Synthesis, characterization, in vitro biological and molecular docking evaluation of N,N'-(ethane-1,2-diyl)bis(benzamides)

Article abstract of DOI:10.1007/s13738-021-02199-8

The present research describes the synthesis, characterization, in vitro biological and docking evaluation of N,N'-(ethane-1,2-diyl)bis(benzamides) (3a-3j). Consequently, in in vitro hRBCs hemolysis assay, only the bis-amide (3d) induced 52.4% hemolysis at higher concentration (1000?μg/mL) that decreased drastically with concentration (250?μg/mL) to 27.9% (CC₅₀ = 400.41). Similarly, the tested bis-amide (3j) was found to be the least toxic with 7.8% hemolysis at higher concentration (1000?μg/mL) that gradually decreases to 6.1% (CC₅₀ = 19,347.83) at lower concentration (250?μg/mL). Accordingly, the tested bis-amides were found to be highly biocompatible against hRBCs at higher concentrations with much higher CC₅₀ values (> 1000?μg/mL). The biocompatible bis-amides (3a-3j) were subjected to in vitro DNA ladder assay to analyze their apoptotic potential. The results obtained suggest the tested bis-amides (3a-3j) are highly degradative toward DNA causing the appearance of more than one bands or complete degradation of DNA except (3a), (3c), (3i) and (3?g). Moreover, the synthesized bis-amides (3a-3j) were tested in in vitro antileishmanial assay to unveil their leishmaniacidal potential. The results obtained clearly indicated that some of the tested bis-amides displayed good dose dependent response. The tested bis-amides were highly active at higher concentration (1000?μg/mL) against the leishmanial promastigotes and their % inhibitory potential decreased drastically with concentration (250?μg/mL). Consequently, at higher concentration (1000?μg/mL), the bis-amide (3f) caused 85% inhibition and was ranked as the most effective leishmaniacidal bis-amides followed by the bis-amide (3?g) with 73.54% inhibition of leishmanial promastigotes. However, in terms of their IC₅₀ values, the best leishmaniacidal potential was displayed by the bis-amide (3f) followed by (3b), (3j) and (3?g) with IC₅₀ values increasing in the order of 633.16, 680.22, 680.22 and 712.93?μg/mL, respectively. Molecular docking studies revealed that bis-amides having electron-donating groups showed good binding potential against antileishmanial target. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s13738-021-02199-8

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-021-02199-8
ORIGINAL PAPER
Synthesis, characterization, in vitro biological and molecular docking
evaluation of N,N’‑(ethane‑1,2‑diyl)bis(benzamides)
Hamid Aziz1 · Aamer Saeed1 · Farukh Jabeen2,3 · Nazif Ullah4 · Ashfaq Ur Rehman5,6
Received: 30 November 2020 / Accepted: 28 January 2021
© Iranian Chemical Society 2021
Abstract
The present research describes the synthesis, characterization, in vitro biological and docking evaluation of N,N’-(ethane-
1,2-diyl)bis(benzamides) (3a-3j). Consequently, in in vitro hRBCs hemolysis assay, only the bis-amide (3d) induced 52.4%
hemolysis at higher concentration (1000 μg/mL) that decreased drastically with concentration (250 μg/mL) to 27.9%
(CC50 =400.41). Similarly, the tested bis-amide (3j) was found to be the least toxic with 7.8% hemolysis at higher concen-
tration (1000 μg/mL) that gradually decreases to 6.1% (CC50 =19,347.83) at lower concentration (250 μg/mL). Accordingly,
the tested bis-amides were found to be highly biocompatible against hRBCs at higher concentrations with much higher CC50
values (>1000 μg/mL). The biocompatible bis-amides (3a-3j) were subjected to in vitro DNA ladder assay to analyze their
apoptotic potential. The results obtained suggest the tested bis-amides (3a-3j) are highly degradative toward DNA causing
the appearance of more than one bands or complete degradation of DNA except (3a), (3c), (3i) and (3 g). Moreover, the
synthesized bis-amides (3a-3j) were tested in in vitro antileishmanial assay to unveil their leishmaniacidal potential. The
results obtained clearly indicated that some of the tested bis-amides displayed good dose dependent response. The tested
bis-amides were highly active at higher concentration (1000 μg/mL) against the leishmanial promastigotes and their %
inhibitory potential decreased drastically with concentration (250 μg/mL). Consequently, at higher concentration (1000 μg/
mL), the bis-amide (3f) caused 85% inhibition and was ranked as the most efective leishmaniacidal bis-amides followed
by the bis-amide (3 g) with 73.54% inhibition of leishmanial promastigotes. However, in terms of their IC50 values, the best
leishmaniacidal potential was displayed by the bis-amide (3f) followed by (3b), (3j) and (3 g) with IC50 values increasing
in the order of 633.16, 680.22, 680.22 and 712.93 μg/mL, respectively. Molecular docking studies revealed that bis-amides
having electron-donating groups showed good binding potential against antileishmanial target.
* Hamid Aziz
hamidazizwazir@yahoo.com
1
Department of Chemistry, Quaid-I-Azam University,
Islamabad 45320, Pakistan
2
Department of Biology, Laurentian University, 935 Ramsey
Lake Road, Sudbury, ON P3E 2C6, Canada
3
Computation, Science, Research and Development
Organization, 1401, 2485 Hurontraio street, Mississauga,
ON L5A2G6, Canada
4
Department of Biotechnology, Abdul Wali Khan University,
Mardan, Khyber Pukhtoonkhwa, Pakistan
5
Department of Bioinformatics and Biostatistics,
Shanghai Jiao Tong University, 800 Dongchuan Road,,
Minhang District, Shanghai 200240, China
6
Department of Biochemistry, Abdul Wali Khan University,
Shankar Campus, Mardan, Khyber Pukhtoonkhwa, Pakistan
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
Graphic abstract
Keywords Agarose gel electrophoresis · Apoptosis · Biocompatibility · Bis-amides · DNA · Leishmania tropica ·
Promastigotes · Human red blood cells
are nitrogen-containing heterocycles formed by coupling
carboxylic acids and amines in the presence of a coupling
reagent or by prior conversion of the carboxylic acid into
reactive acid chlorides. Amide bond is considered as one
of the most stable functionalities in organic chemistry and
is widely distributed in living organisms. Amide bond con-
stitutes key functional group in peptides, natural products
and pharmaceuticals. Amide bonds because of their higher
polarity, stability and conformational diversity constitute
most abundant motif in synthetic organic chemistry [4, 8,
9]. Amide-based heterocycles serve as efcient precursors
and intermediates in organic synthesis to synthesize vari-
ous agrochemicals, lubricants and drugs. The potent antibi-
otic and anti-infammatory drugs valdecoxib (1), celecoxib
(2), and penicillin G (3) contain amide linkages in their
structures [10–12]. Natural and synthetic benzamides are
reported as potent enzyme inhibitors [13, 14], antimicrobials
[15], cytotoxic [16] and anti-infammatory agents [17]. The
development of potent and selective COX-2 inhibitors hav-
ing lower side efects and improved gastric safety profle is a
challenging task for medicinal chemists to deal with. In this
regard, benzamide derivatives are reported as in vitro COX
inhibitors and in vivo anti-infammatory agents [18]. More
Introduction
Leishmaniasis is a neglected vector-borne skin disease being
caused by the bite of obligate intracellular protozoan of the
genus Leishmania. Leishmanial parasite possesses diverse
survival pathways and performs various physiological func-
tions. Species of the genus leishmania transmit easily to its
mammalian host and infects external as well as internal parts
of the human body [1]. Primary therapies of Leishmaniases
include Glucantime and Pentostam. Similarly, Pentamidine
and Amphotericin B are used as second line treatment.
Moreover, the phenomenon of emerging resistance mecha-
nism in the established key targets afects the use of drugs
negatively. This makes the drugs inefcient, troublesome,
and therefore, the development of potent antileishmanial
drugs with alternative and efcient mechanism of action is
required to eradicate this disease [2, 3]. In this context, lit-
erature survey suggests nitrogen-based heterocycles as bio-
logically relevant scafolds having possible leishmaniacidal
as well as biosafe nature [4, 5].
Nitrogen-based heterocycles have immense importance
because of their potential applications as propellants, pyro-
technics, pharmaco and chemotherapeutics [6, 7]. Amides
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Journal of the Iranian Chemical Society
specifcally, peptide derivatives possessing SO2Me and N3
pharmacophores are rationalized as selective COX-2 inhibi-
tors with selectivity index above 500 [19]. N-phenylacet-
amide-based functionalized carbazoles (4) are reported as
novel leading template in the chemotherapeutic treatments
of various bacterial ailments [20]. Amide-based heterocy-
cles ofer opportunity for the development of novel drugs
against tuberculosis. Alanis et al. revealed the synthesis and
potent antimycobacterial activity of α,β-unsaturated amides
[21]. Benzothiazole-based amides are highly biocompatible
and display in vitro antiproliferative potential against cancer
cells [22].
showed good to moderate in vitro leishmaniacidal potential
[4]. Recently, in vitro biocompatible and leishmaniacidal
potential of N,N’-(ethane-1,2-diyl)bis(3-methylbenzamide)
against the promastigotes of Leishmania tropica has been
revealed. The tested bis-amide displayed leishmaniacidal
potential higher than the reference standard Glucantime.
Molecular docking analysis revealed that the bisamide
strongly bind to the active site of Trypanothione reductase,
an enzyme involved in the redox metabolism of the Leishma-
nial parasite [27]. Figure 1 shows some potent heterocyclic
analogues containing amide functionality.
Thus, considering the phenomenon of leishmaniasis, the
lack of efective biocompatible drugs and the leishmani-
acidal potential of heterocyclic amide analogues, there is
a need for the synthesis of biocompatible antileishmanial
amides. In this regard, the present research paper reports
the facile synthesis, spectroscopic characterizations, in vitro
biological and docking evaluation of N,N’-(ethane-1,2-diyl)
bis(benzamides). The synthetic work completes in a sim-
ple and facile way with excellent yields using substituted
organic acids in dry distilled THF at room temperature.
To unveil the leishmaniacidal potential of amide contain-
ing heterocycles, Piplartine (5) has been reported to pos-
sess in vitro leishmaniacidal potential against promastig-
otes of Leishmania donovani. The tested compound showed
89.1 2.9% inhibition at 100 μM concentration with IC50
value of 7.5 μM [23]. Similarly, amide derivatives of isoxa-
zole (6) display promising antileishmanial potential against
Leishmania donovani and could be a possible hit against
leishmaniases [24]. 3-Nitro-1H-1,2,4-triazole as well as
2-nitro-1H-imidazole based amides (7) and sulfonamides
are tested against intracellular amastigotes of Trypano-
soma cruzi. Consequently, most of the tested compounds
were found to be even more potent (up to 58-fold) than the
reference drug Benznidazole [25]. Amides derivatives (8)
extracted from Piper amalago and some of the synthetic
analogues are found to be active against the promastig-
ote and intracellular amastigote forms with IC50 values
in nanomolar range [26]. Chiral amides (9–10) with two
asymmetric centers and C2 axis of symmetry were tested
against Leishmania tropica KWH23 Promastigotes. The
tested compounds particularly their deprotected analogues
Materials and methods
All the reagents and solvents used were purchased from
Sigma-Aldrich and were used without any further purif-
cations. Nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker Avance AVIII spectrometer operating
1
at 400 and 100 MHz for H and 13C NMR, respectively.
Deuterated methanol (CD3OD-d4) was used as a solvent and
TMS as an internal reference. Chemical shifts values are
reported in ppm (δ), relative integral, multiplicity (s, singlet,
Fig. 1 Some literature reported
biologically relevant and potent
amide containing heterocycles
Cl
H
N
NH
CH3
S
O
O
CH3
O
N
O
SO2NH2
N
N
SO2NH2
O
N
O
HO
HO
CH3
F3C
CH3
HN
CH3
(1)
(2)
(3)
(4)
O
N
O
CH3
N
N
O
H3CO
H3CO
O
NO2
N
N
H3C
CH3
O
HN
N
OCH3
(5)
(8)
(6)
(7)
CH3
OH
O
H
O
O
O
N
CH3
OCH3
O
O
O
N
F3CO
NH
O
OH
F3CO
O
OCH3
(9)
(10)
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Journal of the Iranian Chemical Society
d, doublet, dd, doublet of doublets, t, triplet, m, multiplet; b,
broad peak), coupling constant (J, Hz) and the atom assign-
ment. 13C NMR data are reported as the chemical shift value
(δ) and assignment of the atom. FT-IR spectra were recorded
on the Vertex 70 Bruker apparatus. % Elemental analysis
(CHNS) was performed to fnd out the percentage of each
element present in the synthesized compounds. The pro-
gress of the reaction was checked by thin layer chromatog-
raphy (TLC) on 2.0 cm×5.0 cm aluminum sheets precoated
with silica gel 60F254 with a layer thickness of 0.25 mm
(Merck).
2H, J = 8.20 Hz, Ar–H), 7.46 (t, 4H, J = 8.20 Hz, Ar–H),
3.62 (s, 4H, C-H), 3.56 (s, 4H, C-H); 13C NMR (CD3OD-d4,
100 MHz) δ: 170.76 (C=O), 135.62 (Ar), 132.69 (Ar),
129.56 (Ar), 128.30 (Ar), 40.89 (C–C), 39.23 (C-N); Anal.
Calcd. For C18H20N2O2: C, 72.95%; H, 6.80%; N, 9.45%;
Found: C, 72.86%; H, 6.85%; N, 9.49%.
N,N’‑(Ethane‑1,2‑diyl)bis(3‑nirobenzamide) (3c)
Yield (85%): m.p. 225 ℃; Rf; 0.69 (n-hexane/ethyl acetate
1:1); color: white powder; Mol. wt: 358.31; IR (ATR) ν:
3234.45 (NH), 3075.74, 2974.83 (C =C-H), 2865.48 (-C-
H), 1637.83 (C = O, amide), 1623.64, 1559.84, 1523.67,
Synthesis of N,N’‑(Ethane‑1,2‑diyl)bis(benzamides)
(3a‑3j)
1
1450.85 (Ar–C = C stretch), cm−1; H NMR (CD3OD-d4,
400 MHz) δ: 11.09 (b, s, 2H, NH), 8.91 (s, 2H, Ar–H),
8.47 (d, 2H, J=8.60 Hz, Ar–H), 8.37 (d, 2H, J=8.60 Hz,
Ar–H), 7.73 (t, 2H, J=7.80 Hz, Ar–H), 3.52 (s, 4H, C-H);
13C NMR (CD3OD-d4, 100 MHz) δ: 167.63 (C=O), 148.53
(Ar), 135.31 (Ar), 133.63 (Ar), 129.83 (Ar), 124.53 (Ar),
122.43 (Ar), 39.80 (C-N); Anal. Calcd. For C16H14N4O6:
C, 53.63%; H, 3.94%; N, 15.64%; Found: C, 53.58%; H,
3.99%; N, 15.84%.
The synthesis of bis-amides (3a-3j) was performed at room
temperature in dry distilled THF as reported elsewhere [27].
Briefy, substituted organic acids (5 mmol) were treated with
thionyl chloride (5.5 mmol) in dry distilled THF (10 mL) at
room temperature for half an hour (hr). A solution of ethyl-
enediamine (2.50 mmol) dissolved in THF (5 mL) was added
dropwise to the reaction mixture, and the mixture was stirred
for next half an hr. During the course of the reaction, TLC in
n-hexane/ethyl acetate (1:1) solvent system was constantly
used to monitor the progress of the reaction. Once the reac-
tion was completed, THF was rotary evaporated to obtain a
solid product. The solid product obtained was purifed by
washing it with sodium carbonate solution (2%), distilled
water and was later on recrystallized from methanol at room
temperature.
N,N’‑(Ethane‑1,2‑diyl)bis(2‑nirobenzamide) (3d)
Yield (82%): m.p. 256 ℃; Rf; 0.80 (n-hexane/ethyl acetate
1:1); color: white powder; Mol. wt: 358.31; IR (ATR)
ν: 3314.39 (NH), 3080.99, 2946.59 (C=C–H), 2867.60
(–C–H), 1636.53 (C=O amide), 1615.54, 1553.74, 1521.69,
1450.92 (Ar–C=C), cm−1; 1H NMR (CD3OD-d4, 400 MHz)
δ: 11.09 (b, s, 2H, NH), 8.40 (d, 2H, J = 8.29 Hz, Ar–H),
8.24 (d, 2H, J = 8.29 Hz, Ar–H), 7.86 (t, 2H, J= 8.20 Hz,
Ar–H), 7.80 (t, 2H, J=8.20 Hz, Ar–H), 3.63 (s, 4H, C-H);
13C NMR (DMSO-d6, 100 MHz) δ: 170.83 (C=O), 147.23
(Ar), 135.13 (Ar), 133.13 (Ar), 128.43 (Ar), 127.95 (Ar),
121.23 (Ar), 39.73 (C-N); Anal. Calcd. For C16H14N4O6:
C, 53.63%; H, 3.94%; N, 15.64%; Found: C, 53.62%; H,
3.95%; N, 15.64%.
N,N’‑(Ethane‑1,2‑diyl)bis(4‑nirobenzamide) (3a)
Yield (90%): m.p. 250 ℃; Rf; 0.80 (n-hexane/ethyl acetate
1:1); color: white powder; Mol. wt: 358.31; IR (ATR) ν:
3299.63 (NH), 3077.92 (C=C–H), 1636.55 (C=O, amide),
1616.67, 1551.52, 1522.59, 1453.83 (Ar–C=C), cm−1;
1H NMR (CD3OD-d4, 400 MHz) δ: 11.08 (b, s, 2H, NH),
8.40 (d, 4H, J=8.10 Hz, Ar–H), 8.23 (d, 4H, J=8.10 Hz,
Ar–H), 3.53 (s, 4H, C-H); 13C NMR (CD3OD-d4, 100 MHz)
δ: 168.70 (C=O), 151.93 (Ar), 140.33 (Ar), 128.43 (Ar),
121.23 (Ar), 39.73 (C-N); Anal. Calcd. For C16H14N4O6:
C, 53.63%; H, 3.94%; N, 15.64%; Found: C, 53.60%; H,
3.96%; N, 15.65%.
N,N’‑(Ethane‑1,2‑diyl)bis(4‑methylbenzamide) (3e)
Yield (80%): m.p. 237 ℃; Rf; 0.82 (n-hexane/ethyl acetate
1:1); color: white powder; Mol. wt: 296.36; IR (ATR) ν:
3305.57 (NH), 3043.82, 2939.27 (C=C–H), 2815.19,
2853.97 (–C–H), 1628.60 (NCO), 1613.21, 1573.59,
1536.47, 1440.16 (Ar–C=C), cm−1; 1H NMR (CD3OD-d4,
400 MHz) δ: 11.0 (b, s, 2H, NH), 7.74 (d, 4H, J=8.10 Hz,
Ar–H), 7.29 (d, 4H, J=8.10 Hz, Ar–H), 3.62 (s, 4H, C-H),
2.41 (s, 6H, C-H); 13C NMR (CD3OD-d4, 100 MHz) δ:
170.77 (C=O), 143.42 (Ar), 132.75 (Ar), 130.17 (Ar),
128.36 (Ar), 40.92 (C–N), 21.42 (C-H); Anal. Calcd. For
C18H20N2O2: C, 72.95%; H, 6.80%; N, 9.45%; Found: C,
72.88%; H, 6.85%; N, 9.57%.
N,N’‑(Ethane‑1,2‑diyl)bis(phenylacetamide) (3b)
Yield (80%): m.p. 163 ℃; Rf; 0.60 (n-hexane/ethyl acetate
1:1); color: white powder; Mol. wt: 296.36; IR (ATR) ν:
3307.27 (NH), 3027.10 (C=C–H), 2928.33 (–C-–H),
1660.51 (C=O, amide), 1625.13, 1494.37, 1454.38, 1452.37
(Ar–C=C), cm−1; 1H NMR (CD3OD-d4, 400 MHz) δ: 11.0
(b, s, 2H, NH), 7.97 (d, 4H, J = 8.20 Hz, Ar–H), 7.52 (t,
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Journal of the Iranian Chemical Society
N,N’‑(Ethane‑1,2‑diyl)bis(2‑methylbenzamide) (3f)
1583.33, 1546.84, 1490.81, 1460.25 (Ar–C=C), cm−1; 1H
NMR (CD3OD-d4, 400 MHz) δ: 11.10 (b, s, 2H, NH), 6.93
(s, 4H, Ar–H), 3.78 (s, 18H, C-H), 3.53 (s, 4H, C–H);13C
NMR (CD3OD-d4, 100 MHz) δ: 169.63 (C=O), 150.93 (Ar),
142.63 (Ar), 128.53 (Ar), 105.13 (Ar), 56.56 (O–C), 39.73
(C–N); Anal. Calcd. For C22H28N2O8: C, 58.92%; H, 6.29%;
N, 6.25%; Found: C, 58.82%; H, 6.32%; N, 6.32%.
Yield (80%): m.p. 265 ℃; Rf; 0.70 (n-hexane/ethyl acetate
1:1); color: white powder; Mol. wt: 296.36; IR (ATR) ν:
3315.47 (NH), 3056.56, 2952.65 (C=C–H), 2822.15,
2858.86 (–C–H), 1658.56 (C=O, amide), 1618.27, 1577.58,
1565.43, 1442.65 (Ar–C=C), cm−1; 1H NMR (CD3OD-d4,
400 MHz) δ: 11.0 (b, s, 2H, NH), 7.86 (d, 2H, J=8.29 Hz,
Ar–H), 7.28 (t, 2H, J = 8.20 Hz, Ar–H), 7.42 (t, 2H,
J=8.20 Hz, Ar–H), 7.27 (d, 2H, J=8.29 Hz, Ar–H); 3.50
(s, 4H, C-H), 2.27 (s, 6H, C-H); 13C NMR (CD3OD-d4,
100 MHz) δ: 170.10 (C=O), 137.25 (Ar), 135.43 (Ar),
132.41 (Ar), 129.45 (Ar), 127.67 (Ar), 125.93 (Ar), 39.25
(C–N), 17.23 (C–H); Anal. Calcd. For C18H20N2O2: C,
72.95%; H, 6.80%; N, 9.45%; Found: C, 72.90%; H, 6.74%;
N, 9.56%.
N,N’‑(Ethane‑1,2‑diyl)bis(heptylamide) (3j)
Yield (90%): m.p. 254.6 ℃; Rf; 0.50 (n-hexane/ethyl ace-
tate 1:1); color: white powder; Mol. wt: 284.44; IR (ATR)
ν: 3293.90 (NH), 3087.02, 2953.54 (C=C–H), 2819.90,
2856.31 (–C–H), 1635.13 (C=O, amide), 1553.79, 1469.78,
1448.72 (Ar–C=C), cm−1; 1H NMR (CD3OD-d4, 400 MHz)
δ: 11.10 (b, s, 2H, NH), 3.50 (s, 4H, C-H), 2.21 (t, 4H,
J=7.30 Hz, C–H), 1.60 (q, 4H, J=7.20 Hz, C-H), 1.36 (q,
4H, J = 7.20 Hz, C-H), 1.26–1.30 (m, 8H, C-H), 0.90 (t,
6H, J = 7.50 Hz, C-H); 13C NMR (CD3OD-d4, 100 MHz)
δ: 172.73 (C=O), 39.23 (C-N), 36.53 (C-H), 31.73 (C-H),
27.45 (C-H), 24.34 (C-H), 22.83 (C-H), 14.13 (C-H); Anal.
Calcd. For C16H32N2O2: C, 67.56%; H, 11.34%; N, 9.85%;
Found: C, 67.49%; H, 11.40%; N, 9.83%.
N,N’‑(Ethane‑1,2‑diyl)bis(benzamide) (3 g)
Yield (80%): m.p. 175 ℃; Rf; 0.52 (n-hexane/ethyl acetate
1:1); color: white powder; Mol. wt: 268.31; IR (ATR) ν:
3294.04 (NH), 3063.22 (C=C–H), 2945.41 (–C–H), 1631.86
(C=O, amide), 1603.64, 1579.15, 1551.75 (Ar–C=C),
1
cm−1; H NMR (CD3OD-d4, 400 MHz) δ: 11.0 (b, s, 2H,
NH), 7.83–7.80 (m, 4H, Ar–H), 7.54–7.50 (m, 2H, Ar–H),
7.47–7.43 (m, 4H, Ar–H), 3.62 (s, 4H, C–H); 13C NMR
(CD3OD-d4, 100 MHz) δ: 170.76 (C=O), 135.62 (Ar),
132.69 (Ar), 129.56 (Ar), 128.30 (Ar), 40.89 (C–N); Anal.
Calcd. For C16H16N2O2: C, 71.62%; H, 6.01%; N, 10.44%;
Found: C, 71.55%; H, 6.15%; N, 10.43%.
In vitro biological assays
% hRBCs Hemolysis assay
Cytotoxic testing of the synthesized bis-amides (3a-3j) was
performed to confer their biosafe nature using fresh human
red blood cells (hRBCs) hemolysis assay [27]. Briefy, 5 mL
of the hRBCs was diluted with phosphate bufer saline (PBS)
and were centrifuged at 1500 rpm for 10 min (mins) to col-
lect the erythrocytes. The blood suspensions were incubated
for 3 h at 37 ºC using 250 ppm, 500 ppm and 1000 ppm solu-
tions of the bis-amides (3a-3j). The incubated mixtures were
again centrifuged at 1500 rpm for 10 min, and the hemolysis
was observed through naked eye. Hundred microliters of
the supernatant from each concentration was poured into
96-well plate to assess the amount of hemoglobin released
in the supernatants measured at 576 nm using UV–visible
spectrophotometry. Triton X-100 (0.5%) was used as posi-
tive control, while methanol (5%) was taken as negative con-
trol. % Hemolysis was calculated from Eq. (1) given below.
N,N’‑(Ethane‑1,2‑diyl)bis(2‑bromobenzamide) (3 h)
Yield (90%): m.p. 251.60 ℃; Rf; 0.60 (n-hexane/ethyl ace-
tate 1:1); color: white powder; Mol. wt: 426.1; IR (ATR)
ν: 3273.53 (NH), 3069.35 (C=C–H), 2943.68 (–C–H),
1639.99 (C=O, amide), 1589.32, 1538.64, 1464.81, 1451.21
(Ar–C=C), cm−1; 1H NMR (CD3OD-d4, 400 MHz) δ: 11.10
(b, s, 2H, NH), 7.86 (d, 2H, J=8.29 Hz, Ar–H), 7.42 (t, 2H,
J = 8.20 Hz, Ar–H), 7.28 (t, 2H, J= 8.20 Hz, Ar–H), 7.27
(d, 2H, J = 8.29 Hz, Ar–H), 3.50 (s, 4H, C-H);13C NMR
(CD3OD-d4, 100 MHz) δ: 169.63 (C=O), 136.83 (Ar),
134.43 (Ar), 131.83 (Ar), 129.73 (Ar), 127.93 (Ar), 120.72
(Ar), 39.71 (C–N); Anal. Calcd. For C16H14Br2N2O2: C,
45.10%; H, 3.31%; N, 6.57%; Found: C, 45.0%; H, 3.36%;
N, 6.62%.
% Hemolysis
[
N,N’‑(Ethane‑1,2‑diyl)bis(3,4,5‑trimethoxybenzamide) (3i)
]
× 100
OD at576nm in bisamide solution−ODat576nminPBS
ODat576nmin0.5%TritonX − 100−ODat576nminPBS
=
Yield (75%): m.p. 117 ℃; Rf; 0.70 (n-hexane/ethyl acetate
1:1); color: white powder; Mol. wt: 448.71; IR (ATR) ν:
FT-IR (ATR, cm−1): 3353.68 (NH), 3069.35, 2980.43
(C=C–H), 2943.68, 2895.89 (-C-H), 1644.76 (C=O, amide),
((1))
whereas OD = optical density, PBS = phosphate buffer
saline.
1 3

Journal of the Iranian Chemical Society
DNA ladder assay
Molecular docking analysis
DNA extraction DNA was extracted from collected lesion
sample using standard procedure of organic phenol chloro-
form [28]. Accordingly, 750 μL of the blood sample was
mixed with an equal volume of lysis bufer (0.1 mM EDTA,
12 mM NaHCO3 and 155 mM NH4Cl). Proteinase K (Bio-
matik Corporation, Canada) was added to a 0.5 mg/mL of
fnal concentration, and sample was incubated at 56 ºC for
24 h. DNA was extracted successively with one volume of
phenol at pH 8.0, one volume of phenol/chloroform and one
volume of chloroform. The DNA was precipitated from the
aqueous phase with ethanol and the pellet washed with 70%
ethanol. The sample was air-dried and fnally resuspended
in 40 µL of the bufer.
Molecular docking analysis has been shown to play an
essential role in predicting the binding modes of ligands to
proteins. MOE-dock module implemented in the Molecu-
lar Operating Environment (MOE) software package [29]
was used to dock all the synthesized bis-amides (3a-3j)
against antileishmanial target protein to further evaluate
their leishmaniacidal potential. The structural coordinates
of antileishmanial target protein (PDB code 1LML) [30]
were retrieved from Protein Data Bank (www.rcsb.org). All
the solvent molecules were removed and were subjected to
3D protonation and energy minimization up to 0.05 Gradient
using MMFF94s forcefeld implemented in MOE. Since the
1LML is devoid of a co-crystalized ligand so, the active site
was indicated using the site-fnder wizard of MOE by the
creation of an alpha center followed by dummies. The alpha
spheres were created inside the cleft in the form of a com-
pact cluster; similarly, dummy atoms were created at the site
of alpha sphere to carry out docking on the dummy atoms as
the receptor was devoid of its co-crystallized ligand.
DNA treatment The extracted human DNA was treated
with the synthesized bis-amides (3a-3j). Ten microliters
of normal DNA was treated with 1 µL (1000 µg/mL) of
the drugs and was incubated for 1 h at 37 ºC. H2O2 was
used as positive control, and untreated DNA was used as
negative control.
The 3D structural coordinates for all the bis-amides (3a-
3j) were built using Molecular Builder Module in MOE.
Finally, refined structures were used for docking study
using the default parameters of MOE; placement: Triangle
Matcher, rescoring 1: London dG, refnement: Forcefeld,
rescoring 2: GBVI/WSA. Before running the docking pro-
tocol, all the bis-amides (3a-3j) were then docked into pre-
dicted active site of protein and 10 conformations for each
bis-amides were allowed to be generated. The ligands are
fexible during docking so that to obtain the minimal energy
conformation. The ligands were ranked based on docking
score; lower scores indicate more favorable poses. Finally,
the predicted protein–ligand interaction (PLI) was analyzed
for molecular interactions using PyMol v 1.7.
Gel electrophoresis The treated DNA was then visualized
on 1.6% Agarose gel electrophoresis containing 5 µL of
ethidium bromide by keeping the voltage at 72 V for 2 h.
The gel was visualized under Gel documentations system
Bio-Rad.
Antileishmanial assay
Antileishmanial assay of the synthesized bis-amides (3a-
3j) was performed using a literature reported methodology
[27]. For this purpose, Leishmania tropica promastigotes
were cultured in RPMI medium (supplemented with 10%
FBS and 1% antibiotic Streptomycin/Penicillin) unless the
culture count using Neubauer chamber reached 4–5 mil-
lion/mL. The synthesized bisamides (3a-3j) were added to
96-well plate in three diferent concentrations (1000 ppm,
500 ppm, and 250 ppm) screened in triplicates. Ampho-
tericin B was used as a reference drug, while methanol
(5%) being non-toxic to leishmanial parasites was used
as a negative control. A 100 µL of the complete culture
(106 cells/mL) was added to the 96-well plate containing
bisamides (3a-3j) at three diferent concentrations and
incubated at 25 ºC for 72 h. The viability of the promastig-
ote was assessed by tetrazolium-dye (MTT) colorimetric
method. A 100 µL of the MTT dye was added to each well,
and the plates were incubated at 37 ºC for 3 h. Finally, 40
µL of the DMSO was added as stop solution and the read-
ings were taken using an ELISA plate reader at 570 nm.
Results and discussion
Synthesis and chemistry
The synthetic route employed for the synthesis of bis-
amides (3a-3j) is provided below in Scheme 1. Briefy, vari-
ous organic acid chlorides (2a-2j) were synthesized from
organic acids (1a-1j) at room temperature using dry THF
and thionyl chloride by constantly stirring them for half an
hr. To the freshly synthesized acid chlorides (2a-2j), a solu-
tion of ethylenediamine in dry THF was slowly added in situ
and the reaction was allowed to stir at room temperature for
another half an hour to synthesize the corresponding bis-
amides (3a-3j). Upon completion, THF was removed under
reduced pressure to get solid bis-amides (3a-3j) in excellent
yields (75–90%) as given in Table 1. The obtained solid
1 3

Journal of the Iranian Chemical Society
H2N
O
O
O
NH2
SOCl2, THF
H
N
R
R
Cl
R
OH
R
N
H
30 mins stirring, r.t.
THF, 30 mins stirring, r.t.
O
(1a-1j)
(2a-2j)
(3a-3j)
Scheme 1 Synthetic route employed for the synthesis of the target bis-amides (3a-3j)
Table 1 Structures of the synthesized variously substituted bis-
amides (3a-3j)
dry THF (30 mL) at room temperature. The reaction mix-
ture was stirred for 15 min, and then, aniline dissolved in
dry THF (30 mL) was added slowly [31]. The resulting
mixture was refluxed for 3 h to get the crude products in
higher yields (76–91%). Moreover, amides are directly
synthesized from a range of carboxylic acids and amines
using B(OCH2CF3)3. The products obtained can be puri-
fied easily using commercial resins. Briefly, equimolar
solution of the respective acid and amine in acetonitrile
(2 mL, 0.5 M) was stirred at 80 °C for 5–24 h in the
presence of B(OCH2CF3)3 to get the products in up to
99% yields (Entry 3) [32]. Similarly, direct amidation of
carboxylic acids and amines using TiCl4 in pyridine is
reported. The reaction works with a range of substrates
and provides the respective amides in moderate to excel-
lent yields (56–98%). The yield decreases by using steri-
cally hindered carboxylic acid and amines. The method
involves adding TiCl4 (3 mmol) and amine (1 mmol) to a
solution of the corresponding acid (1 mmol) in pyridine
(10 mL). The reaction mixture was then heated at 85 ℃
for 120 min to afford the corresponding amides (Entry 4)
[33]. The highly efficient green synthesis of bis-amides
using polyphosphoric acid supported on silica-coated
NiFe2O4 nanoparticle (NiFe2O4@SiO2-PPA) in methanol
has been described (Entry 5). The optimum conditions
include refluxing aldehydes (1 mmol), amides (2 mmol),
the catalyst NiFe2O4@SiO2-PPA (0.1 gm) and methanol
(1 mL). The synthesized pure symmetrical bis-amide are
obtained in good to excellent yields (93–52%). In this syn-
thetic methodology, aldehydes with electron withdrawing
S. No Compound
R
S. No Compound
R
1
2
3
4
3a
3b
3c
3d
4-NO2-Ar
Ar-CH2
3-NO2-Ar
2-NO2-Ar
6
7
8
9
3f
2-CH3-Ar
Ar
2-Br-Ar
3 g
3 h
3i
3,4,5-triOCH3-
Ar
5
3e
4-CH3-Ar 10
3j
C6H11
bis-amides (3a-3j) were purifed by washing with 2% solu-
tion of sodium carbonate, distilled water and fnally recrys-
tallized from methanol at room temperature.
Efficiency of the current synthetic approach was
compared with some relevant reported methodologies
to argue optimum conditions employed for the synthe-
sis of bis-amides (Table 2). Hence, the ideal conditions
developed included adding ethylenediamine in situ to
the synthesized acid chlorides in THF at room tempera-
ture. The reaction mixture was stirred for 30 min at room
temperature (Entry 1) to obtain the target bis-amides in
excellent yields (75–90%). Table 2 contains compara-
tive efficiency of the current work with some literature
available methodologies employed for the synthesis of
amides. Consequently, direct amidation has been suc-
cessfully achieved using carbonyldiimidazole (Entry
2). The optimum conditions include stirring equimolar
2-naphthoic acid with carbonyldiimidazole dissolved in
Table 2 Comparison of the
S. No
Solvent
Catalyst
Tempera-
Time (hrs)
Yield (%)
Reference
efciency of the current
ture (℃)
methodology adopted for the
synthesis of bis-amides (3a-3j)
with reported methodology
1
2
3
4
5
6
7
8
THF
THF
…
25
66
80
85
0.5
3
75–90
76–91
Up to 99
56–98
5–932
20–74
77–97
45–82
[27]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
Carbonyldiimidazole
B(OCH2CF3)3
TiCl4
NiFe2O4@SiO2-PPA
…
Nanoparticles
…
CH3CN
Pyridine
CH3OH
…
Toluene
CH3OH
5–24
2
0.40–2.1
3.5–5
3–8
24
64
185
110
25
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Journal of the Iranian Chemical Society
groups reacted faster than the aldehydes with electron
donating groups [34]. Similarly, the synthesis of sym-
metrical bis-amides of malonic acid has been performed.
Briefly, dimethyl/ethyl malonate (5.6 mmol) and the cor-
responding amine (11.0 mmol) are stirred at 185 °C for
3.5–5 h to obtain solid products in 20–74% yields (Entry
6). At the optimum conditions, the yield was found to
be highest for unsubstituted aniline and lowest for ani-
lines bearing electron donating groups [35]. Moreover,
silica-bonded S-sulfonic acid nanoparticles are reported
as efficient catalysts for the synthesis of symmetrical bis-
amides (Entry 7). Reaction of aldehydes (1 mmol), amide
(2 mmol) and nanoparticles (0.08 gm) in toluene (7 mL)
heated under reflux affords the symmetrical bis-amides in
good to excellent yields (77–98%). Under optimum con-
ditions, benzaldehydes substituted with either electron-
donating or electron withdrawing groups underwent the
reaction in good to excellent yields. The reaction was
found to be compatible with aliphatic aldehydes [36].
An efficient four-component Ugi synthesis of ferroce-
nyl bis-amides in a mild and neutral conditions has been
successfully described. The optimum conditions of this
synthetic strategy include stirring equimolar mixture of
ferrocene carboxaldehydes, acids and amines in methanol
(3 mL) for 24 h at room temperature (Entry 8). The fer-
rocenyl bis-amides are obtained in good yields (70–45%)
upon completion of the reaction. However, the yield of
the synthesized ferrocenyl bis-amides is improved greatly
(68–82%) by using 2-aminopyridine and 2-aminopyrimi-
dine as heterocyclic amines as well as indole-3-carboxylic
acid as heterocyclic acids [37].
Characterizations
Structural confrmation and assignments of the synthesized
bis-amides (3a-3j) are based on their % elemental analy-
sis (CHNS) data and various spectroscopic technique (FT-
IR, 1H NMR,13C NMR). All of the synthesized bis-amides
(3a-3j) gave satisfactory % analysis data for the elements
C, H and N which was highly aligned with their proposed
structural formulae. The NH protons appears as broader and
intense band at 3305.57 cm−1 in the FT-IR spectrum of the
bis-amide (3e). Similarly, FT-IR spectrum of the bis-amide
(3e) displays sharp band at 1628.60 cm−1 for the amide
bond. This amide bond is clearly visible at 170.77 ppm in the
13C NMR spectrum. Similarly, the p-substituted phenyl ring
resulted in two distinct doublets at 7.29 and 7.74 ppm in the
1H NMR spectrum of bis-amide (3e). The aliphatic chain of
the bisamide bis-amides appears as intense peaks in the 1H
and 13C NMR spectra at 3.62 and 40.92 ppm, respectively.
In vitro biological assays
% hRBCs Hemolysis assay
The cytotoxicity of the tested bis-amides (3a-3j) was inves-
tigated using hRBCs assay as described previously for the
evaluation of cytotoxic efects of the antileishmanial com-
pounds [38]. The release of hemoglobin from hRBCs is an
indication of the cytotoxicity of the bisamide measured by
UV–visible spectroscopy and compared with Triton X-100
(0.5%). The % hemolysis of hRBCs using three diferent
concentrations (1000, 500 and 250 µg/mL) of the tested
bis-amides (3a-3j) and their corresponding CC50 values are
given in Tables 3 and 4. Accordingly, the tested bis-amides
(3a-3j) were found to be highly biocompatible with much
lower % hRBCs hemolysis at lower concentrations (250 µg/
mL). However, with the increase in the concentration of the
tested bis-amides (3a-3j), % hRBCs hemolysis increased
gradually. In terms of % hemolysis at higher concentrations
(1000 µg/mL), the bis-amides (3b) and (3d) induced 55.3%
and 52.4% hRBCs hemolysis, respectively, and were ranked
as highly cytotoxic followed by bis-amides (3 h) and (3e)
as shown in Table 2. Similarly, the bis-amide (3j) induced
7.8% hemolysis at higher concentrations (1000 µg/mL) and
was found to be the least toxic followed by bis-amides (3a),
Subsequently, to compare the efficiency and general-
ize the synthetic methodology employed for the synthesis
of bis-amides, the reaction was performed at optimum
conditions (Entry 1, Table 2). Accordingly, bis-amides
were synthesized in excellent yields (75–90%) by first
stirring organic acids with excess of thionyl chloride at
room temperature for 30 min. Upon completion of the
reaction, a solution of ethylene diamine in dry THF was
added slowly in situ to the synthesized acid chlorides and
the reaction mixture was stirred for next 30 min. After
stirring the reaction mixture for the required time, THF
was removed under reduced pressure to obtain solid pre-
cipitates of bis-amides upon necessary purification.
Table 3 % Hemolysis and CC50
S. No
Compound
% Hemolysis
(1000 µg/mL)
% Hemolysis
(500 µg/mL)
% Hemolysis
(250 µg/mL)
CC50 (µg/mL)
(µg/mL) values obtained for
the slightly toxic bis-amides
(3b, 3d, 3e, 3 h) in hRBCs
hemolysis assay
1
2
3
4
3b
3d
3e
55.3
52.4
40.2
41.9
52.4
43.3
31.2
38.1
30.7
27.9
30.2
5.8
621.77
400.41
1735.71
1089.83
3 h
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Journal of the Iranian Chemical Society
Table 4 % Hemolysis and CC50
(µg/mL) values obtained for the
tested no-toxic bis-amides (3a,
3c, 3f, 3 g, 3i, 3j) in hRBCs
hemolysis assay
S. No
Compound
% Hemolysis
(1000 µg/mL)
% Hemolysis
(500 µg/mL)
% Hemolysis
(250 µg/mL)
CC50 (µg/mL)
1
2
3
4
5
6
3a
3c
3f
3 g
3i
3j
10.4
17
27
11.5
27.4
7.8
7.1
8.3
20
10
24.4
6.6
6.8
5.8
17.1
8.2
21
8794.11
3173.20
2736.84
1.04E+10
131,757.6
19,347.83
6.1
(3 g), (3c), (3f) and (3i) as shown in Table 3. The same
trends are obvious from their CC50 values which show that
the bis-amides (3d) and (3b) (CC50 400.41, 621.77 µg/
mL) are slightly cytotoxic when compared to the remain-
ing bisamides having much higher CC50 values (>1000 µg/
mL) and were ranked as non-toxic to hRBCs.
appearance of the DNA treated with bis-amides (3b) and
(3d) can be observed in the following fgure.
Antileishmanial assay
Antileishmanial potential observed for the tested bis-
amides (3a-3j) is presented in Table 5 in terms of their
% inhibition and IC50 values. In terms of their % inhi-
bition at highest concentration (1000 µg/mL), the bis-
amide (3f) was found to be the most efective one against
promastigotes of Leishmania tropica which caused 85%
growth inhibition followed by 73.54% inhibition of the
bis-amide (3 g). For the tested bis-amides (3a-3j), the per-
cent inhibitory response was found to be highly dependent
upon the concentration of the tested bis-amides (3a-3j)
and decreases drastically at lower concentrations except
for bis-amide (3c) which decreased gradually with the
decrease in concentration. In terms of their IC50 values,
bis-amide (3f) was evaluated to possess good leishmani-
cidal potential (IC50 = 633.16 µg/mL) followed by bis-
amides (3b), (3j) and (3 g). Surprisingly, the IC50 val-
ues were found to be similar (IC50 = 680.22 µg/mL) for
bis-amides (3b) and (3j) though their percent inhibition
was found to be diferent (71.11%, 67.42%) at the tested
concentrations.
DNA ladder assay
DNA ladder assay was adopted to study the efect of the
tested bis-amides (3a-3j) on apoptosis and determine the
possible mode of cellular death (apoptosis). For this pur-
pose, human DNA was exposed to the tested bisamides
for 1 h at 37 ºC. Briefy, one of the hallmarks of the con-
frmation of late apoptosis is the appearance of unclear
DNA fragment of nuclear DNA on agarose gel. Accord-
ing to the results obtained, the well no.1 of the agarose
gel (Fig. 2) shows the normal and untreated human DNA,
while well no. 2 shows DNA treated with H2O2 as posi-
tive control which induces breakage in DNA strands and
result in appearance of diferent bands. In comparison
with the positive control, bis-amides (3a), (3c), (3i) and
(3 g) were found non-toxic to DNA, while the remaining
bisamides were highly degradative toward DNA causing
the appearance of more than one bands and complete deg-
radation (appearance of a smear of DNA). The smear-like
Fig. 2 DNA damage caused
after treatment with the bis-
amides 1: untreated DNA (nega-
tive control), 2: H2O2-treated
DNA (positive control), 3: (3a),
4: (3b), 5: (3d), 6: (3j), 7: (3 h),
8: (3f), 9: (3e), 10: (3c), 11:
(3i), 12: (3 g)
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Journal of the Iranian Chemical Society
Table 5 % Inhibition and IC50
(µg/mL) values obtained for
the tested bis-amides (3a-3j)
against Leishmania tropica
promastigotes
S. No
Compound
% Inhibition
% Inhibition
(500 µg/mL)
% Inhibition
(250 µg/mL)
IC50 (µg/mL)
(1000 µg/mL)
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3f
37.26
71.11
66.56
42.31
45.83
85.89
73.54
13.65
42.32
67.42
31.56
24.56
43.23
25.73
33.38
26.36
25.64
10.92
23.09
36.01
26.94
26.45
38.98
21.36
25.26
24.80
22.97
0.750
18.72
32.23
5673.86
680.22
691.21
1286.48
1146.51
633.16
3 g
3 h
3i
712.93
43,356.53
1258.13
680.22
10
3j
Molecular docking study
enhancing the enzyme potential, i.e., electrically positively,
negatively charged, and polar uncharged side chain residue
R414, D491 and T417 (Fig. 3b) with an average binding
afnity of − 0.7375 kJ/mol. Second- and third-ranked bis-
amides (3b, 3j and 3c) shared almost similar binding mode
with R414 (Fig. 3c–e) with an average binding affinity
of−0.45 kJ/mol, -0.475 kJ/mol and−0.38 kJ/mol. Besides,
bis-amide (3b) showed an additional interaction with T417,
while bis-amide (3j) showed two H-bond with R414. The
highest potential for bis-amide (3f) might be due to the
opposite side attached methyl group which is EDG’s and
have good potential for activating the corresponding com-
pound and hence raising the enzyme activity, while the slight
less potential for bis-amides (3b) and (3j) might be due to
the phenyl group attached at opposite site, which might have
less potential for activating the overall bisamide and hence
The synthesized bis-amides (3a-3j) were evaluated for their
antileishmanial potential by molecular docking approach
using MOE software to explore the possible binding mode
against the antileishmanial target. Generally, the synthesized
bis-amides (3a-3j) possess diferently substituted groups,
e.g., electron-withdrawing groups (EWG’s) and electron-
donating groups (EDG’s). Molecular docking results
revealed good potential for bis-amides which possess EDG’s
against antileishmanial target. The surface representation of
each targeted enzyme with zoomed the active site is depicted
in Fig. 3a. The frst-ranked bis-amide (3f) showed ft-well
binding mode in the predicted active site of the targeted
protein and was observed in adopting favorable interactions
with critical residues which might have important role in
Fig. 3 Protein–ligand Interaction (PLI) profles of the synthesized
bis-amides (3a-3j) against antileishmanial target protein. Figure (3a)
Indicates the surface of the antileishmanial enzyme. Figure (3b-3b)
Indicates PLI profle for the tested bis-amides (3f), (3b), (3j) and (3c).
Double-sided arrows represent the arene–arene interaction
1 3

Journal of the Iranian Chemical Society
less activity. Mostly, the carbonyl oxygen of each bisamide
was observed in adopting essential interaction with active
site residues.
References
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The present research article successfully describes the ef-
cient synthesis, spectroscopic characterizations, in vitro bio-
logical and computational studies of C2 symmetric simple
N,N’-(ethane-1,2-diyl)bis(benzamides) (3a-3j). Biocompat-
ibility of the synthesized bis-amides (3a-3j) was assessed
by analyzing their hemolytic efects on human red blood
cells (hRBCs). The tested bis-amides having low % hemoly-
sis and higher IC50 values were ranked as biocompatible
against hRBCs. Consequently, all the tested bis-amides were
found to be highly biocompatible except (3d) which induced
52.4% hemolysis at 1000 µg/mL (CC50 =400.41 µg/mL) and
hence ranked as slightly toxic. Similarly, DNA ladder assay
was opted to check the apoptotic nature of the tested bis-
amides (3a-3j). The results obtained revealed that most of
the tested bis-amides are highly toxic in nature causing the
complete degradation of DNA except (3a), (3c), (3i) and
(3 g). Leishmaniacidal potential of the tested bis-amides
(3a-3j) against the promastigotes of leishmania tropica was
assessed in terms of their % inhibitions and IC50 values. In
terms of their % inhibitions, the bis-amides (3f) and (3 g)
displayed excellent leishmanicidal potential of 85.0% and
73.54%, respectively. In terms of their IC50 values, (3f) with
IC50 value of 633.16 µg/mL was ranked as the most potent
leishmaniacidal bisamide. However, the bis-amides (3b) and
(3j) received the second highest leishmanicidal potential
with almost similar IC50 values of 680.22 µg/mL. The next
highest leishmanicidal potential was displayed by the bis-
amides (3c) and (3 g) with IC50 values of 691.21 µg/mL and
712.93 µg/mL, respectively. Computational molecular dock-
ing analysis revealed bearing bis-amides bearing electron-
donating groups possess strong binding to the active site and
hence display strong inhibitory potential against antileish-
manial target. Thus, based on the results stated above, the
synthesized bis-amides (3a-3j) could be concluded as a safer
and better antileishmanial scafolds and need to be investi-
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Compliance with ethical standards
Conflict of interest The authors declare no confict of interest.
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