5-Chlorobenzofuran-2-carboxamides: From allosteric CB1 modulators to potential apoptotic antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2019.05.040

Cannabinoids as THC and the CB1 allosteric modulator CBD were reported to have antiproliferative activities with no reports for other CB1 allosteric modulators as the 5-chloroindole-2-carboxamide derivatives and their furan congeners. Based on the antiproliferative activity of two 5-chlorobenzofuran-2-carboxamide allosteric CB1 modulators, a series of novel derivatives was designed and synthesized. The synthesized compounds were tested in a cell viability assay using human mammary gland epithelial cell line (MCF-10A) where all the compounds exhibited no cytotoxic effects and more than 85% cell viability at a concentration of 50 μM. Some derivatives showed good antiproliferative activities against tumor cells as compounds 8, 15, 21 and 22. The most active compound 15 showed equipotent activity to doxorubicin. Compounds 7, 9, 15, 16, 21 and 22 increased the level of active caspase 3 by 4–8 folds, compared to the control cells in MCF-7 cell line and doxorubicin as a reference drug. Compounds 15 and 21, the most activecaspase-3 inducers, increase the levels of caspase 8 and 9 indicating activation of both intrinsic and extrinsic pathways and showed potent induction of Bax, down-regulation of Bcl-2 protein levels and over-expression of Cytochrome C levels in MCF-7 cell lines. Compound 15 exhibited cell cycle arrest at the Pre-G1 and G2/M phases in the cell cycle analysis of MCF-7 cell line. The drug Likeness profile of the synthesized compounds showed that all the compounds were predicted to have high oral absorption complying with different pharmacokinetics filters.

Full text of DOI:10.1016/j.ejmech.2019.05.040

Accepted Manuscript
5-Chlorobenzofuran-2-carboxamides: From allosteric CB1 modulators to potential
apoptotic antitumor agents
Bahaa G.M. Youssif, Ashraf M. Mohamed, Essam Eldin A. Osman, Ola F. Abou-
Ghadir, Dina H. Elnaggar, Mostafa H. Abdelrahman, Laurent Treamblu, Hesham A.M.
Gomaa
PII:
S0223-5234(19)30450-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.040
EJMECH 11350
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 March 2019
Revised Date: 5 April 2019
Accepted Date: 13 May 2019
Please cite this article as: B.G.M. Youssif, A.M. Mohamed, E.E.A. Osman, O.F. Abou-Ghadir, D.H.
Elnaggar, M.H. Abdelrahman, L. Treamblu, H.A.M. Gomaa, 5-Chlorobenzofuran-2-carboxamides:
From allosteric CB1 modulators to potential apoptotic antitumor agents, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.040.
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ACCEPTED MANUSCRIPT
5-chlorobenzofuran-2-carboxamides: from allosteric CB1
modulators to potential apoptotic antitumor agents
Bahaa G.M. Youssif1,2*, Ashraf M. Mohamed3, Essam Eldin A. Osman4, Ola .F.
Abou-Ghadir2, Dina H. Elnaggar3, Mostafa H. Abdelrahman5, Laurent Treamblu6,
Hesham A. M. Gomaa7,8
1Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University,
2
Aljouf, Sakaka 2014, Saudi Arabia; Pharmaceutical Organic Chemistry Department,
3
Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt; Applied Organic
Chemistry Department, National Research Centre, 12622, Dokki, Giza, Egypt;
4Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University,
5
11562 Cairo, Egypt; Department of Pharmaceutical Organic Chemistry, Faculty of
6
Pharmacy, Al-Azhar University, Assiut 71524, Egypt; School of Natural and
Computing Sciences, University of Aberdeen, Meston Building, Aberdeen,
AB243UE, Ireland; 7Pharmacology Department, College of Pharmacy, Jouf
University, Sakaka, Aljouf 2014, Saudi Arabia, 8Biochemistry Department, Faculty of
Pharmacy, Nahda University, Beni-Suef, Egypt,
Short running title: Synthesis and anticancer activity of new benzofuran derivatives
*To whom correspondence should be addressed:
Bahaa G. M. Youssif. Ph.D. Pharmaceutical Organic Chemistry Department, Faculty
of Pharmacy, Assiut University, Assiut 71526, Egypt
Tel.: (00966)-537240294
E-mail address: bgyoussif@ju.edu.sa
Essam Eldin A. Osman. Ph.D. Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Cairo University, 11562 Cairo, Egypt
E-mail address: essamosman@staff.cu.edu.eg

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Abstract:
Cannabinoids as THC and the CB1 allosteric modulator CBD were reported to have
antiproliferative activities with no reports for other CB1 allosteric modulators as the
5-chloroindole-2-carboxamide derivatives and their furan congeners. Based on the
antiproliferative activity of two 5-chlorobenzofuran-2-carboxamide allosteric CB1
modulators, a series of novel derivatives was designed and synthesized. The
synthesized compounds were tested in a cell viability assay using human mammary
gland epithelial cell line (MCF-10A) where all the compounds exhibited no cytotoxic
effects and more than 85% cell viability at a concentration of 50 µM. Some
derivatives showed good antiproliferative activities against tumor cells as compounds
8, 15, 21 and 22. The most active compound 15 showed equipotent activity to
doxorubicin. Compounds 7, 9, 15, 16, 21 and 22 increased the level of active caspase
3 by 4-8 folds, compared to the control cells in MCF-7 cell line and doxorubicin as a
reference drug. Compounds 15 and 21, the most activecaspase-3 inducers, increase
the levels of caspase 8 and 9 indicating activation of both intrinsic and extrinsic
pathways and showed potent induction of Bax, down-regulation of Bcl-2 protein
levels and over-expression of Cytochrome C levels in MCF-7cell lines. Compound 15
exhibited cell cycle arrest at the Pre-G1 and G2/M phases in the cell cycle analysis of
MCF-7 cell line. The drug Likeness profile of the synthesized compounds showed
that all the compounds were predicted to have high oral absorption complying with
different pharmacokinetics filters.
Keywords: benzofuran; carboxamide; CB1; modulators; Anti-proliferative; Apoptotic
assay; Caspases.

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Graphical Abstract
Induce Caspases 3, 8 and 9
Increase Cytochrome c levels
Increase level of Bax
Down regulation of Bcl2
Apoptosis

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Highlights
•
A series of novel 5-chlorobenzofuran-2-carboxamide derivatives 14-25 was
designed and synthesized based on the observed antiproliferative activities of
two allosteric CB1 modulators 7 and 8 in 4 cancer cell lines.
Compounds 8, 15, 21 and 22 were the most active antiproliferative agents.
Compounds 15 and 21 increased the level of active caspase 3 by 6-8 folds.
Results revealed that these compounds showed programmed cell death and
cell cycle arrest
•
•
•
•
The newly synthesized compounds were predicted to have high oral
absorption and good in silico pharmackokinetics profile.

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1. INTRODUCTION
The endocannabinoid system encompasses three components: (a) G-protein-coupled
cannabinoid receptors CB1 and CB2, (b) endocannabinoids (i.e., endogenous CB
receptor ligands), and (c) cellular proteins, enzymes, and carriers involved in the
formation, function and inactivation of the endocannabinoids [1-3]. Growing evidence
suggests the involvement of the endocannabinoid system in a number of physiological
processes and pathological conditions as energy balance [4], obesity [5], appetite
disorders [6], inflammation [7], pain [8], neurodegeneration [9] and cancer [10-12].
The CB1 receptor is thought to exist in multiple active conformations, each of which
may display distinct abilities to regulate individual signaling pathways [13]. While
endogenous ligands as anandamide 1 and exogenous ligands as (-)-∆9-
tetrahydrocannabinols (∆9-THC) 2 are orthosteric agonists of the cannabinoid
receptors, several small molecules were reported to show allosteric modulation of the
CB1 receptor. These CB1 allosteric modulators include the non-psychoactive natural
cannabinoid cannabidiol (CBD) 3 [14], the 5-chloro-indole-2-carbxamide ORG
compounds, ORG27759 4, ORG27569 5, ORG29647 6 [15, 16] and their benzofuran
congeners 7 and 8 [17]. While cannabidiol 3 was shown to be a non-competitive
negative allosteric modulator of the CB1 receptors [14], ORG compounds affect the
CB1 affinity of some orthosteric agonists positively, despite the fact that they inhibit
G-protein coupling induced by these agonists. ORG27569 5 selectively hampers G-
protein signaling and promotes β-arrest in 2-mediated internalization of the receptor
and β-arrest in 1-mediated activation of kinases independently from the occupancy of
CB1 orthosteric site [15, 18]. Allosteric ligand-biased signaling via CB1 may lead to
useful agents without the CNS side effects associated with direct receptor agonism
[19].

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Figure 1:Structures of some orthosteric and allosteric CB1 modulators
Cannabinoids as THC 2 and CBD 3 were shown to affect cancer cells through various
mechanisms including induction of apoptosis [20], inhibition of cell growth [21],
tumor angiogenesis, invasion and metastasis [22-24]. Moreover, Cannabinoids were
claimed to show selective actions against tumor cells and may even protect normal
cells from death [25, 26]. More specifically, CBD 3 was found to induce programmed
cell death, in a concentration-dependent manner, independent of the CB1, CB2, or
vanilloid receptors in both estrogen receptor–positive and estrogen receptor–
negative breast cancer cell lines while having little effect on non-
tumorigenic mammary cells [20]. Furthermore, CBD 3 showed anticancer effects in
other cells as lung [24] and colon cancers [27].

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Other studies demonstrated the antitumor effect of CBD in preclinical models of
breast cancer [21, 28].
No reports, to the best of our knowledge, were describing the screening of other CB1
allosteric modulators as the ORG compounds and their furan congeners, for their
antiproliferative effects despite the strong rationale and attractiveness behind this
endeavor. The indole-2-carboxamide derivatives 5 and its furan congener 8 were
reported to have equilibrium dissociation constant (KB) of 217.3 and 2594 nM,
respectively for the CB1 receptor. Despite the fact that 8 was having much higher KB
value, it has higher binding cooperativity factor (α) with the orthosteric CB1 agonist
compared to 5 [17] and both compounds antagonized the effects of the orthosteric
CB1 agonist on the [35S]GTPγS binding [17]. The 5-chlorobenzfuran-2-carboxamide
derivatives 7 and 8 were selected as a starting point in this study based on their facile
synthesis. Compounds 7 and 8 were synthesized and tested for their antiproliferative
activities in four cancer cell lines, namely lung A549, breast MCF-7, pancreatic Panc-
1 and colon HT-29 cancer cell lines. The promising anticancer activity of compounds
7 and 8 promoted the synthesis and testing of a small library of twelve novel
compounds where the substitution at C3 was varied from ethyl in compounds 14-18 to
methyl in compounds 19-25, Figure 2. The para positions of the phenethyl tails in the
newly synthesized derivatives were either unsubstituted as in 14 and 19 or substituted
with 4-dimethylamino in 20, morpholin-4-yl in 15 and 21, piperidin-1-yl in 22 or
pyrrolidine-1-yl in 16 and 23 to study the effect of substituent variation. The
phenethyl amino carbonyl moieties in 7, 8, 14-16 and 19-23 were modified to 4-
benzylpiperidin-1-yl carbonyl in compounds 17 and 24, or 4-phenylpiperazin-1-yl
carbonyl in compounds 18 and 25 to study the effect of the amidic hydrogen and the
nature of the linker on the anticancer activity.

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Figure 2: Structures of the target compounds 7, 8 and 14-25
2. RESULTS AND DISCUSSION
2.1. Chemistry
The preparation of target compounds 7, 8 and 14-25 having benzofuran nucleus was
outlined in Scheme 1. p-chlorophenol 9 was reacted with propionyl or acetyl chloride
in toluene to afford p-chlorophenyl propionate or acetate 10a-b. The Fries
0
rearrangement of 10a-b in the presence of AlCl3 at 150 C gave the corresponding
phenols 11a-b. The reaction of 11a-b with ethyl bromoacetate in the presence of
K2CO3 afforded 12a-b which were subjected to cyclization using sodium ethoxide to
yield 5-chloro-3-alkylbenzofuran-2-carboxylic acids 13a-b. The carboxylic acids 13a-
b were coupled with amine derivatives using BOP as the coupling reagent in the
presence of DIPEA in DCM to obtain the desired target products 7, 8 and 14-25. The
1H NMR spectrum of 15 as a representative example of this series revealed the
appearance of a signal at 6.66 ppm assigned to amide NH, two sets of doublets at 7.18
and 6.90 ppm with J = 8.4 Hz each, which is indicative of aromatic para-
disubstitution, two signals with two protons integration each at 3.68 (q) and 2.88 (t)
ppm attributed to NHCH2CH2 group and ethyl group signals at δ 3.19 (q, J = 7.2 Hz,

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2H, CH2CH3) and 1.30 (t, J = 8.0 Hz, 3H, CH2CH3) as well as morpholinyl protons.
The structure of 15 was also established by HRESI-MS, which gave a molecular ion
m/z 413.1625 [M + H]+, which is consistent with the molecular formula
C23H26ClN2O3 of the desired product.
0
Reagents and conditions: (a) appropriate acyl chloride, toluene, reflux, 5h; (b) AlCl3, 80 C
0
for 1h, then 150 C for 2h; (c) ethylbromoacetate, K2CO3, acetone, reflux, 6h; (d) NaOEt,
toluene, reflux, 12h, 86%; (e) appropriate amine, BOP, DIPEA, DCM, rt, overnight.
Scheme 1: Synthetic route of 5-chlorobenzofuran-2-carboxamides 7,8 and 14-25.

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2.2. Evaluation of biological Activities
2.2.1. In vitro anticancer activity
2.2.1.1. Cell viability assay
Cell viability assay was carried out using human mammary gland epithelial cell line
(MCF-10A) [28]. 5-Chloro-3-alkylbenzofuran-2-carboxamides 7, 8 and 14-25 were
incubated with MCF-10A cells for 4 days and 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay [29] was used to determine the viability of
cells. All compounds exhibited no cytotoxic effects and more than 85% cell viability
was reported for the majority of the tested compounds at 50 µM.
2.2.1.2. Antiproliferative activity
The antiproliferative activities of 7, 8 and 14-25 against four human cancer cell lines
including pancreas cancer cell line (Panc-1), breast cancer cell line (MCF-7), colon
cancer cell line (HT-29) and epithelial cancer cell line (A-549) using MTT assay and
doxorubicin was used as the reference compound. Graph Pad Prism software (Graph
Pad Software, San Diego, CA, USA) was used to calculate the median inhibition
concentration (IC50) for all compounds. Results were illustrated in Table 1.
The previously described CB1 allosteric modulators 7 and 8 showed promising
antiproliferative activity but was less potent compared to doxorubicin, Table 1. The
4-piperidin-4-yl derivative 8 was slightly more potent than its 4-dimethylamino
derivative 7. Generally, 5-chloro-3-ethylbenzofuran-2-phenethylcarboxamides 7, 8
and 14-16 showed superior antiproliferative activity compared to their methyl
counterparts 19-23, Table 1. For example, the unsubstituted derivatives 14 and 19
showed an average GI50 of 7.37 and 12.25 µM, respectively against the tested cell
lines and the same trend applies to the 4-dimethylamino substituted derivatives 7 and

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20 (mean GI50 = 5.37 and 6.80 µM) or the 4-morpholin-4-yl derivatives 15 and 21
(mean GI50 = 1.35 and 3.45 µM). It worth mentioning that the 3-ethylbenzofuran-2-
carboxamides bearing 4-piperidin-4-yl 8 or 4-pyrrolidin-1-yl 16 in the pendant phenyl
ring were more or less showing the same potency as their methyl congeners 22 and 23
respectively. The para substitution in the phenethyl tail appears to correlate with
higher antiproliferative effects and the activity increased with (R’) in the order of
morpholinyl > piperidyl ≥ pyrrolidinyl > dimethylamino > H.
The 4-morpholin-4-yl phenethyl derivative 15 was the most potent among the
synthesized derivatives, with mean GI50 value of 1.35 µM against the four cell lines,
being equipotent to the reference doxorubicin (mean GI50 = 1.13 µM) and even more
potent than doxorubicin in MCF-7 cell line (GI50 = 0.7 and 0.9 µM, respectively). The
unsubstituted derivative 14 was about 6 folds less potent than 15 showing IC50
ranging from 6.0±1.5 in A-549 to 8.3±2.2 in Panc-1 cell lines (mean GI50 = 7.37 µM)
while the 4-dimethylamino derivative 7 showed mean GI50 = 5.37 µM. Replacement
of the 4-morpholin-4-yl moiety in compound 15 by 4-piperidin-1-yl or 4-pyrrolidin-1-
yl in compound 8 and 16 respectively, resulted in at least 3 folds reduction of the
mean GI50 values. Moreover, the mean GI50 values of the 4-benzylpiperidin-1-yl
carbonyl derivatives 17 and 24, or the 4-phenylpiperazin-1-yl carbonyl derivatives 18
and 25 were the lowest among the tested compounds, suggesting the importance of
the N-phenethyl carboxamide architecture for the antiproliferative activity and
correlating with the previous SAR studies of the CB1 allosteric modulators [30].

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Table 1: Antiproliferative activity of compounds 7, 8, 14-25 and Doxorubicin.
Cell
viability
%
Antiproliferative activity GI50± SEM (µM)
Comp.
R
R’
A-549
MCF-7
Panc-1
HT-29
Average
4.8±1.5
4.7±1.4
5.5±2.2
6.5±2.1
5.375
Et
Et
Et
N(CH3)2
Piperidine
H
85
82
89
91
89
90
82
87
90
84
79
89
96
7
4.5±2.6
6.0±1.5
1.8±0.5
4.9±0.7
27.9±3.6
14.7±2.5
11.4±2.5
6.9±0.3
3.5±0.2
4.4±0.4
5.2±0.6
15.4±1.3
29.5±3.8
3.6±2.2
7.5±1.2
0.7±0.08
4.4±1.0
26.5±2.8
13.6±2.9
12.9±1.8
6.8±1.6
3.1±0.1
4.9±0.6
4.9±0.3
16.4±5.2
35.4±2.7
4.6±2.9
8.3±2.2
1.3±0.2
5.6±0.5
33.7±3.5
13.8±1.9
12.5±2.3
6.6±1.5
3.3±0.2
4.1±0.2
4.8±0.5
18.6±2.5
36±1.9
4.8±1.4
7.7±2.4
1.6±0.2
5.4±1.6
27.8±8.2
13.8±1.6
12.2±1.4
6.9±1.1
3.9±0.6
4.9±0.2
4.9±0.8
16.9±3.7
34.7±3.2
4.375
7.375
1.350
5.075
28.975
13.975
12.250
6.800
3.450
4.575
4.950
16.825
8
14
15
16
17
18
19
20
21
22
23
24
Et Morpholine
Et Pyrrolidine
Et
Et
--
--
H
Me
Me
N(CH3)2
Me Morpholine
Me Piperidine
Me Pyrrolidine
Me
--
33.900
1.136
Me
--
--
--
92
25
-
1.21 ± 0.80 0.90± 0.62 1.41 ± 0.58 1.01 ± 0.82
Doxorubicin

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2.2.3. Apoptosis assay
Previous reports documented the effect of the CB1 allosteric modulator, CBD (3) as
an apoptosis inducer [19]. Therefore, to reveal the proapoptotic potential of our target
compounds, the utmost active compounds 7, 8, 15, 16, 21 and 22 were tested for their
potential ability to induce the apoptosis cascade in MCF-7 breast cancer cell line.
2.2.3.1. Activation of proteolytic caspases cascade
Activation of caspases plays a key role in the instigation and completing of the
apoptotic process [31]. Among the caspases, the executioner caspase-3 is a vital
player that cleaves multiple proteins in the cells, leading to apoptotic cell death [32].
The effect of compounds 7, 8, 15, 16, 21 and 22 on caspase 3 was evaluated and
compared to doxorubicin as a reference drug. The results revealed that the tested
compounds showed an increase in the level of active caspase 3 by 4-8 folds,
compared to the control cells, and that 8, 15 and 21 were the most active compounds
that possessed remarkable over expression of caspase-3 protein level (375.20 ±9.27,
532.2 ±5.13 and 387.40 ±12.20 pg/mL, respectively) compared to doxorubicin (503.2
± 4.22 pg/mL). The most active compound 15 showed an increase in the level of
active caspase 3 by 8 folds compared to the control untreated cells, and induced
caspase 3 higher than doxorubicin (7.66 folds) (Figure 3).

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Figure 3: Caspase-3 level for compounds 7, 8, 15, 16, 21, 22 and doxorubicin in
human breast cancer cell line (MCF-7)
To outline the contribution of the intrinsic and the extrinsic apoptotic pathway in the
antiproliferative effects of compounds 15 and 21, their effect on caspases 8 and
caspases 9 was also evaluated. Result revealed that compound 15 increases the levels
of caspase 8 and 9 by 9.64 and 15.98 folds respectively, while compound 21 showed
5.23 and 13.58 fold increase in the level of caspase 8 and 9, respectively compared to
the control cells which indicates activation of both intrinsic and extrinsic pathways
with more prominent effect on the intrinsic pathway since caspase 9 levels were
higher [33]. Interestingly, these results were in agreement with the previously
described increase in the levels caspase 3 and 9 after CBD (3) treatment[19]. (Table
2).

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Table 2: Effects of compounds 7, 8, 15, 16, 21, 22 and doxorubicin on active
Caspases 3, 8, 9 and Cytochrome C in MCF-7 breast cancer cell line.
Compound
Number
Caspase-3
Conc Fold
change (ng/ml) change (ng/ml) change (ng/ml) change
Caspase-8
Caspase-9 Cytochrome C
Conc Fold Conc Fold Conc Fold
(pg/ml)
254.70 ±3.14
3.87
5.71
8.10
5.17
5.90
4.83
--
--
--
--
--
--
7
375.20 ±9.27
532.20 ±5.13
339.80 ±2.19
387.40 ±12.2
317.35 ±3.92
--
--
--
--
--
--
8
15
16
21
22
1.64
--
9.64 14.87 15.98 0.774 16.83
-- -- -- -- --
5.23 12.63 13.58 0.518 11.26
-- -- -- -- --
0.89
--
503.20 ±4.22
65.64
7.66
1
1.75 10.07 16.23 17.40 0.604 13.13
0.17 0.93 0.046
Doxorubicin
Control
1
1
1
2.2.3.2. Cytochrome C assay
Cytochrome C concentration within the cell has a significant role for activation of
caspases and initiating intrinsic apoptosis pathway [33]. Benzofuran derivatives 15
and 21 were evaluated as Cytochrome C activators in MCF-7 human breast cancer
cell line and the results are listed in Table 2. Compounds 15 and 21 resulted in over-
expression of Cytochrome C levels in MCF-7 human breast cancer cell line about 17
and 11 folds, respectively higher than untreated control cells. The results add another
piece of evidence that apoptosis may be credited to over-expression of Cytochrome C
and activation of the intrinsic apoptotic pathway induced by the tested compounds.

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2.2.3.3. Bax and Bcl-2 levels assay
The most active caspase activators 15 and 21 were further studied for their effect on
Bax and Bacl-2 levels against breast cancer cell line (MCF-7) using doxorubicin as a
reference. The results showed that 15 and 21 elicited remarkable increase in Bax level
compared to doxorubicin Table 3. Compound 15 showed a comparable induction of
Bax (284.70 pg/mL) compared to doxorubicin (276 pg/mL) with 34 fold higher than
control untreated breast cancer cell followed by compound 21 (162.72 pg/mL and
19.7 fold change). Finally, compound 15 caused a down-regulation of Bcl-2 protein
level up to 0.885 ng/mL followed by compound 21 (1.978 ng/mL) in MCF-7 cell line
compared to doxorubicin (0.98 ng/mL).
Table 3: Bax and Bcl-2 levels for compounds 15, 21 and Doxorubicin in MCF-7
breast cancer cell line
Bax
Bcl-2
Fold
(pg/ml) change (ng/ml) change
Compound Number
Conc
Fold
Conc
15
21
284.70
162.72
276.19
34.46
19.7
0.885
1.978
0.983
5.77
2.56
5.10
Doxorubicin
33.42
8.26
1
5.086
1.00
Cont.
2.2.3.4. Flow cytometric cell cycle analysis
Cell cycle analysis was performed in MCF-7 human breast cancer cell line treated
with the most active compound 15. The percentages of cells of MCF-7 cell line in
G0/G1 phase of the cell cycle in the control was 53.71% which recorded a noteworthy
decrease to 24.53% upon treatment with compound 15 while the percentage of cells in
the S phase were slightly reduced with compound 15 (31.49%) compared to the
control (37.42%) (Figure 4 and Figure 5). The percentage of MCF-7 human breast

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cancer cell line at the G2/M phase was apparently increased to 43.98% upon treatment
with 15 compared to the control (8.87%). Furthermore, it is observable that the
apoptotic cell percentage in the Pre-G1 phase was increased from 1.93% for control
untreated MCF-7 human breast cancer cell to 34.29% and 22.17% in cells treated with
15 and doxorubicin, respectively, (Figure 5 and Figure 6). According to the above
results, it is clear that the compound 15 exhibited mainly cell cycle arrest at the Pre-
G1 and G2/M phases. Moreover, it is obvious that the tested compound is not
cytotoxic but antiproliferative causing programmed cell death and cell cycle arrest.
Figure 4: Cell cycle analysis in MCF-7 cell line treated with compound 15
Figure 5. Percentage of apoptosis and necrosis for compounds 15 in MCF-7 cell line.

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Figure 6: Cell cycle analysis and Apoptosis induction analysis using Annexin V/PI of
compound 15 and control untreated MCF-7cell line.
2.3. Drug Likeness Profile
Assessment of absorption, distribution, metabolism and excretion (ADME) became an
early routine in drug discovery programs where the availability of computer models
constitutes valid alternatives to experiments. The drug Likeness profile of the tested
compounds 7, 8 and 14-25 were predicted using SwissADME website [35]. The
results of the drug likeness profile of these compounds are shown in Table 4. All the
compounds were predicted to have high oral absorption and penetrate the blood brain
barrier (BBB). All the tested compounds showed no violation to Lipnski (Pfizer)
filters except one violation for compounds 17 and 25 and no viloations to Ghose [36],
Veber (GSK) [37], Egan (Pharmacia) [38] and Muegge (Bayer) [39] filters. While the

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compounds were free from Brenk proplematic fragments [40], alerts for Pan Assay
Interfering substances (PAINS) [41] were recorded for the tested compounds except
the unsubstituted derivatives 14 and 19 and the 3ry amides 17, 18, 24 and 25 since
they are lacking the dialkylaniline fragment. The potential of the tested compounds to
inhibit CYP enzymes warrants further studying. Surprisingly, CBD (3) was reported
to be an inhibitor of CYP enzymes specially CYP2D6 and CYP2C19 in vitro [42, 43].

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Table 4: Molecular properties of compounds 7, 8 and 14-25 predicted using Swiss
ADME website.
Molecule
MW
7
8
14
15
16
17
18
19
20
21
22
23
24
25
370.87
410.94
327.8
412.91
396.91
381.9
368.86
313.78
356.85
398.88
396.91
382.88
354.83
367.87
#Heavy
atoms
#Aromatic
heavy
26
15
29
15
23
15
29
15
28
15
27
15
26
15
22
15
25
15
28
15
28
15
27
15
25
15
26
15
atoms
Fraction
Csp3
0.29
0.38
0.21
0.35
0.35
0.35
0.29
0.17
0.25
0.32
0.35
0.32
0.25
0.32
#Rotatable
bonds
7
2
1
7
2
1
6
2
1
7
3
1
7
2
1
5
2
0
4
2
0
5
2
1
6
2
1
6
3
1
6
2
1
6
2
1
3
2
0
4
2
0
#H-bond
acceptors
#H-bond
donors
TPSA
45.48 45.48 42.24 54.71 45.48 33.45 36.69 42.24 45.48 54.71 45.48 45.48 36.69 33.45
Consensus
Log P
4.51
5.17
4.5
4.31
4.9
5.19
4.04
4.2
4.2
3.99
4.87
4.62
3.75
4.9
GI
absorption
High
Yes
BBB
permeant
Pgp
substrate
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
CYP1A2
inhibitor
Yes
Yes
Yes
Yes
Yes
Yes
CYP2C19
inhibitor
Yes
Yes
Yes
Yes
CYP2C9
inhibitor
CYP2D6
inhibitor
CYP3A4
inhibitor
Lipinski
#violations
0
0
0
0
0
1
0
0
0
0
0
0
0
1
Ghose
#violations
0
0
0
Veber
#violations
Egan
#violations
Muegge
#violations
1
1
1
1
1
1
1
0
1
1
1
0
1
0
0
0
1
1
0
1
1
1
1
1
0
0
1
0
PAINS
#alerts
Brenk
#alerts
0

ACCEPTED MANUSCRIPT
3- CONCLUSION
In this work, the antiproliferative effect of 5-chlorobenzofuran-2-carboxamide CB1
allosteric modulators was reported for the first time. Based on this, a novel series of
derivatives was synthesized and tested in A549 lung, MCF-7 breast, Panc-1
Pancreatic and HT-29 colon cancer cell lines. Generally, 5-chloro-3-ethylbenzofuran-
2-phenethyl carboxamides 7, 8 and 14-16 showed superior antiproliferative activity
compared to their methyl counterparts 19-23. This trend was observed when the para
position was unsubstituted (14 vs. 19) or substituted with 4-dimethylamino (7 vs. 20)
or morpholin-4-yl (15 vs. 21). The 3-ethyl benzofuran-2-carboxamides bearing 4-
piperidin-4-yl 8 or 4-pyrrolidin-1-yl 16 in the pendant phenyl ring were more or less
showing the same potency as their methyl congeners 22 and 23 respectively. The para
substitution in the phenethyl tail appears to correlate with higher antiproliferative
effects and the activity increased with (R’) in the order of morpholinyl > piperidyl ≥
pyrrolidinyl > dimethylamino > H. The 4-morpholin-4-yl phenethyl derivative 15
was the most potent among the synthesized derivatives, with mean GI50 value of 1.35
µM against the four tested cell lines, being equipotent to the reference standard
doxorubicin (mean GI50 = 1.13 µM) and even more potent than doxorubicin in MCF-7
cell with IC50 of 0.7 and 0.9 µM, respectively. The 4-benzylpiperidin-1-yl carbonyl
derivatives 17 and 24, or the 4-phenylpiperazin-1-yl carbonyl derivatives 18 and 25
showed the lowest activity among the tested compounds, suggesting the importance of
the N-phenethyl carboxamide architecture for the antiproliferative activity and
correlating with the previous SAR studies of the CB1 allosteric modulators.
Compounds 7, 8, 15, 16, 21 and 22 increased the level of caspase 3 in MCF-7 by 4-8
folds compared to the control cells, and 8, 15 and 21 were the most active compounds.
The morpholinyl substituted 3-ethyl derivative 15 increases the levels of caspase 8, 9

ACCEPTED MANUSCRIPT
and Cytochrome C by 9.64, 15.98 and 17 folds, respectively while its methyl
counterpart 21 showed 5.23, 13.58 and 11 folds in MCF-7 breast cancer cell line. 15
and 21 elicited remarkable increase in Bax level and down-regulation of Bcl-2 protein
levels in MCF-7 cell line compared to doxorubicin. The drug Likeness profile of the
synthesized compounds was predicted and while all the compounds were predicted to
have high oral absorption, penetrant the blood brain barrier (BBB) and show no
violation to different pharmacokinetics filters, thier potential to inhibit CYP enzymes
warrants further studying.
4. EXPERIMENTAL
4.1. Chemistry
General Details [See Appendix A]
4.1.1. Synthesis of p-Chlorophenyl propionate (10a)
A mixture of p-chlorophenol 9 (5 g, 38.89 mmol) and propionyl chloride (3.74 mL,
42.78 mmol) in dry toluene (30 mL) was refluxed for 5 h. The toluene was evaporated
in vacuo to give p-chlorophenyl propionate 10a (6.9 g, 96%) as an oil which was used
for further step without purification.
4.1.2. Synthesis of 1-(5-Chloro-2-hydroxyphenyl)propan-1-one (11a)
A mixture of 10a (6.5 g, 35.20 mmol) and AlCl3 (14.07 g, 105.60 mmol) was heated
at 80 0C for 1 h then at 150 0C for 2 h. the resulting residue was decomposed with 3 N
HCl and extracted with EtOAc. The organic layer was washed with brine, dried over
MgSO4, and evaporated under reduced pressure to give crude product which was
purified by flash chromatography on silica gel using a mixture of EtOAc, hexanes
o
(1:30) as eluent to yield 11a (5 g, 77%) as white solid; mp 55-57 C. νmax (KBr
disc)/cm-1 3296 (OH), 2980, 1781 (C=O), 1610, 1455, 1284, 810, 718, 684. 1H NMR
(400 MHz, Chloroform-d) δ 12.21 (s, 1H, OH), 7.70 (d, J = 2.6 Hz, 1H, Ar-H), 7.38

ACCEPTED MANUSCRIPT
(dd, J = 8.9, 2.6 Hz, 1H, Ar-H), 6.92 (d, J = 8.9 Hz, 1H, Ar-H), 3.00 (q, J = 7.2 Hz,
13
2H, CH2CH3), 1.23 (t, J = 7.2 Hz, 3H, CH2CH3). C NMR (100 MHz, CDCl3) δ
206.12 (C=O), 160.78, 135.95, 128.99, 123.46, 120.10, 119.76, 31.64, 7.94. HRESI-
MS m/z calcd for [M+H]+ C9H10ClO2: 185.0364, found: 185.0367.
4.1.3. Synthesis of Ethyl 2-(4-chloro-2-propionylphenoxy)acetate (12a)
A mixture of 11a (4.3 g, 23.29 mmol), ethyl bromoacetate (3.10 mL, 27.95 mmol),
and K2CO3 (6.44 g, 46.58 mmol) in acetone (80 mL) was heated under reflux for 6 h.
After removing of the solvent in vacuo, the residue was extracted with EtOAc,
washed with H2O, brine, dried over MgSO4, and evaporated under reduced pressure to
give a crude product which was purified by flash chromatography on silica gel using a
mixture of EtOAc, hexanes (1:4) as eluent to yield 12a (5.2 g, 83%) as white solid;
mp 60-62 oC. νmax (KBr disc)/cm-1 2981, 1761 (C=O), 1668 (C=O), 1593, 1480, 1287,
822, 756, 703. 1H NMR (400 MHz, Chloroform-d) δ 7.65 (d, J = 2.8 Hz, 1H, Ar-H),
7.34 (dd, J = 8.8, 2.7 Hz, 1H, Ar-H), 6.75 (d, J = 8.8 Hz, 1H, Ar-H), 4.68 (s, 2H,
OCH2CO), 4.26 (q, J = 7.1 Hz, 2H, OCH2CH3), 3.08 (q, J = 7.2 Hz, 2H, CH2CH3),
1.29 (t, J = 7.1 Hz, 3H, OCH2CH3), 1.15 (t, J = 7.2 Hz, 3H, CH2CH3). 13C NMR (100
MHz, CDCl3) δ 201.68 (C=O), 167.80 (OCH2CO),155.01, 132.47, 130.23, 130.09,
127.07, 113.69, 65.73, 61.62, 37.09, 14.10, 8.28. HRESI-MS m/z calcd for [M+H]+
C13H16ClO4: 271.0732, found: 271.0734.
4.1.4. Synthesis of 5-Chloro-3-ethylbenzofuran-2-carboxylic acid (13a)
A mixture of 12a (3.2 g, 11.82 mmol) and sodium ethoxide (20 mL, 21 wt.% solution
in ethanol) in toluene was heated at reflux under N2 atmosphere. After 12 h, the
solvent was removed under reduced pressure and the product was extracted into H2O.
The precipitate formed after acidification of with 3N HCl was filtered and dried to
afford 13a (2.3 g, 86%) as an orange solid; mp 205-207 oC. νmax (KBr disc)/cm-1 3100,

ACCEPTED MANUSCRIPT
2976, 1685 (C=O), 1580, 1459, 1310, 1161, 813, 788, 726, 673. 1H NMR (400 MHz,
DMSO-d6) δ 7.91 (d, J = 2.2 Hz, 1H, Ar-H), 7.67 (d, J = 8.8 Hz, 1H, Ar-H), 7.49 (dd,
J = 8.8, 2.2 Hz, 1H, Ar-H), 3.01 (q, J = 7.5 Hz, 2H, CH2CH3), 1.20 (t, J = 7.6 Hz, 3H,
CH2CH3). 13C NMR (100 MHz, DMSO) δ 161.08 (COOH), 152.54, 130.43, 129.75,
128.26, 128.16, 121.41, 114.17, 17.21, 14.80. HRESI-MS m/z calcd for [M+H]+
C11H10ClO3: 225.0313, found: 225.0315.
4.1.5. Synthesis of Ethyl 2-(2-acetyl-4-chlorophenoxy)acetate (12b)
This compound was prepared as described in preparation of ethyl 2-(4-chloro-2-
propionylphenoxy)acetate 12a
o
Yield % 80, mp 61-63 C. 1H NMR (400 MHz, Chloroform-d) δ 7.70 (d, J = 2.5 Hz,
1H, Ar-H), 7.36 (dd, J = 8.8, 2.8 Hz, 1H, Ar-H), 6.76 (d, J = 8.8 Hz, 1H, Ar-H), 4.69
(s, 2H, OCH2CO), 4.26 (q, J = 7.1 Hz, 2H, OCH2CH3), 2.68 (s, 3H, CH3), 1.29 (t, J =
7.1 Hz, 3H, OCH2CH3). 13C NMR (100 MHz, CDCl3) δ 198.11 (C=O), 167.74
(OCH2CO), 155.45, 132.97, 130.35, 129.82, 127.08, 113.75, 65.72, 61.66, 31.85,
14.10. HRESI-MS m/z calcd for [M+H]+ C12H14ClO4: 257.0575, found: 257.0581.
4.1.6. Synthesis of 5-Chloro-3-methylbenzofuran-2-carboxylic acid (13b)
This compound was prepared as described in preparation of 5-chloro-3-
ethylbenzofuran-2-carboxylic acid 13a
o
Yield % 83, mp 285-287 C. 1H NMR (400 MHz, DMSO-d6) δ 7.81 (d, J = 2.2 Hz,
1H, Ar-H), 7.63 (d, J = 8.8 Hz, 1H, Ar-H), 7.45 (dd, J = 8.8, 2.2 Hz, 1H, Ar-H), 2.48
(s, 3H, CH3). 13C NMR (100 MHz, DMSO) δ 161.56, 152.25, 144.08, 130.90, 128.04,
127.77, 123.36, 121.27, 113.90, 9.48. HRESI-MS m/z calcd for [M+H]+ C10H8ClO3:
211.0156, found: 211.0160.

ACCEPTED MANUSCRIPT
4.1.7. General procedure for coupling of benzofuran carboxylic acids with
amines.
A mixture of benzofuran-2-carboxylic acids 13a-b (1 equiv), BOP (1.5 equiv), and
DIPEA (2 equiv) in DCM (0.05 M) was stirred for 10 mins at rt before addition of the
appropriate amine (1.2 equiv) and the resulting reaction mixture was stirred overnight
at rt. After removing of the solvent in vacuo, the residue was extracted with EtOAc,
washed with 5% HCl, saturated NaHCO3 solution, brine, dried over MgSO4, and
evaporated under reduced pressure to give a crude product which was purified by
flash chromatography on silica gel.
4.1.7.1.
5-Chloro-N-(4-(dimethylamino)phenethyl)-3-ethylbenzofuran-2-
carboxamide (7)
Yield % 83, mp 147-147oC. 1H NMR (400 MHz, Chloroform-d) δ 7.33 (s, 1H, Ar-H),
7.07-7.05 (m, 2H, Ar-H), 6.86 (d, J = 8.5 Hz, 2H, Ar-H), 6.46 (d, J = 8.4 Hz, 2H, Ar-
H), 6.40 (t, J = 6.0 Hz, 1H, amide NH), 3.39 (q, J = 7.0 Hz, 2H, NHCH2CH2), 2.86 (q,
J = 7.8 Hz, 2H, CH2CH3), 2.66 (s, 6H, N(CH3)2), 2.58 (t, J = 7.1 Hz, 2H,
13
NHCH2CH2), 1.03 (t, J = 7.8 Hz, 3H, CH2CH3). C NMR (100 MHz, CDCl3) δ
159.59 (C=O), 151.63, 149.47, 143.54, 130.31, 129.41, 128.69, 127.66, 126.99,
126.49, 120.64, 113.00, 112.68, 40.75, 40.64, 34.80, 17.07, 14.24. HRESI-MS m/z
calcd for [M+H]+ C21H24ClN2O2: 371.1521, found: 371.1519.
4.1.7.2.
5-Chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)benzofuran-2-
carboxamide (8)
o
Yield % 82, mp 142-144 C. νmax (KBr disc)/cm-1 3344 (NH), 2932, 1648 (C=O),
1
1610, 1509, 1442, 1290, 1240, 1156, 920, 805, 731, 642. H NMR (400 MHz,
Chloroform-d) δ 7.60 (s, 1H, Ar-H), 7.34 (d, J = 1.3 Hz, 2H, Ar-H), 7.14 (d, J = 8.6

ACCEPTED MANUSCRIPT
Hz, 2H, Ar-H), 6.91 (d, J = 8.6 Hz, 2H, Ar-H), 6.66 (t, J = 5.9 Hz, 1H, amide NH),
3.68 (q, J = 7.1 Hz, 2H, NHCH2CH2), 3.19 – 3.08 (m, 6H, piperidin-H, CH2CH3),
2.86 (t, J = 7.1 Hz, 2H, NHCH2CH2), 1.75-1.68 (m, 4H, piperidin-H), 1.63 – 1.53
(m, 2H, piperidin-H), 1.30 (t, J = 7.6 Hz, 3H, CH2CH3). 13C NMR (100 MHz, cdcl3)
δ 159.58 (C=O), 151.63, 150.96, 143.50, 130.30, 129.34, 129.13, 128.70, 127.69,
127.01, 120.64, 116.78, 112.69, 50.78, 40.49, 34.90, 25.88, 24.26, 17.07, 14.24.
HRESI-MS m/z calcd for [M+H]+ C24H28ClN2O2: 411.1834, found: 411.1829.
4.1.7.3. 5-Chloro-3-ethyl-N-phenethylbenzofuran-2-carboxamide (14)
Yield % 85, mp 85-87oC. 1H NMR (400 MHz, Chloroform-d) δ 7.61 (s, 1H, Ar-H),
7.39 – 7.21 (m, 7H, Ar-H), 6.69 (t, J = 6.0 Hz, 1H, amide NH), 3.72 (q, J = 6.7 Hz,
2H, NHCH2CH2), 3.13 (q, J = 7.5 Hz, 2H, CH2CH3), 2.96 (t, J = 7.2 Hz, 2H,
13
NHCH2CH2), 1.31 (t, J = 7.6 Hz, 3H, CH2CH3). C NMR (100 MHz, CDCl3) δ
159.63 (C=O), 151.63, 143.41, 138.70, 130.27, 128.79, 128.75, 128.69, 127.84,
127.08, 126.59, 120.67, 112.67, 40.36, 35.89, 17.07, 14.23. HRESI-MS m/z calcd for
[M+H]+ C19H19ClNO2: 328.1099, found: 328.1101.
4.1.7.4. 5-Chloro-3-ethyl-N-(4-morpholinophenethyl)benzofuran-2-carboxamide
(15)
Yield % 80, mp 110-112oC. 1H NMR (400 MHz, Chloroform-d) δ 7.61 (s, 1H, Ar-H),
7.34 (d, J = 1.5 Hz, 2H, Ar-H), 7.18 (d, J = 8.3 Hz, 2H, Ar-H), 6.90 (d, J = 8.4 Hz,
2H, Ar-H), 6.66 (t, J = 6.1 Hz, 1H, amide NH), 3.89-3.85 (m, 4H, morph-H ), 3.68
(q, J = 6.8 Hz, 2H, NHCH2CH2), 3.19 – 3.08 (m, 6H, morph-H, CH2CH3), 2.88 (t, J
= 7.1 Hz, 2H, NHCH2CH2), 1.30 (t, J = 8.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
159.60 (C=O), 151.63, 149.90, 143.45, 130.29, 130.18, 129.53, 128.74, 127.79,

ACCEPTED MANUSCRIPT
127.06, 120.67, 116.02, 112.67, 66.90, 49.53, 40.49, 34.93, 17.07, 14.23. HRESI-MS
m/z calcd for [M+H]+ C23H26ClN2O3: 413.1626, found: 413.1625.
4.1.7.5.
5-Chloro-3-ethyl-N-(4-(pyrrolidin-1-yl)phenethyl)benzofuran-2-
carboxamide (16)
Yield % 79, mp 140-140oC. 1H NMR (400 MHz, Chloroform-d) δ 7.61 (s, 1H, Ar-H),
7.34 (d, J = 1.3 Hz, 2H, Ar-H), 7.12 (d, J = 8.5 Hz, 2H, Ar-H), 6.69 (t, J = 5.9 Hz, 1H,
amide NH), 6.55 (d, J = 8.5 Hz, 2H, Ar-H), 3.67 (q, J = 7.0 Hz, 2H, NHCH2CH2),
3.32 – 3.24 (m, 4H, pyrrolidin-H), 3.14 (q, J = 7.6 Hz, 2H, CH2CH3), 2.85 (t, J = 7.1
Hz, 2H, NHCH2CH2), 2.04 – 1.96 (m, 4H, pyrrolidin-H), 1.31 (t, J = 7.6 Hz, 3H,
13
CH2CH3). C NMR (100 MHz, CDCl3) δ 159.59 (C=O), 146.78, 143.58, 130.32,
129.49, 128.68, 127.62, 126.96, 125.08, 120.63, 112.69, 111.87, 47.68, 40.76, 34.87,
25.46, 17.07, 14.25. HRESI-MS m/z calcd for [M+H]+ C23H26ClN2O2: 397.1677,
found: 397.1668.
4.1.7.6. (4-Benzylpiperidin-1-yl)(5-chloro-3-ethylbenzofuran-2-yl)methanone (17)
Yield % 75, mp 80-82oC. 1H NMR (400 MHz, Chloroform-d) δ 7.58 (d, J = 2.1 Hz,
1H, Ar-H), 7.37 (d, J = 8.8 Hz, 1H, Ar-H), 7.34 – 7.23 (m, 3H, Ar-H), 7.25 – 7.17 (m,
1H, Ar-H), 7.18 – 7.11 (m, 2H, Ar-H), 4.68 - 4.63 (m, 1H, piperidin-H), 4.01 -3.98
(m, 1H, piperidin-H), 3.04 – 2.70 (m, 4H, piperidin-H, CH2CH3), 2.58 (d, J = 7.1
Hz, 2H, PhCH2), 1.86 - 1.74 (m, 4H, piperidin-H), 1.33 - 1.28 (m, 4H, piperidin-H,
13
CH2CH3). C NMR (100 MHz, CDCl3) δ 160.35 (C=O), 151.99, 145.49, 129.68,
129.08, 128.54, 128.33, 126.09, 126.06, 125.01, 120.16, 112.78, 42.93, 38.32, 17.21,
14.23. HRESI-MS m/z calcd for [M+H]+ C23H25ClNO2: 382.1568, found: 382.1569.

ACCEPTED MANUSCRIPT
4.1.7.7.
(18)
(5-Chloro-3-ethylbenzofuran-2-yl)(4-phenylpiperazin-1-yl)methanone
Yield % 76, mp 108-110oC. 1H NMR (400 MHz, Chloroform-d) δ 7.76 (s, 1H, Ar-H),
7.60 – 7.39 (m, 4H, Ar-H), 7.13-7.06 (m, 3H, Ar-H), 4.07-4.02 (m, 4H, piperazin-H),
3.43-3.39 (m, 4H, piperazin-H), 3.06 (q, J = 7.5 Hz, 2H), 1.47 (t, J = 7.6 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ 160.39 (C=O), 151.98, 150.89, 144.74, 129.61, 129.27,
128.73, 126.55, 126.47, 120.64, 120.32, 116.70, 112.82, 49.81, 48.00, 46.69, 42.61,
17.30, 14.24. HRESI-MS m/z calcd for [M+H]+ C21H22ClN2O2: 369.1364, found:
369.1366.
4.1.7.8. 5-Chloro-3-methyl-N-phenethylbenzofuran-2-carboxamide (19)
Yield % 84, mp 80-82oC. 1H NMR (400 MHz, Chloroform-d) δ 7.54 (d, J = 2.0 Hz,
1H, Ar-H), 7.38 – 7.22 (m, 7H, Ar-H), 6.71 (t, J = 5.9 Hz, 1H, amide NH), 3.73 (q, J
= 7.2 Hz, 2H, NHCH2CH2), 2.95 (t, J = 7.2 Hz, 2H, NHCH2CH2), 2.59 (s, 3H, CH3).
13C NMR (100 MHz, CDCl3) δ 159.87 (C=O), 151.46, 143.91, 138.70, 131.11,
128.80, 128.78, 128.69, 127.17, 126.59, 121.73, 120.52, 112.55, 40.39, 35.89, 8.82.
HRESI-MS m/z calcd for [M+H]+ C18H17ClNO2: 314.0942, found: 314.0945.
4.1.7.9.
5-Chloro-N-(4-(dimethylamino)phenethyl)-3-methylbenzofuran-2-
carboxamide (20)
Yield % 79, mp 125-127oC. 1H NMR (400 MHz, Chloroform-d) δ 7.26 (s, 1H, Ar-H),
7.04 (d, J = 2.0 Hz, 2H, Ar-H), 6.83 (d, J = 8.4 Hz, 2H, Ar-H), 6.46 – 6.33 (m, 3H,
Ar-H, amide NH), 3.37 (q, J = 6.8 Hz, 2H, NHCH2CH2), 2.64 (s, 6H, N(CH3)2), 2.55
(t, J = 7.0 Hz, 2H, NHCH2CH2), 2.29 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ
159.82 (C=O), 151.47, 149.47, 144.05, 131.18, 129.41, 128.76, 127.09, 126.48,

ACCEPTED MANUSCRIPT
121.55, 120.52, 113.00, 112.58, 40.74, 40.64, 34.81, 8.82. HRESI-MS m/z calcd for
[M+H]+ C20H22ClN2O2: 357.1364, found: 357.1362.
4.1.7.10.
5-Chloro-3-methyl-N-(4-morpholinophenethyl)benzofuran-2-
carboxamide (21)
Yield % 82, mp 180-182oC. 1H NMR (400 MHz, Chloroform-d) δ 7.57 (d, J = 1.8 Hz,
1H, Ar-H), 7.35 - 7. 33 (m, 2H, Ar-H), 7.17 (d, J = 8.4, 2H, Ar-H), 6.89 (d, J = 8.4
Hz, 2H, Ar-H), 6.66 (t, J = 5.9 Hz, 1H, amide NH), 3.90 – 3.83 (m, 4H, morph-H),
3.68 (q, J = 6.7 Hz, 2H, NHCH2CH2), 3.18 – 3.11 (m, 4H, morph-H), 2.88 (t, J = 7.1
Hz, 2H, NHCH2CH2), 2.59 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 159.83 (C=O),
151.47, 149.97, 143.97, 131.16, 130.09, 129.52, 128.80, 127.16, 121.67, 120.55,
115.98, 112.56, 66.92, 49.48, 40.49, 34.93, 8.82. HRESI-MS m/z calcd for [M+H]+
C22H24ClN2O3: 399.1470, found: 399.1470.
4.1.7.11.
5-Chloro-3-methyl-N-(4-(piperidin-1-yl)phenethyl)benzofuran-2-
carboxamide (22)
Yield % 80, mp 155-157oC. 1H NMR (400 MHz, Chloroform-d) δ 7.56 (d, J = 1.8
Hz, 1H, Ar-H), 7.37 – 7.31 (m, 2H, Ar-H), 7.13 (d, J = 8.6 Hz, 2H, Ar-H), 6.91 (d, J
= 8.6 Hz, 2H, Ar-H), 6.65 (t, J = 5.8 Hz, 1H, amide NH), 3.68 (q, J = 7.1 Hz, 2H,
NHCH2CH2), 3.18 – 3.10 (m, 4H, piperidin-H), 2.86 (t, J = 7.1 Hz, 2H,
NHCH2CH2), 2.59 (s, 3H, CH3), 1.77 – 1.66 (m, 4H, piperidin-H), 1.63 – 1.53 (m,
13
2H, piperidin-H). C NMR (100 MHz, CDCl3) δ 159.81 (C=O), 151.47, 150.98,
144.01, 131.17, 129.34, 129.10, 128.77, 127.11, 121.59, 120.52, 116.77, 112.58,
50.77, 40.49, 34.90, 25.88, 24.27, 8.82. HRESI-MS m/z calcd for [M+H]+
C23H26ClN2O2: 397.1677, found: 397.1673.