Discovery of novel pyrido-pyrrolidine hybrid compounds as alpha-glucosidase inhibitors and alternative agent for control of type 1 diabetes

Article abstract of DOI:10.1016/j.ejmech.2020.112034

A new library of pyrido-pyrrolidine hybrid compounds were designed, developed and screened for their antidiabetic property with α-glucosidase. The design is based on preliminary screening of key fragments identified from literature reported α-glucosidase inhibitors and antidiabetic compounds. The most active fragments were stitched to provide a pyrido-pyrrolidine hybrid molecule as a new motif. A library of these compounds were synthesized and screened against a series of α-glycosidases. Subsequently, compound 3k was the most efficacious analog with IC₅₀ of 0.56 μM. Photoluminescence study and circular dichroism experiments indicated that compound 3k modulates the primary and secondary structure of the enzyme. It successfully brings down the fasting blood glucose level for streptozotocin (STZ, 70 mg/kg, Intraperitoneal) induced type I diabetic male Sprague-Dawley rats (250–320 g). At lower concentration, compound 3k slightly stimulates proliferation of BRIN-BD11 (α-glucose responsive beta cells from rat pancreas islets that secretes insulin) cells.

Full text of DOI:10.1016/j.ejmech.2020.112034

Journal Pre-proof
Discovery of novel pyrido-pyrrolidine hybrid compounds as alpha-glucosidase
inhibitors and alternative agent for control of type 1 diabetes
Tania Luthra, Venkanna Banothu, Uma Adepally, Krishna Kumar, Swathi M, Saikat
Chakrabarti, Srinivas R. Maddi, Subhabrata Sen
PII:
S0223-5234(20)30001-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112034
EJMECH 112034
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 September 2019
Revised Date: 17 December 2019
Accepted Date: 2 January 2020
Please cite this article as: T. Luthra, V. Banothu, U. Adepally, K. Kumar, S. M, S. Chakrabarti, S.R.
Maddi, S. Sen, Discovery of novel pyrido-pyrrolidine hybrid compounds as alpha-glucosidase inhibitors
and alternative agent for control of type 1 diabetes, European Journal of Medicinal Chemistry (2020),
doi: https://doi.org/10.1016/j.ejmech.2020.112034.
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Discovery of novel pyrido-pyrrolidine hybrid compounds as alpha-glucosidase inhibitors and
alternative agent for control of Type 1 diabetes
1
2
2
3
5
3
Tania Luthra, Venkanna Banothu, Uma Adepally, Krishna Kumar, Swathi M, Saikat Chakrabarti,
4
, 1
Srinivas R. Maddi and Subhabrata Sen*
1.
2.
3.
Department of Chemistry, School of Natural Sciences, Shiv Nadar University, Chithera, Dadri, Greater
Noida, UP 201314, India
Institute of Science and Technology, Jawaharlal Nehru Technological University, Kukatpally,
Hyderabad, Telangana 500085, India
Structural Biology and Bio-informatics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja SC
Mullick Road, Kolkata, West Bengal, 700032, India
4
.
.
Acubiosys PVT LTD, TBI, BITS-Pilani Campus , Jawahar Nagar, Hyderabad, Telangana 500078
Center for Innovation in Molecular and Pharmaceutical Sciences, Dr. Reddy’s Institute of Life Sciences,
University of Hyderabad Campus, Gachibowli, Hyderabad-500046
5
Keywords: pyrido-pyrrolidine hybrid; alpha-glucosidase inhibitor; photoluminescence; circular
dichroism; DMPK studies
Abstract
A new library of pyrido-pyrrolidine hybrid compounds were designed, developed and screened for
their antidiabetic property with α-glucosidase. The design is based on preliminary screening of key
fragments identified from literature reported α-glucosidase inhibitors and antidiabetic compounds.
The most active fragments were stitched to provide a pyrido-pyrrolidine hybrid molecule as a new
motif. A library of these compounds were synthesized and screened against a series of α-
glycosidases. Subsequently, compound 3k was the most efficacious analog with IC50 of 0.56 µM.
Photoluminescence study and circular dichroism experiments indicated that compound 3k
modulates the primary and secondary structure of the enzyme. It successfully brings down the
fasting blood glucose level for streptozotocin (STZ, 70 mg/kg, Intraperitoneal) induced type I diabetic
male Sprague-Dawley rats (250–320 g). At lower concentration, compound 3k slightly stimulates
proliferation of BRIN-BD11 (α-glucose responsive beta cells from rat pancreas islets that secretes
insulin) cells.
Introduction
Diabetes or diabetes mellitus (DM) is one of the most debilitating non communicable and recurring
1
metabolic disorders in the present world, where nearly 0.5 billion patients are affected globally. It is
2
a condition which occurs due to accumulation of glucose in blood stream leading to hyperglycemia.
It is responsible for ~ 5 million deaths every year and it aggravates the rise of heart disease,
thrombosis and microvascular complications such as kidney failure, blindness and peripheral

3
neuropathy. Typically it can be induced either due to the destruction of beta cells via autoimmune
pathway that results in complete insulin deficiency (type 1 diabetes mellitus [T1DM]) or due to the
inability of the body for not using insulin appropriately that leads to resistance towards insulin (type
4
2
diabetes mellitus [T2DM]). The medical treatment to control the progress of diabetes focuses on
5
reduction of the concentration of postprandial glucose (PG) level in blood.
α-glucosidase enzyme is located at the brush border of intestine, where it is involved in the
breakdown of dietary sugars and starches to glucose along with two small intestine membrane
bound enzymes maltase-glucoamylase (MGAM) and sucrose-isomaltase (SI). Collectively they are
attractive target for inhibition as a way of managing blood glucose levels among individuals suffering
6
from type 2 diabetes. Among them α-Glucosidase inhibitors (AGI) has been recognized as one of the
6
most important targets for treating T2DM. Few other promising targets include dipeptidyl peptidase
7-9
4
(DPP-4), insulin secretagogues, peroxisome proliferator-activated receptor (PPAR-γ) and etc.
Interestingly, compared to diverse molecular therapy in T2DM, patients suffering from type 1
diabetes (T1DM) insulin is the mainstay for current treatment which, includes maintenance of
optimized timing of the pre-meal insulin injection, application of insulin analogues with a faster
absorption rate and small molecules that can delay nutrient absorption from the intestine. Several
studies have indicated that despite intensive insulin therapy (which involves 3 - 5 insulin injections
daily and monitoring of blood glucose level 4 - 5 times a day) nearly 50% of the T1DM affected
10-11
patients suffer from diabetes related complications such as hypoglycaemia and weight gain.
Consequently this has prompted the diabetes researchers to look for other potential therapies
including α-glucosidase inhibitors (AGIs) to treat T1DM. Recently few reports indeed indicated that
12
AGIs can also treat insulin deficiency caused by T1DM.
This research aims at developing a novel class of α-glucosidase inhibitors (AGIs) which can help in
controlling high blood glucose level in diabetes mellitus in general. Here in we report discovery of a
novel class of molecules based on pyrido-pyrrolidine hybrid for this purpose. The compounds were
conceived via screening of key fragments of known α-glucosidase inhibitor and/ or antidiabetic
compounds from the literature. They were synthesized in a combinatorial fashion via Knoevenagel
condensation of pyridofuranone 1 and pyrrolidine based aldehydes 2a - k. In vitro screening against
α-glucosidase enzyme (yeast origin) provided compound 3k as the most potent inhibitor which
displayed competitive inhibition. Fluorescence spectroscopic investigation revealed that compound
3
k modulated the primary structure of α-glucosidase enzyme. It further attenuates the
concentration of β-sheets with the increase in its concentration thereby attuning the secondary

structure. In vivo efficacy studies on streptozotocin induced T1DM male Sprague-Dawley rats
revealed that compound 3k reduce concentration of fasting blood glucose level.
Results and discussion
Design
In order to design novel inhibitors for α-glucosidase enzyme we manually curated a diverse set of
natural and synthetic molecules from the literature which has demonstrated α-glucosidase
inhibition and / or antidiabetic property. These molecules include natural products 4-dideoxy-1, 4-
imino-D-arabonitol (DAB), N-methyl-α-homonojirimycin, casuarine, kotalanol, broussochalcone,
papyriflavonol A, genistein, dadzein, macluraxanthone, cudraxanthone, cudratricusxanthone F,
radicamine A, radicamine B, emodin, physcion, aloe-emodin, rhein, a pyrrole based alkaloid from the
13-26
fruit peels of Strychnos nux-blanda, fumosorinone, and 3-(Z)-butylidenepthalide (Table 1).
The
list also contained synthetic compounds like densely functionalized dibenzyl ether, diaryl-α, β-
27-30
unsaturated ketones, benzothiazole and C -alkylidene oxoindoles (Table 1).
The fragments
3
extracted from these molecules comprised of substituted pyrrolidine, piperidines,
hexahydropyrrolizine, tetrahydrothiophene, substituted phenols, flavonols, pyranols, anthacenones,
anthracene-diones, stilbene, benzothiazole, substituted pyrrolidine, pyridone and pthalide (A → S).
They were either synthesized in-house or procured commercially (Table 1). Screening of these
fragments against a series of α-glycosidases viz. yeast α-glucosidase, α-mannosidase (jack bean
origin) and α-mannosidase (bovine kidney origin) resulted in the discovery of three fragments Q, R
and S, which were most potent among the fragments screened. Their activity ranged from 5 → 15
µM against α-glucosidase (yeast origin). This double digit micromolar potency may not look
promising however we have to understand these are basic fragments devoid of any functionalities
that can augment binding to α-glucosidase. However these are excellent molecules that could be
either functionalized or transformed into more efficacious chemical entities.
Table 1. Preliminary fragment screening to search suitable fragments for the design of novel
inhibitors against α-glycosidases (NI; no 50% inhibition at 1 mM concentration of inhibitor)
Inhibitory Activity (µM)
Activities as reported
Compds
Fragments α-glucosidase
yeast)
α-fucosidase
α-mannosidase
in the literature
(
(bovine kidney)
(jack bean)
1
,
4-dideoxy-1, 4-
imino-D-arabonitol
(
DAB); Inhibits yeast α-
glucosidase (IC50 = 0.15
µM) and rat intestinal
maltase (IC = 5. 8
58.01 ± 1.21
NI
NI
A
50
13
µM)

N-methyl-α-
homonojirimycin;
Inhibits rat intestinal
maltase (IC = 0.72
50
91.88 ± 0.87
NI
NI
µM) and lowered
B
blood glucose levels in
1
4
healthy mice
Casuarine
Inhibits fungal maltase-
glucosidase (IC = 0.7
50
4
6
1.12 ± 2.33
82
51
µM) and rice α-
C
glucosidase (IC = 1.2
50
15
µM)
Kotalanol
Inhibited rat intestinal
maltase (IC = 0.18
2.31 ± 1.02
71
NI
NI
NI
50
D
16
µM)
Dimethylallyl-
tetrahydroxyflavonol
derivative
NI
Inhibits α-glucosidase
1
7
E
with K of 4.2 µM
i
Broussochalcone
Inhibits α-glucosidase
in a non-competitive
1
01.22 ±
.11
NI
NI
NI
3
fashion with K = 5.3
i
F
17
µM
Papyriflavonol A
Inhibits α-glucosidase
85.89± 0.091
89
1
7
with K = 2.3 µM
i
G
Genistein
Inhibits α-glucosidase
38
with IC = 7 µM
5
0
8
1.03 ± 2.32
54
49
Dadzein
H
Inhibits α-glucosidase
18
with IC = 14 µM
5
0
Macluraxanthone
Inhibits α-glucosidase
1
9
with K = 8.9 µM
i
Cudraxanthone
Inhibits α-glucosidase
1
9
62.31 ± 0.41
NI
NI
with K = 7.4 µM
i
I
Cudratricusxanthone F
Inhibits α-glucosidase
1
9
with K = 7.0 µM
i

Radicamine A
Inhibits α-glucosidase
20
with IC of 6.7 µM
5
0
2
5.21 ± 1.11
41
28
Radicamine B
J
Inhibits α-glucosidase
20
with IC of 9.3 µM
5
0
R = R = R = H,
1
2
3
Emodin, α-glucosidase
yeast), IC = 4.12 µM
(
50
R = R = H; R = Me,
1
3
2
Physcion, α-
glucosidase (yeast),
IC = 5.32 µM
26.43 ± 3.39
32
NI
5
0
R = CH OH, Aloe-
2
K
emodin, α-glucosidase
(
Yeast), IC = 4.56 µM
50
R = COOH, Rhein, α-
glucosidase (Yeast),
21
IC = 5.68 µM
5
0
Natural product,
isolated from Syagmus
Romanzoffiana, lowers
postprandial blood
31.05 ± 0.71
82.67 ± 1.54
NI
NI
22
L
sugar in vivo
a. R = 3-p-tosyl; R = 4-
1
2
OH and 3, 4-di-OH, α-
glucosidase (yeast),
IC = 12 and 16 µM
50
b. R = 4-p-tosyl; R = 4-
91
105
1
2
OH or 3, 4-di-OH, α-
glucosidase (yeast),
IC = 0.98 µM
M
50
2
7
and 0.4 µM
Benzothiazole with
benzohydrazide inhibit
2
0.54 ± 0.06
NI
NI
15
NI
α-glucosidase with IC
50
N
O
28
5
.31 to 53.3 µM
Inhibits yeast α-
glucosidase with IC of
50
5.03 ± 1.87
2
9
0
.16 µM
Inhibits α-glucosidase
(
2
yeast) with IC of
50
1
5.03 ± 3.21
65
21
28
14
.71, 11.41 and 14.2
30
µM
P
A pyrrole alkaloid from
Strychnos nux-blanda
that controls blood
5
.01 ± 0.66
23
Q
sugar

Fumosorinone (Fu)
Inhibits PTP1B activity;
increased insulin
1
4.78 ± 1.68
NI
NI
provoked glucose
uptake in insulin
R
2
5
resistant HepG2 cells
-(Z)-
butylidenepthalide
3
Reduces blood glucose
concen. in NAD-STZ
mediaeddiabetic mice.
Targets α-glucosidase
at the intestinal level
and inhibits at 2.15 µM
non-competitively with
7
.15 ± 0.33
82
41
S
24
Ki 4.86 µM.
Data are average of three independent sets of assays performed replicated thrice
In a bid to stitch fragments Q, R and S together to generate new molecules, the aromatic moiety of
the pthalide fragment S, was replaced with the pyridone fragment R to generate the new molecule
R′. Next the aromatic appendage on R′ was replaced with the pyrrole moiety Q to afford Q′. Finally
the pyrrole nitrogen on Q′ was protected with a range of functionalities to provide the desired
library (3a - k) (Scheme 1).
Scheme 1. Design of novel α-glucosidase inhibitors
Chemistry
The designed compounds were synthesized by Knoevenagel condensation of furopyridine-dione 1
and appropriate pyrrole-2-carbaldehyde derivatives 2a - k. The synthesis of furopyridine-dione 1
31
was based on a literature procedure (Step 1 - 3, Scheme 2). To begin with ethyl-2-ethoxyacetate
was treated with sodium hydride in presence of acetone to afford ethoxy acetylacetone (Step 1,
Scheme 2). It was then treated with 2-cyanoacetamide in piperidine to afford 2-pyridone (Step 2,
Scheme 2) which was then lactonized in presence of conc. hydrochloric acid to generate 1 (Step 3,
Scheme 2). Pyrrole-2-carbaldehydes 2a - k were synthesized via Malliard reaction in which glucose in
31
presence of oxalic acid was treated with diverse amines 4a - k (Step 4, Scheme 2). In the final
reaction, 1 and 2a - k were reacted in presence of piperidine which resulted into precipitation of the

desired products (3a-k) (Step 5, Scheme 2). They were washed with diethyl ether and hexane to
1
afford the pure compounds 3a - k (Scheme 2c). The desired compounds were characterized by H
13
and C NMR analysis and high resolution mass spectroscopy. Single crystal X-Ray of compound 3k
confirmed its structure (Scheme 2) (CCDC# 1934521).
Scheme 2. Synthesis of the hybrid library 3a - k
In vitro screening against α-glucosidase (yeast origin)
The compound library (3a – k) was screened against α-glucosidase of yeast origin. To assess the
extent of selective inhibition of our library molecules they were also screened against α-fucosidase
(
bovine liver origin) and α-mannosidase (jack bean origin). The inhibition was determined in 96-well
plates with 4-nitrophenyl-α-D-glucopyranside (PNPG) as the substrate and according to the
procedure described in the experimental section. Acarbose was the positive control (Table 2). The

results are tabulated in Table 2. In general, the library is selectively potent against α-glucosidase
compared to α-fucosidase or α-mannosidase. Compound 3k was most potent with an IC of 0.56
50
µM which was four times more efficacious than the positive control acarbose (IC = 2.1 µM). The
50
next potent analogue was compound 3d, which was moderately active with an IC of 4.4 µM. It was
50
interesting to see that analogues 3a and 3f - i had substantially reduced activity against α-
glucosidase, with IC in the range of 20 - 33 µM. Compounds 3b, c, e and j demonstrated no activity
50
at all. The structure activity relationship as deduced from this initial screening revealed that pyridine
dione as well as the pyrrolidine moiety is important to evoke any inhibitory response against the
enzyme. Introduction of a tethered heteroaromatic functionality like a C indolyl moiety to the
3
pyrrolidinyl nitrogen (3d) resulted in increased α-glucosidase inhibitory activity compared to that of
a tethered phenyl compound as in compound 3e. In general aromatic substituents at the pyrrolidinyl
nitrogen as exemplified by compound 3f - i, invoked more potency to the resulting molecules than
their aliphatic counterparts 3b and c. Introduction of functionalities on these aromatic rings played a
crucial role as epitomized by the p-CO Et substituent on the benzene ring in compound 3k.
2
Interestingly halogens or hydroxy at the para position (compound 3g - i) or methyl at the ortho
substituent (compound 3j) of the aromatic moiety had negligible effect on the inhibitory efficacity of
the resulting molecules.
Table 2. Screening of compounds 3a-k against various α-glycosidase (NA: no 50% inhibition at 1 mM
concentration of the compound)
IC in µg/ mL
50
Compounds
α-glucosidase
α-fucosidase (bovine
α-mannosidase
(
yeast origin)
20.8 ± 0.65
NA
kidney origin)
51.1 ± 1.21
NA
(jack bean origin)
3
a
b
92.3 ± 2.43
3
NA
NA
3
c
NA
NA
3
d
e
4.4 ± 1.09
NA
12.9 ± 1.11
NA
NA
3
NA
3
f
27.4 ± 0.72
20.7 ± 0.77
21.9 ± 1.34
33.3 ± 2.11
NA
38.4 ± 1.28
NA
61.4 ± 2.38
NA
3
g
3
h
NA
NA
3
i
21.7 ± 1.14
NA
NA
3
j
NA
3
k
0.56 ± 0.11
30.4 ± 0.69
41.2 ± 0.82

Acarbose
2.1 ± 0.78
Data are average of three sets of assays performed
Reaction kinetics
To evaluate the mechanism of binding of the most active inhibitor 3k with α-glucosidase (yeast
origin), kinetics analysis was performed. The type of inhibition and Ki were obtained from the
Lineweaver-Burke plots as shown in figure 1. The experiments were conducted at 37 °C. The Ki was
obtained from Vmax at 2 µM and 10 µM of compound 3k. The experiments showed that compound
3
k was a competitive inhibitor with Ki value of 29.91 µM. The kinetic analysis further suggested
that compound 3k competes with substrate for binding at active site of the α-gluosidase enzyme
yeast origin) (Figure 1a). Figure 1b depicts a linear Lineweaver-Burk plot for the standard drug
(
acarbose.
Figure 1. a) Reactions kinetics experiments to assess the mechanism of action of compound 3k on
the α-glucosidase enzyme (yeast origin). b) Reaction kinetics study of acarbose
Investigation of interaction between a-glucosidase enzyme (yeast origin) and compound 3k with
Fluorescence spectroscopy
Fluorescence spectroscopic investigation of ligand-protein complex determines the alteration of
protein conformation along with binding mechanism (static or dynamic), binding sites and binding
constants. The tryptophan (Trp) residue of α-glucosidase enzyme emit fluorescence upon activation
and hence fluorescence spectroscopy can be harnessed to assess the extent of modulation of α-
32
glucosidase conformation imposed by an inhibitor. In general the resulting fluorescence emission
of an inhibitor bound α-glucosidase enzyme will decrease compared to the free enzyme. This
phenomenon is known as fluorescence quenching. With the steady increment of the concentration
of the inhibitor the fluorescence intensity is further quenched, thereby indicating the extent to
which the ligand binds to the enzyme. Accordingly the fluorescence quenching experiment of
compound 3k with α-glucosidase enzyme (yeast origin) was conducted and the result is depicted in

figure 2. Excitation of α-glucosidase (yeast origin) at a wavelength of 295 nm, displayed a strong
fluorescence emission peak at 330 nm. Inherently compound 3k, had no fluorescence emission and
contributed negligibly to the fluorescence interference. Next, addition of inhibitor 3k at 6.25 ng/
mL and 25 ng/ mL led to the decrease of intrinsic fluorescence spectra of α-glucosidase significantly
at 298 K (Figure 2). This indicated that compound 3k binds to α-glucosidase, and the binding could
be induced through a microenvironment variation of Trp residues in α-glucosidase enzyme.
Figure 2. Fluorescence quenching spectra of compound 3k
Investigation of modulation of 2° structure of a-glucosidase enzyme (yeast origin) by 3k
Circular dichroism (CD) is a technique that can be used to understand the conformational changes of
a protein when modulated by a ligands. Herein, we utilized CD to assess the alteration in secondary
33
structure of α-glucosidase induced by compound 3k (Figure 3). The CD spectrum of α-glucosidase
Figure 3) included 2 negatively humped peaks at 209 and 222 nm which further revealed that α-
(
34
glucosidase has a high percentage of α-helical structures. In the presence of compound 3k, the
intensity of these peaks were reduced when compared to the control (Figure 3). This implied that
the helicity of α-glucosidase was decreased once it interacts with compound 3k. The Secondary
Structure Estimation Program calculated the secondary structural content of α-glucosidase. It was
observed that as the concentration of compound 3k increased, the percentages of β-sheets in α-
glucosidase got decreased, (Figure 3). Hence, the binding of compound 3k to α-glucosidase induced

alterations in the secondary structure of α-glucosidase and modulated the folding of the protein.
This probably contributed to the inhibition pathway whereby compound 3k prevented substrate
binding that lead to the inhibition of α-glucosidase (Figure 3).
Figure 3. Circular Dichroism spectra of compound 3k with α-glucosidase (yeast origin)
Molecular modelling of compound 3k against a homology model of 3D structure of alpha-
glucosidase from Saccharomyces cerevisiae
The 3D structure of alpha-glucosidase from Saccharomyces cerevisiae was generated using
homology modelling approach and further in-silico validation of the model structure was performed
(
see Methods). The compound 3k was docked by the GOLD program onto the CASTp predicted
binding pocket (Figure 4, panel B) of alpha-glucosidase and most plausible solution was identified
based on the CHEMPLP score (69.87) of the GOLD suite. The estimated ∆G of binding was found to
be -15.53. The selected protein - compound 3k complex was further investigated for the interaction
analysis. Accordingly probable interacting residues were identified. Four probable hydrogen bonds
were observed to be formed between protein and compound 3k (Figure 4, panel D).

Figure 4. α-Glucosidase modelling and docking. (A) 3D model structure of α-glucosidase (yeast)
MAL32) protein (PDB ID: 3AJ7) is shown in cartoon representation where domain A (residues: 1-112
(
and 189-509), domain B (residues: 113-188), and domain C (residues: 510-584) are marked in gold,
pink and blue colors, respectively. (B) Largest ligand binding pocket identified by CASTp is marked by
cyan spheres. (C) GOLD predicted most likely complex of compound 3k and yeast alpha-glucosidase
(
MAL32) protein is shown in surface representation where binding pocket is shown in cyan. (D)
Protein-compound 3k interacting residues are shown in cyan and previously reported catalytic
residues D214, E276 and D349 are shown in orange. Probable hydrogen bonds are indicated by
35-38
yellow dotted lines.
DMPK studies
Drug metabolism and pharmacokinetics is essential within drug discovery and development. It
provides information about absorption, metabolism and excretion (ADME) processes of a drug
39
molecule in a physiological system. This information contributes to better designing of drugs and
avoidance of drug-drug interaction (DDI). The primary role of DMPK studies is to subscribe the
optimisation of the compounds for in vivo experiments by considering various physicochemical
properties gathered from in vitro experiments with materials obtained from human beings and in
39
vivo animal studies. Accordingly thermodynamic solubility, log D and PAMPA permeability of
compound 3k was determined. The metabolic stability of compound 3k in mouse, rat and human
liver microsomes were measured. Its ability to bind to plasma in mouse, rat and human plasma were
also measured. Finally pharmacokinetics of compound 3k in mice was investigated.

The physicochemical characterization studies of compound 3k demonstrated that it is a poorly
soluble (solubility ~ 0.1 mg/ mL in phosphate buffer at pH 7.4) and highly permeable compound as
understood from the GIT PAMPA data in table 3. This categorizes it to BCS-2 class of compounds.
Table 3. Thermodynamic solubility data of compound 3k at 1mg/mL in phosphate buffer at pH 7.4
-6
GIT PAMPA at pH 7.4 P(10 cm/s)
Solubility (ug/mL)
.1
Log D
1
5
0
2.4
The high metabolic clearance of compound 3k in human, rat and mouse microsomes as shown in
Table 4 indicated rapid degradation of this compound in liver. The clearance is mainly due to CYP
mediated phase-1 metabolism.
Table 4: Metabolic stability data of compound 3k in liver microsomes of mouse, rat and human
Percent
t1/2
(min)
9
mCLint
CLinvivo
Species
Mouse
Rat
metabolized
99.03 ± 0.01
95.21 ± 0.33
96.33 ± 1.22
(uL/min/kg)
74.11 ± 2.03
54.41 ± 0.09
54.78 ± 1.21
(mL/min/kg)
60.31 ± 0.11
35.22 ± 1.54
15.09 ± 0.91
15
Human
13
The variation in the plasma protein binding from human, mouse and rat may be due to low recovery
of the compound 3k which was explained by its nonspecific binding to the plasma protein (Table 3).
Table 5. Plasma protein binding data of compound 3k at 10 μM in liver microsomes of mouse, rat
and human
Percent free
fraction
8.99 ± 0.20
Percent bound % recovery
fraction
Species
Mouse
Rat
91.01 ± 0.21
98.44 ± 0.32
89.26 ± 2.10
15.02 ± 0.47
1.95 ± 0.40
56.66 ± 4.90
1.56 ± 0.32
Human
10.74 ± 2.10
Following PO administration in swiss albino mouse, compound 3k demonstrated low oral exposure
Figure 5), which could be due to its poor solubility in the intestine which was also evidenced by low
thermodynamic solubility.
(

1
0.0
Compound 3k mouse oral pharmacokinetics
1
.0
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (h)
Figure 5. Pharmacokinetics of compound 3k after oral administration into male Swiss albino mice at
mg/kg
3
The solubility (Kinetic and thermodynamic) and permeability (PAMPA) studies of compound 3k
indicated that it was a poor soluble and high permeable compound which resembles to BCS class II
category. The stability of compound 3k in human, rat and mouse microsomes demonstrated that it
was unstable and revealed high metabolic clearance, indicating that the clearance was mainly due to
CYP mediated phase -1 metabolism. The variation in protein binding of compound 3k from human,
mouse and rat plasma may be due to low recovery of the compound, which could be due to poor
solubility and nonspecific binding of the compound. Investigations are underway to understand the
rapid metabolism of compound 3k and to identify the soft spot responsible for degradation.
In vivo efficacy
Male Sprague Dawley rats (250 - 320g) were utilized in in vivo studies. Diabetes was induced through
administration of a single dose of Streptozotocin (STZ, 70 mg/kg, intraperitoneal) formulated in 0.1
mmol/L citrate buffer, pH 4.5 (Sigma-Aldrich, India). Random blood glucose above 125 mg/dl was
used to consider rats as diabetic. This blood glucose level were compared with normal vehicle
control animals (n=6). Diabetic rats were provided a normal diet throughout the experiment.
Diabetic rats (with similar degree of hyperglycaemia) were divided arbitrarily into three groups and
were assigned as diabetic vehicle, diabetic acarbose (the group treated with acarbose (used as -ve
control), and diabetic 3k (the third group treated with compound 3k) (nꢀ=ꢀ6, in each group).
The fasting blood glucose values of diabetic rats were significantly higher than those of control rats
at week zero (P<0.001), week one (P<0.0001), week two (P<0.0001), week three (P<0.0001) and
week four (P<0.0001) (Figure 6). The same in the diabetic acarbose decreased significantly at week
one (P<0.0001), week two (P<0.0001), week three (P<0.0001), and week four (P<0.0001) compared
to the diabetic vehicle. Interestingly fasting blood glucose values in the group diabetic 3k decreased

significantly at week one (P<0.0001) and week two (P<0.0001), but displayed values above the
diabetic acarbose group at week three (P<0.0001), and week four (P<0.0001) in comparison to the
diabetic group (Figure 6a).
It is noteworthy that both acarbose and compound 3k, were unable to bring down the non-fasting
(
postprandial) blood glucose level during the entire four weeks of investigation, when compared
with the diabetic group (Figure 6b). For acarbose this inability can be explained, as it works only in
TYPE 2 diabetes mellitus , but this efficacy study was against TYPE 1 diabetes mellitus model
(
Streptozotocin induced. Interestingly the mean body weight of diabetic rats significantly reduced
compared to control rats. No significant results were noted between diabetic group and acarbose/
compound 3k treated groups (Figure 6c).
Figure 6. (a) Fasting blood glucose before and after acarbose and compound 3k treatment in male
rats (n = 6 per group) (***P<0.0001 versus the control group; ***P<0.0001 versus the DM group
[
DM = Diabetes Mellites]; Data represents mean ± SD (n=6)). (b) Non-fasting blood glucose prior and
later acarbose and compound 3k treatment in male rats (***P<0.0001 Vs the control group;
**P<0.0001 versus the DM group; Data represents mean ± SD (n=6)). (c) Body weight before and
*
after acarbose and compound 3k treatment in rats (Statistical analysis results are expressed as the
mean ± standard deviation. Statistical analysis was performed with 2-way ANOVA. P<0.0001 was
considered statistically significant. Analysis performed with Graph prism 6.01). Note. There is a
minute statistical difference, hence the error bars not clearly visible in this graph.
β-cell proliferation study

β-cells are found in pancreatic islets and are known to enact a crucial role in metabolism by
modulating glucose homeostasis through the production of insulin. A current prognoses in type 1
diabetes mellitus (T1DM) based on genetic and environmental implication indicates selective
40-41
targeting of β-cells by the immune system that leads to the loss of functional β-cell mass.
Hence
one of the promising therapies for T1D is envisioned as development of small molecules that can
address β-cell dysfunction by promoting regeneration of β-cell mass. Since the in vivo investigation
of compound 3k against T1DM induced S/D rats demonstrated reduction of fasting blood glucose
values, it was further subjected to β-cell proliferation studies. Accordingly MTT assay against BRIN-
BD11 cells (rat beta cell line) was conducted with compound 3k (refer experimental). Cells were
exposed to compound 3k at varying concentration viz. 100, 30, 10, 3, 1, 0.3 and 0.1 µM in 100 µL
medium. Interestingly compound 3k exhibited marginal proliferation of the b-cells at low
concentration (→ 1 µM) (Figure 8) but exerted minimum cytotoxic effect at higher concentration (≥
1
0 µM). However the minor cytotoxic effect remained unchanged with further increase in the
concentration of compound 3k (Figure 7).
Figure 7. Compound 3k induces b-cell proliferation in BRIN-BD11 cells (rat beta cell line)
Discussion
Herein we have reported discovery of a novel series of pyrido-pyrrolidine hybrid compounds as α-
glucosidase inhibitors. Preliminary screening of the fragments of known α-glucosidase inhibitors and
antidiabetic compounds provided the key moieties, which were merged to generate the new
molecules. The compounds were synthesized via Knoevenagel condensation of appropriate
substrates 1 and 2 to provide the compounds. Next the compounds were screened against α-
glucosidase of yeast origin. When compared to human a-glucosidase, they shared similarity in
substrate specificity, pH optimum and towards inhibitor sensitivity. Additionally, modification of the
arginine, tryptophan and cysteine residue of both the enzymes render them inactive. The initial

assay indicated that compound 3k is the most active compound with IC ~ 0.56 µM. It is four times
50
as potent as the standard drug acarbose (IC50 ~ 2 µM). Photoluminescence and CD study
demonstrated that compound 3k modulate primary and secondary structure of the enzyme. From
the reaction kinetics study it was further revealed that 3k is a competitive inhibitor. DMPK
experiments demonstrated that due to poor solubility and high permeability compound 3k belongs
to BCS-2 class of compounds, where its exposure is low when administered orally. α-glucosidase
inhibitors are generally used to remit the blood glucose concentration of patients suffering from
T2DM. Recently they were successfully applied to patients suffering from T1DM. This encouraged us
to assess the efficacy of compound 3k against S-D rats induced with T1DM by streptozotocin.
Accordingly we observed that compound 3k when administered against S-D rats was unable to bring
down the non-fasting (postprandial) blood glucose level during the entire four weeks of
investigation. This was almost comparable with acarbose (standard drug). However, compound 3k
was able to reduce the fasting blood glucose values of diabetic rats which was almost same as
compared with acarbose. Hence in vitro ADME and in vivo efficacy studies of 3k indicated that the
solubility of the compounds need to be increased and permeability needs to be brought down.
Probably the acid analogue of 3k could improve the solubility and appropriate protection of its
hydroxy moiety could provide the appropriate permeability. Research is presently ongoing in our lab
in this direction. Investigation with β-cells revealed that compound 3k induces slight simulation of
the proliferation of β-cells at lower concentration, however at higher concentration there was not
much cytotoxicity observed. This is not unexpected as 3k functioned through inhibition of α-
glucosidase to bring down the blood glucose level. The uniqueness of this investigation is, α-
glucosidase inhibitors (which are typically meant for controlling T2DM) demonstrated reduction of
blood glucose level in T1DM induced S-D rats. To further understand the dose dependent efficacy
of 3k, we have planned to dose with multiple doses in type 1 and type 2 diabetes models as a
future study. We sincerely hope this investigation provides a robust platform to advance such
chemotypes as small molecule therapeutics against T1DM.
Conclusion
In conclusion, a new pyrrolo pyridone hybrid molecule was identified as an α-glucosidase inhibitor
through fragment screening of known α-glucosidase inhibitor. It showed strong and selective
inhibitory activity against yeast α-glucosidase. It presented potent competitive inhibition against the
enzyme. Preliminary structure activity relationship (SAR) studies suggested the existence of pyridine
dione and the pyrrole moiety to play crucial role in the inhibitory activity against α-glucosidase.
3
k, the most potent inhibitor was further evaluated for its in vitro ADME and in vivo efficacy. It
demonstrated efficacious hypoglycemic reaction in streptozotocin induced S-D rats (fasting blood

glucose). However poor solubility, high permeability and low oral exposure of 3k, renders room for
improvement in ADME features of this molecule. However on the basis of these initial investigation
we anticipate 3k could be a potential hit compound for new drug discovery.
Experimental
Chemistry: All reactions were carried out in flame-dried sealed tubes with magnetic stirring. All
experiments were performed under argon atmosphere. All reagents were purchased from Sigma
Aldrich or Alfa Aesar. Reaction was monitored by thin layer chromatography (TLC, Silica gel 60 F254),
1
13
using UV light to visualize the course of the reaction. H NMR and C NMR spectra were recorded
with tetramethylsilane as an internal standard at ambient temperature unless otherwise indicated
1
13
with Bruker 400 MHz instruments at 400 MHz for H NMR and 100 MHz for C NMR spectroscopy.
Splitting patterns are designated as singlet (s), broad singlet (br, s), doublet (d), triplet (t). Splitting
patterns that could not be interpreted or easily visualized are designated as multiplet (m). Mass
spectrometry analysis was done with a 6540 UHD Accurate-Mass QTOF LC- MS system (Agilent
Technologies) equipped with an Agilent 1290 LC system obtained by the Department of Chemistry,
School of Natural Sciences, Shiv Nadar University, Uttar Pradesh 201314, India. HPLC analysis was
conducted using Gradient pump mode with A (ACN) and B (0.1% formic acid in water) as eluent for
1
0 minutes. The retention time (tR) is expressed in min at UV detection of 254 nm. HPLC analysis
was performed on an Agilent Eclipse Plus C18 (4.6 × 50 mm, 3.5 μm) at 30 °C. Flow rate: 0.5 mL/min.
13
Compounds 2a-k were synthesized from a standard protocol reported in the literature.
General Procedure for synthesis of compound 3a-k. To the stirred solution of 6-methylfuro[3,4-
c]pyridine-3,4(1H,5H)-dione (200 mg, mmole) (1 equivalence) in ethanol (10 ml) , piperidine (1
equivalence) was added. To the stirring solution appropriate pyrrole 2a-k (1 equivalence) were
o
added under nitrogen atmosphere and the resulting reaction mixture was refluxed at 80 C for 14
hours within which substantial amount of product precipitates from the reaction mixture. Then
reaction mixture was cooled to room temperature and was filtered and the solid obtained was
washed with diethyl ether and hexane respectively (2 X 5 mL) and desired product was dried.
(
Z)-1-((1-benzyl-5-(hydroxymethyl)-1H-pyrrol-2-yl)methylene)-6-methylfuro[3,4-c]pyridine-
,4(1H,5H)-dione (3a)
Following the general procedure, the desired compound 3a was synthesized from (furopyridindione
200mg, 1.21 mmol), 1-benzyl-5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (260mg, 1.21 mmol)
3
(
and piperidine (0.12 ml, 1.21 mmol). A brown solid was generated with yield of 75%.
1
H NMR (400 MHz; DMSO-d ): δ 2.62 (s, 3H); 4.37 (s, 2H); 5.51(s, 2H); 6.33-6.32 (d, J = 3.92 Hz, 1H);
6
6
.66 (s, 1H); 6.90 (s, 1H); 6.95-6.94 (d, J = 3.88 Hz, 1H); 6.99-6.97 (d, J = 7.40 Hz, 2H); 7.24-7.20 (m,

1
3
1
1
1
H); 7.32-7.27 (m, 2H). C NMR (100 MHz; DMSO-d ): δ 19.48, 39.52, 46.08, 55.36, 96.37, 101.71,
6
04.31, 111.09, 115.66, 125.88, 127.11, 127.17, 128.63, 138.46, 138.56, 138.77, 154.07, 155.38,
-
1
+
57.93, 164.20. IR (neat, ν cm ): 3459, 2920, 1782, 1666, 1610, 1280. [M+H] calculated for
o
(
C H N O ) 363.139 found 363.1447. Melting point: 265 C.
21
18
2
4
(
Z)-1-((5-(hydroxymethyl)-1-(prop-2-yn-1-yl)-1H-pyrrol-2-yl)methylene)-6-methylfuro[3,4-
c]pyridine-3,4(1H,5H)-dione (3b)
Following the general procedure, the desired compound 3b was synthesized from (furopyridindione
(
200mg, 1.21 mmol), 5-(hydroxymethyl)-1-(prop-2-yn-1-yl)-1H-pyrrole-2-carbaldehyde (197mg, 1.21
mmol) and piperidine (0.12 ml, 1.21 mmol). A brown solid was generated with yield of 78%.
1
H NMR (400 MHz; DMSO-d ): δ 2.31 (s, 3H); 3.37 (s, 1H); 4.53 (s, 2H); 5.10 (s, 2H); 6.25-6.24 (d, J =
6
1
3
3
1
1
.76 Hz, 1H); 6.77 (s, 1H); 6.85-6.84 (d, J = 3.84 Hz, 1H); 6.99 (s, 1H). C NMR (100 MHz; DMSO-d ): δ
6
9.98, 33.12, 55.57, 75.81, 80.05, 96.92, 101.80, 104.88, 111.40, 116.12, 127.10, 138.28, 139.40,
-
1
54.73, 155.93, 158.41, 164.67. IR (neat, ν cm ): 3240, 2917, 2110, 1767, 1661, 1612, 1227, 639.
+
o
[
M+H] calculated for (C H N O ) 311.1026 found 311.1019. Melting point: 276 C
17 14 2 4
(
Z)-1-((1-allyl-5-(hydroxymethyl)-1H-pyrrol-2-yl)methylene)-6-methylfuro[3,4-c]pyridine-
,4(1H,5H)-dione (3c)
Following the general procedure, the desired compound 3c was synthesized from (furopyridindione
200mg, 1.21 mmol), 1-allyl-5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (200mg, 1.21 mmol) and
3
(
piperidine (0.12 ml, 1.21 mmol). A orange solid was generated with yield of 80%.
1
H NMR (400 MHz; DMSO-d ): δ 2.30 (s, 3H); 4.43 (s, 2H); 4.80-4.76 (m, 1H); 4.87 (s, 2H); 5.09-5.07
6
(
d, J = 10.32 Hz, 1H); 5.18 (s, 1H); 5.97-5.96 (m, 1H); 6.26-6.25 (d, J = 3.88 Hz, 1H); 6.82-6.76 (m, 2H);
1
3
6
9
1
.87-6.86 (d, J = 3.84 Hz, 1H); 11.86 (s, 1H). C NMR (100 MHz; DMSO-d ): δ 19.95, 45.69, 55.64,
6
6.88, 102.25, 104.74, 111.16, 115.87, 115.98, 127.17, 135.78, 138.60, 139.07, 154.49, 155.91,
-
1
+
58.41, 164.69. IR (neat, ν cm ): 3392, 2928, 1770, 1645, 1615, 905, 970. [M+H] calculated for
o
(
C H N O ) 351.0742 found 351.0737. Melting point: 254 C
17
16
2
4
(
Z)-1-((1-(2-(1H-indol-3-yl)ethyl)-5-(hydroxymethyl)-1H-pyrrol-2-yl)methylene)-6-methylfuro[3,4-
c]pyridine-3,4(1H,5H)-dione (3d)
Following the general procedure, the desired compound 3d was synthesized from (furopyridindione
(
(
200mg, 1.21 mmol), 1-(2-(1H-indol-3-yl)ethyl)-5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde
324mg, 1.21 mmol) and piperidine (0.12 ml, 1.21 mmol). A orange solid was generated with yield of
8
5%.

1
H NMR (400 MHz; DMSO-d ): δ 2.70 (s, 3H); 3.09-3.06 (m, 2H); 4.46-4.40 (m, 3H); 5.18-5.16 (t, J =
6
5
6
.32 Hz, 1H); 5.97 (s, 1H); 6.22-6.21 (d, J = 3.88 Hz, 1H); 6.35 (s, 1H); 6.79-6.78 (d, J = 3.88 Hz, 1H);
.93 (s, 1H); 7.04-7.01 (m, 1H); 7.10-7.07 (m, 1H); 7.30-7.28 (d, J = 7.96 Hz, 1H); 7.61-7.59 (d, J = 7.68
1
3
Hz, 1H); 10.79 (s, 1H); 11.83 (s, 1H). C NMR (100 MHz; DMSO-d ): δ 19.65, 19.92, 27.76, 44.35,
6
5
1
1
5.69, 68.27, 96.52, 99.82, 102.32, 104.46, 109.31, 111.09, 111.29, 111.91, 115.51, 118.65, 118.86,
21.54, 124.19, 127.26, 127.55, 136.68, 138.40, 138.56, 154.08, 155.05, 155.63, 158.36, 164.67,
-
1
+
67.42, 168.73. IR (neat, ν cm ): 3310, 2927, 1754, 1733, 1668, 1607, 1141. [M+H] calculated for
o
(
C H N O ) 416.1605 found 416.1596. Melting point: 238 C
24
21
3
4
(
Z)-1-((5-(hydroxymethyl)-1-phenethyl-1H-pyrrol-2-yl)methylene)-6-methylfuro[3,4-c]pyridine-
,4(1H,5H)-dione (3e)
Following the general procedure, the desired compound 3e was synthesized from (furopyridindione
200mg, 1.21 mmol), 5-(hydroxymethyl)-1-phenethyl-1H-pyrrole-2-carbaldehyde (277mg, 1.21
3
(
mmol) and piperidine (0.12 ml, 1.21 mmol). A yellow solid was generated with yield of 82%.
1
H NMR (400 MHz; DMSO-d ): δ 2.33 (s, 3H); 2.95-2.92 (m, 2H); 4.28-4.27 (d, J = 5.32 Hz, 2H); 4.43-
6
4
2
.36 (m, 2H); 5.15-5.12 (t, J = 5.32 Hz, 1H); 6.19-6.18 (d, J = 3.88 Hz, 1H); 6.78-6.78 (d, J = 9.00 Hz,
13
H); 6.83-6.82 (d, J = 3.84 Hz, 1H); 7.18-7.14 (m, 3H); 7.26-7.23 (m, 2H); 11.91 (s, 1H). C NMR (100
MHz; DMSO-d ): δ 19.98, 37.96, 44.99, 55.60, 97.03, 102.39, 104.67, 111.25, 115.88, 126.86, 126.96,
6
-
1
1
1
28.69, 129.47, 138.54, 138.90, 138.97, 154.24, 155.89, 158.39, 164.69. IR (neat, ν cm ): 3457, 2915,
+
778, 1755, 1665, 1613, 1258. [M+H] calculated for (C H N O ) 377.1496 found 377.1484. Melting
22
20
2
4
o
point: 233 C
(
Z)-1-((5-(hydroxymethyl)-1-phenyl-1H-pyrrol-2-yl)methylene)-6-methylfuro[3,4-c]pyridine-
,4(1H,5H)-dione (3f)
Following the general procedure, the desired compound 3f was synthesized from (furopyridindione
200mg, 1.21 mmol), 5-(hydroxymethyl)-1-phenyl-1H-pyrrole-2-carbaldehyde (243mg, 1.21 mmol)
3
(
and piperidine (0.12 ml, 1.21 mmol). A brown solid was generated with yield of 81%.
1
H NMR (400 MHz; DMSO-d ): δ 2.21 (s, 3H); 4.25-4.20 (m, 2H); 5.05 (s, 1H); 6.06 (s, 1H); 6.28 (s, 1H);
6
6
2
9
1
1
.31 (s, 2H); 6.54-6.44 (d, J = 3.92 Hz, 1H); 7.05-7.04 (d, J = 3.88 Hz, 1H); 7.40-7.38 (d, J = 6.12 Hz,
1
3
H); 7.59-7.56 (m, 2H); 12.11 (s, 1H). C NMR (100 MHz; DMSO-d ): δ 19.65, 55.60, 68.26, 96.38,
6
9.80, 101.83, 104.87, 109.32, 111.54, 116.32, 128.14, 128.92, 129.33, 129.93, 136.54, 139.54,
-
1
39.75, 155.05, 155.29, 158.28, 158.34, 164.52, 167.39, 168.71. IR (neat, ν cm ): 3456, 2919, 1757,
+
o
612, 1271. [M+H] calculated for (C H N O ) 349.1183 found 349.1177. Melting point: 290 C
20
16
2
4

(
Z)-1-((1-(4-fluorophenyl)-5-(hydroxymethyl)-1H-pyrrol-2-yl)methylene)-6-methylfuro[3,4-
c]pyridine-3,4(1H,5H)-dione (3g)
Following the general procedure, the desired compound 3g was synthesized from (furopyridindione
(
200mg, 1.21 mmol), 1-(4-fluorophenyl)-5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (264mg, 1.21
mmol) and piperidine (0.12 ml, 1.21 mmol). A yellow solid was generated with yield of 83%..
1
H NMR (400 MHz; DMSO-d ): δ 2.22 (s, 3H); 4.21-4.19 (d, J = 5.28 Hz, 2H); 5.04-5.01 (m, 1H); 6.08 (s,
6
1
3
1
H); 6.43-6.41 (m, 2H); 7.03-7.02 (d, J = 3.88 Hz, 1H); 7.47-7.39 (m, 4H); 11.93 (s, 1H). C NMR (100
MHz; DMSO-d ): δ 19.66, 55.49, 96.61, 101.74, 104.91, 111.60, 116.24, 116.67, 116.89, 128.48,
6
-
1
1
3
3
31.10, 131.19, 132.88, 139.72, 154.98, 155.37, 158.27, 161.09, 163.53, 164.50. IR (neat, ν cm ):
+
467, 2919, 1760, 1640, 1614, 1273, 996. [M+H] calculated for (C H FN O ) 367.1089 found
20
15
2
4
o
67.1080. Melting point: 303 C
(
Z)-1-((1-(4-chlorophenyl)-5-(hydroxymethyl)-1H-pyrrol-2-yl)methylene)-6-methylfuro[3,4-
c]pyridine-3,4(1H,5H)-dione (3h)
Following the general procedure, the desired compound 3h was synthesized from (furopyridindione
(
200mg, 1.21 mmol), 1-(4-chlorophenyl)-5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (284mg, 1.21
mmol) and piperidine (0.12 ml, 1.21 mmol). A yellow solid was generated with yield of 87%.
1
H NMR (400 MHz; DMSO-d ): δ 2.23 (s, 3H); 4.21-4.20 (d, J = 5.04 Hz, 2H); 5.06-5.03 (m, 1H); 6.13 (s,
6
1
8
1
1
H); 6.46-6.43 (m, 2H); 7.04-7.03 (d, J = 3.88 Hz, 1H); 7.44-7.42 (d, J = 8.60 Hz, 2H); 7.65-7.62 (d, J =
1
3
.60 Hz, 2H); 11.92 (s, 1H). C NMR (100 MHz; DMSO-d ): δ 19.68, 55.46, 96.73, 101.72, 104.94,
6
11.84, 116.42, 128.34, 129.95, 130.75, 133.87, 135.52, 139.56, 139.88, 155.02, 155.40, 158.27,
-
1
+
64.49. IR (neat, ν cm ): 3419, 2920, 1775, 1643, 1615, 1276, 745. [M+H] calculated for
o
(
C H ClN O ) 383.0793 found 383.0782. Melting point: 285 C
20
15
2
4
(
Z)-1-((5-(hydroxymethyl)-1-(4-hydroxyphenyl)-1H-pyrrol-2-yl)methylene)-6-methylfuro[3,4-
c]pyridine-3,4(1H,5H)-dione (3i)
Following the general procedure, the desired compound 3i was synthesized from (furopyridindione
(
200mg, 1.21 mmol), 5-(hydroxymethyl)-1-(4-hydroxyphenyl)-1H-pyrrole-2-carbaldehyde (262mg,
1
.21mmol) and piperidine (0.12 ml, 1.21 mmol). A brown solid was generated with yield of 82%.
1
H NMR (400 MHz; DMSO-d ): δ 2.22 (s, 3H); 4.19-4.17 (d, J = 5.04 Hz, 2H); 4.99 (s, 1H); 5.13 (s, 1H);
6
6
.07 (s, 1H); 6.31 (s, 2H); 6.39-6.38 (d, J = 3.88 Hz, 1H); 6.92-6.89 (d, J = 8.60 Hz, 1H); 7.01-7.00 (d, J =
1
3
3
1
1
.88 Hz, 1H); 7.17-7.15 (d, J = 8.60 Hz, 2H); 9.89 (s, 1H). C NMR (100 MHz; DMSO-d ): δ 19.61,
6
9.65, 55.66, 68.25, 96.35, 99.80, 102.10, 104.77, 109.32, 111.13, 116.07, 116.33, 127.48, 128.36,
30.00, 139.27, 140.11, 154.97, 155.04, 155.26, 158.07, 158.34, 164.56, 167.40, 168.70. IR (neat, ν

-
1
+
cm ): 3429, 2927, 1754, 1612, 1273, 1237. [M+H] calculated for (C H N O ) 365.1132 found
20
16
2
5
o
3
65.1117. Melting point: 278 C
Z)-1-((5-(hydroxymethyl)-1-(o-tolyl)-1H-pyrrol-2-yl)methylene)-6-methylfuro[3,4-c]pyridine-
,4(1H,5H)-dione (3j)
Following the general procedure, the desired compound 3j was synthesized from (furopyridindione
200mg, 1.21 mmol), 5-(hydroxymethyl)-1-(o-tolyl)-1H-pyrrole-2-carbaldehyde (260mg, 1.21 mmol)
(
3
(
and piperidine (0.12 ml, 1.21 mmol). A orange solid was generated with yield of 84%.
1
H NMR (400 MHz; DMSO-d ): δ 1.88 (s, 3H); 2.20 (s, 3H); 4.10-4.09 (m, 2H); 4.94 (s, 1H); 5.81 (s, 1H);
6
6
1
1
1
.21 (s, 1H); 6.46-6.45 (d, J = 3.84 Hz, 1H); 7.05-7.04 (d, J = 3.84 Hz, 1H); 7.30-7.28 (d, J = 7.52 Hz,
1
3
H); 7.42-7.39 (m, 1H); 7.50-7.45 (m, 2H); 11.90 (s, 1H). C NMR (100 MHz; DMSO-d ): δ 17.18,
6
9.62, 55.57, 96.29, 101.21, 104.89, 111.38, 116.12, 127.45, 127.77, 129.59, 130.00, 131.37, 135.60,
-
1
37.16, 139.30, 139.55, 155.05, 155.20, 158.26, 164.46. IR (neat, ν cm ): 3420, 2920, 1751, 1636,
+
1
607, 1347, 1264. [M+H] calculated for (C H N O ) 363.1339 found 363.1336. Melting point: 289
21
18
2
4
o
C
(
Z)-ethyl 4-(2-(hydroxymethyl)-5-((6-methyl-3,4-dioxo-4,5-dihydrofuro[3,4-c]pyridin-1(3H)-
ylidene)methyl)-1H-pyrrol-1-yl)benzoate (3k)
Following the general procedure, the desired compound 3k was synthesized from (furopyridindione
(
200mg, 1.21 mmol)), ethyl 4-(2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl)benzoate (330mg, 1.21
mmol) and piperidine (0.12 ml, 1.21 mmol). A yellow solid was generated with yield of 87%.
1
H NMR (400 MHz; DMSO-d ): δ 1.37-1.34 (t, J = 7.08 Hz, 3H); 2.21 (s, 3H); 4.24-4.22 (s, J = 5.28 Hz,
6
2
2
H); 4.40-4.35 (q, J1 = 7.12 Hz and J2 = 7.08 Hz, 2H); 5.09-5.06 (m, 1H); 6.15 (s, 1H); 6.47-6.45 (m,
13
H); 7.07-7.06 (d, J = 3.88 Hz, 1H); 7.57-7.54 (d, J = 8.52 Hz, 2H); 8.15-8.13 (m, 2H); 11.96 (1.09). C
NMR (100 MHz; DMSO-d ): δ 14.64, 19.67, 55.47, 61.52, 96.75, 101.65, 104.96, 112.09, 116.63,
6
1
28.18, 129.24, 130.39, 130.80, 139.42, 139.97, 140.78, 155.03, 155.42, 158.25, 165.63. IR (neat, ν
-
1
+
cm ): 3400, 2926, 1748, 1713, 1639, 1614, 1274, 1239. [M+H] calculated for (C H N O ) 421.1394
23
20
2
6
found 421.1380. Melting point: 284 °C
Enzymatic assays. The inhibitory effect of the synthesized compounds on α-glucosidase (yeast
origin),
α-fucosidase (bovine kidney origin) and α-mannosidase (jack bean origin) inhibitory activity was
determined in 96-well plates employing the substrate PNPG and 4-nitrophenyl α-D-glucopyranoside
42
according to the procedure previously reported by Ferreres et al. Prior to use, all test compounds
were solubilised in solvent, dimethylsulfoxide (DMSO), and then further diluted in DMSO to acquire
the desired final maximum test concentration. Briefly, each well in 96-well plates contained 100 μL

of 2 mM 4-nitrophenyl α-D-glucopyranoside (PNP-G) in 10 mM potassium phosphate buffer (pH 7.2)
and different test concentrations (2–10 μM). The reaction was initiated by the addition of 5 μL of the
enzyme solution (0.1 IU per well). α-glucosidase (yeast), α-fucosidase (bovine kidney) and α-
mannosidase (jack bean) were purchased from Sigma Aldrich, Bangaluru). The plates were incubated
at 37 °C for 10 min. The absorbance was measured spectrophotometrically at 430 nm (Spectra Max
M5e micro plate reader). The increase in absorbance (ΔA) was compared with that of the control
(
buffer instead of test compound) to compute the inhibitory concentrations (IC50) which was
determined from three independent assays replicated thrice. Acarbose, an illustrious inhibitor of α-
glucosidase was employed as positive control. Inhibition (%) = (ΔAcontrol − ΔAsample/ΔAcontrol ×
1
00).The concentration of compound required to obtain 50% inhibition of α-glycosidase activity
under the assay conditions was defined as the IC value. Data are average of three independent sets
50
of assays performed replicated thrice
Reaction kinetics: The inhibition kinetics against α-glucosidase was determined by adding increasing
concentrations of pNPG (1, 2, 3, 4 and 5 mM respectively) as substrate, in the absence and presence
of compound 3k as inhibitor at 2 and 10 µM for yeast enzyme. The absorbance was measured
spectrophotometrically at 420 nm (Biorad micro elisa plate reader (iMark)). The mode of inhibition
(
i.e. competitive, non-competitive, uncompetitive or mixed type) of the screened compounds was
determined on the basis of the inhibitory effects on Km (Michaelis constant) and Vmax (maximum
reaction velocity) of the enzyme. The study was computed using the primary (Lineweaver–Burk)
plots, which are the double reciprocal plots of enzyme reaction velocities (1/V) versus substrate
concentrations (1/[S]). The Lineweaver–Burk (LB) equation follows as:
where, V is the reaction velocity; Km is Michaelis-Menten constant; Vmax is the maximum velocity
and [S] is the substrate concentration.
Intrinsic fluorescence measurements: α-Glucosidase (1.5U) was pre-treated with certain
o
concentrations of compound 3k (6.25 and 25 ng/ml respectively) for 30 min at 37 C. The intrinsic
fluorescence spectra (300-440 nm) were measured using HORIBA Fluorolog-3 spectrofluorometer
(
Model: FL3-2-IHR) with the excitation wavelength at 260 nm.
Circular dichroism: Circular dichroism (CD) was carried out with a J-1500 spectrometer (Jasco) in the
UV range (195-240 nm) using 0.1 cm quartz cuvette. Different concentration of compound 11 was
added to α-glucosidase (30 U/ml). The CD spectra were recorded with 3 times accumulation. The

data of the secondary structure were dealt with by using the professional software Secondary
Structure Estimation.
Modelling of alpha-glucosidase structure: Three-dimensional (3D) structure of the alpha-
glucosidase (MAL32_YEAST) protein sequence from Saccharomyces cerevisiae was modelled using
isomaltase from Saccharomyces cerevisiae (PDB ID: 3AJ7) as a template which possesses 72%
sequence identity with the alpha-glucosidase (MAL32). MODELLER v9.16 was used to build the
43
structure and top ten models were filtered based on the DOPE score. The filtered models were
subjected to Procheck, Verify3D, and Errat programs in order to check stereo-chemical properties
44-46
and fold compatibility of the predicted models.
Based on these parameters, the most likely 3D
model of S. cerevisiae alpha-glucosidase (MAL32) was selected for further analysis.
Molecular Docking: The predicted alpha-glucosidase structure was subjected to CASTp server in
order to identify the probable binding pocket and the largest pocket was selected to dock the
47
compound 3k. The docking analysis was performed using GOLD docking tool and docking poses
48
were ranked using CHEMPLP scoring function. ∆G of binding was calculated using the MOPAC2016
49
program. The protein-ligand interaction analysis was performed using the Ligplot program to
50
identify interacting residues.
Thermodynamic solubility: Solubility of the compound was determined by incubating 1mg of
compound in 1 mL of phosphate buffer saline for 24h with the vortex of 500 rpm. After incubation
period, samples were centrifuged and supernatant was injected into HPLC to determine the
solubility.
Log D determination: The distribution coefficient was determined by adding compound 3k to
Octanol containing 2 mL deep well plate and vortexed the plate for 10 Min at 1200 g and then
5
00µL of Buffer was added to it. This plate was incubated at room temperature for 1 hr on a plate
shaker at 1200 g. After incubation, the samples were allowed to equilibrate for 20 min and then
samples were centrifuged at 4000 g for 30 min. Octanol and buffer layers were analyzed by HPLC-UV
by adjusting the needle height for each Octanol and buffer.
PAMPA permeability: PAMPA permeability was determined by addition of the compound 3k to the
donor (bottom) plate of the PAMPA sandwich plate and 5 µL of the GIT-0 solution was painted at the
bottom side of the PAMPA acceptor plate by turning the plate upside down. The painted acceptor
plate was placed on top of the donor plate in its normal position. The acceptor sink buffer was
added to the acceptor plate. The PAMPA sandwich plate was kept for incubation for 4 hours. After 4
hours incubation, 150 µL was aliquots from donor and acceptor plate were added to UV analysis
plate and read by PAMPA explorer software.

Metabolic stability in mouse, rat and human liver microsomes: Metabolic stability is defined as the
percentage of parent compound lost over time in the presence of a metabolically active test
system. The incubation mixtures consisted of liver microsomes (1.0 mg microsomal protein
/
mL), compound 3k (1 μM) or positive control (verapamil, 1 μM). The reactions were initiated by
adding 1 mM NADPH. Reactions without NADPH (0 and 60 min) were also incubated to rule out
non-NADPH metabolism or chemical instability in the incubation buffer. All reactions were
terminated using 200 μL of ice-cold acetonitrile containing internal standard (200 ng/mL of
telmisartan) at 0, 5, 15, 30 and 60 min. The vials were centrifuged at 4000 rpm for 20 min. The
supernatants thus obtained were analyzed on LC-MS/MS to monitor the disappearance of
compound 3k.
Plasma protein binding in mouse, rat and human plasma: To evaluate the ability of compound 3k
to bind the plasma proteins, the most common approach of plasma protein binding using rapid
equilibrium dialysis device ( RED inserts) used. Compound 3k was tested at a final concentration of
1
0 μM in mouse, rat and dog plasma. An aliquot of 500 μL plasma containing compound 3k was
added in first half (plasma side) of the well of RED device. An aliquot of 300 μL of 100 mM sodium
phosphate buffer pH 7.4 was added in the second half (buffer side) of the Insert. The plate
containing plasma and buffer was equilibrated at 37 ± 1 °C for 6h, with constant shaking at 500 rpm
on an orbital shaker. Samples were collected from respective halves after the completion of the
incubation time. The proteins were precipitated using organic solvents. The samples were
subjected to centrifugation and the supernatants were analyzed analysis on LC-MS/MS
Pharmacokinetics in mice: Swiss albino mice were obtained from Hylasco, Hyderabad, India. They
were housed under standard conditions and were maintained under a 12 h light/dark cycle in the
laboratory animal resources facilities, Acubiosys Pvt. Ltd, Hyderabad, India (AAALAC accredited).
Mice were fasted overnight before and then until 3 h after dosing. Water was allowed ad libitum
during the fasting period. The bodyweights of individual mice, ranging from 25 to 30 g, were
determined on the morning of the study. Compound 3k was administered orally to twelve mice at
3
mg/kg. Since this is the preliminary pharmacokinetic study, the dosage was based on the initial
solubility studies of 3k. After dosing, 0.1 ml specimens of blood were collected by stagger sampling
with two bleeds per mouse from the retro orbital plexus at 0.25, 0.5, 1, 2, 4, 8, and 24 h after PO
route. After collection, blood was processed to plasma by centrifugation at 10000 rpm for 5 min,
and samples were analysed by LC-MS/MS.
In vivo efficacy in rats: Male Sprague-Dawley rats (250 - 320 g) approximately 8 -10 weeks old were
purchased from the Hylasco Laboratories Hyderabad for the experiments. All the experiments were

performed in accordance with the committee for the purpose of Control and Supervision of
Experiments on Animals (CPCSEA) guidelines. For each treated group six animals were used. Diabetic
rats were administered a single dose of streptozotocin (STZ, 70 mg/kg, Intraperitoneal) formulated
in 0.1 mmol/L citrate buffer, pH 4.5 (Sigma–Aldrich, India). 4 days after the STZ injection, the random
blood glucose level of the diabetic rats was measured to confirm hyperglycaemia. Random blood
glucose above 125 mg/dl was used to define rats as diabetic. Above blood glucose level were
compared with normal vehicle control animals (n=6). Diabetic rats were fed a Normal diet
throughout the experiment. Diabetic rats with a similar degree of hyperglycaemia were randomly
divided into three groups: Diabetic vehicle, Diabetic acarbose, and compound 3k groups (nꢀ=ꢀ6, in
each group).
The typical human daily dose of acarbose is 300 mg/60 kg body weight. According to the formula:
d ꢀ=ꢀd
× 0.71/0.11, the corresponding dose of acarbose for rats is 32.28 mg/kg per day. 60
rat
human
mg/kg per day as high dosages. The control (nꢀ=ꢀ6) and the diabetic group received 0.5% saline,
whereas the Acarbose (TCI Chemical, Chennai, India) and compound 3k groups were given doses of
6
0 and 60 mg/kg in a 0.5% saline solution respectively. The drug was administered once daily for 4
weeks using a gastric gavage. All animals were housed in an environmentally controlled room at
5°C with a 12 h light-dark cycles and were given free access to food and water throughout the
2
experimental period. Fasting animals were allowed free access to water. After 4 weeks of treatment,
blood samples were taken from rats after anaesthesia. The rats were then sacrificed. All procedures
involving animals were approved by the IAEC (institutional animal ethics committee).
Statistical analysis results are expressed as the mean ± standard deviation. Statistical analysis was
performed with 2-way ANOVA. P<0.0001 was considered statistically significant. Analysis performed
with Graph prism 6.01.
Elevated fasting blood glucose levels ware measured after 72h of STZ administration. 50%
Carbohydrate (w/w),2%fat (w/w),18%Protein,8%fiber and water ad libitum. The fasting time was 6h.
Whole blood collected by tail puncture method was checked for blood glucose using Accu-Chek
Active meter (Model GB). Volume of blood sample was 1-2 mL. Route of sampling was tail vein
puncture. The code or number of protocol approval by the Institutional Animal Ethic
BITS/IAEC/2018/13.
Please refer below for the glycaemic value of the control groups in the table below.
Table 6. The glycaemic values of the control groups

2
5/01/2019
Fasting Glucose
(mg/dl)
S. No. Group B.Wt on 25/01/2019
Dose Volume
1
2
3
4
5
300.30
295.21
278.12
281.21
301.21
294.14
1.5 ml
1.5 ml
1.4 ml
1.4 ml
1.5 ml
1.5 mL
95
89
90
91
90
94
Control
6
MTT assay for beta cell proliferation: BRIN-BD11 cells (rat beta cell line) were seeded in a 96-well
4
plate at a density of 10 cells/well in 100µl of RPMI complete medium and were incubated overnight.
Cells were then exposed to different concentrations of compound (100µM, 30µM, 10µM, 3µM,
1
µM, 0.3µM, 0.1µM) in 100µl medium. After 24 h, 10µl off MTT reagent (5mg/ml) was added to
0
each well and cells were incubated at 37 c for 90 mins to allow the formation of formazan crystals
(
MTT is reduced to formazan). Post incubation, medium was replaced with 100µl of DMSO to
dissolve formazan crystals and absorbance at 560nm was measured using flat a microplate reader.
ACKNOWLEDGEMENT
We thank Shiv Nadar University for financial support. We thanks BITS Pilani, Hyderabad to allow us
to use the J-1500 spectrometer (Jasco) to execute Circular dichroism (CD).
ASSOCIATED CONTENT
Supporting information available
1
13
Details of synthetic procedures for compounds 3a - 3k and H and C NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org. The CCDC# for the X-Ray structure of
compound 3k is 1934521. Authors will release the coordinates of the homology model upon article
publication.
AUTHOR INFORMATION
Corresponding Author
*
Phone : +919810250221. Email: subhabrata.sen@snu.edu.in
Notes
The authors declare no competing financial interest
ABBREVIATIONS USED

STZ, streptozotocin; DM, diabetes mellitus; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes
mellitus; PBG, post-prandial blood glucose; AGI, α-glucosidase inhibitors; PPAR-γ, peroxime
proliferator-activated receptor; DAB, 4-dideoxy-1, 4-imino-D_arabonitol; PNPG, p-nitrophenyl-β-D-
glucopyranoside; Trp, tryptophan; CD, circular dichroism; DMPK, drug metabolism and
pharmacokinetics; ADME, adsorption, metabolism and excretion; DDI, drug-drug interaction.
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