Design, synthesis, α-amylase inhibition and in silico docking study of novel quinoline bearing proline derivatives

Article abstract of DOI:10.1016/j.molstruc.2020.127873

α-amylase enzyme hydrolyses carbohydrate into glucose is known to be an important molecular target for type 2 Diabetes mellitus. In the course of developing α-amylase enzyme inhibitors, we designed, synthesized seventeen novel quinoline bearing proline analogs, subsequently physico-chemical properties of designed analogs were also in silico predicted for their drug likeness evaluation. Synthesized compounds were characterized by spectral analysis such as Mass, IR, ¹H NMR, ¹³C NMR and further screened in vitro for α-amylase inhibitory activity using acarbose as standard drug. Seven analogs, 6a, 6b, 6c, 6d, 6g, 10b and 10c showed significant α-amylase inhibitory activity. Eight analogs, 5, 6e, 6f, 6h, 6j, 10a, 10d and 10e showed good to moderate activity while other two analogs, 6i and 9 showed least activity. The molecular docking study of significantly active and weakly active compounds was performed in order to study their putative binding mode of the most and least active compounds (6c and 6i).

Full text of DOI:10.1016/j.molstruc.2020.127873

Journal Pre-proof
Design, synthesis, α-amylase inhibition and in-silico docking study of novel quinoline
bearing proline derivatives
M.S. Ganesan, K. Kanmani Raja, K. Narashimhan, S. Murugesan, Banoth Karan
Kumar
PII:
S0022-2860(20)30197-6
DOI:
https://doi.org/10.1016/j.molstruc.2020.127873
MOLSTR 127873
Reference:
To appear in: Journal of Molecular Structure
Received Date: 6 December 2019
Revised Date: 24 January 2020
Accepted Date: 7 February 2020
Please cite this article as: M.S. Ganesan, K.K. Raja, K. Narashimhan, S. Murugesan, B.K. Kumar,
Design, synthesis, α-amylase inhibition and in-silico docking study of novel quinoline bearing proline
derivatives, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.127873.
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Graphical Abstract

Design, synthesis, α-amylase inhibition and In-silico docking
study of novel quinoline bearing proline derivatives
a*
a*
b
c
M.S.Ganesan, K. Kanmani Raja, K. Narashimhan, S. Murugesan,
c
Banoth karan kumar
a
PG & Research Department of Chemistry, Nandanam Arts College (A), Chennai- 600 035,
Affiliated to Madras University, Tamil Nadu, India.
b
Orchid Pharma Limited, 138-149, SIDCO Industrial Estate, Alathur, Chennai-603110, Tamil
Nadu, India.
c
Medicinal Chemistry Research Laboratory, Department of Pharmacy, Birla Institute of
Technology and Science Pilani, Pilani Campus, Pilani-333031. Rajasthan, India.
*
Corresponding author E-mail: ganeshsarathy@gmail.com
Corresponding author E-mail: kkanmaniraja@gmail.com
*
ABSTRACT
α-amylase enzyme hydrolyses carbohydrate into glucose is known to be an important
molecular target for type 2 Diabetes mellitus. In the course of developing α-amylase enzyme
inhibitors, we designed, synthesized seventeen novel quinoline bearing proline analogs,
subsequently physico-chemical properties of designed analogs were also in-silico predicted
for their drug likeness evaluation. Synthesized compounds were characterized by spectral
1
13
analysis such as Mass, IR, H NMR, C NMR and further screened in vitro for
inhibitory activity using acarbose as standard drug. Seven analogs, 6a, 6b, 6c, 6d, 6g, 10b and
0c showed significant α-amylase inhibitory activity. Eight analogs, 6e, 6f, 6h, 5, 6j, 10a, 10d
α-amylase
1
and 10e showed good to moderate activity while other two analogs, 6i and 9 showed least
activity. The molecular docking study of significantly active and weakly active compounds

was performed in order to study their putative binding mode of the most and least active
compounds (6c and 6i).
Keywords: Proline, Quinoline, Anti diabetic, Molecular docking, α-amylase inhibition.
1
. Introduction
Diabetes mellitus (DM) has been known as third leading cause of death in humans [1].
Study shows that 422 million persons affected with diabetes in 2016 and this number is
expected to increase 844 million in 2030 [2]. There are two primary types of diabetes
mellitus, insulin dependent (Type 1) and non-insulin dependent (Type2) [3]. Type 2 diabetes
is most commonly prevalent over type 1 diabetes [4]. α-amylase is the key enzyme directly
related to type 2 diabetes and found in saliva as well as pancreas [5]. Normal food contains
polysaccharides (Starch and glycogen) [6]. α-amylase enzyme hydrolyses these
polysaccharides into oligosaccharides further hydrolyses into absorbable monosaccharide
(Glucose) and releases it into blood stream that causes sharp increase in the blood glucose
level [7]. Disorders in carbohydrate uptake and immediate digestion by α-amylase enzyme
causes sudden raise in blood glucose level [8]. This can be controlled by inhibition of
carbohydrate hydrolysing α-amylase enzyme is the critical therapeutic area used to control
type 2 diabetes [9]. Acarbose and miglitol are such synthetic oral hyperglycaemic drugs
commercially available in the market but having side effects like discomfort, flatulence,
meteorism and diarrhoea which causes therapy discontinuation [10]. The WHO recommends
to search a safe, potent and non-toxic natural anti-diabetic agent [11]. Ethano botanical
research on traditional herbal remedies has confirmed more than 1200 plants with
hypoglycaemic effect but they cannot be administered directly as a drug [12].

In the search of structures with significant bioactivity, we focussed onto the development
of molecules through combination of different active heterocyclic compounds that may lead
to identification of compounds with improved alpha amylase inhibitory activity.
Among heterocyclic compounds, proline ring is endowed with various activities such as
Antimicrobial, anti-oxidant, anti-carcinogen, anti-HIV and anti-inflammatory, [13-17].
Mahindra Kumar Mishra et al., explored proline as α-amylase inhibitory active moiety [18].
Proline and its analogs isolated from the leaves of koenigii have been used in traditional
medicine particularly for hyperglycaemia [19]. Many heterocyclic amides play a major role
in designing novel chemical entities and also they are well known for their biological activity.
Among them Proline and its substituted phenyl amides have been playing a vital role in drug
discovery design [20].
Quinoline derivatives constitute a vital role in the development of new drug discovery
which possess many biological applications like anti-oxidant, anti-inflammatory, anti-cancer,
anti-viral, anti-diabetic, antimalarial, anti-tubercular and anti-microbial [21-27]. Quinoline
containing compounds have been well known as a drug such as Quinine, bulaquine,
pamaquine and tafenoquine [28]. Quinoline bearing many heterocyclic compounds have been
reported to have potential
α-amylase inhibitory activity [29-30]. Quinoline and their
derivatives isolated from Ephedra pachyclada stem and Ruta chalepensis leaves have shown
significant α-amylase inhibitory activity. [31-32]. 4-Flouro phenyl and cyclopropyl
substituted Quinoline containing chemistry is becoming popular in drug design. Pitavastain
drug contains 4-flouro phenyl and cyclopropyl substituted quinoline in its core structure [33].
Methylene group is an important linker in many drugs [34].

The above observation prompted us, to combine 4-flouro phenyl and cyclopropyl
substituted quinoline, L-proline and Trans hydroxy-L-proline carboxamide via methylene
group and study the structure activity due to substituent variations on the phenyl carboxamide
ring (Fig 1).
Many potent molecules often fail to enter the market because of their unfavourable
pharmacokinetic profiles [35]. Now a days, pharmacokinetic properties of drugs are taken in
to consideration during earlier stages of drug discovery programme and are becoming more
popular [36]. In-silico drug likeness approach reduces time and cost as compared to
experimental methods [37]. Recently many novel α-amylase inhibitors have been identified
and insilco predicted [38]. So, in the current study, physico-chemical properties of the
designed analogs were in-silico predicted for their drug likeness analysis. Designed
1
13
compounds were synthesized, characterized by IR, H NMR, C NMR and Mass
spectroscopy and were screened for in-vitro α-amylase inhibitory activity. Docking study was
also performed in order to predict the putative binding mode of the most (compound 6c) and
least active compound (compound 6i).
2
2
.0 Material and Method
.1 Chemistry
Melting points were recorded on a Buchi melting point B-540 instrument and are
uncorrected. Thin layer chromatography (pre-coated silica gel, Merck) was used to analyse
purity of the compounds. Potassium permanganate solution was used as staining reagent.
Column chromatography was performed using silica gel (60-120 mesh) as packing material,
n-heptane and ethyl acetate as eluent. IR spectrum in KBr pellet method was recorded using
JASCO FT-IR410. The mass spectra were recorded in PE-SCIEX API-3000 LC/MS/MS with

1
13
Turbo ion spray. The H and C NMR spectra were recorded in CDCl on a Bruker Advance
3
4
00MHz Spectrometer with multinuclear BBO probe and TMS as an internal standard.
Synthesis
of
(2R,4S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-4-
hydroxypyrrolidine-2-carboxylic acid (5): N,N-Diisopropylethylamine (0.779 mol) was
added slowly to the stirred solution of methyl (2R, 4S)-4-hydroxypyrrolidine-2-carboxylate
hydrochloride (0.055 mol) and 3-(bromomethyl)-2-cyclopropyl-4-(4-fluorophenyl) quinoline
(0.055 mol) in THF (5 mL) at room temperature. Starting material consumption was
monitored by thin layer chromatography. After completion of reaction, aqueous solution of
potassium hydroxide (0.060 mol in 1 mL water) was added and heated to 50 °C. Progress of
the hydrolysis reaction was monitored by thin layer chromatography. Cooled the reaction
mass to 25 °C, quenched with ice cold water and the resultant product was filtered. White
1
solid; Yield: 97%; H NMR (400 MHz, DMSO-d ): δ 7.857 (d, 1H, J = 8.4), 7.635 (t, 1H, J =
6
7
.4), 7.405-7.353 (m, 5H), 7.127 (d, 1H, J = 8), 4.782 (brs, 1H), 4,097-4.038 (m, 2H), 3.637
(d, 1H, J = 12.8), 3.176-3.122 (m, 2H), 2.948 (t, 1H, J = 7.4), 2.002-1.941 (m, 2H), 1.809-
1
3
1
.789 (t, J = 4.0 1H), 1.276-1.266 (m, 1H), 1.056-0.968 (m, 3H); C NMR (400 MHz,
DMSO-d6): δ 174.355, 163.354, 163.030, 160.598, 146.194, 145.650, 132.621, 132.070,
1
28.861, 128.349, 127.801, 126.038, 125.853, 125.425, 115.314, 68.295, 64.732, 60.810,
1.993, 14.135, 10.709, 9.562; ESI-MS: m/z calculated for C H FN O [M + H]+ 407.17,
5
2
4
23
2
3
found 407.10.
General Procedure for the synthesis of (2R, 4S)-1-((2-cyclopropyl-4-(4-fluorophenyl)
quinolin-3-yl) methyl)-4-hydroxypyrrolidine-2-carboxamide 6(a-j): To the stirred
solution
of
(2R,4S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-4-
hydroxypyrrolidine-2-carboxylic acid (0.0024 mol in 5 mL THF), Triethylamine (0.0027
mol) followed by pivaloyl chloride (0.0024 mol) were added slowly at -45 °C and stirred for

3
0 min. Finally, substituted phenyl amine (0.0024 mol) was added. The progress of the
reaction was monitored by thin layer chromatography. Reaction mass was quenched in an ice
cold water and filtered the product.
(2R,4S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-4-hydroxy-N-
phenylpyrrolidine-2-carboxamide (6a)
-1
White solid; Yield: 97%; IR (KBr) (cm ): 3388 (O-H stretching), 3336 (N-H stretching)
2
1
997 (aromatic C-H stretching), 2864 (Aliphatic C-H stretching), 1668 (C=O stretching),
581, 1514, 1490 (Aromatic C=C/C=N stretching), 1456 (Aliphatic CH bending), 1219 (C-F
stretching), 1155, 1126, 1062 1028 (C-O stretching), 929, 839, 769, 744 (Aromatic CH
1
bending); H NMR (400 MHz, CDCl ): δ 8.679 (s, NH, 1H), 7.811 (d, 1H, J = 8.0), 7.529 (t,
3
1
H, J = 7.6), 7.345-7.325 (m, 1H), 7.296-7.255 (m, 2H), 7.237-7.062 (m, 5H), 7.052 (d, 2H, J
7.6), 6.987 (t, 1H, J = 7.2), 4.365 (t, 1H, J = 5.0), 4.216 (d, benzyl CH 1H, J = 13.6),
=
2
,
4
.055 (d, benzyl CH 1H, J = 13.6), 3.382-3.334 (m, 2H), 2.730-2.652 (m, 2H), 2.171-2.077
2,
1
3
(
m, 2H), 1.553-1.509 (m,1H), 1.288-1.234 (m, 1H), 1.132-1.123 (m, 2H,); C NMR (400
MHz, CDCl ): δ 172.119, 163.725, 162.114, 161.261, 146.988, 146.209, 136.872, 136.743,
3
1
1
1
32.671, 132.365, 131.894, 129.118, 128.806, 127.239, 126.477, 126.259, 125.616, 124.436,
20.365, 116.020, 115.782, 115.533, 71.061, 67.484, 62.933, 55.516, 40.719, 14.472, 11.550,
0.083; ESI-MS: m/z calculated for C H FN O [M + H]+ 482.22, found 482.20.
3
0
28
3
2
(2R,4S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-4-hydroxy-N-(o-
tolyl) pyrrolidine-2-carboxamide (6b)
-1
White solid; Yield: 98%; IR (KBr) (cm ): 3419 (O-H stretching), 2920 (aromatic C-H
stretching), 3334 (N-H stretching), 2920 (aromatic C-H stretching), 1670 (C=O stretching),
1
514, 1490 (Aromatic C=C/C=N stretching), 1406 (Aliphatic CH bending), 1224 (C-F

stretching), 1157, 1126, 1060, 1020 (C-O stretching), 962, 931, 819, 792 (Aromatic CH
1
bending); H NMR (400 MHz, CDCl ): δ 8.587 (s, NH, 1H), 7.836 (d, 1H, J = 8.4), 7.571 (t,
3
1
H, J = 7.6), 7.439-7.400 (m, 1H), 7.305-7.239 (m, 2H), 7.200-7.151 (m, 3H), 7.074 (d, 1H, J
6.8), 6.990 (t, 1H, J = 7.0), 6.921 (t, 1H, J = 7.8), 6.726 (d, 1H, J = 8), 4.390 (t, 1H, J =
.2), 4.215 (d, benzyl CH 1H, J = 13.6), 4.060 (d, benzyl CH 1H, J = 13.2), 3.421-3.355
=
5
2
,
2,
(m, 2H), 2.736-2.698 (dd, 1H), 2.632-2.580 (m, 1H), 2.194-2.096 (m, 2H), 1.985 (s, 3H),
1
.294-1.249 (m, 2H). 1.044-0.971(m, 2H); ESI-MS: m/z calculated for C H FN O [M + H]
31 30 3 2
+
496.23, found 496.20.
(2R,4S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-N-(2,4-
dimethylphenyl)-4-hydroxypyrrolidine-2-carboxamide (6c)
-1
White solid; Yield: 89%; IR (KBr) (cm ): 3325 (O-H stretching), 3286 (N-H stretching)
2
1
974 (aromatic C-H stretching), 2863 (Aliphatic C-H stretching), 1674 (C=O stretching),
597, 1514, 1490 (Aromatic C=C/C=N stretching), 1442 (Aliphatic CH bending), 1220 (C-F
stretching), 1159, 1097, 1062 1022 (C-O stretching), 837, 765, 750 (Aromatic CH bending);
1
H NMR (400 MHz, CDCl ): δ 8.507 (s, NH, 1H), 7.856 (d, 1H, J = 8.4), 7.579 (t, 1H, J =
3
7
.8), 7.415-7.382 (m, 1H), 7.304-7.239 (m, 2H), 7.192-7.150 (m, 3H), 6.883 (s, 1H), 6.722
(
d, 1H, J = 8.4), 6.502 (d, 1H, J = 7.6), 4.391 (t, 1H, J = 5.0), 4.223 (d, benzyl CH 1H, J =
2
,
1
2
1
5
3.2), 4.048 (d, benzyl CH 1H, J = 13.2), 3.397-3.358 (m, 2H), 2.724-2.685 (dd, 1H), 2.629-
2,
.579 (m, 1H), 2.212 (s, 3H), 2.156-2.050 (m, 2H), 1.989 (brs, 1H),1.950 (s, 3H), 1.329-
.159 (m, 2H), 1.023-0.973 (m, 2H); ESI-MS: m/z calculated for C H FN O [M + H]+
3
2
32
3
2
10.25, found 510.20.
(2R,4S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-N-(3,4-
dimethylphenyl)-4-hydroxypyrrolidine-2-carboxamide (6d)

-1
White solid; Yield: 95%; IR (KBr) (cm ): 3259 (O-H stretching), 3066 (N-H stretching)
2
1
989 (aromatic C-H stretching), 2790 (Aliphatic C-H stretching), 1672 (C=O stretching),
602, 1539, 1489 (Aromatic C=C/C=N stretching), 1442 (Aliphatic CH bending), 1218 (C-F
1
stretching), 1157, 1060 (C-O stretching), 929, 891, 839, 759 (Aromatic CH bending); H
NMR (400 MHz, CDCl ): δ 8.573 (s, NH, 1H), 7.836 (d, 1H, J = 8.4), 7.542 (t, 1H, J = 7.0),
3
7
6
1
.327-7.312 (m, 1H), 7.275-7.235 (m, 2H), 7.193-7.147 (m, 3H), 6.892 (d, 1H, J = 8.0),
.800 (d, 1H, J = 7.6), 6.662 (s, 1H), 4.333 (t, 1H, J = 5.0), 4.197 (d, benzyl CH 1H, J =
2
,
3.2), 4.035 (d, benzyl CH 1H, J = 13.2), 3.373-3.295 (m, 2H), 2.708-2.645 (m, 2H), 2.168
2
,
(s, 3H), 2.124-2.097 (m, 2H), 2.075 (s, 3H), 1.503-1.491 (m, 1H), 1.296-1.268 (m, 1H),
1
5
.123-1.097 (m, 2H); ESI-MS: m/z calculated for C H FN O [M + H]+ 510.25, found
32 32 3 2
10.20.
(2R,4S)-N-(3-chlorophenyl)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-
4
-hydroxypyrrolidine-2-carboxamide (6e)
-1
White solid; Yield: 95%; IR (KBr) (cm ): 3404 (O-H stretching), 3263 (N-H stretching)
2
1
918 (aromatic C-H stretching), 2864 (Aliphatic C-H stretching), 1668 (C=O stretching),
604, 1579, 1512, 1490 (Aromatic C=C/C=N stretching), 1450 (Aliphatic CH bending), 1219
(C-F stretching), 1219, 1126, 1089, 1060 (C-O stretching), 962, 893, 817 (Aromatic CH
1
bending), 769 (C-Cl stretching); H NMR (400 MHz, CDCl ): δ 8.678 (s,NH, 1H), 7.813 (d,
3
1
H, J = 8.4), 7.531 (t, 1H, J = 7.6), 7.370-7.330 (m, 1H), 7.297-7.221 (m, 2H), 7.206-7.126
m, 4H), 7.066 (d, 2H, J = 7.6), 6.989 (t, 1H, J = 7.4), 4.382 (t, 1H, J = 4.8), 4.220 (d, benzyl
CH 1H, J = 13.2), 4.059 (d, benzyl CH 1H, J = 13.2), 3.386-3.339 (m, 2H), 2.735-2.654
(
2
,
2,
(m, 2H), 2.154-2.082 (m, 2H), 1.556-1.512 (m, 1H), 1.293-1.254 (m, 1H), 1.135-1.106 (m,
2
H); ESI-MS: m/z calculated for C H ClFN O [M + H]+516.18, found 516.1,[M +
30 27 3 2
2
H]+518.17, found 518.20.

(2R,4S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-N-(3-fluorophenyl)-
4
-hydroxypyrrolidine-2-carboxamide (6f)
-1
White solid; Yield: 90%; IR (KBr) (cm ): 3389 (O-H stretching), 3341 (N-H stretching)
2
1
985 (aromatic C-H stretching), 2871 (Aliphatic C-H stretching), 1660 (C=O stretching),
580, 1574, 1501 (Aromatic C=C/C=N stretching), 1458 (Aliphatic CH bending), 1218 (C-F
stretching), 1155, 1126, 1062 1028 (C-O stretching), 930, 841, 770 (Aromatic CH
1
bending); H NMR (400 MHz, CDCl ): δ 8.688 (s, NH, 1H), 7.792 ( d, 1H J = 8.4), 7.529 (t,
3
1
H, J = 7.3), 7.511-7.359 (m, 1H), 7.353-7.002 (m, 7H), 6.736 (d, 1H, J = 6.4), 6.675 (t, 1H,
J = 7.9), 4.388 (t, 1H, J = 5.0),4.239 (d, benzyl CH 1H, J = 13.6), 4.039(d, benzyl CH 1H, J
2
,
2,
=
13.6), 3.414-3.329 (m, 2H), 2.755-2.717 (m, 1H), 2.765-2.637 (m, 1H), 2.153-2.083 (m,
H), 1.617-1.577 (m, 1H), 1.124-1.216 (m, 1H), 1.154-1.125 (m, 2H); ESI-MS: m/z
calculated for C H F N O [M + H]+ 500.21, found 500.20.
2
3
0
27
2
3
2
(2R,4S)-N-(3-bromophenyl)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-
4
-hydroxypyrrolidine-2-carboxamide (6g)
-1
White solid; Yield: 99%; IR (KBr) (cm ): 3410 (O-H stretching), 3341 (N-H stretching)
2
1
981 (aromatic C-H stretching), 2866 (Aliphatic C-H stretching), 1669 (C=O stretching),
583, 1520, 1450 (Aromatic C=C/C=N stretching), 1456 (Aliphatic CH bending), 1217 (C-F
stretching), 1160, 1130, 1071, 1031 (C-O stretching), 930, 840, 771 (Aromatic CH bending),
1
6
44 (C-Br stretching); H NMR (400 MHz, CDCl ): 8.661 (s, NH, 1H), 7.806 (d, 1H, J
3
=8.28), 7.535 (t, 1H, J = 7.3), 7.359-6.995 (m, 10H), 4.383 (t, 1H, J = 5.0), 4.244 (d, benzyl
CH 1H, J = 13.8), 4.023 (d, benzyl CH 1H, J = 13.8), 3.430-3.392 (m, 1H), 3.354-3.315
2
,
2,
(m, 1H), 2.745-2.654 (m, 2H), 2.099-2.049 (m, 2H), 1.615-1.590 (m, 1H), 1.253-1.208 (m,

1
H), 1.134-0.984 (m 2H). ESI-MS: m/z calculated for C H BrFN O [M + H]+ 562.13,
30 27 3 2
found 562.0, [M + 2H]+563.13, found 563.00.
(2R,4S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-4-hydroxy-N-(4-
methoxyphenyl) pyrrolidine-2-carboxamide (6h)
-1
Pale brown solid; Yield: 89%; IR (KBr) (cm ): 3388 (O-H stretching), 3336 (N-H stretching)
2
997 (aromatic C-H stretching), 2864 (Aliphatic C-H stretching), 1668 (C=O stretching),
581, 1514, 1490 (Aromatic C=C/C=N stretching), 1456 (Aliphatic CH bending), 1219 (C-F
1
stretching), 1155, 1126, 1062 1028 (C-O stretching), 929, 839, 769 (Aromatic CH bending);
1
H NMR (400 MHz, CDCl ): δ 8.550 (s, NH, 1H), 7.834 (d, 1H, J = 8.32), 7.546 (t, 1H, J =
3
7
.5), 7.357-7.152 (m, 6H), 6.910 (d, 2H, J = 8.56), 6.669 (d, 2H, J = 8.48), 4.384 (t, 1H, J =
.0), 4.218 (d, benzyl CH 1H, J = 13.4), 4.037 (d, benzyl CH 1H, J = 13.4), 3.400-3.324
5
2
,
2,
(m, 2H), 3.728 (s, 3H),2.719-2.660 (m, 2H), 2.218-2.105 (m, 2H), 1.4827-1.2931 (m,2H),
0
5
.880-0.836 (m, 2H). ESI-MS: m/z calculated for C H FN O [M + H]+ 512.23, found
31 30 3 3
12.20.
(2R,4S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-4-hydroxy-N-(4-
isopropylphenyl) pyrrolidine-2-carboxamide (6i)
-1
White solid; Yield: 88%; IR (KBr) (cm ): 3341 (O-H stretching), 3340 (N-H stretching)
2
1
989 (aromatic C-H stretching), 2872 (Aliphatic C-H stretching), 1667 (C=O stretching),
578, 1510, 1488 (Aromatic C=C/C=N stretching), 1458 (Aliphatic CH bending), 1220 (C-F
stretching), 1158, 1127, 1060, 1030 (C-O stretching), 930, 840, 768 (Aromatic CH bending);
1
H NMR (400 MHz, CDCl ): 8.624 (s, NH, 1H), 7.825 (d, 1H, J = 8.4), 7.536 (t, 1H, J = 7.5),
3
7
.356-7.322 (m, 1H), 7.291-7.217 (m, 2H), 7.202-7.151 (m, 3H), 7.015-6.964 (m, 4H), 4.364
(
t, 1H, J = 5.0), 4.204 (d, benzyl CH 1H, J = 13.2), 4.055 (d, benzyl CH 1H, J = 13.2),
2
,
2,

3
1
.366-3.327 (m, 2H), 2.811-2.777 (m, 1H),2.724-2.673 (m, 2H), 2.173-2.102 (m, 2H), 1.566-
.521 (m, 1H), 1.293-1.276 (m.1H), 1.179 (s 3H), 1.161(s 3H),1.037-1.108(m, 2H); ESI-MS:
m/z calculated for C H FN O [M + H]+ 524.65, found 524.20.
3
3
34
3
2
(2R,4S)-N-(4-chlorophenyl)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-
4
-hydroxypyrrolidine-2-carboxamide (6j)
-1
White solid; Yield: 99%; IR (KBr) (cm ): 3400 (O-H stretching), 3345 (N-H stretching)
2
1
990 (aromatic C-H stretching), 2864 (Aliphatic C-H stretching), 1668 (C=O stretching),
580, 1520, 1485 (Aromatic C=C/C=N stretching), 1445 (Aliphatic CH bending), 1218 (C-F
stretching), 1166, 1118, 1072, 1020 (C-O stretching), 927, 825, 760 (Aromatic CH bending),
1
7
8
7
40 (C-Cl stretching); H NMR (400 MHz, CDCl ): δ 8.632 (s, NH, 1H), 7.790 (d, 1H, J =
3
.8), 7.539 (t, 1H, J = 7.6), 7.366-7.336 (m, 1H), 7.301-7.203 (m, 2H), 7.197-7.115 (m, 3H),
.077 (d, 2H, J = 7.6), 6.974 (d, 2H, J = 8.8), 4.369 (t, 1H, J = 5.0), 4.240 (d, benzyl CH 1H,
2
,
J =13.6), 4.019 (d, benzyl CH 1H, J = 13.6), 3.429-3.390 (dd, 1H), 3.355-3.315 (t, 1H, J =
2
,
8
), 2.750-2.714 (dd, 1H), 2.658-2.635 (t, 1H, J = 4.6), 2.152-2.063 (m, 2H), 1.563-1.520 (m,
1
H), 1.239-1.207 (m, 1H), 1.130-1.101 (m, 2H); ESI-MS: m/z calculated for
C H ClFN O [M + H]+ 516.18, found 516.1, [M + 2H]+518.17, found 518.20.
30
27
3
2
Procedure for the synthesis of ((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl) methyl)-
L-proline (10): N,N-Diisopropylethylamine (0.1268 mole) was added slowly to a solution of
methyl L- prolinate hydrochloride (0.06037 mole) and 3-(bromomethyl)-2-cyclopropyl-4-(4-
fluorophenyl) quinoline (0.06037 mol) in 5 mL THF at room temperature. Starting material
consumption was monitored by thin layer chromatography. After completion of reaction,
aqueous solution of potassium hydroxide (0.0664 mol in 1 ml water) was added and heated to
5
0 °C. Progress of the hydrolysis reaction was monitored by thin layer chromatography.

Cooled the reaction mass to 25 °C, quenched with ice cold water and filtered the resultant
1
product. White solid; Yield: 99%; H NMR (400 MHz, CDCl ): δ 7.941 (d, 1H, J = 8.32),
3
7
3
1
3
.604 (t, 1H, J = 7.4), 7.373-7.178 (m, 6H), 4.124 (d, 1H, J = 13.0), 3.862d, 1H, J = 13.0),
.1660-3.0514 (m, 2H), 2.7026-2.5266 (m, 2H), 2.137-1.947 (m, 2H), 1.712 (brs, 2H), 1.251-
.237 (m, 2H), 1.187-1.103 (m, 2H). ESI-MS: m/z calculated for C H FN O [M + H]+
24
23
2
2
91.17, found 391.20.
General Procedure for the synthesis of(S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-
-yl)methyl)pyrrolidine-2-carboxamide 10(a-e): To a solution of ((2-cyclopropyl-4-(4-
3
fluorophenyl) quinolin-3-yl) methyl)-L-proline (0.0025mol) in THF, triethylamine (0.0028
mol) was added. Cooled the reaction mass to -45 °C and then pivaloyl chloride (0.0025 mol)
was added slowly at -45 °C, stirred for 30 min. followed by substituted phenyl amine (0.0025
mol) was added. The progress of the reaction was monitored by thin layer chromatography.
Reaction mass was quenched with ice cold water and filtered the product.
(S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-N-phenylpyrrolidine-2-
carboxamide (10a)
-1
White solid; Yield: 99%; IR (KBr) (cm ): 3294 (N-H stretching) 2856 (aromatic C-H
stretching), 2864 (Aliphatic C-H stretching), 1668 (C=O stretching), 1597, 1510, 1490,1440
(Aromatic C=C/C=N stretching), 1411 (Aliphatic CH bending), 1220 (C-F stretching), 927,
1
8
7
7
3
1
93, 769, 755 (Aromatic CH bending); H NMR (400 MHz, CDCl ): δ 8.844 (s, NH, 1H),
3
.834 (d, 1H, J = 8.4), 7.547 (t, 1H, J = 7.2), 7.382-7.368 (m, 1H), 7.313-7.253 (m, 2H),
.218-7.157 (7H m), 7.029-7.012 (t, 1H, J = 7.4), δ 4.146 (d, benzyl CH 1H, J = 13.2),
2
,
.882 (d, benzyl CH 1H, J = 13.2), 3.188-3.178 (m, 1H), 3.100-3.066 (dd, 1H), 2.726 (m,
2
,
H), 2.611-2.549 (q, 1H), 2.185-2.082 (m, 1H), 1.969 (brs, 1H), 1.744-1.733 (m, 2H), 1.570-

1
3
1
1
1
1
.549 (m, 1H), 1.279-1.258 (m, 1H), 1.146-1.120 (m, 2H); C NMR (400 MHz, CDCl ): δ
3
73.003, 163.881, 162.393, 161.418, 147.124, 146.142, 137.179, 132.802, 132.492, 131.878,
29.053, 128.841,127.262, 126.494, 126.326, 125.58, 124.311, 120.397, 116.0, 115.745,
15.482, 67.749, 55.886, 54.072, 31.235, 24.4, 14.41, 11.585, 10.061; ESI-MS: m/z
calculated for C H FN O[M + H]+ 466.22, found 466.20.
3
0
28
3
(S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-N-(2,4-dimethylphenyl)
pyrrolidine-2-carboxamide (10b)
-1
White solid; Yield: 88%; IR (KBr) (cm ): 3275 (N-H stretching) 2997 (aromatic C-H
stretching), 2864 (Aliphatic C-H stretching), 1672 (C=O stretching), 1579, 1502, 1425
(Aromatic C=C/C=N stretching), 1330 (Aliphatic CH bending), 1220 (C-F stretching), 987,
1
9
7
6
3
1
25, 842, 769 (Aromatic CH bending); H NMR (400 MHz, CDCl ): δ 8.723 (s, NH, 1H),
3
.834 (d, 1H, J= 8.4), 7.541 (t, 1H, J = 8.2), 7.346-7.155 (m, 5H), 6.888 (d, 1H, J = 7.6),
.759 (s, 1H), 4.113 (d, benzyl CH 1H, J = 11.8), 3.854 (d, benzyl CH 1H, J = 12.4), 3.478-
2
,
2,
.471 (m, 2H), 3.153-3.028 (m, 2H), 2.694-2.540 (m, 2H),2.136 (s, 3H), 2.106 (s, 3H), 2.144-
.940 (m, 2H), 1.702-1712 (m, 2H), 1.127 (m, 1H), 1.106 (m, 2H); ESI-MS: m/z calculated
for C H FN O[M + H]+494.25, found 494.20.
3
2
32
3
(S)-N-(3-chlorophenyl)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)
pyrrolidine-2-carboxamide (10c)
-1
White solid; Yield: 95%; IR (KBr) (cm ): 3336 (N-H stretching) 2997 (aromatic C-H
stretching), 2864 (Aliphatic C-H stretching), 1668 (C=O stretching), 1581, 1514, 1490
(Aromatic C=C/C=N stretching), 1456 (Aliphatic CH bending), 1219 (C-F stretching), 929,
1
8
39, 769, (Aromatic CH bending); 744 (C-Cl stretching); H NMR (400 MHz, CDCl ):
3
δ8.814 (s, NH, 1H), 7.800 (d, 1H, J = 8.4), 7.525 (t, 1H, J = 7.6), 7.383-7.349 (m, 1H), 7.308-

7
.280 (m, 2H), 7.234-7.116 (m,4H), 7.079-7.039 (m, 1H), 6.911-6.942 (m, 2H), 4.180 (d,
benzyl CH 1H, J = 13.2), 3.803 (d, benzyl CH 1H, J = 13.2), 3.204-3.189 (m, 1H), 3.049-
2
,
2,
3
.014 (dd, 1H), 2.700-2.661 (m, 1H), 2.590-2.550 (q, 1H), 2.144-2.091 (m, 1H), 1.927-1.886
(m, 1H), 1.728-1.711 (m, 2H), 1.626-1.596 (m, 1H), 1.210-1.186 (m, 1H), 1.141-1.131 (m,
2
H); ESI-MS: m/z calculated for C H ClFN O[M + H]+500.18, found 500.20, [M +2
30 27 3
H]+501.19, found 501.20.
(S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-N-(3-fluorophenyl)
pyrrolidine-2-carboxamide (10d)
-1
White solid; Yield: 98%; IR (KBr) (cm ): 3340 (N-H stretching), 2989 (aromatic C-H
stretching), 2872 (Aliphatic C-H stretching), 1666 (C=O stretching), 1580, 1514, 1499
(Aromatic C=C/C=N stretching), 1459 (Aliphatic CH bending), 1210 (C-F stretching), 931,
1
8
42, 755 (Aromatic CH bending); H NMR (400 MHz, CDCl ): δ 8.845(s, NH, 1H), 7.795 (d,
3
1
H, J = 8.1), 7.523 (t, 1H, J = 7.2), 7.359-7.303 (m, 1H), 7.259-7.051 (m, 7H), 6.794 (d, 1H,
J = 7.6), 6.678-6.598 (m, 1H), 4.153 (d, benzyl CH 1H, J = 13.12), 3.830 (d, benzyl CH
2,
2
,
1
H, J = 13.12), 3.192-3.031 (m, 2H), 2.684-2.569 (m, 2H), 2.169-1.921 (m, 2H), 1.724-1.595
m, 3H), 1.215-1.201 (m, 1H), 1.121-1.055 (m, 2H); ESI-MS: m/z calculated for
C H F N O [M + H]+ 484.21, found 484.20.
(
30
27
2
3
(S)-1-((2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methyl)-N-(4-methoxyphenyl)
pyrrolidine-2-carboxamide (10e)
-1
Pale brown solid; Yield: 98%; IR (KBr) (cm ): 3445 ( N-H stretching) 2985 (aromatic C-H
stretching), 2852 (Aliphatic C-H stretching), 1665 (C=O stretching), 1572, 1532, 1481
(Aromatic C=C/C=N stretching), 1448 (Aliphatic CH bending), 1221 (C-F stretching), 915,
1
8
28, 755 (Aromatic CH bending); H NMR (400 MHz, CDCl ): δ 8.692 (s, NH, 1H), 7.833
3

(d, 1H, J = 8.0), 7.537 (t, 1H, J = 8.2), 7.385-7.346 (m, 1H), 7.294-7.234 (m, 2H), 7.217-
7
1
.115 (m, 3H), 6.944 (d, 2H, J = 8.8), 6.681 (d, 2H, J = 9.2), 4.129 (d, benzyl CH 1H, J =
2,
3.2), 3.835 (d, benzyl CH 1H, J = 13.2), 3.721 (s, 3H), 3.172-3.161 (m, 1H), 3.056-3.021
2
,
(
dd, 1H), 2.696-2.664 (m, 1H), 2.562-2.544 (q, 1H), 2.108-2.086 (m, 1H), 1.938-1.931 (m,
H), 1.748-1.689 (m, 2H), 1.514-1.470 (m, 1H), 1.273-1.231(m, 1H), 1.105-1.077 (m, 2H);
ESI-MS: m/z calculated for C H FN O [M + H]+496.23, found 496.20.
1
3
1
30
3
2
2
.2 In silico prediction of physico-chemical parameters
Physicochemical parameters of the designed compounds were in-silico predicted using
Qik-prop module of Schrödinger. The different parameters [39] predicted were molecular
weight (M.Wt.), total solvent accessible surface area (SASA), number of hydrogen bond
donor (HBD), number of hydrogen bond acceptor (HBA), octanol/water partition coefficient
(log P), aqueous solubility (Log S), predicted apparent Caco-2 cell permeability in nm/sec (P
Caco) and number of rotatable bonds (Rot).
2
.3. α-amylase inhibitory activity
Calorimetric method was used to measure in vitro α-amylase inhibitoty activity of all the
synthesized compounds and acarbose was used as the reference compound [40]. α-amylase
0.5 mg/mL), in 2 mM sodium phosphate buffer solution to maintain the pH 6.9 was
(
incubated with and without samples and standard for 10 min at 25 °C. After this
preincubation, starch solution (1%) was slowly added and futher incubated for a period of 30
min at 25 °C. DNSA (3,5-Dinitro salicylic acid) as colour reagent as well as to stop the
enzymatic reaction was added and further incubated for about 5 min in a waterbath at 70 °C.
Experiment temperature was reduced to 25 °C and diluted with distilled water. The
aborbance was measured at 540 nm with the use of spectrophotometer and compared with

that of control experiment. Percentage of inhibition was calculated using the following
formula,
%
of inhibition = 100* (At-Ac/ At)
At= absorbance of test, Ac= absorbance of control
2
.4. Molecular docking studies
Docking studies of the significantly active and weakly active compounds were performed
using Glide module [41] of Schrodinger software [42] installed on Intel Xenon W 3565
processor and Ubuntu enterprise version 14.04 as an operating system. The selected target
protein structure was retrieved from RCSB protein data bank [43]. Targeted ligands were
drawn using Chemdraw 18.0 Perkinelmer software.
Ligand preparation
The ligands used as an input for docking study was sketched by ChemDraw software and
cleaned up the structure for bond alignment. Then, ligands were incorporated into the
workstation and the energy was minimized using OPLS3e (Optimized Potentials for Liquid
Simulations) [44] force field in Ligprep [45] (Version 2019-1, Schrodinger). This
minimization helps to assign bond orders, the addition of hydrogens to the ligands and
conversion of 2D to 3D structure for the docking studies. The generated output file (Best
conformations of the ligands) was further used for docking studies.

Protein preparation
Protein preparation wizard [46] (Version 2019-1, Schrodinger) was the main tool in
Schrodinger to prepare the protein and minimizing the protein. Hydrogen atom was added to
the protein and charges were assigned. Generated Het states using Epik at pH 7.0 ±2.0. Pre-
process the protein and refine, modify the protein by analyzing the workspace water
molecules and other. The critical water molecules remained the same and rest of the
molecules apart heteroatoms from the water was deleted. Finally, the protein was minimized
using OPLS3 force field. A grid was created by considering co-crystal ligand, which was
included in the active site of the protein of the selected target (PDB-4GQR). After the final
step of docking with the co-crystal ligand in XP mode, root mean square deviation (RMSD)
was checked to validate the protein, and the RMSD value lies within the range of 0.46 Å.
Receptor grid generation
A receptor grid was generated around the protein (PDB:4GQR) [47] by choosing the
inhibitory ligand (X-ray pose of the ligand in the protein). The centroid of the ligand was
selected to create a grid box around it and Vander Waal radius of receptor atoms was scaled
to 1.00 Å with a partial atomic charge of 0.25.
Docking and analysis
Molecular docking was performed using the above prepared ligand and protein as input.
The results of the docking study was analyzed with the help of XP Visualiser (Version 2019-
1
, Schrodinger). SMILES format of the compounds was generated by using OSIRIS
Datawarrior [48]. Docking studies of the designed and synthesized molecules were performed
by using Glide module in Schrodinger. All docking calculations were performed using Extra

Precision (XP) mode. A scaling factor of 0.8 and a partial atomic charge of less than 0.15 was
applied to the atoms of the protein. Glide docking score was used to determine the best-
docked confirmation from the output. The interactions of these docked conformations were
investigated further using XP visualizer.
3
3
.0 Results and discussion
.1 Chemistry
Synthesis of L-proline, Trans-4-hydroxy-L-proline derivatives possessing α-amylase
inhibitory activity are shown in scheme 1 and 2. List of synthesized compounds are depicted
in table 1. In the first step, commercially available raw materials such as Trans hydroxy-L-
proline and L-proline were reacted separately with thionyl chloride in methanol at room
temperature for 24 h. Thionyl chloride was distilled completely and afforded their
corresponding methyl ester hydrochloride compounds (2 and 8) with 97% yield [49]. (2-
cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)methanol (compound 3) was brominated using
phophoryl tribromide and key raw material 3-(bromomethyl)-2-cyclopropyl-4-(4-
fluorophenyl)quinoline (compound 4) [50] was obtained. Trans-4-hydroxy-L-proline and L-
proline methyl ester hydrochloride (compound 2 and 8) were N-alkylated with compound 4 in
THF using DIPEA as base yielded corresponding ester compounds and were hydrolysed with
KOH/H O resulted key acid intermediates (compounds 5 and 9). These acid intermediates
2
were activated with pivaloyl chloride and Triethylamine in THF at -40 °C to form reactive
mixed anhydride and then coupled with different substituted phenyl amines afforded the titled
compounds (6a-6j) and (10a-10e).
1
13
All the synthesized compounds were characterized by IR, H, C NMR and Mass
I
spectroscopy. The appearance of singlet at 8.5-8.6 ppm in H NMR confirms amide NH unit

1
3
and one peak appeared around 170 ppm in C NMR confirms the presence of carbonyl
carbon in all the titled derivatives. The IR spectrum yet again confirms the presence of
-1
-1
-1
carbonyl (1668 cm ), NH (3388 cm ) and C-F (1219 cm ) functional groups in all the
1
3
derivatives. Three peaks at 14.47, 11.55 and 10.08 in C NMR confirm isopropyl unit.
1
Appearance of two doublet at 4.146 ppm and another doublet at 3.882 ppm in H NMR
confirm benzylic CH group in all the derivatives. Methyl group of ortho toluidine derivative
2
1
(
Compound 6b) was confirmed in H NMR by the presence of a singlet appeared at 1.985
1
ppm. 2,4-dimethyl derivative (Compund 6c) was confirmed in H NMR by the appearance of
two singlet at 2.610 and 1.989 ppm and so as two singlet at 2.168 and 2.075 confirms 3,4-
dimethyl derivatives (Compund 6d and 11b). OCH of p-methoxy derivatives (Compounds 6h
3
and 11e) were confirmed by the appearance of a singlet at 3.7 ppm and likewise iso propyl
derivative (Compound 6i) methine proton was confirmed by the appearance of multiplet at
1
2
.811-2.77 ppm in H NMR analysis. Chloro and bromo derivatives (Compunds 6e, 6g, 6j
and 11c) were confirmed using mass spectrum by 2 amu variations in the molecular ion peaks
1
with 1:3 and 1:1 peaks intensity, respectively. Peaks between 7.821-6.969 ppm in H NMR
confirm quinoline and phenyl unit aromatic protons.
3
.2 In-silico prediction of physico-chemical parameters
Most of the drugs fail in clinical trial stage or were removed from the market due to poor
ADMET properties and unavoidable side effects. In-silico prediction of ADMET
Absorption, Distribution, Metabolism and Toxicity) properties has reduced the effort of the
researcher to determine it practically for every designed analogs to develop a lead compound
51]. Physico-chemical prediction studies of the titled compounds revealed that the tested
(
[
compounds exhibited the drug-likeness properties such as Mol. Wt, Solvent accessible
surface area (SASA), hydrogen bond donors (HBD), hydrogen bond acceptors (HBA) and

partition co-efficient (log P) within the acceptable range and followed Lipinski rule of five as
that followed by 95% of the market approved drugs. Except compound 5, rest all the titled
compounds possessed significant predicted apparent Caco-2 cell permeability. Parameters
like predicted brain/blood partition co-efficient and number of rotatable bonds are also
obeyed by all the tested analogs. Further, four of the titled compounds (compounds 6c, 6i,
1
0c and 10d) showed low values of predicted log S and their values lied outside the given
range (-6.5 to 0.5). So, these four compounds may possess poor aqueous solubility, but for
the continuation of SAR studies, we included these compounds for further studies. So overall,
based upon the predicted values of these physico-chemical parameters, majority of the titled
compounds possessed the drug-likeness behaviour.
3
.3. α-amylase inhibitory activity
All the synthesized titled compounds (6a-6j) and (10a-10e) were subjected to in vitro α-
amylase inhibitory activity at different concentrations (10, 50, 100, 250, 500 µg/mL) using
acarbose as internal standard. The results of % inhibition at various concentrations I.e. 10, 50,
100, 250, 500 µg/mL are depicted in the Bar diagram (Fig.2). Six compounds 6a, 6b, 6c, 6g,
10b and 10c showed significant α-amylase inhibitory activity. Nine compounds 6d, 6e, 6f, 6h,
5, 6j, 10a, 10d and 10e exhibited moderate to good activity against α-amylase enzyme.
Compounds containing trans-4-hydroxy-L-proline with ortho methyl, 2,4-dimethyl, 3,4-
dimethyl phenyl amides (compounds 6b, 6c, 6d) exhibited significant activity and it showed
electron withdrawing groups may enhance the α-amylase inhibitory activity of these trans-4-
hydroxy-L-proline analogs. While, trans-4-hydroxy-L-proline compounds with electron
withdrawing groups as well as electron donating groups at para position exhibited moderate
activity (compounds 6h, 6i, and 6j). It showed substitutions irrespective of electron
withdrawing and electron donating groups at para position diminishing the α-amylase

inhibitory activity of trans-4-hydroxy-L-proline series of compounds. Among compounds
with electron withdrawing groups (cl, Br and F) at meta position, bromo amide compound
(6g) showed significant activity, flouro amide compound (6f) showed good activity and
chloro amide compound (6e) exhibited moderate activity.
Among L-proline series compounds (10a, 10b, 10c, 10d and 10e), analogs 10b and 10c
showed significant activity and compound 10a exhibited good activity while compound 10d
showed moderate activity. So, overall compounds 6a, 6b, 6c, 6d, 6g, 10b and 10c exhibited
significant α-amylase inhibitory activity and these compounds can be used further as anti-
hyperglycaemic agents. Trans-4-hydroxy-L-proline isopropyl derivative and L-proline
carboxylic acid compound (6i and 9) have shown least activity among all other compounds.
3
.4. Docking studies
To predict the putative binding mode of the significantly active (compound-6c) and least
active compound (compound-6i) with the target protein, docking studuy was carried out. The
docking results of these compounds against human pancreatic α-amylase (HPA) [52]
(retrieved from the Protein Data Bank (PDB ID: 4GQR) revealed that the studied compounds
showed a better correlation between in-vitro activity and in-silico study result. The value of
RMSD obtained between X-ray pose and re-docked pose (Fig. 3) of co-crystallized ligand in
the target protein was found to be 0.46 Å, suggesting that the docking protocol could be
relied on for the docking studies.
By examining the 3D docked pose interactions (Figure 4) and 2D representation (Figure
5
) of Myricetin exhibited maximum interactions with the surrounded amino-acid residues and
water molecules. The co-crystallized ligand disclosed seven hydrogen bond interactions with
the amino-acid residues TRP-59, GLN-63 and ASP-197 (two bonds) including water

molecules HOH-1144, 1244, 1204 and one aromatic bond with the amino-acid residue TRP-
9 (Table-4) of target protein. The docking score and energies of the Myricetin was found to
be -11.0 and -46.50 kcal/mol, respectively.
5
3D docked pose (Figure 6) and 2D representation (Figure 7) of significantly active
compound-6c explained that the hydroxyl group of pyrrolidine nucleus displayed a hydrogen
bond interaction with the NH7680 of amino-acid residue HIE-305 with a distance of 2.01 Å
(Table-5) with docking score and energy of -5.1 and -40.50 kcal/mol, respectively. Apart
from this, compound-6c also showed one aromatic bond between CH44 of ligand and
OH1296 of amino-acid residue TRP-163 with 2.28 Å distance. In addition to this, amino acid
residue TRP-59 displayed two Pi-Pi stacking interactions, respectively with compound-6c.
This compound was well correlated with the in-vitro study result displayed significant
inhibitory activity against α-amylase in increasing order of the tested concentrations I.e.10,
5
0, 100, 250, 500 µg/mL with 20.02, 31.34, 50.23, 67.88, 86.34 % of α-amylase inhibition,
respectively. Same amino-acid residues that are involved in the hydrogen bond and Pi-Pi
stacking interaction with the significantly active compound-6c exhibited their contribution in
the bond formation with the weakly active compound-6i (Figure-8, 9), revealed the docking
score and energy of -5.00 and -44.07 kcal/mol. However, the same amino-acid residue TRP-
5
9 also takes part in the aromatic bond formation with the weakly active compound-6i. Due
to this unwanted additional aromatic interaction of the compound-6i and increased interaction
bond distances for the compound-6i, the slight variation in the decreased docking score was
recognized. This might be the reason for less docking score of the weakly active compound-
6
i. The OH of pyrrolidine of the compound-6i also displayed a hydrogen bond interaction
with NH7680 of amino-acid residue HIE-305 with 2.17 Å distance and one aromatic bond
between CH46 of ligand and CO483 of amino-acid residue TRP-59 with 2.76 Å distance.

Apart from this hydrogen and aromatic bond interactions, the same amino-acid residue TRP-
9 was also displayed two pi-pi interactions with the weakly active compound-6i. An in-vitro
5
study of compound-6i has also disclosed the less % inhibition of α-amylase at various tested
concentrations I.e.10, 50, 100, 250, 500 µg/ml with 07.24, 13.33, 22.47, 44.27 and 64.98%,
respectively in comparison with the standard acarbose. Results of docking studies (i.e.) the
amino acid residues involved in various bond formation with the studied ligands and the
distances of the interaction exhibited by the ligands are depicted in tables 2 and 3,
respectively.
Conclusion
Novel proline based compounds were designed, synthesized by depicted synthetic route,
characterized by appropriate spectral analysis, in silico predicted for their drug likeness
behaviours and screened in vitro for α-amylase inhibitory activity. All the spectral analysis
confirmed the formation of title analogs. In silico physico chemical prediction studies
confirmed that the majority of the title compounds possessed the drug-likeness behaviour.
Finally, in vitro screening results suggested that proline ring with electron donating groups at
th
3
, 4 position (compound 6c) exhibited significant α-amylase inhibitory activity and this
compound can be explored for in vivo activity in the mere future. Docking studies of the
significantly active and least active compounds suggested that compound 6c exhibited
prominent hydrophobic and hydrogen bonding interactions with the target protein when
compared to least active compound 6i which may be responsible for the high potency of
compound 6c over compound 6i in the in vitro studies. These studies will be helpful for
further lead optimisation and designing of new α-amylase inhibitors for the treatment of
diabetes.

Acknowledgements
The authors thank the management, correspondent and the principal, Staff members of
Department of Chemistry, Government Arts College for men, Nandanam, Chennai-600 035
for providing research facility.
Figures and captions
Fig. 1: Design of α-Amylase inhibitors.
Fig. 2: α-amylase inhibitory activity of the synthesized compounds.
Fig. 3: Superimposed view of the native pose of ligand Myricetin (X-Ray
crystallized pose) and docked pose of the same ligand in the active site of
O
the protein (4GQR) (Root mean square deviation 0.46A )
[
Color interpretation: White – X- Ray crystallized pose, Pink – Binding
pose after docking]
Fig. 4: Collaboration of the co-crystallized ligand exhibited various interactions
in the active site of the protein (4GQR)
(Color interpretation: Yellow - Hydrogen bond, Blue – Aromatic bond)
Fig. 5: 2D representation of the docked pose of the co-crystallized ligand
Color interpretation: Magenta- Hydrogen bond)
(

Fig. 6: Collaboration of the significantly active compound-6c exhibited various
interactions in the active site of the protein (4GQR)
[Colour interpretation: yellow- Hydrogen bond, Red – Aromatic bond,
Blue – Pi-Pi-stacking interaction]
Fig. 7: 2D representation of the docked pose of compound-6c
(Colour interpretation: Magenta- Hydrogen bond, Green - pi-pi cationic
bond).
Fig. 8: Collaboration of the weakly active compound-6i exhibited various
interactions in the active site of the protein (4GQR)
[
Colour interpretation: yellow- Hydrogen bond, Red – Aromatic bond,
Blue – Pi-Pi-stacking interaction]
Fig. 9: 2D representation of the docked pose of compound-6i
(Colour interpretation: Magenta- Hydrogen bond, Green - pi-pi cationic
bond)
Tables and Captions
Table. 1: List of synthesized compounds
Table. 2: Docking network between aminoacid residues, water molecules with the
significantly active and weakly active compounds
Table. 3: Atomic level interaction and the distance of various bonds of the significantly
active and weakly active compounds
Schemes and captions
Scheme. 1: Synthetic route followed for the synthesis of titled compounds (6a-6j)

Scheme. 2: Synthetic route followed for the synthesis of titled compounds (10a-10e)
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