Key Engineering Materials Submitted: 2019-06-27

ISSN: 1662-9795, Vol. 840, pp 245-250 Revised: 2019-11-12

doi:10.4028/www.scientific.net/KEM.840.245 Accepted: 2019-11-17
© 2020 Trans Tech Publications Ltd, Switzerland Online: 2020-04-24

Synthesis N-Phenyl Pyrazoline from Dibenzalacetone and Heme

Polymeration Inhibitory Activity (HPIA) Assay
Linda Ekawatia, Bambang Purwonob*
and Muhammad Idham Darussalam Mardjanc
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada,
Jl. Sekip Utara BLS 21, Bulaksumur, Yogyakarta 55281, Indonesia
a
linda.ekawati@mail.ugm.ac.id, bpurwono.bambang@ugm.ac.id, cidham.darussalam@ugm.ac.id

Keywords: Dibenzalacetone, HPIA, N-phenyl pyrazoline

Abstract. The synthesis of 1,5-diphenyl-3-styryl-4,5-dihydro-1H-pyrazole (B1) and 5-(3,4-

dimethoxyphenyl)-3-(3,4-dimethoxystyryl)-1-phenyl-4,5-dihydro-1H-pyrazole (B2) have been
conducted from 1,5-diphenylpenta-1,4-dien-3-on (A1) and 1,5-bis(3,4-dimethoxyphenyl)penta-1,4-
dien-3-one (A2). Heme polymerization inhibitory activity (HPIA) assay of the synthesized
compounds has also been carried out. The first step of reaction was Claisen-Schmidt condensation of
benzaldehyde derivatives and acetone using NaOH 20% and ethanol as solvent. Dibenzalacetone
derivatives were reacted with phenylhydrazine using acetic acid to form N-phenylpyrazoline. The
structure of products was characterized by FT-IR, GC-MS, DI-MS, 1H- and 13C-NMR The result of
heme polymerization inhibitory activity assay showed that IC50 of B1 and B2 1.26 and 0.79 mM while
quinine 1.26 mM. The result indicated that compound B2 was more potent as antimalarial than
quinine.

Introduction
The World Malaria report by the World Health Organization (WHO) in 2017 stated that there were
219 million cases of malaria, of which there were 435 thousand deaths. About 92% of malaria cases
occur in the African region and are followed by Southeast Asian regions with 5% cases and Eastern
Mediterranean regions with 2% [1]. In Indonesia, malaria cases occur in Eastern Indonesia, such as
Papua, West Papua and East Nusa Tenggara [2]. Malaria treatment in the world uses quinoline
derivatives in the form of chloroquine, but in 1950 there were cases of chloroquine resistance.
Mefloquine was used as an antimalarial drug starting in 1983, but a case of mefloquine resistance
emerged in 1995 and 2000 Artesunate-mefloquine and Artesunate-amodiaquine or other combined
drugs were used to treat cases of recurrence that appeared to the existing antimalarial drugs [3].
Artemisinin, which is naturally found in the Artemisia annua plant [4] can be used as Artemisinin
Combination Therapy (ACT) to reduce the high use of artemisinin as single drug [5]. ACT is used as
the main treatment for malaria [6]. The absence of a vaccine for malaria also causes malaria still be a
major cause of death [7]. Therefore, the use of antimalarial drugs is the only way of treatment for
malaria [8,9]. Kalaria et al. reported that heterocyclic compounds with O, S and N atoms could act as
antimalarial compounds; one of them is pyrazoline compound [10]. Substituted pyrazoline in position
1, 3, and 5 has 89% of inhibition; it means having a better antimalarial activity compare with 87%
inhibition of chloroquine [11]. Pyrazoline compounds have a mechanism of action as an antimalarial
through inhibition of heme polymerization [12].
Dibenzalacetone compounds can be obtained through aldol condensation of benzaldehyde or its
derivatives with exes acetone, where acetone has two sides of hydrogen α so it can react with two
benzaldehyde (ratio of benzaldehyde:acetone = 1:2). Dibenzalacetone derivatives are chalcone
compounds with additional carbon-carbon double bonds, as shown in Fig. 1. The dibenzalacetone
was further converted into corresponding pyrazolines to improve activity by pyrazoline ring as found
in curcumin-pyrazoline derivatives. Theoretically, the pyrazoline synthesis pathway can be carried
out by reacting unsaturated ketones (chalcone) with hydrazine in acetic acid [13]. The pyrazoline can
also be obtained from the reaction of dibenzalacetone with phenylhydrazine to produce pyrazoline
N-phenyl compounds [14]. The reaction of the pyrazoline synthesis from dibenzalacetone is shown
in Fig. 2.

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246 Symposium of Materials Science and Chemistry II

R
R
Figure 1. General structure of dibenzalacetone.

O N N
H
N CH3COOH
NH2
R R
R R

Figure 2. Scheme reaction of pyrazoline.

Experimental Section
Materials. The materials employed were 3,4-dimethoxybenzaldehyde (veratraldehyde), acetone,
phenylhydrazine, sodium hydroxide (NaOH), ethanol, glacial acetic acid, dimethyl sulfoxide
(DMSO), quinine hydrochloride dihydrate, n-hexane, and ethyl acetate. All chemicals were purchased
from Merck with high grade and used without any further purification.
Instrumentation. The melting point was determined using Electrothermal 9100 melting point
apparatus and was not corrected. Infra-red spectra were obtained using Shimadzu-Prestige 21
spectrometer. Chromatogram and mass spectra were measured on a Shimadzu QP-2100 GC-MS and
DI-MS spectrometer. 1H and 13C-NMR spectra were obtained on JEOL JNM ECZ500R/S1 (500 MHz
(1H) and 125 MHz (13C)). Equipment for antimalarial assay by polymerization inhibitory activity
assay (HPIA) were micropipette (Rainin and Socorex), microplate 96 well, centrifuge (Thermo
Scientific Sorval LM 17R), vortex (Thermo Scientific), shaker incubator (OSK TW-1116) and ELISA
Reader (Bio-Rad Benchmark).
Synthesis of 1,5-diphenylpenta-1,4-dien-3-on (A1) Benzaldehyde (2) (40 mmol,
4.07 mL) was dissolved in ethanol (20 mL) and then acetone (1) (20 mmol, 1,5 mL) was added. The
solution was vigorously stirred for 15 min and the Erlenmeyer was cooled in an ice bath to maintain
the temperature 1-4 °C. After that, sodium hydroxide solution (20%, 20 mL) was added dropwise.
The reacting mixture was stirred for 1 more hour. The product was neutralized with HCl 10% and the
separated product was filtered, washed with water, and dried. It was recrystallized from ethanol to
give the compound A1 (97.60%) as yellow solid m.p 101-103 °C. FTIR KBr (υ max, cm-1) 3024
(Csp2–H stretching), 1651 (C=O α,β-unsaturated ketone ), 1497 and 1450 (aromatic C=C stretching),
1589 (aliphatic C=C stretching), 988 (HC=CH trans). GC: 100% purity. 1H-NMR (500 MHz,
CDCl3) δ (ppm): 7.09 (1H, d, J = 16 Hz, CH=CH), 7.41-7.43 (3H, m, H-Ar), 7.62-7.64 (2H, m,
H-Ar), 7.74 (1H, d, J = 16 Hz, CH=CH). MS (EI, m/z): 234 (M+), 131, 103, 77 (base peak) and 51.
Synthesis of 1,5-bis(3,4-dimethoxyphenyl)penta-1,4-dien-3-one (A2). This was prepared as
described for A1 from veratraldehyde (3) (10 mmol, 2.75 g) in ethanol (10 mL) with acetone (1)
(5 mmol, 0.5 mL) and the product was recrystallized from ethanol to give the compound A2 (90.90%)
as bright yellow solid m.p 98-101 °C. FTIR KBr (υmax, cm-1): 1643 (C=O α,β-unsaturated ketone),
3009 (Csp2–H stretching), 2924 (Csp3–H stretching), 1512 and 1589 cm-1 (aromatic C=C stretching),
1265 and 1026 (C–O ether), 949 (HC=CH trans). GC: 94.67% purity. 1H-NMR (500 MHz, CDCl3) δ
(ppm): 3.83 (3H, s, –OCH3), 3.84 (3H, s, –OCH3), 6.79 (2H, d, J = 8 Hz, H-Ar), 6.85 (1H, d, J =
16 CH=CH), 7.04 (1H, d, J = 2 Hz, H-Ar), 7.10 (1H, dd, J = 8 and 2 Hz, H-Ar), 7.59 (1H, d, J =
16 CH=CH). MS (EI, m/z): 354 (M+, base peak), 339, 151, 91, 77 and 51.
Synthesis of 1,5-diphenyl-3-styryl-4,5-dihydro-1H-pyrazole (B1) Dibenzalacetone (A1) (4mmol,
1 g) and 15 mL glacial acetic acid were added into a 100 mL of the three-necked flask until it was
 Key Engineering Materials Vol. 840 247

completely dissolved. Phenylhydrazine (4 mmol, 0.4 mL) was added to the solution. The solution
was refluxed for 8 h and cooled to room temperature. Solid pyrazoline was obtained after poured the
solution into ice-aquadest. The separated product was filtered, washed with water and dried. The
residue was recrystallized with ethanol to give the compound B1 (82.17%) as brown solid m.p 121-
123 °C. FTIR KBr (υ max, cm-1): 3024 (Csp2–H stretching), 1597 (C=N), 1497 (aromatic C=C
stretching), 1327 (C–N) and 949 (HC=CH trans). GC-MS: 96.37% purity. 1H-NMR (500 MHz,
DMSO-d6) δ (ppm): 2.94 (1H, dd, J = 17 and 5, –CH2), 3.73 (1H, dd, J = 17 and 10, –CH2), 5.44 (1H,
dd, J = 10 and 5, –CH), 6.73 (1H, d, J = 15, CH=CH), 7.53 (1H, d, J = 15, CH=CH). . 13C-NMR
(125 MHz, DMSO-d6) δ (ppm): 41.7 (–CH2), 62.5 (–CH), 118.5 (CH=CH), 136.3 (CH=CH),
148.7 (C=N). MS (EI, m/z): 324, 247, 220, 104, 91 (base peak), 77 and 51.
Synthesis of 5-(3,4-dimethoxyphenyl)-3-(3,4-dimethoxystyryl)-1-phenyl-4,5-dihydro-1H-
pyrazole (B2). Dibenzalacetone (A2) (2 mmol, 0.71 g) and 15 mL glacial acetic acid were added into
a 100 mL of three-necked flask until it was completely dissolved. Phenylhydrazine (2 mmol, 0.2 mL)
was added to the solution. The solution was refluxed for 11 h and cooled to room temperature. Solid
pyrazoline was obtained after poured the solution into ice-aquadest. The separated product was
filtered, washed with water and dried. The residue was recrystallized with ethanol to give the
compound B (70.45%) as dark red solid m.p 121-123 °C. FTIR KBr (υmax, cm-1): 3001 (Csp2–H
stretching), 2932 (Csp3–H stretching), 1597 (C=N), 1504 (aliphatic C=C stretching), 1466 and
1420 (aromatic C=C stretching), 1258 (C–O), 1134 (C–N) and 957 (HC=CH trans). GC: 81% purity.
1
H-NMR (500 MHz, DMSO-d6) δ (ppm): 2.95 (1H, dd, J = 15 and 5, –CH2), 3.68 (1H, d, J = 10, –
CH2), 3.70 (6H, s, –OCH3), 3,76 (1H, s, –OCH3), 3.81 (1H, s, –OCH3), 5.36 (1H, dd, J = 10 and 5, –
CH), 6.89 (1H, d, J = 11, CH=CH), 7.15 (1H, d, J = 11, CH=CH). 13C-NMR (125 MHz, DMSO-d6) δ
(ppm): 41.7 (–CH2), 55.3 (2C, –OCH3), 55.3 (2C, –OCH3), 62.5 (–CH), 112.7 (CH=CH),
133.2 (CH=CH), 149.1 (C=N). MS (EI, m/z): 444 (M+), 442 (base peak), 151, 91, 77 and 51.
Heme polymerization inhibitory activity assay of pyrazoline derivatives. This test was
conducted by Basilico et al. [15], which modified the dose of hematin solution and the sample used.
A total of 100 μL solution of 1 mM hematin in 0.2 M NaOH was put into the microtube, then added
50 μL of test material with various concentration levels, i.e. 5, 2.5, 1.25, 0.63, 0.31, 0.16 and
0.08 mg/mL. Replication was conducted 3 times for each concentration. To initiate the hem
polymerization reaction, 50 mL glacial acetic acid solution (pH 2.6) was added in the microtube,
which already contains hematin solution and sample, then were incubated at 37 °C for 24 h. The
positive control used was quinine hydrochloride dihydrate, whereas the negative control was mixture
hem and glacial acetic acid without sample.
After incubation, the microtube was centrifuged at 8000 rpm for 10 min. The supernatant was
removed, and the precipitate was washed 3 times with 200 mL DMSO. Each microtube was washed
by centrifugation speed 8000 rpm for 10 min. The precipitate obtained was added with 200 mL
0.2 M NaOH. Each 100 mL of the solution obtained was put in microplate 96 wells and OD values
were read by Elisa reader at a wavelength of 405 nm.
Heme polymerization inhibitory activity values expressed in IC50, i.e., levels that could inhibit
heme polymerization by 50% compared to the negative control. The standard curve was constructed
by making a series of concentrations of hematin (which was dissolved in 0.2 M NaOH). A total of
100 mL of each concentration was added to the wells and 96 wells microcultures OD value was
observed by ELISA reader at a wavelength of 405 nm. Heme polymerization inhibitory IC50 value
was calculated using probit analysis.

Results and Discussion

Dibenzalacetone A1 and A2 were prepared from acetone (1) with benzaldehyde (2) or
veratraldehyde (3) in a base-catalyzed Claisen-Schmidt condensation reaction (Fig. 3). Further, some
of the dibenzalacetone (A1 and A2) were converted to pyrazolines (B1 and B2) using a reported
method [6]. Synthetic methods used were chosen based on their simple procedures with better yields
of products along with their cost-effectiveness. The purity and structures of all synthesized
248 Symposium of Materials Science and Chemistry II

compounds were confirmed by TLC and spectroscopic techniques, respectively. The IR spectra of
dibenzalacetone displayed desired characteristic absorption bands in their respective regions like α,β-
unsaturated ketone (1651 cm-1 for A1 and 1643 cm-1 for A2 ). The pyrazoline B displayed their
characteristic absorption bands due to C=N and C–N in regions 1597 and 1134 cm-1 (B1) while B2 in
regions 1597 and 1134 cm-1. The absence of absorption bands of α,β-unsaturated ketone in pyrazoline
compound confirms that the cyclization of pyrazoline ring was occurred.

O N N
O O
(i) (ii)
H H3C CH3
2 R
R R
R
R
2-3 1 A1-A2 B1-B2

Figure 3. Synthesis of dibenzylideneacetones (A1-A2) and their pyrazoline derivatives (B1-B2).

Reagents and conditions: (i) 20% NaOH, EtOH; (ii) phenylhydrazine, acetic acid, reflux.
The synthesized compounds were tested for in vitro antimalarial activity using HPIA assay.
Quinine hydrochloride was used as a positive control. Plasmodium falciparum in erythrocytes will
break hemoglobin into globin and free heme in the digestive vacuole. Globin will be decomposed
into amino acids as raw materials for the Plasmodium protein synthesis. Free heme, i.e.,
feroprotoporfirin IX will be oxidized to ferriprotoporfirin IX in the acidic digestive vacuole and was
toxic to host cells and Plasmodium. Free heme is highly toxic because it can form radical oxygen
species that can cause death for Plasmodium [8]. By Plasmodium, the free heme is converted to the
inert dimer form, namely hemozoin (malaria pigment). A polymer which is identical to hemozoin, i.e.
β-hematin, can be formed in vitro from hematin under acidic conditions. The amount of β-hematin
crystal formed is inversely proportional to the activity of antimalarial agents inhibiting the heme
polymerization [16]. The IC50 values of the compound, negative and positive controls were listed in
Table 1. The IC50 values of B2 smaller than the positive control, thus B2 displayed heme
polymerization inhibitory activity.
 Key Engineering Materials Vol. 840 249

Table 1. The IC50 values of B1, B2, negative and positive controls based on heme polymerization
inhibitory activity assay.
Average dose of Average
Concentration IC50
Sample hemozoin inhibition
(mg mL-1) (mM)
(mM) (%)
5 27.92 ± 7.84 84.60 ± 4.32
2.5 54.44 ± 8.46 69.97 ± 4.67
1.25 65.76 ± 5.71 63.73 ± 3.15
B1 0.63 89.93 ± 7.35 50.40 ± 4.06 1.26
0.31 92.29 ± 10.85 49.10 ± 5.99
0.16 97.08 ± 5.80 46.46 ± 3.20
0.08 136.11 ± 2.42 24.93 ± 1.33
5 5.97 ±1.98 96.71 ± 1.09
2.5 10.76 ± 4.95 94.06 ± 2.73
1.25 27.22 ± 3.55 84.99 ± 1.96
B2 0.63 35.97 ± 4.39 80.16 ± 2.42 0.79
0.31 83.13 ± 10.31 54.15 ± 5.69
0.16 100.90 ± 4.39 44.35 ± 2.42
0.08 114.58 ± 9.36 36.81 ± 5.16
5 15.42 ± 3.11 91.50 ± 1.72
2.5 18.89 ± 1.89 89.58 ± 1.04
1.25 25.76 ± 1.68 85.79 ± 0.93
Quinine
0.63 84.17 ± 10.54 53.58 ± 5.81 1.26
hydrochloride
0.31 87.99 ± 11.98 51.47 ± 6.61
0.16 121.94 ± 12.21 32.75 ± 6,.73
0.08 135.07 ± 11.34 25.51 ± 6.25
Negative
181.32 ± 3.44 0.00 ± 0.00
control
The activity of the N-phenyl pyrazoline derivative was described in Fig. 4. β-Hematin formation
will be proceeded by the formation of amorphous precipitate of hem, and followed by a slow
conversion into crystalline β-hematin. N-phenyl pyrazoline compound has two nitrogen. The lone
pair at N atom could interact with Fe (III) via coordination bond, while the methoxy group will react
with the acidic site at ferriprotoporfirin IX (carboxylic acid group). Therefore, the polymerization
process might be prevented.

N N
O

N N O O
O
O O
O HO OH

N N OH
FeIII
N N N N
FeIII
O
N N
OH
a
Hydrogen bond
b
Coordination bond

Figure 4. The inhibition mechanism of N-phenyl pyrazoline: (a) B1 and (b) B2 against hematin.
250 Symposium of Materials Science and Chemistry II

Summary
The compounds of 1,5-diphenyl-3-styryl-4,5-dihydro-1H-pyrazole (B1) and 5-(3,4-
dimethoxyphenyl)-3-(3,4-dimethoxystyryl)-1-phenyl-4,5-dihydro-1H-pyrazole (B2) were prepared
from dibenzalacetone (A1 and A2). Results of heme polymerization inhibitory activity showed that
the IC50 values of the samples B1, B2, and quinine were 1.26, 0.79, and 1.26 mM, respectively.

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