Synthesis and Structure-Activity Relationship of 1-(2-Furoyl)Piperazine Bearing Benzamides as Butyrylcholinesterase Inhibitors

Article abstract of DOI:10.1007/s11094-020-02247-2

Four benzamide derivatives (5a, 5b, 8a, 8b) bearing heterocyclic furan and piperazine ring have been synthesized and evaluated for enzyme inhibition and hemolytic activity. Initial 4-(chloromethyl)benzoyl chloride (1) and 3-(chloromethyl)benzoyl chloride

Full text of DOI:10.1007/s11094-020-02247-2

DOI 10.1007/s11094-020-02247-2
Pharmaceutical Chemistry Journal, Vol. 54, No. 6, September, 2020 (Russian Original Vol. 54, No. 6, June, 2020)
SYNTHESIS AND STRUCTURE-ACTIVITY RELATIONSHIP
OF 1-(2-FUROYL)PIPERAZINE BEARING BENZAMIDES
AS BUTYRYLCHOLINESTERASE INHIBITORS
M. A. Abbasi,1,* M. Irshad,2 Aziz-ur-Rehman,1 S. Z. Siddiqui,1
H. M. Junaid,1 S. A. A. Shah,3 and M. Ashraf4
Original article submitted March 1, 1990.
Four benzamide derivatives (5a, 5b, 8a, 8b) bearing heterocyclic furan and piperazine ring have been synthe-
sized and evaluated for enzyme inhibition and hemolytic activity. Initial 4-(chloromethyl)benzoyl chloride (1)
and 3-(chloromethyl)benzoyl chloride (6) were stirred with benzyl amine (2a) and cyclohexyl amine (2b), re-
spectively, in aqueous medium at pH 9 – 10 maintained by aqueous sodium carbonate. The resulting
benzamides (3a, 3b, 7a, 7b) were refluxed with 1-(2-furoyl)piperazine (4) in the presence of K2CO3 and
CH3CN to acquire target compounds (5a, 5b, 8a, 8b). The spectroscopic techniques including 13C NMR, 1H
NMR, IR and EI-MS corroborated the proposed molecular structures of final compounds. Among these, two
compounds (5b, 8b) proved to be considerable inhibitors of butyrylcholinesterase enzyme. Study of the
hemolytic activity potential revealed low toxicity level of compound 5b.
Keywords: benzamides; enzyme inhibition; furan; hemolytic activity; piperazine.
1. INTRODUCTION
types of compounds include peptides or proteins. At present,
about 30% known drugs [14, 15] contain moieties of this
type. When carboxylic acids and amines are coupled, then
amide bond is formed as a key functional group [16 – 19].
The reaction of benzyl chloride with ammonia also yielded
this moiety known as benzoic acid amide. Amide
The bioactive nature of heterocyclic compounds is well
known to researchers. Piperazine is a six membered nitro-
gen-containing heterocyclic compound, which has been
modified structurally and found numerous applications in en-
gineering [1] and polymer [2] fields. Derivatives of this moi-
ety showed inhibition potential against certain microbial
strains [3], various enzymes [4], noroviruses [5] and triple
reuptake [6]. The cannabinoid CB1 receptor agonists [7] and
antagonists for melanocortin-4 receptor [8] have been found
to include piperazine heterocycle. In synthesized com-
pounds, another biologically potent functionality of amides
or benzamides [9 – 13] is inserted to boost up the potential of
piperazine. It was found that biologically significant com-
pounds have amide bonds as a key functional group. These
(RCO-NH ) is a compound which has C=O group and R
2
substituent that may be hydrogen, linear chain or ring struc-
tures. It may also be a compound having metal replacing hy-
drogen in ammonia like sodium amide. Nitrogen admits various
substituents which classify the amides into different classes ex-
hibiting variable physical and chemical behavior [20].
Progress in the medicinal chemistry led to the develop-
ment of many drugs for the cure and treatment of cancer.
Benzamide, its derivatives and its other substituents are con-
sidered to be pharmacologically active. These compounds
have interesting structures containing moieties which show
diverse and remarkable activities. These compounds can be
used for the treatment of different diseases including cancer
[21 – 24]. Metoclopramide is an important benzamide deriv-
ative. This derivative has been reported to enhance the
cisplatin effects [25, 26]. Anacardic acid has been used for
the synthesis of many benzamide derivatives. In particular,
cyano(acrylamido)benzamide is a benzamide derivative pos-
sessing anti-tumor activity against breast cancer cells [27].
Benzamides and their analogs exhibit different types of bio-
1
Department of Chemistry, Government College University, La-
hore-54000, Pakistan.
2
Division of Science and Technology, University of Education, Township
Campus, Lahore-54770, Pakistan.
3
Faculty of Pharmacy & Atta-ur-Rahman Institute for Natural Products
Discovery (AuRIns), Level 9, FF3, Universiti Teknologi MARA, Puncak
Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Ma-
laysia.
4
Department of Chemistry, The Islamia University of Bahawalpur,
Bahawalpur-63100, Pakistan.
*
e-mail: atrabbasi@yahoo.com; abbasi@gcu.edu.pk
596
0091-150X/20/5406-0596 © 2020 Springer Science+Business Media, LLC

Synthesis and Structure-Activity Relationship
597
Scheme 1. Synthesis of N-(2-furoyl)piperazine bearing benzamides (5a, 5b) from 4-(chloromethyl)benzoyl chloride (1). Reagents and condi-
tions: (I) aq. Na CO soln./pH 9 – 10/stirring at RT for 2 – 3 h; (II) acetonitrile/K CO /reflux of 4 for 0.5 h for its activation, followed by addi-
2
tion of respective electrophile (3a, 3b) and final reflux for 3 – 4 h.
3
2
3
logical activities such as anti-convulsant, anti-inflammatory,
analgesic, serotonin, anti-tumor, anti-microbial and anti-de-
pressant [28, 29].
num plates using ethyl acetate and n-hexane (30:70) as sol-
vent system, followed by detection under UV lamp at 254 nm.
2.2. Chemical Synthesis
We have synthesized a series of molecules bearing
piperazine, furan and benzamide and evaluated them for the
inhibition potential against butyrylcholinesterase (BChE) in
search of new drug candidates for treating diseases [30]
caused by this enzyme. In addition, toxicity level was as-
sessed through hemolytic activity analysis.
Synthesis of N-(benzyl/cyclohexyl)-4/3-(chloromethyl)-
benzamides 3a, 3b, 7a, and 7b (Scheme 1). Benzyl amine
(2a) and cyclohexyl amine (2b) (18.0 mmol), respectively,
were dispersed in distilled water (20.0 mL) taken in an io-
dine flask (250 mL). Then, 10% Na CO aqueous solution
2
3
was added to adjust pH at 9 – 10 for proper reaction,
4-(chloromethyl)benzoyl chloride (1) and 3-(chlorome-
thyl)benzoyl chloride (6), respectively, were added dropwise
on strong stirring, and the mixture was further stirred for
2 – 3 h until TLC ascertained the reaction completion.
Finally, the precipitates of title electrophiles (3a, 3b, 7a, 7b)
were filtered, washed with distilled water and dried.
2. EXPERIMENTAL
2.1. Materials and Instruments
1
The H NMR spectra were measured in CDCl using
Bruker spectrometer operating at 600 MHz. The 13C-NMR
3
spectra were measured in CD OD using the same instrument
3
Synthesis of N-(benzyl/cyclohexyl)-4/3-{[4-(2-furoyl)-
1-piperazinyl]methyl}benzamides 5a, 5b, 8a, and 8b
(Scheme 2). 1-(2-Furoyl)piperazine (4) (14.0 mmol) was
mixed with acetonitrile (22 mL) and solid K CO
operating at 150 MHz. The IR spectra were recorded on
Jasco-320-A spectrometer using KBr pellet method. EIMS
spectra were obtained on JMS-HX-110 spectrometer. Sol-
vents (analytical grade) and chemicals (Sigma Aldrich and
Alfa Aesar) were purchased through local supplier. Melting
points were determining using the Griffin-George apparatus
in open capillary tubes and remained uncorrected. Thin layer
chromatography (TLC) was performed on silica coated alumi-
2
3
(14.0 mmol) in a 250 mL round bottom flask. The mixture
was refluxed for 0.5 hour and then electrophiles (3a, 3b, 7a,
7b) were added. The reaction was completed on further re-
flux for 3 – 4 h. After final TLC report, the title compounds

598
M. A. Abbasi et al.
Scheme 2. Synthesis of N-(2-furoyl)piperazine bearing benzamides (8a, 8b) from 3-(chloromethyl)benzoyl chloride (6). Reagents and condi-
tions: (I) aq. Na CO soln./pH 9 – 10/stirring at RT for 2 – 3 h; (II) acetonitrile/K CO /reflux of 4 for 0.5 h for its activation, followed by addi-
2
tion of respective electrophile (7a, 7b) and final reflux for 3 – 4 h.
3
2
3
(5a, 5b, 8a. 8b) were collected through filtration or extrac-
tion by chloroform.
N-Cyclohexyl-4-[4-(2-furoyl)-1-piperazinyl]methyl-
benzamide (5b): white crystalline solid; yield: 94%; m.p:
125 – 127°C; Mol. F.: C H N O ; Mol. Mass: 395; IR
N-Benzyl-4-[4-(2-furoyl)-1-piperazinyl]methylbenz-
amide (5a): brown liquid; yield: 90%; Mol. F.: C H N O ;
23 29
3
3
24 25
3
3
(KBr, cm-1) n : 1109 (C-N-C), 1191 (C-O-C), 1587 (Ar
max
Mol. Mass: 403; IR (KBr, cm-1) n : 1110 (C-N-C), 1195
C=C), 1650 (C=O), 2881 (R C-H), 3079 (Ar C-H), 3409
max
(C-O-C), 1588 (Ar C=C), 1665 (C=O), 2884 (R C-H), 3097
(Ar C-H), 3412 (N-H); 7.85 (d, J = 8.2 Hz, 2H, H-2 & H-6),
7.45 (d, J = 1.0 Hz, 1H, H-5²), 7.38 – 7.27 (m, 5H, H-2¢² to
H-6¢²), 7.21 (d, J = 8.4 Hz, 2H, H-3 & H-5), 7.03 (d,
J = 2.6 Hz, 1H, H-3²), 6.46 (dd, J = 3.4, 1.6 Hz, 1H, H-4²),
3.84 (br.s, 4H, CH 3¢ & CH 5¢), 3.62 (s, 2H, CH 8), 2.54
(N-H); 1H-NMR (600 MHz, CDCl , d / ppm): 7.86 (d,
3
J = 8.2 Hz, 2H, H-2 & H-6), 7.45 (d, J = 0.9 Hz, 1H, H-5²),
7.26 (d, J = 8.4 Hz, 2H, H-3 & H-5), 7.09 (d, J = 2.7 Hz, 1H,
H-3²), 6.41 (dd, J = 3.2, 1.5 Hz, 1H, H-4²), 3.99 – 3.93 (m,
1H, CH-1¢²), 3.87 (br.s, 4H, CH 3¢ & CH 5¢), 3.62 (s, 2H,
2-
2-
2-
2-
2-
CH 8), 2.55 (br.s, 4H, CH 2¢ & CH 6¢), 1.91 – 1.64 (m,
(br.s, 4H, CH 2¢ & CH 6¢), 2.47 (s, 2H, CH 7¢²); 13C-NMR
2-
2-
2-
2-
2-
2-
10H, CH 2¢² to CH 6¢²); 13C-NMR (150 MHz, CD OD, d
2-
2-
3
(150 MHz, CD OD, d in ppm): 167.38 (C-6²), 167.27 (C-7),
3
in ppm): 169.47 (C-6²), 167.79 (C-7), 157.36 (C-3 & C-5),
149.24 (C-2 & C-6), 146.18 (C-3²), 142.53 (C-2²), 136.57
(C-4²), 132.89 (C-5²), 115.22 (C-1), 111.25 (C-4), 65.38
(C-8), 53.24 (C-2¢, C-3¢, C-5¢ & C-6¢), 37.48 (C-1¢²), 36.78
(C-2¢² & C-6¢²), 30.14 (C-4¢²), 29.16 (C-3¢² & C-5¢²); EIMS
(m/z): 395 [M]+, 300 [C H N O]+, 271 [C H N O]+, 268
159.08 (C-3 & C-5), 147.77 (C-2 & C-6), 145.10 (C-3²),
143.73 (C-2²), 141.32 (C-1¢²), 138.41 (C-4²), 133.46 (C-5²),
129.20 (C-2¢² & C-6¢²), 127.20 (C-4¢²), 126.62 (C-3¢² &
C-5¢²), 116.41 (C-1), 111.28 (C-4), 64.24 (C-8), 53.07 (C-2¢,
C-3¢, C-5¢ & C-6¢), 44.00 (C-7¢²); ; EIMS (m/z): 403 [M]+,
308 [C H N O]+, 279 [C H N O]+, 268 [C H N O ]•+,
18 26
3
17 23
2
19 22
3
18 19
2
16 16
2
2
[C H N O ]•+, 217 [C H NO]•+, 179 [C H N O ]+, 118
225 [C H NO]•+, 179 [C H N O ]+, 118 [C H O]•+, 95
16 16
2
2
14 19
9
11
2
2
15 15
9
11
2
2
8
6
[C H O ]+.
[C H O]•+, 95 [C H O ]+.
5
3
2
8 6 5 3 2

Synthesis and Structure-Activity Relationship
599
Control - Test
N-Benzyl-3-{[4-(2-furoyl)-1-piperazinyl]methyl}benz-
amide (8a): off white amorphous solid; yield: 79%; m.p:
130 – 132°C; Mol. F.: C H N O ; Mol. Mass.: 403; IR
Inhibition (%) =
´100.
Control
24 25
3
3
(KBr, cm-1) n : 1118 (C-N-C), 1197 (C-O-C), 1584 (Ar
2.4. Statistical Analysis
max
C=C), 1658 (C=O), 2881 (R C-H), 3080 (Ar C-H), 3406
The results of triple experiments are expressed as mean
1
(N-H); H-NMR (600 MHz, CDCl , d in ppm): 8.20 (s, 1H,
SEM. Microsoft Excel 2010 was utilized for statistical analy-
3
sis and 50% inhibitory concentration (IC ) was calculated
50
H-2), 8.13 (d, J = 7.6 Hz, H, H-6), 7.84 (br.s, 1H, H-5²), 7.82
(distorted d, J = 7.0 Hz, 1H, H-4), 7.79 (t, J = 7.6 Hz, 1H,
H-5), 7.39 – 7.33 (m, 5H, H-2¢² to H-6¢²), 7.33 (d,
J = 3.3 Hz, 1H, H-3²), 6.85 (dd, J = 2.4, 4.1 Hz, 1H, H-4²),
3.91 (br.s, 4H, CH -3¢, CH -5¢), 3.69 (s, 2H, CH -8), 2.85
using EzFit Perrella Scientific Inc. (Amherst, USA) soft-
ware.
3. RESULTS AND DISCUSSION
2
2
2
Two schemes have been used for the synthesis of four
benzamides bearing heterocyclic furan and piperazine moi-
eties (Schemes 1 and 2). The four synthesized compounds
were subjected to screening for inhibition of butyrylcholines-
terase enzyme (Table 1) followed by hemolytic activity anal-
ysis (Table 2).
(br.t, J = 5.1 Hz, 4H, CH -2¢, CH -6¢), 2.47 (s, 2H, CH -7¢²);
2
2
2
13C-NMR (150 MHz, CD OD, d in ppm): 168.04 (C-6²),
3
168.00 (C-7), 159.83 (C-6), 150.90 (C-2), 148.34 (C-5),
141.36 (C-3²), 139.06 (C-2²), 138.11 (C-1¢²), 136.45 (C-4²),
132.55 (C-5²), 131.49 (C-2¢² & C-6¢²), 130.39 (C-4¢²),
129.63 (C-3¢² & C-5¢²), 120.77 (C-3), 115.29 (C-4), 66.35
(C-8), 53.03 (C-2¢, C-3¢, C-5¢ & C-6¢), 44.15 (C-7¢²); EIMS
(m/z): 403 [M]+, 308 [C H N O]+, 279 [C H N O]+, 268
3.1. Chemistry
N-Benzyl-4-[4-(2-furoyl)-1-piperazinyl]methylbenzami
de (5a) was collected as brown liquid after extraction using
chloroform as solvent with 90% yield. The EI-MS spectrum
well supported the molecular formula of this compound as
C H N O with mass of m/z of 403. The significant mass
19 22
3
18 19
2
[C H N O ]•+, 225 [C H NO]•+, 179 [C H N O ]+, 118
16 16
2
2
15 15
9
11
2
2
[C H O]•+, 95 [C H O ]+.
8
6
5
3
2
N-Cyclohexyl-3-{[4-(2-furoyl)-1-piperazinyl]methyl}
24 25
3
3
benzamide (8b): light brown sticky solid; yield: 87%; Mol.
F.: C H N O ; Mol. Mass: 395; IR (KBr, cm-1) n : 1114
fragments are also given for the final synthesized compounds
(5a, 5b, 8a, 8b). The prominent absorptions in IR spectrum
were noted at 3412 (N-H), 3097 (Ar C-H), 2884 (R C-H),
1665 (C=O), 1588 (Ar C=C), 1195 (C-O-C) and 1110
(C-N-C).
23 29
3
3
max
(C-N-C), 1208 (C-O-C), 1573 (Ar C=C), 1650 (C=O), 2877
1
(R C-H), 3063 (Ar C-H), 3421 (N-H); H-NMR (600 MHz,
CDCl , d in ppm): 8.06 (s, 1H, H-2), 8.02 (d, J = 7.7 Hz, H,
3
1
In the typical H-NMR spectrum (Fig. 1), the aromatic
H-6), 7.84 (br.s, 1H, H-5²), 7.78 (d, J = 7.6 Hz, 1H, H-4),
7.72 (t, J = 7.6 Hz, 1H, H-5), 7.30 (d, J = 3.5 Hz, 1H, H-3²),
6.83 (dd, J = 2.0, 4.9 Hz, 1H, H-4²), 4.21 – 4.16 (m, 1H,
H-1¢²), 3.91 (br.s, 4H, CH -3¢, CH 5¢), 3.64 (s, 2H, CH -8),
region well confirmed the presence of three aromatic moi-
eties including furan ring, p-substituted benzoyl group and
2
2-
2
2.86 (distorted t, J = 5.0 Hz, 4H, CH -2¢, CH -6¢),
2
2
TABLE 1. Enzyme Inhibition and Hemolytic Activity of Synthe-
1.72 – 1.59 (m, 10H, CH -2¢² to CH -6¢²); 13C-NMR
sized Compounds
2
2
(150 MHz, CD OD, d in ppm): 171.76 (C-6²), 171.69 (C-7),
Hemolytic
BChE inhibition
activity
3
163.49 (C-6), 150.95 (C-2), 148.29 (C-5), 141.14 (C-3²),
138.92 (C-2²), 136.23 (C-4²), 132.40 (C-5²), 120.72 (C-3),
115.25 (C-4), 66.08 (C-8), 53.40 (C-2¢, C-3¢, C-5¢, C-6¢),
36.61 (C-1¢²), 36.58 (C-2¢², C-6¢²), 29.33 (C-4¢²), 28.98
(C-3¢², C-5¢²); EIMS (m/z): 395 [M]+, 300 [C H N O]+,
Compound
Inhibition (%) at
IC50
(%)
0.5 mmole/L
(mmoles/L)
5a
5.5
2.8
58.13 ± 0.11
74.52 ± 0.11
56.37 ± 0.12
78.52 ± 0.08
82.82 ± 1.09
-
289.61 ± 0.09
54.81 ± 0.06
412.71 ± 0.09
52.36 ± 0.02
0.85 ± 0.0001
-
5b
18 26
3
271 [C H N O]+, 268 [C H N O ]•+, 217 [C H NO]•+,
8a
55.47
19.32
-
17 23
2
16 16
2
2
14 19
179 [C H N O ]+, 118 [C H O]•+, 95 [C H O ]+.
8b
9
11
2
2
8
6
5
3
2
Eserine
Triton-X-100
PBS
2.3. Enzyme Inhibition Assay
100
0.09
The enzyme inhibition activity was assessed using a
method reported for butyrylcholinesterase enzyme [32].
Eserine was used as a positive control for cholinesterase en-
-
-
(IC ): 50% inhibitory concentration calculated through EzFit
50
Perrella Scientific Inc. (Amherst, USA) software from results of
zymes. The inhibition (%) and IC were calculated by the
50
triplicate experiments and expressed as mean ± SEM.
following formula:

600
M. A. Abbasi et al.
1
Fig. 1. H-NMR spectrum of N-benzyl-4-{[4-(2-furoyl)-1-piperazinyl]methyl}benzamide (5a).
13
Fig. 2. C-NMR spectrum of N-benzyl-4-{[4-(2-furoyl)-1-piperazinyl]methyl}benzamide (5a).

Synthesis and Structure-Activity Relationship
601
Fig. 3. General structural features of compounds 5a, 5b, 8a, and 8b.
Fig. 4. Structure – activity relationship of compounds 5a and 5b.
147.77 (C-2 & C-6), 116.41 (C-1) and 111.28 (C-4). The aro-
matic carbons of benzyl groups also presented four signals
for five methine and one quaternary carbons at d 141.32
(C-1¢²), 129.20 (C-2¢² & C-6¢²), 127.20 (C-4¢²) and 126.62
(C-3¢² & C-5¢²). The third moiety resonating in the aromatic
region was furan ring whose three methine carbons and one
quaternary carbon resonated at d 145.10 (C-3²), 143.73
(C-2²), 138.41 (C-4²) and 133.46 (C-5²). The aliphatic re-
gion of this spectrum presented three signals nominated to
methylene carbons of piperazine ring and two open chain
methylene carbons. The entire spectral evidence corrobo-
rated the proposed structures of target compounds.
benzyl group. The two doublets at d 7.85 (d, J = 8.2 Hz, 2H,
H-2 & H-6) and 7.21 (d, J = 8.4 Hz, 2H, H-3 & H-5) were al-
located to four aromatic protons of 1,4-substituted benzoyl
group. The three signals erecting at d 7.45 (d, J = 1.0 Hz,
1H, H-5²), 7.03 (d, J = 2.6 Hz, 1H, H-3²) and 6.46 (dd,
J = 3.4, 1.6 Hz, 1H, H-4²) were designated to three protons
of furan ring. The five aromatic protons of benzyl group ap-
peared at d 7.38 – 7.27 (m, 5H, H-2¢² to H-6¢²) as multiplet.
The two signals resonating at d 3.84 (br.s, 4H, CH -3¢ &
2
CH -5¢) and 2.54 (br.s, 4H, CH -2¢ & CH 6¢) supported the
2
2
2 –
presence of piperazine ring. The four protons attached to
methylene carbons resonated as singlets at d 3.62 (s, 2H,
CH -8) and 2.47 (s, 2H, CH -7¢²).
2
2
3.2. Enzyme Inhibition and Structure – Activity Relationship
The typical 13C NMR spectrum (Fig. 2) showed the most
downfield signals for the quaternary carbons of two carbonyl
groups at d 167.38 (C-6²) and 167.27 (C-7). The p-substi-
tuted benzoyl group presented four signals for four methine
carbons and two quaternary carbons at d 159.08 (C-3 & C-5),
The multi-functional molecules 5a, 5b, 8a, 8b were syn-
thesized and evaluated against butyryl cholinesterase
(BChE). A series of such interrelated compounds have been
previously reported by our research group [31]. The% inhibi-

602
M. A. Abbasi et al.
Fig. 5. Structure – activity relationship of compounds 8a and 8b.
tion and IC values for BChE enzyme are given in Table 1.
parted observable effect which is well illuminated from IC
50
50
Some of the molecules displayed promising inhibiting poten-
tial against this enzyme and well depicted from results.
Though the observed activity is pertained to the cumulative
effect of all functionalities embed in an entire frame work of
molecule, however a brief structure activity relationship
(SAR) was established by examine the effects of different
entities like furoyl, piperazine and benzamide functionalities
were common in these four molecules. Among these com-
pounds, N-cyclohexyl-4-{[4-(2-furoyl)-1-piperazinyl]methyl}-
benzamide (5b) and N-cyclohexyl-3-{[4-(2-furoyl)-1-pipera-
zinyl]methyl}benzamide (8b) remained the most efficient in-
hibitors of BChE enzyme. The other two compounds 5a and
of compound 5a (289.61 ± 0.09 mmoles/L), displayed better
inhibition of enzyme in comparison to 8a presenting inhibi-
tory potential of 412.71 ± 0.09 mmoles/L (Figs. 4 and 5).
Thus, a rational from this structure – activity relationship
studies was framed that molecule 8b bearing cyclohexyl
group and meta orientation of methylene moiety in the vicin-
ity of benzamide part, was the most promising molecule in
inhibiting BChE enzyme. The inhibition potential may be
further enhanced by replacing the cycloalkyl group by a sub-
stituted cycloalkyl or straight chain alkyl groups. Hence,
compounds 5b and 8b might be potent new drug candidates
for BChE enzyme inhibition.
8a remained the least efficient as shown by their higher IC
50
3.3. Hemolytic Activity
values. Eserine was used as reference standard having IC
50
0.85 ± 0.0001 mM. When we look into the structures of these
molecules; we can predict that the difference lies in the
N-substituted groups like aralkyl/aryl and position of methy-
lene group on benzamide moiety. In compounds 5a and 8a,
cyclohexyl group is attached to nitrogen of benzamide func-
tionality; whereas in 5b and 8b benzyl group is present as
N-substituent. In 5a and 5b, the methylene group is oriented
para to benzamide moiety; however it is at 3rd position in
case of molecules 8a and 8b. The general structural features
of studied multifaceted compounds are presented in Fig. 3.
The presence of methylene at 3rd position of benzamide
The hemolytic activity analysis also confirmed the low
toxicity of 5b molecule by showing red blood cells lysis of
only 2.8%, which is much lower as compared to the toxicity
of Triton-X-100 and close to PBS having a low toxicity of
0.09%. Compound 8b exhibited some toxicity but that might
be tolerated, which can be confirmed by further in vivo stud-
ies. Both these molecules should be subjected to in vivo anal-
ysis for further confirmation of these compounds as new
drug candidates for the diseases caused by indicated en-
zymes.
moiety lowers the IC from 252.36 ± 0.02 mmoles/L in 5b to
ACKNOWLEDGEMENTS
50
54.81 ± 0.06 mmoles/L as exhibited by 8b with respect to the
reference standard, Eserine with IC
value of
0.85 ± 0.0001 mmoles/L. In case of 5a and 8a, the occurrence
of methylene group at para position to the benzamide im-
The Higher Education Commission (HEC) of Pakistan is
highly acknowledged by the authors for fiscal aid to conduct
spectral analysis.
50

Synthesis and Structure-Activity Relationship
603
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16. V. Pattabiraman and J. W. Bode, Nature., 480, 471 – 479
(2011).
The authors declare that they have no conflict of interest.
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