Synthesis, characterization, and hypoglycemic efficacy of nitro and amino acridines and 4-phenylquinoline on starch hydrolyzing compounds: an in silico and in vitro study

Article abstract of DOI:10.1007/s11224-020-01529-5

α-Amylase and α-Glucosidase are important therapeutic targets for type II diabetes. The present focus of our study is to elucidate the hypoglycemic activity of novel compounds through in vitro and in silico studies. Here, we synthesized the nitro acridine

Full text of DOI:10.1007/s11224-020-01529-5

Structural Chemistry
https://doi.org/10.1007/s11224-020-01529-5
ORIGINAL RESEARCH
Synthesis, characterization, and hypoglycemic efficacy of nitro
and amino acridines and 4-phenylquinoline on starch hydrolyzing
compounds: an in silico and in vitro study
Lohitha Narayanaswamy1 & Suresh Yarrappagaari2 & Srinivasulu Cheemanapallia3 & Rajeswara Reddy Saddala2
&
V. Vijayakumar1
Received: 17 October 2019 /Accepted: 30 March 2020
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
α-Amylase and α-Glucosidase are important therapeutic targets for type II diabetes. The present focus of our study is to elucidate
the hypoglycemic activity of novel compounds through in vitro and in silico studies. Here, we synthesized the nitro acridines (3a–
3c), amino acridines (4a–4c), and nitro phenylquinoline (3d) and amino phenylquinoline (4d) using a multi-step reaction protocol
in good yields. All the above derivatives were screened for molecular docking, α-Amylase and α-Glucosidase inhibitory
activities utilizing acarbose as standard drug. In silico studies were performed to explore the binding ability of compounds with
the active site of α-Amylase and α-Glucosidase enzymes. The in vitro antihyperglycemic report of 3c exhibits the maximum
inhibitory activity with IC50 values of 200.61 9.71 μmol/mL and 197.76 8.22 μmol/mL against α-Amylase and α-
Glucosidase, respectively. Similarly, the compound 3a exhibits IC50 values of 243.78 13.25 μmol/mL and 296.57
10.66 μmol/mL, and 4c exhibits IC50 values of 304.28 3.51 μmol/mL and 278.86 3.24 μmol/mL with a significant
p < 0.05 in both enzyme inhibitions. In addition, the presence of diverse functional moieties in synthesized compounds may
provide a strong inhibitory action against the abovementioned enzymes compared with standard acarbose inhibition (IC50, 58.74
3.68 μmol/mL and 49.39 4.94 μmol/mL). Also, the docking studies provided an excellent support for our in vitro studies. The
outcome of these studies recommends that the tested compounds might be treated as potential inhibitors for the starch hydro-
lyzing enzymes in type II diabetes.
.
.
.
.
Keywords Nitro and amino acridines Nitro and amino 4-phenylquinoline Antihyperglycemic activity α-Amylase
α-Glucosidase Docking
.
Introduction
pace currently affecting 3% of the world population [1]. This
complex metabolic disease is the most important human
health distress in the world, and it may approximately influ-
ence 300 million people by the year 2025 [2]. This progressive
metabolic disorder is characterized by the constant increase of
blood glucose level which finally leads to macro-vascular
(brain stroke, heart disease, hypertension, peripheral arterial
disease, and foot problems) and microvascular (retinopathy,
cataracts, renal failure, blood vessels, and neuropathy) dam-
ages in several tissues [3]. This results in blood sugar levels, a
circumstance characteristically called diabetes. The body re-
quired insulin to convert sugar, starch, and other food into
energy. The symptoms of this disorder are polydipsia, poly-
phagia, polyuria, weight loss, etc.
Diabetes mellitus is the largely widespread universal endo-
crine disease, and its commonness is mounting at a disturbing
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11224-020-01529-5) contains supplementary
material, which is available to authorized users.
* V. Vijayakumar
kvpsvijayakumar@gmail.com
1
Centre for Organic and Medicinal Chemistry, VIT University,
Vellore, Tamil Nadu 632014, India
2
Department of Biotechnology, Division of Animal Biotechnology,
Dravidian University, Kuppam, Andhra Pradesh 517426, India
Mammalian pancreatic α-Amylase inhibitors propose an
efficient approach to lessen the levels of postprandial high
3
Department of Biochemistry, Sri Krishnadevaraya University,
Anantapuramu, Andhra Pradesh 515003, India

Struct Chem
blood glucose levels through control of starch breakdown.
Pancreatic α-Amylase is responsible for the hydrolysis of
starch to a combination of minor oligosaccharides consisting
of maltose, maltotriose, and a quantity of α-(l-6) and α-(1-4)
oligoglucans [4]. Inhibitors of α-Amylase and α-Glucosidase
postpone the breaking of carbohydrate in the small intestine
and diminish the postprandial blood glucose digression levels
in diabetic patients [5]. Hence, the inhibition of α-Glucosidase
and α-Amylase prominent enzymes has been established as a
functional and efficient strategy to lessen the levels of post-
prandial hyperglycemia.
Several antidiabetic commercial drugs such as acarbose,
voglibose, and miglitol are currently available in the market
in use for the treatment of α-Amylase and α-Glucosidase
enzyme inhibition in diabetes mellitus [6]. No single prescrip-
tion is obtainable as a comprehensive solution to this.
However, these drugs are not wished to be worn constantly
for a long time, due to their rigorous side effects [7].
Therefore, the researchers are focused to develop the safer,
single, and more tolerable α-Glucosidase inhibitors with less-
er side effects for the treatment of the diabetic disorder. To
evaluate the antidiabetic activity of synthetic compounds,
in vitro and in vivo methods are adopted [8].
drug development. In this study, the objective was to synthesize
novel nitro and amino acridines 7-nitro-9-phenyl-3, 4-
dihydroacridine-1(2H)-one (3a), 7-amino-9-phenyl-3, 4-
dihydroacridine-1(2H)-one (4a), 3, 3-dimethyl-7-nitro-9-phe-
nyl-3, 4-dihydroacridine-1(2H)-one (3b), 7-amino-3, 3-dimeth-
yl-9-phenyl-3, 4-dihydroacridine-1(2H)-one (4b), 5,6,7,8-
tetrahydro-2-nitro-9-phenylacridine (3c), 9-phenyl-5, 6, 7, 8-
tetrahydroacridine-2-amine (4c), and nitro phenylquinolines
(Ethyl 2-methyl-6-nitro-4-phenylquinoline-3-carboxylate (3d)
and Ethyl 6-amino-2-methyl-4-phenylquinoline-3-carboxylate
(4d) derivatives) and to evaluate their inhibitory activity against
both α-Amylase and α-Glucosidase with reference to in silico
studies. The findings of the work may lead to develop novel α-
Glucosidase inhibitors with higher potential and reduced risk of
secondary complications which are in general associated with
type2 diabetes mellitus.
Results and discussion
Chemistry
Synthesis and characterization of acridine-
and phenylquinolines-based derivatives
The design of gracious chemical units like acridine and quin-
oline derivatives could lead to the development of new drugs
for the treatment of various diseases. Acridine derivatives are
known for their various chemical and biological activities [9]. It
is also broadly accounted that acridine, an N-donor ligand, has
an affinity to metal complexes, in particular, those of important
metals such as Pt and Pd [10, 11]. Due to their different struc-
tural and remedial actions, thousands of natural or synthetic
acridines have been isolated or developed [12, 13]. The biolog-
ical activities of acridine can mostly depend on its benzene ring
as well as on –NHCH2CH2- or –NHCH2- part [14]. Acridine
core was reported to have antioxidant, antibacterial, anticancer,
antimalarial, and mutagenic properties, mostly associated with
their ability to inhibit the enzymes acting on nucleic acids [15].
Moreover, the derivatization of acridine was demonstrated to
improve the biological effectiveness of acridine and reduce its
side effects subsequently interaction with DNA [16]. Hence,
the synthesis of new derivatives of acridines is a fascinating
field for researchers in their quest to discover novel potent
antidiabetic drugs.
Computer-assisted drug innovation reached significance
mostly because of the dependability in the results and overlays
a new technique for the research hub toward the substitute animal
models. Molecular docking serves this purpose [17]. Protein-
ligand relations are equivalent to the lock-and-key code, in which
the lock encodes the protein and the key is a cluster with the
ligand. The major dynamic forces for binding materialize to be
hydrophobic interaction [18]. Hence, the investigation of com-
pounds with reasonable α-Amylase enzyme inhibitory property
and α-Glucosidase enzyme inhibition is necessary for the new
Initially, the compound 1 was converted to nitro acridines
and nitro phenylquinolines 3a–d according to Scheme 1.
The nitro group of the obtained compounds 3a–d was re-
duced into a corresponding primary amino group using
NH4Cl/Zn dust in dioxane/water (1:1). The newly synthe-
sized compounds 3a–c, 4a–c, 3d, and 4d were character-
ized by one- and two-dimensional NMR and HRMS spec-
tral data (included in the “Experimental” section).
Spectral characterization of compound 4a
The melting point and 1H and 13C NMR spectral data of com-
pounds 3a is found to be in concurrence with literature. The
formation of compound 3a, with the nitro function, was also
inferred based on the absorption peak at 3419 cm−1 and
3348 cm−1 in the IR spectrum, whose disappearance con-
firmed the conversion of 3a into 4a, which is further charac-
1
terized by H NMR, 13C NMR, H, H-COSY, HSQC, and
1
HRMS (ESI) spectral analysis. The H NMR spectrum of
compound 7-amino-9-phenyl-3, 4-dihydroacridine-1(2H)-
one (4a) exhibited the following chemical shifts in the aromat-
ic region: δ ppm 7.87 (d, J = 9.2 Hz, 1H), 7.46 (m, 3H), 7.20
(dd, J = 9.2, 2.4 Hz, 1H), 7.15 (d, J = 7.6 Hz, 2H), and 6.47 (d,
J = 2.4 Hz, 1H). The careful examination of the above signals

Struct Chem
Synthesis of nitro and amino acridines and 4-phenylquinolines
O
O
Zinc dust/NH4Cl
O2N
X
H2N
X
O2N
H2SO4
Acetic acid
12 hours RT
+
R
R
R
R
R
R
X
dioxane water (1;1)
15 minutes RT
N
3a-c
N
NH2
2a-c
1
4a-c
E
o
2a, 3a, 4a = R.R = -H; X= C=O
2b. 3b. 4b= R.R = -CH3; X= C=O
2c. 3c, 4c= R.R = -H; X= -CH2
t
o
h
r
y
t
l
h
a
s
c
p
e
t
h
a
o
o
E
a
1
th
p
c
2
h
e
o
n
T
t
a
h
r
o
r
i
t
s
c
l
e
a
R
c
i
d
O
O
Zinc dust/NH4Cl
O
O2N
H2N
O
dioxane water (1;1)
15 minutes RT
N
N
3d
4d
Scheme 1 Synthesis of nitro and amino acridines and 4-phenylquinolines
clearly illustrated that the signals at δ 7.46 (m, 3H) and 7.15
(d, J = 7.6 Hz, 2H) are due to the phenyl substituent at C-9.
The signal at δ 7.87 (d, J = 9.2 Hz, 1H) is due to the proton at
C-5. The proton at C-6 has two coupling partners at C-5 and
C-8 and hence appeared as a doublet of doublet at δ 7.20 ppm
(dd, J = 9.2, 2.4 Hz, 1H), and hence, the signal at δ 6.47 ppm
(d, J = 2.4 Hz, 1H) is assigned to the proton at C-8. The ali-
phatic region exhibited the following signals at δ 3.85 (s, 2H),
3.30 (t, J = 6.2 Hz, 2H), 2.66 (t, J = 6.4 Hz, 2H), and 2.24–
2.17 (m, 2H). The singlet at δ 3.55 ppm is assigned to two
protons of amino function at C-7, and hence, other signals are
assigned to protons at C-2, C-3, and C-4 carbons. The 13C
NMR spectrum exhibited the following signals: δ 197.4,
157.4, 147.8, 143.5, 143.0, 137.3, 128.6, 127.9, 127.1,
127.0, 126.2, 122.9, 122.8, 106.5, 39.7, 33.2, and 20.5 ppm;
the extreme down-field signal at δ 197.4 ppm was assigned to
the carbonyl carbon at C-1. The three signals appeared at δ
39.7, 33.2, and 20.5 ppm are due to the methylene carbons at
C-2, C-3, and C-4 which was confirmed by DEPT-135 spec-
trum also; the relatively down-field signal at δ 39.7 ppm was
assigned to carbon at C-2 since it may be due to the anisotropy
of the neighboring carbonyl group. The signals at δ 33.2 and
20.5 ppm were assigned to carbons C-4 and C-2, respectively.
All other signals are due to the aryl carbons of phenyl substit-
uent at C-9 and carbons at the acridine ring. All these spectral
data confirmed the formation of the desired molecule. It also
further confirmed by the observation of m/z at 288.1260 in the
mass spectrum (HRMS-ESI (m/z) calculated for C19H16N2O
[M]+= 288.1263, found = 288.1260). The chemical shift
values of the remaining compounds of the series also assigned
in a similar fashion and are included in the Methodology sec-
tion. All the newly synthesized molecules 4a–d were subject-
ed to in vitro antidiabetic evaluations along with 3a–d since
those molecules also are not yet subjected to these studies; the
results are discussed in the “Biology” section.
Biology
In vitro antidiabetic activity of acridine and phenylquinoline
based derivatives
In vitro α-Amylase inhibition study Selectivity of α-
Glucosidase and α-Amylase inhibitors is of enormous signif-
icance, for the reason that unclear inhibition restraint of other
glycosidases, transcendently, pancreatic α-Amylase, may the
reason for the increments of non-processed sugars in the gut,
which in turn results in stomach torment, looseness of the
bowels, and farts [19]. The outcomes in both inhibition and
IC50 values of 3a-c and 4a-c, 3d, 4d, and acarbose against α-
Amylase was represented in Fig. 1a and Table 1 respectively.
These results confirmed a considerable dose-depended dimi-
nution in α-Amylase enzyme activity. The most significant
(p < 0.05) inhibition appears in the 3c (IC50 200.61
9.71 μmol/mL) and 3a (IC50 243.78 13.25 μmol/mL),
whereas 3b (IC50 389.93 13.88 μmol/mL) showed less po-
tency. Comparatively to an acarbose reference compound,
using IC50 value, it is estimated to be 58.74 3.68 μmol/mL
for acarbose (Table 1) activity higher for those compounds
tested. Compounds 4a-c, 3d, and 4d also showed α-
Amylase inhibitory activity (IC50, 366.86 17.29, 332.07
14.87, 304.28 3.51, 302.84 6.42, and 319.60 3.24 μmol/
mL) which had a poorer effect than 3c and 3a derivatives
when compared with the standard drug. However, not any of
the synthesized compounds show a significant α-Amylase
inhibitory activity, whereas the reference drug indicated
71.96% inhibition at a maximum concentration. These out-
comes specify that 3c and 3a compounds were more useful
against α-Amylase.
In vitro α-Glucosidase inhibition study The synthesized amino
and nitro derivatives of acridines (3a–c and 4a–c) and

Struct Chem
Fig. 1 a α-Amylase inhibition and b α-Glucosidase inhibition activity in the acridine- and phenylquinoline-based derivatives (3a-d, and 4a–d)
Table 1 In vitro antidiabetic activity of different acridine- and phenylquinoline-based derivatives (3a-d and 4a-d)
α-Amylase inhibition
activity IC50 values
(μmol/mL)
α-Glucosidase inhibition
activity IC50 values
(μmol/mL)
Test
samples
Molecular
weight
Compound structure
58.74 3.68a
49.39 4.94a
Standard reference
645.60
318.10
Acarbose
3a
243.78 13.25c
296.57 10.66d
389.93 13.88f
200.61 9.71b
302.84 6.42d
366.86 17.29f
332.07 14.87e
369.33 21.29f
197.76 8.22b
279.94 10.31c
370.92 19.67e
370.73 22.20e
346.38
304.34
336.34
288.13
316.40
3b
3c
3d
4a
4b
304.28 3.51d
319.60 3.24e
278.86 3.24c
397.76 23.51g
274.36
306.36
4c
4d
Values in the table are represented as mean SD for triplicates. Mean SD is not allotment a frequent alphabet in the column differ significantly at
p < 0.05 (performed One Way ANOVA followed by Duncan Post Hoc)

Struct Chem
phenylquinolines (3d and 4d) along with acarbose (as reference)
were tested for their α-Glucosidase in vitro inhibition action.
IC50 values and inhibitory activity (%) were given in Table 1
and Fig. 1b. The compound 3c displayed a significant action
against α-Glucosidase (IC50 197.76 8.22 μmol/mL); among
the tested compounds, 4c (IC50 278.86 3.24 μmol/mL) pos-
sessed the best inhibition activity which is found to be better than
the reference acarbose (IC50 49.39 4.94 μmol/mL).
Compounds 3a, 4a, 3b, 4b, 3d, and 4d also showed α-
Glucosidase inhibitory activity (IC50 296.57 10.66, 370.92
19.67, 369.33 21.29, 370.73 22.20, 279.94 10.31, and
397.76 23.51 μmol/mL, respectively) but not better than 3c,
4c, and acarbose. However, none of the synthesized compounds
shows a significant (p < 0.05) activity of α-Glucosidase inhibi-
tion, while acarbose indicated 73.52% inhibition at a maximum
(100 μg/mL) concentration. These results indicate that the com-
pounds 3c and 4c were more effective against α-Glucosidase. No
reports were available about the α-Glucosidase inhibitory activ-
ities of nitro acridines and nitro phenylquinolines in literature.
showing a wide scope of biological activities [20]. The α-
Amylase and α-Glucosidase enzyme inhibition activities were
linearly correlated to compounds nature and its behavior.
Docking studies
Physicochemical and ADMET screening of compounds
To know about the drug-likeliness of all the synthesized com-
pounds, their physiochemical properties were explored. Except
for 3b and 3c, all other compounds found to obey the Lipinski’s
Rule of Five. In silico calculations were made to find the phys-
icochemical properties such as TPSA (polar surface area), mo-
lecular weight, LogP (octanol-water partition coefficient), H-
bond donors/acceptors, and number of rotatable bonds for all
these compounds and are summarized in Table 2. The com-
pounds with perfect ADME properties only will be approved
as a drug in clinical tests, and hence, ADMET analyses of all
these compounds 3a to 4d were predicted using Pre-ADMET
software and found to exhibit satisfactory ADMET results
(Table 3). All these compounds displayed very good intestinal
absorption and modest permeability (except 3c), while they sub-
jected to in vitro study on Caco-2 cells.
Correlation study of acridine- and phenylquinoline-based
derivatives
The synthesized acridines and phenylquinolines were subject-
ed to α-Amylase and α-Glucosidase enzyme inhibition activ-
ity of Pearson correlation analysis and are given in Table 2. In
α-Amylase, 3a exhibited linear correlation with 3b (r = 0.95;
p = 0.18), 3c (r = 0.95; p = 0.18), 4a (r = 0.97; p = 0.62), 4b
(r = 0.85; p = 0.34), 4c (r = 0.93; p = 0.23), and 4d (r = 0.88;
p = 0.31) and a nonlinear correlation with 3d (r = − 0.56; p =
0.62). Compound 3c exhibited a linear correlation in all com-
pounds except 3d (r = − 0.77; p = 0.43), and also 3c signifi-
cantly has a linear correlation with 4a (r = 0.99; p = 0.04) and
4c (r = 0.99; p = 0.04). At the same time, 3d exhibits nonlinear
correlation with all acridine- and phenylquinoline-based de-
rivatives (Table 2).
In α-Glucosidase, 3a exhibited linear correlation with 3c (r =
0.96; p = 0.17), 3d (r = 0.62; p = 0.56), 4a (r = 0.99; p = 0.06),
4b (r = 0.84; p = 0.36), 4c (r = 0.98; p = 0.09), and 4d (r = 0.89;
p = 0.29) and a nonlinear correlation with 3b (r = − 0.59; p =
0.59). At the same time, 3b exhibited a nonlinear correlation in
all compounds except 3c (r = − 0.35; p = 0.77). Compound 3c
exhibits a linear correlation with remaining 3d (r = 0.81; p =
0.39), 4a (r = 0.98; p = 0.10), 4b (r = 0.95; p = 0.18), 4c (r =
0.99; p = 0.07), and 4d (r = 0.98; p = 0.11) derivatives
(Table 2). Compound 4c exhibits the significant linear correlation
with 3c (r = 0.99; p = 0.04) and 4a (r = 0.99; p = 0.03).
The extensive plasma protein binding will increase the
amount of drug that has to be absorbed before effective thera-
peutic levels of unbound drug are reached. The compounds 3a–
4d were studied through ADME to find their binding ability with
plasma proteins; all the compounds 3a–4c were observed to bind
strongly, but the compound 4d exhibited comparatively less
binding ability. The in vitro cell permeability studies were per-
formed using MDCK cells; all the compounds exhibited moder-
ate performance. The CNS active drugs reported to pass across
the BBB; through this study, the side effects caused by CNS
inactive drugs may be avoided. In the in vivo blood-brain barrier,
penetration study of compounds 3a–4d, 3b, and 3d have
displayed poor; 3d, 4a, 4b, 4c, and 4d have displayed moderate;
and 3a and 3c have displayed higher absorption to CNS.
Molecular docking study
To observe the binding mode and possible interactions of com-
pounds with target enzymes (α-Amylase and α-Glucosidase),
docking studies were performed. The 3D models of target en-
zymes were generated using the Swiss model server (shown in
Fig. 2). The electrostatic potential surface models of enzymes
(Fig. 3) reveal the distribution of charged and uncharged residues
in 3D structure and which determines the binding sites for the
activators/inhibitors. Molecular docking studies of compounds
against active site of enzymes revealed that all of them showed
atomic interactions with best docking scores dominated by hy-
drogen bonding with the active site residues. The most straight-
forward way to evaluate the performance of molecular docking is
to analyze the discrepancy between the real and best-scored
On the whole, 3c, 4a, and 3a demonstrated a strong linear
correlation with both α-Amylase and α-Glucosidase enzyme
inhibition in the majority of compounds, while 3d demonstrates
the lesser linear correlation and strong nonlinear correlation. The
pharmacological investigations have demonstrated that the quin-
oline ring framework is available in numerous compounds

Struct Chem
Table 2 Analysis of correlation coefficients of the Pearson test which exhibit a linear correlation between the synthesized compounds α-Amylase and
α-Glucosidase inhibition assays
Test samples
3b
3c
3d
4a
4b
4c
4d
α-Amylase
3a
0.95
0.95
1.00**
− 0.56
− 0.77
− 0.77
0.97
0.85
0.93
0.88
3b
0.99*
0.99*
0.96
0.97
0.99
0.99*
− 0.82
0.98
0.70
0.70
3c
3d
− 0.72
− 0.90
0.94
− 0.10
0.75
4a
4b
0.98
0.51
4c
0.64
α-Glucosidase
3a
3b
3c
3d
4a
4b
4c
− 0.59
0.96
− 0.39
0.62
0.25
0.81
0.99
− 0.50
0.98
0.84
− 0.06
0.95
0.98
− 0.46
0.99*
0.74
0.89
− 0.17
0.98
0.70
0.94
0.90
0.89
0.99*
0.93
0.91
0.99
0.95
Pearson correlation significance between the synthesized compounds considered at *p < 0.05 and **p < 0.01
conformations. The root mean square deviation (RMSD) value
between the real and the best-scored conformations for docked
complexes showed less than 2.0 Å. In all the docking conforma-
tions, the lengths of hydrogen bonds have shown below 5 Å. The
details of docking energy (kcal/mol) and interacting residues of
α-Amylase docked complexes are represented in Table 4, and
the binding mode of compounds with the active site of α-
Amylase has been displayed in Fig. 4. The compound 3c found
to have good docking energy (− 6.79 kcal/mol) with α-Amylase
(compared with the remaining compounds) but exhibited mod-
erate permeability in the in vitro permeability studies with Caco-
2 cells (Table 4).
In all the docked conformations, the α-Amylase active site
residues participated in hydrogen bonding interactions are
Ser353, Asp309, Gln314, Tyr333, Glu397, Arg349, Val350,
Ser352, His30, Gln56, Arg207, Asp312, Asn310, His311,
Trp73, Phe307, and Glu245. The compound 3c has not shown
any hydrogen bond interactions with α-Amylase residues (Figs.
Table 3 ADMET predicted profile for compounds acquired on the webserver of Pre-ADMET
Compound a% of absorption in bnm/s of in vitro Caco-2 cnm/s of in vitro MDCK d% of protein binding ePenetration of in vivo blood-brain
human intestine
cell permeability
cell permeability
in vitro plasma
barrier (C. brain/C. blood)
3a
3b
3c
3d
4a
4b
4c
4d
98.93
98.70
97.72
99.12
96.31
96.46
96.97
96.41
20.76
21.33
1.63
67.57
74.23
78.80
66.05
80.13
87.14
86.04
88.12
93.36
93.86
95.01
94.15
90.47
90.40
99.09
86.45
2.87
0.043
4.54
0.085
1.76
1.62
1.25
1.92
21.54
18.96
23.80
17.91
22.08
a % of absorption in human intestine: 0–20% (reduced absorption); 20–70% (moderate absorption); 70–100% (superior absorption)
b nm/s of in vitro Caco-2 cell permeability: < 4 values are small permeability; > 4–70 values are middle permeability; and > 70 values are elevated
permeability
c nm/s of in vitro MDCK cell permeability: < 25 are short permeability; 25–500 are core permeability; > 500 are elevated permeability
d % of protein binding in vitro plasma: < 90% are weak binding; > 90% are strong binding
e Penetration of in vivo blood brain barrier: < 0.1 (low absorption); 0.1–2.0 (middle absorption); and > 2.0 (higher absorption)

Struct Chem
Fig. 2 3D models of α-Amylase and α-Glucosidase enzymes of rat
2 and 3c). The results of the docking analysis of α-Glucosidase
revealed that compound 3c has shown more docking energy (−
7.89 kcal/mol) compared with others (Table 5). The amino acid
residues participated in hydrogen bond interactions with 3c com-
pound are His295, Phe297, Leu260, Gln633, Glu630, and
Glu262 (Figs. 5 and 3c). The common residues such as
Phe603, His295, and Leu637 were involved in polar interactions
with all compounds (Fig. 5). Based on the docking results, the
compound 3c has shown the highest binding energy than the
remaining compounds with both enzymes, followed by 3a and
4c which have shown more binding energy with amylase and
glucosidase respectively. The active site residues of enzymes
have participated in hydrogen bonding with all compounds ex-
amined in the present study. The interaction was disclosed that
compounds may interfere/inhibit the function of enzymes in dis-
ease conditions.
13CNMR spectra were recorded using Bruker 400 MHz spec-
trometer. High-resolution mass spectra were recorded using a
Bruker MaXis HR-MS (ESI-Q-TOF-MS) instrument.
General procedure for the synthesis of compounds
3a–c and 4a–c
A mixture of compound 1 (0.1 g, 0.0005 M, 1) with either of
cyclo hexan-1, 3-dione, 5, 5-dimethyl cyclohexan-1, 3-dione,
cyclohexadiene, or a catalytic amount of conc. sulphuric acid
was stirred in acetic acid for 12 h. After the completion of the
reaction, the crude was extracted with ethyl acetate, and the or-
ganic layer was dried to obtain 3a–c. The products 3a–c are
reduced to 4a–c respectively by stirring with zinc dust (0.17 g,
0.0026 M) in the presence of NH4Cl (0.16 g, 0.0026 M) in
dioxane/water (1:1) for 15 min. The product was filtered and
purified by column chromatography (basic alumina) using ethyl
acetate/hexane (4:6) as eluent to afford the compound 4a-c.
Methodology
7-nitro-9-phenyl-3, 4-dihydroacridine-1(2H)-one (3a)
Chemistry
Yellow solid, yield 90%, m.p., 233–235 °C; IR ν/cm−1
1552 (NO2), 1695 (C=O, ketone); H NMR (400 MHz,
CDCl3), δ ppm 8.50 (dd, J = 2.8, 9.2 Hz, 1H), 8.42 (d, J =
=
1
Melting points (m.p.) reported herein were recorded using
Elchem microprocessor-based DT apparatus. 1H and
Fig. 3 The electrostatic potential surface models of α-Amylase and α-
Glucosidase enzymes of the rat. Colors on the surface portrayal of the
compound buildings are based on electrostatic possibilities of positive
(blue) and negative (red) charged residues. The electrostatic potential of
enzymes are − 45.869kbT/e to + 45.869kbT/e (α–Amylase) and −
61.985kbT/e to + 61.985kbT/e where kb, T, and e are the Boltzmann
constant, temperature, and the electron charge, respectively

Struct Chem
Table 4 Molecular docking analysis of α-Amylase with compounds
S.
Name of
Kcal/mol of No. of H-
Contributed amino acids
Cluster of
RMSD
Reference of
RMSD
No ligand
binding
energy
bonds
1
2
3
4
3a
3b
3c
3d
− 7.04
− 4.39
− 7.89
− 6.12
8
6
-
Ser353, Asp309, Gln314, Tyr333, Glu397, Arg349
0.00
0.00
0.00
0.47
52.56
53.72
64.99
53.11
His311, Asp312, Arg349, Arg207, Glu245
-
10
Val350, Ser352, His30, Gln56, Arg207, Asp312,
Asn310,His311, Trp73
5
6
7
8
4a
4b
4c
4d
− 5.57
− 4.97
− 6.91
− 6.75
4
3
1
6
Asp311, Arg349, Gln310, Tyr77
0.34
0.00
0.23
0.00
54.66
55.18
54.70
53.87
Asp309, Arg349, Val350
Val350
Phe307, Asn310, Asp312, Arg207, Val350, Glu56
2.4, Hz, 1H), 8.18(d, J = 9.2 Hz, 1H), 7.55 (t, J = 2.8 Hz,3H),
7.18–7.20 (m, 2H), 3.4 (t, J = 6.2 Hz, 2H), 2.75 (t, J = 6.6 Hz,
2H), and 2.26–2.32 (m, J = 6.5 Hz, 2H); 13CNMR (100 MHz,
CDCl3), δ ppm 197.1, 166.1, 153.2, 150.5, 145.6, 135.8,
130.5, 128.6, 128.1, 126.8, 125.3, 125.0, 124.9, 40.5, 34.9,
and 21.0.
7.17–7.20 (m, 2H), 3.31 (t, J = 6.2 Hz, 2H), 2.61 (t, J = 6.4 Hz,
2H), and 1.18 (s, 6H); 13CNMR (100 MHz, CDCl3), δ ppm
197.2, 165.1, 152.9, 150.8, 145.6, 136.8, 130.5, 128.6,
128.55, 128.1, 126.7, 125.1, 124.9, 124.2, 54.1, 48.6, 32.3,
and 28.4.
5,6,7,8-tetrahydro-2-nitro-9-phenylacridine (3c)
3, 3-dimethyl-7-nitro-9-phenyl-3, 4-dihydroacridine-
1(2H)-one (3b)
Yellow solid, yield 85%, m.p., 226–228 °C; IR ν/cm−1
=
1
1552 (NO2), 1695 (C=O, ketone); H NMR (400 MHz,
CDCl3), δ ppm 8.35 (dd, J = 2.4, 9.2 Hz, 1H), 8.29 (d, J =
2.4, Hz, 1H), 8.10 (d, J = 9.2 Hz, 1H), 7.56 (dt, J = 7.2,
2.8 Hz,3H), 7.24 (dd, J = 7.8, 1.4 Hz, 2H) 3.24 (t, J =
6.6 Hz, 2H), 2.65 (t, J = 7.0 Hz, 2H), 2.08–1.965 (m,2H),
Yellow solid, yield 92%, m.p., 230–232 °C; IR ν/cm−1
1552 (NO2), 1693 (C=O, ketone); H NMR (400 MHz,
CDCl3), δ ppm 8.50 (dd, J = 2.8, 9.2 Hz, 1H), 8.43 (d, J =
2.4 Hz, 1H), 8.18 (d, J = 9.2 Hz, 1H), 7.55 (t, J = 3.0 Hz,3H),
=
1
Fig. 4 Molecular interactions of compounds (3a to 4d) with α-Amylase. All compounds and binding filtrates are exposed in deposits which are appeared
in the ball and stick model, respectively. The dotted line represents the hydrogen bond interactions between compounds and residues

Struct Chem
Table 5 Molecular docking analysis of α-Glucosidase with compounds
S.
No
Name of
ligand
Kcal/mol of binding
energy
No. of H-
bonds
Contributed amino acids
Cluster of
RMSD
Reference of
RMSD
1
2
3
3a
3b
3c
− 5.72
− 3.14
− 6.79
3
4
5
Phe603, His295, Leu637
Phe603, His295, Leu637
0.00
0.09
0.00
27.25
27.39
25.47
His295, Phe297, Leu260, Gln633, Glu630,
Glu262
4
5
6
7
8
3d
4a
4b
4c
4d
− 5.61
− 5.37
− 4.65
− 6.47
− 3.83
4
1
1
1
-
Phe603, His295, Leu637
0.47
0.00
0.23
0.00
0.00
27.46
27.21
27.39
27.21
28.13
Leu637
Leu637
Leu637
-
and 1.80–1.86 (m, 2H); 13C NMR (100 MHz,CDCl3), δ ppm
163.6, 148.4, 148.3, 144.9, 135.4, 132.2, 131.6, 130.8, 130.1,
129.2, 129.15, 129.1, 128.94, 128.6, 125.7, 123.0, 212.9,
116.8, 34.6, 29.7, 28.1, 22.7, and 22.5.
122.8, 106.5, 39.7, 33.2, and 20.5; HRMS-ESI (m/z) calcd
for C19H16N2O [M]+ = 288.1263, found = 288.1260.
7-amino-3, 3-dimethyl-9-phenyl-3, 4-dihydroacridine-
1(2H)-one (4b)
7-amino-9-phenyl-3, 4-dihydroacridine-1(2H)-one (4a)
Yellow solid, yield 85%, m.p., 220–222 °C; IR
Reddish brown solid, yield 88%, m.p.:,216–218 °C; IR
ν/cm−1 = 3446, 3305 (NH2), 1680 (C=O, ketone); 1H NMR
(400 MHz, CDCl3), δ ppm 7.87 (d, J = 9.2 Hz, 1H), 7.46 (m,
3H), 7.20 (dd, J = 9.2, 2.4 Hz, 1H), 7.15 (d, J = 7.6 Hz, 2H),
6.47 (d, J = 2.4 Hz, 1H), 3.85 (s, 2H), 3.30 (t, J = 6.2 Hz, 2H),
2.66 (t, J = 6.4 Hz, 2H), and 2.24–2.17 (m, 2H);13C NMR
(100 MHz, CDCl3), δ ppm 197.4, 157.4, 147.8, 143.5,
143.0, 137.3, 128.6, 127.9, 127.1, 127.0, 126.2, 122.9,
1
ν/cm−1 = 3437, 3331 (NH2),1680 (C=O, ketone); H NMR
(400 MHz, CDCl3), δ ppm 7.81 (d, J = 9.2 Hz, 1H), 7.38–7.44
(m, 3H), 7.13 (dd, J = 9.0, 2.2 Hz, 1H), 7.08 (d, J =
6.8 Hz,2H), 6.42 (d, J = 2.0 Hz, 1H), 3.78 (bs, 2H), 3.12 (s,
2H), 2.45 (s,2H), and 1.06 (s, 6H); 1 3 C NMR
(100 MHz,CDCl3), δ ppm 198.4, 157.3, 148.4, 144.5, 144.3,
138.30, 129.7, 128.9, 128.2, 128.1, 127.3, 123.8, 122.9,
Fig. 5 The molecular interactions of compounds 3a–4d with α-Glucosidase. All compounds and binding deposits have appeared in the ball and stick
form, respectively. The dotted line represents the hydrogen bond interactions between compounds and residues

Struct Chem
107.7, 54.3, 48.0, 32.3, and 28.4. HRMS-ESI (m/z) calcd for
C21H20N2O [M]+ = 316.1576, found = 316.1569.
Ethyl 2-methyl-6-nitro-4-phenylquinoline-3-
carboxylate (3d)
9-phenyl-5, 6, 7, 8-tetrahydroacridine-2-amine (4c)
Yellow colored solid, 95% of yielding, m.p., 210–212 °C; IR
ν/cm−1 = 1525 (NO2), 1724 (C=O, ketone); 1H NMR
(400 MHz, CDCl3), δ ppm 8.56 (d, J = 2.4 Hz, 1H), 8.50
(dd, J = 9.2, 2.4 Hz, 1H), 8.22 (d, J = 9.2 Hz, 1H), 7.57 (t,
J = 3.20 Hz, 3H), 7.39 (dd, J = 6.0, 2.4 Hz, 2H), 4.12 (q, J =
7.20 Hz, 2H), 2.85 (s, 3H), and 0.99 (t, J = 7.0, 3H); 13C NMR
(100 MHz, CDCl3), δ ppm 167.5, 158.9, 149.70, 148.0,145.6,
134.1, 130.8, 129.4, 129.3, 129.2, 128.8, 124.5, 123.8,
123.52, 61.80, 24.15, and 13.64.
Solid compound obtained; yellow color appears, yield 80%,
m.p., 218–220 °C; 1H NMR (400 MHz, CDCl3), δ ppm 7.83
(d, J = 8.8 Hz, 1H), 7.50 (t, J = 7.2 Hz, 2H), 7.44 (t, J = 7.4 Hz,
1H), 7.21 (t, J = 6.8 Hz,2H), 7.05 (dd J = 8.8, 2.4 Hz, 1H),
6.38 (d, J = 2.4 Hz 2H), 3.12 (t, J = 6.6 Hz, 2H), 2.53 (t, J =
6.6 Hz, 2H), 1.90–1.96 (m, 2H), and 1.72–1.78 (m, 2H); 13
C
NMR (100 MHz,CDCl3), δ ppm 155.2, 144.3, 143.6, 141.7,
137.7, 129.6, 129.2, 128.8, 128.6, 128.5, 128.1, 127.9, 127.5,
120.5, 106.2, 33.9, 28.1, 23.14, and 23.09; HRMS-ESI (m/z)
calcd for C19H18N2 [M]+ = 274.1470, found = 274.1467.
Ethyl 6-amino-2-methyl-4-phenylquinoline-3-
carboxylate (4d)
General procedure for synthesis of 3d and 4d
Yellow solid, yield 90%, m.p., 206–208 °C; IR ν/cm−1
=
A mixture of 1 (0.1 g, 0.0005 M, 1) with ethyl acetoacetate
and a catalytic amount of ortho-phosphoric acid was stirred in
ethanol for 12 h. After completion of the reaction, the crude
product was poured in ice water; the obtained product 3d was
filtered, dried, and reduced by stirring with zinc dust (0.17 g,
0.0026 M) in presence of ammonium chloride (0.16 g,
0.0026 M) in dioxane/water (1:1) for 1 h. The product was
filtered and purified by column chromatography (basic alumi-
na) using ethyl acetate/hexane (4:6) as eluent.
3462, 3352 (NH2); 1716 (C=O, ketone); 1H NMR
(400 MHz, CDCl3): δ ppm 7.80 (d, J = 8.8 Hz, 1H), 7.37
(m, 3H), 7.26 (d, J = 6.6 Hz, 2H), 7.06 (d, J = 8.0 Hz, 1H),
6.54 (s, 1H), 4.12 (q, J = 7.20 Hz, 2H), 3.79 (s, 2H), 2.63 (s,
3H), and 0.86 (t, J = 7.20 Hz, 3H); 13CNMR (100 MHz,
CDCl3): δ ppm 168.83, 150.50, 144.64, 143.96, 142.81,
136.27, 129.96, 129.38, 128.21, 127.57, 126.55, 122.12,
106.48, 61.21, 23.30, and 13.65. HRMS-ESI (m/z) calcd for
C19H18N2O2 [M]+ = 306.1368, found = 306.1365.
made up to 10 mL using distilled water, and the absor-
bance was measured at 540 nm against blank (without test
sample) in UV-Vis spectrophotometer. Acarbose was used
as a reference drug.
Biological activity
α-Amylase inhibition assay
The α-Amylase enzyme inhibition activity was carried out
by the standard reference method [21]. In a test tube,
1 mL of 1% phosphate buffer prepared in starch solution
was incubated with an α-Amylase enzyme (500 μL) for
10 min at room temperature. About 1 mL of various con-
centrations (20–100 μg/mL) of acridine (3a−c; 4a−c) and
phenylquinoline (3d, 4d) derivatives were added to the
above-incubated enzyme solution; after that 1 mL of
NaOH (2 M) solution was added to freeze the reaction.
Then 1 mL of 3, 5-dinitro salicylic acid (DNS) was added
and maintained in a water bath for 5 min. The contents of
the test tube were cooled in water, the final volume was
Determination of α-Glucosidase inhibition assay
The α-Glucosidase enzyme inhibitory activity was deter-
mined by a slightly modified method [22]. A 1 mg of
Saccharomyces cerevisiae isolated α-Glucosidase enzyme
was dissolved in 100 mL of phosphate buffer saline (pH 6.8)
containing 200 mg of bovine serum albumin. The acridine and
phenylquinoline derivatives were prepared in 20–100 μg/mL
concentration. From that 10 μL of the test solution was taken
and premixed with 490 μL phosphate buffer (pH 6.8) and
250 μL of p-nitro-phenyl-α-d-glucopyranoside (p-NPG)

Struct Chem
(5 mM). After 5 min. of pre-incubation at 37 °C, 250 μL of α-
Glucosidase (0.15 unit/mL) was added and incubated at 37 °C
for further 15 min. To quench the reaction, 2 mL sodium
carbonate (200 mM) was added. The activity of the α-
Glucosidase enzyme was observed at 405 nm using UV-Vis
spectrophotometer by measuring the quantity of p-nitrophenol
released from p-NPG. Acarbose was used as a standard.
The following equation was utilized to measuring the per-
centage of both investigated enzymes inhibition,
triplicate. Graph Pad Prism 5.1 (Graph cushion pro-
gramming, Inc., La Jolla, CA, USA) was used to deter-
mine the IC50 values.
Conclusion
In the present work, we have productively achieved our ob-
jective designate derivatives of 4a–d. All the compounds were
established by appropriate experimental and spectroscopic
techniques. Synthesized derivatives (3a–d and 4a–d) were
evaluated for in vitro α-Amylase and α-Glucosidase inhibito-
ry activities. Among the tested derivatives, 7-nitro-9-phenyl-
1, 2, 4, 4-tetrahydroacridine (3c) compound showed good
inhibitory activity for α-Amylase and α-Glucosidase with
the IC50 values of 200.61 9.71 μmol/mL and 197.76
8.22 μmol/mL, respectively, and 9-phenyl-5, 6, 7, 8-
tetrahydroacridine-2-amine (304.28 3.51 μmol/mL and
278.86 3.24 μmol/mL), and 7-nitro-9-phenyl-3, 4-
dihydroacridine-1(2H)-one (243.78 13.25 μmol/mL and
296.57 10.66 μmol/mL) show moderate inhibitory activity
against α-Amylase and α-Glucosidase. The in silico molecu-
lar docking studies are in good conformity with the in vitro
antidiabetic studies. The molecular docking calculations
showed that hydrogen bonds interactions and desolation ener-
gies play a key role in binding. These features are well thought
out for scheming new inhibitors for α-Amylase and α-
Glucosidase enzymes.
% inhibition of enzyme = [Abs Control – Abs Tested compound
]
/ [Abs Control] × 100
whereas Abs = Absorbance.
Docking studies
Prediction of drug-likeness and ADMET screening
of compounds
For a drug candidate to be sufficiently bio-available and ulti-
mately be a commercial success, its pharmacodynamic activ-
ity needs to be in consistent with its structure and physico-
chemical and biological properties [23–25]. In order to check
the drug-likeness, affinity, and selectivity of lead molecules,
there is necessary to maintain the molecular weight and lipo-
philicity. Lipinski’s Rule of Five was used to predict the in
silico physicochemical properties of all compounds [26] using
the Molinspiration software. Bioavailability involves the in-
testinal absorption and distribution, which are determined by
the stability, dissolution, solubility, permeability, dosage,
first-pass metabolism, and various mechanisms of action of
drug molecule [27].
Acknowledgments The authors are grateful to the VIT University in
Vellore, India, for giving facilities to convey explore work. The authors
are likewise appreciative to the Department of Biotechnology, School of
Herbal Studies, and Nature Sciences in Dravidian University, Kuppam,
India.
Molecular docking studies
Compliance with ethical standards
Homology models of α-Amylase and α-Glucosidase
were generated using Swiss model program according
to the Cheemanapalli et al. (2016) method [28]. The
FASTA sequences of pancreatic alpha amylase (Gen
Bank ID: AAA40725.2) and glucosidase (Gen Bank
ID: AAH61753.1) of Rattus norvegicus were obtained
from NCBI. To make a three dimensional protein
models, the Swiss model program uses template struc-
tures of human enzyme models such as 1B2Y (α-
Amylase) and 5KZW (α-Glucosidase) respectively.
Conflict of interest The authors declare that they have no conflict of
interest
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