Design, synthesis, biological evaluation, molecular docking and QSAR studies of 2,4-dimethylacridones as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2017.02.022

Drug resistance in cancer is an unmet medical challenge and a major drawback for the failure of many chemotherapeutic drugs. Search for targeted, effective drug with minimum toxicity is an urgent need. Acridone which is an alkaloid derivative has been att

Full text of DOI:10.1016/j.ejmech.2017.02.022

Accepted Manuscript
Design, synthesis, biological evaluation, molecular docking and QSAR studies of 2,4-
dimethylacridones as anticancer agents
Manikanta Murahari, Prashant S. Kharkar, Nitin Lonikar, C. Yergeri Mayur
PII:
S0223-5234(17)30087-9
10.1016/j.ejmech.2017.02.022
EJMECH 9218
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 January 2017
Revised Date: 7 February 2017
Accepted Date: 8 February 2017
Please cite this article as: M. Murahari, P.S. Kharkar, N. Lonikar, C.Y. Mayur, Design, synthesis,
biological evaluation, molecular docking and QSAR studies of 2,4-dimethylacridones as anticancer
agents, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.02.022.
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ACCEPTED MANUSCRIPT
Design, synthesis, biological evaluation, molecular docking and QSAR studies of 2,4-
Dimethylacridones as anticancer agents
[
a,b]
[c]
[d]
[c]*
Manikanta Murahari , Prashant S. Kharkar , Nitin Lonikar , Mayur C. Yergeri
[
a]
Department of Pharmaceutical Chemistry, SVKM’s Dr. Bhanuben Nanavati College of
Pharmacy, Mumbai-400 056. India.
[
b]
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, M.S. Ramaiah University
of Applied Sciences, Bangalore-560 054, India.
[
c]
Department of Pharmaceutical Chemistry, SPP School of Pharmacy & Technology
Management, SVKM’s NMIMS, Mumbai-400 056. India.
[
d]
Department of Pharmaceutical Chemistry, AME’s V.L. College of Pharmacy, Raichur-
84103. India
5
Graphical Abstract
Multidrug resistance in cancer is an unmet challenge and drawback for the failure of many
chemotherapeutic drugs. Search for targeted, effective drug with minimum toxicity is an
urgent need. An attempt has been made to explore the chemosensitizing ability of 2,4-
dimethylacridones with alkyl side chain containing substituting tertiary amines at the end of
the side chain. Considering the structural features required for the MDR reversal activity, 2,4-
dimethylacridone with propyl and butyl side chain containing various tertiary amino groups
like morpholinyl, piperidinyl, N-methyl piperazinyl, N,N-diethylamino, N-diethanolamino, β-
hydroxylethyl piperazino at the terminus of the alkyl side chain have been synthesized,
characterized and screened for anticancer and MDR modulation activity.
The 2D interaction diagram of compound 10 with P-gp

ACCEPTED MANUSCRIPT
Cytotoxicity profile of active compounds
Compound IC (µM) ± SD IC (µM) ± SD
Vinblastine
Fold
5
0
50
Code
MCF7
MCF7/ADR
IC (nM) ± SD potentiation
5
0
MCF7/ADR
3.11 ± 0.06
1.25 ± 0.05
1
2e
4 ± 0.05
2 ± 0.03
5.21 ± 0.13
2.56 ± 0.05
Complete
Complete
1
2f

ACCEPTED MANUSCRIPT
Design, synthesis, biological evaluation, molecular docking and QSAR studies of 2,4-
Dimethylacridones as anticancer agents
[
a,b]
[c]
[d]
[c]*
Manikanta Murahari , Prashant S. Kharkar , Nitin Lonikar , Mayur YC
[
a]
Department of Pharmaceutical Chemistry, SVKM’s Dr. Bhanuben Nanavati College of
Pharmacy, Mumbai-400 056. India.
[
b]
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ramaiah University of
Applied Sciences, Bangalore-560 054, India.
[
c]
Department of Pharmaceutical Chemistry, SPP School of Pharmacy & Technology
Management, SVKM’s NMIMS, Mumbai-400 056. India.
[
d]
Department of Pharmaceutical Chemistry, AME’s V.L. College of Pharmacy, Raichur-
84103. India
5
*
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

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Abstract:
Drug resistance in cancer is an unmet medical challenge and a major drawback for the failure of
many chemotherapeutic drugs. Search for targeted, effective drug with minimum toxicity is an
urgent need. Acridone which is an alkaloid derivative has been attributed as molecule in
reversing drug resistance in cancer cells for a long time now. In the present investigation, an
attempt has been made to explore the chemosensitizing ability of 2,4-dimethylacridones with
alkyl side chain containing terminally substituted tertiary amino groups. Considering the
structural features required for the MDR reversal activity, acridone derivatives have been
synthesized with propyl and butyl side chain containing morpholinyl, piperidinyl, N-
methylpiperazinyl, N,N-diethylamino, N-diethanolamino, N-[(β-hydroxylethyl)]piperazino at the
terminus of the alkyl side chain. cLogP values for the synthesized compounds ranged from 2.96
to 4.72 for the propyl derivatives and 3.41 to 5.15 for the butyl derivatives. All the compounds
were screened against breast cancer sensitive MCF7 and resistant MCF7/ADR cell lines.
Compounds 12e and 12f have shown better cytotoxicity profiles with IC50 of 4 ± 0.05 and 2 ±
0
.03 µM against MCF7 cells, 5.21 ± 0.13 and 2.56 ± 0.05 µM against MCF7/ADR cells.
3
Photolabeling studies with [ H]-azidopine and molecular docking studies have identified that
2
,4-dimethylacridones have potential to modulate the P-gp mediated multidrug resistance.
Docking studies identified that compounds have shown favorable interactions with P-gp. QSAR
equation was derived for cytotoxicity vs molecular descriptors of acridone derivatives. Best
models with good predictive ability have been generated with very high square correlation
2
coefficient (R ) values of 0.889, 0.964 and 0.983.
Keywords: Acridones, Chemosensitizers, Drug Resistance, Phase Transfer Catalysis (PTC),
Molecular Docking
1
. Introduction:
The major goal of oncology scientists is to design an effective anticancer agent which is not only
sensitive in normal cancer cells but also against drug resistant cancers. In principle, drug
resistance in cancer treatment has remained as an impediment to successful chemotherapy.
Thirty years of research in cancer and its multidrug resistance has identified a myriad of
mechanism in which tumor cells efflux chemotherapeutic agents. Chemotherapy is baffling the
cancer scientists all over the world, and it has become evident that resistance exists against every
2

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effective anticancer drug including the recently introduced drugs [1-4]. Therefore, the ability to
predict and alter or block the drug resistance mechanism in cancer is likely to improve the
therapeutic index. Acquired drug resistance has become common due to the overexpression of
one or more energy-dependent transporters P-gylcoprotein/ABCB1 that eject anticancer agents
out of cells, which contribute to the failure of chemotherapy. Another important factor is delivery
of anticancer drug to the target tissue which may vary from patient to patient. Penetration of the
drug into tumor cells protected by different mechanisms like altered pH or tumor vasculature,
increased hydrostatic pressure is a major task. Potential reasons for the failure of these drugs
against cancer are attributed due to low affinity, poor specificity and low bioavailability at the
tumor site [5-7].
Acronycine a natural alkaloid was isolated from the bark of Acronychia baurri in 1948 [8].
Acronycine has shown promising activity against wide spectrum of cancers in lab animals.
Glyfoline, alkaloid from Glycosmis citrifolia was found to be potent molecule with an IC of 2.2
5
0
µM in HL60 cells [9]. Tricyclic heterocyclic system, acridone has shown interesting applications
in the area of cancer by multi-targeting mechanisms, antimalarial, drugs acting on CNS with well
identified mechanism and still being explored as an active pharmacophore for various activities
[10-15].
Several targets such as DNA toposiomerases and Protein Kinase C (PKC) which play vital roles
for the normal functioning of cells such as signal transduction, regulatory mechanisms, etc., have
been tried for intervening the tumor progression and metastasis [16]. Of the several classes of
anticancer agents, acridones are an interesting class. Numerous research groups have been
working on the medicinal chemistry and the biological aspects of acridones as chemotherapeutic
agents due to their unique properties. Literature reveals that the acridone alkaloids and their
synthetic derivatives possess a wide range of biological activities like anticancer, MDR
modulators, etc [17]. The acridone derivatives under clinical development are amsacrine (1),
anilinoacridine derivative asulacrine (or CI-921) (2), N-[(2-dimethylamino)ethyl]acridine-4-
carboxamide (DACA) (3), triazoloacridones (C1305 4 and C1533 5) and imidazoacridones (C1311 6)
nd
th
(
Figure 1) [18]. Literature shows that substitution on 2 and 4 position in acridone ring show
nd
good cytotoxicity [19]. Based on this developments, substitution of methyl groups at the 2 and
th
4
positions of the acridone ring has been carried out and this may result in the increased
3

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lipophilicity, which may improve the binding affinity of acridone based derivatives to ABCB1 in
reversing drug resistance. Previously, the author’s research group has reported the synthesis and
screening methods of various acridone derivatives as drug resistant modulators [19].
Several studies have identified that the drug efflux pump P-gp is one of the responsible factors
for promoting multidrug resistance in tumor cells and also for altering the pharmacokinetic
properties of the drugs [20]. ABCB1 belongs to the superfamily of ATP-binding cassette (ABC)
transporters which is encoded by the MDR1 gene. The protein contains 1280 amino acids
arranged in two homologous halves of 610 amino acids joined together by linker of 60 amino
acids chain, each half with six transmembrane sequences followed by the nucleotide-binding
domain. The exact mechanism of action and its selectivity towards efflux of drugs have not been
identified yet. X-ray crystallography studies have identified that the protein has large flexible
binding cavity within the domain [21]. A lot of experiments have been carried out on these
aspects a new direction has been given to the study of acridones and cancer cytotoxicity and drug
resistance mechanism by the author since two decades [19].
Previously the author’s group performed molecular docking studies of chloroacridone derivatives
against calmodulin-dependent cAMP phosphodiesterase to identify the possible binding
interactions. Calmodulin plays vital role in regulating protein kinase activity and other cellular
processes. Studies identified that intracellular calcium concentration was greater in resistant cells
than non-resistant cells. Also calmodulin inhibitors play significant role by interfering cellular
drug accumulation [22]. In the present investigation, authors have designed 2,4-
dimethylacridones to evaluate MDR reversal activity against cancer sensitive and resistant cell
lines. A lot of data has been accumulated only after the establishment of ABCB1 structure. As of
now, not much literature exists about the binding domain with acridones and ABCB1. The
authors have tried to compare the same and how the acridone structural modification has proven
to give more cytotoxic drugs and also in reversing drug resistance in tumor cells.
4

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Amsacrine (1)
Asulacrine (2)
DACA (3)
C1305 (4)
C
1533 (5)
C1311 (6)
5

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Figure 1.
Molecular structures of acridone derivatives under clinical development
2
. Results & Discussion:
2
.1 Chemistry:
2
,4-Dimethylacridone derivatives were synthesized as per general method outlined in Scheme 1.
The synthesis of title compounds begins with Ullmann condensation of 2-halobenzoic acid (7)
and 2,4-dimethylaniline (8) to form 2-[(2,4-dimethylpheny)amino]benzoic acid (9). The starting
materials were added in round bottomed flask, mixed well in isoamyl alcohol and refluxed in the
presence of copper powder and anhydrous potassium carbonate. Compound 9 was further
cyclized with polyphosphoric acid where 2,4-dimethylacridone (10) was obtained in high yield
(86-90 %). N-alkylation i.e., propyl and butyl side chain was attached to compound 10 by the
principle of Phase Transfer Catalysis (PTC) in good yields. Later nucleophilic substitution of N-
1
0
10
alkylated acridones i.e., N -chloropropyl and N -chlorobutyl 2,4-dimethylacridone with
difference secondary amines was performed in anhydrous potassium carbonate.
6

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0
Reagents & Conditions: (i) K
2
CO
3
, Cu, isoamyl alcohol, reflux, 6h; (ii) PPA, 100 C, 3h;
(
iii) 6N KOH, 1-bromo-3-chloropropane/1-bromo-4-chlorobutane, PTC, RT, 24h; (iv) K CO ,
2
3
KI, R-H, reflux, 6h.
Scheme 1: Synthesis of 2,4-dimethylacridone derivatives
7

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N-alkylation of acridone can be performed in strictly anhydrous conditions in the presence of
strong bases like NaNH or NaH because of weakly basic nature of ring nitrogen. The reaction of
2
acridone (10) in anhydrous aniline with chlorobromo alkanes in the presence of strong base
1
0
2
NaNH under reflux yielded respective N -(chloroalkyl) acridones (11-12). However, N-
alkylation requires vigorous experimental conditions with very low yield. But by the applying
the principle of Phase Transfer Catalysis (PTC) compound 10 readily undergoes N-alkylation
with better yields. N-alkylation can be performed at room temperature by stirring acridone
derivative and alkylating agent i.e., 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in a
two phases of tetrahydrofuran and 6N aqueous KOH in the presence of tetrabutylammonium
bromide. Previous step yields 10-(3-chloropropyl) (11) or 10-(4-chlorobutyl) acridone (12) in
good yields. Later, iodide-catalysed nucleophilic substitution of intermediates with various
secondary amines like morpholine, piperidine, pyrrolidine, N-methylpiperazine, N,N-
diethylamine, N-diethanolamine and β-hydroxyethyl piperazine by refluxing in anhydrous
potassium carbonate in acetonitrile yielding the title compounds (11a-f and 12 a-f) as free bases
(Table 1).
All the synthesized compounds were purified by column chromatography packed with silica gel
1
13
and purified compounds were characterized by H and C NMR and Mass spectral methods
Supplementary Material).
(
8

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Table 1.
Physical data of acridone derivatives
1
0
11(a-f)
Molecular Molecular
12(a-f)
Compound
Code
R
Melting
Point ( C)
Yield
%
0
Formula
Weight
1
1
0
1
-H
-Cl
C
15
H
13NO
223.27
318
158
68
46
C
H18ClNO
299.79
350.45
18
11a
C
22
H
26
N
2
O
2
159
168
152
182
58
33
59
42
11b
C H N O
2
348.48
363.50
336.47
3
28
29
28
2
3
2
11c
C
C
23
22
H
H
N
N
O
O
11d
9

ACCEPTED MANUSCRIPT
1
1e
C
22
H
28
N
2
O
3
368.47
393.52
193
223
56
52
1
1f
C H N O
2
24
31
3
1
2
-Cl
C
19
H
20ClNO
313.82
364.48
134
148
52
58
12a
23 28 2 2
C H N O
12b
C
24
H
30
N
2
O
362.51
377.52
350.50
162
183
172
53
59
58
12c
C H N O
24
31
3
12d
C H N O
23
30
2
12e
C
C
23
H
30
N
2
O
3
382.50
407.55
142
181
52
56
1
2f
25
H
33
N
3
O
2
10

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2
2
2
.2 Biological Activity:
.2.1 Cytotoxicity studies:
.2.1.1 In vitro cytotoxicity of acridones in human MCF7 and MCF7/ADR cells
1
0
Newly synthesized N -substituted 2,4–dimethylacridone derivatives were screened for in vitro
anticancer activity in human sensitive MCF7 and resistant MCF-7/ADR cells. Each value
represents the mean of recordings in triplicate and results were shown in Table 2 & Figure 2
and expressed as IC50 i.e., concentration required for 50% inhibition of growth of cells treated
with respect to cells which are untreated.
Table 2.
Cytotoxicity of acridone derivatives on human MCF7 and MCF7/ADR cells and
the potentiation of vinblastine cytotoxicity in drug resistant MCF7/ADR cells.
Compound
Code
IC (µM) ±
IC (µM) ±
Vinblastine
IC (nM) ±
Fold
Potentiation
5
0
50
a
a
SD
MCF7
SD
5
0
a
MCF7/ADR
SD
MCF7/ADR
1
0
1
79 ± 0.12
ND
82.1 ± 0.97
ND
27.12 ± 0.19
1.4
ND
1
ND
1
1a
1b
1c
1d
1e
1f
59.16 ± 0.22
58 ± 0.17
50.21 ± 0.36
29.11 ± 0.18
19.1 ± 0.09
12 ± 0.04
ND
65.3 ± 0.76
61.7 ± 0.69
53.5 ± 0.43
34.2 ± 0.28
22.8 ± 0.15
15.1 ± 0.11
ND
19.9 ± 0.11
17 ± 0.08
17.2 ± 0.13
14.2 ± 0.08
11.22 ± 0.05
8 ± 0.03
6.4
1
7.17
4.75
10.48
9.7
1
1
1
1
16.74
ND
1
2
ND
1
2a
2b
2c
2d
20 ± 0.12
30.10 ± 0.19
21.8 ± 0.19
36.3 ± 0.22
4 ± 0.03
Complete
--
1
5 ± 0.02
1
14.50 ± 0.09
8.3 ± 0.03
16.2 ± 0.31
9.1 ± 0.09
10 ± 0.11
--
1
4.05 ± 0.02
Complete
11

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1
2e
2f
Vinblastine
4 ± 0.05
5.21 ± 0.13
2.56 ± 0.05
3.11 ± 0.06
Complete
Complete
--
1
2 ± 0.03
1.25 ± 0.05
--
3 ± 0.01 nM
69.0 ± 0.2 nM
Note: At IC10 concentration, the modulators were able to reverse drug resistance completely in
MCF-7/ADR cancer cells.
ND= Not Determined
a- Results are average of triplicate analysis
Cytotxicity data of acridone derivatives
1
00
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
1
0
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
MCF7
MCF7/ADR
Figure 2. Experimental IC of acridone derivatives against MCF7 and MCF7/ADR cell lines
50
1
0
Antiproliferative activity of title compounds i.e., N -2,4-dimethylacridone derivatives were
evaluated on human breast adenocarcenoma MCF7 cell line. Results suggests that chemical
nature of the secondary amine attached to alkyl side chain of acridone plays a critical role and
can be considered as pharmacophoric feature for the cytotoxicity. IC50 value of 2,4-
dimethylacridone compound 10 is 79 ± 0.12 µM, found least active among the synthesized
1
2

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compounds indicating that side chain of acridone is essential for cytotoxicity. Considering the
tumor physiology, structural features and physicochemical properties like lipophilicity, pKa,
acridones with propyl and butyl side chain with terminal secondary amines have been
synthesized and screened with an aim of obtaining potent compounds. Title compounds of
acridones with propyl side chain vs butyl side chain have shown appreciable results and also the
study clearly proved that increase in distance from the acridone ring and secondary amine
enhanced the anti-proliferative activity and hydrophobicity. Acridones with propyl side chain
have shown anticancer activity in the range of IC (µM) 59.16 ± 0.22 to 12 ± 0.04 and acridones
5
0
with butyl side chain in the range of 30.10 ± 0.19 to 2 ± 0.03. In the series of acridones with
propyl side chain with secondary amines as morpholinyl (11a), piperidinyl (11b) and N-
methylpiperazinyl (11c) have displayed less cytotoxicity at 59.16 ± 0.22, 58 ± 0.17 and 50.21 ±
0
.36. Compound 11d with secondary amine N,N-diethylamine and compound 11e with N-
diethanolamine have improved the selectivity with cytotoxicity at 29.11 ± 0.18 and 19.1 ± 0.09.
Moreover in the series of acridone with propyl side chain series with -hydroxyethyl piperazine
11f) as secondary amine found more favorable with an IC of 12 ± 0.04. Among the series,
β
(
5
0
acridones containing substituent such as butyl side chain with secondary amines as
β-
hydroxyethyl piperazine (12f), N,N-diethanolamine (12e), N,N-diethylamine (12d) have exerted
better cytotoxicity at IC50 2 ± 0.03, 4 ± 0.05, 8.3 ± 0.03 µM respectively (Table 2). Similar to
the propyl side chain series, compounds with butyl side chain containing secondary amines
morpholinyl (12a), piperidinyl (12b) and N-methyl piperazine (12c) were found less active
against MCF7 with IC50 of 20 ± 0.12, 30.10 ± 0.19 and 14.50 ± 0.09. Compound 12f, acridone
containing butyl alkyl side chains with terminal end of β-hydroxyethyl piperazine was found to
be potent among the series. Interestingly, hydroxyl group containing secondary amines attached
to alkyl side chain of acridones have shown potent and increased the cytotoxicity. These
observations revealed that
β-hydroxyethyl piperazine as secondary amine is essential for
anticancer activity and presence of hydroxyl substituted secondary amines enhance the activity.
The cytotoxicity of 2,4-dimethylacridone derivatives were screened against drug resistant
MCF7/ADR cell line to compare the cytotoxicity and the IC50 values at micromolar
concentration range were presented in Table 2. Acridones with secondary amines at the terminal
end of propyl side chain have exhibited IC (µM) in the range of 82.1 ± 0.97 to 15.1 ± 0.11.
5
0
Similarly acridones with secondary amines and butyl side chain have shown IC50 (µM) in the
13

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range of 21.8 ± 0.19 to 2.56 ± 0.05. Also compound 10 have shown less cytotoxicity against
MCF7/ADR with an IC (µM) of 82.1 ± 0.97 indicating that side chain is essential. Furthermore
5
0
acridones having propyl side chain with secondary amines such as morpholinyl (11a),
piperidinyl (11b) and N-methylpiperazinyl (11c) have displayed reduced cytotoxicity among the
series with an IC50 (µM) values of 65.3 ± 0.76, 61.7 ± 0.69, 53.5 ± 0.43 respectively.
Comparatively acridones with butyl side chain have shown potential cytotoxicity against both
MCF7 and MCF7/ADR cell lines suggesting length of side chain have profound impact. From
the study, compounds 12f, 12e and 12d have exhibited good cytotoxicity profile among the
derivatives with an IC50 (µM) of 2.56 ± 0.05, 5.21 ± 0.13 and 9.1 ± 0.09 respectively. Compound
1
2f having β-hydroxyethyl piperazine as secondary amine with butyl side chain have shown
significant and highest cytotoxicity against both sensitive and resistant MCF7 and MCF7/ADR
cell lines with an IC (µM) of 2 ± 0.03 and 2.56 ± 0.05. Moreover among the series of acridones
5
0
with propyl side chain, compound 11f acridone with
amine and propyl side chain have shown good cytotoxicity with IC50 (µM) of 15.1 ± 0.11. This
highlights the chemical nature of secondary amine plays significant role for cytotoxicity and
hydroxyethyl piperazine is essential, acridone with -hydroxyethyl piperazine as secondary
amine can be taken as promising hit compound for further design of safe and potent compound.
β-hydroxyethyl piperazine as secondary
β-
β
On the other hand, acridones were found more sensitive to MCF7 rather than drug resistant
MCF7/ADR cell line. Multidrug resistance in MCF7/ADR is mediated through many
transporters like P-gp or Breast Cancer Resistance Protein (BCRP). Cytotoxicity profile of title
compounds against MCF7/ADR indicates that 2,4-dimethylacridones have the ability to inhibit
MDR modulation activity mediated through P-gp pump. According to literature, acridones are
studied much as P-gp inhibitors and to a lesser extent of other pump inhibitors of MRP family.
Also it is reported that acridones exhibit anticancer and anti-MDR activity and is might be
through DNA intercalation property [23]. Comparison of IC values of acridones against MCF7
5
0
and MCF7/ADR, clearly identifies that drug resistance in MCF7/ADR cell line is mediated
through overexpression of many pumps like P-gp or Breast Cancer Resistance Protein (BCRP)
and acridones are not substrates of all the pumps. Interestingly title compounds have shown
similar and good cytotoxicity profile against MCF7/ADR cell line, suggesting that 2,4-
dimethylacridones might have ability to modulate MDR mediated by P-gp. Furthermore
14

ACCEPTED MANUSCRIPT
compounds were screened to identify the ability of title compounds to modulate vinblastine
cytotoxicity against MCF7/ADR cell line.
2
.2.1.2 Sensitization of drug resistant MCF7 cells by 2,4-dimethylacridones:
In an attempt to discover novel and potent MDR modulators, the 2,4-dimethylacridones were
evaluated in drug-resistant cancer cell lines. Aim of the study was to investigate the ability of
title compounds to modulate the vinblastine cytotoxicity against MCF-7/ADR cells and to
correlate the structure of acridones with MDR reversal activity. Both doxorubicin and vinca
alkaloids- vincristine and vinblastine are potent cytotoxic agents and substrates of P-gp [24].
According to Tseng et al., compounds daunomycin and vinblastine have shown IC50 values of
7
.12 ± 0.42 µM and 0.0106 ±0.004 µM against MCF7/ADR cell line [25]. Photolabelling studies
against MCF7/ADR cell line too identified that vinblastine is potent cytotoxic agent and also
highly specific to P-gp [26]. Hence, for the sensitization study of acridones against MCF7/ADR
cell line we have used highly competitive binding substrate vinblastine. Cells were treated
continuously with different concentrations of vinblastine ranging from 0-100 nM for 7 days in
the absence or presence of IC concentration of title compounds as MDR modulators. The IC
1
0
50
value of vinblastine in combination with title compounds against MCF-7/ADR cells was
observed in the range of 1.25 ± 0.05 to 27.12 ± 0.19 nM. Each value represents the mean of
recordings in triplicate and results were tabulated in Table 2.
Multidrug resistance is majorly responsible for the failure of many drugs in the therapy of
numerous cancers. Screening of compounds on drug sensitive and resistant cancer cell lines is
the fundamental model to evaluate the behavior of compounds, to test the hypothesis and to
improve the efficacy [27]. To identify the mechanism and resistance modulating ability, title
compounds were screened against cancer sensitive and resistant MCF7 and MCF7/ADR cell
lines. Fold potentiation of acridones was calculated with respect to IC values of MCF7/ADR
5
0
relative to MCF7 cell line. Comparative study to identify the ability of 2,4-dimethylacridones as
MDR modulators in potentiation of cytotoxicity of vinblastine revealed that compounds 12a,
1
2d, 12e & 12f demonstrated the greatest effect. In vitro data of acridones were summarized in
Table 2. Both the series of acridone derivatives have shown appreciable results. Interestingly
acridones with butyl side chain series have displayed better cytotoxicity profile in resistant cell
line MCF7/ADR compared to propyl side chain series, might be because of improved
15

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hydrophobicity. Moreover, acridones containing secondary amines as morpholine (12a), N,N-
diethylamine (12d), N-diethanolamine (12e) and
completely reverse MDR modulation because of hydrophobicity and basicity. In the absence of
,4-dimethylacridone derivatives as modulating agents, vinblastine has shown IC50 value of
β-hydroxyethyl piperazine (12f) were able to
2
about 3.2 ± 0.12 nM against MCF7 and 69.3 ± 0.02 nM against MCF7/ADR cell lines
respectively. This study identified that nature of surrounding tertiary amine and length of alkyl
side chain for 2,4-dimethylacridones have shown profound impact on cytotoxicity, ability to
modulate in drug resistant cell line.
The presence of
β-hydroxyethyl piperazine as secondary amine was found interesting, has shown
IC of 12 ± 0.04 µM with propyl side chain (11f), 2 ± 0.03 µM with butyl side chain (12f)
5
0
against human MCF7 cell line and IC of 15.1 ± 0.11 µM with propyl side chain and 2.56 ± 0.05
5
0
µM with butyl side chain against MCF7/ADR cell line, IC50 of 8 ± 0.03 nM with propyl side
chain and 1.25 ± 0.05 nM with butyl side chain against MCF7/ADR cell line with vinblastine at
IC10 concentration of modulators. Among the series of 2,4-dimethylacridones, compounds with
butyl side chain have shown better cytotoxicity profile compared to propyl side chain.
Comparison of length of alkyl side chain vs cytotoxicity, more interesting thing observed was
acridones with butyl side chain have shown complete reversal of resistance against MCF7/ADR
cell line with vinblastine at IC concentration of modulator, irrespective of secondary amine
1
0
attached. Results suggests that length of alkyl side chain plays a critical role and essential for the
acridones in reversal of MDR mediated through transporter ABCB1. Evidence to that is
increased cytotoxicity against MCF7/ADR cell line of acridones with butyl side chain i.e., long
lipophilic group might be helping in interaction of membrane bound transporter P-gp.
3
2
.2.2 Photolabeling studies with [ H]-Azidopine
Photoaffinity labelling of P-glycoprotein is the best and frequently used method to identify
whether the compound has ability to be a P-gp substrate. In general, compounds which can
compete for the active binding site of transporters responsible for multidrug resistance with
azidopine will block the photolabelling. For example, drugs like vincristine, vinblastine or
verapamil has the ability to completely block the binding of photolabelled azidopine with the
active site of P-gp [28]. In the present study, to identify the possible mechanism and strength of
3
inhibition, competition between [ H]-azidopine and the 2,4-dimethylacridones was observed.
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Literature suggests that KB-VI cells are also one of the multidrug resistant variant which can
exhibits high level resistance to colchicine, Adriamycin or vinblastine mediated through P-
glycoprotein compared to MCF7/ADR cells. Experimental study identified that multidrug
resistant KB cell lines have amplified two related DNA sequences which are homologous to the
Chinese hamster mdr gene. Also resistant KB cell lines have exhibited high specificity to
vinblastine which can be photoactivated to quantify the affinity of acridones to P-gp [29-31].
Bruggemann et al., experimental study identified that P-gp from human KB cells with high
concentration of colchicine contains valine residue at position 185 and with high concentration
of vinblastine or Adriamycin retains glycine residue at position 185. These two cell lines show
different pattern of binding for vinblastine and clearly suggests that amino acid residue at 185
position is essential for binding or transport of drugs by P-gp. Hence, we have performed
azidopine photolabelling of title compounds with well known, selective and potent modulator of
P-gp verapamil [32].
3
Table 3.
Interaction of acridones with P-gp by photolabeling with [ H]-Azidopine
Compound Percentage of
Code
control (%)
1
0
1
33
28
26
34
46
36
40
52
36
36
42
43
1
1
1a
1b
1c
1d
1e
1f
1
1
1
1
1
12
1
2a
2b
2c
1
1
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1
2d
2e
2f
Verapamil
Std.)
54
55
58
61
1
1
(
Our experiment identified that all the synthesized compounds have shown appreciable results by
competing with azidopine at the active site of protein i.e., P-gp and is identified with reduced
photoaffinity (Table 3). Observation of the experimental results suggests that acridone with
butyl side chain have displayed better competition than that of acridones with propyl side chain
might be because of hydrophobicity. Photolabelling studies identified that verapamil has shown
better competition than all of the title compounds. An attempt has been made to identify the
structure activity relationship of 2,4-dimethylacridones with propyl and butyl side chain and
MDR modulation ability. Butyl series of acridones have shown appreciable results compared to
propyl series. To further identify the effect of alkyl side chain length, we are planning for pentyl
and hexyl alkyl side chain of title compounds. Results suggests that MDR modulation of 2,4-
dimethylacridones might be mediated through P-gp transporter mechanism.
2
2
.3 Computational Studies:
.3.1 In silico prediciton of physicochemical properties
Physicochemical properties of title compounds have been predicted virtually based on theoretical
approaches to identify the compounds which violate the optimum parameters required for the
drug likeness and anti-proliferative activity. Log P values obtained were quite different,
acridones with propyl side chain ranging from 2.37 to 4.42 and acridones with butyl side chain
ranging from 2.84 to 4.68. All the title compounds have passed for Lipinski’s rule of five, which
increases the scope for the extension of active compounds to in vivo screening. Descriptors like
Polar Surface Area (PSA), QPlogP o/w (Predicted octanol/water partition coefficient), pKa,
QPPCaco (Predicted apparent Caco-2 permeability in nm/sec), QPlogBB (Predicted brain/blood
partition coefficient), QPPMDCK (Predicted apparent MDCK cell permeability in nm/sec) were
calculated and results were given in Table 4.
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Lipophilicity is one of the fundamental molecular property and important parameter in the design
of drugs. Especially lipophilicity plays a major role in the anticancer activity, but there are potent
and active molecules which exhibit anticancer activity with low lipophilicity and high molecular
weight [33-37]. In the present study, we have calculated the logP by Schrodinger software to
identify the relationship of lipophilicity of acridones and anticancer activity. Here, the series of
acridone derivatives contains propyl and butyl alkyl side chain with tertiary amines as essential
pharmocophoric structures for cytotoxicity like morpholinyl, piperidinyl, N-methylpiperazinyl,
N-diethylamino, N-diethanolamino, N-[(β-hydroxylethyl)]piperazino, etc. Compound 10-(4-N-
piperidinobutyl)-2,4-dimethylacridone (12b) was identified most lipophilic and 10-(3-[N-
diethanolamino] propyl)-2,4-dimethylacridone (11e) less lipophilic. Both the compounds were
not active and thus, the lipophilicity of the title compounds seems to contribute anticancer
activity to some extent.
Table 4. Molecular descriptors of acridone derivatives
1
1a-f
Mol. Wt. PSA
12a-f
QPlog
Compound
Code
R
pKa
QPP
Caco
900.68
QPlogBB
QPP
MDCK
488.79
Po/w
3.278
1
1a
1b
1c
Morpholinyl
Piperidinyl
N-methyl
350.46
348.48
363.50
45.084 7.26
0.272
0.257
0.586
1
35.8
9.4
8.2
4.426
3.115
910.09
195.82
494.31
103.92
1
41.806
piperazinyl
1
1d
N-diethyl amino
N-diethanol
amino
336.47
368.47
33.741
75.6
9.8
9.0
4.18
2.37
913.66
168.91
0.161
496.41
80.06
1
1e
-0.838
1
1f
N-[(
β-
393.52
61.293
7.9
2.583
78.09
-0.041
38.47
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hydroxyethyl)]
piperazino
1
2a
2b
2c
Morpholinyl
Piperidinyl
N-methyl
364.48
362.51
377.52
45.12
7.8
3.677 897.108
4.684 876.67
0.195
0.178
0.499
486.69
474.72
103.39
1
35.101 10.3
41.837 10.7
1
3.521 194.901
piperazinyl
N-diethyl amino
N-diethanol
amino
1
2d
350.50
382.50
34.061 10.7
4.623 927.261
0.085
504.405
81.14
1
2e
76.298
9.3
2.847
2.915
171.02
59.904
-0.979
1
2f
N-[(
β-
407.55
63.592
8.4
-0.266
28.88
hydroxyethyl)
piperazino
pKa is also one of the important and fundamental property to be considered for the design of
effective anticancer compounds. Charge of the molecule i.e., pKa is related to many properties
like solubility, permeability, distribution, protein binding, metabolism, excretion, etc. Physiology
of tumor and pKa of the molecule are essential which in turn effects the cellular uptake and
retention of compounds [38]. In silico method was employed to predict the pKa of acridones by
ACD labs/pKa DB software and observed in the range of 7.26-10.7 [39-43]. Substitution of
acridones with secondary amines have great impact on pKa and makes the molecule exist in both
ionized and unionized forms. Experimental determination of pKa for selected anticancer drugs
have identified that majority of drugs have pKa in the range of 8 to 10. In comparison of pKa
with in vitro cytotoxicity of acridone derivatives, active compounds 12f, 12e, 12d, 11f have pKa
values 8.4, 9.3, 10.7, 7.9. Poor correlation was observed between anticancer activity and pKa.
Experimental observation might give a better correlation and present study suggests that pKa is
one of the essential parameter to be considered for design of effective anticancer agent, might not
be sole property.
Molecular weight and polar surface area of compounds were in the range of 336.47 to 407.55
and 33.74 to 76.29, optimum with respect to number of hydrogen bonding donors and acceptors.
Other descriptors like QPPCaco which predicts the apparent permeability across the Caco-2 cell
20

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membrane which represents the gut-blood barrier through non-active transport were calculated.
Recommended values of QPPCaco as per software are <25 poor permeability and >500 is more
permeability. None of the title compounds have poor Caco permeability and 6 out of 12
compounds have more than 900. QPlogBB represents the brain/blood partition coefficient and
QPPMDCK represents the apparent permeability across the MDCK cells which can be
considered as good mimic for Blood Brain Barrier (BBB) by non-active transport. In summary,
Qikprop predictions suggests that 2,4-dimethylacridone with propyl or butyl chain and
substitution of various secondary amines have optimum parameters for anticancer activity and
can be considered as lead molecule for further modifications.
2
.3.2 Molecular docking
To identify the potential structural features for cytotoxicity and interaction at active site of P-gp,
molecular docking studies have been performed. Compounds have shown better hydrogen
binding interactions and are comparable to control molecules. Along with in vitro results,
docking studies too confirmed that alkyl side chain is essential for anticancer activity and length
of alkyl side chain, chemistry of secondary amine plays crucial role for efficient binding at the
active site. Studies revealed that H-bonding and hydrophobic interactions are the key factors for
binding with P-gp. Molecular docking results indicating docking score, energy, interacting
residues at the binding site of P-gp were summarized in Table 5. Along with that binding
interactions i.e., docked poses of least and most active compounds were represented in Figure 3
and Figure 4.
21

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Figure 3.
2D interaction diagram of compound 10 with P-gp
2
2

ACCEPTED MANUSCRIPT
Figure 4a. 2D interaction diagram of compound 12f with P-gp
2
3

ACCEPTED MANUSCRIPT
Figure 4b. 2D interaction diagram of compound 12e with P-gp
Figure 4. 2D interaction diagram of compounds with P-gp
Binding interactions of all the 12 compounds were analyzed and identified that unsubstituted
acridone 10 was found with least score indicating that side chain is essential (Figure 3). Binding
interactions of compound 12e at the active binding site of P-gp shows that both the hydroxyl
groups of secondary amine N-diethanolamine has formed hydrogen bonds with Glu356 and
Gln357, nitrogen of amine found interacting with Glu356 and Gly379. Presence of two hydroxyl
groups in the secondary amine might have improved the binding affinity at the active site of P-gp
and displayed good docking score -6.426 kcal/mole among the series. Similarly compound 11e,
acridone with propyl side chain containing N-diethanolamine has exhibited better binding
affinity with docking score -6.389 kcal/mole. Two hydroxyl groups of compound formed
2
4

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hydrogen bonding with Glu356 and Gln357, nitrogen of N-diethanolamine with Gly379, π-π
stacking of acridone ring with Tyr352.
The binding interactions of the active compound 12f i.e., acridone with butyl side chain
containing -hydroxy piperazine from in vitro cytotoxicity shows that hydroxyl group of
β
secondary amine forms hydrogen bonding with Ser355, nitrogen of secondary amine with
Asp353 and oxygen of acridone ring with Thr384 (Figure 4a). Molecular docking score of
compound 12f is -5.324 kcal/mol. The binding interaction of active compound 12d i.e., acridone
with butyl side chain containing N-diethylamino shows that nitrogen of secondary amine forms
hydrogen bonding with Glu356 and Gly379. Compounds 11e and 12e with top ranking docking
score have shown similar binding interactions with Gln357, Glu356 and Gly379. Moreover
active compounds of in vitro results have not shown similar binding interactions with that of
compounds 11e and 12e.
The binding interactions of compound 12c i.e., acridone with butyl side chain containing N-
methyl piperazine have displayed least molecular docking score -3.964 kcal/mol shows that
nitrogen of secondary amine forms hydrogen bonding with Glu356 and Gly379, π-π stacking of
acridone ring with Tyr352. Present study identified that chemical nature of secondary amine
substituted at the end of alkyl chain plays an essential role in cytotoxicity and MDR modulation.
1
2e and 11e have shown better docking scores compared to 12f and 11f. 12e and 11e have the
common secondary amine N-diethanolamine whereas 12f and 11f with N-[(
β-
hydroxyethyl)]piperazine. Compounds 12e (Figure 4b) with highest docking score of -6.426
might be because of presence of two hydroxyl groups of secondary amine improved the binding
interactions at the target site and 12f the most active compound with docking score -5.324 at the
active site of P-gp. Similarly compound 11e have shown docking score -6.389 might be because
of secondary amine. Poor correlation was observed between the predicted log P of title
compounds and in vitro cytotoxicity. To further investigate the mechanism, experimental
determination of lipophilicity might help in better understanding. Interestingly title compounds
have shown better interactions than potent and well known P-gp modulator verapamil.
Experimental and molecular studies clearly suggests that acridones with alkyl side chain and
hydroxyl substituted secondary amine are essential for multidrug resistance (MDR) reversing
agents.
25

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Table 5.
Molecular docking results of acridones with the target protein P-gp
10
11a-f
12a-f
Compound
Code
R
Glide SP Glide SP
Amino acid
residues in
Score
emodel
interaction
1
1
1
1a
1b
1c
Morpholinyl
Piperidinyl
N-methyl piperazinyl
-4.688
-4.364
-4.484
-60.097
-51.356
-56.669
Thr384, Asp353
Thr384, Glu356
Tyr352, Glu356,
Gly379
1
1
1d
1e
N-diethyl amino
N-diethanol amino
-4.493
-6.389
-50.986
-69.655
Gly379, Glu356
Tyr352, Gly379,
Glu356, Gln357
Thr384, Glu356
1
1f
N-[(
β-hydroxyethyl)]
-4.809
-4.791
-70.699
-60.478
piperazino
Morpholinyl
1
2a
Tyr352, Asp353,
Glu356
1
1
2b
2c
Piperidinyl
N-methyl piperazinyl
-4.722
-3.964
-58.556
-52.468
Tyr352, Glu356
Tyr352, Glu356,
Gly379
1
2d
2e
2f
Std.
N-diethyl amino
-4.394
-6.426
-5.324
-4.054
-56.913
-73.943
-77.199
-67.145
Tyr352, Glu356,
Asp353
Gln357, Glu356,
Gly379
Thr384, Asp353,
Ser355
Asp353, Tyr352
1
N-diethanol amino
1
N-[(
β-hydroxyethyl)
piperazino
Verapamil
26

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2
.3.3 QSAR descriptors
Correlation of the molecular descriptors and biological activity is essential to identify and
understand the Structure Activity Relationship (SAR) [44-50]. Can be achieved by considering
the most significant and minimum molecular descriptors for the anticancer activity to get a best
fit. A good number of online tools are available to calculate the molecular descriptors i.e.,
chemical descriptors like surface area, volume, charge, dipole moment and molecular property
descriptors like lipophilicity.
2
.3.3.1 Exploring and Selection of descriptors
All the 12 acridone derivatives were prepared by standard protocol using Ligprep module and
submitted to Qikprop module for the prediction of molecular descriptors. Data pertaining to
compounds with ID and name of structure were composed in the software. Under the section of
dependent variable i.e., activity, results obtained from the in vitro cytotoxicity screening against
MCF7 were expressed in the form of log(1/IC ) for convenience. The next critical step is
5
0
selection of essential descriptors to simplify the data and to build a strong model which can
identify the safe and potent molecule. From the data generated by the Qikprop module of
Schrodinger, 15 descriptors [log P, pKa, molecular weight, hydrogen bonding acceptors (HbA),
hydrogen bonding donors (HbD), square of dipole moment divided by the molecular volume
(dip^2/V), Van der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon
0
atoms (PSA), total solvent accessible surface area in angstroms using a probe with 1.4 A radius
(
(
(
SASA), hydrophobic component of the SASA- saturated carbon and attached hydrogen
FOSA), hydrophilic component of the SASA- SASA on N, O, H on heteroatoms and carbonyl C
FISA),
π
component of SASA- carbon and attached hydrogen (PISA), total solvent-accessible
0
volume in cubic angstroms using a probe with 1.4 A radius (volume), globularity descriptor-
2
(4
π
r )/(SASA) (glob), PM3 calculated Ionization potential- negative of HOMO energy (IP) and
PM3 calculated Electron affinity- negative of LUMO energy (EA)] were shortlisted for building
a QSAR model. The abbreviations and description of molecular descriptors were taken from the
Qikprop user manual of Schrodinger software.
27