Development of coumarin-thiosemicarbazone hybrids as aldose reductase inhibitors: Biological assays, molecular docking, simulation studies and ADME evaluation

Article abstract of DOI:10.1016/j.bioorg.2021.105164

The over expression of aldose reductase (ALR2) in the state of hyperglycemia causes the conversion of glucose into sorbitol and initiates polyol pathway. Accumulation of sorbitol in insulin insensitive tissue like peripheral nerves, glomerulus and eyes, i

Full text of DOI:10.1016/j.bioorg.2021.105164

Bioorganic Chemistry 115 (2021) 105164
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Bioorganic Chemistry
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Development of coumarin-thiosemicarbazone hybrids as aldose reductase
inhibitors: Biological assays, molecular docking, simulation studies and
ADME evaluation
,
Aqeel Imrana c, Muhammad Tariq Shehzadb, Taha al Adhamic, Khondaker Miraz Rahmanc,
,
,*
Dilawar Hussaina, Rima D. Alharthyd, Zahid Shafiqb *, Jamshed Iqbala
a Center for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan
b Institute of Chemical Sciences, Bahauddin Zakariya University, Multan 60800, Pakistan
c Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom
d Department of Chemistry, Science and Arts College, Rabigh Campus, King Abdulaziz University, Jeddah, Saudi Arabia
A R T I C L E I N F O
A B S T R A C T
Keywords:
The over expression of aldose reductase (ALR2) in the state of hyperglycemia causes the conversion of glucose
into sorbitol and initiates polyol pathway. Accumulation of sorbitol in insulin insensitive tissue like peripheral
nerves, glomerulus and eyes, induces diabetic complications like neuropathy, nephropathy and retinopathy. For
the treatment of diabetic complications, the inhibition of aldose reductase (ALR2) is a promising approach. A
series of coumarin-based thiosemicarbazone derivatives was synthesized as potential inhibitor of aldose reduc-
tase. Compound N-(2-fluorophenyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)hydrazinecarbiothioamide (3n)
was found to be the most promising inhibitor of ALR2 with an IC50 in micromolar range (2.07 µM) and high
selectivity, relative to ALR1. The crystal structure of ALR2 complexed with 3n explored the types of interaction
pattern which further demonstrated its high affinity. Compound 3n has excellent lead-likeness, underlined by its
physicochemical parameters, and can be considered as a likely prospect for further structural optimization to get
a drugable molecule.
Coumarin
Aldose reductase
Antiglycation
Thiosemicarbazone
In silico studies
1. Introduction
Due to this pathway, advanced glycation end products (AGEs) are
formed, and protein kinase C is activated (PKC). This combination of
Aldose reductase (EC 1.1.1.21, ALR2, AKR1B1) is an important
enzyme of the polyol pathway, and its over-expression in diabetes
mellitus causes diabetic complications [1–3]. The majority of chronic
diabetic patients are suffering from secondary diabetic complications
like retinopathy, nephropathy, neuropathy and other inflammatory
pathologies, including cardiovascular disorders [4,5].The inhibition of
aldose reductase could be a therapeutic option for long-term diabetic
complications as well as in non-diabetic disorders, since the recent
studies suggest that ALR2 is involved in various types of human cancers
such as liver, breast, ovarian, rectal and cervical cancers [6–8]. Several
cardiac disorders, such as congestive heart failure, myocardial ischemia,
cardia hypertrophy and cardiomyopathy, are thought to result from
increased production of reactive oxygen species (ROS) caused by ALR2
[9,10]. In the polyol pathway, ALR2 metabolizes glucose into sorbitol
along with parallel conversion of NADPH to NADP+ (Fig. 1) [11,12].
glycative and oxidative stress synergistically induces the diabetic com-
plications [13–16].
To mitigate or avert the diabetic complications, the design and
development of selective inhibitors of aldose reductase would be a po-
tential target. The most challenging aspect in the development of ALR2
inhibitor is the non-selective inhibition of another enzyme, aldehyde
reductase (ALR1, EC 1.1.1.2), involved in the metabolism of toxic
aldehyde like 3-oxyglucosazone and methylglyoxal. ALR1 shares 65%
homology in its structure with ALR2, and both enzymes belong to the
aldo–keto superfamily of oxidoreductases [17]. There are numerous
aldose reductase inhibitors that have been developed from natural or
synthetic origin [18,19] and entered in clinical trials, but Epalrestat is
the only marketed drug in Japan [20,21]. The well-studied class of
aldose reductase inhibitor is carboxylic acid and hydantoin derivatives,
but the majority of compounds were unsuccessful during clinical trials
* Corresponding authors.
E-mail addresses: zahidshafiq@bzu.edu.pk (Z. Shafiq), drjamshed@cuiatd.edu.pk, jamshediqb@googlemail.com (J. Iqbal).
https://doi.org/10.1016/j.bioorg.2021.105164
Received 19 February 2021; Received in revised form 3 June 2021; Accepted 8 July 2021
Available online 13 July 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
–
–
because of poor pharmacokinetics profile, unwanted side effects and
non-selectivity issues. Owing to these reasons, further development of
selective inhibitors of aldose reductase is required, with better a phar-
macokinetic profile, more selectivity and fewer side effects. In our pre-
vious studies, benzoxazinone and adamantyl based thiosemicarbazones
were synthesized and identified as potent and selective inhibitors of
ALR2 [22,23]. Literature review indicated that the compounds bearing
coumarin nucleus have been synthesized and explored for their inhibi-
tory activity against ALR2 [24,25]. Also, some naturally-occurring
coumarin derivatives have been isolated from plants and studied for
their ALR2 inhibition potential [19,26–28]. The structure of known in-
hibitors of ALR2 are shown in Fig. 2.
to C S were observed in the region of 1156–1228 cmꢀ 1. Similarly, NH
–
and C O stretching appeared at 3207–3371 cmꢀ 1 and 1699–1741 cmꢀ 1
–
respectively. NMR spectra of compounds were recorded in CDCl3 and
DMSO‑d6. In 1H NMR, the methyl protons of the carbonyl moiety
resonate at 2.22–2.35 ppm, whereas the CH3 protons directly attached to
benzene ring resonate between 2.20 and 2.33 ppm. In 3a-3d, 3i-3o and
3q, the thiosemicarbazone NH-CS appeared as a singlet at δ 8.58–11.08,
while in compounds bearing NH-CH2 moiety (3e, 3f, 3h and 3p), it
resonated as triplet in the range from δ 7.84–9.08. In thiosemicarbazone
3g, having cyclohexyl substituent, a doublet was observed for NH-CS at
–
δH 8.1. Similarly, NH-N and coumarin C H signals were observed at
8.74–11.19 and 7.81–9.92 ppm respectively. 13C NMR spectra also fully
supported the structures of the newly synthesized derivatives as iden-
tified by IR and 1H NMR spectra. The methyl carbons of carbonyl moiety
were found to resonate at δC 15.01–16.95, while methyl carbons directly
attached to aromatic ring appeared at δC 17.93–21.21. All the com-
Considering reported inhibitory potential of coumarins and thio-
semicarbazones against ALR2, the hybrid scaffolds were developed
through incorporation of coumarin moiety with thiosemicarbazone
backbone. These hybrid scaffolds were finally investigated for ALR2
inhibitory activity.
–
–
pounds showed signals at δC 153.49–155.98 attributed to C N group.
The most downfield signals were observed at 159.20–159.61
–
–
–
2. Results and discussion
and173.52–179.38 ppm, which were assigned to C O and C S func-
–
tions. In LC-ESI-MS spectra, the molecular ion peaks were appeared as
[M+H]+, which were in total agreement with the molecular weight of
the synthesized derivatives.
2.1. Chemistry
To explore the aldehyde/aldose reductase enzyme inhibition po-
tential, a series of coumarin-based thiosemicarbazones (3a-q) were
synthesized in good to excellent yields (79–90%). A variety of N4-
substituted thiosemicarbazides (1a-q) were reacted with 3-acetyl
coumarin (2) via typical condensation reaction employing catalytic
amount of glacial AcOH (1–2 drops) in methanol to furnish the respec-
tive thiosemicarbazone derivatives (Scheme 1). The reaction conditions
were optimized using phenyl thiosemicarbazide (1a) and respective
ketone in equimolar ratio engaging solvents of variable polarity such as
EtOH, MeOH, THF, DMSO, acetonitrile, 2-propanol and dioxane,
employing various acid and base catalysts such as glacial acetic acid, p-
TsOH and triethylamine. Methanol was found to be the best solvent,
while glacial AcOH was established to perform better than base catalyst.
The structures of target compounds (3a-q) were determined by
elemental and spectral analysis. The infrared (IR) spectra of thio-
2.2. Biological activities and structure activity relationship
The coumarin-based derivatives of thiosemicarbazone were investi-
gated for enzyme inhibition studies against ALR1 and ALR2. The ma-
jority of tested compounds have shown potential enzyme inhibition
against both the enzymes (ALR1 and ALR2), however a few of them have
shown selective inhibition against aldose reductase. Compounds (3a,
3b, 3c, 3d, 3f, 3h, 3i, 3j, 3k, 3l, 3m, 3n) have displayed strong enzyme
inhibitory activity against ALR2, whereas compounds (3e, 3g, 3o, 3p,
3q) have shown moderate inhibition for ALR2. In addition, these de-
rivatives were also tested for their antiglycation activity. The complete
results are shown in Table 1.
Among the investigated derivatives, compound 3n displayed selec-
tive inhibition against ALR2 with an IC50 value of 3.035 µM. Compounds
(3a, 3b, 3d, 3e, 3f, 3h, 3i, 3k, 3l, 3m) have exhibited moderate to good
inhibition of the ALR1 enzyme; among these 3l has shown strong
–
–
semicarbazone derivatives showed absorption bands due to C
N
function to appear at 1590–1618 cmꢀ 1, while characteristic bands due
Fig. 1. Mechanism of polyol pathway for diabetic complications.
2

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Fig. 2. Structures of some known ALR2 inhibitors and newly synthesized coumarin thiosemicarbazones.
inhibition against ALR1 with an IC50 value of 1.376 µM, whereas com-
pounds 3c, 3g, 3j, 3n, 3o, 3p, 3q have shown moderate inhibition
(Figs. 3-6).
enzyme inhibition and selectivity was dependent on different sub-
stituents or functional groups attached to thiosemicarbazone moiety.
The thiosemicarbazone derivatives possessed phenyl ring directly
attached to –NH group of thiosemicarbazone backbone, and have shown
moderate to strong enzyme inhibition against both isozymes - for
example, 3a exhibited inhibition for ALR1 with IC50 value of 15.213 µM
and inhibition for ALR2 with IC50 value of 27.505 µM. For compound
3b, the presence of dimethyl group on phenyl ring at ortho and para-
positions displayed enhanced inhibitory activity against ALR1, with an
IC50 value of 9.379 µM and 11.511 µM for ALR2. On the structure of 3d,
placement of methyl group on phenyl ring at ortho-position caused an
increase in ALR2 inhibitory activity, with an IC50 value of 7.118 µM and
inhibitory activity for ALR1 with IC50 of 4.512 µM. While discussing 3k,
the presence of methyl group at meta- position improved ALR2 inhibi-
tory activity with an IC50 value of 3.883 µM, whereas inhibitory activity
decreased against ALR1 as observed from the IC50 value of 12.394 µM.
The compound 3c, the presence of methoxy group at meta-position on
phenyl ring, enhanced enzyme inhibition as well as selectivity against
Compounds 3c, 3j and 3n exhibited selective enzyme inhibition
against ALR2 in the sub-micromolar range relative to ALR1, whereas
compound 3e have shown significant selectivity of inhibition against
ALR1. Compounds 3f and 3 l were considered as selective inhibitors of
ALR1 while considering their IC50 values. Compound 3e has exhibited
percent inhibition with value of 38.21% against ALR2, but an IC50 of
7.523 µM against ALR1, so it was considered as selective inhibitor of
ALR1. Compound 3f displayed an IC50 value of 15.213 µM for ALR2 and
IC50 value of 4.313 µM for ALR1 whereas 3i showed inhibition for ALR2
with IC50 value of 5.202 µM and for ALR1 with IC50 value of 3.853 µM.
Briefly, it was revealed that 3n was the most selective inhibitor for ALR2
among the tested compounds, and 3l for ALR1. Moreover, the com-
pounds 3g, 3o, 3p and 3q have not shown any significant inhibition
against both enzymes.
The SAR analysis for investigated compounds explained that the
3

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Scheme 1. Synthesis of coumarin derived thiosemicarbazones (3a-q).
ALR2 with an IC50 value of 5.322 µM.
2.4. Molecular docking results
The presence of electron-withdrawing halogen atom(s) (Cl, F, and
Br) on the phenyl moiety has a different impact on enzyme inhibition
against ALR1 and ALR2. The compound 3n, which bears –F atom at
ortho-position on phenyl ring, has shown selective inhibition against
ALR2 with an IC50 value of 3.035 µM, and another similar molecule 3j,
which possessed di-fluoro group at ortho and para position, has also
shown selective inhibition against ALR2 with an IC50 value of 3.527 µM -
whereas in the case of compound 3l, which bears –Cl atom at para-po-
sition on phenyl ring, selective inhibition against ALR1 with IC50 value
of 1.376 µM was observed. The compound 3i, having two –Cl atoms on
phenyl ring at ortho and meta-positions, exhibited strong inhibition
against both enzymes, and compound 3h which bears –Br atom on
phenyl ring at meta position have shown non-selective enzyme inhibi-
tion with IC50 value of 12.155 µM for ALR1 and 28.47 µM for ALR2. In
the case of compound 3e, the presence of benzyl group with –NH group
of thiosemicarbazone has shown selective inhibition against ALR1 with
IC50 value of 7.523 µM, and 3f showed inhibition with IC50 value of
4.313 µM. The compound 3g , which possesses a cyclohexyl group,
exhibited weak inhibition against both isozymes (26.82% and 26.33%,
for ALR2 and ALR1, respectively). The compound 3q bears dimethyl-
amine moiety and 3o bearing isopropyl group on phenyl ring have
shown no significance inhibition against ALR1 and ALR2. Hence, the
placement of the dimethylamine group and isopropyl group on phenyl
group caused significant decrease in enzyme inhibitory activity for both
isozymes.
Based on in vitro potent ALR2 inhibitory activity of compound 3n, a
detailed mechanistic investigation and SAR on ALR2 was performed
with the help of molecular docking studies. Fidarestat bound ALR2
conformation (PDB code: 1PWM) was selected for the docking studies
for compound 3n. The docking behavior of 3l was also explored using
ALR1-NADP+ conformation (PDB code: 3FX4). As shown in Figs. 8-9, 3
3n was tightly bound at active site of ALR2 according to docking results,
where its 2D dimensional conformation has displayed amino acid side
chain interaction with inhibitors (3n). At the specificity pocket of ALR2,
O2 atom of carbonyl carbon (coumarin ring) made conventional
hydrogen bond with side chain Trp111, demonstrating the significant
role of carbonyl for the tight binding of 3n. The oxygen atom of side
chain Gln183 formed a hydrogen bond with –NH group of thio-
semicarbazone moiety. Other significant interactions of 3n with the
active pocket (anion binding site) involved Tyr48 acted as
π-donor
hydrogen bond, whereas Val 47 exhibited hydrogen bond interaction
–
with H N moiety of thiosemicarbazone and π-alkyl interactions with
benzene ring and coumarin ring. At the hydrophobic pocket, the amino
acid residues Phe122 and Trp20 displayed stacking with the benzene
ring. whereasTrp20 also showed -sigma interaction with H- atom of
conjugated bond of coumarin ring and -Sulphur interactions, Gln49
π-π
π
π
displayed interactions with electronegative Fluorine atom. The residues
Phe121, Trp219, Tyr209, Hist110 and Lys21 exhibited the most prom-
inent Van der Waals interactions.
A graphic representing the antiglycation activity of compounds (3a-
q) in terms of % inhibition of AGEs formation is shown in Fig. 7.
2.5. Molecular dynamics simulation
2.3. Mechanism of ALR2 inhibition
The conformational stabilities of the apoenzymes (ALR1 and ALR2),
along with their cognate & test ligand complexes, were performed to
simulate protein flexibility. The structures of proteins (apoproteins)
were first subjected to MD run of 50 ns, and then docked poses of
cognate ligands and selected ligands (holoenzymes) were submitted to
MD run for 50 ns. The time evolution of the radius of gyration of ALR1
(3FX4) and ALR2 (1PWM), apoenzymes and holoenzymes, are shown in
Fig. 10 and Fig. 11, respectively. The graph indicating that the average
The most potent and selective inhibitor of ALR2, 3n, was selected for
investigation of mechanism of inhibition. The type of ALR2 inhibition
was determined through Lineweaver-Burk plots - 3n has shown a mixed
type of enzyme inhibition (Fig. 8).
4

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Table 1
The IC50 values for ALR2, ALR1 and antiglycation activity for compounds 3a-3q.
Codes
R
ALR1
ALR2
I (%, 100 µM) of AGEs formationc
IC50 (µM)a / I (%, 100 µM)b
3a
3b
3c
3d
3e
3f
C6H5
15.213a
27.505a
50.78 ± 1.322
37.12 ± 1.284
62.49 ± 2.076
64.27 ± 2.282
56.34 ± 1.779
61.42 ± 1.610
(7.156–23.27)
(11.35–43.66)
2,4-(CH3)2C6H3
3-OCH3C6H4
2-CH3C6H4
C6H5CH2
9.379a
11.511a
(2.969–15.79)
34.59b
(6.162–16.86)
5.322a
(2.076–8.569)
4.512a
7.118a
(2.575–6.449)
(4.037–10.20)
38.21b
7.523a
(2.94–12.10)
4.313a
4-ClC6H4CH2
15.213a
(2.246–6.38)
(7.156–23.27)
3g
3h
C6H11
26.33b
26.82b
51.24 ± 0.945
47.22 ± 0.505
4-BrC6H4CH2
12.155a
28.47a
(6.791–17.52)
(8.204–48.69)
3i
2,3-(Cl)2C6H3
3.853a
5.202a
64.41 ± 1.035
(1.854–5.852)
(2.623–7.781)
3j
2,4-(F)2C6H3
4-CH3C6H4
4-ClC6H4
23.89b
3.527a
61.81 ± 2.179
39.21 ± 1.283
56.61 ± 1.540
53.08 ± 0.520
68.66 ± 2.111
(2.032–5.022)
3k
3l
12.394a
3.883a
(6.399–18.39)
(2.231–5.535)
13.036a
1.376a
(0.693–2.059)
8.695a
(5.353–20.72)
3m
3n
2,6-(CH3)2C6H3
2-FC6H4
10.916a
(4.54–12.85)
(5.792–16.04)
3.035a
34.78b
(0.819–5.252)
3o
4-isopropylC6H4
C6H5CH2CH2
41.55b
25.20b
38.91 ± 1.522
64.81 ± 0.899
65.09 ± 2.59
n.d.
3p
22.46b
42.27b
3q
4-N(CH3)2C6H4
17.87b
18.69b
Valproic Acidd
49.311a
(9.923–88.40)
n.d.
n.d.
Sorbinile
2.745a
n.d.
(1.088–4.403)
6-aminoguanidinef
74.93 ± 1.266
n.d. -not determined.
a
Half maximal inhibitory concentration IC50 (95% CL) value representing concentration needed to reduce enzyme activity by 50 percent. The results are mean
values ± SEM for three measurements. The same batch for ALR1 and ALR2 was used for measurement of enzymatic activity.
b
Percent inhibition for ALR1/ALR2 measured at 100 µM of inhibitor concentration.
c
d
Percent of inhibition of AGEs formation.
Standard inhibitor for ALR1.
e
Standard inhibitor for ALR2.
f
Standard antiglycative agent.
value of Rg slightly fluctuating between 1.92 and 1.98 nm for ALR1
(3FX4), while for ALR2 (1PWM) the radius of gyration lies between 1.87
and 1.95 nm, signifying a fine degree of compactness for the most part
during the 50 ns simulation run. The radius of gyration for ALR2
(1PWM) and test compound (3n) fluctuates drastically after 35 ns,
indicating an unstable secondary structure after 35 ns as the protein and
ligand complex loses its compactness, prompting an unstable system.
The average values of RMSD over a period 50 ns for 3FX4, apoenzymes
and holoenzymes, exhibited minute fluctuations, reaffirming stable
complex formation between enzymes and test compounds, as shown in
Fig. 12, but in 1PWM the protein & test compound (3n) complex
exhibited greater deviation after 25 ns as the ligand detaches itself from
the complex and moves out of the active pocket, prompting complex
infringement (Fig. 13). RMSF analyzes the portions of structure that are
fluctuating from their mean structure the most (or least). The (RMSF) of
ALR1 and ALR2 were examined, and it was observed that the residues
were found stable in both of the cases (Figs. 14-16).
2.6. Pharmacokinetic studies
Several enzyme inhibitors can prove their in vitro inhibition, while
many got rejected during clinical trials due to a poor pharmacokinetic
profile. Therefore, In-silico techniques were applied for the determina-
tion of physico-chemical parameters for pharmacokinetic studies at
earlier stages of drug development to reduce cost, resources and labor.
The ADME studies of compounds 3a-3q was conducted through in-silico
based methods (SwissADME) that predicted absorptions, distribution,
metabolism and excretion of compounds, and the obtained results are
5

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Fig. 3. Aldose reductase (ALR2) and aldehyde reductase (ALR1) inhibition for compounds 3a, 3b, 3c, 3d.
mentioned in Table 2.
complication through ALR2 inhibition and suppression of formation of
advanced glycation end products.
The boiled egg plot obtained after performing SwissADME gave very
valuable information for gastrointestinal absorption of all synthesized
compounds. This plot gives visual information related absorption and
composed of two part; one with white part of egg presented gastroin-
testinal absorption, inner yellow yolk like portion presented blood brain
barrier permeability of compound. While visualizing boiled-egg plot
(Fig. 17), it is evident that none of any compound (3a-3q) exhibited
blood brain barrier access, and all the synthesized compounds bear good
gastrointestinal absorption properties.
In vitro ALR2 inhibition of coumarin-based thiosemicarbazones (3a-
q) revealed the compounds 3c, 3j, 3k and 3n have shown selective in-
hibition of aldose reductase with IC50 values of 4.21, 5.67, 3.51 and
2.07 µM, respectively. However, compound 3n was identified as the
most potent and selective inhibitor of ALR2 with IC50 value of 2.07 µM
(inhibition for ALR1 was 34.78%). Besides ALR2 inhibition, 3n exhibi-
ted strong antiglycative properties (68.66%) that made it a multifunc-
tional potential drug candidate which might synergistically mitigate the
long-term diabetic complications. The molecular docking and dynamic
simulation studies of 3n has justified in vitro ALR2 inhibition, and hence
it could be presented as a drug lead for diabetic complications.
The boiled egg plot was generated by the SwissADME web server. It
was observed that all synthesized compounds were in line with Lip-
inski’s rule of drug, and no violation of this rule was seen [29]. More-
over, druggability of compounds was evaluated through applying PAINS
(Pan-assay interference compound) filter [30]. Results suggested that all
newly synthesized compounds did not exhibit similarity with the PAINS
and bear unique structures. All compounds (3a-3q) have shown topo-
logical polar surface area (TPSA) between 107.95 Å and 98.72, and
displayed high gastrointestinal absorption. So, after discussing all these
parameters, these compounds can be considered as oral drug candidates
for further optimization.
4. Experimental work
4.1. Materials and methods
The starting materials were synthesized in the laboratory, while
some building blocks including 3-acetyl coumarin were purchased from
Sigma Aldrich and other solvents were purchased from different sup-
pliers and employed in the synthesis. The solvents, i.e. methanol, glacial
acetic acid, ethanol, petroleum ether and ethyl acetate (Merck), were
also purchased and used without any further purification in the reaction
media and TLC. Silica gel 60 aluminum-backed plates were employed to
monitor the progress of the reaction. The IR spectra were recorded in the
range 4000–500 cmꢀ 1 via Bruker Vector-22 spectrometer. 1H and 13C
NMR spectra were taken in DMSO‑d6 and CDCl3 using Bruker
3. Conclusion
A series of coumarin-based thiosemicarbazones were synthesized
with the objective to make hybrids of coumarin and thiosemicarbazone
moiety for the improved ALR2 inhibition and antiglycation activity. This
scaffold has strengthened the ability of compounds to treat diabetic
6

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Fig. 4. Aldose reductase (ALR2) and aldehyde reductase (ALR1) inhibition for compounds 3e, 3f, 3g, 3h.
spectrometer (400 MHz for1H and 125 MHz for 13C) as dilute solutions
at 25 ◦C. The chemical shifts are given in ppm while coupling constants
(J) are expressed in Hertz. Fischer-John melting point apparatus was
used to determine the melting points of the synthesized derivatives and
are uncorrected.
(s, 1H, NH-CS), 9.36 (s, 1H, NH-N), 13C NMR (CDCl3) δ ppm; 15.01
(CH3), 115.63, 116.69, 118.72, 124.15, 124.92, 126.27, 128.69, 132.75,
134.43, 137.75, 141.52, 144.58 (Ar-C and coumarin ring C), 154.04
–
–
–
–
(C N), 159.32 (C O), 173.52 (C S), Anal calcd for C18H15N3O2S
–
–
(337.40); C, 64.08; H, 4.48; N, 12.45, Found C, 64.19; H, 4.54; N, 12.39.
LC-ESI-MS, m/z (%): 338.30 [M+H]+ (100)
4.2. Synthesis of thiosemicarbazones (3a-q)
4.2.2. N-(2,4-dimethylphenyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
hydrazinecarbothioamide (3b)
ꢀ 1
Yield 81%, m.p. 234–236 ◦C, IR ʋ (cm ): 1181 (C S), 1596 (C N),
–
–
–
–
Equimolar amounts i.e., 0.005 mol of each of appropriate thio-
semicarbazide (1a-q) and 3-acetyl coumarin (2) were taken in 50 mL
round bottom flask in 10 mL methanol. A few drops (1–2) of glacial
acetic acid were also added in the flask as catalyst. The reaction mixture
was refluxed for 3–4 h till the completion of reaction was confirmed thin
layer chromatography. After cooling the reaction mixture to room
temperature, the solid thiosemicarbazone derivatives (3a-q) were
filtered and washed with hot methanol followed by ether. The pure
products were finally obtained by recrystallization from ethanol.
The novel thiosemicarbazones are characterized as given below.
1
–
–
–
1720 (C O), 3283 (N H), H NMR (CDCl3) δ ppm; 2.22 (s, 3H, CH3),
2.27 (s, 3H, CH3-Ar), 2.29 (s, 3H, CH3-Ar), 6.99–7.01 (m, 2H, Ar-H),
7.24–7.32 (m, 2H, Ar-H), 7.40–7.42 (m, 1H, Ar-H), 7.50–7.54 (m, 2H,
–
Ar-H), 7.93 (s, 1H, coumarin ring C H), 8.86 (s, 1H, NH-CS), 9.01 (s,
1H, NH-N), 13C NMR (CDCl3) δ ppm; 15.04 (CH3), 17.93 (CH3-Ar), 21.10
(CH3-Ar), 116.66, 118.76, 124.90, 126.10, 127.19, 128.33, 128.66,
131.44, 132.68, 133.67, 133.95, 137.32, 141.38, 144.57 (Ar-C and
–
–
–
–
–
–
coumarin ring C), 154.03 (C N), 159.37 (C O), 177.64 (C S), Anal
calcd for C20H19N3O2S (365.45); C, 65.73; H, 5.24; N, 11.50, Found C,
65.87; H, 5.38; N, 11.38. LC-ESI-MS, m/z (%): 366.50 [M+H]+ (100)
4.2.1. 2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)-N-
phenylhydrazinecarbothioamide (3a)
4.2.3. N-(3-methoxyphenyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
ꢀ 1
Yield 84%, m.p. 220–222 ◦C, IR ʋ (cm ): 1156 (C S), 1593 (C N),
–
–
–
–
hydrazinecarbothioamide (3c)
1
–
ꢀ 1
–
Yield 85%, m.p. 283–285 ◦C, IR ʋ (cm ): 1159 (C S), 1600 (C N),
–
–
–
–
1706 (C O), 3217 (N H), H NMR (CDCl3) δ ppm; 2.29 (s, 3H, CH3),
–
1699 (C O), 3255 (N H), 1H NMR (DMSO‑d6) δ ppm; 2.33 (s, 3H,
–
7.16–7.20 (m, 2H, Ar-H), 7.26–7.35 (m, 3H, Ar-H), 7.52–7.55 (m, 2H,
–
–
–
Ar-H), 7.58–7.63 (m, 2H, Ar-H), 7.93 (s, 1H, coumarin ring C H), 8.78
7

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Fig. 5. Aldose reductase (ALR2) and aldehyde reductase (ALR1) inhibition for the compounds 3i, 3j, 3k, 3l.
CH3), 3.75 (s, 3H, OCH3), 6.76–6.79 (m, 1H, Ar-H), 7.17–7.28 (m, 2H,
Ar-H), 7.35–7.46 (m, 3H, Ar-H), 7.65 (ddd, 1H, Ar-H, J = 1.2 Hz, 8.8
Hz), 7.80 (dd, 1H, Ar-H, J = 0.8 Hz, 7.6 Hz), 8.49 (s, 1H, coumarin ring
–
1H, Ar-H, J = 1.5 Hz, 8.4 Hz), 7.78 (dd, 1H, Ar-H, J = 1.8 Hz, 7.8 Hz),
–
8.35 (s, 1H, coumarin ring C H), 9.04 (t, 1H, NH-CS, J = 6.8 Hz), 10.62
(s, 1H, NH-N), 13C NMR (DMSO‑d6) δ ppm; 16.68 (CH3), 47.17 (CH2),
116.48, 119.27, 125.24, 126.56, 127.23, 127.68, 127.88, 128.61,
128.65, 129.58, 132.88, 139.60, 142.32, 146.78 (Ar-C and coumarin
C
H), 10.12 (s, 1H, NH-CS), 10.88 (s, 1H, NH-N), 13C NMR (DMSO‑d6)
δ ppm; 16.63 (CH3), 55.62 (OCH3), 110.01, 111.19, 116.45, 117.44,
–
–
–
–
119.34, 125.19, 126.06, 129.37, 132.30, 132.61, 138.31, 142.66,
ring C), 153.80 (C N), 159.59 (C O), 179.24 (C S), Anal calcd for
– –
–
–
–
146.61 (Ar-C and coumarin ring C), 153.49 (C N), 159.40 (C O),
C19H17N3O2S (351.42); C, 64.94; H, 4.88; N, 11.96, Found C, 64.88; H,
–
177.28 (C S), Anal calcd for C19H17N3O3S (367.42); C, 62.11; H, 4.66;
4.95; N, 11.80. LC-ESI-MS, m/z (%): 352.30 [M+H]+ (100)
–
–
N, 11.44, Found C, 62.24; H, 4.57; N, 11.57. LC-ESI-MS, m/z (%): 368.30
[M+H]+ (100)
4.2.6. N-(4-chlorobenzyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
hydrazinecarbothioamide (3f)
ꢀ 1
Yield 80%, m.p. 296–298 ◦C, IR ʋ (cm ): 1188 (C S), 1605 (C N),
–
–
–
–
4.2.4. 2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)-N-(o-tolyl)
1
–
–
hydrazinecarbothioamide (3d)
1716 (C O), 3357 (N H), H NMR (CDCl3) δ ppm; 2.22 (s, 3H, CH3),
–
ꢀ 1
Yield 82%, m.p. > 300 ◦C, IR ʋ (cm ): 1156 (C S), 1606 (C N),
4.90 (d, 2H, CH2, J = 6.0 Hz), 7.20–7.33 (m, 7H, Ar-H), 7.45–7.51 (m,
–
–
–
–
1710 (C O), 3280, 3370 (N H), 1H NMR (DMSO‑d6) δ ppm; 2.28 (s,
1H, Ar-H), 7.81 (s, 1H, coumarin ring C H), 7.84 (t, 1H, NH-CS, J = 5.6
–
–
–
–
3H, CH3), 2.33 (s, 3H, CH3-Ar), 7.12–7.18 (m, 2H, Ar-H), 7.37–7.46 (m,
Hz), 8.74 (s, 1H, NH-N), 13C NMR (CDCl3) δ ppm; 15.08 (CH3), 48.50
(CH2), 116.60, 118.71, 124.81, 126.13, 127.77, 127.84, 128.67, 128.78,
128.65, 132.56, 137.37, 141.42, 144.90 (Ar-C and coumarin ring C),
4H, Ar-H), 7.65 (ddd, 1H, Ar-H, J = 1.5 Hz, 7.8 Hz), 7.80 (dd, 1H, Ar-H,
–
J = 1.2 Hz, 7.5 Hz), 8.50 (s, 1H, coumarin ring C H), 10.08 (s, 1H, NH-
CS), 10.83 (s, 1H, NH-N), 13C NMR (DMSO‑d6) δ ppm; 16.67 (CH3),
21.06 (CH3-Ar), 116.47, 116.61, 119.35, 125.24, 125.82, 126.15,
129.11, 129.66, 132.95, 135.04, 136.86, 137.05, 142.72, 146.93 (Ar-C
153.99 (C N), 159.48 (C O), 178.41 (C S), Anal calcd for
–
–
–
–
–
–
C
19H16ClN3O2S (385.87); C, 59.14; H, 4.18; N, 10.89, Found C, 59.27; H,
4.24; N, 11.74. LC-ESI-MS, m/z (%): 386.60 [M+H]+ (100)
–
–
–
–
–
–
and coumarin ring C), 153.82 (C N), 159.50 (C O), 177.56 (C S),
Anal calcd for C19H17N3O2S (351.42); C, 64.94; H, 4.88; N, 11.96, Found
4.2.7. N-cyclohexyl-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
C, 64.81; H, 4.97; N, 11.84. LC-ESI-MS, m/z (%): 352.40 [M+H]+ (100)
hydrazinecarbothioamide (3g)
ꢀ 1
Yield 79%, m.p. 243–245 ◦C, IR ʋ (cm ): 1199 (C S), 1606 (C N),
–
–
–
–
1732 (C O), 3336 (N H), 1H NMR (DMSO‑d6) δ ppm; 1.22–1.38 (m,
–
–
–
4.2.5. N-benzyl-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
hydrazinecarbothioamide (3e)
6H, Cyclohexyl), 1.56–1.72 (m, 2H, Cyclohexyl), 1.86–1.89 (m, 2H,
Cyclohexyl), 2.25 (s, 3H, CH3), 4.17–4.19 (m, 1H, CH-NH), 7.37–7.44
(m, 2H, Ar-H), 7.65 (ddd, 1H, Ar-H, J = 1.5 Hz, 8.4 Hz), 7.83 (dd, 1H,
Ar-H, J = 0.9 Hz, 7.5 Hz), 8.1 (d, 1H, NH-CS, J = 8.4 Hz), 8.31 (s, 1H,
ꢀ 1
Yield 86%, m.p. 261–263 ◦C, IR ʋ (cm ): 1186 (C S), 1592 (C N),
–
–
–
–
1706 (C O), 3217 (N H), 1H NMR (DMSO‑d6) δ ppm; 2.28 (s, 3H,
–
–
–
CH3), 4.85 (d, 2H, CH2, J = 6.3 Hz), 7.22–7.44 (m, 7H, Ar-H), 7.63 (ddd,
8

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Fig. 6. Aldose reductase (ALR2) and aldehyde reductase (ALR1) inhibition for compounds 3m, 3n, 3o, 3p, 3q.
coumarin ring C H), 10.46 (s, 1H, NH-N), 13C NMR (DMSO‑d6) δ ppm;
16.35 (CH3), 24.01, 25.09, 25.34, 25.56, 32.20, 32.31 (Cyclohexyl),
116.43, 119.30, 125.19, 126.32, 129.63, 132.89, 142.56, 146.04 (Ar-C
(DMSO‑d6) δ ppm; 16.71 (CH3), 46.43 (CH2), 115.21, 115.49, 116.49,
–
119.26, 125.25, 126.58, 129.68, 132.90, 135.79, 135.83, 142.33,
–
146.92, 153.80, 159.60 (Ar-C and coumarin ring C), 153.99 (C N),
–
–
–
–
–
–
–
–
–
–
and coumarin ring C), 154.08 (C N), 159.20 (C O), 177.31 (C S),
159.48 (C O), 178.41 (C S), Anal calcd for C19H16BrN3O2S (430.32);
–
Anal calcd for C18H21N3O2S (343.44); C, 62.95; H, 6.16; N, 12.23, Found
C, 53.03; H, 3.75; N, 9.76, Found C, 53.15; H, 3.67; N, 9.89. LC-ESI-MS,
C, 62.84; H, 6.28; N, 12.39. LC-ESI-MS, m/z (%): 344.50 [M+H]+ (100)
m/z (%): 431.30 [M+H]+ (100)
4.2.8. N-(4-bromobenzyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
4.2.9. N-(2,3-dichlorophenyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
hydrazinecarbothioamide (3h)
hydrazinecarbothioamide (3i)
ꢀ 1
ꢀ 1
Yield 83%, m.p. 296–298 ◦C, IR ʋ (cm ): 1210 (C S), 1610 (C N),
Yield 85%, m.p. 279–281 ◦C, IR ʋ (cm ): 1152 (C S), 1603 (C N),
–
–
–
–
–
–
–
–
1741 (C O), 3207, 3310 (N H), 1H NMR (DMSO‑d6) δ ppm; 2.27 (s,
1718 (C O), 3298 (N H), 1H NMR (DMSO‑d6) δ ppm; 2.35 (s, 3H,
–
–
–
–
–
–
3H, CH3), 4.81 (d, 2H, CH2, J = 6.0 Hz), 7.14 (t, 2H, Ar-H, J = 8.6 Hz),
CH3), 7.36–7.48 (m, 3H, Ar-H), 7.57–7.69 (m, 3H, Ar-H), 7.76 (dd, 1H,
–
7.36–7.45 (m, 4H, Ar-H), 7.64 (ddd, 1H, Ar-H, J = 1.5 Hz, 8.4 Hz), 7.78
Ar-H, J = 1.5 Hz, 7.8 Hz), 8.48 (s, 1H, coumarin ring C H), 10.18 (s,
1H, NH-CS), 11.17 (s, 1H, NH-N), 13C NMR (DMSO‑d6) δ ppm; 16.95
–
(dd, 1H, Ar-H, J = 1.5 Hz, 8.4 Hz), 8.35 (s, 1H, coumarin ring C H),
9.08 (t, 1H, NH-CS, J = 6.4 Hz), 10.63 (s, 1H, NH-N), 13C NMR
(CH3), 116.51, 119.28, 125.28, 126.15, 128.13, 128.85, 129.27, 129.64,
9

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Fig. 7. Antiglycation activity (determined as percent inhibition of AGEs formation) of compounds 3a-3q. AG (Aminoguanidine) used as a positive control (100 µM).
The percent inhibition of AGEs is shown at Y-axis and compounds (3a-3q) are oriented at X-axis.
NMR (DMSO‑d6) δ ppm; 16.93 (CH3),104.38, 104.70, 105.05, 111.34,
111.64, 116.52, 119.29, 119.51, 124.19, 124.41, 125.28, 126.22,
129.63, 131.92, 133.00, 142.71, 147.86 (Ar-C and coumarin ring C),
–
–
–
–
153.87 (C N), 159.61 (C O), 179.38 (C S), Anal calcd for
–
–
C
18H13F2N3O2S (373.38); C, 57.90; H, 3.51; N, 11.25, Found C, 57.76; H,
3.62; N, 11.34. LC-ESI-MS, m/z (%): 374.50 [M+H]+ (100)
4.2.11. 2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)-N-(p-tolyl)
hydrazinecarbothioamide (3k)
ꢀ 1
Yield 88%, m.p. 212–214 ◦C, IR ʋ (cm ): 1174 (C S), 1606 (C N),
–
–
–
–
1
–
–
1709 (C O), 3284, 3371 (N H), H NMR (CDCl3) δ ppm; 2.27 (s, 3H,
–
CH3), 2.28 (s, 3H, CH3-Ar), 7.13 (d, 2H, Ar-H, J = 8.0 Hz), 7.26–7.31 (m,
2H, Ar-H), 7.46 (d, 2H, Ar-H, J = 8.4 Hz), 7.52–7.54 (m, 2H, Ar-H), 7.93
–
(s, 1H, coumarin ring C H), 8.79 (s, 1H, NH-CS), 9.26 (s, 1H, NH-N),
13C NMR (CDCl3) δ ppm; 15.03 (CH3), 21.07 (CH3-Ar), 109.40,
116.65, 118.75, 124.42, 124.89, 126.65, 128.70, 129.41, 130.89,
132.68, 135.16, 136.22, 141.51, 143.92 (Ar-C and coumarin ring C),
–
–
–
–
154.03 (C N), 159.36 (C O), 176.58 (C S), Anal calcd for
–
–
C
19H17N3O2S (351.42); C, 64.94; H, 4.88; N, 11.96, Found C, 64.86; H,
4.99; N, 11.81. LC-ESI-MS, m/z (%): 352.20 [M+H]+ (100)
Fig. 8. Lineweaver-Burks plot for inhibition of human AKR1B1(expressed
enzyme in E.coli BL21De3) by compound 3n. The compound 3n was tested in
various concentration 0 µM (blue), 0.1 µM(purple), 1.5 µM(green) and 2.0 µM
(brown) whereas substrate (DL-glyceraldehyde) was used in different concen-
tration (0–60 mM). The concentration of cofactor (NADPH) kept constant at
100 µM. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
4.2.12. N-(4-chlorophenyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
hydrazinecarbothioamide (3l)
ꢀ 1
Yield 82%, m.p. 219–221 ◦C, IR ʋ (cm ): 1189 (C S), 1590 (C N),
–
–
–
–
1705 (C O), 3225 (N H), 1H NMR (DMSO‑d6) δ ppm; 2.34 (s, 3H,
–
–
–
CH3), 7.38–7.46 (m, 5H, Ar-H), 7.44–7.67 (m, 2H, Ar-H), 7.81 (dd, 1H,
–
Ar-H, J = 1.6 Hz, 7.6 Hz), 8.49 (s, 1H, coumarin ring C H), 10.20 (s,
1H, NH-CS), 10.96 (s, 1H, NH-N), 13C NMR (DMSO‑d6) δ ppm; 16.74
(CH3), 116.49, 119.33, 125.27, 126.16, 127.42, 128.37, 128.53, 129.68,
133.01, 138.44, 142.82, 147.53 (Ar-C and coumarin ring C), 153.85
130.05, 132.13, 133.03, 139.05, 142.73, 147.75 (Ar-C and coumarin
–
–
–
–
ring C), 153.87 (C N), 159.53 (C O), 178.44 (C S), Anal calcd for
–
–
C
18H13Cl2N3O2S (406.29); C, 53.21; H, 3.23; N, 10.34, Found C, 53.15;
–
–
–
–
–
–
H, 3.37; N, 10.50. LC-ESI-MS, m/z (%): 440.40 [M+H]+ (100)
(C N), 159.49 (C O), 177.59 (C S), Anal calcd for C18H14ClN3O2S
(371.84); C, 58.14; H, 3.79; N, 11.30, Found C, 58.26; H, 3.63; N, 11.48.
LC-ESI-MS, m/z (%): 372.80 [M+H]+ (100)
4.2.10. N-(2,4-difluorophenyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
hydrazinecarbothioamide (3j)
ꢀ 1
4.2.13. N-(2,6-dimethylphenyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
Yield 81%, m.p. 266–268 ◦C, IR ʋ (cm ): 1160 (C S), 1607 (C N),
–
–
–
–
1719 (C O), 3282 (N H), 1H NMR (DMSO‑d6) δ ppm; 2.33 (s, 3H,
hydrazinecarbothioamide (3m)
–
–
–
ꢀ 1
Yield 90%, m.p. 228–230 ◦C, IR ʋ (cm ): 1181 (C S), 1595 (C N),
–
–
–
–
CH3), 7.11 (ddd, 1H, Ar-H, J = 1.8 Hz, 8.7 Hz), 7.32–7.55 (m, 4H, Ar-H),
1
–
–
1719 (C O), 3282 (N H), H NMR (DMSO‑d6) δ ppm; 2.20 (s, 6H, 2 ×
–
7.63–7.68 (m, 1H, Ar-H), 7.75–7.79 (m, 1H, Ar-H), 9.92 (s, 1H,
coumarin ring C H), 11.08 (s, 1H, NH-CS), 11.19 (s, 1H, NH-N), 13C
CH3-Ar), 2.33 (s, 3H, CH3), 7.08–7.14 (m, 3H, Ar-H), 7.36–7.46 (m, 2H,
–
10

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Fig. 9. Docked inhibitor 3n at active site of ALR2. A) The structure of ALR2 have shown in ribbon representation B) Three dimensional conformation of ALR2
showing interactions of amino acid residue at active site with inhibitor 3n as stick model. The residues are shown in abbreviated form and interactions are rep-
resented through dashed line.
Fig. 10. Two dimensional docked conformation of ALR2 showing types of amino acids interacting with inhibitor 3n. The hydrogen bonds are shown as purple
dashed lines. Amino acids residue showing interaction with inhibitor are TRp111, Gln183, Hist110, Tyr48, Lys77, Trp79, Tyr209, Val47, Trp20 and Phe122. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Ar-H), 7.63–7.67 (m, 1H, Ar-H), 7.74 (dd, 1H, Ar-H, J = 1.6 Hz, 8.0 Hz),
CH3), 7.19–7.46 (m, 5H, Ar-H), 7.56 (ddd, 1H, Ar-H, J = 1.5 Hz, 8.1 Hz),
7.65 (ddd, 1H, Ar-H, J = 1.5 Hz, 7.5 Hz), 7.78 (dd, 1H, Ar-H, J = 1.5 Hz,
–
8.55 (s, 1H, coumarin ring C H), 9.81 (s, 1H, NH-CS), 10.75 (s, 1H, NH-
N), 13C NMR (DMSO‑d6) δ ppm; 15.15 (CH3), 18.04 (CH3-Ar), 21.21
(CH3-Ar), 116.76, 118.86, 125.00, 126.20, 127.15, 127.30, 128.76,
131.54, 132.78, 133.78, 134.05, 137.43, 141.48, 144.67 (Ar-C and
7.8 Hz), 8.50 (s, 1H, coumarin ring C H), 10.01 (s, 1H, NH-CS), 10.11
–
(s, 1H, NH-N), 13C NMR (DMSO‑d6) δ ppm; 16.90 (CH3), 116.06, 116.32,
116.50, 116.63, 119.31, 124.51, 124.56, 125.26, 126.18, 127.45,
127.61, 128.63, 128.74, 129.22, 129.63, 130.46, 132.98, 142.71,
–
–
–
–
coumarin ring C), 154.13 (C N), 159.48 (C O), 177.75 (C S), Anal
–
–
–
calcd for C20H19N3O2S (365.45); C, 65.73; H, 5.24; N, 11.50, Found C,
147.62, 153.86, 154.10 (Ar-C and coumarin ring C), 155.98 (C N),
–
65.89; H, 5.13; N, 11.62. LC-ESI-MS, m/z (%): 366.40 [M+H]+ (100)
159.57 (C O), 178.94 (C S), Anal calcd for C18H14FN3O2S (355.39);
–
–
–
–
C, 60.83; H, 3.97; N, 11.82, Found C, 60.74; H, 3.84; N, 11.98. LC-ESI-
4.2.14. N-(2-fluorophenyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
MS, m/z (%): 356.10 [M+H]+ (100)
hydrazinecarbothioamide (3n)
ꢀ 1
Yield 79%, m.p. 277–279 ◦C, IR ʋ (cm ): 1228 (C S), 1618 (C N),
–
–
–
–
1717 (C O), 3285 (N H), 1H NMR (DMSO‑d6) δ ppm; 2.33 (s, 3H,
–
–
–
11

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Fig. 11. Radius of gyration (Rg) of 3FX4, protein plus cognate ligand (FX4) and protein plus selective compound (3l) during 50 ns MD-simulation run.
Fig. 12. Radius of gyration (Rg) of 1PWM, protein plus cognate ligand (FID) and protein plus selective compound (3n) during 50 ns MD-simulation run.
Fig. 13. Root Mean Square Deviation (RMSD) of 3FX4, protein plus cognate ligand (FX4) and protein plus selective compound (3l) during 50 ns MD-simulation run.
–
4.2.15. N-(3-isopropylphenyl)-2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
Ar-H), 7.92 (s, 1H, coumarin ring C H), 8.77 (s, 1H, NH-CS), 9.28 (s,
hydrazinecarbothioamide (3o)
1H, NH-N), 13C NMR (CDCl3) δ ppm; 15.04 (CH3), 23.96 (2 × isopropyl
CH3), 33.74 (isopropyl CH), 110.22, 116.97, 118.76, 121.67, 124.26,
124.92, 126.83, 128.25, 128.68, 128.94, 132.69, 134.35, 135.27,
ꢀ 1
Yield 87%, m.p. 291–293 ◦C, IR ʋ (cm ): 1193 (C S), 1606 (C N),
–
–
–
–
1716 (C O), 3298 (N H), 1H NMR (CDCl3) δ ppm; 1.18 (d, 6H, 2 ×
–
–
–
–
–
–
CH3, J = 6.8 Hz) 2.28 (s, 3H, CH3), 2.80–2.88 (m, 1H, isopropyl CH),
141.48 (Ar-C and coumarin ring C), 154.15 (C N), 159.35 (C O),
–
–
7.17–7.18 (m, 1H, Ar-H), 7.23–7.32 (m, 3H, Ar-H), 7.46–7.54 (m, 4H,
176.28 (C S), Anal calcd for C21H21N3O2S (379.48); C, 66.47; H, 5.58;
–
12

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Fig. 14. Root Mean Square Deviation (RMSD) of 1PWM, protein plus cognate ligand (FID) and protein plus selective compound (3n) during 50 ns MD-
simulation run.
Fig. 15. Root Mean Square Fluctuation (RMSF) of 3FX4, protein plus cognate ligand (FX4) and protein plus selective compound (3l) during 50 ns MD-simulation run.
Fig. 16. Root Mean Square Fluctuation (RMSF) of 1PWM, protein plus cognate ligand (FID) and protein plus selective compound (3n) during 50 ns MD-
simulation run.
N, 11.07, Found C, 66.61; H, 5.47; N, 11.19. LC-ESI-MS, m/z (%): 380.30
[M+H]+ (100)
4.2.16. 2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)-N-
phenethylhydrazinecarbothioamide (3p)
ꢀ 1
Yield 88%, m.p. 284–286 ◦C, IR ʋ (cm ): 1174 (C S), 1606 (C N),
–
–
–
–
13

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
Table 2
Pharmacokinetic profile for compounds 3a-3q via SwissADME.
Codes
TPSAa
Lipinski violation
PAINSb
WLOGPc
NRBd
HBDe
GI Absorptionf
HBAg
3a
3b
3c
3d
3e
3f
98.72
98.72
107.95
98.72
98.72
98.72
98.72
98.72
98.72
98.72
98.72
98.72
98.72
98.72
98.72
98.72
101.96
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3.31
3.93
3.32
3.62
3.03
3.68
3.31
3.79
4.62
4.43
3.62
3.97
3.93
3.87
4.44
3.22
3.38
5
5
6
5
6
6
5
6
5
5
5
5
5
5
6
7
6
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
3
3
4
3
3
3
3
3
3
5
3
3
3
4
3
3
3
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
a
Topological polar surface area,
b
c
d
e
f
Pan-assay interference,
Logarithm of partition coefficient between n-octanol and water,
Number of rotatable bond,
Hydrogen bond donor,
Gastrointestinal absorption,
g
Hydrogen bond acceptor.
Fig. 17. Boiled egg plot for synthesized compounds 3a-3q.
1709 (C O), 3286, 3369 (N H), 1H NMR (DMSO‑d6) δ ppm; 2.26 (s,
65.73; H, 5.24; N, 11.50, Found C, 65.61; H, 5.39; N, 11.38. LC-ESI-MS,
–
–
–
3H, CH3), 2.90 (t, 2H, CH2, J = 8.1 Hz), 3.77 (q, 2H, CH2, J = 5.7 Hz),
7.19–7.32 (m, 5H, Ar-H), 7.39–7.46 (m, 2H, Ar-H), 7.66 (ddd, 1H, Ar-H,
J = 1.2 Hz, 8.4 Hz), 7.81 (dd, 1H, Ar-H, J = 0.9 Hz, 7.5 Hz), 8.28 (s, 1H,
m/z (%): 364.50 [M+H]+ (100)
4.2.17. N-(3-(dimethylamino)phenyl)-2-(1-(2-oxo-2H-chromen-3-yl)
–
coumarin ring C H), 8.58 (t, 1H, NH-CS, J = 6.8 Hz), 10.54 (s, 1H, NH-
ethylidene)hydrazinecarbothio amide (3q)
ꢀ 1
N), 13C NMR (DMSO‑d6) δ ppm; 16.59 (CH3), 35.14 (CH2), 45.64 (CH2),
Yield 81%, m.p. > 300 ◦C, IR ʋ (cm ): 1192 (C S), 1606 (C N),
–
–
–
–
–
–
116.52, 119.28, 125.30, 126.66, 128.91, 129.08, 129.55, 132.91,
1711 (C O), 3320 (N H), 1H NMR (DMSO‑d6) δ ppm; 2.31 (s, 3H,
–
–
–
139.64, 142.19, 146.45 (Ar-C and coumarin ring C), 153.81 (C N),
CH3), 2.89 (s, 6H, 2 × CH3), 6.69–6.72 (m, 2H, Ar-H), 7.27–7.47 (m, 4H,
–
–
–
159.58 (C O), 178.53 (C S), Anal calcd for C20H19N3O2S (363.45); C,
Ar-H), 7.64 (t, 1H, Ar-H, J = 7.8 Hz), 7.79 (d, 1H, Ar-H, J = 7.5 Hz), 8.50
–
14

A. Imran et al.
Bioorganic Chemistry 115 (2021) 105164
–
(s, 1H, coumarin ring C H), 9.92 (s, 1H, NH-CS), 10.67 (s, 1H, NH-N),
13C NMR (DMSO‑d6) δ ppm; 16.61 (CH3), 30.55 (2 × CH3), 112.28,
116.45, 119.38, 124.94, 125.22, 126.19, 127.16, 128.56, 129.62,
132.88, 142.60, 146.46, 148.94 (Ar-C and coumarin ring C), 153.81
LeadIT reproduced conformation of co-crystalized inhibitor with RMSD
of 0.69 Å.
For each docked ligand, 50 poses were selected and further analyzed
through hyde assessment. The pose having lowest binding energy were
selected and analyzed for 2D, 3D interaction with active site by using
Discovery Studio Visualizer (San Diago, 2019). The free binding en-
ergies (ΔG value) were calculated through hyde assessment tool for
docked inhibitor 3 l (ΔG = -40KJ/mol) for ALR1 and 3n (ΔG = -30KJ/
mol) for ALR2 and correlated with good binding affinities of the
compounds.
–
–
–
–
–
–
(C N), 159.54 (C O), 177.69 (C S), Anal calcd for C20H20N4O2S
(380.46); C, 63.14; H, 5.30; N, 14.73, Found C, 63.29; H, 5.18; N, 14.89.
LC-ESI-MS, m/z (%): 381.30 [M+H]+ (100)
4.3. Enzymatic assay
The enzyme activity for ALR2 was determined at 32 ◦C in the reac-
tion mixture (total assay volume 100 µL), composed of 20 µL of sodium
phosphate buffer (100 mM, pH 6.2), 20 µL of NADPH (0.1 mM), 10 µL of
test compound (1 mM), 30 µL of partially purified enzyme and 20 µL
(0.1 mM) of substrate. Except NADPH (cofactor), the reaction mixture
was incubated at 32 ◦C for 10 min. The NADPH was added for initiation
of reaction and monitored for 5 min. The ALR1 activity was performed at
32 ◦C, the reaction mixture (total assay volume 100 µL) was composed of
20 µL of phosphate buffer (100 mM, pH 7.2), 20 µL of NADPH (0.1 mM),
10 µL of test compound (1 mM), 30 µL of enzyme extract and 20 µL (0.1
mM) of sodium-D-glucuronate (substrate). Except cofactor, the reaction
mixture was subjected to incubation at 32 ◦C for 10 min, and the reac-
tion was initiated with the addition of cofactor and monitored for 5 min.
The compounds 3a-3q were dissolved in DMSO and diluted with
deionized water such that the final concentration of DMSO was kept
0.1% in all incubations. The inhibitory effects of 3a-3q was measured at
concentration of 100 µM (final concentration in the assay). The com-
pounds that showed percentage inhibition>50% were further tested at
different concentration up to 10 nM in triplicate, and IC50 values were
calculated using GraphPad Prism version 8.3.
4.6. In-silico ADME analysis
The SwissADME web server was used for determination of pharma-
cokinetic profile (absorption, distribution, metabolism and excretion)
and physicochemical properties of compounds 3a-3q. With the utility of
Chemdraw professional (2018), chemicals structures were converted to
SMILES format and then uploaded on web server for prediction and
calculation of pharmacokinetic profiles of synthesized compounds.
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.
Acknowledgments
A. Imran is thankful to Higher Education commission, Pakistan for
awarding scholarship under International Research Support Initiative
Program (IRSIP) through award no. 1-8/HEC/HRD/2019/8892.
4.4. Antiglycation assay
Appendix A. Supplementary data
The glycation of bovine serum albumin (BSA) was performed by
following reported method with slight modifications [31]. BSA (1 mg/
mL) was incubated with 0.5 M glucose in 0.1 M PBS (phosphate-buffered
saline) and test compound (1 mM), pH 7.4, at 50 ◦C for 4 days in the
darkness. Aminoguanidine was used as a reference glycation inhibitor.
The glycation of BSA was measured through determining the fluores-
cence intensity at excitation and emission wavelengths 350 nm and 460
nm, respectively. The percentage of AGEs (advanced glycation end
products) inhibition was calculated using the following formula:
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105164.
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