Design, synthesis, biological evaluation, and molecular modeling studies of rhodanine derivatives as pancreatic lipase inhibitors

Received: 25 January 2019

Revised: 27 June 2019

Accepted: 2 July 2019

|

DOI: 10.1002/ardp.201900029

F U L L P A P E R

Design, synthesis, biological evaluation, and molecular

modelingstudiesofrhodaninederivativesaspancreaticlipase

inhibitors

Divya Chauhan¹

Vikramdeep Monga¹

|

Ginson George²

|

S. N. C. Sridhar²

|

Rohit Bhatia¹

|

Atish T. Paul²

|

¹Department of Pharmaceutical Chemistry,

ISF College of Pharmacy, Ghal Kalan, Moga,

Punjab, India

Abstract

A series of rhodanine‐3‐acetic acid derivatives were synthesized via Knoevenagel

condensation of rhodanine‐3‐acetic acid with various substituted aromatic aldehydes.

The synthesized derivatives were screened in vitro for understanding the inhibitory

potential towards pancreatic lipase (PL), a key enzyme responsible for the digestion

of dietary fats. Derivative 8f exhibited a potential inhibitory activity towards PL

(IC₅₀=5.16 µM), comparable to that of the standard drug, orlistat (0.99 µM). An

increase in the density of the aromatic ring resulted in potential PL inhibition. The

enzyme kinetics of 8f exhibited a reversible competitive‐type inhibition, similar to

that of orlistat. Derivative 8f exhibited a MolDock score of ‒125.19 kcal/mol in

docking studies, and the results were in accordance with their PL inhibitory potential.

Furthermore, the reactive carbonyl group of 8f existed at a distance adjacent to

Ser152 (≈3 Å) similar to that of orlistat. Molecular dynamics simulation (10 ns) of the

8f‒PL complex revealed a stable binding conformation of 8f in the active site of PL

(maximum root mean square displacement of ≈2.25 Å). The present study identified

novel rhodanine‐3‐acetic acid derivatives with promising PL inhibitory potential, and

further lead optimization might result in potent PL inhibitors.

²Laboratory of Natural Product Chemistry,

Department of Pharmacy, Birla Institute of

Technology and Science (Pilani campus), Pilani,

Rajasthan, India

Correspondence

Atish T. Paul, Laboratory of Natural Product

Chemistry, Department of Pharmacy, Birla

Institute of Technology and Science (Pilani

campus), Pilani, 333 031 Rajasthan, India.

Email: atish.paul@pilani.bits-pilani.ac.in

Vikramdeep Monga, Department of

Pharmaceutical Chemistry, ISF College of

Pharmacy, Ghal Kalan, Moga, 142001 Punjab,

India.

Email: vikramdeepmonga@gmail.com and

vikramdeepmonga@yahoo.com

K E Y W O R D S

enzyme kinetics, molecular modeling, obesity, pancreatic lipase, rhodanine

|

1

INTRODUCTION

and we are living in a world where obesity kills more people than being

underweight.^[4]With over 2.8 million deaths per year, obesity ranks fifth

Obesity is a complex and multifactorial disease that arises mainly due to

the modern lifestyle and increased consumption of high‐fat diets.^[1]

Obesity is characterized by excessive deposition of lipids in the body,

which results from long term imbalance between the calories consumed

and calories expended.^[2]Moreover, obesity is associated with various

comorbid conditions including diabetes mellitus, musculoskeletal

disorders, and cardiovascular diseases, and so on, posing a major health

risk to obese patients.^[3]As per the World Health Organization (WHO),

the prevalence of obesity has nearly tripled between 1975 and 2016

among global deaths.^[5]Hence, obesity has become a global threat to

developed countries as well as developing countries.

Numerous therapeutic targets and medications are available for the

treatment of obesity. Among them, the reduction in fat digestion that

would reduce the absorption of fat in the GI tract is considered to be a

promising target for the prevention of obesity. Pancreatic lipase (PL) or

triacylglycerol lipase (EC 3.1.1.3), a key enzyme responsible for the

digestion of dietary lipids from the duodenum, has been identified as the

crucial target for regulation of lipid absorption.^[6]Orlistat, a saturated

|

Arch Pharm Chem Life Sci. 2019;e1900029.

1 of 14

https://doi.org/10.1002/ardp.201900029

2 of 14

CHAUHAN ET AL.

|

derivative of lipstatin, remains the only PL inhibitor approved for long‐

term treatment of obesity. However, recent reports from the Food and

Drug Administration indicated severe adverse effects with long term

administration of orlistat, including hepatotoxicity, acute pancreatitis,

and so on.^[7]Thus, the discovery of safer and effective PL inhibitors is

highly desirable.

have designed structural derivatives of thiazolidine‐2,4‐dione, that is,

the rhodanine class of scaffolds possessing a 2‐thioxo group instead

of the 2‐oxo group as present in TZD, and explored them as possible

PL inhibitors. Accordingly, the present study involved the design,

synthesis, characterization, in vitro evaluation, and molecular

modeling studies of novel thioxo‐thiazolidinone acetic acid (rhoda-

nine acetic acid [RAA]) derivatives as potent PL inhibitors.

In recent years, five‐membered nitrogen heterocycles have gained

prominent significance in PL inhibition. Examples include oxadia-

zoles^[8,9]and 1,3‐pyrazoles^[10], and so on, which exhibited potential PL

inhibition. By considering this fact and as a part of our drug discovery

program, we have identified diaryl substituted pyrazole clubbed

thiazolidinediones (TZD) as potential PL inhibitors in our previous

study, wherein the nitrogen‐centered diamide interacted with Ser152

and diaryl pyrazole wing interacted with Phe77 and Phe215 of the lid

domain (Gly76–Lys80 and Leu213–Met217).^[11]

|

2

RESULTS AND DISCUSSION

Chemistry

|

2.1

The synthetic strategy for various arylidene derivatives of rhodanine‐

3‐acetic acid (RAA, 8a‒n) is represented in Scheme 1. Rhodanine‐3‐

acetic acid intermediate (RAA, 3) required for the preparation of

target derivatives was synthesized by cyclizing the glycine with carbon

disulfide and sodium chloroacetate under acidic conditions, using a

previously reported procedure.^[17]Treatment of benzyl bromide with

different commercially available hydroxyl‐substituted aromatic alde-

hydes (5‐chloro‐2‐hydroxybenzaldehyde, 5‐chloro‐2‐hydroxy‐3‐meth-

oxybenzaldehyde, 2‐hydroxy‐5‐nitrobenzaldehyde, and 2‐hydroxy‐1‐

naphthaldehyde) in the presence of anhydrous K₂CO₃in dry acetone

afforded the intermediates 6a‒d. The target compounds 8a‒n (Table 1)

were synthesized by Knoevenagel condensation of rhodanine‐3‐acetic

acid (3) with various substituted aromatic aldehydes (6a‒d and 7). To

simplify the ease of preparation and isolation of various target

derivatives, we explored different reaction conditions involving

various solvents and catalytic bases for this reaction, including acetic

acid/sodium acetate, toluene/piperidine, and ethanol/piperidine.^[18,19]

It was found that the ethanol/piperidine system was the most practical

one, with short reaction times (2‒4 hr) and straightforward product

isolation. Under these conditions, various rhodanine derivatives were

successfully synthesized and in all cases, the product was precipitated

from the ethanolic solution upon cooling at room temperature, and in

turn was collected by simple filtration. Thus, all final derivatives were

obtained as solids in satisfactory yields (53–87%). Remarkably, this

procedure was also applicable to benzaldehydes containing a free

phenolic hydroxyl group (e.g., 4). The Knoevenagel condensation

reaction of RAA with aromatic aldehydes provided only the

Rhodanines and related substances like 2,4‐thiazolidinediones are

often considered as bioisosteric derivatives.^[12]The molecules based on

the 2‐thioxo‐thiazolidine‐4‐one (rhodanine) core, as an imperious

privileged scaffold, are known for their broad range of pharmacological

profiles.^[12–14]The structural diversity among 2‐thioxo‐1,3‐thiazolidine‐

4‐one derivatives is exclusively located in two positions, that is, the first

is the N‐3 position and the second is the nature of arylidene moiety at

the C‐5 position. Introduction of the lipophilic or hydrophilic or aromatic

group at one of the two key positions (or at both) offers a panel of

possibilities for drug design and also for the physicochemical property

studies. Epalrestat (Figure 1) is a rhodanine‐3‐acetic acid derivative and

acts as a potent aldose reductase inhibitor, which has been approved for

the treatment of diabetic complications, such as neuropathy, nephro-

pathy, and cataract. Furthermore, rhodanine derivatives have been

reported as potential PTP1B inhibitors for the treatment of diabetes

and obesity.^[15]Recently, the therapeutic potential of rhodanine

derivatives as potential antidyslipidaemic agents has been demon-

strated by lowering the low‐density lipoprotein cholesterol, very low‐

density lipoprotein cholesterol and triglycerides, while increasing the

level of high‐density lipoprotein cholesterol, thereby decreasing the

atherogenic index.^[16]

In the present study, the derivatives were designed based on the

concept of molecular hybridization. Considering the above facts and

pharmacophoric requirements for the design of PL inhibitors, we

FIGURE 1 Representation for

deciphering the pharmacophoric design of

pancreatic lipase (PL) inhibitors

CHAUHAN ET AL.

3 of 14

|

SCHEME 1 Synthesis of the target

compounds 8a‒n. Reagents and conditions:

(i) NH₄OH, CS₂, KOH, room temperature,

1 hr; ClCOONa, room temperature, 3 hr.

(ii) 1 N HCl, 110°C, reflux, 1 hr. (iii)

Acetone, K₂CO₃, reflux, 2 hr. (iv) EtOH,

AcOH, 80‒90°C, reflux, 3‒4 hr

TABLE 1 Physicochemical data of various rhodanine‐3‐acetic acid derivatives (8a–n)

S.No

1.

Compound number

8a

Structures

Chemical formula

Color

Yield (%)

Melting point (°C)

R_f^a

C₁₂H₈FNO₃S₂

Yellow

67

231–234

0.79

2.

8b

C₁₄H₁₃NO₅S₂

Orange

72

240–243

0.76

3.

4.

8c

8d

C₁₅H₁₅NO₆S₂

Yellow

83

59

193–196

250–252

0.68

0.70

C₁₆H₁₁NO₃S₂

5.

6.

8e

8f

C₁₆H₁₁NO₃S₂

Orange

Brown

63

87

235–237

0.70

0.62

(Lit. 238–239)³

C₁₆H₁₁NO₄S₂

248–251

7.

8g

C₁₂H₈ClNO₄S₂

Yellow

63

245–248

(Lit. 243–247)⁴

0.81

(Continues)

4 of 14

CHAUHAN ET AL.

|

TABLE 1 (Continued)

S.No

8.

Compound number

Structures

Chemical formula

Color

Yield (%)

Melting point (°C)

R_f^a

8h

C₁₃H₁₀ClNO₅S₂

Orange

74

248–250

0.78

9.

8i

C₁₂H₈N₂O₆S₂

Dark brown

82

227–230

0.80

10.

11.

8j

C₁₃H₁₀N₂O₇S₂

Green

Yellow

55

75

245–248

229–231

0.76

0.80

8k

C₁₉H₁₄ClNO₄S₂

12.

8l

C₂₀H₁₆ClNO₅S₂

Yellow

78

244–246

0.74

13.

8m

C₁₉H₁₄N₂O₆S₂

Yellow‐brown

79

212–215

0.82

14.

8n

C₂₃H₁₇NO₄S₂

Yellow

47

233–235

0.76

^aToluene/ethyl acetate/formic acid = 4:0.8:0.2.

thermodynamically more stable Z isomer, as determined by the

chemical shift of the methine proton.^[20,21]The physicochemical

properties of various synthesized derivatives are given in Table 1.

The synthesized derivatives were characterized by FTIR, ¹H, and

¹³C NMR, mass spectroscopy and elemental analysis data, which fully

supported their structural identity. The IR spectrum of title

derivatives (8a‒n) showed strong absorption bands in the range of

1,712–1,722 and 1,692–1,706 cm⁻¹due to two C═O groups

stretching. Derivatives 8a‒n showed typical absorption at

3,410–3,480 cm⁻¹due to OH group stretching, whereas a band due

to C═S group stretching vibrations appeared at 1,317–1,353 cm⁻¹

.

Furthermore, the absorption band appeared in the range of

CHAUHAN ET AL.

5 of 14

|

1,573–1,603 cm⁻¹due to aromatic C═C stretching. In the ¹H NMR

spectrum, the disappearance of a peak due to the methylene group

and appearance of an arylidene (═CH–) peak confirms the

Knoevenagel condensation between various aromatic aldehydes

and rhodanine‐3‐acetic acid. The methine proton of the arylidene

group appeared either as a singlet or multiplet in the range of δ

7.55–7.93. In the ¹H NMR spectrum ═CH– proton was deshielded

more (δ = 7.5–7.9 ppm) as expected in the Z‐form, relative to the

slightly shielded protons of the E‐form (δ = 6.2–6.3 ppm), thus

indicating that all the derivatives were obtained exclusively in Z‐

form. This downfield movement of the methine proton in (Z) C₅‐

arylidene analogs were reported to be due to the deshielding effect

of the adjacent carbonyl group.^[20]Moreover, the Z‐isomer is

thermodynamically more stable because of the intramolecular

hydrogen bond that can be formed between the hydrogen atom of

═CH and the oxygen atom of the carbonyl group in RAA.^[21]A broad

singlet due to the proton of the carboxylic group appeared in the

range of δ 12.98–13.47, whereas the proton of the phenolic group in

derivatives 8f–j appeared as a singlet in the range of δ 10.83–11.33.

Protons of the methylene group (–N–CH₂) in various title derivatives

8a–n resonated as a singlet at δ 4.60–4.86 ppm. In derivatives 8k–n,

another singlet due to the methylene protons of the benzylic group

(–CH₂C₆H₅) resonated at δ = 5.30–5.44 ppm. All other protons were

observed in the expected regions. In ¹³C‐NMR, signals due to two

carbonyl carbons of RAA appeared at δ = 167.51–168.10 and

166.23–166.94 ppm, respectively. The signal due to thiocarbonyl

group resonated at δ = 189.01–193.97. All the derivatives showed

prominent signals at δ = 128.97–136.51 due to arylidene carbon

(–CH═). Furthermore, the derivatives 8a–n also showed character-

istic signals in the range of δ = 45.54–46.70 due to the carbon of the

CH₂group attached to the N‐3 position of the RAA ring. In

derivatives 8k–n, the signal due to benzylic carbon (–CH₂C₆H₅)

appeared in the range of δ = 70.90–75.93 ppm. Finally, the assigned

structures of various derivatives were confirmed by their mass

spectra, where a characteristic [M+H]⁺peak was observed. All

derivatives gave satisfactory elemental analysis.

TABLE 2 PL inhibitory activity of the synthesized rhodanine

derivatives 8a–n

Compound

Ar

IC₅₀(µM)^a

8a

8b

8c

8d

8e

8f

3‐Flurophenyl

47.29 0.91

22.95 0.74

9.34 0.35

16.14 0.45

47.72 1.19

5.16 0.31

11.38 1.52

10.64 0.79

53.19 2.06

13.56 1.17

26.77 1.70

13.52 0.93

2,5‐Dimethoxy phenyl

2,3,4‐Trimethoxy phenyl

2‐Naphthyl

1‐Naphthyl

2‐Hydroxy naphthyl

5‐Chloro‐2‐hydroxyphenyl

5‐Chloro‐2‐hydroxy‐3‐methoxyphenyl

2‐Hydroxy‐5‐nitrophenyl

2‐Hydroxy‐3‐methoxy‐5‐nitrophenyl

2‐(Benzyloxy)‐5‐chlorophenyl

8g

8h

8i

8j

8k

8l

2‐(Benzyloxy)‐5‐chloro‐3‐

methoxyphenyl

8m

2‐(Benzyloxy)‐5‐nitrophenyl

17.51 1.45

25.12 2.54

0.99 0.11

8n

2‐(Benzyloxy)naphthyl

Orlistat

–

Abbreviations: PL, pancreatic lipase; SEM, standard error of mean.

^aAll experiments were performed in triplicate and the results are

expressed as mean SEM.

|

2.2.2

Enzyme kinetics study

To understand the nature of inhibition, an enzyme kinetics study

for the most active derivative (8f) was performed by using a

protocol standardized in our laboratory.^[22]The protocol was

performed at three concentration levels of the inhibitor and a

double reciprocal Lineweaver–Burk plot was acquired. As

represented in Figure 2 and Table 3, the K_mvalues increased in

proportion to inhibitor concentration, indicating a reversible

competitive inhibition similar to that of orlistat from the previous

study. Furthermore, derivative 8f exhibited an inhibition constant

(K_i) value of 2.472 µM as calculated from the Cheng–Prusoff

equation.^[23]

|

2.2

Biological activity

|

2.2.1

In vitro PL inhibitory activity

The synthesized derivatives were subjected to in vitro PL

inhibition assay as per the protocol standardized in our

laboratory.^[22]PL inhibitory activities of various synthesized

derivatives (8a‒n) are represented in Table 2. Derivative 8f

exhibited the most potent PL inhibitory activity of 5.16 µM,

comparable to that of orlistat (0.99 µM), followed by derivative

8c (9.34 µM). Derivatives 8h, 8g, 8l, 8j, 8d, and 8m also exhibited

a potential PL inhibitory activity of 10.64, 11.38, 13.52, 13.56,

16.14, and 17.51 µM, respectively. Derivatives 8b, 8k, and 8n

exhibited moderate activity (>20 µM) and all other derivatives

(such as 8a, 8e, and 8i) exhibited weak PL inhibitory activity

(>40 µM).

|

2.2.3

Structure–activity relationship

Based on the PL inhibitory potential of the synthesized derivatives, a

preliminary structural activity relationship has been derived. As shown

in Figure 3, the potential of the derivatives to inhibit PL varied with

respect to the ring density and the substitutions on the aromatic ring.

Previously, it was reported that the hydrophobic aromatic moieties

attached with the reactive carbonyl groups help in opening the

conformation of the lid domain of PL. For instance, derivative 8f

6 of 14

CHAUHAN ET AL.

|

2

1.5

1

FIGURE 2 Lineweaver‒Burk plot of 8f

representing reversible competitive

inhibition

High Inhibitor (5 µM)

Medium Inhibitor (2.5 µM)

Low Inhibitor (1.25 µM)

No Inhibitor

0.5

0

-0.03

-0.02

-0.01

0

0.01

0.02

0.03

1/S

0.04

0.05

-0.5

TABLE 3 V_maxand K_mvalues calculated from the Lineweaver–

Burk plot for different concentrations of 8f

position compared to derivative 8h exhibited a similar kind of reduction

in the inhibitory activity (IC₅₀=10.64 [8h] to 13.56 µM). In contrast, the

introduction of an additional electron‐donating methoxy group at the

third position of the phenyl ring resulted in compounds 8h and 8j

(IC₅₀= 10.64 and 13.56 µM) with higher PL inhibitory activities

compared to the nonmethoxy counterparts 8g and 8i (IC₅₀=11.38

and 53.19 µM), respectively. Masking of the free hydroxyl group

(in 8g, 8h, and 8f) with benzyl substitution provided derivatives

8k, 8l, and 8n, which display a reduced PL inhibitory potential as

compared to their non benzylated counterpart with the exception of

derivative 8m, in which the inhibitory potency was significantly

improved (IC₅₀= 17.51 µM) compared to its parent counterpart (8i).

I (µM)

0

V_max(µM/min)

0.07401

K_m(µM)

64.120

64.629

65.285

66.289

1.25

2.5

0.07423

0.07535

5.0

0.07652

possessing a dense aromatic ring (bulky 2‐hydroxy naphthyl substitu-

tion) exhibited the maximum PL inhibitory potential (IC₅₀=5.16 µM).

Interestingly, a drastic variation in the activity was observed with the

removal of the 2‐hydroxy group and with respect to the change in the

attachment position of the dense aromatic ring (naphthyl ring).

Derivative 8d, with a 2‐naphthyl ring substitution, exhibited a higher

PL inhibitory potential (IC₅₀= 16.14 µM) as compared to 8e possessing

1‐naphthyl ring substitution (IC₅₀=47.72 µM). Substitution with 2,3,4‐

trimethoxyphenyl ring imparted potential inhibitory activity to the

derivative 8c (IC₅₀=9.16 µM) as compared to that of dimethoxy

substitution (8b, IC₅₀=22.95 µM). Surprisingly, the presence of the Cl

group (8g and 8h) at the fifth position of the phenyl ring resulted in a

significant inhibitory effect with IC₅₀values of 11.38 and 10.64 µM,

respectively. Furthermore, results demonstrated that presence of

electron donating groups (such as –OCH₃, –CH₃, and –OH) on the aryl

rings resulted in potent to good PL inhibitory activity as compared to

that of electron withdrawing groups (such as –NO₂, –F). Replacement of

–Cl (8g) with a more polarized electron withdrawing –NO₂substituent

furnished derivative 8i with weak inhibitory activity (IC₅₀= 53.19 µM).

Apart from this, derivative 8j possessing an additional nitro group at

|

2.3

Docking and molecular dynamics simulations

Docking studies were performed using a Molegro Virtual Docker

(v 6.0) with validated grid parameters (see experimental data) for

understanding the interactions of the derivatives with the active

site amino acids of human PL (PDB ID: 1LPB). Structurally, the

active site of human PL consists of the catalytic triad, Ser152/

Asp176/His263, which is enclosed within

a hydrophobic lid

domain comprised of Gly76/Lys80 and Leu213/Met217.^[11]The

whole series of derivatives docked into the active site of PL, and

MolDock scores along with key interactions exhibited by various

derivatives are summarized in Table 4. The docked poses of these

ligands were according to the hypothesis, wherein substituted

aromatic moieties exhibited a hydrophobic interaction with the

amino acid of the lid domain (Tyr114) while the reactive carbonyl

group of TZD ring existed near the Ser152. The docking scores

were in alignment to the PL inhibitory activity wherein derivative

8f exhibited a potential docking score of −125.19 kcal/mol. The

binding mode of the most active derivative 8f in the active site of

PL is shown in Figure 4a. The major H‐bond interactions included

Gly76, Phe77, His151, and Ser152. Apart from, pi–pi interactions

played a prominent role in the potential PL inhibitory activity

wherein a significant pi–pi interaction of the aromatic ring of 8f

with His263 was observed. Moreover, a variation in the position

and substitution on the aryl ring resulted in a diverse interaction

with the synthesized derivatives. For instance, derivative 8d

exhibited a pi–pi interaction with Phe215 of the lid domain,

FIGURE 3 Preliminary SAR of the synthesized derivatives

depicting the PL inhibitory activity (IC₅₀) versus substitution. PL,

pancreatic lipase; SAR, structure‐activity relationship

CHAUHAN ET AL.

7 of 14

|

TABLE 4 Summary of MolDock scores and interactions exhibited by various synthesized compounds 8a–n

Compounds

MolDock Score (kcal/mol)

−113.29

H bond

Pi–pi

Pi–cation

His263

–

8a

Asp79, His151, Ser152

Gly76, Phe77, His151, Ser152

Phe77, His 151, Ser152, Phe215

Gly76, Phe77, His151, Ser152

Gly76, Phe77, His151

Phe215

Tyr114

Phe215

Tyr114

Phe215

Tyr 114

Tyr114

Phe215

–

8b

8c

−117.21

−121.33

His263

–

8d

8e

−119.73

−110.25

8f

−125.19

Gly76, Phe77, His151, Ser152

Gly76, Phe77, Asp79, Ser152

Gly76, Phe77, His151, Ser152

Gly76, Asp79, Ser152, His263

Gly76, Phe77, His151, His263

Ser152

His263

8g

−119.57

His151, His263

8h

8i

−119.27

–

111.26

–

8j

−120.23

–

8k

8l

−117.67

–

−118.77

Arg256

8m

8n

Orlistat

−122.19

Gly76, Asp79, Ser152, His263

Phe77, Ser152, Leu153

His151, Ser152

Tyr114

Tyr114, Phe215

–

−116.92

His263

−152.58

–

[8,22,24]

which was not observed with 8e. The fact can be further justified

wherein derivative 8d exhibited a potential activity in compar-

ison to 8e. These results are in accordance to previous reports

that highlight the importance of aromatic moieties in hydro-

phobic interactions and the crucial impact of the reactive

carbonyl group in interaction with Ser152.

To understand

the probable role of the reactive carbonyl group in interaction

with Ser152 of the active site of PL, the binding pose of 8f was

superimposed with that of orlistat (Figure 4b) and

a two

dimensional representation of interaction mode of derivative 8f

FIGURE 4 (a) Binding pose of 8f in the

active site of PL; (b) overlay diagram of 8f

(yellow) over orlistat (white) representing

the distance of reactive carbonyl carbon

from Ser152; (c) two‐dimensional

interaction diagram of 8f. PL, pancreatic

lipase

8 of 14

CHAUHAN ET AL.

|

The initial MD run indicated a higher degree of pi–sulfur

interactions with the lid domain of the active site, which vanished

during the course of run time. However, derivative 8f predominantly

made stable pi–pi interactions with the amino acids of the lid domain

(Tyr114 and Phe215). Furthermore, derivative 8f made a variable H‐

bond interaction with Ser152 and Tyr114 throughout the MD.

|

2.3.1

In silico drug‐likeness predictions

Drug likeness is the complex balance between various molecular

properties and structural features that are helpful to predict

how similar is the designed derivative to the known drugs. The

drug‐likeness behavior of the designed compounds was estimated

via predicting their physico‐chemical parameters and this tool is

now widely used by various researchers to correlate the results

of biological findings with the physicochemical properties.^[25,26]

In silico drug likeness prediction of various designed and

synthesized derivatives was carried out by using a Swiss ADME

predictor tool as depicted in Table 6. Percentage absorption (%

ABS) was calculated by using the formula %ABS = 109 ‒ (0.345 ×

TPSA). The synthesized derivatives displayed moderate absorp-

tion in the range of 50‒80%. The results showed (Table 6) that

the ILogP of all the derivatives was below 5.0, LogS values were

found to be around −5.0 with the molecular weight <500. Hence,

the various title derivatives showed no violation of the Lipinski

rule of five. These predictions revealed that the synthesized

analogs could be considered as drug candidates.

FIGURE 5 Ligand root mean square displacement (RMSD) of

derivative 8f acquired through the 10 ns MD run. MD, molecular

dynamics

is given in Figure 4c. Reactive carbonyl groups of the derivatives

existed approximately in equidistance from Ser152 (3.3 and 3.6 Å

for orlistat and derivative 8f, respectively) and also derivative 8f

fitted best within the cavity, which accounts for its highest

activity. Molecular docking results were in accordance with the

PL inhibitory activity exhibited by the derivatives and the binding

pose analysis validated the rationale for designing the rhodanine

based PL inhibitors.

A 10 ns molecular dynamics (MD) simulation of the 8f‒PL complex

was performed for understanding the binding mode and interaction in

a dynamic environment by using the CHARMM27 force field (see

section 4).^[11,24]Derivative 8f exhibited a stable binding conformation

throughout the MD run, as confirmed through the ligand root mean

square displacement (RMSD) and exhibited a maximum of 2.25 Å at

2 ns (Figure 5). The interactions of the ligand with the active site of PL

in the dynamic environment are summarized in Table 5.

|

2.3.2

In silico toxicity prediction

Toxicity prediction is an essential tool to predict the safety of a

derivative. The safety profile of various synthesized derivatives was

predicted using PreADME and ProTox web‐based tools. The

predicted data (Table 7) revealed that the designed derivatives are

noncarcinogenic as shown by their negative values on carcinomouse

and carcinorat models. Medium risk for hERG (human ether‐a‐go‐go‐

related gene) inhibition indicated that the designed derivatives have

a minimum risk on the cardiac action potential. As predicted by

protox, the minimum lethal dose (LD₅₀) for various derivatives was in

the range of 350–1,500 mg/kg that lies under Class 4. Derivatives

belonging to Class 4 are not harmful in this LD₅₀range even if

swallowed. From the toxicity predictions, it is evident that the

designed derivatives were safer for future studies.

TABLE 5 Summary of interactions exhibited by 8f through the

10 ns molecular dynamics run

Time

Pi‐pi

frame (ns) H bond interaction

Pi‐cation

Pi‐sulfur

0

1

Asp79

Phe215

–

Phe77, His151,

His263

Arg256 Tyr114, His263, Ala 178

Leu153

His151, His263

2

3

4

His263

Ser152

–

Tyr114

Phe77

–

Ile 209

Tyr114

Ile209,

Pro180

|

3

CONCLUSION

5

6

7

8

9

Ser152

–

Pro180

Ile209

Pro180

–

Phe77

–

In the current study,

a series of novel substituted 2‐thioxo‐

Tyr114

Ser152

–

thiazolidin‐4‐one acetic acid derivatives were designed, synthesized,

and evaluated for their PL inhibitory activity. Of the derivatives, 8f

exhibited the best in vitro PL inhibitory activity (5.16 0.31 µM)

comparable to that of orlistat (0.99 0.11 µM). Enzyme inhibition

experiments showed that the derivatives acted as reversible

–

Phe77, Tyr114,

Phe215

–

His263

10

–

Phe77

–

CHAUHAN ET AL.

9 of 14

|

TABLE 6 Prediction of in silico drug likeness properties of compounds 8a–n

Compounds

TPSA

MW

RoB

<10

3

HBD

<5

1

HBA

<10

4

ILogP (MlogP)^a

<5

LogS^b

>−4

% ABS

–

Rule

8a

8b

8c

8d

8e

8f

≤140

<500

115.00

133.46

142.69

82.91

297.33

339.39

369.28

299.34

329.39

345.39

329.78

359.86

340.33

370.36

434.94

464.96

430.45

435.52

1.61

−3.40

−3.49

−3.89

−4.43

−4.29

−3.70

−3.79

−3.18

−3.26

−4.83

−4.74

−4.79

−5.87

69.32

62.95

59.77

80.39

69.32

62.34

59.16

46.53

43.35

66.14

62.95

50.33

62.95

5

1

5

2.35

6

1

6

2.55

3

1

3

1.83

115.00

135.23

144.46

181.05

190.28

124.23

133.46

170.05

124.23

3

1

3

2.10

3

2

4

2.04

8g

8h

8i

3

2

4

1.78

4

2

5

2.09

4

2

6

1.05

8j

5

2

7

1.64

8k

8l

6

1

4

2.42

7

1

5

2.32

8m

8n

7

1

6

2.34

6

1

4

2.86

Abbreviations: ABS, percentage absorption; HBD, hydrogen hydrogen bind donors; MW, molecular weight; RoB, number of rotatable bonds; TPSA,

topological polar surface area.

^aLogarithm of compound partition coefﬁcient between n‐octanol and water.

^bLogarithm of water solubility.

competitive inhibitors towards PL. Molecular docking studies of the

synthesized derivatives revealed that the carbonyl group of the

rhodanine moiety interacted with Ser152 whereas various aromatic

substitutions were involved in the interaction with the amino acids of

the lid domain. The obtained molecular docking studies results were

in accordance with the in vitro results. A 10 ns MD simulation of

derivative 8f in the active site of PL revealed a stable binding

confirmation with RMSD ≤ 2.25 Å. Furthermore, in silico drug‐like-

ness of the derivatives followed the Lipinski rule of five and the

toxicity data predicted the derivatives to be safe. To conclude, the

present study identified RAA derivatives as a novel class of potential

PL inhibitors, and further studies with generated hits might be

helpful for the design and development of potent clinical PL

inhibitors.

TABLE 7 In silico toxicity prediction of compounds 8a–n

Compound

Carcinomouse

Negative^a

Carcinorat

Negative^b

hERG inhibition

Medium risk^c

ProTox predicted LD₅₀(mg/kg)

ProTox predicted class

Class 4

8a

8b

8c

8d

8e

8f

350

1,400

450

350

Class 4

8g

8h

8i

Class 4

8j

Class 4

8k

8l

Class 4

8m

8n

Class 4

^aCompound computationally predicted to be safe in carcinomouse.

^bCompound computationally predicted to be safe carcinorat.

^cNo significant effect on the electrical activity of the heart.

Class 4: Not harmful on swallowing at LD₅₀range of 350–1,500 mg/kg.

10 of 14

CHAUHAN ET AL.

|

4

EXPERIMENTAL

COOH), 167.26 (C═O), 44.76 (N–CH₂), 35.94 (CH₂); MS (ESI) m/z (%):

192.0 [M+H]⁺; Anal. calcd. for C₅H₅NO₃S₂: C, 31.40; H, 2.64; N, 7.32.

Found: C, 31.52; H, 2.51; N, 7.24.

|

4.1

Chemistry

|

4.1.1

General

Solvents and organic reagents were purchased from Sigma‐Aldrich,

Merck, and HiMedia and were used without further purification. All

the evaporations were carried out under reduced pressure on a

Buchi‐rotary evaporator. Reactions were monitored by thin‐layer

chromatography (TLC) using commercially available precoated

aluminum plates (Merck Kieselgel 60 F₂₅₄silica, 0.2 mm) with UV

light (254 nm), iodine, and vanillin–sulfuric acid as derivatization

agents. Melting points were recorded with a COMPLAB melting point

apparatus and uncorrected. IR spectra (KBr disks) were recorded on

a NICOLET‐380 FT‐IR spectrophotometer. ¹H NMR and ¹³C NMR

spectra were recorded on a Bruker AM 300 (300 MHz) or a Bruker

Avance III 500 (500 MHz; ¹³C NMR at 75 and 125 MHz) spectro-

meter (Bruker Biosciences) using CDCl₃or DMSO‐d₆as the solvent

at room temperature within the range. Chemical shifts (δ) are

expressed as parts per million (ppm), using tetramethylsilane as the

internal standard. Splitting patterns are designated as follows; s,

singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet.

Chemical shift values (δ) are given in ppm. Mass spectra (MS; please

see the Supporting Information) were taken in ESI mode on an

Agilent 1100 LC–MS (Agilent, Palo Alto, CA). The results of elemental

analyses (% C, H, N) for all the synthesized derivatives were within

0.4% range of the theoretical value.

|

4.1.3

General procedure for the synthesis of

various O‐benzylated aromatic aldehydes

To a solution of aromatic aldehyde (1.52 g) in 8 ml of anhydrous

acetone contained in a round‐bottom flask was added potassium

carbonate (2.76 g) followed by addition of different alkyl/aryl halides

(1.1 eq). The reaction mixture was refluxed at 70°C with continuous

stirring until the completion of the reaction as evidenced by TLC.

Upon completion, the reaction mixture was poured into water and

extracted twice with chloroform (2 × 30 ml). The organic layers were

combined and washed with water (25 ml). The solvent was removed

in vacuum and the solid product so obtained was purified by

recrystallization from absolute ethanol to afford various O‐benzy-

lated aromatic aldehydes in satisfactory yield.

2‐(Benzyloxy)‐5‐chlorobenzaldehyde (6a)

White solid; yield 74%; m.p.: 86–88°C; IR (KBr) ν_max(cm⁻¹): 3,060

(aromatic C–H stretching), 2,921 (aliphatic C–H stretching), 1,710

(C═O), 1,609 (C═C), 1,332 (C═S), 1,262 (C–O stretching), 810 (C–Cl);

¹H‐NMR (300 MHz, DMSO‐d₆): 10.31 (s, 1H, CHO), 7.69–7.65 (m,

2H, Ar–H), 7.62–7.29 (m, 6H, Ar–H), 5.28 (s, 2H, –OCH₂–).

The InChI codes of the investigated derivatives together with

biological activity data are provided as Supporting Information.

2‐(Benzyloxy)‐5‐chloro‐3‐methoxybenzaldehyde (6b)

Off white solid; yield 81%; m.p.: 64–67°C; IR (KBr) ν_max(cm⁻¹): 3,037

(aromatic C–H stretching), 2,943 (aliphatic C–H stretching), 1,703

(C═O), 1,612 (C═C), 1,356 (C═S), 1,278 (C–O stretching), 799 (C–Cl);

¹H‐NMR (300 MHz, DMSO‐d₆): 10.01 (s, 1H, CHO), 7.47–7.32 (m,

6H, Ar‐H), 7.05–7.02 (d, J = 8.4 Hz, 1H, Ar–H), 5.16 (s, 2H, –OCH₂–).

|

4.1.2

General procedure for the

synthesis of rhodanine‐3‐acetic acid

(= 2‐[4‐oxo‐2‐thioxothiazolidin‐3‐yl]acetic acid)

In a round‐bottom flask equipped with a magnetic stirrer, glycine

(24.0 g; 320 mmol) was dissolved with 25% ammonium hydroxide

solution (70 ml) in water (10 ml). Then, carbon disulfide (24.3 g;

320 mmol) was added and the reaction mixture was stirred

vigorously for 1 hr. An aqueous solution of sodium chloroacetate

(37.1 g; 320 mmol) was then added dropwise to the solution over

15 min and stirring was continued at 25°C for 3 hr. Furthermore, the

reaction mixture was acidified with dilute HCl until pH 1.0 and

refluxed for 1 hr and neutralized with saturated NaHCO₃solution.

The resultant solution was acidified again with dilute HCl. The

cyclized product was extracted in ethyl acetate, dried over

anhydrous sodium sulfate, evaporated under reduced pressure, and

the residue was purified by recrystallization from ethyl acetate.

Molecular formula C₅H₅NO₃S₂; yellow solid, yield: 62%; m.p.

144–147°C (Lit. 145–148°C)^[17]; IR (KBr) ν_max(cm⁻¹): 3,513 (–OH

stretching); 2,993 (aliphatic C–H stretching); 1,744 and 1,698 (C═O);

1,345 (C═S); 1,214 (C–N); ¹H NMR (300 MHz, DMSO‐d₆): δ (ppm)

2‐(Benzyloxy)‐5‐nitrobenzaldehyde (6c)

Creamish solid; yield 55%; m.p.: 135–137°C; IR (KBr) ν_max(cm⁻¹):

3,012 (aromatic C–H stretching), 2,932 (aliphatic C–H stretching),

1,697 (C═O), 1,591 (C═C), 1,523 and 1,380 (–NO₂), 1,317 (C═S),

1,282 (C–O stretching); ¹H‐NMR (300 MHz, DMSO‐d₆): 10.64 (s, 1H,

CHO), 8.40–8.37 (d, J = 8.7 Hz, 1H, Ar–H), 8.31 (s, 1H, Ar–H),

7.49–7.37 (m, 6H, Ar–H), 5.42 (s, 2H, OCH₂).

2‐(Benzyloxy)‐1‐naphthaldehyde (6d)

White solid; yield 68%; m.p.:112–114°C; IR (KBr) ν_max(cm⁻¹): 3,048

(aromatic C–H stretching), 2,926 (aliphatic C–H stretching), 1,708

(C═O), 1,601 (C═C), 1,332 (C═S), 1,262 (C–O stretching); ¹H‐NMR

(300 MHz, DMSO‐d₆): 10.84 (s, 1H, CHO), 9.12–9.09 (d, J = 8.7 Hz,

1H, Ar–H), 8.27–8.24 (d, J = 9.3 Hz, 1H, Ar–H), 7.94–7.91 (d,

J = 8.1 Hz, 1H, Ar–H), 7.83–7.78 (m, 2H, Ar–H), 7.69–7.33 (m, 6H,

Ar–H), 5.46 (s, 2H, –OCH₂–).

10.02 (bs, 1H, –COOH); 4.71 (s, 2H, N–CH₂–); 4.12 (s, 2H, –CH₂); ¹³

NMR (DMSO‐d₆, 75 MHz): δ (ppm) 202.68 (C═S), 173.73 (C═O,

C

CHAUHAN ET AL.

11 of 14

|

4.1.4

General procedure for the synthesis of

119.56, 109.32, 62.34 (OCH₃), 61.03 (OCH₃), 56.70 (OCH₃), 45.45

(N–CH₂); MS (ESI) m/z (%): 370.0 [M+H]⁺; Anal. calcd. for

C₁₅H₁₅NO₆S₂: C, 48.77; H, 4.09; N, 3.79. Found: C, 48.64; H, 3.98;

N, 3.86.

arylidene derivatives of rhodanine‐3‐acetic acid

(8a‒n)

A mixture of rhodanine‐3‐acetic acid (20 mmol, 10.9 g) and sub-

stituted aromatic aldehyde (20 mmol) was suspended in absolute

alcohol and to this a catalytic amount of piperidine (16 mmol, 1.4 g)

and glacial acetic acid was added. The mixture was then allowed to

reflux with stirring at 80°C. The completion of the reaction was

monitored with the help of TLC. On completion, the reaction mixture

was concentrated, and poured in ice‐cold water and acidified with

glacial acetic acid to pH 3–4 to afford a solid product, which was

filtered and dried. The crude product was recrystallized from ethanol

to get pure product in satisfactory yield.^[19]

(Z)‐2‐(5‐(Naphthalen‐1‐ylmethylene)‐4‐oxo‐2‐thioxothiazolidin‐3‐

yl)acetic acid (8d)

IR (KBr) ν_max(cm⁻¹): 3,419 (O–H), 3,005 (aromatic C–H stretching),

2,905 (aliphatic C–H stretching), 1,722 (C═O), 1,706 (C═O), 1,594

(C═C), 1,332 (C═S), 1,195 (C–O); ¹H‐NMR (500 MHz, DMSO‐d₆): δ

13.47 (bs; 1H OH), 8.55 (s, 1H, Ar–H), 8.22–8.20 (d, J = 7.8 Hz, 1 H,

Ar–H), 8.15‐8.13 (d, J = 8.2 Hz, 1H; Ar–H), 8.07–8.05 (d; J = 8.2 Hz;

1H, Ar–H), 7.77–7.66 (m, 4H, 3Ar–H, and –CH═), 4.79 (s, 2H,

N–CH₂); ¹³C‐NMR (100 MHz, DMSO‐d₆): δ 194.28 (C=S), 167.76

(COOH), 166.26 (CO), 133.80, 132.13 (–CH═), 131.52, 131.17,

130.44, 129.43, 128.23, 127.76, 127.48, 126.27, 125.70, 123.93,

45.52 (–N–CH₂); MS (ESI) m/z (%): 330.0 [M+H]⁺; Anal. calcd. for

C₁₆H₁₁NO₃S₂: C, 58.34; H, 3.37; N, 4.25. Found: C, 58.57; H, 3.31; N,

4.18.

(Z)‐2‐(5‐(3‐Fluorobenzylidene)‐4‐oxo‐2‐thioxothiazolidin‐3‐yl)acetic

acid (8a)

IR (KBr) ν_max(cm⁻¹): 3,446 (O–H), 2,985 (aromatic C–H stretching),

2,919 (aliphatic C–H stretching), 1,714 and 1,703 (C═O), 1,578

(C═C), 1,323 (C═S), 1,273 (C–O), 1195 (C–N); ¹H‐NMR (300 MHz,

DMSO‐d₆): δ 13.53 (s, 1H, OH), 7.90 (s, 1H, –CH═), 7.62–7.47 (m, 3H,

Ar–H), 7.42–7.36 (m, 1H, Ar–H), 4.75 (s, 2H, N–CH₂); ¹³C‐NMR

(75 MHz, DMSO‐d₆): δ 193.40 (C═S), 167.67 (COOH), 166.69 (C═O),

(Z)‐2‐(5‐(Naphthalen‐2‐ylmethylene)‐4‐oxo‐2‐thioxothiazolidin‐3‐

yl)acetic acid (8e)

IR (KBr) ν_max(cm⁻¹): 3,423 (O–H stretching), 3,093 (aromatic C–H

stretching), 2922 (aliphatic C–H stretching), 1,720 and 1,704 (C═O),

1,593 (C═C), 1,353 (C═S), 1,199 (C–N); ¹H‐NMR (300 MHz, DMSO‐

d₆): δ 13.76 (bs, 1H, OH), 8.21 (s, 1H, Ar–H), 8.10–7.97 (m, 4H, Ar–H

and –CH═), 7.74–7.59 (m, 3H, Ar–H), 4.74 (s, 2H, N–CH₂); ¹³C‐NMR

(75 MHz, DMSO‐d₆): δ 193.73 (C=S), 167.73 (COOH), 166.86 (CO),

134.34, 134.04 (–CH═), 133.23, 132.54, 130.88, 129.67, 129.46,

128.94, 128.22, 127.76, 126.77, 122.54, 45.71 (–N–CH₂); MS (ESI)

m/z (%): 329.9 [M+H]⁺; Anal. calcd. for C₁₆H₁₁NO₃S₂: C, 58.34; H,

3.37; N, 4.25. Found: C, 58.45; H, 3.48; N, 4.27.^[25]

164.40

(C–F),

161.15,

135.58–135.48

(–CH═),

132.87,

132.08–131.97 (d, 1C), 126.66, 123.96, 118.53–117.73 (m, 1C),

46.22 (N–CH₂). MS (ESI) m/z (%): 298.0 [M+H]⁺; Anal. calcd. for

C₁₂H₈FNO₃S₂: C, 48.47; H, 2.71; N, 4.71. Found: C, 48.31; H, 2.63; N,

4.77.

(Z)‐2‐(5‐(2,5‐Dimethoxybenzylidene)‐4‐oxo‐2‐thioxothiazolidin‐3‐

yl(acetic acid (8b)

IR (KBr) ν_max(cm⁻¹): 3,480 (O–H), 3,032 (aromatic C–H stretching),

2,914 (aliphatic C–H stretching), 1,713 and 1,695 (C═O), 1,584

(C═C), 1,329 (C═S), 1,188 (C–N); ¹H‐NMR (300 MHz, DMSO‐d₆): δ

7.89 (s, 1H, –CH═), 7.10 (s, 2H, Ar–H), 6.93 (s, 1H, Ar–H), 4.56 (s, 2H,

N–CH₂), 3.86 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃); ¹³C‐NMR (75 MHz,

DMSO‐d₆): δ 194.01 (C═S), 167.87 (COOH), 167.09 (C═O), 153.66

(C–OCH₃), 153.00 (C–OCH₃), 128.83 (–CH═), 123.10, 122.18,

119.35, 114.68, 113.69, 56.53 (OCH₃), 56.04 (OCH₃), 46.91

(N–CH₂); MS (ESI) m/z (%): 340.0 [M+H]⁺; Anal. calcd. for

C₁₄H₁₃NO₅S₂: C, 49.55; H, 3.86; N, 4.13. Found: C, 49.71; H, 3.72;

N, 4.07.

(Z)‐2‐(5‐((2‐Hydroxynaphthalen‐1‐yl)methylene)‐4‐oxo‐2‐

thioxothiazolidin‐3‐yl)acetic acid (8f)

IR (KBr) ν_max(cm⁻¹): 3,410 (O–H), 3,057 (aromatic C–H stretching),

2,962 (aliphatic C–H stretching), 1,716 and 1,696 (C═O), 1,594

(C═C), 1,338 (C═S), 1,276 (C–O stretching); ¹H‐NMR (500 MHz,

DMSO‐d₆): δ 13.47 (bs, 1H, COOH), 8.59 (s, 1H, Ar–H), 8.26−8.24 (d,

J = 8.1 Hz, 1H, Ar–H), 8.19–8.17 (d, J = 8.7 Hz, 1H, Ar–H), 8.12–8.10

(d, J = 8.4 Hz, 1H, Ar–H), 7.81–7.69 (m, 3H, Ar–H and –CH═), 5.80 (s,

1H, OH), 4.84 (s, 2H, N–CH₂); ¹³C‐NMR NMR (125 MHz, DMSO‐d₆):

δ 193.86 (C═S), 167.72 (COOH), 166.89 (CO), 154.63 (C–OH),

133.72, 133.13 (–CH═), 131.76, 130.48, 129.86, 129.16, 128.64,

127.92, 126.16, 123.47, 118.35, 45.71 (–N–CH₂); MS (ESI) m/z (%):

346.0 [M+H]⁺; Anal. calcd. for C₁₆H₁₁NO₄S₂: C, 55.64; H, 3.21; N,

4.06. Found: C, 55.52; H, 3.42; N, 4.12.

(Z)‐2‐(4‐Oxo‐2‐thioxo‐5‐(2,3,4‐trimethoxybenzylidene)‐

thiazolidin‐3‐yl)acetic acid (8c)

IR (KBr) ν_max(cm⁻¹): 3,440 (O–H), 3,019 (aromatic C–H stretching),

2,929 (aliphatic C–H stretching), 1,703 & 1,697 (C═O), 1,603 (C═C),

1,347 (C═S), 1,209 (C–O); ¹H‐NMR (300 MHz, CDCl₃): δ 13.46 (bs,

1H, OH), 7.87 (s, 1H, –CH═), 7.27–7.24 (d, J = 9.0 Hz, 1H, Ar–H),

7.03–7.00 (d, J = 9.0 Hz, 1H, Ar–H), 4.72 (2H, N–CH₂), 3.89 (s, 6H,

2OCH₃), 3.78 (s, 3H, OCH₃); ¹³C‐NMR (75 MHz, CDCl₃): δ 194.10

(C═S), 167.81 (COOH), 166.97 (C═O), 157.28 (COCH₃), 153.65

(C–OCH₃), 142.18 (C–OCH₃), 129.51 (–CH═), 126.29, 120.14,

(Z)‐2‐(5‐(5‐Chloro‐2‐hydroxybenzylidene)‐4‐oxo‐2‐thioxothiazolidin‐

3‐yl)acetic acid (8g)

IR (KBr) ν_max(cm⁻¹): 3,472 (O–H), 2,985 (aromatic C–H stretching),

2,929 (aliphatic C–H stretching), 1,714 and 1,703 (C═O), 1,573

(C═C), 1,329 (C═S), 785 (C–Cl); ¹H‐NMR (300 MHz, DMSO‐d₆):

12 of 14

CHAUHAN ET AL.

|

δ 13.54 (bs, 1H, COOH), 7.90 (s, 1H, –CH═), 7.47–7.37 (m, 2H, Ar–H),

7.01–6.98 (d, J = 8.6 Hz, 1H, Ar–H), 4.83 (s, 2H, N–CH₂); ¹³C‐NMR

(75 MHz, DMSO‐d₆): δ 193.97 (C═S), 167.72 (COOH), 166.92 (C═O),

156.69 (C–OH), 133.05 (–CH═), 129.51, 128.99, 123.77 (C–Cl),

122.73, 121.87, 118.54, 45.67 (N–CH₂); MS (ESI) m/z (%): 330.7

[M+H]⁺; Anal. calcd. for C₁₂H₈ClNO₄S₂(329.78): C, 43.70; H, 2.45; N,

4.25. Found: C, 43.76; H, 2.32; N, 4.34.^[20]

(Z)‐2‐(5‐(2‐(Benzyloxy)‐5‐chlorobenzylidene)‐4‐oxo‐2‐thioxothiazoli-

din‐3‐yl)acetic acid (8k)

IR (KBr) ν_max(cm⁻¹): 3,423 (O–H), 3,068 (aromatic C–H stretching),

2,953 (aliphatic C–H stretching), 1,718 and 1,698 (C=O), 1,583

(C═C), 1,337 (C═S), 1,284 (C–O); ¹H‐NMR (300 MHz, DMSO‐d6):

δ 7.91 (s, 1H, –CH═), 7.57–7.53 (dd, J = 2.4 and 8.7 Hz, 2H, Ar–H),

7.48–7.26 (m, 6H, Ar–H), 5.30 (s, 2H, OCH₂), 4.63 (s, 2H, N–CH₂);

¹³C‐NMR NMR (75 MHz, DMSO‐d6):

δ 193.56 (C═S), 167.70

(COOH), 166.82 (C═O), 156.44 (C–OCH₂–), 136.44, 132.76

(–CH═), 129.29, 129.06 (2CH), 128.65, 128.16 (2CH), 127.35,

125.65, 124.44, 123.97 (C–Cl), 115.87, 70.90 (O–CH₂), 46.27

(N–CH₂); MS (ESI) m/z (%): 420.0 [M+H]⁺; Anal. calcd. for

C₁₉H₁₄ClNO₄S₂: C, 54.35; H, 3.36; N, 3.34. Found: C, 54.47; H,

3.48; N, 3.26.

(Z)‐2‐(5‐(5‐Chloro‐2‐hydroxy‐3‐methoxybenzylidene)‐4‐oxo‐2‐thiox-

othiazolidin‐3‐yl)acetic acid (8h)

IR (KBr) ν_max(cm⁻¹): 3,473 (O–H), 3,017 (aromatic C–H stretch-

ing), 2,967 (aliphatic C–H stretching), 1,716 and 1,701 (C═O),

1583 (C═C), 1,323 (C═S), 1,273 (C–O stretching); ¹H‐NMR

(300 MHz, DMSO‐d₆): 7.92 (s, 1H, –CH═), 7.19 (s, 1H, Ar–H),

6.96 (s, 1H, Ar–H), 4.70 (s, 2H, N–CH₂), 3.88 (s, 3H, OCH₃);

¹³C‐NMR (75 MHz, DMSO‐d₆): δ 193.92 (C═S), 167.73 (COOH),

166.90 (C═O), 149.53 (C–OMe), 146.51 (C–OH), 128.87 (–CH═),

123.80 (C–Cl), 122.80, 121.29, 120.18, 115.07, 56.99 (OCH₃),

45.69 (N–CH₂); MS (ESI) m/z (%): 359.9 [M+H]⁺; Anal. calcd. for

(Z)‐2‐(5‐(2‐(Benzyloxy)‐5‐chloro‐3‐methoxybenzylidene)‐4‐oxo‐

2‐thioxothiazolidin‐3‐yl)acetic acid (8l)

IR (KBr) ν_max(cm⁻¹): 3,467 (O–H), 3,033 (aromatic C–H stretching),

2,937 (aliphatic C–H stretching), 1,718 and 1,692 (C═O), 1,589

(C═C), 1,345 (C═S), 1,282 (C–O), 796 (C–Cl); ¹H‐NMR (300 MHz,

CDCl₃): δ 12.98 (bs, 1H, OH), 7.68 (s, 1H, –CH═), 7.54–7.37 (m, 5H,

Ar–H), 7.01–6.98 (m, 2H, Ar–H), 5.40 (s, 2H, OCH₂), 4.67 (s, 2H,

N–CH₂), 3.89 (s, 3H, OCH₃); ¹³C‐NMR (75 MHz, DMSO‐d₆): δ 189.01

(C═S), 167.68 (COOH), 166.38 (CO), 154.61 (C–OCH₂–), 149.58

(C–OCH₃–), 145.85, 136.50 (–CH═), 130.75, 129.43–128.49 (5C),

124.84, 118.94, 117.54, 116.29, 75.93 (OCH₂), 57.05 (OCH₃), 45.54

(N–CH₂); MS (ESI) m/z (%): 450.0 [M+H]⁺; Anal. calcd. for

C₂₀H₁₆ClNO₅S₂: C, 53.39; H, 3.58; N, 3.11. Found: C, 53.57; H,

3.64; N, 3.17.

C

₁₃H₁₀ClNO₅S₂: C, 43.40; H, 2.80; N, 3.89. Found: C, 43.32; H,

2.56; N, 3.84.

(Z)‐2‐(5‐(2‐Hydroxy‐5‐nitrobenzylidene)‐4‐oxo‐2‐thioxothiazolidin‐

3‐yl)acetic acid (8i)

IR (KBr) ν_max(cm⁻¹): 3,462 (O–H), 2,995 (aromatic C–H stretching),

2,942 (aliphatic C–H stretching), 1,712 and 1,701 (C═O), 1592 (C=C),

1,510 and 1,371 (–NO₂), 1,322 (C═S), 1262 (C–O stretching); ¹H‐

NMR (300 MHz, DMSO‐d₆): δ 13.10 (1H, bs, COOH), 8.30 (s, 1H,

Ar–H), 8.23–8.21 (d, J = 8.6 Hz, 1H, Ar–H), 7.96 (s, 1H, –CH=),

7.03–7.00 (d, J = 8.6 Hz, 1H, Ar–H), 4.79 (s, 2H, N–CH₂); ¹³C‐NMR

(75 MHz, DMSO‐d₆): δ 193.06 (C═S), 167.24 (COOH), 166.27 (C═O),

162.77 (C–OH), 139.72 (C–NO₂), 128.19 (–CH═), 127.50, 125.89,

123.43, 120.10, 116.70, 44.98 (N–CH₂); MS (ESI) m/z(%): 339.2 [M‒

H]⁺, 341.0 [M+H]⁺; Anal. calcd. for C₁₂H₈N₂O₆S₂: C, 42.35; H, 2.37;

N, 8.23. Found: C, 42.52; H, 2.48; N, 8.33.

(Z)‐2‐(5‐(2‐(Benzyloxy)‐5‐nitrobenzylidene)‐4‐oxo‐2‐thioxothiazoli-

din‐3‐yl)acetic acid (8m)

IR (KBr) ν_max(cm⁻¹): 3,457 (O–H), 3,017 (aromatic C–H stretching),

2,954 (aliphatic C–H stretching), 1,715 and 1,702 (C═O), 1,582

(C═C), 1,547 and 1,342 (–NO₂), 1,317 (C═S), 1,276 (C–O); ¹H‐NMR

(300 MHz, DMSO‐d₆): δ 8.38–8.35 (dd, J = 2.7 and 9.0 Hz, 1H, Ar–H),

8.28–8.27 (m, 1H, Ar–H), 7.92 (s. 1H, –CH=), 7.51–7.34 (m, 6H,

Ar–H), 5.45 (s, 2H, OCH₂), 4.60 (s, 2H, N–CH₂); ¹³C‐NMR (NMR

(75 MHz, DMSO‐d₆): δ 193.17 (C═S), 167.55 (COOH), 166.78 (C═O),

162.10 (C–OCH₂–), 141.44 (C–NO₂), 135.82 (–CH═), 129.16 (2C),

128.91 (2C), 128.35 (2C), 126.07, 125.84, 125.32, 122.80, 114.40,

71.62 (O–CH₂), 46.70 (N–CH₂); MS (ESI) m/z (%): 431.0 [M+H]⁺; Anal.

calcd. for C₁₉H₁₄N₂O₆S₂: C, 53.01; H, 3.28; N, 6.51. Found: C, 53.12;

H, 3.37; N, 6.57.

(Z)‐2‐(5‐(2‐Hydroxy‐3‐methoxy‐5‐nitrobenzylidene)‐4‐oxo‐2‐

thioxothiazolidin‐3‐yl)acetic acid (8j)

IR (KBr) ν_max(cm⁻¹): 3,432 (O–H), 3,043 (aromatic C–H stretch-

ing), 2,923 (aliphatic C–H stretching), 1,719 and 1,706 (C═O),

1,586 (C═C), 1,532 and 1,366 (–NO₂), 1,328 (C═S), 1,269 (C–O

stretching); ¹H‐NMR (300 MHz, DMSO‐d6): δ 13.37 (bs, 1H,

COOH), 7.96–7.95 (m, 1H, Ar–H), 7.88 (s, 1H, –CH═), 7.59 (s, 1H,

Ar–H), 4.72 (s, 2H, N–CH₂), 3.86 (s, 3H, OCH₃); ¹³C‐NMR

(75 MHz, DMSO‐d₆): δ 193.91 (C═S), 167.71 (COOH), 166.92

(C═O), 157.53 (C–OH), 151.22 (C–OMe), 142.97 (C–NO₂),

133.31 (–CH═), 126.37, 123.74, 122.02, 121.17, 116.52, 45.62

(N–CH₂); MS (ESI) m/z (%): 371.0 [M+H]⁺, 393.0 [M+Na⁺]; Anal.

calcd. for C₁₃H₁₀N₂O₇S₂: C, 42.16; H, 2.72; N, 7.56. Found: C,

42.28; H, 2.85; N, 7.63.

(Z)‐2‐(5‐((2‐(Benzyloxy)naphthalen‐1‐yl)methylene)‐4‐oxo‐

2‐thioxothiazolidin‐3‐yl)acetic acid (8n)

IR (KBr) ν_max(cm⁻¹): 3,449 (O–H), 3,033 (aromatic C–H stretching),

2,937 (aliphatic C–H stretching), 1,718 and 1,692 (C═O), 1,589

(C═C), 1,345 (C═S), 1,282 (C–O); ¹H‐NMR (300 MHz, CDCl₃):

δ 9.29–9.26 (d, J = 8.4 Hz, 1H, Ar–H), 8.02–7.99 (d, J = 9.0 Hz, 1H,

Ar–H), 7.76–7.73 (d, J = 7.8 Hz, 1H, Ar–H), 7.63–7.24 (m, 9H, 8Ar–H

and –CH═), 5.31 (s, 2H, OCH₂), 4.72 (s, 2H, N–CH₂); ¹³C‐NMR NMR

CHAUHAN ET AL.

13 of 14

|

(75 MHz, CDCl₃): δ 192.07 (C═S), 164.07 (COOH), 163.19 (C═O),

151.19 (C–OCH₂–), 137.48, 135.96 (–CH═), 131.58, 129.90, 128.82

(3C), 128.72, 128.43, 128.24 (3C), 127.39, 125.03, 124.94, 117.30,

113.99, 71.53 (O–CH₂), 45.54 (N–CH₂); MS (ESI) m/z (%): 436.0

[M+H]⁺; Anal. calcd. for C₂₃H₁₇NO₄S₂: C, 63.43; H, 3.93; N, 3.22.

Found: C, 63.47; H, 4.08; N, 3.34.

ChemBioOffice v16 (Perkin–Elmer). Before docking, the grid para-

meters were validated by redocking the cocrystallized ligand

(subjected to energy minimized), MUP into the active site of PL.

The redocked pose was deviated from the cocrystallized pose by an

RMSD of 1.7 Å. The obtained docked poses were analyzed for their

MolDock scores, while the various interactions exhibited by the

ligands with the active site were visualized in a Discovery Studio

Visualizer (Dassault Systemes Biovia).

|

4.2

Biological assays

|

4.2.1

PL inhibitory assay

|

4.3.2

Molecular dynamics simulations

The assay procedure for method A was performed as per the

protocol optimized in our laboratory.^[22,24]Briefly, 50 mg of porcine

PL was suspended in 10 ml of Tris‐HCl buffer (containing 2.5 mmol of

Tris and 2.5 mmol of NaCl) and adjusted to pH 7.4 with HCl. The

solution was shaken vigorously for 15 min, followed by centrifugation

(4,000 rpm, 291 K for 10 min). The supernatant was collected and

used afresh as the enzyme solution. Stock solutions of orlistat and

derivatives 8a–n were prepared in dimethylsulfoxide (DMSO) at

linear concentrations ranging from 0.78 to 1,000 μg/ml. The final 1 ml

reaction mixture comprised 875 μl of the buffer, 100 μl enzyme and

20 μl of the inhibitors of various stock concentrations. The mixture

was incubated for 5 min at 37°C, followed by addition of 5 μl of the

substrate (4‐nitrophenyl butyrate, 10 mM in acetonitrile). The final

concentration of DMSO in the mixture did not exceed 2%.

Absorbance was detected using an EPOCH microplate spectro-

photometer (BioTek, VT) after 5 min at the absorbance maxima of 4‐

nitrophenol (405 nm). The assay was performed in triplicate and the

percentage inhibition was calculated using the formula

Molecular dynamics represents a useful technique for understanding

the ligand interactions and its binding stability in dynamic

a

environment. Hence, the MD simulations for the most potent ligands

were performed using a GROningen Machine for Chemical Simula-

tions (GROMACS 5.0.4), compiled on a CentOS 7 operating system

equipped with Intel(R) Xeon(R) CPU W3565 and an NVIDIA Quadro

4000 Quad‐Core Processor was used. A CHARMM27 force field was

applied during the MD run and the topology of the ligands was

generated using the online tool provided by the Swiss Institute of

Bioinformatics.^[28,29]Before the initiation of the MD, the complex

was minimized using the Steepest Descent algorithm for 1,000 steps,

followed by stabilization of the system to 310 K and 1 atm pressure

for 50 ps, using the canonical constant Number of atoms, Volume,

and Temperature (NVT) and constant number of atoms, pressure, and

temperature (NPT) ensembles. Parameters like the Particle Mesh

Ewald method for long‐range electrostatics and 14 Å cutoff for van

der Waals and columbic interactions were set during the MD

simulation.^[30]LINCS algorithm was applied for the calculation of

[31]

% Inhibition = ([A_E− A_T]/A_E) × 100

bond length.

Discovery Studio visualizer was used to depict the

graphical representations of the complex.

where A_Eis the absorbance of enzyme control (without inhibitor) and

A_Tis the difference between the absorbance of test samples, with

and without substrate. The IC₅₀values of the inhibitors were

calculated by plotting the linear regression curve (% inhibition vs.

concentration).

|

4.3.3

In silico drug likeness predictions

The drug likeness predicts the oral bioavailability of the derivatives on

the basis of various structural and physicochemical properties. In the

present study, the drug likeness of the synthesized derivatives was

predicted using Lipinski’s rule of five, which describes that for a

molecule to act as a therapeutic candidate, it must possess five

characteristics which are (a) number of hydrogen bond donors should

not be more than 5, (b) number of hydrogen bond acceptors should not

be more than 10, (c) molecular mass should be less than 500 daltons, (d)

LogP (octanol–water partition coefficient) should not be greater than 5.

Drug likeness was predicted by online Swiss ADME tools.

For the enzyme inhibition kinetics, the assay protocol was

repeated using 8f (1.25, 2.5, and 5 μM) at four different substrate

concentrations (25, 50, 100, and 200 µM) and a double reciprocal

Lineweaver–Burk plot was plotted to understand the nature of

inhibition. The inhibition constant, K_i, was calculated from the

Cheng–Prusoff equation.^[23]

|

4.3

Molecular modeling studies

Molecular docking

|

4.3.1

Molecular docking was performed with the aid of Molegro Virtual

Docker 6.0 (CLC Bio, Denmark) and the crystal structure of the open

lid conformation of the human PL was retrieved from the RCSB

Protein Data Bank (PDB Code: 1LPB).^[26,27]Before the docking, the

structures of the ligand were drawn using the ChemDraw 2D module

and the energy minimized structures were obtained using the

molecular mechanics 2 force field in the ChemDraw 3D module of

|

4.3.4

In silico toxicity prediction

The toxicity analysis of the synthesized derivatives was done by using

preADME and protox online software. PreADME predicts the toxicity

profile on carcinorats, carcinomice, and human ether related gene

factor (hERG) models, whereas ProTox predicts the LD₅₀dose as well

as the toxicity class.

14 of 14

CHAUHAN ET AL.

|

ACKNOWLEDGMENTS

[15] Z. Liu, Q. Chai, Y. Li, Q. Shen, L. Ma, L. Zhang, X. Wang, L. Sheng, J. Li,

J. Li, J. Shen, Acta Pharmacol. Sin. 2010, 31, 1005.

[16] A. K. Singh, A. C. Tripathi, A. Tewari, V. Chawla, S. K. Saraf, Med.

Chem. Res. 2017, 26, 1535.

The authors (D. C., R. B., and V. M.) acknowledge the I. S. F. College of

Pharmacy, Moga, Punjab (India) for providing the necessary

laboratory facilities. The authors (G. G., S. N. C., and A. P.)

acknowledge the Birla Institute of Technology and Science, Pilani

(BITS Pilani), Pilani Campus, Pilani, Rajasthan (India) for providing the

necessary laboratory facilities and fellowship to G. G.

[17] S. Wang, Y. Zhao, W. Zhu, Y. Liu, K. Guo, P. Gong, Arch. Pharm. 2012,

345, 73.

[18] Y. Ohishi, T. Mukai, M. Nagahara, M. Yajima, N. Kajikawa, K.

Miyahara, T. Takano, Chem. Pharm. Bull. 1990, 38, 1911.

[19] D. Hardej, C. R. Ashby Jr, N. S. Khadtare, S. S. Kulkarni, S. Singh, T. T.

Talele, Eur. J. Med. Chem. 2010, 45, 5827.

[20] M. Krátký, J. Vinšová, J. Stolaříková, Bioorg. Med. Chem. 2017, 25,

1839.

CONFLICT OF INTEREST

[21] D. K. Aneja, P. Lohan, S. Arora, C. Sharma, K. R. Aneja, O. Prakash,

Org. Med. Chem. Lett. 2011, 1, 15.

The authors declare that there are no conflict of interest.

[22] S. N. C. Sridhar, G. Ginson, P. O. Venkataramana Reddy, M. P. Tantak,

D. Kumar, A. T. Paul, Bioorg. Med. Chem. 2017, 25, 609.

[23] B. T. Burlingham, T. S. Widlanski, J. Chem. Educ. 2003, 80, 214.

[24] S. N. C. Sridhar, S. Mutya, A. T. Paul, Med. Chem. Res. 2017, 26,

1268.

ORCID

Vikramdeep Monga

http://orcid.org/0000-0003-4004-6241

[25] M. J. Naim, M. J. Alam, F. Nawaz, V. G. M. Naidu, S. Aaghaz, M. Sahu,

N. Siddiqui, O. Alam, Bioorg. Chem. 2017, 73, 24.

[26] S. Chander, C. ‐R. Tang, H. M. Al‐Maqtari, J. Jamalis, A. Penta, T. B.

Hadda, H. M. Sirat, Y. T. Zheng, M. Sankaranarayanan, Bioorg. Chem.

2017, 72, 74.

REFERENCES

[1] G. A. Bray, K. K. Kim, J. P. H. Wilding, Obes. Rev. 2017, 18, 715.

[2] WHO Obesity and overweight. http://www.who.int/mediacentre/

factsheets/fs311/en/. (accessed: June 2019).

[27] M. P. Egloff, F. Marguet, G. Buono, R. Verger, C. Cambillau, H. van

Tilbeurgh, Biochemistry 1995, 34, 2751.

[3] L. Khaodhiar, K. C. McCowen, G. L. Blackburn, Clin. Cornerstone 1999,

2, 17.

[28] R. Thomsen, M. H. Christensen, J. Med. Chem. 2006, 49, 3315.

[29] A. D. MacKerell Jr, N. Banavali, N. Foloppe, Biopolymers 2000‐2001,

56, 257.

[4] World Health Organisation. WHO, 2018 http://www.who.int/

mediacentre/factsheets/fs311/en/ (accessed: June, 2019)

[5] https://easo.org/media ‐portal/statistics/ (accessed: June 2019).

[6] G. E. Palade, P. Siekevitz, L. G. Caro, in Ciba Foundation Symposium‐The

Exocrine Pancreas: Normal and Abnormal Functions (Eds: A. de Reuck, M.

Cameron), John Wiley & Sons Ltd, Chichester, UK 2008, pp. 23–55.

[7] http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafety

InformationforPatientsandProviders/ucm213038.htm (accessed:

June, 2019).

[30] V. Zoete, M. A. Cuendet, A. Grosdidier, O. Michielin, J. Comput. Chem.

2011, 32, 2359.

[31] B. Hess, H. Bekker, H. J. C. Berendsen, J. G. E. M. Fraaije, J. Comput.

Chem. 1997, 18, 1463.

[8] V. Point, K. V. P. Pavan Kumar, S. Marc, V. Delorme, G. Parsiegla, S.

Amara, F. Carrière, G. Buono, F. Fotiadu, S. Canaan, J. Leclaire, J. F.

Cavalier, Eur. J. Med. Chem. 2012, 58, 452.

SUPPORTING INFORMATION

[9] V. Point, A. Bénarouche, J. Zarrillo, A. Guy, R. Magnez, L.

Fonseca, B. Raux, J. Leclaire, G. Buono, F. Fotiadu, T. Durand, F.

Carrière, C. Vaysse, L. Couëdelo, J.‐F. Cavalier, Eur. J. Med. Chem.

2016, 123, 834.

Additional supporting information may be found online in the

Supporting Information section.

[10] E. Menteşe, F. Yılmaz, N. Karaali, S. Ülker, B. Kahveci, Russ. J. Bioorg.

Chem. 2014, 40, 336.

How to cite this article: Chauhan D, George G, Sridhar SNC,

Bhatia R, Paul AT, Monga V. Design, synthesis, biological

evaluation, and molecular modeling studies of rhodanine

derivatives as pancreatic lipase inhibitors. Arch Pharm Chem

Life Sci. 2019;e1900029.

[11] S. N. C. S., D. Bhurta, D. Kantiwal, G. George, V. Monga, A. T. Paul,

Bioorg. Med. Chem. Lett. 2017, 27, 3749.

[12] H. Jiang, W.‐J. Zhang, P. H. Li, J. Wang, C. Z. Dong, K. Zhang, H. X.

Chen, Z. Y. Du, Bioorg. Med. Chem. Lett. 2018, 28, 1320.

[13] J. Liu, Y. Wu, H. Piao, X. Zhao, W. Zhang, Y. Wang, M. Liu, Mini Rev.

Med. Chem. 2018, 18, 948.

https://doi.org/10.1002/ardp.201900029

[14] D. Kaminskyy, A. Kryshchyshyn, R. Lesyk, Expert Opin. Drug. Discov.

2017, 12, 1233.

Article Doi

Design, synthesis, biological evaluation, and molecular modeling studies of rhodanine derivatives as pancreatic lipase inhibitors

DOI: 10.1002/ardp.201900029

Source and publish data:

Authors:

Article abstract of DOI:10.1002/ardp.201900029

Full text of DOI:10.1002/ardp.201900029

Products guided by the article

R&D Labs maybe for 486994-24-5

Relevant to this article

Hot Product