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

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,¹Sibusiso T. Malindisa²and Yee Siew Choong^3*

¹Department 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)

²Department 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)

³Institute 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.

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process, which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

10.1111/CBDD.13893

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

(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).

O

N

O

H

N

C

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

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)); ν_max(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)); ν_max(ATR)

630, 692, 821, 1336, 1488, 1612, 1638, 3331, 3406 cm^-1; ¹H-NMR (DMSO-d₆) 7.08 (1H t, J = 7.5

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, J_CF= 19.9 Hz), 118.6, 119.2, 121.4,

3

1

119.2, 121.3, 128.4, 129.0, 133.8, 136.3 (d, J_CF= 7.6 Hz), 145.9, 164.4 (d, J_CF= 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)); ν_max(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)); ν_max

(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)

White solid (0.88 g, 85%), mp. 242–243 °C (Lit. 232.8–236.6 °C (Kamio et al., 2018)); ν_max

(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)); ν_max(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; ν_max(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,

²J_CF= 19.9 Hz), 120.9, 121.1, 128.6, 130.4, 136.3 (d, ³J_CF= 7.6 Hz), 136.4, 145.0, 154.5 (d, ¹J_CF

=

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; ν_max(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.

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; ν_max(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; ν_max(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

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 IC₅₀and 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

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 IC₅₀and 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 IC₅₀and

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% CO₂to 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

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

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

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

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 IC₅₀values (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 IC₅₀value 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 IC₅₀values 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 IC₅₀value 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

of α-amylase inhibitory activities with IC₅₀values 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 IC₅₀value 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 IC₅₀value 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 IC₅₀value 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

IC₅₀value 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 IC₅₀value 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 IC₅₀values 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 IC₅₀values 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.

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 (IC₅₀values 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 IC₅₀values 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 IC₅₀values 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 IC₅₀values 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 IC₅₀values 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 (IC₅₀value of 0.36 ± 0.003 µM). This

compound exhibited the highest NO scavenging activity in this series with an IC₅₀value 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 IC₅₀values 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).

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.

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

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

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 (K_m) value as the velocity of the

reaction (V_max) increases. The Dixon plot (Figure 3b) has straight lines that intersect above the x-

axis with the calculated K_ivalue 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.

The Lineweaver-Burk plot of 2e (Figure 4a) is characterised by decreasing V_max(0.023–0.013

µM/min) and an unchanged K_mvalue of 3.00. Its Dixon plot (Figure 4b), shows several straight

lines that intersect above the x-axis with a K_ivalue 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

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.

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

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

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., Nataraj an, A., Lau, E. Y., Andrei, M., Solano, D. M., Lightst one, F. C., DeNardo,

S. J., Lam, K. S., & Kurth, M. J. (2010). Halogenated benzimidazole carboxamides target

integrin α ₄β₁on 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.

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 H₃₇R_vin 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-

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.

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.

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.

W ang, 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.

Article Doi

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

DOI: 10.1111/cbdd.13893

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Article abstract of DOI:10.1111/cbdd.13893

Full text of DOI:10.1111/cbdd.13893

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