Design, synthesis and in vitro evaluation studies of sulfonyl-amino-acetamides as small molecule BACE-1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.04.023

The identification of a series of sulfonyl-amino-acetamides as BACE-1 (β-secretase) inhibitors for the treatment of Alzheimer's disease is reported. The derivatives were designed based on the docking simulation study, synthesized and assessed for BACE-1 inhibition in vitro. The designed ligands revealed desired binding interactions with the catalytic aspartate dyad and occupance of S1 and S2′ active site regions. These in silico results correlated well with in vitro activity. Out of 33 compounds synthesized, 12 compounds showed significant inhibition at 10 μM concentration. The most active compound 2.17S had IC₅₀ of 7.90 μM against BACE-1, which was concomitant with results of in silico docking study.

Full text of DOI:10.1016/j.bmc.2016.04.023

Accepted Manuscript
Design, synthesis and in vitro evaluation studies of Sulfonyl-amino-acetamides
as small molecule BACE-1 inhibitors
Priti Jain, Pankaj K Wadhwa, Sinduri Gunapati, Hemant R Jadhav
PII:
S0968-0896(16)30257-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.04.023
BMC 12938
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 March 2016
8 April 2016
12 April 2016
Please cite this article as: Jain, P., Wadhwa, P.K., Gunapati, S., Jadhav, H.R., Design, synthesis and in vitro
evaluation studies of Sulfonyl-amino-acetamides as small molecule BACE-1 inhibitors, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.04.023
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Design, synthesis and in vitro evaluation studies of Sulfonyl-amino-acetamides as small molecule
BACE-1 inhibitors
Priti Jaina,b, Pankaj K Wadhwab, Sinduri Gunapatib, Hemant R Jadhava,b*
aComputational Laboratory, Medicinal Chemistry Division, Department of Pharmacy, Birla Institute of
Technology and Science Pilani, Pilani Campus, Rajasthan 333 031
bMedicinal Chemistry Laboratory, Department of Pharmacy, Birla Institute of Technology and Science
Pilani, Pilani Campus, Rajasthan 333 031
*Corresponding authors: Tel: +91-1596-515506
E-mail address: hemantrj@pilani.bits-pilani.ac.in

Abstract
The identification of a series of sulfonyl-amino-acetamides as BACE-1 (β-secretase) inhibitors for the
treatment of Alzheimer’s disease is reported. The derivatives were designed based on the docking
simulation study, synthesized and assessed for BACE-1 inhibition in vitro. The designed ligands revealed
desired binding interactions with the catalytic aspartate dyad and occupance of S1 and S2′ active site
regions. These in silico results correlated well with in vitro activity. Out of 33 compounds synthesized, 12
compounds showed significant inhibition at 10µM concentration. The most active compound 2.17S had
IC50 of 7.90µM against BACE-1, which was concomitant with results of in silico docking study.
Keywords: Alzheimer’s disease, BACE-1 inhibitors, Sulfonyl-amino-acetamide, Docking simulation,
FRET assay
1. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, for which there is no cure. AD is
characterized by decline in cognitive abilities, deterioration of behavioral functions, communication
problems, personality alterations and spatial disorientation.1 It leads to diminished brain functionalities
due to loss of neurons and ultimately leads to death. 5.3 million Americans have been reported to suffer
from AD in 2015 with 5.1 million being from the age group of 65 and older. It bears an annual cost of
approximately $226 billion, ranking it as costliest chronic disease to the society.2-4
Amyloid plaques and neurofibrillary tangles (NFTs) are considered to be the main pathologies for AD.
These are further characterized by neuroinflammation and neuronal dysfunction ultimately leading to
neuronal death. These pathologies are described through amyloid cascade hypothesis, according to which,
Aβ [a fragment of the amyloid precursor protein (APP)] plays the key role.5-6 APP is a large
transmembrane protein whose sequential cleavage by secretases (α, β, and γ) generates Aβ-42. Major
portion of APP is cleaved by α-secretases in non-amyloidogenic pathway while minor portion of APP is
cleaved by β-secretases resulting in the production of soluble APPβ and a 99 amino-acid long C-terminal
fragment. This fragment is further cleaved by γ-secretase to generate Aβ-42.7 As compared to the other
Aβ isoforms (Aβ-39 and Aβ-40), Aβ-42 is more hydrophobic and fibrillogenic. This isoform therefore,
aggregates to form oligomers which accumulate in the brain and cause neuronal dysfunctioning,
neurodegeneration and neuronal fatality. BACE-1 inhibition can lead to reduction of Aβ-42 production.8-
10
BACE-1 knockout mice were not seen to manifest Aβ production and also did not show any negative
health effects.11-12 Furthermore, the crystal structure of BACE-1 co-crystallized with different ligands,
reveals the basic requirements and the key features needed for enzyme inhibition. It proposes the

necessity of a group that acts as H-bond donor, so that it can form H-bonding interactions with catalytic
Asp32 and Asp228. Further, hydrophobic pockets (S1, S2´ and S3) are key regions for β-secretase
inhibition. S1 hydrophobic cleft is formed by the side chains of Tyr71, Phe108, Trp115, Ile118, and
Leu30. Like S1, S3 is also a large hydrophobic pocket formed by side chains of Trp115 and Ile110, as
well as main chains of Gln12, Gly11, Gly230, Thr231, Thr232 and Ser35 while S2´ comprises of Ile126,
Trp76, Val69, Arg128, Tyr198 amino acid residues. Altogether, the evidences gathered from tissue
culture and animal studies and knowledge of active site interactions make BACE-1 the most important
target.9, 11-14
Sulfonamides are known for their extensive pharmacological properties. This moiety is important
component of sulfa drugs having antimicrobial properties. Further, these are also used as anti-bacterials,
antitumor agents, diuretics, carbonic anhydrase inhibitors, hypoglycaemic agents, thyroid inhibitors, and
protease inhibitors.15 In this paper, we report the design and synthesis of low molecular weight sulfonyl-
amino-acetamide derivatives as BACE-1 inhibitors. The synthesized compounds were tested in vitro for
their ability to inhibit BACE-1 and in vitro - in silico correlation was drawn by performing docking
simulations.
2. Design
Sulfonamide derivatives have been reported to be active in central nervous system (CNS) disorders.16-17
Gerritz et al reported acyl guanidine derivatives as BACE-1 inhibitors.18 We replaced acyl guanidine
portion with sulfonyl-amino-acetamide motif (Figure 1) to check its effect on BACE-1 inhibition.
Compound 2.1S, with two aromatic moieties separated by a sulfonyl-amino-acetamide linker was taken as
prototype. Also, naphthyl ring was reduced to p-tolyl and heterocyclic ring was replaced by phenyl ring.
When explored through in silico studies, compound 2.1S revealed important interactions and was
therefore considered for further modifications. When docked over BACE-1 protein (PDB id- 2OHP),
compound 2.1S displayed following interactions (Figure 2): i) hydrogen bonding interaction of
sulfonamide nitrogen (NH) with side chain of catalytic Asp228 and amide nitrogen with Asp32; ii)
sulfonamide nitrogen (-NH-) formed hydrogen bonding interaction with Thr231 and with carbonyl
oxygen of Gly230; iii) Sulfonyl oxygen also interacted with Thr231; iv) ring A with p-methyl substitution
was seen to form hydrophobic interactions with Tyr71 and Tyr 198. It was observed that p-methyl group
and aromatic carbons form π-π stacking interactions; v) ring B displayed hydrophobic contacts with
Leu30, Tyr71, Phe108, Trp115 and Ile118. In short, the compound interacted with aspartate dyad, ring A
occupied part of S2´ active site region whereas ring B occupied S1 active site cavity completely. It is
reported that S3 active site region is also important for BACE-1 inhibition.19 Therefore, we aimed to

introduce substituents that would extend interaction and could also occupy S3 pocket. Based on these
observations, a library of derivatives with various electron donating and electron withdrawing
substitutents was synthesized to study the effect of substituents on BACE-1 inhibition.
Figure 1: Sulfonyl-amino-acetamide motif compared to reported acyl guanidine derivative.
Figure 2: Predicted binding mode of compound 2.1S in the active site of BACE-1 (PDB ID: 2OHP) i:
Chemical structure of compound 2.1S; ii: Docking view of 2.1S showing hydrogen bond in green color
and ligand as sticks with protein colored as shapely residue scheme; iii: Docking view of 2.1S with ligand
as ball and sticks and protein as cartoon assembly. The dashed lines in green represent hydrophobic
interactions.

3. Chemistry
The synthesis of sulfonyl-amino-acetamides is illustrated in scheme 1. The compounds were obtained in
good yield ranging from 68% to 91%. Formation of compound 1 (1.1S-1.33S) is a nucleophilic
substitution reaction which involves the reaction between halide and an amine thereby liberating halogen
acid. Synthesis of compound 2 (2.1S-2.33S) is amide formation reaction from carboxylic acid and amine
using HOBt-EDC.HCl as coupling reagent. The acid reacts with carbodiimide so as to form the key
intermediate; O-acyl isourea which then reacts with amine to produce the desired amide and substituted
urea as byproduct.
Scheme 1: Reagents and conditions: (a) NaHCO3, glycine, 800C, 2-3 h; (b) HOBt, EDC.HCl,
DCM, TEA, substituted aniline, 00C, 12 h.
4. Results and Discussion
Synthesized compounds were tested for BACE-1 inhibition at 10µM concentration and docking was
performed using Glide (Schrodinger). The docking scores and % BACE-1 inhibition values are given in
table 1. Visualization of docked poses helped us to identify the probable differences in potency of the
compounds and establish structure activity relationship (SAR). It was observed that when ring A is not
substituted by any group (compounds 2.2S-2.5S), it failed to occupy the S2′ or S3 region properly and
interacted with only one of the aspartates i.e. Asp32. The low percentage inhibition values were consistent
with docking simulations. Substitution of electron donating group like p-methyl on ring A with different
substitutions on ring B increased the potency of the compounds which correlated well with the docking
scores. It was observed that among compounds 2.6S, 2.7S and 2.8S; 2.8S substituted with o,p-dimethyl on
ring B showed the best interaction pattern and highest inhibition (35.02%) in this group. It displayed: i)
hydrogen bonding interaction with side chain of Asp228 (3.24Å) through sulfonamide nitrogen; ii)
hydrogen bonding interaction with side chain of Asp32 through amide nitrogen; iii) both these nitrogen
also formed hydrogen bonding interactions with Gly230 (3.10Å, 4.81Å); iv) carbonyl oxygen of amide
formed hydrogen bonding interactions with Thr231 (S3 cavity) and also Arg235 (S2 cavity); v) phenyl
ring bearing o,p-dimethyl substitution accommodated S1 cavity; vi) ring A bearing p-methyl substitution
was seen to accommodate S2′ region. Compound 2.6S substituted with p-chloro on ring B showed
hydrogen bonding interactions with aspartate dyad and occupied the cavities well while the 2.7S was seen
to interact with either of the aspartates instead of both. In order to compare methyl substitution on ring A

with other activating groups, we placed moderately activating group like acetamide and strongly
activating group like methoxy at para position on ring with different substituents on ring B.
It was observed that, substitution of p-acetamide on ring A with different substitutions on ring B
enhanced the potency as well as docking scores (2.14S-2.17S). Compound 2.17S was observed to be most
potent. When it (2.17S) was docked in BACE-1 (figure 3), it displayed key interactions with catalytic
aspartate dyad. -NH group of acetamide moiety formed hydrogen bonding interaction with Asp32 (2.59Å)
while -NH of sulfonamide moiety formed hydrogen bond with Asp32 (3.22Å) as well as Asp228 (3.59Å).
Ring A was seen to occupy S2´-S3 active site region through hydrophobic interactions with Gly230,
Tyr71 and Tyr198. Further, the oxygen of substituted acetamide group displayed hydrogen bonding
interaction with Arg128, which also is a key component of S2´ region. Ring B bearing o,p-dimethyl group
revealed strong hydrophobic network with Leu30, Pro70, Tyr71, Phe108 and Trp115 confirming the
occupancy of S1 active site. p-methyl group on ring B was oriented towards Phe108 and Trp115 while o-
methyl was present near Leu30. π electron cloud of ring B interacted with Tyr71 and Pro70 through
strong π-π stacking. The docking results (Glide score -10.93) were concurrent with in-vitro activity data
where it displayed 61.90% BACE-1 inhibition. This implies that p-acetamide on ring A and o,p-dimethyl
group on ring B is preferred for the activity. Further, placing p-methoxy on ring A (compounds 2.30S to
2.33S) did not favour the interactions as needed. Only, compound 2.30S (p-methoxy on ring A and
unsubstituted ring B) and 2.33S (p-methoxy on ring A and o,p-dimethyl on ring B) showed interaction
with Asp 228 and compound 2.30S also occupied both S1 and S3 cavity while 2.33S did not. These
results also were concurrent with inhibition profile where these compounds had lower inhibition than p-
acetamide substitution. Among the series, most active compound 2.32S showed 38% inhibition.
To expand the scope of structure activity relationship, ring A was further substituted with electron
withdrawing group like p-chloro, o-nitro, m-nitro and p-nitro. When p-chloro was placed on ring A
(compounds 2.10S to 2.13S), it displayed better docking score as compared to p-methyl and p-methoxy
group. The in vitro study suggested that the increase in docking score was concomitant to the percentage
inhibition. Significant increase in potency was observed when chlorine was placed at meta position on
ring B. Compound 2.13S having p-chloro at ring A and o,p-dimethyl at ring B had high score as well as
significant inhibition (35.92%). Its docking displayed that amide nitrogen formed hydrogen bonding with
Asp32 and sulfonamide nitrogen formed hydrogen bond with Asp228, ring B was buried into S1 pocket
while ring A was present in S2´ cavity and formed π-cation interaction with Arg128 (S2´ cavity) and its -
NH was seen to form hydrogen bonding interaction with Thr231 (S3 region). From amongst compounds
having m-nitro group on ring A: 2.18S, 2.19S, 2.20S and 2.21S; compounds 2.20S and 2.21S revealed
favorable interactions. Compound 2.20S with m-nitro on ring A and m-chloro on ring B formed key
hydrogen bonding interactions with aspartate dyad; ring A was seen to form π-cation linkage and salt

bridge with Arg128 (S2´ cavity) because of the presence of nitro group. Ring B occupied S1 cavity while
ring A was accommodated in S2´ and S3 pockets. Compound 2.19S with p-chloro on ring B was seen to
interact in the same manner as 2.20S but p-chloro substitution orients ring A away from S3 cavity
towards S2´ cavity. Similarly, 2.21S with o,p-dimethyl on ring B showed desired interactions but failed to
occupy the S3 cavity though the other two (S1 and S2´) were occupied. Placing nitro group at ortho
(compounds 2.22S to 2.25S) and para position (compounds 2.26S to 2.29S) on ring A diminished the
activity as well as interactions. Only compound 2.25S (o-nitro on ring A and o,p-dimethyl on ring B)
showed fair interactions with catalytic aspartate dyad but failed to occupy the S3 and S2´ substrate
binding pockets. The reason may be attributed to o-nitro group being present in proximity of sulfonamide
which tends to move it towards polar region while p-nitro group renders the compound linear and thus
renders improper orientation.
In view of in vitro-in silico data, it can be proposed that among electron donating groups, p-acetamide on
ring A with different substitutions on ring B showed higher % inhibition. Also, among the electron
withdrawing substitutents, p-chloro and m-nitro groups displayed better percentage inhibition, which can
possibly be attributed to the electron pull effect of these groups from electron rich aromatic residues like
Trp115, Thr231, Thr232 and Trp76. However, o-nitro and p-nitro substituted compounds did not show
similar effects. The probable explanation could be, o-nitro at ring A being in close proximity to sulfonyl
thus did not occupy S3 and S2´ cavity while p-nitro, being linear, failed to fit in the active site
(particularly S3 cavity); therefore, these compounds did not show appreciable inhibitory profile.
On the other hand, among ring B substitutents, p-chloro is linearly oriented and seems to move away from
S1 cavity and hence these compounds showed weaker activity while m-chloro group was seen to occupy
the S1 cavity properly. o,p-dimethyl group revealed strong hydrophobic interactions with amino acids of
S1 active site. Therefore, o,p-dimethyl group substituted compounds showed better % inhibition values.
Since compound 2.17S displayed highest %inhibition, it was further studied for calculation of IC50 value
which was found to be 7.90µM.
Further, in order to account for oral bioavailability, few important molecular descriptors and drug
likeliness was calculated using in silico tools. Also the blood brain permeability was assessed using
Online BBB predictor (table 2, in supplementary data). It was observed that calculated Log P values and
molecular weight of all the synthesized compounds suggested good oral bioavailability. Moreover, low
molecular weight (<500 Da) coupled with low polar surface area (< 90Å2) for most of the compounds
also proposes them to have high brain permeability. This was also confirmed by SVM_MACCSFP BBB
score which should be greater than 0.02 to be brain permeable.

C
Figure 3. A. Docking pose of compound 2.17S showing hydrogen bonding interactions in green; B.
Docking pose of compound 2.17S showing hydrophobic interactions in green; C: 2D interaction plot
O
H
N
S
N
R2
R1
O H
O
Table 1: Docking and in vitro assay data for Sulfonyl-amino-acetamide derivatives
Compound
numbers
2.1S
R1
R2
Glide
score
-6.39
-4.72
% Inhibition*
p-CH3
H
21.94 ± 0.2
5.09 ± 0.1
H
p-Cl
2.2S

H
H
m-Cl
o,p-di CH3
H
-5.30
-4.24
-4.28
-6.51
-5.94
-7.21
-4.32
-4.93
-6.74
-8.91
-8.48
-7.17
-8.27
-9.84
-10.93
-4.58
-6.79
-8.57
-8.95
-2.13
-6.94
-7.58
-3.50
-6.18
-5.14
-6.06
-5.18
-6.48
-5.42
-5.37
-7.51
4.83 ± 2.5
5.38 ± 1.3
8.72 ± 0.5
25.31 ± 0.3
31.75 ± 1.5
35.02 ± 0.1
18.54 ± 0.6
22.75 ± 0.3
34.82 ± 1.2
48.63 ± 0.8
35.92 ± 0.2
28.93 ± 0.1
37.09 ± 1.8
52.94 ± 0.6
61.90 ± 0.9
42.05 ± 1.2
29.48 ± 1.1
42.11 ± 0.4
41.04 ± 0.2
14.92 ± 0.2
18.53 ± 1.1
17.48 ± 1.5
21.64 ± 0.6
5.03 ± 0.1
9.89 ± 0.4
17.03 ± 1.1
9.49 ± 0.2
28.59 ± 0.7
35.09 ± 2.3
38.26 ± 0.8
28.45 ± 0.4
2.3S
2.4S
H
2.5S
p-CH3
p-Cl
2.6S
p-CH3
m-Cl
2.7S
p-CH3
o,p-di CH3
H
2.8S
o-CH3
p-Cl
2.9S
H
2.10S
2.11S
2.12S
2.13S
2.14S
2.15S
2.16S
2.17S
2.18S
2.19S
2.20S
2.21S
2.22S
2.23S
2.24S
2.25S
2.26S
2.27S
2.28S
2.29S
2.30S
2.31S
2.32S
2.33S
p-Cl
p-Cl
p-Cl
m-Cl
p-Cl
o,p-di CH3
H
p-NHCOCH3
p-NHCOCH3
p-NHCOCH3
p-NHCOCH3
m-NO2
m-NO2
m-NO2
m-NO2
o-NO2
p-Cl
m-Cl
o,p-di CH3
H
p-Cl
m-Cl
o,p-di CH3
H
o-NO2
p-Cl
o-NO2
m-Cl
o-NO2
o,p-di CH3
H
p-NO2
p-NO2
p-Cl
p-NO2
m-Cl
p-NO2
o,p-di CH3
H
p-OCH3
p-OCH3
p-OCH3
p-OCH3
p-Cl
m-Cl
o,p-di CH3
*% inhibition at 10µM concentration, values are mean ± S.D. of triplicate experiment performed independently

Table 2: Physico-chemical properties of Sulfonyl-amino-acetamides derivatives
Compound
numbers
2.1S
MW HBA HBD PSA LogP M T
I
RE Drug
Drug BBB
likeliness score Score
304.08
324.03
324.03
318.10
290.07
338.04
338.04
332.11
304.08
324.03
357.99
374.02
352.06
347.09
361.10
381.05
375.12
335.05
369.01
369.01
363.08
335.05
369.06
369.01
363.08
335.05
369.01
369.01
363.08
320.08
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
4
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
75.27 2.22
75.27 2.31
75.27 2.31
75.27 2.74
75.27 1.71
75.27 2.83
75.27 2.83
75.27 3.25
75.27 2.22
75.27 2.31
75.27 2.92
75.27 2.83
75.27 3.34
104.37 0.95
104.37 1.46
104.37 1.55
104.37 1.97
118.41 1.65
118.41 2.25
118.41 2.25
118.41 2.68
118.41 1.65
118.41 2.25
118.41 2.25
118.41 2.68
118.41 1.65
118.41 2.25
118.41 2.25
118.41 2.68
84.50 1.55
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
R
G
G
G
R
G
G
G
G
R
G
G
G
R
G
G
G
R
G
G
G
R
G
G
G
R
G
G
G
R
Y
G
G
G
Y
G
G
G
R
G
G
G
R
Y
G
G
R
Y
G
G
R
Y
G
G
R
Y
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
-5.61
-3.04
-3.94
-7.14
-4.53
-2.06
-2.86
-6.02
1.62
0.46
0.45
0.26
0.21
0.45
0.45
0.26
0.20
0.80
0.83
0.76
0.46
0.33
0.87
0.80
0.48
0.63
0.44
0.41
0.24
0.20
0.44
0.43
0.25
0.20
0.44
0.41
0.24
0.20
0.85
0.171
0.145
0.145
0.136
0.177
0.139
0.139
0.119
0.171
0.145
0.145
0.145
0.105
0.143
0.111
0.111
0.107
0.131
0.100
0.100
0.092
0.083
0.050
0.050
0.066
0.131
0.100
0.100
0.092
0.085
2.2S
2.3S
2.4S
2.5S
2.6S
2.7S
2.8S
2.9S
2.78
2.10S
2.11S
2.12S
2.13S
2.14S
2.15S
2.16S
2.17S
2.18S
2.19S
2.20S
2.21S
2.22S
2.23S
2.24S
2.25S
2.26S
2.27S
2.28SS
2.29S
2.30S
4.83
4.83
1.26
4.69
6.74
6.31
3.20
-7.06
-4.45
-5.34
-8.55
-5.35
-2.77
-3.66
-6.84
-6.97
-4.48
-5.29
-8.46
2.14

354.04
354.04
348.11
4
4
4
2
2
2
84.50 2.16
84.50 2.16
84.50 2.58
G
G
G
G
G
R
G
R
O
G
G
G
4.60
3.81
0.64
0.83
0.50
0.33
0.058
0.058
0.045
2.31S
2.32S
2.33S
M=mutagenicity, T= tumorigenic, I= Irritant, RE= reproductive effect. Alphabets in column representing M, T, I and RE imply G=
no indication, Y = medium risk and R = high risk.
5. Conclusion
In conclusion, in this report; we have described the design and synthesis of low molecular weight
sulfonyl-amino-acetamide derivatives as BACE-1 inhibitors. Compound 2.17S emerged as a promising
lead; exhibiting relatively higher potency with drug-like profile. The in vitro activity observed was found
to be consistent with docking studies. We envisaged that the potency of the series could be improved by
introducing substituents that extend interactions to S2´ and S3 regions along with S1 region. Further
occupancy of S1 and S3 active sites can be enhanced by including alkyl chain or aromatic rings on ring A
or B, which would contribute to increased compound affinity for BACE-1. Overall, it was observed that
substituents on ring A matter more than substituent on ring B. It can also be noticed that bulkier the ring
substituent, better is the activity. Both the substituents complement each other and adjusting the bulkiness
would improve BACE-1 inhibition.
6. Experimental Section
6.1. Chemistry
Reaction progress was monitored by TLC using precoated Silica gel G plates (Keiselgel 60F254, Merck)
and visualized using UV light. Melting points were measured with Buchi 530 melting point apparatus and
1
were uncorrected. H NMR spectra were recorded on a Bruker Avance- 400MHz system using CDCl3 or
DMSO as the solvent. Chemical shifts (δ) are reported in parts per millions (ppm) relative to TMS as
internal standard. FTIR spectra were performed on IR Prestige 21 Shimadzu using KBr as standard. Mass
spectra were recorded on Waters-Acquity instrument in electrospray mode. Unless otherwise stated, all
reagents were obtained from commercial suppliers and used without further purification. Compounds
were named as per Chem-Draw Ultra 11.0 software.
6.2 Synthetic Procedure
6.2.1. General procedure for the synthesis of compound 1 (1.1S-1.33S)
To approximately 2.5 equivalent of sodium bicarbonate in water, was added 1 equivalent of glycine. The
solution was stirred at 800C till complete dissolution. It was cooled to around 500C and then 1 equivalent
of substituted phenyl sulfonyl chloride was added. The solution was stirred for 2-3 hours at 800C. The

reaction completion was monitored by TLC. The solution was cooled to 100C, neutralized with 1M HCl
and kept for 30 min for precipitation. The solid compound was filtered out using vacuum and dried.20
6.2.2. General procedure for the synthesis of compound 2 (2.1S-2.33S)
To 1 equivalent of compound 1, was added 2.2 equivalent of HOBt and 2.2 equivalent of EDC.HCl.
About 20 ml dichloromethane was added and stirred. To this, was added triethylamine and 2.5 equivalent
of substituted aniline derivative. The reaction was allowed to stir at 00C overnight. The completion of the
reaction was monitored by TLC and then worked up with DCM, dil. HCl and saturated sodium
bicarbonate solution. The compounds were finally purified by column chromatography.
6.3. Characterization details
2-(4-methylphenylsulfonamido)-N-phenylacetamide (2.1S): Off –white solid, yield: 89%, mp 150-152
0C IR (cm-1, KBr): 3363.86 (sec. N-H stretching), 3253.91 (sec. N-H stretching), 3100.5 (Aromatic C-H
1
stretching), 2967 (Aliphatic C-H stretching), 1093.64 (S=O), H NMR (400 MHz, DMSO-d6, δ ppm):
2.34 (s, 3H, methyl), 3.62 (d, 2H, CH2), 6.90- 7.75 (m, 9H, Ar-H), 7.80 (t, 1H, -NH), 9.78 (s, 1H, -NH);
ESI-MS m/z: 305.16 [M+H]+, 327.10 [M+Na]+
N-(4-chlorophenyl)-2-(phenylsulfonamido)acetamide (2.2S): Light brown solid, yield: 78%, mp: 164-
1660C, IR (cm-1, KBr): 3471.87(sec. N-H stretching), 3379.29 (sec. N-H stretching), 3026.31 (Aromatic
C-H stretching), 2985.81 (Aliphatic C-H stretching), 1684.01 (C=O), 1598.20-1535.67 (aromatic C=C
bend) , 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.69 (d, 2H, CH2), 7.00- 7.77 (m, 9H, Ar-H), 7.85 (t, 1H,
-NH), 9.46 (s, 1H, -NH); ESI-MS m/z: 325.14 [M+H]+
N-(3-chlorophenyl)-2-(phenylsulfonamido)acetamide (2.3S): Light brown solid, yield: 81%%, mp:
162-1640C, IR (cm-1, KBr): 3471.83(sec. N-H stretching), 3379.12 (sec. N-H stretching), 3024.51
(Aromatic C-H stretching), 2985.81 (Aliphatic C-H stretching), 1684.01 (C=O), 1598.20-1535.67
1
(aromatic C=C bend), H NMR (400 MHz, DMSO-d6, δ ppm): 3.65 (d, 2H, CH2), 7.05- 7.82 (m, 9H, Ar-
H), 7.89 (t, 1H, -NH), 9.72 (s, 1H, -NH); ESI-MS m/z: 325.14 [M+H]+
N-(2,4-dimethylphenyl)-2-(phenylsulfonamido)acetamide (2.4S): Off-white solid, yield: 82%, mp:
180-1820C, IR (cm-1, KBr): 3481.83(sec. N-H stretching), 3359.42 (sec. N-H stretching), 3025.81
(Aromatic C-H stretching), 2985.81 (Aliphatic C-H stretching), 2965.51 (Aliphatic C-H stretching),
1
1688.01 (C=O), 1598.20-1535.67 (aromatic C=C bend), H NMR (400 MHz, DMSO-d6, δ ppm): 2.39 (s,
3H, o-CH3), 2.59 (s, 3H, p-CH3), 3.59 (d, 2H, CH2), 7.10- 7.95 (m, 8H, Ar-H), 7.99 (t, 1H, -NH), 9.88 (s,
1H, -NH)
0
N-phenyl-2-(phenylsulfonamido)acetamide (2.5S): Dark yellow powder, yield: 82%, mp: 180-182 C,
IR (cm-1, KBr): 3327.21 (sec. N-H stretching), 3234.62 (sec. N-H stretching), 3132.52 (Aromatic C-H
stretching), 2910.58 (Aliphatic C-H stretching), 1691.57 (C=O), 1604.77-1522.67 (aromatic C=C bend),

1H NMR (400 MHz, DMSO-d6, δ ppm): 3.65 (d, 2H, -CH2), 7.00-7.99 (m, 10H, Ar-H), 8.17 (t, 1H, -
NH), 9.81 (s, 1H, –NH)
N-(4-chlorophenyl)-2-(4-methylphenylsulfonamido)acetamide (2.6S): Off-white powder, yield: 79%,
0
mp 187-190 C, IR (cm-1, KBr): 3363.86 (sec. N-H stretching), 3253.91 (sec. N-H stretching), 3043.5
1
(Aromatic C-H stretching), 2978 (Aliphatic C-H stretching), 1093.64 (S=O); H NMR (400 MHz,
DMSO-d6, δ ppm): 2.36 (s, 3H, p-CH3), 3.62 (d, 2H, CH2), 7.25 (d, 2H, Ar-H), 7.31 (d, 2H, Ar-H), 7.49-
7.68 (m, 4H, Ar-H), 7.99 (t, 1H, -NH), 9.94 (s, 1H, -NH)
N-(3-chlorophenyl)-2-(4-methylphenylsulfonamido)acetamide (2.7S): Light brown solid, yield: 78%,
0
mp 178-180 C, IR (cm-1, KBr): 3332.96 (sec. N-H stretching), 3263.86 (sec. N-H stretching), 3078.35
(Aromatic C-H stretching), 2897 (Aliphatic C-H stretching), 1697.36 (C=O), 1595.13- 1444.68 (aromatic
1
C=C stretch), H NMR (400 MHz, DMSO-d6, δ ppm): 2.56 (s, 3H, p-CH3), 4.45 (d, 2H, CH2), 7.45 (d,
2H, Ar-H), 7.61 (d, 2H, Ar-H), 7.69-7.88 (m, 4H, Ar-H), 7.98 (t, 1H, -NH), 9.69 (s, 1H, -NH)
N-(2,4-dimethylphenyl)-2-(4-methylphenylsulfonamido)acetamide (2.8S): Light yellow solid, yield
83%, mp: 184-1860C, IR (cm-1, KBr): 3311.78 (sec. N-H stretching), 3277.06 (sec. N-H stretching),
3026.31 (Aromatic C-H stretching), 2939 (Aliphatic C-H stretching), 1651.07 (C=O), 1593.20 (aromatic
C=C stretch); 1H NMR (400 MHz, CDCl3, δ ppm): 2.17 (s, 3H, p-CH3), 2.27 (s, 3H, o-CH3), 2.42 (s, 3H,
p-CH3), 3.69 (d, 2H, CH2), 5.29 (t, 1H, -NH), 6.96-7.88 (m , 7H, Ar-H), 7.98 (s, 1H, -NH)
2-(2-methylphenylsulfonamido)-N-phenylacetamide (2.9S): Light yellow solid, yield 79%, mp 183-
184 0C, IR (cm-1, KBr): 3340.71 (sec. N-H stretching), 3275.15 (sec. N-H stretching), 3024.38 (Aromatic
C-H stretching), 2962.66 (Aliphatic C-H stretching), 1651.07 (C=O), 1537.27 (aromatic C=C stretch);1H
NMR (400 MHz, DMSO-d6, δ ppm): 2.62 (s, 3H, CH3), 3.68 (d, 2H CH2), 6.99-7.89 (m, 9H, Ar-H), 7.94
(t, 1H, -NH), 9.78 (s, 1H, -NH)
2-(4-chlorophenylsulfonamido)-N-phenylacetamide (2.10S): Yellow solid, yield 78%, mp: 173-1750C,
IR (cm-1, KBr): 3330.78 (sec. N-H stretching), 3265.15 (sec. N-H stretching), 3034.38 (Aromatic C-H
1
stretching), 1641.07 (C=O), 1527.27 (aromatic C=C stretch); H NMR (400 MHz, CDCl3, δ ppm): 3.49
(d, 2H, CH2), 7.16-7.78 (m , 9H, Ar-H), 8.04 (t, 1H, -NH), 9.62 (s, 1H, -NH)
N-(4-chlorophenyl)-2-(4-chlorophenylsulfonamido)acetamide (2.11S): Off-white solid, yield: 83%,
0
mp 125-127 C, IR (cm-1, KBr): 3353.78 (sec. N-H stretching), 3281.27 (sec. N-H stretching), 3156.25
1
(Aromatic C-H stretching), 1730.15 (C=O), 1597.20-1473.43 (aromatic C=C stretch); H NMR (400
MHz, CDCl3, δ ppm): 3.59 (d, 2H, CH2), 7.36-7.78 (m , 8H, Ar-H), 7.95 (t, 1H, -NH), 8.92 (s, 1H, -NH)
N-(3-chlorophenyl)-2-(4-chlorophenylsulfonamido)acetamide (2.12S): Off-white solid, yield: 89%, mp
0
115-117 C, IR (cm-1, KBr): 3343.58 (sec. N-H stretching), 3261.27 (sec. N-H stretching), 3136.25
1
(Aromatic C-H stretching), 1720.15 (C=O), 1567.20-1479.43 (aromatic C=C stretch); H NMR (400
MHz, CDCl3, δ ppm): 3.39 (d, 2H, CH2), 7.26-8.38 (m , 8H, Ar-H), 8.42 (t, 1H, -NH), 8.98 (s, 1H, -NH)

2-(4-chlorophenylsulfonamido)-N-(2,4-dimethylphenyl)acetamide (2.13S): Off-white to light yellow
0
solid, yield: 81%, mp 142-146 C, IR (cm-1, KBr): 3319.78 (sec. N-H stretching), 3263.56 (sec. N-H
stretching), 3124.68 (Aromatic C-H stretching), 2939 (Aliphatic C-H stretching), 2969 (Aliphatic C-H
1
stretching), 1681.93 (C=O), 1593.20- 1500.62 (aromatic C=C stretch); H NMR (400 MHz, CDCl3, δ
ppm): 2.65 (s, 3H, o-CH3), 2.68 (s, 3H, p-CH3), 3.64 (d, 2H, CH2), 7.16-7.78 (m , 7H, Ar-H), 7.86 (t, 1H,
-NH), 8.92 (s, 1H, -NH)
2-(4-acetamidophenylsulfonamido)-N-phenylacetamide (2.14S): Off-white solid, yield:85%, mp 98-
1000C, IR (cm-1, KBr):
3410.58 (sec. N-H stretching), 3319.49 (sec. N-H stretching), 3217.31
(Aromatic C-H stretching), 2864.29 (Aliphatic C-H stretching), 1716.65 (C=O), 1587.42- 1438.90
1
(aromatic C=C stretch); H NMR (400 MHz, DMSO-d6, δ ppm): 2.49 (s, 3H, -COCH3), 3.61 (s, 2H, -
CH2), 5.89 (s, 1H, -NHCO), 7.02-7.84 (m, 9H, Ar-H), 7.92(t, 1H, -NH), 8.83 (s, 1H, -NH)
2-(4-acetamidophenylsulfonamido)-N-(4-chlorophenyl)acetamide (2.15S): Off-white solid, yield:
0
82%, mp 155-160 C, IR (cm-1, KBr): 3388.98 (sec. N-H stretching), 3300.20, 3275.13 (sec. N-H
stretching), 3054.31 (Aromatic C-H stretching), 2939 (Aliphatic C-H stretching), 1693.50 (C=O), 1670-
1
1535.34 (aromatic C=C stretch), H NMR (400 MHz, DMSO-d6, δ ppm): 2.67 (s, 3H, -COCH3), 3.81 (s,
2H, -CH2), 5.54 (s, 1H, -NHCO), 7.12-7.52 (m, 8H, Ar-H), 7.63 (t, 1H, -NH), 9.21 (s, 1H, -NH), ESI-MS
m/z: 382.12 [M+H]+
2-(4-acetamidophenylsulfonamido)-N-(3-chlorophenyl)acetamide (2.16S): Off-white solid, yield:
91%, mp 164-1680C, IR (cm-1, KBr): 3435.78 (sec. N-H stretching), 3277.06 (sec. N-H stretching),
3026.31 (Aromatic C-H stretching), 2971 (Aliphatic C-H stretching), 1678.07 (C=O), 1598.20-1535.67
1
(aromatic C=C stretch), H NMR (400 MHz, DMSO-d6, δ ppm): 2.73 (s, 3H, -COCH3), 3.24 (s, 2H, -
CH2), 5.21 (s, 1H, -NHCO), 7.05-7.84 (m, 8H, Ar-H), 7.95 (t, 1H, -NH), 9.28 (s, 1H, -NH)
2-(4-acetamidophenylsulfonamido)-N-(2,4-dimethylphenyl)acetamide (2.17S): Off-white solid; yield:
88%, mp 152-1540C, IR (cm-1, KBr): 3317.78 (sec. N-H stretching), 3269.06 (sec. N-H stretching),
3120.31 (Aromatic C-H stretching), 2924 (Aliphatic C-H stretching), 2872.65 (Aliphatic C-H stretching),
1
1662.07 (C=O), 1588.70-1535.67 (aromatic C=C stretch), H NMR (400 MHz, DMSO-d6, δ ppm): 2.08
(s, 3H, o-CH3), 2.10 (s, 3H, p-CH3), 2.22 (s, 3H, -COCH3), 3.64 (s, 2H, -CH2), 6.99 (s, 1H, Ar-H), 7.14-
7.16 (d, 1H, Ar-H), 7.73-7.75 (m, 5H, Ar-H), 9.19 (s, 1H, -NH), 10.35-10.33 (2s, 2H, -NH), 13C NMR
(400 MHz, DMSO-d6, δ ppm): 18.00, 20.92, 24.60, 45.91, 118.96, 125.00, 126.88, 128.31, 131.23,
131.74, 133.58, 134.23, 134.73, 143.34, 166.85, 169.42, ESI-MS m/z: 376.21 [M+H]+
2-(3-nitrophenylsulfonamido)-N-phenylacetamide (2.18S): Off-white solid, yied:79%, mp 172-176 0C,
IR (cm-1, KBr): 3435.78 (sec. N-H stretching), 3290.56 (sec. N-H stretching), 3026.31 (Aromatic C-H
stretching), 1647.21 (C=O), 2985.84 (Aliphatic C-H stretching), 1620.97-1532.81 (aromatic C=C stretch),

1H NMR (400 MHz, DMSO-d6, δ ppm): 3.82 (s, 2H, -CH2), 7.32-7.95 (m, 9H, Ar-H), 8.14 (t, 1H, -NH),
9.08 (s, 1H, -NH); ESI-MS m/z: 336.11 [M+H]+, 358.10 [M+Na]+
N-(4-chlorophenyl)-2-(3-nitrophenylsulfonamido)acetamide (2.19S): Off-white solid, yield: 72%, mp
0
185-187 C, IR (cm-1, KBr): 3350.35 (sec. N-H stretching), 3277.06 (sec. N-H stretching), 3070.68
(Aromatic C-H stretching), 2975.84 (Aliphatic C-H stretching), 1670.35 (C=O), 1598.20-1535.67
1
(aromatic C=C bend), H NMR (400 MHz, DMSO-d6, δ ppm): 3.62 (s, 2H, -CH2), 7.22-7.99 (m, 8H, Ar-
H), 8.13 (t, 1H, -NH), 9.27 (s, 1H, -NH)
N-(3-chlorophenyl)-2-(3-nitrophenylsulfonamido)acetamide (2.20S): Light brown powder, yield:
0
75%, mp 150-154 C, IR (cm-1, KBr): 3367.71 (sec. N-H stretching), 3219.19 (sec. N-H stretching),
1
3101.54 (Aromatic C-H stretching), 1697.36 (Amide C=O), 1593.20-1415.75 (aromatic C=C bend), H
NMR (400 MHz, DMSO-d6, δ ppm): 3.51 (s, 2H, -CH2), 7.05-7.81 (m, 8H, Ar-H), 7.99 (t, 1H, -NH), 8.47
(s, 1H, -NH); ESI-MS m/z: 370.08 [M+H]+
N-(2,4-dimethylphenyl)-2-(3-nitrophenylsulfonamido)acetamide (2.21S): Light brown powder; yield:
71%, mp 158-1600C, IR (cm-1, KBr): 3465.78 (sec. N-H stretching), 3237.06 (sec. N-H stretching),
3006.31 (Aromatic C-H stretching), 2977 (Aliphatic C-H stretching), 1688.07 (C=O), 1588.70-1535.67
(aromatic C=C stretch), 1H NMR (400 MHz, DMSO-d6, δ ppm): 2.43 (s, 3H, o-CH3), 2.51 (s, 3H, p-CH3),
3.52 (s, 2H, -CH2), 7.22-7.96 (m, 7H, Ar-H), 8.98 (s, 1H, -NH)
2-(2-nitrophenylsulfonamido)-N-phenylacetamide (2.22S): Dark brown solid, yield: 73%, mp 160-161
0C; IR (cm-1, KBr): 3445.28 (sec. N-H stretching), 3280.76 (sec. N-H stretching), 3036.37 (Aromatic C-H
stretching), 1645.11 (C=O), 2988.84 (Aliphatic C-H stretching), 1622.97-1530.81 (aromatic C=C stretch),
1H NMR (400 MHz, DMSO-d6, δ ppm): 3.80 (s, 2H, -CH2), 7.12-7.85 (m, 9H, Ar-H), 8.01 (t, 1H, -NH),
9.25 (s, 1H, -NH)
N-(4-chlorophenyl)-2-(2-nitrophenylsulfonamido)acetamide (2.23S): Brown solid, yield: 69%, mp
141-1440C, IR (cm-1, KBr): 3369.64 (sec. N-H stretching), 3217.27 (sec. N-H stretching), 3099.31
(Aromatic C-H stretching), 2873.94 (Aliphatic C-H stretching), 1703.14 (C=O), 1614-1531.48 (aromatic
1
C=C stretch), H NMR (400 MHz, DMSO-d6, δ ppm): 3.62 (s, 2H, -CH2), 6.95-7.72 (m, 8H, Ar-H), 7.91
(t, 1H, -NH), 8.95 (s, 1H, -NH)
N-(3-chlorophenyl)-2-(2-nitrophenylsulfonamido)acetamide (2.24S): Brown solid; yield: 74%, mp
182-1840C, IR (cm-1, KBr): 3357.41 (sec. N-H stretching), 3229.19 (sec. N-H stretching), 3121.54
(Aromatic C-H stretching), 1687.36 (Amide C=O), 1597.10-1415.75 (aromatic C=C bend), 1H NMR (400
MHz, DMSO-d6, δ ppm): 3.57 (s, 2H, -CH2), 6.87-7.82 (m, 8H, Ar-H), 7.90 (t, 1H, -NH), 9.01 (s, 1H, -
NH)
N-(2,4-dimethylphenyl)-2-(2-nitrophenylsulfonamido)acetamide (2.25S): Light yellow solid, yield:
0
73%, mp 165-167 C, IR (cm-1, KBr): 3370.64 (sec. N-H stretching), 3255.84 (sec. N-H stretching),

3043.67 (Aromatic C-H stretching), 2945.30 (Aliphatic C-H stretching), 1616.35 (C=O), 1598.53-
1
1465.87 (aromatic C=C bend), H NMR (400 MHz, DMSO-d6, δ ppm): 2.35(s, 3H, o-CH3), 2.57 (s, 3H,
p-CH3), 3.46 (s, 2H, -CH2), 7.05-7.76 (m, 7H, Ar-H), 7.95 (t, 1H, -NH), 9.12 (s, 1H, -NH)
2-(4-nitrophenylsulfonamido)-N-phenylacetamide (2.26S): Light yellow powder; yield: 83%, mp 175-
1770C, IR (cm-1, KBr): 3465.28 (sec. N-H stretching), 3260.76 (sec. N-H stretching), 3056.37 (Aromatic
C-H stretching), 1655.11 (C=O), 2978.84 (Aliphatic C-H stretching), 1632.97-1530.81 (aromatic C=C
1
stretch), H NMR (400 MHz, DMSO-d6, δ ppm): 3.65 (s, 2H, -CH2), 7.22-7.85 (m, 9H, Ar-H), 8.79 (s,
1H, -NH)
N-(4-chlorophenyl)-2-(4-nitrophenylsulfonamido)acetamide (2.27S): White solid, yield: 79%, mp 90-
920C, IR (cm-1, KBr): 3435.78 (sec. N-H stretching), 3277.06 (sec. N-H stretching), 3026.31 (Aromatic
C-H stretching), 2971 (Aliphatic C-H stretching), 1678.07 (C=O), 1598.20-1535.67 (aromatic C=C
1
stretch), H NMR (400 MHz, DMSO-d6, δ ppm): 3.41 (s, 2H, -CH2), 6.91-7.54 (m, 8H, Ar-H), 7.98 (t,
1H, -NH), 8.87 (s, 1H, -NH)
N-(3-chlorophenyl)-2-(4-nitrophenylsulfonamido)acetamide (2.28S): Off-white solid; yield: 72%, mp
120-1220C, IR (cm-1, KBr): 3357.41 (sec. N-H stretching), 3229.19 (sec. N-H stretching), 3121.54
(Aromatic C-H stretching), 1687.36 (Amide C=O), 1597.10-1415.75 (aromatic C=C bend), 1H NMR (400
MHz, DMSO-d6, δ ppm): 3.42 (s, 2H, -CH2), 7.05-8.10 (m, 8H, Ar-H), 8.25 (t, 1H, -NH), 9.45 (s, 1H, -
NH)
N-(2,4-dimethylphenyl)-2-(4-nitrophenylsulfonamido)acetamide (2.29S): Off-white solid; yield: 84%,
mp 111-1130C , IR (cm-1, KBr): 3370.64 (sec. N-H stretching), 3255.84 (sec. N-H stretching), 3043.67
(Aromatic C-H stretching), 2945.30 (Aliphatic C-H stretching), 1616.35 (C=O), 1598.53-1465.87
1
(aromatic C=C bend), H NMR (400 MHz, DMSO-d6, δ ppm): 2.52 (s, 3H, o-CH3), 2.65 (s, 3H, p-CH3),
3.97 (s, 2H, -CH2), 6.98-7.96 (m, 7H, Ar-H), 8.01 (t, 1H, -NH), 9.65 (s, 1H, -NH)
2-(4-methoxyphenylsulfonamido)-N-phenylacetamide (2.30S): Dark yellow solid, yield: 83%, mp 158-
160 0C, IR (cm-1, KBr): 3369.64 (sec. N-H stretching), 3292.49 (sec. N-H stretching), 3097.68 (Aromatic
C-H stretching), 2883.58 (Aliphatic C-H stretching), 1673.86(C=O), 1598.20-1535.67 (aromatic C=C
1
stretch), H NMR (400 MHz, DMSO-d6, δ ppm): 3.49 (s, 3H, -OCH3), 3.54 (s, 2H, -CH2), 7.05-7.94 (m,
9Ar-H, 1-NH), 9.98 (s, 1H, -NH)
N-(4-chlorophenyl)-2-(4-methoxyphenylsulfonamido)acetamide (2.31S): Light brown powder, yield:
68%, mp 156-1580C, IR (cm-1, KBr): 3327.21 (sec. N-H stretching), 3234.62 (sec. N-H stretching),
3076.46 (Aromatic C-H stretching), 2910.68 (Aliphatic C-H stretching), 1691.57 (C=O), 16-4.77-1552.70
1
(aromatic C=C bend), H NMR (400 MHz, DMSO-d6, δ ppm): 3.55 (s, 3H, -OCH3), 3.84 (s, 2H, -CH2),
7.15-7.95 (m, 8H, Ar-H), 8.05 (t, 1H, -NH), 9.54 (s, 1H, -NH)

N-(3-chlorophenyl)-2-(4-methoxyphenylsulfonamido)acetamide (2.32S): Yellow solid, yield: 74%,
mp 128-1300C, IR (cm-1, KBr): 3350.35 (sec. N-H stretching), 3277.06 (sec. N-H stretching), 3072.31
(Aromatic C-H stretching), 2877.79 (Aliphatic C-H stretching), 1670.35 (C=O), 1597.06- 1492.90
1
(aromatic C=C stretch), 1348.24 (C-O stretch), H NMR (400 MHz, DMSO-d6, δ ppm): 3.65 (s, 3H, -
OCH3), 3.86 (s, 2H, -CH2), 7.12-7.96 (m, 8H, Ar-H), 8.11 (t, 1H, -NH), 9.28 (s, 1H, -NH)
N-(2,4-dimethylphenyl)-2-(4-methoxyphenylsulfonamido)acetamide (2.33S): Light brown solid, yield:
79%, mp 123-1250C , IR (cm-1, KBr): 3350.35 (sec. N-H stretching), 3277.06 (sec. N-H stretching),
3072.60 (Aromatic C-H stretching), 2877.79 (Aliphatic C-H stretching), 1670.35 (C=O), 1597.06-
1
1492.90 (aromatic C=C stretch), 1348.24 (C-O stretch), H NMR (400 MHz, DMSO-d6, δ ppm): 2.49 (s,
3H, o-CH3), 2.55 (s, 3H, p-CH3), 3.55 (s, 3H, -OCH3), 3.41 (s, 2H, -CH2), 7.22-7.96 (m, 8H, Ar-H, -NH),
9.98 (s, 1H, -NH)
6.4. Docking simulation and in-silico physicochemical properties prediction
Protein was prepared using protein preparation wizard of Maestro. The prepared protein was used as an
input file for generating the receptor grid file, which was used as input file for docking simulation. Grid of
active site was created using Maestro. A centroid at on x, y, z axis respectively was used to define the grid
box. Grid box length was set to 67.43 Å, 46.45 Å and 10.57 Å along x, y and z directions respectively.
Preparation of ligands was performed using “LigPrep” module of Schrodinger Suite 2013. All the
compounds were drawn using Chemaxon Marvin Sketch and prepared with ligand preparation wizard of
Maestro. The ionization states in a given pH range of 7 ± 2 were produced by adding or removing protons
from the ligand using EPIK 2.1 module. OPLS 2005 Force Field was selected for energy minimization.
During docking simulation, using different modules of Schrödinger suite, potential of non-polar parts of
ligands was softened by scaling Vander Waals radii of ligand atoms by 0.8 Å with partial charge cut-off
of 0.15. During docking simulation, glide first places the centre of ligand at various grid positions of a 1
Å grid and then by rotating ligand in all the Euler angles it generates various possible conformations
which pass through a filter series composed of initial rough positioning followed by scoring phase. The
docking simulation was performed by allowing flexible torsions in ligands with the use of XP mode. The
parameter selected for docking run was default and a model energy function named Glide score (Gscore)
is used which combines force field and empirical terms for selecting the best docking pose, generated as
output. The molecular docking simulations output file, having all the thermodynamics information in the
form of Glide score, were analyzed using Glide XP visualizer, which enables visualization of ligand-
receptor interactions in an interactive manner.

Validation of docking protocol was done to govern the reliability and reproducibility of the docking
parameters used for the study. The docking strategy revealed that the docked ligand superimposed well
with the reference ligands (co-crystallized ligand) with RMSD value of 0.385Å (docking pose given in
supplementary information).
Oral bioavailability prediction was done using JChem for Excel and drug likeliness was calculated using
OSIRIS property explorer, software of Actelion Pharmaceuticals Ltd., Switzerland, hosted on
http://www.organic-chemistry.org website. Blood brain permeation was predicted using Online BBB
predictor. The physicochemical properties for the synthesized compounds are shown in Table 2 given
below.
6.5 FRET Assay
BACE-1 in-vitro assay was carried out using Fluorescence Resonance Energy Transfer (FRET)
technique. The BACE-1 FRET assay kit was supplied by Pan Vera, Life Technologies (Kit no P2985,
Madison, WI, USA) and the assay was carried out according to the protocol provided by the supplier. The
measure of inhibitory activity of compounds is based upon the reduction in the fluorescence quantum
yield due to inhibition of BACE-1 enzymatic activity. The protease substrate (APP-based peptide)
consists of a fluorescence donor (rhodamine) connected to a quenching acceptor through a peptide
sequence (EVNLDAEFK). The fluorescence is weak intrinsically due to resonance energy transfer but
upon enzymatic cleavage, the quantum yield of donor molecule increases. Inhibitory potential of the
designed compounds can be governed from the reduced fluorescence quantum yield. The assay was
conducted in 50mM sodium acetate buffer (pH 4.5) using 3X BACE-1 enzyme (1U/ml). The screening of
test compounds was performed at 10µM concentration. The test compounds were diluted with DMSO to
prepare the stock solution of 10mM and further diluted with buffer to achieve desired concentration of
10µM. The incubation time was kept 60 minutes following which the fluorescence was read on Tecan
multiwell spectrofluorimeter (excitation: 545 nm; emission: 590 nm). IC50 was determined for the most
active compound using nonlinear regression parameters employing Graph Prism Pad 5.0 software.
Conflict of Interest
The authors confirm that this article content has no conflict of interest.
Supplementary Information: Docking poses of selected compounds and charaterization details of few
compounds are shown in supplementary information.

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Figure captions
Figure 1: Sulfonyl-amino-acetamide motif compared to reported acyl guanidine derivative
Figure 2: Predicted binding mode of compound 2.1S in the active site of BACE-1 (PDB ID: 2OHP) i:
Chemical structure of compound 2.1S; ii: Docking view of 2.1S showing hydrogen bond in green color
and ligand as sticks with protein colored as shapely residue scheme; iii: Docking view of 2.1S with ligand
as ball and sticks and protein as cartoon assembly. The dashed lines in green represent hydrophobic
interactions.
Figure 3. A. Docking pose of compound 2.17S showing hydrogen bonding interactions in green; B.
Docking pose of compound 2.17S showing hydrophobic interactions in green; C: 2D interaction plot

Graphical abstract