Research Article

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Design, synthesis and evaluation of

acridone-2-carbohydrazide derivatives as
p-AKT Ser473 kinase inhibitors
Tanuja T Yadav1 , Maushmi S Kumar1 , Shalini Bajaj3 & Mayur YC*,2
1
Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM’s NMIMS, Mumbai, 400056, India
2
Somaiya Institute for Research & Consultancy, Somaiya Vidyavihar University, Mumbai, 400077, India
3
Truba Institute of Pharmacy, Bhopal, Madhya Pradesh, 462038, India
*Author for correspondence: Tel.: +91 22 6924 6064; [email protected]

Aim: A series of benzylidene- and phenylethylidene-substituted acridone-2-carbohydrazide derivatives

were designed, synthesized and evaluated for their cytotoxicity and response to p-AKT Ser473 . Methods:
The structures of the synthesized compounds were confirmed by spectroscopic techniques and evaluated
for AKT enzyme inhibition activities. Molecular docking and in silico absorption, distribution, metabolism,
elimination and toxicity studies were also performed. Results: Compounds 8k, 8v and 9h demonstrated
good cytotoxicity against breast cancer cell lines. Especially, compounds 8v and 9h exhibited remarkable
inhibition, with IC50 values of 1.75 and 2.40 μM, respectively. These compounds inhibited p-AKT Ser473
more specifically than total AKT in a dose-dependent manner. Moreover, they caused G0/G1-phase cell
cycle arrest and cell apoptosis. Conclusion: This study identified compound 8v as a potent p-AKT Ser473
inhibitor.

Graphical abstract:
Carbohydrazide plays an important role
in anti-cancer activity
O
Significant loss in potency

(MCF-7: lC50 = 38.59 μM)

O O Causes cell cycle arrest
N at G0/G1 phase and induces
N N
H apoptosis
H
N (MCF-7: lC50 = 6.24 μM)
H
Replaced with -CH3 Found to be most potent AKT inhibitor
increases cytotoxicity (lC50 = 1.75 μM)

Order of Reactivity
R 3-OMe > 2-Cl > 2-F > 4-substituted

Replacement with -F, and -OMe at 4th position

of phenyl ring showed slightly decrease in activity

First draft submitted: 12 November 2022; Accepted for publication: 17 March 2023; Published online:
12 May 2023

Keywords: acridone-2-carbohydrazides • anticancer • DNA binding • molecular docking • p-AKT

Protein kinases are intracellular enzymes that regulate the phosphorylation of proteins [1]. They are the largest
group of kinases that phosphorylate at serine, threonine and tyrosine residues, and are involved in several signal
transduction pathways [2]. There are two main types of protein kinases: serine/threonine kinases and tyrosine kinases.
The serine/threonine kinase AKT, which is also referred to as protein kinase B, is a key component of phosphatidyl-

10.4155/fmc-2022-0271
C 2023 Newlands Press Future Med. Chem. (Epub ahead of print) ISSN 1756-8919
Research Article Yadav, Kumar, Bajaj & Mayur

inositol-3-kinase (PI3K), which plays a crucial role in regulating cell growth, metabolism, proliferation, survival,
migration and angiogenesis [3,4]. It is activated by phosphorylation on residue Ser473 or Thr308 and it phosphorylates
a variety of downstream protein substrates [5]. The overexpression or hyperactivation of AKT has been observed
in many cancers and is associated with increased cancer cell survival and proliferation; thus, targeting AKT
could provide an attractive approach for the design and development of small molecules as promising anticancer
agents [5,6].
Acridone is an interesting tricyclic N-heterocycle containing a unique planar structure [7]; it can intercalate with
DNA base pairs [8] and acts as a substrate for multidrug resistance [9,10], efflux pumps like P-glycoprotein [11,12] and
breast cancer resistance protein [13,14]. It has been reported in the literature that this scaffold has the potential to
interact with different biological targets [15,16] such as DNA topoisomerase I/II [17,18], telomerase [19], dihydrofolate
reductase [20] and protein kinases [21], including cyclin-dependent kinases [22] and AKT [23,24]. Hence the acridone
scaffold has emerged as an important anticancer compound and drawn the attention of medicinal chemists aiming
to synthesize novel molecules [25].
Many research scientists have tried to design and synthesize acridone-based scaffolds which would have the ability
to display anticancer activity by interacting with multiple targets. Among them, some studies have discovered that
substituted acridones have the ability to inhibit AKT kinase. For example, Houghton et al. patented the 2-Br/Cl-
N 10 -substituted acridone compounds as AKT inhibitors and revealed that these molecules can inhibit AKT
phosphorylation and kinase activity. Among them, most of the N 10 -butyl-substituted 2-chloroacridones (1a–1e)
were found to be effective against AKT as compared with N 10 -butyl-substituted 2-bromoacridones (except 2a and
2b) at <5 μM concentration [23]. Murahari et al. identified and reported a series of acridone–pyrimidine hybrids as
potent AKT inhibitors. Their study found that these hybrid molecules (3a–3c) showed a good cytotoxicity profile
against A-549 (lung), MCF-7 and MDA-MB-231 (breast) cancer cell lines and exerted their anticancer activity
partly by DNA intercalation. Further, mechanistic studies revealed that the compounds have the potential to inhibit
AKT kinase activity and induce apoptosis. An in vitro AKT kinase assay identified compounds 3a–3c as potent
AKT inhibitors, with an IC50 of <10 nM [24].
There are several AKT inhibitors under clinical investigation, including the ATP-competitive pan-AKT inhibitor
AZD5363, AstraZeneca’s invention, which is also known as capivasertib (Figure 1) [26,27]. It inhibits all AKT isoforms
(AKT1/AKT2/AKT/3) with a potency of <10 nM, and inhibited phosphorylation of AKT substrates in cells
with a potency of approximately 0.3–0.8 μM [28,29]. Currently, this drug is in a phase III clinical trial in two
different combinations: one is with fulvestrant in metastatic hormone receptor-positive/HER2-negative breast
cancer (CAPItello-291) and the second is in combination with palbociclib + fulvestrant in hormone receptor-
positive/HER2-negative advanced breast cancer (CAPItello-292) [27,30].
Helwa et al. designed, synthesized and evaluated 6-morphilino-pyrimidine derivatives as PI3-K inhibitors. Here,
the 6-morphilino-pyrimidine scaffold was linked with aromatic rings through a hydrazinyl linker. All the synthesized
compounds were screened for their cytotoxicity against a panel of NCI-60 cells. Among them, compound 4 showed
the most potent antiproliferative activity against the leukemia SR cell line, with an IC50 of 0.76 μM. The PI3K
kinase inhibition assay found that compound 4 was found to be a promising inhibitor, with IC50 values of 11.73,
6.09 and 11.18 μM against (α), (β) and (γ) of PI3K class IA, respectively. The presence of a hydrazinyl linker and
m-substitution on the phenyl ring resulted in an increase in anticancer activity against a panel of cell lines [31].
Based on previous studies of acridone containing a carbohydrazide linker and the interesting activity of hydrazinyl
compounds, we have synthesized acridone-2-carbohydrazide derivatives and characterized and screened them for
in vitro anticancer activity against human cancer cell lines A-549 (lung), MCF-7 and MDA-MB-231 (breast) and
A-431 (skin). In silico studies were carried out and included molecular docking and prediction of absorption,
distribution, metabolism, elimination and toxicity (ADMET) properties by using computational applications. We
performed molecular docking studies to identify the orientation and binding interactions with essential amino acids
at the active site of AKT1. Acridones have been reported as DNA intercalators [8,32,33]; hence, to estimate the binding
strength of the compounds, we carried out absorption titration against calf thymus DNA (CT-DNA). Quantitative
ELISA analysis of phosphorylated-AKT Ser473 (p-AKT) and total AKT, cell cycle analysis and apoptosis were
carried out for determining the underlying molecular mechanism of the most active derivatives.

10.4155/fmc-2022-0271 Future Med. Chem. (Epub ahead of print) future science group
Design, synthesis & evaluation of acridone-2-carbohydrazide derivatives as p-AKT Ser473 kinase inhibitors Research Article

O
O O
Cl Br

N
N N H H R
N N
R R O N
H
N O

1a R = 4’-Cl O
2a R = 4’-N-Pyrrolidine 3a R = H
1b R = 4’-N-Diethylamine 2b R = 4’-N-Morpholine
1c R = 4’-N-Methypiperazine 3b R = 2-F
1d R = 4’-N-Piperidine 3c R = 4-OMe
1e R = 4’-N-(E-hydroxyethyl)piperazine
Reported AKT inhibitors

O
N
N
O OH
NH2 H
N N N Br
Aromatic Ring HN N
O O
N
H H N
N N N
Acridone ring O N Linker H
H Cl Aromatic Ring
N O O
Aromatic Ring Linker
Linker O Capivasertib
Reported AKT Inhibitor Reported PI3-K inhibitor
(AZD5363)
3c 4

O O
R1 Heterocyclic
N
N Mono-/di-/tri-substituted
H
Aromatic -CH3/-OCH3/-F/
N
H Ring -Cl/ -NO2/-Br/-OH

Acridone Ring Linker

Figure 1. Structures of acridone derivatives as AKT inhibitors with their design approach. (A) Reported AKT
inhibitors. (B) Design approach of acridone-2-carbohydrazide derivatives.

Materials & methods

Reagents & equipment
Reagents such as substituted aldehydes and acetophenones were purchased from (Sigma-Aldrich, Mumbai, India).
Other chemicals were procured such as thionyl chloride (Spectrochem, Mumbai, MH, India), hydrazine hydrate
(Sisco Research Laboratories, Mumbai, MH, India) and glacial acetic acid (SD-Fine Chem, Mumbai, MH, India).
Solvents utilized for the present study were purchased from (Loba Chemie, Mumbai, India). The progress of
reactions was monitored by thin layer chromatography (TLC) using Merck silica gel 60 F254 aluminum sheets
(Merck Millipore, Billerica, MA, USA). The spot identification was performed under UV cabinet (Desaga, Biostep,
Burkhardtsdorf, Germany) at a wavelength of 254 nm. All the compounds were purified by passing through
column chromatography packed with silica gel of 100–200 mesh size. The melting points (mps) of the compounds
were determined on a melting point apparatus (Veego, Mumbai, India). Purity analysis was carried out using the
Shimadzu LC-20AD Prominence high performance liquid chromatography (HPLC) system (Shimadzu, Kyoto,
Japan). The separation process was carried out on a Phenomenex C18 (Sigma-Aldrich) bonded reverse-phase
column. The mobile phase used was methanol and water which was degassed prior to use, and the flow rate was
maintained at 0.7 ml/min. The purity of all final compounds was found to be ≥95%. Pure compounds were
characterized by 1 H NMR and 13 C NMR (Bruker Advance DRX, Billerica) at 400/500 MHz and 100/125
MHz, respectively. In NMR analysis, DMSO-d6 and tetramethyl silane used as a solvent and internal standard
respectively. Chemical shifts relative to deuterated solvents were expressed in ppm. The IR spectra of the molecules

future science group 10.4155/fmc-2022-0271

Research Article Yadav, Kumar, Bajaj & Mayur

were recorded on a Fourier transform infrared (FTIR, Spectrum RXI, Perkin Elmer Spectra, Waltham, MA, USA).
The mass spectra in ESI-MS mode were recorded with 6410 Triple Quad LC/MS (Agilent Technologies Inc, Santa
Clara, CA, USA) using methanol as solvent.

Synthesis of target compounds

Chemistry
Ethyl 9-oxo-9,10-dihydroacridine-2-carboxylate (6)
To 9(10H)-acridone-2-carboxylic acid [14,34] (5) (1 g), thionyl chloride (10 ml) was added and refluxed for 4–5 h.
Then the excess thionyl chloride was distilled and ethanol (30–40 ml) was added slowly and refluxing continued
for 6–8 h. After completion of the reaction, the mixture was left for 2–4 h at room temperature (RT). The
yellow-colored precipitates were collected, filtered and washed with ice-cold saturated sodium bicarbonate solution
and then with cold water.

9(10H)-acridone-2-carbohydrazide (7)
To a stirred solution of compound 6 (1 g, 3.7 mmol) in dry ethanol, hydrazine hydrate (0.337 ml, 1.5 equiv, 6.73
mmol) was added and refluxing continued for 12 h. The reaction mixture was monitored by TLC and evaporated
under reduced pressure to remove the ethanol. The residue was diluted with cold water, filtered and used as such
for further process.

General procedure for compounds 8a–8w & 9a–9h

To a suspended solution of compound 7 (0.2 g, 1 equiv) in ethanol (10 ml) with glacial acetic acid (1 ml),
aldehydes/ketones (1.5 equiv) were added dropwise and the reaction mixture was refluxed for 12–16 h. After
completion of the reaction, the mixture was allowed to cool at RT and then concentrated to dryness. Cold water
was added to the reaction mixture, which was filtered and the crude product was recrystallized from a suitable
solvent to give the desired products (8a–8w) and (9a–9h). Spectral characterization of compounds (8c–8w and
9a–9h) is described in the Supplementary Material (1 H NMR, 13 C NMR, IR, HPLC and MS).

(E)-N -Benzylidene-9-oxo-9,10-dihydroacridine-2-carbohydrazide (8a)

Yellow solid powder, yield: 67.2%; purity (HPLC): 95.33%; mp: 180–182◦ C; ATR (ν/cm-1 ): 3252 (Amide, -NH),
3168 (Ar, -NH), 3106 (Ar, C-H), 3065 (Ar, C-H), 1674 (Amide, C=O), 1578 (Ar, C=O), 1551 (Ar, C=C), 1274
(Ar, C–N); 1 H NMR (500 MHz, DMSO-d6 ) δ: 11.73 (s, 1H), 11.49 (s, 1H), 8.54 (s, 1H), 8.09 (d, J = 8.1 Hz,
2H), 7.93 (d, J = 7.8 Hz, 3H), 7.81 (s, 1H), 7.70 (d, J = 7.3 Hz, 1H), 7.61–7.57 (m, 3H), 7.26 (d, J = 7.5 Hz,
1H), 6.91 (t, J = 7.4 Hz, 1H); 13 C NMR (125 MHz, DMSO-d6 ) δ: 176.4, 163.4, 148.5, 142.4, 140.7, 134.3,
132.6, 130.5, 130.4, 129.2, 128.8, 127.5, 127.3, 126.0, 120.9, 120.0, 119.2, 118.6, 114.8; (ESI): Calcd for [M
+ H]+ 342.11 and for [M + Na]+ 364.11; found: 342.15 and 364.15.

(E)-9-Oxo-N -((E)-3-phenylallylidene)-9,10-dihydroacridine-2-carbohydrazide (8b)

Yellow solid powder, yield: 41.36%; purity (HPLC): 95.62%; mp: 230–233◦ C; ATR (ν/cm-1 ): 3281 (Amide,
-NH), 3205 (Ar, -NH), 3060 (Ar, C-H), 2981 (Al, C-H), 1624 (Amide, C=O), 1546 (Ar, C=O), 1522 (Ar, C=C),
1298 (Ar, C–N), 976 (Al, C=C); 1 H NMR (400 MHz, DMSO-d6 ) δ: 11.99 (s, 1H), 11.95 (s, 1H), 8.83 (s, 1H),
8.26–8.18 (m, 3H), 7.76 (t, J = 7.52 Hz, 1H), 7.61–7.54 (m, 4H), 7.38–7.27 (m, 4H), 7.03 (s, 2H); 13 C NMR
(100 MHz, DMSO-d6 ) δ: 173.7, 162.0, 143.3, 142.4, 140.7, 136.8, 133.4, 132.6, 130.4, 129.7, 129.2, 128.7,
128.2, 127.3, 126.0, 120.9, 120.0, 119.2, 118.6, 116.3; (ESI): Calcd for [M + H]+ 368.13 and for [M + Na]+
390.13; found: 368.20 and 390.20.

Biological activity
In vitro cytotoxicity testing by sulforhodamine B assay
All these compounds were subjected to initial screening to evaluate their in vitro antiproliferative activity. A single
dose (25 μM) of the test compounds was used against a panel of cell lines which included non-small-cell lung
cancer (A-549), skin cancer (A-431) and breast cancer cells (MDA-MB-231 and MCF-7) and normal murine
embryonic fibroblast cells (NIH/3T3). Cells were obtained from the National Centre for Cell Science (Pune,
India) and cultured using Dulbecco’s modified Eagle medium (DMEM) (Genetix-Biotech Asia, New Delhi, India)
supplemented with 10% fetal bovine serum (Himedia, Mumbai, India) and 1% penicillin–streptomycin (Genetix-

10.4155/fmc-2022-0271 Future Med. Chem. (Epub ahead of print) future science group
Design, synthesis & evaluation of acridone-2-carbohydrazide derivatives as p-AKT Ser473 kinase inhibitors Research Article

Biotech Asia, New Delhi, India). The data were represented as percentage growth inhibition (GI%) of treated
cells.
A cytotoxicity study of the synthesized compounds was carried out by sulforhodamine B (SRB) assay, using
doxorubicin as a positive control. Cells were plated onto a 96-well plate with a density of 5000–10,000 cells/well
in a complete growth medium and were incubated for 24 h. They were treated with the tested compounds at
different concentrations (0.1–100 μM) for 72 h and then proceeded to SRB assay [35]. For the assay, 20% TCA
(Trichloro-acetic acid) was used for the fixation of cells and then the cells were stored at 4◦ C for 1 h. SRB reagent
(0.4% in acetic acid) was added for staining purposes and then cells were incubated at RT in the dark for 30 min.
Acetic acid (1%) was utilized to wash the cells for the removal of excess unbound SRB. After drying for 4 h, Tris
base was added to each well to solubilize the bound SRB and the optical density (OD) was measured at 510 nm
using a microplate reader [36].

AKT enzyme inhibition assay

Selected compounds were evaluated for their ability to inhibit AKT kinase activity by using the EKS-400A assay kit
(Enzo Life Sciences, Farmingdale, NY, USA). All the reagents and working standards were prepared according to
the manufacturer’s instructions. The cells were treated with selected acridone derivatives at different concentrations,
and compound 3c (reported AKT inhibitor) was used as a reference compound. The cell lysates were prepared
and assayed for estimation of protein concentration by using a protein assay kit i.e.; bicinchoninic acid (Thermo
Fisher Scientific, MA, USA). The AKT substrate microtiter plate was soaked with kinase assay dilution buffer at
RT for 10 min. A volume of 30 μl of each of the diluted solutions (control, standard and samples) was added
to each empty well in duplicate. The reaction was initiated by adding 10 μl of diluted ATP and was incubated
for 90 min at RT; then 40 μl of phospho-specific substrate antibody, as well as the diluted secondary antibody,
was added and incubated for 30 min. Then 60 μl of 3,3 ,5,5-tetramethylbenzidine was added into each well to
develop color in the wells. After sufficient color development, 20 μl of stop solution was added to the wells. OD
was recorded with a plate reader at 450 nm. Data were analyzed and results expressed as IC50 values using different
concentrations [37,38].
Further, in vitro quantitative estimation of phosphorylated p-AKT Ser473 or total AKT protein was analyzed by
using a human p-AKT Ser473 and total AKT ELISA kit (Raybiotech, Peachtree Corners, GA, USA). Briefly, 100
μl of cell lysates were added into wells of the ELISA kits and incubated for 2.5 h at RT. Then these lysates were
discarded and the wells washed with buffer four times, before 100 μl of primary antibody (p-AKT and total AKT),
as well as secondary antibody solutions, were added to measure phosphorylated or total protein in the cell lysates.
Then, 100 μl of 3,3 ,5,5-tetramethylbenzidine substrate reagent was added, followed by a stop solution. OD was
measured at 450 nm and the activation status of AKT signaling proteins was evaluated according to the formula:
OD value of p-AKT/OD value of total AKT [39].

Cell cycle analysis assay

Flow cytometric analysis was used to observe the cell cycle progression of MCF-7 cells with or without treatment
with the synthesized compounds. The standard propidium iodide (PI) staining method was used to determine the
effect of selected compounds 8k, 8v and 9h on the cell cycle [40,41]. MCF-7 cells were seeded at a density of 2.5
× 105 cells/well in a six-well plate and incubated at 37◦ C for 24 h. Subsequently, the medium was removed and
the cells were treated with compounds at their IC50 concentration for 24 h. Then cells were collected and washed
twice with cold phosphate-buffered saline (PBS) and fixed in ice-cold 70% (v/v) ethanol overnight at 4◦ C. Again,
cells were washed with PBS, resuspended with 100 μg/ml RNase, then the DNA of the cells was stained with 40
μg/ml PI, incubated for 10 min in the dark. The acquisition and analysis of data were performed using a flow
cytometer at the FACS Central Facility at IIT Bombay [42,43].

Apoptosis assay
The annexin V/PI double staining method was used to carry out the apoptosis assay with the help of flow
cytometry [44]. An annexin V–fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (BD Biosciences,
Franklin lakes, NJ, USA) was used for the determination of apoptosis. MCF-7 cells were plated in a six-well plate
with a density of 2.5 × 105 cells/well and incubated for 24 h. Then cells were treated with the selected compounds
(8k, 8v and 9h) at 5 μM concentration for 24 h. Cells were trypsinized and washed with PBS, then resuspended in
1× binding buffer at a concentration of 1.0 × 106 cells/ml. From that, 100 μl of the test solution at a concentration

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Research Article Yadav, Kumar, Bajaj & Mayur

of 1.0 × 105 cells was treated with the mixture of annexin V–FITC (5 μl) and PI (5 μl) and incubated in the dark
for 10–15 min at RT. Finally, 400 μl of binding buffer was added and cells were resuspended again just before the
flow cytometry analysis, performed using a FACSCalibur™ flow cytometer (BD Biosciences) at IIT Powai [31,41].

CT-DNA binding assay

The UV-absorption titration method was utilized for comparison of the DNA-binding affinity of the synthesized
compounds. This study was performed as per the earlier reported method: a fixed concentration of acridones (15
μM) was used with a variable concentration of CT-DNA (0–100 μM) and absorbance was measured using a UV
spectrophotometer [24,45,46]. Their intrinsic binding constants (Kb ) were calculated with the following equation for
studying the DNA-binding affinity of these compounds quantitatively:

[DNA] [DNA] 1
= +
εa − εf εb − εf Kb (εb − εf )

Where [DNA] = concentration of DNA base pairs; Kb = binding constant; and εa, εf, εb are the apparent
extinction coefficients for the complex with DNA, without DNA and fully bound with DNA, respectively. A slope
and an intercept were obtained from the plotted graph of [DNA]/(εa − εf ) versus [DNA] which were equal to 1/(εb
− εf ) and 1/Kb /(εb − εf ), respectively. The Kb was calculated from the ratio of the slope to the intercept [47,48].

Molecular docking
To identify existing binding interactions of the synthesized molecules within the binding site of the target protein,
Autodock Vina software was used [49]. x-ray crystallographic structures of AKT1 (Protein Data Bank [PDB] ID:
4GV1) and DNA (PDB ID: 2GB9) were retrieved from the PDB [50]. A docking study was performed as per the
previously reported method [51–53]. Preparatory steps were carried out before docking analysis using Auto Dock
Tools. The 2D and 3D structures of all the synthesized compounds were generated. Both ligands and receptor
were transformed to the proper format for docking. The molecular docking was performed to observe possible
protein–ligand interactions and binding affinity. The docking score for each compound structure was determined
in kcal/mol. Results were analyzed in BIOVIA/Discovery Studio 2017R2 (Biovia, CA, USA) for the visualization
and structure analysis of the docked complexes of AKT1 and DNA. 2D ‘Receptor–Ligand Interactions’ modules
were used to check various types of binding interactions [54,55].

In silico ADMET studies

For the prediction of absorption, distribution, metabolism and elimination parameters of molecules, the web
tool Swiss-ADME was used [56]. It calculates the physicochemical and significant pharmacokinetic properties of
molecules. In addition to that, toxicity data were predicted using TEST software (TEST, Washington D.C., USA).
Oral rat LD50 (mg/kg) values were predicted for potential molecules [57].

Results & discussion

Chemistry
Synthesis of acridone started with the Ullmann condensation of halobenzoic acid and p-aminobenzoic acid in
the presence of copper as a catalyst. Cyclization with polyphosphoric acid yielded acridone-2-carboxylic acid.
Further esterification and treatment with hydrazine hydrate gave carbohydrazide of acridone in the presence of
solvent ethanol. The final compounds were synthesized by linking the carbohydrazide of acridone with different
substituted aldehydes/ketones in ethanol in the presence of acetic acid which yielded two series, one with substituted
benzaldehydes (8a–8w) and the other with substituted acetophenones (9a–9h). These compounds were synthesized
as per the synthetic route shown in Figure 2. The molecular structures of the newly synthesized compounds were
established and characterized by 1 H NMR, 13 C NMR, IR and mass analytical techniques (see Supplementary
Material).

Biological evaluation
In vitro cytotoxicity assessment
All synthesized compounds were subjected to in vitro antiproliferative assessment against various cancer cell lines
(MCF-7, MDA-MB-231, A-549 and A-431). Compounds were initially screened against the cells at a single dose

10.4155/fmc-2022-0271 Future Med. Chem. (Epub ahead of print) future science group
Design, synthesis & evaluation of acridone-2-carbohydrazide derivatives as p-AKT Ser473 kinase inhibitors Research Article

O O O O

OH (i) OC2H5

N N
H H
5 6

(ii)

O O O O
O OH
N R NH2 (iv) N R
(iii) N N
N
H H H
N N N
H H H
R = Aryl, Heteroaryl R = Aryl

(8a–8w) 7 (9a–9g)

8a=Phenyl 8m=3,4-dimethoxyphenyl 9a=Phenyl

8b=Styryl 8n=2,5-dimethoxyphenyl 9b=4-hydroxyphenyl
8c=2-hydroxyphenyl 8o=3,4-dihydroxyphenyl 9c=4-methoxyphenyl
8d=4-fluorophenyl 8p=4-hydroxy-3-methoxyphenyl 9d=4-methylphenyl
8e=4-Bromophenyl 8q=3,4,5-trimethoxyphenyl 9e=4-fluorophenyl
8f=4-methoxyphenyl 8r=4-hydroxy-3,5-dimethoxyphenyl 9f=4-chlorophenyl
8g=4-chlorophenyl 9g=4-nitrophenyl
8s=4-benzyloxyphenyl
8h=4-methylphenyl 9h=2,4-dichlorophenyl
8t=4-dimethylaminophenyl
8i=2-fluorophenyl
8u=4-cyanophenyl
8j=2-chlorophenyl
8v=4-pyridyl
8k=3-methoxyphenyl
8w=2-furanyl
8l=2-nitrophenyl

Figure 2. Scheme for synthesis of target compounds (8a-8w) and (9a-9h) with reagents and conditions. (i)
SOCl2 ,60◦ C, 3h and ethanol, 80◦ C, 6–8 h; (ii) hydrazine hydrate, 80◦ C, 10 h; (iii) aldehydes, glacial acetic acid, ethanol,
80◦ C, 12–14 h; (iv) ketones, glacial acetic acid, ethanol, 80◦ C, 12–16 h.

of 25 μM concentration and the mean GI% of all compounds was calculated. All the synthesized compounds
(8a–8w and 9a–9h) showed moderate-to-excellent inhibitory activity against all tested cell lines except A-431 at
25 μM. A total of 20 compounds displayed >50% growth inhibition against the MCF-7 cell line (Figure 3). From
them, compounds 8k, 8l, 8o, 8q, 8t, 8u, 8v and 9b–9h exerted good-to-excellent GI% ranging from 63.18 to
80.89%. Similarly, tested compounds displayed moderate inhibitory activity against the MDA-MB-231 cell line.
Compounds 8b, 8f, 8h, 8s, 8u, 8v, 8w, 9b, 9c and 9h demonstrated >50% growth inhibition. For the A-549
cell line, only five compounds (8t, 8w, 9a, 9e, 9g and 9h) had moderate-to-good GI%, ranging from 50.37 to
78.88%. Regarding the breast cancer cell lines, six compounds possessed good inhibitory activity against MCF-7
whereas three compounds displayed good growth inhibition of the MDA-MB-231 cell line.
The cellular toxicity of the selected compounds was also evaluated using normal murine embryonic fibroblast
(NIH/3T3) cell lines at a final concentration of 50 μM to see whether the molecules’ antiproliferative effect had
specificity for cancer cells. The results indicated that these compounds had no significant effect on the growth of
normal cells.
Based on the results of the initial screening of synthesized derivatives, it was found that breast cancer cell lines
MCF-7 and MDA-MB-231 were the most sensitive to the tested compounds, and therefore they were selected for
in vitro antiproliferative activity assessment at different dose levels. The growth inhibitory activity of benzylidene
derivatives (8b, 8c, 8i–8l, 8n, 8o and 8q–8v) and phenylethylidene derivatives (9b–9h) of acridone carbohydrazide
against MCF-7, MDA-MB-231 and A-549 was evaluated by using SRB assay. The results were expressed as half
maximum growth inhibitory concentration (IC50 – the concentration of drug required to exhibit 50% inhibition
of the cell growth as compared with control) and are shown in Table 1.

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In vitro cytotoxicity screening against MCF-7 In vitro cytotoxicity screening against MDA-MB-231
100 100
% growth inhibition (GI %)

% growth inhibition (GI %)

90 90
80 80
70 70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
8u
8v
8w
9a
9b
9c
9d
9e
9f
9g
9h
DOX

8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
8u
8v
8w
9a
9b
9c
9d
9e
9f
9g
9h
DOX
In vitro cytotoxicity screening against A-549 In vitro cytotoxicity screening against A-431
100
100
% growth inhibition (GI %)

% growth inhibition (GI %)

90
90
80
80
70
70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
8u
8v
8w
9a
9b
9c
9d
9e
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9h
DOX

8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
8u
8v
8w
9a
9b
9c
9d
9e
9f
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9h
DOX
Toxicity testing against NIH/3T3
100
90 50 μM
80
% cell viability

70
60
50
40
30
20
10
0
Control
8b
8c
8i
8j
8k
8l
8n
8o
8q
8r
8s
8t
8u
8v
9b
9c
9d
9e
9f
9g
9h

Figure 3. In vitro cytotoxicity screening of acridone-2-carbohydrazide derivative against cancer cell lines at a single dose of 25 μM. (A)
MCF-7 (B) MDA-MB-231 (C) A-549 and (D) A-431. (E) Toxicity testing of selected compounds against normal cells NIH/3T3 at a single dose
of 50 μM.
DOX: Doxorubicin.

The in vitro cytotoxicity study revealed that the substituted phenylethylidene derivatives displayed significant
cytotoxicity against breast cancer cell lines as compared with the substituted benzylidene derivatives. In the first
series (acridone carbohydrazides with benzylidene substitution), most of the compounds showed moderate-to-
good cytotoxic activity against both breast cancer cell lines, indicating that compounds containing the acridone
carbohydrazide with pyridine heterocyclic structure were toxic to cancer cells. Furthermore, the cytotoxic effect
of a few compounds (8b, 8i, 8j, 8k, 8t, 8u and 8v) on MCF-7 cells was much greater than that on MDA-
MB-231 and A-431 cells, indicating that the compounds were selective for AKT kinase. Among them, the most
promising compound 8v showed excellent activity against MCF-7 and MDA-MB-231 cells (IC50 : 6.24 and 9.92
μM, respectively). Compounds 8k, 8t and 8i exhibited good antiproliferative activity against MCF-7 cells, with
IC50 values of 7.92, 8.39 and 10.55 μM respectively, while compounds 8b, 8c, 8h, 8j, 8l, 8n, 8q, 8r and 8u
showed moderate cytotoxicity against MCF-7 cells with IC50 values of 15–30 μM. Among the title compounds
from this series, only the dimethyl amino benzylidene derivative (8t; IC50 : 11.32 μM) and 4-fluoro benzylidene
derivative (8i; IC50 : 19.65 μM) displayed good activity against the A-549 cell line.
In the second series of phenylethylidene derivatives of acridone carbohydrazides, compounds 9b, 9c, 9d and 9h
showed moderate cytotoxicity against MCF-7 and MDA-MB-231 breast cancer cell lines at concentrations <20
μM. Compound 9c showed better activity against MDA-MB-231 (IC50 : 7.45 μM) than against MCF-7 (IC50 :

10.4155/fmc-2022-0271 Future Med. Chem. (Epub ahead of print) future science group
Design, synthesis & evaluation of acridone-2-carbohydrazide derivatives as p-AKT Ser473 kinase inhibitors Research Article

Table 1. In vitro cytotoxicity of synthesized compounds.

Compound ID IC50 (μ M)† (± standard deviation)
MCF-7 MDA-MB-231 A-549
8b 15.64 ± 0.62 23.93 ± 2.04 39.42 ± 1.14
8c 17.82 ± 0.79 45.07 ± 1.50 47.18 ± 1.98
8h 28.32 ± 1.65 18.76 ± 0.68 35.46 ± 0.47
8i 10.55 ± 1.77 50.36 ± 0.49 19.65 ± 0.66
8j 18.73 ± 1.29 41.15 ± 0.46 45.34 ± 0.79
8k 7.92 ± 0.10 33.63 ± 2.23 68.96 ± 0.35
8l 22.63 ± 2.2 46.23 ± 0.77 51.74 ± 1.52
8n 27.36 ± 1.78 39.17 ± 1.42 34.82 ± 0.36
8o 32.21 ± 0.42 45.73 ± 0.92 49.43 ± 1.35
8q 20.93 ± 2.22 38.71 ± 0.23 54.38 ± 1.27
8r 21.83 ± 2.16 43.52 ± 0.18 37.38 ± 1.42
8s 37.62 ± 0.79 16.75 ± 0.62 38.92 ± 0.98
8t 8.39 ± 1.59 26.93 ± 2.04 11.32 ± 0.49
8u 17.40 ± 0.72 45.15 ± 1.65 38.45 ± 0.58
8v 6.24 ± 0.72 9.92 ± 0.10 34.82 ± 0.36
8w 38.59 ± 4.4 29.62 ± 0.12 25.93 ± 0.76
9b 13.68 ± 4.22 18.81 ± 0.81 29.84 ± 0.88
9c 14.47 ± 4.12 7.45 ± 0.17 27.36 ± 0.63
9d 15.29 ± 1.25 17.32 ± 1.76 31.11 ± 0.43
9e 10.39 ± 0.87 21.83 ± 2.16 8.52 ± 0.69
9f 12.86 ± 0.29 23.46 ± 0.43 36.47 ± 0.81
9g 18.69 ± 3.90 32.53 ± 1.45 13.90 ± 0.21
9h 9.60 ± 0.93 17.35 ± 1.38 23.03 ± 0.35
DOX‡ 1.28 ± 1.19 2.6 ± 1.08 4.76 ± 0.15
† IC
50 values are the mean of three separate experiments.
‡ DOX: Doxorubicin, used as positive control.

14.47 μM). Compounds having the phenyl ring substituted with 4-fluoro (9e) or 2,4-dichloro (9h) displayed
better activity against the MCF-7 cell line, whereas phenyl ring substituted with 4-methoxy (9c) showed excellent
activity against MDA-MB-231. Compounds containing an ethylidene acridone carbohydrazide moiety were found
to be more potent against MDA-MB-231 cells, with IC50 values of 7.45–23.46 μM. Among them, 4-fluoro
phenylethylidene derivative 9e (IC50 : 8.52 μM) exhibited better activity against A-549. In comparison, the title
compounds 8v and 9h showed appreciable cytotoxicity against MDA-MB-231 and MCF-7 cell lines.
From these data, the cell proliferation assay determines that acridone carbohydrazide with phenylethylidene
substitution is preferred over benzylidene substitution for anticancer activity. Mono substitution on the benzyli-
dene moiety at either the ortho or meta position showed improved cytotoxicity against cancer cells. Acridone
carbohydrazide containing 4-pyridine derivatives exhibited potential anticancer activity against breast cancer cells.

CT-DNA binding assay by absorption titration

Numerous acridone derivatives have been identified as potential anticancer agents which exert their anticancer
activity through DNA intercalation. Various studies have revealed that acridone derivatives, being a planar aromatic
system, have DNA-binding ability [7,8,45]. Thus, to identify the binding interaction of acridone derivatives with
DNA, absorption titration of compounds with CT-DNA was performed. This is the universally used colorimetric
assay to determine the relative binding affinities of molecules with DNA. The concentration of acridone compounds
was kept constant (15 μM) with varying CT-DNA concentrations (0–100 μM). Using UV-visible absorption
titration, the effect of substitution on the compounds’ DNA-binding affinity was studied and the intrinsic binding
constant (Kb ) was calculated; the results are shown in Supplementary Table 1.
In this study, acridone carbohydrazide derivatives 8k, 8t, 8v, 9e and 9h showed DNA intercalation activity
in the range of Kb 0.87 × 105 M-1 to 3.79 × 105 M-1 . The series of substituted benzylidene derivatives of

future science group 10.4155/fmc-2022-0271

Research Article Yadav, Kumar, Bajaj & Mayur

acridone derivatives showed better DNA-binding affinity compared with the substituted phenylethylidene acridone
compounds. Among these compounds of substituted benzylidene derivatives, the 3-methoxy derivative (8k) and
4-dimethyl amino derivative (8t) showed good intrinsic binding constants of 2.12 × 105 and 3.79 × 105 M-1 ,
respectively. From the series of substituted phenylethylidene acridone compounds, the 4-fluoro derivative (9e) and
2,4-dichloro derivative (9h) showed comparatively low DNA-binding affinity, with Kb values of 1.03 × 105 and
0.77 × 105 M-1 , respectively. From the data obtained from the in vitro cytotoxicity assay results, compounds
containing 3-methoxy and 4-dimethyl amino substitutions showed IC50 values <10 μM. This suggests that these
compounds might exert their cytotoxicity through intercalation. Surprisingly, the most active compounds (8v and
9h) showed a relatively lower binding affinity, with Kb values of 0.87 × 105 and 0.77 × 105 M-1 , respectively. These
acridone derivatives did not display any significant DNA intercalation, which might be because of steric hindrance
due to their bulky structure. These compounds might therefore have produced good cytotoxicity results in the
proliferation assay through another mechanism. Thus further studies are required to find the exact mechanism
behind the cytotoxicity of these types of acridone molecules.

In vitro AKT kinase inhibition assay

In the cell proliferation assay, compounds 8k, 8t, 8v and 9h showed good cytotoxicity profiles against MCF-7 cells,
with IC50 values of <10 μM. Hence these compounds were chosen for the in vitro AKT kinase inhibition assay to
evaluate whether they can truly inhibit AKT activity. Here, the inhibitory rate of selected compounds against AKT
kinase was assayed and then IC50 values were determined. MCF-7 cells were treated with selected compounds (8k,
8t, 8v and 9h) at different concentrations (0, 0.5, 2.5, 5 and 10 μM) for 48 h. The results were then compared
with the reference compound (IC50 : 0.57 μM). All the compounds exhibited decreased inhibitory activity against
AKT kinase, with IC50 values ranging from 1.75 to 7.36 μM. Among them, the inhibitory activities of compounds
8v and 9h were noteworthy, with IC50 values of 1.75 and 2.40 μM, respectively, against AKT kinase; 8k and 8t
also exhibited good AKT kinase inhibitory activity with IC50 values of 5.10 and 7.36 μM, respectively. The results
obtained from the AKT kinase inhibition assay revealed that inhibition of AKT might be one of the reasons behind
the good cytotoxicity profile of compounds 8k, 8v, 8t and 9h against the MCF-7 cell line.

ELISA analysis of p-AKT Ser473 & total AKT

From the in vitro AKT kinase assay, compounds 8k, 8v and 9h were shown to have inhibitory potential toward
AKT kinase in a dose-dependent manner, and therefore they were chosen for elucidating the mechanism behind
the good AKT inhibition. A quantitative determination of the phosphorylated and total AKT levels was done to
evaluate the inhibitory effect of these compounds in MCF-7 cells.
These compounds effectively inhibited the activation of p-AKT Ser473 in MCF-7 cells at 5 μM. Quantitative
ELISA confirmed the above result and showed that these compounds significantly reduced the levels of p-AKT
Ser473 in MCF-7 cells (Figure 4). Among them, compound 8v displayed a significant reduction in levels of p-AKT
Ser473 compared with total AKT at all the tested concentrations, while compounds 8k and 9h showed a significant
reduction of p-AKT at higher concentrations only. These same compounds produced a minimal inhibitory effect
on the levels of total AKT at the tested concentrations. These results suggest that the inhibition of p-AKT Ser473
and total AKT might be responsible for the good activity of compound 8v against the MCF-7 cell line. From the
results, we can conclude that compounds 8k, 8v and 9h were found to have potential anticancer effects due to
p-AKT Ser473 inhibition.

Cell cycle analysis

Cell cycle analysis was carried out to determine the effects of promising compounds on the cell cycle and to identify
the mechanism responsible for the growth inhibition of MCF-7 cells. The cellular DNA content and cell population
in G0/G1, S, G2 and M phases were observed and evaluated using flow cytometry in the MCF-7 cell line. After
treatment with compounds 8k, 8v and 9h, there was a disturbance observed in the phase of the cell cycle of MCF-7
cells. There was a reduction in the percentage of cells in the S and G2/M phases, whereas the percentage of cells in
the G0/G1 phase was increased when compared with the control. Untreated cells with normal phase cycles showed
62.5, 20.8 and 11% of the cells in the G0/G1, S and G2/M phases, respectively; for treated cells, the percentage
of cells in the G0/G1 phase increased from 62.5% (control) to 67.3, 69.1 and 65.9% with compounds 8k, 8v
and 9h, respectively. Thus there was a significant rise in the percentage of cells in the G0/G1 phase with selected

10.4155/fmc-2022-0271 Future Med. Chem. (Epub ahead of print) future science group
Design, synthesis & evaluation of acridone-2-carbohydrazide derivatives as p-AKT Ser473 kinase inhibitors Research Article

Ctrl 1 µM 2.5 µM 5 µM Ctrl 1 µM 2.5 µM 5 µM Ctrl 1 µM 2.5 µM 5 µM

20 70 1.2
18 60 1.0
16 * ns *
p-AKT Ser473 (µg/ml)

50

Total-AKT (µg/ml)

p-AKT Ser473 /AKT

14 **
** ** 0.8
12 * *** ***
40 ** *
** ***
10 *** ** 0.6
30 ** *** *
8 * ** **
*** 0.4 *** ***
6 ** ***
20
4 ***
10 0.2
2
0 0 0.0
8k 8v 9h 8k 8v 9h 8k 8v 9h
Control 8k treated
Control 8k treated

150 150 105 Q1 Q2 105 Q1 Q2

0.033 0.40 11.9 3.31

100 104 104

Count

Count

100

Comp-PE-A

Comp-PE-A
103 103
50 50
SubG1 G0/G1 S G2M SubG1 G0/G1 S G2M
4.01 62.5 20.8 11.0 3.01 67.3 17.7 11.4
0 0
Q3 Q4 Q3 Q4
0 0 -103 99.5 0.11 -103 84.3 0.45
0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K
10 1
10 2
10 10
3 4
10 5
102
10 3
10 4
105
PE-Texas red-A PE-Texas red-A 101
Comp-FITC-A Comp-FITC-A
8v treated 9h treated 8v treated 9h treated
Q1 Q2 Q1 Q2
10 5 105

150 150 17.0 4.42 13.3 2.22

104 104

Comp-PE-A
100
Comp-PE-A

100
Count

Count

103 103
50 50
SubG1 G0/G1 S G2M SubG1 G0/G1 S G2M 0 0
69.1 17.7 8.27 4.29 65.9 19.8
3.92 8.91
Q3 Q4 Q3 Q4
-10 3
77.4 1.10 -103 83.9 0.60
0 0
0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 101 102 103 104 105 101 102 103 104 105
PE-Texas red-A PE-Texas red-A Comp-FITC-A
Comp-FITC-A

Cell cycle analysis Sub-G1 G0/G1 S G2/M

80
70
% cell distribution

60
50
40
30
20
10
0
Control 8k 8v 9h

Figure 4. MCF-7 cells were treated with compounds at different concentrations (0, 1, 2.5 and 5 μM) for 24 h. (A–C) Quantitative ELISA
assay results of compounds 8k, 8v and 9h on (A) levels of p-AKT Ser473 , (B) levels of total AKT and (C) the activation of p-AKT Ser473 . A
significant difference in p-AKT Ser473 was seen between the control group and the 8v-treated group. Compounds inhibit cancer cell
growth by suppressing the activation of AKT signaling. (D) Effect of compounds 8k, 8v and 9h on cell cycle distribution of MCF-7 cells
using flow cytometry. Representation of typical cell cycle profiles. (E) Quantitative analysis of selected compounds on the cell cycle
distribution. (F) Apoptosis assay of compounds 8k, 8v and 9h using flow cytometry after staining with annexin V–fluorescein
isothiocyanate/propidium iodide. MCF-7 cells were treated with 8k, 8v and 9h for 24 h and the results are represented as scatter plots of
propidium iodide (y-axis) versus annexin V (x-axis).
*p < 0.05; **p < 0.01; ***p < 0.001. Error bars: mean ± SD of three independent experiments.
ns: No significant difference.

compounds causing cell cycle arrest, as represented in Figure 4. These compounds were found to have the ability
to inhibit proliferation through cell cycle arrest at G0/G1 phase, which will induce apoptotic cell death [41,42].

Apoptosis induction assay

To enumerate the percentage of cells undergoing apoptosis with the treatment of acridone derivatives, an apoptosis
assay was performed. The apoptotic activity of compounds 8k, 8v and 9h was assayed using the annexin V–
FITC/PI double staining method. Cells that are PI-negative and annexin V-positive are expressed in quadrants 2

future science group 10.4155/fmc-2022-0271

Research Article Yadav, Kumar, Bajaj & Mayur

and 4 (apoptotic cells) (Figure 4), being the early and late apoptotic cells, respectively. The cells that are positive
for both PI and annexin V are expressed in quadrant 1 (necrotic cells), and those that are negative for both PI and
annexin V are expressed in quadrant 3 (living cells).
The obtained results were compared with the untreated MCF-7 cell line as control, and the values of total
apoptosis (early and late apoptotic cells, quadrant 2 + quadrant 4) were calculated. For compound 8v, the total
apoptotic cells significantly increased from 0.51 to 5.52%, which was higher than the percentages seen with 8k and
9h at corresponding concentrations. For compounds 8k and 9h, the total apoptotic cell percentages were 3.76 and
2.82%, respectively. From the data represented in Figure 4, it is indicated that these compounds (8k, 8v and 9h)
increased the percentage of annexin V-positive apoptotic cells in both the early and late stages. These compounds
also showed an increase in the percentage of necrotic cells, which might be due to external environmental factors
such as metabolic stresses including hypoxia. Among them, compound 8v displayed a significant (tenfold) increase
in apoptotic cells as compared with the control. This study revealed that 8v displayed cytotoxicity against the
MCF-7 cell line through the induction of apoptosis.

In silico studies
Molecular docking
To understand the binding pattern of the synthesized compounds, molecular docking studies were performed with
AKT (PDB ID: 4GV1) and DNA (PDB ID: 2B9). Protein–ligand complexes were analyzed by docking score and
their orientation in the active site of the protein structure using Autodock Vina software. The results from the
molecular docking study revealed that all the tested compounds showed good binding energy with AKT rather than
DNA. Values for AKT ranged from -9.0 to -10.7 kcal/mol, whereas compounds showed minimal binding energy
with DNA (ranging from -7.4 to -9.6 kcal/mol). The 2D docking interactions of the most potent compounds are
depicted in Figure 5A & B.
The docking scores and binding poses of all derivatives were compared with those of the reference compound
(3c). A lower binding energy (more negative) score indicates stronger and more favorable binding between the
protein and ligand. Seven compounds (8b, 8k, 8m, 8t, 9d, 9g and 9h) showed lower interaction energies compared
with the reference compound, which showed a binding affinity of -9.5 kcal/mol. It formed two conventional
hydrogen bond interactions and one hydrophobic interaction (π–π T-shaped) with amino acids Lys276 and
Phe161, respectively. It developed some hydrophobic interactions (π–anion and π–cation) with Glu191, Asp292
and Lys179. We found that compounds 9g and 9h exhibited -10.7 and -10.3 kcal/mol binding affinity with the
target enzyme, respectively. These molecules formed many hydrophobic interactions (π–sigma, π–sulfur, π–π T-
shaped and π–alkyl) with Val164, Met281, Phe161, Ala177, Ala230, Leu156 and Phe438. Additionally, compound
9g formed two conventional hydrogen bond interactions with Lys158 and Lys179.
As per the results, the most common hydrophobic interactions were observed between the tricyclic aromatic ring
of the acridone scaffold and amino acid residues such as Val164, Leu156, Ala177, Ala230 and Met281. Most of
the compounds displayed a common hydrogen bonding interaction with amino acid residue Lys158. p-substituted
phenylethylidene derivatives (9c, 9d, 9e and 9f) formed common π–anion interactions with Asp292 and Lys179
as well as π–alkyl interactions with Val164, Ala177 and Met227 amino acid residues. The same type of π–alkyl
interaction was observed with the p-substituted benzylidene derivatives (8d, 8f, 8m and 8t). Additionally, in
molecules 8k and 8m, the benzene ring substituted at the third position with the methoxy group exhibited π–π
T-shaped interactions. Furthermore, in molecules 8i and 8j, the benzene ring substituted at the second position
with –F and –Cl groups showed halogen interactions with Gly162 and a hydrogen bond interaction with Lys276,
respectively (Supplementary Table 2).
Molecular docking studies with DNA revealed that the acridone ring formed π–π T-shaped interactions with
nucleotide base pairs dG A:6, dC A:5 and dG B:2. Along with that, the nitrogen atom of acridone hydrazide
formed two hydrogen bonds, one with nucleotide base pair dG B:2 and one with nucleotide base pair dG A:2 of
DNA. Docking results of compounds were compared with those of doxorubicin, which showed binding energy of
-9.6 kcal/mol. It formed some conventional hydrogen bond interactions with nucleotide base pairs dA A:4, dA
B:4, dC A:5, dG B:2, dT B:3 and π–alkyl interactions with dA A:4. We found that only compounds 9e, 9f and
9g showed better intercalation, with comparable binding energies -9.2, -9.6 and -9.3 kcal/mol, respectively. These
compounds displayed one common hydrogen bond interaction with nucleotide base pair dG B:2. This suggests that
these molecules can intercalate with DNA, which might be partly responsible for their anticancer activity. However,

10.4155/fmc-2022-0271 Future Med. Chem. (Epub ahead of print) future science group
Design, synthesis & evaluation of acridone-2-carbohydrazide derivatives as p-AKT Ser473 kinase inhibitors Research Article

i MET ii ASP
TYR
A:229
A:227 A:292
ALA
THR GLU A:177
A:291 A:234
ALA VAL
PHE MET
A:177 VAL GLY A:164 THR
A:164 A:159 A:161 A:291 A:281
ASP
MET A:292
A:281
LYS PHE MET
A:179 A:161 A:227
GLU
TYR A:198
A:229
LEU
A:181
ALA LYS
LYS A:179 GLY
A:230
GLU A:163 A:157 THR
LEU GLY GLY
A:191 LEU A:211
A:156 A:157 A:162 LYS A:156
PHE LYS A:158
A:438 A:158 LEU PHE
THR GLY
A:181 A:438 ALA
A:195 A:159
ILE A:230
A:186

iii iv THR
A:195
LYS GLU
PHE A:179 A:191 GLU
LEU ASP
A:438 A:198 LEU
A:156 A:292
ASP LYS A:181
ALA ALA VAL A:179 GLU
A:292
A:230 A:177 A:164 PHE A:191
GLY A:161
TYR A:157
A:229 HIS
MET A:194
A:281
GLY
LEU LEU A:294
A:181 A:295 LYS
GLU A:276 GLY
ASP VAL
A:228 GLY A:159
A:274 A:164 A:162
PHE THR
GLY ASN A:161 A:160
MET A:159 A:279 LYS
A:227 THR LYS LYS A:158
A:291 A:158 THR A:163
A:312

v vi
DA
A:4
DA
DC DT B:4 DG DT
B:1 DG B:3 B:2 B:3
DC A:6
A:5 DG
B:2

DA
B:4

DG
A:6
DC
A:5

Figure 5. 2D representation of binding interactions of acridone molecules with AKT1 (PDB ID: 4GV1). (A) (i)
compound 9g, (ii) compound 9h, (iii) compound 8v and (iv) reference compound. (B) 2D representation of binding
interactions of acridone molecules with DNA (PDB ID: 2GB9), (v) compound 9f and (vi) doxorubicin. The dotted lines
in different colors reflect various types of interactions: hydrogen bonding (green), charge or polar interactions
(orange),π–alkyl/alkyl (light pink), π–sulfur (yellow), π–π T-shaped (pink) and π–σ (violet).
PDB: Protein Data Bank.

the most active compounds (8v and 9h) showed different binding patterns than doxorubicin, with less binding
energies (Supplementary Table 3). This indicates that these compounds might not act through DNA intercalation
but rely on a different mechanism for their good cytotoxicity.

In silico ADMET prediction

In drug development and discovery, the prediction of the pharmacokinetic properties of any new compounds is an
important aspect. Hence the ADMET and pharmacokinetic properties of the compounds were calculated using
the online software tool SWISS-ADME to predict their drug-like nature. The drug compounds must obey the

future science group 10.4155/fmc-2022-0271

Research Article Yadav, Kumar, Bajaj & Mayur

Table 2. Data obtained from predicted absorption, distribution, metabolism, excretion and toxicity parameters.
Descriptors Compound ID
8b 8k 8t 8v 9c 9d 9e 9h
MW (g/mol) 367.4 371.39 447.48 342.35 385.42 369.42 373.38 424.28
No. of HBA 3 4 4 4 4 3 4 3
No. of HBD 2 2 2 2 2 2 2 2
TPSA 78.66 87.89 87.89 91.55 87.89 78.66 78.66 79
Consensus LogP 3.4 3.39 4.6 2.68 3.67 4.04 3.97 4.69
ESOL LogS -4.64 -4.7 -6.01 -3.97 -4.9 -5.14 -5.0 -6.02
Gastrointestinal High High High High High High High High
absorption
ESOL class MS MS PS Soluble MS MS MS PS
BBB permeation No No No No No No No No
BA score 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55
Lipinski 0 0 0 0 0 0 0 0
violations
LD50 (mg/kg) 3192.04 1580.4 2228.4 1119.5 3108.5 2900.4 2103.4 2381.2
BA: Bioavailability; BBB: Blood–brain barrier; ESOL class: Solubility class in LogS scale; ESOL LogS: ESOL model logarithm of molar solubility in water; HBA: Hydrogen bond acceptor; HBD:
Hydrogen bond donor; LogP: Logarithm of the partition coefficient; MS: Moderately soluble; MW: Molecular weight; PS: Poorly soluble; TPSA: Topological polar surface area.

Lipinski rule to be developed as orally effective drugs [56]. Any violation of this rule might lead to problems with
pharmacokinetic properties.
As per the results shown in Table 2, all the subjected compounds displayed a molecular weight below 500 g/mol,
which indicates that they can be easily transported. The hydrogen bond acceptor (<10) and donor (<5) numbers
were found to be in the range of 3–4 and 0–2, respectively. Title compounds showed logP values in the range of
2.68–4.69 (values l<5 indicate good permeability). All the compounds showed high gastrointestinal absorption
and no blood–brain barrier permeation.
Results from in silico pharmacokinetic data revealed that all the compounds follow the Lipinski rule with no
violation. These compounds (8b, 8k, 8t, 8v, 9c–9e and 9h) have the desired pharmacokinetic properties and can
be considered as ‘drug-like’ for further development of a lead molecule.

Structure–activity relationship
After the results of the cell proliferation assay and AKT enzyme inhibition studies, compounds 8a–8w and 9a–9h,
bearing the same acridone carbohydrazide moiety but with different substituents on the phenyl ring, were subjected
to structure–activity relationship studies with a few assumptions being made, and the findings are summarized in
Figure 6.
The in vitro cytotoxicity evaluation of substituted acridone carbohydrazides revealed that phenylethylidene
derivatives were found to be more potent against MCF-7 and MDA-MB-231. The presence of electron-withdrawing
and -donating substituents at the o-, p- and m-positions of the phenyl ring plays a significant role in cytotoxicity. In
the series of benzylidene-substituted derivatives, the substitution of the methoxy group at the third position (8k)
of the phenyl ring improved the cytotoxicity against both the cell lines, whereas the di-substitution of the methoxy
group at the third and fourth positions (8m) remarkably reduced the cytotoxicity. However, the cytotoxicity of
the benzylidene acridone carbohydrazide moiety was significantly increased when it was substituted with an m/p-
methoxy group (8f and 8k), and was reduced with p-Cl (8h) and p-F (8d) groups. The presence of –Cl, –OH and
–F at the ortho position enhanced cytotoxicity against MCF-7 cells. When the aromatic ring of the benzylidene of
acridone carbohydrazide was replaced with a heterocyclic ring such as furan, the resultant compound (8w) showed
a significant loss in potency and cytotoxicity. The introduction of a pyridine ring instead of furan in the structure
resulted in compound 8v, which was found to be approximately fivefold more potent than 8w against breast cancer
cell lines and acted as the most potent AKT inhibitor.
In the case of phenylethylidene-substituted derivatives, di-substitution of a halo group (–Cl) at the second and
fourth positions (9h) improved the cytotoxicity against both cell lines as compared with the monosubstituted
compound (9f). Replacement of the benzylidene moiety of acridone carbohydrazide (8c, 8d, 8f, 8g, 8h) with
phenylethylidene (9b, 9c, 9d, 9e, 9f) produced IC50 values of <20 μM and resulted in a significant increase in

10.4155/fmc-2022-0271 Future Med. Chem. (Epub ahead of print) future science group
Design, synthesis & evaluation of acridone-2-carbohydrazide derivatives as p-AKT Ser473 kinase inhibitors Research Article

Carbohydrazide plays an important role

in anti-cancer activity
O
Significant loss in potency

(MCF-7: lC50 = 38.59 μM)

O O
N
N N
H
H
N (MCF-7: lC50 = 6.24 μM)
H
Replaced with -CH3 Found to be most potent AKT inhibitor
increases cytotoxicity (lC50 = 1.75 μM)

Order of Reactivity
R 3-OMe > 2-Cl > 2-F > 4-substituted

Replacement with -F, and -OMe at 4th position

of phenyl ring showed slightly decrease in activity

Figure 6. Structure–activity relationship of the acridone-2-carbohydrazide derivatives as AKT inhibitors and

anticancer agents.

cytotoxicity, suggesting that the presence of a methyl group is needed to enhance the cytotoxic activity of the
compound. These results indicate that the electron-negativity, position and steric effect in the benzene ring may
change the cytotoxic profile of these compounds.

Conclusion
In this study, a series of compounds bearing acridone-2-carbohydrazide scaffolds were designed based on a rational
design approach, synthesized and evaluated against A-549, MCF-7, MDA-MB-231 and A-431 cancer cell lines.
An in vitro cytotoxicity assay revealed that compounds 8k, 8t, 8v and 9h displayed potent antiproliferative activity,
especially in MCF-7 cells, with IC50 values of 7.92, 8.39, 6.24 and 9.60 μM, respectively. Furthermore, these
compounds were found to be nontoxic to normal mouse embryonic fibroblast (NIH/3T3) cells. The in vitro AKT
kinase inhibition assay disclosed that compounds 8v and 9h exhibited strong growth inhibition at IC50 values of
1.75 and 2.40 μM, respectively. Quantitative ELISA showed that compounds 8k, 8v and 9h significantly reduced
the protein levels of p-AKT Ser473 (rather than total AKT) in a dose-dependent manner. All these results help
us conclude that inhibition of p-AKT Ser473 might be the main contributing factor toward the good cytotoxicity
exhibited by compounds 8v and 9h. Compound 8v was found to be a potential anticancer agent because of
selectivity to p-AKT Ser473 rather than total AKT.
Structure–activity relationship studies indicated that the presence of the carbohydrazide group and the hetero-
cyclic group played an important role in the antiproliferative activities of the tested compounds. The m-, o- and
p-substitutions on the phenyl ring of benzylidene and phenylethylidene derivatives of acridone carbohydrazides
gave high growth-inhibitory activity on breast cancer cell lines. Flow cytometry showed that the cell cycle was
arrested at the G0/G1 phase, which led to decreased DNA synthesis and thus induced apoptosis; hence the p-AKT
Ser473 inhibitory effect of selected derivatives might be correlated with changes in the cell cycle. According to
molecular docking studies, the compounds possessed the same type of binding interactions within the active site of
AKT. In silico studies predicted these compounds as promising and orally available compounds with low toxicity
profiles and suitable for further optimization as p-AKT Ser473 inhibitors in the treatment of breast cancer.

Future perspective
As AKT kinase plays a vital role in multiple cellular processes, it is a well-identified molecular target to design AKT
inhibitors as potential anticancer drug candidates. Our study demonstrates the acridone-2-carbohydrazide scaffold
as a lead molecule and opens possibilities for different structural modifications for designing newer anticancer
agents with fewer side effects. We also propose a future study of our compounds to be evaluated in the area of viral
infections and diseases such as malaria, tuberculosis and parasitic diseases, as acridones are reported to have a broad
spectrum of biological activities.

future science group 10.4155/fmc-2022-0271

Research Article Yadav, Kumar, Bajaj & Mayur

Summary points
• Synthesis and characterization and biological evaluation of acridone-2-carbohydrazide derivatives.
• Compounds 8k, 8v and 9h exhibited good cytotoxicity against MCF-7 cells (IC50 <10 μM).
• Compounds 8v and 9h exhibited promising AKT kinase inhibitory activity in MCF-7 cells (IC50 <2.5 μM).
• Quantitative p-AKT and total AKT revealed that compound 8v significantly reduced the levels of p-AKT Ser473
(rather than total AKT) in a dose-dependent manner.
• In a series of acridone-2-carbohydrazides, substituted phenylethylidene derivatives were found to be more
potent than substituted benzylidene derivatives.
• Molecular docking study of compounds showed good binding affinity to AKT kinase.
• Compounds 8k, 8v and 9h caused G0/G1-phase cell-cycle arrest and led to apoptosis.
• The study identifies compound 8v as the most potent agent to inhibit cancer cell growth via suppressing the
activation of AKT signaling through inhibition of p-AKT Ser473 .

Supplementary data
To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/
suppl/10.4155/fmc-2022-0271

Author contributions
T Yadav: study conduction, synthesis, data collection, analysis and interpretation of results, draft manuscript preparation. M Kumar:
methodology suggestions for biological studies, suggestions and supervision, writing (reviewing and editing, critical review of
manuscript). S Bajaj: molecular docking study and manuscript editing. Y Mayur: conceptual design, manuscript reviewing and
editing, approval of final version. All authors reviewed the results and approved the final version of the manuscript.

Acknowledgments
The authors are thankful to the Department of Health Research for providing fellowship. They would also like to acknowledge the
FACS Central Facility, Chemical Engineering Department, IIT Powai, Mumbai for cell cycle and cell apoptosis analysis of samples.

Financial & competing interests disclosure

The authors are grateful to the Department of Health Research (DHR), Government of India, New Delhi, Grant/Award no.
V.25011/547-HRD/2016-HR for providing funding for research. The authors have no other relevant affiliations or financial in-
volvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials
discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

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