Synthesis, characterization and biological evaluation of new 3,5-disubstituted-pyrazoline derivatives as potential anti-Mycobacterium tuberculosis H37Ra compounds

Article abstract of DOI:10.3390/molecules26072081

A total of fourteen pyrazoline derivatives were synthesized through cyclo-condensation reactions by chalcone derivatives with different types of semicarbazide. These compounds were characterized by IR, 1D-NMR (¹H, ¹³C and Distortionless Enhancement by Polarization Transfer-DEPT-135) and 2D-NMR (COSY, HSQC and HMBC) as well as mass spectroscopy analysis (HRMS). The synthesized compounds were tested for their antituberculosis activity against Mycobacterium tuberculosis H37Ra in vitro. Based on this activity, compound 4a showed the most potent inhibitory activity, with a minimum inhibitory concentration (MIC) value of 17 μM. In addition, six other synthesized compounds, 5a and 5c–5g, exhibited moderate activity, with MIC ranges between 60 μM to 140 μM. Compound 4a showed good bactericidal activity with a minimum bactericidal concentration (MBC) value of 34 μM against Mycobacterium tuberculosis H37Ra. Molecular docking studies for compound 4a on alpha-sterol demethylase was done to understand and explore ligand– receptor interactions, and to hypothesize potential refinements for the compound.

Full text of DOI:10.3390/molecules26072081

molecules
Article
Synthesis, Characterization and Biological Evaluation of New
3,5-Disubstituted-Pyrazoline Derivatives as Potential
Anti-Mycobacterium tuberculosis H37Ra Compounds
Kok Tong Wong 1, Hasnah Osman 1, Thaigarajan Parumasivam 2, Unang Supratman 3,
Mohammad Tasyriq Che Omar 4 and Mohamad Nurul Azmi 1,
*
1
School of Chemical Sciences, Universiti Sains Malaysia, Minden, Penang 11800, Malaysia;
koktong_850820@hotmail.com (K.T.W.); ohasnah@usm.my (H.O.)
School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, Penang 11800, Malaysia;
thaigarp@usm.my
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran,
Jatinangor 45363, Indonesia; unang.supratman@unpad.ac.id
2
3
4
Biological Section, School of Distance Education, Universiti Sains Malaysia, Penang 11800, Malaysia;
mtasyriq@usm.my
*
Correspondence: mnazmi@usm.my; Tel.: +60-4653-3562
Abstract: A total of fourteen pyrazoline derivatives were synthesized through cyclo-condensation
reactions by chalcone derivatives with different types of semicarbazide. These compounds were
characterized by IR, 1D-NMR (1H, 13C and Distortionless Enhancement by Polarization Transfer -
DEPT-135) and 2D-NMR (COSY, HSQC and HMBC) as well as mass spectroscopy analysis (HRMS).
The synthesized compounds were tested for their antituberculosis activity against Mycobacterium
tuberculosis H37Ra in vitro. Based on this activity, compound 4a showed the most potent inhibitory
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Wong, K.T.; Osman, H.;
Parumasivam, T.; Supratman, U.; Che
Omar, M.T.; Azmi, M.N. Synthesis,
Characterization and Biological
Evaluation of New
activity, with a minimum inhibitory concentration (MIC) value of 17
synthesized compounds, 5a and 5c–5g, exhibited moderate activity, with MIC ranges between
60 M to 140 M. Compound 4a showed good bactericidal activity with a minimum bactericidal
concentration (MBC) value of 34 M against Mycobacterium tuberculosis H37Ra. Molecular docking
µM. In addition, six other
3,5-Disubstituted-Pyrazoline
Derivatives as Potential
µ
µ
µ
Anti-Mycobacterium tuberculosis
H37Ra Compounds. Molecules 2021,
26, 2081. https://doi.org/10.3390/
molecules26072081
studies for compound 4a on alpha-sterol demethylase was done to understand and explore ligand–
receptor interactions, and to hypothesize potential refinements for the compound.
Keywords: pyrazolines; Mycobacterium tuberculosis; antitubercular agents; molecular docking; alpha-
sterol demethylase
Academic Editor: Fawaz Aldabbagh
Received: 8 March 2021
Accepted: 22 March 2021
Published: 5 April 2021
1. Introduction
Pyrazoline is a five-membered heterocyclic ring that has an endocyclic double bond
with two adjacent nitrogen atoms at position 1- and 2-. The first pyrazoline (4,5-dihydro-
1H-pyrazole) was synthesized by Knorr in 1883 [1]. Pyrazolines are noted for the stability
of their ring system and the reactivity of several sites that permit a series of substitution
reactions to take place. Structural modifications of pyrazolines can be achieved by dec-
orating the stable fragments with different functional groups and aromatic scaffolds to
create benzylhydrazine moieties for the development of new potent compounds possess-
ing biological activities. Previous studies have reported considerable biological activities
when substitution occurs at position 1-, 3- and 5- of the pyrazoline (Figure 1), such as
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anticonvulsant, antimalarial [
2], anti-cardiovascular [3], anticancer [4], anti-amoebic [5],
antimicrobial [ ] and anti-tumor activities [8
6
,
7
]. Meanwhile, carbothioamide derivatives
were found to have significant pharmacological activities, such as monoamine oxidase
(MAO) inhibitors [9,10], antituberculosis [11–13] and anticonvulsant activities [14–16].
Molecules 2021, 26, 2081. https://doi.org/10.3390/molecules26072081
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2081
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Figure 1. The general structure of the pyrazoline at position 1-, 3- and 5-.
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB)
that affects the lungs [17]. According to the World Health Organization (WHO) 2019 Global
Tuberculosis Report, about 1.5 million people from a total of 10 million people with TB
died in 2018 [18]. In addition, WHO has declared that the South-East Asian region is
the most affected with TB cases (44%), followed by Africa (24%), Western Pacific (18%),
Eastern Mediterranean (8%), Americas (3%) and Europe (3%). In Malaysia, the Health
Ministry (2019) has reported about 1500 to 2000 deaths from TB per year, with an average
of six deaths a day in 2018 [19]. The increase in cases is mainly due to delays in seeking
medical treatment and low TB awareness. In addition, the current lengthy treatment
duration impairs patient adherence to the TB medications, which in turn fuels the spread
of highly concerning multi-drug and extensively-drug resistant M. tuberculosis. Hence, this
underscores the importance of developing a new effective anti-TB regimen.
In continuation of our efforts to develop antitubercular agents, here we report the
synthesis of new pyrazoline derivatives, along with their potential anti-TB activity against
M. tuberculosis H37Ra. The attenuated Mycobacterium tuberculosis H37Ra strain was used
due to biosafety constraints with using virulent M. tuberculosis H37Rv and clinical strains.
However, Heinrichs et al. showed that the H37Ra strain is able to equally predict sus-
ceptibility to antibiotics as the H37Rv strain and clinical isolates [20]. This justifies the
selection of avirulent M. tuberculosis for this study. Molecular docking at the active site of
cytochrome P450 14 alpha-sterol demethylase (CYP51) was performed to rationalize the
inhibitory property of potent compounds within the active pocket.
2. Results and Discussion
2.1. Chemistry
The synthesis of the first series of 3,5-disubstituted-4,5-dihydro-N-phenyl-1H-pyrazole-
1-carbothioamide (4a
–g) was synthesized by the cyclo-condensation of chalcone derivatives
(
3a ) with 4-phenyl-3-thiosemicarbazide in the presence of sodium hydroxide as a base
–g
(Scheme 1).
Scheme 1. Synthesis of chalcone and pyrazoline derivatives.

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Compound 4a is the representative compound from the first series. This new com-
pound was synthesized by the cyclo-condensation of 3-(4-methylphenyl)-1-phenyl-2-
propen-1-one (3a) with 4-phenyl-3-thiosemicarbazide in the presence of sodium hydroxide
as a base. Compound 4a formed as a cream-colored precipitate with 26% yield and a melting
◦
point of 133–136 C. Compound 4a was characterized by various spectroscopic techniques
including IR, 1D-NMR, 2D-NMR and HRMS. The i−m1portant absorption bands in the IR
−1
spectrum of compound 4a were observed at 3291 cm (N–H stretching), 1594 cm (C=N
stretching), 1520 cm−1 and 1449 cm−1 (C=C stretching of the aromatic), and 1399 cm−1
(C=S stretching). The absence of C=O and C=C bands, as well as the appearance of new
C=N and C=S bands in the IR spectra of compound 4a suggests the complete formation
of compound 4a via the cyclo-condensation reaction between chalcone 3a and 4-phenyl-3-
thiosemicarbazide (Scheme 1).
The 1H-NMR spectrum of compound 4a showed that two germinal protons, H-4
and H-4 , of the methylene group resonated as a doublet of doublet at δH 3.26 and
δH 4.05, respectively. The appearance of these two signals could be attributed to the non-
equivalent nature of the two germinal protons with a J coupling constant of J4α4β = 18.0 Hz
α
β
,
J4α5 = 3.5 Hz and J4β5 = 11.5 Hz. Meanwhile, the vicinal proton (H-5) also appeared as a
doublet of doublet at a slightly downfield region, δH 6.14, due to vicinal coupling with the
two neighboring germinal protons of the methylene group at position 4- of the pyrazoline
ring. A singlet at δH 9.93 in the downfield region indicated the presence of a thioamide
proton. In compound 4a, some aromatic protons such as H-300, H-400 and H-500 were
observed at the same chemical shift, δH 7.49, due to the overlapping of peaks in the similar
environment. The 1H-NMR spectrum also showed three signals for ring C. A triplet at
δH 7.33 (2H, J = 7.5 Hz) was attributed to H-10 and H-12. In addition, two multiplets at
δH 7.72 (2H) and δH 7.16 were assigned to H-9 with H-13 and H-11. The 13C-NMR spectrum
of carbothioamide compound 4a showed seventeen carbon signals. One carbon signal
each at δC 42.3 and δC 62.3 suggested the presence of pyrazoline ring carbons, which were
assigned to 4-
3- =N in the pyrazoline ring, which is a carbon attached to an electronegative nitrogen by
a double bond and also to a benzene ring. Besides, a signal at δC 174.2 was attributed to
6-
=S. The 2D-NMR correlation of 1H-1H COSY and 1H-13C HMBC spectra were used to
CH2 and 5-CH, respectively. Also, a signal at δC 155.3 was attributed to
C
C
assign the aromatic signals, especially in the case of the pyrazoline ring. The analysis of
1H-1H COSY showed the correlation between of 14-CH3-H-40-H-30 and H-50, 4α-CH with
4
β
-C
with H-500 (Figure 2). The 1H-13C HMBC spectrum of 4a shows the cross-correlations of
7-N with carbons C-9 and C-13, and H-10 with C-9, C-13, C-11, C-12 and C-8 (Figure 2),
H and 5-CH
, H-8 with H-9-H-10-H-11-H-12 and H-200 with H-100-H-600-H-300, H-400
H
and the cross-correlation of 14-CH3 with C-30, C-50 and C-40. The HRMS spectrum of 4a
revealed the molecular ion peak [M + Na]+ at m/z 394.1156, which is consistent with
the molecular formula C23H21N3S (calcd. 394.1354). According to the above spectra
data analysis, compound 4a was identified as a new 2-pyrazoline compound and named
5-(4-methylphenyl)-N,3-diphenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide.
Figure 2. Key COSY/HMBC correlations of compound 4a.

Molecules 2021, 26, 2081
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The second series of new (3,5-disubstituted- 4,5-dihydro-1H-pyrazol-yl) (4-hydroxyph-
enyl)methanone (5a–g) was synthesized by the cyclo-condensation of chalcone derivatives
(3a–g) with 4-hydroxybenzhydrazide in the presence of sodium hydroxide as a base.
Compound 5e was taken as the representative compound from the fourth series, which
◦
formed a pale-yellow powder with 17% yield and a melting point of 237–241 C. Compound
5e was characterized by multiple spectroscopic techniques including IR, 1D-NMR, 2D-
NMR, and HRMS. The important absorption bands in the IR spectrum of compound 5e
were observed at 3137 cm−1 (O–H stretching), 3066 cm−1 (Csp2–H), 2926 cm−1 (Csp3–H),
1737 cm−1 (C=O stretching), 1618 cm−1 (C=N stretching), 1573 cm−1 and 1427 cm−1 (C=C
stretching of the aromatic). Moreover, the hydroxy group of phenol in the para position
is a good activating group [21]. The absence of C=C bands, as well as the appearance
of new C=N and C–O bands in the IR spectrum of compound 5e suggests the complete
formation of the compound 5e via a cyclo-condensation reaction between chalcone 3e and
4-hydroxybenzhydrazide (Scheme 1).
The 1H-NMR spectrum of compound 5e revealed the presence of one, mostly down-
field, sharp singlet at δH 10.11 for the hydroxy proton (13-O
H
). One doublet of doublet at
0
0
δH 7.40 (J = 2.5 and 8.5 Hz) was assigned to H-5 due to its meta coupling with H-3 and ortho
0
0
coupling with H-6 in ring B. A doublet at δH 7.22 (J = 8.0 Hz) was attributed to H-6 due to
its ortho coupling to H-50. In addition, a doublet at δH 7.69 was attributed to H-3 due to its
meta coupling to H-5. On the other hand, a doublet at δH 7.91 (J = 8.0 Hz) integrating to two
protons was assigned to H-9 and H-11, which occurred at the same position and can be con-
sidered chemically equivalent due to symmetry in the structure. The 13C-NMR spectrum
of compound 5e showed that signals corresponded to all twenty-two carbons in the com-
pound. Signals at δC 58.8, 40.1 and 155.3 were assigned to 5-CH, 4-CH2 and 3-C=N of the
pyrazoline ring, respectively, and the presence of the pyrazoline ring was further confirmed
by 2D-NMR spectroscopy. The 1H-1H COSY spectrum of compound 5e showed a very clear
correlation between 5-C
lation between H-8/H-12 (δC 114.9) and H-9/H-11 (δC 132.6) was also observed (Figure 3).
The 1H-13C HMBC spectrum of 5e shows that 4
-C δC 3.13) and 4 -C δC 3.96) were
H (δC 58.8), C-10 (δC 138.9) and 6-
=O (δC 165.2), thus confirming the
H (δC 5.95), 4α-CH (δC 3.13) and 4β -CH (δC 3.96). A strong corre-
α
H
(
β
H (
correlated with 5-
C
C
formation of a pyrazoline ring (Figure 3). The cross-correlation0of H-50 with C-30 and C-10
0
was found, while H-6 was found to correlate with 5-
C
H and C-2 . The HRMS of compound
5e showed a molecular ion peak [M + H]+ at m/z 411.0694 (calcd. 411.0667) which corre-
sponded to the molecular formula C22H17Cl2N2O2. In conclusion, based on the spectral
data, it is proven that the compound 5e, N-(4-hydroxyphenyl)(5-(2,4-dichlorolphenyl)-3-
phenyl-4,5-dihydro-1H-pyrazol-1-yl)methanone, was successfully synthesized.
Figure 3. Key COSY/HMBC correlations of compound 5e.
2.2. Anti-Tuberculosis Activity
Two series of fourteen synthesized pyrazoline derivatives (4a–g and 5a–g) were
screened for antituberculosis activity against M. tuberculosis H37Ra using the Tetrazolium
bromide microplate assay (TEMA) method. Isoniazid was used as a positive control.
Table 1 depicts the results of the antituberculosis activity of these compounds based on
their minimum inhibitory concentration (MIC) and minimum bactericidal concentration
(MBC) values.

Molecules 2021, 26, 2081
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Table 1. In vitro anti-tuberculosis activities of pyrazoline derivatives (4a–g and 5a–g) against My-
cobacterium tuberculosis H37Ra.
MIC
MBC
Compounds
(µg/mL)
(µM)
(µg/mL)
(µM)
4a
4b
4c
4d
4e
4f
4g
5a
6.25
>200
200
>200
200
200
>200
25
17
>517
511
>500
471
511
>517
70
12.5
>200
>200
>200
>200
>200
>200
200
34
>517
>511 (NC)
>500
>471 (NC)
>511 (NC)
>517
562
5b
5c
>200
25
>535
66
>200
100
>535
266
5d
5e
25
25
65
61
200
200
519
488
5f
5g
25
50
0.625
66
134
5
200
200
0.625
532
537
5
Isoniazid (Control)
Results are from three independent experiments performed in duplicate. NC = no bactericidal effect even at the
highest test concentration.
Out of fourteen synthesized compounds, ten compounds exhibited promising activity
against M. tuberculosis, with MIC values in the range of 17
was the most active compound, with the lowest MIC value of 17
the first series 4a , compounds 4a 4c 4e and 4f showed MICs of 17
511 M (~200 g/mL), 471 M (~200 g/mL) and 511 M (~200 g/mL), respectively.
Compound 4a with the para-methyl substitute showed the most activity compared to other
µ
M to 511
M (~6.25
M (~6.25
µ
M. Compound 4a
g/mL). In
g/mL),
µ
µ
–g
,
,
µ
µ
µ
µ
µ
µ
µ
µ
compounds in the same series. In the second series, compounds 5a
5g exhibited moderate activity, with MICs of 70 M (~25 g/mL), 66
65 M (~25 g/mL), 61 M (~25 g/mL), 66 M (~25 g/mL), and 134
,
5c
M (~25
M (~50
,
5d
,
5e
µ
,
5f and
g/mL),
g/mL),
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
respectively. Meanwhile, compound 5b with the para-methoxy group showed no inhibition
against M. tuberculosis.
These ten compounds (4a
bactericidal activity against M. tuberculosis H37Ra. Only seven compounds (4a
5c–g) exhibited bactericidal activity against M. tuberculosis at the tested concentration, with
MBCs of 34 M (~12.5 g/mL), 562 M (~200 g/mL), 266 M (~100 g/mL), 519
(~200 g/mL), 488 M (~200 g/mL), 532 M (~200 g/mL) and 537 M (~200 g/mL),
,
4c
,
4e
,
4f,
5a, and 5c–g) were further evaluated for their
,
5a and
µ
µ
µ
µ
µ
µ
µM
µ
µ
µ
µ
µ
µ
µ
respectively. The remaining compounds did not show any bactericidal effects against
M. tuberculosis H37Ra, even at the highest test concentration of 200 µg/mL.
The in vitro antituberculosis evaluation revealed that the compounds which contained
substituted para methyl (4a and 5a) in two series exhibited activity against M. tuberculosis.
The result confirmed that methyl group substitution at the phenyl ring in pyrazoline ana-
logues has a favorable effect, and a prominent improvement in inhibition was noticed [22].
However, it was found that compounds with a substituted para methoxy (4b and 5b) in
both series showed no inhibition against M. tuberculosis H37Ra, even at the highest tested
concentration of 200 µg/mL. This is not surprising, as previous studies have reported
that (OCH3) group substitution at the phenyl ring in pyrazoline analogues worsens the
antituberculosis activity [23]. Moreover, it was discovered that all compounds (5a and
5c–g) from the second series (2-(4-hydroxyphenyl)-2-oxoethan-1-ide derivatives), except
for compound 5b, exhibited moderate activity against M. tuberculosis H37Ra. These results
confirmed that hydroxylphenyl substitution is necessary for antituberculosis activity [24].
2.3. Molecular Docking
The free binding energy and interaction modes between compound 4a and residues
in the active site of cytochrome P450 14 alpha-sterol demethylase (CYP51) were identified

Molecules 2021, 26, 2081
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by molecular docking studies using AutoDock Vina version 1.14. The crystal structure
of CYP51 complexed with fluconazole (Protein Data Bank ID - PDB ID 1EA1) was used
as a reference structure for the docking process [25]. The structure of isoniazid (PDB
ID: 2VCF) was used as an experimental control to investigate molecular docking with
CYP51. The docking results indicated binding energy between CYP51 and the control
inhibitor (fluconazole) or the experimental control (isoniazid) or compound 4a, ranging
between
−
6.2 to
−
7.1,
−
6.0 to
−
5.0 and
−
6.3 to
−
6.7 respectively (Table 2). The active
site(s) of CYP51 responsible for the binding of compound 4a and the controls (isoniazid
and fluconazole) is shown in Figure 4.
Table 2. Binding energy between cytochrome P450 14 alpha-sterol demethylase (CYP51) with
compound 4a and controls (fluconazole and isoniazid) for all poses.
Enzyme
Compound
Pose
1
Binding
−6.7
−6.6
−6.5
−6.4
−6.3
−7.1
−6.8
−6.6
−6.4
−6.2
−6.0
−5.5
−5.4
−5.3
−5.0
2
4a
3
4
5
1
2
CYP51
(PDB 1EA1)
Fluconazole
Isoniazid
3
4
5
1
2
3
4
5
The interaction modes of compound 4a with CYP51 are shown in Figure 5a–d. These
structures detail the interactions in the active sites that are listed in Table 3. The interaction
modes of the most potent pose in CYP51 (Figure 5a) showed that all phenyl rings at position
1 and 5, including the thioamide group, interacted with ILE27, TRP267, HIS318 and ARG354
via hydrophobic interactions. The second most potent pose (Figure 5b) demonstrated that
the pyrazoline ring and thioamide group interacted with ARG274, GLU424 and TYR426,
respectively, via non-conventional hydrogen bonding. Five hydrophobic interactions were
formed between the thioamide group with ARG274, and at the phenyl rings at position 1
and 3 with TRP267, GLU271, ARG274, and ARG427.

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Figure 4. Active sites of CYP51 for binding of compound 4a and controls (Fluconazole & Isonizid).

Molecules 2021, 26, 2081
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Figure 5. Three-dimensional binding modes of compound 4a (pose 1–5) present at the active sites of CYP51. The interaction
modes of the most potent pose in CYP51: Figure 5a orientation 4a in pose 1, Figure 5b orientation 4a in pose 2, Figure 5c ori-
entation 4a in pose 3, Figure 5d orientation 4a in pose 4 and Figure 5e orientation 4a in pose 5. Pi-cation/Pi-anion/attractive
charge interactions (orange), Pi-sigma interaction (purple), Pi-sulfur (light brown), conventional hydrogen bond interaction
(green), unfavorable interaction (red), Pi-Pi T-shaped interaction (pink).

Molecules 2021, 26, 2081
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Table 3. Binding interactions between compound 4a with CYP51 (poses 1 to 5).
Enzyme
+
Compound
Interacting Unit of
Active Site
Pose
Protein Residue
Type of Interaction
Compounds
Phenyl
Phenyl
Pi–Alkyl
Pi–Pi T-shaped
Pi–Pi T-shaped
Pi–Cation
Unfavorable
Unfavorable
Pi–Pi Stacked
Pi–Anion
ILE27
TRP267
HIS318
ARG354
Phenyl
2
1
Phenyl
Thioamide
Phenyl
Phenyl
TRP267
GLU271
ARG274
Pyrazoline ring
Phenyl
H-bond
Pi–Cation
Unfavorable
Unfavorable
H-bond
Thioamide
2
2
Thioamide
Thioamide
Phenyl
GLU424
TYR426
ARG427
ILE27
H-bond
CYP51
+
Pi–Alkyl
Phenyl
Pi–Alkyl
Compound 4a
Phenyl
ARG274
LEU317
HIS318
ARG354
Pi–Cation
H-bond
Thioamide
Thioamide
Pyrazoline ring
Thioamide
Phenyl
Pi–Sulfur
2
3
H-bond
Unfavorable
Pi–Alkyl
ARG427
HIS430
THR80
GLY84
GLU94
ARG96
THR80
GLU94
ARG96
Thioamide
Phenyl
Pi–Sulfur
Pi–Sigma
Thioamide
Thioamide
Phenyl
H-bond
3
3
4
5
Attractive charge
Pi–Cation
Pi–Sigma
Phenyl
Thioamide
Phenyl
Attractive charge
Pi–Cation
The third pose indicated the compound also docked inside active site 2 (Figure 5c) with
residue thioamide and pyrazoline ring interacted with LEU317 and ARG354, respectively.
Meanwhile, three hydrophobic interactions were occurred between phenyl rings with
ILE27, ARG274, and ARG247. The remaining hydrophobic interactions were established
between the thioamide group and HIS318, ARG354, and HIS430. In contrast, poses 4 and
5 showed that compound 4a preferred to bind at active site 3 of CYP51, with established
interactions between residues THR80 and ARG96 with the phenyl group, and GLU94 with
the thioamide group (Figure 5d–e). Only one non-conventional hydrogen bond was formed
between the thioamide group and GLY84 in pose 4 (Figure 5d).

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3. Materials and Methods
3.1. Experimental
3.1.1. Chemistry
All chemicals and reagents were obtained from Acros Organic (Geel, Belgium), QRec
(Penang, Malaysia, Asia), Sigma-Aldrich Chemical Co. or Merck (Darmstadt, Germany).
Thin-layer chromatography (TLC) was performed on alumina plates pre-coated with silica
gel (Merck 60 F254). The progress of reactions was determined by the appearance of
product and disappearance of reactant spots under UV radiation (
λ
= 254 nm, Muttenz,
max
Switzerland), respectively. Melting points were determined on open capillary tubes and
are uncorrected. All spectral data were obtained on the following instruments: infrared
(IR) spectra were recorded on a Perkin-Elmer System FTIR-ATR spectrometer; 1D- and
2D-NMR spectra were recorded on a 500 FT-NMR Bruker Advance spectrometer (Bruker
Bioscience, Billerica, MA, USA) in CD3COCD3, DMSO-d6, CDCl3 and tetramethylsilane
(TMS) as internal standards. Chemical shifts are reported in part per million (δ-scale) and
the coupling constants, J, are reported in Hertz (Hz). High resolution HRMS mass spectra
were obtained from a Waters Xevo QTOF MS system.
3.1.2. General Procedure for the Preparation of Chalcone Derivatives (3a–g) by the
Claisen–Schmidt Condensation Reaction
Chalcone derivatives (3a
literature. A mixture of acetophenone (
(0.015 mol) in methanol (10 mL) was refluxed in the presence of few drops of piperidine
for 72 h [26 27]. The solution was kept in an ice bath until a solid was obtained, and the
–
g) were synthesized following the method described in the
1) (0.01 mol) and substituted benzaldehyde (2a–g)
,
chalcone compounds were filtered, washed with cold water, dried and recrystallized from
ethanol. The results were compared with the literature [28–31].
(E)-3-(4-methylphenyl)-1-phenylprop-2-en-1-one (3a). Pale yellow crystalline solid; yield: 83%;
m.p.: 94–96 ◦C; FTIR (ATR) max (cm−1): 3025 (Csp2–H), 2918 (Csp3–H), 1656 (C=O), 1595
ν
(–CH=CH–), 1514 and 1449 (aromatic C=C); 1H-NMR (500MHz, CD3COCD3): δH 2.39 (3H,
s, 4-CH3), 7.30 (2H, d, J =008.0 Hz, H-30 and H-50), 7.580(2H, t, J = 7.5 Hz, H-200 and H-600),
0
7.66 (1H, t, J = 7.5 Hz, H-4 ), 7.75 (2H, t, J = 8.0 Hz, H-2 and H-6 ), 7.79 (1H, d, J = 15.5 Hz,
H-2), 7.85 (1H, d, J = 15.5 Hz, H-3) and 8.16 (2H, d, J = 7.5 Hz, H-300 and H-50’); 13C-NMR
(125 MHz; CD3COCD3): δC 20.6 (4-
C
H3)0, 121.0 (C-2), 128.4 (C-300 and C-500), 128.7 (C-200
00
0
0
0
0
00
00
and C-6 ), 128.7 (C-2 and C-6 ), 129.6 (C-3 and C-5 ), 132.4 (C-1 ), 132.7 (C-4 ), 138.4 (C-1 ),
140.9 (C-40), 144.1 (C-3) and 189.1 (1-C=O).
(E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (3b). Yellow crystalline solid; yield: 74%;
m.p.: 73–75 ◦C; FTIR (ATR)
ν
cm−1: 3018 (Csp2–H), 2842 (Csp3–H), 1656 (C=O), 1575
max
1
(–CH=CH–), 1510, 1445 (aromatic C=C), 1253 (C–O); H-NMR (500MHz; CD3COCD3):
δH 3.88 (3H, s, 4-OCH3), 7.03 (2H, d, J = 8.5 Hz, H-30 and H-50), 7.57 (2H, t, J = 7.5 Hz,
H-200 and H-600), 7.65 (1H, t, J = 7.5 Hz, H-400), 7.75 (1H, d, J = 15.5 Hz, H-2), 7.80 (1H, d,
J = 15.5 Hz, H-3), 7.83 (2H, d, J = 7.5 Hz, H-20 and H-60), and 8.15 (2H, d, J = 7.5 Hz, H-300
and H-500); 13C-NMR (125 MHz; 0CD3COCD3); δC 54.90(4-OCH3), 114.4 (C-300and C-500),
0
00
00
119.6 (C-2), 127.7 (C-1 ), 128.3 (C-3 ’ and C-5 ), 128.6 (C-2 ’ and C-6 ), 130.5 (C-2 and C-6 ),
132.6 (C-400), 138.6 (C-100), 143.9 (C-3), 161.9 (C-40) and 189.0 (1-C=O).
(E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (3c). Pale yellow crystalline solid; yield: 82%;
◦
m.p.: 113–115 C; FTIR (ATR)
ν
cm−1: 3059 (Csp2–H), 2931 (Csp3–H), 1653 (C=O),
max
1
1591 (–C=C–), 1563, 1489 (aromatic C=C), 1221 (C–O), 823 (C–Cl); H-NMR (500 MHz,
CD3COCD3): δH 7.52 (2H, d, J = 8.5 Hz, H-30 and H-50), 7.58 (2H, t, J = 8.0 Hz, H-200 and
H-600), 7.68 (1H, t, J = 7.5 Hz, H-400), 7.79 (1H, d, J = 16.0 Hz, H-2), 7.90 (2H, d, J = 8.5 Hz,
H-20 and H-60), 7.93 (1H, d, J = 16.0 Hz, H-3), and 8.17 (2H, d, J = 7.5 Hz, H-300 and H-500);
13C-NMR (125 MHz, CD3COCD3); δC 122.8 (C-2), 128.5 (C-300 and C-500), 128.7 (C-200 and
C-600), 129.1 (C-20 and C-‘6), 130.2 (C-30 and C-50), 132.9 (C-400), 134.0 (C-10), 135.7 (C-40),
138.1 (C-100), 142.4 (C-3) and 188.9 (1-C =O).

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(E)-3-(4-dimethylam◦inophenyl)-1-phenylprop-2-en-1-one (3d). Orange crystalline solid; yield:
57%; m.p.: 113–115 C; FTIR (ATR) ν
max cm−1: 3053.1 (Csp2–H), 2909.8 (Csp3–H), 1646 (C=O),
1611 (–C=C–), 1511, 1457 (C=C aromatic), 1322 (C–N); 1H-NMR (500 MHz; CD3COCD3):
δH 3.07 (6H, s, 4-CH3 and 5-CH3), 6.80 (2H, d, J =9.0 Hz, H-30 and H-50), 7.55 (2H, t,
J =7.5 Hz, H-200 and H-600), 7.61 (1H, d, J = 7.5 Hz, H-400), 7.62 (1H, d, J = 15.5 Hz, H-2),
7.69 (2H, d, J = 9.0 Hz, H-20 and H-60), 7.77 (1H, d, J = 15.5 Hz, H-3) and 8.12 (2H, d,
00
00
J = 7.5 Hz0, H-3 and H-5 ); 13C-NMR (125 MHz; CD3COCD3); δC 39.3 (4-CH3 and 5-CH3),
0
0
00
00
00
0
111.8 (C-3 and C-5 ), 116.3 (C-2), 122.6 (C-1 ), 128.1 (C-3 and C-5 ), 128.5 (C-2 and C-6 ’),
130.5 (C-20 and C-60), 132.2 (C-400), 139.1 (C-100), 145.1 (C-3), 152.3 (C-40) and 188.7 (1-
(E)-3-(2,4-dichlorophenyl)-1-phenylprop-2-en-1-one (3e). Yellow solid; yield: 52%; m.p.: 69–73 ◦C
FTIR (ATR)
cm−1: 3064 (Csp2–H), 2931 (Csp3–H), 1652 (C=O), 1607 (–C=C–), 1514,
C=O).
;
ν
max
1
1475.1 (C=C aromatic); H-NMR (500 MHz, CD3COCD3): δH 7.49 (1H, dd, J = 2.0 Hz
and 8.5 Hz, H-50), 7.59 (2H, t, J = 8.0 Hz, H-200 and H-60’), 7.64 (1H, d, J = 2.0 Hz, H-30),
7.69 (1H, d, J = 7.5 Hz, H-400), 7.96 (1H, d, J = 16.0 Hz, H-2), 8.12 (1H, d, J = 15.5 Hz,
H-3) and 8.18 (3H, t, J = 7.5 Hz, H-60, H-300 and H-500); 13C-NMR (125 MHz, CD3COCD3);
δC 125.2 (C-2), 127.9 (C-50), 128.600(C-30’ and C-500), 128.8 (C-200 and C-600), 129.4 0(C-30),
0
0
0
0
129.6 (C-6 ), 132.0 (C-4 ), 133.2 (C-4 ), 135.6 (C-1 ), 136.0 (C-2 ), 137.6 (C-3), 137.8 (C-1 ’) and
188.7 (1-C=O).
(E)-3-(2-chlorophenyl)-1-phenylprop-2-en-1-one (3f). Pale yellow crystalline solid; yield: 57%;
m.p.: 45–48 ◦C; FTIR (ATR)
ν
cm−1: 3060 (Csp2–H), 2931 (Csp3–H), 1662 (C=O), 1603
max
(–C=C–), 1514, 1463 (C=C aromatic), 748 (C–Cl); 1H-NMR (500 MHz, CD3COCD3): δH 7.47
0
0
0
00
00
(2H, m, H-4 and H-5 ), 7.56 (1H, m, H-3 ), 7.60 (2H, d, J = 7.5 Hz, H-2 and H-6 ), 7.69 (1H,
d, J = 7.5 Hz, H-400), 7.92 (1H, d, J = 15.5 Hz, H-2), 8.14 (1H, d, J = 7.5 Hz, H-60), 8.19 (2H,
d, J = 8.5 Hz, H-300 and H-500) and 8.20 (1H, d, J = 15.5 Hz, H-3); 13C-NMR (125 MHz,
CD3COCD3); δC 124.7 (C-2), 127.6 (C-40), 128.2 (C-60), 128.5 (C-300 and C-500), 128.7 (C-200
and C-600), 130.1 (C-50), 131.6 (C-30), 133.0 (C-10), 133.1 (C-400), 134.9 (C-20), 137.9 (C-100),
139.0 (C-3) and 188.9 (1-C=O).
(E)-3-(2-methoxyphenyl)-1-phenylprop-2-en-1-one (3g). Yellow crystalline solid; yield: 62%;
m.p.: 51–53 ◦C; FTIR (ATR)
ν
cm−1: 3019 (Csp2–H), 2948 (Csp3–H), 1659 (C=O), 1594
max
(–C=C–), 1513, 1439 (C=C aromatic), 1247 (C–O); 1H-NMR (500 MHz; CD3COCD3): δH 3.98
0
0
0
(3H, s, 4-OCH3), 7.05 (1H, t, J = 7.5 Hz, H-5 ), 7.13 (1H, d, J = 8.5 Hz, H-3 ), 7.64 (1H, m, H-4 ),
00
00
00
7.58 (2H, t, J = 7.5 Hz, H-2 and H-6 ), 7.66 (1H, d, J = 7.5 Hz, H-4 ), 7.88 (1H, d, J = 15.5 Hz
,
H-2), 7.90 (1H, t, J = 7.5 Hz, H-60), 8.14 (2H, d, J = 8.5 Hz, H-300 and H-500) and 8.17 (1H,
d, J = 15.5 Hz, H-3); 13C-NMR (125 MHz; CD3COCD3); δC 55.2 (4-OCH3), 111.5 (C-30),
120.7 (C-50), 122.1 (C-2), 123.6 (C-10), 128.4 (C-300 and C-500), 128.6 (C-60), 128.6 (C-200 and
C-600), 132.0 (C-40), 132.7 (C-400), 138.5 (C-100), 139.0 (C-3), 158.8 (C-20) and 189.4 (1-C=O).
3.1.3. General Procedure for the Preparation of the First Series of
3,5-disubstituted-4,5-dihydro-N-phenyl-1H-pyrazole-1-carbothioamide (4a–g)
The carbothioamide compounds (4a–g) were synthesized according to a previously
reported method with slight modifications. The cyclo-condensation of chalcone derivatives
(3a–g) (1 mmol) with 4-phenyl-3-thiosemicarbazide (1.5 mmol) was carried out in ethanol
(6 mL) in the presence of NaOH (3 mmol) for 3 to 8 h [32]. The reaction progress was
observed on a TLC plate. After the reaction was over, the reaction mixture was left at room
temperature overnight. A solid was formed after crushed ice was added. The solid was
filtered, washed with cold water, dried and recrystallized from ethanol. Please refer the
Supplementary Data for full spectra.
5-(4-methylphenyl)-N,3-diphenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (4a). Cream-colored
solid; yield: 26%; m.p.: 133–136 ◦C; FTIR (ATR)
ν
cm−1: 3291 (N–H), 3028 (Csp2–H),
max
2919 (Csp3–H), 1594 (C=N), 1520, 1449 (aromatic C=C), 1399 (C=S); 1H-NMR (500 MHz,
CD3COCD3): δH 2.29 (3H, s, 14-C 3), 3.26 (1H, dd, J = 3.5 Hz and 18 Hz, 4 -C ), 4.05 (1H,
dd, J = 11.5 Hz and 18 Hz, 4 -C ), 6.14 (1H, dd, J = 3.5 Hz and 11.5 Hz, 5-C ), 7.16 (5H,
m, H-20, H-30, H-50, H-60, H-11), 7.33 (2H, t, J = 7.5 Hz, H-10 and H-12), 7.49 (3H, m, H-300,
H
α
H
β
H
H

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H-400 an0d0 H-500), 7.72 (2H, m, H-9 and H-13), 7.98 (2H, dd, J = 2.0 Hz and 7.5 Hz, H-200
and H-6 ), 9.93 (1H, s, 7-NH CH3), 42.3 (4-
); 13C NMR (125 MHz, CD3COCD3): δC 20.2 (14-
C
H2), 63.3 (5-C
H), 124.3 (C-9 and C-13), 124.6 (C-11), 125.5 (C-20 and C-60), 127.2 (C-200
and C-600), 128.0 (C-10 and C-12), 128.7 (C-300 and C-500), 129.1 (C-30 and C-50), 130.7 (C-400),
131.3 (C-100), 136.5 (C-40), 139.8 (C-8), 140.1 (C-10), 155.3 (3-
394.1156 [M + Na]+ {calcd. 394.1354 for C23H21N3SNa}.
C=N), 174.2 (6-C=S); HRMS:
5-(4-methoxyphenyl)-N,3-diphenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (4b). Pale yellow
crystals; yield: 39%; m.p.: 173–175 ◦C; FTIR (ATR) cm−1: 3334 (N–H), 3058 (Csp2–H),
ν
max
2842 (Csp3–H), 1592 (C=N), 1510, 1445 (aromatic C=C), 1396 (C=S), 1244 (C–O); 1H-NMR
(500 MHz, CD3COCD3): δH 3.29 (1H, dd, J = 3.5 Hz and 18.0 Hz, 4 -C ), 3.78 (3H, s,
14-OCH3), 4.05 (1H, dd, J = 11.5 Hz and 18.0 Hz, 4 -C ), 6.13 (1H, dd, J = 3.5 Hz and
), 6.89 (2H, d, J = 9.0 Hz, H-3 and H-5 ), 7.15 (1H, t, J = 7.5 Hz, H-11), 7.22 (2H,
α
H
β
H
0
0
11.5 Hz, 5-C
d, J = 8.5 Hz, H-2 and H-6 ), 7.33 (2H, t, J = 7.5 Hz, H-10 and H-12), 7.50 (3H, m, H-3 , H-4
and H-500), 7.73 (2H, d, J = 7.5 Hz, H-9 and H-13), 7.99 (2H, m, H-200 and H-600), 9.92 (1H, s,
H
0
0
00
00
7-NH CH2), 54.6 (14-OCH3), 63.1 (5-CH),
); 13C NMR (125 MHz, CD3COCD3): δC 42.3 (4-
113.8 (C-30 and C-50), 124.3 (C-9 and C-13),010 24.6 (C-11), 126.9 (C-0200 and C-60), 01027.2 (C-200
00
00
and C-6 ), 128.0 (C-10 and C-12), 128.7 (C-3 and C-5 ), 130.7 (C-4 ), 131.4 (C-1 ), 135.1 (C-
0
0
C=N), 158.9 (C-4 ), 174.2 (6-C
=S); HRMS: 410.1098 [M + Na]+ {calcd.
1 ), 139.8 (C-8), 155.3 (3-
410.1303 for C23H21N3OSNa}.
5-(4-chlorophenyl)-N,3-diphenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (4c). Yellow solid;
◦
yield: 34%; m.p.: 150–153 C; FTIR (ATR)
1538, 1489 (aromatic C=C), 1380 (C=S); 1H-NMR (500 MHz, CD3COCD3): δH 3.33 (1H, dd,
J = 3.5 Hz and 18.0 Hz, 4 -C ), 4.11 (1H, dd, J = 11.5 Hz and 18.0 Hz, 4 -C ), 6.18 (1H,
dd, J = 3.5 Hz and 11.5 Hz, 5-C
), 7.16 (1H, t, J = 7.5 Hz, H-11), 7.35 (6H, m, H-20, H-30,
H-50, H-60, H-10 and H-12), 7.50 (3H, m, H-300, H-400 and H-500), 7.72 (2H, t, J = 7.5 Hz, H-9
ν
max cm−1: 3306 (N–H), 3034 (Csp2–H), 1594 (C=N),
α
H
β
H
H
00
00
and H-13), 7.99 (2H, d, J = 7.0 Hz, H-2 and H-6 ), 9.96 (1H, s, 7-N
H
); 13C-NMR (125 MHz,
CD3COCD3): δC 42.1 (4-CH2), 63.0 (5-C
H), 124.4 (C-9 and C-13), 124.7 (C-11), 127.2 (C-20
and C-60), 127.6 (C-200 and C-600), 128.1 (C-10 and C-12), 128.6 (C-30 and C-50), 128.7 (C-300
and C-500), 130.7 (C-400), 131.2 (C-100), 132.2 (C-40), 139.7 (C-8), 142.0 (C-10), 155.2 (3-C=N),
174.3 (6-C=S); HRMS: 392.0952 [M + H]+ {calcd. 392.0988 for (C22H19ClN3S}.
5-(4-dimethylaminohenyl)-N,3-diphenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (4d). Orange
oil; yield: 12%; FTIR (ATR)
1595 (C=N), 1517, 1446 (aromatic C=C), 1395 (C=S), 1320 (C–N); H-NMR (500 MHz,
ν
cm−1: 3338 (N–H), 3031 (Csp2–H), 2921 (Csp3–H),
max
1
CD3COCD3): δH 2.90(6H, s, 14-CH3 and 15-CH3), 3.09 (1H, dd, J = 3.5 Hz and 18.0 Hz,
4
α
-C
5-C
H-10 and H-12), 7.50 (3H, m, H-3 , H-4 and H-5 ), 7.74 (2H, m, H-9 and H-13), 7.98 (2H, m,
H
), 3.99 (1H, dd, J = 11.5 Hz and 18.0 Hz, 4
β
-C
H), 6.08 (1H, dd, J = 3.5 Hz and 11.0 Hz,
0
0
0
0
H
), 6.69 (2H, d, J = 9.0 Hz, H-3 and H-5 ), 7.13 (3H, m, H-2 , H-6 and H-11), 7.33 (2H, m,
00
00
00
00
00
H-2 and H-6 ), 9.88 (1H, s, 7-N
0 0
15- H3), 42.3 (4- H2), 63.2 (5-CH), 112.4 (C-3 and C-5 ), 124.2 (C-9 and C-13), 124.5 (C-11),
H CH3 and
); 13C-NMR (125 MHz, CD3COCD3): δC 39.8 (14-
C
C
126.6 (C-20 and C-60), 127.2 (C-200 a0n0 d C-600), 128.0 (C-10 and C-12), 128.7 (C-300 and C-500),
00
0
0
130.6 (C-4 ), 130.6 (C-1 ), 131.5 (C-1 ), 139.9 (C-8), 150.0 (C-4 ), 155.3 (3-C=N), 174.1 (6-
HRMS: 401.1848 [M + H]+ {calcd. 401.1800 for C24H25N4S}.
C=S);
5-(2,4-dichlorophenyl)-N,3-diphenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (4e). Yellowish
powder; yield: 15%; m.p.: 175–177 ◦C; FTIR (ATR) cm−1: 3345 (N–H), 3056 (Csp2–H),
ν
max
2925 (Csp3–H), 1589 (C=N), 1510, 1447 (aromatic C=C), 1398 (C=S); 1H-NMR (500 MHz,
CD3COCD3): δH 3.14 (1H, dd, J = 4.0 Hz and 18.0 Hz, 4
α
-CH
), 4.03 (1H, dd, J = 12.0 Hz and
18.0 Hz, 4
β
-C
H
), 6.25 (1H, dd, J = 4.5 Hz and 12.0 Hz, 5-C
H
), 7.04 (2H, m, H-10 and H-12),
7.21 (3H, m, H-50, H-60 and H-11), 7.36 (3H, m, H-300, H-400 and H-500), 7.40 (1H, s, H-30),
7.59 (2H, dd, J = 1.0 Hz and 8.5 Hz, H-9 and H-13), 7.84 (2H, dd, J = 1.5 Hz and 8.0 Hz,
H-200 and H-600), 9.88 (1H, s, 7-N
H
); 13C-NMR (125 MHz, CD3COCD3): δC 40.7 (4-
C
H2),
H), 124.4 (C-10 and C-12), 124.9 (C-11), 127.3 (C-200 and C-600), 127.6 (C-50 and
C-60), 128.1 (C-9 and C-13), 128.7 (C-300 and C-500), 129.2 (C-30), 130.8 (C-400), 131.1 (C-100),
61.1 (5-
C

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132.0 (C-20), 132.9 (C-40), 139.0 (C-8), 139.6 (C-10), 155.4 (3-C=N), 174.3 (6-
426.0573 [M + H]+ {calcd. 426.0599 for (C22H18Cl2N3S}.
C=S); HRMS:
5-(2-chlorophenyl)-N,3-diphenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (4f). Yellow solid;
◦
◦
2
yield: 34%; m.p.: 59–62 C ([33]: 58–60 C); FTIR (ATR)
H), 2936 (Csp3–H), 1588 (C=N), 1516, 1447 (aromatic C=C), 1400 (C=S); 1H-NMR (500 MHz,
CDCl3): δH 3.19 (1H, dd, J = 4.0 Hz and 18.0 Hz, 4 -C ), 3.99 (1H, dd, J = 11.5 Hz and
18.0 Hz, 4 -C ), 6.50 (1H, dd, J = 3.5 Hz and 12.0 Hz, 5-C
), 7.20 (5H, m, H-30, H-40, H-50,
H-60 and H-11), 7.40 (2H, t, J = 7.5 Hz, H-10 and H-12), 7.46 (3H, m, H-300, H-400 and H-500),
7.79 (2H, d, J = 7.5 Hz, H-9 and H-13), 7.83 (2H, m, H-200 and H-600), 9.37 (1H, s, 7-N
);
H), 124.2 (C-9 and C-13), 125.5 (C-
ν –
max cm−1: 3310 (N–H), 3051 (Csp
α
H
H
β
H
H
13C-NMR (125 MHz, CDCl3): δC 41.5 (4-
CH2), 61.3 (5-C
11), 126.9 (C-200 and C-600), 127.2 (C-50), 128.7 (C-10 and C-12), 128.7 (C-60), 128.9 (C-300
and C-500), 129.4 (C-30), 130.0 (C-40), 130.6 (C-400), 131.1 (C-20), 131.3 (C-100), 138.7 (C-10),
138.8 (C-8), 155.4 (3-C=N), 174.1 (6-
C22H18ClN3SNa}.
C
=S); HRMS: 414.0771 [M + Na]+ {calcd. 414.0808 for
5-(2-methoxyphenyl)-N,3-diphenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (4g). Yellow solid;
◦
3
yield: 22%; m.p.: 193–196 C; FTIR (ATR)
H), 1572 (C=N), 1520, 1445 (aromatic C=C), 1379 (C=S); 1H-NMR (500 MHz, CD3COCD3):
δH 3.27 (1H, dd, J = 3.5 Hz and 18.0 Hz, 4 -C ), 3.82 (3H, s, 14-OCH3), 4.06 (1H, dd,
ν –
max cm−1: 3359 (N–H), 3055 (Csp2–H), 2920 (Csp
α
H
J = 11.5 Hz and 18.0 Hz, 4
β
0-C
H
)0, 6.150(1H, dd, J = 3.5 Hz and 11.5 Hz, 5-C
H), 7.16 (5H, q,
0
J = 8.0 Hz and 17.5 Hz, H-3 , H-4 , H-5 , H-6 and H-11), 7.33 (2H, t, J = 7.5 Hz, H-10 and H-
00
00
00
12), 7.50 (3H, m, H-3 , H-4 and H-5 ), 7.73 (2H, d, J = 7.5 Hz, H-9 and H-13), 7.98 (2H, dd, J
00
00
= 1.5 Hz and 8.0 Hz, H-2 and H-6 ), 9.93 (1H, s, 7-N
H
); 13C-NMR (125 MHz, CD3COCD3):
0
δC 42.3 (4- H2), 55.8 (14-O H3), 63.3 (5-CH), 124.3 (C-9 and C-13), 124.6 (C-11), 125.6 (C-5
C
C
and C-60), 127.2 (C-200 and C-600), 128.0 (C-10 and C-12), 128.7 (C-300 and C-500), 129.1 (C-30
and C-40),130.7 (C-400), 131.3 (C-100), 136.5 (C-20), 139.8 (C-8), 140.1 (C-10), 155.3 (3-
174.2 (6-C=S); HRMS: 410.1319 [M + Na]+ {calcd. 410.1303 for C23H21N3OSNa}.
C=N),
3.1.4. General Procedure for the Preparation of (3,5-disubstituted-4,5-dihydro-1H-
pyrazol-yl) (4-hydroxyphenyl)methanone (5a–g)
The new methanone compounds (5a–g) were synthesized by the cyclo-condensation
of chalcone (3a–g) (1 mmol) with 4-hydroxybenzhydrazide (1.5 mmol) in ethanol (6 mL)
in the presence of NaOH (3 mmol) for 3 to 8 h. The reaction progress was observed on a
TLC plate. After the reaction was over, the reaction mixture was left at room temperature
overnight. The reaction mixture was neutralized by adding 1% v/v of HCl and monitored
by pH paper until the precipitate formed. A solid was formed after crushed ice was added.
The solid was filtered, washed with cold water and dried. The products were purified
by CC using silica gel with the eluent n-hexane:ethyl acetate to give the compounds 5a–g
.
Please refer the Supplementary Data for full spectra.
N-(4-hydroxyphenyl)(5-(4-methylphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)methanone (5a).
Pale yellow powder; yield: 29%; m.p.: 224–228 ◦C; FTIR (ATR)
ν
cm−1: 3209 (O–H),
max
3055 (Csp2–H), 2923 (Csp3–H), 1740 (C=O), 1588 (C=N), 1511, 1439 (aromatic C=C), 1230 (C–
O); 1H-NMR (500 MHz, DMSO-d6): δH 2.27 (3H, s, 14-CH3), 3.12 (1H, dd, J = 5.5 Hz and
18.0 Hz, 4
and 11.5 Hz, 5-C
and H-60), 7.47 (3H, m, H-300, H-400 and H-500), 7.75 (2H, m, H-200 and H-600), 7.86 (2H, d,
J = 8.5 Hz, H-9 and H-11), 10.04 (1H, s, 13-O
); 13C-NMR (125 MHz, DMSO-d6): δC 21.1
(14- H3), 41.6 (4- H2), 61.0 (5-
H), 115.0 (C-8 and C-12), 125.3 (C-7), 126.0 (C-200 and
C-600), 127.1 (C-20 and C-60), 129.3 (C-30 and C-50), 129.7 (C-300 and C-500), 130.7 (C-400),
α
-C
H
), 3.87 (1H, dd, J = 12.0 Hz and 18.0 Hz, 4
H
β-CH), 5.71 (1H, dd, J = 5.0 Hz
), 6.85 (2H, d, J = 9.0 Hz, H-8 and H-12), 7.16 (4H, m, H-20, H-30, H-50
H
C
C
C
00
0
0
131.7 (C-1 ), 132.4 (C-9 and C-11), 136.8 (C-4 ), 140.2 (C-1 ), 155.9 (3-
C=N), 160.4 (10-C-OH),
165.2 (6-C=O); HRMS: 357.1591 [M + H]+ {calcd. 357.1603 for C23H21N2O2}.
N-(4-hydroxyphenyl)(5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)methanone (5b).
◦
Pale yellow powder; yield: 30%; m.p.: 228–232 C; FTIR (ATR)
ν
cm−1: 3211.5 (O–
max
H), 3066 (Csp2–H), 2929 (Csp3–H), 1741 (C=O), 1605 (C=N), 1513, 1437 (aromatic C=C),

Molecules 2021, 26, 2081
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1
1236 (C–O); H-NMR (500 MHz, DMSO-d6): δH 3.14 (1H, dd, J = 5.0 Hz and 18.0 Hz,
-C ), 3.72 (3H, s, 14-OCH3), 3.86 (1H, dd, J = 12.0 Hz and 18.0 Hz, 4 -C ), 5.70 (1H,
dd, J = 5.0 Hz and 11.5 Hz, 5-C
4
α
H
β
H
H
), 6.84 (2H, d, J = 9.0 Hz, H-8 and H-12), 6.90 (2H, d,
J = 8.5 Hz, H-20 and H-60), 7.21 (2H, d, J = 8.5 Hz, H-30 and H-50), 7.47 (3H, m, H-300, H-400
and H-500), 7.76 (2H, m, H-200 and H-600), 7.85 (2H, d, J = 9.0 Hz, H-9 and H-11), 10.03 (1H,
s, 13-OH CH2), 55.6 (14-OCH3), 60.7 (5-CH),
); 13C-NMR (125 MHz, DMSO-d6): δC 41.6 (4-
114.5 (C-20 and C-60), 114.9 (C-8 and C-12), 125.4 (C-7), 127.1 (C-200 and C-600), 127.4 (C-30
and C-50), 129.3 (C-300 and C-500), 130.7 (C-400), 131.8 (C-100), 132.4 (C-9 and C-11), 135.2 (C-
10), 155.0 (3-
C C-OH), 165.2 (6-C
=N), 158.9 (C-40), 160.4 (10- =O); HRMS: 373.1583 [M + H]+
{calcd. 373.1552 for C23H21N2O3}.
N-(4-hydroxyphenyl)(5-(4-chlorolphenyl)-3-phenyl-◦4,5-dihydro-1H-pyrazol-1-yl)methanone (5c).
Yellowish needles; yield: 16%; m.p.: 263–266 C; FTIR (ATR)
ν
cm−1: 3255 (O–H),
max
3070 (Csp2–H), 2926 (Csp3–H), 1738 (C=O), 1572 (C=N), 1516, 1438 (aromatic C=C), 1238 (C–
1
O); H-NMR (
δ
/ppm, 500 MHz, DMSO-d6): δH 3.16 (1H, dd, J = 5.5 Hz and 18.0 Hz,
), 3.89 (1H, dd, J = 12.0 Hz and 18.0 Hz, 4 -C ), 5.76 (1H, dd, J = 5.0 Hz and 11.5 Hz,
), 6.85 (2H, d, J = 9.0 Hz, H-8 and H-12), 7.32 (2H, d, J = 8.5 Hz, H-20 and H-60),
7.41 (2H, d, J = 8.5 Hz, H-30 and H-50), 7.47 (3H, m, H-300, H-400 and H-500), 7.75 (2H, m,
H-200 and H-600), 7.87 (2H, d, J = 8.5 Hz, H-9 and H-11), 10.07 (1H, s, 13-O ); 13C-NMR
H), 114.9 (C-8 and C-12), 125.1 (C-
4α
-C
H
β
H
5-CH
H
(δ/ppm, 125 MHz, DMSO-d6): δC 41.4 (4-CH2), 60.7 (5-C
7), 127.2 (C-200 and C-600), 128.1 (C-20 and C-60), 129.1 (C-30 and C-50), 129.3 (C-300 and
C-500), 130.8 (C-400), 131.6 (C-100), 132.2 (C-40), 132.5 (C-9 and C-11), 142.1 (C-10), 155.1 (3-
C=N), 160.5 (10-
C-OH), 165.3 (6-C
=O); HRMS: 377.1030 [M + H]+ {calcd. 376.0979 for
C22H17ClN2O2}.
N-(4-hydroxyphenyl)(5-(4-dimethylaminophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl) methanone
◦
(
5d). Brownish solid; yield: 9%; m.p.: 236–240 C; FTIR (ATR)
ν
cm−1: 3241.4 (O–
max
H), 3065.5 (Csp2–H), 2929 (Csp3–H), 1720 (C=O), 1604 (C=N), 1511, 1423 (aromatic C=C),
1339 (C–N), 1226 (C–O). 1H-NMR (500 MHz, CDCl3): δH 2.77 (6H, s, 14-CH3 and 15-CH3),
3.09 (1H, dd, J = 5.0 Hz and 17.5 Hz, 4α-CH), 3.62 (1H, dd, J = 11.5 Hz and 18.0 Hz, 4β-CH),
5.65 (1H, dd, J = 5.0 Hz and 11.5 Hz, 5-CH), 6.64 (2H, d, J = 8.5 Hz, H-8 and H-12), 6.71 (2H,
d, J = 9.0 Hz, H-20 and H-60), 7.12 (2H, d, J = 8.5 Hz, H-30 and H-50), 7.32 (3H, m, H-300,
H-400 and H-500), 7.63 (2H, m, H-200 and H-600), 7.85 (2H, d, J = 7.5 Hz, H-9 and H-11);
13C NMR (125 MHz, CDCl3): δC 41.1 (14-
CH3 and 15-CH3), 41.4 (4-CH2), 61.1 (5-CH),
113.6 (C-30 and C-50), 114.7 (C-8 and C-12), 125.4 (C-7), 126.8 (C-20 and C-60), 126.9 (C-200
and C-600), 128.7 (C-300 and C-500), 130.4 (C-400), 130.6 (C-10), 131.5 (C-100), 132.5 (C-9 and C-
11), 149.5(C-40), 155.1 (3-C=N), 159.4 (10-C-OH), 166.4 (6-C=O); HRMS: 386.1891 [M + H]+
{calcd. 386.1869 for C24H24N3O2}.
N-(4-hydroxyphenyl)(5-(2,4-dichlorolphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)methanone (5e).
◦
Pale yellow powder; yield: 17%; m.p.: 237–241 C; FTIR (ATR)
(Csp2–H), 2926 (Csp3–H), 1737 (C=O), 1618 (C=N), 1573, 1427 (aromatic C=C), 1244 (C–O);
1H-NMR (500 MHz, DMSO-d6): δH 3.13 (1H, dd, J = 6.0 Hz and 18.0 Hz, 4
-C ), 3.96 (1H,
dd, J = 12.0 Hz and 18.0 Hz, 4 -C ), 5.95 (1H, dd, J = 5.5 Hz and 12.0 Hz, 5-C ), 6.87 (2H, d,
ν
max cm−1: 3137 (O–H), 3066
α
H
β
H
H
0
J = 8.5 Hz, H-8 and H-12), 7.22 (1H, d, J = 8.0 Hz, H-6 ), 7.40 (1H, dd, J = 2.5 Hz and 8.5 Hz,
H-50), 7.0407 (3H, m, H-300, H-400 and H-500), 7.69 (1H, d, J = 2.5 Hz, H-30), 7.75 (2H, m, H-200
and H-6 ), 7.91 (2H, d, J = 8.5 Hz, H-9 and H-11), 10.11 (1H, s, 13-OH
); 13C NMR (125 MHz,
DMSO-d6): δC 40.1 (4-
CH2), 58.8 (5-C
H), 114.9 (C-8 and C-12), 124.7 (C-7), 127.2 (C-200 and
C-600), 128.4 (C-50 and C-60), 129.3 (C-300 and C-500), 129.6 (C-30), 130.9 (C-400), 131.5 (C-100),
0
0
0
132.4 (C-2 ), 132.6 (C-9 and C-11), 133.0 (C-4 ), 138.9 (C-1 ), 155.3 (3-
C=N), 160.7 (10-C-OH),
165.2 (6-C=O); HRMS: 411.0694 [M + H]+ {calcd. 411.0667 for C22H17Cl2N2O2}.
N-(4-hydroxyphenyl)(5-(2-chlorolphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)methanone (5f).
◦
Pale yellow powder; yield: 15%; m.p.: 216–219 C; FTIR (ATR)
(Csp2–H), 2928 (Csp3–H), 1720 (C=O), 1605 (C=N), 1514, 1431 (aromatic C=C), 1240 (C–O);
1H-NMR (500 MHz, DMSO-d6): δH 3.05 (1H, dd, J = 5.5 Hz and 18.0 Hz, 4
-C ), 3.96 (1H,
dd, J = 12.0 Hz and 18.0 Hz, 4 -C ), 5.96 (1H, dd, J = 5.5 Hz and 12.0 Hz, 5-C ), 6.86
ν
max cm−1: 3125 (O–H), 3066
α
H
β
H
H

Molecules 2021, 26, 2081
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(2H, d, J = 9.0 Hz, H-8 and H-12), 7.18 (1H, m, H-50), 7.30 (2H, m, H-30 and H-60), 7.44 (3H,
m, H-300, H-400 and H-500), 7.50 (1H, m, H-40), 7.72 (2H, m, H-200 and H-600), 7.89 (2H, d,
J = 8.5 Hz, H-9 and H-11), 10.27 (1H, s, 13-O
); 13C NMR (125 0M0 Hz, DMSO-d6): δC 40.4
00 0
(4- H2), 59.1 (5-CH), 115.0 (C-8 and C-12), 124.9 (C-7), 127.1 (C-2 , C-6 and C-5 ), 128.2 (C-
H
C
60), 129.3 (C-300 and C-500), 129.5 (C-30), 130.3 (C-40), 130.9 (C-400), 131.4 (C-100 and C-20),
132.5 (C-9 and C-11), 139.6 (C-10), 155.3 (3-
=N), 160.5 (10- -OH), 165.4 (6- =O); HRMS:
377.1011 [M + H]+ {calcd. 377.1057 for C22H18ClN2O2}.
C
C
C
N-(4-hydroxyphenyl)(5-(2-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)methanone (5g).
◦
Pale yellow powder; yield: 22%; m.p.: 220–223 C; FTIR (ATR)
ν
cm−1: 3198 (O–
max
H), 3029 (Csp2–H), 2925 (Csp3–H), 1738 (C=O), 1603 (C=N), 1513, 1429 (aromatic C=C),
1
1245 (C–O); H-NMR (500 MHz, DMSO-d6): δH 3.00 (1H, dd, J = 5.0 Hz and 18.0 Hz,
4
α
-C
dd, J = 4.5 Hz and 12.0 Hz, 5-C
and H-60), 7.26 (1H, m, H-40), 7.45 (3H, m, H-300, H-400 and H-500), 7.74 (2H, m, H-200 and
H-600), 7.89 (2H, d, J = 9.0 Hz, H-9 and H-11), 10.09 (1H, s, 13-O ); 13C NMR (125 MHz,
DMSO-d6): δC 40.1 (4- H2), 56.1 (14-O H3), 56.9 (5-
H), 111.9 (C-60), 114.9 (C-8 and C-
12), 120.9 (C-50), 125.4 (C-7), 125.9 (C-30), 127.1 (C-200 and C-600), 128.9 (C-40), 129.3 (C-300
H
), 3.84 (1H, dd, J = 10.0 Hz and 19.0 Hz, 4
β-CH), 3.85 (3H, s, 14-OCH3), 5.89 (1H,
H
), 6.87 (3H, m, H-50, H-8 and H-12), 7.05 (2H, m, H-30
H
C
C
C
and C-500), 131.0 (C-10), 130.7 (C-400), 131.8 (C-100), 132.4 (C-9 and C-11), 155.5 (3-
156.4 (C-2 ), 160.4 (10-
C
=N),
0
C
-OH), 165.1 (6-
C
=O); HRMS: 373.1599 [M + H]+ {calcd. 373.1552 for
C23H21N2O3}.
3.1.5. Screening of Anti-Tuberculosis Activity against M. tuberculosis H37Ra
The tetrazolium microplate assay (TEMA) method was performed to evaluate the
anti-mycobacterial activity of the derivatives as described by Caviedes et al. with minor
modifications [34]. The assay was performed in 96-well plates in duplicate and at least
three times independently. The derivatives were dissolved in DMSO and serially diluted
to the desired concentration in complete Middlebrook 7H9 media enriched with albumin
dextrose catalase supplement to reduce the DMSO concentration below 1%. DMSO at this
concentration did not inhibit the growth of M. tuberculosis H37Ra (unreported data). Briefly,
200
of Middlebrook 7H9-ADC media into the wells in columns C to G, rows 2 to 11. Then,
100 L of working solution containing the compounds was added into the wells in columns
B and C, rows 2 to 11, in duplicate. A two-fold serial dilution of the compounds was made
by transferring 100 L from the wells in column C to column D, the content was mixed
well, and the dilutions were continued until column G, where 100 L of the excess medium
from the wells in column G was discarded. Log phase M. tuberculosis H37Ra at a density
of (~1.5
107 CFU/mL) was added and incubated at 37 ◦C with 5% CO2 for 5 days. On
day 5, 50 L of tetrazolium reagent mixture was added to all wells, and the plates were
µL of sterile distilled water was added to the outer wells of the microplate and 100 µL
µ
µ
µ
×
µ
re-incubated for 24 h. The results were read visually the following day. The MIC is defined
as the lowest drug concentration that prevented the color change from yellow to purple.
A small volume of the culture from the 96-well plate was transferred into Middlebrook
7H10 agar media and the plates were incubated at 37 ◦C with 5% CO2 for 28 days. The
MBC is defined as the lowest concentration of compound that did not show any bacterial
colony growth.
3.1.6. Molecular Docking
The three-dimensional coordinates of the reference structure, cytochrome P45014
alpha-sterol demethylase (CYP51) complexed with fluconazole (PDB ID: 1EA1), and the
structure of the experimental control, isoniazid (PDB ID: 2VCF), were fetched by ID
from the Protein Data Bank (PDB) database in UCSF Chimera version 1.14 [35]. Before
docking, the crystal PDBs were processed using the Dock Prep tool, starting with the
removal of water molecules and unrelated hetam (i.e., refer to any ions molecules and any
atoms that not belong to the protein) followed by separation of the receptor and inhibitor
from the complexes into individual structures and finally the minimization of individual

Molecules 2021, 26, 2081
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structures by steepest descent steps [36]. The compound 4a was built using ChemDraw
and subsequently converted from cdx format to PDB.
To recognize the binding sites in CYP51, the grid size was set to
−
18,
−
2.6 and 63 along
the X-, Y- and Z-axes, respectively, with a 0.375 Å grid spacing. The grid center along the X-,
Y- and Z-axes was set to 76, 72, and 83 Å. The AutoDock Vina tool was then used to calculate
possible bindings and energies [37]. All poses were combined using UCSF Chimera version
1.14 and further labelled by the GNU Image Manipulation program. The interaction
modes between CYP51 and compound
4 for each pose were further analyzed using Biovia
Discovery Studio Visualizer Client 2020 (Dassault Systèmes BIOVIA, Discovery Studio
Modeling Environment, Release 2017, San Diego: Dassault Systèmes, 2016).
4. Conclusions
In conclusion, two series of pyrazoline derivatives (4a–g and 5a–g) were prepared
by cyclocondensation reaction between chalcone derivatives (3a–g) with a different type
of 4-phenyl-3-thiosemicarbazide/4-hydroxybenzhydrazide in the presence of NaOH as a
catalyst. Nine out of fourteen new compounds synthesized from two series of experiments
were elucidated for structure using FTIR, 1D-NMR, 2D-NMR and HRMS. All seven syn-
thesized compounds (5a–g) from the second series are new compounds. The synthesized
compounds were evaluated for their antituberculosis activity by in vitro study against My-
cobacterium tuberculosis H37Ra. The tested compounds displayed some degree of inhibition,
with MIC values ranging from 17
pound 4a, which had the lowest MIC value of 17
5a 5c 5d 5e 5f and 5g), except for 5b from the second series, exhibited moderate activity
against M. tuberculosis with MICs of 70 M, 66 M, 65 M, 61 M, 66 M and 134 M,
respectively. In the MBC assay, compound 4a showed the strongest killing effect towards
M. tuberculosis H37Ra with a value of 34 M. Molecular docking studies demonstrated that
µM to 535
µM. Significant activity was found for com-
µM. Meanwhile, pyrazoline derivatives
(
,
,
,
,
µ
µ
µ
µ
µ
µ
µ
compound 4a has the best binding capability towards the cytochrome P450 14 alpha-sterol
demethylase (CYP51) complex.
Supplementary Materials: The following are available online. Figures S1–S80: spectrums for com-
pounds 4a–5g.
Author Contributions: Conceptualization, H.O. and M.N.A.; methodology, K.T.W., H.O., M.N.A.
and T.P.; software, M.T.C.O.; validation, M.N.A. and T.P.; formal analysis, K.T.W., T.P. and M.T.C.O.;
investigation, K.T.W., T.P. and U.S.; resources, H.O. and M.N.A.; data curation, K.T.W.; writing—
original draft preparation, K.T.W. and M.N.A.; writing—review and editing, M.T.C.O., H.O., M.N.A.,
U.S. and T.P.; visualization, K.T.W. and M.N.A.; supervision, H.O., M.N.A. and T.P.; project adminis-
tration, H.O. and M.N.A.; funding acquisition, H.O. and M.N.A.; All authors have read and agreed
to the published version of the manuscript.
Funding: This project had been supported by USM RUI grant (1001/PKIMIA/8011072).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or supplementary material. The
data presented in this study are available in this article or supplementary material.
Acknowledgments: The authors would like to acknowledge the financial support from the Ministry of
Higher Education of Malaysia (MyBrain15 Scheme – MyPhD) and USM RUI grant (1001/PKIMIA/8011072).
Conflicts of Interest: The authors declare no conflict of interest.
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