Synthesis and evaluation of curcuminoid analogues as antioxidant and antibacterial agents

Article abstract of DOI:10.3390/molecules22060971

Diazocoupling reaction of curcumin with different diazonium salts of p-toluidine, 2-aminopyridine, and 4-aminoantipyrine in pyridine yielded the arylhydrazones 2a–c. Arylhydrazone of p-toluidine reacted with urea, thiourea, and guanidine nitrate to produc

Full text of DOI:10.3390/molecules22060971

molecules
Article
Synthesis and Evaluation of Curcuminoid Analogues
as Antioxidant and Antibacterial Agents
Dalia R. Emam 1,*, Ahmad M. Alhajoj 2, Khaled M. Elattar 3, Nabila A. Kheder 4,5 and
Ahmed A. Fadda 3,
*
1
Department of Chemistry, Faculty of Science, King Khalid University, Abha 9004, Saudi Arabia
Department of Pharmacology, Faculty of Pharmacy, King Khalid University, Abha 61441, Saudi Arabia;
amalajoj@kku.edu.sa
Department of Chemistry, Faculty of Science, Mansoura University, Mansoura 35516, Egypt;
khaledelattar2@yahoo.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Khalid University, Abha 61441,
Saudi Arabia; nabila.abdelshafy@gmail.com
Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
Correspondence: daliaimam@hotmail.com (D.R.E.); afadda50@yahoo.com (A.A.F.);
Tel.: +966-050-139-0063 (D.R.E.)
2
3
4
5
*
Received: 11 May 2017; Accepted: 7 June 2017; Published: 11 June 2017
Abstract: Diazocoupling reaction of curcumin with different diazonium salts of p-toluidine,
2-aminopyridine, and 4-aminoantipyrine in pyridine yielded the arylhydrazones 2a–c.
Arylhydrazone of p-toluidine reacted with urea, thiourea, and guanidine nitrate to produce
5,6-dihydropyrimidines. Further reaction of 2a with 2,3-diaminopyrdine in sodium ethoxide solution
yielded 1H-pyrido[2,3-b][1,4]diazepine derivative. Bis(2,5-dihydroisoxazole) is obtained from the
reaction of 2a with hydroxylamine hydrochloride, while its reactions with hydrazines afforded the
respective 4,5-dihydro-1H-pyrazoles. The target compounds were evaluated as antioxidant and
antibacterial agents. The tested compounds showed good to moderate activities compared to ascorbic
acid and chloramphenicol, respectively.
Keywords: curcumin; pyrazole; isoxazole; pyrimidine; antioxidant activity; antibacterial activity
1. Introduction
Curcumin (
from Curcuma longa L. (Zingiberaceae) [
pharmacological activities, including anticancer [
activities, and have been used to improve wound healing [9].
1
) is considered a type of natural product compound and is naturally extracted
]. Curcumin analogues have been reported to have variable
], anti-inflammatory [ ], and antibacterial [8]
1
2–
6
7
Previously, Fadda et al. [10] reported the synthesis of 17 curcumin analogues with an evaluation of
the compounds as antitumor agents. The obtained results of both in vitro and in vivo antitumor
activity were excellent (100% dead) compared with 5-fluorouracil at a concentration of 1 M of
the tested compounds. Recently, Bayomi et al. [11] have reported the synthesis and evaluation
of a series of curcumin derivatives as antioxidant agents using the 2,20-azino-bis(3-ethylbenzo-
thiazoline-6-sulphonic acid) (ABTS) method. The compounds that bear o-methoxy substitution to the
4-hydroxy function exhibited significantly higher ABTS+-scavenging.
On the other hand, a review discussing the antibacterial activity of curcumin against Staphylococcus
aureus was reported by Teow et al. [12]. In addition, Moghadamtousi et al. [13] have reported on the
antifungal, antibacterial, and antiviral activities of curcumin.
In connection with our studies in the chemistry of curcumin, ketones, and unsaturated
ketones [14–16], the present work is concerned with extending attempts to synthesize a novel series of
heterocyclic curcumin derivatives in order to evaluate their antioxidant and antibacterial characters.
Molecules 2017, 22, 971; doi:10.3390/molecules22060971
www.mdpi.com/journal/molecules

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2. Resultsand Discussion
2.1. Chemistry
In this work, different series of curcumin derivatives were synthesized as described in Schemes 1–3,
and their antioxidant and antibacterial activities were evaluated.
Diazocoupling of curcumin with the appropriate aryl diazonium chlorides such as p-toluidine,
2-aminopyridine, and 4-aminoantipyrine in a pyridine solution yielded the corresponding
arylhydrazone derivatives 2a
on the basis of elemental analysis and spectral data. The IR spectra of 2a
at
= 3250, 3220, and 3218 cm−1, respectively, corresponding to NH-hydrazo groups. The 1H-NMR
spectra of 2a revealed a singlet signal in the region 10.83–11.03 ppm due to NH proton. The mass
spectra of 2a
showed the molecular ion peaks at m/z 486 (M+, 7.31), 473 (M+, 16.60), and 582 (M+,
–
c
(Scheme 1). The structures of Compounds 2a
–c were elucidated
–c
showed absorption bands
ν
–
c
δ
–
c
20.40), which are compatible with the proposed structures.
Scheme 1. Synthesis of 1,7-bis(4-hydroxy-3-methoxyphenyl)-4-(2-(aryl)hydrazono)hepta-1,6-diene-3,5-diones
(2a–c).
Arylhydrazone derivative of curcumin 2a was used as a starting material to synthesize the target
compounds. Compound 2a has two active symmetric α,β-unsaturated ketones and a 1,3-diketone
moiety. Intermolecular cyclization of 2a with bis-nitrogen nucleophiles (in molar ratios of 1:2),
e.g., urea, thiourea, and guanidine nitrate, in refluxing sodium ethoxide and ethanol yielded
the corresponding 4,40-((2-phenylhydrazono)methylene)bis(6-(4-hydroxy-3-methoxy-phenyl)-5,6-
dihydropyrimidin-2(1H)-one/thione/imine) derivatives 3–5 in good yields. The reaction was
started through initial condensation of the amino group with the carbonyl group of the
arylhydrazonocurcumin skeleton, followed by cyclization to produce the target compounds.
The structures
section). In another route, heating 2a with 2,3-diaminopyridine in a 1:2 molar ratio worked to
furnish bis(2-methyl-2,3-dihydro-1H-pyrido[2,3-b][1,4]diazepine) derivative in good yield (Scheme 2).
The 1H-NMR of Structure
revealed two singlet signals at 10.14 and 11.26 ppm due to NH protons,
3–5 were established by elemental analysis and spectral data (c.f. the experimental
6
6
δ
in addition to other expected protons. The mass spectrum showed a molecular ion peak at m/z 669
(M+ +1, 7.24), which is compatible with the proposed structure.
Next, our attempts were focused to prepare curcumin analogues incorporated five-membered
ring systems in order to study the effect of different ring systems on biological behaviors. In this way,
Compound 2a reacted with hydroxylamine hydrochloride (in a 1:2 molar ratio) in refluxing pyridine to
afford the respective 2,5-dihydro-isoxazole
of 2a with hydrazine derivatives, i.e., hydrazine hydrate, phenyl hydrazine, and ethylhydrazine
afforded the corresponding pyrazole derivatives 10, respectively (Scheme 3). The proposed structures
7–10 are consistent with the analytical and spectral data (c.f. the experimental section).
7 in moderate yield. In addition, intermolecular cyclization
8–

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Scheme 2. Synthesis of (pyrimidin-one/thione/imine) and diazepine derivatives.
O
NH HN
O
NH2OH. HCl (1: 2)
pyridine
Ar
Ar
N
NH
Ar'
O
O
7
Ar
Ar
N
NH
Ar'
2a
H
N
R
R
N
N
N
N
H2N
R (1: 2)
Ar= 3-OCH3-4-OH-C6H3
Ar
Ar
EtOH/ AcOH (1:1)
Ar'= C6H4-CH3-p
N
NH
Ar '
8: R= H
9: R= Ph
10: R= Et
Scheme 3. Synthesis of 2,5-dihydro-isoxazole and pyrazole derivatives.
2.2. Pharmacology
2.2.1. DPPH Antioxidant Assay
The target compounds were tested as antioxidant agents using a 2,2-diphenyl-1-picrylhydrazyl
(DPPH) assay. The antioxidant activity was evaluated by the DPPH method at = 517 nm [17 18].
λ
,
The IC50 values of the tested compounds revealed that the tested compounds exhibited significant
antioxidant properties at a concentration 0.09 mg/mL. In other words, these compounds have the
ability of electron donor to scavenge free radicals. Figure 1 showed a comparison between the values
of IC50 of the tested compounds against ascorbic acid.
The IC50 of antioxidant activity data (Table 1 and Figure 1) indicated that curcumin and its
derivative 2a exhibited the highest antioxidant activity compared to ascorbic acid. Figure 2 shows
a comparison between the inhibition percentage of the tested compounds at concentrations 11.72
and 46.88
µ
g/mL. The compounds at higher concentrations have higher inhibition percentages than
2c , and exhibited no antioxidant activity at low
g/mL), while these compounds have a high activity at higher concentrations
those at low concentration. Compounds 2a
concentrations (11.72
,
,
5
6
µ
(46.88 µg/mL).

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Table 1. IC50 and inhibition percentage of the tested Curcumin derivatives using a 2,2-diphenyl-1-
picrylhydrazyl (DPPH) assay.
Inhibition %
Compound No.
IC50
46.88 µg/mL
11.72 µg/mL
Curcumin
0.252
0.253
1.881
0.548
1.732
0.679
0.477
1.650
1.794
0.615
1.190
4.412
0.022
52.91
49.74
45.64
49.23
45.04
47.61
54.87
44.79
47.61
49.15
48.29
48.80
–
51.11
–
36.50
–
39.40
45.47
–
2a
2b
2c
3
4
5
6
7
–
46.32
47.44
45.98
46.67
–
8
9
10
Ascorbic acid
IC50 = Concentration at 50% DPPH makes scavenging.Inhibition percentage of the target compounds were calculated
at concentrations 46.88 µg/mL and 11.72 µg/mL.
Curcumin
4.5
2a
4
2b
3.5
2c
3
3
2.5
2
4
5
6
1.5
1
7
8
9
0.5
0
10
Compounds
Ascorbic acid
Figure 1. Comparison of IC50 values of the investigated compounds against ascorbic acid.
From the obtained results (Table 1), we conclude the following structure–activity relationships
(SARs): (1) Heterocyclic derivatives of curcumin are less active than curcumin using low concentrations.
(2) The different aryl substituents of compounds 2a
contains a non-substituted benzene ring. (3) By comparing the structures of Compounds
the obtained results, it was found that the most potent was Compound , which had the lowest
electronegative atom. (4) The ring size in the case of Compounds and has no effect on the values of
the antioxidant power. (5) Compounds 10 (R = H, Ph, Et) exhibited variable IC50 values, of which
–c
affect the IC50 values, the best derivative which
3–5
with
5
6
7
8–
the unsubstituted one is preferred. (6) Using higher concentrations of the tested sample increases the
antioxidant power of the tested compounds (higher than 40 µg/mL).

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60
54.87
48.29
52.91
47.61
49.15
47.44
49.23
51.11
46.67
45.98
47.61
45.04
49.74
45.64
48.8
46.32
45.47
50
40
30
20
10
0
44.79
39.4
36.5
0
0
0
0
11.72 µg/ mL
46.88 µg/ mL
COMPOUND NO.
Curcumin 2a 2b 2c
3
4
5
6
7
8
9
10
Figure 2. Comparison of the inhibition percentages of the tested compounds using a DPPH assay.
2.2.2. Antibacterial Activity
Antibacterial activity of the newly prepared target compounds against the Gram-positive (Bacillus
subtilis and Staphylococcus aureus) and the Gram-negative (Escherichia coli and Pseudomonas aeruginosa)
bacterial strains were examined by agar diffusion. Chloramphenicol was used as a positive control.
The inhibition zones (IZ) were measured in mm. The minimum inhibitory concentration (MIC) was
determined for compounds showingsignificant growth of the inhibition zone (>14 mm) using twofold
serial dilution [19]. The MIC (µg/mL) and IZ (diameter) values are shown in Table 2. The inhibition
zones (diameter) values mentioned in Table 2 between brackets are attributed to the tested original
concentration (1 mg/mL) as a preliminary test.
Table 2. Minimal inhibitory concentration (MIC,
µg/mL) and inhibition zone (mm) of the
tested compounds.
MIC a in µg/mL and Inhibition Zone (mm)
Bacteria
Compound No.
Gram-Positive Bacteria
Gram-Negative Bacteria
B. subtilis
S. aureus
E. coli
P. aeruginosa
2a
2b
2c
3
4
5
6
7
100 (15)
80 (20)
95 (15)
25 (30)
3.125 (38)
50 (25)
100 (14)
80 (38)
95 (18)
25 (21)
6.25 (34)
50 (30)
6.25 (41)
6.25 (39)
6.25 (40)
6.25 (41)
6.25 (42)
3.125 (44)
100 (16)
80 (14)
96 (38)
50 (24)
50 (23)
50 (18)
50 (19)
50 (16)
50 (18)
12.5 (37)
25 (35)
6.25 (39)
100 (18)
80 (15)
100 (18)
100 (15)
12.5 (32)
100 (15)
50 (20)
25 (35)
50 (19)
25 (33)
25 (39)
12.5 (38)
25 (30)
8
9
10
12.5 (40)
6.25 (44)
3.125 (45)
3.125 (42)
a
Chloramphenicol
6.25 (38)
MIC: Minimal inhibitory concentration.
The results shown in Table 2 indicate that the majority of the tested compounds showed variable
inhibitory effects on the growth of the tested Gram-positive and Gram-negative bacterial strains.
In general, most of the examined compounds suggested strong antibacterial action against the
Gram-positive rather than the Gram-negative bacteria.

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Moreover, it showed that Compounds
4
and 10 exhibited the highest antibacterial activity
compared to the results of chloramphenicol and the other tested compounds. In addition, it revealed
that the pyrazole derivative of curcumin (10) is more potent than chloramphenicol against B. subtilis,
while the thiopyrimidine analogue
In addition, Compounds , and exhibited moderate activity against B. subtilis, while Compounds
have the same effect as those of Compounds and 10 on the S. aureus cell line. On the other hand,
Compound has the highest activity against Gram-negative bacteria (E. coli), while Compound
4 is nearly equipotent to chloramphenicol against B. subtilis.
6
,
8
9
6
–9
4
9
4
showed the highest activity against P. aeruginosa. Moreover, Compounds 2a–c and 3 showed weak
antibacterial activity compared to chloramphenicol.
By comparing the obtained results (Table 2) with the compound structures, the following SARs
maybe concluded: (1) Compounds that contain six or seven membered rings are more potent than
those with no heterocyclic systems. (2) The presence of high electronegative atom may decrease
antibacterial activity. (3) The presence of H and phenyl substitution (Compounds 8–10) decreases
lipophilicity. (4) The presence of alkyl substituent (Compound 10) enhanced antibacterial activity.
3. Materials and Methods
3.1. Chemistry
3.1.1. General
All melting points were determined on a Gallenkamp electric apparatus and are considered as
uncorrected. The IR spectra were recorded on a Nicolet Magna (model 550 spectrophotometers, LabX,
Markham, Ontario, ON, Canada) using a potassium bromide disk. The nuclear magnetic resonance
spectra were determined in DMSO on a Bruker WPSY 200 MHz spectrometer (Bruker, 15 Fortune Dr,
Billerica, MA 01821, USA) using tetramethylsilane as an internal standard and the chemical shifts (δ)
are in ppm. The mass spectra (energy of ionizing electron 70 electron-volt (eV) with a Varian MAT 311
spectrophotometer (Bremen, Germany). The elemental analyses for C, H, and N were satisfactory for all
synthesized compounds. Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione) (1)
was purchased from Sigma-Aldrich-08511, St. Luis, MO, USA. 1,7-Bis(4-hydroxy-3-methoxy-phenyl)-4-
(2-(p-tolyl)hydrazono)hepta-1,6-diene-3,5-dione (2a) was prepared according to [10]. Antioxidant and
antibacterial evaluation was carried out at Unit of Genetic Engineering and Biotechnology, Faculty of
Science, Mansoura University.
3.1.2. Synthesis of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-(2-(aryl)hydrazono)hepta-1,6-diene-
3,5-diones (2a–c)
General procedure: Preparation of the diazonium salt: 0.2 g of sodium nitrite was dissolved
in 2 mL of water in an ice bath and added drop by drop to a solution of various aromatic amines,
such as p-toluidine (0.01 mol), 2-aminopyridine (0.01 mol), and 4-aminoantipyrine (0.01 mol) in
conc. HCl (3.0 mL). The formed diazonium salt solution was added drop by drop with stirring to
curcumin (1) dissolved in pyridine (20 mL) in an ice bath. The reaction mixture was stirred for an
◦
◦
additional two hours at 0–5 C and left overnight in the refrigerator at 0 C. The precipitated solid was
filtered off, washed with water, dried, and recrystallized from ethanol to yield curcumin derivatives
2a–c, respectively.
1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-(2-(p-tolyl)hydrazono)hepta-1,6-diene-3,5-dione (2a) [10].
1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-(2-(pyridin-2-yl)hydrazono)hepta-1,6-diene-3,5-dione (2b): Yield 56%;
dark red powder, m.p. 176 ◦C; IR (KBr): /cm−1 = 3415 (OH), 3220 (NH), 1682 (C=O), 1635 (C=C).
ν
1H-NMR (DMSO-d6):
δ
/ppm = 3.92 (s, 6H, 2
×
OCH3), 5.84 (s, 2H, 2
×
OH), 6.52 (d, 2H, J = 12.7 Hz, 2
CH=CH),10.83 (s, 1H, NH). MS:
×
CH=CH), 6.83–7.81 (m, 10 H, Ar-H), 7.90 (d, 2H, J = 12.7 Hz, 2
×
m/z (%) = 473 (M+, 16.60), Anal. for C26H23N3O6 (473.48); Calcd. C, 65.95; H, 4.90; N, 8.87%; Found: C,
66.07; H, 4.96; N, 8.96%.

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4-(2-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)hydrazono)-1,7-bis(4-hydroxy-3-
methoxyphenyl)hepta-1,6-diene-3,5-dione (2c): Yield 68%; dark yellow powder, m.p. 184 ◦C; IR
(KBr):
ν
/cm−1 = 3420 (OH), 3218 (NH), 1678 (C=O),1615 (C=C). 1H-NMR (DMSO-d6):
OCH3), 5.35 (s, 2H, 2 OH), 6.67 (d, 2H, J = 12.2 Hz,
CH=CH), 10.83 (s, 1H, NH). MS:
δ/ppm = 2.26
(s, 3H, CH3), 3.11 (s, 3H, N-CH3), 3.83 (s, 6H, 2
×
×
2
×
CH=CH), 6.90–7.37 (m, 11H, Ar-H), 7.70 (d, 2H, J = 12.2 Hz, 2
×
m/z (%) = 582 (M+, 20.40), Anal. for C32H30N4O7 (582.60); Calcd. C, 65.97; H, 5.19; N, 9.62%; Found: C,
65.86; H, 5.26; N, 9.74%.
3.1.3. Synthesis of 4,40-((2-Phenylhydrazono)methylene)bis(6-(4-hydroxy-3-methoxy-phenyl)-5,6-
dihydropyrimidin-2(1H)-one/thione/imine) Derivatives (3–5)
General procedure: Mixtures of compound 2a (0.01 mol) with diamines, i.e., urea (0.02 mol),
thiourea (0.02 mol), or guanidine nitrite (0.02 mol), in sodium ethoxide solutions (0.02 mol sodium, in
50 mL of ethanol) were refluxed for 6–12 h. The reaction mixtures were left to cool at room temperature,
poured into ice-cold water, and acidified with dilute acetic acid. The precipitated solids were filtered,
dried, and recrystallized from ethanol to yield the corresponding pyrimidine derivatives 3–5.
4,40-((2-(p-Tolyl)hydrazono)◦methylene)bis(6-(4-hydroxy-3-methoxyphenyl)-5,6-dihydro-pyrimidin-2(1H)-one)
(3): Yield 41%; m.p. 235 C; IR (KBr): ν
/cm−1 = 3417 (OH), 3225 (NH), 1648 (C=O, amidic), 1618
(C=N). 1H-NMR (DMSO-d6):
δ/ppm = 1.32–1.50 (m, 2H, CH2), 1.66–1.91 (m, 2H, CH2), 2.35 (s, 3H,
CH3),3.85 (s, 6H, 2OCH3), 4.90 (m, 2H, 2CH), 5.41 (s, 2H, 2OH), 6.50–6.98 (m, 10H, Ar-H), 10.28 (s, 2H,
2NH), 11.23 (s, 1H, NH). MS: m/z (%) = 570 (M+, 6.43), Anal. for C30H30N6O6 (570.60): Calcd. C, 63.15;
H, 5.30; N, 14.73%; Found: C, 63.27; H, 5.34; N, 14.81%.
0
4,4 -((2-(p-Tolyl)hydrazono)methylene)bis(6-(4-hydroxy-3-methoxyphenyl)-5,6-dihydropyrimidine-2(1H)-thione)
(4 ν
): Yield 62%; m.p. 260 ◦C; IR (KBr): /cm−1 = 3415 (OH), 3235 (NH), 3223 (NH), 2935 (CH2), 1640
(C=N), 1278 (C=S). 1H-NMR (DMSO-d6):
δ/ppm = 1.76–1.92 (m, 2H, CH2), 2.21–2.35 (m, 2H, CH2),
2.44 (s, 3H, CH3), 3.74 (s, 6H, 2OCH3), 4.85 (m, 2H, 2CH), 5.23 (s, 2H, 2OH), 6.34–6.87 (m, 10H, Ar-H),
10.13 (s, 2H, 2NH), 11.35 (s, 1H, NH). MS: m/z (%) = 602 (M+, 20.15), Anal. for C30H30N6O4S2 (602.73):
Calcd. C, 59.78; H, 5.02; N, 13.94%; Found: C, 59.82; H, 5.13; N, 13.97%.
4-(6-((6-(4-Hydroxy-2-methoxyphenyl)-2-imino-1,2,5,6-tetrahydropyrimidin-4-yl)(2-p-tolylhydrazono)methyl)
-2-imino-2,3,4,5-tetrahydropyrimidin-4-yl)-2-methoxyphenol (5
): Yield 51%; m.p. 240 ◦C; IR (KBr):
ν/cm−1 = 3422 (OH), 3200 (NH), 3180 (NH), 2915 (CH2), 1630 (C=N). 1H-NMR (DMSO-d6):
δ/ppm = 1.53–1.71 (m, 2H, CH2), 2.01–2.24 (m, 2H, CH2), 2.31 (s, 3H, CH3), 3.85 (s, 6H, 2OCH3), 4.88
(m, 2H, 2CH), 5.17 (s, 2H, 2OH), 6.47–6.93 (m, 10H, Ar-H), 10.23 (s, 2H, 2NH), 11.30 (s, 2H, 2NH), 11.45
(s, 1H, NH). MS: m/z (%) = 569 (M+ +1, 12.15), Anal. for C30H32N8O4 (568.63): Calcd. C, 63.37; H, 5.67;
N, 19.71%; Found: C, 63.47; H, 5.72; N, 19.78%.
3.1.4. Synthesis of 4,40-(4,40-((2-(p-Tolyl)hydrazono)methylene)bis(2,3-dihydro-1H-pyrido[2,3-b][1,4]
diazepine-4,2-diyl))bis(2-methoxyphenol) (6)
A mixture of arylhydrazone 2a (0.01 mol) and 2,3-diaminopyridine (0.02 mol) in a sodium ethoxide
solution (0.02 mol sodium, in 40 mL ethanol) was refluxed for 18 h. The reaction mixture was left to cool
at room temperature, poured into ice-cold water, and acidified with dilute acetic acid. The precipitated
solids were filtered,◦dried, and recrystallized from ethanol to yield 1H-pyrido[2,3-b][1,4]diazepine
6.
−1
Yield 67%; m.p. 270 C; IR (KBr):
ν
/cm = 3400 (OH), 3220 (NH), 3160 (NH), 2910 (CH2), 1618 (C=N).
1H-NMR (DMSO-d6):
δ/ppm = 1.74–1.90 (m, 2H, CH2), 2.13–2.30 (m, 2H, CH2), 2.42 (s, 3H, CH3), 3.21
(s, 2H, 2CH), 3.79 (s, 6H, 2
×
OCH3), 5.43 (s, 2H, 2
×
OH), 6.32–6.83 (m, 10H, Ar-H), 7.31–8.22 (m, 6H,
Ar-H), 10.14 (s, 2H, 2NH), 11.26 (s, 1H, NH). MS: m/z (%) = 669 (M+ +1, 7.24), Anal. for C38H36N8O4
(668.74): Calcd. C, 68.25; H, 5.43; N, 16.76%; Found: C, 68.31; H, 5.51; N, 16.83%.

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3.1.5. Synthesis of 4,40-(3,30-((2-(p-Tolyl)hydrazono)methylene)bis(2,5-dihydro-isoxazole-5,3-diyl))bis
(2-methoxyphenol) (7)
A mixture of arylhydrazone 2a (0.01 mol) and hydroxylamine hydrochloride (0.02 mol) in pyridine
(30 mL) was refluxed for 10 h, left to cool, and diluted with water, and the obtained solid products were
crystallized from ethanol to yield isoxazole derivative
7 ν
. Yield 53%; m.p. 200 ◦C; IR (KBr): /cm−1
= 3422 (OH), 3233, 3018 (NH), 1620 (C=N). 1H-NMR (DMSO-d6):
δ/ppm = 2.31 (s, 3H, CH3), 3.76 (s,
6H, 2
×
OCH3), 5.07 (d, 2H, 2CH), 5.22 (d, 2H, 2CH), 5.37 (s, 2H,2
×
OH), 6.32–6.83 (dd, 4H, Ar-H),
7.31–8.22 (m, 6H, Ar-H), 10.14 (s, 2H, 2NH), 10.34 (s, 1H, NH). MS: m/z (%) = 518 (M++2, 13.08), Anal.
for C28H28N4O6 (516.55): Calcd. C, 65.11; H, 5.46; N, 10.85%; Found: C, 65.32; H, 5.54; N, 10.93%.
3.1.6. Synthesis of 4,40-(3,30-((2-(p-Tolyl)hydrazono)methylene)bis(1-substituted/unsubstituted-4,5-
dihydro-1H-pyrazole-5,3-diyl))bis(2-methoxyphenol) Derivatives (8–10)
General procedure: Mixtures of arylhydrazone 2a (0.01 mol) and the appropriate hydrazine
derivative, i.e., hydrazine hydrate (0.02 mol), phenyl hydrazine (0.02 mol), or ethyl hydrazine (0.02 mol),
in mixtures of ethanol/glacial acetic acid (1:1) were refluxed for 4–8 h. The reaction mixtures were left
to cool at room temperature and poured into ice-cold water.The precipitated solids were filtered, dried,
and recrystallized from ethanol to yield bis-pyrazole derivatives (8–10).
4,40-(3,30-((2-(p-Tolyl)hydrazono)methylene)bis(4,5-dihydro-1H-pyrazole-5,3-diyl))bis(2-methoxyphenol) (
8
):
/cm−1 = 3400 (OH), 3212, 3052 (NH), 1605 (C=N). H-NMR
/ppm = 1.61–1.84 (m, 2H, CH2), 2.17–2.28 (m, 2H, CH2), 2.42 (s, 3H, CH3), 3.86 (s, 6H,
◦
1
Yield 82%; m.p. 200 C; IR (KBr):
(DMSO-d6):
ν
δ
2 × OCH3), 3.96 (t, 2H, 2CH, J = 7 Hz), 5.67 (s, 2H, 2
×
OH), 6.71–7.62 (m, 10H, Ar-H), 10.82 (s, 2H,
2NH), 11.16 (s, 1H, NH). MS: m/z (%) = 514 (M+, 25.12), Anal. for C28H30N6O4 (514.58): Calcd. C, 65.35;
H, 5.88; N, 16.33%; Found: C, 65.43; H, 5.91; N, 16.46%.
4,40-(3,30-((2-(p-Tolyl)hydrazono)methylene)bis◦(1-phenyl-4,5-dihydro-1H-pyrazole-5,3-diyl))bis(2-
methoxyphenol) (9): Yield 71%; m.p. 220 C; IR (KBr): ν
/cm−1 = 3422 (OH), 3233 (NH), 1620
(C=N). 1H-NMR (DMSO-d6):
CH3), 3.95 (s, 6H, 2
δ/ppm = 1.74–1.95 (m, 2H, CH2), 2.34–2.57 (m, 2H, CH2), 2.74 (s, 3H,
×
OCH3), 4.17 (t, 2H, 2CH, J = 7 Hz), 5.84 (s, 2H, 2
×
OH), 6.36–7.18 (m, 20H,
Ar-H), 11.06 (s, 1H, NH). MS: m/z (%) = 666 (M+, 17.51), Anal. for C40H38N6O4 (666.77): Calcd. C,
72.05; H, 5.74; N, 12.60%; Found: C, 72.21; H, 5.82; N, 12.74%.
0
0
4,4 -(3,3 -((2-(p-Tolyl)hydrazono)methylene)bis(1-ethyl-4,5-dihydro-1H-pyrazole-5,3-diyl))bis(2-methoxyphenol)
(
◦
1
10): Yield 75%; m.p. 207 C; IR (KBr):
(DMSO-d6): /ppm = 1.13 (t, 6H, 2CH3, J = 7 Hz), 1.68–1.83 (m, 2H, CH2), 2.25–2.49 (m, 2H, CH2), 2.71
(q, 4H, 2CH2, J = 7.2 Hz), 2.98 (s, 3H, CH3), 3.78 (s, 6H, 2 OCH3), 3.85 (t, 2H, 2CH, J = 7 Hz), 5.43 (s,
2H, 2
OH), 6.39–6.84 (m, 10H, Ar-H), 10.08 (s, 1H, NH). MS: m/z (%) = 571 (M+ +1, 33.09). Anal. for
ν
/cm−1 = 3979 (OH), 3207 (NH), 1615 (C=N). H-NMR
δ
×
×
C32H38N6O4 (570.68): Calcd. C, 67.35; H, 6.71; N, 14.73%; Found: C, 67.43; H, 6.76; N, 14.84%.
3.2. Pharmacology
3.2.1. Antioxidant Activity
Several concentrations of each tested sample were prepared in MeOH. One milliliter of DPPH
(0.012%) in MeOH was added to each concentration. The prepared sample test was left to stand in dark
at room temperature for 30 min. The absorbance of each tested sample was measured at λ = 517 nm.
The inhibition percentage of the investigated compounds was calculated from Equation (1):
[(A1 − A2)/A1] × 100.
(1)
A1 = control absorbance.
A2 = sample absorbance.

Molecules 2017, 22, 971
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The inhibitive concentrations were obtained using the inhibition curve by drawing a correlation
between the percentageof DPPH inhibition and the concentrations of each tested sample [20].
3.2.2. Antibacterial Evaluation
The antibacterial activity tests were carried out according to the previously reported
methods [21,22]. What man filter paper disks were obtained with a diameter size of 5.0 mm and
sterilized using screw capped wide mouthed containers. Petri dishes 9 cm in diameter were kept
at 150 ◦C, the agar media were then added to each plate, and the microorganism was seeded. The
sterilized filter paper disks saturated with a solution of each tested sample in DMSO (1 mg/mL)
were added to each plate in triplicates. The used tested microorganisms were different types of
Gram-positive and Gram-negative bacteria, and chloramphenicol was used as a reference drug. DMSO
as a solvent for the tested compound was used as a control has no effect on the bacterial growth. The
◦
plates were incubated at 37 C for 24 h for bacterial growth. Compounds that showed significant
growth inhibition zones (>14 mm) using the twofold serial dilution technique were evaluated for
their MICs.
3.2.3. Minimal Inhibitory Concentration (MIC) Measurement
The MIC values were measured by a determination of the optical density of bacterial cell at a
wavelength (λ) of 600 nm. The experimental method that was used for the determination of MIC
wascarried out according to our previously reported method [23].
4. Conclusions
A novel series of curcumin derivatives were synthesized and subjected to an antioxidant activity
test using a DPPH assay and to antibacterial screening using disc diffusion. Among the newly
synthesized compounds, curcumin and hydrazone derivative 2a exhibited good activity compared
to ascorbic acid, while the rest of the compounds showed moderate antioxidant activities. Results of
antioxidant activity clearly demonstrated that the heterocyclic derivatives of curcumin had less
activity than curcumin. Phenolic antioxidants such as curcumin compounds can inhibit free radical
formation and are electron donors scavenging free radicals. The antibacterial activity of the synthesized
compounds were evaluated against Gram-negative and Gram-positive bacterial strains. The results of
the antibacterial evaluation revealed that the pyrazole derivative of curcumin (10) is more potent than
chloramphenicol against B. subtilis. The synthesized compounds can be applied next on animals such
as mice, in order to evaluate the histological and pathological diagnoses.
Acknowledgments: The authors extend their appreciation to the Deanship of Scientific Research at King Khalid
University for funding this work through General Research Project under grant number (G.R.P-495-38).
Author Contributions: All authors contributed equally to this manuscript.
Conflicts of Interest: The authors declare that there is no conflict of interests regarding the publication of
this paper.
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Sample Availability: Samples of the compounds are available from the authors.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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