5-Benzylidene-2,3-diarylthiazolidine-4-ones: Design, synthesis, spectroscopic characterization, in vitro biological and computational evaluation

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5-Benzylidene-2,3-diarylthiazolidine-4-ones:

Design, synthesis, spectroscopic characterization,

in vitro biological and computational evaluation

Ebrahim Saeedian Moghadam, Bilqees Sameem, Raid Abdel-Jalil,

Mohammad Ali Faramarzi & Mohsen Amini

To cite this article: Ebrahim Saeedian Moghadam, Bilqees Sameem, Raid Abdel-Jalil, Mohammad

Ali Faramarzi & Mohsen Amini (2021): 5-Benzylidene-2,3-diarylthiazolidine-4-ones: Design,

synthesis, spectroscopic characterization, inꢀvitro biological and computational evaluation, Synthetic

Communications, DOI: 10.1080/00397911.2021.1946699

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https://doi.org/10.1080/00397911.2021.1946699

5-Benzylidene-2,3-diarylthiazolidine-4-ones: Design,

synthesis, spectroscopic characterization, in vitro biological

and computational evaluation

Ebrahim Saeedian Moghadam^a,b, Bilqees Sameem^a, Raid Abdel-Jalil^b,

Mohammad Ali Faramarzi^c, and Mohsen Amini^a

^aDepartment of Medicinal Chemistry, Faculty of Pharmacy and Drug Design and Development Research

Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran

b

University of Medical Sciences, Tehran, Iran; Department of Chemistry, College of Science, Sultan

c

Qaboos University, Muscat, Sultanate of Oman; Faculty of Pharmacy, Department of Pharmaceutical

Biotechnology, Tehran University of Medical Sciences, Tehran, Iran

ABSTRACT

ARTICLE HISTORY

Received 29 March 2021

The synthesis and antidiabetic activity of 5-Benzylidene-2,3-diarylthia-

zolidine-4-one derivatives (6a–w) are presented in the current work.

Screening of derivatives 6a–w for their a-glucosidase inhibitory activ-

ity, showed higher inhibitory activity of twenty of the screened com-

pounds (IC₅₀: 105–412mM) in comparison to acarbose (IC₅₀: 750mM)

as a standard. Compounds 6r, 6b, and 6q exerted the best activity

with the IC₅₀value of 105, 110, and 127mM, respectively. Performing

the kinetic studies, revealed the competitive mode of inhibition for

6r. It binds to the active site on the enzyme and competes with the

substrate for binding to the active site. based on molecular docking

studies, 6b, 6q, and 6r interact with HIS280, ASP307, and PRO312

residues, which show the important role of these residues inside the

active site of the enzyme. Cytotoxicity studies also showed

IC₅₀> 750mM for 6a–w on different cell lines namely, NIH3T3, MCF-

7, and HT-29.

KEYWORDS

a-Glucosidase inhibitors;

diabetes mellitus; kinetic

study; molecular docking;

thiazolidinones

GRAPHICAL ABSTRACT

Cl

X

Cl

O

Antidiabetic assay

Cytotoxicity study

S

N

Z

6r

Y

6a-w

Thiazolidin-4-one scaffold

A competitive Inhibitor of α-Glucosidase, IC₅₀: 105 μM

Safe and non-toxic on various cell lines, IC50 > 750 μM

CONTACT Mohsen Amini

moamini@tums.ac.ir Department of Medicinal Chemistry, Faculty of Pharmacy and Drug

Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical

Sciences, Tehran 1417614411, Iran.

Supplemental data for this article is available online at publisher ’s website

ß 2021 Taylor & Francis Group, LLC

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E. SAEEDIAN MOGHADAM ET AL.

Introduction

A complicated mixture of chronic metabolic disorders which is called Diabetes mellitus

(DM), cause high blood sugar levels in patients and result in serious body malfunctions.

There are two types of diabetes namely, type 1 and type 2. In type 1 diabetes (formerly

called insulin-dependent diabetes), the body’s immune system attacks the insulin-pro-

ducing cells of the pancreas, and more than 90% of them are permanently destroyed.

The pancreas, therefore, produces little or no insulin. Only about 5–10% of all people

with diabetes have type 1 disease. Most people who have type 1 diabetes develop the

disease before age 30, although it can develop later in life. In type 2 diabetes (formerly

called non-insulin-dependent diabetes), the pancreas often continues to produce insulin,

sometimes even at higher-than-normal levels, especially early in the disease. However,

the body develops resistance to the effects of insulin, so there is not enough insulin to

meet the body’s needs. As type 2 diabetes progresses, the insulin-producing ability of

the pancreas decreases. Type 2 diabetes is the most common of all diabetes cases

reported all over the world.^[1]

Diabetes is the reason for around 5% of deaths of the human being population.^[2]

Diabetes damages blood vessels, causing them to narrow and therefore restricting blood

flow. Because blood vessels throughout the body are affected, people may have many

complications of diabetes. Many organs can be affected, particularly the following:

Brain, Eyes, Heart, Kidneys, and Nerves which lead to occurring the stroke, blindness,

heart attack, decreased sensation in feet (diabetic nephropathy), and chronic kidney dis-

ease. High blood glucose levels also cause disturbances in the body’s immune system, so

people with diabetes mellitus are particularly susceptible to bacterial and fungal infec-

tions. All of these concerns show the need to develop effective antidiabetic scaffolds to

suppress its occurrence.

Besides health problems, DM causes decreased life quality and disability. DM globally

occurrence is related to many items like simple transportation, severe changes in work

models, reduced physical activity, outstanding changes in lifestyle, fast foods, and high

sugar drinks. Based on WHO statistics, it is expected to have an increase in the popula-

tion of diabetic people by 2.5-fold, in the developing countries from 84 million in 1995

to around 228 million by 2025.^[3,4]

a-Glucosidase is an enzyme, which catalyzes the hydrolysis of an a-glucosidal bond of

carbohydrates. This property increases the release of monosaccharide (a-D-glucose)

which is simply absorbed in the gastrointestinal system. Hyperactivation of a-glucosi-

dase enzyme results in higher blood glucose levels in diabetic patients known as hyper-

glycemia.^[2]Inhibition of a-glucosidase enzyme reduces glucose absorption by delaying

the digestion and absorption of carbohydrates as well as extending digestion time. This

therapeutic strategy is used to control postprandial hyperglycemia in type 2 diabetes.^[5,6]

For this reason, the a-glucosidase enzyme is an important therapeutic target for type 2

diabetes treatment, and some of the common a-glucosidase inhibitors such as acarbose,

miglitol, and voglibose were used in the clinic. In this context, the development of

potent and selective a-glucosidase inhibitors having biosafe nature is an important goal

for medicinal chemists to deal with.^[1]

Heterocyclic chemistry plays an important role in the design and development of

new bioactive agents and a huge number of approved drugs contain heterocycles core.

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(a)

(b)

Figure 1. (a) Reported thiazolidine-4-one based structures with antidiabetic activity. (b) Current work.

Thiazolidine-4-one, a five-membered heterocyclic ring, involving Sulfur and Nitrogen,

exhibited interesting properties as a bioactive scaffold. Thiazolidine-4-one derivatives

showed a diverse range of bioactivity such as anticholinesterase, antimicrobial, antiviral,

anti-inflammatory, antiasthmatic, antiproliferative,^[7–11]and antidiabetic effect^[12–15]

(Fig. 1a).

On the other hand, many cis-diaryl heterocycles are reported as a-glucosidase inhibi-

tors which could be used as a base in the design of potent a-glucosidase inhibi-

tors.^[16–19]regarding these structure-activity relationship facts, and in continuation of

our efforts in the design and development of novel anti-diabetic molecules with a-gluco-

sidase inhibition mechanism,^[20–22]herein, we synthesized a series of 5-Benzylidene-2,3-

diarylthiazolidine-4-one derivatives (6a–w) (Fig. 1b) in two-step reactions. The first step

includes a multicomponent reaction to prepare thiazolidine-4-one rings (4a–f), which is

followed by a condensation reaction between thiazolidine-4-one rings and benzaldehyde

derivatives to obtain 6a–w. The a-glucosidase inhibitory effect of 6a–w was also investi-

gated which led to finding 6r as the most potent compound in the series. In continue,

the cytotoxicity profile of 6a–w was studied using MTT assay to confirm the safety of

synthesized compounds; which results showed 6a–w are mostly nontoxic at their active

a-glucosidase inhibitory concentration. Besides bioassays and enzyme kinetic studies, a

molecular docking study was also done for the most active compounds (6b, 6q, and 6r)

to understand the possible mode of interaction between active structures and active site

residues of the enzyme. Docking studies revealed the most important residues, HIS280,

ASP307, and PRO312, interacting with active compounds.

Results and discussion

Synthesis

The synthetic pathway to target compounds is presented in Scheme 1. At first, benzal-

dehyde derivatives (1) and aniline derivatives (2) were dissolved in Tetrahydrofuran

(THF) as a solvent and then treated with thioglycolic acid (TGA) at 0 ^ꢀC to obtain

4

E. SAEEDIAN MOGHADAM ET AL.

Scheme 1. Synthetic methods and reaction condition used to prepare target compounds 6a–w. i:

selected benzaldehyde derivatives (1), selected aniline derivatives (2), DCC, THF, 0 ^ꢀC to RT, 1 h. ii:

selected intermediates (4a–f), selected benzaldehyde derivatives (5a–r), KOH, EtOH, Reflux, 6 h.

2,3-diarylthazolidin-4-one derivatives (4). All 4a–f derivatives were synthesized in

1

acceptable to good yields (59–82%) and characterized using H-NMR, IR, and melting

point. The presence of a sharp peak in the IR spectrums of 4a–f, around

1

1670–1680 cm^ꢁ1, confirms the desire for amide bond formation. In H-NMR spectrums

related to 4a–f, a singlet peak around 6 ppm related to proton (H_a) attached to chiral

carbon in position two of thiazolidine-4-one ring and two doublet peaks with J value

near 16 around 3.8 to 4 ppm, related to diastereotopic protons (H_band H_c) in position

five of thiazolidine-4-one ring confirms the correctness and validity of synthesized

derivatives (4a–f).

In continuance, selected derivatives (4a–f) were treated with a diverse range of selected

benzaldehyde derivatives (5a–r) in refluxing ethanol to do a condensation reaction to pre-

pare target compounds (6a–w). All compounds were synthesized in acceptable yield

(47–69%). The purification process was very simple using short column chromatography

and all compounds (6a–w) were achieved as pure products. After purification, 6a–w were

characterized using spectroscopic methods namely ¹H-NMR, ¹³C-NMR, MS, and IR besides

elemental analysis and melting point determination. In ¹H-NMR spectrums related to 6a–w,

a singlet peak of a proton (H_a) attached to chiral carbon in position two of thiazolidine-4-

one ring repeated around 6 ppm like intermediates 4a–f, and disappearance of two doublet

peaks related to diastereotopic protons (H_band H_c) in intermediates 4a–f and presence of a

new singlet peak related to vinylic proton (H_d) around 7.8 ppm, specifically confirms the

conversion of intermediates 4a–f to desired products (6a–w) (Table 1).

In vitro biological assays

In vitro a-glucosidase inhibition assay

The synthesized compounds (6a–w) were evaluated for their antidiabetic potential

using in vitro biological assay and the results are given in Table 2, next.

Accordingly, most of the screened compounds, except 6a, 6d, and 6h displayed

higher activity (IC₅₀¼ 105–412 mM) than the reference standard acarbose (IC₅₀

¼

750 mM). Among the screened compounds, 6r, 6b, and 6q displayed the best antidia-

betic potential with the lowest IC₅₀values, that is, 105, 110, and 127 mM,

respectively.

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Table 1. structure of synthesized intermediates (4a–f) and target compounds (6a–w).

Compound

X

Y

Z

4a

4b

4c

4d

4e

4f

6a

6b

6c

6d

6e

6f

H

Me

OMe

F

H

4-OMe

3-OMe

4-OMe

3-OMe

H

4-Me

4-OMe

4-SMe

4-Cl

H

4-F

6g

6h

6i

H

3-Me

3-OMe

3-Cl

6j

3-F

6k

6l

H

Me

OMe

F

H

2-Me

2-OMe

2-Cl

6m

6n

6o

6p

6q

6r

6s

6t

6u

6v

6w

2-F

3,4-di OMe

2,3,4-tri OMe

2,4-di Cl

2,6-di Cl

4-F

4-OMe

3-OMe

4-OMe

3-OMe

4-F

Insertion of different derivatives on benzylidene ring, clearly affected bioactivity of

the screened compounds. The presence of dichloro substituents on the benzylidene part

of the target molecules 6r and 6q led to an increase in activity which might be because

of an increase in lipophilicity in this part of the molecules. Dichloro derivatives 6r and

6q showed better activity in comparison to mono-chloro benzylidene derivatives 6e, 6i,

and 6m. Comparison between 6e, 6i, and 6m derivatives (IC₅₀values of 180, 412, and

148 mM, respectively) with mono-chloro substituent on benzylidene ring shows the

lower activity of meta-substituted derivative (6i) in comparison to ortho (6m), and para

(6e), substituted derivatives (Fig. 2).

This pattern is also repeated for methoxy (6c, 6h, and 6l) with IC₅₀values of 153,

>750, and 164 mM, as para, meta, and ortho-substituted derivatives respectively) and

methyl-substituted derivatives (Fig. 2). The difference in the order of the activity of

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E. SAEEDIAN MOGHADAM ET AL.

Table 2. a-Glucosidase inhibition and cytotoxic activities of 6a–w.

a

b

Compound

a-Glucosidase inhibition (IC₅₀

)

NIH3T3 (IC₅₀

)

MCF-7 (IC₅₀

)

HT-29 (IC₅₀)

6a

6b

6c

6d

6e

6f

6 g

6 h

6i

6j

6k

>750

110.6 4.1

153.0 5.8

>750

184.2 6.4

362.0 7.0

151.4 4.3

>750

412.0 9.5

196.7 6.2

139.5 4.7

164.0 5.5

148.6 4.6

182.3 5.2

193.8 4.1

217.2 6.6

127.0 4.5

105.0 3.6

163.4 4.2

196.1 4.9

210.8 5.5

231.7 6.1

187.3 4.3

750.0 8.6

6 l

6 m

6n

6o

6p

6q

6r

6 s

6t

6 u

6v

6w

Acarbose

^aIn vitro a-Glucosidase inhibition activity (IC₅₀) of synthesized compounds 6a–w; ^bIn vitro cytotoxic activities (IC₅₀) of

synthesized compounds 6a–w on three cell lines (NIH3T3, MCF-7, and HT-29). All IC₅₀are based on micromolar (mM).

derivatives containing electron-donating groups, methyl or methoxy, (para-

> ortho > meta) with derivatives involving electron-withdrawing mono-chloro substitu-

ent (ortho > para > meta) may come from their electronic effects. Electron-donating

groups show certain order of activity but electron-withdrawing substituents, chlorine,

and fluorine have no definite pattern on the activity of synthesized compounds.

Conversion of para methoxy benzylidene derivative (6c) to 3,4-dimethoxy benzylidene

derivative (6o) or 2,3,4-trimethoxy benzylidene derivative (6p) led to reduced activity

(Fig. 2). Altogether, obtained results showed the high potential of the current scaffold as

an a-glucosidase inhibitor.

Enzyme kinetic study

In the next step, a kinetic study was performed to evaluate the mechanism of enzyme

inhibition of the most active compound (6r). According to Figure 3a, the Lineweaver-

Burk plot showed that the K_mgradually increased and Vmax remained unchanged with

increasing inhibitor concentration indicating a competitive inhibition. The results show

6r binds to the active site on the enzyme and compete with the substrate for binding to

the active site. Furthermore, a plot of the K_mversus different concentrations of inhibitor

gave an estimate of the inhibition constant, K_iof 97 mM (Fig. 3b).

Cytotoxicity investigation

For most of the bioactive compounds, except anticancer agents, it is a need to be

safe and nontoxic in the whole body, for this reason, cytotoxicity of the compounds

6a–w was also investigated to check their safety. All 6a–w and acarbose were

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(a)

Cl

O

S

O

S

O

S

N

6q

μ

(IC₅₀:127 M)

6m

μ

(IC₅₀:148 M)

6r

μ

(IC₅₀:105 M)

6e

μ

6i

(IC₅₀:184 M)

(IC₅₀:412 M)

μ

Mono-chloro derivatives

Di-chloro derivatives

(c)

(b)

O

H₃C

O

S

N

O

S

N

6c

μ

(IC₅₀:153 M)

H₃C

O

CH₃

O

CH₃

6b

μ

(IC₅₀:110 M)

CH₃

O

S

O

S

N

6k

μ

(IC₅₀:139 M)

6h

μ

(IC₅₀750 M)

6l

μ

(IC₅₀:164 M)

6g

μ

(IC₅₀:151 M)

Mono-methyl substituted derivatives

Mono-methoxy substituted derivatives

(d)

CH₃

O

CH₃

O

CH₃

O

S

O

S

Reduced activity

N

6p

μ

(IC₅₀:218 M)

6o

μ

(IC₅₀:193 M)

6c

μ

(IC₅₀:153 M)

Di or Tri-methoxy substituted derivatives

Mono-methoxy substituted derivative

Figure 2. Structure activity relationship of different substituted (6a–w) derivatives.

screened on three cell lines namely, NIH-3T3, MCF-7, and HT-29. All cell lines

were treated with 6a–w and acarbose at 750 mM concentration and interestingly, all

of the screened compounds showed extremely low cytotoxic effect with the IC₅₀

above 750 mM on all cell lines. Therefore, it is possible to claim that most of the tar-

get compounds are almost nontoxic with high a-glucosidase inhibitory activity

(Table 2). The low toxicity of target compounds 6a–w makes them more valuable

for further bio-investigation.

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E. SAEEDIAN MOGHADAM ET AL.

(a)

30

20

Concentration of Inhibitor

0 μM

55 μM

80 μM

105 μM

10

0

−1.5

−1.0

−0.5

0.0

0.5

1.0

1/[S](PNPG, mM^-1)

−10

(b)

7

6

5

4

3

2

1

0

−

150

−

100

−

50

0

50

100

150

Compound ESM-12

Figure 3. Kinetics of a-gluosidase inhibition by 6r. (a) The Lineweaver–Burk plot in the absence and

presence of different concentrations of 6r; (b) The secondary plot between K_mand various concentra-

tions of 6r.

Docking study

After biological activity investigation, we were interested to check the possible mode of

interaction of potent compounds with the a-glucosidase enzyme active site. For this rea-

son, we did molecular docking studies. For compound 6b, with a free binding energy of

ꢁ4.34 kcal/mol, docking studies showed that there are interactions between HIS280,

ASP307, PRO312, PHE314, and ARG315 residues and the structure of 6b. There are

also some van der Waals interactions between 6b and THR310, SER311, PHE303,

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Figure 4. Possible mode of interaction between the structures of compounds 6b, 6q, 6r and acarbose

with the enzyme active site.

GLN279, and some other residues inside the active site. In the case of 6r, with a free

binding energy of ꢁ4.64 kcal/mol, the interaction is almost similar to 6b, just with the

difference on HIS280 residue; for 6r, there is stronger interaction between HIS280 resi-

due and the structure of 6r and it seems that it is the reason of higher biological activity

of the compound 6r in comparison to 6b. Like 6b, van der Waals interactions for 6r

are also visible with THR310, SER311, PHE303, GLN279, and some other residues. For

compound 6q, with a free binding energy of ꢁ4.02 kcal/mol, there are interactions

between HIS280, ASP307, PRO312, and ARG315 residues and the structure of 6q,

which are the cause of the biological activity of 6q; and similar to other compounds,

there is also some more weak van der Waals interaction with THR310, SER311,

PHE303, GLN279. As it is distinguishable, there is an important difference between

interactions of 6b and 6r in comparison to the interaction of 6q with enzyme active

site; in both 6b and 6r, the HIS280 residue, has an interaction with the sulfur atom in

the structures but this interaction is missed for 6q. furthermore, both 6b and 6r have

interaction with PHE314 residue which this one is also missed for 6q. altogether these

differences in the mode of interaction between the structures of 6b and 6r in compari-

son to the interaction of 6q with enzyme active site are the causes of higher activity of

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E. SAEEDIAN MOGHADAM ET AL.

6r and 6b with respect to 6q (Fig. 4). In the end, a docking study was also performed

for acarbose. Results showed acarbose binds to the active site of the enzyme with a free

binding energy of 1.47 kcal/mol. Comparison between binding energies of compounds

6r, 6b, and 6q with the binding energy of acarbose confirms the obtained data from

experimental in vitro enzyme inhibition assay. The synthesized compounds interact

more tightly with the active site and as a result, show better bioactivity in comparison

to acarbose in both in vitro assay and docking study.

Experimental

All Chemical reagents and solvents were bought from Merck (Darmstadt, Germany),

synthesis grade, and used without further purification. Melting points were determined

on a Kofler hot-stage instrument (Reichert, Vienna, Austria). The ¹H-NMR spectra

were obtained on a Bruker FT-500 MHz spectrometer (Bruker, Darmstadt, Germany) or

Joel FT-400 MHz spectrometer (Joel, MA, USA) and CDCl₃was used as a solvent. ¹³C-

NMR spectra were also recorded at 125 or 100 MHz. The spin multiplicities are written

as s (singlet), d (doublet), t (triplet), and m (multiplet) and the Coupling constant (J)

values are given in Hertz (Hz). ESI-Mass spectra were done on a 6460 Triple Quad

LCMS instrument (Agilent Technologies, Santa Clara, CA, USA). Elemental analyses

were determined using a Perkin Elmer Model 240-C instrument (PerkinElmer,

Hopkinton, MA, USA). IR spectra were obtained by a Nicolet Magna 550-FT spectrom-

eter (Nicolet Instrument Corporation, Madison, WI, USA).

Chemistry

General procedure for synthesis of 2,3-diarylthiazolidine-4-one derivatives (4)

Benzaldehyde derivatives (1) (2 mmol) and aniline derivatives (2) (1 mmol) were mixed

and stirred for 5 min in THF at 0 ^ꢀC, then TGA (3) (3 mmol) was added to the stirred

solution. After 10 min, N, N-dicyclohexylcarbodiimide (DCC) (1.3 mmol) was added to

the ice-cooled reaction solution and the reaction mixture stirred for a further 50 min at

room temperature. Produced N, N-dicyclohexylurea (DCU) was filtered and the filtrate

was concentrated using a rotary evaporator to dryness. The residue was dissolved in

ethyl acetate and then washed with 10% aq. citric acid, water, and 10% aq. sodium

hydrogen carbonate. Finally, the organic phase was dried over sodium sulfate and the

solvent was evaporated under reduced pressure to get solid. Solid impure compounds

are recrystallized in diethyl ether to collect the pure product (4).^[23]

2,3-Diphenylthiazolidine-4-one (4a). White solid; Isolated yield: 82%; m.p.: 160–161 ^ꢀC.

1

IR (KBr): ꢀ_maxC¼O 1676 cm^ꢁ1; H-NMR (500 MHz, CDCl₃): d 3.85 (d, J ¼ 16.0 Hz,

1H, CH₂), 3.98 (d, J ¼ 16.0 Hz, 1H, CH₂), 6.08 (s, 1H, CH), 7.10–7.29 (m, 10H,

H-Ar).^[23]

General procedure for the synthesis of target compounds (6a–w)

To a stirred solution of selected 2,3-diarylthiazolidine-4-one derivatives (4) (1 mmol) in

ethanol (10 mL), appropriate benzaldehyde derivatives (5) (1 mmol), and KOH

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(1.1 mmol) were added and the solution was refluxed for 6 hrs. After completion of the

reaction, detected by thin-layer chromatography (TLC), the solvent was evaporated and

the crude was washed with water and extracted with ethyl acetate. After drying the

organic layer over sodium sulfate and evaporating the solvent, remained solid was sub-

jected to flash chromatography (n-hexane/Ethyl acetate: 5/1) to obtain pure target com-

pounds (6a–w).

5-Benzylidene-2,3-diphenylthiazolidine-4-one (6a). Yellow solid; Yield: 67%; m.p.:

1

194–195 ^ꢀC. IR (KBr): 3033, 2926, 1679, 1592, 1490 cm^ꢁ1; H-NMR (400 MHz, CDCl₃):

d 6.28 (s, 1H, CH), 7.18 (t, J ¼ 7.0 Hz, 1H, H-Ar), 7.20–7.35 (m, 10H, H-Ar), 7.41 (t,

J ¼ 7.5 Hz, 2H, H-Ar), 7.55 (d, J ¼ 7.1 Hz, 2H, H-Ar), 7.70 (s, 1H, H-Vinyl); ¹³C-NMR

(100 MHz, CDCl₃): d 64.28, 125.52, 125.92, 126.35, 127.21, 127.32, 128.51, 128.87,

129.13, 129.27, 129.33, 129.75, 135.16, 137.57, 138.45, 166.21; ESI-MS (m/z): 366.00

[M þ Na]^þ; Anal. Calcd. for C₂₂H₁₇NOS: C, 76.94; H, 4.99; N, 4.08. Found: C, 76.79; H,

4.98; N, 4.11.

5-(4-Methylbenzylidene)-2,3-diphenylthiazolidine-4-one (6b). Light yellow_ꢁs₁olid; Yield:

60%; m.p.: 190–191 ^ꢀC. IR (KBr): 3024, 2928, 1682, 1595, 1489 cm

;

¹H-NMR

(400 MHz, CDCl₃): d 2.36 (s, 3H, CH₃), 6.26 (s, 1H, CH), 7.18–7.35 (m, 12H, H-Ar),

7.44 (d, J ¼ 8.0 Hz, 2H, H-Ar), 7.66 (s, 1H, H-Vinyl); ¹³C-NMR (100 MHz, CDCl₃): d

21.55, 64.26, 125.91, 126.44, 127.18, 127.25, 128.85, 129.10, 129.24, 129.26, 129.58,

129.73, 132.36, 137.64, 138.59, 138.75, 166.36; ESI-MS (m/z): 379.80 [M þ Na]^þ; Anal.

Calcd. for C₂₃H₁₉NOS: C, 77.28; H, 5.36; N, 3.92. Found: C, 77.16; H, 5.35; N, 3.94.

5-(2,4-Dichlorobenzylidene)-2,3-diphenylthiazolidine-4-one (6q). White solid; Yield: 60%;

1

m.p.: 185–187 ^ꢀC. IR (KBr): 3030, 2929, 1685, 1590, 1495 cm^ꢁ1; H-NMR (500 MHz,

CDCl₃): d 6.18 (s, 1H, CH), 7.15–7.35 (m, 13H, H-Ar), 7.62 (s, 1H, H-Vinyl); ¹³C-NMR

(125 MHz, CDCl₃): d 64.2, 121.0, 125.7, 127.0, 127.1, 127.3, 129.0, 129.10, 129.18, 129.3,

129.4, 129.9, 131.9, 134.2, 135.5, 137.2, 137.9, 165.3; ESI-MS (m/z): 433.75 [M þ Na]^þ;

Anal. Calcd. for C₂₂H₁₅Cl₂NOS: C, 64.09; H, 3.67; N, 3.40. Found: C, 64.20; H, 3.67;

N, 3.39.

5-(2,6-Dichlorobenzylidene)-2,3-diphenylthiazolidine-4-one (6r). Yellow solid; Yield: 51%;

1

m.p.: 182–183 ^ꢀC. IR (KBr): 3035, 2932, 1682, 1597, 1494 cm^ꢁ1; H-NMR (500 MHz,

CDCl₃): d 6.28 (s, 1H, CH), 6.80 (d, J ¼ 12.0 Hz, 2H, H-Ar), 7.05–7.20 (m, 8H, H-Ar),

7.47–7.54 (m, 2H, H-Ar), 7.60 (s, 1H, H-Vinyl); ¹³C-NMR (125 MHz, CDCl₃): d 63.9,

121.4, 125.7, 127.1, 127.3, 128.1, 128.9, 129.1, 129.2, 129.6, 133.4, 133.8, 134.4, 137.3,

138.2, 164.5; ESI-MS (m/z): 449.70 [M þ K]^þ; Anal. Calcd. for C₂₂H₁₅Cl₂NOS: C, 64.09;

H, 3.67; N, 3.40. Found: C, 64.00; H, 3.65; N, 3.41.

Biological investigation

In vitro a-glucosidase inhibition assay

The a-glucosidase inhibitory activity of the synthesized compounds was evaluated using

p-nitrophenyl-a-D-glucopyranoside (pNPG.) as a substrate based on a previously

12

E. SAEEDIAN MOGHADAM ET AL.

reported method. The a-glucosidase enzyme (EC3.2.1.20, Saccharomyces cerevisiae,

20 U/mg) and the substrate pNPG. were purchased from Sigma-Aldrich. The enzyme

was prepared in potassium phosphate buffer (pH 6.8, 50 mM), and 6a–w were dissolved

in DMSO (10% of the final concentration). A volume of 20 mL of various concentration

of the synthesized compounds (6a–w) and 135 mL of phosphate buffer containing 20 mL

of the a-glucosidase solution was added to the 96-well plate and incubated for 10 min at

37 ^ꢀC. After pre-incubation, 25 mL of 4 mM pNPG. solution in 0.05 M phosphate buffer

(pH ¼ 6.8) was added to each well. The reaction mixture was allowed to incubate for

20 min at 37 ^ꢀC. Finally, the reaction was terminated by adding 50 mL of 0.2 M sodium

carbonate solution, and then, the change in absorbance was measured at 405 nm with a

spectrophotometer (Gen5, Power wave xs2, BioTek, America). DMSO and acarbose

were used as a control and reference drug, respectively. The a-glucosidase inhibitory

activity was expressed as the percentage of enzyme inhibition for each synthesized com-

pound and calculated by the following formula:

%Inhibition ¼ ðControlabsorption ꢁ SampleabsorptionÞ=ðControlabsorptionÞ ꢂ 100

The concentration of the sample compounds required to inhibit 50% of the enzyme

activity (IC₅₀) values were obtained from a non-linear regression curve using the

Logit method.^[24–26]

Enzyme kinetic study

The mechanism of inhibition of the most active compound (6r), based on IC₅₀value,

was investigated against the a-glucosidase activity. The enzyme solution (1 U/mL, 20 mL)

was incubated with different concentrations of the most active compound 3 m (20 mL)

for 15 min at 30 ^ꢀC. Different concentrations of pNPG. (1–10 mM) was added as a sub-

strate to initiate the reaction in the absence and presence of compound 6r and change

in the absorbance was determined for 20 min at 405 nm by using a spectrophotometer

(Gen5, Power wave xs2, BioTek, America). A Lineweaver–Burk plot was generated to

identify the type of inhibition and the Michaelis–Menten constant (K_m) value was deter-

mined from the plot between reciprocal of the substrate concentration (1/[S]) and recip-

rocal of enzyme rate (1/V) over various inhibitor concentrations. The experimental

inhibitor constant (K_i) value was constructed by secondary plots of the inhibitor con-

centration [I] versus K_m.^[24]

Cytotoxic activity evaluation

Three cell lines, namely, MCF-7 (breast carcinoma cell lines), HT-29 (colon carcinoma

cell line), and NIH-3T3 (mouse fibroblast cell line) were obtained from the Pasteur

Institute (Tehran, Iran). The cells were cultured in RPMI-1640 medium (Sigma-Aldrich,

St. Louis, MO, USA), supplemented with 10% heat-inactivated fetal bovine serum

(Gibco, Grand Island, NY, USA), penicillin (100 U/mL), and (100 mg/mL) streptomycin

(Roche, Mannheim, Germany) at 37 ^ꢀC in a humidified incubator with 5% CO₂.

All derivatives (6a–w) were screened for cytotoxic effects at 0.01–750 mM concentra-

tions. Cells were seeded in 96-well plates at a density of 8,000–10,000 viable cells per

well and incubated for 24 h to allow cell attachment before treatment. The primary solu-

tions of compounds (75,000 mM) in DMSO were diluted with media and added into

R

SYNTHETIC COMMUNICATIONS^V

13

wells of the plate. After that, cells were incubated for a further 48 h. cell survival was

determined using 3-(4,5-dimethylthiazoyl-2-yl) 2,5-diphenyl tetrazolium bromide

(MTT). For this purpose, cells were washed by phosphate-buffered saline (PBS), and

MTT reagent (20 mL, 5 mg/mL) in PBS was added to each well. After a 4 h incubation at

37 ^ꢀC, the medium was discarded and DMSO (100 mL) was added to each well, and the

solution was continuously mixed to dissolve the purple formazan crystals. The absorb-

ance was measured by a plate reader (Anthous 2020, Biochrom, Cambridge, UK) at a

test wavelength of 550 nm against a standard reference solution at 690 nm. For each

compound, MTT assay was performed in triplicate in three independent experiments,

and the IC₅₀values were specified by nonlinear regression analysis and expressed as

the mean SD.^[27,28]

Molecular docking

Molecular docking studies were carried to get a deeper insight into the binding pattern

of the screened compounds. AutoDock version 4.2.6, AutoDock Tools (ADT) version

1.5.6, and Discovery studio v16.1.0.15350 were used to conduct the docking studies.

The most potent compounds from the series (6b, 6q, and 6r), were generated as a lig-

and with their 3D structure, using the ChemDraw Ultra 12.0.2 version of Cambridge

University. Since there is no report yet about the crystal structure of a-glucosidase from

Saccharomyces cerevisiae for preparing the protein for docking, we retrieved the crystal

structure of isomaltase from Saccharomyces cerevisiae (PDB ID: 3A4A; Resolution

1.6 Å) from Protein Data Bank (www.rcsb.org/pdb/). Then, the protein structure was

prepared by removing the water molecules and original inhibitors. The ligand and pro-

tein pdbqt files were prepared and grid box formation was accomplished using

AutoDock Tools. AutoGrid was used to prepare the grid map using a grid box. The

dimension of the grid size was set to 60 ꢂ 60 ꢂ 60 with a grid spacing of 0.375 Å. The

center of the grid box was designated in coordinates x ¼ 21.277, y ¼ ꢁ0.773, and

z ¼ 18.648. Fifty runs of the AUTODOCK search by the Lamarckian genetic algorithm

were performed for each docked system. To analyze the interactions between the target

enzyme and the inhibitor, the best pose of each ligand was designated and the result

was visualized with Discovery Studio Visualizer v16.1.0.15350 (Accelrys, San

Diego, USA).

Conclusion

The present research presents the successful synthesis and spectroscopic characterization

of twenty-three substituted 5-Benzylidene-2,3-diarylthiazolidine-4-ones (6a-w) in higher

1

yields. All derivatives (6a–w) were characterized using H-NMR, ¹³C-NMR, MS, IR,

elemental analysis, and melting point technics. The synthesized compounds were

screened for their inhibitory potential against a-glucosidase. Consequently, most of the

screened compounds displayed inhibitory potential higher than the reference standard.

Within the screened compounds, (6r, 6b, and 6q) with IC₅₀values of 105, 110, and

127 mM. showed the best inhibitory activity, respectively. All derivatives (6a–w) also

were screened for their cytotoxic effects using MTT assay on different cell lines, and the

14

E. SAEEDIAN MOGHADAM ET AL.

results showed that those are almost nontoxic with IC₅₀> 750 mM on all applied cell

lines. to determine the mechanism of enzyme inhibition, a kinetic study was also done

for 6r. The kinetic studies showed the competitive mode of inhibition for 6r. It binds to

the active site on the enzyme and competes with the substrate for binding to the active

site. At least, molecular docking studies clarified the mode of interactions of the most

potent compounds with enzyme active sites. Compounds 6b, 6q, and 6r have inter-

action with HIS280, ASP307, and PRO312, which play a critical role inside the active

site of the enzyme. Altogether, screened series showed a high potential of 5-

Benzylidene-2,3-diarylthiazolidine-4-one scaffold as an a-glucosidase inhibitor and it is

beneficial to further do further investigation of more derivatives of this family.

1

Full experimental details and copies of H-NMR, ¹³C-NMR, and Mass spectra of syn-

thesized target compounds can be found via the “Supplementary Content” section of

this article’s webpage.

Funding

This work was financially supported by the Research Council of Tehran University of Medical

Sciences and Health Services.

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Article Doi

5-Benzylidene-2,3-diarylthiazolidine-4-ones: Design, synthesis, spectroscopic characterization, in vitro biological and computational evaluation

DOI: 10.1080/00397911.2021.1946699

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