Synthesis, molecular modeling and BACE-1 inhibitory study of tetrahydrobenzo[b] pyran derivatives

Article abstract of DOI:10.1016/j.bioorg.2018.11.023

β-Secretase (BACE1) has been broadly documented as one of the possible therapeutic targets for the treatment of Alzheimer's disease. In this paper, we report the synthesis and the for β-secretase (BACE-1) inhibitory activity of new series of tetrahydroben

Full text of DOI:10.1016/j.bioorg.2018.11.023

BioorganicChemistry84(2019)202–210
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, molecular modeling and BACE-1 inhibitory study of
tetrahydrobenzo[b] pyran derivatives
⁎
Vijaya Bhaskara, , Reshma Chowdaryb, Sheshagiri R. Dixitc, Shrinivas D. Joshic
a Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576104, India
b Department of Medicinal Chemistry, BITS-Pilani Hyderabad Campus, Hyderabad 500078, India
c Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry, SET’s College of Pharmacy, Dharwad 580002, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
β-Secretase (BACE1) has been broadly documented as one of the possible therapeutic targets for the treatment of
Alzheimer’s disease. In this paper, we report the synthesis and the for β-secretase (BACE-1) inhibitory activity of
new series of tetrahydrobenzo [b] pyran derivatives. One-pot synthesis of tetrahydrobenzo [b] pyrans was
carried out by condensing aromatic aldehyde, malononitrile and 1,3-cyclohexanedione using ionic liquid 1-
butyl-3-methyl imidazolium chloride ([bmIm]Cl−) in aqueous alcohol media. The addition of alcohol and water
in the ratio of 1:2 keeps all the reactants in solution which facilitates the reaction and makes the product
formation very easy. The synthesized compounds were subjected to BACE1 inhibition assay and six compounds,
4d, 4e, 4f, 4h, 4i, and 4p have shown significant IC50 values at micromolar level. Among these six active
compounds, 4e was a potential inhibitor with its IC50 value in nanomolar range. All the synthesized compounds
were docked onto the active site of β-Secretase enzyme.
Tetrahydrobenzo [b] pyrans
Ionic liquid
1-Butyl-3-imidazolium chloride
β-secretase
Surflex dock
Molecular docking
1. Introduction
2 (Asp2). It is an important enzyme found early in the cascade of bio-
logical events leading to the progression of the disease [1,4–12]. A
Alzheimer’s disease (AD) is one of the neurodegenerative disorders
of the central nervous system and most common cause of dementia
among old age people. Dementia is a serious brain disorder that affects
daily life and activities through loss of memory, cognitive deficits and
psychiatric symptoms. It is the fourth major cause of death in the de-
veloped world after heart disease, cancer and stroke [1]. Nearly 40
million people worldwide are suffering from AD, out of which 5.5
million are from America and every 70 s someone in America develops
the Alzheimer’s disease. It is expected that by 2050 this disease will
affect 1 in 85 people globally (see Fig. 1).
rational research attempt has been made to reduce Aβ levels in the
brain and it has become an attractive advance for the development of
drugs to curtail AD [13,14].
First identified in 1999, BACE1 belongs to the type-I class of as-
partyl proteases. BACE1 is now considered as the favoured therapeutic
target for reducing brain Aβ levels in the prevention or treatment of AD.
Till date, no molecule has received FDA approval for the clinical
trials. However many molecules have been found with promising
clinical potential inhibitory activity. The potentially invented molecules
have been classified as peptide-mimetic and non peptidic-inhibitors
[15–17]. The research was mainly carried out to investigate for the non-
peptide inhibitors with smaller in size, with less peptidic character,
better metabolic stability, and also better blood-brain-barrier penetra-
tion ability. Many small organic scaffolds have been identified through
computational studies which have BACE1 inhibitory activity [5,18–20].
Based on these studies a small attempt has been made to investigate
further BACE1 inhibition by using tetrahydrobenzo [b] pyran deriva-
tives.
Presently, a range of methods of treatment are aimed to manage
only the symptoms of the disease. No permanent therapy is available for
AD patients. The conclusion of the new research says [2–4] that, the
deposition of an increased level of amyloid-β (Aβ) plaques in the brain
over a long period is a leading cause of AD. Amyloid-β is produced in
neurons from amyloid precursor protein (APP) by the action of an as-
partic protease enzyme called β-secretase. This enzyme is also known as
β-site amyloid precursor protein cleaving enzyme1 (BACE 1) or mem-
brane-associated aspartic protease 2 (memapsin-2) or aspartyl protease
⁎ Corresponding author.
E-mail address: bhaskerkv@gmail.com (V. Bhaskar).
https://doi.org/10.1016/j.bioorg.2018.11.023
Received 3 March 2018; Received in revised form 16 November 2018; Accepted 17 November 2018
Availableonline19November2018
0045-2068/©2018ElsevierInc.Allrightsreserved.

V. Bhaskar et al.
BioorganicChemistry84(2019)202–210
synthesis by using ionic liquids are simple procedure, short reaction
time, excellent yields, room temperature conditions, and easy work-up
procedure. This is
a one pot, three-component model of cyclo-
condensation which involves knoevenagel condensation reaction.
A mixture of an aldehyde, 1, 3-cyclohexanedione (for 4a-4s com-
pounds) and dimedone (for 6a-6t compounds) and malononitrile (ac-
tive methylene compound) was stirred at room temperature in the
presence of an ionic liquid, 1-butyl-3-methyl imidazolium Bromide
([bmIm]Br), for a period of 40–60 min based on the type of aromatic
aldehyde used. The substituents of aromatic aldehydes affect the reac-
tion times and the yield of products. When aromatic aldehydes with
electron-withdrawing groups (such as nitro and halogens) are used the
reaction time shorter than with electron-donating groups (such as
methoxy and hydroxyl groups). Recrystallization of the crude product
from aqueous methanol afforded pure needle shaped crystals of tetra-
hydrobenzo[b]pyrans. The compounds of Scheme 2 were purified by
column chromatography using silica.
Fig. 1. All molecules docked at the active site of the enzyme PDB ID 4L7G.
2. Results and discussion
A variety of substituted aryl and hetero-aryl aldehydes underwent
condensation with active methylene compounds (malononitrile) and 1,
3-cyclohexanedione (or dimedone) by this procedure to provide the
corresponding 4-aryl substituted tetrahydrobenzo[b]pyrans. Their
physical and spectral data are discussed as given below.
2.1. Chemistry
The general procedure for the synthesis of target compounds 4a-4s
and 6a-6t are given in Schemes 1 and 2 and the list of synthesized
compounds are given in Table 2 and 3. The experimental procedure
adopted here is very facile and convenient. The attractive features of
All these reactions are fast and high yielding (85–95%) compared to
the other existing procedures. The recrystallized product gave only a
single spot in TLC. Only 0.05 M of catalyst was sufficient for every one
Scheme 1.
203

V. Bhaskar et al.
BioorganicChemistry84(2019)202–210
Scheme 2.
molar each of the starting materials. The reaction conditions were mild
(only at room temperature) accepting several functional groups, such as
eCH3, eF, eCl, eBr, eOCH3, eNO2, present in molecules. The highly
sensitive functional aldehydes like furyl and thienyl (hetero aldehydes)
participated in this reaction without any difficulty. All the compounds
could be satisfactorily characterized by their spectroscopic (IR, 1H, 13C
NMR) data.
were shown IC50 values in micro molar level as shown in Table 6 and
only one compound 4e had shown in nano molar level.
2.3. Molecular docking studies
All the conformations were minimized using Tripos force field as
shown in Fig. 1. The atomic charges were calculated using MMFF94
(Merck Molecular Force Field) method, while Amber7FF02 was used for
the protein. As depicted in Fig. 2, AKB 20 makes four H-bonding in-
teraction with the amino acid ASP228 i.e., Hydrogen of NH2 at the
ortho-position of coumarin ring interacts with oxygen of amino acid
ASP228 (H-NH2———ASP228, 2.24, 2.55, 2.66 and 2.08 Å), nitrogen
of CN at the meta-position of coumarin ring makes H-bonding with
hydrogen of amino acid THR232 (N-CN————THR232, 2.90 Å) and
oxygen of phenoxy ring at the para-position of coumarin ring makes
hydrogen bonding with amino acid THR232 (O———THR232, 1.93 Å)
The results of molecular docking are listed in Table 1.
2.2. BACE1 in-vitro assay
The percentage inhibitory activities of all the synthesized com-
pounds obtained by subjecting them to in-vitro β-Secretase enzyme in-
hibition assay are given in Table 5.
Initially, the test was performed at 25 μM concentration for all the
compounds. The compounds which showed percentage inhibitions
ranging from 65.4 to 98.8% have been considered for further studies.
The remaining compounds were ignored due to abnormal results or
because of low inhibition.
The compounds which gave percentage of inhibition between 65.4
and 98.8% at 25 μM, were subjected them again for testing at 100 µM,
50 µM, 10 µM, 5 µM, 1 µM, 0.5 µM, 0.1 µM and 0.05 µM. The percen-
tages of inhibition obtained from the above tests have been used to
calculate the IC50 values. The IC50 values were calculated by plotting
logarithmic concentration on the x-axis and percentage of inhibition on
the y-axis as shown in Fig. 3. Five compounds, 4d, 4f, 4h, 4i and 4p
3. Experimental
Melting points were determined using Shital-digital programmable
melting point apparatus and are uncorrected. FTIR spectra in KBr pel-
lets were recorded on a Bruker FTIR spectrophotometer. The 1H and 13
C
NMR spectra were recorded on a Bruker AVANCE II at 400 and 100/
75 MHz, respectively; chemical shifts are expressed in parts per million
204

V. Bhaskar et al.
BioorganicChemistry84(2019)202–210
Fig. 2. Docked view of Molecule AKB 20 at the active site of the enzyme (PDB ID: 4L7G).
Table 1
Surflex-Dock scores (kcal/mol) of Tetrahydrobenzo[b]pyran.
Compound
c
C scorea
Crash scoreb
Polar score
D scored
PMF scoree
G scoref
Chem scoreg
KVB1
4.38
2.93
2.58
3.74
2.59
4.42
2.42
2.68
4.62
3.45
2.63
2.69
2.69
4.76
3.47
4.02
5.21
3.35
3.55
2.83
2.79
3.51
3.90
3.34
2.45
4.13
4.18
4.00
4.71
3.66
3.54
3.74
2.97
2.96
3.07
4.21
6.27
−0.44
−0.59
−0.71
−0.54
−0.63
−0.51
−0.79
−0.40
−1.26
−0.79
−0.23
−0.61
−2.22
−0.98
−0.66
−0.54
−1.14
−0.65
−0.74
−0.74
−0.72
−0.27
−0.97
−1.51
−0.71
−0.65
−0.76
−0.20
−0.65
−0.43
−0.83
−0.56
−0.32
−0.29
−0.47
−1.12
−0.83
0.96
0.00
0.90
2.09
0.22
2.13
0.98
1.11
2.48
1.34
1.67
0.93
2.00
1.67
0.04
2.19
1.14
0.83
1.04
1.04
1.14
3.39
2.07
2.17
0.80
2.14
0.00
2.11
2.22
2.26
1.04
1.95
1.39
1.35
1.95
1.11
2.00
−105.19
−89.64
−61.63
−81.83
−92.83
−86.98
−84.30
−80.65
−94.99
−88.80
−73.53
−62.63
−96.21
−98.33
−92.31
−88.27
−100.12
−87.27
−73.55
−75.00
−79.19
−70.47
−76.06
−95.57
−71.89
−75.15
−86.76
−19.44
−91.28
−77.75
−74.75
−72.62
−82.40
−79.24
−90.13
−93.86
−121.45
−28.49
−10.92
−18.56
−33.07
−14.46
−26.86
−25.72
−10.32
−47.83
−35.77
−1.75
−154.82
−136.34
−111.27
−121.75
−138.52
−114.06
−148.45
−121.39
−162.50
−154.35
−98.31
−22.90
−13.14
−16.32
−23.76
−15.49
−20.54
−21.16
−15.59
−28.58
−20.77
−16.05
−16.16
−21.45
−22.22
−21.42
−20.32
−24.00
−20.69
−17.15
−19.86
−18.42
−21.77
−20.98
−27.17
−17.99
−20.59
−15.66
−22.66
−21.73
−19.68
−17.13
−21.48
−20.76
−20.28
−19.77
−22.50
−26.91
KVB2
KVB3
KVB4
KVB5
KVB6
KVB7
KVB8
KVB9
KVB10
KVB11
KVB12
KVB13
KVB14
KVB15
KVB16
KVB17
AKB1
−15.14
9.21
−112.48
−174.71
−179.41
−169.49
−135.04
−166.10
−163.05
−144.16
−133.66
−148.23
−105.30
−143.49
−156.98
−127.24
−126.28
−162.66
−121.48
−149.96
−124.58
−143.86
−109.98
−148.48
−145.61
−125.90
−167.24
−192.54
−43.75
−48.66
−16.44
−53.45
−46.99
2.64
AKB2
AKB3
−23.03
−9.81
AKB4
AKB5
−56.70
−6.79
AKB6
AKB7
−40.19
−21.88
−9.83
AKB8
AKB9
AKB10
AKB11
AKB12
AKB13
AKB14
AKB15
AKB16
AKB17
AKB18
AKB19
AKB20
−38.99
−22.24
7.29
−3.98
5.37
−47.38
−50.54
−54.62
−17.53
−15.45
−30.34
a
C Score (Consensus Score) integrates a number of popular scoring functions for ranking the affinity of ligands bound to the active site of a receptor and reports
the output of total score.
b
c
Crash-score revealing the inappropriate penetration into the binding site. Crash scores close to 0 are favorable. Negative numbers indicate penetration.
Polar indicating the contribution of the polar interactions to the total score. The polar score may be useful for excluding docking results that make no hydrogen
bonds.
d
D-score for charge and van der Waals interactions between the protein and the ligand (work of Kuntz) [29].
e
f
g
PMF-score indicating the Helmholtz free energies of interactions for protein-ligand atom pairs (Potential of Mean Force, PMF) (work of Muegge and Martin) [30].
G-score showing hydrogen bonding, complex (ligand-protein), and internal (ligand-ligand) energies (work of Willett's group) [31].
Chem-score points for hydrogen bonding, lipophilic contact, and rotational entropy, along with an intercept term (work of Eldridge, Murray, Auton, Paolini, and
Mee) [32].
205

V. Bhaskar et al.
BioorganicChemistry84(2019)202–210
Concentration
100.00
KVB 5
155.000000
101.000000
90.500000
70.400000
70.200000
64.200000
35.000000
14.000000
50.00
25.00
10.00
5.00
1.00
0.50
KVB 5
0.10
200
150
100
50
0
-2
-1
0
1
2
3
IC50
0.9084
log(Concentration in uM)
Fig. 3. Calculation of IC50 value of best molecule with a compound code number KVB-5 (4e).
(δ ppm) relative to TMS. The abbreviations used to describe the peak
patterns are: (b) broad, (s) singlet, (d) doublet, (t) triplet, (q) quartet,
and (m) multiplet (see Fig. 3).
Table 3
The list of compounds prepared from Scheme 2.
Compound
Ar
% Yield
mp (°C)
Mass spectra (MS) were recorded in a JEOL GCMATE II GC-Mass
spectrometer and Schimadzu QP 20105 GC-Mass spectrometer.
Elemental analysis data (performed on Leco Tru Spec CHNS Analyzer)
for C, H, and N were all within 0.4% of the theoretical values.
Analytical thin-layer chromatography (TLC) was performed on the
precoated TLC sheets of silica gel 60 F254 (Merck, Darmstadt, Germany)
visualized by long- and short-wavelength UV lamps. Chromatographic
purifications were performed on Merck aluminium oxide (70–230
mesh) and Merck silica gel (70–230 mesh) (see Tables 2–4).
6a AKB-1
6b AKB-2
6c AKB-3
6d AKB-4
6e AKB-5
6f AKB-6
4-Fluoro
82
75
80
88
86
94
96
86
88
89
91
86
87
90
91
88
81
78
65
86
274
221
210
268
178
222
267
217
245
214
245
224
234
230
201
203
220
210
236
213
2-Fluoro
4-Chloro
3-Bromo
4-Nitro
3-Chloro
3,4-Dichloro
4-Bromo
6g AKB-7
6h AKB-8
6i AKB-9
2-Hydroxy
4-Methyl
1-Naphthyl
2-Naphthyl
4-Trifluro methyl
H
6j AKB-10
6k AKB-11
6l AKB-12
6m AKB-13
6n AKB-14
6o AKB-15
6p KVB-16
6q AKB-17
6r AKB-18
6s AKB-19
6t AKB-20
3.1. General procedure for the synthesis of 2-amino-4-substituted-5-oxo-
5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (4a-s) [21]
P-Methoxy
3-Thienyl
2-Furyl
To mixture of equimolar concentration of substituted aldehydes
(5 mmol), malononitrile (5 mmol) and 1, 3-cyclohexanedione (5 mmol),
a solution ionic liquid (0.25 mmol) in a 1:1 mixture of water and al-
cohol was added, the mixture was stirred at room temperature for
24–30 h. The solid separated was filtered off, washed thrice with water
(10 ml) and treated with charcoal to remove color impurity and are
2-Thienyl
3-Hydroxy
3-Phenoxy
purified by column chromatography on silica gel with petroleum ether/
ethyl acetate (3.5:6.5) as the eluent (see Table 6).
Table 2
The list of compounds prepared from Scheme 1.
3.1.1. 2-Amino-4-(3-hydroxyphenyl)-5-oxo-5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile (4a)
Compound
Ar
% Yield
mp (°C)
4a KVB-1
4b KVB-2
4c KVB-3
4d KVB-4
4e KVB-5
4f KVB-6
3-hydroxy
85
91
84
89
82
83
81
77
88
77
74
88
80
85
80
85
90
88
90
231
198
222
238
226
218
240
229
229
226
230
228
226
215
169
190
–
(Yield 85%). mp 229–231 °C; FTIR (KBr): 3535 (eNH2), 3369
3-phenoxy
(eOH), 2185 (eCN, str), 1683 (C]O), 1163 (CeN, str) cm−1
;
1H NMR
4-fluro
(400 MHz, DMSO)
δ ppm: 1.84–1.99 (m, 2H, chromene-C7-H),
4-hydroxy, 3-methoxy
2-furyl
2.22–2.33 (m, 2H, chromene-C8-H), 2.57–2.60 (t, 2H, chromene-C6-H),
4.07 (s, 1H, chromene-C4-H), 6.54–6.57 (m, 3H, ph-C4, C6-H and eOH),
6.92 (s, 1H, ph-C2-H), 7.02–7.06 (t, 1H, ph-C5-H), 9.25 (s, 2H, eNH2);
13C NMR (100 MHz, DMSO) δ ppm: 19.76, 26.39, 35.20, 36.28, 58.22,
113.48, 113.93, 113.97, 117.67, 119.72, 129.16, 146.09, 157.23,
158.49, 164.20, 195.70; MS (ESI): m/z = found 281 [M+−1]; calcd.
282.29. Anal. Calcd. For C16H14N2O3: C, 68.07; H, 5.00; N, 9.92. Found:
C, 69.75; H, 5.32; N, 10.33.
3-hydroxy, 4-methoxy
2-Thienyl
4g KVB-7
4h KVB-8
4i KVB-9
4-Nitro
3-thienyl
4j KVB-10
4k KVB-11
4l KVB-13
4m KVB-14
4n KVB-15
4o KVB-16
4p KVB-17
4q KVB-18
4r KVB-19
4s KVB-20
5-methyl-3-thienyl
3-methyl-2-thienyl
4-chloro, 3-nitro
4-chloro
4-methyl
p-dimethylamino
4-methoxy
3.1.2. 2-Amino-5-oxo-4-(3-phenoxyphenyl)-5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile (4b)
2-Fluoro
3-Bromo
–
(Yield 91%). mp 196–198 °C; FTIR (KBr): 3329–3398 (eNH2),
4-Nitro
–
2185(eCN, str), 1676 (C]O), 1163 (CeN, str) cm−1
;
1H NMR
206

V. Bhaskar et al.
BioorganicChemistry84(2019)202–210
Table 4
Reaction scheme in biological assay.
Reaction number
1
2
3
4
Assay description
Negative control: no enzyme
Positive control: supplied enzyme activity
Inhibition
78-X µl
20 µl
2 µl
Test: sample enzyme activity
Fluorescent assay buffer
BACE1 substrate solution, 50 µM
BACE1 enzyme solution, 0.3 units/20 µl
Inhibitor solution
80 µl
78 µl
20 µl
2 µl
–
80-Y µl
20 µl
20 µl
–
–
–
–
–
X µl
–
Sample enzyme
–
–
Y µl
–
Assay standard solution, 100 µM
–
–
Total
100 µl
100 µl
100 µl
100 µl
Reaction 1: Negative control reaction with no enzyme is considered as standard curve blank. The “blank” reaction tube reflects fluorescence because of the substrate
alone.
Reaction 2: Supplied enzyme – a positive control.
Reaction 3: Enzyme inhibition test.
Reaction 4: Sample enzyme activity test.
1. The fluorescence was noted immediately after adding the enzyme. This is considered as “time zero” reading.
2. The plate was covered with para-filmand incubated at 37 °C for 2 h.
After the “time zero” recording and the next reading was taken after 2 h. The plate was allowed to cool to room temperature before measuring the florescence.
Table 5
The percentage inhibitory activities of all the synthesized compounds obtained by subjecting them to in-vitro β-Secretase enzyme inhibition assay are given below.
Compound Code
100 µM
50 µM
25 µM
10 µM
5 µM
1 µM
0.5 µM
0.1 µM
0.05 µM
IC50(µM)
4a
4b
4c
4d
4e
4f
–
–
86.9
72.4
65.4
68.8
90.5
98.8
102.6
104.2
102.5
151.7
166.4
66.3
170.6
0.7
103.3
–
–
–
–
–
–
–
5.0
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
11.5
–
–
–
–
–
–
–
32.2
–
–
–
–
–
120
155
–
99.8
64.4
61.5
61.4
64.2
1.5
–
34.0
35.0
–
12.0
14.0
–
1.461
101
70.4
70.2
67.9
–
0.9084
100
69.9
4.042
4g
4h
4i
–
–
79.1
–
–
–
–
104.6
95.0
1.6
1.2
–
1.4
1.3
–
–
–
7.491
–
102.5
85.5
–
–
8.2
4j
–
–
–
–
–
–
4m
4n
4o
4p
6a
6b
6c
6d
6e
6f
–
–
–
–
–
–
–
–
–
–
103.8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
222.2
23.7
43.1
53.5
41.5
38.7
6.0
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
6g
6h
6i
–
–
–
–
–
–
–
–
–
–
-3.9
–
–
–
–
–
–
–
–
158.1
10.2
38.7
36.7
76.0
55.5
67.8
52.2
97.3
48.1
52.1
38.3
–
–
–
–
–
–
6j
–
–
–
–
–
–
–
–
6k
6l
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
6m
6n
6o
6p
6q
6r
–
–
104.7
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
103.8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
100.2
77.4
1.5
–
1.6
–
–
–
8.533
–
–
–
–
–
–
–
–
–
–
–
–
6s
6t
–
–
–
–
–
–
–
–
–
–
(400 MHz, DMSO)
δ ppm: 1.80–1.98 (m, 2H, chromene-C7-H),
Table 6
The calculated IC50 values of selected compounds
are mentioned in the following table.
2.25–2.30 (m, 2H, chromene-C8-H), 2.56–2.59 (t, 2H, chromene-C6-H),
4.17 (s, 1H, chromene-C4-H), 6.76–6.78 (s, 2H, phenoxy-C4, C6-H),
6.90–6.92 (d, 1H, phenoxy-C2-H), 6.97–7.00 (m, 3H, phenoxy-C8, C10
,
Compound
IC50 values
C
12-H), 7.11–7.15 (t, 1H, phenoxy-C5-H), 7.25–7.39 (m, 4H, phenoxy-
C9, C11-H and eNH2); 13C NMR (100 MHz, DMSO) δ ppm: 19.00, 26.00,
113.00, 116.00, 118.00, 119.36, 121.00, 129.00, 146.00, 156.00,
158.57, 164.00, 195.35; MS (ESI): m/z = found 358 [M+−1]; calcd.
358.39. Anal. Calcd. For C16H14N2O3: C, 68.07; H, 5.00; N, 9.92. Found:
C, 69.75; H, 5.32; N, 10.33. GC/MS-m/z, 358(M+281), (M−35),
(M−63), (M−81), 246(100).
4d
4e
4f
1.4610
0.9084
4.0420
7.4910
8.2000
8.5330
4h
4i
4p
207

V. Bhaskar et al.
BioorganicChemistry84(2019)202–210
3.1.3. 2-Amino-4-(4-fluorophenyl)-5-oxo-5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile (4c)
Found: C, 69.75; H, 5.32; N, 10.33. GC/MS-m/z, 311 (M−112),
126(100).
(Yield 84%). mp 220–222 °C; FTIR (KBr): 3417–3334 (eNH2), 2191
(eCN str), 1680 (C]O), 1170 (CeN str) cm−1
;
1H NMR (400 MHz,
3.1.8. 2-Amino-4-(4-nitrophenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-
3-carbonitrile (4h)
DMSO) δ ppm: 1.82–1.98 (m, 2H, chromene-C7-H), 2.19–2.34 (m, 2H,
chromene-C8-H), 2.54–2.64 (m, 2H, chromene-C6-H), 4.19 (s, 1H,
chromene-C4-H), 6.98 (s, 2H, ph-C3, C5-H), 7.06–7.11 (m, 2H, ph-C2,
C6-H), 7.15–7.19 (m, 2H, eNH2); 13C NMR (100 MHz, DMSO) δ ppm:
19.76, 26.39, 35.20, 36.28, 58.22, 113.48, 113.93, 113.97, 119.72,
129.16, 146.09, 157.23, 158.49, 164.20, 195.70. MS (ESI): m/
z = found 358 [M+−1]; calcd. 358.39. Anal. Calcd. For C16H14N2O3:
C, 68.07; H, 5.00; N, 9.92. Found: C, 69.75; H, 5.32; N, 10.33. GC/MS-
m/z, 222, 189 (100).
(Yield 77%). mp 227–229 °C; FTIR (KBr): 3315–3365 (eNH2), 2189
(eCN str), 1681 (C]O), 1066 (CeN str) cm−1
;
1H NMR (400 MHz,
DMSO) δ ppm: 1.84–2.01 (m, 2H, chromene-C7-H), 2.20–2.35 (m, 2H,
chromene-C8-H), 2.60–2.63 (t, 2H, chromene-C6-H), 4.35 (s,1H, chro-
mene-C4-H), 7.13 (s, 2H, ph-C2, C6-H), 7.43–7.46 (m, 2H, ph-C3, C5-H),
8.13–8.16 (m, 2H, eNH2); 13C NMR (100 MHz, DMSO) δ ppm: 19.68,
26.47, 35.53, 36.16, 56.87, 112.70, 119.29, 123.58, 128.53, 146.21,
152.26, 158.50, 165.06, 195.81; MS (ESI): m/z = found 358 [M+−1];
calcd. 358.39. Anal. Calcd. For C16H14N2O3: C, 68.07; H, 5.00; N, 9.92.
Found: C, 69.75; H, 5.32; N, 10.33. GC/MS-m/z, 272 (M−19), (M−56),
(M−65), 207(100).
3.1.4. 2-Amino-4-(4-hydroxy-3-methoxyphenyl)-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile (4d)
(Yield 89%). mp 236-238°C; FTIR(KBr): 3423–3425 (eNH2), 3327
(Phenolic-OH), 2193 (eCN, str), 1676 (C]O), 1128 (CeN, str) cm−1
;
3.1.9. 2-Amino-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile (6a)
1H NMR (400 MHz, DMSO) δ ppm: 1.81–1.99 (m, 2H, chromene-C7-H),
2.20–2.33 (m, 2H, chromene-C8-H), 2.53–2.65 (m, 2H, chromene-C6-
H), 3.29 (s, 3H, eCH3), 4.08 (s, 1H, chromene-C4-H), 6.62 (dd, 1H, ph-
C6-H), 6.64–6.67 (t, 2H, ph-C5-H and eOH), 6.88 (s, 1H, ph-C2-H), 8.76
(s, 2H, eNH2); 13C NMR (100 MHz, DMSO) δ ppm:19.00, 26.00, 34.00,
36.00, 114.00, 115.00, 119.00, 135.00, 145.00, 147.55, 158.00,
164.58. MS (ESI): m/z = found 358 [M+−1]; calcd. 358.39. Anal.
Calcd. For C16H14N2O3: C, 68.07; H, 5.00; N, 9.92. Found: C, 69.75; H,
5.32; N, 10.33. GC/MS-m/z, 312 (M−31), (M−67), (M−83), 245(100).
(Yield 82%). mp 272–274 °C; FTIR (KBr): 3313–3358 (eNH2), 2189
(eCN str), 1680 (C]O), 1151 (CeN str) cm−1
;
1H NMR (400 MHz,
DMSO) δ ppm: 0.93 & 1.01 (s, 6H, chromene-C7-H), 2.11 (d, 2H,
chromene-C8-H), 2.25 (t, 2H, chromene-C6-H), 4.18 (s, 1H, chromene-
C4-CH3), 6.99–7.17 (m, 4H, ph-C2, C3, C5, C6-H), 8.29 (s, 2H, eNH2);
13C NMR (100 MHz, DMSO) δ ppm: 32.37, 37.10, 38.22, 48.90, 112.87,
117.94, 119.30, 130.12, 134.05, 134.14, 149.72, 163.41; MS (ESI): m/
z = found 358 [M+−1]; calcd. 358.39. Anal. Calcd. For C16H14N2O3:
C, 68.07; H, 5.00; N, 9.92. Found: C, 69.75; H, 5.32; N, 10.33. GC/MS-
m/z, 281(M−14), 246(100).
3.1.5. 2-Amino-4-(furan-2-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-
carbonitrile (4e)
(Yield 82%). mp 224–226 °C; FTIR (KBr): 3325–3339 (eNH2), 2187
3.1.10. 2-Amino-4-(2-fluorophenyl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile(6b)
(eCN str), 1678 (C]O), 1163 (CeN str) cm−1
;
1H NMR (400 MHz,
DMSO) δ ppm: 1.88–1.96 (m, 2H, chromene-C7-H), 2.31 (s, 2H, chro-
mene-C8-H), 2.57 (s, 2H, chromene-C6-H), 4.31 (s, 1H, chromene-C4-H),
6.04 (t, 1H, furan-C4-H), 6.29–6.30 (q, 1H, furan-C3-H), 7.03 (s, 1H,
furan-C5-H), 7.46 (d, 2H, eNH2); 13C NMR (100 MHz, DMSO) δ ppm:
19.70, 26.42, 28.94, 36.13, 55.28, 105.04, 110.32, 111.43, 119.48,
141.71, 155.76, 159.25, 165.07, 195.48. MS (ESI): m/z = found 358
[M+−1]; calcd. 358.39. Anal. Calcd. For C16H14N2O3: C, 68.07; H,
5.00; N, 9.92. Found: C, 69.75; H, 5.32; N, 10.33. GC/MS-m/z, 256 (M−
28), (M−45), (M−56), 172 (100).
(Yield 75%). mp 219–221 °C; FTIR (KBr): 3327–3398 (eNH2), 2196
(eCN str),1662 (C]O), 1151 (CeN str) cm−1
;
1H NMR (400 MHz,
DMSO) δ ppm: 0.95 & 1.03 (s, 6H, chromene-C7-CH3), 2.05–2.27 (d,
2H, chromene-C8-H), 2.43–2.56 (d, 2H, chromene-C6-H), 4.43 (s, 1H,
chromene-C4-H), 7.00 (s, 2H, eNH2), 7.07–7.24 (m, 4H, ph-C3, C4, C5,
C6-H); 13C NMR (100 MHz, DMSO) δ ppm: 26.59, 28.45, 31.77, 38.90,
49.88, 56.72, 111.36, 115.49, 119.45, 124.37, 124.41, 128.58, 131.23,
158.73, 161.14, 163.07, 195.53; MS (ESI): m/z = found 358 [M+−1];
calcd. 358.39. Anal. Calcd. For C16H14N2O3: C, 68.07; H, 5.00; N, 9.92.
Found: C, 69.75; H, 5.32; N, 10.33. GC/MS-m/z, 358(M+281), (M−35),
(M−63), (M−81), 246(100).
3.1.6. 2-Amino-4-(3-hydroxy-4-methoxyphenyl)-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile (4f)
(Yield 83%). mp 216–218 °C; FTIR (KBr): 3319–3363 (eNH2), 2191
3.1.11. 2-Amino-4-(3-chlorophenyl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile (6c)
(eCN str), 1681 (C]O), 1066 (CeN str) cm−1
;
1H NMR (400 MHz,
DMSO) δ ppm: 1.81–1.99 (m, 2H, chromene-C7-H), 2.30–2.33 (m, 2H,
chromene-C8-H), 2.56–2.59 (t, 2H, chromene-C6-H), 4.52 (s, 1H,
chromene-C4-H), 6.83–6.84 (d, 1H, phenyl-C3′-H), 6.88–6.90 (m, 1H,
phenyl-C4′-H), 7.08 (s, 2H, NH2), 7.29–7.30 (t, 1H, phenyl-C5′-H); 13C
NMR (100 MHz, DMSO) δ ppm: 20, 26, 30, 36, 58, 114, 120, 124, 127,
149, 159, 164, 196. MS (ESI): m/z = found 358 [M+−1]; calcd.
358.39. Anal. Calcd. For C16H14N2O3: C, 68.07; H, 5.00; N, 9.92. Found:
C, 69.75; H, 5.32; N, 10.33. GC/MS-m/z, 272 (M−66), 206(100).
(Yield 80%). mp 208–210 °C; FTIR (KBr): 3319–3389 (eNH2), 2191
(eCN str), 1678 (C]O), 1151 (CeN str) cm−1
;
1H NMR (400 MHz,
DMSO) δ ppm: 0.96 & 1.04 (s, 6H, chromene-C7-CH3), 2.09–2.28 (m,
2H, chromene-C8-H), 2.51–2.52 (m, 2H, chromene-C6-H), 4.20 (s, 1H,
chromene-C4-H), 7.05 (s, 1H, ph-C6-H), 7.19 (d, 2H, ph-C4, C5-H), 7.36
(d,1H, ph-C2-H), 7.99 (m, 2H, eNH2); 13C NMR (100 MHz, DMSO) δ
ppm: 26.85, 31.77, 35.09, 49.93, 57.77, 112.32, 119.50, 128.25,
129.09, 129.71, 131.08, 143.72, 158.48, 162.58, 195.62; MS (ESI): m/
z = found 358 [M+−1]; calcd. 358.39. Anal. Calcd. For C16H14N2O3:
C, 68.07; H, 5.00; N, 9.92. Found: C, 69.75; H, 5.32; N, 10.33. GC/MS-
m/z, 222, 189 (100).
3.1.7. 2-Amino-5-oxo-4-(thiophen-2-yl)-5,6,7,8-tetrahydro-4H-chromene-
3-carbonitrile (4g)
(Yield 81%). mp 238–240 °C; FTIR (KBr): 3415–3334 (eNH2), 2194
(eCN str), 1683 (C]O), 1068 (CeN str) cm−1
;
1H NMR (400 MHz,
3.1.12. 2-Amino-4-(2,4-dichlorophenyl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile (6g)
DMSO): δ ppm: 1.81–1.99 (m, 2H, chromene-C7-H), 2.30–2.33 (m, 2H,
chromene-C8-H), 2.56–2.59 (t, 2H, chromene-C6-H), 4.52 (s, 1H,
chromene-C4-H), 6.83–6.90 (m, 2H, thiophen-C3, C4-H), 7.08 (s, 2H,
eNH2), 7.29–7.30 (t, 1H, thiophene-C5-H); 13CNMR (100 MHz, DMSO)
δ ppm: 20.23, 26.89, 30.80, 36.71, 58.35, 124.41, 124.84, 127.29,
149.73, 159.48, 164.74, 196.14. MS (ESI): m/z = found 358 [M+−1];
calcd. 358.39. Anal. Calcd. For C16H14N2O3: C, 68.07; H, 5.00; N, 9.92.
(Yield 96%). mp 265–267 °C; FTIR (KBr): 3327–3396 (eNH2), 2196
(eCN, str), 1658 (C]O), 1149 (CeN str) cm−1
;
1H NMR (400 MHz,
DMSO) δ ppm: 0.94 & 1.02 (s, 6H, chromene-C7-CH3), 2.09–2.25 (m,
2H, chromene-C8-H), 2.48–2.51 (m, 2H, chromene-C6-H), 4.23 (s, 1H,
chromene-C4-H), 7.10 (s, 1H, ph-C6-H), 7.37 (d, 1H, ph-C5-H), 7.56 (d,
1H, ph-C3-H), 8.29 (s, 2H, eNH2); 13C NMR (100 MHz, DMSO) δ ppm:
208

V. Bhaskar et al.
BioorganicChemistry84(2019)202–210
32.37, 37.10, 48.90, 54.85, 113.03, 119.47, 125.00, 130.54, 134.13,
146.88, 157.17, 163.41, 164.74; MS (ESI): m/z = found 358 [M+−1];
calcd. 358.39. Anal. Calcd. For C16H14N2O3: C, 68.07; H, 5.00; N, 9.92.
Found: C, 69.75; H, 5.32; N, 10.33. GC/MS-m/z, 312 (M−31), (M−67),
(M−83), 245(100).
2H, chromene-C8-H), 2.39–2.53 (m, 2H, chromene-C6-H), 4.16 (s, 1H,
chromene-C4-H), 6.74 (s, 1H, phenoxy-C4-H), 6.81 (dd, 1H, phenoxy-
C6-H), 6.90–6.99 (M, 4H, phenoxy-C2, C8, C10, C12-H), 7.14 (t, 1H,
phenoxy-C5-H), 7.26–7.38 (m, 4H, eNH2 and naphthyl-C9, C11-H); 13C
NMR (400 MHz, DMSO) δ ppm: 26.70, 28.43, 31.70, 35.40, 49.92,
57.99, 112.36, 116.54, 117.12, 118.59, 119.56, 122.18, 123.45,
129.97, 146.96, 156.37, 156.63, 158.47, 162.64, 195.58; MS (ESI): m/
z = found 358 [M+−1]; calcd. 358.39. Anal. Calcd. For C16H14N2O3:
C, 68.07; H, 5.00; N, 9.92. Found: C, 69.75; H, 5.32; N, 10.33. GC/MS-
m/z, 272 (M−19), (M−56), (M−65), 207(100).
3.1.13. 2-Amino-4-(4-methylphenyl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile (6j)
(Yield 89%). mp 212–214 °C; FTIR (KBr): 3425–3450 (eNH2), 2194
(eCN str), 1676 (C]O), 1145 (eCeN str) cm−1
;
1H NMR (400 MHz,
DMSO) δ ppm: 0.93–1.09 (s, 6H, chromene-C7-H3), 2.05–2.09 (m, 2H,
chromaene-C8-H), 2.21–2.25 (t, 2H, chromene-C6-H), 2.23 (s, 3H, C4′-H
of eCH3), 4.11 (s, 1H, chromene-C4-H), 6.88–6.90 (dd, 1H, phenyl-C3′,
C5′-H), 6.93 (s, 2H, eNH2), 7.06–7.08 (dd, 1H, phenyl-C2′, C6′-H); 13C
NMR (100 MHz, DMSO) δ ppm: 20.56, 31.00, 35.00, 37.00, 49.00,
113.00, 118.00, 120.00, 124.00, 125.00, 129.00, 130.00, 149.58,
163.00. MS (ESI): m/z = found 358 [M+−1]; calcd. 358.39. Anal.
Calcd. For C16H14N2O3: C, 68.07; H, 5.00; N, 9.92. Found: C, 69.75; H,
5.32; N, 10.33. GC/MS-m/z, 256 (M−28), (M−45), (M−56), 172 (100).
3.2. Biological screening studies
3.2.1. BACE1 in-vitro assay
The BACE1 inhibitory assay was carried out by using β-Secretase
activity detection kit purchased from Sigma-Aldrich Company, USA.
The kit is provided with all the necessary reagents, along with the
BACE1 enzyme to be used as a positive control, which is required for
the detection of BACE1 inhibitory activity. The assay is based on the
principle of fluorescence resonance energy transfer (FRET) in which the
fluorescence signal enhancement is observed after the substrate is
cleaved by BACE1.
3.1.14. 2-Amino-7,7-dimethyl-4-(naphthalen-1-yl)-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile (6k)
(Yield 91%). mp 243–245 °C; FTIR (KBr): 3325–3454 (eNH2), 2194
The principle of the assay of BACE1 involves the synthesis of the
peptide substrate with the help of two fluorophores, Viz., fluorescent
donor and a proprietary quenching acceptor. The distance between
these two groups has been assigned, so that upon light excitation, the
donor fluorescence energy is significantly quenched by the acceptor
through a phenomenon known as resonance energy transfer. Upon
cleavage by the protease, the fluorophore will be separated from the
quenching group, restoring the full fluorescence yield of the donor.
Thus, a weak fluorescent peptide substrate becomes highly fluorescent
upon enzymatic cleavage; the increase in fluorescence is linearly related
to the rate of proteolysis [19,22]. The assay was performed in duplicate
on all the compounds.
(eCN str), 1666 (C]O), 1136 (CeN str) cm−1
;
1H NMR (400 MHz,
DMSO) δ ppm: 1.01 & 1.07 (s, 6H, chromene-C7-CH3), 2.06–2.27 (2d,
2H, chromene-C8-H), 2.60 (d, 2H, chromene-C6-H), 5.16 (s, 1H, chro-
mene-C4-H), 6.96 (s, 1H, naphthyl-C2-H), 7.26 (d, 1H, naphthyl-C3-H),
7.44–7.59 (m, 3H, naphthyl-C4, C6, C7-H), 7.79 (d, 1H, naphthyl-C5-H),
7.94 (d, 1H, naphthyl-C8-H), 8.40 (d, 2H, eNH2); 13C NMR (100 MHz,
DMSO) δ ppm: 26.97, 30.25, 31.81, 38.90, 50.00, 58.92, 113.43,
119.60, 123.59, 125.79, 126.94, 128.36, 130.74, 133.32, 141.91,
158.42, 162.73, 195.68. MS (ESI): m/z = found 358 [M+−1]; calcd.
358.39. Anal. Calcd. For C16H14N2O3: C, 68.07; H, 5.00; N, 9.92. Found:
C, 69.75; H, 5.32; N, 10.33. GC/MS-m/z, 272 (M−66), 206(100).
Equipment and reagents required
3.1.15. 2-Amino-7,7-dimethyl-4-(naphthalen-2-yl)-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile (6l)
Fluorometer
•
(Yield 86%). mp 222–224 °C; FTIR (KBr): 3313–3350 (NH2), 1147
Well plate for fluorescence assay
•
(CeN str), 2189 (eCN str), 1680 (C]O) cm−1
;
1H NMR (400 MHz,
Dimethyl sulfoxide (DMSO)
•
DMSO) δ ppm: 0.95 & 1.03 (s, 6H, chromene-C7-CH3), 2.05–2.27 (2d,
2H, chromene-C8-H), 2.48–2.54 (m, 2H, chromene-C6-H), 7.03 (s, 1H,
naphthyl-C1-H), 7.28 (dd, 3H, naphthyl-C3, C5, C6-H), 7.43–7.50 (m,
2H, naphthyl-C4, C8-H), 7.65 (s, 1H, naphthyl-C7-H), 7.82–7.88 (m, 2H,
eNH2); 13C NMR (100 MHz, DMSO): 26.74, 31.79, 35.87, 49.99, 58.12,
112.54, 119.68, 125.62, 126.15, 127.41, 127.62, 128.07, 131.98,
132.83, 142.00, 158.48, 162.54, 195.69; MS (ESI): m/z = found 358
[M+−1]; calcd. 358.39. Anal. Calcd. For C16H14N2O3: C, 68.07; H,
5.00; N, 9.92. Found: C, 69.75; H, 5.32; N, 10.33. GC/MS-m/z, 311
(M−112), 126(100).
Procedure
1. The fluorometer was set on well plate reader mode with excitation
at 320 nm and emission at 405 nm.
2. All the components were brought (except the BACE1 enzyme solu-
tion) to the room temperature.
3. All the components were added to a fluorometer 96 well plate ac-
cording to the following regimen. It was thoroughly mixed. The
BACE1 enzyme was added just before reading.
3.1.16. 2-Amino-4-(3-hydroxyphenyl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile (6s)
3.2.2. Assay of BACE1 inhibitors
Enzyme activity inhibition reactions were set using reactions 1–3
(blank, enzyme activity reaction, and inhibition reaction) as described
in Table 4. Reaction 3 was expanded to include a few wells with dif-
ferent concentrations of the inhibitor ([Asn670, Sta671, Val672]-
Amyloid β/A4 Precursor Protein 770 Fragment 662–675) and this re-
action which was performed at 37 °C for 2 h.
(Yield 65%). mp 234–236 °C; FTIR (KBr): 3311–3454 (NH2), 3315
(eOH), 2198 (eCN str), 1676 (C]O), 1151 (CeN str) cm−1
;
1H NMR
(400 MHz, DMSO) δ ppm: 0.95 & 1.03 (s, 6H, chromene-C7-CH3),
2.47–2.49 (m, 2H, chromene-C6-H), 4.05 (s, 1H, chromene-C4-H),
6.53–6.56 (m, 3H, eOH & ph-C4, C6-H), 6.94 (s, 1H, ph-C2-H), 7.06 (t,
1H, ph-C5-H), 9.28 (s, 2H, eNH2); 13C NMR (100 MHz, DMSO) δ ppm:
26.78, 31.77, 35.43, 49.98, 58.39, 112.87, 113.54, 114.09, 119.73,
129.18, 146.12, 157.28, 158.51, 162.31, 195.56.
3.3. Molecular docking studies
Molecular docking studies were carried out [23] using the Sybyl-X,
version 2.0, run on a Intel® CoreTM i3-2130 CPU @ 3.40 GHz processor
on a Windows-7 professional workstation. When docking was per-
formed with default settings, it revealed a number of possible con-
formations and orientations for the inhibitors at the binding site. Un-
derstanding of the binding site conformations helped us to understand
3.1.17. 2-Amino-7,7-dimethyl-5-oxo-4-(3-phenoxyphenyl)-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile (6t)
(Yield 86%). mp 211–213 °C; FTIR (KBr): 3331–3379 (eNH2), 2191
(eCN str), 1681 (C]O), 1153 (CeN str) cm−1
;
1H NMR (400 MHz,
DMSO) δ ppm: 0.90 & 1.01 (s, 6H, chromene-C7-CH3), 2.07–2.26 (2d,
209

V. Bhaskar et al.
BioorganicChemistry84(2019)202–210
the important interactions that could stabilize ligand-receptor complex.
Surflex-Dock adopted the empirical scoring function using the patented
searching engine [24,25] for molecular docking. The crystal structure
of Diethylaminosulfur trifluoride-mediated intramolecular cyclization
of 2-hydroxy-benzylureas to Fused bicyclic amino oxazoline compounds
and evaluation of their biochemical activity against Beta-Secretase-1
(BACE1) was selected from the Protein Data Bank ((PDB entry code
4L7G) extracted from the Brookhaven Protein Database http://www.
rcsb.org/pdb) and these were used in the initial docking. Co-crystal-
lized ligand and water molecules were removed from the structure,
while the essential hydrogen atoms were added and side chains were
fixed during the protein preparation. The 3D structures of pyrrole de-
rivatives were constructed using the standard geometric parameters of
Sybyl-X 2.0 software and the structure was subjected to energy mini-
mization. The MMFF94 (Merk Molecular Force Field) charges were
calculated for the ligand, while Amber7FF02 was used for the protein.
The model was then subjected to energy minimization following the
gradient termination of Powell method for 3000 iterations using the
Tripos force field [26] with non-bonding cut-off value set at 8.0 and
dielectric constant set at 1.0. Then, ligand-based docking was in-
troduced to generate the “protomol”, and all the inhibitors were docked
within the prepared protein. In order to identify the ligand-protein in-
teractions, the top pose and protein were loaded into the work area and
MOLCAD (Molecular Computer Aided Design) program was employed
to visualize the binding mode between the protein and the ligand.
MOLCAD calculates and exhibits the surfaces of channels and cavities as
well as separating surface between the protein subunits [27,28].
MOLCAD program provides to create molecular surface by using fast
Connolly method, a marching cube algorithm to engender the surface.
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Acknowledgements
We thank, Dr. Akkattu T Biju, Scientist from NCL, Pune, for all his
guidance and support in synthesis of compounds. We are also thankful
for Dr. D. Sriram, Professor from BITS, Hyderabad, for his help in
biological screening and finally we are extremely thankful to Principal,
Manipal College of Pharmaceutical Sciences, Manipal, for supporting in
accomplishing this project.
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