Synthesis of novel 5-(aroylhydrazinocarbonyl)escitalopram as cholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.06.036

A novel series of 5-(aroylhydrazinocarbonyl)escitalopram (58–84) have been designed, synthesized and tested for their inhibitory potential against cholinesterases. 3-Chlorobenzoyl- (71) was found to be the most potent compound of this series having IC₅₀ 1.80 ± 0.11 μM for acetylcholinesterase (AChE) inhibition. For the butyrylcholinesterase (BChE) inhibition, 2-bromobenzoyl- (76) was the most active compound of the series with IC₅₀ 2.11 ± 0.31 μM. Structure-activity relationship illustrated that mild electron donating groups enhanced enzyme inhibition while electron withdrawing groups reduced the inhibition except o-NO₂. However, size and position of the substituents affected enzyme inhibitions.. In docking study of AChE, the ligands 71, 72 and 76 showed the scores of 5874, 5756 and 5666 and ACE of ?64.92,-203.25 and ?140.29 kcal/mol, respectively. In case of BChE, ligands 71, 76 and 81 depicted high scores 6016, 6150 and 5994 with ACE values ?170.91, ?256.84 and ?235.97 kcal/mol, respectively.

Full text of DOI:10.1016/j.ejmech.2017.06.036

Accepted Manuscript
Synthesis of novel 5-(aroylhydrazinocarbonyl)escitalopram as cholinesterase
inhibitors
Mehr-un Nisa, Munawar A. Munawar, Amber Iqbal, Asrar Ahmed, Muhammad Ashraf,
Qurra-tul-Ann A. Gardener, Misbahul A. Khan
PII:
S0223-5234(17)30482-8
10.1016/j.ejmech.2017.06.036
EJMECH 9531
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 April 2017
Revised Date: 16 June 2017
Accepted Date: 21 June 2017
Please cite this article as: M.-u. Nisa, M.A. Munawar, A. Iqbal, A. Ahmed, M. Ashraf, Q.-t.-A.A.
Gardener, M.A. Khan, Synthesis of novel 5-(aroylhydrazinocarbonyl)escitalopram as cholinesterase
inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.036.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT

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Synthesis of novel 5-(aroylhydrazinocarbonyl)escitalopram as
cholinesterase inhibitors
Mehr-un-Nisaa,b, Munawar A. Munawara , Amber Iqbalc, Asrar Ahmedb, Muhammad
Ashrafc, Qurra-tul-Ann A. Gardenerd, Misbahul A. Khana,c.
aInstitute of Chemistry, University of the Punjab, Lahore 54590, Pakistan.
bDepartment of Chemistry, Division of Science & Technology, University of Education,
Township, Lahore 54770, Pakistan.
cDepartment of Chemistry, The Islamia University of Bahawalpur, Bahawalpur 63100,
Pakistan.
dSchool of Biological Sciences, University of the Punjab, Lahore 54590, Pakistan.
Corresponding author. Tel.: +92-42-99230997; fax: +92-42-99230998;
E-mail: mamunawar.chem@pu.edu.pk (M. A. Munawar).
Keywords:
Acetylcholinesterase; Alzheimer’s disease; Butyrylcholinesterase; Enzyme inhibition;
Escitalopram.

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Abstract
A novel series of 5-(aroylhydrazinocarbonyl)escitalopram (58-84) have been designed,
synthesized and tested for their inhibitory potential against cholinesterases. 3-Chlorobenzoyl-
(71) was found to be the most potent compound of this series having IC50 1.80±0.11ꢀM for
acetylcholinesterase (AChE) inhibition. For the butyrylcholinesterase (BChE) inhibition, 2-
bromobenzoyl- (76) was the most active compound of the series with IC50 2.11±0.31 ꢀM.
Structure-activity relationship illustrated that mild electron donating groups enhanced
enzyme inhibition while electron withdrawing groups reduced the inhibition except o-NO2.
However, size and position of the substituents affected enzyme inhibitions. . In docking study
of AChE, the ligands 71, 72 and 76 showed the scores of 5874, 5756 and 5666 and ACE of -
64.92,-203.25 and -140.29 kcal/mol, respectively. In case of BChE, ligands 71, 76 and 81
depicted high scores 6016, 6150 and 5994 with ACE values -170.91, –256.84 and -235.97
kcal/mol, respectively.
Abbreviations:
ACE, atomic contact energy; AChE, acetylcholinesterase; AD, Alzheimer’s disease; BChE,
butyrylcholinesterase; DTNB, 5,5'-Dithiobis-(2-nitrobenzoic acid); RCSB, the research
collaboratory for structural bioinformatics.

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1. Introduction
Augustintion and Nachmansohn introduced the term acetylcholinesterase (AChE) in 1949 for
the cholinesterase which can hydrolyze acetylcholine quicker than esterases. It hydrolyses the
neurotransmitter acetylcholine into choline and acetate, hence playing an important role in
cholinergic neurotransmission [1]. AChE is directly associated with various neuromuscular
disorders e.g., myasthenia gravis, glaucoma [2], and cholinergic deficiency related to
Alzheimer’s disease (AD) [3].
Butyrylcholinesterase (BChE) is also a serine hydrolase that is interrelated to AChE [4]. For
BChE, butyrylcholine is the best substrate, neither exist naturally, nor physiologically
necessary for humans [5]. BChE function has not been fully been elucidated, however, it is
proposed that it scavenges anti-cholinesterase agents and defends synaptic AChE from
inhibition. BChE has been explored as a possible stoichiometric bioscavenger in
organophosphorus nerve agent poisoning [6]. It is extensively dispersed in the mammal’s
nervous system, indicating its likely participation in the neural functions and in
neurodegenerative diseases [7]. Certain selective BChE inhibitors have been described to
increase acetylcholine concentrations and to decrease the creation of abnormal amyloid
originate in AD [8,9].
Previous studies have revealed that citalopram is an important inhibitor of cholinestrases. The
selective serotonin re-uptake inhibitor antidepressant citalopram affects cholinesterase
inhibition with potency comparable to that of therapeutically important donepezil and
galantamine, the reversible cholinesterase inhibitors [10]. The clinical and imaging studies
have indicated that the treatment of cognitive symptoms of AD with galantamine may have
an extra benefit when treated with citalopram [11]. These studies have demonstrated two
possibilities about better effects of galantamine after treatment with citalopram. The
citalopram increases cholinesterase inhibition by inhibiting galantamine, the un-activated

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enzyme-substrate complex that is not affected by galantamine. Moreover, these drug
combinations have been claimed to generate an inhibitory effect on BChE higher than either
of the drug alone [12]. BChE synergistic inhibition results in the higher brain acetylcholine
levels. BChE remains at normal [13,14] or higher levels [15], in contrast to AChE which is
reduced in AD. Conversely, BChE enzyme may truly be the core factor in the reduction of
acetylcholine levels in AD [12].
In this ongoing study, we have designed and synthesized a novel series of 5-
(aroylhydrazinocarbonyl)escitalopram (58-84) and have screened for their potential role in
the inhibition of AChE and BChE.
2. RESULTS AND DISCUSSION
2.1.
Design
Escitalopram (1) is very effective to inhibit cholinestrases with potency comparable to AD
(e.g., donepezil and galantamine) other than depression, migraine, schizophrenia etc. [10].
Escitalopram triazoles (1a) and tetrazole (1b) have also been reported as cholinesterase
inhibitors (Figure 1-2) [16]. Recently, we have synthesized novel 5-
(aroylhydrazinocarbonyl)escitalopram (58-84) derivatives and screened for their
cholinesterase inhibition.
R = H, 2-NH2, 3-NH2, 4-NH2, 2-OH, 3-OH, 4-OH, 2-NO2, 3-NO2, 4-NO2, 2-CH3, 3-CH3, 4-
CH3, 2-Cl, 3-Cl, 4-Cl, 2-F, 3-F, 4-F, 2-Br, 3-Br, 4-Br, 2-I, 3-I, 4-I, 2-OCH3, 3-OCH3, 4-OCH3,
4-OH-3-OCH3.
Figure 1.
Structure of escitalopram triazoles, 1a [16].

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Figure 2. Structure of escitalopram tetrazole, 1b [16].
2.2. Chemistry
Escitalopram (1) was functionalized by partial hydrolysis of nitrile moiety into amide (2) by
following classical method [17]. Later, amide (2) was converted into 5-carboxyescitalopram
(3) using reported method (Scheme1) [18]. Benzoic acid and its derivatives (4-30) were
converted into benzohydrazides (31-57) by one pot conventional method [16]. 5-
(Aroylhydrazinocarbonyl) escitalopram (58-84) were synthesized by one pot coupling
(Scheme 2) [19]. A mixture of 5-carboxyescitalopram (3) and a benzohydrazide (31-57) was
dissolved in DMF and stirred for few minutes, HBTU was incorporated. Then
diisopropylethylamine
(DIEA)
was
added
and
stirred.
The
5-
(aroylhydrazinocarbonyl)escitalopram (58-84) were purified with column chromatography.
O
O
NC
N
HO
H2N
N
N
(i)
(ii)
O
O
O
F
(1)
F
(3)
F
(2)
Scheme 1. Synthetic strategy of 5-carboxyescitalopram (3). Reagents and conditions: (i)
H2O2, DMSO, K2CO3, rt. stirring; (ii) Ethylene glycol, KOH, refluxed for 3 h.

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4, 31, 58 (R = H),
5, 32, 59 (R = 2-NH2),
9, 36, 63 (R = 4-OH),
13, 40, 67 (R = 2-CH3),
17, 44, 71 (R = 3-Cl),
21, 48, 75 (R = 4-F),
25, 52, 79 (R = 2-I),
6, 33, 60 (R = 4-NH2),
7, 34, 61 (R = 2-OH),
11, 38, 65 (R = 3-NO2),
15, 42, 69 (R = 4-CH3),
19, 46, 73 (R = 2-F),
23, 50, 77 (R = 3-Br),
27, 54, 81 (R = 4-I),
8, 35, 62 (R = 3-OH),
12, 39, 66 (R = 4-NO2),
16, 43, 70 (R = 2-Cl),
20, 47, 74 (R = 3-F),
24, 51, 78 (R = 4-Br),
28, 55, 82 (R = 2-OCH3),
10, 37, 64 (R = 2-NO2),
14, 41, 68 (R = 3-CH3),
18, 45, 72 (R = 4-Cl),
22, 49, 76 (R = 2-Br)
26, 53, 80 (R = 3-I),
30, 57, 84 (R = 4-OH-3-OCH3)
29, 56, 83 (R = 4-OCH3),
Scheme 2. Syntheses of 5-(aroylhydrazinocarbonyl)escitalopram (58-84). Reagents and
conditions: (i) C2H5OH, 3N H2SO4, reflux 6 h, NaHCO3, hydrazine hydrate,
AcOH, reflux 3-6 h (ii) HBTU, DIEA, DMF, rt. stirring for 1 h.
2.3.
Biological Evaluation
The screening of novel 5-(aroylhydrazinocarbonyl)escitalopram (58-84) for cholinesterase
inhibition was performed by Ellman method using eserine as positive control. The inhibitor
interacts with AChE and BChE, and inhibits the hydrolysis of acetylthiocholine iodide,
butyrylthiocholine chloride. The inhibitor lessens the enzyme activity in a dose-dependent
manner. The unbound enzyme hydrolyzes the substrates and ultimately percent inhibition is
calculated by quantification of the product of its reaction with DTNB (5,5′-dithiobis-(2-
nitrobenzoic acid)) spectrophotometrically.
2.3.1. Acetylcholinesterase inhibition
In the present study, a series of compounds (58-84) were screened for AChE inhibition
activities (Table 1). The AChE inhibition of this series follows the following pattern
71>72>76>82>73>84>75>67>77>70>63>68>59>78>83>66>80>60>65>74>79>62>81>64
>58>69>61 which corresponds the following substituents on the aroyl group: 3-Cl>4-Cl>2-
Br>2-OCH3>2-F>4-OH-3-OCH3>4-F>2-CH3>3-Br>2-Cl>4-OH>3-CH3>2-NH2>4-Br>4-
OCH3>4-NO2>3-I>4-NH2>3-NO2>3-F>2-I>3-OH>4-I>2-NO2>H>4-CH3>2-OH. The results
of this study indicate that 3-chlorobenzoyl- (71) is the most potent compound of the series

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having IC50 1.80±0.11ꢀM which is 45-fold less active than the standard eserine (IC50
0.04±0.0001ꢀM). Two other members of the series have also shown good inhibition <10 ꢀM
e.g., 4-chlorobenzoyl- (72, IC50 6.71±0.32 ꢀM) and 2-bromobenzoyl- (76, IC50 7.81±0.33
ꢀM). While other compounds of the series 82, 73, 84, 75, 67, 77, 70, 63, 68, 59, 78, 83, 66,
80, 60, 65, 74, 79, 62, 81, 64, 58, 69, 61 have shown good to moderate inhibition with IC50
values 45.81-295.53 ꢀM.
The SAR of the series reveals that the mild electron donating groups increase AChE
inhibition. It’s worth noting that the substituents size and position are also important with the
increased inhibition. Among ortho, meta, and para positions, the meta substitutions remained
the most critical. In meta substituted ligands, the observed decreasing order of their inhibition
has been found as follow 3-Cl>3-Br>3-CH3>3-I>3-NO2>3-F>3-OH. Among the ortho
substituted ligands, 2-Br (76) has been found better inhibitor than other ligands 2-OCH3>2-
F>2-CH3>2-Cl>2-NH2>2-I>2-NO2>2-OH. Similarly, among the para substituted ligands, 4-
Cl (72) has shown more AChE inhibition than 4-OH-3-OCH3>4-F>4-OH>4-Br>4-OCH3>4-
NO2 >4-NH2>4-I>4-CH3.
Most of the compounds of the series have shown good AChE activities that are comparable to
that of standard eserine. However, it is very hard to establish SAR of the studied 5-
(aroylhydrazinocarbonyl)escitalopram (58-84) possibly because of multiple factors, i.e. size,
shape, polarizability, and electronegativity of a ligand playing crucial role in enzyme
inhibition [16].
2.3.2. Butyrylcholinesterase inhibition
BChE inhibition studies of the series of 5-(aroylhydrazinocarbonyl)escitalopram (58-84) with
their IC50 values are as given (Table 2). The decreasing order of BChE inhibition by the
compounds is 76>71>81>60>59>70>80>73>58>64>84>79>68>61>62>77>82>72>69>66>
75>74>78>63>83>67. The effect of substituents attached on the inhibition has been observed

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in the following order of 2-Br>3-Cl>4-I>4-NH2>2-NH2>2-Cl>3-I>2-F>H>2-NO2>4-OH-3-
OCH3>2-I>3-CH3>2-OH>3-OH>3-Br>2-OCH3>4-Cl>4-CH3>4-NO2>4-F>3-F>4-Br>4-OH
>4-OCH3>2-CH3. The 2-bromobenzoyl- (76) has been found to be the most potent compound
of the series with lowest IC50 2.11±0.31 ꢀM. While three other compounds of the series have
depicted IC50 values < 10 ꢀM, e.g., 3-chlorobenzoyl- (71, IC50 3.90±0.66 ꢀM), 4-
iodobenzoyl- (81, IC50 5.88±0.27 ꢀM), 4-aminobenzoyl- (60, IC50 5.98±0.29 ꢀM). All these
four compounds have been found highly active against BChE. Twenty one other compounds
59>70>80>73>58>64>84>79>68>61>62>77>82>72>69>66>75>74>78>63>83
have
demonstrated moderate inhibitions with IC50 values in the range of 68.21-196.22 ꢀM,
exhibiting selectivity from 0.04- to <35.66-fold more towards BChE than AChE. The 2-
methylbenzoyl- (67) has shown the weak inhibition with IC50 232.81±0.13 ꢀM. The SAR has
shown the same but comparatively decreased trend of BChE inhibition as in case of AChE.
The results indicate that the electron donating groups at the ortho, meta and para positions
increase BChE inhibition, while electron withdrawing groups, e.g., nitro group reduces the
inhibition except at ortho position [16].
A comparison of BChE inhibition among the ortho substituted ligands has shown the
following trend of inhibition: 2-Br>2-NH2>2-Cl>2-F>2-NO2>2-I>2-OH>2-OCH3>2-CH3.
Among the meta substituted ligands, 3-Cl (71) has been found to be the best inhibitor as
compared to 3-I (80), 3-CH3 (68), 3-OH (62), 3-Br (77) and 3-F (74). Similarly, the effect of
para substituents on BChE inhibition have been found in the order of 4-I>4-NH2>4-OH-3-
OCH3>4-Cl>4-CH3>4-NO2>4-F>4-Br>4-OH. In spite of that, it is difficult to establish SAR
of the compounds (58-84) against BChE because of the multiple factors involved between
ligand and enzyme interactions. The different behavior of the ligands toward AChE and
BChE is most likely due to Van der Waal’s weaker interactions between BChE and the
ligands than those between AChE and the ligands perhaps because of a larger active site

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gorge and less significant peripheral anionic site for AChE when compared to BChE [16,20-
22]. It is also evident from the results that these compounds have selectivity; the compounds
that have been active against AChE were not significantly active against BChE and vice
versa. The difference in selectivity may be due to the difference in structural motifs in the
both enzymes [16,11-12].
In this manuscript, the results markedly indicate that the novel 5-
(aroylhydrazinocarbonyl)escitalopram (58-84) have shown good inhibitory activity against
cholinesterase, especially AChE, and can be used as a significant starting point for further
exploration of the possible use of these ligands in the drug discovery against
neurodegenerative diseases.

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Table 1. Determination of IC50 values of AChE of 5-(aroylhydrazinocarbonyl)escitalopram.
AChE
Inhibition
(%) at 0.5
mM
AChE
IC50 (µM)a
Compd. No.
-R
Compd.
No.
-R
IC50 (µM)a
Inhibition
(%) at 0.5
mM
H
69.78±0.13
267.90±0.23
3-Cl
93.51±0.51
1.80±0.11
58
71
2-NH2
4-NH2
2-OH
84.48±0.35
83.23±0.56
59.02±0.22
117.87±0.32
129.22±0.17
295.53±0.11
4-Cl
2-F
3-F
86.07±0.15
84.38±0.35
77.09±0.16
6.71±0.32
110.97±0.82
140.51±0.33
59
60
61
72
73
74
3-OH
4-OH
2-NO2
64.43±1.16
84.58±0.25
70.30±0.11
196.51±0.24
116.87±0.32
255.67±0.55
4-F
2-Br
3-Br
86.58±0.25
85.15±0.19
80.11±0.29
111.97±0.22
7.81±0.33
62
63
64
75
76
77
114.97±0.99
3-NO2
4-NO2
79.29±0.46
75.75±0.67
136.51±0.31
125.36±0.33
4-Br
2-I
78.30±0.11
81.75±0.67
117.67±0.95
145.66±0.23
65
66
78
79
2-CH3
3-CH3
83.36±0.35
82.58±0.36
114.87±0.12
117.22±0.42
3-I
4-I
83.73±0.26
76.38±0.63
127.22±0.17
209.66±0.33
67
68
80
81
4-CH3
2-Cl
69.73±0.16
81.39±0.35
91.27±1.17
268.10±0.23
115.87±0.42
0.04±0.0001
2-OCH3
4-OCH3
88.58±0.25
76.37±0.21
81.35±0.65
45.81±0.38
123.57±0.75
111.67±0.11
69
70
82
83
84
4-OH-3-
OCH3
Eserine Std.
aResults are mean of three independent experiments (n = 3) ± S.E.M.

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Table 2. Determination of IC50 values of BChE of 5-(aroylhydrazinocarbonyl)escitalopram.
BChE
Selectivity
for
BChE
Selectivity
for BChEb
Compd.
No.
– R
Compd.
No.
– R
Inhibition
IC50 (µM)a
Inhibition
IC50 (µM)a
BChEb
(%) at 0.5
mM
(%) at 0.5
mM
H
81.54±0.65
75.81±0.24
68.21±0.15
5.98±0.29
3.53
1.72
3-Cl
4-Cl
2-F
84.01±0.41
3.90±0.66
0.46
0.04
1.55
58
59
60
71
72
73
2-NH2 84.44±0.27
4-NH2 85.52±0.63
78.37±0.15 138.11±0.27
85.49±0.29 71.38±0.29
21.60
2-OH
3-OH
85.69±0.78
88.99±0.56
3.32
1.52
3-F
4-F
73.32±0.27 184.56±0.33
74.58±0.12 178.90±0.13
0.76
0.63
61
62
74
75
88.23±0.67 129.16±0.29
73.25±0.24 191.52±0.18
4-OH
0.61
3.35
0.74
0.74
0.49
1.32
1.69
1.67
2-Br
3-Br
84.09±0.34
2.11±0.31
3.70
0.88
0.62
1.87
1.81
35.66
0.35
0.63
1.45
63
64
65
66
67
68
69
70
76
77
78
79
80
81
82
83
84
2-NO2 84.13±0.15
76.34±0.39
88.76±0.79 130.12±0.25
76.22±0.25 190.27±0.11
3-NO2 73.32±0.27 184.56±0.33
4-NO2 74.58±0.12 168.90±0.13
2-CH3 72.42±0.52 232.81±0.13
4-Br
2-I
82.58±0.67
85.44±0.26
85.52±0.63
77.89±0.23
70.21±0.17
5.88±0.27
3-I
3-CH3 85.68±0.91
4-CH3 76.60±0.16 157.90±0.44
2-Cl 85.44±0.26 69.26±0.45
82.82±1.09 0.85±0.0001
88.73±0.31
4-I
2-OCH3
4-OCH3
88.77±0.66 130.44±0.27
72.22±0.25 196.22±0.11
4-OH-3- 84.09±0.11
OCH3
76.90±0.32
Eserine
Std.
aResults are mean of three independent experiments (n = 3) ± S.E.M.,
bSelectivity for BChE = IC50 (AChE)/ IC50 (BChE).

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2.3.3. Molecular Docking Studies
The crystal structures of AChE and BChE proteins were retrieved from the Research
Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB ID 4m0e and
4bds) accordingly. The experimental (in-vitro) enzyme inhibition studies were carried out
against AChE (EC 3.1.1.7) from Electrophorus Electricus, and BChE (EC 3.1.1.8) from
Equine Serum, and docking study against human AChE (PDB ID: 4m0e at 2.0 Å resolution)
and human BChE (PDB ID: 4BDS at 2.1 Å).
The coordinate files were subjected to Discovery Studio 4.5 Visualizer for pre-docking
receptor preparation by removing water molecules and adding hydrogen atoms. Ligands 58-
84 were docked with AChE (4m0e) and BChE (4bds) by PatchDock
(http://bioinfo3d.cs.tau.ac.il/ PatchDock/). PatchDock is a molecular docking tool, targeted to
find docking transformations that produce good molecular shape complementarity based on
shape complementarity principles [23]. The input files include the receptor protein and ligand
in PDB format.
PatchDock server provides multiple solutions and the “solution 1” was chosen as it enclosed
the most important residues as binding pocket for docking analyses designated in crystal
structure of AChE receptor (4m0e) [24]. Similarly, “solution 1” enclosed the preferred
binding pocket of BChE (4bds) as it comprised most critical amino acid residues in crystal
structure of BChE receptor (4bds) [25]. The docked structures were examined by using
Discovery Studio 4.5 Visualizer.
The binding affinities of the docked ligands were evaluated as scores and ACE (atomic
contact energy) of the docked complexes. The hydrogen bonding and hydrophobic
interactions of each ligand was assessed within binding pocket of receptor protein. The
conformation of the ligands which illustrated the highest biological activities is showed in
Table 3 & 4 and Figure 1 & 2 with their favorable contacts in the binding pockets.

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To get qualitative evaluation and to recognize molecular basis of the calculated biological
activities (IC50), the docked complexes of ligands 58-84 were investigated.
In the initial assessment of the docked complexes of AChE (58-84) revealed that ligands 71,
72 and 76 showed significant interaction patterns. Ligand 71 showed most potent interaction
with AChE with a score of 5874 and an ACE of -64.92. The interacting residues of this
complex are Arg463, Arg90 (Figure 1). Ligand 71 has shown a potential hydrogen bond
between 3-chlorophenyl- group and Arg90 while second hydrogen bond has formed between
NH attached with carbonyl group adjacent to the isobenzofuran ring and Arg463. The lengths
of the two hydrogen bonds were ≤ 3.19 Å indicating significant interaction. Similarly, ligand
71 exhibited hydrophobic contact potential with pocket amino acids Val132, Ile457, Phe65 and
also depicted arene cation contact potential with Glu81.
Interestingly, ligands 72 and 76 showed no hydrogen bond interaction with AChE receptor
but instead found significant geometric fit of these ligands in the receptor and hence scoring
in PatchDock being based on shape complementarity principles it gave a score of 5756 and
5666 with AChE. Ligands 72 and 76 showed hydrophobic contact potential with pocket
amino acids Pro537, Ala412, Ala505 and Pro410, Pro235, His405 respectively. Ligand 72 has shown
potential for arene-cation contact with Arg534.
In case of BChE, ligands 71, 76 and 81 depicted highest scores 6016, 6150 and 5994 with an
ACE values -170.91, –256.84 and 235.97 kcal/mol, respectively. Visual inspection of these
complexes predicts a binding conformation of ligand 76 showed significant interaction with
BChE compared to the other 25 ligands. This ligand exhibited hydrophobic contact potential
with pocket amino acids Pro303, Val233 and also depicted arene cation contact with Arg242,
Glu238 and can play an important part to give this ligand better binding affinity than other
ligands of this series. Ligand 71 has shown potential for van der Waals contact and such
interactions involve the hydrophobic contact with Pro281, Leu286. This ligand also depicted

ACCEPTED MANUSCRIPT
arene cation contact with Arg242 and was unable to show polar interactions with the pocket
amino acids. Ligand 81 in which para-fluorophenyl group directly attached with the
isobenzofuran scaffold shows a potential to accept a hydrogen bond from the backbone
amino group of Tyr128 with an average distance of 3.23 Å. It also exhibited potential for
hydrophobic contact with pocket amino acids Trp82, Gly115, Gly116, Val280.
Our docking experiments have shown that ligands 71, 72 and 76 for AChE and ligands 71, 76
and 81 for BChE could possibly show the inhibitory potential against cholinesterases (ChE).
This is consistent with the IC50 values of the ligands (58-84).

ACCEPTED MANUSCRIPT
(a)
(d)
Ligand 71
(b)
(e)
Ligand 72
(c)
(f)
Ligand 76
Figure 1. 2D representation of the docked ligands 71a, 72b, 76c within AChE receptor
pocket showing their hydrogen bond interactions with the enzyme and 3D orientation of
docked ligands 71d, 72e, 76f with PatchDock, depicting unfavorable bump (red), carbon
hydrogen bond (light green), pi-alkyl (light pink), pi-cation (brown) and amide pi-stack
(pink) interactions.

ACCEPTED MANUSCRIPT
(g)
(j)
Ligand 71
(h)
(k)
Ligand 76
(i)
(l)
Ligand 81
Figure 2. 2D representation of the docked ligands 71g, 76h, 81i within BChE receptor pocket showing their hydrogen bond
interactions with the enzyme and 3D orientation of docked ligands 71j, 72k, 76l (with PatchDock), showing unfavorable
bump (red), carbon hydrogen bond (light green), pi-alkyl (light pink), pi-cation (brown), conventional hydrogen bond
(green) and amide pi-stack (pink) interactions.

ACCEPTED MANUSCRIPT
Table 3. Docking results for the highest ranked biologically active ligands (AChE).
Amino acids
show
hydrophobic
contacts
Amino acids
show arene-
cation
Compd.
No.
ACE
Amino acids show
Distance
(Å)
IC50 (µM)
Score
(kcal/mol) hydrogen bond contacts
contacts
58
59
60
267.90±0.23
117.87±0.32
129.22±0.17
5539
5366
5390
-543.63
-423.55
-461.74
-
-
Trp86
Glu81
-
Trp86, Tyr124, Ser125
2.15, 1.54, 2.20
-
-
-
-
Trp86
61
62
295.53±0.11
196.51±0.24
5577
5435
-580.46
-492.36
Ser203
1.97
-
-
Trp86
Trp86
Ser125, Ala127, Tyr133
Ser203
Tyr124, Ser125, Tyr133
Ser203, His447
-
,
2.92, 3.18,
2.85, 2.81
2.20, 2.81,
1.81, 2.51, 2.95
-
63
116.87±0.32
5370
-420.53
,
Trp86
-
64
65
255.67±0.55
136.51±0.31
5525
5396
-535.37
-455.36
Trp86
Trp86
Trp86
-
Ser125, Tyr133
2.16, 2.80
2.08
66
67
125.36±0.33
114.87±0.12
5385
5377
-444.75
-411.77
Ser125
Trp86
Trp86
Trp86
-
Ser125, Ala127, Tyr337
2.98, 2.40, 3.10
68
69
117.22±0.42
268.10±0.23
5369
5546
-421.88
-547.46
-
-
Trp86
-
Glu81
Trp86
Trp86, Ser203
1.18, 2.02
70
71
115.87±0.42
1.80±0.11
5386
5874
-416.46
-64.92
Trp86, Ser125, Tyr337
Arg463, Arg90
1.11, 2.75, 3.27
3.19, 2.83
-
Trp86
Glu81
Val132, Ile457
Phe65
,
72
6.71±0.32
5756
-203.25
-
-
Pro537, Ala412
,
Arg534
Ala505
-
73
74
75
76
110.97±0.82
140.51±0.33
111.97±0.22
7.81±0.33
5409
5398
5387
5666
-398.66
-469.63
-403.03
-140.29
Trp86
Tyr133
Tyr337
-
1.06
2.19
3.19
-
-
Trp86
Trp86
Glu81
Trp86
-
Pro410, Pro235
,
His405
-
77
78
79
80
81
82
83
84
114.97±0.99
117.67±0.95
145.66±0.23
127.22±0.17
209.66±0.33
45.81±0.38
123.57±0.75
111.67±0.11
5380
5373
5407
5389
5441
5489
5381
5390
-407.55
-427.83
-472.55
-448.44
-499.56
-209.45
-441.34
-403.11
Tyr337
2.57
Trp86
Trp86
-
Tyr124
2.23
-
Ser125
1.82
Trp86
Trp86
Trp86
-
-
-
Glu81
-
Asn87
2.34
-
-
Trp86
Trp86
-
Gly121, Ser125
Tyr133, Tyr337
2.83, 3.01
2.88, 2.53
-
Trp86
No. = Specific code assigned to ligand; ACE = Atomic contact energy calculated by
Patchdock (kcal/mol); IC50 = experimental calculation of inhibitory constant (µM); Distance
= hydrogen bond length calculated from docked pose by using Ligand interaction tool of
PatchDock.

ACCEPTED MANUSCRIPT
Table 4. Docking results for the highest ranked biologically active ligands (BChE).
Amino acids
show hydrogen
bond contacts
Amino acids
show arene-
cation
Compd.
No.
IC50 (µM)
Score
(ACE)
(kcal/mol)
Distance
(Å)
Amino acids show hydrophobic
contacts
contacts
Pro285, Phe329
Tyr332
75.81±0.24
5423
-467.44
Ser287, Gly116
3.18, 2.70
58
Pro285, Phe329, Trp82, Ala328
Pro285, Ala277, Phe329, Trp82
Pro285, Trp82
Tyr332
Tyr332
Tyr332
68.21±0.15
5.98±0.29
88.99±0.56
5376
6002
5463
-438.37
-161.55
-483.88
-
Gln119
-
-
2.41
-
59
60
61
129.16±0.29
191.52±0.18
5477
5551
-467.44
-549.66
Gln119
-
2.01
-
62
63
Pro285, Phe329, Ala328
-
Pro285, Phe329, Trp82, Ile69
Tyr332
Pro285, Ala277, Trp82
Pro285, Phe329, Ala277, Trp82, Ala328
Trp82
-
76.34±0.39
5432
5541
-459.55
-537.66
-
-
64
65
184.26±0.35
Ser287, Gly116
2.67, 3.13
Pro285, Phe329, Ala277, Trp82
Tyr332
168.90±0.13
5523
-523.54
-
-
66
232.81±0.13
88.73±0.31
157.90±0.44
69.26±0.45
5602
5458
5510
5390
-345.66
-479.38
-508.55
-450.44
-
Asn289
Ser287, Gly116
-
-
2.49
67
68
69
70
Pro285, Phe329, Ala277, Trp82, Ala328
Pro285, Phe329, Ala328
Pro285
-
Tyr332
Trp82
-
2.38, 2.00
-
Pro285, Phe329, Ala277
3.90±0.66
138.11±0.27
71.38±0.29
184.56±0.33
6016
5498
5411
5539
-170.91
-497.47
-469.66
-541.27
-
-
71
72
73
74
Pro281, Leu286
Pro285, Phe329, Ala277, Trp82, Ala328
Pro285, Ala277
Arg242
-
-
-
-
-
Trp82
-
Pro285, Phe329, Ala328, Ala277
Asn289
2.19
Pro285, Phe329, Trp82
Pro303, Val233
-
178.90±0.13
2.11±0.31
5527
6150
-567.55
-256.84
-
-
-
-
75
76
Arg242, Glu238
130.12±0.25
190.27±0.11
77.89±0.23
70.21±0.17
5.88±0.27
5491
5567
5439
5399
5994
5488
-490.66
-567.77
-481.88
-462.33
-235.97
-483.77
Ser287, Gly116
-
2.08, 3.00
-
77
78
79
80
81
82
Pro285, Phe329, Ala277, Ala328
Pro285, Phe329, Trp82, Ile69
Pro285, Phe329, Ala328
Trp82
-
Gln119
Asn289
Tyr128
Asn289
2.51
2.59
3.23
2.94
-
Pro285, Phe329, Ala328
Tyr332
-
Trp82, Gly115, Gly116, Val280
Pro285, Phe329, Ala277, Ala328
Trp82
130.44±0.27
Pro285, Phe329, Ile69
Pro285, Phe329, Ala328
-
196.22±0.11
76.90±0.32
5573
5431
-587.78
-478.44
-
-
83
84
Tyr332
Asn289
2.79
No. = Specific code assigned to ligand; ACE = Atomic contact energy calculated by
PatchDock (kcal/mol); IC50 = experimental calculation of inhibitory constant (µM); Distance =
hydrogen bond length calculated from docked pose by using Ligand interaction tool of
PatchDock.
18

ACCEPTED MANUSCRIPT
3. Conclusions
In the current investigation, a novel series of 5-(aroylhydrazinocarbonyl)escitalopram (58-84)
has been designed, synthesized and tested for their inhibitory potential against cholinesterases. In
in vitro studies of inhibition, 3-chlorobenzoyl-(71) derivative exhibited potent AChE inhibition
(IC50 1.80±0.11ꢀM) in this series. In case of BChE inhibition, 2-bromobenzoyl- (76) has been
found to be the most potent compound of the series with IC50 2.11±0.31ꢀM. SAR studies
declared that the mild electron donating groups increased enzyme inhibition while electron
withdrawing group decreased their inhibition except o-NO2 group. In conclusion, size and
position of the substituents also matter the cholinesterase inhibition. Docking studies showed the
potential role of these synthesized ligands in in vitro as well as in silico studies. In case of AChE,
the ligands 71, 72 and 76 showed the scores of 5874, 5756 and 5666 and an ACE of -64.92,-
203.25 and -140.29 respectively. In case of BChE, ligands 71, 76 and 81 exhibited highest scores
6016, 6150 and 5994 with an ACE values -170.91, –256.84 and -235.97 kcal/mol,
correspondingly.
4. Material and methods
All chemicals were purchased from Sigma Aldrich Co. and Alfa Aesar and were used as
1
received. H NMR and 13C spectra were recorded on a Bruker Advance-300 and 400
spectrometer. All NMR chemical shifts are reported as ppm relative to tetramethylsilane (TMS: δ
= 0.00). Coupling constants (J) are reported in hertz. Spectra were processed with MestreNova
and MestreC softwares. Mass spectra were obtained using ESI LC TOF 6224 (Agilent), under
positive ion mode. High resolution mass spectra were obtained on the 9.4 T Bruker FT-ICR-MS
spectrometer in MeOH: MeCN (1: 1). The purity of compounds was determined by HPLC that
19

ACCEPTED MANUSCRIPT
was >98% and completed on a Hewlett Packard 1100 [C-18, Microsorb-MVTM, 4.6 mm x 250
mm, 5 ꢀm]. 10-90% of methanol and acetonitrile with 0.1% TFA within 30 min and up to 100%
within extra 5 min, 1 mL/min, and TLC was determined on silica gel plates (Analtech 02521),
solvent system EtOAc: hexanes (8: 2) and CHCl3: CH3OH (1: 1).
4.1.
1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-
carboxamide (2)
1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carboxamide (2)
was afforded from free amine of escitalopram (1) by using the method described by Katritzky et
al. [17] for conversion of nitrile to amide. Purified free amine of escitalopram (1, 2.0 g, 6 mmol)
was dissolved in ~ 8.5 mL of DMSO and K2CO3 (0.1 g) was added and the reaction mixture was
stirred for half an hour. After cooling in ice bath, H2O2 (30%, 1.25 mL) was added slowly into it.
The reaction mixture was stirred overnight under N2 atmosphere. Ice cold water (50 mL) was
added and stirred for half an hour. The reaction mixture was extracted with ethyl acetate (3 x 25
mL) and the combined organic layer was kept over anhydrous MgSO4 overnight. The ethyl
acetate layer was filtered and evaporated under reduced pressure. Yield: 2.0 g (95%); mp: 125-
o
o
1
126 C (lit. [26] 126.4 C); H NMR (400 MHz, CDCl3) δH: 7.76-7.74 (m, 1H), 7.70-7.69 (m,
1H), 7.47-7.39 (m, 2H), 7.33 (d, J = 8.0 Hz, 1H), 6.99 (t, J = 8.0 Hz, 2H), 6.60-6.38 (m, 2H),
5.20-5.08 (m, 2H), 2.99-2.98 (m, 4H), 2.24 (t, J = 8.0 Hz, 2H), 2.13 (s, 6H); 13C NMR (100
MHz, CDCl3) δC:169.23, 163.08, 160.64, 148.06, 140.41, 139.75, 133.45, 127.05, 126.85,
120.68, 115.19, 90.91, 71.59, 59.53, 45.35, 42.64, 22.20; MS-(ESI) m/z: [M+H]+, 343; HRMS:
Calcd. for C20H24FN2O2: 343.1798; found: 343.1792.
4.2.
1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-
carboxylic acid (3)
20

ACCEPTED MANUSCRIPT
1-[3-(Dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro isobenzofuran-5-carboxylic acid
(5-carboxyescitalopram, 3) was achieved by standard procedure [18]. The KOH (0.84 g, 500
mmol) was dissolved in ethylene glycol (6 mL) under gentle heating. After cooling, 1-[3-
(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carboxamide (2, 1.70 g,
5 mmol) was added and reaction contents were refluxed for 3 hours. The ethylene glycol was
o
distilled under reduced pressure (1 mm Hg) at 130-140 C. The residue was neutralized with
CH3COOH (0.9 mL) and extracted with dichloromethane (3 x 25 mL). The combined organic
layer was dried over anhydrous MgSO4 overnight and evaporated under reduced pressure to get
light brown oil. Yield: 1.65 g (96 %); 1H NMR (400 MHz, CDCl3) δH:10.61 (br s, 1H), 7.92 (d, J
= 8.0 Hz, 1H), 7.82 (s, 1H), 7.47 (t, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 1H), 6.95 (t, J = 8.0 Hz,
2H), 5.13 (dd, J = 12.0, 4.0 Hz, 2H), 3.04-2.87 (m, 2H), 2.64 (s, 6H), 2.26-2.2 (m, 2H), 1.72-1.59
(m, 2H); 13C NMR (100 MHz, CDCl3) δC: 175.67, 171.37, 160.56, 146.03, 140.27, 138.73,
134.95, 129.39, 126.78, 121.29, 115.19, 90.30, 71.67, 63.74, 57.28, 42.31, 22.02; MS-(ESI)
m/z:[M+H]+, 344; HRMS: Calcd. for C20H23FNO3: 344.1658; found: 344.1656.
4.3. Synthesis of Benzohydrazides
4.3.1.General procedure:
Benzohydrazides (31-57) were synthesized by one pot conventional method [16] (see
Supplimentary Information).
4.4. Synthesis of 5-(aroylhydrazinocarbonyl)escitalopram
4.4.1. General Procedure:
5-(Aroylhydrazinocarbonyl)escitalopram (58-84) were synthesized by the coupling reaction
conditions mostly used in peptide synthesis [19]. 5-Carboxyescitalopram (3, 0.001 mmol) and
21

ACCEPTED MANUSCRIPT
benzohydrazide (31, 0.001 mmol) were dissolved in DMF (15 mL). After five minutes stirring,
HBTU (0.0012 mmol) was incorporated and stirred for 20 minutes. Then DIEA (0.002 mmol)
was added and stirred for 1 hour. Saturated solution of NH4Cl (1 x 10 mL) was added into the
reaction mixture and extracted with ethyl acetate (3 x 10 mL). The ethyl acetate layer was
washed with solution of NaHCO3 (5 %, 2 x 10 mL) followed by brine (1 x 10 mL) and dried
over anhydrous MgSO4 overnight. After the removal of ethyl acetate under reduced pressure, the
residue was purified using column chromatography.
4.4.1.1.
N'-Benzoyl-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzo
furan-5-carbohydrazide (58)N'-Benzoyl-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
di hydroisobenzofuran-5-carbohydrazide (58) was synthesized from 5-carboxyescitalopram (3,
0.343 g, 0.001 mmol) and benzohydrazide (31, 0.136 g, 0.001 mmol). Off white amorphous;
o
Yield: 0.450 g (98%); mp: 172-174 C;1H NMR (300 MHz, DMSO-d6) δH: 7.78 (br s, 2NH),
7.77-7.70 (m, 4H), 7.68-7.55 (m, 4H), 7.52-7.47 (m, 3H), 7.19-7.11 (m, 2H), 5.22-5.10 (m, 2H),
2.17-2.16 (m, 2H), 2.04 (s, 6H), 1.32-1.20 (m, 4H); MS-(ESI) m/z:[M+H]+, 462; HRMS: Calcd.
for C27H29FN3O3: 462.2189; found: 462.2199; HPLC analysis: retention time = 12.1 min; peak
area, 99.08%.
4.4.1.2. N'-(2-Aminobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro
o
1
isobenzofuran-5-carbohydrazide (59) Yield: 0.462 g (97%); mp: >311 C (decompose); H
NMR (300 MHz, DMSO-d6) δH: 7.88 (br s, 2NH), 7.68-7.56 (m, 5H), 7.48-7.40 (m, 3H), 7.14 (t,
J = 8.7 Hz, 3H), 6.83 (br s, -NH2), 5.23-5.12 (m, 2H), 2.71-2.67 (m, 2H), 2.24 (s, 6H), 1.92-1.84
(m, 2H), 1.17-1.12 (m, 2H); MS-(ESI) m/z: [M+H]+, 477; HRMS: Calcd. for C27H30FN4O3:
477.2298; found: 477.2306; HPLC analysis: retention time = 12.4 min; peak area, 99.41%.
22

ACCEPTED MANUSCRIPT
4.4.1.3. N'-(4-Aminobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro
isobenzofuran-5-carbohydrazide (60) Brown amorphous; Yield: 0.466 g (98%); mp: > 296
1
oC(decompose) H NMR (300 MHz, DMSO-d6) δH: 7.79 (br s, 2NH), 7.76-7.73 (m, 4H), 7.60-
7.57 (m, 4H), 7.19-7.14 (m, 3H), 6.83 (br s, -NH2), 5.24-5.13 (m, 2H), 2.73-2.67 (m, 2H), 2.20
(s, 6H), 1.54-1.51 (m, 2H), 1.45-1.42 (m, 2H); MS-(ESI) m/z: [M+H]+, 477; HRMS: Calcd. for
C27H30FN4O3: 477.2300; found: 477.2310; HPLC analysis: retention time = 12.3 min; peak area,
99.23%.
4.4.1.4.
N'-(2-Hydroxybenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (61) Colourless crystalline; Yield: 0.468 g (98%);
mp: 120-123 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.80 (br s, 2NH), 7.70-7.54 (m, 5H), 7.48-
7.40 (m, 3H), 7.17-7.11 (m, 2H), 6.85-6.83 (m, 1H), 5.24-5.12 (m, 2H), 4.13 (s, 1H), 2.71-2.67
(m, 2H), 2.24 (s, 6H), 1.92-1.84 (m, 2H), 1.17-1.12 (m, 2H); MS-(ESI) m/z: [M+H]+, 478;
HRMS: Calcd. for C27H29FN3O4: 478.2140; found: 478.2147; HPLC analysis: retention time =
12.7 min; peak area, 99.66%.
4.4.1.5.
N'-(3-Hydroxybenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (62) Colourless crystalline; Yield: 0.465 g (97%);
mp: 137-139 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.81 (br s, 2NH), 7.79-7.65 (m, 5H), 7.61-
6.57 (m, 3H), 7.19-7.13 (m, 3H), 5.24-5.12 (m, 2H), 4.14 (s, 1H), 2.81-2.77 (m, 2H), 2.21 (s,
6H), 1.50-1.23 (m, 4H); MS-(ESI) m/z: [M+H]+, 478; HRMS: Calcd. for C27H29FN3O4:
478.2137; found: 478.2150; HPLC analysis: retention time = 12.5 min; peak area, 99.49%.
4.4.1.6.
N'-(4-Hydroxybenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (63) Colourless crystalline; Yield: 0.47 g (98%); mp:
23

ACCEPTED MANUSCRIPT
125-127 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.76 (br s, 2NH), 7.74-7.70 (m, 5H), 7.58-7.53
(m, 3H), 7.15 (t, J = 9.0 Hz, 3H), 5.20-5.10 (m, 2H), 4.11 (s, 1H), 2.14-2.09 (m, 2H), 2.00 (s,
6H), 1.84-1.76 (m, 2H), 1.27-1.15 (m, 2H); MS-(ESI) m/z (deconvoluted): [M+]+, 477; HRMS:
Calcd. for C27H29FN3O4: 478.2141; found: 478.2156; HPLC analysis: retention time = 12.4 min;
peak area, 99.52%.
4.4.1.7.
N'-(2-Nitrobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro
isobenzofuran-5-carbohydrazide (64) Yellow amorphous; Yield: 0.493 g (97%); mp: 120-122
oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.77 (br s, 2NH), 7.75-7.72 (m, 4H), 7.58-7.55 (m, 4H),
7.16-7.11 (m, 3H), 5.19-5.10 (m, 2H), 2.16-2.10 (m, 4H), 2.00 (s, 6H), 1.22-1.16 (m, 2H); MS-
(ESI) m/z (deconvoluted): [M+]+, 506; HRMS: Calcd. for C27H28FN4O5: 507.2040; found:
507.2051; HPLC analysis: retention time = 13.3 min; peak area, 99.41%.
4.4.1.8.
N'-(3-Nitrobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro
isobenzofuran-5-carbohydrazide (65) Yellow amorphous; Yield: 0.491 g (97%); mp: > 308 oC
(decompose) 1H NMR (300 MHz, DMSO-d6) δH: 7.81 (br s, 2NH), 7.80-7.85 (m, 4H), 7.62-7.58
(m, 4H), 7.19-7.15 (m, 3H), 5.25-5.14 (m, 2H), 2.90-2.86(m, 2H), 2.15-2.11 (m, 2H), 2.55 (s,
6H), 1.27-2.23 (m, 2H), 1.57-1.45 (m, 2H); MS-(ESI) m/z: [M+H]+, 507; HRMS: Calcd. for
C27H28N4O5: 507.2037; found: 507.2048; HPLC analysis: retention time = 13.1 min; peak area,
99.34%.
4.4.1.9.
N'-(4-Nitrobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro
isobenzofuran-5-carbohydrazide (66) Yellow amorphous; Yield: 0.496 g (98%); mp: > 246
1
oC(decompose); H NMR (300 MHz, DMSO-d6) δH: 7.80 (br s, 2NH), 7.79-7.73 (m, 4H), 7.60-
7.56 (m, 4H), 7.18-7.14 (m, 3H), 5.23-5.13 (m, 2H), 2.79-1.67 (m, 2H), 2.22 (s, 6H), 1.54-1.42
24

ACCEPTED MANUSCRIPT
(m, 2H), 1.34-1.29 (m, 2H); MS-(ESI) m/z: [M+H]+, 507; HRMS: Calcd. for C27H28FN4O5:
507.2042; found: 507.2059; HPLC analysis: retention time = 13.4 min; peak area, 99.47%.
4.4.1.10.
N'-(2-Methylbenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (67) Off white crystalline; Yield: 0.458 g (96%); mp:
129-131 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.77 (br s, 2NH), 7.76-7.72 (m, 4H), 7.58-7.55
(m, 3H), 7.15-7.11 (m, 4H), 5.19-5.09 (m, 2H), 2.49 (s, 3H),2.31-2.30 (m, 2H), 2.00 (s, 6H),
1.29-1.15 (m, 4H); MS-(ESI) m/z: [M+H]+, 476; HRMS: Calcd. for C28H31FN3O3: 476.2345;
found: 476.2362; HPLC analysis: retention time = 12.7 min; peak area, 99.59%.
4.4.1.11.
N'-(3-Methylbenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (68) Off white crystalline; Yield: 0.462 g (97%); mp:
122-124 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.77 (br s, 2NH), 7.76-7.72 (m, 4H), 7.58-7.55
(m, 4H), 7.15-7.11 (m, 3H), 5.19-5.10 (m, 2H), 2.49 (s, 3H), 2.14-2.13 (m, 2H), 2.11 (s, 6H),
1.26-1.14 (m, 4H); MS-(ESI) m/z: [M+H]+, 476; HRMS: Calcd. for C28H31FN3O3: 476.2347;
found: 476.2359; HPLC analysis: retention time = 12.8 min; peak area, 99.87%.
4.4.1.12.
N'-(4-Methylbenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (69) Off white crystalline; Yield: 0.466 g (98%); mp:
110-112 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.76 (br s, 2NH), 7.73-7.72 (m, 4H), 7.58-7.55
(m, 4H), 7.16-7.11 (m, 3H), 5.20-5.10 (m, 2H), 2.49 (s, 3H),2.28-2.24 (m, 2H), 2.18-2.13 (m,
2H), 2.10 (s, 6H), 1.00-0.98 (m, 2H); MS-(ESI) m/z (deconvoluted): [M+]+, 475; HRMS: Calcd.
for C28H31FN3O3: 476.2352; found: 476.2367; HPLC analysis: retention time = 12.9 min; peak
area, 99.92%.
25

ACCEPTED MANUSCRIPT
4.4.1.13.
N'-(2-Chlorobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (70) White crystalline; Yield: 0.486 g (98%); mp:
85-87 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.81 (br s, 2NH), 7.79-7.73 (m, 5H), 7.59-7.56 (m,
3H), 7.17 (t, J = 8.7 Hz, 3H), 5.24-5.12 (m, 2H), 2.73-2.68 (m, 2H), 2.44 (s, 6H), 2.22-2.17 (m,
2H), 1.58-1.44 (m, 2H); MS-(ESI) m/z: [M+H]+, 496, 498; HRMS: Calcd. for C27H28ClFN3O3:
496.1800; found: 496.1812; HPLC analysis: retention time = 13.2 min; peak area, 99.66%.
4.4.1.14.
N'-(3-Chlorobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (71) White crystalline; Yield: 0.471 g (95%); mp:
130-132 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.75 (br s, 2NH), 7.73-7.69 (m, 3H), 7.57-7.52
(m, 4H), 7.14-7.08 (m, 4H), 5.18-5.07 (m, 2H), 2.14-2.10 (m, 4H), 2.20 (s, 6H), 1.28-1.11 (m,
2H); MS-(ESI) m/z (deconvoluted): [M+]+, 495, 497; HRMS: Calcd. for C27H28ClFN3O3:
496.1802; found: 496.1821; HPLC analysis: retention time = 13.3 min; peak area, 99.71%.
4.4.1.15.
N'-(4-Chlorobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (72) White crystalline; Yield: 0.47 g (98%); mp:
197-200 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.81 (br s, 2NH), 7.80-7.75 (m, 4H), 7.62-7.58
(m, 4H), 7.19-7.15 (m, 3H), 5.25-5.14 (m, 2H), 2.91-2.87 (m, 2H), 2.55 (s, 6H), 2.27-2.23 (m,
2H), 1.58-1.46 (m, 2H); MS-(ESI) m/z (deconvoluted): [M+]+, 495, 497; HRMS: Calcd. for
C27H28ClFN3O3: 496.1798; found: 496.1817; HPLC analysis: retention time = 13.1 min; peak
area, 99.78%.
4.4.1.16.
N'-(2-Fluorobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (73) White amorphous; Yield: 0.469 g (98%); mp:
26

ACCEPTED MANUSCRIPT
146-148 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.78 (br s, 2NH), 7.77-7.75 (m, 4H), 7.60-7.56
(m, 4H), 7.18-7.12 (m, 3H), 5.22-5.10 (m, 2H), 2.17-2.16 (m, 2H), 2.04 (s, 6H), 1.32-1.20 (m,
4H); MS-(ESI) m/z: [M+H]+, 480; HRMS: Calcd. for C27H28F2N3O3: 480.2094; found: 480.2105;
HPLC analysis: retention time = 13.5 min; peak area, 99.65%.
4.4.1.17.
N'-(3-Fluorobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (74) White amorphous; Yield: 0.465 g (97%); mp:
125-127 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.81 (br s, 2NH), 7.79-7.73 (m, 4H), 7.61-7.57
(m, 4H), 7.19-7.13 (m, 3H), 5.25-5.12 (m, 2H), 2.81-2.77 (m, 2H), 2.21 (s, 6H),1.50-1.45 (m,
2H), 1.28-1.23 (m, 2H); MS-(ESI) m/z (deconvoluted): [M+]+, 479; HRMS: Calcd. for
C27H28F2N3O3: 480.2097; found: 480.2109; HPLC analysis: retention time = 13.4 min; peak
area, 99.21%.
4.4.1.18.
N'-(4-Fluorobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (75) White amorphous; Yield: 0.47 g (98%); mp:
1
115-117oC; H NMR (300 MHz, DMSO-d6) δH: 7.81 (br s, 2NH), 7.79-7.73 (m, 5H), 7.61-7.56
(m, 3H), 7.20-7.14 (m, 3H), 5.24-5.12 (m, 2H), 2.73-2.68 (m, 2H), 2.44 (s, 6H), 2.22-2.17 (m,
2H), 1.55-1.47 (m, 2H); MS-(ESI) m/z (deconvoluted): [M+]+, 479; HRMS: Calcd. for
C27H28F2N3O3: 480.2100; found: 480.2117; HPLC analysis: retention time = 13.3 min; peak
area, 99.33%.
4.4.1.19.
N'-(2-Bromobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (76) Lemon yellow amorphous; Yield: 0.525 g
o
1
(97%); mp: 118-120 C; H NMR (300 MHz, DMSO-d6) δH: 7.76 (br s, 2NH), 7.75-7.71 (m,
4H), 7.58-7.54 (m, 4H), 7.16-7.10 (m, 3H), 5.20-5.08 (m, 2H), 2.14-2.00 (m, 4H), 1.99 (s, 6H),
27

ACCEPTED MANUSCRIPT
1.76-1.74 (m, 2H); MS-(ESI) m/z (deconvoluted): [M+]+, 539, 541; HRMS: Calcd. for
C27H28BrFN3O3: 540.1300; found: 540.1317; HPLC analysis: retention time = 13.2 min; peak
area, 99.23%.
4.4.1.20.
N'-(3-Bromobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (77) Light yellow amorphous; Yield: 0.520 g (96%);
mp: 135-139 oC; 1H NMR (300 MHz, DMSO-d6) δH: 7.77 (br s, 2NH), 7.75-7.71 (m, 4H), 7.58-
7.54 (m, 3H), 7.15-7.11 (m, 4H), 5.19-5.10 (m, 2H), 2.31-2.29 (m, 2H), 2.12 (s, 6H), 1.01-2.97
(m, 2H), 1.26-1.19 (m, 2H); 13C NMR (75.45 MHz, DMSO-d6) δC: 165.57, 164.42, 160.35,
147.93,141.23, 139.09, 134.92, 132.25, 130.87, 130.76, 127.06, 126.96, 123.62, 121.96, 120.77,
118.97, 115.11, 114.84, 90.41, 71.22, 58.85, 45.02, 38.54, 21.89; MS-(ESI) m/z: [M+H]+, 540,
542; HRMS: Calcd. for C27H28BrFN3O3: 540.1302; found: 540.1311; HPLC analysis: retention
time = 13.1 min; peak area, 99.42%.
4.4.1.21.
N'-(4-Bromobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbohydrazide (78) Lemon yellow powder; Yield: 0.535 g (99%);
o
mp: > 298 C(decompose); 1H NMR (300 MHz, DMSO-d6) δH: 7.78 (br s, 2NH), 7.76-7.72 (m,
4H), 7.60-7.50 (m, 4H), 7.17 (t, J = 9.0 Hz, 3H), 5.23-5.10 (m, 2H), 3.20 (t, J = 8.1 Hz, 2H),
2.99-2.96 (m, 2H), 2.16 (s, 6H), 1.63-1.56 (m, 2H); MS-(ESI) m/z: [M+H]+, 540, 542; HRMS:
Calcd. for C27H28BrFN3O3: 540.1299; found: 540.1308; HPLC analysis: retention time = 13.3
min; peak area, 99.24%.
4.4.1.22. N'-(2-Iodobenzoyl)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro
o
isobenzofuran-5-carbohydrazide (79) White amorphous; Yield: 0.562 g (96%); mp: 83-85 C;
1H NMR (300 MHz, DMSO-d6) δH: 7.78 (br s, 2NH), 7.77-7.75 (m, 4H), 7.60-7.58 (m, 4H),
28