Design and synthesis of phenylpiperazine derivatives as potent anticancer agents for prostate cancer

Article abstract of DOI:10.1111/cbdd.13575

Novel thiourea (5a, 5b) and thiazolidinone derivatives (6a, 6b) were synthesized by hybridizing molecules starting from the compound 6-(4-phenylpiperazin-1-yl)pyridin-3-amine (4) which is known to show anticancer activity. The synthesis of the leading compound was carried out by using 1-(5-nitropyridin-2-yl)-4-phenylpiperazine (3) which was obtained by a novel method of the reaction of 2-chloro-5-nitropyridine (1) and N-phenylpiperazine (2). The structures of the compounds were confirmed using FTIR, ¹H NMR, ¹³C NMR, HRMS spectroscopic methods and elemental analysis. The organic molecules were tested for their anticancer activities against prostate cancer (PC) cell lines: DU 145, PC-3 and LNCaP. As the compound 5a exerted the highest cytotoxic activity, IC₅₀ concentrations of compound 5a were further investigated in terms of morphology, colony-forming ability, RNA expression, fragmented DNA and cell cycle distributions of PC cell lines. Overall data revealed that compound 5a treatment induces apoptosis and DNA fragmentation in PC cell lines and inhibits cell cycle progression resulting in the accumulation of cells in either the G1 or the S phases.

Full text of DOI:10.1111/cbdd.13575

DR AYŞEGÜL DOĞAN (Orcid ID : 0000-0003-4160-2270)
Article type : Research Article
Design and Synthesis of Phenylpiperazine Derivatives as Potent Anticancer Agents for
Prostate Cancer
Serpil Demirci1*, Taha Bartu Hayal2, Binnur Kıratlı2, Hatice Burcu Şişli2, Selami Demirci3, Fikrettin
Sahin2, Ayşegül Doğan2*
1Giresun University, Vocational High School of Health Services, Department of Medical Services and
Techniques, Giresun, Turkey
2Yeditepe University, Department of Genetics and Bioengineering, Faculty of Engineering, Istanbul,
Turkey
3National Heart, Lung, and Blood Institute (NHLBI), Sickle Cell Branch, Cellular and Molecular
Therapeutics National Institutes of Health (NIH)
Running title: Phenylpiperazine Derivatives as Anticancer Agents
Acknowledgments
The support provided by Scientific and Technological Research Council of Turkey (TUBITAK,
Project no: 116Z932) is gratefully acknowledged.
Authors do not have any conflict of interest
ABSTRACT
Novel thiourea (5a, 5b) and thiazolidinone derivatives (6a, 6b) were synthesized by hybridizing
molecules starting from the compound 6-(4-phenyl-piperazin-1-yl)pyridine-3-ylamine (4) which is
known to show anticancer activity. The synthesis of the leading compound was carried out by using 1-
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13575
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(5-nitropyridin-2-yl)-4-phenylpiperazine (3) which was obtained by a novel method of the reaction of
2-chloro-5-nitropyridine (1) and N-phenylpiperazine (2). The structures of the compounds were
1
confirmed using FT IR, H NMR, 13C NMR, HRMS spectroscopic methods and elemental analysis.
The organic molecules were tested for their anticancer activities against prostate cancer cell lines; DU
145, PC-3 and LNCaP. As the compound 5a exerted the highest cytotoxic activity, IC50
concentrations of 5a were further investigated in terms of morphology, colony forming ability, RNA
expression, fragmented DNA and cell cycle distributions of prostate cancer cell lines. Overall data
revealed that 5a treatment induces apoptosis and DNA fragmentation in prostate cancer cell lines and
inhibits cell cycle progression resulting in the accumulation of cells in either the G1 or S phases.
Keywords: Antitumor activity, apoptosis, cell cycle, DNA fragmentation, thiourea, thiazolidinone
INTRODUCTION
Prostate cancer (PC) is the leading noncutaneous cancer type among men in US with an expected
174,650 new cases and 31,620 deaths in 2019. PC is the second lethal cancer with a 10% mortality
ratio after lung cancer (24%) (Siegel, Miller, & Jemal, 2019). As a multistep and complicated cancer
type, PC progression is regulated by androgen activity at the cellular level which is the basis of
hormone deprivation therapy at early stages when PC cells are still responsive to androgen
suppression. (Gregory et al., 2001) While prostatectomy followed by radiation therapy is the standard
therapy in clinics for early diagnosed cases (Sriprasad, Feneley, & Thompson, 2009), patients with
advanced stage disease remain incurable due to resistance to chemotherapy, acquired androgen-
refractory phenotype, and inefficient radiation therapies (Jarvis, Nelius, Martinez Marin, Sennoune,
& Filleur, 2018; Oudard, 2013; Seruga, Ocana, & Tannock, 2011). Treatment at late stage of PC
could only be possible by combining surgical removal, radiotherapy and chemotherapy options based
on clinical priority to monitor adverse outcomes (Nicolae, Venugopal, & Ravi, 2016; Teply & Hauke,
2016). Although heterogenous nature of PC restricts the treatment, chemotherapy is generally
considered as a prior treatment regimen with both adjuvant and neo-adjuvant options (Beer &
Raghavan, 2000). Combination of different chemotherapeutic agents is a new therapeutic modality
but still remains insufficient for improved life expectancy and quality, a goal that is demanding for
current clinical trials (Demirci et al., 2017). Therefore, development of novel chemotherapeutics
displaying targeted cytotoxicity to PC cells is still focus of international research.
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Substantial advances in the technology and synthetic chemistry coupled with traditional
methodology is expected to enable identification of various novel drug molecules in near future. In
this manner, combinatorial chemistry based on the use of building blocks, scaffolds and libraries of
standard chemical reactions, in solid or solution phases by simple addition and incubation steps, has
an important role in the synthesis of small organic compounds. Of those organic compounds, thiourea
derivatives, used as precursors for the synthesis of different classes of biologically active acyclic and
heterocyclic compounds, have recently gained importance thanks to their broad biological activities
such as anti-inflammatory, antimicrobial, antimalarial and pesticidal functions (Ghorab et al., 2016).
Additional biochemical and pharmacological studies have confirmed that these molecules are also
effective against various cancer cell lines including breast cancer (Lv, Li, Sun, Zhou, & Zhu, 2010),
nasopharyngeal carcinoma (Peng et al., 2011), lung and liver cancer cell lines (Hu et al., 2017)
through mainly inhibiting receptor tyrosine kinases (RTKs), protein tyrosine kinases (PTKs), and
DNA topoisomerases (Kumar & Chimni, 2015).
Thiazolidinone derivatives, another group of biologically active organic compounds which have
different substituents around the core nucleus, are also considered as potential anticancer agents. Their
strong affinity for various biological targets including JNK-stimulating phosphatase-1 (JSP-1)
(Cutshall, O’Day, & Prezhdo, 2005), non-membrane protein tyrosine phosphatase (SHP-2)
(Geronikaki et al., 2008), tumour necrosis factor-α (Carter et al., 2001), and antiapoptotic biocomplex
Bcl-xL/BH3 (Degterev et al., 2001) might be potential reasons for the anticancer activity. Anti-cancer
activities for various thiazolidinone compounds against breast cancer (Wu et al., 2014), glioblastoma
(Avdieiev et al., 2014) and drug resistant lung cancer (Zhou et al., 2008) have been reported in the
literature.
Here we synthesized and characterized novel thiourea and thiazolidinone derivatives as anti-cancer
agents. The anticancer activities of the compounds were evaluated against PC cell lines. Cancer cells
treated with the highest activity displaying compound, 5a, were examined in terms of cell
morphology, RNA expression profile, DNA fragmentation and cell cycle distribution to explore
possible action of mechanism of the organic molecule.
EXPERIMENTAL SECTION
1. Chemistry
1.1. General Methods
All chemicals were purchased from FlukaChemie AG Buchs (Switzerland). Melting points of the
synthesized compounds were determined in open capillaries on a Buchi B-540 melting point
apparatus and are uncorrected. Reactions were monitored by thin-layer chromatography (TLC) on
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0.25 mm silicagel plates (GF254) and visualized under UV light. FT IR spectra were recorded using a
Mattson 1000 FT-IR spectrometer. 1H NMR and 13C NMR spectra were recorded on Bruker Avene II
400 MHz NMR spectrometer (chemical shift in ppm downfield from TMS as an internal reference).
The elemental analysis was performed on a Leco CHNS-932 (Michigan, USA) elemental analyzer.
The mass spectra were obtained at a high-resolution mass spectra (HRMS) TOF (1200/6210, Agilent)
by electrospray ionization.
1.2. 1-(5-Nitropyridin-2-yl)-4-phenylpiperazine (3)
2-Chloro-4-nitropyridine (1) (0.63 g, 4 mmol) was added dropwise to N-phenylpiperazine (2) (1.62 g,
10 mmol) at 0-5 °C over 20 min. The temperature was gradually raised to 135 °C and refluxed for 4h.
The reaction was cooled at room temperature and water was added. The enthusiast was filtered in the
fridge overnight. Crystallization with EtOAc-hexane gave a pale brown product. Yield, 60% (1.68 g);
1
mp, 155-156 °C; H-NMR (400 MHz, CDCl3): δ 9.06 (d, 1H, J: 2.7 Hz), 8.23 (dd, 1H, J: 9.5, 2.7
Hz), 7.32-7.27 (m, 2H), 6.96-6.90 (m, 3H), 6.61 (d, 1H, J: 9.5 Hz), 3.94 (d, 4H, J: 5.14 Hz), 3.42 (d,
4H, J: 5.14 Hz); APT (13C-NMR) (100 MHz, DMSO-d6): δ 165.30,155.80, 151.29, 139.59, 138.08,
134.23, 124.46, 120.86, 110.92, 53.44, 49.49; FT IR (cm–1): 3072 (ar-CH), 1593-1290 (NO2);
Elemental analysis for C15H16N4O2 calculated; C, 63.37; H, 5.67; N, 19.71; found: C, 63.28; H, 5.85;
N, 19.75; HRMS (APCI): m/z calculated: C15H16N4O2 (M+ + H): 285.32; found: 285.13.
1.3. 6-(4-Phenylpiperazin-1-yl)pyridin-3-amine (4)
Compound (3) (2.85 g, 10 mmol) was refluxed by the addition of Pd/C (0.53 g, 5 mmol) in a two
necked flask and stirring in n-butanol in an oil bath. After the reflux started, 60% hydrazine hydrate
(2.5 g, 4.04 ml, 50 mmol) was added dropwise over 20 min. It was refluxed for 15 h. At the end of the
time the hot reaction mixture was filtered through the celite. The solvent was removed by evaporation.
Crystallization with EtOAc-hexane gave a dark red product. Yield, 95% (2.41 g); mp,136-137 °C; 1H-
NMR (400 MHz, DMSO-d6): δ 7.64 (d, 1H, J: 2.7 Hz), 7.23 (t, 2H, J: 7.8 Hz), 7.00-6.94 (m, 3H),
6.80 (t, 1H, J: 7.21 Hz), 6.70 (d, 1H, J: 8.76 Hz), 4.59 (s, 2H, NH2), 3.36-3.49 (m, 4H), 3.26-3.16 (m,
4H); APT (13C-NMR) (100 MHz, DMSO-d6): δ 151.34,134.92, 134.72, 129.16, 126.48, 120.00,
116.39, 116.37, 108.86, 49.29, 46.91; FT IR (cm–1): 3309 and 3201 (NH2), 3008 (ar-CH); Elemental
analysis for C17H16O2 calculated: C, 70.84; H, 7.13; N, 22.03, found: C, 70.49; H, 7.27; N, 21.99;
HRMS (APCI): m/z calculated: C15H18N4 (M+ + H): 255.34; found: 255.15.
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1.4. General Method for the Synthesis of Compounds 5a, 5b
Phenylisothiocyanate (for 5a) (10 mmol), benzylisothiocyanate (for 5b) (10 mmol) were added to the
solution of compound 4 (10 mmol) in a dried DCM drop-wise and the mixture was refluxed for 8-18h.
The solid precipitate was collected by filtration and recrystallized from suitable solvent to afford the
desired compound.
1-Phenyl-3-(6-(4-phenylpiperazin-1-yl)pyridin-3-yl)thiourea (5a)
1
Crystallized EtOAc; dark pink solid product. Yield 51% (1.95 g); mp, 144-145 °C; H-NMR (400
MHz, DMSO-d6): δ 9.72 (s, 1H, NH), 9.49 (s, 1H, NH), 8.09 (d,1H, J: 2.5 Hz), 7.62 (dd, 1H, J: 8.9,
2.5 Hz),7.48 (d, 2H, J: 8.1 Hz), 7.33 (t, 2H, J: 7.8 Hz), 7.25 (t, 2H, J: 7.9 Hz), 7.13 (t, 1H, J: 7.4 Hz),
7.01 (d, 2H, J: 8.2 Hz), 6.90 (d, 1H, J: 9.0 Hz), 6.81 (t, 1H, J: 7.2 Hz), 3.65-3.62 (m, 4H), 3.26-3.23
(m, 4H); APT (13C-NMR) (100 MHz, DMSO-d6): δ 181.25, 157.29, 151.71, 145.05, 140.10, 136.34,
129.68, 129.16, 127.36, 125.15, 124.47, 119.89, 116,45, 107.29, 48.89, 45.63; FT IR (KBr,cm–1):
3199 (NH), 3023 (ar-CH), 1227 (C=S); Elemental analysis for C22H23N5S calculated: C, 67.84; H,
5.95; N, 17.98; S: 8.23, found: C, 67.89; H, 5.91; N, 17.89; S, 7.98; HRMS (APCI): m/z calculated:
C22H23N5S (M+ + H): 390.52; found: 390.42.
1-Benzyl-3-(6-(4-phenylpiperazin-1-yl)pyridin-3-yl)thiourea (5b)
1
Crystallized EtOAc; dark green solid product. Yield 85% (3.43 g); mp, 139-140 °C; H-NMR (400
MHz, DMSO-d6): δ 9.35 (s,1H, NH), 8.06 (bs, 2H, arH+NH), 7.55 (d, 1H, J: 8.8 Hz), 7.34-7.32 (m,
4H), 7.24 (t, 3H, J: 8 Hz), 7.0 (d, 2H, J: 8.1 Hz), 6.90 (d, 1H, J: 9.0 Hz), 6.81 (t, 1H, J: 7.2 Hz), 4.72
(d, 2H, J: 4.5 Hz), 3.63 (d, 4H, J: 4.0 Hz), 3.23 (d, 4H, J: 4.3 Hz); APT (13C-NMR)(100 MHz,
DMSO-d6): δ 182.41, 157.16, 151.47, 144.93, 139.69, 136.16, 129.45, 128.69, 127.80, 127.25,
126.51, 119.64, 116.21, 107.35, 48.66, 47.80, 45.36; FT IR (cm–1): 3258, 3181 (2NH), 3028 (ar-CH),
1226 (C=S); Elemental analysis for C23H25N5S calculated: C, 68.46; H, 6.24; N, 17.35; S, 7.95,
found: C: 68.43, H: 6.27, N: 17.67, S: 7.62; HRMS (APCI): m/z calculated: C23H25N5S (M+ + H):
404.55; found: 404.19.
1.5. General Method for the Synthesis of Compounds 6a, 6b
A mixture of compound 5a (for 6a), 5b (for 6b) (10 mmol) and ethyl bromoacetate (10 mmol) in
absolute ethanol allowed to reflux in the presence of dried sodium acetate (50 mmol) for 15-18 h. The
reaction mixture was then cooled to room temperature. After the solvent was removed under reduced
pressure. Then water was added to the solution that led to solid structure formation. This crude
product was recrystallized from a suitable solvent to afford the desired compound.
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3-Phenyl-2-((6-(4-phenylpiperazin-1-yl)pyridin-3-yl)imino)thiazolidin-5-one (6a)
Crystallized EtOAc; light brown solid product. Yield %53 (2.27 g); mp, 163 °C; 1H-NMR (400 MHz,
CDCl3): δ 8.22 (d, 1H, J: 2.5 Hz) 7.52 (dd, 1H, J: 9.0, 2.6 Hz), 7.33-7.27 (m, 4H), 7.12 (t, 1H, J: 7.4
Hz),6.97 (d, 2H, J: 7.4 Hz), 6.92-6.87 (m, 3H), 6.78 (d, 1H), 3.98 (s,2H), 3.77 (t, 4H, J: 5.2 Hz), 3.29
(t, 4H, J: 5.2 Hz); APT (13C-NMR) (100 MHz, DMSO-d6): δ 172.25, 158.59, 156.50, 151.43,
148.58, 147.39, 137.98,129.71, 129.45, 124.67, 122.39, 121.14, 119.65, 116.23, 107.35, 48.64, 45.03,
33.20; FT IR (cm–1): 3023 (ar-CH), 1729 (C=O); Elemental analysis for C24H23N5OS calculated: C,
67.11; H: 5.40; N, 16.30; S, 7.46, found: C, 67.78; H, 5.36; N, 15.96; S, 7.20; HRMS (APCI): m/z
calculated C24H23N5OS (M+ + H): 430.54; found: 430.15.
3-Benzyl-2-((6-(4-phenylpiperazin-1-yl)pyridin-3-yl)imino)thiazolidin-5-one (6b)
1
Crystallized EtOAc; light yellow solid product. Yield 93% (4.43 g); mp, 140-141 °C; H-NMR (400
MHz, DMSO-d6): δ 7.83 (s, 1H),7.36-7.32 (m, 4H), 7.29-7.23 (m, 4H), 6.99 (d, 2H, J: 8.0 Hz), 6.91
(d, 1H, J:8.8 Hz), 6.80 (t, 1H, J: 6.8 Hz), 4.92 (bs, 2H), 4.12 (s, 2H), 3.60 (bs, 4H), 3.23 (bs, 4H);
APT (13C-NMR)(100 MHz, DMSO-d6): δ 172,37, 156.83, 155.73, 151.47, 140.31, 136.66, 135.51,
131.37, 129.44, 128.90, 128.21, 127.91, 119.61, 116.19,107.92, 48.63, 45.92, 45.49, 33.07; FT IR
(cm–1): 3014 (ar-CH), 1721 (C=O), 1619 (C=N); Elemental analysis for C25H25N5OS calculated: C,
67.70; H, 5.68; N, 15.79; S, 7.23 found: C, 67.64; H, 5.84; N, 15.62; S, 7.31; HRMS (APCI): m/z
C25H25N5OS calculated (M+ + H): 444.57; found: 444.19.
2.
Anticancer Activity
2.1. Cell lines and proliferation assay
Human prostate cancer cell lines, PC-3 (CRL-1435), DU 145 (HTB-81), and LNCaP (CRL-1740),
were purchased from ATCC (Rockville, Maryland, USA). All cell lines were incubated in Dulbecco`s
Modified Eagle Media (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS,
Invitrogen) and 1% penicillin-streptomycin-amphotericin (PSA, Invitrogen) in a humidified incubator
at 37°C and 5% CO2 conditions. Anticancer activity of newly synthesized hybrid molecules against
prostate cancer cell lines were assessed with 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) assay and NCI60 response parameters was used at the end of 24h incubation period.
Results are given as % cell inhibition. Optic density for the cells treated only with solvent (DMSO)
was accepted as 100%. Reponse parameters, IC50 (50% cell viability inhibition), GI50 (50% growth
inhibition), TGI (total growth inhibition), and LC50 (50% cell death) were anayzed based on National
Institutes of Health (NIH) definitions using logaritmic fonctions (González-Vallinas et al., 2013).
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2.2. Colony-forming Unit (CFU) Assay
CFU assay was performed to determine whether compound 5a affects the colony-forming ability of
cells as described previously (Demirci et al., 2018). Briefly, PC-3, DU145 and LNCaP cells were
seeded at a cell density of 100 cell per well of 6-well plates in the presence of respective IC50
concentrations of 5a for each cell line. Media were replaced with growth medium after 24h compound
5a treatment. Cells were incubated for an additional 13 days at 37 °C in a humidified incubator.
Colonies were stained with crystal violet, counted and imaged with Zeiss PrimoVert light microscope
with an AxioCam ERc5 s camera and Zen 2011 software (Carl Zeiss Microscopy, LLC, Thornwood,
NY, USA).
2.3. Morphological Analyses: light microscope
Cell morphology was determined by light microscope analysis after 5a exposure for 24 h. Briefly, 105
cells/well were seeded onto 12 well plates and exposured to IC50 concentrations of 5a. Apoptotic
morphology was determined by images taken after 24 h using Zeiss PrimoVert light microscope with
an AxioCam ERc5 s camera and Zen 2011 software (Carl Zeiss Microscopy, LLC, Thornwood, NY,
USA).
2.4. Quantitative RT-PCR (q-PCR) analysis
q-PCR experiments were performed according to the previously described protocol (Doğan, Demirci,
Apdik, Apdik, & Şahin, 2017). Primers for p21, MDM2, Caspase-3, Caspase-7, AKT, PI3K and
GAPDH were designed using Primer-BLAST software from the National Center for Biotechnology
(Bethesda,MD, USA) and synthesized by Macrogen (Seoul, Korea). GAPDH was used as
housekeeping gene. Total RNA from 5a treated PC-3, DU145 and LNCaP cells were isolated using
RNAeasy plus mini kit (Qiagen, Hilden, Germany) and cDNA was synthesized using High Fidelity
cDNA synthesis kit (Roche, USA). q-PCR analyses performed by using SYBR method and CFX96
RT-PCR system (Bio-Rad, Hercules, CA).
2.5. DNA Fragmentation
DNA fragmentation, as an indicator of DNA damage and apoptosis, was performed according to the
previously described protocol (Ji et al., 2012). In short, cells were seeded onto 6 well plates (Corning
Plasticware, Corning, NY) at a cell density of 3×105 cells/well. Cells were treated with compound 5a
for 24h. DNA was isolated from 5a treated cells by using DNA isolation kit (Invitrogen, USA). DNA
samples were loaded to 1% agarose gel in Tris-borate buffer, stained with ethidium bromide (EtBr,
Sigma, USA) and images were obtained using the luminometer system (Bio-Rad).
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2.6.
Cell Cycle Analysis
The cell cycle distribution of PC-3, DU145 and LNCaP cells after 5a treatment was measured using
propidium iodide (PI) staining. Cells were fixed with 70% cold ethanol and incubated with 1 ml PI
staining solution containing 40 µg/ml PI, 10 µg/ml RNase A, and 0.1% NP-40 for 30 min at 37 °C.
Cell cycle analyses were performed using a Becton Dickinson FACSCalibur flow cytometry system
(Becton Dickinson, San Jose, CA, USA).
2.7. ADMET Analysis
In silico ADME study, which is one of the computational calculations, was applied for the synthesized
compounds. In this study, we calculated molecular volume (MV), molecular weight (MW), logarithm
of partition coefficient (miLogP), number of hydrogen bond acceptors (n-ON), number of hydrogen
bonds donors (n-OHNH), topological polar surface area (TPSA), number of rotatable bonds (n-
ROTB) and Lipinski's rule of five using Molinspiration online property calculation toolkit
(Molinspiration Cheminformatics, Bratislava, Slovak Republic, http://www.molinspiration.com/cgi-
bin/properties). Absorption (% ABS) was calculated by: % ABS = 109
et al., 2002).
̶
(0.345xTPSA) (Y. H. Zhao
2.8. Statistical Analysis. The data were statistically analyzed using one-way analysis of variance.
The values of p ≤ 0.05 were considered statistically significant.
RESULTS AND DISCUSSION
Chemistry
The synthetic strategies adapted to obtain the target compounds are depicted in Scheme 1. To
obtain 6-(4-phenylpiperazin-1-yl)pyridin-3-amine (4), which is also a commercially available organic
molecule, previously published protocol was followed (Walsh, Sneddon, & Moody, 2013). However,
we could not be able to synthesize the 1-(5-Nitropyridin-2-yl)-4-phenylpiperazine (3) molecule using
the same methodoloy. Unlike the proposed protocol, we presented an optimal synthesis method for
the organic molecule, which was performed in a solvent-free environment in an oil bath.
Spectroscopic data for the compound 3 have been shown for the first time (see Suplementary file 1).
Synthesis of thiourea derivatives (5a, 5b) were accomplished by nucleophilic attack of the amine
compound (4) to the alkylisothiocyanates (Scheme 1). In the FT IR spectra, the signals originated
from –C=S function of compounds 5a and 5b appeared between 1226-1227 cm-1. In the 13C NMR
spectra of these compounds (5a, 5b), –C=S function resonated between 182.41-181.25 ppm (see
Suplementary file 1). Further evidence for the formation of carbothioamides was the presence of four
1
–NH- signals at 9.72– 8.06 ppm in the H NMR spectra. The elemental analysis data of carbon,
hydrogen and nitrogen were formed to be satisfactory and within the permissible limit error.
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Reaction and conditions for the synthesis of compounds 3-6. i: 2-Chloro-4-nitropyridine in
acetonitrile, 4 h, ruflux; ii: H2NNH2.H2O in But-OH, Pd/C, 15 h, ruflux; iii: Phenylisothiocyanate (for
5a) or benzylisothiocyanate (for 5b) in DCM, 8 h, ruflux; iv: ethyl bromoacetate, dry CH3COONa in
absolute ethanol, 13-15 h, ruflux.
Synthesis of thiazolidinone derivatives (6a, 6b) were carried out starting from intermediates of
thiourea (5a, 5b). Compounds (6a, 6b) were characterized by the presence of additional strong band
at 1729-1721 cm-1 in the FT IR spectrum corresponding to additional carbonyl function which arose
from ring-closure. This was considered as a confirmation of thiazolidinone nucleus formation.
1
Moreover, H NMR, 13C NMR spectrums, the elemental analyses and mass spectral data were
compatible with the suggested structure (see Suplementary file 1).
ADMET Prediction
All synthesized compounds exhibited a good % ABS (91.05-94.02%) based on all parameters (Table
1). It was monitored that none of the com-pound violated Lipinski's rule of five (Table 1). It can be
suggested that these two compounds may be developed as good drug candidates. A molecule probably
to be evolved as an orally active drug candidate should exhibit no more than one violation of the
following four criteria: logP (octanole
̶ water partition coefficient) ≤5, molecular weight ≤500,
number of hydrogen bond acceptors ≤10 and number ofhydrogen bond donors ≤5 (Ertl, Rohde, &
Selzer, 2000). Since the obtained compounds (5a and 6a) followed this rule for orally active drug and
therefore can be promoted as oral drug candidates.
Compound 5a contains thiosemicarbazone group and compound 6a is a thiazolidinone derivative.
Thiosemicarbazone derivatives has been reported for their anticancer activities in the literature
(Altıntop et al., 2016). Similarly, thiazolidinone derivatives such as Ciglitazone, Rosiglitazone,
Pioglitazone demonstrated anti-cancer activity (Chinthala et al., 2013). The study has demonstrated
the synthesis of some novel 1-(5-nitropyridin-2-yl)-4-phenylpiperazine (3) derivatives with anticancer
activities by incorporating several pharmacophore groups such as structures of phenylisothiocyanate,
benzisothiocyanate and thiazolidinone rings.
Anticancer activity
5a Reduces the Cell Proliferation Rate And CFU Ability of PC Cells.
After characterization of the molecules, possible GI50, TGI, LC50 and IC50 levels were determined
against PC cell lines. Table 2 shows that the thiourea and thiazolidinone derivatives displayed growth
inhibitory effects against PC cell lines comparable to 5-fluorouracil which is a validated anticancer
drug against several solid tumors (Longley, Harkin, & Johnston, 2003).
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While GI50 levels of all organic molecules remained at low concentrations (3.34-64.49 µM for DU
145, 4.62-74.66 µM for PC-3 and 4.71-279.14 µM for LNCaP), LC50 and TGI concentrations were
higher than 500 µM (the highest concentration tested) for most of the compunds. IC50 levels, used as a
clinically relevant concentration to determine the potency of a given drug, were mostly over the test
range for thiazolidnone derivatives while concentrations for thiourea compounds were comparable to
5-FU (102.95-175.35 µM for DU 145, 14.90-160.41µM for PC-3 and 301.13->500 µM for LNCaP
(Table 2). The thiourea derivatives (5a and 5b) exhibited robust inhibitory activity against DU 145
and PC-3 but they remained relatively weak against LNCaP. LnCaP is an androgen responsive cell
line, but the canonical androgen pathway does not work for PC-3 and DU145 cells (Marchiani et al.,
2010), indicating that thiourea derivatives might target a different proliferation pathway other than the
androgen pathway. As 5a was more effective against PC cell lines compared to compound 5b, we
selected 5a for further action of mechanism studies.
To further evaluate the viability effect of 5a, PC cells were treated with respective IC50 concentrations
of the organic molecule for 24 h and CFU abilities were investigated after a 14-days incubation
period. CFU capacity of PC-3 and DU 145 cells were not significantly affected by 5a treatment, while
colonies become irregular and smaller in size, showing anti-proliferative effect of the compound
rather than affecting colony formation capacity (Figure 1).
On the other hand, CFUs in LNCaP cells exposed to 5a significantly reduced, which is consistent with
the proliferation assay showing a different mechanism of inhibitory action on androgen-responsive
and unresponsive cells.
5a Treatment Diminishes Cancer Cell Growth by Inducing Apoptosis and DNA Damage.
PC cell lines were tested for RNA expression profile and morphology to see whether the 5a treatment
triggers apoptotic pathway. Incubation with 5a lead to apoptotic cell formation in PC-3 and DU 145
cells (Figure 2A). In line with this, live cell imaging confirmed high prevalence of apoptotic cancer
cells treated with the compound during the culture period (Supplementary video 1, 2, and 3). Having
this finding, to propose a possible mechanism, q-PCR assay was conducted to analyze apoptosis-
related RNA levels in PC cells. The results revealed that caspase-3 was induced in all three cells,
while there was a slight change in caspase-7, proposing that apoptosis might be executed through
caspase-3 dependent pathway (Figure 2B). Apoptosis induction using various approaches is one of
the leading ways of current clinical practice to cope with cancers. In this manner, cancer progression
or metastasis are controlled either introducing apoptotic intermediate enhancing factors such as
reactive oxygen species (Redza-Dutordoir & Averill-Bates, 2016) or decreasing the levels of anti-
apoptotic proteins such as Bcl-2, Bcl-XL, and Bcl-w (Sarosiek & Letai, 2016). Thus, apoptosis
induction in all cells treated with 5a would be promising, if it is proven safe and effective in animal
models.
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PI3K/AKT pathway manages several cell responses including cell cycle progression, survival,
proliferation and activates protein biosynthesis (Xia & Xu, 2015). It has been stated that almost every
major components of the pathway is mutated in a large variety of solid tumors resulting in permanent
activation of AKT (Carnero & Paramio, 2014). As expected AKT levels were lower in 5a treated PC
cells that might be the possible explanation for survival inhibitory activity (Figure 2B). In normal
cells, PTEN is upstream element of the pathway, inhibiting the activation of AKT (Carnero, Blanco-
Aparicio, Renner, Link, & Leal, 2008). Given the fact that while DU 145 cells express functional
PTEN, PC-3 cells are PTEN-null and LNCaP cells do not have active PTEN (Jäntti et al., 2018),
downregulation of AKT would have been PTEN-independent way. Correspondingly, inherited and
spontaneous tumors with PTEN deficiency or loss has been presented (Cantley & Neel, 1999),
suggesting that AKT activation can also be in the absence of PTEN or in the presence of an PTEN
analog. Likewise, integrin-linked kinase (ILK) 1-associated AKT phosphorylation was reported in
PC-3 and LNCaP cells (Persad et al., 2000). Although q-PCR results propose that apoptotic pathway
is triggered, and the survival pathway is inhibited by 5a treatment, additional protein studies are
needed to validate this observation and explore the exact inhibitory mechanism.
To evaluate whether the apoptotic activity is related with DNA damaging activity of the organic
molecule, we conducted DNA fragmentation assay for 5a treated PC cells (Figure 3). The results
confirmed that although control cells also have DNA fragmentation, 5a treatment augmented DNA
breaks. To address if this is an indirect effect or direct interaction of the compound with DNA,
additional interaction experiments should be performed.
5a Inhibits Cell Cycle Progress.
5a exposed cells were subjected to flow cytometry analysis to evaluate possible cell cycle
dysregulation. Figure 4 demonstrates that there is an increase of cells in the S phase and a
corresponding decrease of cells in the G2/M phases in 5a treated PC-3 and DU 145 cells. The reason
for S phase arrest is most likely due to the inhibitory action of thiourea derivatives on DNA
topoisomers (Esteves-Souza, Pissinate, da Graça Nascimento, Grynberg, & Echevarria, 2006; Y.
Zhao, Wang, Wu, Fang, & Zhu, 2012) that overwind or underwind of DNA during replication. This
observation also explains antiproliferative effect of 5a treatment on PC-3 and DU 145 cells as CFU
results showed same colony numbers in treated and control groups but smaller colonies 5a treated
cells. On the other hand, 5a treatment resulted in G1 arrest in LNCaP cells which is in line with the
hypothesis that cytotoxic effect of 5a on these cells is through different mechanisms.
CONCLUSION
This study reports synthesis of some new 1-(5-nitropyridin-2-yl)-4-phenylpiperazine (3) derivatives
by incorporating several biologically important heterocyclic moieties; phenylisothiocyanate,
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benzisothiocyanate and thiazolidinone rings. The structures of new compounds were confirmed by FT
1
IR, H-, 13C NMR, mass spectroscopic and elemental analysis techniques. These organic molecules
were tested for their anticancer activities against androgen-responsive and unresponsive PC cell lines.
Thiourea derivatives exhibited higher cytotoxicity; and of those, 5a was found to be the highest
anticancer activity displaying organic molecule. Molecular mechanism analysis revealed that 5a
exerted its activity through induction of DNA damage, apoptosis and cell cycle arrest. This study
highlights the importance of novelthiourea derivative 5a, as a potential anti-cancer agent to improve
PC chemotherapy by inducing apoptosis. Further studies are required to explore its exact action of
mechanism that may allow us to elucidate functional groups’ roles in the anticancer activity.
Moreover, this organic molecule might be modified to specifically target cancer cells for clinical
perspective. Combination with known anticancer agents to increase clinical benefits would be another
option for chemothraphy resistant cancer types.
Acknowledgments
The support provided by Scientific and Technological Research Council of Turkey (TUBITAK,
Project no: 116Z932) is gratefully acknowledged.
Research data are not shared.
Conflict of Interest
Authors do not have any conflict of interest.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: aguldgn@gmail.com (A. Doğan); demirciserpil17@gmail.com (S. Demirci).
TBH provided and cultured cancer cells and conducted prolideraion analyses, BK and HBŞ conducted
all remaining in vitro experiments, FŞ and SD helped analyzing the data. Serpil Demirci synthyzed
and characterized the chemicals. Serpil Demirci and Ayşegül Doğan designed the experimens and
organized as a manuscript.
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ORCID
Ayşegül Doğan: 0000-0003-4160-2270; Serpil Demirci: 0000-0002-6579-4273
SUPPLEMENTARY MATERİAL
Supplementary File. 1 H- and 13C-NMR spectra of each reference compound.
Supplementary Video 1. Live imaging for DU 145 cells treated with 5a
Supplementary Video 2. Live imaging for PC-3 cells treated with 5a
Supplementary Video 3. Live imaging for LNCaP cells treated with 5a
ABBREVIATIONS USED
1
FT IR, Fourier transform infrared spectroscopy; KBr, potassium bromide; H NMR, proton nuclear
magnetic resonance; 13C NMR, carbon nuclear magnetic resonance; APT, Attached proton test;
HRMS, high-resolution mass spectrometry; TLC, thin-layer chromatography; TMS, tetramethylsilane;
DNA, deoxyribonucleic acid; RNA, ribonucleic acid; IC50, half-maximum inhibitory concentration;
PC, Prostate cancer; RTKs, receptor tyrosine kinases; PTKs, protein tyrosine kinases; JSP-1, JNK-
stimulating phosphatase-1; SHP-2, non-membrane protein tyrosine phosphatase; DMSO-d6,
deuterated dimethyl sulfoxide; CDCl3, deuterated chloroform; Pd/C, palladium on carbon; EtOAc,
ethyl acetate; DCM, dichloromethane; H2NNH2.H2O, hydrazine hydrate; CH3COONa, sodium
acetate; s, singlet; d, doublet; dd, doublet of doublets; m, multiplet; t, triplet; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; DMEM, dulbecco`s modified eagle media;
FBS, fetal bovine serum; PSA, penicillin-streptomycin-amphotericin; GI50, 50% growth inhibition;
TGI, total growth inhibition; LC50, 50% cell death; NIH, National Institutes of Health; CFU, colony-
forming unit; q-PCR, Quantitative RT-PCR.
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Figure Legends
Figure 1: Effect of compund 5a on colony formation ability of cancer cell lines. Colony-forming
capacity of PC-3, DU145 and LNCaP cells were demonstrated in comparison with 5-Fluorouracil (5-
FU). Colonies were stained with crystal violet and counted. The numbers of colonies were
significantly low only in LNCaP cells. The diameters of colonies were small in compund 5a treated
cancer cell lines which was shown in colony morphology images. Notes: * p<0.05, Scale bar: 100 µm,
5-FU: 5-Fluorouracil.
Figure 2: Morphological observation of apoptotis and gene expression analyses of
apoptotic/proliferative pathway genes. (A) Light microscope analyses of 24h compund 5a treated PC-
3, DU 145 and LNCaP. Apoptotic cells with round morphology were observed after 24 h exposure in
both compund 5a and 5-Fluorouracil (5-FU) treated groups. (B) Gene expression analyses of
compund 5a treated PC-3, DU 145 and LNCaP cells. Expression of apoptotic/proliferative genes
exerted a diverse pattern in three different cell types indicating the potential cell type spesific action.
Notes: Scale bar: 200µm, 5-FU: 5-Fluorouracil.
Figure 3: Effect of compund 5a on DNA fragmentation. Compund 5a and 5-Fluorouracil (5-FU)
treatment induced DNA damage in PC-3, DU145 and LNCaP cells which was detected by agrose
DNA gel electrophoresis. DNA fragmentation was determined by 1.0% agarose gel electrophoresis of
genomic DNA isolated from Compund 5a and 5-FU treated prostate cancer cell lines. Notes: 5-FU: 5-
Fluorouracil.
Figure 4: Effect of compund 5a treatment on cell cycle progression in PC-3 (A), DU145 (B) and
LNCaP (C) cells. Compund 5a treatment resulted in cell accumalion at S phase for PC-3 and DU145
cells. In contrast, LNCaP cells were not able to pass through to the S and G2/M phases followed by
compund 5a treatment. Notes: 5-FU: 5-Fluorouracil.
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Table 1. Pharmacokinetic parameters important for good oral bioavailability of synthesized
compound 5a and 6a.
Entry
%
ABS
TPSA
(A2)
n-
ROTB
MV
MW
miLogP n-ON
acceptors
n-OHNH
donors
Nviolations
-
-
-
-
≤500
≤5
<10
<5
2
≤1
0
94.02
91.05
43.42
52.04
6
4
357.30 389.53 3.37
383.75 429.55 4.87
5
6
5a
6a
0
0
% ABS: percentage absorption, TPSA: topological polar surface area, n-ROTB: number of
rotatable bonds, MV: molecular volume, MW: molecular weight, miLog P: logarithm of
partition coefficient of compound between n-octanol and water, n-ON acceptors: number of
hydrogen bond acceptors, n-OHNH donors: number of hydrogen bonds donors.
Table 2. GI50, TGI and LC50) and IC50 values of the test molecules determined by MTT test
DU145
GI50
PC3
LnCaP
GI50
TGI
LC50
>500
>500
IC50
GI50
TGI
LC50
>500
>500
IC50
TGI
>500
>500
LC50
>500
>500
IC50
No
3
17.23
9.44
>500
>500
74.66
18.48
>500
>500
79.14
18.09
>500
>500
4
174.9
6
175.3
5
160.4
1
160.4
1
5a 3.34
469.5
5
>500
>500
102.9
5
4.62
15.40
>500
>500
14.90
6.16
4.71
301.1
3
>500
>500
301.13
85.99
5b 13.63
>500
>500
21.06
>500
>500
118.7
0
6a 64.49
6b 45.76
>500
>500
>500
>500
>500
>500
15.60
6.54
>500
50.72
>500
>500
>1000 13.50
36.75 13.07
>500
>500
>500
>500
>500
>500
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Scheme 1. The synthetic route of key intermediates 3-6a
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