Design, synthesis, 3D pharmacophore, QSAR, and docking studies of carboxylic acid derivatives as Histone Deacetylase inhibitors and cytotoxic agents

Accepted Manuscript

Design, synthesis, 3D Pharmacophore, QSAR, and docking studies of carbox-

ylic acid derivatives as cytotoxic agents

Mona M. Abdel-Atty, Nahla A. Farag, Shaymaa E. Kassab, Rabah A.T. Serya,

Khaled A. Abouzid

PII:

S0045-2068(14)00073-X

DOI:

http://dx.doi.org/10.1016/j.bioorg.2014.08.006

YBIOO 1754

Reference:

Bioorganic Chemistry

To appear in:

Received Date:

19 May 2014

Please cite this article as: M.M. Abdel-Atty, N.A. Farag, S.E. Kassab, R.A.T. Serya, K.A. Abouzid, Design,

synthesis, 3D Pharmacophore, QSAR, and docking studies of carboxylic acid derivatives as cytotoxic agents,

Bioorganic Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bioorg.2014.08.006

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Design, synthesis, 3D Pharmacophore, QSAR, and docking studies of carboxylic acid derivatives as

cytotoxic agents

Mona M. Abdel-Atty^a, Nahla A. Farag^a, Shaymaa E.Kassab^b, Rabah A. T. Serya^cand Khaled A.

Abouzid^c,*

^aPharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, Cairo, Egypt 11431

^bPharmaceutical Chemistry Department, Faculty of Pharmacy, Damanhur University, Damanhur, Egypt

^cPharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt 11566

*Corresponding author. Tel: +201222165624; e-mail: abouzid@yahoo.com

Abstract

In this study, five series of (E)-6-(4-substituted phenyl)-4-oxohex-5-enoic acids IIb-f (E) , (E)-3-(4-

(substituted)-phenyl)acrylic acids IIIa-g (E), 4-(4-(substituted)phenylamino)-4-oxobutanoic acids VIa,b,e,

5-(4-(substituted)phenylamino)-5-oxopentanoic

acids

VIIa,f

and

2-[(4-(substituted)phenyl)

carbamoyl]benzoic acids VIIIa,e were designed and synthesized. Selected compounds were screened in

vitro for their cytotoxic effect on 60 human NCI tumor cell lines. Compound IIf (E) displayed significant

inhibitory activity against NCI Non-Small Cell Lung A549/ATCC Cancer cell line (68% inhibition) and

NCI-H460 Cancer cell line (66% inhibition). Moreover, the final compounds were evaluated in vitro for

their cytotoxic activity on HepG2 Cancer cell line in which histone deacetylase (HDAC) is overexpressed.

Compounds IIc (E), IIf (E), IIIb (E), and IIIg (E) exhibited the highest cytotoxic activity against HepG2

human cancer cell lines with IC₅₀ranging from 2.27-10.71 μM. In addition, selected compounds were

tested on histone deacetylase isoforms (HDAC1-11). Molecular docking simulation was also carried out

for HDLP enzyme to investigate their HDAC binding affinity. In addition, generation of 3D-

pharmacophore model and quantitative structure activity relationship (QSAR) models were combined to

explore the structural requirements controlling the observed cytotoxic properties.

Key words:

Synthesis, Histone Deacetylase, 3D QSAR Pharmacophore Model, Cytotoxic activity

1. Introduction

Histone acetylation has been shown to be one of the major regulatory mechanisms for gene expression.

[1] Control of expression is dependent on the balance between the competing activities of histone acetyl

transferases (HATs) and histone deacetylases (HDACs) on the regulation of chromatin structure by

acetylation of lysines on histone tails. [2] Acetylation of the N-terminal tails of lysine residues is done by

HATs which removes positive charges, thereby reducing the affinity between histones and negatively

charged DNA. This facilitates access of the transcriptional machinery to the DNA template. [3]

Conversely, HDACs remove the acetyl group from the histone tails, reversing the effects of HATs and

altering transcription. In general, acetylation is associated with a transcriptionally active state and

deacetylation with gene silencing. [4, 5] Studies demonstrated that these two enzymes are not only

involved in the regulation of chromatin structure and gene expression, but they can also regulate cell-

cycle progression and carcinogenic process. [6] HDACs therefore play a pivotal role in the regulation of

gene expression, cell growth and proliferation. [7] Over expression of HDACs has been linked to the

development of cancers in human. [8] Eighteen mammalian HDACs have been identified and categorized

into four structural and functionally distinct classes. [9] Class I (HDAC1–3 and 8), class II (HDAC4–7, 9

and 10) and class IV (HDAC11). HDACs share conserved residues in the catalytic core regions and

require zinc ion for deacetylation, while class III (SIRT1–7) HDACs are unrelated sirtuin deacetylases

and require NAD⁺for their enzymatic activity. [9, 10]

HDAC inhibitors present a promising class of anticancer agents. [11, 12] HDAC inhibition induces

transcriptional events involved in growth arrest, cell proliferation, and apoptosis. [13] There are numbers

of HDACIs emerging as an exciting novel class of antitumor agents as shown in (Fig. 1), Trichostatin A

(TSA) is an effective natural hydoxamate HDACI and Suberoylanilide hydroxamic acid (Vorinostat,

SAHA) is the first HDACI approved by FDA in 2006. [14-16] Belinostat and Panobinostat are also

hydroxamate HDACIs which induce acetylation of histone at nanomolar concentrations. [17]

Mocetinostat and Entinostat are benzamide HDACIs with limited cardiac toxicity in preclinical studies.

[18]

During the past 15 years, a number of structurally diverse HDAC inhibitors have been identified

which can be divided into structural classes including hydroxamates, cyclic peptides, benzamides,

electrophilic ketones and carboxylic acids.[19, 20] Despite their different chemical structures, these

agents have a common pharmacophore composed of three portions: (a) zinc binding group (ZBG), which

chelates zinc ion at the bottom of pocket, (b) linker (scaffold), usually hydrophobic which occupies the

narrow channel, (c) surface recognition moiety (SRM), which interacts with hydrophobic interactions

with residues on the rim of active site, (Fig. 1). [21, 22] The common linkers are aliphatic chain, aromatic

chain and vinyl-aromatic chain. [21] Any HDACI with its strong ZBG can substitute the native substrate

and preserve the ‘His-Asp’ charge transfer system within the enzyme complex. [23] This ‘three-

component’ structural template has been widely realized as a successful strategy for the design of

efficient new HDACI's. [24-26]

Although Hydroxamic acid derivatives from the class of HDAC inhibitors is effective in nanomolar

dose (TSA IC50: 12 nM and SAHA IC50: 110–370 nM), [27, 28] it has problems such as poor

pharmacokinetic properties, severe toxicities and non-selectivity.[29] The carboxylic acid class is the least

investigated inhibitor group probably due to poor HDAC inhibition activity.[19, 22] The carboxylic acid

group is thought to be a metal-binding functional group. [28] A limited number of compounds such as

VPA (valproic acid), BA (butyric acid) and PBA (phenyl butyric acid) are identified as carboxylic acid

class HDAC inhibitors, (Fig. 2). [30] All these inhibitors are in phase trials with high micromolar doses,

where they have anti-proliferative, anti-cancer and anti-convulsant effects, and their toxicity studies have

been completed. [31, 32] They are used for treatment of cancer and also neurodegenerative diseases such

as spinal muscular atrophy (SMA). [33] Most carboxylic acids reported to date have simple alkyl chains

and the structure activity relationship studies for this group have been very limited. Therefore, advances

are needed in designing new carboxylic acid derivatives with improved HDAC inhibition activity.

Linker

SRM

ZBG

H

O

S

O

H

N

OH

N

O

H

O

H₃C

N

CH₃

Trichostatin A (TSA)

Vorinistat (SAHA)

Belinostat (PXD101)

O

N

O

N

OH

H

N

H

N

O

N

NH₂

N

NH₂

HN

O

N

H

Entinostat (MS275)

Panabinostat (LBH589)

Mocetinostat (MGCD0103)

Figure 1. Examples of histone deacetylase inhibitors as antitumor agents and the three essential pharmacophore features shown

in color codes.

O

OH

O

Valproic acid

Phenylbutyric acid

Butyric acid

Figure 2. Examples of carboxylic acids based histone deacetylase inhibitors.

2. Rational and design

The design of the newly synthesized compounds was based on earlier study of carboxylic acid as zinc

binding functional group in histone deacetylase metallo-enzymes, [28] directing the design towards the

synthesis of carboxylic acid derivatives as HDAC inhibitors in an attempt to increase their potency by

manipulating the carboxylic acid moiety as zinc binding group to be directly attached to the linker (n = 0)

as in series III or expand the chain (n = 2,3) as in series II, VI, VII or rigidify the side chain through

incorporation of phenyl moiety as in series VIII. In addition, modifying the SRM to be heterocyclic or

aromatic amine derivatives which are essential for recognizing and binding to the rim of the active pocket

of the HDAC enzymes and also developing the hydrocarbon linker which occupies the hydrophobic

channel with styrene as in series II, III or benzamide as in series VI, VII, VIII which are approaches to

enrich and optimize HDAC inhibition activity [21], as shown in (Fig. 3).

Design of the target compounds was based on molecular docking study which was carried out on x-ray

crystal structure of HDLP enzyme (histone deacetylase like protein) PDB code (1C3S) complexed with

its binding inhibitor SAHA (suberoylanilide hydroxamic acid) to predict the binding affinities and

orientation of the target compounds at the active site of HDAC. In addition, we describe the structural

requirements and structure-activity relationships on carboxylic acid derivatives as cytotoxic agents by

successfully generating a valid 3D-pharmacophore model which was then combined to generate a

validated 2D-QSAR model to explore the structural requirements controlling the observed cytotoxic

activity estimated by the effect of the synthesized compounds on HepG₂cancer cell line.

Selected compounds were evaluated for their cytotoxic effect on sixty human tumor cell lines, under

the drug discovery program of the NCI and their enzyme inhibitory effect on different HDAC isoforms.

Target compounds were also in vitro tested for their cytotoxic effect on Liver HepG2 Cancer cell line.

O

II

OH

H

N

O

S

N

OH

N

O

III

H

N

O

Belinostat (PXD101)

N

O

VI

N

O

OH

N

H

N

NH₂

H

N

O

N

VII

N

O

OH

Mocetinostat (MGCD0103)

N

Linker

OH

O

VIII

Cl

O

OH

SRM

Phenyl butyric acid

ZBG

Figure 3. Design strategy and structures of target compounds

3. Chemistry

The route adopted for the synthesis of the novel (E)-6-(4-(substituted) phenyl)-4-oxohex-5-enoic acids

IIb-f (E) and (E)-3-(4-(substituted) phenyl)-acrylic acids IIIa-g (E) is depicted in Scheme 1. The key

intermediate 4-(substituted) benzaldehyde derivatives Ia-g were prepared following the literature methods

as illustrated in Scheme 1. [34] 4-(substituted) benzaldehyde derivatives Ia-g were synthesized in good

yield through the reaction of p-flurobenzaldehyde 1 with the appropriate amine 2a-g using potassium

carbonate and aliquat 336 as a catalyst mixed in dimethylformamide (DMF) under reflux affording the

corresponding benzaldehydes Ia-g. (E)-6-(4-(substituted) phenyl)-4-oxohex-5-enoic acids IIb-f (E) were

prepared by the condensation of the appropriate aldehyde Ib-f and levulinic acid using catalytic amounts

of piperidine and acetic acid in benzene under reflux, with azeotropic removal of water using a deanstark

trap [35] to give novel hexenoic acid derivatives IIb-f (E) as presented in Scheme 1. Under these

conditions, the carbanion formed at the α-methyl group of levulinic acid adds to the aldehyde carbon

1

followed by dehydration to afford the arylidine ketoacid derivatives IIb-f (E). H-NMR shows the

formation of E-isomers with minor amounts of Z-isomers which were detected by TLC as minor spots that

are removed upon recrystallization from the given solvent. Exceptionally, during synthesis of hexenoic

acid derivatives IIb (E) and IIf (E), the appropriate Z-isomers were obtained in good yields with their E-

isomers and were separated by column chromatography using hexane/ethylacetate 8:2 as the elution

system for separation to produce the pure (E)-6-(4-(pyrrolidin-1-yl)phenyl)-4-oxo-hex-5-enoic acid IIb

(E) , (Z)-6-(4-(pyrrolidin-1-yl)phenyl)-4-oxo-hex-5-enoic acid IIb (Z), (E)-6-(4-(4-phenylpiperazin-1-yl)

phenyl) -4-oxohex-5-enoic acid IIf (E) and (Z)-6-(4-(4-phenylpiperazin-1-yl)phenyl)-4-oxohex-5-enoic

acid IIf (Z).

1

Compound IIb (E) was confirmed as an E-isomer by H-NMR, where the spectrum of its E-isomer

1

revealed coupling constants of -CH=CH- higher than that of its Z-isomer. HNMR spectrum of IIb (E)

revealed a doublet signal with an integration of one proton –CH=CH-CO at δ6.56-6.62 ppm (1H) with J =

16.2 Hz which was coupled with the other proton –CH=CH-CO appearing as doublet signal at δ7.56-7.61

1

ppm (1H) with J = 15.9 Hz, while compound IIb (Z) was confirmed as a Z-isomer by HNMR spectrum

where –CH=CH-CO appeared as doublet signal at δ6.56-6.62 ppm (1H) with J = 8 Hz which was coupled

with –CH=CH-CO appearing as doublet signal at δ7.52-7.54 ppm (1H) with J = 8 Hz. Similarly compound

1

IIf (E) was confirmed as an E-isomer by H-NMR spectrum, where the coupling constants of -CH=CH-

1

where higher than its Z isomer. HNMR of –CH=CH-CO appeared as doublet signal at δ6.61-6.66 ppm

(1H) with J = 16.2 Hz which was coupled with –CH=CH-CO appearing as doublet at δ7.53-7.58 ppm (1H)

1

with J = 16.2 Hz, while compound IIf (Z) was confirmed as a Z-isomer by HNMR where –CH=CH-CO

appeared as singlet signal at δ6.82 ppm (1H) and CH=CH-CO appearing as doublet signal at δ7.52-7.54

(1H) with J = 6.4 Hz.

(E)-3-(4-(substituted)-phenyl)acrylic acids IIIa-g (E) were prepared by mixing of the appropriate

aldehyde Ia-g and malonic acid in pyridine under reflux using catalytic amount of piperidine as presented

1

in Scheme 1. E-isomers were obtained as a sole product and confirmed by H-NMR where the coupling

constant of –CH=CH-COOH appeared as doublet at δ 6.20-6.40 ppm (1H) with J = 16.2 Hz which was

coupled with –CH=CH-COOH appearing as doublet at δ 7.40-7.60 (1H) with J = 16.2 Hz.

The pathway applied for the synthesis of 4-(4-(substituted)phenylamino)-4-oxobutanoic acids VIa,b,e,

5-(4-(substituted)-phenylamino)-5-oxopentanoic

acids

VIIa,f

and

2-((4-substituted)phenyl)

carbamoyl)benzoic acids VIIIa,e were presented in Scheme 2. The key intermediates 4-(substituted)

aniline Va,b,e,f were prepared following the literature methods, [36, 37] Where 1-(amino substituted)-4-

nitrobenzene IVa,b,e,f were synthesized through the reaction of para nitro chlorobenzene 3 with the

appropriate amine 2a,b,e,f using potassium carbonate and aliquat 336 as a catalyst mixed in dimethyl

formamide under reflux affording 1(amino substituted) 4-nitrobenzene IVa,b,e,f. Reduction of the

synthesized nitro derivatives IVa,b,e,f to their amino derivatives using sodium dithionite in ammonia to

yield the corresponding intermediate 4-(substituted) aniline Va,b,e,f. Finally stirring of 4(substituted)

aniline Va,b,e,f with the appropriate anhydride (succinic anhydride, glutaric anhydride and phthalic

anhydride) in methylene chloride at room temperature afforded 4-(4-(substituted)phenylamino)-4-

oxobutanoic acids VIa,b,e, 5-(4-(substituted)phenylamino)-5-oxopentanoic acids VIIa,f, and 2-((4-

substituted)phenyl)carbamoyl) benzoic acids VIIIa,e, respectively.

COOH

R

iii

R

i

R

OHC

F

N

HN

N

CHO

R

IIIa-g (E)

1

Ia-g

2b-f

ii

OH

COOH

O

R

N

O

R

N

O

R

IIb-f (E)

IIb,f (Z)

IIIa

IIb / IIIb

IIc / IIIc

IId / IIId

IIe / IIIe

IIf / IIIf

IIIg

R

O

N

Cl

N

H₃C N

N

Scheme 1: Synthesis of (E)-6-(4-(substituted)phenyl)-4-oxohex-5-enoic acids IIb-f (E), (Z)-6-(4-(substituted)phenyl)-4-oxohex-5-

enoic Acids IIb,f (Z) and (E)-3-(4-(substituted) phenyl)acrylic acids IIIa-g (E). Reagents and conditions: i) potassium carbonate,

aliquat 336 in dimethylformamide, reflux, 24 hrs. ii) levulinic acid, piperidine and acetic acid in benzene, reflux, 6 hrs. iii) malonic

acid, piperidine in pyridine, reflux, 4 hrs.

R

i

N

NO₂

O₂N

Cl

HN

R

3

2a,b,e,f

IVa,b,e,f

ii

R

N

NH₂

R

Va,b,e,f

COOH

O

iii

v

iv

COOH

R

O

N

NH

O

R

N

NH

N

NH

R

HOOC

VIa,b,e

VIIa,f

VIIIa,e

b

e

f

a

R

N

H₃C N

N

Scheme 2: Synthesis of of 4-(4-(substituted) phenylamino)-4-oxobutanoic acids VIa,b,e, 5-(4-(substituted) phenylamino) -5-

oxopentanoic acids VIIa,f and 2-((4-substituted)phenyl)carbamoyl)benzoic acids VIIIa-e. Reagents and conditions: i)

potassium carbonate, aliquat 336 in dimethylformamide, reflux, 24 hr. ii) sodium dithionite in ammonia, reflux, 6 hr. iii)

succinic anhydride in methylene chloride, stir at room temperature. iv) glutaric anhydride in methylene chloride, stir at room

temperature. v) phthalic anhydride in methylene chloride, stir at room temperature.

4. Results and discussion

4.1. Biological evaluation

4.1.1. In vitro anticancer activity

4.1.1.1. Primary single dose (10^-5M) full NCI 60 cell panel in vitro assay

Ten structures of the final carboxylic acid based products were submitted to National Cancer Institute

“NCI” (www.dtp.nci.nih.gov), Bethesda, Maryland, USA, and eight compounds were selected on the basis

of degree of structure variation and computer modeling techniques for evaluation of their antineoplastic

activity. The tumor growth inhibition properties of the eight compounds IIb-f (E), IIIf (E), VIb and VIIf

with their respective NCI codes: NSC D-780150/1, NSC D-780151/1, NSC D-780154/1-, NSC D-

780152/1, NSC D-780153/1, NSC D-780155/1, NSC D-780157/1 and NSC D-780156/1, were selected

among the synthesized compounds IIb-f (E), IIIb,c,f, VIb, VIIf by the National Cancer Institute (NCI),

USA and were screened on human tumor cell lines, under the drug discovery program of the NCI.

Primary in vitro one dose (10^-5M concentration) anticancer assay was performed in full NCI 60 cell

panel. Results for each compound were reported as a mean graph of the percent growth of the treated cells

when compared to the untreated control cells. Analysis of historical Development Therapeutics

Programme (DTP) was performed and results are represented in Table 1.

Compounds IIf (E) and IIIf (E) among the tested compounds showed selectivity and sensitivity against

different NCI cell panel.

In series II, compound IIf (E) showed remarkably lowest cell growth promotion against Non-Small

Cell Lung A549/ATCC Cancer cell line (cell growth promotion 32%, inhibition 68%) and NCI-H460

Cancer cell line (cell growth promotion 34%, inhibition 66%). Also it exhibited broad spectrum cell

growth inhibition against Leukemia SR Cancer cell line (cell growth promotion 44.38%, inhibition

55.62%), K-562 Cancer cell line (cell growth promotion 65.61%, inhibition 34.39%), Breast T-47D

Cancer cell line (cell growth promotion 75.68%, inhibition 24.32%), Non-small cell Lung NCI-H23

Cancer cell line (cell growth promotion 77.6%, inhibition 22.4%). Compound IIc (E) is the second active

compound from series II, it was found to be active mostly against breast cancer T-47D cell line (cell

growth promotion 61.28%, inhibition 38.72%). Compound IIc (E) showed inhibition activity also against

Leukemia HL-60(TB) (cell growth promotion 77.66%, inhibition 22.34%) and K-562 (cell growth

promotion 72.14%, inhibition 27.86%) Cancer cell lines. Compound IIb (E) showed lower inhibition activity,

where it shows activity mostly against Breast T-47D Cancer cell line (cell growth promotion 76.6%, inhibition

23.4%). Compounds IId (E) and IIe (E) showed lowest inhibition activity among the tested series II

compounds.

In series III, compound IIIf (E) was found to be most active against Breast cancer MDA-MB-468 cell

line (cell growth promotion 42.66%, inhibition 57.34%). Compound IIIf (E) showed broad spectrum of

inhibition activity against Non-small lung cancer NCI-H226 cell line (cell growth promotion 73.05%,

inhibition 26.95%), Breast cancer T-47D cell line (cell growth promotion 74.03%, inhibition 25.96%),

ovarian cancer OV-CAR8 cell line (cell growth promotion 78.16%, inhibition 21.84%) and Melanoma

UACC-257 cell line (cell growth promotion 79.21%, inhibition 20.79%).

While in series VI and VII, compounds VIb and VIIf showed insignificant activity against most of the

60 tested Cancer cell lines.

Table 1. Sixty human tumor cell line anticancer screening data at single dose assay (10^-5M concentration) as percent cell growth

promotion of IIb-f (E), IIIf (E), VIb, VIIf

IIb (E)

IIc (E)

IId (E)

IIe (E)

95.52

101.86

82.67

92.88

95.69

98.14

IIf (E)

86.34

81.41

65.61

84.32

90.76

44.38

IIIf (E)

VIb

VIIf

Leukemia

CCRF-CEM

HL-60(TB)

K-562

MOLT-4

RPMI-8226

SR

89.13

94.03

93.17

91.73

95.12

107.76

81.60

77.66

72.14

80.57

92.58

79.02

97.30

97.58

92.03

95.50

101.79

103.18

98.34

96.78

95.20

86.96

95.16

81.99

98.77

96.63

92.23

100.62

106.09

103.70

113.81

97.89

96.64

99.81

103.14

98.17

Non-Small Cell Lung Cancer

A549/ATCC

HOP-62

93.88

100.21

92.08

91.39

93.60

101.29

105.08

86.95

90.22

93.66

81.93

95.21

87.90

97.35

103.95

82.81

90.92

108.32

97.38

97.91

97.77

96.15

102.79

96.06

98.09

99.48

91.20

99.97

98.85

102.97

108.55

96.39

32.24

95.65

83.75

83.73

77.60

82.41

34.23

86.85

99.76

92.29

89.75

73.05

97.35

91.99

102.50

82.01

98.62

103.57

97.30

105.53

102.24

97.79

111.03

104.08

96.00

101.27

103.12

98.85

HOP-92

NCI-H226

NCI-H23

NCI-H322M

NCI-H460

NCI-H522

Colon Cancer

COLO 205

HCC-2998

HCT-116

HCT-15

99.78

111.91

106.22

103.33

.

116.44

90.57

93.80

108.64

85.70

105.42

94.10

85.31

101.66

89.58

99.22

98.20

115.18

85.84

92.97

102.95

99.79

103.65

107.87

110.00

100.11

103.48

103.69

102.37

104.48

103.26

113.98

84.64

80.56

87.37

94.59

95.08

102.80

116.74

106.23

86.84

103.19

100.78

102.94

103.69

107.53

111.41

101.56

99.41

107.90

104.42

104.18

104.05

100.29

103.85

94.90

104.30

106.92

98.51

HT29

KM12

SW-620

100.85

101.15

CNS Cancer

SF-268

SF-295

SF-539

SNB-19

91.19

111.04

104.24

101.18

107.51

88.17

95.60

99.70

103.57

101.61

97.69

87.51

98.41

103.99

95.58

107.29

96.55

94.56

102.78

105.74

102.50

103.15

92.04

107.93

77.80

106.95

103.85

107.80

86.26

103.58

100.61

99.37

109.28

106.83

88.50

108.91

99.17

101.16

107.95

83.02

99.70

94.90

94.06

107.91

80.89

102.63

SNB-75

U251

100.31

104.93

Melanoma

LOX IMVI

MALME-3M

M14

MDA-MB-435

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

Ovarian Cancer

IGROV1

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

NCI/ADR-RES

SK-OV-3

Renal Cancer

786-0

94.74

105.59

97.37

103.72

105.23

107.18

97.61

94.28

96.11

97.42

110.11

103.83

103.61

95.91

88.73

91.07

97.10

96.47

92.80

112.05

101.13

100.66

100.35

90.69

99.56

90.33

91.78

89.14

102.53

101.79

96.41

101.35

98.83

90.66

88.34

87.87

91.37

100.81

107.02

99.85

106.30

111.07

101.69

100.25

96.91

96.99

100.54

104.41

103.68

105.88

101.72

104.07

101.16

102.69

101.54

109.23

102.70

107.25

102.06

92.40

105.10

112.86

110.19

111.91

93.89

84.00

91.26

79.21

97.80

104.22

99.41

111.37

98.36

97.94

98.00

103.04

97.91

97.49

108.7

91.31

97.29

104.46

97.62

89.90

98.54

98.50

103.69

101.20

110.24

94.43

101.77

102.70

116.30

104.87

110.76

108.94

104.46

102.14

103.53

101.52

106.13

99.33

97.49

114.57

96.72

92.91

94.86

96.60

102.98

118.31

78.16

100.41

100.91

112.15

109.23

108.74

101.36

102.86

101.46

105.39

108.27

113.88

105.96

96.74

102.59

110.34

100.86

111.43

99.06

105.01

101.03

95.96

101.09

104.65

88.54

98.70

100.75

107.18

106.10

97.76

100.25

84.50

96.35

109.65

104.19

115.14

106.06

99.13

94.90

109.71

108.13

107.29

99.89

92.22

107.98

105.26

114.99

98.11

87.80

109.98

110.43

92.21

106.33

93.80

93.23

106.95

91.52

106.75

105.33

107.24

101.66

99.29

100.30

94.33

104.71

104.38

107.41

94.30

ACHN

CAKI-1

RXF 393

SN12C

TK-10

UO-31

95.82

95.05

91.97

90.71

86.56

89.23

Prostate Cancer

PC-3

DU-145

94.10

94.92

89.51

95.64

97.88

109.30

99.49

105.09

87.52

94.75

101.75

101.80

97.11

109.07

102.26

109.53

Breast Cancer

MCF7

MDA-MB 231/ATCC

HS 578T

BT-549

T-47D

99.69

106.10

100.67

85.01

76.60

96.70

88.17

88.23

100.21

88.83

61.28

101.29

95.55

110.81

104.72

100.03

100.53

102.91

99.07

103.34

105.59

99.14

94.68

95.99

80.56

98.70

105.52

96.45

75.68

87.29

86.51

103.17

109.41

88.90

74.03

42.66

96.10

89.28

109.55

100.17

98.74

106.87

104.53

113.92

102.28

102.40

103.93

103.33

MDA-MB-468

O 10-20% growth inhibition, O 20-30% growth inhibition, O 30-40% growth inhibition, O 50-60% growth inhibition, O 60-

70% growth inhibition

4.1.1.2. In vitro cytotoxicity assay on HepG2 human liver cancer cell line

Furthermore, 21 synthesized compounds were evaluated for their cytotoxic activity on human liver

cancer cell line HepG2 in which HDAC1 is over expressed [38]. Cytotoxicity assay on HepG2 cancer cell

line was determined using sulforhodamine B assay method (SRB). The concentration of the test

compounds causing 50% inhibition (IC₅₀, µM) was calculated from the concentration–inhibition response

curve (triplet determinations) and are represented in Table 2.

In series II and series III, it was found that compounds IIc (E), IIf (E), IIIg (E), IIIb (E) displayed

potent cytotoxic activities with IC₅₀values 2.27, 10.21, 10.63 and 10.71 µM respectively, comparable with

SAHA having IC₅₀value of 63.48 µM [39]. Compounds IIIc (E), IIIa (E), IIIf (E), IIId (E), and IIb (E)

displayed significant cytotoxic activity with IC₅₀values 18.04, 21.01, 32.85, 45.69 and 63.58 µM

respectively. Other compounds IId (E), IIe (E) and IIIe (E) displayed IC50 100 µM. While series VI,

VII, and VIII displayed poor cytotoxic activity compared to series II and III carboxylic acids derivatives.

Interestingly compounds IIb (Z) and IIf (Z) which are representing the Z-isomers of 6-(4-(pyrrolidin-1-

yl)phenyl)-4-oxo-hex-5-enoic acid and 6-(4-(4-phenylpiperazin-1-yl)phenyl)-4-oxohex-5-enoic acid

respectively, displayed poor cytotoxic activity with IC₅₀values of 413.9 and 849.2 µM respectively,

compared with their E-isomers IIb (E) and IIf (E) with IC₅₀value of 63.48 and 10.21 µM respectively.

In conclusion, it is found that series II and series III displayed significant cytotoxic activity utilizing

styrene as a linker to be considered essential for its biological effect. On the other hand, insignificant

cytotoxic effect was observed in series VI, VII and VIII carboxylic acid derivatives which utilizes

benzamide as a linker. Exceptionally, compounds IId (E) (NHR₂= morpholine), IIe (E) (NHR₂=

methylpiperazine) and IIIe (E) (NHR₂= methylpiperazine) exhibited poor cytotoxic effect.

Table 2. Data of the In vitro anticancer activity on HepG2 cancer cell line.

Test Compound

IC₅₀^a(µM)

63.58

413.9

2.27

IIb (E)

IIb (Z)

IIc (E)

IId (E)

IIe (E)

IIf (E)

IIf (Z)

IIIa (E)

IIIb (E)

IIIc (E)

IIId (E)

IIIe (E)

IIIf (E)

IIIg (E)

VIa

138.1

904.8

10.21

849.2

21.02

10.71

18.04

45.69

430.7

32.85

10.63

468.2

997.2

672.7

629.5

725.2

548.1

525.7

63.48³⁹

VIb

VIe

VIIa

VIIf

VIIIa

VIIIe

SAHA

^aIC₅₀value is the compound concentration required to produce 50% inhibition of HepG2 human liver cancer cell line.

4.1.2. In vitro HDAC enzyme inhibition assay

Nine synthesized carboxylic acid compounds IIb,c,f (E), IIIb,f,g (E), VIa, VIIa and VIIIa were

selected as representatives for the five series in the present study to be in vitro tested for HDAC1 enzyme

inhibition. The percentage inhibition at 10 µM concentration of the synthesized compounds against

HDAC1 is summarized in Table 3. Synthesized Compounds were expected to inhibit HDACs according

to molecular docking study that was performed in the present study on HDAC class I homologue (HDAC-

like protein, HDLP)/SAHA complex (PDB: 1C3S) which shares 35.2% sequence identity with HDAC1

[23, 10]. Results show that at 10 µM concentration, none of the tested compounds showed significant

inhibitory activity against HDAC1.

Further in vitro enzyme inhibition assay was carried out on 3 synthesized carboxylic acid compounds

selected as representatives for three series presented in this study, series II ((4-(substituted)phenyl)-4-

oxohexanoic acids), series III (4-(substituted)phenyl) acrylic acids) and series VII 5-(4-(substituted)

phenylamino)-5-oxopentanoic acid to identify the series endowed with HDAC inhibitory activity on

different HDAC isoforms (HDAC2-11). Compounds IIf (E), IIIf (E) and VIIa were randomly selected to

test their percentage inhibition at 50 µM concentration on different HDAC isoforms (2-11). The

percentage inhibition of the tested compounds at 50 µM concentration is summarized in Table 4. Results

show that surprisingly newly synthesized compound IIf (E) showed remarkable increase in HDAC activity

against different HDAC isoforms instead of the expected inhibitory activity against class I HDACs, based

on the validated docking study that was performed prior to synthesis in the present study (-19% inhibition

against HDAC2, -73% inhibition against HDAC3, -53% inhibition against HDAC8). Compound IIIf (E)

showed potential enzyme inhibitory activity at 50 µM concentration against different HDAC isoforms

(27% inhibition against HDAC6, 25% inhibition against HDAC8, 20% inhibition against HDAC5 and

15% inhibition against HDAC2), while compound VIIa showed no significant inhibitory activity at 50 µM

concentration.

The enzyme inhibitory assay results were in consistency with the fact that carboxylic acid derivatives

are poor HDAC inhibitors showing potential HDAC inhibitory activity at high doses. [31, 32] Results

implicates a potent zinc binding group is crucial for HDAC inhibitory activity. This work provided some

insights to the binding interactions of inhibitors with HDAC and could facilitate development of novel

hydroxamate and/or benzamide HDAC inhibitors from the novel synthesized carboxylic acid derivatives

in this study in an attempt to synthesize more potent histone deacetylase inhibitors with stronger chelating

groups.

Table 3. The percentage inhibition of synthesized compounds at 10 µM concentration against HDAC1.

Test compound

at (10 µM)

HDAC1

IIb (E)

IIc (E)

IIf (E)

IIIb (E)

IIIf (E)

IIIg (E)

VIa

VIIa

VIIIa

SAHA

(500nM)

84

-10

-6

-5

2

-3

1

4

3

5

%Inhibition

Table 4. The percentage inhibition of compounds IIf (E), IIIa (E) and VIIa at 50 µM concentration against (HDAC2 - 11).

ND^a: not done

%Inhibition

at 50 µM

IIf (E)

IIIf (E)

VIIa

HDAC2 HDAC3

HDAC4

HDAC5

HDAC6

HDAC7

HDAC8

HDAC9 HDAC10

HDAC11

-19

15

6

-73

7

-1

-37

4

-75

20

1

ND

78

-27

27

-1

97

ND

-65

9

2

ND

90

-53

25

1

ND

89

-38

-5

1

-29

1

3

-42

-2

1

ND

84

2

SAHA(500nM)

TSN(30 µM)

74

ND

90

ND

ND^a

88

ND

87

ND

85

4.2. Molecular modeling studies

4.2.1. Docking study:

Molecular docking was performed using Accelrys Discovery studio 2.5 software using the Dock

ligands (CDOCKER) protocol which is an implementation of the CDOCKER algorithm. CDOCKER is a

grid-based molecular docking method that employs CHARMm-based molecular dynamics (MD) scheme

to dock ligands into a receptor binding site. Random ligand conformations are generated using high-

temperature MD. It allows you to run a refinement docking of any number of ligands with a single protein

receptor. Various scoring functions were applied to the ligands including -CDOCKER_ENERGY

(CHARMm energy: interaction energy plus ligand strain) and -CDOCKER_INTERACTION_ENERGY:

interaction energy only). Poses are sorted by CHARMm energy and the top scoring poses are calculated.

Molecular docking was based on crystal structures of HDLP (PDB ID: 1C3S) which is class I HDAC

homologue complexed with SAHA (hydroxamic acid inhibitor of HDAC in crystal complex). Protein was

prepared using prepare protein parameter which cleans up common problems in the input protein structure

in preparation for further processing, where it inserts missing atoms in residues as hydrogen which was

added and minimized and it also removes alternate conformations. All the waters were removed as well as

all ions except for catalytic zinc ion. Synthesized compounds IIb-f (E), IIb,f (Z), IIIa-g (E), VIa,b,e,

VIIa,f and VIIIa,e were prepared from ligands prepare tool which adds hydrogen, fix bad valencies and

generates a 3D coordinates using catalyst. Docking was performed using (CDOCKER) protocol. Pose

cluster radius was set to 0.5 Å, top hits were set to 10 and other docking parameters were kept at default.

Results were analyzed according to -CDOCKER_ENERGY.

4.2.1.1. Docking results

The binding modes of the synthesized carboxylic acids into the active site of histone deacetylase like

protein (PDB ID: 1C3S) were in consistency with the proposed design and rational as described in Table

5. CDOCKER energy scores indicated favorable binding modes of SAHA and the newly synthesized

compounds IIc (E) and IIIg (E) into the active site of HDLP (PDB ID: 1C3S) as shown in (Fig. 4).

In (A), it was found that the CDOCKER energy score of the original ligand SAHA was -46.07 forming

three hydrogen bond interactions between oxygen of hydroxamic hydroxyl group and histidine (His131)

with a distance of 2.41 Å, hydrogen of hydroxamic NH and histidine (His132) with a distance of 1.90 Å

and oxygen of amidic carbonyl group and tyrosine (Tyr297) with a distance of 1.79 Å. The distance

between oxygen atom of amidic carbonyl group and oxygen of hydroxyl group with zinc ion were 1.79 Å

and 1.84 Å, respectively. This was found to retrieve the reported [23] binding mode of SAHA in the X-ray

crystal structure of HDLP (PDB ID: 1C3S).

In (B), the newly synthesized compound IIc (E) showed the best binding affinity with CDOCKER

energy score of -51.03 and the most potent cytotoxic activity against HepG2 cancer cell line (in which

histone deacetylase enzyme is overexpressed) with measured IC₅₀of 2.27 µM. Binding mode was

consistent with SAHA, since compound IIc (E) forms two hydrogen bond interactions between oxygen of

ketonic carbonyl group and tyrosine (Tyr297) with a distance of 2.33 Å and oxygen of acidic carbonyl

group and histidine (His131) with a distance of 2.18 Å. Phenyl ring of styrene shows additional two pi-pi

interaction with the phenyl rings of phenylalanine (Phe141) and phenylalanine (Phe198). The distance

between the oxygen atom of ketonic carbonyl group and oxygen atom of acidic carbonyl group with zinc

ion were 2.24 and 2.22 Å, respectively.

In (C), the newly synthesized compound IIIg (E) from series III showed the best binding affinity with

CDOCKER energy score of -40.02 and the most significant cytotoxic activity with measured IC₅₀of 10.63

µM. Binding mode was consistent with SAHA forming three hydrogen bond interactions between oxygen

of acidic carbonyl group and tyrosine (Tyr297) with a distance of 2.39 Å, oxygen of acidic hydroxyl group

and histidine (His131) with a distance of 2.43 Å and hydrogen of acidic hydroxyl group and histidine

(His132) with a distance of 2.15 Å. Phenyl ring of styrene shows additional pi-pi interaction with phenyl

ring of phenylalanine (Phe198). The distance between oxygen atom of acidic carbonyl group and oxygen

atom of acidic hydroxyl group with zinc ion were 2.15 and 2.64 Å, respectively.

Table 5. Docking results for the synthesized compounds as well as SAHA in the active site of HDLP enzyme PDB (1C3S)

showing the H-bonds, Pi-interactions formed with the binding site residues and also distance of atoms related to zinc metal.

Name

^aIC₅₀

µM

CDOCKER

energy

Hydrogen bonds

Hydrogen

bond

Pi-Pi interactions

Distance related to zinc

distance

SAHA

63.48

-46.07

HE2 of HIS131 - O of OH

2.41

none

1.79 (C=O, amide)

NE2 of HIS132 - H of hydroxamic 1.90

1.84 (OH, hydroxamic)

NH

1.79

HH of TYR297 - O of amidic C=O

IIb (E)

63.58

-43.27

HE2 of HIS131 - O of acidic C=O

HH of TYR297 - O of ketonic C=O

HN1 of GLY295 - O of acidic OH

2.14

2.32

2.06

PHE141 - phenyl ring of styrene

PHE198 - phenyl ring of styrene

2.19 (C=O, ketonic)

2.27 (C=O, acidic)

IIb (Z)

IIc (E)

413.9

2.27

-21.96

-51.44

HH of TYR297 - O of acidic OH

2.00

PHE198 - phenyl ring of styrene

2.15 (C=O, ketonic)

HE2 of HIS131 - O of acidic C=O

HH of TYR297 - O of ketonic C=O

2.18

2.33

PHE141 - phenyl ring of styrene

PHE198 - phenyl ring of styrene

2.24 (C=O ketonic)

2.22 (C=O acidic)

IId (E)

138.1

-33.33

O of GLY129 - H of acidic OH

2.38

2.14

PHE141 - phenyl ring of styrene

PHE198 - phenyl ring of styrene

2.18 (OH, acidic)

IIe (E)

IIf (E)

904.8

10.21

-43.80

-48.8

PHE141 - phenyl ring of styrene

PHE198 - phenyl ring of styrene

2.65 (C=O, ketonic)

3.21 (C=O, acidic)

HE2 of HIS131 – O of acidic C=O

HH of TYR297 – O of ketonic C=O

2.14

2.35

PHE141 - phenyl ring of styrene

PHE198 - phenyl ring of styrene

2.21 (C=O, ketonic)

2.27 (C=O, acidic)

IIf (Z)

849.2

21.02

-28.40

-28.60

HH of TYR297 – O of ketonic C=O

2.46

PHE141 - phenyl ring of styrene

PHE198 - phenyl ring of styrene

7.44 (C=O, acidic)

IIIa (E)

HH of TYR297- O of acidic OH

2.42

PHE141 - phenyl ring of styrene

1.53 (C=O, acidic)

PHE198 - phenyl ring of styrene and 2.19 (OH acidic)

imidazole ring

IIIb (E)

IIIc (E)

IIId (E)

IIIe (E)

10.71

18.04

45.69

430.7

-28.14

-37.68

-37.23

-31.14

HH of TYR297 – O of acidic C=O

NE2 of HIS132 - H of acidic OH

1.84

2.44

none

2.88 (C=O, acidic)

1.76 (OH, acidic)

HH of TYR297 – O of acidic OH

O of GLY140- H of acidic OH

HH of TYR297- O of acidic OH

2.82

2.94

2.38

PHE141 - phenyl ring of styrene

PHE198 - phenyl ring of styrene

2.86 (C=O, acidic)

2.43 (OH acidic)

PHE141 - phenyl ring of styrene

PHE198 - phenyl ring of styrene

2.46 (C=O, acidic)

1.83 (OH, acidic)

PHE141 - phenyl ring of styrene

PHE198 - phenyl ring of styrene

2.14 (OH acidic)

IIIf (E)

IIIg (E)

32.85

10.63

-37.48

-40.02

PHE141 - phenyl ring of styrene

PHE198 - phenyl ring of styrene

2.53 (C=O, acidic)

2.48 (OH, acidic)

HH of TYR297 - O of acidic C=O

HE2 of HIS131 - O of acidic OH

NE2 of HIS132 - H of acidic OH

2.39

2.43

2.15

PHE198 - phenyl ring of styrene

2.15 (C=O, acidic)

2.64 (OH, acidic)

VIa

468.2

-30.35

O of GLY140:O – H of acidic OH

2.39

PHE141 - phenyl ring of benzamide

PHE198 - phenyl ring of benzamide

2.16 (C=O, acidic)

VIb

VIe

997.2

672.7

-28.82

-33.44

O of GLY140 - H of acidic OH

none

2.45

PHE141 - phenyl ring of benzamide

2.15 (C=O, acidic)

2.14 (C=O, acidic)

none

PHE141 - phenyl ring of benzamide

PHE198 - phenyl ring of benzamide

VIIa

VIIf

629.5

725.2

-30.35

-44.75

O of GLY140 - H of acidic OH

O of GLY140 – N of amide

2.41

2.26

PHE141 - phenyl ring of benzamide

2.17 ( C=O, acidic)

2.27 (C=O, amide)

PHE141 - phenyl ring of benzamide

PHE198 - phenyl ring of benzamide

VIIIa

VIIIe

548.1

526.9

-14.50

-17.95

HE2 of HIS170 - O of amidic C=O

2.10

PHE141 - phenyl ring of pthalic acid

PHE198 - phenyl ring of phthalic

acid

not significant

PHE200 - phenyl ring of benzamide

none

PHE141 - phenyl ring of benzamide

PHE198 - phenyl ring of benzamide

Figure 4. Molecular modeling of original ligand (SAHA) and two newly synthesized compounds IIc (E) and IIIg (E) to

compare binding interactions within the active site of HDLP (PDB ID: 1C3S). In (A) binding mode of original ligand (SAHA)

with HDLP, (B) binding mode of newly synthesized compound IIc (E) with HDLP, (C) binding mode of newly synthesized

compound IIIg (E) with HDLP. All interactions are described as H-bonds shown in dotted lines, Pi-Pi interactions shown in

orange solid lines and metal coordination distance shown in blue lines. Amino acids involved in interactions and zinc metal

were labelled.

4.2.1.2. Docking validation

Validation of docking algorithm was achieved by docking of the selected lead compound, SAHA in the

active site of histone deacetylase enzyme homologue (PDB ID:1C3S), this was found to retrieve the

reported [23] binding mode of SAHA in the X-ray crystal structure of histone deacetylase enzyme

homologue PDB (1C3S) as shown in (Fig. 4) (A). Nevertheless, the root mean square difference (RMSD)

between the top docking pose and original crystallographic geometry of SAHA was 0.88 Å. This

successful validation provides sufficient confidence in drawing meaningful conclusions from the docking

study.

4.2.2. Pharmacophore modeling

In this study, pharmacophore generation was performed using Discovery Studio 2.5 software. The 3D

QSAR Pharmacophore Generation protocol (Catalyst HypoGen algorithm) was used to derive structure

activity relationship hypothesis models (3D-QSAR pharmacophore models) from a set of ligands with

known activity values on a given biological target. [40]

The training set is composed of twenty one synthesized compounds from the present study with

measured IC₅₀against HepG2 cancer cell line for the generation of pharmacophore models considered as

the certainty value. The uncertainty value was set as 1.5 instead of the default value 3.0. The

pharmacophore features used are hydrogen bond acceptor (HBA), hydrogen bond donor (HBD),

hydrophobic (HYP), ring aromatic (RA) and negative ionizable features. Though the Zn ion binding

function is not included in the catalyst feature dictionary, it can be represented by the hydrogen bond

acceptor or donor. [40] Fisher validation was applied and set to 95% significance. Pharmacophores were

then generated in HypoGen module and the top nine scoring hypotheses were exported. HypoGen

identifies features common to the active compounds and excludes features common to the inactive

compounds within conformational allowable regions of space. It further estimates the activity of our newly

synthesized and tested compounds using regression parameters. The parameters were computed by

regression analysis using the relationship of geometric fit value versus the activity. The better the

geometric fit the greater the activity prediction of the compound.

4.2.2.1. Pharmacophore study results

Nine predictive pharmacophore models (hypothesis) were generated via aligning different

conformations of the represented training set ligands to bind with the generated pharmacophore models.

These pharmacophore models were exported for further studies. All of the generated pharmacophore

models contained at least four chemical features. Five out of nine pharmacophore models had two HBA,

one HYP, and one RA feature.

The top pharmacophore model generated, Hypothesis 5, was developed having two HBA, one HYP, and

one RA feature as shown in (fig. 5) with constraint distances and angles between its features as described

in Table 6.

Figure 5. Constraint distances and angles between features of the generated top pharmacophore model with the features

considered hydrogen bond donor (HBA) colored in green, ring aromatic (RA) colored in orange, hydrophobic (HYP) colored in

cyan. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article).

Table 6. Constraint distances and angles between features of the generated top pharmacophore model.

Constraint distances (Å)

(HBA_1) - (HBA_2), 2.984;

(HBA_1) -(RA_4), 5.842;

(HBA_2) - (RA_4), 4.940;

(HYP_3) - (HBA_1),10.435;

(HYP_3) -(RA_4), 4.631;

(HYP_3) - (HBA_2), 9.306

Constraint angles (^o)

(HBA_1) - (HBA_2) - (HYP_3), 103.95;

(RA_4) - (HBA_2) - (HBA_1), 91.60;

(HBA_2) - (HYP_3) - (RA_4), 13.98;

(RA_4) - (HYP_3) - (HBA_1), 5.50

In (fig. 6), SAHA and newly synthesized compound IIc (E) are represented with the best generated

pharmacophore model. Where In (A) SAHA has fit value of 5.90 and its measured IC₅₀is 63.48 µM [39]

against HepG2 Cancer cell line. Three features out of four were considered, where carbonyl group of

hydroxamic acids fits with (HBA_1), hydroxyl group of hydroxamic acid fits with (HBA_2) and phenyl

ring fits with (HYP). While in (B) compound IIc (E) has highest fit value of 7.59 and highest cytotoxic

activity against Human Liver HepG2 Cancer cell line with IC₅₀of 2.27 µM. Four pharmacophore features

were considered, where oxygen of ketonic carbonyl group fits with (HBA_1), oxygen of hydroxyl group

fits with (HBA_2), pyridine ring fits with (HYP) and additionally the phenyl ring of styrene fits with (RA).

Features of the generated pharmacophore model mapped with the 21 synthesized compounds as well as

SAHA are summarized in Table 7.

Figure 6. The best generated pharmacophore model with the features considered hydrogen bond acceptor (HBA) colored in

green, ring aromatic (RA) colored in orange, hydrophobic (HYP) colored in cyan where A) shows SAHA fitted in the

pharmacophore with fit value 5.90 (B) shows synthesized structure compound IIc (E) fitted in the pharmacophore with fit value

7.59, (For interpretation of the references to color in this figure, the reader is referred to the web version of this article).

Table 7. The pharmacophore features (hydrogen bond acceptor HBA_1, hydrogen bond acceptor HBA_2, hydrophobic feature

HYP_3, and ring aromatic feature (RA)) mapped with the synthesized compounds and SAHA, as well as their fit values.

Fit

Compd.

HBA_1

HBA_2

HYP_3

RA_4

value

6.09

5.29

7.59

5.67

5.60

6.76

5.03

6.96

6.67

6.63

5.45

6.45

6.61

5.26

5.04

5.14

5.13

5.09

5.19

5.21

IIb (E)

IIb (Z)

IIc (E)

IId (E)

IIe (E)

IIf (E)

IIf (Z)

IIIa (E)

IIIb (E)

IIIc (E)

IIId (E)

IIIe (E)

IIIf (E)

IIIg (E)

VIa

VIb

VIe

VIIa

VIIf

VIIIa

VIIIe

CO of ketone

--------------------------

CO of ketone

OH of carboxylic acid

--------------------------

OH of carboxylic acid

CO of carboxylic acid

OH of carboxylic acid

--------------------------

OH of carboxylic acid

--------------------------

OH of carboxylic acid

CO of amide

pyrrolidine

piperidine

morpholine

4-CH₃-piperazine

piperazine

phenyl ring of styrene

phenyl ring of benzamide

CO of ketone

piperazine

imidazole

CO of carboxylic acid

OH of carboxylic acid

CO of carboxylic acid

OH of carboxylic acid

--------------------------

CO of amide

pyrrolidine

piperidine

morpholine

4-CH₃-piperazine

piperazine

-------------

pyrrolidine

4-CH₃-piperazine

imidazole

piperazine

--------------

piperazine

OH of carboxylic acid

--------------------------

CO of amide

CO of carboxylic acid

CO of amide

--------------------------

OH of carboxylic acid

--------------------------

OH of carboxylic acid

SAHA

5.90

CO of hydroxamic acid

OH of hydoxamic acid

phenyl ring

--------------------------

4.2.2.2. Pharmacophore validation

The pharmacophore models generated should be statistically significant, able to predict the activities of

new chemical compounds and retrieve active compounds from the database. The selection of the generated

pharmacophore model (hypothesis 5) was be based on its validation using four methods; cost analysis,

activity prediction, mapping of SAHA as a reported external reference to the generated pharmacophore

and Fisher validation test.

HypoGen selects the best hypothesis by applying a cost analysis. The overall cost of each hypothesis is

calculated by summing three cost factors, a weight cost, an error cost, and a configuration cost. However,

configuration cost is the main contributor. HypoGen also calculates two theoretical costs, the null and

fixed costs that can be used to determine the significance of the selected hypothesis. The fixed cost is the

lowest possible cost representing a simplest hypothetical model that fits all data perfectly and the null cost

represents the maximum cost of a pharmacophore with no features and estimates the activity to be the

average activity of the training set compounds and the total cost for every pharmacophore. A larger

difference between the fixed and null costs than that between the fixed and total costs of each hypothesis

signifies the quality of a pharmacophore model. The significance of the hypothesis also depends on the

cost difference which is measured between the null cost and the total cost of a given hypothesis. The larger

the value of the cost difference, the more statistically significant the hypothesis is believed to be.

The quality of the generated pharmacophore hypothesis (hypothesis 5) was evaluated by considering

the cost functions calculation by HypoGen module during hypothesis generation. In detail, the null cost

and fixed cost of the nine pharmacophore hypothesis were equal to 278.4 and 61.2, respectively.

Hypothesis 5 is the best generated pharmacophore hypothesis as it is characterized by the lowest total cost

(105.7), the highest cost difference between null and total hypothesis cost (172.7), and the best correlation

coefficient (0.88) which indicates the capability of the pharmacophore model to predict the activity of the

training set compounds.

The pharmacophore model (hypothesis 5) was also validated through activity prediction of the

synthesized structures as training set. Further validation for the pharmacophore model (hypothesis 5)

generated was performed through mapping of SAHA using ligand pharmacophore mapping protocol,

comparing its fit value with the fit values of the tested compounds which was detected as 5.90 and

comparing its predicted activity with its experimental activity. The predicted activities of SAHA and the

training set through the pharmacophore model as well as their fit values are represented in Table 8. It

should be noted that the predicated anti-proliferative activities by the 3D Pharmacophore QSAR model

were very close to those experimentally observed, indicating that these models can be safely applied for

prediction of cytotoxic activity of newly synthesized compounds.

Fischer validation is another approach for pharmacophore model validation. The Fischer validation

confidence level for hypothesis 5 is 95%. This validation method checks the correlation between the

chemical structures and biological activity. This method generates pharmacophore hypothesis using the

same parameters as those used to develop the original pharmacophore hypothesis by randomizing the

activity data of the training set compounds.

Table 8. Fit values and predicted activities for the synthesized compounds mapped with the generated 3D-pharmacophore

model. SAHA mapping on the generated pharmacophore was used for further validation of the model.

Compound

Predicted

activity

(IC₅₀µM)

68.08

437.1

2.18

Experimental

activity

(IC₅₀µM)

63.58

Fit

value

IIb (E)

IIb (Z)

IIc (E)

IId (E)

IIe (E)

IIf (E)

IIf (Z)

IIIa (E)

IIIb (E)

IIIc (E)

IIId (E)

IIIe (E)

IIIf (E)

IIIg

VIa

VIb

VIe

VIIa

VIIf

VIIIa

VIIIe

6.09

5.29

7.59

5.67

5.60

6.76

5.03

6.96

6.68

6.67

6.63

5.45

6.42

6.61

5.26

5.04

5.14

5.13

5.09

5.19

5.21

5.90

413.9

2.27

138.1

904.8

10.21

849.2

21.02

10.71

18.04

45.69

430.7

32.85

10.63

468.2

997.2

672.7

180.1

212.1

14.73

785.7

9.364

17.88

18.05

19.81

302.2

32.14

20.79

468.3

763.7

611.3

627.8

685.1

549.2

526.9

72.26

629.5

725.2

548.1

525.7

SAHA

63.48³⁹

4.2.2.3. Conclusion

In conclusion, results of cytotoxic activity against Liver HepG2 Cancer cell line were consistent with

pharmacophore fit score values and docking score energies. Where it was observed that compound IIc (E)

((E)-6-(4-(piperidin-1-yl) phenyl)-4-oxo-hex-5-enoic acid) with the lowest IC₅₀of 2.27 µM (i.e most

potent), showed the best fit value of 7.59 and the highest binding affinity with CDOCKER energy score of

-50.27.

Interestingly, 3D QSAR pharmacophore model created to explore the observed cytotoxic activity against

HepG2 cancer cell line for the E and Z isomers of the newly synthesized compound IIf coincides with

docking. This enabled an explanation for the increase of cytotoxic activity of compound IIf (E) having an

IC₅₀of 10.21 µM when compared with its Z isomer IIf (Z) having an IC₅₀of 849.2 µM.

In (Fig. 7), compound IIf (E) has CDOCKER energy score of –48.71, where its two oxygen atoms of

ketonic and acidic carbonyl accepts two hydrogen atoms from tyrosine (Tyr297) and histidine (His131)

respectively. And its phenyl ring of styrene forms two pi-pi interaction with phenylalanine (Phe141) and

phenylalanine (Phe198). The respective distance between the oxygen atoms of ketonic and acidic carbonyl

with zinc ion were 2.21 and 2.27 Å, which enabled the coordination of zinc ion. Also compound IIf (E)

has fit value of 6.76 fitting with the four features of the generated pharmacophore model. Where oxygen of

ketonic carbonyl group fits with (HBA_1), oxygen of acidic carbonyl fits with (HBA_2), piperazine fits

with (HYP_3) and phenyl ring of styrene fits with RA_4.

Figure 7. Docking model (A) coincides with 3D QSAR pharmacophore model (B) for E-isomer of newly synthesized

compound IIf (E) having a measured IC₅₀against HepG2 cancer cell line of 10.21 µM. In (A) compound IIf (E) was docked in

the active site of HDAC (PDB code:1C3S) with a CDOCKER energy score of -48.71, interactions are described as H-bonds

shown in dotted lines, Pi-Pi interactions shown in orange solid lines and metal coordination shown in blue lines ,amino acids

involved in interactions and zinc metal were labelled. In (B) compound IIf (E) fitted in the pharmacophore model with fit value

6.76. Features considered are hydrogen bond acceptor (HBA) colored in green, ring aromatic (RA) colored in orange and

hydrophobic (HYP) colored in cyan.

While Compound IIf (Z) has CDOCKER energy score of -28.4 score, where oxygen of ketonic

carbonyl accepts one hydrogen atom from tyrosine (Tyr297) and its phenyl ring of styrene forms two pi-pi

interaction with Phe141 and Phe198. The measured distance of its ketonic carbonyl with zinc ion was 7.44

Å, which showed inability to coordinate the zinc ion. Also, compound IIf (Z) has the lowest fit value of

5.03 and fits with only three features of the pharmacophore model generated. Where oxygen of ketonic

carbonyl group fits with (HBA_1), piperazine fits with (HYP_3) and phenyl ring of styrene fits with

(RA_4) as shown in (Fig. 8).

Figure 8. Docking model (A) coincides with 3D QSAR pharmacophore model (B) for Z-isomer of newly synthesized

compound IIf (Z) having a measured IC₅₀of 849.2 µM. In (A) compound IIf (Z) was docked in the active site of HDAC (PDB

code:1C3S) with a CDOCKER energy score of -28.4, interactions are described as H-bonds shown in dotted lines, Pi-Pi

interactions shown in orange solid lines, amino acids involved in interactions and zinc metal were labelled. In (B) compound IIf

(Z) fitted in the pharmacophore model with fit value 5.03. Features considered hydrogen bond acceptor (HBA) colored in green,

ring aromatic (RA) colored in orange, hydrophobic (HYP) colored in cyan.

From series III, compound IIIg (E) ((E)-3-(4-(4-(4-chlorophenyl)piperazin-1-yl)phenyl)acrylic acid)

with the lowest measured IC₅₀of 10.63 µM, has the best fit value of 6.61 and highest binding affinity with

CDOCKER energy score of -40.02.

While all synthesized compounds of series VI (4-(4-(substituted)phenylamino)-4-oxobutanoic acid),

VII (5-(4-(substituted)phenylamino)-5-oxopentanoic acid) and VIII (2-((4-substituted)phenyl) carbamoyl)

benzoic acid) have insignificant cytotoxic activity IC₅₀100 µM, lowest binding affinity and lowest fit

value ranging from 5.04-5.26.

4.2.3. 2D QSAR study:

The QSAR study was performed using Discovery Studio 2.5 Software. The training set was composed

of the twenty one synthesized compounds from the present study with their measured pIC₅₀(-Log IC₅₀)

against HepG2 cancer cell line for QSAR modeling. “Calculate Molecular Properties” module was used

for calculating the 2D and 3D molecular properties as well as energies of highest occupied and lowest

unoccupied molecular orbitals (HOMO and LUMO) [41] of the training set compounds.

2D Descriptors involved: AlogP, fingerprints, molecular properties, surface area and volume, as well as

topological descriptors, and the 3D descriptors: dipole, jurs descriptors, principle moments of inertia, and

shadow indices.

Furthermore, the training set compounds were fitted against representative pharmacophores and their fit

values were added as additional descriptors. The fit values of the training set compounds were calculated

automatically using the previously described “3D QSAR Pharmacophore Generation” module.

Multiple linear regression (MLR) analysis was employed to search for optimal QSAR models that

combine high quality binding pharmacophores with other molecular descriptors and being capable of

correlating bioactivity variation across the used training set collection. Compound IIf (Z) was identified as

a statistical outlier. QSAR model was validated employing leave one-out cross-validation by setting the

folds to a number much larger than the number of samples , r²(squared correlation coefficient value) and

r²prediction (predictive squared correlation coefficient value), residuals between the predicted and

experimental activity of the test set and training set. [42]

4.2.3.1. QSAR study results

Equation 1 represents the best performing QSAR model

-logIC₅₀= -1260.8 ALogP – 4.059 pKa[1] – 3.313 pKa[2] – 109.98 Molecular_Fractional Polar

Surface Area + 2042.3

Abbreviations used: -logIC₅₀is the negative logarithmic value of the concentration required to produce

50% inhibition of HepG2 cancer cells.; ALogP is a measure of the hydrophobicity of the molecule; it is

calculated in Discovery Studio as the Log of the octanol-water partition coefficient using Ghose and

Crippen's method [43]; pKa is the pKa value of all ionizable sites; Molecular_Fractional Polar Surface

Area is the ratio of the polar surface area divided by the total surface area of the molecule.

According to Equation 1 QSAR model is represented graphically by scattering plots of the experimental

versus the predicted bioactivity values –logIC₅₀for the training set compounds as shown in (Fig. 9).

The method used to build the model was Least-Squares, r²= 0.948, r²(adj) 1.017, r²(pred) 0.215, Least-

squared error 0.033155, where r²(adj) is r²adjusted for the number of terms in the model; r²(pred) is the

prediction r², equivalent to q²from a leave-1-out cross-validation.

Figure 9. Shows the corresponding scatter plots of the experimental versus the predicted bioactivity values -logIC₅₀for the

training set compounds according to

Equation 1. (r²= 0.948).

Knowledge of pka values together with partition coefficient can be used for estimating the extent to

which the compound enters the bloodstream. Enzyme kinetics are dependent on the the pKa values of

many acids and bases and is also a prerequisite for a quantitative understanding of the interaction between

acids and bases and metal ions to form complex. In conclusion, Equation 1 describes that the cytotoxic

activity of synthesized compounds on Liver HepG2 cancer cell line is affected by three molecular

descriptors AlogP, pKa and molecular fractional polar Surface, where cytotoxic activity is increased by

decreasing the hydrophobicity (ALogP), pKa value of all ionizable sites and decreasing the molecular

fractional polar surface area of the synthesized compounds.

4.2.3.2. QSAR validation

QSAR models were validated internally employing leave-1-out cross-validation where r²(squared

correlation coefficient value) which is 0.948, r²(pred) is the prediction r², equivalent to q²from a leave-1-

out cross-validation which is 0.215. Validation was also employed by measuring the residuals between the

experimental activity and the predicted activity of the training set. The experimental activities and those

predicted by QSAR studies were presented in Table 9. It should be noted that the predicated anti-

proliferative activities by our QSAR models were very close to those experimentally observed, indicating

that these models can be safely applied for prediction of more effective hits having the same skeletal

framework.

Table

compounds against

Equation 1^a.

9.

Experimental activity of the synthesized

the predicted activity according to

Compd.

Experimental

Activity

(-log IC₅₀)

-1.803

-2.616

-0.356

-2.140

-2.956

-1.009

-1.32

Predicted

activity

(-log IC₅₀)

-2.149

-0.508

-2.063

-2.973

-0.942

-1.32

-1.041

-1.083

-1.725

-2.458

-1.765

-1.026

-2.432

-3.056

-3.013

Residual

IIb (E)

IIb (Z)

IIc (E)

IId (E)

IIe (E)

IIf (E)

IIIa (E)

IIIb (E)

IIIc (E)

IIId (E)

IIIe (E)

IIIf (E)

IIIg (E)

VIa

0.346

-0.467

0.152

-0.076

0.016

-0.066

0.000

0.011

-0.172

0.065

-0.175

0.249

0.000

-0.237

0.057

0.186

-1.029

-1.256

-1.659

-2.634

-1.516

-1.026

-2.67

VIb

VIe

-2.998

-2.827

VIIa

VIIf

VIIIa

VIIIe

-2.79

-2.860

-2.730

-2.720

-3.014

-2.634

-2.748

-2.701

0.224

-0.226

0.018

-0.018

5. Conclusion

In summary, it was found that docking coincides with 3D QSAR pharmacophore model generated for

the displayed cytotoxic activity of the synthesized compounds against HepG2 Cancer cell line.

It was revealed that E-isomers for compounds IIb, IIf displayed significant cytotoxic activity compared

to their Z-isomers which showed insignificant cytotoxic activity. Compounds IIc (E), IIf (E), IIIb (E),

and IIIg (E) displayed the highest cytotoxic activity against HepG2 human cancer cell lines with IC₅₀

ranging from 2.27-10.71 μM. Compound IIf (E) (NHR₂= phenylpiperazine) displayed significant

cytotoxic activity against NCI Non-Small Cell Lung A549/ATCC Cancer cell line (68% inhibition

activity), NCI-H460 Cancer cell line (66% inhibition activity) and Liver HepG2 Cancer cell line (IC₅₀,

10.21 µM). And unpredictably, compound IIf (E) showed remarkable stimulation of HDAC activity

towards different HDAC isoforms instead of the expected inhibitory activity against class I HDACs. It is

suggested that the significant cytotoxic effect of compound IIf (E) is maybe due to another mechanism of

action. Compound IIIf (E) (NHR₂= phenylpiperazine) showed significant inhibitory activity (57.34%)

against NCI Breast MDA-MB-468 Cancer cell line and potential HDAC inhibitory activity against

different HDAC isoforms (20% HDAC5, 27 % HDAC6 and 25% HDAC8).

It was found that the major structural factors affecting the potency of these compounds were related to

their basic skeleton. The results indicated that the carboxylic acid derivatives of series II and III could

serve as promising lead compounds for further optimization.

6. Experimental protocols

6.1. Chemistry

Starting materials and reagents were purchased from Sigma-Aldrich and Merck and used without further

purification. Melting points were recorded on Stuart Scientific apparatus and were uncorrected. Reactions

were monitored using thin layer chromatography (TLC), performed on 0.255 mm silica gel plates, with

visualization under U.V. light (254 nm). FT-IR spectra were recorded on a 4100 Jasco spectrophotometer

using KBr disc in the Microanalytical center, Cairo University. 1H NMR spectra were recorded in δ scale

given in ppm on Varian Mercury VX-300 NMR spectrometer in NMR Lab, Chemistry Department,

Faculty of Science, Cairo University and Varian Mercury VX-400 NMR spectrometer in NMR Lab,

Chemistry Department, Faculty of Pharmacy, Cairo University. Coupling patterns are described as

follows: s, singlet; d, doublet; t, triplet; m, multiplet; and 1H, 2H, 3H, etc. J describes a coupling constant.

The coupling constants were rounded off to one decimal place. MS spectra mass were recorded on

Shimadzu GCMS-QP 1000 EX gas chromatograph mass spectrometer in the Microanalytical center, Cairo

University. Elemental analyses were performed at the Microanalytical Center, Azhar University.

6.1.1. General method for preparation of 4-(substituted) benzaldehyde (Ia-g) [34]

A mixture of p-fluorobenzaldehyde 1 (25.0 g, 0.200 mol) and appropriate amine 2a-g (0.300 mol) and

anhydrous potassium carbonate (40.0 g) were mixed in DMF (300 mL), after which catalytic amount of

∘

Aliquat 336 reagent was added. The mixture was then refluxed for 24 hrs at 100 C. The mixture was

concentrated under low pressure and left to cool. The mixture was then poured into ice water and left

overnight. The formed solid was filtered, washed with water and crystallized with methanol to yield

compounds Ia-g.

6.1.1.1. 4-(1H-imidazol-1-yl) benzaldehyde Ia.

Yield 92% as yellow crystals, mp 152⁰C, (as reported) [34, 44]

6.1.1.2. 4-(pyrrolidin-1-yl) benzaldehyde Ib.

Yield 98% as yellow crystals, mp 85⁰C, (as reported) [45, 46]

6.1.1.3. 4-(piperidin-1-yl) benzaldehyde Ic.

Yield 98% as yellow crystals, mp 64⁰C, (as reported) [47, 48]

6.1.1.4. 4-morpholinobenzaldehyde Id.

Yield 89% as yellow crystals, mp 69^oC, (as reported) [49, 50]

6.1.1.5. 4-(4-methylpiperazin-1-yl) benzaldehyde Ie.

Yield 87% as yellow crystals, mp 64⁰C, (as reported) [45, 51]

6.1.1.6. 4-(4-phenylpiperazin-1-yl) benzaldehyde If.

Yield 98% as yellow crystals, mp 134⁰C, (as reported) [52]

6.1.1.7. 4-(4-(4-chlorophenyl) piperazin-1-yl) benzaldehyde Ig.

Yield 92% as yellow crystals, mp 133⁰C. IR: ( max, cm^-1): 1685 (C=O, CHO), 2724-2822 (CHO). ¹H-

NMR (300 MHz)(CDCl₃) δ: 3.26 (t, 4H, piperazine H₃, H₅, J = 5.1 Hz), 3.56 (t, 4H, piperazine H₂, H₆, J =

5.1 Hz), 6.91-7.04 (m, 6H, aromatic Hs), 7.79 (d, 2H, aromatic H_3’, H_5’, J = 9 Hz), 9.82 (s, 1H, CHO).

MS: m/z (%): 300 (M⁺, 89%) & base peak at 105 (100%).

6.1.2. General method for preparation of (E)-6-(4-(substituted)phenyl)-4-oxohex-5-enoic acid IIb-f and

(Z)-6-(4-(substituted) phenyl)-4-oxohex-5-enoic acid IIb,f [35]

A mixture of the respective aldehyde Ib-f (0.003 mol) and levulinic acid (0.003 mol) were dissolved in

benzene (100 mL) containing acetic acid (3 mL) and piperidine (1 mL). The solution was heated under

reflux using Dean-Stark water trap under nitrogen until the theoretical amount of water had been collected

(~6 hrs). The solvent was then evaporated under vacuum and the formed solid product was washed twice

with 10 mL of diethyl ether and then twice with 15 mL of 2 M HCl. The product was dried and purified

using solvent crystallization or column chromatography (as illustrated below).

6.1.2.1. (E)-6-(4-(pyrrolidin-1-yl) phenyl) -4-oxo-hex-5-enoic acid IIb (E).

Purified by silica gel column chromatography using hexane/ethyl acetate 8:2 eluent with E/ Z ratio 1 to 3

respectively. Yield 25% as red solid, mp 155^oC. IR: ( max, cm^-1): 1600 (C=C), 1651 (C=O, ketonic),

1

1712 (C=O, COOH), 2949-3429 (OH, COOH). H-NMR (300 MHz)(CDCl₃) δ: 2.04 (t, 4H, pyrrolidine

H₃, H₄, J = 6.6 Hz), 2.73 (t, 2H, CH₂-CH₂-COOH, J = 6.6 Hz), 3.03 (t, 2H, CH₂-CH₂-COOH, J = 6.6 Hz),

3.40 (t, 4H, pyrrolidine H₂, H_6,J = 6.6 Hz), 6.61 (d, 1H, CH=CH-CO, J = 16.2 Hz), 6.61 (d, 2H, aromatic

H₂, H₆, J = 8.4 Hz), 7.45 (d, 2H, aromatic H₃, H₅, J = 8.4 Hz), 7.61 (d, 1H, CH=CH-CO, J = 15.9 Hz).

MS: m/z (%): 273 (M⁺, 100%), 275 (M⁺+2, 8%). Anal. Calcd. For C₁₆H₁₉NO₃: C 70.31, H 7.01, N 5.12;

Found: C 70.42, H 7.13, N 5.19.

6.1.2.2. (Z)-6-(4-(pyrrolidin-1-yl) phenyl) -4-oxo-hex-5-enoic acid IIb (Z).

Purified by silica gel column chromatography using hexane/ethylacetate 8:2 eluent with Z/ E ratio 3 to 1

respectively. Yield 75% as yellow solid, mp 188^oC. IR: ( max, cm^-1): 1600 (C=C), 1644 (C=O, ketonic),

1

1709 (C=O, COOH), 2964-3445 (OH, COOH). H-NMR (400 MHz)(DMSO) δ: 1.95 (t, 4H, pyrrolidine

H₃, H₄, J = 6.4 Hz), 2.50 (t, 2H, CH₂-CH₂-COOH, J = 6.4 Hz), 2.88 (t, 2H, CH₂-CH₂-COOH, J = 6.4 Hz),

3.32 (t, 4H, pyrrolidine H₂, H₆, J = 6.4 Hz), 6.58 (d, 1H, CH=CH-CO, J = 8.8 Hz), 6.59-6.63 (m, 2H,

aromatic H₂, H₆), 7.50 (m, 2H, aromatic H₃, H₅), 7.54 (d, 1H, CH=CH-CO, J = 8.8 Hz), 12.05 (s_br, 1H,

COOH). MS: m/z (%): 273 (M⁺, 40%), 274 (M⁺+1, 28%) & base peak at 64 (100%). Anal. Calcd. For

C₁₆H₁₉NO₃: C 70.31, H 7.01, N 5.12; Found: C 70.42, H 7.13, N 5.19.

6.1.2.3. (E)-6-(4-(piperidin-1-yl) phenyl) -4-oxo-hex-5-enoic acid IIc (E).

Yield 60% as reddish purple crystals (recrystallized from ethanol), mp 176^oC. IR: ( max, cm^-1): 1600

(C=C), 1627 (C=O, ketonic), 1751 (C=O, COOH), 2928-3440 (OH, COOH). ¹H-NMR (300 MHz)(CDCl₃)

δ: 1.69 (s_br, 8H, piperidine H₃, H₄, H₅and CH₂-CH₂-COOH), 3.37 (s_br, 6H, piperidine H₂, H₆and CH₂-

CH₂-COOH), 6.61 (d, 1H, CH=CH-CO, J = 16.2 Hz), 6.89 (d, 2H, aromatic H₂, H₆, J = 8 Hz), 7.17 (d, 1H,

CH=CH-CO, J = 16.2 Hz), 7.51-7.54 (d, 2H, aromatic H₃, H₅, J = 8 Hz). MS: m/z (%): 287 (M⁺, 17.8%),

288 (M⁺+1, 5.5%) & base peak at 213 (100%). Anal. Calcd. For C₁₇H₂₁NO₃: C 71.06, H 7.37, N 4.87;

Found: C 71.22, H 7.43, N 4.93.

6.1.2.4. (E)-6-(4-morpholinophenyl) -4-oxo-hex-5-enoic acid IId (E).

Yield 56% as yellow crystals (recrystallized from ether/petroleum ether), mp 101^oC. IR: ( max, cm^-1):

1507 (C=C), 1644 (C=O, ketonic), 2964-3440 (OH, COOH). ¹H-NMR (300 MHz)(CDCl₃) δ: 2.69 (t, 2H,

CH₂-CH₂-COOH, J = 6.6 Hz), 3.08 (t, 2H, CH₂-CH₂-COOH, J = 6.6 Hz), 3.58-3.70 (m, 8H, morpholine

H), 6.75 (d, 1H, CH=CH-CO, J = 15.9 Hz), 7.08 (d, 2H, aromatic H₂, H₆, J = 8.7 Hz), 7.55 (d, 2H,

aromatic H₃, H₅, J = 8.7 Hz), 7.60 (d, 1H, CH=CH-CO, J = 15.9 Hz). MS: m/z (%): 287 (M⁺-2, 1.58%) &

base peak at 149 (100%). Anal. Calcd. For C₁₆H₁₉NO₄: C 66.42, H 6.62, N 4.84; Found: C 66.61, H 6.70,

N 4.89.

6.1.2.5. (E)-6-(4-(4-methylpiperazin-1-yl) phenyl)-4-oxo-hex-5-enoic acid IIe (E).

Yield 60% as yellow crystals (recrystallized from methanol), mp 190^oC. IR: ( max, cm^-1): 1590 (C=C),

1

1680 (C=O, ketonic), 2955-3423 (OH, COOH). H-NMR (300 MHz)(CDCl₃) δ: 2.44 (s, 3H, CH₃-

piperazine), 2.58 (t, 2H, CH₂-CH₂-COOH, J = 6.3 Hz), 2.73 (t, 4H, piperazine H₃, H₅, J = 5.1 Hz), 3.01 (t,

2H, CH₂-CH₂-COOH, J = 6.3 Hz), 3.35 (t, 4H, piperazine H₂, H₆, J = 4.8 Hz), 6.64 (d, 1H, CH=CH-CO, J

= 16.2 Hz), 6.87 (d, 2H, aromatic H₂, H₆, J = 8 Hz), 7.43 (d, 2H, aromatic H₃, H₅, J = 8 Hz), 7.54 (d, 1H,

CH=CH-CO, J = 16.2 Hz). MS: m/z (%): 302 (M⁺, 100%), 304 (M⁺+2, 2.06%). Anal. Calcd. For

C₁₇H₂₂N₂O₃: C 67.53, H 7.33, N 9.26; Found: C 67.68, H 7.41, N 9.34.

6.1.2.6. (E)-6-(4-(4-phenylpiperazin-1-yl) phenyl) -4-oxohex-5-enoic acid IIf (E).

Purified by silica gel column chromatography using hexane/ethylacetate 8:2 eluent with E/ Z ratio 1 to 2

respectively. Yield 35% as yellow needle crystals, mp 171^oC. IR: ( max, cm^-1): 1596 (C=C), 1693 (C=O,

1

ketonic), 1701 (C=O, COOH), 2954-3429 (OH, COOH). H-NMR (300 MHz)(CDCl₃) δ: 2.68 (t, 2H,

CH₂-CH₂-COOH, J = 6.6 Hz), 3.02 (t, 2H, CH₂-CH₂-COOH, J = 6.3 Hz), 3.25 (t, 4H, piperazine H₃, H₅, J

= 5.4 Hz), 3.42 (t, 4H, piperazine H₂, H₆, J = 5.7 Hz), 6.66 (d, 1H, CH=CH-CO, J = 16.2 Hz), 6.92 (d, 2H,

aromatic H_2’, H_6’, J = 7.5 Hz), 6.95-7.00 (m, 2H, aromatic H₃, H₅), 7.27-7.28 (m, 1H, aromatic H_4’), 7.34

(d, 2H, aromatic H_3’, H_5’, J = 7.8 Hz) 7.50 (d, 2H, aromatic H₂, H₆, J = 6.6 Hz), 7.58 (d, 1H, CH=CH-CO,

J = 16.2 Hz). MS: m/z (%): 364 (M⁺, 17%), 365 (M⁺+1, 21.86%) & base peak at 105 (100%). Anal. Calcd.

For C₂₂H₂₄N₂O₃: C 72.50, H 6.64, N 7.69; Found: C 72.59, H 6.69, N 7.82.

6.1.2.7. (Z)-6-(4-(4-phenylpiperazin-1-yl) phenyl) -4-oxohex-5-enoic acid IIf (Z).

Purified by silica gel column chromatography using hexane/ethylacetate 8:2 eluent with Z/ E ratio 2 to 1

respectively. Yield 62.5% as orange solid, mp 230^oC. IR: ( max, cm^-1): 1509 (C=C), 1592 (C=O,

1

ketonic), 2966-3446 (OH, COOH). H-NMR (300 MHz)(CDCl₃) δ: 2.28 (t, 2H, CH_2-CH_2-COOH), 2.99

(t, 2H, CH_2-CH_2-COOH), 3.45 (s, 4H, piperazine H₃, H₅), 3.56 (s, 4H, piperazine H₂, H₆), 6.82 (d, 1H,

CH=CH-CO), 7.00-7.14 (m, 5H, phenyl piperazine aromatic H’s), 7.25 (d, 1H, CH=CH-CO, J = 6.4 Hz),

7.65 (d, 2H, aromatic H₃, H₅, J = 8.4 Hz), 7.76 (d, 2H, aromatic H₂, H₆, J = 8.2 Hz). MS: m/z (%): 364

(M⁺, 3.81%) & base peak at 65 (100%). Anal. Calcd. For C₂₂H₂₄N₂O₃: C 72.50, H 6.64, N 7.69; Found: C

72.59, H 6.69, N 7.82.

6.1.3. General method for preparation of (E)-3-(4-substituted phenyl)acrylic acid IIIa-g [53]

A mixture of 0.208 g. (0.002 moles) of malonic acid and 0.166 g. (0.001 mol) of 4-substituted

benzaldehyde Ia-g and (5 mL) of pyridine was prepared. Malonic acid is dissolved by shaking and

warming on a steam bath. Catalytic amount of piperidine was then added and the mixture was heated at

80-85°C for 1 hr. After which the mixture was heated under reflux (109–115°C) for an additional 3 hrs.

The reaction mixture was then cooled and poured into cold water. The mixture was then acidified by slow

addition of concentrated hydrochloric acid with stirring. The formed light crystals were separated by

filtration and washed 4 times with cold water. The crude acid was dissolved in a solution of 5% sodium

hydroxide. The resulting solution was filtered, diluted with an additional water, and acidified by adding

concentrated hydrochloric acid. The formed solid was filtered and washed with cold water. The solid was

further purified by using methanol as the solvent of crystallization to yield titled compounds IIIa-g.

6.1.3.1. (E)-3-(4-(1H-imidazol-1-yl)phenyl) acrylic acid IIIa (E).

Yield 75% as pale white solid, mp 326^oC, (as reported) [54, 55]

6.1.3.2. (E)-3-(4-(pyrrolidin-1-yl)phenyl) acrylic acid IIIb (E).

Yield 85% as yellow solid, mp 265^oC, (as reported) [54, 56]

6.1.3.3. (E)-3-(4-(piperidin-1-yl)phenyl) acrylic acid IIIc (E).

Yield 87% as reddish purple solid, mp 110^oC. IR: ( max, cm^-1): 1594 (C=C), 1755 (C=O, COOH), 2928-

1

3420 (OH, COOH). H-NMR (400 MHz)(DMSO) δ: 1.54 (m, H, piperidine H₃, H₄, H₅), 3.39 (t, 4H,

piperidine H₂, H₆), 6.47 (d, 1H, CH=CH-COOH, J = 16 Hz), 7.01 (d, 2H, aromatic H₃, H₅, J = 8.8 Hz),

7.50 (d, 1H, CH=CH-COOH, J = 15.8 Hz), 7.65 (d, 2H, aromatic H₂, H₆, J = 8.8 Hz). MS: m/z (%): 231

(M⁺, 77%), 232 (M⁺+1, 58%) & base peak at 209 (100%). Anal. Calcd. For C₁₄H₁₇NO₂: C 72.70, H 7.41,

N 6.06; Found: C 72.84, H 7.48, N 6.19.

6.1.3.4. (E)-3-(4-morpholinophenyl) acrylic acid IIId (E).

Yield 75% as orange solid, mp 198^oC, (as reported) [57]

6.1.3.5. (E)-3-(4-(4-methyl piperazin-1-yl)phenyl) acrylic acid IIIe (E).

Yield 90% as yellow solid, mp 228^oC, (as reported) [54]

6.1.3.6. (E)-3-(4-(4-phenylpiperazin-1-yl)phenyl) acrylic acid IIIf (E).

Yield 90% as yellow solid, mp 240^oC. IR: ( max, cm^-1): 1600 (C=C), 1759 (C=O, COOH), 2920-3452

1

(OH, COOH). H-NMR (400 MHz)(DMSO) δ: 3.24 (t, 4H, piperazine H₃, H₅, J = 4.8 Hz), 3.48 (t, 4H,

piperazine H₂, H₆, J = 4.8 Hz), 6.25 (d, 1H, CH=CH-COOH, J = 15.6 Hz), 6.74-7.21 (m, 5H, phenyl

piperazine aromatic Hs), 7.46 (d, 1H, CH=CH-COOH, J = 15.6 Hz), 6.71 (d, 2H, aromatic H₃, H₅, J = 8.8

Hz), 7.69 (d, 2H, aromatic H₂, H₆, J = 8.8 Hz). MS: m/z (%): 310 (M⁺+2, 34%), 311 (M⁺+3, 23%) & base

peak at 282 (100%). Anal. Calcd. For C₁₉H₂₀N₂O₂: C, 74.00, H 6.54, N 9.08; Found: C 74.13, H 6.41, N

9.17.

6.1.3.7. (E)-3-(4-(4-(4-chlorophenyl)piperazin-1-yl)phenyl)acrylic acid IIIg (E).

Yield 95% as pale yellow solid, mp 201^oC. IR: ( max, cm^-1): 1594 (C=C), 1699 (C=O, COOH), 2918-

1

3434 (OH, COOH). H-NMR (300 MHz)(DMSO) δ: 3.23 (t, 4H, piperazine H₃, H₅, J = 4.5 Hz), 3.53 (t,

4H, piperazine H₂, H₆, J = 4.8 Hz), 6.32 (d, 1H, CH=CH-COOH, J = 16.2 Hz), 7.02-7.13 (m, 4H, aromatic

H₃, H₅, H_2’, H_6’), 7.48 (d, 1H, CH=CH-COOH, J = 16.2 Hz), 6.7 (d, 2H, aromatic H_3’, H_5’, J = 8.4 Hz),

7.82 (d, 2H, aromatic H₂, H₆). MS: m/z (%): 342 (M⁺, 42%), 344 (M+2, 9%) & base peak at 64 (100%).

Anal. Calcd. For C₁₉H₁₉ClN₂O₂: C 66.57, H 5.59, N 8.17; Found: C 66.68, H 5.64, N 8.29.

6.1.4. General method for preparation of 1-(substituted)-4-nitrobenzene IVa,b,e,f [34]

A mixture of p-nitro chlorobenzene 3 (25.0 g, 0.200 mol), appropriate secondary amine 2a,b,e,f (0.300

mol) and anhydrous potassium carbonate (40.0 g) were mixed in DMF (300 mL), and then catalytic drops

of Aliquat 336 reagent were added. The mixture was heated from 24-36 hrs at 100^oC. The reaction

mixture was then concentrated under vacuum, cooled and poured into ice water and left aside overnight.

The formed solid was filtered, washed with water and crystallized from ethanol to yield titled compounds

IVa,b,e,f.

6.1.4.1. 1-(4-nitrophenyl)-1H-imidazole IVa.

Yield 65% as pale yellow solid, mp 203^oC, (as reported) [58, 59]

6.1.4.2. 1-(4-nitrophenyl)pyrrolidine IVb.

Yield 98% as orange solid, mp 169^oC, (as reported) [60, 61]

6.1.4.3. 1-methyl-4-(4-nitrophenyl)piperazine IVe.

Yield 98% as orange solid, mp 104^oC, (as reported) [62]

6.1.4.4. 1-(4-nitrophenyl)-4-phenylpiperazine IVf.

Yield 92% as red solid, mp184^oC, (as reported) [63]

6.1.5. General method for preparation of 4-(substituted)aniline Va,b,e,f [36, 37]

To a solution of 1-(substituted) 4-nitrobenzene IVa,b,e,f (0.01mol) in NH₄OH (20 mL, 30%), a solution

of sodium dithionite (7g, 0.04 mol) in water (30 mL) was quickly added, the reaction mixture was refluxed

for 15 min. After cooling, the crude product was filtered, washed and crystallized from methylene chloride

to yield target compounds Va,b,e,f.

6.1.5.1. 4-(1H-imidazol-1-yl) aniline Va.

Yield 70% as grey solid, mp 147^oC, (as reported) [64, 65]

6.1.5.2. 4-(pyrrolidin-1-yl) aniline Vb.

Yield 80% as yellow oil, (as reported) [66]

6.1.5.3. 4-(4-methylpiperazin-1-yl) aniline Ve.

Yield 75% as off white acicular solid, mp 90⁰C, (as reported) [62, 67, 68]

6.1.5.4. 4-(4-phenylpiperazin-1-yl) aniline Vf.

Yield 80% as white solid, mp 128-130^oC, (as reported) [63]

6.1.6. General method for preparation of 4-(4-(substituted) phenylamino)-4-oxobutanoic acid VIa,b,e

[69]

A mixture of 4-(substituted) aniline Va,b,e (0.003 mol) and succinic anhydride (0.30 g., 0.003 mol)

were mixed in 10 (mL) methylene chloride as a solvent. The mixture was allowed to stir on cold for 24

hrs till the product is precipitated. The solid was then filtered, washed with methylene chloride and

crystallized with diethyl ether to yield the required product VIa,b,e.

6.1.6.1. 4-(4-(1H-imidazol-1-yl)phenylamino)-4-oxobutanoic acid VIa.

Yield 98% as greyish white solid, mp 238^oC. IR: ( max, cm^-1): 1654 (C=O, amide), 1715 (C=O, COOH),

1

3295 (NH), 2965-3220 (OH, COOH). H-NMR (400 MHz)(DMSO) δ: 2.56 (t, 2H, CH₂CH₂COOH, J =

5.4 Hz), 2.63 (t, 2H, CH₂CH₂COOH, J = 5.5 Hz), 7.02 (s, 1H, imidazole H₂), 7.52 (d, 2H, aromatic H₃, H₅,

J = 8.2 Hz), 7.61 (d,1H, imidazole H₄), 7.66 (d, 2H, aromatic H₂, H₆, J = 8.2 Hz), 8.11 (d, 1H, imidazole

H₅), 10.07 (s, 1H, NH), 12.08 (s_br, 1H, COOH). MS: m/z (%): 259 (M⁺, 55%), 260 (M⁺+1, 17%) & base

peak at 157 (100%). Anal. Calcd. For C₁₃H₁₃N₃O₃: C 60.22, H 5.05, N 16.21; Found: C 60.47, H 5.11, N

16.37.

6.1.6.2. 4-(4-(pyrrolidin-1-yl)phenylamino)-4-oxobutanoic acid VIb.

Yield 87% as greyish white solid, mp 195^oC. IR: ( max, cm^-1): 1652 (C=O, amide), 1713 (C=O, COOH),

3290 (NH), 2839-3115 (OH, COOH). ¹H-NMR (300 MHz)(DMSO) δ: 1.91-1.95 (m , 4H, pyrrolidine H₃,

H₄), 2.48-2.62 (m, 4H, CH₂CH₂COOH), 3.19 (t, 4H, pyrrolidine H₂, H₅, J = 6.3 Hz), 6.48 (d, 2H, aromatic

H₃, H₅, J = 8 Hz), 7.37 (d, 2H, aromatic H₂, H₆, J = 8.7 Hz), 9.58 (s, 1H, NH), 12.00 (s_br, 1H, COOH).

MS: m/z (%): 262 (M⁺, 48%), 263 (M⁺+1, 26%) & base peak at 161 (100%). Anal. Calcd. For

C₁₄H₁₈N₂O₃: C 64.10, H 6.92, N 10.68; Found: C 64.23, H 7.01, N 10.81.

6.1.6.3. 4-(4-(4-methylpiperazin-1-yl)phenylamino)-4-oxobutanoic acid VIe.

Yield 85% as greyish white solid, mp 168^oC. IR: ( max, cm^-1): 1659 (C=O, amide), 1710 (C=O, COOH),

1

3297 (NH), 2954-3212 (OH, COOH). H-NMR (400 MHz)(DMSO) δ: 2.19 (t , 4H, piperazine H₃, H₅),

2.34 (s, 3H, CH₃-piperazine), 2.44 (t, 2H, CH₂CH₂COOH, J = 6.6 Hz), 2.99 (t, 2H, CH₂CH₂COOH, J =

6.6 Hz), 3.34 (t , 4H, piperazine H₂, H₆), 6.81 (d, 2H, aromatic H₃, H₅, J = 8.8 Hz), 7.36 (d, 2H, aromatic

H₂, H₆, J = 8.8 Hz), 9.65 (s, 1H, NH ). MS: m/z (%): 291 (M⁺, 38%), 292 (M⁺+1, 12%) & base peak at

189 (100%). Anal. Calcd. For C₁₅H₂₁N₃O₃: C 61.84, H 7.27, N 14.42; Found: C 62.01, H 7.36, N 14.65.

6.1.7. General method for preparation of 5-(4-(substituted) phenylamino)-5-oxopentanoic acid VIIa,f

[70]

A mixture of 4 (substituted) aniline Va,f (0.003 mol) and glutaric anhydride (0.34 g., 0.003 mol) were

dissolved in (10 mL) methylene chloride. The solution was allowed to stir on cold for 24 hrs till the

product is precipitated. The solid was then filtered, washed with methylene chloride and crystallized with

diethyl ether to yield the required product VIIa,f.

6.1.7.1. 5-(4-(1H-imidazol-1-yl)phenylamino)-5-oxopentanoic acid VIIa.

o

Yield 90 % as greyish white solid, mp 230 C.IR: ( max, cm^-1): 1659 (C=O, amide), 1708 (C=O,

1

COOH), 3295 (NH), 2899-3186 (OH, COOH). H-NMR (400 MHz)(DMSO) δ: 1.78 (t, 2H, CH₂-CH₂-

CH₂-COOH, J = 7.2 Hz), 2.24 (t, 2H, CH₂-CH₂-CH₂-COOH, J = 7.2 Hz ), 2.30 (t, 2H, CH₂-CH₂-CH₂-

COOH, J = 7.2 Hz), 7.02 (s, 1H, imidazole H₂), 7.49 (d, 2H, aromatic H_3,H₅, J = 8.8 Hz), 7.61 (d, 1H,

imidazole H₄), 7.64 (d, 2H, aromatic H₂, H₆, J = 8.8 Hz), 8.11 (d , 1H, imidazole H₅), 10.00 (s, 1H, NH ),

12.04 (s_br, 1H, COOH). MS: m/z (%): 273 (M⁺, 49%), 274 (M⁺+1, 18%) & base peak at 84 (100%). Anal.

Calcd. For C₁₄H₁₅N₃O₃: C 61.53, H 5.53, N 15.38; Found: C 61.76, H 5.59, N 15.52.

6.1.7.2. 5-(4-(4-phenylpiperazin-1-yl)phenylamino)-5-oxopentanoic acid VIIf.

Yield 95% as greyish white solid, mp 223^oC. IR: ( max, cm^-1): 1653 (C=O, amide), 1701 (C=O, COOH),

3291 (NH), 2962-3181 (OH, COOH). ¹H-NMR (300 MHz)(DMSO) δ: 1.77-1.82 (t, 2H, CH₂-CH₂-CH₂-

COOH, J = 7.2 Hz), 2.24-2.33 (m, 4H, CH₂-CH₂-CH₂-COOH), 3.20-3.27 (m, 8H, piperazine ), 6.78 (t, 1H,

aromatic H_4’,J = 7.2 Hz), 6.92 (d, 2H, aromatic H₃, H₅, J = 8 Hz), 7.01 (d, 2H, aromatic H_2’, H_6’, J = 8.1

Hz), 7.26 (d, 2H, aromatic H_3’, H_5’, J = 7.2 Hz), 7.44 (d, 2H, aromatic H₂, H₆, J = 8 Hz), 9.69 (s, 1H, NH

), 12.00 (s_br, 1H, COOH). MS: m/z (%): 367 (M⁺, 37%), 368 (M⁺+1, 29%) & base peak at 80 (100%).

Anal. Calcd. ForC₂₁H₂₅N₃O₃: C 68.64, H 6.86, N 11.44; Found: C 68.78, H 6.56, N 11.59.

6.1.8. General method for the preparation of 2-((4-substituted)phenyl)carbamoyl) benzoic acid VIIIa,e

[71]

A mixture of 4 (substituted) aniline Va,e (0.003 mol) and phthalic anhydride (0.44 g., 0.003 mol) were

dissolved in (10 mL) methylene chloride. The solution was allowed to stir on cold for 24 hrs till the

product is precipitated. The solid was then filtered, washed with methylene chloride and crystallized with

diethyl ether to yield the required product VIIIa,e.

6.1.8.1. 2-((4-(1H-imidazol-1-yl)phenyl)carbamoyl) benzoic acid VIIIa.

Yield 95% as greyish white solid, mp 264^oC. IR: ( max, cm^-1): 1659 (C=O, amide), 1711 (C=O, COOH),

3295 (NH), 2972-3214 (OH, COOH). ¹H-NMR (400 MHz)(DMSO) δ: 7.06 (s, 1H, imidazole H₂), 7.50-

7.63 (m, 4 H, phthalic), 7.57 (d, 2H, aromatic H_3,H₅, J = 8.8 Hz), 7.65 (d ,1H, imidazole H₄), 7.76 (d, 2H,

aromatic H₂, H₆, J = 8.8 Hz), 8.17 (d , 1H, imidazole H₅), 10.43 (s, 1H, NH). MS: m/z (%): 307 (M⁺,

55%), 308 (M⁺+1, 17%) & base peak at 121 (100%). Anal. Calcd. For C₁₇H₁₃N₃O₃: C 66.44, H 4.26, N

13.67; Found: C 66.6, H 4.32, N 13.79.

6.1.8.2. 2-((4-(4-methylpiperazin-1-yl)phenyl)carbamoyl) benzoic acid VIIIe.

Yield 89% as off white solid, mp 192.5^oC. IR: ( max, cm^-1): 1650 (C=O, amide), 1702 (C=O, COOH),

3298 (NH), 2994-3221 (OH, COOH). ¹H-NMR (400 MHz)(DMSO) δ: 1.95 (t, 4H, piperazine H_3,H₅), 2.30

(s, 3H, CH₃-piperazine), 3.22 (t, 4H, piperazine H_3,H₅), 6.91 (d, 2H, aromatic H_3,H₅, J = 8.8 Hz), 7.46 (d,

2H, aromatic H₂, H₆, J = 8.8 Hz), 7.44-7.60 (m, 4H, phthalic), 10.09 (s, 1H, NH). MS: m/z (%): 339 (M⁺,

60%), 340 (M⁺+1, 28%) & base peak at 189 (100%). Anal. Calcd. For C₁₉H₂₁N₃O₃: C, 67.24, H 6.24, N

12.38; Found: C 67.38, H 6.29, N 12.51.

6.2. Cell growth inhibitory activity in cancer cells

6.2.1. Evaluation for cytotoxic activity against a panel of sixty human cancer cell lines

National Cancer Institute (NCI) for in vitro anticancer assay evaluates different compounds for their

anticancer activity. The screening is a two-stage process, beginning with the evaluation of all compounds

against the full NCI 60 cell lines panel representing leukemia, Non-Small Cell Lung Cancer, melanoma,

colon cancer, CNS cancer, breast cancer, ovarian cancer, renal cancer and prostate cancer at a single dose

of 10^-5M. The output from the single dose screen is reported as a mean graph and is available for analysis

by the COMPARE program. Compounds which are selected are evaluated against the 60 cell panel at five

concentration levels.

6.2.1.1. Assay protocol

Methodology of assay is as reported. [72, 73] The human tumor cell lines of the cancer-screening panel

are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical

screening experiment, cells are inoculated into 96 well microtiter plates in 100 ml at plating densities

ranging from 5000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell

inoculation, the microtiter plates are incubated at 37^oC, 5% CO2, 95% air and 100% relative humidity for

24 hrs prior to addition of experimental drugs. After 24 hrs, two plates of each cell line are fixed in situ

with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition

(Tz). Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test

concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate

is thawed and diluted to twice the desired final maximum test concentration with complete medium

containing 50 mg/ml Gentamicin. Additional four, 10-fold or ½log serial dilutions are made to provide a

total of five drug concentrations plus control. Aliquots of 100 ml of these different drug dilutions are

added to the appropriate microtiter wells already containing 100 ml of medium, resulting in the required

final drug concentrations. Following drug addition, the plates are incubated for an additional 48 h at 37⁰C,

5% CO2, 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by the addition

of cold TCA. Cells are fixed in situ by the gentle addition of 50 ml of cold 50% (w/v) TCA (final

concentration, 10% TCA) and incubated for 60 min at 4⁰C. The supernatant is discarded, and the plates are

washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 ml) at 0.4% (w/v)

in 1% acetic acid is added to each well, and plates are incubated for 10 min at room temperature. After

staining, unbound dye is removed by washing five times with 1% acetic acid and the plates are air dried.

Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an

automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same

except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 ml

of 80% TCA (final concentration, 16% TCA).

6.2.1.2. Data analysis

Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the

presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of the

drug concentrations levels. Percentage growth inhibition is calculated as: [(Ti - Tz)/(C - Tz)] -100 for

concentrations for which Ti>/ ¼ Tz and [(Ti - Tz)/Tz] -100 for concentrations for which Ti < Tz: three

dose response parameters are calculated for each experimental agent. [74]

6.2.2. Evaluation for cytotoxicity against HepG2 Cancer cell line:

6.2.2.1. Cell culture

HepG2 human hepatocellular carcinoma cells were grown in RPMI-1640 medium, supplemented with

10% heat inactivated FBS, 50 units/mL of penicillin and 50 mg/mL of streptomycin and maintained at 37°

in a humidified atmosphere containing 5% CO₂. The cells were maintained as “monolayer culture” by

serial subculturing.

6.2.2.2. SRB cytotoxicity assay

Cytotoxicity was determined using SRB method as previously described by Skehan et al. [75]

Exponentially growing cells were collected using 0.25% Trypsin-EDTA and seeded in 96-well plates at

1000-2000 cells/well in RPMI-1640 supplemented medium. After 24 h, cells were incubated for 72 h with

various concentrations of the tested compounds. Following 72 h treatment, the cells will be fixed with

10% trichloroacetic acid for 1 h at 4 ºC. Wells were stained for 10 min at room temperature with 0.4%

SRB dissolved in 1% acetic acid. The plates were air dried for 24 h and the dye was solubilized with Tris-

HCl for 5 min on a shaker at 1600 rpm. The optical density (OD) of each well was measured

spectrophotometrically at 564 nm with an ELISA microplate reader (ChroMate-4300, FL, USA).

6.2.3. Data analysis

The IC₅₀values were calculated according to the equation for Boltzmann sigmoidal concentration–

response curve using the nonlinear regression fitting models (Graph Pad, Prism Version 5).

6.3. Histone deacetylase inhibitory activity

6.3.1. Materials and methods

The histone deacetylase activity was carried out by BPS Bioscience (www.bpsbioscience.com). Single

dose concentration of 10 µM HDAC substrate 3 (BPS#50037) of HDAC1 (BPS#50051), HDAC2

(BPS#50002), HDAC3 (BPS#50003), HDAC6 (BPS#50006) and HDAC10 (BPS#50010) was used in

testing. And also it was carried on 2 µM HDAC substrate 1 (BPS#50040) of HDAC4 (BPS#50004),

HDAC5 (BPS#50005), HDAC7 (BPS#50007), HDAC8 (BPS#50008), HDAC9 (BPS#50009) and

HDAC11 (BPS#50021). Other materials are used as HDAC Assay Buffer (BPS#50031), HDAC Assay

Developer (BPS#50030), SAHA as a reference compound purchased from sigma (St. Louis, MO

#SML0061) and TSN as another reference compound purchased from Selleckcom.com (#S1045).

6.3.2. Assay protocols

6.3.2.1. In vitro HDAC1 inhibitory assay

A 100µM solution of each compound was prepared with 10% DMSO in HDAC assay buffer and 5µl

of the dilution was added to a 50µl reaction so that the final concentration of DMSO is 1% in all of

reactions. The enzymatic reactions for the HDAC enzymes were conducted in duplicate at 37ºC for 30

minutes in a 50µl mixture containing HDAC assay buffer, 5µg BSA, HDAC substrate 3, HDAC1 enzyme

and a test compound (10 µM). After enzymatic reactions, 50μl of 2 x HDAC Developer was added to each

well for the HDAC enzymes and the plate was incubated at room temperature for an additional 15

minutes. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm

using a Tecan Infinite M1000 microplate reader.

6.3.2.2. In vitro HDAC2-11 inhibitory assay

A 500µM solution of each compound was prepared with 10% DMSO in HDAC assay buffer and 5µl

of the dilution was added to a 50µl reaction so that the final concentration of DMSO is 1% in all of

reactions. The enzymatic reactions for the HDAC enzymes were conducted in duplicate at 37ºC for 30

minutes in a 50µl mixture containing HDAC assay buffer, 5µg BSA, HDAC substrate 1/ 3, a HDAC 2-11

enzyme and a test compound (50 µM).After enzymatic reactions, 50μl of 2 x HDAC Developer was added

to each well for the HDAC enzymes and the plate was incubated at room temperature for an additional 15

minutes. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm

using a Tecan Infinite M1000 microplate reader.

6.3.3 Data analysis

HDAC activity assays were performed in duplicates at each compound. The fluorescent intensity data

were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the

fluorescent intensity (F_t) in each data set was defined as 100% activity. In the absence of HDAC, the

fluorescent intensity (F_b) in each data set was defined as 0% activity. The percent activity in the presence

of each compound was calculated according to the following equation: %activity = (F-F_b)/(F_t-F_b), where

F= the fluorescent intensity in the presence of the compound. % Inhibition was calculated as: % inhibition

= 100 (%) _ % activity.

Acknowledgments

The authors are grateful to The National Cancer Institute, Maryland, USA for performing anticancer

activity and would like to thank BPS Bioscience for EGFR binding assay. And also would like to thank

Dr. Ahmed Essmat (Pharmacology &Toxicology Department, Faculty of Pharmacy, AinShams University,

Cairo, Egypt 11566) for performing the in vitro cytotoxicity assays against Liver HepG2 Cancer cell line.

References and notes

[1]

[2]

[3]

[4]

[5]

[6]

W. D. Cress, E. Seto, J. Cell. Physiol. 184 (2000) 1-16.

B. D. Strahl, C. D. Allis, Nature 403 (2000) 41-45.

V. Morales, H. Richard-Foy, Mol. Cell. Biol. 20 (2000) 7230-7237.

K. Struhl, Genes Dev. 12 (1998) 599-606.

P. A. Wade, Hum. Mol. Genet. 10 (2001) 693-698.

A. J. de Ruijter, A. H. van Gennip, H. N. Caron, S. Kemp, , A. B. van Kuilenburg, Biochem. J. 370

(2003) 737-749.

[7]

[8]

C. Hildmann, D. Riester, A. Schwienhorst, Appl. Microbiol. Biotechnol. 75 (2007) 487-497.

D. C. Drummond, C. O. Noble, D. B. Kirpotin, Z. Guo, G. K. Scott, C. C. Benz, Annu. Rev.

Pharmacol. Toxicol. 45 (2005) 495-528.

[9] H. Lu, Y. Chen, B. Yang, Q. You, Acta Pharma. Sin. B 1 (2011) 240-247.

[10] A.Vannini, C. Volpari, P. Gallinari, P. Jones, M. Mattu, A. Carfi, , R. De Francesco, C.

Steinkuhler, S. Di Marco, EMBO Rep. 8 (2007) 879-884.

[11] K. B. Glaser, Biochem. Pharmacol. 74 (2007) 659-671.

[12] M. Dokmanovic, P. A. Marks, J. Cell. Biochem. 96 (2005) 293-304.

[13] S. Ropero, M. Esteller, Mol. Oncol. 1 (2007) 19-25.

[14] M. Duvic, R. Talpur, X. Ni, C. Zhang, P. Hazarika, C. Kelly, J. H. Chiao, J. F. Reilly, J. L. Ricker,

V. M. Richon, S. R. Frankel, Blood 109 (2007) 31-39.

[15] P. A. Marks, R. Breslow, Nat. Biotechnol. 25 (2007) 84-90.

[16] X. Kong, Z. Lin, D. Liang, D. Fath, N. Sang, J. Caro, Mol. Cell. Biol. 26 (2006) 2019-2028.

[17] A. Grassadonia, P. Cioffi, F. Simiele, L. Iezzi, M. Zilli, C. Natoli, Cancers (Basel) 5 (2013) 919-

942.

[18] B. Venugopal, T. R. Evans, Curr. Med. Chem. 18 (2011) 1658-1671.

[19] O. Moradei, C. R. Maroun, I. Paquin, A. Vaisburg, Curr. Med. Chem. Anticancer Agents 5 (2005)

529-560.

[20] J. E. Bolden, M. J. Peart, R. W. Johnstone, Nat. Rev. Drug Discov. 5 (2006) 769-784.

[21] F. Wang, W. Lu, T. Zhang, J. Dong, H. Gao, P. Li, S. Wang, Zhang, J. Bioorg. Med. Chem. 21

(2013) 6973-6980.

[22] T. A. Miller, , D. J. Witter, Belvedere, S. J. Med. Chem. 46 (2003) 5097-5116.

[23] M. S. Finnin, J. R. Donigian, A. Cohen, V. M. Richon, R. A. Rifkind, P. A. Marks, R. Breslow, N.

P. Pavletich, Nature 401 (1999) 188-193.

[24] Y. D. Chen, Y. J. Jiang, J. W. Zhou, Q. S. Yu, Q. D. You, J. Mol. Graph. Model 26 (2008) 1160-

1168.

[25] M. Jung, G. Brosch, D. Kolle, H. Scherf, C. Gerhauser, P. Loidl, J. Med. Chem. 42 (1999) 4669-

4679.

[26] S. M. Sternson, J. C. Wong, C. M. Grozinger, S. L. Schreiber, Org. Lett. 3 (2001) 4239-4242.

[27] B. Gopalan, T. Ponpandian, V. Kachhadia, K. Bharathimohan, R. Vignesh, V. Sivasudar, S.

Narayanan, B. Mandar, R. Praveen, N. Saranya, S. Rajagopal, S. Rajagopal, Bioorg. Med. Chem. Lett. 23

(2013) 2532-2537.

[28] A. V. Bieliauskas, S. V. Weerasinghe, M. K. Pflum, Bioorg. Med. Chem. Lett. 17 (2007) 2216-

2219.

[29] E. Hahnen, J. Hauke, C. Trankle, I. Y. Eyupoglu, B. Wirth, I. Blumcke, Expert. Opin. Investig.

Drugs 17 (2008) 169-184.

[30] G. Bora-Tatar, D. Dayangac-Erden, A. S. Demir, S. Dalkara, K.Yelekci, H. Erdem-Yurter, Bioorg.

Med. Chem. 17 (2009) 5219-5228.

[31] J. S. Chen, D. V. Faller, R. A. Spanjaard, Curr. Cancer Drug Targets 3 (2003) 219-236.

[32] M. Gottlicher, S. Minucci, P. Zhu, O. H. Kramer, A. Schimpf, S. Giavara, J. P. Sleeman, F. Lo

Coco, C. Nervi, P. G. Pelicci, T. Heinzel, EMBO J. 20 (2001) 6969-6978.

[33] J. Pearn, Lancet 1 (1980) 919-922.

[34] Z. Q. Liang, C. X. Wang, J. X. Yang, H.W. Gao, Y. P. Tian, X. T. Tao, M. H. Jiang, New J. Chem.

31 (2007) 906-910.

[35] K. Abouzid, P. Frohberg, J. Lehmann, M. Decker, Med. Chem. 3 (2007) 433-438.

[36] S. Mowafy, N. A. Farag, K. A. M. Abouzid, Eur J. Med. Chem. 61 (2013) 132-145.

[37] H. A. A. Shady, S. L. El-Ansary, , D. A. A. El-Ella, N. A. Farag, Bull. Fac. Pharm. Cairo Univ. 42

(2004) 31-42.

[38] S. Ray, C. Lee, T. Hou, I. Boldogh, A. R. Brasier, Nucleic Acids Res. 36 (2008) 4510-4520.

[39] H. Su, A. Nebbioso, V. Carafa, Y. Chen, B. Yang, L. Altucci, Q. You, Bioorg. Med. Chem 16

(2008) 7992-8002.

[40] H. Li, J. Sutter, R. Hoffmann, HypoGen: An Automated System for Generating Predictive 3D

Pharmacophore Models, in: Pharmacophore Perception, Development, and use in Drug Design. O. F.

Güner (Ed.), International University Line, La Jolla, CA 2000 pp171–187.

[41] R. F. George, N. S. M. Ismail, J. Stawinski, A. S. Girgis Eur J. Med. Chem. 68 (2013) 339-351.

[42] A. S. Girgis, J. Stawinski, N. S. M. Ismail, H. Farag, Eur J. Med. Chem. 47 (2012) 312-322.

[43] A. K. Ghose, G. M. Crippen, J. Comput. chem. 7 (1986) 565-577.

[44]

L. Zhu, G. Li, L. Luo, P. Guo, J. Lan, J. You, J. Org. chem. 74 (2009) 2200-2202.

[45] D. Kumar, K. K. Raj, M. Bailey, T. Alling, T. Parish, D. S. Rawat, Bioorg. Med. Chem. Lett. 23

(2013) 1365-1369.

[46] M. Sarma, T. Chatterjee, S. Ghanta, S. K. Das, J. Org. chem. 77 (2012) 432-444.

[47]

N. R. Eibergen, I. Im, N. Y. Patel, P. Hergenrother, J. Chembiochem : A Eur. J. chem. biology 13

(2012) 574-584.

[48]

P. G. Mandhane, R. S. Joshi, D. R. Nagargoje, A. V. Chate, C. H. Gill, Phosphorus, Sulfur Silicon

Relat. Elem. 186 (2010) 149-158.

[49] R. S. Joshi, P. G. Mandhane, S. D. Diwakar, S. K. Dabhade, C. H. Gill, Bioorg. Med. Chem. Lett.

20 (2010) 3721-3725.

[50] M. Meciarova, S. Toma, P. Magdolen, Ultrason. Sonochem. 10 (2003) 265-270.

[51] S. Lieber, F. Scheer, W. Meissner, S. Naruhn, T. Adhikary, S. Muller-Brusselbach, W. E.

Diederich, R. Muller, J. Med. Chem. 55 (2012) 2858-2868.

[52]

L. Heping, G. Cancheng, R. Jianming, H. Boyun, Chinese J. Org. Chem. 24 (2004) 783-787.

[53]

S. R. Byeon, Y. J. Jin, S. J. Lim, J. H. Lee, K. H. Yoo, K. J. Shin, S. J. Oh, D. Kim, J. Bioorg.

Med. Chem. Lett. 17 (2007) 4022-4025.

[54] A. Bombrun, U.S. Patent 6, 306, 870B1, 2001.

[55] I. Sircar, B. L. Duell, J. A. Bristol, R. E. Weishaar, D. B. Evans, J. Med. Chem. 30 (1987) 1023-

1029.

[56] S. Sasmal, D. Balasubrahmanyam, H. R. Kanna Reddy, G. Balaji, G. Srinivas, S. Cheera, C.

Abbineni, P. K. Sasmal, I. Khanna, V. J. Sebastian, V. P. Jadhav, M. P. Singh, R. Talwar, J. Suresh, D.

Shashikumar, K. Harinder Reddy, V. Sihorkar, T. M. Frimurer, O. Rist, L. Elster, T. Hogberg, Bioorg.

Med. Chem. Lett. 22 (2012) 3163-3167.

[57] J. Guo, W. Dong, W. Liu, Z. Yan, N. Wang, Z. Liu, Eur J. Med. Chem. 62 (2013) 670-676.

[58] K.Yang, Y. Qiu, Z. Li, Z. Wang, S. Jiang, J. Org. Chem. 76 (2011) 3151-3159.

[59] Q. Zhang, J. Luo, Y. Wei, Synth. Commun. 42 (2011) 114-121.

[60] R. N. Prabhu, R. Ramesh, Tetrahedron Lett. 54 (2013) 1120-1124.

[61] B. Sreedhar, R. Arundhathi, P. L. Reddy, M. L. Kantam,. J. Org. Chem. 74 (2009) 7951-7954.

[62] M. I. Husain, S. K. Agarwal, Ind. J. Chem. 13 (1975) 1238-1239.

[63] M. Khatri, S. K. Rai, S. Alam, A. Vij, M. Tiwari, Bioorg. Med. Chem. 17 (2009) 1890-1897.

[64] J. M. Smallheer, R. S. Alexander, J. Wang, S. Wang, S. Nakajima, K. A. Rossi, A. Smallwood, F.

Barbera, D. Burdick, J. M. Luettgen, R. M. Knabb, R. R. Wexler, P. K. Jadhav, Bioorg. Med. Chem. Lett.

14 (2004) 5263-5267.

[65] J. Engel-Andreasen, B. Shimpukade, T. Ulven, Green Chem. 15 (2013) 336-340.

[66] S. K. Singh, M. Srinivasa Reddy, M. Mangle, K. Ravi Ganesh, Tetrahedron 63 (2007) 126-130.

[67] P. Xiang, T. Zhou, L. Wang, C. Y. Sun, J. Hu, Y. L. Zhao, L. Yang, Molecules 17 (2012) 873-883.

[68] Desai, D. Akshay, E-J. Chem. 2 (2005) 15-20.

[69] Y. Liu, L. Shang, H. Fang, H. Zhu, J. Mu, Q. Wang, X. Wang, Y. Yuan, W. Xu, Bioorg. Med.

Chem. 17 (2009) 7398-7404.

[70] Y. Xu, N. Yamamoto, D. I. Ruiz, D. S. Kubitz, K. D. Janda, Bioorg. Med. Chem. Lett. 15 (2005)

4304-4307.

[71] A. M. Granados, R. H. de Rossi, J. Org. Chem. 66 (2001) 1548-1552.

[72] R. H. Shoemaker, Nat. Rev. Cancer 6 (2006) 813-823.

[73] M. R. Grever, S. A. Schepartz, B. A. Chabner, Semin. Oncol. 19 (1992) 622-638.

[74] M. C. Alley, D. A. Scudiero, A. Monks, M. L. Hursey, M. J. Czerwinski, D. L. Fine, B. J. Abbott,

J. G. Mayo, R. H. Shoemaker, M. R. Boyd, Cancer Res. 48 (1988) 589-601.

[75] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J. T. Warren, H. Bokesch,

S. Kenney, M. R. Boyd, J. Natl. Cancer Inst. 82 (1990) 1107-1112.

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Design, synthesis, 3D pharmacophore, QSAR, and docking studies of carboxylic acid derivatives as Histone Deacetylase inhibitors and cytotoxic agents

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