Modulation of DNA damage response by targeting ATM kinase using newly synthesized di-phenoxy acetamide (DPA) analogs to induce anti-neoplasia

Article abstract of DOI:10.1007/s43440-021-00292-6

Background: Imbalance and instability in the structure of the DNA have become major characteristics of cancer. In response to DNA damage, DNA damage response (DDR) protein, ataxia telangiectasia mutated (ATM), plays a pivotal role in the modulation of regulatory regions responsible for inhibition of apoptosis, thereby neoplastic progression. Methods: A new series of DPA (7a–t) were synthesized, characterized. Anti-proliferative studies to identify the lead compound were carried out by LDH and MTT assay. Apoptosis/DNA damage was measured through FACS, Annexin-v staining, TUNEL and Comet assay. Elucidation of molecular mechanism through immunoblot and further validation of the drug effect through in vivo approaches. Results: Initial in vitro anti-proliferative screening of Compounds DPA (7a–t) against multiple cancer cell lines identified Compound DPA (7n) as a potent cytotoxic molecule with IC₅₀ value of 4.3?μM. Down the line, in vitro and in vivo evaluation of Compound DPA (7n) inferred that it has apoptotic inducing potentiality. Further, evaluation of molecular mechanism inferred that Compound DPA (7n) effectively modulates ATM phosphorylation only, eventually altering downstream signalling pathways. Conclusions: Compound DPA (7n) emerged as a potent proapoptotic and anti-neoplastic agent by inhibiting ATM kinase activity both in vitro and in vivo. The conferring results ascertain that the drug could be developed as a new ATM kinase inhibitor with anti-cancer capacity. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s43440-021-00292-6

Pharmacological Reports
https://doi.org/10.1007/s43440-021-00292-6
ARTICLE
Modulation of DNA damage response by targeting ATM kinase using
newly synthesized di‑phenoxy acetamide (DPA) analogs to induce
anti‑neoplasia
Fares Hezam Al‑Ostoot1,2 · Ankith Sherapura3 · Vikas H. Malojirao3,4 · Prabhu Thirusangu3,5
Tahani I. Al‑Muhimeed6 · Shaukath Ara Khanum1 · B. T. Prabhakar3
·
Received: 3 February 2021 / Revised: 21 May 2021 / Accepted: 31 May 2021
© Maj Institute of Pharmacology Polish Academy of Sciences 2021
Abstract
Background Imbalance and instability in the structure of the DNA have become major characteristics of cancer. In response
to DNA damage, DNA damage response (DDR) protein, ataxia telangiectasia mutated (ATM), plays a pivotal role in the
modulation of regulatory regions responsible for inhibition of apoptosis, thereby neoplastic progression.
Methods A new series of DPA (7a–t) were synthesized, characterized. Anti-proliferative studies to identify the lead com-
pound were carried out by LDH and MTT assay. Apoptosis/DNA damage was measured through FACS, Annexin-v staining,
TUNEL and Comet assay. Elucidation of molecular mechanism through immunoblot and further validation of the drug efect
through in vivo approaches.
Results Initial in vitro anti-proliferative screening of Compounds DPA (7a–t) against multiple cancer cell lines identifed
Compound DPA (7n) as a potent cytotoxic molecule with IC50 value of 4.3 μM. Down the line, in vitro and in vivo evalu-
ation of Compound DPA (7n) inferred that it has apoptotic inducing potentiality. Further, evaluation of molecular mecha-
nism inferred that Compound DPA (7n) efectively modulates ATM phosphorylation only, eventually altering downstream
signalling pathways.
Conclusions Compound DPA (7n) emerged as a potent proapoptotic and anti-neoplastic agent by inhibiting ATM kinase
activity both in vitro and in vivo. The conferring results ascertain that the drug could be developed as a new ATM kinase
inhibitor with anti-cancer capacity.
Graphic abstract
Shaukath Ara Khanum and B.T. Prabhakar contributed equally.
Extended author information available on the last page of the article
Vol.:(0123456789)
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F. H. Al-Ostoot et al.
Keywords Di-phenoxy acetamide analogs · ATM kinase · DDR · Apoptosis · Anti-neoplasia
as a selective target against neoplasia by promoting apopto-
sis through inhibiting ATM kinase activity. Overall, we can
anticipate that Compound DPA (7n) could be developed into
a target-specifc therapeutic drug.
Introduction
Cellular homeostasis is maintained through, proper control
of cell division, cell cycle arrest and induction of apoptosis
[1]. Invariably cells are challenged by thousands of lesions
imposed on their DNA every day leading to DNA damage
[2]. If such lesions are left unrepaired, they may prove fatal
to the cells and give rise to damaging mutations, leading to
the establishment of cancer. The cellular response to DNA
damage is one of the coordinated repairs and restraint of pro-
liferation [3]. Cells respond to DNA damage by activating
a complex network of signal transduction pathways. These
DDR pathways include sensors responsible for recognizing
the genotoxic insult, transducers responsible for relaying
the signal and efectors that induce the appropriate cellular
response. As a consequence, genomic stability is established
with coordinated DNA repair for cell cycle progression [4].
The phosphatidylinositol 3-kinase (PI3K)-related kinase
(PIKK) family member, ATM is a protein kinase, plays a
critical role in the maintenance of genome integrity and sta-
bility by detaining the cell cycle [5, 6]. The cellular func-
tions of these protein kinases range from regulation of the
DDR to cell survival, proliferation, metabolism, diferentia-
tion, motility, setting this as a central regulator of the DDR.
The ATM, also known as tumour suppressor gene, is fre-
quently mutated in a broad range of solid tumour making its
activation signal as a barrier to chemotherapeutic activity.
Many reports have correlated these kinase families to cancer
cell survival and subsequent therapy, rendering these kinases
potential targets for the treatment of cancer. However, the
development of selective inhibitors for these kinases is con-
sidered to be difcult due to the high degree of homology in
the kinase domain [7].
Materials and methods
Chemistry
General procedure for the synthesis of ester analogs (3a–g)
Compounds (3a–g) were synthesized as per the earlier
reported method from our group [18]. To a solution of
substituted phenols (1a–g, 0.08 mol) and chloro-acetic
acid ethyl ester (2, 0.080 mol) in acetone (25 ml), anhy-
drous potassium carbonate (0.075 mol) was added and then
refuxed for 9–11 h in a water bath. The mixture was cooled
to room temperature and the solvent was removed by distil-
lation. The residual mass was treated with ice-cold water to
remove potassium carbonate and then extracted with ether
(3×25 ml). The organic layer has been washed with a 10%
sodium hydroxide solution (3× 25 ml) followed by water
(3×30 ml) and dried over anhydrous sodium sulphate. Then
the solvent has been evaporated to get crude Compounds
(3a–g) and fnally Compounds (3a–g) were purifed by the
recrystallization method using methanol.
General procedure for the synthesis of phenoxy acetic acid
analogs (4a–g)
Compounds (4a–g) were synthesized as per the earlier
reported method from our group [19]. To a solution of phe-
noxy ester Compounds (3a–g, 0.05 mol) in ethanol (15 ml),
sodium hydroxide (0.04 mol) was added. The mixture was
then refuxed for 7–10 h using heating mental. After the
completion of the hydrolysis reaction [monitored by TLC
using the solvent system hexane:ethyl acetate:methanol
(6:3:1)], the mixture was cooled to room temperature and
then acidifed with 2 N hydrochloric acid. The precipitate
was fltered out, washed with distilled water and fnally
recrystallized from methanol to aford desired phenoxy ace-
tic acid analogs (4a–g).
With this rationale, we focus on the design, synthesis and
development of selected drug molecules that target a spe-
cifc gene for an efective response [8–10]. In this co-action,
acetamide moiety and its derivatives are the most studied
group of compounds in medicinal chemistry which have
shown signifcant biological activity with greater stability
[11, 12]. Both natural and synthetic analogs of acetamide
are also important therapeutic drugs with a broad range of
pharmacological activities like anti-infammatory [13, 14],
anti-oxidant [13], anti-ulcerative [15], anti-microbial [16]
and anti-angiogenic [17]. Considering their broad spectrum
of biological properties, we have synthesized and charac-
terized the title Compounds DPA (7a–t). Initial in vitro
screening, identifed Compound DPA (7n) as a lead mol-
ecule with IC50 value of ˜ 5 µM. The results are further vali-
dated through in vitro and in vivo experiments. Overall, the
study compiles the identifcation of Compound DPA (7n)
General procedure for the synthesis of phenoxyacetamide
analogs (6a–g)
Compounds (6a–g) were synthesized as per the earlier
reported method from our group [20].To a solution of Com-
pounds (4a–g, 0.009 mol), in 10 ml dry dichloromethane
1 3

Modulation of DNA damage response by targeting ATM kinase using newly synthesized di-phenoxy…
(DCM), lutidine (1.3 vol.) was added at room temperature
followed by 1, 2-diaminobenzene (5, 0.009 mol). Then,
the reaction mixture was stirred for 30 min, then cooled to
0–4 °C and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetrameth-
ylammonium tetrafluoroborate (TBTU, 0.02 mol) was
added for a period of 30 min maintaining the temperature
below 4 °C. The reaction mixture was stirred for 14–16 h
and monitored using TLC with the mobile phase system
[hexane:ethyl acetate (4:1)]. After the reaction, the reaction
mixture was diluted with 25 ml of DCM and then treated
with 2N hydrochloric acid solution (30 ml) to remove unre-
acted 1,2-diaminobenzene. The organic layer was washed
with distilled water (3×25 ml) and then with a brine solu-
tion (3 × 25 ml). Finally, the organic layer was dried over
anhydrous sodium sulphate and concentrated to aford crude
Compounds (6a–g) which were purifed by recrystallization
with ethanol.
Ar–H), 4.87 (s, 4H, OCH2), 3.31 (m, 1H, CH), 1.13 (d,
6H, CH3); 13C NMR (DMSO-d6) δ:167.85, 167.21, 156.60,
155.32, 150.42, 137.06, 130.57, 129.83, 127.08, 126.30,
126.12, 125.70, 121.74, 117.90, 114.25, 112.40, 68.05,
67.45, 26.38, 23.15, 20.14; LC–MS m/z 470 [M+], 472
[M+2]. Anal.Calcd.for C25H24ClFN2O4(470): C, 63.76; H,
5.14; N, 5.88. Found: C, 63.55; H, 4.96; N, 5.65%.
2‑(4‑Bromophenoxy)‑N‑(2‑(2‑(2‑chloro‑6‑fluorophenoxy)
acetamido)phenyl)acetamide (7c) Yield: 75%; M.P.
82–84 ºC; FT-IR (KBr, νmaxcm−1); 3122–3340 (NH), 1682
1
(amide, C=O); H NMR (400 MHz, DMSO-d6) δ (ppm):
9.68 (s, 1H, NH), 9.56 (s, 1H, NH), 6.78–7.72 (m, 11H,
Ar–H), 4.91 (s, 4H, OCH2); 13C NMR (DMSO-d6) δ: 167.84,
167.20, 156.64, 155.30, 150.44, 137.00, 130.55, 129.83,
127.07, 126.30, 126.14, 125.72, 121.73, 117.93, 114.25,
112.41, 68.05, 67.46; LC–MS m/z 508 [M+], 510 [M+2],
512 [M+4]. Anal.Calcd.for C22H17BrClFN2O4(508): C,
52.04; H, 3.37; N, 5.52. Found: C, 51.83; H, 3.19; N, 5.29%.
General procedure for the synthesis
of di‑phenoxyacetamide benzene analogs DPA (7a‑t)
2‑(4‑Chloro‑2‑methylphenoxy)‑N‑(2‑(2‑(2‑chloro‑6‑fluo‑
Compounds (4a–g, 0.002 mol) were added to Compounds
(6a–g, 0.002 mol) in dry DCM (10 ml) at 25–30 ºC followed
by the addition of lutidine (0.001 mol). The mixture was
stirred at 25–30 ºC for 30 min and then cooled to 0–5 ºC in
an ice bath and TBTU (0.003 mol) was added for a period
of 20 min while maintaining the temperature below 4 ºC.
The reaction was stirred for 14–16 h and monitored by TLC
using a hexane and ethyl acetate (3:1) system. When the
reaction is over, the mixture was diluted with 20 ml of DCM
and then washed with a solution of 10% sodium bicarbonate
(3 × 25 ml) followed by distilled water (3 × 25 ml). After
that, the organic layer was dried over anhydrous sodium
sulphate and concentrated to yield crude title Compounds
DPA (7a–t). Finally, these title Compounds DPA(7a–t) were
recrystallized with ethanol to achieve pure samples.
rophenoxy)acetamido)phenyl)acetamide
(7d) Yield:
82%; M.P. 91–93 ºC; FT-IR (KBr, νmaxcm−1); 3128–3342
(NH),1672 (amide, C=O);1H NMR (400 MHz, DMSO-d6)
δ (ppm): 9.74 (s, 1H, NH), 9.68 (s, 1H, NH), 6.88–7.66
(m, 10H, Ar–H), 4.95 (s, 4H, OCH2), 2.41 (s, 3H, CH3);
13C NMR (DMSO-d6) δ: 167.85, 166.91, 156.54, 155.31,
150.40, 137.05, 130.56, 129.80, 127.09, 126.30, 126.12,
125.73, 121.75, 117.92, 114.26, 112.42, 68.08, 67.44,
14.56; LC–MS m/z 476 [M+], 478 [M+2], 480 [M+4].
Anal.Calcd.for C23H19Cl2FN2O4(476): C, 57.88; H, 4.01; N,
5.87. Found: C, 57.67; H, 3.83; N, 5.64%.
2‑(4‑Bromophenoxy)‑N‑(2‑(2‑(2‑isopropylphenoxy)aceta‑
mido)phenyl)acetamide (7e) Yield: 79%; M.P. 102–104 ºC;
FT-IR (KBr, νmaxcm−1); 3138–3336 (NH), 1664 (amide,
1
C=O); H NMR (400 MHz, DMSO-d6) δ (ppm): 9.77 (s,
N,N’‑(1,2‑Phenylene)bis(2‑(2‑chloro‑6‑fluorophenoxy)
acetamide) (7a) Yield: 88%; M.P. 92–94 ºC; FT-IR (KBr,
νmaxcm−1); 3123–3345 (NH), 1670 (amide, C=O); 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 9.70 (s, 1H, NH),
9.55 (s, 1H, NH), 6.71–7.43 (m, 10H, Ar–H), 4.58 (s, 2H,
OCH2); 13C NMR (DMSO-d6) δ: 167.81, 152.53, 149.22,
132.81, 126.64, 124.91, 124.56, 124.34, 124.10, 114.73,
67.65; LC–MS m/z 480 [M+], 482 [M+2]. Anal.Calcd.for
C22H16Cl2F2N2O4(480): C, 54.90; H, 3.35; N, 5.82. Found:
C, 54.69; H, 3.17; N, 5.59%.
1H, NH), 9.67 (s, 1H, NH), 6.76–7.55 (m, 12H, Ar–H),4.86
(s, 4H, OCH2), 3.32 (m, 1H, CH), 1.14 (d, 6H, 2CH3);
13C NMR (DMSO-d6) δ: 167.84, 167.23, 156.63, 155.32,
137.06, 130.57, 129.83, 127.08, 126.31, 126.12, 125.73,
121.74, 117.91, 114.25, 112.40, 68.04, 67.45, 26.37, 23.16,
20.15; LC–MS m/z 496 [M+], 498 [M+2]. Anal.Calcd.for
C25H25BrN2O4(496): C, 60.37; H, 5.20; N, 5.63. Found: C,
60.16; H, 5.02; N, 5.40%.
2‑(4‑Chloro‑2‑methylphenoxy)‑N‑(2‑(2‑(2‑isopropylphe‑
noxy)acetamido)phenyl)acetamide (7f) Yield: 80%; M.P.
95–97 ºC; FT-IR (KBr, νmaxcm−1); 3132–3342 (NH), 1665
2‑(2‑Chloro‑6‑fluorophenoxy)‑N‑(2‑(2‑(2‑isopropylphe‑
noxy)acetamido)phenyl)acetamide (7b) Yield: 81%; M.P.
88–90 ºC; FT-IR (KBr, νmaxcm−1); 3130–3340 (NH), 1665
1
(amide, C=O); H NMR (400 MHz, DMSO-d6) δ (ppm):
9.68 (s, 1H, NH), 9.55 (s, 1H, NH), 6.79–7.50 (m, 11H,
Ar–H), 4.58(s, 4H, OCH2), 3.38 (m, 1H, CH), 2.21 (s, 3H,
CH3), 1.13 (d, 6H, CH3); 13C NMR (DMSO-d6) δ: 167.80,
1
(amide, C=O); H NMR (400 MHz, DMSO-d6) δ (ppm):
9.72 (s, 1H, NH), 9.68 (s, 1H, NH), 6.51–7.80 (m, 11H,
1 3

F. H. Al-Ostoot et al.
167.20, 156.63, 155.26, 137.02, 130.56, 129.91, 127.07,
126.37, 126.17, 125.79, 121.86, 117.92, 114.23, 112.35,
68.01, 67.48, 26.38, 23.12, 20.18; LC–MS m/z 466 [M+],
468 [M+2]. Anal.Calcd.for C26H27ClN2O4(466): C, 65.88;
H, 4.83; N, 6.00. Found: C, 65.67; H, 4.65; N, 5.77%.
2‑(2‑Isopropylphenoxy)‑N‑(2‑(2‑(2‑methoxyphenoxy)aceta‑
mido)phenyl)acetamide (7 k) Yield: 74%; M.P. 99–101 ºC;
FT-IR (KBr, νmaxcm−1); 3130–3340 (NH), 1664 (amide,
1
C=O), 1260 (O–C); H NMR (400 MHz, DMSO-d6) δ
(ppm): 9.63 (s, 1H, NH), 9.56 (s, 1H, NH), 6.73–7.36 (m,
12H, Ar–H), 4.56 (s, 4H, OCH2), 3.71 (s, 3H, OCH3), 3.34
(m, 1H, CH), 1.30 (d, 6H, CH3); 13C NMR (DMSO-d6) δ:
167.82, 167.18, 157.60, 150.21, 148.40, 135.00, 130.54,
129.92, 127.02, 126.34, 126.15, 125.79, 121.80, 117.93,
114.21, 112.30, 68.05, 67.47, 56.17, 26.36, 23.11, 20.15;
LC–MS m/z 449 [M+1]. Anal.Calcd.for C26H28N2O5(448):
C, 70.77; H, 6.29; N, 6.25. Found: C, 70.56; H, 6.11; N,
6.02%.
N,N’‑(1,2‑Phenylene)bis(2‑(4‑bromophenoxy)acetamide)
(7g) Yield: 64%; M.P. 110–112 ºC;FT-IR (KBr, νmaxcm−1);
3133–3344 (NH), 1672 (amide, C=O);1H NMR (400 MHz,
DMSO-d6) δ (ppm): 9.52 (s, 1H, NH), 6.73–7.66 (m, 6H,
Ar–H), 4.68 (s, 2H, OCH2); 13C NMR (DMSO-d6) δ:
167.42, 155.10, 136.74, 131.41, 127.36, 125.63, 121.45,
119.46, 115.55, 112.41, 68.83; LC–MS m/z 534 [M+], 536
[M+2], 538 [M+4]. Anal.Calcd.for C22H18Br2N2O3(534):
C, 49.95; H, 3.45; N, 5.24. Found: C, 49.74; H, 3.27; N,
5.01%.
2‑(4‑Bromophenoxy)‑N‑(2‑(2‑(2‑methoxyphenoxy)aceta‑
mido)phenyl)acetamide (7l) Yield: 84%; M.P. 106–108 ºC;
FT-IR (KBr, νmaxcm−1); 3145–3320 (NH), 1670 (amide,
1
N,N’‑(1,2‑Phenylene)bis(2‑(4‑chloro‑2‑methylphenoxy)
acetamide) (7h) Yield: 81%; M.P. 103–105 ºC; FT-IR (KBr,
νmaxcm−1); 3120–3335 (NH), 1677 (amide, C=O); 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 10.14 (s, 1H, NH), 6.96–
7.32 (m, 5H, Ar–H), 4.58 (s, 2H, OCH2), 2.25 (s, 3H, CH3);
13C NMR (DMSO-d6) δ: 167.00, 157.01, 156.91, 136.87,
129.88, 125.72, 125.45, 117.88, 117.01, 114.50, 66.72,
20.21; LC–MS m/z 472 [M+], 474 [M+2], 476 [M+4].
Anal.Calcd.for C24H22Cl2N2O4(472): C, 60.90; H, 4.68; N,
5.92. Found: C, 60.69; H, 4.50; N, 5.69%.
C=O), 1262 (O–C); H NMR (400 MHz, DMSO-d6) δ
(ppm): 9.71 (s, 1H, NH), 9.61 (s, 1H, NH), 6.80–7.53(m,
12H, Ar–H), 4.63 (s, 4H, OCH2), 3.74 (s, 3H, OCH3);
13C NMR (DMSO-d6) δ: 167.84, 167.10, 157.18, 149.81,
147.58, 132.55, 130.84, 130.23, 126.27, 126.02, 125.37,
122.83, 121.07, 117.47, 115.28, 113.26, 113.00, 68.94,
67.46, 56.06; LC–MS m/z 484 [M+], 486 [M+2]. Anal.
Calcd.for C23H21BrN2O5(484): C, 56.92; H, 4.36; N, 5.77.
Found: C, 56.71; H, 4.18; N, 5.54%.
2‑(4‑Chloro‑2‑methylphenoxy)‑N‑(2‑(2‑(2‑methoxyphe‑
noxy)acetamido)phenyl)acetamide (7m) Yield: 75%; M.P.
87–89 ºC; FT-IR (KBr, νmaxcm−1); 3140–3334 (NH), 1675
(amide, C=O), 1264 (O–C);1H NMR (400 MHz, DMSO-
d6) δ (ppm): 9.72 (s, 1H, NH), 9.60 (s, 1H, NH), 6.86–7.50
(m, 11H, Ar–H), 4.79 (s, 4H, OCH2), 3.72 (s, 3H, OCH3),
2.28 (s, 3H, CH3); 13C NMR (DMSO-d6) δ: 167.84, 167.10,
157.18, 150.01, 147.54, 131.55, 130.82, 130.21, 126.27,
126.12, 125.37, 122.81, 121.07, 117.45, 115.28, 113.25,
113.02, 68.94, 67.45, 56.06, 20.15; LC–MS m/z 454 [M+],
456[M+2]. Anal.Calcd.forC24H23ClN2O5(454): C, 63.37;
H, 5.10; N, 6.16. Found: C, 63.16; H, 4.92; N, 5.93%.
2‑(4‑Bromophenoxy)‑N‑(2‑(2‑(4‑chloro‑2‑methylphenoxy)
acetamido)phenyl)acetamide (7i) Yield: 71%; M.P.
92–94 ºC; FT-IR (KBr, νmaxcm−1); 1682 (amide, C=O),
3128–3325 (NH); 1H NMR (400 MHz, DMSO-d6) δ (ppm):
9.75 (s, 1H, NH), 9.68 (s, 1H, NH), 6.90–7.50 (m, 11H,
Ar–H), 4.64 (s, 4H, OCH2), 2.21 (s, 3H, CH3); 13C NMR
(DMSO-d6) δ: 167.82, 167.22, 156.60, 155.25, 137.00,
130.55, 129.92, 127.04, 126.34, 126.17, 125.79, 121.86,
117.92, 114.21, 112.35, 68.03, 67.47, 20.17; LC–MS
m/z 504 [M+], 506 [M+2], 508 [M+4]. Anal.Calcd.for
C23H20BrClN2O4(504): C, 54.84; H, 4.00; N, 5.56. Found:
C, 55.05; H, 4.18; N, 5.79%.
N,N’‑(1,2‑Phenylene)bis(2‑(2‑methoxyphenoxy)acetamide)
(7n) Yield: 83%; M.P. 106–108 ºC; FT-IR (KBr, νmaxcm−1);
2‑(2‑Chloro‑6‑fuorophenoxy)‑N‑(2‑(2‑(2‑methoxyphenoxy)
acetamido)phenyl)acetamide (7j) Yield: 77%; M.P. 89–91
ºC; FT-IR (KBr, νmaxcm−1); 1265 (O-C), 3135–3335 (NH),
1670 (amide, C=O);1H NMR (400 MHz, DMSO-d6) δ
(ppm): 9.73 (s, 1H, NH), 9.65 (s, 1H, NH), 6.88–7.54
(m, 11H, Ar–H), 4.63 (s, 4H, OCH2),3.81 (s, 3H, OCH3);
13C NMR (DMSO-d6) δ: 167.80, 167.20, 156.63, 155.23,
153.43, 151.00, 130.55, 129.92, 127.04, 126.34, 126.17,
125.79, 121.85, 117.90, 114.21, 112.33, 68.05, 67.47,
56.17; LC–MS m/z 458 [M+], 460 [M+2]. Anal.Calcd.for
C23H20ClFN2O5(458): C, 60.20; H, 4.39; N, 6.10. Found: C,
59.99; H, 4.21; N, 5.87%.
1
3150–3323 (NH), 1688 (amide, C=O), 1260 (O–C); H
NMR (400 MHz, DMSO-d6) δ (ppm): 9.54 (s, 1H, NH),
6.79–7.51 (m, 6H, Ar–H), 4.57 (s, 2H, OCH2), 3.74 (s, 3H,
OCH3); 13C NMR (DMSO-d6) δ: 169.67, 151.80, 149.85,
132.56, 128.00, 127.60, 124.73, 123.01, 117,35, 114.98,
70.88, 57.99; LC–MS m/z 437 [M+1]. Anal.Calcd.for
C24H24N2O6(436): C, 66.04; H, 5.54; N, 6.42. Found: C,
65.83; H, 5.36; N, 6.19%.
2‑(2‑Chloro‑6‑fluorophenoxy)‑N‑(2‑(2‑(4‑fluorophenoxy)
acetamido)phenyl)acetamide (7o) Yield: 74%; M.P.
1 3

Modulation of DNA damage response by targeting ATM kinase using newly synthesized di-phenoxy…
85–87 ºC; FT-IR (KBr, νmaxcm−1); 3140–3352 (NH), 1655
(s, 4H, OCH2), 3.74 (s, 3H, OCH3); 13C NMR (DMSO-d6)
1
(amide, C=O); H NMR (400 MHz, DMSO-d6) δ (ppm):
δ: 167.84, 167.10, 157.18, 155.81, 149.58, 136.55, 130.84,
130.21, 126.27, 126.02, 125.37, 122.83, 121.07, 117.45,
115.28, 113.26, 113.03, 68.95, 67.46, 56.04; LC–MS m/z
425 [M+1]. Anal.Calcd.for C23H21FN2O5(424): C, 65.09;
H, 4.89; N, 6.60. Found: C, 65.30; H, 5.07; N, 6.83%.
9.78 (s, 1H, NH), 9.68 (s, 1H, NH), 6.77–7.51 (m, 11H,
Ar–H), 4.58 (s, 4H, OCH2); 13C NMR (DMSO-d6) δ: 167.82,
167.14, 157.14, 156.81, 150.58, 149.85, 130.85, 130.21,
126.27, 126.06, 125.38, 122.81, 121.07, 117.45, 115.25,
113.26, 113.04, 68.94, 67.45; LC–MS m/z 446 [M+], 448
[M+2]. Anal.Calcd.for C22H17ClF2N2O4(446): C, 59.14; H,
3.83; N, 6.27. Found: C, 58.93; H, 3.65; N, 6.04%.
N,N’‑(1,2‑Phenylene)bis(2‑phenoxyacetamide) (7t) Yield:
82%; M.P. 113–115 ºC; FT-IR (KBr, νmaxcm−1); 3140–3348
(NH), 1675 (amide, C=O); 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 9.72 (s, 2H, NH), 6.90–7.51 (m, 14H, Ar–H), 4.61
(s, 4H, OCH2); 13C NMR (DMSO-d6) δ: 167.52, 157.85,
130.25, 129.94, 126.12, 125.71, 121.83, 115.15, 67.37; LC–
MS m/z 377 [M+1]. Anal.Calcd.for C22H20N2O4(376): C,
69.35; H, 5.28; N, 7.44. Found: C, 69.56; H, 5.46; N, 7.67%.
2‑(4‑Fluorophenoxy)‑N‑(2‑(2‑(2‑isopropylphenoxy)aceta‑
mido)phenyl)acetamide (7p) Yield: 72%; M.P. 111–113 ºC;
FT-IR (KBr, νmaxcm−1): 3140–3328 (NH), 1665 (amide,
1
C=O); H NMR (400 MHz, DMSO-d6) δ (ppm): 9.60 (s,
1H, NH), 9.58 (s, 1H, NH), 6.76–7.54 (m, 12H, Ar–H),
4.63 (s, 4H, OCH2), 3.36 (m, 1H, CH), 1.14 (d, 6H, CH3);
13C NMR (DMSO-d6) δ: 167.81, 167.11, 157.11, 155.82,
153.58, 136.85, 132.85, 130.21, 126.27, 126.05, 125.37,
122.83, 121.04, 117.42, 115.24, 113.26, 113.02, 68.98,
67.46, 26.35, 23.14, 20.12; LC–MS m/z 437 [M+1]. Anal.
Calcd.for C25H25FN2O4(436): C, 68.79; H, 5.77; N, 6.42.
Found: C, 68.58; H, 5.59; N, 6.19%.
Biology
Materials
Listed cell lines, MCF-7, A375, HepG2, ACHN, B16-F10,
HEK293 and NIH3T3 cells were procured from NCCS,
Pune, India. MTT, protease inhibitor cocktail from Sigma-
Aldrich, USA. Cell culture grade DMEM, antibiotic–anti-
mycotic, trypsin–EDTA and FBS from Invitrogen (Gibco),
2‑(4‑Bromophenoxy)‑N‑(2‑(2‑(4‑fuorophenoxy)acetamido)
phenyl)acetamide (7q) Yield: 75%; M.P. 103–105 ºC;
FT-IR (KBr, νmaxcm−1); 3123–3345 (NH), 1668 (amide,
USA. Antibodies ATM, p-ATM (s1981), ATR, p-ATR(t1989)
,
1
C=O); H NMR (400 MHz, DMSO-d6) δ (ppm): 9.65 (s,
p53, γ-H2AX, CHK2, p-CHK2 from Cell Signalling Tech-
nologies, USA. The 5-Ethynyl-2′-deoxyuridine (EdU),
KU-55933 from Sigma-Aldrich, USA. The Anti BAX, anti-
BAD, anti-β-Actin antibodies, Annexin-V FITC fuorescence
microscopy kit (Catalogue No 550911) from BD Biosci-
ence, USA Click-it TUNEL Alexa Fluor Imaging Assay kit
(Catalogue No C10245) was purchased from Thermo Scien-
tifc, USA and COMET assay reagents were purchased from
Sigma-Aldrich. All bright feld and fuorescence images
were captured with 100 µm at 40X magnification from
EVOS FL cell imaging, Thermo Scientifc, USA. Blotting
images were documented using Bio-Rad Gel Documentation
™ XR+Imaging system (Bio-Rad, USA) and densitometry
analysis was performed. All the photographs were taken
using a Canon power shot Sx500 IS camera.
1H, NH), 9.54(s, 1H, NH), 6.74–7.56 (m, 12H, Ar–H), 4.63
(s, 4H, OCH2); 13C NMR (DMSO-d6) δ: 167.80, 167.17,
157.10, 155.81, 154.53, 136.85, 132.87, 130.28, 126.27,
126.00, 125.34, 122.83, 121.03, 117.41, 115.24, 113.27,
113.02, 68.93, 67.45; LC–MS m/z 472 [M+], 474 [M+2].
Anal.Calcd.for C22H18BrFN2O4(472): C, 55.83; H, 3.83; N,
5.92. Found: C, 56.04; H, 4.01; N, 6.15%.
2‑(4‑Chloro‑2‑methylphenoxy)‑N‑(2‑(2‑(4‑fluorophenoxy)
acetamido)phenyl)acetamide (7r) Yield: 70%; M.P.
97–99 ºC; FT-IR (KBr, νmaxcm−1); 3145–3335 (NH), 1686
1
(amide, C=O); H NMR (400 MHz, DMSO-d6) δ (ppm):
9.67 (s, 1H, NH), 9.58 (s, 1H, NH), 6.71–7.67 (m, 11H,
Ar–H), 4.63 (s, 4H, OCH2), 2.22 (s, 3H, CH3); 13C NMR
(DMSO-d6) δ: 167.83, 167.15, 157.14, 155.84, 154.53,
136.88, 132.85, 130.24, 126.67, 126.10, 125.34, 122.73,
121.03, 117.41, 115.24, 114.27, 113.02, 68.93, 67.45,
20.14; LC–MS m/z 442 [M+], 444 [M+2]. Anal.Calcd.for
C23H20ClFN2O4(442):C, 62.38; H, 4.55; N, 6.33. Found: C,
62.59; H, 4.73; N, 6.56%.
Methods
Cell culture and in vitro anti‑proliferative studies The listed
cell lines were cultured and maintained in DMEM supple-
mented with 10% FBS, sodium bicarbonate, anti-mycotic
and antibiotics in 98% humidifed atmosphere supplemented
with 5% CO2 at 37 °C. The cells were treated with vary-
ing concentrations (0, 10, 20, 50 and 100 μM in DMSO)
of Compounds DPA (7a–t) for various time intervals up to
48 h by keeping the appropriate vehicle and positive control
(5-fuorouracil). Anti-proliferative studies to determine the
2‑(4‑Fluorophenoxy)‑N‑(2‑(2‑(2‑methoxyphenoxy)aceta‑
mido)phenyl)acetamide (7s) Yield: 83%; M.P. 105–107 ºC;
FT-IR (KBr, νmaxcm−1); 3160–3342 (NH), 1670 (amide,
1
C=O); H NMR (400 MHz, DMSO-d6) δ (ppm): 9.69 (s,
1H, NH), 9.57 (s, 1H, NH), 6.74–7.55(m, 12H, Ar–H), 4.68
1 3

F. H. Al-Ostoot et al.
IC50 value were carried out by two diferent methods, MTT
and LDH release assays for Compounds DPA (7a–t).
cells were cultured and treated with Compound (7n) (0,
3, and 5 µM) for 48 h. About 1×104 cells were smeared
on 1.2% agarose pre-coated glass slides and slides were
immersed in alkaline lysis solution (1.2 M NaCl, 100 mM
Na2EDTA, 0.1% sodium lauryl sarcosinate, 0.26 M NaOH
(pH>13)) for 1 h. Further cells were electrophorized at
30 V for 15 min in electrophoresis solution (0.03 M NaOH,
2 mM Na2EDTA (pH ∼12.3) and washed in 2×distilled
water. Cells were stained with DAPI (3 µg/ml) and COMET
were detected by a fuorescence microscope and analysed by
Casplab software. The tail moment was used to evaluate the
degree of DNA damage.
Clonogenic assay The confuence B16F10 cells were treated
with and without lead Compound DPA (7n) and clonogenic
assay was performed as reported earlier [21]. In brief, the
cells were exposed with Compound DPA (7n) for 6 h, rinsed
with fresh medium and cultured further for 12 days. Colo-
nies were fxed with methanol, stained with crystal violet
(0.4 g/L), counted and photographed.
In vitro drug treatment and IB studies To assess the
molecular mechanism, the confuence B16F10 cells were
prior exposed with or without EdU to induce DNA dam-
age, (20 µM) for 6 h and treated with and without Com-
pound DPA (7n) (0, and 5 µM), Compound DPA (7i), (0
and 60 µM), KU55933 (0 and 5 µM) for 0, 2, 4 and 6 h for
phosphorylation studies. For downstream diferential gene
expression studies, the cells were treated with Compound
DPA (7n) (0, 3 and 5 µM) for 48 h. Fromharvestedcells,
whole cell lysate was prepared by sonication using RIPA
bufer (100 mM Tris pH-7.5,1% Triton X-100,0.1% SDS,
140 mM NaCl, 0.5% sodium deoxycholate, 5 mM EDTA,
0.5 mM PMSF and protease inhibitor cocktail). The lysate
was quantifed and 20 µg of proteins were resolved in gra-
dient gel (4–16% of gels). Immunoblot (IB) was carried
out by transferring the gel into PVDF (polyvinylidene dif-
luoride) membrane for 1 h at 100 V. The membrane was
kept for blocking using blocking bufer (3% BSA in 10 mM
Tris Base, pH-7.5, 100 mM NaCl, 0.1% Tween-20) probed
with p-ATM (1:500), ATM (1:500), p-Chk2 (1:500),Chk2
(1:500), p-ATR (1:500), ATR (1:500), γh2ax (1:1000), BAX
(1:1000), BAD (1:1000), p53 (1:1000) dilution and β-actin
(1:5000 dilution) for overnight in an incubation bufer (3%
BSA in TBST). Further blots were probed with appropri-
ate secondary antibodies (1:5000 dilution) and developed
as reported earlier [22]. The blots were photographed and
documented. The band intensity was densitometrical meas-
ured using Bio-Rad Gel Documentation ™ XR+Imaging
system (Bio-Rad, USA) for expression of the protein.
Animal tumor models and treatment Healthy BALB/c
mice weighing (25–30 g) were used in the entire study
for in vivo experiments [25] and were maintained as per
Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA) with the permission of
Institutional Animal Ethics Committee (Ref No. KSHEMA/
IAEC/01/2020 Date: 04.06.2020). The anti-tumoral activity
of Compound DPA (7n) was tested by injecting the Dalton’s
lymphoma ascites (DLA) cells into the thigh region of the
mice to induce solid tumors. After the tumor establishment,
mice were divided into three experimental group (compris-
ing each group n=6) and treated with Compound DPA
(7n) (0, 20 and 40 mg/kg b.w) on every two bufer days for
six doses and tumor regression was examined with all the
growth inhibition parameters [26]. Dissected tumor tissues
were subjected to lysate preparation and analyzed through
IB analysis by probing with appropriate antibodies.
Statistical analysis The obtained data were checked for
normal distribution through Shapiro–Wilk test. All in vitro
experiments were repeated three times independently and
statistically analysed through one-way ANOVA followed
by Schefe’s post hoc test. All in vivo experiments involved
six animals per group and were repeated three times inde-
pendently. Data were analysed using one-way ANOVA
with repeated measures. The results are expressed as mean
standard deviation (SD). Statistical analysis was done
through SPSS version 16.
Apoptotic assays Confuent B16F10 cells were treated with
either Compound DPA (7n) (0, 3 and 5 µM) or Compound
DPA (7i) (5 µM), for 48hr to induce cytotoxicity. The per-
centage of proliferating/apoptotic cells and mode DNA
damage were measured through FACS analysis. The cells
were either stained with Hoechst 33,342 and results were
analysed using WinMDI version 2.9 software [23]. The
Annexin-FITC and TUNEL assay were performed as per kit
manufacturer guidelines [21]. To assess the specifcity of
elevated DNA damage, Compound DPA (7n)-induced DNA
damage was measured by alkaline comet assay according
to the previously published methods [24]. In brief, B16F10
Results and discussion
Chemistry
The synthetic method for the new DPA (7a–t) analogs is
illustrated in Scheme 1. The preparation of title Compounds
DPA (7a–t) began with commercially available substituted
phenols (1a–g) which easily refuxed with ethyl chloroac-
etate (2) in the presence of anhydrous potassium carbon-
ate and acetone as a suitable solvent. TLC was used to
1 3

Modulation of DNA damage response by targeting ATM kinase using newly synthesized di-phenoxy…
Scheme 1 Synthesis of DPA (7a–t) analogs:(i) ClCH2COOC2H5/
dry acetone, K2CO3, refux at 60 °C for 9–11 h, yield: 70–90%. (ii)
Aq.NaOH/ethanol, refux, for 7–10 h, yield: 69–91%. (iii) TBTU/luti-
dine, dry DCM, stirring at 0–4 °C for 30 min, continues for 14–16 h
at RT, and yield: 72–88%. (iv) TBTU/lutidine, dry DCM, stirring at
0–4 °C for 30 min, continue for 14–16 h at RT, and yield: 64–88%
monitor the reaction to achieve the corresponding phenoxy
esters (3a–g). Then Compounds (3a–g) were subjected to
hydrolysis with an ethanolic solution of sodium hydroxide
aforded the appropriate phenoxy acetic acid analogs (4a-
g) as shown in Scheme 1. Moreover, the coupled product
phenoxyacetamide derivatives (6a–g) were synthesized by
coupling Compounds (4a–g) with 1,2-diamino benzene (5)
at a low temperature (around 5 °C) using DCM as a suitable
solvent, 2,6 lutidine and TBTU as a coupling agent. Finally,
the title Compounds DPA (7a–t) were synthesized by the
treatment of Compounds (4a–g) with (6a–g) using the above
condition for the coupling reaction to afrm the expected
title Compounds DPA (7a–t) with the yield in the range of
69–91%. Furthermore, all the synthesized compounds have
been proved by FTIR, 1H NMR, 13C NMR and mass spectra
characterization.
1 3

F. H. Al-Ostoot et al.
Concerning the synthesized series (7a–t), Compound
DPA (7n) was taken as an illustrative example to explain
the characterizations that support the formation of the com-
pounds. The FTIR spectrum of Compound DPA (7n) proved
that NH stretching appeared between 3150 and 3223 cm−1
and both carboxylic and amino groups stretching bands of
Compounds (4e) and (6e) disappeared, respectively (Sup-
plementary Fig. 1). Interestingly, Compound DPA (7n) is a
symmetric compound and, therefore, in 1HNMR spectrum,
integration for six protons instead of twelve protons was dis-
played. Besides, it became clear that, in Compound DPA
(7n), the protons of the carboxyl group as well as the amino
group of Compounds (4e) and (6e), respectively, disap-
peared and a new proton for the amide group at δ 9.54 ppm
appeared (Supplementary Fig. 2). Compound DPA (7n) was
also supported by inspecting the 13CNMR and mass spec-
trum with a signifcant M+1 peak at m/z437 (Supplemen-
tary Figs. 3 and 4).
The detailed structure–activity relationship (SAR) of
Compounds DPA (7a–t) is depicted in Table 1a, b. The
result indicated that the IC50 value for Compound DPA (7n)
with two electron-donating methoxy groups at ortho posi-
tions in rings A and C exhibited a maximum inhibitory efect
of ̴ 4.3 and ̴ 5.1 µM on B16F10 and A375 cells, respec-
tively, than other cells as verifed by MTT and LDH release
assays (Fig. 1). The experimental evidence highlighted that,
among the series of Compounds DPA (7a–t), Compounds
DPA (7j–m and 7 s) with one methoxy electron-donating
group in diferent positions on ring A and C have shown
moderate to minimal activity compared to other series of
compounds. Furthermore, Compounds DPA (7a–i, 7o–r,
and 7t) without the methoxy group have not shown signif-
cant cytotoxicity compared to Compound DPA (7n). This
highlights the fundamental role of the methoxy group in
phenoxy acetamides’ anti-neoplastic action and Compound
DPA (7n) was identifed as a lead compound. The clono-
genic assay further indicates long term cytotoxic efect of
Compound DPA (7n) towards the B16F10 cells by inhibit-
ing the colony formation capability up to 80 to 90% in a
dose-dependent manner(Fig. 1C). Thus, it is evident that the
presence of two methoxy groups at ortho positions in rings
A and C in Compound DPA (7n) and it is vital for the anti-
neoplastic activity since no other substitutions in the remain-
ing compounds have exhibited signifcant cytotoxicity.
Biology
Compound DPA (7n) as a lead molecule and its SAR
The ATM protein plays an important role in cancer by
contributing several favourable factors in many stages of
tumor development by inhibiting apoptosis. Targeting the
ATM molecule can be an important therapeutic target for
the deactivation of the tumor progression [27, 28]. A new
series of DPA (7a–t) analogs has drawn up attention over the
past decades due to their wide range of pathological condi-
tions including cancer [29]. Extensive research based on the
development of novel cancer drugs indicates that acetamide
analogs combined with various specifc bioactive molecules
have shown potential anti-cancer efciency [17, 30–33].
With this notion, in the current investigation, acetamide was
integrated with phenoxy moiety bearing various electron-
donating (methoxy, methyl, and isopropyl) and withdrawing
(fuoro, chloro, and bromo) groups at diferent positions in
rings A and C and assessed for antiproliferation studies. To
authenticate cytotoxicity, two diferent experiments, MTT
and LDH (each n= 3) were performed and repeated three
independent times. Screening results inferred that Com-
pounds DPA (7j–n and 7 s) were shown moderate to mini-
mum cytotoxicity (Table 1), whereas Compound (7n) has
Compound DPA (7n) induces DNA damage response.
DNA damage response which leads to apoptosis is one of the
important physiological phenomena [34]. The drug-induced
cell death will activate the sequential hallmark of apoptosis
without damaging the cell through necrosis which can be
translated into an efective chemotherapeutic agent [35]. The
cytotoxic evaluation of Compound DPA (7n) displayed that
it specifcally activates cellular and biochemical hallmarks
of apoptosis like the loss of membrane integrity, membrane
fipping and DNA breakage (Fig. 2). Furthermore, Com-
pound DPA (7n) typically exhibited the cell cycle arrest in
G2/M phase in a dose-dependent manner and no peak eleva-
tion in sub-G1 phase was observed (Fig. 2A).
Being a typical hallmark of apoptosis, translocation of
phosphatidylserine, from the inner membrane to the outer
environment, can be measured by Annexin-V-FITC stain-
ing. Compound DPA (7n) proved an efcient ability to
induce the membrane fip-fopping feature in B16F10 cells
in a concentration-dependent manner. The intensity of the
fuorescence is the direct measurement of Compound DPA
(7n) ability to induce apoptosis which was observed under a
microscope (Fig. 2B). As supportive evidence, DNA damage
was further confrmed by the TUNEL assay. The specifc
breakage in the DNA of treated cells with Compound DPA
(7n) was detected by observing the fuorescence emission of
displayed maximum sensitivity at ̴ 5 µM against melanoma
cell lines of both murine and human origin. The results were
additionally represented in graphical illustration (Fig. 1A,
B). None of the compounds exhibited cytotoxicity against
normal cells counterpart HEK293 and fbroblast cell line
NIH3T3 (Table 1). The growth inhibition profle of varied
cell lines with an increase in cytotoxicity is B16F10>A37
5>ACHN>HepG2>MCF7 cells. The detailed results are
tabulated in Table 1.
1 3

Modulation of DNA damage response by targeting ATM kinase using newly synthesized di-phenoxy…
1 3

F. H. Al-Ostoot et al.
̴
31 and
̴
45% in 3 and 5 µM treatment, respectively, against
control cells which did not show any signifcant TUNEL
positive nuclei damage (Fig. 2C).To validate the more spe-
cifc action of Compound DPA (7n) on inhibition of DDR
response, COMET assay was performed. The technique
allowed for the quantifcation of DNA damage in an indi-
vidual cell treated with DNA-damaging agents. Exposure of
Compound DPA (7n) induces DNA damage which resulted
in increased COMET tail, whereas untreated showed undam-
aged high molecular weight intact COMET head which
indicates that Compound DPA (7n) induced DNA damage.
The result indicates the relaxation of supercoils and migra-
tion of the broken ends toward the anode, producing the
COMET tails. It was noteworthy that higher tail moment
values were found in Compound DPA (7n) Treated cells in
a dose-dependent manner, compared to the control group
(Fig. 2D). All the experiments were conducted with least
IC50 molecule Compound DPA (7i), to rule out the specifc
action of Compound DPA (7n). Results inferred that Com-
pound DPA (7i) does not induce any apoptotic hallmark and
thereby served as negative control for the experiments.
Compound DPA (7n) targets ATM kinase activity
The DDR protein, “ATM” is frequently mutated in many of
human solid tumors [36]. The ATM appears to be critical
for the induction of many cellular events, orchestrating the
response by the downstream signalling to other response
factors [37]. By understanding the important role of ATM
in tumor development, it’s been considered as a therapeutic
target in the treatment of cancer. The key focus of the current
investigation is to target ATM kinase in cancer conditions
and disrupt its downstream cellular events which can lead
to induction of apoptosis and there by establishing a thera-
peutic regime.
On the other hand, genotoxic agents are prior footstep in
the DNA damage induction and which are highly benef-
cial in the detection of the drug efcacy towards the geno-
toxic induced DNA damage. The EdU is one of the most
widely used genotoxic agents [38]. Once treated with EdU,
it induces DNA damage particularly triggering the phospho-
rylation of ATM on Ser1981. EdU specifcally damages the
double strand of DNA and as a result, ATM phosphorylation
gets triggered as a protective barrier for cell survival [38].
Our experimental results highlighted that, when Compound
DPA (7n) was treated with and without genotoxic agent
EdU, to induce elevated DDR response, it was efciently
able to inhibit the phosphorylation of ATM at ser 1981 in
both the cases (Fig. 3A, and 3B, respectively). Moreover,
Compound DPA (7n) efciently competes with the known
ATM inhibitor, KU55933 in inhibition of ATM phospho-
rylation [39]. Further experiments with negative control,
Compound DPA (7i) indicated the inefciency in inhibition
1 3

Modulation of DNA damage response by targeting ATM kinase using newly synthesized di-phenoxy…
Fig. 1 Cytotoxic assessment of lead Compounds DPA (7j–n and
s): Compounds DPA (7j–n and s) were treated to confuent Bl6F10
and A375 with various concentrations (0, 10, 20, 50 and 100 μM
in DMSO) for 48 h, followed by MTT and LDH release assays. A
Graphical representation of Compound DPA (7n) showing IC50 value
A375 cell lines. C Anti-clonogenic efect of Compound DPA (7n) on
B16F10 cells. The photographs were taken using a Canon power shot
Sx500 IS camera. All experiments were repeated three times indepen-
dently (n=3). Bars represent means
SD. Statistical analysis was
performed using one-way ANOVA followed by Schefe’s post hoc test
of ̴4.3 μM against B16F10 cell lines. B IC50 value of ̴ 5.1 μM against
of ATM kinase activity (Fig. 3C). Breakage in the double-
stranded DNA activates the cellular sensor response for
the repair and survival, the trinity of the DDR involves the
signalling mechanism interconnected with ATM, ATR and
DNA PKs was always a crucial focus in the DNA damage
1 3

F. H. Al-Ostoot et al.
Fig. 2 Compound DPA (7n) induces apoptotic hallmark in B16F10
cells: TheB16F10 cells were treated with Compound DPA (7n) at
various concentrations (0, 3 and 5 μM) and Compound DPA (7i) (0
and 60 μM) for 48 h and examined. A Histogram showing cell cycle
arrest at G2/M phase and altered variation in sub-G1 phase on Com-
pound DPA (7n) and DPA (7i) treatment. B Annexin-V immune
staining of bright feld and fuorescent images showing changes
in membrane integrity with quantifed cell death. C Changes in the
DNA double-strand breaks in treated and untreated cells stained with
TUNEL and counterstained with Hoechst with its quantifed cell
death. D COMET assay shows the DNA breakdown in Compound
DPA (7n) and Compound DPA (7i) treated cells by showing the elon-
gated tail movement with a graph of tail length of the individual cell.
All bright feld and fuorescence images were captured with100 µm at
40X magnifcation from EVOS FL cell imaging, Thermo Scientifc,
USA. Bars represent means SD (n=3). Statistical analysis was per-
formed using one-way ANOVA followed by Schefe’s post hoc test
repair. [40]. Further, experimental evidence suggested the
specifcity of Compound DPA (7n) against ATM kinase
only, but not against the other DDR response kinase ATR
(Fig. 3B).
rationale, the development of target-specifc therapeutics
with more relevant clinical outcomes, the anti-tumor poten-
tiality of Compound DPA (7n) has been tested against solid
tumor models. Dalton’s lymphoma cell lines were chosen
for the induction of solid tumor models to investigate the
primary anti-tumor efectiveness of Compound DPA (7n).
The in vitro results confer the inhibition of phosphoryla-
tion of ATM at S1981eventually refects in the downregula-
tion of phosphorylation of its principal substrate Chk2 in
DDR signalling of the cell cycle thereby induces the apopto-
sis through the cell cycle arrest [41]. This molecular modula-
tion is obviously responsible for the induction of apoptosis
(Fig. 3A). Inhibition of phosphorylation of ATM results in
induction of apoptosis by altering its downstream signalling
event [27]. As a consequence, proapoptotic genes such as
BAX, BAD, tumor suppressor gene p53 and DDR signal-
ling component γh2ax are diferentially altered (Fig. 3E) by
giving molecular evidence in support of apoptosis (Fig. 2).
Drastic reduction in the tumor volume to
recorded in a dose-dependent manner at 0, 20 and 40 mg/
kg b.w, respectively, as compared to control where 14 g
̴ 9 g and 4 g was
̴
of tumor volume was noted (Fig. 4A). As a consequence
of tumor regression, twofold increases in the survivability
of animals were observed (Fig. 4B). Any drug with target
specifc in action should exhibit the minimum side efects is
the key criteria in the drug development process. Evaluation
of the toxicological side efect of Compound DPA (7n) on
serum and haematological profling of tumor-bearing and
normal mice does not induce signifcant changes (Supple-
mentary Fig. 5). Upon verifying the mechanism of tumor
inhibition, it is on par with in vitro studies (Fig. 3A, B). The
results authenticated the in vitro IB results, in an in vivo
system which refected in the modulation of ATM signal-
ling (Fig. 4C). To our knowledge, the current investigation
work is the frst to evaluate a small molecule approach to
Compound DPA (7n) regresses in vivo tumor growth.
The initial development of anti-tumor drugs primarily
depends on the efcacy of the drug on animal tumor models.
The solid tumors models have been measured as the most
suitable in vivo model in pre-clinical testing [42]. With this
1 3

Modulation of DNA damage response by targeting ATM kinase using newly synthesized di-phenoxy…
Fig. 2 (continued)
target ATM. The in vitro and in vivo results reinforce the
view that the ATM protein kinase represents a tractable and
highly attractive target for the development of novel types
of anti-cancer agents.
Conclusion
The efcacy of drugs always depends on their target-specifc
action along with their small molecules in nature which ulti-
mately leads to the development of translational therapeutics
with mechanisms of action and function. As an approach,
Statistical results are tabulated in the Supplementary
Material, Table 1.
1 3

F. H. Al-Ostoot et al.
1 3

Modulation of DNA damage response by targeting ATM kinase using newly synthesized di-phenoxy…
◂Fig. 3 Compound DPA (7n) modulates the ATM signalling: the
series of DPA (7a–t) analogs was synthesized, character-
B16F10 cells were prior exposed with or without genotoxic agent
EdU (20 µM for 6 h) to induce DNA damage and further treated with
or without Compound DPA (7n) or (7i) or KU55933 for various time
intervals (0, 2, 4 and 6 h) for phosphorylation’s studies. The whole
cells lysate was subjected to IB analysis and probed with p-ATM,
ATM, p-ATR, ATR, p-Chk2, and Chk2. A Modulatory efect of
Compound DPA (7n) on phosphorylation of ATM and Chk2 genes
in presence of genotoxic agent EdU, along with densitometry quanti-
fcation. B Variations induced by Compound DPA (7n) on phospho-
rylation of ATM and ATR genes with densitometry graph. C Efect of
positive control KU55933 and negative control Compound DPA (7i)
on ATM phosphorylation. D Comparison of KU55933 vs Compound
DPA (7n) through densitometric analysis: B16F10 cells were treated
with or without Compound DPA (7n) (0, 3 and 5 µM) for 48 h and
protein lysates were subjected to IB analysis by probing with p-ATM,
ATM antibodies. E Altered gene expression profle ATM downstream
singling pathway genes such as BAD, p53, γH2Ax, BAX and β-actin.
The band images were documented using Bio-Rad Gel Documenta-
tion ™ XR+Imaging system (Bio-Rad, USA) and densitometry anal-
ized and screened for identifcation of anti-proliferative
activity. Compound DPA (7n) was identifed as a lead mol-
ecule exhibiting and proapoptotic activity. Elucidation of the
molecular mechanism revealed that Compound DPA (7n)
efectively modulated the ATM phosphorylation thereby
modulating its downstream signalling pathway. This could
be refected in pathological changes like a regression of the
tumor. The experimental data provided the characterization
of a novel Compound DPA (7n) which is not only acting as a
chemo-sensitizer. It also described the molecular tool which
can be used to study the many signalling facts of ATM in the
cellular responses to DNA damage. The data provided the
frst step in the development of more potent and pharmaceu-
tically acceptable inhibitors of ATM that maybe ultimately
helpful in clinical usage for cancer treatment.
ysis was performed. Bars represent means
SD (n=3). Statistical
analysis was performed using one-way ANOVA with repeated meas-
ures for (A–D) or a one-way ANOVA for (E) followed by Schefe’s
post hoc test
Fig. 4 Compound DPA (7n) inhibits in vivo DL Solid tumor (DLS):
DLA cells (5X106 cells/thigh sc) were injected into the thigh of to
induce solid tumor. After the onset of the tumor, mice were grouped
and administered with Compound DPA (7n) (0, 20 and 40 mg/kg
b.w) (ip) for 6 times on every two alternative days. At the end of the
35th day, tumor parameters were examined and cell lysate was sub-
jected to the gene expression analysis. A Graphical representation
of tumor volume inhibition, the morphology of mice bearing solid
tumor and reduction of solid tumor mass. The photographs were
taken using a Canon power shot Sx500 IS camera. B Prolonged sur-
vivability. C IB studies with p-ATM, ATM, p-Chk2, Chk2, p53, BAX
and β-actin antibodies representing modulation of gene expression.
The band images were documented using Bio-Rad Gel Documenta-
tion ™ XR+Imaging system (Bio-Rad, USA) and densitometry anal-
ysis was performed. Bars represent means
SD (n=3). Statistical
analysis was performed using one-way ANOVA with repeated meas-
ures followed by Schefe’s post hoc test
1 3

F. H. Al-Ostoot et al.
12. Al-Ostoot FH, Geetha DV, Mohammed YH, Akhileshwari P,
Sridhar MA, Khanum SA. Design-based synthesis, molecular
docking analysis of an anti-infammatory drug, and geometrical
optimization and interaction energy studies of an indole acetamide
derivative. J Mol Struct. 2020;1202:127244.
Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s43440-021-00292-6.
Acknowledgements Fares Hezam Al-Ostoot is thankful to the gov-
ernment of Yemen and Al-Baydha University, Yemen, for providing
fnancial assistance under the teacher’s fellowship and also thankful to
the University of Mysore, Mysore, India. Ankith Sherapura is thankful
to the Lady Tata Memorial Trust for providing the fnancial support
(LTMT-JRF 2020-2021) and B.T. Prabhakar gratefully acknowledges
the grant support by VGST(VGST/P-2/CISEE/GRD-231/2013-14) and
SERB [EMR/2017/00088/dated 3/01/2019]. Tahani I. Al-Muhimeed
is thankful to King Saud University, Riyadh, Saudi Arabia. Shaukath
Ara Khanum thankfully acknowledges the fnancial support provided
by VGST, Bangalore, under CISEE Program [Project sanction order:
No.VGST/CISEE/282/2012-13].
13. Alasmary FA, Awaad AS, Alafeefy AM, El-Meligy RM, Alqa-
soumi SI. Novel quinazoline and acetamide derivatives as safe
anti-ulcerogenic agent and anti-ulcerative colitis activity. Saudi
Pharm J. 2018;26:138–43.
14. Önkol T, Dogruer DS, Ito S, Sahin MF. Synthesis and antinocic-
eptive activity of (5-Chloro-2-benzothiazolinon-3-yl) acetamide
derivatives. Arch Pharm. 2000;333:337–40.
15. Pattanashetty SH, Hosamani KM, Shettar AK, Mohammed
SR. Design, synthesis and computational studies of novel car-
bazole N-phenylacetamide hybrids as potent antibacterial,
anti-infammatory, and antioxidant agents. J Heterocycl Chem.
2018;55:1765–74.
Author contributions FHA and AS performed all the experiments.
VHM, PT and TIA assisted in experimental design. SAK and PBT
designed the experiments, wrote the manuscript and monitored the
entire investigation.
16. Al-Ostoot FH, Mohammed YH, Zabiulla AN, Khanum SA. Syn-
thesis, in silico study and in vitro anti-microbial evaluation of
some new N-benzoyl-N’-[2-(4-chloro-phenoxy)-acetyl]-hydrazide
analogs. J Appl Pharm. 2019;9:042–9.
17. Zabiulla NHS, Begum AB, Prabhakar BT, Khanum SA. Design
and synthesis of diamide-coupled benzophenones as potential
anticancer agents. Eur J Med Chem. 2016;115:342–51.
18. Al-Ostoot FH, Sherapura A, Vigneshwaran V, Basappa G, Vivek
HK, Prabhakar BT, Khanum SA, Targeting HIF-1α by newly syn-
thesized Indolephenoxyacetamide (IPA) analogs to induce anti-
angiogenesis-mediated solid tumor suppression. Pharmacol Rep.
2021;1–6. https://doi.org/10.1007/s43440-021-00266-8.
19. Khamees HA, Mohammed YH, Ananda S, Al-Ostoot FH, San-
gappa Y, Alghamdi S, et al. Efect of o-difuoro and p-methyl sub-
stituents on the structure, optical properties and anti-infammatory
activity of phenoxy thiazole acetamide derivatives: Theoretical
and experimental studies. J Mol Struct. 2020;1199:127024.
20. Geetha DV, Al-Ostoot FH, Mohammed YH, Sridhar MA, Kha-
num SA, Lokanath NK. Synthesis, Elucidation, Hirshfeld sur-
face analysis, and DFT calculations of 4-chloro-N-[2-(2–1H-
indol-3-yl-acetylamino)-phenyl]-benzamide. J Mol Struct.
2019;1178:384–93.
Declarations
Conflict of interest The authors declare that they have no known com-
peting fnancial interests or personal relationships that could have ap-
peared to infuence the work reported in this paper.
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Authors and Afliations
Fares Hezam Al‑Ostoot1,2 · Ankith Sherapura3 · Vikas H. Malojirao3,4 · Prabhu Thirusangu3,5
Tahani I. Al‑Muhimeed6 · Shaukath Ara Khanum1 · B. T. Prabhakar3
·
* Shaukath Ara Khanum
Sahyadri Science College, Kuvempu University,
Shivamogga, Karnataka, India
shaukathah@ycm.uni-mysore.ac.in
4
5
6
* B. T. Prabhakar
Department of Neurosurgery, Houston Methodist, Fannin
Street, Houston, TX, USA
prabhakarbt1@kussc.org
Department of Experimental Pathology and Laboratory
Medicine, Mayo Clinic, Rochester, MN, USA
1
Department of Chemistry, Yuvaraja’s College, University
of Mysore, Mysuru, India
Department of Chemistry, College of Science, King Saud
University, Riyadh, Saudi Arabia
2
Department of Biochemistry, Faculty of Education
and Science, Al-Baydha University, Al Bayda, Yemen
3
Molecular Biomedicine Laboratory, Postgraduate
Department of Studies and Research in Biotechnology,
1 3