Facile one-pot synthesis, butyrylcholinesterase and α-glucosidase inhibitory activities, structure–activity relationship, molecular docking and DNA–drug binding analysis of Meldrum’s acid derivatives

Article abstract of DOI:10.1007/s11164-020-04100-3

Meldrum’s acid derivatives were facile synthesized by one-pot condensation process and characterized by NMR (¹H, ¹³C, DEPT-90 and DEPT-135) and EI-MS. The synthesized compounds were screened for their potential to inhibit butyrylcholinesterase (BChE) and α-glucosidase enzymes. Interestingly, the derivative 3a showed potent α-glucosidase inhibitory activity, with the IC₅₀ value equal to 2.1?mg/mL as compared to standard acarbose (IC₅₀ = 4.7?mg/mL), whereas, in terms of BChE inhibitory activity investigation, the derivatives 3a and 3c showed novel results, with the IC₅₀ values equal to 1.2 and 2.9?mg/mL, respectively, as compared to standard galantamine hydrobromide (IC₅₀ = 4.7?mg/mL), making derivative 3a a dual inhibitor of both enzymes. Further, structure–activity relationship, comparative molecular docking analysis and the DNA–drug binding interaction were studied to investigate relationship between the chemical structure and its biological activity, inhibition of mechanism, interaction of compounds, DNA binding constant and Gibbs free energy. Structural insights into inhibitor binding to the α-glucosidase and BuChE revealed significant contribution of hydrophobic regions and significant residues of active sites. Comparative molecular docking studies showed that the residues of oxyanion hole, catalytic triad and hydrophobic pocket were actively engaged in interaction with the inhibitor. DNA binding constant was found in the order K_b 3e > K_b 3c > K_b 3a > K_b 3b > K_b 3d, while Gibbs free energy was found in the order ?G 3e > ?G 3a > ?G 3b > ?G 3c > ?G 3d.

Full text of DOI:10.1007/s11164-020-04100-3

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-020-04100-3
Facile one‑pot synthesis, butyrylcholinesterase
and α‑glucosidase inhibitory activities, structure–activity
relationship, molecular docking and DNA–drug binding
analysis of Meldrum’s acid derivatives
Haroon Mehfooz, et al. [full author details at the end of the article]
Received: 18 July 2019 / Accepted: 7 February 2020
© Springer Nature B.V. 2020
Abstract
Meldrum’s acid derivatives were facile synthesized by one-pot condensation pro-
cess and characterized by NMR (1H, 13C, DEPT-90 and DEPT-135) and EI-MS.
The synthesized compounds were screened for their potential to inhibit butyryl-
cholinesterase (BChE) and α-glucosidase enzymes. Interestingly, the derivative
3a showed potent α-glucosidase inhibitory activity, with the IC50 value equal to
2.1 mg/mL as compared to standard acarbose (IC50 =4.7 mg/mL), whereas, in terms
of BChE inhibitory activity investigation, the derivatives 3a and 3c showed novel
results, with the IC50 values equal to 1.2 and 2.9 mg/mL, respectively, as compared
to standard galantamine hydrobromide (IC50 =4.7 mg/mL), making derivative 3a a
dual inhibitor of both enzymes. Further, structure–activity relationship, compara-
tive molecular docking analysis and the DNA–drug binding interaction were studied
to investigate relationship between the chemical structure and its biological activ-
ity, inhibition of mechanism, interaction of compounds, DNA binding constant and
Gibbs free energy. Structural insights into inhibitor binding to the α-glucosidase and
BuChE revealed signifcant contribution of hydrophobic regions and signifcant resi-
dues of active sites. Comparative molecular docking studies showed that the resi-
dues of oxyanion hole, catalytic triad and hydrophobic pocket were actively engaged
in interaction with the inhibitor. DNA binding constant was found in the order Kb
3e>Kb 3c>Kb 3a>Kb 3b>Kb 3d, while Gibbs free energy was found in the order
∆G 3e>∆G 3a>∆G 3b>∆G 3c>∆G 3d.
Keywords Meldrum’s acid · α-Glucosidase · Butyrylcholinesterase · Molecular
docking · DNA–drug interaction · DFT calculations
Introduction
Alzheimer’s disease (AD), a type of dementia, is a progressive neurodegenerative
disorder commonly occurring in the elderly people above 40s. The most common
form of dementia is dementia with Lewy bodies (DLB) and is characterized by loss
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H. Mehfooz et al.
of memory, behavior and other cognitive defciencies. Pathologically, AD is char-
acterized by loss of cholinergic neurons, deposition of plaque and neurofbrillary
tangles (NFTs) within the central nervous system. This deposition of plaque and
neurofbrillary tangles takes place within the cortex and hippocampus, areas of the
brain associated with higher cognitive functions and memory [1, 2]. These plaques
are mainly consisted of amyloid β-peptide (Aβ42), and interestingly, cholinergic
synaptic function is considered to be particularly susceptible to β-amyloid peptide
toxicity. Thus, the loss of synaptic vesicles on axon terminals may cause cholinergic
neuronal loss [3, 4]. In order to treat AD, various drugs have been disclosed in the
literature such as multi-targetable chalcone analogs [5], amino acids/peptides con-
jugated heterocycles [6], chloro-containing molecules [7], sulfur (SVI)-containing
motifs [8], benzisoxazole [9], tetralone derivatives [10], α,β-unsaturated carbonyl-
based cyclohexanone derivatives [11], tetramethylpyrazine-based chalcone deriva-
tives [12], podophyllotoxin derivatives [13] and ligustrazine-based cyclohexanone
and oxime analogs [14].
Butyrylcholinesterase (BChE, EC 3.1.1.8) belongs to the family of enzymes
known as cholinesterase. It plays an important role in the hydrolysis of acetylcholine
(ACH), a neurotransmitter in the brain, which results in the termination of impulse
transmission at cholinergic synapses [15]. As discussed earlier, Alzheimer’s disease
is characterized by the loss of cholinergic neurons, resulting in decreased levels of
BChE; this lack of butyrylcholine manifests itself in diferent symptoms such as loss
of memory, impaired intellectual abilities and cognitive dysfunction [16]. Thus, cho-
linesterase is an important target in the treatment of AD and various cholinesterase
inhibitors (ChEI) have been developed, i.e., donepezil, rivastigmine and tacrine, and
they act by increasing the availability of ACh in central synapses [17].
Diabetes mellitus continues to be a serious threat to human health. Medicinal
chemists have devoted their eforts to uncover the complications associated with
lethal disease, diabetes [18, 19]. It is believed that advanced glycation end prod-
ucts (AGEs) mediate several diabetic complications. Glycation of non-enzymatic
lipids or proteins produces the AGEs via condensation, fragmentation and oxida-
tive modifcations by reducing sugars. The rise in these AGEs signifcantly increases
the chances of diabetes and other cardiovascular diseases. Thus, inhibition of pro-
tein glycation can be an efective approach to minimize complications of diabetes.
Currently available medication for treatments of diabetes is insulin and various oral
antidiabetic agents, such as biguanides, sulfonylureas and glucosidase inhibitors,
but most of these accessible antidiabetic agents have few serious adverse efects
[20–24]. To fully cope with the diabetes, the development of efcient and risk-free
diabetic agents is the need of the hour.
Meldrum’s acid derivatives are remarkable organic synthon (Fig. 1) and are
widely employed in the synthesis of natural products and heterocyclic cores [25,
26]. Besides being versatile synthon, Meldrum’s acid derivatives exhibit encompass-
ing spectrum of biological activities. They show antibacterial, antimicrobial, anti-
malarial, antioxidant and HIV-1 inhibitor activities [27, 28]. The well-recognized
synthetic and biological signifcance of Meldrum’s acid prompted us to synthesize
C-5-substituted novel derivatives by condensation reaction of diferent substituted
anilines with Meldrum’s acid.
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Facile one‑pot synthesis, butyrylcholinesterase and…
Fig. 1 Synthetic utility of Meldrum’s acid in organic synthesis
Due to excellent activities, in this research work, we have synthesized Mel-
drum’s acid derivatives by one-pot simple condensation process and charac-
terized these derivatives by NMR and EI-MS. The synthesized compounds
were screened for their potential to inhibit butyrylcholinesterase (BChE) and
α-glucosidase enzymes. Structure–activity relationship, comparative molecular
docking analysis, the DNA–drug binding interaction and computational investi-
gations have also been done.
Experimental
Methods and materials
All the reagents and chemicals were purchased commercially and were further
purifed prior to use. Thin-layer chromatography was performed using silica gel-
coated aluminum sheets (Merck). Melting point was taken three times using a
Gallenkamp apparatus. Mass spectra were recorded with JEOL MS route oper-
1
ated with electron ionization mode with Varian MAT312. H NMR spectra were
recorded in d6-DMSO with Bruker AM 300 and AM 400 spectrometers (Rhein-
stetten-Forchheim, Germany) operating at 300 MHz and 400 MHz, respectively.
13C NMR spectra were recorded in DMSO-d6 with Bruker AM 300 spectrometer
(Rheinstetten-Forchheim, Germany) operating at 75.5 MHz, respectively. Tetra-
methylsilane was taken as an internal standard in NMR spectra.
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H. Mehfooz et al.
General procedure for synthesis of (3a–3e)
In a 50-mL round-bottom fask, Meldrum’s acid (0.3 g, 0.002 mol) and equimolar
respective amino compounds were allowed to react in the presence of slightly excess
triethyl orthoformate (0.98 g, 0.06 mol) in 2-butanol (5 mL) as a solvent. The reac-
tion mixture was refuxed for 3–5 h. The solid formed in hot state was collected
by suction fltration. Washing with ethanol furnished TLC pure compound in excel-
lent yield. The purity of the synthesized compounds was checked by thin-layer chro-
matography using ethyl acetate and n-hexane (2:8), dichloromethane and n-hexane
(4:6) and ethyl acetate and methanol (1:9) composition solvent systems.
Experimental data
2,2‑Dimethyl‑5‑{[3‑(trifuoromethylphenyl]amino}methylene)‑1,3‑dioxane‑4,6‑dione
(3a)
1
Yield: 80%, yellow, m.p. 148 °C. H NMR (300 MHz, DMSO-d6 δ:11.3 (1H, s,
CH–NH), 8.6 (1H, s, CH–NH), 8.1 (1H, s, ArH), 7.9 (1H, d, J 8, ArH), 7.5–7.6 (2H,
m, ArH), 1.6 (6H, s, 2xCH3); 13C NMR (75.5 MHz, DMSO-d6) δ: 163.5, 162.6,
153.8, 139.5, 130.5, 129.5, 125.1, 123.1, 122.4, 122.3, 104.1, 87.5, 26.4; EI-MS
(EI): m/z 314.8 (M+ 23.4%), 257.8 (39.2), 256.7(64.5), 211.9 (100), 184.9 (65.8),
115.9 (14.9), 52.9 (62.9). Anal Calc: C14H12F3NO4; C, 53.34, H, 3.85, N, 4.65.
Found, C, 54.37, H, 4.53, N, 5.04.
4‑{[(2,2‑Dimethyl‑4,6‑dioxo‑1,3‑dioxan‑5‑ylidene)methyl]amino}‑2‑hydroxybenzoic
acid (3b)
1
Yield: 77%, brown, m.p. 192 °C. H NMR (300 MHz, DMSO-d6) δ:11.1 (1H, d,
CH–NH), 10.8 (1H, s, COOH), 8.5 (1H, d, CH–NH), 7.2 (1H, t, J 8, ArH), 6.9
(1H, d, ArH), 6.8 (1H, s, ArH), 6.6 (1H, m, OH), 1.7 (6H, s, 2xCH3); 13CNMR
(75.5 MHz, DMSO-d6) δ: 163.8 (C), 162.7 (C), 158.3 (C), 152.8 (CH), 139.5 (C)
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Facile one‑pot synthesis, butyrylcholinesterase and…
131.7 (C), 113.4 (CH),109.4 (CH), 105.7 (CH), 105 (C), 104.1 (C), 86.4 (C), 26.4
(CH3); MS (EI): m/z 262.8 (M+ 18.5%), 204.9 (48.3), 159.9 (68.3), 132.9 (100),
104.9 (38.4), 65.0 (34.5). Anal Calc: C14H13NO7; C, 54.84, H, 4.35, N, 5.65. Found,
C, 55.37, H, 5.53, N, 5.04.
2,2‑Dimethyl‑5‑({[2‑(trifuoromethyl)phenyl]amino}
methylidene)‑1,3‑dioxane‑4,6‑dione (3c)
Yield: 73%, dark yellow, m.p. 120 °C. 1H NMR (300 MHz, DMSO-d6) δ: 11.6 (1H,
d, J 12.8, CH–NH), 8.7 (1H, d, J 12.8, CH–NH), 7.9 (1H, d, J 8, ArH),7.4 (1H, m,
J 7.7, ArH), 7.7 (1H, m, ArH), 1.7 (6H, s, 2CH3); 13C NMR (300 MHz, DMSO-d6)
δ: 164.3 (C), 162.0 (C), 154.8 (CH), 135.8 (C) 134.4 (CH), 126.7(CH),126.6 (CH),
126.6 (C), 120.8 (CH), 118.6 (C), 104.1 (C), 88.5 (C), 26.4 (CH3); MS (EI): m/z
314.7 (M+ 7.7%), 256.7 (65.6), 211.9 (48.8), 191.9 (22.6), 113.8 (10.9), 82.9 (100),
53 (37.3). Anal Calc: C14H12F3NO4; C, 53.34, H, 3.85, N, 4.65. Found, C, 54.37, H,
4.53, N, 5.04.
2,2‑Dimethyl‑5‑{[(2‑methyl‑3‑nitrophenyl)amino]methylid}
ene)‑1,3‑dioxane‑4,6‑dione (3d)
Yield: 83%, light yellow, m.p. 160 °C. 1H NMR (300 MHz, DMSO-d6) δ: 11.3 (1H,
d, J 13.8, CH–NH), 8.5 (1H, d, J 14.1, CH–NH), 7.9 (1H, d, J 7.8, ArH), 7.7 (1H,
m, J 7.8, ArH), 7.5 (1H, m, J 8.1, ArH), 2.4 (3H, s, CH3), 1.7 (6H, s, 2CH3); 13C
NMR (300 MHz, DMSO-d6) δ: 164.5 (C), 162.4 (C), 155.5 (CH), 150.8 (C), 139.3
(C) 127.9 (CH), 123.8 (CH),123.6 (C), 121.6 (CH), 104.4 (C), 87.5 (C), 26.4 (CH3),
12.9 (CH3); MS (EI): m/z 305.8 (M+ 10.6%), 247.8 (83.7), 188.9 (47.9), 158.9
(19.9), 76.9 (100). Anal Calc: C14H14N2O6; C, 54.91, H, 4.65, N, 9.15. Found, C,
55.37, H, 5.53, N, 10.04.
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H. Mehfooz et al.
5‑{[(3‑Fluorophenyl)amino]methylidene)‑2,2‑dimethyl‑1,3‑dioxane‑4,6‑dione (3e)
Yield: 79%, light yellow, m.p. 144 °C. 1H NMR (300 MHz, DMSO-d6) δ: 11.2 (1H,
s, CH–NH), 8.5 (1H, s, CH–NH), 7.5 (1H, m, ArH), 7.4-7.5 (2H, m, ArH), 7.0-
7.1 (1H, m, ArH), 1.7 (6H, s, 2CH3); 13C NMR (300 MHz, DMSO-d6) δ:164.2 (C),
160.9 (C), 153.4 (CH), 140.5 (C), 140.3 (C), 131.3 (CH), 115.1 (CH), 112.9 (CH),
106.7 (CH), 104.2 (C), 86.4 (C), 26.4 (2CH3); MS (EI): m/z 265 (M+ 16.3%), 249.9,
207 (64.2), 162 (92.8), 135 (100), 53 (56.4). Anal Calc: C13H12FNO4; C, 58.88, H,
4.57, N, 5.35. Found, C, 59.37, H, 5.53, N, 6.04.
Methods of bioassay
α‑Glucosidase assay
α-Glucosidase enzyme inhibition assay was performed according to the previously
reported method [20]. For experiment, 25 µL p-nitrophenyl–α-d-glucopyranoside,
65 µL phosphate bufer (pH 6.8) and 5 µL α-glucosidase enzyme (0.05 U/mL) were
mixed in a 96-well microtiter plate. Five microliters compound with the fnal con-
centrations of 500, 250 and 125 µg/mL was added in respective wells. Acarbose and
DMSO were used as positive and negative controls, respectively. Plates were incu-
bated at 37 °C for 30 min, followed by the addition of 0.5 mM sodium bicarbonate
(100 µL) as a stopping agent. Absorbance was measured at 405 nm using a micro-
plate reader (BioTek Elx-800, USA), and IC50 was calculated by using GraphPad
Prism 5.
Butyrylcholinesterase assay
Ellman’s method was used to determine the butyrylcholinesterase enzyme inhibition
potential of the compound [21, 22]. The compound (5 μL) with the fnal concen-
trations of 500, 250 and 125 µg/mL was mixed with 20 μL of 100 µM phosphate
bufer (pH 8.0) and 5 μL BChE enzyme (0.05 U/mL). Then, 10 μL butyrylthiocho-
line iodide (4 mM) and 60 μL DTNB (3 mM) were added. Galantamine hydrobro-
mide and DMSO served as positive and negative controls, respectively. The reaction
mixtures were then incubated for 30 min at 37 °C. After incubation, absorbance was
measured at 405 nm using a microplate reader (BioTek Elx-800, USA) and IC50 was
calculated by using GraphPad Prism 5.
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Facile one‑pot synthesis, butyrylcholinesterase and…
Methodology for molecular docking
The crystal structures of α-glucosidase and butyrylcholinesterase (PDB IDs: 4LBS
and 1A27) were retrieved through PDB (http://www.rcsb.org). 2D structures of
compounds were generated by ChemDraw (http://www.cambridgesoft.com) tool,
followed by geometrical optimization with LigandScout [23]. Each compound was
virtually docked against α-glucosidase and butyrylcholinesterase (BuChE) through
AutoDock 4.2 [24] suit of PyRx to achieve an optimal complementarity of steric and
physiochemical properties. The number of runs for each docking was set to 100. The
Lamarckian genetic algorithm (LGA) was applied to the following parameters: ini-
tial population of 150 randomly placed individuals, a maximum number of 27,000
generations, a mutation rate of 0.02, 2.5×106 energy evaluations and a crossover
rate of 0.80, while the remaining docking parameters were set to default. For the
purpose of attaining the best binding pose, these inhibitors were comparatively
docked through PatchDock [25]. Afterward, hydrophobic and electrostatic interac-
tions were mapped using LigPlus [26] and visualized by UCSF Chimera 1.9.0 [27].
Results and discussion
Chemistry
Synthesis
Compounds (3a–3e) were obtained in good yield ranging from 73 to 83%, by treat-
ing diferent aromatic amino compounds with Meldrum’s acid in the presence of
triethyl orthoformate using 2-butanol as shown in Scheme 1 [29–31].
Scheme 1 Synthetic route toward the synthesis of novel Meldrum’s acid derivatives (3a–3e)
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H. Mehfooz et al.
Spectroscopic characterization
Structures of all these compounds were elucidated by spectroscopic technique NMR
1
(1H, 13C, DEPT-90 and 135) and mass spectrometry. H NMR spectra of com-
pounds showed that (=CH–NH) proton appeared as doublet at δ 11.1–11.6. Simi-
larly, (=CH–NH) proton of compounds also displayed doublet at δ 8.4–8.7. Both
have same J value, which proves that these are coupling partners. An intense singlet
displayed at δ 1.6–1.7 of six protons integral for two methyl groups attached at C2.
The 13C NMR spectra of compounds (3a–3e) showed that both cyclic quaternary
carbonyl carbons (C-2 and C-6) in all compounds appeared at δ 151.9–162.7 and δ
152.8–164.9, respectively. In all the compounds, methylidene carbon displayed sig-
nal at δ 152.8–155.5. DEPT-90 proved it and appeared downfeld due to the direct
attachment of methylidene carbon with inductively electron-withdrawing nitrogen
atom and also due to conjugation with carbonyl group. Similarly, C-2 quaternary
carbon displayed signal at δ 104.1–174.7, while C-5 quaternary carbon showed sin-
glet at δ 86.1–88.5. The two methyl groups exhibited signal at δ 26.4–26.4. DEPT-
135 confrmed the presence of methyl groups and CH groups by the appearance of
positive signals.
The mass spectrometry analysis also provided evidence of formation of products.
The two methyl groups of Meldrum’s acid were confrmed by the loss of acetone
by Retro–Diels–Alder fragmentation pattern, resulting in the cyclic lactone having
m/z=207. The loss of CO from cyclic lactone resulted in the formation of acyclic
carboxylic acid having a triple bond (Scheme 2).
Biological activity
α‑Glucosidase assay
The antidiabetic potential of the compounds was evaluated by using commercial
purifed α-glucosidase enzyme. The assay was performed in triplicate, and results
are presented in Table 1. Acarbose was used as a positive control, and DMSO served
as a negative control. The results showed that 3a compound showed α-glucosidase
inhibitory activity with the IC50 value of 2.1 mg/mL which is higher than the stand-
ard acarbose (IC50 =4.7 mg/mL). The other four derivatives were also found as
potent derivatives.
Butyrylcholinesterase assays
The synthesized compounds were tested for their activity against butyrylcholinester-
ase enzyme. The assay was performed in triplicate, and results are shown in Table 2.
Galantamine hydrobromide was used as a positive control. The results showed that
the highest activity was exhibited by compounds 3a and 3c (IC50 values 1.2 and
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Facile one‑pot synthesis, butyrylcholinesterase and…
Scheme 2 EI-MS fragmentation of compound 3e
Table 1 Results of
S. no.
Compound codes
IC50 (mg/mL)
α-glucosidase activities analysis
(IC50 values of Meldrum’s
1
2
3
4
3a
2.1
12
10
13.6
7.9
4.7
acid derivatives (3a–3e) using
3b
acarbose as standard)
3c
3d
5
3e
Standard
Acarbose
1 3

H. Mehfooz et al.
Table 2 Results of
S. no.
Compound codes
IC50 (mg/mL)
butyrylcholinesterase activities
analysis (IC50 values of
Meldrum’s acid derivatives
(3a–3e) using acarbose as
standard)
1
2
3
4
3a
3b
3c
3d
3e
1.2
19
2.9
6.3
7.5
4.7
5
Standard
Galantamine hydrobro-
mide
2.9 mg/mL, respectively) which is higher than the standard galantamine hydrobro-
mide (IC50 =4.7 mg/mL). Interestingly, compound 3a was found to be a dual inhibi-
tor of both these enzymes.
Molecular docking analysis
Through comparative docking analysis, we selected the best docked conformers with
the lowest binding energy and characterized their detailed binding pattern (Fig. 2).
The calculated binding energies obtained through stable docked conformations of
inhibitors in complex with α-glucosidase and BuChE are shown in Table 3.
To monitor the binding behavior thoroughly, detailed analysis of adjacent amino
acids lying at the active site was carried out. Trp82 residue of BuChE which partici-
pates in controlling the peripheral anionic activity was actively involved in inhibi-
tor binding. Similarly, residues of the oxyanion hole (Gly116 and Gly117), cata-
lytic triad (Ser198 and His438) and anionic site (Tyr332) were also participated in
Fig. 2 Analysis of α-glucosidase and BuChE specifc binding pockets. a α-Glucosidase pocket insight;
b BuChE pocket insight. Labeled residues in stick representation indicate peripheral anionic site:
Trp82 and Tyr332 (blue); catalytic triad: Ser198 and His438 (yellow); and oxyanion hole: GLY116 and
GLY117 (red). Interacting residues of α-glucosidase and BuChE are depicted in coral color, while sur-
face is shown in white color. The bound inhibitors are indicated by wire representation. Blue, inhibitor
3a; orange, inhibitor 3b; green, inhibitor 3c; cyan, inhibitor 3d; and black, inhibitor 3e. (Color fgure
online)
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Facile one‑pot synthesis, butyrylcholinesterase and…
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H. Mehfooz et al.
Fig. 3 Binding mode analysis. Best docked complexes of (3a) BuChE–inhibitor-1, (3b) BuChE–inhibi-
tor-2, (3c) BuChE–inhibitor-3, (3d) BuChE–inhibitor-4 and (3e) BuChE–inhibitor-5. BuChE is shown
in gray ribbon, while interacting residues are represented in coral sticks. Inhibitors are shown in stick
representation
the inhibitor binding, thereby competing with substrate binding [32] (Figs. 2 and
3). Gly116, Gly117, Ser198, His438, Trp82 and Phe329 residues of BuChE were
involved in hydrogen bonding. Despite these polar contacts, multiple hydrophobic
interactions were also observed through Tyr128, Glu197, Val288, Ala328, Phe398
and Trp430 residues that appeared to assist in binding.
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Facile one‑pot synthesis, butyrylcholinesterase and…
Furthermore, we evaluated the interactions of specifed inhibitors with
α-glucosidase. Majority of inhibitors exhibited binding at the Ala509–Asp777
region. It was observed that the residues of oxyanion hole, catalytic triad and hydro-
phobic pocket were actively engaged in interaction with the inhibitors (Figs. 2
Fig. 4 Binding modes of inhibitor-bound α-glucosidase complexes. Best docked complexes of (3a)
α-glucosidase–inhibitor-1, (3b) α-glucosidase–inhibitor-2, (3c) α-glucosidase–inhibitor-3, (4d)
α-glucosidase–inhibitor-4 and (4e) α-glucosidase–inhibitor-5. α-Glucosidase is shown in gray ribbon,
while interacting residues are represented in coral sticks. Inhibitors are shown in stick representation
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H. Mehfooz et al.
and 4). Except inhibitor 3a, others showed binding at similar binding cavity of
α-glucosidase. Ala285, Ala509, Arg520, Ser521, Phe522, Ile523, Lys534, Phe535,
Ala536, Ala537, Met567 and Lys776 residues of α-glucosidase were involved in
binding. Overall, structural insights into inhibitor binding to the α-glucosidase and
BuChE revealed signifcant contribution of hydrophobic regions and signifcant
residues of active sites. LigPlots of BuChE–inhibitors complexes are investigated
(Fig. 5), BuChE with inhibitors 1–5. BuChE residues that involved in H-bonding
(green dotted lines) are shown in ball-and-stick representation, while brown semicir-
cles represent hydrophobic residues of BuChE. All the results are comparable with
experimental values.
Drug–DNA interaction studies
Concentration of DNA was determined by UV–visible spectrophotometer at 260 nm
and was found 1.4×10−4 M. Spectroscopic titrations were done under normal body
temperature (37 °C). The concentration of each synthesized compound (3a–3e)
was prepared as 1.14×10−4 M. The absorbance measurements were taken by keep-
ing the concentration of synthesized compounds (3a–3e) constant (1.4×10−4 M)
in the sample cell, while varying the concentration of dsDNA in the sample cell.
The change in absorbance was measured before and after the addition of various
Fig. 5 LigPlots of BuChE–inhibitors complexes
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Facile one‑pot synthesis, butyrylcholinesterase and…
concentrations of DNA. Solutions were allowed to stay for few minutes before each
measurement so that equilibrium could be achieved between compound and DNA
complex formation. Sample solutions were kept within the cell cavity for few sec-
onds to assure the required temperature (37 °C).
The electronic absorption spectroscopy is one of the most useful techniques to
study the drug–DNA interactions and provides a useful complement to other tech-
niques used for DNA binding studies [33]. Commonly, hypochromic efect along
with red or blueshift is observed in the absorption spectra of molecules if they inter-
calate with DNA.
UV spectra of fve C-5-substituted Meldrum’s acid were recorded separately by
adding varying concentrations of DNA, and concentration efect of DNA on opti-
mized concentration (1.4×10−4 M) of all the compounds was observed at body
temperature (37 °C). The addition of DNA in aliquots was resulted in decrease
in absorption peak intensity of the compounds with a slight blueshift of 1.0 nm,
1.2 nm, 0.1 nm and 0.4 nm, respectively, for compounds 3a, 3b, 3c, 3d and 3e,
respectively. A decrease in the peak intensities of 3a, 3b, 3c, 3d and 3e in the pres-
ence of DNA was evaluated as 11.2%, 16.3%, 16.5%, 18.1% and 19.0%, respectively,
using the following equation:
Afree − Abound
H% =
× 100.
(1)
Afree
The observed hypochromic efect along with slight blueshift upon addition of
DNA may correspond to the binding of compounds 3a–3e with dsDNA through
intercalative mode of interactions [34]. The decrease in peak intensity is related to
decreased transition probabilities as coupling π-orbital is partially flled by elec-
trons, which resulted in hypochromism in the spectra. On the other hand, a blueshift
arises in the spectra due to improper coupling (conformational changes) of π*-orbital
of intercalated part of the compound with the π-orbital of the base pairs [34]. This
distortion in the π-orbital of the base pairs and π*-orbital of intercalated molecules
resulted in unstacking of base pairs with hypochromic efect.
Determination of binding constants and free energy changes of compound–DNA
complexes
Variation in absorbance of a compound in UV spectra in the presence of DNA leads
to determine the binding constant “Kb” of compound–DNA complex using Ben-
esi–Hildebrand equation [1, 33].
Ao
ꢀG
ꢀG
1
=
+
ꢀH−G − ꢀG ꢀH−G − ꢀG Kb[DNA]
(2)
A − Ao
where Kb is the binding constant, Aο and A are the absorbance of the free and DNA-
bound complex and εG and εH–G are their molar extinction coefcients, respectively.
From the plot of Ao/(A−Ao) against 1/[DNA], the ratio of the intercept to the slope
furnished the value of binding constant, Kb (Fig. 6).
1 3

H. Mehfooz et al.
2
1
0
1/[DNA] (µM)-1
1/[DNA] (µM)-1
0.01 0.02
a
h
0
0.05
0.1
0
-10
-20
-30
-40
-50
-60
2
1
0
0
-5
a
i
0
0.03
K
= -21.562 KJ.Mol-1
=5.44 x 103M-1
∆G
k= 1.121x103M-1
ΔG=-15.937Kj/mol
-10
-15
-20
-25
-30
-35
300
400
300
400
nm
nm
3a
3b
2
2
1/[DNA] (µM)-1
0
1/[DNA] (µM)-1
0
-10
-20
-30
-40
-50
a
j
0
0.02
0.03
0.01k= 5.01x102M-1
a
0
0.005
0.01
0.015
0.02
0.025
0.03
-10
-20
-30
-40
-50
-60
k= 8.75x102M-1
ΔG=-15.37Kj/mol
ΔG=-14.109Kj/mol
h
1
0
1
0
300
400
280 290 300 310 320 330 340 350 360 370 380 390 400
nm
3c
nm
3d
2
1
0
1/[DNA] (µM) -1
a
0
0
0.01
0.02
0.03
-5
-10
-15
-20
-25
-30
k= 2.98x102M-1
ΔG=-12.93Kj/mol
j
nm
300
400
3e
Fig. 6 DNA binding constant and Gibbs free energy calculations of synthesized compounds 3a–3e. The
arrow indicates the increasing concentration of DNA
The binding constant values (Kb) were evaluated for all the fve compounds under
physiological temperature (37 °C) and given in fgures. Kb values for all compounds
with DNA were found in the range of 102–03 M−1. Binding constant values and UV
spectral changes observed during ligand–DNA complex formation, i.e., hypochro-
mic efect and hypochromic shift in the present study may further be credited to
the small structure of molecules whose planer parts may possibility be intercalated
between the adjacent DNA base pairs. Drug–DNA binding studies and Gibbs free
1 3

Facile one‑pot synthesis, butyrylcholinesterase and…
energy calculations were carried out using various concentrations of µM DNA such
as 40, 80, 120, 160, 200, 240 and 280.
The inset graph represents the plot of A0/A−Ao versus 1/[DNA] (µM)−1 for
the calculation of the binding constant (k) and Gibbs free energy (ΔG). Since
greater binding constant values are the measure of the complex stability, the val-
ues evaluated for all the synthesized compounds for their binding with DNA
were found signifcant and may be inferred to the formation of stable com-
pound–DNA complex [35–37]. Compound 3e showed the comparatively greater Kb
value (5.44×103 M−1). The order of binding constants of Meldrum’s acid deriva-
tives (3a–3e) was found as follows:
Kb3e > Kb3c > Kb3a > Kb3b > Kb3d.
Further, Gibbs free energies (∆G) of novel Meldrum’s acid DNA complexes were
calculated by using the values of binding constant (Kb) in the following equation:
ΔG = − RT ln Kb.
Free energy changes were evaluated as negative values, indicating that all com-
pounds 3a–3e interacted spontaneously with DNA during compound–DNA com-
plex formation. However, 3e bound to the DNA more spontaneously as compared
to other compounds as evident from its comparatively greater ∆G value. The order
of complex spontaneity is not same as for binding constant: ∆G 3e>∆G 3a>∆G
3b>∆G 3c>∆G 3d.
Structure–activity relationship
As explained above, compounds with diferent substitutions on the aromatic ring
linked with Meldrum’s acid via olefnic bond were designed and synthesized (3a–3e).
Electron-withdrawing group, such as CF3, enhanced inhibitory against both enzymes
Fig. 7 Structure–activity relationship of the most potent and dual inhibitor of α-glucosidase and butyryl-
cholinesterase
1 3

H. Mehfooz et al.
(α-glucosidase and BChE). On the contrary, electron-donating groups, such as methyl
and hydroxyl, decreased the enzyme inhibition activity (Fig. 7). The derivative in 3a
and 3c bears CF3 groups and in 3a CF3 group is at meta position and showed higher
activity compared to 3c against butyrylcholinesterase enzyme. However, NO2 group
was found to decrease the inhibitory activity. To be more precise, CF3 group at meta
position in compound 3a exhibited excellent activity against both enzymes and found
to be a dual inhibitor of both enzymes. The induction of fuorine atom in organic mol-
ecules enhances the lipophilic character which as a result increases the rate of cell pen-
etration and transport of a drug to an active site [38, 39]. The higher polarizability due
to the C–F bond may give new possibilities for binding to the receptor. Overall, the CF3
groups at meta position are the main cause of excellent activity of 3a.
Conclusions
A novel small set of compounds 2,2-dimethyl-5{[3(substituted-phenyl]amino}
methylene)-1,3-dioxane-4,6-dione was designed and synthesized in excellent yield
(73%-83%). The synthesized compounds (3a–3e) were subjected to enzyme inhibi-
tion activities (α-glucosidase and butyrylcholinesterase). The derivative 3a showed
higher activity compared to standards used (acarbose and galantamine hydrobro-
mide) and was coincidently found to be most potent dual inhibitor of both enzymes.
The derivatives 3a and 3c showed activity higher than the standard in butyrylcho-
linesterase enzyme inhibition, whereas compound 3a showed excellent inhibition
(IC50 value 2.1 which was found to be higher than the standard acarbose IC50 4.7)
against α-glucosidase. The other compounds showed moderate inhibition against
these two enzymes. Comparative molecular docking studies were performed, and
it was observed that the residues of oxyanion hole, catalytic triad and hydrophobic
pocket were actively engaged in interaction with the inhibitor. The DNA–drug bind-
ing studies were performed to evaluate the interaction of compounds and calculate
DNA binding constant and Gibbs free energy. DNA binding constant was found in
the order Kb 3e>Kb 3c>Kb 3a>Kb 3b>Kb 3d, while Gibbs free energy was found
in the order ∆G 3e>∆G 3a>∆G 3b>∆G 3c>∆G 3d. Density functional theory
(DFT) calculations were done to investigate chemical insights (HOMO–LOMO) and
charge density.
Acknowledgements Authors are thankful to Quaid-i-Azam University, Islamabad, Pakistan, for provid-
ing healthy research environment.
Compliance with ethical standards
Confict of interest The authors declare no confict of interest.
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Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional afliations.
Afliations
Haroon Mehfooz1 · Aamer Saeed1 · Muhammad Faisal1 · Fayaz Ali Larik1 ·
Urooj Muqadar1 · Saira Khatoon1 · Pervaiz Ali Channar1 · Hammad Ismail3 ·
Salma Bilquees1 · Sajid Rashid4 · Shagufta Shafque4 · Bushra Mirza2 ·
Erum Dilshad2 · Fawad Ahmad1
*
*
*
Aamer Saeed
aamersaeed@yahoo.com
Muhammad Faisal
mfaisal4646@gmail.com
Fayaz Ali Larik
fayazali@chem.qau.edu.pk
1
2
3
Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
Department of Biochemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
Department of Biochemistry and Molecular Biology, University of Gujrat, Gujrat 50700,
Pakistan
4
National Centre for Bioinformatics, Quaid-I-Azam University, Islamabad 45320, Pakistan
1 3