Synthesis and cellular bioactivities of novel isoxazole derivatives incorporating an arylpiperazine moiety as anticancer agents

Article abstract of DOI:10.1080/14756366.2018.1504041

In our endeavour towards the development of effective anticancer therapeutics, a novel series of isoxazole-piperazine hybrids were synthesized and evaluated for their cytotoxic activities against human liver (Huh7 and Mahlavu) and breast (MCF-7) cancer ce

Full text of DOI:10.1080/14756366.2018.1504041

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and cellular bioactivities of novel
isoxazole derivatives incorporating an
arylpiperazine moiety as anticancer agents
Burcu Çalışkan, Esra Sinoplu, Kübra İbiş, Ece Akhan Güzelcan, Rengül Çetin
Atalay & Erden Banoglu
To cite this article: Burcu Çalışkan, Esra Sinoplu, Kübra İbiş, Ece Akhan Güzelcan, Rengül Çetin
Atalay & Erden Banoglu (2018) Synthesis and cellular bioactivities of novel isoxazole derivatives
incorporating an arylpiperazine moiety as anticancer agents, Journal of Enzyme Inhibition and
Medicinal Chemistry, 33:1, 1352-1361, DOI: 10.1080/14756366.2018.1504041
To link to this article: https://doi.org/10.1080/14756366.2018.1504041
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UK Limited, trading as Taylor & Francis
Group.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1352–1361
https://doi.org/10.1080/14756366.2018.1504041
ORIGINAL ARTICLE
Synthesis and cellular bioactivities of novel isoxazole derivatives incorporating an
arylpiperazine moiety as anticancer agents
a
b
a
b
b
a
_
€
€
€
Burcu C¸alıs¸kan , Esra Sinoplu , Kubra Ibis¸ , Ece Akhan Guzelcan , Rengul C¸etin Atalay and Erden Banoglu
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Ankara, Turkey; Department of Bioinformatics, Middle East
Technical University, Ankara, Turkey
ABSTRACT
ARTICLE HISTORY
Received 4 June 2018
Accepted 20 July 2018
In our endeavour towards the development of effective anticancer therapeutics, a novel series of isoxa-
zole-piperazine hybrids were synthesized and evaluated for their cytotoxic activities against human liver
(Huh7 and Mahlavu) and breast (MCF-7) cancer cell lines. Within series, compounds 5l-o showed the most
potent cytotoxicity on all cell lines with IC50 values in the range of 0.3–3.7 lM. To explore the mechanistic
aspects fundamental to the observed activity, further biological studies with 5m and 5o in liver cancer
cells were carried out. We have demonstrated that 5m and 5o induce oxidative stress in PTEN adequate
Huh7 and PTEN deficient Mahlavu human liver cancer cells leading to apoptosis and cell cycle arrest at
different phases. Further analysis of the proteins involved in apoptosis and cell cycle revealed that 5m
and 5o caused an inhibition of cell survival pathway through Akt hyperphosphorylation and apoptosis
and cell cycle arrest through p53 protein activation.
KEYWORDS
Isoxazole; piperazine; liver
cancer; oxidative stress;
cytotoxicity
Introduction
4,5-diarylisoxazole amides have been developed as potent heat
shock protein (HSP90) inhibitors, exemplified by NVP-AUY922
(Luminespib)13, which was active against a variety of tumor xeno-
grafts and has been evaluated in phase II clinical trials14. Currently
marketed immunosuppresory drug Leflunomide, which has
recently been identified as a potential anticancer drug15, is also
an isoxazole derivative. Isoxazole derivatives as comberastatin A-4
analogues are also successfully described as tubulin polymerisa-
tion inhibitors with antiproliferative activities towards various cell
lines16,17. Hewings and others reported the 3,5-dimethylisoxazole
moiety as an effective acetylated lysine (KAc) mimic, which was
used for developing bromodomain inhibitors with anticancer
activity18–21. In addition, a naturally occurring diarylisoxazole
derivative has recently been reported as a new chemical tool with
efficacy against AR-expressing breast cancer cells22.
Another widely occurring structural fragment in anticancer com-
pounds is the piperazine moiety and a large number of compounds
have appeared in the literature having piperazine motif with cyto-
toxicity against various cancer cells23–27. For instance, studies on an
arylpiperazine derivative naftopidil28,29, a well-known a1-adrenergic
receptor antagonist, and several other arylpiperazines have shown
significant cytotoxicity against prostate cancer cells30–32. A series of
piperazine derivatives have also been demonstrated to bear potent
antiproliferative activities against various cancer cells including
colon, prostate, breast, lung, and leukaemia as well as to suppress
experimental tumours in small animal models by a mechanism
involving inhibition of microtubule synthesis, inhibition of cell-cycle
progression and angiogenesis33,34. Recently, incorporation of arylpi-
perazine moiety in purine nucleoside analogues resulted in com-
Cancer is one of the leading cause of deaths globally, and can be
classified as a multifactorial disease, which is diligently orches-
trated by a combination of genetic, epigenetic, and environmental
factors working together towards the progression of tumours1,2
.
Hepatocellular carcinoma (HCC) is the most common type of liver
cancer accounting for about 75% of all primary liver cancers and
is the 6th most frequent and the 2nd deadly cancer worldwide3.
Etiological factors, which are associated with HCC are chronic
infection by hepatitis B virus (HBV) and hepatitis C virus (HCV),
alcohol consumption, obesity, and aflatoxin exposure4,5. Primary
liver cancer is extremely resistant to conventional chemotherapeu-
tics, only 7% of patients have five-year survival6. Sorafenib and
Regorafenib are the only FDA approved agents for advanced liver
cancer cases, which extend patient survival approximately 3–7
months7,8. One of the biggest challenge in anticancer drug devel-
opment is that HCC cells are reported to display high levels of cel-
lular heterogeneity9, which hampers significantly the development
of new cancer therapeutics and causes failures in clinical studies
since many candidate drugs do not produce clinical benefit in the
overall population10. Consequently, an endless effort has continu-
ously been devoted to the discovery and development of new
and more effective anticancer agents that are capable to intervene
with this complex disease.
Diverse azaheterocyclic ring systems have been in the centre
of medicinal chemists, and used as versatile tools and building
blocks for the synthesis of small molecule cancer therapeutics11.
Among them, one of the most constantly growing area was the
investigation of the antitumor properties of compounds contain-
ing isoxazole core structure12. For example, a series of resorcinylic pounds with senescence-induced cell death in liver cancer cells26.
CONTACT Erden Banoglu
banoglu@gazi.edu.tr
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Ankara, Turkey
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1
1353
Other recent progress on anticancer piperazine derivatives can be d 52.88, 52.98, 53.29, 62.26, 99.60, 124.20 (q, JC-F ¼ 270.0 Hz),
found elsewhere35.
125.15 (q, JC-F ¼ 3.9 Hz), 125.77, 127.51, 128.92, 129.18, 129.44
3
2
In the course of our ongoing research interest concerning bio-
active heterocycles36–40, we have relied on the aforementioned
data for the design, synthesis, and biological evaluation of novel
isoxazole derivatives containing in their structural framework an
aryl piperazine residue. Since primary liver cancer incidence is
expected to increase due to the obesity associated non-alcoholic
fatty liver disease4, we think that the synthesis of novel anticancer
agents as reported herein will contribute not only the mechanistic
bioactivity analysis of these isoxazole-piperazine hybrids, but also
the future treatment options for HCC. In this context, we hereby
report the straightforward synthesis of the novel isoxazole-pipera-
zine hybrids, which were evaluated for their antitumor activities.
(q, JC-F ¼ 30.0 Hz), 130.08, 142.26, 161.68, 169.97. HRMS (m/z)
[M þ H]þ calcd for C22H23F3N3O: 402.1793, found, 402.1794.
5-(4-Fluorophenyl)-3-((4-(4-(trifluoromethyl)benzyl)piperazin-1-
yl)methyl)isoxazole (5b)
Purified by flash column chromatography (0% ! 10% MeOH in
DCM). Yield 74.0%; mp 110.1–111.0 ꢂC. 1H NMR (CDCl3):
d
2.60–2.72 (8H, m), 3.64 (2H, s), 3.72 (2H, s), 6.58 (1H, s), 7.12–7.18
(2H, m), 7.49–7.52 (2H, m), 7.58 (2H, d, J ¼ 8.4 Hz), 7.74–7.79 (2H,
m). 13C NMR (CDCl3) d 53.06, 53.19, 53.46, 62.47, 99.60, 116.37
2
4
1
(d, JC-F ¼ 21.8 Hz), 124.05 (d, JC-F ¼ 3.2 Hz), 124.40 (q, JC-F
¼
3
3
270.5 Hz), 125.38 (q, JC-F ¼ 3.8 Hz), 128.04 (d, JC-F ¼ 8.4 Hz), 129.41,
1
142.46, 162.05, 163.90 (d, JC-F ¼ 250.0 Hz), 169.20. HRMS (m/z)
[M þ H]þ calcd for C22H22F4N3O: 420.1699, found, 420.1686.
Experimental
Chemistry
Starting materials were purchased from commercial suppliers and
used without further purification. 1H and 13C NMR spectra were
recorded in CDCl3 or DMSO-d6 on a Varian Mercury 400 MHz spec-
trometer (Agilent Technologies, Santa Clara, CA, USA) using tetra-
methylsilane as the internal standard. All chemical shifts were
recorded as d (ppm), coupling constants are reported as Hertz.
High resolution mass spectra data (HRMS) were collected using
Waters LCT Premier XE Mass Spectrometer (high sensitivity orthog-
onal acceleration time-of-flight instrument) operating in ESI (þ) or
ESI (ꢀ) method, also coupled to an AQUITY Ultra Performance
Liquid Chromatography system (Waters Corporation, Milford, MA,
USA) using a UV detector monitoring at 254 nm. Purity for all final
compounds were >95%, according to the UPLC-MS method using
(A) water þ 0.1% formic acid and (B) acetonitrile þ 0.1% Formic
Acid; flow rate ¼ 0.3 mL/min, Column: Aquity BEH C18 column
(2.1 ꢁ 100 mm, 1.7 mm; Waters Corporation, Milford, MA, USA). Flash
chromatography on silica gel was performed on RediSep prepacked
disposable silica gel columns using Teledyne Isco Combiflash.
Preparative liquid chromatography was performed on Vydac Denali
C18 Column (150 ꢁ 20 mm, 5 m; Grace, Columbia, MD, USA) using
5-(4-Chlorophenyl)-3-((4-(4-(trifluoromethyl)benzyl)piperazin-1-
yl)methyl)isoxazole (5c)
Purified by flash column chromatography (0% ! 20% EA in DCM).
Yield 68.1%; mp 126.2–126.9 ꢂC. 1H NMR (CDCl3): d 2.52 (4H, bs),
2.59 (4H, bs), 3.57 (2H, s), 3.66 (2H, s), 6.55 (1H, s), 7.41–7.45 (4H,
m), 7.56 (2H, d, J ¼ 7.6 Hz), 7.69 (2H, d, J ¼ 8.4 Hz). 13C NMR (CDCl3)
1
d 52.80, 52.94, 53.21, 62.23, 99.95, 124.19 (q, JC-F ¼ 270.6 Hz),
3
125.15 (q, JC-F ¼ 3.8 Hz), 125.91, 127.03, 129.24, 129.28, 136.18,
141.95, 161.79, 168.86. HRMS (m/z) [M þ H]þ calcd for
C22H22ClF3N3O: 436.1404, found, 436.1402.
5-(p-Tolyl)-3-((4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)methyl)-
isoxazole (5d)
Purified by flash column chromatography (0% ! 10% MeOH in
1
DCM). Yield 84.0%; mp 141.0–142.3 ꢂC. H NMR (CDCl3): d 2.40 (3H.
s), 2.61 (4H, bs), 2.69 (4H, bs), 3.63 (2H, s), 3.71 (2H, s), 6.57 (1H, s),
7.27 (2H, d, J ¼ 8.0 Hz), 7.48 (2H, d, J ¼ 7.6 Hz), 7.58 (2H, d,
J ¼ 7.6 Hz), 7.66 (2H, d, J ¼ 8.0 Hz). 13C NMR (CDCl3) d 21.66, 53.07,
1
53.17, 53.52, 62.48, 99.22, 124.41 (q, JC-F ¼ 269.8 Hz), 124.98,
3
Reveleris
PREP
purification
system.
All
microwave
Biotage
125.37 (q, JC-F ¼ 3.8 Hz), 125.91, 129.41, 129.82, 140.60, 142.50,
161.81, 170.36. HRMS (m/z) [M þ H]þ calcd for C23H25F3N3O:
irradiation experiments were carried out in
a
Initiator þ microwave apparatus with Biotage sealed microvawe vials.
Melting points of the synthesized compounds were determined by
SMP50 automatic melting point apparatus and uncorrected.
Experimental data for all intermediate compounds can be found in
Supporting Information.
416.1950, found, 416.1948.
3-((4-(4-(Trifluoromethyl)benzyl)piperazin-1-yl)methyl)-5-(4-
(trifluoromethyl) phenyl)isoxazole (5e)
Purified by flash column chromatography (0% ! 10% MeOH in
1
DCM). Yield 44.0%; mp 110.1–111.5 ꢂC. H NMR (CDCl3): d 2.53 (4H,
Synthesis of Compounds 5a-o
bs), 2.61 (4H, bs), 3.58 (2H, s), 3.68 (2H, s), 6.67 (1H, s), 7.45 (2H, d,
J¼ 8.2Hz), 7.56 (2H, d, J¼ 8.2 Hz), 7.72 (2H, d, J¼ 7.8Hz), 7.88 (2H, d,
J¼ 7.8 Hz). 13C NMR (CDCl3) d 52.83, 52.99, 53.19, 62.23, 101.08,
The mixture of the appropriate bromide derivative (4a-o)
(0.5 mmol, 1 eq), 4-trifluoromethylbenzylpiperazine (0.6 mmol, 1.2
eq) and DIEA (1 mmol, 2 eq) in DMF (2 ml) was heated by micro-
wave irradiation at 80 ꢂC for 20 min. Then, it was poured into ice-
water and formed precipitate was filtrated. The crude product was
purified by flash chromatography.
1
1
3
123.71(q, JC-F ¼ 270.6 Hz), 124.19 (q, JC-F ¼ 270.5 Hz), 125.17 (q, JC-
3
2
¼ 3.9 Hz), 125.99 (q, JC-F ¼ 3.8 Hz), 126.03, 129.19, 129.50 (q, JC-
F
2
¼ 31.0 Hz), 130.58, 131.84 (q, JC-F ¼ 32.7 Hz), 142.18, 162.01, 168.33.
F
HRMS (m/z) [Mþ H]þ calcd for C23H22F6N3O: 470.1667, found, 470.1667.
5-(4-Isopropylphenyl)-3-((4-(4-(trifluoromethyl)benzyl)piperazin-1-
yl)methyl) isoxazole (5f)
5-Phenyl-3-((4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)methyl)isox-
azole (5a)
Purified by flash column chromatography (0% ! 10% MeOH in
Purified by flash column chromatography (0% ! 10% MeOH in
1
1
DCM). Yield 87.0%; mp 112.6–113.9 ꢂC. H NMR (CDCl3): d 2.51 (4H, DCM). Yield 67.0%; mp 111.8–112.2 ꢂC. H NMR (CDCl3): d 1.27 (6H,
bs), 2.59 (4H, bs), 3.57 (2H, s), 3.66 (2H, s), 6.55 (1H, s), 7.42–7.48 d, J ¼ 6.8 Hz), 2.51 (4H, bs), 2.58 (4H, bs), 2.93–2.96 (1H, m), 3.57
(5H, m), 7.56 (2H, d, J ¼ 8.0 Hz), 7.76–7.78 (2H, m). 13C NMR (CDCl3) (2H, s), 3.65 (2H, s), 6.51 (1H, s), 7.31 (2H, d, J ¼ 8.2 Hz), 7.44 (2H, d,

1354
B. C¸ALIS¸KAN ET AL.
J ¼ 7.8 Hz), 7.56 (2H, d, J ¼ 7.8 Hz), 7.69 (2H, d, J ¼ 8.2 Hz). 13C NMR t, J ¼ 7.4 Hz), 1.80–1.85 (2H, m), 2.51 (4H, bs), 2.58 (4H, bs), 3.56
(CDCl3) d 23.76, 34.08, 52.83, 52.94, 53.30, 62.25, 99.06, 124.21 (q, (2H, s), 3.64 (2H, s), 3.96 (2H, t, J ¼ 6.4 Hz), 6.42 (1H, s), 6.95 (2H, d,
3
1JC-F ¼ 270.0 Hz), 125.10, 125.18 (q, JC-F ¼ 3.8 Hz), 125.84, 127.04, J ¼ 8.8 Hz), 7.43 (2H, d, J ¼ 7.8 Hz), 7.56 (2H, d, J ¼ 7.8 Hz), 7.68 (2H,
d, J ¼ 8.8 Hz). 13C NMR (CDCl3) d 10.46, 22.48, 52.82, 52.92, 53.30,
2
129.23, 129.40 (q, JC-F ¼ 34.0 Hz), 142.15, 151.30, 161.53, 170.19.
HRMS (m/z) [M þ H]þ calcd for C25H29F3N3O: 444.2263,
found, 444.2265.
1
62.25, 69.64, 98.17, 114.85, 120.07, 124.21 (q, JC-F ¼ 270.6 Hz),
3
2
125.18 (q, JC-F ¼ 3.8 Hz), 127.33, 129.24, 129.40 (q, JC-F ¼ 32.7 Hz),
142.10, 160.66, 161.48, 170.07. HRMS (m/z) [M þ H]þ calcd for
C25H29F3N3O2: 460.2212, found, 460.2213.
5-(4-(Trifluoromethoxy)phenyl)-3-((4-(4-(trifluoromethyl)benzyl)pi-
perazin-1-yl)methyl)isoxazole (5g)
5-(4-(Allyloxy)phenyl)-3-((4-(4-(trifluoromethyl)benzyl)piperazin-1-
yl)methyl)isoxazole (5l)
Purified by flash column chromatography (0% ! 10% MeOH in
1
DCM). Yield 85.0%; mp 103.2–103.9 ꢂC. H NMR (CDCl3): d 2.59 (4H,
Purified by flash column chromatography (0% ! 10% MeOH in
bs), 2.67 (4H, bs), 3.63 (2H, s), 3.71 (2H, s), 6.62 (1H, s), 7.31 (2H, d,
J ¼ 8.4 Hz), 7.47 (2H, d, J ¼ 8.0 Hz), 7.58 (2H, d, J ¼ 8.0 Hz), 7.81 (2H,
d, J ¼ 8.4 Hz). 13C NMR (CDCl3) d 53.05, 53.21, 53.44, 62.46, 100.30,
1
DCM). Yield 78.5%; mp 107.1–107.4 ꢂC. H NMR (CDCl3): d 2.51 (4H,
bs), 2.58 (4H, bs), 3.56 (2H, s), 3.64 (2H, s), 4.58 (2H, dt, J ¼ 5.6 Hz,
1.6 Hz), 5.33 (1H, dq, J ¼ 10.4, 1.4 Hz), 5.44 (1H, dq, J ¼ 17.2, 1.6 Hz),
6.01-6.10 (1H, m), 6.43 (1H, s), 6.97 (2H, d, J ¼ 9.0 Hz), 7.44 (2H, d,
J ¼ 7.8 Hz), 7.56 (2H, d, J ¼ 7.8 Hz), 7.69 (2H, d, J ¼ 9.0 Hz). 13C NMR
(CDCl3) d 52.66, 52.78, 53.20, 62.14, 68.86, 98.35, 115.12, 118.05,
1
1
120.55 (q, JC-F ¼ 257.0 Hz), 121.53, 124.40 (q, JC-F ¼ 270.5 Hz),
3
125.39 (q, JC-F ¼ 3.8 Hz), 126.28, 127.61, 129.41, 142.50, 150.50,
162.15, 168.76. HRMS (m/z) [M þ H]þ calcd for C23H22F6N3O2:
486.1616, found, 486.1600.
1
3
120.38, 124.18 (q, JC-F ¼ 270.6 Hz), 125.23 (q, JC-F ¼ 3.8 Hz), 127.36,
129.32, 132.70, 142.02, 160.07, 161.25, 170.04. HRMS (m/z)
[M þ H]þ calcd for C25H27F3N3O2: 458.2055, found, 458.2061.
5-(4-(Methylsulfonyl)phenyl)-3-((4-(4-(trifluoromethyl)benzyl)pipera-
zin-1-yl)methyl)isoxazole (5h)
Purified by flash column chromatography (0% ! 10% MeOH in
1
DCM). Yield 84.0%; mp 163.5–164.1 ꢂC. H NMR (CDCl3): d 2.51 (4H,
5-(4-((3-Methylbut-2-en-1-yl)oxy)phenyl)-3-((4-(4-(trifluoromethyl)-
benzyl)piperazin-1-yl)methyl)isoxazole (5m)
bs), 2.59 (4H, bs), 3.08 (3H, s), 3.57 (2H, s), 3.68 (2H, s), 6.73 (1H, s),
7.44 (2H, d, J ¼ 7.8 Hz), 7.56 (2H, d, J ¼ 7.8 Hz), 7.96 (2H, d,
J ¼ 8.8 Hz), 8.04 (2H, d, J ¼ 8.8 Hz). 13C NMR (CDCl3) d 44.43, 52.83,
Purified by flash column chromatography (0% ! 10% MeOH in
1
DCM). Yield 66.8%; mp 100.0–100.3 ꢂC. H NMR (CDCl3): d 1.76 (3H,
1
s), 1.81 (3H, s), 2.53 (4H, bs), 2.60 (4H, bs), 3.58 (2H, s), 3.66 (2H, s),
4.55 (2H, d, J ¼ 6.8 Hz), 5.47–5.51 (1H, m), 6.44 (1H, s), 6.97 (2H, d,
J ¼ 8.8 Hz), 7.45 (2H, d, J ¼ 8.0 Hz), 7.56 (2H, d, J ¼ 8.0 Hz), 7.69 (2H,
53.00, 53.17, 62.24, 101.94, 124.19 (q, JC-F ¼ 269.8 Hz), 125.20 (q,
3JC-F ¼ 3.6 Hz), 126.52, 128.20, 129.23, 132.11, 141.55, 142.09,
162.21, 167.69. HRMS (m/z) [M þ H]þ calcd for C23H25F3N3O3S:
480.1569, found, 480.1567.
d, J ¼ 8.8 Hz). 13C NMR (CDCl3) d 18.23, 25.81, 52.86, 52.95, 53.31,
1
62.26, 64.93, 98.21, 115.05, 119.15, 120.18, 124.10 (q, JC-F
¼
3
270.6 Hz), 125.18 (q, JC-F ¼ 3.2 Hz), 127.33, 129.23, 138.72, 160.38,
170.04. HRMS (m/z) [M þ H]þ calcd for C27H31F3N3O2: 486.2368,
found, 486.2362.
4-(3-((4-(4-(Trifluoromethyl)benzyl)piperazin-1-yl)methyl)isoxazol-5-
yl)phenol (5i)
Purified by flash column chromatography (0% ! 10% MeOH in
1
DCM). Yield 71.6%; mp 154.2–156.0 ꢂC. H NMR (CDCl3): d 2.56 (4H,
3,5-Dimethyl-4-((4-(3-((4-(4-(trifluoromethyl)benzyl)piperazin-1-
yl)methyl)isoxazol-5-yl)phenoxy)methyl)isoxazole
hydrochloride (5n)
bs), 2.66 (4H, bs), 3.57 (2H, s), 3.67 (2H, s), 6.22 (1H, s), 6.70 (2H, d,
J ¼ 8.4 Hz), 7.42–7.47 (4H, m), 7.56 (2H, d, J ¼ 8.4 Hz). 13C NMR
(CDCl3): d 52.83, 53.18, 62.47, 98.64, 116.31, 119.78, 124.20 (q,
3
1JC-F ¼ 270.0 Hz), 125.46 (q, JC-F ¼ 3.1 Hz), 127.65, 129.62, 142.10,
Purified by flash column chromatography (0% ! 10% MeOH in
DCM). Yield 58.4%; mp 200.4–201.2 ꢂC (decomp). 1H NMR (DMSO-
d6): d 2.23 (3H, s), 2.28 (3H, s), 2.43 (3H, s), 3.40 (8H, bs), 4.33 (2H,
s), 4.47 (2H, s), 5.02 (2H, s), 7.09 (1H, s), 7.19 (2H, d, J ¼ 8.8 Hz),
7.80–7.85 (4H, m), 7.90 (2H, d, J ¼ 8.0 Hz). 13C NMR (DMSO-d6) d
9.70, 10.65, 47.99, 48.01, 49.68, 57.38, 59.23, 100.13, 110.08,
158.19, 160.44, 170.43. HRMS (m/z) [M þ H]þ calcd for
C22H23F3N3O2: 418.1742; found, 418.1736.
5-(4-Methoxyphenyl)-3-((4-(4-(trifluoromethyl)benzyl)piperazin-1-
yl)methyl)isoxazole (5j)
1
3
115.70, 119.50, 124.00 (q, JC-F ¼ 243.6 Hz), 125.59 (q, JC-F ¼ 3.8 Hz),
2
Purified by flash column chromatography (0% ! 10% MeOH in
127.36, 129.61, 130.08 (q, JC-F ¼ 32.1 Hz), 132.25, 159.54, 159.92,
1
167.60, 169.81. HRMS (m/z) [M þ H]þ calcd for C28H30ClF3N4O3:
DCM). Yield 59.4%; mp 117.9–118.2 ꢂC. H NMR (CDCl3): d 2.49 (4H,
bs), 2.57 (4H, bs), 3.55 (2H, s), 3.63 (2H, s), 3.85 (3H, s), 6.42 (1H, s),
527.2270, found, 527.2275.
6.96 (2H, d, J ¼ 9.2 Hz), 7.43 (2H, d, J ¼ 7.8 Hz), 7.55 (2H, d,
J ¼ 7.8 Hz), 7.70 (2H, d, J ¼ 9.2 Hz). 13C NMR (CDCl3) d 52.94, 53.05,
1
5-(4-((1,3-Dimethyl-1H-pyrazol-4-yl)methoxy)phenyl)-3-((4-(4-
(trifluoromethyl)benzyl)piperazin-1-yl)methyl)isoxazole (5o)
Purified by flash column chromatography (0% ! 10% MeOH in
53.37, 55.37, 62.33, 98.24, 114.34, 120.35, 124.23 (q, JC-F
¼
3
270.0 Hz), 125.14 (q, JC-F ¼ 3.8 Hz), 127.35, 129.17, 129.32 (q,
2JC-F ¼ 32.0 Hz), 142.40, 161.03, 161.72, 169.90. HRMS (m/z)
[M þ H]þ calcd for C23H25F3N3O2: 432.1899, found, 432.1891.
1
DCM). Yield 58.0%; mp 131.4–131.9 ꢂC. H NMR (CDCl3): d 2.52 (3H,
s), 2.51 (4H, bs), 2.58 (4H, bs), 3.57 (2H, s), 3.64 (2H, s), 3.84 (3H, s),
5.03 (2H, s), 6.11 (1H, s), 6.45 (1H, s), 7.02 (2H, d, J ¼ 8.8 Hz), 7.44
(2H, d, J ¼ 7.8 Hz), 7.56 (2H, d, J ¼ 7.8 Hz), 7.71 (2H, d, J ¼ 8.8 Hz).
5-(4-Propoxyphenyl)-3-((4-(4-(trifluoromethyl)benzyl)piperazin-1-
yl)methyl)isoxazole (5k)
13C NMR (CDCl3) d 13.38, 36.46, 52.40, 52.56, 53.05, 60.62, 61.98,
1
Purified by flash column chromatography (0% ! 10% MeOH in 98.71, 106.88, 115.18, 120.99, 124.13 (q, JC-F ¼ 270.6 Hz), 125.31 (q,
1
DCM). Yield 79.0%; mp 103.0–103.5 ꢂC. H NMR (CDCl3): d 1.05 (3H, 3JC-F ¼ 3.8 Hz), 127.50, 129.45, 137.26, 147.38, 159.50, 160.95, 169.91.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1355
HRMS (m/z) [M þ H]þ calcd for C28H31F3N5O2: 526.2430, superoxide production in the cells42. Compound 5o impaired
found, 526.2429.
Mahlavu cells a lot, so 10,000 events were analysed for treated cells,
2000 events could be done in 5o treated Mahlavu cells. In parallel,
Huh7 cells were plated into six-well plates for 24 h followed by treat-
ment with 5o (1 mM) or 5m (1 mM) or DMSO or selenium deficient
serum-free medium. After 24 h, 48 h, 72 h treatment periods, sam-
ples were washed three times with 1 ꢁ PBS, then they were incu-
bated with dichloro-dihydro-fluorescein diacetate (DCFH-DA)
solution (10 mM glucose, 0.5 mM DCFH-DA, 10 mM HEPES dissolved
in 1 ꢁ PBS) in order to detect ROS (particularly H2O2) in the cells for
15 min in humidified chamber in dark at 37 ꢂC. The solution was
aspirated and cells were washed with PBS two times. The staining
was analysed in situ with fluorescence microscope.
Biology
Cell culture
Huh7 (epithelial-like) and Mahlavu (mesenchymal-like) human hep-
atocellular cancer cell lines and MCF7 human breast cancer carcin-
oma cells were grown in Dulbecco’s Modified Eagles
Medium(DMEM) supplemented with %10 fetal bovine serum
(Gibco, Invitrogen, Carlsbad, CA, USA), 1% non-essential amino
acids (Gibco, Invitrogen) and 100 units/ml penicillin/streptomycin
(Gibco, Invitrogen). Cells were maintained at 37 ꢂC in a humidified
incubator under 5% CO2.
Flow cytometry for cell cycle analysis
Huh7 and Mahlavu cells were seeded onto 100 mm culture dishes.
After 24 h, cells were treated with 5o (1 mM for Huh7 and 4 mM
for Mahlavu) or 5m (1 mM for Huh7 and Mahlavu) or DMSO as a
negative control. The end of 24 h, 48 h, and 72 h of incubation
period, cells were fixed with ice-cold 70% ethanol for 3 h at
ꢀ20 ꢂC. Cell cycle analysis was carried out by PI (propidium iod-
ide) staining using MUSE Cell Analyzer according to the man-
ufacturer’s recommendations (Millipore).
NCI-60 sulforhodamine B assay
Huh7, MCF7 (2500 cell/well in 150 ml/well) and Mahlavu (1000
cell/well in 150 ml/well) cells were plated in 96-well plates and
were grown in incubator for 24 hours. The compounds were dis-
solved in DMSO (Sigma, St Louis, MO, USA) as 20 mM stock solu-
tion. The compounds which were below 2.5 mM were tested in a
concentration range of starting from 2.5 mM to 0.015 mM. Cells
were fixed using 10% (v/v) trichloroacetic acid (Sigma ) for an
hour after the end of 72 h incubation time. The fixed plates were
dried and fixed cells were stained with sulforhodamine B (SRB)
solution (Sigma) (50 ml of a 0.4% (m/v) of SRB in 1% acetic acid
solution (Sigma)) for 10 min. In order to remove unbound SRB
dye, cells were washed with 1% acetic acid three times and left
for air-drying. The protein bound SRB dye was dissolved in 10 mM
Tris-base (Sigma) and absorbance was measured with 96-well
plate reader at 515 nm. The IC50 values were calculated and the
cells treated with DMSO alone were used as control. All experi-
ments were done in triplicate. Data with R2 values >0.9 was con-
sidered significant.
Immunofluorescence staining
Huh7 (50,000 cells/well) and Mahlavu (35,000 cells/well) cells were
inoculated on cover slides in 6-well plates after 24 h, cells were
treated with 5o (1 mM for Huh7 and 4 mM for Mahlavu) or 5m
(1 mM for Huh7 and Mahlavu) or DMSO control for 24 h, 48 h, and
72 h. After incubation time periods, the cells were washed three
times with 1 ꢁ PBS and fixed with %100 ice-cold methanol. Then,
the cells were stained with 1 mg/ml Hoechst (#33258, Sigma).
Finally, the cells were analysed under a fluorescent microscope.
Western blot analysis
Real-time cell growth surveillance by cell electronic sensing
(RT-CES)
Cells were treated with the 5o (1 mM for Huh7 and 4 mM for
Mahlavu), 5m (1 mM for Huh7 and Mahlavu) and with DMSO as
control for 72 h. After 72 h incubation, the cells were collected
with scraper, their total proteins were isolated and protein con-
centrations were calculated with Bradford assay. Bio-Rad protein
Real-time cell growth analysis was performed using the
xCELLigence System (Roche Applied Sciences, Penzberg,
Germany). The Huh7, MCF7 (2500 cell/well) and Mahlavu (1000
cell/well) cells were seeded in E-Plates 96. In proliferation step,
the cellular growth was analysed with cell index measurements in
every 30 min for 24 h. After 24 h of incubation, when cells reached
the log growth phase, they were treated with 5m and 5o starting
from 10 mM and 1/2 folds’ serial dilutions three times. The cell
index values (CI) were initially monitored every 10 min for 24 h
and then CI were recorded in 30 min intervals. After 72 h of incu-
R
electrophoresis (Mini-PROTEANV TetraCellSystems and TGXTM pre-
cast gels, Bio-Rad, Hercules, CA, USA) and transfer system (Trans-
R
BlotV TurboTransfer System, Bio-Rad, Hercules, CA, USA) were
used according to the manufacturer’s protocol for all the Western
blotting analyses. About 20–40 mg of protein were used per well.
Proteins were transferred to a PVDF membrane. For immunoblot-
ting, PARP (#9532S, Cell Signaling), p21/WAF1/Cip1 (#05-345,
Millipore), p53 (#05-224, Millipore), phospho-p53Ser15 (#9286S, Cell
Signaling), Rb (#9309, Cell Signaling), and phospho-RbSer807/811
(#9308S, Cell Signaling), a-phospho-AktSer473 (Cell Signaling,
#9271), and AKT (#9272, Cell Signaling) antibodies were used in
1:100 to 1:500 5% BSA-TBS-T. b-actin (#A5441, Sigma) antibody
was used in 1:1000 concentration for equal loading control.
bation, the cellular growth ratios were calculated by CIdrug/CIDMSO
.
Oxidative stress assay
Mahlavu (35000 cells/well) and Huh7 (50000 cells/well) cells were
inoculated into 6-wells plate for 24 h. Then cells were treated with
5o (1 mM for Huh7 and 4 mM for Mahlavu) and 5m (1 mM for
Huh7 and Mahlavu). One group of cells did not receive the com-
pounds, but they were grown in selenium-deficient serum-free
medium as positive control for oxidative stress41. Fourth group
was treated with DMSO as negative control. At the end of 24 h,
48 h, and 72 h incubation period, the cells were collected and ana-
R
Proteins were visualized using a C-DigitV imaging system (Ll-COR)
Results and discussion
Chemistry
lysed by MUSE Oxidative Stress Kit (MCH100111, Merck Millipore, Compounds 5a–o was prepared following the reaction sequence
Burlington, MA, USA), which uses dihydroethium to monitor illustrated in Schemes 1 and 2 using the known general methods.

1356
B. C¸ALIS¸KAN ET AL.
Scheme 1. Synthesis of compounds 5a-j. Reagents and conditions: i. NaOEt, EtOH ii. NH2OH.HCl, EtOH iii. LiAlH4, THF or NaBH4, THF/MeOH iv. CBr4/PPh3, DCM
v. 4- (trifluoromethyl)benzylpiperazine, DIEA, DMF.
Scheme 2. Synthesis of compounds 5k-o. Reagents and conditions: i. R-Br, K2CO3 ii. CBr4/PPh3, DCM iii. 4- (trifluoromethyl)benzylpiperazine, DIEA, DMF.
Table 1. In vitro cytotoxic activities of 5a-o with 72 h of treatment.
Hence, diethyloxalate has been treated with substituted acetophe-
nones in the presence of a base to obtain b-ketoesters 1a–j. These
intermediates (1a–j) were subsequently cyclized with hydroxyl-
amine hydrochloride to provide isoxazole esters 2a–j. Reduction of
2a–j with LAH or NaBH4 followed by bromination with CBr4/PPh3
provided isoxazole methylbromides (4a–j). Finally, these intermedi-
ate alkyl bromides were treated with 4-trifluoromethylbenzylpipera-
IC50 (lM)
Compd No
R
Huh7
Mahlavu
MCF7
zine to achieve target compounds 5a–j. For the synthesis of 5a
–H
–F
–Cl
–CH3
–CF3
–i-Pr
–OCF3
–SO2CH3
–OH
17.1
5.3
9.6
14.1
6.2
19.9
11.9
14
9.4
9.5
9.8
10.2
2.9
8.3
5b
compounds 5k–o, alkylation of phenolic hydroxyl of the intermedi-
ate 3i with appropriate alkyl bromides was first accomplished, and
5c
5d
26.8
>40
>40
>40
11.0
26.0
7.5
24
then used to produce desired final compounds 5k–o following the
reaction sequence shown in Scheme 2. All compounds were puri-
fied by automated flash chromatography and checked for purity by
TLC and UPLC before being tested in biological assays (purity was
97% based on the peak area percentage of UPLC analysis). The
structure of synthesized compounds was confirmed by means of 1H
NMR, 13C NMR and high-resolution mass spectrometry (HRMS).
5e
5f
5g
5h
5i
5j
5k
19.9
20.8
3.6
4.2
3.8
14.6
8.8
–OCH3
7.6
7.7
14.8
4.9
5l
2.0
1.3
1.8
0.3
3.5
2.7
5m
5n
5o
1.2
2.8
1.6
Biological evaluation
Cytotoxicity of isoxazole-piperazine hybrids 5a–o in liver and
breast cancer cells
0.3
3.7
1.2
The newly synthesized compounds (5a–o) were evaluated for their
antitumor activities against human liver (Huh7 and Mahlavu), and
breast (MCF7) carcinoma cell lines using the sulforhodamine B
(SRB) assay43. Data are expressed as IC50 values, defined as the
half maximal inhibitory concentration, and are shown in Table 1.
Among the isoxazole analogues, compound having a non-sub-
stituted phenyl attachment at 5-position of the isoxazole nucleus
(5a) showed the least potent cytotoxic activity for all the three
IC50 values were calculated from the cell growth inhibition percentages obtained
with five different concentrations in triplicates (R2 > 0.9).
substitution improved the cytotoxic activity, and 5b appeared to
be equally efficient in both liver cancer cells (IC50 ¼ 5.3 mM for
Huh7 and 6.2 mM for Mahlavu) but was less efficient in breast can-
cer cells (IC50 ¼ 11.9 mM for MCF7), while the chlorine substituted
cell lines (IC50 ¼ 14.1
– 19.9 mM). However, para fluorine derivative 5c was 4.3-fold less potent in Mahlavu as compared to

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1357
Figure 1. RT-CES analysis of liver cancer cell lines (Huh7 and Mahlavu) and breast cancer cell line (MCF7) treated with compounds 5o (dark red, red and orange for
10, 5, and 2.5 mM, respectively) and 5m (dark blue, blue and turquoise for 10, 5 and 2.5 mM, respectively) with DMSO control (0.1%) at different concentrations (black,
dark grey and grey for 10, 5 and 2.5 mM, respectively) for 120h. The experiment was conducted in triplicate and was normalized to DMSO controls.
5b (Table 1). Compounds 5d–f having p-alkyl substituents showed investigated in detail with PTEN adequate epithelial like Huh7 cells
and PTEN deficient mesenchymal like Mahlavu cells46.
only moderate cytotoxic activity against MCF7 breast cancer cells
with IC50 values of 9.4–9.8 mM but proved to be less potent in
liver cancer cells such as Huh7 (IC50 values of 19.9–24 mM) and
Mahlavu (IC50 >40 mM). Introducing polarity to p-phenyl (5g–j)
clearly improved the cytotoxic potency for both Huh7 (IC50 ¼ 3.6 –
Oxidative stress induced by compounds 5m and 5o
Reactive oxygen species (ROS), depending on their dose, can alter
cellular pathways and promote cell cycle arrest and apoptosis in liver
5.4 mM) and MCF7 cells (IC50 ¼ 2.9 – 10.2 mM) along with a slight
improvement in Mahlavu cells (IC50 ¼ 7.5 – 26 mM). Next, O-alky-
lated analogues of compound 5i were evaluated for their anti-pro-
liferative activity. As compared to the compounds 5g–j, cytotoxic
potency significantly increased by allyl (5l), prenyl (5m), 3,5-dime-
thylisoxazol-4-ylmethyl (5n) and 1,3-dimethylpyrazol-5-ylmethyl
(5o) substitutions with IC50 values between 0.3 and 3.7 mM,
depending on the cell line (Table 1). Based on the promising cyto-
toxic activities, 5m and 5o were selected for further biological
studies in order to understand the underlying mechanisms of their
anticancer activities.
cancer cells41. Therefore, induction of oxidative stress by compounds
5m and 5o was analyzed in HCC cells (Mahlavu and Huh7) at differ-
ent time periods (Figure 2). Selenium-deficient serum free medium
grown cells were used as experimental positive control for ROS gen-
eration41. It is shown that while PTEN deficient Mahlavu cells can tol-
erate selenium deficient serum free medium, PTEN adequate Huh7
cells are strongly affected41. For visualization of in situ presence of
oxidative stress, dichloro-dihydro fluorescein diacetate (DCFH-DA)
assay was performed on these cells, which were treated with 5m/5o
for 24 h, 48h and 72 h (Figure 2(A)). In the presence of oxidative
stress, DCFH-DA dye was oxidized to a green fluorescent molecule,
DCF. Fluorescent microscopy images represented that oxidative
stress was triggered by compounds 5m and 5o. While compounds
5m and 5o started to affect Mahlavu cells after 24 h, 5o and 5 m
treated Huh7 cells displayed a raise in ROS (þ) cells at 24h (Figure
2(B)), which were in parallel to cell death as determined by RT-CES
assay. We illustrated that 5o leads to an increase in ROS (þ) cells
with 40% and 13% for 48 h and 85% and 15% for 72 h in Mahlavu
and Huh7 cells, respectively, when compared to DMSO controls
(Figure 2(B)). In addition, compound 5m increased ROS (þ) cells
with 16% for 48 h and 25% for 72 h in Huh7, and it also caused a
rise in ROS (þ) cells with 32% in Mahlavu cells for 48 h (Figure 2(B)).
Metabolic stress induces cell death through ROS-induced apop-
tosis and Akt is one of the primary effectors in response to meta-
bolic stress47. Akt protein, which is hyperactivated in many
tumours, plays a major role in both cell survival and resistance to
tumor therapy48. While Akt pathway is hyperactive in Mahlavu cells
due to PTEN deletion, the pathway is normoactive in PTEN
adequate Huh7 cells49. Therefore, the poorly differentiated Mahlavu
cells are considered as aggressive HCC phenotype. In addition, Akt
signaling was illustrated to be involved in the oxidative stress
Real-time cellular response of cancer cells upon treatment with
compounds 5m and 5o
Time-dependent cytotoxic activities of 5m and 5o were scruti-
nized with real time cell electronic sensing (RT-CES)44 by monitor-
ing dynamic cell proliferation of Huh7, Mahlavu, and MCF7 cells.
RT-CES assay revealed that 5m/5o significantly reduced the
growth rate of cells as compared to DMSO control. This real-time
growth pattern confirmed that 5m and 5o displayed time and
dose-dependent growth inhibitory effects in all cells (Figure 1).
Cytotoxic effects of 5m and 5o on all three cell lines could be
observed after 24 h of compound treatment, and reached to its
highest values upon 72 h. As a result of cell cycle arrest or oxida-
tive stress, this real-time growth pattern proposed growth inhib-
ition in which the cells were neither proliferating nor dying, while
the DMSO treated cells sustained to increase their number until
they reached confluence45. In general, the RT-CES results were
consistent with values obtained with the SRB assay. In light of this
information, the molecular mechanisms underlying the cytotoxic
activities of these isoxazole-piperazine hybrids were further induced cellular pathways50. Based on the findings that the

1358
B. C¸ALIS¸KAN ET AL.
Figure 2. Oxidative stress induced by 5o and 5m liver cancer cells, which were treated with 5o (1 mM for Huh7 and 4 mM for Mahlavu) and 5m (1 mM for Huh7 and
Mahlavu) or DMSO control for 24 h, 48 h, and 72 h. Selenium deficient serum-free medium was used as a positive control of ROS induction. A. DCFH-DA staining of the cells
under oxidative stress with fluorescent microscope (20ꢁ) for 24 h, 48 h, and 72 h. (B) Cytometric analysis of oxidative stress induction. ROS positive cells are indicated with
orange and ROS negative cells are shown in blue. (C) AKT and phospho-AKT in Mahlavu and Huh7 cells treated with 5o and 5m for 72h. Actin was used for equal loading.
compounds 5m and 5o caused ROS generation in HCC cells, Akt chromatin condensation, nuclear fragmentation and apoptotic
and phospho(p)-Akt protein levels comparatively analyzed in PTEN bodies were detected in treated cells for different time periods.
The activation of apoptotic pathways through 5m and 5o
treatments were further confirmed with apoptosis-associated
PARP protein levels. There was a significant decrease in total PARP
protein in 5m/5o treated Mahlavu and Huh7 cells while an
increase in PARP cleavage in the 5m treated Huh7 cells was iden-
tified at 72 h. These results supported the increased cytotoxic
effects of the compounds on liver cancer cells (Figure 3(B) and
3(C)). Our data indicates that the 5m/5o-induced ROS leading to
the cell death characterized with apoptosis. We then analyzed the
proteins involved in apoptosis and cell cycle with the aim of fur-
ther characterization of cell death mechanism.
deficient Mahlavu and PTEN adequate Huh7 cells (Figure 2(C)).
Significant decrease in the levels of Akt and p-Akt proteins was
observed in 5m/5o treated Mahlavu cells as compared to DMSO
control. However, compounds treated Huh7 cells did not result in
a significant decrease in Akt protein levels. Although Mahlavu cells
have hyperactive Akt signal pathway due to the deletion of PTEN
gene, which leads to more drug resistant phenotype, more than
80% of Mahlavu cells were ROS positive upon 5o treatment
(Figure 2(B)). Under normal conditions Mahlavu cells exhibit cryp-
tic resistance to intrinsic oxidative stress-induced apoptosis due to
selenium deficiency. However, treatment with 5m and 5o clearly
induces oxidative stress along with cell death that the latter being
more effective. Hence, we analyzed the nature of cell death
induced with these compounds in liver cancer cells.
Induction of cell cycle arrest and analysis of cellular pathways
targeted by 5m and 5o
Initially, cell cycle arrest was analysed by flow cytometry analysis
using propidium iodide (PI) staining of DNA. Huh7 cells treated
5m and 5o induce apoptosis
Compounds 5m and 5o induced apoptotic morphological changes with 5m and 5o showed an increase in entry to G2/M and G1
as observed by nuclear staining with Hoechst in Huh7 and phases in 48 h (Figure 4(C)) and 72 h (Figure 4(D)), respectively.
Mahlavu cells. Following treatment with 5m/5o for 24 h, 48 h, and While no arrest was observed in Mahlavu cells treated with 5m
72 h, cells were analyzed under fluorescent microscopy. As shown and 5o for 48 h (Figure 4(A)), the cells treated with 5m and 5o
in Figures 3(A) and S1, typical morphological changes such as represented a higher cell population in S and G2/M phases for

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1359
Figure 3. Characterization of cell death using fluorescent microscopy and Western blotting. (A) Hoechst staining of 5o and 5m treated Mahlavu and Huh7 cells with
apoptotic nuclei at 24 h and 72 h. (B) PARP in Mahlavu and Huh7 cells treated with 5o and 5m for 72 h. Actin was used for equal loading. (C) The bar graphs repre-
senting relative band intensities of PARP and cleaved-PARP, which were normalized with their actin loading controls.
Figure 4. Detection of cell cycle arrest. Cell cycle analysis of Mahlavu for (A) 48 h (B) 72 h and Huh7 for (C) 48 h and (D) 72 h after treatment with compounds 5o and
5m, and DMSO controls following 48 h and 72 h of treatment. Orange, blue, yellow and black show GO/S1, S, G2/M, and Sub-G1, respectively. (E) Rb, p53, phospho-
Rb, and phospho-p53 in Mahlavu and Huh7 cells with 5o and 5m for 72h. Actin was used for equal loading.
72 h (Figure 4(B)). Quantitative results of cell cycle analysis were Western blot analyses. Proteins p53, p21, Rb are inhibitory regula-
also revealed in Table S1.
tors of the cell cycle, and are effectors of responses to cellular
stress leading to apoptosis and cell cycle arrest. Since 5m and 5o
induce cellular stress-associated cell death, we examined the levels
of these proteins upon compound treatment. Compound 5o
Based on the finding that 5m/5o caused ROS accumulation,
cell cycle arrest and apoptosis, several targets involved in these
pathways at the protein level were further investigated by

1360
B. C¸ALIS¸KAN ET AL.
treated Mahlavu cells exhibited significant alteration in protein
levels of Rb, phosphorylated form of Rb (Ser807/811), p53 and
phosphorylated form of p53 (Ser15) while p21 protein keeps its
expression practically unchanged (Figure 4(E)). The phosphory-
lated from of p53 tumor-suppressor protein has critical roles for
cell cycle arrest in response to DNA injury. It is a known fact that
reactive oxygen species damage several complex biomolecues in
the cell incuding DNA. Upon DNA damage, p53 is stabilized by
phosphorylation and activates the expression of target genes.
Primary among these is p2151, which provides a direct link
between p53 and pRb for cell cycle arrest. The p21 retains pRb in
Funding
This work was supported by the Scientific and Technological
Research Council of Turkey [TUBITAK Grant No: 214S062].
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