Biological evaluation the 2-aryl-2,3-dihydrobenzodiazaborinin-4(1H)-ones as potential dual α-glucosidase and α-amylase inhibitors with antioxidant properties

Article abstract of DOI:10.1111/cbdd.13893

The 2-aryl-2,3-dihydrobenzodiazaborinin-4(1H)-ones (azaborininone) were synthesized as analogues of the 2-arylquinazoline-4-ones and screened through enzymatic assay in vitro for inhibitory effect against α-glucosidase and α-amylase activities. These azaborininones exhibited moderate to good inhibitory effect against these enzymes compared to acarbose used as a reference standard. The results are supported by the enzyme-ligand interactions through kinetics (in vitro) and molecular docking (in silico) studies. The test compounds also exhibited significant antioxidant activity through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) free radical scavenging assays. These azaborininone derivatives exhibited no effect on the viability of the human lung cancer (A549) cell line after 24?hr and were also not toxic towards the Vero cells.

Full text of DOI:10.1111/cbdd.13893

Article type : Research Article
Biological evaluation the 2-aryl-2,3-dihydrobenzodiazaborinin-4(1H)-ones as potential dual
α-glucosidase and α-amylase inhibitors with antioxidant properties
Malose J. Mphahlele,1* Nontokozo M. Magwaza,1 Sibusiso T. Malindisa2 and Yee Siew Choong3*
1 Department of Chemistry, College of Science, Engineering and Technology, University of South
Africa, Private Bag X06, Florida 1710, South Africa. E-mail: magwan@unisa.ac.za (NMM)
2 Department of Life & Consumer Sciences, College of Agriculture and Environmental Sciences,
University of South Africa, Private Bag X06, Florida1710, South Africa. E-mail:
malinst@unisa.ac.za (STM)
3 Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang
11800, Malaysia
* Authors for correspondence: mphahmj@unisa.ac.za (MJM) and yeesiew@usm.my (YSC)
Abstract: The 2-aryl-2,3-dihydrobenzodiazaborinin-4(1H)-ones (azaborininone) were synthesized
as analogues of the 2-arylquinazoline-40ones and screened through enzymatic assay in vitro for
inhibitory effect against α-glucosidase and α-amylase activities. These azaborininones exhibited
moderate to good inhibitory effect against these enzymes compared to acarbose used as a reference
standard. The results are supported by the enzyme-ligand interactions through kinetics (in vitro)
and molecular docking (in silico) studies. The test compounds also exhibited significant
antioxidant activity through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) free
radical scavenging assays. These azaborininone derivatives exhibited no effect on the viability of
the human lung cancer (A549) cell line after 24 h, and were also not toxic towards the Vero cells.
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Keywords:
2-Aryl-2,3-dihydrobenzodiazaborinin-4(1H)-ones;
α-glucosidase;
α-amylase;
antioxidant; toxicity; drug-receptor interactions
1. Introduction
The synthesis of boron-based heteroaromatic compounds has fascinated synthetic chemists for
some time to understand their aromaticity, and to expand molecular diversity particularly through
developments in the Suzuki–Miyaura coupling reactions (Churches & Hutton, 2016). The situation
has since changed because of the emergence of boron-based drugs that show unique modes of
inhibition and activity against various biological and biochemical targets (Barker et al., 2009;
Smoum et al., 2012). Boron containing compounds exhibit unique biological properties and have
been found to be generally nontoxic (Barker et al., 2009). Replacement of covalent carbon-carbon
bonds with boron-nitrogen bonds has in many cases provided compounds with significantly
improved biological and electronic properties. Moreover, the boron center can be converted from
neutral trigonal planar (sp2) to tetrahedral (sp3) hybridization under proper physiological conditions
to form strong interactions with the receptor (Özçayan, 2019). Previous studies on the 2,1-
borazaronaphthalenes, for example, have demonstrated strong correlation of their biological
properties in vitro as inhibitors of phosphodiesterase 10A (PDE10A) (Vlasceanu et al., 2015) and
β2 receptor (Rombouts et al., 2015) compared to the reference standards used for the assays.
Benzodiazaborine scaffolds, on the other hand, serve as hydrophobic arene mimics (C=C versus
B–N bond) in a biological context (Barker et al., 2009; Davis et al., 1998; Liu et al., 2009). The
athranilamide moiety in the case of the 2-aryl-2,3-dihydrobenzodiazaborinin-4(1H)-one
(azaborininone) scaffolds (A) shown in Figure 1 has been used extensively as a protecting group in
the cross-coupling reactions, and also as an ortho-directing group in the o-C–H functionalization
reactions to afford complex arylboronic acid derivatives that would be difficult to synthesize
otherwise (Ihara et al., 2011). Although several methods for the synthesis of the 1,3,2-
diazaboracyclohexane structures (A) have been described in the literature (Davies et al., 2017;
Settepani, et al. 1970; Wang et al., 2019), only a few examples of these compounds and their N-
substituted derivatives have previously been evaluated for biological activity particularly as insect
chemosterilants, and were found to exhibit significant sterilizing activities against the house flies,
Musca domestica I., compared to the corresponding benzeneboronic acids (Settepani et al., 1970).
The molecular construct of these azaborininones resembles that of the 2-arylquinazolin-4(3H)-ones
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(B) or their 2,3-dihydroquinazolin-4(1H)-one derivatives (C), which exhibit a wide range of
biological and pharmacological properties as anti-virus, anti-bacterial, anti-inflammatory, anti-
cancer, anti-allergic, anti-fungal, anti-rheumatic, anti-convulsant, and central nervous system
(CNS) depressant activities (Hemalatha & Mahumitha, 2016). The 2-arylquinazolin-4(3H)-ones
(Javaid et al., 2015; Wei et al., 2017) and the 2,3-dihydroquinazolin-4(1H)-ones (Barmak et al.,
2019) have received extended attention as new classes of α-glucosidase inhibitors, which
complements the activity of the pancreatic α-amylase. α-Amylase plays an important role in the
early breakdown of complex carbohydrates such as large starch and glycogen molecules into
simple absorbable disaccharides and polysaccharides (Dan et al., 2019; Imran et al., 2017; Rafique
et al., 2020a). These sugars are, in turn, converted into glucose by α-glucosidase present in the
brush border surface membrane of the intestinal cells for the absorption into the blood stream in
the small intestine (Alqahtani, et al. 2020). This process results in an increased level of glucose in
blood (hyperglycemia), which is a primary indication for diabetes mellitus (T2DM). Chronic
hyperglycemia may result in oxidative stress due to the interaction of scavenger receptors, such as
RAGE, with advanced glycoxidation end-products (AGEs) formed from the non-enzymatic
glycation of proteins, lipids and nucleic acids with reducing sugars and with the products of
glucose metabolism and their oxidation products (Chetan et al., 2015). Inhibition of α-glucosidase
and/or α-amylase represents an effective strategy for the treatment of T2DM to suppress
carbohydrate digestion and delay glucose uptake to lead to reduced blood sugar levels (Alqahtani
et al., 2020; Rafique, et al. 2020b; Vieira et al., 2019). T2DM has become a serious global health
concern and in the absence of adequate interventions, this metabolic disorder may lead to death or
complications such as stroke, coronary heart disease or cancer (Rafique et al., 2020b;
Tangvarasittichai, 2015). Most of the complications associated with diabetes have been found to
result from free radical-mediated oxidative stress, due to the ability of free radicals to damage
every class of biological macromolecules, including proteins, lipids, carbohydrates and nucleic
acids (Rani et al., 2019). Targeting and inhibiting the activities of the enzymes implicated in the
pathogenesis and/or progression of T2DM such as α-amylase and/or α‐glucosidase as well as
oxidative stress is considered to represent the most effective therapeutic strategy for the treatment
of this metabolic disorder compared to combination therapies (Rafique et al., 2020a).
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O
N
O
O
H
H
H
N
C
N
N
B
or
Ar
N
H
Ar
N
H
Ar
A
B
C
Figure 1: Azaborininone (A), quinazolin-4(3H)-one (B) and 2,3-dihydroquinazolin-4(1H)-one (C)
scaffolds.
The replacement of C=C bond with isoelectronic B–N bonds has received considerable
attention since the synthesis of 1,2-azaborine in 2008 and, this strategy is being explored for
applications of organoboron derivatives in pharmaceutical and material science (Dixit et al., 2017).
We considered the structural resemblance of the 1,3,2-diazaboracyclohexane derivatives A to those
of quinoline-4-ones B, which have been found to inhibit α-glucosidase activity in vitro, and
decided to synthesize these boron-based heterocycles. The main aim was to evaluate them through
enzymatic assays in vitro for inhibitory effect against α-glucosidase and α-amylase activities. Due
to the effects of oxidative stress on the induction of diabetic complications (Dixit et al., 2017;
Rafique et al., 2020b; Tangvarasittichai, 2015) and considering the good antioxidant activities of
boric acid esters and arylboronic acid derivatives (König et al., 1988), we also evaluated the test
compounds for antioxidant potential through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the
nitric oxide (NO) free radical scavenging assays. Kinetic studies have been performed on the most
active derivative against α-glucosidase and α-amylase complemented with molecular docking (in
silico) to determine plausible protein–ligand interactions on a molecular level. Their ADME
(absorption, distribution, metabolism, and excretion) properties have also been simulated to
establish their drug-likeness at theoretical level. Since metabolic diseases require a stable long-
term therapy, well tolerated and low toxicity, the test compounds were also assayed for toxicity
against the African green monkey kidney (Vero) cells to establish their safety profile in vitro.
Moreover, their potential antiproliferative effect was assayed against the human lung cancer
(A549) cell line.
2. Materials and Methods
2.1. General considerations
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The melting point values of the test compounds were recorded on a Thermocouple digital
melting point apparatus (Mettler Toledo LLC, Columbus, OH, USA). The infrared (IR) spectra
were recorded using the thin-film method on a Bruker VERTEX 70 FT-IR Spectrometer (Bruker
Optics, Billerica, MA, USA) equipped with an ATR (diamond attenuated total reflectance)
accessory. Merck kieselgel 60 (0.063–0.200 mm) (Merck KGaA, Frankfurt, Germany) was used as
1
a stationary phase for column chromatography. The H-NMR and 13C-NMR spectra of the
prepared compounds were obtained as dimethyl sulfoxide-d6 (DMSO-d6) solutions using Agilent
500 MHz NMR spectrometer (Agilent Technologies, Oxford, UK) operating at 500 MHz and 125
1
MHz for H and 13C, respectively. The chemical shifts are quoted relative to tetramethylsilane
(TMS) used as an internal reference standard (δ = 0.00 ppm) or to residual protonated solvent. The
high-resolution mass spectra were recorded at an ionization potential of 70 eV using Micromass
Autospec-TOF (double focusing high resolution) instrument (Waters Corp., Milford, MA, USA).
α-Glucosidase type 1 from baker’s yeast (G5003), p-nitrophenyl-α-D-glucopyranoside (N1377)
and acarbose (A8980) were purchased from Sigma Aldrich (Pty) Ltd. (Modderfontein,
Johannesburg, South Africa). α-Amylase inhibitor screening kit was sourced from Biovision via
Biocom Africa (Pty) Ltd (Centurion, Pretoria, South Africa).
2.2. Typical procedure for the synthesis of the 1,3,2-diazaboracyclohexane derivatives 2a–j
A stirred mixture of anthranilamide derivative 1 (1 equiv.), phenylboronic acid derivative (1
equiv.) and p-toluenesulfonic acid (0.1% equiv.) in toluene was refluxed for 2 h under Dean-Stark
water collector conditions. The mixture was concentrated under reduced pressure and the solid
obtained was filtered, washed with cold water and recrystallized from ethyl acetate to afford 2.
Compounds 2a–j below were prepared in this fashion.
2-Phenyl-2,3-dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-one (2a)
White solid (0.55 g, 85%), mp. 209–210 °C (Lit. 206−208 °C (Davies et al., 2017), 212–213
(Wang et al., 2019), 210–211 °C (Chissick et al., 1961)); νmax (ATR) 686, 747, 1257, 1485, 1612,
1
1634, 3244, 3335 cm-1; H-NMR (DMSO-d6) 7.11 (1H, t, J = 7.5 Hz, H-7), 7.42–7.49 (4H, m,
Ar), 7.57 (1H, dt, J = 1.5 and 8.5 Hz, H-6), 8.04–8.08 (3H, m, Ar), 9.34 (1H, s, NH), 9.72 (1H, s,
13
NH); C-NMR (DMSO-d6) 120.8, 121.4, 123.4, 130.4, 130.6, 133.1, 134.9, 135.9, 136.0, 148.1,
169.0; HRMS (ES): m/z [M + H]+ calc for C13H12BN2O: 223.1043; found 223.1044.
2-(4-Fluorophenyl)-2,3-dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-one (2b)
White solid (0.45 g, 65%), mp. 252–254 °C (Lit. 252–253 °C (Wang et al., 2019)); νmax (ATR)
630, 692, 821, 1336, 1488, 1612, 1638, 3331, 3406 cm-1; 1H-NMR (DMSO-d6) 7.08 (1H t, J = 7.5
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Hz, H-6), 7.40 (1H, d, J = 7.5 Hz, H-8), 7.62 (2H, t, J = 8.5 Hz, H-3՛,5՛), 7.55 (1H, t, J = 7.5 Hz, H-
5), 8.02 (1H, d, J = 8.0 Hz, H-5), 8.17 (2H, dt, J = 5.7 Hz and J = 7.5 Hz, H-2՛,6՛), 9.32 (1H, s,
2
NH), 9.73 (1H, s, NH); 13C-NMR (DMSO-d6) 115.2 (d, JCF = 19.9 Hz), 118.6, 119.2, 121.4,
3
1
119.2, 121.3, 128.4, 129.0, 133.8, 136.3 (d, JCF = 7.6 Hz), 145.9, 164.4 (d, JCF = 246.6 Hz),
166.8; HRMS (ES): m/z [M + H]+ calc for C13H11BFN2O: 241.0948; found 241.0956.
2-(4-Chlorophenyl)-2,3-dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-one (2c)
White solid (0.67 g, 77%), mp. 255–257 °C (Lit. 156–157 °C (Wang et al., 2019)); νmax (ATR)
692, 722, 813, 1335, 1488, 1612, 1635, 3307, 3406 cm-1; 1H-NMR (DMSO-d6) 7.09 (1H, t, J = 8.0
Hz, H-6), 7.40 (1H, d, J = 8.0 Hz, H-8), 7.50 (2H, J = 8.0 Hz, H-3՛,5՛), 7.55 (1H, dt, J 1.5 and 8.5
Hz, H-5), 8.00 (1H, d, J = 7.5 Hz, H-5), 8.05 (2H, d, J = 8.5 Hz, H-2՛,6՛), 9.36 (1H, s, NH), 9.74
13
(1H, s, NH); C-NMR (DMSO-d6) 118.6, 119.3, 121.4, 128.3, 128.4, 131.5, 133.9, 135.7, 136.1,
145.8, 166.7; HRMS (ES): m/z [M + H]+ calc for C13H11BClN2O: 257.0653; found 257.0653.
2-(4-Methoxyphenyl)-2,3-dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-one (2d)
White solid (0.79 g, 82%), mp. 232–234 °C (Lit. 230.1–233.3 °C (Kamio et al., 2018)); νmax
(ATR) 752, 1179, 1273, 1569, 1640, 3299, 3359 cm-1; 1H-NMR (DMSO-d6) 3.79 (3H, s, -OCH3),
6.99 (2H, d, J = 8.5 Hz, H-3՛,5՛), 7.07 (1H, t, J = Hz, H-6), 7.41 (1H, d, J = 8.0 Hz, H-8), 7.54 (1H,
dt, J = 1.5 and 8.5 Hz, H-7), 8.01 (1H, d, J = 8.5 Hz, H-4), 8.03 (2H, d, J = 8.5 Hz, H-2՛,6՛), 9.19
(1H, s, NH), 9.61 (1H, s, NH); 13C-NMR (DMSO-d6) 55.4, 118.5, 119.1, 121.0, 124.0, 28.4, 133.8,
135.5, 146.1, 161.8, 166.8; HRMS (ES): m/z [M + H]+ calc for C14H14BN2O2: 253.1148; found
253.1148.
2-(4-Hydroxyphenyl)-2,3-dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-one (2e)
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White solid (0.88 g, 85%), mp. 242–243 °C (Lit. 232.8–236.6 °C (Kamio et al., 2018)); νmax
(ATR) 529, 821, 1354, 1488, 1612, 1640, 3306, 3406 cm-1; 1H-NMR (DMSO-d6) 6.83 (2H, d, J =
8.5 Hz, H-2՛,6՛), 7.05 (1H, t, J = 7.5 Hz, H-6), 7.40 (1H. d, J = 8.5 Hz, H-8), 7.52 (1H, dt¸J = 1.5
and 8.5 Hz, H-7), 7.90 (2H, d, J = 8/0 Hz, H-3՛,5՛), 8.00 (1H, d, J = 7.5 Hz, H-5), 9.11 (1H, s, NH),
9.51 (1H, s, NH), 9.73 (1H, s, OH); 13C-NMR (DMSO-d6) 115.4, 118.4, 119.0, 120.9, 122.3,
128.4, 133.7, 135.6, 146.2, 160.2, 166.8. HRMS (ES): m/z [M + H]+ calc for C13H12BN2O2:
239.0992; found: 239.0989.
6-Bromo-2-phenyl-2,3-dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-one (2f)
White solid (1.02 g, 76%), mp. 275–278 °C (Lit. 282–284 °C (Yale, 1971)); νmax (ATR) 529,
670, 828, 1487, 1512, 1610, 1642, 3300, 3336 cm-1; 1H-NMR (DMSO-d6) 7.40 (1H, d, J = 9.0 Hz,
H-8), 7.11–7.50 (3H, m, Ph), 7.70 (1H, dd, J = 2.5 and 9.0 Hz, H-7), 8.01 (2H, dd, J = 1.5 and 8.5
Hz, Ph), 8.06 (1H, d, J = 2.5 Hz, H-5), 9.43 (1H, s, NH), 9.84 (1H, s, NH); 13C-NMR (DMSO-d6)
112.9, 121.1, 127.8, 128.3, 130.4, 131.1, 132.3, 133.8, 134.5, 136.4, 145.1, 165.6; HRMS (ES):
m/z [M + H]+ calc for C13H11BBrN2O: 301.0145; found 301.0148.
6-Bromo-2-(4-fluorophenyl)-2,3-dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-one (2g)
White solid (0.99 g, 74%), mp. 301–302 °C; νmax (ATR) 530, 692, 1354, 1488, 1612, 1640,
3306, 3406 cm-1; 1H-NMR (DMSO-d6) 7.26 (2H. t, J = 8.5 Hz, H-2՛,6՛), 7.36 (1H, d, J = 8.5 Hz, H-
8), 7.69 (1H, dd, J = 2.5 and 9.0 Hz, H-7), 8.05 (1H, d, J = 2.5 Hz, H-5), 8.08 (2H, dt, J = 5.7 Hz
and J = 7.5 Hz, H-3՛,5), 9.43 (1H, s, NH), 9.87 (1H, s, NH); 13C-NMR (DMSO-d6) 112.9, 115.3 (d,
2JCF = 19.9 Hz), 120.9, 121.1, 128.6, 130.4, 136.3 (d, 3JCF = 7.6 Hz), 136.4, 145.0, 154.5 (d, 1JCF
=
246.5 Hz), 165.6; HRMS (ES): m/z [M + H]+ calc for C13H10BBrFN2O: 319.0054; found
319.0054.
6-Bromo-2-(4-chlorophenyl)-2,3-dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-one (2h)
White solid (0.56 g, 68%), mp. 261–263 °C; νmax (ATR) 530, 693, 722, 813, 1488, 1515,
1612, 1635, 3307, 3400 cm-1; 1H-NMR (DMSO-d6) 7.36 (1H, d, J = 8.5 Hz, H-8), 7.50 (2H, d, J =
7.5 Hz, H-2՛,6՛), 7.70 (1H, dd, J = 1.5 and 8.5 Hz, H-7), 8.02 (2H, d, J = 8.5 Hz, H-3՛,5), 8.05 (1H,
d, J = 1.5 Hz, H-5), 9.48 (1H, s, NH), 9.89 (1H, s, NH); 13C-NMR (DMSO-d6) 113.0, 121.0, 121.1,
128.4, 130.4, 135.7, 136.3, 136.5, 144.9, 165.5; HRMS (ES): m/z [M + H]+ calc for
C13H10BBrClN2O: 334.9758; found 334.9745.
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6-Bromo-2-(4-methoxyphenyl)-2,3-dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-one (2i)
White solid (1.02 g, 85%), mp. 268–270 °C; νmax (ATR) 524, 720, 821, 1212, 1489, 1605,
1
1643, 3303, 3398 cm-1; H-NMR (DMSO-d6) 6.98 (2H, J = 8.5 Hz, H-3՛,5՛), 7.37 (1H. d, J = 8.5
Hz, H-8), 7.68 (1H, dd, J= 2.0 and 8.5 Hz, H-7), 7.98 (2H, d, J = 8.5 Hz, H-2՛,6՛), 8.04 (1H, d, J=
2.0 Hz, H-5), 9.31 (1H, s, NH), 9.75 (1H, s, NH); 13C-NMR (DMSO-d6) 55.5, 112.6, 113.9, 120.8,
121.0, 123.7, 130.4, 131.2, 135.6, 136.4, 145.2, 161.9, 165.2; HRMS (ES): m/z [M + H]+ calc for
C14H13BBrN2O2: 331.0253; found 331.02490.
6-Bromo-2-(4-hydroxyphenyl)-2,3-dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-one (2j)
White solid (1.23 g, 90%), mp. 315–317 °C; νmax (ATR) 529, 692, 821, 1213, 1354, 1487,
1611, 1642, 3304, 3406 cm-1; 1H-NMR (DMSO-d6) 6.81 (2H, d, J = 7.5 Hz, H-3՛,5՛), 7.35 (1H, d, J
= 8.7 Hz, H-8), 7.67 (2H, dd, J = 1.2 and 8.7 Hz, H-6), 7.87 (2H, d, J = 7.5 Hz, H-2՛,6՛), 8.04 (1H,
13
d, J = 1.2 Hz, H-5), 9.24 (1H, s, NH), 9.66 (1H, s, NH), 9.74 (1H, s, OH); C-NMR (DMSO-d6)
112.4, 115.4, 120.7, 120.9, 122.3, 130.4, 135.7, 136.3, 145.3, 160.4, 165.6; HRMS (ES): m/z [M +
H]+ calc for C13H11BBrN2O2: 317.0097; found 317.0105.
2.3. Inhibition of α-glucosidase and α-amylase activity of 2a–j
2.3.1. α-Glucosidase inhibitory activity assay of compounds 2a–j
The α-glucosidase inhibitory activity of compounds 2a–j was assayed following a method
described in our previous study (Mphahlele et al., 2020). The stock solutions of the test compounds
(100 µM) prepared in DMSO and acarbose as positive control in DMSO were diluted with 100
mM phosphate buffer to obtain the concentrations 1, 1.5, 5, 10, 25 and 50 µM. α-Glucosidase (0,96
U/mL) was diluted to 0.48 U/mL using a 100 mM phosphate buffer of pH 6.8. The enzyme (0.48
U/mL in 100 mM phosphate buffer of pH 6.8, 17 µL) was incubated with 17 µL of varying
concentrations of the test compounds and acarbose as positive control in DMSO (1, 1.5, 5, 10, 25,
50 µM) at 37 °C for 10 min. Then 17 µL of 2 mM PNP-G was added to all the well containing
reaction mixtures to initiate the reaction. Five different absorbance readings were recorded for each
triplicate run at a wavelength of 400 nm using Varioskan flash microplate spectrophotometer
(Thermo Scientific, Waltham, MA, USA). The concentrations of the compounds that inhibited
50% of α-glucosidase activity (IC50) was determined by nonlinear regression analysis and
expressed as the mean standard deviation (SD) of three distinct experiments using graph pad prism.
2.3.2. α-Amylase inhibitory activity assay of compounds 2a–j
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The α-amylase assay was performed in triplicate using a 96-well plate following the
manufacturer’s protocol as outlined in the α-Amylase Inhibitor Screening Kit (Catalog No. K482;
Bio Vision). The stock solution (100 µM) of the test compounds (2a–j), specific α-amylase
inhibitor from Triticum aestivum included in the kit and acarbose were prepared in DMSO, and
further diluted with α-amylase assay buffer to obtain final concentrations of 1, 1.5, 5, 10, 25 and
50 µM. α-Amylase inhibitor from Triticum aestivum (10 µL) and assay buffer (40 µL) were added
to 3 wells of the 96-well microplate to represent inhibitor control. The assay buffer (50 µL) was
added to the other three wells to represent the enzyme control. The test compounds (50 µL) were
added to the remaining designated wells of the plate. A solution of α-amylase enzyme (50 µL)
prepared by adding 490 µL of assay buffer to 10 µL of α-amylase enzyme was added to each of
the wells containing the reaction mixture to initiate the reaction. The plate was incubated for 10
minutes at room temperature in the dark. Five different absorbance readings were recorded for
each triplicate run at a wavelength of 405 nm using Varioskan flash microplate spectrophotometer
(Thermo Scientific, Waltham, MA, USA). The IC50 and SD values were calculated using graph
pad prism.
2.4. Kinetic studies on 2e against α-glucosidase and α-amylase
2.4.1. Kinetic study on 2e against α-glucosidase
The kinetics study on 2e was performed according to the reaction conditions in 2.3.1 with
inhibitor concentrations of 1, 1.5, 5, 10 µM and the ranges of final substrate concentrations of 0.5
10 µM. The type of inhibition was determined using Lineweaver-Burk plot (the inverse of velocity
(1/v) against the inverse of the substrate concentration (1/[S]). The inhibitor constant was obtained
by Dixon plot of the inverse of velocity (1/v) against concentration of inhibitor at each substrate
concentration.
2.4.2. Kinetic study on 2e against α-amylase.
Compound 2e was selected for the kinetic study with various substrate concentrations of 0.5–
1.5 µM following the experimental conditions outlined in 2.3.2. The selected inhibitor
concentrations were 5, 10, 25 and 50 µM. The type of inhibition was determined by using
Lineweaver-Burk plot and the inhibitor constant was obtained by Dixon plot (Cengiz et al., 2010).
2.5. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays of 2a–j
The DPPH radical scavenging activity of the test compounds 2a–j was evaluated by following
a literature method described in our previous study (Mphahlele et al., 2020). Briefly the test
compounds and ascorbic acid at various concentrations (1, 5, 10, 25, 50 and 100 µM µM) in
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DMSO were mixed with a solution of DPPH (0.20 mM) in methanol and incubated in the dark for
45 min. Five absorbance readings were recorded at 512 nm using Varioskan flash microplate
spectrophotometer reader. The average values obtained from the absorbance readings were used to
determine the IC50 and standard deviation values.
2.6. Nitric oxide radical scavenging activity of compounds 2a–j
Nitric oxide was generated from sodium nitroprusside and measured by Griess’ reaction
following the literature method using ascorbic acid as a positive control (Mohana, & Kumar, 2013).
A mixture of sodium nitroprusside (10 mM), phosphate buffer saline and Griess reagent (1.00 g of
sulphanilic acid + 0.10 g naphthylethylene diamine dihydrochloride). Sodium nitroprusside (20 µL),
phosphate buffer (5 µL) and the test compound (5 µL) were incubated at 25 °C for 2.5 h. After
incubation, 20 µL of Griess reagent was added to the previous mixture and allowed to stand for 30
min. The absorbance of the colour developed during diazotization of nitrite with sulphanilamide and
its subsequent coupling with napthylethylenediamine hydrochloride. Five absorbance readings were
recorded at 550 nm using Varioskan flash microplate spectrophotometer. The average values
obtained from the absorbance readings of the triplicate runs were used to determine the IC50 and
standard deviation values.
2.7. Cytotoxicity Studies Using the MTT Assay
Cytotoxicity was measured in treated human lung cancer (A549) and African green monkey
kidney (Vero) cell lines using the standard 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) assay. 10 000 cells per well (100 µL) were seeded in a 96-well microtiter plates
and incubated for 24 h at 37 °C and 5% CO2 to allow the cells to attach to the bottom of the wells.
The cells were treated with compound concentrations ranging from 0–50 µM. The microtitre plates
were further incubated for 24 h. After the 24 h incubation period, the MTT reagent (10 µL) was
added to a final concentration of 0.5 mg/mL and the plate was further incubated for another 4 h.
After 4 h, 100 µL of DMSO was added to each well and the plate was allowed to stand for 20
minutes to dissolve the tetrazolium salts. Absorbance readings were measured at 570 nm using the
Varioskan Flash plate reader (Thermo Fisher Waltham, Massachusetts, USA).
2.8. Molecular docking studies of 2a–j against α-glucosidase
The structures of α-glucosidase and α-amylase were obtained from RCSB PDB with accession
number 5NN8 (Roig-Zamboni et al., 2017) and 5E0F, respectively. The polar hydrogen atoms of
the proteins were added using AutoDockTools (Morris et al., 2009). The Kollman-Amber united
atom partial charges and solvation parameters were then assigned using AutoDockTools. The
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initial structure of the test compounds 2a–j were prepared using Avogadro (Hanwell et al., 2012).
The polar hydrogen atoms of the test compounds were retained while the Gasteiger charges and
torsional angles were added using AutoDockTools. The grid box was centered at -12.000, -35.000,
88.000 (x, y z coordinates) for α-glucosidase and, -7.000, 10.000, -21.000 (x, y z coordinates) for
α-amylase with 50 × 50 × 50 points and 0.375 Å as grid spacing. Prior to docking calculation, the
boron atom was replaced with carbon atom as boron parameters are not validated in Autodock4
(Morris et al., 2009). A total of 100 docking runs was performed using Lamarckian genetic
algorithm employed in AutoDock4.2.6 (Morris et al., 2009) together with the following
parameters: 2,500,000 energy evaluation, 27,000 generation, 150 population, 0.8 crossover rate
and 0.02 mutation rate. The conformation in the most populated cluster with most favourable
binding free energy was used for further interaction analysis.
2.9. Drug likeliness prediction of test compounds
The drug likeliness and pharmacokinetic properties of compounds 2a–j were calculated using
Molinspiration (www.molinspiration.com).
3. Results and Discussion
3.1. Chemical synthesis and characterization
The synthesis of the test compounds 2a–e and 2f–j was achieved via cyclocondensation-
dehydration of anthranilamide derivatives 1a (R = H) and 1b (R = Br) with arylboronic acids in the
presence of p-toluenesulfonic acid as catalyst in toluene under Dean-Stark conditions as
represented in Scheme 1. The compounds were isolated by filtration without evidence of any side
products, recrystallized from ethyl acetate, and then characterised using a combination of NMR, IR
and high-resolution mass spectroscopic techniques. Crystals of quality suitable for X-ray
diffraction were obtained for 2e, and the molecular structure of these 1,3,2-diazaboracyclohexane
derivatives was distinctly confirmed by X-ray diffraction. The crystallographic numbering has
been used in the context of the X-ray analysis and it differs from systematic numbering for this
class of compounds. The asymmetric unit shows molecules of 2e held in chains via intermolecular
hydrogen bonding interaction between the hydroxyl oxygen atom (O-H) of one molecule and
hydrogen atom of N-H or C(O)N-H of the adjacent molecules (Figure 2). The molecular construct
is essentially planar with a slight twist of the 2-phenyl group from the coplanarity of the
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dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-one framework with torsion angle C(13)-C(8)-B(1)-
N(1) or C(9)-C(8)-B(1)-N(2) of -6.9° and -6.2°, respectively.
O
O
R
R
H
NH2
N
B
ArB(OH)2, tolune, reflux, 2 h
NH2
N
H
Ar
1a (R = H)
1b (R = Br)
2a–e
2f–j
Scheme 1: Synthesis of the 2-aryl-2,3-dihydrobenzo[d][1,3,2]diazaborinin-4(1H)-ones 2a–j.
Figure 2: Molecular structure of 2e determined from single-crystal X-ray data, showing the atom-
labelling scheme.
The trivalent boron is known to provide alternative electrostatic contacts for enzymatic targets
(Liu et al., 2009, Zhao et al., 2017) and it also enables compounds to adopt favourable geometry to
form strong interactions with the binding pocket of the enzyme (Vlasceanu et al., 2015). We
envisaged that the relatively planar conformation of the test 1,3,2-diazaboracyclohexane
derivatives would allow for increased conjugative effect resulting in stronger carbohydrate
hydrolysing enzyme inhibition and antioxidant activity. Moreover, compounds 2f–j contain
bromine atom, which is known to contain a region with positive charge responsible for this atom’s
directional and stabilizing characteristics on the drug molecule (Carpenter et al., 2010). With these
assumptions in mind, we evaluated the carbonyl-bearing boron-heterocyclic scaffolds 2a–j for
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inhibitory effect in vitro against α-glucosidase and α-amylase activities, and for free radical
scavenging properties as described in the next section.
3.2. Biological evaluation
3.2.1. Inhibitory effect on α-glucosidase and α-amylase, as well as antioxidant activity of 2a–j.
Enzyme inhibitors have become of special interest in drug design and discovery because
altering the activity of an enzyme has immediate and defined effects (Copeland et al., 2007).
Compounds 2a–j were screened through enzymatic assays in vitro for inhibitory effect against α-
glucosidase and α-amylase activities. The IC50 values (μM) defined as the concentration of
compound exhibiting 50% inhibition of enzyme activity represented in Table 1 below were
calculated from log dose inhibition curves, and are expressed as means ± standard deviation (SD)
of three independent experiments. We employed α-glucosidase from Saccharomyces cervisiae,
which is generally used as a target protein for screening the activity of α-glucosidase inhibitors.
The 2-phenyl substituted derivative 2a exhibited significant inhibitory effect against α-glucosidase
activity compared to acarbose (IC50 = 1.12 ± 0.003 µM) with an IC50 value of 2.72 ± 0.024 µM.
The presence of a moderately π-electron delocalizing 2-(4-fluorophenyl) group resulted in higher
activity for 2b (IC50 = 1.73 ± 0.006 µM) compared to 2c (IC50 = 3.10 ± 0.005 µM) substituted with
a 4-chlorophenyl group at the C-2 position. Compounds 2d and 2e substituted with the strongly π-
electron delocalizing and more lipophilic methoxy or hydroxyl group at the para position of the 2-
phenyl ring were found to be the most active against α-glucosidase within this series with IC50
values of 1.15 ± 0.013 µM and 1.35 ± 0.005 µM, respectively. The presence of bromine atom at
the C-6 position of the azaborininone scaffold resulted in relatively reduced α-glucosidase
inhibitory effect for compounds 2f and 2g with IC50 values of 3.24 ± 0.009 µM and 3.68 ± 0.003
µM, respectively. However, a combination of the electron withdrawing bromine and 2-(4-
chlorophenyl) substituent on the framework of 2h resulted in significant inhibitory effect against α-
glucosidase with an IC50 value of 2.09 ± 0.005 µM. The presence of bromine at the C-6 position of
compounds 2i and 2j also resulted in reduced inhibitory effect against α-glucosidase activity. α-
Glucosidase inhibitors are also targeted by medicinal chemists for the treatment of other
carbohydrate mediated diseases such as cancer, human immunodeficiency virus (HIV) and virus
infection, and obesity (Gamblin et al., 2009).
α-Amylase inhibitory assay was carried out following protocol enclosed in BioVision’s α-
Amylase Inhibitor Screening kit (Catalog # K482-100) against acarbose (IC50 = 11.71 ± 0.051 µM)
as a reference standard. The compounds within the two series of analogues showed varying degree
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of α-amylase inhibitory activities with IC50 values in the range 2.66 ± 0.056 µM to 28.14 ± 0.010
µM (2a–e) and 5.12 ± 0.036 µM to 23.42 ± 0.032 µM (2f–j). The 2-phenyl substituted derivative
2a exhibited increased activity against this enzyme with an IC50 value of 4.77 ± 0.011 µM, which
is comparable to that of the reference standard. Compound 2b which exhibited increased inhibitory
effect against α-glucosidase was found to be less active against α-amylase with an IC50 value of
28.14 ± 0.010 µM. However, the 2-(4-chlorophenyl) analogue 2c with moderate activity against α-
glucosidase was found to be relatively more active against α-amylase than 2b with an IC50 value of
11.15 ± 0.062 µM. The presence of a strongly π-electron delocalizing methoxy group at the para
position of the 2-phenyl substituent resulted in increased activity for 2d against α-amylase with an
IC50 value of 10.41 ± 0.027 µM. Compound 2e substituted with a strongly hydrogen bonding
hydroxyl group at the para position of the 2-phenyl ring was found to be the most active derivative
among the test compounds against α-amylase with an IC50 value of 2.66 ± 0.056 µM. Compounds
2d and 2e exhibit dual inhibitory effect against α-glucosidase and α-amylase activities, and these
compounds have potential to suppress carbohydrate digestion and delay glucose uptake to lead to
reduced blood sugar levels.
A combination of bromine atom at the C-5 position and 2-phenyl group in 2f or a 2-(4-
chlorophenyl) group in 2h resulted in significantly reduced activity for these compounds compared
with IC50 values of 23.05 ± 0.023 µM and 23.42 ± 0.032 µM, respectively. These 6-bromo
substituted derivatives are less active against this enzyme compared to their C-6 unsubstituted
precursors 2a and 2c. However, a combination of the 6-bromo atom and 2-(4-fluorophenyl) group
resulted in significantly increased activity for 2g (IC50 = 11.74 ± 0.090 µM) compared to 2b. The
most active compounds within the 6-bromo substituted series 2f–j are 2i and 2j substituted with the
lipophilic and strongly π-electron delocalizing methoxy or hydroxyl group at the para position of
the 2-phenyl ring with IC50 values of 5.81 ± 0.082 µM and 5.12 ± 0.036 µM, respectively. These
oxygen-based substituents are capable of forming non-covalent interactions such as hydrogen
and/or halogen bonds with protein residues of the receptor. Compounds 2b, 2f and 2h with
significantly reduced activity against α-amylase and significant inhibitory effect against α-
glucosidase could serve as first-line drugs for the treatment of T2DM to prevent the digestion of
carbohydrates in the intestine and defer glucose absorption, in turn, suppress post-prandial
hyperglycaemia (PPHG). The other derivatives in both series have potential to serve as dual
inhibitors of both carbohydrate hydrolysing enzymes to delay the glucose absorption and lower the
postprandial blood glucose level.
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Laboratory studies have revealed that a compound exhibiting a higher glucose-lowering effect
also has good antioxidant properties (Famuyiwa, et al. 2019; Rafique et al., 2020b; Rani, et al.,
2019) As a prelude to organoboron-based 6-membered heterocycles with dual carbohydrate
hydrolysing inhibitory and antioxidant activities, the test compounds were evaluated for
antioxidant properties through the DPPH and NO free radical scavenging assays with ascorbic acid
as a reference standard (IC50 values of 9.25 ± 0.003 µM and 7.40 ± 0.019 µM, respectively) for the
assays. Within the series 2a–e, only compounds 2a and 2b exhibited reduced DPPH scavenging
activity compared to ascorbic acid with IC50 values of 28.81 ± 0.002 µM and 36.42 ± 0.013 µM,
respectively. However, both compounds exhibited increased NO scavenging activity compared to
the reference standard with IC50 values of 5.16 ± 0.050 µM and 2.73 ± 0.027 µM, respectively.
Compound 2a exhibited significant α-glucosidase and increased α-amylase inhibitory activities as
well as NO scavenging activity. The presence of a 4-chlorophenyl group on the scaffold of 2c, on
the other hand, resulted in increased antioxidant activity in the DPPH assay, but moderate NO
scavenging activity with IC50 values of 2.11 ± 0.003 µM and 13.19 ± 0.090 µM, respectively.
Increased antioxidant activity was also observed for 2d substituted with a strongly π-electron
delocalizing 4-methoxyphenyl group at position 2, and the corresponding IC50 values are 0.28 ±
0.006 µM and 2.09 ± 0.024 µM in the DPPH and NO radical scavenging assays, respectively.
Increased propensity for the phenolic group to donate a hydrogen atom to the DPPH radical also
resulted in increased free radical scavenging effect for 2e (IC50 value of 0.36 ± 0.003 µM). This
compound exhibited the highest NO scavenging activity in this series with an IC50 value of 0.45 ±
0.019 µM. The phenyl derivative 2f within the 6-bromo substituted series 2f–j exhibited relatively
reduced antioxidant activity in both assays with IC50 values of 19.84 ± 0.006 µM and 24.4 ± 0.030
µM, respectively. A combination of the 6-bromo atom and 2-(4-fluorophenyl) group in 2g, on the
other hand, resulted in increased DPPH (IC50 = 4.67 ± 0.017 µM) and NO (IC50 = 7.27 ± 0.009
µM) radical scavenging activity compared to ascorbic acid. The presence of bromine atom at C-6
position of compounds 2h, 2i and 2j also resulted in increased antioxidant activity in both assays
compared to the reference standard.
Considering that metabolic diseases require a stable long-term treatment, well tolerated and
low toxicity, the test compounds were assayed for toxicity against the African green monkey
kidney (Vero) cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay
to establish their safety profile at least in vitro. The MTT assay revealed that the test compounds
were not toxic to the African green monkey kidney (Vero) cells after 24 h (refer to Fig. S2 of SI).
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The Vero cells continue to be routinely used as a part of screening programs to evaluate the
toxicity of compounds of different nature, either chemical or microbial toxins (Menezes et al.,
2013). Additionally, increased glucose metabolism plays an important role in supporting cancer
cell proliferation (Vander Heiden, et al. 2009), and therefore compounds that alter glucose
metabolism are important candidates to be tested for anti-cancer properties. Consequently,
compounds were assayed for cytotoxicity against the human lung cancer (A549) cell line as a
representative model to establish their antiproliferative properties and selectivity between the
normal and cancer cells. The preliminary cytotoxicity assay revealed that these compounds have
little or no effect on the viability of A549 cell line. However, these preliminary cytotoxicity results
require further tests on different cell lines to confirm further the safety of these compounds.
Compound 2e with dual inhibitory activity against α-glucosidase and α-amylase was selected for
mechanism-based enzyme inhibition studies as described in the next section.
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Table 1: Inhibition of 2a–j on α-glucosidase and α-amylase, and their free radical scavenging potential.
IC50 (µM, SD)
2a–l
R
H
H
H
H
H
Br
Br
Br
Br
Br
-
Ar
α-Glucosidase α-Amylase
DPPH
NO
2a
C6H5-
2.72 ± 0.024
1.73 ± 0.006
3.10 ± 0.005
4.77 ± 0.11
28.14 ± 0.01
11.15 ± 0.06
10.41 ± 0.03
2.66 ± 0.06
23.05 ± 0.02
11.74 ± 0.09
23.42 ± 0.03
5.81 ± 0.08
5.12 ± 0.04
11.71 ± 0.05
-
28.81 ± 0.002
36.42 ± 0.013
2.11 ± 0.003
0.28 ± 0.006
0.36 ± 0.003
19.84 ± 0.006
4.67 ± 0.017
14.4 ± 0.017
5.14 ± 0.006
6.43 ± 0.004
-
5.16 ± 0.05
2.73 ± 0.03
13.19 ± 0.09
2.09 ± 0.02
0.45 ± 0.02
24.4 ± 0.03
7.27 ± 0.01
8.12 ± 0.05
10.48 ± 0.02
6.01 ± 0.01
-
2b
4-FC6H4-
4-ClC6H4-
2c
2d
4-MeOC6H4- 1.15 ± 0.013
2e
4-HOC6H4-
C6H5-
1.35 ± 0.005
3.24 ± 0.009
3.68 ± 0.003
2.09 ± 0.005
2f
2g
4-FC6H4-
4-ClC6H4-
2h
2i
4-MeOC6H4- 4.08 ± 0.017
2j
4-HOC6H4-
4.44 ± 0.005
1.12 ± 0.003
-
Acarbose
Ascorbic acid
-
-
-
9.25 ± 0.003
7.40 ± 0.02
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3.2.2 Kinetic study
The Lineweaver-Burk plot of 1/V versus 1/[S] in the presence of different concentrations of
compound 2e in the case of α-glucosidase gave a series of straight lines that intersect on the x-axis
(Figure 3a). The plot shows an unchanged Michaelis constant (Km) value as the velocity of the
reaction (Vmax) increases. The Dixon plot (Figure 3b) has straight lines that intersect above the x-
axis with the calculated Ki value of 0.42 ± 0.11 µM. The observed trends are consistent with a
mixed mode of inhibition of this compound against α-glucosidase activity.
0.5 µM
1.5 µM
2700
2200
1700
1200
700
2.0 µM
1.0 µM
200
-0.5
0
0.5
1
1.5
2
2.5
-300
1/[S] mM
(a)
2700
2200
1700
1200
700
5.0 mM
1.5 mM
0.5 mM
1.0 mM
200
-2
0
2
4
6
-300
µM
[INHIBITOR]
(b)
Figure 3: Lineweaver-Burk plot (a) and Dixon plot (b) of 2e against α-glucosidase.
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The Lineweaver-Burk plot of 2e (Figure 4a) is characterised by decreasing Vmax (0.023–0.013
µM/min) and an unchanged Km value of 3.00. Its Dixon plot (Figure 4b), shows several straight
lines that intersect above the x-axis with a Ki value of 5.22 ± 0.23 µM. The observed trends are
consistent with a mixed mode of inhibition of this compound against α-amylase activity. This
compound probably binds to the active site and other sites of the enzyme to affect its activity.
700
25 µM
600
50 µM
500
5 µM
400
10 µM
300
200
100
0
-2
0
2
-100
1/[S] mM
(a)
0.5 mM
1.5 mM
0.7 mM
1.3 mM
700
600
500
400
300
200
100
0
-45
-25
-5
15
µM
35
55
-100
[INHIBITOR]
(b)
Figure 4: Lineweaver-Burk plot (a) and Dixon plot (b) of 2e against α-amylase.
We speculated that the N-H, C=O, -OH and OCH3, halogen atoms, and the π-electrons of the
aromatic rings in these compounds may have hydrophobic effects, hydrogen bonded salt bridges,
electrostatic interactions, π-stacking interactions, and other noncovalent bonds with the amino acid
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residues of the target enzymes. Consequently, we subjected these compounds to molecular docking
(in silico) studies to determine plausible protein–ligand interactions on a molecular level, and to
establish their drug likeness at theoretical level.
3.2.3 Molecular docking into α-glucosidase and α-amylase active sites
The test compounds were docked into the active sites of α-glucosidase (PDB code: 5NN8) and
α-amylase (PDB code: 5E0F). The conformation in the most populated cluster with most
favourable free binding energy (BE) was used for further interaction analysis (refer to Table S1 in
SI for BE values). The most active derivative 2e from series 2a–e against both enzymes, and
compound 2h from series 2f–j which exhibited significant activity against α-glucosidase and
reduced inhibitory effect against α-amylase were chosen as representative models for the docking
poses into α-glucosidase (Figure 5) and α-amylase (Figure 6) active sites. Both 2e and 2h form
three hydrogen bonds with α-glucosidase. Three hydrophobic contacts and a π-cation interaction
are also predicted between 2e and α-glucosidase. Compound 2h, on the other hand, has five
hydrophobic contacts that support it in the binding pocket of α-glucosidase.
The favourable binding affinity of 2e against α-amylase could be contributed by the hydroxyl
group of the phenol region which forms as many as four hydrogen bonds with the protein residues
in the active site of this enzyme. Two hydrogen bonds are also predicted to form between the
azaborininone region of 2e with α-amylase. The azaborininone region of 2h make the same
number of hydrogen bond with α-amylase while the bromine atom forms two halogen bonds with
α-amylase Arg195. Both 2e and 2h form hydrophobic contacts with the residues Trp58, Trp59 and
Tyr62, and the two compounds are also involved in π-π stacking interaction with Tyr62.
Figure 5. The interaction analysis between compound 2e and 2h with α-glucosidase.
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Figure 6. The interaction analysis between compound 2e and 2h with α-amylase.
3.2.4 Drug-likeness predictions
The drug-likeness of the test compounds was predicted at theoretical level through the
Lipinski rule of five which states that for a molecule to act as a therapeutic candidate it must
possess four characteristics, which are (i) number of hydrogen bond donors should not be more
than 5, number of hydrogen bond acceptors should not be more than 10, the molecular mass should
be less than 500 Da, and LogP (octanol-water partition coefficient) should not be greater than 5
(Table 2). The test compounds fulfill all the four druglikeliness characteristics (refer to Table S2 in
SI). The predicted absorption rate of nearly 90% for the highest active derivative 2e against both
enzymes has also suggested possible oral adminstration for this compound.
4.
Conclusions
The lipophilic and strongly π-electron delocalizing methoxy (2d and 2i) or hydroxy (2e and 2j)
group increase the electron density of the these azaborininones, and therefore the ability of this
scaffold to engage in hydrogen bonding and hydrophobic interactions with the protein residues in
the receptor. Compounds 2d, 2e, 2i and 2j with dual inhibitory effect against α-glucosidase and α-
amylase activities, and strong free radical scavenging activity have potential to serve as multi-
target ligands to reduce blood sugar level and probably ameliorate complications associated with
oxidative stress. These compounds showed little or no effect on the viability of A549 cell line and
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were also not toxic to the Vero cells. These preliminary results support the potential of the 1,3,2-
diazaboracyclohexane derivatives as bioisosteric replacements for the 2-arylquinazolin-4-ones in
antidiabetic drug discovery programs. More analogues will be synthesised to optimize the
inhibition effects of this 1,3,2-diazaboracyclohexane scaffold. Moreover, further cellular-based in
vitro and in vivo studies including bioavailability and cell permeability would help to clarify the
mechanism of action of these compounds in the body, and to establish their safety profile as
potential multi-target agents against the pathogenesis and progression of T2DM. It is envisaged
that the results of this study will offer medicinal chemists an opportunity to explore these carbonyl-
bearing boron-heterocyclic scaffolds and their analogues for other modes of inhibition against a
variety of biochemical and biological targets, and pioneer new areas of drug discovery.
1
13
Supplementary Information: Figure S1: Copies of the H- and C-NMR spectra of compounds
2a–j; Table S1: Estimated binding free energy obtained from docking simulation of compound 2a–
j towards α-glucosidase (PDB id 5NN8) and α-amylase (PDB id 5E0F); Table S2. The
pharmacokinetics properties predictions of test compounds 2a–j; and Fig. S2. Cytotoxicity results
of compounds 2a–j against A549 and Vero cell lines.
Author Contributions: Conceptualization, M.J.M.; Methodology, data curation and analyses,
M.J.M.; bioassays, NMM & STJ; Writing–review & editing, M.J.M. and Y.S.C.
Funding: This project was funded by the University of South Africa and the National Research
Foundation (NRF) in South Africa (NRF GUN: 118554) as well as Universiti Sains Malaysia (RUi
grant: 1001/CIPPM/8011051). The views and opinions expressed herein are those of the authors
and not of the funding bodies.
Data Availability Statement: The CIF file containing complete information on the studied
structure was deposited with the Cambridge Crystallographic Data Center, CCDC 2032015, and is
freely available upon request from the following website: www.ccdc.cam.ac.uk/datarequest/cif or
by contacting the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ,
UK; fax: +44-1223-336033; email: deposit@ccdc.cam.ac.uk.
Acknowledgements: We are grateful to the University of Stellenbosch Central Analytical Facility
(CAF) and the University of the Witwatersrand for mass spectrometric and X-ray data,
respectively.
Conflict of interest: The authors declare no conflict of interest.
References
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Alqahtani, A. S., Hidayathulla, S., Rehman, M. T., ElGamal, A. A., Al-Massarani, S., Razmovski-
Naumovski, V., Alqahtani, M. S., El Dib, R. A., & AlAjmi, M. F. (2020). Alpha-amylase
and alpha-glucosidase enzyme inhibition and antioxidant potential of 3-oxolupenal and
katononic acid isolated from Nuxia oppositifolia. Biomolecules, 10, 61.
DOI:10.3390/biom10010061.
Barker, S. J., Ding, C. Z., Akama, T., Zhang, Y. -K., Hernandez, V., & Xia, Y. (2009). Therapeutic
potential of boron-containing compounds. Future Medicinal Chemistry, 1, 1275–1288.
DOI:10.4155/fmc.09.71.
Barmak, A., Niknam, K., & Mohebbi, G. (2019). Synthesis, structural studies, and α-glucosidase
inhibitory, antidiabetic, and antioxidant activities of 2,3-dihydroquinazolin-4(1H)-ones
derived from pyrazol-4-carbaldehyde and anilines. ACS Omega, 4, 18087–18099.
DOI:10.1021/acsomega.9b01906.
Carpenter, R. D., Natarajan, A., Lau, E. Y., Andrei, M., Solano, D. M., Lightstone, F. C., DeNardo,
S. J., Lam, K. S., & Kurth, M. J. (2010). Halogenated benzimidazole carboxamides target
integrin α 4β1 on T-cell and B-cell lymphomas. Cancer Research, 70, 5448–5456.
DOI:10.1158/0008-5472.
Cengiz, S., Cavas, L., & Yurdakoc, K. (2010). Alpha-amylase inhibition kinetics by caulerpenyne.
Mediterranean Marine Science, 93–103. DOI:10.12681/mms.93.
Chetan, S., Amarjeet, K., Thind, S. S., Baljit, S., & Shiveta, R. (2015). Advanced glycation End-
products (AGEs): An emerging concern for processed food industries. Journal of Food
Science and Technology, 52, 7561–7576. DOI:10.1007/s13197-015-1851-y.
Chissick, S. S., Dewar, M. J. S., & Maitlis, P. M. (1961). New heteroaromatic compounds. XIV.
Boron-containing analogs of purine, quinazoline and perimidine. Journal of the American
Chemical Society, 83, 2708–2711. DOI:10.1021/ja01473a025.
Churches, Q. I., & Hutton, C. A. (2016). Introduction, interconversion and removal of boron
protecting groups. ACS Symposium Series, 1236, 357−377. DOI:10.1021/bk-2016-
1236.ch011.
Copeland, R. A., Harpel, M. R., & Tummino, P. J. (2007). Targeting enzyme inhibitors in drug
discovery.
Expert
Opinion
on
Therapeutic
Targets,
11,
967–978.
DOI:10.1517/14728222.11.7.967.
This article is protected by copyright. All rights reserved

Dan, W. -J., Zhang, Q., Zhang, F., Wang, W. -W., & Gao, J- M. (2019). Benzonate derivatives of
acetophenone as potent α-glucosidase inhibitors: synthesis, structure–activity relationship
and mechanism. Journal of Enzyme Inhibition and Medicinal Chemistry, 34, 937–945.
DOI:10.1080/14756366.2019.1604519.
Davies, G. H. M., Mukhtar, A., Saeednia, B., Sherafat, F., Kelly, C. B., & Molander, G. A. (2017).
Azaborininones: Synthesis and structural analysis of a carbonyl containing class of
azaborine. Journal of Organic Chemistry, 82, 5380–5390. DOI:10.1021/acs.joc.7b00747.
Davis, M. C., Franzblau, S. G., & Martin, A. R. (1998). Syntheses and evaluation of
benzodiazaborine compounds against M. tuberculosis H37Rv in vitro. Bioorganic &
Medicinal Chemistry Letters, 8, 843–846.
Dixit, V.A., Goundry, W. R. F., & Tomasi, S. (2017). C=C/B–N substitution in five membered
heterocycles. A computational analysis. New Journal of Chemistry, 41, 3619–3633.
DOI:10.1039/C7NJ00950J.
Famuyiwa, S. O., Sanusi, K., Faloye, K. O., Yilmaz, Y., & Ceylan, Ü. (2019). Antidiabetic and
antioxidant activities: Is there any link between them? New Journal of Chemistry, 43, 13326–
13329. DOI:10.1039/C9NJ01251F.
Gamblin, D. P., Scanlan, E. M., & Davis, B. G. (2009). Glycoprotein synthesis: An update.
Chemical Reviews, 109, 131−163. DOI:10.1021/cr078291i.
Hanwell, M. D., Curtis, D. E., Lonie, D. C., Vandermeersch, T., Zurek, E., & Hutchison, G. R.
(2012). Avogadro: An advanced semantic chemical editor, visualization, and analysis
platform. Journal of Cheminformatics, 4, 17. http://www.jcheminf.com/content/4/1/1.
Hemalatha, K., & Madhumitha, G. (2016). Synthetic strategy with representation on mechanistic
pathway for the therapeutic applications of dihydroquinazolinones. European Journal of
Medicinal Chemistry, 123, 596–630. DOI:10.1016/j.ejmech.2016.08.001.
Ihara, H., Koyanagi, M., & Suginom, M. (2011). Anthranilamide: A Simple, removable ortho-
directing modifier for arylboronic acids serving also as a protecting group in cross-coupling
reactions. Organic Letters, 13, 2662–2665. DOI:10.1021/ol200764g.
Imran, S., Taha, M., Selvaraj, M., Ismail, N. H., Chigurupati, S., & Mohammad, J. I. (2017).
Synthesis and biological evaluation of indole derivatives as α-amylase inhibitor.
Bioorganic Chemistry, 73, 121–127. DOI:10.1016/j.bioorg.2017.06.007.
Javaid, K., Saad, S. M., Rasheed, S., Moin, T., Sued, N., Fatima, I., Salar, U., Khan, K. M.
Perveen, S., & Choudhary, M. I. (2015). 2-Arylquinazolin-4(3H)-ones: A new class of α-
This article is protected by copyright. All rights reserved

glucosidase
inhibitors.
Bioorganic
Chemistry
23,
7417–7421.
DOI:10.1016/j.bmc.2015.10.038.
Kamio, S., Kageyuki, I., Osaka, I, Hatano, S., Abe, M., & Yoshida, H. (2018). Anthranilamide
(aam)-substituted diboron: palladium-catalyzed selective B(aam) transfer. Chemical
Communication, 54, 9290–9293. DOI:10.1039/c8cc05645e.
König, T., Mannel, D., Häbicher, W. D., & Schwertlick, K. (1988). Organoboron antioxidants: Part
1—Boric acid derivatives as primary antioxidants. Polymer Degradation and Stability, 22,
137–145. DOI:10.1016/0141-3910(88)90037-7.
Liu, L., Marwitz, A. J. V., Matthews, B. W., & Liu, S. -Y (2009). Boron mimetics: 1,2-Dihydro-
1,2-azaborines bind inside a nonpolar cavity of T4 lysozyme. Angewandte Chemie
International Edition, 48, 6817–6819. DOI:10.1002/anie.200903390.
Menezes, C., Valério, E., & Dias, E. (2013). The kidney Vero-E6 cell line: A suitable model to
study the toxicity of microcystins. In New Sights into Toxicity and Drug Testing, Vol. 2.
Rijeka, Croatia: InTechOpen, pp. 29–48. DOI:10.5772/54463.
Mohana, K.N., & Kumar, C. B. P. (2013). Synthesis and antioxidant activity of 2-amino-5-
methylthiazol derivatives containing 1,3,4-oxadiazole-2-thiol moiety. ISRN Organic
Chemistry, 8, 620718. DOI:10.1155/2013/620718.
Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D. S., & Olson. A.
J. (2009). AutoDock4 and AutoDockTools4: Automated docking with selective receptor
flexibility. Journal of Computational Chemistry, 40, 2785–2791. DOI:10.1002/jcc.21256.
Mphahlele, M. J., Magwaza, N. M., Gildenhuys, S., & Setshedi, I. B. (2020). Synthesis, α-
glucosidase inhibition and antioxidant activity of the 7-carbo–substituted 5-bromo-3-
methylindazoles. Bioorganic Chemistry, 97, 103702. DOI:10.1016/j.bioorg.2020.103702.
Özçayan, G. (2019). Synthesis of organoboron amide-ester branched derivatives of poly(itaconic
anhydride-alt-2-vinyl-1,3-dioxolane) and cancer cells interaction studies. Boron, 4, 159–
171. DOI:10.30728/boron.515517.
Rafique, R., Khan, K. M., Arshia, Chigurupati, S., Wadood, A., Rehman, A. U., Salar, U.,
Venugopal, V., Shamim, S., Taha, M., & Perveen, P. (2020a). Synthesis, in vitro α-amylase
inhibitory, and radicals (DPPH & ABTS) scavenging potentials of new N-sulfonohydrazide
substituted
indazoles.
Bioorganic
Chemistry,
94,
105410.
DOI:10.1016/j.bioorg.2019.103410.
This article is protected by copyright. All rights reserved

Rafique, R., Khan, K. M., Arshia, Kanwal, Chigurapati, S., Wadood, A., Rehman, A. U.,
Karunanidhi, A., Hammed, S., Taha, M., & Al-Rashida, M. (2020b). Synthesis of new
indazole based dual inhibitors of α-glucosidase and α-amylase enzymes, their in vitro, in
silico
and
kinetics
studies.
Bioorganic
Chemistry,
94,
103195.
DOI:10.1016/j.bioorg.2019.103195.
Rani, V., Deep, G., Singh, R. K., Palle, K., & Yadav, U. C. S. (2016). Oxidative stress and
metabolic disorders: Pathogenesis and therapeutic strategies. Life Sciences, 1148, 183–193.
DOI:10.1016/j.lfs.2016.02.002.
Roig-Zamboni, V., Cobucci-Ponzano, B., Iacono, R., Ferrara, M. C., Germany, S., Bourne, Y.,
Parenti, G, Moracci, M., & Sulzenbacher, G. (2017). Structure of human lysosomal acid
alpha-glucosidase– a guide for the treatment pf Pompe disease. Nature Communications, 8,
1111. DOI:10.1038/s41467-017-01263-3.
Rombouts, F. J. R., Tovar, F., Austin, N., Tresadern, G., & Trabanco, A. A. (2015).
Benzazaborinines as novel bioisosteric replacements of naphthalene: Propranolol as an
example.
Journal
of
Medicinal
Chemistry,
58,
9287−9295.
DOI:10.1021/acs.jmedchem.5b01088.
Settepani, J. A., Stokes, J. B., & Bokkovec, A. B. (1970). Insect chemosterilants. VIII. Boron
compounds. Journal of Medicinal Chemistry, 13, 128–131. DOI:10.1021/jm00295a032.
Smoum, R., Rubinstein, A., Dembitsky, V. M., & Srebnik, M. (2012) Boron containing
compounds as protease inhibitors. Chemical Reviews, 112, 4156–4220.
DOI:10.1021/cr608202m.
Tangvarasittichai, S. (2015). Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes
mellitus. World Journal of Diabetes, 6, 456–480. DOI:10.4239/wjd.v6.i3.456.
Vander Heiden, M. G., Cantley, L. C., & Thompson, C. B. (2009). Understanding the Warburg
effect: the metabolic requirements of cell proliferation. Science, 324, 1029–1033. DOI:
10.1126/science.1160809.
Vieira, R., Souto, S. B., Sánchez-López, E., Machado, A. L., Severino, P., Jose, S., Santini, A.,
Fortuna, A., Garcia, M. L., Silva, A. M., & Souto, E. B. (2019). Sugar-lowering drugs for
type 2 diabetes mellitus and metabolic syndrome– Review of classical and new
compounds: Part-I. Pharmaceuticals, 12, 152. DOI:10.3390/ph12040152.
This article is protected by copyright. All rights reserved

Vlasceanu, A., Jessing, M., & Kilburn, J. P. (2015) BN/CC isosterism in borazoranaphthalenes
towards phosphodiesterase 10A (PDE10A) inhibitors. Bioorganic & Medicinal Chemistry,
23, 4453–4461. DOI:10.1016/j.bmc.2015.06.019.
Wang, H. ‐J., Zhang, M., Li, W. -J., Ni, Y., Lin, J., & Zhang, Z. -H. (2019). An efficient Ni/Pd
catalyzed chemoselective synthesis of 1,3,2‐benzodiazaborininones from boronic acids and
anthranilamides. Advanced Synthesis
&
Catalysis, 361, 5018–5024. DOI:
10.1002/adsc.2019.
Wei, M., Chai, W. -M., Wang, R., Yang, Q., Deng, Z., & Peng, Y. (2017). Quinazolinone
derivatives: Synthesis and comparison of inhibitory mechanisms on α-glucosidase.
Bioorganic & Medicinal Chemistry, 25, 1303–1308. DOI:10.1016/j.bmc.2016.09.042.
Yale, H. L. (1971). Novel boron heterocycles. I. 2,3‐dihydro‐1,3,2‐benzodiazaborin‐4(1H)‐ones
and 1,2‐dihydro‐1,3,2‐benzodiazaborines. Journal of Heterocyclic Chemistry, 8, 193–204.
DOI:10.1002/jhet.5570080203.
Zhao, P., Nettleton, D. O., Karki, R. G., Zécri, F. J., & Liu, S. -L. (2017). Medicinal chemistry
profiling
of
monocyclic
1,2‐azaborine.
ChemMedChem,
12,
358–361.
DOI:10.1002/cmdc.201700047.
This article is protected by copyright. All rights reserved