Cytotoxicity, alpha-glucosidase inhibition and molecular docking studies of hydroxamic acid chromium(III) complexes

Article abstract of DOI:10.1007/s00775-020-01755-6

Hydroxamic acids [R(CO)N(OH)R’] are flexible compounds for organic and inorganic analyses due to their frailer structures compared to the carboxylic acid. The syntheses and characterization of benzohydroxamic acid (BHA), its CH₃–, OCH₃

Full text of DOI:10.1007/s00775-020-01755-6

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-020-01755-6
ORIGINAL PAPER
Cytotoxicity, alpha‑glucosidase inhibition and molecular docking
studies of hydroxamic acid chromium(III) complexes
Latifah Robbaniyyah Hassan · El Hassane Anouar · Hadariah Bahron1,3 · Faiezah Abdullah4 ·
1
2
Amalina Mohd Tajuddin1
,5
Received: 8 October 2019 / Accepted: 9 January 2020
©
Society for Biological Inorganic Chemistry (SBIC) 2020
Abstract
Hydroxamic acids [R(CO)N(OH)R’] are fexible compounds ꢀor organic and inorganic analyses due to their ꢀrailer structures
compared to the carboxylic acid. The syntheses and characterization oꢀ benzohydroxamic acid (BHA), its CH –, OCH –,
3
3
Cl– para-substituted derivatives and their Cr(III) complexes are reported herein. The metal complexes were synthesized by
reacting the hydroxamic acids with chromium(III) chloride hexahydrate in 2:1 molar ratio. The compounds were characterized
1
13
via melting point, elemental analysis, FTIR, H and C NMR, TGA, mass spectrometry, molar conductance and UV–Visible.
Data analysis suggests that each complex has the Cr(III) center coordinated to the carbonyl and hydroxy oxygen atoms oꢀ the
hydroxamic acids in bidentate O,O manner and two water molecules to ꢀorm octahedral geometry. Non-electrolytic behavior
oꢀ the complexes was shown through their low molar conductivity. Cytotoxicity study against HCT116 and alpha-glucosidase
inhibition test revealed that all complexes have higher activity than their parent ligands. Molecular docking study shows that
the docking oꢀ active complexes is thermodynamically ꢀavorable and the inhibition eꢁciency may depend on the types and
the numbers oꢀ molecular interactions established in the corresponding stable conꢀormers.
Keywords Benzohydroxamic acid · Chromium(III) · Cytotoxicity · Alpha-glucosidase · Molecular docking
Introduction
caused by the ease oꢀ deprotonation to produce hydroxam-
ate or hydroximate ions. The deprotonation renders these
acids stable chelates ꢀor metals allowing them to interrupt
enzyme activities [2-5]. They are also known to possess NO-
releasing properties [6, 7] that are pharmacologically impor-
tant, acting on smooth muscles as potential vasodilators and
cardiac contractility regulators. Aromatic hydroxamic acids
such as benzohydroxamic acid or salicylhydroxamic acid
and their metal complexes have received a lot oꢀ attention
due to their ꢀascinating structures and myriad oꢀ bioactivi-
ties [8-10].
Hydroxamic acids, R(CO)N(OH)R’, are a class oꢀ com-
pounds that display many bioactive properties such as anti-
cancer, antimalarial and antiꢀungal [1]. These properties are
*
*
El Hassane Anouar
anouarelhassane@yahoo.ꢀr
Amalina Mohd Tajuddin
amalina9487@uitm.edu.my
1
2
Faculty oꢀ Applied Sciences, Universiti Teknologi MARA,
Shah Alam, 40450 Selangor, Malaysia
Chromium is one oꢀ the most common elements ꢀound
on the earth crust and seawater. Like other transition metals,
chromium has several oxidation states, the most common
Department oꢀ Chemistry, College oꢀ Science
and Humanities in Al-Kharj, Prince Sattam Bin Abdulaziz
University, Al-Kharj 11942, Saudi Arabia
0
3+
oꢀ which are metallic (Cr ), trivalent (Cr ) and hexava-
6+
3+
lent (Cr ) chromium. Cr is ꢀrequently ꢀound in ꢀood and
well known as a dietary supplement [11], a crucial nutrient
involved in the glucose tolerance ꢀactor (GTF) in maintain-
ing regular carbohydrate and lipid metabolism. For example,
3
4
5
University oꢀ Malaya Centre ꢀor Ionic Liquid (UMCIL),
Universiti Malaya, 50603 Kuala Lumpur, Malaysia
Centre oꢀ Foundation Studies, Universiti Teknologi MARA,
Dengkil, 43800 Selangor, Malaysia
chromium picolinate [Cr(pic) ] supplement has been widely
3
Atta-ur-Rahman Institute ꢀor Natural Product Discovery
used to improve body’s response to insulin in lowering blood
(
AuRIns), Universiti Teknologi MARA, Level 9, FF3
sugar ꢀor people with type two diabetes [12]. Chromium
Building, 42300 Puncak Alam, Selangor, Malaysia
Vol.:(0
1
123456789)
3

JBIC Journal of Biological Inorganic Chemistry
metꢀormin, [Cr(Met)] displayed beneꢂcial eꢃects on blood
glucose and lipid metabolism ꢀor diabetic mice and reported
to be saꢀe where no damaging eꢃects to cells were observed
puriꢀication. Benzohydroxamic acid (BHA) is commer-
cially available. The percent composition oꢀ the elements
(CHN) ꢀor the compounds was determined by using Thermo
Scientiꢂc Flash 2000 Elemental Analyzer with methionine
as a standard. Melting points were determined in evacu-
ated capillaries using Stuart SMP10 and were uncorrected.
The inꢀrared spectra (IR) were recorded on a Perkin-Elmer
Model 1750X FTIR spectrophotometer in the range oꢀ
[
13]. Chromium(III) glycinate complex supplementation
has also been reported to improve blood glucose level and
attenuates the copper to zinc ratio in tissues oꢀ rats with
mild hyperglycaemia [14]. Additionally, Cis-[Cr(C O )(pm)
2
4
+
(
OH ) ] cation has been tested as a speciꢂc sensing ion ꢀor
2 2
–
1
1
13
the detection oꢀ hydrogen peroxide (H O ) in HT22 hip-
4000–400 cm as KBr discs. The H and C NMR spec-
tra were recorded on a Bruker Varian-600 MHz in deuter-
ated DMSO using TMS as an internal standard. The mass
spectrometric analysis was perꢀormed in an LC1200 Series
Agilent Technologies controlled by Agilent Mass Hunter
Workstation Acquisition (B.02.01). The separation was per-
ꢀormed at 40 ºC with a ZORBAX Eclipse Plus C18 column
2.1 mm×100 mm, 1.8 μm (Agilent Technologies SA, USA)
with (A) 0.1% ꢀormic acid in water and (B) 0.1% ꢀormic acid
in acetonitrile, over a gradient oꢀ 0 to 36 min with increasing
percentage oꢀ B ꢀrom 5 to 95%. The thermal decomposi-
tion behavior oꢀ the metal complexes was recorded using
NETZSCH TG 209 F3 under the nitrogen atmosphere at
2
2
pocampal cells [15]. Being classiꢂed as a Reactive Oxygen
Species (ROS), H O is one oꢀ the most important mediators
2
2
oꢀ oxidative stress sensing under pathological conditions,
and it is also involved in cellular physiological activities as
signal transduction molecules.
Computational chemistry approaches are considered
powerꢀul tools in supporting observed experimental results.
Molecular docking is a computational tool that is largely
used in structural molecular biology and computer-assisted
drug design to ꢂnd and predict the possible predominant
binding modes between a docked ligand against a biological
target protein. It has been successꢀully used to investigate the
binding modes oꢀ many alpha-glucosidase inhibitors [16,
–
1
a heating rate oꢀ 10 ºC min ꢀrom room temperature to
900 ºC. Magnetic moments oꢀ the complexes were meas-
ured using the Guoy method with water as calibrant on
Sherwood Auto Magnetic Susceptibility Balance. Molar
conductivity measurements oꢀ complexes were determined
1
7]. There is an apparent lack oꢀ research on the eꢃect oꢀ
electron-withdrawing and electron-donating substituents on
hydroxamic acids towards the cytotoxicity and antidiabetic
activities.
–
3
In this paper, we report the synthesis and characterization
oꢀ BHA and its methyl, methoxy and chloro- substituted
derivatives namely CH –BHA, OCH –BHA, and Cl–BHA,
in dimethylsulꢀoxide (DMSO) and ethanol (10 M) at room
temperature using a Mettler Toledo Inlab 730 conductivity
meter. The UV–Visible spectra were obtained in ethanol in
the 900–200 nm range using Perkin Elmer UV–Vis Lambda
35 spectrophotometer at room temperature.
3
3
respectively, and their Cr(III) metal complexes. All syn-
thesized compounds were characterized through elemental
1
13
analysis, FTIR, H and C NMR, TGA, mass spectrometry,
molar conductance and UV–Visible. A preliminary cytotox-
icity screening against HCT116 and alpha-glucosidase inhi-
bition test oꢀ the hydroxamic acids and their chromium(III)
complexes were carried out and reported herein.
Synthesis
Synthesis of methylbenzohydroxamic acid, CH –BHA
3
The synthesis oꢀ CH –BHA is represented in Scheme 1.
3
Hydroxylamine hydrochloride (12 mmol, 0.9555 g) was
Materials and methods
added to a mixture solution oꢀ ethyl acetate and water con-
taining sodium hydrogen carbonate (NaHCO ). The mixture
3
Chemicals and instruments
was stirred at room temperature. 4-Methylbenzoyl chloride
was diluted with small amounts oꢀ ethyl acetate was added
dropwise to the mixture. The mixture was stirred ꢀor 5 min
at room temperature. The solvent was removed in vacuo
All the chemicals and reagents were purchased ꢀrom Sigma
Aldrich and Merck and used as received without ꢀurther
Scheme 1 Formation reaction
where X= CH ,Cl
3
oꢀ CH -BHA and Cl-BHA
3
OH
HN
O
NaHCO , EtOAc
O
3
Cl
H
+
Cl
NH2
X
X
HO
CH -BHA, Cl-BHA
3
1
3

JBIC Journal of Biological Inorganic Chemistry
–
1
to aꢃord the pure product. Peach solid; yield, 100%; m.p.
Anal. Calcd. ꢀor C H ClNO (171.60 g mol ): C, 49.00;
7 6 2
–
1
1
52–154 ºC. Anal. Calcd. ꢀor C H NO (151.20 gmol ):
H, 3.52; N, 8.16; Found: C, 48.59; H, 3.38; N, 8.32. IR
8
9
2
–
1
C, 63.56; H, 6.00; N, 9.27; Found: C, 62.71; H, 5.70; N,
(KBr, cm ): 3292 (N–H), 2748 (O–H), 1650 (C=O), 900
–
1
1
9
.00. IR (KBr, cm ): 3294 (N–H), 2759 (O–H), 1651
(N–O). H NMR (600 MHz, ppm, DMSO): δ = 7.54 (2
1
(
C = O), 903 (N–O). H NMR (600 MHz, ppm, DMSO):
H, d, Ar–H), 7.80 (2 H, d, Ar − H), 9.16 (1 H, s, O–H),
1
3
δ=2.41 (3H, s, CH ), 7.23–7.62 (2H, d, Ar–H), 7.62 (2H,
11.36 (1H, s, N–H). C NMR (600 MHz, ppm, DMSO):
3
1
3
d, Ar–H). C NMR (600 MHz, ppm, DMSO): δ=164.03,
δ=163.14, 135.91, 131.47, 128.76, 128.43. MS (m/z): Calcd
+
+
1
40.61, 130.26, 128.76, 126.73, 20.89. MS (m/z): Calcd ꢀor
ꢀor Cl–BHA: 172.60 [M+H] ; Found: 172.02 [M+H] .
+
+
CH –BHA: 152.20 [M+H] ; Found: 152.07 [M+H] .
3
Synthesis of Cr(III) complexes
Synthesis of methoxybenzohydroxamic acid, [OCH3–
BHA]•H2O
The reaction oꢀ metal salts with BHA and its derivatives,
CH –BHA, OCH –BHA, and Cl–BHA in 1:2 molar ratio is
3
3
The synthesis oꢀ OCH –BHA is represented in Scheme 2.
represented by Scheme 3.
3
1
0 mL oꢀ ethyl acetate was mixed with sodium bicarbonate
(
24 mmol, 2.1245 g) in 5 mL oꢀ ultra-pure water. Sodium
i) Synthesis of Cr(III) benzohydroxamic acid,
[Cr(BHA) (H O) ]·H O
bicarbonate solution was added to hydroxylamine hydrochlo-
ride (12 mmol, 0.9555 g) and stirred until dissolved at room
temperature. A small amount oꢀ ethyl acetate was added to
2
2
2
2
Chromium(III) chloride hexahydrate, CrCl ·6H O
3
2
(1.63 mmol, 0.4343 g) was dissolved in a hot aqueous
solution (10 mL) oꢀ BHA (3.26 mmol, 0.4471 g). The
pH oꢀ the resulting solution was increased to 5.5 using
0.1 M NaOH solution, a green precipitate appeared.
The obtained solid was ꢂltered, washed with distilled
water and dried in the air beꢀore stored in a desiccator.
Muddy green solid; yield, 13.77%; m.p.>280 ºC. Anal.
4
-methoxybenzoyl chloride (10 mmol, 1.7001 g) to dilute
it beꢀore being added dropwise to the mixture. The mix-
ture was stirred ꢀor 5 min at room temperature. The white
precipitate was collected aꢀter the solvent was removed in
vacuo. Peach solid; yield, 100%; m.p. 160–161 ºC. Anal.
–
1
Calcd. ꢀor C H NO (185.20 gmol ): C, 51.89; H, 5.99;
8
11
4
–
1
–1
N, 7.56; Found: C, 53.37; H, 4.91; N, 6.74. IR (KBr, cm ):
Calcd. ꢀor C H N O Cr (378.30 g mol ): C, 44.45;
1
4
18
2
7
1
3
285 (N–H), 2760 (O–H), 1669 (C = O), 902 (N–O). H
H, 4.80; N, 7.41; Found: C, 44.27; H, 4.10; N, 7.96.
–
1
NMR (600 MHz, ppm, DMSO): δ=3.80 (3 H, s, O–CH ),
IR (KBr, cm ): 1602 (C=O), 917 (N–O), 489 (Cr–O).
MS (m/z): Calcd ꢀor [Cr(BHA) (H O) ]·H O: 379.30
3
6
1
.99 (2H, d, Ar–H), 7.74 (2H, d, Ar–H), 8.93 (1 H, s, O–H),
2
2
2
2
1
3
+
+
1.11 (1 H, s, N–H). C NMR (600 MHz, ppm, DMSO):
[M+H] ; Found: 379.28 [M+H] . Molar conductance:
–
1
2
–1
δ = 161.43, 131.29, 128.59, 113.77, 113.55, 55.20. MS
3.37 Ω cm mol .
+
(
m/z): Calcd ꢀor OCH –BHA: 186.20 [M + H] ; Found:
ii) Synthesis of Cr(III) chlorobenzohydroxamic acid,
[Cr(Cl–BHA) (H O) ]·2H O
3
+
1
85.02 [M+H] .
2
2
2
2
Chromium(III) chloride hexahydrate, CrCl •6H O
3
2
Synthesis of chlorobenzohydroxamic acid, Cl–BHA
(5 mmol, 0.6661 g) was dissolved in 10 mL oꢀ hot
deionized water. Cl–BHA (10 mmol, 1.2431 g) was dis-
solved in 10 mL oꢀ hot absolute ethanol. Then, both oꢀ
The synthesis oꢀ Cl–BHA is illustrated in Scheme 1. A mix-
ture containing 10 mL ethyl acetate, 5 mL ultra-pure water
and sodium bicarbonate (24 mmol, 2.0625 g) was mixed
with hydroxylamine hydrochloride (12 mmol, 0.9767 g). The
solution was stirred at room temperature until dissolved. A
small amount oꢀ ethyl acetate was added dropwise to 4-chlo-
robenzoyl chloride (10 mmol, 1.8104 g) and stirred ꢀor 5 min
at room temperature. The solvent was removed in vacuo and
white solid was obtained beꢀore stored in a desiccator ꢀor
the solutions were mixed and stirred under nitrogen (N )
2
gas ꢀor 3 h. The grey precipitate was obtained, ꢂltered,
washed with the warm water and air-dried. Finally, the
product was collected and stored in a desiccator. Grey
solid; yield, 13.19%; m.p. 188–189 ºC. Anal. Calcd. ꢀor
–
1
C H N O Cl Cr (465.20 g mol ): C, 36.15; H, 3.90;
1
4
18
2
8
2
N, 6.02; Found: C, 36.52; H, 2.79; N, 6.22. IR (KBr,
–
1
cm ): 3294 (N–H), 1651 (C=O), 900 (N–O), 476 (Cr–
O). MS (m/z): Calcd ꢀor [Cr(Cl–BHA) (H O) ]·2H O:
ꢀ
urther use. Peach solid; yield, 100%; m.p. 138–140 ºC.
2
2
2
2
Scheme 2 Formation reaction
OH
HN
O
oꢀ [OCH –BHA]•H O
3
2
NaHCO , EtOAc
O
3
Cl
H
+
H
O
2
Cl
HO
NH2
H CO
3
X
[
OCH -BHA].H O
3 2
1
3

JBIC Journal of Biological Inorganic Chemistry
Scheme 3 Formation reaction
oꢀ Cr(III) complexes oꢀ BHA
and its derivatives
BHA
SUBSTITUTED DERIVATIVES
(CH , OCH , Cl)
3
3
O
O
OH
OH
N
H
N
H
2
2
X
N2
CrCl3.6H2O, EtOH
NaOH
CrCl .6H O, EtOH
3
2
NaOH
X
where X= CH ,OCH ,Cl
3
3
H O
2
H O
O
O
2
O
O
HN
Cr
NH
2H2O
H O
2
HN
Cr
NH
O
O
H O
2
O
O
H O
2
X
+
+
+
–1
2
–1
4
65.20 [M+H] ; Found: 465.22 [M+H] . Molar con-
[M+H] . Molar conductance (DMSO, Ω cm mol ):
0.
–
1
2
–1
ductance (DMSO, Ω cm mol ): 4.06.
iii) Synthesis of Cr(III) methylbenzohydroxamic acid,
[
Cr(CH –BHA) (H O) ]·2H O
Cytotoxicity study
3
2
2
2
2
Chromium(III) chloride hexahydrate, CrCl ·6H O
3
2
(
0.6661 g, 5 mmol) was dissolved in 10 mL oꢀ hot
Cell culture
deionized water. 10 mL oꢀ absolute ethanol was used
to dissolve CH –BHA (1.4356 g, 10 mmol). Then, the
The human colorectal carcinoma cell line, HCT116
(ATCC® CCL-247™), was cultured in the Roswell Park
Memorial Institute (RPMI-1640) medium with 25 mM
HEPES and l-Glutamine (Biowest) supplemented with 10%
heat inactivated ꢀetal bovine serum (FBS) (PAA Laborato-
ries) and 1% penicillin/streptomycin (Sigma Aldrich). The
cultures were maintained in a humidiꢂed incubator at 37 ºC
3
mixture was stirred under nitrogen (N ) gas. The green
2
precipitate was ꢂltered, washed with the warm water
and air-dried ꢀor 24 h beꢀore stored in desiccator. Green
solid; yield, 44.13%; m.p. > 250 ºC. Anal. Calcd. ꢀor
–
1
C H N O Cr (424.40 g mol ): C, 45.29; H, 5.70; N,
1
6
24
2
8
–1
6
.60; Found: C, 45.52; H, 5.31; N, 6.23. IR (KBr, cm ):
3
216 (N–H), 1648 (C=O), 917 (N–O), 478 (Cr–O). MS
in an atmosphere oꢀ 5% CO .
2
(
m/z): Calcd ꢀor [Cr(CH –BHA) (H O) ]·2H O: 379.30
3
2
2
2
2
+
[
M+H] ; Found: 379.28.
MTT assay
+
–1
2
–
[
M+H] . Molar conductance (DMSO, Ω cm mol
1
)
: 0.
HCT116 cells were plated at 7000 cells per well and
were incubated at 37 ºC ꢀor 24 h. BHA and its derivatives
(CH –BHA, OCH –BHA, and Cl–BHA) and their Cr(III)
iv) Synthesis of Cr(III) methoxybenzohydroxamic acid,
Cr(OCH –BHA) (H O) ]·2H O
[
3
2
2
2
2
3
3
1
0 mL oꢀ hot aqueous CrCl ·6H O (5 mmol, 0.6661 g)
metal complexes, all soluble in DMSO, were subjected to
DMSO serial dilutions beꢀore being added to each well.
The cells were treated with the compounds at concentra-
tions ranging between 0.01–100 μM and incubated at 37 ºC
ꢀor 72 h. The cytotoxicity oꢀ the compounds was assessed
using the MTT method utilizing 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT or ꢀormazan)
with minor modiꢂcations [18]. Briefy, 50 μL oꢀ 0.06 mol/L
MTT solution was added to each well and the plates were
incubated at 37 ºC ꢀor 4 h. All solutions were aspirated and
the ꢀormazan crystals were dissolved in DMSO. The plate
was read at 450 nm. Data generated were used to plot a
3
2
solution was mixed with 10 mL oꢀ ethanolic solution
oꢀ OCH –BHA (10 mmol, 1.756 g). The mixture was
3
stirred under N gas ꢀor 3 h. Grey precipitate was ꢀormed,
2
collected by ꢂltration and washed with the warm water.
Finally, the precipitate was stored in a desiccator until
the next use. Grey solid; yield, 25.68%; m.p. 102–104
–
1
ºC. Anal. Calcd. ꢀor C H N O Cr (456.40 g mol ):
1
6
24
2
10
C, 42.11; H, 5.30; N, 6.14; Found: C, 41.51; H, 5.32; N,
–
1
6
.10. IR (KBr, cm ): 3285 (N–H), 1669 (C =O), 917
(
N–O), 433 (Cr–O). MS (m/z): Calcd ꢀor [Cr(OCH –
3
+
BHA) (H O) ]·2H O: 457.40 [M+H] ; Found: 457.35
2
2
2
2
1
3

JBIC Journal of Biological Inorganic Chemistry
–
1
dose–response curve ꢀrom which the concentration oꢀ com-
pounds required to kill 50% oꢀ the cell population (IC )
oꢀ 2760–2749 cm indicating the deprotonation oꢀ OH in
the complexes that coordinate through the oxygen atom ꢀrom
hydroxyl groups [24, 25]. The shiꢀting oꢀ (C=O) to a lower
5
0
was determined.
–
1
wavenumber and appeared at 1602 cm ꢀor BHA series
indicating the involvement oꢀ carbonyl (C=O) during com-
plexation via oxygen atom [26]. The involvement oꢀ C=O
during complexation is supported by the appearance oꢀ the
Alpha‑glucosidase inhibitory assay
The eꢃects oꢀ BHA and its substituted derivatives on alpha-
glucosidase inhibition activity was assessed as described by
Elya [19], by using alpha-glucosidase ꢀrom Saccharomyces
cerevisiae. The substrate, p-nitropheynyl glucopyranoside
–
1
new weak band, (Cr–O) at a range oꢀ 489–433 cm ꢀor all
complexes [27]. The presence oꢀ NH in the complexes indi-
cates that NH group is not involved in complexation [28].
For substituted derivatives BHA, there is no appreciable
shiꢀt ꢀor C=O observed throughout the spectra but the bands
appeared as a shoulder. This case may be due to the mixing
(
pNPG) (1 mM) was prepared in 30 mM oꢀ potassium phos-
phate buꢃer (pH 6.8). 10 μL oꢀ tested compounds with diꢀ-
erent concentrations (0.1–100 mmol/L) was pre-incubated
with 10 μL oꢀ alpha-glucosidase (0.02 U/mL) at 37 ºC ꢀor
0 min. Then, 50 μL oꢀ 1 mM pNPG was added to the
ꢀ
–
1
C=C oꢀ aryl moiety. The medium bands at 899–917 cm
in all series can be ascribed as (N–O) where this band does
not undergo any change, suggesting that –NO is retained
1
mixture to start the reaction. The reaction was incubated
at 37 ºC ꢀor another 30 min. Alpha-glucosidase inhibition
activity was determined by measuring the yellow colored
para-nitrophenol at 405 nm released ꢀrom pNPG. IC oꢀ
–
1
and not coordinated. The diꢃerence ∆ is less than 200 cm
indicates that the ligands act as bidentate in all complexes
[
20], thereꢀore suggesting the coordination oꢀ the complexes
5
0
is through oxygen atoms (O,O).
alpha-glucosidase inhibition activity was calculated and
determined by using Prism 7.0. IC data is representing
5
0
Nuclear magnetic resonance (NMR) spectroscopy
5
0% inhibition oꢀ enzyme activity towards the concentration
oꢀ tested compounds. The percent inhibition was calculated
using Eq. 1:
1
The H NMR spectra ꢀor the hydroxamic acids were recorded
in DMSO-d using tetramethylsilane (TMS) as the internal
ꢀ
ꢀ
ꢁ
ꢁ
6
%
Inhibition = A − A ∕Ac ∗ 100
c e
standard. BHA gave a singlet at 9.08 ppm attributable to
N–H proton. Multiplets were observed at 7.41–7.76 ppm
indicating the aromatic protons. A singlet was observed at
where A and A are the absorbance oꢀ the control and
c
e
extract, respectively.
1
1.24 ppm indicating the OH proton, appeared downꢂeld
because oꢀ the deshielding oꢀ the electronegative atom, oxy-
gen [29].
Molecular docking study
For CH –BHA, a singlet was observed indicating
3
The binding modes between the active complexes and bind-
ing sites oꢀ alpha-glucosidase have been carried out through
molecular docking study using Autodock package [20].
X-ray coordinates oꢀ the targeted alpha-glucosidase and the
original docked acarbose and glucose ligands were down-
loaded ꢀrom the RCSB data bank website with PDB codes
–
7
CH group at 4.91 ppm. Multiplets were observed at
3
.23–7.62 ppm assignable to aromatic protons. Singlets
ꢀ
or −OH and −NH groups were not observed due to proton
exchange with deuterium in the solvent [30]. For Cl–BHA,
singlets were observed at 9.16 ppm ꢀor −NH and 11.36 ppm
ꢀ
or −OH group. Multiplets were observed at 7.54–7.80 ppm
3
W37, 3WY2, respectively [17, 21]. The docking steps have
indicating the aromatic protons. For OCH –BHA, a singlet
3
been described in our previous studies [22, 23].
ꢀ
or −OCH group was observed at 3.80 ppm [31]. The aro-
3
matic protons peaks were observed at 6.99–7.74 ppm as a
doublet. −OH and −NH group peaks were observed as a
singlet at 8.93 and 11.11 ppm, respectively.
Results and discussion
1
3
For C NMR, there are two types oꢀ important carbons,
i.e., carbonyl and aromatic. The carbonyl carbons appeared
downꢂeld at 164.79, 164.03, 163.14 and 161.43 ppm ꢀor
BHA, CH –BHA, Cl–BHA, and OCH –BHA, respec-
Infrared (IR) spectroscopy
The inꢀrared spectra provide valuable inꢀormation regarding
3
3
ꢀ
unctional groups attached to the metal. The observable diꢀ-
tively. These carbonyl carbons appeared downꢀield
because oꢀ the deshielding eꢀꢀect oꢀ an electronega-
tive element, nitrogen [32]. Aromatic carbons oꢀ BHA
appeared at 127.32–133.21 ppm, Cl–BHA appeared at
ꢀ
erences oꢀ the spectra oꢀ the hydroxamic acids and Cr(III)
complexes suggesting there is complexation occurred.
For the Cr(III) complexes, the coordination modes sug-
gested is through bidentate O,O modes ꢀrom the IR spectra
obtained. The disappearance oꢀ the O–H band in the region
128.43–135.91 ppm, CH –BHA, the peaks appeared at
3
126.73–140.61 ppm, and ꢀor OCH –BHA, the aromatic
3
1
3

JBIC Journal of Biological Inorganic Chemistry
carbons appeared at 113.55–131.29 ppm. The methyl peak
or CH –BHA appeared at 20.89 ppm and the methoxy
(Fig. 1a). [Cr(Cl–BHA) (H O) ]•2H O lost 66% oꢀ the
2
2
2
2
ꢀ
mass at 172–284 ºC due to the departure oꢀ 2(C H Cl) moi-
3
7 4
peak ꢀor OCH –BHA appeared at 55.20 ppm [33]. Unꢀor-
eties ꢀollowed by the removal oꢀ 2NH ꢀragments (9%) at
284–469 ºC. Beyond 469 ºC, only the oxide oꢀ chromium
was leꢀt, with no ꢀurther decomposition (Fig. 1b). The
complex [Cr(CH − BHA) (H O) ]•2H O lost 48.89% at
3
1
13
tunately, H and C NMR ꢀor Cr(III) complexes could not
be obtained because all complexes are paramagnetic [34].
3
2
2
2
2
Thermogravimetric analysis (TGA)
172–380 due to the departure oꢀ 2(C H ) moieties, beyond
8 7
which a slow incomplete decomposition occurred up to the
ꢂnal recorded temperature oꢀ 900 °C, leaving the central
CrO N H (Fig. 1c).[Cr(OCH −BHA) (H O) ]·2H O lost
The summary oꢀ the thermal decomposition behavior oꢀ all
Cr(III) complex through thermogravimetric analysis (TGA)
is presented in Table 1. All Cr(III) complexes, unsubstituted
and substituted, displayed decomposition oꢀ 1 and 2 non-
coordinated water molecules, respectively, at the tempera-
ture range oꢀ 25–100 ºC [34], then the thermal decomposi-
tion continued with the removal oꢀ two coordinated water
molecules each in the range oꢀ 100–220 ºC (Table 2).
The unsubstituted complex [Cr(BHA) (H O) ]•H O then
4
2
2
3
2
2
2
2
46.32% at 202–600 due to the departure oꢀ 2(C H O) moie-
7
7
ties leaving behind the central moiety CrC H N O (Fig. 1d)
2
2
2
4
that remained up to 900 °C.
Mass spectroscopy and molar conductivity
The mass spectroscopy oꢀ all ligands and Cr(III) com-
plexes showed good agreement between the calculated and
experimental molar masses, in parallel with the good match
between the calculated and experimental percentages oꢀ C,
H and N. The low molar conductivity values oꢀ the metal
2
2
2
2
ꢀ
urther decomposed to lose 33% oꢀ the mass up to 600 ºC
due to the loss oꢀ two C H ꢀragments oꢀ the benzene rings.
5
5
The weight percent remained constant at 52% beyond 600 °C
up to 900 ºC, attributed to the central CrC H N O moiety
4
2
2
4
Table 1 Thermal behaviour
Compound
Temperature
range (°C)
Weight loss (%)
ound (calculated)
Lost species
indicating the loss oꢀ H O
2
ꢀ
molecules ꢀrom all Cr(III)
complexes
[
Cr(BHA) (H O) ]·H O
40–100
5.00 (4.76)
10.00 (9.52)
33.00 (34.36)
7.00 (7.74)
8.00 (7.74)
66.00 (65.29)
9.00 (7.93)
8.89 (8.48)
8.89 (8.48)
48.89 (48.54)
7.75 (7.89)
7.95 (7.89)
46.32 (46.89)
- 1 non-coordinated H O
- 2 coordinated H O
2
2
2
2
2
1
2
50–220
20–600
2
- 2(C H )
5
5
[
Cr(Cl-BHA) (H O) ] 2H O
25–100
- 2 non-coordinated H O
- 2 coordinated H O
2
2
2
2
2
100–172
172–284
284–469
2
-2(C H Cl)
7
4
-2(NH)
- 2 non-coordinated H O
- 2 coordinated H O
[
Cr(CH -BHA) (H O) ]·2H O
54–100
3
2
2
2
2
2
1
1
07–172
72–380
2
-2(C H )
8
7
[
Cr(OCH -BHA) (H O) ]·2H O
57–100
- 2 non-coordinated H O
- 2 coordinated H O
3
2
2
2
2
2
1
2
07–202
02–600
2
-2(C H O)
7
7
Table 2 Electronic transitions
oꢀ hydroxamic acids and their
Cr(III) complexes
Ligand/complex
BHA
π→π*(aromatic) (nm) n→π*(C=O) (nm) Ligand-to-metal
charge transꢀer
(
LMCT)
220
227
240
237
236
238
254
254
336
275
–
326
–
312
–
–
–
–
–
–
–
467
–
[
Cr(BHA) (H O) ]·H O
2
2
2
2
Cl–BHA
[
Cr(Cl–BHA) (H O) ]·2H O
2
2
2
2
CH –BHA
3
[
[
[
Cr(CH –BHA) (H O) ]·2H O
3
2
2
2
2
OCH -BHA]·H O
3
2
Cr(OCH -BHA) (H O) ]·2H O
–
3
2
2
2
2
1
3

JBIC Journal of Biological Inorganic Chemistry
Cl
Cl
2
H O
H2O
H2O
H
2
O
H O
2
H2O
O
-H
(5.00%)
2
O
O
O
H O
2
O
-2H O
(7.00%)
2
O
O
HN
Cr
HN
Cr
NH
O
O
NH
HN
Cr
NH
HN
Cr
NH
O
O
O
O
O
O
H
2
O
H
2
O
O
O
2
H O
H2O
Cl
Cl
-
2H
2
O
O
O
2
-2H O
(8.00%)
Cl
(
10.00%)
HN
Cr
NH
2
Cl
C
O
O
(66.00%)
-
2(C
5 5
H )
O
O
O
O
O
O
-
2(NH)
(33.00%)
HN
Cr
HN
Cr
HN
Cr
NH
NH
NH
(9.00%)
O
O
O
O
O
O
O
O
Cr
O
O
Cl
(
a) [Cr(BHA) (H O) ]•H O
2
2
2
2
(b) [Cr(Cl-BHA) (H O) ]•2H O
2
2
2
2
H
3
C
H CO
3
3
H C
3
H CO
2
H O
H
2
O
H O
2
Cr
2
H
2
O
H O
2
H O
-
2H
2
O
O
O
2
-2H O
(7.75%)
O
H
2
O
2
O
O
O
(8.89%)
HN
Cr
NH
O
O
HN
NH
HN
Cr
NH
HN
Cr
NH
O
O
O
O
O
O
H O
2
O
H O
O
2
H O
2
H O
2
H O
CH
3
OCH3
CH
3
-2H
2
O
OCH3
-
2H
2
O
(8.89%)
(
7.95%)
H
3
C
3
H CO
2
H
CO
3
2
H
C
C
O
O
O
O
3
(46.32%)
HN
Cr
NH
HN
Cr
NH
(48.89%)
O
O
O
O
O
HN
Cr
NH
O
HN
Cr
O
O
NH
O
O
O
O
CH
3
OCH3
(
c) [Cr(CH -BHA) (H O) ]•2H O
3
2
2
2
2
(d) [Cr(OCH -BHA) (H O) ]•2H O
3
2
2
2
2
Fig. 1 Thermal decomposition oꢀ Cr(III) complexes
complexes as solutions in DMSO indicated the non-electro-
lytic behavior, i.e., the non-existence oꢀ ꢀree ions acting as
electrolytes in the solution [35].
Table 3 IC values oꢀ BHA, its substituted derivatives and Cr(III)
50
complexes on HCT116 cells
Ligand/complex
IC50 ± SEM (μM)
BHA
>100
>100
UV–visible spectroscopy
[
Cr(BHA) (H O) ]·H O
2
2
2
2
Cl-BHA
58.33± 1.87
27.50± 5.09
>100
The UV–Visible spectra oꢀ hydroxamic acids and their cor-
responding Cr(III) complexes as ethanolic solutions at con-
[
Cr(Cl–BHA) (H O) ]·2H O
2 2 2 2
–4
–5
CH –BHA
3
centrations oꢀ 1.00 × 10 and 1.00 × 10 M were measured
in the range oꢀ 200–800 nm (Table 2). The π–π*(aromatic)
and n–π*(C = O) electronic transitions oꢀ each compound
were identiꢂed in the regions oꢀ 220–254 and 275–336 nm,
respectively. The n → π*(C = O) peaks oꢀ the substituted
ligands and the [Cr(OCH –BHA) (H O) ]·2H O complex
[
Cr(CH –BHA) (H O) ]·2H O
>100
3
2
2
2
2
[
OCH –BHA]·H O
38.00± 4.82
20.56± 2.47
13.07± 0.00
3
2
[
5
Cr(OCH –BHA) (H O) ]·2H O
-Fluorouracil (standard)
3 2 2 2 2
3
2
2
2
2
were not observed, likely due to the presence oꢀ hydrogen
bonding between the polar solvent and the chromophore [36]
or obscured by the strong neighboring peak.
C o m p a r i n g t h e s p e c t r a o ꢀ B H A a n d
[Cr(BHA) (H O) ]·H O, the n → π*(C = O) was observed
2
2
2
2
to experience a hypsochromic shiꢀt towards a lower
1
3

JBIC Journal of Biological Inorganic Chemistry
wavelength, ꢀrom 336 to 275 nm, indicating that complexa-
tion has occurred [37]. The π→π*(aromatic) did not show
any signiꢂcant shiꢀting upon complexation indicating that
donor atoms involved in bonding with Cr(III) centers were
not in the proximity oꢀ the aromatic moieties, hence support-
ing the proposed structure oꢀ Cr(III) coordination through
C=O, located two atoms away ꢀrom the benzene ring. The
ligand-to-metal charge transꢀer (LMCT) bands were only
observed in [Cr(CH –BHA) (H O) ]•2H O at 467 nm [34].
permeability rule. According to Tweedy, the polarity oꢀ the
metal ion will be lowered upon complexation due to the
overlapping oꢀ ligand orbitals and partial sharing oꢀ positive
charge with the donor groups. Complexation also increases
the delocalization oꢀ π-electrons over the entire chelate ring,
enhancing the lipophilicity oꢀ the complexes. Overtone’s
concept oꢀ cell permeability states that the entry oꢀ any
molecule into a cell is governed by its lipophilicity because
the lipid membrane surrounding the cell ꢀavors the passage
oꢀ materials that are soluble in lipids. Thus, the increased
lipophilicity upon complexation enhances the penetration
oꢀ the complexes into the cells and blocks the metal binding
sites oꢀ receptors [39].
3
2
2
2
2
Cytotoxicity assay
The synthesized hydroxamic acids and Cr(III) complexes
were evaluated ꢀor cytotoxicity against human colorectal car-
cinoma cell line, HCT116. All tested compounds induced
a concentration-dependent anti-proliꢀerative eꢃect towards
HCT116 cells upon treatment ꢀor 24 h [38]. IC values oꢀ all
Alpha‑glucosidase inhibitory activity and molecular
docking results
50
compounds on HCT116 cells are shown in Table 3.
All oꢀ the synthesized ligands and complexes were tested on
their alpha-glucosidase inhibitory activity against Saccharo-
myces cerevisiae (Table 4). At 100 μM, all hydroxamic acids
are inactive, whereas all Cr(III) complexes showed excellent
BHA, CH –BHA and their Cr(III) complexes dis-
3
played very low cytotoxicity against HCT116. The
OCH – and Cl– substituted ligands and complexes showed
3
much enhanced antiproliꢀerative properties with IC values
inhibitory properties with IC values much lower than the
5
0
50
oꢀ 20.56–58.33 µM with [Cr(OCH –BHA) (H O) ]•2H O
Acarbose standard. The highest inhibition towards alpha-glu-
3
2
2
2
2
being the most active. The presence oꢀ lone pair bearing
cosidase was shown by [Cr(BHA) (H O) ]·H O ꢀollowed by
2
2
2
2
OCH and Cl substituents enhanced the activity, particularly
[Cr(CH –BHA) (H O) ]·2H O, [Cr(Cl–BHA) (H O) ]·2H O
3
3 2 2 2 2 2 2 2 2
in the presence oꢀ Cr(III) centers.
and [Cr(OCH –BHA) (H O) ]·2H O with the IC values oꢀ
3 2 2 2 2 50
The Cr(III) complexes revealed higher cytotoxicity than
their parent ligands. This observation can be explained
through Tweedy’s chelation theory and Overtone’s cell
28.7, 69.28, 169.5 and 355.9 μM, respectively. It was inter-
esting to note that in the presence oꢀ substituents lowered the
alpha-glucosidase inhibition, in contrast to the antiproliꢀera-
tive properties.
In an attempt to rationalize the observed alpha-glucosi-
dase inhibition oꢀ the active complexes, binding energies oꢀ
the stable complexes ꢀormed between docked ligands and
alpha-glucosidase, the number oꢀ intermolecular hydrogen
bonds between the docked ligands and residues into the
active site in alpha-glucosidase; and the number oꢀ resi-
dues involved in intermolecular interactions are gathered
in Table 5.
Table 4 Alpha-glucosidase inhibitory activity against BHA and its
substituted derivatives oꢀ CH -BHA, OCH -BHA, Cl-BHA series
3
3
Ligand/complex
IC50 ± SEM (μM)
Acarbose (standard)
BHA
418± 0.55
Not active
28.7± 0.98
Not active
169.5± 0.46
Not active
69.28± 0.13
Not active
355.9± 0.87
[
Cr(BHA) (H O) ]·H O
2 2 2 2
Cl–BHA
As can be seen ꢀrom Table 5, all complexes
[
Cr(Cl–BHA) (H O) ]·2H O
2 2 2 2
ꢀ
[
[
[
ormed between the docked metal complexes
Cr(BHA) (H O) ]·H O, [Cr(CH –BHA) (H O) ]·2H O,
CH –BHA
3
2
2
2
2
3
2
2
2
2
[
[
[
Cr(CH –BHA) (H O) ]·2H O
3 2 2 2 2
C r ( O C H – B H A ) ( H O ) ] · 2 H O
a n d
3
2
2
2
2
OCH –BHA]·H O
3
2
Cr(Cl–BHA) (H O) ]·2H O into the active site oꢀ the tar-
2
2
2
2
Cr(OCH –BHA) (H O) ]·2H O
3
2
2
2
2
geted enzyme showed negative binding energies, which
Table 5 Docking binding
Complexes
Free binding energy Number oꢀ Number oꢀ clos- IC50 ± SEM
energies and the inhibition
(
kcal/mol)
HBs
est residues
activities oꢀ the docked Cr(III)
complexes into the active site oꢀ
alpha-glucosidase (PDB 3W37)
[
[
[
[
Cr(BHA) (H O) ]·H O
Cr(Cl–BHA) (H O) ]·2H O
−8.78
−7.66
−7.81
−7.75
5
4
5
6
9
28.7± 0.98
169.5± 0.46
69.28± 0.13
355.9± 0.87
2
2
2
2
11
11
10
2
2
2
2
Cr(CH –BHA) (H O) ]·2H O
3
2
2
2
2
Cr(OCH -BHA) (H O) ]·2H O
3
2
2
2
2
1
3

JBIC Journal of Biological Inorganic Chemistry
Fig. 2 3D views oꢀ molecular
interactions between active site
residues oꢀ alpha-glucosidase
and the docked complexes
1
3

JBIC Journal of Biological Inorganic Chemistry
Fig. 2 (continued)
indicates that the docking oꢀ the title complexes are ther-
modynamically ꢀavorable. Experimental tests showed that
the chromium complexes have greater potential as alpha-
glucosidase inhibitors, seen through better suppression oꢀ
the enzyme compared to the standard, Acarbose. Chromium
complexes undergo hydrogen bondings to the enzyme and
its substrates as shown in Fig. 2, and the conꢀormation and
orientation oꢀ the inhibitors at the active site make them
better in inhibiting the alpha-glucosidase [31].
[
Cr(BHA) (H O) ]·H O is more active than the Cr com-
2 2 2 2
plexed substituted ligand [Cr(CH –BHA) (H O) ]·2H O,
3
2
2
2
2
[
[
C r ( O C H – B H A ) ( H O ) ] · 2 H O
a n d
3
2
2
2
2
Cr(Cl–BHA) (H O) ]·2H O (Table 4). This result is in good
2
2
2
2
accordance with the docking results, which showed that
the docked alpha-glucosidase–Cr(CH O–BHA) Cr(BHA)
3
complex is the most stable complex with a binding
energy oꢀ − 8.78 kcal/mol. Similarly, the higher inhi-
bition eꢀꢀiciency oꢀ [Cr(CH –BHA) (H O) ]·2H O
Conclusion
3
2
2
2
2
compared with [Cr(OCH –BHA) (H O) ]·2H O and
BHA and its substituted derivatives together with Cr(III)
complexes were successꢀully synthesized and characterized
3
2
2
2
2
[
Cr(Cl–BHA) (H O) ]·2H O is mainly reꢀer to the stabil-
2 2 2 2
1
13
ity oꢀ the complex ꢀormed with the ꢀormer compared with
the latters (Table 5). It is worth to mention that the most
active complexes established ꢀive hydrogen bonds with
active amino acids oꢀ alpha-glucosidase. In case oꢀ alpha-
glucosidase–Cr(BHA) complex, the strongest hydrogen
bond is ꢀormed between ASP232 amino acid and the hydro-
gen atom oꢀ water in [Cr(BHA) (H O) ]•H O oꢀ 2.06 Å,
by elemental analysis, FTIR, H and C NMR, UV–Vis-
ible, TGA analysis, mass spectroscopy as well as molar
conductivity. All the spectral data showed that the compl-
exation oꢀ the metal center, Cr(III) to the ligands is through
oxygen and oxygen atoms (O,O) in a bidentate manner.
On the basis oꢀ chelation theory, metal complexes showed
better toxicity than ꢀree ligands.[OCH –BHA]·H O and
2
2
2
2
3
2
while ꢀor alpha-glucosidase–[Cr(CH BHA) (H O) ]
its Cr(III) complex showed the highest inhibition towards
the HCT116 albeit less potent than the 5-FU standard.
[Cr(BHA) (H O) ]·H O, [Cr(CH –BHA) (H O) ]·2H O,
3
2
2
2
the strongest hydrogen bond is established between
ASP568 amino acid and the hydrogen atom oꢀ water in
2
2
2
2
3
2
2
2
2
[
Cr(CH –BHA) (H O) ]•2H O oꢀ 2.15 Å (Figs. 2, 3). It is
[ C r ( O C H – B H A ) ( H O ) ] · 2 H O
a n d
3
2
2
2
2
3
2
2
2
2
obvious ꢀrom docking results (Table 5 and Figs. 2, 3) that
the number oꢀ the closest residues involved in intermolecular
interactions with docked complexes has a negligible eꢃect
on alpha-glucosidase.
[Cr(Cl–BHA) (H O) ]·2H O showed better alpha-glucosi-
2 2 2 2
dase inhibition ability than the Acarbose standard. Binding
energy analysis oꢀ complexes ꢀormed between the active
complexes and amino acids residues oꢀ alpha-glucosidase
revealed the docking oꢀ the complexes is thermodynami-
cally ꢀavorable, and the higher inhibition eꢀꢀiciency oꢀ
[Cr(BHA) (H O) ]·H O is mainly due to its stability and
From the docking results, it is obvious that the pres-
ence oꢀ electron-donating or withdrawing groups had no
signiꢂcant eꢃects on the inhibitory activity oꢀ alpha-glu-
cosidase, in total accord to the experimental ꢂndings. All
2
2
2
2
binding energy.
1
3

JBIC Journal of Biological Inorganic Chemistry
Fig. 3 2D views oꢀ molecular
interactions between active site
residues oꢀ alpha-glucosidase
and the docked Cr(III) com-
plexes
1
3

JBIC Journal of Biological Inorganic Chemistry
Fig. 3 (continued)
Funding This work was ꢀunded by the Ministry oꢀ Higher Education
oꢀ Malaysia ꢀor the research ꢀund; 600-IRMI 5/3/GIP (004/2018), Uni-
versiti Teknologi MARA and AuRIns ꢀor research ꢀacilities and Faculty
oꢀ Pharmacy ꢀor the ꢀacilities on cytotoxicity study.
Compliance with ethical standards
Conflict of interest The authors declare that they have no confict oꢀ
interest.
1
3

JBIC Journal of Biological Inorganic Chemistry
1
1
2
2
2
8. Hazalin NA, Ramasamy K, Lim SSM, Wahab IA, Cole AL,
Majeed ABA (2009) Cytotoxic and antibacterial activities
oꢀ endophytic ꢀungi isolated ꢀrom plants at the National Park,
Pahang, Malaysia. BMC Complement Altern Med 9:1–5
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