Dihydropyridines as potential α-amylase and α-glucosidase inhibitors: Synthesis, in vitro and in silico studies

Article abstract of DOI:10.1016/j.bioorg.2020.103581

Dihydropyridine derivatives 1–31 were synthesized via one-pot solvent free condition and screened for in vitro against α-amylase and α-glucosidase enzyme. The synthetic derivatives 1–31 showed good α-amylase inhibition in the range of IC₅₀ = 2.

Full text of DOI:10.1016/j.bioorg.2020.103581

Journal Pre-proofs
Dihydropyridines as potential α-Amylase and α-Glucosidase Inhibitors: Syn-
thesis, In Vitro and In Silico Studies
Hina Yousuf, Shahbaz Shamim, Khalid Mohammed Khan, Sridevi
Chigurupati, Kanwal, Shehryar Hameed, Muhammad Naseem Khan,
Muhammad Taha, Minhajul Arfeen
PII:
S0045-2068(19)31311-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.103581
YBIOO 103581
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
9 August 2019
30 November 2019
11 January 2020
Please cite this article as: H. Yousuf, S. Shamim, K. Mohammed Khan, S. Chigurupati, Kanwal, S. Hameed, M.
Naseem Khan, M. Taha, M. Arfeen, Dihydropyridines as potential α-Amylase and α-Glucosidase Inhibitors:
Synthesis, In Vitro and In Silico Studies, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.103581
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Dihydropyridines as potential α-Amylase and α-Glucosidase Inhibitors: Synthesis, In
Vitro and In Silico Studies
Hina Yousuf,a Shahbaz Shamim,a Khalid Mohammed Khan,a,b Sridevi Chigurupati,c
Kanwal,a Shehryar Hameed,a Muhammad Naseem Khan,d Muhammad Taha,b Minhajul
Arfeenc
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi, 75270, Pakistan
bDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations
(IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 1982,
Dammam, 31441, Saudi Arabia
cDepartment of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Qassim
University, Buraidah, 52571, Saudi Arabia
dDepartment of Chemistry, COMSATS University Islamabad, Abbottabad campus,
Abbottabad (22010) Pakistan
Abstract: Dihydropyridine derivatives 1-31 were synthesized via one-pot solvent free
condition and screened for in vitro against α-amylase and α-glucosidase enzyme. The synthetic
derivatives 1-31 showed good α-amylase inhibition in the range of IC50 = 2.21 ± 0.06 - 9.97 ±
0.08 µM, as compared to the standard drug acarbose (IC50 = 2.01 ± 0.1 µM) and α-glucosidase
inhibition in the range of IC50 = 2.31 ± 0.09 - 9.9 ± 0.1 µM as compared to standard acarbose
(IC50 = 2.07 ± 0.1 µM), respectively. To determine the mode of binding interactions of synthetic
molecules with active sites of enzyme, molecular docking studies were also performed.
Different spectroscopic techniques such as 1H-, 13C-NMR, EI-MS, and HREI-MS were used to
characterize all the synthetic compounds.
Keywords: Dihydropyridine; synthesis; α-amylase; α-glucosidase; in vitro; structure-activity
relationship (SAR); in silico studies
Introduction
Heterocyclic chemistry have become very familiar in the pharmaceutical chemistry and organic
synthesis. Additionally, science and technology are moving towards the environmental and
economical caring, and supportable methods [1]. In the recent past the use of non-toxic and
environment friendly reagents have attracted a great considerations of synthetic chemist to
avoid the basic environmental toxins that have been harmful for the living organisms [2]. The
solvent free one-pot multicomponent reactions are one of the basic approach in green chemistry
*Corresponding Authors: khalid.khan@iccs.edu, drkhalidhej@gmail.com, Tel.: +922134824910, Fax:
+922134819018
1

to attain the desired products with minimal usage of harmful chemicals in short time and being
economical [3]. The nitrogen containing heterocycles acquired much importance due to their
role in biological systems. Dihydropyridines (DHPs) are a prolific source of pharmacological
and biological actions. The 1,4-dihydropyridine nucleus is known as one of the most important
heterocyclic framework that is found in many pharmaceuticals and drugs [2]. They are
commercially used as calcium channel blocker [4].
Diabetes is a serious health disorder which in the 6th leading cause of global mortality and
growing at alarming rates around the world [5]. Diabetes mellitus which is caused by deficient
insulin secretion is known as type I DM, however, decrease utilization of insulin leads to type
II DM [6]. Insulin is a peptide hormone that decreases blood glucose level by increased
utilization of glucose thus reduces gluconeogenesis [7]. Type II diabetes is the most common
form of diabetes, almost 80% to 90% of all the cases are suffered from this type [8].
α-Amylase is a common key enzyme found in pancreatic juice and saliva. α-Amylase
effectively targets the control of postprandial hyperglycemia [9]. It acts randomly on internal
α-1,4-glucosidic linkages of starch, glycogen, and related polysaccharides giving various kinds
of oligosaccharides as end product. α-Amylase belongs to the family of glycoside hydrolases,
and lies under glycoside hydrolases family 13. It can be isolated from different sources such as
microbes, plants, and animals. Microbial amylases have advantages like space, cost and time
effectiveness, and easy optimization of production process. Bacterial α-amylase can be easily
engineered for the desired characteristics to enhance their industrial demands [10]. Unlike α-
glucosidase that catalyses last step of disaccharides and starch hydrolysis, α-amylase involves
in the hydrolysis of large starch molecules in to absorbable molecules. Therefore, it is an
effective approach to inhibit pancreatic α-amylase to treat diabetes [9]. Acarbose, a
tetrasaccharide mimic with potent α-amylase inhibitory activity is used to treat T2DM. Various
α-amylase inhibitors have been isolated from nature and reported, however, selective and
potent α-amylase inhibitors are still in search [7].
α-Glucosidase is present in the brush border surface membrane of the intestinal cells. It is one
of the most important carbohydrate digestive enzyme [11]. It hydrolyzes the 1,4-linked α-
glucose residues from the non-reducing end of polysaccharide to give a single α-glucose
molecule. It performs an important role in the final step of carbohydrate digestion, therefore,
inhibition of α-glucosidase can effectively control postprandial hyperglycemia. α-Glucosidase
is also responsible for other diseases like viral infections and cancer [12]. Various α-
glucosidase inhibitors such as acarbose, voglibose, and miglitol have been clinically approved.
2

These inhibitors are usually taken orally, acting as an antibiotic drug by delaying the absorption
of sugar and suppressing the digestion of carbohydrate. It leads plasma glucose to be
maintained at a steady level [13]. Unfortunately, α-glucosidase inhibitors cause flatulence,
abdominal pain, diarrhea, and liver disorder which is most common in noncompliance.
Therefore, research for finding new α-glucosidase inhibitors with minor or no side effects is an
important task [14].
Our research group has identified many heterocyclic organic molecules of different classes in
search for their potential uses in medicinal chemistry [15-20]. We have been working on the
identification of small molecules that may act as lead compounds and may serve as potential
antidiabetic agents. Nevertheless, recently we had designed, synthesized, identified, and
reported dihydropyridine derivatives as potential α-glucosidase inhibitors [21] (Figure-1).
Currently Synthesized Scaffold (1-31)
NO2
R
O
O
O
O
H3C
O
O
CH3
O
CH3
H3C
N
CH3
H3C
H3C
H
N
CH3
H
-Glucosidase Inhibitiors [21]

IC50 = 2.21 ± 0.06 - 9.97 ± 0.08
M

Different Substituents on both sides of dihydropyridine ring
Structural Similarity
NO2
R
O
O
O
O
H3C
O
O
CH3
O
CH3
H3C
H3C
H3C
N
CH3
H
N
CH3
H
-Glucosidase Inhibitiors [21]

IC50 = 2.21 ± 0.06 - 9.97 ± 0.08
M

Figure-1: Rationale of the current study
Keeping in mind the antidiabetic potential of dihydropyridines, synthesized compounds were
also subjected for α-amylase and α-glucosidase inhibition. The synthetic compounds were
structurally different from the previous compounds having two different substituents on either
3

side of dihydropyridine ring. The difference in structures resulted in better inhibitory action
against α-amylase and α-glucosidase enzymes. Fortunately, we had identified dual and
potential inhibitors of α-amylase and α-glucosidase enzymes that would be an ideal therapy for
treatment of diabetes.
Results and Discussion
Chemistry
Ethyl-2,7,7-trimethyl-5-oxo-4-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate
derivatives 1-31 were synthesized by reacting ethyl acetoacetate, dimedone, ammonium
acetate, and different aryl aldehydes in “one pot”. The reaction was catalyzed with copper
º
nitrate trihydrate (Cu(NO3)2.3H2O) in solvent free condition at 80-90 C (Scheme-1). The
reaction mixture was refluxed until a solid mass was obtained. Thin layer chromatography
(TLC) was used to monitor the completion of reaction. After completion of reaction, the solid
mass was filtered and washed with excess of distilled water followed by washing with hexane.
Ethanol was used for crystallization to get pure products. Different spectroscopic techniques
like 1H-, 13C-NMR, EI-MS, and HREI-MS were used to characterize the structures of synthetic
compounds 1-31 (Table-1).
R
O
O
R
NH4OAc
O
O
H
+
Cu(NO3)2 .3H2O,
80 °C, 20-30 min
H3C
H3C
O
CH3
O
O
O
H3C
H3C
N
CH3
H
H3C
O
CH3
Scheme-1:
Ethyl-2,7,7-trimethyl-5-oxo-4-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate derivatives (1-31)
4

In vitro α-amylase and α-glucosidase inhibitory activities:
All synthetic ethyl-2,7,7-trimethyl-5-oxo-4-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate derivatives 1-31 were subjected for their in vitro α-amylase inhibitory activity. It
is noteworthy that all synthetic compounds displayed good and moderate inhibitory activity
against α-amylase with IC50 values in the range of 2.21 ± 0.06 - 9.97 ± 0.08 µM and α-
glucosidase in the range of IC50 = 2.31 ± 0.09 - 9.9 ±0.1 µM as compared to the standard
acarbose (IC50 = 2.01 ± 0.1 µM) (Table-1). The general structural features of the molecule
comprise of pyrimidine ring, acetyl group, dimedone segment, and an aryl ring (R) (Figure-2).
All these parts are playing important role in the activity, however, slight variation in the activity
of synthetic molecules was might be due to variability in the positions and nature of substituents
on ring (R).
Aryl ring
Ethyl Acetoacetate
O
R
O
Dimedone
O
CH3
H3C
H3C
Pyrimidine ring
N
H
CH3
Figure-2: General structure of synthetic compounds
Table-1: In vitro α-amylase, α-glucosidase inhibitory activity of ethyl-2,7,7-trimethyl-5-
oxo-4-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate derivatives (1-31)
α-Amylase Inhibition α-Glucosidase Inhibition
Compounds No.
R
IC50 ± SEMa (µM)
IC50 ± SEMa (µM)
1
9.97 ± 0.08
9.9 ± 0.1
OMe
OMe
2
3
2.58 ± 0.09
3.90 ± 0.18
2.88 ± 0.06
4.01 ± 0.02
OMe
OMe
MeO
4
5
4.31 ± 0.3
6.2 ± 0.03
4.82 ± 0.12
6.4 ± 0.12
MeO
MeO
OMe
5

OMe
OMe
6
6.11 ± 0.12
6.17 ± 0.21
OMe
OMe
MeO
7
8
7.41 ± 0.12
6.51 ± 0.13
6.91 ± 0.12
3.22 ± 0.05
7.71 ± 0.05
6.9 ± 0.22
7.31 ± 0.01
3.02 ± 0.05
OMe
OEt
OMe
F
9
F
10
OMe
OMe
11
12
13
5.11 ± 0.23
4.65 ± 0.08
9.01 ± 0.14
5.22 ± 0.02
4.95 ± 0.08
9.14 ± 0.1
F
Cl
Cl
Cl
OMe
14
15
16
4.1 ± 0.11
5.91 ± 0.03
4.91 ± 0.13
4.22 ± 0.2
5.92 ± 0.12
4.95 ± 0.02
Br
Br
F
F
17
18
7.31 ± 0.12
2.21 ± 0.06
7.61 ± 0.01
2.31 ± 0.09
Br
Br
OMe
OMe
OMe
Br
OMe
19
3.98 ± 0.08
3.08 ± 0.02
6

MeO
Br
20
6.31 ± 0.13
6.44 ± 0.22
OH
OH
21
22
5.21 ± 0.3
5.42 ± 0.32
6.14 ± 0.22
6.11 ± 0.13
OH
23
24
4.0 ± 0.18
4.1 ± 0.11
4.08 ± 0.02
4.22 ± 0.12
OMe
Br
OH
25
26
27
2.33 ± 0.05
4.31 ± 0.03
4.11 ± 0.13
2.55 ± 0.05
4.55 ± 0.32
4.9 ± 0.32
O
O
O
OMe
SMe
CH3
28
29
2.36 ± 0.1
6.01 ± 0.13
6.61 ± 0.23
2.77 ± 0.1
6.34 ± 0.21
7.01 ± 0.02
N
CH3
NO2
30
31
CF3
6.71 ± 0.11
2.01 ± 0.1-
7.11 ± 0.05
2.07 ± 0.1
Acarbose (Standard)b
SEMa (Standard error of mean); Standardb (Inhibitor for α-amylase, α-glucosidase enzymes).
7

Structure-Activity Relationship of α-Amylase Inhibitory Activity
All synthetic derivatives 1-31 were subjected for their in vitro α-amylase inhibitory activity.
Limited structure-activity relationship was established based on variation in the substitution at
ring “R”. Compound 1 (IC50 = 9.97 ± 0.08 µM) bearing benzene ring without any substitution
displayed least activity amongst the series. Nevertheless, the substituted derivatives displayed
good inhibitory activities in comparison with compound 1 showing that substitution on benzene
ring increases the inhibitory potential of compounds. Among dimethoxy substituted
derivatives, compound 2 (IC50 = 2.58 ± 0.09 µM) with two methoxy groups ortho to each other
showed similar activity to acarbose (IC50 = 2.01 ± 0.1 µM). However, compounds 3 (IC50 =
3.90 ± 0.18 µM) and 4 (IC50 = 4.31±0.3 µM) bearing two methoxy substituents meta to each
other and compound 5 (IC50 = 6.2 ± 0.03 µM) having two methoxy para to each other were
found to be less active as compared to compound 2. It showed that methoxy residues adjacent
to each other displayed good a-amylase inhibitory activity and it may be capable to attain such
conformation that will easily interact with enzyme. Among trimethoxy analogs, compounds 6
(IC50 = 6.11 ± 0.12 µM) and 7 (IC50 = 7.41 ± 0.12 µM) showed moderate activities as compared
to the standard, might be the presence of the three methoxy groups resulted in increased steric
hindrance thus having decrease interaction within enzyme’s active site. Compound 8 (IC50 =
6.51 ± 0.13 µM) possessing methoxy and ethoxy groups showed decreased activity in
comparison with dimethoxy substituted compounds 2-5, nonetheless, were more active then
trimethoxy substituted compounds 6 and 7 (Figure-3).
8

H3CO
O
OCH3
O
OCH3
H3CO
H3CO
O
OCH3
O
OC2H5
CH3
O
O
N
H
3
OC2H5
OC2H5
CH3
N
H
CH3
N
H
IC50 = 3.90 ± 0.18

4
2
IC50 = 4.31 ± 0.3

IC50 = 2.58 ± 0.09
OCH3

O
O
OCH3
H3CO
OC2H5
C2H5O
O
O
O
O
N
H
1
CH3
OC2H5
CH3
OC2H5
H3C
H3C
N
H
N
H
CH3
IC50 = 9.97 ± 0.08

5
8
IC50 = 6.2 ± 0.03

IC50 = 6.51 ± 0.13

H3CO
OCH3
OCH3
OCH3
H3CO
O
H3CO
O
O
O
OC2H5
CH3
OC2H5
N
H
6
N
H
7
CH3
IC50 = 7.41 ± 0.12

IC50 = 6.11 ± 0.12

Figure-3: Structure-activity relationship of compounds 1-8
The halogen substituted compounds also showed good to moderate inhibitory activity.
Compound 9 (IC50 = 6.91 ± 0.12 µM) in which fluoro group is present at ortho position showed
lesser activity. However, compound 12 (IC50 = 4.65 ± 0.08 µM) bearing chloro substituent at
para position was more active as compared to compound 13 (IC50 = 9.01 ± 0.14 µM) with
chloro group at ortho position. The chloro group at ortho position is near to side chain group
might be creating steric hindrance thus results in reduced activity. The para bromo substituted
compound 15 (IC50 = 5.91 ± 0.03 µM) exhibited decreased activity as compared to chloro
substituted analog 12. Compound 16 (IC50 = 4.91 ± 0.13 µM) with addition of fluorine meta to
bromine displayed enhanced activity, nevertheless, compound 17 (IC50 = 7.31 ± 0.12 µM) with
bromine and fluorine para to each other resulted in reduced activity. The reduced activity of
compound 17 might be due to position of these two halogens on aryl ring (Figure-4).
9

Br
Br
Br
O
F
F
O
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
CH3
N
H
CH3
N
H
CH3
15
16
17
IC50 = 5.92 ± 0.12
Cl
M

IC50 = 4.95 ± 0.02 M

IC50 = 7.61 ± 0.01 M

F
Cl
O
O
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
CH3
N
CH3
N
H
CH3
H
12
13
9
IC50 = 9.14 ± 0.1 M

IC50 = 7.31 ± 0.01
M

IC50 = 4.95 ± 0.08
M

Figure-4: Structure-activity relationship of compounds 9, 12, 13, 15-17
The synthetic compounds with combination of methoxy and halogens also displayed good
inhibition against α-amylase enzyme. The inhibitory potential of compound 9 might be
compared with compounds 10 (IC50 = 3.22 ± 0.05 µM) bearing fluoro and methoxy groups
para to each other. Compound 10 displayed almost two folds increased inhibition might be the
addition of methoxy group resulted in better interactions of compounds with the active site of
enzyme. The change in positions of fluoro and methoxy groups in compound 11 (IC50 = 5.11
± 0.23 µM) resulted in decreased activity in comparison with compound 10, however, it is more
active than compound 9. Compound 14 (IC50 = 4.1 ± 0.11 µM) with addition of one methoxy
group ortho to chlorine displayed enhanced activity in comparison with its other chloro
substituted analogs. Compounds 18, 19, and 20 with the combination of bromo and methoxy
groups displayed potent inhibitory activity. Compound 18 (IC50 = 2.21 ± 0.06 µM) with bromo
and two methoxy groups which are present at meta and para positions, respectively, displayed
most activity among the whole library of compounds and activity of this compound might be
due to better interaction of compound with enzyme. Compound 19 (IC50 = 3.98 ± 0.08 µM)
having a bromo in between two methoxy groups showed less activity than compound 18.
However, compound 20 (IC50 = 6.31 ± 0.13 µM) in which bromo and methoxy groups are
10

present ortho to each other displayed reduced activity which showed that position of bromo
and methoxy groups displaying an important effect on the activity (Figure-5).
OCH3
OCH3
Br
H3CO
O
OCH3
H3CO
O
Br
O
Br
O
O
O
O
CH3
O
CH3
O
CH3
N
H
CH3
N
CH3
N
H
CH3
H
18
20
19
IC50 = 6.44 ± 0.22 M

IC50 = 2.31 ± 0.09
M
IC50 = 3.08 ± 0.02 M


OCH3
F
H3CO
H3CO
Cl
F
O
O
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
CH3
N
H
CH3
N
H
CH3
10
11
14
IC50 = 4.22 ± 0.2 M
IC50 = 3.02 ± 0.05 M

IC50 = 5.22 ± 0.02 M

Figure-5: Structure-activity relationship of compounds 10, 11, 14, 18-20.
Compound 21 (IC50 = 5.21 ± 0.3 µM) with hydroxy at para position showed moderate activity.
Switching the position of hydroxy group from para to meta in compound 22 (IC50 = 6.11 ±
0.13 µM) resulted in declined activity. Combination of other groups with hydroxyl displayed
increased activity such as compound 23 (IC50 = 4.0 ± 0.18 µM) having methoxy ortho to
hydroxy group resulted in increased activity might be due to excessive hydrogen bonding of
both polar groups within the active site of enzyme. Compound 23 displayed comparable
activity with compound 24 (IC50 = 4.1 ± 0.11 µM) having bromine ortho to hydroxy. Amongst
the benzyloxy substituted molecules 25-27, compound 25 (IC50 = 2.33 ± 0.05 µM) in which
benzyloxy is present at meta position showed two folds more potent activity than compounds
26 (IC50 = 4.31 ± 0.03 µM) and 27 (IC50 = 4.11 ± 0.13 µM) having benzyloxy present at ortho
and meta, positions, respectively (Figure-6).
11

OCH3
OH
HO
O
HO
O
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
CH3
N
H
CH3
N
H
CH3
22
23
21
IC50 = 4.0 ± 0.18 M

IC50 = 6.11 ± 0.13
M

IC50 = 5.21 ± 0.3
M

OH
Br
O
O
O
O
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
CH3
N
H
CH3
N
H
CH3
25
26
24
IC50 = 2.33 ± 0.05
M

IC50 = 4.3 1± 0.03
M

IC50 = 4.1 ± 0.11
M

OCH3
O
O
O
O
CH3
N
H
CH3
27
IC50 = 4.11 ± 0.13
M

Figure-6: Structure-activity relationship of compounds 21-27
Compound 28 (IC50 = 2.36 ± 0.1 µM) bearing a thiomethyl substitution displayed potent
inhibition, the thiol group might have better interactions with the enzyme active site.
Compound 29 (IC50 = 6.01 ± 0.13 µM) with dimethyl amino group displayed moderate activity.
Reduced activity might be due to streric hindrance by the two methyl groups on nitrogen.
Compound 30 (IC50 = 6.61 ± 0.23 µM) having nitro group showed further decreased activity
might be due to electron withdrawing nature of nitro group. Compound 31 (IC50 = 6.71 ± 0.11
µM) having trifluoro methyl substitution also displayed further decreased activity (Figure-7).
12

H3C CH3
N
NO2
SCH3
O
O
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
CH3
N
H
CH3
N
H
CH3
29
30
28
IC50 = 6.61 ± 0.23
M

IC50 = 2.36 ± 0.1 M
IC50 = 6.01 ± 0.13
CF3
M


O
O
O
CH3
N
H
CH3
31
IC50 = 6.71 ± 0.11
M

Figure-7: Structure activity relationship of compounds 28-31
Structure-Activity Relationship of α-Glucosidase Inhibitory Activity
All synthetic derivatives 1-31 exhibited good to moderate α-glucosidase inhibitory activity in
the range of IC50 = 2.31 ± 0.09 - 9.9 ±0.1 µM as compared to the standard acarbose IC50 = 2.07
± 0.1 µM. Compound 1 (IC50 = 9.9 ± 0.1 µM) with unsubstituted benzene ring displayed least
activity among all compounds of the library. Among dimethoxy substituted derivatives,
compound 2 (IC50 = 2.88 ± 0.06 µM) bearing two methoxy groups which are present ortho to
each other showed good inhibitory potential and was found to be more active in comparison
with compounds 3 (IC50 = 4.01 ± 0.02 µM) and 4 (IC50 = 4.82 ± 0.12 µM) bearing two methoxy
groups meta to each other. Nevertheless, compound 5 (IC50 = 6.4 ± 0.12 µM) having two
methoxy groups para to each other was least active amongst di methoxy substituted
compounds. Compound 8 (IC50 = 6.9 ± 0.22 µM) bearing methoxy and ethoxy groups ortho to
each other was less active in comparison with compound 2 which suggests that methoxy groups
at ortho positions are actively participating in the activity (Figure-8).
13

OCH3
H3CO
H3CO
O
OCH3
O
H3CO
O
OCH3
O
O
O
OC2H5
OC2H5
CH3
OC2H5
CH3
N
H
2
CH3
N
H
3
N
H
4
IC50 = 4.01 ± 0.02

IC50 = 2.88 ± 0.06
OCH3

IC50 = 4.82 ± 0.12

OCH3
C2H5O
H3CO
O
O
O
O
O
O
OC2H5
OC2H5
OC2H5
CH3
N
H
CH3
N
H
CH3
N
H
8
1
5
IC50 = 6.4 ± 0.12

IC50 = 6.9 ± 0.22

IC50 = 9.9 ± 0.1

Figure-8: Structure-activity relationship of compounds 1-5, 8
The trimethoxy substituted compounds were less active than dimethoxy compounds. Among
trimethoxy substituted derivatives, compound 6 (IC50 = 6.17 ± 0.21 µM) was more active than
compound 7 (IC50 = 7.71 ± 0.0 µM), since in compound 7 three methoxy groups were adjacent
to the bulky group of benzene ring so it increases hindrance and decreases chance of binding
with active site of enzyme, therefore, the activity was decreased (Figure-9).
OCH3
OCH3
OCH3
H3CO
H3CO
O
H3CO
O
O
O
OC2H5
OC2H5
CH3
N
H
CH3
N
H
7
6
IC50 = 7.71 ± 0.05
IC50 = 6.17 ± 0.21


Figure-9: Structure-activity relationship of compounds 6-7
Compound 9 (IC50 = 7.31 ± 0.01 µM) with fluoro group exhibited weak inhibitory potential as
compared to the standard acarbose. Among chloro substituted derivatives, compound 12 (IC50
= 4.95 ± 0.08 µM) bearing chloro group at para position was two folds more active than ortho
chloro substituted compound 13 (IC50 = 9.14 ± 0.1 µM). Compound 15 (IC50 = 5.92 ± 0.1 µM)
with para substituted bromo group also exhibited decreased activity as compared to chloro
14

substituted compound 12. Compound 16 (IC50 = 4.95 ± 0.02 µM) bearing bromo and fluoro
substituents meta to each other was more active as compared to compound 17 (IC50 = 7.61 ±
0.01 µM) in which bromo and fluoro groups were para to each other (Figure-10).
Cl
F
Cl
O
O
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
13
CH3
N
H
12
CH3
N
H
9
CH3
IC50 = 9.14 ± 0.1 M

IC50 = 7.31 ± 0.01
Br
M
IC50 = 4.95 ± 0.08
Br
M


Br
F
F
O
O
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
CH3
N
H
CH3
N
H
CH3
15
17
16
IC50 = 7.61 ± 0.01 M

IC50 = 5.92 ± 0.12
M

IC50 = 4.95 ± 0.02 M

Figure-10: Structure-activity relationship of compounds 9, 12-17
Compounds 10 (IC50 = 3.02 ± 0.05 µM) and 11 (IC50 = 5.22 ± 0.02 µM) bearing methoxy
groups that are present meta and ortho to fluoro, respectively, displayed good inhibitory
activity in comparison with mono fluoro substituted compound 9. The combination of chloro
with methoxy also exhibited enhanced activity in compound 14 (IC50 = 4.22 ± 0.2 µM) as
compared to its chloro substituted derivatives. Compound 18 (IC50 = 2.31 ± 0.09 µM) having
bromo and two methoxy groups was most potent compound of this series and it displayed
comparable activity with the standard acarbose (IC50 = 2.07 ± 0.1 µM). However, changing the
positions of bromo and methoxy groups in compound 19 (IC50 = 3.08 ± 0.02 µM) resulted in
decreased activity. Compound 20 (IC50 = 6.44 ± 0.22 µM) with one methoxy group along with
bromo showed less activity than compound 18 and 19. It was observed that addition of methoxy
with bromo displayed increased activity (Figure-11).
15

F
OCH3
H3CO
O
H3CO
Cl
O
F
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
11
CH3
N
H
14
CH3
N
H
10
CH3
IC50 = 4.22 ± 0.2 M

IC50 = 5.22 ± 0.02 M
IC50 = 3.02 ± 0.05 M


Br
OCH3
OCH3
H3CO
O
OCH3
H3CO
Br
Br
O
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
19
CH3
N
H
20
CH3
N
H
18
CH3
IC50 = 3.08 ± 0.02 M

IC50 = 2.31 ± 0.09
M
IC50 = 6.44 ± 0.22 M


Figure-11: Structure-activity relationship of compounds 10, 11, 14, 18-20
Among mono hydroxy substituted derivatives, compound 21 (IC50 = 5.42 ± 0.32 µM) with
para substitution showed more activity as compared to compound 22 (IC50 = 6.14 ± 0.22 µM)
having meta substitution. It was found that hydroxy substitution at meta is creating hindrance
with bulky group of benzene thus it will not easily attain such conformation which will interact
well with enzyme. Compound 23 (IC50 = 4.08 ± 0.02 µM) in which methoxy group was present
ortho to hydroxy exhibited increased activity as compared to compound 24 (IC50 = 4.22 ± 0.12
µM) bearing bromo group ortho to hydroxy group. Compound 25 (IC50 = 2.55 ± 0.05 µM)
having benzyloxy group at meta position displayed potential inhibitory activity. Shifting of
benzyloxy group from meta to ortho showed decreased activity in compound 26 (IC50 = 4.55
± 0.32 µM) because bulky benzyloxy at ortho position creates more hindrance with another
bulky group of benzene ring so it will not easily interact with active site of enzyme. Compound
27 (IC50 = 4.9 ± 0.32 µM) in which methoxy group was added ortho to benzyloxy group
exhibited further decreased activity (Figure-12).
16

OH
OCH3
HO
O
HO
O
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
21
CH3
N
H
22
CH3
N
H
23
CH3
IC50 = 5.42 ± 0.32
M

IC50 = 6.14 ± 0.22
M
IC50 = 4.08 ± 0.02
M


OH
Br
O
O
O
O
O
O
O
O
O
CH3
O
CH3
CH3
O
CH3
N
H
CH3
N
H
N
H
CH3
24
25
26
IC50 = 4.22 ± 0.12
M

IC50 = 2.55 ± 0.05
M

IC50 = 4.55 ± 0.32
M

OCH3
O
O
O
O
CH3
CH3
N
H
27
IC50 = 4.9 ± 0.32
M

Figure-12: Structure-activity relationship of compounds 21-27
Compound 28 (IC50 = 2.77 ± 0.1 µM) with para thiomethyl substitution displayed good activity
as compared to the standard acarbose. Compound 29 (IC50 = 6.34 ± 0.21 µM) having dimethyl
amino substitution showed decreased activity as compared to compound 28. Compound 30
(IC50 = 7.01 ± 0.02 µM) with nitro group was found to be moderately active might be due to
electron withdrawing effect of nitro group. Compound 31 (IC50 = 7.11 ± 0.05 µM) bearing
trifluoromethyl substitution displayed weak inhibitory potential (Figure-13).
17

H3C CH3
N
SCH3
NO2
O
O
O
O
O
O
O
CH3
O
CH3
O
CH3
N
H
29
CH3
N
H
28
CH3
N
H
30
CH3
IC50 = 2.77 ± 0.1
M
IC50 = 6.34 ± 0.21
CF3
M


IC50 = 6.34 ± 0.21
M

O
O
O
CH3
N
H
CH3
31
IC50 = 7.11 ± 0.05
M

Figure-13: Structure-activity relationship of compounds 28-31
Docking studies
2.3.
In present study, molecular docking studies were carried out using Autodock Vina for 1-31
synthetic compounds against α-amylase and α-glucosidase by targeting their active sites and
investigating the binding poses adopted by the ligands. The docking parameters for α-amylase
were validated by re-docking acarbose in the binding site of crystallographic protein (PDB ID:
3BAJ) with same parameters as used for the docking of synthesized ligands in the active site
of α-amylase. As shown in Figure-14, the re-docked (blue) acarbose showed interaction in
almost similar manner like its matching crystallographic conformation (red), declaring the
selected parameters to be acceptable. While, the Autodock Vina parameters selected for α-
glucosidase were verified in our previous study.
18

Figure-14: Overlap of re-docked (blue) and crystallographic (red) conformations of acarbose
in α-amylase (3BAJ).
Study carried out with α-amylase displayed significant interactions between the synthesized
ligands and side chain amino acids of the active site i.e. Ala198, Lys200, His201, Trp58, Trp59,
Tyr151, Asp197, Glu233, Leu162, Thr163, Arg195, and Asp300. These are the most active
compounds of the series i.e. 18, 25, 28, and 2 displayed numerous strong interactions through
residues which are present on side chain of the active site of α-amylase. Compound 18 (IC50 =
2.21 ± 0.06 µM) (Figure-15a) which is the most active compound, its graphical investigation
displayed that oxygen atom of ketonic group mediated a hydrogen bond interaction with side
chain imidazole -NH of His305 (3.00 Å), however, the bromine atom at ortho position of
phenyl group was found making hydrogen bond interaction with the hydroxyl group of side
chain Thr163 (3.26 Å). Sidewise, following three carbon hydrogen bond interactions were
spotted between the docked ligand and the side chain residues of the active site; i) between
para substituted methoxy of phenyl ring and hydroxyl group of side chain Tyr151 (3.54 Å), ii)
between alkyl chain of ester group and carboxylate group of side chain Glu233 (3.51 Å) and
iii) between meta substituted methoxy group and carboxylate group of side chain Asp300 (3.01
Å), respectively. Other interactions which were associated in stabilization of the complex were
the alkyl interaction between bromine atom of phenyl group and side chain Leu162 and π-
sigma interactions between the methyl groups of 18 and indole rings of Trp59.
Binding manner of 25, which displayed as second most active derivative (IC50 = 2.33 ± 0.05
µM) revealed this compound was involved in forming almost the same interactions as that
formed by 18 including one hydrogen bond interaction between ketonic group of quinoline and
19

side chain imidazole-NH of His305 (3.05 Å) as shown in Figure-15b. Whilst, the second
hydrogen bond formed by the bromine atom of compound 18 and side chain Thr163 was not
found in this case. Although, the phenyl ring of benzyloxy group was found in forming
numerous interactions including π-anion, π-sigma, π-π T-Shaped and π-alkyl interactions with
the side chain Glu233, Ile235, His201, Lys200 and Ala198, respectively. These interactions
could be the main reason for stabilizing the ligand receptor complex and making the derivative
second most active analog among the series.
The binding manner of another active molecule of the series, compound 28 (IC50 = 2.33 ± 0.05
µM) (Figure-15c) displayed that this compound was found in forming a single strong hydrogen
bond interaction between the NH group of quinoline and the carboxylate oxygen atom of side
chain Asp300 (2.19 Å). A carbon hydrogen bond which was found between the ethyl group of
ester and carbonyl oxygen of side chain of Tyr62 showed a bond distance of 3.71 Å. Other
interactions which could be resulting in decreasing the energy of the complex were the π-sulfur
and π-sigma interactions between the methylthio group of compound 28 and the indole ring of
side chain Trp59 and alkyl interactions between the methyl group of ligand and alkyl part of
side chain Ile235.
Docking results of fourth most active analog of the series, compound 2 (IC50 = 2.58 ± 0.09 µM)
displayed its involvement in formation of two hydrogen bond interactions between the oxygen
atom of ester group and side chain imidazole-NH of His305 (3.13 Å) and between carbonyl
oxygen of ketonic group and hydroxyl group of side chain Thr163 (2.73 Å), respectively
(Figure-15d). Also, a carbon hydrogen bond of 3.65 Å was found between the methoxy group
at para position of phenyl ring and carboxylate oxygen which is present on side chain Asp300.
Among hydrophobic interactions, the two methyl groups of this compound were also mediating
a π-alkyl interaction with the side chain Trp59.
Ultimately, in the docking study of α-amylase inhibition, the interactions of least active
compounds i.e. 13 (IC50 = 9.01 ± 0.14 µM) and 1 (IC50 = 9.97 ± 0.08 µM) were also analyzed
and it was found that these two compounds failed to develop hydrogen bond interactions with
the residues of the active site (Figure-15e and Figure-15f). Furthermore, compound 13 bearing
chlorine atom at ortho position resulted in making more hydrophobic interactions with the
amino acids which are present on the side chain of the active site as compared to 1. Therefore,
the phenyl ring substitution greatly influenced the interaction of ligands and the active site
residues. Although, the substituted phenyl ring itself was not involved in making important
20

interactions with catalytic residues but it resulted the parent molecule to interact and make
stronger interactions with the catalytic residues.
Figure-15: Docked conformation of synthesized compounds with the side chain residues of α-
amylase, Ligands shown in green color, side chain amino acids in violet color, and distances
(Angstrom) colored in black; A) Interactions of compound 18; B) Interactions of compound
25; C) Interactions of compound 28; D) Interactions of compound 2; E) Interactions of
compound 13 and F) Interactions of compound 1.
Molecular docking studies was also performed to support the in vitro inhibition potential
studies of synthetic derivatives against α-glucosidase in which Autodock Vina is used. All
21

synthetic compounds were docked against established homology model of α-glucosidase. The
most active molecule of the series showed significant interactions with the side chain amino
acids of the homology modeled α-glucosidase. The most active compound 18 (Figure-16a)
was found in establishing three hydrogen bond interactions with the side chain residues of the
active site i.e. i) between carbonyl oxygen of ester group and NH group of side chain Asn241
(1.78 Å), ii) between oxygen of ethoxy group and the side chain imidazole-NH of His279 (2.33
Å) and iii) between carbonyl oxygen of ketonic group and side chain NH groups of Arg312
(1.95 Å and 2.64 Å), respectively. Other residues which were found in making hydrophobic
interactions with compound 18 were the involvement of Phe157, Pro309 and Phe310 in
forming carbon hydrogen bonds, Phe157 and Phe300 forming π-alkyl interactions and Glu304
spotted in creating halogen bonding with the bromine atom which is present at ortho position.
The predicted binding modes of compound 25 which is the second most active compound in
the whole series displayed that the compound was involved in various interactions as shown in
Figure-16b. The carbonyl oxygen of ketonic group was found in developing two hydrogen
bond interactions with the NH groups of side chain Arg312 with the bond distances of 2.43 Å
and 2.47 Å, respectively. While, the carbonyl group of the ester was found in forming hydrogen
bond interaction with the NH group of side chain His279 (2.00 Å). Other interactions which
could have resulted in stabilizing the ligand receptor complex are the formation of hydrophobic
interactions which are found between the ligand and side chain residues including Phe157,
Phe177, Pro240 and Arg312, respectively.
The third and fourth most active compounds 28 and 2 (Figure-16c and Figure-16d) were also
involved in forming hydrogen bond interactions with side chain amino acids of the active site.
The carbonyl oxygen of compound 28 was found forming two hydrogen bonds with the NH
groups of side chain Arg312 having a bond distance of 2.75 Å and 2.76 Å, respectively. While,
compound 2 was found forming three hydrogen bond interactions i.e. i) between methoxy
oxygen at para position and NH group of side chain His239 (2.33 Å), ii) between ethoxy
oxygen of ester group and NH group of side chain His279 (2.14 Å) and iii) between carbonyl
oxygen of ester group and NH group of side chain Arg312 (2.82 Å), respectively. Meanwhile,
the sulfur atom of compound 28 was also found in forming π-sulfur interaction with the phenyl
ring of side chain Phe311. Other interactions resulting in minimizing the energy of the complex
formed by compound 28 and 2 were the carbon hydrogen interactions of the ethyl chains of the
ester group of both compounds with the side chain Glu304 and hydrophobic interactions
22

including alkyl-alkyl interactions, π-alkyl interactions and π-sigma interactions with the side
chain residues including Lys155, Phe158, Phe177, and Phe300, respectively.
The least active derivatives among the series were also analyzed by studying their predicted
binding modes as shown in Figure-16e and Figure-16f. Both compound 13 and 1 were found
forming less interactions with the residues of active site as those formed by the active
compounds. Compound 13 was found in forming interactions with only four residues of the
active site including one hydrogen bond interaction between the carbonyl oxygen of ester group
and NH group of side chain His239 (2.78 Å). While, compound 1 was also found in forming a
single hydrogen bond interaction between the NH group of quinoline and carbony oxygen of
side chain Phe157 (1.82 Å). Although, these compounds show inhibitory potential, but their
least activeness as compared to the most active compounds of the series may be due to the
establishment of less interactions with the active site of the enzyme. Therefore, it is concluded
that those derivatives which contained substituents which are present on the meta and para
position of the substituted phenyl ring resulted the docked complex to form more interactions
as compared to those derivatives which contained either no substitution or were ortho
substituted.
23

Figure-16: Docked conformation of synthesized compounds with the side chain residues of α-
glucosidase, Ligands shown in green color, side chain amino acids in violet color, and distances
(Angstrom) colored in black; A) Interactions of compound 18; B) Interactions of compound
25; C) Interactions of compound 28; D) Interactions of compound 2; E) Interactions of
compound 13 and F) Interactions of compound 1.
Conclusion
Ethyl-2,7,7-trimethyl-5-oxo-4-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylates (1-31)
were synthesized and evaluated for their antidiabetic potential. The synthetic molecules were
subjected for their in vitro α-amylase and α-glucosidase inhibitory activities and displayed
24

potential inhibition against α-amylase in the range of (IC50 = 2.21 ± 0.06 - 9.97 ± 0.08 µM) as
compared to the standard acarbose (IC50 = 2.01 ± 0.1 µM). The synthetic derivatives also
exhibited good to moderate α-glucosidase inhibition in the range of (IC50 = 2.01 ± 0.1 - 2.31 ±
0.09 - 9.9 ± 0.1 µM) as compared to the standard acarbose (IC50 = 2.07 ± 0.1 µM). Limited
structure-activity relationship deduced that almost all structural features are contributing in the
α-amylase and α-glucosidase inhibition, however, compounds with functional groups such as
F, Cl, Br, OH, and OMe were actively participating in the activity. To identify the structural
features that were involved in the binding interactions with the active site of enzyme in silico
study was also performed. This study has showed various chemical spaces that may be use as
lead molecules for future research to discover good α-amylase and α-glucosidase inhibitors for
the treatment of diabetes patients.
Experimental
General procedure for the synthesis of Ethyl-2,7,7-trimethyl-5-oxo-4-phenyl-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate derivatives (1-31)
In a 100 mL round-bottomed flask dimedone (1 mmol), ethyl acetoacetate (1 mmol),
ammonium acetate (1 mml) and different aryl aldehyde derivative (1 mmol) were added.
Copper nitrate trihydrate (Cu(NO3)2.3H2O (10 mol%) was added as catalyst in this flask and
heated at 80-90 ºC with vigorous mixing. Thin layer chromatography (TLC) was used to predict
reaction progress. As the reaction was completed, the solid product was washed with distilled
water then hexane after that with hot hexane to get pure product. Ethanol was used for
crystallization of obtained product to get pure product. Compounds 4, 8, 17, 19, 20, 24, and 27
found to be new while remaining compounds are already reported in literature [19-26]. Various
spectroscopic techniques were used to characterize the synthetic compounds. A complete
spectral data of all compounds is as follows.
Ethyl-2,7,7-trimethyl-5-oxo-4-phenyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (1)
[27]
State: solid; Color: light green; Yield: 65%; M.P.: 156-159 ºC; Rf: 0.48
1
(dichloromethane/methanol, 9.6:0.4); H-NMR (300 MHz, DMSO-d6): δH 9.04 (s, 1H, NH),
7.19 (m, 5H, H-2', H-3', H-4', H-5', H-6'), 4.83 (s, 1H, H-4), 3.99 (q, JCH2,CH3 = 6.9 Hz, 2H,
CH2), 2.43 (d, JHa,Hb = 17.1Hz, 1H, Ha), 2.29 (ovp, 1H, Hb), 2.26 (s, 3H, CH3), 2.17 (d, JHa',Hb'
= 16.2 Hz, 1H, Ha'), 1.98 (d, JHb',Ha' = 15.9 Hz, 1H, Hb'), 1.13 (t, JCH3,CH2 = 7.2 Hz, 3H, CH3),
25

0.99 (s, 3H, CH3), 0.82 (s, 3H, CH3); EI-MS m/z (% rel. abund.): 339 (M+, 34), 310 (19), 294
(14), 262 (100), 234 (59), 178 (10), 150 (5), 115 (2), 105 (3) 77 (1).
Ethyl-4-(3,4-dimethoxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (2) [28]
State: solid; Color: white; Yield: 65%, M.P.: 198-200 ºC; Rf: 0.43 (dichloromethane/methanol,
9.6:0.4); 1H-NMR (300 MHz, DMSO-d6): δH 9.00 (s, 1H, NH), 6.76 (ovp, 2H, H-2', H-5'), 6.62
(dd, J6',2' = 1.8 Hz, J6',5' = 6.6 Hz, 1H, H-6'), 4.77 (s, 1H, H-4), 4.02 (q, JCH2,CH3 = 6.9 Hz, 2H,
CH2), 3.65 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 2.44 (d, JHa,Hb = 17.1 Hz, 1H, Ha), 2.29 (ovp,
1H, Hb), 2.25 (s, 3H, CH3), 2.19 (d, J Ha',Hb' = 16.2 Hz, 1H, Ha'), 2.00 (d, JHb',Ha' = 16.2Hz, 1H,
Hb'), 1.17 (t, JCH3,CH2 = 7.2 Hz, 3H, CH3), 1.00 (s, 3H, CH3), 0.87 (s, 3H, CH3); EI-MS m/z (%
rel. abund.): 399 (M+, 93), 370 (27), 354 (12), 326 (22), 262 (100), 252 (25), 234 (35), 178
(10), 83 (3).
Ethyl-4-(3,5-dimethoxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (3) [27]
State: Solid; Color: white; Yield: 70%, M.P.: 152-155 ºC; Rf: 0.42 (dichloromethane/methanol,
9.6:0.4); 1H-NMR (400 MHz, DMSO-d6): δH 9.03 (s, 1H, NH), 6.27 (d, J2',4'/6',4' = 2.0 Hz, 2H,
H-2', H-6'), 6.23 (d, J4',2'/4',6' = 2.4 Hz, 1H, H-4'), (m, 1H, H-4' ), 4.79 (s, 1H, H-4), 4.02 (q,
JCH2,CH3 = 7.2 Hz, 2H, CH2), 3.64 (s, 6H, (OCH3)2), 2.42 (d, JHa,Hb = 17.2 Hz,1H, Ha), 2.30
(ovp, 1H, Hb), 2.25 (s, 3H, CH3), 2.18 (d, JHa',Hb' = 16.0 Hz, 1H, Ha'), 2.01 (d, JHb',Ha' = 16.0 Hz,
1H, Hb'), 1.16 (t, JCH3,CH2 = 6.8 Hz, 3H, CH3), 1.00 (s, 3H, CH3), 0.88 (s, 3H, CH3); EI-MS m/z
(% rel. abund.): 399 (M+, 83), 384 (3), 370 (13), 354 (16), 326 (26), 296 (5), 262 (100), 246
(5), 234 (83), 178 (14), 150 (5), 138 (8), 77 (2).
Ethyl-4-(2,6-dimethoxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (4)
State: Solid; Color: White; Yield: 65%, M.P.: 220-223 ºC; Rf: 0.45 (dichloromethane/methanol,
9.6:0.4); 1H-NMR (400 MHz, DMSO-d6): δH 8.77 (s, 1H, NH), 6.99 (t, J4',3'/4',5' = 8.4 Hz, 1H,
H-4'), 6.48 (d, J3',4'/5',4' = 8.4 Hz, 2H, H-2', H-5'), 5.44 (s, 1H, H-4), 3.85 (q, JCH2,CH3 = 7.2 Hz,
2H, CH2), 3.68 (s, 6H, (OCH3)2), 2.33 (d, JHa,Hb = 16.4 Hz, 1H, Ha'), 2.15 (ovp, 1H, Hb'), 2.14
(s, 3H, CH3), 2.06 (d, JHa',Hb' = 16.0 Hz, 1H, Ha'), 1.82 (d, JHb',Ha' = 16.0 Hz, 1H, Hb'), 1.04 (t,
JCH3,CH2 = 7.2 Hz, 3H, CH3), 0.96 (s, 3H, CH3), 0.78 (s, 3H, CH3); 13C-NMR (75 MHz, DMSO-
26

d6): δ 193.4, 150.9, 150.1, 144.8, 142.9, 126.6, 123.0 108.0, 103.9, 101.2, 58.3, 55.4, 50.6,
39.7, 31.7, 29.3 26.6, 25.9, 18.1, 13.9; EI-MS m/z (% rel. abund.): 399 (M+, 100), 384 (11),
370 (86), 354 (14), 322 (16), 262 (56), 257 (29), 241 (34), 234 (20), 189 (14), 115 (3), 83 (5);
HREI-MS Calcd for C23H29O5N1: m/z = 399.20424, Found 399.2046.
Ethyl-4-(2,5-dimethoxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (5) [29]
State: Solid; Color: White; Yield: 62%, M.P.: 169-172 ºC; Rf: 0.44 (dichloromethane/methanol,
9.6:0.4); 1H-NMR (300 MHz, DMSO-d6): δH 8.93 (s, 1H, NH), 6.75 (d, J3',4' = 8.4 Hz, 1H, H-
3') ), 6.62 (ovp, 2H, H-4', H-6'), 4.97 (s, 1H, H-4), 3.92 (q, JCH2,CH3 = 2.1 Hz, 2H, CH2), 3.63
(s, 3H, OCH3), 3.61 (s, 3H, OCH3), 2.41 (d, JHa,Hb = 16.8 Hz, 1H, Ha), 2.25 (d, JHb,Ha = 18.0
Hz, 1H, Hb), 2.16 (s, 3H, CH3), 2.13 (d, JHa',Hb' = 16.2Hz, 1H, Ha'), 1.91 (d, JHb',Ha' = 15.9 Hz,
1H, Hb'), 1.12 (t, JCH3,CH2 = 6.9 Hz, 3H, CH3), 0.98 (s, 3H, CH3), 0.84 (s, 3H, CH3); EI-MS m/z
(% rel. abund.): 399 (M+, 55), 366 (46), 354 (6), 322 (7), 262 (76), 257 (100), 234 (16), 200
(18), 189 (3), 138 (5), 83 (11), 55 (4).
Ethyl-2,7,7-trimethyl-5-oxo-4-(3,4,5-trimethoxyphenyl)-1,4,5,6,7,8-hexahydroquinoline-
3-carboxylate (6) [30]
State: Solid; Color: White; Yield: 65%, M.P.: 178-180 ºC; Rf: 0.43 (dichloromethane/methanol,
9.6:0.4); 1H-NMR (300 MHz, DMSO-d6): δH 9.06 (s, 1H, NH), 6.39 (s, 2H, H-5', H-6'), 4.80
(s, 1H, H-4), 4.06 (q, JCH2,CH3 = 6.9 Hz, 2H, CH2), 3.65 (s, 6H, (OCH3)2), 3.57 (s, 3H, OCH3),
2.41 (ovp, 1H, Ha), 2.33 (d, JHb,Ha = 17.4 Hz, 1H, Hb), 2.25 (s, 3H, CH3), 2.21 (d, JHa',Hb' = 16.2
Hz, 1H, Ha'), 2.03 (d, JHb',Ha' = 16.2 Hz, 1H, Hb'), 1.20 (t, J CH3,CH2 = 7.2 Hz, 3H, CH3), 1.01 (s,
3H, CH3), 0.92 (s, 3H, CH3); EI-MS m/z (% rel. abund.): 429 (M+, 76), 400 (14), 384 (10), 356
(12), 262 (100), 234 (29), 189 (2), 168 (9), 150 (3).
Ethyl-2,7,7-trimethyl-5-oxo-4-(2,3,4-trimethoxyphenyl)-1,4,5,6,7,8-hexahydroquinoline-
3-carboxylate (7)
State: Solid; Color: White; Yield: 65%, M.P.: 159-162 ºC; Rf: 0.43 (dichloromethane/methanol,
9.6:0.4); 1H-NMR (400 MHz, DMSO-d6): δH 9.89 (s, 1H, NH), 6.78 (d, J6',5' = 8.8 Hz, 1H, H-
6'), 6.59 (d, J5',6' = 8.8 Hz, 1H, H-5'), 4.95 (s, 1H, H-4), 3.92 (q, JCH2,CH3 = 2.4 Hz, 2H, CH2),
3.76 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.63 (s, 3H, OCH3), 2.38 (d, JHa,Hb = 17.2 Hz, 1H, Ha),
2.25 (d, JHb,Ha = 17.2 Hz, 1H, Hb), 2.17 (s, 3H, CH3), 2.12 (d, JHa',Hb' = 16.0 Hz, 1H, Ha'), 1.93
27

(d, JHb',Ha' = 16.0 Hz, 1H, Hb'), 1.13 (t, JCH3,CH2 = 7.2 Hz, 3H, CH3), 0.98 (s, 3H, CH3), 0.87 (s,
3H, CH3); EI-MS m/z (% rel. abund.): 429 (M+ , 30), 409 (100), 398 (59), 384 (4), 352 (9), 325
(3), 273 (8), 262 (35), 234 (12), 189 (3), 161 (2), 83 (4).
Ethyl-4-(3-ethoxy-4-methoxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate (8)
State: Solid; Color: White; Yield: 60%, M.P.: 173-176 ºC; Rf: 0.40 (dichloromethane/methanol,
9.6:0.4); 1H-NMR (300 MHz, DMSO-d6):δH 8.99 (s, 1H, NH), 6.75 (ovp, 2H, H-2', H-5'), 6.61
(d, J6',5' = 8.1 Hz, 1H, H-6'), 4.76 (s, 1H, H-4), 4.01 (q, JCH2,CH3 = 6.9 Hz, 2H, CH2), 3.89 (q,
JCH2',CH3' = 5.1 Hz, 2H, CH2'), 3.65 (s, 3H, OCH3) 2.43 (d, JHa,Hb = 17.4 Hz,1H, Ha), 2.28 (ovp,
1H, Hb), 2.25 (s, 3H, CH3), 2.18 (d, JHa',Hb' = 15.9 Hz, 1H, 8-Ha), 1.99 (d, JHb',Ha' = 16.2 Hz,
1H, Hb'), 1.29 (t, JCH3',CH2' = 6.9 Hz, 3H, CH3'), 1.16 (t, JCH3,CH2 = 6.9 Hz, 3H, CH3), 0.99 (s,
3H, CH3), 0.85 (s, 3H, CH3); 13C-NMR (75 MHz, DMSO-d6): δc 194.2, 166.9, 149.2, 147.1,
147.0, 144.5, 140.3, 119.2, 113.0, 111.5, 110.0, 103.7, 63.5, 58.9, 55.3, 50.2, 39.5, 35.0, 32.0,
29.1, 26.3, 18.1, 14.7, 14.1; EI-MS m/z (% rel. abund.): 413 (M+ ,89), 384 (26), 368 (13), 340
(17), 302 (19), 273 (7), 262 (100), 234 (25), 218 (2), 189 (3), 178 (8), 152 (8), 83 (8), 54 (3);
HREI-MS Calcd for C24H31O5N1: m/z = 413.2225, Found 413.2202.
Ethyl-4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (9)
State: Solid; Color: Light green; Yield: 59%, M.P.: 160-162 ºC; Rf: 0.37
1
(dichloromethane/methanol, 9.6:0.4); H-NMR (400 MHz, DMSO-d6): δH 9.04 (s, 1H, NH),
7.20 (t, J4',3'/4', 5' = 7.2 Hz, 1H, H-4'), 7.09 (m, 1H, H-3') 7.02 (overlap multiplet, 2H, H-5', H-
6'), 5.03 (s, 1H, H-4), 3.93 (q, JCH2,CH3 = 7.2Hz, 2H, CH2), 2.42 (d, JHa,Hb = 17.2 Hz, 1H, Ha),
2.27 (ovp, 1H, 6-Hb), 2.23 (s, 3H, CH3), 2.15 (d, JHa',Hb' = 15.6 Hz, 1H, Ha'), 1.92 (d, JHb,Ha
=
16.4 Hz, 1H, Hb'), 1.09 (t, JCH3,CH2 = 7.2Hz, 3H, CH3), 0.99 (s, 3H, CH3), 0.82 (s, 3H, CH3) ;
EI-MS m/z (% rel. abund.): 357 (M+, 26), 336 (4), 328 (16), 312 (7), 284 (9), 262 (100), 234
(30), 198 (3), 178 (4), 157 (3), 133 (1).
Ethyl-4-(2-fluoro-4-methoxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate (10)
State: Solid; Color: Green; Yield:52%, M.P.: 180-182 ºC; Rf: 0.32 (dichloromethane/methanol,
9.6:0.4); 1H-NMR (400 MHz, DMSO-d6): δH 9.00 (s, 1H, NH), 7.07 (t, J H-3', F = 8.4Hz, 1H, H-
28

3'), 6.60 (overlap multiplet 2H, H-5', H-6'), 4.94 (s, 1H, H-4), 3.94 (q, JCH2,CH3 = 7.2 Hz, 2H,
CH2), 3.67 (s, 3H, OCH3), 2.41 (d, JHa,Hb = 16.8 Hz, 1H, Ha), 2.25 (ovp, 1H, Hb), 2.22 (s, 3H,
CH3), 2.14 (d, JHa',Hb' = 16.0 Hz, 1H, Ha'), 1.92 (d, JHb',Ha' = 16.0 Hz, 1H, Hb'), 1.11 (t, J CH3,CH2
= 7.2 Hz, 3H, CH3), 0.99 (s, 3H, CH3), 0.83 (s, 3H, CH3); EI-MS m/z (% rel. abund.): 387 (M+,
37), 358 (14), 342 (9), 314 (17), 262 (100), 234 (15), 189 (4), 178 (4), 150 (1), 126 (2), 83 (1).
Ethyl-4-(4-fluoro-3-methoxyphenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate (11)
State: Solid; Color: Green; Yield: 52%, M.P.: 212-215 ºC; Rf: 0.32 (dichloromethane/methanol,
9.6:0.4); 1H-NMR (400 MHz, DMSO-d6): δH 9.06 (s, 1H, NH), 7.02 (m, 1H, H-5'), 6.89 (dd,
J6',2' = 1.6 Hz, J6',5' = 6.8 Hz, 1H, H-6'), 6.66 (m, 1H, H-2'), 4.82 (s, 1H, H-4), 4.01 (q, JCH2,CH3
= 6.8 Hz, 2H, CH2), 3.73 (s, 3H, OCH3), 2.43 (d, JHa,Hb = 17.2 Hz, 1H, Ha), 2.30 (ovp, 1H, Hb),
2.27 (s, 3H, CH3), 2.18 (d, JHa',Hb' = 16.0 Hz, 1H, Ha'), 2.00 (d, JHb',Ha' = 16.0 Hz, 1H, Hb'), 1.15
(t, JCH3,CH2 = 7.2 Hz, 3H, CH3), 1.00 (s, 3H, CH3), 0.85 (s, 3H, CH3); EI-MS m/z (% rel. abund.):
387 (M+, 22), 358 (7), 342 (3), 314 (5), 262 (100), 234 (15), 178 (3), 150 (1), 77 (1).
Ethyl-4-(4-chlorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (12) [31]
State: Solid; Color: White; Yield: 67%, M.P.: 224-226 ºC; Rf: 0.40 (dichloromethane/methanol,
9.6:0.4); 1H-NMR (400 MHz, DMSO-d6): δH 9.08 (s, 1H, NH), 7.24 (d, J3',2'/5',6' = 8.4 Hz, 2H,
H-3', H-5'), 7.14 (d, J2',3'/6',5' = 8.4 Hz, 2H, H-2', H-6'), 4.82 (s, 1H, H-4), 3.98 (q, JCH2,CH3 = 7.2
Hz, 2H, CH2), 2.42 (d, JHa,Hb = 17.2 Hz, 1H, Ha), 2.27 (s, 3H, CH3), 2.24 (ovp, 1H, Hb), 2.17
(d, JHa',Hb' = 16.4 Hz, 1H, Ha'), 1.98 (d, JHb',Ha' = 16.4 Hz, 1H, Hb'), 1.12 (t, JCH3,CH2 = 6.8 Hz,
3H, CH3), 0.99 (s, 3H, CH3), 0.81 (s, 3H, CH3); EI-MS m/z (% rel. abund.): 373 (M+, 90.3),
344 (29), 328 (24), 262 (100), 252 (15), 234 (68), 178 (10), 150 (6), 105 (2), 83 (2).
Ethyl-4-(2-chlorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (13) [32]
State: Solid; Color: White; Yield: 60%, M.P.: 169-172 ºC; Rf: 0.41 (dichloromethane/methanol,
9.6:0.4); 1H-NMR (400 MHz, DMSO-d6): δH 9.05 (s, 1H, NH), 7.27 (d, J3',4' = 7.2 Hz, 1H, H-
3'), 7.21 (ovp, 2H, H-4', H-6'), 7.07 (t, J5',4'/5',6' = 7.6 Hz, 1H, H-5'), 5.16 (s, 1H, H-4), 3.93 (q,
JCH2,CH3 = 4.4 Hz, 2H, CH2), 2.42 (d, JHa,Hb = 16.8 Hz, 1H, Ha'), 2.26 (ovp, 1H, Hb'), 2.22 (s,
3H, CH3), 2.14 (d, JHa',Hb'= 16.4 Hz, 1H, Ha'), 1.91 (d, JHb',Ha' = 16.0 Hz, 1H, Hb'), 1.08 (t, JCH3,CH2
29