Syntheses of novel pyrazolines as antibacterial agents from natural product vanillin

Article abstract of DOI:10.14233/ajchem.2017.20247

A series of pyrazoline derivatives have been synthesized from natural vanillin and tested as antibacterial agents by diffusion method. Pyrazolines were synthesized from vanillin via chalcone intermediate. The structure of all the products was elucidated b

Full text of DOI:10.14233/ajchem.2017.20247

Asian Journal of Chemistry; Vol. 29, No. 2 (2017), 454-456
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2017.20247
Syntheses of Novel Pyrazolines as Antibacterial Agents from Natural Product Vanillin
1
1,*
2
AMRI SETYAWATI , TUTIK DWI WAHYUNINGSIH and BAMBANG PURWONO
1
2
Chemistry Study Program, Universitas Islam Indonesia, Yogyakarta, Indonesia
Chemistry Department, Universitas Gadjah Mada, Yogyakarta, Indonesia
*Corresponding author: E-mail: tutikdw@ugm.ac.id
Received: 17 August 2016; Accepted: 24 October 2016;
Published online: 30 November 2016;
AJC-18179
A series of pyrazoline derivatives have been synthesized from natural vanillin and tested as antibacterial agents by diffusion method.
Pyrazolines were synthesized from vanillin via chalcone intermediate. The structure of all the products was elucidated by FTIR, GC-MS,
1
13
H NMR and C NMR spectrometers.All the tested compounds showed their potential as antibacterial agents in broad spectrum. Inhibition
value of all the active compounds was compared with standard drug tetracycline and DMSO. The highest antibacterial activity of pyrazoline
derivatives was found against Staphylococcus aureus (1000 ppm, 7.25 mm). Antibacterial tests of pyrazoline derivatives showed that
N-acetyl pyrazoline increased the antibacterial activity at Gram-positive bacteria but decreased at Gram-negative ones.
Keywords: Vanillin, Chalcone, Pyrazoline, Antibacterial.
INTRODUCTION
EXPERIMENTAL
Bacterial pathogens are the main cause of human disease
All chemicals were procured from E. Merck with pro-
analysis (p.a) quality such as vanillin, 2-hydroxycetophenone,
hydrazine monohydrate, potassium bromate, sodium
thiosulphate, sodium hydroxide, hydrochloric acid, glacial acetic
acid, ethanol, methanol, n-hexane, ethyl acetate and dimethyl
sulfoxide 99.9 %.
in developing countries and can cause, for example, malaria,
tuberculosis, cholera, pneumonia, respiratory problems and
diarrhea [1]. Treatment of these infections became more difficult
because the emergence of drug resistant strains [2,3].
Compounds with C=N and N-N groups, such as pyra-
zolines, have many pharmacological activities. Pyrazolines are
well known five membered heterocyclic compounds with anti-
bacterial properties [4,5]. In addition, pyrazoline derivative
compounds have analgesic and anti-inflammatory properties
Melting points were measured using Electrothermal 9100
melting point apparatus and are uncorrected. Infrared spectra
were recorded with a Shimadzu Prestige-21 FTIR spectrometer
1
13
using KBr disc. H NMR and C NMR spectra were obtained
on a JEOL JNMECA (500 MHz) spectrometer. Mass spectra
were recorded on Agilent GC-6890 and MS-5973 (EI). The
purity of the compounds was confirmed by GC and thin layer
chromatography.
[6].
Several methods exist for the synthesis of pyrazolines.
One of the most important methods is the reaction of α,β-
unsaturated ketones with 1,2-binucleophile by refluxing [7].
Several pyrazoline compounds have been modified to be more
active as antibacterial agents by the addition of bromide [8,9],
hydroxy and methoxy groups [10]. Designing and exploring a
new a synthetic method should consider the availability of
materials. Vanillin and wintergreen oil are an aldehyde source
from available natural resources.
3-Bromo-4-hydroxy-5-methoxybenzaldehyde (1):
Vanillin was brominated by Schatz method [11] under acidic
condition using KBrO
3
as bromide source and CH COOH as
3
acid. The white crystal was yielded (98 %) with m.p.: 164 °C,
-1
IR spectrum (KBr, cm ): 3333 (OH), 2846 and 2746 (C-H),
1
1674 (C=O), 1589 (C=C), 1357 (C-H). H NMR spectrum (500
Several studies focused on the influence of substituents
on the phenyl group of pyrazoline on its antibacterial ability.
But the effect of substituents on N-pyrazoline has not been
widely reported. This research focuses on the syntheses of N-
acetylated pyrazoline derived from vanillin and wintergreen
oil and testing of the antibacterial properties of this compound.
MHz, CDCl
Ar-H), 7.72 (d, 1H, J = 1.3 Hz, CH
NMR spectrum (125 MHz, CDCl ): δ 29.88 (OCH
149.78 (Ar-C), 190.43 (CHO. MS: 232 (M+2).
(E)-3-(3-Bromo-4-hydroxy-5-methoxyphenyl)-1-(2-
hydroxyphenyl)prop-2-en-1-one (2): 5-Bromovanillin (1.07 g,
3
): δ 3.90 (s, 3H, OCH ), 7.41 (d, 1H, J = 1.3 Hz,
3
13
α
), 9.77 (s, 1H, CHO). C
), 56.73-
3
3

Vol. 29, No. 2 (2017)
Syntheses of Novel Pyrazolines as Antibacterial Agents from Natural Product Vanillin 455
5
mmol) was dissolved in methanol and solution of 2-hydroxy-
stirred for 6 h then poured onto crushed ice. The precipitate
was filtered off, washed with ethanol and dried to get white
powder (Scheme-I).Yield 88.88 %, m.p. 106-111 °C, IR (KBr,
acetophenone (0.67 mL, 5 mmol) in 12 mL NaOH 20 % was
added. The mixture was refluxed for 24 h then poured into
crushed ice and acidified with HCl 10 % to form a precipitate.
The precipitate was filtered off and recrystallized from ethanol
to give yellow solid crystal.Yield 78 %, m.p. 168 °C, IR spectrum
-1
cm ): 3425 (OH), 2924 (CH
3
-sym), 2854 (CH
(C=O), 1597 (C=N), H NMR (500 MHz, CDCl
3H, CH ), 3.28 J = 17.82 Hz; 5.2 (dod, 1H, CH), 3.85 J =
17.82 Hz; 11,7 (dod, 1H, CH), 5.44 J = 12.05 Hz; 5.2 (dod,
3
-asym), 1643
1
3
): δ 2.39 (s,
3
-1
(
KBr, cm ): 3387 (OH), 1635 (C=O), 979 (C-H trans-bend).
1
13
H NMR spectrum (500 MHz, CDCl
6
J = 15.55 Hz, CH
3
): δ 3.99 (s, 3H, CH
.22 (s, 1H, OH), 7.5 (d, 1H, J = 14, 95 Hz, CH ), 7.79 (d, 1H,
). C NMR spectrum (125 MHz, CDCl ): δ
3
),
1H, CH), C NMR (125 MHz, CDCl
3
): δ 29.88 (CH ), 42.91
3
α
(CH), 56.60 (C-N), 58.47 (C-O-C), 147.51 (C=N). GC 90 %,
MS: 406 (M+2).
13
β
3
2
9.88 (CH
C=O). MS: 350 (M+2).
-Bromo-4-[3-(2-hydroxyphenyl)-4,5-dihydro-1H-
pyrazol-5-yl]-6-methoxyphenol (3a): Chalcone (2) (0.35 g,
mmol) was dissolved in methanol (10 mL) to which hydrazine
3
), 109.05 (C-Br), 144.37 & 163.78 (C-OH), 193.50
(
RESULTS AND DISCUSSION
2
The pyrazolines derivatives were screened for the anti-
bacterial activity against five bacterial strains, i.e. three Gram-
positive bacteria (Staphylococcus aureus FNCC 0047, Bacillus
substilis FNCC 0041 and Bacillus cereus FNCC 0040) and
two Gram-negative bacteria (Eschericia coli FNCC 0091 and
Shigella flexnerri ATCC 12022). Tetracycline (100 ppm/disc)
was used as standard antibiotic references and dimethyl sulfoxide
(DMSO) was used as negative control for the comparison
purpose. After incubation the diameter of zone of inhibition
formed around the cavities and disc of standard drugs was
accurately measured in mm. The observed zones of inhibition
are presented in Table-1.
According to these results, pyrazoline showed a weak to
moderate activity. Pyrazoline compounds 3a and 3b have the
highest activity against Bacillus cereus (1000 ppm: 6.1 mm);
Staphylococcus aureus (1000 ppm: 7.25 mm) and Bacillus
subtilis (1000 ppm: 6.95 mm). It was observed that change of
hydrogen atom into acetyl group at N-pyrazoline increased
1
monohydrate (0.1 mL, 2 mmol) was added. The mixture was
refluxed for 24 h, then poured onto crushed ice and allowed
to stand in the refrigerator for over night. The precipitate was
filtered off, washed with cold water and dried to get grey powder
-1
(
(
(
Scheme-I).Yield 87.04 %, m.p. 251 °C, IR (KBr, cm ) : 3333
1
NH), 1589 (C=N), H NMR (500 MHz, CDCl
dod,1H, J = 7.15; 9.57 Hz, CH), 3.56 (dod, 1H, J = 5.85; 11
), 4.81 (t, 1H, CH), 5.95 (d, 1H, NH),
3
): δ 3,07
Hz, CH), 3.92 (d, 1H, CH
3
13
7
4
8
.10 and 10.94 (s, 1H, OH). C NMR (125 MHz, CDCl
2.11 (CH ), 56.60 (C-N), 62.49 (C-O-C), 147.71 (C=N). GC
7.04 %, MS: 364 (M+2).
-[5-(3-Bromo-4-hydroxy-5-methoxyphenyl)-3-(2-
hydroxyphenyl]-4,5-dihydro-1H-pyrazol-1-yl)ethanone
3b): Chalcone (2) (0.35 g, 1 mmol) was dissolved in glacial
acetic acid (6 mL) and hydrazine monohydrate (0.1 mL,
3
): δ
2
1
(
2
mmol) was added. The mixture was heated to 60-70 °C and
Br
O
O
OH
O
OH
(
1)
KBrO3
OH
Br
OCH3
3
CH COOH
NaOH
OCH3
Vanillin
+
OH
(
2)
OH
O
OCH3
CH3
.
NH NH H O
2
2
2
.
NH NH H O
2
2
2
CH COOH
3
CH OH
3
OH
H
N
N
OH
O
N
N
Br
Br
OH
(3a)
O
H
OCH3
(3b)
OCH3
Scheme-I: Synthesis of pyrazolines (3a and 3b)

456 Setyawati et al.
Asian J. Chem.
TABLE-1
ANTIBACTERIAL DATA OF PYRAZOLINE DERIVATIVES (3a and 3b)
Zone of inhibition (mm)
Conc.
Compound 3a
Gram-positive
S. aureus B. subtilis B. cereus
Compound 3b
Gram-positive
(
ppm)
Gram-negative
E. coli S. flexnery
Gram-negative
S. aureus B. subtilis B. cereus
E. coli
S. flexnery
1
3
5
00
00
00
2.40
4.00
3.45
4.77
16.37
–
5.37
3.75
5.65
5.63
12.67
–
5.50
4.42
5.50
6.10
11.03
–
3.37
3.00
1.25
2.40
15.33
–
2.55
3.25
3.35
4.58
15.12
–
3.25
4.45
6.60
7.25
16.00
–
4.67
6.65
6.77
6.95
12.8
–
5.00
5.45
5.00
5.50
11.68
–
–
–
–
–
2.75
2.75
–
1000
–
Control (+)
Control (–)
16.35
–
15.05
–
the antibacterial activity at Gram-positive bacteria. In contrast,
antibacterial activity at Gram-negative was decreased.
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2
In the present study, two pyrazolines (3a and 3b) have
been synthesized and yielded in 87.04 and 88.83 %, respec-
tively. Antibacterial test of pyrazoline derivatives showed that
N-acetyl pyrazoline increased the antibacterial activity at Gram-
positive but decreased at Gram-negative. The highest antibac-
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