A Swift One-Pot Solvent-Free Synthesis of Benzimidazole Derivatives and Their Metal Complexes: Hydrothermal Treatment, Enzymatic Inhibition, and Solubilization Studies

Article abstract of DOI:10.1134/S107036322008023X

Abstract: Three benzimidazole derivatives, 1-(1H-benzimidazol-2-yl)ethanol (HBE), 1H-benzimidazol-2-yl(diphenyl)methanol (BDM) and 1,2-bis(1H-benzimidazol-2-yl)ethane-1,2-diol (BHBED), have been synthesized following the one-pot rapid green protocol. Complexes of benzimidazole derivatives with six 3d transition metals, Cu(II), Mn(II), Zn(II), Fe(II), Co(II), and Ni(II), have been synthesized by free hydrothermal method. The synthesized products have been characterized by FTIR, ¹H, and ¹³C NMR, and mass spectroscopy, and CHN analysis, and 2:1 ligand to metal stoichiometry has been confirmed. The synthesized ligands and metal complexes have been tested for antioxidant potential (DPPH), inhibitory activity including inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), lipoxygenase (LOX), α-glucosidase. Micellar solubilization of the metal complexes has been studied in sodium dodecyl sulphate (SDS) by UV-Vis spectroscopy and conductivity. The selected complexes of nickel, zinc and cobalt have demonstrated interaction with SDS, and the value of critical micellar concentration increased in all cases.

Full text of DOI:10.1134/S107036322008023X

ISSN 1070-3632, Russian Journal of General Chemistry, 2020, Vol. 90, No. 8, pp. 1533–1543. © Pleiades Publishing, Ltd., 2020.
A Swift One-Pot Solvent-Free Synthesis
of Benzimidazole Derivatives and Their Metal Complexes:
Hydrothermal Treatment, Enzymatic Inhibition,
and Solubilization Studies
M. B. Taja,b,*, A. Raheelc, W. Alelwanid, D. Hajjard, A. Makkid, A. M. Alnajeebid,
N. A. Babteend, S. A. Tırmizic, and S. Noore,**
a Department of Chemistry, Islamia University, Bahawalpur, 63100 Pakistan
b Department of Chemistry, University of Sahiwal, Sahiwal, 57000 Pakistan
c Department of Chemistry, Quaid-e-Azam University, Islamabad, 44000 Pakistan
d Department of Biochemistry, College of Science, University of Jeddah, Jeddah, 80203 Saudi Arabia
e Department of Chemistry, University of Agriculture, Faisalabad, 38000 Pakistan
e-mail: *drbabartaj@gmail.com; **sadiaa613@gmail.com
Received May 31, 2020; revised July 20, 2020; accepted August 5, 2020
Abstract—Three benzimidazole derivatives, 1-(1H-benzimidazol-2-yl)ethanol (HBE), 1H-benzimidazol-
2-yl(diphenyl)methanol (BDM) and 1,2-bis(1H-benzimidazol-2-yl)ethane-1,2-diol (BHBED), have been synthe-
sized following the one-pot rapid green protocol. Complexes of benzimidazole derivatives with six 3d transition
metals, Cu(II), Mn(II), Zn(II), Fe(II), Co(II), and Ni(II), have been synthesized by free hydrothermal method.
The synthesized products have been characterized by FTIR, 1H, and 13C NMR, and mass spectroscopy, and CHN
analysis, and 2:1 ligand to metal stoichiometry has been confirmed. The synthesized ligands and metal complexes
have been tested for antioxidant potential (DPPH), inhibitory activity including inhibition of acetylcholinesterase
(AChE), butyrylcholinesterase (BChE), lipoxygenase (LOX), α-glucosidase. Micellar solubilization of the metal
complexes has been studied in sodium dodecyl sulphate (SDS) by UV-Vis spectroscopy and conductivity. The
selected complexes of nickel, zinc and cobalt have demonstrated interaction with SDS, and the value of critical
micellar concentration increased in all cases.
Keywords: anionic surfactant, benzimidazole derivatives, hydrothermal treatment, inhibition activity, micellar
solubilization, 3d metal complexation
DOI: 10.1134/S107036322008023X
INTRODUCTION
studies of their complexes biological and enzymatic
activities [9]. However, synthetic approaches to such
compounds are retarded by stringent reaction conditions,
multistep procedures, low yields, and unavoidable
side reactions. Therefore, search for efficient synthetic
approaches to such compounds is still of considerable
importance.
Benzimidazole drivatives have been explored as
poisons in non-nucleoside topoisomerase-I and inhibitors
of reverse transcriptase against HIV-1 and DNA-gyrase
[1]. Benzimidazoles can act as ligands in binding with
transition metals for experimental modelling in the
biological systems [2–4]. Numerous routes to their
synthesis, including the coupling of o-phenylenediamines
with carboxylic acids [5], aldehydes [6] and o-esters under
catalysis by Lewis acids [7] are reported. Phthalic acid
attached to polymer-supported 4-fluoro-3-nitrobenzoic
acid and polyethylene glycol ether in solid phase has been
reported as a precursor for 2-substituted benzimidazoles,
benzoxazoles and benzothiazoles [8]. High selectivity of
transition metals, especially 3d, 4d, and 5d, stimulated
The structural similarities of micelles and biological
membranes allow the former ones to be used as substitute
models for in vitro study of drug-membrane interactions
[10–14]. Some valuable information on solubilization of
transition metal complexes with nitrogen-donor ligands
has been presented [15–19].
Herein, we present the green one-pot solvent-free
synthesis of benzimidazole derivatives, their complexation
with different 3d metals and some biological properties.
1533

1534
TAJ et al.
Scheme 1.
RESULTS AND DISCUSSION
because of proton removal from the amino group of
ligands as confirmed by the presence of singlets at 3350,
3360, and 3360 cm–1 for HBE, BDM, and BHBED,
respectively. Formation of ligands was confirmed by
C–N peaks at 1655, 1630, and 1630 cm–1, respectively.
The bands of O–H were not recorded in the spectra of
complexes indicating involvement of oxygen in the
complexation. The bands of C–N of the complexes were
The pure ligands L1–L3 were synthesized by a highly
efficient one-pot solvent-free method (Scheme 1). Their
structural and physical characteristics were in close
agreement with those reported for the known compounds.
FTIR spectra confirmed formation of the ligands and
their complexes. The doublet of NH2 at 3450 cm–1 in
the spectrum of o-phenylenediamine was not recorded
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A SWIFT ONE-POT SOLVENT-FREE SYNTHESIS
1535
Table 1. Biological activities of the synthesized ligands and their complexes
DPPH Radical Scavenging activity
Sample Ligand–complex Inhibition, %
IC50
Sample Ligand–complex Inhibition, %
IC50
L1
1d
1e
2c
HBE
(HBE)2Fe
(HBE)2Co
87.02±0.71
85.23±0.25
86.85±0.62
104.11±0.11
81.71±0.18
141.81±0.16
(BDM)2Zn
Control
72.89±0.11
Quercetin
208.91±0.41
16.96±0.14
Acetylcholinesterase (AChE) inhibition activity
L1
L2
1a
1c
1f
2b
2d
2f
HBE
BDM
(HBE)2Cu
(HBE)2Zn
(HBE)2Ni
(BDM)2Cu
61.81±0.71
56.45±0.65
51.93±0.45
41.71±0.18
70.63±0.44
56.11±0.25
192.61±0.21
212.61±0.71
423.11±0.32
423.11±0.15
183.51±0.34
212.91±0.33
(BDM)2Mn
(BDM)2Fe
(BDM)2Ni
(BHBED)2Mn
Control
41.71
±
0.18
423.11±0.15
427.31±0.1
145.71±0.11
409.11±0.14
0.04±0.01
53.60±0.32
77.34±0.34
55.61±0.01
Eserine
3b
2a
Butyrylcholinesterase (BChE) inhibition activity
1f
2f
3c
(HBE)2Ni
(BDM)2Ni
64.61±0.11
60.50±0.21
238.21±0.44
278.91±0.34
(BHBED)2Zn
Control
82.09±0.31
Eserine
186.51±0.05
0.85±0.001
Lipoxygenase (LOX) inhibition activity
L3
1b
3d
3e
3f
BHBED
(HBE)2Mn
(BHBED)2Fe
53.71±0.35
59.89±0.1
51.86±0.28
412.31±0.11
376.11±0.58
<400
(BHBED)2Co
(BHBED)2Ni
Control
49.24±0.41
29.30±0.34
Baicalein
–
–
22.4±1.3
α-Glucosidase activity
2c
3c
(BDM)2Zn
(BHBED)2Zn
70.49±0.82
81.33±0.98
134.20±0.25
244.57±0.74
Anti-urease activity
3b
Control
Acarbose
36.20±0.05
1c
2c
(HBE)2Zn
(BDM)2Zn
77.79±0.82
75.49±0.84 134.3±60.56
61.34±0.25
(BHBED)2Mn
Control
86.12±0.98
Acarbose
244.57±0.74
21.25±0.15
recorded in lower field region 1429–1547 cm–1 than those
characterized by the higher inhibition activity than the
other complexes and ligands. The complex (HBE)2Mn
and the complexes of ligand BHBED with Fe, Co and Ni
were the most potent inhibitors of lipoxygenase (LOX).
The highest α-glucosidase activity was determined for the
complexes (BDM)2Zn, BHBED)2Mn and BHBED)2Zn,
whereas promising results of anti-urease activity were
achieved with the complexes (HBE)2Zn, (BDM)2Zn and
(BHBED)2Mn.
of the ligands (1655–1630 cm–1).
1H NMR and mass spectra clearly supported formation
of the ligands and their corresponding complexes.
Biological tests of the synthesized compounds.
The ligands and their metal complexes were screened
for biological activities including antioxidant DPPH
activity, inhibition of acetylcholinesterase (AChE),
butyrylcholinesterase (BChE), lipoxygenase (LOX),
α-glucosidase, and urease [20–27]. According to the
initial biological tests, the metal complexes demonstrated
higher biological potential than their ligands due to
availability of metal-ligand bond for the corresponding
processes of inhibition (Table 1).
Electrical conductivity. Values of CMC (critical
micellar concentration) and other thermodynamic
parameters were studied in the presence of metal
complexes in the micellar media. Gradual dilution led
to noticeable changes in the conductivity. The effect
of temperature changes on micellization and CMC of
surfactants on its interaction with metal complexes was
tested (Fig. 1). The CMC was calculated at the intersection
of two straight lines in the plots [15–19, 28–31]. The
presence of metal complexes led to an increment in CMC
with increasing temperature of the medium.
The ligand HBE and its complexes with iron, cobolt
and nickel were characterized by the highest antioxidant
potential in DPPH radical scavenging assay. In the case
of acetylcholinesterase (AChE) inhibition, the complexes
(HBE)2Cu, (BDM)2Mn and (BHBED)2Mn exhibited the
highest activity as compared to other counterparts. In
terms of butyrylcholinesterase (BChE) inhibition, the
complexes (HBE)2Ni, (BDM)2Ni and (BHBED)2Zn were
The thermodynamic parameters summarized in Table 2
clearly indicated the significant interaction between
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TAJ et al.
Fig. 1. Graphical representation of electrical conductivity at various temperatures [(1) 45, (2) 35, (3) 25°C]. (a) (HBE)2Ni, (b) (BDM)2Ni,
(c) (BHBED)2Ni, (d) (BDM)2Zn, (e) (BHBED)2Zn, and (a) (HBE)2Co.
complex and micelles of SDS at different temperatures.
The process was entropy-driven as confirmed by the
positive values of ∆Sm due to transfer of hydrophobic tails
to core micellar region from aqueous medium. Significant
hydrogen bonding around the non-polar tail ends
differentiated hydrophobic hydration and conventional
solute-solvent interactions [15–19]. Screening of
metal complexes (1.5 mmol) [(HBE)2Ni; (BDM)2Ni;
(BHBED)2Ni; (BDM)2Zn; (BHBED)2Zn; [(HBE)2Co]
was carried out at the micellar concentration of SDS equal
to 8.2 mmol. The similar trend was observed in the study
of UV-Vis absorbance (Table 2).
of SDS due to gradual incorporation of the complex
within micelles, and the critical micellar concentration
increased in all cases. The bathochromic shift indicated
interaction and association of nickel complexes with the
surfactant [15–19].
The actual starting point of micellization was hard to
standardize but this change could be measured by means
of some physical parameters such as UV-Vis absorbance,
electrical conductivity, surface tension, light scattering,
and solubilization. These physical properties could be
used for approximating the value of CMC [9–19, 28–31]
(Table 3).
UV-Vis spectra. Interaction of metal complexes with
surfactant SDS was studied by UV-Vis spectroscopy and
its differential version. The values of λmax of aqueous
solutions of complexes were recorded in the presence
and absence of the surfactant and then plotted against the
concentration of SDS (Fig. 2). The extent of interaction
of complexes with SDS was assessed by the shift of λmax
in the spectra. It was speculated that the prevalence of
electrostatic and hydrophobic interactions could cause
incorporation of molecules of metal complexes in the
proximity of micelles of SDS. The value of absorbance
gradually increased upon the increase of concentration
Increase of differential absorption was growing with
SDS concentration and indicated constant incorporation
of metal complex molecules in the micellar system. In
the presence of (HBE)2Ni the CMCs of SDS shifted from
8.2 to 10.1 mmol, similarly (BDM)2Ni increased CMC
to 10.51 mmol, (BHBED)2Ni to 10.57 mmol; (BDM)2Zn
to 9.26 mmol; (BHBED)2Zn to 9.55 mol, and (HBE)2Co
to 10.03 mmol. Penetration of metal complexes into SDS
micelles led to higher values of CMC in comparison with
pure SDS.
The intermolecular hydrogen bond between the units
of metal complexes and water molecules led to adsorption
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A SWIFT ONE-POT SOLVENT-FREE SYNTHESIS
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Table 2. Micellar and thermodynamic parameters determined for the selected metal complex in SDS
Complex
(HBE)2Ni
T, °C
25
35
CMC, mmol
10.00
∆Gm, kJ/mol
–39.814
–39.962
∆Hm, kJ/mol
–3.49
∆Sm, J mol–1 K–1
121.91
β
0.14
0.19
10.55
–3.61
118.04
45
25
35
45
25
35
45
25
35
45
25
35
45
25
35
45
11.53
10.42
11.02
12.16
10.59
10.51
11.53
9.45
–40.473
–36.116
–35.568
–37.491
–37.060
–33.010
–34.940
–34.420
–24.330
–29.020
–36.350
–35.310
–35.870
–37.730
–35.070
–33.760
–3.88
–9.98
–10.40
–10.70
–5.35
–5.10
–5.24
–5.99
–5.11
–4.70
–3.16
–3.27
–3.40
–11.40
–10.40
–11.90
115.07
88.24
81.74
84.16
106.43
90.62
93.40
95.40
62.39
76.49
1011.36
104.04
105.26
88.26
79.97
68.61
0.18
0.30
0.32
0.37
0.25
0.44
0.5
0.40
0.72
0.90
0.31
0.36
0.40
0.23
0.49
0.38
(BDM)2Ni
(BHBED)2Ni
(BDM)2Zn
(BHBED)2Zn
(HBE)2Co
10.01
10.46
9.52
9.96
11.00
10.12
11.98
12.04
Table 3. Solubilization parameters for selected metal complex in the micellar media of SDS
Complex
(HBE)2Ni
Kx, dm3/mol
3920
∆Gp, kJ/mol
–20.510
–19.376
–23.550
–18.770
–6.010
Kb, dm3/mol
110.0
12.50
6.67
31.40
125.00
476.00
∆Gb, kJ/mol
–11.65
–9.81
–4.71
–8.54
–11.96
–15.22
(BDM)2Ni
(BHBED)2Ni
(BDM)2Zn
(BHBED)2Zn
(HBE)2Co
2490
13400
1950
11.3
332
–24.330
of complexes in the peripheral region of SDS micelles.
The change in adsorption of metal complexes upon their
binding with the surfactant micelles helped to calculate
the binding and partition constants (Kb and Kx) [15–19].
the partitioning coefficient, the greater value indicated
their location in the outer region of the micelle and the
molecule having the lowest value concentrated close to
the core region (Scheme 2).
Comparative interaction of selected metal
complexes with SDS. UV-Vis spectroscopy and electrical
conductivity data demonstrated the close agreement
with the CMC in all cases. The highest partitioning
constant (13400 dm3/mol) of (BHBED)2Ni/SDS system
among all other complexes indicated its highest extent
of partitioning between aqueous and micellar medium.
On the contrary, the binding constant of (HBE)2Co/SDS
system (476 dm3/mol) indicated its lowest partitioning
tendency. Therefore, (BHBED)2Ni complex molecules
were positioned near the peripheral region of micelles,
while all other complexes were distributed in the other
areas of micelles. The highest negative value ∆Gp
(–24.33 kJ/mol) of (BHBED)2Ni/SDS system exhibited
its stability and spontaneity of the process. Position
of the solubilizates in the micelles was determined by
EXPERIMENTAL
All chemicals were purchased from the authorized
chemical suppliers and used without additional
purification. Elemental analysis was carried out on a
Perkin-Elmer elemental analyzer. FTIR spectra were
recorded on a Bio-Rad Merlin spectrophotometer in
KBr disc. H and 13C NMR spectra were measured
1
on a Bruker AM-250 spectrometers using CDCl3 as
a solvent and TMS as an internal reference. UV-Vis
absorption was recorded on a Perkin-Elmer Lambda-25
spectrophotometer [32,33]. The absorbance of biological
assays was recorded on a Synergy HT microplate reader.
Synthesis of ligands L1–L3. The equimolar amounts
of o-phenylenediamine and carboxylic acids (C1–C3)
were ground with a pestle in a mortar at room temperature
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TAJ et al.
Fig. 2. UV-Vis absorbance of selected metal complexes in SDS. (I) Simple absorbance and (II) differntial absorbance; (a) (HBE)2Ni,
(b) (BDM)2Ni, (c) (BHBED)2Ni, (d) (BDM)2Zn, (e) (BHBED)2Zn, and (f) (HBE)2Co.
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A SWIFT ONE-POT SOLVENT-FREE SYNTHESIS
1539
Scheme 2.
N
H
H
HO
CH3
H
O
N
N
N
H
CH3
CH3
N
N
N
O
H
N
Ni
Ni
Ni
Ni
O
O
H
CH3
Ni
H
N
Ni
N
CH3
O
O
N
N
N
H
N
N
H
OH
N
CH3
H
H
HO
N
H
N
N
N
H
CH3
CH3
H
O
CH3
N
N
N
O
H
N
Zn
Zn
O
Co
Zn
H
N
Co
Zn
CH3
O
O
H
CH3
N
O
N
N
N
N
H
N
H
N
H
OH
CH3
Interaction of Selected complexes with SDS
Na+
OSO3Na
Na+
Na+
Na+
SO3
SO3
SO3
O
O
O
SO3
Na+
O
O
SO3
Na+
Ni
O
SO3
O
Na+
Zn
Ni
SO3
O
O
Na+
Zn
Na+
SO3
SO3
Co
O
Ni
SO3
O
Na+
SO3
Na+
O
SO3
O
SO3
O
O
O
Na+
Na+
SO3
SO3
SO3
Na+
Na+
Na+
until the mixture melt. The molten mixture was heated at
140°C for ca. 1–2 h. Formation of ligands was monitored
by TLC. Upon completion of the reaction, the molten
mixtures were washed, and the products were purified
in cold water.
0.8 (30% EtOAc–hexane). IR spectrum, ν, cm–1: 3470
(N–H), 3050 (O–H), 1658 (C=N). H NMR spectrum,
δ, ppm: 2.9 (CH3), 5.1 (O–H), 6.9–7.0 (phenyl), 8.84
(N–H). 13С NMR spectrum, δС, ppm: 25, 71, 100, 110,
118, 126, 128, 138, 149, 154. Found, %: C 66.40; 5.79;
N 17.10. C18H18N4O2. Calculated, % C 67.07; H 5.63;
N17.38. MS: m/z: 322 [M]+.
1
1-(1H-Benzimidazol-2-yl)ethanol (L1). White
amorphous solid, yield 65%, mp 184–186°C, Rf 0.7 (50%
EtOAc–hexane). IR spectrum, ν, cm–1: 3400 (N–H),
Synthesis of metals complexes. The mixture of 25 mL
of aqueous solution of a synthesized ligand (L1–L2–L3,
1 equiv) with 25 mL of an aqueous solution of a metal
salt (Cu2+–Mn2+–Zn2+–Fe2+–Co2+–Ni2+; 2 equiv) was
heated in Teflon lined stainless steel autoclave at 100°C.
Upon completion of the process, the product was filtered
off, washed with distilled water and purified by ethanol.
It was impossible to produce crystals appropriate for
X-ray analysis.
1
3235 (O–H), 1655 (C=N). H NMR spectrum, δ, ppm:
4.3 (O–H), 6.9–7.5 (phenyl), 10.7 (N–H). 13С NMR
spectrum, δС, ppm: 25, 70, 120, 135, 156. Found, %: C
64.92; H 6.01; N 16.92, C9H10N2O. Calculated, %: C
66.65; H 6.21; N 17.27. MS: m/z: 162 [M]+.
1H-Benzimidazol-2-yl(diphenyl)methanol (L2).
Light blue amorphous solid, yield 83%, mp 210–211°C,
Rf 0.8 (50% EtOAc–hexane). IR spectrum, ν, cm–1: 3410
1
(N–H), 3250 (O–H), 1645 (C=N). H NMR spectrum,
(HBE)2Cu (1a). Light grey solid, yield 78%, mp 245–
247°C. IR spectrum, ν, cm–1: 3300 (N–H), 1660 (C=N).
Found, %: C 57.10; H 4.40; N 13.45. C18H18N4O2Cu.
Calculated, %: C 56.02; H 4.70; N 14.52.
δ, ppm: 4.6 (N–H), 5.2 (O–H), 6.9–7.6 (phenyl). 13С
NMR spectrum, δС, ppm: 102, 118, 119, 124, 135, 142,
147. Found, %: C, 80.01; H, 5.30; N, 9.50. C20H16N2O.
Calculated, %: C 79.98; H 5.37; N 9.33. MS: m/z: 300
[M]+.
(HBE)2Mn (1b). Off white solid, yield 70%, mp 235–
236°C. IR spectrum, ν, cm–1: 3330 (N–H), 1662 (C=N).
Found, %: C 58.36; H 5.10; N 13.92. C18H18N4O2Mn.
Calculated, %: C 57.30; H 4.81; N 14.85.
1,2-Bis(1H-Benzimidazol-2-yl)ethane-1,2-diol (L3).
Grey amorphous solid, yield 75 %, mp 189–191°C, Rf
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TAJ et al.
(HBE)2Zn (1c). White solid, yield 65%, mp 225–
(BDM)2Ni (2f). Green solid, yield 76%, mp 245–
247°C, Rf 0.8 (50% EtOAc–hexane). IR spectrum, ν,
cm–1: 3357 (N–H), 1659 (C=N). 1H NMR spectrum, δ,
ppm: 7.5–7.9 (phenyl), 8.3 (N–H). 13С NMR spectrum,
δС, ppm: 92, 113, 117, 128, 135, 146, 157. Found, %: C
68.12; H 4.39; N 7.51. C40H30N4O2Ni. Calculated, %: C
67.09; H 4.22; N 7.82.
227°C. IR spectrum, ν, cm–1: 3300 (N–H), 1665 (C=N).
1H NMR spectrum, δ, ppm: 7.5–8.9 (phenyl), 9.3 (N–H).
13С NMR spectrum, δС, ppm: 20, 65, 121, 125, 139, 156.
Found, %: C 56.85; H 4.98; N 13.80. C18H18N4O2Zn.
Calculated, %: C 55.76; H 4.68; N 14.45.
(HBE)2Fe(1d). Brownish black solid, yield 54%,
mp 250–253°C. IR spectrum, ν, cm–1: 3310 (N–H),
1657 (C=N). Found, %: C 58.22; H 4.48; N 14.70.
C18H18N4O2Fe. Calculated, %: C 57.16; H 4.80; N 14.81.
(BHBED)2Cu (3a). Bluish green solid, yield 75%, mp
221–223°C, Rf 0.7 (50% EtOAc–hexane). IR spectrum,
ν, cm–1: 3347 (N–H), 1645 (C–N). Found, %: C 60.15; H
4.81; N 16.01. C36H34N8O4Cu. Calculated, %: C 61.22;
H 4.85; N 15.87.
(HBE)2Co (1e). Light pink solid, yield 59%, mp 241–
243°C. IR spectrum, ν, cm–1: 3340 (N–H), 1665 (C=N).
Found, %: C 56.60; H 4.50; N 14.80. C18H18N4O2Co.
Calculated, %: C 56.70; H 4.76; N 14.69.
(BHBED)2Mn (3b). Light pink, yield 69%, mp
220–223°C, Rf 0.73 (50% EtOAc–hexane). IR spectrum,
ν, cm–1: 3353 (N–H), 1665 (C–N). Found, %: C 61.50; H
4.99; N 16.45. C36H34N8O4Mn. Calculated, %: C 61.98;
H 4.91; N 16.06.
(HBE)2Ni (1f). Green solid, yield 70%, mp 209-
210°C. IR spectrum, ν, cm–1: 3352 (N–H), 1658 (C=N).
1H NMR spectrum, δ, ppm: 6.5–6.9 (phenyl), 10.45
(N–H). 13С NMR spectrum, δС, ppm: 23.4, 72, 127,
136, 145, 147. Found, %: C 57.20; H 5.20; N 13.95.
C18H18N4O2Ni. Calculated, %: C 56.74; H 4.76; N 14.70.
(BHBED)2Zn (3c). White, yield 83%, mp 235–237°C,
Rf 0.66 (50% EtOAc–hexane). IR spectrum, ν, cm–1:
1
3360 (N–H), 1670 (C=N). H NMR spectrum, δ, ppm:
2.9 (N–H), 6.1 (O–H), 7.1–7.9 (phenyl), 9.3 (N–H), 10.1
(N–H). 13С NMR spectrum, δС, ppm: 25, 57, 68, 110,
123, 125, 129, 132, 136, 147, 159. Found, %: C 60.62; H
4.79; N 15.67. C36H34N8O4Zn. Calculated, %: C 61.06;
H 4.84; N 15.82.
(BDM)2Cu (2a). Greyish black solid, yield 70%, mp
255–257°C, Rf 0.9 (50% EtOAc–hexane). IR spectrum,
ν, cm–1: 3351 (N–H), 1650 (C–N). Found, %: C 72.55;
H 4.55; N 8.41. C40H30N4O2Cu. Calculated, %: C 72.60;
H 4.57; N 8.46.
(BHBED)2Fe (3d). Dark red solid, yield 65%, mp
248–251°C, Rf 0.83 (50% EtOAc–hexane). IR spectrum,
ν, cm–1: 3349 (N–H), 1654 (C–N). Found, %: C 62.01; H
4.82; N 16.41. C36H34N8O4Fe. Calculated, %: C 61.90;
H 4.91; N 16.04.
(BDM)2Mn (2b). Off white solid, yield 65%, mp
217–220°C, Rf 0.6 (50% EtOAc–hexane). IR spectrum,
ν, cm–1: 3370 (N–H), 1657 (C–N). Found, %: C 72.40;
H 4.64; N 8.55. C40H30N4O2Mn. Calculated, %: C 72.55;
H 4.57; N 8.46.
(BHBED)2Co (3e). Pink solid, yield 69%, mp 253–
254°C, Rf 0.58 (50% EtOAc–hexane). IR spectrum, ν,
cm–1: 3348 (N–H), 1656 (C–N). Found, %: C 60.70; H
4.93; N 15.97. C36H34N8O4Co. Calculated, %: C 61.63;
H 4.88; N 16.36.
(BDM)2Zn (2c). White solid, yield 73%, mp 230–
232°C, Rf 0.85 (50% EtOAc–hexane). IR spectrum, ν,
cm–1: 3345 (N–H), 1652 (C=N). 1H NMR spectrum, δ,
ppm: 7.5–8.7 (phenyl), 9.9 (N–H). 13С NMR spectrum,
δС, ppm: 23, 83, 117, 124, 127, 138, 158. Found, %: C
72.24; H 4.50; N 8.65. C40H30N4O2Zn. Calculated, %: C
72.35; H 4.55; N 8.44.
(BHBED)2Ni (3f). Greenish black solid, yield
73%, mp 250–253°C, Rf 0.6 (50% EtOAc–hexane).
1
IR spectrum, ν, cm–1: 3343 (N–H), 1668 (C=N). H
(BDM)2Fe (2d). Pink solid, yield 60%, mp 236–
238°C, Rf 0.62 (50% EtOAc–hexane). IR spectrum, ν,
cm–1: 3353 (N–H), 1660 (C–N). Found, %: C 73.42; H
4.44; N 8.77. C40H30N4O2Fe. Calculated, %: C 73.40; H
4.62; N 8.56.
NMR spectrum, δ, ppm: 2.6 (N–H), 6.3 (O–H), 6.8–8.1
(phenyl), 10.3 (N–H), 11.6 (N–H). 13С NMR spectrum,
δС, ppm: 23, 49, 56, 98, 112, 119, 126, 129, 134, 136,
138, 151, 156. Found, %: C 60.89; H 4.72; N 15.46.
C36H34N8O4Ni. Calculated, %: C 61.63; H 4.88; N 15.97.
(BDM)2Co (2e). Orange solid, yield 71%, mp 247–
248°C, Rf 0.58 (50% EtOAc–hexane). IR spectrum, ν,
cm–1: 3362 (N–H), 1633 (C–N). Found, %: C 73.18; H
4.73; N 8.60. C40H30N4O2Co. Calculated, %: C 73. 06;
H 4.60; N 8.52.
Biological evaluation of metal complexes. DPPH
antioxidant activity. The appropriate amount of a
compound was dissolved in the solvent (10 μL), and
90 μL of 100 μM methanolic DPPH solution were added
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A SWIFT ONE-POT SOLVENT-FREE SYNTHESIS
1541
to make a final volume of 100 μL in 96-well plates.
The mixtures were thoroughly mixed and incubated for
30 min at 37°C and their absorbance was determined
at 517 nm on a microplate reader. L-Ascorbic acid and
quercetin were used as the standard antioxidants [20,
21]. IC50 values were calculated using EZ-Fit5 Perrella
Scientific Inc. Amherst US software.
α-Glucosidase inhibition activity. 100 μL of the
reaction mixture containing 70 μL of phosphate buffer
(50 mM; pH 6 .8) was used in this assay. The enzyme
(10 μL; 0.057 units) was added to 10 μL (0.5 mM) of
the test sample and incubated at 37°C for ca 10 min.
Absorbance was measured at 400 nm. The substrate
“p-nitrophenyl glucopyranoside” (10 μL; 0.5 mM) was
then transferred to the reaction mixture to activate the
reaction. Acarbose was used as a positive control. After
incubating the reaction mixture for 30 min at 37°C, its
absorbance was measured again to calculate percent
inhibition [26].
Acetylcholinesterase (AChE) inhibition activity.
Acetylcholinesterase inhibition was studied using a
total of 100 μL of the reaction content, which contained
60 μL of sodium phosphate buffer (50 mM; pH 7.7). The
reaction mixture ca 10 μL of the metal complex (0.5 mM
per well) was mixed with 10 μL of enzyme. Absorbance
was measured at 405 nm and then pre-incubated for
10 min at 37°C. The reaction was initiated with 10 μL
of acetylthiocholine iodide (0.5 mM/well) and incubated
for 15 min at 37°C. Post-incubation absorbance of the
reaction mixture was recorded at 405 nm. Triplicate
experiments were carried out with eserine (0.5 mM/well)
used as a positive control [22–24].
Anti-urease activity. The 85 μL of the reaction
mixture was mixed with 10 μL of sodium phosphate
buffer (pH 7.0), and it was added to 96-well plate.
The sample solution (10 μL) and the enzyme solution
(25 μL; 0.134 units) were added subsequently. The
reaction mixture was pre-incubated for 5 min at 37°C, then
40 μL of urea stock solution (20 mM) was transferred
into wells and incubated for an additional 10 min at the
same temperature. Each well was loaded with 115 μL
of freshly prepared phenol hypochlorite (45 μL phenol
reagent mixed in 70 μL of alkali reagent). The reaction
content was further incubated under the same conditions
for 10 min, the colour and absorbance were measured at
625 nm. The urease inhibition was computed [27].
Butyrylcholinesterase (BChE) inhibition activity.
Butyrylcholinesterase (BChE) inhibition was studied
using 100 μL of the reaction mixture, which contained
60 μL of sodium phosphate buffer (50 mM; pH 7.7).
The reaction mixture (ca 10 μL of the metal complex,
0.5 mM per well) was mixed with 10 μLof enzyme BChE.
Absorbance of the content was measured at 405 nm, and
then it was pre-incubated at 37°C for 10 min. The reaction
was initiated with 10 μL of butyrylthiocholine chloride
(0.5 mM/well), followed by addition of 10μL DTNB
and then incubated for 15 min at 37°C. Post-incubation
absorbance was also measured at 405 nm. Triplicate
experiments were performed with eserine (0.5 mM/well)
used as a positive control, and the percent inhibition was
calculated [24].
Solubilization of synthesized complexes in SDS.
The micellar solubilization of metal complexes with
SDS was studied in aqueous solutions of metal
complexes in deionized water for their conductometric
and spectroscopic measurements [28–31]. For initial
screening, the metal complexes were divided into three
groups in accordance with the nature of the corresponding
ligands 1a–1f, 2a–2f, 3a–3f.
Electrical conductivity. Electrical conductivity
measurements were recorded on a Hanna-Cond HI-99301
(capacity 0.01–199.9 mS) equipped with a platinum
black electrode to subside polarization. Calibration of
the electrode was carried out with KClaq over a certain
range of SDS concentration. Measurements of specific
conductivities were accomplished at three different
temperatures with 10°C increment (25–45°C). All
measurements of electrical conductivity were carried out
in pre-micellar to post-micellar surfactant concentrations
(7–15 mmol; CMC of pure SDS at 25°C, 8.2 mmol)
[15–19, 34, 35]. Thermodynamic and other micellar
parameters were derived from electrical conductivity data.
Lipoxygenase (LOX) inhibition activity. Lipoxygenase
inhibition was studied by using 200 μL of the reaction
mixture containing 150 μL of sodium phosphate buffer
(100 mM; pH 8.0), 10 μL of the test complexes and
15 μL of purified lipoxygenase enzyme (600 units per
well). Absorbance of the reaction content was measured
at 234 nm, and it was incubated at 25 °C for 10 min,
followed by addition of substrate solution (25 μL) to
initiate the reaction.Absorbance was measured again after
6 min of incubation at 234 nm. The assay was performed
in triplicate with baicalin used as a positive control. The
percent inhibition of samples was calculated [25].
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1542
TAJ et al.
UV-Vis spectra. In the first step, aqueous primary
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