Acridone-pyrimidine hybrids- design, synthesis, cytotoxicity studies in resistant and sensitive cancer cells and molecular docking studies

Article abstract of DOI:10.1016/j.ejmech.2017.08.023

Hybrid systems of acridones with substituted pyrimidines were designed with an objective of discovering next generation anticancer agents targeting multiple mechanisms in the cancer cell. Hybrid compounds were synthesized by simple and convenient methods

Full text of DOI:10.1016/j.ejmech.2017.08.023

Accepted Manuscript
Acridone-pyrimidine hybrids- design, synthesis, cytotoxicity studies in resistant and
sensitive cancer cells and molecular docking studies
Manikanta Murahari, Karanam Vanitha Prakash, Godefridus J. Peters, Y.C. Mayur
PII:
S0223-5234(17)30626-8
10.1016/j.ejmech.2017.08.023
EJMECH 9667
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 May 2017
Revised Date: 26 July 2017
Accepted Date: 8 August 2017
Please cite this article as: M. Murahari, K.V. Prakash, G.J. Peters, Y.C. Mayur, Acridone-pyrimidine
hybrids- design, synthesis, cytotoxicity studies in resistant and sensitive cancer cells and molecular
docking studies, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.08.023.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Acridone-pyrimidine hybrids- Design, Synthesis, Cytotoxicity studies in resistant and
sensitive cancer cells and molecular docking studies
[
a]
[b]
,
[c]
Manikanta Murahari , Karanam Vanitha Prakash Godefridus J. Peters , Mayur C.
[
a,d]*
Yergeri
[
a]
Department of Pharmaceutical Chemistry, SVKM’s Dr. Bhanuben Nanavati College of
Pharmacy, Mumbai-400 056. India.
[
b]
Department of Pharmaceutical Chemistry, SSJ College of Pharmacy, Hyderabad-500 075,
India.
[
c]
Department of Medical Oncology, VU University Medical Center, Amsterdam, The
Netherlands
[
d]
Department of Pharmaceutical Chemistry, SPP School of Pharmacy & Technology
Management, SVKM’s NMIMS- Deemed to be University, Mumbai-400 056. India.
Graphical abstract

ACCEPTED MANUSCRIPT
Acridone-pyrimidine hybrids- Design, Synthesis, Cytotoxicity studies in resistant and
sensitive cancer cells and molecular docking studies
[
a]
[b]
[c]
[d]*
Manikanta Murahari , Karanam Vanitha Prakash Godefridus J. Peters , Mayur YC
,
[
a]
Department of Pharmaceutical Chemistry, SVKM’s Dr. Bhanuben Nanavati College of
Pharmacy, Mumbai-400 056. India.
[
b]
Department of Pharmaceutical Chemistry, SSJ College of Pharmacy, Hyderabad-500 075,
India.
[
c]
Department of Medical Oncology, VU University Medical Center, Amsterdam, The
Netherlands
[
d]
Department of Pharmaceutical Chemistry, SPP School of Pharmacy & Technology
Management, SVKM’s NMIMS- Deemed to be University, Mumbai-400 056. India.
*
Corresponding Author:
Dr. Mayur C. Yergeri
Department of Pharmaceutical Chemistry,
SPPSPTM
SVKM’s NMIMS University
Vile Parle (West), Mumbai- 400 056. India.
Tel: (+91)- 9930713869,
Fax: 0091-22-26132905
E-mail: mayuryc@rediff.com, mayuryc@hotmail.com
1

ACCEPTED MANUSCRIPT
ABSTRACT
Hybrid systems of acridones with substituted pyrimidines were designed with an objective of
discovering next generation anticancer agents targeting multiple mechanisms in the cancer cell.
Hybrid compounds were synthesized by simple and convenient methods in the lab, characterized
by NMR and Mass spectral methods and screened for cytotoxicity against A549 (lung), Hela
(cervical), MCF7 (breast) and MDA-MB-231 (breast) cancer cell lines respectively. Evaluation
of compounds for cell proliferation identified active compounds 11b, 11d and 11h against
MCF7, MDA-MB-231 and A549 cell lines. Further absorption titrations with CT-DNA and gel
electrophoresis identified that hybrid molecules displayed anticancer activity partly by DNA
intercalation. Also further results of western blotting assay with Akt kinase identified that hybrid
compounds have the ability to inhibit the Akt kinase activity and induce apoptosis, with ABCC1
suggests that active compounds too have the ability to modulate multidrug resistance (MDR)
associated with ABCC1/MRP1. Selective Akt1 kinase assay have identified 11a, 11b, 11d and
1
1h as potential inhibitors. Molecular docking studies identified the orientation and binding
interactions at the active site of Akt1 and DNA. Compounds 12e and 12f have shown good
cytotoxicity profile against lung cancer cell lines of sensitive and resistant type. Acute toxicity
study of compound 12f at the dose of 5000 mg/kg has identified no signs of clinical toxicity.
Prediction of ADMET properties and oral toxicity of the drug likeness features of new hybrid
systems were carried out using software’s. This experimental data suggests that hybrid systems
of acridone with substituted pyrimidines can be taken as a lead for the design of efficient
inhibitors and active compounds which can be taken up for further studies.
KEY WORDS: acridone, pyrimidine, carbohydrazide, hybrid molecules, anticancer activity
2

ACCEPTED MANUSCRIPT
1
. Introduction
Cancer is one of the leading cause of death in the world till today. Advancement of technology,
climate changes and life style has aggravated the situation of cancer as a disease, due to its
abnormal cell mutations that can cause in any human at any given time through multiple
mechanisms in different environment compared to normal healthy human cell [1-4]. If a drug
molecule acting against cancer has the ability to reach target site and specific to cancer cell,
capable of binding to multiple targets might be beneficial and may also prevent in acquiring drug
resistance [5-7]. Based on the above parameters, acridone is a versatile heterocyclic structure
having unique chemical properties like planar structure which exists in both protonated and
unprotonated forms, ability to intercalate with DNA, also as a substrate to P-glycoprotein which
was one of the essential target for multidrug resistance (MDR) in cancer. Because of this ideal
and unique properties, acridone has drawn attention of medicinal chemists and few of its
derivatives are currently under clinical trials [8-11]. Pyrimidine is a six membered heterocyclic
ring with nitrogen atoms, building block of nucleotides DNA and RNA, exhibits wide variety of
pharmacological activities and reported extensively for the treatment of cancer. Also this unique
chemical structure of pyrimidine shows anticancer property through multiple mechanisms and
hase the ability to interact with diverse target sites i.e., enzymes, receptors, DNA, protein targets,
etc. Recent literature suggests that 59 patents have been published on pyrimidine based
derivatives as anticancer agents between the years 2009-2014 across the globe. Almost 32
patents have been published in the period 2012 itself which further highlights the potential of
pyrimidine scaffolds potency to display anticancer activity [12-13]. Based on this we have tried
to combine acridone and pyrimidine scaffolds and synthesized a series of novel hybrid molecules
which would have ability to display anticancer activity by interacting with multiple targets. The
linker used for the conjugation of acridone with pyrimidines was an hydrazide. Hydrazide and
substituted hydrazides have been used as versatile building blocks for the conjugation of
heterocylic systems. Carbohydrazide containing molecules have shown diverse pharmacological
activities. Hydrazide of acridone is synthesized by treating ester of acridone with hydrazine
hydrate. The study also helps in identifying the potential of acridones with hydrazide linker
against sensitive and resistant cancer decreases the ability of cancer cells acquiring resistance.
3

ACCEPTED MANUSCRIPT
For examples, Zhang et al have reported the series of pyridyl acridone derivatives as potent DNA
binding and apoptosis inducing agents. Compounds have shown good cytotoxicity profile against
K562 (human leukemia cells) by MTT assay and identified active compound (1) with an IC of
5
0
0
.46 µM (Figure 1). Further mechanistic studies identified that active compound was exhibiting
cytotoxicity through intercalation of DNA, inhibition of topoisomerase I and induction of
apoptosis through mitochondrial pathway. Study identified the effect of length of alkyl chain,
carboxamide group, N,N-dimethylamino group, number of pyridyl groups and position of
nitrogen atom in pyridine ring has suggested that pyridyl acridones can be considered as lead for
design of potent DNA binding agents [14]. Maryam et al have reported the in vitro cytotoxicity
and apoptosis induction of acridone with 1,2,3-triazoles. Also literature suggests that triazole
moiety has high dipole moment, metabolic stability, hydrogen bonding ability. To explore the
cytotoxicity of hybrid systems, compounds were designed, synthesized and screened against
human breast cancer cell lines of MCF7, MDA-MB-231 and T-47D. These compounds have
shown good cytotoxicity profile and identified active compound (2) with an IC50 of 11.0 µM
against MCF7 which is more potent than etoposide (IC - 12.4 µM). The study confirmed the
5
0
apoptosis induction of active compound by flow cytometric analysis [15].
4

ACCEPTED MANUSCRIPT
(1)
(2)
Figure 1. Structures of acridone hybrid systems as anticancer agents
Shao et al have reported the in vitro anticancer activity of pyrimidine-benzimidazole hybrids
against MCF7, MGC-803, EC-9706 and SMMC-7221 human cancer cell lines. The compounds
have shown good cytotoxicity profile against MCF7 and MGC-803 and identified two active
compounds (3 and 4) with an IC values ranging from 1.40 to 10.58 µM and 1.06 to 9.70 µM
5
0
against all four cell lines respectively, exhibiting cytotoxicity more potent than 5-fluoro uracil.
Also flow cytometry analysis of compound 4 (Figure 2) identified that cytotoxicity in MGC-803
was mediated through apoptosis induction and cell cycle arrest at G2/M phase [16]. Li-Ying et al
have reported in vitro cytotoxicity of 1,2,3-triazole-pyrimdine hybrids against MGC-803, EC-
1
09, MCF-7 and B16-F10 human cancer cell lines. Compounds have displayed moderate to good
cytotoxicity profile against all the four cell lines. Study identified active compound 5 exhibiting
IC values ranging from 1.42 to 6.52 µM, interestingly compound was exhibiting 7 fold more
5
0
5

ACCEPTED MANUSCRIPT
potency than 5-flouro uracil against EC-109. Also flow cytometry analysis confirmed the
apoptosis and cell cycle arrest at G2/M phase [17].
HN
O
N
N
Cl
N
S
CN
N
H
(
4)
6

ACCEPTED MANUSCRIPT
Figure 2. Structures of pyrimidine hybrid systems as anticancer agents
Based on our previous studies and in view of interesting biological profiles of novel hybrid
systems, we have made an attempt to synthesize hybrid molecules of acridone with substituted
pyrimidines (Figure 3), characterized and screened for in vitro anticancer activity against human
cell lines A549 (lung), MCF7 and MDA-MB-231 (breast), Hela (cervical) respectively.
Acridones have been reported as DNA intercalators and to interpret the strength of binding we
carried out absorption titration against CT-DNA (Calf Thymus DNA) and also gel
electrophoresis technique of active compounds to observe the pattern of intercalation
experimentally [18-24]. From the preliminary studies, active compounds were further taken up
for screening against a panel of nine sensitive and resistant cancer cell lines. With this we
performed molecular docking studies to identify the orientation, binding pattern and essential
amino acids at the active site of Akt1 responsible for apoptosis and also with DNA to identify the
interacting atoms of hybrid molecules with DNA. This study also highlights the lipophilicity,
ADMET properties, Oral toxicity studies by using computational applications of novel hybrid
systems for being potential candidates as anti-proliferative agents. Also the active compound was
evaluated for in vivo acute toxicity study and to correlate with predicted LD values.
5
0
7

ACCEPTED MANUSCRIPT
O
R1
O
R2
N
O
N
H
Cl
N
CH3
NH2
O
N
H
acid hydrazide of acridone
chloro derivative of substituted pyrimidines
O
R1
O
N
H
R2
N
O
H
N
acridone ring
O
N
H
N
CH3
pyridmidine ring
carbohydrazide linker
Figure 3. Design approach of “acridone-pyrimidine” hybrids
2
2
. RESULTS AND DISCUSSION
.1 Chemistry
Based on the literature review, anticancer activity has not been reported for hybrid structures of
acridone nucleus with substituted pyrimidines and acridone hydrazide. Synthesis of acridone
starts with the Ulmann condensation of halobenzoic acid and anthranilic acid in the presence of
copper as a catalyst. Cyclization with polyphosphoric acid yields substituted acridone-1-
carboxylic acid. Further esterification and treating with hydrazine hydrate gives carbohydrazide
of acridone in the presence of solvent ethanol (Scheme 1a).
Synthesis of pyrimidine ring started with the well reported Biginelli reaction by one pot synthesis
using monochloroacetic acid as catalyst under solvent free conditions gave good yields. Two
8

ACCEPTED MANUSCRIPT
series of pyrimidines were prepared using substituted benzaldehydes, urea and ethyl acetoacetate
(5a-5h) and substituted benzaldehydes, urea and methyl acetoacetate (8a-8g). Further oxidation
with 60% HNO yielded 6a-6h and 9a-9g. Later chlorination with phosphorus oxychloride
3
yielded different chloro substituted pyrimidine derivatives gave 7a-7h and 10a-10g (Scheme
1
b). Then acridone-pyrimidine hybrids were synthesized by linking carbohydrazide of acridone
with different chloro substituted pyrimdines in acetonitrile in the presence of anhydrous
potassium carbonate, yielded the hybrid derivatives 11a-11h and 12a-12g (Scheme 1c).
The novel substituted acridone-pyrimidine hybrids were synthesized by the outlined
scheme. Molecular structure of the newly synthesized compounds were established and
1
13
characterized by HNMR, CNMR and Mass analytical techniques (Supporting Information).
The physico-chemical properties of title compounds are depicted in Table 1.
Reagents & Conditions: (i) SOCl , Reflux for 4 h (ii) EtOH, Reflux for 6 h (iii) EtOH,
2
NH .NH , Reflux for 12 h.
2
2
Scheme 1a. Synthesis of acridone hydrazide (2)
9

ACCEPTED MANUSCRIPT
0
Reagents & Conditions: (i) ClCH COOH, 100 C, 3h (ii) 60% HNO , 10-30 min (iii) POCl ,
2
3
3
reflux, 1h
Scheme 1b. Synthesis of ethyl/methyl ester of substituted pyrimidines
10

ACCEPTED MANUSCRIPT
3 2 3
Reagents & Conditions: (iv) CH CN, anhydrous K CO , Reflux 4-6 h.
Scheme 1c. Synthetic scheme for hybrid molecules of acridone with ethyl/methyl ester of
substituted pyrimidines
11

ACCEPTED MANUSCRIPT
Table 1. Physico-chemical properties of acridone-pyrimidine hybrid molecules
(11a-11h)
(12a-12g)
S. No.
1a
1b
1c
1d
1e
1f
R
Mol. Formula Mol. Wt. % Yield
λmax (nm)
1
H
C
28
H
23
N
5
O
4
493.51
511.50
511.50
523.54
523.54
553.57
507.54
473.52
479.49
497.48
497.48
509.51
509.51
539.54
493.51
55
50
52
60
65
55
60
80
55
60
58
65
60
58
75
408.0, 263.0
408.0, 262.5
407.5, 260.5
408.5, 262.0
408.0, 260.0
408.0, 259.5
408.0, 258.5
406.5, 258.5
407.5, 261.0
407.5, 263.0
407.5, 258.0
407.5, 258.5
408.0, 259.5
407.5, 257.0
407.5, 259.0
1
2-fluoro
4-fluoro
C H FN O
28 22 5
4
1
C H FN O
28 22 5
4
1
4-methoxy
3-methoxy
3,4-dimethoxy
4-methyl
isobutyl
C H N O
2
9
25
5
5
1
C
29
H
25
N
5
O
5
6
4
4
4
1
C H N O
30 27 5
1
1
1g
1h
C H N O
29 25 5
C H N O
26
27
5
1
2a
2b
2c
2d
2e
2f
2g
H
C
27
H
21
N
5
O
1
2-fluoro
C H FN O
27 20 5
4
1
4-fluoro
C H FN O
27 20 5
4
1
4-methoxy
3-methoxy
3,4-dimethoxy
4-methyl
C H N O
2
8
23
5
5
1
C
28
H
23
N
5
O
5
6
4
1
C H N O
29 25 5
1
C H N O
28 23 5
12

ACCEPTED MANUSCRIPT
2
2
.2 Biological Evaluation
.2.1 Cell proliferation assay
The synthesized compounds (11a-11h, 12a-12g) were screened for in vitro cytotoxicity against
human cancer cell lines of A549 (lung cancer), MCF7 and MDA-MB-231 (breast cancer), Hela
(cervical cancer). From Table 2 & Figure 2, some of the hybrid compounds have showed good
cytotoxicity against A549 (lung carcinoma), MCF7 and MDA-MB-231 (breast carcinoma) cell
lines. Hybrid compounds were found to be inactive or less cytotoxic against Hela cell line.
Erlotinib was taken as standard for A549 and Doxorubicin for MCF7, MDA-MB-231 and Hela
cell lines. Among the synthesized compounds, 2-fluoro derivative (11b, IC50 = 3.125 µM), 4-
methoxy derivative (11d, IC50 = 3.26 µM), isobutyl derivative (11h, IC50 = 3.9 µM) have
exhibited good cytotoxicity against A549 with IC less than 5 µM. 2-fluoro derivative (11b, IC
5
0
50
=
7.3 µM), isobutyl derivative (11h, IC50 = 4.32 µM) have exhibited good activity against MCF7
with IC less than 10 µM. 4-methyl derivative (11g, IC = 5.6 µM), 2-fluoro derivative (11b,
5
0
50
IC50 = 6.28 µM), 4-methoxy derivative (11d, IC50 = 7.12 µM), isobutyl derivative (11h, IC50
=
8
.61 µM) have exhibited good activity against MDA-MB-231 with IC50 less than 10 µM. In
comparison, title compounds 11b, 11d and 11h have shown appreciable cytotoxicity against
A549, MDA-MB-231 and MCF7 cell lines.
From the two series of acridone hybrids i.e., acridone with ethyl ester derivative of substituted
pyrimidines and acridone with methyl ester derivative of substituted pyrimidines, ethyl ester
derivative of substituted pyrimidine hybrids have shown significant cytotoxicity. Among them,
acridone-pyrimidine hybrids (11a-11h) were found active against A549 lung cancer cell lines
with cytotoxicity less than 5 µM. Among the series, aryl substituted pyrimidine compounds with
2
-fluoro (11b), 4-methoxy (11d), 4-methyl (11g) and isobutyl (11h) substituted pyrimidines have
exhibited better activity against all the three cell lines. Hybrid molecules of acridone-pyrimidine
rd
have shown poor cytotoxicity profile against Hela cell line. Substitution of methoxy group at 3
position (11e) of phenyl pyrimidine ring has lowered the cytotoxicity against all the four cell
rd
th
lines. But on di-substitution of methoxy group at 3 and 4 position (11f) has remarkably
lowered the cytotoxicity. Among the title compounds, hybrid of acridone with isobutyl
pyrimidine derivative (11h) displayed better activity against multiple cell lines. In the second
series, acridone with methyl ester derivative of substituted pyrimidines, compounds (12a-12g)
13

ACCEPTED MANUSCRIPT
have shown poor cytotoxicity profile against all the four cell lines i.e., A549, MDA-MB-231,
MCF7 and Hela with IC of more than 50 µM. The compounds 12a, 12b, 12e and 12f have
50
shown cytotoxicity in the range of 20-30 µM against A549 cell line. From this data, cell
proliferation assay demonstrates that acridone with aryl and alkyl substituted pyrimidines is
nd
th
favored for anticancer activity preferably monosubstitution on phenyl ring either at 2 or 4
position. Also ethyl ester substitution on pyrimidine is more favored than methyl ester.
Four active compounds 11f, 12c, 12e and 12f were identified from the preliminary cytotoxicity
by MTT assay and DNA binding studies and screened for anticancer activity against sensitive
and resistant cancer cell lines of ovarian, lung and breast cancer type. Results of the study were
depicted in Table 3 & Figure 3. Surprisingly none of the compounds have found active against
ovarian and breast cancer type. Among the four compounds, 3-methoxy derivative (12e, IC = 5
5
0
µM) and 3,4-dimethoxy derivative (12f, IC50 = 4 µM) have exhibited good activity against SW
573 WT of lung cancer type. Also 3-methoxy derivative (12e, IC = 10 µM) and 3,4-dimethoxy
1
5
0
derivative (12f, IC50 = 12 µM) have shown good cytotoxicity against SW 1573 2R 160 P-gp of
lung cancer type. Current study once again clearly indicates that acridones are selective and
specific to the target. Structural similarity among the active compounds 12e and 12f is methoxy
rd
at 3 position on aryl substituted pyrimidines, might be essential for cytotoxicity.
Also, title compounds were evaluated for cytotoxicity against Vero cell line. Most of the
compounds have shown cytotoxicity above 70 µM which is non-toxic. Study revealed that active
hybrid molecules 11f, 12c, 12e and 12f have shown CC50 values 84.28, 78.15, 95.06 and 85.70
µM respectively were non-cytotoxic. Furthermore, hybrid of acridone with alkyl substituted
pyrimidines can be taken as lead to explore the different alkyl substitutions, length of chain, etc
for design of better compounds on multiple cell lines. Similarly, concerning the cytotoxicity
profile of two series, effect of propyl and butyl ester of substituted pyrimdines can be studied to
further understand the SAR of hybrid compounds.
14

ACCEPTED MANUSCRIPT
Table 2. Cell proliferation assay of acridone-pyrimdine hybrid molecules
a
S. No.
R
IC50 (µM) ± SD
A549
8.49 ± 0.11
3.125 ± 0.23
> 100
MDA-MB-231 Hela
MCF7
VERO (CC )
50
1
1a
1b
1c
1d
1e
1f
H
19.6 ± 0.26
6.28 ± 0.35
> 100
45.8 ± 0.34 15.6 ± 0.17
52.6 ± 0.17 7.3 ± 0.41
92.80
61.34
58.20
82.34
64.13
84.28
72.31
78.25
80.00
76.12
78.15
86.24
95.06
85.70
64.25
--
1
2-fluoro
4-fluoro
4-methoxy
3-methoxy
1
47.1 ± 0.57
74.5 ± 0.36 10.5 ± 0.19
>100 >100
>100
1
3.26 ± 0.14
> 100
7.12 ± 0.67
> 100
1
1
3,4-dimethoxy 41.73 ± 0.05
47.3 ± 0.25
5.6 ± 0.06
8.61 ± 0.09
47.4 ± 0.21
41.6 ± 0.34
56.2 ± 0.45
74.5 ± 0.89
58.3 ± 0.38
62.9 ± 0.18
23.7 ± 0.07
0.75 ± 0.03
--
57.5 ± 0.23 26 ± 0.12
48.5 ± 0.12 11.4 ± 0.22
39.1 ± 0.07 4.32 ± 0.06
56.7 ± 0.16 36.2 ± 0.21
67.3 ± 0.11 25.7 ± 0.36
44.5 ± 0.19 33 ± 0.12
37.4 ± 0.27 52.1 ± 0.23
72.6 ± 0.45 32 ± 0.13
63.5 ± 0.26 65 ± 0.34
76.4 ± 0.31 45.2 ± 0.22
1.2 ± 0.07 0.63 ± 0.04
1
1
1g
1h
4-methyl
isobutyl
H
9.74 ± 0.11
3.9 ± 0.01
1
2a
2b
2c
2d
2e
2f
2g
20.38 ± 0.32
20.55 ± 0.06
36.48 ± 0.15
44.83 ± 0.61
27.16 ± 0.42
1
2-fluoro
4-fluoro
4-methoxy
3-methoxy
1
1
1
1
3,4-dimethoxy 29.14 ± 0.16
1
4-methyl
Doxorubicin
Erlotinib
40.52 ± 0.56
10.2 ± 0.02
21.3 ± 0.11
Std.1
Std.2
--
--
--
a- Results are average of triplicate analysis
15

ACCEPTED MANUSCRIPT
Cytotoxicity of acridone hybrids
1
1
20
00
8
6
4
2
0
0
0
0
0
1
1a
11b
11c
11d
11e
11f
11g
11h
12a
12b
12c
12d
12e
12f
12g
A549
MDA-MB-231
MCF7
Hela
Figure 2. Cytotoxicity profile of acridone-pyrimidine hybrids by MTT assay
Table 3. Cell proliferation assay of acridone-pyrimdine hybrid molecules against cancer
sensitive and resistant cell lines
Cancer
type
Name of tumour cell
line
IC50 of active compounds with codes in µM
1
1f
12c
12e
34 ± 0.12
74 ± 0.41
72 ± 0.18
5 ± 0.02
>100
12f
26 ± 0.19
42 ± 0.12
65 ± 0.32
4 ± 0.01
>100
Ovarian
cancer
2008 WT
15 ± 0.23
25 ± 0.19
19 ± 0.08
45 ± 0.16
23 ± 0.31
24 ± 0.11
27 ± 0.13
75 ± 0.29
>100
2
2
008 MRP1 Clone 4
008 MRP1 Clone 6
Lung
SW 1573 WT
cancer
SW 1573 2R 120 MRP1 38 ± 0.21
SW 1573 2R 160 P-gp 62 ± 0.36
62 ± 0.15
10 ± 0.05
12 ± 0.17
16

ACCEPTED MANUSCRIPT
Breast
cancer
MCF7 WT
MCF7/MR
MCF7/Doxo
26 ± 0.12
38 ± 0.09
30 ± 0.17
33 ± 0.12
30 ± 0.08
45 ± 0.17
32 ± 0.13
30 ± 0.18
78 ± 0.22
65 ± 0.34
78 ± 0.41
78 ± 0.33
Ovarian Cancer
8
6
4
2
0
0
0
0
0
1
1f
12c
12e
12f
2
008 WT 2008 MRP1 Clone 4 2008 MRP1 Clone 6
Breast Cancer
1
00
8
6
4
2
0
0
0
0
0
1
1f
12c
12e
12f
MCF7 WT
MCF7/MR
MCF7/Doxo
17

ACCEPTED MANUSCRIPT
Lung Cancer
1
1
20
00
8
6
4
2
0
0
0
0
0
1
1f
12c
12e
12f
SW 1573 WT SW 1573 2R 120 MRP1 SW 1573 2R 160 P-gp
Figure 3. Cytotoxicity profile of active acridone-pyrimidine hybrids (11f, 12c, 12e and 12f)
against a panel of nine sensitive and resistant cancer cell lines of Ovarian. Breast and Lung
cancer
2
.2.2 CT-DNA binding assay by Absorption titration
Chemotherapy is the best and ideal strategy for treating cancer and numerous potential
candidates have been identified. DNA intercalating agents are considered as one of potent class
of compounds and a prime target. Anticancer drugs such as doxorubicin, Adriamycin,
daunorubicin, mitoxantrone, amsacrine etc, are approved by FDA which act by forming Drug-
DNA complexes. Interaction of small molecules with DNA can be categorized as intercalators
and groove binders. Studies identified that acridone derivatives being a planar polyaromatic
system exhibit anticancer activity by DNA intercalating activity. Absorption titration is the
universally employed method to identify the binding pattern and interaction of small molecules
with DNA. It is a simple colorimetric assay to determine the relative binding affinities of small
molecules with DNA. To correlate the results of cell proliferation assay and to study the effect of
substitution on DNA affinity, absorption titration of acridone derivatives with CT-DNA was
performed and the values are depicted in Table 4.
From the present study, hybrids of acridone with aryl substituted pyrimidines exhibited DNA
5
-1
5
-1
intercalation activity in the range of K 0.251 X 10 M to 2.334 X 10 M . From the series of
b
18

ACCEPTED MANUSCRIPT
hybrid derivatives of ethyl ester substituted pyrimidines have shown better DNA affinity
compared to methyl ester substituted pyrimidines. Among the title compounds of ethyl ester
5
-1
substituted pyrimidine derivatives, 4-methoxy derivative (11d, K = 2.334 X 10 M ), 2-fluoro
b
5
-1
5
-1
derivative (11b, K
b b
= 1.461 X 10 M ) and 3,4-dimethoxy derivative (11f, K = 1.429 X 10 M )
have shown better intercalation with DNA. With respect to in vitro cytotoxicity, compound 11f
rd
th
with di-substitution of methoxy at 3 and 4 position has shown less K value. From the series of
5
-
methyl ester substituted pyrimidine derivatives, 4-methoxy derivative (12d, K = 1.759 X 10 M
b
1
5
-1
)
and 3-methoxy derivative (12e, K = 1.574 X 10 M ) showed good intercalation with DNA.
b
In comparison with cell proliferation study of hybrid derivatives, compounds containing 2-fluoro
and 4-methoxy substitutions have shown IC less than 5µM. This indicates that compounds
5
0
might have shown cytotoxicity through intercalation. Surprisingly, hybrid derivatives of acridone
with isobutyl pyrimidine derivative was found most active among the title compounds, but
5
-1
showed less binding constant (11h, K = 0.251 X 10 M ) with DNA.
b
Hybrids of acridone derivatives have not shown any significant DNA intercalation, might be
because of their bulky structure and steric hindrance. On the other hand, the title compounds
might have shown better cytotoxicity profile through another mechanism. Further studies need to
be done to identify the exact mechanism of these kind of hybrid molecules.
Table 4. Absorption titrations of CT-DNA with acridone-pyrimidine hybrid molecules
5
-1
S. No.
1a
1b
1c
1d
1e
1f
R
λ max free
λ max bound ∆ λ max K x10 M
b
1
H
408.5, 244.0
409.0, 255.5
410.0, 243.5
412.5, 243.0
407.5, 256.0
408.5, 244.5
0, 0.5
1.085
1.461
0.931
2.334
1.098
1.429
0.752
0.251
1
2-fluoro
4-fluoro
4-methoxy
3-methoxy
408.5, 256.0 0.5, 0.5
409.5, 244.5 0.5, 1.0
410.5, 243.5 2.0, 0.5
407.0, 256.5 0.5, 0.5
1
1
1
1
3,4-dimethoxy 409.0, 257.0
410.0, 257.0
407.0, 258.0 0.5, 0.5
407.5, 256.0 0, 0
1.0, 0
1
1g
1h
4-methyl
isobutyl
407.5, 257.5
407.5, 256.0
1
19

ACCEPTED MANUSCRIPT
1
2a
2b
2c
2d
2e
2f
2g
H
408.0, 256.5
408.5, 257.0 0.5, 0.5
408.0, 244.5 0, 0.5
1.163
0.735
1.335
1.759
1.574
1.167
0.691
1
2-fluoro
4-fluoro
4-methoxy
3-methoxy
408.0, 244.0
408.5, 244.5
408.5, 243.5
408.5, 243.0
1
408.0, 245.0 0.5, 0.5
407.5, 244.5 1.0, 1.0
1
1
408.5, 245.0
408.5, 244.0 0.5, 0.5
408.5, 256.5 0, 0.5
0, 2.0
1
3,4-dimethoxy 408.0, 243.5
4-methyl 408.5, 256.0
1
2
.2.3 Cell apoptosis study by Gel Electrophoresis
Apoptosis is a series of programmed events that leads to cell death. Apoptotic cells can be
characterized by several biochemical events like DNA breakdown, protein cleavage, protein
cross linking, etc. During the process of apoptosis, DNA undergoes extensive fragmentation and
this can be characterized by several techniques. Gel electrophoresis is widely used, easy-to-
perform and cost effective technique to study the apoptosis. Conventional DNA fragmentation
assay i.e., phenol-chloroform method having certain limitations like require large amount of
sample, loss of sample during the process of separation and consumes more time. The present
method is very sensitive, simple, requires less time for processing without any wastage and more
accurate than conventional method. Acridone-pyrimidine hybrids have shown better cytotoxicity
profile against MDA-MB-231 cell line in the cell proliferation assay.
From the experimental data, based on the cell proliferation assay and CT-DNA binding assay by
absorption titrations, active compounds 11b, 11d, 11h and doxorubicin as standard, were
selected to study the DNA fragmentation pattern (Figure 4). From the DNA ladder assay, active
compounds have shown slightly lesser intense band compared to the standard doxorubicin. This
study suggests that active compounds showed cytotoxicity against A549, MCF7 and MDA-MB-
2
31 cell lines partially by DNA intercalation. Further mechanistic studies has to be conducted for
identifying mechanism of cytotoxicity.
20

ACCEPTED MANUSCRIPT
Figure 4. DNA ladders of MDA-MB-231 cells obtained by DMSO-SDS-TE method Lane 1:
Compound 11b, Lane 2: Compound 11d, Lane 3: Compound 11h, Lane 4: Doxorubicin
(Standard), Lane 5: DNA Ladder
2
2
.2.4 Western blotting
.2.4.1 Blotting analysis of Akt
Western blotting, an important technique to separate and identify specific proteins from cell
extract in cellular and molecular biology. By using specific antibodies to identify the protein of
interest and band thickness corresponds to the concentration of the protein present. Ideal
technique to detect the specific protein and also to quantify its expression. The Akt is serine-
threonine kinase which gets activated by growth factors through phosphatidylinositol-3-kinases
(PI3Ks) for the growth and survival of cell. Studies identified that overexpression or activation
of Akt is an observed phenomenon in variety of human cancers like breast, lung, ovarian,
21

ACCEPTED MANUSCRIPT
pancreatic, gastric cancers and are also associated with resistance to chemotherapy of many
anticancer agents.
Based on the previous experimental studies, MDA-MB-231 cells were selected and treated with
different acridone-pyrimidine hybrid derivatives for 48 hours, after incubation the cells were
harvested in western lysis buffer. Protein extracts were isolated by using cold centrifuge. Protein
concentrations were estimated using Bradford assay. Then samples were boiled and loaded into
1
0% SDS gel. From the previous experimental studies 11a, 11b, 11d, 11h and doxorubicin as
standard, tubulin as loading control were taken for the analysis to identify the ability of active
compounds to reduce the expression of pAkt and tubulin served as a loading control. However,
total Akt levels were unchanged across the samples. Blotting experiment suggests that active
compounds 11a, 11b, 11d and 11h have the ability to reduce the expression of pAkt which in
turn reduces the cell survival (Figure 5). Surprisingly, study couldn’t differentiate the expression
of pAkt of active hybrid molecules and further enzyme inhibition studies can be carried out to
identify the structure activity relationship which helps in design of safe and potent Akt inhibitors.
1.0
0.8
0.6
0.4
0.2
0.0
*
*
***
***
***
***
Figure 5. Western blot analysis of phospho-Akt in MDA-MB-231 cells treated with active
compounds for 48 hrs
Lane 1: Untreated; Lane 2: Doxorubicin treated (Standard); Lane 3: Compound 11a; Lane 4:
Compound 11b; Lane 5: Compound 11d; Lane 6: Compound 11h
Results were interpreted by GraphPad Prism software and presented as mean ± SD for three
independent trails; P < 0.001 (***), P < 0.01 (**) relative to control.
2
2

ACCEPTED MANUSCRIPT
2
.2.4.2 Blotting analysis of ABCC1 (MRP1)
Tumor cells display resistance towards majority of chemotherapeutic agents which might be
because of overexpression of transporter of ATP binding cassette like P-glycoprotein (P-gp) or
multidrug resistance protein 1 (MRP1). Similarly, based on the experimental studies MDA-MB-
2
31 cells were treated with active compounds 11a, 11b, 11h, 11d and doxorubicin added as
standard, tubulin as loading control to analyze that hybrid systems have the potential to modulate
MDR. Literature suggests that controlling the expression of ABCC1 plays a major role in the
regulation of ABC transporters in MDR. The data also suggests that active compounds have the
ability to permeate through cellular membrane and regulate the expression of ABC transporters
(Figure 6). Interestingly, blotting study could identify that hybrid molecule 2-fluoro derivative
compound 11b could reduce the expression of ABCC1/MRP1 when compared to active
compounds 11a, 11d and 11h. Hence hybrid compounds have the ability to reverse MDR by
indirectly perturbing the lipid matrix in which P-gp is embedded. Further optimization should be
done to improve the activity and hybrid of acridone with substituted pyrimidines can be taken as
better lead for investigating MDR and other related cancers.
Figure 6. Western blot analysis of ABCC1 in MDA-MB-231 cells treated with active
compounds for 48 hrs
Lane 1: Untreated; Lane 2: Doxorubicin treated (Standard); Lane 3: Compound 11a; Lane 4:
Compound 11b; Lane 5: Compound 11d; Lane 6: Compound 11h
2
3

ACCEPTED MANUSCRIPT
Results were interpreted by GraphPad Prism software and presented as mean ± SD for three
independent trails; P < 0.001 (***), P < 0.01 (**) relative to control.
2
.2.5 Akt1 kinase assay
Acridone-pyrimidine hybrid molecules have shown good selectivity to Akt1 and identified IC50
values in single digit nanomolar range (Table 5). Blotting studies identified that compounds 11a,
1
1b, 11d and 11h have remarkably reduced the expression of pAkt. Selective Akt1 kinase assay
too identified that same compounds were potent. 4-methoxy derivative compound 11d have
rd
shown most potent and selective to Akt1 with an IC50 of 1.72 nM. Substitution of methoxy at 3
rd
th
position and di-substitution at 3 and 4 position have reduced the selectivity to Akt1. Similarly
nd
substitution of fluorine at 2 position 11b was found selective to Akt1 with an IC50 of 8.2 nM
th
and substitution at 4 position have decreased the selectivity with an IC50 of 176 nM. On
th
substitution of methyl group at 4 position has further reduced the selectivity and displayed IC
5
0
with 980 nM. Second series (12a-12g) of hybrid molecules have shown IC50 values in the range
of 56 to 738 nM and found less selective compared to first series (11a-11h). In comparison with
reported cell proliferation assay, compounds 11a, 11b, 11d and 11h have shown good
cytotoxicity profile might be because of selectivity to Akt1. Compounds have exhibited good
correlation between Akt1 kinase activity and anti-proliferative activity. Present study suggests
that acridone-pyrimidine hybrid molecules are selective and have potential to inhibit Akt1. Also
th
nd
substitution of methoxy group at 4 position and fluorine group at 2 position of
phenylpyrimidine ring might be beneficial.
Table 5. Enzyme assay results for hybrid molecules
a
Comp.
Code
R
Akt1 IC50 (nM)
1
1a
1b
1c
1d
H
4.7
8.2
1
2-fluoro
4-fluoro
4-methoxy
1
176
1.72
1
24

ACCEPTED MANUSCRIPT
1
1e
3-methoxy
3,4-dimethoxy
4-methyl
isobutyl
251
115
980
12
1
1f
1
1
1g
1h
1
2a
2b
2c
2d
2e
2f
2g
H
56
1
2-fluoro
432
313
640
482
311
738
1
4-fluoro
1
4-methoxy
3-methoxy
3,4-dimethoxy
4-methyl
1
1
1
a
All experiments were tested under 10 µM ATP concentration and values are mean of three
experiments.
.2.6 Acute oral toxicity
2
Compound 12f was selected for acute oral toxicity as the toxicity prediction tool- Protox server
has identified its LD as 4000 mg/kg. Active compound was evaluated for oral toxicity as per
5
0
OECD guidelines by administering compound suspended in normal saline with Tween 80 in
normal saline through oral route with a maximum dose of 5000 mg/kg. Rats were kept for
overnight fasting before the day of dosing and for 3h after dosing. On the first day of dosing, rats
were monitored periodically for every hour upto 12 hours for mortality, clinical signs and
behavioral changes. And thereafter observed twice in a day for 14 days and body weight was
recorded daily (Table 6 & Supporting information). No clinical sign of toxicity was observed
during the period of 14 days under observation among the groups of control and tested
compound. The gain in body weight of rats was found to be normal in the groups of control and
tested compound. Present study suggests that compound 12f is safe for oral administration of
upto 5000 mg/kg in single dose to albino Wistar rats of female and also in correlation with
predicted LD values by Protox server.
5
0
25

ACCEPTED MANUSCRIPT
Table 6. Effect of compound 12f on rat body weight
Dose (mg/kg)
Body weight (g)
Day 7
Day 0
160 ± 6
180 ± 8
183 ± 6
186 ± 10
Day 14
191 ± 18
208 ± 20
213 ± 17
228 ± 16
Control
175 ± 10
191 ± 15
195 ± 19
206 ± 17
300
2
5
000
000
2
2
.3 COMPUTATIONAL STUDIES
.3.1 Lipophilicity
The term lipophilicity is traditionally expressed by distribution or partition of molecule between
organic and aqueous phase. According to the principles of medicinal chemistry, n-octanol and
water can be considered as standard and good solvent for experimental determination of
compound or drug. As biological activity of a compound is based on chemical structure and its
physico-chemical properties. By replacing the atoms or substituents present in a compound
modifies the biological activity. Establishing a good relationship between the physicochemical
properties and biological activity helps in the better design of compounds and majority of drug
molecules failed because of poor bioavailability. Lipophilicity of hybrid derivatives of acridones
with substituted pyrimidines was determined using computational programs Chem Draw 15.0,
ALGOPS 2.1 and Qikprop module of Schrodinger software. Lipophilicity prediction of
compounds was done by three computational applications to identify and compare the accuracy
and sensitivity. Prediction of lipophilicity by computational programs by considering the
molecular descriptors of compound i.e., atomic number, type of bonds, number of rings,
aromaticity of rings, molecular mass, molecular volume, dipole moment etc by logarithmic
26

ACCEPTED MANUSCRIPT
calculations. Every software has its own method or fashion of calculating the lipophilicity of a
compound. On comparing the predicted log P values of hybrid derivatives by computational
applications, Chem Draw 15.0 and ALGOPS 2.1 have shown values almost similar with minor
differences. But Chem Draw software of version 15.0 have shown same Log P values for 11b (2-
fluoro) and 11c (4-fluoro), 11d (4-methoxy) and 11e (3-methoxy), 12b (2-fluoro) and 12c (4-
fluoro), 12d (4-methoxy) and 12e (3-methoxy). And poor correlation was observed for predicted
log P values by Qikrop module of Schrodinger software with other softwares (Supporting
Information).
Lipophilicity is one of the most important parameter to be considered especially for anticancer
drugs as it plays major role and is responsible for permeation and accumulation of drug inside
the cancer cell. Based on the structure and chemistry of acridones, majority of the acridone
derivatives exists as protonated and unprotonated forms. By considering the predicted log P
values by ALGOPS 2.1, lipophilicity of hybrid compounds of acridone with substituted ethyl
ester pyrimidines ranges from 4.30 to 4.65 and for acridone with substituted methyl ester
pyrimidines ranges from 4.22 to 4.43. Analysis of relationship between the lipophilicity and
anticancer activity of hybrid compounds have shown a poor correlation. Among the ethyl ester
series, active compounds 11h, 11b and 11d have log P values 4.30, 4.60 and 4.50 i.e., compound
with least and near to highest log P values have displayed better cytotoxicity profile against
A549, MCF7 and MDA-MB-231 cell lines. From the methyl ester series, compounds have
shown poor cytotoxicity and further modifications have to be performed to understand the
relationship between the structure, lipophilicity and cytotoxicity for the design of better lead
molecules (Supporting Information). Therefore, study concludes that lipophilicity of hybrid
derivatives of acridone with substituted pyrimidines might be essential but not the sole property
for cytotoxicity.
2
.3.2 Prediction of ADME properties
Poor pharmacokinetic profile is one of the major limitation for the failure of many drugs. Ideally
drug should be easily absorbed from the site of administration, transported to active site without
non-specific interactions, maximum efficacy and safety, easily metabolized and eliminated
without giving any toxic metabolites from the body and collectively termed as ADMET
properties. Predicting the ADMET properties by computational programs in the advanced stages
27

ACCEPTED MANUSCRIPT
of drug discovery helps in the design of safe and effective drugs, reduces cost and saves lot of
time. Nowadays many pharma companies are relying on the computational applications for the
design of drugs and also working to design a theoretical model which can actually simulate the
process or chemical reactions of the human body. Several number of software tools are available
for the prediction of AMDET properties of drug candidates.
ADMET related properties of the hybrid derivatives were predicted by the Qikprop module of
Schrodinger software and the results displayed in Table 7. According to Lipinski criteria for the
drug candidate should have molecular weight < 500, log P < 5, hydrogen bonding donors (HBD)
≤
5 and hydrogen bonding acceptors (HBA) ≤ 10. It is noteworthy to mention that many of the
active and potent compounds from plant origin, antibiotics from microorganisms, anticancer
drugs, etc have molecular weight in the range of 500 -1000. Also the same high molecular
weight compounds have more flexibility and rigidness with respect to HBD and HBA. Here
hybrid derivatives of acridone with ethyl ester derivatives of substituted pyrimidines have
molecule weight, more than 500 except compounds 11a and 11h. Interestingly compounds 11b
and 11d with molecular weight 511.51 and 523.54 have displayed very good cytotoxicity profile
below 5 µM against A549 and below 10 µM against MCF7 and MDA-MB-231 cell lines
respectively. But still 11b and 11d have the potential of cytotoxic ability.
The other part of ADMET properties involves the prediction of QPlogHERG, QPPCaco,
QPlogBB which represents the apparent cell permeability of the compounds. For every
parameter, software gives a cut-off limit and gives an idea about the acceptance or rejection of
compounds for further studies. Caco-2 cell is best model and widely used to predict the apparent
cell permeability of drug candidates. Recommended value by software is in the range of 50-500
i.e., less than 50 is poor and greater than 500 have high permeability. Predictions are confined to
non-active transport and all the hybrid compounds have QPPCaco in the range of 190.55 to
2
53.152. Interestingly, active compound 11h have highest Caco permeability value 253.15.
Blocking of human Ether-á-go-go Related Gene (hERG) potassium ion channels is essential and
important cause for the occurrence of ventricular arrhythmia which can lead to cardiac arrest.
After doing several trials of active compounds, developers have fixed the value below -5 to be
concerned. None of the hybrid derivatives have value -5, but are in the range of -6.065 to -6.88.
28