Discovery of novel N-substituted thiazolidinediones (TZDs) as HDAC8 inhibitors: in-silico studies, synthesis, and biological evaluation

Journal Pre-proofs

Discovery of novel N-substituted thiazolidinediones (TZDs) as HDAC8 in-

hibitors: in-silico studies, synthesis, and biological evaluation

Neha Upadhyay, Kalpana Tilekar, Niklas Jänsch, Markus Schweipert, Jessica

D. Hess, Luca Henze Macias, Piotr Mrowka, Renato J. Aguilera, Jun-yong

Choe, Franz-Josef Meyer-Almes, C.S. Rama

PII:

S0045-2068(20)30740-9

DOI:

https://doi.org/10.1016/j.bioorg.2020.103934

YBIOO 103934

Reference:

To appear in:

Bioorganic Chemistry

Received Date:

Revised Date:

Accepted Date:

31 March 2020

11 May 2020

Please cite this article as: N. Upadhyay, K. Tilekar, N. Jänsch, M. Schweipert, J.D. Hess, L. Henze Macias, P.

Mrowka, R.J. Aguilera, J-y. Choe, F-J. Meyer-Almes, C.S. Rama, Discovery of novel N-substituted

thiazolidinediones (TZDs) as HDAC8 inhibitors: in-silico studies, synthesis, and biological evaluation,

Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103934

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Discovery of novel N-substituted thiazolidinediones (TZDs) as HDAC8

inhibitors: in-silico studies, synthesis, and biological evaluation

Neha Upadhyay¹, Kalpana Tilekar¹, Niklas Jänsch², Markus Schweipert², Jessica D. Hess³, Luca Henze

Macias³, Piotr Mrowka^4,5, Renato J. Aguilera³, Jun-yong Choe^6,7, Franz-Josef Meyer-Almes^*2, C S

Ramaa*¹

¹Department of Pharmaceutical Chemistry, Bharati Vidyapeeth’s College of Pharmacy, Navi Mumbai, India.

²Department of Chemical Engineering and Biotechnology, University of Applied Science, Darmstadt, Germany.

³The Cellular Characterization and Biorepository Core Facility & Border Biomedical research Centre & Department of

Biological Sciences, The University of Texas at El Paso, El Paso, Texas, USA.

⁴Department of Biophysics and Human Physiology, Medical University of Warsaw, Chalubinskiego, Warsaw, Poland.

⁵Institute of Hematology and Blood Transfusion, Indira Gandhi St., Warsaw, Poland.

⁶East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27834, USA.

⁷Department of Biochemistry and Molecular Biology, The Chicago Medical School, Rosalind Franklin University of Medicine

and Science, North Chicago, Illinois, USA.

*Corresponding authors

1. C S Ramaa; E-mail address: sinharamaa@yahoo.in; Tel.: +91 22 27572131/1122; Fax: +91 22 27574515.

2. Franz-Josef Meyer-Almes; E-mail address: franz-jose f.m eye r -almes@h-da.de; Phone: +49 6151168406; Fax:

+49 6151168404.

Abstract

Epigenetics plays a fundamental role in cancer progression, and developing agents that regulate epigenetics is

crucial for cancer management. Among Class I and Class II HDACs, HDAC8 is one of the essential epigenetic

players in cancer progression. Therefore, we designed, synthesized, purified, and structurally characterized novel

compounds containing N-substituted TZD (P1-P25). Cell viability assay of all compounds on leukemic cell lines

(CEM, K562, and KCL22) showed the cytotoxic potential of P8, P9, P10, P12, P19, and P25. In-vitro screening

of different HDACs isoforms revealed that P19 was the most potent and selective inhibitor for HDAC8 (IC₅₀– 9.3

μM). Thermal shift analysis (TSA) confirmed the binding of P19 to HDAC8. In-vitro screening of all compounds

on the transport activity of GLUT1, GLUT4, and GLUT5 indicated that P19 inhibited GLUT1 (IC₅₀– 28.2 μM).

P10 and P19 induced apoptotic cell death in CEM cells (55.19% and 60.97% respectively) and P19 was less

cytotoxic on normal WBCs (CC₅₀– 104.2 μM) and human fibroblasts (HS27) (CC₅₀– 105.0 μM). Thus, among

this novel series of TZD derivatives, compound P19 was most promising HDAC8 inhibitor and cytotoxic on

leukemic cells. Thus, P19 could serve as a lead for further development of optimized molecules with enhanced

selectivity and potency.

Keywords

N-substituted thiazolidinediones; HDAC; GLUT; Leukemia; Docking.

1. Introduction

Epigenetics is the major contributor to cancer initiation, progression, and metastasis [1]. Histone modifications, a

central epigenetics mechanism, include acetylation, methylation, phosphorylation, and many others. Histone

acetylation unlocks chromatin and promotes gene transcription. Two enzymes control the acetylation of histones:

histone acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylation of lysine residues by HATs on

histone tails leaves the chromatin structure intact and accessible and is responsible for transcription activation. In

contrast, HDAC removes acetyl groups, leading to a more condensed chromatin structure, and, thus, repressing

gene transcription [2]. HDACs are also involved in the regulation of cell differentiation, apoptosis, and cell cycle.

Malignant cells have higher levels of HDACs compared with healthy cells, which leads to an abnormal histone

acetylation pattern.

There are 18 human HDACs classified into four groups based on their structural homology: Class I comprised of

HDAC 1-3 and 8, found in the nucleus; Class II comprised of HDAC 4-7, and 10, which are in the nucleus and

cytoplasm; Class III comprised of Sirtuins (SIRT) 1-7, found in the nucleus, cytoplasm and/or mitochondria; and

Class IV including the newly discovered HDAC 11. HDAC inhibitors approved by FDA for different types of

cancer include: Vorinostat, Belinostat, Panobinostat, Romidepsin, and many others are in clinical studies [3–5].

Currently, FDA-approved HDAC inhibitors are generally pan-inhibitors that target more than one HDAC isoform

and are associated with severe side effects [6–8]. Hence, isoform-selective HDAC inhibitors with fewer side effects

and enhanced potency efforts have become a significant research focus.

HDAC8 is one of the essential epigenetic player in cancer progression and overexpresses in different cancer types,

including cancers of the colon, breast, lung, gastric, and pancreas, acute myeloid leukemia (AML), and acute

lymphocytic leukemia (ALL). Upregulation of HDAC8 inhibited apoptosis and induced cancer cell proliferation

[9–12]. Therefore, targeting HDAC8 could counteract cancer and combat the adverse effects associated with pan-

inhibitors.

O

OH

O

N

H

HO

N

H

1

2

O

OH

N

H

S

N

O

N

4

OH

O

N

H

Br

3

O

Cl

N

NH₂

N

O

F

5

F

Figure 1. Reported selective HDAC8 inhibitors: PCI34051 (1), linkerless hydroxamic acid (2), ortho-aryl N-

hydroxycinnamide (3), NCC149 (4), azetidinone (5) [13–16].

There is a big challenge to develop HDAC8-selective inhibitors, since the active site is largely conserved among

HDACs in general and class I HDACs including HDAC8 in particular. Therefore, linear hydroxamate inhibitors

like SAHA are rather unselective on HDAC isoenzymes. However, HDAC8 contains several structural

characteristics that allowed the development of different types of inhibitors with selectivity for this enzyme. First

of all, HDAC8 shows significantly higher structural flexibility around the binding pocket, particularly at the outer

rim between L1-, L2- and L6-loops as well as at the bottom of the catalytic site at the transition to the acetate

release channel. Furthermore, crystal structures of three distinct major conformations of HDAC8-ligand complexes

demonstrate the extraordinary malleability of HDAC8 around the active site pocket. There is a transition between

the closed (PDB-ID 1T69), a sub-open conformation with a second transient binding pocket (PDB-ID 1T64) and

a wide-open protein structure (PDB-ID 1VKG) of HDAC8. These structural features have been exploited by a

variety of HDAC-inhibitors with selectivity for HDAC8. Most reported isoenzyme selective HDAC8 inhibitors

contain a hydroxamate group as zinc chelating warhead. The selectivity of hydroxamate inhibitors was recently

attributed to their L-shape and binding to a subpocket between L1- and L6-loop at the outer rim of the active site

pocket that is unique for HDAC8 [14]. The selectivity of other non-hydroxamate inhibitors was explained by the

occupation of the transition area between the active site and the acetate release channel at the bottom of the

conserved binding pocket [17]. This binding mode is also proposed for recently discovered non-hydroxamate

inhibitors with benzothiazine scaffold [18,19]. So called linkerless HDAC8 inhibitors (Fig. 1, compound 2) have

been shown to induce the formation of a unique subpocket in HDAC8 by splitting F152 and M274 [20].

Additionally, the transient side pocket, which is observed in crystal structure of PDB-ID 1T64 (trichostatin A)

would also be

a

good candidate for the design of selective HDAC8 inhibitors [21,22].

We have previously reported N-substituted-2,4-thiazolidinedione (TZD) derivative SRR2 as HDAC inhibitor (Fig.

2), wherein, TZD was at the tail portion of the compound, and docking revealed its interaction with zinc [11]. In

our recent attempt to develop specific HDAC8 inhibitors, we designed naphthylidene TZD derivatives, with

naphthalene as the linker, wherein compound 3h (Fig. 2) inhibited HDAC8 potently (IC₅₀– 6.7 μM), though it was

hypothesized that the TZD moiety would act as zinc binding group (ZBG), the docking analysis shown that the

carboxylate group interacted with zinc, and TZD occupied the hydrophobic pocket [23]. Thus, based on our

previously reported HDAC inhibitors, we designed novel HDAC8-selective inhibitors. The nature of CAP plays

significant role to enhance the potency of compounds, there are variety of functionalities reported which could

serve the purpose [24] and thus, we placed variously substituted aryl//heteroaryl/fused heterocyclic aromatic rings

at the CAP portion. The next essential moiety is the hydrophobic linker which could be linear or cyclic [25,26],

hence, similar to our previous HDAC inhibitor SRR2, we retained linear linker but reduced the length to actually

see the difference in the activity (Fig. 2). The next part is ZBG, which is the essential game changer and could

exert significant effects on the inhibitory activity of the compounds [27]. Thus, we retained N-substituted TZD

moiety in the compound to gain HDAC inhibitory potential and attached it to the linker. Moreover, –CH=CH–

functionality attached to ZBG were reported to show promising HDAC8 inhibitory activities, and some of them

are currently under clinical investigations [24,28,29]. Therefore, similar to compound 3h, we attached TZD ring

to pyridine ring with –CH=CH– which led to a completely novel ZBG, pyridin-2-ylmethylene TZD. Interestingly,

the designing located the TZD ring at the end of the linker, which will help us to determine the effects of TZD on

the activity. Also, this unique structural design of novel HDAC8 inhibitors with pyridin-2-ylmethylene TZD

provided L-shape to the compounds and hence we expect them to show HDAC8 inhibitory potential.

Docking studies of the compounds at the active site of HDAC8 showed zinc binding interactions with the carbonyl

group of the TZD ring or the nitrogen atom of the pyridine ring or both (supplementary file). Hence by placing the

TZD ring centrally, we expected that these newly designed pyridin-2-ylmethylene TZD derivatives might exhibit

potential HDAC8 inhibitory activity. Based on these considerations, in the present work, we designed and

synthesized 25 novel compounds (P1-P25) and studied their in-vitro antiproliferative activity.

Figure 2. Designing aspects of novel HDAC8 inhibitors [11,13,14,23].

HDACs are overexpressed in different leukemia subtypes such as acute promyelocytic leukemia (APL) [9,30,31],

neuroblastoma [32], and T-cell lymphoma [33]. HDACs overexpression leads to the deacetylation of histones.

Increased histone deacetylation causes an increase in the rate of cell cycle and a decrease in the differentiation and

apoptosis of leukemic cells. These processes could be important in the identification and treatment of leukemias.

Moreover, as our selective HDAC8 inhibitors (naphthylidene TZDs) have proved their in-vitro antiproliferative

potential in leukemic cells, we decided to evaluate these newly designed HDAC8 inhibitors in leukemic cell lines.

2. Results and Discussion

2.1. Chemistry

Scheme I illustrates the synthesis of compounds. Synthesis of final compounds P1-P25 proceeded via four steps.

The first step in the synthesis of variously substituted amides was as previously reported [23,34–37]. Substituted

amines 1a-1y were stirred overnight with chloroacetyl chloride in the presence of potassium carbonate (K₂CO₃)

and dichloromethane (DCM) as a solvent to produce 2a-2y. The second step included knoevenagel condensation

of pyridine-2-carboxaldehyde 3 and 2,4-thiazolidinedione 4 in the presence of acetic acid and sodium acetate to

yield intermediate 5. By refluxing 5 with 2a-2y in the presence of methanol did not produce P1-P25, so salt of 5

was synthesized in the third step by refluxing 5 with potassium hydroxide in the presence of ethanol to yield

knoevenagel salt i.e. 6. In the final step, refluxing vigorously 6 and 2a-2y led to the compounds of interest P1-

P25. Knoevenagel salt 6 was common for the synthesis of all the final compounds; therefore, it was synthesized

in bulk. The purity of the final compounds P1-P25 was >95% by HPLC. All the synthesized compounds were

1

confirmed structurally by different methods viz. H-NMR, ¹³C-NMR, FTIR, UV Spectroscopy, and Mass

Spectrometry (Supplementary file). The detailed synthesis procedure and other spectral observations are in the

experimental section.

2.2. Biological evaluation

2.2.1. In-vitro cytotoxicity assay

To evaluate the cytotoxic potential of this series of compounds, we performed the Differential Nuclear Staining

(DNS) assay after exposing human cancer cell lines to each compound for 48 h. The DNS assay is typically the

first experiment for novel compounds because it provides simple and reliable measure of viability that is

quicker/easier to obtain yet robust and comparable to what would be seen if the viability were assessed by flow

cytometry. It uses two fluorescent nuclear stains, Hoechst 33342 and Propidium iodide (PI), to selectively label

living and dead cells [38]. An initial screening of all 25 compounds, at 50 μM concentration, was conducted on K-

562, KCL-22, and CEM leukemia cells. Solutions of each compound were prepared in DMSO, and the desired

concentration of each compound was added to cell-containing wells at a final concentration of 1% (v/v) DMSO.

Six compounds demonstrated activity in previous biological analysis and considerable cell death in one or more

cancer cell lines. These six compounds were selected and assessed with a concentration gradient of 100-0.5 μM

via secondary DNS assays on CEM, K-562, and KCL-22 to estimate their CC₅₀values. Cytotoxicity is reported

as CC₅₀(cytotoxic concentration 50%, concentration of compound needed to kill 50% of the cell population)

because in the DNS assay, living and dead cells are differentiated based on their membrane integrity, not on

metabolic activity like the popular MTT assay. The CC₅₀values were estimated by linear interpolation of the two

concentrations nearest 50% cell death. Compounds P8, P9, P10, P19, and P25 displayed CC₅₀values in the mid-

micromolar range (Table 1). Out of these six compounds, P12 was the least cytotoxic and did not approach 50%

cell death on any cell line at the concentrations used.

Table 1. Cytotoxic concentration 50% (CC₅₀) of compounds P8, P9, P10, P12, P19, and P25 in leukemic cell

lines (CEM, K562 and KCL22). Cells were exposed to different concentrations of compounds for 48 h.

Compound

P8

Cell Line

CEM

CC50 (µM)

36.9±1.11

K562

40.28% cell death at 100 μM

29.22% cell death at 100 μM

4.80% cell death at 100 μM

101.8±2.45

KCL22

CEM

P9

K562

KCL22

CEM

6.54% cell death at 100 μM

2.39% cell death at 100 μM

27.06% cell death at 100 μM

17.4±2.4

P10

P12

P19

P25

K562

KCL22

CEM

38.67% cell death at 100 μM

39.42% cell death at 100 μM

24.81% cell death at 100 μM

79.9±3.74

K562

KCL22

CEM

K562

85.4±3.25

KCL22

CEM

43.2±0.23

39±1.69

K562

65.6±1.13

KCL22

82.2±0.32

2.2.2. In-vitro HDAC enzyme inhibition assay

2.2.2.1. Primary screening on HDAC4 and HDAC8

To determine the HDAC inhibitory potential of the target compounds, we assessed all of them on two isoforms of

HDAC viz. HDAC4 and HDAC8. These two isoforms were selected because they possess high deacetylation

activity, and belong to two diverse classes of deacetylase: class I (HDAC 8) and class II (HDAC4). The potency

and selectivity of the target compounds for the two HDAC isoforms were examined and compared.

The primary screening was done with 50 µM concentration of P1-P25 on HDAC4 and HDAC8. Percent relative

activities for all compounds on both HDAC isoforms were determined (Fig. 3A, 3B, and Table 2). Only two

compounds, P5 and P12, showed HDAC4 inhibition, with IC₅₀of 50 µM (Fig. 3C). Interestingly, compounds P10,

P12, and P19 displayed moderate to good HDAC8 inhibition, with IC₅₀values of 23 µM, 47 µM, and 9.3 µM,

respectively (Fig. 3D). Among all compounds, P19 and P10 were more selective on HDAC8 over HDAC4, with

HDAC8-IC₅₀in the sub-micromolar range. The presence of the benzothiazole ring may be responsible for the more

potent activity of P19 on HDAC8; it was suggested that compounds incorporating the benzothiazole ring had good

HDAC8 inhibition [39–43]. Thus, we assume that the benzothiazole ring contributed to the selectivity and activity

of P19 against HDAC8. The thermal shift assay (TSA) was performed to examine the effects of P5, P9, P10, P12,

and P19 binding to HDAC8 (Supplementary file – Fig. S5). Thermal shift data (∆T) suggested that the tested

compounds did not confer significant stability to the protein; only compound P19 had a weak stabilizing effect of

about 0.3 °C.

Table 2. Primary screening of P1-P25 on two different classes of HDAC isoforms, HDAC4 and HDAC8.

Code HDAC4 IC₅₀HDAC8 IC₅₀Residual HDAC4

Residual HDAC8

activity at 50 µM

(μM)

activity at 50 µM

P1

P2

>50

100 %

89 %

92 %

>50

100 %

P3

P4

>50

50

>50

100 %

59 %

53 %

85 %

80 %

82 %

61 %

82 %

38 %

17 %

n.d.

P5

>50

P6

>50

50

>50

100 %

76 %

P7

>50

P8

>50

P9

>50

P10

P11

P12

P13

P14

P15

P16

P17

P18

P19

P20

P21

P22

P23

P24

P25

23±1.1

>50

100 %

97 %

47±1.1

>50

75 %

91 %

67 %

94 %

n.d.

>50

88 %

99 %

22 %

92 %

54 %

64 %

54 %

68 %

89 %

>50

9.3±1.0

>50

100 %

85 %

>50

100 %

86 %

>50

IC₅₀-values were determined in triplicates, N=3. Means and standard deviations are provided. n.d. – not

determined.

Figure 3. Screening of P1-P25 on two different classes of HDAC isoforms, HDAC4 and HDAC8. Primary

screening of all target compounds at 50 μM against HDAC4 (A) and HDAC8 (B). (C) Dose response curves of

P5, P9, P10, P12, and P19 on HDAC4. (D) Dose response curves of P5, P9, P10, P12, and P19 on HDAC8.

2.2.2.2. HDAC-profiling on a panel of HDACs (HDAC1-8)

In the primary screening on HDAC4 and HDAC8, compounds P10, P12, and P19 were HDAC8 inhibitors (Table

2). P10 and P19, the best HDAC8 inhibitors, were screened on a panel of HDACs (HDAC1-8) to establish if they

were selective for HDAC8 (Table 3, Fig. 4). Testing P10 and P19 on a panel of HDACs revealed that P10 inhibited

multiple HDAC isoforms such as HDAC2, HDAC5, HDAC6, and HDAC8 in mid-micromolar range. In contrast,

P19 displayed HDAC8 inhibition with IC₅₀in single-digit micromolar range as compared to other HDACs.

Table 3. IC₅₀of P10 and P19 on a panel of HDAC.

Compounds HDAC1 HDAC2 HDAC3 HDAC4 HDAC5 HDAC6 HDAC7 HDAC8

P10

P19

>50

3.0

45

41

45

>50

38

>50

10

43

26

17

18

>50

-

23

9.3

>50

PCI-34051*

0.024

*Kleinschek, A., et al. (2016). "Potent and Selective Non-hydroxamate Histone Deacetylase 8 Inhibitors."

ChemMedChem 11 (23): 2598-2606.

Figure 4. Dose response curve of P10 and P19 on different HDAC isoforms.

2.2.3. GLUT 1, 4, and 5 inhibition assay

The therapeutic effects of HDAC inhibitors stem from their capacity to alter gene expression and acetylation status

of histones. Lately, substantial attention has focused on the ability of HDAC inhibitors to modify cellular

metabolism; they inhibit glucose transporter 1 (GLUT1)-mediated glucose transport by downregulating GLUT1

expression [44,45]. This downregulation has been linked to a time-dependent decrease in glucose uptake in

multiple myeloma cell lines (H929).

Compounds containing N-substituted TZD have been reported as GLUT1 inhibitors [46,47]. Our designed HDAC

inhibitors (P1–P25) also hold N-substituted TZD and have exhibited HDAC8 inhibitory activity. Considering that

HDAC inhibitors also interfere with GLUT1 expression and to assess their inhibitory potential on GLUTs, we

screened all target compounds (P1-P25) for their effect on the transport activity of GLUT 1, 4, and 5 at 50 μM

concentration (Fig. 5). For the transport assay, we used the GLUT-specific systems provided by the hexose

transport null (hxt⁰) yeast strains engineered to express a particular human GLUT [48,49]; the only glucose or

fructose uptake in these cells is through the recombinant human GLUTs.

Among all tested compounds, P19 showed significant inhibition of GLUT1 (IC₅₀= 28.2 ± 1.8 μM) and remained

ineffective on the other two GLUT isoforms (Fig. 5). The rest of the compounds exhibited >50% relative activity

at 50 μM concentration on all three GLUT members. Thus, P19 was selective for HDAC8, among other HDAC

isoforms, and was also specific for GLU1, compared with GLUT1 and GLUT4. Moreover, our studies also suggest

that cancers depending on GLUT1 for the production of energy and glycolysis might be tackled with HDAC

inhibitors.

Figure 5. Effect of the test compounds on the relative transport activity of GLUT 1, 4, and 5. Percent relative

activity of GLUT1 (A), GLUT4 (C), and GLUT5 (D) in the presence of 50 μM concentration of test compound.

(B) Dose response curve of P19 in GLUT1. Transport assay was initiated by the addition of 5 mM C¹⁴-glucose

(for GLUT1 and GLUT4) or 10 mM C¹⁴- fructose (GLUT5) to hxt⁰yeast cells expressing GLUT1, GLUT4, or

GLUT5 (see Material and Methods for details). The transport activity was measured in triplicates and means with

S.D. are shown.

2.2.4. Apoptosis studies by flow cytometry

HDAC8 inhibitors have been found to induce apoptosis in solid and hematological cancer cells through various

mechanisms. PCI-34051, a HDAC8 inhibitor have caused apoptosis in T-cell lymphoma cells by caspase activation

[7]. In gastric carcinoma cells, HDAC8 inhibition induced apoptosis facilitated by Bcl-2-modifying factor (BMF)

and STAT3 [50]. Selective HDAC8 inhibitors have been found to cause dose-dependent selective apoptosis of

CD34⁺leukemic stem cells and progenitor cells [7]. Our recently reported selective HDAC8 inhibitor has found

to induce apoptosis in CEM, lymphoid leukemia CEM cell line [23]. Also, the up-regulation of HDAC8 have been

found to inhibit apoptosis in hepatocellular carcinoma, indicating a role of HDAC8 inhibitors in induction of

apoptosis [51]. Thus HDAC8 inhibitors have ability to cause apoptotic cell death, and hence to determine if the

cytotoxicity was induced by P10 and P19 was apoptosis-related, we stained CEM cells with annexin V-FITC/PI

and determined by flow cytometry the percent of cell apoptosis. Incubation of the CEM cells with P10 and P19 at

their CC₅₀concentrations caused an increase in the cell population in the apoptotic region compared with the

control. In P10-treated cells, the percent of apoptotic cells was 55.19%, and live cells were only 29.89% when

compared with that of control (Fig. 6A, 6C), which was 92.15% (Supplementary file – Fig. S6). P19-treated cells

had 60.97% apoptotic cells; the living cells were only 30.86% compared with the control (Fig. 6B, 6C). Thus, as

seen with reported HDAC8 inhibitors, compounds P10 and P19 can induce apoptotic death in the CEM cell line.

Figure 6. Analysis of apoptosis induced by compounds P10 and P19 on CEM cells. (A-B) Cytograms of test

compounds P10 and P19, respectively. CEM cells were treated with IC₅₀concentration of compounds P10 (A) and

compound P19 (B) for 24 h. (C) Graphical representation of apoptotic events of control (1% DMSO), P10-treated

and P19-treated cells.

2.2.5. Cell viability of P19-treated non-cancerous cells by MTT and DNS assays

To ascertain the cytotoxicity of P19 on normal and cancerous cells, we performed both MTT and DNS assays to

compare their cytotoxicity concentration 50% by two different assays. We determined the cell viability of CEM

cell lines and normal white blood cells (WBCs) with the MTT assay and that of CEM cell lines and normal human

fibroblast (HS27) with the DNS assay (Supplementary file – Fig. S7). By MTT assay CC₅₀of P19 in normal WBCs

was 104.2 μM, whereas, in CEM cells it was 11.91 μM (Table 4). DNS assay of HS27 showed that CC₅₀of P19

was 105.0 μM, and of CEM cells was 79.9 μM. Thus, compound P19 could be safe for non-cancerous cells.

Table 4. Viability of P19-treated cells by MTT and DNS assays.

P19

MTT assay (CC₅₀in μM)

DNS assay (CC₅₀in μM)

CEM

11.91

79.9

Non-cancerous cells

104.2 (WBCs)

105.0 (HS27)

2.3. In-silico studies

2.3.1. Molecular docking

Flexible docking was performed with MOE software, using the crystal structures of HDAC8 (PDB ID 1T69) and

HDAC4 (PDB ID 2VQJ). To validate the docking protocol, we determined the Root Mean Square Deviation

(RMSD) between the crystal pose vs. the docked pose. The corresponding alkyl chains with zinc-chelating

hydroxamate warhead of SAHA bound to HDAC8 superpose excellently within the binding pocket of HDAC8

with an RMSD value of 0.9 Å. Similarly, the buried thiophene linker and trifluoromethyl warhead of the redocked

ligand in PDB ID 2VQJ showed an excellent RMSD value of 0.6 Å with respect to the crystal structure. The

aromatic head groups of SAHA in complex with HDAC8 and the trifluoromethylketone ligand in HDAC4 reach

out of the binding pocket were freely rotatable and therefore not considered for the calculation of RMSD. P19

showed the highest experimental activity and the best docking score against HDAC8 (Tables 5). The 2D and 3D

ligand-enzyme interaction diagrams illustrating the major interactions of the representative compound P19 with

amino acid residue at the active site of HDAC8 are shown in Fig. 7 (Other images in supplementary file). P19

fitted well in the binding pocket of HDAC8 forming hydrogen bonds with the backbone oxygen of G151 and to

the side chain of Y306 at the bottom of the active site. Interestingly, P19 interacted with the catalytic zinc ion

through carboxamide carbonyl oxygen and not with our hypothesized TZD carbonyl oxygen. The relatively bulky

benzothiazole moiety of P19 was inserted between A37 and W141 and protruded into the acetate release channel.

The amino acids of the canonical binding tunnel were highly conserved among all classes of zinc dependent

HDACs with one major exception: Y306 in class I HDAC8, which was pivotal for the enzyme mechanism, was

exchanged against a histidine that was swung away from the catalytic site. However, the amino acids flanking the

acetate release channel differed widely, e.g. W141 is unique for HDAC8. Since the bulky benzothiazole group of

P19 fitted perfectly into the specific acetate release channel of HDAC8, we hypothesized that this feature together

with the hydrogen bond between P19 and Y306, which was not present in HDAC4, could be the reason for the

observed inhibitory activity and selectivity towards HDAC8 (Table 2). To visualize the structural differences

between HDAC4 and HDAC8, we superposed the best-scoring docking poses of P19 within the crystal structures

of both enzymes (Fig. 7C). At first glance, the corresponding conserved amino acids of the binding pockets in

HDAC8 and HDAC4 (F208/F227, F152/F168, H142/H158, H143/H159, respectively) showed no perfect overlap

but rather significant shifts. Also it was seen that H332, which corresponds to Y306 in HDAC8, was flipped away

from the catalytic site opening a selectivity side pocket that is characteristic to all class IIa HDACs. Therefore,

H332, unlike Y306 in HDAC8, was not able to form interactions with P19. Most strikingly, HDAC4 did not show

an open subpocket at the transition area to the acetate release channel like HDAC8. Consequently, the

benzothiazole moiety of P19, which protruded into the acetate release channel of HDAC8, would clash with the

surface of HDAC4 in a similar docking pose. The unbeneficial docking pose of P19 at the upper surface of the

binding pocket of HDAC4 was in agreement with the observed absent activity against this enzyme (Fig. 7C).

Table 5. Docking scores target compounds docking to the HDAC8 crystal structure.

Compound Code

GBVI/WSA dG score

Compound Code

GBVI/WSA dG score

SAHA (redocked)

-11.2

-8.0

-7.9

-8.2

-7.8

-8.0

-8.4

-7.9

-8.4

-7.5

-8.0

P13

P14

P15

P16

P17

P18

P19

P20

P21

P22

P23

P24

P25

-8.0

-7.8

-7.9

-7.3

-8.4

-8.6

-7.4

-7.9

-7.5

-7.8

-8.4

-8.1

P1

P2

P3

P4

P5

P6

P7

P8

P9

P10

P11

P12

Figure 7. Docking analysis of compound P19 on HDAC8 (PDB ID 1T69) and HDAC4 (PDB ID 2VQJ). A) 3D

binding pose of P19 (stick-and-ball model) within the binding pocket of HDAC8. B) 2D ligand interactions

between P19 and HDAC8. Hydrogen bonds are indicated by dashed green lines (A) or dotted green or blue arrows

(B) and metal ion contacts are shown as dotted magenta lines. C) Superposition of docking poses of P19 in the

binding pocket of HDAC8 (PDB ID 1T69, orange) and HDAC4 (PDB ID 2VQJ, magenta). Green spheres represent

the catalytic zinc ion of both structures. The amino acids are annotated according to the respective PDB file. The

meshed surfaces indicate the respective binding pocket surfaces of HDAC8 (orange) and HDAC4 (magenta).

2.3.2. SwissADME predictions

The therapeutic action of a compound depends on it reaching the desired target site in the body at sufficient

concentration described by its ADME (absorption, distribution, metabolism, and excretion) properties. Lipinski’s

rule-of-five outlines the probability of a drug to be orally active (i.e., the drug-likeness) and the correlation between

the pharmacokinetic and physicochemical parameters. SwissADME has computational models that give fast yet

robust predictive data about the physicochemical properties, pharmacokinetics, and drug-likeness along with user-

friendly submission and straightforward result interpretation [52]. We used it to predict the physicochemical

properties and bioavailability of synthesized compounds P1-P25. All 25 compounds obey Lipinski’s rule of five

(Table 6) with no violations, indicating their drug-likeliness. All compounds have an acceptable range of clogP

value of 2.00-5.00. Topological polar surface area (TPSA) is a physicochemical descriptor of the surface sum of

all the polar atoms in the molecules, and the acceptable region for suitable absorption is between 20 and 130 Å².

Four compounds exhibited higher TPSA than 130 Å², and the rest fell in the acceptable zone. Compounds solubility

ranged from soluble to moderately soluble. The bioavailability score for all compounds was 0.55. In summary, the

compounds had drug-likeliness and were good candidates for passive oral absorption.

Table 6. SwissADME prediction of physicochemical properties and bioavailability of compounds P1-P25.

Code TPSA^aLog

P^b

Log

S^c

ESOL^dClass

GI^e

absorption

Lipinski

#violations

Bioavailability

Score

P1

P2

P3

P4

104.67 2.08

-3.4

Soluble

High

0

0.55

104.67 2.64 -3.56

104.67

3.4

-4.47

Moderately

soluble

P5

P6

104.67 3.19 -3.72

104.67 4.25 -4.26

Soluble

High

0

0.55

Moderately

soluble

P5

P8

104.67

4.9

-4.86

Moderately

soluble

High

0

0.55

104.67 3.38 -4.59

Moderately

soluble

P9

104.67

131.6

4.9

-4.86

Moderately

soluble

P10

1.87 -4.21

Moderately

soluble

P11 104.67 2.64 -3.56

P12 104.67 2.38 -3.7

Soluble

High

0

0.55

P13

113.9

3.87 -4.86

Moderately

soluble

P14

113.9

2.08 -3.47

Soluble

High

0

0.55

P15 104.67 3.96 -4.63

P16 150.49 1.98 -3.47

Moderately

soluble

High

0

0.55

Soluble

Low

High

0

0.55

P17 104.67 3.04

-4.3

Moderately

soluble

P18

145.8

2.69 -4.29

Moderately

soluble

Low

0

0.55

P19

P20

P21

130.7

145.8

1.37 -2.86

1.53 -3.03

1.84 -3.35

Soluble

High

Low

High

0

0.55

P22 104.67 2.84 -4.31

Moderately

soluble

P23 117.56 1.47 -2.94

Soluble

High

0

0.55

P24

113.9

2.08 -3.47

3.6 -5.22

P25 104.67

Moderately

soluble

^aTopological polar surface area; ^bLog of the partition coefficient (P); ^cLog solubility; ^dEstimated aqueous solubility

in mg/mL; ^eGastrointestinal.

BOILED-Egg is an intrinsic model in SwissADME for predicting brain access or passage of the brain blood

barriers (BBB) and passive gastrointestinal absorption (HIA). It is a proficient method that depends on two

descriptors WLOGP (lipophilicity) and TPSA (apparent polarity). The white region of BOILED-Egg represents a

high probability of passive absorption by the gastrointestinal tract, and the yellow region represents a strong

possibility of reaching the brain. Whether the candidate is (PGP+) or not (PGP-) a substrate for P-gp, a multidrug

resistance efflux pump responsible for drug clearing, is indicated by points colored red or blue, respectively [53].

BOILED-Egg was plotted for all the 25 compounds (Supplementary file – Fig. S1). None of them were in the

yellow region, implying that they may not penetrate the brain. Twenty-one compounds containing the substituted

phenyl rings were in the white region, indicating proper absorption. Four compounds (P16, P18, P20, and P21)

were outside the egg, suggesting that they neither absorb nor cross the BBB and may have poor bioavailability.

These four compounds are substituted five/six-membered aromatic/ heteroaromatic or fused ring viz. nitrophenyl

(P16), methylisoxazole (P18), thiazole (P20), or 5-methylthiazole (P21). Accordingly, the biological screening of

the compounds showed that these four compounds were inactive or nearly inactive at the receptor site.

2.3.3. Computation of toxic hazards

Checking the alerts for Cramer rules and cytotoxicity by P450-mediated drug metabolism for the compound P19

resulted in high Class III and four sites of metabolism (Supplementary file – Fig. S2 and Fig. S3). Verbose

explanation stated that, “Compound has heterocyclic ring with complex substituents (Q7 & 11)”, which could be

the reason for being under Class III, and that there are four sites of CYP450-mediated metabolism viz. S-oxidation,

N-oxidation, and aromatic hydroxylation. Even though P19 is under Class III and predicted to display toxicity, the

possible sites of metabolism by CYP450 could reduce the toxicity by enhancing the compound metabolism.

3. Materials and methods

3.1. In-silico studies

3.1.1. Molecular docking

Molecular docking of the target compounds in the HDAC8 crystal structure was performed with MOE 2019

software (Chemical Computing Group ULC, Canada). The crystal structures of HDAC8 (PDB-ID: 1T69) and

HDAC4 (PDB-ID: 2VQJ) were obtained from RCSB Protein Data Bank. The PDB-files were subjected to structure

preparation including 3D-protonation for subsequent docking. The partial charges of all protein and ligand atoms

were calculated using the implemented Amber14 force field. Molecular docking was performed choosing the

triangle matcher for placement of the ligand in the binding site and ranked with the London dG scoring function.

The best 30 poses were passed to the refinement and energy minimization in the pocket using the induced fit

method and then rescored with the GBVI/WSA dG scoring function.

3.1.2. SwissADME predictions tool

SwissADME is a reliable free online tool [52] that predicts the physicochemical properties of the compounds. The

bioavailability and pharmacokinetic parameters of any synthetic compound is obtained by inputting its structure

on the website http://www.swissadme.ch/index.php#.

3.1.3. Computation of toxic hazards

Toxtree is a free, user-friendly, and flexible open-source application tool, developed by IDEA Consult Ltd. (Sofia,

Bulgaria). It assesses the toxic hazards of a compound by applying a decision tree approach. Toxtree decision tree

can be used for applying the Cramer rules, Cramer rules with extensions, Verhaar scheme, Skin irritation and

corrosion prediction, eye irritation, and corrosion prediction, Benigni/Bossa rule base (for mutagenicity and

carcinogenicity), Michael Acceptors, Skin sensitization alerts, START biodegradability, cytochrome P450-

mediated drug metabolism, DNA binding alerts, protein binding alerts etc. It can evaluate the Threshold of

Toxicological Concern (TTC) of the material or their potential toxicity. Cramer rules predict toxicological dangers

of the molecules when administered orally. In this, molecules are grouped into three classes based on a decision

tree. This involves 33 structural rules, and places the assessed molecules into one of three classes: Class I (Low)

molecules with low oral toxicity (simple chemical structures with efficient modes of metabolism); Class II

(Intermediate) molecules with intermediate toxicity; and Class III (High) molecules that have reactive functional

groups and possible significant toxicity. SMARTCyp is a reactivity model that estimates the molecule site(s) labile

to metabolism by Cytochromes P450 isoform 3A4 [54,55].

3.2. Chemistry

All reagents, solvents, and chemicals were purchased from commercial sources viz. Sigma Aldrich, S.D. Fine

Chem. Ltd., Himedia, and VWR. Reactions were monitored by Thin Layer Chromatography (TLC) using Merck

precoated silica gel 60 F-254 plates under short wavelength UV-light (254nm) to detect the UV absorbing spots to

ensure the completion of the reaction and also the purity of compounds at each step. All intermediates were purified

by recrystallization with suitable solvents such as chloroform, methanol, ethanol, etc. The final compounds were

purified by either recrystallization or column chromatography. Column chromatography was performed on silica

gel 60 (60 to 120 mesh) with combinations of suitable solvents. The purity of all final products (≥95%) was

determined using an Agilent 1200 high-performance liquid chromatography (HPLC) system; software- EZ chrome

Elite. The chromatographic column was HemochromIntsil A31 C18 5U 150 mm × 4.6 mm Sn-B180127, with

detection at 300 nm. UV−visible detector was used with the flow rate of 1 mL/min. The oven temperature was

30°C; gradient elution with a run time of 10 min using Methanol: Formic Acid (1%) (Formic acid: in 1000 mL

double distilled water 1mL formic acid was added) in 80:20 ratio. The melting points of all the intermediates and

final compounds were determined with VEEGO, MODEL: VMP-DS Melting Point apparatus.

Structural characterization of the intermediates was done by FTIR and ¹H-NMR, and of the final step products by

FTIR, ¹H-NMR, ¹³C-NMR, and mass spectrometry. IR was recorded with JASCO FT/IR- 4100 typeA spectrometer

using direct sampling technique. ¹H-NMR spectra were recorded on Bruker Avance 400 MHz Spectrometer using

DMSO-d₆as solvent.¹³C-NMR was recorded on a Bruker Avance Spectrometer at 100 MHz using DMSO-d₆as

solvent. All shifts of ¹H NMR are in δ (ppm) units relative to the signals for the solvent DMSO (δ- 2.50 ppm). All

coupling constants (J values) are in hertz (Hz). NMR abbreviations are: s, singlet; d, doublet; t, triplet; q, quartet;

m, multiplet; and dd, doublet of doublets. The mass spectrum was recorded on LC-MS Agilent Technologies 1260

Infinity instrument. To determine the absorption of synthesized compounds at different wavelengths, we performed

UV Spectroscopy (Supplementary file).

The 25 new derivatives of pyridyl containing N-substituted TZDs were synthesized as shown in Scheme 1.

Variously substituted chloroacetylated amides (2a-2y) were prepared as previously reported [34–37,56,57]. All

chloroacetylated amides were purified by recrystallization with suitable solvents and then used for the next reaction

steps. The compound 5-(pyridin-2-ylmethylene)thiazolidine-2,4-dione (5) was synthesized by following the

Knoevenegel condensation. Synthesis of potassium salt of (5) was done by refluxing potassium hydroxide in

ethanol to produce potassium-2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-ide (6). The substituted

chloroacetylated amides (2a-2y) and 6 were added to methanol (10 ml) in round-bottom flask. This solution was

then refluxed and after completion the reaction was stopped and the mixture was cooled. The solid precipitated out

was collected and purified to produce the final products.

3.2.1. Synthesis of 5-(pyridin-2-ylmethylene)thiazolidine-2,4-dione (5): 2-Pyridinecarboxaldehyde 3 (5 ml, 0.046

mol), thiazolidine-2,4-dione (4) (5 g, 0.0427 mol) and sodium acetate (3 g, 0.036 mol) were added to acetic acid

(5 ml) and refluxed for 5 h. The reaction mixture was then cooled to RT and the precipitated solid was collected

by filtering under vacuum, washed several times with water, and dried under RT. Crude solid was purified by

recrystallization from an appropriate solvent to obtain brown shiny crystals (5). Yield 9 g (90%); Brown shiny

1

crystal; M.P. 232 °C (Charred); IR (cm^-1): 3136, 1737, 1614. H-NMR (400 MHz, DMSO-d₆, δ ppm) 7.41-7.42

(m, 1H), 7.80-7.84 (m, 2H), 7.90-7.94 (m, 1H), 8.73-8.74 (m, 1H), 12.40 (s, 1H).

3.2.2. Synthesis of potassium-2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-ide (6): To the solution of

potassium hydroxide (5 g, 0.089 mol) in ethanol (25 ml), (5) (10 g, 0.048 mol) was added with stirring, and the

reaction mixture was refluxed for 3 h. The fine solid obtained after cooling the reaction mixture was collected by

filtering and washed with cold ethanol to obtain potassium salt 6. Yield 10 g (66%); Light brown color solid; M.P.

292.4 °C (Charred); IR (cm^-1): 3037, 1626, 1662. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm). 7.18-7.21 (m, 1H), 7.26

(s, 1H), 7.52 (d, J=8Hz, 1H), 7.74-7.78 (m, 1H), 8.61-8.62 (m, 1H).

3.2.3.

Synthesis

of

N-(substitutedaryl/heteroaryl)-2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-

yl)acetamide (P1-P25): Intermediate 6 (1 g, 0.004 mol) and variously substituted amides (2a-2y) (0.004 mol) were

dispensed together in a round-bottom flask along with 10 mL methanol. This mixture was refluxed and the reaction

was monitored for completion by TLC using appropriate mobile phase of Hexane: Ethyl acetate (1:1). After 5-6 h

the reaction was stopped and the mixture was cooled. The precipitated solid was collected by filtering under

vacuum and purified by recrystallization (chloroform: methanol, 2:1) or column chromatography (hexane: ethyl

acetate, 1.5: 0.5) with an appropriate solvent to give the corresponding final products (P1-P25).

O

a

Cl

Ar-NH₂

Cl

Ar-NH

1a-1y

Chloroacetyl chloride

2a-2y

N

O

S

N

O

b

H

NH

O

3

5

4

O

c

S

O

N

Cl

N

S

Ar-NH

O

N

O

2a-2y

NH

Ar

O

NK⁺

d

O

P1 - P25

6

0

Scheme 1. Synthetic route of P1-P25. Reaction conditions: a) DCM, K₂CO₃, 0 C, stirring; b) AcOH, Sodium

acetate, reflux 5 h; c) EtOH, KOH, reflux 3 h; d) Methanol, reflux 6 h.

3.2.3.1. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-phenylacetamide (P1): Yield 0.9 g (56%); M.P.

282.8 °C (Charred); buff white color solid; IR (cm^-1): 3267, 1739, 1656, 1683, 1620, 1599, 1388. ¹H-NMR (400

MHz, DMSO-d₆, δ ppm) 4.50 (s, 2H), 7.08 (s, 1H), 7.32 (s, 2H), 7.46 (s, 1H), 7.55 (s, 2H), 7.91-8.00 (m, 3H), 8.79

13

(s, 1H), 10.39 (s, 1H). C-NMR (400 MHz, DMSO-d₆): 43.355, 119.175, 123.714, 124.264, 125.266, 128.002,

128.854, 129.461, 137.635, 138.320, 149.342, 151.035, 163.880, 165.544, 170.979. UV- Spectrum (10 ppm, λ_max

– 331.8 nm). Theoretical mass: 339, LC-MS (m/z, I %): 338 [(M-H)⁺, 100%]. HPLC Purity: % Area 98.32, RT

2.99 mins.

3.2.3.2. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(2-fluorophenyl)acetamide (P2): Yield 1 g

(60%); M.P. 293.9 °C (Charred); buff white color solid; IR (cm^-1): 3263, 1737, 1666, 1686, 1616, 1546, 1386,

1298. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.58 (s, 2H), 7.16-7.18 (m, 2H), 7.25-7.30 (m, 1H), 7.46 (t, J= 5.8

Hz, 1H), 7.85-7.91 (m, 2H), 7.94-8.00 (m, 2H), 8.78 (d, J= 4 Hz, 1H), 10.26 (s, 1H). ¹³C-NMR (400 MHz, DMSO-

d₆): 43.192, 115.476, 115.669, 123.862, 124.274, 124.476, 125.250, 128.007, 129.472, 137.645, 149.347, 151.028,

164.503, 165.510, 170.955. UV- Spectrum (10 ppm, λ_max– 331.7 nm). Theoretical mass: 357, LC-MS (m/z, I %):

355.9 [(M-2H)⁺, 100%]. HPLC Purity: % Area 97.54, RT 2.97 mins.

3.2.3.3. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(3-fluorophenyl)acetamide (P3): Yield 0.85 g

(75%); M.P. 250.4 °C (Charred); buff color solid; IR (cm^-1): 3306, 1739, 1685, 1666, 1602, 1543, 1388, 1325. ¹H-

NMR (400 MHz, DMSO-d₆, δ ppm) 4.52 (s, 2H), 6.916 (t, J= 7.6 Hz, 1H), 7.27-7.29 (m, 1H), 7.35 (q, J= 13 Hz,

13

1H), 7.44-7.53 (m, 2H), 7.89-7.98 (m, 2H), 8.00 (s, 1H), 8.78 (d, J= 3.2 Hz, 1H), 10.63 (s, 1H). C-NMR (400

MHz, DMSO-d₆): 43.372, 105.891, 106.153, 110.118, 110.326, 114.943, 124.283, 125.186, 128.019, 129.542,

130.519, 130.612, 137.638, 139.928, 140.039, 149.341, 151.006, 160.897, 163.298, 164.251, 164.334, 165.500,

170.962. UV- Spectrum (10 ppm, λ_max– 331.8 nm). Theoretical mass: 357, LC-MS (m/z, I %): 356.1 [(M-H)⁺,

100%]. HPLC Purity: % Area 97.28, RT 3.34 mins.

3.2.3.4. N-(4-bromo-2-fluorophenyl)-2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)acetamide (P4):

Yield 0.6 g (52%); M.P. charred above 300 °C; light gray color solid; IR (cm^-1): 3306, 1737, 1672, 1597, 1602,

1543, 1386, 1213, 686. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.58 (s, 2H), 7.38 (d, J= 8.4 Hz, 1H), 7.46 (q, J=

6.6 Hz, 1H), 7.60-7.63 (dd, J= 10.6 Hz, 1H), 7.83.-7.86 (m, 1H), 7.87-7.91 (m, 1H), 7.94-7.96 (m, 1H), 7.98 (s,

1H), 8.78 (d, J= 4.4 Hz, 1H), 10.33 (s, 1H). ¹³C-NMR (400 MHz, DMSO-d₆): 43.214, 118.813, 119.041, 124.287,

125.034, 125.204, 127.583, 128.014, 129.517, 137.649, 149.344, 151.009, 164.661, 165.478, 170.938. UV-

Spectrum (10 ppm, λ_max– 331.8 nm). Theoretical mass: 434, LC-MS (m/z, I %): 433.9 [(M-H)⁺, 100%], 435.9

[(M-H)⁺, 90%]. HPLC Purity: % Area 97.72, RT 4.4 mins.

3.2.3.5. N-(2,4-difluorophenyl)-2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)acetamide (P5): Yield

0.95 g (77%); M.P. 284.9 °C (Charred); white color solid; IR (cm^-1): 3273, 1737, 1672, 1552, 1614, 1505, 1384,

1222. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.55 (s, 2H), 7.06 (t, J= 7.8 Hz, 1H), 7.31 (t, J = 8.8 Hz, 1H), 7.45

(t, J= 5.8 Hz, 1H), 7.77-7.80 (q, J= 11 Hz, 1H), 7.87-7.89 (m, 1H), 7.93-7.95 (m, 1H), 7.98 (s, 1H), 8.77 (d, J= 3.6

Hz, 1H), 10.27 (s, 1H). ¹³C-NMR (400 MHz, DMSO-d₆): 43.056, 104.201, 111.350, 124.296, 125.223, 127.983,

129.481, 137.648, 149.334, 150.968, 164.590, 165.493, 170.981. UV- Spectrum (10 ppm, λ_max– 331.8 nm).

Theoretical mass: 375, LC-MS (m/z, I %): 374 [(M-H)⁺, 100%]. HPLC Purity: % Area 95.82, RT 3.31 mins.

3.2.3.6. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(3-(trifluoromethyl)phenyl)acetamide (P6):

Yield 1.2 g (80%); M.P. 244.5 °C (Charred); buff white color solid; IR (cm^-1): 3273, 1741, 1685, 1666, 1616,

1545, 1390, 1214. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.54 (s, 2H), 7.44 (t, J= 8.8 Hz, 2H), 7.58 (t, J= 8 Hz,

1H), 7.72-7.74 (m, 1H), 7.88-7.90 (m, 1H), 7.94-7.96 (m, 1H), 7.99 (s, 1H), 8.03 (s, 1H), 8.78 (d, J= 4 Hz, 1H),

10.76 (s, 1H). ¹³C-NMR (400 MHz, DMSO-d₆): 43.365, 115.260, 120.123, 122.800, 124.305, 125.176, 128.004,

129.560, 130.177, 137.649, 138.993, 149.338, 150.967, 164.631, 165.506, 170.997. UV- Spectrum (10 ppm, λ_max

– 332.4 nm). Theoretical mass: 407, LC-MS (m/z, I %): 407 [(M+)⁺, 100%]. HPLC Purity: % Area 97.59, RT 2.98

mins.

3.2.3.7.

N-(4-chloro-2-(trifluoromethyl)phenyl)-2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-

yl)acetamide (P7): Yield 0.8 g (64%); M.P. charred above 300 °C; cream color solid; IR (cm^-1): 3217, 1743,

1620, 1668, 1608, 1537, 1309, 1282, 779. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.52 (s, 2H), 7.45 (t, J= 5.8 Hz,

1H), 7.49-7.51 (m, 1H), 7.76-7.78 (m, 1H), 7.82 (s, 1H), 7.87-7.89 (m, 1H), 7.93-7.95 (m, 1H), 7.98 (s, 1H), 8.77

(d, J= 3.6 Hz, 1H), 10.18 (s, 1H). ¹³C-NMR (400 MHz, DMSO-d₆): 43.416, 113.673, 117.503, 119.575, 119.948,

121.114, 123.837, 126.396, 131.352, 131.797, 133.114, 133.448, 147.955, 149.093, 164.934, 165.442, 167.703.

UV- Spectrum (10 ppm, λ_max– 332 nm). Theoretical mass: 441, LC-MS (m/z, I %): 439.9 [(M-H)⁺, 100%]. HPLC

Purity: % Area 97.99, RT 3.99 mins.

3.2.3.8. N-(3,4-dichlorophenyl)-2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)acetamide (P8): Yield 1.1

g (84%); M.P. 269.4 °C (Charred); white color solid; IR (cm^-1): 3336, 1737, 1668, 1620, 1585, 1531, 1325, 779.

¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.51 (s, 2H), 7.45 (d, J= 6.4 Hz, 2H), 7.55-7.58 (m, 1H), 7.87-7.89 (m,

2H), 7.93-7.97 (m, 2H), 8.77 (s, 1H), 10.72 (s, 1H). ¹³C-NMR (400 MHz, DMSO-d₆): 43.349, 119.285, 120.487,

124.294, 125.143, 125.316, 127.996, 129.569, 130.791, 131.124, 137.629, 138.280, 149.320, 150.946, 164.534,

165.475, 170.982. UV- Spectrum (10 ppm, λ_max– 332.2 nm). Theoretical mass: 406, LC-MS (m/z, I %): 405.9

[(M-H)⁺, 100%]. HPLC Purity: % Area 98.68, RT 3.44 mins.

3.2.3.9.

N-(2-chloro-5-(trifluoromethyl)phenyl)-2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-

yl)acetamide (P9): Yield 0.5 g (45%); M.P. 282.6 °C (Charred); white color solid; IR (cm^-1): 3306, 1741, 1676,

1618, 1583, 1548, 1327, 1130, 781. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.65 (s, 2H), 7.46 (t, J= 6 Hz, 1H),

7.57 (d, J= 8.4 Hz, 1H), 7.77 (d, J= 8.4 Hz, 1H), 7.88-7.90 (m, 1H), 7.94-7.98 (m, 1H), 7.99(s, 1H), 8.14 (s, 1H),

8.77 (d, J= 4 Hz, 1H), 10.35 (s, 1H). ¹³C-NMR (400 MHz, DMSO-d₆): 43.222, 121.459, 122.823, 124.317,

125.155, 128.009, 129.566, 130.908, 135.101, 137.658, 149.345, 150.957, 165.307, 165.477, 170.974. UV-

Spectrum (10 ppm, λ_max– 331.8 nm). Theoretical mass: 441, LC-MS (m/z, I %): 439.9 [(M-H)⁺, 100%]. HPLC

Purity: % Area 98.70, RT 4.48 mins.

3.2.3.10. N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-(2,4-dioxo-5-(pyridin-2-ylmethylene)

thiazolidin-3-yl)acetamide (P10): Yield 0.93 g (73%); M.P. 254.6 °C (Charred); light yellow color solid; IR (cm^-

¹): 3304, 3052, 1743, 1658, 1618, 1585, 1550, 1300. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 2.10 (s, 3H), 3.04 (s,

3H), 4.46 (s, 2H), 7.33 (d, J= 7.2 Hz, 3H), 7.34-7.51 (m, 3H), 7.86-7.88 (m, 1H), 7.93-7.84 (m, 1H), 7.97 (s, 1H),

8.76 (d, J= 3.2 Hz, 1H), 9.57 (s, 1H). ¹³C-NMR (400 MHz, DMSO-d₆): 11.024, 35.725, 42.835, 106.286, 123.804,

124.246, 125.357, 126.512, 127.948, 129.107, 129.331, 134.759, 137.625, 149.327, 151.012, 152.074, 161.452,

164.916, 165.520, 170.971. UV- Spectrum (10 ppm, λ_max– 331.5 nm). Theoretical mass: 449, LC-MS (m/z, I %):

448.1 [(M-H)⁺, 100%].. HPLC Purity: % Area 97.32, RT 3.01 mins.

3.2.3.11. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(4-fluorophenyl)acetamide (P11): Yield 1 g

(80%); M.P. 274.5 °C (Charred); buff white color solid; IR (cm^-1): 3265, 1737, 1681, 16758, 1616, 1548, 1330,

1298. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.49 (s, 2H), 7.15 (t, J= 7.8 Hz, 2H), 7.45 (s, 1H), 7.56 (s, 2H), 7.88

(d, J= 7.2 Hz, 1H), 7.94 (d, J= 7.2 Hz, 1H), 7.98 (s, 1H), 8.78 (s, 1H), 10.46 (s, 1H). ¹³C-NMR (400 MHz, DMSO-

d₆): 43.249, 115.319, 115.542, 121.033, 121.110, 124.276, 125.230, 127.981, 129.476, 134.648, 137.630, 149.326,

150.981,157.026, 159.413, 163.871, 165.530, 171.008. UV- Spectrum (10 ppm, λ_max– 332 nm). Theoretical mass:

357, LC-MS (m/z, I %): 356 [(M-H)⁺, 100%]. HPLC Purity: % Area 95.74, RT 3.14 mins.

3.2.3.12. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(p-tolyl)acetamide (P12): Yield 1.3 g (90%);

M.P. 287.3 °C (Charred); white color solid; IR (cm^-1): 3250, 3047, 1737, 1681, 1651, 1614, 1548, 1323. ¹H-NMR

(400 MHz, DMSO-d₆, δ ppm) 2.25 (s, 3H), 4.48 (s, 2H), 7.13 (d, J= 8.32 Hz, 2H), 7.42-7.48 (m, 3H), 7.91-7.93

(m, 1H), 7.95-7.99 (m, 1H), 8.01 (s, 1H), 8.79 (d, J= 4.96 Hz, 1H), 10.28 (s, 1H). ¹³C-NMR (400 MHz, DMSO-

d₆): 43.803, 113.675, 117.543, 119.165, 119.565, 119.927, 129.214, 132.680, 135.822, 147.948, 149.116, 163.547,

165.083, 167.854. UV- Spectrum (10 ppm, λ_max– 332.3 nm). Theoretical mass: 353, LC-MS (m/z, I %): 351.8

[(M-2H)⁺, 100%]. HPLC Purity: % Area 98.56, RT 4.48 mins.

3.2.3.13. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(2-phenoxyphenyl)acetamide (P13): Yield

0.75 g (80%); M.P. 212.3 °C (Charred); gray color solid; IR (cm^-1): 3265, 1739, 1688, 1668, 1620, 1585, 1107,

1230, 1327. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.53 (s, 2H), 6.68 (s, 1H), 7.03 (d, J= 7.2 Hz, 2H), 7.10-7.18

(m, 3H), 7.39-7.97 (m, 3H), 7.87 (d, J= 6.8 Hz, 1H), 7.97 (s, 3H), 8.77 (s, 1H), 10.05 (s, 1H). ¹³C-NMR (400 MHz,

DMSO-d₆): 43.255, 118.463, 118.660, 123.269, 123.561, 123.638, 124.268, 125.224, 125.288, 127.978, 128.952,

129.442, 129.950, 137.629, 147.401, 149.331, 150.989, 156.428, 164.419, 165.515, 170.963. UV- Spectrum (10

ppm, λ_max– 331.7 nm). Theoretical mass: 431, LC-MS (m/z, I %): 430.1 [(M-H)⁺, 100%]. HPLC Purity: % Area

97.97, RT 3.34 mins.

3.2.3.14. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(4-methoxyphenyl)acetamide (P14): Yield 1.2

g (89%); M.P. 284.8 °C (Charred); buff white color solid; IR (cm^-1): 3252, 1739, 1685, 1658, 1618, 1583, 1107,

1257, 1327. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 3.06 (s, 3H), 4.57 (s, 2H), 7.38 (d, J=8 Hz, 2H), 7.44 (d, J=5.6

Hz, 1H), 7.60 (d, J=10 Hz, 1H), 7.81-7.89 (m, 2H), 7.93-7.98 (m, 2H), 8.77 (d, J=3.2 Hz, 1H), 10.39 (s, 1H). ¹³C-

NMR (400 MHz, DMSO-d₆): 43.179, 118.811, 119.033, 124.309, 125.023, 125.163, 127.550, 128.010, 129.531,

137.657, 149.341, 150.955, 151.978, 164.664, 165.482, 170.982. UV- Spectrum (10 ppm, λ_max– 331.8 nm).

Theoretical mass: 369, LC-MS (m/z, I %): 368.2 [(M-H)⁺, 100%]. HPLC Purity: % Area 97.11, RT 2.9 mins.

3.2.3.15.

N-(4-bromo-2,6-difluorophenyl)-2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)acetamide

(P15): Yield 0.8 g (70%); M.P. charred above 300 °C; buff white color solid; IR (cm^-1): 3250, 1749, 1541, 1678,

1610, 1583, 1384, 1234, 779. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.52 (s, 2H), 7.44 (s, 2H), 7.57 (d, J= 5.6

Hz, 1H), 7.87-7.98 (m, 3H), 8.77 (s, 1H), 10.22 (s, 1H). ¹³C-NMR (400 MHz, DMSO-d₆): 42.668, 116.086,

124.295, 125.343, 127.979, 129.432, 137.665, 149.346, 151.612, 164.704, 165.383, 170.850. UV- Spectrum (10

ppm, λ_max– 331.8 nm). Theoretical mass: 452, LC-MS (m/z, I %): 453.8 [(M-H)⁺, 100%], 452 [(M-H)⁺, 90%].

HPLC Purity: % Area 99.03, RT 2.94 mins.

3.2.3.16. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(4-nitrophenyl)acetamide (P16): Yield 0.55 g

(40%); M.P. 289.4 °C (Charred); cream color solid; IR (cm^-1): 3259, 1741, 1564, 1678, 1614, 1593, 1379, 1317.

¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.57 (s, 2H), 7.46 (t, J= 6 Hz, 1H), 7.79 (d, J= 8.8 Hz, 2H), 7.88-7.90 (m,

1H), 7.95 (m, 1H), 7.99 (s, 1H), 8.23 (d, J= 8.8 Hz, 2H), 8.78 (d, J= 4 Hz,1H), 11.04 (s, 1H). ¹³C-NMR (400 MHz,

DMSO-d₆): 43.500, 119.039, 124.344, 125.040, 125.086, 128.040, 129.649, 137.670, 142.604, 144.318, 149.346,

150.933, 165.019, 165.467, 170.983. UV- Spectrum (10 ppm, λ_max– 332.5 nm). Theoretical mass: 384, LC-MS

(m/z, I %): 383.1 [(M-H)⁺, 100%]. HPLC Purity: % Area 99.16, RT 3.81 mins.

3.2.3.17. N-(3-chloro-4-methylphenyl)-2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)acetamide (P17):

Yield 1 g (83%); M.P. 273.5 °C (Charred); buff white color solid; IR (cm^-1): 3246, 2949, 1737, 1581, 1656, 1614,

1579, 1388, 779. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 2.26 (s, 3H), 4.49 (s, 2H), 7.27-7.34 (q, J= 16.8 Hz, 2H),

7.45 (t, J= 6 Hz, 1H), 7.71 (s, 1H), 7.87-7.89 (m, 1H), 7.93-7.95 (m, 1H), 7.98 (s, 1H), 8.77 (d, J= 4 Hz, 1H),

13

10.52 (s, 1H). C-NMR (400 MHz, DMSO-d₆): 18.863, 43.303, 117.835, 119.192, 124.303, 125.186, 128.004,

129.513, 130.508, 131.320, 133.067, 137.330, 137.651, 149.341, 150.966, 164.114, 165.515, 171.005. UV-

Spectrum (10 ppm, λ_max– 332 nm). Theoretical mass: 387, LC-MS (m/z, I %): 386 [(M-H)⁺, 100%]. HPLC Purity:

% Area 96.36, RT 4.68 mins.

3.2.3.18. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(4-methylisoxazol-3-yl)acetamide (P18):

Yield 0.68 g (50%); M.P. 272.6 °C (Charred); buff white color solid; IR (cm^-1): 3219, 2982, 1743, 1529, 1687,

1616, 1568, 1325. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 2.36 (s, 3H), 4.51 (s, 2H), 6.55 (s, 1H), 7.45 (t, J= 6 Hz,

13

1H), 7.87-7.88 (m, 1H), 7.93-7.95 (m, 1H), 7.97 (s, 1H), 8.77 (d, J= 4.6 Hz, 1H), 11.40 (s, 1H). C-NMR (400

MHz, DMSO-d₆): 12.034, 43.053, 96.126, 124.300, 125.180, 127.993, 129.537, 137.641, 149.325, 150.944,

157.537, 164.506, 165.431, 169.985, 170.963. UV- Spectrum (10 ppm, λ_max– 332 nm). Theoretical mass: 344,

LC-MS (m/z, I %): 343 [(M-H)⁺, 100%]. HPLC Purity: % Area 98.69, RT 2.67 mins.

3.2.3.19. N-(benzo[d]thiazol-2-yl)-2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)acetamide (P19): Yield

1.3 g (89%); M.P. 263.9 °C (Charred); white color solid; IR (cm^-1): 3227, 1735, 1602, 1666, 1614, 1583, 1383.

¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.68 (s, 2H), 7.32 (t, J= 7.4 Hz, 1H), 7.44-7.48 (m, 2H), 7.77 (d, J= 8 Hz,

13

1H), 7.89-7.90 (m, 1H), 7.93-7.98 (m, 2H), 8.00 (s, 1H), 8.78 (d, J= 4.4 Hz, 1H), 11.40 (s, 1H). C-NMR (400

MHz, DMSO-d₆): 30.782, 35.806, 42.934, 120.655, 121.781, 123.893, 124.358, 125.134, 126.309, 128.048,

129.693, 131.336, 137.683, 149.357, 150.933, 162.465, 165.420, 165.666, 170.996. UV- Spectrum (10 ppm, λ_max

– 331.7 nm). Theoretical mass: 396, LC-MS (m/z, I %): 394.9 [(M-2H)⁺, 100%]. HPLC Purity: % Area 97.45, RT

4.5 mins.

3.2.3.20. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(thiazol-2-yl)acetamide (P20): Yield 0.85 g

(68%); M.P. charred above 300 °C; buff white color solid; IR (cm^-1): 3246, 1726, 1602, 1678, 1579, 1473, 1386.

¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.61 (s, 2H), 7.23 (d, J= 3.6 Hz, 1H), 7.45-7.49 (m, 2H), 7.88-7.90 (m,

13

1H), 7.94-7.98 (m, 1H), 7.99 (s, 1H), 8.78 (d, J= 4.4 Hz, 1H), 12.59 (s, 1H). C-NMR (400 MHz, DMSO-d₆):

42.691, 114.045, 124.346, 125.162, 128.034, 129.617, 137.684, 149.362, 150.946, 165.435, 170.984. UV-

Spectrum (10 ppm, λ_max– 332.3 nm). Theoretical mass: 346, LC-MS (m/z, I %): 344.9 [(M-2H)⁺, 100%]. HPLC

Purity: % Area 98.34, RT 2.78 mins.

3.2.3.21. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(5-methylthiazol-2-yl)acetamide (P21): Yield

0.67 g (60%); M.P. charred above 300 °C; buff white color solid; IR (cm^-1): 3338, 2841, 1735, 1500, 1678, 1579,

1465, 1381. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 2.32 (s, 3H), 4.58 (s, 2H), 7.15 (s, 1H), 7.45 (t, J= 6 Hz, 1H),

7.87-7.89 (m, 1H), 7.93-7.95 (m, 1H), 7.98 (s, 1H), 8.77 (d, J= 4.4 Hz, 1H), 12.38 (s, 1H). ¹³C-NMR (400 MHz,

DMSO-d₆): 11.027, 42.659, 124.322, 125.175, 126.887, 128.013, 129.584, 137.660, 149.341, 150.945, 165.427,

170.975. UV- Spectrum (10 ppm, λ_max– 332 nm). Theoretical mass: 360, LC-MS (m/z, I %): 358.9 [(M-2H)⁺,

100%]. HPLC Purity: % Area 97.49, RT 3.73 mins.

3.2.3.22. N-(4-bromophenyl)-2-(2,4-dioxo-5-(pyridin-2-yl methylene)thiazolidin-3-yl)acetamide (P22): Yield 0.9

g (88%); M.P. 279.3 °C (Charred); buff color solid; IR (cm^-1): 3254, 1734, 1548, 1676, 1581, 1410, 1386, 727.

¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 4.50 (s, 2H), 7.43-7.53 (m, 5H), 7.86-7.88 (m, 1H), 7.92-7.94 (m, 1H),

7.97 (s, 1H), 8.76 (d, J= 3.6 Hz, 1H), 10.55 (s, 1H). ¹³C-NMR (400 MHz, DMSO-d₆): 43.337, 115.394, 121.171,

124.280, 125.178, 127.988, 129.518, 131.668, 137.616, 149.319, 150.952, 164.123, 165.507, 171.007. UV-

Spectrum (10 ppm, λ_max– 332.7 nm). Theoretical mass: 416, LC-MS (m/z, I %): 418 [(M+2H)⁺, 100%], 416

[(M+)⁺, 75%]. HPLC Purity: % Area 97.69, RT 4.28 mins.

3.2.3.23. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(pyridin-2-yl)acetamide (P23): Yield 1.1 g

(66%); M.P. 256.3 °C (Charred); buff white color solid; IR (cm^-1): 3265, 1739, 1543, 1678, 1620, 1577, 1386. ¹H-

NMR (400 MHz, DMSO-d₆, δ ppm) 4.57 (s, 2H), 7.13 (s, 1H), 7.45 (s, 1H), 7.78 (s, 1H), 7.87-7.98 (m, 4H), 8.34

(s, 1H), 8.78 (s, 1H), 10.94 (s, 1H). ¹³C-NMR (400 MHz, DMSO-d₆): 43.329, 113.530, 119.898, 124.303, 125.205,

128.002, 129.509, 137.653, 138.417, 148.083, 149.342, 150.972, 151.244, 164.796, 165.519, 171.012. UV-

Spectrum (10 ppm, λ_max– 331.4 nm). Theoretical mass: 340, LC-MS (m/z, I %): 339 [(M-H)⁺, 100%]. HPLC

Purity: % Area 97.76, RT 2.77 mins.

3.2.3.24. 2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)-N-(3-methoxyphenyl)acetamide (P24): Yield

0.92 g (85%); M.P. 249.3 °C (Charred); buff white color solid; IR (cm^-1): 3252, 1741, 1556, 1681, 1604, 1581,

1386. ¹H-NMR (400 MHz, DMSO-d₆, δ ppm) 3.71 (s, 3H), 4.49 (s, 2H), 6.65 (d, J= 7.2 Hz, 1H), 7.07 (d, J= 6.8

13

Hz, 1H), 7.20-7.24 (m, 2H), 7.44 (s, 1H), 7.86-7.97 (m, 3H), 8.77 (s, 1H), 10.42 (s, 1H). C-NMR (400 MHz,

DMSO-d₆): 43.331, 54.953, 104.867, 109.289, 111.396, 124.271, 125.202, 127.980, 129.492, 129.711, 137.617,

139.449, 149.322, 150.965, 159.528, 163.947, 165.532, 171.024. UV- Spectrum (10 ppm, λ_max– 332 nm).

Theoretical mass: 369, LC-MS (m/z, I %): 368 [(M-H)⁺, 100%]. HPLC Purity: % Area 97.25, RT 3.09 mins.

3.2.3.25. N-(3,4-dibromophenyl)-2-(2,4-dioxo-5-(pyridin-2-ylmethylene)thiazolidin-3-yl)acetamide (P25): Yield 1

g (78%); M.P. 272 °C (Charred); buff white color solid; IR (cm^-1): 3340, 1737, 1531, 1666, 1618, 1583, 1381. ¹H-

NMR (400 MHz, DMSO-d₆, δ ppm) 4.51 (s, 2H), 7.44-7.46 (m, 2H), 7.56-7.58 (m, 1H), 7.87-7.90 (m, 2H), 7.93-

7.95 (m, 1H), 7.98 (s, 1H), 8.77 (s, 1H), 10.73 (s, 1H). ¹³C-NMR (400 MHz, DMSO-d₆): 43.344, 119.277, 120.466,

124.313, 125.123, 125.308, 128.012, 129.578, 130.806, 131.120, 137.646, 138.275, 149.332, 150.937, 164.543,

165.477, 170.991. UV- Spectrum (10 ppm, λ_max– 331.9 nm). Theoretical mass: 494, LC-MS (m/z, I %): 496.9

[(M+2H)⁺, 100%],496 [(M+2H)⁺, 70%]. HPLC Purity: % Area 95.73, RT 5.61 mins.

3.3. Biological Evaluation

3.3.1. In-vitro cytotoxicity assays

The Differential Nuclear Staining (DNS) assay is a live cell imaging-based assay, which utilizes two nuclear dyes

to easily label living and dead cells [38]. An initial assessment of cytotoxicity on this series of 25 compounds was

conducted via the DNS assay upon exposure of CEM, K-562 and KCL-22 human cancer cell lines to 50 μM of

compounds for 48 h. Compounds were dissolved and diluted in Dimethyl sulfoxide (DMSO) to reach the desired

concentration. DMSO, Hydrogen peroxide, and untreated cells were utilized as vehicle, positive, and negative

controls, respectively, in all experiments. Cells were seeded in a 96-well plate at a density of 10,000 cells/well in

100 μL of complete culture media. Images of stained cells were collected with the GE Healthcare Life Sciences

IN Cell Analyzer 2000. Two h prior to imaging, Hoechst and Propidium iodide (PI) stains were added to each

well to distinguish between living and dead/dying cells. Hoechst is a dye able to permeate the membranes of all

cells within a sample, whereas PI only penetrates into cells with compromised membranes. Colocalization of

Hoechst (blue) and PI (red) signals indicate the dead cell population. In this initial screening, experimental samples

were assessed singularly, and control samples in quadruplicate. Subsequent DNS assays on the CEM, K-562,

KCL-22 cell lines, were then performed to determine the CC₅₀values of the compounds that showed cytotoxicity

in the initial screening and activity in other biological analyses. Cells were seeded in an identical manner but were

treated with a concentration gradient of compound (P8, P9, P10, P12, P19, and P25) ranging from 0.5 μM to 100

μM. Experimental samples and controls were evaluated for cytotoxicity in triplicate after 48 h of incubation.

3.3.1.1. Statistical analysis

For each sample group, the average of replicates and their corresponding standard deviations are displayed.

Statistical significance was determined by two-tailed Student’s t-test comparing experimental treatments to vehicle

controls (DMSO). P values < 0.05 were deemed significant and graphically displayed using asterisks (*=P<0.05,

**=P<0.01, ***=P<0.001).

3.3.2. HDAC enzyme activity assay

Recombinant HDAC4 and 8 were produced as in [58]. The other HDAC isoenzymes were purchased from BPS

Bioscience. Serial dilutions of the inhibitor in the assay buffer (25 mM Tris-HCl, pH 8.0, 75 mM KCl, 0.001 %

Pluronic F-127) were incubated with HDAC in a black 96-well microtiter half-area plate (Greiner) for 60 min at

30 °C. Subsequently the reaction was started by the addition of 20 μM Boc-Lys(trifluoroacetyl)-AMC (Bachem)

as a substrate for HDAC4, 5 and 8, and 50 μM Boc-Lys(acetyl)-AMC as substrate for HDAC1, 2, 3, and 6. After

incubation for 60 min at 30 °C, the reaction was stopped by the addition of 1.7 μM SATFMK for HDAC4, 5, and

8, and 4.17 µM suberoylanilide hydroxamic acid (SAHA, Cayman Chemical Company) for HDAC1, 2, 3, and 6.

The deacetylated substrate was transformed into a fluorescent product by the addition of 0.4 mg/ml trypsin

(Applichem). The release of AMC was followed in a microplate reader (PheraStar Plus, BMG Labtech) at 450 nm

(λEx = 350 nm) and correlated to enzyme activity. Dose-response curves were produced with GraphPad Prism and

fitted to a four parameters logistic function to obtain IC₅₀values [59]

(퐸_푚푎푥― 퐸₀)

퐸퐴 = 퐸₀+

)

(

)

log 퐼퐶₅₀― 푥 ∗ ℎ

1 + 10

Where,

EA is the enzyme activity at a given inhibitor concentration x; E_maxand E₀are the enzyme activities determined at

zero and complete inhibition, respectively; IC₅₀represents the inhibitor concentration at which half the enzyme

molecules are inhibited; h is the slope of the curve.

3.3.3. GLUT 1, 4 and 5 transport inhibition assay

Synthesized compounds were examined for their effect on the transport activity of GLUT1 [48], GLUT4 [48], and

GLUT5 [49] expressed in hexose transporter null yeast cells (hxt⁰). Yeast cell culturing was done at 30 ºC with

shaking (180 rpm). VW4000fgy1 yeast cells expressing GLUT1 [48] were cultured for 2-3 days in the synthetic

complete media without uracil (SC-uracil) with 2% (w/v) maltose. Cells were washed once in SC-uracil, 2% (w/v)

glucose media, transferred in the same media so that OD_600nm~0.5, and grown further for 1-2 days.

VW4000fgy1erg4 yeast cells expressing GLUT4 [48] were cultured like GLUT1, but in media with lower

concentrations of maltose and glucose: SC-uracil, 1% (w/v) maltose media for the initial cell culture, and SC-

uracil, 0.2% (w/v) glucose media for the final cell culture. VW4000 yeast cells expressing GLUT5 [49] were

cultured for one day in YEP [1% (w/v) yeast extract and 2% (w/v) peptone], 2% (w/v) maltose media, 100 µg/ml

geneticin G418. Cells were washed once in YEP, 2% (w/v) fructose media transferred in the same media so that

initial OD_600nm~0.5, and grown further for 1-2 days. For transport assay, cells in the hexose media were centrifuged

(1000 x g, 5 minutes), washed once in PBS buffer (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 2.7 mM KCl, 137 mM

NaCl, pH 7.4), then resuspended in PBS buffer at an OD_600nm~ 10; each assay contained 100 µl of this cell solution.

The transport assay was started by the addition of C¹⁴-hexose to a final concentration of 5 mM glucose for GLUT1

or GLUT4, and 10 mM fructose for GLUT5. Transport assay was stopped after 10 minutes by the addition of 3-

ml ice-chilled Quench buffer (0.1 M KPi, 0.1 M LiCl, pH 5.5) to the assay, followed by filtration under vacuum

on a glass fiber filter (GC50; Advantec, Tokyo, Japan), another wash with 3-ml Quench buffer, and one more

filtration. The filtration membranes containing the cells were transferred to scintillation vials, combined with 10

ml of Scintillation buffer (BioSafeII; Research Products International, Mount Prospect, IL, USA), and, after brief

vortexing, radioactivity was measured with a scintillation counter (Tri-carb 2900TR, Perkin Elmer, USA). As all

synthesized compounds were solubilized in DMSO, controls for calculating the relative activity included 1% (v/v)

DMSO -to account for DMSO concentration in the transport assay due to inhibitor addition- and saturating

concentrations of known inhibitors for GLUTs [200 µM phloretin for GLUT1 and GLUT4 [60], and 100 µM N-

[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA) for GLUT5 [61]. Initial inhibition

screening was done at 100 µM concentration; for the compounds that decreased the relative activity by 50% or

more, inhibitor concentration was varied in the transport assay from 0.1 to 100 µM to determine inhibitor IC₅₀.

Data were analyzed with GraphPad Prism (San Diego, CA, USA).

3.3.4. Apoptosis studies by flowcytometry

The initiation of apoptosis by compounds P10 and P19 was studied by flow cytometry. Cells were seeded in a 24-

well flat-bottom micro-plate and incubated overnight at 37 °C in a CO₂incubator for 24 h. The media was replaced

with fresh media, and the cells were incubated with IC₅₀concentration of P10 and P19 for 24 h. Untreated cells

were the negative control. Post incubation, cells were washed with PBS, centrifuged for 5 min at 500 x g at 4 °C,

and the supernatant was discarded. Cell pellets were re-suspended in ice-cold 1X Binding Buffer, to which 1 µL

of annexin V-FITC solution and 5 µL PI (propidium iodide). Tubes were kept in ice and incubated for 15 min in

the dark, 400 µL of ice-cold 1X binding buffer was added. Cell preparations were analyzed by flow cytometry

(BD Accuri^TMC5 flow cytometer, BD Biosciences, CA, USA). Cytometry data were analyzed by FlowJo software

(version 10.1, Ashland, OR, USA) [62].

3.3.5. Cell viability on CEM cell line and normal WBCs by MTT assay

3.3.6.1. WBCs isolation procedure

A volume of 2.5 mL HiSep was transferred to a 15 mL clean centrifuge tube and overlayed with 7.5 ml diluted

blood, followed by centrifugation at 400 x g at room temperature for 30 minutes. The supernatant containing most

of the plasma and platelet was discarded by aspiration. The mononuclear cells were transferred to a clean centrifuge

tube to which 10 ml of isotonic phosphate buffered saline was added and mixed by gentle aspiration. This was

followed by centrifugation at 200x g, at room temperature for 10 minutes. The cells were washed again with

isotonic phosphate buffered saline and re-suspended in an RBC lysis for 5 mins. The tube was then centrifuged at

300x g for 5 mins, and the supernatant was discarded. Cells were washed with PBS twice and again centrifuged at

300x g for 5 mins. The cells were re-suspended in RPMI 1640 medium, and maintained in a CO₂incubator at 37

ºC (95% humidity and 5% CO₂) until completion of MTT assay experiments.

3.3.6.2. Cytotoxicity determination by MTT assay

The effect of compound P19 on the viability of CEM cell lines and normal human WBCs were determined by the

MTT assay. MTT is a colorimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-

diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The reduction of MTT can only

occur in metabolically active cells. The level of P19 activity was determined by measuring cell viability. The cells

were seeded at a density of approximately 5×10³cells/well in a 96-well flat-bottom micro plate, and maintained at

37 ºC in 95% humidity and 5% CO₂overnight. The cells were incubated with different concentrations of P19 (100,

75, 50, 25, 10, and 2.5 μM) for another 48 h. The cells in each well were washed twice with phosphate buffer

solution, and 20 µL of the MTT staining solution (5 mg/ml in phosphate buffer solution) was added to each well,

and the plate was incubated at 37 ºC. After 4 h, 100 µL of dimethyl sulfoxide (DMSO) was added to each well to

dissolve the formazan crystals, and the absorbance at 570 nm was recorded with a micro-plate reader [63]. Graph

Pad Prism Version 5.1 was used to calculate the CC₅₀. The DMSO concentration used for the experiments was less

1.5%. All the concentrations were used in duplicates.

The formula used for determining the cell viability:

Surviving cells (%) = [Mean OD of cells treated with the test compound/Mean OD of Negative control]×100

4. Conclusion

We synthesized, purified, and characterized the structures of all 25 compounds through various methods (FTIR,

¹H-NMR, ¹³C-NMR, and mass spectrometry). Notably, these compounds do not contain a hydroxamate or

trifluoromethylketone zinc-binding group typical for canonical HDAC inhibitors used in clinical therapy, trials or

academic research. Two compounds, P10 and P19 showed moderate activity against HDAC8 and a remarkable

selectivity against other HDAC isoenzymes. Docking analysis of P19 with best activity (IC₅₀of 9.3 μM) and

docking score against HDAC8 revealed significant binding interactions with the catalytic zinc ion and amino acid

residues such as Y306, and G151 in the active site pocket of HDAC8 as well as partial occupation of the specific

acetate release channel of HDAC8. This finding is in agreement with the observed experimental isoenzyme

selectivity. However, the proposed binding mode has to be confirmed by detailed crystallographic studies. Most

interestingly, six compounds (P8, P9, P10, P12, P19, and P25) showed cytotoxicity against leukemic cell lines

(K562, CEM, and KCL22) in mid-micromolar range. Moreover, the best active compound P19 was less cytotoxic

for non-cancerous cells (WBCs IC₅₀– 104.2 μM and HS27 IC₅₀– 105.0 μM). P10 and P19 exhibited apoptotic

death in CEM cells. As HDAC inhibitors can affect the expression of GLUT1, we also checked the effect of the

compounds on GLUT1, and two other GLUT isoforms GLUT4, and GLUT5. We found that among all 25

compounds only P19 significantly inhibited GLUT1 (IC₅₀28.2 μM), and it did so without affecting GLUT4 or

GLUT5. Thus, P19 is a promising anti-cancer agent, with selectivity and potency against two cancer targets -

HDAC8 and GLUT1, offering a starting point for the development of novel non-hydroxamate compounds with

enhanced HDAC8 inhibitory potency.

Acknowledgments.

This research work was supported by the grant, “Indo-Poland Joint Research Programme” from the Department of

Science and Technology (DST), Government of India. Project Reference Number DST/INT/Pol/P-27/2016.

In addition, the cytotoxicity work was supported by NIMHD grant 5U54MD007592 to the Border Biomedical

Research Center (BBRC) at UTEP.

Abbreviations.

TZDs – Thiazolidinediones; HDAC – Histone Deacetylase; GLUT – Glucose Transporter; HATs – Histone

acetylases; AML – Acute myeloid leukemia; ALL – acute lymphocytic leukemia; APL – Acute promyelocytic

leukaemia; ZBG – Zinc Binding Group; RMSD – Root Mean Square Deviation; TPSA – Topological Polar Surface

Area; AcOH – Acetic acid; EtOH – Ethanol; DCM – Dichloromethane; K₂CO₃– Potassium carbonate; ¹H-NMR

– Proton Nuclear Magnetic Resonance; Hz – Hertz; J – Coupling Constant; ¹³C-NMR – Carbon Nuclear Magnetic

Resonance; FTIR – Fourier-transform infrared spectroscopy; UV – Ultraviolet Spectroscopy; HPLC – High

Performance Liquid Chromatography; M.P. – Melting Point; DNS – Differential Nuclear Staining; DMSO –

Dimethyl sulfoxide; TSA – Thermal Shift Assay; MTT – 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium

bromide.

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Discovery of novel N-substituted thiazolidinediones (TZDs) as HDAC8 inhibitors: in-silico studies, synthesis, and biological evaluation

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