Distinctive inhibition of alkaline phosphatase isozymes by thiazol-2-ylidene-benzamide derivatives: Functional insights into their anticancer role

Article

Distinctive Inhibition of Alkaline Phosphatase Isozymes by thiazol-2-ylidene-

†

Benzamide Derivatives: Functional insights into their Anticancer Role

Syeda Abida Ejaz,¹Aamer Saeed,^*2Syed Jawad Ali Shah,¹Abdul Hameed,³Joanna Lecka,^4,5

Jean Sévigny,^4,5Jamshed Iqbal^*1

¹Centre for Advanced Drug Research, COMSATS Institute of Information Technology,

Abbottabad, Pakistan

²Department of Chemistry, Quaid-I-Azam University, 45320, Islamabad, Pakistan

3

H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological

Sciences, University of Karachi, Karachi-75270, Pakistan

⁴Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université

Laval, Québec, QC, Canada

⁵Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, Canada

^*corresponding authors; Centre for Advanced Drug Research, COMSATS Institute of

Information Technology, Abbottabad, Postal Code 22060, Pakistan. Tel: +92-992-383591-96

Fax: +92-992-383441. e-mail: drjamshed@ciit.net.pk / jamshediqb@googlemail.com

^†This article has been accepted for publication and undergone full peer review but has not been through

the copyediting, typesetting, pagination and proofreading process, which may lead to differences between

this version and the Version of Record. Please cite this article as doi: [10.1002/jcb.26692]

Additional Supporting Information may be found in the online version of this article.

Received 29 September 2017; Revised 29 December 2017; Accepted 22 January 2018

Journal of Cellular Biochemistry

DOI 10.1002/jcb.26692

Abstract

In the recent years, the role of alkaline phosphatase (AP) isozymes in the cause of neoplastic

diseases such as breast, liver, renal and bone cancer has been confirmed and, thus they represent

a novel target for the discovery of anticancer drugs. In this study different derivatives of thiazol-

2-ylidene-benzamide were evaluated for their potential to inhibit alkaline phosphatase (AP)

isozymes. Their anticancer potential was assessed using human breast cancer (MCF–7), bone-

marrow cancer (K–562) and cervical cancer (HeLa) cell lines in comparison to normal cells from

baby hamster kidney BHK–21. The results suggested that in comparison to other derivatives,

compounds 2i, 2e and 2a showed more sensitivity towards human tissue non-specific alkaline

phosphatase (h-TNAP). Among these, 2′′-chloro-N-(3-(4′-fluorophenyl)-4-methylthiazol-2(3H)-

ylidene) benzamide (2e) was found as the most potent and selective inhibitor for h-TNAP with

an IC₅₀value of 0.079 ± 0.002 µM. Moreover, a significant correlation was observed between

the enzyme inhibition profile and cytotoxic data. The compounds exhibiting maximum

anticancer

potential also induced maximum

apoptosis

in

the

respective

cell

lines. Furthermore, the DNA interaction studies exhibited the non–covalent mode of interaction

with the herring sperm–DNA. Molecular docking studies also supported the in vitro inhibitory

activity of potent compounds. Our findings suggested that potent and selective inhibitors might

be useful candidates for the treatment or prevention of those diseases associated with the higher

level of AP. Moreover, the study can be useful for the researcher to explore more molecular

mechanisms of such derivatives and their analogues with the exact findings. This article is

Keywords: ALKALINE PHOSPHATASE; HUMAN BREAST CANCER CELL LINE (MCF–7); CERVICAL

CANCER CELL LINE (HeLa); BONE-MARROW CANCER CELL LINE (K–562); MOLECULAR DOCKING;

THIAZOL-2-YLIDENE-BENZAMIDE

1. INTRODUCTION

Among different ecto-nucleotidases; alkaline phosphatase (AP; EC 3.1.\3.1) contributes as an

important family which is responsible to catalyze various transphosphorylation reactions to keep

the balance of phosphate inside and outside the cell (De prada et al., 1996; Zappa et al., 2001;

Ghosh and Sil, 2007). Alkaline phosphatase contains three metal ions, one Mg²⁺and two Zn²⁺

within its active site which are mainly responsible for its catalytic activity (Kim and Wyckoff,

1991). There is only one alkaline phosphatase found in Escherichia coli, but four types of

alkaline phosphatase isozymes have been isolated from human. In humans, out of the four

isozymes, three are considered as tissue-specific (placental, intestinal and germ cells) while the

fourth one is tissue non-specific which is present throughout the body with its maximum

expression in liver, kidney and bone. The tissue specific isozyme shares 80-90% similarities

within the group while TNAP exhibits 50% structural similarities to the other three isozymes

(Weiss et al., 1986; Millan and Manes, 1988; Millan, 2006). In bone tissues, the major function

of TNAP is the hydrolysis of inorganic pyrophosphate (PPi), an inhibitor of mineralization, to

inorganic phosphate for the normal bone growth (Hessle et al., 2002; Johnson et al., 2003).

TNAP gene is present on chromosome 1p34–36 (Greenberg et al., 1990) and any mutation in the

gene resulted in hypophosphatasia; a hereditary disorder which is characterized by defective

bone mineralization (Whyte, 1994).

Among the tissue specific type; intestinal alkaline phosphatase (IAP) is present on the epithelial

linings of the intestine where its functions have been described as a lipopolysaccharide (LPS)-

detoxifying enzyme (Poelstra et al., 1997). High level of IAPs is associated with various

multifactorial disorders such as atherosclerosis, rheumatoid arthritis (RA), sepsis, inflammatory

bowel disease (IBD) and multiple sclerosis (MS) (Vincent et al., 1992; Petal et al., 1995; Bentala

et al., 2002).

Among different isozymes, liver and bone APs play an important role in normal physiological

functions in human body and their serum level have been applied extensively in routine

diagnosis. Normal range of AP is 20 to 140 U/L (levels are signiﬁcantly higher in children and

pregnant women) while its elevated level is associated with different disease conditions

especially in cancer (Sharma et al., 2014). The elevated level of AP in the cancer patients is the

clear indication that the disease has spread to the patient liver or bone. The malignancies of the

thyroid, breast, lung, colon, prostate and other organs can metastasize to liver or bone (Anderson

et al., 2004). It is worth noting that the primary cancers in various organs can cause the elevation

of AP even without metastasis. Such types of tumors normally elevates specific forms of AP

isozymes. Higher APs level have been reported in different cancer patients (Schepeler et al.,

2008). IAP is highly expressed in hepatocellular carcinoma (Lalles, 2014) while increased level

of TNAP in plasma may correlates with breast cancer, osteosarcomas, Paget’s disease and

osteoblastic bone metastases. Moreover, the metabolic role of these APs in the cancer is

associated with the abrupt hydrolysis of extracellular nucleotides into nucleosides and other

associated molecules which altered the normal functioning of purinergic signaling pathways.

These pathways are responsible for many cellular functions such as cell adhesion, cell

proliferation and cell growth (Lazarowski et al., 2000; Zimmermann, 2000). So many

pathological conditions can be attributed to higher level of APs as discussed above.

Due to the high structural similarity between the two isozymes; IAP and TNAP, a very few

selective inhibitors of APs have been described. Histamine, L-phenylalanine, theophylline and

imidazole are uncompetitive inhibitors of alkaline phosphatase while phenylphosphonate,

phosphoethanolamine and phosphate inhibit alkaline phosphatase competitively (Gosh and

Fishmen, 1966; Farley et al., 1987; Hoylaerts et al., 1992). Previously we have reported few

synthetic polyoxometalates (Raza et al., 2012) and few isolated compound from the leaves of

Olea ferruginea Royle (Hashmi et al., 2015) plant as the potential alkaline phosphatase inhibitors

with anticancer effects but their mechanism was not fully explored. Consequently, it has

compelled us to design new small molecules as inhibitors of APs having the ability to affect or

revoke these pathological conditions. Here in the present study, we were interested to identify

more effective and selective inhibitors of alkaline phosphatase with strong anticancer profile

against those cell lines which have high expression of both APs. For this, we evaluated a series

of small molecules as h-TNAP and h-IAP inhibitors.

Their structure activity and structure selectivity relationships have been proposed for both h-

TNAP and h-IAP. In comparison to h-IAP, these derivatives were found more sensitive and

selective towards h-TNAP. Keeping in mind the role of h-TNAP in various cancers, the

anticancer potential of these derivatives was also evaluated using three different cancer cell lines

i.e., MCF–7, K–562 and HeLa in comparison to normal cell line i.e., BHK–21. The potent and

selective inhibitors obtained might be useful candidates for the treatment or prevention of some

diseases.

2. MATERIALS AND METHODS

2.1. CHEMISTRY

Synthetic protocol of all the compounds has been reported in our previous publication (Saeed et

al., 2011) (See supplementary information for detail).

2.2. BIOCHEMICAL ASSAYS:

2.2.1. CELL TRANSFECTION WITH HUMAN APs

Lipofectamine was used for transfection of COS-7 cells with plasmids expressing APs: h-TNAP

and h-IAP (Bravo et al., 2014) in 10-cm plates. When cells became confluent, they were allowed

to incubate in DMEM/F-12 media without fetal bovine serum (FBS), containing lipofectamine

reagent (24 µL) and plasmid DNA (6 µg) for 5 h at 37 °C. After incubation, transfection reaction

was stopped by adding same volume of DMEM/F-12 with 20% FBS. Finally cells were

collected after 48–72 h.

2.2.2. PREPARATION OF MEMBRANE FRACTIONS

After harvesting the transfected cells, they were washed with Tris–saline buffer in triplicate at 4

°C and separated from the harvesting buffer (95 mM NaCl, 0.1 mM PMSF, and 45 mM Tris

o

buffer, pH 7.5) by scraping. The cells were allowed to centrifuge at 300×g for 5 min at 4 C for

washing purpose (Kukulski et al., 2005). Then cells were again suspended in the harvesting

buffer comprising aprotinin (10 µg/mL) and sonicated. After 10 min of centrifugation at 300×g

o

at 4 C, the cellular and nuclear debris were removed. Finally glycerol (7.5%) was added to the

supernatant solution and kept at -80 °C until further used. Protein concentration was estimated by

using the Bradford microplate assay (Bradford, 1976), considering the bovine serum albumin as

a standard protein.

2.2.3. ALKALINE PHOSPHATASE INHIBITION ASSAY (h-TNAP AND h-IAP)

The assay was carried out according to the previously reported method (Sergienko and Millan,

2010). According to this, the inhibitory activity was measured by using diethanolamine (DEA)

buffer (pH 9.8), comprises of 0.05 mM ZnCl₂and 2.5 mM MgCl_2.The total assay volume of 50

µL consisted of 10 µL compound (20 µM final concentrations) and 20 µL of h-TNAP (46

ng/well) or h-IAP (57 ng/well). After incubation of 10 min at 37^oC, the luminescence signals

were measured using microplate reader (BioTek FLx800^TM, Instruments, Inc. USA). Then CDP-

star (20 µL, final concentration of 110 µM) was mixed to start the enzymatic reaction. After 10

min of incubation, the change in the luminescence was observed. The compounds exhibiting

≥50% inhibition of enzymes activity were further selected for their IC₅₀values. All experiments

were performed in triplicate and non-linear regression analysis of program PRISM 5.0

(GraphPad, San Diego, California, USA) was used for the interpretation of results.

2.2.4. COMPUTATIONAL STUDIES

Docking Studies on Alkaline Phosphatase Inhibitors: The putative binding of the most potent

compounds (2d) and (2e) was determined by using FlexX utility of LeadIT v2.1.8 from

BioSolveIT GmbH, Germany (LeadIT, 2014). Prior to molecular docking, chemical structures of

the compounds were drawn and 3D structures were optimized using ACD/ChemSketch

(ACD/ChemSketch, 2015). Homology models of h-TNAP and h-IAP (Khan et al., 2015) were

used as enzyme templates for molecular docking of the compounds. Using Dock Prep utility of

Chimera v1.10 (Pettersen et al., 2004) the previously modelled protein structures were made

ready for docking studies. The essential steps in preparing modelled proteins included addition of

Gasteiger charges, addition of hydrogen atoms and energy minimization steps. Net charge of +2

was added to the metal ions present in the modelled protein structures. The active site was

determined by selecting the amino acid residues in 7.5 Å radius around the phosphate ion and

zinc metal ions. Chemical structures of compounds which were previously sketched and

optimized were loaded into compound library for docking. For docking calculations, default

docking parameters were used. Top 10 highest ranking poses were generated and further

visualized in Discovery Studio Visualizer v4.0 (Visualizer, 2005).

DNA Docking Protocol: DNA docking study was carried out to investigate the binding

orientation of compound structure within DNA molecule. Molecular operating environment-dock

(MOE-dock) by Chemical Computing Group Inc (MOE version 2014), was used to perform this

study. Before final docking; the binding pockets in the minor and major groves of the DNA were

identified by the known compounds. Moreover the intercalating sites in the DNA were also

identified. The docking protocol was further optimized and the optimized DNA structure was

used for docking purpose. The compound structures were drawn using builder toll of MOE

2014.09. The 3D structures of compounds were energy minimized and optimized through semi-

empirical method PM3 inbuilt in MOE. The X-ray crystallographic DNA structure was obtained

from Protein Data Bank (PDB ID: IBNA) (Drew et al., 1981). The structure of DNA was 3D

protonated with its standard geometry; followed by energy optimization tool using MOPAC 7.0.

Whole DNA structure was selected as binding site to search systematic conformational at default

parameters with RMS gradient of 0.01 kcalmol^-1. Tolat 50 poses per compound were generated

in order to get a final binding position as accurate as possible. The best conformation was

selected based on energetics ground. Final poses with lowest binding free energies were selected

and then imported to Discovery studio [3] for visualization purpose.

2.3. ANTICANCER ASSAYS:

2.3.1. CELL VIABILITY ASSAYS (MTT ASSAY)

The cytotoxic activity of the compounds was investigated against the breast cancer cells i.e.,

MCF-7, cervical cencer i.e., HeLa and bone marrow cells i.e., Lymphoblast cells (k-562). The in-

vitro MTT (Dimethyl–2–thiazolyl–2,5–diphenyl–2H–tetrazolium bromide)–based cell viability

assay was originally described by Mosmann (1983) and later on modified by Niks and Otto

(1990). In this assay, 90 μL of the cell culture containing 2.5 × 10⁴cells/ mL were seeded in 96–

well flat–bottom plates. After that, test compound solution (10 μL, at final concentration of 100

o

µM) was added to the respective wells and plates were incubated for 24 h at 37 C. A blank

control wells was also seeded containing only 100 μL of culture medium without any compound.

Carboplatin was utilized as a standard drug. After incubation, MTT reagent (10 μL) was added to

each well and allowed to incubate for 4 h at 37 ˚C. To stop the enzyme reaction, stopping reagent

(100 µL, 50% isopropanol and 50 mL of 10% sodium dodecyl sulfate) was added. The plates

were incubated under agitation at room temperature for an additional 30 min. Finally change in

absorbance was measured at 570 nm after subtracting the background signal (690 nm), using a

96–well microplate reader (Bio–Tek ELx 800^TM, Instruments, Inc. Winooski, VT, USA). The

activity of mitochondrial dehydrogenases of metabolically active cells resulted in formation of

formazan that represented the number of viable cells. All experiments were performed in

triplicate and results were reported as percent inhibition values with mean of three independent

experiments (± SEM). IC₅₀values of potential inhibitors (≥50%) were determined with the help

of non–linear regression analysis program PRISM 5.0 (GraphPad, San Diego, California, USA).

2.3.2. CELL CYCLE ANALYSIS ASSAY

The cell cycle analysis was performed by using BD accuri flow cytometer accordingly to the

previous reported method (Saito et al., 2010). According to this method the confluent cells were

treated with compounds and allowed to incubate for 24 h. Then cells pellet was formed and

resuspended in 200 µL of PBS solution containing 3% FBS. After that these cells were treated

with propidium iodide (5 µL, 250 µg/mL), 10% Triton X–100 (5 µL) and ribonuclease A (25 µL,

10 mg/mL) and allowed to incubate at room temperature for 1 h in dark environment. The 10,000

events were observed for each sample. The collected data was analyzed by using BD Accuri

software.

2.3.3. MICROSCOPIC ANALYSIS OF APOPTOSIS

The cells were cultured in DMEM High glucose media containing 10% FBS and allowed to

grow until confluent. These confluent cells were seeded in 96 well plate with density of 2 × 10⁵

cells per well. The cells were again incubated in CO₂incubator at 37 ^oC for 24 h. Then cells were

treated with potent compounds at a final concentration of 0.1 mM and incubated again for 24 h

under the same conditions. Then medium was removed and cells were washed with ice cold

PBS. Finally cells were fixed with 4% formalin solution and allowed to become permeable with

0.1% Triton X–100. Then propidium iodide (PI) or 4',6-diamidino-2-phenylindole (DAPI) dye

was used to stain the nuclear material and kept for 10 min. in dark. Fluorescent microscope was

used to observe and capture the image with excitation at 350 nm and emission at 460 nm (Lin et

al., 2013).

2.3.4. DNA INTERACTION STUDIES

The DNA interaction studies were carried out with slight modification of previously reported

method (Sirajuddin et al., 2014). The interaction studies of potent compounds were performed at

the final concentration of 100 µM. The concentration of Herring–Sperm DNA (HS–DNA) was

determined at 260 nm by using micro–plate reader FLUOstar Omega (Germany). The stock

solution of DNA was prepared by dissolving 5 mg /10 mL in deionized water and 4.15 ×10^–4M

concentration was determined by using molar absorption coefficient of 6600 M^–1cm^–1. Then

different concentrations of DNA i.e., 0, 40, 90, 140, 190, 240, 290 and 340 µM were used to

interact with fixed concentrations of compounds. The same amount of DNA was also added in

their respective reference solutions. The plates were incubated for 30 min. at room temperature.

The absorption spectra were measured with blank correction using 96–well plates of 5.5 mm

path length. All the experiments were performed in triplicate. The mode of interaction of

compounds was determined by using Omega– Data Analysis Software, Program Version: 3.00

R3.

3. RESULTS AND DISCUSSION

3.1. CHEMISTRY

Synthetic layout of the desired thiazol-2-ylidene-benzamides 2(a-j) is present in the Scheme-1.

Compounds 2(a-j) were prepared by simply condensing the corresponding thiourea derivatives

with acetone in the presence of bromine and triethyl amine, heated at reflux. Synthetic procedure

has been reported in detail in our previous publication (Saeed et al., 2011).

3''

R²

O

2''

4''

O

B

H

₃C CH₃

5''

1''

acetone

O

S

6''

R²

N

S

, Et

N

H

Br_{2 3}N

6'

R¹

N

1'

H

R¹₄⁵_'^'

heated at reflux

(1)

⁴CH₃

A

2'

Compounds

R₁= 2'-F; R₂= 4''-F (82%)*

3'

2a)

* yield

2(a-j)

= 2'-F; R₂= 2''-Cl (88%)*

= 3'-F; R₂= 2''-Cl (91%)*

R₁= 3'-F; R₂= 4''-F (84%)*

R₁= 4'-F; R₂= 2''-Cl (84%)*

2b) R¹

2c) R¹

2d)

2e)

2f)

2g)

2h)

2i)

R₁= 2'-F; R₂= 3'', 4'', 5''-tri-OMe (84%)*

R₁= 3'-F; R₂= 3'', 4'', 5''-tri-OMe (90%)*

R₁= 4'-F; R₂= 3'', 4'', 5''-tri-OMe (88%)*

R₁= 3'-Cl-4'-F; R₂= 3'', 4'', 5''-tri-OMe (86%)*

R₁= 3'-Cl-4'-F; R₂= 2'' Cl (83%)*

2j)

Scheme-1: Synthesis of thiazol-2-ylidene-benzamide derivatives 2(a-j) (Saeed et al., 2011)

3.2. BIOACTIVITY: IN VITRO HUMAN ALKALINE PHOSPHATASE INHIBITION

AND STRUCTURE ACTIVITY RELATIONSHIP (SAR)

Thiazol-2-ylidene-benzamide derivatives were assayed in vitro as human tissue non-specific

alkaline phosphatase (h-TNAP) and human intestinal alkaline phosphatase (h-IAP) inhibitors.

Levamisole and L-phenylalanine were used as reference inhibitors against h-TNAP and h-IAP

with IC₅₀values of 19.2 ±.001 and 80.2 ± 0.001 µM, respectively. Bioactivity data in Table-1

showed that in the case of h-TNAP inhibition, all the compounds in the series exhibited IC₅₀

values ranging from 0.079 ± 0.002 µM to 0.83 ± 0.07 µM. Compound (2e) 2′′-chloro-N-(3-(4′-

fluorophenyl)-4-methylthiazol-2(3H)-ylidene)benzamide was found to be the most potent

inhibitor of h-TNAP with an IC₅₀value of 0.079 ± 0.002 µM.

Compound (2e) was >240 folds more potent to inhibit h-TNAP when compared with the

reference inhibitor, levamisole. The detailed comprehensive study of the structure of this

compound suggested that the activity of (2e) might be due to the presence of 4′-F substitution on

phenyl ring A. It was further justified when the substitutional position changed from 4′ to

position 3′ and 2′. For example the compound (2c) having 3′-F substituent and compound (2b)

having 2′-F substituent exhibited reduced activity with an IC₅₀value of 0.22 ± 0.01 and 0.43 ±

0.07 µM, respectively. Moreover, difluoro derivative 4′′-fluoro-N-(3-(3′-fluorophenyl)-4-

methylthiazol-2(3H)-ylidene) benzamide (2d) exhibited comparatively higher inhibition with an

IC₅₀value of 0.17 ± 0.02 µM. While in comparison to compound 2d, the activity of other

difluoro substituted compound (2a) reduced to almost half (0.33 ± 0.03 µM). This again might

be due to the change in position of functional group on phenyl rings (R¹= 2′-F) as in the case of

(2a). Thiazol-2-ylidene-benzamide derivatives (2f), (2g), and (2h) have R²= 3′′, 4′′, 5′′-tri-OMe

substituents and R¹= F substituent in variable positions on the phenyl ring A, respectively.

Among these compounds, 4′-fluoro substituted compound (2h) showed the highest activity with

an IC₅₀value of 0.21 ± 0.01 µM. Among trimethoxy derivatives, 2′-fluoro derivative (2f)

exhibited an intermediate IC₅₀value of 0.35 ± 0.01 µM while 3′-fluoro derivative (2g) showed

the lowest IC₅₀value of 0.83 ± 0.07 µM. Further, 4′-fluoro substituted derivatives (2i) with an

addition of 3′-Cl substituents increased the activity with an IC₅₀value of 0.16 ± 0.01 µM.

Substituting 3′′, 4′′, 5′′-trimethoxy groups with 2′′-Cl group (2j) resulted in reduced activity (IC₅₀

value 0.51 ± 0.03 µM).

Screening of thiazol-2-ylidene-benzamide derivatives against h-IAP showed that (2a), (2b), (2g),

(2h) and (2j) exhibited good activities with IC₅₀values ranging between 0.11 ± 0.001 to 0.57 ±

0.11 µM. Compound (2d) was the most potent among the tested series against h-IAP with an

IC₅₀value of 0.11 ± 0.001 µM. It showed ~ 725 folds more activity compared to the standard

inhibitor L-phenylalanine. Compound (2h) having 4-F′ and 3′′, 4′′, 5′′-trimethoxy substituents

showed almost comparable IC₅₀value with slight decline in value (IC₅₀value of 0.19 ± 0.03

µM). Replacement of 4′-F with 3′-F group (2g), reduced the activity to almost twice with IC₅₀

value of 0.35 ± 0.01 µM. Compound (2j) and (2a) with different substituents showed further

decline in the activity against h-IAP with IC₅₀values of 0.44 ± 0.02 and 0.57 ± 0.11 µM,

respectively. The bioactivity results revealed 2′′-chloro-N-(3-(4′-fluorophenyl)-4-methylthiazol-

2(3H)-ylidene) benzamide (2e) as a selective inhibitor of h-TNAP and 4′′-fluoro-N-(3-(3′-

fluorophenyl)-4-methylthiazol-2(3H)-ylidene) benzamide (2d) as dual inhibitors of alkaline

phosphatases (Table -1). Both these compounds (2d and 2e) could serve as potential lead for the

development of novel alkaline phosphatase inhibitors.

F

O

N

Cl

N

S

CH₃

S

CH₃

F

2′′-chloro-N-(3-(4′-fluorophenyl)-4-methyl

thiazol-2(3H)-ylidene)benzamide (2e)

h–TNAP (IC₅₀±SEM =0.079 ± 0.002)

(Z)–4–fluoro–N–(3–(4–fluorophenyl)–4–methyl

thiazol–2(3H)–ylidene)benzamide (2d)

h–IAP (IC₅₀±SEM =0.11 ± 0.001)

3.3. KINETICS STUDY

In order to determine the type of inhibition, the inhibitor (2e) and (2d) were selected for kinetics

study with h-TNAP and h-IAP, respectively. The obtained results showed that compounds (2d)

and (2e) exhibited competitive mode of inhibition against both h-TNAP (fig.1 (a)) and h-IAP by

showing the same y–intercept for uninhibited and inhibited enzyme (Fig.1 (b)). Moreover,

Michaelis-Menten kinetic parameters K_mand V_maxof h-TNAP and h-IALP inhibition were also

determined in the presence and absence of inhibitor 2e and 2d. In both cases, the maximal

velocity (V_max)found almost constant even by using different concentrations of respective

inhibitors, however the Michaelis constant (K_m) was found in increasing order as depicted in

Figures 1a-b.

3.4. COMPUTATIONAL STUDIES

Molecular docking studies revealed important interactions of the most potent compounds with

the enzyme active site. Compound (2e) was the most potent compound of the series against h-

TNAP (figure 2a). Its molecular docking was performed inside h-TNAP active site. The

important interactions identified by the docking studies include hydrogen bonding of catalytic

amino acid Ser93 and amino acid Arg167 with thiazol-2-ylidene ring (green dashed line) of

compound (2e). Moreover, phenyl ring adjacent to the carbonyl group was involved in making

one hydrogen bond with amino acid His324 (green dashed line) and two π– π stacking

interaction with amino acid His321 (pink dashed line). Additionally, amino acids His437 and

Arg167 also formed π-stacked interactions (golden dashed line) with phenyl ring attached with

the thiazol-2-ylidene ring. Oxygen of benzamide ring of compound (2e) formed one hydrogen

bond with His154 (green dashed line).

Binding interactions of potent compound 2d within the active pocket of h-IAP revealed five

different interaction as shown in figure 2 (b). Oxygen atoms of the benzamide ring was involved

in making one hydrogen bond (green dashed line) with amino acid residue of Ser92. Two π

interactions (pink-dashed line) were also formed by carbonyl group of compound 2d with amino

acids His317 and His153. Briefly, phenyl ring adjacent to the thiazol-2-ylidene ring was

involved in making one π-π interaction with amino acid His317 and fluorine atom present on this

phenyl ring was involved in making conventional hydrogen bond with His153 as shown in figure

2 (b).

3.5. ANTICANCER ASSAYS:

3.5.1. CELL VIABILITY BY MTT ASSAY: As illustrated in Table 1, all of the target thiazol–

2–ylidene–benzamide derivatives had some cytotoxicity on 4 different cancer cell lines and

exhibited less toxicity against normal BHK–21 cells. In general, most of the compounds showed

significant selectivity toward MCF–7 cells over K–562 cells. However, it was worth noting that

only compound 2e exhibited maximum selectivity towards K–562 cells with a potency in lower

micro–molar range (Table 2). Moreover, the % growth inhibition results of both cancer and

normal cells demonstrated that the tumor cells were more sensitive than the normal cells i.e.,

BHK–21. Analysis of Table 1 reveals that against MCF–7, the cytotoxicity effect (% inhibition)

of benzamide derivatives 2a–e was higher than those of the other derivatives i.e., 2h, 2i and 2j

(fig. 3). It was concluded from structure activity relationship (SAR) point of view, different

functional groups at R1 and R2 position can affect cytotoxicity of compounds. In case of MCF–7

cells, the compounds having electron–withdrawing halide atoms at both 2, 3, position (R1 and

R2 position) of both rings were found superior (2a–e) to those with electron–donating groups

such as methoxy substituents (2h, 2i and 2j). On the other hand, in case of K–562 cells, inverse

behavior was observed. The activity of compound 2i against K–562 might be due to the presence

of two halogens i.e., Cl and F at 3 and 4 positions, respectively. The most potent derivatives were

further selected for the evaluation of their growth inhibitory concentration values (GI₅₀) and the

results obtained were in lower mico-molar range as given in Table 2.

3.5.2. EFFECT OF POTENT DERIVATIVES ON CELL CYCLE USING DIFFERENT

CELL LINES BY FLOW CYTOMETRY: Cell cycle arrest is the most common mechanism

in anticancer drug treatment which can be estimated by DNA content. In each cell cycle, the G0

and G1 phase represent two set of Chromosomes (2N DNA) which replicate in S–phase and are

represented by 4N DNA in the G2 and M phase. In order to find the effect of most potent

derivatives from different amide series on cell cycle progression and apoptosis, flow cytometric

analysis was carried out using human breast cancer cell line (MCF–7), bone marrow lymphoblast

cell lines (K–562) and human cervical cancer cell line (HeLa). The selected derivatives were

treated at final concentration of 100 µM with respective cell line i.e., MCF–7, k–562 or HeLa.

The results after 24 hr treatment of cells are shown in figure 4. Carboplatin was used as positive

control and also treated at final concentration of 100 μM. Untreated cells, MCF–7, K–562 or

HeLa in the G0/G1, S and G2/M phases were considered 100% (control). The obtained results

suggested that the anti-cancer effect was depended on the structure of the treated derivatives (fig.

4).

The most potent derivative from thiazol–2–ylidene–benzamides i.e., 2i exhibited remarkably

effective cytotoxic effect on the MCF–7 cell line. Compound 2e was found as most sensitive

towards K–562 and against HeLa cells compound 2a was found to be the most potent. Against

MCF–7, compound 2i induced 47.7% apoptotic potential which was comparable with the

positive control used i.e., carboplatin which exhibited approximately 52% apoptotic potential.

The detailed cell cycle analysis of 2i suggested that it interfered and inhibited the mitotic

spindling that ultimately resulted in DNA damage and cell arrest in G2 phase. Therefore on

MCF–7 cells this compound led to accumulation of cells at G2 phase. Against K–562, compound

2e induced comparable apoptosis i.e., 54.3% with respect to positive control, carboplatin

(58.1%). The cell cycle analysis of this derivative suggested that the more cell death occurred in

S phase (approx. 22%), with the overall apoptosis of 54.3%, as compared to other two phases

(Go and G2). Against HeLa, compound 2b induced 22.2% apoptosis that was comparable with

carboplatin which induced approx. 25% apoptosis in HeLa cells. This compound also caused S-

phase apoptotic cell death. It can be suggested these compound intercalated with DNA molecule

and undergone programmed cell death (apoptosis). The effect of 2i, 2e and 2a on different cell

cycle phases in respective cell lines was also analyzed and the percentages of Go– G1, S, and

G2/ M phase cells are presented graphically in Fig. 5.

3.5.3. MICROSCOPIC ANALYSIS OF THE MOST POTENT DERIVATIVES USING

DAPI & PI STAINING:

The results from flow cytometry led to the fluorescent microscopic analysis, to further confirm

the nuclei fragmentation using nuclear staining dyes (DAPI, PI). Three different cancer cells

were cultured and used for microscopic analysis. The compounds 2i, 2e and 2a were treated with

MCF–7, K–562 and HeLa cells, respectively, at final concentration of 100 μM.

After 24 h, the images revealed the characteristic apoptotic changes like higher percentage of

rounded cells, condensed and fragmented nuclei unlike untreated control. The nuclei in the

viable cells have a regular, oval shape with homogeneous chromatin, whereas in the apoptotic

cells, shrinking and fragmentation of the nuclei could be observed. The cells that were treated

with 100 μM carboplatin were considered as apoptosis–positive control. The microscopic images

with DAPI staining (fig. 6) showed bluish intact nuclei in the control and bright fragmented

nuclei in derivatives (2i, 2e and 2a) treated cells. Similarly another dye, PI, is impermeable to

nuclei of living cells because of intact nuclear membrane (Fig. 7). While in dead cells, the

nuclear membrane is compromised that allows the permeability of PI with resultant image of red

nuclei in front of black background. Among different derivatives (2i, 2e and 2a), the most potent

derivative 2e induced maximum apoptosis as indicated by DAPI and PI stained images in their

respective cell lines as compared to carboplatin, positive control. The results were in correlation

with flow cytometric analysis and suggested that there is a significant increase in DNA damage

in each case. While other derivatives exhibited less apoptotic activity.

3.5.4. DNA INTERACTION STUDIES:

The mechanism of action of the cytotoxic derivatives against MCF–7 cells (2i), K–562 cells (2e)

and HeLa cells (2a) was further evaluated by their interaction studies with HS–DNA in UV–

visible range, using Omega FLUOstar, microplate reader. The absorption spectra of all the potent

samples in the absence and presence of increasing concentration of HS–DNA was observed as

depicted in fig. 8. In each case with the increase in HS–DNA concentration, increase in

absorbance with no shift of band positions was observed. Thus the hyperchromic effect indicated

the non–covalent interaction of different derivatives with HS–DNA. Among the potent

derivatives of K–562 compound 2e exhibited maximum growth reduction and it depicted

maximum DNA interactions having Gibbs free energy Δ–17.88 KJ/mol. Compound 2a and 2i

also exhibited maximum interactions with Gibbs free energy Δ–17.48, –17.16 KJ/mol,

respectively (Fig. 8). Analyzing the binding orientation of compounds with DNA revealed that

almost all the potent compounds were docked favorably inside the minor groove of DNA.

4. CONCLUSIONS

In conclusion, these results showed that all compounds of this series inhibited h-TNAP while

only five compounds inhibited the h-IAP. From this series, 2′′-chloro-N-(3-(4′-fluorophenyl)-4-

methylthiazol-2(3H)-ylidene)benzamide (2e) displayed the most potent inhibition of the h-TNAP

(IC₅₀±SEM = 0.11 ± 0.001 µM) while 4′′-fluoro-N-(3-(3′-fluorophenyl)-4-methylthiazol-2(3H)-

ylidene)benzamide (2d) was found as the potent and dual inhibitor of both h-TNAP and h-IAP

having the IC₅₀value of 0.11 ± 0.001 and 0.17 ± 0.02 µM, respectively. The compounds

exhibiting maximum anticancer potential also induced maximum growth reduction in the

respective cell lines. Molecular docking studies were also supported the experimental results.

Therefore it was concluded from the study that the compounds having effective inhibitory values

against enzyme as well as cancer cell lines, provided a great hope to search new anticancer

mechanism for the development of future novel drugs. These derivatives can be further selected

for in–vivo studies in order to explore the effect of these derivatives for pharmaceutical point of

view.

ACKNOWLEDGEMENTS

J. Iqbal is thankful to the Organization for the Prohibition of Chemical Weapons (OPCW), The

Hague, The Netherlands and Higher Education Commission of Pakistan for the financial support

through Project No. 20-3733/NRPU/R&D/ 14/520. JS received support from the Canadian

Institutes of Health Research (CIHR) and was also a recipient of a “Chercheur National”

research award from the Fonds de recherche du Québec – Santé (FRQS).

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TABLE I. Alkaline Phosphatase Inhibitory Effect and Anticancer Potential of Thiazol-2-

ylidene-Benzamide Derivatives 2 (a-j)

Structure

R1, R2

Enzyme Inhibition

h–TNAP

IC₅₀(µM)^a±SEM

Anticancer Activity

K–562 HeLa

Growth reduction in %^b

MCF–7

h–IAP

BHK–21

Codes

62%^b

57%^b

64%^b

9%^b

69%^b

62%^b

56%^b

66%^b

71%^b

64%^b

–––

47%^b

49%^b

66%^b

42%^b

92%^b

53%^b

43%^b

70%^b

24%^b

53%^b

–––

71%^b

66%^b

62%^b

55%^b

66%^b

62%^b

63%^b

64%^b

67%^b

61%^b

–––

15%^b

19%^b

47%^b

25%^b

5%^b

18%^b

8%^b

24%^b

18%^b

4%^b

2'–F, 4''–F

0.33 ± 0.03 ^a

0.57 ± 0.11 ^a

2a

2b

2c

2d

2e

2f

2'–F, 2''–Cl

0.43 ± 0.07 ^a

0.22 ± 0.01 ^a

0.17 ± 0.02 ^a

0.079 ± 0.002^a

0.35 ± 0.01 ^a

0.83 ± 0.07 ^a

0.21 ± 0.01 ^a

0.16 ± 0.01 ^a

0.51 ± 0.03 ^a

>100 ^a

0.11 ± 0.001 ^a

>100 ^a

3'–F, 2''–Cl

3'–F, 4''–F

4'–F, 2''–Cl

2'–F, 3'',4'',5''–tri–OMe

3'–F, 3'',4'',5''–tri–OMe

4'–F, 3'',4'',5''–tri–OMe

3'Cl– 4'–F, 3'',4'',5''–tri–OMe

3'–F, 2''–Cl

>100 ^a

0.35 ± 0.01 ^a

0.19 ± 0.03 ^a

>100 ^a

2g

2h

2i

0.44 ± 0.02 ^a

2j

19.2 ± 0.1 ^a

–––

80.2 ± 1 ^a

–––

Levamisole

–––

L–Phenyl alanine

–––

90%^b

82%^b

85%^b

18%^b

Carboplatin

a

IC₅₀is the concentration at which the compound reduces the enzyme activity by 50%. Alkaline phosphatase (h-

b

TNAP, h-IAP) inhibition assay was performed at the final concentration of 200 µM. The anticancer effect was

measured at the final concentration of 100 μM of tested compounds; SEM = Standard error of mean

TABLE II. Cytotoxic Efficacies of the Most Potent Compounds on Different Cell Lines

MCF–7

K–562

GI₅₀±SEM (μM)

>100

HeLa

Code

13.1 ± 1.77

14.2 ± 1.89

10.2 ± 1.07

3.91 ± 0.32

2a

11.5 ± 0.15

17.1 ± 2.11

16.4 ± 1.78

5.13 ± 0.45

2e

2i

0.86 ± 0.05

>100

Carboplatin

4.11 ± 0.38

GI₅₀denotes compound concentrations that result in a 50% decrease in the cell number compared to non–treated

controls and were derived after 24 h treatment. Results from three independent experiments are presented.

Figure 1

Article Doi

Distinctive inhibition of alkaline phosphatase isozymes by thiazol-2-ylidene-benzamide derivatives: Functional insights into their anticancer role

DOI: 10.1002/jcb.26692

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