Development of pyrazole and spiropyrazoline analogs as multifunctional agents for treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bioorg.2019.103080

Cholinergic hypothesis of Alzheimer's disease has been advocated as an essential tool in the last couple of decades for the drug development. Here in, we report de novo fragment growing strategy for the design of novel 3,5-diarylpyrazoles and hit optimization of spiropyrazoline derivatives as acetyl cholinesterase inhibitors. Both type of scaffolds numbering forty compounds were synthesized and evaluated for their potencies against AChE, BuChE and PAMPA. Introduction of lipophilic cyclohexane ring in 3,5-diarylpyrazole analogs led to spiropyrazoline derivatives, which facilitated and improved the potencies. Compound 44 (AChE = 1.937 ± 0.066 μM; BuChE = 1.166 ± 0.088 μM; hAChE = 1.758 ± 0.095 μM; P_e = 9.491 ± 0.34 × 10^?6 cm s¹) showed positive results, which on further optimization led to the development of compound 67 (AChE = 0.464 ± 0.166 μM; BuChE = 0.754 ± 0.121 μM; hAChE = 0.472 ± 0.042 μM; P_e = 13.92 ± 0.022 × 10^?6 cm s¹). Compounds 44 and 67 produced significant displacement of propidium iodide from the peripheral anionic site (PAS) of AChE. They were found to be safer to MC65 cells and decreased metal induced Aβ_1-42 aggregation. Further, in-vivo behavioral studies, on scopolamine induced amnesia model, the compounds resulted in better percentage spontaneous alternation scores and were safe, had no influence on locomotion in tested animal groups at dose of 3 mg/kg. Early pharmacokinetic assessment of optimized hit molecules was supportive for further drug development.

Full text of DOI:10.1016/j.bioorg.2019.103080

Accepted Manuscript
Development of pyrazole and spiropyrazoline analogs as multifunctional agents
for treatment of Alzheimer’s disease
Gopichand Gutti, Devendra Kumar, Pankaj Paliwal, Ankit Ganeshpurkar,
Khemraj Lahre, Ashok Kumar, Sairam Krishnamurthy, Sushil Kumar Singh
PII:
S0045-2068(19)30593-0
https://doi.org/10.1016/j.bioorg.2019.103080
103080
DOI:
Article Number:
Reference:
YBIOO 103080
Bioorganic Chemistry
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Accepted Date:
17 April 2019
11 May 2019
18 June 2019
Please cite this article as: G. Gutti, D. Kumar, P. Paliwal, A. Ganeshpurkar, K. Lahre, A. Kumar, S. Krishnamurthy,
S.K. Singh, Development of pyrazole and spiropyrazoline analogs as multifunctional agents for treatment of
Alzheimer’s disease, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103080
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Development of pyrazole and spiropyrazoline analogs as multifunctional
agents for treatment of Alzheimer’s disease
Gopichand Gutti, Devendra Kumar, Pankaj Paliwal, Ankit Ganeshpurkar, Khemraj Lahre, Ashok
Kumar, Sairam Krishnamurthy, Sushil Kumar Singh*.
Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology
(Banaras Hindu University), Varanasi 221005, India.
*Corresponding author. Tel.: +91 542 6702736; Fax: + 91 542 368428.
E-mail address: sksingh.phe@iitbhu.ac.in

Abstract:
Cholinergic hypothesis of Alzheimer’s disease has been advocated as an essential tool in the last
couple of decades for the drug development. Here in, we report de novo fragment growing
strategy for the design of novel 3,5-diarylpyrazoles and hit optimization of spiropyrazoline
derivatives as acetyl cholinesterase inhibitors. Both types of scaffolds numbering forty
compounds were synthesized and evaluated for their potencies against AChE, BuChE and
PAMPA. Introduction of lipophilic cyclohexane ring in 3,5-diarylpyrazole analogs led to
spiropyrazoline derivatives, which facilitated and improved the potencies. Compound 44 (AChE
= 1.937 ± 0.066 µM; BuChE = 1.166 ± 0.088 µM; hAChE = 1.758 ± 0.095 µM; Pe = 9.491±0.34
x 10-6 cm s1) showed positive results, which on further optimization led to the development of
compound 67 (AChE = 0.464 ±0.166 µM; BuChE = 0.754 ± 0.121 µM; hAChE = 0.472 ± 0.042
µM; Pe = 13.92±0.022 x 10-6 cm s1). Compounds 44 and 67 produced significant displacement of
propidium iodide from the peripheral anionic site (PAS) of AChE. They were found to be safer
to MC65 cells and decreased metal induced Aβ1-42 aggregation. Further, in-vivo behavioral
studies, on scopolamine induced amnesia model, the compounds resulted in better percentage
spontaneous alternation scores and were safe, had no influence on locomotion in tested animal
groups at dose of 3mg/kg. Early pharmacokinetic assessment of optimized hit molecules was
supportive for further drug development.
Key words: Central nervous system, Alzheimer’s disease, Acetylcholinesterase, Aamyloid beta,
De novo drug design, and Spiropyrazoline.

1
INTRODUCTION
Alzheimer’s disease (AD) is the most common cause of age-related dementia, which usually
starts in the late middle age or in the old age. Major symptoms of the disease include
progressive memory loss, impaired thinking, cognitive decline, disorientation, sudden
fluctuations in personality and mood, and noticeable deterioration of brain neurons, particularly
in cerebral cortex[1, 2]. Inordinate symptoms of anxiety may, in fact, be an early cause of AD by
enhancing the amyloid beta (Aβ1-42) protein deposition [3-5]. Research endeavors in drug
discovery strategies have yielded some exciting outcomes in the past two decades[6, 7].
Donepezil (DNZ), galantamine, rivastigmine and memantine are the currently approved and
marketed drugs for the treatment of AD-associated dementia. Out of these, first three act on
central nervous system (CNS) cholinergic pathways, whereas memantine serves as N-methyl-D-
aspartate (NMDA) receptor antagonist[2]. The cholinergic abnormalities and Aβ1-42 aggregation
have been observed in neurodegenerative conditions. The peripheral anionic site (PAS) of
Acetylcholinesterase (AChE) catalyzes Aβ1-42 aggregation and plays crucial role in senile
plaques formation. A large number of potential therapeutic interventions have been developed to
counteract the loss of presynaptic cholinergic functions [8-10].
AChE and butyrylcholinesterase (BuChE) are two major forms of cholinesterases present in
mammalian brain. Both of these are diverse in genetic makeup, structure, and their kinetic
activities. AChE is a serine hydrolase enzyme that catalyzes the breaking down of acetylcholine
into choline and acetic acid and prevents neurotransmission at cholinergic synapses. In human
brain, BuChE is present in neurons, glial cells, as well as in amyloid plaques and neurofibrillary
tangles in AD patients. AChE activity decreases progressively in the brain of AD patients,
whereas, BuChE activity shows some increase[11]. An ideal drug candidate should inhibit the

enzymes; produce potent activity against AD pathology and cross the blood brain barrier (BBB).
Additionally, it should be non toxic to neuronal cells as well as normal cells. Further, good
adsorption, distribution, metabolism, excretion and toxicity (ADMET) profiles form the major
criteria for a clinical candidate to reach the patient from the laboratory. Chalcone, [12]
flavonoid,[13] pyrazole, N-benzylpiperidine[10] and piperazinedione[14] derivatives are the
major scaffolds involved in the preclinical studies acting through cholinesterase hypothesis.
Pyrazole derivatives are progressively gaining immense significance in medicinal chemistry[15].
The therapeutic voyage of pyrazole derivatives and the presence of scaffold in various category
of drugs viz. anti-Alzheimer’s[16], anti-Parkinson’s[17, 18], anti-psychotic[19],
neuroprotective[20], anti-inflammatory[21], anti-tumor[22], anti-tubercular[23] etc. have made
it an indispensable anchor for the design and development of new druglike molecules.
Many of the traditional and in-silico drug discovery techniques have played a major role in
picking out novel chemical entities at early stages. Among them, computational de novo drug
design is the most viable method as it allows the target specific design and highly efficient drug
like scaffolds with structural flexibility[24]. The present study includes, de novo drug design
(fragment growing strategy) to develop cholinesterase inhibitors using AChE as a paradigm
LigBuilder 2 was used to grow fragments from our in-house chemical library[25]. Fragments
were developed into small molecule inhibitors through iterative cycles of generations using
genetic algorithm. The designed compounds were selected for the synthesis based on their
molecular interactions within the active site. The compounds were synthesized and evaluated for
their inhibitory activities and other parameters. The obtained hit molecule was further optimized
to improve lipophilicity and pharmacokinetic profiles. The hit (compound 44) and hit optimized
molecule (compound 67) were further evaluated for neuro-protective property on MC65 (human

neuroblastoma) cell line, Aβ1-42 aggregation assay, in-vivo studies using scopolamine induced
amnesia models to evaluate the working memory, learning response and brain pharmacokinetic
profile in mice.
2
RESULTS AND DISCUSSION
2.1 Design and synthesis
2.1.1 De novo design and in-silico molecular docking studies
The overall designing strategy is shown in figure 1. In order to achieve successful de novo drug
design, a fragment-growing strategy was utilized. For accomplishing this, LigBuilder 2.0 was
employed (http://repharma.pku.edu.cn/ligbuilder/download.html). This package facilitates
automatic build ligand molecules based on three dimensional structure of the target protein
within the binding pocket and subsequently screen them. Human acetylcholinesterase (PDB ID:
4EY7) with DNZ as co-crystallized ligand was considered as a model target. Small fragments
from known ligands of single target are usually well grounded to become lead structures. The in-
house small fragment library was developed on the basis of following criteria: (1) the molecular
weight should be less than 250 Da, (2) lipophilic groups, and (3) The number of aromatic rings
should be equal or greater than one. In the initial stage, various in-house fragments were docked
using Glide XP module of Schrödinger Maestro 2018.1. The first stage generation revealed three
fragments with good binding interactions (S4, figure S3, supporting information). The fragment-
1 (S4-figure S3 A) showed hydrogen bonding interaction (TYR124) and hydrophobic
interactions (TRP286, TYR341) at peripheral anionic site (PAS) of AChE. It also showed
interaction with HIS447 residue of catalytic active site (CAS) of AChE through polar interaction.
Consequently, fragment-1, which showed good docking poses was selected and used as a seed
for the de novo designing of molecules.

As the fragments were small and their potencies were expected to be low, a second stage of seed
growing was applied. Eventually, we obtained a virtual hit (compound 43, 3,5-diarylpyrazole
derivative) which had a better docking score and an improved interaction pose at the CAS and
PAS of AChE (S4-figure S3 E). The obtained virtual hit was successfully synthesized and
chemically modified with various electron donating (EDG, methyl and methoxy etc.) and
withdrawing groups (EWG, chloro, bromo, and trifluoromethyl etc.) to establish the potencies
based on biological assays. The Glide score of compound 44 was found to be -9.5 Kcal/mole.
The p-chlorophenylpyrazole part of the molecule was aligned towards PAS and benzamide group
of compounds 44 was oriented towards CAS of AChE. The p-chlorophenyl ring interacted
hydrophobically with TYR124 and formed π-π stacking interactions with TRP286 and TYR341
residues of PAS-AChE. At CAS, benzamide part formed polar interactions with HIS447 and
SER203 residues. 3,5-Diarylpyrazole derivatives (43-62) showed satisfactory inhibitory
activities and high BBB permeability (explained in sections 2.3 and 2.5 respectively). Compound
44 was found to be a suitable hit molecule (Hit compound 1) for further investigation. Based on
the findings of the protein-ligand interactions of compound 44 with AChE active sites, the
following modifications (and combinations thereof) were explored in the next round of
optimization step: (1) slight increase in total polar surface area (tPSA) and (2) increase in
molecular weight and lipophilicity by incorporation of cyclohexane ring. The designed
molecules retained the binding pose of previous hit molecules with characteristic interactions.
Thus, a new series of spiropyrazoline derivatives (66-85) of the previous hit molecules, were
designed and synthesized auspiciously. The docking study of compound 67 showed better Glide
score of -11.5 Kcal/mole with improved binding pose at PAS-AChE compared to compound 44.
The benzamide functionality of compound 67 was oriented towards PAS and forming

hydrophobic (TYR72, TYR124, TRP286), electrostatic (ASP72), and π-π stacking interactions
(TYR341) with active site residues. Moreover, spiropyrazoline —NH of compound 67 formed
hydrogen bonding interaction to HIS447 and polar interaction with SER203 at CAS-AChE.
Additionally, compounds 44 and 67 interacted with anionic subsite (TRP86, GLU202, PHE338),
oxyanion hole (GLY120, GLY121), and acyl binding pocket (PHE295, PHE297) residues. The
effective binding modes of compounds 44 and 67 are depicted in figure 2. Hit compound
(compound 44) and potent optimized hit (compound 67) were further subjected to biological
evaluations.
2.1.2 Synthesis
The synthesis of 3,5-diaryl-1H-pyrazole derivatives 43-62 is illustrated in scheme 1. Formation
of N-(3-acetylphenyl)benzamide (2) from 3-aminoacetophenone (1), followed by Claisen-
Schmidt condensation with various substituted aromatic aldehydes, afforded corresponding key
α,β-unsaturated chalcone intermediates (23-42). Introduction of pyrazole ring (43-62) system
was made feasible by refluxing 23-42 with 5.0 equivalent of hydrazine hydrate in methanol,
which was followed by dehydrogenation by refluxing with catalytic molecular iodine in
1
13
DMSO[26]. H and C NMR showed appearance of characteristic signals of 4th position of the
pyrazole ring (43-62) at δ 7.0 -7.2 and 95 – 105 ppm respectively.
The synthetic route for spiropyrazoline derivatives 66-85 is delineated in scheme 2. Compound
64 was prepared by reacting 1.5 equivalent of hydrazine hydrate with 4-methylbenzenesulfonyl
chloride (63), which was refluxed with equivalent cyclohexanone to obtain cyclic ketone N-
tosylhydrazone (65) in excellent yields. Stereoselective synthesis of spiropyrazolines was
reported earlier [27-29]. Compound 65 underwent 1,3-cycloaddition with 23-42, followed by a
1,3-hydrogen shift to afford spiropyrazolines (66-85) with high selectivity and excellent yields.

In 1H and 13C NMR, appearance of the tertiary carbon and its corresponding proton signals at 4.0
- 4.5 and 50 – 57 ppm with other characteristic signal of derivatives confirmed formation of
target compounds (66-85). Amidic –NH and pyrazole –NH were established by D2O exchange
analysis (figure S10). Cyclohexanone ring protons appeared as complex multiplet at aliphatic
1
13
region in H NMR and five distinct signals in aliphatic region of C NMR spectra. Quaternary
carbon atom at C-5 position of pyrazole ring was identified at 69.0 – 69.99 ppm for all
derivatives. The above said signals were further reconfirmed with 2D NMR (figure S11, S12)
and DEPT 135° experiments (figure S13). To observe the configuration of the compound,
specific rotation, differential scanning calorimetry and X-ray crystal structure analysis was
performed for one representative compound 73 (3-Fluoro substituted). Specific rotation (
[∝]589
32.0) (table 1) and melting temperature (figure S1) were observed as +213.25° and 260.4 °C
respectively. We were fortunate that compound 73 formed large crystals upon slow evaporative
crystallization from ethylacetate and ethanol (1:1), allowing for structure determination through
X-ray crystallography (figure 3).
2.2 In-vitro cholinesterase inhibitory activity and SAR studies
The pharmaceutical drug discovery and development efforts, at present, are focused on
optimizing new chemical entities that act through specific enzyme inhibition. In AD, cholinergic
hypothesis is an epitomized therapeutic strategy to produce effective agents[30]. The virtual hit
(compound 43) obtained from our de novo drug design was initially evaluated for preliminary
enzyme inhibition on AChE and BuChE at concentrations of 50 and 100 µM. Our first goal was
to ascertain the significance of phenyl ring at 3rd position of the pyrazole scaffold.
Encouragingly, compound 43 showed better inhibition at these concentrations, this driving for
further investigations. In the second stage, six different concentrations of (0.01µM - 100 µM) the

compound were used to determine the IC50. On AChE and BuChE, IC50 of the compound was
determined as 4.048 ± 0.115 µM and 8.633 ± 0.108 µM respectively. This may be due to the π-π
interactions with PAS residue i.e TYR72. The phenyl ring attached to pyrazole was of interest
for various structural modifications. Therefore, compound 43 was further explored with
introduction of multiple EDG and EWG at phenyl ring. After successful synthetic assignment,
compounds 43-62 were obtained and the role of substitutions in enzyme inhibition was
evaluated. Fascinatingly, out of 20 derivatives, compound 44 (para-chloro) was found to be
potent on both AChE and BuChE with two folds increase in IC50 (table 2; AChE = 1.937 ± 0.066
µM; BuChE = 1.166 ± 0.087 µM). Among 3,5-diarylpyrazole series (43-62), halogen containing
compounds (weak EWD groups; chloride, bromide, and fluoride) showed moderate to good IC50
values and remaining derivatives showed satisfactory activities (table 2). However, increasing
the bulkiness by adding another phenyl ring as in α-napthyl (62, AChE = 27.78 ± 0.107 µM;
BuChE = 22.30 ± 0.068 µM) exhibited highest IC50 values. Interestingly, in all derivatives (43-
62), para substituted analogs were slightly potent, when compared with other analogs. Bulky
substituted analogs at para position (para-methoxy (51)-AChE = 8.451 ± 0.069 µM; BuChE =
7.305 ± 0.087 µM; para-trifluoromethoxy (56)-AChE = 6.092 ± 0.184 µM; BuChE = 6.755 ±
0.077 µM) of the phenyl ring showed moderate activities. Surprisingly, in spiropyrazoline series
(66-85), same pattern of IC50 values were observed in most of the analogs. Compound 67 (para-
chloro) exhibited most potent inhibitory activities (AChE = 0.464 ±0.166 µM; BuChE = 0.754 ±
0.121 µM). Compound 72 (para-fluoro) showed good inhibition at 0.948 ± 0.096 µM for AChE,
where as in case of BuChE (IC50 = 1.959 ± 0.082 µM) it was more than double. Compound 74
(para-methoxy, AChE = 2.319 ± 0.147 µM; BuChE = 3.549 ± 0.116 µM) and 79 (para-
trifluoromethoxy, AChE = 2.240 ± 0.122 µM; BuChE = 7.792 ± 0.066 µM) derivatives of

spiropyrazoline were potent in comparison to 3,5-diarylpyrazole derivatives (51 and 56) but not
with para-chloro (67) of spiropyrazolines. Bulkiness was also not favored in spiropyrazoline
analog (85, AChE = 29.190 ± 0.117 µM; BuChE = 35.49 ± 0.061 µM). The enzyme inhibition
studies of both 3,5-diarylpyrazoles (43-62) and spiropyrazolines (66-85) on cholinesterase
enzymes (AChE and BuChE) constructively developed the potency profiles of all the synthesized
analogs. Weak EWD substituted derivatives showed good inhibitory activities. Compounds 44
and 67 showed significant IC50 values among them and were also evident from molecular
docking studies. The SAR studies suggested that substitution at para position of phenyl ring will
be beneficial for activity where as ortho/meta substitutions in the same analogs were found to be
satisfactory. Further, bulkiness on the phenyl group led to decrease in activity.
To assess the selectivity towards human AChE (hAChE), four representative compounds (44, 46,
67 and 69) were selected and studied further. Compounds 44, 46, 67 and 69 exhibited IC50 1.758
± 0.095 µM, 1.027 ± 0.062 µM, 0.472 ± 0.042 µM and 0.693 ± 0.062 µM respectively (Standard
DNZ = 0.022 ± 0.031 µM). To demonstrate the worthiness of drug design approach, compounds
44 and 67 were additionally evaluated for enzyme kinetic assay on AChE.
2.3 AChE Enzyme kinetic assay
To discover a potential analog, it is crucial that selective inhibitors be identified, which requires
evaluation of candidate for binding pattern against selected target. The mechanistic role of AChE
inhibition by compounds 44 and 67 was explored through enzyme kinetics parameters like
maximal velocity (Vmax), Michaelis–Menten / dissociation constant (Km), and inhibitory
concentration (ki). The reciprocal Lineweaver-Burk plot of compound 67 (figure 4) suggested
decreased pattern of Vmax and Km with increase in inhibitor concentrations and the intersection
point of trendlines fell in the second quardrant. This result demonstrated that compound 67

inhibited the AChE enzyme non-competitively. The Dixon plot showed that it has Ki = 2.65µM
(figure 5).
2.4 In-vitro Blood-Brain barrier permeation assay
BBB and its penetration by neurotherapeutics is the gate way towards CNS activity[31]. To
screen the BBB permeability of all synthesized compounds (43-62 & 66-85), a parallel artificial
membrane permeation assay (PAMPA) was employed as described in previous reports[14]. Nine
representative commercially available drugs were selected and evaluated for BBB permeability
(Pe, S3, table S2). The Pe values of the tested compounds are listed in table 3. As expected, the
BBB penetration potential of spiropyrazoline series (compound 67 Pe = 13.92±0.022 x 10-6 cms-
1), was improved significantly as compared to 3,5-Diarylpyrazole series (Compound 44 Pe =
9.491±0.34 x 10-6 cms-1). This could be explained by the introduction of lipophilic cyclohexane
ring. Compound 82 (4-methyl substituted derivative) showed greater BBB permeability with Pe
value of 14.745±0.01 x 10-6 cms-1. Moreover, all tested compounds could cross BBB in vitro,
with excellent permeation potentials.
2.5 Propidium iodide displacement assay.
Molecular docking studies suggested that compounds 44 and 67 demonstrated significant
interactions with PAS residues. Further, enzyme inhibition and PAMPA assays manifest potent
inhibition of AChE and BBB permeation. Therefore, PAS-binding affinity of compounds 44 and
67 was evaluated by propidium iodide displacement assay at 10 and 50 µM concentrations (table
4). The binding of compounds 44 and 67 to PAS-AChE resulted into decreased fluorescence
intensity. Compound 67 exhibited considerably equal displacement of propidium iodide at a
concentration of 10 µM (21.24%) but higher in case of 50 µM (41.10%) compared to donepezil
(10 µM = 21.30%; 50 µM = 38.23%). Compound 44 (10 µM = 15.68%; 50 µM = 28.04%)

appeared to have lesser displacement of propidium iodide from PAS-AChE. The results of
propidium iodide displacement assay are in concurrence with molecular docking studies of
compounds 44 and 67.
2.6 Aβ1-42 aggregation assay (Thioflavin T assay) and confocal fluorescence imaging
Compounds 44 and 67 were further evaluated by metal induced Aβ1-42 aggregation assay to
establish their potency. Aβ1-42, when incubated with the metal showed 100% aggregation. DNZ,
at a dose of 20 µM, showed significant inhibition of metal induced Aβ1-42 aggregation.
Compound 44, inhibited nearly 50% as of Aβ1-42 aggregation, whereas in compound 67 it was
more than 50% compared with Fe+2 + Aβ1-42 and Aβ1-42 groups (figure 7.A). Recently, Avinash
S. Kumbhar and coworkers demonstrated the use of Thioflavin T (ThT) based confocal imaging
experiments to monitor conformational changes of Aβ1-42 aggregation in presence of Ru(II)
polypyridyl complexes[32] . Confocal imaging was carried out to understand the interaction of
Aβ1-42, Fe+2, compound 44, and 67 at molecular level after 10 days of incubation. Fluorescent
background was obtained using ThT dye (figure 3.B). Aβ1-42 aggregates were obtained when
incubated and treated with ThT (figure 7.C), whereas no fluorescence was observed in Aβ1-42
alone (figure 7.D) and Aβ1-42 along with the metal were incubated without ThT (figure 7.E).
These blank images explain that neither Aβ1-42 and metal nor their combinations showed any
background noises in absence of ThT. Aβ1-42 incubated with metal showed vigorous plaques
deposition (figure 7.F), while the plaques were decreased upon treatment with compounds 44
and 67 (figure 7.G and 7.H). These results suggest that test compounds possibly inhibit or
lessened the Aβ1-42 aggregation at early stages of fibril formation.

2.7 Neuroprotection studies on MC65 cell lines
Cell line studies provide a system for ready and rapid evaluation[33]. The use of cell cytotoxicity
analysis is a valuable tool to study issues of clinical relevance, especially those related to
diseases, and cell toxicity mechanisms. MC65 is a versatile in vitro model in neurobiology and it
conditionally expresses a C-terminal derivative of the amyloid β precursor protein (AβPP)
termed SβC (a fusion protein composed of the amino-17 and carboxyl-99 residues of βPP),
which further induces ROS generation. The cell line is accompanying with oxidative stress and
Aβ induced cellular toxicity in tetracycline (TC) removal condition (TC-)[34]. Neurotherapeutic
likeliness and toxicity profiles of the potent derivatives (compounds 44 and 67) were ascertained
by MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compounds 44
and 67 at concentrations of 50, 40, 30, 20, 10, and 1µM were used in the experiment and
compared to that of tetracycline (TC+). Significant decrease in the Aβ production was observed
with respect to TC- cells (figure 6).
2.8 Behavioral Studies by Y-Maze test
The evaluation of a chemical molecule for potential in-vivo efficacy requires a robust approach,
which screens for efficacy in appropriate cognitive domains and monitors behavior to appraise
cognition. The spontaneous alternation behavior in Y-maze, as sign of short-term memory, is a
fruitful method to screen new molecules against amnesic rodent model[35]. Effects of
compounds 44 and 67 on scopolamine-induced impairment of spontaneous alternation behavior
and number of arm entries were assessed by administering scopolamine hydrobromide (3 mg/kg)
by intraperitoneal injection. Scopolamine hydrobromide showed markedly impaired spontaneous
alternation behavior and significant difference (p < 0.05) with respect to control, vehicle and
DNZ treated groups. (figure 8.A). Compound 44 at a dose of 1.5 mg/kg, exhibited no significant

difference with scopolamine hydrobromide treated group, whereas at doses of 3mg/kg and 6
mg/kg showed significant difference. Notable differences were observed among compound 67
treated groups, relative to the Scopolamine hydrobromide 3mg/kg group. There was a dose
dependent increase in the percentage of spontaneous alternation among compound 44 treated
groups. No significant difference in the percentage of spontaneous alternation was observed
between DNZ treated group and compound 67 (at dose of 3 mg/kg and 6 mg/kg) treated groups.
Neophobia and recognizing behaviors of all groups were assessed by monitoring their novel arm
entries. Scopolamine hydrobromide (3 mg/kg) showed remarkable decrease in novel arm entries
whereas, DNZ (3 mg/kg) exhibited no significant deference with respect to control and vehicle
groups. Compound 44 at a dose of 1.5 mg/kg showed significant difference with respect to
scopolamine and DNZ groups. Dose dependent increase in novel arm entries was observed until
a dose of 3mg/kg, before hitting a plateau at 6mg/kg. Same pattern was observed with compound
67, which at a dose of 3 mg/kg showed no significant difference compared to DNZ. Increase in
novel arm entries by spiropyrazoline derivative (compound 67 at 3mg/kg) was mainly because of
greater availability of drug in the brain as compared to 3,5-diarylpyrazole analog (compound 44).
In case of % total arm entries, all groups are significantly different with respect to scopolamine
group. This suggests that none of the compounds influenced the locomotor activity of the
animals.
2.9 Neurochemical level estimation
The animals used in the behavioral study were further evaluated for neurochemical (AChE) level
in brain by earlier described protocols [14]. High levels of AChE were observed in scopolamine
treated animals (figure 9.A), but was significantly lessened with DNZ treatment. Compounds 67
(3 mg/kg), 44 (3 mg/kg) showed slightly higher levels of AchE as compared to DNZ and

compounds 67. Catalase (CAT) is a very important enzyme in protecting the cells from oxidative
damage by reactive oxygen species (ROS). CAT levels in the brain of animals were also
determined after the behavioral studies (figure 9.B) and compounds 44 and 67 maintained its
normal level at a dose of 3 mg/kg (figure 9). The brain tissue pattern in the normal, toxic and
treated groups was further evaluated by histopathological examination (figure 8.E) of the brain
samples. Some abnormal cell morphology was observed in the scopolamine treated animal, while
the standard pattern of the brain tissues in control, compounds 44 and 67 treated animals
evidently deduce that the test compounds were safer for brain tissues.
2.10 In-vivo mice brain pharmacokinetic profiles of compounds 44 and 67
For a potent CNS drug, it is necessary to understand the brain penetration and ratio of brain and
plasma concentration[36]. In view of the appreciative potency in in-vitro BBB permeation and
scopolamine induced amnesia models, compounds 44 and 67 were further evaluated for brain
pharmacokinetic profile in mice. As shown in table 5, compound 67 showed better brain
penetration and it was increased by 2.05 times compared to compound 44. Moreover, it showed
acceptable terminal half-life (t1/2 = 2.61 h) after oral administration.
3
SUMMARY AND CONCLUSIONS
We described de novo drug design based on fragment growing strategy for the identification of
potent chemical entities for AChE inhibition. The development of potent scaffolds and
evaluation was the focus of present study by using tiny fragment molecules. Fragments which
were having good binding interaction to AChE active site were selected and allowed to grow in
LigBuilder 2. The obtained optimized hit was chemically modified to get improved binding
interactions. Increase in potency from hit to optimized hit compound have proved the rationality
of design strategy and inspired to search compounds with superior ChEs inhibitory profile. Using

this approach, we designed, synthesized and evaluated 40 compounds through in-vitro assays and
identified that compound 67 (AChE = 0.464 ±0.166 µM; BuChE = 0.754 ± 0.121 µM; hAChE =
0.472 ± 0.042 µM; Pe = 13.92±0.022 x 10-6 cm s1) was most potent and showed displacement of
propidium iodide from PAS-AChE. We also characterized all derivatives with sophisticated
analytical techniques. The X-ray crystal structure of compound 73 established formation of
spiropyrazoline derivatives. Hit (compound 44) and optimized hit (compound 67) were further
assessed to support the design approach. At 50 µM, compounds 44 and 67 exhibited near 90 %
cell viability and diminished the metal induced Aβ1-42 aggregation effectively at 20 µM. In-vivo
behavioral analysis of compound 67 demonstrated better spontaneous alternation score and novel
arm entries without influencing the locomotor activity. Rise in brain penetration of compound 67
by 2.05 times compared to compound 44, reinforce the design strategy. Considering the ChE’s
inhibitory potency, BBB permeability, promising data from the MC65 cell line, propitious in-
vivo behavioral and brain penetration analysis, compound 67 represents a beneficial hit for
development of new chemical entities against Alzheimer’s disease.
4
EXPERIMENTAL SECTION
4.1 Chemistry
4.1.1 General method
All commercially available chemicals were purchased from sigma Aldrich, TCI Co., ltd, and
Avra synthesis pvt.Ltd. The solvents used for the study were dried by proper techniques and
were used anhydrous unless otherwise stated. Experiments were carried out in oven-dried
glassware under dry N2 atmosphere and standard vacuum techniques were used. Purifications
were carried out by using column chromatography on silica gel 60 (Avra), particle size =
0.140−0.25 mm (60−120 mesh), as the stationary phase. All reactions were monitored by silica

gel F254 TLC aluminium sheets (Merck) and ultraviolet light (254 nm) or iodine vapors were
used for visualization of spots. Melting points were determined by using automated melting point
apparatus (Bamstead Electrothermal, UK). All intermediates and target compounds were
1
characterized by H NMR, 13C NMR and mass spectrometry. NMR spectra were recorded in
13
Bruker-500 (1H 500 MHz, C 125.8 MHz) instrument using CDCl3 and DMSO-d6 as solvents.
Chemical shift was measured in the ppm (δ) and coupling constant (J) was measured in Hz. 1 H
NMR spectra are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, dd = doublet of doublet, td = triplet of doublet, bs = broad
singlet), coupling constant (J) in Hertz (Hz), integration, and proton assignment.
Massspectrometric analysis was performed using Waters Q-TOF premier-HAB213 instrument
equipped with APCI and ESI multimode ionization source. Optical rotation was measured on a
Fisher scientific, model ADP-45 automatic polarimeter using a light-emitting diode (LED) lamp,
589
emitting light at a wavelength of 589 nm and a 1 dm polametric tube. Specific rotation
,
[∝]32.0
589
was calculated using the equation: α/lc, where α is the observed optical rotation
, l is the
∝
32.0
path length of the cell in dm, and c is the concentration of test compound in g/ml. Differential
scanning calotimetric analysis was performed by using heat flux type Shimadzu DSC-60 plus
instrument with Chromel-Alumel thermocouple detector. X-ray data collection was performed
with Bruker D8 VENTURE Kappa Apex III CMOS PHOTON 100 diffractometer equipped with
graphite monochromated Mo (K) ( = 0.71073 Å) radiation. The structures were solved by
SHELXT-2014/5 (Sheldrick, 2014) and refined by full-matrix least squares techniques using
SHELXL-2014, (Sheldrick, 2014) computer program. Molecular graphics were drawn using
ORTEP3 (Farrugia, 1997).

Purity of the compounds was determined by high-performance liquid chromatography (HPLC-
Agilent 1260 infinity II Quaternary LC). Isocratic mobile phase was delivered by quaternary
pump with flow rate of 1ꢀml/min. The mobile phase composition was phase A (Water) and phase
B (methanol) in ratio of 1:9. 5ꢀμL samples were injected into the HPLC column through auto
sampler. Diode-array detectors (DAD HS G7115A) detector was used at 310ꢀnm for the
detection of the compounds. Agilent ZORBAX Eclipse plus C8 column (5μm, 4.6 ×ꢀ250ꢀmm)
was used. The purity of the compounds was found to be above 99%.
4.1.2 Synthesis of N-(3-acetylphenyl)benzamide (2).
Compound 2 was synthesized according to a previously described method[37] and obtained as
off white solid. Yield - 94.6%, M.P.- 108-109 °C,1H NMR (500 MHz, DMSO-d6) δ 10.46 (s, 1H.
amide), 8.38 (s, 1H, acetylphenyl C2), 8.09 (d, J = 8 Hz ,1H, acetylphenyl C6), 8.00 (d, J = 7.5
Hz, 2H, benzamide C2, C6), 7.73 (d, J = 8Hz,1H, acetylphenyl C4), 7.63-7.60 (m, 1H, benzamide
13
C4), 7.57-7.50 (m, 3H, benzamide C3, C5 & acetylphenyl C5), 2.59 (s, 3H, methyl). C NMR
(125 MHz, DMSO-d6) δ 198.2 (-C=O, acetyl), 166.52 (-C=O, amide), 139.64 (acetylphenyl C3),
138.26 (acetylphenylC1), 135.42 (benzamide C1), 131.57 (acetylphenyl C5), 129.62 (benzamide
C4), 128.76 (benzamide C3, C5), 127.23 (benzamide C2, C6), 125.53 (acetylphenyl C6), 123.27
(acetylphenyl C4), 119.53 (acetylphenyl C2), 27.22 (-CH3, methyl).

4.1.3 General Procedure for the Synthesis of 23-42. To a solution of compound 2 (1.0 eq) in
ethanol (10ml) were added aromatic aldehyde (3-22, 1.0 eq) and 1M NaOH solution (1ml) at
below 25°C. The reaction mixture was stirred at room temperature for 6 h. After completion of
the reaction (monitored by TLC), reaction mixture was kept refrigeration. Then the precipitated
compound was filtered off and washed with ice cold ethanol (8 to 10ml) to afford titled
compound.
1
N-(3-cinnamoylphenyl)benzamide(23): White solid, yield - 89%, M.P.- 115-116 °C, H NMR
(500 MHz, DMSO-d6) δ 10.51 (s, 1H, amide NH), 8.48 (s, 1H, phenyl C2), 8.16 (d, J = 7.5 Hz,
1H, β =CH ), 8.03 (d, J = 7 Hz, 2H, benzamide C2, C6), 7.96 (d, J = 7.5 Hz, 1H, phenyl C6 ), 7.89
- 7.87 (m, 3H, phenyl C4, C5, benzamide C4), 7.80 (d, J = 16 Hz, 1H, α =CH), 7.63 - 7.54 (m, 4H,
benzamide C3, C5, cinnamoyl C2, C6), 7.47 (s, 3H, cinnamoyl C3, C4, C5). 13C NMR (125 MHz,
DMSO-d6) δ 189.64 (-C=O, cinnamoyl), 166.24 (-C=O, amide), 144.64 (β =CH), 140.17 (phenyl
C3), 138.51 (phenyl C1), 135.09 (cinnamoyl C1), 132.25 (benzamide C1), 131.18 (benzamide C4),
129.64 (benzamide C3, C5), 129.45 (cinnamoyl C3, C5), 129.30 (Cinnamoyl C2, C6), 128.92
(benzamide C2, C6), 128.19 (phenyl C6, cinnamoyl C4), 125.36 (phenyl C5), 124.47 (phenyl C4),
122.63 (α =CH), 120.58 (Phenyl C2); MS (ESI+): m/z calculated for C22H17NO2: 327.38, found –
328.4 (M+1).
N-(3-(3-(4-chlorophenyl)acryloyl)phenyl)benzamide(24): White solid, yield - 93%, M.P.- 105-
1
106 °C, H NMR (500 MHz, DMSO-d6) δ 10.49 (s, 1H, amide NH), 8.47 (t, J = 2 Hz, 1H,
phenyl C2), 8.15 (d, J = 7 Hz, 1H, β =CH ), 8.02 - 8.00 (d, J = 7 Hz, 2H, benzamide C2, C6), 7.97
- 7.90 (m, 4H, benzamide C2, C6, phenyl C4, C6), 7.78 (d, J = 15.5Hz, 1H,α =CH), 7.64 - 7.53
13
(m, 6H, chlorophenyl C2, C6, C3, C5, benzamide C4, phenyl C5 ). C NMR (125 MHz, DMSO-
d6) δ 189.50 (-C=O, acryloyl), 166.21 (-C=O, amide), 143.14 (β =CH), 140.18 (phenyl C3),

138.40 (phenyl C1), 135.64 (phenyl C6), 135.07 (chlorophenyl C4), 134.09 (chlorophenyl C1),
132.25 (benzamide C1), 131.01 (benzamide C4), 129.63 (benzamide C3, C5), 129.47
(chlorophenyl C2, C6), 128.91 (chlorophenyl C3, C5), 128.18 (benzamide C2, C6), 125.46 (phenyl
C5), 124.56 (phenyl C4), 123.34 (α =CH), 120.56 (Phenyl C2); MS (ESI+): m/z calculated for
C22H16ClNO2: 361.83, found – 362.15 (M+), 364.12 (M+2).
N-(3-(3-(2-chlorophenyl)acryloyl)phenyl)benzamide(25): Off white solid, yield - 92%, M.P.-
119-120 °C, 1H NMR (500 MHz, DMSO-d6) δ 10.50 (s, 1H, amide NH), 8.48 (s, 1H, phenyl C2),
8.20 (d, J = 7.5 Hz, 1H, chlorophenyl C3), 8.16 (d, J = 8 Hz, 1H, phenyl C6), 8.08 (d, J = 15.5
Hz, 1H, β =CH), 8.02 (d, J = 7.5 Hz, 2H, benzamide C2, C6), 7.98 (d, J = 7.5 Hz, 1H, benzamide
C4 ), 7.95 (d, J = 15.5 Hz, 1H, α =CH), 7.63 - 7.54 (m, 5H, benzamide C3, C5, phenyl C4, C5,
13
chlorophenyl C6), 7.51 - 7.45 (m, 2H, chlorophenyl C4, C5). C NMR (125 MHz, DMSO-d6) δ
189.41 (-C=O, acryloyl), 166.24 (-C=O, amide), 140.23 (β =CH), 139.15 (phenyl C3), 138.20
(phenyl C1), 135.06 (chlorophenyl C2), 134.83 (benzamide C1), 132.75 (chlorophenyl C1),
132.53 (phenyl C6), 132.25 (benzamide C4) , 130.55 (chlorophenyl C3), 129.68 (chlorophenyl
C4), 129.01 (chlorophenyl C6), 128.91 (benzamide C3, C5), 128.22 (chlorophenyl C5), 128.18
(benzamide C2, C6), 125.63 (phenyl C5), 125.38 (phenyl C4), 124.63 (α =CH), 120.60 (Phenyl
C2); MS (ESI+): m/z calculated for C22H16ClNO2: 361.83, found – 362.05 (M+) 363.7 (M+2).
N-(3-(3-(2,4-dichlorophenyl)acryloyl)phenyl)benzamide(26): Off white solid, yield - 94%, M.P.-
1
126-127 °C, H NMR (500 MHz, DMSO-d6) δ 10.49 (s, 1H, amide NH), 8.47 (t, J = 2Hz, 1H,
phenyl C2), 8.22 (d, J = 8.5Hz, 1H, β =CH), 8.15 - 8.13 (dd, J = 8Hz, 1.5Hz, 1H, phenyl C6),
8.01 - 7.99 (m, 2H, benzamide C2, C6), 7.97 - 7.96 (m, 3H, phenyl C4, benzamide C4, α =CH),
7.74 (d, J = 2Hz, 1H, dichlorophenyl C5), 7.62 - 7.53 (m, 5H, benzamide C3, C5, phenyl C5,
13
dichlorophenyl C3, C6). C NMR (125 MHz, DMSO-d6) δ 188.77 (-C=O, acryloyl), 165.75 (-

C=O, amide), 139.77 (β =CH), 137.62 (phenyl C3), 137.40 (phenyl C1), 135.67 (dichlorophenyl
C2), 135.15 (benzamide C1), 134.57 (phenyl C5), 131.77 (benzamide C4), 131.35 (dichlorophenyl
C1), 129.78 (dichlorophenyl C6), 129.53 (dichlorophenyl C3), 129.18 (dichlorophenyl C5), 128.43
(benzamide C3, C5), 127.97 (phenyl C6), 127.70 (benzamide C2, C6), 125.41 (dichlorophenyl C4),
125.24 (phenyl C4), 124.21(α =CH), 120.12 (phenyl C2); MS (ESI+): m/z calculated for
C22H15Cl2N2O2: 396.27, found – 396.5 (M+), 398.6 (M+2).
N-(3-(3-(4-bromophenyl)acryloyl)phenyl)benzamide(27): Whitish brown solid, yield - 92%,
M.P.- 105-106 °C, 1H NMR (500 MHz, DMSO-d6) δ 10.49 (s, 1H, amide NH), 8.47 (t, J = 2Hz,
1H, phenyl C2), 8.15 (dd, J = 8 Hz, 1.5 Hz, 1H, phenyl C6), 8.02 (s, 1H, benzamide C2), 8.01 (s,
1H, benzamide C6), 7.96 (d, J = 8Hz, 1H, benzamide C4), 7.93 (d, J = 15.5Hz, 1H, β =CH), 7.86
(d, J = 8.5Hz, 2H,bromophenyl C3, C5), 7.76 (d, J = 15.5Hz, 1H, α =CH), 7.68 (d, J = 8.5Hz, 2H,
13
bromophenyl C2, C6 ), 7.64 - 7.54 (m, 4H, phenyl C4, C5, benzamide C3, C5). C NMR (125
MHz, DMSO-d6) δ 189.52 (-C=O, acryloyl), 166.21 (-C=O, amide), 143.22 (β =CH), 140.18
(phenyl C3), 138.40 (phenyl C1), 135.07 (benzamide C1), 134.42 (beomophenyl C1), 132.40
(bromophenyl C3, C5), 132.24 (phenyl C5), 131.21 (benzamide C3, C6), 129.63 (benzamide C4),
128.91 (bromophenyl C2, C6), 128.18 (benzamide C2, C6), 125.47 (phenyl C6), 124.55 (phenyl
C4), 124.51 (bromophenyl C4), 123.41 (α =CH), 120.58 (Phenyl C2); MS (ESI+): m/z calculated
for C22H16NO2: 406.28, found – 405.9 (M+) 407.9 (M+2).
N-(3-(3-(3-bromophenyl)acryloyl)phenyl)benzamide(28): Whitish brown solid, yield - 94%,
M.P.- 109-110 °C, 1H NMR (500 MHz, DMSO-d6) δ 10.47 (s, 1H, amide NH), 8.46 (t, J = 2Hz,
1H, phenyl C2), 8.19 (s, 1H, bromophenyl C2), 8.15 - 8.13 (dd, J = 8Hz, 1.5Hz, 1H, phenyl C6),
8.01 - 7.94 (m, 4H, benzamide C2, C6, β =CH, benzamide C4), 7.87 (d, J = 8Hz, 1H, phenyl C4 ),
7.75 (d, J = 15.5Hz, 1H, α =CH), 7.65 - 7.54 (m, 5H, benzamide C3, C5, bromophenyl C4, C5,

13
C6), 7.44 (t, J = 8Hz, 1H, bromophenyl C5). C NMR (125 MHz, DMSO-d6) δ 189.00 (-C=O,
acryloyl), 165.72 (-C=O, amide), 142.31 (β =CH), 139.68 (phenyl C3), 137.83 (bromophenyl
C1), 137.16 (phenyl C1), 134.58 (benzamide C1), 133.07(phenyl C5), 131.75 (bromophenyl C2),
130.96 (benzamide C4), 130.82 (bromophenyl C4), 129.12 (bromophenyl C5), 128.42 (benzamide
C3, C5), 128.12 (bromophenyl C6), 127.69 (benzamide C2, C6), 125.08 (phenyl C6), 124.20
(phenyl C4), 123.66 (bromophenyl C3) ,122.39 (α =CH), 120.13 (phenyl C2); MS (ESI+): m/z
calculated for C22H16NO2: 406.28, found – 405.9 (M+) 407.9 (M+2).
N-(3-(3-(4-fluorophenyl)acryloyl)phenyl)benzamide(29): White solid, yield- 96%, M.P.- 126-127
°C, 1H NMR (500 MHz, DMSO-d6) δ 10.50 (s, 1H, amide NH), 8.47 (s, 1H, phenyl C2), 8.15 (d,
J = 8Hz, 1H, phenyl C6), 8.02 - 7.95 (m, 5H, benzamide C2, C6, β =CH, fluorophenyl C2, C6),
7.87 - 7.76 (m, 2H, α =CH, benzamide C4), 7.63 - 7.54 (m, 4H, benzamide C3, C5, phenyl C4,
13
C5), 7.33 (t, J = 9Hz, fluorophenyl C3, C5). C NMR (125 MHz, DMSO-d6) δ 189.52 (-C=O,
acryloyl), 166.21 (-C=O, amide), 164.92 (fluorophenyl C4), 143.39 (β =CH), 140.17 (phenyl C3),
138.49 (phenyl C1), 135.07 (benzamide C1), 132.24 (phenyl C5), 131.72 (benzamide C4),
131.65(fluorophenyl C1), 129.61 (fluorophenyl C2, C6), 128.91 (benzamide C3, C5), 128.18
(benzamide C2, C6), 125.38 (phenyl C6), 124.51 (phenyl C4), 122.51(α =CH), 120.56 (phenyl
C2), 116.54 (fluorophenyl C3), 116.37 (fluorophenyl C5); MS (ESI+): m/z calculated for
C22H16FNO2: 345.37, found – 346.52 (M+ 1).
N-(3-(3-(3-fluorophenyl)acryloyl)phenyl)benzamide(30): White Solid, yield - 91%, M.P.- 128-
129 °C, 1H NMR (500 MHz, DMSO-d6) δ 10.49 (s, 1H, amide NH), 8.47 (t, J = 2Hz, 1H, phenyl
C2), 8.16 - 8.14 (m, J = 8, 2, 1Hz, phenyl C6), 8.02 - 7.95 (m, 4H, benzamide C2, C6, benzamide
C4,β =CH), 7.86 - 7.83 (dt, J = 10, 2Hz, 1H, phenyl C5), 7.79 (d, J = 15.5Hz, 1H, α =CH), 7.72
(d, J = 7.5Hz, phenyl C4), 7.64 - 7.49 (m, 5H, benzamide C3, C5, fluorophenyl C2, C4, C5), 7.32 -

13
7.29 (td, J = 8.5, 2Hz, 1H, fluorophenyl C5). C NMR (125 MHz, DMSO-d6) δ 189.54 (-C=O,
acryloyl), 166.21 (-C=O, amide), 163.95 (fluorophenyl C3), 143.13 (β =CH), 140.19 (phenyl C3),
138.32 (fluorophenyl C1), 137.73 (phenyl C1), 135.06 (benzamide C1), 132.25 (phenyl C5),
131.41 (benzamide C4), 131.34 (fluorophenyl C5), 129.64 (phenyl C6), 128.92 (benzamide C3,
C5), 128.18 (benzamide C2, C6), 125.96 (fluorophenyl C5), 125.55 (fluorophenyl C6), 124.66
(phenyl C4), 124.08 (α =CH), 120.60 (phenyl C2), 117.88 (fluorophenyl C4), 115.27
(fluorophenyl C2); MS (ESI-): m/z calculated for C22H16FNO2: 345.37, found – 344.3 (M-1).
N-(3-(3-(4-methoxyphenyl)acryloyl)phenyl)benzamide(31): Off white solid, yield- 91%, M.P.-
125-126 °C, 1H NMR (500 MHz, DMSO-d6) δ 10.48 (s, 1H, amide NH), 8.45 (s, 1H, phenyl C2),
8.14 (dd, J = 8 Hz,1.5 Hz, 1H, β =CH ), 8.02 (s,1H, benzamide C2), 8.01 (s, 1H, benzamide C6),
7.93 (d, J = 8Hz, 1H, α =CH), 7.86 (d, J = 8.5Hz, 2H, methoxyphenyl C2, C6), 7.75 (d, J = 2Hz,
2H, phenyl C4, C5), 7.63 - 7.54 (m, 4H, benzamide C3,C6,C4, Phenyl C6), 7.04 (d, J = 9Hz, 2H,
13
methoxyphenyl C3, C5 ), 3.83 (s, 3H, -OMe). C NMR (125 MHz, DMSO-d6) δ 189.44 (-C=O,
acryloyl), 166.19 (-C=O, amide), 161.91 (methoxyphenyl C4), 144.65 (β =CH), 140.11 (phenyl
C3), 138.81 (phenyl C1), 135.10 (benzamide C1), 132.23 (phenyl C5), 131.23 (benzamide C4),
129.56 (benzamide C3, C5), 128.91 (methoxyphenyl C2, C6), 128.18 (benzamide C2, C6),
127.73(methoxyphenyl C1), 125.12 (phenyl C6), 124.34 (phenyl C4), 120.49 (α =CH), 120.07
(phenyl C2), 114.94 (methoxyphenyl C3, C5), 55.87 (-OMe); MS (ESI+): m/z calculated for
C23H19NO3: 357.41, found – 358.1 (M+1), 359.2 (M+2).
N-(3-(3-(3-methoxyphenyl)acryloyl)phenyl)benzamide(32): White solid, yield - 93%, M.P.- 127-
128 °C, 1H NMR (500 MHz, DMSO-d6) δ 10.48 (s, 1H, amide NH), 8.47 (t, J = 2Hz, 1H, phenyl
C2), 8.16 - 8.14 (m, J = 8 Hz, 1.5 Hz, 1H, phenyl C6), 8.02 (m, 2H, benzamide C2, C6 ), 7.97 -
7.96 (dt, 1H, benzamide C4), 7.90 - 7.87 (d, J = 15.5 Hz, 1H, β =CH), 7.77 - 7.74 (d, J = 16 Hz, α

=CH), 7.64 - 7.54 (m, 4H, benzamide C3, C5, phenyl C4, C5) 7.48 - 7.37 (m, 2H, methoxyphenyl
C2,C6), 7.40 (t, J = 8Hz, methoxyphenyl C5), 7.06 - 7.04 (m, J = 1H, methoxyphenyl C4), 3.84 (s,
3H, -OMe). 13C NMR (125 MHz, DMSO-d6) δ 189.68 (-C=O, acryloyl), 166.21 (-C=O, amide),
160.16 (methoxyphenyl C3), 144.62 β =CH), 140.17 (phenyl C3), 138.51 (phenyl C1), 136.53
(methoxyphenyl C1), 135.09 (benzamide C1), 132.23 (phenyl C5), 130.46 (benzamide C4),
129.61 (methoxyphenyl C5), 128.91 (benzamide C3, C5), 128.18 (benzamide C2, C6), 125.38
(phenyl C6), 124.54 (phenyl C4), 122.95 (α =CH), 121.97 (methoxyphenyl C6), 120.57 (phenyl
C2), 117.16 (methoxyphenyl C4), 114.02 (methoxyphenyl C2), 55.79 (-OMe); MS (ESI+): m/z
calculated for C23H19NO3: 357.41, found – 358.4 (M+1) 359.2 (M+2).
N-(3-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)benzamide(33): Off white solid, yield - 95%,
M.P.- 122-123 °C, 1H NMR (500 MHz, DMSO-d6) δ 10.48 (s, 1H, amide NH), 8.43 (t, J = 2Hz,
1H, phenyl C2), 8.14 - 8.12 (dd, J = 8Hz, 1.5Hz, 1H, phenyl C6), 8.02 (d, J = 7Hz, 2H,
benzamide C2, C6), 7.96 (d, J = 8Hz, 1H, β =CH), 7.80 – 7.72 (m, 2H,α =CH, benzamide C4),
7.64 - 7.54 (m, 5H, benzamide C3, C5, phenyl C4, C5, dimethoxyphenyl C2), 7.42 - 7.40 (dd, J =
8.5Hz, 2Hz, dimethoxyphenyl C5), 7.05 (d, J = 8.5Hz, 1Hdimethoxyphenyl C6), 3.87 (s, 3H, -
13
OMe C3), 3.83 (s, 3H, -OMe C4). C NMR (125 MHz, DMSO-d6) δ 189.54 (-C=O, acryloyl),
166.20 (-C=O, amide), 151.82 (dimethoxyphenyl C3), 149.50 (dimethoxyphenyl C4), 145.21 (β
=CH), 140.10 (phenyl C3), 138.87 (phenyl C1), 135.09 (benzamide C1), 132.24 (phenyl C5),
129.52 (benzamide C4), 128.91(benzamide C3, C5), 128.18 (benzamide C2, C6), 127.93 (phenyl
C6), 125.17(dimethoxyphenyl C1), 124.47 (phenyl C4), 124.32 (dimethoxyphenyl C6), 120.46 (α
=CH), 120.23 (phenyl C2), 112.08 (dimethoxyphenyl C5), 111.36 (dimethoxyphenyl C2), 56.22 (-
OMe), 56.09 (-OMe); MS (ESI+): m/z calculated for C24H21NO4: 387.44, found – 388.08 (M+
1).

N-(3-(3-(4-(trifluoromethyl)phenyl)acryloyl)phenyl)benzamide(34): White Solid, yield - 97%,
1
M.P.- 144-145 °C, H NMR (500 MHz, DMSO-d6) δ 10.50 (s, 1H, amide NH), 8.49 (s, 1H,
phenyl C2), 8.16 (d, J = 8Hz, 1H, phenyl C6 ), 8.12 (d, J = 8Hz, 2H, benzamide C2, C6), 8.03 -
7.98 (m, 4H, benzamide C4, β =CH, phenyl C4, phenyl C5), 7.84 - 7.81 (m, 3H, α =CH,
(trifluoromethyl)phenyl C2, C6), 7.63 - 7.54 (m, 4H, (trifluoromethyl)phenyl C3, C5, benzamide
C3, C5). 13C NMR (125 MHz, DMSO-d6) δ 189.53 (-C=O, acryloyl), 166.23 (-C=O, amide),
142.56 (β =CH), 140.21 (phenyl C3), 139.13 ((trifluoromethyl)phenyl C1), 138.20 (phenyl C1),
135.05 (benzamide C1), 132.25 (phenyl C5), 130.70 (benzamide C4), 130.45
((trifluoromethyl)phenyl C4), 129.86 (-CF3,), 129.67 (phenyl C6), 128.91 (benzamide C3, C5),
128.18 (benzamide C2, C6), 126.21 ((trifluoromethyl)phenyl C2), 126.18 ((trifluoromethyl)phenyl
C6), 125.62 ((trifluoromethyl)phenyl C5), 125.26 ((trifluoromethyl)phenyl C3), 124.67 (phenyl
C4), 123.43 (α =CH), 120.60 (phenyl C2); MS (ESI+): m/z calculated for C23H16F3NO2: 395.38,
found – 396.2 (M+1).
N-(3-(3-(3-(trifluoromethyl)phenyl)acryloyl)phenyl)benzamide(35): White solid, yield - 96%,
1
M.P.- 141-142 °C, H NMR (500 MHz, DMSO-d6) δ 10.50 (s, 1H, amide NH), 8.48 (s, 1H,
phenyl C2), 8.32 (s, 1H, phenyl C6), 8.19 - 8.15 (dd, J = 14Hz, 8Hz, 2H, benzamide C2, C6), 8.08
(d, J = 16Hz, 1H, β =CH), 8.02 (d, J = 7.5Hz, 3H, benzamide C3, C5, phenyl C5), 7.87 (d, J =
16Hz, 1H, α =CH), 7.81 (d, J = 7.5Hz, 1H, phenyl C4), 7.71 (t, J = 8Hz, 1H,
(trifluoromethyl)phenyl) C5), 7.63 - 7.54 (m, 4H, benzamide C4, (trifluoromethyl)phenyl) C2, C4,
C6). 13C NMR (125 MHz, DMSO-d6) δ 189.55 (-C=O, acryloyl), 166.23 (-C=O, amide), 142.75
(β =CH), 140.18 (phenyl C3), 138.27 (phenyl C1), 136.30 ((trifluoromethyl)phenyl) C1), 135.06
(benzamide C1), 133.21 (phenyl C5), 132.25 (benzamide C4), 130.44 ((trifluoromethyl)phenyl)
C6), 130.21 ((trifluoromethyl)phenyl) C3), 129.62 (phenyl C6), 128.91 (benzamide C3, C5,

(trifluoromethyl)phenyl) C2), 128.18 (benzamide C2, C6), 127.19 (-CF3), 125.63
((trifluoromethyl)phenyl) C4), 124.78 (phenyl C4), 124.57 ((trifluoromethyl)phenyl) C2), 123.44
(α =CH), 120.60 (phenyl C2); MS (ESI+): m/z calculated for C23H16F3NO2: 395.38, found –
396.5 (M+1).
N-(3-(3-(4-(trifluoromethoxy)phenyl)acryloyl)phenyl)benzamide(36): White solid, yield - 92%,
M.P.- 136-137 °C, 1H NMR (500 MHz, DMSO-d6) δ 10.49 (s, 1H, amide NH), 8.48 – 8.47 (t, J
= 2Hz, 1H, phenyl C2), 8.15 - 8.14 (dd, J = 1, 1.5 Hz, 1H, phenyl C6), 8.06 - 8.00 (m, 4H,
benzamide C2, C6, phenyl C4, phenyl C5), 7.98 - 7.96 (d, J = 8Hz, 1H, benzamide C4), 7.94 - 7.91
(d, J = 16Hz, 1H, β =CH), 7.81 - 7.78 (d, J = 15.5Hz, 1H, α =CH), 7.64 - 7.54 (m, 4H,
13
(trifluoromethoxy)phenyl C2, C3, C5, C6), 7.48 - 7.46 (d, J = 8Hz, 2H, benzamide C3, C5). C
NMR (125 MHz, DMSO-d6) δ 194.28 (-C=O, acryloyl), 170.95 (-C=O, amide), 154.83
((trifluoromethoxy)phenyl C4), 147.56 (β =CH), 144.96 ((trifluoromethoxy)phenyl C3), 143.10
((trifluoromethoxy)phenyl C5), 139.82 (phenyl C3), 139.22 (phenyl C1), 137.00 (benzamide C4),
136.06 (benzamide C1), 134.37 (phenyl C5), 133.66 ((trifluoromethoxy)phenyl C2), 132.94
((trifluoromethoxy)phenyl C6), 130.27 (-OCF3), 129.34 (phenyl C6), 128.57 (α =CH), 126.59
(benzamide C5), 126.52 (benzamide C3), 126.25 (phenyl C2), 125.36 (benzamide C2), 125.25
(benzamide C6), 124.21 (phenyl C4); MS (ESI+): m/z calculated for C23H16F3NO3: 411.38, found
– 412.2 (M+1).
N-(3-(3-(4-cyanophenyl)acryloyl)phenyl)benzamide(37): Off white solid, yield - 92%, M.P.-
122-123 °C, 1H NMR (500 MHz, DMSO-d6) δ 10.48 (s, 1H, amide NH), 8.47 (t, J = 1.5Hz, 1H,
phenyl C2), 8.15 - 8.13 (dd, J = 8Hz, 1.5Hz, 1H, phenyl C6), 8.09 (d, J = 8Hz, 2H, benzamide C2,
C6), 8.04 - 7.97 (m, 4H, β =CH, benzamide C4, cyanophenyl C3, C5), 7.94 (d, J = 8.5Hz, 2H,
benzamide C3, C5), 7.81 (d, J = 16Hz, 1H, α =CH), 7.63 - 7.53 (m, 4H, phenyl C4, C5,

cyanophenyl C2, C6). 13C NMR (125 MHz, DMSO-d6) δ 189.03 (C=O, acryloyl), 165.74 (-C=O,
amide), 141.83 (β =CH), 139.73 (cyanophenyl C1), 139.20 (phenyl C3), 137.68 (phenyl C1),
134.56 (benzamide C1), 132.73 (cyanophenyl C3,C5), 131.76 (phenyl C5), 129.38 (benzamide C3,
C5), 129.19 (benzamide C4), 128.42 (cyanophenyl C2,C6), 127.69 (benzamide C2, C6), 125.43
(phenyl C6), 125.20 (phenyl C4), 124.22(α =CH), 120.14 (phenyl C2), 118.60 (-CN), 112.35
(cyanophenyl C4); MS (ESI+): m/z calculated for C23H16N2O2: 352.39, found – 353.0 (M+1).
N-(3-(3-(3-cyanophenyl)acryloyl)phenyl)benzamide(38): Off white solid, yield - 93%, M.P.-
1
115-116 °C, H NMR (500 MHz, DMSO-d6) δ 10.49 (s, 1H, amide NH), 8.48 (t, J = 2Hz, 2H,
phenyl C2, cyanophenyl C2), 8.20 (d, J = 8Hz, 1H, cyanophenyl C4), 8.16 - 8.14 (dd, J = 8Hz,
1Hz, 1H, phenyl C6), 8.07 (d, J = 16Hz, 1H, β =CH), 8.02 - 8.01 (m, 3H, benzamide C2, C6,
cyanophenyl C6), 7.91 - 7.89 (dt, 1H, benzamide C4), 7.80 (d, J = 16Hz, 1H,α =CH), 7.69 (t, J =
13
8Hz, 1H, phenyl C5), 7.64 - 7.54 (m, 4H, benzamide C3, C5, cyanophenyl C5, phenyl C4). C
NMR (125 MHz, DMSO-d6) δ 189.43 (-C=O, acryloyl), 166.21 (-C=O, amide), 142.09 (β =CH),
140.21 (phenyl C3), 138.23 (phenyl C1), 136.45 (cyanophenyl C1), 135.05 (benzamide C1),
134.04 (phenyl C5), 134.00 (cyanophenyl C6), 132.41 (benzamide C4), 132.25 (cyanophenyl C4),
130.60 (cyanophenyl C2), 129.63 (cyanophenyl C5), 128.91 (benzamide C3, C5), 128.18
(benzamide C2, C6), 125.70 (phenyl C6), 124.89 (phenyl C4), 124.75 (α =CH), 120.62 (phenyl
C2), 118.93 (-CN), 112.65 (cyanophenyl C3); MS (ESI+): m/z calculated for C23H16N2O2: 352.39,
found – 353.5 (M+1).
N-(3-(3-(p-tolyl)acryloyl)phenyl)benzamide(39): White solid, yield - 89%, M.P.- 118-119 °C, 1H
NMR (500 MHz, DMSO-d6) δ 10.47 (s, 1H, amide NH), 8.46 (t, J = 2Hz, 1H, phenyl C2), 8.15 -
8.13 (dd, J = 8Hz, 1.5 Hz, 1H, phenyl C6), 8.02 (d, J = 7Hz, 2H, benzamide C2, C6), 7.94 (d, J =
7.5Hz, 1H,benzamide C4), 7.84 - 7.74 (m, 4H, β =CH, phenyl C4, C5, α =CH), 7.63 - 7.54 (m,

13
4H, benzamide C3, C5, p-tolyl C2, C6 ), 7.30 (d, J = 8Hz, 2H, p-tolyl C3, C5), 2.36 (-CH3). C
NMR (125 MHz, DMSO-d6) δ 189.57 (-C=O, acryloyl), 166.21 (-C=O, amide), 144.71 (β =CH),
141.28 (phenyl C3), 140.15 (p-tolyl C4), 138.64 (phenyl C1), 135.10 (benzamide C1), 132.39
(phenyl C5), 132.23 (p-tolyl C1), 130.07 (benzamide C3, C5), 129.60 (benzamide C4), 129.33 (p-
tolyl C3, C5), 128.91 (p-tolyl C2, C6), 128.18 (benzamide C2, C6), 125.26 (phenyl C6), 124.40
(phenyl C4), 121.55 (α =CH), 120.54 (phenyl C2), 21.57 (-CH3); MS (ESI+): m/z calculated for
C23H19NO2: 341.41, found – 342.5 (M+1).
N-(3-(3-(o-tolyl)acryloyl)phenyl)benzamide(40): White solid, yield - 91%, M.P.- 132-133 °C, 1H
NMR (500 MHz, DMSO-d6) δ 10.48 (s, 1H, amide NH), 8.48 (t, J = 2Hz, phenyl C2), 8.16 - 8.14
(dd, J = 8Hz, 1Hz, 1H, phenyl C6), 8.03 - 8.00 (m, 3H, benzamide C2, C6, β =CH), 7.97 (d, J =
7.5Hz, benzamide C4), 7.94 (d, J = 8Hz, phenyl C5), 7.76 (d, J = 15.5Hz, α =CH), 7.64 - 7.54 (m,
4H, o-tolyl C5, C6, benzamide C3, C5), 7.38 - 7.35 (m, 1H, phenyl C4), 7.32 - 7.30 (m, 2H, o-tolyl
C3, C4), 2.46 (s, 3H, -CH3). 13C NMR (125 MHz, DMSO-d6) δ 189.66 (-C=O, acryloyl), 166.24
(-C=O, amide), 141.73 (β =CH), 140.18 (phenyl C3), 138.52 (phenyl C1), 135.11 (benzamide
C1), 133.76 (o-tolyl C1), 132.23 (o-tolyl C2), 131.32 (phenyl C5), 130.92 (benzamide C4), 129.65
(o-tolyl C4, C6), 128.91 (benzamide C3, C5), 128.18 (benzamide C2, C6), 127.25 (phenyl C6),
126.90 (o-tolyl C3), 125.31 (o-tolyl C5), 124.42 (phenyl C4), 123.58 (α =CH), 120.60 (phenyl
C2), 19.82 (-CH3); MS (ESI+): m/z calculated for C23H19NO2: 341.41,found – 342.5 (M+1).
N-(3-(3-(4-isopropylphenyl)acryloyl)phenyl)benzamide(41): Pale yellow solid, yield - 98%,
1
M.P.- 134-135 °C, H NMR (500 MHz, DMSO-d6) δ 10.49 (s, 1H, amide NH), 8.47 (s, 1H,
phenyl C2), 8.15 (d, J = 7.5Hz, 1H, phenyl C6), 8.02 (d, J = 7.5Hz, 2H, benzamide C2, C6), 7.94
(d, J = 8Hz, 1H, β =CH), 7.83 - 7.74 (m, 4H, benzamide C3, C4, C5, phenyl C4), 7.63 - 7.54 (m,
4H, Phenyl C5, α =CH, , isopropylphenyl C2, C6), 7.34 (d, J = 7.5Hz, isopropylphenyl C3, C5),

2.95 - 2.90 (m, 1H, isopropyl CH), 1.22 (d, J = 6.5Hz, 6H, isopropyl -CH3). 13C NMR (125
MHz, DMSO-d6) δ 189.61 (-C=O, acryloyl), 166.22 (-C=O, amide), 152.03 (isopropylphenyl
C4), 144.73 (β =CH), 140.15 (phenyl C3), 138.63 (phenyl C1), 135.08 (benzamide C1), 132.79
(phenyl C5), 132.24 (benzamide C4), 129.61 (isopropylphenyl C1), 129.46 (benzamide C3, C5),
128.91 (isopropylphenyl C2, C6), 128.18 (benzamide C2, C6), 127.43 (isopropylphenyl C3, C5),
125.26 (phenyl C6), 124.40 (phenyl C4), 121.65 (α =CH), 120.53 (phenyl C2), 33.90 (isopropyl
CH), 24.07 (isopropyl -CH3); MS (ESI+): m/z calculated for C22H23NO2: 369.46, found –370.6
(M+1).
N-(3-(3-(naphthalen-1-yl)acryloyl)phenyl)benzamide(42): White solid, yield - 95%, M.P.- 121-
122 °C, 1H NMR (500 MHz, DMSO-d6) δ 10.52 (s, 1H, amide NH), 8.61 (d, J = 15.5Hz, 1H, β
=CH), 8.55 (s, 1H, phenyl C2), 8.33 (d, J = 8Hz, 1H, naphthalen-1-yl C4), 8.24 (d, J = 7.5Hz, 1H,
naphthalen-1-yl C5), 8.19 (d, J = 8Hz, 1H, naphthalen-1-yl C8), 8.09 (d, J = 8Hz, 1H,
naphthalen-1-yl C2), 8.03 - 7.93 (m, 5H, benzamide C2, C6, phenyl C6, C4,α =CH, phenyl C6),
7.68 - 7.55 (m, 7H, benzamide C3, C4, C5, naphthalen-1-yl C7, C3, C6, phenyl C4). 13C NMR (125
MHz, DMSO-d6) δ 189.53 (-C=O, acryloyl), 166.24 (-C=O, amide), 140.62 (phenyl C3), 140.22
(phenyl C1), 138.47 (β =CH), 135.10 (benzamide C1), 133.86 (phenyl C5), 132.25 (naphthalen-1-
yl C1), 131.86 (naphthalen-1-yl C10), 131.67 (naphthalen-1-yl C9), 131.39 (benzamide C4),
129.70 (naphthalen-1-yl C5), 129.28 (naphthalen-1-yl C4), 128.92 (benzamide C3, C5), 128.20
(benzamide C2, C6), 127.77(phenyl C6), 126.84 (naphthalen-1-yl C3), 126.19 (naphthalen-1-yl
C6), 126.12 (naphthalen-1-yl C7), 125.37 (naphthalen-1-yl C8), 125.22 (naphthalen-1-yl C2),
124.51 (phenyl C4), 123.56 (α =CH), 120.65 (phenyl C2); MS (ESI+): m/z calculated for
C26H19NO2: 377.44, found – 378.3 (M+1).