Targeting HIF-1α by newly synthesized Indolephenoxyacetamide (IPA) analogs to induce anti-angiogenesis-mediated solid tumor suppression

Article abstract of DOI:10.1007/s43440-021-00266-8

Background: Hypoxic microenvironment is a common feature of solid tumors, which leads to the promotion of cancer. The transcription factor, HIF-1α, expressed under hypoxic conditions stimulates tumor angiogenesis, favoring HIF-1α as a promising anticancer agent. On the other hand, synthetic Indolephenoxyacetamide derivatives are known for their pharmacological potentiality. With this background here, we have synthesized, characterized, and validated the new IPA (8a–n) analogs for anti-tumor activity. Methods: The new series of IPA (8a–n) were synthesized through a multi-step reaction sequence and characterized based on the different spectroscopic analysis FT-IR, ¹H, ¹³C NMR, mass spectra, and elemental analyses. Cell-based screening of IPA (8a–n) was assessed by MTT assay. Anti-angiogenic efficacy of IPA (8k) validated through CAM, Rat corneal, tube formation and migration assay. The underlying molecular mechanism is validated through zymogram and IB studies. The in vivo anti-tumor activity was measured in the DLA solid tumor model. Results: Screening for anti-proliferative studies inferred, IPA (8k) is a lead molecule with an IC₅₀ value of _?5?μM. Anti-angiogenic assays revealed the angiopreventive activity through inhibition of HIF-1α and modulation downstream regulatory genes, VEGF, MMPs, and P53. The results are confirmative in an in vivo solid tumor model. Conclusion: The IPA (8k) is a potent anti-proliferative molecule with anti-angiogenic activity and specifically targets HIF1α, thereby modulates its downstream regulatory genes both in vitro and in vivo. The study provides scope for new target-specific drug development against HIF-1α for the treatment of solid tumors. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s43440-021-00266-8

Pharmacological Reports
https://doi.org/10.1007/s43440-021-00266-8
ARTICLE
Targeting HIF‑1α by newly synthesized Indolephenoxyacetamide
(IPA) analogs to induce anti‑angiogenesis‑mediated solid tumor
suppression
Fares Hezam Al‑Ostoot1,2 · Ankith Sherapura3 · Vigneshwaran V3,4 · Giridhara Basappa3 · Vivek H.K.5 · Prabhakar B.
T3 · Shaukath Ara Khanum1
Received: 2 February 2021 / Revised: 8 April 2021 / Accepted: 10 April 2021
© Maj Institute of Pharmacology Polish Academy of Sciences 2021
Abstract
Background Hypoxic microenvironment is a common feature of solid tumors, which leads to the promotion of cancer.
The transcription factor, HIF-1α, expressed under hypoxic conditions stimulates tumor angiogenesis, favoring HIF-1α as a
promising anticancer agent. On the other hand, synthetic Indolephenoxyacetamide derivatives are known for their pharma-
cological potentiality. With this background here, we have synthesized, characterized, and validated the new IPA (8a–n)
analogs for anti-tumor activity.
Methods The new series of IPA (8a–n) were synthesized through a multi-step reaction sequence and characterized based
on the diferent spectroscopic analysis FT-IR, 1H, 13C NMR, mass spectra, and elemental analyses. Cell-based screening of
IPA (8a–n) was assessed by MTT assay. Anti-angiogenic efcacy of IPA (8k) validated through CAM, Rat corneal, tube
formation and migration assay. The underlying molecular mechanism is validated through zymogram and IB studies. The
in vivo anti-tumor activity was measured in the DLA solid tumor model.
Results Screening for anti-proliferative studies inferred, IPA (8k) is a lead molecule with an IC50 value of ˜5 μM. Anti-
angiogenic assays revealed the angiopreventive activity through inhibition of HIF-1α and modulation downstream regulatory
genes, VEGF, MMPs, and P53. The results are confrmative in an in vivo solid tumor model.
B.T. Prabhakar and Shaukath Ara Khanum contributed equally.
* Prabhakar B.T
prabhakarbt1@kussc.org
* Shaukath Ara Khanum
shaukathah@ycm.uni-mysore.ac.in
1
Present Address: Department of Chemistry, Yuvaraja’s
College, University of Mysore, Mysuru, India
2
Department of Biochemistry, Faculty of Education
and Science, Al-Baydha University, Mecca, Yemen
3
Molecular Biomedicine Laboratory, Postgraduate
Department of Studies and Research in Biotechnology,
Sahyadri Science College, Kuvempu University,
Shivamogga, Karnataka, India
4
Present Address: Department of Pharmacology, University
of Illinois at Chicago, Chicago, USA
5
Faculty of Natural Sciences, Adichunchanagiri
University-Center for Research and Innovation,
Adichunchanagiri University, BGSIT Campus, B.G. Nagara,
Mandya, Karnataka 571448, India
Vol.:(0123456789)
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F. H. Al-Ostoot et al.
Conclusion The IPA (8k) is a potent anti-proliferative molecule with anti-angiogenic activity and specifcally targets HIF1α,
thereby modulates its downstream regulatory genes both in vitro and in vivo. The study provides scope for new target-specifc
drug development against HIF-1α for the treatment of solid tumors.
Graphic abstract
Keywords Indolephenoxyacetamide · Angiogenesis · HIF-1α · Solid tumor
to a specific interest in HIF-1α targeted therapy [5].
Therefore, developing novel molecules that specifically
counteract tumor cell growth with target specificity and
spread potency is critical for the treatment of angiogenic-
dependent neoplasia.
Introduction
Angiogenesis is a complex and multistep process stimu-
lated by various biological signals regardless of its physi-
ological or pathological action. Many proangiogenic fac-
tors including VEGF are activated in response to signals
leading to damage in blood vessels, infraction, and reduc-
tion in the flow of blood that lead to a decrease in the
supply of oxygen [1]. The condition is termed as hypoxia,
which mediates angiogenesis, invasion and, metastasis
in most of the solid tumors and creates indulgence for all
the cancer cells with respect to adequate oxygenation [3].
The hypoxic microenvironment triggers the angiogenic
process by stimulating proangiogenic factors triggered
by the upstream signaling cascade, led by the overexpres-
sion of the transcription factor hypoxia-inducible factor
(HIF)-1α [2].
The heterocyclic structures are deemed to be signif-
cantly useful to medicinal chemists in that they ofer a real
break to the designing and establishment of one library
depending on a specifc central scafold and then assess it
against several target receptors [6]. In indole medications,
being rich with electrons in pyrole moiety along with other
qualities can utilize the non-covalent interactions with
many other molecules by creating hydrogen bonding in
the NH group using the π–π system and is considered a
privileged scafold in pharmaceutical research and drug
development [7], such as anti-oxidant [8], anti-microbial
[9], anti-HIV [10] and anti-cancer [11]. In particular, some
indoles have been found to constrain the growth of sev-
eral cancers along with boosting apoptosis and stopping
tumor invasion and metastasis [12]. With this rationale,
the investigation of new indole analogs carrying acetamide
moiety can improve and also display a pleasing variety
of biological efects against a diversity of health issues
[13]. As an approach, in the current investigation, new
series of substituted phenoxy acetic acids bonded at the
ortho position of diaminobenzene which then conjugated
to potent indole acids backbone have been synthesized
and evaluated for potential anti-cancer activity. Studies
Highly stabilized HIF-1α involved in the tolerance of
tumor cells to a hypoxic environment, translocates into
the nucleus and forms heterodimer, thereby regulating
oxygen concentration which is tightly associated in the
pathophysiology of solid tumors [4]. This results in the
activation of the tumor-promoting proangiogenic genes,
such as VEGF, MMPs and angiopoietins, etc. Overex-
pression of HIF-1α, significantly promotes aggressive
angiogenesis-mediated tumorigenesis, contributing
1 3

Targeting HIF‑1α by newly synthesized Indolephenoxyacetamide (IPA) analogs to induce…
of the structure–activity relationship (SAR) have shown
that the substituents of halogen at para position infuence
the activity of the indole derivatives with signifcant anti-
angiogenesis-mediated anti-cancer activity.
General procedure for the synthesis of phenoxyacetic acid
analogs (4a–h)
To the solution of compounds (3a–h, 0.05 mol) in etha-
nol (15 ml), alcoholic sodium hydroxide solution (3 ml,
0.04 mol) was added. The reaction mixture was allowed
to refux for 8–10 h, and the completion of the reaction
was monitored using TLC hexane: ethyl acetate: methanol
system (6:3:1). The mixture was cooled and acidifed with
2 N hydrochloric acid. The precipitate was fltered, washed
with distilled water, and fnally recrystallized from metha-
nol to aford desired compounds (4a–h) [15].
Materials and methods
Experimental section
The chemicals, solvents, and reagents were procured
from Sigma Aldrich Chemicals Pvt. Ltd. with 90–99%
purity. Using the instrument Chemi Line CL-725 Micro-
controller-based melting point and boiling point with a
digital thermometer, the melting points and boiling point
were determined. The progress and completion of reac-
tions were monitored using thin-layer chromatography
(TLC) performed on aluminium-backed silica plates and
the spots were detected using UV lamp at λ = 254 nm. The
IR spectra were recorded by the potassium bromide pellet
method on Cary 630 FTIR Agilent spectrophotometer. The
NMR (1H and 13C) spectra were recorded on a VNMRS-
400,100 MHz Agilent-NMR spectrophotometer in DMSO
and mass spectra were obtained on Mass Lynx SCN781
spectrometer TOF mode. Elemental analysis was obtained
using Thermo Finnigan Flash EA 1112 CHN analyser and
the fndings are within 0.4% of the calculated value.
General procedure for the synthesis
of N‑(2‑Amino‑phenyl)‑2‑phenoxy‑acetamide analogs
(6a–h)
The syntheses of compounds of N-(2-aminophenyl)-2-phe-
noxyacetamide analogs (6a–h), were accomplished using the
condensation procedure. To a solution of (4a–h, 0.009 mol),
in dry dichloromethane (DCM) (10 ml), lutidine (1.3 vol.) was
added, followed by the addition of o-phenylenediamine (5,
0.009 mol), the mixture was stirred at room temperature for
30 min. The reaction mixture was cooled to 0–5 °C, TBTU
(0.02 mol) was added, over a period for 30 min while main-
taining the temperature below 5 °C. The reaction was allowed
to stir overnight. After completion of the reaction which was
monitored by TLC using a mobile-phase system [hexane: ethyl
acetate (3:1)], the mixture was diluted with 20 ml of DCM
and treated with 2 N hydrochloric acid solution (20 ml). The
organic layer was washed with water (3×25 ml) and brine
(3×25 ml). Finally, the organic layer was dried over anhydrous
sodium sulfate and concentrated to aford compounds (6a–h).
Chemistry
General procedure for the synthesis of phenoxy ester
analogs (3a–h)
To the solution of substituted phenols (1a–h, 0.080 mol)
and ethyl chloroacetate (2, 0.080 mol) in dry acetone
(25 ml), anhydrous potassium carbonate (0.075 mol) is
added and the reaction mixture was refuxed for 10–12 h.
The mixture was then allowed to cool at room tempera-
ture and the solvent was removed by distillation. The
residual mass was triturated with cold water to remove
potassium carbonate, and the product was extracted with
ether (3 × 25 ml). The ether layer was washed with a 10%
sodium hydroxide solution (3 × 25 ml) followed by water
(3 × 25 ml) and dried over anhydrous sodium sulfate, then
the solvent was evaporated to aford compounds (3a–h).
Finally, compounds (3a–h) were purifed by the distilla-
tion method [14].
General procedure for the synthesis of indolephenoxyacet‑
amid analogs IPA (8a–n)
To the solution of compounds (6a–h, 0.002 mol), in dry
DCM (10 ml), the (1H-Indol-3-yl)-acetic acid, 4-(1H-indol-
3-yl) butanoic acid, and 1H-indole-2-carboxylic acid (7a-
c, 0.002 mol) were added at room temperature followed
by the addition of lutidine (0.002 mol). The reaction mix-
ture was stirred for 30 min and the mixture was cooled
to 0–5 °C, then, the TBTU (0.003 mol) was added over a
period for half an hour while maintaining the temperature
between 0 and 5 °C. The reaction was stirred overnight
and monitored by TLC using hexane and ethyl acetate (3:1)
system. The reaction mixture was diluted with (20 ml) of
DCM and treated with 10% of sodium bicarbonate solu-
tion (3×25 ml). The organic layer was washed with water
(3×25 ml), dried over anhydrous sodium sulfate, and con-
centrated to yield the title compounds IPA (8a–n). The
1 3

F. H. Al-Ostoot et al.
Scheme 1 Reaction pathway for the synthesis of the title compounds IPA (8a–n)
1 3

Targeting HIF‑1α by newly synthesized Indolephenoxyacetamide (IPA) analogs to induce…
schematic representation of the synthesized compounds
N‑[2‑(2‑1H‑Indol‑3‑yl‑acetylamino)‑phenyl]‑2‑phenoxy‑
acetamide (8d)
IPA (8a–n) is shown in Scheme 1.
Synthesis of N‑[2‑(2‑1H‑In‑
dol‑3‑yl‑acetylamino)‑phenyl]‑2‑phenoxyacetamide
analogs IPA (8a‑d)
Yield: 75%; M.P. 181–183 °C; FT-IR (KBr, νmaxcm−1);
1
1633 (amide, C=O), 3210–3320 (amide CO–NH). H
NMR (400 MHz, DMSO-d6) δ (ppm): 3.70 (s, 2H, CH2),
4.47 (s, 2H, OCH2), 6.89–7.58 (m, 14H, Ar–H), 9.57 (s,
1H, NH), 9.68 (s,1H, NH), 10.92 (s, 1H, NH indole); 13
C
N‑(2‑(2‑(1H‑indol‑3‑yl)acetamido)
phenyl)‑2‑(4‑bromophenoxy)acetamide (8a)
NMR (DMSO-d6) δ: 171.04, 167.07, 157.80, 136.65,
131.15, 130.59, 129.94, 127.62, 125.86, 125.76, 125.57
125.30, 124.64, 121.81, 121.49, 118.90, 115.18, 111.86,
108.37, 67.33, 33.57; LC–MS m/z 400 [M + 1]. Anal.
Calcd. for C24H21N3O3(399): C, 72.16; H, 5.30; N, 10.52.
Found: C, 72.10; H, 5.24; N, 10.47%.
Yield: 79%; M.P. 186–188 °C; FT-IR (KBr, νmaxcm−1);
1635 (amide, C=O), 3215–3325 (amide CO–NH). 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 3.70 (s, 2H, CH2), 4.45 (s, 2H,
OCH2), 6.85–7.60 (m, 13H, Ar–H), 9.55 (s, 1H, NH), 9.65
(s, 1H, NH), 10.88 (s, 1H, NH indole); 13C NMR (DMSO-
d6) δ: 171.07, 167.04, 157.82, 136.63, 131.16, 130.57,
129.95, 127.61, 125.85, 125.73, 125.55 125.32, 124.63,
121.84, 121.47, 118.91, 115.16, 111.87, 108.36, 67.35,
33.52; LC–MS m/z 477 [M+], 479 [M+2]. Anal. Calcd. for
C24H20BrN3O3(477): C, 60.26; H, 4.21; N, 8.78. Found: C,
60.20; H, 3.95; N, 8.74%.
Synthesis of 4‑(1H‑Indol‑3‑yl)‑N‑[2‑(2‑phe‑
noxy‑acetylamino)‑phenyl]butyramide analogs IPA
(8e‑g)
N‑{2‑[2‑(4‑Bromo‑phenoxy)‑acetylamino]‑phenyl}‑4‑(1H‑in
dol‑3‑yl)‑butyramide (8e)
Yield: 76%; M.P. 176–178 °C; FT-IR (KBr, νmaxcm−1),
2‑(2‑Chloro‑6‑fuoro‑phenoxy)‑N‑[2‑(2‑1H‑indol‑3‑yl‑acety‑
lamino)‑phenyl]acetamide (8b)
1
1635 (amide, C=O), 3210–3325 (amide CO–NH). H
NMR (400 MHz, DMSO-d6) δ (ppm):1.82 (m, 2H, CH2),
2.33 (t, 2H, CO-CH2), 2.51 (t, 2H, CH2), 4.64 (s, 2H,
OCH2), 6.78–7.65 (m, 13H, Ar–H), 9.66 (s, 1H, NH), 9.75
(s,1H, NH), 10.88 (s, 1H, NH indole); 13C NMR (DMSO-
d6) δ: 171.12, 168.03, 157.84, 136.60, 131.13, 130.51,
129.97, 127.61, 125.83, 125.71, 125.58 125.32, 124.62,
121.84, 121.45, 118.90, 114.14, 111.88, 108.36, 67.35,
33.56, 25.28, 25.11; LC–MS m/z 505 [M +], 507 [M + 2].
Anal. Calcd. for C26H24BrN3O3(505): C, 61.67; H, 4.78;
N, 8.30. Found: C, 61.61; H, 4.72; N, 8.30%.
Yield: 81%; M.P; 187–190 °C; FT-IR (KBr, νmaxcm−1);
1630 (amide, C=O), 3220–3320 (amide CO–NH). 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 3.66 (s, 2H, CH2), 4.47 (s, 2H,
OCH2), 6.80–7.85 (m, 12H, Ar–H), 9.53 (s, 1H, NH), 9.66
(s, 1H, NH), 10.76 (s, 1H, NH indole);13C NMR (DMSO-
d6) δ: 172.05, 167.03, 157.81, 136.65, 131.15, 130.57,
129.93, 127.65, 125.83, 125.70, 125.54 125.33, 124.63,
121.84, 121.46, 118.91, 114.16, 110.87, 108.36, 67.35,
33.50; LC–MS m/z 451 [M +], 453[M + 2]. Anal. Calcd.
forC24H19ClFN3O3(451): C, 63.79; H, 4.24; N, 9.30. Found:
C, 63.66; H, 4.18; N, 9.25%.
N‑{2‑[2‑(4‑Chloro‑2‑methyl‑phenoxy)‑acetylamino]‑phenyl}
‑4‑(1H‑indol‑3‑yl)‑butyramide (8f)
2‑(4‑Chloro‑phenoxy)‑N‑[2‑(2‑1H‑indol‑3‑yl‑acetylamino)
‑phenyl]acetamide (8c)
Yield: 78%; M.P. 186–188 °C; FT-IR (KBr, νmaxcm−1);
1
1635 (amide, C=O), 3215–3322 (amide CO–NH). H
Yield: 78%; M.P. 174–177 °C; FT-IR (KBr, νmaxcm−1);
1640 (amide, C=O), 3212–3322 (amide CO–NH). 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 3.72 (s, 2H, CH2), 4.45 (s, 2H,
OCH2), 6.81–7.83 (m, 13H, Ar–H), 9.41 (s, 1H, NH), 9.68
(s, 1H, NH), 10.72 (s, 1H, NH indole); 13C NMR (DMSO-
d6) δ: 171.03, 167.05, 157.84, 136.63, 131.12, 130.55,
129.95, 127.65, 125.84, 125.71, 125.56 125.32, 124.60,
121.86, 121.49, 118.91, 115.12, 110.87, 107.36, 67.35,
33.52; LC–MS m/z 433 [M+], 435[M+2]. Anal. Calcd.
forC24H20ClN3O3(433): C, 66.44; H, 4.65; N, 9.68. Found: C,
66.40; H, 4.60; N, 9.61%.
NMR (400 MHz, DMSO-d6) δ (ppm): 1.91 (m, 2H, CH2),
2.40 (t, 2H, CO-CH2), 2.53 (t, 2H, CH2), 4.71 (s, 2H,
OCH2), 6.72–7.69 (m, 12H, Ar–H), 9.68 (s, 1H, NH), 9.79
(s,1H, NH), 10.85 (s, 1H, NH indole); 171.05, 167.03,
157.82, 136.62, 131.15, 130.55, 129.94, 127.60, 125.83,
125.71, 125.53 125.30, 124.63, 121.84, 121.47, 118.91,
115.12, 111.83, 108.35, 67.35, 33.51, 25.25, 25.09, 16.34;
LC–MS m/z 475 [M +], 477 [M + 2]. Anal. Calcd. for
C27H26ClN3O3(475): C, 68.13; H, 5.51; N, 8.83. Found:
C, 68.02; H, 5.46; N, 8.80%.
1 3

F. H. Al-Ostoot et al.
N‑{2‑[2‑(4‑Chloro‑phenoxy)‑acetylamino]‑phenyl}‑4‑(1H‑in
1H‑Indole‑2‑carboxylic acid {2‑[2‑(2‑methoxy‑phenoxy)‑ace
dol‑3‑yl)‑butyramide (8 g)
tylamino]‑phenyl}‑amide (8j)
Yield: 81%; M.P. 184–186 °C; FT-IR (KBr, νmaxcm−1);
Yield: 85%; M.P. 195–197 °C; FT-IR (KBr, νmaxcm−1);
1632 (amide, C=O), 3215–3325 (amide CO–NH). 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 3.88 (s, 3H, OCH3), 4.62 (s,
2H, OCH2), 6.85–7.81 (m, 13H, Ar–H), 9.68 (s, 1H, NH),
10.22 (s,1H, NH), 11.79 (s, 1H, -NH indole); 13C NMR
(DMSO-d6) δ: 166.94, 160.61, 156.42, 150.35, 131.48,
131.15, 130.122, 129.68, 129.45, 127.48, 126.42, 126.35,
125.84, 125.53, 124.47, 122.25, 120.45, 116.88, 116.65,
113.91, 112.72, 67.77, 56.21; LC–MS m/z 416 [M + 1].
Anal. Calcd. for C24H21N3O4 (415): C, 69.39; H, 5.10; N,
9.54. Found: C, 69.31; H, 5.02; N, 9.50%.
1
1645 (amide, C=O), 3210–3330 (amide CO–NH). H
NMR (400 MHz, DMSO-d6) δ (ppm):1.65 (m, 2H, CH2),
2.39 (t, 2H, CO-CH2), 2.49 (t, 2H, CH2), 4.71 (s, 2H,
OCH2), 6.63–7.69 (m, 13H, Ar–H), 9.11 (s, 1H, NH), 9.55
(s,1H, NH), 10.91 (s, 1H, NH indole); 13C NMR (DMSO-
d6) δ: 171.07, 167.00, 157.83, 136.61, 131.19, 130.53,
129.95, 127.64, 125.84, 125.71, 125.56 125.32, 124.60,
121.86, 121.50, 118.91, 115.20, 110.77, 107.35, 67.35,
33.51, 25.34, 25.19; LC–MS m/z 461 [M +], 463 [M + 2].
Anal. Calcd. for C26H24ClN3O3(461): C, 67.60; H, 5.24;
N, 9.10. Found: C, 67.51; H, 5.18; N, 9.04%.
1H‑Indole‑2‑carboxylic acid {2‑[2‑(4‑chloro‑phenoxy)‑acety
lamino]‑phenyl}‑amide (8k)
Synthesis of 1H‑Indole‑2‑carboxylic acid [2‑(2‑phe‑
noxy‑acetylamino)‑phenyl] amide analogs IPA
(8 h‑n)
Yield: 89%; M.P. 206–208 °C; FT-IR (KBr, νmaxcm−1);
1
1635 (amide, C=O), 3212–3320 (amide CO–NH). H
NMR (400 MHz, DMSO-d6) δ (ppm): 4.72 (s, 2H, OCH2),
6.88–7.78 (m, 13H, Ar–H), 9.64 (s, 1H, NH), 10.16 (s, 1H,
NH), 11.82 (s, 1H, NH indole);13C NMR (DMSO-d6) δ:
166.95, 160.64, 156.45, 137.42, 131.52, 131.12, 130.11,
129.66, 129.46, 127.50, 126.42, 126.36, 125.82, 125.54,
124.42, 122.26, 120.47, 116.88, 116.62, 112.92, 104.71,
67.77; LC–MS m/z 419 [M +], 422 [M + 2]. Anal. Calcd.
for C24H21N3O4 (419): C, 69.40; H, 5.09; N, 10.02. Found:
C, 69.33; H, 5.04; N, 10.01%.
1H‑Indole‑2‑carboxylic acid
{2‑[2‑(4‑bromo‑phenoxy)‑acetylamino]‑phenyl}‑amide (8 h)
Yield: 88%; M.P. 176–178 °C; FT-IR (KBr, νmaxcm−1);
1
1630 (amide, C=O), 3215–3320 (amide CO–NH). H
NMR (400 MHz, DMSO-d6) δ (ppm): 4.73 (s, 2H, OCH2),
6.88–7.78 (m, 13H, Ar–H), 9.64 (s, 1H, NH), 10.11 (s,1H,
NH), 11.80 (s, 1H, NH indole); 13C NMR (DMSO-d6) δ:
166.94, 160.61, 156.42, 137.35, 131.48, 131.15, 130.122,
129.68, 129.45, 127.48, 126.42, 126.35, 125.84, 125.53,
124.47, 122.25, 120.45, 116.88, 116.65, 112.91, 104.72,
67.77;LC–MS m/z 463 [M +], 466 [M + 2]. Anal. Calcd.
for C23H18BrN3O3 (463): C, 59.50; H, 3.91; N, 9.05.
Found: C, 59.44; H, 3.87; N, 9.01%.
1H‑Indole‑2‑carboxylic acid [2‑(2‑phe‑
noxy‑acetylamino)‑phenyl]‑amide (8 l)
Yield: 74%; M.P. 177–179 °C; FT-IR (KBr, νmaxcm−1);
1
1635 (amide, C=O), 3215–3325 (amide CO–NH). H
NMR (400 MHz, DMSO-d6) δ (ppm): 4.74 (s, 2H, OCH2),
6.87–7.75 (m, 14H, Ar–H), 9.66 (s, 1H, NH), 10.21(s,1H,
NH), 11.80 (s, 1H, NH indole);13C NMR (DMSO-
d6) δ: 166.99, 161.05, 156.42, 137.42, 131.50, 131.11,
130.10, 129.67, 129.48, 127.43, 126.40, 126.37, 125.83,
125.53, 124.41, 122.24, 120.48, 117.87, 116.53, 112.90,
111.71, 67.66; LC–MS m/z 386 [M + 1]. Anal. Calcd. for
C23H19N3O3 (385): C, 71.67; H, 4.97; N, 10.90. Found: C,
71.61; H, 4.94; N, 10.88%.
1H‑Indole‑2‑carboxylic acid {2‑[2‑(4‑chloro‑2‑me‑
thyl‑phenoxy)‑acetylamino]‑phenyl}‑amide (8i)
Yield: 74%; M.P. 171–173 °C; FT-IR (KBr, νmaxcm−1)
1
1635 (amide, C=O), 3215–3325 (amide CO–NH). H
NMR (400 MHz, DMSO-d6) δ (ppm): 2.54 (s, 3H, CH3),
4.67 (s, 2H, OCH2), 6.83–7.77 (m, 12H, Ar–H), 9.63 (s,
1H, NH), 10.22 (s,1H, NH), 11.84 (s, 1H, NH indole);
13C NMR (DMSO-d6) δ: 167.57, 161.63, 156.41, 137.42,
131.55, 131.13, 130.11, 129.65, 129.45, 127.52, 126.46,
126.34, 125.85, 125.51, 124.44, 122.25, 120.48, 116.87,
116.63, 106.90, 103.75, 67.66, 15.11; LC–MS m/z 433
[M +], 435 [M + 2]. Anal. Calcd. forC24H20ClN3O3 (433):
C, 66.44; H, 4.65; N, 9.68. Found: C, 60.44; H, 4.59; N,
9.63%.
1H‑Indole‑2‑carboxylic acid {2‑[2‑(2‑bromo‑phenoxy)‑acety
lamino]‑phenyl}‑amide (8 m)
Yield: 77%; M.P. 180–182 °C; FT-IR (KBr, νmaxcm−1);
1
1630 (amide, C=O), 3210–3330 (amide CO–NH). H
NMR (400 MHz, DMSO-d6) δ (ppm): 4.73 (s, 2H, OCH2),
6.86–7.74 (m, 13H, Ar–H), 9.64 (s, 1H, NH), 10.16 (s,1H,
NH), 11.80 (s, 1H, NH indole); 13C NMR (DMSO-d6) δ:
1 3

Targeting HIF‑1α by newly synthesized Indolephenoxyacetamide (IPA) analogs to induce…
167.06, 162.66, 155.41, 137.43, 131.54, 131.13, 130.10,
129.67, 129.48, 127.50, 126.41, 126.36, 125.83, 125.54,
124.41, 122.25, 120.47, 116.87, 116.63, 112.95, 104.73,
66.89; LC–MS m/z 463 [M +], 465 [M + 2]. Anal. Calcd.
forC23H18BrN3O3 (463): C, 59.50; H, 3.91; N, 9.05. Found:
C, 59.47; H, 3.88; N, 9.01%.
Cell culture and in vitro anti‑proliferative studies
Listed cell lines A549, MCF-7, A375, LLC and BEAS-2B
were grown in DMEM with 10% FBS, enriched in antibi-
otic–anti-mycotic solution (100 μg/ml), sodium carbonate
(0.37%) and incubated at 37 °C in 5% CO2, with ≥ 98%
humidity. The anti-proliferative efciency of IPA (8a–n) (0,
2, 5, 10, 25, and 50 μM in DMSO) was evaluated through
MTT release assay. The average IC50 values were calculated
as described earlier [16]. The DMSO and 5-fuorouracil were
used as a vehicle and positive control, respectively.
1H‑Indole‑2‑carboxylic acid {2‑[2‑(2‑isopropyl‑phenoxy)‑ac
etylamino]‑phenyl}‑amide (8n)
Yield: 79%; M.P. 174–176 °C; FT-IR (KBr, νmaxcm−1);
1630 (amide, C=O), 3215–3322 (amide CO–NH). 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 1.20 (d, 6H, CH3), 3.10 (m,
H, CH), 4.75 (s, 2H, OCH2), 6.85–7.75 (m, 13H, Ar–H), 9.65
(s, 1H, NH), 10.15 (s, 1H, NH), 11.83 (s, 1H, NH indole);
13C NMR (DMSO-d6) δ: 166.96, 160.65, 156.44, 137.40,
131.51, 131.10, 130.10, 129.67, 129.48, 127.49, 126.43,
126.37, 125.83, 125.55, 124.44, 122.25, 120.48, 116.87,
116.63, 112.90, 111.71, 67.76, 27.50, 23.80; LC–MS m/z
428 [M+1]. Anal. Calcd. for C26H25N3O3 (427): C, 73.05;
H, 5.89; N, 9.83. Found: C, 73.01; H, 5.84; N, 9.80%.
The BrdU cell proliferation assay was performed as per
manufacturer protocol. In brief, cultured confuence cells
were treated with or without IPA (8k) (0, 3, and 5 µM)
for 48 h, followed by 10 µM BrdU incorporation. Cells
were washed, fxed, permeabilized, and treated with anti-
BrdU primary antibody. Cells were counterstained with
4′,6-diamidino-2-phenylindole (DAPI) and visualized under
the microscope, documented.
Chorioallantoic membrane (CAM) assay
The in vivo anti-angiogenic efect of IPA (8k) was per-
formed as reported earlier [17]. In brief, 6 days of pre-
incubated eggs (each groups containing n= 6 eggs) were
surface-sterilized, made window, and induced with recom-
binant VEGF-10 ng/CAM (rVEGF16). The growing CAM
was treated with IPA (8k) (0, 3, 5 μg/CAM). After 48 h,
variation in the blood vessel formation was visually docu-
mented and quantifed.
Pharmacology
Materials and methods
Cancer cell lines of human origin, Adenocarcinoma (A549),
Breast Carcinoma (MCF-7), Melanoma (A375), mouse can-
cer cell line, such as Lewis Lung Carcinoma (LLC) and Nor-
mal human lung epithelial cells BEAS-2B, HUVEC (human
umbilical vein endothelial cell), were obtained from NCCS,
Pune, India. Cell culture reagents, DMEM, EGM, antibi-
otic–anti-mycotic solution, Trypsin–EDTA solution, fetal
bovine serum (FBS), poly HEMA (poly hydroxyl meth-
acrylate) from invitrogen (Gibco). Cell cultures were from
Eppendorf, Germany. The MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide), and BrdU (Bromo-
deoxyuridine) cell proliferation assay kit were from Sigma
Aldrich. Antibodies against HIF-1α (hypoxia-inducible
factor-1 alpha), VEGF (vascular endothelial growth factor),
MMP-2 (Matrix Metallo Proteinase-2), MMP-9 (Matrix
Metallo Proteinase-9), were procured from Santa Cruz Bio-
technology Inc, USA. The immunostaining kit was from
Leica Biosystems, Germany. Eggs for CAM were procured
from Indian Veterinary Research Institute (IVRI) Bengaluru,
Karnataka, India. All other chemicals including solvents
used were purchased from Hi-Media, Mumbai, India. All
bright feld and fuorescence images were documented in
EVOS FL cell imaging, Thermo Scientifc, USA. Quantif-
cation of angiogenesis was carried out by ImageJ software
(NIH, Bethesda, MD, USA).
Rat corneal micro‑pocket assay
To evaluate the angioinhibitory efect of IPA (8k), the rat
corneal micro-pocket assay was performed as reported ear-
lier [18]. In short, the sprouting angiogenesis was induced
in rat cornea by applying rVEGF165 (10 ng/pellet) and
treated with IPA (8k) (0, 3, 5 μg/pellet) using formulated
poly HEMA. Corneal angiogenesis and re-modulation were
measured in histopathological studies (H and E). The MVD
(Micro Vessel Density) number was quantifed and graphi-
cally represented.
Hypoxic conditions, in vitro treatment and lysate
preparations
To examine the IPA (8k) efcacy on the anti-proliferative
mechanism, A549 cells were treated at diferent concentra-
tions (0, 3 and 5 µM) under normoxic, pseudohypoxic and
hypoxic environments. The pseudohypoxic microenviron-
ment was created by treating the cells with CoCl2 (100 μM)
for 48 h before IPA (8k) treatment. For hypoxia, cells were
grown in a hypoxia chamber with a gas supply containing
1 3

F. H. Al-Ostoot et al.
1% O2, 5% CO2 and 94% N2, and exposed with IPA (8k).
The cells were harvested and whole cell lysate was used
for further Immunoblot (IB) studies. Cell-free conditioned
medium was used for gelatin zymogram and ELISA studies
[18, 19].
Serum collected was used for measurement of VEGF, alka-
line phosphatase (ALP), creatinine, urea, RBC and WBC
from each group. Survivability of the mice bearing DLS
tumor-treated mice (n=10 each) was monitored separately.
Immunohistochemistry (IHC)
HUVEC cell migration and tube formation assay
The histopathological assessments of MVD and IHC analy-
sis were performed for HIF-1α using dissected DLS tumor
tissue [23]. The IHCs were independently evaluated by two
pathologists and documented.
The confuence monolayer HUVECs grown in a 96-well
plate and 1 mm size wounds were made by scratching with
a microtip and exposed with rVEGF165 (30 ng/ml) and IPA
(8k) (0, 3, 5 µM) for 48 h. The migrated cells locations
were photographed at identical locations and analyzed by
comparing fnal gap width to initial gap width. ECM gel
was polymerized for 30 min at 37 °C for coating 96-well
plate. HUVECs suspended in EGM medium was added to
each well coated with ECM gel and treated with or without
rVEGF165 and IPA (8k) (0, 3, 5 µM) for 24 h. Inhibition in
the formation tube and its lengths was documented [18].
VEGF‑ELISA
The in vitro and in vivo secretions of VEGF levels were
quantifed using anti-VEGF antibody through ELISA [24].
In brief, 100 μl of conditioned media (in vitro) and serum
(in vivo) was coated in coating bufer at 4 °C overnight and
incubated with anti-VEGF-A antibody, followed by re-incu-
bation with ALP conjugated secondary antibody. Quantifca-
tion of secreted VEGF-A was measured at 405 nm.
Gelatin zymography
Gelatin zymography is one of the most suitable assays to
quantify the secreted MMP-2 and MMP-9 concentration
[20]. In brief, conditioned media from the cultured A549
cells were treated with or without IPA (8k) (0, 3, and 5 μM)
for 48 h and resolved in 8% SDS-PAGE gel containing 0.1%
(w/v) dissolved gelatin and the zymogram was developed.
The decrease of gelatin lysis zone was documented using
Bio-rad Gel Documentation™ XR+Imaging System.
Immunoblots
Immunoblot (IB) analysis was performed for HIF-1α, p53,
MMP-2, MMP-9, and β-actin proteins as mentioned earlier
[25]. The protein lysate prepared from IPA (8k)-treated and
-untreated conditions was resolved on SDS-PAGE and trans-
ferred to nitrocellulose membrane. The blots were probed
with appropriate antibodies and images were densitometri-
cally quantifed.
Animal models and ethics
Statistical analysis
Healthy BALB/c mice weighing (27–30 g) were used in
in vivo studies [21]. All the experiments performed on ani-
mals were maintained as per CPCSEA guidelines with the
permission of the Institutional Animal Ethics Committee
(Ref No. KSHEMA/IAEC/01/2018).
All in vitro experiments were repeated three times indepen-
dently and statistically analysed through one-way ANOVA
followed by Brown–Forsythe test. The results are expressed
as mean standard deviation (SD). All in vivo experiments
involved six variables per group and were repeated three
times independently. Data were analysed using one-way
ANOVA followed by Brown–Forsythe test. The results are
expressed as mean standard deviation (SD). Statistical sig-
nifcance was set at *p < 0.05 to ****p < 0.0001. All data
analyses were performed using Graph pad prism version 8.0.
Animal tumor models and treatment
Dalton’s lymphoma (DL) solid tumor was developed by
adopting the earlier reported procedure [22]. In brief, DLA
(5×106 cells) was injected into the thigh region of the mice
subcutaneously (sc) to develop a solid tumor (DLS). After
the onset of tumor development to 100 mm3 in size, mice
were administered with IPA (8k) (0, 30 and 50 mg/kg) bw
(n = 6 each) for six doses (ip) for every alternate day. At
the end of the 35th day from tumor implantation, the tumor
tissues were dissected and photographed. The tissue lysate
from treated and untreated group mice were prepared for
IB and gelatin zymography analysis. Tumour tissues were
further processed immunohistochemistry (IHC) analysis.
Results and discussion
Chemistry
Scheme 1 showed the synthetic methods for the new IPA
(8a–n) analogs. The preparations of the title compounds IPA
(8a–n) were started with commercially available substituted
1 3

Targeting HIF‑1α by newly synthesized Indolephenoxyacetamide (IPA) analogs to induce…
Fig. 1 IPA (8k) shows induces
cytotoxicity against the A549
cells: the various cancer cell
lines were treated with varied
concentrations of IPA (8a–n)
for 48 h and cell proliferation
rate was measured by MTT
assay. a IPA (8k) showing the
IC50 value of ˜ 5 µM against the
A549 cell with potent cytotoxic-
ity activity more or less similar
to 5-Fu. b, c Antiproliferative
efcacy of IPA (8k) in BrdU
proliferation assay with prolif-
eration inhibition graph. Graphs
were plotted by taking mean
(n=3) SD. Statistical analysis
was performed using one-way
ANOVA followed by Brown-
Forsythe test and statistical
signifcance was set at *p<0.05
to ****p<0.0001
phenols (1a–h) which easily refuxed with ethyl chloroac-
etate (2) in the presence of anhydrous potassium carbonate
and acetone as a suitable solvent. The reaction mixture was
monitored using TLC until the completion of the reaction to
achieve corresponding phenoxy esters (3a–h). In the second
step, hydrolysis of the compounds (3a–g) was carried out
1 3

F. H. Al-Ostoot et al.
Table 1 MTT IC50 values of compounds IPA (8a–n)
Cell
lines
IPA
IPA
IPA
IPA
IPA
IPA
IPA IPA IPA IPA IPA IPA IPA IPA
5-FU
3.9
(8K) (8C) (8G) (8I)
(8H) (8A) (8E) (8F) (8B) (8M) (8J) (8N) (8L) (8D)
5.0 46.21 48.83 52.6
51.25 32.35 45.6 53.21 62.35 64.9 62.5 48.5 54.5 71.23 0.59
A549 0.59
1.83 1.85
1.85 2.5 1.2 1.75 1.5 1.25 0.8 2.3 1.8 1.6 1.97
4.2
MCF
14.2
42.23 55.29 56.2
59.52 45.56 44.6 55.23 55.56 66.7 64.2 55.8 62.1 72.25
2.2 2.8 2.25 2.2 2.2 0.9 2.1 2.5 1.9 1.93 0.26
53.15 46.16 46.7 58.56 66.16 64.6 68.8 64.6 68.6 73.25 3.8
-7
0.26
2.8
42.52 56.59 54.9
1.25 2.25 1.85
38.25 45.56 52.23 49
2.39
1.67
16.2
A375 0.98
7.5
2.5
1.1 1.04 1.8
51.29 53.59 59.6
1.63 2.37 1.78
49.2
1.8 0.9
61.29 65.62 65.21 65.95 71.2 74.5 4.2
0.58 1.98 1.59 1.69 1.68 1.5 0.51
83.7 73.2 92.5 79.8 75.78 5.0 1
1.8
1.9
1.5
2.87 0.98
LLC
0.59
1.95
56.29
1.1
2.29 0.9
1.85
58.33
2.3
49.3
61.2
BEA
61.41
52.31
1.6
58.59 92.12
1.38
1.86
2.98
1.5
1.3
2.5
1.2
2.85 .38
S-2B
2.02
1.2
2.65
using an ethanolic solution of sodium hydroxide to aford the
appropriate phenoxy acetic acid analogs (4a–h). Moreover,
the coupled product phenoxy acetamide derivatives (6a–h)
were synthesized by condensation of compounds (4a–h)
with O-phenylenediamine (5), at a low temperature around
5 °C using DCM as a suitable solvent and 2,6 lutidine and
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafu
oroborate (TBTU) used as a coupling agent. Finally, the title
compounds IPA (8a–n) were synthesized by the treatment of
the compounds (4a–h) with (6a–h) using the above condi-
tion to afrm the expected title product IPA (8a–n) with the
yield in the range of 74–89%.
target. Based on the investigation results, one of the major
drawbacks in establishing such cancer therapy is failing to
reach the translational side either due to their non-speci-
ficity or adverse side effects [26]. In this view, designing
a molecule that specifically counteracts these problems is
the foremost essential requirement. With this notion, we
have synthesized IPA (8a–n) analogs which are known
to be pharmacologically active molecules against various
pathological conditions such as solid tumors [27]. The
development of an active drug always relies on screening
against various cancer cell lines with a different origin for
better evaluation [28]. Therefore, newly synthesized IPA
(8a–n) analogs were initially screened for their in vitro
cytotoxic and anti-proliferative activity against cell lines,
such as A549, MCF-7, A375, LLC and normal BEAS-2B.
Among IPA (8a–n) analogs, IPA (8k) showed promis-
ing anti-proliferative potency with an IC50 value of 5 μM
specifically against A549 cells as verified by MTT assay
(Fig. 1a) without affecting normal cell counterpart. Our
preliminary investigation infers that the IPA (8k) with
electron-withdrawing chloro group at para position of the
Furthermore, all the synthesized compounds have been
1
proved by FT-IR, H NMR, 13C NMR and mass spectra
characterization. For instance, among the synthesized series
IPA (8a–n), the compound (8d) was taken as an example.
The FT-IR spectrum of compound IPA (8d) proved by the
appearance of two CO–NH stretching bands in the range
3210–3320 cm−1 and disappearance of both carboxylic and
amino groups stretching bands of compounds (7a) and (6d),
respectively (Supplementary S1). Interestingly, the 1H NMR
spectrum of compound IPA (8d) showed the disappearance
of the protons of the carboxyl group as well as the amino
group of the compounds (7a) and (6d), respectively, and
also the appearance of new protons for two NH groups at δ
9.57 ppm and 9.68 ppm (Supplementary S2). The compound
IPA (8d) was also supported by inspecting the 13C NMR
and mass spectrum with a signifcant M+1 peak at m/z 400
(Supplementary S3 and 4).
◂
Fig. 2 IPA (8k) attenuates the neovessel formation in non-tumoral
angiogenic models: The growing CAM and rat cornea was stimu-
lated for angiogenesis with rVEGF165 and treated with or without IPA
(8k) for 48 h. a The in vivo CAM results symbolize the inhibition
of neovessel formation by declining the MVD density with graph. b
Modulation in the sprouting of neovessel formation in rat cornea with
decreased MVD with respective graph. c H&E staining shows the
efect of the compound IPA (8k) in the normalization of the cornea
with decrease in the MVD. Antimigratory and tube formation efect
of endothelial cells were measure in HUVEC’s cells treated with or
without IPA (8k) for 48 h. d Regression of capillary tube formation
ability of the HUVECs. e Inhibition in the migration of the endothe-
lial cells to heal the wound with quantifed representation. Graphs
were plotted by taking mean (n=6 for Fig. 2a, b and n=3 for Fig. 2d,
e) SD. Statistical analysis was performed using one-way ANOVA
followed by Brown-Forsythe test and statistical signifcance was set at
*p<0.05 to ****p<0.0001
Biology
IPA (8k) as a lead compound and SAR
The current investigation mainly focused on identifying
and developing a new active anti-prolifeartive/cytotoxic
molecule inhibitor for tumor angiogenesis with specific
1 3

Targeting HIF‑1α by newly synthesized Indolephenoxyacetamide (IPA) analogs to induce…
phenoxy ring has potent cytotoxicity against the A549
cells. The results are on par with the standard drug 5-fluo-
rouracil (Table1). Analyzing the activity data revealed
that other IPA (8c, 8g, 8i, 8h, 8a, 8e and 8f) analogs with
electron-withdrawing halo groups at para position showed
moderate anti-proliferative potency in decreasing order.
1 3

F. H. Al-Ostoot et al.
While the other compounds with different substituents
have not shown significant activity (Table 1). The anti-
proliferative effect of IPA (8k) was further validated and
confirmed by BrdU cell proliferation assay with a sig-
nificant reduction in the rate of proliferation (Fig. 1b, c).
participating in the tumor progression. This cross-interaction
of HIF-1α and proangiogenic factors is the base for the cap-
illary formation under hypoxic conditions. Initiation of the
angiogenesis under hypoxic conditions primarily activated
by HIF-1α and mainly dependent on VEGF followed by the
MMPs [31]. All these create the hyper-recognition towards
HIF-1α and its downstream pathway as an attractive target in
cancer research. Even though several small molecules with
diferent chemotypes and intense biological potency were
reported to be promising HIF-1α inhibitors, but none of the
available drugs were clinically approved [3]. In this connec-
tion, eforts in this direction are necessary to establish, tar-
geted therapy towards the HIF-1α inhibition. In the current
investigation, IPA (8k) efcacy in inhibition of the HIF-1α
was evaluated. The hypoxic microenvironment is created by
treating the cells with CoCl2 before IPA (8k) treatment in a
concentration-dependent manner. The results imply that IPA
(8k) efectively inhibits the HIF-1α expression which cre-
ates an obstacle to the tumor cells to overcome by hypoxic
condition (Fig. 3a). The HIF-1α-dependent expression of
the MMP-2 and MMP-9 in the treated cell was reduced
signifcantly as verifed in both IB and zymogram analysis
(Fig. 3b). Down the line, anti-tumoral gene p53 was elevated
in concentration-dependent manner (Fig. 3a). One of the
major angiogenic initiating factors VEGF-A expression level
has direct refection with the aggressive tumor angiogen-
esis [31]. The treatment of IPA (8k) drastically reduces the
VEGF-A secretion and thereby attenuates the angiogenesis
promotion (Fig. 3c).
IPA (8k) induces angioinhibiton
The process of angiogenesis in neoplasia involves several
basic steps, such as hypoxia, ischemia, blood vessel damage
and elevated expression of proangiogenic growth factors and
resulting in the formation of the new blood vessels [29]. The
VEGF being one such predominant growth factor plays an
important role in the stimulation of angiogenesis and used in
reliable non-tumor angiogenesis assay models, such as CAM
and rat corneal micro-pocket assay, which are well authenti-
cated for its target-specifc action towards neovascularization
[25, 30]. The mechanism underlying the anti-proliferative
efcacy of IPA (8k) was evaluated in a non-tumorigenic
in vivo CAM model to detect the inhibition of angiogenesis.
The results interpreted that, IPA (8k) drastically reduced
the rVEGF165-induced MVD in a concentration-dependent
manner up to 40 and 60%, respectively, when compared with
rVEGF165-induced control CAM (Fig. 2a). The anti-angio-
genic efcacy of IPA (8k) was additionally evaluated by the
corneal neovascularization inhibition potency by an enthus-
ing cornea with rVEGF165. Results inferred that sprouting
concentration of the neoangiogenesis through the cornea was
blocked up to 50 and 70% when compared to the control
corneal neovascularization concentration-dependent man-
ner. Also, IPA (8k) was able to improve the remodulation
of corneal neoangiogenesis gesture (Fig. 2b, c).
IPA (8k) suppresses solid tumor
Further, HUVECs capillary tube formation assay results
postulated that IPA (8k) evidently stops the human func-
tional endothelial cell migration adjacent linkage and capil-
lary tube formation efciency by exhibiting the irregular
cellular morphology up to 65 and 80% in their respective
treatment manner. Tube length and branch number were
also drastically reduced by IPA (8k) (Fig. 2d). Supportive
to this, the efect of IPA (8k) on the invasive and migration
property of A549 cells was evaluated. The relative migration
capability of A549 cells to heal the wound distance was sig-
nifcantly shorter in IPA (8k) treated cells with 25.65% and
17.32% in 3 and 5 μM concentrations, respectively (Fig. 2E).
Tumor hypoxia is an important hurdle to the effective
treatment of cancer. Hypoxia exists in most solid tumors
due to insufficient oxygen supply of the abnormal vas-
culature formation which results in the high demands of
oxygen by rapidly proliferating cancer cells. The capa-
bility of the drug efficacy was always confirmed by the
reflecting results as shown in an in vitro condition, in the
development of the novel potent pharmacophores. DLA
Solid (DLS) tumor model is the most widely used vital
model for the investigation of drug efficacy, within the
natural tumor hypoxic condition as a hypoxia inhibitor.
Based on the evidential results from in vitro data, the
anti-tumoral effect of the IPA (8k) was reconfirmed in
an in vivo DLS tumor model system. The drug dosage
concentrations (0, 30 and 50 mg/kg b.w) for the treat-
ment of the compound IPA (8k) for an in vivo study were
determined by LD50 assay, by simultaneously assessing
the possible impact on hematological and hepatological
side effects. The results referred that IPA (8k) does not
induce any significant toxicological effect in the murine
model system (Supplementary S20).
IPA (8k) inhibits HIF‑1α and its downstream signaling
The HIF-1α is key transcription factor expressed under a
solid tumor microenvironment. The associations between
HIF-1α with angiogenesis for tumor growth promotion
have been well established in many solid tumor studies. The
HIF-1α takes part in every step of angiogenesis by inter-
acting with several cellular signaling molecules, thereby
1 3

Targeting HIF‑1α by newly synthesized Indolephenoxyacetamide (IPA) analogs to induce…
Fig. 3 IPA (8k) targets HIF-1α
gene expression and modu-
lates its downstream signal-
ing. Whole cell lysate and
conditioned media from the
IPA (8k) treated A549 (0, 3,
5 µM) cells were subjected to
IB and gelatin zymography
analysis. a Suppression of
HIF-1α and its downstream
MMP-2 and MMP-9 gene
expression with elevation in
the p53 gene expression and
their respective densitometry
graph. b Decreased gelati-
nase activity of MMP-2 and
MMP-9. c Decrease in the
production of the VEGF-A in
the conditioned media. Graphs
were plotted by taking mean
(n=3) SD. Statistical analysis
was performed using one-way
ANOVA followed by Brown-
Forsythe test and statistical
signifcance was set at *p<0.05
to ****p<0.0001
The treatment of IPA (8k) on the DLS tumor model
attenuates the tumor progression in a dose-dependent man-
ner which is refected in the enhanced survival period with
noticeable and improved physiology (Fig. 4 A, B, C and
D). The inhibitory efect of solid tumor progression is due
to the inhibition of MVD as verifed by H and E staining.
The MVD count was signifcantly down in IPA (8k) treated
cells in a concentration-dependent manner (Fig. 4e, f). Since
MVD strongly relies on HIF-1α-dependent VEGF levels,
we verifed the expression of HIF-1α in both IHC and IB
analysis (Fig. 4g–i). Results reconfrmed the in vitro data.
Eventually, modulation in the expression levels of its rel-
evant genes MMP-2, MMP-9, P53 and VEGF as verifed by
zymogram, IB and ELISA (Fig. 4j, k). Overall, the IPA (8k)
could be developed into an inhibitor of HIF-1α with minimal
cytotoxicity and provides the mechanism-based evidence for
the possible drug development for cancer therapy.
Conclusion
One of the most terrifying health issues is cancer, which
has driven chemists and other concerned parties to strug-
gle for the development of efficient medicines with new
strategies. Clinical development of anti-angiogenic mol-
ecules for cancer therapy was a success and translated
into approved treatment in many cancers. Tumor hypoxia
acts as a prevalent and major barrier to effective can-
cer treatment. A diversity of different methods has been
employed to reverse tumor hypoxic environment and to
date, there are no Food and Drug Administration (FDA)
approved treatments to reverse tumor hypoxia. As a con-
tinued effort, in the present investigation, IPA (8k) with
chloro group at the para position can induce cell-specific
cytotoxic effects and can effectively inhibit neovascu-
larization in both in vitro and in vivo by inhibiting the
HIF-1α, a master protein that plays a critical role in the
tumor cell survival. Overall, our results postulate IPA
(8k) has the potential to inhibit the solid tumor progres-
sion by blocking one of the major hallmarks of cancer
by specifically targeting HIF-1α and provides scope for
anti-angiogenic-based therapies.
Statistical results
Each experiment was statistically analyzed and results are
expressed in terms of f, df, and p values and tabulated (Sup-
plementary Table 1).
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F. H. Al-Ostoot et al.
Fig. 4 IPA (8k) inhibits in vivo DL Solid tumor (DLS) with HIF-1α
inhibitory mechanism. DLA cells (5×106 cells/thigh sc) were
injected into the thigh of to induce solid tumor. After onset of the
tumor, mice were grouped and administered with IPA (8k) (0, 30
and 50 mg/kg b.w (ip) for 6 doses on every two alternative days. At
the end of 35th day, tumor parameters were examined and cell lysate
was subjected to the gene expression analysis. a, b Morphology of
solid tumor bearing mice with tumor section. c Percentage of mass
of tumor inhibition. d Kaplan Meyer graph for extended mice sur-
vivality. e, f H&E staining for MVD and its quantifcation. g, h IHC
studies shows the expression levels of the HIF-1α and its relative
expression level i. Suppression of HIF-1α expression and downstream
MMP-2 and MMP-9 genes expression with elevation in the anti-
tumor p53 gene expression with their respective densitometry graph.
j Decreased gelatinase activity of MMP-2 and MMP-9. k Decrease
in the production of the VEGF-A in the serum. Graphs were plotted
by taking mean (n=6) SD. Statistical analysis was performed using
one-way ANOVA followed by Brown-Forsythe test and statistical sig-
nifcance was set at *p<0.05 to ****p<0.0001
1 3

Targeting HIF‑1α by newly synthesized Indolephenoxyacetamide (IPA) analogs to induce…
Fig. 4 (continued)
Supplementary Information The online version contains supplemen-
References
tary material available at https://doi.org/10.1007/s43440-021-00266-8.
1. Zimna A, Kurpisz M. Hypoxia-inducible factor-1 in physiological
and pathophysiological angiogenesis: applications and therapies.
BioMed Res Int. 2015;2015:549412.
Acknowledgements Fares Hezam Al-Ostoot is thankful to the govern-
ment of Yemen and Al-Baydha University, Yemen, for providing fnan-
cial assistance under the teacher’s fellowship and also thankful to the
University of Mysore, Mysore, India. Ankith Sherapur thanks the Lady
Tata Memorial Trust for JRS (2020–2021) and B.T. Prabhakar grate-
fully acknowledges the grant support by VGST (VGST/P-2/CISEE/
GRD-231/2013-14) and SERB [EMR/2017/00088/dated 3/01/2019].
Shaukath Ara Khanum thankfully acknowledges the fnancial support
provided by VGST, Bangalore, under CISEE Program [Project sanction
order: No. VGST/CISEE/282/2012-13].
2. Semenza GL. Hypoxia-inducible factor 1 and cardiovascular dis-
ease. Annu Rev Physiol. 2014;76:39–65.
3. Ma Z, Xiang X, Li S, Xie P, Gong Q, Goh BC, et al. Targeting
hypoxia-inducible factor-1, for cancer treatment: recent advances
in developing small-molecule inhibitors from natural compounds.
Semin Cancer Biol. 2020. https://doi.org/10.1016/j.semcancer.
2020.09.011.
4. Fuse S, Suzuki K, Kuchimaru T, Kadonosono T, Ueda H, Sato
S, et al. Design, synthesis, and evaluation of indeno [2, 1-c]
pyrazolones for use as inhibitors against hypoxia-inducible
factor (HIF)-1 transcriptional activity. Bioorganic Med Chem.
2020;28:115207.
Author contributions FHA and AS performed all the experiments, VV
and HKV assisted in experiments, GB supported through statistical
analysis, SAK and PBT designed the experiments, wrote the manu-
script and monitored the entire investigation.
5. Jin X, Dai L, Ma Y, Wang J, Liu Z. Implications of HIF-1 in the
tumorigenesis and progression of pancreatic cancer. Cancer Cell
Int. 2020;20:1–11.
Declarations
6. Gurupadaswamy HD, Thirusangu P, Avin BV, Vigneshwaran V,
Kumar MP, Abhishek TS, et al. DAO-9 (2, 5-di (4-aryloylaryloxy-
methyl)-1, 3, 4-oxadiazole) exhibits p53 induced apoptogenesis
through caspase-3 mediated endonuclease activity in murine car-
cinoma. Biomed Pharmacother. 2014;68:791–7.
Conflict of interest The authors declare that there are no conficts of
interest.
1 3

F. H. Al-Ostoot et al.
response in vivo by counteracting angiogenic gene expressions.
Clin Exp Immunol. 2017;189:21–35.
7. Shimazaki Y, Yajima T, Takani M, Yamauchi O. Metal complexes
involving indole rings: structures and efects of metal–indole
interactions. Coord Chem Rev. 2009;253:479–92.
20. Avin BV, Prabhu T, Ramesh CK, Vigneshwaran V, Riaz M,
Jayashree K, et al. New role of lupeol in reticence of angiogen-
esis, the cellular parameter of neoplastic progression in tumo-
rigenesis models through altered gene expression. Biochem
Biophys Res Commun. 2014;448:139–44.
8. Demurtas M, Baldisserotto A, Lampronti I, Moi D, Balboni G,
Pacifco S, et al. Indole derivatives as multifunctional drugs:
synthesis and evaluation of antioxidant, photoprotective and
antiproliferative activity of indolehydrazones. Bioorganic Chem.
2019;85:568–76.
21. Zabiulla Z, Malojirao VH, Mohammed YH, Thirusangu P,
Prabhakar BT, Khanum SA. Synthesis, molecular docking, and
apoptogenic efcacy of novel N-heterocycle analogs to target
B-cell lymphoma 2/X-linked inhibitors of apoptosis proteins to
regress melanoma. Med Chem Res. 2019;28:1132–60.
22. Vigneshwaran V, Thirusangu P, Madhusudana S, Krishna V,
Pramod SN, Prabhakar BT. The latex sap of the ‘Old World
Plant’Lagenaria siceraria with potent lectin activity mitigates
neoplastic malignancy targeting neovasculature and cell death.
Int Immunopharmacol. 2016;39:158–71.
9. Lakshmi NV, Thirumurugan P, Noorulla KM, Perumal PT. InCl3
mediated one-pot multicomponent synthesis, anti-microbial, anti-
oxidant and anticancer evaluation of 3-pyranyl indole derivatives.
Bioorganic Med Chem Lett. 2010;20:5054–61.
10. Merino I, Monge A, Font M, de Irujo JJ, Alberdi E, Santiago E,
et al. Synthesis and anti-HIV-1 activities of new pyrimido [5, 4-b]
indoles. IlFarmaco. 1999;54:255–64.
11. Brew CT, Aronchik I, Hsu JC, Sheen JH, Dickson RB, Bjeldanes
LF, et al. Indole-3-carbinol activates the ATM signaling pathway
independent of DNA damage to stabilize p53 and induce G1 arrest
of human mammary epithelial cells. Int J Cancer. 2006;118:857–68.
12. Meng Q, Qi M, Chen DZ, Yuan R, Goldberg ID, Rosen E, et al.
Suppression of breast cancer invasion and migration by indole-
3-carbinol: associated with up-regulation of BRCA1 and E-cad-
herin/catenin complexes. J Mol Med. 2000;78:155–65.
13. 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.
23. Thirusangu P, Vigneshwaran V, Ranganatha VL, Avin BV,
Khanum SA, Mahmood R, et al. A tumoural angiogenic gate-
way blocker, Benzophenone-1B represses the HIF-1α nuclear
translocation and its target gene activation against neoplastic
progression. Biochem Pharmacol. 2017;125:26–40.
24. Prabhakar BT, Khanum SA, Shashikanth S, Salimath BP.
Antiangiogenic efect of 2-benzoyl–phenoxy acetamide in EAT
cell is mediated by HIF-1α and down regulation of VEGF of
in-vivo. Invest New Drugs. 2006;24:471–8.
25. Prabhakar BT, Khanum SA, Jayashree K, Salimath BP, Shashi-
kanth S. Anti-tumor and proapoptotic efect of novel synthetic
benzophenone analogues in Ehrlich ascites tumor cells. Bioor-
ganic Med Chem. 2006;14:435–46.
14. Al-Ostoot FH, Grisha S, Mohammed YH, Vivek HK, Khanum SA.
Molecular docking and synthesis of cafeic acid analogous and its
anti-infammatory, analgesic and ulcerogenic studies. Bioorganic
Med Chem Lett. 2021;33:127743.
26. Maj E, Papiernik D, Wietrzyk J. Antiangiogenic cancer treat-
ment: The great discovery and greater complexity. Int J Oncol.
2016;49:1773–84.
15. Khamees HA, Mohammed YH, Swamynayaka A, Al-Ostoot FH,
Sert Y, Alghamdi S, et al. Molecular structure, DFT, vibrational
spectra with fuorescence efect, hirshfeld surface, docking simu-
lation and antioxidant activity of thiazole derivative. Chemis-
trySelect. 2019;4:4544–58.
27. Xu D, Xu Z. Indole Alkaloids with potential anticancer activity.
Curr Top Med Chem. 2020;20:1938–49.
28. Mohammed YH, Thirusangu P, Vigneshwaran V, Prabhakar BT,
Khanum SA. The anti-invasive role of novel synthesized pyri-
dazine hydrazide appended phenoxy acetic acid against neoplastic
development targeting matrix metallo proteases. Biomed Pharma-
cother. 2017;95:375–86.
16. Ranganatha VL, Avin BV, Thirusangu P, Prashanth T, Prabhakar
BT, Khanum SA. Synthesis, angiopreventive activity, and in vivo
tumor inhibition of novel benzophenone–benzimidazole analogs.
Life Sci. 2013;93:904–11.
29. Chung AS, Lee J, Ferrara N. Targeting the tumour vascula-
ture: insights from physiological angiogenesis. Nat Rev Cancer.
2010;10:505–14.
17. Vijay Avin BR, Thirusangu P, Ranganatha VL, Firdouse A, Prab-
hakar BT, Khanum SA. Synthesis and tumor inhibitory activity
of novel coumarin analogs targeting angiogenesis and apoptosis.
Eur J Med Chem. 2014;75:211–21.
30. Rogers MS, Birsner AE, D’amato RJ. The mouse cornea micro-
pocket angiogenesis assay. Nat Protoc. 2007;2:2545–50.
31. Shivakumar S, Prabhakar BT, Jayashree K, Rajan MG, Sali-
math BP. Evaluation of serum vascular endothelial growth factor
(VEGF) and microvessel density (MVD) as prognostic indicators
in carcinoma breast. J Cancer Res Clin Oncol. 2009;135:627–36.
18. Thirusangu P, Vigneshwaran V, Prashanth T, Avin BV, Malo-
jirao VH, Rakesh H, et al. BP-1T, an antiangiogenic benzo-
phenone-thiazole pharmacophore, counteracts HIF-1 signalling
through p53/MDM2-mediated HIF-1? proteasomal degrada-
tion. Angiogenesis. 2017;20:55–71.
19. Vigneshwaran V, Thirusangu P, Vijay Avin BR, Krishna V,
Pramod SN, Prabhakar BT. Immunomodulatory glc/man-
directed Dolichos lablab lectin (DLL) evokes anti-tumour
1 3