Novel 5-(1-aryl-1H-pyrazol-3-yl)-1H-tetrazoles as glycogen phosphorylase inhibitors: An in vivo antihyperglycemic activity study

Article abstract of DOI:10.1002/ddr.21606

In this study, we report the ring transformation of 3-arylsydnone into 1-aryl-1H-pyrazole-3-carbonitriles via [3 + 2] cycloaddition with acrylonitrile. 1-Aryl-1H-pyrazole-3-carbonitrile underwent [2 + 3] cycloaddition with sodium azide to afford 5-(1-aryl-1H-pyrazol-3-yl)-1H-tetrazoles which were further subjected to N-alkylation with aryl/heteroaryl alkyl halides to afford 1,5- and 2,5-disubstituted tetrazoles. Furthermore, the title compounds were screened for in vivo antihyperglycemic activity using albino Wistar rats of either sex. Compounds 4a, 6b, 7a, 7b, 8b, and 9b showed maximum fall in the blood glucose levels in streptozotocin-induced diabetic rats after 5–7 days of administration. In support of antidiabetic activity, we also performed the experimental in vivo studies, namely, effect of compounds on enzymes (serum glutamic oxaloacetic transaminase, serum glutamic-pyruvic transaminase, creatinine, urea, and total protein), antihyperlipidemic, and histopathology. Moreover, the molecular docking study has been performed for potent molecules among the series with glycogen phosphorylase as target enzyme, and this study corroborated the experimental in vivo results.

Full text of DOI:10.1002/ddr.21606

Received: 8 May 2019
Revised: 12 July 2019
Accepted: 4 August 2019
DOI: 10.1002/ddr.21606
R E S E A R C H A R T I C L E
Novel 5-(1-aryl-1H-pyrazol-3-yl)-1H-tetrazoles as glycogen
phosphorylase inhibitors: An in vivo antihyperglycemic activity
study
Pramod P. Kattimani1 | Shilpa M. Somagond1 | Praveen K. Bayannavar1
|
Ravindra R. Kamble1
Shrinivas D. Joshi4
| Subhas C. Bijjaragi2 | Raveendra K. Hunnur3
|
1Department of Studies in Chemistry,
Karnatak University, Dharwad, Karnataka,
India
Abstract
In this study, we report the ring transformation of 3-arylsydnone into 1-aryl-1H-
pyrazole-3-carbonitriles via [3 + 2] cycloaddition with acrylonitrile. 1-Aryl-1H-
pyrazole-3-carbonitrile underwent [2 + 3] cycloaddition with sodium azide to afford
5-(1-aryl-1H-pyrazol-3-yl)-1H-tetrazoles which were further subjected to N-
alkylation with aryl/heteroaryl alkyl halides to afford 1,5- and 2,5-disubstituted
tetrazoles. Furthermore, the title compounds were screened for in vivo
antihyperglycemic activity using albino Wistar rats of either sex. Compounds 4a, 6b,
7a, 7b, 8b, and 9b showed maximum fall in the blood glucose levels in
streptozotocin-induced diabetic rats after 5–7 days of administration. In support of
antidiabetic activity, we also performed the experimental in vivo studies, namely,
effect of compounds on enzymes (serum glutamic oxaloacetic transaminase, serum
glutamic-pyruvic transaminase, creatinine, urea, and total protein), antihyperlipidemic,
and histopathology. Moreover, the molecular docking study has been performed for
potent molecules among the series with glycogen phosphorylase as target enzyme,
and this study corroborated the experimental in vivo results.
2Department of Studies in Zoology, Karnatak
University, Dharwad, Karnataka, India
3APL Research Ltd., Batchupally, Telangana,
India
4Novel Drug Design and Discovery
Laboratory, Department of Pharmaceutical
Chemistry, S.E.T.'s College of Pharmacy,
Dharwad, Karnataka, India
Correspondence
Ravindra R. Kamble, Department of Studies in
Chemistry, Karnatak University, Dharwad
580003, Karnataka, India.
Email: kamchem9@gmail.com
Funding information
University Grants Commission
K E Y W O R D S
3-arylsydnone, glycogen phosphorylase, in vivo antihyperglycemic activity
prevalence is driven by several factors such as diet, obesity, stress,
urbanization, and lack of physical activity (World Health Organization,
1
| INTRODUCTION
2016). Type 2 diabetes mellitus (T2DM) is a severe sickness with giant
economic consequences, which is considerably underdiagnosed and
incompletely treated within the general population (Zimmet, Alberti, &
Shaw, 2001; Diamond (2003). In the treatment of diabetic patients,
control of blood sugar levels is a key factor and these patients are
most frequently prescribed with modification of diet and exercise, one
or more oral hypoglycemic agents, as well as insulin. In spite of the
provision of various categories of hypoglycemic medicine, present
treatments are often unable to attain an intensive degree of blood
Diabetes is one of the foremost serious life style disorder and meta-
bolic chronic disease that has attacked the world at an alarming wide
spread rate. It is characterized by high blood glucose level due to the
defects in insulin secretion or insulin action or both (Matschinsky,
1996). A survey from the International Diabetes Federation (IDF)
reveals that diabetes has affected approximately 425 million individ-
uals in 2017, and this range is anticipated to rise to 629 million by
2045 (IDF Diabetes Atlas, 2018). It is grouped as Type 1 and Type
2 that happened due to insulin resistance in body tissues, and its
Drug Dev Res. 2019;1–15.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

2
KATTIMANI ET AL.
sugar management to cut back effectively the occurrence and brutal-
ity of diabetic complications (Wagman & Nuss, 2001).
Wittenberger, 1994). However, 1,5-disubstituted tetrazoles can be
used as isosteres of cis-amide bond of peptides (May & Abell, 2001;
May & Abell, 2002). Substances containing 1,5-disubstituted tetrazole
fragment are being well studied as they exhibit anti-inflammatory
(Rajasekaran & Thampi, 2004), antiviral, (Muraglia et al., 2006),
antitubercular (Karabanovich et al., 2015), anticonvulsant, antimicro-
bial (Rostom, Ashour, Abd El Razik, Abd El Fattah, & El-Din, 2009),
antiparasitic (Pedro et al., 2014), antiulcer (Uchida et al., 1989), antihy-
pertensive (Ashton et al., 1992), antimalarial (Pandey et al., 2013),
anticancer (Arshad et al., 2014), and COX-2 inhibitory (Al-Hourani
et al., 2011) properties. 2,5-Disubstituted tetrazoles have been proved
to exhibit antiviral (Chang et al., 2005), antimicrobial (Rostom, Ashour,
Abd El Razik, Abd El Fattah, & El-Din, 2009), and glutamate receptor
modulating (Vieira et al., 2005) properties. Synthesized tetrazole hav-
ing N-glycosides as SGLT2 inhibitors and evaluated for their hypogly-
cemic effects in vivo by mice oral glucose tolerance test (Gao et al.,
2010). 1-{5-[(4-Ethoxyphenyl)methyl]-2H-tetrazol-2-yl}-1-deoxy-β-D-
glycopyranose 4 was found to be most active against standard drug
Dapagliflozin 6 (Gao et al., 2010). Vieira et al. (2005) reported a new
series of 5-[(5-aryl-1H-pyrazol-3-yl) methyl]-1H-tetrazoles 5 and eval-
uated for their in vivo antihyperglycemic activity by sucrose loaded
model. These compounds showed significant antihyperglycemic activ-
ity (Sharon et al., 2005a, 2005b) (Figure 2).
In Type 2 diabetic patients, the hepatic glucose output is increased
and the current evidence indicates that abnormal high production of
glucose by liver is due to the glycogenolysis (release of monomeric
glucose from the glycogen polymer storage form). One of the pharma-
cological approaches to reduce this elevated hepatic glucose in Type
2 diabetes is to inhibit glycogen phosphorylase (GP) enzyme (Baker,
Greenhaff, & Timmons, 2006; Henke & Sparks, 2006; Loughlin, 2010;
Oikonomakos, 2002; Oikonomakos & Somsák, 2008; Somsak et al.,
2008). GP is primarily expressed in the liver and is responsible for gly-
cogen breakdown to release glucose and connected metabolites for
energy supply (glycogenolysis), thus inhibiting GP will limit glycogenol-
ysis and subordinate hepatic glucose production (Kurukulasuriya et al.,
2003). Hence, GP is a molecular therapeutic target for treating hyper-
glycemia associated with T2DM (Henke & Sparks, 2006; Hoover
et al., 1998; Oikonomakos, 2002; Somsak et al., 2008; Treadway,
Mendys, & Hoover, 2001).
The occurrence of a heterocycle functioning as central part of
drug molecules is an incident that takes place commonly in many nat-
ural and synthetic drug molecules. Particularly, five- and six-
membered heterocyclic systems are motifs of many efficacious drugs
(Figure 1). Pyrazoles are well-known examples of aromatic five-
membered heterocyclic organic compounds containing two adjacent
nitrogen atoms endowed with diverse biological activity on humans
such as antimicrobial (Sridhar et al., 2004), antidiabetic, anticancer
(Altıntop, Ozdemir, Ilgın, & Atli, 2014), anti-inflammatory (Raffa et al.,
2010; Ren et al., 2018), antiviral (El-Sabbagh et al., 2009), anticonvul-
sant, and antidepressant (Abdel-Aziz, El-Din, Abuo-Rahma, & Hassan,
2009). Furthermore, the pyrazole derivatives are used as synthon for
the development of condensed heterocyclic scaffolds and thus signify
as interesting template for combinational chemistry (Singh, Paul, &
Holzer, 2006; Tiwari, Ameta, Ranwal, Ameta, & Punjabi, 2013).
Pyrazole-containing remogliflozin etabonate is found to lower the
blood glycemic levels and is now under clinical trials (Fujimori
et al., 2008).
3-Arylsydnones undergo ring transformation reactions yielding
pharmaceutically important heterocycles (Angadiyavar
& George,
1971; Browne & Harrity, 2010; Mallur & Badami, 2000; Wu, Fang,
Larock, & Shi, 2010). This fact has triggered sydnone as the prominent
synthon among all the mesoionic compounds. In view of this, we have
attempted [3 + 2] cycloaddition reaction of 3-arylsydnone with acry-
lonitrile to afford 1-aryl-1H-pyrazole-3-carbonitrile (2a–c) which
served as dipolarophile for subsequent [3 + 2] cycloaddition with
sodium azide to get 5-(1-aryl-1H-pyrazol-3-yl)-1H-tetrazoles (3a–c).
Tetrazole derivative 3a–c was then subjected to N-alkylation with
aryl/heteroaryl alkyl halides (i–iv) in the presence of anhydrous potas-
sium carbonate to get the title Compounds 4–9 (a–d) (Scheme 1). The
title Compounds 4–9 (a–d) were further evaluated for in vivo
antihyperglycemic activity using Wistar albino rats of either sex and
carried out the histopathological, biochemical parameters, and lipid
profile studies.
5-Substituted-1H-tetrazoles serve as nonclassical isostere for the
carboxylic acid moiety (RCO2H) in biologically active molecules
(Butler, 1996; Le Bourdonnec et al., 2000; Lipinski, 1986; Nisa et al.,
2015; Sharon et al., 2005a, 2005b; Subramanian et al., 2015;
2
| METHODS
2.1 | X-ray diffraction studies
Single-crystal X-ray diffraction (XRD) study was carried out for Com-
pound 4b. Data collection: SMART (Bruker, 2001); cell refinement:
SAINT (Bruker, 2001); data reduction: SAINT; program(s) used to
solve structure: SHELXS97 (Sheldrick, 1997); molecular graphics:
ORTEP-3 (Farrugia, 2012); software used to prepare material for pub-
lication: SHELXL97 (Sheldrick, 1997). The ORTEP, unit cell, hydrogen
bonding, dihedral angles, and packing diagrams were obtained by
MERCURY software.
FIGURE 1 Several drug molecules that include pyrazole as a core
moiety

KATTIMANI ET AL.
3
FIGURE 2 Some examples of
tetrazole-bearing potent
antihyperglycemic-reported
molecules
SCHEME 1 Synthetic route for the title Compounds 4–7 (a–d)
2.2 | In vivo antihyperglycemic assay
2.2.2 | Experimental protocols
2.2.1 | Streptozotocin-induced diabetic rat model
The animals were further divided into 10 groups of 6 animals each as
follows:
Animals were supplied by the Department of Zoology, Karnatak Uni-
versity, Dharwad, Karnataka, India, and the animal house registration
number is 639/02/A/CPCSEA, dated: July 19, 2002. Diabetes was
induced in fasted rats (12 hr) by intraperitoneal injection of
streptozotocin monohydrate at 120 mg/kg body weight (b.w.) dis-
solved in 0.90 M saline. Food was provided 30 mins after the adminis-
tration of streptozotocin. Diabetes was confirmed 48 hr later by
glucometer (One Touch Ultra, Johnson & Johnson Co. LifeScan Scot-
land Ltd., Beechwood Park North, Inverness IV2 3ED, UK) using tail
vein blood sample. Animals with a blood glucose level above 200 mg/
dL were considered diabetic and were included in the experiment.
Group I: Normal (vehicle).
Group II: Dimethylsulphoxide (DMSO) control (0.2 ml).
Group III: Diabetic control.
Group IV: Diabetic animals treated with glibenclamide (0.25 mg/
kg b.w.).
Group V: Diabetic animals treated with 4a (100 mg/kg b.w. in
0.2 ml DMSO).
Group VI: Diabetic animals treated with 6b (100 mg/kg b.w. in
0.2 ml DMSO).
Group VII: Diabetic animals treated with 7a (100 mg/kg b.w. in
0.2 ml DMSO).

4
KATTIMANI ET AL.
SCHEME 2 Mechanism of [3 + 2]
cycloaddition for the formation of
Compound 2a–c
Group VIII: Diabetic animals treated with 7b (100 mg/kg b.w. in
0.2 ml DMSO).
Group IX: Diabetic animals treated with 8b (100 mg/kg b.w. in
0.2 ml DMSO).
Group X: Diabetic animals treated with 9b (100 mg/kg b.w. in
0.2 ml DMSO).
Animals of Group I were untreated and considered as normal control.
2.2.4 | Tissue preparation
Animals of all the groups were sacrificed by the administration of
40 mg/kg b.w. pentobarbital and dissected to get pancreas tissue for
histopathological studies. The collected tissues were preserved in 4%
paraformaldehyde for 48 hr and then dehydrated sequentially in gradi-
ent ethanol–water (50, 70, 80, 90, and 95%, and finally in 100% alco-
hol). Samples were immersed in xylene for 30 min and incubated with
paraffin at 65 ꢀC overnight. After embedding in wax, tissues were cut
into ultrathin sections of 5 ?m thickness by Leica RM 2255 microtome
(Leica, Nussloch, Germany) and spread on the glass slides. Sections
were deparaffinated with fresh xylene for 30 min, hydrated with gradi-
ent ethanol (100, 90, 80, and 70%), and then washed with double dis-
tilled water for three times. The sections were stained with
hematoxylin and eosin and mounted in deparaffinated xylene for the
observation of microscopic cells for necrosis, fat degradation, and pan-
creatic degeneration. The sections were examined and micrographs
were captured using Axio Imager M.2 microscope for histological
evaluation.
Animals of Group II were given an equal amount of DMSO. Compounds
4, 5, 6, 7, 8, and 9 (a–d) and glibenclamide were orally administered
through gavage daily for 28 days. The blood glucose level of treated
Groups IV–X was determined at 30, 60, 90, 120, 180, and 240 min and
12 hr using glucometer on the first day of administration of synthesized
compounds. From second day onwards, the blood glucose level was
determined at every 6-hr time interval for 28 days. Animals not found
diabetic after 24-h posttreatment with streptozotocin were not consid-
ered and omitted from the calculations. The treated compounds that did
not show any fall in the blood glucose level of animals were also omitted
from the calculations and considered as inactive. Food but not water
was withheld from the cages at the time of experiment.
At the end of experimental period (28 days), the animals were
fasted overnight and the blood sample of experimental animals was
withdrawn through retro-orbital plexus puncture. The serum was sep-
arated by centrifuging at 3000 rpm for 20 min and used for various
biochemical estimations.
2.2.5 | Statistical analysis
All the values were expressed as mean SE. One-way analysis of vari-
ance was used to detect statistical significance followed by post hoc
multiple comparisons (Tukey's test). A value of p <.05 was considered
to be significant.
2.2.3 | Biochemical parameters
2.3 | Docking simulation
The blood serum creatinine, urea, and total protein were estimated
according to the standardized procedures. The serum lipid profiles
such as total cholesterol (TC), triglyceride (TG), and high-density lipo-
protein (HDL) were estimated using commercially available kits
(Accurex Biomedicals, Pvt. Ltd., Mumbai, India). The serum low-
density lipoprotein (LDL) and very low-density lipoprotein (VLDL)
The crystal structure used was that of human liver GP, complexed with
caffeine, N-acetyl-beta-D-glucopyranosylamine, and CP-403700 (Protein
Data Bank [PDB] ID: 1L5Q; B chain) for docking studies, obtained from
the PDB. The protein was designed for docking by adding polar hydrogen
atom with the Gasteiger–Huckel charges (Gasteiger and Marsili, 1980)
and water molecules were removed. The three-dimensional structure of
the ligands was generated by the SKETCH module implemented in the
SYBYL program (Tripos Inc., St. Louis, MO) and its energy-minimized con-
formation was obtained with the help of the Tripos force field using the
were calculated using Friedewald formula (Friedewald, Levy,
&
Fredrickson, 1972). Serum glutamic oxaloacetic transaminase (SGOT)
and serum glutamic-pyruvic transaminase (SGPT) activities were
determined by the method developed by Reitman and Frankel (1957).

KATTIMANI ET AL.
5
TABLE 1 Structures of the synthesized compounds
Sl. no.
R
R0
2,5-Disubstituted compound
1,5-Disubstituted compound
1
C6H5
C6H5
N
N
N
N
N
N
N
N
N N
N
N
4a
7a
2
C6H5
p-BrC6H4
Br
Br
N
N
N
N
N
N
N
N
N
N
N
N
7b
4b
3
C6H5
C6H4CO
N
N
N
N
N
N
O
4c
O
N
N
N
N
N N
7c
4
C6H5
benzimidazol-2-yl
N
N
N
N
N
N
N
N
N
HN
H
N
N
N
N
N
N
4d
7d
5
p-ClC6H4,
C6H5
N
N
N
N
N
N
N
N N
Cl
N N
N
Cl
5a
8a
6
p-ClC6H4
p-BrC6H4
Br
Br
N
N
N
N
N
N
N
Cl
N
N
N
Cl
Cl
N N
8b
5b
7
p-ClC6H4
C6H4CO
O
N
N
N
N
O
N
N
N
N
5c
N
N
Cl
N
N
8c
(Continues)

6
KATTIMANI ET AL.
TABLE 1 (Continued)
Sl. no.
R
R0
2,5-Disubstituted compound
1,5-Disubstituted compound
8
p-ClC6H4
Benzimidazol-2-yl
N
N
N
N
N
N
Cl
N
N
HN
N
H
N
N
N
N
5d
Cl
N
N
N
8d
9
p-CH3O-C6H4
C6H5
N
N
N
N
N
N
N
N
O
N
O
N
N
6a
9a
10
p-CH3O-C6H4,
p-BrC6H4
Br
Br
N
N
N
N
N
N
N
O
N
N
N
O
O
N N
9b
6b
11
p-CH3O-C6H4
C6H4CO
O
N
N
N
N
O
N
N
N
N
N
6c
N
O
N
N
9c
12
p-CH3O-C6H4
Benzimidazol-2-yl
N
N
N
N
N
N
O
N
N
HN
N
H
N
N
N
N
6d
O
N
N
9d
Gasteiger-Huckel charges, and molecular docking was performed with
Surflex-Dock program that is interfaced with Sybyl-X 2.0 (Tripos Interna-
tional, 2012), and other miscellaneous parameters were assigned with the
default values given by the software.
chloranil (II) resulting in the formation of 2a–c (Scheme 2). The obtained
1-aryl-1H-pyrazole-3-carbonitrile (2a–c) was subjected to [3 + 2] cycload-
dition with sodium azide in the presence of triethylamine hydrochloride
to afford corresponding 5-(1-aryl-1H-pyrazol-3-yl)-1H-tetrazole (3a–c).
It is well known that 5-substituted-1H-tetrazole (RCHN4) con-
taining a free N H bond exists mainly in 1H and 2H tautomeric
forms. Consequently, N-alkylation of 5-(1-aryl-1H-pyrazol-3-yl)-1H-
tetrazole (3a–c) with aryl/heteroaryl alkyl halides (i–iv) in the presence
of anhydrous potassium carbonate afforded two regioisomers,
namely, 2,5-disubstituted (4–6 [a–d]) and 1,5-disubstituted (7–9 [a–
d]) tetrazoles nearly in 1:1 ratio. These regioisomers were successfully
separated by column chromatographic technique using hexane:
ethylacetate (9:1) solvent mixture as an eluent. All the synthesized
3
| RESULTS AND DISCUSSION
3.1 | Chemistry
To start with, 3-arylsydnone (1a–c) was ring transformed into 1-aryl-1H-
pyrazole-3-carbonitrile (2a–c) via [3 + 2] dipolar cycloaddition with acrylo-
nitrile in the presence of chloranil. The reaction was initiated with the loss
of carbon dioxide (I) followed by rearomatization of intermediate by

KATTIMANI ET AL.
7
FIGURE 3 (a) Molecular structure of Compound 4b; displacement ellipsoids are drawn at the 50% probability. (b) Unit cell packing diagram of
the molecules of 4b; hydrogen atoms are shown as sphere of arbitrary radius. (c) Planes of the molecule 4b
TABLE 2 In silico pharmacokinetics data of the title Compounds 4a–d, 5a–d, 6a–d, and 7a–d as predicted by Molsoft and admet SAR online
servers
Hydrogen
Bond Acceptor
(accptHB)
< 10c
Hydrogen
Bond Donor
(donorHB)
< 5d
Topological
polar surface
area (TPSA)
< 140 Åe
Molecular
weight
Drug
Blood Brain
Barrier
Entry
no.
Lipinsk's
violation
likeliness
score
Log P < 5a
3.26
4.11
2.61
2.67
3.97
4.83
3.32
3.38
3.35
4.20
2.70
2.76
2.86
3.71
2.21
2.27
<500 amub
permeability
Probability
.98
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
302.13
380.04
330.12
342.13
336.09
414.00
364.08
376.10
332.14
410.05
360.13
372.14
302.13
380.04
330.12
342.13
4
4
5
5
4
4
5
5
5
5
6
6
4
4
5
5
0
0
0
1
0
0
0
1
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
52.58
52.58
65.83
72.86
52.58
52.58
65.83
72.86
60.13
60.13
73.37
80.41
53.41
53.41
66.66
73.69
−0.69
−0.58
−0.77
−0.54
−0.44
−0.92
−0.51
−0.16
−0.70
−0.64
−0.69
−0.43
−0.69
−0.58
−0.77
−0.54
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
.98
.99
.98
.98
.98
.98
.97
.99
.98
.98
.98
.98
.98
.98
.98
aLogarithm of partition coefficient.
bAtomic Mass Unit.
cHydrogen Bond Acceptor.
dHydrogen Bond Donor.
eTopological Polar Surface Area.
compounds (Table 1) were characterized by 1H NMR, 13C NMR, mass
spectral, and elemental analyses. And also we have carried out the
single-crystal XRD studies for Compound 4b.
In case of 1H NMR analysis, the C5 proton of pyrazole ring has
resonated as doublet in the range δ 7.82–8.74 ppm, whereas the C4
proton of pyrazole ring has resonated as doublet at δ 6.98–7.25 ppm.
The C5 carbon of tetrazole ring showed signals between δ 159 and δ
161 ppm. The signal in the range δ 141–142 ppm was attributed to
C3 carbon of pyrazole ring of 2,5-disubstituted tetrazoles and δ
148–149 ppm for 1,5-disubstituted tetrazoles. The C5 and C4 car-
bons of pyrazole ring gave signals around 126–128 and
δ
δ
107–109 ppm, respectively. It was interesting to know that both 2,5-
and 1,5-disubstituted tetrazole derivatives showed slightly different
chemical shift values particularly for the methylene group. Methylene
protons in the case of 2,5-disubstituted tetrazoles were resonated at
slightly upfield values in the range δ 5.77–6.22 ppm when compared
to 1,5-disubstituted tetrazole derivatives which have shown signals at
δ 6.05–6.28 ppm. In case of 13C NMR, the methylene carbon of

8
KATTIMANI ET AL.
TABLE 3 Blood glucose level (mg/dl)
of each group
Entry
Control
Diabetic
4a
0 hr
95
Day 1
107
434
397
434
405
397
423
421
409
461
397
360
392
387
434
384
477
448
386
402
378
407
415
418
397
399
380
Day 2
123
430
382
418
402
386
417
429
415
458
389
348
396
390
412
376
475
449
389
390
395
400
414
390
388
404
320
Day 3
101
455
327
426
397
400
419
418
401
460
404
311
399
394
367
342
466
451
384
365
409
398
409
363
392
395
210
Day 4
97
Day 5
114
441
181
411
407
397
422
418
412
452
404
178
410
386
311
272
471
463
382
315
415
417
420
300
390
393
155
Day 6
102
446
154
427
408
398
425
417
411
457
401
144
406
390
230
220
472
458
389
245
410
410
423
221
395
399
142
Day 7
117
447
126
416
401
402
428
419
406
455
402
113
401
392
144
137
475
460
386
151
406
416
425
138
392
395
131
440
425
444
390
393
417
419
399
454
400
387
398
378
460
396
472
450
382
425
367
411
409
444
395
393
400
457
255
431
415
404
421
420
399
456
406
273
405
389
359
327
469
457
386
337
411
412
417
334
391
383
171
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
G
Note: G represents glibenclamide.
TABLE 4 Effect of compounds on biochemical parameters
Groups/parameter
SGOT
SGPT
Creatinine
Urea
Total protein
7.99 0.27
4.75 0.17
5.57 0.13
5.40 0.20
6.24 0.65
6.15 0.07
6.32 0.11
6.72 0.21
Control
Diabetic
4a
130.33 1.05
214.33 1.81
165.00 3.72
152.33 1.99
138.34 1.45
145.02 1.91
149.17 1.78
155.82 2.41
82.50 0.98
161.50 2.18
117.51 1.43
106.34 1.47
105.52 1.61
94.33 1.12
105.02 1.06
108.01 2.04
0.73 0.35
2.41 0.170
1.41 0.598
1.00 0.027
0.98 0.028
1.16 0.148
1.15 0.155
0.90 0.030
36.23 0.59
86.48 1.21
65.10 1.25
61.33 0.63
55.75 0.94
63.16 0.92
65.83 0.62
65.32 0.61
6b
7a
7b
8b
9b
Note: The data represent the mean SE (n = 6).
Abbreviations: SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic-pyruvic transaminase.
2,5-disubstituted tetrazoles gave signals at
δ 46–57 ppm, while
3.2 | X-ray crystallographic structure analysis of 4b
1,5-disubstituted tetrazoles gave signals at δ 49–56 ppm. All other sig-
nals were in accordance with the proposed structures. The mass spec-
tral analysis of all the title compounds has shown the m/z values
which correspond to their molecular mass.
Crystals of Compound 4b were developed by slow evaporation of dry
acetone at room temperature. The asymmetric unit of Compound 4b
is shown in Figure 3b. The dihedral angle (Figure 3c) between

KATTIMANI ET AL.
9
tetrazole ring and pyrazole and two terminal benzene rings of mole-
cules are 4.21ꢀ, 71.69ꢀ, 4.76ꢀ, respectively. In the structure, all bond
lengths and angles are within normal the range (Zhang, 2009). The
crystal structure is further stabilized by intermolecular C Hꢁ ꢁ ꢁN and
intramolecular C Hꢁ ꢁ ꢁN hydrogen bonds (Figure 3).
3.3 | Pharmacology
3.3.1 | Drug likeliness parameters
To be an effective antidiabetic drug, the candidate compounds must
be able to cross the Blood Brain Barrier (BBB) to exert their action.
They should also possess good pharmacokinetic and oral bioavailabil-
ity properties (Kothayer, Ibrahim, Soltan, Rezq, & Mahmoud, 2018).
The drug-like properties of the designed compounds were predicted
using the web tool Molinspiration (www.molinspiration.com) based on
Lipinski's (2004) “Rule of five”. Furthermore, the compounds with
BBB permeability were predicted via the admet structure activity rela-
tionship (SAR) server (http://lmmd.ecust.edu.cn/admetsar1/predict/).
The data are listed in Table 2. As seen from the data, all the com-
pounds have a high probability of crossing the BBB. None of the com-
pounds violate Lipinski's rules, making our compounds excellent
candidates as Central Nervous System (CNS)-active compounds.
3.3.2 | Biological testing
FIGURE 4 Effect of compounds on SGOT, SGPT, and urea.
Values are given as mean SE. SGOT, serum glutamic oxaloacetic
transaminase; SGPT, serum glutamic-pyruvic transaminase
In vivo antihyperglycemic activity
The in vivo antihyperglycemic activity of the newly synthesized Com-
pounds 4a–d, 5a–d, 6a–d, 7a–d, 8a–d and 9a–d was examined against
streptozotocin-induced hyperglycemic rats using antihyperglycemic
FIGURE 5 Effect of compounds on creatinine and total protein
parameters. Values are given as mean SE
FIGURE 6 Effect of compounds on lipid profiles (values are given
as mean SE)
TABLE 5 Effect of compounds on lipid profile
Groups/parameters
TC
TG
HDL
LDL
VLDL
Control
Diabetic
4a
133.50 1.11
259.83 5.44
185.11 4.17
164.33 2.61
146.17 3.02
152.67 1.49
148.50 1.91
146.17 2.13
114.17 1.40
184.83 2.93
155.33 2.83
144.50 2.45
139.40 4.37
136.67 2.10
132.83 1.77
144.17 2.65
58.10 0.55
29.58 1.56
34.50 1.47
45.16 1.35
45.83 2.08
52.50 1.23
45.33 1.60
46.83 1.16
76.33 0.96
259.33 3.97
158.67 5.70
143.50 1.36
106.17 1.19
106.17 1.66
113.33 0.88
114.83 1.88
45.19 0.82
86.16 1.04
58.00 1.54
63.50 2.55
64.66 0.88
72.50 1.23
62.50 1.87
62.00 1.36
6b
7a
7b
8b
9b
Note: The data represent the mean SE (n = 6).
Abbreviations: HDL, high-density lipoprotein; LDL, low-density lipoprotein; TC, total cholesterol; TG, triglyceride; VLDL, very low-density lipoprotein.

10
KATTIMANI ET AL.
FIGURE 7 Histology of pancreas of normal control, diabetic control, and diabetic-treated rats
assay (using Wistar albino rats of either sex having average b.w. of
190 20 g). Glibenclamide included in the experiment as a standard
drug. The results were expressed as mg/dl reduction in blood glucose
level (Table 3). Most of the synthesized molecules showed excellent
to modest antihyperglycemic activity against streptozotocin-induced
hyperglycemic rats.
Effect of compounds on blood glucose level
The antihyperglycemic activity of synthesized compounds was affi-
rmed by measuring blood glucose level for 28 days. As shown in
Table 3, the blood glucose levels of diabetic control rats increased sig-
nificantly after the injection of streptozotocin, compared with normal
(Group I) rats. Administration of Compounds 4a, 6b, 7a, 7b, 8b, and

KATTIMANI ET AL.
11
TABLE 6 Surflex docking score (kcal/mol) of the title Compounds 8(k–z) on enzyme glycogen phosphorylase (PDB ID: 1L5Q; B chain)
Entry no.
C-scorea
4.43
Crash scoreb
−0.96
Polar scorec
0.33
D-scored
PMF scoree
−5.70
4.46
G-scoref
−222.21
−247.38
−200.51
−234.76
−209.09
−248.45
Chem scoreg
4a
6b
7a
7b
8b
9b
−4,976.23
−5,050.07
−4,828.30
−4,947.20
−4,729.18
−5,201.23
−30.35
−38.77
−27.33
−35.06
−28.55
−36.55
6.05
−2.47
2.75
4.28
−0.36
1.13
−5.13
−2.12
9.94
4.48
−1.58
1.57
4.47
−0.56
0.81
6.41
−1.79
2.17
3.21
aC-score integrates a number of popular scoring functions for ranking the affinity of ligands bound to the active site of a receptor and reports the output of
total score.
bCrash score revealing the inappropriate penetration into the binding site. Crash scores close to 0 are favorable. Negative numbers indicate penetration.
cPolar score indicating the contribution of the polar interactions to the total score. The polar score may be useful for excluding docking results that make
no hydrogen bonds.
dD-score for charge and van der Waals interactions between the protein and the ligand.
ePotential of mean force (PMF) score indicating the Helmholtz free energies of interactions for protein–ligand atom pairs.
fG-score showing hydrogen bonding, complex (ligand–protein), and internal (ligand–ligand) energies.
gChem score points for H-bonding, lipophilic contact, and rotational entropy, along with an intercept term.
9b daily caused gradual lowering of blood glucose level and finally
reached to normal in 5–7 days which was comparable with the stan-
dard drug glibenclamide. While rats treated with other than these
compounds did not show any significant decrease in blood glucose
levels (<200 mg/dl).
TG, LDL, and VLDL levels and increased HDL level compared to
untreated diabetic rats (Figure 6).
Histopathological studies
Histology of pancreas in experimental rats was determined after
28 days of treatment. Normal control rats' normal architecture of
islets of Langerhans are presented in Figure 7 (1). Diabetic control
group-pancreatic sinusoid necrosis and degranulated islet cells
with vanishing of nuclei resulting shrunken islets of Langerhans
are presented in Figure 7 (2), diabetic rats treated with Compound
4a, and the pancreas showing regeneration of islets of Langerhans
are shown in Figure 7 (3). Diabetic rats treated with Compound 6b
and islets with beta cells containing more cytoplasm and normal
exocrine acini are shown in Figure 7 (4). Diabetic rats treated with
Compound 7a and islets with beta cells containing more cyto-
plasm and normal exocrine acini are presented in Figure 7 (5). Dia-
betic rats treated with Compound 7b and pancreas showing
regeneration of islets of Langerhans are shown in Figure 7 (6).
Diabetic rats treated with Compound 8b and hence pancreas
showing regeneration of islets of Langerhans are shown in
Figure 7 (7). Diabetic rats treated with Compound 9b islets with
beta cells containing more cytoplasm and normal exocrine acini
are presented in Figure 7 (8).
Effect of compounds on SGOT, SGPT, creatinine, urea, and total
protein
The SGOT and SGPT are reliable markers of liver function which are
released during liver necrosis in diabetic-induced rats. An increase in
the activities of SGOT and SGPT in the plasma is due to the leakage
of these enzymes from the liver cytosol into the blood stream which
gives the indication of hepatic dysfunctions. The serum creatinine and
urea will be high in diabetic-induced rats. However, the total protein
will be reduced due to insufficient insulin release which stimulates
protein synthesis and retards protein degradation.
As shown in Table 4, streptozotocin-induced diabetic rats showed
significant increase in SGOT, SGPT, creatinine, and urea levels, while
total protein was significantly reduced. Oral administration of Com-
pounds 4a, 6b, 7a, 7b, 8b, and 9b has decreased the concentrations of
serum SGOT, SGPT, creatinine, and urea, and increased the total pro-
tein content in diabetic rats compared to untreated diabetic rats
(Figures 4 and 5).
Effect of compounds on lipid profiles
Structure activity relationship
DM is associated with changes in the plasma lipid profile and is con-
sidered to be major risk for coronary heart disease. In diabetic individ-
ual, LDL and VLDL carry cholesterol to the peripheral tissues where it
is deposited, while HDL carry cholesterol from peripheral tissue to
liver, thus facilitating its excretion and metabolism.
As outlined in the rationale, we aimed at studying the SAR of
5-(1-aryl-1H-pyrazol-3-yl)-1H-tetrazoles 4a–d, 5a–d, 6a–d, 7a–d, 8a–
d, and 9a–d hybrids as potential antihyperglycemic agents targeting
GP enzyme. Observing the results of different in vivo biological exper-
iments, we could presume valuable data about the SARs. The obtained
results of in vivo antihyperglycemic activity in Table 4 shows that 4a–
d, 5a–d, 6a–d, 7a–d, 8a–d, and 9a–d exhibited moderate-to-excellent
antihyperglycemic activity. Among the tested compounds, Com-
pounds 4a (2,5-disubstituted tetrazole containing N-substituted p-
Entries of Table 5 indicated that in streptozotocin-induced rats,
TC, TG, LDL, and VLDL levels were increased and HDL level was
decreased compared to normal rats. Administration of Compounds 4a,
6b, 7a, 7b, 8b, and 9b showed significant reduction in elevated TC,

12
KATTIMANI ET AL.
FIGURE 8 Docked view of Compound 9b at the active site of the enzyme glycogen phosphorylase enzyme
phenyl side chain, N-phenyl pyrazole), 6b (2, 5-disubstituted tetrazole
3.4 | Docking study
containing N-substituted p-bromobenzyl side chain, N-substituted p-
Docking simulation has grown to be a significant part of drug discov-
ery analysis in the present days and it imparts information of the
structural and functional aspects of proteins and ligands. In this
regard, with in vivo antihyperglycemic activity results in hand, it was
appropriate to carry out in silico studies to support the in vivo activity.
The potent antihyperglycemic compounds, namely, 4a, 6b, 7a, 7b, 8b,
and 9b were docked into the active site of the GP (PDB ID: 1L5Q; B
chain). The predicted binding energies and the observed consensus
score (C-score) values of potent molecules are ranging from 4.28 to
6.41 (Table 6). The C-score values representing the summary of all
forces of interaction between ligands and the GP. It may be concluded
that all potent compounds have exhibited very good docking score
against target enzyme.
anisyl pyrazole), 7a (1,5-disubstituted tetrazole containing N-
substituted p-phenyl side chain, N-phenyl pyrazole), 7b
(1,5-disubstituted tetrazole containing N-substituted p-bromobenzyl
side chain, N-substituted p-anisyl pyrazole), 8b (1,5-disubstituted
tetrazole containing N-substituted p-bromobenzyl side chain, N-
substituted p-chlorophenyl pyrazole), and 9b (1,5-disubstituted
tetrazole containing N-substituted p-bromobenzyl side chain, N-
substituted p-anisyl pyrazole) have shown potent in vivo
antihyperglycemic activity.
From the above results, it is clear that several 1,5-disubstituted
tetrazole derivatives comprising N-substituted p-bromobenzyl side
chain (viz., 6b, 7b, 8b, and 9b) have shown maximum fall in the blood
glucose levels and exhibited significant decrease in SGOT, SGPT, urea,
and creatinine coupled with increase in the total protein levels. Also,
these compounds have exhibited antihyperlipidemic activities as
evidenced by decrease in TC, TG, LDL, and VLDL coupled with the
elevation of HDL levels. Moreover, the introduction of a halogen sub-
stituent at the phenyl ring resulted in enhanced antihyperglycemic
activity. The rise in the in vivo activity with respect to the substituents
follows the order as follows: Br > Cl > OCH3 > H. Interestingly,
in vivo antihyperglycemic activity revealed that the 1,5-regiosomers
have shown more inhibition compared to 2,5-regioisomers rep-
resenting the regioselectivity of an enzyme. These results were fur-
ther confirmed by docking studies.
Among docked compounds, Compound 9b acts as very good
inhibitor of GP enzyme. As depicted in Figure 8, Compound 9b
makes six hydrogen bonding interaction at the active site of the
enzyme (PDB ID: 1L5Q; B chain), among them four interactions
came from the tetrazole ring, out of which three hydrogen bonding
interactions were raised from nitrogen atoms of tetrazole ring with
hydrogen of ARG569 ( Nꢁ ꢁ ꢁH-ARG569) and another one with
hydrogen of LYS574 ( Nꢁ ꢁ ꢁH-LYS574). Also, nitrogen atom of
pyrazole ring makes a hydrogen bonding interaction with hydrogen
of LYS574 ( Nꢁ ꢁ ꢁH-LYS574), and oxygen atom of methoxy group
present at the fourth position of aromatic ring makes hydrogen

KATTIMANI ET AL.
13
bonding interaction with hydrogen of ASN696
ASN696).
(
OCH3ꢁ ꢁ ꢁH-
ORCID
Ravindra R. Kamble
https://orcid.org/0000-0002-0384-655X
4
| CONCLUSIONS
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