Utilization of the common functional groups in bioactive molecules: Exploring dual inhibitory potential and computational analysis of keto esters against α-glucosidase and carbonic anhydrase-II enzymes

Article abstract of DOI:10.1016/j.ijbiomac.2020.11.170

Diabetes mellitus, a progressive chronic disease, characterized by the abnormal carbohydrate metabolism is associated with severe health complications including long term dysfunction or failure of several organs, cardiovascular and micro-angiopathic problems (neuropathy, nephropathy, retinopathy). Despite the existence of diverse chemical structural libraries of α-glucosidase inhibitors, the limited diabetic treatment due to the adverse side effects such as abdominal distention, flatulence, diarrhoea, and liver damage associated with these inhibitors encourage the medicinal research community to design and develop new and potent inhibitors of α-glucosidase with better pharmacokinetic properties. In this perspective, we demonstrate the successful integration of common functional groups (ketone & ester) in one combined pharmacophore which is favorable for the formation of hydrogen bonds and other weaker interactions with the target proteins. These keto ester derivatives were screened for their α-glucosidase inhibition potential and the in vitro results revealed compound 3c as the highly active inhibitor with an IC₅₀ value of 12.4 ± 0.16 μM compared to acarbose (IC₅₀ = 942 ± 0.74 μM). This inhibition potency was ~76-fold higher than acarbose. Other potent compounds were 3f (IC₅₀ = 28.0 ± 0.28 μM), 3h (IC₅₀ = 33.9 ± 0.09 μM), 3g (IC₅₀ = 34.1 ± 0.04 μM), and 3d (IC₅₀ = 76.5 ± 2.0 μM). In addition, the emerging use of carbonic anhydrase inhibitors for the treatment of diabetic retinopathy (a leading cause of vision loss) prompted us to screen the keto ester derivatives for the inhibition of carbonic anhydrase-II. Compound 3b was found significantly active against carbonic anhydrase-II with an IC₅₀ of 16.5 ± 0.92 μM (acetazolamide; IC₅₀ = 18.2 ± 1.23 μM). Compound 3a also exhibited comparable potency with an IC₅₀ value of 18.9 ± 1.08 μM. Several structure-activity relationship analyses depicted the influence of the substitution pattern on both the aromatic rings. Molecular docking analysis revealed the formation of several H-bonding interactions through the ester carbonyl and the nitro oxygens of 3c with the side chains of His348, Arg212 and His279 in the active pocket of α-glucosidase whereas 3b interacted with His95, -OH of Thr197, Thr198 and WAT462 in the active site of carbonic anhydrase-II. Furthermore, evaluation of ADME properties suggests the safer pharmacological profile of the tested derivatives.

Full text of DOI:10.1016/j.ijbiomac.2020.11.170

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Utilization of the common functional groups in bioactive
molecules: Exploring dual inhibitory potential and computational
analysis of keto esters against α-glucosidase and carbonic
anhydrase-II enzymes
Imtiaz Khan, Ajmal Khan, Sobia Ahsan Halim, Majid Khan,
Sumera Zaib, Balqees Essa Mohammad Al-Yahyaei, Ahmed Al-
Harrasi, Aliya Ibrar
PII:
S0141-8130(20)35095-9
DOI:
https://doi.org/10.1016/j.ijbiomac.2020.11.170
BIOMAC 17333
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
23 October 2020
21 November 2020
24 November 2020
Please cite this article as: I. Khan, A. Khan, S.A. Halim, et al., Utilization of the common
functional groups in bioactive molecules: Exploring dual inhibitory potential and
computational analysis of keto esters against α-glucosidase and carbonic anhydrase-II
enzymes, International Journal of Biological Macromolecules (2018), https://doi.org/
10.1016/j.ijbiomac.2020.11.170
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Utilization of the common functional groups in bioactive molecules: Exploring dual inhibitory
potential and computational analysis of keto esters against α-glucosidase and carbonic
anhydrase-II enzymes
Imtiaz Khan,a,* Ajmal Khan,b Sobia Ahsan Halim,b Majid Khan,b,c Sumera Zaib,d
Balqees Essa Mohammad Al-Yahyaei,b Ahmed Al-Harrasi,b,* Aliya Ibrar,e,*
aDepartment of Chemistry and Manchester Institute of Biotechnology, The University of Manchester,
131 Princess Street, Manchester M1 7DN, United Kingdom
bNatural and Medical Sciences Research Center, University of Nizwa, P.O Box 33, Postal Code 616,
Birkat Al Mauz, Nizwa, Oman
cH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan
dDepartment of Biochemistry, Faculty of Life Sciences, University of Central Punjab, Lahore-54590,
Pakistan
eDepartment of Chemistry, Faculty of Natural Sciences, The University of Haripur, Haripur, KPK-
22620, Pakistan
*Correspondence: imtiaz.khan@manchester.ac.uk (I. Khan); aharrasi@unizwa.edu.om (A. Al-
Harrasi); jadoonquaidian@gmail.com (A. Ibrar).
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Abstract
Diabetes mellitus, a progressive chronic disease, characterized by the abnormal carbohydrate
metabolism is associated with severe health complications including long term dysfunction or failure of
several organs, cardiovascular and micro-angiopathic problems (neuropathy, nephropathy, retinopathy).
Despite the existence of diverse chemical structural libraries of α-glucosidase inhibitors, the limited
diabetic treatment due to the adverse side effects such as abdominal distention, flatulence, diarrhoea,
and liver damage associated with these inhibitors encourage the medicinal research community to
design and develop new and potent inhibitors of α-glucosidase with better pharmacokinetic properties.
In this perspective, we demonstrate the successful integration of common functional groups (ketone &
ester) in one combined pharmacophore which is favourable for the formation of hydrogen bonds and
other weaker interactions with the target proteins. These keto ester derivatives were screened for their
α-glucosidase inhibition potential and the in vitro results revealed compound 3c as the highly active
inhibitor with an IC50 value of 12.4 ± 0.16 µM compared to acarbose (IC50 = 942 ± 0.74 µM). This
inhibition potency was ~76-fold higher than acarbose. Other potent compounds were 3f (IC50 = 28.0 ±
0.28 µM), 3h (IC50 = 33.9 ± 0.09 µM), 3g (IC50 = 34.1 ± 0.04 µM), and 3d (IC50 = 76.5 ± 2.0 µM). In
addition, the emerging use of carbonic anhydrase inhibitors for the treatment of diabetic retinopathy (a
leading cause of vision loss) prompted us to screen the keto ester derivatives for the inhibition of
carbonic anhydrase-II. Compound 3b was found significantly active against carbonic anhydrase-II with
an IC50 of 16.5 ± 0.92 µM (acetazolamide; IC50 = 18.2 ± 1.23 µM). Compound 3a also exhibited
comparable potency with an IC50 value of 18.9 ± 1.08 µM. Several structure-activity relationship
analyses depicted the influence of the substitution pattern on both the aromatic rings. Molecular
docking analysis revealed the formation of several H-bonding interactions through the ester carbonyl
and the nitro oxygens of 3c with the side chains of His348, Arg212 and His279 in the active pocket of
α-glucosidase whereas 3b interacted with His95, -OH of Thr197, Thr198 and WAT462 in the active
site of carbonic anhydrase-II. Furthermore, evaluation of ADME properties suggests the safer
pharmacological profile of the tested derivatives.
Keywords: Diabetes mellitus; α-Glucosidase; Carbonic anhydrase; Keto esters; Diabetic retinopathy;
PAINS; ADME properties; Structure-activity relationships.
1. Introduction
Diabetes mellitus (DM), a multifactorial chronic metabolic disorder, largely characterized by the
abnormal carbohydrate metabolism remained as one of the rapidly growing serious global health
threats [1]. According to International Diabetes Federation (IDF) and World Health Organization
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(WHO), the expected number of diabetic patients could be up to 693 million in 2045 and this steady
increase contributes enormously towards the overall risk of dying prematurely [2]. Type II diabetes
mellitus contributes about 80-90% of all diabetic cases where body cannot use the insulin properly
resulting in the poor control over glucose metabolism [3-5], thus rendering the diabetic patients more
vulnerable to severe health complications such as increased risk of common infections, cancer, kidney
failure, leg amputation, vision loss, and the development of micro-angiopathic problems such as
neuropathy, nephropathy and retinopathy [6-10]. Current anti-diabetic drugs available for the treatment
of type 2 diabetes include insulin secretagogues (sulfonylureas, biguanides, thiazolidinediones) and
insulin mimetics (glucagon-like peptide analogues and agonists, α-glucosidase, sodium-glucose
cotransporter, and DPP IV inhibitors) [11]. Despite intensive lifestyle modifications combined with
oral medication, there remained a challenge in the scientific community to develop more potent and
less toxic clinical agents with reduced side effects to treat diabetic complications more effectively.
As such the inhibition of α-glucosidase (EC 3.2.1.20), an intestinal cell membrane enzyme responsible
for the hydrolysis of polysaccharides into monosaccharides, is considered as an essential target for the
discovery of novel inhibitors for the treatment of non-insulin-dependent diabetes mellitus (type II)
[12,13]. These inhibitors slow down the digestion of carbohydrates which in turn suppress the
postprandial hyperglycemia thereby reducing the absorption of gut glucose into the blood [14,15].
Several α-glucosidase inhibitors including metformin, acarbose, voglibose, and miglitol are
commercially available for the treatment of type 2 diabetes (Fig. 1). However, the serious side effects
such as abdominal distention, flatulence and diarrhoea have inspired the medicinal research community
to design and develop several synthetic heterocyclic and non-heterocyclic inhibitors with better
pharmacological profile [16-22].
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Fig. 1. Examples of clinical drugs for type 2 diabetes mellitus.
Diabetic retinopathy (DR), characterized by retinal ischemia, proliferation of retinal vessels and
increased retinal vascular permeability, is the leading cause of vision loss among working-age
populations in developed countries [23,24]. Despite having limited treatment options such as tight
glycaemic, blood pressure control and destructive laser surgery, this disease is prevalent in almost all
patients with type 1 diabetes and over 60% with type 2 diabetes [25,26]. Among the emerging effective
treatment options, the inhibition of carbonic anhydrases (EC 4.2.1.1), zinc-containing metalloenzymes,
is considered as promising therapeutic approach by lowering the intraocular pressure [27]. These
ubiquitous enzymes are actively involved in the rapid conversion of carbon dioxide to bicarbonate and
protons, thus playing an important role in several physiological and pathological processes [28,29]. Out
of 16 isozymes, CA I, II, IV and XIV have been identified in the human eye [30] and are responsible
for bicarbonate secretion regulating aqueous humor production and intraocular pressure. Well-
recognized CA II inhibitors including acetazolamide, methazolamide and dichlorphenamide showed
promising effects in treating glaucoma, characterized by excessive aqueous humor and elevated
intraocular pressure (IOP) through multiple mechanisms [23,31] (Fig. 2). Therefore, design and
development of new small molecule inhibitors offer therapeutic promise for the treatment of diabetic
retinopathy.
Inspired by the above-mentioned literature precedents and in continuation of our efforts on the
development of α-glucosidase inhibitors [32,33], in the present study we investigated a series of keto
esters which are versatile intermediates in organic chemistry, agrochemicals, and pharmaceuticals [34-
44]. To the best of our knowledge, this motif has not been evaluated for the identification of α-
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glucosidase and carbonic anhydrase inhibitors. Therefore, the utilization of the common functional
groups (keto esters) in bioactive molecules was explored while investigating their scope as potential
inhibitors of α-glucosidase and carbonic anhydrase for the treatment of diabetes mellitus and diabetic
retinopathy, respectively. In vitro biological activity results were rationalized using molecular docking
approach where the binding interactions of the potent inhibitors with the specific amino acid residues
were the key stabilizing factor in the active pocket of the enzyme. Furthermore, evaluation of ADME
properties of all the screened derivatives suggests their safer pharmacological profile.
Fig. 2. Schematic illustration of pathophysiologic characteristics of diabetic retinopathy and
mechanisms of anti-CA therapy in each stage (color pattern depicts the events at different stages).
Reproduced with permission from Ref. [23]. Copyright 2009, Elsevier Ltd.
2. Results and discussion
2.1. Synthetic chemistry
The synthetic route for the synthesis of keto esters 3a–m is depicted in Scheme 1. Halogenated acids
1a,b were successfully coupled with diversely substituted phenacyl bromides 2a‒h in the presence of
triethylamine in DMF at room temperature [45-47]. The phenacyl bromides used were either purchased
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from commercial source or prepared via the bromination of acetophenones [48]. The desired keto esters
3a–m were furnished in good yields. The structural diversity was inherited from acetophenones which
possess a range of electron-donating and electron-withdrawing groups on the aromatic ring. The
condensation of aromatic acids with phenacyl bromides was evidenced by the evaluation of FTIR
spectra where characteristic stretching bands for two carbonyls (vC=OKeto and vC=Oester) were observed
in the range of 1702–1682 cm–1 and 1732–1717 cm–1, respectively. Additionally, the spectra were
1
devoid of stretching absorption for COOH in the range of 3400–2400 cm–1. H NMR analysis also
revealed the successful coupling of aromatic acids with phenacyl bromides where methylene signals
were observed in the range of 5.75–5.53 ppm as singlet alongside the disappearance of characteristic
COOH proton. Further evidence was acquired from 13C NMR data where the two distinctive signals for
keto and ester carbonyl carbons appeared in the range of 195.3–182.9 ppm and 167.9–163.3 ppm,
respectively. The methylene carbon also resonated in the range of 66.8–66.4 ppm. Moreover, the mass
spectroscopic data also confirmed the formation of keto ester products.
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Scheme 1. Synthesis and scope of keto esters 3a‒m.
2.2. Enzymatic activity
The use of small molecule inhibitors with dual mode of action in drug discovery programs remained a
driving force for the identification of new classes of compounds incorporating medicinally relevant
pharmacophores. The presence of various functional groups (FGs) as bioactive pharmacophores
contribute enormously to the physicochemical properties and intrinsic reactivity of the parent molecule.
These functional groups have actively been involved in the formation of different interactions with
target proteins. Thus, the combination of more than one functional group in a specific molecule offers a
pressing opportunity to increase the drug discovery space by introducing new combinatorial libraries of
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compounds. These indispensable literature findings on the use of various functional groups in
medicinal chemistry and drug discovery arena prompted us to generate new structures 3a‒m
incorporating ketone and ester functionalities and assess their α-glucosidase and carbonic anhydrase-II
inhibitory potential. The in-vitro screening results are presented in Table 1. Acarbose and
acetazolamide were used as standard inhibitors with IC50 values of 942 ± 0.74 and 18.2 ± 1.23 µM,
against α-glucosidase and carbonic anhydrase-II, respectively. The results demonstrated a striking
variation in the inhibitory activities with IC50 values ranging from 12.4‒381.7 and 16.5‒103.8 µM,
against α-glucosidase and carbonic anhydrase-II enzymes, respectively. In case of α-glucosidase, all
active compounds (3b‒i) showed much higher activity than the standard acarbose while compound 3b
was found significantly active against carbonic anhydrase-II with IC50 of 16.5 ± 0.92 µM. The
predictive structure-activity relationship (SAR) of the tested compounds was investigated considering
the attached functional groups at different positions of both aryl rings as the key contributing
functionalities towards the inhibition of the corresponding enzyme.
2.3. Structure-activity relationship analyses and molecular docking studies
The role of various substituents on the aromatic rings connected with both ketone and ester
functionalities on the inhibition of α-glucosidase and carbonic anhydrase-II enzymes was investigated
and several structure-activity relationship analyses were observed. In vitro activity data presented in
Table 1 for the inhibition of α-glucosidase enzyme revealed compound 3c as the highly active inhibitor
with an IC50 value of 12.4 ± 0.16 µM followed by 3f (IC50 = 28.0 ± 0.28 µM), 3h (IC50 = 33.9 ± 0.09
µM), 3g (IC50 = 34.1 ± 0.04 µM), and 3d (IC50 = 76.5 ± 2.0 µM). The most potent compound 3c
incorporates a bromo substituent at the ortho-position of the phenyl ring derived from carboxylic acid
component whereas a highly polarizable electron-deficient nitro group was present at the para-position
of the aromatic ring (R2). Keeping the acid derived component unchanged, the nitro group was replaced
with hydrophobic electron-donating aromatic (phenyl) and aliphatic (methyl) groups and a descending
trend in the inhibitory potential was observed (3c>3h>3d) (Fig. 3).
Table 1. α-Glucosidase and carbonic anhydrase-II inhibitory potential of keto esters 3a–m.
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Carbonic
anhydrase-II
inhibition
α-Glucosidase
Substituents
inhibition
Compound
R1
R2
IC50 ± SEM (µM)
2-Br
2-Br
4-F-Ph
NA
18.9 ± 1.08
16.5 ± 0.92
44.0 ± 1.12
37.0 ± 2.48
103.8 ± 2.88
60.0 ± 0.72
NA
3a
4-Cl-Ph
381.7 ± 8.1
12.4 ± 0.16
76.5 ± 2.0
112.4 ± 9.6
28.0 ± 0.28
34.1 ± 0.04
33.9 ± 0.09
168.8 ± 4.0
NA
3b
2-Br
4-NO2-Ph
4-Me-Ph
4-OMe-Ph
3,4-diCl-Ph
Naphthyl
Biphenyl
4-F-Ph
3c
2-Br
3d
2-Br
3e
2-Br
3f
2-Br
3g
2-Br
NA
3h
3-Cl,4-F
3-Cl,4-F
3-Cl,4-F
3-Cl,4-F
3-Cl,4-F
NA
3i
4-Cl-Ph
53.3 ± 0.86
90.9 ± 2.83
40.4 ± 0.28
58.8 ± 1.95
―
3j
4-Me-Ph
4-OMe-Ph
3,4-diCl-Ph
NA
3k
3l
NA
NA
3m
―
―
942 ± 0.74
―
Acarbose
Acetazolamide
18.2 ± 1.23
NA = Not active.
Fig. 3. Decreasing effect of substitution pattern on the α-glucosidase inhibition.
Similarly, swapping the nitro group with an electron-rich methoxy (3e; IC50 = 112.4 ± 9.6 µM) as well
as introduction of a di-substitution in the form of chloro group at meta- and para-position (3f; IC50 =
28.0 ± 0.28 µM) also produced reduced inhibition of α-glucosidase compared to 3c (IC50 = 12.4 ± 0.16
µM), however, all the compounds were significantly more active than standard drug (acarbose).
Compound 3g bearing a bulky naphthyl group instead of a 4-nitrophenyl ring also furnished the
excellent inhibitory results comparable to 3h while ~28-folds higher potency compared to acarbose
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(Fig. 4). Moreover, second set of compounds derived from di-halogenated acid were less productive
and only compound 3i showed activity against α-glucosidase enzyme with an IC50 value of 168.8 ± 4.0
µM (Fig. 4). Compound 3b was identified as the least active inhibitor with an IC50 value of 381.7 ± 8.1
µM.
Fig. 4. Decreasing effect of substitution pattern on the α-glucosidase inhibition.
To understand the observed in vitro inhibitory activity results, role of various substitution patterns and
binding interactions in the active site of α-glucosidase, molecular docking analysis was performed. For
this purpose, we used the homology model of S. cerevisiae α-glucosidase due to the unavailability of
the crystal structure. The most potent compound 3c was found to be well accommodated in the active
site of α-glucosidase. The binding mode of 3c showed that the ester carbonyl and the nitro oxygens
mediated multiple H-bonding with the side chains of His348, Arg212 and His279. Additionally, it was
also observed that the keto esters derived from 3-chloro-4-fluorobenzoic acid did not show favorable
binding interactions and both substituents cause steric hindrance in the active site of α-glucosidase
enzyme, thus rendering these derivatives surface exposed.
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Similarly, the side chains of Arg212 and His348 provided H-bonds to the oxygen of ester carbonyl in
compound 3f. Likewise, the ester moieties of 3h and 3g were found to be H-bonded with the side chain
of Arg212. Additionally, a water molecule and Phe157 also offered H-bond and hydrophobic
interaction to compound 3h whereas the ester carbonyls of 3d and 3e were stabilized by the side chain
of His348 through H-bonding. Similarly, the side chains of Arg212 and His348 offered H-bonds to the
ester moiety of the least active inhibitors 3i and 3b. The docked view of the most active hit is shown in
Fig. 5.
Fig. 5. The active site residues of α-glucosidase enzyme are shown in the box. The binding modes of
compounds 3b‒i are depicted in the upper right panel, while the docked view of the most active hit 3e
is shown in the lower right panel. The hydrogen bonds are presented in black lines.
The synthesized compounds were also tested against carbonic anhydrase-II and the in vitro results
revealed that several compounds (3a‒d, 3f‒j, 3l, 3m) possess significant inhibitory affinities when
compared to the standard drug (acetazolamide). Among the screened compounds, 3a and 3b exhibited
highest potency with IC50 values of 18.9 ± 1.08 µM and 16.5 ± 0.92 µM, respectively. Both derivatives
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incorporate halogens (F & Cl) at the para-position of the aryl ring and in combination with the ortho-
bromophenyl ring impart significant influence on the biological inhibition of CA-II enzyme. The loss
of potency was observed when inductively withdrawing chloro substituent was replaced with
moderately electron-donating (methyl) and highly polarizable electron-withdrawing (nitro) groups.
These derivatives (3d & 3c) demonstrated IC50 values of 37.0 ± 2.48 µM and 44.0 ± 1.12 µM,
respectively (Fig. 6). However, presence of a strongly electron-rich (methoxy) substituent imparted
deleterious effect on the inhibition of CA-II enzyme (3e; IC50 = 103.8 ± 2.88 µM). The inhibitory
activity was regained when a bromophenyl ring in 3e was replaced with a di-halogenated (3-Cl,4-F)
phenyl ring (Fig. 7).
Fig. 6. Decreasing effect of substitution pattern on carbonic anhydrase-II inhibition.
Fig. 7. Effect of substitution pattern on carbonic anhydrase-II inhibition.
Furthermore, the introduction of an additional chloro group at the meta-position of the phenyl ring
already equipped with the same substituent at the para-position also proved detrimental, resulting in
moderate inhibitory efficacy (3f; IC50 = 60.0 ± 0.72 µM). The use of sterically demanding biphenyl and
naphthyl groups as R2 (3g & 3h) remained completely impotent. In contrast, the keto ester derivatives
(3j‒m) derived from the di-halogenated acids were identified as the selective inhibitors of CA-II
enzyme and showed no activity against α-glucosidase enzyme. In this set of compounds, electron-rich
(methoxy) and halogen substituted derivatives (3j, 3l, 3m) produced moderate inhibitory efficacy
demonstrating IC50 values in the range of 40.4-58.8 µM (Fig. 8). Compounds 3k (IC50 = 90.9 ± 2.83
µM) and 3e (IC50 = 103.8 ± 2.88 µM) were observed as the least active inhibitors of CA-II.
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Fig. 8. Decreasing effect of substitution pattern on carbonic anhydrase-II inhibition.
For better understand of the binding of the potent inhibitors into the target enzyme (CA-II) and to
rationalize the observed in vitro inhibitory activity results and influence of the different substituents,
molecular docking studies were performed. For this purpose, we used the X-ray crystallographic
structure of CA-II (PDB: 1V9E). The binding mode of 3b revealed the formation of excellent binding
interactions within the active site where the ester carbonyl moiety of 3b mediated multiple H-bonding
with the side chains of His95, -OH of Thr197 and a conserved water molecule, and a metal-ligand
interaction with the Zn2+ ion in the active site of enzyme. The carbonyl oxygen of the compound was
also found to be H-bonded with the side chain of Thr198 and WAT462. The docked view of 3b is
shown in Fig. 9. Similarly, the ester carbonyl of compound 3a was bound with Zn2+ ion through
metallic interactions while the side chains of His95 and Gln91 provided H-bond to the oxygen atom of
the ester carbonyl. Additionally, His63 offered hydrophobic interaction to the compound. The binding
modes of 3a and 3b were found to be nearly similar. On the other hand, compounds 3g-i were not
active in the carbonic anhydrase-II assay. The naphthyl, biphenyl and 4-fluorophenyl moieties as R2 did
not fit into the active site of CA-II. Moreover, the presence of 3-chloro and 4-fluoro substituents in 3i
also causes clashes with the active site residues.
The binding mode of 3d and 3l showed that the ester carbonyl mediated H-bonding with the side chain
of Asn66 and the amino nitrogen of Thr197, respectively. The carbonyl oxygen of 3c and the ester
oxygen of 3j interacted with the side chain of Gln91 through H-bonding while the side chain of His93
provided π-π interactions to these compounds. The ester oxygen of 3m mediated multiple H-bonds with
the side chains of His63 and Asn66 and hydrophobic interaction with the side chain of Phe129.
Similarly, the side chain of Asn66 donated H-bond to the ester oxygen of 3f. However, the side chains
of Gln91 and His93 stabilized the carbonyl moiety of 3k via H-bonding and hydrophobic interaction,
respectively. Compound 3e was the least active inhibitor of CA-II. The side chains of His63 and His93
established H-bonding and hydrophobic interaction, respectively, with the compound 3e. It was
observed that the substitution of more bulky groups as R2 substituent is not favorable for the binding of
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the compound and produced steric hinderance in the active site of CA-II, thus decrease the biological
activities of the compounds. The docking scores and docking interactions of compounds are well
correlated with the inhibitory affinities of the compounds. The binding interactions of 3a‒m in the
active sites of α-glucosidase and CA-II are tabulated in Table S1.
Fig. 9. The active site residues of carbonic anhydrase-II are shown in the box. The binding modes of
compounds 3a‒f and 3j‒m are depicted in the upper right panel, while the docked view of the most
active hit 3b is shown in the lower right panel. The hydrogen bonds are presented in black lines while
metal-ligand coordination is demonstrated in red dotted lines.
2.4. Kinetics studies
To investigate the type of inhibition and dissociation constant of the tested compounds, kinetics studies
of the most active compounds 3b (against bCA-II) and 3c (against α-glucosidase) were performed at
different concentrations of compounds and substrates. Kinetics study of compound 3b against bCA-II
showed that compound 3b inhibited the bCA-II enzyme in a concentration-dependent manner with Ki
value of 17.05 ± 0.021 µM. From the kinetics studies, it was also inferred that the compound 3b is a
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competitive inhibitor for bCA-II. The type of inhibition was determined by Lineweaver-Burk plots, the
reciprocal of the rate of reaction was plotted against the reciprocal of substrate concentrations to
monitor the effect of inhibitor on both Km and Vmax. The Lineweaver-Burk plots of 3b against bCA-II
clearly showed that the mode of inhibition of 3b is competitive (Fig. 10A). In competitive inhibition,
the Km of enzyme increases without affecting Vmax. The Lineweaver-Burk plots also showed that in the
presence of compound 3b, the Km of bCA-II was increased significantly, while the Vmax remains
constant indicating the competitive inhibition.
On the other hand, Kinetics study of compound 3c against α-glucosidase enzyme showed that
compound 3c is a mixed-type inhibitor with Ki value of 18.01 ± 0.018 µM. In a mixed-type inhibition,
both the Km and Vmax are changed. The high Km and low Vmax of α-glucosidase in the presence of
compound 3c indicated a mixed-type of inhibition (Fig. 11A).
The Ki values were determined by secondary replots of Lineweaver-Burk plots by plotting the slope of
each line in the Lineweaver-Burk plots against different concentrations of compounds 3b and 3c (Figs.
10B and 11B). The Ki values were confirmed by Dixon plot after plotting the reciprocal of the rate of
reaction against different concentrations of compounds 3b and 3c (Figs. 10C and 11C).
A
B
C
Fig. 10. The mode of inhibition of compound 3b against bCA-II; (A) Lineweaver–Burk plot of the
reciprocal rate of reaction (velocities) vs. reciprocal of substrate (p-nitrophenol acetate) in the absence
(Δ), and presence of 14 µM (■), 17 µM (□), 20 µM (●), and 23 µM (○) of compound 3b. (B)
Secondary re-plot of Lineweaver–Burk plot between the slopes of each line on Lineweaver–Burk plot
vs. different concentrations of compound 3b. (C) Dixon plot of the reciprocal rate of reaction
(velocities) vs. different concentrations of compound 3b.
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A
B
C
Fig. 11. The mode of inhibition of compound 3c against α-glucosidase; (A) Lineweaver–Burk plot of
the reciprocal rate of reaction (velocities) vs. reciprocal of substrate (p-nitrophenyl-α-D-
glucopyranoside) in the absence (▲), and presence of 8 µM (■), 13 µM (□), 18 µM (●), and 23 µM (○)
of compound 3c. (B) Secondary re-plot of Lineweaver–Burk plot between the slopes of each line on
Lineweaver–Burk plot vs. different concentrations of compound 3c. (C) Dixon plot of the reciprocal
rate of reaction (velocities) vs. different concentrations of compound 3c.
2.5. ADME properties
ADME properties predict the impact of therapeutic compounds to access the target considering some
parameters. These properties can be evaluated using several prediction tools [49-51]. These properties
help in the determination of the drug-likeness of compounds being used for drug discovery and
development by sorting out new druggable candidates that are safer and follow the effective rules used
for determination of these parameters. Below are some important ADME properties compiled using
web service and its underlying methodologies (SwissADME) [49]. The results for all the designed
compounds are reported in Table S2. The properties suggested that our derivatives are safer to use as
drug candidates and have high probability of blood brain penetration and absorption.
2.5.1. Physicochemical properties
Some physicochemical and molecular descriptors are included in the properties such as molecular
weight, number of specific atom types, molecular refractivity and polar surface area (calculated by
fragmental technique called topological polar surface area (TPSA)), taking sulfur and phosphorus in
account as polar atoms [52]. These descriptors are useful in prediction of ADME properties, with
reference to biological barrier penetration like absorption and brain access [49].
2.5.2. Lipophilicity
The partition coefficient between water (log Po/w) and n-octanol demonstrates the classical descriptor
of lipophilicity and some more freely available predictive models, like XLOGP3 (an atomistic method
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including corrective factors and knowledge-based library), WLOGP (our own implementation of a
purely atomistic method based on the fragmental system), MLOGP (an archetype of topological
method relying on a linear relationship with 13 molecular descriptors), SILICOS-IT (an hybrid method
relying on 27 fragments and 7 topological descriptors) and finally iLOGP (our in-house physics-based
method relying on free energies of solvation in n-octanol and water calculated by the Generalized-Born
and solvent accessible surface area (GB/SA) model).
2.5.3. Water solubility
Solubility is an important property enhancing absorption that greatly facilitates many drug development
activities of molecules for discovery projects targeting oral administration. Two very well-established
topological methods used for the prediction of water solubility in SwissADME [49], including an
implementation of the ESOL model. These methods demonstrate strong linear correlation between
predicted and experimental values (R2 = 0.69 and 0.81, respectively). Moreover, another predictor for
solubility was developed by SILICOS-IT. All predicted values are the decimal logarithm of the molar
solubility in water (log S) providing the SwissADME solubility in mol/l and mg/ml.
2.5.4. Pharmacokinetics
The parameters in this section include the predictions for passive human gastrointestinal absorption
(HIA) and blood-brain barrier (BBB) permeation, both are demonstrated in the BOILED-Egg model
(Fig. 12) [51], in addition to the knowledge about compounds being substrate or non-substrate of the
permeability glycoprotein (P-gp). The overexpression of P-gp is an important parameter to monitor in
case of central nervous system and some tumor cells to investigate multidrug-resistance.
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Fig. 12. The BOILED-Egg model predicts the position of molecules in the WLOGP versus TPSA [51].
The BOILED-Egg model predicts the intuitive evaluation of human intestinal absorption and brain
penetration with reference to the position of molecules in the TPSA versus WLOGP. The model
demonstrates the high probability of passive absorption by intestinal tract in white region, while yellow
region demonstrates the high probability of blood brain penetration. Moreover, the red dots indicate
that the molecules act as non-substrate of P-glycoprotein (PGP‒). All the compounds were predicted as
brain-penetrant (lies inside the yolk) and PGP‒, except 3c which is well absorbed but inaccessible to
the brain (lies inside the white) and is not subject to active efflux (PGP‒).
Another important parameter is the knowledge of molecules with reference to interactions with
cytochromes P450 (CYP). This superfamily of isoenzymes is a key player in drug elimination through
metabolic biotransformation. Literature showed that therapeutic molecules are substrate of five major
isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4) and inhibition of these isoenzymes is
certainly one major cause of pharmacokinetics-related drug-drug interactions. It is therefore of great
importance for drug discovery to predict the tendency with which the molecule will cause significant
drug interactions through inhibition of CYPs, and to determine which isoforms are affected.
2.5.5. Drug-likeness
Drug-likeness is a qualitative analysis of a molecule to become oral drug-candidate with enhanced
bioavailability. The SwissADME properties include five different rule-based filters, with diverse
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properties considering a molecule as drug-like. These filters often originate from analyses by major
pharmaceutical companies aiming to improve the quality of their proprietary chemical collections.
2.5.6. Medicinal chemistry
The main purpose of these recognition methods is the identification of the potentially problematic
fragments in a molecule. PAINS (for pan assay interference compounds) are molecules containing
substructures showing potent response in assays irrespective of the protein target. Moreover, lead-
likeness is a similar concept to drug-likeness and focuses on molecular entity acceptable for
optimization and modification to enhance the size and lipophilicity, if required.
2.5.7. Toxicity
All the tested derivatives (3a‒m) have been evaluated for their toxicology profile [53]. The compounds
exhibited non-toxic liver profile and no cytotoxicity was noted (Table S2).
3. Conclusions
In summary, we have synthesized a series of keto ester derivatives 3a‒m through a facile coupling
reaction of halogenated carboxylic acids and diversely substituted phenacyl bromides. The title keto
esters were isolated in 72‒82% yield. The in vitro inhibitory activities against α-glucosidase and
carbonic anhydrase-II enzymes were evaluated and the results revealed that most of the compounds
exhibit potent biological potential. Compound 3c was identified as the lead inhibitor with an IC50 value
of 12.4 ± 0.16 µM, 76-folds higher inhibitory efficacy compared to acarbose (IC50 = 942 ± 0.74 µM).
However, 3b demonstrated the best result against carbonic anhydrase-II with an IC50 of 16.5 ± 0.92 µM
(acetazolamide; IC50 = 18.2 ± 1.23 µM). The bioactivity results, influence of the substitution pattern
and binding modes of the tested compounds were analyzed through molecular docking approach. The
stabilization of the inhibitors in the active site of both enzymes was achieved through the formation of
several conspicuous H-bonding interactions between the keto and ester functional groups and the
amino acid residues. The evaluation of ADME properties such as physicochemical, pharmacokinetics,
drug-likeness and medicinal chemistry affability by different methods such as iLOGP and BOILED-
Egg, etc. suggests the safer pharmacological profile of all the screened derivatives.
4. Experimental
4.1. General chemistry methods
Unless otherwise noted, all materials were obtained from commercial suppliers (Aldrich and Merck
companies) and used without further purification. Thin layer chromatography (TLC) was performed on
Merck DF-Alufoilien 60F254 0.2 mm precoated plates. Product spots were visualized under UV light at
254. Melting points were recorded on a Stuart melting point apparatus (SMP3) and are uncorrected.
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Infra–red (IR) spectra were recorded on FTS 3000 MX, Bio–Rad Merlin (Excalibur model)
1
spectrophotometer. H NMR spectra were recorded on a Bruker Avance (300 MHz) spectrometer.
Chemical shifts () are quoted in parts per million (ppm) downfield of tetramethylsilane, using residual
solvent as internal standard (CDCl3 at 7.24 ppm). Abbreviations used in the description of resonances
13
are: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), Ar (aromatic). Proton–decoupled C
NMR spectra were recorded on a Bruker Avance (75 MHz) spectrometer using deuterated solvent as
internal standard (CDCl3 at 77.23 ppm). Electron-spray ionization high resolution mass spectrometry
(ESI-HRMS, m/z) data was acquired on an Agilent spectrometer (6530, Accurate Mass Q-TOF
LC/MS).
4.2. General procedure for the preparation of keto ester derivatives (3a‒m)
To a stirred solution of corresponding benzoic acids (1a,b) (1.0 mmol) in N,N-dimethylformamide (4
mL) was added triethylamine (3-4 drops) at room temperature. After stirring for 30 min, the
corresponding phenacyl bromide (2a‒h) (1.0 mmol) were added and the reaction mixture was stirred at
room temperature for 2 h. After completion of reaction (monitored by TLC), the reaction mixture was
washed with water and extracted with ethyl acetate (3 × 10 mL). The combined organic fractions were
washed with brine (20 mL), dried over anhydrous MgSO4, filtered and evaporated in vacuo. The
resultant solids were purified by recrystallization in ethanol to afford the corresponding keto esters (3a‒
m) [45-47]. The data was in full agreement to those reported in our previous work [47].
4.2.1. 2-(4-Fluorophenyl)-2-oxoethyl 2-bromobenzoate (3a)
Yield: 78%; m.p.: 98–99 °C; Rf: 0.35 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3015, 2950, 2840, 1732
1
(C=Oester), 1697 (C=Oketo), 1594, 1506 (C=C); H NMR (300 MHz, CDCl3): δH 8.02–7.99 (m, 3H,
ArH), 7.73–7.70 (m, 1H, ArH), 7.46–7.36 (m, 2H, ArH), 7.24–7.18 (m, 2H, ArH), 5.57 (s, 2H, OCH2);
13C NMR (75 MHz, CDCl3): δC 192.9, 161.7 (d, J = 255 Hz), 163.0, 134.5, 133.1, 132.0, 131.0, 130.6,
130.5, 127.3, 122.1, 116.2 (d, J = 21.7 Hz), 66.5; HRMS (ESI+ve): exact mass calculated for
C15H10BrFNaO3 [M+Na]+: 358.96950; found, 359.19800.
4.2.2. 2-(4-Chlorophenyl)-2-oxoethyl 2-bromobenzoate (3b)
Yield: 79%; m.p.: 94–96 °C; Rf: 0.25 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3085, 2932, 2868, 1724
1
(C=Oester), 1698 (C=Oketo), 1584, 1471 (C=C); H NMR (300 MHz, CDCl3): δH 7.91–7.90 (m, 3H,
ArH), 7.73–7.70 (m, 1H, ArH), 7.53–7.50 (m, 2H, ArH), 7.49–7.36 (m, 2H, ArH), 5.57 (s, 2H, OCH2);
13C NMR (75 MHz, CDCl3): δC 192.9, 163.3, 140.6, 136.0, 134.5, 133.1, 132.4, 131.0, 129.3, 129.2,
127.3, 122.1, 66.5; HRMS (ESI+ve): exact mass calculated for C15H10BrClNaO3 [M+Na]+: 374.93995;
found, 374.94533.
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4.2.3. 2-(4-Nitrophenyl)-2-oxoethyl 2-bromobenzoate (3c)
Yield: 72%; m.p.: 92–94 °C; Rf: 0.14 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3045, 2946, 2837, 1725
1
(C=Oester), 1698 (C=Oketo), 1574, 1491 (C=C); H NMR (300 MHz, CDCl3): δH 8.37–8.32 (m, 2H,
ArH), 8.29–8.24 (m, 2H, ArH), 8.07–8.03 (m, 1H, ArH), 7.97–7.93 (m, 2H, ArH), 7.70–7.66 (m, 1H,
13
ArH), 5.57 (s, 2H, OCH2); C NMR (75 MHz, CDCl3): δC 190.1, 165.3, 148.8, 146.5, 138.7, 134.4,
133.3, 132.6, 132.1, 131.0, 129.9, 127.3, 66.4; HRMS (ESI+ve): exact mass calculated for
C15H10BrNNaO5 [M+Na]+: 385.96400; found, 386.04910.
4.2.4. 2-Oxo-2-p-tolylethyl 2-bromobenzoate (3d)
Yield: 75%; m.p.: 91–93 °C; Rf: 0.27 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3031, 2924, 2844, 1728
(C=Oester), 1685 (C=Oketo), 1603, 1481 (C=C); 1H NMR (300 MHz, CDCl3): δH 7.88 (d, 2H, J = 8.4 Hz,
ArH), 7.72–7.69 (m, 2H, ArH), 7.45–7.37 (m, 2H, ArH), 7.36–7.28 (m, 2H, ArH), 5.57 (s, 2H, OCH2),
13
2.45 (s, 3H, CH3); C NMR (75 MHz, CDCl3): δC 192.6, 165.4, 145.1, 134.4, 133.0, 132.0, 131.6,
131.2 (2× C), 127.9, 127.3, 122.1, 66.5, 22.6; HRMS (ESI+ve): exact mass calculated for
C16H13BrNaO3 [M+Na]+: 354.99458; found, 354.99304.
4.2.5. 2-(4-Methoxyphenyl)-2-oxoethyl 2-bromobenzoate (3e)
Yield: 80%; m.p.: 107–109 °C; Rf: 0.20 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3011, 2946, 2842,
1730 (C=Oester), 1682 (C=Oketo), 1598, 1571 (C=C); 1H NMR (300 MHz, CDCl3): δH 7.98–7.96 (m, 1H,
ArH), 7.95–7.94 (m, 2H, ArH), 7.72–7.69 (m, 1H, ArH), 7.46–7.35 (m, 2H, ArH), 7.02–6.97 (m, 2H,
13
ArH), 5.58 (s, 2H, OCH2), 3.90 (s, 3H, OCH3); C NMR (75 MHz, CDCl3): δC 191.3, 165.5, 143.8,
141.0, 134.4, 133.0, 132.0, 131.3, 130.2, 127.3, 122.1, 114.1, 66.5, 55.6; HRMS (ESI+ve): exact mass
calculated for C16H13BrNaO4 [M+Na]+: 370.98949; found, 370.99396.
4.2.6 2-(3,4-Dichlorophenyl)-2-oxoethyl 2-bromobenzoate (3f)
Yield: 75%; m.p.: 84–86 °C; Rf: 0.23 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3101, 2926, 2844, 1728
(C=Oester), 1701 (C=Oketo), 1584, 1489 (C=C); 1H NMR (300 MHz, CDCl3): δH 8.07 (d, 1H, J = 2.1 Hz,
ArH), 8.04–8.01 (m, 1H, ArH), 7.80 (dd, 1H, J = 8.4, 1.8 Hz, ArH), 7.73–7.70 (m, 1H, ArH), 7.60 (s,
1H, ArH), 7.46–7.37 (m, 2H, ArH), 5.54 (s, 2H, OCH2); 13C NMR (75 MHz, CDCl3): δC 189.9, 165.3,
134.8, 134.6, 133.8, 133.6, 133.5, 133.2, 132.3, 132.0, 131.1, 130.8, 129.9, 127.3, 66.5; HRMS
(ESI+ve): exact mass calculated for C15H9BrCl2NaO3 [M+Na]+: 408.90098; found, 408.93258.
4.2.7. 2-(Naphthalen-2-yl)-2-oxoethyl 2-bromobenzoate (3g)
Yield: 74%; m.p.: 88–90 °C; Rf: 0.34 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3054, 2925, 2847, 1732
(C=Oester), 1687 (C=Oketo), 1625, 1590 (C=C); 1H NMR (300 MHz, CDCl3): δH 8.11–7.90 (m, 5H, Ar-
H), 7.73–7.58 (m, 3H, ArH), 7.47–7.37 (m, 3H, ArH), 5.76 (s, 2H, OCH2); 13C NMR (75 MHz,
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CDCl3): δC 191.6, 165.5, 136.0, 134.5, 133.0, 132.4, 132.0, 131.5, 131.2, 129.7, 129.0, 127.9, 127.3,
127.1, 123.3, 122.1 (2 × C), 118.4, 66.8; HRMS (ESI+ve): exact mass calculated for C19H13BrNaO3
[M+Na]+: 390.99458; found, 391.09437.
4.2.8. 2-(Biphenyl-4-yl)-2-oxoethyl 2-bromobenzoate (3h)
Yield: 80%; m.p.: 101–103 °C; Rf: 0.48 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3037, 2933, 2842,
1732 (C=Oester), 1692 (C=Oketo), 1600, 1560 (C=C); 1H NMR (300 MHz, CDCl3): δH 8.06–8.04 (m, 4H,
ArH), 7.77–7.68 (m, 3H, ArH), 7.54–7.39 (m, 6H, ArH), 5.65 (s, 2H, OCH2); 13C NMR (75 MHz,
CDCl3): δC 191.4, 165.4, 146.7, 139.6, 134.5, 133.1 (2 × C), 132.8, 132.1, 131.2, 129.1, 128.5, 128.4,
127.6, 127.3, 122.1, 66.7; HRMS (ESI+ve): exact mass calculated for C21H15BrNaO3 [M+Na]+:
417.01023; found, 417.01350.
4.2.9. 2-(4-Fluorophenyl)-2-oxoethyl 3-chloro-4-fluorobenzoate (3i)
Yield: 77%; m.p.: 121–123 °C; Rf: 0.61 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3054, 2955, 2834,
1723 (C=Oester), 1702 (C=Oketo), 1592, 1496 (C=C); 1H NMR (300 MHz, CDCl3): δH 8.25 (dd, 1H, J =
13
8.7, 2.1 Hz, ArH), 8.08–7.99 (m, 3H, ArH), 7.29–7.19 (m, 3H, ArH), 5.57 (s, 2H, OCH2); C NMR
(75 MHz, CDCl3): δC 190.1, 166.3 (d, J = 255 Hz), 164.1, 161.4 (d, J = 256 Hz), 132.8, 130.6, 130.5,
130.4, 126.5 (d, J = 3 Hz), 121.7 (d, J = 18 Hz), 116.8 (d, J = 21.8 Hz), 116.3 (d, J = 21.8 Hz), 66.5;
HRMS (ESI+ve): exact mass calculated for C15H9ClF2NaO3 [M+Na]+: 333.01060; found, 333.17520.
4.2.10. 2-(4-Chlorophenyl)-2-oxoethyl 3-chloro-4-fluorobenzoate (3j)
Yield: 77%; m.p.: 121–123 °C; Rf: 0.61 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3052, 2939, 2845,
1717 (C=Oester), 1698 (C=Oketo), 1588, 1494 (C=C); 1H NMR (300 MHz, CDCl3): δH 8.24 (dd, 1H, J =
13
7.2, 2.1 Hz, ArH), 7.93–7.90 (m, 2H, ArH), 7.28–7.22 (m, 4H, ArH), 5.56 (s, 2H, OCH2); C NMR
(75 MHz, CDCl3): δC 190.6, 164.1, 161.4 (d, J = 255 Hz), 140.7, 132.6 (d, J = 39 Hz), 130.5, 130.4,
129.4, 129.2, 126.5 (d, J = 3.8 Hz), 121.7 (d, J = 18.8 Hz), 116.8 (d, J = 21.8 Hz), 66.6; HRMS
(ESI+ve): exact mass calculated for C15H9Cl2FNaO3 [M+Na]+: 348.98105; found, 349.16330.
4.2.11. 2-Oxo-2-p-tolylethyl 3-chloro-4-fluorobenzoate (3k)
Yield: 82%; m.p.: 137–139 °C; Rf: 0.42 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3054, 2936, 2841,
1722 (C=Oester), 1694 (C=Oketo), 1593, 1494 (C=C); 1H NMR (300 MHz, CDCl3): δH 8.83-8.22 (d, 2H,
J = 7.5 Hz, ArH), 7.87 (d, 2H, J = 8.1 Hz, ArH), 7.34–7.22 (m, 3H, ArH), 5.59 (s, 2H, OCH2), 2.46 (s,
13
3H, CH3); C NMR (75 MHz, CDCl3): δC 191.2, 164.2, 161.3 (d, J = 255 Hz), 145.1, 132.8, 131.5,
130.5, 130.4, 129.7, 127.9, 126.7 (d, J = 3.8 Hz), 116.8 (d, J = 21.8 Hz), 66.7, 22.6; HRMS (ESI+ve):
exact mass calculated for C16H12ClFNaO3 [M+Na]+: 329.03567; found, 329.19800.
4.2.12. 2-(4-Methoxyphenyl)-2-oxoethyl 3-chloro-4-fluorobenzoate (3l)
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Yield: 78%; m.p.: 134–136 °C; Rf: 0.35 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3048, 2941, 2844,
1720 (C=Oester), 1687 (C=Oketo), 1594, 1573 (C=C); 1H NMR (300 MHz, CDCl3): δH 8.26 (dd, 1H, J =
6.9, 2.1 Hz, ArH), 8.09–8.04 (m, 1H, ArH), 7.98–7.93 (m, 2H, ArH), 7.26 (d, 1H, J = 8.7 Hz, ArH),
13
7.02–6.97 (m, 2H, ArH), 5.56 (s, 2H, OCH2), 3.91 (s, 3H, OCH3); C NMR (75 MHz, CDCl3): δC
190.0, 164.2, 161.3 (d, J = 255 Hz), 132.8, 130.3 (d, J = 8 Hz), 130.1, 127.1, 126.8, 126.7, 121.6 (d, J =
18 Hz), 116.8 (d, J = 21 Hz), 114.2, 66.5, 55.6; HRMS (ESI+ve): exact mass calculated for
C16H12ClFNaO4 [M+Na]+: 345.03058; found, 345.04408.
4.2.13. 2-(3,4-Dichlorophenyl)-2-oxoethyl 3-chloro-4-fluorobenzoate (3m)
Yield: 75%; m.p.: 110–112 °C; Rf: 0.53 (20% EtOAc/n-hexane); IR (ATR, cm–1) 3064, 2973, 2938,
1
1718 (C=O), 1699 (C=Oketo), 1582, 1494 (C=C); H NMR (300 MHz, CDCl3): δH 8.24–8.17 (m, 1H,
ArH), 8.08–8.02 (m, 2H, ArH), 7.80 (dd, 1H, J = 8.4, 2.1 Hz, ArH), 7.63 (d, 1H, J = 8.4 Hz, ArH),
7.29–7.23 (m, 1H, ArH), 5.54 (s, 2H, OCH2); 13C NMR (75 MHz, CDCl3): δC 189.8, 164.0, 161.7 (d, J
= 255 Hz), 138.9, 133.9, 133.5, 133.1, 132.9, 131.2, 130.5 (d, J = 8.3 Hz), 129.9, 126.5 (d, J = 37.5
Hz), 121.8 (d, J = 18.7 Hz), 116.9 (d, J = 21.8 Hz), 66.5; HRMS (ESI+ve): exact mass calculated for
C15H8Cl3FNaO3 [M+Na]+: 382.94208; found, 382.92015.
4.3. Bioassay protocols
4.3.1. In vitro α-glucosidase inhibition assay
In vitro α-glucosidase inhibitory activity was determined according to the previously developed
fluorimetric protocol [54]. The total assay solution comprised of enzyme (0.2 unit/well) prepared in
phosphate buffered saline (PBS) (100 mM, pH 6.8) with different concentrations of samples (1-0.0312
mM) at 37 °C were incubated for 15 min. All test compounds were solubilized in HPLC grade DMSO
(7.5% final volume) along with negative and positive controls. After pre-incubation, the reaction was
started by adding 20 µL of substrate p-nitrophenyl-α-D-glucopyranoside (0.7 mM) and continuous
changes in absorbance was measured at 400 nm for 30 min by microplate spectrophotometer
(xMark™, BIO-RAD, California, LA, United States). Acarbose (specific inhibitor) with IC50 value of
942.2 ± 0.74 µM was used as a reference drug. The IC50 values were calculated by using different
concentrations of tested compounds. The results were processed and analyzed by MS-Excel and Ez-fit
software programs. The % inhibition of each compound was calculated using the following formula:
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4.3.2. In vitro carbonic anhydrase-II inhibition assay
In-vitro bCA-II activity was measured by following the spectrophotometric method described by
Pocker and Meany with slight modifications [55,56]. The spectrophotometric assay was conducted in
HEPES-Tris buffer of pH 7.4 (20 mM) at 25 °C. Each inhibitory well consists of 140 µL of HEPES-
Tris buffer solution, 20 µL of bCA-II enzyme solution (0.1 mg/mL HEPES-Tris buffer) and 20 µL of
test compound in HPLC grade DMSO (maintain 10% of the final concentration). The mixture solution
was pre-incubated for 15 min at 25 °C. Substrate p-nitrophenol acetate (0.7 mM) was prepared in
HPLC grade methanol and the reaction was started by adding 20 µL to well in 96-well plate. The
continuous measurement of amount of product formed at 400 nm for 30 min at 1 min interval in 96-
well plate, using xMARK microplate spectrophotometer, Bio-Rad (USA). The activity of controlled
compound was taken as 100%. All experiments were carried out in triplicate of each used
concentration, and results are represented as mean of the triplicate. The % inhibition of each compound
was calculated using the following formula:
4.3.3. Statistical Analysis
The EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA) was employed to
calculate the IC50 values. All graphs were plotted using GraFit program (1999). Values of the
correlation coefficients, intercepts, slopes, and their standard errors were calculated by the linear
regression analysis using the same program. Each point in the constructed graphs represents the mean
of the three experiments [57].
4.4. Molecular docking protocol
The molecular modeling studies were carried out using Molecular Operating Environment (MOE,
2014.14). The structures of the compounds were prepared by ChemDraw and minimized on MOE until
an RMSD gradient of 0.1 kcal∙mol‒1Å‒1 was achieved with MMFF94x force field. During
minimization, partial charges were automatically calculated, and the structures were transformed into
three-dimensional form. The X-ray crystallographic structure of CA-II (PDB ID: 1V9E, resolution:
1.95Å) was downloaded from the RCSB Protein Data Bank (https://www.rcsb.org), while the 3D-
structure of Saccharomyces cerevisiae α-glucosidase was prepared by homology modeling. The
detailed procedure is discussed in our previous reports [56,58]. The enzyme structures were then
prepared for the molecular docking simulation using Protonate 3D protocol in MOE with its default
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parameters. The docking was performed using Triangle Matcher placement method and London dG
scoring function.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
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Graphical abstract
The present study investigates the dual inhibitory potential of a series of keto ester derivatives against
α-glucosidase and carbonic anhydrase-II enzymes.
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CRediT authorship contribution statement
Imtiaz Khan: Conceptualization, Supervision, Synthesis, Writing – review & editing original draft, Formal
analysis, Investigation, Visualization, Ajmal Khan: Biological activities, Sobia Ahsan Halim: Computational
analysis, Majid Khan: Biological activities, Sumera Zaib: ADME properties, Balqees Essa Mohammad Al-
Yahyaei: Biological activities, Ahmed Al-Harrasi: Supervision, Resources, Aliya Ibrar: Synthesis &
characterization, Writing, Data investigation.
29