Design and synthesis of novel pyrazole-phenyl semicarbazone derivatives as potential α-glucosidase inhibitor: Kinetics and molecular dynamics simulation study

Article abstract of DOI:10.1016/j.ijbiomac.2020.10.263

A series of novel pyrazole-phenyl semicarbazone derivatives were designed, synthesized, and screened for in vitro α-glucosidase inhibitory activity. Given the importance of hydrogen bonding in promoting the α-glucosidase inhibitory activity, pharmacophore modification was established. The docking results rationalized the idea of the design. All newly synthesized compounds exhibited excellent in vitro yeast α-glucosidase inhibition (IC₅₀ values in the range of 65.1–695.0 μM) even much more potent than standard drug acarbose (IC₅₀ = 750.0 μM). Among them, compounds 8o displayed the most potent α-glucosidase inhibitory activity (IC₅₀ = 65.1 ± 0.3 μM). Kinetic study of compound 8o revealed that it inhibited α-glucosidase in a competitive mode (Ki = 87.0 μM). Limited SAR suggested that electronic properties of substitutions have little effect on inhibitory potential of compounds. Cytotoxic studies demonstrated that the active compounds (8o, 8k, 8p, 8l, 8i, and 8a) compounds are also non-cytotoxic. The binding modes of the most potent compounds 8o, 8k, 8p, 8l and 8i was studied through in silico docking studies. Molecular dynamic simulations have been performed in order to explain the dynamic behavior and structural changes of the systems by the calculation of the root mean square deviation (RMSD) and root mean square fluctuation (RMSF).

Full text of DOI:10.1016/j.ijbiomac.2020.10.263

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Design and synthesis of novel pyrazole-phenyl semicarbazone
derivatives as potential α-glucosidase inhibitor: Kinetics and
molecular dynamics simulation study
Fateme Azimi, Jahan B. Ghasemi, Homa Azizian, Mohammad
Najafi, Mohammad Ali Faramarzi, Lotfollah Saghaei, Hojjat
Sadeghi-aliabadi, Bagher Larijani, Farshid Hassanzadeh,
Mohammad Mahdavi
PII:
S0141-8130(20)34916-3
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.10.263
BIOMAC 17153
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
25 September 2020
29 October 2020
31 October 2020
Please cite this article as: F. Azimi, J.B. Ghasemi, H. Azizian, et al., Design and synthesis
of novel pyrazole-phenyl semicarbazone derivatives as potential α-glucosidase inhibitor:
Kinetics and molecular dynamics simulation study, International Journal of Biological
Macromolecules (2018), https://doi.org/10.1016/j.ijbiomac.2020.10.263
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Design and synthesis of novel pyrazole-phenyl semicarbazone derivatives as potential α-
glucosidase inhibitor: Kinetics and molecular dynamics simulation study
a
b
c
d
Fateme Azimi , Jahan B. Ghasemi , Homa Azizian , Mohammad Najafi , Mohammad Ali
Faramarzi , Lotfollah Saghaei a,*1, Hojjat Sadeghi-aliabadi , Bagher Larijani , Farshid
e
a
f
Hassanzadeh a,*, Mohammad Mahdavi f,*
a Department of Medicinal Chemistry, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran
b Department of Chemistry, Faculty of Sciences, University of Tehran, Tehran, Iran
c Department of Medicinal Chemistry, School of Pharmacy-International Campus, Iran University of Medical
Science, Tehran, Iran
d Department of Chemistry, Isfahan University of Technology, Isfahan, Iran
e Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran
University of Medical Sciences, Tehran, Iran
f Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Research Institute, Tehran
University of Medical Sciences, Tehran, Iran
Abstract
A series of novel pyrazole-phenyl semicarbazone derivatives were designed, synthesized, and
screened for in vitro α-glucosidase inhibitory activity. Given the importance of hydrogen
bonding in promoting the α-glucosidase inhibitory activity, pharmacophore modification was
established. The docking results rationalized the idea of the design. All newly synthesized
compounds exhibited excellent in vitro yeast α-glucosidase inhibition (IC50 values in the range of
65.1 - 695.0 μM) even much more potent than standard drug acarbose (IC50=750.0 μM). Among
them, compounds 8o displayed the most potent α-glucosidase inhibitory activity (IC50 = 65.1 ±
0.3 µM). Kinetic study of compound 8o revealed that it inhibited α-glucosidase in a competitive
mode (Ki = 87.0 μM). Limited SAR suggested that electronic properties of substitutions have
little effect on inhibitory potential of compounds. Cytotoxic studies demonstrated that the active
compounds (8o, 8k, 8p, 8l, 8i, and 8a) compounds are also non-cytotoxic. The binding modes of
the most potent compounds 8o, 8k, 8p, 8l and 8i was studied through in silico docking studies.
Molecular dynamic simulations have been performed in order to explain the dynamic behavior
*
Corresponding authors.
E-mail addresses: saghaie@pharm.mui.ac.ir (L. Saghaie), hassanzadeh@pharm.mui.ac.ir (F. Hassanzadeh), momahdavi@tums.ac.ir (M.
Mahdavi)

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and structural changes of the systems by the calculation of the root mean square deviation
(RMSD) and root mean square fluctuation (RMSF).
Keywords:
α-Glucosidase inhibitor; Synthesise; Pyrezole; Structure–activity relationship; Enzyme kinetic
study; Molecular dynamic simulation
1. Introduction
Diabetes mellitus (DM) is a chronic metabolic disorder resulting from defects in insulin action
(type 2 diabetes), insulin secretion (type 1 diabetes), or both, which affects the metabolic
functions of the endocrine system. Diabetes is undoubtedly one of the major public health
problems worldwide [1, 2]. According to WHO reports, more than 420 million people are
suffering from diabetes and this number is expected to be more than 642 million by 2040 [2, 3].
Type-2 diabetes is the most common type and accounts for approximately 80-90% of all diabetic
cases, which is responsible for around 5% death globally [4-6]. This dysfunction leads to high
blood glucose concentration or hyperglycemia. Prolonged hyperglycemia causes a wide variety
of critical complications, including diabetic neuropathy [7, 8], cancer [9, 10], retinopathy [11],
stroke [12, 13], amputations [14, 15], microangiopathy, and cardiovascular diseases [16-18].
One of the critical strategies to control diabetes complications is by managing the blood
glucose level [19]. α-Glucosidase inhibitors prevent the hydrolysis α-glucosidic bond of complex
carbohydrates and delay monosaccharide (α-D-glucose) absorption, which is mainly responsible
to cause hyperglycemia [20]. Thus, α-glucosidase inhibitors have been considered as a most
attractive therapeutic target for drug discovery in the treatment of type 2 diabetes [21-23].
Beside, α-glucosidase is biocatalyst involved in a series of relevant processes like glycolipid and
glycoprotein metabolic routes, cell-to-cell signaling, and virus recognition [24, 25]. Therefore,
glucosidase inhibitors have attracted plenty of interest by the pharmaceutical industry as novel
agents to treat other carbohydrate mediated diseases including viral infections, hepatitis, cancer,
pompe disease, and other degenerative diseases such as nojirimycin and castanospermine [25-
30]. Currently, glycosidic based α-glucosidase inhibitors such as acarbose (Glucobay), miglitol
(Glyset), and voglibose (Volix, Basen) are clinically used in the treatment of type-2 diabetes

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mellitus [31]. Although these medications have a certain therapeutic effect on diabetes, they are
associated with serious adverse. As the result, they have to be used in combination with other
anti-diabetic agents to improve the effectiveness [32]. On the other hand, the synthesis of other
similar derivatives of mentioned α- glucosidase inhibitors is tedious and need complicated multi-
step procedures due to the presence of sugar moieties in their structures [33]. So, it is still an
appealing and interesting area of medicinal chemistry research to discovery and develop efficient
small molecules capable of possessing potent α-glucosidase inhibitory activities.
The heterocyclic scaffolds have attracted considerable attention of medicinal chemists due to
their exceptional chemical and versatile biological profiles. In this category, pyrazole and their
derivatives represent one of the key structural units in numerous medicinally important
compounds with various pharmacological effects including anti-inflammatory, analgesic,
antimicrobial, neuroprotective, antihypercholestrolemia, antimalarial, anti-Alzheimer,
antihypertensive, antitubercular, antiviral, and antihyperglycaemic activities [34-36]. Pyrazole
scaffold has also reported to show selective and potent inhibitory activity against lung, colon,
renal, prostate, and breast cancer cells [37, 38]. Furthermore, pyrazole derivatives possess the
LRRK2 inhibitory activity to identify new drugs to treat Parkinson's disease [39]. Several
pyrazole containing drug molecules with diverse therapeutic activities such as Celecoxib,
sildenafil, Crizotinib, Surinabant, Rimonabant, and Pyrazofurin are now available in the market
[34]. Historically, different substituted pyrazole had been reported as in vivo anti hypoglycemic
agent [40]. Also, recent studies have confirmed that several entities of the pyrazole have been
possessed antidiabetic [41-43] and hypoglycemic [44-46] activity (Fig. 1). In addition to the
mentioned successful studies, significant metabolic stability and pharmacological efficiency of
pyrazole derivatives as antidiabetic agents and approved the ‘‘Teneligliptin”, a pyrazole motif
containing antidiabetic drug, encouraged us to further study on pyrazole scaffolds[47].
Benzohydrazides are important classes of compounds due to its wide range of activities,
including antiglycation, antioxidant, antileishmanial, antitumor, antibacterial, and anticonvulsant
[48-53]. In recent years, several compounds possessing benzohydrazide scaffolds with high α-
glucosidase inhibitory activity have been reported [25, 54]. for instance, benzothiazole hybrids
having benzohydrazide moiety (Fig. 2 compound C) were reported by Taha et al. as a potent α-
glucosidase inhibitor (IC50 = 5.31 ± 0.03 µM comparing with acarbose, 906 ± 6.3 µM) [54].

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Molecular hybridization has been a strong tool to design and develop new biologically active
compounds with improved potency. In this work, in continuation of our efforts for developing
new α-glucosidase inhibitors using molecular hybridization and endeavoring to extend the
chemical space, we designed a new scaffold in order to identify lead candidates for more
advanced research in future [55-57]. Steps for the rational design of new hybrid are depicted in
Fig. 3 Enormous importance of hydrogen bonding in promoting the α-glucosidase inhibitory
activity has inspired the modification of benzohydrazide to the phenyl semicarbazide moiety,
which introduced extra nitrogen atom into the structure as hydrogen bond donor (Fig. 2
compound D). Modifications were implemented in the designed hybrid in order to enhance the
key factors required to promote the glucosidase inhibitory such as p-stacking, hydrogen bonding,
and hydrophobicity [58, 59]. Hydrazone linker was included in the design of the new hybrid as
part of the initial design. It was hypothesized that the addition of the hydrazine linkage could
substantially decrease the entropic penalty for the formation of the enzyme-inhibitor complex
(Fig. 2 compound B) [25]. Accordingly, our research team designed and synthesized a new
hybrid moiety that incorporated pyrazole, phenyl semicarbazide, and hydrazone in a single
structural entity and evaluated them against α-glucosidase enzyme for the first time. The docking
results rationalized the idea of conjugation of pyrazole with arylsemicarbazone moiety. The
designed hybrid packed in the catalytic hydrophobic pocket of active site whereas nitrogen and
oxygen atoms of the semicarbazide formed hydrogen bonds with the enzyme (Fig. 2E).
Apart from in vitro assessment of target compounds, in silico study and mechanism
underlying enzymatic inhibition were also investigated to have a better understanding of the
interactions of the title compounds with α-glucosidase. Moreover, molecular dynamic simulation
was performed to explain of the dynamic behavior and structural changes of the system.
Please insert Fig. 1 about here
Please insert Fig. 2 about here
2. Result and discussion
2.1. Chemistry

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The pathway for the synthesis of pyrazole-phenyl semicarbazone hybrids 8a-p has been
depicted in Scheme 1. Different substituted acetophenones 1 reacted with phenyl hydrazine or 4-
methyl phenyl hydrazine 2 in the presence of sulfuric acid in absolute ethanol to afford the
hydrazone intermediates 3a-p. The synthesized hydrazone intermediates 3a-p then undergo a
Vilsmeier–Hack reaction in the presence of DMF and POCl3 to form pyrazole carbaldehydes 4a-
p. On the other hand, 4-phenyl semicarbazide 7 was constructed according to the known urea
preparation method [60]. Aniline 5 was treated with sodium cyanate in the presence of glacial
acetic acid to yield phenyl urea 6. The 4-phenyl semicarbazide 7 was obtained upon refluxing
phenyl urea 6 with hydrazine hydrate in ethanol in the presence of sodium hydroxide. Finally,
pyrazole-phenyl semicarbazone hybrids 8a-p were synthesized by condensing equimolar
amounts of 4-phenyl semicarbazide with different aldehydes in the presence of catalytic amount
of acetic acid [61, 62].
Please insert Scheme 1 about here
2.2. In vitro α-glucosidase inhibitory activity and structure–activity relationships (SAR)
study
All synthesized new hybrids 8a-p were screened for their in vitro α-glucosidase inhibitory
potential. The IC50 values were determined and acarbose as marketed α-glucosidase inhibitor was
used for comparison purposes. In order to optimize the anti-α-glucosidase activity and better
understanding of structure-activity relationship, substituent acetophenone , phenylhydrazine, and
4-methyl phenylhydrazine were used to synthesize various derivatives of the designed scaffold.
The in vitro α-glucosidase inhibitory activity of tested compounds as IC50 values are summarized
in Table 1. Obtained results revealed that all the target compounds had excellent inhibitory
activity in the range of IC50 =65.1 ± 0.3 - 695.0 ± 9.5 µM and all of them were more potent than
acarbose (IC50 = 750.0 ± 10. 0 µM). High inhibitory potential of designed compound suggested
that all structural features such as pyrazole core, hydrazone linkage, and semicarbazide moiety
are cordially playing their part in demonstrating the inhibitory potential; however, the variation
of the substituents in the phenyl rings on the pyrazole moiety is responsible for variation in the
inhibitory activity. Based on substituents in R2, synthesized compounds can be categorized into
two categories and SAR was interpreted based on substitution in R1 in each series (Table 1).

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Compound 8a-8i of “Category A” (IC50 = 92.0 ± 0.7- 695.0 ± 9.5 µM), having no substitution on
R2. In this category, compound 8i (IC50 = 92.0 ± 0.7 µM) with para trifluoromethyl substitution
was found to be the most active compound with more than eight-fold higher activity than
acarbose. Removal of trifluoromethyl group of compound 8i was associated with a slight
decrease in activity as observed in the second most active analog in this series 8a (IC50 = 96.3 ±
0.8 µM), whereas replacement of this group with other substituents (8b-8h), resulted in a
moderate to significant decrease in the inhibitory activity in the range of IC50=157.3 ± 1.9-695.0
± 9.5 µM. Despite the decrease in the activity of remaining compounds, their inhibitory potential
was around 1–7 fold more than acarbose. Therefore, the activity pattern showed that other groups
are less participating in the activity compared to the trifluoromethyl group. Interestingly,
compounds 8f (IC50 = 130.7 ± 1.5 µM) and 8g (IC50 = 134.5 ± 1.6 µM) with nitro (electron
withdrawing) and hydroxyl (electron donating) substituents on the para position of phenyl,
respectively, showed almost similar inhibitory activity against α- glucosidase. These compounds
were found to be the third most active in this category and it seemed that the electronic
properties of substitutions did not have substantial effect on inhibitory potential. Replacing
hydroxyl with methyl also decreased activity in compound 8b (IC50 = 157.3 ± 1.9 µM), while
methylation of the hydroxyl group in compound 8g further decreased the activity as observed in
compound 8c (IC50 = 182.6 ± 2.3 µM). Halogenated derivatives also displayed variable
inhibition against α-glucosidase. By substitution of Cl or Br at the para positions (compounds 8d
and 8e), identical inhibitory potential was observed, however, lower than compound 8i with CF3
substitution. Replacement of Cl or Br by F resulted in a significant deterioration in activity in
compound 8h. As a result, the IC50 value of compound 8c reached 695.0 ± 9.5 µM, which was
more than four-fold lower than 8d or 8e. The interesting point about halogenated compounds was
that 4-CF3 derivative was the most potent analog, whereas 4-fluoro derivative was the least
active molecule of all halo-substituted compounds. These results suggested that the inhibitory
activity of halogenated compounds may be affected by the size of substitution. Compounds 8j-8p
belong to Category-B having methyl group as R2 showed their inhibitory activity in the range of
IC50 = 65.1 ± 0.3- 506.2 ± 8.5 µM. In this group, compound 8o with a 4-nitro substituent showed
the most inhibitory activity (IC50 = 65.1 ± 0.3 µM), which was approximately 12 times more
potent than acarbose (IC50 = 750.0 ± 10.0 µM). This compound also was found to be the most
active derivative among all of the synthesized compounds. On the other hand, compound 8o

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showed two-fold higher activity compared to its counterpart 8f (IC50 = 130.7 ± 1.5 µM) in
Category A. Compound 8k (IC50 = 77.0 ± 0.5 µM) with methyl substitution was identified as the
second most potent derivative in this series and also among all synthesized compounds. This
compound was also showed two-fold higher activity in comparison to its counterpart 8b in
Category A. Therefore, as emphasized in the Category A, it seems that electronic properties have
no significant effect on the inhibitory activity. The introduction of a hydroxyl and methoxy
group as R1 in compounds 8p (IC50 = 86.3 ± 0.6 µM) and 8l (IC50 = 89.6 ± 0.7 µM), respectively,
led to a slight reduction in inhibitory activity. These compounds were the third most active
analogs among all screened compounds and compared with their counterparts 8g and 8c in
Category A, about 1.5-2 fold increased activity were observed. In the case of halogenated
compounds in this series, the replacement of the hydroxyl or methoxy groups with an electron
withdrawing chloro group in compound 8m (IC50 = 116.7 ± 1.3 µM) reduced the inhibitory
efficacy. On the other hand, 8n was more active than its counterpart 8e in category A. Switching
from chloro to bromo group in compound 8n (IC50 = 506.2 ± 8.5 µM) led to a dramatic decrease
in the inhibitory activity which, in contrast to Category A, reduced its activity by more than four-
fold comparison to chlorinated compound 8m. It is also interesting to note that the unsubstituted
derivative 8j (IC50 = 387.7 ± 5.0 µM) in series B demonstrated less inhibitory activity and its
activity was four-fold lower than its counterpart in Category A (8a).
According to the comparison of IC50 values of derivatives in two groups, except analogs 8j
and 8n (un-substituted and 4-bromo derivatives, respectively), all other derivatives in Category B
were more active than their counterparts in the first category. Thus, based on the limited
structure-activity relationship, it can be concluded that the presence of a methyl group in R2
played a more effective role in in vitro α-glucosidase inhibitory activity compared with effect of
the change of substituent in the R1 position. It seems that the presence of the methyl group led to
a better fit of the phenyl group in the pocket of α-glucosidase enzyme. In silico molecular
modeling was also performed in order to obtain a better understanding of the effect of different
structural motifs of these novel derivatives in their binding modes with the active site of α-
glucosidase enzyme.
Please insert Table 1 about here

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2.3. Inhibition kinetics of α-glucosidase
In order to gain further insight into the inhibition mechanism of the synthesized compounds
on α-glucosidase, kinetic study was performed on the most potent compound 8o according to our
previous report [63-66]. The mode of inhibition was determined by Lineweaver–Burk plots and
then Ki was calculated with secondary re-plot of Lineweaver–Burk plots. The analysis of
obtained Lineweaver–Burk plots showed that, with increasing concentrations of compound 8o,
Vmax value remained unchanged while the Km increased. It indicated that compound 8o was a
competitive inhibitor and competed with the substrate for binding to the active site of α-
glucosidase (Fig. 3a). Moreover, secondary re-plot of mentioned Lineweaver–Burk plots against
the different concentrations of compound 8o provided an estimate of the inhibition constant, Ki
values of 87.0 µM for compound 8o (Fig. 3b).
Similarly, recently, new bis-azo-containing Schiff base possessed nitro groups was
synthesized which also competitively inhibited the α-glucosidase [67]. Interestingly, to the best
of our knowledge, none of the reported pyrazole-based inhibitor competitively inhibited the
enzyme activity.
Please insert Fig. 3 about here
2.4. Cytotoxic activity
In order to determine the toxicities of the most potent compounds (8o, 8k, 8p, 8l, 8i, and 8a),
The MTT assay was conducted on normal 3T3 cell line. The results of cytotoxic activity were
expressed as the IC50 (µM) and outlined in Table 2. Results revealed that the selected compounds
depicted no cytotoxic activity against 3T3 cells with IC50 value > 50 µM, showing that these
compounds have a high level of safety profile.
[Please insert Table 2 about here]
2.5. Computational methods
2.5.1. Homology modeling

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Since the crystallographic structure of the protein has not been obtained yet, the homology
modeling technique was used to generate three-dimensional (3D) structure of the protein.
Molecular docking was performed by modeled 3D structure. The primary sequence of
Saccharomyces cerevisiae α-glucosidase was retrieved from the Uniprot in FASTA format
(Access code P53341) and searched in Protein BLAST against the PDB structures to get
template [68]. As a result, the X-ray crystal structure of α-D-glucose bound isomaltase from S.
cerevisiae (PDB ID: 3A4A) was chosen as a template for the homology modeling due to its
highest sequence identity among the other templates [69]. Before the modeling process,
alignment of the target and template sequences were performed using the multiple sequence
alignment method. The selected template showed sequence identity and similarity of 71.4 % and
86.7 %, respectively (Fig. S1 in Supplementary). The 3D structural model of α-glucosidase for S.
cerevisiae was obtained using the “Build Homology Models” module in Discovery Studio 4.1. A
set of 10 intermediate models were generated. The best model was selected based on the lowest
PDF Total Energy (3270.6404) and DOPE Scores (-73110.257813) and evaluated for further
validation (see details in Supplementary Data).
2.5.2. Molecular docking study
Docking simulation was employed to identify the possible binding mode of the most active
inhibitors in the active site of the developed homology model of α-glucosidase by employing the
GOLD 5.3.0 tool. Before docking and analysis of the binding mode, docking method was
validated by redocking the co-crystallized acarbose and α-D-glucopyranose in binding site of α-
glucosidase from Sugar beet (PDB code: 3W37) and isomaltase from Baker's yeast (PDB code:
3A4A), respectively. The RMSD value between best docked and actual pose of acarbose and α-
D-glucopyranose were found to be 1.12 Å and 0.96 Å, respectively, which indicated the
reliability of the method (Fig. S2 in Supplementary).
Acarbose as standard drug and the synthesized compounds were docked in the active site of
modeled α-glucosidase. The docking results revealed that all the synthetic compounds showed
significant in silico inhibitory activities and exhibited prominent gold fitness docking scores
ranging from 36.72 to 62.78 that showed the best accommodation of all the compounds in the
active site of the enzyme. Investigation of ligand-protein interactions of synthetic derivatives

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indicated that the compounds in which the substituent has either EWG or EDG showed good in
silico inhibition. Analysis of the predicted binding conformations showed that diphenyl pyrazole
moiety could be completely inserted into the hydrophobic site pocket through pi-pi or pi-cation
interactions with residues of the enzyme. Besides, carbonyl oxygen atoms and NH of hydrazone
linker form hydrogen bonds to improve the docking affinity with α-glucosidase. The detailed
binding mode of acarbose revealed that this drug formed hydrogen bonding interactions with
residues Asn241, Gln181, His348, Glu276, Asp68 and hydrophobic interaction with residues
Ala278 and Phe157 (Fig. 4b). For more investigation, detailed binding modes of most potent
compounds 8o, 8k, 8p, 8l, and 8i were also shown in Fig. 4c-f. Compound 8o is the most potent
compound in all of the synthesized compounds and well fitted into the binding cavity of α-
glucosidase (Fig. 4c). Several hydrophobic interactions were observed between 1, 3-diphenyl
pyrazole moiety and the active site residues, viz., Arg439, Tyr71, Phe157, Phe177, and Ala278
which stabilizes the binding of compound 8o in the active site of the enzyme. Asp68 formed a
strong hydrogen bond with oxygen of the nitro substituent. Whereas, residues Ala278 and
His279 formed a pi-alkyl interaction with methyl substituent on the phenyl ring. This interaction
seems to justify partial high potency of Category B compared to Category A. Asp408 formed H-
acceptor interaction with nitrogen atom of hydrazone linker and an H-donor interactions were
observed between Arg439 and Tyr313 and carbonyl oxygen atoms which further stabilized the
enzyme-inhibitor complex. Moreover, Arg312 and Arg349 involved in aren-alkyl interaction
with the phenyl ring of the semicarbazide moiety and a pi-hydrogen interaction was also found
between Arg312 and pi system of the ring. Compound 8k, the second most active compound,
showed considerable hydrogen bonding interactions with the active site residues. The binding
mode of compound 8k is shown in Fig. 4d. Asp349 formed hydrogen acceptor interaction with
the NH groups of semicarbazide moiety while Arg212 and Asp214 formed H-donor interactions
with the oxygen atom of the carbonyl group. In addition, compound 8k also established some
important hydrophobic interactions with Ala278, Phe157, Phe300, Arg 312, Arg 439, Tyr313,
and Leu437. Furthermore, 4-methyl substituent in R1 position established pi-alkyl interactions
with Ala278, Phe157, and His245. The third and the fourth most potent compounds 8p (4-
hydroxy on R1 position) and 8l (4-methoxy on R1 position) showed almost similar interaction
with the active site, as shown in Fig. 4e. This observation confirmed by the obtained results of in
vitro assay. Comparing the binding mode of compound 8p and 8l, it was revealed that the lone

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pair of oxygen atom, in both hydroxyl and methoxy substitutions, interacted with Tyr71 while in
compound 8l Asp68 formed an H-acceptor interaction with the CH3 of methoxy substituent.
Compound 8i is the fourth most active among all of the synthesized compounds and most active
in Category A. Similar to other compounds, 1, 3-diphenyl pyrazole moiety is positioned in a
hydrophobic pocket formed by Phe177, Phe157, Ala278, Glu276, Leu218, and Ala278 while
CF3 substituent interacted with residues As68, Arg439 and Tyr71. Gln350 formed an H-acceptor
interaction with the NH of semicarbazide moiety and Arg312 displayed a hydrogen donor
interaction with the carbonyl of the same moiety. Also, terminal phenyl group showed pi-alkyl
interaction with Arg439 (Fig. 4f).
Comparing the binding mode of A and B categories indicated that both series showed
interactions with almost similar active site residues. Based on docking results, more biological
activity of Category B might be due to the interaction with more active site residues as compare
to Category A. The slight difference in the binding modes of compounds in A and B series was
in good agreement with those results obtained in in vitro assay. The molecular docking study
showed that all synthesized compounds had a good binding affinity towards the binding site of α-
glucosidase enzyme. The higher binding affinity for these compounds is presumably attributed to
strong binding contributed by the higher number of stable hydrogen bonds and hydrophobic
interactions between the reactive pharmacophore and amino acids in the active site of the
enzyme. As previous studies have confirmed, hydrogen bonding and hydrophobic interactions
may be the important factors affecting the enhancement of inhibitory activity [22].
The minimum energy of interaction between the ligand and active site of α-glucosidase
enzyme was obtained as gold fitness as a scoring function. The calculated docking score of most
potent compounds was significantly more than that of acarbose (35.71). The compounds 8o, 8k
and 8l displayed best docking scores with values of 62.78, 61.24 and 58.59 which are highly
correlated with in vitro α- glucosidase inhibition activity.
Please insert Fig. 4 about here
2.5.3. Molecular dynamic investigation

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Additionally MD simulation was performed in order to understand the effect of the most
active compound over the enzyme active site. For this purpose, the structural perturbations
incurred by the most potent compound (compounds 8o) have been investigated through study the
RMSD and RMSF and its effect on the active site environment in comparison to acarbose as α-
glucosidase standard inhibitor.
Root mean square deviation (RMSD) of the enzymes’s Cα was analyzed over 20 ns MD
simulation in order to study the stability of the protein-ligand complex. It showed that the RMSD
of the apo α-glucosidase is lower than the two enzyme complexes and its values decreased after
about 5 ns and became more stable at 1.5 Å with lower fluctuation towards the end of the
simulation. This means that the RMSD of the bounded state enzymes deviate from the initial
structure of apo enzyme in the early part of the simulation as a result of structural flexibility. It is
expected that compound 8o had higher RMSD than acarbose for its higher number of rotatable
bond and flexibility which makes it more deviate from the initial structure. α-glucosidase
complexed with compound 8o and acarbose maintained an overall stability for the most part of
simulation time with fluctuation stabilizing at an average of 2.63 Å and 2.1 Å, respectively (Fig.
S3 in Supplementary). Such observation indicated that the employed simulation time was enough
to obtain an equilibrium structure over the simulation time. Thus, the structures at the MD
equilibrium state was used to investigate the structural specificity of the ligand-protein
complexes.
The RMSF, which indicated the flexibility of the protein structure, was referred to the
fluctuation of the Cα atom from its average position throughout the simulation time. Comparing
RMSF values of α-glucosidase-compound complexes show that the residues of the conserved
region I (106-111), II (210-28), III (276-279), and IV (344-349) would have the same RMSF
value in α-glucosidase-acarbose and α-glucosidase-8o complexes (Fig. 5). Based on the observed
result of RMSF plot, although α-glucosidase-acarbose complex showed residues at the entrance
region loop covering the active site (310-315) have lower RMSF value than in α-glucosidase-8o
complex (green box), the other lid loop covering the active site consists of residues 230-236 [70]
showed significantly lower RMSF value in α-glucosidase-8o complex (pink box) rather than α-
glucosidase-acarbose complex.

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Fig. 6a and 6b represent the detailed orientation and interactions that occurred more than 30%
of the simulation time during the equilibrated phase over α-glucosidase complexed with
compound 8o. The interaction analysis depicted that compound 8o oriented horizontally at the
bottom of the active site and stabilized the enzyme domains by interacting with Tyr71, Arg443,
Asp349 and Arg439 from domain A and Phe158 and Phe177 from domain B (Fig. 6a). On the
other hand, acarbose oriented vertically and formed non-binding interaction with the Phe311,
Asn241, Arg439, Asp68, His245, Asp349, and Asp214 which belong to the domain A of the
enzyme (Fig. 6c). This observation may propose the contribution to the higher α-glucosidase
inhibition activity.
Comparing the MD simulation of compound 8o and acarbose revealed a long lasting non-
binding interaction with Asp439, Asp68 and Arg439 proposed to have a significant role on the
inhibition activity of the mentioned compounds (Fig. 6d).
Fig. 6b represented compound 8o formed H-bond interaction through the semicarbazone
linker with the conserved catalytic residue Asp349 and Glu304 for about 96% of MD simulation
time which is similar to the behavior of the NH2 group in the acarbose. Moreover, the para nitro
phenyl group oriented toward the interface of domain A and B through strong ionic interaction of
the nitro group with Asp68 and Arg439, π-cation and π-π hydrophobic interaction with Phe177
and Arg443 at the wall of the active site, and Tyr71 at the bottom of the active site. It was
obvious from the MD study that H-bonding and hydrophobic interactions had critical role in
stabilizing compound 8o at the bottom of the active site during the simulation time and caused α-
glucosidase inhibition activity.
In addition to the interaction analysis, the Prime/MM-GBSA module was used to estimate the
strengths of interactions between the ligand-protein complexes which generated by the clustering
method. ΔGbind of α-glucosidase/compound 8o complex and α-glucosidase/acarbose complex
were estimated to be -74.83 and -62.49 kcal/mol, respectively, revealing stronger binding
interaction of compound 8o than acarbose which also supported by experimental assay.
Please insert Fig. 5 about here
Please insert Fig. 6 about here

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2.6. Conclusion
In conclusion, a novel series of pyrazole-phenyl semicarbazone hybrids were designed and
synthesized as potential α-glucosidase inhibitors. The docking results rationalized the idea of
hybridization. As a result, designed hybrid packed in the catalytic hydrophobic pocket of the
active site while nitrogen and oxygen atoms of the semicarbazide formed hydrogen bonds
with the enzyme. It is worth mentioning that inhibitory activity of all new designed
compounds in the range of 1-12 fold were better than acarbose. Among them, the most potent
compound
was
1-((3-(4-nitrophenyl)-1-p-tolyl-1H-pyrazol-4-yl)methylene)-4-
phenylsemicarbazide 8o with IC50 value of 65.1 µM is around 12-fold more active than
acarbose as the standard drug. Also, analysis of the kinetics of enzyme inhibition indicated
that this compound can inhibit α-glucosidase via a competitive mechanism. The analysis of
the structure-activity relationship revealed that electronic properties of substitutions in R1 did
not have much effect on inhibitory potential. The cytotoxicity assays confirmed that most
potent compounds (8o, 8k, 8p, 8l, 8i, and 8a) showed no cytotoxicity against 3T3 cell line.
The docking study predicted that most potent compounds bind to the active site of the
modeled α-glucosidase through the multiple hydrogen bonds and hydrophobic interactions. In
addition, MD simulations showed compound 8o oriented horizontally at the bottom of the
active site and stabilized the enzyme domains by interacting with the interface of domain A
and domain B while acarbose oriented vertically and formed non-binding interactions with the
residue belong to the domain A of the enzyme. Moreover, semicarbazone linker provided
such a strategic point with the ability to interact with the Asp349 which has the catalytic role
for α-glucosidase activity.
Based on these studies, it is assumed that the newly identified inhibitors may serve as lead
molecules for further research for getting powerful α-glucosidase inhibitors.
3. Experimental
All reagents and solvents used in this study were purchased from Sigma Aldrich (USA) and
Merck (Germany) and were used without further purification. All the reactions were monitored
on pre-coated silica gel aluminum plates (Merck silica gel 60, F254) and visualized using UV
lamp at 254 nm (UVGL-58; Upland, USA). Melting points of the target compounds 8a-p were

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determined with a Kofler hot stage apparatus and are uncorrected. The IR spectra recoded with
1
13
Nicolet FT-IR Magna 550 spectrographs using KBr pellets from 400 to 4000 cm−1. H and C
NMR spectra were recorded on a Bruker FT-500 in DMSO-d6 using TMS as internal standard.
Elemental analysis was carried out on Elementar Analysen system GmbH VarioEL CHNS mode.
3.1. General procedure for the synthesis of pyrazole-4-carbaldehydes 4a-p
Different substituted acetophenone 1 (10 mmol), phenyl hydrazine or 4-methyl phenyl
hydrazine 2 (12 mmol) and 1 ml of sulfuric acid as catalyst was being dissolved in 15 mL of
ethanol and refluxed for 8-12 h. Once the completion of the reaction was confirmed by TLC,
the reaction mixture was poured into ice bath to complete the crystallization. The precipitated
hydrazone intermediate 3a-p were filtered off, washed with water, and purified by
recrystallization in ethanol. In the next step, resulted hydrazone derivative (10 mmol) (3a–p)
was added to a solution of DMF (6 mL) and POCl3 (30 mmol) and the reaction was heated at
60 °C for 5-8 h. The reaction was monitored by TLC. After completing the reaction, the
mixture was cooled to room temperature and added 20 mL of ice-cold water. After
neutralizing the mixture with a saturated solution of sodium hydroxide, the solid product was
filtered, washed with water, dried and recrystallized from ethanol [22].
3.2. General procedure for the synthesis of phenyl urea 6
50 mmol of sodium cyanate in 80 ml of warm water was added to a stirring solution of aniline
5 (50 mmol) in 20 ml of glacial acetic acid and 10 ml of water. The reaction mixture was allowed
0
to stand for 30 min at 0-5 C in ice-water bath. Then, the obtained precipitate was filtered and
recystilized from boiling water to obtain pure phenyl urea 6 [60, 71].
3.3.General procedure for the synthesis of phenyl semicarbazide 7
A mixture of 50 mmol phenyl urea 6 and equimolar quantities of hydrazine hydrate (2.5 mL)
in ethanol (50 mL) was refluxed for 12 h in the presence of 50 mmol sodium hydroxide. After
completion of reaction (checked by TLC), the two-third volume of ethanol were evaporated and

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then poured into ice. The resultant precipitate was filtered, dried, and then crystallized from
ethanol and yielded phenyl semicarbazide 7 [60, 71].
3.4. General procedure for the synthesis pyrazole-phenyl semicarbazone hybrids 8a-p
The final step involves the condensation of various pyrazole carbaldehydes with phenyl
semicarbazide. A solution of substituted pyrazole-4-carbaldehydes 4a–p (0.5 mmol), phenyl
semicarbazide 7 (0.6 mmol) in 15 mL of ethanol was heated at reflux in the presence of a
catalytic amount of glacial acetic acid for 8-12 h. Progress monitored using TLC and after
completion of the reaction, two-third volume of solvent was being removed using rotary
evaporator. The mixture was cooled down to room temperature. The resulting precipitate was
filtered off, washed with ethanol, and purified by recrystallization in ethanol.
3.4.1.
1-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)-4-phenylsemicarbazide (8a)
Pale yellow crystals; yield: 83%, mp 213-215 ºC. IR (KBr, cm-1): 3390 (N-H), 3141, 2883
1
(Ar-H), 1683 (C=O), 1610 (C=N), 1577, 1454 (C=C). H NMR (400 MHz, DMSO-d6) δ 10.65
(s, 1H, CO-NH), 9.21 (s, 1H, N-NH), 8.65 (s, 1H, H5- pyr), 8.08 (s, 1H, -CH=), 7.98 – 7.93 (m,
2H, Ar-H), 7.73 – 7.68 (m, 2H, Ar-H ), 7.64 – 7.46 (m, 8H, Ar-H), 7.43 – 7.37 (m, 1H, Ar-H),
7.35 – 7.28 (m, 2H, Ar-H), 7.06 – 7.00 (m, 1H, Ar-H). Anal. Calcd for C23H19N5O: C, 72.42; H,
5.02; N, 18.36; O, 4.19. Found: C, 72.36; H, 4.83; N, 19.10.
3.4.2.
4-phenyl-1-((1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)methylene)semicarbazide (8b)
Pale yellow crystals; yield: 78%, mp 212-214 ºC. IR (KBr, cm-1): 3378 (N-H), 3067, 2933
1
(Ar-H), 1683 (C=O), 1597 (C=N), 1526, 1439 (C=C). H NMR (400 MHz, DMSO-d6) δ 10.80
(s, 1H, CO-NH), 9.32 (s, 1H, N-NH), 8.75 (s, 1H, H5- pyr), 8.20 (s, 1H, -CH=), 8.09 – 8.05 (m,
2H, Ar-H), 7.75 – 7.68 (m, 6H, Ar-H), 7.55 – 7.41 (m, 5H, Ar-H), 7.19 – 7.13 (m, 1H, Ar-H),
2.53 (s, 3H, CH3). 13C NMR (101 MHz, DMSO) δ 152.71 (C=O), 151.02 (C3-pyr), 139.06,
138.87(-CH=), 137.95, 133.38, 129.65, 129.47, 129.26, 128.54, 128.14, 127.65, 126.83 (C5-pyr),
122.44, 119.40, 118.51, 117.08 (C4-pyr), 20.85 (CH3). Anal. Calcd for C24H21N5O: C, 72.89; H,
5.35; N, 17.71; O, 4.05. Found: C, 73.13; H, 4.96; N, 17.35.
3.4.3.
1-((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4
phenylsemicarbazide (8c)

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Pale yellow crystals; yield: 75%, mp 288-290 ºC. IR (KBr, cm-1): 3397 (N-H), 3110, 2920
1
(Ar-H), 1694 (C=O), 1616 (C=N), 1599, 1523 (C=C), 1250 (C-OCH3). H NMR (400 MHz,
DMSO-d6) δ 10.79 (s, 1H, CO-NH), 9.31 (s, 1H, N-NH), 8.76 (s, 1H, H5- pyr), 8.20 (s, 1H, -
CH= ), 8.09 – 8.05 (m, 2H, Ar-H), 7.79 – 7.67 (m, 6H, Ar-H), 7.55 – 7.49 (m, 1H, Ar-H), 7.48 –
7.42 (m, 2H, Ar-H), 7.25 – 7.20 (m, 2H, Ar-H), 7.20 – 7.13 (m, 1H, Ar-H), 3.96 (s, 3H, OCH3).
13C NMR (101 MHz, DMSO) δ 159.47 (C-O), 152.72 (C=O), 150.88 (C3-pyr), 139.07, 138.89 (-
CH=), 133.47, 129.64, 129.56, 128.53, 127.56, 126.76 (C5-pyr), 124.65, 122.44, 119.41, 118.45,
116.92 (C4-pyr), 114.11, 55.21 (CH3O-). Anal. Calcd for C24H21N5O2: C, 70.06; H, 5.14; N,
17.02; O, 7.78. Found: C, 70.55; H, 5.73; N, 16.76.
3.4.4.
1-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4
phenylsemicarbazide (8d)
Pale yellow crystals; yield: 84%, mp 287-289 ºC. IR (KBr, cm-1): 3230 (N-H), 3123, 3050
(Ar-H), 1670 (C=O), 1593 (C=N), 1561, 1420 (C=C), 1012 (C-Cl). 1H NMR (400 MHz, DMSO-
d6) δ 10.86 (s, 1H, CO-NH), 9.38 (s, 1H, N-NH), 8.77 (s, 1H, H5- pyr), 8.22 (s, 1H, -CH=), 8.12
– 8.06 (m, 2H, Ar-H), 7.90 – 7.85 (m, 2H, Ar-H), 7.77 – 7.68 (m, 6H, Ar-H), 7.56 – 7.50 (m, 1H,
13
Ar-H), 7.48 – 7.41 (m, 2H, Ar-H), 7.19 – 7.13 (m, 1H, Ar-H). C NMR (101 MHz, DMSO) δ
152.67 (C=O), 149.68 (C3-pyr), 138.95, 138.89 (-CH=), 133.29, 133.04, 131.20, 129.99, 129.67,
128.75, 128.55, 128.09, 127.01 (C5-pyr), 122.42, 119.36, 118.59, 117.27 (C4-pyr). Anal. Calcd
for C23H18ClN5O: C, 66.43; H, 4.36; Cl, 8.52; N, 16.84; O, 3.85. Found: C, 66.79; H, 3.86; N,
15.21.
3.4.5.
1-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-
phenylsemicarbazide (8e)
Pale yellow crystals; yield: 86%, mp > 300 ºC. IR (KBr, cm-1): 3301 (N-H), 3139, 2920 (Ar-
H), 1617 (C=O), 1593 (C=N), 1552, 1490 (C=C). 1H NMR (400 MHz, DMSO-d6) δ 10.82 (s,
1H, CO-NH), 9.34 (s, 1H, N-NH), 8.71 (s, 1H, H5- pyr), 8.19 (s, 1H, -CH=), 8.12 – 8.03 (m, 2H,
Ar-H), 7.92 – 7.78 (m, 4H, Ar-H), 7.72 (m, 4H, Ar-H), 7.57 – 7.41 (m, 3H, Ar-H), 7.21 – 7.13
(m, 1H, Ar-H). 13C NMR (101 MHz, DMSO) δ 152.65 (C=O), 149.74 (C3-pyr), 138.94, 138.83
(-CH=), 133.01, 131.66, 131.57, 130.28, 129.69, 128.58, 128.13, 127.04 (C5-pyr), 122.46,

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121.97, 119.35, 118.60, 117.23 (C4-pyr). Anal. Calcd for C23H18BrN5O: C, 60.01; H, 3.94; Br,
17.36; N, 15.21; O, 3.48. Found: C, 60.55; H, 3.42; N, 14.82.
3.4.6.
1-((3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-
phenylsemicarbazide (8f)
Pale yellow crystals; yield: 82%, mp > 300 ºC. IR (KBr, cm-1): 3375 (N-H), 3138, 2960 (Ar-
1
H), 1677 (C=O), 1605 (C=N), 1598, 1446 (C=C), 1534, 1350 (C-NO2). H NMR (400 MHz,
DMSO-d6) δ 10.87 (s, 1H, CO-NH), 9.40 (s, 1H, N-NH), 8.75 (s, 1H, H5- pyr), 8.55 – 8.47 (m,
2H, Ar-H), 8.25 (s, 1H, -CH=), 8.20 – 8.14 (m, 2H, Ar-H), 8.14 – 8.07 (m, 2H, Ar-H), 7.78 –
7.67 (m, 4H, Ar-H), 7.56 (t, J = 7.4 Hz, 1H), 7.44 (t, J = 7.9 Hz, 2H), 7.20 – 7.13 (m, 1H, Ar-H).
13C NMR (101 MHz, DMSO) δ 152.65 (C=O), 148.59 (C-NO2), 147.12 (C3-pyr), 138.82,
138.80 (-CH=), 132.65, 129.73, 129.25, 128.58, 128.54, 127.35 (C5-pyr), 123.91, 122.51,
119.41, 118.78, 117.92 (C4-pyr). Anal. Calcd for C23H18N6O3: C, 64.78; H, 4.25; N, 19.71; O,
11.26. Found: C, 64.22; H, 4.52; N, 20.12.
3.4.7.
1-((3-(4-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-
phenylsemicarbazide (8g)
Pale yellow crystals; yield: 86%, mp 204-206 ºC. IR (KBr, cm-1): 3360 (N-H), 1688 (C=O),
1
1599 (C=N), 1536, 1442 (C=C). H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H, CO-NH), 9.90
(s, 1H, OH), 9.28 (s, 1H, N-NH), 8.78 (s, 1H, H5- pyr), 8.19 (s, 1H, -CH=), 8.06 (d, J = 8.0 Hz,
2H, Ar-H), 7.80 – 7.59 (m, 6H, Ar-H), 7.48 (dt, J = 7.6, 25.3 Hz, 3H, Ar-H), 7.16 (t, J = 7.3 Hz,
1H, Ar-H), 7.05 (d, J = 8.2 Hz, 2H, Ar-H). 13C NMR (101 MHz, DMSO) δ 157.84 (C-OH),
152.75 (C=O), 151.31 (C3-pyr), 139.10, 138.93 (-CH=), 133.67, 129.62, 129.55, 128.54, 127.34,
126.68 (C5-pyr), 122.96, 122.41, 119.43, 118.39, 116.78 (C4-pyr), 115.46. Anal. Calcd for
C23H19N5O2: C, 69.51; H, 4.82; N, 17.62; O, 8.05. Found: C, 69.85; H, 4.24; N, 16.80.
3.4.8.
1-((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-
phenylsemicarbazide (8h)
Pale yellow crystals; yield: 90%, mp 228-230 ºC. IR (KBr, cm-1): 3129 (N-H), 3015, 2913
(Ar-H), 1697 (C=O), 1613 (C=N), 1530, 1499 (C=C), 1243 (C-F). 1H NMR (400 MHz, DMSO-
d6) δ 10.80 (s, 1H, CO-NH), 9.34 (s, 1H, N-NH), 8.77 (s, 1H, H5- pyr), 8.19 (s, 1H, -CH=), 8.08
(d, J = 8.0 Hz, 2H, Ar-H), 7.89 (dd, J = 5.5, 8.4 Hz, 2H, Ar-H), 7.78 – 7.48 (m, 7H, Ar-H), 7.45
(t, J = 7.7 Hz, 2H, Ar-H) 7.17 (t, J = 7.4 Hz, 1H, Ar-H). 13C NMR (101 MHz, DMSO) δ 162.26
(d, J=246.4 Hz, C-F), 152.69 (C=O), 150.06 (C3-pyr), 138.99, 138.86 (-CH=), 133.13, 130.41,

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130.32, 129.67, 128.77, 128.74, 128.54, 127.72, 126.97 (C5-pyr), 122.47, 119.44, 118.56, 117.15
(C4-pyr), 115.79, 115.57. Anal. Calcd for C23H18FN5O: C, 69.16; H, 4.54; F, 4.76; N, 17.53; O,
4.01. Found: C, 64.18; H, 4.93; N, 16.95.
3.4.9.
4-phenyl-1-((1-phenyl-3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-
yl)methylene)semicarbazide (8i)
Pale yellow crystals; yield: 92%, mp 258-260 ºC. IR (KBr, cm-1): 3233 (N-H), 3018, 2950
(Ar-H), 1699 (C=O), 1605 (C=N), 1500, 1496 (C=C), 1303 (C-F). 1H NMR (400 MHz, DMSO-
d6) δ 10.82 (s, 1H, CO-NH), 9.38 (s, 1H, N-NH), 8.72 (s, 1H, H5- pyr), 8.23 (s, 1H, -CH=), 8.13
– 8.01 (m, 6H, Ar-H), 7.77 – 7.68 (m, 4H, Ar-H), 7.56 (td, J = 1.2, 7.3 Hz, 1H, Ar-H), 7.47 –
13
7.40 (m, 2H, Ar-H), 7.20 – 7.13 (m, 1H, Ar-H). C NMR (101 MHz, DMSO) δ 152.64 (C=O),
149.36 (C3-pyr), 138.90, 138.80 (-CH=), 136.41, 132.86, 129.72, 129.37, 128.98, 128.84,
128.53, 128.34, 127.19 (C5-pyr), 125.63, 125.59, 125.55, 122.49, 119.35, 118.69, 117.56 (C4-
pyr). Anal. Calcd for C24H18F3N5O: C, 64.14; H, 4.04; F, 12.68; N, 15.58; O, 3.56. Found: C,
63.70; H, 4.52; N, 16.09.
3.4.10.
4-phenyl-1-((3-phenyl-1-p-tolyl-1H-pyrazol-4-yl)methylene)semicarbazide (8j)
Pale yellow crystals; yield: 81%, mp 223-225 ºC. IR (KBr, cm-1): 3325 (N-H), 3073, 2923
1
(Ar-H), 1702 (C=O), 1574 (C=N), 1501, 1480 (C=C). H NMR (400 MHz, DMSO-d6) δ 10.78
(s, 1H, CO-NH), 9.30 (s, 1H, N-NH), 8.80 (s, 1H, H5- pyr), 8.21 (s, 1H, -CH=), 8.00 – 7.94 (m,
2H, Ar-H), 7.85 – 7.80 (m, 2H, Ar-H), 7.79 – 7.73 (m, 2H, Ar-H), 7.71 – 7.58 (m, 3H, Ar-H),
7.54 – 7.42 (m, 4H, Ar-H), 7.17 (tt, J = 1.2, 7.3 Hz, 1H, Ar-H), 2.51 (s, 3H, CH3). 13C NMR (101
MHz, DMSO) δ 152.74 (C=O), 150.79 (C3-pyr), 138.90 (-CH=), 136.85, 136.31, 133.38,
132.30, 130.02, 128.71, 128.53, 128.45, 128.21, 127.35 (C5-pyr), 122.45, 119.50, 118.44, 116.97
(C4-pyr), 20.48 (CH3). Anal. Calcd for C24H21N5O: C, 72.89; H, 5.35; N, 17.71; O, 4.05. Found:
C, 73.41; H, 5.82; N, 17.33.
3.4.11.
1-((1,3-dip-tolyl-1H-pyrazol-4-yl)methylene)-4-phenylsemicarbazide (8k)
Pale yellow crystals; yield: 85%, mp 276-279 ºC. IR (KBr, cm-1): 3365 (N-H), 3139, 2924
(Ar-H), 1685 (C=O), 1596 (C=N), 1578, 1420 (C=C). 1H NMR (400 MHz, DMSO-d6) δ 10.78
(s, 1H, CO-NH), 9.27 (s, 1H, N-NH), 8.75 (s, 1H, H5- pyr), 8.20 (s, 1H, -CH=), 7.98 – 7.92 (m,

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2H, Ar-H), 7.72 (dd, J = 7.2, 8.4 Hz, 4H, Ar-H), 7.54 – 7.41 (m, 6H, Ar-H), 7.20 – 7.12 (m, 1H,
13
Ar-H), 2.52 (s, 3H, CH3), 2.50 (s, 3H, CH3). C NMR (101 MHz, DMSO) δ 152.73 (C=O),
150.78 (C3-pyr), 138.89 (-CH=), 137.87, 136.86, 136.21, 133.46, 130.00, 129.52, 129.24,
128.54, 128.12, 127.39 (C5-pyr), 122.43, 119.41, 118.38, 116.84 (C4-pyr), 20.85 (CH3), 20.48
(CH3). Anal. Calcd for C25H23N5O: C, 73.33; H, 5.66; N, 17.10; O, 3.91. Found: C, 73.83; H,
5.15; N, 16.80.
3.4.12.
1-((3-(4-methoxyphenyl)-1-p-tolyl-1H-pyrazol-4-yl)methylene)-4-
phenylsemicarbazide (8l)
Pale yellow crystals; yield: 80%, mp 224-226 ºC. IR (KBr, cm-1): 3372 (N-H), 3125, 2910
1
(Ar-H), 1670 (C=O), 1590 (C=N), 1580, 1468 (C=C), 1244 (C-OCH3). H NMR (400 MHz,
DMSO-d6) δ 10.77 (s, 1H, CO-NH), 9.25 (s, 1H, N-NH), 8.76 (s, 1H, H5- pyr), 8.19 (s, 1H, -
CH=), 7.98 – 7.93 (m, 2H, Ar-H), 7.79 – 7.72 (m, 4H, Ar-H), 7.53 – 7.41 (m, 4H, Ar-H), 7.25 –
7.20 (m, 2H, Ar-H), 7.19 – 7.13 (m, 1H, Ar-H), 3.96 (s, 3H, OCH3), 2.50 (s, 3H, CH3). 13C NMR
(101 MHz, DMSO) δ 159.44 (C-O), 152.74 (C=O), 150.65 (C3-pyr), 138.91 (-CH=), 136.90,
136.13, 133.59, 129.98, 129.54, 128.52, 127.33 (C5-pyr), 124.75, 122.43, 119.42, 118.36, 116.68
(C4-pyr), 114.09, 55.21 (OCH3), 20.47 (CH3). Anal. Calcd for C25H23N5O2: C, 70.57; H, 5.45; N,
16.46; O, 7.52. Found: C, 70.97; H, 5.88; N, 15.92.
3.4.13.
1-((3-(4-chlorophenyl)-1-p-tolyl-1H-pyrazol-4-yl)methylene)-4-
phenylsemicarbazide (8m)
Pale yellow crystals; yield: 91%, mp 287-289 ºC. IR (KBr, cm-1): 3349 (N-H), 3014, 2919
(Ar-H), 1694 (C=O), 1613 (C=N), 1580, 1452 (C=C), 1092 (C-Cl). 1H NMR (400 MHz, DMSO-
d6) δ 10.79 (s, 1H, CO-NH), 9.29 (s, 1H, N-NH), 8.72 (s, 1H, H5- pyr), 8.19 (s, 1H, -CH=), 7.99
– 7.94 (m, 2H, Ar-H), 7.90 – 7.84 (m, 2H, Ar-H), 7.77 – 7.70 (m, 4H, Ar-H), 7.54 – 7.41 (m, 4H,
Ar-H), 7.16 (t, J = 7.3 Hz, 1H, Ar-H), 2.51 (s, 3H, CH3). 13C NMR (101 MHz, DMSO) δ 152.67
(C=O), 149.46 (C3-pyr), 138.84 (-CH=), 136.75, 136.45, 133.23, 133.09, 131.25, 130.04,
129.97, 128.74, 128.56, 127.78 (C5-pyr), 122.46, 119.39, 118.48, 117.03 (C4-pyr), 20.49 (CH3).
Anal. Calcd for C24H20ClN5O: C, 67.05; H, 4.69; Cl, 8.25; N, 16.29; O, 3.72. Found: C, 66.71;
H, 4.15; N, 16.67.
3.4.14.
1-((3-(4-bromophenyl)-1-p-tolyl-1H-pyrazol-4-yl)methylene)-4-
phenylsemicarbazide (8n)

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Pale yellow crystals; yield: 89%, mp > 300 ºC. IR (KBr, cm-1): 3435 (N-H), 3156, 2915 (Ar-
H), 1700 (C=O), 1610 (C=N), 1560, 1439 (C=C). 1H NMR (400 MHz, DMSO-d6) δ 10.76 (s,
1H, CO-NH), 9.26 (s, 1H, N-NH), 8.68 (s, 1H, H5- pyr), 8.19 (s, 1H, -CH=), 7.99 – 7.93 (m, 2H,
Ar-H), 7.90 – 7.83 (m, 2H, Ar-H), 7.84 – 7.78 (m, 2H, Ar-H), 7.75 – 7.69 (m, 2H, Ar-H), 7.54 –
7.41 (m, 4H, Ar-H), 7.20 – 7.13 (m, 1H, Ar-H), 2.51 (s, 3H, CH3). 13C NMR (101 MHz, DMSO)
δ 152.66 (C=O), 149.49 (C3-pyr), 138.84 (-CH=), 136.74, 136.45, 133.08, 131.64, 130.25,
130.03, 128.57, 127.86 (C5-pyr), 122.45, 121.89, 119.37, 118.48, 117.01 (C4-pyr), 20.49 (CH3).
Anal. Calcd for C24H20BrN5O: C, 60.77; H, 4.25; Br, 16.84; N, 14.76; O, 3.37. Found: C, 59.95;
H, 4.62; N, 14.33.
3.4.15.
1-((3-(4-nitrophenyl)-1-p-tolyl-1H-pyrazol-4-yl)methylene)-4-
phenylsemicarbazide (8o)
Pale yellow crystals; yield: 84%, mp > 300 ºC. IR (KBr, cm-1): 3392 (N-H), 3079, 2938 (Ar-
1
H), 1682 (C=O), 1598 (C=N), 1518, 1444 (C=C), 1572, 1332 (C-NO2). H NMR (400 MHz,
DMSO-d6) δ 10.86 (s, 1H, CO-NH), 9.36 (s, 1H, N-NH), 8.77 (s, 1H, H5- pyr), 8.53 – 8.47 (m,
2H, Ar-H), 8.28 (s, 1H, -CH=), 8.19 – 8.13 (m, 2H, Ar-H), 8.02 – 7.97 (m, 2H, Ar-H), 7.76 –
7.69 (m, 2H, Ar-H), 7.55 – 7.49 (m, 2H, Ar-H), 7.47 – 7.38 (m, 2H, Ar-H), 7.16 (t, J = 7.2 Hz,
13
1H, Ar-H), 2.51 (s, 3H, CH3). C NMR (101 MHz, DMSO) δ 152.65 (C=O), 148.29 (C3-pyr),
147.03 (C-NO2), 138.87 (-CH=), 136.77, 136.63, 132.74, 130.07, 129.19, 128.52, 128.37 (C5-
pyr), 123.88, 122.46, 119.41, 118.63, 117.74 (C4-pyr), 20.50 (CH3). Anal. Calcd for
C24H20N6O3: C, 65.45; H, 4.58; N, 19.08; O, 10.90. Found: C, 64.93; H, 4.85; N, 18.65.
3.4.16.
1-((3-(4-hydroxyphenyl)-1-p-tolyl-1H-pyrazol-4-yl)methylene)-4-
phenylsemicarbazide (8p)
Pale yellow crystals; yield: 80%, mp 208-210ºC. IR (KBr, cm-1): 3320 (N-H), 1672 (C=O),
1580 (C=N), 1526, 1430 (C=C). 1H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H, CO-NH), 9.96
(s, 1H, OH), 9.25 (s, 1H, N-NH), 8.81 (s, 1H, H5- pyr), 8.20 (s, 1H, -CH=), 7.95 (d, J = 8.1 Hz,
2H, Ar-H), 7.77 (d, J = 8.0 Hz, 2H, Ar-H), 7.64 (d, J = 8.3 Hz, 2H, Ar-H), 7.53 – 7.40 (m, 4H,
Ar-H), 7.16 (t, J = 7.3 Hz, 1H, Ar-H), 7.06 (d, J = 8.4 Hz, 2H, Ar-H), 2.50 (s, 3H, CH3). 13C
NMR (101 MHz, DMSO) δ 157.85 (C-OH), 152.76 (C=O), 151.08 (C3-pyr), 138.98 (-CH=),

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136.92, 136.01, 133.79, 129.97, 129.49, 128.52, 127.11 (C5-pyr), 122.97, 122.37, 119.43,
118.27, 116.53 (C4-pyr), 115.47, 20.47 (CH3). Anal. Calcd for C24H21N5O2: C, 70.06; H, 5.14;
N, 17.02; O, 7.78. Found: C, 70.42; H, 5.61; N, 16.79.
3.5. In vitro α-glucosidase inhibition assay
The in vitro α-glucosidase inhibitory activities of pyrazole-phenyl semicarbazone hybrids 8a-
p were carried out according to the protocol previously reported [72]. α-Glucosidase enzyme
((EC3.2.1.20, Saccharomyces cerevisiae, 20 U/mg) and substrate (p-nitrophenyl
glucopyranoside) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Enzyme (50
mM) and the desired concentrations of nitrophenol-α-D-glycopyranoside (PNP) were prepared in
potassium phosphate buffer (pH= 6.8). The stock solutions (10 mM) of standard and all tested
compounds were dissolved in DMSO and further diluted with potassium phosphate buffer to
prepare sample solutions in various concentrations. Firstly, the various concentrations of
compounds 8a-p (20 μL), enzyme solution (20 μL) and potassium phosphate buffer (135 μL) was
pre-incubated at 37 ºC for 10 min in a 96-well plate. After that, 25 μL of substrate (p-
nitrophenyl-α-Dglucopyranoside, 4 mM) was added to each well and then incubated at 37 ºC for
20 min. The reaction was monitored spectrophotometrically by measuring the change in
absorbance at 405 nm. DMSO and acarbose were used as the control and standard inhibitor,
respectively. The percentage of enzyme inhibition for each entry was calculated by using the
following formula:
% Inhibition=[(Abs control-Abs sample)/Abs control]×100
IC50 values of tested compounds were obtained from the nonlinear regression curve (logit
method). All the tests were performed in triplicate and the results are expressed as the mean ±
SD

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3.6. Kinetic studies
The mechanism of inhibition of the most potent compound 8o was investigated with different
concentrations of p-nitrophenyl α-D-glucopyranoside as substrate in the absence and presence of
compound 8o at different concentrations. The α-glucosidase solution (1 U/mL, 20 μL) was
incubated with different concentrations (0, 30, 45, and 65 μM) of compound 8o (20 μL) for the
period of 15 min at 30 ºC. Then, change in absorbance was measured for 20 min at 405 nm using
spectrophotometer (Gen5, Power wave xs2, BioTek, America) following the adding different
concentrations of p-nitrophenyl glucopyranoside (2–10 mM) as substrate. The inhibition types
and the Michaelis–Menten constant (Km) value were determined by Lineweaver-Burk plots. The
experimental inhibitor constant (Ki) value was constructed by secondary plots of the inhibitor
concentration [I] versus Km.
3.7. Cytotoxicity evaluation of the most potent compounds on 3T3 cell line
The 3T3 cells were suspended in Dulbecco's modified Eagle medium (DMEM) formulated
with 10% fetal bovine serum (FBS), 100 IU/mL streptomycin, and 100 IU/mL penicillin. The
cells were plated at a concentration of 2× 105 cells/mL and incubated overnight at 37 ºC under
5% CO2. Then supernatant was removed and 200 mL fresh medium along with different
concentrations of the selected compounds 8o, 8k, 8p, 8l, 8i, and 8a (0.04 to 50 µM) were added
and then further incubated for 48 h at 37 °C in CO2 incubator. After treatment, the medium was
aspirated carefully, and 50 μL of MTT solution (2 mg/mL in PBS) was added to all wells. After
4 h of incubation, MTT solution was aspirated and 100 µL DMSO was added to each well in
order to dissolve the blue insoluble MTT formazan produced by the action of mitochondrial.
Finally, the absorbance was recorded at 570 nm with a multi-well plate reader (Rayto, China)
after agitating at room temperature for 10 min and readings were processed using MS Excel
software. All the experiments were performed in triplicate.
3.8. Homology modeling

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The amino acid sequence of Saccharomyces cerevisiae α-glucosidase was retrieved from
UniProtKB database (Uni- ProtKB, http://www.uniprot.org/) with accession number P53341.
Hence, the sequence similarity search was performed using NCBI BLAST server (https://
blast.ncbi.nlm.nih.gov/Blast.cgi) based on sequence identity. The crystal structures of isomaltase
from Saccharomyces cerevisiae (PDB ID: 3A4A) selected as suitable template and retrieved
from the Protein Data Bank. A sequence alignment between query and template was performed
by Align sequence to template protocol in Discovery Studio (Accelrys, San Diego, CA) (DS).
Finally, Homology modeling was performed with MODELER inbuilt in DS package. The
number of models was set to 10 and the optimization level was set to “high”, whereas default
values were set for other parameters.
DOPE score and profile-3D for preliminary evaluation of developed model were carried out
using verify protein protocol in DS. Ramachadran plot and Verify3D with PROCHECK program
(http://servicesn.mbi.ucla.edu/PROCHECK) were also applied to estimate the quality of the
established model [73, 74]. Eventually, ProSA server was used for evaluation of the consistency
between the modeled structure and the selected template [75].
3.9. Docking simulation
Molecular docking approach was performed using genetic algorithm based docking program
(GOLD), which has been incorporated into Discovery Studio (DS) 4.1 to dock synthetic
compounds against the α-glucosidase enzyme. 3D structure of internal ligands (acarbose and α -
D-glucopyranose) and final synthesized derivatives were drawn using Chem Draw Ultra 12.0
software and were transferred into DS. Compounds were typed with CHARMm force field and
partial charges were calculated by Momany-Rone option. Subsequently, resulting structures were
minimized with Smart Minimizer algorithm which contains 1000 steps of steepest descent with a
RMS gradient tolerance of 3, followed by Conjugate Gradient minimization. Later, 3D modeled
structure of the α-glucosidase was subjected to protein preparation and minimization. The
complex was typed with CHARMm force field, hydrogen atoms were added and pH of the
protein was adjusted to almost neutral, 7.4, using protein preparation protocol in DS. A 9 A ˚
radius sphere was defined as the binding region for docking study. Other parameters were set by
default protocol settings [76]. All synthesized derivatives were docked into the active site of the

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α-glucosidase and generated binding modes for each compound ranked by GOLD score fitness
function.
3.10. Molecular dynamic simulation
Molecular dynamic (MD) simulation of this study was performed by using the Desmond v5.3
module implemented in Maestro interface (from Schrödinger 2018‐4 suite) [77]. The appropriate
pose for MD simulation procedure of the compounds was achieved by docking method.
In order to build the system for MD simulation, the protein-ligand complexes were solvated
with SPC explicit water molecules and placed in the center of an orthorhombic box of
appropriate size in the Periodic Boundary Condition. Sufficient counter‐ions and a 0.15 M
solution of NaCl were also utilized to neutralize the system and to simulate the real cellular ionic
concentrations, respectively. The MD protocol involved minimization, pre-production, and
finally production MD simulation steps. In the minimization procedure, the entire system was
allowed to relax for 2500 steps by the steepest descent approach. Then the temperature of the
system was raised from 0 to 300 K with a small force constant on the enzyme in order to restrict
any drastic changes. MD simulations were performed via NPT (constant number of atoms,
constant pressure i.e. 1.01325 bar and constant temperature i.e. 300 K) ensemble. The Nose‐
Hoover chain method was used as the default thermostat with 1.0 ps interval and Martyna‐
Tobias‐Klein as the default barostat with 2.0 ps interval by applying isotropic coupling style.
Long‐range electrostatic forces were calculated based on Particle‐mesh‐based Ewald approach
with the he cut‐off radius for columbic forces set to 9.0 Å. Finally, the system subjected to
produce MD simulations for 20 ns for protein-ligand complex. During the simulation every 1000
ps of the actual frame was stored. The dynamic behavior and structural changes of the systems
were analyzed by the calculation of the root mean square deviation (RMSD) and RMSF.
Subsequently, the energy-minimized structure calculated from the equilibrated trajectory system
was evaluated for investigation of each ligand-protein complex interaction.
3.11. Prime MM-GBSA

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The ligand binding energies (ΔG Bind) were calculated for compound 8o and acarbose using
Molecular mechanics/generalized born surface area (MM‐ GBSA) modules (Schrödinger LLC
2018) (75) based on the following equation;
ΔG Bind = E Complex – [E Receptor + E Ligand
]
Where ΔG Bind is the calculated relative free energy which includes both ligand and receptor
strain energy. E Complex is the MM-GBSA energy of the minimized complex, and E is the
Ligand
MM-GBSA energy of the ligand after removing it from the complex and allowing it to relax. E
Receptor is the MM-GBSA energy of relaxed protein after separating it from the ligand. The MM-
GBSA calculation was performed based on the clustering method for energy calculation.
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