Design, synthesis and biological evaluation of small molecules as potent glucosidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.04.059

Abstract Herein we have reported design, synthesis and in vitro biological evaluation of a library of bicyclic lactams that led to the discovery of compounds 6 and 7 as a novel class of α-glucosidase inhibitors. They inhibited α-glucosidase (yeast origin)

Full text of DOI:10.1016/j.ejmech.2015.04.059

Accepted Manuscript
Design, synthesis and biological evaluation of small molecules as potent glucosidase
inhibitors
Santanu Hati, Sanjay M. Madurkar, Chandramohan Bathula, Chiranjeevi Thulluri,
Rahul Agarwal, Faiza Amber Siddiqui, Poonam Dangi, Uma Adepally, Ashutosh
Singh, Shailja Singh, Subhabrata Sen
PII:
S0223-5234(15)30016-7
10.1016/j.ejmech.2015.04.059
EJMECH 7874
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 December 2014
Revised Date: 23 April 2015
Accepted Date: 25 April 2015
Please cite this article as: S. Hati, S.M. Madurkar, C. Bathula, C. Thulluri, R. Agarwal, F.A. Siddiqui,
P. Dangi, U. Adepally, A. Singh, S. Singh, S. Sen, Design, synthesis and biological evaluation of small
molecules as potent glucosidase inhibitors, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2015.04.059.
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ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of small molecules as potent glucosidase
inhibitors
1
§
2§
1
3
Santanu Hati, Sanjay M. Madurkar, Chandramohan Bathula, Chiranjeevi Thulluri, Rahul
4
5
4
3
4
Agarwal, Faiza Amber Siddiqui, Poonam Dangi, Uma Adepally, Ashutosh Singh, Shailja
4
2
Singh* and Subhabrata Sen*
1
Department of Chemistry, School of Natural Sciences, Shiv Nadar University, UP 201314,
India
2
Department of Chemistry, Mahatma Gandhi University, Ri Bhoi, Meghalaya, India
3
Institute of Science, Jawaharlal Nehru Technical University, Kukatpally, Hyderabad,
Telangana, India
4
Department of Life Science, School of Natural Sciences, Shiv Nadar University, UO 201314,
India
5
International Centre for Genetic Engineering and Biotechnology, New Delhi, India
§
Equal contributor
Abstract. Herein we have reported design, synthesis and in vitro biological evaluation of a
library of bicyclic lactams that led to the discovery of compounds 6 and 7 as a novel class of α-
glucosidase inhibitors. They inhibited α−glucosidase (yeast origin) in a mixed type of inhibition
with an IC50 of ~150 nM. Molecular docking studies further substantiated screening results.
Interestingly phenotypic screening of this library against the human malaria parasite revealed 7
as a potent antiplasmodial agent.
1
. Introduction.
1
-2
Diabetes mellitus is one of the most common non-communicable diseases of the globe. The
prevalence of diabetes is estimated to double by 2030 with 69% increase in developing countries
and 20% increase among adults in developed countries. Despite phenomenal progress in medical
3
science, diabetes continues to be a major killer.
Diabetes is a disease characterized by chronic hyperglycemia that leads to development of macro
4
and micro vascular complication. Hence, one of the therapeutic approaches in type 2 diabetes is
to reduce the demand for insulin by lowering the corresponding postprandial hyperglycemic
5
levels via inhibition of enzymes in the digestive organs such as the α-glucosidases. Research has
1

ACCEPTED MANUSCRIPT
shown that inhibition of α-glucosidase enzyme located at the intestinal brush border of the
5
intestine may play a role in the lowering of postprandial hyperglycemia.
To date, the only reported inhibitors of glycosidase enzymes are either complex natural products,
like Validoxylamine A, MDL 25637 (α-homonojirimycin-7-O-β-D-glucopyranoside), trehazolin,
acarbose, nojirimycin, castanospermineetc., or synthetic analogs like DNJ, N-butyl-DNJ, DANA
6
and others. Iminosugars and thiosugar derivatives are also one of the most promising
7
glycosidase inhibitors. However there are not very many non-sugar synthetic compounds as
8
-15
potent glycosidase inhibitors.
Hence, discovery of such molecules will have a niche appeal
and applicability in the pharmaceutical world.
Herein we report our investigation involving identification of a small molecule inhibitor of α-
glucosidase via routine glucosidase profiling of small molecule libraries unbiased towards any
specific targets. This exercise resulted in generating a potential hit compound 1 possessing
bicyclic lactam motif. Design and synthesis of a focused library of bicyclic lactams around this
motif with diverse functionalities followed by in vitro screening against yeast α-glucosidase
enzyme generated 6 and 7 as most potent compounds with IC50 ~150 nM. Reaction kinetics
suggested that they are mixed inhibitors.
In addition phenotypic screening against 3D7 strain of Plasmodium falciparum revealed decent
antiplasmodial activity of 7.
2
2
. Results and discussion
.1 Initial screening,
To begin with we subjected our in-house compound collection, unbiased towards any specific
targets in a routine glycosidase panel profiling. The exercise resulted in generating 1 as an
interesting hit molecule with IC50 of 2µM against yeast α-glucosidase. 1 was obtained by the
1
6
dihydroxylation of the previously reported cyclic ene-lactam 2.
2
.2 Library design and synthesis
In a bid to generate the library from 1 we envisioned 8, the diastereomer of 1, as a library
member followed by 6-membered bicyclic lactams 9 and 10 as the higher homologs of 1.
Additionally, we choose 3 and 5 where oxygen is located differently than in 1. Also included in
2

ACCEPTED MANUSCRIPT
the library are the dehydroxylated analog 6 and the unsaturated analogs 7 and 2 (the immediate
precursor of 1). We further envisioned that epoxide analogs 4 and 11 will streamline the SAR,
hence they were synthesized too (fig. 4). In a bid to diversify the aromatic moiety of the bicyclic
lactams, benzyl and indoline substituted bicyclic lactams 12 and 13 were incorporated. Finally
we envisioned a set of dimeric bicyclic lactams 14-17 in our library. By virtue of possessing
diverse biological activity the dimeric compounds have lately gained lot of importance in the
pharmaceutical world and that prompted their inclusion in this focused library (figure 1).
(Figure 1)
1
7-19
Synthesis of compounds 3, 4, 5 and 10 were accomplished following literature procedures.
Compound 2 was obtained from bicyclic lactam 18, by phenyl selenium bromide mediated
addition-reduction followed by oxidation of the arylselenium intermediate with hydrogen
peroxide. 18 in turn was obtained by condensing S-pyroglutaminol and benzaldehyde with
catalytic p-toluenesulfonic acid (PTSA) in refluxing toluene. Dihydroxylation of 2 in presence of
N-methylmorpholine-N-Oxide and osmium tetraoxide in 1:1 acetone: water afforded 1. In a
different reaction, epoxidation of 2 with meta-perbenzoic acid (mCPBA) resulted in 4. In a
similar effort compound 6 (synthesized from R-pyroglutaminol and benzaldehyde) was
converted to 7. And a similar dihydroxylation of 7 yielded 8. Epoxidation of 7 provided 11.
Simple condensation of S-phenylalaninol and S-Tryptophanol with 2-(2-nitrophenyl)-4-
oxopentanoic acid in toluene in catalytic p-toluene sulfonic acid furnished 12 and 13. Finally
oxidative homoenolate coupling of bicyclic lactams 5, 18, 19 and 20 in presence of lithium
bis(trimethylsilyl)amide (2.1 eq.) and 2.1 eq. of the oxidant iodine (I ) afforded 14-17. The
2
synthesis of the library was designed in a way where each member of the library can be obtained
in minimum number of steps and with high steps/molecule ratio (~1.7), thereby making the
library generation more facile (figure 2).
(Figure 2)
2
.3 In vitro assay against α-glucosidase (yeast origin)
We screened all the compounds against
α-glucosidase enzyme of yeast origin along with
acarbose, a known -glucosidase inhibitor, as the positive control. The percentage
α
inhibition on the α-glucosidase was measured at a concentration of 10 µM to 50 µM
3

ACCEPTED MANUSCRIPT
(
refer SI for experimental details). To assess the potency of our compounds, the
inhibitory activities were plotted against the concentrations using non-linear regression
approach (sigmoid curves) (cf. SI) from which their IC values were computed (Table 1).
50
Among the compounds screened,
6
and
7
exhibited strong inhibition against α-
M respectively, faring much better than
also bind effectively with IC ranging
glucosidase with IC s of 0.17 and 0.15
µ
50
acarbose control (900nM). Compounds
between 0.19 & 0.21
obtained their dose responses against
of -glucosidase was reduced by majority of our compounds (
reliant fashion thereby indicating their strong affinity towards the enzyme (Figure 3).
Table 1
Figure 3)
2
and
8
50
µ
M. To further probe the inhibitory effect of our compounds, we
-glucosidase. The results showed that the activity
and ) in a dose-
α
α
2,
6,
7
8
(
)
(
2.4 Molecular Docking and modelling
With the IC50 values in hand, we wanted to understand the binding interactions of the initial
compound 1 and most active compounds 6 and 7 with α−glucosidase. This would enable us to
design more active analogues of 1. Even though the X-ray crystallographic structure of α-
glucosidase has been reported for some bacteria, the three dimensional structural information is
not available for the yeast α-glucosidase enzyme. Hence we constructed the 3D structure of α-
glucosidase by homology modeling. The sequence of α-glucosidase was downloaded from
Uniprot (ID: P10253). BLASTP ® against protein data bank was performed in order to identify
the template for sequence alignment. Human Maltase-Glucoamylase (PDB ID: 2QLY, 3L4T)
2
0
were showing good similarity to our query sequence. Residues starting from 89 are aligning to
these PDB and showing 44% identity. These two protein structures were therefore selected to
model α-glucosidase using homology modelling. The homology model of α-glucosidase was
built using Modeller v9.14. Three models were generated using Modeller v9.14 and the model
2
1
having best DOPE score is selected for further studies. The energy of the model was minimized
using MOE energy minimization algorithm using Force Field MMFF94x. The stereochemical
quality of this model was validated by the Ramachandran plot using the PROCHECK program.
9
8.1% residues are in the favored and allowed regions, only 1.9% are outliers and no active site
residue lie in this outlier region. Further the model is also validated using verify 3D program
figure 3).
(
4

ACCEPTED MANUSCRIPT
(Figure 4)
Molecular modelling of 1 revealed that it was bound deeply into the binding cavity of α-
glucosidase and showed interaction with the residues Lys 609, Asp 724, Leu 613, Gln 688, Ile
7
26, Ala 610, Thr 725, Gln 575, Phe 579, Val 728 & Asp 686 residues. 1 was found to form a
hydrogen bond with the δ-amino moiety of Lys 609 and carbonyl group of amide moiety of Asp
24 (figure 2).
7
(Figure 5)
Next both compounds
6
and
7
were docked against the protein (figure 6 and 7).
6 and 7
bind to the active site residues of modelled
α
-glucosidase protein by forming one
hydrogen bond and few other non-bonded interactions. For 6, the hydrogen bond is
formed with Asp 686 residue while His 729, Asn 727, Val 728, Thr 725, Pro 722, Ile 726,
Gln 688, Leu 719, Pro 720 residues shared non-bonded interactions. Similarly
the active site residues of modelled -glucosidase protein by the hydrogen bond formed
with Asp 686 residue while Thr 725, Gln 688, His 729, Pro 722, Ile 726, Val 728, Asn
27, Leu 719, Pro 720 residues shared non-bonded interactions. and showed similar
binding with the receptor but the energy of the docked model is different. showed
stronger binding as compared to and but the stability of docked model is better in case
of and . This may be the reason why
Figures 6a and b).
7 binds to
α
7
6
7
1
6
7
6
7
6
and
7
demonstrated better activity than
1
(
(
Figure 6a
)
(
Figure 6b
)
2
.5 Reaction kinetics study
Enzyme kinetic assays were conducted with the most active compounds viz.
understand their mode of inhibition to
primary (Lineweaver-Burke) and secondary plots of inhibition kinetics (Figures 7 [a] and
6
and
7
to
α
-glucosidase. This was determined by employing
[
b]) for the compounds. As revealed from the Lineweaver-Burke plot, the data points of
6
and intersected in the second quadrant thereby indicating a mixed type of inhibition.
7
5

ACCEPTED MANUSCRIPT
The mixed inhibition is an intermediate of the competitive and uncompetitive inhibition
and can be represented as shown in the equation below (eq. 1).
(
Equation 1
were able to bind to either the free α-glucosidase (E) or the
α-glucosidase-substrate (pNPG) complex (E+S). The two inhibition constants in the
)
As mixed inhibitors,
6 and 7
mixed inhibition, K and K signified the equilibrium constant of dissociation of the
ia
ib
ꢀ
ꢁ ꢂ (inhibitor-enzyme) complex and the ꢀ ꢁ ꢂꢃ (inhibitor-bound enzyme substrate)
complex. These values for and were computed from the secondary plots of the slope
and Y-intercept from the Lineweaver-Burke plot versus inhibitor concentration (refer to
6
7
SI). K and K of
ia
ib
6
were determined to be 11.2µM & 17.5µM and those for
7
were
2
2.6µM & 33.7µM respectively. These constants show that both the compounds have
strong potency to bind to the free enzyme.
(
Figure 7)
2.5 Phenotypic screening against malaria phenotype (3D7).
Since -glucosidase inhibitors possess versatile mode of action against multiple
α
therapeutic areas (viz. cancer, viral infection, malaria and etc.) other than diabetes we
subjected the library of compounds to a phenotypic screening against the malaria parasite,
Compound
7 showed remarkable inhibition against 3D7 strain of Plasmodium
Falciparum). The parasites were treated at the ring stage and the percent parasitemia was
calculated at the trophozoite stage of the second cycle (i.e. after 72 hours of incubation).
The percent inhibition of invasion was calculated w.r.t the untreated control and nearly
6
5% inhibition was observed in presence of
7 (figure 8).
(
Figure 8
)
The fact that the same compound 7 showed activity against α-glucosidase and 3D7 strain of
malaria parasite forced us to rethink about any connection between α-glucosidase and malaria
parasite life cycle. However this is a preliminary result. Perhaps there is a plausible connection
between the two but no direct evidence for such an observation is evident yet. Much exhaustive
investigation is required to connect diabetes and malaria. It is an interesting avenue to explore for
6

ACCEPTED MANUSCRIPT
the medicinal chemists. Further work on this is ongoing in our lab
. Conclusion
.
3
Herein we have discussed design and synthesis of a focused library of compounds based on
bicyclic lactam 1. It was identified from a random unbiased screening of our in-house library. In
vitro screening of the library against yeast α-glucosidase generated two novel inhibitors 6 and 7,
with IC50 in the range of 150 – 200 nM. Reaction kinetics indicated that both of them have mixed
type inhibition against α-glucosidase. Molecular docking of 6 and 7 against the homology model
of α-glucosidase rationalized their activity. Interestingly, phenotypic screening of the library
yielded 7 as a potent inhibitor of 3D-7 strain of Malaria Parasite Plasmodium Falciparum. Both
antiplasmodial and anti α-glucosidase property of the 7, opens a possibility of development of
this compound as a potential treatment option for both the diseases.
4. Materials and methods
General Experimental
Reagents were obtained from Aldrich Chemical (www.sigma-aldrich.com) or Acros
Organics (www.fishersci.com) and used without further purification. Optima grade
solvents were obtained from Fisher Scientific (www.fishersci.com), degassed with Argon,
and purified on a solvent drying system as described, unless otherwise indicated. Lithium
hexamethyl disilylamide (LHMDS) was obtained from Aldrich in SureSeal bottles.
Phenyl selenium bromide was obtained from Acros. Compounds
6
and
3-5, 9-11 and 18-
17-19
20 are prepared from the literature procedure.
The single crystal X-Ray of 11a (the
amine compound from 11), confirms the relative configuration of 11. All reactions were
performed in flame-dried glassware under positive Argon pressure with magnetic stirring
unless otherwise noted. Rubber septa and syringes were used for the transfer of liquid
reagents and solutions. Syringes were purged with argon prior to use. TLC was performed
on 0.25 mm E. Merck silica gel 60 F254 plates and visualized under UV light (254 nm) or
by staining with potassium permanganate (KMnO
iodine (I ), or -anisaldehyde. Silica flash chromatography was performed on E. Merck
30-400 mesh silica gel 60. IR spectra were recorded on a Perkin Elmer Spectrum 100
4
), phosphomolybdic acid (PMA),
p
2
2
FTIR spectrometer with peaks reported in cm-1. NMR spectra were recorded on Varian
7

ACCEPTED MANUSCRIPT
4
00MR and Varian 300 instruments at 24°C in CDCl
3
unless otherwise indicated. Spectra
were processed using VNMR J 3.2 software, and chemical shifts are expressed in ppm
1
1
13
relative to TMS ( H, 0 ppm) or solvent signals: CDCl ( H, 7.26; C, 77.0 ppm);
3
coupling constants are expressed in Hz. Mass spectra were obtained at the Shiv Nadar
University Analytical Facility on an Agilent 6540QTOF by electrospray (ESI) ionization.
(
3R,7aS)-3-phenyl-1,7a-dihydropyrrolo[1,2-c]oxazol-5(3H)-one (2).
To the solution of bicyclic lactam 18 (900mg, 1eq) in THF (9mL) was added 1M solution of
LiHMDS in THF (8.8mL, 2eq) at -78°C, pale yellow solution was allowed to stir 30 min. After
that solution of PhSeBr (1.15g, 1.1 eq.) in THF was added drop-wise over 10 minutes. Reaction
mixture was further stirred for 30 min at -78°C. Reaction progress was monitored by TLC.
Reaction mixture was quenched by adding saturated aqueous NH Cl solution, and then warmed
4
to rt and extracted in diethyl ether (50 mL*3). The organic extracts were separated and
concentrated to afford phenyl selenylated product.
Phenyl selenylated product dissolved in dichloromethane (40 mL) and treated with 30%
H
2
O
2
(6 eq.) at 0°C. The resulting heterogeneous mixture was allowed to warm to rt while
stirring vigorously for 16 h. The aqueous phase was extracted with CH Cl , and the
2
2
organic extracts were combined and washed successively with 1N HCl and saturated
aqueous NaHCO . The organic phase was dried (Na SO ) and concentrated under reduced
3
2
4
pressure to afford a crude compound, which further purified by column chromatography
1
(
(
30% EA in
n
-Hexane) to afford alpha beta unsaturated compound
): 7.57-7.35 (m, 7H), 6.18-6.17 (d,
.78-4.74 (t, J1 = 7.6 Hz, J2 = 15.6 Hz, 1H), 4.27-4.24 (t, J1 = 7.2 Hz, J2 = 15.2 Hz, 1H),
2
(600mg). H NMR
300 MHz, DMSO-D
6
δ
J = 5.2 Hz, 1H), 5.95 (s, 1H),
4
3
1
13
.34-3.30 (m, 1H). C NMR (75 MHz, DMSO-D
6
) 176.95, 147.82, 138.51, 129.17,
+
28.61, 128.42, 126.13, 87.37, 68.04, 65.11 ppm. AQUITY UPLC (MS ESI): [M + H]
calcd for C H NO 202.22, found 202.10
12
11
2
(
3R,6R,7R,7aR)-6,7-dihydroxy-3-phenyltetrahydropyrrolo[1,2-c]oxazol-5(3H)-one
Compound (200 mg, 1 eq.) and 50% solution of acetone in water (4mL) purged with
nitrogen and then it was charged with 50% aqueous N-methylmorpholine-N-oxide
NMO) (0.4mL) and 2% solution of OsO in butanol (0.4mL) at 25°C. Reaction mixture
(1).
2
(
4
was stirred for 16h at rt. Once TLC indicates complete consumption of starting material
8

ACCEPTED MANUSCRIPT
2 3
the reaction mixture was quenched with aq. solution of Na SO . It was extracted with
ethyl acetate (4 X 25mL), Organic layer dried over sodium sulphate and distilled off on
1
rotary evaporator to afford white solid compound
DMSO-D ):
1
(150mg). H NMR (300 MHz,
6
δ
7.40-7.34 (m, 5H), 6.08 (s, 1H), 5.88-5.87 (m, 1H), 5.52-5.38 (m, 1H),
13
4
.27-4.22 (m, 1H), 4.08-3.96 (m, 3H), 3.95-3.66 (m, 1H). C NMR (75 MHz, DMSO-D )
6
1
74.78, 137.22, 128.92, 128.58, 125.99, 110.01, 87.00, 74.23, 71.00, 69.24, 65.07 ppm.
+
20
AQUITY UPLC (MS ESI): [M + H] calcd for C12
46.3 (c 1, CH OH)
3S,7aR)-3-phenyl-1,7a-dihydropyrrolo[1,2-c]oxazol-5(3H)-one
4 D
H13NO 236.24, found 236.14. [α]
+
3
(
(
7
).
, Reaction done on 800 mg scale from
): 7.61-7.59 (d, J = 8Hz, 2H),
Reaction procedure is same as
2
8 to afford 500mg
1
of desired compound (
7
). H NMR (300 MHz, DMSO-D
6
δ
7
1
=
.41-7.28 (m, 3H), 7.26-7.19 (m, 1H), 6.19- 6.17 (m, 2H), 4.65-4.61 (t, J1 = 7.2 Hz, J2 =
5.2 Hz, 1H), 4.29-4.21 (dd, J1 = 7.6 Hz, J2 = 17.2 Hz, 1H), 3.45-3.41 (t, J1 = 8.4 Hz, J2
13
16.8 Hz, 1H). C NMR (75 MHz, DMSO-D ) 176.94, 150.59, 139.07, 128.54, 128.33,
6
+
1
27.75, 126.39, 86.95, 67.88, 65.27 ppm. AQUITY UPLC (MS ESI): [M + H] calcd for
C
12
H
11NO
2
202.22, found 202.05
3S,6S,7S,7aS)-6,7-dihydroxy-3-phenyltetrahydropyrrolo[1,2-c]oxazol-5(3H)-one
Reaction procedure is same as , reaction done on 230mg scale to afford 220mg of
generate desired compound as white solid. H NMR (300 MHz, DMSO-D
(
(8).
1
8
to
7.38-7.37
m, 5H), 6.08 (s, 1H), 6.04-5.89 (m, 1H), 4.52-4.49 (m, 1H), 4.08-3.69 (m, 2H), 3.67-3.33
1
6
): δ
(
(
1
3
m, 1H). C NMR (75 MHz, DMSO-D ) 175.55, 175.24, 138.94, 138.68, 128.58, 128.45,
6
1
28.37, 126.13, 86.29, 86.20, 74.82, 73.78, 71.01, 70.03, 69.22, 64.40, 64.18, 58.81ppm.
+
20
AQUITY UPLC (MS ESI): [M + H] calcd for C H NO 236.24, found 236.0. [
α
]
-
D
12
13
4
4
0.3 (c 1, CH OH)
3
(3S,6R,7aR)-3-benzyl-7a-methyl-6-(2-nitrophenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one
(12).
2-(2-nitrophenyl)-4-oxopentanoic acid (12 g, 50.6 mmol), (S)-phenyl alaninol (7.3 g, 52
mmol) and PTSA (12 mg, cat) were dissolved in toluene (120 mL). The flask was
equipped with a Dean-Stark trap, and the solution was heated to reflux. After 16 h the
3
solution was cooled, washed with saturated aqueous NaHCO , dried, and concentrated.
Purification by flash column chromatography on silica gel (hexane/EtOAc, 1:1) generated
9

ACCEPTED MANUSCRIPT
the desired compound which was triturated with diethyl ether to afford 12 (7.4 g, 41.5%)
1
as white solid. H NMR (300 MHz, DMSO-D
6
):
δ
7.98-7.95 (d,
J
= 8.1 Hz ,1H),7.59-7.36
(
m, 3H), 7.32-7.22 (m, 6H), 7.15-7.02 (m, 1H), 4.68-4.48 (m, 1H), 4.40-4.28 (m, 1H),
.25-4.16 (t, J1 = 8.7 Hz, J2 = 18Hz, 1H) , 4.01-3.89 (m, 1H), 3.15-3.11(m, 1H), 2.88-
4
2
1
1
9
13
.72 (m, 2H), 2.49-2.41 (t, J = 12Hz, 1H), 1.55 (s, 3H). C NMR (75 MHz, DMSO-D )
6
75.28, 149.58, 136.93, 133.24, 132.23, 131.44, 129.62, 129.32, 128.57, 128.35, 126.76,
26.54, 125.46, 125.15, 124.26, 122.67, 122.57, 120.47, 120.25, 119.95, 111.78, 111.38,
7.18, 55.89, 47.98, 44.36, 39.99, 27.72, 25.02 ppm. AQUITY UPLC (MS ESI): [M +
+
20
H] calcd for C H NO 353.39, found 353.30. [
α
]
+21.5 (c 1, CH OH)
D 3
12
13
4
(
3R,6S,7aS)-3-((1H-indol-3-yl)methyl)-7a-methyl-6-(2-nitrophenyl)tetrahydropyrrolo[2,1-
b]oxazol-5(6H)-one (13).
-(2-nitrophenyl)-4-oxopentanoic acid (6 g, 25 mmol), (S)-tryptophanol (4.94 g, 26
2
mmol) and PTSA (12 mg, cat) were dissolved in toluene (120 mL). The flask was
equipped with a Dean-Stark trap, and the solution was heated to reflux. After 16 h the
3
solution was cooled, washed with saturated aqueous NaHCO , dried, and concentrated.
Purification by flash column chromatography on silica gel (hexane/EtOAc, 1:1) generated
the desired compound which was triturated with diethyl ether to afford 13 (3.4 g, 35%) as
1
colourless solid. H NMR (300 MHz, DMSO-D
6
):
= 1.6Hz, 1H), 7.17-7.13 (m, 1H), 7.10-7.06
= 1.2Hz, 1H), 4.92 (s, 2H), 4.34-4.31 (m, 2H), 4.20-4.11 (m, 1H),
.93-3.89 (m, 1H), 3.17-3.08 (m, 1H), 2.96-2.91 (m, 1H), 2.10-2.04 (m, 1H), 1.51 (s, 3H).
δ 10.90 (s, 1H), 7.59-7.56 (m, 1H),
7.37-7.35 (d,
J = 8Hz, 1H), 7.29-7.28 (d, J
(
m, 1H), 7.01-6.98 (t,
J
3
+
20
AQUITY UPLC (MS ESI): [M + H] calcd for C H N O 392.42, found 392.78. [α]
D
22
24
3
4
+
11.3 (c 1, CH OH)
3
Procedure for the synthesis of dimeric bicyclic lactam 14:
(
3aR,3'aR,4aS,4'aS,9bR,9'bR)-3a,3'a-dimethyl-3,3a,3',3'a,4a,4'a,5,5'-octahydro-1H,1'H-
2,2'-biindeno[1,2-d]pyrrolo[2,1-b]oxazole]-1,1'(2H,2'H,9bH,9'bH)-dione 14).
[
(
To a solution of bicyclic lactam 19 (10.0 g, 43.6 mmole) in THF (100 ml) was added
LiHMDS (1.0 M solution in THF, 47.97 ml, 47.97 mmole) at -78°C in a drop wise
manner. The reaction mixture was stirred for 30 min at that temperature. A solution of
10

ACCEPTED MANUSCRIPT
iodine (11.07g, 43.6 mmole) in THF(10 ml) was added to the reaction mixture in drop
wise manner over a period of 30 min. The reaction mixture was further stirred for 2 hours
at -78°C and the warmed upto room temperature. Reaction progress was monitored by
thin layer chromatography [eluent: 30% EA in
n-hexane]. After completion of the
reaction, the reaction mixture was cooled to -78°C and quenched by saturated ammonium
chloride solution. The crude product was extracted in ethyl acetate (200 ml*2). Organic
layer was washed with aqueous sodium thiosulphate. 5H O solution (250 ml), water (250
2
ml) and brine solution (250 ml). And then dried over sodium sulphate and distilled off on
a rotary evaporator to afford the crude solid which was further purified by column
chromatography (silica:100-200 mesh, eluent: 16% ethyl acetate in
n
-hexane) to afford
7.41-7.31
= 8Hz, 1H), 4.92-4.89 (m, 1H), 3.49-
1
off white solid compound (9.5 g, 47.7%). H NMR (300 MHz, DMSO-D ):
δ
6
(
t,
J
= 8Hz, 1H), 7.29-7.23 (m, 4H), 5.68-5.66 (d,
J
13
3
.45 (m, 1H), 3.28-3.14 (m, 2H), 2.33-2.28 (m, 1H), 2.11-1.60 (m, 1H), 1.03 (s, 3H). C
NMR (75 MHz, DMSO-D ) 179.2, 131.2, 130.4, 130.2, 126.6, 125.8, 72.1, 58.5, 52.3,
6
+
4
6.4, 42.8, 21.2 ppm. AQUITY UPLC (MS ESI): [M + H] calcd for C H N O 457.53,
28
28
2
4
20
found 457.2. [
α
]
+34.8 (c 1, CH OH).
D 3
(3R,3'R)-3,3'-diphenyloctahydro-[6,6'-bipyrrolo[2,1-b]oxazole]-5,5'(6H,6'H)-dione (15).
The above procedure was followed with bicyclic lactam
LiHMDS (2.75mL), I
CDCl ):
.87-3.83 (m, 2H), 3.64-3.59 (m, 1H), 2.68-2.60 (m, 2H), 1.99-1.92 (m, 2H). C NMR
5
(0.5g, 2.46mmol), 1M
1
2
(0.63g, 2.46 mmol) and THF (10mL). H NMR (300 MHz,
3
δ 7.38-7.28 (m, 10H), 5.31-5.30 (m, 2H), 5.29-5.08 (m, 2H), 4.60-4.56 (m, 2H),
1
3
3
(
75 MHz, CDCl ) 181.2, 128.2, 126.4, 126.3, 124.2, 67.3, 62.8, 61.9, 60.4, 42.1, 40.1,
3
+
2
4
8.1, 24.2 ppm. AQUITY UPLC (MS ESI): [M + H] calcd for C H N O 405.46, found
24
24
2
4
05.0.
(3R,3'R,7aR,7'aR)-7a,7'a-dimethyl-3,3'-diphenyloctahydro-[6,6'-bipyrrolo[2,1-b]oxazole]-
5
,5'(6H,6'H)-dione (16)
The above procedure was followed with bicyclic lactam 20 (0.5g, 2.30mmol), 1M
1
LiHMDS (2.75mL), I (0.6g, 2.30 mmol) and THF (10mL). H NMR (300 MHz, CDCl ):
2
3
11

ACCEPTED MANUSCRIPT
δ
7.37-7.33 (t, = 8Hz, 15.2Hz, 1H), 4.62-
J
= 8Hz, 2H), 7.29-7.24 (m, 2H), 5.14-5.11 (t,
J
4.57 (t, J = 8.4 Hz, 16.8Hz, 1H), 4.11-4.07 (t, J = 7.2 Hz, 16Hz, 1H), 3.70-3.66 (t, J = 8.8
13
Hz, 17.6Hz, 1H), 2.45-2.40 (m, 1H), 2.21-2.15 (m, 1H), 1.49 (s, 3H). C NMR (75 MHz,
+
CDCl ). AQUITY UPLC (MS ESI): [M + H] calcd for C H N O 433.5, found 433.0.
3
26 28
2
4
(3S,3'S)-3,3'-diphenyloctahydro-[6,6'-bipyrrolo[1,2-c]oxazole]-5,5'(3H,3'H)-dione (17)
The above procedure was followed with bicyclic lactam 18 (0.5g, 2.46mmol), 1M
1
LiHMDS (2.75mL), I (0.63g, 2.46 mmol) and THF (10mL). H NMR (300 MHz,
2
CDCl3):
δ 7.50-7.26 (m, 10H), 6.36 (s, 2H), 4.39-4.36 (m, 2H), 4.19-4.16 (m, 2H), 3.41-
13
3
.40 (m, 4H), 3.39-3.25 (m, 2H). C NMR (75 MHz, CDCl ). AQUITY UPLC (MS
3
+
28 2 4
ESI): [M + H] calcd for C26H N O 405.46, found 405.1.
In vitro assay of α-Glucosidase inhibitory activity
The inhibitory potency of the compounds against α-glucosidase activity was assessed in 96-
well plates using PNPG (4-nitrophenyl α-D-glucopyranoside) as a substrate according to the
2
2
procedure reported by Ferreres et al. Prior to screen, all the test compounds were solubilized in
a suitable solvent, Dimethylsulfoxide (DMSO) and eventually diluted to attain the desired
concentration. Briefly, each well was comprised of 100µL of substrate (2mM, PNPG dissolved
in 2mM phosphate buffer at a pH of 7.2) and different test concentrations (10-100 µM). Then the
final volume of the reaction mixture was made up to 200µl with 2mM phosphate buffer (pH 7.2).
The hydrolytic reaction was commenced by the addition of α-glucosidase enzyme (0.5 IU/mL)
(obtained from Sigma Aldrich, Bangalore) and the plates were incubated at 37°C for 15 min. The
reaction was terminated by the addition of 50µL of 2N Na CO solution. The absorbance was
2
3
measured spectrophotometrically at 400 nm (Epoch reader; version 2.00.18). The
decrease/increase in absorbance (∆A) was compared with that of control (buffer instead of test
compound) to compute the inhibitory profile of enzyme. The data used for the determination of
IC50 concentrations were fitted by non-linear regression fitting and the variance analysis was
carried out by using MINITAB 15 software (trail version). The concentration of inhibition
required for 50% of α-glucosidase activity under the assay conditions was defined as the IC50
value. The half maximal inhibitory (IC ) concentrations were determined from two independent
5
0
12

ACCEPTED MANUSCRIPT
assays, performed in duplicate. Acarbose, an eminent α-glucosidase inhibitor, was employed as a
positive control.
Inhibition (%) = ∆Acontrol -∆Asample)/ ∆Acontrol x 100%
One unit (IU) is defined as the amount of enzyme (α-glucosidase) which produces 1 µmol of
PNP (p-nitro phenol) per min at 37°C and pH 7.2 under the conditions described above.
Inhibition-kinetic studies
The kinetic mode of inhibition of selected active compounds against α-glucosidase was
determined as said above by preparing a series of test solutions in which the concentration of the
substrate (PNPG) was varied in the presence of different concentrations of the inhibitors (10µM-
1
00µM). The mode of inhibition (i.e. competitive, non-competitive, uncompetitive or mixed-
type) of the test compounds was evaluated on the basis of the inhibitory effects on Km
2
3
(
dissociation constant) and Vmax (maximum reaction velocity) of the enzyme. This was
determined using the primary (Lineweaver-Burke plot) plot, which is the double reciprocal plot
of enzyme reaction velocity (V) versus substrate (pNPG) concentration (1/V versus 1/[pNPG]).
Analysis of the same data by secondary plots of slope versus [Inhibitor] and Y-intercept versus
[
Inhibitor] were also performed. The Lineweaver-Burke equation follows as,
1
ꢄ
1
ꢊꢇ
1
ꢅ
ꢁ
ꢋ
ꢆ
ꢆ
ꢇꢈꢉ
ꢌSꢍ
ꢇꢈꢉ
Malaria-growth inhibition assays (GIA):
P. falciparum culture 3D7 was maintained in vitro at pH 7.4 in plastic petri-dishes using human
+
O erythrocytes, at 4% hematocrit, in RPMI-HEPES medium supplemented with 5% Albumanx,
5
0 µg/ml hypoxanthine, 25 mM NaHCO3 and 20 µg/ml gentamicin maintained in an atmosphere
2
4
of 1% O2, 4% CO2, and 95% N2 at 37°C, as previously described (Ref). Parasite cultures were
synchronized using the sorbitol treatment. The GIA was put at ring stage at 0.3% parasitemia and
2
% hematocrit in 96-well plates that were incubated in a sealed, humidified, gassed box. The
assay plate was read after 72 hours i.e., at the trophozoite stage of the second cycle of invasion.
0 compounds were tested at two different concentrations of 10 µM and 50 µM in the GIAs.
1
Parasitemia was determined using flow cytometry. For flow cytometry, 100 µl of 10-µg/ml
ethidium bromide in phosphate-buffered saline (PBS; pH 7.3) was added to each well and
incubated for 1 h in dark at room temperature. After centrifugation, the supernatant was
discarded, cells were washed with PBS and re-suspended in 200 µl/well of PBS, and the samples
13

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were processed using a FACS Calibar flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
Parasitemia was evaluated using FlowJo software (Tree Star, Inc., Ashland, OR) by first gating
for intact erythrocytes by side scatter and forward scatter parameters and subsequently
determining the proportion of ethidium bromide-positive cells indicating the percent parasitemia.
All compounds were tested in duplicate.
5
. Acknowledgement
We thank Shiv Nadar University for the support
. References
6
1
2
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Satya, J. K. Kumar, F. Khan, A. K. Tiwari, G. Paramjit, Synthesis, biological evaluation
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0. S. Wang, J. Yan, X. Wang, Z. Yang, F. Lin, T. Zhang, Synthesis and evaluation of the α-
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2. K. Takao, R. Ishikawa, Y. Sugita, Chemical and Pharmaceutical Bulletin, 62 (2014), 810-
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5. T. Damsud, S. Adisakwattana, P. Phuwapraisirisan, Three new phenylpropanoyl amides
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(
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7
. Figure Captions
Figure 1. The library of bicyclic lactams designed around 1
Figure 2. The synthetic scheme to access the small molecules depicted in the design (figure 1)
Table. 1. The in vitro activity of the library of pyrrolidines against yeast α-glucosidase
Figure 3. Inhibition of α-glucosidase enzyme activity as a function of inhibitors (from 10
µM to 50 µM) with Acarbose, as a reference standard
Figure 4. Ramachandran plot of the modelled
α
-glucosidase enzyme
-glucosidase enzyme
Figure 5. Predicted binding mode of in modelled
1
α
Figure 6a. Predicted binding mode of
Figure 6b. Predicted binding mode of
Equation 1. Mixed inhibition
6
7
in modelled
in modelled
α
α
-glucosidase enzyme
-glucosidase enzyme
Figure 7. Double-reciprocal (Lineweaver-Burke) plot of the inhibition kinetics of α-
glucosidase by compound 6 (A) and 7 (B); α-Glucosidase (activity 0.5IU/mL) was
subjected with compound
-nitrophenyl α-D-glucopyranoside (PNPG). IU: Defined as the amount of enzyme (
glucosidase) which produces 1
under the conditions described in SI
Figure 8. Malaria parasite growth inhibition assay
6 & 7 at 37°C for 10min, followed by varying concentrations of
4
α
-
µ
mol of PNP (p-nitrophenol) per minute at 37°C and pH
.
17

ACCEPTED MANUSCRIPT
Figure 1. The library of bicyclic lactams designed around 1
1
8

ACCEPTED MANUSCRIPT
1
9

ACCEPTED MANUSCRIPT
Figure 2. The synthetic scheme to access the small molecules depicted in the design (figure 1)
Table. 1. The in vitro activity of the library of pyrrolidines against yeast α-glucosidase
α-glucosidase
Entry
Compound
(yeast)
a
IC (µmol)
50
1
2
3
4
5
6
7
8
9
2
0.19
c
3
-
53
-
4
c
5
6
7
8
0.17
0.15
0.21
-
c
9
1
1.70
-
c
1
1
1
1
1
1
1
1
1
0
1
3
4
5
6
7
8
9
10
11
95
-
12
13
213
-
14
15
-
16
-
17
121
0.093
b
Acarbose
a
Concentration of compound that reduced enzyme activity by 50% in an Activity
(
The values furnished were the means of two independents)
The concentration was observed in our laboratory assay method
b
c
>
50� 50 concentration was observed in our laboratory
20

ACCEPTED MANUSCRIPT
Figure 3. Inhibition of α-glucosidase enzyme activity as a function of inhibitors (from 10
µM to 50 µM) with Acarbose, as a reference standard
21

ACCEPTED MANUSCRIPT
Figure 4. Ramachandran Plot of our α-glucosidase homology model
22

ACCEPTED MANUSCRIPT
Figure 5. Predicted binding mode of 1 in modeled protein in a 3D representation along with 2D
representation
23

ACCEPTED MANUSCRIPT
Figure 6a. Predicted binding mode of 6 in modelled protein
Figure 6b. Predicted binding mode of 7 in modelled protein
24

ACCEPTED MANUSCRIPT
Equation 1. Mixed inhibition
Figure 7. Double-reciprocal (Lineweaver-Burke) plot of the inhibition kinetics of α-
glucosidase by compound 6 (A) and 7 (B); α-Glucosidase (activity 0.5IU/mL) was
subjected with compound
-nitrophenyl α-D-glucopyranoside (PNPG). IU: Defined as the amount of enzyme (
glucosidase) which produces 1
under the conditions described in SI
6 & 7 at 37°C for 10min, followed by varying concentrations of
4
α
-
µ
mol of PNP (p-nitrophenol) per minute at 37°C and pH
.
25

ACCEPTED MANUSCRIPT
Figure 8. Malaria parasite growth inhibition assay
26

ACCEPTED MANUSCRIPT
Graphical Abstract
A bicyclic lactam has been discovered as a novel mixed inhibitor of α-glucosidase (yeast)
enzyme that also inhibits 3D-7 strain of Plasmodium Falciparum.
27

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
•
We executed virtual screening of a library against a homology model of α-glucosidase.
We designed and synthesized a molecular library.
In vitro screening identified two compounds as potent α-glucosidase inhibitors
Reaction kinetics suggested mixed type inhibition.
Phenotypic screening of one of the compounds also indicated antimalarial properties
28

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of small molecules as potent glycosidase
inhibitors and antimalarials
Analysis of novel compounds
1
5
5
2
Sanjay M. Madurkar, Santanu Hati, Chandramohan Bathula, Chiranjeevi Thulluri, Rahul
,3
4
3
2
3
3
Agarwal Faiza Baig, Poonam Dangi, Uma Adepally, Ashutosh Singh, Shailja Singh and
5
Subhabrata Sen*
1
2
Department of Chemistry, Mahatma Gandhi University, Ri Bhoi, Meghalaya, India
Institute of Science, Jawaharlal Nehru Technical University, Kukatpally, Hyderabad,
Telengana, India
3
Department of Life Science, Shiv Nadar University, PO Shiv Nadar University, Uttar Pradesh,
India
4
International Centre for Genomics Engineering and Biotechnology, New Delhi, India
5
Department of Chemistry, Shiv Nadar University, PO Shiv Nadar University, Uttar Pradesh,
India