Rational design, synthesis and in vitro evaluation of allylidene hydrazinecarboximidamide derivatives as BACE-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.11.044

BACE-1 (β-secretase) is considered to be one of the promising targets for treatment of Alzheimer's disease as it catalyzes the rate limiting step of Aβ-42 production. Herein, we report a novel class of allylidene hydrazinecarboximidamide derivatives as moderately potent BACE-1 inhibitors, having aminoguanidine substitution on allyl linker with two aromatic groups on either side. A library of derivatives was designed based on the docking studies, synthesized and evaluated for BACE-1 inhibition in vitro. The designed ligands displayed interactions with the catalytic aspartate dyad through guanidinium functionality. Further, the aromatic rings placed on either side of the linker occupied S1 and S3 active site regions contributing to the activity. These ligands were also predicted to follow Lipinski rule and cross blood brain barrier. Compound 2.21, having high docking score, was found to be most active with IC₅₀ of 6.423 μM indicating good correlation with docking prediction.

Full text of DOI:10.1016/j.bmcl.2015.11.044

Bioorganic & Medicinal Chemistry Letters 26 (2016) 33–37
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Rational design, synthesis and in vitro evaluation of allylidene
hydrazinecarboximidamide derivatives as BACE-1 inhibitors
Priti Jain a,b, Pankaj K. Wadhwa b, Shilpa Rohilla b, Hemant R. Jadhav a,b,
⇑
a Computational Chemistry Laboratory, Department of Pharmacy, Birla Institute of Technology and Science Pilani, Pilani Campus, Rajasthan 333 031, India
b Medicinal Chemistry Laboratory, Department of Pharmacy, Birla Institute of Technology and Science Pilani, Pilani Campus, Rajasthan 333 031, India
a r t i c l e i n f o
a b s t r a c t
Article history:
BACE-1 (b-secretase) is considered to be one of the promising targets for treatment of Alzheimer’s disease
as it catalyzes the rate limiting step of Ab-42 production. Herein, we report a novel class of allylidene
hydrazinecarboximidamide derivatives as moderately potent BACE-1 inhibitors, having aminoguanidine
substitution on allyl linker with two aromatic groups on either side. A library of derivatives was designed
based on the docking studies, synthesized and evaluated for BACE-1 inhibition in vitro. The designed
ligands displayed interactions with the catalytic aspartate dyad through guanidinium functionality.
Further, the aromatic rings placed on either side of the linker occupied S1 and S3 active site regions con-
tributing to the activity. These ligands were also predicted to follow Lipinski rule and cross blood brain
Received 22 September 2015
Revised 9 November 2015
Accepted 14 November 2015
Available online 14 November 2015
Keywords:
Alzheimer’s disease
BACE-1
Allylidene hydrazinecarboximidamide
Docking simulation
barrier. Compound 2.21, having high docking score, was found to be most active with IC50 of 6.423 lM
indicating good correlation with docking prediction.
Ó 2015 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder. It leads to memory loss, steady deterioration of cognition,
and dementia. AD is the sixth leading cause of death in United
States whereby every 1 in 3 elderly dies with Alzheimer’s.1 By
2025, about 7.1 million people of age 65 and older will suffer from
AD, which represents a 40% increase from the present affected pop-
ulation. It bears an annual cost of approximately $226 billion
which is proposed to be over $1.1 trillion in 2050. Hence, it is
ranked as the costliest chronic disease.2–5
while minor portion of APP is cleaved by b-secretases resulting in
production of soluble APPb and a C-99 C-terminal fragment. This
fragment is further cleaved by c-secretase within the transmem-
brane domain to generate Ab-42. Among the various Ab isoforms
(Ab-39, Ab-40 and Ab-42), Ab-42 is more hydrophobic and fibrillo-
genic, which renders it the ability to aggregate and form oligomers,
accumulate in the brain and initiate a cascade of events resulting in
neuronal dysfunctioning, neurodegeneration and neuronal fatality.
Silencing BACE-1 can reduce Ab-42 production and therefore it
represents a viable therapeutic strategy.8–10 Furthermore, the crys-
tal structures of BACE-1, co-crystallized with different ligands,
reveal the basic requirements and key features needed for its inhi-
bition. It proposes the necessity of a group that acts as H-bond
donor, so that it can form H-bonding interactions with catalytic
aspartate dyad that is, Asp32 and Asp228. Further, large hydropho-
bic pockets (S1, S3, S20) are key regions and demand hydrophobic
groups to fill these pockets. S1 hydrophobic cleft is formed by
the side chains of Tyr71, Phe108, Trp115, Ile118, and Leu30. Like
S1, S3 is also largely hydrophobic pocket formed by side chains
of Leu30, Tyr71, Trp115, and Ile110, as well as main chain of
Gln12, Gly11, Gly230, Thr231, Thr232 and Ser35 while S20
comprises of Ile126, Trp76, Val69, Arg128, Tyr198 amino acid
residues. Earlier reported inhibitors were transition-state mimetics
having ADME problems, structural complexities and poor blood
brain barrier penetration.10–13 There is need of small non-peptidic
inhibitors that would bind to BACE-1 active site and demonstrate
efficacy.
Despite of large population suffering from AD, the number of
therapeutic options in market remain a few. At present, four drugs
(tacrine, donepezil, rivastigmine and galantamine) have been
approved for AD treatment.6 These drugs, however, are not able
to alter or prevent disease progression. They are, instead, palliative
in alleviating the symptoms of disease. No disease-modifying ther-
apy is available yet. One of the major characteristic and patholog-
ical hallmarks of AD is represented by the senile plaques chiefly
composed of cytotoxic amyloid-b peptide (Ab-42), of which
production and deposition is the central event in pathogenesis of
Alzheimer’s disease (AD). It is further characterized by neuroin-
flammation and neuronal dysfunction ultimately leading to
neuronal death. Ab-42 is excised from the amyloid precursor pro-
tein (APP) through sequential actions of
a
, b, and
c
secretases.7
Cleavage by
a-secretases governs the non-amyloidogenic pathway
⇑
Corresponding author. Tel.: +91 1596 515506.
E-mail address: hemantrj@pilani.bits-pilani.ac.in (H.R. Jadhav).
http://dx.doi.org/10.1016/j.bmcl.2015.11.044
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

34
P. Jain et al. / Bioorg. Med. Chem. Lett. 26 (2016) 33–37
Scheme 1. Reagents and conditions: (a) NaOH, EtOH, rt, 4–6 h and (b) aminoguanidine–HCl, concd HCl, THF, reflux 20–24 h.
In this paper, we report the design, synthesis and in vitro eval-
residues on either side to occupy S1 and S3 cavities; and a hydro-
uation of low molecular weight allylidene hydrazinecarboximi-
damide derivatives as BACE-1 inhibitors. Compounds 2.1–2.24
were synthesized as per the synthetic route outlined in Scheme 1.
Docking studies were performed for these compounds to predict
their relative binding affinities and binding modes in the active site
of the BACE-1 enzyme. The blood–brain barrier (BBB) permeation
was predicted using online BBB prediction software available at
http://www.cbligand.org/BBB/. Further, in vitro BACE-1 inhibition
was measured using fluorescence resonance energy transfer (FRET)
assay to draw structure activity relationship (SAR).
Considering the structural requirements for BACE-1 inhibition,
we initially aimed at identifying a potential moiety that could
interact with catalytic aspartate dyad and position the ring sys-
tems in such a manner that S1 and S3 active sites could be occu-
pied via hydrophobic interactions. Earlier, we had designed
sulfonyl amino acetamide derivatives and 2-amino pyrimidine
derivatives, which indicated that 3 atom linker with two aromatic
gen bond donor to bind aspartate dyad is essential (unpublished
data from our lab). Among the possible scaffolds, allylidene
hydrazinecarboximidamide positioned with two aromatic rings
on its either side was considered. A prototype (compound 2.1)
bearing unsubstituted phenyl rings was initially docked. The dock-
ing simulation revealed the following interactions (Fig. 1): (i) –NH–
of imine formed hydrogen bond interaction with Asp32 and
Asp228 (3.31 Å, 3.28 Å, respectively); (ii) –NH2 group displayed
hydrogen bonding interactions with Asp32 (3.18 Å, 3.37 Å); (iii)
ring A was seen to occupy S3 cavity and (iv) ring B was seen to
occupy S1 cavity forming hydrophobic interactions with Phe108,
Ile110, Leu30. On this basis, we synthesized a library of allylidene
hydrazinecarboximidamides by introducing different substituents
on aromatic ring systems.
The synthesis of allylidene hydrazinecarboximidamides is
illustrated in Scheme 1. Benzylidene acetophenones (compounds
1.1–1.24) were prepared by condensation of substituted
Figure 1. Docking pose of the prototype compound 2.1. (A) Chemical structure of compound 2.1; (B) docked view of compound 2.1 with ligand represented as sticks and
protein colored as per shapely residue scheme and (C) 2D interaction plot of compound 2.1.

P. Jain et al. / Bioorg. Med. Chem. Lett. 26 (2016) 33–37
35
Table 1
and high (48%) inhibition. The reason may be explained through
strong interactions observed in docking simulation: (i) ring A occu-
pied S3 active site region with the nitro group hydrogen bonded to
Gly11 and Thr232; (ii) aminoguanidinium formed interactions
with catalytic aspartate dyad; (iii) imine formed hydrogen bond
with Gly230 and (iv) guanidinium group also formed interaction
with Thr231. Introducing 2,5-dimethoxy (2.7) and p-dimethy-
lamino (2.9) group gave less potent molecules probably due to
weaker interactions with the enzymatic active site.
Docking and in vitro assay data for allylidene hydrazinecarboximidamide derivatives
Further, to expand the scope of SAR, we incorporated sub-
stituents on both the rings synthesizing 4 series of derivatives. In
first series, ring A was substituted with m-nitro group and sub-
stituents on ring B were changed. It was observed that p-nitro
group substitution (compound 2.11) displayed high potency with
62% inhibition. In docking studies, it displayed good interaction
pattern (Fig. 2i). Its guanidinium moiety formed hydrogen bonding
interactions with the key aspartate dyad (Asp228: 2.99 Å, 3.16 Å,
Asp 32:2.75 Å, 2.99 Å) and also with Gly230 (3.52 Å and 3.23 Å).
Ring B protruded well within the S1 cavity as nitro group helps
Code
R1
R2
Glide score
% inhibitiona
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.9
2.10
2.11
2.12
2.13
2.14
2.15
2.16
2.17
2.18
2.19
2.20
2.21
2.22
2.23
2.24
H
H
H
H
H
ꢀ5.62
ꢀ6.12
ꢀ8.47
ꢀ4.93
ꢀ4.18
ꢀ6.41
ꢀ5.18
ꢀ4.14
ꢀ8.94
ꢀ7.85
ꢀ9.59
ꢀ9.17
ꢀ7.43
ꢀ6.16
ꢀ3.48
ꢀ5.21
ꢀ10.09
ꢀ10.25
ꢀ10.53
ꢀ10.38
ꢀ7.38
ꢀ6.03
ꢀ9.58
10.84 0.03
13.50 0.01
ND
23.40 0.18
18.00 0.08
47.93 0.05
32.58 0.32
IA
42.13 0.27
62.14 0.07
11.04 0.09
5.40 0.56
10.76 0.55
10.83 0.07
23.95 0.40
27.97 0.21
23.94 0.43
10.33 0.09
61.18 0.04
78.23 0.09
45.83 0.14
21.83 0.52
25.94 0.06
p-NO2
m-OH
m-NO2
p-Cl
H
H
H
m-NO2
2,5-di-OCH3
p-NMe2
m,p-di-OCH3
m-NO2
m-NO2
m-NO2
in extending deeper in the pocket and also formed
p–p stacking
H
H
with Tyr71. m-Nitro of ring A was seen to occupy S3 pocket with
hydrogen bonding interactions with Thr232 (3.16 Å, 3.23 Å,
2.23 Å). It also formed hydrogen bond with water molecule since
it was solvent exposed. Placing p-methoxy (2.12), o,p-dichloro
(2.13) or m-Br (2.14), although showed good interactions in dock-
ing, were less active, which may be attributed to poor hydrophobic
interactions in S1 region.
p-NO2
p-OCH3
o,p-diCl
m-Br
p-CH3
p-NO2
m-NO2
m-Br
p-OCH3
p-NO2
p-Cl
m-NO2
p-NMe2
p-NMe2
p-NMe2
2,5-di-OCH3
2,5-di-OCH3
m,p-di-OCH3
m,p-di-OCH3
m,p-di-OCH3
m,p-di-OCH3
m,p-di-OCH3
In the second series, p-dimethylamino group was substituted on
ring A with alterations on ring B. It was observed that all three
compounds (2.15, 2.16 and 2.17) had low docking scores and weak
activity. It can be proposed that polar nature of p-dimethylamino
group having high electron density bars it from eliciting hydropho-
bic and van der Waal interactions and hence ring A fails to com-
pletely occupy S3 region. The third series with 2,5-di-methoxy
group on ring A and m-bromo (2.18) and p-methoxy (2.19) on ring
B also had poor inhibitory profile. It was observed that 2,5-di-
methoxy group on ring A changes the conformation in such as
way that ring B is pushed out of S1 active site (Fig. 2iv). However,
docking scores were good because the substituents formed hydro-
gen bonds with amino acids outside the cavity. In the last series
with m,p-di-methoxy group on ring A, favorable interaction pat-
tern was seen with high docking scores and BACE-1 inhibitory
potential (compounds 2.20–2.24). It was observed that para elec-
tron withdrawing substitution on ring B is preferred over o,p- or
meta. Compound 2.20 (p-nitro on ring B) and 2.21 (p-chloro on ring
B) had highest inhibition of 61.18% and 78.23%, respectively. The
enhanced potency of compound 2.21 could be rationalized from
its docking pose. The docking pose for most active compound
(2.21) displayed following interactions (Fig. 2ii): (i) methoxy group
at para position formed hydrogen bond with Thr232; (ii) guani-
dinium functionality showed strong hydrogen bonding with cat-
alytic aspartate dyad; (iii)–NH2 formed hydrogen bonding with
Thr231 and Gly230; (iv) ring B occupied S1 active site cleft through
m-NO2
o,p-di-Cl
m-Br
a
% inhibition at 10 lM concentration, values are mean SD of triplicate exper-
iment performed independently; IA = inactive, ND = not done.
benzaldehydes and acetophenones in the presence of base. The
reaction commences with formation of enolate ion by removal of
-hydrogen in presence of hydroxide. The nucleophilic enolate
a
then attacks the carbonyl carbon of aromatic aldehyde to form
an intermediate alkoxide (nucleophilic addition reaction) which
then deprotonates producing hydroxide ion and b-hydroxyketone,
the aldol product. Finally, hydroxide removes acidic b-hydrogen
giving the reactive enolates. The electrons associated with a nega-
tive charge of the enolate are used to form a carbon–carbon double
bond (C@C) and displace a leaving group, regenerating the hydrox-
ide giving the final product, the conjugated ketone. It then reacts
with aminoguanidine hydrochloride to form carbinolamine inter-
mediate, which liberates a water molecule and ultimately results
in the formation of allylidene hydrazinecarboximidamide (2.1–
2.24).
Synthesized compounds were tested for BACE-1 inhibition at
10 lM concentration and docking was performed using Glide
(Schrodinger). The docking scores and % BACE-1 inhibition values
are given in Table 1. Exploring the compounds through docking
studies revealed the occupance of substrate binding cavities and
interactions with active site amino acids. Substituting ring B alone
with electron poor and electron rich moieties was not favorable for
BACE-1 inhibition. Among ring B substituted compounds, 2.2 with
p-nitro group had high docking score compared to the compounds
2.3, 2.4 and 2.5. This may be because p-nitro group is engulfed
deep in the S1 region while m-nitro group (2.4) was slightly sol-
vent exposed. In the in vitro study, however, contrary to docking
scores, compound 2.4 had higher inhibition as compared to others.
When ring A alone was substituted (compounds 2.6–2.10), it was
observed that compound 2.6 with m-nitro had high docking score
hydrophobic dispersion interactions and also revealed p–p stack-
ing with Tyr71; (v) ring A occupied S3 active site region. o,p-
Dichloro and m-bromo make the ring B solvent exposed rendering
the molecules less active. It can be proposed that molecules pos-
sessing electron withdrawing or moderately activating sub-
stituents capable of interacting with S3 region amino active
residues on ring A and electron poor functionalities on ring B cap-
able of forming
p–p stacking with Tyr71 (S1 amino acid residue)
display high inhibitory profile.
Compound 2.21 was studied further and was found to have IC50
of 6.423 lM, which confirms our hypothesis that for BACE-1 inhi-
bition, 3 atom linker with substituted aromatic residues on either
side and hydrogen bond donor to bind aspartate dyad is crucial.

36
P. Jain et al. / Bioorg. Med. Chem. Lett. 26 (2016) 33–37
Figure 2. Docked poses of representative compounds. (i) Docking view of 2.11; (ii) docking view of 2.21; (iii) secondary view of protein showing superimposition of 2.20
(chrome), 2.21 (gray), 2.18 (brown) and (iv) electrostatic surface showing superimposed compounds 2.19 and 2.21 (more poses can be seen in Supplementary data).
In light of these promising in vitro–in silico results, drug likeli-
ness and few molecular descriptors accounting for oral bioavail-
ability and blood brain barrier permeability were determined
(Table 2 in Supplementary Data). It was observed that molecular
weight for all the compounds was less than 405 Da, logP was
within the range of 2–5 and polar surface area for many com-
pounds was less than 95 Å2 and overall less than 140 Å2. As has
been reported, compounds having PSA less than 95 Å2 will be able
to cross BBB though all the compounds with PSA less than 140 Å2
will be highly absorbed through oral route (>90%).14 All these
parameters are indicative of good oral bioavailability and improved
capability to cross blood brain barrier. Therefore, BBB permeability
was predicted using online BBB permeation prediction software. As
per the software, any compound having SVM_MACCSFP BBB Score
of more than 0.02 will be able to cross BBB. It was observed that
except three compounds (2.15, 2.20 and 2.22), all compounds were
having score above 0.02 and hence were predicted to be BBB pen-
etrant. All the compounds obeyed Lipinski rule of five and also
were devoid of mutagenicity, tumorigenicity, reproductive effects
and irritant effects except compound 2.9. The drug score for all
the compounds was a positive value implying that the designed
ligands are potential candidate to serve as drug.
To support the above prediction, cell viability assay was per-
formed to study the cytotoxic effect of these compounds in Hep3D
liver carcinoma cell line. It was found that the compounds do not
have any cytotoxicity at 10 lM concentration.
In summary, based on our prior experience, we have attempted
to design low molecular weight, small molecules as BACE-1 inhibi-
tors. A series of allylidene hydrazinecarboximidamide derivatives
were designed and synthesized with an aim to identify drug like
inhibitors possessing potential for good oral bioavailability and
CNS penetration. Diverse array of compounds were investigated
for BACE-1 inhibition through FRET assay. Compound 2.21 was
found to be most potent with an IC50 of 6.423 lM. In the perspec-
tive of SAR studies, it can be suggested that moderately flexible 3
atom linker with substituted aromatic residues on both side and
hydrogen bond donor to bind aspartate dyad may results in active
compounds. It would be beneficial to incorporate electron poor
groups on para position of ring B which renders capability to form
strong p–p stacking with Tyr71. Inclusion of functional groups on

P. Jain et al. / Bioorg. Med. Chem. Lett. 26 (2016) 33–37
37
ring A interacting with S3 region amino acids (Thr231, Thr232,
References and notes
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