Original Article Thai Journal of Pharmaceutical Sciences

Microwave-assisted synthesis, in silico

studies and in vivo evaluation for the
antidiabetic activity of new brominated
pyrazoline analogs
Jasril Jasril1, Ihsan Ikhtiarudin2, Syilfia Hasti2, Annisa Indah
Reza1, Neni Frimayanti2
1
Department of Chemistry, Faculty of Mathematic and Natural Sciences, Universitas
Riau, Jalan H.R. Subrantas Km. 12.5, Pekanbaru, 28293, Indonesia, 2Department of
Pharmacy, Sekolah Tinggi Ilmu Farmasi, Jalan Kamboja, Pekanbaru, 28293 Indonesia
Corresponding Author:
Neni Frimayanti, Department
of Pharmacy, Sekolah Tinggi
Ilmu Farmasi, Jalan Kamboja, ABSTRACT
Pekanbaru, 28293 Indonesia.
E-mail: nenifrimayanti@gmail. Introduction: Pyrazolines have played a crucial role in the drug discovery researches and have
com been used widely as important pharmacophores or intermediate for the synthesis of bioactive
compounds. Objective: The aim of this work is to explore the potential of new brominated
Received: Aug 10, 2018 pyrazoline analogs as antidiabetic. Materials and Methods: Synthesis of brominated
Accepted: Mar 12, 2019 pyrazoline analogs has been conducted under microwave irradiation, in silico studies were
Published: Jun 05, 2019 performed using AutoDock 1.5.4 software packages and nanoscale MD Program 2.9, and the
animal used in the in vivo evaluation is male albino mice (Mus musculus L.). Results: The
in silico studies for antidiabetic activity showed that compound 3a is the most active compound
and furthermore it can be developed as new active agents for antidiabetic. The in vivo evaluation
showed that compound 3a has a good ability to increase the percentage of change in blood
glucose level and weight loss prevention, decreasing of the drinking water and also decreasing
of the urine volume, significantly (P < 0.05) compared with negative control with dosage of 25,
50, and 100 mg/kg of the body weight. The oral administrations of compound 3a with dosage
50 and 100 mg/kg of body weight did not show the significant difference (P > 0.05) in the
ability to increase the percentage of change in the loss of weight prevention compared with the
glibenclamide. Then, the oral administrations of compound 3a with a dosage of 25, 50, and
100 mg/kg of body weight did not show the significant difference (P > 0.05) in the ability to
decrease the drinking and urine volume compared with the glibenclamide. In addition, the oral
administration of compound 3a also did not show the significant effect (P > 0.05) on change
in the relative weight of organs of treated diabetic mice, compared to the normal control in the
given dosages. Conclusion: This strategy reflects a logical progression for early-stage drug
discovery that can be used to successfully identify drug candidates for antidiabetic agents.

Keywords: Antidiabetic activity, brominated pyrazolines, in vivo evaluation, microwave-assisted synthesis,

molecular docking studies, molecular dynamic simulation

INTRODUCTION people who are infected by diabetic in the world are expected

D
to approximately double between 2000 and 2030, based solely
iabetes mellitus is a complex metabolic syndrome.
on demographic changes. In 2000, the number of people with
It is caused by the abnormality of the carbohydrate
diabetes is 171 million people. The latest estimates showed
metabolism which is linked to low blood insulin level
a global prevalence of 366 million people with diabetes in
or insensitivity of target organs to insulin.[1] It is a major
2030[2] and expected to rise to 592 million on 2035.[3]
of human health concern in the world over, a common and
very prevalent disease affecting to the citizens in the world. Pyrazolines have played a crucial role in the drug
Assuming that age-specific prevalence remains constant, the discovery researches and they have been used widely as

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 Jasril, et al.: Microwave-assisted synthesis, in silico studies and in vivo evaluation

MATERIALS AND METHODS

General Information’s
The materials used to synthesize the brominated
pyrazoline analogs included 1-acetyl naphthalene, 2-acetyl
naphthalene, 2-bromobenzaldehyde, potassium hydroxide,
hydrochloric acid, hydrazine hydrate, phenylhydrazine,
glacial acetic acid, and some organic solvents, such as
ethanol, n-hexane, and ethyl acetate were produced by
Merck and or Sigma-Aldrich. The materials used to evaluate
the antidiabetic activity included alloxan (Aldrich), 10%
Figure 1: Scheme of the synthesis new brominated pyrazoline glucose solution (Otsuka), sodium carboxymethyl cellulose
analogs (NaCMC) (Brataco), sodium chloride solution (Otsuka),
and glibenclamide (IndoFarma).
important pharmacophores or intermediate for the synthesis All the synthesis reactions were performed in a Samsung
of bioactive compounds.[4] Scheme of the synthesis new ME109F domestic microwave oven. The melting point was
brominated pyrazoline analogs is presented in Figure 1. determined on a SMP 11 apparatus (Stuart®) (uncorr).
Pyrazolines have numerous prominent effects, such as Thin-layer chromatography analysis was performed using
antimicrobial, antiamoebic, anti-inflammatory, anticancer, GF254 (Merck Millipore) under UV Lamp 254/366 nm
antidepressant, antiandrogenic, and antiviral activities.[5] (Camag™). High-performance liquid chromatography (HPLC)
Some patented pyrazolines have been used for the treatment chromatograms were recorded on Shimadzu LC solution.
of food disorders, including obesity or metabolic syndrome in UV spectrum was recorded on Genesys™ 10S UV visible
patients with developed diabetes[6] and treatment of diabetes spectrophotometer (Thermo Scientific™), Fourier transform-
and also related diseases, especially Type II diabetes.[7] infrared (FT-IR) spectra were recorded in KBr powder on a
In addition, the presence of atom bromine in an organic
Shimadzu® FT-IR Prestige-21 spectrophotometer (Shimadzu
compound has been reported can enhance the hypoglycemic
Corporation), and 1H and 13C nuclear magnetic resonance
activity. Introducing atom bromine to coumarin sulfonylurea
(NMR) spectral data were recorded on a JEOL JNM ECA at 500
compound has been reported can enhance their hypoglycemic
and 125 MHz, respectively. Mass spectral data were recorded
activity.[8] Another work reported that some brominated
on a LC-mass spectrometry (MS) (Mariner Biospectrometry).
pyrazoles are also shown the higher hypoglycemic activity
The blood glucose levels were measured on GlucoDr™ Blood
than antidiabetic drug.[9] In the view of the importance
Glucose Test Meter (All Medicus).
pharmacological of pyrazolines and bromo substituent, it
was thought worthwhile to construct some new pyrazoline
analogs containing with bromo substituent. General Procedures
There are some methods that have been used to synthesize Synthesis of brominated pyrazoline analogs
the pyrazolines analogs, such as grinding, stirring, and reflux. Preparation of compounds 1a and 1b
However, the microwave-assisted synthesis is a more attractive Acetylnaphthalene (5 mmol) and 2-bromobenzaldehyde (5
method for researchers.[10] This method has been widely mmol) were dissolved in absolute ethanol (10 mL) in an
applied to synthesis some various heterocyclic compound[11] Erlenmeyer and potassium hydroxide 1N (5 mL) was added.
and showed some advantages, such as reduce in reaction time, Then, the mixture was irradiated using a domestic microwave
increase the yield by increasing the selectivity of reaction (180 W) for 3 min. After the reaction was completed, cold
and it is a very easy and simple method when combined with distilled water (10 mL) was added into the mixture and
supporting reagents[12] and appropriate irradiation power.[13] was neutralized with hydrochloric acid 1N to afford the
Thus, so far, computational approaches can aid in drug discovery precipitate. The precipitate was filtered in vacuo, washed
studies in various ways. In this study, we are interested to using cold n-hexane, dried in a desiccator, and re-crystallized
explore the potential of some brominated pyrazoline analogs with mixture of ethyl acetate and n-hexane to get pure 1a
as antidiabetic, considering that the prevalence of diabetes and 1b.
mellitus patients is increasing every year. The pyrazoline
analogs have been synthesized under microwave irradiation Preparation of compounds 2a, 2b, 3a, and 3b
for then can be used as an antidiabetic agent with the aid of Compounds 1a and 2b (1 mmol) were dissolved in absolute
in silico way (i.e., molecular docking and molecular dynamic ethanol (15 mL) in a closed reaction vessel using an
[MD] simulation). In addition, in vivo evaluation has been also ultrasonicator. Then, hydrazine compound (5 mmol) and
conducted to evaluate the results from in silico studies and five drops of glacial acetic acid were added. The mixture was
to confirm the effect of oral administration of compound 3a irradiated using a domestic microwave (180 W) for 2 min. After
on the percentage of change in the blood glucose level and the reaction was completed, the mixture was cooled in the ice
also weight loss prevention, decreasing the drinking and urine bath to afford the precipitate. The precipitate was filtered in
volume and to obtain the effect of the oral administration on vacuo, washed using cold n-hexane, dried in a desiccator, and
the change of relative weight of liver, kidney, and heart of re-crystallized in a mixture of ethyl acetate and n-hexane to
treated diabetic mice. get pure 2a, 2b, 3a, and 3b.

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 Jasril, et al.: Microwave-assisted synthesis, in silico studies and in vivo evaluation

In silico studies for antidiabetic activity acclimated for a week. The mice were declared as healthy if
they did not show 10% changing of the body weight and also
Preparation of protein
did not show any healthy problem symptom, visually. In this
Molecular docking was conducted using AutoDock 1.5.4
study, the animal used is followed by the Ethical Clearance
software packages. Docking was performed after finishing the
No. 178/UN.19.5.1.1.8/UEPKK/2018. There are 48 mice were
preparation of protein and ligand. Protein was prepared with
used in this study, they were divided into eight mice for the
retrieving the three-dimensional of the crystal structure. This
normal control group, eight mice for the negative control group,
crystal structure was downloaded from PDB database with
PDB ID 2QMJ (α-glucosidase) for then it can be used as the eight mice for the positive control group, and 24 mice were used
macromolecule for molecular docking. This protein must be in treated groups (i.e. 25, 50, and 100 mg/kg of the body weight).
kept rigid. For then, all hydrogen atoms were added, merging Preparation of dosage and suspension
non-polar hydrogen atoms, checking and repairing missing Compound 3a was oral administration to the diabetic mice
atoms, adding Gasteiger charges, checking and fixing total with three dosages (25, 50, and 100 mg/kg of body weight).
charges on residues, and assigning atom types to the protein Volume of oral administration is 1% of mice weight was
structure. Autogrid 4 software was used to generate a grid made in 5 mL of 1% NaCMC suspension. Glibenclamide
box of the protein structure with default atom types (carbon, was oral administration to the diabetic mice with dosage
hydrogen, oxygen, and nitrogen), grid spacing of 0.35 Å, with 5 mg/kg of body weight with conversion factor to mice dosage
the dimensions of 122 × 116 × 120 points along the X-, Y-, 5 mg/kg of body weight × 0.0026 = 0.013 mg/20 g of body
and Z-axes and centered on the protein for the docking. weight = 0.65 mg/kg of body weight. Alloxan was injected in
Preparation of ligands a dosage 175 mg/kg of body weight.
These six compounds were used as the ligands and drawn Inducing to diabetic
using ChemDraw as depicted in Figure 2. In this case, these The albino mice were fasted for 18 h before induced to the
ligands must be kept flexible. All the ligands were required to diabetic. The mice were injected with alloxan solution,
minimize the energies for then the minimized structures were intraperitoneally. The blood glucose level before and after
subsequently prepared with the detected root of torsion and inducing to the diabetic was measured. After alloxan injection,
number of torsions for flexible-ligand docking using AutoDock 10% glucose solution was given to the mice for 2 days. The blood
Tools 1.5.4 software. glucose level was measured 2 days after alloxan induction.
Analyzing and output visualization For 3rd until the 7th day, drinking water was given to the
The docking poses were ranked based on the docking scores. mice and each mouse was moved into a cage, where a cage
The binding affinity of one ligand to the receptor molecule can contained a mice and the blood glucose level of each mouse
be predicted using the scoring function in AutoDock. The lowest was also measured. If mice were diabetic (blood glucose
binding affinity was selected as the best docking conformation level >200 mg/dL), they were treated with suspension of
for then it can be used for analysis after the docking process. compound 3a, orally. The blood glucose level of treated mice
The molecular visualization of the docked complexes was
was measured every 2 days (1st, 3rd, 5th, and 7th day).
performed using the Discovery Studio Visualizer (Biovia).
Treating with compound 3a
MD simulation
Randomly, the albino mice were divided into six groups. Each
Preliminary study for MD was performed using NAMD
group contained five mice. The first group is normal mice
(Nanoscale MD Program; v 2.9), it was used for modeled
(did not induced to diabetic and did not treated). The second
protein for then all the files were generated using VMD (visual
group is negative control (just treated with 1% NaCMC). The
MD).[14] The protein was solvated with a TIP3P water box with
third group is positive control (treated with glibenclamide
a 2.0 Å layer of water for each direction of the coordinate
in a dosage of 0.65 mg/kg of body weight). Group four-six
structure, and a Chemistry at Harvard Macromolecular
were treated with compound 3a in dosages of 25, 50, and
Mechanics was used as a force field.
100 mg/kg of body weight, respectively. The mice were treated
In vivo evaluation for antidiabetic activity with compound 3a, orally, 1 time a day for a week.
The animal used in this in vivo evaluation is male albino mice Measurement of blood glucose level, weight loss, drinking, and
(Mus musculus L.), 20–30 g weight. Before used, the mice were urine volume
Blood was taken from mice tail. The blood was placed on the
GlucoDr™ Blood Glucose Test Strips and the blood glucose
level on the 1st, 3rd, 5th, and 7th days after diabetic condition
was measured with a GlucoDrTM Blood Test Meter. The loss
of mice weight was measured on the 1st, 3rd, 5th, and 7th days
after diabetic condition with an analytical balance. Drinking
volume was measured on the 1st, 3rd, 5th, and 7th days after
diabetic condition. Mice were given some water with known
initially volume. Then after 24 h, the volume residue of water
was measured. The difference of water volume with residue
of water volume was calculated as drinking volume. The
volume of urine was measured by summing up of urine was
Figure 2: Structure of the six ligands collected for 24 h on the 1st, 3rd, 5th, and 7th days after diabetic

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 Jasril, et al.: Microwave-assisted synthesis, in silico studies and in vivo evaluation

condition. Each mouse was transferred to the metabolic cage Compound 2b was obtained as yellow solid in 83% yield.
with equipped using filter on the bottom of cage. Mp: 87–89°C. HPLC: tR = 13.9 min. UV (MeOH) λmax (nm): 220
and 309. FT-IR (KBr) ʋ̅ (cm−1): 3323, 3052, 2960, 1601, 1583,
Measurement of relative weight of heart, liver, and kidney
1462, 1030, 683. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.03 (d,
On the 8th day, all mice were sacrificed. The mice were
1H), 7.86 (d, 1H), 7.84 (m, 3H), 7.66 (d, 1H), 7.61 (d, 1H), 7.49
sacrificed by neck dislocation. This process was performed
(m, 2H), 7.34 (t, 1H), 7.17 (t, 1H), 5.37 (t, 1H, J1 = J2 =10 Hz),
according to good animal test protocol. Then, the heart, liver,
3.80 (dd, 1H, J1 = 16 Hz, J2 =10.5 Hz), 3,03 (dd, 1H, J1 = 16 Hz,
and kidney were collected. The organs were dried on the
J2 = 9.5 Hz). 13C NMR (125 MHz, CDCl3) δ (ppm): 151.2, 141.5,
filter paper and weight for each organ was measured using
133.5, 133.2, 132.9, 130.3, 129.1, 128.2, 128.2, 128.0, 127.8,
analytical balance.
127.4, 126.5, 126.4, 125.7, 123.4, 122.9, 63.4, 40.0. MS (ESI):
m/z [M-H]+ = 349.5; m/z [(M+2)-H]+ = 351.2.
RESULTS AND DISCUSSION
Compound 3a was obtained as yellow solid in 97% yield.
Synthesis of Brominated Pyrazoline Mp: 154–155°C. HPLC: tR = 16.07 min. UV (EtOAc) λmax (nm):
Analogues 220 and 295. FT-IR (KBr) ʋ̅ (cm−1): 2902, 1595, 1499, 1329,
665. 1H NMR (500 MHz, CDCl3) δ (ppm): 9.54 (d, 1H), 7.88
In this work, we have successfully applied the microwave (d, 1H), 7.81 (d, 1H), 7.69 (t, 1H), 7.64 (t, 1H), 7.56 (t,1H),
irradiation to synthesize some brominated pyrazolines analogs 7.47 (d, 1H), 7.42 (t, 1H), 7.28 (d, 1H), 7.25 (t, 2H), 7.20
by two-step reactions. The first step is synthesis of brominated (t, 1H), 7.13 (t, 1H), 7.07 (d, 2H), 6.84 (t, 1H), 5.64 (dd,
chalcone analogs 1a and 2a through Claisen-Schmidt 1H, J1 = 6.5 Hz, J2 = 12.5 Hz), 4.19 (dd, 1H, J1 = 12 Hz,
condensation. The second step is synthesis of brominated J2 = 17 Hz), 3.26 (dd, 1H, J1 = 6.5 Hz, J2 = 17 Hz). 13C NMR
pyrazolines analogs through Michael addition and followed (125 MHz, CDCl3) δ (ppm): 147.5, 144.3, 140.7, 134.2, 133.2,
by intramolecular cyclization of brominated chalcone with 130.6, 129.7, 129.2, 128.9, 128.7, 128.4, 127.4, 126.9, 126.2,
hydrazine hydrate or phenylhydrazine. Both reactions are 124.9, 121.9, 119.4, 113.3, 62.4, 44.5. HRMS (ESI): m/z
taken in a short time about 2–3 min of radiation time and we [M-H]+ = 425.3671; [(M+2)-H]+ = 427.3742.
obtained a good yield about 70–97% yield.
Compound 3b was obtained as bright yellow solid in 70%
Compound 1a was obtained as yellow soli in 73% yield. yield. Mp: 163–164°C. HPLC: tR = 16.6 min. UV (EtOAc)λmax
Mp: 102–103°C. HPLC: tR = 16.2 min. UV (MeOH) λmax (nm): (nm): 240 and 307. FT-IR (KBr) ʋ̅ (cm−1): 3037, 1593, 1496,
212 and 380. FT-IR (KBr) ʋ̅ (cm−1): 3064, 1655, 1597, 1506, 1335, 689. 1H NMR (CDCl3) δ (ppm): 8.18 (dd, 1H), 7.82 (m,
649. 1H NMR (500 MHz, CDCl3) δ (ppm): 8.38 (d, 1H), 8.00 4H), 7.65 (dd, 1H), 7.47 (m, 2H), 7.23 (m, 4H), 7.14 (td,
(t, 1H), 7.98 (d, 1Hα, J = 15.5 Hz), 7.91 (d, 1H), 7.81 (d, 1H), 1H), 7.05 (d, 2H), 6.83 (t, 1H), 5.68 (dd, 1H, J1 = 12 Hz,
7.70 (d, 1H), 7.57 (m, 4H), 7.35 (t, 1H), 7.24 (d, 1H), 7.22 (d, J2 = 6.5 Hz), 4.11 (dd, 1H, J1 = 17 Hz, J2 = 12.5 Hz), 3.17
1Hα, J = 15,5 Hz). 13C NMR (125 MHz, CDCl3) (ppm): 195.1, (dd, 1H, J1 = 17 Hz, J2 = 7 Hz). 13C NMR (125 MHz, CDCl3)
144.3, 136.6, 134.9, 134.0, 133.6, 132.1, 131.5, 130.6, 129.7, δ (ppm): 147.1, 144.3, 140.9, 133.6, 133.4, 133.2, 130.3,
128.6, 128.0, 127.9, 127.7, 127.6, 126.6, 126.0, 125.8, 124.5. 129.2, 128.4, 128.3, 128.2, 127.9, 127.6, 126.6, 126.5, 125.3,
HRMS (ES+): m/z [M+H]+ = 337.3219 and [(M+2)+H]+ 123.5, 121.8, 119.4, 113.3, 63.9, 42.0. HRMS (ESI): m/z
339.3165. [M-H]+ = 425.4034; [(M+2)-H]+ = 427.4004.
Compound 1b was obtained as yellow solid in 92% yield.
Mp: 102–104°C. HPLC: tR = 16.8 min. UV (MeOH) λmax (nm): 261 In silico Studies for Antidiabetic Activities
and 363. FT-IR (KBr) ʋ̅ (cm-1): 3059, 1651, 1600, 1467, 661. 1H
In silico docking studies were performed to evaluate the
NMR (CDCl3) δ (ppm): 8.53 (s, 1H), 8.19 (d, 1Hα, J = 15.5 Hz),
potential of those compounds as antidiabetic. Docking study is
8.10 (dd, 1H), 7.98 (d, 1H), 7.93 (d, 1H), 7.89 (d, 1H), 7.79 (dd,
a computational technique for the exploration of the possible
1H), 7.64 (dd, 1H), 7.60 (dt, 1H), 7.57 (d, 1Hα, J = 15.5 Hz),
binding modes of a ligand to a given receptor, enzyme, or other
7.56 (dt, 1H), 7.38 (t, 1H), 7.25 (td, 1H). 13C NMR (125 MHz,
binding site. Docking can be used to predict the preferred and
CDCl3) δ (ppm): 190.3, 143.2, 135.6, 135.3, 133.7, 132.6, 131.4,
also the best orientation of the molecule (i.e., ligand) to a
130.3, 129.7, 128.7, 128.6, 128.0, 127.9, 127.8, 126.9, 126.0,
protein when they bound each other to form a stable and good
125.2, 124.6. HRMS (ES+): m/z [M+H]+ = 337.2925; m/z
complex.
[(M+2)+H]+ = 339.2951.
Six compounds (1a, 1b, 2a, 2b, 3a, and 3b) were docked
Compound 2a was obtained as white solid in 82% yield.
into protein (PDB ID: 2QMJ). Based on the docking results,
Mp: 102–104°C. HPLC: tR = 14,5 min. UV (MeOH) λmax (nm):
there are three compounds (1a, 3a, and 2b) are find to be
225 and 317. FT-IR (KBr) ʋ̅ (cm−1): 3328, 3053, 2861, 1592,
active as antidiabetic. Compound 1a showed three hydrophobic
1465, 1319, 565. 1H NMR (500 MHz, CDCl3) δ (ppm): 9.19 (d,
interactions with the residues Lys513, Lys534, and Lys776.
1H), 7.86 (d, 1H), 7.82 (d, 1H); 7.69 (dd, 1H), 7.58 (t, 2H),
This molecule also constructed Van der Waals interaction with
7.51 (t, 2H), 7.43 (t, 1H), 7.33 (t, 1H), 7.15 (td, 1H), 5.33 (t,
Glu510 and Asp777. In addition, hydrogen bonding was also
1H, J1 = 10 Hz, J2 = 9.5 Hz), 3.83 (dd, 1H, J1 = 16 Hz, J2 =
observed with the residues Lys513. Based on this, probably
10 Hz), 3.11 (dd, 1H, J1 = 16 Hz, J2 = 9 Hz). 13C NMR (125
the reason the molecule (1a) can be assumed as an active
MHz, CDCl3) δ (ppm): 42.8, 62.7, 123.1, 124.9, 126.1, 127.0,
compound.[10]
127.1, 127.4, 128.0, 128,5. 129.1, 129.7, 130.9, 133.0, 134.1,
141.5, 151.9. HRMS (ES-): m/z [M-H]+ = 349.2994; m/z Another compounds assumed active compounds
[(M+2)-H]+ = 351.2969. are compounds 3a and 2b. Based on the docking results,

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 Jasril, et al.: Microwave-assisted synthesis, in silico studies and in vivo evaluation

compound 3a seems to have one hydrogen bonding with the In Vivo Evaluation for Antidiabetic
residue Arg547, two hydrophobic interactions with Lys765 Activity of Compound 3a
and Glu767. There is Van der Waals interaction between the
ligand and residue Glu767. Compound 2b has four Van der in vivo evaluation was also conducted to confirm the effect
Waals interactions with the residues Lys513, Arg520, Lys534, of oral administration of compound 3a with dosages 25, 50,
and Lys775. In addition, compound 2b also has hydrophobic and 100 mg/kg of body weight on percentage of change in
interaction with amino acid Asp777. The spatial arrangement blood glucose level and in weight loss prevention, decreasing
of compounds 1a, 3a, and 2b is depicted in Figure 3 and the the drinking, and urine volume and to obtain the effect of the
interaction of the ligands with the amino acid is presented in oral administration on the change in relative weight of heart,
Table 1. liver, and kidney of treated diabetic mice.

MD simulation was performed with the main purpose The % change in blood glucose level was calculated using
is to explore the first stage of docking by assessing the equation below.
binding pose prediction by MD simulation and also to
% Change = (Li−Lo)/Li×100% (e.q. 1)
demonstrate that MD simulation can provide useful
information to complement the docking prediction.[15] MD Where Li is the initial level of blood glucose of diabetic mice
simulation starting from a stable energy minimum, the and Lo is observed level of blood glucose (every 2 days after
system fluctuates around the initial conformation and the administration of tested compound). The oral administration
ligand continues to have initial binding modes.[16] In this of compound 3a with dosages 25, 50, and 100 mg/kg of body
study, the MD simulations were performed at temperature weight showed that compound 3a has ability to increase
300 K to see the affinity of the ligand to the binding site. In the change of percentage in blood glucose level and in loss
general, the conformation of this active ligand (1a, 3a, and of weight prevention of treated diabetic mice, significantly
2b) is maintained to binding with the important residues as (P < 0.05) compared than negative control as depicted in
presented like a visualization of MD simulation is presented Figure 5. These results shown that compound 3a has ability to
in Figure 4. Based on the estimated free energy of binding reduce the blood glucose level. However, the ability is lower
and MD simulation, it seems that molecule 3a is the most than glibenclamide. Figure 5a showed that on the 7th day,
active compound and furthermore it can be used as new compound 3a with dosages of 25, 50, and 100 mg/kg body
active agents for antidiabetic. weight was able to increase the change of percentage in blood
glucose level by 26.26%, 37.38%, and 38.13%, respectively,
whereas the glibenclamide as the antidiabetic drug was able
to increase the change of percentage in blood glucose level
by 47.91%. Then, Figure 5b showed that on the 7th day,
compound 3a with dosages of 25, 50, and 100 mg/kg body
weight was able to increase the change of percentage in loss of
weight prevention by 3.38%, 5.66%, and 5.84%, respectively,
whereas the glibenclamide was able to increase the change of
percentage in loss of weight prevention by 8.60%. Based on
a b
the statistical analysis, the oral administration of compound
3a with the dosage of 50 and 100 mg/kg body weight did not
show the significant difference (P > 0.05) in ability to prevent
the weight loss of treated diabetic mice, compared than the
glibenclamide.
Oral administration of compound 3a also showed that
compound 3a has an ability to decrease the drinking volume in
c 24 h for a week and it is also has ability to decrease the urine
Figure 3: Spatial arrangement of three assumed active compounds volume in 24 h for a week, significantly (P < 0.05) compared
(a) compound 1a, (b) compound 3a, (c) compound 2b than negative control as depicted in Figure 6. Figure 6a showed

Table 1: Docking output

Ligands Estimated free energy of Interactions
binding (kcal/mol)
Van der Waals Hydrophobic H‑bonding
1a −6.58 Glu510, Asp777 Lys513, Lys534, Lys776 Lys513
2a −4.36 Glu114, Asp777 His645, Lys534, Lys778 ‑
3a −7.87 Glu767 Lys765 and Glu767 Arg547
1b −4.23 Glu11 His50 Lys45
2b −5.08 Lys513, Arg520, Lys534 and Lys775 Asp777 ‑
3b −3.45 Glu276 Arg283, Arg647 ‑

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 Jasril, et al.: Microwave-assisted synthesis, in silico studies and in vivo evaluation

not show the significant effect (P > 0.05) on the change in

that on the 7th day, compound 3a with dosages of 25, 50, and
relative weight of heart, liver, and kidney of treated diabetic
100 mg/kg body weight was able to reduce the drinking volume
mice compared to the normal control. The result shown that
from 5.98 mL to 4.40 mL, 4.24 mL, and 4.12 mL, respectively,
the oral administration of the compound 3a to the diabetic
whereas the glibenclamide was able to reduce the drinking
mice had no effect on damage that interfered the functional
volume to 3.92 mL. Then, Figure 6b showed that on the 7th day,
process of heart, liver, and kidney of the treated diabetic mice.
compound 3a with dosages of 25, 50, and 100 mg/kg body
It was done by determination of the relative weight of organs
weight was able to reduce the urine volume from 1.16 mL
and is performed as a preliminary illustration to observe
to 0.72 mL, 0.60 mL, and 0.56 mL, respectively, whereas the
whether the tested compound gives the potential toxic effects
glibenclamide was able to reduce the urine volume to 0.60 mL.
on the organs or not.
Based on the statistical analysis, the oral administration of
compound 3a with the dosage of 25, 50, and 100 mg/kg body
weight did not show the significant difference (P > 0.05) in CONCLUSION
ability to reduce the drinking and urine volume of treated Some new analogs of brominated pyrazolines has been
diabetic mice compared than the glibenclamide. synthesized under microwave irradiation. The in silico studies
Figure 7 showed that the oral administration of compound showed that three compounds (1a, 3a, and 2b) have a good
3a with dosages of 25, 50, and 100 mg/kg of body weight did potency as antidiabetic agent and based on the in vivo evaluation,
compound 3a was able to increase the percentage of decreasing
in blood glucose level and in weight loss prevention, decreasing
the drinking, and urine volume, significantly (P < 0.05) in
dosages 25, 50, and 100 mg/kg of body weight compared to the
negative control. Then, the oral administrations of compound
3a with dosages 50 and 100 mg/kg of body weight did not
show the significant difference (P > 0.05) in ability to increase
the percentage of change in loss of weight prevention compared

a b

c
Figure 4: Visualization of binding mode of active compounds a
(a) compound 1a, (b) compound 3a, and (c) compound 2b

b
Figure 6: The effect of oral administration on (a) drinking volume
a and (b) urine volume, in 24 h for a week after oral administration

b
Figure 5: The effect of oral administration on (a) the percentage of
change in blood glucose level and (b) in weight loss prevention for a Figure 7: The effect of oral administration for a week on the change
week of relative weight of mice organs.

88  http://www.tjps.pharm.chula.ac.th TJPS 2019, 43 (2): 83-89

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89  http://www.tjps.pharm.chula.ac.th TJPS 2019, 43 (2): 83-89