. It is known that quinones are
used as drugs to treat human cancer. Most of the quinones
having antitumor activity can expose reversible enzymatic
reduction and oxidation. The antitumor activity of quinones
is related to DNA damage caused by alkylating species or
oxygen radicals [38]. Therefore, this formation of quinones
may be advantageous to treat some disease.
The synthesized compounds include the indole ring,
phenyl ring and the imine moiety as a linker between these
rings. The presence of this imine group allows for conju-
gation between two aromatic rings and thus increases the
resonance stabilization over the whole molecule. The high
electron conjugation makes the formed radical more stable.
Consequently, it considers that this feature enhances the
ability of the synthesized compounds for acting as a potent
antioxidant [36, 37]. The predicted mechanism of the syn-
thesized compounds to act as a scavenger can propose as
1 3

Molecular Diversity
4. Moving the halogen atoms (F, Cl or Br) on the phenyl
ring from ortho-position to meta- and para-positions
led to an increase in ABTS·+ and CUPRAC activities
(comparing 5j (R = 2-F, IC50 = 1.25 µM for ABTS·+
and A0.50 =29.38 µM for CUPRAC) with 5k (R=3-F,
IC50 = 1.06 µM for ABTS·+ and A0.50 = 24.76 µM
for CUPRAC) and 5l (R = 4-F, IC50 = 0.98 µM for
ABTS·+ and A0.50 = 16.99 µM for CUPRAC); com-
paring 5m (R = 2-Cl, IC50 = 2.43 µM for ABTS·+ and
A0.50 = 26.31 µM for CUPRAC) with 5n (R = 3-Cl,
IC50 = 1.48 µM for ABTS·+ and A0.50 = 18.13 µM
for CUPRAC) and 5o (R = 4-Cl, IC50 = 1.28 µM for
ABTS·+ and A0.50 = 16.67 µM for CUPRAC); com-
paring 5p (R = 2-Br, IC50 = 1.85 µM for ABTS·+ and
A0.50 = 20.92 µM for CUPRAC) with 5q (R = 3-Br,
IC50 = 1.45 µM for ABTS·+ and A0.50 = 16.90 µM for
CUPRAC) and 5r (R=4-Br, IC50 =1.24 µM for ABTS·+
and A0.50 =16.15 µM for CUPRAC)). On the other hand,
the compounds (5k (R = 3-F, IC50 = 137.75 µM for
DPPH), 5n (R=3-Cl, IC50 =178.91 µM for DPPH) and
5q (R = 3-Br, IC50 = 204.90 µM for DPPH)), contain-
ing halogens at the meta-position of the phenyl ring,
showed lower DPPH activity than the compounds (5j
(R = 2-F, IC50 = 125.40 µM for DPPH), 5m (R = 2-Cl,
IC50 = 129.40 µM for DPPH) and 5p (R = 2-Br,
IC50 = 143.90 µM for DPPH)), containing halogens
at the ortho-position. In addition, the compounds (5l
(R = 4-F, IC50 = 90.15 µM for DPPH), 5o (R = 4-Cl,
IC50 = 110.95 µM for DPPH) and 5r (R = 4-Br,
IC50 = 127.16 µM for DPPH)), containing halogens at
the para-position of the phenyl ring, exhibited the high-
est DPPH activity among the halogen series.
Structure–activity relationships (SAR)
Generally, the synthesized compounds had higher ABTS.+
activity than DPPH and CUPRAC activities. Moreover, the
compounds (5c and 5d), containing two hydroxyl groups,
exhibited the strongest antioxidant activities for all assays.
The following structure–activity relationship (SAR)
observations can also be drawn from data of Table 1:
1. The presence of OH group on the phenyl ring increased
the antioxidant activities (comparing 5a (R = H,
IC50 =171.74 µM and 18.45 µM for DPPH and ABTS·+,
respectively; A0.50 = 75.87 µM for CUPRAC) with 5b
(R=3-OH, IC50 =94.81 µM and 1.93 µM for DPPH and
ABTS·+, respectively; A0.50 =18.76 µM for CUPRAC)).
In addition, the binding of the second –OH group to
the phenyl ring caused a signifcant increase for all the
antioxidant activities (comparing 5b (R = 3-OH) with
5c (R = 2,3-di-OH, IC50 = 4.49 µM and 0.39 µM for
DPPH and ABTS·+, respectively; A0.50 = 0.42 µM for
CUPRAC) and 5d (R = 2,5-di-OH, IC50 = 18.65 µM
and 0.86 µM for DPPH and ABTS·+, respectively;
A0.50 = 1.35 µM for CUPRAC)). On the other hand,
the presence of the third –OH group on the phenyl
ring decreased the antioxidant activities (comparing
5c (R = 2,3-di-OH) and 5d (R = 2,5-di-OH) with 5e
(R = 2,4,6-tri-OH, IC50 = 64.03 µM and 1.57 µM for
DPPH and ABTS·+, respectively; A0.50 =12.99 µM for
CUPRAC)); however, 5e still had higher antioxidant
activities than 5a (R=H) and 5b (R=3-OH).
2. The compounds (5b (R=3-OH), 5c (R=2,3-di-OH) and
5d (R = 2,5-di-OH)), containing one or two hydroxyl
groups, exhibited better antioxidant activities than
the compounds (5f (R = 4-OCH3, IC50 = 145.00 µM
and 6.83 µM for DPPH and ABTS·+, respectively;
A0.50 =28.74 µM for CUPRAC), 5 g (R=3,4-di-OCH3,
IC50 =130.00 µM and 1.45 µM for DPPH and ABTS·+,
respectively; A0.50 = 28.06 µM for CUPRAC) and 5 h
(R = 2,5-di-OCH3, IC50 = 81.78 µM and 0.95 µM for
DPPH and ABTS·+, respectively; A0.50 =17.59 µM for
CUPRAC)), containing one or two methoxy groups. On
the other hand, the presence of the second –OCH3 group
on the phenyl ring decreased the antioxidant activities
(comparing 5f (R = 4-OCH3) with 5c (R = 2,3-di-OH)
and 5d (R=2,5-di-OH)).
5. The DPPH and CUPRAC activities seem to be strongly
dependent on the size and polarizability of the halogen
substituent at the ortho-, meta- and para-positions of the
phenyl ring. The DPPH activity of the compounds con-
taining halogens decreased with the growing size of the
halogens, whereas CUPRAC activity of them increased
(for size and polarizability, Br > Cl > F; for DPPH
activity, 5j (R=2-F, IC50 =125.40 µM)>5m (R=2-Cl,
IC50 = 129.40 µM) > 5p (R = 2-Br, IC50 = 143.90 µM);
5k (R = 3-F, IC50 = 137.75 µM) > 5n (R = 3-Cl,
IC50 = 178.91 µM) > 5q (R = 3-Br, IC50 = 204.90 µM);
5 l (R = 4-F, IC50 = 90.15 µM) > 5o (R = 4-Cl,
IC50 =110.95 µM)>5r (R=4-Br, IC50 =127.16 µM); for
CUPRAC activity, 5p (R=2-Br, A0.50 =20.92 µM)>5m
(R = 2-Cl, A0.50 = 26.31 µM) > 5j (R = 2-F,
A0.50 =29.38 µM); 5q (R=3-Br, A0.50 =16.90 µM)>5n
(R = 3-Cl, A0.50 = 18.13 µM) > 5 k (R = 3-F,
A0.50 =24.76 µM); 5r (R=4-Br, A0.50 =16.15 µM)>5o
(R = 4-Cl, A0.50 = 16.67 µM) > 5l (R = 4-F,
A0.50 =16.99 µM)). This relation was not observed for
the ABTS·+ activity.
3. The exchanging methoxy group at para-position of
phenyl ring with dimethylamine group increased the
antioxidant activities (comparing 5f (R = 4-OCH3,
IC50 =145.00 µM and 6.83 µM for DPPH and ABTS·+,
respectively; A0.50 = 28.74 µM for CUPRAC) with 5i
(R = 4-N(CH3)2, IC50 = 112.33 µM and 1.56 µM for
DPPH and ABTS·+, respectively; A0.50 =15.38 µM for
CUPRAC)).
1 3

Molecular Diversity
Table 2 FMO energies of the selected compounds
Comp.
R
EHOMO (eV)
ELUMO (eV)
ΔE (eV)
A (eV)
η(eV)
S (eV)−1
χ (eV)
Etot(au)
5c
5d
5h
5j
5k
5l
2,3-di-OH
2,5-di-OH
2,5-di-OCH3
2-F
3-F
4-F
−5.905
−5.660
−5.605
−5.905
−5.986
−5.959
−5.959
−5.905
−2.068
−1.823
−1.769
−2.041
−2.068
−2.122
−2.095
−1.932
3.837
3.837
3.836
3.864
3.918
3.837
3.864
3.973
2.068
1.823
1.769
2.041
2.068
2.122
2.095
1.932
1.919
1.919
1.918
1.932
1.959
1.919
1.932
1.987
0.521
0.521
0.521
0.518
0.510
0.521
0.518
0.503
3.987
3.742
3.687
3.973
4.027
4.041
4.027
3.919
−1090.91
−1142.13
−1220.75
−1142.15
−1090.91
−1090.91
−1451.27
−3565.19
5o
5r
4-Cl
4-Br
A electron afnity, η chemical hardness, χ electronegativity, S chemical softness, E total energy
6. Moving the nitro groups on the phenyl ring from ortho-
position to meta- and para-positions led to an increase in
DPPH and ABTS·+ activities (comparing 5s (R=2-NO2,
IC50 =162.91 µM and 19.20 µM for DPPH and ABTS·+,
respectively) with 5t (R=3-NO2, IC50 =126.62 µM and
1.56 µM for DPPH and ABTS·+, respectively) and 5u
(R=4-NO2, IC50 =118.86 µM and 1.38 µM for DPPH
and ABTS·+, respectively)); however, it led to a decrease
in the CUPRAC activity (comparing 5s (R = 2-NO2,
A0.50 = 15.12 µM for CUPRAC) with 5t (R = 3-NO2,
A0.50 = 19.23 µM for CUPRAC) and 5u (R = 4-NO2,
A0.50 =26.03 µM for CUPRAC)).
–OCH3 at the same position, 5j, 5k and 5l were chosen due
to comparing the efects of the substituent position, and
5l, 5o and 5r were selected to investigate halogen series
at the same position.
It is well known that the molecular chemical stability,
chemical reactivity and spectroscopic properties of the com-
pounds are connected with the relationship between energy
gap and the frontier molecular orbitals (FMOs). Generally,
highest occupied molecular orbital (HOMO) and lowest
unoccupied molecular orbital (LUMO) energies are related
to electron afnity. These FMOs are associated with the
molecule’s reactivity. HOMO energy is closely related to
susceptibility to electrophilic attack, while LUMO energy
is closely related to susceptibility to nucleophilic attack [39,
40].
In addition, some electronic parameters (the highest
occupied molecular orbital and the lowest unoccupied
molecular orbital (HOMO–LUMO) energy levels, elec-
tronegativity, chemical hardness and softness, total energy
and dipole moments) of the selected compounds were cal-
culated to support and enrich the SAR of obtained experi-
mental results (Table 2). For quantum chemical calcula-
tions, 5c was selected because of the best inhibitor, 5d
and 5h were selected to compare the efects of –OH and
Rising the LUMO energies decreased the antioxi-
dant activities (comparing 5c (ELUMO = − 2.068 eV) with
5d (ELUMO = − 1.823 eV) and 5h (ELUMO = − 1.769 eV),
5c > 5d > 5h for each antioxidant activity; comparing 5j
(ELUMO =−2.041 eV) with 5k (ELUMO =−2.068 eV) and 5l
(ELUMO =−2.122 eV), 5l>5k>5j for ABTS and CUPRAC
activity, 5l > 5j ~ 5k for DPPH activity; comparing 5l
1 3

Molecular Diversity
(ELUMO = − 2.122 eV) with 5o (ELUMO = − 2.095 eV) and
5r (ELUMO = − 1.932 eV), 5l > 5o > 5r for DPPH activity,
5l > 5o ~ 5r for ABTS activity; 5l ~ 5o ~ 5r for CUPRAC
activity). It is known that low LUMO energy increases the
acceptor properties of the molecules, and they can get elec-
trons easily.
structure–activity relationship was presented and also it was
supported with quantum chemical calculations. All the syn-
thesized compounds exhibited higher ABTS cation radical
scavenging ability than DPPH and CUPRAC activities. 5c,
containing two hydroxyl groups on the phenyl ring, had the
most potent antioxidant activity according to three diferent
antioxidant activity methods. The SAR study revealed that
the antioxidant activity of the synthesized compounds could
also be infuenced by the type and position of substituent on
the phenyl ring. In particular, the presence of the hydroxyl
groups and the position and size of the halogens on the phe-
nyl ring were seen to play a key role for antioxidant activity.
The quantum chemical calculations showed that the LUMO
energies, electronegativity and chemical softness of the mol-
ecules were efective on their antioxidant activities.
The electronegativity of the molecules showed a linear
relationship with the increasing antioxidant activity (com-
paring 5c (χ = 3.987 eV) with 5d (χ = 3.742 eV) and 5h
(χ = 3.687 eV), 5c > 5d > 5h for each antioxidant activity;
comparing 5j (χ=3.973 eV) with 5k (χ=4.027 eV) and 5l
(χ=4.041 eV), 5l>5k>5j for ABTS and CUPRAC activity,
5l>5j~5k for DPPH activity; comparing 5l (χ=4.041 eV)
with 5o (χ=4.027 eV) and 5r (χ=3.919 eV), 5l>5o>5r
for DPPH activity, 5l>5o~5r for ABTS activity; 5l~5o~5r
for CUPRAC activity). This relationship can be explained
that the high electronegativity increases the electron afnity
of the molecule and so its antioxidant activity is enhanced.
Also, the high electronegativity of molecule can give stabil-
ity to occurring radical.
Experimental
General methods
It was seen that 5c, 5d and 5h have same chemical
hardness (η) and softness (S). On the other hand, increas-
ing chemical softness and conversely decreasing chemi-
cal hardness enhanced the antioxidant activity comparing
5j (η=1.932 eV, S=0.518 (eV)−1)) with 5k (η=1.959 eV,
S=0.510 (eV)−1)) and 5l (η=1.919 eV, S=0.521 (eV)−1)),
5l > 5j > 5k for DPPH activity, 5l> 5k > 5j for ABTS and
CUPRAC activity; comparing 5l (η = 1.919 eV, S = 0.521
(eV)−1)) with 5o (η = 1.932 eV, S = 0.518 (eV)−1)) and 5r
(η = 1.987 eV, S = 0.503 (eV)−1)), 5l > 5o > 5r for DPPH
activity, 5l > 5o ~ 5r for ABTS activity; 5l ~ 5o ~ 5r for
CUPRAC activity). According to “hard and soft (Lewis)
acids and bases” (HSAB) theory, soft acids react faster and
form stronger bonds with soft bases, whereas hard acids
react faster and form stronger bonds with hard bases [41].
Radicals are soft both as an acceptor or donor [42]. There-
fore, raising softness of the molecule can increase its anti-
oxidant activity as expected.
Melting points were measured on a Barnstead Electrother-
mal 9200. IR spectra were measured on a Shimadzu Pres-
tige-21 (200 VCE) spectrometer. 1H and 13C NMR spectra
were measured on a Varian Infnity Plus spectrometer at 300
1
and at 75 Hz, respectively. H and 13C chemical shifts are
referenced to the internal deuterated solvent. Mass spectra
were obtained using MICROMASS Quattro LC–MS–MS
spectrometer. The elemental analyses were performed with
a Leco CHNS-932 instrument. Spectrophotometric analy-
ses were performed by a BioTek Power Wave XS (BioTek,
USA). The chemicals and solvents were purchased from
Fluka Chemie, Merck, Alfa Easer and Sigma-Aldrich.
Synthesis procedures and spectral data
5‑Nitroindoline‑2,3‑dione (2) 3 mmol of KNO3 and
H2SO4 was stirred in an ice bath. 3 mmol of isatin was
slowly added to this mixture. The mixture was stirred for
2 h at room temperature. Finally, it was poured into ice, the
precipitate was fltered of and dried under vacuum.
Yellow powder, 96% yield, 1H NMR (DMSO-d6,
300 MHz) δ/ppm: 7.07 (1H, d, J = 8.7 Hz), 8.18 (1H, d,
J=2.0 Hz), 8.41-8.43 (1H, dd, J=2.0, 8.5 Hz), 11.66 (1H,
s, NH). 13C NMR (DMSO-d6, 75 MHz) δ/ppm: 113.2, 118.8,
120.3, 133.8, 143.2, 155.8, 160.6, 183.0.
These quantum chemical calculations support struc-
ture–activity relationship of the compounds including simi-
lar group; however, considering all molecules, there are
some contradictions. It is clear that the antioxidant activities
of the compounds not only depend on their LUMO energies,
electronegativity or chemical hardness/softness, but also on
resonance stability of the formed radical signifcant efects
on antioxidant activity.
Conclusion
5′‑Nitrospiro[[1,3] dioxolane‑2,3′‑indolin]‑2′‑one
(3) 15 mmol of 5-nitroindoline-2,3-dione (2), 60 mmol
of ethylene glycol and 3 mmol of p-toluene sulphonic acid
(PTSA) in benzene were stirred in a Dean–Stark apparatus
at refux temperature for 24 h. The mixture was cooled and
In conclusion, a series of 21 novel Schiff base-substi-
tuted spiro-isatin derivatives (5a–u) was synthesized and
their antioxidant activities were evaluated. In addition,
1 3

Molecular Diversity
1
evaporated in vacuum. The residue was extracted with ethyl
acetate (3×50 mL), and the organic phase was washed with
aqueous NaHCO3. It was dried with MgSO4 and evaporated
in vacuum.
683 cm−1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.25–
4.29 (2H, m),4.33–4.38 (2H, m), 6.88 (2H, td, J1=2.4 Hz,
J2=7.3 Hz), 7.27-7.35 (5H, m), 8.57 (1H,s), 9.68 (1H,s,OH),
10.51 (1H,s,NH); 13C NMR (DMSO-d6, 75 MHz) δ/ppm:
66.3, 102.4, 111.7, 111.8, 118.0, 111.3, 120.9, 126.2, 126.4,
130.5, 138.2, 141.7, 146.8, 158.3, 159.8, 175.2; LC–MS
(m/z): 311.2070 [MH+]. Anal. Calcd. for C17H14N2O4: C,
65.80; H, 4.55; N, 9.03; found: C, 65.85; H, 4.51; N, 9.07.
Yellowish powder, 83% yield, 1H NMR (CDCl3 +DMSO-
d6, 300 MHz) δ/ppm: 4.34–4.42 (2H, m), 4.50–4.58 (2H,
m), 6.96 (1H, d, J=8.8 Hz), 8.19 (1H, d, J=2.3 Hz), 8.22–
8.25 (1H, dd, J=2.3, 8.8 Hz), 10.60 (1H, s, NH); 13C NMR
(CDCl3 + DMSO-d6, 75 MHz) δ/ppm: 66.2, 101.4, 111.0,
121.3, 125.6, 128.5, 143.4, 149.4, 175.5.
(E)‑5′‑((2,3‑dihydroxybenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5c) Dark brown powder,
85.8% yield, mp. 265.5–266.9 °C; IR: 3440, 3092, 2981,
2905, 1727, 1627, 1467, 1213, 1066, 1028, 938, 729 cm−1;
1H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.28–4.33 (2H,
m),4.36–4.40 (2H, m), 6.78 (1H, t, J =7.6 Hz), 6.92–6.95
(2H, m), 7.07–7.09 (1H, dd, J1= 1.2 Hz, J2= 7.8 Hz),
7.41–7.44 (1H, dd, J1= 2.0 Hz, J2= 8.2 Hz), 7.55 (1H, d,
J = 2.0 Hz), 8.95 (1H, s), 9.18 (1H, s, OH), 10.61 (1H,s,
NH), 13.17 (1H, s, OH); 13C NMR (DMSO-d6, 75 MHz) δ/
ppm: 66.3, 102.3, 111.9, 118.1, 119.0, 119.4, 120.1, 123.4,
126.5, 126.7, 142.3, 143.5, 146.2, 149.7, 162.9, 175.2; LC–
MS (m/z): 349.1845 [MNa+]. Anal. Calcd. for C17H14N2O5:
C, 62.57; H, 4.32; N, 8.59; found: C, 62.54; H, 4.36; N, 8.55.
5′‑Aminospiro[[1,3] dioxolane‑2,3′‑indolin]‑2′‑one
(4) 7.5 mmol of 5′-nitrospiro[[1,3] dioxolane-2,3′-indolin]-
2′-one (3) was dissolved in anhydrous ethanol. 37 mmol of
cyclohexene and 3.975 g of 10% Pd–C were added to this
solution. The mixture was refuxed for 2 h. It was cooled and
fltered to separate solid Pd–C. The solution was evaporated,
and the product was recrystallized with ethanol.
Brown powder, 87% yield, IR: 3398, 3374, 3319, 3161,
2902, 2812, 1741, 1716, 1697, 1622, 1490, 1477, 1211,
1080, 993, 742, 640 cm−1; 1H NMR (DMSO-d6, 300 MHz)
δ/ppm: 4.16–4.24 (2H, m), 4.27–4.35 (2H, m), 4.85 (2H,
s, NH2), 6.52 (2H, d, J=1.5 Hz), 6.59 (1H, d, J=1.5 Hz),
9.99 (1H, s, NH); 13C NMR (DMSO-d6, 75 MHz) δ/ppm:
66.0, 102.9, 111.5, 112.2, 116.7, 125.8, 132.7, 145.0, 174.9;
LC–MS (m/z): 207.13 [MH+].
(E)‑5′‑((2,5‑dihydroxybenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5d) Brown powder,
87.2% yield, mp. 279.9–281.4 °C; IR: 3394, 3151, 2968,
2896, 1739, 1626, 1486, 1276, 1220, 1188, 993, 757 cm−1;
1H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.30–4.32 (2H,
m),4.35–4.40 (2H, m), 6.77–6.87 (2H, m), 6.90 (1H, d,
J = 8.2 Hz), 7.01(1H, d, J = 2.6 Hz), 7.38–7.41 (1H, dd,
J1= 2.3 Hz, J2= 8.2 Hz), 7.52(1H, d, J = 1.7 Hz), 8.88
(1H, s), 9.10 (1H, s, OH),10.59 (1H, s, NH), 12.30 (1H, s,
OH);13C NMR (DMSO-d6, 75 MHz) δ/ppm: 66.3, 102.3,
111.9, 117.6, 117.8, 118.2, 120.0, 121.5, 126.5, 126.6,
142.2, 143.9, 150.3, 153.7, 162.2, 175.2; LC–MS (m/z):
327.2204 [MH+]. Anal. Calcd. for C17H14N2O5: C, 62.57;
H, 4.32; N, 8.59; found: C, 62.53; H, 4.35; N, 8.56.
Synthesis of 5′‑(benzylideneamino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one derivatives (5a–
u) 10 mmol of 5′-aminospiro[[1,3] dioxolane-2,3′-indolin]-
2′-one (4), 12 mmol of various aldehyde derivatives and
0.05 mmol of Et3N in EtOH were refuxed for 8 h. The
mixture was cooled and evaporated in vacuum. The crude
product was washed with chloroform and dried in vacuum.
( E ) ‑ 5 ′ ‑ ( b e n z y l i d e n e a m i n o ) s p i r o [ [ 1 , 3 ]
dioxolane‑2,3′‑indolin]‑2′‑one (5a) Dark brown pow-
der, 75% yield, mp. 204.3–205.8 °C; IR: 3232, 2993, 2907,
1728, 1697, 1623, 1479, 1286, 1216, 1066, 834, 753, 691,
1
612 cm−1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.25–
(E)‑5′‑((2,4,6‑trihydroxybenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5e) Dark brown powder,
95% yield, mp. 257.3–257.6 °C; IR: 3174, 2970, 1726, 1619,
1579, 1492, 1281, 1197, 1170, 1135, 1072, 941, 814 cm−1;
1H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.27–4.31 (2H, m),
4.33–4.37 (2H, m), 5.82 (2H, s), 6.86 (1H, d, J =8.5 Hz),
7.27-7.25 (1H, dd, J1= 2.0 Hz, J2= 8.5 Hz), 7.35 (1H, d,
J = 2.0 Hz), 8.90 (1H, s), 10.10 (1H, br, OH), 10.55 (1H,
s, NH);13C NMR (DMSO-d6, 75 MHz) δ/ppm: 66.3, 94.8,
102.1, 102.3, 111.9, 117.9, 125.2, 126.7, 141.3, 144.2,
157.1, 164.2, 164.8, 175.2; LC–MS (m/z): 381.3689 [MK+].
Anal. Calcd. for C17H14N2O6: C, 59.65; H, 4.12; N, 8.18;
found: C, 59.68; H, 4.10; N, 8.14.
4.29 (2H, m), 4.30–4.35 (2H, m), 6.89 (1H, d, J=8.2 Hz),
7.31–7.34 (1H, dd, J1= 2.0 Hz; J2= 8.2 Hz), 7.39 (1H, s),
7.51–7.53 (3H, m), 7.92 (2H, t, J = 3.8 Hz), 8.69 (1H,s),
10.55 (1H, s, NH); 13C NMR (DMSO-d6, 75 MHz) δ/ppm:
66.3, 102.4, 111.7, 118.0, 126.3, 126.4, 129.2, 129.5, 131.9,
136.9, 141.7, 146.8, 159.8, 175.2; LC–MS (m/z): 295,1135
[MH+]. Anal. Calcd. for C17H14N2O3: C, 69.38; H, 4.79; N,
9.52; found: C, 69.34; H, 4.76; N, 9.57.
(E)‑5′‑((3‑hydroxybenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5b) Brown powder,
83% yield, mp. 248.5-249.6 °C; IR: 3395, 3175, 2970,
2904, 1736, 1628, 1578, 1489, 1221, 1186, 992, 777,
1 3

Molecular Diversity
(E)‑5′‑((4‑methoxybenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5f) Brown powder, 79%
yield, mp. 218.8-220.2 °C; IR: 3140, 3042, 2894, 2839,
1721, 1630, 1606, 1510, 1474, 1246, 1162, 1070, 1027, 995,
827, 607 cm−1; 1H NMR (DMSO-d6, 300 MHz) δ/ppm: 3.81
(3H, s), 4.27–4.34 (4H, m), 6.85 (1H, d, J=8.2 Hz), 7.05
(2H, d, J=8.5 Hz), 7.26 (1H, d, J=8.2 Hz), 7.32 (1H, s),
7.86 (2H, d, J= 8.5 Hz), 8.57 (1H, s), 10.50 (1H, s, NH);
13C NMR (DMSO-d6, 75 MHz) δ/ppm: 56.1, 66.2, 102.4,
111.7, 114.9, 117.9, 126.0, 126.4, 129.7, 130.9, 141.2,
147.1, 159.0, 162.4, 175.2; LC–MS (m/z): 325.1615[MH+].
Anal. Calcd. for C18H16N2O4: C, 66.66; H, 4.97; N, 8.64;
found: C, 66.60; H, 4.95; N, 8.68.
NMR (DMSO-d6, 75 MHz) δ/ppm: 40.3, 66.2, 102.5, 111.6,
112.1, 117.8, 124.5, 125.6, 126.4, 130.8, 140.6, 147.8,
152.9, 159.3, 175.2; LC–MS (m/z): 360.2523[MNa+]. Anal.
Calcd. for C19H19N3O3: C, 67.64; H, 5.68; N, 12.46; found:
C, 67.60; H, 5.70; N, 12.43.
(E)‑5′‑((2‑fluorobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5j) Brown powder,
62% yield, mp. 244.9–247.4 °C; IR: 3104, 3038, 2896,
1729, 1627, 1474, 1452, 1278, 1213, 1074, 1000, 834, 763,
1
634 cm−1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.26-
4.30 (2H, m), 4.32–4.37 (2H, m), 6.87 (1H, d, J=8.2 Hz),
7.30–7.36(3H, m), 7.41(1H, d, J=2.0 Hz), 7.53-7.60 (1H,
qd, J1=1.8 Hz, J2=5.5 Hz), 8.04–8.09 (1H, td, J1=1.6 Hz,
J2= 7.6 Hz), 8.79 (1H, s), 10.55 (1H, s, NH);13C NMR
(DMSO-d6, 75 MHz) δ/ppm: 66.3, 102.3, 111.7, 116.7,
116.9, 118.3, 124.3, 125.6, 126.3, 128.3, 134.1, 142.1,
146.6, 152.3, 164.4, 175.2; LC–MS (m/z): 335.2516[MNa+].
Anal. Calcd. for C17H13FN2O3: C, 65.38; H, 4.20; N, 8.97;
found: C, 65.35; H, 4.22; N, 8.96.
(E)‑5′‑((3,4‑dimethoxybenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5g) Orange powder,
84% yield, mp. 134.8-136.9 °C; IR: 3242, 2902, 2835, 1729,
1631, 1581, 1512, 1263, 1193, 1142, 1068, 1022,943, 752,
1
629 cm−1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 3.84
(6H, s), 4.29-4.31 (2H, m), 4.34–4.36 (2H, m), 6.87 (1H,
d, J=8.2 Hz), 7.09 (1H, d, J=8.5 Hz), 7.27-7.29 (1H, dd,
J1=1.8 Hz, J2=8.2 Hz), 7.35(1H, d, J=1.8 Hz), 7.45(1H, d,
J=8.2 Hz), 7.54 (1H, s), 8.57 (1H, s), 10.52 (1H, s, NH);13C
NMR (DMSO-d6, 75 MHz) δ/ppm: 56.0, 56.3, 66.3, 102.4,
109.9, 111.7, 111.9, 117.9, 124.5, 126.0, 126.4, 129.8,
141.2, 147.1, 149.6, 152.3, 159.3, 175.2; LC–MS (m/z):
355.2162 [MH+]. Anal. Calcd. for C19H18N2O5: C, 64.40;
H, 5.12; N, 7.91; found: C, 64.44; H, 5.15; N, 7.87.
(E)‑5′‑((3‑fluorobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5k) Brown powder,
62% yield, mp. 227.5–229.1 °C; IR: 3232, 2974, 2907,
1728, 1697, 1625, 1482, 1450, 1215, 1066, 993, 941, 751,
1
685,617 cm−1; H NMR (DMSO-d6, 300 MHz) δ/ppm:
4.29–4.34 (4H, m), 6.88 (1H, d, J=8.2 Hz), 7.31–7.40 (3H,
m), 7.52–7.58(1H, q, J = 5.8 Hz), 7.67 (1H, s), 7.73 (1H,
t, J = 7.3 Hz), 8.70 (1H, s), 10.55 (1H, s, NH);13C NMR
(DMSO-d6, 75 MHz) δ/ppm: 66.3, 102.3, 111.7, 114.7,
114.9, 118.0, 118.8, 125.7, 126.5, 126.7, 131.5, 139.4,
142.0, 146.2, 158.4, 175.2; LC–MS (m/z): 313.3289 [MH+].
Anal. Calcd. for C17H13FN2O3: C, 65.38; H, 4.20; N, 8.97;
found: C, 65.36; H, 4.23; N, 8.98.
(E)‑5′‑((2,5‑dimethoxybenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5h) Brown powder, 71%
yield, mp. 183.8–186.1 °C; IR: 3139, 3103, 2954, 2905,
2834, 1724, 1624, 1493,1275, 1213, 1022, 946, 787, 707,
1
634 cm−1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 3.75
(3H, s), 3.83 (3H, s), 4.27–4.29 (2H, m), 4.31–4.34 (2H,
m), 6.85(1H, d, J = 7.9 Hz), 7.08(2H, d, J = 2.7 Hz), 7.25
(1H, d, J=8.2 Hz), 7.28 (1H, s), 7.50 (1H, s), 8.80 (1H, s),
10.51(1H, s, NH);13C NMR (DMSO-d6, 75 MHz) δ/ppm:
56.1, 56.9, 66.3, 102.4, 110.7, 111.8, 114.2, 118.3, 119.9,
125.0, 125.7, 126.5, 141.6, 147.4, 153.8, 154.4, 159.5,
175.2; LC–MS (m/z): 377.2217[MNa+]. Anal. Calcd. for
C19H18N2O5: C, 64.40; H, 5.12; N, 7.91; found: C, 64.45;
H, 5.16; N, 7.86.
(E)‑5′‑((4‑fluorobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5l) Dark brown pow-
der, 65% yield, mp. 206.5–207.9 °C; IR: 3281, 2982, 2901,
1746, 1706, 1630, 1480, 1197, 1168, 1132, 998, 831, 728,
1
609 cm−1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.26–
4.31 (2H, m), 4.33–4.35 (2H, m), 6.87 (1H, d, J=8.2 Hz),
7.27–7.37 (4H, m), 7.95–7.98 (2H, q, J=5.9 Hz), 8.66 (1H,
s), 10.53 (1H, s, NH);13C NMR (DMSO-d6, 75 MHz) δ/ppm:
66.3, 102.4, 111.7, 116.4, 116.7, 117.9, 126.4, 131.4, 131.5,
133.5, 141.7, 146.6, 158.5, 175.2; LC–MS (m/z): 313.2348
[MH+]. Anal. Calcd. for C17H13FN2O3: C, 65.38; H, 4.20;
N, 8.97; found: C, 65.33; H, 4.24; N, 8.94.
(E)‑5′‑((4‑(dimethylamino)benzylidene)amino)
spiro[[1,3] dioxolane‑2,3′‑indolin]‑2′‑one (5i) Orange
powder, 94.4% yield, mp. 252.6–254.4 °C; IR: 3220, 2899,
1737, 1698, 1589, 1475, 1364, 1198, 1166, 1065, 940,
1
816 cm−1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 3.00
(E)‑5′‑((2‑chlorobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5m) Yellowish powder,
75.4% yield, mp. 271.5–273.3 °C; IR: 3148, 3106, 2967,
2904, 1700, 1627, 1478, 1274, 1200, 1073, 993, 755 cm−1;
1H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.30–4.33 (4H,
(3H, s), 3.04 (3H, s), 4.28–4.31(2H, m), 4.33–4.36 (2H, m),
6.77 (2H, d, J=8.8 Hz), 6.83 (1H, d, J=8.2 Hz), 7.20-7.22
(1H, dd, J1=2.1 Hz, J2=8.2 Hz), 7.28 (1H, d, J=2.0 Hz),
7.73 (2H, d, J=8.8 Hz), 8.45 (1H, s), 10.46 (1H, s, NH);13C
1 3

Molecular Diversity
m), 6.89 (1H, d, J=7.9 Hz), 7.33–7.56 (5H, m), 8.13 (1H,
d, J = 6.5 Hz), 8.87 (1H, s), 10.58 (1H, s, NH);13C NMR
(DMSO-d6, 75 MHz) δ/ppm: 66.3, 102.3, 111.9, 118.4,
118.6, 126.3, 126.5, 128.3, 128.9, 130.8, 133.4, 135.6, 142.2,
146.5, 155.3, 175.3; LC–MS (m/z): 351.1948 [MNa+]. Anal.
Calcd. for C17H13ClN2O3: C, 62.11; H, 3.99; N, 8.52; found:
C, 62.15; H, 3.97; N, 8.54.
1704, 1629, 1480, 1210, 1168, 1070, 994, 821, 779 cm−1;
1H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.25–4.30 (2H, m),
4.32–4.37(2H, m), 6.88 (1H, d, J=8.2 Hz), 7.31–7.34 (1H,
dd, J1=2.3 Hz, J2=8.2 Hz), 7.40 (1H, d, J=2.1 Hz), 7.45
(1H, t, J = 7.9 Hz), 7.69 (1H, d, J = 7.9 Hz),7.89 (1H, d,
J=8.0 Hz), 8.08 (1H, s), 8.67 (1H, s), 10.57 (1H, s, NH);13C
NMR (DMSO-d6, 75 MHz) δ/ppm: 66.3, 102.3, 111.7,
118.0, 122.8, 126.5, 126.7, 128.2, 131.4, 131.7, 134.4,
139.1, 142.0, 146.2, 158.1, 175.2; LC–MS (m/z): 375.1272
[MH+]. Anal. Calcd. for C17H13BrN2O3: C, 54.71; H, 3.51;
N, 7.51; found: C, 54.75; H, 3.54; N, 7.47.
(E)‑5′‑((3‑chlorobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5n) Brown powder,
74.3% yield, mp. 184.8–186.1 °C; IR: 3323, 2972, 2905,
1742, 1704, 1630, 1481, 1283, 1199,1169, 1074, 1030, 781,
1
679 cm−1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.25–
(E)‑5′‑((4‑bromobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5r) Orange powder,
60% yield, mp. 205.6–206.8 °C; IR: 3138, 3081, 3041,
2892, 1722, 1630, 1486, 1281, 1199, 1162, 1066, 992,
4.30 (2H, m), 4.32–4.37 (2H, m), 6.88 (1H, d, J=8.2 Hz),
7.31–7.34 (1H, dd, J1= 2.0 Hz, J2= 8.2 Hz), 7.40 (1H, d,
J=1.8 Hz), 7.49–7.57 (2H,m), 7.85 (1H, d, J=6.7 Hz), 7.93
(1H, s), 8.68 (1H, s), 10.57 (1H, s, NH);13C NMR (DMSO-
d6, 75 MHz) δ/ppm: 66.3, 102.3, 111.7, 118.1, 126.5, 126.6,
127.8, 128.4, 131.4, 131.5, 134.3, 138.9, 142.1, 146.2,
158.2, 175.2; LC–MS (m/z): 329.2433 [MH+]. Anal. Calcd.
for C17H13ClN2O3: C, 62.11; H, 3.99; N, 8.52; found: C,
62.14; H, 3.96; N, 8.55.
1
892, 778, 604 cm−1; H NMR (DMSO-d6, 300 MHz) δ/
ppm: 4.28-4.30 (2H, m), 4.32–4.38 (2H, m), 6.87 (1H, d,
J = 8.2 Hz), 7.29–7.32 (1H, dd, J1= 2.1 Hz, J2= 8.2 Hz),
7.39 (1H, d, J=1.8 Hz), 7.69 (2H, d, J=8.5 Hz), 7.83 (2H,
d, J = 8.5 Hz),8.66 (1H, s), 10.55 (1H, s, NH); 13C NMR
(DMSO-d6, 75 MHz) δ/ppm: 66.3, 102.3, 111.7, 118.0,
125.4, 126.4, 126.5, 130.9, 132.5, 136.0, 141.9, 146.4,
158.5, 175.2; LC–MS (m/z): 375.1052 [MH+]. Anal. Calcd.
for C17H13BrN2O3: C, 54.71; H, 3.51; N, 7.51; found: C,
54.73; H, 3.55; N, 7.46.
(E)‑5′‑((4‑chlorobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5o) Brown powder, 75%
yield, mp. 260.1–262.4 °C; IR: 3137, 3042, 2890, 2843,
1724, 1631, 1490, 1281, 1201, 1072, 993, 779, 607 cm−1;
1H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.28–4.34 (4H,
m), 6.87 (1H, d, J=8.2 Hz), 7.31 (1H, d, J=8.2 Hz), 7.32
(1H,s), 7.56 (2H, d, J = 6.9 Hz), 7.92 (2H, d, J = 6.9 Hz),
8.68 (1H, s), 10.54 (1H, s, NH);13C NMR (DMSO-d6,
75 MHz) δ/ppm: 66.3, 102.3, 111.7, 118.0, 126.4, 126.5,
129.6, 130.8, 135.7, 136.5, 141.9, 146.4, 158.4, 175.2; LC–
MS (m/z): 329.2477 [MH+]. Anal. Calcd. for C17H13ClN2O3:
C, 62.11; H, 3.99; N, 8.52; found: C, 62.13; H, 3.95; N, 8.56.
(E)‑5′‑((2‑nitrobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5s) Orange powder,
74% yield, mp. 258.8–260.0 °C; IR: 3140, 3101, 2970, 2900,
1720, 1622, 1516, 1481, 1344, 1216, 1169, 1068, 1027, 994,
1
733 cm−1; H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.25–
4.31 (2H, m), 4.33–4.38(2H, m), 6.90 (1H, d, J =8.0 Hz),
7.31–7.34 (1H, dd, J1= 2.0 Hz, J2= 8.2 Hz), 7.37 (1H, d,
J = 2.0 Hz),7.70–7.76 (1H, td, J1= 1.8 Hz, J2= 7.6 Hz),
7.81–7.87 (1H, td, J1=1.0 Hz, J2=7.6 Hz), 8.06–8.09 (1H,
dd, J1=1.2 Hz, J2=8.2 Hz), 8.12–8.15 (1H, dd, J1=1.2 Hz,
J2= 8.0 Hz), 8.89 (1H, s), 10.59 (1H, s, NH); 13C NMR
(DMSO-d6, 75 MHz) δ/ppm: 66.3, 102.3, 111.8, 118.3,
125.2, 126.4, 126.7, 130.1, 130.7, 132.3, 134.3, 142.5,
146.0, 149.9, 155.6, 175.2; LC–MS (m/z): 362.1700[MNa+].
Anal. Calcd. for C17H13N3O5: C, 60.18; H, 3.86; N, 12.38;
found: C, 60.15; H, 3.83; N, 12.40.
(E)‑5′‑((2‑bromobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5p) Yellowish powder,
76% yield, mp. 216.9–218.2 °C; IR: 3178, 3106, 2968, 2902,
1728, 1627, 1476, 1271, 1201, 1072, 1028, 992, 754 cm−1;
1H NMR (DMSO-d6, 300 MHz) δ/ppm: 4.28–4.40 (4H,
m), 6.94 (1H, d, J = 7.9 Hz), 7.34–7.38 (2H, m), 7.44–
7.55 (2H, m), 7.75 (1H, d, J=7.6 Hz), 8.10–8.13 (1H, dd,
J1= 1.8 Hz, J2= 7.4 Hz), 8.81 (1H, s), 10.64 (1H, s, NH);
13C NMR (DMSO-d6, 75 MHz) δ/ppm: 66.3, 102.3, 111.9,
118.4, 125.9, 126.0, 126.5, 128.8, 129.4, 133.7, 133.9,
134.7, 142.2, 146.5, 157.7, 175.2; LC–MS (m/z): 397.0669
[MNa+]. Anal. Calcd. for C17H13BrN2O3: C, 54.71; H, 3.51;
N, 7.51; found: C, 54.76; H, 3.53; N, 7.48.
(E)‑5′‑((3‑nitrobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5t) Dark brown pow-
der, 88.8% yield, mp. 214.3–216.0 °C; IR: 3174, 2963,
2897, 1735, 1622, 1524, 1476, 1348, 1199, 1072, 996,
1
813, 730, 613 cm−1; H NMR (DMSO-d6, 300 MHz) δ/
ppm: 4.29–4.32 (2H, m), 4.33–4.36 (2H, m), 6.90 (1H, d,
J=8.2 Hz),7.38 (1H, d, J=8.2 Hz),7.47 (1H, s), 7.77 (1H,
t, J=7.0 Hz),8.30 (2H, d, J=7.6 Hz),8.69 (1H, s), 8.85 (1H,
s), 10.59 (1H, s, NH); 13C NMR (DMSO-d6, 75 MHz) δ/
(E)‑5′‑((3‑bromobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5q) Dark brown powder,
89% yield, mp. 271.9–272.9 °C; IR: 3322, 2969, 2901, 1741,
1 3

Molecular Diversity
ppm: 66.3, 102.3, 111.8, 118.2, 123.1, 125.9, 126.5, 126.9,
131.1, 135.1, 138.4, 142.4, 145.8, 148.8, 157.5, 175.2; LC–
MS (m/z): 340.25 [MH+]. Anal. Calcd. for C17H13N3O5: C,
60.18; H, 3.86; N, 12.38; found: C, 60.14; H, 3.84; N, 12.41.
concentration of 1000 µg/mL. The absorbance was meas-
ured at room temperature at 734 nm, after 6 min from
ABTS·+ addition. The decrease in the absorption was used
for the calculation. The results were calculated as IC50.
Propyl alcohol was used as a solvent to controls.
(E)‑5′‑((4‑nitrobenzylidene)amino)spiro[[1,3]
dioxolane‑2,3′‑indolin]‑2′‑one (5u) Orange powder,
83.5% yield, mp. 268.8–270.4 °C; IR: 3286, 2963, 2898,
1748, 1709, 1632, 1512, 1485, 1338, 1198, 1165, 995,
Cupric reducing antioxidant capacity assay
(CUPRAC)
1
845, 728, 605 cm−1; H NMR (DMSO-d6, 300 MHz) δ/
Cupric reducing antioxidant capacities of the synthesized
compounds were determined according to the literature
method [35]. The solutions of compounds and stand-
ards were prepared in n-propanol at a concentration of
1000 µg/mL. Diferent volumes (1000 mg/L and 54.5 mL)
of the sample were added to a solution prepared by adding
61.0 µL of 10 mM CuCl2, 61.0 µL of 7.5 mM neocuproine
and 61.0 µL of 1.0 mM NH4CH3COO bufer (pH = 7),
respectively. The absorbance was measured at room tem-
perature at 450 nm, after an hour. The results were cal-
culated as A0.50. Propyl alcohol was used as a solvent to
controls.
ppm: 4.25–4.30 (2H, m), 4.32–4.37 (2H, m), 6.89 (1H, d,
J = 8.2 Hz), 7.37–7.41 (1H, dd, J1= 2.4 Hz, J2= 8.2 Hz),
7.47 (1H, d, J=2.0 Hz), 8.13 (2H, d, J=8.8 Hz), 8.33 (2H,
d, J = 8.8 Hz), 8.85 (1H, s), 10.59 (1H, s, NH); 13C NMR
(DMSO-d6, 75 MHz) δ/ppm: 66.5, 102.5, 112.0, 118.3,
118.8, 124.9, 126.7, 127.4, 130.3, 142.6, 146.0, 149.5,
157.8, 175.4; LC–MS (m/z): 381.3993[MK+]. Anal. Calcd.
for C17H13N3O5: C, 60.18; H, 3.86; N, 12.38; found: C,
60.16; H, 3.85; N, 12.37.
Antioxidant activity assays
In CUPRAC assay, the absorbance values were used to
calculate the A0.50, but in ABTS and DPPH assay, inhibi-
tion (%) values were used to calculate the IC50.
Computational assay
Quantum chemical calculations were carried out by using
the Q-CHEM 4.3, and the output fles were visualized via
IQmol program [43]. The optimized molecular structures
of compounds in the ground states were obtained at the
B3LYP/6-311G(d,p) [44] level of density functional theory
(DFT). Afterwards, the FMOs (the frontier molecular orbit-
als: HOMO and LUMO called highest occupied and lowest
unoccupied molecular orbitals) calculations were performed
at the same level. Finally, the chemical hardness (η), soft-
ness (S) and electronegativity (χ) parameters associated
with FMO energies were obtained by using same level. The
calculation of FMO energies and related parameters (the
electronegativity (χ), chemical hardness (η) and softness
(S) parameters) for compounds was performed by using
B3LYP/6-311G(d,p) level. The χ, η and S parameters were
obtained by using χ=(IP+EA)/2, η=(IP–EA)/2 and S=1/η
equations [45].
DPPH (1,1‑diphenyl‑2‑picrylhydrazyl) free radical
scavenging assay
Free radical scavenging activities are determined using
1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical [33].
1000 μM stock solutions of the materials were prepared.
Of these stock solutions, 2, 5, 10 and 20 μL was taken
and completed to 40 μL with ethanol, and then 160 μL of
0.1 mM DPPH solution was added. The absorbance values
of the prepared solutions were measured at 517 nm after
30 min of incubation in the dark at room temperature.
Inhibition values (%) of the samples were calculated from
the obtained absorbance values. The absorbance values of
the samples were evaluated against the control.
ABTS cation radical decolourization assay
Acknowledgements This work was supported by the Sakarya Research
Fund of the Sakarya University (Project Number: 2014-50-01-036).
ABTS·+ scavenging activities of the synthesized com-
pounds were determined according to the literature method
[34]. The solution of ABTS·+ radical was generated by
dissolving 19.2 mg of 2,2′-azino-bis(3-ethylbenzothiazo-
line-6-sulphonic acid) (7 mM ABTS) and 3.3 mg K2S2O3
in distilled water (5 mL). The solution was kept in dark
for 24 h at room temperature, and the absorbance of the
solution was fxed to ~ 0.70 at 734 nm by dilution. The
solutions of the samples were prepared in n-propanol at a
Compliance with ethical standards
Conflict of interest The authors declare they have no confict of inter-
est.
1 3

Molecular Diversity
derivatives. Bioorg Med Chem Lett 28(2):113–116. https://doi.
org/10.1016/j.bmcl.2017.11.047
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