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

Bioorganic Chemistry 115 (2021) 105164

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Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Development of coumarin-thiosemicarbazone hybrids as aldose reductase

inhibitors: Biological assays, molecular docking, simulation studies and

ADME evaluation

,

Aqeel Imran^{a c}, Muhammad Tariq Shehzad^b, Taha al Adhami^c, Khondaker Miraz Rahman^c,

,

,*

Dilawar Hussain^a, Rima D. Alharthy^d, Zahid Shaﬁq^{b *}, Jamshed Iqbal^a

^aCenter for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan

^bInstitute of Chemical Sciences, Bahauddin Zakariya University, Multan 60800, Pakistan

^cInstitute of Pharmaceutical Science, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom

^dDepartment 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 IC₅₀in 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

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 cm^{ꢀ 1}. Similarly, NH

–

and C O stretching appeared at 3207–3371 cm^{ꢀ 1}and 1699–1741 cm^{ꢀ 1}

–

respectively. NMR spectra of compounds were recorded in CDCl₃and

DMSO‑d₆. In ¹H NMR, the methyl protons of the carbonyl moiety

resonate at 2.22–2.35 ppm, whereas the CH₃protons 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-CH₂moiety (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

–

δ_H8.1. 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 δ_C15.01–16.95, while methyl carbons directly

attached to aromatic ring appeared at δ_C17.93–21.21. 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 δ_C153.49–155.98 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 IC₅₀value 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 cm^{ꢀ 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 IC₅₀value 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 IC₅₀value of 15.213 µM

and inhibition for ALR2 with IC₅₀value 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

IC₅₀value 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 IC₅₀value of 7.118 µM and

inhibitory activity for ALR1 with IC₅₀of 4.512 µM. While discussing 3k,

the presence of methyl group at meta- position improved ALR2 inhibi-

tory activity with an IC₅₀value of 3.883 µM, whereas inhibitory activity

decreased against ALR1 as observed from the IC₅₀value 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 IC₅₀values. Compound 3e has exhibited

percent inhibition with value of 38.21% against ALR2, but an IC₅₀of

7.523 µM against ALR1, so it was considered as selective inhibitor of

ALR1. Compound 3f displayed an IC₅₀value of 15.213 µM for ALR2 and

IC₅₀value of 4.313 µM for ALR1 whereas 3i showed inhibition for ALR2

with IC₅₀value of 5.202 µM and for ALR1 with IC₅₀value 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

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A. Imran et al.

Bioorganic Chemistry 115 (2021) 105164

Scheme 1. Synthesis of coumarin derived thiosemicarbazones (3a-q).

ALR2 with an IC₅₀value 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 IC₅₀value 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 IC₅₀value 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 IC₅₀value

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 IC₅₀value 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

IC₅₀value of 7.523 µM, and 3f showed inhibition with IC₅₀value 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,

O₂atom 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

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).

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A. Imran et al.

Bioorganic Chemistry 115 (2021) 105164

Table 1

The IC₅₀values for ALR2, ALR1 and antiglycation activity for compounds 3a-3q.

Codes

R

ALR1

ALR2

I (%, 100 µM) of AGEs formation^c

IC₅₀(µM)^a/ I (%, 100 µM)^b

3a

3b

3c

3d

3e

3f

C₆H₅

15.213^a

27.505^a

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

9.379^a

11.511^a

(2.969–15.79)

34.59^b

(6.162–16.86)

5.322^a

(2.076–8.569)

4.512^a

7.118^a

(2.575–6.449)

(4.037–10.20)

38.21^b

7.523^a

(2.94–12.10)

4.313^a

4-ClC₆H₄CH₂

15.213^a

(2.246–6.38)

(7.156–23.27)

3g

3h

C₆H₁₁

26.33^b

26.82^b

51.24 ± 0.945

47.22 ± 0.505

4-BrC₆H₄CH₂

12.155^a

28.47^a

(6.791–17.52)

(8.204–48.69)

3i

2,3-(Cl)₂C₆H₃

3.853^a

5.202^a

64.41 ± 1.035

(1.854–5.852)

(2.623–7.781)

3j

2,4-(F)₂C₆H₃

4-CH₃C₆H₄

4-ClC₆H₄

23.89^b

3.527^a

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

12.394^a

3.883^a

(6.399–18.39)

(2.231–5.535)

13.036^a

1.376^a

(0.693–2.059)

8.695^a

(5.353–20.72)

3m

3n

2,6-(CH₃)₂C₆H₃

2-FC₆H₄

10.916^a

(4.54–12.85)

(5.792–16.04)

3.035^a

34.78^b

(0.819–5.252)

3o

4-isopropylC₆H₄

C₆H₅CH₂CH₂

41.55^b

25.20^b

38.91 ± 1.522

64.81 ± 0.899

65.09 ± 2.59

n.d.

3p

22.46^b

42.27^b

3q

4-N(CH₃)₂C₆H₄

17.87^b

18.69^b

Valproic Acid^d

49.311^a

(9.923–88.40)

n.d.

Sorbinil^e

2.745^a

n.d.

(1.088–4.403)

6-aminoguanidine^f

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 R_gslightly 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

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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 IC₅₀values 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 IC₅₀value 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 cm^{ꢀ 1}via Bruker Vector-22 spectrometer. ¹H and ¹³C

NMR spectra were taken in DMSO‑d₆and CDCl₃using 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, CH_2,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, CH_2,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

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, CH_2,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

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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

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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,

–

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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

TPSA^a

Lipinski violation

PAINS^b

WLOGP^c

NRB^d

HBD^e

GI Absorption^f

HBA^g

3a

3b

3c

3d

3e

3f

98.72

107.95

98.72

101.96

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

6

5

6

5

6

5

6

7

6

2

High

3

4

3

5

3

4

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, CH_2,J = 8.1 Hz), 3.77 (q, 2H, CH_2,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 IC₅₀values 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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16

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