Synthesis and free radical scavenging activity of new hydroxybenzylidene hydrazines

Article abstract of DOI:10.3390/molecules22060894

Hydroxybenzylidene hydrazines exhibit a wide spectrum of biological activities. Here, we report synthesis and free radical scavenging activity of nine new N-(hydroxybenzylidene)-N0-[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazines. The chemical structures

Full text of DOI:10.3390/molecules22060894

molecules
Communication
Synthesis and Free Radical Scavenging Activity of
New Hydroxybenzylidene Hydrazines
1
, ,†
2,†
3
2
1
Frantisek Sersen * , Fridrich Gregan , Peter Kotora , Jarmila Kmetova , Juraj Filo ,
Dusan Loos 4 and Juraj Gregan
5,6,
*
1
Institute of Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovicova 6,
42 15 Bratislava, Slovakia; filo@fns.uniba.sk
Department of Chemistry, Faculty of Natural Sciences, Matej Bell University, Tajovskeho 40,
74 01 Banska Bystrica, Slovakia; frido.gregan@gmail.com (F.G.); Jarmila.Kmetova@umb.sk (J.K.)
Institute of Process Engineering, Faculty of Mechanical Engineering, Slovak University of Technology
in Bratislava, Namestie slobody 17, 812 31 Bratislava, Slovakia; kotora@fns.uniba.sk
Department of Organic Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava,
Ilkovicova 6, 842 15 Bratislava, Slovakia; loosdus@gmail.com
8
2
3
4
5
6
9
Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovicova 6,
8
42 15 Bratislava, Slovakia
Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9,
030 Vienna, Austria
1
*
Correspondence: f.sersen@gmail.com (F.S.); juraj.gregan@univie.ac.at (J.G.);
Tel.: +421-2-602-96 (ext. 412) (F.S.); +421-2-602-96 (ext. 447) (J.G.); Fax: +421-2-654-29-064 (J.G. & F.S.)
These authors contributed equally to this work.
†
Academic Editor: Derek J. McPhee
Received: 7 April 2017; Accepted: 26 May 2017; Published: 29 May 2017
Abstract: Hydroxybenzylidene hydrazines exhibit a wide spectrum of biological activities.
Here, we report synthesis and free radical scavenging activity of nine new N-(hydroxybenzylidene)-
0
N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazines. The chemical structures of these compounds
1
13
19
were confirmed by H-NMR, C-NMR, F-NMR, IR spectroscopy, LC-MS, and elemental analysis.
The prepared compounds were tested for their activity to scavenge 2,2-diphenyl-1-picrylhydrazyl
0
(
(
DPPH), galvinoxyl radical (GOR), and 2,2 -azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid
ABTS) radicals. The free radical scavenging activity expressed as SC50 values of these compounds
varied in a wide range, from a strong to no radical scavenging effect. The most effective
radical scavengers were hydroxybenzylidene hydrazines containing three hydroxyl groups in the
benzylidene part of their molecules. The prepared compounds were also tested for their activity to
inhibit photosynthetic electron transport in spinach chloroplasts. IC50 values of these compounds
varied in wide range, from an intermediate to no inhibitory effect.
0
Keywords: N-(hydroxybenzylidene)-N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazines; radical
scavenging activity; DPPH scavenging; GOR scavenging; ABTS scavenging; photosynthetic
electron transport
1
. Introduction
N-Hydroxybenzylidene hydrazines, also known as hydrazones, are N-arylmethylidene-
0
0
N -arylhydrazines with a diazamethylidene group C=N-NH [
1,2
]. The fact that N-arylmethylidene-
N -arylhydrazines represent an important class of compounds for new drug development motivates
0
researchers to synthesize and test new N-arylmethylidene-N -arylhydrazines. These compounds can
be prepared from the corresponding aromatic aldehydes and substituted arylhydrazines in alcohol
(
ethanol, methanol), acetic acid, or another solvents [
1
,2]. Synthesis without solvents using acidic
Molecules 2017, 22, 894; doi:10.3390/molecules22060894
www.mdpi.com/journal/molecules

Molecules 2017, 22, 894
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ionic liquids such as choline chloride and oxalic acid has also been reported [
3]. The combination of
diazamethylene group with other functional groups leads to compounds with interesting physical and
chemical characteristics [4].
0
The synthesis of novel N-arylmethylidene-N -arylhydrazines and their derivatives is of
great interest because of their potential use in the biopharmaceutic industry. These compounds
possess various biological and pharmacological properties, including antimicrobial, analgesic,
antifungal, anti-tubercular, antiviral, anticancer, antimalarial, antihelmintic, anti-trypanosomal,
0
and antischistosomiasis properties. In addition, N-arylmethylidene-N -arylhydrazines are used
as pigments, dyes, catalysts, ligands in organometallic complexes and polymer stabilizers [5–12]
N-arylmethylidene-N -arylhydrazines are also important for the synthesis of heterocyclic compounds
.
0
such as indoles and pyrazoles [13,14]. Derivatives of benzylidene hydrazine are potent inhibitors
of fungal growth with little mammalian cell toxicity, making them promising new targets for
0
future therapeutic development [
exhibit amoebicidal activity with an IC50 of 0.84
cell growth inhibition potency with respect to metronidazole (IC50 = 6.3
,4-dinitrophenylhydrazone betulinic acid derivatives showed significant cytotoxicity and selectivity
1]. Several derivatives of N-nitrobenzylidene-N -phenylhydrazines
µM, which represents a sevenfold increase in
µM) [12]. Several novel
2
against some tumor cell lines [15]. Due to their strong chemical stability, N-arylmethylidene-
0
N -arylhydrazines are also an attractive material in optoelectronics technologies and development of
potential chemosensors, optical switching devices, and organic light emission devices (OLEDs) [16].
0
However, N-arylmethylidene-N -arylhydrazines also show adverse effects. Some N-arylmethylidene-
0
N -arylhydrazines induce DNA fragmentation [17] and damage photosynthesis in chloroplasts [18
–
21].
The main aim of this work was to synthesize new hydroxybenzylidene hydrazines and
0
determine their free radical scavenging activity. We prepared nine new N-(hydroxybenzylidene)-N -
[
2,6-dinitro-4-(trifluoromethyl)]phenylhydrazines with OH groups at different positions of the benzene
ring and analyzed their ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH), galvinoxyl radicals
0
(
GOR), and 2,2 -azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS).
2
. Results and Discussion
2
.1. Chemistry
All compounds prepared in this study (5a–5i) contain the 2,4-dinitro-4-(trifluoromethyl)phenyl
group and an additional hydroxyphenyl group (5a, R = 4-hydroxyphenyl, 5b, R = 2,3-dihydroxyphenyl,
5
5
c
, R = 2,4-dihydroxyphenyl, 5d, R = 2,5-dihydroxyphenyl, 5e, R = 3,5-dihydroxyphenyl,
f
, R = 2,3,4-trihydroxyphenyl, 5g, R = 2,4,6-trihydroxyphenyl, 5h, R = 3,4,5-trihydroxyphenyl, and 5i
,
R = phenyl) (Scheme 1).
The starting compounds for the synthesis of hydroxybenzylidene hydrazines 5a–5i were
2
,6-dinitro-4-(trifluoromethyl)phenylhydrazine
compound for the preparation of 2-methoxy-1,3-dinitro-5-(trifluoromethyl)benzene
-chloro-1,3-dinitro-4-(trifluoromethyl)benzene . The yield of the compound
3 and aromatic aldehydes 4a–4i (Scheme 1). The starting
2
(Scheme 1) was
◦
2
1
2
was 76%, M.p. 61 C [22].
For the synthesis of compounds 5a
–
5i, we used trifluoroacetic acid (TFA) as acidic catalyst and
◦
ethanol as solvent. The reaction time was 3–4 h, reaction temperatures were 20–25 C, and yields
were 70–85%. The purity of prepared compounds and the course of reactions were monitored
by TLC. The crude hydrazines 5a–5i were purified by column chromatography on silica gel in
hexane/ethylacetate (4:1) as the mobile phase. All prepared hydroxybenzylidene hydrazines 5a–5i
as well as their solutions in organic solvents were dark red. The dark red color changed to dark
blue upon the increase in pH, suggesting that these compounds are sensitive to pH. The melting
◦
points of these compounds are relatively high (226–280 C). The chemical structures of prepared
1
13
19
compounds were confirmed by H-NMR, C-NMR, F-NMR, IR spectroscopy, LC-MS and elemental
analysis. Elemental analyses agreed with theoretical values ( 0.3). The IR spectra revealed several
characteristic absorption bands. Two absorption bands were observed in the region stretching NO
±
2

Molecules 2017, 22, 894
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vibration. The more intense band appearing at higher wave numbers (1538–1503 cm 1) corresponds to
−
−1
assymetric NO vibrations, and the less intense one, appearing at a lower wave number (1279–1257 cm ),
corresponds to symmetric vibrations. The absorption band at 3277–3257 cm corresponds to stretching
N–H vibrations. The wave number of this absorption band is affected by the mesomeric effect of
2
−
1
−
1
OH groups on the benzene ring. The absorption band at 1619–1616 cm corresponds to stretching
19
13
vibrations in the C=N group. The presence of CF group in molecules was confirmed by F- C splitting
3
13
0
2
to quartet in C NMR spectra of corresponding derivatives [4 (115.82–116.71 ppm, J = 35.1–35.4 Hz);
0
0
3
1
3
,5 (127.189–127.72 ppm, J = 3.2–3.5 Hz); CF3 (122.67–123.20 ppm, J = 271.3–271.6 Hz)].
The double bond between C and N in C=N–NH– group of benzylidene hydrazines allows for the
formation of two stereoisomers, namely (E) and (Z). The stereoisomerism of these compounds has not
been studied in detail, but we predict that (E) is the more abundant form [2,23].
i
ii
1
2
3
iii
3
4a - 4i
5a - 5i
Scheme 1. Synthesis of 2,6-dinitro-4-(trifluoromethyl)phenylhydrazine
3 and N-(hydroxybenzylidene)-
0
N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazines 5a
–5i.
5a R = 4-hydroxyphenyl, 5b R = 2,3-
dihydroxyphenyl, 5c R = 2,4-dihydroxyphenyl, 5d R = 2,5-dihydroxyphenyl, 5e R = 3,5-
dihydroxyphenyl, 5f R = 2,3,4-trihydroxyphenyl, 5g R = 2,4,6-trihydroxyphenyl, 5h R = 3,4,5-
trihydroxyphenyl, 5i R = phenyl. Reactants and reaction condition: (i) H C-ONa, methanol,
3
◦
room temperature, 1 h; (ii) H N-NH2
·
H O, EtOH, 0 C, 1 h; (iii) EtOH, trifluoroacetic acid (TFA),
room temperature, 3 h. Structure of all newly prepared compounds was confirmed by H-NMR,
2
2
1
13
19
C-NMR, F-NMR, IR spectroscopy, and elemental analysis.
2
.2. Free Radical Scavenging Activity Assay
SC50 values for scavenging DPPH, GOR, and ABTS radicals are shown in the Table 1. These results
suggest that the majority of studied hydroxybenzylidene hydrazines exhibit free radical scavenging
activity. Almost all studied benzylidene hydrazines (with the exception of compound 5i) scavenged
ABTS radical in a water solution. The most effective scavengers of ABTS radicals were compounds 5b
and 5e with two OH groups in positions 2, 3 and 3, 5, respectively. Hydroxybenzylidene hydrazines
with three hydroxyl groups (5b
scavengers of DPPH radicals were hydroxybenzylidene hydrazines with three OH groups (5h and 5f)
The molecules with two OH groups (5b 5c 5g) were weaker scavengers of DPPH radicals.
, 5f, 5h and 5g) also scavenged ABTS radicals. The most effective
.
,
,
Hydroxybenzylidene hydrazine with one OH group (5a) exhibited very weak scavenger activity,
and the compound with OH groups in positions 3 and 5 (5e) as well as the compound without OH
group (5i) did not scavenge DPPH radicals.
The compound with three OH groups (5f) in positions 2, 3, and 4 was the most effective scavenger
of GOR radicals. The compounds 5b, 5d, 5g, and 5h exhibited moderate scavenger activity, and the
compound 5a (with one OH group) exhibited very low activity to scavenge GOR radicals. Similarly as
observed with DPPH radicals, the compounds 5e and 5i showed no scavenging of GOR.
Taken together, we conclude that molecules with three hydroxyl groups (5b
, 5f, and 5h)
exhibited high free radical scavenging activities. The most effective compound was the compound 5f
,

Molecules 2017, 22, 894
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which contains OH groups in positions 2, 3, and 4. Interestingly, the compound with no hydroxyl group
(
5i) showed no scavenging activity. Some of the studied compounds (5f and 5h) exhibited higher free
3
radical scavenging activity than that reported for ascorbic acid (SC5 = 12.55
µmol/dm ) [24], resveratrol
0
3
3
(SC50 = 26.37 µmol/dm ) [25], and esculetin (SC50 = 8.64 µmol/dm ) [26]. We speculate that the ability to
release hydrogen atom or proton from hydroxyl groups of the hydroxybenzylidene hydrazine molecule
contributes to the mechanism of radical scavenging of the studied hydroxybenzylidene hydrazines.
Next, we tried to find a possible correlation between free radical scavenging activity and energy
necessary for releasing of hydrogen atom or proton. We used the method PM6 (see the Experimental
Section) to calculate the energy associated with the release of hydrogen atom (bond dissociation
enthalpy, BDE), the proton relaxation (proton dissociation enthalpy, PDE), release of electron (ionization
potential, IP), and the energy associated with the combined transfer of the electron and proton
(ETE and PA, respectively) for the studied benzylidene hydrazines. The values of these energies
in methanol or in water are presented in Tables S1–S8. Figure 1 shows the dependence of SC50 of
DPPH, GOR, and ABTS scavenging on the sum of PA + ETE values and BDE, respectively. The results
presented in the Figure 1A,B suggest that the ability of scavenging of DPPH and GOR radicals at higher
2
BDE and PE + ETE enthalpy decreases—the dependence is almost linear (the square deviation r = 0.87
2
for DPPH and r = 0.70 for GOR). This tendency is confirmed by the fact that the inactive substances
(5a and 5e) have the greatest values of BDE and PA + ETE (Table 1). On the other hand, the dependence
of the scavenging of ABTS radicals on BDE or PA + ETE shows no dependence (Figure 1C).
Table 1. Antiradical and PET inhibition activities of studied hydroxybenzilidene hydrazines expressed
as SC50 and IC50 values, respectively.
BDE
PA + ETE
BDE
PA + ETE
IC50 µM
PET
Inhibition
SC50 µM
DPPH
SC50 µM
GOR
SC50 µM
ABTS
Compound
(
kJ/mol) in
CH3OH
(kJ/mol) in
CH3OH
(kJ/mol) (kJ/mol) in
in Water
Water
5
a
144.4
16.2
48.9
168.1
25.4
25.8
17.3
2.6
9.4
7.8
3.5
5.4
7.4
5.4
182.9
1003
42.8
14.7
408.8
387.2
339.1
309.8
421.3
305.3
351.6
299.5
638.9
540.2
569.2
539.9
651.7
535.5
590.8
529.7
408.7
387.0
339.3
309.8
421.1
305.5
364.4
299.9
654.1
632.2
584.7
555.2
666.5
550.8
612.3
545.2
5b
5
c
5d
13.7
15.4
93
NDA 1
10.8
NDA 1
5.1
NDA 1
NDA 1
263
5
5
e
f
5g
38.5
16.7
NDA 1
344
5h
8.2
10.8
NDA 1
NDA 1
5
i
1
NDA (no detectable activity).
30
25
C
B
1
1
1
40
20
00
A
25
20
15
10
5
0
20
15
80
60
40
20
0
10
5
0
300
400
500
600
700
3
00
400
500
600
700
300
BDE
400
500
600
700
BDE
PA+ETE [kJ/mol]
BDE
PA+ETE [kJ/mol]
PA+ETE [kJ/mol]
Figure 1. Dependence of SC50 of DPPH (A), GOR (B), and ABTS (C) scavenging on BDE (red squares)
or PA + ETE (black circles).
2
.3. Inhibition of Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
The prepared compounds were also tested for their activity to inhibit PET in spinach chloroplasts.
3
IC50 values of these compounds varied in a wide range, from an intermediate (14.7
µ
mol/dm ) to

Molecules 2017, 22, 894
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3
no inhibitory effect (Table 1). The most effective were compounds 5c (IC50 = 14.7
µ
mol/dm ) and
3
5
b
(IC50 = 42.8
µ
mol/dm ). These activities are relatively low as compared to the values reported
for the classical herbicide diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCMU) with an IC50 of
3
1
.9 µmol/dm [27] and are unlikely to be of industrial interest.
3
. Materials and Methods
3
.1. General Information
2-Chloro-1,3-dinitro-4-(trifluoromethyl)benzene 1 (Scheme 1), galvinoxyl free radical (GOR),
and organic solvents were purchased from Alfa Aesar (Ward Hill, MA, USA) and used without
0
further purification. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2 -azino-bis(3-ethylbenzothiazoline-
6-sulfonic acid) diammonium salt (ABTS) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Methyl alcohol p.a., TRIS, MgCl , saccharose and dimethylsulfoxide p.a. (DMSO) were purchased
2
from Centralchem (Bratislava, Slovakia).
Melting points were determined on a Boetius apparatus and are uncorrected. IR spectra
were obtained on a NICOLET iS50 FT-IR spectrophotometer using an ATR technique in the
−
1
region 4000–400 cm . Elemental analyses were obtained on an Elemental Analyzer Carlo Erba
CHNS-OEA 1108. NMR spectra were performed on a Spectrometer Varian VNMRS 300 MHz (300 MHz
1
13
19
for H, 75 MHz for C, 282 MHz for F) and on a Spectrometer Varian VNMRS 600 MHz (600 MHz for
1
13
H and 150 MHz for C) in DMSO-d , with tetramethylsilane (TMS) as an internal standard. The purity
6
of prepared compounds and the course of reactions were checked on Merck TLC Silica gel 60 F254
plates in ethyl acetate–n-hexane as the mobile phase. The numbering of atoms for the evaluation of
NMR spectra of measured compounds
recorded by a Genesis 6 spectrophotometer (Thermo–Scientific, Waltham, MA, USA). FTIR spectra
in solid phase) were recorded on a Nicolet 6700 spectrometer (Thermo–Scientific (Nicolet), Waltham,
3 and 5a–5i is given in the formulae. Absorption spectra were
(
MA, USA) using the ATR technique.
MS spectra were recorded by LC-MS spectrometer consisting of an Agilent 1200 HPLC (Walbron,
Germany), with an MSD 6110 MS detector (Agilent Technologies, Santa Clara, CA, USA).
3
.2. Synthesis
3
.2.1. Synthesis of 2,6-Dinitro-4-(Trifluoromethyl)Phenylhydrazine (3)
A solution of hydrazine monohydrate (0.75 g, 15 mmol) in anhydrous ethanol (5 mL) was
added dropwise to a solution of 2-methoxy-1,3-dinitro-5-(trifluoromethyl)benzene 2 (3.2 g, 12 mmol)
◦
in anhydrous ethanol (17 mL) under an argon atmosphere. The reaction mixture was stirred at 0 C for
1
h and monitored by TLC. The solvent was removed under vacuum, and crude product was purified
by column chromatography on silica gel in hexane/ethylacetate (4:1) as the mobile phase. This resulted
◦
in 2.8 g (88%) of 2,6-dinitro-4-(trifluoromethyl)phenylhydrazine 3 (yellow solid), M.p. 125–126 C.
The previously published M.p. for this compound is 124 C [23].
◦
O N 2
2
3
H
1
4
H N N
CF3
2
1
2
6
5
O N
2
◦
,6-dinitro-4-(trifluoromethyl)phenylhydrazine (3). Yield 88%; red solid, M.p. 125–126 C, Anal. Calcd.
2
for C H F N O (266.14) C, 31.59; H, 1.89: N, 21.05. Found: C, 31.71; H, 1.74; N, 20.86%. IR: 3248,
7
5
3
4
4
υ
(N–H); 1636,
= 9.65 (s, 1H, NH-2), 8.40 (s, 2H, H-3, H-5), 4.84 (s, 2H, NH-1). C NMR (75 MHz, DMSO): δ = 142.97,
υ(C=N); 1536, υas(NO ); 1263, υs(NO ); 1118, υ
(CF3). 1H NMR (300 MHz, DMSO):
2 2
13
δ

Molecules 2017, 22, 894
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1
36.75, 127.87 (q, J = 3.5 Hz), 123.32 (q, J = 271.0 Hz), 113.76 (q, J = 35.3 Hz); negative LC-MS m/z: 265.0
−
−
[M − H] calc. for C H F N O , 265.018, found 265.0.
7
4
3
4
4
0
3
(
.2.2. Synthesis of N-Hydroxybenzylidene-N -[2,6-Dinitro-4-(Trifluoromethyl)]Phenylhydrazines
0
5a–5h) and N-(Benzylidene)-N -[2,6-Dinitro-4-(Trifluoromethyl)]Phenylhydrazine (5i)
A solution of 2,6-dinitro-4(trifluoromethyl)phenylhydrazine 3 (1 mmol) in ethanol (3 mL) was
added to a solution of aldehydes 4a–4i in ethanol (5 mL) and trifluoroacetic acid (0.5 mL). The reaction
mixture was stirred for 3–4 h at room temperature in argon atmosphere and monitored by TLC.
◦
The reaction mixture was cooled to 0 C, and the red solid product was filtered off, washed with
ethanol/ether (1:5), and dried. The product was purified by column chromatography on silica gel in
hexane/ethylacetate (3:1) as the mobile phase.
0
N-(4-Hydroxybenzylidene)-N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazine (5a). Yield 72%; red solid,
◦
M.p. 246–248 C, Anal. Calcd. for C H F N O (370.24) C, 45.42; H, 2.45: N, 15.13. Found: C, 45.30;
7
9
3
4
5
H, 2.38; N, 15.10%. IR: 3507,
υ
(O–H); 3277,
υ
(N–H); 1636, υ(C=N); 1537, υas(NO ); 1261, υs(NO2);
2
= 11.46 (s, 1H, NH), 10.05 (s, 1H, OH), 8.53 (s, 2H,
1
1
125,
υ
(CF ). H NMR (600 MHz, DMSO):
δ
3
0
0
H-3 , H-5 ), 8.41 (s, 1H, H-a), 7.39 (d, J = 8.6 Hz, 2H, H-2, H-6), 6.84 (d, J = 8.6 Hz, 2H, H-3, H-5).
13
C NMR (151 MHz, DMSO):
δ
= 160.34, 149.53, 137.47, 136.16, 129.54, 127.64 (q, J = 3.3 Hz), 125.11,
−
1
23.17 (q, J = 271.5 Hz), 116.37 (q, J = 35.1 Hz), 116.32; negative LC-MS m/z: 369.0 [M
−
H] calc. for
−
C H F N O , 369.045, found 369.0.
7
8
3
4
5
0
N-(2,3-Dihydroxybenzylidene)-N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazine (5b). Yield 79%; red solid,
◦
M.p. 226–228 C, Anal. Calcd. for C H F N O (386.24) C, 43.54; H, 2.35: N, 14.51. Found: C, 43.64;
14
9
3
4
6
H, 2.39; N, 14.63%. IR: 3528, 3377
υ
(O–H); 3274,
υ
(N–H); 1633,
υ
(C=N); 1537, υas(NO ); 1279, υs(NO2);
2
1
1
8
125,
υ
(CF ). H NMR (600 MHz, DMSO):
δ
= 11.56 (s, 1H, NH), 9.63 (s, 1H, OH-b), 9.03 (s, 1H, OH-c),
3
0 0
.79 (s, 1H, H-a), 8.51 (s, 2H, H-3 , H-5 ), 6.93 (dd, J = 7.9, 1.5 Hz, 1H, H-6), 6.81 (dd, J = 7.9, 1.5 Hz, 1H,
13
H-4), 6.65 (dd, J = 7.9 Hz, 1H, H-5). C NMR (151 MHz, DMSO):
δ
= 146.29, 146.19, 146.15, 137.71,
1
1
36.11, 127.63 (q, J = 3.4 Hz), 123.15 (q, J = 271.4 Hz), 121.24, 119.81, 117.36, 116.71 (q, J = 35.4 Hz),
−
−
6
16.44; negative LC-MS m/z: 385.0 [M − H] calc. for C H F N O , 385.04, found 385.0.
14
8
3
4
0
N-(2,4-Dihydroxybenzylidene)-N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazine (5c). Yield 73%; red solid,
◦
M.p. 229–230 C, Anal. Calcd. for C H F N O (386.24) C, 43.54; H, 2.35: N, 14.51. Found: C, 43.66;
14
9
3
4
6
H, 2.24; N, 14.40%. IR: 3528, 3377
υ
(O–H); 3274,
υ
(N–H); 1633,
υ
(C=N); 1537, υas(NO ); 1279, υs(NO2);
2

Molecules 2017, 22, 894
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1
1
8
6
1
1
125,
υ
(CF ). H NMR (600 MHz, DMSO):
δ
= 11.46 (s, 1H, NH), 9.94 (s, 1H, OH-b), 9.88 (s, 1H, OH-c),
3
0
0
.63 (s, 1H, H-a), 8.48 (s, 2H, H-3 , H-5 ), 7.27 (d, J = 8.6 Hz, 1H, H-6), 6.31 (d, J = 2.2 Hz, 1H, H-3),
.28 (dd, J = 8.6, 2.2 Hz, 1H, H-5). 13C NMR (151 MHz, DMSO):
= 161.69, 158.89, 146.59, 137.36,
36.07, 127.82, 127.67 (q, J = 3.2 Hz), 123.20 (q, J = 271.4 Hz), 115.93 (q, J = 35.3 Hz), 112.09, 108.66,
02.73; negative LC-MS m/z: 385.0 [M − H] calc. for C H F N O , 385.04, found 385.0.
δ
−
−
14
8
3
4
6
0
N-(2,5-Dihydroxybenzylidene)-N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazine (5d). Yield 83%; red solid,
◦
M.p. 233–234 C, Anal. Calcd. for C H F N O (386.24) C, 43.54; H, 2.35: N, 14.51. Found: C, 43.41;
14
9
3
4
6
H, 2.42; N, 14.62%. IR: 3517, 3346
υ
(O–H); 3257,
υ
(N–H); 1635,
υ
(C=N); 1536, υas(NO ); 1270, υs(NO2);
2
1129,
υ
(CF3). 1H NMR (600 MHz, DMSO):
δ
= 11.55 (s, 1H, NH), 9.40 (s, 1H, OH-b), 8.95 (s, 1H,
0
0
OH-c), 8.73 (s, 1H, H-a), 8.54 (s, 2H, H-3 , H-5 ), 6.85 (d, J = 2.1 Hz, 1H, H-6), 6.73 (s, 2H, H-4,
H-3). 13C NMR (151 MHz, DMSO):
δ = 150.42, 150.34, 146.27, 137.74, 136.10, 127.64 (q, J = 3.5 Hz),
1
23.15 (q, J = 271.6 Hz), 120.70, 120.21, 117.42, 116.71 (q, J = 35.3 Hz), 111.37; negative LC-MS m/z: 385.1
−
−
6
[M − H] calc. for C H F N O , 385.04, found 385.1.
14
8
3
4
0
N-(3,5-Dihydroxybenzylidene)-N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazine (5e). Yield 78%; red solid,
◦
M.p. 264–266 C, Anal. Calcd. for C H F N O (386.24) C, 43.54; H, 2.35: N, 14.51. Found: C, 43.66;
14
9
3
4
6
H, 2.23; N, 14.38%. IR: 3539, 3347
υ
(O–H); 3270,
υ
(N–H); 1637,
υ(C=N); 1536, υas(NO ); 1268, υs(NO2);
2
1
1
126,
υ
(CF ). H NMR (300 MHz, DMSO):
δ
= 11.45 (s, 1H, NH), 9.49 (s, 2H, OH-b, OH-c),
3
0
0
8
1
1
.55 (s, 2H, H-3 , H-5 ), 8.34 (s, 1H, H-a), 6.42 (d, J = 2.1 Hz, 2H, H-2, H-6), 6.32 (dd, J = 2.1 Hz,
H, H-4). 13C NMR (75 MHz, DMSO):
δ
= 158.63, 149.63, 137.33, 135.66, 135.20, 127.19 (q, J = 17.8 Hz),
−
22.67 (q, J = 271.5 Hz), 116.60 (q, J = 35.4 Hz), 105.48, 105.01; negative LC-MS m/z: 385.0 [M
−
H]
−
calc. for C H F N O , 385.04, found 385.0.
14
8
3
4
6
5
6
Ha O N
2
1
2´ 3´
dHO
H
4
1´
4´
3
2
N N
CF3
c
HO
OH
b
6
´
5´
O N
2
0
N-(2,3,4-Dihydroxybenzylidene)-N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazine (5f). Yield 85%; red solid,
◦
M.p. 232–233 C, Anal. Calcd. for C H F N O (402.24) C, 41.80; H, 2.26: N, 13.93. Found: C, 41.70;
14
9
3
4
7
H, 2.18; N, 14.05%. IR: 3532, 3481, 3359
υ
(O–H); 3272,
υ
(N–H); 1633,
υ(C=N); 1530, υas(NO2);
1
1
269, υs(NO ); 1128,
υ
(CF ). H NMR (300 MHz, DMSO):
δ
= 11.48 (s, 1H, NH), 9.70 (s, 1H, OH-b),
2
3
0
0
9
1
1
.03 (s, 1H, OH-d), 8.67 (s, 1H, OH-c), 8.54 (s, 1H, H-a), 8.50 (s, 2H, H-3 , H-5 ), 6.85 (d, J = 8.6 Hz,
= 160.34, 149.53, 137.47, 136.16,
29.54, 127.64 (q, J = 3.3 Hz), 125.11, 123.17 (q, J = 271.5 Hz), 116.37 (q, J = 35.1 Hz), 116.32; negative
H, H-6), 6.37 (d, J = 8.6 Hz, 1H, H-5). 13C NMR (151 MHz, DMSO):
δ
−
−
LC-MS m/z: 401.0 [M − H] calc. for C H F N O , 401.035, found 401.0.
14
8
3
4
7

Molecules 2017, 22, 894
8 of 11
0
N-(2,4,6-Dihydroxybenzylidene)-N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazine (5g). Yield 84%; red solid,
◦
M.p. 238–240 C, Anal. Calcd. for C H F N O (402.24) C, 41.80; H, 2.26: N, 13.93. Found: C, 41.96;
14
9
3
4
7
H, 2.30; N, 13.80%. IR: 3323
υ
(O–H); 3262,
υ
(N–H); 1633, υ(C=N); 1532, υas(NO ); 1263, υs(NO2);
2
1
1
126,
υ
(CF ). H NMR (300 MHz, DMSO):
δ
= 11.44 (s, 1H, NH), 10.02 (s, 1H, OH-c), 9.83 (s, 2H, OH-b,
3
0
0
13
OH-d), 8.89 (s, 1H, H-a), 8.54 (s, 2H, H-3 , H-5 ), 5.86 (s, 2H, H-3, H-5). C NMR (75 MHz, DMSO):
δ = 162.57, 159.57, 150.75 (q, J = 14.5 Hz), 137.13, 135.08, 127.72 (q, J = 18.9 Hz), 122.68 (q, J = 271.5 Hz),
−
−
,
1
4
15.91 (q, J = 35.4 Hz), 98.57, 94.47; negative LC-MS m/z: 401.0 [M
01.035, found 401.0.
−
H] calc. for C H F N O
7
14
8
3
4
0
N-(3,4,5-Trihydroxybenzylidene)-N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazine (5h). Yield 71%; red solid,
◦
M.p. 279–280 C, Anal. Calcd. for C H F N O (402.24) C, 41.80; H, 2.26: N, 13.93. Found: C, 41.62;
14
9
3
4
7
H, 2.15; N, 13.80%. IR: 3428
υ
(O–H); 3266,
υ
(N–H); 1634, υ(C=N); 1537, υas(NO ); 1265, υs(NO2);
2
1
1125,
υ
(CF ). H NMR (300 MHz, DMSO):
δ
= 11.37 (s, 1H, NH), 9.15 (s, 2H, OH-b, OH-d),
3
0
0
13
8.78 (s, 1H, OH-c), 8.53 (s, 2H, H-3 , H-5 ), 8.26 (s, 1H, H-a), 6.53 (s, 2H, H-2, H-6). C NMR (75 MHz,
DMSO): δ = 150.27, 146.09, 137.01, 136.57, 135.66, 127.23 (q, J = 3.5 Hz), 123.76, 122.73 (q, J = 271.5 Hz),
− −
H] calc. for C H F N O , 401.035,
14 8 3 4 7
1
15.82 (q, J = 35.4 Hz), 106.85; negative LC-MS m/z: 401.0 [M
−
found 401.0.
5
3
6
H
a
2
O N
2´
1
3´
4
H
1´
4´
N N
CF3
2
6
´
5´
O N
2
0
◦
N-(Benzylidene)-N -[2,6-dinitro-4-(trifluoromethyl)]phenylhydrazine (5i). Yield 70%; red solid, M.p. 231–232 C,
Anal. Calcd. for C H F N O (354.24) C, 47.47; H, 2.56: N, 15.82. Found: C, 47.65; H, 2.48; N, 15.71%.
14
9
3
4
4
1
IR: 3265,
DMSO):
υ
(N–H); 1631,
υ
(C=N); 1538, υas(NO ); 1267, υs(NO ); 1121,
υ
(CF ). H NMR (600 MHz,
2
0
2
3
0
δ
= 11.57 (s, 1H, NH), 8.57 (s, 2H, H-3 , H-5 ), 8.53 (s, 1H, H-a), 7.55 (dd, J = 7.7, 1.4 Hz, 2H,
13
H-2, H-6), 7.50 – 7.42 (m, 3H, H-3, H-4, H-5). C NMR (151 MHz, DMSO):
δ = 149.02, 137.84, 136.05,
1
34.15, 130.94, 129.44, 127.64 (q, J = 3.4 Hz), 127.58, 123.10 (q, J = 271.6 Hz), 117.29 (q, J = 35.3 Hz);
−
−
4
negative LC-MS m/z: 353.0 [M − H] calc. for C H F N O , 353.05, found 353.0.
14
9
3
4
3
.3. Free Radical Scavenging Activity Assay
The free radical scavenging activity of the prepared hydroxybenzylidene hydrazines was carried
out according to our previous work [25]. Various amounts of tested compounds were added into
a methanol solution of DPPH or GOR and the final DPPH or GOR concentration was kept constant
−
4
−3
) or into a water solution of ABTS. The free radical scavenging activity was
(
c = 10 mol·dm
evaluated using the values SC , i.e., the concentration of the studied compound, which causes
50

Molecules 2017, 22, 894
9 of 11
a 50% decrease in absorbance at 517 nm (for DPPH), 862 nm (for GOR), or 734 nm (for ABTS) as
compared to the control sample. Methanol or water was used as a blank. All samples were measured
in triplicate. Standard square deviations were in a range from 0.91 to 0.99.
3
.4. Photosynthetic Electron Transport (PET) Study
PET was monitored in spinach chloroplasts prepared according to our previous work [4,27].
PET through PSII was monitored by the Hill reaction with DCPIP as an artificial electron acceptor.
DCPIP photoreduction was determined spectrophotometrically. The chlorophyll (Chl) concentration in
3
these experiments was 30 mg/dm . The inhibitory activities of the studied compounds were expressed
by IC50 values, i.e., molar concentrations of the compounds causing a 50% decrease of absorbance at
6
00 nm compared to the control sample. Each sample was measured in triplicate and standard square
deviations were in the 0.88–0.94 range.
3
.5. Molecular Calculations
The prepared hydroxybenzylidene hydrazines, their anions and radicals were studied using the
quantum chemical method PM6 [28], which is part of the program MOPAC2012 [29]. Optimal structures
of compounds were calculated (keyword PRECISE). The effect of solvents on the above mentioned
compounds were studied by COSMO-method [30], which is also part of MOPAC2012 [31]. Ionization
potentials and enthalpy of formations used for the calculation of PDE, BDE, PA, and ETE according to
our previous work [25].
Supplementary Materials: The following are available online: Table S1: Proton dissociation energy of prepared
N-hydroxybenzylidene hydrazines in methanol; Table S2: Dissociation energy of hydrogen and electron of prepared
N-hydroxybenzylidene hydrazines in methanol; Table S3: Proton affinity of prepared N-hydroxybenzylidene
hydrazines in methanol; Table S4: Electron transfer enthalpy of prepared N-hydroxybenzylidene hydrazines in
methanol; Table S5: Proton dissociation energy of prepared N-hydroxybenzylidene hydrazines in water; Table S6:
Dissociation energy of hydrogen and electron of prepared N-hydroxybenzylidene hydrazines in water; Table S7:
Proton affinity of prepared N-hydroxybenzylidene hydrazines in water; Table S8: Electron transfer enthalpy of
prepared N-hydroxybenzylidene hydrazines in water.
Acknowledgments: This work was supported by the Austrian Science Fund (FWF): (P23609, P21437, and F34),
the Slovak Grant Agency VEGA (1/0196/14) and by the European Community’s Seventh Framework Programme
(
FP7/2007–2013) under grant agreement number PCIG11-GA-2012-322300. We want to thank our colleagues from
the Department of Chemistry, Faculty of Natural Sciences, Matej Bell University for measuring IR spectra and
from the Research Institute of Molecular Pathology, Vienna, Austria, for their support.
Author Contributions: F.S., F.G. and P.K. performed experiments. F.S., F.G., J.K., J.F., D.L. and J.G. analyzed
results and wrote the manuscript. All authors contributed to the paper and approved the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).