Nitric oxide releasing acridone carboxamide derivatives as reverters of doxorubicin resistance in MCF7/Dx cancer cells

Article abstract of DOI:10.1016/j.bioorg.2015.11.007

A series of nitric oxide donating acridone derivatives are synthesized and evaluated for in vitro cytotoxic activity against different sensitive and resistant cancer cell lines MCF7/Wt, MCF7/Mr (BCRP overexpression) and MCF7/Dx (P-gp expression). The resu

Full text of DOI:10.1016/j.bioorg.2015.11.007

Bioorganic Chemistry 64 (2016) 51–58
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Nitric oxide releasing acridone carboxamide derivatives as reverters
of doxorubicin resistance in MCF7/Dx cancer cells
a
b
c
b
V.V.S. Rajendra Prasad a,b, , G. Deepak Reddy , Ietje Kathmann , M. Amareswararao , G.J. Peters
⇑
a Medicinal Chemistry Research Division, Vishnu Institute of Pharmaceutical Education and Research, Narsapur, India
b Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands
c Clinical Research Department, Emcure Pharmaceuticals Ltd., Pune, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of nitric oxide donating acridone derivatives are synthesized and evaluated for in vitro cytotoxic
activity against different sensitive and resistant cancer cell lines MCF7/Wt, MCF7/Mr (BCRP overexpres-
sion) and MCF7/Dx (P-gp expression). The results showed that NO-donating acridones are potent against
both the sensitive and resistant cells. Structure activity relationship indicate that the nitric oxide donat-
ing moiety connected through a butyl chain at N10 position as well as morpholino moiety linkage through
an amide bridge on the acridone ring system at C-2 position are required to exert a good cytotoxic effect.
Further, good correlations were observed when cytotoxic properties were compared with in vitro nitric
oxide release rate, nitric oxide donating group potentiated the cytotoxic effect of the acridone derivatives.
Exogenous release of nitric oxide by NO donating acridones enhanced the accumulation of doxorubicin in
MCF7/Dx cell lines when it was coadministered with doxorubicin, which inhibited the efflux process of
doxorubicin. In summary, a nitric oxide donating group can potentiate the anti-MDR property of
acridones.
Received 15 September 2015
Revised 23 November 2015
Accepted 25 November 2015
Keywords:
Nitric oxide
Acridone
Doxorubicin
Breast cancer
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
drugs commonly arises in the cancer cells due to genetic and
epigenetic alterations that affect drug sensitivity [1].
Tumor cell resistance to a wide spectrum of anticancer drugs
continues to be a major concern for effective cancer chemotherapy.
Many different mechanisms have been suggested to explain the
development of multidrug resistant (MDR) phenotype in cancer
cells, such as alterations in the drug’s specific target, reduced
uptake or increased efflux of the drug, reduced capacity to enter
apoptosis, increase in the ability to repair DNA damage, dissimilar
compartmentalization, and an increased rate of detoxification of
the drug [1]. One of the mechanisms by which tumors acquire drug
resistance is the overexpression of membrane transporters such as
drug efflux pump P-glycoprotein (P-gp/ABCB1) which transports
structurally and functionally unrelated compounds. The potential
role of P-gp in clinical drug resistance stimulated the investigation
of various P-gp inhibitors in clinical trials [2,3]. Anthracyclines
such as doxorubicin, epirubicin and their derivatives are exten-
sively used in the treatment of solid and hematological cancers.
However, their efficacy is limited because of their dose dependent
cardiotoxicity, an important adverse effect which impairs the
patient’s outcome and survival [4]. Moreover, resistance to these
It has been demonstrated that HMG-CoA reductase inhibitors
such as statins revert doxorubicin resistance in different cancer cell
lines [5,6]. Statins, by inhibiting RhoA and its effector Rho kinase,
can activate the IKK/NF-kB pathway [7]. By activating NF-kB, sta-
tins enhance the transcription of the inducible NO synthase (iNOS)
[8], one of the three NOS isoforms, which catalyze the formation of
L-citrulline from L-arginine by releasing nitric oxide with a 1:1 sto-
ichiometry [9]. Nitric oxide (NO) is a signaling molecule involved in
the control of cellular growth, differentiation and apoptosis [10]. In
oxygenated living systems, NO is rapidly converted into nitrite and
nitrate [11]. High levels of NO and its metabolic derivatives, the
reactive nitrogen species (RNS) can modify functional proteins by
S-nitrosylation, nitration, disulfide formation leading to bio-
regulation, inactivation and cytotoxicity particularly in tumor cells
[12]. It has been suggested that doxorubicin evokes a significant
increase of NO synthesis in murine breast cancer cells and exert
its therapeutic effect via an NO-dependent mechanism [13]. Nitric
oxide production is quite different in doxorubicin resistant and
sensitive cells; restoration of NO production in resistant cells could
reverse the MDR in cancer [5]. It was also observed that NO inhibits
the efflux of doxorubicin, thus decreasing the resistance of the cell
to the drug, further, inducers of NO synthesis such as atorvastatin
⇑
Corresponding author at: Medicinal Chemistry Research Division, Vishnu
Institute of Pharmaceutical Education and Research, Narsapur 502313, India.
E-mail address: rajendraprasad.vvs@viper.ac.in (V.V.S. Rajendra Prasad).
http://dx.doi.org/10.1016/j.bioorg.2015.11.007
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

52
V.V.S. Rajendra Prasad et al. / Bioorganic Chemistry 64 (2016) 51–58
and NO donors (GSNO, SNAP and sodium nitroprusside) increased
drug accumulation in doxorubicin resistant cells [5]. Further,
enhanced histone glutathionylation and reversal of drug resistance
was observed after GSNO treatment of MCF7/Dx cells in compar-
ison with MCF7 cells [14].
decent yield (95%) was synthesized by Ullmann condensation reac-
tion of 2-chlorobenzoic acid with p-amino benzoic acid to produce
2,40-iminodibenzoic acid (I), which was then cyclized with
polyphosphoric acid instead of sulfuric acid in a water bath at
100 °C for three hours to give acridone-2-carboxylic acid (II).
Acridone-2-carboxamides were synthesized by a reaction between
an activated form of acridone-2-carboxylic acid (II) and an
appropriate amine. A convenient technique for carboxylic group
activation was developed that allowed us to synthesize the
products in high yields. We prepared an essentially complete set
of acridone-2-carboxamides (1–6) whose amide fragments were
formed by the alkyl groups or aryl ring systems bearing exocyclic
groups at different ring positions.
Further, N-alkylation of the different acridone-2-carboxamides
(1–6) was achieved by using strong base sodium hydride because
the nitrogen atom of the acridone nucleus is normally resistant
to undergo N-alkylation with alkyl halides due to their weakly
basic nature. Compounds (7–10) in good yields obtained by stirring
of acridone-2-carboxamides with alkylating agent 1-bromo-3-
chloropropane or 1-bromo-4-chloro butane under nitrogen in the
presence of potassium iodide at room temperature for 24 h. To
introduce the nitric oxide donating moiety, N-alkylated acridone-
2-carboxamides (7–10) were treated with silver nitrate in dry
acetonitrile for three hours. Physical characterization data of all
synthesized compounds are shown in Table 1.
Nitric oxide plays an important role in numerous physiologic
and pathologic processes, including in nonspecific antitumor
immune response. New types of NO-releasing hybrid compounds
need to be developed to obtain a tissue-specific NO-related func-
tion. This creates the possibility that new drugs can be developed
which supplement NO exogenously when the body cannot gener-
ate sufficient amounts to allow normal biological functions. As a
sequel in our research to discover new MDR modulators, we
hypothesized that introduction of a NO donating group into a
P-gp and/or BCRP inhibitor acridone carboxamide moiety would
be capable to release NO in a controlled manner and might reverse
the doxorubicin resistance in cancer. In the present investigation
new set of acridone derivatives are designed based on earlier
observations: (i) Acridone-carboxamide analogues are potent inhi-
bitors of the P-gp and/or ABCG2 pumps (ii) Controlled NO release
in MCF7/Dx cells reverts doxorubicin resistance via enhanced his-
tone glutathionylation (iii) NO donors could affect doxorubicin
accumulation through the nitration of tyrosine residues in the drug
efflux pumps.
All the compounds were separated and purified by column
chromatography and/or recrystallization method and dried under
high vacuum for more than 12 h. Purified molecules were charac-
terized by using 1H NMR, 13C NMR and Mass spectrometric studies.
The 1H NMR spectrum of acridone carboxylic acid (II) showed
seven aromatic protons and were resonated at d7.31–8.52 ppm
as multiplet; a singlet at d11.15 ppm was assigned to N–H proton
and one proton of the carboxylic group resonated as singlet at
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of compounds (1–14)
Nitric oxide releasing acridone derivatives were synthesized
according to Scheme 1. Initially acridone-2-carboxylic acid (II) with
Scheme 1. Synthesis of nitric oxide donating acridone carboxamide derivatives.

V.V.S. Rajendra Prasad et al. / Bioorganic Chemistry 64 (2016) 51–58
53
d12.63. Thus, a combination of chemical shift, spin–spin couplings
and integration data permitted the identification of individual
hydrogen atoms at each side in the aromatic ring. The assignment
of protons in all the compounds is fully supported by the integra-
tion curves and all the derivatives displayed the characteristic
chemical shifts for the acridone nucleus. The 13C NMR spectrum
of NO donating acridone carboxamide (11–14) showed 20 signals
representing 20 magnetically dissimilar environmental carbons.
Synthesized NO releasing acridone carboxamides were ana-
lyzed by mass spectra under ESI conditions. Molecular ions were
observed in the form of M + H. The data indicates that as such there
is no difference in the fragmentation pattern among the set of
acridone series. Overall, mass spectral features of these acridones
were similar and straight forward. Most of the compounds yield
abundant molecular ions in the form of M + H. All the bonds in
the N10-side chain portion are prone to cleavage. This means, that
the data presented in this article also demonstrate the usefulness
of MS for characterization of acridone derivatives. In conclusion,
1H, 13C NMR and mass spectral data were consistent with the
proposed structures.
2.2. Biological
2.2.1. In-vitro cytotoxic activity
The in vitro cytotoxic effects of the NO-acridones in comparison
with reference drugs doxorubicin (Dx) and mitoxantrone (Mr) were
studied by using Sulphorhodamine-B (SRB) assay [15] in the human
breast cancer cell line MCF7 and its two drug cross-resistant pheno-
type sublines (P-gp and BCRP), doxorubicin resistant MCF7/Dx
(P-gp expression) and mitoxantrone resistant MCF7/Mr (BCRP
Table 1
Physical characterization data of nitric oxide donating acridones.
.
Molecule
R1
H
R
MW
321
Volume
885.573
LogP
Yield (%)
71
M.P (0C)
328–330
1
1.81
2
3
H
H
308
306
824.525
956.458
1.65
2.73
70
77
332–334
321–325
4
H
334
1007.481
1.98
74
297–299
5
6
H
H
383
425
1195.715
1321.636
4.39
3.78
73
70
307–311
336–339
7
8
–(CH2)3–C1
397
384
411
398
424
411
438
425
1091.580
1031.632
1147.389
1086.715
1264.463
1163.379
1338.013
1247.593
2.89
2.76
3.31
3.16
1.08
1.22
1.53
1.642
76
68
66
71
62
69
69
71
239–241
248–251
237–240
233–237
230–231
252–256
219–221
284–287
–(CH2)3–C1
9
–(CH2)4–C1
10
11
12
13
14
–(CH2)4–C1
–(CH2)3–ONO2
–(CH2)3–ONO2
–(CH2)4–ONO2
–(CH2)4–ONO2

54
V.V.S. Rajendra Prasad et al. / Bioorganic Chemistry 64 (2016) 51–58
Table 2
Cytotoxic activity of acridone carboxamide derivatives against various drug sensitive and resistant cancer cell lines.
Compound
Cell lines/IC50
MCF7/Wt1
(l
M)a
MCF7/Mr2
MCF7/Dx3
SW13984
WiDr5
LS174T6
3
4
5
6
7
8
9
10
11
12
13
14
22.0 1.8
30.0 1.5
4.5 0.8
8.5 1.5
18.2 0.9
17.5 0.8
12.6 1.1
10.5 1.6
1.6 0.2
1.2 0.4
1.4 0.6
0.8 0.1
32.0 1.4
55.0 1.1
5.1 1.1
10.2 0.9
26.0 1.3
30.1 2.1
22.4 0.6
20.3 1.9
2.1 0.2
1.9 0.1
1.7 0.1
2.7 0.1
30.0 1.9
60.0 1.7
6.5 1.2
9.5 0.5
28.2 0.8
26.7 2.0
21.7 1.4
18.5 1.6
3.2 0.4
2.9 0.4
4.2 0.2
1.9 0.1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2.9 0.1
11.0 0.7
1.7 0.2
4.5 0.8
19.1 1.5
2.8 0.7
3.8 1.0
18.2 3.1
3.1 0.3
Mitoxantrone (Mr)
Doxorubicin (Dx)
0.090
0.098
3.0
–
–
3.7
–
–
–
–
–
–
a
1
2
3
4
5
6
Inhibitory concentrations are presented in Mean SEM.
MCF7/Wt: Human breast cancer cell line.
MCF7/Mr: Mitoxantrone resistant MCF7 cells (BCRP overexpression).
MCF7/Dx: Doxorubicin resistant MCF7 cells (P-gpexpression).
SW1398: Colorectal cancer cell line.
WiDr: Human colon adenocarcinoma.
LS174T: Human colorectal cancer cell lines.
expression) cell lines. Selected molecules were also screened for
their cytotoxic properties against three colorectal cancer cell lines
(SW1398, WiDr and LS174T). Each cell line was incubated with
4
Dx
Dx+Compound14
eight concentrations (0.5–100 lM) for each compound and was
3
2
1
0
used to create concentration versus growth inhibition curves. The
response parameter IC50 was calculated for each cell line and
tabulated in Table 2.
In the present study, the newly synthesized compounds exhib-
ited an individual potential pattern of selectivity as well as broad-
spectrum cytotoxicity against both sensitive and resistant cell
lines. Most of the nitric oxide releasing acridones showed activity
against the wild type MCF7 cells with IC50 values varying from
0.8
with BCRP expression were sensitive towards the compounds 11,
12, 13 and 14 with IC50values of 1.7–2.7 M comparable to mitox-
antrone (3.0 M). Regarding doxorubicin resistant MCF7/Dx cells
lM to 1.6 lM (Table 2). Mitoxantrone resistant MCF7/Mr cells
MCF7/Wt
MCF7/Dx
l
Compounds (180 min)
l
with P-gp expression, compounds fused with NO donor, such as
compound 11, 12, 13, and 14 exhibited higher cytotoxicity with
Fig. 1. Intracelular accumulation of doxorubicin in the presence and absence of
compound 14 in MCF7 cell lines. To measure the doxorubicin efflux, cell
IC50 values of 1.9
lM (compound 14)–4.2
lM (compound 13) com-
monolayers were treated with 4
lM of doxorubicin with or without nitric oxide
parable to doxorubicin IC50 (3.7
l
M). Additionally, compounds 12
donating acridone (compound 14, 50
l
M) for 30 min. An extracellular doxorubicin
concentration was measured at 180 min by using spectrofluorometer Intragroup
statistical calculation are performed using t-test and two-tailed P-values are
reported. Significant increase in the intracellular concentration of doxorubicin is
seen in MCF7/Dx cell lines in presence of compound 14 at 180 min (P = .0076). No
significant difference in intracellular doxorubicin in MCF7/Wt cell lines with or
without compound 14 at 180 min (P = .3125).
and 14 also possessed better cytotoxicity against SW1398, WiDr
and LS174T cell lines compared to compound 13. However, the
cytotoxicity potential of acridone derivatives that were not bearing
nitric oxide releasing group was found to be inferior compared to
NO donating acridones at lower concentrations against the most
of the studied cell lines.
2.2.2. Doxorubicin accumulation and efflux
the effect of nitric oxide on the doxorubicin efflux process in P-gp
expressing cancer cells, in the present study, newly designed nitric
oxide releasing acridones were evaluated for their effects on
doxorubicin efflux in MCF7/wt and MCF7/Dx (P-gp expressing) cell
lines. Results of doxorubicin accumulation and efflux in doxoru-
bicin sensitive (MCF7/Wt) and resistant (MCF7/Dx) with and
without compound 14 is shown in Figs. 1 and 2 respectively, and
the rates of efflux of doxorubicin in combination with and without
compound 14 in MCF7/Dx cell lines is shown in Figs. 3 and 4.
During 180 min of exposure time, the intracellular levels of dox-
orubicin significantly increased (P < .001). We have also observed
a significant reduction (P = .0004) in efflux of the doxorubicin into
Earlier studies suggested that doxorubicin sensitive and dox-
orubicin resistant human colon cancer cells exhibit a different
capacity to produce nitric oxide [5] and the doxorubicin resistance
is reversed by inducers of NO production, such as cytokines and
atorvastatin, or NO releasing compounds. Another study reported
implication of NO in the doxorubicin cytotoxicity in HT29 cells
and reverts doxorubicin resistance, via the tyrosine nitration of
P-gp and MRP3, ABC transporters which recognize doxorubicin as
a substrate [16]. NO can also inhibit the efflux of doxorubicin
(NO donor SNAP), thus decreases the resistance of the cell to the
drug. This hypothesis was confirmed by kinetic data [5]. Considering

V.V.S. Rajendra Prasad et al. / Bioorganic Chemistry 64 (2016) 51–58
55
Dx+Compound 14
Dx
5
4
3
2
1
0
4
3
2
1
Dx
Dx+Compound14
0
MCF7/Wt
MCF7/Dx
30
60
90
120
150
180
Time (min)
Fig. 2. Efflux of doxorubicin in the presence and absence of compound 14 in MCF7
cell lines. To measure the intracellular doxorubicin concentration, cell monolayers
were treated with 4
acridone (compound 14, 50
l
M of doxorubicin with or without nitric oxide donating
M) for 30 min. An extracellular doxorubicin concen-
Fig. 4. Time dependent efflux of doxorubicin in the presence and absence of
compound 14 in MCF7/Dx. To measure the doxorubicin efflux, cell monolayers were
l
tration was measured at 180 min by using spectrofluorometer. Intragroup statis-
tical calculation are performed using t-test and two-tailed P-values are reported. No
significant difference in doxorubicin efflux concentration in MCF7/Wt cell lines
with or without compound 14 at 180 min (P = .76). Significant decrease in the efflux
concentration of doxorubicin is seen in MCF7/Dx cell lines in presence of compound
14 at 180 min (P = .021).
treated with 4
(compound 14, 50
l
M of doxorubicin with or without nitric oxide donating acridone
M) for 30 min. An extracellular doxorubicin concentration was
l
measured at different time intervals (30–180 min). Inter-group analysis is per-
formed using ANOVA test. Significant reduction (P = .0004) in doxorubicin efflux
concentration is seen in presence of compound 14.
Table 3
Exogenous release of nitrite by NO-acridones in MCF7 cancer cells.
Compound
Nitrite concentration was expressed as
nano moles of nitrite per 24 h/mg cell
protein
4
Dx
MCF7/Wt
MCF7/Dx
Dx+Compound 14
11
12
14
5.21
4.65
7.02
4.31
3.49
6.64
3
2
1
0
2.2.3. Nitric oxide release
Exogenous release of nitric oxide by NO-donating acridones
might improve the cytotoxicity of acridones as well as affect the
role of NO in doxorubicin efflux and influx. Therefore we
determined the release of nitric oxide by NO donating acridones
by measuring nitrite production in MCF7/Wt and MCF7/Dx (P-gp
expressing) cell lines. Compounds 11, 12 and 14 were studied for
their ability of nitric oxide release (Table 3). Results indicate that
nitrite levels in both cell lines were between 3.49 and 7.02 nmol/mg
of protein per 24 h. Among the derivatives compound 14 had a
higher NO release in MCF7/Wt (7.02 nmol/mg per 24 h) and
MCF7/Dx (6.64 nmol/mg per 24 h) cells compared to other
investigated molecules. Furthermore, the release of nitric oxide
in sensitive and resistant cell line was identical in the experimental
condition. Moreover, good associations were observed when
cytotoxic data of these molecules compared with exogenous nitrite
release.
30
60
90
120
150
180
Time (min)
Fig. 3. Time dependent intracellular accumulation of doxorubicin in the presence
and absence of compound 14 in MCF7/Dx. To measure the doxorubicin influx, cell
monolayers were treated with 4
donating acridone (compound 14, 50
concentration was measured at different time intervals (30–180 min). Significant
difference in intracellular doxorubicin concentration is seen every time interval in
presence of compound 14. Statistical calculations are performed suing t-test (two-
tailed P value at 30 min is .00998, .02 at 60 min, <.001 at 90, 120, 150 and 180 mins).
l
M of doxorubicin with or without nitric oxide
M) for 30 min. An intracellular doxorubicin
l
2.2.4. Molecular docking and binding energy calculations
the extracellular medium in presence of compound 14. When cell
lines were treated with doxorubicin along with fixed concentration
of nitric oxide releasing acridone for same time intervals, there was
no effect observed on doxorubicin accumulation in MCF7/Wt cells
(P = .31; P = .76). However, nitric oxide releasing acridones signifi-
cantly increased the intracellular concentration of doxorubicin in
MCF7/Dx cells (P = .0076) by inhibiting the doxorubicin efflux pro-
cess. In addition, acridone derivatives without nitric oxide donat-
ing moiety (1–10) are unable to alter the doxorubicin efflux.
These investigations demonstrated that NO donating acridones sig-
nificantly restored the doxorubicin susceptibility in P-gp expressed
MCF7/Dx cells by inhibiting the doxorubicin efflux process.
Molecular docking studies indicated that the TYR 310 and GLN
725 amino acids of P-gp protein were associated in binding with
newly synthesized molecules (protonated after dissociation of NO
group). Ligands such as compound 10 and compound 1 interacted
with protein residues GLN 990 and SER 222 respectively. Com-
pound 14 reported highest docking score (ꢀ8.610) (Fig. 5). Earlier
studies conducted by Dolghih E et al. also reported binding of var-
ious P-gp inhibitors at the same pocket [17]. Post docking
calculation include estimation of ligand binding energy with
protein, suggested compound 14 has shown highest binding energy
of -83.57 kcal/mol. Protein ligand interactions along with binding
energies of acridone derivatives with P-gp were shown in Table 4.

56
V.V.S. Rajendra Prasad et al. / Bioorganic Chemistry 64 (2016) 51–58
Fig. 5. Interactions of compound 14 with target protein P-glycoprotein. Prominent hydrophobic interactions are seen between compound 14 and PHE 343 and other amino
acid of docked P-glycoprotein.
Table 4
4. Experimental
Protein ligand interactions and post docking calculations of novel acridones with
P-glycoprotein.
4.1. Materials and methods
Compound
Dock
score
No of
H-bonds
Interacting
amino acids
H-bond
distance
(Å)
Binding
energy
All chemicals and solvents were supplied by Sigma Aldrich, S.D.
Fine Chemicals Ltd, Bangalore India. Reactions were monitored by
thin layer chromatography (TLC) and compounds were purified by
using column chromatography with silica gel Merck Grade 60
(230–400 mesh, Merck, Germany). Melting points were recorded
on a Tempirol hot-stage with microscope (AGA International,
India) and were uncorrected. Nitric oxide releasing acridone
derivatives were characterized by 1H and 13C NMR in DMSO-d6
solution in a 5 mm tube on a Brukerdrx 500 Fourier transform
spectrometer (Bruker Bioscience, USA) and tetra methyl silane
(TMS) was used as an internal standard. Chemical shifts were
expressed as d (ppm) values. The spectrometer was internally
locked to deuterium frequency of the solvent. Acridones were also
characterized by ESI-MS spectrometry. Collision-induced dissocia-
tion (CID) spectra were acquired in the positive ion mode on an
MDS Sciex (Concord, Ont., Canada) API 4000 triple quadrupole
mass spectrometry with direct infusion of each acridone at a con-
14
13
5
10
3
6
4
9
12
2
ꢀ8.610
ꢀ7.411
ꢀ6.825
ꢀ6.718
ꢀ6.274
ꢀ6.196
ꢀ6.097
ꢀ6.178
ꢀ5.93
0
1
0
1
0
0
0
1
0
0
1
2
–
–
1.83
–
1.91
–
–
–
2.23
–
ꢀ83.571604
ꢀ76.915953
ꢀ70.954276
ꢀ68.425796
ꢀ67.854175
ꢀ62.197424
ꢀ66.741288
ꢀ60.745826
ꢀ59.271147
ꢀ55.412975
ꢀ56.124834
ꢀ53.943285
GLN 725
–
GLN 990
–
–
–
TYR 310
–
–
TYR 310
TYR 310
GLN 725
–
ꢀ5.739
ꢀ5.618
ꢀ5.482
–
7
11
2.08
2.36
2.71
–
8
1
ꢀ5.198
ꢀ5.014
0
1
ꢀ59.215846
ꢀ54.158622
SER 222
1.84
3. Conclusions
centration of 10 lM in 50% methanol, at flow rate of 25 lL/min.
The instrument was operated with a spray voltage of 5.5 kV, a
declustering potential of 50 eV, a source temperature of 100 °C, a
GSI value of 50 and the curtain gas set at 10. Ultra-pure nitrogen
was used as both curtain gas and collision gas. MS/MS spectra of
the protonated molecule of each drug were acquired and multiple
reaction monitoring (MRM) transition for important fragments
were monitored as the collision energy was ramped from 5 V to
100 V (step size 0.5 V). The data for the fragment-ion curves repre-
sent an average of five consecutive experiments. The nitrate/nitrite
release rates from individual derivatives in the cells were deter-
mined by a colorimetric assay using the nitrate/nitrite colorimetric
assay kit (Sigma Aldrich, The Netherland), according to the
manufacturer’s instructions.
Several newly developed nitric oxide donating acridone carbox-
amides were active in drug sensitive and resistant cell lines and
could reverse MDR, which might be related the nitric oxide release
rate. We used the fluorescent nature of doxorubicin to determine
the altered abilities of MCF7/Wt and MCF7/Dx (P-gp expressed)
cells to accumulate doxorubicin during 180 min of exposure in
the presence and absence of nitric oxide releasing acridone. The
results from the cytotoxicity assays, in vitro nitrite release, and
drug accumulation studies clearly indicate that NO donating acri-
dones had a remarkable rate of nitrite release and cytotoxic effects.
Moreover, the present studies also demonstrated that exogenous
release of nitric oxide by NO donating acridones enhanced the
accumulation of doxorubicin in MCF7/Dx cell lines when it is coad-
ministered with doxorubicin by inhibiting efflux process. It is also
clear that presence of NO group potentiated the cytotoxic effect of
the acridone derivatives. Further, nitric oxide donating group could
potentiate the N10-substituted acridones to reverse the doxoru-
bicin resistance in MCF7/Dx cells.
4.2. In-vitro cytotoxic studies by SRB assay
The nitric oxide releasing acridone derivatives were
evaluated for their cytotoxic activity against different cancer cells

V.V.S. Rajendra Prasad et al. / Bioorganic Chemistry 64 (2016) 51–58
57
(MCF7/Wt, MCF7/Dx, MCF7/Mr, SW1398, WiDr, LS174T) by using
measured at 540 nm with a Tecan microplate reader. Then50 lL
the sulforhodamine B (SRB) assay [15]. In brief, cells were cultured
in RPMI 1640 supplemented with 10% fetal calf serum, and cultures
were passaged once or twice a week using trypsin EDTA to detach
the cells from their culture flasks. The fast-growing cells were
harvested, counted and plated at suitable concentrations in
96-well microplates. Cells were allowed to adhere for 24 h.
Thereafter one plate was fixed to determine the initial absorbance
(T0). To the other plates compounds dissolved in the culture med-
ium were added to the culture wells in triplicate and incubated
further for 72 h at 37 °C under 5% CO2 atmosphere. The cultures
were fixed with cold TCA and stained with0.4% SRB dissolved in
of the cell suspension was used for measurement of cellular pro-
teins. A blank was prepared for each experimental condition in
the absence of cells, and its absorbance was subtracted from that
obtained in the presence of cells. Nitrite concentration was
expressed as nanomoles of nitrite per 24 h/mg of cellular protein.
4.5. Molecular docking and binding energy calculations
In silico binding interactions of P-gp & newly synthesized acri-
dones was performed by using Glide XP algorithm of Schrodinger
Suite. Digital crystal structure of P-gp protein was retrieved from
the PDB website (PDB ID: 4Q9H) and was processed by removing
non protein components and adding missing amino acids and
hydrogen atoms to satisfy the valence and optimized by using
OPLS-2005 force field. Binding pockets were identified and a recep-
tor grid was generated and ligands (energy minimized) were
docked [18]. The terminal NO group of each compound is replaced
with protonation in order to understand the binding pattern of the
acridone which is protonated after dissociation of NO donor group.
Later, to the docked complexes MM/GBSA calculations were
performed to determine the binding energy [18]. The total free
energy of binding is then expressed in the form below mentioned
equation:
1% acetic acid. After dissolving the bound stain with 150 lL of
10 mM unbuffered Tris base (Tris(hydroxymethyl)aminomethane)
solution using gyratory shaker, absorbance was measured at
540 nm using a microplate reader (Tecan). Growth was calculated
by correcting the control plate (without drugs) cultured for 72 h
(T72 control) with the T0 plate. Growth inhibition was calculated
as: (T72 drug – T0)/(T72 control – T0) ⁄ 100%. The efficacy of the
compounds was compared by estimating the concentration
required to inhibit cellular growth by 50% (i.e., IC50) from each
separate growth inhibition curve. Each value represents the mean
of triplicate experiments.
4.3. Doxorubicin accumulation and efflux studies by
spectrofluorometer
D
Gbind = Gcomplex – (Gprotein + Gligand) where
D
Gbind is ligand
binding energy
In other terms,
Doxorubicin sensitive and resistant MCF7 cells (7 ꢁ 105/well)
were grown in 35 mm diameter Petri dishes. Before every test, cells
were washed and cultured in fresh medium without doxorubicin
for 24 h. Later, cells were incubated in 2 ml medium containing
D
Gbind
¼
D
Egas
þ
D
Gsolvation ꢀ T
DS
where gas phase molecular mechanical energy is
D
Egas (i.e. Contri-
butions from the van der Waals energy, electrostatic energy, and
internal energy), the solvation free energy are Gsolvation (polar
and non-polar contributions) and T S is the entropy [19].
4
l
mol/L doxorubicin with or without nitric oxide donating acri-
D
dones (50 M) for different time intervals (30–180 min), washed
l
D
twice in ice-cold PBS and harvested using trypsin/EDTA
(0.05/0.02% v/v). Cells were centrifuged for 3 min at 15,000 rpm
(4 °C) and re-suspended in 1 mL of a 1:1 mixture of ethanol/0.3 N
4.6. Synthesis and chemical characterization
HCl. Then 50
lL of the cell suspension was used for measurement
4.6.1. Preparation of 2,40-iminodibenzoic acid (I): Ullmann
Condensation
of cellular proteins. The remaining part was checked for the dox-
orubicin content using a Fluor-max spectrofluorimeter. Excitation
and emission wavelengths were 475 nm and 553 nm, respectively.
A blank was prepared in the absence of cells in every set of exper-
iments and its fluorescence was subtracted from that obtained in
the presence of cells. Fluorescence was converted in nanomoles
of doxorubicin per milligram of cellular protein, using a calibration
curve prepared previously. To measure the drug efflux, cell mono-
To a mixture of o-chloro benzoic acid (7.8 g, 0.05 mol), p-amino
benzoicacid (6.85 g, 0.05 mol) and copper powder (0.2 g) in 60 mL
isoamyl alcohol, dry potassium carbonate (5 g) was slowly added
and the contents were allowed to reflux for 6–8 h on an oil bath
at 160 °C. The isoamyl alcohol was removed by steam distillation
and the mixture was poured into one liter of hot water and acidi-
fied with concentrated hydrochloric acid. Precipitate was filtered,
washed with hot water and collected. The crude acid was dissolved
in aqueous sodium hydroxide solution, boiled in the presence of
activated charcoal and filtered. On acidification of the filtrate with
concentrated hydrochloric acid, a light yellowish precipitate was
obtained which was washed with hot water and recrystallized
from aqueous methanol to give light yellow solid (yield 91%,
mp 187 °C).
layers were incubated with 4 lmol/L doxorubicin for 30 min; cells
were washed twice with PBS and incubated in fresh 5 ml of PBS,
then 0.5 mL of supernatant was taken at different time intervals
(15–180 min) and checked for doxorubicin associated fluorescence
as described above.
4.4. Nitrite measurement in vitro
The levels of nitrite formed from individual compounds in the
cells were determined by a colorimetric assay using the nitrate/
nitrite colorimetric assay kit (Sigma Aldrich, The Netherland),
according to the manufacturer’s instructions. Confluent cell mono-
layers (6 ꢁ 105/well) in 35 mm diameter Petri dishes were treated
4.6.2. Synthesis of acridone-2-carboxylic acid (II)
Five grams of 2,40-iminodibenzoic acid (I) was put into a round
bottom flask and 50 grams of polyphosphoric acid was added. The
reaction mixture was shaken well and heated on a water bath at
100 °C for 3 h. Appearance of yellow color indicated the completion
of the reaction. Then, it was poured into one liter of hot water and
made alkaline by liquor ammonia. The yellow precipitate was
filtered and collected. The acridone-2-carboxylic acid (II) was
recrystallized from acetic acid (yield 89%, mp 326–328 °C). Further,
purity of the compound was checked by TLC and the purified
product was characterized by spectral methods.
in triplicate with 25 lM NO releasing acridone and incubated for
24 h under the experimental conditions. The cells were washed
twice in ice-cold PBS and harvested using trypsin/EDTA
(0.05/0.02% v/v). Cells were centrifuged for 5 min at 15,000 rpm
(4 °C) and re-suspended in 1 mL of a 1:1 mixture of ethanol/0.3 N
HCl. Nitrite production was measured by mixing 100
lysates with 100 L of Griess reagent in a 96 well plate and after
30–300 min incubation at 37 °C in the dark, absorbance was
lL of cell
l
1H NMR (DMSO-d6) d ppm: 12.63 (s, 1H, OH), 11.15 (s, 1H, NH),
7.31–8.52 (m, 7H, Ar–H). 13C NMR (DMSO-d6) d ppm: 172.4, 169.4,

58
V.V.S. Rajendra Prasad et al. / Bioorganic Chemistry 64 (2016) 51–58
156.2, 143.5, 140.4, 137.4, 134.3, 132.4, 131.2, 128.3, 126.3, 125.5,
122.4. ESI-MS (m/z, %): 239.21 (100).
12: 1H NMR (DMSO-d6) d ppm: 7.51–8.59 (m, 7H, Ar–H), 2.81 (t,
4H, CH2–N–CH2), 2.92 (t, 4H, CH2–O–CH2), 3.03 (t, 2H, N–CH2–
CH2–CH2–ONO2), 4.63 (t, 2H, N–CH2–CH2–CH2–ONO2), 2.15 (m,
4.6.3. General method of preparation of acridone carboxamides (1–6)
To the suspension of compound II (1 gm, 4.18 mmol) in 15 ml of
dry toluene, thionyl chloride (0.42 ml, 5.81 mmol) and freshly
dried pyridine (0.46 ml, 5.81 mmol) were added. The reaction mix-
ture was stirred at room temperature for 3–4 h and then combined
with excess of secondary amines and triethylamine (1.72 ml,
12.37 mmol) and stirred for another 3 h. The reaction was moni-
tored by TLC. The following day the solvent was removed under
vacuum and water was added to the solid residue. The precipitate
was filtered, washed with water and dried. The crude product was
crystallized from n-butanol-DMF (19:1).
2H, N–CH2–CH2–CH2–ONO2). 13C NMR (DMSO-d6) d ppm: 171.6,
162.4, 158.1, 143.7, 141.5, 139.4, 136.6, 135.1, 133.4, 129.7,
128.1, 126.4, 124.9, 68.4, 59.5, 58.3, 55.2, 54.2, 49.5, 48.2. ESI-MS
(m/z,%): 412.8 (40).
14: 1H NMR (DMSO-d6) d ppm: 7.32–8.45 (m, 7H, Ar–H), 2.65 (t,
4H, CH2–N–CH2), 2.89 (t, 4H, CH2–O–CH2), 3.16 (t, 2H, N–CH2–
CH2–CH2–CH2–ONO2), 4.58 (t, 2H, N–CH2–CH2–CH2–CH2–ONO2),
2.27 (m, 4H, N–CH2–CH2–CH2–CH2–ONO2). 13C NMR (DMSO-d6)
d ppm: 172.1, 163.5, 158.4, 143.3, 142.4, 139.5, 136.2, 134.4,
133.6, 128.4, 127.1, 126.6, 125.3, 65.5, 59.4, 58.4, 57.9, 55.3, 54.1,
50.5, 49.3. ESI-MS (m/z,%): 426.7 (50).
1: 1H NMR (DMSO-d6) d ppm: 10.85 (s, 1H, NH), 7.38–8.57
(m, 7H, Ar–H), 2.94 (t, 8H, CH2–N–CH2–CH2–N–CH2), 2.98 (s, 3H,
N–CH3). 13C NMR (DMSO-d6) d ppm: 173.1, 168.2, 159.5, 141.4,
140.5, 136.1, 135.7, 134.7, 130.8, 127.2, 126.4, 124.7, 123.6, 56.8.
54.3, 47.8, 46.3.ESI-MS (m/z,%): 321.7 (80).
Acknowledgments
Dr. V.V.S. Rajendra Prasad would like to acknowledge the fund-
ing support from SERB, Department of Science and Technology
(DST), Government of India under ‘‘Fast Track Scheme” [SR/FT/LS-
175/2009] and Laboratory Medical Oncology, VU University
Medical Center (VUMC), Amsterdam, The Netherlands for provid-
ing necessary research facilities to carry out the cytotoxicity
studies.
4.6.4. N-alkylation of acridone carboxamide (7–10)
Substituted acridone-carboxamide (310 mg, 1 mM) was dis-
solved in 25 ml of dry DMF and then added NaH (120 mg). Then
the reaction mixture was stirred under nitrogen at room tempera-
ture for 60 min and 1-bromo-3-chloropropane or 1-bromo-4-
chloro butane (2 mM), potassium iodide (150 mg) added slowly
into the reaction mixture and stirred for 24 h at room temperature.
Water was slowly added under ice bath with rapid stirring. The
crude yellow color product was collected and purified.
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7: 1H NMR (DMSO-d6) d ppm: 7.41–8.53 (m, 7H, Ar–H),
2.71–2.82 (t, 8H, CH2–N–CH2–CH2–N–CH2),2.91 (t, 2H, N–CH2–
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ppm: 172.5,164.9, 156.3, 141.6, 140.1,137.3, 136.2, 135.2, 133.1,
129.1, 128.6, 126.2, 124.5, 61.2, 59.2, 58.6. 56.3, 55.9, 49.4, 46.4,
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4.6.5. Nitration of N-alkylated acridone carboxamide (11–14)
A solution of the appropriate haloalkyl derivative in dry ace-
tonitrile (2 ml) was treated portion wise with a solution of AgNO3
(0.34 g, 2 mmol) in dry acetonitrile (5 ml) and the whole mixture
was stirred at room temperature for 3 h; the reaction was moni-
tored by TLC. The mixture was then filtered, evaporated to dryness
then residue was recrystallized.
11: 1H NMR (DMSO-d6) d ppm: 7.48–8.51 (m, 7H, Ar–H), 2.68–
2.91 (t, 8H, CH2–N–CH2–CH2–N–CH2), 3.12 (t, 2H, N–CH2–CH2–
CH2–ONO2), 4.42 (t, 2H, N–CH2–CH2–CH2–ONO2), 2.02 (m, 2H,
N–CH2–CH2–CH2–ONO2), 3.12 (s, 3H, N–CH3). 13C NMR (DMSO-
d6) d ppm: 172.4, 161.6, 157.6, 144.6, 143.2, 137.1, 136.3, 135.3,
132.5, 128.4, 127.4, 126.3, 124.3, 68.6, 59.6, 58.2, 55.5, 53.3, 48.7,
47.8, 37.3. ESI-MS (m/z,%): 425.6 (68).
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