Synthesis, Characterisation, Molecular Docking, Anti-microbial and Anti-diabetic Screening of Substituted 4-indolylphenyl-6-arylpyrimidine-2-imine Derivatives

Article abstract of DOI:10.1055/s-0043-106444

The purpose of the research is to synthesise a novel series of (E)-2-(4-(1H-indol-3-yl)-6-p-substituted phenylpyrimidin-2-yl)dimethylguanidine derivatives since 3-(1H-indol-3-yl)-1-p-substituted phenylprop-2-en-1-one and evaluate their molecular docking s

Full text of DOI:10.1055/s-0043-106444

Thieme
Original Paper
Synthesis, Characterisation, Molecular Docking, Anti-microbial
and Anti-diabetic Screening of Substituted 4-indolylphenyl-6-
arylpyrimidine-2-imine Derivatives
Authors
Supporting Information for this article is available online at
Veerasamy Ramya1, Santhirakasu Vembu1, Ganesan
Ariharasivakumar2, Manathusamy Gopalakrishnan1
http://www.thieme-connect.de/products
AbStrAct
Affiliations
1
Department of Chemistry, Annamalai University, Annamalai
The purpose of the research is to synthesise a novel series of (E)-2-(4-
(1H-indol-3-yl)-6-p-substituted phenylpyrimidin-2-yl)dimethylguani-
dine derivatives since 3-(1H-indol-3-yl)-1-p-substituted phenylprop-
2-en-1-one and evaluate their molecular docking studies,
antimicrobial, and anti-diabetic activities. Among all the synthesized
compounds (11a-g), compound 11a exhibits excellent CDOCKER
energy (−11.36kcal/mol). The entire compounds (11a-g) confirm very
good antimicrobial activity towards the tested microorganisms. In the
in vitro anti-diabetic studies, compounds (11a, 11c, and 11g) confirm
higher alpha-amylase and alpha-glucosidase inhibition activity. In the
in vivo anti-diabetic activities, the synthesized compounds (11a-g)
(10 mg/kg, p.o.) investigated by the streptozotocin (60 mg/kg, ip) –
nicotinamide (120 mg/kg, p.o.) – induced model in adult male albino
Wistar rat and these derivatives show considerable fasting blood glucose
level when compared to metformin hydrochloride a potent and well-
known anti-diabetic drug as a reference.
Nagar, Tamil Nadu, India
2
Department of Pharmacology, KMCH College of Pharmacy, Kovai
Estate, Tamil Nadu, India
Key words
Indole-3-carboxaldehyde, Metformin, Molecular docking, Type 2
diabetes, In vitro enzyme inhibition, Streptozotocin
Bibliography
DOI http://dx.doi.org/10.1055/s-0043-106444
Published online: 2017
Drug Res
© Georg Thieme Verlag KG Stuttgart · New York
ISSN 2194-9379
Correspondence
M. Gopalakrishnan
Department of Chemistry
Annamalai University
Annamalai Nagar
Chidambaram 608 002
Tamil Nadu
India
Tel.: + 91/0944/2389 644,
mgkrishnan61@gmail.com
Abbreviation
tion, adult blindness, neuropathy, kidney failure, strokes, and heart
attack. The collective evidence has been used to divide diabetes into
four types namely, insulin dependent diabetes mellitus (type 1,
IDDM), non-insulin dependent diabetes mellitus (type 2, NIDDM),
malnutrition-related diabetes and other types of diabetes [3]. In
type 1 diabetes, there is destruction of beta-cells of the pancreas,
with consequent insulin deficiency. The signs of IDDM are polydip-
sia, polyurea, and weight loss. In type-2 diabetes the pancreas pro-
duces insulin, but the body does not take the insulin correctly. This
may be due to peripheral tissue insulin obstruction where insulin
receptors or other intermediates in the insulin signalling pathways
within body cells are insensitive to insulin and consequently, glu-
cose does not readily enter the tissues leading to hyperglycemia or
elevated blood glucose concentrations [4, 5].
DM
Diabetes Mellitus
MIC
GK
Minimum Inhibitory Concentration
Glucokinase
DMSO
STZ
Dimethylsulfoxide
Streptozotocin
p.o
per oram
i.p
Intraperitonial
NIDDM
TMS
Non –Insulin-Dependent Diabetes Mellitus
Tetramethylsilane
Introduction
Diabetes mellitus (DM) is a metabolic disorder [1] characterized by
chronic hyperglycemia with disturbances in fat, protein, and car-
bohydrate metabolism, resulting from defects in insulin action, in-
sulin secretion, or both [2]. Diabetes Mellitus is the major effect of
final-stage renal disease, non-traumatic lower extremity amputa-
Obesity is a common problem for this type, and most patients
with NIDDM are obese [6] and will require multiple antihypergly-
cemic agents to maintain glycemic control [7]. Four types of oral
anti-diabetic agents (▶Fig. 1) are available namely, insulin secre-
Ramya V et al. Type-2 Diabetic Potential Compounds… Drug Res

Thieme
Original Paper
O
N
N
N
H
HN
C
NH2
NH
NH NH
CH 3
N
O2S
NH
CH 3
H
N
NH2
HN
O
N
H
N
H
NH2
H3C
C
NH
N
NH
NH
H
2
3
4
1
H
N
O
OH
N
N
O
H
O2
S
N
OCH 3O
N
H
N
H
CHO
HO
O
OH
N
H
OH
Cl
5
7
6
▶Fig. 1 Chemical structure of some known anti-diabetic agents and some other compounds.
tagogues (sulfonylureas), biguanides (phenformin, buformin, and
metformin), thiazolidinediones (pioglitazone) and alpha-glucosi-
dase inhibitors. The main function of sulfonylureas (tolbutamide,
glimepiride, and glipizide) is to stimulate pancreatic insulin secre-
tion. Metformin originates from Galega officinalis, reduces the sign
of diabetes. The active compound of metformin is gelatine, guan-
idine derivatives. Metformin is not only an inexpensive drug but
also has several other beneficial pharmacological effects which in-
clude reduction and stabilization of weight [8], reduces the chang-
es of hypoglycemia [9] and other beneficial vascular effects. Met-
formin is not metabolized; it's important sites of concentration are
the intestinal mucosa and the salivary glands. The mode of action
of metformin is not fully understood. It has been postulated that
metformin might potentiate the impact over insulin then that
would possibly decorate the impact about insulin in the peripheral
receptor site. This accelerated sensitivity follows an increase in
number of insulin receptors on cell surface membranes.
The hetero cyclic molecules, which possess indole [10] moie-
ties, exhibit an extensive extent of biological activities [11]. Indole
alkaloids prove to be medicinally essential natural compounds.
From the above discussion, of considering the biological proper-
ties of the indole ring, it is planned to synthesize a novel series of
4-indolylphenyl-6-arylpyrimidine-2-imine derivatives having a side
chain with different structures; such derivatives could possess very
useful and important anti-diabetic activity.
various substituted 3-(1H-indol-3-yl)-1-phenylprop-2-en-1-one
(9a-g) derivatives are carried out by the condensation of indole-
3-carboxaldehyde 7 by p-substituted acetophenones 8 and the
products are purified by recrystallising ethyl alcohol. In the second
scheme synthesis of 4-indolylphenyl-6-arylpyrimidine-2-imines
(11a-g) is carried out by the reaction of substituted 3-(1H-indol-
3-yl)-1-phenylprop-2-en-1-one (9a-g) derivatives with metformin
hydrochloride 10 and sodium methoxide in absolute ethanol and
the products are purified by recrystallisation from ethyl alcohol as
a suitable solvent. The structures of the synthesized products are
established by FT-IR, 1H & 13C NMR, mass spectrometry, and ele-
mental analysis. In FT-IR spectra, the disappearance of the C = O
band in the characteristic range and appearance of the C= N band
in the range of 1 543–1 606 cm− 1 are evidence for ring closure of
the pyrimidine ring of compounds (11a-g). The synthesized com-
pounds (11a-g) also show the presence of -NH2 band in the region
of 3 162–3 463 cm− 1 and the compounds (11a-g) reveal charac-
teristic bands at 3107–3426cm− 1 (NH-indole), 2342–2957cm− 1
(aromatic C-H), 2 106–2 853 cm − 1 (aliphatic C-H). The 1H NMR
spectra of compounds (11a-g) reveal the following signals: a sin-
glet equivalent to one proton in the δ 12.17–10.76ppm range char-
acteristic of an indole -NH proton, a singlet equivalent to two pro-
tons at δ 8.75-8.41ppm characteristic of -NH2 protons, a multiplet
at δ 8.58-6.44 ppm characteristic for the aromatic protons, a dou-
blet at δ 8.21–8.65 ppm characteristic of the pyrimidine ring pro-
ton which confirmed the cyclization of the chalcone into the py-
rimidine ring. The singlet between δ 3.82-2.50ppm due to -N(CH3)2
protons, besides the presence of two singlets at δ 3.82ppm and δ
2.49ppm corresponding to the methyl protons of –OCH3 and –CH3
respectively. In the 13C NMR spectra of compounds the chemical
shift values of carbon atoms appear between δ 168.8-158.0 ppm
due to pyrimidine ring ipso carbons, δ 158.0-157.4 ppm due to
guanidine group ipso carbon, δ 137.6-111.3ppm due to aromatic
Result and Discussion
Chemistry
The novel series of 4-indolylphenyl-6-arylpyrimidine-2-imines
(11a-g) are synthesized according to the method shown in
▶Fig. 2 (Scheme 1 & Scheme 2). In the first scheme synthesis of
Ramya V et al. Type-2 Diabetic Potential Compounds… Drug Res

Scheme 1
R
O
H
N
C
C
CH
9a, 11a R = H
(i)
H
+
9b, 11b R = CH3
9c, 11c R = OCH3
9d, 11d R = NO2
9e, 11e R = F
CHO
N
H
C
8
7
H3C
O
9a – g
R
9f, 11f R = Cl
9g, 11g R = Br
Reagents and conditions;
(i) Ethanol, NaOCH3, Reflux, 12 h, 80 – 85 %
(7) Indole-3-carboxaldehyde
(8) p-Substituted acetophenone
R
Scheme 2
O
H3C
CH3
C
C
CH
N
NH
H
(i)
.
HCl
+
6
1N
2
NH2
H
5
4
HN
N
H
N
H
CH3
3
N
N
H
N
N
9a – g
R
3
CH3
N
H
11a – g
Reagents and conditions;
(i) NaOCH3 Ethanol, Reflux,72 h, 70 – 75 %
(3) Metformin hydrochloride
▶Fig. 2 Synthetic scheme of 4-indolylphenyl-6-arylpyrimidine-2-imine derivatives (11a-g).
▶table 1 Docking results of the designed compounds (11a-g) towards
1v4s.
carbon atoms, δ 106.7-103.9ppm due to the one proton attached
carbon of the pyrimidine ring, δ 37.3-36.7 ppm due to guanidine
group -N(CH3)2 carbon atoms and δ 27.3 ppm, δ 55.2 ppm due to
phenyl ring attached –CH3, -OCH3 respectively. Moreover, the el-
emental analysis of compounds (11a-g) agrees with the proposed
structure. The mass spectra of compounds (11a-g) are observed
as characteristic molecular ion peaks corresponding to their mo-
lecular formula.
S.NO
ENtrY
ꢀ−ꢀCDOCKERꢀ
ENErGY kcal/mol
ꢀ−ꢀCDOCKERꢀ
INtErActION
ENErGY kcal/mol
1
2
3
4
5
6
7
8
11a
11b
11.36
8.77
4.87
4.84
5.43
5.93
9.13
21.60
44.70
42.08
41.17
39.75
36.47
42.53
41.16
28.75
11c
11d
Molecular docking studies
11e
Molecular docking study conveys out over glucokinase (1v4s) as
much an anti-diabetic protein and synthesized compounds (11a-g)
are performed using a CDOCKER protocol over Discovery Studio
(v 16.1.0.15350). The results are analyzed for docking interaction
[12] of the human glucokinase (1v4s) with compounds (11a-g).
Glucokinase [13] is found only in pancreatic beta cells and liver per-
forms a key role in the regulation of glucose metabolism. In the
beta cells, glucokinase is believed to be in accordance with the re-
maining portion of the glucose absorbance and to be involved in
the regulation of insulin release. The diabetic mice are treated along
11f
11g
Metformin
3-HMX active principle beyond plant expanded glucokinase activ-
ity [14]. From that report, the compounds (11a-g) increased glu-
cokinase activity by binding with the metformin. Therefore, in-
creasing the application of glucose is preferred to reduce blood
glucose level. The ligands have been ranked after docking, based
Ramya V et al. Type-2 Diabetic Potential Compounds… Drug Res

Thieme
Original Paper
▶Fig.ꢀ5 Docking interactions of glucokinase (1v4s) protein with
compound 11a.
(1v4s) structure of the active site residues is performed by discov-
ery studio. The structure of 11a shows interaction with the resi-
dues MET A: 235; MET A: 210; PRO A: 66; ARG A: 63; ILF A: 211; VAL
A: 452; THR A: 65; GLN A: 98; TYR A: 215. The interaction of the
best-docked structure 11a is shown in the ▶Fig. 4,ꢀꢁ5. It is evident
from this analysis that the best inhibitors are located in the center
of the active site and is stabilized by H-bonding interactions. Out
of seven docked complexes, only one best docking compound
(11a) is got which shows lowest CDOCKER energy with the amino
acid residues of the receptor molecule.
▶Fig. 3 Two-dimensional diagram of compound 11a docked with
1v4s protein.
In vitro antimicrobial activity
The review of literature [15] indicates that the metformin is an an-
tihyperglycemic drug having antimicrobial activity, for diabetic pa-
tients it would be an additional advantage. It could produce more
immunity in the body of the diabetic patient. Hence one should ex-
amine the synthesized compounds (11a-g) containing a metform-
in moiety which also been possesses the same microbial activity or
not. Hence the antimicrobial activities of the synthesized com-
pounds (11a-g) are reported. In vitro antimicrobial activity [16]
was evaluated by measuring the minimum inhibitory concentra-
tion (MIC in µg/ml) and the diameter zone of inhibition (DZI in mm).
▶table 2, 3 illustrate the screening test of compounds (11a-g) on
five fungal strains (Candida albicans, Candida glabrata, Candida
krusei, Candida tropicalis and Candida parapsilosis) and two Gram-
positive (Staphylococcus aureus, Bacillus subtilis), three Gram-neg-
ative bacterial strains (Escherichia coli, Vibrio cholerae and Proteus
vulgaries). Compounds 11a, 11c, 11e, 11f, and 11g exhibits sig-
nificantly high antibacterial activity at a diameter of inhibition
zones ( > 21 mm) and minimum inhibition concentration ( < 6.25)
against the bacterial strains as compared to standard drug Cipro-
floxacin. Compounds 11b and 11c are exhibit moderate antibac-
terial activity at MIC (12.5 µg/mL) and DIZ ( < 20 mm) towards Vi-
▶Fig. 4 The structure of glucosidase (1v4s) protein in complex with
compound 11a.
on their binding and CDOCKER energy. The final docked results of
selected ligands are given in the ▶table 1. From that result, the
synthesized compound 11a shows a good cdocker energy
( − 11.36 kcal/mol) and cdocker interaction energy ( − 44.70 kcal/
mol). The cdocker energy value of compounds 11b and 11g
are −8.77kcal/mol and −9.13kcal/mol respectively. A two-dimen-
sional structure of the synthetic ligand (11a) is produced using dis-
covery studio (v 16.1.0.15350) (▶Fig. 3). In the two-dimensional
structure, the pale green dotted lines represent the Van der Waals
interaction, dark green dotted lines represent the conventional hy-
drogen bonding, pale blue dotted lines represent the Carbon-Hy-
drogen bond and pink dotted lines represent the Pi-alkyl interac-
tion. Docking of these optimized compounds against a glucokinase
Ramya V et al. Type-2 Diabetic Potential Compounds… Drug Res

Ramya V et al. Type-2 Diabetic Potential Compounds… Drug Res

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Original Paper
▶table 4 In vitro anti-diabetic activity of compounds (11a-g).
compounds α-glucosidaseꢀinhibitoryꢀactivityꢀConcentrationsꢀ Ic 50ꢀ(μg/ml)
α-amylaseꢀinhibitoryꢀactivityꢀConcentrationsꢀ
Ic 50ꢀ(μg/ml)
(µg/ml)
(µg/ml)
20
40
60
80
100
89.21
87.65
86.67
79.43
88.12
89.02
91.21
20
40
60
80
100
11a
11b
11c
11d
11e
11f
20.01
22.12
21.23
20.32
19.33
22.21
21.32
37.21
33.33
30.23
29.34
30.32
32.13
31.21
56.21
54.43
54.32
38.43
57.32
49.21
59.32
68.32
68.32
65.32
48.44
62.27
59.54
67.43
55.98
56.94
58.25
66.01
57.46
60.73
56.27
30.33
31.22
31.21
30.21
32.56
32.45
31.76
56.34
54.32
55.45
56.32
50.59
58.32
52.58
63.32
67.43
65.66
61.98
67.77
60.33
68.56
78.31
71.21
74.44
75.33
77.56
74.78
75.44
83.23
92.22
93.43
90.21
91.56
90.78
91.12
49.50
51.02
50.09
53.53
49.94
52.61
49.90
11g
Values are expressed as mean ± SD (n = 3)
brio cholerae, Staphylococcus aureus, and Escherichia coli. Simi-
larly compounds 11a, 11e and 11g exhibit better antifungal
activity against Candida glabrata, Candida krusei, Candida tropi-
calis and Candida parapsilosis at MIC ( < 12.5 µg/mL) and DIZ
( > 15 mm) as compared to the standard drug Amphotericin B. The
screening consequences revealed as the newly designed com-
pounds (11a-g) containing halogen groups have shown significant
antibacterial [17] and antifungal activity compared to correspond-
ing standard drugs. It reveals that the metformin residue and the
indole site attached to the compounds may increase the antimi-
crobial activities [18–23] of the entire compounds (11a-g).
▶Fig. 6 Alpha-glucosidase inhibition activity of compounds (11a-g).
In vitro enzyme inhibition activity assay
The novel series of 4-indolylphenyl-6-arylpyrimidine-2-imines (11a-
g) hold definite promises in the remedy regarding diabetes mellitus
(DM). Two carbohydrates hydrolysing enzymes [24] (alpha-amylase
and alpha-glucosidase) are responsible for postprandial hyperglyce-
mia. Alpha-glucosidase catalyzes the disaccharides to a monosac-
charide, leading to postprandial hyperglycemia and alpha-amylase
begins the process of carbohydrate metabolism by hydrolysis of 1,
4-glycosidic linkage of polysaccharides (starch, glycogen) to disac-
charides [25,26]. Hence, inhibitors of alpha-amylase [27] and alpha-
glucosidase are beneficial within the control of hyperglycemia as they
delay carbohydrate metabolism, which consequently reduces the
postprandial blood glucose level. In the current study, compounds
(11a-g) have been evaluated because of their inhibitory effect on
alpha-amylase and alpha-glucosidase [28] enzymes by in vitro meth-
od. The inhibitory activity of the designed compounds against alpha-
amylase and alpha-glucosidase are proved within ▶table 4, ▶Fig.
6,ꢀꢁ7. Compounds of different concentrations are used to assess their
inhibitory potential against alpha-glucosidase. Hence the com-
pounds 11a and 11g show alpha-glucosidase inhibitory activity with
an IC50 value of 55.98µg/ml and 56.27µg/ml respectively (▶Fig. 8).
Among the seven synthesized compounds, compound 11a, 11e, and
11g exhibit the highest inhibition of alpha-amylase with an IC50 value
of 49.50µg/ml, 49.94µg/ml, and 49.90µg/ml respectively (▶Fig.
9). Overall, our result suggests that compounds of 4-indolylphenyl-
6-arylpyrimidine-2-imines (11a-g) show maximum potent as natu-
ral alpha-amylase and alpha-glucosidase inhibitors decrease the me-
tabolism of dietary starch.
▶Fig. 7 Alpha-amylase inhibition activity of compounds (11a-g).
▶Fig. 8 Alpha-glucosidase IC50 values of compounds (11a-g).
Ramya V et al. Type-2 Diabetic Potential Compounds… Drug Res

▶Fig. 9 Alpha-amylase IC50 values of compounds (11a-g).
In vivo anti-diabetic activity
Administration over streptozotocin [29] causes diabetes by the
rapid destroy of pancreatic beta cells, thereby brings about a re-
duction concerning insulin release and induction of insulin resist-
ance, both of which are associated with type 2 diabetes[30]. In the
present study, an increase in blood glucose level among diabetic ani-
mals confirms the installation of diabetes mellitus. The anti-diabetic ef-
fect about more than a few doses of compounds (11a-g) in normal
(Group 1) and streptozotocin (60mg/kg, ip) -nicotinamide (120mg/kg
p.o.) -induced (Group 2) diabetic rats are assessed at different time pe-
riods. The oral administration of a single dose of designing compounds
brought about a massive reduction of serum glucose in diabetic rats.
Blood glucose level analysis
The anti-diabetic recruited on synthesized compounds (11a-g) is in-
vestigated in streptozotocin (60mg/kg, ip) - nicotinamide (120mg/
kg, p.o.) - induced diabetic adult male albino Wistar rats [31]. The
change of blood glucose level from the initial to 28th day fasting gly-
cemia is shown in the ▶Tableꢀ5. ▶Fig. 10 indicates the change in
blood glucose level in control and experimental normal rats received
(11a-g) (10mg/kg, p.o.) and standard drug metformin (10mg/kg,
p.o.) [32]. The highest blood glucose level is noted on the 28th day
of the test for the experimental positive and negative control rats.
Hence, the compound (11a, 10mg/kg, p.o. 152.23mg/dl) and the
positive control (metformin, 10mg/kg, p.o. 154.23mg/dl) retained
[33] the blood glucose level to the fasting glycemia after the 15th
day. However for compounds 11b, 11c, and 11d the blood glucose
level on the final day is 182.5± 11 mg/dl, 180.232± 12 mg/dl, and
181.32 ± 12 mg/dl respectively are less potent and take a longer
period during the experiments. The moderate blood glucose
levels are observed in the compounds 11e (170.21 mg/dl), 11f
(167.45 mg/dl) and 11g (173.44 mg/dl) on the last day of experi-
ments. The compounds (11a-g) steadily exert an anti-diabetic
effect at the tested doses when compared to the negative control
(diabetes mice). The improved diabetic control between oral glucose
tolerance tests with the aid of the 4-indolylphenyl-6-arylpyrimidine-
2-imine derivatives suggests that the compounds lower the blood
glucose levels also in normal rats. The effect of decreasing blood
glucose level in normal healthier rats is due to the increased efficien-
cy of the peripheral tissues for the uptake of glucose from the blood.
Ramya V et al. Type-2 Diabetic Potential Compounds… Drug Res

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Original Paper
Conclusions
Body weight analysis
After Streptozotocin (60 mg/kg, ip) - nicotinamide (120 mg/kg,
p.o.) administration, the body weight of rats [34, 35] is observed
after compounds (11a-g) (10 mg/kg. p.o.) and in standard drug
metformin (10 mg/kg, p.o.) administration when compared to di-
abetic control rats. Normal body weight gain is an indication of ef-
ficient glucose homeostasis; but among hyperglycemic, glucose is
not available; consequently the cells make use of alternative pro-
teins for energy; Due to excessive breakdown of tissue protein, a
loss in body weight occurs. Results on the effect of compounds
(11a-g) on body weight regarding streptozotocin (60mg/kg, ip) -
nicotinamide (120mg/kg, p.o.) induce diabetic among the animals
after 4 weeks of treatment are summarized in ▶table 6. At the end
of 4th week, diabetic rats gain less body weight with significant de-
crease in the normal healthy control animals. The novel series of
4-indolylphenyl-6-arylpyrimidine-2-imines (11a-g) improve the
body weight of diabetic rats with a significant increase compared
to the diabetic control rats (▶Fig. 11). This investigation thus indi-
cates that the compounds 11a (153.89g) and 11g (155.76g) sig-
nificantly increase the body weight [36, 37] of nicotinamide
In conclusion, the docking studies of the seven ligands (11a-g) with
target protein 1v4s show that compound 11a and 11g are good
molecules which docks well with target correlated to diabetes mel-
litus. Based on a biological evaluation resulting data, the entire
compounds (11a-g) show good results to moderate antimicrobial
activity against tested microorganism by both MIC and disc diffu-
sion method. The compounds (11a-g) exhibit significant in vitro
anti-diabetic activity using alpha-amylase and alpha-glucosidase
enzymes as well as in vivo anti-diabetic activity using streptozo-
tocin-nicotinamide-induced animal model. This study reveals that
compounds (11a and 11g) an in vivo anti-diabetic examination,
bringing back the blood glucose and body weight normal in dia-
betic rats. With the results, it is observed that the presence of in-
dole ring and the halogen (Br) substitution of an aryl ring attached
to the pyrimidine system enhances the anti-diabetic activity. Thus
the synthesised compounds (11a-g) can be considered for devel-
oping a potent anti-diabetic drug.
(120mg/kg, p.o.) – streptozotocin (60mg/kg, ip) - induced diabet- Experimental
ic rats almost near to the normal healthier rats (154.12g) compared
General
to all other synthesized compounds.
All the melting points are uncorrected and determined by an open
capillary tube. IR absorption spectra are recorded within the 4000-
400 cm− 1 range of an Agilent Cary 650 FT-IR spectrometer using
KBr pellets.1H NMR (400 MHz) and 13C NMR (100 MHz) spectra are
recorded within DMSO-d6 with a Bruker Avance III spectrometer
using TMS as an internal standard. The splitting patterns are pre-
cise as follows; s-singlet, d-doublet, t-triplet, m-multiplet. Mass
spectra have been recorded on an API 3000 series mass spectrom-
eter. Elemental analysis has been performed on a Vario Micro
V2.2.0 C H N analyzer.
General procedure for synthesis of 4-indolylphenyl-
6-arylpyrimidine-2-imine derivatives (11a-g)
▶Fig. 10 Blood glucose level on compounds (11a-g) in diabetic rats
A solution of substituted 3-(1H-indol-3-yl)-1-phenylprop-2-en-1-
one (0.01 mole) and metformin hydrochloride (0.01 mole) within
▶table 6 Effect of compounds (11a-g) on body weight in diabetic rats.
Groups
body Weight (g)
Initial body Weight
body Weight
1st week
body Weight 2nd week
body Weight 3rd week
body Weight
4th week
Normal
125.322 ± 1.341
138.21 ± 1.09
132.21 ± 1.823
140.23 ± 1.244
147.22 ± 1.321
120.21 ± 1.212 * *
148.213 ± 3.564 *
154.12 ± 2.13
Only STZ
141.21 ± 0.251 * *
145.32 ± 3.21 * *
130.32 ± 0.983 * * *
140.32 ± 0.213 * * *
112.32 ± 1.312 *
157.67 ± 1.231
STZ + Metformin
10 mg/kg
136.29 ± 2.0113 * * *
STZ + 11a10 mg/kg
STZ + 11b 10 mg/kg
STZ + 11c10 mg/kg
STZ + 11d 10 mg/kg
STZ + 11e10 mg/kg
STZ + 11f10 mg/kg
STZ + 11g10 mg/kg
130.34 ± 2.312 *
133 ± 1.095 * * *
129.23 ± 1.323
137.32 ± 1.22 * *
122.32 ± 1.42
142.87 ± 1.002 * *
125.33 ± 1.520 *
134.21 ± 0.321 *
143.22 ± 1.22 *
146.85 ± 1.221 * * *
130.5 ± 1.147 * *
139.21 ± 3.22
149.21 ± 2.11
153.89 ± 0.213 * *
146.83 ± 1.077 * *
147.21 ± 1.22 * *
159.32 ± 1.44 * * *
146.54 ± 0.88
140.16 ± 0.980
144.85 ± 1.322 * * *
153.23 ± 2.11 * * *
143.21 ± 1.33 *
151.66 ± 1.66 *
148.99 ± 0.77
149.23 ± 1.22 * *
137.21 ± 1.22 * * *
146.54 ± 1.77 * *
138.55 ± 1.54
130.44 ± 0.34 * * *
139.54 ± 1.07 * *
132.70 ± 0.44 * * *
131.43 ± 1.99 * *
127.86 ± 0.88 * * *
160.34 ± 2.08 * *
155.76 ± 0.33 * *
Values are expressed as the mean± S.D; Statistical significance (p) calculated by one way ANOVA followed by Dennett’s * * * P < 0.001, * * P < 0.01,
* P < 0.05 calculated by comparing treated group with control group
Ramya V et al. Type-2 Diabetic Potential Compounds… Drug Res

163.1, 162.4, 157.9, 137.0, 132.3, 130.3, 128.2, 127.7, 125.13,
122.6, 120.4, 118.7, 114.1, 113.8, 111.3, 105.0, 55.2, 36.8. MS
(m/z): 387(m+H). Elemental analysis for C22H22N6O (%): calculated:
C, 68.39; H, 5.70; N, 21.76; found: C, 68.42; H, 5.74; N, 21.80.
(E)-2-(4-(1H-indol-3-yl)-6-(4-nitrophenyl)pyrimidin-2-yl)
dimethylguanidine (11d)
Yield: 0.79 g (75 %); Mp.: 96–100 °C; Color: pale yellow; FT-IR (KBr,
cm− 1); 3162(-NH2), 3107(-NH -indole), 2918(Ar, -CH str), 2343(Al,
1
-CH str), 1543(-C = N str), 1516(Ar, -C = C); H NMR (400 MHz,
CDCl3): (δ, ppm) 11.63(s, 1H), 8.54(s, 2H), 8.52(s, 1H), 8.46-
6.56(m, 7H), 3.10(s, 6H); 13C NMR(100 MHz, CDCl3); (δ, ppm),
168.8, 166.6, 165.4, 157.8, 137.1, 136.9, 129.2, 128.9, 127.6,
125.1, 123.0, 122.0, 121.3, 120.8, 114.2, 112.1, 103.9, 36.9. MS
(m/z): 402(m + H). Elemental analysis for C21H19N7O2 ( %): calcu-
lated: C, 62.84; H, 4.74; N, 24.44; found: C, 62.88; H, 4.77; N,
24.47.
▶Fig. 11 Body weight analysis of compounds (11a-g) in diabetic rats
25ml ethyl alcohol is added 0.5 g of anhydrous sodium methoxide.
The reaction mixture is refluxed, thin layer chromatography (TLC)
confirmed the reaction is completed after 72h at a solvent ratio of
pet-ether: ethyl acetate (2:1) ratio. After finishing about the reac-
tion, the reaction mixture is poured into beaten ice; precipitate fil-
tered, washed with water until the basic catalyst is removed and
recrystallized from ethanol to give 4-indolylphenyl-6-arylpyrimi-
dine-2-imine derivatives (11a-g) obtained in moderate yields (70–
75 %).
(E)-2-(4-(4-fluorophenyl)-6-(1H-indol-3-yl)pyrimidin-2-yl)
dimethylguanidine (11e)
Yield: 0.78 g (72 %); Mp.: 74–76 °C; Color: pale brown; FT-IR (KBr,
cm− 1); 3311(-NH2), 3053(-NH -indole), 2915(Ar, -CH str), 2847(Al,
1
-CH str), 1594(-C = N str), 1452(Ar, -C = C); H NMR (400 MHz,
CDCl3): (δ, ppm) 10.76(s, 1H), 8.46(s, 2H), 8.45(d, 1H), 8.40-
6.99(m, 7H), 3.82(s, 6H); 13C NMR(100 MHz, CDCl3); (δ, ppm),
166.1, 165.8, 164.8, 157.9, 137.1, 136.2, 133.8, 130.8, 128.2,
126.1, 123.7, 122.1, 120.5, 117.1, 112.0, 111.4, 106.6, 36.8. MS
(m/z): 375(m + H). Elemental analysis for C21H19N6F ( %): calculat-
ed: C, 67.38; H, 5.08; N, 22.46; found: C, 67.40; H, 5.12; N, 22.48.
(E)-2-(4-(1H-indol-3-yl)-6-phenylpyrimidin-2-yl)dimethyl-
guanidine (11a)
Yield: 0.79 g (75 %); Mp.: 96–98 °C; Color: pale yellow; FT-IR (KBr,
cm− 1); 3316(-NH2), 3201(-NH -indole), 2342(Ar, -CH str), 2106(Al,
1
-CH str), 1594(-C = N str), 1493(Ar, -C = C); H NMR (400 MHz,
(E)-2-(4-(4-chlorophenyl)-6-(1H-indol-3-yl)pyrimidin-2-yl)
dimethylguanidine (11f)
CDCl3): (δ, ppm) 12.17(s, 1H), 8.64(s, 2H), 8.54(d, 1H), 8.53-7.22
(m, 8H), 3.18(s, 6H); 13C NMR(100 MHz, CDCl3); (δ, ppm), 163.8,
163.3, 162.9, 157.4, 137.1, 136.6, 130.8, 128.8, 126.9, 125.1,
122.3, 121.9, 120.9, 113.3, 112.2, 105.6, 37.3. MS (m/z):
357(m + H). Elemental analysis for C21H20N6 ( %): calculated: C,
70.79; H, 5.62; N, 23.19; found: C, 70.83; H, 5.67; N, 23.21.
Yield: 0.80 g (75 %); Mp.: 68–70 °C; Color: pale yellow; FT-IR (KBr,
cm− 1); 3449(-NH2), 3426(-NH -indole), 2957(Ar, -CH str), 2853(Al,
1
-CH str), 1550(-C = N str), 1491(Ar, -C = C); H NMR (400 MHz,
CDCl3): (δ, ppm) 11.82(s, 1H), 8.41(s, 2H), 8.21(d, 1H), 8.20-
7.18(m, 7H), 3.08(s, 6H); 13C NMR(100 MHz, CDCl3); (δ, ppm),
161.8, 159.3, 158.0, 157.9, 136.2, 135.1, 129.8, 128.8, 128.7,
128.5, 126.2, 123.8, 121.1, 116.3, 114.5, 111.3, 106.7, 36.7. MS
(m/z): 391(m+ H). Elemental analysis for C21H19N6Cl (%): calculat-
ed: C, 64.61; H, 4.87; N, 21.54; found: C, 64.65; H, 4.92; N, 21.57.
(E)-2-(4-(1H-indol-3-yl)-6-p-tolylpyrimidin-2-yl)dimethyl-
guanidine (11b)
Yield: 0.64 g (71 %); Mp.: 72–74 °C; Color: pale brown; FT-IR (KBr,
cm− 1); 3414(-NH2), 3154(-NH -indole), 2850(Ar, -CH str), 2316(Al,
1
-CH str), 1606(-C = N str), 1571(Ar, -C = C); H NMR (400 MHz,
(E)-2-(4-(4-bromophenyl)-6-(1H-indol-3-yl)pyrimidin-2-yl)
dimethylguanidine (11g)
CDCl3): (δ, ppm) 11.77(s, 1H), 8.43(s, 2H), 8.41(d, 1H), 8.40-
7.18(m, 7H), 3.08(s, 3H), 2.49(s, 6H); 13C NMR(100 MHz, CDCl3);
(δ, ppm), 164.2, 163.8, 163.1, 157.8, 137.6, 135.3, 131.5, 129.2,
129.2, 128.2, 126.6, 124.3, 122.5, 121.2, 114.6, 112.9, 104.9,
36.9, 27.3. MS (m/z): 371(m + H). Elemental analysis for C22H22N6
( %): calculated: C, 71.35; H, 5.94; N, 22.70; found: C, 71.40; H,
5.98; N, 22.72.
Yield: 0.74 g (74 %); Mp.: 78–80 °C; Color: pale brown; FT-IR (KBr,
cm− 1); 3463(-NH2), 3299(-NH -indole), 2921(Ar, -CH str), 2313(Al,
1
-CH str), 1548(-C = N str), 1486(Ar, -C = C); H NMR (400 MHz,
CDCl3): (δ, ppm), 10.77(s, 1H), 8.48(s, 2H), 8.46(d, 1H), 8.33-
6.72(m, 7H), 3.02(s, 6H); 13C NMR(100 MHz, CDCl3); (δ, ppm),
163.7, 162.4, 161.9, 158.0, 136.5, 135.7, 131.7, 130.2, 129.9,
128.8, 127.1, 125.1, 124.0, 122.1, 120.8, 111.3, 105.6, 36.8. MS
(m/z): 437(m+H). Elemental analysis for C21H19N6Br (%): calculat-
ed: C, 57.80; H, 4.36; N, 19.27; found: C, 57.81; H, 4.38; N, 19.31.
(E)-2-(4-(1H-indol-3-yl)-6-(4-methoxyphenyl)pyrimidin-
2-yl)dimethylguanidine (11c)
Yield: 0.64 g (73 %); Mp: 74–76 °C; Color: pale yellow; FT-IR (KBr,
cm− 1); 3380(-NH2), 3211(-NH -indole), 2928(Ar, -CH str), 2314(Al,
-CH str), 1571(-C=N str), 1512(Ar, -C=C); 1H NMR (400MHz, CDCl3):
(δ, ppm) 11.95(s, 1H), 8.75(s, 2H), 8.65(d, 1H), 8.58-6.44(m, 7H),
3.82 (s, 3H), 2.50(s, 6H); 13C NMR(100MHz, CDCl3); (δ, ppm), 163.6,
Molecular docking
Molecular docking study of target protein 1v4s is carried out using
a CDOCKER docking protocol of Discovery studio (CHARMm-based
Ramya V et al. Type-2 Diabetic Potential Compounds… Drug Res

Thieme
Original Paper
DOCKER) is a molecular dynamics based docking algorithm. It uses
the CHARMm family about the force field and offers full flexibility
to ligand including dihedral, angles, and bonds. Docking helps to
predict best binding compounds based on a variety of scoring func-
tions. For the active site, ligands are docked to get the best inter-
action poses. The top poses along with CDOCKER energy and in-
teraction energy are calculated. The best-docked poses are select-
ed as the ones with the lowest CDOCKER energy the more negative
the cdocker energy, the more favourable the binding.
growth is taken as the MIC when compared with the growth of the
control. The compounds under tests are dissolved in DMSO serial-
ly diluted in growth medium, inoculated and incubated at 37 °C.
Growth MIC is determined at 48 h for the fungal and bacterial
strains.
Anti-diabetic activity assay
In vitro anti-diabetic activity assay
In vitro alpha-amylase inhibition assay This assay is carried out
using alpha-amylase (0.5 mg/mL) premixed along the sample at
different concentrations (20–100 µg/ml) and 0.5 % starch as a
starch solution is added to start the reaction. The process is carried
out at 37 °C for 10 min. The reaction is continued by adding 2 mL
of DNS (3, 5-dinitrosalicylic acid) reagent and further incubated in
boiling water bath for 10min and cooled to room temperature. The
content of each test tube is diluted with 10mL of distilled water in
an ice bath and the absorbance is measured at 540 nm from the
spectrometer. The alpha-amylase inhibitory activity is calculated
as in eq. (1)
Protein structure preparation
The protein choosing for the present investigation is one of the
most crucial aspects. 1v4s proteins are selected as target receptor
due to its significant role in human diabetes. The three-dimension-
al structure of 1v4s is retrieved from the Protein Data Bank (PDB)
(http://www.pdb.org/). The choice protein is prepared formerly in
imitation of the docking studies by using correcting the chemistry
of the missing hydrogens and the unfilled valence atoms. Thereaf-
ter, the protein is once subjected, according to energy minimiza-
tion by making use of the CHARMm force field until the best gradi-
ent tolerance is obtained.
⎧
⎪
⎫
⎪
⎡
⎤
)
⎦
Abs540 control − Abs
test compound
(
)
(
540
⎣
%Inhibition =
100 (1)
⎨
⎬
Ligand preparation
Abs540 control
(
)
⎪
⎩
⎪
⎭
A quantity of seven synthesized compounds is chosen in conform-
ity with a challenge against the 1v4s protein receptor molecule.
These compounds have been drawn in Chem Draw and their cor-
responding 3D structures are generated on the Discovery Studio
(v 16.1.0.15350). The ligand preparation consists of a series of
steps so much operate conversions, request corrections after the
structures, eliminate unwanted structures, and optimize the struc-
tures. Many of the steps are optional and are managed by select-
ing choices from the Ligand Preparation panel. The process of con-
verting the structure format, pick out the structures, assemble hy-
drogen atoms, remove undesirable molecules, neutralize charged
groups, grow ionization states, generate low energy ring confor-
mations to get the output file.
The concentration of the test compounds to inhibit 50 % of al-
pha-amylase enzyme activity (IC50) is determined graphically using
Microsoft Excel 2007.
In vitro alpha-glucosidase inhibition assay The effect of the
compound on alpha-glucosidase activity is determined by premix-
ing alpha-glucosidase (0.07 Units) with the compound at various
concentrations 20–100µg/ml. The substrate solution, p-nitrophe-
nyl glucopyranoside 3mM is added. The reaction mixture is pre-in-
cubated at 37°C for 10min and 2mL of sodium carbonate is added
to terminate the reaction. By measuring the release of p-nitrophe-
nyl from p-nitrophenyl glucopyranoside at 400 nm, the activity of
alpha-glucosidase is determined. The results ( % Inhibition) are ex-
pressed as percentage of control as in eq. (2)
In vitro antimicrobial activity assays
⎧
⎪
⎫
⎪
⎡
⎤
)
⎦
Abs540 control − Abs
test compound
(
)
(
540
⎣
% Inhibition =
100 (2)
⎨
⎬
Disc diffusion method
Abs540 control
(
)
⎪
⎩
⎪
⎭
The in vitro antimicrobial (antibacterial and antifungal) activities
of synthesized seven compounds (11a-g) are screened by the filter
paper disc diffusion method. The various bacterial species
(Gram + ve and Gram − ve) and fungal species are first incubated
at 48°C for 24h. The sterile filter paper discs (6mm) have been im-
pregnated along a variety of test compounds and standard anti-
bacterial and antifungal agents Ciprofloxacin and Amphotericin B
are dried at 48 °C. The discs have been positioned below the nutri-
ent agar Petri plates previously seeded with a suspension of each
bacterial and fungal strain. The diameters of zone of inhibition are
measured at 35 °C after 24 h. The results are given in ▶table 3.
The concentration of the test compounds to inhibit 50 % of
alpha- glucosidase enzyme activity (IC50) is determined graphical-
ly using Microsoft Excel 2007.
In vivo anti-diabetic assay
Experimental animals Adult healthy albino male Wistar rats of
aged 6 weeks and weighing between 150 to 200g (Sri Venkatesh-
wara Enterprises, Pvt. Ltd, Bangalore) with no prior drug treatment
is used in the present investigation. The mice are housed within
pure polypropylene cages and a well-ventilated temperature con-
trolled along a constant 12 h dark and 12 h light schedule and fed
on rodent pellets and water ad libitum. The experimental animals
and procedures used in this investigation are approved by the eth-
ical committee and in agreement with the recommendation for the
proper use of laboratory animals.
Minimal inhibitory concentration method
The in vitro antifungal and antibacterial activity of all tested com-
pounds are measured by means of the minimal inhibitory concen-
tration (MIC) using the serial dilution method with 96-well Micro
test plates. The lowest concentration at which there is no visible
Ramya V et al. Type-2 Diabetic Potential Compounds… Drug Res

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Ethical statement All the experimental protocols and procedures
used in this investigation have been approved by the Ethics Com-
mittee KMCH College of Pharmacy, Department of Pharmacology,
Coimbatore (registration number KMCRET/PhD/01/2016-17).
Induction of diabetes mellitus The male albino Wistar rats are
randomly divided into 10 groups of six animals each. Diabetes is
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dissolved in normal saline and streptozotocin is dissolved in a
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weight. Diabetes is supported by the elevated level of blood sugar
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Group 4: Animals receive Nicotinamide 120 mg/kg (po) and
Streptozotocin (60mg/kg) with compound 11a 10mg/kg. (po).
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